PHYTOCHEMICAL ANALYSIS OF TRICHOSANTHES … · The phytochemical analysis of the stem, ... presence of following bioactive compounds by using the standard qualitative procedures (Sofowora,

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Indo American Journal of Pharmaceutical Research, 2013 ISSN NO: 2231-6876

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INDO AMERICAN

JOURNAL OF

PHARMACEUTICAL

RESEARCH

PHYTOCHEMICAL ANALYSIS OF TRICHOSANTHES CUCUMERINA L.

Shyamsundarachary Rudroju, Samatha Talari, Rajinikanth Marka, Srinivas Penchala and Rama Swamy

Nanna*1

1Plant Biotechnology Research Lab, Department of Biotechnology, Kakatiya University, Warangal 506 009 (A P) India.

Corresponding author

Rama Swamy Nanna* Plant Biotechnology Research Lab, Department of Biotechnology, Kakatiya University,Warangal 506 009 (A P) India. e.mail:

[email protected], Tel: +91-870-2567137 (R), 2461455(O), Mobile: +91-9390101665.

Copy right © 2013 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical

Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ARTICLE INFO ABSTRACT

Article history Received 07/04/2013

Available online

28/04/2013

Keywords Trichosanthes

cucumerina,

Phytochemical

analysis, Fluorescence

analysis

Trichosanthes cucumerina L. is an important medicinal plant belonging to the family

Cucurbitaceae. It is an annual climber. It contains wide range of medicinal properties like

anti-diabetic, anthelmintic, anti-cardiac failure, hypoglycemic, anti-fertility, anti-

inflammatory and against to HIV. Preliminary phytochemical investigations of leaves, stem,

fruit wall and seed extracts have been carried out by using different solvents (methanol,

petroleum ether, benzene, chloroform and aqueous) for the presence of various

phytoconstituents. Phytochemical analysis of various solvent extracts of leaf, stem, fruit wall

and seed had shown the presence of alkaloids. Glycosides were found to be present strongly

in only leaf extracts followed by feebly in seed extracts. Tannins were absent in all the solvent

extracts of stem and seeds. Flavonoids were found only in leaf and stem. Sterols were absent

in leaf, stem and fruit wall extracts. Whereas lignins were present in leaf extracts. Phenols

were found in stem and fruit wall extracts. Lignins, saponins and quinones were absent in the

solvent extracts of leaf, stem, fruit wall and seeds. Thus, the species T. cucumerina contains

alkaloids, glycosides, tannins, flavonoids, phenols and sterols which play a role in

pharmaceutical industry. As the plant T. cucumerina possesses medicinally important

secondary metabolites, these can be employed in the treatment of various diseases.

Please cite this article in press as Rama Swamy Nanna et.al. Phytochemical Analysis of Trichosanthes cucumerina L.

Indo American Journal of Pharm Research.2013:3(4).

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mailto:[email protected]

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Vol 3, Issue 4, 2013. Rama Swamy Nanna et al. ISSN NO: 2231-6876

INTRODUCTION

The use of plants and plant products as medicine can be traced as far back beginning of human

civilization. Medicinal plants are the richest bio-resource of drugs of traditional system of medicine, modern

medicine, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediate and lead compounds in

synthetic drugs (Ncube et al., 2008). Phytochemicals are non-nutritive plant chemicals that are more complex

and specific and exert their action by resembling endogenous metabolites. These natural constituents can be

derived from any part of the plant such as leaves, flowers, roots, fruits, seeds and bark, etc. As the synthetic

drugs are associated with side effects, plant derived antimicrobials have been employed now a day as they

possess the therapeutic value to cure infectious diseases. In view of the importance of these phytochemicals, we

have undertaken the present investigation to screen the bioactive compounds in Trichosanthes cucumerina L.

T. cucumerina belongs to Cucurbitaceae. It is an annual climber. It is commonly called as snake gourd.

It is highly bitter in taste and is being known to contain wide range of medicinal properties (Choudhary, 1967).

All the parts are being used as medicine. It is also used in Ayurveda, Siddha and Allopathy. Roots are used for

its anti-diabetic (Arawwawala et al., 2009), anthelmintic (Nadkarni, 2002), leaf juice rubbed over the liver in

remittent fever (Kirtikar and Basu, 2000), skin diseases (Chopra et al., 1969), anti-spasmodic, emetic,

hypoglycaemic acivity (Kar et al., 2003). It has long been used as appetizer, laxative, aphrodisiac and blood

purifier (Shivarajan and Indira, 1994) and also in cardiac failure (Pullaiah, 2006). The stem is used for bilious

disorders, skin diseases. The fruit is considered to be antihelmintic, hepatoprotective (Sathes Kumar, 2009). The

dried seeds are used for its anthelmintic, anti-diarrhoeal, anti-bacterial and insecticidal properties. The seed

possess anthelmintic, antifibrile and haemoagglutinating activity (Chakravarthy, 1982). The whole plant used for

antifertility activity (Devendra and Kage, 2009), and also used in prevent to infections and malignancies

(Kongtun et al., 2009). This plant material has potent action against HIV because of its ribosome inactivating

activity (Jian-Hua Wang et al., 2002). T. cucumerina is a rich source of protein, fat, fibre, vitamins A,C&E and

some of the elements such as potassium, phosphorus and cucurbitacin B, cucurbitacin E, Isocucurbitacin B, 23-

24-dihydroisocucurbitacin (Sandhya et al., 2010). The aqueous extract of root exhibited significant anti-

inflammatory activity (Kolte et al., 1997).

MATERIALS AND METHODS

Plant Material

The plant material of T. cucumerina was collected from Kesamudram Mandal, Warangal district of

Andhra Pradesh, India in the month of August, 2012 and it was authenticated by Prof. VS Raju, Depertment of

Botany, Kakatiya University, Warangal. The plant material was washed thoroughly with distilled water and was

shade dried for two months. Each sample of the material was ground separately into fine powder and stored in

airtight containers at ambient temperature.

Extraction of Phytochemicals

Five gram of each sample was soaked in conical flask containing 50 ml petroleum ether, methanol,

benzene, chloroform and water separately for 1hour. The extracts were filtered through Whatman No.1 filter

paper. The supernatants were collected, covered labeled and used for the screening of various phytochemicals.

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Phytochemical Analysis

The phytochemical analysis of the stem, leaves, fruit wall and seeds was carried out to determine the

presence of following bioactive compounds by using the standard qualitative procedures (Sofowora, 1993;

Harborne, 1998; Samatha et al., 2012; Archana et al., 2012).

Tests Performed for the Presence of Phytoconstituents

a) Tests for Alkaloids

1. Dragendorff’s test: To 1ml of each of the sampale solution taken in a test tube few drops of

Dragendorff’s reagent (potassium bismuth iodide solution) was added. A reddish brown precipitate was

observed indicating the presence of alkaloids.

2. Meyer’s test: To 1ml of each of the sample solution few drops of Meyer’s reagent (potassium mercuric

chloride solution) was added. A creamish white precipitate was formed indicating the presence of

alkaloids.

3. Wagner’s test: To few ml of each of the sample solution, Wagner’s reagent (iodine in potassium iodide)

was added, which resulted in the formation of reddish brown precipitate indicating the presence of

alkaloids.

4. Hager’s test: To 1ml of each of the sample few drops of Hager’s reagent (Picric acid) was added.

Yellow precipitate was formed reacting positively for alkaloids.

5. Tannic acid test: When few ml of 10% Tannic acid was added to 1ml of each sample, a buff colour

precipitate was formed giving positive result for alkaloids.

6. FeCl3 test: One drop of FeCl3 solution was added to each of the test sample, formation of yellow

precipitate was resulted reacting positively for alkaloids.

b) Tests for Glycosides

1. Raymond’s test: Test solution when treated with dinitrobenzene in hot methanolic alkali giving a violet

colour

2. Legal’s test: When the test samples were treated with pyridine and sodium nitroprusside solution blood

red colour appears

3. Bromine water test: When treated with bromine water test solution gives yellow precipitate.

4. Kellar Kiliani test: 1 ml of concentrated sulphuric acid was taken in a test tube then 5 ml of

extract and 2 ml of glacial acetic acid with one drop of ferric chloride were added, formation of a blue

colour.

5. Concentrated Sulphuric acid test: Conc.H2SO4 was added to test sample which resulted in appearance of

reddish colour

6. Molisch test: When alpha naphthol and concentrated H2SO4 were added to test samples resulted reddish

violet ring at junction of two layers.

c) Tests for Tannins and Phenolic Compounds

1. Ferric chloride test: When few drops of ferric chloride were added to sample solution a blackish

precipitate appears.

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2. Gelatin test: When gelatin and water were added to test samples resulted the formation of white

precipitate.

3. Lead acetate: Few ml of test samples were taken in different test tubes followed by the addition of

aqueous basic lead acetate. It results in the formation of reddish brown bulky preceipitate.

4. Alkaline reagent: When sodium hydroxide solution was added to the sample solution results in the

formation of yellow to red precipitate.

5. Mitchell’s test: Tannins give a water soluble iron-tannin complex with iron and ammonium citrate or

iron and sodium tartarate.

6. Ellagic acid test: When 5% glacial acetic acid and 5% sodium nitrite were added to test samples a

muddy niger brown colour appears, which is a positive result for phenols.

d) Tests for Flavonoids

1. Zinc Hydrochloride reduction test: To test the sample solution for the flavonoids added a mixture of zinc

dust and concentrated hydrochloric acid results in red colour.

2. Lead acetate test: When aqueous basic lead acetate was added to test sample produces reddish brown

precipitate.

3. Ferric chloride test: To few ml of test samples taken separately, few drops of ferric chloride were added

which resulted in the formation of blackish red precipitate.

4. Shinoda test (Magnesium hydrochloride reduction test): To the test solution few fragements of

magnesium ribbon and concentrated hydrochloric acid were added drop wise resulted reddish to pink

colour.

5. Alkaline reagent test: When sodium hydroxide solution was added to the test samples formation of

intense yellow colour which turns to colour less on addition of few drops of dilute acid indicates the

presence of flavonoids.

e) Tests for Sterols

1. Libermann-Buchard test: When samples were treated with few drops of acetic anhydride, boiled and few

drops of concentrated sulphuric acid from the sides of the test tube were added, shows a brown ring at

the junction of two layers and the upper layer turns green which shows the presence of steroids.

2. Salkowski test: Few drops of concentrated sulphuric acid were added to the test samples in chloroform,

a red colour appears at the lower layer indicates the presence of sterols.

f) Tests for Fats and Oils

1. Stain test: Press the small quantity of each extract between two filter papers, the stain on filter papers

indicates the presence of oils.

2. Saponification test: Added a few drops of 0.5N alcoholic potassium hydroxide to various extracts with a

drop of phenolphthalein separately and heat on water bath for 1-2hours, formation of soap or partial

neutralization of alkali indicates the presence of oils and fat.

g) Tests for Lignins

1. Labat test: When gallic acid is added to the test sample, it results in the formation of olive green colour.

2. Furfuraldehyde test: When furfuraldehyde is added to the test sample a red colour appears indicating the

presence of lignin.

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h) Tests for Quinones

1. Alcoholic KOH test: When alcoholic KOH was added to the test samples red to blue colour appears

reacting positively for quinones.

i) Tests for Saponins

1. Foam test: 5 ml of extract was shaken vigorously to obtain a stable persistent froth. The froth was then

mixed with three drops of olive oil and observed for the formation of an emulsion, which indicates the

presence of saponins.

Table 1: Showing the fluorescence analysis of various extracts of T. cucumerina

under normal and UV light.

Name of the

extracts

Colour of the extract under normal light Colour of the extract under UV light

Leaf Stem Fruit wall Seed Leaf Stem Fruit wall Seed

Aqueous Brown Brown Muddy brown White Brown Yellowish Dark brown Light blue

Benzene Muddy Dark Light brown Light Yellowish Brown Dark brown Brown

green green yellow green

Chloroform Dark Blackish Brown Light Dark Dark Yellowish Grenish

green green brown green brown brown white

Methanol Light Green Grenish Yellow Green Dark Yellowish Brown

green brown green brown

Petroleum Dark Muddy Grenish White Blackish Dark Brown Light blue

ether green brown brown green brown

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Table 2: Analysis of phytochemicals from leaf extracts of T. cucumerina

Tests for Phytoconstituents Aqueous Benzene Chloroform Methanol Petroleum

extract extract extract extract ether extract

Tests for alkaloids

1) Dragendorff’s test + + + + -

2) Mayer’s test + + + + -

3) Wagner’s test + + + + +

4) Hager’s test + + + + +

5) Tanicacid test + + + + +

Tests for glycosides

1) Raymond’s test + + + + -

2) Legal’s test - - - - -

3) Bromine water test - - - - -

4) Kellar Kiliani test + + + + -

5) Conc. H2SO4 test + + + + -

6) Molisch test + + + + -

Tests for tannins

1) FeCl3 test + + - + -

2) Gelatin test + + - + -

3) Lead acetate test + + - + -

4) Alkaline reagent test + + - + -

5) Mitchell’s test + + - + -

Tests for flavonoids

1) Zn-HCl reduction test + - - + -

2) Lead acetate test + - - + -

3) FeCl3 test + - - + -

4) Shinoda’s test + - - + -

5) Alkaline reagent test + - - + -

Tests for sterols

1) Libermann Burchard test - - - - -

2) Salkowski test - - - - -

Tests for fats & oils

1) Stain test - - - - -

2) Saponification test - - - - -

Tests for phenols

1) FeCl3 test - - - - -

2) Elagic acid test - - - - -

Tests for lignins

1) Labat test - - - - -

2) Lignin (furfuraldehyde) test + + + + +

Tests for quinones

1) Alcoholic KOH test - - - - -

Tests for saponins

1) Foam test - - - - -

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Table 3: Analysis of phytochemicals from stem extracts of T. cucumerina



Tests for alkaloids

1) Dragendorff’s test + + + + +

2) Mayer’s test + + + + +





1) Raymond’s test - - - - -



4) Kellar Kiliani test - - - - -

5) Conc. H2SO4 test - - - - -

6) Molisch test - - - - -

Tests for tannins

1) FeCl3 test + + + + +

2) Gelatin test - - - - -

3) Lead acetate test - - - - -

4) Alkaline reagent test - - - - -

5) Mitchell’s test - - - - -


1) Zn-HCl reduction test + - - + -

2) Lead acetate test + + + + +

3) FeCl3 test + + + + +

4) Shinoda’s test + - - + +

5) Alkaline reagent test + + - + -

Tests for sterols




1) Stain test - - - - -


Tests for phenols

1) FeCl3 test + + + + +

2) Elagic acid test + + + + +

Tests for lignins

1) Labat test - - - - -

2) Lignin (furfuraldehyde) test - - - - -

Tests for quinones


Tests for saponins

1) Foam test - - - - -

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Table 4: Analysis of phytochemicals from fruit wall extracts of T. cucumerina



Tests for alkaloids











5) Conc. H2SO4 test - - - - -

6) Molisch test - - - - -

Tests for tannins

1) FeCl3 test + + + + +

2) Gelatin test - + + + -

3) Lead acetate test + - - - +

4) Alkaline reagent test + - - - -

5) Mitchell’s test - + - + +


1) Zn-HCl reduction test - - - - -


3) FeCl3 test - - - - -

4) Shinoda’s test - - - - -


Tests for sterols




1) Stain test - - - - -


Tests for phenols

1) FeCl3 test + + + + +

2) Elagic acid test + + + + +

Tests for LIGNINS

1) Labat test - - - - -


Tests for QUINONES


Tests for SAPONINS

1) Foam test - - - - -

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Table-5: Analysis of phytochemicals from seed extracts of T. cucumerina



Tests for alkaloids








2) Legal’s test - + + - +



5) Conc. H2SO4 test - + + - +

6) Molisch test - + + - +

Tests for tannins

1) FeCl3 test - - - - -

2) Gelatin test - - - - -



5) Mitchell’s test - - - - -


1) Zn-HCl reduction test - - - - -


3) FeCl3 test - - - - -

4) Shinoda’s test - - - - -


Tests for sterols


2) Salkowski test + + + + +


1) Stain test + + + + +


Tests for phenols

1) FeCl3 test - - - - -

2) Elagic acid test - - - - -

Tests for lignins

1) Labat test - - - - -


Tests for quinones


Tests for saponins

1) Foam test - - - - -

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Figure1: Showing habit, fruit and seeds of T. cucumerina

a) Plant, b) Mature fruit, c) Fruit split open, d) Fresh seeds

RESULTS

The preliminary phytochemical screening of leaf, stem, fruit wall and seed extracts of different solvents

viz., aqueous, benzene, chloroform, methanol and petroleum ether of T. cucumerina have been carried out for the

identification and analysis of biologically active phytochemicals by using standard procedures (Sofowora, 1993;

Harborne, 1998; Samatha et al., 2012; Archana et al., 2012). Fluorescence analysis of all the extracts under UV

and normal light (Table-1) has been carriedout. Various solvents have shown the different colors under UV and

normal light fluorescence.

Leaf Extracts

Phytochemical analysis of various solvent extracts of leaf of T. cucumerina revealed the presence of

alkaloids, glycosides, tannins, flavonoids and lignins. Whereas, sterols, fats & oils, phenols, quinones and

saponins were completely absent (Table-2). The leaf extracts of aqueous, benzene, chloroform and methanol

showed rich in alkaloids, whereas in petroleum ether extracts less amount was present. Glycosides were feebly

present but absent in petroleum ether extracts. Tannins were strongly present in aqueous, benzene and methnol

extracts. Whereas completely absent in chloroform and petroleum ether extracts. Aqueous and methanol extracts

showed rich in flavonoids whereas absent in benezene, chloroform and petroleum ether extracts. Lignins were

feebly present in all the extracts.

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Stem Extracts

Phytochemical analysis of all the solvent extracts of stem revealed the presence of alkaloids, flavonoids,

and phenols. Whereas glycosides, tannins, sterols, fats & oils, lignins, quinones and saponins were completely

absent (Table-3). Analysis of alkaloids and phenols in stem extracts of T. cucumerina were strongly positive in

all the extracts. Flavonoids were strongly present in aqueous and methanol extracts. Whereas feebly present in

benzene, chloroform and petroleum ether extracts.

Fruit wall Extracts

Different solvent extracts of fruit wall of T. cucumerina showed the presence of high concentration of

alkaloids and phenols. Whereas glycosides, flavonoids, sterols, fats & oils, lignins, quinones and saponins were

completely absent (Table-4). Tests performed for alkaloid and phenols analysis of aqueous, benzene,

chloroform, methanol and petroleum ether extracts were strongly positive. All solvent extracts of fruit wall

revealed the feebly presence of tannins.

Seed Extracts

Phytochemical analysis of various solvent extracts of seeds of T. cucumerina indicated the presence of

alkaloids, glycosides, sterols, fats & oils. Whereas tannins, flavonoids, phenols, lignins, quinones and saponins

were completely absent (Table-5). Alkaloids were strongly positive in all the tests performed of all the solvent

extracts. Tests conducted for the presence of glycosides were weakly detected in benzene, chloroform and

petroleum ether extracts. Whereas absent in aqueous and methanol extracts. Sterols, fats & oils were weakly

detected in all the solvent extracts in tests performed for their presence.

DISCUSSION

Phytochemical analysis conducted in the plant extracts revealed the presence of constituents which are

known to exhibit medicinal as well as physiological activities (Sofowora, 1993). The various parts of leaf, stem,

fruit wall and seed extracts of T. cucumerina revealed the presence of phytochemicals such as alkaloids,

glycosides, tannins, flavonoids, sterols, fats & oils, phenols and lignins. The present analysis of alkaloids was

present in all the parts viz., leaf, stem, fruit wall and seed extracts of T. cucumerina. Alkaloids have been

associated with medicinal uses for centuries and one of their common biological properties is their cytotoxicity

(Nobori et al., 1994). Several workers have reported the analgesic (Antherden, 1969) antispasmodic and

antibacterial (Stray, 1998; Okwu and Okwu, 2004) properties of alkaloids. Glycosides were present in leaf and

seed extracts of T. cucumerina. Glycosides are known to lower the blood pressure according to many reports

(Nyarko and Addy, 1990). The results obtained in this study thus suggest the identified phytochemical

compounds may be the bioactive constituents and these plants are proving to be an increasingly valuable

reservoir of bioactive compounds of substantial medicinal merit. The phenolic compounds are one of the largest

and most ubiquitous groups of plant metabolites (Singh et al., 2007) and they are present in stem and fruit wall

of T. cucumerina. They possess biological properties such as antiapoptosis, antiaging, anticarcinogen,

antiinflammation, antiatherosclerosis, cardiovascular protection and improvement of endothelial function, as

well as inhibition of angiogenesis and cell proliferation activities (Han et al., 2007). Several studies have

described the antioxidant properties of medicinal plants which are rich in phenolic compounds (Brown and Rice-

Evans, 1998; Krings and Berger, 2001). Natural antioxidant mainly comes from plants in the form of phenolic

compounds such as flavonoid, phenolic acids, tocopherols etc. (Ali et al., 2008). Tannins were present in leaf

and fruit wall extracts of T. cucumerina. Tannins bind to proline rich protein and interfere with protein

synthesis.

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The extracts of T. cucumerina leaf and stem extracts were also revealed to contain flavonoids. They are

hydroxylated phenolic substances known to be synthesized by plants in response to microbial infection and they

have been found to be antimicrobial substances against wide array of microorganisms in vitro. Their activity is

probably due to their ability to complex with extracellular and soluble proteins and to complex with bacterial cell

wall (Marjorie, 1996). They are effective antioxidants and show strong anticancer activities (Del-Rio et al.,

1997; Okwu, 2004). Sterols have been reported to have antibacterial properties (Raquel, 2007) and they are very

important compounds especially due to their relationship with compounds such as sex hormones (Okwu, 2001).

Sterols are present in seeds of T.cucumerina which are useful in antibacterial activity.

CONCLUSION

The preliminary phytochemical screening of various parts prepared in different solvents of T.

cucumerina indicated that the plant possesses medicinally important phytochemicals hence can be employed as

herbal medicine for primary health care needs as the conventional chemical drugs have many side effects.

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PHYTOCHEMICAL ANALYSIS OF TRICHOSANTHES … · The phytochemical analysis of the stem, ... presence of following bioactive compounds by using the standard qualitative procedures (Sofowora,