Effect of Solvent on the Phytochemical Extraction and GC-MS Analysis of Gymnema sylvestre

Since time immemorial, various parts of the plants such as leaves, roots, stem etc. are being used to treat number of diseases and infections. India is blessed with and is a source for variety of herbal plants with medicinal properties. Gymnema sylvestre is a woody climber shrub from the family of Apocynaceae. Gymnema genus has 50 species in the genus. It is native to India, Australia, Africa and China. It is found to be grown well in the tropical regions. Gmnema sylvestre is an important plant with medicinal properties. In local languages of India, it is called as Sakkarai kolli which literally means “destroyer of sugar”1. Because of the medicinal importance, this plant is used in the preparation of formulated medicine for treating various health ailments. In Indian Ayurvedic medicine system, G.sylvestre is used for treating diabetes. Therapeutically, crude extract of this plant is used as a diuretic, to cure stomachic, eye complaints, asthma, chronic cough, cardiopathy, constipation, piles etc. Apart from these, various pharmacological and biological activities such as antibacterial, antiviral, antifungal, antiinflammatory, anticancer has been reported2-5.


INTRODUCTION
Since time immemorial, various parts of the plants such as leaves, roots, stem etc. are being used to treat number of diseases and infections. India is blessed with and is a source for variety of herbal plants with medicinal properties. Gymnema sylvestre is a woody climber shrub from the family of Apocynaceae. Gymnema genus has 50 species in the genus. It is native to India, Australia, Africa and China. It is found to be grown well in the tropical regions. Gmnema sylvestre is an important plant with medicinal properties. In local languages of India, it is called as Sakkarai kolli which literally means "destroyer of sugar" 1 . Because of the medicinal importance, this plant is used in the preparation of formulated medicine for treating various health ailments. In Indian Ayurvedic medicine system, G.sylvestre is used for treating diabetes. Therapeutically, crude extract of this plant is used as a diuretic, to cure stomachic, eye complaints, asthma, chronic cough, cardiopathy, constipation, piles etc. Apart from these, various pharmacological and biological activities such as antibacterial, antiviral, antifungal, antiinflammatory, anticancer has been reported 2-5 . Due to the side effects associated with allopathic medicines, in recent years, research interests have turned towards plant-based phytochemicals in treating various diseases 6 . Phytocompounds from medicinal plants are used in formulations of various healthcare nutraceuticals and cosmetics products. The phytocompounds of G. sylvestre has been effective in controlling diabetes 7 . The phytochemical compounds like phenols, flavonoids, terpenoids, saponins, tannins of the plants are the base for modern day allopathic medicines. The active components of allopathic drugs constitute about 25 -40% of plant-based origins 8 . The literature survey revealed that no work has been done to compare the effect of solvents on biochemical constituents of G.sylvestre plant extracts. To the best of our knowledge, no study has been conducted to study the effect of agroclimatic location on the antioxidant activity of G. sylvestre leaves. In this study, the plant samples collected from different regions of Tamil Nadu were evaluated for their antioxidant activities and the sample showing high level of antioxidant activity was extracted with six different solvents like ethyl acetate, ethanol, methanol, chloroform, hexane and benzene. The phytochemical constituents of the crude extracts of G. sylvestre of the different solvents were characterized by GCMS analysis. This study will reveal the relationship between the effect of sampling locations to the quantity and quality of the phytocompounds and its antioxidant activities of the plant sample and also the effect of solvents on the phytochemical availability in the crude extract. This will help to select the suitable solvent based on the actual application of the extracts.

Effect of sampling location on antioxidant activity
The effect of locations on antioxidant activity of G.sylvestre plants was estimated by DPPH method following the method 9 of Blois,1958. The

DPPH radical scavenging activity
The influence of source of plant collection on the antioxidant activity was studied by DPPH method. The antioxidant activity % is presented in the Table 1. Significant influence of the location on antioxidant activity was found. Variation in the activity was witnessed with highest activity observed in the plants collected from Shenbagadevi falls at Coutrallam on the Western Ghats in Tirunelveli District with 73.40% followed by Anthyodhaya sangham at Trichy 66.10%. The plants collected from Gandhigram Trust at Dindigul district showed the least activity of 36.70%.

Chemical constituents of various extracts
The leaves obtained from coutrallam is used for the extraction and identification of phytochemicals using different solvents such as hexane, benzene, chloroform, ethyl acetate, methanol and ethanol. The results pertaining to GC-MS analysis (Figure 3) of the hexane crude extract of G.sylvestre leaves was analysed using GC-MS which lead to the identification of 36 different organic compounds is listed in the Table  2  reaction mixture (0.1mM DPPH and extract) was vortexed, incubated and its absorbance was measured at 517 nm. The scavenging ability of the plant extracts was calculated using the following equation (1) (1) Where, Abs control is the absorbance of DPPH without sample; Abs sample is the absorbance of DPPH with sample 10 Cieśla et al.

Extraction procedure
The plant sample obtained from Coutrallam is used for extraction. G.sylvestre leaf powder 50g was extracted with 500ml ethanol by cold maceration method for 72h. After extraction, it was filtered using Whatman N0. 41 filter paper to obtain solid particle free extract and the solvent was evaporated to dryness under vacuum using a rotary evaporator. The crude extract obtained was stored at 4 °C for further usage. The same procedure was followed for all other solvents like methanol, benzene, hexane, ethyl acetate and chloroform.

Estimation of chemical constituents by GC-MS
To determine the various volatile bioactive compounds present in each solvent extract, GC-MS analysis was conducted using SHIMADZU, QP2010 PLUS following the injecting temperature at 250°C, column temperature at 50 °C, pressure at 29.7 kPa and column flow rate at 0.72 ml/min. The total running time for the sample was 50 minutes. Based on the retention time the phytochemical compounds in various solvent extracts were identified by matching MS with available standards using NIST and Willey library.
The GC-MS analysis ( Figure 5) results of the chloroform extract of G.sylvestre showed the presence of 32 compounds in it. The compounds present in the chloroform extract is given in the Table 4.

DISCUSSION
In the current study, out of various locations (Districts of Tamilnadu), the plant samples collected from Shenbagadevi falls, Courtallam showed maximum antioxidant activity and it was found to be in the order of Courtallam > Thirunelveli > Trichy > Coimbatore > Arni (Thirvannamalai) > Padavedu (Thiruvannamalai) > Chengalpattu > Dindigul. Significant variations in the antioxidant activities with respect to the sampling locations were observed. The dynamics of variation in the antioxidant content is possibly associated with the expression of variety of genes during various developmental stages of the plant or because of the environmental factors arising from seasonal variations [11][12][13] . In general, environmental factors like variations in the altitude, temperature, precipitation, etc varies rapidly. The sampling locations of this study falls between 36 to 1778m elevations representing diverse climatic conditions, which is associated with antioxidant activity 14 . Literature review also suggests that antioxidant activity is influenced by various species of compounds. This implies that the intake of antioxidant compounds by the G.sylvestre plants will have vital impact on the antioxidant activity of the plant samples collected from diverse locations. The variation of intake depends on the texture of the soil and the seasonal conditions 15 . It is noteworthy that the observed antioxidant activity is much superior to that of the total leaf extract reported recently 16 .
The compounds identified in the crude extracts of G.sylvestre are mostly belongs to terpenes, alcohols, hydrocarbons, alkaloids and its derivatives. From the literature search, these compounds are found to be known for their therapeutic properties and are previously reported in many different medicinal plants. Some of these compounds are separately isolated in extracts and are used as antimicrobial and radical scavenging agents in medicine formulations. This study shows that the chemical compounds isolated in different crude extracts of G.sylvestre could be used as a vital source of antioxidant for food and pharmaceutical industry.
The crude extracts from the G.sylvestre leaves were subjected for GCMS analysis for identifying compounds. Various studies using GCMS has revealed the influence of different solvents in isolating the phytochemical constituents with medicinal values from crude extracts of medicinal plants 17 . The major chemical compounds identified in the G.sylvestre crude extracts such as Inositol, 1-deoxy-found in methanol extract, 2-Pentanone, 3,3,4,4-tetramethyl found in methanol and ethanolic extracts, Tetratriacontane and Hexadecane found in benzene, chloroform, ethyl acetate and hexane extracts, Eicosane form benzene, chloroform and ethyl acetate extracts, Heneicosane found in benzene, ethyl acetate and hexane extracts, Phthalic acid, di(2-propylpentyl) ester found in hexane and methanol extracts, Squalene, Phytol, n-Hexadecanoic acid and Stigmasterol found in all the extracts are chemically or biologically active compounds (Table  8).

S. No.
Compound Name Structure Nature and Therapeutic activity

CONCLUSION
The present study revealed a number of compounds isolated in different solvents and its efficiency. Also, the effect of locations on the antioxidant activity exhibited by the plant was revealed. The whole plant can be used as a good source of antioxidant. Future research will be taken up for the isolation and characterization of individual compounds from the crude extracts of Gymnema sylvestre and tested for in-vivo studies for further understanding the activities of plant compounds.