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CHAPTER ONE

1.0 INTRODUCTION OF PHYTOCHEMSTRY

Phytochemistry is the study of chemicals derived from plants (phytochemicals). Phytochemicals


exists as long as plants exist but we only know about hundred years about their existence.
Medicinal plants are widely used by traditional cultures all over the world. It is likely that the
knowledge of traditional medicine developed through trial and error over many centuries. The
Chinese have the oldest medicine system. More than 5000 years ago, the Chinese based their
medicine on the influence of yin and yang, and on the five elements.

During the 19th and 20th century, the main strategy of the scientists was to discover the active
ingredients, which had medicinal or pesticidal properties. Examples of these discoveries are
salicylic acid, morphine and pyrethroids (pesticides). During the 1980s many laboratories started
to identify phytochemicals in plants that might be used as medicines. Many of these discovered
phytochemicals seems to fight diseases such as cancer, heart attack and stroke. At the same time
other scientist conducting epidemiological studies to determine the relationship between the
consumption of phytochemicals and human health. Most studies showed that diets rich in plants
gave lower rates of cancer and heart disease.

Today, most new pharmaceuticals are not discovered in plants but are new synthetic creations.
Recently there is a renewed interest in the discovery of phytochemicals. This renewed interest is
our awareness has already developed many chemicals, which still have to be discovered. New
modern laboratory techniques have made it easer to discover and identify new phytochemicals.

Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive
properties. There are more than thousand known phytochemicals. It is well-known that plant
produce these chemicals to protect itself but recent research demonstrate that they can protect
humans against diseases. Some of the well-known phytochemicals are lycopene in tomatoes,
isoflavones in soy and flavanoids in fruits.

1.1 CHEMISTRY OF PHYTOCHEMICALS

List of some phytochemicals and their Sources.

• Alkaloids
• Carotenoids
• Flavonoids
• Hydroxycinnamic Acids
• Monophenols
• Terpenes
• Organosulfides
• Phenolic Acids
1.1.0Alkaloids:

Alkaloids are a group of naturally occurring chemical compounds which mostly


contain basic nitrogen atoms. This group also includes some related compounds with neutral (A.
D. McNaught and A. Wilkinson 1997) and even weakly acidic properties (R. H. F. Manske
1965). Also some synthetic compounds of similar structure are attributed to alkaloids (Robert
Alan Lewis 1998). Beside carbon, hydrogen and nitrogen, molecules of alkaloids may
contain sulfur and rarely chlorine, bromine orphosphorus.
Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants,
and animals and are part of the group of natural products (also called secondary metabolites).
An alkaloid like Caffeine is a water-soluble alkaloid. Pure caffeine is a white odourless
crystalline powder with a very bitter taste. Caffeine is closely related to other alkaloids such as
theophylline (mainly found in tea) and theobromine (mainly found in cacao beans). The
difference between these three molecules is the position of the methyl groups.

Molecular Weight.194.19

Formula: C8H10N4O2

1.1.1 Carotenoid:

Carotenoids are fat-soluble phytochemicals with a Vitamin-A-like structure that have strong antioxidant
and other potentially protective properties. Carotenoids are found in many fruits and vegetables.
Although there are more than 600 carotenoids, six account for most of those found in the human diet:
alpha-carotene, beta-carotene, beta-cryptoxanthin, lycopene, lutein and zeaxanthin.
Beta-Carotene
Molecular Weight: 536.87
Formula: C40H56

1.1.2 Flavonoids

Flavonoids, a subclass of polyphenols, are a group of phytochemicals that are among the most
potent and abundant antioxidants in our diet. The flavonoids are further divided into subclasses
based on slightly different chemical structures. Although more than 4000 flavonoids have been
identified, several appear to be important components of many fruits and vegetables. These
flavonoids are listed below after the subclass under which they fall.

• Flavonols: myricetin and quercetin


• Flavones: apigenin and luteolin
• Flavanones: hesperetin and naringenin
• Flavan-3-ols: catechin, epicatechin, epicatechin gallate, epigallocatechin,
epigallocatechin gallate
• Anthocyanins and Anthocyanidins: cyanidin, delphinidin, malvidin, pelargonidin,
peonidin

I. Flavan-3-ols

Flavan-3-ols (sometimes referred to as flavanols) are a class of flavonoids that use the 2-phenyl-
3,4-dihydro-2H-chromen-3-ol skeleton. These compounds include the catechins and the catechin
gallates.

Catechin is a flavonoid that is associated with a lower risk of coronary heart disease and certain
cancers and with healthy lung function. (Arts IC et al, 2002) Catechin is found in: tea, red wine,
cocoa powder, dark chocolate, grapes, and plums.

The epicatechins (epicatechin, epicatechin gallate epigallocatechin, epigallocatechin gallate)


have been linked to lower risk for cardiovascular disease and cancer. (Leake DS., 1997) They are
found in: variety of teas, fruits, legumes.
II. Anthocyanins:

Anthocyanins are water-soluble vacuolar pigments that may appear red, purple, or blue
according to pH. They belong to a parent class of molecules called flavonoids synthesized via
the phenylpropanoid pathway; they are odorless and nearly flavorless, contributing to taste as a
moderately astringent sensation. Anthocyanins occur in all tissues of higher plants,
including leaves, stems, roots, flowers, and fruits. Anthoxanthins are their clear, white to yellow
counterparts occurring in plants. Anthocyanins are derivatives of anthocyanidins which include
pendant sugars. E.g. Cyanidin.

Cyanidin belongs to the group of anthocyanins and has the typical C6-C3-C6 structure.
Cyanidin is a water-soluble pigment. The colour of cyanidin will depend on the pH of the
solution. Cyanidin is red when pH is below 3, blue at pH higher than 11 and violet at neutral
pH. In plants the cyanidin is bound to a sugar molecule to form cyanidin 3-O-beta-Glucoside.

Molecular Weight: 286

Formula: C40H56O6

1.1.3 Monophenols

In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds
consisting of a hydroxyl group (-OH) bonded directly to an aromatic hydrocarbon group. The
simplest of the class is phenol (C6H5OH).

E.g Hydroxytyrosol
Hydroxytyrosol is believed to be the antioxidant with the highest free radical scavenging
capacity: double that of quercetin and more than 3 times that of epicatechin. The wastewaters
generated during olive processing contain a high levels hydroxytyrosol, most of which can be
recovered to produce hidroxytyrosol extracts. Studies by (Francesco Visioli et al, 2000) showed
that a low dose of hydroxytyrosol reduces the consequences of sidestream smoke-induced
oxidative stress in rats.

Molecular Weight: 154.1


Formula: C8H10O3

4 Terpenes

Terpenes are a large and varied class of hydrocarbons, produced primarily by a wide variety of
plants, particularly conifers, though also by some insects such as termites or swallowtail
butterflies, which emit terpenes from their osmeterium.

They are the major components of resin, and of turpentine produced from resin. The name
"terpene" is derived from the word "turpentine". In addition to their roles as end-products in
many organisms, terpenes are major biosynthetic building blocks within nearly every living
creature. The following types of terpenes are discussed below;

I. Monoterpenes: consist of two isoprene units and have the molecular formula C10H16.
Examples of monoterpenes are: geraniol, limonene and terpineol.

Geraniol is acyclic monoterpene-alcohol. Pure geraniol is a colourless oily liquid, with a sweet rose-like
scent. When oxidized, geraniol becomes geranial or citral.

Molecular Weight: 154.14


Formula: C10H16O
II. Triterpenoids: Triterpenes are terpenes consisting of six isoprene units and have the
molecular formula C30H48. The pentacyclic triterpenes can be classified
into lupane, oleanane or ursane groups.

Animal- and plant-derived triterpenes exist, such as:

 squalene
 ambrein (a tricyclic triterpene alcohol)
 ganoderic acid (quad cyclic)

Triterpenoids are thought of as modified triterpenes, such as lanosterol.

Ursolic acid is a is a pentacyclic triterpenoid, used in cosmetics, that is also capable of inhibiting
various types of cancer cells by inhibiting the STAT3 activation pathway and human
fibrosarcoma cells by reducing the expression of matrix metalloproteinase-9 by acting through
the glucocorticoid receptor. As medicine, it is well tolerated and can be used topically and orally.

Molecular Weight: 456.68


Formula: C30H48O3

1.1.5 Organosulfides

Allium vegetables include garlic, onions, shallots, chives and leeks. These vegetables contain
organosulfur compounds that are thought to protect against cancer. Studies on garlic also show
that it has the potential to lower many risk factors for cardiovascular disease. (Bianchini F et al,
2001). Chives, leeks, garlic, onions, shallots

E.g Allicin

Allicin is garlic's defence mechanism against attacks by pests. When the garlic plant is attacked
or injured it produces allicin by an enzymatic reaction. The enzyme alliinase, converts the
chemical alliin to allicin, which is toxic to insects and microorganisms. The antimicrobial
acivity of allicin was discovered in 1944 by Cavallito. Purified allicin is not sold commercially
because it is not stable and has an offensive odour. Allicin extracted from garlic loses its
beneficial properties within hours and turns into other sulphur containing compounds. Diallyl
trisulfade, which is similar to allicin but is chemically produced, is stable and is used for
treatment bacterial, fungal and parasitic infections.

Molecular Weight: 127.27


Formula: C6H10OS2

1.1.6 Phenolic Acids


Ellagic acid falls into a broader class of phytochemicals called polyphenols. Ellagic acid acts as
an antioxidant and may reduce the risk of certain types of cancer. (Hannum SM. 2004) Ellagic
acid is found in nuts and fruits including: blueberries, blackberries, raspberries, red grapes,
strawberries

Molecular Weight: 302.19


Formula: C14H6C8

1.2 METHODS OF ANALYSIS IN PHYTOCHEMISTRY

• Introduction
• Methods of Extraction and Isolation
• Methods of Separation
• Methods of Identification
• Applications

1.2.0 Introduction

The subject of phytochemistry, or plant chemistry, has developed in recent years as a distract
discipline, somewhere in between natural product organic chemistry and plant biochemistry and is
closely related to both- It is concerned with the enormous variety of organic substances that are
elaborated and accumulated by plants and deals with the chemical structures of these substances,
their biosynthesis, turnover and metabolism, their natural distribution and their biological
function.
In all these operations, methods are needed for separation, purification and identification of the
many different constituents present in plants. Thus, advances in our understanding of
phytochemistry are directly related to the successful exploitation of known techniques, and the
continuing development of new techniques to solve outstanding problems as they appear. One of
the challenges of phytochemistry is to carry out all the above operations on vanishingly small
amounts of material. Frequently, the solution of a biological problem in, say, plant growth
regulation, in the biochemistry of plant-animal interactions, or in understanding the origin of
fossil plants depends on identifying a range of complex chemical structures which may only be
available for study in microgram amounts.

1.2.1 Methods of Extraction and Isolation

Ideally, fresh plant tissues should be used for phytocbemical analysis and the material should be
plunged into boiling alcohol within minutes of its collection. Sometimes, the plant under study is
not at hand and material may have to be supplied by a collector living in another continent. In
such cases, freshly picked tissue, stored dry in a plastic bag, will usually remain in good condition
for analysis during the several days required for transport by airmail.

Alternatively, plants may be dried before extraction. If this is done, it is essential that the drying
operation is carried out under controlled conditions, to avoid too many chemical changes
occurring. It should be dried as quickly as possible, without using high temperatures, preferably in
a good air draft.

Ultimately, the goal in surveying plants for biologically active or medicinally useful compounds
should be to isolate the one or more constituents responsible for a particular activity. Hence with
the selection of a specific plant for phytochemical investigation either on the basis of one or more
approaches set forth under phytopharmacologic approaches, or through some other avenue,
phytochemical screening techniques can be a valuable aid.

1.2.1.0 Phytochemical screening approaches

A. Extraction, using soxhelet extractor and hydrous methanol.

Phytochemical screenings of plant materials as samples can be performed by extracting with


soxhelet extractor and hydrous methanol. The following method can be applied.

I. Alkaloids

The alcoholic extract (corresponding to 2.5 g of plant material) should be evaporated to dryness
and heating the residue on a boiling water bath with 2N HCl (5 ml). After cooling, the mixture is
then filtered and the filtrate should be divided into two equal portions. One portion to be treated
with a few drops of Mayer's reagent and the other with equal amounts of Wagner's reagent (Rizk
A M, 1982). The samples can then observed for the presence of turbidity or precipitation. if the
reagent produced only a slight opaqueness then A (+) score can be recorded; if a definite turbidity
occurs then a (++) score can be recorded, but if a definite heavy precipitate or flocculation is
produced(Salehi Surmaghi M H, 1992) then no flocculation is observed and a (+++) score
recorded

II. Flavonoids

Taking 5 ml of alcoholic extract, corresponding to 1 g of plant material to be treated with a few


drops of concentrated HCl and magnesium turnings (0.5 g). If pink or magenta-red color
developed within 3 min then the presence of flavonoids is indicated. (Somolenski S J. et al 1972).

III. Saponins

Extract 2.5 g of the plant material with boiling water. After cooling, the extract should be shaken
vigorously to froth and then allow to stand for 15-20 min and can be classified for saponin content
as follows: no froth = negative; froth less than 1 cm = weakly positive; froth 1.2 cm high =
positive; and froth greater than 2 cm high = strongly positive). (Somolenski S J, et al 1974) ;
( Kapoor L D et al 1969).

IV. Tannins

Evaporate alcoholic extract (corresponding to 1 g of plant material) and extract the residue with
10 ml of hot 0.9% NaCl solution, filter and divide into 3 equal portions. A sodium chloride
solution should be added to one portion of the text extract, 1% gelatin solution to a second portion
and the gelatin-salt reagent to a third portion. Precipitation with the latter reagent or with both the
second and third reagent will indicate the presence of tannins. The addition of FeCl3 solution to
the extract resulting to a characteristic blue, blue-black, green or blue-green color and precipitate
(phenolic compounds) confirms a positive test. (Segelman A B et al 1969).

B. Standard procedures

Standard procedures to identify the constituents contained in the aqueous extract and in the
powdered specimens by carrying out a chemical test as described by Sofowara (1993), Trease and
Evans (1989) and Harborne (1973).

I. Test for steriods: Add two ml of acetic anhydride was to 0.5g ethanolic extract of
each sample with 2 ml H2S04. When colour changes from violet to blue or green in
some samples it indicates the presence of steroids.
II. Test for phlobatannins: Appearance of a red precipitate is deposited when an
aqueous extract of the plant sample is boiled with 1% aqueous hydrochloric acid
which is taken as evidence for the presence of phlobatinins.
III. Test for terpenoids (Salkowski test): Mix 5ml of each extract in 2ml of chloroform,
and add concentrated H2S04 (3ml) carefully to form a layer. A reddish brown
colouration of the interface forms to show positive results for the presence of
terpenoids.

IV. Test for tannins: Boil about 0.5 g of the dried powdered samples in 20 ml of water in
a test tube and then filter. A few drops of 0.1% ferric chloride should be added and
observed for browrish green or a blue-black colouration.
V. Teat for flavonoids: Three methods can be use to determine the presence of
flavonoids in the plant sample (Sofowara, 1993; Harbrone, 1973). Add 5 ml of dilute
ammonia solution to a portion of the aqueous filtrate of each plant extract followed by
addition of concentrated H2S04. A yellow colouration will be observed in each
extract, indicating the presence of flavonoids.

The yellow colouration disappeared on standing. Add few drops of 1% aluminium


solution to a portion of each filtrate. A yellow colouration will be observed,
indicating the presence of flavonoids.

A portion of the powdered plant sample in each case should be heated with 10ml of
ethyl acetate over a steam bath for 3mins. Filter the mixture and shake 4ml of the
filtrate with 1ml of dilute ammonia solution. A yellow colouration will be observed,
indicating a positive test for flavonoids.

VI. Test for cardiac glycosides (Keller-Killani test): 5ml of each extracts should be
treated with 2 ml of glacial acetic acid containing one drop of ferric chloride solution,
then underlay with 1ml of concentrated sulphuric acid. A brown ring of the interface
indicates a deoxysugar characteristic of cardenolides and violet ring may appear also
below the brown ring, while in the acetic acid layer, a greenish ring may form just
gradually throughout thin layer.

VII. Test for saponin: Boil 2 g of the powdered sample in 20 ml of distilled water in a
water bath and filter. Mix 10ml of the filtrate with 5 ml of distilled water and shake
vigorously for a stable persistent froth. The frothing should be mixed with 3 drops of
olive oil and shaken vigorously, and then observe for the formation of emulsion.

1.2.2 Methods of Separation

The separation and purification of plant constituents is mainly carried out using one or other, or a
combination, of three chromatographic techniques: paper chromatography (PC), thin, layer
chromatography (TLC) and gas liquid chromatography (GLC). The choice of technique depends
largely on the solubility properties and volatilities of the compounds to be separated. PC is
particularly applicable to water soluble plant constituents, namely the carbohydrates, amino acids,
nucleic acid bases, organic acids and phenolic compounds. TLC is the method of choice for
separating all lipid soluble components, i.e. the lipids, steroids, carotenoids, simple quinones and
chlorophylls. By contrast, the third technique GLC finds its main application with volatile com-
pounds, fatty acids, mono- and sesquiterpenes, hydrocarbons and sulphur compounds. However,
the volatility of higher boiling plant constituents can be enhanced by converting them to esters
and/or trimethylsilyl ethers so that there are few classes which are completely unsuitable for GLC
separation. Finally, it should be pointed out that there is considerable overlap in the use of the
above techniques and often a combination of PC and TLC or TLC and GLC may be the best
approach for separating a particular class of plant compound.

All the above techniques can be used both on a micro and a macro scale. For preparative work,
TLC is carried out on thick layers of adsorbent and PC on thick sheets of filter paper. For
isolation on an even larger scale than this, it is usual to use column chrornatography coupled with
automatic fraction collecting. This procedure will yield purified components in gram amounts.

One further technique which has fairly wide application in phytochemistry is electrophoresis. In
the first instance, this technique is only applicable to compounds which carry a charge, i.e. amino
acids, some alkaloids, amines, organic acids and proteins. However, in addition, certain classes of
neutral compounds (sugars, phenols) can be made to move in an electric field by converting them
into metal complexes (e.g. by use of sodium borate). Sargent(1969)

Besides the techniques so far mentioned, a few others are used occasionally in phytochemical
research. Separation by simple liquid-liquid extraction; is still of some value in the carotenoid
field. The means for automatic liquid-liquid extraction, as embodied in the Craig counter current
distribution apparatus, has been available for some time but it tends only to be used as a last resort
when other techniques fail. Separation of plant proteins and nucleic acids often requires special
techniques not otherwise used, such as filtration through Sephadex and differential centrifugation.
(Stahl, E. 1969 ; Bobbitt and Truter 1963) .

1.2.3 Methods of Identification


In identifying a plant constituent, once it has been isolated and purified, it is necessary first to
determine the class of compound and then to find out which particular substance it is within that
class. Its homogeneity must be checked carefully beforehand, i.e. it should travel as a single spot
in several TLC and/or PC systems. The class of compound is usually dear from its-response to
colour tests, its solubility and Rf properties'' and its UV spectral characteristics. Biochemical tests
may also be invaluable: presence of a glucoside can be confirmed by hydrolysis with β-
gIucosidase, of a mustard oil glycoside by hydrolysis with myrosinase and so on. For growth
regulators, a bioassay is an essential part of identification.

Complete identification within the class depends on measuring other properties and then
comparing these data with those in the literature. These properties include melting point (for
solids), boiling point (for liquids), optical rotation (for optically active compounds) and Rf or RRt
(under standard conditions). However, equally informative data on a plant substance are its
spectral characteristics: these include ultraviolet (UV), infra-red (IR), nuclear magnetic resonance
(NMR) and mass spectral (MS) measurements. A known plant compound can usually be
identified on the above basis. Direct comparison with-authentic material (if available), should be
carried out as final confirmation. If authentic material, is not available, careful comparison .with
literature data may suffice for its: identification. If a new compound is present, all the above data
should be sufficient to characterize it. With new compounds, however, it is preferable to confirm
the identification through chemical degradation or by preparing the compound by laboratory
synthesis. (Brand and Eglinton, 1965)

1.2.4 APPLICATION AND USES OF PHYTOCHEMICALS

These are some possible actions of phytochemicals with their different works

I. Antioxidant
Most phytochemicals have antioxidant activity and protect our cells against oxidative damage
and reduce the risk of developing certain types of cancer. Phytochemicals with antioxidant
activity: allyl sulfides (onions, leeks, garlic), carotenoids (fruits, carrots), flavonoids (fruits,
vegetables), polyphenols (tea, grapes).

II. Hormonal action


Isoflavones, found in soy, imitate human estrogens and help to reduce menopausal symptoms
and osteoporosis.

III. Stimulation of enzymes


Indoles, which are found in cabbages, stimulate enzymes that make the estrogen less effective
and could reduce the risk for breast cancer. Other phytochemicals, which interfere with enzymes,
are protease inhibitors (soy and beans), terpenes (citrus fruits and cherries).

IV. Interference with DNA replication


Saponins found in beans interfere with the replication of cell DNA, thereby preventing the
multiplication of cancer cells. Capsaicin, found in hot peppers, protects DNA from carcinogens.
V. Anti-bacterial effect
The phytochemical allicin from garlic has anti-bacterial properties

VI. Physical action


Some phytochemicals bind physically to cell walls thereby preventing the adhesion of pathogens
to human cell walls. Proanthocyanidins are responsible for the anti-adhesion properties of
cranberry. Consumption of cranberries will reduce the risk of urinary tract infections and will
improve dental health.

1.3 INTRODUCTION OF THE PLANT “SENNA TORA LINN”

Senna tora (originally described by Linné as Cassia tora) is a legume in the subfamily
Caesalpinioideae. It grows wild in most of the tropics and is considered a weed in many places;
its native range is not well known but probably South Asia. It is often confused with Chinese
Senna or Sicklepod, S. obtusifolia. If it is given a distinct common name at all, it is called Sickle
Wild Sensitive-plant. (Nature Serve, 2007).

Figure 1.0 Pictorial Diagram of Senna tora Linn.

1.3.0 SCIENTIFIC CLASSIFICATION OF “SENNA TORA LINN”

Kingdom: Plantae

Division: Magnoliophyta
Class: Magnoliopsida

Subclass: Rosidae

Order: Fabales

Family: Fabaceae

Subfamily: Caesalpinioideae

Tribe: Cassieae

Local name: Epa-ikun (Ijesha Land, Osun state), Epaja Abo, Jeleun benu
(ondo state)

English name: Foetid senna, the sickle senna, Ringworm Plant

Subtribe: Cassiinae

Genus: Senna

Species: S. tora

Synonyms: Cassia tora

1.3.1 CHEMISTRY OF THE PLANT “SENNA TORA LINN”

Senna tora Linn (Leguminacea) is a medicinal plant traditionally used as laxative, for the
treatment of leprosy and various skin disorders. Preliminary phytochemical analysis of leaf
showed the presence of polyphenols (3.7 mg gallic acid equivalent per gram dried leaves). The
presence of phenolic compound enables evaluation of its antioxidant and antiproliferative
potential. In the present study Senna tora L. methanolic leaf extract should evaluated for its nitric
oxide scavenging activity and reducing power assays using Rutin and BHT as standards.

The extract should then be studied for its lipid peroxidation inhibition assay using an animal liver
and brain (preferably, Rat). It must be observed that in all assays, there should be a correlation
existing between concentration of extract and percentage inhibition of free radical, reducing
power and inhibition of lipid peroxidation. The antiproliferative activity of S. tora methanolic
leaf extract usually called the CTME (i.e C. tora methanolic leaf extract) with Cisplatin,
anticancer drug can the be studied using human cervical cancer cells (HeLa). Proliferation of
HeLa should be measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide
assay, cell DNA content by modified diphenylamine method and apoptosis by Caspase 3
activity. The plant extract will induce a marked concentration dependent inhibition on
proliferation, reducing DNA content and apoptosis in HeLa. Therefore, results will clearly
indicate that C. tora is effective against free radical mediated diseases (Rejiya C. S., Cibin T. R.,
Abraham Annie, et al 2009).

Chemical constituents of senna tora linn are:


I. Roots:
1,3,5-trihydroxy-6-7-dimethoxy-2-methylanthroquinone and beta-sitosterol.
II. Seeds:
Naptho-alpha-pyrone-toralactune, chrysophanol, physcion, emodin, rubrofusarin,
cchrysophonic acid-9-anthrone.
III. Leaves:
Emodin, tricontan-1-0l, stigmasterol, b-sitosteral-b-D-glucoside, freindlen, palmitic,
stearic, succinic and d-tartaric acids uridine, quercitrin and isoquercitrin. Pankaj Oudhia
(2002)

1.3.2 APPLICATION AND USES OF “SENNA TORA LINN” TRADITIONALLY

I. Senna tora linn is a medicinal plant traditionally used as laxative, for the treatment of
leprosy and various skin disorders.

II. Recommended for reclamation of saline, alkaline and brackish soils.

III. Used as green manure crop in acidic soils. The aqueous extracts of whole plant and
leaves produces inhibitory allelopathic effects on common weeds specially on
Parthenium hysterophorus.

IV. Organic farms of India, Cassia tora are used as natural pesticide.

V. The leaves and seeds are acrid, laxative, antiperiodic, anthelmintic, ophthalmic, liver
tonic, cardiotonic and expectorant. The leaves and seeds are useful in leprosy, ringworm,
flatulence, colic, dyspepsia, constipation, cough, bronchitis, cardiac disorders.

1.3.3 BIOLOGICAL STUDIES OF THE PLANT “SENNA TORA LINN”

 Antifungal: Chrysophanic acid-9-anthrone from C. tora reported to have fungicidal


activity. Study showed ethanol extract of CT to have potent antifungal activities against
Microsporum canis and C albicans, suggesting a potential as a antifungal agent.

 Immunomodulatory: Anthraquinones of edible wild vegetable Cassia tora stimulate


proliferation of human CD4+ T lymphocytes and secretion of interferon-gamma or
interleukin 10
 Antioxidant: chemical components and antioxidant activity of the volatile oil from cassia
tora linn seed prepared by supercritical fluid extraction: The study showed antioxidant
activity of potential use for hyperlipidemia, hypertension and inflammatory disease.

 Lipid Effects: Effects of Cassia tora Fiber Supplement on Serum Lipids in Korean
Diabetic Patients: Cassia tora fiber supplement can help improved serum lipids in
T2DM. Ethanolic extract of seeds of CT decreased total and LDL cholesterol,
triglycerides and increased HDL.

 Hypotensive / Vagal Reflex: A possible reflex mechanism of hypotensive action of


extract from Cassia tora seeds: Study suggests a possible vagal reflex that alters the
vasomotor tone of the sympathetic NS.

 Anthelmintic: Study demonstrated the anthelmintic activity of alcohol and aqueous


extracts of Cassia tora.

 Antimicrobial: Study on various extracts of Cassia tora, Calendula officinalis and


Mormodica charantia showed activity against all tested bacteria, Staph aureus being more
susceptible to the aqueous extracts.

 Antihypertensive: Study of the metahnol extracts from the raw and roasted seeds of
Cassia tora exhibited significant inhibitory properties against ACE (angiotensin
converting enzyme).

 Aldose Reductase / Diabetes: Study of seed extracts of CT isolated nine anthraquinones,


with compounds 6 and 8 exhibiting inhibitory activities on protein glycation and aldose
reductase
CHAPTER 2

2.0 LITERATURE REVIEW OF “SENNA TORA LINN”

2.1 Hypolipidemic activity of seeds of Cassia tora Linn.


Ethanolic extract of seeds of Cassia tora L. and its fractions were investigated for hypolipidemic
activity on triton induced hyperlipidemic profile. Ethanolic extract and its ether soluble and
water soluble fraction was said to decrease serum level of total cholesterol by 42.07, 40.77 and
71.25%, respectively. On the other hand ethanolic extract, ether soluble fraction and water
soluble fraction increases the serum HDL-cholesterol level by 6.72, 17.20 and 19.18%,
respectively. Ethanolic extract, ether fraction and water fraction decreased triglyceride level by
26.84, 35.74 and 38.46%, respectively. The reduction in LDL-cholesterol level by ethanolic
extract, ether soluble fraction and water soluble fraction were 69.25, 72.06 and 76.12%,
respectively (Patil, U. K., Saraf, et al 2006).

2.2 Leaves of Cassia tora as a novel cancer therapeutic - An in vitro study.


They reported that Cassia tora Linn (Leguminacea) is a medicinal plant traditionally used as
laxative, for the treatment of leprosy and various skin disorders. Preliminary phytochemical
analysis of leaf showed the presence of polyphenols (3.7 mg gallic acid equivalent per gram
dried leaves). The presence of phenolic compound prompted them to evaluate its antioxidant and
antiproliferative potential. In the present study C tora methanolic leaf extract (CTME) was
evaluated for its nitric oxide scavenging activity and reducing power assays using Rutin and
BHT as standards. The extract was studied for its lipid peroxidation inhibition assay using rat
liver and brain. In all assays, a correlation existed between concentration of extract and
percentage inhibition of free radical, reducing power and inhibition of lipid peroxidation. The
antiproliferative activity of CTME with Cisplatin, anticancer drug was studied using human
cervical cancer cells (HeLa). Proliferation of HeLa was measured by MTT assay, cell DNA
content by modified diphenylamine method and apoptosis by Caspase 3 activity. The plant
extract induced a marked concentration dependent inhibition on proliferation, reduced DNA
content and apoptosis in HeLa. These results clearly indicate that C. tora is effective against free
radical mediated diseases (Rejiya C. S., Cibin T. R., Abraham Annie, et al 2009).

2.3 Endosperm in Cassia tora Linn.


During the course of a comparative study of several members of the Leguminosæ, he found an
interesting type of endosperm formation in Cassia tora Linn. (family Cæsalpiniaceæ), a common
roadside plant in India. The primary endosperm nucleus, by repeated division, gives rise to a
number of free nuclei which are at first more or less uniformly distributed throughout the
embryo-sac. Afterwards, one of the nuclei situated near the chalazal end becomes more
prominent than the rest of the endosperm. Wall formation takes place only in the micropylar part
of the endosperm. The chalazal part, which remains free nuclear,becomes a narrow tube with
denser cytoplasm in its elongated lower end. As the mass of endosperm tissue increases, the
lower tubular process becomes irregularly coiled and twisted. The sac-like portion at its tip is
often displaced so that it is sometimes found lying on one side of the cellular zone or superposed
on it. Microtome sections naturally fail to give any clear or complete picture of this interesting
tubular process; but whole mounts of the endosperm showed it quite clearly (M. Anantaswamy
Rau, 1950).

2.4 Effects of Cassia tora Fiber Supplement on Serum Lipids in Korean Diabetic
Patients.
Sung-Hee Cho and others, reported that Cassia tora fiber supplement consisting of 2 g of soluble
fiber extracted from Cassia semen (C. tora L.), 200 mg of α-tocopherol, 500 mg of ascorbic acid,
and 300 mg of maltodextrin was formulated in a pack, and given to 15 type II diabetic subjects
(seven men and eight women 57.1 ± 2.9 years old) with instructions to take two packs per day
for 2 months. Placebo contained maltodextrin only with a little brown caramel color. Lifestyle
factors and dietary intakes of the subjects were not altered during the 2-month period. Serum
total cholesterol was moderately (P < .1) decreased in the C. tora group compared with the age-
and gender-matched placebo group, as was the ratio of apolipoprotein B to apolipoprotein A1 (P
< .1). Levels of serum triglycerides and low-density lipoprotein-cholesterol tended to decrease
more in the C. tora-supplemented group than in the placebo group. Serum α-tocopherol was
increased (P < .01) but lipid peroxides were not significantly lower in the C. tora group. Fasting
blood glucose, hemoglobin A1c, blood urea nitrogen, creatinine, and activities of serum aspartate
aminotransferase and alanine aminotransferase were not changed by the fiber supplement. We
concluded that C. tora supplements can help improve serum lipid status in type II diabetic
subjects without serious adverse effects (Sung-Hee Cho, Tae-Hee Kim, et al 2005).

2.5 In vitro antifungal properties of cassia tora (gelenggang kecil) extracts


Cassia tora Linn (family :Leguminosae) is a shrub, extensively used in traditional medicine in
tropical and warm substropical countries. Cassia tora commonly found in waste grounds and
secondary forest. Decoctions of parts of Cassia tora are uses as an analgesic, anticonvulsant,
antipyretic, antifungal, antihelmint, diuretic, expectoran, laxatif, purgatif, treatment of glaucoma
and hypertention, treatment of skin disease, ringworm and itch (Perry, 1980). Their objective is
to evaluate the effects of Cassia tora extracts on the growth of Aspergillus fumigatus,
Microsporum canis and Candida albicans in vitro. Crude ethanol and aqueous extract of leaves
and seeds from Cassia tora were tested for antifungal activity in vitro against three fungi,
Aspergillus fumigatus, Microsporum canis and Candida albicans using the disc diffusion method
test. Discs (6mm diameter from Oxoid, UK) were impregnated with six different concentrations
(5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, and 30 mg/ml) of both extracts. Ethanol
extracts of Cassia tora seeds shows positive results for Candida albicans. Clear inhibition zones
at 25 mg/ml and 30 mg/ml of Cassia tora seeds extracts were observed. The inhibition zone at 25
mg/ml concentration of Cassia tora seeds extract is 8.8 mm and at 30 mg/ml is 11.1 mm in
diameter. Ethanol extract of Cassia tora leaves and aqueous extracts of Cassia tora seeds and
leaves did not show any inhibition zone. Ethanol extracts Cassia tora leaves inhibited the
Microsporum canis growth. There were no macroconidia of Microsporum canis observed under
microscope (40 x). Ethanol extracts of Cassia tora seeds had no effects on Microsporum canis
growth. The ethanol and aqueous extracts of Cassia tora seeds and leaves were not effective
towards inhibiting the growth of Aspergillus fumigatus. This current study shows that ethanol
extracts of Cassia tora have potent antifungal activities against Microsporum canis and Candida
albicans and low potency against Aspergillus fumigatus. It can be concluded that Cassia tora has
the potential as an antifungal agent. (Omar, R., Ali Rahman et al. 2002)
2.6 Antishigellosis activity of the root extracts of Cassia tora Linn.

Awal M.A , M. Shamim Hossain and other investigated that the root of Cassia tora exhibited
substantial antishigellosis activity. The ethylacetate fraction of the crude extract showed
maximum activity with the zone of inhibition ranging between 23-25 mm at the concentration of
200 μg disc-1. The minimum inhibitory concentration (MIC) of ethylacetate, chloroform and
ethanol extracts was found between 32-64 μg ml-1 whereas the methanol and petroleum fractions
showed MIC values between 128-512 μg ml-1. Thus the results suggest that the ethylacetate
fraction may have some chemical constituents which could be useful as antishigellosis agents in
modern clinical practice. Our effort is going on to isolate the potent antishigellosis constituents
from the root extracts of Cassia tora with the aim of adding new therapeutic agents to fight
against shigellosis problem in Bangladesh. (Awal M.A , M. Shamim Hossain, et al 2004).
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