Azole-synergistic anti-candidal activity of altenusin, a biphenyl metabolite of the endophytic fungus Alternaria alternata isolated from Terminalia chebula Retz.

In this study, a tropical endophytic fungus, Alternaria alternata Tche-153 was isolated from a Thai medicinal plant Terminalia chebula Rezt. The ethyl acetate extract prepared from the fermentation broth exhibited significant ketoconazole-synergistic activity against Candida albicans. Bioassay-directed fractionation of the ethyl acetate extract led to the isolation of altenusin (1), isoochracinic acid (2), and altenuic acid (3) together with 2,5-dimethyl-7-hydroxychromone (4). Using the disc diffusion method and the microdilution chequerboard technique, only altenusin (1) in combination with each of three azole drugs, ketoconazole, fluconazole or itraconazole at their low sub-inhibitory concentrations exhibited potent synergistic activity against C. albicans with the fractional inhibitory concentration index range of 0.078 to 0.188. This first discovery of altenusin (1) as a new azole-synergistic prototype possessing a biphenyl structure is of significance for further development of new azole-synergists to treat invasive candidiasis.     

<Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz. Fruit Extracts Inhibit Bacterial Triggers of Some Autoimmune Diseases and Potentiate the Activity of Tetracycline

Terminalia chebula Retz. is a northern Indian plant species known for its anti-inflammatory and antimicrobial properties. T. chebula fruit powder was extracted with solvents of varying polarity and screened for bacterial growth inhibition by disc diffusion assay. The minimum inhibitory concentration (MIC) was quantified by both liquid dilution and disc diffusion techniques. To screen for combinatorial effects, the T. chebula fruit extracts were combined with a range of conventional antibiotics and tested against each bacteria using a liquid dilution assay. Where synergy was detected, the optimal ratios were determined using isobologram analysis. Toxicity was examined using Artemia nauplii and HDF bioassays. T. chebula fruit methanolic, aqueous and ethyl acetate extracts displayed strong antimicrobial activity against the bacterial triggers of all autoimmune inflammatory diseases except K. pneumoniae, for which only moderate inhibition was observed. Indeed, MIC values as low as 195 μg/mL were measured for the aqueous extract against a resistant strain of P. aeruginosa. Of further note, both the aqueous and ethyl acetate extracts interacted synergistically in combination with tetracycline against K. pneumoniae (Σ FIC 0.38 and 0.25 respectively). All extracts were nontoxic in the Artemia and HDF toxicity assays, further indicating their potential for medicinal use.     

Inhibitory effect of Torilis anthriscus on growth of microorganisms

Antibacterial and antifungal activities of aqueous, ethanol and ethyl acetate extract of Torilis anthriscus (L.) Gmel. (Apiaceae) were tested in vitro against ten species of bacteria and five of fungi. Antimicrobial properties were determined by disk diffusion and broth tube dilution method. In the minimum inhibitory concentrations (MICs), the ethanol extract showed the highest activity, followed by the ethyl acetate extract and the aqueous extract against bacterial species, while the extracts were inactive against the tested fungi species. The most active extract was chosen to examine the effects of its combinations with commercial antibiotics by checkerboard method. The obtained results showed that the interactions between ethanol extract/streptomycin and ethanol extract/chloramphenicol were additive and indifferent against the tested human-pathogenic bacteria. Synergism and antagonism were not observed.     

In vitro kinetics and antifungal activity of various extracts of Terminalia chebula seeds against plant pathogenic fungi

The aim of the present study was to examine the efficacy of various seed extracts of Terminalia chebula as an antifungal potential against certain important plant pathogenic fungi. The organic extracts of methanol, ethyl acetate and chloroform at the used concentration of 1500 ppm/disc revealed remarkable antifungal effect as a fungal mycelial growth inhibitor against Fusarium oxysporum, Fusarium solani, Phytophthora capsici and Botrytis cinerea, in the range of 41.6–61.3%, along with MIC values ranging from 62.5 to 500 μg/ml. Also, the extracts had a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of B. cinerea. The results obtained from this study suggest that the natural products derived from Terminalia chebula could become an alternative to synthetic fungicides for controlling such important plant pathogenic fungi.     

Screening of antifungal activity of various plant leaves extracts from Indian plants

The present study was designated to evaluate the antifungal activity and to root out the antifungal plant leaf extracts from this Indian folk-flore. The in vitro antifungal assay was performed by agar diffusion test and minimum inhibitory concentration (MIC) for hexane, ethyl acetate, methanol and distilled water plant leaf extracts. Extraction of 17 different plant leaves was carried out in different solvents such as hexane, ethyl acetate, methanol and distilled water. Among them extractive yield of methanol was maximum than the rest of the three solvents. These extracts were screened for their antifungal activity against nine different fungi. Among these ethyl acetate extracts of Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia exhibited maximum antifungal activity against Alternaria sp., Aspergillus parasi, Aspergillus nidulans, Trichoderma harzianum and Aspergillus flavus with MIC of 80, 40 and 20 ppm against Aspergillus nidulans and Alternaria sp. Ethyl acetate extracts showed promising antifungal activity against Adhatoda vasica, Ocimum sanctum and Holoptelea integrifolia against Aspergillus nidulans, and Alternaria sp. might be applicable as fungicide against fungal plants disease.     

Inhibition activities of colchicum luteum baker on lipoxygenase and other enzymes

In vitro enzymes inhibition activities of the crude methanolic extract and various fractions of Colchicum luteum Baker (Liliaceae) including chloroform, ethyl acetate, n-butanol and aqueous were carried out against actylcholinesterase, butyrylcholinesterase, lipoxygenase and urease enzymes. A significant enzyme inhibition activity (89%) is shown by the crude methanolic extract and its fractions against lipoxygenase, while low to significant activity (32–75%) was evident against butyrylcholinesterase. The crude methanolic extract and its various fractions demonstrated low activity (29–61%) against acetylcholinesterase and no activity against urease.     

Inhibitory effects of some invasive alien species leaf extracts against tomato (Lycopersicon esculentum Mill.) bacterial wilt (Ralstonia solanacearum)

The inhibitory activity of selected invasive alien species leaf extracts against tomato bacterial wilt was studied in vitro and greenhouse. In vitro inhibitory activity was undertaken in a disc diffusion sensitivity test. Furthermore, aqueous extracts of Eichhorina crassipes, Mimosa diplotricha and Lantana camara and methanolic extract of Prosopis juliflora which showed better inhibitory effect in vitro were evaluated against R. solanacearum in greenhouse on tomato. The plant extracts were applied and evaluated at three different times of application (at the time of inoculation; two days before pathogen inoculation; and two days after pathogen inoculation). The result showed that most of the treatment combinations significantly reduce disease incidence and area under disease progress curve and increase biomass of tomato plants, but the effectiveness of tested plant species depends on the type of plant species and application time. The application of plant extracts at the time of pathogen inoculation resulted in highest reduction of disease development on tomato plants. Aqueous extract of E. crassipes was found to be the most effective plant extract in disease suppression and increase above-ground biomass compared to inoculated control. The study revealed that the tested plant species have a potential of inhibiting the development of bacterial wilt on tomato.     

Protective effects of luteolin-7-O-β-D-glucuronide methyl ester from the ethyl acetate fraction of Lycopi Herba against pro-oxidant reactive species and low-density lipoprotein peroxidation

In this study the potent scavenging activity of “Lycopi Herba” (LH) extract was studied using the following: evaluation of the total phenolics, measuring the antioxidant activity by Trolox equivalent antioxidant concentration, measuring the scavenging effects on reactive oxygen species, on reactive nitrogen species, and measuring the inhibitory effect on Cu²⁺ induced human low-density lipoprotein oxidation in vitro. The ethyl acetate fraction from the LH extracts were found to have a potent scavenging activity against all of the reactive species tested, as well as an inhibitory effect on LDL oxidation. Therefore, we isolated and identified luteolin-7-O-β-D-glucuronide methyl ester as the major compound from the ethyl acetate fraction of LH and their antioxidant activities were evaluated.     

<Emphasis Type="Italic">In vitro</Emphasis> susceptibility testing of <Emphasis Type="Italic">Microsporum gypseum</Emphasis> isolated from healthy cattle and soil samples against itraconazole,terbinafine, fluconazole and topical veterinarian drugs

The present study evaluated in vitro susceptibility testing of dermatophytes isolates from healthy cattle and soil samples against three antifungal agents and three topical veterinarian drugs. Itraconazole and terbinafine showed a higher in vitro fungicidal activity than fluconazole. The veterinarian drugs LEPECID^® and iodine 5% were more active in vitro than the UNGÜENTO^® spray. All drugs showed fungicidal activity against Microsporum gypseum, and they may be considered as efficient agents for the topical treatment of dermatophytoses in cattle.     

Characterization of medicinal plant-associated biocontrol Bacillus subtilis (SSL2) by liquid chromatography-mass spectrometry and evaluation of compounds by in silico and in vitro methods

Abstract

This study explores the antimicrobial properties of bioactive secondary metabolites extracted from the medicinal plant (Solanum surattense)-associated Bacillus subtilis strain SSL2. The secondary metabolites were extracted from B. subtilis (SSL2) using ethyl acetate, acetone, butanol, chloroform and methanol solvents. The crude extract was tested against two wilt causing pathogens: Ralstonia solanacearum and Fusarium oxysporum. The results revealed that the ethyl acetate extract has maximum inhibition against both the pathogens tested in this study. Furthermore, liquid chromatography-mass spectrometry (LC-MS) analysis of ethyl acetate extract identified 80 different compounds based on mass-to-charge ratio, database difference, resolution of mass spectrum and so on. Among the 80 compounds, citrulline (m/z?=?158.0917), chloramphenicol (m/z?=?195.075) and carnitine (m/z 162.11) were further selected based on m/z ratio for in silico and in vitro analyses. The in silico analysis revealed that citrulline, chloramphenicol and carnitine inhibited the virulent genes phcA (R. solanacearum) and ste12 (F. oxysporum). Further, under in vitro condition, citrulline and chloramphenicol were found to inhibit the growth of R. solanacearum and F. oxysporum. On the basis of the biocontrol activity of B. subtilis (SSL2) in in silico and in vitro conditions, the bacteria could be used as a biocontrol agent against both bacterial and fungal wilt-causing pathogens. However, this needs to be tested in pot studies or field conditions before being used as biocontrol agents.

Communicated by Ramaswamy H. Sarma     

Milk microbiome signatures of subclinical mastitis-affected cattle analysed by shotgun sequencing

Aims: Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next‐generation sequencing 454 GS‐FLX technology to elucidate the microbial community structure of cattle milk. Methods and Results: Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web‐based tool MG‐RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt–zinc–cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed. Conclusions: The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows. Significance and Impact of the Study: The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.     

Isolation and Characterization of Antimicrobial Compounds in Plant Extracts against Multidrug-Resistant Acinetobacter baumannii

The number of fully active antibiotic options that treat nosocomial infections due to multidrug-resistant Acinetobacter baumannii (A. baumannii) is extremely limited. Magnolia officinalis, Mahonia bealei, Rabdosia rubescens, Rosa rugosa, Rubus chingii, Scutellaria baicalensis, and Terminalia chebula plant extracts were previously shown to have growth inhibitory activity against a multidrug-resistant clinical strain of A. baumannii. In this study, the compounds responsible for their antimicrobial activity were identified by fractionating each plant extract using high performance liquid chromatography, and determining the antimicrobial activity of each fraction against A. baumannii. The chemical structures of the fractions inhibiting >40% of the bacterial growth were elucidated by liquid chromatography/mass spectrometry analysis and nuclear magnetic resonance spectroscopy. The six most active compounds were identified as: ellagic acid in Rosa rugosa; norwogonin in Scutellaria baicalensis; and chebulagic acid, chebulinic acid, corilagin, and terchebulin in Terminalia chebula. The most potent compound was identified as norwogonin with a minimum inhibitory concentration of 128 µg/mL, and minimum bactericidal concentration of 256 µg/mL against clinically relevant strains of A. baumannii. Combination studies of norwogonin with ten anti-Gram negative bacterial agents demonstrated that norwogonin did not enhance the antimicrobial activity of the synthetic antibiotics chosen for this study. In conclusion, of all identified antimicrobial compounds, norwogonin was the most potent against multidrug-resistant A. baumannii strains. Further studies are warranted to ascertain the prophylactic and therapeutic potential of norwogonin for infections due to multidrug-resistant A. baumannii.     

Plant growth promotion and fungicidal activity in a siderophore-producing strain of Proteus sp

AProteus strain inhibited mycelial growth ofFusarium oxysporum in vitro. Seed bacterization showed significant plant growth promotion andFusarium-wilt suppression activity ofPhaseolus mungo in a gnotobiotic system. The culture filtrate of this strain exhibited three prominent bands in UV-VIS spectra between 300 and 400 nm. The growth promotion assay of the extracted compound against different indicator organisms indicated the production of a compound related to a 2-oxoacid-type siderophore. The HPLC of the purified ethyl acetate extract of the strains and standard 4-methyl-2-oxopentanoate (2-oxoisocaproate) revealed a single peak, similarly as the coinjection of the extract and the standard. The production of siderophore, probably 2-oxoisocaproate, was demonstrated.     

Enzyme inhibition and radical scavenging activities of aerial parts of Paeonia emodi Wall. (Paeoniaceae)

The ethanolic extract derived from aerial parts of an indigenous medicinal plant Paeonia emodi was screened for enzyme inhibition activities against Urease (jack bean and Bacillus pasteurii) and α-Chymotrypsin. The extract was also investigated for its radical scavenging activity using DPPH assay. The crude extract was found to possess significant enzyme inhibition activities against jack bean (74%) and Bacillus pasteurii (80%) urease and a moderate activity (54%) against α-Chymotrypsin. The extract also displayed excellent (83%) radical scavenging activity. On the basis of these results, the crude extract was subsequently fractionated into n-hexane, chloroform, ethyl acetate, n-butanol and water fractions and tested independently for the aforesaid activities. Significant inhibitory activity against urease enzyme was observed for the ethyl acetate, n-butanol and water fractions while the n-hexane and chloroform fractions were devoid of any such activity. In the α-Chymotrypsin enzyme inhibition studies the activity was concentrated into the ethyl acetate fraction. All the fractions displayed potent radical scavenging activity. The crude extract and fractions thereof were also subjected to total phenolic content determination. A correlation between radical scavenging capacities of extracts and total phenolic content was observed in the majority of cases.     

Antifungal activity of herbal extracts against plant pathogenic fungi

The present study was designed to evaluate in vitro antifungal activity of herbal extracts against three plant pathogenic fungi (viz. Rhizopus stolonifer, Botrytis cinerea and Colletotrichum coccodes). Extracts of leaves and rinds of Garcinia indica, rhizomes of Curcuma aromatica, roots of Glycyrrhiza gahliae, leaves of Nyctanthes arbour-tristis and seeds of Vernonia anthelmintica were used for screening. Screening was done using poisoned food technique. Relatively potent extracts were shortlisted from this study and were further assayed to find out their minimum fungicidal concentration (MFC). From the above studies, it was observed that ethyl acetate extract of rhizomes of C. aromatica and unripe fruit rinds of G. indica have shown the lowest MFC values amongst the other tested plant extracts. This study indicates that the potential of these plant extracts in the management of diseases caused by plant pathogenic fungi.     

Hydroperoxy-cycloartane triterpenoids from the leaves of Markhamia lutea, a plant ingested by wild chimpanzees

In the framework of the phytochemical investigation of plant species eaten by wild chimpanzees in their natural environment in Uganda, leaf samples of Markhamia lutea were selected and collected. The crude ethyl acetate extract of M. lutea leaves exhibited significant in vitro anti-parasitic activity and low cytotoxicity against MRC5 and KB cells. Fractionation of this extract led to six cycloartane triterpenoids, musambins A–C and their 3-O-xyloside derivatives musambiosides A–C. The structures were elucidated on the basis of spectral studies including mass spectroscopy and extensive 2D NMR. Most of the compounds exhibited mild anti-leishmanial and anti-trypanosomal activities.     

Antiproliferative activity of Pterodon pubescens Benth. seed oil and its active principle on human melanoma cells

On a preliminary screening, relevant in vitro antiproliferative activity was observed to the crude ethanolic extract of Pterodon pubescens seed oil against the human melanoma cell line SK MEL 37. The diethyl ether fraction from crude ethanolic extract which exhibited stronger activity was submitted to fractionation by gradient elution with hexane/ethyl acetate. Subfraction A, eluted by hexane/ethyl acetate (80:20), was essentially the most active between all the assayed subfractions with an IC₅₀ of 37 μg/ml calculated by the MTT colorimetric method. At this concentration, subfraction A caused morphological features and internucleosomal DNA fragmentation pattern of apoptosis. Through chromatographic separation, the furane diterpene 1 was isolated from this active subfraction and identified by spectral techniques. Compound 1 showed an IC₅₀ value of 32 μM and fluorescence staining with DAPI revealed some typical nuclear changes which are characteristic of apoptosis. These findings support a role for diterpenoids vouacapan-type skeleton as a model to develop new anticancer agents.     

Antifungal and enzymatic evaluation of plant crude extracts derived from cinnamon and rosemary against Sclerotinia carrot rot

The objective of this study was to assess the antifungal potential of plant crude extracts derived from cinnamon and rosemary against three isolates of Sclerotinia sclerotiorum under in vitro and in vivo conditions. The crude extracts were obtained using two different solvents including ethyl acetate (EA) and ethanol. The results showed that crude extracts of cinnamon are able to reduce mycelial growth of isolate 2 at volatile and contact phase by 35.4% and 78.2%, respectively. Furthermore, sclerotial myceliogenic at contact phase and carpogenic germination of isolate 2 were inhibited by 94.3% and 68.1%, respectively. In general, rosemary extracts showed less inhibitory efficacy than cinnamon. As the most effective treatment, EA extract of cinnamon was analysed using Gas Chromatography/Mass Spectrometry. The results showed the presence of 33 components and the major constituents were E‐cinnamaldehyde (66.4%) followed by Alpha‐Muurolene (4.86%), Alpha‐Copaene (4.73%) and 2H‐1‐Benzopyran‐2‐one (3.72%). The enzyme analysis showed that the activity of phenylalanine ammonia lyase, polyphenoloxidase and peroxidase decrease in the inoculated carrots after application of plant crude extracts indicating that they cannot be considered as resistance inducers against Sclerotinia carrot rot. In conclusion, cinnamon extract was found to be more effective against the pathogen. Although crude extracts of cinnamon and rosemary were able to reduce severity of carrot rot during storage, EA extract of cinnamon (2 g L^?1) was found to have practically significant effect against the disease.     

Antibacterial activity of certain plant extracts against bacterial wilt of tomato

Abstract

Five isolates of Ralstonia solanacearum were isolated from a naturally wilted root of tomato plants grown in Assiut governorate. The antibacterial activity of extract of Datura, Garlic and Nerium were tested in controlling R. solanacearum in vitro and in vivo. Garlic exhibited the strongest antibacterial activity against bacterial wilt in vitro and in vivo followed by Datura and then Nerium. Cold water extracts of these plant species were more effective than hot water extract in the development of the disease in vivo. In greenhouse experiments, the application of the tested plant extract to soil at the time of inoculation, two days before inoculation and two days after inoculation the pathogen, significantly reduced the disease index of wilt on Super Marmande tomato cultivars. The application of plant extracts at the same time as inoculation resulted in the highest reduction of disease index.     

Susceptibility of some clinical isolates of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> to fractions from the aerial parts of <Emphasis Type="Italic">Leuzea carthamoides</Emphasis>

The antimicrobial activity of the dichloromethane extract from aerial parts of Leuzea carthamoides DC. was tested in vitro against 19 Staphylococcus aureus strains (ATCC 25923, CNCTC Mau 43/60, clinical isolates). The extract was fractionated by column chromatography on silica gel into six fractions (petroleum ether, toluene, dichloromethane, ethyl acetate, methanol and water). The minimum inhibitory concentrations (MICs) of the fractions ranged from 64 to 1024 μg/mL. An ethyl acetate fraction (EA 1) with the widest range of activity inhibited all of the strains with MIC in the range 128–512 μg/mL. This fraction exhibited potent activity against strains which showed associated resistance to oxacillin, ciprofloxacin and erythromycin.