EXTRACTION AND SCREENING OF PHYTOCHEMICALS FROM THE LEAVES OF SELECTED FERN SPECIES

Gabriel Angelo Celebria Raphael Yturralde Bryan Atas

Phytochemicals
• Naturally occurring, non-nutritive plant chemicals • any chemical, nutrient, or antioxidant that contain protective, disease preventing compounds derived from a plant source • Determines the medicinal value of the plant

Ferns
• Spore-forming plant of the family Pteridophyta • Used as ornamental plants, vegetable, herbal medicines, building materials

Significance of the study
Results would provide information • regarding the presence of phytochemicals in ferns • Types of phytochemicals in each fern species

Statement of the problem
• Do the leaf extracts of ten species of ferns contain alkaloids, flavonoids, saponin, and tannins?

Objectives of the study
• To collect leaf samples from ten selected species of ferns. To obtain extracts from the leaves of ten selected species of ferns. To determine the presence of alkaloids, flavonoids, saponins and tannins in the leaf extracts of ferns.

• • • •

Alkaloids Flavonoids Saponins Tannins Our study will be concerned with only four types of phytochemicals

Alkaloids
• usually containing at least one nitrogen atom in a heterocyclic ring Protects plants from herbivores
» McKey(1974) e.g. ephedrine

• •

anti-cholinergenic, anti-inflammatory, anti-spasmodic, hyperglycemic, sedative, tranquilizer and vasodilator
» (www.shaman-australis.com/~auxin/heimia.html).

Flavonoid
• class of plant secondary metabolite based around a phenylbenzopyrone • produce pigmentation on plants
e.g. flavone

• protect against UV-B radiation in apples
» (Solovhcenko A. and Schimitz-Eilberger M. 2003)

• anti-allergic, anti-inflammatory, anti-microbial, anti-oxidative and anti-cancer activity

Saponins
• glycosides of steroids, steroid alkaloids or triterpenes found in plants • form a waxy protective coating on plant skins • protect against cancers, decrease blood lipids, lower blood glucose response, inhibit dental caries, inhibit platelet aggregation, anti-microbial, and lowers cholesterol
» (www.eas.com/glosary/glossary.asp?glos_pk=278)

Tannins
• astringent, bitter-tasting plant polyphenols that bind and precipitate proteins Hydrolysable Condensed • Anti-microbial » Have polyol(Yao K and others• 2006) polymers of 2 to 50 •• Divided into hydrolysable and condensed
carbohydrate (or more) flavonoid molecule at units that are joined • the center plants againstcarbon-carbon defend the by herbivores by decreasing protein availability bonds • Not susceptible to hydrolysis

Ferns
• common name of any cryptogamous or spore-forming plants. • Belongs to the family Pteridophyta

Ferns used in the study
• Asplenium nidus • A. musifolium • Davallia solida • Drynaria quercifolia • Microsorium heterocarpum • M. punctatum ‘Grandiceps’ • Nephrolepis exaltata • N. biserrata • N. hirsutula • Phlebodium aureum

Asplenium nidus • epiphyte • leaves are simple, stiff, oblanceolate, light green, wavy margins with shiny surface.

A. musifolium • epiphyte • leaves are simple, stiff, oblanceolate, and light green, crowded

Davallia solida • epiphytic, dimorphic fern
– Sterile leaves are pinnate, sub opposite, broadly deltoid long – Fertile leaves are lobed, with each lobe bearing several sporangia.

Drynaria quercifolia • epiphytic fern, with 2 kinds of leaves
– nest leaves which are concave, resembling the oak-leaf – foliage leaves, used for photosynthesis

• used in haemoptysis and is anti-helminthic

Microsorium heterocarpum • short creeping rhizomes • simple, narrowly decurent almost to the base, and ovatelanceolate fronds

M. punctatum ‘Grandiceps’ • leaves are bright green, elongated, more or less ruffled at the margin, and forking in various segments at the apex.

Phlebodium aureum • a creeping fern with thick rhizomes covered with rusty brown hair like scales

Nephrolepis biserrata • sori pinnae are about 5cm long, fresh green, leathery at the ends of the veins

N. hirsutula • leaflets are variably spaced ,linearlanceolate. • sori are dot like and are near the margins.

N. exaltata • leaflets are overlapping with each other and are tiny

Methodology

Preparation and collection of samples

Preparation of extracts

Flavonoid screening Alkaloid analysis Tannin screening

Saponin screening

Preparation and collection of samples
• All ferns (except Drynaria quercifolia) were taken from WVSU • Drynaria quercifolia was taken from Mandurriao • Additional leaves of A. nidus and N. bisserata were taken from PSHS-WV

Flavonoid screening
Flavonoid screening

Bate-Smith and Metcalf

Wilstatter “cyanidin” test

test for leucoanthocyanins

test for y-benzopyrone nucleus

Alkaloid screening
Alkaloid screening (fresh)

Saponin screening
•Libermann – Buchard Test

Tannin screening
Test tube method

Ferric chloride test

Gelatin test

Results and Discussion

Table 1. Presence and absence of alkaloids, flavonoids, leucoanthocyanins, triterpenoid saponin, condensed tannin and hydrolyzable tannin in the leaves selected fern species.
flavonoids Fern species A. musifolium A. nidus D. solida D. quercifolia M. heterocarpum M. punctatum N. bisserata N. exaltata N. hirsutula P. aureum alkaloid Batesmith & Metcalf positive positive negative positive positive positive positive positive positive positive Wilstatter Cyanidin positive positive negative positive positive positive positive positive positive positive

Results
tannin gelatin positive positive positive positive positive positive positive positive positive positive Ferric chloride Both* Both* Both* condensed condensed Both* Both* Both* Both* Both*

saponin

positive positive positive positive positive positive positive positive positive positive

negative positive positive positive positive positive negative positive positive positive

*both- condensed and hydrolyzable tannins are present

Discussion
• Phytochemicals are present in the plants structures because they provide defensive capabilities to plants.

Alkaloid
• Alkaloids provide plants with defense against alkaloid herbivores, viruses, bacteria and fungi. Fern species
A. musifolium positive

•A. nidus positive alkaloids are concentrated in the plant parts D. solida are most susceptible to attacks, such as positive that D. quercifolia positive leaves and stems M. heterocarpum positive
M. punctatum N. bisserata N. exaltata N. hirsutula P. aureum

» McKey (1974).
positive positive positive positive positive

Flavonoids
flavonoids Fern species A. musifolium A. nidus D. solida D. quercifolia M. heterocarpum M. punctatum N. bisserata N. exaltata N. hirsutula P. aureum Bate-smith & Metcalf positive positive negative positive positive positive positive positive positive positive Wilstatter Cyanidin positive positive negative positive positive positive positive positive positive positive

Flavonoids
• are present in all the leaves of selected ferns except the giant hare’s foot fern (D. solida)
• D. solida usually grows in places near bodies of water and away from direct sunlight.

• protect plants from UV-B radiation
» (Solovchenko A. and Schimitz-Eilberger 2003)

Saponins
Fern species A. musifolium A. nidus D. solida D. quercifolia M. heterocarpum M. punctatum N. bisserata N. exaltata N. hirsutula P. aureum saponin negative positive positive positive positive positive negative positive positive positive

Saponin
• Saponin is one of the most common phytochemical in plants
» (Cornel University 2004).

• Only the leaves of sword fern (N. biserrata) and the dwarf bird’s nest fern (A. musifolium) lack saponins

Tannin
tannin Fern species A. musifolium A. nidus D. solida D. quercifolia M. heterocarpum M. punctatum N. bisserata N. exaltata N. hirsutula P. aureum gelatin positive positive positive positive positive positive positive positive positive positive Ferric chloride Both* Both* Both* condensed condensed Both* Both* Both* Both* Both*

Tannins
• protect plants from ruminants or herbivores
» (Robins C. 1987).

• All leaf samples contain tannin

Summary, Conclusion and Recommendation

Summary
• The tests show that 7 of 10 ferns show the presence of all the phytochemicals tested in this study; specifically, alkaloids, flavonoids, saponins and tannins. At most one phytochemical is absent from the fern leaves.

Conclusion
• Most of the fern species contain the tested phytochemicals in their leaves. • Of the ten fern species tested, only three lacked one type phytochemical.

• All ferns contain alkaloids and tannins in their leaves; which, implies that the ferns have a potential medicinal value. • The presence of flavonoids in all fern species, except the hare’s foot fern, implies that most of the fern leaves have anti-oxidative properties. • The presence of saponin in all of the tested fern leaves, except for A. musifolium and N. bisserata, suggest that eight out of ten tested fern species have anti-microbial properties in their leaves.

Recommendation
• Further test regarding it should be performed
– Anti-microbial – Anti-oxidative – Anti-cancer – quantification

Alkaloid screening
• N. hirsutula alkaloid screening • showing the formation of a white precipitate

Flavonoid

• Flavonoid screening showing Bate-Smith Metcalf and Wilstatter “cyanidin’’ test on the extracts of Phelbodium aureum

Saponin
• Saponin screening showing the darker control on the right • A. nidus

Tannin

• Ferric chloride test, showing the tannic acid on the center

END Thank You