ABSTRACT The objective of this study was to perform preliminary screening of phytochemical compounds and quantification of major phenolics and flavonoid markers in Italian ryegrass extract using HPLC-DAD. Previously, LC–MS analysis has identified different phenolic acids, including caffeic acid, ferulic acid, p-coumaric acid, chlorogenic acid, dihydroxy benzoic acid, propyl gallate, catechin, and six flavonoids including rutin hydroxide, luteolin, kaemferol, vitexin, narcissoside, and myricetin from Italian ryegrass extract. In the present study, Italian ryegrass silage powder was extracted with ethanol: water for 20 min at 90 °C. The extract targeted optimum yield of phenolic acids and flavonoids. Crude phenolic acid and flavonoids were then purified by solid phase extraction method. Purified fractions were then injected into HPLC with a diode-array detector. Quantified concentrations of isolated phenolic acids and flavonoids ranged from 125 to 220 µg/g dry weight. Limits of detection and limits of quantification for all standards (unknown compounds) ranged from 0.38 to 1.71 and 0.48 to 5.19 µg/g dry weight, respectively. Obtained values were compared with previous literatures, indicating that our HPLC-DAD quantification method showed more sensitivity. This method showed better speed, accuracy, and effectiveness compared to previous reports. Furthermore, this study could be very useful for developing phenolic acids and flavonoids from compositions in Italian ryegrass silage feed for pharmaceutical applications and ruminant animals in livestock industries.

Background: Xylanase from bacteria finds use in prebleaching process and bioconversion of lignocelluloses into feedstocks. The xylanolytic enzyme brings about the hydrolysis of complex biomolecules into simple monomer units. This study aims to optimize the cellulase-free xylanase production and cell biomass of Bacillus tequilensis strain ARMATI using response surface methodology (RSM). Results: Statistical screening of medium constituents and the physical factors affecting xylanase and biomass yield of the isolate were optimized by RSM using central composite design at N = 30, namely 30 experimental runs with 4 independent variables. The central composite design showed 3.7 fold and 1.5 fold increased xylanase production and biomass yield of the isolate respectively compared to 'one factor at a time approach',inthe presence of the basal medium containing birchwood xylan (1.5% w/v) and yeast extract (1% w/v), incubated at 40°C for 24 h. Analysis of variance (ANOVA) revealed high coefficient of determination (R2)of0.9978 and 0.9906 for the respective responses at significant level (p < 0.05). The crude xylanase obtained from the isolate showed stability at high temperature (60°C) and alkaline condition (pH 9) up to 4 h of incubation. Conclusions: The cellulase-free xylanase showed an alkali-tolerant and thermo-stable property with potentially applicable nature at industrial scale. This statistical approach established a major contribution in enzyme production from the isolate by optimizing independent factors and represents a first reference on the enhanced production of thermo-alkali stable cellulase-free xylanase from B. tequilensis.

Background: Agro-wastes were used for the production of fibrinolytic enzyme in solid-state fermentation. The process parameters were optimized to enhance the production of fibrinolytic enzyme from Bacillus halodurans IND18 by statistical approach. The fibrinolytic enzyme was purified, and the properties were studied. Results: A two-level full factorial design was used to screen the significant factors. The factors such as moisture, pH, and peptone were significantly affected enzyme production and these three factors were selected for further optimization using central composite design. The optimum medium for fibrinolytic enzyme production was wheat bran medium containing 1% peptone and 80% moisture with pH 8.32. Under these optimized conditions, the production of fibrinolytic enzyme was found to be 6851 U/g. The fibrinolytic enzyme was purified by 3.6-fold with 1275 U/mg specific activity. The molecular mass of fibrinolytic enzyme was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis, and it was observed as 29 kDa. The fibrinolytic enzyme depicted an optimal pH of 9.0 and was stable at a range of pH from 8.0 to 10.0. The optimal temperature was 60°C and was stable up to 50°C. This enzyme activated plasminogen and also degraded the fibrin net of blood clot, which suggested its potential as an effective thrombolytic agent. Conclusions: Wheat bran was found to be an effective substrate for the production of fibrinolytic enzyme. The purified fibrinolytic enzyme degraded fibrin clot. The fibrinolytic enzyme could be useful to make as an effective thrombolytic agent.

Light emitting diode (LED) lights play an important role in the plant physiology and alter the metabolites in a significant manner. Glucosinolates (GSLs), polyphenols, flavonoids and antioxidant properties of Chinese cabbage and kale cultivated in varying LED lights were investigated. Analysis revealed 7 aliphatic, 3 indolyl and 1 aromatic GSLs in Chinese cabbage and kale. The total GSL content ranged from 1.5-19.08 and 1.85-24.87 µmol/g DW, and glucobrassicanapin was the predominant GSL (3) in Chinese cabbage, whereas; sinigrin (3.49 µmol/g DW) was in kale. Blue and red LED lights produced significantly higher amount of GSLs in Chinese cabbage and kale respectively. Results revealed higher amount of total polyphenol (3.845 µg/mL) and total flavanoids (3.939 μg/mL) in Chinese cabbage. Chinese cabbage and kale showed significant antioxidant activities when compare with positive control, and the antioxidant assays were slightly correlated with total GSLs, polyphenols and flavanoids contents. The influence of LED lights on glucobrassicin in Chinese cabbage and kale should be studied extensively, because GSL is the precursor of indole-3-carbinol, a potent anticancer isothiocyanate.

BACKGROUND: From ancient times, marine algae have emerged as alternative medicine and foods, contains the rich source of natural products like proteins, vitamins, and secondary metabolites, especially Chlorella vulgaris (C. vulgaris) contains numerous anti-inflammatory, antioxidants and wound healing substances. Type 2 diabetes mellitus is closely associated with adipogenesis and their factors. Hence, we aimed to investigate the chemical constituents and adipo-genic modulatory properties of C. vulgaris in 3T3-L1 pre-adipocytes. RESULTS: We analysed chemical constituents in ethanolic extract of C. vulgaris (EECV) by LC-MS. Results revealed that the EECV contains few triterpenoids and saponin compounds. Further, the effect of EECV on lipid accumulation along with genes and proteins expressions which are associated with adipogenesis and lipogenesis were evaluated using oil red O staining, qPCR and western blot techniques. The data indicated that that EECV treatment increased differentiation and lipid accumulation in 3T3-L1 cells, which indicates positive regulation of adipogenic and lipogenic activity. These increases were associated with up-regulation of PPAR-γ2, C/EBP-α, adiponectin, FAS, and leptin mRNA and protein expressions. Also, EECV treatments increased the concentration of glycerol releases as compared with control cells. Troglitazone is a PPAR-γ agonist that stimulates the PPAR-y2, adiponectin, and GLUT-4 expressions. Similarly, EECV treatments significantly upregulated PPAR-γ2, adiponectin, GLUT-4 expressions and glucose utilization. Further, EECV treatment decreased AMPK-α expression as compared with control and metformin treated cells. CONCLUSION: The present research findings confirmed that the EECV effectively modulates the lipid accumulation and differentiation in 3T3-L1 cells through AMPK-α mediated signalling pathway.

Effects of cold storage and three common cooking practices, blanching, sauteing, and microwave cooking at different time intervals, on the content of glucosinolate (GSL) anticancer components in six Brassica vegetables were investigated. Eleven GSLs including progoitrin, glucoraphanin, sinigrin, glucoalyssin, gluconapin, glucobrassicanapin, glucoerucin, glucobrassicin, 4-methoxyglucobrassicin, gluconasturtiin, and neoglucobrassicin were quantified using LC-MS and HPLC. Storage at 4 ºC indicated no significant loss of GSLs in broccoli, kohlrabi, and cabbage, and approximately 90-100% of the total concentration of aliphatic and indolyl GSLs were detected. Interestingly, glucoraphanin and glucobrassicin, known as a cancer prevention agents, increased approximately above 50% in broccoli, kohlrabi, and cabbage, while the amount of glucobrassicin decreased by 5% in cauliflower for 5 days at 4 ºC. Blanching of broccoli at 120 sec significantly (36%) decreased total GSLs; however, sautéing and microwaving decreased by13-26%. Individual GSLs have different response at blanching. These findings suggest that different processing methods for each vegetable would be preferred to preserve the nutritional qualities.