CHEMICAL AND BIOLOGICAL STUDIES OF HONEY BEE PROPOLIS COLLECTED FROM ZANGO KATAF LOCAL GOVERNMENT AREA OF KADUNA STATE, NIGERIA
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ABSTRACT
Honeybee Propolis from Apis mellifera (Linn), has been used in ethno medicine as an emollient in the treatment of ringworm, measles, chickenpox and with reported biological activities, such as antitumor, antioxidant, immunomodulatory action and anti-inflammatory action among other ailments, was subjected to chemical and biological studies. The chemical techniques employed were column chromatography and preparative thin layer chromatography. The antioxidant activity was studied using 1,1- Diphenyl-2-Picrylhydrazyl (DPPH) free radical scavenging activity while the antimicrobial activity was studied using Agar diffusion and Broth dilution methods and the microorganism used were clinical isolates and these includes; Gram- negative strains (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia, Shigella dysenteriae and Proteus mirabilis), Gram-positive strains (Staphyloccocus aureus, Bacillus subtilis, Streptococcus pyrogenes and Corynebacterium ulcerans) and fungal strains (Candida albicans, Candida krusei and Candida tropicalis). Preliminary phytochemical screening of the crude ethanol extract revealed the presence of flavonoids, phenolic compounds, saponins, steroids and triterpenes while phytochemical screening of the n-hexane soluble fraction revealed steroids, triterpenoids, and phenolic compound. Extensive chromatographic separation of the n-hexane soluble fraction using silica gel column chromatography followed by preparative thin layer chromatography led to the isolation of a steroid, stigmast-5-en-3β-ol (β-sitosterol). Its structure was determined by spectral analysis including 1D and 2D NMR. The in-vitro DPPH free radical scavenging activity of ethanol extract (CR) and sub- fractions hexane fraction (HH), chloroform fraction (CC), ethyl acetate fraction (EE), butanol fraction ix (BB) were determined, with ethyl acetate fraction (EE) having significant antioxidant activity of IC50 value of 1.78 ± 0.01ug/ml in comparism to other extracts and standard ascorbic acid of 2.54 ± 0.01ug/ml at p ≤ 0.05. The order of decreasing antioxidant activity of the extracts was EE>CR>CC>BB>HH. The in-vitro antimicrobial activity of propolis ethanol extracts (CR, HH, CC, EE, and BB) and isolated compound X1 assayed using some clinical isolates as test organisms shows that, the crude ethanol extract showed activity against S. aureus, B. subtilis, P. aeruginosa, C. krusei and C. tropicalis, while hexane fraction (HH) showed activity only on B. subtilis and C. krusei. The chloroform fraction (CC), ethyl acetate fraction (EE) and butanol fraction (BB) showed same activity on susceptible organism to the ethanol crude extract with the exception of P. aeruginosa which was not susceptible to only the butanol fraction (BB). Isolated compound X1 (β-sitosterol) showed activity on S aureus, B.substilis, S. pyrogenes, K. pneumonia, S. dysenteriae and C. krusei with the exception of P. aeruginosa and C. topicalis which are susceptible to the ethanol crude extract. The isolated compound J1 has zone of inhibition ranging from (27-34mm), while the ethanol extract and its sub-fractions exhibit zone of inhibition ranging (20-27mm). The results suggest that the propolis ethanol extract and its sub fractions possess antimicrobial compounds that may be useful in treatment of infections and isolated β- sitosterol (100ug/mL) has an antimicrobial activity even though lower than the standard antibiotic drug (Sparfloxacin, Ciprofloxacin) and anti-fungal agent (Fluconazole) at (5ug/mL) with zones of inhibition ranging (32- 47mm) on the same susceptible organism. The result of this study has added new knowledge on the chemical and biological studies of honey bee propolis from Northern Nigeria and confirmed the rationale of the ethno medicinal use of the propolis.
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