Xanthine oxidase inhibitory activity of compounds from Chythrantus claneianus

  • Anar Sahib Gojayev Department of Biochemistry and Biotechnology, Faculty of Biology, Baku State University, AZ 1148, Azerbaijan; I.C.C.B.S., International Center for Chemical and Biological Sciences, University of Karachi 75270, Pakistan.
  • Jean Jules Kezetas Bankeu Department of Chemistry, Faculty of Science, University of Bamenda, P. O. Box 39 Bambili, Cameroon.
  • Angelbert Fusi Awantu Department of Chemistry, Faculty of Science, University of Bamenda, P. O. Box 39 Bambili, Cameroon.
  • Eric René Sieliatchom Nkanwen Chemistry Department, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon.
  • Muhammad Shaiq Ali I.C.C.B.S., International Center for Chemical and Biological Sciences, University of Karachi 75270, Pakistan.
  • Bruno Ndjakou Lenta Higher Teacher’s Training College, University of Yaoundé I, P.O. Box 47, Yaoundé, Cameroon.
  • Akif Alakbar Guliyev Department of Biochemistry and Biotechnology, Faculty of Biology, Baku State University, AZ 1148, Azerbaijan.
  • Didérot Tchamo Noungoué TWAS Research Unit, Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P. O. Box 812 Yaoundé, Cameroon.
  • Silvère Augustin Ngouela TWAS Research Unit, Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P. O. Box 812 Yaoundé, Cameroon.
  • Etienne Tsamo TWAS Research Unit, Department of Organic Chemistry, Faculty of Science, University of Yaoundé I, P. O. Box 812 Yaoundé, Cameroon.
Keywords: Chythrantus claneianus, rheumatism, xanthine oxidase inhibitor
DOI: 10.3329/bjp.v8i1.13485

Abstract

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 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%.

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 antioxidant 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 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). Melting points were obtained on a Gallenkamp melting point apparatus. Optical rotations were measured with a JASCO DIP-360 polarimeter. The structures of compounds were elucidated using spectral methods UV–vis, fourier transform infrared spectroscopy, 1D and 2D NMR, HR-ESIMS and HR-FABMS. Ultraviolet spectra were recorded on a Hitachi UV 3200 spectrophotometer. A JASCO 320-A spectrophotometer was used for scanning IR spectroscopy using KBr pellets. 1D and 2D NMR spectra were run on a Bruker spectrometer operating at 75, 100, 125, 150, 300, 400, 500, and 600 MHz, where chemical shifts (δ) were expressed in ppm with reference to the solvent signals. Electron impact mass spectra were obtained on a Varian MAT 311A mass spectrometer. Fast atom bombardment mass spectra were measured on a JEOL-HX 110 mass spectrometer. Column chromatography was performed on silica gel 230-400 mesh (Merck). Fractions were monitored by thin layer chromatography using precoated aluminum-backed silica gel 60 F 254 sheets. Spots were visualized under ultraviolet light (254 and 365 nm) or using ceric sulfate reagent.

Plant material: The stem bark and the trunk of C. klaineanus were collected in April 2008 from Mount Kala in the Centre Region of the Republic of Cameroon. The plant was identified by Mr. Nana Victor, botanist at the National Herbarium of Cameroon, where a voucher specimen (no. HNC 1489) has been deposited.

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 precoated 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). Fraction F3 was eluted with an n-hexane/EtOAc mixture of increasing polarity (5: 5 to 1: 9) to yield 5 (230 mg). Fraction F4 was subjected to column chromatography over silica gel (Merck, 70-230 mesh) with a CH2Cl2/MeOH mixture (9.5: 0.5 to 8: 2) to yield 6 (320 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:

Inhibition = (%)= 100- (OD test Compound)/(OD control)x 100

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.

Result 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.

Compound 6 was isolated as yellow crystals. It reacted positively to the Molish test, indicating its glycoside nature. The molecular formula C29H32O17, implying 14 degrees of unsaturation, was deduced from NMR and its HR-ESIMS which showed the pseudo-molecular ion peak at m/z = 675.15465 [M+Na]+ (calcd. for C29H32O17 + Na+, 675.15686). The ultraviolet spectrum in MeOH exhibited absorption bands at λmax 256, 360 nm, suggesting an ellagic acid derivative skeleton (El-Toumy et al., 2003, Pakulski and Budzianowski, 1996). The IR spectrum of 6 showed absorptions for hydroxy groups (3431 cm−1) and lactone functions (1751 cm−1), aromatic C=C groups (1600 cm−1), and glycosidic C–O bonds (1091 cm−1). The broad band decoupled 13C NMR of compound 6 displayed 29 carbon signals, which were sorted by DEPT and HSQC experiments into twelve methine groups [including two aromatic methine carbons at δC 107.5 (C-5′) and 111.7 (C-5), and ten oxymethine carbons], one oxymethylene at δC 60.3, twelve quaternary carbons [including two lactone carbons at δC 158.2 (C-7) and 158.3 (C-7′)], three methoxy groups and one methyl group at δC 18.0 (C-6‴)] (Table I). Its 1H NMR spectrum exhibited two deshielded singlets, characteristic of an ellagic acid moiety at δH 7.65 (1H, s, H-5′) and 7.84 (1H, s, H-5) (6), ten oxymethine and two oxymethylene protons at δH 3.18 (1H, t, J = 9.4 Hz, H-4‴), 3.25 (1H, t, J = 8.8 Hz, H-4″), 3.37 (1H, brs, H-2‴), 3.47 (2H, m, H-3″ and H-6″), 3.55 (1H, brt, J = 8.8 Hz, H-5″), 3.64 (2H, m, H-2″ and H-6″), 3.72 (2H, m, H-3‴ and H-5‴), 5.26 (1H, d, J = 1.3 Hz, H-1‴) and 5.46 (1H, d, J = 7.5 Hz, H-1″) indicating the presence of two sugar units in 6 (6). The spectrum also exhibited three methoxy groups protons at δH 4.01 (3H, s, 4′-MeOH), 4.05 (3H, s, 3′-MeOH) and 4.11 (3H, s, 3-MeOH) as well as six hydroxy protons. The two sugar units obtained from the acid hydrolysis were identified as glucose and rhamnose by comparing with standard sugar samples on thin layer chromatography plates using n-BuOH-Me2CO-H2O (4: 5: 1) solvent system as well as the carbon and proton chemical shifts and the coupling constant pattern of the protons of the two sugar units compare to those of literature (Soh et al., 2009, El-Toumy et al., 2003).

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

Position δC δH [m, J (Hz)] δC δH [m, J (Hz)]
1 113.7   112.6  
2 141.9   141.7  
3 140.8   142.4  
4 151.1   151.6  
5 111.7 7.84 (s) 112.2 7.83 (s)
6 112.2   114.2  
7 158.2   158.6  
1′ 113.7   113.1  
2′ 141.2   141.6  
3′ 141.1   141.3  
4′ 154.3   154.8  
5′ 107.5 7.65 (s) 108.0 7.65 (s)
6′ 112.6   113.3  
7′ 158.3   158.8  
1″ 98.5 5.46 (d, J = 7.5) 99.0 5.46 (d, J = 7.5)
2″ 76.2 3.64 (m) 76.7 3.63 (m)
3″ 77.2 3.47 (m) 77.5 3.46 (m)
4″ 69.6 3.25 (t, J = 8.8) 70.1 3.24 (t, J = 8.8)
5″ 77.0 3.55 (brt, J = 8.8) 77.7 3.56 (brt, J = 8.8)
6a″ 60.3 3.64 (m) 60.8 3.65 (m)
6b″   3.47 (m)   3.48 (m)
1‴ 100.2 5.26 (d, J = 1.3) 100.7 5.26 (d, J = 1.3)
2‴ 70.4 3.37 (brs) 70.9 3.37 (brs)
3‴ 70.3 3.72 (m) 70.8 3.72 (m)
4‴ 71.8 3.18 (t, J = 9.4) 72.3 3.18 (t, J = 9.4)
5‴ 68.5 3.72 (m) 69.0 3.70 (m)
6‴ 18.0 1.11 (d, J = 6.3) 18.5 1.11 (d, J = 6.3)
3-OMe 61.3 4.11 (s) 62.2 4.11 (s)
3′-OMe 61.7 4.05 (s) 61.8 4.05 (s)
4′-OMe 56.8 4.01 (s) 57.2 4.01 (s)

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 α-rhamnose 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).

Figure 1:HMBC correlations in compound 6

The structures of the five other known compounds were established as β-sitosterol (1) (Xiao et al., 2002), umbelliferone (2) (Ngadjui et al., 1991), scopoletin (3) (Cardona et al., 1992), benjaminamide (4) (Simo et al., 2008) and β-sitosterol-3-O-β-D-glucopyranoside (5) (Nguyena et al., 2004) by comparing of their spectral and physical data with those published before (Figure 2).

Figure 2:Structures of isolates 1-6

β-sitosterol (compound 1): Colourless needles, mp 139─140 °C. 13C NMR (100 MHz, CDCl3) δC 38.6 (C-1), 30.7 (C-2), 73.8 (C-3), 39.7 (C-4), 141.9 (C-5), 122.9 (C-6), 33.1 (C-7), 33.3 (C-8), 51.7 (C-9), 37.9 (C-10), 22.2 (C-11), 41.1 (C-12), 43.5 (C-13), 58.2 (C-14), 25.3 (C-15), 29.4 (C-16), 57.4 (C-17), 12.3 (C-18), 19.9 (C-19), 37.5 (C-20), 19.3 (C-21), 35.1 (C-22), 27.1 (C-23), 47.3 (C-24), 30.3 (C-25), 20.2 (C-26), 19.4 (C-27), 24.1 (C-28), 12.4 (C-29). EI-MS m/z 414 (64), 396 (40), 382 (14), 303 (23), 255 (20), 159 (23), 81 (37), 43 (100).

Umbelliferone, 7-hydoxycoumarin (compound 2): Yellowish solids, mp 231-233 °C. RMN 1H (500 MHz, DMSO-d6) δH 6.20 (d, J = 9.5 Hz, H-3), 7.94 (d, J = 9.5 Hz, H-4), 7.53 (d, J = 8.8 Hz, H-5), 6.79 (dd, J = 8.5, 1.9 Hz, H-6), 6.71 (d, J = 1.9 Hz, H-8), 10.57 (7-OH). 13C NMR (125 MHz, DMSO-d6) δC 161.2 (C-2), 113.1 (C-3), 144.5 (C-4), 111.2 (C-4a), 129.7 (C-5), 111.4 (C-6), 160.4 (C-7), 102.1 (C-8), 155.4 (C-8a). EI-MS m/z 78 (31), 105 (23), 134 (100), 162 (87).

Scopoletin, 7-hydroxy-6-methoxycoumarin (compound 3): Yellow crystals, mp 201-203 °C. RMN 1H (500 MHz, DMSO-d6) δH 6.22 (d, J = 9.5 Hz, H-3), 7.91 (d, J = 9.5 Hz, H-4), 7.21 (s, H-5), 6.78 (s, H-8), 3.82 (s, OMe-6), 10.31 (7-OH). 13C NMR (125 MHz, DMSO-d6) δC 161.1 (C-2), 112.1 (C-3), 144.9 (C-4), 111.0 (C-4a), 110.0 (C-5), 151.6 (C-6), 145.7 (C-7), 103.2 (C-8), 149.9 (C-8a), 56.4 (OMe-6). EI-MS m/z 69 (38), 121 (24), 149 (53), 177 (64), 192 (100).

Benjaminamide, (2S,3S,4R,12E)-2-{[(2R)-2-hydroxytetracosanoyl]amino}octadec-12-ene-1,3,4-triol (compound 4): Colorless crystals, mp 141-142 °C, [a] +34 (c 0.08, pyridine). IR (KBr) νmax 3200-3500 (OH), 1633 (NH─C=O), 1H NMR (500 MHz, pyridine-d5) δH 8.55 (d, J = 9 Hz, NH), 4.69 (dd, J = 9.9, 6.6 Hz, H-1a), 4.49 (dd, J = 9.9, 6.6 Hz, H-1b), 5.27 (m, H-2), 4.44 (overlapped dd, H-3), 4.18 (overlapped dd, H-4), 2.27 (m, H-5a), 1.90 (m, H-5b and H-6), 1.22 (brs, H-7 to H-10), 2.04 (m, H-11), 5.45 (m, H-12), 5.48 (m, H-13), 2.20 (m, H-14), 1.22 (brs, H-15 to H-17), 0.84 (t, J = 7.2 Hz, H-18), 4.55 (brdd, H-2′), 2.17 (m, H-3a′), 1.98 (m, H-3b′), 1.90 (m, H-4′), 1.29 (brs, H-5′ to H-23′), 0.84 (t, J = 7.2 Hz, H-24′). 13C NMR (125 MHz, pyridine-d5) δC 62.3 (C-1), 51.7 (C-2), 75.9 (C-3), 72.5 (C-4), 33.9 (C-5), 26.6 (C-6), 29.6-33.9 (C-7 to C-10), 27.5 (C-11), 130.2 (C-12), 130.4 (C-13), 27.9 (C-14), 22.9-33.0 (C-15 to C-17), 14,3 (C-18), 175.7 (C-1′), 72.4 (C-2′), 35.5 (C-3′), 26.7 (C-4′), 29.3-33.0 (C-5′ to C-22′), 22.9 (C-23′), 14.2 (C-24′). (+)HR-FABMS m/z 681.6280 (calcd. 681.6271 for C42H83NO5, [M+H]+).

β-sitosterol-3-O-β-D-glucopyranoside (compound 5): Colorless crystals; mp 260-261 °C. 13C NMR (100 MHz, Pyridine-d5) δC 37.4 (C-1), 30.2 (C-2), 78.5 (C-3), 39.3 (C-4), 140.9 (C-5), 121.8 (C-6), 32.1 (C-7), 32.0 (C-8), 50.3 (C-9), 36.9 (C-10), 21.2 (C-11), 39.9 (C-12), 42.4 (C-13), 56.8 (C-14), 24.4 (C-15), 28.4 (C-16), 56.2 (C-17), 11.9 (C-18), 19.0 (C-19), 36.3 (C-20), 19.2 (C-21), 34.2 (C-22), 26.4 (C-23), 46.0 (C-24), 29.4 (C-25), 19.9 (C-26), 19.4 (C-27), 23.3 (C-28), 12.1 (C-29), 102.5 (C-1′), 75.2 (C-2′), 78.3 (C-3′), 71.7 (C-4′), 78.2 (C-5′), 62.8 (C-6′). EI-MS m/z 414 (5), 396 (63), 381 (24), 255 (77), 159 (39), 81 (99), 55 (100).

Panconoside B [3,3′,4′-tri-O-methylellagic acid 4-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside] (compound 6): Yellow crystals, mp 257-259 °C. UV (MeOH) λmax (logε) 256 (3.72), 360 (2.47). [a] −80.0°(c 0.5, MeOH). IR (KBr) νmax 3431 (O─H), 1751 (C=O), 1600 (C=C), 1091 cm1. 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) spectroscopic data, see Table I. (+)-HR-ESIMS m/ z 675.15465 (calcd. 675.15686 for C29H32O17 + Na+). EI-MS m/z 344 (100), 329 (20), 286 (17).

In vitro xanthine oxidase inhibitory assay All the isolated compounds and the extract were tested in vitro for their inhibitory effects towards the xanthine oxidase enzyme, which is a target in gouty arthritis and uric acid nephrolithiasis drug research. As depicted in Table II, results showed that, overall, all the pure compounds exhibited weaker xanthine oxidase inhibitory activity compared to the reference drug, allopurinol.

Table II:In vitro inhibition of xanthine oxidase by natural compounds from Chythrantus claneianus

  Sample % Inhibition IC50 ± SEM (µM)a
1 β-sitosterol - ND
2 Umbelliferone 47 >500
3 Scopoletin 52 475 ± 3.7
4 Benjaminamide 11 ND
5 β-sitosterol-3-O-β-D-glucopyranoside - ND
6 Panconoside B 82 307±2.3
  MeOH Extract 43 ND
  Allopurinolb 98.8 13.7 ± 0.2
aIC50: Concentrations that inhibited 50% of enzymes relative to negative control; ND: Not determined; SEM: standard mean error. bStandard used for the assay

We found for the first time that panconoside B (6) inhibited XO activity. The highest values were obtained with compound 6 (82%) and 3 (52%). The isolates showed IC50 values ranging from 307 μM (compound 6) to 475 μM (compound 3), whereas the IC50 value of the reference drug allopurinol was 13.70 μM.

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).

All constituents of Conyza bonariensis (L.) including β-sitosterol (1) were tested for their activity towards XOD inhibition. The in vitro enzyme assay demonstrated that this compound didn’t display XOD inhibitory activity (Kong et al., 2001). In another study 3β-hydroxysitosterol from snake fruit (Salacca edulis Reinw.) was mentioned to be inactive (Priyatno et al., 2007). We reported previously that β-sitosterol (1), β-sitosterol-3-O-β-D-glucopyranoside (5) and benjaminamide (4) were inactive (Gojayev et al., 2011).

Conclusion

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.

Acknowledgements

The authors wish to thank the Third World Academy of Sciences (TWAS) and the International Center for Chemical and Biological Studies (ICCBS). Baku State University is also acknowledged. The interest and support of Prof. M. I. Choudhary is greatly acknowledged by Anar Gojayev.

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Published
2013-02-13

Apply citation style format of Bangladesh Journal of Pharmacology

Section
Research Articles
Financial Support
Self-funded
Conflict of Interest
Authors declare no conflict of interest