Pipermint

Journal of Ethnopharmacology 87 (2003) 123142
Preliminary comparative analysis of medicinal plants used in the traditional medicine of Bulgaria and Italy
Maria Lucia Leporatti a, , Stephanie Ivancheva b
a
Department of Vegetal Biology University La Sapienza, 00185 Roma, Italy b Bulgarian Academy of Science, Soa, Bulgaria
Received 12 February 2002; received in revised form 6 February 2003; accepted 6 February 2003
Abstract Despite their geographical, historical and cultural differences, Bulgaria and Italy share a surprisingly similar patrimony as regards the popular uses of medicinal plants. The extensive knowledge acquired over the centuries by people living in these countries and engaged in agriculture, derives from continuous contact with natural resources. This paper compares approximately 250 medicinal plants present in both countries and used in popular medicine. From this comparison it emerges that knowledge of medicinal plants and their uses are well founded. In fact, more than 80% of the plants are employed in identical or similar kinds of ailments, their preparation also showing marked similarities. The remaining 20% have very different uses, several of these being particularly noteworthy. The role played by edible plants, moreover, is important, about 30% being employed as medicine. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Ethnobotany; Bulgaria; Italy
1. Introduction Bulgaria and Italy are very different countries from several points of view, historical, phytogeographic and even religious but, over the centuries, the people living in them have resorted equally to their respective patrimonies regarding the knowledge of medicinal plants. The landscape of both countries alternates imposing mountains with atlands, where people have been engaged in agricultural activities since ancient times. Such knowledge is, therefore, the result of experience developed through continuous contact with natural resources down through the ages.
2. Methodology The present work consists of a preliminary comparative analysis of a large number of plants claimed to have medicinal properties in the popular traditional medicine of Bulgaria and Italy. About 250 plants of different hierarchic
Corresponding
author.
order (species and/or subspecies), among the very numerous examples belonging to both oras, have been examined in relation to their availability and the frequency of their use in the two countries. The analysis was carried out rstly in accordance with the respective oras: Bulgarian Flora (Stojanov et al., 1967) and Flora dItalia (Pignatti, 1982). The Bulgarian Flora records 3700 species in all, whilst 5599 species are recorded in Flora dItalia, the respective geographical differing greatly. The specic literature of the respective countries was then compared. As regards Italian ethnobotanical literature, papers were mainly consulted which relate to whole regions or large geographic areas (Gastaldo, 1987; Guarrera, 1990; Tammaro, 1984) but also specic contributions about individual, smaller areas (Bruno et al., 1959; Corsi and Pagni, 1978; Lentini et al., 1987/1988, 1994, 1994/1995, 1995, 1996; Lentini and Raimondo, 1990; Leporatti et al., 1985a,b; Pagni and Corsi, 1979; Pieroni, 2000; Raimondo and Lentini, 1990; Uncini Manganelli and Tomei, 1999a,b; Reuther and Reuther, 1988). For the Bulgarian Flora we consulted Iordanov et al. (1977), Isaiev et al. (1977), Ivancheva and Stantcheva (2000), Kitanov (1953), Petkov (1982), Iordanov et al. (1969), Isaiev et al. (1992), and Petkov (1982).
0378-8741/03/$ see front matter 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00047-3
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M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142
Table 1 Most numerously represented families Families Asteraceae Lamiaceae Apiaceae Rosaceae Fabaceae Number of taxa 24 19 16 17 11 Families Brassicaceae Liliaceae Ranunculaceae Scrophulariaceae Solanaceae Number of taxa 9 9 8 6 6
3. Discussion The species examined belong to 72 families and 72 genera. Many families are present only in a small number of species, e.g. Caryophyllaceae, Rhamnaceae and Thymeleaceae. Table 1 shows the families having a larger number of species.
4. Results Table 2 presents a commented list of the species examined and their uses.
contrary, Crataegus laevigata is acclaimed in Bulgaria for its anti-Herpes activity but unknown in Italy. Also the hepatoprotective and cholagogue action of Chelidonium majus is unknown in Italy where this plant has only external uses. Most of the plants in both the countries, however, have the same or comparable therapeutic uses, only about 20% of them being employed in a completely different way. For example, Astragalus glycyphyllos used in Bulgaria against menstrual pains is, instead, known in Italy as a diuretic and antirheumatic, or again Betonica ofcinalis used as an antiseptic in Bulgaria and for its hepatoprotective, cholagogue and diuretic properties in Italy. In Bulgaria, Galanthus nivalis is said to ght the effects of curare poisoning, but in Italy has only an external use against foruncolosis and abscesses; also Sedum acre in Italy is only used externally for cicatrizing sores and infected wounds whilst in Bulgaria it is employed as a hypotensive (in Table 2 the plants having different therapeutic properties are marked with the symbol ). Other species present several common properties and differ in only one or two uses. Several plants present interesting therapeutic properties (Table 3). In particular: - Viola odorata employed in cases of particular dermatitis: only those related to the presence of arthritic illness; this reects notable intuition as, in fact, arthritis and dermatitis are often closely related as symptoms of some autoimmune diseases. - Ecballium elaterium and Verbascum sinuatum considered active against psoriasis (this is also an autoimmune disease). The prescription from the Italian region of Sicily is characteristic: The aerial parts of the plants are crushed and soaked in cold water and untreated wine. The suspension is boiled by heating with a hot ame until half has evaporated. It is left to stand for 12 h at room temperature and then ltered. The ltrate must be kept in a refrigerator until required for use. (Amenta et al., 2000). One could hypothesise that these plants, given the abundance of long stiff hair on the aerial parts, would have a largely mechanical and abrasive action on the affected skin, but the complex process which the recipe demands would suggest rather more than this. - Daucus carota and Pastinaca sativa subsp. sativa considered as coronary dilators and capillarotrophic agents. - Corylus avellana used against benign prostatic hypertrophy (BPH). - Crataegus laevigata is considered to be effective against Herpes simplex. - Chelidonium majus and Solanum melongena as hepatoprotective agents are also interesting. Each one of these plants and their uses deserves careful consideration so as to stimulate further investigation. In both countries the most frequently used methods for preparing traditional medicines are infusion, decoction and maceration in wine. For the small number of plants (5% ca.)
5. Discussion and conclusion From attentive examination of the results, some points may be drawn: people have mainly resorted to the use of spontaneously occurring plants (wild or cultivated), exotic plants accounting for only 10 cases (Aloe, Cannabis, Eucalyptus, etc.). Some general considerations may be extrapolated regarding the different plant species or subspecies used in the two countries. For example, in Bulgaria only Origanum heracleoticum is used, whilst in Italy both O. heracleoticum and Origanum vulgare are employed, this latter being more common than the former; Heracleum sphondilium is used in Italy without distinction of subspecies, whereas in Bulgaria only the subspecies sibiricum is employed in popular medicine. The same applies to Helleborus odorus used in Bulgaria, whilst in Italy several species of this genus are equally employed, as is also the case with Aristolochia sp. div. This may be due to the abundance or availability of these plants in the respective countries, but also to well-grounded botanical knowledge of the plants and of their therapeutic properties. This is the case of the three species of Potentilla: anserina, erecta and reptans considered effective in relation to three different kinds of disease. Often therapeutic properties which have been well known since ancient times in one of the two popular medicines are completely unknown in the other, e.g.: the anti-helmintic and antipyretic properties of Artemisia absinthium, or the hypotensive activity of Crataegus laevigata, common in traditional Italian medicine are unknown in Bulgaria. On the
Table 2 List of examined species

Botanical name Family Local name in Bulgaria Kokitche Local name in Italy Bucaneve Therapeutic use in Bulgaria Anticurare Therapeutic use in Italy Part used in Bulgaria Aerial parts Part used in Italy Bulb Bulgarian manipulation Source of nivalin Maceration in hot water Crushed and boiled fruits Extract with white wine Fresh juice (for Vitamin C content), infusion Infusion Italian manipulation Notes
Galanthus nivalis L.
Amaryllidaceae
Abscesses, furunculosis, arthritis, synovitis As mouth wash Appetiser, spasmolytic, diuretic, carminative Stimulates digestion, tonic, carminative Antiscorbutic, diuretic, expectorant, appetiser carminative, (seeds) Antiseptic for skin and mucosa, to promote peripheral circulation, externally applied to increase milk secretion, appetiser, uterotonic Aromatic to avour food (raw seeds), spasmolytic, to stimulate digestion, antiseptic Nutritive, carminative, depurative, soothes the skin, coronary dilating (seeds) Anti-oedema, diuretic, cholagogue, choleretic, to promote perspiration Carminative, diaphoretic, diuretic Sedative for CNS, against nervous depression
Cataplasm, poultice, locally applied ointment Infusion Infusion Elixir Infusion
Toxic
Cotinus coggygria Scop. Anethum graveolens L. Angelica archangelica L. Apium graveolens L.
Anacardiaceae Apiaceae Apiaceae Apiaceae
Smradlika Kopar Letchbna Pichtjalka Zelina
C` otino, Sc` otano Aneto Angelica Sedano
Astringent, anti-inammatory Carminative, spasmolytic Sedative, spasmolytic Diuretic
Leaves Fruits, oil Roots, fruits Roots, fruits
Leaves, owers, bark Fruits Roots Leaves, fresh stems, seeds, (fruits) Fruits
External use. Contact can cause skin ulcers
Dried fruits are used to avour food Employed in making candies and liqueurs Aerial parts are eaten as salad or boiled in soups
Carum carvi L.
Apiaceae
Kim
Cumino
Carminative, appetiser, spasmolytic stimulates gastric secretion, cholagogue, uterotonic
Fruits
Decoction
Dried fruits used to avour food
Coriandrum sativum L.
Apiaceae
Koriandar
Coriandolo
Stimulates gastric secretion spasmolytic, carminative Anti-ascaridic, source of carotenoids Prostatitis, diuretic, spasmolytic Carminative, stimulates gastric secretion -------Hypotensive, spasmolytic, appetiser, antidiarrhoeic, gastrointestinal diseases
Fruits
Fruits
Infusion
Tincture, infusion
Dried fruits used to avour food
Daucus carota L.
Apiaceae
Morkov
Carota
Roots, seeds
Roots
Juice of fresh root, seeds Infusion
Juice as drink, cataplasm of fresh root pulp Decoction, tincture
Roots are eaten boiled or raw as vegetable
Eryngium campestre L.
Apiaceae
Vetrogon
Calcatreppola
Roots
Roots
Foeniculum vulgare ssp. vulgare Miller et F.v. ssp. piperitum (Ucria) Coutinho Heracleum sphondilium L. Heracleum sphondilium L. ssp. sibiricum (L.) Simonkai
Apiaceae
Rezee
Finocchio
Fruits
Fruits
Infusion
Infusion
Base of leaves of cultivated species eaten as vegetable. Seeds used to avour food Toxic. Subspecies used in Italy without distinction The only subspecies growing in Bulgaria
Apiaceae Apiaceae
-------Sibirski divisil
Spondilio, Panace
-------Roots, fruits
Aerial parts, fruits
-------Maceration in cold water
Tincture
Levisticum ofcinale Koch Pastinaca sativa L. ssp. sativa
Apiaceae Apiaceae
Letcheben selim Pachtarnaksta
Sedano di monte Pastinaca
Diuretic, stimulates gastric secretion Cardio-tonic, spasmolytic, hypotensive, coronary dilator, capillarotrophic agent
Diuretic, carminative, reduces intestinal gas Dietetic, diuretic, cholagogue
Roots Roots, seeds
Roots Roots, leaves
Decoction Decoction
Decoction Infusion
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Table 2 (Continued )
Botanical name Family Local name in Bulgaria Magdanoz Local name in Italy Prezzemolo Therapeutic use in Bulgaria Diuretic, spasmolytic, abortive Therapeutic use in Italy Part used in Bulgaria Fruits, roots Part used in Italy Roots, leaves Bulgarian manipulation Maceration, infusion Italian manipulation Notes
Petroselinum crispum Mill.
Apiaceae
Diuretic, emmenagogue (abortive!). Nutritive being rich in Vitamins A and E. For insect stings (juice of leaves rubbed locally) Astringent, emmenagogue Aperitive, aromatic, digestive, spasmolytic Expectorant, against catarrh Tonic, haemostatic, diuretic, antiscorbutic, hypotensive Rheumatic pains, against gout, anti-inammatory for gastrointestinal and respiratory tract Appetizer, diuretic, insecticidal, antibacterial Expectorant, antitussive, antineuralgic, anaesthetic in rheumatic and arthritic pains (external use) to clean wounds, to darken hair Vulnerary
Juice or decoction from roots or from fresh leaves
Fresh leaves are used to avour food
Peucedanum ofcinale L. Pimpinella anisum L. Pimpinella saxifraga L. Vinca minor L. Arum maculatum L.
Apiaceae Apiaceae Apiaceae Apocynaceae
Samodivska treva Anason Bedreniza Malak zimzelen Zmijarnik
Finocchio di porco Anice verde Tragoselino, Becchino Pervinca Aro, Gigaro, Pan di serpe
Cardio-tonic Antitussive, to stimulate gastric secretion Antitussive Hypotensive, sedative, astringent Diseases of gastrointestinal tract, lung diseases, haemorrhoids Appetizer, antidiarrhoeic Anti-inammatory, antitussive
Roots, fruits Fruits, oil Fruits Leaves Tuber
Roots Fruits Roots Aerial parts Tuber
Decoction Infusion Maceration Decoction Maceration in cold water
Infusion Infusion, tincture Decoction Infusion, tincture Tincture Toxic plant Used in making candies and liqueurs External use for eye diseases (Bg)
Araceae
Acorus calamus L. Hedera helix L.
Araceae Araliaceae
Blaten air Brachljan
Calamo aromatico Edera
Rhizome Leaves
Rhizome Leaves
Decoction Decoction
Decoction Infusion, decoction Toxic. The fruits can cause severe allergies. Internal use in Italy now abandoned
Aristolochia clematitis L.
Aristolochiaceae
Valtcha jabalka
Strallogi
Inamed wounds
Roots
Rhizome
Maceration, decoction
Decoction
Toxic plant; only external use in Italy; also used A. rotunda L. and A. pistolochia L. In Bulgaria, particularly against chronic alcoholism; abandoned due to its toxicity in Italy
Asarum europaeum L.
Aristolochiaceae
Kopitnik
Erba Renella
Antitussive, sedative, expectorant
Expectorant, emetic (strong doses)
Roots
Rhizome, leaves
Infusion
Periploca graeca L. Asplenium trichomanes L.
Asclepiadaceae Aspleniaceae
Garbatch Iztravnitche
Topa Erba Rugginina
Cardiotonic Antitussive
Cardiotonic, hypertensive, diuretic, purgative (leaves) Expectorant, emollient, externally for dandruff Expectorant, diuretic astringent. Externally applied on burns and inamed mucosa, anti-helmintic. Externally used as cicatrizing agent To improve blood circulation, haemostatic, to aid digestion, against menstrual pains, bitter-tonic, haemorrhoids Cicatrizing agent in skin diseases, antibacterial, antimycotic Anti-helmintic, diuretic, emmenagogue, antipyretic, mouth wash, antiseptic
Bark Aerial parts
Bark, leaves Aerial parts
Tincture Infusion
Decoction, infusion (leaves) Decoction
Scolopendrium ofcinale Sm. (=Phyllitis s.) (L.) Newmann
Aspleniaceae
Volski ezik
Lingua di cervo, Lingua di cane
Antitussive
Aerial parts
Leaves, rhizome
Infusion
Infusion, decoction
Achillea millefolium L.
Asteraceae
Bel ravnez
Millefoglio, Erba Livia
Astringent, haemostatic
Aerial parts
Aerial parts
Infusion
Infusion
Arctium lappa L. and A. nemorosum Lej.
Asteraceae
Repei
Bardana
Diuretic, ulcer
Roots
Leaves, roots
Decoction
Decoction, cataplasm of leaves is locally applied Decoction
In Italy A. minusBernh are used equally. Boiled leaves and stalks are eaten as a vegetable Leaves and owering tops are used to give aroma to liqueurs
Artemisia absinthium L.
Asteraceae
Pelin
Assenzio
Appetizer, to stimulate gastric secretion, mouth wash
Aerial parts
Aerial parts
Infusion
Artemisia vulgaris L.
Asteraceae
Div pelin
Assenzio selvatico, Canapaccio
Appetizer, astringent, sedative, haemostatic
Tonic, aromatic, emmenagogue
Aerial parts (not to be used in cases of gastric ulcer) Capitula (heads) Capitula (heads) Roots
Aerial parts
Infusion
Decoction
Bellis perennis L. Calendula ofcinalis L.
Asteraceae Asteraceae
Paritchka Neven
Pratolina Fiorrancio
Inamed wounds Spasmolytic, inamed wounds, analgesic Diuretic, anti-inammatory, for urinary tract Appetizer, tonic, diuretic Cholagogue stimulant for gastric secretion, hypoglycaemic Appetizer, stimulates gastric secretion Appetizer
Emollient, vulnerary, against sores and bruises Soothing and emollient for skin, against menstrual pains, against warts To promote sweating and secretions, diuretic, cholagogue As mouth and eye wash, against coughs, tonic Choleretic, hepatoprotective against jaundice mild laxative (water after boiling), hypoglycaemic Antipyretic, to aid digestion, tonic cholagogue, expectorant, anti-acid Dietetic, to promote digestion and diuresis. Mild sedative (seeds) Source of inuline and betaine, dietetic, hypoglycaemic against uric acid Diuretic, astringent, antipyretic, anti-inammatory Against bronchitis, catarrh and coughs; bitter-tonic, choleretic, diuretic, externally applied for exanthemata and itching Mild sedative, cicatrizing agent, emollient and soothing for the skin, spasmolytic for gastrointestinal tract In the past claimed as anticarcinoma Sedative, for CNS, anti-asthma and cough, diuretic, to promote perspiration Digestive troubles, amenorrhoea, dysmenorrhoea Choleretic, cholagogue, hepatoprotective
Capitula (heads) Leaves and owering tops Roots
Maceration Infusion
Infusion Decoction
Carlina acanthifolia All.
Asteraceae
Rechetka
Carlina
Infusion, alcoholic extract Infusion Decoction
Decoction, alcoholic extract Infusion Decoction, squashed fresh leaves In Italy the boiled leaves are mainly eaten as a vegetable
Centaurea cyanus L. Cichorium intybus L.
Sinja metlitchina Sinja zlatchka
Fiordaliso Cicoria
Flowers Roots, aerial parts
Flowers Leaves
Cnicus benedictus L.
Asteraceae
Presetchka
Cardo santo
Aerial parts
Aerial parts
Infusion
Infusion
Helianthus annus L.
Asteraceae
Slantchogled
Girasole
Leaves
Flowering tops, seeds Tuber, oil from the seeds Aerial parts
Infusion
Infusion, tincture
In Italy the toasted seeds are enjoyed as snacks. Oil is used in cooking Mainly ornamental; edible tuber eaten boiled or roasted
Helianthus tuberosus L.
Asteraceae
Gulia
Topinambur, Patata del Canada
Provitamin A
Leaves
Infusion
Hieracium pilosella L.
Asteraceae
Michi uchi
Pilosella
Diuretic, astringent
Aerial parts
Infusion
Infusion
Inula helenium L.
Asteraceae
Bjal oman
Enula campana
Anti-inammatory, antitussive anti-ascaridic
Roots
Roots
Maceration
Infusion, decoction, tincture
Matricaria chamomilla L. (=Ch. recutita (L.) Rauschert)
Asteraceae
Laika
Camomilla, Capomilla
Anti-inammatory antiseptic, spasmolitic in colitis and gastritis, choleretic, mouth wash in tooth ache Diuretic, stimulant of gastric secretion Spasmolytic
Flowers (heads)
Capitula (heads)
Infusion
Infusion
Used in aromatizing liqueurs
Onopordon acanthium L. Petasites hybridus L.
Magarechki bodil Letchebna ovtcharka
Cardo asinino Farfaraccio
Aerial parts Leaves
Juice from the fresh leaves Capitula (heads), leaves, rhizome Flowering plant Fruits
Infusion Infusion
Abandoned Infusion, decoction, tincture
Senecio vulgaris L. Silybum marianum (L.) Gaertn.
Sporez Bjal tran
Calderugia, Verzellina Cardo Mariano, Cardo di Maria
Cholinolitic, uterotonic Choleretic cholagogue, hepatoprotective urinary and bladder diseases
Aerial parts Seeds, fruits
Decoction Decoction
Decoction Decoction
Toxic
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128
Botanical name Family Local name in Bulgaria Gorski entchez Local name in Italy Verga doro Therapeutic use in Bulgaria Diuretic, antitussive, urinary diseases (nephritis) Anti-ascaridic, antiseptic, anti-inammatory Cholagogue, choleretic Therapeutic use in Italy Part used in Bulgaria Aerial parts Part used in Italy Flowering tops, rhizome Aerial parts, fruits Rhizome Bulgarian manipulation Infusion Italian manipulation Notes
Solidago virga-aurea L.
Asteraceae
Emollient, anti-inammatory, astringent, diuretic, in case of nephritis Anti-helmintic
Infusion
Tanacetum vulgare L.
Asteraceae
Vratiga
Tanaceto
Aerial parts
Infusion
Infusion
Toxic
Taraxacum ofcinale Weber
Asteraceae
Gluchartche
Tar` assaco, Dente di leone, Sofone F` arfara, Tassilaggine
Diuretic, cholagogue, choleretic, eupeptic, digestive depurative Expectorant, emollient for catarrh, against cough, soothing for the skin
Roots
Infusion
Decoction
Leaves are often eaten mixed in salad
Tussilago farfara L.
Asteraceae
Podbel
Antitussive (whooping cough), anticatarral, respiratory disease, (silicosis, emphysema, tubercular cough) antiseptic, anti-inammatory Cholagogue, diuretic, against diarrhoea and dysentery Astringent, antidiarrhoeic Diuretic, cholagogue
Leaves
Leaves, capitula (heads)
Infusion
Infusion
Raw leaves are eaten as salad
Berberis vulgaris L.
Berberidaceae
Kisel tran
Crespino
Haemostatic, diuretic astringent Mouth wash, anti-inammatory Antipyretic, diuretic, cholagogue, diaphoretic, in skin diseases Emollient, lenitive, mild laxative, diuretic Diuretic, expectorant, vitaminic, to promote perspiration Astringent, antidiarrhoeic, vulnerary To promote digestion. As spices in food, rubefacient
Roots, fruits
Roots, bark, leaves, fruits Bark
Infusion
Decoction
Fruits are rich in vitamins, employed in preparing home-made jams
Alnus glutinosa (L.) Gaertner
Betulaceae
Tchema elcha
Ont` ano
Crushed cones or bark in water Bark
Decoction
Decoction
Betula pendula Roth (=B. alba L.) Borago ofcinalis L.
Betulaceae
Breza
Betulla
Bark
Infusion, decoction Maceration
Infusion, decoction
Boraginaceae
Krasta vitchna treva Meduniza
Borrana, Borragine
Diuretic, to promote perspiration Antitussive, anti-inammatory, diuretic, expectorant Inamed wounds Appetizer, to stimulate gastric secretion, antiscorbutic To stimulate ow of blood
Aerial parts
Leaves, owering tops Leaves, owering tops Roots, leaves, owering tops Roots
Infusion, decoction
In Italy the aerial parts raw or boiled are eaten as a vegetable
Pulmonaria ofcinalis L.
Boraginaceae
Polmonaria
Aerial parts
Infusion
Decoction, infusion
Symphitum ofcinale L. Armoracia rusticana Gaertner, Meyer and Scherb
Boraginaceae Brassicaceae
Tcherenoman Hrjan
Consolida maggiore Cren, Barbaforte
Roots Roots
Decoction Decoction, crushed roots mixed with honey Crushed seeds with honey
Infusion Fresh roots, cataplasm Used as spice on meat and sh
Brassica nigra (L.) Koch
Brassicaceae
Sinap, hardal
Senape nera
To stimulate blood-ow, as cataplasm locally applied on painful joints, catarrh, bronchial diseases Antiscorbutic, cicatrizing agent for sores and burns
Seeds
Crushed seeds
Cataplasm powdered seeds boiled in water
External use
Brassica oleracea L.
Brassicaceae
Zele
Cavolo
Choleretic, anti-ulcer
Aerial parts
Leaves crushed or scalded Aerial parts
Fresh
Cicatrizing cataplasm, cicatrizant, leaves raw or boiled as antiscorbutic Decoction, juice of fresh plant or dried + powdered plant
Boiled leaves, buds or the whole plant are eaten as a vegetable
Capsella bursa-pastoris L.
Brassicaceae
Ovtcharska torbitchka Poretch Tcherna rjapa
Borsa di pastore, Borsacchina Nasturzio Ravanello Radicette
Astringent, menstrual diseases Source of vitamins Cholagogue, antitussive
Haemostatic, diuretic, astringent, for menstrual pain Source of Vitamins A, B, and C, antiscorbutic, diuretic Digestive, to stimulate blood-ow
Aerial parts
Maceration
Nasturtium ofcinale R. Br. Raphanus sativus L.
Brassicaceae Brassicaceae
Aerial parts Roots
Whole plant Roots
Fresh plant Fresh plant
To be eaten as vegetable Fresh roots are eaten as vegetable
Sinapis alba L.
Brassicaceae
Bjal sinap
Senape bianca
Laxative
Laxative (whole entire seeds), aromatic stimulates digestion, for painful joints (e.u.) -------Sedative for CNS and sexual apparatus, bitter-tonic Diuretic, antirheumatic, laxative antineuralgic, anti-inammatory Diuretic, antirheumatic, laxative antineuralgic, anti-inammatory, emollient, dislocated bones (e.u.) Uterotonic, anti-abortive Diuretic, in case of gall-stones Expectorant, to promote secretion of respiratory and gastrointestinal tract Astringent, diuretic, vulnerary As cicatrizing agent (e.u.), emollient, mild laxative, anti-anaemic (rich in iron) Laxative, diuretic, against furunculosis Mild sedative, cholagogue, carminative Antidiarrhoeic, anti-haemorrhagic, antipyretic Antiseptic, intestinal astringent, for haemorrhoids (piles), antiseptic for minor wounds and sores Externally applied to sores, infected wounds, warts, anti-atherosclerosis Diuretic, purgative, externally applied on painful joints, or on furuncles Drastic purgative, against psoriasis
Seeds
Seeds
Crushed
Decoction of crushed, powdered seeds applied locally -------Infusion, decoction Decoction, infusion
Seeds are used in preparing sauces
Cannabis sativa L. Humulus lupulus L. Sambucus ebulus L.
Cannabaceae Cannabaceae Caprifoliaceae
Konop Chmel Bazak
Canapa L` uppolo Ebbio
Anti-inammatory, spasmolytic Sedative Diuretic, antiseptic, antitussive Diuretic, to promote perspiration
Seeds Cones (female inorescence) Roots, fruits, owers Flowers, fruits
-------Cones (female inorescence) Roots, fruits, owers, leaves Flowers, fruits
Maceration Infusion Infusion
Illegal use in Italy Mainly used to amortise beer Toxic
Sambucus nigra L.
Caprifoliaceae
Tcheren baz
Sambuco
Infusion, decoction
Infusion, squashed leaves locally applied Infusion (high doses are toxic) Infusion
Inorescence are fried and eaten, fruits are employed in preparing home-made jams
Viburnum opulus L. Herniaria glabra L.
Caprifoliaceae Caryophyllaceae
Tchervena kalinka Golo izsipli-vtche
Pallone di maggio Erniaria
Uterotonic, astringent, haemostatic Diuretic, spasmolytic
Roots Aerial parts
Bark The whole plant with the root Roots
Infusion Infusion
Toxic
Saponaria ofcinalis L.
Caryophyllaceae
Letchebno sapuntche Vrabtchovitchrevza Tchuven
Saponaria
Antitussive, diuretic, stimulates sweating Anti-inammatory Inamed wounds
Roots
Infusion
Decoction
Toxic
Stellaria media L.
Caryophyllaceae Chenopodiaceae
Centocchio Buon enrico, Colubrina Vilucchio
Aerial parts Roots
Aerial parts Flowering plant Aerial parts, roots The whole plant Fruits fresh bark, fresh pulp of the fruits Fruits, bark, leaves
Infusion Infusion
Infusion, juice of fresh plant Decoction In Italy, the boiled aerial parts are also eaten as a vegetable
Chenopodium bonus-henricus L.
Convolvulus arvensis L.
Convovulaceae
Polska povetiza, gramofontche Kukuvitcha prezda
Strong laxative, hypertensive (owers), furunculosis Laxative, diuretic, spasmolytic Astringent
Aerial parts
Tincture, cataplasm Decoction
Tincture, infusion, cataplasm Infusion, tincture
Cuscuta europaea L. and related species Cornus mas L.
Convolvulaceae
Cuscuta
Aerial parts
Cornaceae
Drjan
Corniolo
Fruits
Decoction
Decoction
Fruits are used in home-made jams or liqueurs
Corylus avellana L.
Corylaceae
Obik novena leska
Nocciolo
Cardiac diseases, prostatic hypertrophy
Fruits, bark
Infusion
Decoction
Sedum acre L.
Crassulaceae
Ljutivatlastiga
Erba pignola
Hypotensive, stimulant
Aerial parts
Aerial parts
Infusion
Poultice of fresh leaves Cataplasm of fresh leaves (e.u.), infusion Infusion, decoction evaporated then ltered
Under medical supervision
Bryonia alba L. and Bryonia dioica Jacq. Ecballium elaterium (L.) Richard
Cucurbitaceae
Diva tikva
Vite bianca
Diuretic, laxative, to stimulate ow of blood Laxative
Roots
Leaves
Decoction
Toxic
Cucurbitaceae
Luda krastaviza
Cocomero asinino
Fruits
Fruits, aerial part
Infusion
Toxic
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Botanical name Family Local name in Bulgaria Local name in Italy Therapeutic use in Bulgaria Therapeutic use in Italy Part used in Bulgaria Part used in Italy Bulgarian manipulation Italian manipulation Notes
Juniperus communis L.
Cupressaceae
Sinja hvoina
Cipresso
Diuretic, antiseptic for urinary tract
Balsamic, diuretic, antiseptic for urinary tract, antipyretic, antidiarrhoeic, muscular pains (e.u.) Diuretic. Locally applied against warts Emetic, purgative, revulsive
Pseudofruits
Young stem with leaves and pseudofruits Leaves Roots
Infusion, decoction, pseudofruits maceration in olive oil Decoction Crushed and mixed with oil Infusion Infusion
Infusion, decoction or tincture in wine
Pseudofruits are used in aromatizing liqueurs (I)
Thuja occidentalis L. Tamus communis L.
Cupressaceae Dioscoreaceae
Bozjo darvo Brej
Tuja Tamaro
Against corns To stimulate ow of blood Lung diseases Diuretic, astringent, source of microelements Astringent, anti-inammatory, hypoglicemic
Leaves Roots
Decoction, tincture Decoction, tincture Toxic. External use only
Drosera rotundifolia L. Equisetum telmateja Ehrh. (=E. arvense L.)
Droseraceae Equisetaceae
Rosjanka Polski hvocht
Rosolida Coda cavallina
Against cough, bronchial spasmolytic Diuretic, reconstituent to cartilage in lungs. To strengthen hair and nails, source of microelements Astringent, antiseptic, to protect retina and capillary vessels, anti-inammatory for the skin, hypoglicemic (leaves) Diuretic, antiseptic, anti-inammatory Diuretic, rheumatic pains, against gout Hypoglicemic, increasing milk secretion Diuretic, laxative, emetic Antitussive, expectorant, emollient, sedative for stomach-ache, antiprostatic adenoma Cholagogue, choleretic, laxative Anti-inammatory eye wash and nostril wash, mild antiseptic and antipyretic, diuretic, sedative See Ononis spinosa Diuretic, anti-inammatory for the urinary tract and for acne, mouth wash Emollient, cholagogue (owers), laxative or drastic purgative depending on dosage Regulating mucosa secretion
Aerial parts Aerial parts
Aerial parts, roots Aerial parts, owering tops
Decoction Infusion Use must be avoided in case of urinary problems, can provoke haematuria Fresh fruits are used in preparing jams
Vaccinum myrtillus L.
Ericaceae
Tchema borovinka
Mirtillo nero
Leaves, fruits
Leaves, fruits
Decoction
Juice, pulp
Vaccinium vitis-ideae L.
Ericaceae
Tchema borovinka
Mirtillo rosso
Astringent anti-inammatory, hypoglicemic Menstrual pains Hypoglicemic Diuretic, laxative Ulcer, antitussive, laxative, gastric disorders To facilitate respiration
Leaves, fruits
Leaves, fruits
Decoction
Juice from fruit pulp Decoction Infusion, tincture Infusion Infusion
Fresh fruits are used in preparing jams
Astragalus glycyphyllos L. Galega ofcinalis L. Genista tinctoria L. Glycirrhyza glabra L.
Fabaceae Fabaceae Fabaceae Fabaceae
Klinavitche Zja blek Bagrilna zjaltura Sladak koren
Liquerizia bastarda Galega, Capraggine Ginestrella Liquerizia
Aerial parts Aerial parts Aerial parts Roots
Roots, leaves Flowering tops Seeds Roots
Decoction Infusion Infusion Infusion
Dried roots are chewed for sweet taste
Laburnum anagyroides Medicus (=Cytisus l. L.) Melilotus ofcinalis (L.) Lam.
Fabaceae
Zlaten dajd
Maggiociondolo
Seeds
Leaves
Decoction
Infusion
Toxic
Fabaceae
Letchebna komuniga
Meliloto, Trifoglio cavallino
Sedative
Aerial parts
Flowering tops
Infusion
Infusion
Ononis arvensis L. Ononis spinosa L.
Fabaceae Fabaceae
Gramotran Bodliv gramotan
See Ononis spinosa Arrestabuoi, Stanca bue Robinia
Diuretic Diuretic, kidney stones, urinary diseases Antitussive, laxative
Roots Roots
See Ononis spinosa Roots
Maceration Infusion, tincture Infusion
See Ononis spinosa Decoction
This species is rare in Italy
Robinia pseudoacacia L.
Fabaceae
Bjal salkam
Flowers, bark, leaves
Flowers bark, leaves
Infusion (owers), decoction (bark)
Trifolium pratense L.
Fabaceae
Livadna detelina
Trifoglio
Antitussive, diuretic
Flowers
Flowers
Infusion
Infusion
External use, anti-inammatory
Trigonella foenum-graecum L. Quercus petraea (Mattuschka) Liebl (=Q. sessiliora L.) Quercus robur L.
Fabaceae Fagaceae
Grazki sminduh Zimen dab, Gorun
Fieno greco Quercia
Appetizer, anabolic, sedative Astringent, for dermatitis Astringent, for dermatitis Appetizer
Dietetic, nutrient, appetizer, reconstituent Astringent, anti-inammatory, mild antiseptic, anti-haemorrhagic See Quercus petraea
Seeds Bark
Seeds Bark, fruits
Powder mixed with gum Decoction
Powdered seeds, decoction Decoction External use. Roasted seeds used as coffee substitute. Acorns as food for pigs External use. Roasted seeds used as coffee substitute. Acorns as food for pigs
Fagaceae
Leten dab
See Quercus petraea Biondella, Cacciafebbre
Bark
See Quercus petraea Aerial parts
Decoction
See Quercus petraea
Centaurium erythraea Ran.
Gentianaceae
Tcherven kantarion
Antipyretic, cicatrizing agent for sores and minor wounds, appetizer, digestive, hepatoprotective, hypoglicemic Appetizer, bitter-tonic digestive, antipyretic, cicatrizing agent for minor wounds (e.u. of the leaves) Astringent, diuretic, haemostatic, antidiarrhoeic, soothing for the skin, anti-oedema Stomatitis as mouth wash, astringent
Aerial parts
Infusion
Infusion
Gentiana lutea L.
Gentianaceae
Zjalta tintjava
Genziana maggiore, Genziana gialla Cicutaria
Appetizer
Roots, aerial part
Roots, aerial part
Maceration
Decoction, tincture
Roots highly valued in preparing elixirs, bitter-tonic
Erodium cicutarium (L.) LHer
Geraniaceae
Zikutovo tchasovnitche
Astringent
Aerial parts
Aerial parts
Infusion
Infusion
Geranium sanguineum L.
Geraniaceae
Kraven zdravez
Sanguinaria Malvaccini
Hypotensive, antivirus, immuno-stimulant sedative, CNS depressant Vitaminic, astringent, anti-inammatory, diuretic Ulcers, anti-inammatory Haemorrhoids Antitussive, anti-inammatory, stomach-ache Antitussive, anti-inammatory, stomach-ache -------Neurodermatitis
Roots
Aerial parts
Maceration
Infusion
Ribes nigrum L.
Grossulariaceae
Kasis
Ribes
Vitaminic, aperitive, diuretic, astringent, anti-inammatory protects blood vessels and retina Cicatrizing agent, against Herpes simplex cholagogue Anti-oedema, against varicose veins and bruises See Iris germanica
Fruits, leaves
Fruits, leaves
Fresh plant, infusion
Pulp or juice of the fruits, infusion
Fruits used in preparing jams
Hypericum perforatum L.
Guttiferae
Zjalt kantarion
Erba cacciadiavoli, Erba di S. Giovanni Ippocastano Iris, Ireos, Giaggiolo Iris, Ireos, Giaggiolo Ireos, Iris, Giaggiolo ` coro Falso a
Aerial parts
Flowering tops Seeds See Iris germanica Rhizome
Infusion
Infusion or maceration, decoction Decoction See Iris germanica
This plant has a long tradition in magic ritual beliefs
Aesculus hippocastanum L. Iris orentina L.
Hippocastanaceae Iridaceae
Konski kesten Bjala perunika
Bark, fruits Roots
Tincture
Iris germanica L.
Iridaceae
Sinja perunika
Against coughs and catarrh, emollient for the skin Expectorant, cholagogue Astringent, emetic, purgative applied externally as haemostatic Bitter-tonic, digestive, antiseptic hypoglicemic, depurative. Externally applied on reddened mucosa or skin; to darken hair (husk)
Roots
Maceration
Decoction
Iris pallida Lam. Iris pseudacorus L.
Iridaceae
-------Vodna perunika
-------Roots
Rhizome Rhizome
-------Maceration
Decoction, tincture Decoction, tincture
Iridaceae
Juglans regia L.
Juglandaceae
Oreh
Noce
Astringent, anti-inammatory
Leaves
Leaves, walnut husk
Decoction
Decoction
Seeds valued dry fruits. Husk is used to make Nocino liqueur (I)
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Betonica ofcinalis L. (=Stachys ofcinalis (L.) Trevisan Galeopis tetrahit L. Glechoma hederaceae L. Lamium album L.
Lamiaceae Lamiaceae Lamiaceae Lamiaceae
Ranilist Budariza Samobaika Bjala martva kopriva Lavandula
Betonica Erba giudaica, Canapa selvitca Edera terrestre Ortica bianca
Antiseptic for inamed wounds Antitussive Bronchitis Astringent, diuretic, tonic Sedative, spasmolytic
Diuretic, cholagogue, hepatoprotective Diuretic, against catarrah Astringent, tonic, diuretic, against catarrh Astringent, haemostatic, diuretic, uterotonic, skin abscesses and burns (e.u.) Sedative, spasmolytic, externally applied antiseptic, antihysteric Mild cardiotonic, hypotensive and sedative for CNS. Anti-depressive Choleretic, digestive, expectorant, antipyretic, anti-anaemic, antirheumatic, on painful joints (e.u.), against tachycardia Sedative, spasmolytic, digestive, choleretic, externally for headache and neuralgia Choleretic, spasmolytic, antiseptic, anaesthetic, digestive refreshing, balsamic, hepatoprotective, against jaundice See Mentha piperita
Aerial parts Aerial parts Aerial parts Flowers
Leaves Flowering tops Aerial parts
Infusion Decoction Infusion Infusion
Infusion Infusion, tincture Infusion Infusion
Lavandula angustifolia Miller
Lamiaceae
Lavanda
Fruits, oil
Flowers
Infusion
Infusion
Leonurus cardiaca L.
Lamiaceae
Djavolska usta
Cardiaca
Sedative, cardiotonic
Aerial parts
Aerial parts
Maceration
Infusion, tincture
Marrubium vulgare L.
Lamiaceae
Ptchelnik
Marrobio
Cholagogue, spasmolytic, antitussive, menstrual disorders, hepatoprotective Sedative, hypotensive, spasmolytic
Aerial parts
Flowering tops, leaves
Maceration
Infusion, tincture, cataplasm of boiled aerial part
Melissa ofcinalis L.
Lamiaceae
Matotchina
Melissa, Erba limoncina
Leaves, aerial parts
Leaves, aerial parts
Infusion
Infusion
Mentha piperita L.
Lamiaceae
Ljutiva menta
Menta
Choleretic spasmolytic, antiseptic, anaesthetic
Leaves oil
Leaves, owering tops
Infusion
Infusion, tincture
Fresh or dried leaves are used as herb in food
Mentha pulegium L.
Lamiaceae
Blatna menta, divdjodjen
Puleggio
Choleretic spasmolytic, antiseptic, anaesthetic, jaundice, hepatoprotective Choleretic spasmolytic, antiseptic, anaesthetic Antiviral Antitussive, to stimulate gastric secretion
Leaves oil
Leaves, owering tops
Infusion
Infusion
Fresh or dried leaves are used as herb in food (I)
Mentha spicata L. Nepeta cataria L.
Lamiaceae
Djodjen Kotcha treva Bjal rigan
Menta Erba gattaia Origano
See Mentha piperita Mild sedative, spasmolytic, aromatic, digestive Aperitive, digestive. Mild antiseptic, spasmolytic, expectorant, in case of inamed lungs Aperitive, digestive. Mild antiseptic, spasmolytic, expectorant, in case of inamed lungs Aperitive, digestive, aromatic, diuretic, balsamic, antiseptic, skin tonic (promoting cutaneous blood circulation), cholagogue, choleretic, against perspiration
Leaves Aerial parts Aerial parts
Leaves, owering tops Flowering tops Aerial parts
Infusion Infusion Infusion
Infusion Infusion, tincture Infusion, tincure
Fresh or dried leaves are used as herb in food (I)
Lamiaceae Lamiaceae
Origanum heracleoticum L.
Dried leaves are used as herb in food (I)
Origanum vulgare L.
Lamiaceae
Tcherven rigan
Origano
Antitussive, stimulates gastric secretion, choleretic, cholagogue To stimulate gastric secretion, diuretic, choleretic
Aerial parts
Aerial parts
Infusion
Infusion, tincture
Dried leaves are used as herb in food
Rosamarinus ofcinalis L.
Lamiaceae
Rosmarin
Rosmarino
Leaves oil
Leaves
Infusion
Infusion
Fresh leaves are used as herb in food
Salvia ofcinalis L.
Lamiaceae
Gradinski tchai
Salvia
Anti-inammatory
Eupeptic, digestive, cholagogue, antiseptic, expectorant, emmenagogue, as mouth wash, tooth ache Spasmolytic, stimulating digestion, eupeptic, depurative; by gargling against sore throat and as mouth wash, antimycotic Choleretic, stimulant, digestive, antiseptic for gastrointestinal tract Aperitive, digestive, bitter-tonic, antiseptic Antibacterial, expectorant, balsamic, digestive, depurative, aromatic, to stimulate blood-ow, gargling as mouth and tooth wash Hypoglicemic
Leaves
Leaves and owering tops
Infusion
Infusion
Leaves are used in cooking
Satureja hortensis L.
Lamiaceae
Gradinska tchubriza
Santoreggia
Against intestinal gas, spasmolytic, antiviral, astringent, hypotensive, hepatoprotective, digestive Dermatitis ab insectis
Aerial parts
Aerial parts
Infusion
Infusion
The fresh or dried leave are used to avour food
Satureja montana L.
Lamiaceae
Planinska tchubriza
Santoreggia
Aerial parts
Aerial parts
Fresh plant
Infusion, fresh plant
Teucrium chamaedrys L.
Lamiaceae
Tcherveno poddabitche Machterka
Camedrio
Stomach-ache, antimicrobial activity Antitussive, antiviral, spasmolytic
Aerial parts
Flowering tops Aerial parts
Infusion
Infusion
Thymus sp. pl.
Lamiaceae
Timo
Aerial parts
Infusion
Infusion
Leaves are used to avour meat
Allium cepa L.
Liliaceae
Kromid Juk
Cipolla
Appetizer, stomach-ache Anti-atherosclerosis, hypotensive, antiviral Anti-atherosclerosis, antiviral Stimulant, laxative, choleretic
Bulb
Bulb
Fresh bulb
Fresh bulb
Sliced raw bulb is used mixed in salads, also boiled or roasted Sliced cloves are used in several recipes Sliced cloves are mixed in salad In Italy, no longer used in popular medicine. Other species are rarely employed
Allium sativum L.
Liliaceae
Tchesan
Aglio
Anti-atherosclerosis, hypotensive, anti-helmintic, antibacterial, antiviral Stimulates digestion, rubefacient, hypotensive Aperitive, laxative
Bulb
Bulb
Fresh cloves
Fresh cloves
Allium ursinum L.
Liliaceae Liliaceae
Levurda Aloe
Aglio ursino Aloe
Aerial parts, bulb Fresh leaves
Flowering tops, bulb --------
Fresh cloves Extract, infusion (under medicinal control) Decoction
Fresh cloves --------
Aloe arborescens Miller
Asparagus ofcinalis L.
Liliaceae
Asparagus
Asparago
Diuretic
Diuretic
Roots
Rhizome
Decoction
Boiled young stems (turioni) commonly eaten as vegetable (I) Toxic External use. In ancient times, it was believed to be useful in healing broken bones Not used in cases of pyelo-nephritis
Convallaria majalis L. Polygonatum odoratum (=P. ofcinale All.)
Liliaceae Liliaceae
Momina salza Momkova salza
Mughetto Sigillo di Salomone
Cardiotonic Astringent diuretic, anti-inammatory
Cardiotonic Astringent, diuretic, anti-inammatory
Aerial parts Roots
Flowers Rhizome
Tincture Decoction, juice or pulp from squashed rhizome Tincture
Infusion Infusion
Ruscus aculeatus L.
Liliaceae
Michi tchimchir
Pungitopo
Diuretic, haemorrhoids (piles)
Diuretic, antigout. To promote perspiration astringent, anti-inammatory, protects capillary vessels, against haemorrhoids (piles) Applied externally as analgesic on painful joints Emollient, anti-inammatory for the skin, against itching, to regulate intestine (whole seeds)
Roots
Rhizome
Decoction, tincture
Veratrum album L.
Liliaceae
Bjala tchemerika
Veratro, Falso elleboro Lino
Hypotensive
Roots
Rhizome
Tincture
Ointment (in olive oil) Decoction or cataplasm of crushed seeds; also crushed seeds in water are drunk for intestinal problems
Toxic. No longer used in Italy
Linum usitatissimum L.
Linaceae
Len
Laxative, anti-inammatory for gastric and urinary tract
Seeds
Seeds
Maceration
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Viscum album L. Lycopodium clavatum L.
Loranthaceae Lycopodiaceae
Bjal imel Plavun
Vischio Licopodio
Hypotensive Mild skin emollient, uroseptic, diuretic, antipyretic Antitussive, anti-inammatory for respiratory system Spasmolytic, antitussive, sedative, cystitis, cholagogue Appetizer, purgative, in gastric disorders Laxative, anti-inammatory, antitussive Hypoglicemic, hypotensive, diuretic, antitussive Hypoglicemic, hypotensive, diuretic, antitussive Spasmolytic, against bronchitis
Hypotensive, diuretic, spasmolytic Mild emollient for inamed skin Emollient, soothing for the skin, as mouth wash against tooth ache, expectorant, mild laxative Emollient, soothing, mild laxative and expectorant Bitter-tonic, antiscorbutic, diuretic, cholagogue, source of Vitamin A and Fe, Mn, I Laxative, anti-inammatory, antitussive
Leaves Aerial parts, spores Roots
Young stems, leaves Spores
Infusion Infusion
Infusion Powder of spores
Althaea ofcinalis L.
Malvaceae
Rouza
Altea
Roots
Decoction
Decoction
Malva sylvestris L.
Malvaceae
Gorski slez
Malva
Flowers, leaves --------
Flowers leaves Leaves
Infusion
Infusion
Young leaves are boiled to make soups
Menyanthes trifoliata L.
Menyanthaceae
--------
Trifoglio brino, Trifoglio dacqua Fico
--------
Infusion, fresh leaves juice Infusion or decoction of dried fruits, boiled with milk Syrup from the fruits Caustic latex seeping from the green parts is used to remove warts and corns Fruits are eaten fresh or used in preparing jams and ices Fresh fruits are eaten or employed in preparing jams and ices Used areas given water in Italy in marshy to ght malaria, its ability to drain from the soil
Ficus carica L.
Moraceae
Smokinja
Fruits
Dried or fresh fruits are also eaten Fruits, leaves, bark
Boiled with milk
Morus alba L.
Moraceae
Bjala tcherniza
Gelso bianco
Expectorant laxative (fruits), astringent, hypoglicemic (leaves), diuretic, anti-helmintic (bark) Expectorant laxative (fruits), astringent, hypoglicemic (leaves), diuretic, anti-helmintic (bark) Balsamic, expectorant, anticatarrhal
Fruits, leaves
Decoction
Morus nigra L.
Moraceae
Tcherniza
Gelso
Fruits, leaves
Fruits, leaves, bark
Decoction
Syrup from the fruits
Eucalyptus globulus Labill. and E. chamaldulensis Dehnh.
Myrtaceae
Evkalipt
Eucalipto
Leaves, oil
Leaves
Infusion
Infusion, smoke of burning leaves is inhaled Decoction, syrup from owers Decoction, syrup
Nymphaea alba L.
Nympheaceae
Bjala vodnarosa
Ninfea
To attenuate freckles
Narcotic, sedative, astringent (raw rhizome) Laxative, antitussive
Flowers
Flowers, rhizome Juice exuding from the trunk Bark Fruits, oil, leaves
Decoction
The rhizome rich in starch was eaten in times of famine as source of carbohydrate
Fraxinus ornus L.
Oleaceae
Mazdrjan
Orniello Avorniello
Astringent, anti-inammatory, haemostatic -------Cholagogue, hypotensive, cardiotonic, antiarhytmic Antipyretic, appetizer Appetizer
Bark
Infusion
Fraxinus excelsior L. Olea europea L.
Oleaceae Oleaceae
-------Maslina
Frassino Olivo
Antirheumatic antigout Cholagogue, vitaminic, hypotensive (leaves), anticholesterolemic. Locally applied to soothe and heal burns, mild laxative Antipyretic, astringent, to soothe the skin (owers) Depurative, diuretic, astringent, thirst quenching
-------Fruits, oil
-------Tincture oil
Infusion The oil used as seasoning in food, decoction of leaves Pickled fruits as snacks. The oil is highly valued in cookery for its nutritional value
Syringa vulgaris L.
Oleaceae
Ljuljak
Lill` a
Flowers, leaves Aerial parts
Bark, fruits, leaves Leaves
Crushed and boiled in water Fresh state
Decoction, oil maceration (owers) Juice from fresh leaves, decoction Leaves are also eaten as salad
Oxalis acetosella L.
Oxalidaceae
Kiselitche
Acetosella
Paeonia ofcinalis L. Paeonia peregrina Mill. Chelidonium majus L.
Paeoniaceae Paeoniaceae Papaveraceae
-------Tcherven bojur Zmijsko mljako
Peonia -------Celidonia, Erba da porri
Sedative, spasmolytic Sedative, spasmolytic Cholagogue, spasmolytic, hypotensive analgesic, hepatoprotective Cholagogue, spasmolytic, hypotensive Antitussive
Sedative -------To remove warts and corns (e.u.)
-------Roots, owers Aerial parts
Petals, ripe seeds, root -------Latex
-------Infusion Infusion
Infusion -------Latex is locally applied Not used for children
Fumaria ofcinalis L. related species also used Glaucium avum Crantz
Papaveraceae
Letcheben, Rosopas Zjaltmak
Fumastrello
Depurative, to promote circulation and breathing Antiseptic, cicatrizing, to heal minor sores and wounds
Aerial parts
Aerial parts
Maceration
Infusion
Small doses stimulate breathing and circulation, high doses inhibit them Also used in Italy is Glaucium corniculatum J.H. Rudolph
Papaveraceae
Papavero cornuto
Aerial parts
Latex exuding from the broken stem (in June August) Petals --------
Infusion
External use
Papaver rhoeas L. Papaver somniferum L.
Papaveraceae Papaveraceae
Polski mak Sanotvoren mak
Papavero, Rosolaccio Papavero da Oppio
Antitussive Analgesic
Mild sedative, against cough, bronchitis --------
Flowers Opium
Infusion Opium
Infusion --------
Toxic Only under medical supervision. The use and cultivation in Italy is forbidden The seeds are eaten raw and also appreciated in pastry and as snacks The most highly valued species in Italy is P. major L. Leaves are boiled and mixed in soups Seed our was once used in making bread (I)
Pinus sylvestris L.
Pinaceae
Bjal bor
Pino
Antitussive, antiseptic, diuretic, anti-inammatory Emollient, to soothe the skin, bronchial diseases and coughs, mild laxative (seeds) Sedative for CNS
Balsamic expectorant, diuretic Emollient for the skin, laxative, for coughs, soothing, eye wash, mouth wash Sedative emollient for the skin (bath), diuretic in bladder and urinary pains, painful joints (e.u.) Diuretic, emollient for the skin, mouth wash, in renal and urinary problems, emollient for gastrointestinal tract Diuretic
Turiones Pini (young stems) Leaves, aerial parts
Buds, young stems, resin Seeds, leaves
Decoction
Decoction, infusion
Plantago sp. pl.
Plantaginaceae
Tsenolisten zilovljak
Piantaggine
Infusion
Infusion, decoction. Seeds mixed in water drunk as laxative Maceration, decoction
Avena sativa L.
Poaceae
Oves
Avena, biada
Flour, seeds
Flour, seeds
Maceration decoction, cataplasm Decoction
Cynodon dactylon (L.) Pers.
Poaceae
Troskot
Gramigna, Zizzania
Diuretic, laxative
Roots
Roots
Decoction, infusion
Elytrigia repens (L.) Nevski (=Agropyron repens (L.) P.S.) Zea mays L.
Poaceae
Pyrei
Gramigna
Antitussive, anti-inammatory, diuretic Diuretic, cholagogue, astringent
Underground stems Stigmata
Rhizome
Decoction
Decoction, infusion
Poaceae
Zareviza
Granoturco, Mais
Diuretic, depurative, sudoric, hypotensive
Stigmata
Decoction
Infusion, tincture
Seed our commonly used in preparing polenta (I). Oil has anticholesterol properties
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Persicaria bistorta (L.) Samp. (=Polygonum bistorta L.)
Polygonaceae
Karvavitche
Bistorta, Serpentaria
Astringent anti-inammatory, in case of stomatitis Diuretic, astringent, haemostatic Laxative, cholagogue Antiscorbutic, diuretic, tonic Anti-ascaridic
Astringent, anti-inammatory antidiarrhoeic, to wash greasy hair Diuretic, cicatrizing agent, astringent Aperitive, depurative laxative, cholagogue Diuretic, antipyretic, as mouth wash for gingivitis and stomatitis Anti-helmintic (mainly against Taenia solium) Expectorant, diuretic, choleretic, laxative, anti-inammatory, anti-helmintic Diuretic, astringent, antitussive, to stimulate ow of bile, expectorant, stimulates glandular secretion, soothes swollen legs and feet Drastic purgative, emmenagogue, anti-helmintic Diuretic, spasmolytic, expectorant, sedative for CNS, externally applied (decoction) is useful in case of painful joints Anti-helmintic, astringent, thirst quenching (seeds), anticatarrhal, in gingivitis and pyorrhoea Cardiac diseases, diuretic Against rheumatic pains and gout, revulsive ( the fresh plant locally applied can cause skin ulcers), as wash for vein ulcers and furuncolosis
Roots
Leaves, rhizome
Decoction
Infusion, powdered rhizome
Polygonum aviculare L.
Polygonaceae
Patcha treva
Corrigiola Centinodia Rabarbaro Acetosa
Aerial parts
Aerial parts
Infusion
Infusion
Being rich in Si, it was used in the past as a palliative in tuberculosis
Rheum palmatum L. Rumex acetosa L.
Polygonaceae Polygonaceae
Reven Kiselez
Roots Aerial parts
Rhizome Aerial parts
Tincture Decoction
Decoction, powder
Decoction
Fresh leaves are eaten as salad Given its toxicity it is no longer used in Italy
Dryopteris lix-mas L. (=Aspidium f.m. L.) Polypodium vulgare L.
Polypodiaceae
Mazka paprat
Felce maschio
Roots
Rhizome
Decoction
Decoction
Polypodiaceae
Sladka paprat
Felce dolce
Antitussive
Roots
Rhizome
Maceration
Decoction
Anagallis arvensis L.
Primulaceae
Ognivtche
Mordigallina, Centocchio
Diuretic, antitussive, inamed wounds
Aerial parts
Flowering tops
Decoction (poisonous plant)
Infusion, foot bath (not in case of ulcerated skin)
Under medical supervision
Cyclamen hederifolium Aiton and C. repandum Sm. Primula veris L. (=P. ofcinalis L.)
Primulaceae
Ciklama
Ciclamino
Sedative
Tuber
Tuber
Infusion
Powdered tuber
Toxic, tuber contains substances not resistant to heat. No longer used in Italy A mixture with Althaea ofcinalis and Verbascum thapsiforme calms tubercular coughs Flowers can be used in medicines to improve avour. Fresh seeds are refreshing No longer present in Italy Toxic; external use. Several different species, such as C. recta L. or C. ammula L. are used in Italy. Young leaves and buds are smoked as tobacco substitute. Young stems can be boiled or fried with omelettes Toxic
Primulaceae
Zjaltaiglika
Primula, Primavera
Antitussive, expectorant, anti-inammatory for lungs Anti-helmintic (mainly against tapeworm)
Roots, owers
Roots, owers, leaves
Infusion
Decoction, infusion
Punica granatum L.
Punicaceae
Nar
Melograno
Bark
Bark of the root, stem, peel, seeds Aerial parts Leaves
Infusion
Decoction of seeds with sugar drunk for catarrh -------Maceration of crushed leaves in alcohol mixed with fat, tincture
Adonis vernalis L. Clematis vitalba L.
Ranunculaceae
Gorizvet Obiknoven povet
Adonide giallo, Occhio del diavolo Vitalba
Cardiac diseases, diuretic, sedative Revulsive
Aerial parts Leaves, roots, owers
Tincture Infusion
Ranunculaceae
Consolida regalis S.F. Gray (=Delphinium c. L.) Helleborus odorus Waldst et Kit
Ranunculaceae
Obiknovena raliza
Speronella, Erba cornetta Elleboro
Curare like activity
Diuretic
Aerial parts
Aerial parts, seeds Roots
Infusion
Infusion
Ranunculaceae
Kuturiak
--------
Drastic cardiotonic, anaesthetic, active on CNS
--------
Decoction
--------
Not frequent in Italy, no longer used in popular medicine
Helleborus sp. pl.
Ranunculaceae
--------
Elleboro
--------
--------
Cardiotonic anti-helmintic, to promote blood-ow (leaves) Seeds
--------
--------
Decoction
Toxic
Nigella sativa L.
Ranunculaceae
Polskatchelebitka
Damigella, Fanciullaccia Pulsatilla Frangola, Alno nero Eupatoria
Carminative, laxative, anti-ascaridic Sedative, anaphrodisiac Laxative
Diuretic, carminative, emmenagogue, increasing milk secretion Antineuralgic, promotes blood-ow Laxative
Seeds
Decoction
Decoction
Pulsatilla vulgaris Miller Frangula alnus Miller
Ranunculaceae Rhamnaceae
Sassanka Elchoviden zarnastez Kamchik
Aerial parts Bark
Flowering tops Bark
Tincture Decoction or powdered bark
Infusion
No longer used in Italy given its toxicity
Agrimonia eupatoria L.
Rosaceae
Astringent, anti-inammatory in urinary and liver diseases Astringent, inamed wounds Antivirus effect against Herpes simplex
Anti-helmintic, astringent, as mouth wash, anticatarrhal, hepatoprotective, mild hypotensive and antistaminic Against catarrh, against menstrual pains Cardiac diseases, coronary protecting, against tachycardia hypotensive, sedative for CNS, emollient for the skin, antipyretic (bark), atherosclerosis See Crataegus laevigata
Aerial part, roots
Leaves and owering tops
Infusion
Fresh leaves on sores as cataplasm
Alchemilla vulgaris L. Complex Crataegus laevigata (Poiret) DC. (C. oxyacantha All.)
Rosaceae Rosaceae
Chapitche Obiknoven glog
Erba stella, Erba ventaglina Biancospino
Aerial part Flowers, leaves, fruits
Leaves Flowers, fruits, bark
Infusion Decoction
Infusion Infusion (owers), decoction (fruits, bark)
Crataegus monogyna Jacq.
Rosaceae
Glog
Biancospino
Cardiac diseases, coronary diseases, myocardial ischemia Antitussive, astringent
Flowers, leaves, fruits Seeds
See Crataegus laevigata Fresh fruit pulp, seeds, leaves Flowering tops Fruits, leaves, rhizome
Infusion
See Crataegus laevigata Decoction Fresh pulp is used in making home-made jams
Cydonia oblonga Miller
Rosaceae
Djulja
Cotogno
Anti-inammatory for the skin (seeds), astringent, dietetic, mild sedative (leaves) Rheumatic pains, nephritis, gout, oedema Emollient for the skin (fruits) astringent
Decoction
Filipendula ulmaria (L.) Maxim Fragaria moschata Duchesne
Rosaceae Rosaceae
Blaten taznik Gradinska jagoda
Filipendula, Erba peperina Fragola
Diuretic, against rheumatism Diuretic, astringent, anti-inammatory, anti-sclerosis Diuretic, astringent, anti-inammatory, anti-atherosclerosis Diuretic, astringent, anti-inammatory, anti-sclerosis Anti-inammatory, astringent, antimicrobial Intestinal gas, dismenorrhea, stomach-ache Astringent, anti-inammatory for mouth and throat (by gargling), in gastric and digestive disorders
Aerial parts Fruits, leaves
Maceration Infusion
Infusion Infusion (leaves, rhizome) squeezed pulp of fresh fruits locally applied Decoction (rhizome), squeezed fresh fruit is locally applied Infusion (leaves, rhizome) Infusion, tincture Mainly valued as edible fruits, may cause skin allergy. Used in making jams and jellies Mainly valued as edible fruit, but it can cause skin allergy. Used in making jams and jellies Used less than the preceding two species
Fragaria vesca L.
Rosaceae
Gorska jagoda
Fragola
Emollient for the skin (fruits) astringent
Pulp of the fruits, leaves, rhizome Fruits, leaves
Fruits, leaves
Infusion
Fragaria viridis Duchesne
Rosaceae
Planiza
Fragola
See Fragaria vesca
Fruits, leaves rhizome Rhizome, aerial parts Aerial parts, rhizome See Potentilla anserina
Infusion
Geum urbanum L.
Rosaceae
Omajnitche
Cariollata, Ambretta Argentina
Astringent, anti-inammatory, antipyretic Astringent, antipyretic, antidiarrhoeic See Potentilla anserina
Roots, aerial parts Aerial parts
Infusion
Potentilla anserina L.
Rosaceae
Patchiotchibolez
Infusion
Decoction
Potentilla erecta (L.) Rauschel
Rosaceae
Buturak
Tormentilla
Roots
Infusion
See Potentilla anserina
137
138
Potentilla reptans L. Prunus spinosa L.
Rosaceae Rosaceae
Galitcheva treva Tranka
Cinquefoglio Prugnolo
Diarrhoea Laxative, diuretic, astringent
See Potentilla anserina Astringent, anti-inammatory, depurative, diuretic, anti-asthma, hypoglycemic Antiscorbutic, diuretic, astringent, anti-inammatory for the skin, nutritive
Roots Flowers, fruits
See Potentilla anserina Bark, fruits, owers
Infusion Infusion, decoction
See Potentilla anserina Decoction Fruits are also used in preparing home-made liqueurs In the past hips were collected as source of vitamins. They are still used in making syrups, jams and jellies Fruits are eaten raw, or used in preparing home-made jams Fruits are eaten or used in making home-made jams
Rosa canina L.
Rosaceae
Chipka
Rosa di macchia
Antiscorbutic, cholagogue, diuretic, astringent
Cynorrhods (hips)
Leaves, cynorrhods, (hips), petals
Decoction
Decoction
Rubus sp. pl.
Rosaceae
Kapina
Rovo
Astringent, antiseptic
Astringent, anti-inammatory, antiscorbutic, diuretic, thirst quenching, laxative Astringent, anti-inammatory for intestinal tract Astringent, anti-haemorrhagic, against haemorrhoids Dietetic, soothing for throat and skin See Sorbus aucuparia Diuretic, spasmolytic, sedative Spasmolytic, sedative, mild hypnotic, astringent for chapped skin (e.u.) Diuretic, sedative for CNS; externally used in case of inamed skin Kidney stones, gall stones choleretic, cholagogue, locally applied on inamed skin To stimulate digestion
Roots, leaves, fruits
Leaves, fruits
Infusion
Decoction, syrup by fruits, fresh leaves locally applied Decoction
Rubus idaeus L.
Rosaceae
Malina
Lampone
Astringent anti-inammatory Astringent, anti-inammatory Antirheumatic, astringent, diuretic -------Diuretic, laxative, analgesic Diuretic, antitussive
Roots, leaves, fruits Roots
Leaves, fruits
Infusion
Sanguisorba ofcinalis L.
Rosaceae
Letchebna dinka
Salvastrella
Whole plant
Decoction
Decoction
Sorbus aucuparia L.
Rosaceae
Oka, kalina
Sorbo degli uccellatori, Sorbo rosso Sorbo Attacca veste Stellina odorosa
Fruits
Fruits
Decoction
Juice, decoction
Sorbus domestica L. Galium aparine L.
Rosaceae Rubiaceae Rubiaceae
-------Lepka Lazarkinja
-------Aerial parts Aerial parts
See Sorbus aucuparia Flowering tops Flowering tops Flowery tops
-------Infusion Infusion
See Sorbus aucuparia Infusion Infusion
Fruits are eaten
Galium odoratum (L.) Scop. (=Asperula odoratum L.) Galium verum L.
Rubiaceae
Enjovtche
Caglio
Diuretic, laxative, analgesic Spasmolytic, kidney stones
Aerial parts
Infusion
Infusion
It is also used in dyeing clothes Used after meals
Rubia tinctorum L.
Rubiaceae
Broch
Robbia
Roots
Roots, rhizome
Tincture
Infusion, decoction
Dictamnus albus L.
Rutaceae
Rocen, samodivsko bile Sedeftche
D` ttamo
Diuretic
Roots
Flowering tops Tops before owering
Infusion
Infusion
Toxic
Ruta graveolens L.
Rutaceae
Ruta
Sedative, anti-inammatory abortive, anti-ascaridic Astringent diuretic, antiseptic
Protecting blood vessels, emmenagogue, abortive, anti-helmintic. To aromatize liqueurs Antirheumatic, astringent bitter-tonic, diuretic, diaphoretic, absorbs intestinal gas (char wood)
Aerial parts
Maceration
Rarely used given its toxicity and ability to iname skin Decoction
Fresh stems are put into rooms infested by mice and eas to repel them
Populus alba L.
Salicaceae
Bjala topola
Pioppo, Gattice
Gemmae (buds)
Bark, gemmae (buds)
Tincture
Populus nigra L. Populus tremula L. Salix alba L.
Salicaceae Salicaceae Salicaceae
Tcherna topola Trepetlika Bjala varba
Pioppo nero, Pioppo cipressino Pioppo tremulo Salice
Astringent diuretic, antiseptic Astringent diuretic, antiseptic Antirheumatic, antipyretic Astringent, anti-inammatory antiviral, eye diseases Laxative, diuretic Diuretic, laxative, haemorrhoids Antitussive, anti-inammatory, spasmolytic Hypotensive, spasmolytic --------
Anti-arthritis, antigout, bronchial sedative See Populus alba Antirheumatic, antipyretic, antineuralgic Emollient, eye wash
Gemmae (buds) Gemmae (buds) Roots
Bark See Populus alba Bark
Tincture Tincture Decoction
Decoction See Populus alba Decoction Used externally in gynaecology (Bg)
Euphrasia ofcinalis L. s.l.
Scrophulariaceae
Otchanka
Eufrasia
Aerial parts
Whole plant
Decoction
Gauze soaked in infusion, or decoction, are locally applied Infusion, decoction Infusion, cataplasm of the crushed drug See Verbascum thapsus L. See Verbascum thapsus L. Toxic
Gratiola ofcinalis L. Linaria vulgaris Mill.
Scrophulariaceae Scrophulariaceae
Letchebna sirotiza Lulitchka
Graziella Gratia-Dei Linaiola
Cardiac stimulant, diuretic Astringent, anti-inammatory, laxative, anti-haemorrhoids See Verbascum thapsus L.
Aerial parts Aerial parts
Leaves Flowering tops See Verbascum thapsus L. See Verbascum thapsus L. Aerial parts, leaves
Infusion Infusion
Verbascum densiorum Bertol.
Scrophulariaceae
Visok lopen
Verbasco, Tasso barbasso Verbasco, Tasso barbasso Verbasco, Tasso barbasso
Leaves
Infusion
Verbascum phlomoides L.
Scrophulariaceae
Lopen
See Verbascum thapsus L.
Leaves
Component of the remedy Verbaskan Decoction evaporated then ltered. Cataplasm of crushed leaves used on wounds Cataplasm, ointment (in olive oil), decoction Infusion
Verbascum sinuatum L.
Scrophulariaceae
--------
Against psoriasis, haemorrhoids (piles), to wash infected wounds
--------
Verbascum thapsus L.
Scrophulariaceae
--------
Verbasco, Tasso barbasso
Antitussive
Emollient against catarrh and coughs. Cataplasm applied externally to soothe gangrenous sores Appetizer, anti-inammatory, soothing agent, diuretic, antitussive Spasmolytic, sedative in gastric and intestinal troubles, externally for rheumatism (alcohol rub) To stimulate digestion, antiscorbutic, against haemorrhoids (piles), painful rheumatism in joints Sedative for CNS, antispasmodic, chilblains (foot or hand bath) Spasmolytic, against asthma, anaesthetic, analgesic, hypnotic, rheumatic pains Spasmolytic, against asthma, anaesthetic, analgesic, hypnotic
--------
Flowers
Veronica ofcinalis L.
Scrophulariaceae
Letchebno velikdentche
Veronica
Appetizer, antitussive, anti-inammatory expectorant, asthma, pharingitis Spasmolytic, in Parkinsons disease Cura Bulgarica Stimulates blood-ow, appetizer
Aerial parts
Aerial parts
Infusion
Atropa belladonna L.
Solanaceae
Ludobile
Belladonna
Leaves, owers, roots
Leaves, owers, roots
Tincture, alcoholic extract Tincture
Infusion, decoction
Capsicum annum L.
Solanaceae
Piper
Peperoncino
Fruits
Raw fruits
Cataplasm of fresh or dried and crushed fruits mixed with vinegar Tincture, infusion
Mixed with foods to give avour
Datura stramonium L.
Solanaceae
Tatul
Stramonio
Spasmolytic, anti-asthmatic Spasmolytic, against asthma Spasmolytic, against asthma
Leaves
Leaves, owers, seeds Leaves, seeds
Tincture
Toxic
Hyosciamus albus L.
Solanaceae
Bel bojour
Giusquiamo
Leaves
Infusion
Infusion, maceration in olive oil locally applied Infusion
Toxic. Seeds used to make decayed teeth to fall out Toxic
Hyosciamus niger L.
Solanaceae
Tcheren bljan
Giusquiamo
Leaves
Leaves, seeds
Infusion
139
140
Physalis alkekengi L.
Solanaceae
Mechunka
Alkekengi
Diuretic, anti-inammatory To promote perspiration Antibiotic
Diuretic, in case of kidney and gall stones antipyretic, rheumatism and gout Hypnotic, anaphrodisiac, anti-asthma Hepatoprotective, diuretic, depurative Spasmolytic, sedative, antalgic, sliced fresh pulp externally applied in skin diseases, itching and painful joints Anti-acid, anti-inammatory. Sliced fresh pulp is externally applied to minor burns and reddened skin Revulsive Mild sedative, diaphoretic, soothes the mucosa, antitussive See Tilia cordata
Fruits
Fruits
Decoction
Infusion, decoction powdered fruits Decoction Decotion
Toxic
Solanum dulcamara L. Solanum melongena L.
Solanaceae Solanaceae
Razvodnik Sin domat
Dulcamara Melanzana
Aerial parts Fruits
Young stems, aerial parts Fruit peel with outer part of pulp Aerial parts
Decoction Juice
Toxic The frruit is mainly eaten as vegetable Toxic
Solanum nigrum L.
Solanaceae
Tchemo kutchechko grodze
Erba morella
Spasmolytic, sedative
Aerial parts
Tincture
Infusion
Solanum tuberosum L.
Solanaceae
Kartof
Patata
Anti-acid, anti-inammatory
Tuber
Tuber
Juice
Pulp
It is used mainly as food
Daphne mezereum L. Tilia cordata Miller
Thymeleaceae Tiliaceae
Bjasnodarvo Drebnolistna lipa
Fior di stecco Tiglio
Revulsive To promote perspiration, anti-inammatory To promote perspiration, anti-inammatory, sedative Astringent, against diarrhoea, anti-inammatory Stimulant, astringent, diuretic
Barks Flowers
Barks Flowers and bracts See Tilia cordata
Powder mixed with fat Infusion
Decoction Infusion
Toxic. External use only
Tilia platiphyllos Scop.
Tiliaceae
Edrolistna lipa
Tiglio
Flowers
Infusion
See Tilia cordata
Ulmus minor Miller
Ulmaceae
Kolski brjast
Olmo
Diuretic, depurative, anti-inammatory Diuretic, depurative, soothing for the intestinal tract, antigout to strengthen hair and ght dandruff, haemostatic Sedative for CNS Diuretic, bitter-tonic depurative, anti-pyretic, antirheumatic, antineuralgic, to protect liver and spleen, trigeminal neuralgia, against psoriasis Emollient, expectorant for coughs, sudoriferous, diuretic, mild laxative Used against dermatitis when arthritic pain is also present. Against psoriasis
Barks
Bark
Decoction
Decoction
Urtica dioica sp. pl.
Urticaceae
Kopriva
Urtica
Leaves
Leaves
Fresh plant, infusion
Infusion, decoction
Boiled leaves are eaten in soups or in omelettes
Valeriana ofcinalis L. Verbena ofcinalis L.
Valerianaceae Verbenaceae
Diljanka Varbinka
Valeriana Verbena
Sedative for CNS To promote perspiration, antipyretic, sedative
Roots Aerial parts
Roots Flowering tops
Decoction Infusion
Decoction Infusion, fresh squeezed pulp, decoction evaporated and then ltered
Viola odorata L.
Violaceae
Gorska temenuzka
Viola mammola
Antitussive, diuretic, against atherosclerosis Antitussive, diuretic, against dermatitis, against atherosclerosis
Aerial parts, roots Aerial parts
Flowers rhizome Flowering plant
Infusion
Infusion
Flowers are used in making candies Flowers are used in making candies
Viola tricolor L.
Violaceae
Trizvetna temenuzka
Viola tricolore
Infusion
Cataplasm with infusion, decoction evaporated and then ltered
M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142 Table 3 Noteworthy therapeutic uses Antibiotic Antipyretic Antiviral Solanum melongena Cornus mas Crataegus laevigata Euphrasia ofcinalis Geranium sanguineum Geum urbanum Hypericum perforatum (only against Herpes simplex) Nepeta cataria Satureja hortensis Thymus sp. pl. Geranium sanguineum Sambucus ebulus Sambucus nigra Verbena ofcinalis (only trigeminal neuralgia) Sedum acre Viola tricolor Viola odorata Coronary dilating and capillarotrophic Cardiotonic Geranium macrorrhizum Corylus avellana
141
Daucus carota Pastinaca sativa subsp. sativa
Jaundice
Mentha pulegium Mentha spicata
Antiviral and immuno-stimulant Antineuralgic
Hypotensive Hepatoprotective
Centaurium erytrhaea Chelidonium majus Solanum melongena Mentha spicata Corylus avellana Ecballium elaterium Verbascum sinuatum
Anti-atherosclerosis Against dermatitis when arthritic pains are also present
Prostatic hypertrophy Psoriasis
used externally, the direct employment of fresh plant juice is common. This use is probably a form of rst aid in cases of burns or small cuts, or as an anti-inammatory and soothing agent (Scolopendrium ofcinale, Sedum acre, Quercus sp. pl., Fragaria sp. pl., Sorbus aucuparia, Solanum tuberosum, Petroselinum crispum, etc.). An important role is played by edible plants, which constitute about 30% and are used as food medicine, showing the close interconnection between therapy and nutrition. These plants and their fruits are employed not only as spices or herbs and in preparing liqueurs or jams, but are usually eaten in soups, salads or even more simply as vegetables. This role is mainly played by plants with depurative and anti-anaemic properties, such as Cichorium intybus, Borago ofcinalis, Chenopodium bonus-henricus, etc. or diuretics, such as Silybum marianum, a source of microelements, particularly iron, and with hepatoprotective qualities, etc. This preliminary comparative analysis strengthens the rm belief that ethnobotanical ndings represent an important shared heritage not to be relegated to a narrow historical, cultural context, but to be exploited in order to provide new, useful and important knowledge. Today, in fact, many invaluable drugs have taken their place in contemporaryat least as prototypes of efcient, synthetic drugsas a result of this kind of research. It is equally clear why a comprehensive programme aimed at discovering more efcient drugs must carefully consider every new biologically active compound with potential healing found in plants.
References
Amenta, R., Camarda, I., Di Stefano, V., Lentini, F., Venza, F., 2000. Traditional medicine as a source of new therapeutic agents against psoriasis. Fitoterapia 71, 1320. Bruno, F., Di Martino, A., Bonomo, R., 1959. Le piante ofcinali spontanee della Sicilia e dellArcipelago delle Pelagie, vol. XVII. Lav Ist. Bot. E Giard. Col., Palermo. Corsi, G., Pagni, A.M., 1978. Studi sulla Flora e vegetazione del Monte Pisano (Toscana NordOccidentale). 1. Le piante della medicina popolare nel versante pisano. Webbia 33 (1), 159204. Gastaldo, P., 1987. Compendio della Flora Ofcinale Italiana. Piccin (Ed.). Padova. Guarrera, P.M., 1990. Il Patrimonio Etnobotanico del Lazio. Iordanov, D., Nicolov, P., Boichinov, A., 1969. Phytotherapy in Bulgaria. Med. and Phyzicultura, Soa, p. 360. Isaiev, I., Landjev, I., Nechev, G., 1977. Herbs in Bulgaria. Zemizdat, Soa, p. 406. Ivancheva, S., Stantcheva, B., 2000. Ethnobotanical Inventory of medicinal plants in Bulgaria. Journal of Ethnopharmacology 69, 165172. Kitanov, B., 1953. Bulgarian Folk Medicine. pp. 3251. Lentini, F., Raimondo, F.M., 1990. Indagini Etnobotaniche in Sicilia. IV. Luso tradizionale delle piante nel territorio di Mistretta (Messina). Quad. Bot. Ambiente Appl. 1, 103117. Lentini, F., Catanzaro, F., Aleo, M., 1987/1988. Indagini Etnobotaniche in Sicilia. III. Luso tradizionale delle piante nel territorio di Mazara del Vallo (Trapani). Atti. Acc. Sc. Lett. Ar., Palermo, pp. 129. Lentini, F., Di Martino, A., Amenta, R., 1994. Contributo alla conoscenza della Flora popolare dellisola di Ustica. Quad. Bot. Ambiente Appl. 5, 4754. Lentini, F., Faqi, A.S., Amenta, R., 1994/1995. Relazioni Etnobotaniche tra Sicilia e Nord Africa. Atti. Acc. Sc. Lett. Ar., Palermo, pp. 81115. Lentini, F., Giani, S., Amenta, R., 1995. Luso tradizionale delle piante nelle Isole Eolie (Sicilia). Acta Technologiae et Legis Medicamenti 351355.
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Anti-inammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene from Zingiber cassumunar Roxb.

Rattima Jeenapongsa a, , Krongtong Yoovathaworn b , Kittima M. Sriwatanakul b , Ubonwan Pongprayoon c , Kampon Sriwatanakul b
b
Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Muang, Phitsanulok 65000, Thailand Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, Thailand c Thailand Institute of Scientic and Technological Research, Bangkok, Thailand
Received 3 September 2002; received in revised form 22 February 2003; accepted 13 March 2003
Abstract This study aimed to investigate the anti-inammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD), isolated from Zingiber cassumunar Roxb., using in vivo and in vitro models. The results show that DMPBD dose-dependently inhibited the rat ear edema induced by ethyl phenylpropiolate (EPP), arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) and it was more potent than any other standard drugs being used. In EPP-induced edema IC50 of DMPBD and oxyphenbutazone were 21 and 136 nmol per ear, respectively. The IC50 of DMPBD and phenidone were 60 and 2520 nmol per ear, respectively, in AA-induced edema whereas DMPBD was 11 times more potent than diclofenac in TPA-induced edema (IC50 = 660 and 7200 pmol per ear, respectively). DMPBD and diclofenac inhibited the rat paw edema induced by carrageenan but not by platelet activating factor (PAF). In in vitro study DMPBD, aspirin and phenidone inhibited collagen-induced platelet aggregation with IC50 of 0.35, 0.43 and 0.03 mM, respectively. Whereas IC50 of these agents in ADP, AA and PAF inductions were 4.85, 3.98 and 1.30 mM; 0.94, 0.13 and 0.04 mM; and 1.14, 6.96 and 2.40 mM, respectively. These results indicate that DMPBD possesses a potent anti-inammatory activity through the inhibition of CO and LO pathways and seems to have more prominent effects on the LO pathway. 2003 Published by Elsevier Science Ireland Ltd.
Keywords: Zingiber cassumunar; DMPBD; Anti-inammatory activity; Platelet aggregation; Ear edema; Paw edema
1. Introduction In many Asian countries, Zingiber cassumunar Roxb. (Zingiberaceae) is widely used in folklore remedies as a single plant or as a component of herbal recipes. Compounds with known chemical structures have been isolated from Zingiber cassumunar Roxb. Among these the hexane extract seemed to possess a potent anti-inammatory activity. Compound D, isolated from the hexane extract, exhibited a strong inhibitory activity on the edema formation in carrageenan-induced rat paw edema. In rat pleurisy model, it exerted markedly inhibitory activity on the exudates formation, the accumulation of leukocytes and the
author. Tel.: +66-55-261000-4 Ext 3620; fax: +66-55-261057. E-mail address: rattimaj@yahoo.com (R. Jeenapongsa).
Corresponding
prostaglandin-like activity of the exudates (Panthong et al., 1990). (E)-1-(3,4-Dimethoxyphenyl) butadiene (DMPBD) was isolated from the hexane extract of Zingiber cassumunar Roxb. by Thailand Institute of Scientic and Technological Research (TISTR). Its chemical structure is shown in Fig. 1. Preliminary studies suggested that DMPBD is an active ingredient of the essential oil derived from Zingiber cassumunar Roxb. Our in vivo preliminary study revealed the effect of DMPBD on both cyclooxygenase (CO) and lipoxygenase (LO) pathways. Therefore, this study aimed to characterize the anti-inammatory activity of DMPBD using in vivo and in vitro models. Several inducers and standard drugs were employed so that possible mechanisms of action of DMPBD on arachidonic acid (AA) metabolic pathway could be postulated.
0378-8741/03/$ see front matter 2003 Published by Elsevier Science Ireland Ltd. doi:10.1016/S0378-8741(03)00098-9
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R. Jeenapongsa et al. / Journal of Ethnopharmacology 87 (2003) 143148
Fig. 1. Chemical structure of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD).
The in vivo studies were carried using rat ear edema and rat paw edema models whereas platelet aggregation model was employed as an in vitro study.
2. Materials and methods 2.1. Animals Male SpragueDawley rats, 5070 and 120130 g, were obtained from the National Laboratory Animal Center of Mahidol University. Before conducting any studies animals were acclimatized for 1 week at 2426 C with food and water ad libitum. Adult male and female albino rabbits weighing 35 kg were supplied by the Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. 2.2. Materials DMPBD was supplied by TISTR in the form of white crystal. Ethyl phenylpropiolate (EPP), AA, 12-O-tetradecanoylphorbol 13-acetate (TPA), carrageenan, platelet activating factor (PAF), oxyphenbutazone, diclofenac, phenidone, salbutamol, collagen and adenosine diphosphate (ADP) were obtained from Sigma (St. Louis, MO). All other solvents and chemicals employed were of analytical grade. Compounds were prepared as solutions in acetone (reagent grade) just prior to application. 2.3. In vivo study
the experiment. Prior to any treatment, ear thickness was determined as a basal value. In the EPP- and AA-induced ear edema, ear thickness was determined at 0.5, 1 and 2 h and 0.5, 1, 1.5, 2, 3 and 4 h after the treatment, respectively. In the TPA treated group, the thickness was determined at 2, 4, 6, 8, 10 and 12 h after the TPA application. Drug was applied simultaneously with EPP or AA. Oxyphenbutazone or DMPBD was dissolved in the 5% EPP solution to obtain the nal drug concentrations of 0.005, 0.05, 0.5 and 5%. In the AA treatment group, phenidone or DMPBD was dissolved in 10% AA solution to obtain the drug concentration of 5%. In the TPA-induced edema, diclofenac was prepared in a mixture of ethanol and acetone (4:6) and DMPBD was prepared in acetone. The solution of diclofenac or DMPBD was applied on the ear at 1 h after the TPA application. 2.3.2. Rat paw edema Paw edema was induced in male SpragueDawley rats weighing 120130 g by subplantar injection of 0.1 ml carrageenan or PAF by a modied method described by Winter et al. (1962) with some modications. Carrageenan (1%, w/v) in 0.9% normal saline solution was injected into the left hind paw. Paw volume was measured before and at 1, 2, 3, 4, 5 and 6 h after the injection of carrageenan. Diclofenac or DMPBD was dissolved in acetone and 0.06 ml of the drug was applied over the upper and lower surfaces of the injected paw at 1 h after the carrageenan injection. Edema volume was determined plethysmographically at 1, 2, 3, 4, 5 and 6 h after the treatment. PAF was dissolved in 0.9% normal saline to obtain a concentration of 8 g/ml. Diclofenac and DMPBD were prepared at the same concentration as those used in the study induced by carrageenan. A solution of salbutamol (5%) was obtained by dissolving salbutamol in a mixture of methanol and acetone (1:1). Then 5% salbutamol was diluted with acetone to give the concentrations of 1, 0.5 and 0.05%. Drug solutions were applied over the upper and lower surfaces of the paw 30 min before the injection of PAF. Edema volume was determined at 0.5, 1, 1.5, 2 and 3 h after the PAF injection. 2.3.3. Platelet aggregation
2.3.1. Rat ear edema Ear edema was induced in male SpragueDawley rats weighing 5070 g, according to the modied method of Brattsand et al. (1982) and Pongprayoon et al. (1991) with some modications. The edema was induced by an application of a solution of EPP, AA or TPA in acetone at concentrations of 5, 10 and 0.02%, respectively. Each solution in volume of 0.01 ml was applied to each of inner and outer surfaces of both ears by means of an automatic micropipette. For ear thickness determination, a dial caliper (no. 7039, Mitutoyo) was applied to the tip of the ear. In order to minimize technical variations among measurements, the same investigator performed the measurements throughout
2.3.3.1. Preparation of platelet suspension. Blood was taken from the central ear artery of the rabbits. An aliquot (9 ml) of blood sample was placed into a polystyrene tube containing 1 ml of 3.2% (w/v) sodium citrate as an anti-coagulant. Citrated blood was centrifuged at 160 g for 10 min and the supernatant was removed as platelet rich plasma (PRP). The remaining blood was further centrifuged at 4000 g for 10 min to obtain the supernatant as platelet-poor plasma (PPP). Platelet concentration of the PRP was adjusted to 3105 to 5105 /ml by diluting with the PPP as required. Platelet aggregation tests were performed during 30 min and 3 h after the blood had been drawn.
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2.3.3.2. Preparation of chemical reagents. Collagen and ADP were dissolved in distilled water at the concentration of 10 g/ml and 0.2 mM, respectively. AA was dissolved in a small volume of absolute ethanol (not higher than 5% in nal concentration) and then adjusted with 0.9% normal saline to obtain the nal concentration of 12.38 mM. PAF was prepared by evaporating the solution of PAF in chloroform under nitrogen gas and reconstitute with normal saline to achieve a nal concentration of 10 mg/ml. Aspirin was dissolved in distilled water at the concentrations of 1 and 5 mg/ml. Phenidone (1%, w/v) was prepared by dissolving the drug in propylene glycol (60% in nal concentration) and then the nal volume was adjusted with normal saline. DMPBD was dissolved in propylene glycol (40% in nal concentration) and adjusted the volume with normal saline. 2.3.3.3. Platelet aggregation study. PRP (450 l) was placed into siliconized glass cuvettes. The aggregating agent was added into the stirred platelet suspension to induce platelet aggregation. When studying the inhibitory effects of aspirin, phenidone and DMPBD on platelet aggregation induced by these four agents, the studied compound was added into the stirred PRP 2 min before the addition of the aggregating agent. The extent of platelet aggregation was shown as a percentage of change in light transmission through the cuvette comparing with that of PPP suspension. The inhibitory effect was observed as the decrease in light transmission comparing with that observed in the aggregation without an inhibitor. Absence of platelet aggregation was dened by having no changes in light transmission after the period of 5 min. 3. Results 3.1. DMPBD inhibits ear edema induced by EPP, AA and TPA Topical application of 5% EPP produced ear swelling within 30 min. Erythema was also observed. The maximum effect of EPP was at 1 h, thereafter the ear swelling decreased
gradually. DMPBD and oxyphenbutazone when applied concomitantly with 5% EPP concentration-dependently inhibited the EPP-induced edema formation with IC50 of 21 and 136 nmol per ear, respectively (Table 1). DMPBD was about six times more potent than oxyphenbutazone. AA produced a rapid edema formation with a maximum effect at 3060 min and the edema then gradually decreased. DMPBD and phenidone exhibited concentration-related inhibitory prole with the maximal activity at 30 min after the topical administration. DMPBD (IC50 of 60 nmol per ear) was much more potent than phenidone (IC50 2520 nmol per ear) in inhibiting AA-induced ear edema in rats. TPA-induced edema formation was clearly observed at 2 h after the topical application and reached its maximum effect at 8 h after the application. DMPBD and diclofenac applied 1 h after TPA application inhibited ear edema with the maximum activity at 2 h. DMPBD (IC50 of 660 pmol per ear) was approximately 11 times more potent than diclofenac (IC50 of 7200 pmol per ear) in inhibiting TPA-induced ear edema. 3.2. DMPBD inhibits paw edema induced by carrageenan but not by PAF The injection of carrageenan resulted in paw edema within 1 h and the maximum effect was reached at 3 h. This reaction was inhibited by topical application of diclofenac in a concentration-dependent fashion with an IC50 at 3 h of 14 mol per paw. The topical application of DMPBD also showed a concentration-dependent effect with an IC50 at 3 h of 22 mol per paw. The subplantar injection of PAF into a rat hind paw rapidly induced edema formation. The topical application of diclofenac or DMPBD showed no signicant inhibition on PAF-induced paw edema. However, salbutamol signicantly inhibited the PAF-induced paw edema in a dose-related manner with IC50 of about 1.9%. 3.3. DMPBD inhibits platelet aggregation induced by collagen, ADP, AA and PAF Collagen-induced platelet aggregation in a concentration-dependent manner with a lag period of 23 min.
Table 1 Inhibitory effects of DMPBD and standard drugs on rat ear edema and rat paw edema. Data are expressed as IC50 values IC50 DMPBD Oxyphenbutazone Ear edema EPP AA TPA Paw edema Carrageenan PAF is non-applicable. 21 nmol/ear 60 nmol/ear 660 pmol/ear 22 mol/paw No effect 136 nmol/ear Standard drugs Phenidone 2520 nmol/ear Diclofenac 7200 pmol/ear 14 mol/paw No effect Salbutamol 1.9%
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Table 2 Inhibitory effects of DMPBD, aspirin and phenidone on platelet aggregation induced by collagen, ADP, AA and PAF Aggregating agents DMPBD Standard drugs Aspirin Collagen ADP AA PAF 0.35 4.85 0.94 1.14 0.43 3.98 0.13 6.96 Phenidone 0.03 1.30 0.04 2.40
Data are shown as IC50 (mM) values.
Preincubation of platelets with aspirin, phenidone and DMPBD inhibited collagen-induced platelet aggregation with IC50 of 0.43, 0.03 and 0.35 mM, respectively (Table 2). The lag phases were prolonged by these agents. The inhibition was concentration-related and among these phenidone was the most potent inhibitor. ADP-induced platelet aggregation differently from that of collagen, in that the lag period was not observed with ADP. The aggregation was observed immediately after the addition of ADP solution into the platelet suspension. The response to ADP increased as ADP concentration was increased. Higher concentrations of aspirin, phenidone and DMPBD were needed to inhibit platelet aggregation induced by ADP than those required in the collagen induction. The IC50 of aspirin, phenidone and DMPBD were 3.98, 1.30 and 4.85 mM, respectively. Platelet aggregation prole induced by AA was similar to that induced by ADP. The platelets aggregated immediately after the addition of the aggregating agents. Aggregation proles in the presence of the inhibitors were similar but it was slightly delayed when DMPBD was used. Aspirin, phenidone and DMPBD concentration-dependently inhibited AA-induced platelet aggregation with IC50 of 0.13, 0.04 and 0.94 mM, respectively. Platelet response occurred immediately after the addition of PAF solution into the stirred PRP suspension. Aspirin, phenidone and DMPBD inhibited platelet aggregation with IC50 of 6.96, 2.40 and 1.14 mM, respectively.
4. Discussion Murine models of inammation provide a relative system for studying the physiological and biochemical mechanisms of anti-inammatory agents (Jacobson and Jacobs, 1992). In this study, DMPBD was clearly shown to exert its effects on all models tested, except in PAF-induced rat paw edema. DMPBD showed a marked inhibitory activity on EPPinduced ear edema. This action is more potent than oxyphenbutazone. Immediately after the application of EPP, the vascular permeability was increased (Patrick et al., 1987). The combined actions of kinins and PGs on vascular permeability seemed to be involved in the early response to EPP while increased in inammatory cells in the tissues
provided a new source of mediators that contributed to the modulation of the blood ow. The response to EPP during the rst hour was partially suppressed by pretreatment with indomethacin, aprotinin and mechlorethamine (Patrick et al., 1987). The topical application of TPA onto a rat ear induced a long-lasting edema formation. The majority of TPA actions appear to involve or be dependent on AA release and metabolism (Patrick et al., 1987). At the biochemical level, cAMP, PGI2, PGE2 and PGF2 are elevated within minutes to hours after TPA application (Ashendel and Boutwell, 1979; Furstenberger and Marks, 1980). Phospholipase inhibitor, CO inhibitors and LO inhibitors as well as corticosteroids are effective at edema suppression after topical application of high dose of TPA (Young et al., 1983; Carlson et al., 1985), conrming a role of AA release and metabolism. DMPBD, if possessing the same pharmacological effect as diclofenac, should possess inhibitory activity on the CO pathway. However, when comparing the IC50 of both compounds in inhibiting TPA-induced ear edema, DMPBD with IC50 of 660 pmol per ear is more potent than diclofenac. The topical application of AA provokes a rapid and intense inammatory response of the ear. This response is not affected by CO inhibitors (Young et al., 1983) but is inhibited by agents that inhibit both CO and LO pathways of AA metabolism (Young et al., 1984). It was found that AA-induced ear edema is predominantly mediated by leukotriene C4 and other LO products while PGE2 (in the presence of LTs) acts to facilitate ear swelling (Inoue et al., 1988). In this study DMPBD and phenidone, a dual CO and LO inhibitor, inhibited AA-induced ear edema with IC50 of 60 and 2520 nmol per ear, respectively, suggesting that DMPBD may also acts on the LO pathway. In the rat paw edema model, carrageenan induces edema formation in three distinct phases according to the mediators involved (Di Rosa et al., 1971). The initial phase occurring during the rst hour after subplantar injection of carrageenan into rat hind paw is mediated by histamine and 5-HT. After that the increased vascular permeability in the second phase is maintained by kinin release up to 2.5 h. Then the third phase, from 2.5 to 6 h, the mediators involved are PGs (Di Rosa et al., 1971; Hwang et al., 1986). The biosynthesis of LTs is suggested to be involved in the fourth phase of carrageenan-induced edema formation (Holsapple and Yim, 1984). All non-steroidal anti-inammatory drugs (NSAIDs) are effective in inhibiting edema formation especially in the late phase in which PGs involves (Niemegeers et al., 1964). In this study, diclofenac showed the maximum inhibitory effect on edema formation at 5 h. This corresponds with the period of PG phase (Di Rosa and Willoughby, 1971). DMPBD also inhibited edema formation with maximum activity at the same period as diclofenac. These results support the suggestion from the rat ear edema model that DMPBD may interfere with the CO pathway of AA metabolism. It has been observed that the injection of PAF into rat hind paw provokes an inammatory response characterized by an
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acute edema (Cordeiro et al., 1986). The subplantar injection of PAF to rat hind paw-induced dose-related edema formation with the maximum response at 1 h (data not shown). The results are in agreement with the study reported by Castro-Faria-Neto et al. (1990). In this study, PAF-induced paw edema was suppressed by 2 -adrenergic agonist, salbutamol. It strongly inhibited paw edema while diclofenac and DMPBD did not, suggesting that DMPBD and diclofenac do not affect the pathways involved in PAF-induced paw edema. The platelet aggregation study represents an attempt to explore the effects of DMPBD on AA metabolism in platelet, since AA metabolites, PGs and thromboxane A2 (TBA2 ) are involved in the platelet aggregation. The effects of the anti-platelet aggregating agents, aspirin and phenidone, on collagen-induced platelet aggregation produced characteristics similarly to that reported in the previous study (Karniguian et al., 1990). Collagen-induced platelet aggregation in a concentration-related fashion and a lag phase was observed before the aggregation occurred. The earlier study suggested that the lag phase is the time that is needed for collagen to activate phosphatidylinositol 4,5-diphosphate-specic phospholipase C (Karniguian et al., 1990). The results are in agreement with the previous studies that aspirin could inhibit collagen-induced aggregation (OBrien, 1968; Kuster and Frolich, 1986). Phenidone, a CO and LO inhibitor, was slightly more potent than aspirin. In this study, phenidone was the most potent inhibitor of collagen-induced platelet aggregation. Regarding the greater potent anti-aggregating effects of phenidone compared to aspirin, it may be postulated that LTs, which are synthesized via the LO pathway, may also be involved in the platelet aggregation. DMPBD may affect both CO and LO pathways but it is less potent than phenidone in the collagen-induced aggregation. NSAIDs including aspirin are reported to be effective inhibitors of ADP-induced platelet aggregation (Zucker and Peterson, 1968; Chars et al., 1977). In this study, phenidone seemed to have a higher potency than aspirin and DMPBD. This observation suggests that both CO and LO pathways are involved in the activity of ADP and DMPBD may exert its inhibitory effect on these pathways. In the AA-induced platelet aggregation, AA is converted to endoperoxides and thromboxanes by platelets and these compounds are further responsible for platelet aggregation (Kinlough-Rathbone et al., 1976). Aspirin, phenidone and DMPBD inhibited the effect of AA on platelets but with different potency. Phenidone and aspirin were more potent than DMPBD (Table 1). Based on the action of aspirin and phenidone, it may be postulated that DMPBD may affect the common pathways of anti-platelet aggregation with that of aspirin and phenidone, possibly CO and LO pathways. PAF-induced platelet aggregation accompanies with the increase in inositol phosphate metabolism (Dhar et al., 1990). When binding to its receptors, PAF activates phos-
pholipase C and phosphorylation of several proteins occurs (Dhar et al., 1990). TXA2 , an AA metabolic product, is not found to be important in the mechanism of PAF-induced aggregation (McCulloch and Vandongen, 1990). Aspirin and other NSAIDs do not affect this response (Kuster and Frolich, 1986). In this study, DMPBD strongly inhibited platelet response to PAF with IC50 of 1.14 mM. Aspirin and phenidone also inhibited aggregation with lower potency than DMPBD (Table 1). Aspirin was effective only at much higher concentration than those of DMPBD and phenidone. This may suggest that the effects of aspirin on PAF action may occur via pathways other than CO inhibitor and DMPBD may also affect pathways other than the CO and LO pathways in PAF-induced aggregation. The results obtained from the in vivo models of inammation suggest that DMPBD exerts a clear anti-inammatory effect. The differences in the potency of DMPBD in inhibiting platelet aggregation induced by each inducer point out the specicity of DMPBD on the pathways involved in platelet aggregation. The in vivo data suggest that DMPBD may inhibit CO and LO activities and seems to have more prominent effects on the LO pathway. However, further studies should be performed to explore the activities of DMPBD on the enzymatic levels of the inammation before any nal conclusion can be made. This study is the rst to demonstrate that DMPBD is a unique topically active anti-inammatory agent. Possibly it is a modulator of AA metabolism having activities on both CO and LO enzymes. It has a potential for local therapeutic applications in inammatory diseases. A drug development program should be systematically initiated to determine the possibility of making this compound a new pharmacological agent for the treatment of inammatory disorders.
Acknowledgements This work is supported by the University Developing Commissions Grant, Ministry of University Affairs, and the Faculty of Graduate Studies, Mahidol University, Thailand. The authors would like to thank all the staff at TISTR for their technical assistance and valuable suggestion.
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Comparative effect of Prunus persica L. BATSCH-water extract and tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) on concentration of extracellular acetylcholine in the rat hippocampus
Yeon-Kye Kim a,b , Byung-Soo Koo b , Dae-Jong Gong b , Young-Choon Lee c , Jeong-Heon Ko d , Cheorl-Ho Kim a,b,
a
National Research Laboratory for Glycobiology (NRLG), Korean Ministry of Science and Technology, Kyungju City, Kyungbuk 780-714, South Korea b Department of Biochemistry, Molecular Biology and Neurobiology, DongGuk University COM, Kyungju City, Kyungbuk 780-714, South Korea c Faculty of Life Science and Bio-Resources, Dong-A University, Saha-Ku, Pusan 608-714, South Korea d Proteomic Research Laboratory, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-600, South Korea Received 8 November 2002; received in revised form 13 March 2003; accepted 13 March 2003
Abstract Prunus persica L. BATSCH seed-water extract (PPE) has been used in the treatment of the degenerative disorders, such as hypermenorrhea and dysmenorrhea, in Taiwan, China, Japan and Korea. In this study, the effects of oral administration of PPE on the extracellular acetylcholine concentration in the hippocampus of rats were evaluated, and compared to that of tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride), a well-known and centrally acting acetylcholinesterase (AChE) inhibitor, which had been developed for the treatment of Alzheimers disease. We measured the inhibition of brain AChE. PPE at 2.5 g/kg and tacrine at 5 mg/kg showed signicant effects for more than 6 h. At these doses, the maximum increases were observed at about 1.5 h after administration of PPE, and at about 2 h with tacrine, and were 454 and 412% of the pre-level, respectively. The results suggest that oral administration of PPE and tacrine increases acetylcholine concentration in the synaptic cleft of the hippocampus mostly through AChE inhibition, and that PPE has a potent and long-lasting effect on the central cholinergic system. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Semen Persicae (Prunus persica L. BATSCH); Acetylcholinesterase inhibitor; Acetylcholine; Microdialysis; Tacrine
1. Introduction Semen Persicae (Prunus persica L. BATSCH) water extract (PPE) has long been used as an agent in China (Tounin and Taoren in Chinese), Japan (Donin in Japanese) and Korea (Doin in Korean) in the treatment of the degenerative disorders, such as hypermenorrhea, dysmenorrhea, leiomyoma and infertility (Sakamoto et al., 1988, 1992; Wang et al., 1998; Ge et al., 1983). It is frequently used as an ingredient in a variety of traditional prescriptions, particularly those used to treat womens diseases (Fukuda et al., 2003). It was also reported that Semen Persicae showed a relatively strong anti-tumour promoter and anti-Oketsu syndrome (stagnation of blood circulations) effects (Okuyama et al.,
Corresponding
author. Tel.: +82-54-770-2663; fax: +82-54-770-2281. E-mail address: chkimbio@dongguk.ac.kr (C.-H. Kim).
1995; Kosuge et al., 1985). The chemical constituents of the herb include the cyanogenic glycosides, amygdalin and prunasin as major components, along with the glycerides and sterols (Kosuge et al., 1985; Ministry of Health and Welfare, 2001; Arichi et al., 1985; Isoza et al., 2001; He and Li, 1988). Preliminary pharmacological studies in vitro have revealed that PPE is comprised of constituents having reversible and non-competitive cholinesterase inhibitory activity. PPE produces marked and long-lasting inhibition of brain acetylcholinesterase (AChE) and increases the brain content of acetylcholine in vivo. Tacrine is the rst drug approved by the US FDA for the treatment of Alzheimers disease, although it has adverse effects related to its actions on the peripheral nervous system and to hepatic toxicity (Beermann, 1993; Kosasa et al., 1999). Alzheimers disease is a progressive neurodegenerative disease characterised by decits in memory and cognitive function. One of the most
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pronounced changes in the brain of Alzheimers disease patients occurs in the cholinergic systems. Cholinesterase inhibitors are the only class of drugs currently approved for the treatment of Alzheimers disease. Since the extracellular acetylcholine concentration measured by the intracerebral microdialysis technique reects the acetylcholine concentration in the synaptic cleft, it is a useful parameter with which to compare the potential efcacy of various cholinesterase inhibitors in the central cholinergic system. The effects of PPE and tacrine on extracellular acetylcholine concentration have been studied by microdialysis. In the present study, we have examined and compared the effects of orally administered PPE and tacrine on the basal concentration of extracellular acetylcholine in the hippocampus of rats under the same experimental conditions and they were compared. Moreover, in order to validate the microdialysis data, we measured the inhibition of brain AChE and the brain concentrations of these drugs.
extracted three times with 1 l of distilled water in the hot water bath for 5 h, and after ltration the ltrate was concentrated at reduced pressure for convenience. The extract solution was stored at 4 C. 2.3. Implantation of microdialysis probe Rats were anaesthetised with pentobarbital Na (50 mg/kg, i.p.) and placed in a stereotaxic frame (Narishige, Tokyo, Japan). The skull was exposed and a hole was drilled for implantation of a microdialysis probe. In order to increase the recovery of acetylcholine, a probe with a long membrane (3.0 mm membrane length, 0.5 mm diameter; I-4-03 Eicom, Kyoto, Japan) was used. The probes were implanted according to the methods of Maysinger et al. (1988) into the right lateral hippocampus at 4.5 mm lateral and 5.1 mm posterior to the bregma and to a depth of 8.0 mm from the brain surface according to the atlas of Paxinos and Watson (1986). The probe was then xed with dental cement. After surgery, the rats were housed in their home cages. Placement of the probe within the hippocampus was conrmed by visual inspection of the probe track at the end of the experiment. 2.4. Microdialysis
2. Materials and methods 2.1. Animals and reagents Male Wistar rats (210290 g) at 6 weeks of age were purchased from Experimental Animal Station, Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology (Daejon, South Korea). They were allowed at least 1 week to adapt to the environment (25 3 C, 55 5% humidity and a 12 h light/dark cycle, and start at 07:00 h) for at least 1 week before experiments. The animals were given free access to food and water. All animal experiments were approved by the Animal Care and Use Committee of DongGuk University Medical and Oriental Medical Hospital. Tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) was obtained from Sigma Co. (St. Louis, MO, USA). All other chemicals were commercial products of reagent grade. Tacrine was dissolved in distilled water and administered by gavage. 2.2. Water-extract preparation from Prunus persica L. BATSCH Prunus persica L. BATSCH-water extract (300 g) was obtained from the Oriental Medical Hospital (Specimen No. P-75), DongGuk University College of Oriental Medicine, Kyungju City, Kyungbuk, South Korea, as an oral administration grade for human. Also, for a large scale production in our laboratory, Prunus persica L. BATSCH samples from South Korea were purchased from Kyungju market, Kyungju, South Korea. A voucher specimen has been deposited at the Kyungju Oriental Medical Hospital, DongGuk University, Kyungju City, Kyungbuk, South Korea, under acquisition number P-W-17. Sliced seeds (200 g) nely was
One day after surgery, the microdialysis probe was perfused with Ringers solution (145 mM NaCl, 4.5 mM KCl, 0.5 mM MgSO4 , 2.5 mM CaCl2 , 5.0 mM HEPES) at a ow rate of 1.0 l/min. The perfusate was discarded during the rst 1 h of perfusion and then collected at 20-min intervals into microtubes containing 5 l of 0.1 M phosphate buffer (pH 3.5). Tacrine or PPE was orally administered after three fractions had been collected. The perfusate was collected until 6 h after administration. 2.5. Acetylcholine assay Acetylcholine concentration in dialysis samples was measured by high-performance liquid chromatography (HPLC) with electrochemical detection (Kosasa et al., 1999). As an internal standard, 500 fmol of isopropylhomocholine was added to the sampling tubes, and the mixture was subjected to HPLC. The HPLC system (Shimazu LC-930A) consisted of a degasser, an HPLC pump system, an autosampler, a column temperature controller, and an electrochemical detector with a platinum electrode (5 mm in diameter). A guard column and an enzyme reactor (3.0 mm 4.0 mm; AC-Enzympak, Eicom) were placed before and after the analytical column (2.0 mm 160 mm; Shimpak AC-Gel, Shimadzu CO., Tokyo, Japan), respectively. Following its separation from the analytical column, acetylcholine was converted to hydrogen peroxide inside the enzyme reactor. The analytical and enzyme columns and the electrode were kept at 30 C in a column temperature controller. The mobile phase was 0.1 M phosphate buffer (pH 8.3)
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Fig. 1. Effect of oral administration of PPE on the basal concentration of extracellular acetylcholine in the hippocampus of rats. Data are expressed as percentages of the pre-levels (average of three samples prior to administration = 100%). Values are means S.E.M. (n = 6). Pre-levels were: control: 102.6 19.1 fmol/tube; 0.625 g/kg: 75.4 6.3 fmol/tube; 1.25 g/kg: 88.2 17.1 fmol/tube; 2.5 g/kg: 73.2 6.2 fmol/tube. P < 0.05 vs. control (Dunnetts multiple comparison test).
containing 150 mg/l sodium 1-decanesulphonate, 50 mg/l tetramethylammonium chloride and 40 mg/l EDTA2Na. The ow rate of the mobile phase was 0.1 ml/min. Peaks were recorded on a Powerchrom integrator (Shimazu). Acetylcholine in the dialysate sample was quantied by the internal standard method. The data are expressed as percentages of the pre-level (average of three samples prior to administration).
2.6. Measurement of AChE activity Rats were decapitated at 0.5, 1, 2, 4, 8 and 12 h after receiving PPE (2.5 g/kg) and tacrine (10 mg/kg) by oral administration. Control animals received no treatment. The whole brain, excluding cerebellum and olfactory bulb, was removed and the two hemispheres were split for assays of AChE inhibition and brain concentrations of the drugs.
Fig. 2. Effect of oral administration of tacrine on the basal concentration of extracellular acetylcholine in the hippocampus of rats. Data are expressed as percentages of the pre-levels (average of three samples prior to administration = 100%). Values are means S.E.M. (n = 6). Pre-levels were: control: 101.5 15.4 fmol/tube; 1.25 mg/kg: 90.2 9.5 fmol/tube; 2.5 mg/kg: 81.3 7.5 fmol/tube; 5 mg/kg: 74.2 12.1 fmol/tube. P < 0.05 vs. control (Dunnetts multiple comparison test).
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AChE activity was measured using the radiometric method, as described by Thomsen et al. (1989) and Sherman (1991). [3 H]Acetylcholine iodide (Amersham Korea, Seoul, South Korea) was used as a substrate. In order to minimise the dilution effect which is seen in ex vivo assays of the inhibition by reversible cholinesterase inhibitors, the brain hemisphere was homogenised in 4 vol. of buffer, and nally 10 mg of brain tissue was diluted in 100 l of assay solution. Data are presented as percentages of the intact level.
ing Dunnetts multiple comparison test was applied between doses for individual fractions. Cholinesterase inhibition data were analysed with Dunnetts multiple comparison test. A P value of less than 0.05 was considered signicant. Statistical analysis was conducted using the software package SAS ver. 6.12 (SAS Institute Japan, Tokyo, Japan), available on the statistical analysis support system.
4. Results 3. Statistical analysis The data are expressed as means S.E.M. Microdialysis data were analysed using a repeated measures analysis of variance with dose and fraction as factors. If a signicant dose fraction interaction existed, post hoc analysis us4.1. Comparative effects of PPE and tacrine on hippocampal extracellular acetylcholine concentration We examined the effects of PPE and tacrine on the extracellular acetylcholine concentration in the hippocampus of rats. These cholinesterase inhibitors produced
Fig. 3. Comparison of the hippocampal extracellular brain AChE inhibition induced by PPE (A) and tacrine (B). Extracellular acetylcholine data are expressed as percentages of the pre-level (average of three samples prior to administration = 100%). Values are means S.E.M. (n = 6). AChE activity is expressed as a percentage of the intact level shown at 0 h (100%). Values are means S.E.M. (n = 5). P < 0.05 vs. intact (Dunnetts multiple comparison test).
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dose-dependent increases in the extracellular acetylcholine concentration (Figs. 1 and 2). PPE had a signicant effect at a dose of 2.5 g/kg. The maximum increase produced by this dose was seen at about 1.5 h, when acetylcholine reached 465% of the pre-treatment level. The duration of the acetylcholine increase produced by the drug at this dose was more than 6 h (Fig. 1). The increase in the extracellular acetylcholine concentration produced by tacrine at 5 mg/kg was maximum at 2 h when the level was 418% of the pre-treatment level. The acetylcholine-increasing action at this dose continued for more than 6 h (Fig. 2). 4.2. Effects of PPE and tacrine on rat brain AChE activity Fig. 3 shows the effects of PPE (2.5 g/kg) and tacrine (10 mg/kg) on rat brain AChE activity ex vivo. PPE produced maximal inhibition at 1 h after administration, when AChE activity was 44% of the intact level. AChE activity gradually recovered thereafter, and reached 78% of the intact level at 12 h after administration. The maximal inhibition of AChE by tacrine was observed at 12 h after administration, although AChE inhibition remained at a plateau for 0.58 h after administration (0.5 h, 41%; 1 h, 40%; 2 h, 41%; 4 h, 42%). The AChE activity was 73% of the intact level at 12 h after administration. Acetylcholine elevation in the hippocampus was a mirror image of AChE inhibition.
less potent than those of tacrine at 10 mg/kg. Moreover, PPE and tacrine showed long-lasting effects on both the extracellular acetylcholine concentration and the brain AChE activity. These results are consistent with the idea that the effect of these compounds on the extracellular acetylcholine concentration is based on the inhibition of AChE in the brain.
6. Conclusion The ndings of this study demonstrated that centrally acting cholinesterase inhibitors, PPE and tacrine, potently increase the extracellular acetylcholine concentration in the synaptic cleft of the hippocampus of rats mostly through AChE inhibition, and that PPE has a potent activity and a long-lasting effect on the central cholinergic system. PPE may be one of the more useful cholinesterase inhibitors for the treatment of Alzheimers disease.
Acknowledgements This work was in part supported by National Research Laboratory Program, KISTEP, MOST, South Korea (No. M10203000024-02J0000-01300 to C.-H. Kim).
References 5. Discussion Semen Persicae (Prunus persica L. BATSCH) water extract are well known as a traditional medicine in China, Japan, Korea and other Asian countries to treat womens diseases (Sakamoto et al., 1988, 1992; Wang et al., 1998). As a part of investigation to develop the function of the Semen Persicae, we examined the effect on concentration of extracellular acetylcholine in rat hippocampus. The results in this study clearly demonstrate that PPE has a potent activity and a long-lasting effect on the central cholinergic system, in terms of the basal concentration of extracellular acetylcholine in the hippocampus and the AChE activity in the brain of rats. Oral administration of PPE dose-dependently increased the basal concentration of extracellular acetylcholine in the hippocampus of rats. PPE caused a marked and dose-dependent increase in extracellular acetylcholine concentration. Our data show that extracellular acetylcholine is increased by PPE even when the oral administration route is used. PPE at a dose of 2.5 g/kg was somewhat more potent than tacrine at a dose of 5 mg/kg. Kawashima et al. (1994) also examined the effect of tacrine (1.65 and 5 mg/kg, i.p.) under the same experimental conditions. The acetylcholine-increasing action in the rat hippocampus produced by PPE and tacrine was well correlated to the ex vivo AChE inhibition in the rat brain at the same dose of each drug. Both the acetylcholine-increasing action and the AChE inhibition of PPE at 2.5 g/kg were somewhat
Arichi, S., Kubo, M., Tani, T., Nakamura, H., Motoyoshi, S., Ishii, K., Imazu, C., Seto, Y., Kadokawa, T., Nagamoto, N., 1985. Studies on Persicae Semen. II. Pharmacological activity of water soluble compositions of Persicae Semen. Yakugaku Zasshi 105, 886894. Beermann, B., 1993. Side effects of long-acting cholinesterase inhibitor. Acta Neurologica Scandinavica 149, 5354. Fukuda, T., Ito, H., Mukainaka, T., Tokuda, H., Nishino, H., Yoshida, T., 2003. Anti-tumor promoting effect of glycosides from Prunus persica seeds. Biological and Pharmaceutical Bulletin 26, 271273. Ge, R.Y., Zhou, C.H., She, Y.C., 1983. Inuences of Stigma Croci and Semen Persicae on function of ovary-uterus in pseudopregnant rats. Journal of Traditional Chinese Medicine 3, 2326. He, L.Y., Li, B.M., 1988. MicroHLPC determination of amygdalin in Semen pruni armeniacae and Semen pruni persicae. Biomedical Chromatogrphy 2, 271273. Isoza, T., Matano, Y., Yamamoto, K., Kosaka, N., Tani, T., 2001. Quantitative determination of amygdalin epimers by cyclodextrin-modied micellar electrokinetic chromatography. Journal of Chromatography: A 923, 249254. Kawashima, K., Sato, A., Yoshizawa, M., Fujii, T., Fujimoto, K., Suzuki, T., 1994. Effects of the centrally acting cholinesterase inhibitors tetrahydroaminoacridine and E2020 on the basal concentration of extracellular acetylcholine in the hippocampus of freely moving rats. Naunyn-Schmiedebergs Archives of Pharmacology 350, 523528. Kosasa, T., Kuriya, Y., Matsui, K., Yamanishi, Y., 1999. Effect of donepezil hydrochloride (E2020) basal concentration of extracellular acetylcholine in the hippocampus of rats. European Journal of Pharmacology 380, 101107. Kosuge, T., Ishida, H., Ishii, M., 1985. Studies on active substances in the herbs used for oketsu (stagnant blood) in Chinese medicine. II. On the anticoagulative principle in persicae semen. Chemical and Pharmaceutical Bulletin 33, 14961498.
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Y.-K. Kim et al. / Journal of Ethnopharmacology 87 (2003) 149154 Sakamoto, S., Yoshino, H., Shirahata, Y., Shimodairo, K., Okamoto, R., 1992. Pharmacotherapeutic effects of kuei-chih-fu-ling-wan (keishi-bukuryo-gan) on human uterine myomas. American Journal of Chinese Medicine 20, 313317. Sherman, K.A., 1991. Pharmacodynamics of oral E2020 and tacrine in humans: novel approaches. In: Becker, R., Giacobini, E. (Eds.), Cholinergic Basis for Alzheimer Therapy. Birkhauser, Boston, MA, pp. 321328. Thomsen, T., Kewitz, H., Pleul, O., 1989. A suitable method to monitor inhibition of cholinesterase activities in tissues as induced by reversible enzyme inhibitors. Enzyme 42, 219224. Wang, D., Wang, Z., Yu, C., 1998. Endometriosis treated by the method of resolving blood stasis to eliminate obstruction in the lower-jiao. Journal of Traditional Chinese Medicine 18, 711.
Maysinger, D., Herrera-Marschitz, M., Carlsson, A., Garofalo, L., Cuello, A.C., Ungerstedt, U., 1988. Striatal and cortical acetylcholine release in vivo in rats with unilateral decortication: effects of treatment with monosialoganglioside GM1. Brain Research 461, 355360. Ministry of Health and Welfare, 2001. In: Ministry of Health, Labour and Welfare, Tokyo, Japan (Ed.), The Japanese Pharmacopoeia, 14th ed. pp. D-803D-806. Okuyama, T., Matsuda, M., Kishi, N., Lee, S.N., Baba, M., Okda, Y., Nishino, H., 1995. Natural Medicines, vol. 49. pp. 261265. Paxinos, G., Watson, C., 1986. The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney, Australia. Sakamoto, S., Kudo, H., Kawasaki, T., Kuwa, K., Kasahara, N., Sassa, S., Okamoto, R., 1988. Effects of a Chinese herbal medicine, keishi-bukuryo-gan, on the gonadal system of rats. Journal of Ethnopharmacology 23, 151158.
The use of medicinal plants in self-care in rural central Ethiopia

Teferi Gedif , Heinz-Jrgen Hahn
Institute of Pharmacoepidemiology and Pharmaco Economics, School of Pharmacy, Martin-Luther University, Wolfgang-Langenbeck Street 4, Room # 107, 06120 Halle/Saale, Germany Received 17 July 2002; received in revised form 20 January 2003; accepted 14 March 2003
Abstract Medicinal plants are an important element of Ethiopian traditional medicine. This questionnaire survey examined the extent and type of medicinal plants used in self-care by rural Ethiopian community. Six hundred mothers were interviewed using a semi-structured questionnaire. The prevalence of the use of herbal drugs in self-care was found to be 12.5%. Twenty-ve plant species belonging to 21 families were reported, each with local names, methods of preparation and parts used. This study showed that self-care using medicinal plants is a major part of health care options in Butajira community. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Medicinal plants; Self-care; Prevalence; Rural Ethiopia
1. Introduction The use of plants as medicines predates written human history and almost all cultures in the world have a body of expertise concerned with the therapeutic properties of the local ora (Houghton, 1995). Ethiopian traditional medicine is composed of a number of specic skills, namely, the use of plants, animal products and minerals as well as magic and superstition (Pankrust, 1976). The main body, however, is based on the use of ethnobotany (Vicchiato, 1993). Though most practices and treatments in herbal medicine require specialists or professionals which are referred generally to as herbalists, self-care using plants is common in Ethiopia (Kitaw, 1987; Gedif, 1995). Although few studies on the medicinal plant resources of Ethiopia have been conducted (Abebe and Ayehu, 1993; Tadesse and Demissew, 1992; Abebe, 1986; Jansen, 1981), the extent and types of herbs used in self-care by the vast majority of the population particularly in rural areas has not been documented. Knowing factors involved in the selection of different treatment options at household level is also important for health service planning and to incorporate herbal medicine in a countrys health care delivery system. Mothers in most rural
Corresponding
communities of the developing countries including Ethiopia are the de facto healers of the family treating accidents and ailments with medicinal plants (Lambert et al., 1997). The present research, therefore, attempted to document the medicinal plants used in self-care in a rural Ethiopian community using mothers as informants.
2. Subjects and methods 2.1. Description of study area Butajira is a district located 130 km south of Addis Ababa, capital city of Ethiopia (Fig. 1). The population size extrapolated from 1994 census is estimated to be 257,000. Generally the demographic pattern is typical of developing countries where children below the age of 15 constitute the majority (Berhane, 2000). The dominant ethnic group is Gurage of meskan dialects. Farming is the main economic activity and the main cash crops being pepper, coffee and khat. The estimated size of the district is 797 km2 and lies at an average of 2100 m above sea level, ranging from 1750 in the lowlands to 3400 m in mountainous areas (Berhane et al., 1999). During the study time, the district had 1 health centre, 2 health stations, 11 private clinics, 11 health posts and 8 drug retail outlets. Although the health centre is the highest level of health institutions in the district, it has
author. Fax: +49-345-55-2-72-92. E-mail address: Gedif66@yahoo.com (T. Gedif).
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T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155161
for health related researches (Berhane, 2000; Shamebo, 1993). 2.2. Data collection and analysis Information on demographic characteristics, prevalence of perceived illness, the extent and type of herbs used in self-care; and factors associated with the choice of treatment options were collected by using a semi-structured questionnaires from mothers (or woman who assumed the role of a mother) in 600 households. When mother in a household was absent in the rst visit, repeated visits were made up to three times. The 600 households were selected using systematic random sampling technique. Enumerators who are workers of Butajira Rural Health Project were given additional training for two days on the data collection instrument. Before the initiation of the interview, oral consent was obtained from each respondent who participated in the study. EPI-INFO Version 6.0 statistical software was used for data entry and analyses. 3. Results 3.1. Perceived illness The distribution of illness and the corresponding action taken against the illness by background factors is presented in Table 1. One hundred thirty-six persons were reported to have an illness episode during a four weeks recall period preceding the interview date. Females were reported to
Fig. 1. Map of Ethiopia showing the location of the Butajira district.
no capacity to manage surgical and obstetric emergencies (Berhane, 2000). Communicable diseases including malaria, ARI, diarrhoeal diseases and tuberculosis are the major public health problems of the district. This study was carried out in villages that are under a continuous demographic surveillance by the Butajira Rural Health Programme (BRHP). There are 10 study communities under BRHP which were selected based on probability proportional to size out of 84 rural and 4 urban kebeles (the lowest administrative units) of Butajira sub-district (Fig. 2). The demographic surveillance has been going on since 1987 to provide sampling frame
Fig. 2. Location of the 10 study villages in the district.
T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155161 Table 1 Types of action taken by those with perceived illness in four weeks recall period Background factors Sex Male Female Age in years 04 514 1554 55+ Marital statusa Married Others Religion Muslim Christian, others Educationb Illiterate Literate Household size 14 59 10+
a b
157
Modern health facilities (%) 77.6 71.3 82.5 82.1 72.3 46.7 65.4 71.4 73.3 74.0 64.8 81.8 69.6 89.2 83.3
Herbal medicine (%) 14.3 16.1 14.3 15.4 46.7 21.2 21.4 16.3 14.0 22.5 3.0 21.4 10.8 6.7
No action (%) 8.2 12.6 3.6 17.9 12.3 6.7 13.5 7.1 10.5 12.0 12.7 15.2 8.9 13.5
Total ill 49 87 28 28 65 15 52 28 86 50 71 33 56 74 6
Excludes too young to get married. Excludes too young to be literate.
have more morbidity than males. Being illiterate was also associated with high morbidity. Febrile illnesses including malaria, cough/cold, diarrhoea, and skin disorders were the most frequently reported illnesses in the area.
3.2. Health care options The overall action taken for those with reported perceived illness was 89%; out of which 17.4% used herbal medicine
Table 2 Self-care with herbal drugs and factors associated with it in Butajira community, 2000 Category Sex Male Female Age in years 014 1554 55+ Religion Muslim Christian, others Marital Married Others statusa 52 28 71 33 56 80 19.2 17.9 29.4 3.0 17.9 8.8 0.03 1 NS Number ill 49 87 56 65 15 86 50 Self-care with herbs (%) 10.2 13.8 3.6 13.8 40.0 14.0 10.0 Chi-square d.f. P-value
0.43
NS
44.3
0.0000
0.43
NS
Educationb Illiterate Literate Household size 14 5+

a
23.25
0.000014
2.74
NS
Excludes too young to get married. b Excludes too young to be literate; NS = P > 0.05.
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Table 3 Medicinal plants used in self-care by Butajira community, south central Ethiopia Family/species Acanthaceae Adhatoda schimperiana Hochst. ex Nees Alliaceae Allium sativum Local name Sensel Part used Leaf Citation 6 Uses, preparation For scabies, fresh leaves are crushed and macerated in water, and then the affected area is washed with the macerate To treat diarrhoea and malaria, the bulb are chewed and swallowed. For cough/cold treatment, the bulb is crushed and boiled with tea and drunk For skin rashes, fresh leaves are crushed and macerated in water, then wash the affected area with the macerate To treat diarrhoea, headache and fever, the juice of fresh squeezed leaves is drunk For diarrhoea and dysentery, most said grind the seed and make pellet with honey and swallowed; few said solution in water is drunk. To treat skin disorders, the powdered seed is mixed with fresh butter and applied topically To treat skin problems, the juice from the apical parts of the plant is applied on the affected area To treat stomachache, vomiting and fever, the root is pounded in water and the juice is drunk For diarrhoea and malaria, decoction of the fresh leaves is drunk; for treating wound, the leaves are pounded into a paste and is applied on the affected part Decoction or infusion of pounded leaves is drunk to treat respiratory problems To treat diarrhoea, amoeba (diarrhoea with blood) and cough/cold, fresh leaves are pounded in water and the juice is drunk To treat diarrhoea and amoeba, the water solution of toasted and powdered seed is drunk in an empty stomach For eczema, the leaves are pounded and the paste applied on the affected area To expel tape worm, the ground fruit is macerated in tela (local alcoholic beverage) or water and left over night, and then the macerate is drunk in an empty stomach To treat cough/cold, the fresh leaves are decocted and the steam is inhaled To treat diarrhoea, rice water is drunk To treat scabies, chopped leaves are immersed in water and then the affected part is washed with the solution To treat amoeba, the solution of the dried and powdered leaves is drunk
Nech-shinkurt
Bulb
97
Compositae Vernonia amygdalina Delile Cucurbitaceae Zehneria scabra Sond. Crucifereae Lepidum sativum
Girawa
Leaf
Areg-ressa
Leaf
Feto
Seed
43
Euphorbiaceae Croton macrostachyus Hoechst Fabaceae Taverniera abyssinica A. Rich Labiatae Ajuga integrifolia Buch-Ham Thymus serrulatus Hoechst ex. Benth Ocimum lamiifolium Hochest ex. Benth Linaceae Linum usitatissimum L. Malvaceae Sida ovata Forsk Myrsinaceae Embelia schimperi Vatke Myrtaceae Eucalyptus globulus Labill Poaceae Oryza sativa L. Phytolaccaceae Phytolacca dodecandra LHerit Polygonaceae Rumex helpalensis
Bissana
Leaf; areal part
Dingetegna
Root
90
Anamaro/(armagussa)
Leaf
26
Tosegne Damkesse
Leaf Leaf
6 195
Telba
Seed
118
Chifrig
Leaf
Enkoko
Fruit
Nech-bahirzaf
Leaf
27
Ruz Endod
Seed Leaf
3 5
Tult
Leaf
T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155161 Table 3 (Continued ) Family/species Rumex nervosus Vahl. Rosaceae Hagenia abyssinica (Bruce) Gmel. Rubiaceae Coffea arabica Linn. Rutaceae Ruta chalepensis L. Solanaceae Datura stramonium L. Solanum campylacanthum Hochst Verbenaceae Verbena ofcinalis subsp. africana Zingiberaceae Zingiber ofcinale Rosc. Local name Embatcho/dengago Part used Leaf Citation 9 Uses, preparation
159
To treat skin disorders, leaves are crushed and mixed with butter, and then it is applied on the affected area To expel tape worm, water/local alcoholic extract of the ower is drunk in the morning in an empty stomach or the ower is pasted with honey and taken Roasted and powdered coffee is pasted with honey and taken orally for the treatment of diarrhoea and amoebiasis A decoction of the leaves mixed with tea is drunk against headache, fever, and cough/cold For treating eczema, powdered leaves is mixed with butter or fat; and the ointment is applied on the affected area To treat eczema and scabies, the juice inside the fruit is applied The crushed roots are left in water for some time and then the extract is drunk to stop diarrhoea To treat stomachache, the rhizome is chewed; for cough/cold the decoction is mixed with tea and drunk
Kosso
Flower
70
Buna
Seed
18
Tenadam
Leaf
97
Itsefaris/astenagir Emboye
Leaf Fruit
6 7
Atuch
Root
Zingibel
Rhizome
68
and 82.4% modern medicine. There was a relatively higher rate of action taken among males than females. Belief on efcacy (57.2%), and economic (28.5%) and geographical inaccessibility of modern health care (14.3%) were mentioned as reasons for choosing herbal medicine as the rst line of health care option in Butajira area. Proportions of those with perceived illness and chose to use herbal medicine in self-care were compared between subgroups (e.g. among different ages, males versus females, illiterate versus literate, etc.) using chi-square tests (Table 2). Females were more likely to use herbal medicine than males but the association was not statistically signicant (P > 0.05). Age was found to have a signicant association with the use of herbal medicine (P < 0.0001). Chi-square for linear trend for age also indicated that the tendency to use herbal medicine increases with age. Illiterates (who are not able to read and write) were signicantly more likely to use herbal medicine than literates (P < 0.0001). 3.3. Self-care with herbal drugs The prevalence of self-care with herbal drugs in Butajira community in four weeks recall period was 12.5%. Twenty-ve species belonging to 21 families were claimed to be used in self-care. Taverniera abyssinica A. Rich. (Fabaceae), Ocimum lamiifolium Hochst. ex Benth (Labiatae), Allium sativum (Alliaceae), Ruta chalepensis (Rutaceae), Linum usitatissimum L. (Linaceae), Hagenia abyssinica (Bruce) Gmel (Rosaceae), Zingiber ofcinale Rosc. (Zingiberaceae) and Lepidum sativum (Crucifereae)
were the most frequently used plants (Table 3). Leaves were the part of the plant commonly used.
4. Discussion and conclusions Perceived morbidity is the main initiator for seeking health care. The perception of an illness by a person in a household was, therefore, used as a starting point of our inquiry to reveal the extent and types of herbs taken against illnesses in Butajira community. The morbidity pattern reported in this study concurs with what was reported by Shamebo et al. (1994) and other similar study done in the rift valley (Kitaw, 1987). Like other studies, this study showed that the burden of illness is less pronounced in males than females (Kitaw, 1987; Gedif, 1995). This might have a strong association with the status of women in Butajira community. As documented by Berhane (2000), women in rural Butajira are in a very disadvantaged position because of very low literacy levels, high workloads and low access to modern health facilities. This study also showed that perceived efcacy, economic and geographic accessibility are main reasons for popularity of herbal medicine and its practitioners in Butajira community. Although the association was not statistically signicant, females were more likely found to use herbal medicine in self-care than males. This is consistent to the result of previous study done in Hong Kong about the general use of herbal medicine (Wong et al., 1997). It was also interesting to note that education and age had a signicant association
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with the use of herbal medicine. In this regard, illiterates and older residents are signicantly more likely to use herbal medicine than literate and younger people, respectively. A study conducted in rural Tanzania also showed that age and education were the main factors that seemed to inuence the choice of health care (Satimia et al., 1998). Almost 1/3rd of the medicinal plants reported to be used in self-care in this study have also long been used as food and/or spices in food and widely sold in Ethiopian markets (Kloos et al., 1978). Recognising their frequent use there is a need to promote phytochemical and pharmacological investigations on these plants.
Acknowledgements The nancial assistance from Katholischer Akademischer Auslnder-Dienst (KAAD) is highly acknowledged. We extended our deepest appreciation to Professor Dr. R. Neubert for his all rounded support to make this study a reality. Ethiopian Science and Technology Commission and Butajira Rural Health Project are acknowledged for facilitating the eld work. Special thanks go to enumerators who skilfully collected the data. Finally, we wish to express our gratitude to mothers in Butajira who shared with us information about their personal lives and experiences.
Appendix A
References
Abebe, D., Ayehu, A., 1993. Medicinal Plants and Enigmatic Health Practices of Northern Ethiopia. Addis Ababa, Ethiopia. Abebe, W., 1986. A survey of prescriptions used in traditional medicine in Gondar region, north western Ethiopia: general pharmaceutical practice. Journal of Ethnopharmacology 18, 147165. Berhane, Y., 2000. Womens Health and Reproductive Outcome in Rural Ethiopia. Ume University Medical Dissertation, New Series No. 675. UmU Press, Ume. Berhane, Y., Wall, S., Kebede, D., Emmelin, A., Enqueselassie, F., et al., 1999. Establishing an epidemiological eld laboratory in rural areas-potential for public health research and interventions: Butajira Rural Health Project. Ethiopian Journal of Health Development 13, 147. Gedif, T., 1995. Self-Medication and Its Determinants in Butajira, Southern Ethiopia. Addis Ababa University Masters Thesis, Addis Ababa. Houghton, P.J., 1995. The role of plants in traditional medicine and current therapy. Journal of Alternative and Complementary Medicine 1, 131143. Jansen, P.C.M., 1981. Spices, Condiments and Medicinal Plants in Ethiopia, Their Taxonomy and Agricultural Signicance. Centre for Agricultural Publishing and Documentation, Wageningen. Kitaw, Y., 1987. Self care: a study of three communities in Ethiopia. Ethiopian Journal of Health Development 2, 175.
T. Gedif, H.-J. Hahn / Journal of Ethnopharmacology 87 (2003) 155161 Kloos, H., Tekle, A., Yohannes, L.W., Yosef, A., Lemma, A., 1978. Preliminary studies of traditional medicinal plants in nineteen markets in Ethiopia: use patterns and public health aspects. Ethiopian Medical Journal 16, 3343. Lambert, J., Srivastava, J., Vietmeyer, N., 1997. Medicinal Plants: Rescuing a Global Heritage, World Bank Technical Paper No. 355. The World Bank, Washington. Pankrust, R., 1976. Historical reections on the Traditional Ethiopian Pharmacopoeia. Journal of Ethiopian Pharmaceutical Association 2, 2933. Satimia, F., McBride, S.R., Leppard, B., 1998. Prevalence of skin disease in rural Tanzania and factors inuencing the choice of health care, modern or traditional. Archives of Dermatology 134, 13631366. Shamebo, D., 1993. Epidemiology for Public Health Research and Action in a Developing Society: Butajira Rural Health Project in Ethiopia. Ume University PhD Dissertation, Ume.
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Shamebo, D., Muhe, L., Freij, L., Sandstrm, A., Wall, S., 1994. The Butajira Rural Health Project: health needs and care in Ethiopiaa case for improved and equitable services. Ethiopian Journal of Health Development 8, VI1VI14. Tadesse, M., Demissew, S., 1992. Medicinal Ethiopian plants: inventory, identication and classication. In: Sue, E., Zemede, A. (Eds.), Plants Used in African Traditional Medicine as Practised in Ethiopia and Uganda. Botany 2000: East and Central Africa. NAPRECA Monograph Series No. 5, Addis Ababa University, Addis Ababa, pp. 119. Vicchiato, N.L., 1993. Traditional medicine. In: Helmut, K., Zein Ahmed, Z. (Eds.), The Ecology of Health and Disease in Ethiopia. West View Press, Boulder, pp. 157178. Wong, T.W., Yu, T.S., Liu, J.L.Y., Lee, N.L., Lloyd, O.L., 1997. Factors associated with the utilisation of traditional Chinese medicine in a small town in Hong Kong. American Journal of Chinese Medicine 15, 367373.
Biological activity of extracts from Catalpa bignonioides Walt. (Bignoniaceae)

D. Muoz-Mingarro a, , N. Acero b , F. Llinares c , J.M. Pozuelo d , A. Galn de Mera b , J.A. Vicenten b , L. Morales e , L.F. Alguacil e , C. Prez e
Seccin de Qu mica Anal tica, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU, Apartado 67, 28660 Boadilla del Monte, Madrid, Spain b Seccin de Biolog a Vegetal, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU, Apartado 67, 28660 Boadilla del Monte, Madrid, Spain c Seccin de Microbiolog a, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU, Apartado 67, 28660 Boadilla del Monte, Madrid, Spain d Seccin de Biolog a Celular, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU, Apartado 67, 28660 Boadilla del Monte, Madrid, Spain Seccin de Farmacolog a y Toxicolog a, Facultad de Ciencias Experimentales y de la Salud, Universidad San Pablo CEU, Apartado 67, 28660 Boadilla del Monte, Madrid, Spain Received 19 February 2002; received in revised form 17 March 2003; accepted 21 March 2003
a
Abstract Catalpa bignonioides Walt. (Bignoniaceae) is a species that belongs to a tropical family but has been introduced in many countries as ornamental. Although this plant is consumed by indigenous cultures of South America for medical uses, experimental studies of the biological properties of Catalpa bignonioides are lacking. The aim of this work was to study the biological activity of crude extracts from either pods, seeds or leaves of Catalpa bignonioides which were collected in Spain. Ethyl ether, butanolic and aqueous fractions of the pod extract were also prepared and studied. We have examined the antimicrobial activity against ve bacteria and one yeast, the cytotoxic activity against HepG2 cells and the anti-inammatory and antinociceptive effects in rodents. A preliminary phytochemical analysis of the extracts and fractions was also conducted. Results showed no antimicrobial or antitumoral effects, but prominent anti-inammatory and antinociceptive actions of the extracts. These last activities may be a result of the presence of either of saponins, sterols or phenols, mainly found in the leaves and pods of the plants. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Catalpa bignonioides; Extracts; Biological activities
1. Introduction Species belonging to the Bignoniaceae are widely used for many different purposes in medicinal practice by the indigenous cultures of South America (Rasadah and Houghton, 1998). Some of these species, like Jacaranda micranta Cham., Tabebuia caraiba (Mart.) Bureau, Tecoma sambucifolia Kunth and Tecoma stans (L.) Juss. ex Kunth are known for the anti-inammatory, antirheumatic, antinociceptive, narcotic or antisyphilitic activity of their extracts. The Catalpa Scop. genus, which belongs to this family, comprises 11 species of trees and shrubs native to East Africa and North and South America. Outside the tropics, several
Corresponding
author.
species are found in cultivation as greenhouse ornamentals. Some extracts from the Catalpa species have shown interesting biological properties: extracts from Catalpa ovata G. Don fructus have mutagenic activity towards Salmonella typhimurium (Nozaka et al., 1989), while its stem-bark extracts have antitumoral activity (Fujiwara et al., 1998). Regarding properties and uses of Catalpa bignonioides, commonly known as Bean-tree, which were already mentioned in the physiomedical dispensatory of Cook (1869), the bark of Catalpa bignonioides is cited as a stimulating tonic in syrup form, the decoction of the pods as a demulcent with relaxant and stimulant properties and the leaves are described as useful for irritable ulcers. Catalpa bignonioides has also been described for the treatment of respiratory diseases (decoction of pods and seeds), irritable scrofulous ulcers (cataplasms of bruised leaves), strumous
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D. Muoz-Mingarro et al. / Journal of Ethnopharmacology 87 (2003) 163167
ophthalmia, cutaneous affections (juice of leaves and roots), scrofulous maladies and helmintic infections (decoction or powder of barks). Despite all these potential benecial effects, experimental studies of the biological properties of Catalpa bignonioides extracts are lacking. Phytochemical studies (phytochemDB) show the presence of several classes of compounds in extracts of Catalpa bignonioides, which could be linked to some of the traditional uses of Bignoniaceae. These include fats, sugars, terpenes, alkaloids, tannins and, particularly, avonoid and phenolic compounds, which were detected in high amounts. Previously Rau (1870; Kings American Dispensatory) made an examination of the inner bark and found it to contain tannin and a nauseating matter soluble in ether. Sugar, tannin, resin and xed oil are constituents of the seeds. In this paper we report a preliminary phytochemical analysis and biological screening on bactericidal, cytotoxic, anti-inammatory and antinociceptive activities of aqueous extracts from leaves, pods and seeds of Catalpa bignonioides. These activities were chosen in part on the basis of previous ndings of our team regarding the biological activity of Tecoma sambucifolia, another Bignoniaceae plant (Alguacil et al., 2000). The pod extract was further fractionated in order to approach the isolation of the possible active principles.
and sterols by using the methods previously described by Tona et al. (1998). 2.4. In vitro cytotoxicity assay The cell line used was human hepatoma cell line (HEpG2) obtained from the American Type Culture Collection (ATCC, HB 8065). The cell line was grown on Dulbecos Mem medium supplemented with 10% fetal bovine serum (FBS), and incubated at 37 C in an atmosphere of 5% CO2 in air (95% humidity). At a log phase of their growth cycle, the cells were treated in triplicate with various concentrations of the extracts (between 25 and 0.1 mg/ml) with a nal dimethylsulfoxide (DMSO) concentration of 0.1%. This concentration of DMSO did not affect cell viability. The assay was performed using a 96-well plate which contained 2500 cells per well incubated with the corresponding extract, during 72 h, as previously described. Test wells containing the cells alone, free of plant extracts were used as controls. The MTT assay, according to Borenfreund et al. (1988), was used to obtain the effective dose that inhibits 50% growth after the incubation period (IC50 ). 2.5. Antimicrobial assays The bacterial growth inhibition assays were performed using cultures of Escherichia coli (ATCC 35219), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC 24213), Enterococcus faecalis (ATCC 29212), Salmonella tiphymurium (ATCC 13311), and the yeast Candida albicans (ATCC 10231). Bacteria strains were maintained on Mueller-Hinton broth and the yeast on Sabourands dextrose agar. The diluted extract suspension was homogenized and the screening was then performed according to the liquid dilution method (Vanden Berghe and Vlietinck, 1991). Minimum inhibitory concentration (MIC) was determined by incorporating various amounts (1200 mg/ml) of reconstituted extract solution into the medium. The MIC was interpreted as the lowest concentration of the extracts that did not permit any visible growth when compared with that of the control. 2.6. In vivo pharmacological assays 2.6.1. Animals and sample preparation Male SpragueDawley rats (200250 g; San Pablo-CEU University breeding) and male OF1 mice (2530 g, Iffa-Credo, France) were used. The animals were housed in cages with water and food available ad libitum, kept in a controlled environment (temperature, 2022 C; dark/light, 12 h/12 h, humidity, 4555%) and randomly assigned to the different experimental groups. The aqueous extracts were dissolved in physiological saline for i.p. administration (10 ml/kg) at a dose of 1 g/kg. The doses of the fractioned
2. Methodology 2.1. Plant material Catalpa bignonioides specimens were collected in Boadilla del Monte (Madrid, Spain) and identied by Dr. Galn de Mera, USP (San Pablo University), Madrid. A voucher specimen was deposited in the USP herbarium under the number 250399. Collected material was dried under darkroom conditions. 2.2. Plant extracts Crude extracts of leaves, pods, and seeds were prepared by decoction of 10 g of each pulverized material in 200 ml of water for 30 min. The resultant extracts were then ltrated and concentrated to dryness under reduced pressure. The yield was 22.9% (w/w) for pods, 14.1% (w/w) for leaves and 5.3% (w/w) for seeds. The concentrated aqueous extract of pods was fractionated by successive extraction with ethyl ether (3 100 ml) and butanol (3 100 ml). An ethyl ether (P-E, 3.94% w/w), a butanol (P-B, 28% w/w) and an aqueous fraction (P-A, 55.2% w/w) were obtained. 2.3. Preliminary phytochemical analysis Plant materials were screened for the presence of saponins, tannins, total phenols, anthraquinones, avonoids
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pod extracts were calculated to provide an amount of the different substances equivalent to that of the crude pod extract, i.e. 40 mg/kg for P-E, 300 mg/kg for P-B and 555 mg/kg for P-A. 2.6.2. Acetic acid writhing in the mouse The test was performed as described by Koster et al. (1959). Nociception induced by acetic acid was characterized by the presence of abdominal constrictions, consisting of the contraction of the ank muscles associated with inward movements of the hind limbs or with whole body stretching. Animals were injected with the extracts from Catalpa bignonioides, saline or indomethacin (3 mg/kg; Sigma, Spain), which was used as a reference compound. Thirty minutes after treatment, all the animals received 2% acetic acid i.p., and 10 min afterwards, the number of abdominal constrictions was recorded for 15 min by visual observation of the animals. 2.6.3. Carrageenan-induced hind paw edema in the rat Carrageenan-induced inammation was initially described by Winter et al. (1962) and has become a widely used model for screening anti-inammatory agents. At the beginning of the study, the baseline paw volume was determined by submerging the right hind paw up to the tibiotarsal joint into a water cell of a plethysmometer (Digital Pletysmomet, L37500; Letica). The volume of displacement, which is equal to the paw volume, was then read on a digital display. Each determination was the average of three repeated measures. After baseline determination, the rats were injected with the extracts from Catalpa bignonioides, saline or indomethacin (5 mg/kg; Sigma), which served as a positive control. One hour afterwards, the rats were subcutaneously injected with 0.1 ml of 1% lambda carrageenan (Sigma) into the surface of the right hind paw, and the effect on paw volume was studied 1, 2 and 3 h post injection.
Table 1 Phytochemical screening and cytotoxic activity, against HEpG2 cells, of crude aqueous extracts and fractions from Catalpa bignonioides Anth. Crude extract Seeds Leaves Pods Pod fractions P-A P-B P-E Sap. Este. Tan. T. Ph. Flav. IC50 (mg/ml) 28 23.6 10.9 3.5 0.68 3.58
+ + + +
+ + + + +
+ +
+ + + + +
+ + +
Anth.: anthraquinones, Sap.: saponins, Este.: sterols, Tan.: tannins, T. Ph.: total phenols, Flav.: avonoids. P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction.
4.2. Cytotoxicity assay The IC50 (mg/ml) values obtained in the cytotoxicity assay against the HepG2 cell line are shown in Table 1. A very low toxicity was found in all extracts and fractions. The highest toxicity was found in the crude extract of pods and fraction P-B. 4.3. Antimicrobial activity The inhibitory bacterial growth by extracts, indicated by their MIC values, is summarized in Table 2. None of the extracts examined showed a signicant bactericidal activity, since the lower MIC (showed by pods against Salmonella tiphymurium) was 3.12 mg/ml. No effects were observed in yeast analysis, as the MIC obtained was higher than 200 mg/ml in all cases. 4.4. Anti-inammatory activity The results of this study are shown in Table 3. The extracts of leaves and pods exhibited a signicant anti-inammatory activity of similar magnitude 2 and 3 h after carrageenan injection, while the extract of seeds was devoid of signicant effects during all the periods considered. Although the three pod fractions exhibited some activity in the test, only P-A provided a marked and sustained effect throughout the evaluation period considered. P-E only produced a transient decrease of paw edema, and P-B also provided a slight effect that achieved statistical signicance 3 h post injection. 4.5. Antinociceptive activity The effects obtained in the antinociception experiments were qualitatively similar to those of the anti-inammatory study (Fig. 1). Although the three crude extracts exhibited some pharmacological activity, the extract of seeds was the least potent and the extracts of pods and leaves behaved similarly. Concerning the pod fractions, P-A was again the
3. Statistical analysis Statistical analysis was performed with ANOVA followed by multiple range tests (least-squares difference test, LSD). Differences were considered signicant at P < 0.05.
4. Results 4.1. Phytochemical study The results of our assay on the crude extracts and fractions are shown in Table 1. All crude extracts showed the presence of saponins (triterpenic and steroidic), sterols and phenols and were negative for anthraquinones. Regarding pod fractions, saponins were detected in P-A, sterols in P-A and P-B and avonoids in P-B and P-E.
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Table 2 Antibacterial activity of crude aqueous extracts and fractions from Catalpa bignonioides (MIC, mg/ml) Escherichia coli Crude extract Seeds Leaves Pods Pod fractions P-A P-B P-E 100 6.25 6.25 25 >200 >200 Pseudomonas aeruginosa 100 100 50 50 >200 >200 Staphylococcus aureus 50 12.5 12.5 25 100 100 Enterococcus faecalis 50 12.5 12.5 25 >200 100 Salmonella tiphymurium 50 6.25 3.12 6.25 >200 >200
P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction. Table 3 Anti-inammatory effect of crude aqueous extracts and fractions from Catalpa bignonioides on carrageenan-induced hind paw inammation in the rat Treatment n Paw volume (ml) after carrageenan injection Baseline Saline Indomethacin Crude extract Seeds Leaves Pods Pod fractions P-A P-B P-E 15 14 6 6 16 7 9 7 1.53 0.02 1.55 0.02 1.56 0.01 1.59 0.04 1.61 0.02 1.51 0.04 1.53 0.03 1.48 0.02 1h 2.00 0.03 1.79 0.03 1.92 0.07 1.93 0.03 1.92 0.04 1.77 0.06 1.88 0.04 1.81 0.04 2h 2.64 0.10 2.18 0.04 2.62 0.20 2.15 0.10 2.10 0.11 2.15 0.13 2.35 0.10 2.33 0.07 3h 2.91 0.10 2.43 0.04 2.86 0.25 2.31 0.22 2.25 0.11 2.37 0.15 2.56 0.12 2.68 0.12
P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction. P < 0.05 vs. saline.
Fig. 1. Effect of aqueous extracts of Catalpa bignonioides on abdominal constrictions produced by acetic acid. Bars are means S.E.M. P < 0.05 vs. saline. (P-A: aqueous fraction, P-B: butanol fraction, P-E: ethyl ether fraction).
most active; on the other hand, the effect of P-E injection did not reach statistical signicance.
5. Discussion and conclusions The antitumoral activity of species such as Catalpa ovata (Kingston and Rao, 1982; Fujiwara et al., 1998) has been related to phenolic compounds like naphtoquinones. Since the
phytochemical analysis of aqueous extracts from Catalpa bignonioides show the presence of phenolic compounds, the cytotoxic effect observed is considerably low and has little relevance against the human hepatoma cell line (HEpG2), it has been suggested that the reputed antimicrobial activity of some species of Bignoniaceae like Kigelia pinnata (Jacq.) DC (Akunyili et al., 1991), Jacaranda mimosifolia D. Don, Tecoma stans, Crescentia cujete L. (Binutu and Lajubutu, 1994) Tabebuia spectabilis (Planch. and Linden)
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G. Nicholson, and Oroxylum indicum (L.) Vent. (Rasahad and Houghton, 1998) could be linked to another kind of substances, the iridoids. Although these compounds have previously been reported in Catalpa bignonioides (Iwagawa et al., 1991; McDaniel, 1992), the isolation of iridoids was not favored in our study and therefore would explain the very low effect obtained. All these results indicate that the potential antitumoral and antimicrobial activities of Catalpa bignonioides extracts appear to be of lesser therapeutic value than those of other Bignoniaceae; however, this conclusion can be strictly applied only to the Spanish specimen of this species and we cannot rule out that other tropical plants might contain higher amounts of active substances or even different compounds of interest. In contrast, the anti-inammatory and antinociceptive effects of the extracts studied were quite signicant. These ndings are coherent with the similar pharmacological prole of the extracts obtained from other Bignoniaceae, such as Tecoma sambucifolia (Alguacil et al., 2000). The crude extracts of pods and leaves were more active with respect to those of seeds. This result, combined with the phytochemical analysis, suggests that the biological activity can be attributed either to saponins, sterols or phenolic compounds, while the contribution of avonoids does not seem probable. On the other hand, the aqueous fraction of the pods is the one that retains the highest pharmacological activity. In this sense, the substances that could be expected to play a signicant role in the case of Catalpa bignonioides are steroidic saponins, which possess hydro-aromatic ring systems similar to those of steroids (Gupta et al., 1969). In summary, neither antitumoral nor antimicrobial activities were detected in crude aqueous extracts of Catalpa bignonioides but it has an anti-inammatory and antinociceptive potential, which is probably related to the saponins, sterols or phenols contained in its leaves and pods. Further fractioning experiments are in progress to isolate the active principles responsible for these biological activities. Acknowledgements We wish to express our gratitude to Linda Hamalainen for her linguistic help.
References
Akunyili, D.N., Houghton, P.J., Raman, A.R., 1991. Antimicrobial activities of the stem-bark of Kigelia pinnata. Journal of Ethnopharmacology 35, 173177. Alguacil, L.F., Galn de Mera, A., Gmez, J., Llinares, F., Morales, Muoz-Mingarro, D., Pozuelo, J.M., Vicente Orellana, J.A., 2000. Tecoma sambucifolia: anti-inammatory and antinociceptive activities, and in vitro toxicity of extracts of the huarumo of Peruvian Incas. Journal of Ethnopharmacology 70, 227233. Binutu, O.A., Lajubutu, B.A., 1994. Antimicrobial potentials of some plant species of the Bignoniaceae family. African Journal of Medical Sciences 23, 269273. Borenfreund, E., Babich, H., Martin-Alguacil, N., 1988. Comparison of two in vitro cytotoxicity assays: the neutral red (NR) and tetrazolium (MTT) tests. Toxicology in Vitro 2, 16. Cook, W., 1869. The Physio-Medical Dispensatory: A Treatise on Therapeutics, Materia Medica, and Pharmacy in Accordance with the Principles of Physiological Medication. Cincinnati. Fujiwara, A., Mori, T., Iida, A., Ueda, S., Hano, Y., Nomura, T., Tokuda, H., Nishimo, H., 1998. Antitumor-promoting naphthoquinones from Catalpa ovata. Journal of Natural Products 61, 629632. Gupta, M.B., Bhalla, T.N., Gupta, G.P., Mitra, C.R., Bhargava, K.P., 1969. Anti-inammatory activity of natural products (I) Triterpenoids. European Journal of Pharmacology 6, 6770. Iwagawa, T., Hamada, T., Kurogi, S., Hase, T., Okubo, T., Kim, M., 1991. Iridoids from Catalpa bignonioides. Phytochemistry 30, 40574060. Kingston, D.G., Rao, M.M., 1982. Plant anticancer agents. XII. Isolation and structure elucidation of new cytotoxic quinones from Tabebuia cassinoides. Journal of Natural Products 45, 600604. Koster, R., Anderson, M., de Beer, E.J., 1959. Acetic acid for analgesic screening. Federation Proceedings 18, 412. McDaniel, C.A., 1992. Major antitermitic components of the heartwood of southern catalpa. Journal of Chemical Ecology 18, 359369. Nozaka, T., Watanabe, F., Ishino, M., Morimoto, I., Kondoh, H., Koyama, K., Natori, S., 1989. A mutagenic new iridoid in the water extract of catalpae fructus. Chemical and Pharmaceutical Bulletin 37, 28382840. Rasadah, M.A., Houghton, P.J., 1998. Antimicrobial activity of some species of Bignoniaceae. ASEAN Review of Biodiversity and Enviromental Conservation (ARBEC). Article III. Rau, E.A., 1870. In: Wickes, H., Lloyd, J., (Eds.), American Journal of Pharmacology. Kings American Dispensatory, Cincinnati. Tona, L., Kambu, K., Ngimbi, N., Cimanga, K., Vlietnick, A.J., 1998. Antiamoebic and phytochemical screening of some Congolese medical plants. Journal of Ethnopharmacology 61, 5765. Vanden Berghe, D.A., Vlietinck, A.J., 1991. In: Dey, P.Mp., Harborne, J.B., (Eds.), Methods in Plant Biochemistry: screening Methods for Antibacterial and Antiviral Agents from Higher Plants. Academic Press, London, pp. 4769. Winter, C.A., Risley, E.A., Nuss, G.W., 1962. Carrageenan-induced edema in hind paw of the rat as an assay for anti-inammatory drugs. In: Proceedings of the Society for Experimental Biology and Medicine III, pp. 554547.
Comparison of the gastroprotective effect of a diterpene lactone isolated from Croton cajucara with its synthetic derivatives
Patricia Silva Melo a , Nelson Durn b,c , Cllia Akiko Hiruma-Lima d , Alba Regina Monteiro Souza-Brito e , Marcela Haun a,
Departamento de Bioqu mica, Instituto de Biologia, Universidade Estadual de Campinas, cp. 6110, CEP 13081-970, Campinas, SP, Brazil Laboratrio de Qu mica Biologica, Instituto de Qu mica, Universidade Estadual de Campinas, cp. 6154, CEP 13081-970, Campinas, SP, Brazil c Ncleo de Ci encias Ambientais, Universidade Mogi das Cruzes, CEP 08790-911, Mogi das Cruzes, SP, Brazil Departamento de Fisiologia, Instituto de Biociencias, Universidade Estadual Paulista, cp. 510, Rubio Junior s/n, CEP 18618-000, Botucatu, SP, Brazil e Departamento de Fisiologia e Biof sica, Instituto de Biologia, Universidade Estadual de Campinas, cp. 6109, CEP 13084-970, Campinas, SP, Brazil
b a
Received 30 November 2002; received in revised form 27 March 2003; accepted 27 March 2003
Abstract The effect of three new derivatives from dehydrocrotonin (DHCcompound I) on gastric damage in different animal models including gastric ulceration induced by a necrotic agent and hypothermic restrained-stress was studied: compound II (produced by reducing the cyclohexenone moiety of DHC with NaBH4 ); compound III (produced by reducing the carbonyls with LiAlH4 ); and compound IV (produced by transforming the lactone moiety into an amide). Their structures were conrmed on the basis of chemical and physicochemical evidence. When previously administered (p.o.) at a dose of 100 mg/kg, compound II signicantly (P < 0.01) reduced gastric injury induced by HCl/ethanol (78%) and indomethacin (88%) better than did reference compound I (48 and 43%, respectively). But the anti-ulcerogenic activity of compound II was completely abolished by the stress-induced ulcer. Reduction of carbonyls with LiAlH4 (compound III) caused decreased activity, markedly when no protective effect in any of the models was applied (P > 0.05). However, compound IV, in which the lactone moiety was changed into an amide, when administered at the same dose (100 mg/kg, p.o.), was more effective. The presence of a lactone moiety or Michael acceptor is probably essential for the anti-ulcerogenic effect of these compounds. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Gastroprotective activity; DHC; Croton cajucara
1. Introduction In Brazil Croton cajucara Benth. (Euphorbiaceae), popularly known as Sacaca is used in popular medicine as a cytoprotective agent for gastric ulcers and to treat several illnesses, such as diarrhea, diabetes, and liver inammation (Di Stasi et al., 1989). As a start for our studies on anti-ulcer substances, we reported the anti-ulcerogenic activity of trans-dehydrocrotonin (DHCcompound I, Fig. 1), the primary compound (nor-clerodane diterpene lactone) isolated from Croton cajucara bark, in different ulcerogenic models, using mice and rats (Souza-Brito et al., 1998). The protective effect of DHC on induced gastric lesions occur through the suppression of acid secretion and
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through protection of the gastric mucosa by an increase of prostaglandins-E2 (Hiruma-Lima et al., 1999). Clerodane diterpenes with anti-ulcerogenic activity have also been isolated from Croton sublyratus (Kitasawa et al., 1980) and Aparisthmium cordatum (Hiruma-Lima et al., 2000, 2001). The etiology of gastroduodenal ulcers is inuenced by various aggressive and defensive factors, such as acid-pepsin secretion, parietal cell, mucosal barrier, mucus secretion, blood ow, cellular regeneration, and endogenous protective agents, such as prostaglandins and epidermic growth factors (Repetto and Llesuy, 2002). It is well known that peptic ulcers are the outcome of an imbalance between defensive factors (forces of mucosal resistance) and aggressive factors (gastric acid and pepsin secretion) (Yamamoto et al., 1998). There are many products used for the treatment of gastric ulcers, such as antacid, proton pump inhibitor or H2 -blockers, but most of these drugs produce some adverse reactions (Brunton, 1996). Therefore, numerous efforts have
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Fig. 1. DHC and derivatives.
been pursued to nd a novel class of anti-ulcer agent which strengthens defensive mechanisms with less toxic effect. Studies of the acute toxicity of DHC in vivo (Souza-Brito et al., 1998) have shown that this compound has a relatively low oral toxicity when administered for a short period of time. Moreover, in subchronic toxicity experiments, a signicant dose-dependent increase in liver weight was observed, whereas a signicant reduction in both plasma alkaline phosphatase and cholesterol levels, as well as an increase in -glutamyl transpeptidase were observed in the DHC-treated animals. Despite the good anti-ulcerogenic activity of DHC, the long-term use of this compound can induce liver damage based on in vitro and in vivo evaluation (Rodriguez and Haun, 1997, 1999). According to Melo et al. (2001, 2002), derivatives of DHC differ from DHC in their toxicity on V79 cells and hepatocytes. Therefore, the present research was undertaken to determine the anti-ulcerogenic activity of three DHC derivatives in different experimental models of gastric ul-
cers in mice with the aim to compare the structureactivity relationship.
2. Material and methods 2.1. Animals All experiments were performed using male albino Swiss mice (30 3.0 g) from the Central Animal House of the State University of Campinas (CEMIB/UNICAMP). The mice were fed a standard chow (Nuvilab CR-a, Nuvital) and had free access to water under xed conditions of illumination (12/12 h light/dark cycle), humidity (55 1.0%), and temperature (22 1.0%). The mice were fasted before all assays, because standard drugs were administered orally (by gavage) using a 12% solution of Tween 80 (10 ml/kg) as vehicle. Experimental Protocols were approved by the Ethic Committee for Animal Research (UNICAMP), and the
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experiments were conducted according to the recommendations of the Canadian Council on Animal Care (Olfert et al., 1993). 2.2. DHC and derivatives DHC was obtained from Croton cajucara bark as described by Souza-Brito et al. (1998). The lactone DHC molecule was opened through a dimethylamine reaction to yield compound IV, as described by Cromwell and Cook (1958): 0.5 g of DHC was dissolved in 2.0 ml of MeOH and 3.0 ml of aqueous 25% solution of dimethylamine heated in a sealed tube at 75 C for 72 h. The reaction mixture was evaporated to dryness, and the product puried by thin-layer chromatography to yield compound IV (Fig. 1) as a yellow oil. The reduction of the cetone group in DHC was done as described by Itokawa et al. (1989), using a methanol solution of DHC (100 mg) treated with an excess of sodium borohydride (NaBH4 , 30 mg). After work-up in the usual way, the product was puried by thin-layer chromatography (n-hexaneEtOAcMeOH, 7:2:1 v/v), affording compound II (Fig. 1) as a white powder (Itokawa et al., 1989). For compound III, DHC (300 mg) in 50 ml of tetrahydrofuran (THF) was added to a solution of LiAlH4 (130 mg) in THF. The mixture was reuxed by stirring for 2 h to give a crude crystalline material which was separated by thin layer chromatography (EtOAchexane, 1:1 v/v), yielding a white powder (Shimoma et al., 1998). The purity and structure of compounds IIIV was characterized by NMR, UV, IR, and MS techniques (Melo et al., 2002). 2.3. Drugs The drugs and reagents were prepared immediately before use. The following drugs were used: cimetidine and indomethacin from Sigma Chemical Co. (St. Louis, MO); Tween 80 (Synth) obtained commercially in Brazil. All other reagents used were of analytical grade. 2.4. Anti-ulcerogenic activity The gastroprotective activity of DHC derivatives was assessed on three experimentally induced gastric ulcer models. 2.4.1. HCl/ethanol-induced ulcers The anti-ulcerogenic activity of DHC and DHC derivatives on HCl/ethanol-induced gastric ulcers was assessed in mice as described by Mizui and Doteuchi (1983). Mice were divided into groups of eight animals and fasted for 24 h prior to receiving an oral dose of the vehicle solution (12% Tween 80, 10 ml/kg), cimetidine (100 mg/kg), and compounds I, II, III, and IV (100 mg/kg). Fifty minutes later, all groups were dosed orally with 0.2 ml of a 0.3 M HCl/60% ethanol solution (HCl/ethanol) to induce gastric ulcers. Animals were killed by cervical dislocation 1 h after the administration
of HCl/ethanol, and the extent of the gastric lesions was determined, expressed as a lesion index, corresponding to the sum of the lesions. The results were expressed as an ulcerative index (UI) as described by Szeleyi and Thiemr (1978). 2.4.2. Hypothermic restraint-stress ulcers The anti-ulcerogenic activity of DHC and DHC derivatives in this model was assessed in mice using the method of Levine (1971) with some modications. After 24 h of starvation, the animals were given an oral dose of compounds I, II, III or IV (100 mg/kg). One hour after treatment, ulceration was induced by immobilizing the animals in a closed, cylindrical cage at 4 C. After 3 h, the mice were killed by cervical dislocation, and the stomachs removed and examined for ulcers as described earlier. 2.4.3. Indomethacin-induced ulcers Mice (8/group) were randomly divided into four groups and fasted for 24 h. Thirty minutes after the oral administration of compounds I, II, III or IV (100 mg/kg), cimetidine (100 mg/kg) or 12% Tween 80 (10 ml/kg), 30 mg/kg of indomethacin was administered subcutaneously to the mice from each group as described by Hayden et al. (1978). The animals were killed 4 h later, the stomachs removed and opened and the gastric lesions determined as described earlier. 2.5. Statistical analysis The results were expressed as mean S.D. Statistical signicance was determined by one-way ANOVA followed by Dunnets pairwise test, with the level of signicance set at P < 0.05.
3. Results 3.1. DHC and derivatives Bark of Croton cajucara were collected in our experimental plantation in Benca, near Belm, Par, Brazil. A voucher specimen number 247 has been deposited in the Herbarium of Museu Paraense Em lio Goeldi. Powdered plant material (20 kg dry bark) was extracted with hexane in a Soxhlet apparatus, and the crude crystals formed in a concentrate hexane solution were recovered after a few days. A pure compound (111 g) was obtained after repeated crystallization from isopropanol, showing physicochemical properties in perfect accordance with reported data for the structure of trans-DHC, as depicted in Fig. 1. Compounds II, III, and IV (Fig. 1) were obtained at good yields (80, 70, and 30%, respectively) and were characterized by standard spectroscopic methods, such as IR, UV-Vis, MS, and 1 H NMR according to the literature (Melo et al., 2002).
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P.S. Melo et al. / Journal of Ethnopharmacology 87 (2003) 169174 Table 1 Comparative effects of compounds I (DHC), II, III, and IV on the different methods of induced gastric lesions in mice Treatment (p.o.) Ulcer inhibition (%) HCl/ethanol Control Cimetidine Compound Compound Compound Compound 49 48 78 22 89 Indomethacin 70 43 88 21 90 Stress 75 53 14 18 74
I II III IV
All compounds were administered at 100 mg/kg. Data are expressed as mean S.D. ANOVA followed by Dunnetts test. P < 0.01.
Fig. 2. Effects of compounds I (DHC), II, III, and IV on gastric ulcer induced in mice by HCl/ethanol (positive control: cimetidine), hypothermicstress (positive control: cimetidine), and indomethacin (positive control: cimetidine). Results are expressed as mean S.D. Asterisks indicate signicant difference from corresponding control (ANOVA followed by Dunnetts test, where P < 0.01).
3.2. HCl/ethanol-induced ulcers The effects of the DHC and DHC derivatives in the three assays of gastric lesions are shown in Fig. 2. All the tested compounds were administered orally at a dose of 100 mg/kg. This dose was chosen because it is the same as our standard drug, cimetidine, thus allowing us to compare the potencies of the compounds. Compounds II, III, and IV obtained in this study were evaluated for anti-ulcer activity in the HCl/ethanol model. In this method, cimetidine, as well as compounds I, II, and IV signicantly protected the gastric mucosa against HCl/ ethanol (15 5 mm, 13 6 mm, 9 4 mm, and 5 3 mm, P < 0.05) when compared with the control group. Meanwhile, the treatment with compound III resulted in the total disappearance of the anti-ulcer activity (54 5 mm, P > 0.05). 3.3. Hypothermic restraint-stress ulcers The oral administration of compound IV inhibited ulcer formation in the hypothermic restraint-stress model (Fig. 2). The derivative IV presented good activity which was equipotent with that of cimetidine and two-folds more active than DHC at the same dose. But at this model, the anti-ulcerogenic activities of compounds II and III were markedly abolished. These results indicate that reducing the cyclohexenone moiety of DHC with NaBH4 , or reducing the carbonyls with LiAlH4 , resulted in loss of gastroprotective activity. 3.4. Indomethacin-induced ulcers The anti-ulcerogenic effects of DHC and DHC derivatives were assayed against gastric lesions induced by in-
domethacin. Cimetidine, as well as compound I, signicantly protected the gastric mucosa against gastric lesion induced by indomethacin (12 5 and 26 6, P < 0.05) when compared with the control group. In this model, the activity of compound IV was more potent than that of the reference compound, cimetidine, at the same dose. The same results were also obtained with compound II. However, the derivative III also abolished the anti-ulcerogenic activity markedly. Compounds II and IV signicantly decreased the lesions induced by indomethacin. 3.5. Anti-ulcerogenic activity The relative potency of DHC (compound I) and its derivatives were observed in Table 1 at a xed oral dose of 100 mg/kg. This allowed direct comparison of the potency of these compounds. In this table, we can observe that the oral administration of compound III presented less activity than did other derivatives (P > 0.05) on the ulceration caused by stress, ethanol or indomethacin (Fig. 2) when compared with control animals. However, in this series, the most active compound was IV which yielded inhibition of ulcers induced by stress (74%), HCl/ethanol (89%), and indomethacin (90%) at the test dose (Table 1).
4. Discussion In this study, we changed synthetically the DHC moiety with the aim to improve the anti-ulcerogenic activity, as well as to reduce the toxic effects of DHC. The three DHC derivatives obtained were tested against various acute ulcer models in animals. Research on new gastroprotective agents commonly include the use of different models of experimentally induced gastric lesions that act by different mechanism of ulcerogenesis. These models allow the determination of the potential of anti-ulcer substances, and they represent the rst approach to the possible mechanism involved in their gastroprotective activity. The 60% ethanol/0.3 M HCl induces an acute ulcer model that allows the evaluation of the capacity of the drug to
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protect the gastric mucosa. Ethanol induces the formation of gastric ulcers by a direct action on the mucosa, with little involvement of gastric acid in the formation of this lesion; the presence of HCl only accelerates the process. Moreover, ethanol induces solubilization of mucus constituents in the stomach with a concomitant decrease in the transmucosal potential difference, and increases the Na+ and K+ ux into the lumen, while enhancing pepsin secretion, the loss of H+ ions, and the histamine content in the lumen (Szabo, 1987). The results obtained in this work in the HCl/ethanol model suggest that compounds II and IV may enhance gastric-mucosal defensive factors, such as mucus and prostaglandin production. The cytoprotective activities of the compounds were also examined on the hypothermic restraint-stress model. This assay induces the formation of gastric ulcer through disturbances in the gastric-mucosal microcirculation, alteration in gastric secretion, abnormal motility, and gastric-mucus depletion (Koo et al., 1986). However, the most important factor in the genesis of stress ulcers is an increase in gastric acid secretion, often termed the aggressive factor (Goa and Monk, 1987). Compound IV was the most effective to protect the gastric mucosa in this induced-ulcer model. Anti-inammatory agents, such as indomethacin, reduce gastric cyclooxygenase activity and decrease endogenous prostaglandin levels. There is increasing evidence that an elevation of certain endogenous prostaglandins can enhance gastric-mucosal resistance to ulcerogenic agents, such as NSAID (Vane and Botting, 1995), and that a rise in prostaglandin levels signicantly increases blood ow in the stomach (Droy-Lefaix, 1988). Compounds II and IV signicantly decreased the lesions induced by indomethacin (88 and 90%, respectively). These results also suggest a probable involvement of compound II and, primarily, compound IV in prostaglandin synthesis and/or release. Hiruma-Lima et al. (1999) also suggested that the excellent preventive effect of DHC on gastric ulcers occurred mainly by the suppression of acid secretion through non-competitive antagonism with receptors involved in gastric acid secretion, and by protecting the gastric mucosa through an increase in PGE2 levels. According to Giordano et al. (1992), structureactivity studies of sesquiterpene lactones provided theoretical and experimental evidence that the presence of a non-sterically hindered Michael acceptor is an essential structural requirement for the cytoprotective activity of these compounds. The mechanism of gastric cytoprotection would, thus be mediated by an action on prostaglandin biosynthesis (Robert et al., 1979) and by a Michael reaction between the SH-containing compounds of the mucosa on the Michael acceptors present in anti-ulcerogenic compounds (Giordano et al., 1992; Mar a et al., 1995). These results provide insight into the structural requirements for cytoprotective activity of sesquiterpene lactones and may be helpful in designing compounds with this pharmacological activity. Our results with compound IV indicate that, despite change
in the lactone, the molecule retained its anti-ulcerogenic activity because of the presence of a Michael acceptor substitute [(C(O)N(CH3 )2 )]. In contrast, compound III lost its anti-ulcerogenic activity due to the absence of a Michael acceptor. In recent report about the cytotoxic effects of these compounds, it was demonstrated that compounds II, III, and IV differed in toxicity from DHC (Melo et al., 2001, 2002). The data obtained with rat cultured hepatocytes further indicated that compound II, a derivative with a lactone moiety, produced similar toxicological manifestations to those seen with DHC. However, compound IV showed lower hepatotoxic effect than DHC and compound II, when evaluated in rats cultured hepatocytes (Melo et al., 2002). A balance between the therapeutic versus toxicological effects of a compound is an important parameter, when verifying its applicability as a pharmacological drug. Cell culture can be used to evaluate basal cytotoxicity and in some cases, may provide information on the lethal dose in vivo. According to recent results, compound IV was less toxic in the cell models used (Melo et al., 2001, 2002) than the other compounds; so it is a potential compound to be investigated in relation to its pharmacological potential, since it was also the better anti-ulcer derivative. In conclusion, compounds II and IV had signicant anti-ulcer activity following oral administration. This effect could be related to an increase of gastric-mucosal defensive mechanisms. The presence of the lactone ring or a Michael acceptor substitute is probably essential for the anti-ulcer activity of these compounds. Further experiments are currently underway to determine the anti-ulcer mechanisms of these DHC derivatives and to assess their structureactivity relationships.
Acknowledgements This work was supported by the Brazilian foundations CAPES, FAPESP and PADCT-FINEP. The authors thank Nelson A. Rosa by identication of the voucher specimen used in the study.
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P.S. Melo et al. / Journal of Ethnopharmacology 87 (2003) 169174 Mizui, T., Doteuchi, M., 1983. Effect of polyamines on acidied ethanol-induced gastric lesion in rats. Japanese Journal of Pharmacology 33, 939945. Olfert, E.D., Cross, B.M., McWilliam, A.A., 1993. Guide to the Care and Use of Experimental Animals, vol. 1. Canadian Council on Animal Care, Ottawa, Ont., pp. 1213. Repetto, M.G., Llesuy, S.F., 2002. Antioxidants properties of natural compounds used in popular medicine for gastric ulcers. Brazilian Journal of Medical and Biological Research 35, 523534. Robert, A., Nezamis, J.E., Lancaster, C., Hanchar, A.J., 1979. Cytoprotection by prostaglandins in rats. Prevention of gastric necroses produced by alcohol, HCl, NaOH, hypotonic and thermal injury. Gastroenterology 77, 433443. Rodriguez, J.A., Haun, M., 1997. P-450 mediated dehydrocrotonin toxicity on rat hepatocyte cultures. FASEB Journal 11, P169. Rodriguez, J.A., Haun, M., 1999. Cytotoxicity of trans-dehydrocrotonin from Croton cajucara on V79 cells and rat hepatocytes. Planta Medica 65, 15. Shimoma, F., Kusaka, H., Wada, K., Azami, H., Yasunami, M., Susuki, T., Hagiwara, H., Ando, M., 1998. A novel synthetic method of the (+)-(3a,8a)-ethyl 8-hydroxy-6-methyl-2-oxooctahydor2H-cyclohepta[b]furan-3-carboxylate and its chemical transformation to (+)-(3a,8a)-3,6-dimethyl-3,3a,4,5,6,8a-hexahydro-2H-cyclohepta[b]furan-2-one, (+)- and ()-7-(2-acetoxy-1-methylethyl)-4methyl-2-cyclohepten-1-ol, and ()-7-(2-acetoxy-1-methylethyl)4-methyl-2-cyclohepten-1-one. Possible common synthetic intermediates for pseudoguaianolides, 4,5-secopseudoguaianolides, guaianolides, 4,5-secoguaianolides, and octalactins. Journal of Organic Chemistry 63, 920929. Souza-Brito, A.R.M., Rodriguez, J.A., Hiruma-Lima, C.A., Haun, M., Nunes, D.S., 1998. Anti-ulcerogenic activity of trans-dehydrocrotonin from Croton cajucara Benth. Planta Medica 64, 126129. Szabo, S., 1987. Mechanisms of mucosal injury in the stomach and duodenum: time-sequence analysis of morphologic, functional, biochemical and histochemical studies. Scandinavian Journal of Gastroenterology 22, 2128. Szeleyi, I., Thiemr, K., 1978. Distension ulcer as a model for testing of drugs for ulcerogenic side effects. Archives of Toxicology 41, 99105. Vane, J.R., Botting, R.M., 1995. New insights into the mode of action of anti-inammatory drugs. Inammation Research 44, 110. Yamamoto, K., Kakegawa, H., Ueda, H., Matsumoto, H., Sudo, Y., Miki, T., Satoh, T., 1998. Gastric cytoprotective anti-ulcerogenic actions of hydroxychalcones in rats. Planta Medica 58, 389393.
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Effect of Holotrichia diomphalia larvae on liver brosis and hepatotoxicity in rats

Woo-Yong Oh a , Suhkneung Pyo a , Kang-Ro Lee a , Bum-Koo Lee a , Dae-Hee Shin b , Sung Ig Cho c , Sun-Mee Lee a,
a
College of Pharmacy, Sungkyunkwan University, 300 Chonchon-dong, Changan-gu, Suwon 440-746, Kyunggi-do, South Korea b Institute of Complementary Medicine, Conmaul Oriental Hospital & Medical Clinic, Seoul 137-881, South Korea c Department of Pharmacology, Ajou University School of Medicine, Suwon 442-749, South Korea Received 30 April 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract Holotrichia diomphalia larvae, one of the most widely used Korean folk medicinal preparations, have long been used for the treatment of chronic liver cirrhosis. The present study was undertaken to clarify whether extract of Holotrichia diomphalia larvae could prevent acute liver damage and liver brosis in rats. A single administration of Holotrichia diomphalia protected rats from acute liver damage induced by carbon tetrachloride (200 l/kg, i.p.) and -d-galactosamine (600 mg/kg, i.p.). This was evidenced by the lowered serum aminotransferase (ALT, AST) activities in rats treated with Holotrichia diomphalia. The hepatic cirrhosis was induced by 28 days of bile duct ligation/scission in rats. The four-week treatment with Holotrichia diomphalia reduced the serum ALT, AST, alkaline phosphatase activities, and hydroxyproline content in the liver and improved the histological appearance of the liver sections. The present results led us to conclude that Holotrichia diomphalia larvae can reduce the degree of hepatocellular damage and may become a promising antibrotic agent for liver brosis/cirrhosis. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Holotrichia diomphalia larvae; Acute hepatotoxicity; Liver cirrhosis
1. Introduction Recently, the number of cases of liver disease has tended to increase year by year, and more than 15,000 deaths from this disease occur annually in Korea. Hepatic brosis is an important feature of, and a common sequel to, most forms of chronic liver disease and is an essential component in the development of cirrhosis (Friedman, 1993). Therefore, the prevention or suppression of brotic changes in the liver or protection from and treatment of cirrhosis is important. At present, there is no effective therapy to cure cirrhosis or to prevent its complications. Furthermore, there are no drugs to suppress brosis, therefore, it is important to prevent the development of hepatic brosis. Holotrichia diomphalia (Coleoptera, Scarabaeidae) larvae (Scarab beetle) have been traditionally used in Korea for the treatment of liver cirrhosis, contusion, edema, furuncle and apoplexy. Recently, potent antibacterial proteins have been isolated from Holotrichia diomphalia larvae (Lee
et al., 1994). Prophenoloxidase from the hemolymph of Holotrichia diomphalia larvae has also been puried and characterized (Kwon et al., 1997). Furthermore, our own recent studies have shown that Holotrichia diomphalia larvae augment macrophage function and stimulate the synthesis and release of cytotoxic mediators (Kang et al., 2002). However, the hepatoprotective effects of Holotrichia diomphalia larvae have not been investigated. The present study was undertaken to investigate the hepatoprotective effect of Holotrichia diomphalia larvae against acute liver injury and liver brosis.
2. Materials and methods 2.1. Animals and treatments Male, SpragueDawley rats weighing 240 20 g were obtained from Jeil Animal Breeding Company of Korea and were acclimated to the laboratory conditions at Sungkyunkwan University for at least one week. During this period, food (Samyang Co., Korea) and tap water were supplied ad libitum. The experimental animals were kept in
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author. Tel.: +82-31-290-7712; fax: +82-31-292-8800. E-mail address: sunmee@yurim.skku.ac.kr (S.-M. Lee).
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a temperature- and humidity-controlled room (25 1 C, 55 5%, respectively) with a lightdark cycle of 12 h, and were fasted 18 h before the experiment. 2.2. Chemicals
p.o.) and Holotrichia diomphalia (100, 300 mg/10 ml/kg, p.o.). After 48 h -d-galactosamine administration, blood was taken from the abdominal aorta for the assay of serum aminotransferase activity. 2.5. Liver cirrhosis
Carbon tetrachloride, -d-galactosamine hydrochloride, silymarin, p-dimethylaminobenzaldehyde, and p-toluenesulfon-chloramide (chloramine T) were supplied by the Sigma Chemical Co., USA. All the other chemicals used in this study were reagent grades and are locally and commercially available. 2.3. Preparation of crude extracts Holotrichia diomphalia larvae (1 kg) were purchased at the herbal drug market in Cheju-Do, Korea, in August 1999 and identied by Dr. B.G. Lee of the Institute for Traditional Medicine, Sungkyunkwan University, Suwon, Korea. A voucher specimen (SKK-H001) is deposited in the College of Pharmacy at Sungkyunkwan University. Air-dried and chopped Holotrichia diomphalia larvae (1 kg) were reuxed with 70% ethanol (2 l) two times for 8 h. The materials were ltered, and the clear supernatant was then concentrated under reduced pressure at 40 C with a vacuum rotary evaporator. The concentrated ethanol extract (100 g) was partitioned between water (1 l) and n-hexane (0.5 l 2, 20 g). After removing the n-hexane fraction, the aqueous layer was partitioned again with methylene chloride (0.5 l 2, 10 g), followed by n-butanol (0.5 l 2, 40 g). The extract was evaporated and the residue was used for the experiment. 2.4. Acute hepatotoxicity Five groups of animals were studied. All animals were treated humanely under Sungkyunkwan University Animal Care Committee guidelines. The rats in group I (vehicle) received only olive oil (2 ml/kg, i.p.). In groups IIV, carbon tetrachloride (CCl4 ) was dissolved in olive oil (1:9) (v/v), then intraperitoneally administered (nal concentration: 200 l/kg). Four hours after the CCl4 treatment, groups I (vehicle) and II (control) were treated with saline (10 ml/kg, p.o.), and groups IIIV were treated with silymarin (positive control, 300 mg/10 ml/kg, p.o.) and Holotrichia diomphalia (100, 300 mg/10 ml/kg, p.o.). Following 24 h CCl4 administration, blood was taken from the abdominal aorta for the assay of serum aminotransferase activity. In order to induce hepatitis, ve other groups of rats were given an intraperitoneal injection of 600 mg/kg of -d-galactosamine dissolved in saline. Group I (vehicle) was given an intraperitoneal injection of saline (1 ml/kg). Four hours after the -d-galactosamine treatment, group II (control) was treated with saline (10 ml/kg, p.o.), and groups IIIV were treated with silymarin (300 mg/10 ml/kg,
Secondary biliary cirrhosis was induced by double ligation and division of the common bile duct (Kountouras et al., 1984; Gross et al., 1987). Under ether anesthesia, a midline incision was made to the abdomen, the common bile duct was isolated, and the proximal bile duct was held with two silk sutures and then dissected between the double ligations. In sham-operated group, the bowel and mesentery were manipulated and replaced. After operation, saline (10 ml/kg), silymarin (12.5 mg/10 ml/kg) or Holotrichia diomphalia (6.25, 12.5 mg/10 ml/kg) were fed orally to each group of rats once a day for four weeks. At four weeks, blood was taken from the abdominal aorta for the assay of serum aminotransferases and alkaline phosphatase activities. The medium and left lobes of the liver were removed and used for histology and hydroxyproline measurements. 2.6. Assay of serum ALT, AST and ALT activities and hydroxyproline content The serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) activities were determined by spectrophotometric procedures, using Sigma Kits (Sigma Chemical Co., St. Louis, MO, USA). The hydroxyproline (HOPro) contents in the liver were measured according to the method of Jamall et al. (1981). Briey, the liver tissue was homogenized in 6N hydrochloride (HCl) and then hydrolyzed at 110 C for 18 h. After cooling, chloramine T was added to the hydrolysate. After 5 min, p-dimethylaminobenzaldehyde was added and the mixture was incubated for 30 min at 60 C. The samples were read at 560 nm against a reagent blank, which contained the complete system without added tissue, using a spectrophotometer (Milton Roy Spectonic 1201, USA). 2.7. Histology A small piece of liver tissue from the anterior portion of the left lateral lobe was taken for light microscopy. Parafn blocks were prepared after xation in 10% neutral formalin and stained with hematoxylin and eosin. The degree of necrosis and brosis was assessed according to Frei et al. (1984). The severity of liver alteration was semi-quantitatively graduated on a scale of 0 to IV (0: absent; I: minimal; II: mild; III: modest; IV: severe), and separate scores were obtained for each of the following: cell necrosis, inammatory cell inltration, brosis and steatosis. The bile duct proliferation was rated as present or absent.
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2.8. Statistical analysis All results were expressed as means S.E.M. The unpaired Students t-test was used to analyze the difference between groups. Values of P < 0.05 were considered to be signicant.
Table 1 Effect of Holotrichia diomphalia on serum aminotransferase activities in carbon tetrachloride-induced acute hepatic injury Group Vehicle CCl4 Control Holotrichia diomphalia Silymarin Dose (mg/kg) ALT (U/l) 46 4 871 71 402 156+ 187 31,++ 286 64,++ AST (U/l) 74 6 1310 146 577 45,++ 410 51,++ 778 144,+
100 300 300
3. Results 3.1. Acute hepatotoxicity The serum levels of ALT and AST in the vehicle-treated rats were 46 4 U/l and 74 6 U/l, respectively, which were similar to those of the normal rats. In the CCl4 -treated control group, the ALT and AST increased to 871 71 U/l and 1310 146 U/l, respectively. These higher levels were markedly suppressed in a dose-dependent manner by Holotrichia diomphalia. The increase in ALT and AST was also attenuated by silymarin (Table 1). In the galactosamine-treated control group, the ALT and AST increased to 1638 282 U/l and 2342 408 U/l, respectively. Holotrichia diomphalia and silymarin treatments prevented the elevations of serum aminotransferases. The hepatoprotective effect of Holotrichia diomphalia was stronger than that of silymarin (Table 2). 3.2. Liver cirrhosis The liver of the sham-operated rats was normal. Histology of the bile duct ligation/scission (BDL/S) rat liver showed excessive bile duct proliferation, inammation and
Each value is the mean S.E.M. for six to ten rats per group. (, ) signicantly different (P < 0.05, P < 0.01) from vehicle-treated group. (+, ++) signicantly different (P < 0.05, P < 0.01) from control group.
Table 2 Effect of Holotrichia diomphalia on serum aminotransferase activities in -d-galactosamine-induced hepatitis Group Vehicle -d-Galactosamine Control Holotrichia 100 diomphalia 300 Silymarin 300 Dose (mg/kg) ALT (U/l) 46 4 1638 282 704 82,+ 368 99,++ 615 75,+ AST (U/l) 74 6 2342 408 694 204,+ 552 132,++ 1266 124
Each value is the mean S.E.M. for six to ten rats per group. (, ) signicantly different (P < 0.05, P < 0.01) from vehicle-treated group. (+, ++) signicantly different (P < 0.05, P < 0.01) from control group.
Table 3 Quantitative summary of histological observation on Holotrichia diomphalia protection of BDL/S-induced liver cirrhosis Histological changes (%) Sham BDL/S Control Holotrichia diomphalia 6.25 mg/kg Necrosis No change Grades III Grades IIIIV Inammatory inltration No change Grades III Grades IIIIV Fibrosis No change Grades III Grades IIIIV Bile duct proliferation 100 0 0 100 0 0 100 0 0 Absent 0 20 80 0 30 70 0 20 80 Present 0 60 40 0 60 40 0 50 50 Present 12.5 mg/kg 0 70 30 0 70 30 0 60 40 Present 0 40 60 0 50 50 0 40 60 Present Silymarin
Scores are the numerical values of individual 10 rats per group. Necrosis and brosis were assessed according to Frei et al. (1984). Inammatory inltration grading was made according to ve severity grades (0: absent, I: minimal, II: mild, III: modest and IV: severe). Bile duct proliferation was rated as present or absent.
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Table 4 Serum biochemical values in rats with cirrhosis induced by BDL/S treated with Holotrichia diomphalia Group Sham BDL Control Holotrichia diomphalia Silymarin Dose (mg/kg) ALT (U/l) 24 4 308 141 126 241 11 26,++ 11,++ 33 AST (U/l) 177 39 665 318 356 592 52 55++ 65,++ 35 ALP (U/l) 157 10 929 576 670 750 31 87,++ 20,++ 39,++
6.25 12.5 12.5
Each value is the mean S.E.M. for seven to ten rats per group. (, ) signicantly different (P < 0.05, P < 0.01) from sham-operated group. (++) signicantly different (P < 0.01) from control group.
Fig. 1. Hydroxyproline content of liver from cirrhotic rats with BDL/S treated with Holotrichia diomphalia. () signicantly different (P < 0.01) from sham-operated group. (++) signicantly different (P < 0.01) from control group. Values are means S.E.M. for seven to ten rats per group.
connective tissue deposition resulting in destruction of the lobular architecture. In the Holotrichia diomphalia-treated rats, there was a tendency towards less pronounced destruction of the liver architecture. In the silymarin-treated BDL/S rats, the liver histology was similar to that of the BDL/S rats (Table 3). The serum biochemical parameters of the BDL/S rats are shown in Table 4. The levels of serum ALT, AST and ALP were signicantly elevated in the control BDL/S rats. In the Holotrichia diomphalia-treated rats, the serum ALT, AST and ALP levels were significantly lower relative to the control BDL/S rats. In the BDL/S rats treated with silymarin, the serum level of ALP was signicantly reduced, and there were no signicant changes in the serum ALT and AST levels compared with those of the control BDL/S rats. BDL/S increased the hydroxyproline content about three-fold. Compared with the control BDL/S group, treatments with Holotrichia diomphalia and silymarin reduced the hydroxyproline content in the liver by up to 64 and 68%, respectively (Fig. 1).
4. Discussion and conclusions In the present study, the hepatoprotective effect of Holotrichia diomphalia larvae in three experimental liver injury models was investigated. The sequence of events after CCl4 administration has been well characterized by many investigators (Slater, 1966; Recknagel, 1967). After administering CCl4 , the histological changes of the liver include ballooning degeneration, fatty metamorphosis in the adjacent hepatocyte, cell necrosis, cell inammation and the inltration of lymphocytes and Kupffer cells around the central vein. The mechanism of CCl4 -induced liver damage is considered to be due to the enzymatic activation (cytochrome P450) of CCl4 into the trichloromethyl free radical ( CCl3 ) within the membrane of the endoplasmic reticulum. This is followed by chloromethylation, saturation, peroxidation and progressive destruction of the unsaturated fatty acid of the endoplasmic reticulum membrane phospholipids (Reynolds and Moslen, 1979). These processes are known as lipid
179
peroxidation, leading to its functional and structural disruption (Recknagel, 1983). In the CCl4 -treated control of the present study, the serum aminotransferases levels were elevated signicantly. The hepatoprotective effects against CCl4 -induced hepatic injury were clearly demonstrated in the rats treated with Holotrichia diomphalia. This effect may be attributable to the inhibition of cytochrome P450 activity as well as the prevention of lipid peroxidation. -d-Galactosamine-induced acute liver injury was considered in an experimental model of hepatitis. Decker and Keppler (1974) have shown that the metabolite of -dgalactosamine, uridindiphosphogalactosamine (Rasenack et al., 1980), may deplete several uracil nucleotides including UDP-galactose, UDP-glucose and UTP, impairing mRNA and glycoprotein synthesis and altering the composition of the cellular membranes. These phenomena may lead to cellular damage and cellular inammation resulting in a histological and biochemical picture closely resembling viral hepatitis (Lin et al., 1996). The results presented here show a signicant increase in ALT and AST activities after administration of -d-galactosamine. In contrast to the control rats, the elevated serum aminotransferase levels were reduced by treatment with Holotrichia diomphalia. Furthermore, the hepatoprotective effect of Holotrichia diomphalia seemed to be higher than that of silymarin, which has been used as a potent hepatoprotective agent. The present results of the CCl4 and -d-galactosamine-induced liver injuries suggest that Holotrichia diomphalia may have potential clinical application for treating liver diseases. Therapy for hepatic brosis should affect the production of hepatic connective tissue proteins in particular (Schuppan, 1991). The best therapeutic strategies can be designed only with a full understanding of the mechanism involved in brogenesis, which has yet to be completed. Nonetheless, any intervention that blocks collagen deposition will probably be effective in reducing hepatic brosis, regardless of the mechanisms involved. In this study, we used BDL/S cirrhotic rats. Cirrhosis induced by BDL/S showed a slight decrease in body weight due to the operation during the rst week, and then returned to normal weight afterwards. Between the control BDL/S rats and Holotrichia diomphalia-treated BDL/S rats, there was no signicant difference in body weight. In the BDL/S rats, the liver weight increased markedly 28 days after biliary obstruction. The liver-to-body weight ratio of the Holotrichia diomphalia-treated BDL/S rats was slightly lower than that of the control BDL/S rats, but the difference was not signicant (data not shown). We were able to show that this procedure resulted in biliary brosis, as demonstrated by the histology and moderately increased serum aminotransferases and alkaline phosphatase. Furthermore, a signicant increase in the hydroxyproline content in the liver was observed. Treatment with Holotrichia diomphalia reduced the serum levels of
ALT, AST, ALP and also lowered the hydroxyproline in the liver, and improved its morphology. In conclusion, treatment using Holotrichia diomphalia prevents hepatocellular damage and improves liver brosis. Our results suggest that Holotrichia diomphalia could be a promising antibrotic agent. Further study is needed to fully understand the mechanism by which Holotrichia diomphalia larvae inhibit collagen deposition and to establish its clinical applicability.
Acknowledgements This work was supported by research grant (99-B5-2) from the Kyunggi Pharmaceutical Research Center, Korea.
References
Decker, K., Keppler, D., 1974. Galactosamine hepatitis: key role of the nucleotide deciency period in the pathogenesis of cell injury and cell death. Reviews of Physiology, Biochemistry and Pharmacology 71, 77106. Frei, A., Zimmermann, A., Weigand, K., 1984. The N-terminal propeptide of collagen type III in serum reects activity and degree of brosis in patients with chronic liver disease. Hepatology 4, 830834. Friedman, S.L., 1993. Seminars in medicine of the Beth Israel Hospital Boston. The cellular basis of hepatic brosis. Mechanisms and treatment strategies. The New England Journal of Medicine 328, 1828 1835. Gross Jr., J.B., Reichen, J., Zeltner, T.B., Zimmermann, A., 1987. The evolution of changes in quantitative liver function tests in a rat model of biliary cirrhosis: correlation with morphometric measurement of hepatocyte mass. Hepatology 7, 457463. Jamall, I.S., Finelli, V.N., Que, S.S., 1981. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues. Analytical Biochemistry 112, 7075. Kang, N.S., Park, S.Y., Lee, K.-R., Lee, S.-M., Lee, B.G., Shin, D.-H., Pyo, S., 2002. Modulation of macrophage function activity by ethanolic extract of larvae of Holotrichia diomphalia. Journal of Ethnopharmacology 79, 8994. Kountouras, J., Billing, B.H., Scheuer, P.J., 1984. Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. British Journal of Experimental Pathology 65, 305311. Kwon, T.H., Lee, S.Y., Lee, J.H., Choi, J.S., Kawabata, S., Iwanaga, S., Lee, B.L., 1997. Purication and characterization of prophenoloxidase from the hemolymph of coleopteran insect, Holotrichia diomphalia larvae. Molecules and Cells 7, 9097. Lee, S.Y., Moon, H.J., Kurata, S., Kurama, T., Natori, S., Lee, B.L., 1994. Purication and molecular cloning of cDNA for an inducible antibacterial protein of larvae of a coleopteran insect, Holotrichia diomphalia. Journal of Biochemistry (Tokyo) 115, 8286. Lin, S.C., Lin, C.C., Lu, F.J., Lin, Y.H., Chen, C.H., 1996. Protective and therapeutic effects of huanglian-jie-du-tang on hepatotoxin-induced liver injuries. American Journal of Chinese Medicine 24, 219 229. Rasenack, J., Koch, H.K., Nowack, J., Lesch, R., Decker, K., 1980. Hepatotoxicity of d-galactosamine in the isolated perfused rat liver. Experimental and Molecular Pathology 32, 264275. Recknagel, R.O., 1967. Carbon tetrachloride hepatotoxicity. Pharmacological Reviews 19, 145208.
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W.-Y. Oh et al. / Journal of Ethnopharmacology 87 (2003) 175180 Schuppan, D., 1991. Connective tissue polypeptides in serum as parameters to monitor antibrotic treatment in hepatic brogenesis. Journal of Hepatology 13, 1725. Slater, T.F., 1966. Necrogenic action of carbon tetrachloride in the rat: a speculative mechanism based on activation. Nature 209, 3640.
Recknagel, R.O., 1983. A new direction in the study of carbon tetrachloride hepatotoxicity. Life Sciences 33, 401408. Reynolds, E.S., Moslen, M.T., 1979. Environmental liver injury: halogenated hydrocarbons. In: Farber, E., Fisher, M. (Eds.), Toxic Injury of the Liver. Marcel Dekker Inc., New York and Basel.
Evaluation of immunomodulatory activity of Ipomoea carnea on peritoneal cells of rats

I.M. Hueza a , E.S.M. Fonseca a , C.A. Paulino b , M. Haraguchi c , S.L. Grniak a,
a
Research Centre for Veterinary Toxicology (CEPTOX), Department of Pathology, School of Veterinary Medicine, University of So Paulo, SP, CEP 05508-900, Brazil b University Bandeirante of So Paulo, SP, CEP 02071-013, Brazil c Biological Institute of So Paulo, SP, CEP 04014-002, Brazil Received 31 July 2002; received in revised form 28 March 2003; accepted 28 March 2003
Abstract In the present study, animals of the experimental groups were treated with an aqueous fraction (AF) of Ipomoea carnea diluted in drinking water in order to obtain daily doses of 3 g dry leaves/kg/body weight (bw) and 15 g/kg/bw for 14 and 21 days, or by gavage 15 g/kg/bw administered for 14 days, respectively. Peritoneal macrophages were collected and submitted to the spreading, phagocytosis, and hydrogen peroxide release tests. AF administration in drinking water for 14 and 21 days promoted increased macrophage phagocytosis activity and hydrogen peroxide release. However, the administration of 15 g/kg/bw of AF by gavage for 14 days resulted in no alteration in macrophage activity. These results suggest that low dosages of Ipomoea carnea induced enhanced phagocytosis activity and hydrogen peroxide production by macrophages. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Ipomoea carnea; Swainsonine; Macrophages; Immunomodulation
1. Introduction Ipomoea carnea Jacq. spp. stulosa (Choisy) D.F. Austin (Convolvulaceae) is a toxic plant found throughout Brazil and other tropical countries (Austin and Huaman, 1996). Natural intoxication occurs when different animal species, such as cattle, sheep, and goats, chronically ingest the plant (Hoehne, 1939). Such intoxication is clinically characterized by inappetence, soft feces, and weight loss, and by central nervous system (CNS) signs, such as head shaking, hyperesthesia, and death (De Balogh et al., 1999). The toxic principles of the plant have been recently identied as two nortropane alkaloids (De Balogh et al., 1999), calystegines B2 and C1, and indolizidine alkaloid, swainsonine (SW). The rst two are powerful glycosidase inhibitors affecting -glucosidase and - and -galactosidases, leading to the production of phenocopies of the human genetic lysosomal storage defects, Gauchers and Fabrys disease, respectively (Dorling, 1984). The latter alkaloid has also been isolated from other Leguminosae plants, such as Swainsona canescens (Australia; Dorling et al., 1978) and certain
author. Tel.: +55-11-30917693; fax: +55-11-30917829. E-mail address: gorniak@usp.br (S.L. G orniak).
Corresponding
species known as locoweed, belonging to the genera Astragalus and Oxytropis (North America; van Kampen and James, 1969). The SW mechanism of intoxication has been extensively investigated and has been found to be due to the potent inhibition of the lysosomal -mannosidase which causes a similar genetic disease, -mannosidosis, described principally in Angus cattle and felines, but rarely in humans (Jolly and Walkley, 1997). The effect of the acquired lysosomal -mannosidase inhibition, a characteristic lysosomal storage disease, showing lysosomal accumulation of incompletely processed oligosaccharides, loss of cellular function, and ultimately cell death (Tulsiani et al., 1988) has been observed in different organs, including thyroid, liver, kidneys, and mainly CNS (Stegelmeier et al., 1998; De Balogh et al., 1999). SW also inhibits Golgi mannosidase II, which is involved in the N-linked glycoprotein metabolism (Elbein, 1989). The resulting alteration in the synthesis, processing, and transport of glycoproteins causes impairment and dysfunction of cell adhesion molecules, circulating hormones, and various membrane receptors (Stegelmeier et al., 1998). This effect is seen clinically as abnormal embryogenesis (Nelson et al., 1980), abnormal endocrine (Stegelmeier et al., 1995) and gastrointestinal functions (Pan et al., 1993), and alteration of the immune system (Karasuno et al., 1992).
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The modication resulting from glycoprotein metabolism has received much attention because of its potential immunomodulatory properties. Studies involving eld observations reported that SW causes immunodeciency in animals exposed to plants containing this compound (Stegelmeier et al., 1998). Animals also became susceptible to the occurrence of pneumonia, foot rot, or pink eye (Sharma et al., 1984). Experimental studies conducted in vitro, however, showed that SW has potential immunomodulatory properties, such as increasing murine natural-killer (NK) activity (Humphries et al., 1988), enhancing the generation of lymphokine-activated killer (LAK) cell activity (Bowlin et al., 1989), increasing human large granular lymphocytes citotoxicity against NK-resident colon carcinoma cells (Yagita and Saksela, 1990), and activation of resident tissue macrophages (Das et al., 1995). The purpose of the present study was to evaluate in vivo the effect of Ipomoea carnea on macrophage activity in rats, specically, macrophage spreading, phagocytosis, and hydrogen peroxide (H2 O2 ) production by peritoneal cells after lipopolysaccharide (LPS) activation.
2.3. Animals and experimental design Fifty-one female Wistar rats aged about 60 days (150 200 g), inbred in the Departament of Pathology, School of Veterinary Medicine, were used. The animals were housed in temperature-controlled (2426 C) and articially lighted rooms on a 12-h light/12-h dark cycle (lights on at 07:00 h) with free access to food and water, and used in accordance with the guidelines of the National Research Council, USA. The animals were divided at random into eight groups: ve experimental groups (DW14A, DW14B, DW21A, DW21B, and G) and three control groups (CDW14, CDW21, and CG). Animals from the DW groups were treated with AF diluted in drinking water in order to obtain the target doses of 3 or 15 g/kg/day of Ipomoea carnea dry leaves. Rats from the DW14A and DW21A groups were to receive 3 g/kg/day of the plant leaves for 14 or 21 days, respectively, and those from the DW14B and DW21B groups were to ingest 15 g/kg/day of the plant leaves for 14 or 21 days, respectively. Animals from the G group were dosed by gavage for 14 consecutive days with 15 g/kg of Ipomoea carnea leaves. Control groups received only tap water for 14 days (CDW14) or 21 days (CDW21). CG rats received tap water by gavage for 14 days. Every day the amount of AF administered in the drinking water was adjusted to the body weight and to the water consumption, and a fresh solution of AF was provided. Twenty-four hours before euthanasia, the rats were given an intraperitoneal (i.p.) injection of LPS (1 mg/kg) in order to induce peritoneal inammatory cell activation. The macrophage activity was evaluated using the protocols previously described by Rabinovitch and DeStefano (1973a,b). 2.4. Evaluation of spreading and phagocytosis of peritoneal macrophages Peritoneal exudate cells were obtained by lavage of the peritoneal cavity 24 h after injection of LPS. The cells were centrifuged and resuspended in PBS and adjusted to 2 106 cells/ml. To study macrophage spreading, 200 ml of each cell suspension was prepared in duplicate on glass slide monolayer that was kept in multiwell (6 wells) tissue culture plate (Corning Costar). Within 20 min, the wells were washed several times with cold PBS (4 C); RPMI-1640-supplemented medium was added to each well. The culture plates were incubated for 60 min at 37 C in a humidied atmosphere of 5% CO2 . After incubation, the wells were rinsed with cold (4 C) PBS and the adherent cells xed with 0.5% glutaraldehyde for 10 min. These cells were then counted with a phase contrast microscope (Nikon, Inc.) at 40 magnication. Using an ocular grid, 200 macrophages were scored as either round or spread. An index of macrophage spreading (SI) was then calculated for each monolayer of each well: SI = number of spreading macrophages 100/200 adherent cells counted; hence, SI = percent of spreading macrophages.
2. Material and methods 2.1. Plant material Leaf sample of Ipomoea carnea was collected from the cultivated plants at the Research Centre for Veterinary Toxicology (CEPTOX), University of So Paulo (USP), Pirassununga, Brazil, in May 2000. A voucher herbarium specimen was deposited in the Botanical Institute of So Paulo (SP), Brazil, under number SP-360911. Taxonomic identication was performed by Dr. Rosangela Simo Bianchini, Botanical Institute of So Paulo. Dry leaf sample (800 g) was macerated in 96% ethanol (3 l). After total solvent evaporation under reduced pressure at 50 C, a dark green extract was obtained (140 g, 17.5% w/w), which was suspended in water to remove the waxy residue (60.2 g, 7.5% w/w) and consecutively fractionated with n-butanol saturated with water (25.2 g, 3.2% w/w). The remaining aqueous solution was lyophilized to give an aqueous fraction (AF), which by previous assay, revealed the presence of the active principles. 2.2. Reagents Trypan Blue Stain, RPMI-1640 culture medium supplemented with 10 mM HEPES, 11 mM sodium bicarbonate, 2 mM l-glutamine, and 10% fetal bovine serum (FBS) were used to maintain the cell cultures; these reagents were purchased from Gibco. Zymosan A, LPS 0127:B8 100 mg, and phenol red (0.5%) were purchased from Sigma Chemical Co. Glutaraldehyde-25% was purchased from Nuclear, So Paulo. Phorbol myristate acetate (PMA) was purchased from ICN and hydrogen peroxide was purchased from Merck, So Paulo.
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Macrophage phagocytosis was performed using the same method as described above. One milligram of zymosan A solution (5.0 mg/ml) was added to each well 1 h before the 60-min incubation period (37 C). Using the same microscope, and the same objective and methods described above, an index of phagocytosis (PI) was then obtained: PI = number of macrophages with phagocytic activity 100/200 adherent cells counted; hence, PI = percent of macrophages with zymosan particles phagocytized. The mean of four counts obtained from the two slides of each rat was used to express the SI or PI index. 2.5. Hydrogen peroxide release Spontaneous and PMA-induced H2 O2 release from macrophages was measured by a method described previously (Russo et al., 1989). Briey, the peritoneal cells adjusted to 2 106 cell/ml were centrifuged for 10 min and resuspended in 1 ml of phenol red solution (PRS, containing 140 mM NaCl, 10 mM potassium-phosphate buffer, pH 7.0, 0.5 mM dextrose, 0.28 mM phenol red, and 8.5 U/ml HRPO) for H2 O2 detection. The solutions were prepared as described elsewhere (Pick and Mizel, 1981). The cell suspensions were added to 96-well at-bottom microplates and incubated in a humidied 5% CO2 atmosphere at 37 C for 1 h. Subsequently, the wells containing PRS received 10 l of 1N NaOH to stop the reaction. Hydrogen peroxide-dependent phenol red oxidation was measured spectrophotometrically at 620 nm with a Titertek Multiscan apparatus (Flow Laboratories). The same procedure was employed to determine H2 O2 release after stimulation with PMA, with 10 l of 10 ng/ml PMA added to each well before incubation. The concentration of H2 O2 was calculated from absorbance measurements as described previously (Pick and Mizel, 1981). Spontaneous and PMA-induced H2 O2 production experiments were repeated four times for each rat in each group, and the mean value of the four counts was used for H2 O2 determination.
2.6. Statistical analysis Students t-test was used to compare two groups and ANOVA followed by Dunnets post hoc test was used for more than two groups, with the level of signicance set at P < 0.05. Data are expressed as mean S.D.
3. Results 3.1. Daily consumption of AF and weight gain The actual dose of AF received from DW14A and DW21A was near the target dose (3 g/kg/day); however, animals from the DW14B and DW21B groups showed a dramatic depression of water consumption resulting in a much lower ingestion of the plant extract than expected (P < 0.05). Exposure to the higher concentration of Ipomoea carnea in drinking water or Ipomoea carnea administered by gavage reduced the food consumption of the experimental rats as compared to their respective controls. In addition, body weight gain was lower in the animals from groups DW14B, DW21A, and DW21B compared to their control groups (P < 0.05), whereas animals from the G group did not show a significant difference in body weight gain compared to the CG group (Table 1). 3.2. Number of peritoneal cells harvested The number of peritoneal cells collected from animals from the DW groups ranged from 2.4 106 to 12.4 106 cells/ml peritoneal exudate. This variability among animals in the number of cells harvested from the peritoneum under the same experimental conditions did not show signicant differences between these groups (data not shown). However, G group rats showed a signicant decrease (P < 0.05) in the number of peritoneal cells harvested (1.90 1.03) when compared to CG animals (5.30 3.20).
Table 1 Target and actual doses, food consumption, water consumption, and total weight gain of rats treated with aqueous fraction of Ipomoea carnea dry leaves Group n Treatment (days) Dose (g/kg/day) Target CDW14 DW14A DW14B CDW21 DW21A DW21B CG G
a
Actual 0.0 2.34 7.63 0.0 1.90 10.60 0.0 15.0 0.14 0.16 0.47 0.64
Food consumption (g/day) 16.60 16.37 13.77 17.79 16.14 14.74 16.37 14.72 1.32 0.98 1.10a 1.37 1.02 1.11a 0.44 0.46a
Water consumption (ml/day) 31.66 23.48 15.26 32.28 19.0 21.20 29.74 27.58 3.14 1.40a 1.67a 4.82 4.78a 6.45a 2.02 2.69
Total weight gain (g) 18.85 17.28 2.83 20.2 4.48 0.40 12.56 8.83 6.31 5.56 5.87a 6.67 6.74a 7.89a 6.83 3.73
7 7 6b 6 6 6 6 6
14 14 14 21 21 21 14 14
0.0 3.0 15.0 0.0 3.0 15.0 0.0 15.0
Students t-test was used when comparing two groups and for more than two groups data were analyzed statistically by ANOVA followed by Dunnets post hoc test, with the level of signicance set at P < 0.05. Data are expressed as mean S.D. b One animal of the DW14B group died on the last day of treatment.
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Fig. 1. Effect of administration of aqueous fraction of Ipomoea carnea on macrophage spreading index (SI) in rats treated with daily doses of 3 g/kg/body weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with the daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw (DW21B) for 21 days (B), and with a dose of 15 g/kg/bw (G) for 14 days (C).
Fig. 2. Effect of administration of Ipomoea carnea aqueous fraction on macrophage phagocytosis index (PI) in rats treated with daily doses of 3 g/kg/body weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw (DW21B) for 21 days (B), and with a dose of 15 g/kg/bw (G) for 14 days (C). Data are mean S.D. of n 6 animals. P < 0.05, compared with the respective control groups.
3.3. Effect of AF administration on macrophage spreading and phagocytosis activity The spreading activity of peritoneal macrophages from animals of all experimental groups was not affected by Ipomoea carnea administration (Fig. 1). On the other hand, rats from groups DW14A, DW14B, DW21A, and DW21B showed increased macrophage phagocytosis (P < 0.05). No difference in this parameter was found between animals from the G and CG groups (Fig. 2).
macrophages of rats treated with AF by gavage for 14 days (G group) did not show any signicant increase in H2 O2 release, with PMA stimulation or not, when compared to the CG group (Fig. 3).
4. Discussion and conclusion Our rst attempt was to deliver the plant sample in drinking water since this route permits administration without any stress. However, apparently the AF of Ipomoea carnea has a very poor palatability and experimental rats drastically refused to ingest water containing AF, especially those from group DW14B (target dose of 15 g/kg/day) that consumed about half the expected amount of Ipomoea carnea AF. This would have probably led the animals from the DW14B and DW21B groups to ingest small quantities of food, with a consequent reduction in body weight gain. For this reason, we administered the plant material by gavage (G group) which assured the ingestion of 15 g/kg/day of the AF of Ipomoea carnea. However, while no signicant difference in water
3.4. Effect of AF administration on spontaneous and induced H2 O2 release by peritoneal macrophages Administration of AF for 14 and 21 days (DW14A, DW14B, DW21A, and DW21B groups) increased the spontaneous H2 O2 release by peritoneal macrophages in all of these experimental groups (P < 0.05) compared to their respective untreated controls (CDW14 and CDW21); the same was observed when peritoneal macrophages were induced to release H2 O2 by PMA (P < 0.05). Nevertheless,
185
Fig. 3. Effect of administration of Ipomoea carnea aqueous fraction on spontaneous and induced H2 O2 release by peritoneal macrophages in rats treated with daily doses of 3 g/kg/body weight (bw) (DW14A) and 15 g/kg/bw (DW14B) for 14 days (A), with daily doses of 3 g/kg/bw (DW21A) and 15 g/kg/bw (DW21B) for 21 days (B), and with a dose of 15 g/kg/bw (G) for 14 days (C). Data are mean S.D. of n 6 animals. P < 0.05, compared with the respective control groups.
intake or body weight gain was detected in these rats from the G group, a decrease in food consumption was observed. These results signify that a decrease of food intake is related to a direct toxic effect of the plant. In fact, it was well demonstrated that SW, the main toxic principle of Ipomoea carnea, produces anorexic effects (Pritchard et al., 1990). While several in vitro investigations have shown unequivocally that SW produces immune stimulation in different effector cells, such as lymphocytes, NK cells (Humphries et al., 1988; Bowlin et al., 1989), and peritoneal macrophages (Das et al., 1995), there is a lack of information in the literature showing this effect in an in vivo trial. The present study reveals that the administration of AF at moderate doses (about 210 g/kg/day) produces stimulatory effects on macrophage phagocytosis and hydrogen peroxide production by peritoneal cells, even without PMA stimulation, during both periods of 14 and 21 days of administration. It is known that the immune responses are largely regulated by cell surface and secreted glycoproteins. In addition, interaction between sugars and lectins might be functionally involved in the immune recognition and activation, including the receptors involved in phagocytosis (Linehan et al., 2000). Thus, one explanation for the enhanced phagocytosis activity of macrophages observed here could be the alteration of the synthesis and processing of a hybrid type of oligosaccharide caused by SW. Further supporting this hypothesis was the observation that peritoneal macrophages of rats treated with Ipomoea carnea showed enhanced peroxide production of the same intensity as that observed when PMA was added. It is known that PMA increases hydrogen peroxide production acting directly on protein kinase C (PKC). PKC participation could be involved in the transduction of phagocytic signals generated by various receptors (Kwiatkowska and Sobota, 1999). Since Breton et al. (1990) suggested that SW indirectly mediates the same event as that induced by PMA in the modulation of PKC activity,
we propose that receptor alterations promoted by SW could be responsible for the enhanced activation of these effector cells. An important nding of the present study was the absence of immune stimulation veried in rats treated with the highest dose of Ipomoea carnea AF (CG group). A possible explanation for this lack of activation of peritoneal cells is given by the dual effects produced by SW. Thus, since SW inhibits not only Golgi mannosidase II but also lysosomal -mannosidase, the impairment of the latter enzyme could lead to vacuolization of different tissue cells, including those of the myeloid system, the monocytes, as observed in -mannosidosis disease (Alroy et al., 1989). Hence, this monocyte vacuolization in animals that received the highest dose of the plant extract could be the responsible factor for the unsuccessful recruitment elicitation of macrophages after LPS activation. This hypothesis is supported by the decrease in the number of harvested cells found in the peritoneum of rats from the G group. The disparity of the results obtained for rats that received lower doses and rats in the gavaged group could also help explain the discrepancy between eld observations, which shows that animals intoxicated with plants containing SW are more susceptible to infections (Sharma et al., 1984; Stegelmeier et al., 1998), and in vitro experiments, which show the immune stimulation produced by SW. Thus, immune stimulation or suppression is probably directly related to the dose of SW ingested. However, to better investigate this theory, future experiments will be conducted in our laboratory, administering higher doses than that used here (15 g/kg/kg) to verify the possible immunosuppressive effects of Ipomoea carnea. In conclusion, this study shows that the administration of AF of Ipomoea carnea at moderate doses, for a period of up to 3 weeks, clearly up-regulates phagocytosis in macrophages, as well as metabolic pathways yielding H2 O2 .
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I.M. Hueza et al. / Journal of Ethnopharmacology 87 (2003) 181186 Jolly, R.D., Walkley, S.U., 1997. Lysosomal storage diseases of animals: an essay in comparative pathology. Veterinary Pathology 34, 527548. Karasuno, T., Kanayama, Y., Nishiura, T., Nakao, H., Yonezawa, T., Tarui, S., 1992. Glycosidase inhibitors (castanospermine and swainsonine) and neuraminidase inhibit pokeweed mitogen-induced b-cell maturation. European Journal of Immunology 22, 20032008. Kwiatkowska, K., Sobota, A., 1999. Signaling pathways in phagocytosis. BioEssays 21, 422431. Linehan, S.A., Mart nez-Pomares, L., Gordon, S., 2000. Macrophage lectins in host defense. Microbes and Infection 2, 279288. Nelson, B.K., James, L.F., Sharma, R.P., Cheney, C.D., 1980. Locoweed embryotoxicity in rats. Clinical Toxicology 16, 149166. Pan, Y.T., Ghidoni, J., Elbein, A.D., 1993. The effects of castanospermine and swainsonine on the activity and synthesis of intestinal sucrase. Archives of Biochemistry and Biophysics 303, 134144. Pick, E., Mizel, D., 1981. Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay. Journal of Immunological Methods 46, 211226. Pritchard, D.H., Huxtable, C.R., Dorling, P.R., 1990. Swainsonine toxicosis suppresses appetite and retards growth in weanling rats. Research in Veterinary Science 48, 228230. Rabinovitch, M., DeStefano, M.J., 1973a. Macrophage spreading in vitro. I. Inducers of spreading. Experimental Cell Research 77, 323334. Rabinovitch, M., DeStefano, M.J., 1973b. Macrophage spreading in vitro. II. Manganese and other metals as inducers or as co-factor for induced spreading. Experimental Cell Research 79, 423430. Russo, M., Teixeira, H.C., Marcondes, M.C.G., Barbuto, J.A.N., 1989. Superoxide independent hydrogen peroxide release by activated macrophages. Brazilian Journal of Medical and Biological Research 22, 12711273. Sharma, R.P., James, L.F., Molineux, R.J., 1984. Effect of repeated locoweed feeding on peripheral lymphocytic function and plasma protein in sheep. American Journal of Veterinary Research 45, 2090 2093. Stegelmeier, B.L., Molyneux, R.J., Elbein, A.D., James, L.F., 1995. The lesions of locoweed (Astragalus mollissimus), swainsonine, and castanospermine in rats. Veterinary Pathology 32, 289298. Stegelmeier, B.L., Snyder, P.D., James, L.F., Panter, K.E., Molyneux, R.J., Ralphs, M.H., Pster, J.A., 1998. The immunologic and toxic effects of chronic locoweed (Astragalus lentiginosus) intoxication in cattle. In: Garland, T., Barr, A.C. (Eds.), Toxic Plants and Other Natural Toxicants. CAB International, Wallingford Oxon, pp. 285 290. Tulsiani, D.R., Broquist, H.P., James, L.F., Touster, O., 1988. Production of hybrid glycoproteins and accumulation of oligosaccharides in the brain of sheep and pigs administered swainsonine or locoweed. Archives of Biochemistry and Biophysics 264, 607617. van Kampen, K.R., James, L.F., 1969. Pathology of locoweed poisoning in sheep. Pathologia Veterinaria 6, 413423. Yagita, M., Saksela, E., 1990. Swainsonine, an inhibitor of glycoprotein processing, enhances cytotoxicity of large granular lymphocytes. Scandinavian Journal of Immunology 31, 275282.
On the other hand, at higher doses the plant extract apparently causes non-specic immunosuppression.
Acknowledgements We thank Professors Drs. H.S. Spinosa and M.L.Z. Dagli for their comments on the manuscript and for reviewing this paper. This research was supported by grants from FAPESP, Brazil.
References
Alroy, J., Freden, G.O., Goyal, V., Raghavan, S.S., Schunk, K.L., 1989. Morphology of leukocytes from cats affected with alpha-mannosidosis and mucopolysaccharidosis VI (MPS VI). Veterinary Pathology 26, 294302. Austin, D.F., Huaman, Z., 1996. A synopsis of Ipomoea (Convolvulaceae) in the Americas. Taxon 45, 338. Bowlin, T.L., McKown, B.J., Kang, M.S., Sunkara, P.S., 1989. Potentiation of human lymphokine-activated killer cell activity by swainsonine, an inhibitor of glycoprotein processing. Cancer Research 49, 41094113. Breton, P., Assefa, A., Grzegorzewski, K., Akiyama, S.K., White, S.L., Cha, J.K., Olden, K., 1990. Swainsonine modulation of protein kinase C activity in murine peritoneal macrophages. Cancer Communication 2, 333338. Das, P.C., Roberts, J.D., White, S.L., Olden, K., 1995. Activation of resident tissue-specic macrophages by swainsonine. Oncology Research 7, 425433. De Balogh, K.I.M., Dimande, A.P., van der Lugt, J.J., Molyneux, R.J., Naud, T.W., Welman, W.G., 1999. A lysosomal storage disease induced by Ipomoea carnea in goats in Mozambique. Journal of Veterinary Diagnostic Investigation 11, 266273. Dorling, P.R., 1984. Lysosomal storage diseases in animals. In: Dingle, J.T., Dean, R.T., Sly, W. (Eds.), Lysosomes in Biology and Pathology. Elsevier, Amsterdam, The Netherlands, pp. 347379. Dorling, P.R., Huxtable, C.R., Vogel, P., 1978. Lysosomal storage in Swainsona spp. toxicosis: an induced mannosidosis. Neuropathology and Applied Neurobiology 4, 285295. Elbein, A.D., 1989. The effects of plant indolizidine alkaloids and related compounds in glycoprotein processing. In: James, L.F., Elbein, A.D., Molyneux, R.J., Warren, C.D. (Eds.), Swainsonine and Related Glycosidase Inhibitors. Iowa University Press, Ames, pp. 87155. Hoehne, F.C., 1939. Plantas e Subst ancias Vegetais Txicas e Medicinais. O Estado de So Paulo, So Paulo, p. 32. Humphries, M.J., Matsumoto, K., White, R., Molyneux, R.J., Olden, K., 1988. Augmentation of murine natural killer cell activity by swainsonine, a new antimetastatic immunomodulator. Cancer Research 48, 1410.
In vitro antioxidant and antithrombotic activity of Hemidesmus indicus (L) R.Br.

N.K. Mary, C.R. Achuthan, B.H. Babu, J. Padikkala
Amala Cancer Research Centre, Amala Nagar, Thrissur, Kerala 680 553, India Received 4 July 2002; received in revised form 31 March 2003; accepted 2 April 2003
Abstract The methanolic extract of Hemidesmus indicus (L) R.Br. (Asclepiadaceae) roots was found to inhibit lipid peroxidation and scavenge hydroxyl and superoxide radicals in vitro. The amount required for 50% inhibition of lipid peroxide formation was 217.5 g/ml. The concentrations needed to scavenge hydroxyl and superoxide radicals were 73.5 and 287.5 g/ml, respectively. The intravenous administration of this extract (5 mg/kg body weight) in rabbits delayed the plasma recalcication time and enhanced the release of lipoprotein lipase enzyme signicantly. The extract also inhibited ADP-induced platelet aggregation in vitro (50250 g), which was comparable to commercial heparin. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Hemidesmus indicus; Antioxidant activity; Antithrombotic activity; Antiplatelet aggregation; Anticoagulant activity; Lipoprotein lipase
1. Introduction The traditional Indian medicine and the use of plant drugs against various diseases receive considerable attention nowadays. Hemidesmus indicus (L) R.Br. is a twining shrub which has been used as folk medicine and as ingredient in Ayurvedic and Unani preparations against diseases of blood, inammation, etc. (Vaidya and Kulkarni, 1991). It has also been used in combination with other drugs for snake bite (Kirthikar and Basu, 1935; Mors, 1991). Recently, this plant was used to treat viper venom (haemotoxic)-induced lethality (Alam et al., 1996) and against hypercholesterolaemia in hyperlipidaemic rats (Bopanna et al., 1997). Coronary heart diseases are primarily lipid disorders; yet being initiated and associated with the increased intracellular generation of reactive oxygen species leading to tissue injury with a variety of pathological processes like ischaemia, inammation, atherosclerosis, and thrombosis. Hence compounds, which can scavenge the excess of free radicals formed or inhibit their production or protect membranes from peroxidation are of wide therapeutic value (Diaz et al., 1997). The hypolipidaemic and anticoagulant agents are also playing major roles in preventing cardiovascular diseases. Although several chemicals and drugs are generally used against atherosclerosis and related heart dis Corresponding
eases, the indigenous drugs with long descended heritage of traditional use, are of most signicance, in this period of high rate of the disease. Present study is a scientic approach to reestablish the traditional uses of the plant Hemidesmus indicus and evaluates its antioxidant and antithrombotic properties.
2. Materials and methods 2.1. Plant material The plant Hemidesmus indicus was collected locally and identied by Dr. Sasidharan, Taxonomist, Kerala Forest Research Institute (KFRI), Thrissur, Kerala, India. A voucher specimen was kept in the herbarium of our Institute (ACRH-24). The dried root of the plant was used for the experiment. 2.2. Preparation of the drug Dried and powdered roots of Hemidesmus indicus (10 g) were extracted twice with 70% MeOH by continuous stirring. The extract was pooled and evaporated to dryness under reduced pressure. The yield of the extract was 14% (w/w). The extract was re-suspended in water and subjected to various studies after preliminary phytochemical screening (Wagner et al., 1984) which showed positive reaction
author. Fax: +91-487-211020. E-mail address: jpadikkala@rediffmail.com (J. Padikkala).
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for avonoids, terpenoids, polyphenols, and coumarins (Harbone, 1976; Stahl, 1969). Alkaloids are not found in the extract. 2.3. Animals Male New Zealand White rabbits and Male Wistar rats were purchased from Veterinary College, Mannuthy, Thrissur. They were housed in ventilated cages and fed with pellet diet (Lipton India Ltd.) and water ad libitum. 2.4. Superoxide radical scavenging activity Superoxide radical scavenging activity was determined by the Nitroblue tetrazolium (NBT) reduction method of Mc Cord and Fridovich (1969). The reaction mixture contained EDTA (0.1 M) containing 0.0015% NaCN, riboavin (0.12 mM), NBT (1.5 mM), various concentrations of the extract (10500 g/ml), and phosphate buffer (M/15, pH 7.5) in a nal volume of 3 ml. The tubes were uniformly illuminated under an incandescent lamp for 15 min and the optical density was measured at 530 nm before and after illumination. The percentage inhibition of superoxide generation was evaluated by comparing the absorbance values of the control and experimental tubes. A known antioxidant, curcumin (1100 g) was used as reference. 2.5. Inhibition of lipid peroxide formation 2.5.1. Induction by Fe2+ /ascorbate system The peroxide formation was measured by the method of Ohkawa et al. (1979) by measuring the colour of thiobarbituric acid reactive substances (TBARS) formed at the end of the reaction. Malonaldehyde (MDA), which is formed, as the end product in lipid peroxidation will react with thiobarbituric acid (TBA) to give TBARS, which is pink in colour, measured at 530 nm. The reaction mixture contained rat liver homogenate (0.1 ml, 25% (w/v)) in TrisHCl buffer (20 mM, pH 7.0), KCl (150 mM), ferrous ammonium sulphate (0.8 mM), ascorbic acid (0.3 mM) and various concentrations of the drug (10500 g) in a nal volume of 0.5 ml, was incubated for 1 h at 37 C (Bishayee and Balasubramanian, 1971). The incubated reaction mixture (0.4 ml) was treated with 0.2 ml of 8% sodium dodecyl sulphate (SDS), thiobarbituric acid (1.5 ml, 8%) and acetic acid (1.5 ml, 20%, pH 3.5). The total volume was then made up to 4 ml by adding distilled water and kept in a water bath at 100 C for 1 h. After cooling, 1 ml of distilled water and 5 ml of a mixture of n-butanol-pyridine (15:1 (v/v)) were added and shaken vigorously. The absorbance of the organic layer was measured at 560 nm after centrifugation. The percentage inhibition of lipid peroxide formation was determined by comparing the results of the drug-treated and untreated samples. Curcumin (1100 g) was used as reference.
2.6. Hydroxyl radical scavenging activity Hydroxyl radical scavenging was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe3+ /ascorbate/EDTA/ H2 O2 system. The hydroxyl radicals attack deoxyribose, which eventually results in TBARS formation (Elizabeth and Rao, 1990). The reaction mixture contained deoxyribose (2.8 mM), FeCl3 (0.1 mM), EDTA (0.1 mM), H2 O2 (1 mM), ascorbate (0.1 mM), K H2 PO4 KOH buffer (20 mM, pH 7.4), and various concentrations of the drug (10500 g/ml) in a nal volume of 1 ml. The reaction mixture was incubated for 1 h at 37 C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al. (1979) and percentage inhibition was calculated. Curcumin (1100 g) was used as reference. 2.7. Anticoagulant activity by plasma recalcication method Blood was collected from normal rabbits through the ear vein in EDTA (0.1 M) added tubes. The plasma was separated by centrifugation (1000 rpm 5 min). 200 microlitre of M/100 CaCl2 was added to 100 l of the plasma prewarmed at 37 C. The time taken for the formation of a rm clot was noted immediately by the help of a stopwatch (Achuthan et al., 1997). Similarly the plasma recalcication time was noted 10 min after the intravenous administration of the drug (5 mg/kg body weight) and compared with the anticoagulant heparin (1 mg/kg) as reference. 2.8. Antiplatelet aggregation activityADP induced Platelet rich plasma (PRP) was prepared by centrifugation (1000 rpm 5 min) of blood collected from normal aspirin free blood bank donors. 1.5 millilitre of acid citrate dextrose (ACD) was used as anticoagulant for every 8.5 ml of blood. PRP was taken into siliconized glass cuvettes. Platelet poor plasma (PPP) collected by centrifugation (3000 rpm 5 min) was kept as reference. The cuvettes were incubated at 37 C for 5 min. The aggregation was initiated by adding 20 l of ADP (10 M) to 1 ml of PRP. The aggregation was recorded for 5 min using spectrophotometer at 600 nm. The effect of different concentrations (50250 g) of extract was studied by incubation of PRP and the drug at 37 C for 5 min before the addition of ADP. Commercial heparin (20 g/ml) was used as reference (Subramaniam and Satyanarayana, 1989). 2.9. Lipoprotein lipase releasing activity The lipoprotein lipase releasing activity of the drug was determined by the method of Korn (1962). Blood was collected from normal rabbits through the ear vein in EDTA (0.1 M) added tubes. The plasma was collected by centrifugation (3000 rpm 5 min) and used as the enzyme source. The substrate was lipimic serum obtained from rabbits 3 h
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after feeding 35 g of saltless butter. The substrate (0.1 ml), enzyme (0.10.4 ml), albumin (20%, 0.4 ml, pH 8.5), and (NH4 )2 SO4 (0.1 ml) were mixed at a low temperature and made up to a nal volume of 1 ml. The mixture was incubated at 37 C for 1 h by transferring 0.05 ml of aliquots at 0 h and at the intervals of 30 min into centrifuge tubes containing 0.1 ml of 1N H2 SO4 for glycerol determination. The samples treated with 0.1 ml of sodium periodate (0.05 M) and 0.1 ml of sodium arsenate (0.05 M) were kept in boiling water bath for 30 min after adding 9 ml of chromotropic acid. The volume was adjusted to 10 ml and after cooling the optical density was measured at 570 nm. Heparin (1 mg/kg) was used as reference. The assay was standardized with glycerol solution of known molarity and the glycerol liberated was calculated. The same experiment was repeated after the administration of the drug (5 mg/kg) for a period of 10 min. The glycerol liberated was calculated and compared with normal untreated groups.
Table 2 Anticoagulant activity of Hemidesmus indicus Sample Normal rabbit blood Heparin Hemidesmus indicus (5 mg/kg) Values are mean S.D. of triplicates. Plasma recalcication time (s) 58 2 142 4 130 5
of the extract needed for 50% inhibition was 217.5 g/ml where as curcumin needed was only 8.9 g/ml (Table 1). 3.4. Hydroxyl radical scavenging activity Degradation of deoxyribose mediated by hydroxyl radicals generated by the Fe3+ /ascorbate/EDTA/H2 O2 system was also found inhibited by Hemidesmus indicus root extract. The concentration of root extract needed for 50% inhibition was 287.5 g/ml and that of curcumin was 2.7 g/ml (Table 1). 3.5. Effect on plasma recalcication time Normal plasma recalcication time noticed was 58 2 s. Administration of heparin could delay the time to 142 4 s (Table 2). The methanolic extract of Hemidesmus indicus delayed the time to 130 5 s, which is much greater than the recalcication time of normal plasma. Compared to heparin this drug is more or less equally effective. 3.6. Effect on platelet aggregation Addition of ADP to a suspension of washed human platelets caused a marked decrease in optical density at 600 nm indicating the aggregation of platelets. The aggregation effect was greater at 37 C compared to that at room temperature. The methanol extract of Hemidesmus indicus (50250 g/ml) interestingly inhibited platelet aggregation (Fig. 1). Greater inhibition of aggregation was noticed with greater concentrations. 3.7. Lipoprotien lipase releasing activity The drug-treated animals for a period of 10 min showed increased production of glycerol as an index of the greater release and activity of enzyme from the arterial intima. The glycerol liberated in the drug-treated animals was found to
Table 3 Effect of Hemidesmus indicus on lipoprotein lipase releasing activity Sample Normal rabbits Heparin (1 mg/kg) Hemidesmus indicus (5 mg/kg) Values are mean S.D. of triplicates. Glycerol liberated (mg/dl) 3.6 0.2 12.7 1.7 9.0 0.7
3. Results 3.1. Phytochemical screening The phytochemical screening of the root extract showed positive reaction for avonoids, terpenoids, polyphenols and coumarins. Alkaloids are not found in the extract. 3.2. Superoxide scavenging activity The root extract of Hemidesmus indicus was found to scavenge the superoxide generated by photoreduction of riboavin. The concentration needed for 50% scavenging of superoxide was found to be 73.5 and 6.3 g/ml for Hemidesmus indicus and curcumin, respectively (Table 1). 3.3. Inhibition of lipid peroxidation The generation of lipid peroxides by Fe2+ /ascorbate in rat liver homogenate was found inhibited by the addition of root extract of Hemidesmus indicus. The concentration
Table 1 Effect of Hemidesmus indicus on free radical generation Percentage inhibition 10 20 30 40 50 50 (curcumin) 60 70 Hydroxyl radical (g/ml) 17.5 35.5 50.0 170.2 287.5 2.7 375.3 427.7 0.7 1.2 1.3 2.1 1.7 0.07 2.5 2.7 Superoxide radical (g/ml) 9.5 20.9 43.0 56.9 73.5 6.3 90.2 106.7 0.05 0.6 0.9 0.5 1.3 0.06 1.2 2.1 Lipid peroxide (g/ml) 15.0 33.7 47.5 128.7 217.5 8.9 220.5 302.2 0.2 0.4 0.8 0.7 1.5 0.05 1.7 1.9
Values are mean S.D. of triplicates.
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Fig. 1. Effect of methanolic extract of Hemidemus indicus on the aggregation of human platelets (washed).
be 9 mg/dl where in the normal animals it was only 3.6 mg/dl (Table 3).
4. Discussion India recently increased research in Traditional Ayurvedic Herbal Medicines after observations that they are effective for conditions to which they have traditionally been applied. The present investigation has explored the use of one such plant Hemidesmus indicus, abundantly found in the Indian continent, for preventing coronary artery diseases. The vascular endothelium is the principal site of action of cardiovascular risk factors and early atherogenesis (Ross, 1993). The imbalance between prooxidants and antioxidants in the development of atherosclerosis has prompted the investigation of antioxidants as a possible therapy (Khan and Butler, 1998). The process of atherogenesis is initiated by the oxidation of lipids in low-density lipoproteins (LDL), termed lipid peroxidation (Diaz et al., 1997; Camejo et al., 1976). The screening of the antioxidant activity of this plant has revealed its capacity to scavenge the superoxide and hydroxyl radicals at low concentrations. The lipid peroxidation was also found inhibited by low concentrations of the root extract. Platelets play an important role in the process of atherothrombosis by adhering to the damaged regions (caused by reactive oxygen species) of the endothelial surface. The activated platelets form platelets to platelets bonds, binds also to leucocytes bringing them into a complex process of plaque formation and growth (Prentice, 1999). The antiplatelet therapy constitutes the best available tool
for ameliorating the mechanisms related to atherogenesis (Cimmniello and Toschi, 1999) and this drug has interestingly inhibited platelet aggregation. Hemidesmus indicus root extract incubated with viper venom antagonized coagulant and haemorrhagic activity (Alam et al., 1996). The plasma recalcication time was also delayed signicantly by the intravenous administration of the root extract. Lipoprotein lipase has been reported in the post-heparin plasma of rabbits and has a major role in the transport and metabolism of triglycerides of exogenous origin (Korn, 1962). It is the key enzyme, the metabolic gatekeeper regulating the disposal of lipid fuels in the body (Fielding and Frayn, 1998). The glycerol liberated by the action of lipoprotein lipase enzyme in the drug-treated animals was found to be three times greater than the normal untreated animals. This drug thus has an enhancing role of releasing and activating the enzyme, resulting in the metabolic degradation of lipids. Bopanna et al. (1997) have also reported the hypolipidaemic effect of the cell culture derived Hemidesmus indicus. The various in vitro studies have shown earlier that certain avonoids are the potent inhibitors of the oxidative modication of LDL by macrophages (Havsteen, 1983). The avonoids (Mors, 1991), terpenoids (Gupta et al., 1992), polyphenols and coumarins (Nandkarni, 1976) present in the plant extract may account for the above-mentioned properties. With all these wide spectrum of the antioxidant, anticoagulant, hypolipidaemic, antiplatelet aggregation, antihaemorrhagic and lipoprotein lipase releasing properties, Hemidesmus indicus can be considered an effective antiatherogenic agent preventing coronary artery diseases.
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Havsteen, B., 1983. Flavonoids, a class of natural products of high phamacological potency. Biochemistry and Pharmacology 32, 11411148. Khan, F., Butler, R., 1998. Free radicals in cardiovascular diseases. Asian Journal of Clinical Cardiology 1, 5260. Kirthikar, K.R., Basu, B.D., 1935. Indian Medicinal Plants, vol. III. Periodical Experts, New Delhi, pp. 15961598. Korn, E.D., 1962. Lipoprotein lipase (clearing factor). Methods in Enzymology V, 542545. Mc Cord, J.M., Fridovich, I., 1969. Superoxide dismutase, an enzymatic function for erythrocuprein. Journal of Biological Chemistry 244, 60496055. Mors, W.B., 1991. Plants active against snake bite. In: Economic and Medicinal Plant Research, vol. 5. Academic Press, New York, pp. 353373. Nandkarni, A.K., 1976. Indian Materia Medica, 3rd ed., vol. 1. Popular Prakashan, Bombay. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Annals of Biochemistry 95, 351358. Prentice, C.R.M., 1999. Platelets and Atherosclerosis. European Heart Journal Supplements 1, A3A7. Ross, R., 1993. The pathogenesis of Atherosclerosis, a perspective for the 1990. Nature 362, 301309. Stahl, E., 1969. Thin Layer Chromatography: A Laboratory Hand Book. Springer, New York, pp. 855905. Subramaniam, A., Satyanarayana, M.N., 1989. Inuence of certain dietary plant constituents on platelet aggregation. Journal of Food Safety 9, 201214. Vaidya, K., Kulkarni, P.H., 1991. A study of an Ayurvedic formula viz Jivak. Deerghaya International 7, 20. Wagner, H., Bladt, S., Zgainski, E.M., 1984. Plant Drug Analysis. Springer, Berlin/New York, pp. 126169.
Inhibition of experimental gastric lesion and inammation by Phyllanthus amarus extract

K. Regi Raphael, Ramadasan Kuttan
Amala Cancer Research Centre, Thrissur, Kerala 680 553, India Received 16 July 2002; received in revised form 28 March 2003; accepted 2 April 2003
Abstract Methanolic extract of Phyllanthus amarus Shum & Thonn (Euphorbiaceae) 50, 200, and 1000 mg/kg body weight signicantly inhibited gastric lesions, induced by intragastric administration of absolute ethanol (8 ml/kg). Mortality, increased stomach weight, ulcer index, and intraluminal bleeding were reduced signicantly by Phyllanthus amarus. Biochemical analysis indicated that reduced glutathione (GSH) of gastric mucosa produced by ethanol administration was signicantly elevated by treatment with Phyllanthus amarus extract. Aqueous and methanol extracts of Phyllanthus amarus produced an inhibition of rat paw edema up to 42% compared to control in 3 h and continued up to 8 h. Anti-oxidant activity of the extract as well as presence of tannins in the extract may be responsible for these observed activities. 2003 Elsevier Ireland Ltd. All rights reserved.
Keywords: Phyllanthus amarus; Ethanol toxicity; Gastric lesion; Anti-inammatory
1. Introduction Phyllanthus amarus is traditionally used to treat u, dropsy, diabetes, and jaundice (Foo, 1993). It is also being used to treat hepatic and urolitic diseases and have diuretic activity. Phyllanthus amarus inhibited hepatitis B virus polymerase activity and decreased episomal hepatitis B virus DNA content and suppressed viral release into the culture medium (Lee et al., 1996). Simultaneous administration of Phyllanthus amarus extract along with carcinogen has been reported to inhibit the hepatocellular carcinoma development induced by NDEA and increased the life span of hepatocellular carcinoma harbouring animals (Joy and Kuttan, 1998; Rajesh and Kuttan, 2000). In chemically induced liver toxicity models Phyllanthus amarus signicantly protected the liver tissue (Prakas et al., 1995). Phyllanthus amarus has potent free radical scavenging activity and could scavenge superoxides and hydroxyl radicals and can inhibit lipid peroxides (Joy and Kuttan, 1995). As the inammation is mainly produced by the oxidative burst of the macrophages, many anti-oxidants may be effective to reduce the inammation. Infusion of the young shoots of Phyllanthus amarus has been recommended to lessen the edematous swelling and ulcers (Mhaskar et al., 2000). In the present study, we have checked anti-inammatory
Corresponding
activity of Phyllanthus amarus using experimental paw edema produced by carrageenan administration. We have also looked the protection of gastric lesions by Phyllanthus amarus extract.
2. Materials and methods 2.1. Extraction of Phyllanthus amarus Leaves and stems of Phyllanthus amarus were collected from Thrissur district of Kerala and were dried at 50 C. A voucher specimen of the plant was identied by Regi Raphael K, Botanist, Amala Cancer Research Centre, Thrissur, Kerala (Voucher No: EUP. 9) and has been kept at Amala Ayurvedic Hospital and Research Centre. 2.1.1. Preparation of alcoholic extract Dried parts of Phyllanthus amarus were powdered and this powder was extracted twice in ve volumes of 75% methanol by stirring overnight and centrifuged at room temperature. This supernatant was evaporated to dryness at 50 C under reduced pressure using a rotary evaporator. The yield of the extract was 8%. 2.1.2. Preparation of aqueous extract Powdered Phyllanthus amarus (50 g) was extracted twice overnight with 250 ml of distilled water at room temperature.
author. Fax: +91-487-307020. E-mail address: amalaresearch@rediffmail.com (R. Kuttan).
0378-8741/03/$ see front matter 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00120-X
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The supernatant was collected and evaporated to dryness at 50 C under reduced pressure. The yield of the extract was 10%. Both methanolic and aqueous extracts had almost similar TLC pattern. Major extracted products in both cases were tannins as seen by ferric chloride test. However, yield of the aqueous extract was higher than methanol extract. Extracts were prepared freshly before each experiment. 2.2. Induction of ethanol-induced gastric lesion Adult male Wistar rats weighing 120 g were used for the experiment. They were grouped into four groups of ve animals each. All the animals were fasted for 16 h and deprived of water for 12 h prior to the experiments. Group I acted as animals treated with alcohol alone. Group IIIV were treated with 1000, 200, and 50 mg of Phyllanthus amarus extract/kg body weight as a single dose 30 min prior to the experiment. Acute gastric lesion was induced by absolute ethanol (Robert et al., 1979). Briey, absolute ethanol (8 ml/kg body weight) was administered intragastrically to control and drug-treated animals. Each animal was sacriced by ether overdose 1 h after administration of ethanol and stomach was excised, opened along the greater curvature and washed gently with ice cold solution. The stomach weight and intraluminal bleeding were recorded. The extent of erosion of stomach mucosa was assessed from a scoring system designed by MerazziUberti Turba as follows: 0, no erosions; 1, one to three small erosions (4 mm or smaller); 2, more than three small erosions or one large erosion; 3, two large erosions; 4, three to four large erosions; and 5: more than four large erosions or lesion proliferation (Giordano et al., 1990). The results were expressed in terms of an ulcer index, which is the average severity of erosions per rat each group on the scale from 0 to 5. 2.3. Biochemical analysis The mucosa of glandular stomach was removed by scraping with a blunt knife and 10% homogenate was prepared. Reduced glutathione (GSH) in the gastric mucosa was determined by Ellmans reaction using 5 5 -dithio-bis-2-nitro benzoic acid (DTNB) was described by Moron et al. (1979). Briey 125 l of 25% trichloro acetic acid (TCA) was added to 0.5 ml of homogenate to precipitate proteins. The tubes were cooled in ice for 5 min and the mixture was further diluted with 0.6 ml of 5% TCA and centrifuged at 9000 g for 10 min. 0.3 ml of the supernatant was taken for estimation. For this purpose the volume of the aliquot was made up to 1 ml with 0.2 M sodium phosphate buffer (pH 8.0) and 2 ml of freshly prepared DTNB solution (0.6 mM in 0.2 M phosphate buffer pH 8.0) was added to the tubes. After 10 min the intensity of the yellow color formed was read at 412 nm in a spectrophotometer. Reduced glutathione (Sisco Research Laboratories, Mumbai, India) was used as a standard. The protein content of the gastric mucosa was quantied by the
method of Lowry et al. (1951) with bovine serum albumin as the standard. 2.4. Histopathology A portion of the stomach from each group was xed in 10% formalin. The formalin xed specimens were embedded in parafn and sectioned (35 m) and stained with hematoxylin and eosin and histochemical sections were evaluated by light microscopy. 2.5. Statistical evaluation The values are expressed as mean standard deviations. The results were analyzed statistically by analysis of variance. Values of P less than 1% (P < 0.01) were considered to be statistically signicant. 2.6. Determination of anti-inammatory activity Anti-inammatory activity was determined by carrageenan-induced mice paw edema method of Langrange et al. (1974). Female inbred strains of Balb/c mice weighing 2528 g (67 weeks old) were used for the experiment. They were divided into four groups of three animals each. Group 1 acted as control. Groups 24 received 500, 250, and 100 mg/kg body weight of Phyllanthus amarus extract orally as a single dose 1 h prior to the experiment. Paw edema was induced by injecting carageenan (200 g/20l) into the sub-plantar region of the left paw. The thickness of paw edema was measured by venire calipers before treatment and after injection with carageenan. Measurement was continued at 60 min intervals up to 8 h and further at the 24th hour. The inhibition of paw edema was calculated by comparing the difference in paw thickness of the control and treated group. Experiment was repeated twice and average values were taken.
3. Results 3.1. Effect of Phyllanthus amarus extract in gastric lesion The present investigation indicate that rat mucosal gastric injury induced by ethanol was signicantly and dose dependently reduced by methanolic extract of Phyllanthus amarus (Table 1). Administration of absolute ethanol to fasted rats resulted in severe gastric damage visible from the outside of the stomach as thick reddish-black lines. After opening, the gastric lesions were found in the mucosa and consisted of elongated bands, 110 mm long, usually parallel to the long axis of the stomach. They were located mostly in the corpus (the portion of the stomach secreting acid and pepsin). No gross lesions developed in the fore stomach (the nonsecretory part of the stomach). Intragastric administration of the absolute ethanol to rats resulted in 50% mortality due to
K.R. Raphael, R. Kuttan / Journal of Ethnopharmacology 87 (2003) 193197 Table 1 Effect of Phyllanthus amarus administration on mortality, stomach weight, and intraluminal bleeding of rats treated with absolute ethanol Treatment Dose (mg/kg) Mortality Number Normal rats Ethanol Ethanol + Phyllanthus amarus Ethanol + Phyllanthus amarus Ethanol + Phyllanthus amarus
a,b,c,d
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% 0 50 20 0 0
Stomach weight (g/100 g body weight S.D.) 0.68a 0.05 1.02d 0.13 0.85c 0.10 0.79b,c 0.05 0.70a,b 0.08
Intraluminal bleeding Number 0/5 5/5 2/4 0/5 0/5 % 0 100 50 0 0
0 0 50 200 1000
0/5 5/10 1/5 0/5 0/5
Result of the signicance test done by ANOVA method.
acute reaction of the alcohol and its metabolites. Increased mortality in the controls were found to be aggrevated due to the fasting (16 h) and deprivation of water (12 h). The rats, which died, had perforated lesions and severe intraluminal bleeding. Stomach weight in alcohol-treated rats was increased to 1.02 0.013 g/100 g body weight as compared to normal rat stomach weight 0.68 0.05 g/100 g body weight possibly due to inammation. In the treated animals because of scavenging of the oxygen radicals generated by ethanol, the mortality rate and increase in stomach weight induced by ethanol was found to be signicantly less (Table 1). All animals treated with absolute ethanol caused intraluminal bleeding in the glandular portion of the stomach, while all animals in the Phyllanthus amarus (200 and 1000 mg/kg) pretreated group were found to be signicantly protected from intraluminal bleeding. Ethanol administration to rats produced gastric damage with an ulcer index of 4.75 0.5 and 48.8% reduction in gastric mucosal GSH. Phyllanthus amarus pretreatment (50, 200, and 1000 mg/kg) signicantly reduced the ulcer index to 3.5 0.6, 2.0 0.5, and 0.6 0.5, respectively and reduced the depletion of GSH to 36, 16.5, and 5.5%, respectively (Table 2). Histological analysis of ethanol-treated rat stomach revealed the presence of necrotic debrii in the lamina propria
of the mucosa which are inltered with polymorphonuclear leucocytes. The depth of the lesion extended up to the muscularis mucosae with red blood corpuscles extravasation. The submucosa of the corpus was markedly thickened by edema but devoid of polymorphonuclear leucocytes. Histologically, the stomach of the Phyllanthus amarus pretreated groups (250 and 1000 mg/kg) showed supercial erosion in the mucosa and moderate degree of sub-mucosal edema with neutrophilic inltration. 3.2. Anti-inammatory activity of Phyllanthus amarus Development of paw edema was observed in both control and treated group after carrageenan injection. Thickness of the paw was found to be increased initially upon injection of carrageenan due to volume effect. Difference in the thickness of mice paw edema was further increased during the time interval of 60180 min in control group. Water extract of Phyllanthus amarus (100, 250, and 500 mg/kg) produced an inhibition of 26, 33, and 39%, respectively at 3 h (Fig. 1). While the methanol extract of Phyllanthus amarus (100, 250, and 500 mg/kg) produced an inhibition of 29, 37, and 42%, respectively at 3 h (Fig. 2) and signicant inhibition of paw oedema was observed throughout the course of the experiment up to 8 h.
Table 2 Effect of Phyllanthus amarus administration on the ulcer index and glutathion (GSH) content of the mucosa of rats treated with absolute alcohol Treatment Normal rats Ethanol Ethanol + Phyllanthus amarus Ethanol + Phyllanthus amarus Ethanol + Phyllanthus amarus
a,b,c,d
Dose (mg/kg) 0 0 50 200 1000
Ulcer index S.D. 0 4.75a 0.5 3.5b 0.6 2.0c 0.5 0.6d 0.5
Inhibition (%) 100 26.3 57.9 87.4
GSH (nmol/mg (%) reduction protein) in GSH 12.7a 0.8 6.5b 0.6 8.1c 1.1 10.6d 1.1 12.0a 0.5 48.8 36 16.5 5.5
Result of the signicance test done by ANOVA method.
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Fig. 1. Anti-inammatory activity of water extract of Phyllanthus amarus. () Control treated with the extract; () 100 mg/kg; () 250 mg/kg; () 500 mg/kg.
Fig. 2. Anti-inammatory activity of methanolic extract of Phyllanthus amarus. () Control treated with the extract; () 100 mg/kg; () 250 mg/kg; () 500 mg/kg.
4. Discussion In the present study we have checked the anti-lesion and anti-inammatory activity of Phyllanthus amarus extract which is a very important plant in the herbal medicine practice. Phyllanthus amarus has been shown to be an effective medicine against viral hepatitis as it has been shown to suppress the mRNA transcription of hepatitis B virus (Lee et al., 1996). It has been shown to be useful to reduce the Hbs/Ag antigen found in human hepatitis carriers (Unander and Blumberg, 1992). Our recent ndings indicate that the extract had a signicant activity to suppress the chemical carcinogenesis induced by chemicals such as 3-methyl cholanthrene (Rajesh and Kuttan, 2001) and N-nitroso diethylamine (Joy and Kuttan, 1998). The mechanism of action of the extract seems to be (a) suppression of prolif-
eration, (b) suppression of activation of carcinogen, and (c) anti-oxidant activity of the extract. The present study indicating that the extract has significant effect in reducing inammation and lesion is again reective of its activity as scavenging oxygen radicals. Reactive oxygen species has been shown to have signicant effect on the cellular system, damages its structure, and induces alteration especially in its high molecular weight components. Large doses of ethanol has been shown to specically effect the inner lining of the stomach producing erosion. In liver ethanol converted to ethanal (aldehyde) which is more toxic. Ethanal has been shown to reduce GSH levels in liver tissues. Fall in GSH increases lipid peroxidation. Pretreatment with the Phyllanthus amarus extract may either produce a protective lining on the stomach and reduces oxygen radical production and thereby reduces
197
its effect on the liver. Similar mechanism could also be postulated for its anti-inammatory activity of Phyllanthus amarus. Inammation produced by carrageenan is mainly due to macrophage activation and there by the producing of oxygen radicals. Phyllanthus amarus extract could inhibit the oxygen radicals and there by reduces the inhibition. Another possible mechanism for the activity of the extract to inhibit gastric lesion produced by alcohol may be due to the formation of a protective layer of the polyphenolic compounds present in the extract with the protein of the stomach lining by hydrophobic interaction. Moreover the extract may inhibit the prostaglandin synthesis like nonsteroidal anti-inammatory drugs. It has been shown that Phyllanthus amarus extract could inhibit the onset of diarrhea induced by castor oil and reduced frequency of defecation and also reduced gut meal travel distance signicantly (Odetola and Akojenu, 2000). This effect has been attributed to the inhibition of prostaglandin synthesis. Extract may also inhibit the macrophage migration which can reduce the inammatory response produced by carrageenan. Several active compounds have been identied in Phyllanthus amarus extracts. Most common among them are lignans like phyllanthin and hypophyllanthin (Somanabandhu et al., 1993), avonoids like quercetin, astragalin (Nara et al., 1977), ellagitannins like amarinic acid (Foo, 1995) as well as amarin (Foo, 1993) and phyllanthisiin D (Foo and Wong, 1992). Hydrolysable tannins puried from Phyllanthus amarus were found to be potent inhibitors of rat liver cyclic AMP-dependent protein kinases. These hydrolyzable tannin inhibitors are the most specic and potent plant-derived inhibitors of cyclic AMP-dependent protein kinase catalytic sub unit yet found (IC50 0.217 fM) (Polya et al., 1995). It also showed anti-genotoxic properties (Gowrishankar and Vivekanandan, 1994). Phyllanthin, a diaryl butane lignan, isolated from Phyllanthus amarus showed a signicant protection against CCl4 -induced elevation in transferase levels and signicantly increased protein level (Syamasundar et al., 1985). Anti-viral agents: repandusinic acid (Ogata et al., 1992) and niruriside (Qian-Cutrone et al., 1996) isolated from Phyllanthus amarus were shown to inhibit HIV transcription in tissue culture. Active compounds responsible for the anti-ulcerogenic and anti-inammatory activity produced by the extract have not been clearly understood.
References
Foo, L.Y., 1993. Amariin, a di-dehydro hexahydroxy diphenoyl hydrolysable tannin from Phyllanthus amarus. Phytochemistry 33, 487 491. Foo, L.Y., 1995. Amarinic acid and related ellagitannins from Phyllanthus amarus. Phytochemistry 39, 217224. Foo, L.Y., Wong, H., 1992. Phyllanthisiin, an unusual hydrolysable tannins from Phyllanthus amarus. Phytochemistry 31, 711713. Giordano, O.S., Guerreiro, E., Pestchanker, M.J., 1990. The gastric cytoprotective effect of several sesquiterpene lactones. Journal of Natural Products 53, 803809.
Gowrishankar, B., Vivekanandan, O.H., 1994. In vivo studies of a crude extract of Phyllanthus amarus L by tannery efuents. Mutation Research 322, 185192. Joy, K.L., Kuttan, R., 1995. Antioxidant activity of selected plant extracts. Amala Research Bulletin 15, 6871. Joy, K.L., Kuttan, R., 1998. Inhibition by Phyllanthus amarus of hepatocarcinogenesis induced by N-nitrosodiethylamine. Journal of Clinical Biochemistry and Nutrition 24, 133139. Langrange, P.H., Mackaness, G.B., Miller, T.E., 1974. Inuence of dose and route of antigen injection on the immunological induction of T-cells. Journal of Experimental Medicine 139, 528530. Lee, C.D., Ott, M., Thyagarajan, S.P., Shfritz, D.A., Burk, R.D., Gupta, S., 1996. Phyllanthus amarus down regulates hepatitis B virus m RNA transcription and translation. European Journal of Clinical Investigations 26, 10691076. Lowry, H.D., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. Journal of Biological Chemistry 193, 265275. Mhaskar, K.S., Blatter, E., Caius, J.F., 2000. Kirtikar and Basus Illustrated Indian Medicinal Plants, vol. 9. Sri Satguru Publications, Delhi, India, p. 3074. Moron, M.A., Depierre, J.W., Mannervick, B., 1979. Levels of glutathion, glutathion S-transferase activities in rat liver. Acta Biochemistry and Biophysics 582, 6768. Nara, T.K., Glyeye, J., Cerval, E.L., Stanislan, E., 1977. Flavonoids of Phyllanthus niruri, Phyllanthus urinaria, Phyllanthus orbiculatus. Plantes Medicinales et Phytotherapie 11, 8286. Odetola, A.A., Akojenu, S.M., 2000. Anti-diarrhoeal and gastrointestinal potentials of the aqueous extract of Phyllanthus amarus (Euphorbiaceae). African Journal of Medicine and Medical Sciences 29, 119 122. Ogata, T., Higuchi, H., Mochida, S., Matsumoto, H., Kato, A., Endo, T., Kaji, A., Kaji, H., 1992. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri. AIDS Research in Human retroviruses 11, 1937 1944. Polya, G.M., Wang, B.H., Fooly, L.Y., 1995. Inhibition of signal regulated protein kinases by plant derived hydrolysable tannins. Phytochemistry 38, 307314. Prakas, A., Satyan, K.S., Washi, S.P., Singh, R.P., 1995. P. urinaria, P. niruri and P. simplex, on carbon tetrachloride induced liver injury in the rat. Phytotherapy Research 9, 594596. Qian-Cutrone, J., Huang, S., Trimble, J., Li, H., Lin, P.F., Alam, M., Klohr, S.E., Kadow, K.F., 1996. Niruriside, a new HIV REV/RRE binding inhibitor from Phyllanthus niruri. Journal of Natuaral Products 59, 196199. Rajesh Kumar, N.V., Kuttan, R., 2000. Phyllanthus amarus extract administration increases the life span of rats with hepatoprotective carcinoma. Journal of Ethnopharmacology 73, 215 219. Rajesh Kumar, N.V., Kuttan, R., 2001. Cancarea herbal formulation inhibits chemically induced tumors in experimental animals. Indian Journal of Experimental Biology 39, 654659. Robert, A., Nezamis, J.E., Lancster, C., Hanchar, A.J., 1979. Cytoprotection by prostaglandins in rats. Prevention of gastric necrosis produced by alcohol, HCl, NaOH, hypertonic NaCl and thermal injury. Gastroenterology 77, 433443. Somanabandhu, A., Nityangkuru, S., Mahidol, C., 1993. 1 H and 13 C NMR assignments of phyllanthin and hypophyllanthin lignans that enhance cytotoxic responses with cultured multidrug-resistant cells. Journal of Natural Products 56, 233239. Syamasundar, K.V., Singh, B., Thakur, R.S., Husain, A., Kiso, Y., Hino, H., 1985. Antihepatotoxic principles of Phyllanthus niruri herbs. Journal of Ethnopharmacology 14, 4144. Unander, D.W., Blumberg, B.S., 1992. In-vitro activity of Phyllanthus (Euphorbiaceae) species against the DNA polymerase of hepatitis viruses: effects of growing environment and intra- and inter-specic differences. Economic Botany 45, 225242.
Anti-inammatory and analgesic activities of mature fresh leaves of Vitex negundo

M.G. Dharmasiri, J.R.A.C. Jayakody, G. Galhena, S.S.P. Liyanage, W.D. Ratnasooriya
Department of Zoology, University of Colombo, Colombo 3, Sri Lanka Received 8 November 2002; received in revised form 6 February 2003; accepted 3 April 2003
Abstract This study conrmed the oral anti-inammatory, analgesic and antihistamine properties of mature fresh leaves (MFL) of Vitex negundo L. (Verbenaceae) claimed in the Ayurveda medicine by orally treating a water extract of the leaves to rats. The early phase (2 h) of carrageenan-induced rat paw oedema was signicantly (P < 0.01) suppressed in an inversely does-dependent (r2 = 1, P < 0.01) manner by MFL. The EC50 was 2 g/kg of MFL. In the formaldehyde-induced rat paw oedema test, the 2.5 and 5 g/kg leaves signicantly (P < 0.05) suppressed the inammation on days 46 of the test. In the hot plate test, 2.5 and 5 g/kg of MFL showed a signicant (P < 0.05) and directly dose-dependent analgesic activity at 1 h of treatment while the activity was absent in the tail ick test in rats. The EC50 for the analgesic activity was 4.1 g/kg. In the formalin test, 1.25, 2.5 and 5 g/kg of MFL signicantly (P < 0.05) suppressed the pain in both the phases of the test like aspirin. The leaves showed an inversely dose-dependent in vivo antihistamine and in vitro prostaglandin (PG) synthesis inhibition, membrane stabilising and antioxidant activities. Naloxone did not abolish the analgesic activity in the hot plate test. A 5 g/kg of MFL did not impair muscle strength and co-ordination and did not induce sedation. The treatment of 5 g/kg of MFL did not show signs of acute toxicity or stress. Fourteen-day oral treatment of 5 g/kg of MFL signicantly increased the serum activity of AST. Flowering of the tree did not abolish the analgesic and anti-inammatory activities of the leaves. These observations revealed that the fresh leaves of Vitex negundo have anti-inammatory and pain suppressing activities possibly mediated via PG synthesis inhibition, antihistamine, membrane stabilising and antioxidant activities. The antihistamine activity can produce the anti-itching effect claimed in Ayurveda medicine. 2003 Published by Elsevier Science Ireland Ltd.
Keywords: Vitex negundo fresh leaves; Anti-inammatory activity; Analgesic activity; Antihyperalgesic activity; Antihistamine activity; Prostaglandin synthesis inhibition activity; Antioxidant activity; Membrane stabilisation
1. Introduction As a result of adverse side effects, like gastric lesions, caused by NSAIDs and tolerance and dependence induced by opiates, the use of these drugs as anti-inammatory and analgesic agents have not been successful in all the cases. Therefore, new anti-inammatory and analgesic drugs lacking those effects are being searched all over the world as alternatives to NSAIDs and opiates. During this process, the investigation of the efcacy of plant-based drugs used in the traditional medicine have been paid great attention because they are cheap, have little side effects and according to WHO still about 80% of the world population rely mainly on plant-based drugs (Kumara, 2001). Vitex negundo L. (Verbenaceae), Nika in Sinhala, is a small tree of which water extract of fresh mature leaves are used in Ayurveda medicine as anti-inammatory, anal Corresponding
gesic and anti-itching agents internally and externally (Gunatillake, 1994). The tree is distributed in Sri Lanka, India, Malaya, The Philippine Islands and East Africa. Flowers occur throughout the year (Jayaweera, 1981). However, the claimed activities of the leaves have not been investigated using controlled experiments in detail. The purpose of this study was to investigate in rats (a) the effectiveness of the leaves as anti-inammatory, analgesic and antihistaminic agents, (b) whether owering of the tree abolishes the anti-inammatory and analgesic activities and (c) any possible toxic effect caused after short-term use of the leaves.
2. Materials and methods 2.1. Chemicals Carrageenan (Sigma Chemical Co., St. Louis, MO, USA), aspirin, indomethacin, chlorpheniramine (State Pharmaceutical Corporation, Colombo, Sri Lanka), naloxone
author. Tel.: +94-1-503-399. E-mail address: sd@chem.cmb.ac.lk (W.D. Ratnasooriya).
0378-8741/03/$ see front matter 2003 Published by Elsevier Science Ireland Ltd. doi:10.1016/S0378-8741(03)00159-4
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hydrochloride, formaldehyde (Fluka, Buchs, Switzerland), assay kits (Randox Laboratories Ltd., Co., Antrim, UK). 2.2. Collection of plants and preparation of extracts Mature fresh leaves (MFL) of Vitex negundo at 13 weeks before owering and 12 weeks after owering were collected from a tree in the campus garden of the University of Colombo, Colombo, Sri Lanka, between March and July 2002 and authenticated by Prof. A.S. Seneviratne, Department of Botany, University of Colombo. Voucher specimen (23-VN) has been deposited at the museum of the Department of Zoology, University of Colombo. Fifty grams of MFL was macerated with 100 ml of distilled water (Jayasinghe, 1975) using an electric blender for 3 min, squeezed through muslin cloth and 50% (w/v) extract was obtained. Doses were determined according to the information provided by an Ayurvedic physician, Dr. S. Ediriweera, and animals were orally treated with the extract so that they received MFL 1.25, 2.5 and 5 g/kg of body weight (5 g/kg dose per rat is approximately equal to the human dose when extrapolated). The extract was prepared every day for the experiments. 2.3. Animals Wistar male and female rats (150200 g) housed under standardised animal house conditions were used in all the experiments. They had access to pelleted food (Vet House Ltd., Colombo, Sri Lanka) and water ad libitum. The animals were assigned to different groups to be treated in experiments. 2.4. Anti-inammatory activity 2.4.1. Carrageenan-induced paw oedema Male rats (n = 9/group) were treated with 1.25, 2.5 and 5 g/kg of MFL at preowering stage of the tree, 5 g/kg of MFL at owering stage, 5 ml/kg water and 5 mg/kg indomethacin, respectively (Forestieri et al., 1996). After 1 h, these rats were injected with 0.05 ml of 1% carrageenan suspension into the foot pad of left hind paw (Winter et al., 1962). The paw volumes of these rats were measured using a Plethysmometer (Letica Scientic Instruments, Barcelona, Spain) at 1 h before, and 2 and 4 h after the carrageenan injection and the paw oedema was calculated. 2.4.2. Formaldehyde-induced paw oedema Male rats (n = 9/group) were treated with 1.25, 2.5 and 5 g/kg/day of MFL and 5 ml/kg/day of water for 7 consecutive days. After 1 h on days 1 and 3 of treatment, these rats were injected with 0.1 ml of 2% formaldehyde into the foot pad of left hind paw (Selye, 1949). Paw oedema was measured 1 h before formaldehyde injection and at 4 h after the injection on day 1 and everyday at 1 h after the treatment for 7 consecutive days.
2.5. Analgesic activity 2.5.1. Hot plate and tail ick tests Female rats (n = 6/group) were treated with 1.25, 2.5 and 5 g/kg of MFL at preowering stage, 5 g/kg of MFL at owering stage, 5 ml/kg of water and 100 mg/kg aspirin. The reaction times of these rats were measured 1 h prior to the treatment, 1 and 3 h after the treatment using hot plate and tail ick techniques as described by Langerman et al. (1995). In the hot plate test, the rat was placed in a hot plate analgesia meter (Model MK 35 A, Muromachi Kikai Co. Ltd., Tokyo, Japan) at 50 C and the time taken to lick the hind paw or to jump was recorded. In the tail ick test, the tail of the rat 45 cm from its tip was immersed in a water bath at 55 C and the time taken to ick the tail was recorded. Rats showing a pre-treatment reaction time greater than 15 s in the hot plate test and 5 s in the tail ick test were not used in the experiment. A cut off time of 25 s was set to avoid tissue damage. The hot plate test was repeated with naloxone (5 mg/kg s.c.) and 5 g/kg dose. 2.5.2. Formalin test Rats of either sex (n = 10/group) were treated respectively with 1.25, 2.5 and 5 g/kg of MFL, 5 ml/kg of water and 100 mg/kg of aspirin. One hour later, these rats were injected with 0.05 ml of 2.5% formaldehyde in normal saline into the foot pad of one of the hind paw (Dubuisson and Dennis, 1977), immediately placed in a transparent plastic cage separately and the licking time and frequency of the injected paw were recorded from the rst 0 to 5 and 20 to 25 min (Hunskaar et al., 1985). 2.6. Mechanisms of anti-inammatory and analgesic activities 2.6.1. Prostaglandin (PG) synthesis inhibition The experiment was carried out according to Lindsey et al. (1999) and Dharmasiri et al. (2003). One centimetre portions of isolated dioestrous rat uteri were suspended in a 50 ml organ bath containing KrebsHenseleit solution (pH 7.4), maintained at 37 C and aerated with 5% CO2 and 95% O2 gas mixture. The spontaneous contractions of the uteri were recorded using an isometric sensor (Star Medicals, Tokyo, Japan) for 10 min and then, the organ bath was treated in triplicate with the extracts so that the nal concentrations of MFL in the organ bath became 100, 200, 300 and 400 g/ml and the contractions were recorded for further 1015 min. Aspirin was used as the positive reference drug. The latent period for the initiation of contractions and the percent reduction of the amplitude and frequency of contractions with respect to the normal contractions was calculated. 2.6.2. Membrane stabilising activity The experiment was done using heat-induced haemolysis of rat erythrocytes in vitro as described by Perez et al. (1995)
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and Dharmasiri et al. (2003). Vials containing 20 l fresh rat blood in 1 ml of phosphate-buffered saline were treated in triplicate with the extract so that the nal concentration of the MFL in the vials became 2.5, 5, 7.5 and 10 mg/ml. Fifteen microlitres of saline was used as the control while aspirin was used as the positive reference drug. The vials were then incubated for 15 min at 37 C followed by 54 C for 25 min, centrifuged and the absorbance of the supernatant was measured at 540 nm spectrophotometrically. The percent inhibition of haemolysis with respect to the control was calculated. 2.6.3. Antioxidant activity The experiment was carried out using thiobarbitiuric acid reactive substances assay as described by Dorman et al. (1995). The vials containing the reagents were treated in triplicate with the extract so that the nal concentrations of the MFL in the vials became 1.25, 1.875, 2.5 and 3.125 mg/ml. A 100 g/ml of butylated hydroxytoluene (BHT) was used as the positive reference and distilled water was used in the control. The vials were mixed well and incubated at 95 C for 60 min, allowed to cool, 5 ml of butanol was added, mixed well and centrifuged at 1500 g. The absorbance of the butanol layer was measured at 532 nm and the antioxidant index was calculated as follows: Antioxidant index = 1 T C 100
strength) followed by the bridge test (to evaluate muscle co-ordination) and the latency to fall and slide off was determined, respectively (Plaznic et al., 1993). 2.7. Toxicity Male rats (n = 6/group) were treated either with 5 g/kg/day of MFL or 5 ml/kg/day of water for 14 consecutive days. After the treatment, these rats were continuously observed for 1 h for overt clinical signs of acute toxicity or stress. They were daily observed for overt signs of toxicity or stress during the period of treatment. The rats were weighed using an animal balance (MP 6000, Chyo Balance Corporation, Tokyo, Japan), prior to the start of the experiment and on day 1 of the post-treatment. On day 1 of the post-treatment, blood was taken out from the tail under mild ether anaesthesia, serum separated out. The serum activities of aspartate and alanine transaminases, serum concentrations of glucose, urea and creatinine were determined using Randox assay kits. Then, the rats were killed, stomachs were taken out, opened and examined for macroscopic haemorrhagic gastric lesions. 2.8. Analysis of data Data are given as meansS.E.M. Statistical analyses were done by using Students t-test, linear regression analysis and Pearsons correlation analysis, one-way ANOVA followed by Tukeys Family Error Rate test. P = 0.05 was considered as signicant.
where T is the absorbance of test and C absorbance of control. 2.6.4. Antihistamine activity Rats of either sex (n = 9/group) of which fur on posterior lateral side have been shaved 24 h earlier were treated with 1.25, 2.5 and 5 g/kg of MFL, 0.67 mg/kg of chlorpheniramine and 5 ml/kg of water, respectively. After 1 h, these rats were subcutaneously injected with 0.05 ml of 200 g/ml histamine dihydrochloride into the fur removed area of the skin (Spector, 1956) under mild ether anaesthesia and the area of the wheal formed after 1.5 min was calculated. 2.6.5. Sedative activity Rats of either sex (n = 9/group) were treated either with 5 g/kg of MFL or 5 ml/kg of water. After 1 h, all these rats were tested on rat hole-board apparatus to determine sedation as described by File and Wardill (1975). The rats were individually placed in the centre of rat hole-board apparatus and the number of rears, number of head dips, locomotory activity and the number of faecal boluses produced were recorded for 7.5 min. The time spent per head dip was then calculated. 2.6.6. Muscle strength and co-ordination Rats of either sex (n = 9/group) were treated either with 5 g/kg of MFL or water. One hour later, these rats were subjected to bar holding test (to evaluate muscle
3. Results 3.1. Anti-inammatory activity 3.1.1. Carrageenan-induced paw oedema test When compared with the control, treatment with MFL signicantly (P < 0.05) and dose-dependently reduced the paw oedema only at the 2 h after carrageenan injection while there was no signicant suppression at 4 h. There was no signicant difference between the paw oedema of rats treated with MFL at preowering and owering stages of the tree (data not shown). However, 5 mg/kg indomethacin signicantly suppressed paw oedema at both 2 and 4 h (Table 1). The suppression of paw oedema by MFL was inversely dose related (r 2 = 1, P < 0.01). The EC50 for the suppression of paw oedema by MFL was 2 g/kg. 3.1.2. Formaldehyde-induced paw oedema test The treatment with 2.5 and 5 g/kg/day of MFL for 7 days signicantly (P < 0.05) reduced the paw oedema from days 4 to 6 when compared with the control. By day 7 of the treatment, the paw oedema in control and treated groups had almost disappeared. However, the treatment with 1.25 g/kg/day did not signicantly suppressed the
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Table 1 Effect of oral treatment of Vitex negundo mature fresh leaves (MFL) on the carrageenan-induced paw oedema of rats Dose Paw oedema (ml) 2h 5 ml/kg water 1.25 g/kg MFL 2.5 g/kg MFL 5 g/kg MFL 5 mg/kg indomethacin 0.52 0.23 0.28 0.38 0.11 0.05 0.03 0.04 0.03 0.02 4h 0.49 0.45 0.49 0.43 0.28 0.02 0.04 0.06 0.04 0.03
trol. The frequency of licking was signicantly (P < 0.05) reduced only at the late phase. However, aspirin signicantly (P < 0.05) reduced the licking time and frequency at both the phases of the test. The licking time was signicantly (P < 0.05) higher at the early phase than the late phase in both MFL- and aspirin-treated rats (Table 4). 3.3. Mechanisms of the anti-inammatory and analgesic activities 3.3.1. PG synthesis inhibition activity The treatment with MFL does-dependently reduced the amplitude and frequency of spontaneous contractions of isolated dioestrous rat uterus indicating a PG synthesis inhibition (Table 5). The percent inhibition was inversely proportional to the concentration (for amplitude, r = 0.98, P < 0.05; for frequency, r2 = 0.88, P < 0.05). For aspirin, percent inhibition of amplitude (r = 0.96, P < 0.05) and frequency (r = 0.98, P < 0.05) was directly proportional to the concentration. The EC50 for the inhibition of amplitude: MFL versus aspirin: 228.1 g/ml versus 3.0 g/ml and the frequency: MFL versus aspirin: 257.7 g/ml versus 3.6 g/ml. The latent period for the initiation of the inhibition: MFL versus aspirin: 228.0 30.6 s versus 202.8 33.0 s. 3.3.2. Membrane stabilising activity MFL dose-dependently inhibited the heat-induced haemolysis of rat erythrocytes in vitro indicating a membrane stabilising activity as seen with aspirin (Table 6). However, the activity was inversely related to the concentration (r 2 = 0.9, P < 0.05) with MFL while with aspirin it was directly related to the concentration. EC50 for the membrane stabilising activity: MFL versus aspirin: 2.6 mg/ml versus 44.4 g/ml. 3.3.3. Antioxidant activity MFL showed a dose-dependent (r2 = 0.68) antioxidant activity as indicated by the antioxidant index. However, BHT showed a higher antioxidant index than MFL (Table 7). The doseresponse relationship was curvilinear (r 2 = 0.68) and the EC50 for the antioxidant activity was 2.3 mg/ml MFL.
Values are means S.E.M. (n = 9). P < 0.05 as compared with the control (Students t-test). P < 0.01 as compared with the control (Students t-test).
paw oedema (Table 2). The paw oedema suppression showed a bell-shaped doseresponse relationship. 3.2. Analgesic activity 3.2.1. Hot plate and tail ick tests As compared with the controls, the treatment with MFL produced a signicant and dose-dependent (r = 0.92, P < 0.05) increase in the reaction time of rats at 1 h after treatment in the hot plate test showing an analgesic effect. A signicant difference could not be observed between the reaction times of rats treated with MFL at preowering and owering stages of the tree (data not shown). The increase of the reaction time was signicant only with the 2 and 5 g/kg doses. The EC50 for the increase in reaction time was 4.1 g/kg. However, aspirin produced a signicant increase in the reaction time at 1 and 3 h after treatment. There was no signicant increase in the reaction time with 1.25 g/kg in the hot plate test and any of the doses in the tail ick test (Table 3). The treatment with 5 mg/kg of naloxone did not signicantly reduce the reaction time of MFL-treated rats excluding an opioid receptor-mediated action (reaction time: MFL + naloxone versus MFL + saline: 12.8 1.4 s versus 13.6 0.9 s). 3.2.2. Formalin test The treatment with MFL signicantly (P < 0.05) reduced the licking time of the formalin-injected paw both in the early and late phases of the test as compared with the con-
Table 2 Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the formaldehyde-induced paw oedema of rats Dose Paw oedema (ml) Day 1 5 ml/kg/day water 1.25 g/kg/day MFL 2.5 g/kg/day MFL 5.0 g/kg/day MFL 0.26 0.31 0.41 0.46 0.03 0.03 0.03 0.04 Day 2 0.24 0.35 0.21 0.22 0.05 0.04 0.04 0.04 Day 3 0.22 0.15 0.25 0.32 0.05 0.05 0.04 0.04 Day 4 0.46 0.47 0.26 0.31 0.05 0.08 0.03 0.05 Day 5 0.39 0.36 0.18 0.24 0.02 0.03 0.02 0.05 Day 6 0.31 0.29 0.13 0.18 0.05 0.04 0.02 0.04 Day 7 0.16 0.09 0.11 0.11 0.03 0.04 0.03 0.03
M.G. Dharmasiri et al. / Journal of Ethnopharmacology 87 (2003) 199206 Table 3 Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the reaction time of rats Reaction time(s) Hot plate Pre-treatment 5 ml/kg water 1.25 g/kg MFL 2.5 g/k MFL 5 g/kg MFL 100 mg/kg aspirin 11.8 12.2 10.9 12.2 9.5 0.8 1.0 0.5 0.6 1.2 1h 11.1 9.1 15.2 18.8 15.3 0.7 0.8 1.5 1.6 1.7 3h 12.3 11.1 11.3 13.3 16.4 0.9 0.8 2.4 1.6 1.4 Tail ick Pre-treatment 1.5 1.7 1.8 1.5 1.5 0.2 0.2 0.1 0.2 0.1 1h 1.8 1.4 1.9 1.2 1.3 0.2 0.8 0.2 0.1 0.1 3h 1.6 1.8 1.7 1.3 1.1
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0.1 0.2 0.1 0.1 0.1
Table 4 Effect of oral treatment of mature fresh leaves (MFL) of Vitex negundo on the licking time and frequency of rats in the formalin test Dose Early (rst) phase (05 min) Licking time (s) 5 ml/kg water 1.25 g/kg MFL 2.5 g/kg MFL 5 g/kg MFL 100 mg/kg aspirin 59.3 37.9 43.0 39.4 20.8 5.3 4.5 6.1 6.0 3.0 Licking frequency (min1 ) 12.4 8.4 12.0 8.8 7.0 1.4 0.8 1.4 1.0 1.5 Late (second) phase (2025 min) Licking time (s) 48.8 21.9 14.8 14.2 8.3 7.4 5.1 5.2 4.1 2.2 Licking frequency (min1 ) 10.1 6.0 9.0 4.1 4.9 1.2 1.9 4.0 0.9 1.5
3.3.4. Antihistamine activity As compared with the control treatment with MFL significantly (P < 0.01) and dose-dependently reduced the area of the wheal formed on the rat skin by the injection of histamine indicating an antihistamine activity. A 0.67 mg/kg of chlorpheniramine also signicantly (P < 0.01) reduced the area of the wheal (Fig. 1). The antihistamine activity of MFL is inversely dose related (r = 0.98, P < 0.05). EC50 for the antihistamine activity was 0.99 g/kg of MFL.
Table 5 Prostaglandin synthesis inhibition activity of different concentrations of mature fresh leaves (MFL) of Vitex negundo and aspirin as indicated by the reduction of spontaneous contractions of isolated rat uterus at dioestrus with respect to normal contraction Concentration (g/ml) 100 MFL 200 MFL 300 MFL 400 MFL 2 aspirin 4 aspirin 6 aspirin % reduction of amplitude 71.3 57.5 51.1 37.2 36.9 65.4 79.7 3.1 6.8 15.1 6.5 3.5 9.9 5.7 % reduction of frequency 70.1 50.9 48.9 33.8 19.4 50.8 70.8 9.0 6.3 7.6 10.0 4.0 8.0 4.2
3.3.5. Sedative activity The treatment with MFL failed to signicantly alter the parameters of rat hole-board test compared to control (data not shown), showing that there is no sedative action in MFL. 3.3.6. Muscle relaxation and co-ordination When compared with the control, the treatment with MFL failed to signicantly alter the latency to fall in the bar holding test (control versus treatment: 60.0 0.1 versus
Table 6 Membrane stabilising effect of different concentrations of Vitex negundo mature fresh leaves (MFL) and aspirin as indicated by the inhibition of heat-induced haemolysis of rat erythrocytes in vitro Concentration 2.5 mg/ml MFL 5 mg/ml MFL 7.5 mg/ml MFL 10 mg/ml MFL 5 g/ml aspirin 10 g/ml aspirin 15 g/ml aspirin 20 g/ml aspirin % inhibition 31.2 26.8 16.5 6.6 17.6 20.9 25.9 31.9 0.9 2.8 2.5 2.6 1.6 1.8 0.5 0.6
Values are means S.E.M. (n = 3/concentration). There was a signicant relationship (P < 0.05) between the concentration and percent reduction (linear regression, Pearsons correlation).
Values are means S.E.M. There was a signicant (P < 0.05) relationship between the concentration and percent inhibition (linear regression analysis).
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Fig. 1. Antihistamine activity of Vitex negundo mature fresh leaves as indicated by the area of the wheal. Data are given as means S.E.M. P < 0.01 as compared with the control (one-way ANOVA, Tukeys Family Error Rate test).
56.9 2.1) or latency slide off in the bridge test (control versus treatment: 58.3 1.7 versus 55.6 2.2). 3.4. Toxicity The treatment with 5 g/kg/day of MFL for 14 days failed to produce any overt clinical signs of toxicity or stress. The treatment also did not signicantly alter the body weights (control versus treatment: 232.0 8 g versus 251.8 5.6 g), serum creatinine (control versus treatment: 1.7 0.3 mg/dl versus 1.1 0.2 mg/dl), urea (control versus treatment: 33.6 3.0 mg/dl versus 38.6 1.2 mg/dl), random glucose (control versus treatment: 136.2 8.0 mg/dl versus 141.5 6.6 mg/dl) and the activity of ALT (control versus treatment: 12.0 2.1 U/l versus 15.0 2.0 U/l). However, the treatment caused a signicant (P < 0.05) increase in serum activity of AST (control versus treatment: 24.3 5.1 U/l versus 74.7 5.5 U/l). The treatment also did not cause haemorrhagic lesions in the gastric mucosa after 14 days of treatment.
4. Discussion Experimental investigations revealed that the MFL of Vitex negundo have dose-dependent activity against inammation as revealed in the carrageenan and formaldehyde models. Further, hot plate test and the formalin test revealed that MFL can also suppress acute pain. However, the anti-inammatory activity is 1.7 times lower than indomethacin in the carrageenan model while the analgesic activity is 1.2 times lower than aspirin in the formalin test. MFL demonstrated a dose-dependent PG synthesis inhibition, membrane stabilising, antihistamine and antioxidant activities. The inverse doseresponse relationship shown by acute anti-inammatory, antihistamine, PG synthesis inhibition and membrane stabilising activities may be due to reduction of the effectiveness of the active principle at its high concentrations. However, further investigations are needed to conrm this suggestion. This type of relationship indicates that the concentrations used in these tests are within the therapeutic window of MFL in which certain drugs exert their maximum curative effect (Tripathi, 1994). According to Vinegar et al. (1987) and Antonio and Brito (1998) in the carrageenan model, the early phase (12 h) is mainly mediated by histamine, serotonin and the increase of PG synthesis in the surroundings of the damaged tissues while the late phase is mainly mediated by bradykinin, leukotrienes, polymorphonuclear cells and PGs produced in tissue macrophages. In this experiment, the suppression of inammation at the early phase of inammation can be contributed by PG synthesis inhibition and antihistamine activities shown by MFL. The lack of anti-inammatory activity at the second phase may indicate the short duration
Table 7 Antioxidant activity of different concentrations of mature fresh leaves (MFL) of Vitex negundo Concentration (mg/ml) 1.25 MFL 1.875 MFL 2.5 MFL 3.125 MFL 0.1 BHT Values are means S.E.M. Antioxidant index 40.2 38.8 33.7 45.6 57.4 0.1 1.2 0.5 0.1 1.2
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of action of MFL and the increase of the leukotriene at the second phase caused by the inhibition of PG synthesis in the rst phase because inhibition of PG synthesis diverts the reaction towards increase in leukotrienes synthesis (Mayes, 1996). Although opioid receptor-mediated activities can suppress the inammation in the carrageenan-induced paw oedema (Planas et al., 2000), such activity can be excluded here because the naloxone test revealed that the leaves do not act via opioid receptors. In the formaldehyde-induced paw oedema model, the anti-inammatory activity was evident from days 4 to 6 of the treatment, indicating that MFL is effective against the establishment of chronic inammation which happens at the later stage of acute inammation (Hanna and Poste, 1991). This action can be contributed by the PG synthesis inhibition, membrane stabilising and antioxidant activities of MFL as reduced PG synthesis and oxidation and the membrane stabilisation play key roles in countering the establishment of chronic inammation (Perez et al., 1995; Rang et al., 1995). As seen in this experiment, the ability of a drug to suppress inammation when it is applied after the onset of inammation is likely to be due to the genuine anti-inammatory activity of the drug (Duwiejua et al., 1994). These observations provide the support for the use of MFL of Vitex negundo as anti-inammatory agent in the Ayurveda medicine while the strong antihistamine activity conrms the use of MFL to counter skin itching. The analgesic activity shown only in the hot plate test reveals that the activity is supraspinally mediated (Hough et al., 1999) and can be brought about by the PG synthesis inhibition activity of MFL. In the formalin test, the pain in the early phase is caused due to the direct stimulation of the sensory nerve bres by formalin while the pain in the late phase is due to inammatory mediators, like histamine, PGs, serotonin and bradykinin (Murray et al., 1988; Tjolsen et al., 1992). The pain suppression at the early phase by MFL as well as aspirin may be due to a local anaesthetic activity caused by the membrane stabilising activity of them because membrane stabilising agents produce local anaesthesia reducing the sensation of acute pain (Rang et al., 1995). The considerably higher pain suppression at the late phase than the early phase could be due to the concerted action of PG synthesis inhibition, membrane stabilisation and antihistamine activities of MFL. Formalin develops a hyperalgesic state in the late phase of the test (Kaufmann et al., 1997). The pain suppression at the second phase therefore indicates the antihyperalgesic activity of the leaves which is useful in controlling acute pains. NSAIDs when acting supraspinally reduce the pain at both the phases of the formalin test (Martindale et al., 2001) which was also observed in this experiment with MFL as well as with aspirin. Sedatives produce analgesia (Rang et al., 1995). However, MFL did not have sedative action in the rat hole-board test. Therefore, a contribution from sedation to the analgesic activity can be excluded. As naloxone failed to reverse
analgesic activity in the hot plate test, analgesic action operating through opioid receptors can be ruled out. Muscle relaxants can produce false positive results in the hot plate test indicating analgesic activity (Gracioso et al., 1998). However, there was no muscle relaxant activity induced by MFL in the bar holding test. Stress can produce analgesia (Ganong, 1995). There was no sign of stress observed in the rats treated with the MFL. The anti-inammatory and analgesic activities of the leaves did not disappear after the owering of the tree in contrast to Anisomeles indica which lost these activities after owering of the plant (Dharmasiri et al., 2002, 2003). The treatment of MFL for 14 days did not produce detectable toxic effect in terms of body weight, serum concentrations of urea, creatinine, glucose and serum activity of ALT. The treatment did not cause gastric lesions which is a benecial effect compared to the modern NSAIDs. The reason for the increase of serum activity of AST has to be further investigated. Therefore, people should be cautious when the leaves are used in oral preparations. In conclusion, these observations provide evidence for the anti-inammatory and analgesic properties of mature fresh leaves of Vitex negundo claimed in Ayurveda medicine. Also, it uncovered some of the possible mechanisms of these actions. Further studies will be undertaken to correlate the pharmacological activities with the chemical constituents.
Acknowledgements Dr. G.A.S. Premakumara of Industrial Technology Institute, Sri Lanka, is acknowledged for testing the antioxidant activity of leaves.
References
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Hypoglycaemic effect of water extracts of Aegle marmelos fruits in streptozotocin diabetic rats
N. Kamalakkannan, P. Stanely Mainzen Prince
Department of Biochemistry, Annamalai University, Annamalai Nagar, Tamil Nadu 608002, India Received 8 August 2002; received in revised form 12 February 2003; accepted 16 April 2003
Abstract Aegle marmelos Corr. (Rutaceae) is widely used in Indian Ayurvedic medicine for the treatment of diabetes mellitus. The hypoglycaemic effect of the water extract of the fruits of Aegle marmelos was examined in streptozotocin-induced diabetic Wistar rats. Oral administration of the water extract (125 and 250 mg kg1 ) twice a day for 4 weeks resulted in signicant reductions in blood glucose, plasma thiobarbituric acid reactive substances, hydroperoxides, ceruloplasmin and -tocopherol and a signicant elevation in plasma reduced glutathione and Vitamin C in diabetic rats. The effect of the extract at a dose of 250 mg kg1 was more effective than glibenclamide in restoring the values of these parameters. The results of this study clearly shows the hypoglycaemic activity of the fruit extract. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Aegle marmelos; Streptozotocin diabetes; Fruit extract; Plasma lipid peroxides; Antioxidants
1. Introduction Diabetes mellitus is a metabolic disease as old as mankind and its incidence is considered to be high (45%) all over the world (Pickup and Williams, 1997). Active oxygen metabolism plays a key role in the normal functioning of the -cells of pancreas. Free oxygen radicals and oxidative stress appears to be an important element of the production of secondary complications in diabetes (Thomson and McNeil, 1993; Thornalley et al., 1996). Hyperglycaemia generates reactive oxygen species which in turn cause lipid peroxidation and membrane damage (Hunt et al., 1988). Elevated levels of circulating lipid peroxides have been registered in diabetic patients and experimental diabetic animals (Srinivasan et al., 1997; Stanely Mainzen Prince and Menon, 1998). Circulating lipid peroxides were shown to be capable of initiating atherosclerosis which is a well known late feature of diabetes (Hessler et al., 1983). Many indigenous drugs have been used by practitioners of Ayurvedic system for the treatment of diabetes mellitus in India. Different parts of Aegle marmelos Corr. (Rutaceae) (leaves and ripe fruits) have been used in the treatment
of diarrhoeas, dysenteries and diabetes mellitus (Chopra et al., 1958; Saheb Dass, 1969). Preliminary reports indicate Aegle marmelos leaf extract exhibits antidiabetic action in glucose-induced hyperglycaemic rats (Sachdewa et al., 2001) and in alloxanized diabetic rats (Ponnachan et al., 1993a,b). Karunanayake et al. (1984) have reported that aqueous decoction of root bark exhibits hypoglycaemic action in rats. There are no available reports on the pharmacological actions of Aegle marmelos fruit extract. This investigation has been carried out to see, whether the aqueous extract of Aegle marmelos fruits (AMFEt) exert any effect on blood glucose, plasma lipid peroxides and non-enzymatic antioxidants in streptozotocin (STZ)-induced diabetic rats. The effect of the extract was compared with glibenclamide, a well-known hypoglycaemic drug.
2. Materials and methods 2.1. Plant extract An aqueous Aegle marmelos fruit extract (brown dry powder) was received as a gift from Chemiloids (manufacturers and exporters of herbal extracts), Vijayawada, Andhra Pradesh (India). Herb-to-product ratio is 8:1. The extract was suspended in distilled water prior to use.
Corresponding
author. Tel.: +91-4144-238343. E-mail address: psmprince@rediffmail.com (P.S.M. Prince).
0378-8741/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00148-X
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2.2. Drugs and chemicals All the drugs and biochemicals used in this experiment were purchased from Sigma, St. Louis, MO, USA. All other chemicals were of analytical grade. 2.3. Induction of diabetes in rats Female albino Wistar rats of body weight 160190 g procured from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University were used in the present study. The rats were fed on a standard pellet diet (Kamadhenu Agencies, Bangalore, India) and water was freely available. Diabetes was induced in rats by a single intraperitoneal injection of freshly prepared streptozotocin (45 mg kg1 body weight) in 0.1 M citrate buffer (pH = 4.5) in a volume of 1 ml kg1 (Siddique et al., 1987). Forty-eight hours after STZ administration, blood glucose level of each rat was determined. Rats with a blood glucose range of 250300 mg/ 100 ml were considered diabetic and included in the study. 2.4. Experimental design In the experiment, a total of 30 rats (6 normal; 24 STZ diabetic surviving rats) were used. The rats were divided into ve groups of six rats each. Group 1: Group 2: Groups 3 and 4: Normal untreated rats. STZ-treated diabetic rats. STZ-treated diabetic rats administered AMFEt orally (125 and 250 mg kg1 body weight) in distilled water using an intragastric tube twice a day for 4 weeks. STZ-treated diabetic rats given glibenclamide orally (300 g kg1 body weight) in distilled water using an intragastric tube twice a day for 4 weeks.
et al. (1973), respectively. Plasma ceruloplasmin and -tocopherol were determined according to the procedures of Ravin (1961) and Desai (1984), respectively. 2.7. Statistical analysis Statistical analysis was performed using one way analysis of variance (ANOVA) followed by Duncans Multiple Range Test (DMRT). Results were expressed as mean S.D. from six rats in each group. P-values <0.05 were considered signicant. 3. Results Glucose levels measured in blood of normal and experimental rats are given in Table 1. STZ-treated diabetic rats showed signicantly increased levels of blood glucose. Aqueous Aegle marmelos extract treatment showed a signicant decrease in blood glucose in diabetic rats. The levels of lipid peroxides, thiobarbituric acid reactive substances and hydroperoxides in plasma in normal and experimental animals are depicted in Table 2. Diabetic animals showed signicantly increased levels of lipid peroxides and hydroperoxides. Aqueous Aegle marmelos extract treatment resulted in signicantly lower levels of lipid peroxides and hydroperoxides in diabetic rats. Vitamin C and reduced glutathione levels measured in plasma of normal and experimental animals are shown in Table 3. Diabetic rats had signicantly lower levels of plasma Vitamin C and reduced glutathione. Aqueous Aegle
Table 1 Effect of Aegle marmelos extract on blood glucose in diabetic rats Group Blood glucose (mg/dl) Initial Normal Streptozotocin-treated Streptozotocin + extract (125 mg) Streptozotocin + extract (250 mg) Streptozotocin + glibenclamide (300 g) 83.7 268.1 260.6 262.3 270.6 5.4 15.2 17.9 20.1 19.3 Final 87.6 309.8 198.2 98.7 129.0 6.2a 31.5b 19.9c 8.3a 8.2d
Group 5:
After 4 weeks of treatment, all the rats were decapitated after fasting overnight. Blood was collected in potassium oxalate and sodium uoride containing tubes for the estimation of fasting blood glucose. Plasma was separated for the estimation of thiobarbituric acid reactive substances (TBARS), hydroperoxides (HP), Vitamin C, reduced glutathione (GSH), ceruloplasmin and -tocopherol. 2.5. Estimation of blood glucose and lipid peroxides Fasting blood glucose was estimated by the method of Sasaki et al. (1972). Plasma thiobarbituric acid reactive substances and hydroperoxides were determined by the methods of Yagi (1976) and Jiang et al. (1992), respectively. 2.6. Determination of non-enzymatic antioxidants Plasma Vitamin C and reduced glutathione were estimated by the methods of Omaye et al. (1979) and Rotruck
Each value is the mean S.D. of six rats in each group. Values not sharing a common superscript differ signicantly at P < 0.05 (DMRT). Table 2 Effect of Aegle marmelos extract on plasma TBARS and HP in diabetic rats Group Normal Streptozotocin-treated Streptozotocin + extract (125 mg) Streptozotocin + extract (250 mg) Streptozotocin + glibenclamide (300 g) TBARS (nM/ml) 2.2 3.9 3.2 2.7 3.0 0.2a 0.3b 0.3c 0.3d 0.2c,d HP (values 105 mM/dl) 7.2 11.3 9.3 7.5 8.1 0.3a 0.7b 0.9c 0.5a,d 0.5d
Each value is the mean S.D. of six rats in each group. Values not sharing a common superscript differ signicantly at P < 0.05 (DMRT).
N. Kamalakkannan, P.S.M. Prince / Journal of Ethnopharmacology 87 (2003) 207210 Table 3 Effect of Aegle marmelos extract on plasma Vitamin C and GSH in diabetic rats Group Normal Streptozotocin-treated Streptozotocin + extract (125 mg) Streptozotocin + extract (250 mg) Streptozotocin + glibenclamide (300 g) Vitamin C (mg/dl) 1.5 0.9 1.0 1.2 1.1 0.1a 0.1b 0.0b,c 0.1d 0.1c GSH (mg/dl) 23.2 14.7 19.4 22.8 21.4 1.8a 1.1b 1.8c 1.6a 1.5a,c
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Each value is the mean S.D. of six rats in each group. Values not sharing a common superscript differ signicantly at P < 0.05 (DMRT). Table 4 Effect of Aegle marmelos extract on plasma ceruloplasmin and -tocopherol in diabetic rats Group Normal Streptozotocin-treated Streptozotocin + extract (125 mg) Streptozotocin + extract (250 mg) Streptozotocin + glibenclamide (300 g) Ceruloplasmin (mg/dl) 20.7 36.3 27.8 22.0 24.9 2.9a 2.8b 1.9c 1.7a,d 1.9c,d -Tocopherol (mg/dl) 1.6 3.6 2.4 1.9 2.1 0.1a 0.3b 0.2c 0.0d 0.1e
Each value is the mean S.D. of six rats in each group. Values not sharing a common superscript differ signicantly at P < 0.05 (DMRT).
marmelos extract treatment resulted in signicant increase in plasma Vitamin C and reduced glutathione in diabetic rats. The levels of plasma ceruloplasmin and -tocopherol in normal and experimental animals are illustrated in Table 4. Diabetic rats had signicantly increased levels of ceruloplasmin and -tocopherol. Aqueous Aegle marmelos extract treatment resulted in signicant reduction in plasma ceruloplasmin and -tocopherol in diabetic rats. For all the parameters studied, Aegle marmelos at a dose of 125 mg kg1 showed a signicant effect and 250 mg kg1 showed a highly signicant effect and brought back all the parameters to near normal levels. The effect at a dose of 250 mg kg1 was better than that of glibenclamide.
4. Discussion Oral administration of an aqueous extract of Aegle marmelos fruit has shown hypoglycaemic effect against streptozotocin-induced diabetes in rats. The extract lowered signicantly the levels of blood glucose, plasma thiobarbituric acid reactive substances, hydroperoxides, ceruloplasmin and -tocopherol and signicantly increased the levels of plasma GSH and Vitamin C. To our knowledge, this is the rst study in which a follow up was made on the effect of Aegle marmelos extract in the levels of glucose, lipid peroxides and non-enzymatic antioxidants in diabetic animals. In our study, we observed elevated levels of blood glucose in streptozotocin diabetic rats. Ad-
ministration of Aegle marmelos fruit extract decreases blood glucose in diabetic rats. This shows the hypoglycaemic effect of the fruit extract. Phytochemical studies have shown the presence of coumarins such as marmelosin, alloimperatorin, -sitosterol and d--phellandrene in Aegle marmelos fruits (Chopra et al., 1958). Shani et al. (1974) have reported that coumarins exhibit hypoglycaemic action in diabetic rats. The hypoglycaemic effect may be due to the presence of coumarins in the fruit extract. We also observed elevated levels of thiobarbituric acid reactive substances and hydroperoxides in plasma of STZ-induced diabetes in rats. Elevated levels of peroxides observed in plasma are a consequence of increased production and liberation of lipid peroxides into the circulation due to pathological changes (Stanely Mainzen Prince and Menon, 1998). Similar ndings were reported for STZ-induced diabetes in rats (Kinalski et al., 2000). Alterations in the non-enzymatic antioxidants were observed in diabetic animals. The plasma protein, ceruloplasmin is a powerful non-enzymatic antioxidant that inhibits lipid peroxidation by binding to copper (Halliwell and Gutteridge, 1990). Dormandy (1980) has shown that ceruloplasmin levels increase under conditions leading to the generation of superoxide radicals and hydrogen peroxide. The observed increase in plasma ceruloplasmin in diabetic animals may be due to elevated lipid peroxides. Analogous ndings were observed in experimental diabetes (Stanely Mainzen Prince and Menon, 1998). -Tocopherol is the most important lipid soluble, radical scavenging antioxidant. It reduces lipid hydroperoxides generated during the process of peroxidation and protects the cell structures against damage (Kinalski et al., 2000). The elevated levels of -tocopherol noted in diabetic rats play a protective role against increased peroxidation in diabetes (Stanely Mainzen Prince and Menon, 1998). Similar results have been observed by other workers for STZ-induced diabetes in rats (Asayama et al., 1994). Glutathione protects the tissues from free radical damage. It inhibits free radical-mediated lipid peroxidation (Meistor and Anderson, 1983). We observed a decrease in the levels of GSH in plasma in diabetic rats. It appears that generation of oxygen radicals by increased levels of glucose causes utilization of GSH and thus diminishes GSH levels in the plasma in diabetes (Stanely Mainzen Prince and Menon, 1998). In this context, Sajithlal et al. (1998) also reported diminished levels of serum GSH in experimental diabetic rats. Vitamin C is an excellent hydrophilic antioxidant in plasma, because it disappears faster than other antioxidants when plasma is exposed to reactive oxygen species (Frei et al., 1986). The observed decrease in plasma Vitamin C may be due to increased utilization as an antioxidant defence against increased reactive oxygen species or due to a decrease in GSH level, since GSH is required for the recycling of Vitamin C (Inefers and Sies, 1988). Similar observations were reported in STZ-induced diabetes (Sajithlal et al., 1998).
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N. Kamalakkannan, P.S.M. Prince / Journal of Ethnopharmacology 87 (2003) 207210 Pickup, J.C., Williams, G., 1997. Epidemiology of diabetes mellitus. In: Text Book of Diabetes, 2nd ed., Vols. 12. Blackwell, Oxford, pp. 3.13.28. Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993a. Hypoglycaemic effect of alkaloid preparation from leaves of Aegle marmelos. Amala Research Bulletin 13, 3741. Ponnachan, P.T.C., Paulose, C.S., Panikkar, K.R., 1993b. Effect of leaf extract of Aegle marmelos in diabetic rats. Indian Journal of Experimental Biology 31, 345347. Ravin, H.A., 1961. An improved colorimetric enzymatic assay of ceruloplasmin. Journal of Laboratory and Clinical Medicine 589, 161 168. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swason, A.B., 1973. Selenium: biochemical role as a component of glutathione peroxidase. Science 179, 588590. Sachdewa, A., Raina, D., Srivatsava, A., Khemani, L.D., 2001. Effect of Aegle marmelos and Hibiscus rosa sinensis leaf extract on glucose tolerance in glucose induced hyperglycemic rats (Charles foster). Journal of Environmental Biology 22, 5357. Saheb Dass, G., 1969. Aegle marmelos. In: Pt. Krishna Prasad Trivedi (Ed.), Dhanvantari. Dhanvantari Karyalaya, Aligarh, India, p. 204. Sajithlal, G.B., Chithra, P., Chandrakasan, G., 1998. Effect of curcumin on the advanced glycation and cross-linking of collagen in diabetic rats. Biochemistry and Pharmacology 56, 16071614. Sasaki, T., Matsy, S., Sonae, A., 1972. Effect of acetic acid concentration on the colour reaction in the o-toluidine-boric acid method for blood glucose estimation. Rinsho Kagaku 1, 346353. Shani, J., Goldschmied, A., Joseph, B., Ahronson, Z., Sulman, F.G., 1974. Hypoglycaemic effect of Trigonella foenum graecum and Lupinus terminis (Leguminosae) seeds and their major alkaloids in alloxan diabetic and normal rats. Archives internationals de Pharmacodynamic et de Therapie 210, 2731. Siddique, O., Sun, Y., Lin, J.C., Chein, Y.W., 1987. Facilitated transdermal transport of insulin. Journal of Pharmacological Science 76, 341 345. Srinivasan, K.N., Pugalendi, K.V., Sambandam, G., Ramakrishna Rao, M., Menon, V.P., 1997. Diabetes mellitus, lipid peroxidation and antioxidant status in rural patients. Clinica Chimica Acta 259, 183 186. Stanely Mainzen Prince, P., Menon, V.P., 1998. Effect of Syzigium cumini in plasma antioxidants on alloxan-induced diabetes in rats. Journal of Clinical Biochemistry and Nutrition 25, 8186. Thomson, K.H., McNeil, J.H., 1993. Effect of vanadyl sulfate feeding on susceptibility to peroxidative changes in diabetic rats. Research Communications in Chemical Pathology and Pharmacology 80, 180 200. Thornalley, P.J., McLellan, A.C., Lo, T.W., Bern, J., Sonksen, P.H., 1996. Negative association between reduced glutathione concentration and diabetic complications. Medical Science 91, 575582. Yagi, K., 1976. A simple uorometric assay for lipid peroxide in blood plasma. Biochemical Medicine 15, 212216.
In conclusion, aqueous extract of Aegle marmelos fruit exhibited hypoglycaemic effect in streptozotocin-induced diabetes in rats. The effect of the extract on plasma antioxidants is due to reduction in blood glucose concentration in diabetic rats. The extract at a dose of 250 mg kg1 was more effective than glibenclamide. Further studies are necessary to determine the exact nature of the active principle and the mechanism of action of the fruit extract. References
Asayama, K., Nakanae, T., Uchida, W., Hayashibe, K., Dobashi, K., Nakazawa, S., 1994. Serum antioxidant status in streptozotocin-induced diabetic rat. Hormone and Metabolic Research 26, 313315. Chopra, R.N., Chopra, I.C., Handa, K.L., Kapur, L.D., 1958. In: Dhar, V.N., Sons, V.N. (Eds.), Indigenous Drugs of India. Calcutta, India, pp. 267270. Desai, I.D., 1984. Vitamin E analysis methods for animal tissue. Methods in Enzymology 105, 138142. Dormandy, T.L., 1980. Free radical reactions in biological systems. Annals of the Royal College of Surgeons of England 62, 188194. Frei, B., England, L., Ames, B.N., 1986. Ascorbate is an outstanding antioxidant in human blood plasma. Proceedings of National Academic Science United States of America 86, 63776381. Halliwell, B., Gutteridge, J.M.C., 1990. The antioxidant of human extracellular uids. Archives of Biochemistry and Biophysics 280, 18. Hessler, J.R., Morel, D.W., Lewis, L.J., Chrisolm, L.W., 1983. Lipoprotein oxidation and lipoprotein induced toxicity. Arteriosclerosis 3, 215. Hunt, J.V., Dean, R.T., Wolff, S.P., 1988. Hydroxyl radical production and autoxidative glycosylation. Glucose autoxidation as the cause of protein damage in the experimental glycation model of diabetes and aging. Biochemistry Journal 256, 205212. Inefers, H., Sies, H., 1988. The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E. European Journal of Biochemistry 174, 353357. Jiang, Z.Y., Hunt, J.V., Wolff, S.P., 1992. Ferrous ion oxidation in the presence of Xylenol orange for detection of lipid hydroperoxide in low density lipoprotein. Annals of Biochemistry 202, 384387. Karunanayake, E.H., Welihinda, J., Sirimanne, S.R., Sinnadorai, G., 1984. Oral hypoglycaemic activity of some medicinal plants of Sri Lanka. Journal of Ethnopharmacology 11, 223231. Kinalski, M., Sledziewski, A., Telejko, B., Zarzycki, W., Kinalska, I., 2000. Lipid peroxidation and scavenging enzyme activity in streptozotocin induced diabetes. Acta Diabetologia 37, 179183. Meistor, A., Anderson, M.E., 1983. Glutathione. Annual Reviews in Biochemistry 52, 711760. Omaye, S.T., Turnabull, J.C., Sanberlick, H.E., 1979. Selected methods for the determination of ascorbic acid in animal cells, tissues and uids. Methods in Enzymology 62, 311.
Potential risk of acute hepatotoxicity of kodo poisoning due to exposure to cyclopiazonic acid
Mary Antony a , Yogeshwar Shukla b , K.K. Janardhanan c,
a
National Environmental Engineering Research Institute (NEERI), CSIR Complex South Kalamassery, Cochin 693 109, India b Industrial Toxicology Research Centre, M.G. Marg, Lucknow 226 001, India c Amala Cancer Research Centre, Amala Nagar, Trichur 680 553, India Received 19 August 2002; received in revised form 9 April 2003; accepted 16 April 2003
Abstract Kodo millet (Paspalum scrobiculatum L.) is a staple food of some sections of people of North India. Consumption of Kodo millet is often found to cause intoxication and poisoning. The grains are frequently infested with Aspergillus tamarii Kita, which produced substantial amount of a mycotoxin, cyclopiazonic acid (CPA). Investigations were carried out to evaluate the hepatotoxic/preneoplastic changes in rat liver following single and multiple dose administration of CPA. Results showed a marked increase in the activity of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) following CPA exposures, suggesting acute hepatotoxicity. Signicant increase was also observed in gamma glutamyl transpeptidase (GGT) activity following CPA exposures, indicating preneoplastic changes in the liver. The results reveal that Kodo poisoning might cause acute hepatotoxicity in men and animals. The ndings thus suggest that the consumption of contaminated Kodo millet is a serious health hazard due to exposure to CPA produced by Aspergillus tamarii associated with the millet. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Kodo millet; Aspergillus tamarii; Cyclopiazonic acid; Hepatotoxic; Hepatocarcinogenic
1. Introduction Kodo millet (Paspalum scrobiculatum L.) is a staple food of some sections of people in parts of North India. However, Kodo poisoning is a toxic syndrome encountered in men and animals in areas where this millet is grown and consumed. The consumption of Kodo millet is often reported to cause intoxication and poisoning. The millet is reported to be toxic to animals when consumed (Bazlur, 1960). The symptoms of Kodo poisoning in the affected people are characterized by nausea, vomiting, delirium, depression, intoxication, and unconsciousness. Kodo millet is often found heavily infested with Aspergillus tamarii Kita. Janardhanan et al. (1984) isolated fumigaclavin A from Aspergillus tamarii associated with Kodo millet and suggested that the compound might be responsible for part of Kodo poisoning symptoms. The fungus was also reported to produce signicant amount of a mycotoxin, cyclopiazonic acid (CPA; Fig. 1) (Dorner, 1983; Rao and Husain, 1985). CPA has been reported to be isolated from a number of fungi namely Penicillium cyclopium, Penicillium camembertii, Aspergillus versicolor,
Corresponding
Aspergillus oryzae, Aspergillus avus (Holzapfel, 1968; Ohmomo et al., 1973; Orth, 1977; Luk et al., 1977; Still et al., 1978). Morrissey et al. (1985) reported that CPA was acutely toxic to rats and produced focal necrosis in many organs. In chickens, CPA exposure caused reduced weight gain and produced proventricular lesions (Dorner et al., 1983). Neuhring et al. (1985) found that main target organs of CPA-induced toxicity in dogs were gastrointestinal tract and kidneys. In this communication, we report the acute hepatotoxicity and preneoplastic changes in rat liver following oral administration of CPA, isolated from Aspergillus tamarii associated with Kodo millet collected from Uttar Pradesh, India, in the month of September. The voucher specimen of Aspergillus tamarii was deposited in CAB International Mycological Institute, UK (IMI-260311).
2. Materials and methods Gamma glutamyl-p-nitroanilide hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO). Glycylglycine and 2,4-dinitrophenyl hydrazine were obtained from BDH, UK. The rest of the chemicals used were of analytical grade.
author. Fax: +91-487-2307020. E-mail address: kkjanardhanan@yahoo.com (K.K. Janardhanan).
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(5 mg in 200 l vegetable oil) once; Group III: vegetable oil (200 l/animal) three times alternate days; Group IV: CPA (5 mg in 200 l vegetable oil/animal) three times alternate days. 2.3. Assay for biochemical parameters
Fig. 1. Structure of CPA.
2.1. Isolation of CPA from Aspergillus tamarii CPA was isolated by the method described by Rao and Husain (1985) with some modications. The fungus was grown in several 1 l Erlenmeyer asks containing 200 ml modied Richards medium (g/l: KNO3 10.0, KH2 PO4 5.0, MgSO4 7H2 O 2.5, FeCl3 0.01, sucrose 50.0, and yeast extract 1.0) for 10 days at 2527 C. After the incubation period, the cultures were harvested, centrifuged at 4000 rpm for 15 min, and mycelial biomass and culture ltrate were separated. The mycelium was washed, dried at 4550 C for 48 h, and powdered. The culture ltrate (10 l) and dried mycelial powder were extracted separately with chloroform. The chloroform extract of culture ltrate and mycelium were pooled and dried over anhydrous Na2 SO4 . The solvent evaporated to dryness under vacuum. The residue, crude toxin (3.5 g), was obtained. The crude toxin was analyzed for the presence of CPA by TLC on silica gel G plates impregnated with 0.4N oxalic acid using chloroform:methylethylketone (80:20) as solvent system. The plates were sprayed with Ehrlich reagent (1 g p-dimethyl benzaldehyde in 30 ml of 50% HCl). CPA was detected as violet-colored spot (Rao and Husain, 1985). The crude toxin was further puried by column chromatography using cellulose powder impregnated with formamide:oxalic acid and then eluted with hexane followed by hexane:benzene mixture (Holzapfel, 1968). The hexane:benzene mixture fractions were combined and the solvent evaporated completely at low temperature. Further purication of the toxin was done by preparatory TLC on silica gel G impregnated with 0.4N oxalic acid using chloroform:methylethylketone (80:20) solvent system (Rao and Husain, 1985). CPA band was removed carefully eluted with chloroformacetone. The fraction was washed several times with water to remove oxalic acid. The solvent was completely evaporated and the residue contained chromatographically pure (99%) CPA (280 mg). 2.2. Treatment schedule Male Wistar rats (100 5 g) maintained under environmentally controlled conditions and standard solid pellet diet were used for the experiments. The CPA (99% pure) was dissolved in vegetable oil and administered at a dose of 50 mg/kg body weight (5 mg in 200 l vegetable oil/animal). The animals were divided in four groups. Group I: untreated control (vegetable oil 200 l/animal) once; Group II: CPA
All animals were sacriced 48 h after the last dose by stunning and their blood was collected by cardiac puncture into clean dry centrifuge tubes. The livers were immediately taken out, cleaned, and weighed. The blood was allowed to clot at 810 C and clear serum was collected after centrifugation. One-fourth of the liver was homogenized (10% w/v) in 0.05 M Tris buffer (pH 7.0) for the estimation of enzyme, gamma glutamyl transpeptidase (GGT), and another part was homogenized in 0.25 M sucrose for the assay of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), and lipid peroxidation. Third portion of the liver was homogenized in 0.15% KCl containing 1 nmol nicotinamide and used for the assay for aryl hydrocarbon hydroxylase (AHH) and aminopyrene-N-demethylase. The fraction used for non-protein thiols (NPSH) was homogenized in mannitolEDTA. GGT activity was assayed according to the method of Roomi and Goldenburg (1981) in liver and in sera according to the method of Naftalin et al. (1969). GOT and GPT were assayed by the method of Wootton (1964) in liver and serum. AHH was estimated by the method of Dehnean et al. (1973) and aminopyrene-N-demethylase was estimated according to Schenkman et al. (1967). Non-protein thiols were estimated according to the method of Sedlak and Lindsay (1968). Lipid peroxidation was measured by the method of Utley et al. (1967) and protein content of the samples was estimated by the method of Lowry et al. (1951). 2.4. Statistical analysis Experimental data were expressed as mean S.D. The Students t-test was applied for detecting the signicance. P < 0.05 was regarded as signicant.
3. Results and discussion Both single and multiple dose administration of CPA had shown some behavioral changes like breathlessness and lethargy, reluctance to move even on provocation. But a few hours after treatment, they became apparently normal. An oral dose of 50 mg/kg body weight CPA has been reported to cause severe liver lesions and cell necrosis in rats (Purchase, 1974). Hence, this dose was selected for the studies. Activity of aminopyrene-N-demethylase was found to be decreased in animals exposed to CPA (Table 1). The induction of aminopyrene-N-demethylase is considered to be related with synthesis of cytochrome P-450 (Pelissier and Albrecht, 1976). A signicant increase in the AHH
M. Antony et al. / Journal of Ethnopharmacology 87 (2003) 211214 Table 1 Effect of CPA on hepatic aryl hydrocarbon hydroxylase (AHH) and aminopyrene-N-demethylase Parameter Group I AHH(nmol 3-OH B (a) P formed/h/mg protein) N-demethylase (nmol formaldehyde formed/min/mg protein) Each value represents mean S.D. of six rats. P < 0.01. P < 0.001. 2.90 0.11 3.90 0.57 II 4.80 3.45 0.48 0.31 III 2.50 0.26 4.40 0.45 IV
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5.60 0.22 2.00 0.25
activity was observed in both single (Group II) and multiple dose (Group IV)-treated animals (Table 1) which indicated that there might be some involvement of polycyclic aromatic hydrocarbon (PAH)-specic modulation of aminopyrene-N-demethylase. The two-fold increase in AHH activity indicated the possibility of CPA to induce only CYP1A1- and CYP1A2-dependent induction, which might convert CPA to its toxic metabolite forms that could bind to the macromolecules inside the cell (Ioannides et al., 1984). Therefore, it may be suggested that the AHH thus induced may activate CPA to its intermediate forms for the expression of their mutagenic/preneoplastic/carcinogenic potential. A signicant increase in the activity of GGT, which is a marker enzyme for the assessment of preneoplastic changes (Hanigan and Pitot, 1985; Periano et al., 1983), was observed in both liver and serum in the animals of the Groups II and IV (Table 2). GGT is known to catalyze the transfer of gamma glutamyl group of compounds containing this group to a wide variety of amino acceptors (Meister, 1973). Ab-
normally high levels of GGT were also observed in tumors of a variety of tissues including hepatocellular carcinomas (Brelsterili, 1979). The parameters examined to evaluate hepatic damage in this study were tissue and serum glutamate oxaloacetate transaminase (GOT and SGOT) and tissue and serum glutamate pyruvate transaminase (GPT and SGPT). Signicant increase in the activity of both the enzymes in tissue and serum of CPA-exposed animals were observed. Induction in the activity of these two enzymes are reported to be associated with generalized hepatotoxicity. The levels of lipid peroxidation in the liver of CPA-exposed animals did not show any appreciable variation. This indicates that free radicals are not involved in CPA-induced hepatotoxicity (Table 3). However, signicant increase in the non-protein thiols was found in animals exposed to CPA (Table 3). The increase in the levels of non-protein thiols may be due to the formation of deoxyribonucleoside diphosphate (dNdp). The formation of dNdp in relatively more efcient manner appears to be of metabolic
Table 2 Effect of CPA on gamma glutamyl transpeptidase (GGT), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) in liver and serum of rats Parameter Group I GGT (nmol p-nitroaniline liberated/min/mg protein) GOT (mol/min/g tissue) GPT (mol/min/g tissue) SGGT (nmol p-nitroaniline liberated/min/mg protein) SGOT (mol/min/g protein) SGPT (mol/min/g protein) Each value represents mean S.D. of six rats. P < 0.05. P < 0.01. P < 0.001. Table 3 Non-protein thiols and lipid peroxidation in liver of rats following exposure to CPA Parameter Group I Non-protein thiols (nmol/mg protein) Lipid peroxidation (nmol malonaldehyde formed/h/mg protein) Each value represents mean S.D. of six animals. P < 0.05. 17.80 0.97 0.478 0.024 II 21.40 1.02 0.56 0.370 III 20.2 1.18 0.475 0.04 IV 34.3 1.62 0.60 0.04 3.89 7.84 39.87 29.42 0.306 3.269 0.35 0.75 5.1 1.42 0.029 0.250 II 6.44 9.24 48.4 35.49 0.459 4.68 0.45 0.82 2.7 1.30 0.017 0.23 III 4.5 6.22 38.91 27.33 0.497 2.89 1.1 0.43 3.20 1.7 0.013 0.17 IV 18.0 16.33 98.53 61.24 0.752 4.949 2.7 1.77 6.30 2.98 0.025 0.15
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M. Antony et al. / Journal of Ethnopharmacology 87 (2003) 211214 Ioannides, C., Lum, P.Y., Parke, D.V., 1984. Cytochrome P 448 and the activation of toxic chemicals and carcinogens. Xenobiotica 14, 119 127. Janardhanan, K.K., Sattar, A., Husain, A., 1984. Production of fumigaclavin A by Aspergillus tamarii Kita. Canadian Journal of Microbiology 30, 247250. Lowry, O.H., Rosenbrough, N.D., Furr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. Journal of Biological Chemistry 193, 265275. Luk, K.C., Kobbe, B., Townsend, J.M., 1977. Production of cyclopiazonic acid by Aspergillus avus Link. Applied and Environmental Microbiology 33, 211212. Meister, A., 1973. On the enzymology of amino acid transport. Science 180, 3339. Morrissey, E.R., Norred, P.W., Cole, J.R., Dorner, J., 1985. Toxicity of mycotoxin, cyclopiazonic acid to SpragueDawley rats. Toxicology and Applied Pharmacology 77, 9497. Naftalin, L., Sexton, M., Whitaker, T., Tracy, D.A., 1969. Routine procedure for estimating serum gamma glutamyl transpeptidase activity. Clinica Chemica Acta 26, 293296. Neuhring, L.P., Rowland, G.N., Harrison, L.R., Cole, R.J., Dorner, J.W., 1985. Cyclopiazonic acid mycotoxicosis in the canine. American Journal of Veterinary Research 46, 16701676. Ohmomo, S.M., Sugita, M., Ahe, H., 1973. Isolation cyclopiazonic acid, cyclopiazonic acid imine and bis secodehydro cyclopiazonic acid from the cultures of Aspergillus versicolor (Vull). Tirahoschi Journal of Agricultural Chemical Society Japan 47, 8389. Orth, R., 1977. Mycotoxins of Aspergillus oryzae strains for use in the food industry as starters and enzyme producing moulds. Annals of Nutrition and Ailment 31, 617624. Pelissier, M.A., Albrecht, R., 1976. Teneur Minmail Du Regime en lindane induisant les monoxygenases microsomalos chez le rats. Food Chemistry and Toxicology 14, 297301. Periano, C., Richards, W.L., Stevens, F.J., 1983. Multistage hepatocarcinogenesis. Environmental Health Perspectives 56, 143. Purchase, I.F.H., 1974. Pencillium cyclopium. In: Purchase, I.F.H. (Ed.), Mycotoxins. Elsevier Scientic Publishing Company, New York, pp. 149162. Rao, B.L., Husain, A., 1985. Presence of cyclopiazonic acid in Kodo millet (Paspalum scrobiculatum) causing Kodua poisoning in man and its production by associated fungi. Mycopatholgia 89, 177180. Roomi, M.W., Goldenburg, D.M., 1981. Comparison of gamma GTP induction by phenobarbital in rats. Biochemical Pharmacology 30, 15631571. Schenkman, J.B., Remmer, H., Estabrook, R.W., 1967. Spectral studies of drug interaction with hepatic microsomal cytochrome. Molecular Pharmacology 3, 113123. Sedlak, J., Lindsay, R.H., 1968. Estimation of total protein bound and non-protein sulfhydryl groups in tissues with Ellmans reagent. Annals of Biochemistry 25, 192205. Still, P.E., Eckardt, C., Leister, L., 1978. Bilding von cyclopiazonsaure durch Penicillium camemberti isolate von Kase. Fleischwirtschaft 58, 876877. Utley, H.G., Bernheim, F., Hochstein, P., 1967. Effects of sulfhydril reagents on peroxidation of microsomes. Archives of Biochemistry and Biophysics 118, 2932. Wootton, I.P., 1964. Micromethods. In: Medicinal Biochemistry, 4th ed. Churchill, London.
importance in cell proliferation subsequent to cellular damage (Holmgren, 1976). The present investigations reveal that single or repeated oral administration of CPA may exert hepatotoxic/ hepatocarcinogenic risk to the exposed population. Kodo millet is often found contaminated with Aspergillus tamarii, which is reported to produce signicant amount of CPA. The extract of the infected millet with the fungus has also been found to produce this mycotoxin (Rao and Husain, 1985). The ndings thus suggest that consumption of contaminated Kodo millet is a serious health hazard to humans because of the risk of exposure to CPA. Although Aspergillus tamarii isolated from kodo millet has been reported to produce an indole alkaloid, fumigalavin A (Janardhanan et al., 1984), no information is available on the involvement of this compound in hepato-toxicity.
Acknowledgements Part of the work was done in Industrial Toxicology Research Centre, Lucknow and the authors are thankful to the Director for the facilities. We also thank Nayana Jose and T.A. Ajith for their help in the preparation of the manuscript.
References
Bazlur, M., 1960. Probable mona grass (Penicillium commersoni) poisoning. Indian Veterinary Journal 37, 3134. Brelsterili, U., 1979. Gamma-glutamyl transpeptidase (GGT): an early marker for hepatocarcinogens in rats. Trends in Pharmaceutical Sciences 1, 4749. Dehnean, W., Torringas, R., Roos, J.A., 1973. Modied method for the assay of benzo (a) pyrene hydroxylase. Annals of Biochemistry 53, 373378. Dorner, J.W., 1983. Production of cyclopiazonic acid by Aspergillus tamarii Kita. Applied and Environmental Microbiology 46, 1435 1437. Dorner, J.W., Cole, R.J., Lomax, L.G., Gosser, H.S., Diener, U.L., 1983. Cyclopiazonic acid production by Aspergillus avus and its effect on broiler chickens. Applied and Environmental Microbiology 46, 697 703. Hanigan, M.H., Pitot, H.C., 1985. Gamma glutamyl transpeptidaseits role in hepatocarcinogenesis. Carcinogenesis 6, 165172. Holmgren, A., 1976. Hydrogen donor system for Escherichia coli ribonucleoside diphosphate reductase dependent upon glutathione. Proceedings of National Academy of Sciences U.S.A. 73, 2275 2279. Holzapfel, C.W., 1968. The isolation and structure of cyclopiazonic acid, a toxic metabolite of Penicillium cyclopium Westling. Tetrahedron 24, 21012119.
Antioxidant and antimicrobial activity of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae)
Ferda Candan a, , Mehmet Unlu b , Bekta s Tepe c , Dimitra Daferera d , d Moschos Polissiou , Atalay Skmen c , H. A skn Akpulat c
a
Department of Chemistry, Faculty of Science and Literature, Cumhuriyet University, Sivas 58140, Turkey b Department of Microbiology, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey c Department of Biology, Faculty of Science and Literature, Cumhuriyet University, Sivas 58140, Turkey Laboratory of General Chemistry, Agricultural University of Athens, Iera Odos 75, Athens 118 55, Greece Received 6 October 2002; received in revised form 14 April 2003; accepted 16 April 2003
Abstract The in vitro antimicrobial and antioxidant activities of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium Afan. (Asteraceae) were investigated. GC-MS analysis of the essential oil resulted in the identication of 36 compounds constituting 90.8% of the total oil. Eucalyptol, camphor, -terpineol, -pinene, and borneol were the principal components comprising 60.7% of the oil. The oil strongly reduced the diphenylpicrylhydrazyl radical (IC50 = 1.56 g/ml) and exhibited hydroxyl radical scavenging effect in the Fe3+ EDTAH2 O2 deoxyribose system (IC50 = 2.7 g/ml). It also inhibited the nonenzymatic lipid peroxidation of rat liver homogenate (IC50 = 13.5 g/ml). The polar phase of the extract showed antioxidant activity. The oil showed antimicrobial activity against Streptococcus pneumoniae, Clostridium perfringens, Candida albicans, Mycobacterium smegmatis, Acinetobacter lwofi and Candida krusei while water-insoluble parts of the methanolic extracts exhibited slight or no activity. This study conrms that the essential oil of Achillea millefolium possesses antioxidant and antimicrobial properties in vitro. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Achillea millefolium; Antioxidant activity; Antimicrobial activity; Methanol extracts; Essential oil; GC-MS
1. Introduction It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. Recently, there is a growing interest in oxygen-containing free radicals in biological systems and their implied roles as causative agents in the aetiology of a variety of chronic disorders. Accordingly, attention is focused on the protective biochemical functions of naturally occurring antioxidants in the cells of the organisms containing them (Larson, 1988; Halliwell, 1997). Antioxidants in the oils are important in the stabilization of free fatty acids (Six, 1994; Baldioli et al., 1996). The antioxidant activity of phenols and other compounds present in oils has been well and widely studied by several authors (Litridou et al., 1997; Visioli et al., 1998; Yoshida and Takagi, 1999). Besides, reports concerning the local uses, pharmacological features of the extracts and the
author. Tel.: +90-346-219-1010x1417; fax: +90-346-219-1606. E-mail address: candan@cumhuriyet.edu.tr (F. Candan).
Corresponding
essential oils of several Achillea species have been cited in the literature. (Rustaiyan et al., 1998; Chalchat et al., 1999; Simic et al., 2000). The genus Achillea (Family: Asteraceae, Section: Santolinoidea) is represented by about 85 species mostly found in Europe and Asia and a handful in North America (Knemann, 1999). Forty species of Achillea are widely distributed in Turkey (Davis, 1982). As far as ethnopharmacologic background is concerned, Achillea millefolium is a well-known species amongst the members of Achillea (Asteraceae) (Chandler et al., 1982). It is known as Civanperemi and used in folk remedies as an appetizer, wound healer, diuretic, carminative or menstrual regulator (Baytop, 1999). To the best of our knowledge, information concerning the in vitro antioxidant features of Achillea millefolium subsp. millefolium has not been found in the literature. As a part of our continuing research project on the in vitro antimicrobial and antioxidant activities of several Achillea species, we previously reported antimicrobial activities of the essential oils isolated from Achillea setacea and Achillea teretifolia (nl et al., 2002). The aim of this study was
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to evaluate the in vitro antioxidant and antimicrobial properties of the essential oil and methanol extracts of Achillea millefolium subsp. millefolium (Asteraceae).
2.5. Antioxidant activity 2.5.1. Hydroxyl radical scavenging activity Hydroxyl radical scavenging was carried out by measuring the competition between deoxyribose and the extract or crude oil for hydroxyl radicals, which attack to deoxyribose leading to the formation of thiobarbituric acid reaction system (TBARS), generated from the Fe3+ ascorbateEDTAH2 O2 system (Kunchandy and Rao, 1990). The formed TBARS were measured by using the method described elsewhere (Ohkawa et al., 1979). Experiments were carried out in triplicate. All reagents were prepared freshly. Inhibition (I) of deoxyribose degradation in percent was calculated in following way: I= A0 A1 A0 100
2. Materials and methods 2.1. Collection of plant material The herbal parts of Achillea millefolium subsp. millefolium were collected in Kzlda g Pass, 130 km east of Sivas, when owering late-July 2001. The voucher specimens have been deposited at the Herbarium of the Department of Biology, Cumhuriyet University, Sivas, Turkey (CUFH-Voucher No.: ED 6358). 2.2. Preparation of the methanolic extracts The air-dried and nely ground sample was extracted by using the method as described elsewhere (Skmen et al., 1999). The resulting extract (19.78%, w/w) was suspended in water and partitioned with chloroform (CHCI3 ) to separate less polar, water-insoluble compounds. The water-soluble (16.36%, w/w) and non-soluble parts (3.42%, w/w) were then lyophilised and kept in the dark at +4 C until tested. 2.3. Extraction of the essential oil The air-dried and ground aerial parts of plants collected were submitted for 3 h to water-distillation using a Clevenger-type apparatus to produce an oil in 0.6% (v/w) yield. Oil was dried over anhydrous sodium sulphate and, after ltration, stored at +4 C until tested and analyzed. 2.4. GC-MS analysis The analysis of the essential oil was performed using a Hewlett Packard 5890 II GC, equipped with a HP-5 MS capillary column (30 m 0.25 mm i.d., 0.25 m) and a HP 5972 mass selective detector. For GC-MS detection, an electron ionisation system was used with ionisation energy of 70 eV. Helium was the carrier gas, at a ow rate of 1 ml/min. Injector and MS transfer line temperatures were set at 220 and 290 C, respectively. Column temperature was initially at 50 C, then gradually increased to 150 C at a 3 C/min rate, held for 10 min and nally increased to 250 C at 10 C/min. Diluted samples (1/100 in acetone) of 1.0 l were injected manually and splitless. The components were identied based on the comparison of their relative retention time and mass spectra with those of NBS75K library data of the GC-MS system, literature data (Adams, 2001) and standards of the main components. The results were also conrmed by the comparison of the compounds elution order with their relative retention indices on non-polar phases reported in the literature (Adams, 2001).
where A0 is the absorbance of the control reaction (containing all reagents except the test compound), and A1 is the absorbance of the test compound. The IC50 value represented the concentration of the compounds, that caused 50% inhibition. 2.5.2. Inhibition of superoxide radicals Superoxide radical generated by the xanthinexanthine oxidase system was determined spectrophotometrically by monitoring its ability to reduce nitroblue tetrazolium (NBT) (Robak and Gryglewski, 1988). Percent scavenging of superoxide was calculated from the optical density of the treated and control samples. 2.5.3. DPPH assay Radical scavenging activity was determined by a spectrophotometric method based on the reduction of a methanol solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH) as described elsewhere (Cuendet et al., 1997). Tests were carried out in triplicate. 2.5.4. Inhibition of lipid peroxide formation The reaction mixture contained 0.1 ml of 25% (w/v) rat liver homogenate in 40 mM, pH;7.0 TrisHCl buffer, 30 mM KCl, 0.16 mM ferrous iron, various concentrations of the extract, and positive controls; BHT, curcumin, 0.06 mM ascorbic acid in a nal volume of 0.5 ml. As positive controls, BHT and curcumin had their own control reactions containing all related reagents except the test compounds. The mixture was then incubated at 37 C for 1 h (Bishayee and Balasubramanian, 1971). The lipid peroxide formation was measured by using the method described elsewhere (Ohkawa et al., 1979). The percentage inhibition of lipid peroxidation was determined by comparing the results of the test compounds with those of controls not treated with the extracts. Calculations were done as mentioned in hydroxyl radical scavenging method.
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2.6. Antimicrobial activity 2.6.1. Microbial strains The methanolic extracts (both water-soluble and water-insoluble parts) and the essential oil of Achillea millefolium were individually tested against a panel of microorganisms including Staphylococcus aureus ATCC #25923 and for minimum inhibitory concentration (MIC) test, ATCC #29213, Streptococcus pneumoniae ATCC #49619, Moraxella catarrhalis ATCC #49143, Bacillus cereus ATCC #11778, Acinetobacter lwofi ATCC #19002, Enterobacter aerogenes ATCC #13043, Escherichia coli ATCC #25922, Klebsiella pneumoniae ATCC #13883, Proteus mirabilis ATCC #7002, Pseudomonas aeruginosa ATCC #27853, Clostridium perfringens KUKENS-Turkey, Mycobacterium smegmatis CMM 2067, Candida albicans ATCC #10239 and Candida krusei ATCC #6258. Bacterial strains were cultured overnight at 37 C in Mueller Hinton agar (MHA), with the exception of Streptococcus pneumoniae (MHA containing 50 ml citrate blood/l) and Clostridium perfringens (in anaerobic conditions). Yeasts were cultured overnight at 30 C in Sabouraud dextrose agar. 2.6.2. Antimicrobial screening Two different methods were employed for the determination of antimicrobial activities; agar well-diffusion method for the methanol extracts (water-soluble and water-insoluble parts) and agar disc diffusion method for the essential oil. MICs of the essential oil against the test organisms were determined by broth microdilution method. All the tests were performed in duplicate and repeated twice. Modal values were selected. 2.6.3. Agar well-diffusion method The water-soluble extracts were weighed and dissolved in phosphate buffer saline (PBS; pH 7.07.2), 10 mg/ml, water-insoluble parts were dissolved in dimethylsulphoxide (DMSO), 10 mg/ml. Both extracts were lter-sterilised using a 0.45 m membrane lter. Each microorganism was suspended in sterile saline and diluted at ca. 106 colony forming unit (cfu)/ml. They were ood-inoculated onto the surface of MHA. The wells (8 mm in diameter) were cut from the agar and 0.06 ml of extract solution was delivered into them. After incubation for 24 h at 37 C, all plates were examined for any zones of growth inhibition, and the diameter of these zones were measured in millimetres. 2.6.4. Disc diffusion method Disc diffusion method was employed for the determination of antimicrobial activities of the essential oil (NCCLS, 1997). Briey, a suspension of the tested microorganism (0.1 ml of 108 cells per ml) was spread on the solid media plates. Filter paper discs (6 mm in diameter) were impregnated with 15 l of the oil or the fraction and placed on the inoculated plates. These plates, after staying at 4 C for 2 h,
were incubated at 37 C for 24 h for bacteria and at 30 C for 48 h for the yeasts. The diameters of the inhibition zones were measured in millimetres. Amikacin, clindamycin and ciprooxacin were individually used as positive controls for bacteria. 2.6.5. Determination of minimum inhibitory concentration (MIC) A broth microdilution broth susceptibility assay was used, as recommended by NCCLS, for the determination of MIC (NCCLS, 1999). All tests were performed in Mueller Hinton broth (MHB; BBL) supplemented with Tween 80 detergent (nal concentration of 0.5% (v/v), with the exception of the yeasts (Sabouraud dextrose broth-SDB + Tween 80). Bacterial strains were cultured overnight at 37 C in MHA and the yeasts were cultured overnight at 30 C in SDB. Test strains were suspended in MHB to give a nal density of 5 105 cfu/ml and these were conrmed by viable counts. Geometric dilutions ranging from 0.036 mg/ml to 72.00 mg/ml of the essential oil were prepared in a 96-well microtiter plate, including one growth control (MHB + Tween 80) and one sterility control (MHB + Tween 80 + test oil). Plates were incubated under normal atmospheric conditions at 37 C for 24 h for bacteria and at 30 C for 48 h for the yeasts. The MIC of amikacin, clindamycine and ciprooxacine was individually determined in parallel experiments in order to control the sensitivity of the test organisms. The bacterial growth was indicated by the presence of a white pellet on the well bottom.
3. Results and discussion 3.1. Chemical composition of the essential oil Thirty-six compounds were identied and constituted 90.8% of the total oil. The essential oil of Achillea millefolium was characterized by a high number of monoterpenes. Eucalyptol (24.6%), camphor (16.7%), -terpineol (10.2%), -pinene (4.2%), and borneol (4.0%) were the principal components comprising the 59.7% of the essential oil (Table 1). Changes in the composition of Achillea millefolium essential oil were reported as being related to maturation of the plant, with increasing amounts of monoterpenes in relation of the sesquiterpenes (Rohloff et al., 2000). Eucalyptol, camphor, and/or -terpineol have been found as major compounds in many other Achillea species (Rustaiyan et al., 1998; Chalchat et al., 1999; Simic et al., 2000). The studied essential oil displayed different chemical prole from those observed from Achillea millefolium plants of other geographical origin. Achillea millefolium essential oil from Cuba (Pino et al., 1998) was found to include caryophyllene oxide at a percentage of 20%, while Achillea millefolium oil from ve clones from Russia (Orth
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Table 1 Chemical composition of the essential oil from Achillea millefolium subsp. millefolium Compounda Thujene -Pinene Camphene Benzaldehyde Sabinene -Pinene 2,3-Dehydro-1,8-cineole -Terpinene Eucalyptol -Terpinene Terpinolene Linalool Camphor Borneol Terpinen-4-ol -Terpineol Myrtenol Fragranol 7-Methyl-3-methylene-6-octen-1-ol 3,7-Dimethyl-3,6-octadien-1-ol Chrysanthenyl acetate Bornyl acetate Myrtenyl acetate Eugenol -Copaene Caryophyllene -Farnesene -Curcumene Zingiberene Nerolidol Caryophyllene oxide -Eudesmol -Eudesmol Bisabolol oxide II Bisabolone oxide -Bisabolol Others not identied (33) Total
a b
Rt b 9.412 9.669 10.323 10.948 11.523 11.592 12.355 13.545 14.496 15.666 17.123 17.807 20.166 21.117 21.692 22.515 22.663 23.486 23.694 24.408 25.716 26.866 28.888 30.196 31.019 32.991 34.002 35.588 36.262 39.464 40.604 43.647 44.945 45.381 47.354 47.443
Composition (%) 0.1 2.4 2.4 0.2 2.8 4.2 0.6 0.5 24.6 1.0 0.2 0.6 16.7 4.0 2.8 10.2 0.3 0.5 0.2 0.6 0.8 0.1 0.1 0.2 0.1 0.4 0.3 0.2 0.3 0.1 0.7 1.8 1.6 3.8 3.3 2.1 9.2 100
Compounds listed in order of elution from a HP-5 MS column. Retention time (as minutes).
lipid peroxides, superoxides, and hydroxyl radicals. Since the water-insoluble part of the extract is partly soluble in aqueous test media and its colour interfered the spectroscopic measurements, only water-soluble part and essential oil could be tested for their antioxidative capacity. All results are reported in Table 2. As can be seen from Table 2, the extract improved 50% inhibition at higher concentrations, indicating lesser antioxidant capacity than positive controls, but in the superoxide radical inhibition case, it was found to be more effective than ascorbic acid. Hydroxyl radical scavenging and lipid peroxidation were not tested with ascorbic acid since this chemical was already present in the test medium. On the other hand, results in Table 2 demonstrated the strong ability of the essential oil to act as a donor for hydrogen atoms or electrons. The reduction of the stable radical DPPH to yellow coloured diphenylpicrylhydrazine was obtained with an IC50 = 1.56 g/ml instead of 3.90, 7.92, 19.30 g/ml for ascorbic acid, curcumine, and BHT, respectively. Both hydroxyl radical scavenging and the lipid peroxidation inhibition of the oil were also better than curcumine and BHT. This could be assigned to the presence of some phenolic compounds (Table 1). The antioxidative effectiveness in natural sources was reported to be mostly due to phenolic compounds (Hayase and Kato, 1984). Phenolic compounds were reported to play an important role in inhibiting autoxidation of the oils (Ramarathnam et al., 1986). In order to determine the antioxidant nature of the oil, its main components, e.g. eucalyptol, camphor, -pinene, borneol, terpinen-4-ol, -pinene were all tested individually and none exhibited antioxidative activity in all methods employed. Antioxidant activity of eucalyptol and terpinen-4-ol were previously reported using two different methods; aldehyde/carboxylic acid assay and lipid peroxidation (Lee and Shibamoto, 2001) with prolonged incubation periods (30 days and 18 h, respectively) in contrast to our experimental procedure employing 3060 min incubation period. These results implicate that the main components in the total oil might synergize each other or other components involve in these activities. 3.3. Antimicrobial activity Water-insoluble parts of the methanolic extracts were found to have moderate activity against Clostridium perfringens and the yeasts. No activity was exhibited by the water-soluble parts. The water-insoluble parts exhibits, in many cases, greater activity than the water-soluble (aquatic) ones (Skmen et al., 1999). The essential oil possessed stronger antimicrobial activity than the extracts tested. The oil exhibited moderate activity against Streptococcus pneumoniae, Clostridium perfringens and Candida albicans, and weak activity against Mycobacterium smegmatis, Acinetobacter lwofi and Candida krusei (Table 3). The growth inhibitions of test microorganisms ranged from 4.5 mg/ml (w/v) to 72.00 mg/ml (w/v) with
et al., 1999) were characterized by sesquiterpenes with high chamazulene contents (4674%) in the four clones and high -caryophyllene content (3845%) in the one clone. The fraction of sesquiterpenes in the essential oil of Achillea millefolium (section Santolinoidea) of Turkey origin has been found in remarkable amounts, but was qualitatively different from the mentioned above (Table 1). 3.2. Antioxidant activity The antioxidant activity of Achillea millefolium extract (water-soluble part) was examined by comparing it to the activity of known antioxidants such as curcumin, ascorbic acid and BHT by the following four in vitro assays; inhibition of DPPH radical and the oxygen radicals such as
219
Table 2 Effects of methanolic extracts (water-soluble part) and essential oil of Achillea millefolium and positive controls on the in vitro free radical (DPPH, superoxide and hydroxyl) and lipid peroxidation generation Sample IC50 (g/ml) DPPH Extract Oil Curcumin Ascorbic acid BHT nt: not tested. 45.60 1.56 7.92 3.90 19.30 1.30 0.03 0.30 0.15 0.05 Superoxide 304.00 5.10 nt 11.04 0.17 1390.00 2.90 nt Hydroxyl 407.30 2.70 14.28 nt 32.00 4.05 0.03 0.08 1.20 Lipid peroxidation 892.67 13.50 40.83 nt 17.80 13.0 0.07 0.15 0.04
Table 3 Antimicrobial activity of the essential oil and the methanolic extracts of Achillea millefolium subsp. millefolium Microorganism Essential oil DDa Staphylococcus aureus Streptococcus pneumoniae Moraxella catarrhalis Bacillus cereus Acinetobacter lwofi Enterobacter aerogenes Escherichia coli Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa Clostridium perfringens Mycobacterium smegmatis Candida albicans Candida krusei
a b
MeOHc MICb 72.00 4.50 na 72.00 18.00 72.00 na 72.00 na na 4.50 9.00 4.50 18.00 H2 O nae na na na na na na na na na na na na na CHCl3 na na na 10 na na na na na na 12 na 12 12
The MIC of Antibioticsd AK 2.00 nt nt nt nt nt 2.00 nt nt 1.00 nt nt nt nt CF 0.25 nt nt nt nt nt 0.015 nt nt 0.25 nt nt nt nt CM ntf 0.125 nt nt nt nt nt nt nt nt 0.25 nt nt nt FC nt nt nt nt nt nt nt nt nt nt nt nt 128.00 64.00
8 14 na 10 15 7 na 9 na na 12 12 21 16
DD, disc diffusion method as recommended by NCCLS. Diameter of zone of inhibition (mm) including disk diameter of 6 mm. MIC, minimum inhibitory concentration. Values given as mg/ml (for the essential oil) and as g/ml (for antibiotics). c MeOH, methanolic extracts. Diameter of zone of inhibition (mm) including well diameter of 8 mm. d AK, amikacin; CF, ciprooxacin; CF, clindamycin, FC, uconazole. e na, not active. f nt, not tested.
the lowest MIC value against Streptococcus pneumoniae, Clostridium perfringens, Candida albicans at 4.5 mg/ml (w/v). Eucalyptol (1,8-cineole) and camphor are well-known chemicals with their pronounced antimicrobial potentials (Pattnaik et al., 1997; Tzakou et al., 2001). Antimicrobial activities of borneol have also been previously reported by other investigators (Knobloch et al., 1989; Tabanca et al., 2001). In conclusion, our observations conrm that oil of Achillea millefolium possess strong antioxidative activity but low antimicrobial activity in vitro. Acknowledgements Financial support from the Research Council of Cumhuriyet University, Sivas, Turkey (Project No.: F-88) is gratefully acknowledged.
References
Adams, R.P., 2001. Identication of Essential Oils components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, Illinois, USA. Baldioli, M., Servili, M., Perretti, G., Montedoro, G.F., 1996. Antioxidant activity of tocoferols and phenolic compounds of virgin olive oil. Journal of American Oil Chemistry Society 73, 15891593. Baytop, T. (Ed.), 1999. Trkiyede bitkiler ile tedavi (treatment with plants in Turkey). Istanbul University Publications No.: 3255:40, Istanbul, p. 176. Bishayee, S., Balasubramanian, A.S., 1971. Lipid peroxide formation in rat brain. Journal of Neurochemistry 18, 909920. Chalchat, J.C., Gorunovic, M.S., Petrovic, S.D., 1999. Aromatic plants of Yugoslavia. I. chemical composition of oils of Achillea millefolium L. ssp. pannonica (Scheele) Hayak, A. crithmifolia W. et K., A. serbica Nym. and A. tanacetifolia all. Journal of Essential Oil Research 11, 306310. Chandler, R.F., Hooper, S.N., Harvey, N.J., 1982. Ethnobotany and phytochemistry of yarrow. Achillea millefolium, Compositae. Economic Botany 36, 203215.
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F. Candan et al. / Journal of Ethnopharmacology 87 (2003) 215220 Pino, J.A., Rosado, A., Fuentes, V., 1998. Chemical composition of the leaf oil of Achillea millefolium L. grown in Cuba. Journal of Essential Oil Research 10, 427428. Ramarathnam, N., Osawa, T., Namiki, M., Tashiro, T., 1986. Studies on the relationship between antioxidative activity of rice hull and germination ability of rice seeds. Journal of the Science of Food and Agriculture 37, 719726. Robak, J., Gryglewski, R.J., 1988. Flavonoids are scavengers of superoxide anions. Biochemical Pharmacology 37, 837841. Rohloff, J., Skagen, E.B., Steen, A.H., Iversen, T.-H., 2000. Production of yarrow (Achillea millefolium L.) in Norway: essential oil content and quality. Journal of Agricultural and Food Chemistry 48, 6205 6209. Rustaiyan, A., Komeilizadeh, H., Shariatpanahi, M.S., Jassbi, A., Masoudi, S., 1998. Comparative study of the essential oils of three Achillea Species from Iran. Journal of Essential Oil Research 10, 207 209. Simic, N., Andjelkovic, S., Palic, R., Vajs, V., Milosavicevic, S., 2000. Composition and antibacterial activity of Achillea chrysocoma essential oil. Journal of Essential Oil Research 12, 784787. Six, P., 1994. Current research in natural food antioxidants. International News on Fats, Oils & Related Materials 5, 679688. Skmen, A., Jones, B.M., Ertrk, M., 1999. The in vitro antibacterial activities of Turkish medicinal plants. Journal of Ethnopharmacology 67, 7986. Tabanca, N., Kirimer, N., Demirci, B., Demirci, F., Ba ser, K.H.C., 2001. Composition and antimicrobial activity of the essential oils of Micromeria cristata subsp. phyrgia and the Enantiomeric Distribution of Borneol. Journal of Agricultural and Food Chemistry 49, 43004303. Tzakou, O., Pitarokili, D., Chinou, I.B., Harvala, C., 2001. Composition and antimicrobial activity of the essential oil of Salvia ringens. Planta Medica 67, 8183. nl, M., Daferera, D., Dnmez, E., Polissiou, M., Tepe, B., Skmen, A., 2002. Compositions and the in vitro antimicrobial activities of the essential oils of Achillea setacea and Achillea teretifolia. Journal of Ethnopharmacology 83, 117121. Visioli, F., Bellomo, G., Galli, C., 1998. Free radical-scavenging properties of olive oil polyphenols. Biochemical and Biophysical Research Communications 247, 6064. Yoshida, H., Takagi, S., 1999. Antioxidative effects of sesamol and tocopherols at various concentrations in oils during microwave heating. Journal of the Science of Food and Agriculture 79, 220226.
Cuendet, M., Hostettmann, K., Potterat, O., 1997. Iridoid glucosides with free radical scavenging properties from Fagraea blumei. Helvetica Chimica Acta 80, 11441152. Davis, P.H., 1982. Flora of Turkey and the East Aegean Islands, vol.5. University Press, Edinburgh, p. 244. Halliwell, B., 1997. Antioxidants and human disease: a general introduction. Nutrition Revews 55, S44S52. Hayase, F., Kato, H., 1984. Antioxidative components of sweet potatoes. Journal of Nutritional Science and Vitaminology 30, 3746. Knemann, 1999. Botanica: The Illustrated A-Z of over 10,000 Garden Plants and How to Cultivate Them. Gordon Cheers Publication, Hong Kong, pp. 5153. Knobloch, K., Pauli, A., Iberi, B., Wegand, H., Weis, N., 1989. Antibacterial and antifungal properties of essential oil components. Journal of Essential Oil Research 1, 119128. Kunchandy, E., Rao, M.N.A., 1990. Oxygen radical scavenching activity of curcumin. International Journal of Pharmacognosy 58, 237240. Larson, R.A., 1988. The antioxidants of hinger plants. Phytochemistry 27, 969978. Lee, K.G., Shibamoto, T., 2001. Antioxidant activities of volatile components isolated from Eucalyptus species. Journal of the Science of Food and Agriculture 81, 15731579. Litridou, M., Linssen, J., Schols, H., Bergmans, M., Posthumus, M., Tsimidou, M., Boskou, D., 1997. Phenolic compounds in virgin oilve oils: fractionation by solid-phase extraction and antioxidant activity assessment. Journal of the Science of Food and Agriculture 74, 169 174. NCCLS (National Committee for Clinical Laboratory Standards), 1997. Performance standards for antimicrobial disk susceptibility test, 6th ed. Approved Standard, Wayne Pa., M2-A6. NCCLS (National Committee for Clinical Laboratory Standards), 1999. Performance standards for antimicrobial susceptibility testing; 9th International Supplement, Wayne Pa. M100-S9. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351358. Orth, M., Czygan, F.C., Dedkov, V.P., 1999. Variation in essential oil composition and chiral monoterpenes of Achillea millefolium s.i. from Kaliningrad. Journal of Essential Oil Research 11, 681687. Pattnaik, S., Subramanyam, V.R., Bapaji, M., Kole, C.R., 1997. Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios 89, 3946.
In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected Ivorian plants

Catherine Vonthron-Sncheau a , Bernard Weniger b, , Modibo Ouattara c , Fezan Tra Bi d , Alphonse Kamenan a , Annelise Lobstein b , Reto Brun e , Robert Anton b
b a UFR des Sciences et Technologie des Aliments, Universit dAbobo-Adjam, 02 BP 801 Abidjan 02, Ivory Coast Unit de Pharmacognosie, UMR ULP/CNRS 7081, Facult de Pharmacie, Universit Louis Pasteur, Strasbourg, France c UFR des Sciences Pharmaceutiques et Biologiques, Universit de Cocody, 01 BP V34 Abidjan, Ivory Coast d UFR des Sciences de la Nature, Universit dAbobo-Adjam, 02 BP 801 Abidjan 02, Ivory Coast e Swiss Tropical Institute, Medical Parasitology and Infection Biology, Socinstrasse 57, CH-4002 Basel, Switzerland
Received 3 January 2003; received in revised form 9 April 2003; accepted 16 April 2003
Abstract Eight extracts from four Ivorian medicinal plants, traditionally used to treat malaria, were tested for their antiplasmodial activity in vitro by assessing their ability to inhibit the uptake of [3 H]hypoxanthine into the Plasmodium falciparum K1 chloroquine-resistant strain. The most active extract was the methylene chloride extract of Anogeissus leiocarpus which exhibited an IC50 value of 3.8 g/ml. Inhibition of the growth of Plasmodium falciparum was also observed with the methylene chloride extract of Cochlospermum planchonii and Microdesmis keayana as well as with both methylene chloride and methanolic extracts of Hymenocardia acida. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Malaria; Traditional medicine; Ivory Coast
1. Introduction Malaria remains the rst world endemic parasitic disease and is responsible for the death of over 1 million people annually in the belt of Africa, within which lies Ivory Coast (Snow et al., 1999). Most malaria mortality in this country is caused by Plasmodium falciparum, the most common species in the highly malaria endemic areas of Africa. Present drugs have become ineffective because Plasmodium falciparum is developing resistance to most of them, particularly to chloroquine, the cheapest and previously very effective antimalarial drug, and to pyrimethamine (Peters, 1998; Wellems and Plowe, 2001). Thus, there is an urgent need to discover new antiplasmodial agents. History shows that biodiversity and traditional medicine knowledge are useful to open new ways in the eld of antimalaria therapy, as it was the case for artemisinin (Quinghaosu Antimalarial Coordinating Research Group, 1979).
Corresponding
author. Tel.: +33-390-244238; fax: +33-390-244311. E-mail address: weniger@pharma.u-strasbg.fr (B. Weniger).
In this framework, data on plants traditionally used in the treatment of malaria in the Savannah and the forest regions of Ivory Coast were collected, by personal contact with local traditional healers during an ethnobotanical survey of medicinal plants, conducted from April to August 2001. A bibliographical survey permitted to retain four native plant species among the most used by traditional healers and for which antiplasmodial activity had never been evaluated (Anogeissus leiocarpus (DC.) Guill. and Perr., Hymenocardia acida Tul. and Microdesmis keayana Leon.) or at least only partially studied (Cochlospermum planchonii Hook. f. ex Planch.). Apolar (methylene chloride) and polar (methanol) extracts were prepared from the part of each selected plant indicated as traditionally used in decoction by the traditional healers, i.e. leaves for Anogeissus leiocarpus, Hymenocardia acida and Microdesmis keayana and roots for Cochlospermum planchonii. Each extract was then evaluated for antiplasmodial activity in vitro, by assessing its ability to inhibit the uptake of [3 H]hypoxanthine by the parasite. The cytotoxicity of the most active extracts was evaluated using L6 cells.
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Table 1 Plant species analyzed for antiplasmodial activity Botanical name/family Anogeissus leiocarpus (DC.) Guill. and Perr./Combretaceae Cochlospermum planchonii Hook. f. ex Planch./Cochlospermaceae Hymenocardia acida Tul./Euphorbiaceae Microdesmis keayana J. Leon./Pandaceae Traditional names K` er` ek` et` e (M) Tourougbebourou (M) Djoun (M) Ibr el egni (B) Collection area S S S A Part used Leaf Root Leaf Leaf Voucher no. CNF CNF CNF CNF 14798 14643 8705 17789
M: Malinke; B: Bowle; S: Savannah region near Seguela (Northern Ivory Coast); A: forest region near Abidjan (Southern Ivory Coast).
2. Methodology 2.1. Plant material The selected plants (Table 1) were collected between June and August 2001 in their natural habitats in Ivory Coast, i.e. in the Savannah region in Bouandougou near Seguela (Northern Ivory Coast) for Anogeissus leiocarpus, Cochlospermum planchonii and Hymenocardia acida and in the forest region near Abidjan (Southern Ivory Coast) for Microdesmis keayana. Botanical determination was performed by Dr. L. Ak Assi (Centre National de Floristique, Universit de Cocody, Abidjan). Voucher specimens are deposited at the Herbarium of the Centre National de Floristique (CNF). 2.2. Preparation of crude extracts
ing the methods of Trager and Jensen (1976). Plant extracts were tested on K1 strain (multidrug pyrimethamine/ chloroquine-resistant strain; Thaithong and Beale, 1981). Initial concentration of the plant extracts was 30 g/ml diluted with two-fold dilutions to make seven concentrations, the lowest being 0.47 g/ml. After 48 h incubation of the parasites with the extracts at 37 C, [3 H]hypoxanthine (Amersham, UK) was added to each well and the incubation was continued for another 24 h at the same temperature. The extract concentrations at which the parasite growth (=[3 H]hypoxanthine uptake) was inhibited by 50% (IC50 ) was calculated by linear interpolation between the two drug concentrations above and below 50% (Huber and Koella, 1993). Chloroquine and artemisinin were used as positive references. The values given in Table 2 are means of two independent assays; each assay was run in duplicate. 2.4. Cytotoxicity assay
Air dried plant material of each species (10 g) was ground (0.2 mm sieve) and defatted with hexane (200 ml) overnight at room temperature. The plant material was extracted twice for 30 min with methylene chloride (60 ml) at 40 C, then twice for 30 min with methanol (60 ml) at 64 C, in a semi-automated Soxhlet extractor (Soxtec Avanti 2055 apparatus, Foss Tecator AB, Hgans, Sweden). The ltrates were taken to dryness under vacuum and the residues were stored at room temperature until testing. Tannins were removed from the crude methanolic extracts using Sephadex LH-20 exclusion chromatography, according to the method described by Houghton and Raman (1998). Methylene chloride and methanol extract (after tannin removal) yields were 5 and 0.7% for Anogeissus leiocarpus; 2.3 and 0.4% for Cochlospermum planchonii; 7.7 and 0.8% for Hymenocardia acida; 14.3 and 6.7% for Microdesmis keayana, respectively. 2.3. Antimalarial assay Quantitative assessment of antimalarial activity in vitro was determined by means of the microculture radioisotope technique based upon the method previously described by Desjardins et al. (1979) and modied by Ridley et al. (1996). The assay uses the uptake of [3 H]hypoxanthine by parasites as an indicator of viability. Continuous in vitro cultures of asexual erythrocytic stages of Plasmodium falciparum were maintained follow-
Cytotoxicity assay of the plant extracts was done following the method of Pag and Noel (1993) with the modication of Ahmed et al. (1994). Cell line L6 (rat skeletal muscle myoblasts) were seeded in 96-well Costar microtiter plates at 2.2105 cells/ml, 50 l per well in MEM supplemented with 10% heat inactivated fetal bovine serum (FBS). A three-fold serial dilution ranging from 500 to 0.07 g/ml of crude extracts in test medium was added. Plates with a nal volume of 100 l per well were incubated at 37 C for 72 h in a humidied incubator containing 5% CO2 . Alamar Blue was added as viability indicator according to Ahmed et al. (1994). After an additional 2 h of incubation, the plate was measured with a uorescence scanner using an excitation wavelength of 536 nm and an emission wavelength of 588 nm (SpectraMax GeminiXS, Molecular Devices). IC50 values were calculated from the sigmoidal inhibition curve.
3. Results Traditional healers in the area of Seguela and Abidjan were consulted directly in collecting the basic ethnobotanical information about the plants studied. Questions concentrated mainly on plants which are traditionally used against malaria. Based on this information, different databases (NAPRALERT Database, MEDLINE/PubMed, ISI Web of
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Table 2 In vitro antiplasmodial activity of the selected plant extracts against K1-resistant strain of Plasmodium falciparum and in vitro cytotoxicity towards L6 cells for the extracts exhibiting IC50 below 20 g/ml Plant species Plant part Extract Antiplasmodial activity (Plasmodium falciparum K1 strain) IC50 (g/ml) Anogeissus leiocarpus Cochlospermum planchonii Hymenocardia acida Microdesmis keayana Chloroquine Artemisinin Leaf Root Leaf Leaf Db Mc D M D M D M 3.8 >20 4.4 >20 6.9 10.7 12.2 >20 0.064 0.0015 95% Condence intervala 2.55.1 3.15.7 5.97.9 7.114.3 10.414 Cytotoxic activity (L6 cells) IC50 (g/ml) 71.9 67.3 65.4 71.8 nd Selectivity index (SI) 19 15 10 6 nd
Selectivity index, ratio of cytotoxic activity on L6 cells to antiplasmodial activity against K1 strain of Plasmodium falciparum; nd: not determined; IC50 values are means of two experiments. a 95% condence intervals were determined with Students t-test (Snedecor and Cochran, 1980), P < 0.05. b Methylene chloride extract. c Methanolic extract after tannins removal.
Science, Chemical abstracts and PRELUDE Database) were consulted to survey the literature concerning these plants. Consequently, four plant species, belonging to four genera and four families, were chosen and collected in the eld (Table 1). Traditional uses of these species are listed in Section 4. The part of the selected plant species traditionally used was extracted to yield eight extracts, which were tested for antiplasmodial activity. In vitro antiplasmodial activity of these eight extracts is summarized in Table 2. Finally, active plant extracts (IC50 < 20 g/ml) were investigated for their cytotoxicity towards L6 cells (rat skeletal muscle myoblasts). The strongest antiplasmodial activities were found in the dichloromethane extract of Anogeissus leiocarpus (IC50 = 3.8 g/ml) and of Cochlospermum planchonii (IC50 = 4.4 g/ml). Methanolic and methylene chloride extracts of Hymenocardia acida as well as the methylene chloride extract of Microdesmis keayana showed greater IC50 values, ranging from 6.9 to 12.2 g/ml. Methanolic extracts of Anogeissus leiocarpus, Cochlospermum planchonii and Microdesmis keayana exhibited IC50 values greater than 20 g/ml. Table 2 also summarizes the cytotoxicity towards L6 cells and the selectivity index (SI) of extracts which exhibited IC50 < 20 g/ml in the antiplasmodial assay. SI is dened as the ratio of cytotoxicity to biological activity. Cytotoxic activities of the four extracts tested are all in the same range, the highest cytotoxic activity was found for the methanolic extract of Hymenocardia acida (IC50 = 65.4 g/ml) and the lowest for the methylene chloride extracts of Hymenocardia acida and Anogeissus leiocarpus (IC50 = 71.9 and 71.8 g/ml, respectively). The best SI was found for the methylene chloride extract of Anogeissus leiocarpus (SI = 19) and the lowest one for the methylene chloride extract of Hymenocardia acida (SI = 6).
4. Discussion Anogeissus leiocarpus (DC.) Guill. and Perr. (Combretaceae): Leaves are used in Nigeria and in Guinea as antimalarial (Adjanohoun et al., 1991; Bhat et al., 1990; Gbile et al., 1990; Pobeguin, 1911). Other medicinal uses in Africa are reported, like emmenagogue (Berhault, 1974), antitussive (Baoua et al., 1976), disinfection of syphilitic ulcers, against diarrhea after parturition, against leprosy (Vasileva, 1969) and for cleaning teeth (Bhat et al., 1990). This species showed antimicrobial activity against Haemophilus inuenzae, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and Moraxella catarrhalis (Sanogo et al., 1998). The presence of 3,4,3 -tri-O-methylavellagic acid and its glucoside were recently described in this species. This glucoside showed antimicrobial activity on Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans (Adigun et al., 2000). Cochlospermum planchonii Hook. f. ex Planch. (Cochlospermaceae): The rhizome of this species is used as antimalarial in Niger (Adam et al., 1972; Dakuyo, 1989). The related species Cochlospermum trinctorium and Cochlospermum angolense are used for the same purpose in Ivory Coast (Benoit-Vical et al., 1995) and in Angola (Presber et al., 1992). Essential oil, long-chain triacylbenzenes and unusually high levels of manganese and zinc have been found in this species (Addae-Mensah et al., 1985; Aliyu et al., 1995). A crude leaf extract and essential oil prepared from the leaves of this species showed both antiplasmodial activity and the leaf oil yielding the best antimalarial effect (IC50 = 2235 g/ml) (Benoit-Vical et al., 1999). The rhizome is also used in decocotion by native medical practitioners in the treatment of jaundice in Northern Nigeria (Ak Assi and Guinko, 1991). Aliyu et al. (1995) isolated a zinc salt from an aqueaous extract of the rhizome which acts
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as an hepatoprotective agent by inhibiting the cytochrome P450 enzymes. Hymenocardia acida Tul. (Euphorbiaceae): The roots are used as antimalarial in Nigeria (Ainslie, 1937). Other reports mention the traditional use of this species against sleeping sickness (Kerharo, 1974), in case of skin diseases, diabetes, anemia, cough, infected wounds, urinary tract infections and dental caries (Muanza et al., 1994) and as an aphrodisiac (Muanza et al., 1994; Bouquet and Debray, 1974; Alvaro Viera, 1959). A peptide alkaloid, hymenocardine, was isolated from the root bark of this species (Pais et al., 1968). The methanol extract from the same part of the plant exhibited cytotoxic activity against a panel of human tumor cell lines (Muanza et al., 1995), antimicrobial activity against Streptococcus mutans and Salmonella typhimurium (Muanza et al., 1994) and spasmolytic activity (Kambu et al., 1990). The ethanolic extract exhibited antitrypanosomal activity (Freiburghaus et al., 1997) and antiinamatory effect (Sackeyo, 1998). Microdesmis keayana J. Leon. (Pandaceae): A related species, Microdesmis puberula was used in Ivory Coast as an emmenagogue and as an aphrodisiac (Bouquet and Debray, 1974). Neither biological nor chemical data about this species could be found in the literature. All these four plant species, selected for their traditional medicinal use in the treatment of malaria, showed in vitro antiplasmodial activity. These results support the traditional use of these species as antimalarial agents. The Anogeissus leiocarpus dichloromethane extract was the most active one with an IC50 of 3.8 g/ml, comparable to activity reported in the literature for ethanolic extracts of Artemisia annua (IC50 = 3.9 g/ml; K1-resistant strain) and of Azadirachta indica (IC50 = 4.17.2 g/ml; FcB1-resistant strain) in the in vitro microdilution test (ONeill et al., 1985; Benoit-Vical et al., 1996; Udeinya, 1993). In spite of a large range of uses in traditional medicine, this is the rst report of antiplasmodial activity for extracts from species of Anogeissus and Hymenocardia genera. Concerning Cochlospermum planchonii, weak in vitro antiplasmodial activities against another resistant strain of Plasmodium falciparum had already been reported, but for an aqueous extract and for essential oil prepared from the leaves, with IC50 values = 2235 g/ml and 2575 g/ml, respectively (Benoit-Vical et al., 1999). Whereas here, in our assay, a methylene chloride extract prepared from the leaves of the same species showed good antiplasmodial activity (IC50 = 4.4 g/ml), thus pointing to the hydrophobic nature of the putative active principles. The methylene chloride extract of Microdesmis keayana, a species for which, as far as we know, no biological or chemical information is available, showed mild antiplasmodial activity in vitro (IC50 = 12.2 g/ml). In vitro microdilution tests are convenient and rapid but they give no information about the selectivity of the toxicity, so we tried to clarify the potential of the tested plants for clinical use. Thus, the selectivity indexes (SI = ratio of cytotoxicity to biological activity) of the most active extract
were determined. It is generally considered that biological efcacy is not due to in vitro cytotoxicity when SI 10. In this study, three extracts out of four showed SI 10; only the methanolic extract of Hymenocardia acida showed poor selectivity (SI = 6). The three plant extracts that showed both signicant antiplasmodial activity and good selectivity in in vitro tests have been selected for bioguided fractionation. Phytochemical studies of these plants are being performed in order to identify the most effective fraction or compound, bearing in mind that the optimal product may not always correspond to single compounds.
Acknowledgements The authors wish to thank the traditional healers from Ivory Coast for their willingness to share with us their knowledge about plants, especially A. Kamgate, L. Ouattara and N. Samake in Bouandougou, near Seguela. Special thanks to retired Professor L. Ak Assi who kindly performed the botanical determinations.
References
Adam, J.G., Echard, N., Lescot, M., 1972. Plantes mdicinales Hausa de lAder (Rpublique du Niger). Journal dAgriculture Tropicale et de Botanique Applique 19, 259399. Addae-Mensah, I., Waibel, R., Achenbach, H., 1985. Novel long-chain triacylbenzenes from Cochlospermum planchonii. Justus Liebigs Annalen der Chemie 6, 12841287. Adigun, J.D., Amupitan, J.D., Kelly, D.R., 2000. Isolation and investigation of antimicrobial effect of 3,4,3 -tri-O-methylavellagic acid and its glucosid from Anogeissus. Bulletin of the Chemical Society of Ethiopia 14, 169174. Adjanohoun, E., Ahyi, M.R.A., Ak Assi, L., 1991. Contribution to Ethnobotanical and Floristic Studies in Western Nigeria. CSTR-OUA, p. 220. Ahmed, S.A., Gogal, R.M., Walsh, J.E., 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3 H]thymidine incorporation assay. Journal of Immunological Methods 170, 211224. Ainslie, J.R., 1937. A List of Plants Used in Native Medicine in Nigeria. Imperial Forestry Institute, University of Oxford, Institute Paper No. 7, p. 107. Ak Assi, L., Guinko, S., 1991. Plants Used in Traditional Medicine in West Africa. Roche Editions, Basel, Switzerland, p. 62. Aliyu, R., Okoye, Z.S., Shier, W.T., 1995. The hepatoprotective cytochrome P450 enzyme inhibitor isolated from the Nigerian medicinal plant Cochlospermum planchonii is a zinc salt. Journal of Ethnopharmacology 48, 8997. Alvaro Viera, R., 1959. Subsidio para o estuda da ora medicinal da Guinea portuguesa. Agencia Geral do Ultramar, Lisboa, Portugal. Baoua, M., Fayn, J., Bassiere, J., 1976. Preliminary phytochemical testing of some medical plants of Niger. Plantes Mdicinales et Phytothrapie 10, 251266. Bhat, R.B., Eterjere, E.O., Olapido, V.T., 1990. Ethnobotanical studies from Central Nigeria. Journal of Economic Botany 44, 382390. Benoit-Vical, F., Valentin, A., Pelissier, Y., Marion, C., Dakuyo, Z., Malli, M., Yapo, A., Bastide, J.M., 1995. Antimalarial activity in vitro
C. Vonthron-S en echeau et al. / Journal of Ethnopharmacology 87 (2003) 221225 of Cochlospermum trinctorium tubercule extracts. Transactions of the Royal Society of Tropical Medicine and Hygiene 89, 217218. Benoit-Vical, F., Valentin, A., Pelissier, Y., Diafouka, F., Marion, C., Kon-Bamba, D., Malli, M., Yapo, A., Bastide, J.M., 1996. In vitro antimalarial activity of vegetal extracts used in Western African traditional medicine. American Journal of Tropical Medicine and Hygiene 54, 6771. Benoit-Vical, F., Valentin, A., Pelissier, Y., Malli, M., Bastide, J.M., Bessire, J.M., 1999. In vitro antimalarial activity and cytotoxicity of Cochlospermum trinctorium and Cochlospermum planchonii leaf extracts and essential oils. Planta Medica 65, 378381. Berhault, J., 1974. Flore illustre du Sngal. II. Gouvernement du Sngal, Ministre du Dveloppement Rural, Division des Eaux et For ets, Dakar, p. 46. Bouquet, A., Debray, M., 1974. Plantes mdicinales de la C ote dIvoire. Travaux et Documents de lOrstom 32, 1232. Dakuyo, P.Z., 1989. Recettes de la mdecine traditionnelle. Bulletin de Mdecine Traditionnelle et Pharmacope 3, 8384. Desjardins, R.E., Caneld, C.J., Haynes, J.D., Chilay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro was determined by a semi-automated microdilution technique. Antimicrobial Agents and Chemotherapy 16, 710718. Freiburghaus, F., Jonker, S.A., Nkunya, M.H.H., Mwasumbi, L.B., Brun, R., 1997. In vitro trypanocidal activity of some rare Tanzanian medicinal plants. Acta Tropica 66, 7981. Gbile, Z.O., Adayemi, F.A., Odewa, T.K., 1990. Nigerial ora and its pharmaceutical potential no. 3. Mitteilungen des Instituts fr Allgemeine Botanik 23b, 10331038. Houghton, P.J., Raman, A., 1998. Laboratory Handbook for the Fractionation of Natural Extracts. Chapman & Hall, London, p. 49. Huber, W., Koella, J.C., 1993. A comparison of the three methods of estimating EC50 in studies of drug resistance of malaria parasite. Acta Tropica 55, 257261. Kambu, K., Tona, L., Kaba, S., Cimanga, K., Mukala, N., 1990. Antispasmodic activity of extracts proceeding of plant antidiarrheic traditional preparations used in Kinshasa, Zaire. Annales de Pharmacie Franaise 48, 200208. Kerharo, J., 1974. Historic and ethnopharmagnosic insight into the belief and traditional practices in the treatment of sleeping sickness in West Africa. Bulletin de la Socit de Mdecine dAfrique Noire et de Langue Franaise 19, 400410. Muanza, D.N., Kim, B.W., Euler, K.L., Williams, L., 1994. Antibacterial and antifungal activities of nine medicinal plants from Zaire. International Journal of Pharmacognosy 32, 337345. Muanza, D.N., Euler, K.L., Williams, L., Newman, D.J., 1995. Screening for antitumor and anti-HIV activities of nine medicinal plants from Zaire. International Journal of Pharmacognosy 33, 98106. ONeill, M.J., Bray, D.H., Boardman, P., Phillipson, J.D., Warhurst, D.C., 1985. Plants as sources of antiplasmodial drugs. Part 1. Planta Medica 61, 394398.
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Pag, M., Noel, C., 1993. A new uorimetric assay for cytotoxicity measurements in vitro. International Journal of Oncology 3, 473 476. Pais, M., Marchand, J., Ratle, G., Jarreau, F.X., 1968. Peptidic alkaloides. VI. Hymenocardine, alkaloid from Hymenocardia acida Tul. Bulletin de la Socit Chimique de France 7, 29792984. Peters, W., 1998. Drug resistance in malaria parasites of animals and man. Advances in Parasitology 41, 14. Pobeguin, M., 1911. Plantes mdicinales de la Guine franaise. Agriculture Pratique des Pays Chauds T1, 279295, 387394, 484496; T2, 3745, 133144, 233238. Presber, W., Hegenscheid, B., Hernandez-Alvarez, H., Herrmann, D., 1992. Inhibition of the growth of Plasmodium falciparum and P. berghei in vitro by an extract of Cochlospermum angolense (Welw.). Acta Tropica 50, 331338. Quinghaosu Antimalarial Coordinating Research Group, 1979. Antimalaria studies on qinghaosu. Chinese Medical Journal 92, 811 816. Ridley, R.G., Werner, H., Hugues, M., Catherine, J., Arnulf, D., Raffaello, M., Synese, J., Wolfgang, F.R., Alberto, G., Maria, A.G., Heinrich, U., Werner, H., Sodsri, T., Wallace, P., 1996. 4-Aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum. Antimicrobial Agents and Chemotherapy 40, 18461854. Sackeyo, A.C., 1998. Inhibition of adjuvant arthrisis in the rat and pinnal inammation in the mouse by an extract of Hymenocardia acida. Phytotherapy Research 21, 4245. Sanogo, R., Crisa, G., Germano, M.P., De Pasquale, R., Bisignane, G., 1998. Evaluation of Malian traditional medicines: screening for antimicrobial activity. Phytotherapy Research Supplement 12, S154 S156. Snedecor, G.W., Cochran, W.G., 1980. Statistical Methods, 7th ed. Iowa State University Press, Hemisphere. Snow, R.W., Craig, M., Deichmann, U., Marsh, K., 1999. Estimating mortality, morbidity and disability due to malaria among Africas non-pregnant population. Bulletin of the World Health Organization 77, 624640. Thaithong, S., Beale, G.H., 1981. Resistance of 10 Thai isolates of Plasmodium falciparum to chloroquine and pyrimethamine by in vitro tests. Transactions of the Royal Society of Tropical Medicine and Hygiene 75, 271273. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673675. Udeinya, I.J., 1993. Anti-malarial activity of Nigerian neem leaves. Transactions of the Royal Society of Tropical Medicine and Hygiene 87, 471. Vasileva, B., 1969. Plantes mdicinales de Guine Conakry (Rpublique de Guine). University of Moscow, p. 11. Wellems, T., Plowe, C., 2001. Chloroquine-resistant malaria. The Journal of Infectious Diseases 184, 770776.
Antioxidant activity of Thespesia populnea bark extracts against carbon tetrachloride-induced liver injury in rats
Raju Ilavarasan a, , Mani Vasudevan a , Sockalingam Anbazhagan a , Subramanian Venkataraman b
a
Department of Pharmacology, C. L. Baid Metha College of Pharmacy, Jyothi Nagar, Old Mahabalipuram Road, Thorapakkam, Chennai 600096, India b Department of Pharmacology and Environmental Toxicology, Dr. A. L. Mudaliar P.G. Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600113, India
Received 7 January 2003; received in revised form 14 April 2003; accepted 16 April 2003
Abstract Antioxidant activity of the aqueous (AET) and methanolic extracts (MET) of the Thespesia populnea bark was investigated in rats by inducing liver injury with carbon tetrachloride:olive oil (1:1). The extracts exhibited signicant antioxidant activity showing increased levels of glutathione peroxidase (GPX), glutathione S-transferase (GST), glutathione reductase (GRD), superoxide dismutase (SOD) and catalase (CAT) and decreased level of lipid peroxidation (LPO). Thespesia populnea bark extracts, AET and MET, at a dose level of 500 mg/kg showed signicant antioxidant activity against carbon tetrachloride-induced liver injury in rats. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Thespesia populnea; Carbon tetrachloride; Marker enzymes in liver; Antioxidant activity
1. Introduction Thespesia populnea Soland ex Correa (Family: Malvaceae) is a large avenue tree found in the tropical regions and coastal forests in India. The bark, leaves, owers and fruits are useful in cutaneous infections, such as scabies, psoriasis, eczema, ringworm and guinea worm. A decoction of the bark is commonly used for the treatment of skin and liver diseases. Oil of bark mixed with vegetable oil is useful in urethritis and gonorrhea. The astringent bark, roots and fruits were used in dysentery, cholera and hemorrhoids; the mashed bark is employed as a poultice or hot fomentation for wounds. The leaves were reported to be employed locally as anti-inammatory in swollen joints (Anonymous, 1995). Based on the above reports, the present study has been undertaken to investigate the antioxidant activity of aqueous (AET) and methanolic extracts (MET) of the bark of Thespesia populnea in CCl4 -intoxicated liver injury in rats.
2. Materials and methods 2.1. Plant material The fresh bark of Thespesia populnea was collected from Chennai, India in the month of August and the plant was identied and authenticated by the Research Ofcer (Botany), Central Research Institute (Siddha), Ministry of Health and Family Welfare, Govt. of India. The voucher specimen has been kept in the Department of Pharmacology, C. L. Baid Metha College of Pharmacy, Chennai, India. 2.2. Preparation of aqueous and methanolic extracts The freshly collected bark of this plant was chopped, shade dried and coarsely powdered. The powder was then successively extracted with methanol and distilled water using a Soxhlet extractor. The aqueous and methanolic extracts were dried under reduced pressure using a rotary ash evaporator and they were kept under refrigeration. Both the extracts were administered to the animals as a suspension in propyleneglycol.
Corresponding
author.
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2.3. Chemicals Reduced glutathione (GSH), 1-chloro-2,4-dinitrobenzene (SD Fine Chem. Ltd., Mumbai), 5,5 -dithio-bis-2-nitrobenzoic acid, nicotine adenine dinucleotide phosphate, epinephrine (Sigma, MO, USA), ethylenediamine-tetra-acetic acid, hydrogen peroxide (Qualigens Fine Chemicals, Mumbai), thiobarbituric acid (Rolex Laboratory, Mumbai) were used for the study. All other reagents used were of analytical grade. 2.4. Animals Adult Wistar rats (120200 g) of either sex (King Institute of Preventive Medicine, Chennai) were maintained in well-ventilated room temperature with natural daynight cycle in large polypropylene cages. They were fed balanced rodent pellet diet (Poultry Research Station, Tamilnadu Veterinary and Animal Sciences University, Chennai) and water ad libitum through out the experimental period. The animals were quarantined for one week, prior to the experiments to acclimatize to laboratory conditions. The study protocol was approved by the IAEC (Institutional animal ethics committee of CPCSEA, Govt. of India). 2.5. Antioxidant activity The adult Wistar rats of either sex were divided into seven groups of six animals each. Group I received only propyleneglycol (5 ml/kg per day p.o.) for seven days and served as control. Group II animals received single dose of equal mixture of carbon tetrachloride and olive oil (50% v/v, 5 ml/kg i.p.) on the seventh day. Group III and IV animals were treated with AET at a dose level 250 and 500 mg/kg per day p.o., respectively, for seven days. On the seventh day, a single dose of equal mixture of carbon tetrachloride and olive oil was given (50% v/v, 5 ml/kg i.p.). Group V and VI animals were treated with MET at doses of 250 and 500 mg/kg per day p.o., respectively, for seven days and on the seventh day, a single dose of equal mixture of carbon tetrachloride and olive oil (50% v/v, 5 ml/kg i.p.) was administered. Group VII animals were treated with silymarin (25 mg/kg per day p.o.) for seven days and on the seventh day, a single dose of equal mixture of carbon tetrachloride and olive oil (50% v/v, 5 ml/kg i.p.) was administered. All animals were sacriced by cervical decapitation under light ether anesthesia on the eighth day. Immediately after sacrice, the liver were dissected out, washed in the ice-cold saline, and the homogenate was prepared in 0.1 M TrisHCl buffer (pH 7.4). The homogenate was centrifuged and the supernatant was used for the assay of marker enzymes namely glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GRD) by reported methods (Necheles et al., 1968; Habig et al., 1974; Dubler and Anderson, 1981, respectively). The activities of superoxide dismutase (SOD), catalase (CAT) were
determined by reported methods (Misra and Fridovich, 1972; Bergmeyer et al., 1994). LPO was estimated by the methods of Ohkawa et al. (1979). The total protein content was estimated by biuret methods (Doumas et al., 1971). 2.6. Statistical analysis The statistical analysis was carried out using one way analysis of variance (ANOVA) followed by Dunnets t-test. P-values <0.05 were considered as signicant.
3. Results The results of antioxidant activity of AET and MET on CCl4 -intoxicated rats are shown in Table 1. 3.1. Glutathione peroxidase (GPX) GPX activity in liver homogenates was signicantly (P < 0.001) reduced in CCl4 -treated animals when compared to control. The AET and MET treatment (500 mg/kg dose level) signicantly increased (P < 0.001) the GPX levels when compared to CCl4 -treated animals. However, AET and MET (250 mg/kg) showed less signicant increase in GPX levels (P < 0.02 and P < 0.01, respectively) in liver homogenate when compared to CCl4 -treated animals. Silymarin (25 mg/kg p.o.)-treated animals also showed signicant (P < 0.001) increases of GPX level in the liver homogenate compared with CCl4 -treated animals. 3.2. Glutathione S-transferase (GST) GST level in liver was signicantly reduced (P < 0.001) in CCl4 -treated animals when compared with normal animals. Treatment with AET and MET at 500 mg/kg dose showed signicant increase (P < 0.001) in GST when compared to CCl4 -treated group. AET and MET (250 mg/kg) also showed less signicant (P < 0.05 and P < 0.01) increase of GST in liver homogenate. A signicant (P < 0.001) increase of GST in the liver homogenate of Silymarin-treated animals was observed. 3.3. Glutathione reductase (GRD) In the present study, GRD activity was signicantly decreased (P < 0.001) in CCl4 -treated animals when compared to control. A signicant increase (P < 0.001) in the level of GRD was observed in AET and MET (500 mg/kg)treated rats when compared with CCl4 -treated animals. AET and MET (250 mg/kg) also showed less signicant (P < 0.02 and P < 0.01) increase in the GRD when compared with CCl4 -treated animals. Silymarin-treated group also showed signicant (P < 0.001) increase in the level of GRD when compared to CCl4 -treated animals.
229
Silymarin (25 mg/kg) + CCl4
3.4. Superoxide dismutase (SOD)

9.6b,c 12b,c 0.8b,c 3.5b,c 4.7b,c 0.7b,c
500
289.84 252.45 18.42 73 150.5 6.5
SOD level was signicantly reduced (P < 0.001) in CCl4 -treated animals when compared with normal animal. The AET (500 mg/kg) and MET (250 and 500 mg/kg) showed signicant increase (P < 0.001) in SOD when compared to CCl4 -treated animals; AET at 250 mg/kg showed only less signicant increase (P < 0.01) in SOD in liver homogenate compared with CCl4 -treated animals. Silymarintreated animals also showed signicant (P < 0.001) increase in SOD when compared to CCl4 -treated animals. 3.5. Catalase (CAT)
MET (mg/kg)
8.9b,e 1.3c,e 4.9b,c 3.7c,e 0.9c,e
4.2c,e
17.2b,c 5.1b,c 0.9b,c 4.3b,c 3.9b,c 0.8b,c
302.4 279.1 21.8 77.2 165 5.8
Thespesia populnea bark extracts + CCl4
CAT activity was signicantly (P < 0.001) reduced in CCl4 treatment when compared to control. AET and MET at 500 mg/kg dose signicantly increased (P < 0.001) CAT in liver homogenate when compared to CCl4 -treated animals. Treatment of AET and MET at 250 mg/kg dose also showed less signicant (P < 0.02 and P < 0.01) increase of CAT when compared with CCl4 -challenged animals. Silymarin-treated group also showed signicant (P < 0.001) increase of CAT when compared to CCl4 -treated animals. 3.6. Lipid peroxidation (LPO) LPO level of liver homogenates signicantly increased (P < 0.001) in CCl4 -challenged rats when compared to control rats. Treatment with AET and MET of 500 mg/kg dose showed signicant (P < 0.001) decrease in LPO when compared with CCl4 -treated animals. AET and MET at 250 mg/kg dose also showed less signicant (P < 0.01) decrease in LPO in liver homogenate when compared with CCl4 -treated animals. The silymarin-treated group showed a signicant (P < 0.001) decline in the LPO when compared to CCl4 -treated animals.
250 500 250
AET (mg/kg)
Table 1 Antioxidant activity of Thespesia populnea bark extracts against CCl4 -induced liver damage
CCl4 -treated group
Control
314.6 292.4 22.3 83.6 198 4.2
22.7 26.8 1.5 4.4 6.7 0.8
189.1 162.9 10.8 40.2 48 13.5
21.5a,b
16.9a,b 0.9a,b 2.3a,b 4.8a,b 1.3a,b
249.9 207.7 14.6 56.9 68.7 9.1
5.2c,f 1.2c,d 3.1c,e 2.1c,f 0.6c,e
12.7c,d
271.4 240.4 17.2 65.8 140 7.4
10.9b,c 0.9b,c 3.5b,c 9.9b,c 1.5b,c
13.1b,c
256.2 229.8 15.6 59.8 74.8 8.7
4. Discussion
Control compared with CCl4 -treated animals. P < 0.001. c CCl -treated animals compared with AET and MET. 4 d P < 0.02. e P < 0.01. f P < 0.05.
GPX plays a pivotal role in H2 O2 catabolism (Eaton, 1991) and the detoxication of endogenous metabolic peroxides and hydroperoxides which catalyses GSH (Floka, 1971). GPX activity was signicantly reduced after CCl4 treatment when compared to control. The reversal of the GPX activity to normal after pretreatment with AET and MET is due to antioxidant activity by scavenging/ detoxifying the endogenous metabolic peroxides generated after CCl4 injury in the liver tissues. Many investigators have suggested that GST offers protection against LPO by promoting the conjugation of toxic electrophiles with GSH (Jakoby, 1988). GST plays a physiological role in initiating the detoxication of potential alkylating agents. Chemicals like chloroform and CCl4 alter the hepatic GST activity (Aniya and Anders, 1985). GST level was signicantly reduced in CCl4 -treated animals and
GPX (nmol of GSH oxidized/min/mg protein) GST (nmol of CDNB conjugate formed/min/mg protein) GRD (nmol of GSSG utilized/min/mg protein) SOD (Kat/g protein) CAT (nmol of H2 O2 decomposed/min/mg protein) LPO (nmol of MDA/mg protein)
Parameters
230
upward reversal was observed after the treatment with AET and MET. This may be attributed to a direct action of the extract on the hepatic GST activation, the mechanism of which is not known. An increase in GRD activity implies that AET and MET protect the liver tissue from oxidative damage by GSH regenerated from its oxidized form (GSSG). In the present study, the SOD activity is signicantly reduced in CCl4 -intoxicated rats. The SOD activity was brought to near normal after treatment with the extracts in CCl4 -intoxicated rats. Decreased activity of CAT was observed in animals treated with CCl4 . Presumably, a decrease in CAT activity could be attributed to cross-linking and inactivation of the enzyme protein in the lipid peroxides. Decreased CAT activity is linked up to exhaustion of the enzyme as a result of oxidative stress caused by CCl4 . The CAT activity was restored to normal after treatment with extracts evidently shows the antioxidant property of the extracts against oxygen free radicals (OFRs). The level of LPO is a measure of membrane damage and alterations in structure and function of cellular membranes. The level of thiobarbituric acid relative substance (TBARS) is an indirect measurement of lipid peroxidation (Halliwell et al., 1995). Lipid peroxide levels in tissue were found to be signicantly elevated in CCl4 -challenged rats. This toxic effect is the consequence of CCl4 activation by cytochrome P450 to trichloromethyl radical (CCl3 ) which readily reacts with oxygen to form trichloromethyl peroxyl (CCl3 O2 ) radical (Tappel, 1973). These free radicals trigger cell damage through two mechanisms namely covalent binding to cellular macromolecules and lipid peroxidation which affect the ionic permeability of the membrane preventing the disintegration and solubilization of membrane structure. The diminished LPO activity after treatment with the extracts may be attributed to the antioxidant activity of the plant by scavenging the CCl3 radical generated due to the metabolic transformation of CCl4 in the liver. Free radical reactions are implicated in the progression of cancer, inammation, atherosclerosis, hepatocellular damage and the biological process of aging. The hepatoprotective action combined with antioxidant activity has a synergistic effect to prevent the process of initiation and progress of hepatocellular diseases (Wilkinson, 1962).
Although the precise mechanism of action of AET and MET has not been elucidated, it can be safely assumed that the lowering of enzyme levels is responsible for cell injury and enhancing the enzymes responsible for antioxidant activity. It can be concluded that the AET and MET possess denite antioxidant activities either through stabilization of cellular membrane or antiperoxidase activity.
References
Aniya, Y., Anders, M.W., 1985. Alteration of hepatic glutathione-Stransferase and release into serum after treatment with bromobenzene and carbon tetrachloride. Biochemical Pharmacology 39, 42394244. Anonymous, 1995. The Wealth of India. Publication and Information Directorate (CSIR), New Delhi, pp. 223275. Bergmeyer, H.V., Gawehn, K., Grassik, M., 1994. Methods of enzymatic analysis. In: Bergmeyer, H.V., Chemie, V., Weinhein, S. (Eds.). Academic Press, New York, p. 348. Doumas, B.T., Watson, W.A., Biggs, A.G., 1971. Estimation of total protein. Clinical Chemistry Acta 31, 8796. Dubler, R.E., Anderson, B.M., 1981. Simultaneous inactivation of the catalytic activities of yeast glutathione reductase by N-alkyl meleimimdes. Biochemica Biophysica Acta 659, 70. Eaton, J.W., 1991. Catalase, glutathione peroxidase and hydrogen peroxidase. Journal of Laboratory and Clinical Medicine 118, 34. Floka, L., 1971. Glutathione peroxidase enzymologic and biological aspects. Klin Nochenschr 32, 49. Habig, W.H., Pabst, M.J., Jocoby, W.B., 1974. The rst enzyme step in mercaptouric acid formation. Journal of Biological Chemistry 249, 71307139. Halliwell, B., Aeschbach, R., Loligger, J., Aruoma, O.I., 1995. The characteristic of antioxidants. Federal Chemical Toxicology 33, 601617. Jakoby, W.B., 1988. Detoxication, conjugation and hydrolysis in liver biology and pathology. In: Arias, I.M., Jakoby, W.B. (Eds.), Raven Press, New York, pp. 375385. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry 247, 31703185. Necheles, T.F., Boles, T.A., Allen, D.M., 1968. Glutathione peroxidase assay method. Journal of Pediatrics 72, 319. Ohkawa, H., Onishi, N., Yagi, K., 1979. Assay of lipid peroxidation in animal tissue by thiobarbituric acid reaction. Analytical Biochemistry 95, 351354. Tappel, A.C., 1973. Lipid peroxidation damage to cell components. Federal Proceedings 32, 18701874. Wilkinson, J.H., 1962. An introduction to diagnostic enzymology. In: Arnold, E. (Ed.). Academic press, London, p. 84.
Screening and comparison of antioxidant activity of solvent extracts of herbal medicines used in Korea
Dae Gill Kang a , Chi keun Yun b , Ho Sub Lee a,
a
Department of Herbal Resources, Professional Graduate School of Oriental Medicine and Medicinal Resources Research Center (MRRC), Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea b Department of Health Administration, Wonkwang University, Iksan, Jeonbuk 570-749, Republic of Korea Received 20 March 2002; received in revised form 7 April 2003; accepted 17 April 2003
Abstract The hexane, ethylacetate, n-butanol, and water extracts of 10 Korean herbal medicines were screened and compared for their antioxidant activities in a range of lipid peroxidation system using rat brain homogenates, antihemolysis assay of red blood cells, and other in vitro assays to determine their ability to scavenge superoxide and hydroxyl radicals. All of the 10 Korean herbal medicines have potent antioxidant activities. Among the four solvent extracts, the antioxidant activities of more-polar solvent extracts (BuOH and water extracts) were relatively higher than that of non-polar solvent extracts (hexane and EtOAC extracts). These results will be useful to further analyze those herbal medicines that contain the most antioxidant activity in order to identify the active principles. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Antioxidant; Korean herbal medicines; Solvent extracts; Screening
1. Introduction Reactive oxygen species (ROS), such as hydroxyl radical, hydrogen peroxide, and superoxide anions, are produced as byproducts in aerobic organisms, and have been implicated in the pathology of a vast variety of human diseases including cancer, atherosclerosis, diabetic mellitus, hypertension, AIDS, and aging (Halliwell and Gutteridge, 1984; Wallace, 1999; Lee et al., 2000). Therefore, antioxidant activity is an important in-view of the free radical theory of aging and associated diseases. It has long been recognized that naturally occurring substances in higher plants have antioxidant activity. A great number of plant origin substances have been suggested to appear as antioxidants. Flavonoids and other phenolic compounds of plant origin have been reported as scavenger ROS, thus they are viewed as promising therapeutic drugs for free radical pathologies (Parshad et al., 1998; Lee et al., 2000). The antioxidant activity of plant origin is dependent on the type and polarity of the extracting solvent as well as on the test system and the substrate to be protected by the antioxidant (Heinonen et al., 1998; Moure et al., 2000; Kang
and Lee, 2001). Solvent extraction is frequently used for isolation of the antioxidants and both extraction yield and antioxidant activity of the extracts are strongly dependent on the solvent, due to the different antioxidant potentials of compounds with different polarity. For these reasons, comparative studies for selecting the optimal solvent providing maximum antioxidant activity are required for each substrate. Although the use of different polarity substances can provide more exhaustive information on the properties of the extracts, the literature contains few reports of the polarity-based solvent extraction of medicinal plants. The present study was undertaken to perform the screening of antioxidant properties of 10 traditional medicines grown in Korea, and we selected hexane, ethylacetate, n-BuOH, and water as extract solvents which permits comparison of the antioxidant properties among the polaritybased solvent extracts of medicinal plants.
2. Materials and methods 2.1. Chemicals Hexane, ethylacetate (EtOAC), and n-butanol (n-BuOH) were purchased from Merck (Darmstate, Germany). Phenazine methosulfate (PMS)--nicotinamide adenine
Corresponding
author. Tel.: +82-63-850-6841; fax: +82-63-850-7324. E-mail address: host@wonkwang.ac.kr (H.S. Lee).
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Table 1 Latin names, herbarium voucher specimen numbers, plant parts, and uses in Korea Herbal medicines Inula helenium L. Astragalus membranaceus BUNGE Atratylodes koreana NAKAI Gardenia jasminoides for. Grandiora MAKINO Magnolia liliora DESR. Scutellaria baicalensis GEORGI Siegesbeckia orientalis L. Sinomenium acutum REHDER et WILS. Sorbus amurensis KOEHNE Xanthium strumarium L. Voucher specimen numbers DH-43 DH-14 DH-07 DH-08 DH-34 DH-05 DH-57 DH-15 DH-41 DH-58 Plant parts Root Root Root Fruit Flower Root Whole Root Bark Fruit Uses in Korea Antibacterial, disinsection Cardiotonic, diuretic Sedative, hypoglycemic Sedative, cholagogue Antihypertensive Antipyretic, antiallergy Antihypertensive Analgesia, antiinammation Expectorant Analgesia
dinucleotide (reduced form, NADH), nitroblue tetrazolium (NBT), l-ascorbic acid, cytochrome c, dl-dithiothreitol, thiobarbituric acid (TBA), Butylated hydroxyanisole (BHA), and 2,2 -azo-bis-(2-amidinopropane)dihydrochloride (AAPH) were purchased from Sigma (St. Louise, MO, USA). All other unstated chemicals and reagents were of analytical grade. 2.2. Plant material
USA). l-Ascorbic acid was used as a positive control. The superoxide radical scavenging ratio (%) was calculated using the following formula: Superoxide radical scavenging ratio (%) = A A1 100 A
where A is the absorbance of positive control, and A1 is the absorbance of the test samples. 2.5. Scavenging activities of hydroxyl radicals
All herbal medicines were purchased from a herbal market in Iksan, Jeonbuk Province, South Korea. Dr. Kyu Kwan Jang at the Botanical Garden of Wokwang University identied plant materials. Herbarium voucher specimens were prepared and deposited in the herbarium of the Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk, South Korea (Table 1). 2.3. Extraction For the partitioning by solvent, the Korean herbal medicines (100 g) were air-dried at room temperature and reduced to ne powder by milling. The resulting powder was subjected to extraction with 200 ml of methanol, three times, 24 h each. The methanol extract was evaporated and resuspended in H2 O, and sequentially partitioned with n-hexane, EtOAC, and BuOH. 2.4. Scavenging activities of superoxide radicals Scavenging activity of superoxide radicals was determined by the Liu and Ng method (2000), which was slightly modied by Kang and Lee (2001). Superoxide radicals were generated in a PMS--nicotinamide adenine dinucleotide (reduced form, NADH) system by oxidation of NADH and were assayed by the reduction of NBT in the microplate. The superoxide radicals were generated in 200 l of 16 mM TrisHCl buffer (pH 8.0), which contained 78 M NADH, 50 M NBT, 10 M PMS and samples (10 l) to be tested at different concentrations. The color reaction between superoxide radicals and NBT was detected at A560 nm using a microplate reader (Molecular Devices, Synnyvate, CA,
Scavenging activity of hydroxyl radicals was determined by the Liu and Ng method (2000), which was slightly modied by Kang and Lee (2001). The hydroxyl radicals were generated in a l-ascorbic acidCuSO4 system by reduction of Cu2+ and were assayed by the oxidation of cytochrome c in the 96-well microplate. The hydroxyl radicals were generated in 200 l of 10 mM sodium phosphate buffer (pH 7.4), containing 100 M l-ascorbic acid, 100 M CuSO4 , 12 M cytochrome c and the samples to be tested at different concentrations. The reduced cytochrome c was produced by addition of excess dl-dithiothreitol and followed by Sephadex G-15 chromatography (bed volume, 10 ml) to remove excess dl-dithiothreitiol. The change in absorbance caused by the oxidation of cytochrome c was measured at 550 nm using a microplate reader (Molecular Devices). Thiourea was used as a positive control. The scavenging activity of hydroxyl radical by 500 g/ml of thiourea was taken as 100%. The scavenging activity of hydroxyl radical was calculated using the following formula: Hydroxyl radical scavenging activity (%) = A A0 100 AT A 0
where A is the absorbance of samples, and AT and A0 are the absorbance of the thiourea and the control, respectively. 2.6. Inhibitory effects on erythrocyte hemolysis Whole blood was obtained carefully by cannulation of femoral artery in SpragueDawley rats and collected in heparinized tubes. Erythrocytes were isolated from plasma by centrifugation (1000 g for 20 min) and washed three times
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with 10 volumes of saline solution. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system (Niki et al., 1988). A 10% suspension of erythrocytes in 10 mM of phosphate-buffered saline (PBS, pH 7.4) was added to the same volume of 200 mM AAPH in PBS solution containing samples to be tested at different concentrations. The reaction mixture was shaken gently while being incubated at 37 C for 2 h. The reaction mixture was then removed by centrifugation (1000 g for 20 min), diluted with eight volumes of PBS and centrifuged at 1000 g for 20 min. The absorbance (A) of supernatant was read at 540 nm using spectrophotometer (Milton Roy, Rochester, NY, USA). Similarly, the reaction mixture was treated with eight volumes of distilled water to achieve complete hemolysis, and the absorbance (B) of the supernatant obtained after centrifugation was measured at 540 nm. The inhibition of hemolysis (%) was calculated by the equation (1 A/B) 100. l-Ascorbic acid was used as a positive control. 2.7. Inhibitory effects on lipid peroxide (LPO) production in brain homogenates For the in vitro studies, the brains of normal Sprague Dawley rats were isolated and homogenized with Polytron homogenizer (Switzerland) in ice-cold TrisHCl buffer (20 mM, pH 7.4) to produce a 10% (w/v) homogenate. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant (0.5 ml) was incubated with the test samples in the presence of 10 M FeSO4 and 0.1 mM ascorbic acid at 37 C for 1 h. The reaction was stopped by addition of 0.5 ml trichloroacetic acid (TCA, 28%, w/v) and 0.75 ml thiobarbituric acid (TBA, 1%, w/v) in succession, and the solution was then heated at 100 C for 15 min. After centrifugation to remove precipitated protein, the color of the malondialdehyde (MDA)-TBA complex was detected at OD 532 nm using spectrophotometer (Milton Roy). BHA was used as a positive control. The inhibition ratio (%) was calculated using the following formula: Inhibition ratio (%) = A A1 100 A
3.1. Scavenging effects of solvent extract on O2 radical The four solvent extracts of 10 herbal medicines were screened for their superoxide-scavenging activity in the PMS/NADHNBT system, and the results are shown in Table 2. In the PMS/NADHNBT system, superoxide anion derived from dissolved oxygen by PMS/NADH coupling reaction reduces NBT. The decrease of absorbance at 560 nm with antioxidants thus indicates the consumption of superoxide anion in the reaction mixture. There was a difference in the overall scavenging ability among the extract solvents from the 40 extracts and even among the same species. Seven of the extracts, at 200 g/ml assay, displayed scavenging activities that were greater than 50%, while seven extracts exhibited a nearly zero-scavenging activity of the superoxide radical. Among them, the water extract of Sinomenium acutum showed the highest scavenging activity of superoxide radical in this system. 3.2. Scavenging effects of solvent extract on OH radical Table 3 shows the scavenging effect of solvent extracts of 10 Korean herbal drugs on hydroxyl radicals generated by l-ascorbic acid/CuSO4 cytochrome c system. The hydroxyl radicals were generated in a l-ascorbic acid/CuSO4 system by reduction of Cu2+ and were assayed by the oxidation of cytochrome c. High-scavenging activity (50% at 200 g/ml assay) was found in the BuOH extracts of Astragalus membranaceus, Siegesbeckia orientalis, and Sorbus amurensis, and EtOAC extracts of Scutellaria baicalensis and Sinomenium acutum, and hexane extracts of Sorbus amurensis. 3.3. Inhibitory effects on erythrocyte hemolysis The azo compound generates few radicals by its unimolecular thermal decomposition. The rate of generation of peroxyl radicals can be easily controlled and measured by adjusting the concentration of AAPH (Miki et al., 1987). Therefore, hemolysis induced by AAPH must provide suitable means for studying the oxidative erythrocyte membrane damage by peroxyl radical attack from the outside of the membrane (Ng et al., 2000). Of the extracts studied, BuOH extracts of Astragalus membranaceus, Atratylodes koreana, Magnolia liliora, Xanthium strumarium, and Scutellaria baicalensis, and water extracts of Gardenia jasminoides and Sinomenium acutum, and EtOAC extracts of Scutellaria baicalensis, tested at 100 and 200 ug/ml, markedly inhibited erythrocyte hemolysis in this system (Table 4). 3.4. Inhibitory effects on LPO production in brain homogenates Quantication of MDA, one of the products of lipid peroxidation, with TBA is the most common assay used for determination of the rate extent of lipid peroxidation.
where A is the absorbance of control, and A1 is the absorbance of the test samples.
3. Results and discussion The methanol extracts of 10 Korean herbal drugs were divided into four fractions with different polarities by partitioning it in various solvents such as hexane, EtOAC, n-BuOH, and H2 O. Then these solvent extracts were tested for their antioxidant activity in a range of lipid peroxidation systems using rat brain homogenates, red blood cells and other in vitro assay to determine their ability to scavenge ROS.
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Table 2 Effect of solvent extracts of Korean herbal medicines on superoxide radicals generated by PMS/NADH system Samples Concentration (g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 7.5 4.2 18.2 3.7 nz nz nz nz 17.1 7.0 45.1 1.4 nz nz nz 16.7 1.0 9.4 2.7 16.7 2.4 6.1 1.2 8.2 2.1 nz nz nz nz EtOAC 5.76 2.3 12.4 2.8 nz nz nz nz 18.8 3.5 47.9 3.2 5.8 1.1 12.4 2.2 65.9 1.4 83.8 1.3 nz 20.0 6.3 13.9 2.9 16.0 0.1 nz 5.5 7.8 14.7 3.5 31.7 1.3 n-BuOH 21.7 1.8 31.6 9.1 20.2 5.8 40.1 6.5 38.2 1.3 56.5 3.6 29.8 3.5 44.4 5.6 27.9 1.2 37.9 3.4 56.8 1.7 75.6 1.7 30.7 5.6 49.6 2.7 nz 14.3 3.1 60.1 1.6 78.1 3.5 31.9 6.5 61.7 2.6 H2 O 22.3 0.9 23.2 8.3 29.0 8.0 33.2 4.3 6.5 7.0 13.5 3.2 54.6 0.4 68.8 1.2 19.8 3.6 52.5 1.5 26.3 3.8 30.1 1.1 53.5 5.5 67.6 0.6 90.2 0.5 91.7 0.1 34.6 1.1 41.1 4.1 14.6 4.0 49.1 4.1
Results show mean S.E. (n = 3) of the inhibition of superoxide radical (%); nz: nearly zero.
Table 3 Effect of solvent extracts of Korean herbal medicines on hydroxyl radicals generated by l-ascorbic acid/Cu2+ system Samples Concentration (g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 12.4 1.9 16.7 3.7 11.4 2.7 15.7 3.7 15.8 5.0 27.6 6.1 nz nz 12.9 1.5 29.3 2.9 25.8 2.5 38.7 1.9 9.2 0.9 19.1 1.6 5.5 2.3 13.6 1.6 32.8 0.8 51.9 8.8 13.4 2.9 14.9 2.4 EtOAC 31.5 2.6 45.8 2.5 23.5 2.2 28.8 2.6 13.7 7.0 24.3 5.3 32.1 4.6 46.7 0.9 10.0 0.8 28.8 1.0 32.5 4.8 63.4 3.7 25.3 3.6 28.1 2.2 34.7 3.8 85.5 3.7 10.7 1.1 15.0 1.5 24.5 6.0 28.2 1.5 n-BuOH 6.7 0.4 17.1 0.3 40.6 7.7 49.8 4.6 19.1 2.1 31.4 5.9 16.7 7.0 40.2 4.1 12.8 1.9 30.9 0.9 34.0 2.8 44.8 1.0 67.1 0.3 84.5 4.4 27.0 4.4 33.6 0.5 28.1 2.0 48.9 3.1 11.1 2.8 30.2 0.5 H2 O 5.7 1.9 10.3 0.5 20.7 3.3 31.5 7.3 6.2 1.0 21.1 2.2 24.8 1.4 39.1 1.0 5.23 1.2 9.96 1.6 11.0 1.2 10.5 2.8 26.5 9.7 41.4 2.0 34.6 1.7 41.8 3.7 7.1 3.4 16.3 1.7 5.5 1.4 10.2 1.4
Results show mean S.E. (n = 3) of the inhibition of hydroxyl radical (%); nz: nearly zero.
D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231236 Table 4 Effect of solvent extracts of Korean herbal medicines on the inhibition of hemolysis by AAPH system Samples Concentration (g/ml) Solvents Hexane Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 100 200 18.1 3.5 28.2 0.7 6.5 4.8 24.4 2.8 8.7 6.8 12.0 0.1 23.8 3.4 24.5 2.0 12.2 2.0 16.1 0.6 9.8 0.2 12.2 0.8 21.7 1.1 21.6 0.9 17.0 2.9 25.2 3.5 11.4 1.5 16.4 2.7 8.6 5.6 12.7 2.3 EtOAC 20.9 2.3 40.8 2.2 8.4 1.9 14.6 0.3 12.2 2.5 13.2 0.2 37.6 3.3 65.9 7.4 13.4 1.7 25.2 0.4 96.9 0.9 97.6 1.6 16.1 2.8 22.1 1.0 32.2 3.8 43.9 2.7 13.5 3.3 20.9 1.5 43.2 0.5 58.3 1.1 n-BuOH 58.8 2.0 73.6 0.2 94.9 1.0 94.5 0.4 96.4 1.1 97.8 0.1 56.3 0.8 79.3 1.6 97.2 1.0 98.7 0.2 87.9 1.4 97.1 0.3 56.3 3.9 76.3 0.2 11.4 3.6 30.2 5.2 64.6 2.0 78.9 2.1 94.8 0.4 95.3 0.1 H2 O
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12.3 3.7 21.6 2.9 38.8 2.5 48.6 2.3 7.2 5.9 14.3 4.9 92.8 0.2 96.7 0.4 33.3 2.5 55.2 1.3 26.9 2.2 54.5 0.7 41.1 4.6 75.2 2.1 93.2 1.3 98.5 0.2 23.0 0.2 28.9 1.3 43.0 1.4 69.4 1.2
Results show mean S.E. (n = 3) of the inhibition of erythrocyte hemolysis (%).
Our experiment proved that incubation of the rat brain homogenate with Fe2+ /ascorbate at pH 7.4 causes rapid peroxidation, detectable by the TBA method. Table 5 shows the activities of the representative 10 extracts, which showed high-antihemolysis activity, against lipid peroxidation of the rat brain homogenate. All the extracts markedly inhibited LPO production in the brain homogenates (Table 5). Medicinal herbs are known to contain a variety of antioxidants. Numerous substances have been suggested to appear as antioxidants. The most detailed investigations so far were concerned with reactions involving phenolic compounds, ranging from polymer chemistry to biochemistry
Table 5 Inhibitory effect of solvent extracts of Korean herbal medicines on the lipid peroxide production in brain homogenates Plants Inula helenium Astragalus membranaceus Atratylodes koreana Gardenia jasminoides Magnolia liliora Scutellaria baicalensis Siegesbeckia orientalis Sinomenium acutum Sorbus amurensis Xanthium strumarium Concentration (g/ml) 200 200 200 200 200 200 200 200 200 200 Solvents BuOH BuOH BuOH H2 O BuOH EtOAC BuOH H2 O BuOH BuOH Inhibition (%) 88.8 90.8 90.9 83.8 93.7 96.2 90.5 91.2 90.7 88.8 0.2 0.1 1.5 0.8 0.1 0.0 1.1 0.2 0.7 1.4
Results show mean S.E. (n = 3) of the inhibition of production of LPO.
and food chemistry (Bravo, 1998). It has been revealed that various phenolic antioxidants, such as avonoids, tannins, coumarins, xanthones and more recently procyanidins scavenge radicals dose-dependently, thus they are viewed as promising therapeutic drugs for free radical pathologies (Lee et al., 2000). Flavonoids are 15-carbon aromatic pigments found in green plants and include chalcones, avonones, avones, biavonoids, dihydroavonols, anthrocyanidins, and avonols (VanderJagt et al., 2002). More than 4000 naturally occurring avonoids have been described (Hollman and Katan, 1998). The present study suggests that more-polar components present in herbal medicinal extracts contributed towards the increased ROS-scavenging activity. Although there are no direct evidences in this study, the antioxidant activities shown by BuOH extracts and/or water extracts of herbal medicines could be related to the presence of phenolic compounds such as tannins and avonoids because they contain an aromatic hydroxyl moiety (Yesilada et al., 2000; Ramezani et al., 2001). As well known, polyphenols are very important constituents because of their scavenging ability with ROS and chelating ability with divalent cations due to their hydroxyl groups (de las Heras et al., 1998; Hatano et al., 1989; Lopes et al., 1999). Although the active principles responsible for the antioxidant activity of the tested extracts have not yet been isolated in this study, it will be useful to further analyze those herbal medicines that contain the most antioxidant activity
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D.G. Kang et al. / Journal of Ethnopharmacology 87 (2003) 231236 damage after transient global ischemia in gerbils. Neuroscience Letter 287, 191194. Lee, Y.M., Kim, H., Hong, E.K., Kang, B.H., Kim, S.J., 2000. Water extract of 1:1 mixture of Phellodendron cortex and Aralia cortex has inhibitory effects on oxidative stress in kidney of diabetic rats. Journal of Ethnopharmacology 73, 429436. Liu, F., Ng, T.B., 2000. Antioxidative and free radical scavenging activities of selected medicinal herbs. Life Sciences 66, 725735. Lopes, G.K., Schulman, H.M., Hermes-Lima, M., 1999. Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions. Biochimica Biophysica Acta 1472, 142 152. Miki, M., Tamai, H., Mino, M., Yamamoto, Y., Niki, E., 1987. Free-radical chain oxidation of rat red blood cells by molecular oxygen and its inhibition by alpha-tocopherol. Archives of Biochemisty and Biophysics 258, 373380. Moure, A., Franco, D., Sineiro, J., Dominguez, H., Nunez, M.J., Lema, J.M., 2000. Evaluation of extracts from Gevuina avellana hulls as antioxidants. Journal of Agricultural and Food Chemistry 48, 3890 3897. Ng, T.B., Liu, F., Wang, Z.T., 2000. Antioxidative activity of natural products from plants. Life Sciences 66, 709723. Niki, E., Komuro, E., Takahashi, M., Urano, S., Ito, E., Terao, K., 1988. Oxidative hemolysis of erythrocytes and its inhibition by free radical scavengers. Journal of Biological Chemistry 263, 19809 19814. Parshad, R., Sanford, K.K., Price, F.M., Steele, V.E., Tarone, R.E., Kelloff, G.J., Boone, C.W., 1998. Protective action of plant polyphenols on radiation-induced chromatid breaks in cultured human cells. Anticancer Research 18, 32633266. Ramezani, M., Hosseinzadeh, H., Daneshmand, N., 2001. Antinociceptive effect of Elaeagnus angustifolia fruit seeds in mice. Fitoterapia 72, 255262. VanderJagt, T.J., Ghattas, R., VanderJagt, D.J., Crossey, M., Glew, R.H., 2002. Comparison of the total antioxidant content of 30 widely used medicinal plants of New Mexico. Life Sciences 70, 10351040. Wallace, D.C., 1999. Mitochondrial diseases in man and mouse. Science 283, 14821488. Yesilada, E., Tsuchiya, K., Takaishi, Y., Kawazoe, K., 2000. Isolation and characterization of free radical scavenging avonoid glycosides from the owers of Spartium junceum by activity-guided fractionation. Journal of Ethnopharmacology 73, 471478.
in order to identify the active principles. Then it would lead to a better understanding of the kinds of antioxidants used in Korea as herbal medicines.
Acknowledgements This study was supported by the Brain Korea 21 Project and grant of the Oriental Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea (HMP-00-CO03-0003).
References
Bravo, L., 1998. Polyphenols: chemistry, dietary sources, metabolism, and nutritional signicance. Nutrition Reviews 56, 317333. Halliwell, B., Gutteridge, J.M., 1984. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1, 13961397. Hatano, T., Edamatsu, R., Haramatsu, M., Mori, A., Fujita, Y., Yasyhara, T., Yoshida, T., Okuda, T., 1989. Effects of the interaction of tannins with co-existing substances VI. Effects of tannins and related polyphenols on superoxide anion radical, and on DPPH radical. Chemical Pharmaceutical Bulletin 37, 20162021. Heinonen, I.M., Lehtonen, P.J., Hopia, A.I., 1998. Antioxidant Activity of Berry and Fruit Wines and Liquors. Journal of Agricultural and Food Chemistry 46, 2531. de las Heras, B., Slowing, K., Benedi, J., Carretero, E., Ortega, T., Toledo, C., Bermejo, P., Iglesias, I., Abad, M.J., Gomez-Serranillos, P., Liso, P.A., Villar, A., Chiriboga, X., 1998. Antiinammatory and antioxidant activity of plants used in traditional medicine in Ecuador. Journal of Ethnopharmacology 61, 161166. Hollman, P.C., Katan, M.B., 1998. Bioavailability and health effects of dietary avonols in man. Toxicology Supplement 20, 237248. Kang, D.G., Lee, H.S., 2001. An improved method in screening of superoxide and hydroxyl radical scavenging activities of plant medicinal extracts. Korean Journal of Pharmacognosy 32, 253256. Lee, S., Suh, S., Kim, S., 2000. Protective effects of the green tea polyphenol (-)-epigallocatechin gallate against hippocampal neuronal
Antihepatotoxic activity of seeds of Cichorium intybus

Bahar Ahmed a,b, , Tawfeq A. Al-Howiriny b , Abu B. Siddiqui a
a
Antihepatotoxic Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India b Department of Pharmacognosy, Medicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia Received 5 June 2002; received in revised form 2 April 2003; accepted 17 April 2003
Abstract The different fractions of alcoholic extract and one phenolic compound AB-IV of seeds of Cichorium intybus Linn were screened for antihepatotoxic activity on carbon tetrachloride (CCl4 )-induced liver damage in albino rats. The degree of protection was measured using biochemical parameters like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALKP), and total protein (TP). The methanol fraction and compound AB-IV were found to possess a potent antihepatotoxic activity comparable to the standard drug Silymarin (Silybon-70). The histopathological study of the liver was also carried out, wherein the methanolic fraction and compound AB-IV showed almost complete normalization of the tissues as neither fatty accumulation nor necrosis was observed. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Cichorium intybus; Antihepatotoxic activity; Histopathology; Silymarin
1. Introduction The crude extracts of about 100 Indian medicinal plants belonging to 40 families are used in the herbal formulations for the treatment of various diseases of the liver. In addition, about 600 commercial preparations, mainly plant crude extracts with claimed liver-protecting activity, are available all over the world (Handa et al., 1986). The plant Cichorium intybus Linn (Family: Compositae, Asteraceae) commonly known as Chicory or Kasni is also used as liver tonic, cardiotonic, diuretic, stomachic, cholagogue, depurative, emmenagogue, hepatomegaly, cephalalgia, inammations, anorexia, dyspepsia, atulence, colic, jaundice, splenomegaly, amenorrhea dysmenorrhea, and asthma, etc. (The Wealth of India, 1992; Sala, 1994). The seed of the plant is also one of the main ingredients of Jigrine, a commercial product of Hamdard (Waqf) Dawakahna, New Delhi, which is used for the treatment of various diseases of liver. The literature survey revealed that neither the systematic assessment of antihepatotoxic activity nor phytochemical investigation of the seeds of the plant have been done so far. We have recently reported one new and one rare sterol from
author. Tel.: +966-1-467-7210; fax: +966-1-467-7245. E-mail addresses: drbahmed@rediffmail.com, bahmed@ksu.edu.sa (B. Ahmed).
Corresponding
ethyl acetate fraction of the seeds, namely cichosterol, characterized as 13,14-seco-stigma 5(6),14(15)-diene-3--ol and stigma 5(6)-ene-3--O-(-d-glucopyranoside), respectively (Ahmed et al., 2002). We now report a thorough pharmacological screening for the antihepatotoxic activity of different fractions of the seeds of the plant on carbon tetrachloride (CCl4 )-induced liver damage in rats. The study showed different degrees of activity on measuring the different biochemical parameters like aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALKP), and total protein (TP), wherein the methanol fraction and compound AB-IV were found to be most active. The histopathological study of the liver of the methanolic fraction and compound AB-IV (isolated from methanol fraction) also showed almost complete normalization of the liver tissues as neither fatty accumulation nor necrosis was observed. The central vein appeared clearly indicating a potent antihepatotoxic activity.
2. Materials and methods 2.1. Plant material The seeds of Cichorium intybus L. were procured from Khari Bawli market, Delhi, India and were identied by a taxonomist, Department of Botany, Hamdard University,
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New Delhi, where a voucher specimen no. 765 has been deposited for future reference. 2.2. Preparation of the plant extracts The dried seeds were crushed to a coarse powder (9.0 kg) and were exhaustively extracted with ethanol by cold percolation. The crude alcoholic extract was concentrated under reduced pressure to get a viscous mass (950 g). It was dissolved in boiling methanol and kept at room temperature, solidied fats (250 g) were removed by suction, and the ltrate so obtained was concentrated to get a viscous solid, and then subsequently fractionated into petroleum ether (6080 C) (200 g), ethyl acetate (250 g), and methanol soluble (150 g) fractions. The petroleum ether fraction could not be analyzed for its chemical components, since it was an oily material. The ethyl acetate fraction afforded a new steroid named as cichosterol and a rare steroid namely stigma (Ahmed et al., 2002). The methanol fraction (100 g) on column chromatography (CHCl3 MeOH = 3:2) afforded a phenolic compound AB-IV (5.0 g) as viscous solid, which gave a positive test for phenols. All fractions and isolated compounds were ltered using a 0.2 m nylon lter. The purity of all compounds was checked by TLC and HPLC. After evaporating the solvents, the above fractions, compound AB-IV and the total alcoholic extract (100 g) were prepared with gum acacia (1:1) for oral administration to albino rats. 2.3. Experimental animals The study was carried out on Wistar albino rats (150 200 g) of either sex as reported in the literature (Handa and Singh, 1995). The rats were bred in a colony in the Central Animal House of Jamia Hamdard. They were fed with a standard pellet diet (Gold Mohar, Lipton India Ltd., Kolkata) and water ad libitum. Before their use in the experiment, the rats were kept in standard environmental conditions (2528 C, 6070% relative humidity and 12/12 h light/dark cycle). Eight animals in each group were used in all sets of experiments. 2.4. Testing of antihepatotoxic activity Animals were divided into eight groups of six rats in each for all the experiment. The rst group served as vehicle control and received normal saline only. The second group served as CCl4 -intoxicated control and received by gavage vehicle (normal saline) and CCl4 diluted with liquid parafn (1:1, single dose). The third group was given standard drug Silymarin at the dose of 70 mg/kg body weight and the remaining groups were given different extracts at the dose of 500 mg/kg body weight (group 8 received 250 mg/kg) and CCl4 . The vehicle (1% gum acacia in distilled water) or test drugs were administered orally for 5 days. CCl4 diluted with liquid parafn (1:1) was administered in a dose
of 1.5 ml/kg by oral route. After 24 h of CCl4 administration, blood of rats was obtained by puncturing retro-orbital plexus. The livers of animals from each group were taken for histopathological studies. The blood samples of each animal were taken and allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm at 30 C for 15 min and analyzed for various biochemical parameters. 2.5. Assessment of the liver function The biochemical parameters such as AST, ALT, and AKLP were estimated by reported methods (Reitman and Frankel, 1957; Kind and King, 1954). The TP was also measured according to the reported methods (Wooton, 1964; Dumas et al., 1971). 2.6. Statistical analysis The results of the biochemical estimations are reported as mean S.E. Total variation present in a set of data was estimated by one-way ANOVA, students t-test and Dennetts test were used for determining the signicance (Woolson, 1987; Dennett, 1964). 2.7. Histopathological studies of the liver The histopathological studies were carried out by reported method (Luna, 1968). The rats were sacriced under light ether anesthesia after 24 h of the last dosage, and the livers removed and washed with normal saline. Small pieces of liver tissues were processed and embedded in parafn wax. Sections of 56 m in thickness were cut, stained with hematoxylin and eosin, and then studied under an electron microscope.
3. Results and discussions As shown in Table 1, activities of liver enzymes, ALT, AST, and ALKP, were markedly elevated, while the TP level was decreased in CCl4 -treated animals in comparison to normal values. Administration of different extracts of the seeds of Cichorium intybus markedly prevented CCl4 -induced elevation of AST, ALT and ALKP, and diminution of TP. The petroleum ether, ethyl acetate, methanol, total alcoholic extracts, and compound AB-IV decreased the levels of ALT, AST, and ALKP, while the level of TP increased against CCl4 -intoxicated control. The methanol fraction and compound AB-IV were found to be most active at the dose levels of 500 and 250 mg/kg, respectively, exhibiting a decrease in ALT, AST, and ALKP as compared to standard drug Silymarin against intoxicated control in comparison to normal values. The level of TP was increased by all fractions in different proportions, wherein methanol and compound AB-IV were found to be most active as compared to
B. Ahmed et al. / Journal of Ethnopharmacology 87 (2003) 237240
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Table 1 Effect of different fractions and the isolated compound of the total alcoholic extract of the seeds of Cichorium intybus on biochemical parameters in albino rats intoxicated with CCl4 Groups 1 2 3 4 5 6 7 8 Treatment Normal (control) CCl4 (toxicity control) Silymarin (standard drug) Petroleum ether fraction Ethyl acetate fraction Methanol fraction Total alcoholic extract Compound AB-IV Dosea Normal saline only 1.5 ml/kg (p.o.) 70 mg/kg (p.o.) 500 mg/kg (p.o.) 500 mg/kg (p.o.) 500 mg/kg (p.o.) 500 mg/kg (p.o.) 250 mg/kg (p.o.) ALT (units/l) 65.50 248.16 38.88 105.55 99.99 24.0 108.88 29.99 173 16.17 21.17 8.89 12.61 3 21.17 5.77 AST (units/l) 76.66 180.83 39.66 39.66 26.83 35.0 32.66 35.0 5.77 17.61 4.04 4.04 2.02 7 4.04 0 ALKP (units/l) 31.75 59.33 31.25 30.77 32.30 31.25 35.12 27.53 2.05 4.04 2.16 3.35 3.84 2.17 3.63 5.60 TP (g/dl) 9.41 5.68 7.48 6.55 6.84 8.89 6.72 8.59 0.36 0.141 0.29 0.32 0.26 0.66 0.40 1.35
Values are mean S.E. of six rats. ALT, alanine transaminase; AST, aspartate transaminase; ALKP, alkaline phosphatase; TP, total protein; p.o., per oral. a Single dose of CCl on rst day and daily dose of drug/extract were given for 5 days. 4 P < 0.001. P < 0.01. P < 0.1 vs. intoxicated control using Students t-test. Table 2 Histopathological studies of liver tissues Groups 1 2 Treatment Normal (control) CCl4 (toxicity control) Observations On rst day: The section of its liver showed normal hepatocytes without any necrosis and fatty depositions. On fth day: Neither the change nor any sign of degeneration was observed in the liver sections. On second day of CCl4 administration: The liver showed centrilobular necrosis with prominent and enlarged central vein. Fatty depositions were also seen. The section showed a classic view of degenerating liver. After 5 days: The liver did not show any signicant recovery. The section showed focal and centrilobular fatty change with necrosis. The section showed good recovery with absence of necrosis and fatty depositions. The central vein was clear. The liver section showed partial disappearance of fatty deposits and necrosis. Hepatic cells in focal areas showed various degrees of degenerative changes like cloudy swelling and hydropic degeneration. However, other areas showed hepatic cell with prominent nucleus and nucleolus indicating mild antihepatotoxic activity. The liver section did not show any signicant activity. It showed signicant recovery with disappearance of fatty deposition and necrosis, the central vein appeared clearly indicating a potent antihepatotoxic activity. There was a signicant recovery except mild fatty change. The section revealed a tremendous progress with disappearance of fatty deposits and necrosis. It showed superiority as compared with other groups.
3 4
Silymarin (standard drug) Petroleum ether fraction
5 6 7 8
Ethyl acetate fraction Methanol fraction Total alcoholic extract Compound AB-IV
standard drug Silymarin against CCl4 -intoxicated control in comparison to normal values. In addition, decrease in liver enzymes and increase in TP was also observed by other fractions (Table 1). The histopathological studies of the liver showed swelling and necrosis in hepatocytes in CCl4 -treated rats in comparison to normal control rats. Administration of different extracts of the plant exhibited a signicant recovery of hepatocytes in different sections of the liver, wherein the methanolic fraction and compound AB-IV showed almost complete normalization of the tissues as neither fatty accumulation nor necrosis was observed. The central vein appeared clearly indicating a potent antihepatotoxic activity. The compound AB-IV showed superiority as compared with other groups. Other fractions also showed a considerable recovery of the liver tissues (Table 2). Thus, the different extracts/fractions of the seeds of Cichorium intybus have various degrees of antihepatotoxic activity. The methanol fraction and compound AB-IV possessed better antihepatotoxic activity. Further, they did not produce
any gross behavioral changes or mortality. It can, therefore, be said to be non-toxic and may be used as a safe drug.
4. Conclusion The above observations lead to the conclusion that the methanolic fraction possessed most active chemical component, which has also been isolated and named as AB-IV. The preliminary identication has shown it to be a phenolic compound. Further elucidation of its structure is under progress in the laboratory.
Acknowledgements The authors are thankful to the Head, Department of Pharmaceutical Chemistry for providing necessary research facilities, and to the in-charge, Central Animal Facility, Jamia Hamdard, New Delhi for providing rats and related
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B. Ahmed et al. / Journal of Ethnopharmacology 87 (2003) 237240 Handa, S.S., Sharma, A., Chakarborti, K.K., 1986. Natural products and plants as liver protecting drugs. Fitoterapia 57, 307351. Kind, P.R.N., King, E.J.J., 1954. Estimation of plasma phosphatase by determination of hydrolyzed phenol with aminopyrines. Journal of Clinical Pathology 7, 332. Luna, L.G., 1968. Manual of Histology: Staining Methods of Armed Force Institute of Pathology, 3rd ed. McGraw-Hill, New York. Reitman, S., Frankel, S.A., 1957. Colorimetric method for the determination of serum glutamic oxalo acetic acid and glutamic pyruvic transaminases. American Journal of Clinical Pathology 28, 5663. Sala, A.V. (Ed.), 1994. Indian Medicinal Plants: A Compendium of 500 Species, 1st ed. Origent Longmen Ltd., Chennai, p. 74. The Wealth of India, vol. III, 1992. Publication & Information Directorate, Council of Scientic and Industrial Research, New Delhi, p. 555. Woolson, R.F., 1987. Statistical Methods for the Analysis of Biomedical Data. Wiley, New York. Wooton, I.D.P., 1964. Microanalysis in Medical Biochemistry, 4th ed. Churchill, London, pp. 138140.
facilities. One of the authors (A.B.S.) is also thankful to UGC, New Delhi for awarding GATE Scholarship. References
Ahmed, B., Bawa, S., Siddiqui, A.B., Alam, T., Alam, S.A., 2002. Components from seeds of Cichorium intybus Linn. Indian Journal of Chemistry 41B, 27012705. Dennett, C.W., 1964. New tables for multiple comparisons with a control. Biometrics 20, 482491. Dumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and the measurement of serum albumin with bromocresol green. Clinica Chimica Acta 31, 8796. Handa, S.S., Singh, A., 1995. Hepatoprotective activity of Apium graveolens and Hygrophila auriculata against paracetamol and thioacetamide intoxication in rats. Journal of Ethnopharmacology 49, 119126.
Screening of antimutagenicity via antioxidant activity in Cuban medicinal plants

A. Ramos , A. Visozo, J. Piloto, A. Garc a, C. A. Rodr guez, R. Rivero
Centro de Investigacin y Desarrollo de Medicamentos, Avenue 26, No. 1605, Nuevo Vedado, Ciudad de La Habana, CP 10600, Cuba Received 11 September 2002; received in revised form 23 April 2003; accepted 25 April 2003
Abstract The reducing activity on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, OH radical scavenging potential, in vitro inhibition of lipid peroxidation and modulation of mutagenicity induced by ter-butyl hydroperoxide (TBH) in Escherichia coli were sequentially screened in 45 species of plants used with medicinal purposes in Cuba, in a search for antioxidant agents which protect DNA against oxidative stress. Five species, e.g. Tamarindus indica L., Lippia alba L., Pimenta dioica (L.) Merr, Rheedia aristata Griseb. and Curcuma longa L. displayed IC50 < 30 g/ml in the DPPH radical reduction assay and IC50 < 32 g/ml in lipid peroxidation inhibition testing. Pimenta dioica and Curcuma longa L. showed also a 20% inhibition of the in vitro induced OH attack to deoxyglucose. Further antimutagenesis assay in Escherichia coli IC 188 evidenced that only Pimenta dioica prevents DNA damage by TBH to the test bacteria. A role of antioxidant enzymes is presumed in this case, as judged by a different response in the isogenic Escherichia coli IC 203 decient in catalase and alkyl hydroperoxide reductase and the discrete inhibition of oxidative mutagenesis also observed when pre-treatment of the extract was assayed. Eugenol, the main constituent of the essential oil of Pimenta dioica, also inhibited oxidative mutagenesis by TBH in Escherichia coli, at concentrations ranging from 150 to 400 g/plate. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Antioxidant; Lipid peroxidation; Escherichia coli; Antimutagenesis; Pimenta dioica; Eugenol
1. Introduction The inhibition of permanent (transmissible) damage of DNA in living organisms, known as antimutagenesis, encompasses several enzymatic and non-enzymatic mechanisms (De Flora and Ramel, 1988) to counteract the modication of DNA components such as nitrogen bases and sugars by electrophiles. Free radicals released during oxidative stress are among the most widespread intracellular DNA modiers (Hochstein and Atallah, 1988; Mller and Wallin, 1998). Their involvement in carcinogenesis, inammation, diabetes, atherosclerosis, brain and heart ischaemia, ageing, etc. has been intensely addressed during the last years. Search for novel antimutagens acting in chemoprevention of relevant biomolecules oxidation is a promising eld in phytotherapy. This study presents the screening for antioxidant activity leading to inhibition of DNA damage of 45 botanical species traditionally used in Cuba as herbal remedies, in a sequential strategy including three non-enzymatic
Corresponding
in vitro assays followed by testing of the best candidates for bacterial reversion in Escherichia coli.
2. Materials and methods 2.1. Plant material Medicinal plants screened in this study were collected in 2001 at the Medicinal Plants Experimental Station Dr. Juan Toms Roig, in Gira de Melena, La Habana. The species are listed alphabetically in Table 1, indicating the parts of the plant studied. A voucher specimen (number indicated in the table) was deposited at the herbarium of this institution. 2.2. Extracts preparation The species collected were dried and 1 kg was subjected to hydroalcoholic percolation (Martin and Cook, 1961), yielding 1 l of extract. It was concentrated under reduced pressure to obtain a crude, kept in sealed containers at 4 C. Before testing, the crude was dissolved in 70% methanol (v/v) up to 5 mg/ml and appropriately diluted.
author. E-mail address: arruiz2001@yahoo.com (A. Ramos).
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A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241246
Table 1 Medicinal plants screened for antioxidant antimutagenicity IC50 values in the DPPH reduction assay of the hydroalcoholic extracts screened No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 15 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 35 34 36 37 38 39 40 41 42 43 44 45 Species Anethum graveolens L. Annona reticulata L. Artemisia absinthium L. Bidens pilosa L. Boerhavia erecta L. Bromelia pinguin L. Brugmansia candida Pers. Calendula ofcinalis L. Capsicum frutescens L. var. frutescens Cassia grandis L. Chrysantellum americanum (L.) Vatke Curcuma longa L. Cyperus rotundus L. Hamelia patens Jacq. Indigofera suffructicosa Mill. Justicia pectoralis Jacq. var. Pectoralis Lepidium virginicum L. Lipia alba (Mill) NE Brown Matricaria recutita L. Melia azederach L. Melissa ofcinalis L. Mentha spicata L. Mentha x piperita L. Momordica charantia L. var. abreviata Ser. Musa x paradisiaca L. Nerium oleander L. Ocimum basilicum L. Ocimum tenuiorum L. Parthenium hysterophorus L. Pedilanthus tithymaloides (L.) Poit Petiveria alliacea L. Pimenta dioica (L.) Merr. Piper aduncum L. s.l. Plantago lanceolata L. Plantago major L. Plectranthus amboinicus (Lour) Spreng. Portulaca oleraceae L. Pouteria mammosa (L.) Cronquist Rheedia aristata Griseb. Ruta graveolens L. Stachitarpheta jamaicensis (L.) Vahl Tamarindus indica L. Teloxys ambrosioides (L.) W.A. Weber Thuja occidentalis L. Zebrina pendula Schnizl Family Apiaceae Annonaceae Asteraceae Asteraceae Nyctaginaceae Bromeliaceae Solanaceae Asteraceae Solanaceae Caesalpinaceae Asteraceae Zingiberaceae Cyperaceae Rubiaceae Fabaceae Acanthaceae Brassicaceae Verbenaceae Asteraceae Meliaceae Lamiaceae Lamiaceae Lamiaceae Cucurbitaceae Musaceae Apocynaceae Lamiaceae Lamiaceae Asteraceae Euphorbiaceae Phytolaccaceae Myrtaceae Piperaceae Plantaginaceae Plantaginaceae Lamiaceae Portulacaceae Sapotaceae Clusiaceae Rutaceae Verbenaceae Caesalpinaceae Chenopodiaceae Cupressaceae Commelinaceae Voucher ID ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG ROIG 4644 5663 4640 4598 4642 4667 4691 4625 4650 4692 4699 4693 4688 4684 4594 4636 4626 4611 4692 4687 4586 4621 4590 4694 4695 4665 4638 4675 4626 4697 4678 4609 4679 4588 4589 4579 4685 4683 4698 4630 4641 4670 4639 4632 4674 Parts used Seed Aerial parts Aerial parts Aerial parts Aerial parts Leaves, fruits Leaves, owers Flowers Fruit Seed Aerial parts Rhizome Whole plant Leaves Aerial parts Aerial parts Aerial parts Aerial parts Flowers Aerial parts Aerial parts Aerial parts Aerial parts Aerial parts Juice Aerial parts Aerial parts Aerial parts Aerial parts Stem Whole plant Aerial parts Whole plant Aerial parts Aerial parts Leaves Whole plant Fruit, seed Bark Aerial parts Aerial parts Bark Whole plant Aerial parts Aerial parts IC50 (g/ml) 87 54 121 127 51 645 314 76 418 1108 109 47 72 116 341 368 752 23 351 732 33 4571 90 169 918 104 109 76 254 251 255 21 346 534 290 181 253 1253 41 459 79 16 153 112 48
2.3. DPPH reduction assay Reduction of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) by the crude was performed in 96-well microplates. The absorbance at 515 nm of a methanolic solution of DPPH was recorded at different concentrations of the extract, according to Navarro et al. (1992). IC50 values were derived from the inhibition curves obtained. 2.4. Lipid peroxidation assay Inhibition of lipid peroxidation was assessed as described by Ramos et al. (2001).
2.5. Scavening of hydroxyl radical activity Inhibition of hydroxyl radical damage to deoxyglucose was assayed according to Jodynis-Liebert et al. (1999). OD at 535 was recorded and the inhibition of OH attack at 100 g/ml of extract expressed as a percentage of control. Mannitol (50 mM) was used as reference OH radical scavenger. 2.6. Antimutagenesis assay in Escherichia coli Inhibition of bacterial mutagenesis reversion was tested in Escherichia coli WP2 trp65 uvrA rfa/pKM 101 (IC 188)
A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241246 Table 2 In vitro hydroxyl radical scavenging and lipid peroxidation inhibition values of some species screened for antioxidant activity No. Species % Inhibition
OH
243
Scavenging
Lipid peroxidationa 2.23 100.00 100.00 0.00 100.00 100.00 100.00 25.57 (6.13) (31.30) (29.30) (14.83) (18.70)
5 12 18 21 32 39 42 45
Boerhavia erecta Curcuma longa Lippia alba Melissa ofcinalis Pimenta dioica Rheedia aristata Tamarindus indica Zebrina pendula
0.00 22.52 9.06 3.84 23.00 1.37 6.23 0.00
a Assayed for 100 g/ml of extract; values in parentheses indicate IC 50 in g/ml.
and its isogenic oxyR30 derivative (IC 203), kindly sent by Dr. Manuel Blanco (Instituto de Investigaciones Citolgicas, Valencia, Espaa). The basic plate incorporation protocol described by Blanco et al. (1998) was followed. Briey, a mixture of fresh overnight culture of the strain, plant extract (typically 010 mg/ml) and tert-butylhydroperoxide (TBH) at 100 g/plate was poured in minimal agar plates and incubated at 37 C. Revertants were scored 48 h later and expressed as percentage to the untreated control (without extract), an indicator of oxidative mutagenesis inhibition. Concurrent testing to discriminate cytotoxicity from true antimutagenesis was performed by treating bacteria with the extract only. Pre- and post-treatment assays were performed with the inclusion of a pre-incubation step (Yahagi et al., 1977). For pre-treatment, Escherichia coli was treated with 05 mg/ml of extract during 4 h at 37 C with shaking and plated at 100 g/plate of TBH. On the other hand, challenging of the cells with 100 g/ml TBH was followed by post-treatment with 05 mg/plate of the extract.
to test suppression of the mutagenic response. Results are shown in Fig. 1, where a plot was shown for each species. The Tamarindus indica extract did not exert any effect upon Escherichia coli mutagenicity by TBH. However, a lowering to 2550% in the revertant count was observed when Escherichia coli was treated with the extracts of Lippia alba L. and Rheedia aristata in the concentration ranging from 2.5 to 10 mg/plate, even in the absence of TBH, which indicates some toxicity by the species assayed. This effect was even more drastic in the case of Curcuma longa, where a decrease of 25% was recorded at 4 mg/plate. A different response was observed with the extract of Pimenta dioica. A clear reduction up to 25% in the revertant frequency was observed at no appreciable cytotoxicity. The effect was conrmed when bacteria were pre-treated (5 mg/ml) with the Pimenta dioica extract before TBH challenge, a moderate 35% inhibition of oxidative mutagenesis being recorded. On the other hand, post-treatment did not result in a signicant decrease in mutagenicity. Escherichia coli strain IC 203, unable to express key antioxidant enzymes such as catalase and alkylhydroperoxide reductase, proved sensitive to the Pimenta dioica extract alone, with less than a 25% revertant survival at 10 mg/plate. Eugenol, the main component of the Pimenta dioica essential oil, was also tested in the Escherichia coli IC 188 system and the results were similar than those observed for the extract (Fig. 2). At low levels of cytotoxicity in a range of 300400 g/plate, a signicant decrease of 50% in the oxidative mutagenesis by TBH was recorded.
4. Discussion Antimutagenic properties elicited by plant species have a full range of prospective applications in human healthcare. Herbal remedies and phytotherapy drugs containing active principles are currently developed to protect against electrophile (e.g. free radical) attack to DNA and its widespread outcomes such as ageing and cancer. Even for populations which use herbs traditionally, encouraging the use of species with chemopreventive actions could be helpful as part of life expectancy improvement strategies: costs are signicantly low, herbs have usually little or no toxicity during long-term oral administration and are relatively available at large scale. Such is the case of curcumin, a polyphenol isolated from Curcuma sp. widely known in Asiatic medicinal practice, though a dietary component also (Commandeur and Vermeulen, 1996). Evidence of antioxidative properties has been gathered previously for some medicinal species scored as positive in our screening. Three active components (2-hydroxy-3 , 4 -dihydroxyacetophenone, methyl 3,4-dihydroxybenzoate, 3,4-dihydroxyphenyl acetate and ()-epicatechin) were isolated by Tsuda et al. (1994) from the coat seed of Tamarindus indica. There is a wide amount of literature supporting the use of Curcuma longa in the chemoprevention of pathologies
3. Results DPPH reduction by the extracts is shown in Table 1. Eight extracts (Boerhavia erecta L., Melissa ofcinalis L., Zebrina pendula L., Tamarindus indica L., Lippia alba L., Pimenta dioica (L.) Merr., Rheedia aristata Griseb. and Curcuma longa L.) exhibit IC50 values below 30 g/ml, indicating good potential as free radical scavengers. When the screening proceeded to in vitro lipid peroxidation only ve extracts out of the above mentioned were really active as inhibitors of oxidative injury (Table 2). Besides, Curcuma longa and Pimenta dioica displayed some inhibition (approximately 20%) of the hydroxyl radical attack to deoxyglucose at 100 g/ml, a relevant endpoint for DNA protection against damage induced by free radicals. Antimutagenicity was then assayed in the ve extracts displaying a positive response in at least two of the former biochemical testing systems, using Escherichia coli reversion upon oxidative damage induced by TBH as a model
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Fig. 1. Inuence of hydroalcoholic extracts of Pimenta dioica, Curcuma longa, Tamarindus indica, Lippia alba and Rheedia aristata upon the oxidative mutagenesis induced by TBH in Escherichia coli strains IC 188 and IC 203.
associated to free radical damage (Arujo and Len, 2001). Those effects have been attributed to curcumin and related compounds (Commandeur and Vermeulen, 1996), well-known hydroxyl radical scavengers and inhibitors of lipid peroxidation in vitro (Joe and Lokesh, 1994; Ruby et al., 1995). Finally, methanolic extracts of Pimenta dioica were also reported as antioxidant by Oya et al. (1997), activity being attributed to the presence of pimentol and biorin. Apart from eugenol, Kikuzaki et al. (1999) isolated ve phenilpropanoids with antioxidant activity from the berries of Pimenta dioica.
Results of the screening tests indicate that antioxidant potential estimated in biochemical assays, though demonstrated in 18% of the species tested, does not confer protection to the cellular genome per se against oxidative stress in the experimental conditions described. Out of the ve species which positively reduced the DPPH radical and inhibited lipid peroxidation, only two were moderate hydroxyl scavengers and just one of them, Pimenta dioica, resulted in an effective antimutagen in Escherichia coli. The case with Curcuma longa is particularly surprising, due to the huge amount of experimental data accumulated
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Fig. 2. Effect of eugenol on the oxidative mutagenesis induced by TBH in Escherichia coli IC 188.
which supports its antioxidant potential (Arujo and Len, 2001). However, as in other phenolic phytochemicals (e.g. avonoids), the question of reactive intermediaries and auto-oxidation through redox cycling, leading to a pro-oxidant state, should be addressed (De Groot and Rauen, 1988). In fact, Kelly et al. (2001) have reported that 25 M curcumin, the main biologically active principle of Curcuma longa, damages the DNA of Jurkat-T-lymphocytes as measured by single cell gel electrophoresis (comet assay). As for Pimenta dioica, eugenol seems to be involved in the protective response against the genotoxic injury by TBH, though the contribution from closely related phytochemicals (e.g. phenylpropanoids) should not be excluded. As TBH releases reactive oxygen species (ROS) intracellularly (Kappus, 1987), inactivating enzyme induction could be involved in the lower levels of bacterial reversion observed, apart from chemical interaction between ROS and the extract components. In this sense, results with strain IC 203 differed markedly, indicating a cytotoxic effect of the extract alone that might be due to its inability to cope with reactive species released after extract uptake. Additionally, it should be pointed out that pre-treatment with the extract also lead to a moderate inhibitory response in the strain IC 188. In fact, it has been reported that eugenol induces phase II enzymes in mammals (Yokota et al., 1986), though data on metabolism in prokaryotes is not available. On the other hand, eugenol was not mutagenic in the bacterial reversion (Ames) assay at concentrations of 3.3333.3 g/plate, assayed in the standard battery of Salmonella strains (Haworth et al., 1983) and its clastogenic potential in vitro and in vivo is conned to doses near toxicity (Armstrong et al., 1992; Ellahuee et al., 1994). Besides, its antimutagenic capacity is not clear, in vivo assays with transgenic mice being negative (Rompelberg et al., 1996). So the response described herein deserves further attention. At present, experiments are carried out in peripheral blood lymphocytes to get more insight on the extract potentiality as antioxidative antimutagen, by measuring another endpoint (micronuclei) in quite a different scenario such as peripheral lymphocytes in culture.
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A. Ramos et al. / Journal of Ethnopharmacology 87 (2003) 241246 Ruby, A.J., Kuttan, G., Babu, K.D., Rajasekharan, K.N., Kuttan, R., 1995. Anti-tumour and antioxidant activity of natural curcuminoids. Cancer Letters 94, 7983. Tsuda, T., Watanabe, M., Ohshima, K., Yamamoto, A., Kawakishi, S., Osawa, T., 1994. Antioxidative components isolated from the seed of tamarind (Tamarindus indica L.). Journal of Agricultural and Food Chemistry 42, 26712674. Yahagi, T., Nagao, M., Seino, Y., Matsushima, T., Sugimura, T., Okada, M., 1977. Mutagenicities of N-nitrosamines on Salmonella. Mutation Research 48, 121130. Yokota, H., Hoshino, J., Yuasa, A., 1986. Suppressed mutagenicity of benzopyrene by the liver S9 fraction and microsomes from eugenol treated rats. Mutation Research 172, 231236.
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Evaluation of antioxidant activity of leaf extract of Seabuckthorn (Hippophae rhamnoides L.) on chromium(VI) induced oxidative stress in albino rats
S. Geetha a , M. Sai Ram a , S.S. Mongia a , Virendra Singh b , G. Ilavazhagan a , R.C. Sawhney a,
a
Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, Delhi 110 054, India b H.P. Agriculture University, Palampur, Himachal Pradesh, India Received 12 December 2002; received in revised form 17 April 2003; accepted 25 April 2003
Abstract The present study reports the antioxidant activity of Seabuckthorn (Hippophae rhamnoides), family Elaegnaceae, on chromium induced oxidative stress in male albino rats. Oxidative stress was induced in the rats by force-feeding of potassium dichromate equivalent to a dose of 30 mg/kg body weight (BW) of chromium(VI) for 30 days. Administration of chromium decreased the body weight and increased organ to body weight ratio signicantly. Chromium treatment signicantly decreased reduced glutathione (GSH), and increased malondialdehyde (MDA) and creatine phosphokinase (CPK) levels; further it also enhanced glutamate oxaloacetate transferase (GOT) and glutamate pyruvate transferase (GPT) levels in the serum. Different doses of the alcoholic leaf extract of Seabuckthorn were evaluated for the protection against the chromium induced oxidative stress. The results show that the leaf extract at a concentration of 100 and 250 mg/kg BW protected the animals from the chromium induced oxidative injury signicantly. 2003 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Seabuckthorn; Chromium; Oxidative stress; Antioxidants
1. Introduction Cellular exposure to exogenously or endogenously generated oxidants causes macromolecular damage, including protein oxidation, lipid peroxidation, and nucleic acid instability, and mutation (Ames and Shigenaga, 1992; Halliwell, 1998). Chromium is a naturally occurring heavy metal found commonly in the environment in the trivalent Cr(III) and hexavalent Cr(VI) forms. Cr(VI) compounds have been declared as potent occupational carcinogens among workers in chrome plating, stainless steel, and pigment industries. The reduction of Cr(VI) to Cr(III) results in the formation of reactive intermediates together with the oxidative tissue damage and a cascade of cellular events, including modulation of apoptosis regulatory gene p53, and contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds (Flores and Perez, 1999; Bagchi et al., 2001). In recent years, the clinical importance of the herbal drugs has received considerable attention. As many syn-
Corresponding
author. Tel.: +91-11-394-6546; fax: +91-11-393-2869. E-mail address: em dipas@yahoo.com (R.C. Sawhney).
thetic antioxidants have been shown to have one or the other side effects (Musk et al., 1994; Nocentini et al., 2001), there has been an upsurge of interest in the therapeutic potential of medicinal plants as antioxidants in reducing free radical induced tissue injury (Siddique et al., 2000; Engelhart et al., 2002; Koleva et al., 2002). Numerous plant products have been shown to have the antioxidant activity (Aruoma and Cuppelt, 1997; De-Groot and Rauen, 1998; Koleva et al., 2002; Scartezzini and Speroni, 2000), and the antioxidant vitamins, avonoids, and polyphenolic compounds of the plant origin have been extensively reported as scavengers of free radicals and inhibitors of lipid peroxidation (Hanasaki et al., 1994; Formica and Regelson, 1995; Tapiero et al., 2002). Seabuckthorn (Hippophae rhamnoides L., Elaegnaceae) is a thorny nitrogen xing deciduous shrub, native to Europe and Asia (Rousi, 1971). All parts of the plant are considered to be a good source of a large number of bioactive substances. The ripe fruit has been reported to be a rich source of Vitamins A, C, E, and K, carotenoids, and organic acids (Chen et al., 1990; Yao and Tigerstedt, 1992; Pintea et al., 2001; Kallio et al., 2002). Many medicinal effects of Seabuckthorn against u, cardiovascular diseases, mucosal
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injuries, and skin disorders (Xiao, 1980; Beveridge et al., 1999; Eccleston et al., 2002,) have been suggested to be due to the high contents of antioxidant substances present in this plant. In an earlier study (Geetha et al., 2002), we reported that the alcoholic leaf extract has a signicant antioxidant and immunomodulatory activity against the chromium induced oxidative stress, in vitro, in rat lymphocytes. Alcoholic leaf extract at a concentration of 500 g/ml provided signicant cytoprotection against the chromium induced free radical production, apoptosis, and DNA fragmentation. To conrm whether the antioxidant activity obtained from in vitro applies to in vivo also, a study has been carried out to evaluate the antioxidant activity of the leaf extract against the chromium induced oxidative injury using SpragueDawley albino rats as a model system.
dislocation and weight of various organs of the body was determined. 2.4. Determination of biochemical parameters Reduced glutathione (GSH) was estimated in the blood by the method of Kum-Talt and Tan (1974). Malondialdehyde (MDA) was determined by the method of Dousset et al. (1983). Superoxide dismutase (SOD), glutathione peroxidase (GPx), serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) activity in erythrocytes was determined as per the manufacturers instructions using kits obtained from M/s Randox. All the experiments were conducted on two different occasions and data were analyzed using Student t-test. Since there was no signicant difference in any of the parameters in the animals fed with the leaf extract alone, the data have not been incorporated.
2. Methodology 2.1. Plant material Seabuckthorn (Hippophae rhamnoides) leaves were collected from the hilly regions of Western Himalayas, in the month of September where the plant grows wildly under natural conditions. The fresh leaves were cleaned and dried under shade. The extraction was carried out using powdered leaf (10 g) by reuxing with 500 ml of 70% ethanol at 80 C in a Soxhlet for 10 h. The extract was ltered and the ltrate was dried at 50 C under reduced pressure in a rotary evaporator and dissolved in 70% alcohol to provide a concentration of 100 mg/ml. The crude extract was then diluted in sterile saline containing 0.1% Tween-80. 2.2. Animals The experiments were conducted on male Sprague Dawley albino rats weighing 180200 g maintained at 25 2 C with food and water ad libitum. The animals were housed three rats per cage, and maintained on 12 h day and night cycle. Three different concentrations of the leaf extract were given orally, 50, 100, and 250 mg/kg body weight (BW) with the help of gastric canula and the control group was maintained on saline containing 0.1% Tween-80. The leaf extract was administered an hour prior to the chromium feeding. The study was approved by the Institutes Animal Ethical Committee and conrms to National guidelines on the care and use of laboratory animals. 2.3. Oxidative stress Oxidative stress was induced in rats by force-feeding of 1 ml potassium dichromate equivalent to a dose of 30 mg/kg BW of chromium(VI) for 30 days. Food and water intake by the animals was monitored daily and body weight was measured weekly. The animals were sacriced by cervical
3. Results 3.1. Body weight and food and water consumption The effect of alcoholic leaf extract of Seabuckthorn on body weight changes during the chromium induced oxidative stress is shown in Fig. 1. Chromium feeding resulted in a signicant decrease in the body weight with the duration of treatment; however, in animals fed with both the leaf extract and chromium, there was no signicant change as compared to the control group. Administration of chromium did not cause any signicant change in the food and water intake (data not shown). 3.2. Organ to body weight ratio Table 1 shows organ to body weight ratio in the chromium- and Seabuckthorn-treated animals. Administration of chromium caused a signicant increase in the
Fig. 1. Body weight changes in the chromium- and Seabuckthorn leaf extract (LE)-treated animals.
S. Geetha et al. / Journal of Ethnopharmacology 87 (2003) 247251 Table 1 Organ to body weight ratio in the chromium- and Seabuckthorn leaf extract (LE)-treated animals Control Heart Liver Lung Spleen Kidney
a b
249
Chromium 0.03 0.06 0.75 0.3 0.78 4.37 0.05 5.56 2.51 9.45 0.21a 0.01a 0.49 0.31a 0.48a
LE (50 mg) + chromium 3.41 0.030 5.41 1.67 7.20 0.12b 0.01b 0.19 0.26b 0.54b
3.52 0.03 5.25 1.7 7.26
P < 0.05 vs. control. P < 0.05 vs. chromium.
heart, liver, spleen, and kidney to body weight ratio in all the animals. However, the lung to body weight ratio was not altered in the chromium-treated animals. Pretreatment with Seabuckthorn leaf extract in all the doses (50,100, and 250 mg) maintained the organ to body weight ratio comparable to that of control values. 3.3. GSH and MDA levels Erythrocyte GSH levels were signicantly decreased following the chromium treatment, whereas a signicant increase in plasma MDA levels was observed (Fig. 2). Administration of leaf extract in 100 and 250 mg/kg BW dose reverted the GSH and MDA levels to that of control values. 3.4. GPx and SOD activity Administration of chromium caused a signicant increase (P < 0.05) in the erythrocyte GPx levels but did not affect SOD levels (Fig. 3). The leaf extract in a dose of 100 and 250 mg/kg BW was able to restore the GPx levels to that of control values. 3.5. SGOT, SGPT, and creatine phosphokinase (CPK) activity CPK, SGOT, and SGPT levels were increased (P < 0.05) in all the animals treated with chromium (Fig. 4). Administration of 100 and 250 mg/kg BW dose of the Seabuckthorn leaf extract signicantly inhibited the chromium induced increase in enzyme levels and restored to that of control values
Fig. 3. Glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in the chromium- and Seabuckthorn leaf extract-treated animals.
4. Discussion Three different concentrations of the leaf extract, 50, 100, and 250 mg/kg BW, were evaluated for the antioxidant activity against the chromium induced oxidative stress in male albino rats. The results of the present study demonstrate that the ethanolic leaf extract of Seabuckthorn at a concentration of 100 and 250 mg/kg BW protected the animals signicantly from the chromium induced oxidative damage. Oral feeding of chromium resulted in a signicant decrease in body weight and increase in organ to body weight ratio. Chromium(VI) compounds are well-known oxidizing agents capable of directly inducing tissue damage and possess carcinogenic, mutagenic, and teratogenic potency (Sugiyama, 1992). Chromium(VI) compounds are easily taken up by the cells and are subsequently reduced to Cr(III) species. This reduction generates free radicals, which play a major role in the adverse biological effects of these compounds (Shi et al., 1999). Administration of chromium signicantly increased the lipid peroxidation as evident by the increase in MDA levels. To cope with the oxidative stress, there was a signicant increase in GPx activity in the erythrocytes, which in turn caused a marked reduction in the GSH levels. No signicant change in the SOD activity was observed in the chromium-treated animals and our re-
Fig. 2. Effect of various doses of the leaf extract (LE) of Seabuckthorn on reduced glutathione (GSH) and malondialdehyde (MDA) levels.
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Fig. 4. Serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and creatinine phosphokinase (CPK) activity in the chromium- and Seabuckthorn leaf extract-treated animals.
sults fall in conrmation with earlier studies (Bagchi et al., 1995). Besides activating the oxidative stress, chromium also caused a marked increase in CPK, SGOT, and SGPT activity suggesting that the chromium treatment also causes hepatic damage. Many workers have also demonstrated the hepatotoxic effects of chromium (Ueno et al., 1995; Dartsch et al., 1998), which is mainly due to the lipid peroxidation. These adverse effects of chromium could be signicantly curtailed by pretreating the animals with the leaf extract of Seabuckthorn. In animals fed with 50 mg/kg BW of the leaf extract, no signicant protection was observed against the chromium induced oxidative stress. This was revealed by enhanced MDA and lowered GSH levels compared to the control animals. However, at higher concentrations (100 and 250 mg/kg BW), the extract inhibited the chromium induced increase in plasma MDA levels and restored the intracellular antioxidants, like GSH and GPx, levels to that of control. The extract also protected the animals signicantly from the hepatotoxicity induced by the chromium as revealed by the decreased CPK, SGOT, and SGPT activity compared to the chromium-treated animals. The results of the present investigation conrm our earlier in vitro observations in which the leaf extract has been demonstrated to have a potent antioxidant activity against the chromium induced oxidative stress in rat lymphocytes (Geetha et al., 2002). Many studies conducted earlier revealed that the fruits of Seabuckthorn have potent antioxidant (Eccleston et al., 2002; Suleyman et al., 2002), antiulcerative (Suleyman et al., 2001), and radioprotective (Goel et al., 2002) properties. In the present investigation, we found that the leaf extract too possesses a potent antioxidant activity. Leaves of Seabuckthorn are claimed to be a rich source of many antioxidant substances, like avonoids and other polyphenolic compounds. The main antioxidant components of leaves are avonoids, quercetin, isorhamnetin, and avonols, like epicatechin and leucoanthocyanidins (Novruzov, 2001). Further investigations are in progress to develop the extract as a nutraceutical. Detailed studies on the antioxidant properties, hepatoprotective potentials,
and phytochemical analysis are also in progress in this laboratory.
Acknowledgements The authors are grateful to Director, DIPAS, for providing all the facilities to carry out the experimental work. Secretarial assistance of Mr. Parveen Kumar is also acknowledged.
References
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Short communication
Antimalarial activity of Cinchona-like plants used to treat fever and malaria in Brazil
V.F. Andrade-Neto a , M.G.L. Brando b , J.R. Stehmann c , L.A. Oliveira a , A.U. Krettli a,
a Laboratory of Malaria, Centro de Pesquisas Ren Rachou/FIOCRUZ and Department of Parasitology, Universidade Federal de Minas Gerais (UFMG), Av. Augusto de Lima 1715, Belo Horizonte, CEP 30190-002 MG, Brazil b Laboratory of Pharmacognosy, Faculty of Pharmacy, UFMG, Belo Horizonte, Brazil c Department of Botany, UFMG, Belo Horizonte, Brazil
Received 30 September 2002; received in revised form 27 March 2003; accepted 27 March 2003
Abstract For centuries, malaria was treated with the bark of Cinchona calisaya and Cinchona succirubra plants named quinas in Brazil, from which the quinine molecule was isolated. Other plant species known also as quinas are used to treat fever and malaria, like Deianira erubescens (roots and leaves), Strychnos pseudoquina (bark), and Remijia ferruginea (bark). Based on this popular knowledge, we evaluated the in vivo antimalarial activity of the ethanol crude extracts of these plant species in mice infected with Plasmodium berghei. Only Remijia ferruginea showed antimalarial activity, reducing parasitaemia and mortality at the highest dose tested. Its phytochemical analysis showed the presence of alkaloids but not quinine. The other two plant species were inactive. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Antimalarial tests; Ethnopharmacology; Cinchona; Remijia ferruginea; Plasmodium berghei
1. Introduction Malaria is a human protozoan disease widespread in the Amazon region. In Brazil, most malaria cases at present are caused primarily by Plasmodium vivax, followed by Plasmodium falciparum (Ministry of Health/Funasa, 2002). Several derivatives of the quinine molecule are used to treat acute symptomatic malaria including chloroquine, amodiaquine and meoquine or, to prevent Plasmodium vivax late relapses, as primaquine and other 8-aminoquinolines (Greenwood, 1995; Ridley, 2002). At present, malaria chemotherapy is complicated by drug-resistant strains of Plasmodium falciparum, including in the Amazon region (Segurado et al., 1997), thus, new antimalarials are needed. Quinine is an alkaloid rst identied and isolated from the barks of the Peruvian plants Cinchona calisaya Wedd. and Cinchona succirubra Pav. ex Klotzsch (Rubiaceae) (Bruce-Chwatt, 1988), popularly known as quinas in Brazil. Quinine has been used to treat human malaria for
over 350 years (Meshnick, 1997; Meshnick and Dobson, 2001), especially in cases of chloroquine-resistant Plasmodium falciparum (Ridley, 2002). The antimalarial activity of about 40 medicinal plant species used in Brazil have been tested in vivo and in vitro (Carvalho et al., 1991; reviewed in Krettli et al., 2001). In this paper, we studied species known as quinas used to treat fever and malaria, namely, Deianira erubescens Cham. and Schltdl. (Gentianaceae; roots and leaves), Remijia ferruginea (A.St.-Hil.) DC. (Rubiaceae; bark) and Strychnos pseudoquina A.St.-Hil. (Loganiaceae; bark) (Wasick, 1944; Correa, 1975; Balbach, 1980) for their antimalarial activity.
2. Material and methods 2.1. Plant samples The roots and leaves of Deianira erubescens and the bark of Remijia ferruginea and Strychnos pseudoquina, popularly used as remedies, were collected and dried at room temperature. Plant samples were collected in the cerrado (savanna-like) vegetation of the Serra do Cabral, northern Minas Gerais, in July 2000. Voucher herbarium specimens
author. Tel.: +55-31-3295-3566; fax: +55-31-3295-3115. E-mail address: akrettli@cpqrr.ocruz.br (A.U. Krettli).
Corresponding
0378-8741/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00141-7
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were prepared and deposited in the herbarium BHCB, Federal University of Minas Gerais. Plants were identied by one of us (J.R.S.) as Deianira erubescens Cham. and Schltdl. (BHCB 47133), Remijia ferruginea (A.St.-Hil.) DC. (BHCB 47136), and Strychnos pseudoquina A.St.-Hil. (BHCB 4720). 2.2. Ethanol extract preparation Plant samples (100 g each) were pulverized, then extracted by percolation with 80% ethanol, as described earlier by Brando et al. (1997). The dried extracts (4.82 g from root and 23.1 g from leaves of Deianira erubescens, 6.75 g from Remijia ferruginea bark, and 11.2 g from Strychnos pseudoquina bark) were stored at 4 C and tested as a fresh water suspension in Tween 20 at 1% nal concentration as antimalarial drug. 2.3. Phytochemical screening For identication of alkaloids, the dried extract was solubilized in ethanol and water (1:2), alkalinized to pH 9.0 with NH4 OH and extracted with CH2 Cl2 . The extracts were chromatographed (TLC) using concentrated dichloromethane phase and applied on silica gel. The solvent system used was tolueneethyl acetatediethylamine (7:2:1) or chloroformdiethylamine and Draggendorff/NaNO2 or 10% ethanol/H2 SO4 (and UV-365 nm) as a spray reagent. Quinine (Sigma Ref. 1125, St. Louis, MO, USA) was used as the reference compound. For the identication of avonoids, saponins and tannins, solvent systems and detection methods as described by Wagner and Bladt (1996) were used. 2.4. Malaria parasites The Plasmodium berghei, strain NK-65 was originally received from the New York University Medical School. It was maintained through weekly blood passages in mice, by intraperitoneal route (i.p.), using 3.8% sodium citrate as anticoagulant and also in liquid nitrogen with glycerolyte. 2.5. Antimalarial tests We used the traditional suppressive test of Peters (1965) as modied by Carvalho et al. (1991). Briey, adult Swiss albino mice weighing 1820 g were inoculated by i.p. route with 1 105 Plasmodium berghei-infected red blood cells. The mice were randomly divided in groups of ve per cage, and treated during 4 consecutive days with daily doses of the extracts, by oral route. Two control groups were used in each experiment, one treated with chloroquine at low non-curative doses (25 mg/kg, orally), the other group was kept untreated. On the 5th day after parasite inoculation, blood smears were prepared from all mice, xed with methanol, stained with Giemsa, then microscopically exam-
ined (1000 magnication). Parasitaemia was determined in coded blood smears by randomly counting 20006000 erythrocytes in the case of low parasitaemia (10%); or up to 1000 erythrocytes in the case of higher parasitaemia. Overall mortality was monitored daily in all groups during a period of four weeks following inoculation. The inhibition of parasite growth in the drug-treated groups was calculated as follows: parasitaemia in the control (non-treated) group minus parasitaemia in the drug-treated group, divided by parasitaemia in the control (non-treated) group, expressed as percentages. The extracts were considered partially active if parasitaemia was reduced by 30% or more (Carvalho et al., 1991). All extracts were tested in three independent experiments at daily doses of 500 and 1000 mg/kg body weight. 2.6. Statistical analysis The ANOVA and Students t-test were used for comparison of average parasitaemia and the KruskalWallis test by the Biostat 1.0 Software for Windows (MCT-CNPq) for survival analysis. 2.7. Ethical approval The present work was approved by the Ethical Committee for Using Animals at Fundao Instituto Oswaldo Cruz, FIOCRUZ (CEUA P0094-01).
3. Results and discussion The phytochemical screening of quina plants showed the presence of avonoids and tannins in Deianira erubescens leaves, and tannins in its root; alkaloids were detected in the Strychnos pseudoquina and Remijia ferruginea barks. The antimalarial activity of these plants in Plasmodium berghei-infected mice is shown in Table 1 for one representative experiment in a total of three performed. Extracts from the bark of Strychnos pseudoquina and the root and leaves of Deianira erubescens were inactive and caused no signicant reduction in parasitaemia or mortality. Extracts from Remijia ferruginea bark caused up to 48% inhibition of parasite growth at the dose of 1000 mg/kg, but only a borderline activity at the dose of 500 mg/kg in one experiment. Malaria mortality was slightly but not signicantly reduced by Remijia ferruginea in relation to the mortality in non-treated mice (P > 0.05). However, the mice survival time was reduced in groups treated with Deianira erubescens extracts from leaves and root (Table 1). Indeed, one dose of 4000 mg/kg of Deianira erubescens ethanol extract caused 20% mortality to non-malaria mice, whereas, similar doses of Strychnos pseudoquina and Remijia ferruginea bark extracts caused no mortality (not shown). Chloroquine, a standard antimalarial used in non-curative doses to mice, caused a signicant reduction of parasitaemia (5195%) and mortality (Table 1) in all experiments.
V.F. Andrade-Neto et al. / Journal of Ethnopharmacology 87 (2003) 253256 Table 1 Antimalarial activity of ethanolic extracts of Cinchona-like plants and chloroquine in mice infected with Plasmodium berghei Plant species/part used or reference drug Strychnos pseudoquina Bark Dose (mg/kg) orally 4 1000 500 0c 1000 500 0c 1000 500 0c 1000 500 0c 25 12.5 0c Reduction of parasitaemia (%)a 10 0 Half-survival time in days S.D. 10 2.0 10 1.5 10 2.0 12 2.6 11 2.4 10 2.1 9 9 10 8 9 11 1.2 2.3 0.6 1.7+ 2.6 0.6
255
Antimalarial activityb None
Remijia ferruginea Bark
48 34
Partial
Deianira erubescens Leaves
0 0 0 18 95 51
None
Root
None
Chloroquine (control)
28 3.0++ 18 1.5+++ 11 1.0

P
Active Partial = 0.0001;

P
S.D.: standard deviation. The signicance of parasitaemia inhibition evaluated by the Students t-test was P = 0.045; and, of mortality by the KruskalWallis test was + P = 0.015; ++ P = 0.007; +++ P = 0.04. a Reduction of parasitaemia in relation to control non-treated mice. b Extracts which reduce 30% parasitaemia are considered partially active. c Control non-treated group.
= 0.001;
Cinchona species, a source of quinine, do not occur in Brazil. Remijia ferruginea known as quina has been used as a substitute of quinine to treat malaria (Wasick, 1944; Souza, 1951). Other plants named quinas are used in traditional medicine to treat fever and malaria. In the present study, the antimalarial effect of three species of quinas tested in rodent malaria proved to be disappointing. Only one, Remijia ferruginea, displayed some activity; Strychnos pseudoquina and Deianira erubescens were totally inactive. It has been previously shown that Strychnos pseudoquina extracts tested in Plasmodium cathemerium, an avian malaria, were inactive (Wasick, 1944). In an attempt to characterize the active compounds in Remijia ferruginea, we performed TLC tests of the alkaloid extract. We found no quinine. Other alkaloids are described in Remijia plants (McCalley, 2002), but further work has to be undertaken to elucidate whether they are responsible for the antimalarial activity of Remijia ferruginea.
Acknowledgements We express thanks to the Brazilian Agencies (CNPq and CAPES) for fellowships, to PRONEX, FAPEMIG and FIOCRUZ (PAPES-II) for nancial support.
References
Balbach, A., 1980. A Flora nacional na Medicina Domstica, 17th ed., vol. 2. Edicaes do Lar, So Paulo, 921 p.
Brando, M.G.L., Krettli, A.U., Soares, L.S.R., Nery, C.G.C., Marinuzzi, H.C., 1997. Antimalarial activity of extracts and fractions from Bidens pilosa and other Bidens species (Asteraceae) correlated with the presence of acetylene and avonoid compounds. Journal of Ethnopharmacology 57, 131138. Bruce-Chwatt, L.J., 1988. Cinchona and its alkaloids: 350 years later. New York State Journal of Medicine 88, 318322. Carvalho, L.H., Brando, M.G.L., Santos-Filho, D., Lopes, J.L.C., Krettli, A.U., 1991. Antimalarial activity of crude extracts from Brazilian plants studied in vivo in Plasmodium berghei-infected mice and in vitro against Plasmodium falciparum in culture. Brazilian Journal of Medical and Biological Research 24, 1113 1123. Correa, M.P., 1975. Dicionrio de Plantas uteis do Brasil e das Exticas e Cultivadas, 3 ed., vol. 16. Instituto Brasileiro de Desenvolvimento Florestal, Rio de Janeiro. Greenwood, D., 1995. Conicts of interest: the genesis of synthetic antimalarial agents in peace and war. Journal of Antimicrobial Chemotherapy 36, 857872. Krettli, A.U., Andrade-Neto, V.F., Brando, M.G.L., Ferrari, W.M.S., 2001. The search for new antimalarial drugs from plants used to treat fever and malaria or plants randomly selected: a review. Memrias do Instituto Oswaldo Cruz 96, 10331042. McCalley, D., 2002. Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques. Journal of Chromatography A 967, 119. Meshnick, S.R., 1997. Why does quinine still work after 350 years of use? Parasitology Today 13, 8990. Meshnick, S.R., Dobson, M., 2001. The history of antimalarial drugs. In: Rosenthal, P. (Ed.), Antimalarial Chemotherapy. Mechanisms of Action, Resistance, and New Directions in Drug Discovery. Humana, Totowa, New Jersey, pp. 1516. Ministry of Health, Brazil/Funasa, 2002. Casos de Malria no Brasil. Available at www.funasa.gov.br/index. Peters, W., 1965. Drug resistance in Plasmodium berghei I. Chloroquine resistance. Experimental Parasitology 17, 8089.
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V.F. Andrade-Neto et al. / Journal of Ethnopharmacology 87 (2003) 253256 Souza, A.H., 1951. Quinas e falsas Quinas. Tribuna Farmac eutica 8, 127 130. Wagner, H., Bladt, S., 1996. Plant Drug Analysis. A Thin Layer Chromatography Atlas, 2nd ed. Springer-Verlag, Berlin, p. 384. Wasick, R., 1944. O problema da quina e de seus alcalides no Brasil. Anais da Associao Qu mica do Brasil 3, 4459.
Ridley, R.G., 2002. Medical need, scientic opportunity and the drive for antimalarial drugs. Nature 415, 686693. Segurado, A.A., Di Santi, S.M., Shiroma, M., 1997. In vivo and in vitro Plasmodium falciparum resistance to chloroquine, amodiaquine and quinine in the Brazilian Amazon. Revista do Instituto de Medicina Tropical de So Paulo 39, 8590.

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Journal of Ethnopharmacology 87 (2003) 123142

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142

Table 2 List of examined species

Cataplasm, poultice, locally applied ointment Infusion Infusion Elixir Infusion

Cotinus coggygria Scop. Anethum graveolens L. Angelica archangelica L. Apium graveolens L.

Anacardiaceae Apiaceae Apiaceae Apiaceae

Smradlika Kopar Letchbna Pichtjalka Zelina

C` otino, Sc` otano Aneto Angelica Sedano

Astringent, anti-inammatory Carminative, spasmolytic Sedative, spasmolytic Diuretic

Leaves Fruits, oil Roots, fruits Roots, fruits

External use. Contact can cause skin ulcers

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142

Carminative, appetiser, spasmolytic stimulates gastric secretion, cholagogue, uterotonic

Dried fruits used to avour food

Dried fruits used to avour food

Juice of fresh root, seeds Infusion

Juice as drink, cataplasm of fresh root pulp Decoction, tincture

Roots are eaten boiled or raw as vegetable

Aerial parts, fruits

-------Maceration in cold water

Levisticum ofcinale Koch Pastinaca sativa L. ssp. sativa

Letcheben selim Pachtarnaksta

Sedano di monte Pastinaca

Diuretic, carminative, reduces intestinal gas Dietetic, diuretic, cholagogue

Roots Roots, seeds

Roots Roots, leaves

Petroselinum crispum Mill.

Juice or decoction from roots or from fresh leaves

Fresh leaves are used to avour food

Apiaceae Apiaceae Apiaceae Apocynaceae

Samodivska treva Anason Bedreniza Malak zimzelen Zmijarnik

Roots, fruits Fruits, oil Fruits Leaves Tuber

Roots Fruits Roots Aerial parts Tuber

Decoction Infusion Maceration Decoction Maceration in cold water

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142

Acorus calamus L. Hedera helix L.

Blaten air Brachljan

Calamo aromatico Edera

Antitussive, sedative, expectorant

Expectorant, emetic (strong doses)

Periploca graeca L. Asplenium trichomanes L.

Topa Erba Rugginina

Bark Aerial parts

Bark, leaves Aerial parts

Decoction, infusion (leaves) Decoction

Scolopendrium ofcinale Sm. (=Phyllitis s.) (L.) Newmann

Lingua di cervo, Lingua di cane

Millefoglio, Erba Livia

Arctium lappa L. and A. nemorosum Lej.

Decoction, cataplasm of leaves is locally applied Decoction

Appetizer, to stimulate gastric secretion, mouth wash

Assenzio selvatico, Canapaccio

Appetizer, astringent, sedative, haemostatic

Tonic, aromatic, emmenagogue

Bellis perennis L. Calendula ofcinalis L.

Capitula (heads) Leaves and owering tops Roots

Carlina acanthifolia All.

Infusion, alcoholic extract Infusion Decoction

M.L. Leporatti, S. Ivancheva / Journal of Ethnopharmacology 87 (2003) 123142

Centaurea cyanus L. Cichorium intybus L.

Sinja metlitchina Sinja zlatchka

Flowers Roots, aerial parts

Topinambur, Patata del Canada