Evaluation of the Antibacterial and Antibiofilm Effects of Ethyl Acetate Root Extracts from Vernonia adoensis (Asteraceae) against Pseudomonas aeruginosa.

There is an increase in mortality and morbidity in the health facilities due to nosocomial infections caused by multidrug-resistant nosocomial bacteria; hence, there is a need for new antibacterial agents. Vernonia adoensis has been found to possess medicinal value. Plant phytochemicals may have antimicrobial activity against some resistant pathogens. The antibacterial efficacy of root extracts against Staphylococcus aureus and Pseudomonas aeruginosa was investigated using the microbroth dilution method. All extracts from the roots had an inhibitory effect on the growth of both bacteria, with the most susceptible being P. aeruginosa. The most potent extract was the ethyl acetate extract which caused a percentage inhibition of 86% against P. aeruginosa. The toxicity of the extract was determined on sheep erythrocytes, and its effect on membrane integrity was determined by quantifying the amount of protein and nucleic acid leakage from the bacteria. The lowest concentration of extract used, which was 100 µg/ml, did not cause haemolysis of the erythrocytes, while at 1 mg/ml of the extract, 21% haemolysis was observed. The ethyl acetate extract caused membrane impairment of P. aeruginosa, leading to protein leakage. The effect of the extract on the biofilms of P. aeruginosa was determined in 96-microwell plates using crystal violet. In the concentration range of 0-100 µg/ml, the extract inhibited the formation of biofilms and decreased the attachment efficiency. The phytochemical constituents of the extract were determined using gas chromatography-mass spectrometry. Results of analysis showed the presence of 3-methylene-15-methoxy pentadecanol, 2-acetyl-6-(t-butyl)-4-methylphenol, 2-(2,2,3,3-tetrafluoropropanoyl) cyclohexane-1,4-dione, E,E,Z-1,3,12-nonadecatriene-5,14-diol, and stigmasta-5,22-dien-3-ol. Fractionation and purification will elucidate the potential antimicrobial compounds which are present in the roots of V. adoensis.

Ozoroa insignis reticulata (Baker f.) R. Fern. & A. Fern. Root Extract Inhibits the Production of Extracellular Proteases by Staphylococcus aureus.

Treatment of infections caused by S. aureus has become a challenge due to the emergency of resistant strains. Ozoroa reticulata root extracts have been used in traditional medicine to treat throat and chest pains in Zimbabwe. The objective of the study was to determine the effects of O. reticulata root bark extracts on the production of extracellular proteases by S. aureus. The root barks were collected, dried, and crushed into powder. To obtain different phytoconstituents, plant extractions were performed. Extractions were carried out using two solvent mixtures: ethanol : water (50 : 50 v/v) and dichloromethane : methanol (50 : 50 v/v). Serial exhaustive extractions were also performed using methanol, ethanol, dichloromethane, acetone, ethyl acetate, hexane, and water. The broth microdilution assays were used to assess the antibacterial effects of the Ozoroa reticulata root bark extracts against S. aureus. Ciprofloxacin was used as a positive control. Qualitative screening for extracellular protease production by S. aureus on BCG-skim milk agar plates using the most potent extract was carried out. The proteolytic zones were measured and expressed as the ratio of the diameter of the colony to the total diameter of the colony plus the zone of hydrolysis (P z values). The ethyl acetate extract was found to be the most potent inhibitor of the growth of S. aureus with 99% inhibition and a minimum inhibitory concentration (MIC) of 100 µg/mL. Inhibition of extracellular protease production was directly proportional to the concentration of the extract. At 100 µg/mL, the ethyl acetate extract had a P z value of 0.84, indicative of mild proteolytic activity. A P z value of 0.94 was observed at a concentration of 200 µg/mL and signified weak proteolytic activity. In conclusion, the extract inhibited the production of extracellular proteases in S. aureus. Further work on the isolation and purification of bioactive compounds responsible for inhibiting the production of extracellular proteases is of importance in the discovery of agents with antivirulent effects on S. aureus.

The Effects of Tormentic Acid and Extracts from Callistemon citrinus on Candida albicans and Candida tropicalis Growth and Inhibition of Ergosterol Biosynthesis in Candida albicans.

Candida albicans and Candida tropicalis are the leading causes of human fungal infections worldwide. There is an increase in resistance of Candida pathogens to existing antifungal drugs leading to a need to find new sources of antifungal agents. Tormentic acid has been isolated from different plants including Callistemon citrinus and has been found to possess antimicrobial properties, including antifungal activity. The study aimed to determine the effects of tormentic and extracts from C. citrinus on C. albicans and C. tropicalis and a possible mode of action. The extracts and tormentic acid were screened for antifungal activity using the broth microdilution method. The growth of both species was inhibited by the extracts, and C. albicans was more susceptible to the extract compared to C. tropicalis. The growth of C. albicans was inhibited by 80% at 100 µg/ml of both the DCM: methanol extract and the ethanol: water extract. Tormentic acid reduced the growth of C. albicans by 72% at 100 µg/ml. The effects of the extracts and tormentic acid on ergosterol content in C. albicans were determined using a UV/Vis scanning spectrophotometer. At concentrations of tormentic acid of 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml, the content of ergosterol was decreased by 22%, 36%, 48%, and 78%, respectively. Similarly, the DCM: methanol extract at 100 µg/ml and 200 µg/ml decreased the content by 78% and 88%, respectively. A dose-dependent decrease in ergosterol content was observed in cells exposed to miconazole with a 25 µg/ml concentration causing a 100% decrease in ergosterol content. Therefore, tormentic acid inhibits the synthesis of ergosterol in C. albicans. Modifications of the structure of tormentic acid to increase its antifungal potency may be explored in further studies.

Antibiofilm Activity of Extract and a Compound Isolated from Triumfetta welwitschii against Pseudomonas aeruginosa.

Triumfetta welwitschii has been used as a traditional medicine in Africa. It is documented as a rich source of phytochemicals with antibacterial activities. To further explore the antibacterial potential of these phytochemical components, the phytochemical profile of the dichloromethane: methanol leaf extract from T. welwitschii was investigated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Compounds were isolated from the extract using column chromatography and thin-layer chromatography. Compound B1 was isolated from the fraction eluted by 90 hexane:10 ethyl acetate using column chromatography. The antibacterial activity of B1 against Pseudomonas aeruginosa was evaluated in vitro using the broth microdilution method and the iodonitrotetrazolium (INT) colorimetric assay. The antibiofilm activities of the extract and B1 against P. aeruginosa were determined by quantifying the biofilms using crystal violet. The effect of the extract and B1 on capsular polysaccharide and extracellular DNA content of biofilm formed by P. aeruginosa was determined using phenol-sulphuric acid and propidium iodide, respectively. A total of 28 peaks were detected and identified using UPLC-MS/MS. The three most abundant phytochemicals identified were catechin, umbelliferone, and a luteolin derivative. B1 showed antibacterial activity against P. aeruginosa with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of 25 µg/ml. Only 38% and 6% of the biofilms were formed in the presence of the extract and B1, respectively. The extract and B1 reduced the capsular polysaccharide content in biofilms formed in P. aeruginosa by 40% and 65%, respectively. The extract and B1 significantly reduced the extracellular DNA content of biofilms by 29% and 72%, respectively. The results of this study provide evidence of the antibacterial and antibiofilm activities of B1 and leaf extracts from T. welwitschii. Future work should identify the chemical structure of B1 using nuclear magnetic resonance and mass spectrometry.

The Effects of Combining Cancer Drugs with Compounds Isolated from Combretum zeyheri Sond. and Combretum platypetalum Welw. ex M.A. Lawson (Combretaceae) on the Viability of Jurkat T Cells and HL-60 Cells.

Combretum zeyheri and Combretum platypetalum have been shown to have anticancer, antibacterial, antituberculosis, and antifungal effects in both in vivo and in vitro studies. This study sought to evaluate the antiproliferative effects of compounds isolated from C. zeyheri and C. platypetalum on Jurkat T and HL-60 cancer cell lines in combination with doxorubicin and/or chlorambucil. At their GI50 concentrations, the isolated compounds were combined with the corresponding GI50 of chlorambucil and doxorubicin. The cytotoxic effects of the combined compounds were determined on BALB/c mouse peritoneal cells. All the 4 isolated compounds had significant cytotoxic effects on Jurkat T cells. Compounds CP 404 (1), CP 409 (2), CZ 453 (3), and CZ 455 (4) had GI50s on Jurkat T cells of 3.98, 19.33, 6.82, and 20.28 µg/ml, respectively. CP 404 (1), CP 409 (2), CZ 453 (3), and CZ 455 (4) showed GI50s of 14.18, 28.69, 29.87, and 16.46 µg/ml on HL-60 cancer cell lines, respectively. The most potent combination against Jurkat T cells was found to be CP 404 (1) and chlorambucil. This combination showed no cytotoxic effects when tested on BALB/c mouse peritoneal cells. It was concluded that the compounds extracted from C. zeyheri and C. platypetalum inhibit the growth of Jurkat T cells in vitro. The combination of the compounds with anticancer drugs enhanced their anticancer effects. The combination of CP 404 (1) and chlorambucil was found not to be toxic to normal mammalian cells. Therefore, CP 404 (1), 3-O-ß-L-rrhamnopyranosyl-5,7,3'4',5'-pentahydroxyflavone, has the potential to be a source of lead compounds that can be developed for anticancer therapy. Further structure-activity relationship studies on this compound are warranted.

Determination of the Cytotoxic Effect of Different Leaf Extracts from Parinari curatellifolia (Chrysobalanaceae).

Despite plants being a rich source of useful chemical compounds with different pharmacological properties, some of these compounds may be toxic to humans. Parinari curatellifolia, among its other important pharmacological activities, has been shown to have significant antiproliferative activity on cancer cell lines. Toxicity studies are required to determine the safety profile of P. curatellifolia in the consideration of its potential pharmaceutical benefits as a source of lead compounds in cancer therapy. The effects of P. curatellifolia on both the integrity of the erythrocyte membrane and on normal cells were determined. The dried leaf powder of P. curatellifolia was used in serial exhaustive extraction procedures using hexane, dichloromethane, ethyl acetate, acetone, ethanol, methanol, and water as solvents in addition to extraction using DCM: methanol in equal ratio. Alkaloids, flavonoids, and saponins were isolated from the ethanol extract. The leaf extracts were tested for haemolytic activity on sheep erythrocytes at concentrations of 0.625 to 5 mg/ml. The extracts were also tested for toxicity activity on normal mammalian cells such as the BALB/c mice peritoneal cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at the concentrations of 6.3 to 50 µg/ml. In the haemolysis assays, none of the plant extracts had a significant haemolytic activity with the saponin-enriched extract having the maximum haemolytic activity of 12.2% for a concentration of 5 mg/ml. In the MTT cell viability assay, none of the 11 plant extracts had significant cytotoxicity. The water extract, however, had significant (p < 0.01) proliferative activity towards the murine immune cells at all concentrations. P. curatellifolia leaf extracts were, therefore, not toxic to both erythrocytes and immune cells, and the water extract may have immunostimulatory effects. It is concluded that P. curatellifolia leaf extracts are not toxic in vitro and, therefore, our results support the use of the plant for ethnomedicinal use.

Phytochemical Fingerprinting and Activity of Extracts from the Leaves of Dolichos kilimandscharicus (Fabaceae) on Jurkat-T Cells.

Plants are a source of over a quarter of the prescription drugs currently in use worldwide. Zimbabwe has a rich plant biodiversity with only a limited number reported for the treatment of cancer. The leaf extracts of Dolichos kilimandscharicus were selected for the screening of their antiproliferative efficacy and cytotoxicity effects. This plant has increasingly been used by local folk as a treatment for cancer or cancer-related symptoms though its bioactivity has not been scientifically determined. This investigation also sought to identify constituent compounds in the crude extract preparations responsible for their antiproliferative efficacy. The antiproliferative effects of six-leaf extracts on Jurkat-T in vitro were investigated using the Trypan blue exclusion assay. The extracts were tested with increasing concentration, using chlorambucil as a standard anticancer drug. Cytotoxicity of extracts was determined against RAW 264.7 cells using a colorimetric tetrazolium-based assay. In additionthe ability of the extracts to induce apoptosis was determined for the most potent leaf extracts. The order of potency of the leaf extracts of D. kilimandscharicus against Jurkat-T cell line was found to be MeOH < Ethyl Acetate < DCM: MeOH < EtOH with IC50s of 33.56, 30.44, 22.93, and 21.59 µg/mL, respectively. Furthermore, the most potent extracts exhibited very low cytotoxicity against all the tested cells. D. kilimandscharicus leaf extracts induced apoptosis in the Jurkat-T cells as was shown by DNA fragmentation. UPLC-MS analysis of crude extracts led to the identification of 23 compounds from the ethanol extract and these may be responsible for the observed antiproliferative effects. Rutin, quercetin, luteolin, apigenin, hispidulin, kaempferol derivatives, as well as caffeoylquinic acid are some of the compounds identified in the extracts. The results of this study showed that the ethanol and ethyl acetate leaf extracts of D. kilimandscharicus have antiproliferative activity against Jurkat-T cells and may act by inducing apoptosis.. The current findings offer supporting evidence for the use of these plant species in the treatment of cancer in ethnomedicinal practices.

Antibacterial activities, proposed mode of action and cytotoxicity of leaf extracts from Triumfetta welwitschii against Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa has become a main cause of Gram-negative infection, particularly in patients with compromised immunity. High rates of resistance to antibiotics are associated with nosocomial infections caused by P. aeruginosa strains. The search for novel antimicrobials has been necessitated by the emergence of antimicrobial resistance in some bacteria Plant-based antimicrobials has great potential to combat microbial infections using a variety of mechanisms. Triumfetta welwitschii plant roots are traditionally used to treat symptoms of diarrhoea and fever, suggesting that it possess antimicrobial and immunomodulatory effects. Since research investigating antimicrobial properties of the roots of Triumfetta welwitschii has been explored, there is need to investigate the antimicrobial activity of its leaf extracts in order to probe their prospective use as new antimicrobial agents that can be used to combat nosocomial infections. The objective of this study was to evaluate the antibacterial activities, the mode of action and cytotoxicity of T. welwitschii leaf extracts. METHOD: Extracts of T. welwitschii leaves were obtained using eight different solvents, the serial exhaustive extraction method and the cold maceration technique. In vitro antibacterial activity evaluation of the extracts was done on eight bacterial isolates using the broth microdilution method. The mode of action for the most potent extracts was investigated using the rhodamine 6G efflux assay and the propidium iodide-based membrane damage assay. Toxicity of the extracts was evaluated using the haemolytic and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. RESULTS: The results showed that acetone, ethanol and dichlorometane: methanol extracts had the most potent antibacterial activities against Pseudomonas aeruginosa (ATCC 27853). All three extracts caused membrane disruption of P. aeruginosa as shown by nucleic acid leakage. All three extracts were unable to inhibit efflux pumps. CONCLUSION: The presence of antibacterial activities and low toxicity shown by the extracts indicates that the plant may be a source of effective antibacterial against some bacterial infections caused by P. aeruginosa. The disruption of membrane integrity is one possible mode of action of antibacterial activity of the potent extracts.

Evaluation of antimicrobial activity of chondrillasterol isolated from Vernonia adoensis (Asteraceae).

BACKGROUND: Bacteria have developed resistance to most of the current antibiotics. There is evidence suggesting that plant-derived compounds have a potential for interacting with biological processes. One of the plants commonly used in African ethnomedicine is Vernonia adoensis from the Asteraceae family. The leaves of the plant have been reported to have antimicrobial activity. Hence, the aim of this study was to isolate the bioactive compounds from the leaf extract and evaluate their antibacterial activity on Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, the effect of the isolated compound on biofilms of P. aeruginosa was determined. METHODS: Isolation of phytochemicals from the leaves of V. adoensis was done using column chromatography. Preparative TLC was used to further isolate mixed compounds in the fractions. Nuclear magnetic resonance spectroscopy and mass spectrometry was used to identify the isolated pure compounds. The broth microdilution assay was carried out to evaluate the antibacterial activity of the isolated compound on P. aeruginosa, S. aureus and K. pneumoniae. Crystal violet staining technique was used to evaluate the effect of the isolated compound on biofilms of P. aeruginosa. RESULTS: The compound isolated from V. adoensis was identified as chondrillasterol. Chondrillasterol exhibited 25, 38 and 65% inhibition of growth on S. aureus, K. pneumoniae and P. aeruginosa respectively. At 1.6 µg/mL chondrillasterol completely disrupted mature biofilm of P. aeruginosa while at 100 µg/mL the compound completely inhibited formation of biofilms of the bacteria. CONCLUSION: Chondrillasterol isolated from V. adoensis has antibacterial properties against S. aureus, K. pneumoniae and P. aeruginosa. The compound also has biofilm inhibition and disruption activity against P. aeruginosa biofilms. Thus, the active phytochemical could be a useful template for the development of new antimicrobial agents with both antibacterial and antibiofilm activity.

Inhibition of biofilm formation in Mycobacterium smegmatis by Parinari curatellifolia leaf extracts.

BACKGROUND: Tuberculosis (TB) is a serious public health problem worldwide. Mycobacterium tuberculosis (M. tuberculosis) grows as drug tolerant pellicles. Agents that inhibit biofilm formation in M. tuberculosis have the potential to reduce the disease treatment period and improve the quality of tuberculosis chemotherapy. Parinari curatellifolia (P. curatellifolia) leaf extracts are claimed to treat symptoms similar to tuberculosis in ethnomedicinal practices. Mycobacterium smegmatis (M. smegmatis) is a surrogate organism used in antimycobacterial drug discovery assays. In this study, the effect of the leaf extracts of P. curatellifolia on M. smegmatis growth and biofilm formation was investigated in order to determine the basis of its use in traditional medicinal use. METHODS: Phytochemicals from P. curatellifolia leaves were prepared using a mixture of 50% dichloromethane (DCM): 50% methanol and by serial exhaustive extraction using different solvents of decreasing polarity. The solvents were used in the following order, hexane > dichloromethane > ethyl acetate > acetone >ethanol > methanol > water. The micro-broth dilution method was used as an antimycobacterial susceptibility test to screen for the extract that effectively inhibited M. smegmatis growth and biofilm formation. Biofilm quantification was performed by staining the biofilms with crystal violet and determining the amount of the stain using a spectrophotometer. In addition, the effects of combining the most active extract with kanamycin were also investigated. RESULTS: The minimum inhibitory concentrations (MIC) of the extracts were found to be 6.2 µg/ml for the acetone extract, 12.5 µg/ml for both the ethanol and the total extract and 50 µg/ml for both the methanol and ethyl acetate extracts. The ethanol extract, dichloromethane extract and water extract were the only extracts that effectively inhibited biofilm formation in M. smegmatis. Combining the ethanol extract with kanamycin enhanced the effect of the ethanol extract in terms of inhibition of biofilm formation. CONCLUSIONS: P. curatellifolia leaves contain phytochemicals that have the potential to be used both as antimycobacterial and anti-biofilm formation compounds.

Alkaloid extracts from Combretum zeyheri inhibit the growth of Mycobacterium smegmatis.

BACKGROUND: Current tuberculosis regimens have failed to combat the issue of drug resistance and ethno medicines may represent a possible source of antimycobacterial agents. Combretum species are well known in African traditional medicines and used for various ailments including pneumonia, venereal diseases like syphilis, mental problems, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis. Alkaloids function as either hydrogen-acceptor or hydrogen-donor in hydrogen bonding critical for the interaction between targets thus, potentiating effects of curative agents on diseases. Alkaloid extracts from leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum, were assessed for antimycobacterial activity to establish rationale for their use in traditional medicines for various ailments including pneumonia, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis. METHODS: Alkaloids were extracted from the leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum. The broth microdilution method was used for the screening of growth inhibitory activity. The standard drug rifampicin was used as the positive control. Alkaloid extracts from the most potent plant species, Combretum zeyheri were further investigated for time-kill dependency effects on drug transport in Mycobacterium smegmatis. RESULTS: Using the broth microdilution susceptibility method, C. zeyheri alkaloid extract, was found to have the most antimycobacterial effects with an MIC value of 125 µg/ml whilst MICs for C. molle and C. platypetalum were above 1000 µg/ml. An MBC value of 250 µg/ml was observed with alkaloid extracts from Combretum zeyheri whilst the remaining three Combretum species showed no bactericidal activity. It was also shown that C. zeyheri had potential efflux pump inhibitory activity. Determination of the time-kill kinetics of extracts from C. zeyheri showed not only a concentration-dependent activity but time-dependent bactericidal effect as well. CONCLUSIONS: Alkaloid extracts from the leaves of C. zeyheri have potential as a source of lead compounds that may be developed further into antimycobacterial compounds. The mechanism of action of may be due to inhibition of transport across the cell membrane. Further work needs to be done to isolate the active components in these extracts.

Multiple cellular effects of leaf extracts from Parinari curatellifolia.

BACKGROUND: Parinari curatellifolia is a prominent plant in folk medicine in Sub-Saharan Africa. The plant decoctions are used to treat various ailments, including the treatment of cancer, pneumonia, fever, microbial infections and anti-inflammation. The aims of the study were to investigate the effects of P. curatellifolia leaf extracts on cell inflammatory and proliferative activity. METHODS: Parinari curatellifolia fresh leaves were collected from Centenary in Mashonaland Central Province of Zimbabwe. Plant extracts were prepared using methanol, water, acetone and ethanol. Firstly, the effects of the extracts were determined on xanthine oxidase activity. Kinetic constants were determined for the extracts that showed inhibitory effects. Then the effects of Parinari curatellifolia water extract on LPS, menadione and hydrogen peroxide-activated nitric oxide production in RAW 264.7 cells was determined by quantifying the amount of nitrites formed. Finally, the effects of P. curatellifolia on the proliferation of Jurkat-T cells as well as its modulation of cisplatin-induced cell- cytotoxicity was investigated on a Jurkat human T-cell lymphoma cell line. RESULTS: There was significant XO inhibitory activity by the ethanol and methanol extracts at 15.6 µg/ml and 3.9 µg/ml respectively. The IC50 determination for allopurinol, ethanol extract and methanol extract were 0.43 µg/ml, 1.38 µg/ml and 2.19 µg/ml respectively. The kinetic results showed that the ethanol and methanol extracts were allosteric inhibitors of XO. The water extract of P. curatellifolia inhibited NO production in RAW cells when LPS was used as an activator. P. curatellifolia and cisplatin showed dose-dependent cytotoxicity on Jurkat-T cells. Isolated DNA from the cells showed that there was DNA cleavage on cells exposed to P. curatellifolia indicating that apoptosis may be a mechanism by which P. curatellifolia exerts its cytotoxicity on Jurkat-T cells. CONCLUSIONS: These results scientifically support the use of P. curatellifolia leaf extracts in the management of pain, inflammatory and neoplastic conditions. P. curatellifolia thus has multiple biological effects, thus, validating its use in traditional medical uses.

In vivo and in vitro inhibition of rat liver glutathione transferases activity by extracts from Combretum zeyheri (Combretaceae) and Parinari curatellifolia (Chrysobalanaceae).

BACKGROUND: Parinari curatellifolia and Combretum zeyheri are medicinal plants used in Zimbabwe and other Southern African countries for stomach ailments, fever, body aches, wound healing, cancer and tuberculosis. Glutathione transferases (GSTs) are mammalian enzymes that play a significant role in the detoxification and metabolism of many xenobiotic and endogenous compounds and as such can interact with many exogenous compounds including herbal medicines. The effects of Parinari curatellifolia and Combretum zeyheri leaf extracts on glutathione transferases of male Sprague-Dawley rats were investigated in vivo and in vitro after oral administration of either leaf ethanol or water extracts of each plant. METHODS: For Parinari curatellifolia, 18 male Sprague-Dawley rats were administered with 0, 500 and 1000 mg/kg body weight of the leaf extracts in corn oil or saline. Animals were sacrificed after 96 h and the kidney and liver samples were removed and used to prepare the cytosolic fractions. GST activity was determined using 1-chloro-2, 4-dinitrobezene. For Combretum zeyheri, twenty four male Sprague-Dawley rats were randomly divided into two groups. These two groups were further divided into three groups of four animals each. They were given either the aqueous or ethanol extract at doses of C. zeyheri at 0, 50 mg/kg body weight and 200 mg/kg body weight. The extracts were administered orally by oral gavage for four consecutive days and the rats were sacrificed by cervical dislocation on the fifth day. RESULTS: In animals administered with C. zeyheri, GST activity was significantly increased by the 200 mg/kg aqueous extract in the kidneys and livers in vivo whilst the ethanolic extract at 200 mg/kg decreased enzyme activity significantly both organs. Both the ethanol and aqueous extracts inhibited GST activity in vitro with the ethanol extract being more potent inhibitor than ethacrynic acid, a standard GST inhibitor. The increased GST activity in vivo and versus inhibition in vitro suggests that metabolites may be responsible for the effects observed in vivo. For P. curatellifolia, a dose-dependent decrease in GST activity was observed in vivo for the animals given the aqueous extract but no changes were observed with the ethanol extract. There was a concentration-dependent inhibition of cytosolic GSTs when P. curatellifolia leaf extracts in vitro. The ethanol extract of P. curatellifolia exhibited GST-inhibitory activity in the liver with an IC50 value of 12 µg/mL and for ethacrynic acid, the IC50 was found to be 10 µg/mL. This showed that this extract was a potent inhibitor of GSTs in vitro. CONCLUSIONS: C. zeyheri had an inductive effect on GST activity when administered in aqueous solution but inhibited GST in vitro whilst P. curatellifolia inhibited GST activity in vivo. Induction of GSTs would be cytoprotective against the toxic effects electrophilic chemicals. Since GSTs are responsible for the synthesis of prostaglandins, the inhibition of GST activity of by these two plants in vivo maybe one of the reasons that makes the plants important for use in the treatment pain and fever in ethnopharmacology.

In vitro toxicity determination of antifungal constituents from Combretum zeyheri.

BACKGROUND: Candida albicans is one of the organisms living on the human body symbiotically, but, in hosts with low immunity it becomes one of the most pathogenic fungal organisms. Combretum zeyheri has been reported to have antifungal, antibacterial and antioxidant activities. Medicinal plants are believed to be non-toxic by the general public. Toxicity studies, however, have indicated that they are capable of causing numerous side effects, therefore, evaluation of safety is required. The objective of this study was to determine the toxicity of the antifungal constituents of Combretum zeyheri on mammalian cells. METHODS: Alkaloids, saponins, flavonoids-enriched extracts and crude ethanol extracts were prepared from the leaves of Combretum zeyheri. The broth microdilution method was used to investigate for antifungal activity, with miconazole used as the positive control. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell viability of the Candida albicans cells. The most potent extracts; the ethanol extract, alkaloids and saponins respectively, were further tested for their toxicity on sheep erythrocytes, mouse peritoneal macrophages and Jurkat T cells. RESULTS: All Combretum zeyheri extracts displayed a dose-dependent antifungal activity and had IC50 values ranging from 16 µg/ml to 159 µg/ml for Candida albicans. The alkaloids, saponins and ethanol extracts were found to be non-toxic towards mouse peritoneal cells and Jurkat T cells. In the haemolysis assay, all extracts were haemolytic at varying degrees and showed their greatest haemolytic activity at the highest concentration of 5 mg/ml. The saponins were the least haemolytic, followed by the ethanol extracts and the alkaloids respectively. Although these extracts were haemolytic to some extent, they may considered safe at therapeutic concentrations since there was a large difference between the antifungal IC50 and haemolysis EC50 values, hence a large therapeutic window. CONCLUSIONS: Combretum zeyheri antifungal constituents are, therefore, a potential source of lead compounds which can be developed into antifungal drugs of natural origin owing to Combretum zeyheri's effective antifungal activity and low toxicity to mammalian cells.

Antiproliferative Activity of T. welwitschii Extract on Jurkat T Cells In Vitro.

Triumfetta welwitschii is a plant used traditionally for the treatment of fever and diarrhoea. Previous work has shown that T. welwitschii has antibacterial activity. The purpose of this study was to investigate T. welwitschii extract for anticancer activity against Jurkat T cells. The Jurkat T cell line is used to study acute T cell leukaemia. An antiproliferation assay, determination of induction of apoptosis, the determination of the effect of the combination of the extract and GSH, and effects of the extract on DNA leakage were conducted. T. welwitschii was found to decrease cell viability in a dose- and time-dependent manner. T. welwitschii caused apoptosis in the Jurkat T cells as shown by DNA fragmentation. When T. welwitschii was combined with reduced GSH, it was found that the growth of the Jurkat T cells was significantly reduced compared to untreated cells after 72 h of treatment. This was unexpected, as cancer cells have elevated levels of GSH compared to normal cells. The results of this study show that T. welwitschii is a potential source of compounds that may serve as leads for anticancer compounds.

Isolation and characterization of an antifungal compound 5-hydroxy-7,4'-dimethoxyflavone from Combretum zeyheri.

BACKGROUND: Combretum zeyheri, belongs to the family Combretaceae and is one of the most popular herbal plants in tropical and subtropical countries. The leaves of Combretum zeyheri have been used as herbal medicine and have been reported to have pharmacological activity which includes anti-bacterial, anti-fungal, anticancer and antioxidant properties. The goal of this study was to isolate, identify and characterize compounds from C. zeyheri leaves which are responsible for its antifungal activity. METHODS: The preliminary isolation of C. zeyheri active compounds was carried out using chromatographic techniques which include sephadex gel column chromatography, silica gel column chromatography and thin-layer chromatography (TLC). The isolated compounds were then investigated for their antifungal activity using broth dilution assay. The combined effect of the most potent compound and an antifungal drug miconazole was investigated using the checkerboard assay. Time-kill assays were conducted for the combinations using the colony counting method. The mechanism of action of 5-hydroxy-7,4'-dimethoxyflavone as a potent antifungal agent was investigated by determining its inhibitory activity on Candida albicans drug efflux pumps using the ciprofloxacin assay. The ability of 5-hydroxy-7,4'-dimethoxyflavone to inhibit antioxidant enzymes as well as the biosynthesis of ergosterol were also investigated. RESULTS: A total of four pure compounds (A-D) were isolated from C. zeyheri leaf extract. Compound B (5-hydroxy-7,4'-dimethoxyflavone) was found to be active against Candida albicans using broth dilution method. This compound was also found to have synergistic activity on growth of C. albicans when combined with miconazole, completely inhibiting growth after only 4 hrs of incubation. Analysis of ergosterol content from Candida albicans showed a time-dependent decrease to 91 % and 63 % at 16 and 24 hrs respectively, in cells treated with ½ MIC of 5-hydroxy-7,4'-dimethoxyflavone. The compound 5-hydroxy-7,4'-dimethoxyflavone also showed inhibition of both the drug efflux pumps (with IC50 = 51.64 µg/ml) and the antioxidant enzymes (at 5 µM). CONCLUSION: The compound 5-hydroxy-7,4'-dimethoxyflavone may be partly responsible for the reported antifungal activity of C. zeyheri, and may serve as a potential source of lead compounds that can be developed as antifungal phytomedicines.

Evaluation of cell membrane integrity as a potential antimicrobial target for plant products.

BACKGROUND: There is urgent need to discover new antimicrobial compounds with diverse chemical structures and mechanisms of action due to increasing new and re-emerging infectious diseases. Additionally, appearance of undesirable side effects of certain antibiotics and increasing resistance to antibiotics in current clinical use is also a cause for concern. Bacterial cell membranes are a possible target for developing new antibacterial drugs since membrane-based efflux pump systems play an important role in bacterial pathogenicity and antimicrobial resistance in bacteria. Hence, the objective of our study was to evaluate bacterial membrane integrity of two species of bacteria; Staphylococcus aureus and Pseudomonas aeruginosa, in the presence of ethanolic leaf extracts of two plant species Callistemon citrinus and Vernonia adoensis from Zimbabwe. METHODS: Bacterial efflux pump inhibition using both leaf extracts was determined by monitoring the transport of Rhodamine 6 G (R6G) across the cell membrane and IC50 values were obtained. Membrane permeabilizing properties of both extracts were also evaluated using the membrane potential sensitive dye 3'3 dipropylthiadicarbocyanine (diSC3-5). Haemolysis effect of both extracts on sheep erythrocytes was also investigated. RESULTS: Both extracts inhibited bacterial efflux pumps which resulted in the accumulation of R6G inside the cell. The IC50 values for C. citrinus and V. adoensis against S. aureus were 1.44 mg/ml and 1.61 mg/ml, respectively. Both leaf extracts however, showed similar IC50 values of 1.64 mg/ml against P. aeruginosa. Both plant extracts showed some significant effects on permeability of the bacterial membrane when a 24-28% increase of diSC3-5 dye release was observed for S. aureus and 45-53% of dye was released from P. aeruginosa cell membrane after a 60 minute incubation period. In addition, both extracts exhibited haemolytic effects on sheep erythrocytes at concentrations greater than 2.5 mg/ml. CONCLUSIONS: These plant extracts may provide new lead compounds for developing potential efflux pump inhibitors (EPIs) or permeabilising agents that could aid the transport of antibacterial agents into bacterial cells.

Inhibition of hematopoietic prostaglandin D2 synthase (H-PGDS) by an alkaloid extract from Combretum molle.

BACKGROUND: Hematopoietic prostaglandin D2 synthase (H-PGDS, GST Sigma) is a member of the glutathione S-transferase super family of enzymes that catalyses the conjugation of electrophilic substances with reduced glutathione. The enzyme catalyses the conversion of PGH2 to PGD2 which mediates inflammatory responses. The inhibition of H-PGDS is of importance in alleviating damage to tissues due to unwarranted synthesis of PGD2. Combretum molle has been used in African ethno medicinal practices and has been shown to reduce fever and pain. The effect of C. molle alkaloid extract on H-PGDS was thus, investigated. METHODS: H-PGDS was expressed in Escherichia coli XL1-Blue cells and purified using nickel immobilized metal affinity chromatography. The effect of C. molle alkaloid extract on H-PGDS activity was determined with 1-chloro-2, 4-dinitrobenzene (CDNB) as substrate. The effect of C. molle alkaloid extract with time on H-PGDS was determined. The mechanism of inhibition was then investigated using CDNB and glutathione (GSH) as substrates. RESULTS: A specific activity of 24 µmol/mg/min was obtained after H-PGDS had been purified. The alkaloid extract exhibited a 70% inhibition on H-PGDS with an IC50 of 13.7 µg/ml. C. molle alkaloid extract showed an uncompetitive inhibition of H-PGDS with Ki = 41 µg/ml towards GSH, and non-competitive inhibition towards CDNB with Ki = 7.7 µg/ml and Ki' = 9.2 µg/ml. CONCLUSION: The data shows that C. molle alkaloid extract is a potent inhibitor of H-PGDS. This study thus supports the traditional use of the plant for inflammation.

Antiproliferative activity of the isofuranonaphthoquinone isolated from Bulbine frutescens against Jurkat T cells.

Cancer is a major public health burden in both developed and developing countries. The quinone moiety has been shown to possess antitumor activity and several cancer drugs in clinical use contain this entity. The effect of isofuranonaphthoquinone isolated from Bulbine frutescens on Jurkat T cells was determined. Cells were exposed to the isofuranonaphthoquinone (IFNQ) at different concentrations. Significant antiproliferative effects were observed which were comparable to that of the anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). A combination of IFNQ with BCNU produced synergistic effects which were observed after 72 hrs. It was also observed that combining IFNQ with reduced glutathione abolished the anticancer activity of the compound. It is, therefore, proposed that the isofuranonaphthoquinone may exert part of its effect by producing reactive oxygen species resulting in death of the cells as the effects of this compound were antagonized by reduced glutathione. An investigation on the effects of isofuranonaphthoquinone on glutathione transferase (GST) activity and drug efflux pumps showed that this compound exhibited inhibitory effects on both the GST and the drug efflux pumping activities. Thus, the isofuranonaphthoquinone showed cytotoxicity, works through inhibition of some cellular mechanisms, and could present a potential source of lead compounds for anticancer drug development.

The evaluation of novel natural products as inhibitors of human glutathione transferase P1-1.

Glutathione transferase P1-1 is over expressed in some cancer cells and contributes to detoxification of anticancer drugs, leading to drug-resistant tumors. The inhibition of human recombinant GSTP1-1 by natural plant products was investigated using 10 compounds isolated from plants indigenous to Southern and Central Africa. Monochlorobimane and 1-chloro-2,4-dinitrobenzene were used to determine GST activity. Each test compound was screened at 33 and 100 µM. Isofuranonapthoquinone (1) (from Bulbine frutescens) showed 68% inhibition at 33 µM, and sesquiterpene lactone (2) (from Dicoma anomala) showed 75% inhibition at 33 µM. The IC(50) value of 1 was 6.8 µM. The mode of inhibition was mixed, partial (G site) and noncompetitive (H site) with K(i) values of 8.8 and 0.21 µM, respectively. Sesquiterpene 2 did not inhibit the CDNB reaction. Therefore, isofuranonapthoquinone 1 needs further investigations in vivo because of its potent inhibition of GSTP1-1 in vitro.