Isolation of -amyrin from the Stems of Solenostemma argel, Elucidation of its Structure and Determination of Antimicrobial Activity

New triterpenoid alcohol was isolated from the active fraction of the extract of stems of Solenostemma argel and elucidated by physical (m. p.) and spectroscopic methods (UV, IR, 1 H and 13 CNMR and EIMS), as β-amyrin. The cup-plate agar diffusion method was used to estimate the inhibition of the activity of four types of bacteria: two Gram-positive Bacillus subtilis (B.s) and Staphylococcus aureus (S.a), and two Gram-negative Escherichia coli (E.c) and Pseudomonas aeruginosa (p.s), as well as two types of fungal Aspergillus niger (A. n) and Candida albicans (C. a). Among the other solvents used for the extractions (ethyl acetate, nhexane, n-butanol and water), chloroform extract demonstrated the highest antimicrobial activity.


INTRODUCTION
Solenostemma argel that belongs to the Asclepiadaceae family, known for used as anti-seizure (Idris et al., 2011& Dall et al., 2011, anti-rheumatic and anti-inflammatory agent (Shayoub et al., 2013). It is used in the treatment of some diseases such as diabetes mellitus, jaundice, measles and cold cough (El-Kamali et al., 1997) and in addition has insecticidal effect (Awad et al., 2012).

MATERIALS AND METHODS General Nuclear
Magnetic Resonance spectra ( 1 HNMR, 500 MHz; 13 C NMR, 125 MHz) were recorded on a Bruker Spectrometer 500 MHz's. Infrared (IR) spectra was specified with a Perkin -Elmer FTIR Spectrometer Model 1600, American Spray Probe, Ultra violet absorption was specified with Unicum Heyios UV Visible Spectrophotometer. Melting points specified with a thermo system FP800 Mettler FP80. EIMS were obtained using Shimadzu-Liquid Chromatography-Mass Spectrometry (LC/MS).
Column chromatography (CC) and Thin-layer chromatography (TLC) were used Silica gel 60/230-400 mm. and 60 F254 (Merck), respectively. Plant material Solenostemma argel stems were taken from Albakheet-Northern State of Sudan during 2017, stems were dried, cleaned and grinded. The powder obtained was stored in room conditions. Extraction, fractionation and isolation 2.5 Kg of dried stems of Solenostemma argel were extracted and fractionated, bioactivity of the different fractions against the microbial activity revealed that the chloroform extract (8.9 gram) was the most active. The active chloroform fraction was examined by column chromatography using gel silica (3.50 cm i.d., 350.75 gm). Elution started with 100% chloroform followed by chloroform/ethyl acetate mixtures with increasing the amounts of ethyl acetate. TLC monitored fractions and similar fractions were pooled to produce 33 fractions. Fraction 6 eluted with ethyl acetate: methanol, 75:25, (1.087mg) was further purified by silica gel thin-layer chromatography.

Antimicrobial activity Testing for antibacterial Activity
The antibacterial activity of extracts of the stems of Solenostemma argel was estimated the inhibition of the activity of four types of pathogenic bacteria: (E.c), (p.s), (B.s) and (S.a), by using cup-plate agar diffusion method (Kavanagh, 1972). 100ml of molten sterile nutrient agar were added to the one ml of stock suspension of standardized bacterial (108 -109 C.F.U/ ml).
After incubated of Inoculated nutrient agar at 45 ºC; 20 ml were diffused into sterile petri-dishes, which were cut with a sterile cork in the middle, and by using automatic micro liter pipette drills were filled with 0.1 ml of samples, which were diluted in methanol. after were diffused about two hours; petridishes were incubated at 37 ºC for 18 hours, each extract against each of the test organisms were replicated two times. The growth of inhibition zones were measured, averaged and tabulated (Table (1). Figure (1), (2) and (3) show the active samples.

Testing for antifungal activity
The antifungal activity of the extract of the stems of Solenostemma argel was estimated by using the cup-plate agar diffusion method (Kavanagh, 1972). The inoculated medium of Sabouraud dextrose agar was incubated at 25 ºC, for A. n. ( Three days) and C. a.( two days). Preliminary screening for antifungal activity of various extracts of Solenostemma argel stems were tabulated.

RESULTS AND DISCUSSION
Crude extracts and their fractions against microbial activity are shown in Table (1) obtained results signify a low effectiveness of crude extract, n-hexane and aqueous extract against the four bacteria. Ethyl acetate and chloroform extract showed high effectiveness against B.s & S.a. Here again n-hexane and the aqueous extract showed low activity against the standard fungi. An increase in the zone of suppress was noted with chloroform extract. Triterpene compound was isolated from Solenostemma argel stems subjected to the spectral data and resulting of testing of Lieberman -Borchard reagent that were clarified the compound. The UV spectrum of compound recorded in methanol shows an absorption peak at 231 nm. The IR spectrum of compound shows broadband centered at Copyright © Nov.-Dec., 2020; IJPAB 149 3510cm 1 , 2904 cm -1 and 1639 cm -1 ; that is evidence of the existence of O-H, CH3 and C = C groups, respectively. The proton NMR spectrum shows the presence of methyl signals at δH 0.89 at C23, C24, C26 and C27, δH 0.84 at C25, δH 0.70 at C28 and δH 0.87 at C29 and C30.

β-amyrin compound
The proton signal at δH 3.34 ppm as double triplet correlated to the carbon signal at δC 32.08 ppm is assigned to C-3. The proton signal at δH 5.12 corresponding to the carbon signal at δC 109.35 ppm along with the quaternary carbon signal at δC 150.90 ppm are assigned to the C = C functional group between C-12 and C-13. According Vesterberg (Vesterberg et al., 2010), the compound was elucidated as 3b-hydroxylolean-12-ene its beta amyrin (Figure 2).