S.F. Van Vuuren

Antimicrobial activity of South African medicinal plants

This paper reviews the antimicrobial research undertaken on South African medicinal plants during the period 1997-2008. Antimicrobial methods (disc diffusion, minimum inhibitory concentration (MIC), bio-autography) are briefly discussed and an analysis of the publications reviewed indicates that the majority of papers use MIC assays for antimicrobial determination. Antimicrobial investigations on extracts are presented where the most active plants are identified from screening publications. A summary of some bioactive compounds are given with data restricted to papers reporting quantitative antimicrobial activity equivalent to or below 200 microg/ml. Antimicrobial activities on the essential oils of indigenous medicinal aromatic plants are also reviewed. An overview is given on what activities (extracts, compounds and oils) should be considered noteworthy for publication. Studies focusing on geographical ethnobotany, specific pathogenesis, formulation aspects and in vivo investigations are examined. Future recommendations to consider include pathogen selection, interactive studies and dosage administrations.

The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials.

Aims:  Due to the emergence of multi‐drug resistance, alternatives to conventional antimicrobial therapy are needed. This study aims to investigate the in vitro pharmacological interactions between essential oils (considered valuable as natural therapeutic treatments) and conventional antimicrobials (ciprofloxacin/amphotericin B) when used in combination.

An antimicrobial investigation of plants used traditionally in southern Africa to treat sexually transmitted infections

AIM OF THE STUDY Eighteen plants were assessed for antimicrobial activity against pathogens associated with prevalent urogenital/sexually transmitted infections. Plant selection was based on information obtained from the ethnobotanical literature. MATERIALS AND METHODS Dried plant material was submerged in a 1:1 mixture of methanol and dichloromethane for 24 h. Aqueous extracts were prepared by submerging dried plant material in sterile distilled water for 24 h followed by lyophilization. Essential oils were distilled from the two aromatic plant species (Tarchonanthus camphoratus and Croton gratissimus). Antimicrobial activity was assessed using the micro-well minimum inhibitory concentration (MIC) assay with specific modifications to facilitate fastidious growth of pathogens. RESULTS Tarchonanthus camphoratus (solvent extract) showed the most significant broad spectrum activity with MIC values ranging between 0.50 and 0.70 mg/ml against five of the six pathogens tested. Other noteworthy activity was found for Hypericum aethiopicum (root) at 0.3 mg/ml (Neisseria gonorrhoeae).Polygala fruticosa and the solvent root extract of Hypericum aethiopicum showed highest sensitivities towards Gardnerella vaginalis at 0.2 mg/ml. Efficacy of the solvent extracts against the pathogen Oligella ureolytica demonstrated antimicrobial activity (MIC values <or=1.0 mg/ml) for seven plant species. The highest activity noted against Ureaplasma urealyticum was for Psidium guajava (solvent extract) at 0.8 mg/ml. In general the aqueous extracts displayed mostly poor anti-STI activity. The most noteworthy susceptibility for the aqueous extracts was noted with the plant extract Syzygium cordatum (MIC value 0.1 mg/ml against Candida albicans). The most noteworthy activity for the essential oils was observed for Tarchonanthus camphoratus (0.8 mg/ml) against Oligella ureolytica. CONCLUSIONS Antimicrobial activity was observed for a number of the plant samples against at least one or more pathogen, thus validating the ethnobotanical use as an anti-infective to treat sexually transmitted diseases.

Medicinal plants used for the treatment of diarrhoea in northern Maputaland, KwaZulu-Natal Province, South Africa.

AIM OF THE STUDY To document the ethnobotanical knowledge on antidiarrhoeal plant use by lay people in a rural community in northern Maputaland. MATERIALS AND METHODS Interviews were conducted amongst homestead inhabitants, using structured questionnaires. The focus was on the medicinal plants which are growing in and around the immediate vicinity of the homesteads. RESULTS Twenty-three plant species are used in the study area to treat diarrhoea. Four plants (Acacia burkei, Brachylaena transvaalensis, Cissampelos hirta and Sarcostemma viminale) are recorded for the first time globally as an antidiarrhoel. The three antidiarrhoeal plants most frequently used in the study area are Psidium guajava, Catharanthus roseus and Melia azedarach (all three are exotic to South Africa), followed by Sclerocarya birrea and Strychnos madagascariensis which are indigenous. Seven of the 23 plant species are used in five different plant combinations for increased antidiarrhoeal efficacy. CONCLUSION The wide variety of plants that are used to treat diarrhoea in this area supports the traditional value that medicinal plants have in the primary health care system of the rural people in northern Maputaland, KwaZulu-Natal.

Plants used for treating respiratory infections in rural Maputaland, KwaZulu-Natal, South Africa

ETHNOPHARMACOLOGICAL RELEVANCE Traditional remedies are frequently used in treating various respiratory ailments, and are very important in the primary health care of the people living in rural Maputaland, KwaZulu-Natal, South Africa. Novel information gathered from surveys like the present study is important in preserving indigenous knowledge. AIM OF THE STUDY To explore the knowledge that the lay people of a rural community in northern Maputaland have about medicinal plants used in the vicinity to treat respiratory infections. MATERIALS AND METHODS Interviews were conducted among 80 homestead inhabitants, using structured questionnaires where convenience sampling was used. The focus was on plants used in treating respiratory infections. Some of the main topics discussed during the interviews were vernacular plant names, plant parts used, harvested amounts, preparation methods, dosage forms and quantities, use of plants in combination as well as the related symptomatic relief associated with respiratory infections. RESULTS The study documented 30 plant species (18 families) which are used to treat respiratory infections by the rural people in the study area. Decoctions made with these plants are mostly taken orally, combined with the use of steaming. To the best of our knowledge, Acanthospermum glabratum, Aloe marlothii, Krauseola mosambicina, Ozoroa obovata, Parinari capensis and Plectranthus neochilus are recorded for the first time globally as medicinal plants used for treating respiratory infections and related symptoms. The indigenous aromatic shrub, Lippia javanica was by far the most frequently used plant species, followed by Eucalyptus grandis (an exotic), Tetradenia riparia and then Senecio serratulloides. Twenty-four different plant combinations were used where the most frequently used combination encountered was Eucalyptus grandis with Lippia javanica. CONCLUSION The large number of different plant species traditionally used against respiratory infections supports previous research on the importance of traditional medicine in the primary health care of this remote area. The finding of new vernacular plant names and plant uses in the current survey shows the importance of the documentation of such ethnobotanical knowledge.

In vitro evidence of phyto-synergy for plant part combinations of Croton gratissimus (Euphorbiaceae) used in African traditional healing

AIM OF THE STUDY Despite the extensive traditional use of Croton gratissimus Burch. var. gratissimus for medicinal purposes, scientific studies validating the therapeutic properties of this indigenous plant are lacking. As the bark, roots and leaves of C. gratissimus are used separately as well as in combination, this study focused on determining antimicrobial efficacies of the plant parts independently and in combination to assess possible pharmacological interactions (e.g. synergy, antagonism). MATERIAL AND METHODS The hydro-distilled leaf essential oil and extracts of bark, root and leaf were comparatively assessed for antimicrobial activity by means of microdilution minimum inhibitory concentration (MIC). The fractional inhibitory concentrations (FIC) were determined for the leaf and root (1:1), bark and root (1:1), leaf and bark (1:1) combination. Isobolograms were plotted to demonstrate interactions between various ratios of the roots and leaves. RESULTS The MIC and FIC results indicated variable efficacies for the various plant part combinations, the greatest of which was noted for Cryptococcus neoformans in the root and leaf combination (MIC 0.4 mg/ml and FIC of 0.4). Isobolograms indicated the greatest synergy for Bacillus cereus, Candida albicans and Cryptococcus neoformans. CONCLUSION The observed synergistic interactions clearly indicate that the reductionist approach may often be short-sighted and that biological activity may be improved through combination therapy, where different complex metabolic pools collectively contribute to the enhanced effect.

The in vitro biological activity of selected South African Commiphora species

Ten South African Commiphora (Burseraceae) species were investigated to validate their use in traditional healing rites. The leaf and stem extracts of each species were analysed for the anti-oxidant (ABTS and DPPH assays), antimicrobial (MIC and death kinetic assays), anti-inflammatory (5-LOX assay), anticancer (SRB assay) properties, as well as the cytotoxic effects (tetrazolium-based assay). The best anti-oxidant activity (ABTS assay) was observed for the stem extracts of Commiphora tenuipetiolata IC(50)=5.10 microg/ml), Commiphora neglecta (IC(50)=7.28 microg/ml) and Commiphora mollis (IC(50)=8.82 microg/ml). Extracts generally exhibited poor anti-oxidant activity in the DPPH assay, with the exception of Commiphora schimperi (stem), Commiphora neglecta (stem), Commiphora tenuipetiolata (stem and leaf), and Commiphora edulis (stem), with IC(50) values ranging between 7.31 and 10.81 microg/ml. The stem extracts exhibited moderate to good 5-LOX inhibitory activity with Commiphora pyracanthoides (stem) displaying the greatest inhibitory effect (IC(50)=27.86+/-4.45 microg/ml). For the antimicrobial (MIC) assay, a greater selectivity was exhibited by the extracts against the Gram-positive bacteria (0.01-8.00 mg/ml) and the yeasts (0.25-8.00 mg/ml) than against the Gram-negative bacteria (1.00-8.00 mg/ml). Using death kinetic studies (time-kill studies), the rate at which Commiphora marlothii (stem) kills Staphylococcus aureus over a 24h period was determined. Mostly, a concentration-dependent antibacterial activity was observed beginning after ca. 30 min. All concentrations exhibited antibacterial activity, with complete bactericidal effect achieved by the 24(th) hour. The most active Commiphora species against the HT-29 cells (SRB anticancer assay) were Commiphora glandulosa (leaf and stem) and Commiphora marlothii (leaf). The MCF-7 cells (SRB anticancer assay) exhibited the highest sensitivity to indigenous Commiphora species, with Commiphora edulis (leaf and stem), Commiphora glandulosa (leaf and stem), Commiphora marlothii (leaf), Commiphora pyracanthoides (leaf and stem), Commiphora schimperi (stem), and Commiphora viminea (stem) all possessing a percentage inhibition greater than 80% at 100 microg/ml. Commiphora glandulosa (leaf and stem) and Commiphora pyracanthoides (leaf and stem) were the two most active species against the SF-268 cells (SRB anticancer assay), with IC(50) values ranging between 68.55+/-2.01 and 71.45+/-1.24 microg/ml. The majority of the Commiphora extracts were largely non-cytotoxic against Graham human kidney epithelial cells when investigated in the MTT assay.

Phytochemistry and in vitro pharmacological activities of South African Vitex (Verbenaceae) species

AIM OF THE STUDY The in vitro phytochemical and pharmacological investigation of the non-volatile extracts of five South African Vitex species (Verbenaceae); V. obovata ssp. obovata, V. obovata ssp. wilmsii, V. pooara, V. rehmannii and V. zeyheri were investigated in order to validate their traditional use to treat a wide range of ailments such as malaria, wounds, skin diseases and body pains. MATERIAL AND METHODS The antimicrobial activity was assessed using the minimum inhibitory concentration assay. Through bioactivity-guided fractionation, the fraction responsible for the antimicrobial activity was determined. The toxicity profile, anti-oxidant and anti-inflammatory activity was evaluated using the tetrazolium cellular viability, 2,2-diphenyl-1-picrylhydrazyl and 5-lipoxygenase assays respectively. The antimalarial activity of the extracts and isolated compound from V. rehmannii was also investigated on the chloroquine-resistant Gambian FCR-3 strain of Plasmodium falciparum using the tritiated hypoxanthine incorporation assay. RESULTS Mostly good antimicrobial inhibition was evident against Gram-positive bacteria (0.02-8.00 mg/ml) and lower activity against the Gram-negative bacteria and the yeast (0.50-8.00 mg/ml). The fraction responsible for antimicrobial activity of V. rehmannii was purified to give a labdane diterpene as an inseparable epimeric mixture of 12S,16S/R-dihydroxy-ent-labda-7,13-dien-15,16-olide. Cirsimaritin was also isolated and identified from V. rehmannii. All the species, apart from V. zeyheri, exhibited scavenging activity (IC50: 22.14+/-1.74 to 33.06+/-1.68 microg/ml) in the anti-oxidant assay. None of the species displayed any anti-inflammatory activity at 100 microg/ml. All the extracts and the labdane diterpene exhibited good antimalarial activity, with the labdane diterpene being the most active (IC50: 2.39+/-0.64 microg/ml). The test extracts were shown to be highly toxic, displaying safety index values ranging from 0.53 to 2.59. CONCLUSION Of all the pharmacological investigations, the antimalarial and antimicrobial activity exhibited greatest activity and may provide a scientific basis for the ethnomedical use of Vitex species.

An antimicrobial evaluation of plants used for the treatment of respiratory infections in rural Maputaland, KwaZulu-Natal, South Africa

ETHNOPHARMACOLOGICAL RELEVANCE Abundant availability of medicinal plants in the study area offers low cost health care, but scientific validation is needed in order to lend credibility to the traditional use against respiratory infections. AIM OF THE STUDY This study focussed on determining the antimicrobial efficacies of 30 plant species (independently and in various combinations) used for respiratory related infections in rural Maputaland. MATERIALS AND METHODS In vitro minimum inhibitory concentration (MIC) assays were undertaken on dichloromethane-methanol (CH(2)Cl(2): MeOH) and aqueous extracts, as well as the hydro-distilled essential oils (for aromatic plants). Selected plant parts were assessed for antimicrobial activity against a range of respiratory pathogens i.e. Cryptococcus neoformans (ATCC 14116), Klebsiella pneumoniae (ATCC 13883), Moraxella catarrhalis (ATCC 23246), Mycobacterium smegmatis (ATCC 14468) and Staphylococcus aureus (ATCC 6538). The sum of the fractional inhibitory concentrations (∑FIC) was determined for plants traditionally used in combination. Isobolograms represent MIC values for a selection of interactions where two plants were combined in various ratios. RESULTS The most antimicrobially active aqueous extracts were that of Ozoroa obovata and Sclerocarya birrea (0.10 mg/ml) while organic extracts from Parinari capensis subsp. incohata and Tetradenia riparia demonstrated the most noteworthy (0.03 mg/ml) activity. Although both Lippia javanica and Eucalyptus grandis were by far the most popular plants traditionally used for respiratory infections, the antimicrobial activity was mostly only moderate. Furthermore, the traditional use in a 1:1 combination did not display strong antimicrobial interactions, but isobolograms demonstrate (against some test organisms) that when combined in various ratios, predominant additive interactions are evident where E. grandis was present in larger proportions. The combination of E. grandis with O. obovata demonstrated synergism against both C. neoformans and K. pneumoniae, with ∑FIC values of 0.34 and 0.28 respectively. Various ratios of these two plants also demonstrated a predominantly synergistic profile. CONCLUSION Although this in vitro study supports the traditional use of some plants independently and in combination for the treatment of respiratory ailments in rural Maputaland, results demonstrate that the traditional selection of plants in higher frequency do not necessarily correlate with higher antimicrobial efficacy.

Plants traditionally used individually and in combination to treat sexually transmitted infections in northern Maputaland, South Africa: antimicrobial activity and cytotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE Although medicinal plants are used extensively to treat sexually transmitted infections (STIs) in rural northern Maputaland, KwaZulu-Natal, the efficacy and safety of these plants have not previously been evaluated. AIM OF STUDY A study was designed to investigate the in vitro antimicrobial activity and cytotoxicity profiles of a selection (individual plants and selected combinations) of traditionally used plants in this study area. MATERIALS AND METHODS Aqueous and organic (dichloromethane: methanol, 1:1) extracts were prepared. Antimicrobial activity was assessed using the minimum inhibitory concentration (MIC) assay against the STI associated pathogens; Candida albicans ATCC 10231, Ureaplasma urealyticum clinical strain, Oligella ureolytica ATCC 43534, Trichomonas vaginalis clinical strain, Gardnerella vaginalis ATCC 14018 and Neisseria gonorrhoeae ATCC 19424. For the combination study, interactions were assessed using the fractional inhibitory concentration (ΣFIC). The plant species were assessed for safety using the 3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay on the human embryonic kidney epithelial (Graham, HEK-293) cell line. RESULTS For the antimicrobial studies, U. urealyticum was the most sensitive of the six test organisms, with the aqueous extract of Ranunculus multifidus (0.02mg/ml) and the organic extract of Peltophorum africanum (0.04mg/ml) being the most antimicrobially active plant species studied. Sclerocarya birrea was found to have the broadest spectrum of activity (mean MIC of 0.89mg/ml). The only plant species to exhibit some degree of cytotoxicity against the kidney epithelial cell line was Kigelia africana (100µg/ml), with 22% and 16% cell death for the aqueous and organic extracts, respectively. Of the 13 combinations studied, several synergistic combinations were evident, the most prominent being the combination of Albizia adianthifolia and Trichilia dregeana (aqueous extract) with an ΣFIC value of 0.15 against O. ureolytica. Synergistic interactions were observed regardless of the ratio of the aqueous mixtures of the two plants. Syzygium cordatum and S. birrea (aqueous extract) was also a combination of interest, demonstrating synergistic (ΣFIC=0.42) interactions against O. ureolytica. This combination, however, also displayed some cytotoxicity towards the human epithelial cell line. CONCLUSION This study demonstrated that anecdotal evidence of plant use does not always correlate with in vitro activity. Furthermore, the toxicological profiling is of utmost importance as if not combined in its correct ratio can lead to potential adverse effects.