Influence of environmental parameters on efficacy of herbal medicines

Thesis (PhD)--University of Pretoria, 2012.

It is evident that herbal medicines continue to be the mainstay of healthcare systems and source of livelihoods of many local communities in South Africa and other developing countries. As a result, there is an overwhelming dependence on medicinal products harvested from natural populations. This dependence has led to local extinction of some important medicinal plants that include Warburgia salutaris and Cassine transvaalensis in South Africa. Cultivation has great potential to relieve the pressure on natural populations. However, some traditional practitioners and scientists believe that cultivation may weaken medicinal properties and that increased secondary metabolites may form only under stress conditions, respectively. This is certainly true in some cases especially where infections with pathogens, browsing by herbivores or competition takes place in nature. It is however not clear how true this is with environmental stresses. The overall aim of this study was to evaluate to what degree different environmental conditions influenced antimicrobial and antioxidant activities of plants cultivated outside their natural environment. In order to address the aim of the study, exploratory and in-depth studies were undertaken. The exploratory study comprised long-lived Combretum collinum Fresen. (Combretacea), Terminalia sericea Burch. ex DC. (Combretaceae) and Sclerocarya birrea (A. Rich.) Hochst. (Anacardiaceae). Short-lived herbaceous Tulbaghia violacea Harv. (Alliaceae) and Hypoxis hemerocallidea Fish, C.A.Mey,&Avé- Lall, (Hypoxidaceae), were included as part of the exploratory study. The in depth studies were further undertaken, also with short-lived herbaceous Leonotis dysophylla Benth. (Lamiaceae), Bulbine frutescens (L.) Willd. (Asphodelaceae) and T. violacea. Acetone leaf extracts of all plants were studied for antimicrobial activity against bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis) and fungi (Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus). Extracts were also studied for antioxidant activity against Trolox and L-ascorbic acid standard oxidants using 2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and 2,2- diphenyl-2-picryl-hydrazyl (DPPH) free radicals, respectively. The exploratory study tested the effect of different rates of annual rainfall (≥870 mm/year, 651 mm/year and 484 mm/year) on the antibacterial activity of C. collinum, T. sericea and S. birrea growing in nature. The minimum inhibitory concentration (MIC) of acetone extracts of air-dried leaves was determined by using microplate serial dilution technique. Thin layer chromatography (TLC) and bioautography determined chemical constituents and antibacterial activity of extracts, respectively. The majority of extracts had low MIC values, which indicated good antibacterial activity against test bacteria (MIC of 240 μg/ml - 60 μg/ml). Leaf extracts of C. collinum and S. birrea against S. aureus (range of 390 – 100 μg/ml), E. coli (310 -70 μg/ml) and P. aeruginosa (520 - 70 μg/ml) had antibacterial activity increased significantly with low rate of annual rainfall. However, extracts of T. sericea against P. aeruginosa (240 - 100 μg/ml) and E. faecalis (150 - 820 μg/ml) had antibacterial activity significantly increased and decreased, respectively. Extracts of C. collinum and S. birrea against E. faecalis as well as T. sericea against S. aureus and E. coli did not show any clear correlation between activity and different rates of annual rainfall. Inconsistent results suggest that other factors in nature such as genetic variability, age difference, pathogens, herbivores or allelopathy (competition) might have influenced the antibacterial activity of extracts. The results indicate that the antimicrobial activity of plants growing in nature may be highly variable. In order to eliminate possible effect of those factors common in nature, another exploratory study was undertaken using clone T. violacea and H. hemerocallidea of similar age (Chapter 3). Plants were grown under controlled conditions that included irrigation with 1000 ml of distilled water in intervals of 3, 14 and 21 days outside natural environment. Dry mass of all plants was reduced significantly (P≤0.05) with watering interval of 21 days, which indicated the effect of water stress. Air-dried leaves of all plants were finely ground and extracted with acetone. Extracts had good antibacterial activity as attested by low MIC values (< 1 mg/ml) across watering intervals. Differences in the antibacterial activity of the extracts against test bacterial between water treatments were not statistically significant (P≤0.05). Furthermore, there was no clear correlation between the activity of extracts and water treatments in terms of the MIC and total activity values or chemical constituents. The results in general suggest that cultivation under optimal watering intervals may not necessarily weaken the biological activity of extracts. To complement the above findings, in depth studies were also undertaken with clone L. dysophylla, T. violaceae and B. frutescens of similar age growing under controlled conditions outside natural environment. The studies determined the influence of a wide range of water (50 ml – 500 ml) and temperature (15°C and 30°C) treatments on antibacterial, antifungal and antioxidant of extracts. With the exception of a crassulacean acid metabolism (CAM) plant, B. frutescens, transpiration, dry mass and leaf areas of the other two plants were reduced significantly (P 0.05) under high temperature of 30°C and lowest water supply of 50 ml. Acetone leaf extracts had some biological activity. Differences in the majority of antibacterial and antifungal activities of extracts between water and temperature treatments were not statistically significant. With the exception of the influence of temperature, the majority of the antioxidant activity of extracts was almost similar between water treatments. However, the significant reduction of the antioxidant activity of all extracts under high temperature of 30°C was indicative of great sensitivity to high temperatures. The overall findings suggest that the biological activity of plants is more likely to vary widely in nature than under controlled conditions outside the natural environment. This is an indication that natural environment cannot always guarantee high and stable biological activity. As a result, beliefs by some traditional practitioners and scientists that cultivation weakens medicinal properties and good secondary metabolites form only under stress, respectively, cannot be widely substantiated. Therefore, the study encourages cultivation of medicinal plants. It has potential to optimise yield of biomass production, and ensure uniform and quality biological activity as well as reduce misidentification.

Paraclinical Sciences

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