Phytochemical

Phytochemical
Phytochemicals, are chemical compounds that occur naturally in plants (phyto means "plant" in Greek), are responsible for color and organoleptic properties, such as the deep purple of blueberries and smell of garlic. The term is generally used to refer to those chemicals that may have biological significance, for example antioxidants, but are not established as essential nutrients.

Phytochemical screening
This is a process of tracing plant constituents. There are various secondary metabolites eg. alkaloids (a plant constituent) can be screened phytochemically through chemical tests like Mayer's and Wagner's test etc. There are various plant constituents like: 1. Alkaloids 2. Glycosides Anthraquinones Saponin glycosides Cyanogenic glycosides Cardiac glycosides Cardenolides and Bufadionolides 3. Flavonoids 5. Tannins and Polyphenolic compounds
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6. Resins 7. Fat and oils

Cardiac glycosides Cardenolides and bufadienolides

Cardenolides and bufadienolides are steroids containing deoxysugars and unsaturated lactone ring. These compounds are referred to as cardiac glycosides since they affect the muscles of the heart. (Malmis, 1999) The medical application of cardenolides and bufadienolides is associated with a high risk, due to their toxicity and the small difference between the therapeutic and toxic doses.

The structures of bufalin (a), cinobufagin (b), resibufogenin (c) and diphenhydramine (d).

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General structure bufadienolide

Phytochemical screening of cardiac glycosides: Tests for cardiac glycosides Tests for cardenolides Tests for bufadienolides
Liedermanns test (test for bufateniloids)

1. 2. 2ml of extract. 3. desire quantity of chloroform. 4. few drops of acetic anhydride. two drops of H2SO4. 3

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Result- frist red then blue and then green colour appears..

SIGNIFICANCE OF PHYTOCHEMICAL SCREENING OF BUFADIENOLIDES

The purpose of this sudy was to examine the toxic effects of bufadienolides of antimicrobial peptides and alkaloids. Amphibian skin secretions are considered a rich source of biologically active compounds and are known to be rich in peptides, bufadienolides and alkaloids. Bufadienolides are
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cardioactive steroids from animals and plants that have also been reported to possess antimicrobial activities. Amphibian skin contains a remarkable spectrum of biologically active compounds, including biogenic amines, peptides, proteins, bufadienolides, tetrodotoxins and lipophilic alkaloids[1-7]. The lipophilic alkaloids include the samandarines and an incredible array of piperidine-based, pyrrolidine-based, and steroidal alkaloids. Such an array of over four hundred new alkaloids has been detected in skin extracts from four genera of dendrobatid frogs of New World tropics, the bufonid genus Melanophryniscus of Southeastern South America, the mantelline genus Mantella of Madagascar, and the myobatrachid genus Pseudophryne of Australia. Many frogs contain mild toxins that make them unpalatable to potential predators. For example, all toads have large poison glands (the parotoid glands) located behind the eyes, on the top of the head. Some frogs, such as some poison dart frogs, are especially toxic. The chemical makeup of toxins in frogs varies from irritants to hallucinogens, convulsants, nerve poisons, and vasoconstrictors. Many predators of frogs have adapted to tolerate high levels of these poisons. Others, including humans, may be severely affected. Oophaga pumilio, a poison dart frog, contains numerous alkaloids which deter predators Some frogs obtain poisons from the ants and other arthropods they eat; others, such as the Australian Corroboree Frogs (Pseudophryne corroboree and Pseudophryne pengilleyi), can manufacture an alkaloid not derived from their diet. Poisonous frogs tend to advertise their toxicity with bright colours, an adaptive strategy known as aposematism. There are at least two nonpoisonous species of frogs in tropical America (Eleutherodactylus gaigei and Lithodytes lineatus) that mimic the colouration of dart poison
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frogs' coloration for self-protection (Batesian mimicry). Because frog toxins are extraordinarily diverse, they have raised the interest of biochemists as a "natural pharmacy". The alkaloid epibatidine, a painkiller 200 times more potent than morphine, is found in some species of poison dart frogs. Other chemicals isolated from the skin of frogs may offer resistance to HIV infection. Arrow and dart poisons are under active investigation for their potential as therapeutic drugs. Bufadienolides and cardenolides are described as cardiacglycosides owing to the similarity in their biological activity, the increase in the contractile force of the heart by inhibiting the enzyme Na+, K+ATPase. The enzyme is the only receptor for the cardiac glycosides and is responsible for the active extrusion of intercellular Na+ in exchange for extracellular K+. Cardiac glycosides contain a perhydrophenanthrene nucleus substituted at C-17 with a pentadienolide and butenolide for the bufadienolides (e.g. bufalin 1) and cardenolides (e.g. digitoxigenin 2), respectively. Over 20 major structural alkaloid classes, several of which may co-occur in a single frog, have been detected in anuran skin. Such alkaloids include the batrachotoxins (sodium channel activators), the histrionicotoxins (noncompetitive blockers of nicotinic channels), the pumiliotoxin, allopumiliotoxin, and homopumiliotoxin group decahydroquinolines, various izidines, epibatidine (a potent nicotinic agonist), the tricyclic coccinellines, the pseudophrynamines and spiropyrrolizidines (potent noncompetitive blockers of nicotinic channels). Structures of some alkaloids from amphibian skin are shown in Fig. 1. In this study, the toxicological effects of amphibian bufadienolides and peptides was evaluated..
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Materals and methods

Animals were collected while living from cold spring sources at approximately 20 km away from Mu, Turkey.One hundred live animals (Gammarus pulex) were used for each experiment. Experimental groups were arranged as 15 groups for bufadienolides and 20 groups for Amphibia peptides. Chemicals concerning with Amphibian bufadienolides and peptides purchased were purchased commercially (Sigma,Merck). Percentage of deaths within one hour of animals were recorded. Completely stopped the movement of animals dying.The concentrations 30% of these agents were tested on animals. Results were considered statistically and analysis of varians was performed.

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SAPARATION AND ISOLATION OF CONSTITUENTS

As the instrumentation for the structure elucidation of organic compounds becomes ever mor efactive and allow the use of incresingly small amount of material ,the most difficult operation in phytochemacal research becomes that of isolation and purification of plant constituents

Method of separation. Sublimation Distillation Farctional liberation Fractional crystallization Adsorption chromatography Partition chromatography

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Method of isolation
General method of extraction and isolation of glycosides
1. powdered drug extracted in suitable apparatus with alcohol 2. alcoholic extracted treated with lead acetate to pricipited tannin and non glycocsidal impurities 3. H2S pass through the solution to pricipited the excess lead acetate as lead sulphide 4. Extract filtered and concentrated to get crude glycosides

test for cardinoloides

1. Prepare aquous or alcoholic extract 2. Add 1ml of pyridine 3. Add 1ml of sodium nitro pussoide
Result- pink and red colour

Paljetes test
Thick section shows yellow to orange colour with sodium picrate.

Keller killioni test (test for de-oxy sugar)

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2ml extract + glycial acetic acid+ acetic acid(conc.) Reddish brown

colour appear at junction.

Liedermanns test (test for bufateniloids)

5. 6. 7. 8.

Take 2ml of extract. Add desire quantity of chloroform. Add few drops of acetic anhydride. Add two drops of H2SO4.

Result- frist red then blue and then green colour appears..

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