Isolation & Characterisation of Beta-sitosterol from the rhizomes of Arisaema utile and its Evaluation for Antioxidant Activity

The aim of this study is isolation, identification and characterized the bioactive compounds from the rhizomes of arisaema utile. Preliminary Phytochemical screening of the rhizome extract of arisaema utile revealed the presence of Steroids, terpenoids, Flavonoids, Alkaloids, Saponins & Carbohydrates. The air dried rhizomes were pulverized to powder, subjected to Soxhlet extraction and compound Isolation. The isolated compound was colorless crystalline, which was further subjected to IR, 13 CNMR and 1 HNMR for proper characterization and elucidation of the structure. The compound was concluded as βSitosterol. Antioxidant activity of the isolated compound was measured by DPPH assay under in-vitro condition. The isolated compound showed most promising radical scavenging activity at concentration of 10μg/ml.


INTRODUCTION
A substantial part of all drugs are still based on compounds originally isolated from nature. The plant kingdom is a treasure house of potential drugs and in the recent years there has been an increasing awareness about the importance of medicinal plants. Drugs from the plants are easily available, less expensive, safe, and efficient and rarely have side effects. Arisaema is a genus of about 150 species in the flowering plant family Araceae, native to eastern Africa, central Africa, Asia and eastern North America. Asiatic species are often called cobra lilies, while western species are often called jack-in-the-pulpit. It can be found growing on rocky slopes at an altitude of 2,400-4,600 meters. It grows in shady, moist, well-drained and fertile soil. Arisaemas are tuberous perennials that die back to the ground in winter. Arisaema utile emerges in spring. Most of the species from genus Araceae have a history of use in folk medicine for the treatment of various infectious diseases. Rhizomes of few species of Arisaema have long history of use in Traditional Medicine, especially in Asian countries like Arisaema jacquemontiana for Muscular strength and Skin infections, Arisaema propinquum for Skin eruption or rashes etc [1]. Many medicinal plants have been screened extensively for their antimicrobial potential worldwide [2, 3, 4]. Further, plant phenolic compounds have been found to possess potent antioxidant [4-9], antimicrobial and anticancer activities [10,11]. As an individual plant, Arisaema utile is used for treating various infections in the blood, liver and bile which correlates to the signs and symptoms of parasitic and microbial infections, cancer and inflammatory conditions. A lectin was also purified from tubers of Himalayan cobra lily Arisaema utile [12]. One of the most exiting properties resulting out of the interaction of lectins with lymphocytes is mitogenicity, i.e. the triggering of quiescent, non-dividing lymphocytes into a state of growth and proliferation. The discovery of first mitogenic lectin Nowell [13] led to the detection of many other such lectins, most notably concanavalin A [14], Wheat germ agglutinin [15] and Pokeweed mitogen [16]. The crude extracts of this plant as mentioned above showed significant antimicrobial, antioxidant and prominent cytotoxic activities against few cancer cell lines. The plant has reports of being used in traditional medicines by the tribal people of Jammu and Kashmir for curing various diseases. Keeping in view global and national scenario of medicinal plants, Encouraged by these finding, we carried out in-depth phytochemical isolation and further investigate the antifungal and antioxidant activities of the isolated compound from arisaema utile especially existing at high altitudes of Jammu and Kashmir with proven folklore medicinal claim.

Collection of Plant material and processing:
The Arisaema utile plant material was collected in the month of July from Gulmarg area of district Budgam of Jammu & Kashmir state, India. Voucher specimen of Arisaema utile bearing specimen no 27911, was Identified and deposited at KASH herbarium in

Extraction and purification:
Eight hundred grams (800g) of powdered rhizome of arisaema utile was subjected to sequential extraction using soxhlet apparatus from non polar to polar solvents such as n-hexane < ethyl acetate < methanol. The solvent was recovered under reduced pressure using rotary evaporator under vacuum condition and the residue was stored in the refrigerator. Hexane extract was chromatographed on a silica gel column and eluted with solvent mixtures of increasing polarity, composed of hexane, ethyl acetate and methanol. All the fractions were monitored on TLC. Fractions collected with 20:80 ethyl acetate/Hexane were pulled together as these fractions showed a single spot on TLC. Further these combined fractions were kept in refrigerator overnight for crystallization which resulted in the formation of crystalline needle shaped compound 1. The structure of the isolated compound was established on the basis of elemental analysis and spectroscopic evidences (IR, 1 HNMR, 13 CNMR). The structure was simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon.

Spectroscopic characterization of Compound 1
The various spectroscopic methods like FT-IR, DEPT, 1 H-NMR, and 13 C-NMR were used to elucidate the structure of isolated compounds. The Fourier Transform-Infrared (FTIR) spectroscopy was carried out on a Perkin Elmer FT-IR fitted with Spectrum software version 10.3.2 using a liquid sampler. 1 H-NMR (400MHz) and 13 C-NMR (400MHz) were recorded using CDCl 3 as solvent in MeOD on Bruker, Avance (400MHz) NMR spectrometer.

Determination of antioxidant activity
The in-vitro antioxidant potential of the isolated compounds was measured in terms of hydrogen donating or free radical scavenging ability using the stable radical DPPH according to the standard procedure [17]. 0.1mM solution of DPPH in Methanol was prepared and 1.0ml of this solution was added to 1.0 ml of the test solution in methanol at different concentrations of Isolated Compounds (2, 4, 8, 10 & 12µg/mL). The reaction mixture was incubated at 37 • C for 30 min in darkness. The absorbance of the sample at 517 nm was measured and then compared with that of a control solution containing the reaction mixture amended methanol instead of Isolated Compounds. Ascorbic acid (2, 4, 8, 10 & 12µg/mL) was used as the standard reference compound, and the percentage of DPPH free radical scavenging activity was calculated using the following equation: % scavenging activity = ((A 0 -A) / A 0 × 100). Where A 0 was the absorbance of the control (blank, without compound) and A was the absorbance of the reaction mixture. All the tests were performed in triplicate and the graph was plotted with the mean values.

A. Fourier Transform-Infrared (FT-IR) spectroscopy
The IR absorption spectrum of Compound 1 showed absorption peaks at 3332.   Escudero et al., 1985 [19, 20]. (Table 1) shows the 1 H-NMR and 13 C-NMR values in comparison with the previous data available. 13  The 13 C-NMR of compound 1 has shown recognizable signals at 140.8 and 121.7 ppm, which are assigned C5 and C6 double bonds respectively. The value at 24.32 ppm corresponds to angular carbon atom (C-15). Spectra show twenty nine carbon signal including six methyls, nine methylenes, eleven methane and three quaternary carbons. The alkene carbons appeared at 140.8 and 121.7 ppm. The structure was simulated using ACD/NMR program to obtain the chemical shifts of both proton and carbon. The above spectral features are in closed agreement to those observed for β -Sitosterol according to Manoharan et al., 2005 andEscudero et al., 1985 [19, 20]. On comparison the standard data matched with the simulated data which supports the proposed structure of this compound as β -Sitosterol. ii). Liebermann burchard reaction: A few crystals of compound 1 were dissolved in chloroform and a few drops of concentrated sulfuric acid were added to it followed by addition of 2-3 drops of acetic anhydride. Solution turned violet blue and finally green or green-blue coloured after a few minutes thus positive indication of steroid ring [21].

C-NMR and DEPT
Phytochemical tests (Salkowski's test and Lieberman-Burchard test) of the compound 1 confirm its steroidal nature. Since, the NMR machine indicated steroidal nucleus and the compound gives positive test for steroids so all of the other structures other than steroids were rejected. Based upon the functional group analysis it was found that the nature of oxygen was hydroxyl, also supported by IR spectroscopy (Perkin Elmer FT-IR). This implies presence of one double bond in the structure. So, the steroids with other functional groups were rejected. Also on considering the nature of oxygen as hydroxyl and presence of one double bond, the general formula for the compound is C n H 2n-8 O.
The exact molecular mass for the formula is found to be 414.37 and the chemical formula could be tentatively C 29 H 50 O. Therefore it must be a tetra cyclic compound.
Based on the analysis of spectral data (IR, 13 C-NMR, 1 HNMR and DEPT) the structure of the isolated compound 1 is proposed as (Fig. 1);

Fig. 1. β-sitosterol (Stigmast-5-en-3β-ol) (Compound 1)
β-sitosterol is a natural micro-nutrient which is found in the cells and membranes of all oil producing plants, fruit, vegetables, grains, seeds and trees. It has been proven to be a safe, natural and effective nutritional supplement and has shown amazing potential benefits in many diverse applications.  (Table  2). Compound 1 showed most promising radical scavenging activity at concentration of 8μg/ml. These results are plotted in the form of graph (Fig. 2)

IV. CONCLUSION
In our study an attempt was made to isolate the compounds for the first time from rhizomes of Arisaema utile and highlight antioxidant potential of the isolated compounds. In our study a steroid was isolated and reported for the first time from rhizomes of Arisaema utile and its antioxidant potential was evaluated. The structure of the isolated compound was identified beta-sitosteroid on the basis of spectroscopic methods and by comparing their physical properties reported in the literature. Beta-sitosterol is mainly known and used for its cholesterol lowering property. But studies have shown that the phytochemical may have other health benefits: reducing risk of cancer, prevention of fungal infections and prevention of oxidative damage through its antioxidant activity. The results of the present study suggest that the isolated compound (Beta-sitosterol) from Arisaema utile shows promising antioxidant activity. Therefore this plant can hopefully be considered in future for more clinical evaluations and possible applications. We should maintain our efforts in considering and valorizing our natural patrimony as well as conducting more research on Arisaema utile with an aim to isolate some Novel compounds with promising pharmacological aspects.