Xanthine oxidase inhibitory activity of compounds from Chythrantus claneianus

Phytochemical investigation of the stem bark and the trunk of Chythrantus claneianus led to the isolation of six known compounds named β-sitosterol (1), umbelliferone (2), scopoletin (3), benjaminamide (4), β-sitosterol-3-O-β-D-glucopyranoside (5) and Panconoside B (6). All these compounds were isolated for the first time from this plant species. The chemical structures of isolates were elucidated on the basis of 1 and 2 D-NMR spectra and other spectroscopic techniques including UV–vis, FT-IR, HR-ESIMS and HR-FABMS. The isolates were tested in vitro for their inhibitory properties towards xanthine oxidase enzyme. Compounds 2, 3 and 6 showed weak inhibi-tory activities on the enzyme with IC 50 values ranging from 307 µM for com-pound 6 to 475 µM for compound 3, while the extract and compounds 1, 4 and 5 showed extremely weak activities with inhibition percentage less than 50%.


Introduction
Plants of the Sapindaceae family are trees or shrubs generally found in tropical or subtropical regions.In Cameroon, they are used in traditional medicine for the treatment of several ailments such as skin diseases, dysentery and rheumatism (Abdou-Shoer et al., 1993).
Previous phytochemical studies on plants of this family reported the presence of various classes of secondary metabolites including flavonoids, coumarins, ellagic acids, ceramides, sterols, and saponins as major constituents (Aubreville et al., 1973, Huang et al., 2008, Li et al., 2007, Rangkadilok et al., 2005, Soh et al., 2009).Some of these compounds were shown to exhibit interesting pharmacological properties including antiplasmodial, hemolytic, antibacterial and anti-oxidant activities (Huang et al., 2008, Mesquita et al., 2005, Voutquenne et al., 2005).Chythrantus klaineanus is a tree or shrub of about 1-5 m high, found in Central Africa (Abdou-Shoer et al., 1993).No previous reports on the chemical constituents or biological properties of this species have been reported.As part of our ongoing search for bioactive metabolites from Cameroonian plants of the Sapindaceae family, we have investigated the MeOH extract from the mixture of the stem bark and the trunk of C. klaineanus.
Xanthine oxidase (XO, EC 1.1.3.22) is a complex metalloflavoprotein which catalyzes the oxidation of hypoxanthine and xanthine into uric acid, plays a vital --- Xanthine oxidase inhibitory activity of compounds from

Chythrantus claneianus
Phytochemical investigation of the stem bark and the trunk of Chythrantus claneianus led to the isolation of six known compounds named β-sitosterol (1), umbelliferone (2), scopoletin (3), benjaminamide (4), β-sitosterol-3-O-β-Dglucopyranoside (5) and panconoside B (6).All these compounds were isolated for the first time from this plant species.The chemical structures of isolates were elucidated on the basis of 1 and 2 D-NMR spectra and other spectroscopic techniques including UV-vis, FT-IR, HR-ESIMS and HR-FABMS.The isolates were tested in vitro for their inhibitory properties towards xanthine oxidase enzyme.Compounds 2, 3 and 6 showed weak inhibitory activities on the enzyme with IC50 values ranging from 307 µM for compound 6 to 475 µM for compound 3, while the extract and compounds 1, 4 and 5 showed extremely weak activities with inhibition percentage less than 50%.
role in disorders such as hyperuricemia and gout.XO inhibitors may be useful for treatment of these diseases.
For the treatment of hyperuricemia and gout allopurinol, oxypurinol, and febuxostat have been used widely (Higgins et al., 2011;Nguyen et al., 2005).

Materials and Methods
General: Isolation of compounds was done with normal phase column chromatography using silica gel (70-230 mesh, Merck, Germany Extraction and isolation: 2.5 kg of air-dried and powdered mixture of the stem bark and the trunk of C. klaineanus were extracted with MeOH (5 L) at room temperature for 48 hours and filtered.The extract was concentrated to dryness under vacuum to give 40 g of a dark brownish crude extract.The extract was extracted selectively with EtOAc.The EtOAc-soluble fraction was evaporated to dryness under vacuum to give a dry residue (13 g) which was then subjected to medium pressure liquid chromatography over silica gel 230-400 mesh (Merck) eluted successively with n-hexane, mixtures of n-hexane/EtOAc, EtOAc, and EtOAc/ MeOH of increasing polarities.Subfractions (119), of 500 mL each, were collected and combined according to their thin layer chromatography profiles on pre-coated silica gel 60 F254 plates developed with n-hexane/EtOAc mixtures to yield 4 fractions (F1-4).Fraction F1 was subjected to column chromatography over silica gel (Merck, 70-230 mesh) eluted with an n-hexane/EtOAc mixture (9.5: 0.5 to 7: 3).This resulted in the isolation of 1 (21 mg) and 2 (63 mg).Fraction F2 was also subjected to successive column chromatography over silica gel (Merck, 70-230 mesh) eluted with a mixture of n-hexane and EtOAc (6: 4) to give 3 (33 mg) and 4 (151 mg).
Acid hydrolysis of compound 6: A solution of 10 mg of compound 6 in 10 mL MeOH/H2O (1 : 1) mixed with 5 mL 2N HCl was refluxed at 100°C for 3 hours.After evaporation of the organic solvent under vacuum and threefold extraction of the aqueous phase with EtOAc, the aglycone was identified as 3,3′,4′-tri-O-methylellagic acid using NMR techniques.The neutralized aqueous portion resulted in the detection of glucose and rhamnose by comparison with standard sugar samples on thin layer chromatography plates using n-BuOH/ Me2CO/H2O (4 : 5 : 1) as solvents and using (NH4) 6Mo7O24Ce(SO4)2H2SO4 reagent for visualization.
In vitro xanthine oxidase inhibitory activity assay: Xanthine (X-0626) and xanthine oxidase (EC 1.1.3.22)(from butter milk) were obtained from Sigma Aldrich, (Japan).The xanthine oxidase inhibitory activity of compounds was determined by measuring the rate of hydroxylation of the substrate (xanthine) with the formation of uric acid, which is a colorless product of the reaction which shows absorption at 295 nm (Lee et al., 1998).Briefly, the reaction mixture containing 10 μL of 1 mmol/L of isolates was dissolved in dimethyl sulfoxide, 150 μL of phosphate buffer (0.05 mmol/L, pH 7.4), 0.003 units of xanthine oxidase dissolved in buffer (20 μL), and 20 μL of 0.1 mmol/L xanthine as substrate for enzyme.After addition of xanthine oxidase, the mixture was incubated for 10 min at room temperature and pre-read in the ultraviolet region at 295 nm.The substrate was added to reaction mixture, and subsequent continuous reading for 15 min at an interval of 1 min was observed (on Spectra MAX-340).The percentage inhibitory activity by the samples were determined against a DMSO blank and calculated using the following formula: The IC50 of the compounds as calculated using EZ-Fit windows-based software (Perrella Scientific Inc. Amherst, USA).To compare the inhibitory activities of the compounds, allopurinol was used as standard.The reaction for each compound was performed in triplicate.

Results and Discussion
The MeOH extract of the stem bark and the trunk of C. klaineanus was fractionated and purified on silica gel and sephadex LH-20 columns, successively, to afford six known compounds.
The configuration of the anomeric protons [5.26 (1H, d, J = 1.3 Hz, H-1 ) and 5.46 (1H, d, J = 7.5 Hz, H-1″)] were determined based on their coupling constant to be αrhamnose and β-glucose, respectively (Soh et al., 2009, El-Toumy et al., 2003).HMBC spectrum was used to determine the junction between the rhamnose and glucose and between the glucose and the ellagic acid moiety (Figure 1).This spectrum was also used to determine the position of the different methoxy groups on the ellagic acid moiety.In fact, cross-peaks were observed in the HMBC spectrum between the anomeric proton of the glucose moiety at δH 5.46 (1H, d, J = 7.5 Hz, H-1″) and the carbon C-4 (δC 151.1), and between the anomeric proton of the rhamnose moiety at δH 5.26 (1H, d, J = 1.3 Hz, H-1 ) and the carbon C-2″ (δC 76.2).In addition, cross-peaks were observed between the three methoxy groups protons at δH 4.11 (3H, s, 3-MeOH), 4.05 (3H, s, 3′-MeOH) and 4.01 (3H, s, 4′-MeOH), and carbons C-3 (δC 140.8),C-3′ (δC 141.1) and C-4′ (δC 154.3), respectively.The position of the glucose unit was also supported by the observation of a NOE between protons H-5 (δH 7.84) and H-1″ (δH 5.46) in the NOESY spectrum.On the basis of the above spectral data and by comparing of these values with those reported in literature, the identity of compound 6 was confirmed as panconoside B which was recently isolated from Pancovia pedicellaris, a plant of the same family (Soh et al., 2009).
Lin et al. investigated the effect of the coumarin derivatives on the inhibition of xanthine oxidase (XO) activity, and the structure-activity relationships of these derivatives against xanthine oxiase activity.They reported that scopoletin (3) and umbelliferone (2) showed lower (IC50 values of >100 mM) XO inhibitory activity (Hsiu-Chen et al., 2007).
It is interesting to note that the active compounds were coumarins and their derivatives.Our results can justify the use of C. klaineanus by Cameroonian traditional healers in the treatment of inflammatory diseases such as rheumatism.

Table I
Comparative 1 H and 13 C NMR spectral data of 6 with those of panconoside B

Table II
In vitro inhibition of xanthine oxidase by natural compounds from Chythrantus claneianus a IC50: Concentrations that inhibited 50% of enzymes relative to negative control; ND: Not determined; SEM: standard mean error.b Standard used for the assay