Phytochemical Investigation on Vinca rosea by Thin Layer Chromatography

Gomathi B.and Anuradha R.*

PG and Research Department of Biochemistry, S.T.E.T. Women’s College, Sundarakkottai, Mannargudi -614001, Tamil Nadu, India.

ABSTRACT:

A preliminary phytochemical analysis was carried out of Vinca rosea a very useful and much exploited medicinal plant in Tamil Nadu. This study revealed that the methanol, petroleum ether, ethanol, and aqueous extract of leaves of Vinca rosea Linn were analyzed through qualitative and quantitative identification of secondary metabolites. The phytochemical constituents like alkaloids, flavonoids, Sterols, Phenols, Tannins and glycosides were screened through TLC.

Keywords: Phytochemical analysis, Vinca rosea, TLC.

INTRODUCTION:

India is one of the world’s two leading Biodiversity centers with the presence of over 45,000 different plant species. India is perhaps the largest product of medicinal herbs and rightly calling the “Botanical garden of the world” medicinal herbs have been in use for thousands of years, in one form or another, under the indigenous system of medicine like Siddha, Ayurvedha and Unnani. Since independence in 1947 (Kakkar, 1956).

Secondary metabolites are organic compounds that are not directly involved in the normal growth, development, or reproduction of organisms. (Fraenkel, 1959).Unlike primary metabolites, absence of secondary metabolites does not result in immediate death, but rather in long-term impairment of the organism's survivability, fecundity, or aesthetics, or perhaps in no significant change at all. Secondary metabolites are often restricted to a narrow set of species within a phylogenetic group (Stamp, 2003). Secondary metabolites often play an important role in plant defense against herbivores and other interspecies defenses. In the present study, phytochemical constituents were done by thin layer chromatography in plant of Vinca rosea.

Catharanthus roseus (Madagascar periwinkle) is a species of Catharanthus native and endemic to Madagascar. Synonyms include Vinca rosea (the basionym), Ammocallis rosea, and Lochnera rosea; other English names occasionally used include Cape Periwinkle, Rose Periwinkle, Rosy Periwinkle, and "Old-maid". In the wild, it is an endangered plant; the main cause of decline is habitat destruction by slash and burn agriculture. It is also however widely cultivated and is naturalized in subtropical and tropical areas of the world. It is an evergreen sub shrub or herbaceous plant growing to 1 m tall. The leaves are oval to oblong, 2.5–9 cm long and 1–3.5 cm broad, glossy green, hairless, with a pale midrib and a short petiole 1–1.8 cm long; they are arranged in opposite pairs. The flowers are white to dark pink with a darker red centre, with a basal tube 2.5-3 cm long and a corolla 2–5 cm diameter with five petal-like lobes. The fruit is a pair of follicles 2–4 cm long and 3 mm broad.

MATERIALS AND METHODS:

QUANTITATIVE ANALYSIS

TLC

Thin layer chromatography is one of the valuable and versatile methods for analysis of wide rang biomolecules. TLC is nothing but a modification of paperchromatography

Where the sheet of paper is replaced by thin layer of absorbent material. Therefore the separation in TLC is also due to the differential partition of solutes between the stationary and mobile phases. The sample is applied as a band across the layer rather than as a spot.

Alkaloids

The samples of Vinca rosea was wetted with a half diluted NH₄OH and lixiviated with EtOAc for 24 hrs at RT. The organic phase is separated from the acidified filtrate and basified with NH₄OH (pH 11-12). It is extracted with chloroform (3 xs), condensed by evaporation and used for chromatography. The alkaloid spots were separated using the solvent mixture chloroform and methanol in the ratio of 15:1. The color and R_f value of the separated alkaloids were recorded both under Ultra Violet (UV 254 nm) and visible light after spraying with Dragendroff’s reagent (Wagner and Bladt, 1996).

Flavonoids

The Samples of Vinca rosea was extracted with 10ml methanol on water bath (60^oC/5 min). The filtrate was condensed by evaporation, added a mixture of water and EtOAc in the ratio of 10:1 and mixed thoroughly. The EtOAc phase thus retained is used for chromatography. The flavonoids spots were separated using chloroform and methanol solvent mixture in the ratio of 19:1. The color and R_fvalue of these spots were recorded under ultraviolet (UV 254 nm) light (Stanl, 1997).

Glycosides

The Samples of Vinca rosea was extracted with 70% EtOH on rotary shaker (180 thaws/min) for 10 hrs. 70% lead acetate was added to the filtrate and centrifuged at 5000 rpm/10min. Then the Supernatant was further centrifuged by adding 6.3% Na₂Co₃ at 10000 rpm/10 min. The retained supernatant was dried, redissolved in chloroform and used for chromatography. The glycosides were separated using EtOAc-MeOH-H₂O solvent mixture in the ratio of 80:10:10. The color and R_f values of these spots were recorded by observing under ultraviolet (UV 254 nm).

Phenols

The Samples of Vinca rosea was lixiviated in methanol on rotary shaker (180 thaws/min) for 24h. The condensed filtrate was used for chromatography. The phenols were separated using chloroform and methanol solvent mixture in the ratio of 27:0:3. The color and R_f values of these phenols spots were recorded under visible light after spraying the plates with Folin-Ciocalteu’s reagents heating at 80^oC/10min (Haborne, 1998).

Saponins

The Samples of Vinca rosea was extracted with 10 ml 70% EtOH by refluxing for 10 min. The filtrate was condensed, enriched with saturated n-BuOH, and thoroughly mixed. The butanol was retained, condensed and used for chromatography. The Saponins were separated using chloroform, glacial acetic acid, methanol and water solvent mixture in the ratio of 64:34:12:8. The color and R_f values of these spots were recorded by exposing chromatogram to the iodine vapours (Stanl, 1997).

Sterols

The Samples of Vinca rosea was extracted with 10 ml methanol in water bath (80^oC/15 min). The condensed filtrate is used for chromatography. The sterols were separated using chloroform, glacial acetic acid, methanol and water solvent mixture in the ratio of 64:34:12:8. The color and R_f values of these spots were recorded under visible light after spraying the plates with anaisaldehyde-sulphuric acid reagent and heating (100^oC/6 min) (Wagner and Bladt, 1996).

RESULTS:

The result in phytochemical investigation of qualitative and qualitative analysis of Vinca rosea extract have been presented and discussed here

Qualitative phytochemical analysis

The preliminary qualitative analysis of phytochemical investigation revealed the presence of alkaloids, flavonoids, tannins, glycosides, carbohydrates, Chlorogenic acid, anthocyanins and pseudo tannins in ethanolic extract of plant vinca rosea as showed in Table-1 Thus the preliminary screening test may be useful in the detection of the bioactive compounds.

Quantitative phytochemical analysis by TLC

The TLC profile of secondary metabolites (Alkaloids, flavonoids, glycosides, phenols, saponins and sterols) are tabulated in the table-2 Among the six groups of phytochemical constituents are determined from the samples of Vinca rosea that is Phenols, flavonoids, were found to be the most abundant one followed by alkaloids and while saponins , sterols , glycosides were low in concentration.

DISSCUSION:

Alkaloids are major chemical compound present in leaves of Vinca rosea (color) on TLC plate. Alkaloids are important defence of the plant against pathogenic organism and herbivores. It also toxin for insects which further modify the alkaloids and incorporate them into their own defence secretion (Khanuja, 2002). Flavonoids have been reported to expert multiple biological effects such as, anti-inflammatory, anti-allergies, anti-viral and anti-cancer activities (Havesteen, 1991).Tannins are an important ingredient in the process of tanning leather. Oak bark, mimosa and quebracho tree have traditionally been the primary source of tannery tannin, though inorganic tanning agents are also in use today and account for 90% of the world's leather production (Marion Kite and Roy Thomson, 2006). Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with Saponins found in particular abundance in various plant species. Specifically, they are amphipathic glycosides grouped phenomenological by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic triterpens derivative (Hostettmann,1995). A ready and therapeutically relevant example is the cardio-active agent digoxin, from common foxglove. Phenols were observed on the both extract of this plants showed as blue color spots on TLC plates. Generally phenolic pigments are visibly colored and they are particularly easily monitor their isolation and purification (Reberu-Gayan, 1972).

In the present study, screening of phytochemical constituents present in the dried leaf of Vinca rosea and was carried out by qualitative analysis of secondary metabolites such as alkaloids, carbohydrates, flavonoids, tannins, steroids and glycosides were present in Vinca rosea. But protein, flavones, Catechin, anthocyanins are absent.

Secondary metabolites on Vinca rosea using various solvents like ethanol, methanol, petroleum ether and aqueous extracts through thin layer chromatography. From these analysis six compounds such as Alkaloids, flavonoids, glycosides, sterols, Saponins, phenols were obtained. Finally it concluded that, the leaf of Vinca rosea have a highest amount of flavonoids, alkaloids and phenolic compounds and low amount of sterols, saponins and glycoside compounds.

Table 1: Preliminary phytochemical analysis in leaves of Vinca rosea

Sl. No.	Name of the Test	Phytochemical constituents	Ethanolic Extract of Vinca rosea
1.	Mayer’s reagent	Alkaloids	+++
2.	Benedicts Test	Carbohydrates	++
3.	Benedicts Test	Glycosides	+
4.	Foam Test	Saponins	_
5.	Lead Acetate	Tannins	+++
6.	Gelatin	Pseudo tannins	+
7.	Hcl	Catechin	_
8.	Ammonia	Chlorogenic acid	+
9.	H₂So₄	Anthocyanin	++
10	Saponinsglycosides	steroids	_
11	Test For Flavanoids	Flavanoids	+
12	Shinoda’s Test	Flavones	_
13	Ferric chloride	Phenols	_
14	Sodium chloride	coumarin	_

+++ : High ++ : Moderate + : Present - : Absent

Table 2: TLC Profile on phytochemicals in leaves of vinca rosea

Sl.No	Phytochemical constituents	R_f Values
		Alcoholic extract		Petroleum ether Extract	Aqueous Extract
		Ethanol Extract	Methanol Extract	Petroleum ether Extract	Aqueous Extract
1.	Alkaloids	0.92	0.87	0.97	0.92
2.	Flavonoid	0.83	0.53	0.88	0.83
3.	Glycoside	0.35	0.35	0.32	0.35
4.	Phenols	0.97	0.670	0.97	0.97
5.	Saponins	0.48	0.54	0.45	0.48
6.	terols	0.63	.73	.66	0.63

ACKNOWLEDGEMENT:

The authors are grateful to the management of STET Women’s College, Mannargudi for providing laboratory facilities.

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Received on 14.01.2012

Modified on 01.02.2012

Accepted on 12.02.2012

Research Journal of Pharmacognosy and Phytochemistry. 4(2): March-April 2012, 89-91