Isolation and Characterization of Β -Sitosterol from Methanol Extracts of the Stem Bark of Large- Leaved Rock Fig ( Ficus Abutilifolia Miq)

: This work was aimed at isolating and characterizing some bioactive constituent(s) from the stem bark of Ficus abutilifolia. The phytochemical analysis of the crude methanol extract indicates the presence of flavonoids, alkaloids, cardiac glycosides, saponnins, tannin, steroids and triterpenes. The extract was subjected to column chromatography which resulted in the isolation and purification of a white needle-like crystalline substance with a melting point of 134 – 136 0 C, the 13 CNMR shows recognizable peak at 71.98 ppm assignable to the beta hydroxyl group and the 1 HNMR spectrum which shows an olefinic proton at δ 5.37 was identified as β -sitosterol. Identification of the compound was purely by spectral evidence.

Nature in general, has provided mankind with a complete store house of remedies to cure most human ailments Yadav and Singh, (2011). Medicinal plants contain several secondary metabolites such as alkaloids, saponnins, flavonoids, carbohydrates, tannins and anthraquinones which exert biological effects such as antioxidant, anti malaria, antibacterial and anti-inflammatory activities against external agents especially microorganisms Salem et al., (2013). The Ficus species belongs to the family, Moraceae often called the Mulberry family or Fig family is a family of flowering plants comprising about 53 genera and over 1,400 species. Most are widespread in tropical and subtropical regions but a few extend into the temperate regions Singh et al., (2008). F. abutilifolia is a small-medium sized, deciduous-semideciduous tree up to 50m high. The bark is whitish yellowish or white and smooth, powdery or somewhat flaking and it is this conspicuous bark that is immediately apparent to the eye. The trunk is usually twisted or contorted, branchless, short and glabrous; lacking hairs and marked with leaf and stipular scars Banso and Adeyemo, (2006). F. abutilifolia miq is known by the common names large leaves rock fig or rock wild fig. The leaves of F. abutilifolia are used traditionally to treat various ailments such as typhoid fever, chronic dysentery, sexually transmitted infections, malaria, infertility and the milky latex is used to remove skin warts. The stem bark decoction is taken by men as a strengthening tonic by indigenous communities especially in south eastern and northern parts of Nigeria Ukwulibile, (2014). This research is aimed at the isolation and characterization of the bioactive compound(s) from the stem bark of Ficus abutilifolia.

MATERIALS AND METHOD
Plant collection and methods: The stem bark of Ficus abutilifolia Miq was collected fresh from Samaru, Zaria in Kaduna State, Nigeria in March, 2017. Taxonomical identification was done at the Herbarium of the Biological Sciences Department, Ahmadu Bello University, Zaria, Nigeria and the voucher number of its deposited specimen in the herbarium is 900742. The plant bark was air-dried under a shade, segregated and pulverized by manual pounding using wooden mortar and pestle. The pulverized plant material was stored in a cool dry place to avoid moisture from the environment.
Extraction and isolation: Five hundred gram (500 g) of the pulverized stem bark was extracted by cold maceration using 2 liters of methanol at room temperature for 48 hours. The supernatant was filtered through Whatman No.1 filter paper. The extract was dried under reduced pressure using the rotatory evaporator at 40 0 C. The quantity extracted was 20 g. The crude extract was subjected to the qualitative phytochemicals analysis using standard methods of Harborne, (1998) (Table 1). Phytochemical screening of the extracts indicated the presence of flavonoids, saponins, glycosides, tannins, saponnins, alkaloids, steroids and terpenoids. A small portion of the crude methanol extract was dissolved in n-hexane and the solution was spotted on precoated TLC plates. The TLC plates were developed by specific solvent systems and were viewed individually under UV light at 236 nm and also sprayed with the Vanillin-H2SO4 reagent. Through several pilot test experiments it was found out that the compounds of this methanol extract were separated by the solvent system of n-hexane and ethyl acetate in the proportion of 9:1 and the separation was better at 4:1 solvent system. The methanol extract (5 g) was subjected to column chromatography using silica gel (70-230 mesh). The column was packed and eluted with 4:1 n-hexane and ethyl acetate mixture to obtain 32 fractions of 50 mls each. Collected fractions were spotted on a TLC plate and a single spot was observed for A10 and further purification was carried out on it using preparative TLC with a solvent system of 1:4 ethyl acetate and n-hexane to obtain a homogenous A10 compound. This was a white crystalline substance with a melting point of 134 -136°C. The substance was further subjected to 1 H-NMR and 13 C-NMR spectroscopy.

RESULT AND DISCUSSION
Phytochemical analysis test: The results of the phytochemical analysis of the crude methanol extracts revealed the presence of alkaloids, flavonoids, steroids/triterpenes, tannins, saponnins and cardiaic glycoside. Isolation, purification and characterization: the column separation of the methanol extract with a solvent system of 4:1 of n-hexane and ethyl acetate led to the isolation of a compound (A10), which was identified as beta-sitosterol (structure 1) with the following physical properties, chemical and spectral data.
Physical properties: Compound A10 was isolated as a white needle -like crystalline substance with a melting point of 134 -136 0 C and a molecular formula of C29H50O.
Chemical test of the isolated compound: the isolated compound showed positive tests for Salkowski and Liebermann-Burchard test thus confirming the compound as steroid/triterpenoid.
Spectroscopic analysis : 1 H NMR (CDCl3, 400 MHz); The 1 H NMR spectrum of the compound (ppm) showed the presence of six methyl signals that appeared as two methyl sinlgets at δ 0.70, and 1.03 (Fig 1) which corresponds to the angular methyl singlets. Three methyl doublets appeared at δ 0.95, 0.84, and 0.85, and a methyl triplet at δ 0.87correspounding to the methyl protons. The 1 H NMR spectrum of this substance also showed one olefinic proton at δ 5.37. Its 1 H NMR spectrum showed a proton corresponding to the hydroxyl group which appeared as a multiplet at δ 3.55ppm.
Structure 1. Chemical structure of β -sitosterol Molecular formula: C29H50O; Molecular weight: 414g/mol; Melting point: 134 -136 0 C 13 C NMR (CDCl3, 100 MHz); The 13 C-NMR showed recognizable signals at 140.88 ppm and 121.80 ppm which are typical of alkene double bonds (Fig 2). The values at 19.18 and 12.12ppm correspond to angular methyl carbon atoms. The signal at 71.98 ppm is assignable to the beta hydroxyl group attached to the carbon at position 3 Structure 1. The proton and carbon spectral are shown in Figures 1 and 2. The 13 C-NMR spectrum also showed twenty-nine carbon signals made up of six methyl, eleven methylenes, nine methine and three quaternary carbon signals. Based on these results and also by comparism with existing literature the spectral peaks are typical of βsitosterol Arjun et al., (2010). The isolated sitosterol which is a known compound has been reported to possess pharmacological activities such as antifungal and antibacterial, antioxidant and antidiabetic properties, anticancer activities, antihelmitic and antimutagenic activities and anti-inflammatory activity in human systems Soodabeh et al., (2014). The protons and carbons were assigned on the basis of their 1 H and 13 C NMR shifts given in Table 2. Conclusion: Compound A10 isolated from the methanol stem bark extracts of Ficus abulitifolia is a phytosterols named β -sitosterol. The structure of the isolated compound were identified on the basis of spectroscopic methods and by comparing their physical properties reported in the literature. This is the first time beta-sitosterol is isolated from the plant of Ficus abutilifolia Miq.