INTRODUCTION:

Biological screening is necessary to provide a scientific basis for validating the traditional utilization of medicinal plants. A great number of screening programs are going on worldwide for new plant based bioactive molecules. Gas Chromatography (GC) and Mass Spectroscopy (MS) can be used to study Traditional Medicines and characterize the compound of interest. The Fabaceae family (= Leguminosae) consists of approximately 650 genera and 18,000 species; it is one of the largest Angiosperm families (Polhill et al., 1981; Judd et al., 1999). Many plants of this family have been used in traditional systems of medicine. Still, several potent plants of Fabaceae are unexplored which deserve attention and research. Tephrosia villosa (L.) Pers. is such plant which has not been explored extensively by the scientific world so far. The genus Tephrosia is a pantropical taxa with about four hundred species distributed throughout the world (Gillett, 1971). About twenty four species of Tephrosia were recorded in India (Gamble and Fischer, 1918; Saldanha and Singh, 1984). Most of the Tephrosia species are herbs to under shrubs and grow as weeds. The genus is well known for its richness in prenylated flavonoids and is considered to possess insect repellant, larvicidal, piscicidal, antimicrobial and anticancer properties (Sarin Jagat 1976; Chen Yuh-Lin, 1978; Bentley et al., 1987).

Tephrosia villosa (L.) Pers. is an erect white tomentose viscid under shrub distributed in dry lands. The plant is commonly called as Poonai Kolinji in Tamil. Fresh roots are considered as hypoglycemic (Yoganarasimhan, 2000). Leaf juice is used for dropsy (Yoganarasimhan, 2000; Rahmatullah Qureshi et al., 2010) and roots are used in preparation of toothpaste (Jayaweera, 1980 – 1982). Root, leaves and bark are used as anthelmintic and antipyretic and also cure diseases of liver, spleen, heart, blood and leprosy (Varaprasad Bobbarala et al. 2009). Root powders are used to cure stomachache (Giday et al., 2009). Juice of the leaves is given in dropsy and also useful in diabetes (Chopra et al., 1956).

MATERIALS AND METHODS:

Plant material

The medicinal plant Tephrosia villosa (L.) Pers. was collected from Wolf Hill (70m MSL), Sivanthipatti – a small hillock 7 Km south east of Tirunelveli, Tamil Nadu, India. The identified plant species was confirmed with Voucher specimen No: 4303 available in the Survey of Medicinal Plant Unit (SMP), Govt. Siddha Medical College, Palayamkottai.

Soxhlet extraction

About 60 g dried sample was refluxed with 250 ml of the ethanol for 5 hour on a steam bath. The extract was collected and concentrated.

Procedure

The GC - MS analyses were carried out in a Shimadzu GC – MS - QP 2010 gas chromatograph fitted with a DB1 (methylphenylsiloxane, 30 m × 0.25 mm i.d.) capillary column. Carrier gas, helium with a flow rate of 0.7 mL/min; column oven temperature 70^o C, 5 min in 180°C, 180-260°C at 3°C/min, 5 min in 260°C, 260-280°C at 0.2°C/min, and finally 5 min in 280°C; injector temperature, 280°C detector temperature, 290°C, Volume injected, 1 μL of TMS ether derivatives in n-hexane (2%); Split ratio, 3:0. The MS operating parameters were as follows: ionization potential 70 eV; ion source temperature 200°C; quadrupole 100°C, Solvent delay 6.0 min , scan speed 2000 amu/s and scan range 30-600 amu, eV voltage 3000 volts.

The concentrated extract is injected into the GC/MS instrument (Hewlett Packard 5890 GC/MS with Mass Selective Detector with an HP-1 glass capillary column). The sample is volatilized at the injection port and eluted through a capillary column under increasing temperature. As the sample moves through the column, various components are separated due to their affinity for the stationary phase of the column and can be identified by retention time (the time it takes for a compound to pass through the column and gas chromatograph system). Each chemical component in a sample has a distinct retention time measured in minutes, shown in a peak on a graph which measures abundance on the ordinate against retention time on the abscissa. The integrated peak is correlated to the concentration of the chemical. A mass selective detector breaks up each chromatographic component into fragment ions, which are shown by their abundance, with each ion represented as a vertical line in increasing molecular weight. The height of each line corresponds to the abundance of that ion. The resulting mass spectrum is unique to that chemical. This mass spectrum forms a ‘‘fingerprint’’ that can identify the compound by a computer search of mass spectra. A computer search of the mass spectra corresponding to all the chromatographic peaks for a sample should yield a statistical match for nicotine at a 12·9 min retention time value if they were present two modes of GC/MS were possible with this instrumental method. First, there is a ‘‘Scan’’ mode which looks at all the constituents of a sample, listing whatever chemical components are present.

Table: 1. Composition of the methanolic extract of the whole plant of Tephrosia villosa (L.) Pers. (Peak Report TIC)

Peak #	R.Time	Area	Area %	Name
1.	11.015	158269	0.27	4,11,11-Trimethyl-8-methylenebicyclo[7.2.0] undec-4-ene
2.	11.459	97990	0.17	ALPHA.-Caryophyllene
3.	12.409	10207512	17.71	exo-2-Hydroxycineole
4.	12.609	227186	0.39	o-Menth-8-ene-4-methanol, .alpha.,.alpha.-dimethyl-1-vinyl-,(1S, 2S,4R)-(-)-
5.	13.117	438743	0.76	Hexadecane
6.	13.758	406012	0.70	Gamma.-Eudesmol
7.	13.870	445864	0.77	Beta.-selinenol
8.	13.900	433001	0.75	Gamma.-Eudesmol
9.	14.062	1062283	1.84	2-(4,8-Dimethyl-3,7-Cyclodecadien-1-YL)-2-Propanol
10.	14.233	2880532	5.00	Bulnesol
11.	15.343	357498	0.62	Heptadecane
12.	15.565	393971	0.68	Cedran-8-OL
13.	15.681	642838	1.12	Rosifoliol
14.	15.830	198733	0.34	(-)-Spathulenol
15.	15.872	213109	0.37	Cedran-8-OL
16.	16.104	135770	0.24	Diisobutyl phthalate
17.	16.448	368735	0.64	Elemol
18.	17.012	8459645	14.68	n-Hexadecanoic acid
19.	17.307	928627	1.61	Ethyl Hexadecanoate
20.	18.092	426449	0.74	2-Methyl-5-(2,6,6-Trimethyl-1-Cyclohexen-1-YL)-2,3-Pentanediol
21.	18.461	741112	1.29	3,7,11,15-Tetramethylhexadec-2-EN-1-OL
22.	18.693	7459996	12.94	Oleic Acid
23.	18.893	1725594	2.99	Octadec-9-Enoic Acid
24.	18.943	1112421	1.93	Ethyl Octadec-9-Enoate
25.	19.165	183199	0.32	Stearic Acid Ethyl Ester
26.	22.145	956111	1.66	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester
27.	23.674	947068	1.64	2-Tert-Butyl-4-Hexylphenol
28.	24.209	1525499	2.65	trans-squalene
29.	27.524	1216837	2.11	5-Methyl-3-Phenyl-1H-indazole
30.	27.596	511962	0.89	Ergost-5-EN-3-OL
31.	27.931	3901349	6.77	Stigmasterol
32.	28.053	652838	1.13	15-Tetracosenoic acid, Methyl ester
33.	28.583	3785097	6.57	Stigmast-5-EN-3-OL
34.	29.380	1189631	2.06	Lupenone
35.	29.673	2516148	4.37	Lupeol
36.	31.515	726063	1.26	Neophytadiene
		57633692	100.00

Compound Identification

Components of the methnolic extracts were identified by comparison of their mass spectra and retention indices with those published in the literature and contained in the NIST ’98 MS computer library (Wiley).

RESULTS AND DISCUSSION:

The results of the GC-MS analyses are leading to the identification of a number of compounds in T. villosa. Thirty six components were identified in the powder extract of T. villosa. Hydrocarbons, fatty acids and their esters were found as the dominant groups in this fraction. The major components were exo-2-2 Hydroxycineole (17.71%), n-Hexadecanoic acid (14.68%) and Oleic Acid (12.94%). The chemical constituents of the analysed extract, the percentage composition of the individual constituents, and the major classes and subclasses of the identified components are presented in Table: 1and Fig: 1, 2 and 3 respectively. The chromatogram of the constituents indicated three major peaks (Fig: 4).

Fig 1: Mass spectrum for Tephrosia villosa

Fig 2: Mass spectrum for Tephrosia villosa

Different types of sterols were present in considerable amounts in the chosen species. Gamma-sitosterol and stigmasterol were found in this fraction. Sterols are important constituents of all eukaryotes and play vital role in plant cell membranes. Plant sterols possess valuable physiological activities; they are biogenetic precursors of many hormones and oviposition stimulants of some insects (Harborne, 2001).

Fig 3: Mass spectrum for Tephrosia villosa

Fig 4: Chromatogram for Tephrosia villosa

Stigmasterol was found to markedly inhibit tumor promotion in two-stage carcinogenesis in mice (Yasukawa et al., 1991; Kasahara et al., 1994) and to exhibit significant inhibitory effect on HIV reverse transcriptase (Akihisa et al., 2001). A mixture of stigmasterol and sitosterol was shown to possess anti-inflammatory activity after topical application (Gomez et al., 1999). Therefore, the presences of these sterols in chosen species are of practical importance. Sitosterol possesses antihyperlipoproteinaemic, antibacterial and antimycotic activity and has been shown to act as inhibitor of tumor promotion in vivo (Yasukawa et al., 1991) and to inhibit carcinogenesis (Raicht et al., 1980).

The fatty acids are well known active metabolites. They serve as an important energetic substrate for the cells. Linolenic acid is essential for maintenance of growth and α-linolenic acid for neural functions. Both acids were shown to be potent cycloxygenase-2 (COX-2) catalyzed prostaglandin biosynthesis inhibitors (Ringbom et al., 2001). Pain-relieving activity of a plant may be due to the anti-inflammatory effect of stigmasterol (Garcia et al., 1999; Gomez et al., 1999). Traditional use of the T. villosa for pain relief is well supported by the presence of stigmasterol and lupenol. Lupeol and b-amyrin both have a hepatoprotective effect (Sunitha et al., 2001; Oliveira et al., 2005) and lupeol also has a nephroprotective effect (Nagaraj, 2000). Some of main constituents identified in study are reported to have antibacterial property.Therefore, antibacterial constituents from T. villosa methanol extract could hold promise for future application in therapy. Further experiments, are planned to establish the influence of the components of these mixtures on the pharmacological activity.

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Received on 06.09.2012 Modified on 18.09.2012

Asian J. Research Chem. 5(11): Nov., 2012; Page 1331-1334