Microplate Dilution Method Research Articles

Antiplasmodial, Antimicrobial and Cytotoxic Activities of Extracts from Selected Medicinal Plants Growing in Tanzania

This paper reports on the evaluation of antiplasmodial, antimicrobial and cytotoxic activities of extracts from eleven plant species traditionally used by some Tanzanian coastal communities for treatment of malaria, microbial infections and related ailments. Crude extracts from selected plant species namely Acacia zanzibarica, Danais xanthorrhoea, Diospyros loureiriana ssp. rufescens, Erythrina sacleuxii, Newtonia paucijuga, Pentas lanceolata, Scorodophloeus fischeri, Stuhlmannia moavi, Tarenna pavettoides, Tessmannia burttii and Toussaintia orientalis growing in Tanzania were investigated using an imaging-based assay (antiplasmodial), well diffusion and microplate dilution methods (antimicrobial) and human embryonic kidney cells (HEK 293) and brine shrimp larvae assays (toxicity). The extracts exhibited activities of varying potencies and cytotoxicity with IC50 values ranging from 0.45 ± 0.09 to 75.70 ± 24.19 μg/mL against Plasmodium falciparum (3D7 strain), MIC ranging from 0.25 to 2.0 mg/mL (against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans and Cryptococcus neoformans), LC50 ranging from 0.75 to 1000 μg/mL against brine shrimp larvae (Artemia salina) and IC50 ranging from 4.02 ± 1.05 to more than 289 μg/mL against HEK 293 cells. The observed bioactivities of some of the investigated plant extracts validate their ethnomedicinal use and are indicative of the presence of bioactive ingredients for further phytochemical investigations.

Synthesis, Characterization and Evaluation of Liponiosome Containing Ginger Extract as a New Strategy for Potent Antifungal Formulation

Aflatoxins are natural fungous toxins made by Aspergillus species. The toxins can cause tissue necrosis. In this study, nanoliponiosomes containing ginger extract were synthetized and their antifungal effect on the growth and Aflatoxin production of Aspergillus flavus and Aspergillus parasiticus fungi was studied. Physiochemical characteristics of the sample were determined using XRD, FTIR, AFM, DLS and SEM. The resultant nanostructure can operation as an angiogenesis promoter and provide antifungal nanocarrier for improving the wound healing process. Antifungal activity of ginger extract was determined by disc diffusion and microplate dilution methods. The inhibitory effect of extract on Aflatoxin-B1 production was investigated using MIC method. Ginger concentration, cholesterol%, SPAN-60 and DPPC% were optimized by response surface method. FTIR analysis showed that ginger extract and nanoliponiosome had no chemical interaction resulting in the change of functional groups. SEM observation showed the spherical morphology of particles and the average size of 95 nm. It was also found out that ginger extract had a stronger antifungal effect against Aspergillus flavus fungus compared to Aspergillus parasiticus fungus. The results of this study showed that the ginger extract loaded nanoliponiosomes can be promising antifungal agents for improving wound healing process with high antifungal effects and low side effects.

Different Combretum molle (Combretaceae) leaf extracts contain several different antifungal and antibacterial compounds

Combretum molle occurs widely in Africa and is used traditionally to treat many different ailments including microbial infections. Some work has been done on the antimicrobial activity of bark extracts, but not on leaf extracts, a sustainable resource.Dried ground leaves were extracted with methanol, butanol, acetone, ethyl acetate, chloroform, dichloromethane and n-hexane. The antibacterial and antifungal activities were determined by using a serial microplate dilution method and the number of antimicrobial compounds were determined by bioautography. Both animal (Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus) and plant (Penicillium janthinellum, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Rhizoctonia solani) pathogenic fungal species were examined. Four important nosocomial bacterial species (Staphylococcus aureus, Escherichia coli, Enterococcus faecalis and Pseudomonas aeruginosa) were also examined.Butanol extracted the highest mass of plant material (41 mg) and hexane and dichloromethane extracted the lowest masses, 8 and 9 mgl, respectively. In bioautograms developed with BEA sprayed with Aspergillus fumigatus, all the extracts had clear zones of inhibition at R values of c. 0.12, 0.23, and 0.40 for the different fungi.The MIC values of the extracts ranged from 0.04 to 0.77 mg/ml after 12 h incubation. The acetone leaf extract was the most active with an MIC of 0.04 mg/ml against P. janthinellum, an important plant fungal pathogen. The total activity of 775 ml/g (indicating that the extract obtained from 1 g of leaves can be diluted to 775 ml and will still inhibit the growth) means that the acetone leaf extract could be used to control P. janthinellum infections on plants.

Deciphering the role of cyanobacteria in water resistome: Hypothesis justifying the antibiotic resistance (phenotype and genotype) in Planktothrix genus

The importance of environmental microorganisms in the emergence and dissemination of antibiotic resistance is an undeniable fact. However, cyanobacteria are not seen yet as putative players in the dynamic of environmental resistome, despite their ubiquity in water environments, where they are exposed to antibiotic pollution and in straight contact with native and pathogenic bacteria harboring antibiotic resistance genes (ARGs). In this work we evaluated the susceptibility of 8 strains of Planktothrix agardhii (from surface freshwaters reservoirs) and 8 strains of Planktothrix mougeotii (from a wastewater treatment plant) to several classes of antibiotics, using a microplate dilution method previously described by us. We also search for ARGs in those strains by molecular methods. None of the 16 tested strains were susceptible to trimethoprim, nalidixic acid and norfloxacin, from 0.0015–1.6 mg/L, but all were susceptible to streptomycin, gentamicin, kanamycin, ceftazidime and ceftriaxone. The minimum inhibitory concentrations (MICs) ranged between 0.05–0.8 mg/L for the aminoglycosides and 0.4–1.6 mg/L for the two β‑lactams. Major differences were found in the susceptibility to amoxicillin and tetracycline, with P. agardhii being susceptible (MIC of 0.05 mg/L and 0.4 mg/L, respectively) and P. mougeotii not susceptible. These distinct responses might be due to differences between species. However, the lower susceptibility of wastewater strains suggests that antibiotic resistance phenotype of cyanobacteria is related with their habitat. The failure to detect acquired genes conferring resistance to trimethoprim/quinolones, strongly supports the hypothesis that cyanobacteria are intrinsically resistant to these antibiotics. Interestingly, we detected a class-1-type integron and a sul1 gene in 3 strains of both P. agardhii and P. mougeotii, which supports the possibility of cyanobacteria to acquire and transfer antibiotic resistance determinants. In conclusion, the identification of ARGs and related integrons, as well as the reduced susceptibility to some antibiotics, suggests that cyanobacteria may play a role on environmental resistome.

Terminalia laxiflora and Terminalia brownii contain a broad spectrum of antimycobacterial compounds including ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols

Ethnopharmacological relevanceTerminalia laxiflora Engl. & Diels, (Sudanese Arabic name: Darout الدروت) and Terminalia brownii Fresen (Sudanese Arabic name: Alshaf ألشاف) (Combretaceae) are used in Sudanese traditional folk medicine and in other African countries for treatment of infectious diseases, TB and its symptoms, such as cough, bronchitis and chest pain. Aim of studyBecause of the frequent use of T. laxiflora and T. brownii in African traditional medicine and due to the absence of studies regarding their antimycobacterial potential there was a need to screen extracts of T. laxiflora and T. brownii for their growth inhibitory potential and to study the chemical composition and compounds in growth inhibitory extracts. Materials and methodsThe plant species were collected in Sudan (Blue Nile Forest, Ed Damazin Forestry areas) and selected according to their uses in traditional medicine for the treatment of bacterial infections, including TB. Eighty extracts and fractions of the stem bark, stem wood, roots, leaves and fruits of T. laxiflora and T. brownii and nine pure compounds present in the active extracts were screened against Mycobacterium smegmatis ATCC 14468 using agar diffusion and microplate dilution methods. Inhibition zones and MIC values were estimated and compared to rifampicin. HPLC-UV/DAD, GC/MS and UHPLC/Q-TOF MS were employed to identify the compounds in the growth inhibitory extracts. ResultsThe roots of T. laxiflora and T. brownii gave the best antimycobacterial effects (IZ 22–27 mm) against Mycobacterium smegmatis. The lowest MIC of 625 µg/ml was observed for an acetone extract of the root of T. laxiflora followed by methanol and ethyl acetate extracts, both giving MIC values of 1250 µg/ml. Sephadex LH-20 column chromatography purification of T. brownii roots resulted in low MIC values of 62.5 µg/ml and 125 µg/ml for acetone and ethanol fractions, respectively, compared to 5000 µg/ml for the crude methanol extract. Methyl (S)-flavogallonate is suggested to be the main active compound in the Sephadex LH- 20 acetone fraction, while ellagic acid xyloside and methyl ellagic acid xyloside are suggested to give good antimycobacterial activity in the Sephadex LH-20 ethanol fraction. RP-18 TLC purifications of an ethyl acetate extract of T. laxiflora roots resulted in the enrichment of punicalagin in one of the fractions (Fr5). This fraction gave a five times smaller MIC (500 µg/ml) than the crude ethyl acetate extract (2500 µg/ml) and this improved activity is suggested to be mostly due to punicalagin. 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone and β-sitosterol were found in antimycobacterial hexane extracts of the stem bark of both studied species. Of these compounds, 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone have not been previously identified in T. brownii and T. laxiflora. Moreover, both plant species contained friedelin, betulinic acid, β-amyrine and two unknown oleanane-type triterpenoids. Of the listed compounds, friedelin, triacontanol and sitostenone gave a MIC of 250 µg/ml against M. smegmatis, whereas stigmasterol and β-sitosterol gave MIC values of 500 µg/ml. ConclusionsOur results show that T. laxiflora and T. brownii contain antimycobacterial compounds of diverse polarities and support the traditional uses of various parts of T. laxiflora and T.brownii as decoctions for treatment of tuberculosis. Further investigations are warranted to explore additional (new) antimycobacterial compounds in the active extracts of T. laxiflora and T. brownii.

Antitrypanosomal, antiplasmodial, and antibacterial activities of extracts from selected Diospyros and Annonaceae species

Aim: To screen methanol extracts from root bark, leaves, and stem bark of selected plant species from the genus Diospyros and some Annonaceae species for antitrypanosomal, antiplasmodial, and antibacterial activities against selected test organisms. Methods: Antitrypanosomal and antiplasmodial assays of methanol extracts from selected plant species were carried out in single concentration screens and in dose-response for active extracts. The minimum inhibitory concentration (MIC) values of selected plant extracts against selected bacterial strains were determined by microplate dilution method in sterile 96-well microtiter plates. Results: In the dose-response antitrypanosomal assay, the most potent extracts tested exhibited activities against Trypanosoma brucei brucei (Lister 427 strain) with IC50 values ranging from 1.28 to 7.85 μg/ml, with methanol extract of Diospyros verrucosa stem bark being the most active with IC50 value of 1.28 μg/ml. In the dose-response antiplasmodial assay, three extracts exhibited activities against Plasmodium falciparum(strain 3D7) with IC50 values ranging from 4.55 to 24.22 μg/ml, with methanol extract of Diospyros capricornuta root bark being the most potent with IC50 value of 4.55 μg/ml. In the antibacterial assay, the investigated extracts exhibited a wide range of activities against Staphylococcus aureus [American Type Culture Collection (ATCC) strain 25923], Bacillus cereus (ATCC strain 11775), and Escherichia coli (ATCC strain 8740) with MIC values ranging from 0.00125 to 0.00625 mg/ml (more active), 0.125 to 0.500 mg/ml (moderately active), and 1.00 to 8.00 mg/ml (less active) while some extracts were inactive at the highest concentration tested of 16.00 mg/ml. Conclusions: Methanol extracts obtained from root bark, leaves, and stem bark of selected plant species from the genus Diospyros and some Annonaceae species that showed good activities in antitrypanosomal, antiplasmodial, and antibacterial assays corroborate reported literature about the traditional medicinal uses of the members of genus Diospyros and some Annonaceae species.

Antimicrobial Activities of Syzygium aromaticum (L.) Merr. and L.M. Perry (Myrtaceae) Fruit Extracts on Six Standard Microorganisms and Their Clinical Counterpart

Syzygium aromaticum is used in combination with other plants as an al-coholic infusion by traditional practitioners to treat infections. It has been selected for evaluation for its antimicrobial properties to justify its use in traditional pharmacopoeia. The fruits were used as plant material while the microbial germs consisted of six reference strains: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Salmonella typhimurium ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Shigella flexneri ATCC 12022, Candida albicans ATCC 35659 and their clinical counterparts. The extracts were obtained by aqueous decoction, hydroethanolic and ethanolic macerations. The phytochemical screening was performed by chemical staining tests. The antibiotic susceptibility test was performed using the well diffusion method and the MIC and MBC or MFC were determined using the 96-well microplate dilution method. The results showed that 66.67% of the strains tested were sensitive to the aqueous extract with inhibition diameters ranging from 15 to 21 mm and MIC and MCB or MFC between 0.0976 - 0.3906 mg/mL and 0.1953 - 07812 mg/mL respectively, thus determining bacteriostatic activity. 100% of the germs tested were sensitive to hydroethanolic and ethanolic extracts. The inhibition diameters range from 12 - 28 mm for hydroethanolic extract with MIC and MBC or MFC ranging from 0.0488 - 0.3906 mm and 0.0488 - 0.7812 mm respectively. The ethanolic extract gave inhibition diameters of 12 - 26 mm; MIC and MBC or MFC ranging from 0.0976 - 0.7812 mm. Hydroethanolic extract gave three (3) bactericidal/fungicidal activities compared to four (4) as for ethanolic extract. These results prove the use of S. aromaticum among traditional recipes for treating infections in the pharmacopoeia but further studies remain important to produce traditionally improved drugs.

Anti-mycobacteria potential and synergistic effects of combined crude extracts of selected medicinal plants used by Bapedi traditional healers to treat tuberculosis related symptoms in Limpopo Province, South Africa

BackgroundTuberculosis is an infectious communicable disease and the causative agent of the disease has over the years developed resistance to streamline chemotherapeutic agents with dire consequences and there is a need for development of new and more potent alternatives.MethodsConstituents of leaves material of Combretum heroroense, Citrus lemon and Apodytes dimidiata were serially extracted using solvents of varying polarity. TLC finger print profile of the different extracts were determined by spraying eluted plates with vanillin sulphuric acid and 2, 2- diphenylpicryl hydrazyl (DPPH) for the presence of antioxidant constituents. Presence of different phytochemicals was determined using standard chemical test. Bioautography was used to determine the number of compounds present in sub-fractions active against Mycobacterium smegmatis. Minimum inhibitory concentration (MIC) values extract and sub-fractions were determined using serial microplate dilution method against M. smegmatis (ATCC 1441), M. tuberculosis (ATCC H37Rv) and multi-drug resistant TB (MDR-TB) field strain. Synergy of the crude extracts of the three plants was determined using microplate dilution method against M. smegmatis.ResultsMass extracted by different solvents was less than 6% dry weight for all the plants. Phlobatannins were not detected in A. dimidiata, C. heroroense and C. lemon as well as cardiac glycosides in C. lemon and A. dimidiata, and saponins in C. heroroense. Sub-fractions of the different plants were shown to contain constituents with antioxidant activity with the highest number detected in C. heroroense. Bioautography results reveal the presence of a compound(s) in the ethyle acetate sub-fraction of C. heroroense and butanol, methanol/water, ethyl acetate and water no.2 subfractions of A. dimidiata, active against M. smegmatis that were not shown to have antioxidant capacity. MIC results for different crude extracts of the three plants against M. smegmatis ranges from 0.1 to 3 mg/ml. The average MIC for the synergistic effect of the plants ranged from 0.04 mg/ml to 1.25 mg/ml. An activity greater than that obtained for the reference drugs was shown for the butanol and hexane fractions of A. dimidiata (0.47 mg/ml) against the field strain of MDR-TB while that obtained for the M.TB (ATCC H37Rv) was 0.31 mg/ml.ConclusionA significant finding shown in this study reveals the potent anti-mycobacteria potential of sub-fractions of A. dimidiata against MDR-TB field strain that can lead to the isolation of compounds that can be used to counter resistant strains of tuberculosis.

Epigallocatechin Gallate, a Green Tea Polyphenol Inhibits Mycobacterium smegmatis: In silico and In vitro Studies

Presence of multidrug resistant strains makes treatment of tuberculosis difficult. Conventional frontline antituberculosis therapy has high incidence of treatment failure due to non-adherence and severe side effects, and therefore warrants alternative approaches using safe, natural compounds. (-) Epigallocatechin gallate, a green tea catechin with significant antimicrobial properties, and its derivatives have been studied for their inhibitory effects on Mycobacterium smegmatis. Enoyl reductase (Inh A, PDB ID: 2NV6), an important drug target inhibited by the antituberculosis drug, isoniazid, was docked with geometrically optimized conformation of epigallocatechin gallate and two of its derivatives using AutoDock software. Perpropionate and permethyl derivatives of epigallocatechin gallate were synthesized and, along with epigallocatechin gallate, evaluated for their inhibitory and cytotoxic properties against M. smegmatis using microplate dilution method and alamarBlue® assay, respectively. Ames test was performed to assess the mutagenic potential of epigallocatechin gallate. It was docked successfully onto the InhA with docking energy of –9.38 kcal mol-1. Analysis of neighboring groups revealed 11 amino acid residues common to both isoniazid and epigallocatechin gallate. The minimum inhibitory concentrations for epigallocatechin gallate, perpropionate and permethyl were found to be 128 (58.2% inhibition), 8 (32.9%) and 4 (12.5%) µg/ml, respectively. Epigallocatechin gallate showed a cytotoxicity of 18.6% at a concentration of 8 µg/ ml, while the two derivatives did not show any toxicity, even at 64 µg/ml. Ames test revealed the absence of mutagenicity for epigallocatechin gallate, in presence and absence of metabolic activation. Non-mutagenic epigallocatechin gallate inhibits the growth of M. smegmatis and warrants further investigation as an adjunct therapy for pathogenic mycobacteria.

Cytotoxicity, antimicrobial and antidiarrhoeal activities of standardised aqueous root extract of Elephantorrhiza elephantina (Burch.)

Elephantorrhiza elephantina is used in Basotho traditional medicine to treat infectious diseases including diarrhoea. This study evaluated its aqueous extract for cytotoxicity, antimicrobial and antidiarrhoeal activities. While brine shrimp lethality assay was used for the cytotoxicity study, the antimicrobial activity was determined via a micro-plate dilution method. Using castor oil, and gastrointestinal motility models, the antidiarrhoeal activity was also investigated. The extract inhibited all the tested bacterial and Candida strains at minimum inhibitory concentrations of 0.78–3.13, and 0.78–1.56 mg/mL, respectively. For the antidiarrhoeal study, the extract dose dependently and significantly prolonged the onset time of diarrhoea, decreased the characteristic faeces features, increased intestinal Na+-K+-ATPase activity, attenuated nitric oxide content and effectively inhibited gastrointestinal motility in experimental animals. In all the antidiarrhoeal models, the extract (at 400 mg/kg) elicited effects that compared favourably with loperamide (standard drug). Judging by the LC50 value of 42.04 μg/mL for the cytotoxicity assay, the extract could be adjudged highly potent and effective. The results from the present study have not only presented E. elephantina as being endowed with chemotherapeutic constituents which could be potentially useful for the development of drugs against bacterial infections, but also validate its Basotho traditional application against diarrhoea.

Antimicrobial activity of Albizia gummifera (J.F.Gmel.) C.A.Sm leaf extracts against four Salmonella serovars

In the past few decades, non-typhoidal serovars of Salmonella have gained resistance to several classes of antibiotics including third generation Cephalosporins and Fluoroquinolones. There has also been an increase in the number of infections caused by antimicrobial resistant Salmonella serotypes including Salmonella Typhimurium DT 104 which is resistant to at least five antimicrobial agents. Therefore, the aim of this study was to investigate Albizia gummifera (Fabaceae), a native tree species of Africa that has been used extensively in African traditional medicine for the treatment of stomach infection, skin diseases and malaria for antimicrobial activity against four Salmonella serovars. Dichloromethane, methanol and acetone leaf extracts of A. gummifera were screened for antimicrobial activity against Salmonella Typhimurium, S. Enteritidis, S. Dublin, and S. Gallinarum using the microplate dilution method. The minimal inhibitory concentration (MIC) of the extracts ranged between 0.125 and 1mg/ml, while minimal bactericidal concentrations (MBC) ranged between 0.25 and >2.00mg/ml. The best activity was exhibited by the methanol extract against all the tested strains. The mutagenic and cytotoxic potential of the most active methanol extract were also determined. The extract was not mutagenic in the Ames tester strains TA98 and TA100 with and without metabolic activation. However, the methanol extract was cytotoxic to both Human hepatocellular carcinoma (C3A) and Madin-Darby Bovine kidney (MDBK) cell lines. The results obtained in this study validate in part the use of this plant in African traditional medicine for the treatment of stomach infection.

Antimicrobial, anti-inflammatory activity and cytotoxicity of Funtumia africana leaf extracts, fractions and the isolated methyl ursolate

Funtumia africana is used to treat and manage diverse ailments including fever, inflammation, malaria, cancer and urinary incontinence in South Africa. In this study, the antibacterial, antifungal, anti-inflammatory activities and cytotoxicity of the crude extracts, fractions and an isolated compound were determined. Serial microplate dilution and bioautography methods were used to determine the antimicrobial activities. The bacteria tested were ATCC reference strains of Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus. The fungal test organisms used were three clinical isolates (Aspergillus fumigatus, Cryptococcus neoformans and Candida albicans), C. albicans ATCC 10231 and three phytopathogenic fungi (Fusarium oxysporum, Penicillium janthinellum and Rhizoctonia solani). The anti-inflammatory activity was determined using cyclooxygenase (COX) enzymes and the MTT assay was used to determine cellular toxicity against Vero and human liver (C3A) cells. The crude extract had MICs as low as 80μg/ml against both bacteria and fungi. The chloroform fraction had the lowest MIC of 20μg/ml against P. aeruginosa. The hexane and chloroform fractions had MIC of 40μg/ml against C. albicans ATCC 10231. The crude extract, hexane and chloroform fractions had moderate activity against both COX-1 and COX-2. The chloroform fraction was more active than the crude extract (59.7%) with an inhibition of 68.2% against COX-1. Bioassay-guided fractionation using column chromatography led to the isolation of methyl ursolate (MU) with an MIC of 62.5μg/ml against F. oxysporum. It was relatively toxic against Vero cells with an IC50 of 10.4μg/ml. The antimicrobial and anti-inflammatory activities of the crude extract provide some support for the traditional use of the plant.

Synthesis, Spectral and Biological Studies of Schiff Bases Derived from 3-Aminophenol and Substituted Benzaldehydes

Increased reports of hospital acquired infections as a result of microbial resistance reported to known antimicrobial and antiseptic agents have led the search of new highly active agents. A series of Schiff bases (L1-L8) derived from 3-aminophenol and substituted benzaldehydes; 2-methoxybenzaldehyde, 2-hydroxybenzaldehyde, 2-chlorobenzaldehyde, 2-nitrobenzaldehyde, 4-methoxybenzaldehyde, 4-hydroxybenzaldehyde, 4-chlorobenzaldehyde and 4-nitrobenzaldehyde  have been synthesized and characterized based on elemental analysis, FT-IR, 1H NMR and UV-Vis spectroscopy. The Schiff base ligands were screened for their in-vitro antibacterial activity against six pathogenic bacteria; Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus cereus, Enterococcus faecalis and Klebsiella pneumoniae  using the disc diffusion method and 96-well micro-plate dilution method with ampicillin and sodium hypochlorite used as reference compounds. The antibacterial activity exhibit a dependence on both nature and position of substituent with compounds derived from 4- chlorobenzaldehyde (L7) and 4-nitrobenzaldehyde (L8) showing highest activity against the tested bacterial strains better than the reference compounds. These compounds can be employed as active ingredients in development of antibacterial/antispetic agents against these pathogenic organisms.

Development and evaluation of new microemulsion-based hydrogel formulations for topical delivery of fluconazole.

The aim of the present investigation was to develop and evaluate microemulsion-loaded hydrogels (MEHs) for the topical delivery of fluconazole (FZ). The solubility of FZ in oils, surfactants and cosurfactants was evaluated to identify the components of the microemulsion. The pseudo-ternary phase diagrams were constructed using the novel phase diagram by micro-plate dilution method. Carbopol EDT 2020 was used to convert FZ-loaded microemulsions into gel form without affecting their structure. The selected microemulsions were assessed for globule size, zeta potential and polidispersity index. Besides this, the microemulsion-loaded hydrogel (MEH) formulations were evaluated for drug content, pH, rheological properties and in vitro drug release through synthetic membrane and excised pig ear skin in comparison with a conventional hydrogel. The optimised MEH FZ formulations consisting of FZ 2%, Transcutol P 11.5% and 11%, respectively, as oil phase, Lansurf SML 20-propyleneglycol 52% and 50%, respectively, as surfactant-cosurfactant (2:1), Carbopol EDT 2020 1.5% as gelling agent and water 34.5% and 37%, respectively, showed highest flux values and high release rate values, and furthermore, they had low surfactant content. The in vitro FZ permeation through synthetic membrane and excised pig ear skin from the studied MEHs was best described by the zero-order and first-order models. Finally, the optimised MEH FZ formulations showed similar or slightly higher antifungal activity as compared to that of conventional hydrogel and Nizoral® cream, respectively. The results suggest the potential use of developed MEHs as vehicles for topical delivery of FZ, encouraging further in vitro and in vivo evaluation.

Anti-bacterial and anti-oxidant activities of leaf extracts of Combretum vendae (Combretecacea) and the isolation of an anti-bacterial compound.

Combretum vendae A.E. van Wyk (Combretaceae) is used for the treatment of bacterial related infections and oxidative related diseases by indigenous people of South Africa. Dried leaves extracts of C. vendae were investigated for bioactivity against a variety of bacterial strains and their antioxidant potential evaluated. Constituents of leaf material were serially extracted using solvents of varying polarities, TLC chromatograms of the fractions were sprayed with 2,2 diphenyl-1-picrylhydrazyl (DPPH) to determine the presence of antioxidant compounds. Bio-autography was used to determine the number of antibacterial compounds active against Staphylococcus aureus, Enterococcus faecalis, Eschericha coli and Pseudomonas aeruginosa. Minimum inhibitory concentration (MIC) values were determined using serial microplate dilution method. The chloroform fraction was subjected to bio-assay guided column chromatography to isolate the active compound. The mass extracted by different solvents was below 10% dry weight. MIC values for different extracts against different pathogens ranges from 0.08 to 0.64 mg/ml. The compound isolated was identified as acacetin having an Rf value of 0.28 following elution in the Ethanol: Methanol: Water [E: M: W (10: 1.35: 1 v/v). Acacetin had MIC values ranging from 0.16 to 0.35 mg/ml. We report for the first time the isolation of acacetin as the main antibacterial compound from the leaves of Combretum vendae.

Antimicrobial activity, toxicity and selectivity index of two biflavonoids and a flavone isolated from Podocarpus henkelii (Podocarpaceae) leaves.

BackgroundDifferent parts of Podocarpus henkelii have been used in many cultures around the world to treat ailments such as cholera, stomach diseases, rheumatism, cancer, canine distemper in dogs and gall sickness in cattle. The aim of this study was to evaluate the biological activity and toxicity of isolated compounds from Podocarpus henkelii after an earlier study indicated a promising activity in crude extracts against viral pathogens of veterinary importance.MethodsThe antibacterial and antifungal activity of two biflavonoids 7, 4’, 7”, 4”’-tetramethoxy amentoflavone (TMA), isoginkgetin (IGG) and podocarpus flavone–A (PFA) isolated from the leaves of Podocarpus henkelii were determined using a serial microplate dilution method with tetrazolium violet as growth indicator. The cytotoxicity of compounds TMA and IGG were determined on different cell types using a tetrazolium-based colorimetric cellular assay (MTT). The Ames test was used to determine their mutagenic activities.ResultsTMA had reasonable antifungal activity against Aspergillus fumigatus (MIC = 30 μg/ml). IGG had a wide spectrum of activity against four bacterial and two fungal pathogens with much higher selectivity index values obtained for A. fumigatus and Cryptococcus neoformans (SI > 30). PFA had a broad spectrum of activity against Enterococcus faecalis and Pseudomonas aeruginosa (SI > 15) and less activity against the two fungal pathogens. In both the cytotoxicity assays and Ames mutagenicity test using Salmonella typhimurium strains TA98 and TA100, TMA and IGG had no deleterious effect on the different cell types and did not induce mutations in the Ames test.ConclusionAlthough the antimicrobial activities of the isolated compounds were not that exciting, the compounds had no cytotoxic activity at the highest concentration (1000 μg/ml) tested against all three cell lines. IGG was the most active against E. coli, S. aureus, A. fumigatus and C. neoformans, exhibiting both antibacterial and antifungal activity with good selectivity index values. PFA had a broad spectrum of activity against E. faecalis and P. aeruginosa. The two compounds isolated had low toxicity and no genotoxic activity in the Ames test.

Reactivity of Crystalline ZnO Superstructures against Fungi and Bacterial Pathogens: Synthesized Using Nerium oleander Leaf Extract

For the first time, different morphologies of zinc oxide (ZnO) superstructures are synthesized by a simple and environmental friendly route using Nerium oleander leaf extract as fuel. Powder X-ray diffraction, scanning electron microscopy, UV–visible spectroscopy, and photoluminescence studies are performed to ascertain the formation and characterization of ZnO. X-ray diffraction confirmed the crystalline nature of the compound with hexagonal Wurtzite structure. When the concentration of the leaf extract is varied, different morphologies are formed. ZnO are tested for antifungal using soybean seed-borne fungi by food-poison method and antibacterial activity against bacterial human pathogens by a broth microplate dilution method using 96-well plates. Among the screened soybean seed-borne fungi, Fusarium equisiti was found to be more susceptible, which was followed by Macrophomina phaseolina for ZnO nanoparticles (NPs) prepared using 0.2188 mol/dm3 N. oleander leaf extract. It was observed that NPs exhibited pronounced antifungal activity in a dose-dependent manner with a relatively high percentage of mycelial inhibition. ZnO obtained with the concentration of 0.2188 mol/dm3 leaf extract showed both minimum inhibitory concentration and minimum bactericidal concentration effectiveness compared to other synthesized compounds. It is observed that the samples with small crystallite size show greater antibacterial activity than those of larger crystallite size. Further, we found that crystallite size and morphology significantly affects the antibacterial activity of ZnO. Prepared compounds showed significant inhibition against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeurginosa. Among the tested bacteria, P. aeurginosa is more susceptible and E. coli is the least effective against bacterial pathogens. The antibacterial activities of the as-formed ZnO are preliminarily studied against Gram-positive (B. subtilis and S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria and are found to be dependent on the shape of the nanostructures.

Extraction and Purification of Flaxseed Proteins and Studying their Antibacterial Activities

Flaxseed or linseed (Linum usitatissimum L.) is an annual herb belongs to the Linaceae family. It is cultivated worldwide and has been used for its oil seed and fiber since ancient times in Egypt, Rome and Greece. Nowadays it is considered as a medicinal plant in Asia, Europe and North America. Therapeutic effects of flaxseed fixed oil have been reported in several publications, but there are only few reports on biological activities of the flaxseed proteins. In the present study antimicrobial activities of the flaxseed proteins have been investigated. Hemogenized flaxseed flour was at first defatted and demucilaged, and its protein content was extracted in distilled water. After centrifugation, proteins of supernatant were isolated by adjusting pH. Gel filtration and ion-exchange chromatography on the protein extact gave fairly pure protein fractions, which antibacterial activities were examined against several microorganisms using the microbial micro-plate dilution method. The results showed that flaxseed proteins have an inhibitory activity on bacteria especially against Entrococcus foecalis, Salmonella typhimurium and Escherichia coli. Our experiment also revealed that mucilage composition as carbohydrates would not contribute in the inhibitory effect, but in fact, might cover and inhibit the antibacterial activity of flaxseed proteins. Moreover, it is possible that the carbohydrate components of flaxseed promote the microorganism growth.

Antibacterial synergy between quercetin and polyphenolic acids against bacterial pathogens of fish

ObjectiveTo evaluate combinations of quercetin with gallic acid, p-anisic acid and cinnamic acid in vitro for synergistic activity against common Gram-negative bacterial pathogens of fish viz., Aeromonas hydrophila, Aeromonas salmonicida and Edwardsiella tarda. MethodsAntibacterial activity of quercetin, gallic acid, p-anisic acid and cinnamic acid was determined against selected bacterial pathogens individually, followed by combination of quercetin with polyphenolic acids using serial microplate dilution method measuring minimum inhibitory concentrations. Fractional inhibitory concentration indices were calculated. ResultsQuercetin and other polyphenolic compounds exhibited antibacterial action against the selected fish pathogens with mean minimum inhibitory concentrations ranging from 0.83 to 2.5 mg/mL. It was observed that fractional inhibitory concentration indices for combination of quercetin with gallic acid, p-anisic acid or cinnamic acid against Aeromonas salmonicida were less than 0.5, indicating synergistic interaction. However, the above combinations produced additive antimicrobial activity against Aeromonas hydrophila and Edwardsiella tarda. ConclusionsPositive antibacterial interaction was evident between quercetin and selected polyphenolic acids in vitro.

Evaluation of pharmacological activities, cytotoxicity and phenolic composition of four Maytenus species used in southern African traditional medicine to treat intestinal infections and diarrhoeal diseases.

BackgroundMicrobial infections and resulting inflammation and oxidative stress are common pathogenesis of gastrointestinal tract (GIT) disorders. In South Africa, several species of the genus Maytenus are used in traditional medicine to treat various infectious diseases. Most of the previous work on this genus was focused on nonpolar extracts from the root and bark. In this study, leaf extracts of polar extracts of Maytenus peduncularis, Maytenus procumbens, Maytenus senegalensis and Maytenus undata were evaluated for antimicrobial, anti-inflammatory and antioxidant activities to determine their efficacy as therapeutic agents in GIT disorders.MethodsPhenolic-enriched leaf extracts and fractions were prepared by extracting with acidified 70% methanol and solvent-solvent fractionation. The activities of the fractions against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as well as clinical isolates of Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans were determined using a serial microplate dilution method. Antioxidant activities were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH) radical scavenging and linoleic acid peroxidation inhibitory assays. The phenolic composition as well as the cytotoxicity against Vero cell lines of the crude extracts was evaluated using various standard protocols.ResultsThe antimicrobial activities were concentrated in the non-polar fractions of hexane, dichloromethane and ethyl acetate (MICs 19–312 μg/ml). The crude extracts and polar fractions (butanol and water) had moderate to poor antimicrobial activity (MICs 312 to above 2500 μg/ml). The crude extracts and polar fractions had good antioxidant activity (EC50 values varied from 1.22 to 607 μg/ml, 1.71 to 312 μg/ml and 23 to 284 μg/ml for DPPH, ABTS and OH respectively. Linoleic acid peroxidation inhibition EC50 values of the crude extracts ranged between 27 and 39 μg/ml with relatively low toxicity against Vero cell lines (IC50 values 87 to 187 μg/ml). Fractionation of a crude extract with low activity could lead to fractions with more potent activity.ConclusionThis study justifies the traditional use of leaf crude extracts and fractions from these four plants to remedy gastrointestinal disorders resulting from infection, inflammation and oxidative stress complications. The study also provides rationale for the use of leaf extracts with same beneficial effects in place of unsustainable root and bark harvest.