Chemical studies and evaluation of some pharmacological activities of propolis from three geographycal regions of Cameroon.

Abstract

As part of an ongoing investigation of bioactive metabolites from natural sources, extracts of propolis samples from different regions of Cameroon were prepared and screened for their anti-ulcer, antioxidant and antimicrobial properties. Some secondary metabolites were isolated and their structures elucidated. Thirty-two natural products were isolated: ten compounds and a triterpene fraction from the acetone extract of Nkambe propolis; ten from the ethanol extract of Foumban propolis; six from the hexane extract of Ndian propolis and six from the ethyl acetate extract of Ndian propolis using routine separation and chromatographic techniques. The structures of twenty-four of these compounds were elucidated by interpretation of their physical and spectroscopic data (IR, MS, melting point, 1D & 2D NMR) and by comparison of the latter with similar data reported in literature and due to similarity between some compounds, the number reduced to 20 distributed as follows: ➢ 16 triterpenes, two of which are novel derivatives namely: 3β,27-dihydroxycycloart-24-en-26-oic acid methyl ester (138) and 3-oxo-6,22-dihydroxy-4,4,14-trimethylspinast-7,23-dien-29-al (133). The 14 known triterpenoids are: lupeol acetate (82), betulinic acid (140), oleanolic acid (139), lupenone (81), 27-hydroxymangiferonic acid (135), mangiferonic acid (78), betulin (136), 3β-hydroxylanostan-9,24-dien-21-oic acid (137), 3β-hydroxycycloart-12,25-diene (86), 3β-O-acetylbetulin (144), lup-12,20(29)-dien-3-ol (142), and a mixture of lupeol and stigmasterol (88 and 134), 24-methylenecycloartenol and stigmasterol (141 and 134), lupeol and β–amyrine (88 and 143). ➢ 1 new mono-ether of glycerol: 1’-O-eicosanyl glycerol (145) ➢ 2 fatty acid esters: heptadecyl butanoate (148) and eicosanyl butanoate (146) ➢ 1 fatty alcohol: n-heptatriacontanol (147) It is observed that, lupane and oleanane type triterpenoids could be possible chemical markers of Cameroonian propolis. Also, six benzylic esters (three of lupeol and three of β-amyrine) were synthesized. It resulted from these syntheses that the yield depended on the activating effect of the methoxyl substituents on the benzene ring of the benzoylchloride used. The greater the number of methoxyl groups, the greater the inductive effect and the greater the yield. β-amyrine was also oxidized to amyrenone. A triterpene fraction was isolated and analysed by GC-MS as a mixture of lanosterol, α-amyrine, 28-norolean-12-en-3-ol, Cycloartenol, 3-epi-α-amyrine, lupeol and 24-methylenecycloartenol. GC-MS profiles of 13 extracts from different samples of propolis were established and revealed the presence of over 50 compounds belonging to different structural groups but containing mainly triterpenes as would be expected of propolis from tropical and subtropical zones. Characteristically, GC-MS profiles of Cameroonian propolis revealed alkenyl phenols and resorcinols, fatty acids, triterpenes and sugars. The presence of some of these compounds indicated that Mangifera indica is the major plant from which bees collect propolis. Samples from Northern part of Cameroon were richer in triterpenes, void of alkenyl phenols and resorcinols and had averagely higher antimicrobial activity while alkenyl phenols and resorcinols and some phenolic compounds were found in samples of North-West and Western regions which showed averagely higher antiradical activity on DPPH. This difference is explained by the variation in vegetation and plants foraged by bees. The methanol, acetone and hexane extracts of Nkambe propolis (200-600 mg/kg) dose-dependently prevented the formation of ethanol-induced gastric lesions (% inhibition, 61, 54 and 55%, respectively, at the dose of 600 mg/kg). Increasing doses of the extracts inhibited pylorus ligation–induced lesions by 64.5, 73.1 and 16.2 %, respectively, for the highest dose but none of them showed antisecretory activity as compared with controls. The most further significantly (P<0.01) reduced HCl/ethanol-induced ulcer indices from 4.33+ 0.32 in cytoprotective (acetone) extract (56.6-73.1 % inhibition under highly acidic gastric environments) to 1.25 +0.53 and 0.6 + 0.04 at the dose of 400 and 600 mg/kg, respectively (% inhibition: 71-86%). Furthermore, upon pretreatment of the rats with indomethacin prior to HCl/ethanol, the acetone extract significantly (P<0.001) decreased ulcer index from 5.55 + 0.73 in the controls to 1.89 + 0.15 at the dose of 600 mg/kg. Although pretreatment with indomethacin reduced the protective effect of the acetone extract by 23 to 27%, cytoprotection remained high (62-66% inhibition). The cytoprotective action of the most active (acetone) extract may involve the mediation of endogenous prostaglandins. The order of decreasing radical scavenging activity was Hexane extract of Foumban propolis (IC50 = 5.6 mg/mL), Hexane extract of Ndian propolis (IC50 = 4.00 mg/mL), Ethyl acetate extract of Ndian propolis (IC50 = 1.65 mg/mL), Ethyl acetate extract of Foumban propolis (IC50 = 1.40 mg/mL), 3β-hydroxylanostan-9,24-dien-21- oic acid (IC50 = 1.22 mg/mL), mangiferonic acid (IC50 = 1.09 mg/mL), Methanol extract of Foumban propolis (IC50 = 1.07 mg/mL),methyl-3β,27-dihydroxycycloart-24-en-26-oate (IC50 = 0.98 mg/mL), 1’-O-eicosanyl glycerol (IC50 = 0.93 mg/mL), Vitamin C (IC50 = 0.80 mg/mL) and Gallic acid (IC50 = 0.30 mg/mL). Although none of the samples showed antiradical activity greater than that of the standards, their activities remained nevertheless closer to those of the standard antioxidants Gallic acid and vitamin C. It is observed that, all the pure compounds showed higher DPPH• radical scavenging activity than the extracts except for methanol extract of Foumban propolis that was more active than mangiferonic acid and 3β-hydroxylanostan-9,24-dien-21-oic acid.