GC-MS analysis of bioactive compounds in methanolic extract of tubers of Pueraria tuberosa (Roxb. ex Willd.) DC. - Fabaceae

The present experiment was designed to determine the bioactive constituents from tuber extracts of Pueraria tuberosa (Roxb. ex Willd.) DC. of the family Fabaceae. The medicinal value of a plant species is dependent upon its various phytochemical constituents. The chemical compositions of the methanolic extract of tubers of P. tuberosa were investigated using Gas chromatography-Mass spectrometry and about nineteen bioactive phytochemical compounds were identified. The prevailing compounds were 2, 3-Dimethylaziridine; 2-Cyclopenten-1-one, 2-hydroxy-; 2-Hydroxy-gamma-butyrolactone; 3-Methyl-1,2-cyclopentanedione; 2,5- Dimethyl-4-hydroxy-3 (2H) – furanone; Butane 2-methyl; Oxetane; Maltol; 1, 5-Anhydro-6-deoxyhexo-2,3-diulose; 2, 3-Dihydro-2, 5-dihydroxy-6-methyl-4H-pyran-4-One; 5-Hydroxymethylfurfural, Phenol,2,6-dimethoxy; Dodecanoic Acid; Guanosine; Tetradecanoic acid; Myo-inositol; Hexadecanoic Acid; 9, 12-Octadecadienoic acid, methyl ester and Cis-vaccenic acid. This was the first report on identification of bioactive compounds from methanolic extract of tubers of P. tuberosa.


INTRODUCTION
From ancient times plants are best sources of bioactive compounds having interesting biological activities. Literature studies represent the medicinal plants as reservoir of effective chemotherapeutants, play a principal role in the maintenance of human health. Knowledge on the phytoconstituents of plants is highly desirable for disclosing the actual significance of folkloric remedies, Milne, (1993). The secondary metabolites of plants have a variety of structural arrangements and properties, De-Fatima et al., (2006). Phytochemicals of natural drugs have overlapping and complementary mechanism of action; hence thorough validation of natural drugs was prioritized and emphasized. Mass Spectrometry coupled with Gas Chromatography is normally used for the direct analysis of chemical constituents present in plant based medicine. GC-MS analysis is a highly commended analysis for non-polar components, fatty acids, volatile essential oil, lipids, Jie and Choi, (1991) and alkaloids, Betz et al., (1997).
The tubers of P.tuberosa are widely used in ayurveda and in ethanomedicine. It has been recommended for the treatment of menopausal syndrome, sexual debility, cardiovascular diseases, fertility disorders, hepatosplenomegaly and spermatorrhoea and has been used as antiaging, spermatogenic and immune booster, Amal et al., (2014). There has been tremendous progress in medicinal plant research which involve the isolation and identification of secondary metabolites of plants and their use as active principles in therapeutics , Mary et al., (2013). Literature studies indicate that no reports on GC-MS analysis P. tuberosa has so far been undertaken to provide enough data in favour of its traditional uses. As part of the endeavor for the study of therapeutic properties of P. tuberosa we herein reported the GC-MS analysis of methanol extract of the tubers.

Collection of plant sample
The tubers (Plate 1) of the P. tuberosa (Roxb. ex Willd.) DC. were collected from Nelliyampathy forests of Palakkad district, Kerala state. The tubers were authenticated by Dr. P.S. Udayan, Sree Krishna College, Guruvayur and the voucher specimens were preserved for further reference.

Preparation of powder and extract
Collected tubers were thoroughly washed in running tap water for 10 minutes. These were cut into pieces and were air dried in shade so as to prevent decomposition of active principle and made fine powder by using mechanical grinder. Then the powder was extracted using methanol as a solvent. Twenty gram of dried powder was weighed and subjected to extract successively with 200 ml methanol in soxhlet extractor. The extract was condensed and preserved in refrigerator in air tight bottles.

GC-MS analysis of bioactive compounds
The methanolic extracts obtained were subjected to GC-MS analysis for the determination of various bioactive volatile compounds in P. tuberosa. The analysis was carried out using Shimadzu Make QP-2010 with nonpolar 60 M RTX MS column, operating in electron impact mode at 70 eV. Helium was used as the carrier gas and an injection volume of 0.5μl was employed in split less mode at injection temperature 260ºC; ion-source temperature 200ºC. The oven temperature programming was set with a rate of 10 ºC with an initial oven temperature at 60º C and final temperature at 280º C, held for 8 minutes. The total running time for the sample was 25 minutes. The chemical constituents of the methanolic tuber extracts of plant samples were identified by comparing the retention times of peaks using NIST Library to relative retention indices. The relative percentage of each of the component in the extract was calculated by comparing its average peak area to the total areas.
It has been reported that tuberosin, one of the active principles of P. tuberosa inhibits lipopolysaccharide (LPS) induced inflammatory changes in macrophages and directly scavenges various species of free radicals, Panday and Tripati, (2010). P.tuberosa showed significant dose dependent ulcer protective effect due to its antioxidant activities and it was comparable to the reference drug OMP, Sumalatha et al., (2010). In the present experiment different compounds displayed similar activity and the presence of various radical scavenging and antiinflammatory compounds in the methanolic extract of P.tuberosa may be the responsible for its antioxidant properties.

V. CONCLUSION
The GC-MS studies carried out on methanol extract of tubers of P. tuberosa showed the presence of chemical components responsible for its potent medicinal activity. Further work regarding specific activity of various identified compound will provide more ins ight about the use of the tuber.