ISOLATION AND CHARACTERISATION OF SECONDARY METABOLITES AND ESSENTIAL OILS FROM Costus lucanusianus J. Braun & K. Schum AND THEIR ANTIMICROBIAL ACTIVITY

Abstract

The resistance of microorganisms to antimicrobial agents has resulted into a huge challenge in the cure of infectious diseases. Some currently used synthetic antimicrobial drugs have side effects such as skin irritation and vomiting. This necessitated a search for potent antimicrobial compounds from natural sources. Costus lucanusianus has been identified to be bioactive against infectious diseases from Nigeria ethnomedicine. However, the isolation of its bioactive chemical constituents to support the uses has not been reported. The study was designed to isolate and characterise bioactive compounds from Costus lucanusianus and investigate the in vitro antimicrobial activity. Aerial parts (inflorescence, leaf, stem) and rhizome of Costus lucanusianus were collected in Ibadan and authenticated at Forestry Research Institute Herbarium, Ibadan. The dried and pulverised samples from the aerial and rhizome parts were cold macerated with methanol separately to obtain crude extracts which were successively partitioned with n-hexane and ethyl acetate. The leaf, stem and rhizome parts were hot extracted with n-hexane and methanol successively and methanol extracts partitioned with ethyl acetate. The extracts were subjected to phytochemical screening, column and thin layer chromatography using standard methods. Fresh samples (390 g each) were subjected to hydrodistillation and essential oils obtained were analysed using gas chromatography-mass spectrometry. The compounds obtained were characterised using infra-red (IR), 1H and 13C nuclear magnetic resonance (NMR) and mass spectroscopy. The cold extracts, essential oils and isolated compounds were evaluated for in vitro antimicrobial activity against clinical strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Candida albicans obtained from the Pharmaceutical microbiology laboratory, University of Ibadan by Agar well diffusion techniques at concentrations of 12.5-100 µg/mL. Gentamicin and Tioconazole were used as positive standards. Minimum inhibitory concentration (MIC) of the compounds was determined using Agar dilution method. Data were analysed using descriptive statistics.

The cold and hot extracts yields were 0.01-1.20% and 0.07-3.4%, respectively. Saponins, tannins, flavonoids, phlobatannins, anthraquinone, phenol, alkaloids, resin, terpenoids and reducing sugar were detected, which indicate bioactive potential of the plant. Three new (3,27-dihydroxy-1-methoxy-22-cholest-5-enone, β-sitosterol-3-O-β-D-3-deoxyxylo-4-hydroxy4,5-dimethyl-pent-2-one and 20-N-methoxyaminolucanu-1,4,6-triene-3-xylo-2-pero-4-methylpentan-2-one) compounds were isolated from the extracts. 3,27-dihydroxy-1-methoxy-22-cholest-5-enone (yield=20mg), showed two strong IR absorption bands at 3500 and 1655 cm-1 indicating hydroxyl and olefinic functional groups, respectively. The 1H NMR spectrum showed the presence of four methyl singlets between δ 0.9 and 1.60 ppm and a singlet at δ 3.90 due to methoxy group. 13C NMR and DEPT 135 revealed five methyl, ten methylene, nine methine and four quaternary carbons. The mass spectrum showed a protonated molecular ion peak at m/z 446.30 which corresponded to C28H45O4. The yields of essential oils ranged from 0.05-0.18% (w/w) with 11-octadecenoic acid, methyl ester (41.0%) and heptacosane (30.2%), 3',8,8'-trimethoxy-3-piperidyl-2,2'-binaphthalene-1,1',4,4'-tetrone (22.1%) and 4-(1,3,3-Trimethyl-bicyclo[4.1.0]hept-2-yl)-but-3-en-2-one (28.1%) as major components. The rhizome hexane extract and its essential oil showed the highest antimicrobial activity (10-31 mm) at 100 µg/mL. 3, 27-dihydroxy-1-methoxy-22-cholest-5-enone and β-sitosterol-3-O-β-D-3-deoxyxylo-4-hydroxy4,5-dimethyl-pent-2-one showed inhibition zones (18-28 mm) with MIC value of 25-50 µg/mL.

Three new compounds and essential oils were obtained from Costus lucanusianus and they all displayed antimicrobial activity.