Lingarao & Savithramma

ISSN-2249-6335

ISOLATION AND CHARACTERIZATION OF SITOSTEROL FROM LEAF METHANOL EXTRACT OF SVENSONIA HYDEROBADENSIS A RARE MEDICINAL PLANT TAXON
M Lingarao, N Savithramma*
Department of Botany, S.V. University, Tirupati, A.P. India

Science Instinct Publications

Abstract

Phytochemical screening of the methanolic leaf extract of Svensonia hyderobadensis revealed that the presence of steroids, terpenoids, phenolic compounds, saponins, tannins and flavonoids. The aim of the present study was to isolate and characterize the bioactive compound from S. hyderobadensis a rare medicinal plant. Dried leaf powder of S. hyderobadensis was extracted with various solvents and subjected to column chromatography and characterized by IR, LCMS, 1H NMR and 13C NMR data analysis. The results were obtained by methanolic and chloroform solvent system (9:1), based on the spectral data analysis and chemical reactions, the compound have been established as -sitosterol. Keywords: Svensonia hyderobadensis, -sitosterol, leaf, chromatography

Corresponding Author: N. Savithramma Department of Botany, S.V. University, Tirupati, A.P. India

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Introduction Plant secondary metabolites consist of low molecular weight compounds that are regarded as not essential for sustaining life, but as crucial for the survival of the production organism [1]. More than 50,000 structures have been identified in plants by NMR, MS and X-ray analysis. However, as only less than 20% of all plants have been studied, it is very likely that the actual number of secondary metabolites or bioactive compounds in the plant kingdom would exceed 100,000 structures [2]. Secondary metabolites are produced in specific pathway and sites of synthesis can differ between types of compounds and between plant species. Natural products have been considered anecdotal to the effective maintenance of good health. -sitosterol is a known plant sterol, the sterol in plant is called phytosterols and it is a waxy substance which is white in colour. -sitosterol has also reported to be abundant in wheat germ oil, cotton seed oil, corn oil and soybean oil [3]. Its efficacy is reported as follows in the literature review.

Fig. 1. Structure of -sitosterol The structures of -sitosterol and cholesterol are quite similar. It is reasonable that sitosterol can inhibit the absorbing of cholesterol in the body [4] and thus reduce the cholesterol levels in the plasma [5]. The liver function activity can be improved with sitosterol [6] and this can reduce prostate cancer and colan-cancer cell growth [7, 8]. sitosterol can also be found in vegetables and fruits, the presence of -sitosterol in soybean foods has been reported to suppress carcinogenesis. It can also be the factor used to form the lymphocells and NK in the immunity process circulation [9]. -sitosterol can be found in vegetables such as peanut oil. It is used in experiments for treating breast cancer and prostate cancer -sitosterol in soybean oil has been reported to reduce Cholesterol levels [10]. Natural compounds can be lead compounds, allowing the design and rational planning of new drugs, biomimetic synthesis development and the discovery of new therapeutic properties not yet attributed to known compounds [11]. In addition, compounds such as muscarine, physostigmine, cannabinoids, yohimbine, forskolin, colchicines and phorbol esters, all obtained from plants, are important tools used in pharmacological, physiological and
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biochemical studies [12]. Svensonia hyderobadnesis is belongs to the family Verbenaceae. As the plant is a rare medicinal herb and has hepato protective [13], the Phytochemical [14], pharmacongostical [15] and antimicrobial studies were carried out. It is an ideal plant for the synthesis of silver nanoparticles [16]. An attempt had been made to isolate the bioactive compound from the leaves of Svensonia hyderobadensis as the maiden step. Material and methods Isolation by silica gel column chromatography The dry crude methanol extract (25 g) was adsorbed on silica gel (Acmaes 60-120 mesh) and loaded on a silica gel (Acmaes 100-200 mesh) by dry packing method. The column (100 cm x 3.5 cm i.d.) was eluted initially (in hexane, then followed by chloroform: methanol in gradient manner) with continuous suitable system and gradually with increasing the polarity of mixture of solvents. Fractions collected were monitored on silica gel TLC. The visualization of spots on TLC plates was carried out either in UV Light or exposing TLC plates to iodine vapours. Thin layer chromatography Silica gel 25 g (Acmaes) was dissolved in 50 ml of double distilled water, stirred well and then poured in the spreader. The spreader was gently pulled along the plates, (2-3 mm thickness) to get an even application adopting the method of Jaworska and Nybom [17]. The plates were activated at 100C for about 45 min, allowed to cool, removed from the oven and subjected to use. The fractions were applied with micropipette, 2 cm above the base of the plate. The material was applied in the form of spot and was allowed to dry with hair dryer. The solvent was allowed to move to 15 cm, then plates were removed, allowed to dry and subjected to the different spray reagents, then the compounds were visualized. In this way numbers of silica gel coated plates were developed in different solvent systems. The outline of the compounds was marked with needle on the unsprayed silica gel plates. The compounds were eluted into the corresponding solvent systems and the eluates were preserved for physical analyses. Characterization and structural determination of the compounds were established mainly on the basis of spectral studies. Ultra-violet (UV) spectra were recorded on Beckman 25 and Shimadzu UV-240 spectrophotometers and values were given in nm. Infrared spectroscopy (IR) involves the energy transition between different vibrational energy levels. Mainly IR can be used to find the functional groups present in a pure or mixture of compounds, because IR gives details of strong absorption pattern at a particular frequency of a particular functional group. Infra red spectrums were recorded on IR AFFINITY-1 model shimadzu FT-IR spectrophotometer by the KBr pallet method. Nuclear magnetic resonance (NMR) sepectroscopy NMR is used as analytical tool for predicting the structure of the molecule based on the different environments of hydrogen atom by measuring the magnetic moments of hydrogen atom. Purified sample was subjected to NMR studies. Tetra Methyl Saline (TMS) was used as standard, which shows chemical shift value at zero on the scale. 1H and 13C NMR spectra were recorded with a Bruker-AMX400 (400 MHz) NMR Spectrometer (H1), 400 MHz (C13) and chemical shifts were recorded in ppm.
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Mass Spectrometry (MASS) Mass spectra (MS) were recorded on LC-MSD-Trape-SL (Agilent Technologies). Structure elucidations of all these compounds were presented separately in results. All the chemicals and reagents used were obtained in high purity from E. Merck Pvt. Ltd., Bombay, India. Result and discussion Methanolic extract on concentration yielded a brown viscous residue (40 g). This residue was subjected to column chromatography over silica gel using the chloroform: Methanol mixtures in increasing polarity. The bioactive fraction was collected on 1: 9 ratio of chloroform and Methanolic solvent system and examined. Fraction Physical properties Amount isolated Melting point Molecular formula Compound : White crystalline substance : 500 mg : 144-1460C : C29H50O : -sitosterol

IR (KBr) Vmax: 3389.08, 2973.40, 2935.78, 1693.57, 1560.48, 1463.07, 1428.35, 1368.55, 1297.18, 1075.36, 879.58, 814.96, 781.20 cm-1
1

H NMR (400 MHz MeOD): 3.51 (m), 5.248 (br, s), 0.68 (s), 1.07 (s), 0.90 (d, j = 6.4), 0.812 (d, j = 6.5), 0.838 (d, bj = 6.5), 0.845 (d, j = 7.5), 3.55 (m), 5.239 (br, 5), 0.656 (s), 1.14 (d, j = 7.5), 0.892 (d, j = 6.5), 0.854 (d, j = 6.5), 0.797 (t, j = 7.5).
13

C NMR (400 MHz MeOD): 140.8 (C-5), 121.7 (C-6), 71.72 (C-3), 58.14 (C-14), 56 (C17), 51.75 (C-9), 45.9 (C-24), 42.3 (C-13), 42.2 (C-4), 39.8 (C-12), 37.3 (C-1), 36.5 (C-10), 36.2 (C-20), 33.9 (C-22), 31.9 (C-8), 31.9 (C-7), 30.76 (C-2), 29.2 (C-25), 28.3 (C-16), 26.1 (C-23), 24.3 (C-15), 23.1 (C-28), 21.35 (C-11), 19.8 (C-26), 19.4 (C-19), 19.3 (C-27), 18.49 (C-21), 11.9 (C-18), 12.2 (C-29). The IR absorption spectrum showed absorption peaks at 3389.08 cm-1 (O-Hstr.); 2973.49 cm1 (- HC == CH cyclic); 2935.78 cm-1 (C-Hstr.); 1693.57 cm-1 (C-Hstr.); 1560.48 cm-1 (C=C absorption peak); other absorption peaks includes 1428 cm-1 ([CH2] n); 1368 cm-1 (OH def.) and 1075 cm-1 (cycloalkane).
1

HNMR has given signals at 3.51 (m), 5.248 (br, s), 0.68 (s), 1.07 (s), 0.90 (d, J = 6.4), 0.812 (d, J = 6.5), 0.838 (d, bj = 6.5) and 0.845 (d, j = 7.5) ppm. Other peaks are observed at 3.51 (m), 5.239 (br, 5), 0.656 (s), 1.14 (d, j = 7.5), 0.892 (d, j = 6.5), 0.854 (d, j = 6.5) and 0.797 (t, j = 7.5) ppm.

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Table-1: 13C and 1H NMR data of -sitosterol S. NO. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
13 13

37.3 30.76 71.72 42.2 140.8 121.7 31.9 31.9 51.75 36.5 21.35 39.8 42.3 58.14 24.3 28.3 56 11.9 19.4 36.2 18.49 33.9 26.1 45.9 29.2 19.8 19.3 23.1 12.2 0.812 (d, 6.5) 0.812 (d, 6.5) 0.838 (d, 6.5) 0.90 (d, 6.4) 0.680 s 1.07 s 5.248 br s 3.51 m

NMR has given signal at 140.8 and 121.7 ppm for C5=C6 double bond respectively, 71.8 for C3 -hydroxyl group 19.4 and 11.9 for angular methyl carbon atoms for C19 and C18 respectively (Table-1). LCMS Spectrometry showed the molecular ion peaks at 414 correspond to the molecular formula C29H50O. Ion peaks were also observed at m/z 379, 341, 271, 253, 313, 159, 133, 81 and 57. -sitosterol is a white crystalline substance with a melting point of 144-1460C. -sitosterol gave a positive test to Liebermann Buchard reagent
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for steroidal nucleus. On subjection to I.R. Spectroscopic analysis, the observed absorption bands are 3389 cm1 (b) that is characteristic of O-H stretching. Absorption at 2973 cm1 is due to cyclic olefinic HC = CH str, 2935 cm1 is due to = CH str. and 1693 cm1 assigned to C-H str. Other absorption frequencies include 1560 cm1 as a result of C=C absorption, however, this band is weak [18]. The proton NMR has revealed the existence of signals for Olefinic proton at 5.358 (br, s.), Angular methyl proton at 0.68 (s), 0.699 (s) and 1.01 (s) corresponding to C18 and C19 proton respectively. The 13CNMR has shown recognizable signals 140.8 and 121.7 ppm, which are assigned C5 and C6 double bonds respectively as in 5 spirostene [19]. The value at 71.0 ppm is due to C.3 hydroxyl group [18]. The signals at 19.4 and 11.9 ppm correspond to angular carbon atom (C19 and C18 respectively). The value for C18 is lower due to -gauche interaction that increases the screening of the C18 hence lower chemicals shift. However, the loss of H in C6 results in decrease in screening of the C19 leading to increase in 13C chemicals shift to higher frequency [20]. The above IR, 1 HNMR, 13CNMR and LCMS spectral data and a comparison of the 13CNMR signal with those described in the literatures [18, 19, 21] showed the structure of -sitosterol (Fig-1).

Acknowledgement Authors are very much thankful to Saif, IIT Madras and Prof. C. Venkata Rao, Department of Chemistry, S.V. University, Tirupati for structural analysis.

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