Screening Of Ten Selected Medicinal Plants For Antibacterial And Antifungal Activity And The Isolation And Characterisation Of Bioactive Constituents From Alectra Sessiliflora (vahl) Kuntze Andblumea Axillaris (LAM.) DC.

Abstract

Introduction: A host of opportunistic infections add to the present endemic state of some already existing infections with most of the morbidity and mortality in AIDS cases resulting from opportunistic infections. Opportunistic infections are caused by pathogens such as bacteria, viruses, protozoa or fungi in individuals whose immune system is compromised. Most opportunistic infections are however well managed to some extent with the available specific standard drug regimens. The emergence of fungi and bacteria as ever-increasing causes of morbidity and mortality as well as the complications in their drug management has led to the need for exploration, discovery and development of new antifungal and antibacterial drugs with better safety and efficacy profile. Search for antifungal and antibacterial agents from natural sources is one area that is currently drawing a lot of attention. Problem statement and study justification: Despite the availability and use of the standard antifungal and antibacterial drugs, some therapeutic problems still persist, in particular the emergence of new pathogenic fungal species, variable drug bioavailability, drug toxicity, lack of desired and appropriate dosage form preparations, significant drug interactions for some agents and development of resistance or breakthrough infections. The search for new molecules from other sources such as plants that are useful as lead models for and/or as medicines that can handle these setbacks must continually be pursued. Study objectives: The main objective of this study was to investigate the potential antibacterial and antifungal activity of ten selected plants of medical importance with a view to providing scientific basis for the traditional use in treating of mouth and skin fungal infections. The specific objectives were to determine the in vitro antibacterial and antifungal activity of crude extracts; determine and isolate the constituents of the two most bioactive

plants, Alectra sessiliflora Vahl. Kuntze and Blumea axillaris Lam. DC.; elucidate the structure of the isolated compounds and to determine the in vitro antifungal and antibacterial activity of the isolated compounds. Methodology: The following plants were screened in the study: Sida tenuicarpa Vollesen (Malvaceae), Spilanthes mauritiana (Rich. ex Pers.) DC. (Asteraceae), Fuerstia africana T.C.E.Fries (Labiatae), Chamaecrista mimosoides (L.) Greene (Caesalpiniaceae), Alectra sessiliflora (Vahl.) Kuntze (Scrophulariaceae), Blumea axillaris (Lam.) DC. (Asteraceae), Cucumis anguria L. var. longaculeatus J.H.Kirkbride (Cucurbitaceae), Hibiscus fuscus Garcke (Malvaceae),Physalis peruviana L.(Solanaceae) and Lantana trifolia L. (Verbenaceae). Preparation of reagents was carried out as per published protocols or val idated methods. In preliminary investigations the chloroform, methanol and water extracts were screened for antimicrobial activity against microbes of medical importance. The antibacterial and antifungal activity of the plant extracts, at fixed specified concentrations per well, against standardized inoculum of Staphylococcus aureus and Candida albicans, respectively, was tested by the disk diffusion method. The bio-autography procedure was then employed to determine the active fractions of the active extracts. Arising from the screening results, Alectra sessiliflora Vahl. Kuntze and Blumea axillaris Lam. DC were investigated further through Soxhlet extraction, fractionation, compound isolation and characterisation. The fractionation of plant extracts was carried out using a combination of various physical techniques namely solvent extraction and open column chromatography on silica gel, thin layer chromatography being used to follow purity of isolated fractions, macroporous resin and size exclusion chromatography. Chemical structure elucidation was based on spectroscopic data iacluding IR, lH-NMR, 13C_NMR and MS and GC-MS. Results and discussion: Thirty extracts were obtained from the ten plant species. Of the extracts, 90 % inhibited bacterial growth, while 70 % inhibited fungal growth when screened

in vitro. With the exception of only one extract, water extract of Chamaecrista mimosoides, all the zones of inhibitions diameters were less than those of the standard reference drugs, chloramphenicol and nystatin, tested at 50 ug/well, A total of 89 spots were visualized using the 3-(4, 5-dimethylthiazo-2-yl)-2, 5-diphenyltetrazolium bromide (MIT) spray reagent on the bio-autographs of the thirteen most active extracts with 52 spots (58.4 %) showing antimicrobial bioactivity. Five compounds, a sphingoid base, P-sitosterol; two phenolic acids, p-coumaric acid and 3, 4 dihydroxybenzoic acid; a flavonoid, luteolin were isolated from Alectra sessiliflora Vahl. Kuntze. In addition,two phytosterols, stigmasterol and p-sitosterol as well as two liquid isolates were realised from Blumea axillaris Lam. DC. The liquid isolates were found to compromise of a total of eight major constituents, 2-methyl-4- hydroxycyclobutanone, tert-pentylamine, diethylacetoacetamide, 2-(1, I-dimethylethyl)-l ,4- dimethoxybenzene, 2-dodecanone , 4-methylester octanoic acid, butanoic acid octyl ester and 9-tetradece-l-ol acetate. Of the isolated compounds, p-coumaric acid, 3,4-dihydroxybenzoic acid, luteolin and the two liquid isolates obtained from Blumea axillaris exhibited appreciable antibacterial and antifungal activity (MIC<25.0 ug/well) against tested bacteria Pseudomonas aeruginosa, Escherichia coli and Bacillus pumilus as well as the fungi Candida albicans, Aspergillus niger and Cryptococcus neoformans. Conclusion and recommendations: All the screened plant extracts showed some level of antimicrobial activity, with some plants possessing antifungal constituents some of which were isolated. From literature, this is the first report of in vitro antibacterial and anti-fungal activity and isolation of the bioactive compounds from Alectra sessiliflora Vahl. Kuntze. The isolation of the stigmasterol and p-sitosterol from B. axillaris Lam. DC.is also reported for the first time although -the compounds had earlier been reported from other Blumea species. These compounds could possibly be a basis of chemotaxonomy of the genus. The composition of the two liquid isolates realised is reported from B. axil/aris for the first time.

The antibacterial and antifungal activity reported in this study may be due to some of the isolated compounds acting either singly or in combination. The results obtained from this work may inform the continued use of the plants as herbal remedies or use of the isolated constituents as possible lead compounds in the development of new antifungal agents. Further scientific work is however recommended especially toxicity studies and use of combinational and computational techniques in synthesis of new leads that may lead to more useful compound to tackle the current or any future emerging fungal infections.