Solvent-solvent Fractionation Research Articles

Use of Saponinosomes from Ziziphus spina-christi as Anticancer Drug Carriers.

Saponins are plant glycosides with different structures and biological activities, such as anticancer effects. Ziziphus spina-christi is a plant rich in saponin, and this compound is used to treat malignant melanoma in the present study. Nanophytosomes can be used as an advantageous nanodrug delivery system for plant extracts. The aim of this work is to use the saponin-rich fraction (SRF) from Z. spina-christi and prepare SRF-loaded nanophytosomes (saponinosomes) and observe the in vitro and in vivo effects of these carriers. First, the SRF was obtained from Z. spina-christi by a solvent–solvent fractionation method. Then, Fourier transform infrared (FTIR) analyses were performed to confirm the presence of saponins in the extracted material. Subsequently, the saponinosomes were prepared by the solvent injection method (ether injection method) using a 1:1:1 ratio of lecithin/cholesterol/SRF in the mixture. Characterization of the prepared saponinosomes was performed by FTIR, dynamic light scattering (DLS), field-emission scanning electron microscopy (FE-SEM), and atomic force microscopy (AFM) analyses. In addition, a UV–vis spectrophotometer was used to determine the entrapment efficiency (EE) and in vitro release of the SRF. Finally, cell cytotoxicity of the different formulations was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay on both mouse melanoma cells (B16F10) and fibroblasts (L929). Using DLS, AFM, and FE-SEM analyses, the particle size was determined to be 58 ± 6 nm with a zeta potential of −32 ± 2 mV. The calculated EE was 85 ± 3%. The results of the in vitro release profile showed that 68.2% of the SRF was released from the saponinosome after 48 h. The results of the MTT assay showed that the SRF and saponinosomes have high toxicity on B16F10 melanoma cells, but saponinosomes showed a significant decrease in cytotoxicity on L929 fibroblast cells compared with that of the SRF. Our results indicate that the SRF from Z. spina-christi has anticancer activity, and the saponinosomes prepared in this work can control tumor growth, improve therapeutic efficacy, and reduce the side effects of saponins.

Isolation and characterization of antimicrobial and anti-inflammatory triterpenoids from the acetone extract of Grewia flava DC. (Malvaceae) roots

Grewia flava root acetone extracts were assessed for antimicrobial activity against a range of pathogenic microorganisms using the broth microdilution and bioautography assays. Cytotoxicity against human dermal fibroblast (HDF) and bovine dermis (BD) cell lines was also investigated. Solvent-solvent fractionation was carried out on the acetone extract to yield fractions of different polarities. Bioautograms from fractions showed four antimicrobial compounds in the carbon tetrachloride fraction, with activity against Escherichia coli. Two compounds from carbon tetrachloride, hexane and butanol fractions were active against Candida albicans, Bacillus cereus, Klebsiella pneumoniae and Escherichia coli. The aqueous fraction had the lowest minimum inhibitory concentration (MIC) values of 40 µg/ml against Cryptococcus neoformans, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. Two triterpenoid compounds, namely taraxerol and lupeol, were isolated and had potent antimicrobial activity. Lupeol had better antimicrobial activity than vancomycin (control drug) with MIC of 10 µg/ml against Mycobacterium smegmatis, Mycoplasma hominis and Escherichia coli. Both taraxerol and lupeol exhibited better anti-inflammatory activity than quercetin (positive control) in the soybean lipoxygenase (15-LOX) inhibition assay and by inhibiting nitric oxide release from RAW264.7 macrophages. Although fractions and isolated compounds exhibited potent antimicrobial, antioxidant and anti-inflammatory activity, there is a need to explore the modes of action thereof. The study supports the use of the plant species in the treatment and management of various microbial infections posing danger to human health.

Isolation and identification of few fatty acid esters from the aerial roots of Rhaphidophora aurea twined over different host trees

Plants are the store houses of various secondary metabolites and bio-essential natural products. Several therapeutic agents are isolated from Rhaphidophora aurea. The aerial roots of the mentioned plant twined over different trees were explored. Sequential extraction (Polar to non-polar solvents) was carried out for the aerial roots of Rhaphidophora aurea twined over different host trees. Each extract was subjected to solvent-solvent fractionation. Through Thin-layer chromatography and column chromatography, the fatty acid compounds were identified and isolated. The isolated compounds were characterized through recording UV, IR, GC-MS/MS (Thermo), and 1D and 2D NMR techniques. Six fatty acids viz. 4-oxo-tricosanoic acid icosyl ester, butyl octadecanoate, dodecanoic acid dodec-3-enyl ester, octacosa-23, 26-dien-12-one, ethyl cis-6-octadecenoate and 15,18- dotriacontadienoic acid and methyl ester was isolated and characterized by analytical characterization techniques.

Antioxidant Activities and Phenolic Compounds of Extract from Hallasan Wormwood

Background: Many Artemisia species are used in traditional medicine to treat gastrointestinal disorders and various other ailments. However, the antioxidant and phenolic compounds present in Hallasan wormwood (Artemisia japonica var. hallaisanensis) have not been investigated yet.<BR>Methods and Results: Extracts of Hallasan wormwood (aerial parts and roots) were extracted using methanol and sequentially treated with solvent-solvent fractionation using n-hexane, ethyl acetate, and water-saturated butanol, to yield fractions with variations in polarity. The phenolic compounds present in the extracts and fractions were determined using high performance liquid chromatography (HPLC) and colorimetry. The antioxidant activities were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), and oxygen radical antioxidant capacity (ORAC) methods. The results revealed that the ethyl acetate fraction had high total phenolic and total flavonoid contents. Furthermore, the ethyl acetate fraction of roots showed the highest antioxidant activity.<BR>Conclusions: Metanolic extracts from the roots and aerial parts of Hallasan wormwood showed different antioxidant activities; nine phenolic acids and one flavonoid were detected in Hallasan wormwood. Thus, the finding of this study provide valuable data for research on functional substances from plants.

Bioactivities and GC-MS profiling of Malewana Madhumeha Choorna polyherbal hot infusion

Although traditional practitioners prescribe Malewana Madhumeha Choorna hot infusion (MMCHI) in Sri Lanka to combat diabetes, its antidiabetic properties have yet to be explored. We examined the in vitro hypoglycemic activity of MMCHI using α-amylase, α-glucosidase enzymes, and glucose uptake assays. Further, in vitro antioxidant activity was determined using DPPH (2,2 diphenyl-1-picrylhdrazyl) and H2O2 free radical scavenging activities. To identify the phytochemicals present in the hot infusion, we conducted solvent-solvent fractionation (hexane, chloroform, ethyl acetate, methanol) procedures and subjected them to gas chromatography-mass spectrometry (GC-MS). Both MMCHI and fractions exhibited a dose-dependent inhibition of glucose uptake and the enzymes α-amylase and α-glucosidase. The hot infusion exhibited enzyme inhibitory activities more or less similar to the standard drug acarbose. Enzyme inhibition was especially pronounced with α-amylase. Glucose uptake by MMCHI was more potent than that of the standard drug, especially at the 125 µg/mL dose. Strong antioxidant scavenging activity, comparable to that of the standard drug, was observed in the MMCHI, further indicating its antidiabetic potential. The presence of organic compounds and essential oils in the extract was responsible for potent antidiabetic activities via inhibition of α-amylase enzyme. Also, MMICH exhibited strong free radical scavenging abilities. The current study justifies the use of MMCHI to manage diabetes.

Effects of solvent-solvent fractionation on the total terpenoid content and in vitro anti-inflammatory activity of Serevenia buxifolia bark extract.

Severinia buxifolia (Rutaceae) is often used as a traditional medical plant. The present study was carried out to estimate the effects of solvents (petroleum ether and hexane: ethyl acetate) used in liquid–liquid extraction to total terpenoid content (TTC) and in vitro anti‐inflammatory activity of the extracts obtained from S. buxifolia bark. The results showed that solvent fractionation increased the TTC compared with crude extracts. The hexane: ethyl acetate bark extract fraction (HEF) had the highest TTC (731.48 µg/ml) in comparison with the petroleum ether bark extract fraction (PEF) (564.81 µg/ml) and the crude extract (CE) (184.26 µg/ml). In addition, one of composition of terpenoid of S. buxifolia, namely ursolic acid, was determined by HPLC method from the crude CE and the fractions PEF and HEF: 2.44 μg/g DW, 3.56 μg/g DW and 5.04 μg/g DW, respectively. The samples had an in vitro anti‐inflammatory activity comparable with that of two reference standards (aspirin and indomethacin). Particularly, the HEF fraction had the highest in vitro anti‐inflammatory activity (i.e., albumin denaturation: IC50 = 147.91 μg/mL, heat‐induced hemolysis: IC50 = 159.91 μg/mL, proteinase inhibition: IC50 = 117.72 μg/mL, and lipoxygenase activity: IC50 = 90.45 μg/mL). Besides, the preliminary experiments of this study were conducted to determine the influences of maceration factors (solvent type, temperature, and time) for S. buxifolia bark extract. The TTC ranged from 453.70 to 842.59 mg linalool/g DW, and the extraction yield from 2.40% to 5.120% in all extracts. Based on TTC and EY, the hexane: acetone mixture is recommended as the optimal solvent to obtain the crude bark extract (CE) at 46°C for 24 hr of maceration. Extracts of S. buxifolia bark are a promising source for the treatment of inflammatory diseases.

Bronchodilator effect of apigenin and luteolin, two components of Dracocephalum kotschyi on isolated rabbit trachea

Introduction: Dracocephalum kotschyi is a native Iranian plant with antispasmodic activities on smooth muscles such as ileum and uterus. However, so far antispasmodic effect of D. kotschyi on tracheal smooth muscle has not been reported. Therefore, the objective of this research was to investigate antispasmodic activity of D. kotschyi extract and two of its components luteolin and apigenin on rabbit tracheal contraction in vitro. Methods: Rabbits were euthanized by carbon dioxide and the trachea was dissected and immersed in a Tyrode’s solution. Tracheal rings were prepared and mounted vertically in an organ bath at 37°C and gassed continuously with O2. The tracheal ring preparations were contracted with acetylcholine (ACh) and KCl. The isotonic tension was recorded before and after addition of aminophylline, apigenin, luteolin or flavonoids rich extract of D. kotschyi. Flavonoids rich extract were prepared from D. kotschyi using solvent-solvent fractionation technique. Results: Standard drug aminophylline, prevented tracheal ring preparation contracted with ACh. Cumulative addition of aminophylline also attenuated tonic contraction induced by KCl on tracheal smooth muscle. D. kotschyi extract at concentration ranges of 32-512 μg/mL in a concentration dependent manner inhibited KCl and ACh induced tracheal contraction. Apigenin and luteolin (range 16–512 μg/mL) relaxed KCl and ACh-induced contraction of tracheal smooth muscle in vitro in a concentration-dependent manner. Conclusion: This study demonstrated that D. kotschyi extract is a relaxant of tracheal smooth muscle. The relaxant effect of D. kotschyi extract could be due to its flavonoids component such as apigenin and luteolin.

Evaluation of Bioactive Metabolites Isolated from Endophytic Fungus Chaetomium cupreum of the Plant Mussaenda luteola

Abstract: Background: Endophytic fungi are one of the most promising resources of natural bioactive products. In the present study, secondary metabolites were isolated from the endophytic fungus Chaetomium cupreum and evaluated in vitro for pharmaceutical potentials. Objective: To characterize the secondary metabolites obtained from the endophytic fungus Chaetomium cupreum from the plant Mussaenda luteola and to determine its antioxidant, cytotoxicity and anti-mycobacterial activities. Methods: Three secondary metabolites were isolated by column chromatography and solvent-solvent fractionation method from the ethyl acetate and methanol extract of C. cupreum. The structures were elucidated by spectroscopic studies includes FT-IR, NMR, MS analysis. Anti-mycobacterial activity of metabolites was determined by Microtitre Plate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis H37Rv (ATCC27294) and also evaluated the antioxidant potentials using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging assay. The cytotoxicity test was performed against breast cancer cell line MCF-7 using MTT assay. Results: Three secondary metabolites were characterized as 6- (heptacosa- 18ˈ Z enyl)- 2- (- 18ˈˈ hydroxyl- 1ˈˈ enyl- 19ˈˈ oxy)- 3 hydroxy benzoquinone (1), (3β –5α– dihydroxy –6β– phenyl acetyloxy – ergosta –7, 22 – diene) (2) and 2- dodecanol (3). The results of bio-assays revealed that the compound (1) and (2) exhibit significant inhibition of mycobacterium with MIC of 6.25μg/ml and 25μg/ml respectively, which is similar to the standard streptomycin drug. It also exhibit good DPPH scavenging potential of 72.07±1.95 and 71.63±1.40 % respectively. The cytotoxicity was tested against breast cancer cell line MCF-7 and found compound 1 and 2 had cytotoxicity of 52% and 49% respectively at 100μg/mL. Conclusion: The present study indicated that the isolated secondary metabolites from the endophytic fungus C. cupreum could be a potential lead molecule of antimicrobial agents. Key words: Endophytic fungi, Chaetomium cupreum, Secondary metabolites, Antimycobacterial, Antioxidant.

English

Traditional medicine is widely practiced in Ethiopia. Here we investigate the toxicity of extracts of seven medicinal plants traditionally used to treat breast cancer in Ethiopia. These plants, Sideroxylon oxyacanthum, Zanthoxylum chalybeum, Clematis simensis, Clematis longicauda, Dovyalis abyssinica, Vernonia leopoldi, and Clerodendrum myricoides, were selected based on recommendations by traditional healers and on the frequency of use. After harvesting the plant material, the water content was determined and the powder was subjected to methanol extraction resulting in crude extracts which were tested for cytotoxicity in dose response assay. Then the methanol extract of the most toxic plants was subjected to further solvent-solvent fractionation to gain petroleum ether, hexane, chloroform, ethyl acetate, and water fractions and these were also tested for cytotoxicity in dose response assays. Extracts of Z. chalybeum and C. myricoides were not toxic. The crude extracts of S. oxyacanthum, C. simensis, and D. abyssinica showed cytotoxicity with half maximal inhibitory concentration 50% (IC50) below 1 &mu;g/ml in the human breast cancer cell lines JIMT-1, MCF-7, and HCC1937. The ethyl acetate fraction of V. leopoldi was the most cytotoxic fraction of all fractions tested with an IC50 of 0.87 &mu;g/ml in JIMT-1 cells. The aqueous fraction of S. oxyacanthum and the chloroform fraction of C. simensis were also cytotoxic. In conclusion, our data show a wide difference in in vitro toxicity of medicinal plants used to treat breast cancer patients, which may guide the use of traditional medicine and the choice of plants for isolation of new compounds for cancer treatment. Key words: Cancer, Ethiopia, in vitro cytotoxicity, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT), traditional medicine. &nbsp

Solvent-solvent fractionation can increase the antifungal activity of a Melianthus comosus (Melianthaceae) acetone leaf extract to yield a potentially useful commercial antifungal product

Fungal infections of plants cause major difficulties in agricultural food production and in storage of food especially due to the development of fungal resistance and the negative environmental impact of some chemical fungicides. The aim of this study was to develop a plant-based product that could be used to control plant fungal pathogens in agriculture. Melianthus comosus is a weedy shrub endemic to southern Africa. Its acetone leaf extract had good activity against plant fungal pathogens. We investigated the efficiency of ten extractants with varying polarities. Acetone was the best with a 52 times higher selectivity for antifungal activity against eight important fungal pathogens than methanol. The average minimum inhibitory concentration (MIC) was 100μg/ml varying from 20 to 310μg/ml against eight fungal pathogens. By using open column silica gel chromatography and bioautography the major antifungal compound was isolated and characterized as oleanolic acid. Two methods based on selective extraction and solvent–solvent extraction were used to potentize the activity of the extract by removing inactive polar and non-polar compounds. The product had an average MIC of 66μg/ml varying between 20 and 160μg/ml against 10 fungal pathogens. The product had low water solubility and was stable for a month at 55ᵒC. When used in a field trial on comfrey (Symphytum officinale) with a natural rust infection it was much more effective at a concentration of 0.2mg/ml than the commercial product containing dicarboximide at a concentration of 1.5mg/ml. The Melianthus extract treatment led to 50 infected leaves in the bed compared to 250 infected leaves of the dicarboximide treatment and extensive infection in the negative control. It appears that this product has the potential to be used in agricultural practice. This plant may become a new crop that may be useful for organic cultivation.

Isolation and characterization of the compounds responsible for the antimutagenic activity of Combretum microphyllum (Combretaceae) leaf extracts

BackgroundMutations play a major role in the pathogenesis and development of several chronic degenerative diseases including cancer. It follows, therefore that antimutagenic compound may inhibit the pathological process resulting from exposure to mutagens. Investigation of the antimutagenic potential of traditional medicinal plants and compounds isolated from plant extracts provides one of the tools that can be used to identify compounds with potential cancer chemopreventive properties. The aim of this study was to isolate and characterise the compounds responsible for the antimutagenic activity of Combretum microphyllum.MethodsThe methanol leaf extract of C. microphyllum was evaluated for antimutagenicity in the Ames/microsome assay using Salmonella typhimurium TA98. TA100 and TA102. Solvent-solvent fractionation was used to partition the extracts and by using bioassay-guided fractionation, three compounds were isolated. The antimutagenic activity of the three compounds were determined in the Ames test using Salmonella typhimurium TA98, TA100 and TA102. The antioxidant activity of the three compounds were determined by the quantitative 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging method. The cytotoxicity was determined in the MTT assay using human hepatocytes.ResultsA bioassay-guided fractionation of the crude extracts for antimutagenic activity led to the isolation of three compounds; n-tetracosanol, eicosanoic acid and arjunolic acid. Arjunolic acid was the most active in all three tested strains with a antimutagenicity of 42 ± 9.6%, 36 ± 1.5% and 44 ± 0.18% in S. typhimurium TA98, TA100 and TA102 respectively at the highest concentration (500 μg/ml) tested, followed by eicosanoic acid and n-tetracosanol. The antioxidant activity of the compounds were determined using the quantitative 2,2 diphenyl-1-picryhydrazyl (DPPH)-free radical scavenging method. Only arjunolic acid had pronounced antioxidant activity (measured as DPPH-free scavenging activity) with an EC50 value of 0.51 μg/ml. The cytotoxicity of the isolated compounds were determined in the MTT assay using human hepatocytes. The compounds had low cytotoxicity at the highest concentration tested with LC50 values >200 μg/ml for n-tetracosanol and eicosanoic acid and 106.39 μg/ml for arjunolic acid.ConclusionsBased on findings from this study, compounds in leaf extracts of C. microphyllum protected against 4-NQO and MMC induced mutations as evident in the Ames test. The antimutagenic activity of arjunolic acid may, at least in part, be attributed to its antioxidant activity resulting in the detoxification of reactive oxygen species produced during mutagenesis.

Antibacterial activity of 3,3',4'-Trihydroxyflavone from Justicia wynaadensis against diabetic wound and urinary tract infection.

The present investigation was designed to study the effect of an active compound isolated from Justicia wynaadensis against multi drug resistant organisms (MDRO's) associated with diabetic patients. The drug resistant pathogens implicated in wound and urinary tract infection of diabetic patients were isolated and identified by molecular sequencing. Solvent–solvent fractionation of crude methanol extract produced hexane, chloroform, ethyl acetate and methanol–water fraction, among which chloroform fraction was found to be potent when compared with other three fractions. Further, chloroform fraction was subjected to preparatory HPLC (High-Performance Liquid Chromatography), that produced four sub-fractions; chloroform HPLC fraction 1 (CHF1) through CHF4. Among the sub-fractions, CHF1 inhibited the pathogens effectively in comparison to other three sub-fractions. The purity of CHF1 was found to be >95%. Therefore, CHF1 was further characterized by NMR and FTIR analysis and based on the structure elucidated, the compound was found to be 3,3′,4′-Trihydroxyflavone. The effective dose of this bioactive compound ranged from 32 μg/mL to 1.2 mg/mL. Thus, the present study shows that 3,3′,4′-Trihydroxyflavone isolated from J. wynaadensis is an interesting biopharmaceutical agent and could be considered as a source of antimicrobial agent for the treatment of various infections and used as a template molecule for future drug development.

EVALUATION OF PHYTOCHEMICAL ANALYSIS AND ANTIOXIDANT AND ANTIFUNGAL ACTIVITY OF PITHECELLOBIUM DULCE LEAVES’ EXTRACT

ABSTRACTObjective: Diseases due to pathogenic fungi represent a critical problem to human health which is a major cause of morbidity and mortalityworldwide. Synthetic drugs such as Amphotericin B mitigate many of the side effects associated with liver and kidney dysfunctions. Widely distributedtraditional medicinal plants-based antimicrobial drugs are cost effective in the treatment of infectious diseases having no side effects. Pithecellobiumdulce (Roxb.) belongs to the family of Leguminosae, is an evergreen tree widely distributed in the greater part of India which has been widely used intraditional medicine, but lacks scientific scrutiny. The present study revealed P. dulce extract effectiveness against A. fumigates and Aspergillus nigerand showed antioxidant activity.Methods: Leaves’ extract was prepared according to the polarity of solvent, i.e., hexane, chloroform, acetone, methanol, and water. Phytochemicalanalysis, antioxidant activity, and minimum inhibitory concentration (MIC) were tested against selected fungus.Results: Phytochemical analysis of the extract revealed the presence of alkaloids, flavonoids, saponins, coumarin, tannins, anthocyanin, andtriterpenoids. Methanol and water extract was found to contain significant amounts of alkaloids, phenols, and flavonoids. Methanolic extract showedMIC against tested fungus, and extract was further fractionated by solvent-solvent fractionation and MIC was tested. MIC for A. fumigatus was0.62 mg/ml and for A. niger was 1.25 mg/ml, and the results were comparable with effective synthetic drug Amphotericin B.Conclusion: Extract possesses potent fungicidal activity which, in turn, may be due to the presence of biologically active ingredient.Keywords: Antioxidant activity, Phytochemical activity, Antifungal activity, Minimum inhibitory concentration, Fractionation.

Antibacterial Activity of the Bioactive Fractions of Cyathula uncinulata (Amaranthaceae)

Cyathula uncinulata (Schrad.) Schinz is used in ethnomedicine in various parts of the world. This study isolated and characterized a bioactive compound from C. uncinulata based on its antibacterial activities. Separation of the bioactive compound from the ethyl acetate fractions of the plant was by solvent-solvent fractionation followed by repeated column chromatography. The structure of the compound was elucidated by nuclear magnetic resonance and mass spectroscopic Original Research Article Bisi-Johnson et al.; EJMP, 10(1): 1-9, 2015; Article no.EJMP.18649 2 methods. The NMR spectra of the isolated compound showed that the compound with formula C22H38O7 and molecular weight 414.5329 has a long aliphatic chain made up of sugar and fatty acyl moiety. The minimum inhibitory concentration (MIC) of fractions of extracts ranges from 0.39 to 2.5 mg/ml. The MIC of the final purified compound was 0.34 mg/ml compared to the MIC of 0.19 of kanamycin indicating a reasonable bioactivity. This study thus supports the traditional use of C. uncinulata in the treatment of bacterial infections.

Some Strychnos spinosa (Loganiaceae) leaf extracts and fractions have good antimicrobial activities and low cytotoxicities.

BackgroundStrychnos spinosa Lam. is a deciduous tree used in traditional medicine to treat infectious diseases. This study is designed to determine the antimicrobial, antioxidant and cytotoxic activities of extracts and fractions from leaves of S. spinosa.MethodsExtracts were obtained by maceration with acetone, methanol and dichloromethane/methanol (1/1) while fractions were prepared by liquid-liquid fractionation of the acetone extract. A broth serial microdilution method with tetrazolium violet as growth indicator was used to determine the minimum inhibitory concentration (MIC) against fungi, Gram-positive and Gram-negative bacteria. The antioxidant activity was determined using free-radical-scavenging assays, and the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay was used to determine cytotoxicity.ResultsFour extracts and five fractions had good to weak antimicrobial activity with MICs ranging from 0.04 to >1.25 mg/ml against both fungi and bacteria. The chloroform and ethyl acetate fractions had an MIC of 0.08 mg/ml against Aspergillus fumigatus. The n-butanol fraction had an MIC of 0.04 mg/ml against Cryptococcus neoformans. The hexane and chloroform fractions had an MIC of 0.08 mg/ml against Staphylococcus aureus. The antioxidant activities were much lower than that of the positive controls. Except for the alkaloid extract, all the extracts and fractions had free-radical-scavenging activity (IC50 ranging from 33.66 to 314.30 μg/ml). The cytotoxicity on Vero cells was reasonable to low with LC50 values ranging between 30.56 and 689.39 μg/ml.ConclusionThe acetone extract and the chloroform fraction had the highest antibacterial activity. By solvent-solvent fractionation it was possible to increase the activity against A. fumigatus and to decrease the cytotoxicity leading to a potentially useful product to protect animals against aspergillosis. Our results therefore support the use of S. spinosa leaves in traditional medicine to treat infectious diseases.

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.

Preliminary screening of some fractions of Loxostylis alata (Anacardiaceae) for antimicrobial and antioxidant activities

An acetone extract of Loxostylis alata was separated into six fractions based on polarity by a solventsolvent fractionation procedure and the different fractions were screened for antimicrobial and antioxidant activities. The number of antimicrobial compounds in the carbon tetrachloride (CCl 4 ), chloroform (CC), aqueous methanol (AM) and butanol (BT) fractions were determined by bioautography. Each fraction was loaded onto thin layer chromatography (TLC) plates and eluted using two different mobile solvent systems, namely chloroform/ethyl acetate/formic acid (5:4:1): (CEF) and ethyl acetate/methanol/water (40:5.4:5) (EMW). Relative front (R f ) values of active compounds against bacteria ranged from 0.4 - 0.9 in CEF and EMW solvent system. Similarly, the Rf values of compounds active against fungi were in the range of 0.7 - 0.9. Hexane, carbon tetrachloride, chloroform, aqueous methanol and butanol fractions showed areas of inhibition against bacterial organisms, while only hexane and carbon tetrachloride fractions depicted areas of fungal growth inhibition on their chromatograms. The CCl 4 extract was active against six out of the nine microbial strains used and was particularly active against Staphylococcus aureus, Enterococcus faecalis, Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Microsporum canis with minimum inhibitory concentration (MIC) of 0.04, 0.04, 0.1, 0.1, 0.06 and 0.03 mg/ml, respectively. M. canis was the most sensitive organism with the lowest average MIC of 0.16 mg/ml. Qualitative antioxidation using diphenylpicryl- hydrazyl (DPPH) and qualitative assay using both 2,2'-azinobis-3-ethylbenzothiazoline-6- sulfonate (ABTS) and DPPH radicals revealed the presence of three antioxidant compounds in the AM and BT fractions, while the water fraction had only one antioxidant compound. However, the concentration of the antioxidant compounds was more in the AM and BT fractions as revealed by their pronounced colour intensity on the TLC plates. The water fraction had more free radical scavenging effect against DPPH with EC 50 value of 0.62 ± 0.03 μg/ml. The lower the EC 50 value of an extract, the more effective its antioxidant activity. In a similar manner, TEAC value of water fraction was 2.97, which further indicates superior free radical scavenging effect of the fraction against ABTS radical. The greatest reduction of reactive oxygen species (ROS) production induced by diclofenac was also achieved by the water fraction which on overall shows that the water fraction contains more antioxidants. The presence of antimicrobial and antioxidant activities in the fractions of L. alata suggests that this plant may be a source of pharmacologically active substances with beneficial actions towards disease control and treatment in animals and humans. Key words : Medicinal plant, antibacterial, antifungal, serial microdilution, minimum inhibitory concentration, antioxidants.

Pharmacological and histological effects of Centaurea bruguierana ssp. belangerana on indomethacin-induced peptic ulcer in rats

The species Centaurea bruguierana (DC.) Hand.-Mazz. ssp. belangerana (DC.) Bornm. (CBB) (Asteraceae), known as "Baad-Avard" in Borazjan, Bushehr Province, southern Iran, is used in folk medicine as a hypoglycemic herb in diabetes and as a remedy for peptic ulcer disorders. Total 80% EtOH extract and petroleum ether, CHCl(3), EtOAc, n-BuOH, and remaining fractions obtained by solvent-solvent fractionation of dried aerial flowering parts of the plant were investigated for anti-ulcer activity against indomethacin-induced ulcerogenesis in rats. Anti-ulcer activity was evaluated by measuring the ulcer index (UI) and ulcer inhibition. The UI was significantly reduced in all treated animals. A dramatic decrease in the UI was observed following the administration of total extract (100 mg/kg, p < 0.001) and CHCl(3) fraction (42 mg/kg, ***p < 0.001) in comparison with the control group. The percentage ulcer inhibition with total extract at a dose of 100 mg/kg (97.66%) and CHCl(3) fraction at a dose of 42 mg/kg (96.96%) was found to be higher (p < 0.001) than the reference group (cimetidine 100 mg/kg) (87.08%). The pharmacological and histological results of the present study proved that the aerial flowering parts of CBB possess preventive activity against peptic ulcer, supporting the traditional assertion in southern Iranian folk medicine.

Larvicidal Activity of Centaurea bruguierana ssp. belangerana Against Anopheles stephensi Larvae.

In this study, the total 80% of MeOH extract and also petroleum ether, CHCl3, EtOAc, n-BuOH, and the remaining MeOH fractions obtained by solvent-solvent fractionation of the whole flowering samples of Centaurea bruguierana (DC.) Hand.-Mzt. ssp. belangerana (DC.) Bornm. (Asteraceae), namely "Baad-Avard", collected from Borazjan in Bushehr Province (Bushehr, Iran) were investigated for larvicidal activity against malaria vector, Anopheles stephensi Liston, according to WHO methods. The mortality rate of total extract and petroleum ether fraction in concentration of 40 ppm were 28% and 86% respectively and the other fractions were inactive. The probit regression analysis for the dose-response to petroleum ether fraction treatment of larvae exhibited the LC50 and LC90 values of 15.7 ppm and 48.3 ppm, respectively. As results showed, the larvicidal activity of the petroleum ether fraction would be due to the nonpolar compounds in the plant which further isolation and purification would obtain the more active compounds in lower concentrations useful for preparation of biological insecticides.

Pharmacological and histological study of Centaurea bruguierana ssp. belangerana on indomethacin-induced peptic ulcer in rats

The species Centaurea bruguierana (DC.) Hand.-Mzt. ssp. belangerana (DC.) Bornm. (CBB) (Asteraceae) [1], namely „Baad-Avard“ in Bushehr province, south of Iran, is used in folk medicine as hypoglycemic in diabetes and remedy for peptic ulcer disorders. The inhibitory effects of total extract and fractions of dried flowering samples of CBB collected from Borazjan, Bushehr Province, Iran on indomethacin-induced peptic ulcer in rats were studied. The 80% EtOH extract and petroleum ether, CHCl3, EtOAc, n-BuOH, and remaining MeOH fractions obtained by solvent-solvent fractionation of dried flowering samples of CBB was investigated for anti-ulcer activity against indomethacin-induced ulcerogenesis in rats. The percentage inhibition for total extract with dose of 100mg/kg (99%) and CHCl3 fraction with dose of 42mg/kg (98.95%) were found to be more prominent (***p&lt;0.001) compared to the reference group (cimetidine 100mg/kg, 87.62% ulcer inhibition). A dramatic decrease in ulcer index was observed following the administration of total extract (100mg/kg, ***p&lt;0.001) and CHCl3 fraction (42mg/kg, ***p&lt;0.001) compared to control group. Pharmacoilogical and histological results of the present study proved that the aerial flowering parts of CBB possess preventive activity against peptic ulcer supporting the folkloric assertion in southern Iranian folk medicine. However, these effects are not limited to this species and previous studies have revealed that some other Centaurea species are active as well [2,3,4].