Verification of the folkloric and anecdotal antidiabetic effects of Hypoxis hemerocallidea (Fisch., C.A. Mey. & Avé-Lall) and isolated, ß-sitosterol using early-stage type II spontaneous diabetic mutant BKS-Leprdb mice.

BACKGROUND: Previous studies in our laboratory in ex vivo assays have demonstrated H. hemerocallidea extract as potential antidiabetic agent through increased insulin release from pancreatic beta cells. Thus, for this study the early stage type II spontaneous diabetic mutant mice model was used to evaluate and determine the degree of the antidiabetic efficacy of H. hemerocallidea. METHODS: Eight-weeks-old type II spontaneous pre-diabetic mutant BKS-Leprdb mice were fed with feed supplemented with either H. hemerocallidea extract, isolated compound (ß-sitosterol) or chlorpropamide (positive control) for 4 weeks. The haematological parameters, clinical chemistry, glucose tolerance, feed intake, faecal output and body weights were measured. RESULTS: The blood glucose concentrations of all the animals treated with plant extract, ß-sitosterol compound and non-treated pre-diabetic animals did not return to baseline levels. Only the ß-sitosterol treatment and positive control groups resulted in a respective small decrease of 5.8 and 5.2% in the mouse weights over the study period, with no significant changes (p > 0.05) in food intake. However, there was a general trend for decrease in faecal output for all the groups. Albumin, triglycerides, and total cholesterol levels in ß-sitosterol and chlorpropamide-treated animals were lower, relative to untreated-animals. Animals fed with plant extract showed large amounts of internal fat. There were no significant changes (p > 0.05) in total serum protein, globulin, alanine aminotransferase, alkaline phosphatase, urea nitrogen and creatinine attributed to administration of treatments. In all groups, some animals showed lesions associated with cardiac puncture. Few animals except animals treated with plant extract, showed presence of a left-ventricular hypertrophic cardiomyopathy. The liver and kidneys for all groups appeared macroscopically normal and the thymuses were small (±2 mg). There were pathological signs in some of the animals particularly in myocardial fibres, renal tubular, glomerular, hepatocyte granularity and pancreas islets. However, there was no significance trend between the groups. CONCLUSION: Based on the results, none of the treatments could be considered highly effective for the management of type II pre-diabetes as sole therapeutic intervention.

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus.

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.

THE USE OF PLANTS TO PROTECT PLANTS AND FOOD AGAINST FUNGAL PATHOGENS: A REVIEW.

BACKGROUND: Plant fungal pathogens play a crucial role in the profitability, quality and quantity of plant production. These phytopathogens are persistent in avoiding plant defences causing diseases and quality losses around the world that amount to billions of US dollars annually. To control the scourge of plant fungal diseases, farmers have used fungicides to manage the damage of plant pathogenic fungi. Drawbacks such as development of resistance and environmental toxicity associated with these chemicals have motivated researchers and cultivators to investigate other possibilities. MATERIALS AND METHODS: Several databases were accessed to determine work done on protecting plants against plant fungal pathogens with plant extracts using search terms "plant fungal pathogen", "plant extracts" and "phytopathogens". Proposals are made on the best extractants and bioassay techniques to be used. RESULTS: In addition to chemical fungicides, biological agents have been used to deal with plant fungal diseases. There are many examples where plant extracts or plant derived compounds have been used as commercial deterrents of fungi on a large scale in agricultural and horticultural setups. One advantage of this approach is that plant extracts usually contain more than one antifungal compound. Consequently the development of resistance of pathogens may be lower if the different compounds affect a different metabolic process. Plants cultivated using plants extracts may also be marketed as organically produced. Many papers have been published on effective antimicrobial compounds present in plant extracts focusing on applications in human health. More research is required to develop suitable, sustainable, effective, cheaper botanical products that can be used to help overcome the scourge of plant fungal diseases. CONCLUSIONS: Scientists who have worked only on using plants to control human and animal fungal pathogens should consider the advantages of focusing on plant fungal pathogens. This approach could not only potentially increase food security for rural farmers, lead to commercial rewards, but it is also much easier to test the efficacy in greenhouse or field experiments. Even if extracts are toxic it may still be useful in the floriculture industry.

Novel Mycobacterium avium Complex Species Isolated From Black Wildebeest (Connochaetes gnou) in South Africa.

A study was undertaken to isolate and characterize Mycobacterium species from black wildebeest suspected of being infected with tuberculosis in South Africa. This led to the discovery of a new Mycobacterium avium complex species, provisionally referred to as the Gnou isolate from black wildebeest (Connochaetes gnou). Sixteen samples from nine black wildebeest were processed for Mycobacterium isolation. Following decontamination, samples were incubated in an ordinary incubator at 37°C on Löwenstein-Jensen slants and in liquid medium tubes using the BACTEC™ MGIT™ 960 system, respectively. Identification of the isolate was carried out by standard biochemical tests and using the line probe assay from the GenoType® CM/AS kit (Hain Lifescience GmbH, Nehren, Germany). The DNA extract was also analysed using gene sequencing. Partial gene sequencing and analysis of 16S rRNA gene, and 16S-23S rRNA (ITS), rpoB and hsp65 and phylogenetic analyses by searching GenBank using the BLAST algorithm were conducted. Phylogenetic trees were constructed using four methods, namely Bayesian inference, maximum likelihood, maximum parsimony and neighbour-joining methods. The isolate was identified as Mycobacterium intracellulare using the GenoType® CM/AS kit and as Mycobacterium avium complex (MAC) by gene sequencing. The gene sequence targeting all the genes, ITS, 16S rRNA, rpoB and hsp65 and phylogenetic analyses indicated that this isolate presented a nucleotide sequence different from all currently published sequences, and its position was far enough from other MAC species to suggest that it might be a new species.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against Plasmodium falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF THE STUDY: To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS: Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20µg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS: Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes' viability at 10 and 20µg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10µg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION: This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.

Anti-inflammatory, anticholinesterase and antioxidant activity of leaf extracts of twelve plants used traditionally to alleviate pain and inflammation in South Africa.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative stress and inflammatory conditions are among the pathological features associated with the central nervous system in Alzheimer׳s disease. Traditionally, medicinal plants have been used to alleviate inflammation, pains and also other symptoms possibly associated with Alzheimer׳s disease. Therefore, the present study was designed to determine the in vitro anti-inflammatory, antioxidant and anticholinesterase activity of twelve South African medicinal plants traditionally used to alleviate pain and inflammation. MATERIALS AND METHODS: Nitric oxide (NO) production in LPS-activated RAW 264.7 macrophages and 15-lipoxygenase (LOX) inhibitory assay were used to evaluate the anti-inflammatory activity. Acetylcholinesterase inhibition was assessed by using a modification of the Ellman׳s method. Antioxidant activity, total phenolic and total flavonoids contents were determined using standard in vitro methods. RESULTS: The extract of Burkea africana had the highest anti-15-lipoxygenase activity with 85.92% inhibition at 100µg/mL. All the extracts tested inhibited nitric oxide (NO) production in a dose dependant manner in LPS-stimulated RAW 264.7 macrophages. However, extracts from Leucaena leucocephala, Lippia javanica inhibited the production of NO by 97% at a concentration of 25µg/mL. In addition, both Leucaena leucocephala and Englerophytum magaliesmontanum had strong activity against acetylcholinesterase with IC50 values of 118µg/mL and 160µg/mL respectively. Hight levels of phenolics and flavonoids were found in Leucaena leucocephala, Lippia javanica and Burkea africana. The correlation with antioxidant activities was not strong indicating that other metabolites may also be involved in antioxidant activity. CONCLUSIONS: The results obtained in this study validate the use of leaf extracts of these plants in South African traditional medicine against inflammation. Extracts of these plants species might be of value in the management of various diseases emerging from oxidative stress and related degenerative disorders.

Evaluation of six plant species used traditionally in the treatment and control of diabetes mellitus in South Africa using in vitro methods.

CONTEXT: Numerous plants are used by the local communities of South Africa for the treatment and management of type II diabetes. OBJECTIVES: For this study, we undertook a survey of the plants sold for the management of diabetes in the town of Newcastle, South Africa. Identified plants were subsequently evaluated for their in vitro antidiabetic activity. MATERIALS AND METHODS: Plants were identified through an interview with a herbalist at the market. Antidiabetic activity of extracts of purchased plants was evaluated using in vitro α-amylase and α-glucosidase activity, as well as islets of Langerhans excretory activity. RESULTS: Senna alexandrina Mill. (Fabaceae), Cymbopogon citrates Stapf. (Poaceae), Cucurbita pepo L. (Cucuribitaceae), Nuxia floribunda Benth. (Stilbaceae), Hypoxis hemerocallidea Fisch. and Mey (Hypoxidaceae), and Cinnamomum cassia Blume (Lauraceae) were identified. The hexane extract of S. alexandrina (EC50=0.083 mg/ml), ethyl acetate extract of H. hemerocallidea (EC50=0.29 mg/ml), and methanol extracts of Cymbopogon citratus (EC50=0.31 mg/ml) and Cinnamomum cassia (EC50=0.12 mg/ml) had the highest α-amylase inhibitory activity, albeit lower than acarbose (EC50=0.50 mg/ml). All the plants had good α-glucosidase inhibitory activity (>50%) with the exception of some methanol (Cinnamomum cassia, N. floribunda, and Cymbopogon citratus) and acetone extracts (Cucurbita pepo and N. floribunda). Only the H. hemerocallidea acetone extract had an insulin stimulatory effect (2.5 U/ml at 8 µg/ml). CONCLUSION: All the evaluated plants demonstrated inhibitory activity against the specific GIT enzyme systems evaluated. Only H. hemerocallidea had insulin secretory activity, adding evidence to the traditional use of these purchased plants in the management of the type II diabetic post-prandial hyperglycemia.

Isolation of seselin from Clausena anisata (Rutaceae) leaves and its effects on the feeding and development of Lucilia cuprina larvae may explain its use in ethnoveterinary medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Clausena anisata are used traditionally to expel maggots from wounds of animals in Zimbabwe. We have previously proved in the laboratory that the plant certainly affects the behaviour and growth of blowfly larvae. The objective of this study was to isolate and identify the active compounds responsible for this activity. MATERIALS AND METHODS: The acetone extract of Clausena anisata leaf powder was separated by solvent-solvent partition into five fractions. The n-hexane fraction was the most active in the larvicidal assay and therefore subjected to open column chromatography on silica gel. RESULTS: The isolated compound was identified by nuclear magnetic resonance (NMR) and mass spectroscopy (MS) as the pyranocoumarin, seselin, chemically known as 2',2'-dimethylpyranocoumarin. It inhibited feed intake in the first and second instars of blowfly larvae at the minimum concentration tested of 1 ppm resulting in significant lower mass pupae (13.5±0.5 mg and 22.4±0.4 mg for the first and second instar larvae respectively) compared to the solvent control group (26.19±0.8 mg) (p<0.05). CONCLUSIONS: This is the first report of the isolation of seselin from the leaves of Clausena anisata and the first report of the compound having an effect against blowfly larvae.

Antifungal activity and cytotoxicity of isolated compounds from leaves of Breonadia salicina.

ETHNOPHARMACOLOGICAL RELEVANCE: Breonadia salicina is used traditionally to treat wounds, ulcers, fevers, headaches, and fungal infections. The aim of this study was to investigate the antifungal activity of the plant extract and compounds isolated there from. MATERIALS AND METHODS: Leaf extracts of Breonadia salicina were screened for antifungal activity against seven plant pathogens: Aspergillus niger, Aspergillus parasiticus, Colletotrichum gloeosporioides, Trichoderma harzianum, Penicillium expansum, Penicillium janthinellum and Fusarium oxysporum. Bioautography assay was used to determine the presence and number of antifungal compounds of the plant extracts. Bioassay-guided fractionation using column chromatography of the chloroform extract led to the isolation of four antifungal compounds. Nuclear Magnetic Resonance (NMR) spectroscopy, Mass Spectrometry (MS) and Electron Impact Mass Spectrometry (EIMS) were used for the identification of antifungal compounds. Cytotoxicity of the chloroform crude extract and isolated compounds was determined using the MTT (3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium bromide) assay against Vero monkey kidney cells. RESULTS: Compound 1 was identified as ursolic acid, while compounds 2, 3 and 4 were not identified conclusively owing to the presence of mixtures of long chain fatty acids. Compounds 3 and 4 had good antifungal activity against Aspergillus parasiticus and Penicillium janthinellum with MIC values of 10 and 16 µg/ml respectively. Compound 2 and ursolic acid had some activity with MIC values ranging between 20 and 250 µg/ml. The crude extract was less toxic to the Vero cells (LC50=82 µg/ml) than ursolic acid (LC50=25 µg/ml). Compounds 2 and 3 were not toxic at the highest concentration tested (LC50=200 µg/ml). Compound 4 was the most toxic to the cells with an LC50 of 35 µg/ml. CONCLUSIONS: The results support the traditional use of Breonadia salicina for antifungal applications, and demonstrate the potential value of developing antifungal compounds from plant natural products. Indications of toxicity should be evaluated at an early stage as the selectivity of the product in affecting fungi preferentially to plant or mammalian cells should be identified when assessing the potential usefulness of the product.

Evaluation of plant species used traditionally to treat myiasis for activity on the survival and development of Lucilia cuprina and Chrysomya marginalis (Diptera: Calliphoridae).

Myiasis is a common parasitic problem of livestock responsible for severe economic losses in developing and developed countries. There are a number of challenges with the current control strategy, which depends largely on the use of pharmaceutical chemicals. These include inaccessibility, the increasing concern about pesticide accumulation in the environment and potential development of insecticide resistance in the devastating myiasis-causing flies. Consequently the search for alternatives is important. The use of plants in the treatment of wound myiasis in livestock as an alternative to commercial insecticides has been reported in resource poor areas worldwide. We therefore, undertook a study to establish the biological activity of seven plant species used against blowflies in southern Africa. A larvicidal assay was carried out in which third instar larvae of blowfly were fed meat treated with acetone leaf extracts of selected plant species. Four of the species, Aloe zebrina, Clausena anisata, Erythrina lysistemon and Spirostachys africana, induced developmental anomalies in the blowfly such as paralysis, prolongation of the prepuparium stage, reduced pupation rates, pupal malformations and reduced adult emergence. These results suggest that the plants may contain compounds that interfere with the neuroendocrine control mechanisms in the blowfly.

Antibacterial activity of two biflavonoids from Garcinia livingstonei leaves against Mycobacterium smegmatis.

Amentoflavone and 4' monomethoxy amentoflavone were previously isolated from Garcinia livingstonei leaves. These compounds had good activities (MIC 6 and 8 µg/ml) against some nosocomial bacteria. In this study, the activity of these purified compounds were tested against fast-growing non-pathogenic Mycobacterium smegmatis. Amentoflavone was the most active compound, with an MIC of 0.60 ± 0.70 mg/ml. The MIC of 4' monomethoxy amentoflavone and the positive control isoniazid against Mycobacterium smegmatis were similar 1.40 ± 1.56 and 1.30 ± 1.70 mg/ml respectively. Although, Mycobacterium smegmatis is a non-pathogenic fast growing mycobacterium the activities of these compounds may also be useful in combating infections by pathogenic Mycobacterium spp.

Anthelmintic and cytotoxic activities of extracts of Markhamia obtusifolia Sprague (Bignoniaceae).

The anthelmintic activity of Markhamia obtusifolia Sprague (Bignoniaceae) leaf extracts was evaluated against the ruminant gastrointestinal nematode parasite Trichostrongylus colubriformis (Nematoda: Strongylida) using the in vitro egg hatch test. Also, the cytotoxic activity of aqueous extracts of M. obtusifolia was evaluated in cell line cytotoxicity assays. The results indicated that the effective concentration (EC(50)) for the water extract of M. obtusifolia leaves (0.46 mg/mL; Confidence Interval [CI] 0.3-0.5mg/mL) was significantly lower than the EC(50) for the acetone extract of M. obtusifolia (0.8 mg/mL; CI 0.7-1mg/mL). Aqueous extracts were twice as potent as the acetone extracts. The EC(90) (0.2mg/mL; CI 0.1-0.02) for thiabendazole (positive control) was significantly lower than the EC(90) for the water extract of M. obtusifolia (10.7 mg/mL; CI 8.3-13.7 mg/mL). In the cytotoxicity bioassay, the lethal concentration (LC(50)) for the aqueous extract of M. obtusifolia was 0.476 mg/mL, which was relatively high (low toxicity) in comparison to the highly toxic berberine (LC(50)=9.80 µg/mL). The current study showed that M. obtusifolia plant extracts possess anthelmintic activity and are relatively non-cytotoxic, thus providing support for their use in traditional veterinary practices.

Direct anthelmintic effects of Cereus jamacaru (Cactaceae) on trichostrongylid nematodes of sheep: in vivo studies.

Following claims of anthelmintic activity of Cereus jamacaru DC (Cactaceae) by a commercial farmer, in vivo studies were conducted to determine the possible direct anthelmintic effects of the plant on ovine gastrointestinal nematodes. Eighteen sheep were infected with 4000 Haemonchus contortus and 6000 Trichostrongylus colubriformis larvae given in three divided doses over a period of three days. Once the infections were patent, the sheep were allocated to three groups and were drenched once a week for six weeks with fresh blended C. jamacaru plant material at a single (32.3g/sheep) or double dose (64.6g/sheep) or they remained as undrenched controls. Faeces were collected from individual animals on the day of treatment and three days thereafter on a weekly basis for seven weeks for faecal egg count. While there were no statistically significant differences in the egg counts between the groups, a double dose of C. jamacaru was effective in reducing the egg counts in the sheep by 18-65% over the 49 days of the experiment. Given that all animals remained in good health throughout the course of the experiment, with no adverse events occurring during the study, further experiments using higher doses or administering the plant material for a longer period of time than in the present study would be warranted.

Ovicidal and larvicidal activity of Cassia alata leaf acetone extract and fractions on Haemonchus contortus: in vitro studies.

CONTEXT: The failure of modern anthelmintics to control nematode parasites of sheep and goats is a reality on many farms in the tropical/subtropical regions of the world. This necessitates chemotherapeutic control alternatives and plant secondary metabolite with activity is one of those potential solutions. OBJECTIVE: This study was design to evaluate the efficacy of solvent: solvent fractions of Cassia alata Gelenggang Besar (Leguminosae) leaf acetone extract against Heamonchus contortus Rudolphi (Trichostrongylidae). MATERIALS AND METHODS: C. alata leaf was extracted with 70% acetone and fractions were obtained by solvent: solvent group separation procedures. The acetone extract and the fractions were tested by egg hatch assay (EHA) and larval development and viability assay to assess relative bioactivity against H. contortus eggs and larvae. RESULTS: The extracts inhibited egg hatchability and killed infective larvae of H. contortus in a concentration-dependent manner. The best-fit LC(50) values were 0.562, 0.243, 0.490, 0.314, and 0.119 mg/mL for the acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against nematode eggs. The best-fit LC(50) values were 0.191, 0.505, 1.444, 0.306, and 0.040 mg/mL for acetone extract, chloroform, hexane, butanol and 35% water in methanol fractions, respectively, when tested against larvae. The 35% water in methanol fraction was the most active against the larvae and eggs of H. contortus demonstrating the lowest LC(50) values DISCUSSION AND CONCLUSION: This study demonstrates that the leaf extracts of C. alata have anthelmintic activity; therefore it could find application in the control of helminths in livestock.

Anthelminthic activity of acetone extract and fractions of Vernonia amygdalina against Haemonchus contortus eggs and larvae.

The current control of parasitic nematodes in small ruminants relies on the use of chemical anthelminthics, but the development of resistance and the problem of drug residues require research into alternatives. Acetone extract and solvent-solvent fractions of Vernonia amygdalina Del. (Compositae) were evaluated in vitro for potential anti-parasitic effects against the eggs and larvae of Haemonchus contortus. Significant effects were obtained with the extract and fractions but differences were observed depending on the parasitic stage. The acetone extract and fractions inhibited egg hatching and inhibited larval development and killed larvae of H. contortus in a concentration-dependent manner. Best-fit 50% lethal concentration (LC(50)) values were 957.0, 76.0, 524.0, 309.0 and 224.0 µg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively, when tested against nematode eggs. Best-fit LC(50) values for the larval viability test were 508.2, 485.5, 569.3, 348.9 and 196.6 µg/ml for the acetone extract, and the butanol, hexane, chloroform and 35% water in methanol fractions, respectively. The butanol fraction was most active against nematode eggs while the 35% water in methanol fraction was the most active on nematode larvae, although differences in activity between fractions were not significant (p > 0.05). Overall, these in vitro results suggest that V. amygdalina, traditionally used by small farmers in Western Africa, does possess anti-parasitic properties.

A simplified but effective method for the quality control of medicinal plants by planar chromatography.

Three of the factors limiting the rational use of herbal medicine are uncertainty on effectivity, uncertainty on safety and variation in quality of the product. Because many herbal medicines have been used over centuries by indigenous peoples, the safety and effectivity is frequently not such a big concern. With more people collecting and distributing herbal medicine, the offered product is however, frequently not what the label indicates either through a genuine mistake, but also through fraud especially where expensive herbal medicine is concerned. Some wrong identifications have already led to serious side effects and deaths. Planar chromatography or thin layer chromatography [TLC] is widely used to verify the identity of plant extracts by determining the chemical fingerprint of the extracts. In a leading publication 17 different extractants, 41 solvent systems and 44 spray reagents have been used to verify the identity of important herbal preparations. We investigated whether a simplified system could not be developed to aid small laboratories in identifying different herbal medicines. We compared the efficacy of different extractants, identified and developed three TLC solvent systems that would separate compounds with low, medium and high polarity and then also investigated the use of several spray reagents. With acetone as extractant and benzene:ethanol:ammonia [9:1:0.1], chloroform:ethylacetate:formic acid [5:4:1] and ethylacetate:methanol:water [10:1.35:1] as TLC solvent system and vanillin-sulphuric acid as spray reagent the identity of 81 samples of more than 50 herbal preparations could be verified on the basis of the chromatograms. The same product from different suppliers usually gave similar chromatograms. More importantly in several cases it was clear that products with the same label were so different that a mistake must have occurred in the labelling. This method has found application in the quality control of the most important African medicinal plants in the recently published African Herbal Pharmacopoeia produced by the Association for African Medicinal Plant Standards (AAMPS).

In vitro anthelmintic effect of Anogeissus leiocarpus (DC.) Guill. & Perr. leaf extracts and fractions on developmental stages of Haemonchus contortus.

The anthelmintic effect of acetone leaf extract and fractions of Anogeissus leiocarpus was investigated to determine the relative efficacy of the components as anthelmintic against Haemonchus contortus (Rudolphi). The fractions were obtained by solvent-solvent group separation of the leaf extract. The fractions were evaluated for ovicidal and larvicidal activity by egg hatch inhibition assay and larval development viability assay. Best-fit LC(50) values for egg hatch test were 0.360, 0.316, 0.093, 0.219 and 0.196 mg/ml for the crude acetone extract, hexane, chloroform, butanol, and 35% water in methanol fractions, respectively. While the best-fit LC(50) values for larval development and viability test were 0.509, 0.162, 0.186, 0.288 and 0.130 mg/ml for the crude acetone extract, hexane, chloroform, butanol, and 35% water in methanol fractions, respectively. The 35% water in methanol fractions was the more active on larvae, although differences in activity between fractions were not significant (p>0.05). A. leiocarpus leaf extracts could find application in anthelmintic therapy in veterinary practice.

Attraction response of adult Rhipicephalus appendiculatus and Rhipicephalus pulchellus (Acari: Ixodidae) ticks to extracts from Calpurnia aurea (Fabaceae).

Experiments were carried out to investigate the response of two tick species Rhipicephalus pulchellus Gerstaker, 1873 and Rhipicephalus appendiculatus Neumann, 1901 to three different extracts (acetone, aqueous and oil) of the dried leaves of Calpurnia aurea (Aiton) Benth in both an inverted glass tube and a dual choice T-olfactometer. The oil extract at 50 and 100mg/ml attracted 46.7% and 65.9% of R. appendiculatus, respectively, in the inverted glass tube assay, which was comparable to 47.8% of the attraction-aggregation-attachment pheromone (AAAP) used as positive control. At a dose of 100mg/ml the oil extract attracted 52.4% of R. pulchellus in the T-olfactometer bioassay. The relative attraction of both tick species to plant extract was also tested in semi-field plot experiments using a trap baited with different concentrations of emulsifiable extract of C. aurea. A dose of 100mg/ml attracted 52.2% of R. pulchellus and 44.4% of R. appendiculatus from a distance of 1m while 14.4% of R. pulchellus and 12.2% of R. appendiculatus were attracted from 5m distance at the same dose. Addition of CO(2) to the plant extract-baited-trap at the dose of 100mg/ml increased the range of attraction of adult R. pulchellus (44.4% from 5m distance) and up to 33.3% of adult R. appendiculatus tick from a distance of 4m. The results of this study suggest that extracts from C. aurea can potentially be used as baits in a trap for the control of ticks in the field.

Antimycoplasmal activity of some plant species from northern Nigeria compared to the currently used therapeutic agent.

CONTEXT: Mycoplasma spp. are obligate parasites of humans and animals. But due to the special requirements needed to culture Mycoplasma in the laboratory, little or no research has been done to evaluate the efficacy of medicinal plants on the organism. OBJECTIVE: To screen medicinal plants traditionally used to treat infections for possible antimycoplasmal and cytotoxic activities. MATERIALS AND METHODS: Acetone extracts of 21 Nigerian medicinal plants were analyzed for antimycoplasmal and cytotoxicity activities using the metabolic inhibition and colorimetric methods, respectively. The extract with the best antimycoplasmal activities was also analyzed for its phytochemical constituents using the desktop method. RESULTS: Calotropis procera (Aiton) R.Br (Asclepiadaceae) extract had the best antimycoplasmal effect with a minimum inhibitory concentration (MIC) of 80 µg/mL and minimum mycoplasmacidal concentration (MMC) of 160 µg/mL. This extract contained saponins, tannins, cardiac glycosides, alkaloids, and flavonoids. The extract of Vernonia amygdalina Delile (Compositae) was the most cytotoxic with median lethal concentration (LC(50)) of approximately 17 µg/mL, and that of Anacardium occidentale L. (Anacardiaceae) was the least cytotoxic with an LC(50) of approximately 1919 µg/mL. DISCUSSION: Calotropis procera is a promising plant for an alternative antimycoplasmal agent because the crude acetone extract had a higher mycoplasmacidal activity than the conventional drug tylosin, which is currently used in treatment of the disease in Nigeria. CONCLUSION: The crude extract of Calotropis procera is worth investigating for the development of a potent agent against cattle Mycoplasma, which has long defied solution by conventional chemotherapy.

Antifungal efficacy of ten selected South African plant species against Cryptococcus neoformans.

Fungal infections are a major threat to public health care. Cryptococcosis in humans and animals, caused by Cryptococcus neoformans, is a life-threatening disease. In a random antifungal screening of acetone leaf extracts of 400 tree species against Cryptococcus neoformans, the following plant species had good activity: Zanthoxylum capense (Thunb.) Harv. (Rutaceae), Morusmesozygia Stapf (Moraceae), Calodendrum capense (L.f.) Thunb. (Rutaceae), Catha transvaalensis Codd (Celastraceae), Cussonia zuluensis Strey (Araliaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae), Croton sylvaticus Hochst. ex C. Krauss (Euphorbiaceae), Maytenus undata (Thunb.) Blakelock (Celastraceae), Celtis africana Burm.f. (Ulmaceae), and Cassine aethiopica Thunb. (Celastraceae). Hexane, dichloromethane, acetone, and methanol extracts of these 10 plants were tested against Cryptococcus neoformans using bioautography and microdilution assays. Acetone extracted the highest quantity of plant material. Dichloromethane and hexane extracts of Maytenus undata showed clear bands in bioautography while the other species did not produce good results in bioautography. Maytenus undata extracts had promising antifungal activity against C. neoformans, with average minimum inhibitory concentration (MIC) of 0.09 mg/mL after 24 h and 0.18 mg/mL after 48 h incubation. Croton sylvaticus and Catha transvaalensis extracts also had good activity, with average MIC values of 0.07 mg/mL and 0.09 mg/mL, respectively. Because of the clear bands on bioautograms and low MIC values compared to the other plant species investigated, M. undata was identified as a good candidate for further studies.