Jacobus Nicolaas Eloff

Which extractant should be used for the screening and isolation of antimicrobial components from plants

Freeze dried and finely ground leaves of two plants with known antimicrobial activity, Anthocleista grandiflora and Combretum erythrophyllum were extracted with acetone, ethanol, methanol, methylenedichloride, methanol/chloroform/water and water at a 1 to 10 ratio in each case. The quantity and diversity of compounds extracted, number of inhibitors extracted, rate of extraction, toxicity in a bioassay, ease of removal of solvent and biological hazard were evaluated for each extractant. An arbitrary scoring system was developed to evaluate the above parameters for the different extractants. Acetone gave the best results with these plants with an arbitrary value of 102 followed by methanol/chloroform/water (81), methylene dichloride (79), methanol (71), ethanol (58) and water (47). Four five minute sequential extractions of very finely ground A. grandiflora shaking at a high rate extracted 97% of the total antimicrobial activity.

The biological activity and chemistry of the southern African Combretaceae

AIM OF THE STUDY Members of the Combretaceae family are widely traded in the traditional medicine market in southern Africa. The family is also used for medicinal purposes in the rest of Africa and Asia for close to 90 medicinal indications. Many of these indications are related to treating infections. This contribution summarizes work done to date and identifies avenues for future research. MATERIALS AND METHODS Substantial work has already been done on the chemistry of especially Combretum and Terminalia species over many years. During the last decade we have focussed on bio-assay guided isolation of biologically active compounds with the aim of producing new effective antimicrobial products. RESULTS AND DISCUSSION Methods developed to facilitate this process and data on 25 compounds isolated from 7 species are presented. The large majority of compounds isolated were known, but the biological activities were not known. In practically all cases the antibacterial or antifungal activity of compounds isolated were much lower than expected from the activity of the crude extracts. It appears that synergism plays a role in antimicrobial activity of plant extracts and that the hope of isolating a single compound that can be used as a new agent to address antibiotic resistance has been frustrated. By simple manipulation such as selective extraction the activity of some crude extracts could however, be increased substantially and this offers a new approach to address antibiotic resistance via the herbal medicine industry. Practically all extracts obtained using intermediate polarity extractants had reasonable to very good activity with MICs as low as 40 microg/ml, validating the traditional use for infectious diseases. Aqueous extracts however, generally had hardly any activity. CONCLUSIONS The Combretaceae contains a diversity of antimicrobial compounds. Because poor people usually have only water available as extractant, it raises the question how plants growing in poor rural communities can be used to treat infections more effectively, and what the mechanism of activity of aqueous extracts used to treat infections in traditional medicine are.

The potential of South African plants against Mycobacterium infections

ETHNOPHARMACOLOGICAL RELEVANCE In South Africa, tuberculosis (TB) caused by Mycobacterium tuberculosis is the most commonly notified disease and the fifth largest cause of mortality, with one in ten cases of TB resistant to treatment in some areas. Many plants are used locally in traditional medicine to treat TB-related symptoms. AIM OF THE STUDY The aim was to summarize currently available knowledge on South African plants used to treat TB symptoms, and antimycobacterial efficacy of plant-derived extracts and compounds. MATERIALS AND METHODS The traditional uses of plants for respiratory ailments and TB were collated and tabulated. The antimycobacterial activity tests of extracts and chemical constituents of several of these plants and others using different methods and target organisms were summarized. RESULTS Almost 180 plants used for TB-related symptoms in South African traditional medicine were documented. About 30% of these have been tested for antimycobacterial efficacy, mostly against fast-growing, non-pathogenic Mycobacterium species. CONCLUSIONS Many plant species are used in traditional South African medicine to alleviate symptoms of TB, and several interesting leads have originated for further inquiry following in vitro antimycobacterial activity evaluation. However, much work remains to be done on the systematic assessment of anti-TB efficacy of local plants against pathogenic Mycobacterium species, both in vitro and in vivo.

Inflammation: the foundation of diseases and disorders. A review of phytomedicines of South African origin used to treat pain and inflammatory conditions.

Great interest in herbal medicine as a potential source of phytopharmaceuticals has created the need to review common factors responsible for major diseases and body disorders. This review shows one such common factor in inflammation and the role herbal medicine can play. Traditional medicinal herbal remedies in the southern African region have long been used to treat various pain- or inflammation-related symptoms. Although the precise mechanisms of action of many herbal drugs have yet to be determined, some of them have been shown to exert anti-inflammatory and/or antioxidant effects in a variety of cells in the human and animal bodies. There is increasing evidence to indicate that both peripheral and central nervous system cells play a prominent role in the chronic inflammatory responses in the body system and anti-inflammatory herbal medicine and its constituents are being proved to be a potent protector against various pro-inflammatory mediators in diseases and disorders. These mediators have therefore been suspected of being the functional basis of diseases and disorders. The structural diversity of these medicinal herbs makes them a valuable source of novel lead compounds against the therapeutic molecular targets, cytokines and mediators, that have been newly discovered by the platforms of genomics, proteomics, metabolomics and highthroughput technologies. This article reviews the basic mechanisms of inflammation and the potential of 123 southern African plant species to be effective as chronic inflammatory disease preventive agents. With one third of these species there are no indications of the chemical composition, indicating possible subjects for further research.

An investigation on the biological activity of Combretum species.

Leaf extracts of 20 Combretum species, many of which are used in southern African traditional medicine, were screened for anti-inflammatory, anthelmintic, anti-bilharzia (antischistosomal) and DNA-damaging activity. Significant activity in more than one bioassay was exhibited by Combretum apiculatum, Combretum hereroense, Combretum molle and Combretum mossambicense. Ethyl acetate extracts were generally most active, followed by acetone and then water extracts.

Antibacterial activity of marula (Sclerocarya birrea) (A. Rich.) Hochst. subsp. caffra (Sond.) Kokwaro (Anacardiaceae) bark and leaves

Marula bark is widely used for bacteria-related diseases by indigenous cultures in Africa. This study was undertaken to investigate whether the ethnobotanical use can be validated by laboratory studies. Bark and leaves were extracted with acetone and MIC values were determined using a microplate serial dilution technique with Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as test organisms. All extracts were active with MIC values from 0.15 to 3 mg/ml. Based on minimum inhibitory concentration values, inner bark extracts tended to be the most potent followed by outer bark and leaf extracts, but the differences were not statistically significant. There were two major bioactive components visible after bioautography of leaf extracts: one strongly polar and the other highly non-polar. The bioactive components could be separated from 92% of the non-active dry matter by solvent-solvent fractionation into the carbon tetrachloride, chloroform and n-butanol fractions; these fractions, however, still contained many different compounds. Using bark may be detrimental to the plant, but leaf material can also be used for antibacterial application.

Ethnoveterinary use of southern African plants and scientific evaluation of their medicinal properties

AIM OF THE STUDY Livestock keepers in many developing countries with restricted access to orthodox veterinary healthcare services commonly use traditional remedies to treat their animals when disease is encountered. This review collates the documented use of plants in South Africa for healing various ailments in domestic animals, and records bioactivity testing that has been carried out on these plants. MATERIALS AND METHODS A literature survey was conducted on the use of plants in South African ethnoveterinary medicine (EVM), as well as on biological activity investigations relating to their ethnoveterinary use where available. RESULTS The ethnoveterinary application of plants, and results of screening studies of EVM plant extracts in various bioassays is presented. For diseases such as coughs, wounds, skin diseases, mild diarrhoea and reproductive disorders, EVM may be a cheap and easily accessible alternative to expensive pharmaceuticals. Studies on biological activity of EVM plants can provide indications of promising leads for extracts that can be developed into standardized medications to be used on a commercial basis. Isolation studies on active plants may yield pure active compounds that could be chemically modified to optimize medicinal value and reduce possible toxic effects. CONCLUSION In South Africa, a large proportion of the population relies on traditional remedies to treat themselves and their animals for common diseases. Only a small percentage of EVM plants have been analysed for biological activity or toxic effects, and hence research in this field offers fertile possibilities for future investigation.

The value of plant extracts with antioxidant activity in attenuating coccidiosis in broiler chickens

Coccidiosis remains one of the most important diseases in the poultry industry and results in the annual loss of millions of US dollars by the poultry industry. In South Africa and other developing countries where a large percentage of the population is unemployed, cheap food production is necessary. If the control of the coccidian parasite could be made more economical, these savings could be passed on to the consumer. In Europe, where the economics are different, people are becoming more aware of the potential dangers of using antimicrobials in producing animal protein. A solution to both these problems could be the use of plant products that function by mechanisms other than those of chemotherapeutics, with the additional advantage of a natural origin. Antioxidant compounds could hold promise for the control of Eimeria infections due to the association of coccidial infection with lipid peroxidation of the intestinal mucosa. Four plant extracts with antioxidant activity were screened for their anticoccidial activity in vivo with toltrazuril as the positive control. Combretum woodii (160 mg/kg) proved to be extremely toxic to the birds, while treatment with Tulbaghia violacea (35 g/kg), Vitis vinifera (75 mg/kg) and Artemisia afra (150 mg/kg) resulted in feed conversion ratios similar to toltrazuril, and higher than the untreated control. T. violacea also significantly decreased the oocyst production in the birds. From this study we conclude that antioxidant-rich plant extracts have potential benefits in treating coccidial infections. The promising results obtained with T. violacea justify further studies on the potential value of the plant as a therapeutic or prophylactic anticoccidial agent.

The preliminary isolation of several antibacterial compounds from Combretum erythrophyllum (Combretaceae).

Freeze dried ground leaves of Combretum erythrophyllum were extracted with different extractants to determine if they contain inhibitors of pathogenic bacteria. Acetone yielded the most compounds inhibiting the growth of Staphylococcus aureus using bioautography of thin layer chromatography plates. Acetone also extracted many different compounds and group separation was attempted by solid phase extraction on reverse and normal phase silica gel. Both techniques separated the bioactive components to a degree. The best group separation results, however, were obtained using liquid/liquid extraction. The six fractions obtained inhibited the four test organisms to different degrees. S. aureus was the most sensitive (100%) followed by Enterococcus faecalis (36%), Escherichia coli (11%) and Pseudomonas aeruginosa (3%). With S. aureus as test organism, the chloroform soluble fraction contained by far the largest quantity of inhibiting components (100%), followed by the fractions soluble in water (23%), 35% methanol in water (18%), butanol (5%), carbon tetrachloride (2%) and hexane (traces). The lowest minimum inhibitory concentration for S. aureus was 0.05 mg/ml at this stage of purification compared to MIC values of 0.08 and 0.16 mg/ml for ampicillin and chloramphenicol. There were at least 14 different inhibitors with a wide range of polarities present in the different fractions. The polar components apparently did not contain polysaccharides and were probably basic according to the chromatographic behaviour.

Four pentacyclic triterpenoids with antifungal and antibacterial activity from Curtisia dentata (Burm.f) C.A. Sm. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE Curtisia dentata is used in southern Africa to treat various diseases of bacterial and fungal origin in humans and animals to such a degree that the species is vulnerable and declining [Dold, A.R., Cocks, M.L., 2001. Traditional veterinary medicine in the Alice district of the Eastern Cape Province, South Africa. South African Journal of Science 97, 375-379]. Preliminary studies indicated good activity against Candida albicans. AIM OF THE STUDY The phytochemistry and antimicrobial activity of this plant species has not been extensively investigated, and a closer examination of the compounds responsible for antimicrobial activity was warranted. MATERIALS AND METHODS Broth microdilution assay and bioautography were used to evaluate antibacterial and antifungal activity in Curtisia dentata leaf extracts and fractions. Bioassay-directed fractionation using column chromatography yielded four compounds characterised by spectroscopic methods. RESULTS Lupeol (1), betulinic acid (2), ursolic acid (3) and 2alpha-hydroxyursolic acid (4) were isolated from Curtisia dentata leaves. Betulinic acid, ursolic acid and 2alpha-hydroxyursolic acid appreciably inhibited fungal growth with MIC values ranging from 8 to 63mug/mL. CONCLUSIONS The isolation of four antibacterial and antifungal triterpenoids is reported for the first time from Curtisia dentata. This study provides information on the antimicrobial compounds of this species, as well as a preliminary rationale for the use in traditional South African medicine.