Bioactive stilbenes from a Bacillus sp. N strain associated with a novel rhabditid entomopathogenic nematode.

AIMS: The aim of the present study was to purify and characterize a natural antimicrobial compound from Bacillus sp. strain N associated with a novel rhabditid entomopathogenic nematode. METHODS AND RESULTS: The cell-free culture filtrate of a bacterium associated with a novel entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp. exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by column chromatography, and two bioactive compounds were isolated and their chemical structures were established based on spectral analysis. The compounds were identified as 3,4',5-trihydroxystilbene (1) and 3,5-dihydroxy-4-isopropylstilbene (2). The presence of 3,4',5-trihydroxystilbene (resveratrol) is reported for the first time in bacteria. Compound 1 showed antibacterial activity against all the four test bacteria, whereas compound 2 was effective against the Gram-positive bacteria only. Compounds 1 and 2 were active against all the five fungi tested and are more effective than bavistin, the standard fungicide. The antifungal activity of the compounds against the plant pathogenic fungi, Rhizoctonia solani is reported for the first time. CONCLUSIONS: Cell-free extract of the bacterium and isolated stilbenes demonstrated high antibacterial activity against bacteria and fungi especially against plant pathogenic fungi. We conclude that the bacterium-associated EPN are promising sources of natural bioactive secondary metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Stilbene compounds can be used for the control of fungi and bacteria.

Antimicrobial activity of Curcuma zedoaria and Curcuma malabarica tubers.

The antimicrobial activity of extracts of Curcuma zedoaria and Curcuma malabarica tubers was tested against six bacterial and two fungal strains using the agar well diffusion and broth dilution methods. Petroleum ether, hexane, chloroform, acetone and ethanol extracts exhibited antibacterial as well as antifungal activity. Acetone and hexane extracts of both tubers showed comparable antimicrobial activity as indicated by minimum inhibitory concentration (MIC) values, but other extracts of Curcuma malabarica showed significantly lower activity than those of Curcuma zedoaria. The MIC values for different strains and extracts ranged from 0.01 to 0.15 mg/ml in Curcuma zedoaria and from 0.01 to 0.94 mg/ml in Curcuma malabarica. Staphylococcus aureus (Gram positive) was inhibited by Curcuma malabarica but not by Curcuma zedoaria. This study is the first report of the antimicrobial properties of Curcuma malabarica. The findings also support the use of Curcuma zedoaria tubers in traditional medicine for the treatment of bacterial and fungal infections.

Cassava latex as a source of linamarase for determination of linamarin.

A major constraint in the enzymatic assays for determination of linamarin in cassava is the preparation of purified linamarase. Cassava latex, which exhibits high linamarase activity, was tried as an alternate source of the enzyme. Enzyme yield from latex was compared with that from rind and leaf. Preparations from latex had significantly higher linamarase activity (approximately 300-fold) compared to leaf and rind. The purification of the enzyme was easier since homogenization of large quantities of tissues could be avoided. A 1 g amount of latex could yield enough enzyme for >3000 assays.

Characterization of linamarase of latex and its localization in petioles in cassava.

The distribution of linamarase in latex and its purification, characterization, and immunocytochemical localization in petioles were studied in order to get an insight into the process of cyanogenesis in cassava. Crude latex exudate exhibited low linamarase activity, but on dilution the activity increased about fivefold. Assay using petiole latex collected in isotonic medium showed that the enzyme was distributed in vesicle-like structures. In vitro studies showed that about 50% activity was released from the vesicle into the medium within an hour. Latex linamarase was purified by ammonium sulfate fractionation and DEAE-cellulose chromatography and was characterized with respect to its amino acid composition and kinetic properties. Gel filtration and SDS-PAGE analysis showed that the enzyme was made up of a 70,000-Da peptide. Immunocytochemical studies on the localization of linamarase in cassava petioles showed sporadic positive staining in the phloem and intense staining in the thickened corners of the collenchyma cells of the cortex, suggesting the distribution of linamarase in laticifers as well as in the cell wall.

Enzyme inhibitors in tuber crops and their thermal stability.

Tubers of Cassava (Manihot esculenta), yams (Dioscorea esculenta), aroids (Amorphophallus campanulatus, Colocasia esculenta, Xanthosoma sagittfolium) and Coleus (Solenostemon rotundifolius) were screened for inhibitory activities against amylase, trypsin and chymotrypsin. Coleus tuber possessed the highest anti-amylase activity, whereas Colocasia tuber was the most potent source of anti-tryptic and anti-chymotryptic activity. Xanthosoma tubers exhibited amylase inhibitory activity and Amorphophallus tubers antiprotease activity. Dioscorea esculenta had low levels of amylase and chymotrypsin inhibitors, while Cassava tubers were totally free of inhibitors. When tubers were processed by pressure cooking, there was significant reduction/complete elimination in inhibitory activity. Partial retention of inhibition was observed in the case of amylase inhibitor in Dioscorea, chymotrypsin inhibitor in Colocasia and trypsin inhibitor in Colocasia, Coleus and Amorphophallus. In vitro experiments on heat stability of the different inhibitors revealed almost similar pattern of inactivation.

Spectrophotometric determination of cyanoglucosides in cassava.

A new method is reported for determination of cyanoglucosides in cassava. The method is simple, rapid, and sensitive. Ten g cassava tuber is homogenized with warm (65-70 degrees C) 80% ethanol (1 + 6, w/v) to extract cyanoglucosides (CNG). The ethanol is evaporated, and an aliquot of the extract (0.1-0.2 mL) is incubated with added linamarase in pH 6.0 phosphate buffer for 15 min at 30 degrees C. The reaction is stopped by adding 0.2 N sodium hydroxide, the solution is neutralized, and cyanide is estimated by adding chloramine T and barbituric acid-pyridine reagent and measuring the absorbance at 570 nm. Complete CNG extraction and rapid inactivation of endogenous linamarase is possible with 80% ethanol. There is no interference from extractives in the linamarase reaction or in the estimation of cyanide. Recovery of added linamarin (as cyanide) is 98% by this assay. The minimum detection limit of cyanide in the assay is 0.1 micrograms/mL.

Ascorbic acid metabolism in rats fed high fat cholesterol diet.

The synthesis and catabolism of ascorbic acid has been studied in rats fed an atherogenic diet as also has the relation between the concentration of ascorbic acid and concentration of lipids in the tissues. The concentration of ascorbic acid was found to decrease in the serum, liver, spleen and adrenals, but not in the kidney, in the animals fed an atherogenic diet when compared with those fed normal diet. An inverse relationship was found between the concentration of ascorbic acid in the tissue and the concentration of lipids. The synthesis of ascorbic acid from D-glucuronalactone did not significantly differ in the liver and kidney, but decreased in the spleen in the animals fed the atherogenic diet. On the other hand, the catabolism of ascorbic acid significantly increased in the liver and spleen.

Ascorbic acid and glycosaminoglycan and lipid metabolism in guinea pigs fed normal and atherogenic diets.

The effect of low and high doses of ascorbic acid on glycosaminoglycan and lipid metabolism was studied in guinea pigs fed both normal and atherogenic diets. The high dose of ascorbic acid (25 mg/100 g body weight/day) decreased the cholesterol level in the liver and aorta but not in the serum in animals fed the normal diet in comparison with those fed the low dose of ascorbic acid (0.1 mg/100 g body weight/day). In animals fed the atherogenic diet, cholesterol decreased in the serum and liver, but not in the aorta. Serum triglycerides were not affected by the dose of ascorbic acid in the group on the normal diet, but in the animals receiving the atherogenic diet, the high dose of ascorbic acid caused serum triglycerides to decrease when compared with the low dose. Hepatic and aortic triglycerides decreased in groups on normal and atherogenic diets receiving the high dose of ascorbic acid. Lipoprotein lipase activity was not affected in the aorta by the dose of ascorbic acid either in the normal or atherogenic diet group. It was increased in the liver and heart in both the groups receiving the low dose of ascorbic acid but decreased in the high dose group. The concentration of all the glycosaminoglycans significantly increased in the aorta of animals on normal diet receiving the high dose of ascorbic acid when compared with the low dose group. In the group on the atherogenic diet, hyaluronic acid was not affected, but all the sulphated glycosaminoglycans increased in the animals receiving the high dose when compared with those receiving the low dose. In the liver all the sulphated glycosaminoglycans increased while hyaluronic acid decreased in both the normal and atherogenic diet groups receiving the high rather than the low dose of ascorbic acid. L-Glutamine:D-fructose-6-phosphate aminotransferase and UDPG dehydrogenase, two key enzymes in the biosynthesis of precursors of glycosaminoglycans, were studied in relation to the dose of ascorbic acid. Hepatic aminotransferase activity was higher both in the normal and atherogenic diet groups when receiving the high rather than the low dose of ascorbic acid. UDPG dehydrogenase was not affected by the dose of ascorbic acid. The activities of the degrading enzymes -- hyaluronidase, beta-glucuronidase, beta-hexosaminidase and aryl sulphatase -- significantly increased both in the normal and atherogenic diet groups when receiving the low rather than the high dose of ascorbic acid. The concentration of PAPS, sulphate activity and sulphotransferase activity were all increased in both the normal and atherogenic diet groups receiving the high dose of ascorbic acid.