Oligocephlate, a new a-Glucosidase inhibitory Neohopane Triterpene from Vernonia oligocephala.
The genus Vernonia belongs to the family Asteraceae consists of approximately 1000 species distributed in Africa, Southeast Asia, South and North America . Most of the species of this genus are used for the treatment of malaria , gastrointestinal disorders, as a tonic and appetite stimulant, for skin diseases and as a medication for fever, dysentery, malaria, diabetics and constipation  as astringent and cure asthma and bronchitis . Vernonia oligocephala is a perennial herb which is traditionally used to treat abdominal pain and colic. The roots of this plant are used to treat ulcerative colitis . Literature survey revealed that sesquiterpene lactones have been reported from this plant .
Herein we report the isolation and structure elucidation of a new triterpene 3b-acetoxyneohop-13(18)-ene (1) together with b-sitosterol (2) , 3b-hydroxylup-20,29-ene (3) , 3b-hydroxyolean-12- en-28-oic acid (4)  and b-sitosterol-3-O-b-D- glucopyranoside (5) , respectively.
a-Glucosidase (EC 184.108.40.206) comprises a family of enzymes, hydrolases, located in the brush- border surface membrane of small intestinal cells. Its major function is to hydrolyze the glycosidic linkage and produce glucose and other monosaccharide [10,11]. a-Glucosidase inhibitors are used as oral anti-diabetic drugs for patients with type-2 diabetic mellitus. Postprandial hyperglycemia has a vital role in the development of type-2 diabetes and complications associated with disease such as nephropathy, neuropathy, micoangiopathy, macroangipathy [12,13]. The inhibitors of this enzyme can retard the liberation of glucose and delay glucose absorption, resulting in reduced postprandial hyperglycemia [14,15]. Therefore, inhibition of a- glucosidase is considered important in managing type-2 diabetes.
Acarbose, voglibose and miglitol are commerical a-glucosidase inhibitors that are considered as first-line treatment for diabetic individuals with post-prandial hyperglycemia. The objective of this study is to identify new metabolites which can be considered as target molecule for future anti-diabetic drugs.
Results and Discussion
The methanolic extract of the whole plant of V. oligocephala was subsequently divided into n- hexane, ethyl acetate, n-butanol and water-soluble fraction. The n-hexane soluble fraction was subjected to silica gel column chromatography using different mobile phases (see Experimental) and finally compounds 1-5 (Fig. 1) were purified which were characterized using different spectroscopic and spectrometric techniques.
Compound 1 was isolated as colorless amorphous solid. The IR spectrum showed the peaks for ester (1730 cm-1) and unsaturation (1640 cm-1).
The high resolution electron impact mass spectrometry (HREIMS) determined the molecular formula C32H52O2 through a molecular ion peak [M]+ at m/z 468.3980 (calcd. for C32H52O2, 468.3968) having seven double bond equivalence (DBE).
The 1H NMR spectrum of 1 (Table-1) showed eight methyl signals including six tertiary and two secondary methyls at d 0.76, 0.83, 0.84, 0.94, 0.97, 1.05 (3H each, s) and 0.87 (3H, d, J = 6.4 Hz), 0.93 (3H, d, J = 6.4 Hz), respectively. This observation indicated the presence of pentacyclic triterpenoid . A methyl singlet at d 2.02 (3H, s) is attributed to acetyl group in the molecule. An oxymethine proton was resonated at d 4.50 (1H, dd, J = 11.6, 5.6 Hz) is assigned to H-3 and its larger coupling constant value confirmed it axial and a in orientation . The 13C NMR spectra (BB and DEPT) of 1 (Table-1) showed 32 carbon signals for nine methyl, ten methylene five methine and eight quaternary carbons.
The downfield carbon at d 171.0 assigned to acetyl group and d 141.0 and 131.0 to olefinic quaternary carbons. The positions of both acetyl group and double bond was done by HMBC correlations in which H-3 showed correlation with ester carbonyl at d 171.0, CH3-27 (d 1.05) with C-13 (d 131.0) and CH3-28 (d 0.76) with C-18 (d 141.0) confirming the position of acetyl group at C-3 and double bond between C-13and C-18.
The above data showed close resemblance to the data reported for boehmeryl acetate . The relative stereochemistry at C-17 was determined through 13C NMR chemical shift of C-28 (d 17.9)  confirmed CH3-28 as axial and a and missing of its NOESY correlations with H-21 confirmed the orientation of isopropyl group at C-21 as a. Based on these evidences compound 1 was 3b-acetoxyneohop- 13(18)-ene and named as oligocephlate.
Column chromatography was carried out using silica gel of 70-230 and 230-400 mesh. Aluminium sheets precoated with silica gel 60 F254 (20x20 cm, 0.2 mm thick; E-Merck) were used for TLC to check the purity of the compounds and were visualized under UV light (254 and 366 nm) followed by ceric sulfate as spraying reagent.
IR spectra were recorded on Shimadzu IR-460 spectrophotometer (n in cm[?]1). EIMS and HREIMS spectra were recorded on Jeol JMS-HX 110 spectrometer with data system. The 1H NMR spectra were recorded on Bruker AMX-400 MHz instruments using TMS as an internal reference. The chemical shift values are reported in ppm units and the scalar coupling constants (J) are in Hz. The 13C NMR spectra were recorded at 100 MHz on the same instrument.
The whole plant of Vernonia oligocephala (10 kg) was collected from Lal Sohanra (District Bahawalpur) in April 2008 and identified by Dr.
Muhammad Arshad (late), Plant Taxonomist, Cholistan Institute for Desert Studies (CIDS), The Islamia University of Bahawalpur, where a voucher specimen is deposited (VO-CIDS-21/08).
Extraction and Isolation
The shade dried, ground whole plant of the V. oligocephala (10 kg) was extracted with MeOH (3 x 25 L). The combined methanolic extract (430 g) was dissolved in water and partitioned with, n- hexane, ethyl acetate and n-butanol. The n-hexane soluble fraction (40 g) was subjected to column chromatography (CC) over silica gel eluting with with n-hexane, n-hexane:chloroform, chloroform, chloroform:methanol and methanol in increasing order of polarity to get 8 fractions (1-8). The fraction 2 obtained at n-hexane:chloroform (8.5:1.5) was subjected for further purification on CC to get 1. The fraction 3 obtained at n-hexane:chloroform (8.0:2.0) was chromatographed over silica gel eluting with n- hexane-chloroform (7.8:2.2) to purify b-sitosterol (2).
The fraction 4 obtained from n-hexane:chloroform (7.0:3.0) on further purification afforded lupeol (3). The fraction 5 obtained from n-hexane-chloroform (1:1) was subjected to column chromatography eluted eluted with same solvent sstem yielded oleanolic acid (4). The fraction 6 obtained from 100% chloroform was further subjected to silica gel column chromatography and yielded b-sitosterol-3-O-b-D- glucopyranoside (5) at chloroform:methanol (9.8:0.2).
Colorless amorphous solid (35 mg); IR (KBr) nmax cm-1: 1730, 1640, 1390, 1380, 960, 840. The 1H and 13C NMR data, see Table-1; HREIMS m/z: 468.3980 (calcd. for C32H52O2, 468.3967).
a-Glucosidase Inhibition Assay
The a-glucosidase inhibition assay was performed with slight modifications as done by Pierre et al. . Total volume of 100 uL reaction mixture contained 70 uL 50 mM phosphate buffer, pH 6.8, 10 uL (0.5 mM) test compound, followed by the addition of 10 uL (0.0234 units, Sigma Inc.) enzyme. The contents were mixed, preincubated for 10 min at 37oC and pre-read at 400 nm.
The reaction was initiated by the addition of 10 uL of 0.5 mM substrate (p-nitrophenyl glucopyranoside, Sigma Inc.). After 30 min of incubation at 37oC, absorbance of the yellow color produced due to the formation of p- nitrophenol was measured at 400 nm using Synergy HT (BioTek, USA) using 96-well microplate reader. Acarbose was used as positive control. The percent inhibition was calculated by the following equation Inhibition (%) = (abs of control - abs of test / abs of control) x 100
IC50 values were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).
The authors are thankful to Alaxander von Humboldt (AvH) Foundation, Germany and Third World Academy of Science (TWAS), Italy for providing some of the basic lab facilities in Chemistry Department of IUB.
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|Author:||Riaz, Naheed; Feroze, Kanwal; Saleem, Muhammad; Misaddiq, Sara; Ashraf, Muhammad; Alam, Umber; Musta|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Jun 30, 2013|
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