Phytochemical analysis and antibacterial evaluation of the ethyl acetate extract of the stem bark of Bridelia micrantha.

BACKGROUND: Plant cells fundamentally are chemical factories containing a rich supply of therapeutically useful phytocompounds that have the potential of being developed into potent antimicrobial agents. AIM OF THE STUDY: To investigate the antibacterial activity of fractionated extracts of the ethyl acetate extract of the stem bark of Bridelia micrantha (Hochst., Baill., Euphorbiaceae). MATERIALS AND METHODS: Thin-layer chromatography and column chromatography were used to purify the extracts and antimicrobial activity performed on reference and clinical strains of Staphylococcus aureus, Shigella sonnei, Salmonella Typhimurium, and Helicobacter pylori using direct and indirect bioautographic methods respectively. Furthermore, the eluted compound fractions were then assayed for minimum inhibitory concentration (MIC50) using the 96-well micro dilution technique. RESULTS: Better separation of phytocompounds was obtained from the non-polar Benzene/Ethanol/Ammonia (BEA) and intermediate-polar Chloroform/Ethyl acetate/Formic acid (CEF) eluents compared to the polar Ethanol/Methanol/Water (EMW). Bioautography revealed the presence of three bioactive compounds (Rf values; 0.12, 0.20, and 0.42) on the BEA plates, designated fractions 3, 7, and 8 with MIC50 values; 0.0048mg/mL to 1.25mg/mL (fraction 3), 0.0024mg/mL to 5 mg/mL (fraction 7), and 0.0024mg/mL to 2.5mg/mL (fraction 8). CONCLUSION: Our findings demonstrate that ethyl acetate extract of the stem-bark of B. micrantha possess potent bioactive phytocompounds that may be developed into new antimicrobials.

Evaluation of the antibacterial and antifungal potential of Peltophorum africanum: toxicological effect on human Chang liver cell line.

We assessed the in vitro antimicrobial activity of Peltophorum africanum by means of the agar well and macrodilution methods. The toxicity on a normal human liver cell (Chang liver cell) was determined using the CellTiter-Blue cell viability assay, and the compounds contained in the fractions were identified using GC-MS. Zone diameter of inhibition of the extract ranged from 12.5 ± 0.7 to 32 ± 2.8 mm for bacteria and from 7.5 ± 0.7 to 26.4 ± 3.4 mm for yeast. Marked activity of the extract was observed against Plesiomonas shigelloides ATCC 51903, with MIC and MLC values of 0.15625 and 0.3125 mg/mL, respectively. The extract was both bactericidal (MIC(index) ≤ 2) and bacteriostatic/fungistatic (MIC(index) > 2) in activity. Lethal dose at 50 (LD50) showed 82.64 ± 1.40 degree of toxicity at 24 hrs, and 95 percentile of cell death dose activity ranged from log 3.12 ± 0.01 to 4.59 ± 0.03. The activity of the eight fractions tested ranged from 1.0 ± 0.5 to 3.7 ± 1.6 mg/mL (IC50) and from 2.1 ± 0.8 to 6.25 ± 0 mg/mL (IC90). The extract was toxic to human Chang liver cell lines.

Identification and antibacterial evaluation of bioactive compounds from Garcinia kola (Heckel) seeds.

We assessed the bioactivity of G. kola seeds on Streptococcus pyogenes, Staphylococcus aureus, Plesiomonas shigelloides and Salmonella typhimurium. The crude ethyl acetate, ethanol, methanol, acetone and aqueous extracts were screened by the agar-well diffusion method and their activities were further determined by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays. The extracts were fractionated by Thin Layer Chromatography (TLC). Bioautography was used to assess the activity of the possible classes of compounds present in the more active extracts. Column chromatography was used to purify the active compounds from the mixture, while GC-MS was used to identify the phytocomponents of the fractions. The inhibition zone diameters of the extracts ranged from 0-24 ± 1.1 mm, while MIC and MBC values ranged between 0.04-1.25 mg/mL and 0.081-2.5 mg/mL, respectively. The chloroform/ethyl acetate/formic acid (CEF) solvent system separated more active compounds. The MIC of the fractions ranged between 0.0006-2.5 mg/mL. CEF 3 (F3), CEF 11 (F11) and CEF 12 (F12) revealed the presence of high levels of linoleic acid, 1,2-benzenedicarboxylic acid and 2,3-dihydro-3,5-dihydroxy-6-methyl ester, respectively. The results obtained from this study justify the use of this plant in traditional medicine and provide leads which could be further exploited for the development of new and potent antimicrobials.

Evaluation of the Acetone and Aqueous Extracts of Mature Stem Bark of Sclerocarya birrea for Antioxidant and Antimicrobial Properties.

We assayed the antimicrobial activity of acetone and aqueous extracts of the stem bark of Sclerocarya birrea on some selected bacteria and fungi species including; Streptococcus pyogenes, Plesiomonas shigelloides, Aeromonas hydrophila, Salmonella typhimurium, Cryptococcus neoformans, Candida glabrata, Trichosporon mucoides, and Candida krusei using both agar well diffusion and minimum inhibitory concentration (MIC) assays. Based on the levels of activity, the acetone extract was examined for total polyphenolic content, radical scavenging and antioxidant activities. Total phenols of the extract were determined spectrophotometrically. The antioxidant activity was determined by the DPPH, ABTS and reducing power. All the bacteria and fungi species were susceptible to the plant extracts. The acetone extract was the most active for the bacterial species with MIC (0.156-0.625 mg/mL) while the aqueous extract was the most active for the fungi species with MIC (0.3125-1.25 mg/mL). The polyphenolic compounds were found as 27.2 mg/g tannic acid equivalent, 25.2 mg/g quercetin equivalent, 9.1 mg/g quercetin equivalent for phenols, flavonoid and flavonols respectively. The acetone extract exhibited a remarkable ability to scavenge radicals, strong reducing ability and a potential source of natural antioxidants. Both the acetone and aqueous extracts of S. birrea may provide a target for drug discovery.

Antifungal activity of Arctotis arctotoides (L.f.) O. Hoffm. and Gasteria bicolor Haw. against opportunistic fungi associated with human immunodeficiency virus/acquired immunodeficiency syndrome.

BACKGROUND: In South Africa where many patients are immunocompromised as a result of the AIDS pandemic, opportunistic fungal infections such as candidiasis caused mainly by Candida albicans are common. Arctotis arctotoides and Gasteria bicolor are two plants which are frequently and commonly used in traditional medicine in the treatment of HIV patients. AIM: The aim of this study was to investigate the antifungal activity of A. arctotoides and G. bicolor against opportunistic fungi common in HIV/AIDS patients. MATERIALS AND METHODS: The agar diffusion and micro-dilution methods were used to determine the antifungal activities of the medicinal plant extracts against 10 opportunistic fungi. RESULTS: All the hexane and acetone extracts were active against at least one of the fungi with zones of inhibition varying from 8 to 32 mm, while none of the aqueous extracts was active against any of the fungi. The inhibitory activity of the active extracts, based on the overall mean inhibition diameters, was in the order: A. arctotoides (hexane) > A. arctotoides (acetone) > G. bicolor (hexane) > G. bicolor (acetone). The most susceptible fungi, based on the overall mean diameter of growth inhibition, were Candida glabrata, C. krusei, and Microsporum canis, while Cyptococcus neoformans, Trycophyton tonsurans, and Microsporum gypseum were not susceptible to any of the extracts even at 5 mg/ml which was the highest concentration used. CONCLUSION: This study validates the use of these plants in traditional medicine in the treatment of secondary fungal infections in HIV/AIDS patients.

Inhibitory and bactericidal potential of crude acetone extracts of Combretum molle (Combretaceae) on drug-resistant strains of Helicobacter pylori.

Infection with Helicobacter pylori is strongly associated with a number of gastroduodenal pathologies. Antimicrobial resistance to commonly-used drugs has generated a considerable interest in the search for novel therapeutic compounds from medicinal plants. As an ongoing effort of this search, the susceptibility of 32 clinical strains of H. pylori and a reference strain-NCTC 11,638-was evaluated against five solvent extracts of Combretum molle, a plant widely used for the treatment of gastric ulcers and other stomach-related morbidities in South Africa. The extracts were screened for activity by the agar-well diffusion method, and the most active one of them was tested against the same strains by micro-broth dilution and time kill assays. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. The solvent extracts all demonstrated anti-H. pylori activity with zone diameters of inhibition between 0 and 38 mm. The most potent anti-H. pylori activity was demonstrated by the acetone extract, to which 87.5% of the clinical strains were susceptible. The minimum inhibitory concentration (MIC90) values for this extract ranged from 1.25 to 5.0 mg/mL while those for amoxicillin and metronidazole ranged from 0.001 to 0.94 mg/mL and from 0.004 to 5.0 mg/mL respectively. The acetone extract was highly bactericidal at a concentration of 2.5 and 5.0 mg/mL, with complete elimination of the test organisms in 24 hours. Its inhibitory activity was better than that of metronidazole (p<0.05) as opposed to amoxicillin (p<0.05). The results demonstrate that C. molle may contain therapeutically-useful compounds against H. pylori, which are mostly concentrated in the acetone extract.

Aqueous and organic solvent-extracts of selected south African medicinal plants possess antimicrobial activity against drug-resistant strains of Helicobacter pylori: inhibitory and bactericidal potential.

The aim of this study was to identify sources of cheap starting materials for the synthesis of new drugs against Helicobacter pylori. Solvent-extracts of selected medicinal plants; Combretum molle, Sclerocarya birrea, Garcinia kola, Alepidea amatymbica and a single Strychnos species were investigated against 30 clinical strains of H. pylori alongside a reference control strain (NCTC 11638) using standard microbiological techniques. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. All the plants demonstrated anti-H. pylori activity with zone diameters of inhibition between 0 and 38 mm and 50% minimum inhibitory concentration (MIC(50)) values ranging from 0.06 to 5.0 mg/mL. MIC(50) values for amoxicillin and metronidazole ranged from 0.001 to 0.63 mg/mL and 0.004 to 5.0 mg/mL respectively. The acetone extracts of C. molle and S. birrea exhibited a remarkable bactericidal activity against H. pylori killing more than 50% of the strains within 18 h at 4× MIC and complete elimination of the organisms within 24 h. Their antimicrobial activity was comparable to the control antibiotics. However, the activity of the ethanol extract of G. kola was lower than amoxicillin (P < 0.05) as opposed to metronidazole (P > 0.05). These results demonstrate that S. birrea, C. molle and G. kola may represent good sources of compounds with anti-H. pylori activity.

Preliminary phytochemical screening and in vitro anti-Helicobacter pylori activity of acetone and aqueous extracts of the stem bark of Sclerocarya birrea (Anacardiaceae).

Helicobacter pylori antibiotic resistance and other problems associated with combination therapy have generated a considerable interest in the search for alternative therapeutic agents. In order to identify novel sources of such agents, the antimicrobial activity of five solvent extracts of the stem bark of Sclerocarya birrea was investigated against 30 clinical strains of H. pylori and a reference strain NCTC 11638 using standard microbiological techniques. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. The active phytocomponents were detected by TLC and indirect bioautography. All the extracts exhibited anti-H. pylori activity with zone diameters of inhibition between 0 and 21 mm. The acetone and aqueous extracts showed potent anti-H. pylori activity with minimum inhibitory concentration (MIC(90)) values ranging from 0.06-2.50 mg/mL, whereas those for the control antibiotics ranged from 0.001-5.0 mg/mL. The acetone extract was highly bactericidal at 1.2 mg/mL with complete elimination of the organisms within 18 h. The activity of both acetone and aqueous extracts was better than metronidazole (p<0.05). Most of the active phytocomponents were located in the acetone extract; R(f)≤0.62 with >90% inhibition. These results demonstrate that the acetone and aqueous extracts of S. birrea may contain compounds with therapeutic activity; therefore, they may represent potential sources of new anti-H. pylori regimen.

Preliminary phytochemical screening and in vitro anti-helicobacter pylori activity of extracts of the stem bark of Bridelia micrantha (Hochst., Baill., Euphorbiaceae).

Helicobacter pylori is a major risk factor for gastritis, ulcers and gastric cancer. This study was aimed to determine the antimicrobial activity of the stem bark of Bridelia. micrantha on H. pylori isolated in South Africa. Extracts and clarithromycin were tested against 31 clinical strains, including a standard strain (NCTC 11638) of H. pylori, by measuring the diameters of the corresponding inhibition zones, followed by determination of the Minimum Inhibitory Concentration (MIC) (using metronidazole, and amoxicillin as control antibiotics) and the rate of kill. Preliminary phytochemical screening was also done. Inhibition zone diameters which ranged from 0-23 mm were observed for all five of the extracts and 0-35 mm for clarithromycin. Marked susceptibility of strains (100%) was noted for the acetone extract (P < 0.05), followed by ethyl acetate extract (93.5%). The MIC50 values ranged from 0.0048 to 0.156 mg/mL for the ethyl acetate extract and 0.0048 to 0.313 mg/mL for the acetone extract. The MIC90 values ranged from 0.0048 to 2.5 mg/mL for the ethyl acetate extract and 0.078 to > 0.625 mg/mL for the acetone extract, respectively. Insignificant statistical difference in potency was observed when comparing the crude ethyl acetate extract to metronidazole and amoxicillin (P > 0.05). Complete killing of strain PE430C by the ethyl acetate extract was observed at 0.1 mg/mL (2 × MIC) and 0.2 mg/mL (4 × MIC) at 66 and 72 h. For strain PE369C, 100% killing was observed at 0.1 mg/mL (2 × MIC) in 66 and 72 h. The ethyl acetate extract could thus be a potential source of lead molecules for the design of new anti-Helicobacter pylori therapies as this study further confirmed the presence of phytochemicals including alkaloids, flavonoids, steroids, tannins and saponins.

Characterization of n-Hexane sub-fraction of Bridelia micrantha (Berth) and its antimycobacterium activity.

BACKGROUND: Tuberculosis, caused by Mycobacterium tuberculosis (MTB), is the most notified disease in the world. Development of resistance to first line drugs by MTB is a public health concern. As a result, there is the search for new and novel sources of antimycobacterial drugs for example from medicinal plants. In this study we determined the in vitro antimycobacterial activity of n-Hexane sub-fraction from Bridelia micrantha (Berth) against MTB H37Ra and a clinical isolate resistant to all five first-line antituberculosis drugs. METHODS: The antimycobacterial activity of the n-Hexane sub-fraction of ethyl acetate fractions from acetone extracts of B. micrantha barks was evaluated using the resazurin microplate assay against two MTB isolates. Bioassay-guided fractionation of the ethyl acetate fraction was performed using 100% n-Hexane and Chloroform/Methanol (99:1) as solvents in order of increasing polarity by column chromatography and Resazurin microtiter plate assay for susceptibility tests. RESULTS: The n-Hexane fraction showed 20% inhibition of MTB H37Ra and almost 35% inhibition of an MTB isolate resistant to all first-line drugs at 10 µg/mL. GC/MS analysis of the fraction resulted in the identification of twenty-four constituents representing 60.5% of the fraction. Some of the 24 compounds detected included Benzene, 1.3-bis (3-phenoxyphenoxy (13.51%), 2-pinen-4-one (10.03%), N(b)-benzyl-14-(carboxymethyl) (6.35%) and the least detected compound was linalool (0.2%). CONCLUSIONS: The results show that the n-Hexane fraction of B. micrantha has antimycobacterial activity.

Crude ethanolic extracts of Garcinia kola seeds Heckel (Guttiferae) prolong the lag phase of Helicobacter pylori: inhibitory and bactericidal potential.

Problems associated with current treatment regimens have generated a considerable interest in alternative approaches for the eradication of Helicobacter pylori infections using phytochemical compounds. In an attempt to identify potential sources of such compounds, the antimicrobial activity of five solvent extracts of Garcinia kola seeds were investigated against 30 clinical strains of H. pylori and a standard control strain, NCTC 11638, using standard microbiological techniques. Metronidazole and amoxicillin were included in these experiments as positive control antibiotics. All the extracts tested exhibited anti-H. pylori activity with zone diameters of inhibition between 0 and 25 mm. The ethanol extract demonstrated considerable anti-H. pylori activity with a percentage susceptibility of 53.3% and minimum inhibitory concentration for 50% susceptibility (MIC50) values ranging from 0.63 to 5.0 mg/mL. Ranges of MIC50 values for amoxicillin and metronidazole were 0.01-0.63 mg/mL and 0.04-5.0 mg/mL, respectively. The inhibitory activity of the ethanol extract was similar to that of metronidazole (P > .05) as opposed to amoxicillin (P < .05). The extract caused a 12-hour extension of the lag phase of H. pylori at 1.25 mg/mL. The same observations were recorded when this concentration was doubled and quadrupled alongside a killing rate of 80.1% and 93.7%, respectively, after 24 hours and of 100% after 30 hours. These results demonstrate that the ethanol extract of G. kola may contain therapeutically useful compounds against H. pylori.

The Effect of the acetone extract of Arctotis arctotoides (Asteraceae) on the growth and ultrastructure of some opportunistic fungi associated with HIV/AIDS.

In this study, the effect of the acetone extract of Arctotis arctotoides (L.f.) O. Hoffm. (Asteraceae) on the growth and ultrastructure of some opportunistic fungi associated with HIV/AIDS was analyzed by means of scanning electron microscope (SEM). Remarkable morphological alterations in the fungal mycelia which were attributed to the loss of cell wall strength ranged from loss of turgidity and uniformity, collapse of entire hyphae to evident destruction of the hyphae. The elements responsible for giving the fungi their characteristic virulence were detected and quantified by energy dispersive X-ray microanalysis techniques. X-ray microanalysis showed the specific spectra of sodium, potassium and sulfur as the principal intersection of the four pathogenic fungi studied. Since these ions have the potential of fostering fungal invasion by altering the permeability of hosts' membranes, their presence was considered inherent to the pathogenicity of the opportunistic fungi. Hence, these findings indicate the potential of the crude extract of A. arctotoides in preventing fungal invasion and subsequent infection of host's membranes.

Inhibitory properties of selected South African medicinal plants against Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB) is the most commonly notified disease and the fifth largest cause of mortality. One in 10 cases is resistant to treatment in some areas. Several plants are used locally to treat TB-related disease. AIMS OF THE STUDY: The aim was to screen selected South African medicinal plants used to treat TB and related symptoms by traditional healers for antimycobacterial activity. MATERIALS AND METHODS: Ethnobotanical information on these plants was obtained. Crude acetone, methanol, hexane and ethanol extracts of 21 selected medicinal plants obtained in Venda, South Africa were screened for their ability to inhibit MTB H(37)Ra and a clinical strain resistant to first-line drugs and one second-line drug using tetrazolium microplate assay to determine the minimum inhibitory concentration (MIC). Results were analyzed using Microsoft Excel 2007 and One way ANOVA; p<0.05 was considered for statistical significance. RESULTS: Few acetone extracts were active against MTB with MIC under 100 microg/mL. Four plants showed lower MIC values; Berchemia discolor Klotzsch Hemsl 12, 5 microg/mL on H(37)Ra and 10.5 microg/mL on the clinical isolate, Bridelia micrantha Hochst. Baill (25 microg/mL), Warbugia salutaris Bertol. F Chiov (25 microg/mL), and Terminalia sericea Burch ex D. F (25 microg/mL) on both H(37)Ra and clinical isolate. However, the roots of Ximenia caffra Sond. Var. caffra, barks of Sclerocarya birrea (A Rich) Hochst, Asclepias fruticosa L, tubers of Allium sativum L, leaves of Carica papaya L, Solanum panduriforme E. Mey C, and roots of Securidaca longepedunculata Fresen gave MIC greater than 100 microg/mL. CONCLUSION: The acetone extracts of Berchemiadiscolor, Bridelia micrantha, Terminalia sericea and Warbugia salutaris could be important sources of mycobactericidal compounds against multidrug-resistant MTB.

In vitro anti-Helicobacter pylori activity of extracts of selected medicinal plants from North West Cameroon.

ETHNOPHARMACOLOGICAL RELEVANCE: Helicobacter pylori, a gram negative microaerophilic bacterium is a major etiological agent in duodenal, peptic and gastric ulcers. The growing problem of antibiotic resistance by the organism demands the search for novel compounds from plant based sources. AIM OF STUDY: The present study is aimed at evaluating the antimicrobial activity of some selected medicinal plants on clinical isolates of H. pylori circulating in Cameroon in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. MATERIALS AND METHODS: Gastric biopsy samples were obtained from patients presenting with gastroduodenal complications. H. pylori was isolated from the specimens following standard microbiology procedures. The disk diffusion method was used to determine the susceptibility of 15 isolates to ten methanol plant extracts (Ageratum conyzoides, Scleria striatinux, Lycopodium cernua, Acanthus montanus, Eryngium foetidium, Aulutandria kamerunensis, Tapeinachilus ananassae, Euphorbia hirta, Emilia coccinea and Scleria verrucosa). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the most active plant extracts were also determined by the agar dilution method. Results were analyzed statistically by the Fisher's exact test. RESULTS: All the plants tested demonstrated antimicrobial activity with zone diameters of inhibition ranging from 0-30mm. Of these, A. conyzoides, S. striatinux and L. cernua showed very potent antibacterial activity on the isolates. The lowest MIC and MBC recorded were 0.032mg/mL and 0.098mg/mL respectively. However, the MIC of the extracts ranged from 0.032-1.0mg/mL for S. striatinux; 0.063-0.5mg/mL for L. cernua and 0.063-1.0mg/mL for A. conyzoides. The MBC of the extracts ranged from 0.098-15.0mg/mL for S. striatinux; 0.098-12.5mg/mL for A. conyzoides, and 0.195-12.5mg/mL for L. cernua. The extracts had a wide spectrum of activity. The three most potent extracts possessed significant (P<0.05) inhibitory activities. CONCLUSION: The plant extracts may contain compounds with therapeutic activity.