Defense Power Point

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.

Our study will be concerned with only four types of phytochemicals

• Alkaloids

• Flavonoids

• Saponins

• Tannins

Alkaloids

• usually containing at least one nitrogen atom in a heterocyclic ring

• Protects plants from herbivores» McKey(1974)

• anti-cholinergenic, anti-inflammatory, anti-spasmodic, hyperglycemic, sedative, tranquilizer and vasodilator

» (www.shaman-australis.com/~auxin/heimia.html).

e.g. ephedrine

Flavonoid

• class of plant secondary metabolite based around a phenylbenzopyrone

• produce pigmentation on plants

• 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

e.g. flavone

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

• Divided into hydrolysable and condensed

Hydrolysable• Have polyol

carbohydrate molecule at the center

Condensed• polymers of 2 to 50

(or more) flavonoid units that are joined by carbon-carbon bonds

• Not susceptible to hydrolysis

• Anti-microbial» (Yao K and others 2006)

• defend the plants against herbivores by decreasing protein availability

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 ovate-lanceolate 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 ,linear-lanceolate.

• 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

Alkaloid analysis

Flavonoid screening

Saponin screening

Tannin 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 screeningFlavonoid screening

Bate-Smith and Metcalf

test for leucoanthocyanins

Wilstatter “cyanidin” test

test for y-benzopyrone nucleus

Alkaloid screeningAlkaloid screening (fresh)

Saponin screening•Libermann – Buchard Test

Tannin screeningTest tube method

Gelatin testFerric chloride test

Results and Discussion

ResultsTable 1. Presence and absence of alkaloids, flavonoids, leucoanthocyanins, triterpenoid saponin, condensed tannin and hydrolyzable tannin in the leaves selected fern species.

Fern speciesalkaloid

flavonoids

saponin

tannin

Bate-smith & Metcalf

Wilstatter Cyanidin

gelatinFerric

chloride

A. musifolium positive positive positive negative positive Both*

A. nidus positive positive positive positive positive Both*

D. solida positive negative negative positive positive Both*

D. quercifolia positive positive positive positive positive condensed

M. heterocarpum positive positive positive positive positive condensed

M. punctatum positive positive positive positive positive Both*

N. bisserata positive positive positive negative positive Both*

N. exaltata positive positive positive positive positive Both*

N. hirsutula positive positive positive positive positive Both*

P. aureum positive positive positive positive positive Both*

*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

herbivores, viruses, bacteria and fungi.

• alkaloids are concentrated in the plant parts that are most susceptible to attacks, such as leaves and stems

» McKey (1974).

Fern speciesalkaloid

A. musifolium positive

A. nidus positive

D. solida positive

D. quercifolia positive

M. heterocarpum positive

M. punctatum positive

N. bisserata positive

N. exaltata positive

N. hirsutula positive

P. aureum positive

Flavonoids

Fern species

flavonoids

Bate-smith & Metcalf

Wilstatter Cyanidin

A. musifolium positive positive

A. nidus positive positive

D. solida negative negative

D. quercifolia positive positive

M. heterocarpum positive positive

M. punctatum positive positive

N. bisserata positive positive

N. exaltata positive positive

N. hirsutula positive positive

P. aureum 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 speciessaponin

A. musifolium negative

A. nidus positive

D. solida positive

D. quercifolia positive

M. heterocarpum positive

M. punctatum positive

N. bisserata negative

N. exaltata positive

N. hirsutula positive

P. aureum 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

Fern species

tannin

gelatinFerric

chloride

A. musifolium positive Both*

A. nidus positive Both*

D. solida positive Both*

D. quercifolia positive condensed

M. heterocarpum positive condensed

M. punctatum positive Both*

N. bisserata positive Both*

N. exaltata positive Both*

N. hirsutula positive Both*

P. aureum positive 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

ENDThank You