Biological Activity and Food Analysis ofCyttaria spp. (Discomycetes)

The biological activity and nutritional composition of Chilean collections ofCyttaria berteroi C. darwinii, C. espinosae, C. harioti andC. johowii have been determined. The crude protein, lipid, ash, and carbohydrate content of the samples examined were similar to that of other edible fungi. Amino acid analysis of ChileanCyttaria showed that proteins of all species are deficient in methionine and cysteine and excepting oneC. espinosae collection, all samples proved to be below the WHO values for the essential amino acids valine, isoleucine, leucine, and lysine. The acute oral toxicity test ofC. espinosae in rats showed that doses up to 2.5 g extract/kg body weight (corresponding to 25.7–38.7 g fresh weight/kg) did not produce mortality or macroscopic damage in the organs examined of the test animals. Cyttaria extracts assayed at 50 fig/ml were inactive or moderately active as inhibitors towards the enzymes xanthine oxidase (0-31%) and β-glucuronidase (0-65%), and lacked antifungal and antibacterial effects in a battery of antimicrobial assays. When administered intravenously at 2.5 mg/kg, the water-soluble extract ofCyttaria produced a hypotensive response in rats (-16 to -21%). Furthermore, most of the aqueous extracts ofC. espinosae andC. harioti showed DNA binding activity. The main sterols fromCyttaria espinosae andC. berteroi were identified as dihydrobrassicasterol derivatives. Our study suggests that edibleCyttaria species do not represent an acute toxicity risk for consumers and that their nutritional value is similar to that of other edible, cultivated mushrooms. The preservation ofCyttaria spp. as a food resource is linked to the protection of the temperateNothofagus forests.     

<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.     

Instant green synthesis of silver nanoparticles using Terminalia chebula fruit extract and evaluation of their catalytic activity on reduction of methylene blue

A novel green approach for the synthesis and stabilization of silver nanoparticles (AgNPs) using water extract of Terminalia chebula (T. chebula) fruit under ambient conditions is reported in this article. The instant formation of AgNPs was analyzed by visual observation and UV–visible spectrophotometer. Further the effect of pH on the formation of AgNPs was also studied. The synthesized AgNPs were characterized by FT-IR, XRD, HR-TEM with EDS and DLS with zeta potential. Appearance of brownish yellow color confirmed the formation of AgNPs. In the neutral pH, the stability of AgNPs was found to be high. The stability of AgNPs is due to the high negative values of zeta potential and capping of phytoconstituents present in the T. chebula fruit extract which is evident from zeta potential and FT-IR studies. The XRD and EDS pattern of synthesized AgNPs showed their crystalline structure, with face centered cubic geometry oriented in (1 1 1) plane. HR-TEM and DLS studies revealed that the diameter of stable AgNPs was approximately 25 nm. Moreover the catalytic activity of synthesized AgNPs in the reduction of methylene blue was studied by UV–visible spectrophotometer. The synthesized AgNPs are observed to have a good catalytic activity on the reduction of methylene blue by T. chebula which is confirmed by the decrease in absorbance maximum values of methylene blue with respect to time using UV–visible spectrophotometer and is attributed to the electron relay effect.     

Efficacy Assessment Of Trichogramma Evanescens And T. Embryophagum ( Bdhym.: Trichogrammatidae), For Control Of Stored Products Moth Pests In Bulk Wheat

The potential ofTrichogramma evanescens Westwood andT. embryophagum Quednau to controlEphestia kuehniella andE. elutella was evaluated under laboratory conditions. BothTrichogramma species parasitised eggs of the twoEphestia spp. in bulk wheat at 1, 2 and 5 cm depth. The release ofT. evanescens was more effective than that ofT. embryophagum resulting in higher rates of parasitism, predation and parasitoid induced mortality. At 17?C and 26?C, the mortality ofEphestia due to the release ofT. evanescens was 67, 78% and ofT. embryophagum 27 and 38%, respectively. The difference in host finding efficiency between the two species was large, at 5 cm depth and 26?C,T. evanescens parasitised about four times more host eggs compared to T.embryophagum.     

Virus-induced virus inhibitors from systemically virus-infected plants

After infection ofNicotiana tabacum cv. Samsun with tobacco mosaic virus (TMV) crude extracts from dark-green spots of upper leaves had a more strongly marked inhibitory effect upon TMV addedin vitro than crude extracts from the surrounding light-green tissue. Likewise, crude extracts from leaves ofNicotiana tabacum cv. Samsun showing recovery after infection with tobacco ringspot virus (TRV) were seen to have a marked inhibitory effect on TMV addedin vitro. The results obtained suggest that virus inhibitors are produced after virus infections not only in hypersensitive hosts but also in systemic hosts. Necrotizing processes are not an indispensable prerequisite of the production of virus-induced virus inhibitors.     

Genome size and polyploidy variation in the tropical hardwood genusTerminalia (Combretaceae)

Chromosome complements and 2C DNA amounts of six species ofTerminalia have been studied.Terminalia oliveri, T. myriocarpa andT. arjuna are diploid (2n = 24),T. chebula andT. bellirica are tetraploid (2n = 48),T. muelleri shows a triploid number (2n = 36). Two well demarcated groups of species are recognizable on the basis of chromosome length and 2C DNA values which range from 3.60 pg (T. oliveri) to 12.80 pg (T. bellirica) showing a 3.5-fold difference. Differences of DNA per basic genome or per chromosome are greatest (1.97-fold) betweenT. oliveri andT. arjuna. Two species groups (1)T. oliveri andT. chebula, and (2)T. myriocarpa, T. arjuna, T. muelleri, T. bellirica, therefore are well differentiated by DNA per basic genome, irrespective of polyploidy. The mean values of the two groups are 1.81 pg and 3.34 pg, respectively, showing a 1.84-fold difference. Within diploids and tetraploids there is 1.97-fold and 1.76-fold variation, respectively.     

Dithiothreitol reactivates desacetoxycephalosporin C synthetase after inactivation

Dithiothreitol (DTT) has been found to stimulate the desacetoxycephalosporin C synthetase (expandase) activity of a frozen crude extract of Streptomyces clavuligerus, even in the presence of an optimum concentration of ascorbate. Catalase, both native and heat-denatured, and bovine serum albumin (BSA) also stimulated the enzyme activity but were less active than DTT. Although fresh extracts were sporadically stimulated, frozen extracts or extracts inactivated by shaking at 37°C in air were consistently activated. It is felt that DTT functions as a reactivating, rather than an activaing, agent. Similar effects were observed with extracts of Cephalosporium acremonium.     

Natural insecticides fromHippocratea excelsa andHippocratea celastroides

Hippocratea excelsa andHippocratea celastroides have therapeutic and insecticide applications in Mexican traditional medicine. The toxicity ofH. excelsa root cortex has been previously demonstrated against the stored grain pest Sitophilus zeamais. To identify the active compounds, several extracts (petroleum ether, CH₂Cl₂, acetone, methanol, and water) and compounds were obtained from the roots, and tested (1% w/w) with a force-feeding assay againstS. zeamais. AllH. excelsa extracts showed high antifeedant activity, and elicited moderate mortality. The triterpenoid pristimerin and a mixture of sesquiterpene evoninoate alkaloids, isolated from the hexane and methanol extracts, respectively, strongly reduced the insect feeding capacity. Other triterpenoids (friedelin, β-sitosterol, canophyllol) isolated from the hexane extract, and the alditol galactitol obtained from the water extract, were innocuous or its activity was not statistically significant. The organic extracts fromH. celastroides only showed moderate antifeedant activity, while the water extract was innocuous. Galactitol was also obtained from this extract.     

Antioxidant and antimicrobial properties of ethanolic extract fromLepista nuda (Bull.) Cooke

Antioxidant capacity and antimicrobial activities ofLepista nuda (Bull.) Cooke extracts obtained with ethanol were investigated. Four complementary test systems, namely DPPH free radical scavenging, β-carotene/linoleic acid systems, total phenolic compounds and total flavonoid concentration, have been used. Linoleic, acid inhibition values ofL. nuda ethanolic extract, BHA and α-to copherol standards were found to be 84.3% 98.9% and 99.2% respectively in the concentration of 160μg/ml. Total flavonoid amount was 8.21 ± 0.56 μg mg^?1 quercetin equivalent while the phenolic compound amount was 48.01 ± 0.29 μg mg^?1 pyrocatechol equivalent in the extract. The antimicrobial activity ofL. nuda extract was testedin vitro by using the agar-well diffusion method. TheL. nuda extract showed antibacterial activity againstMicrococcus flavus, Micrococcus luteus, Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Salmonella enteritidis andEscherichia coli. TheL. nuda extract did not exhibit antican didal activity againstCandida albicans. The extracts could be suitable as antimicrobial and antioxidativeagents in the food industry.     

Antimicrobial activity of volatile component and various extracts ofEnteromorpha linza (Linnaeus) J. Agardh from the coast of Izmir, Turkey

The methanol, dichloromethane, hexane, chloroform and volatile components ofEnteromorpha linza were testedin vitro for their antimicrobial activity against five Gram-positive, four Gram-negative bacteria andCandida albicans ATCC 10239. GC-MS analysis of the volatile components ofE. linza resulted in the identification of 35 compounds which constituted 84.76% of the total compounds. The volatile components ofE. linza consisted of n-tetratriacontane (8.45%), 1-heptadecanamine (6.65%) and docosane (6.46%) as major components. The methanol and chloroform extracts showed more potent antimicrobial activity than hexane and dichloromethane extracts. The volatile oils of these algae did not remarkably inhibit the growth of tested microorganisms.     

Effect of media composition and growth conditions on production of cellulase and β-glucosidase by a mixed fungal fermentation

The effect of growth temperature, medium composition and pH were examined in shake-flask-scale studies to determine the optimum conditions for cellulase production in mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis. The optimum temperature and pH were 27°C and pH 4.6. Decreases in medium complexity and cost were achieved through reductions in the concentration of Ca, Mg, and K salts. Ten litre fermentations were implemented to study the kinetics of substrate utilization and product formation. The pH at which the fermentation was controlled appeared to be the critical parameter for batch growth of mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis.     

Comparative study of genotoxicity and antimutagenicity of methanolic extracts from Teucrium chamaedrys and Teucrium montanum in human lymphocytes using micronucleus assay

Since Teucrium chamaedrys and Teucrium montanum are the most popular plants used in the treatment of many diseases, we evaluated genotoxic potential of their methanolic extracts on cultured human peripheral blood lymphocytes (PBLs) using cytokinesis-block micronucleus (MN) assay. Cultures were treated with four concentrations of both plants (125, 250, 500 and 1,000 μg/ml), both separately and in combination with mitomycin C (MMC). The results revealed that extract of T. chamaedrys administered at the tested concentrations did not significantly affect the mean MN frequency in comparison to untreated cells. Methanolic extract of T. montanum increased the mean MN frequency in PBL at the tested concentrations, but significantly only at the concentration of 1,000 μg/ml. In all tested concentrations, the extract of T. chamaedrys significantly reduced the MMC-induced MN frequency, in a dose dependent manner (r = − 0.687, p < 0.01). The extract of T. montanum decreased the MMC-induced MN frequency at the tested concentrations, but statistically only at 125 μg/ml. Both extracts administered alone did not significantly affect the nuclear division index (NDI) at the tested concentrations. In the combined treatments with MMC, the extract obtained from T. chamaedrys in the concentrations of 500 and 1,000 μg/ml significantly decreased NDI values in comparison to MMC-treated cells alone, while the extract of T. montanum significantly decreased NDI at all tested concentrations. Both extracts nonsignificantly decreased NDI at all tested concentrations in comparison to untreated cells. Our results suggest the important function of T. chamaedrys extract in cancer therapy, this methanolic extract may prevent genotoxic effects of chemotherapy in PBLs.     

The selective allelopathic interaction of a pasture-forest intercropping in Taiwan

An allelopathic interaction of a pasture-forest intercropping system was evaluated by field and greenhouse experiments and by laboratory assays. A study site was situated in the farm of Hoshe Forestry Experiment Station at Nantou County, Taiwan. After deforestation of Chinese fir (Cunninghamia lanceolata), a split plot design of 8 treatments was set up: open ground without planting as control, planted with kikuyu grass, planted with kikuyu grass andAlnus formosana, planted with kikuyu grass andZelkova formosana, planted with kikuyu grass andCinnamomum camphora, planted withA. formosana, planted withZ. formosana, and planted withC. camphora. Field measurements showed that weeds grew luxuriantly six months after treatment in plots which had not been planted with kikuyu grass. However, the growth of weeds was significantly retarded but that of woody plants was not affected when the plots were planted with kikuyu grass. As compared with the tap water control, the aqueous leachate of kikuyu grass stimulated the seedling growth ofC. camphora andA. formosana, but the extract stimulated the growth ofC. camphora and inhibited that ofA. formosana. In contrast, the aqueous extracts of three hardwood plants exhibited variable inhibition on the root initiation of kikuyu grass, and the extract ofZ. formosana revealed the highest phytotoxic effect. The aforementioned extracts and leachates were bioassayed against seed germination and radicle growth of four test plants, namely annual rye grass, perennial rye gras, tall fescue, and Chinese cabbage and against seedling growth ofMiscanthus floridulus. The extract ofZ. formosana revealed the highest phytotoxic effect on the test species while that of kikuyu grass showed the least effect. By means of paper chromatography and high performance liquid chromatography, phytotoxic phenolics were identified and the amount of phytotoxins present was highest in the extract ofZ. formosana but was lowest in that of kikuyu grass. The degree of phytotoxicity and amount of phytotoxins was in good correlation, indicating that a selective allelopathic effect was involved. The findings suggest that allelopathy may contribute benefits in the intercropping system to reduce the need for herbicides and to lessen the labour cost for weed control.Paper No. 346 of the Scientific Journal Series of the Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China. This study was supported in part by grants of Academia Sinica, Taipei, and Council of Agriculture, Executive Yuan of the Republic of China.     

On the long-term survival of keratinophilic fungi in non-sterile soil

Sixty samples belonging to various types of soil have been stored for 10 years (1959–1969), to obtain further data on the long-term survival of keratinophilic fungi in non-sterile soil. The most resistant proved to beC. keratinophilum, which could be detected even after 10 years, thenK. (T.) ajelloi) 8 years survival, followed byT. terrestre andC. (T.) evolceanui, both showing almost the same capacity of resistance. Only 4 years of survival has been noted forM. gypseum, which was able to resist even in the sandy soil. Interesting was the late finding ofC. keratinophilum, C. evolceanui andC. indicum in some soil samples mixed with old bones.     

Effect of some plant extracts on two coccinellid predators of the cowpea aphid,Aphis craccivora (Hom.: Aphididae)

This study was conducted to examine the effects of some aphicidal biologically active plant compounds on the natural enemies of the cowpea aphid,Aphis craccivora Koch. In the laboratory, acetone extracts ofCymbopogon citratus (DC) Stapf.,Momordica charantia L.,Zingiber officinale Rose andAframomum melegueta (Rose) K. Schum, were tested for toxicity to two coccinellid species,Cheilomenes lunata (Fabricius) andCheilomenes vicina (Mulsant) predaceous on the aphid. All the extracts were highly ovicidal to eggs of the two coccinellid predators. None of the extracts caused mortality of eitherC. lunata orC. vicina fourth-instar larvae after 24 h. Larvae treated with the extracts consumed fewer aphids in 24 h than untreated larvae. Ability to pupate was also significantly reduced in larvae treated with the extracts. No morphological deformities were observed in adults emerging from treated larvae.     

Development of the bitterling,Paracheilognathus himantegus (Cyprinidae), with a note on minute tubercles on the skin surface

The development of the eggs and larvae and minute tubercles on the skin surface ofParacheilognathus himantegus larvae were observed. The egg began to hatch approximately 68 hours after insemination and the larvae reached the free-swimming stage 23 days after hatching at water temperature of 22±1°C. The larval development and minute tubercles on the skin surface of this species were similar to those ofAcheilognathus lanceolata, A. limbata, A. signifer andTanakia tanago. However, the shape of the ripe eggs ofP. himantegus differed from those of the four species. As regards the shape of eggs, there was a common characteristic amongP. himantegus, Rhodeus uyekii andA. limbata from Korea. As regards larval development,P. himantegus had two characters also found inRhodeus. These facts seem to suggest thatP. himantegus is closely related toA. lanceolata, A. limbata, A. signifer andT. tanago but is more specialized than these four species, except forA. limbata from Korea.     

Allelopathic effects ofEupatorium riparium on population regulation of two species ofGalinsoga and soil microbes

Summary Allelopathic effect ofEupatorium riparium Regel, a dominant ruderal weed at higher altitudes in Meghalaya state in north-eastern India, was studied on two common sympatric annual weeds,Galinsoga ciliata (Raf.) andG. parviflora Cav. and on soil microbes. Seed germination and radicle and plumule growth of both species ofGalinsoga were suppressed by the aqueous extract and leachate ofE. riparium. Although the leaf leachate, leaf and litter extracts and litter bed caused considerable reduction in leaf area and seed and dry matter production in both species ofGalinsoga, the effect was much more pronounced onG. parviflora. The inhibitory effect was directly correlated with the concentration of the extract and leachate. The soil microbial population and growth of theGalinsoga spp. declined considerably in the experimental pots where the soil had earlier received leachate of different plant parts ofE. riparium growing in it. The presence of the partly decomposed litter ofE. riparium in the pots reduced soil microbial population and growth of the two weeds much more strongly as compared to the litter in the advanced stages of decomposition. The study also revealed that the abundance and colony growth of the two test fungiviz. Trichoderma viride andAspergillus flavus were differentially affected by the allelopathy ofE. riparium; T. viride being favoured andA. flavus inhibited.     

Aqueous Extracts of the Marine Brown Alga Lobophora variegata Inhibit HIV-1 Infection at the Level of Virus Entry into Cells

In recent years, marine algae have emerged as a rich and promising source of molecules with potent activities against various human pathogens. The widely distributed brown alga Lobophora variegata that is often associated with tropical coral reefs exerts strong antibacterial and antiprotozoal effects, but so far has not been associated with specific anti-viral activities. This study investigated potential HIV-1 inhibitory activity of L. variegata collected from different geographical regions, using a cell-based full replication HIV-1 reporter assay. Aqueous L. variegata extracts showed strong inhibitory effects on several HIV-1 strains, including drug-resistant and primary HIV-1 isolates, and protected even primary cells (PBMC) from HIV-1-infection. Anti-viral potency was related to ecological factors and showed clear differences depending on light exposition or epiphyte growth. Assays addressing early events of the HIV-1 replication cycle indicated that L. variegata extracts inhibited entry of HIV-1 into cells at a pre-fusion step possibly by impeding mobility of virus particles. Further characterization of the aqueous extract demonstrated that even high doses had only moderate effects on viability of cultured and primary cells (PBMCs). Imaging-based techniques revealed extract effects on the plasma membrane and actin filaments as well as induction of apoptosis at concentrations exceeding EC₅₀ of anti-HIV-1 activity by more than 400 fold. In summary, we show for the first time that L. variegata extracts inhibit HIV-1 entry, thereby suggesting this alga as promising source for the development of novel HIV-1 inhibitors.     

Neutral proteinase activity ofCandida albicans

Neutral proteinase activity (pH optimum 6–7.5) was demonstrated in cell extracts of the yeast form ofCandida albicans H-317. Proteolytic activity which required activation by treatment with sodium dodecyl sulfate was measured by a colorimetric assay for Azocoll hydrolysis. The activity was inhibited by antipain, chymostatin, phenylmethylsulfonyl fluoride, andp-chloromercuribenzoate, but was not affected by ethylenediaminetetraacetate, leupeptin, or pepstatin A. These results give the first evidence of a neutral proteinase inC. albicans.     

Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

Background

Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and Results

Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C₁₂HSL and C₄HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C₄HSL. F7 also showed antagonistic activity against 3-oxo-C₁₂HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract.

Conclusions

This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.