Enzyme-treated red rice (Oryza sativa L.) bran extracts mitigate inflammatory markers in RAW 264.7 macrophage cells and exhibit anti-inflammatory efficacy greater/comparable to ferulic acid, catechin, γ-tocopherol, and γ-oryzanol.

ETHNOPHARMACOLOGICAL RELEVANCE: Rice (Oryza sativa L.), a staple food for a significant portion of the global population, has been recognized for its traditional medicinal properties for centuries. Rice bran, a by-product of rice milling, contains many bioactive compounds with potential pharmaceutical and therapeutic benefits. In recent years, research has highlighted the anti-inflammatory potential of rice bran, contributed by the bioactive components concentrated in their bran but, unfortunately, entrapped in the bran matrix, with limited bioavailability. Previous studies have reported that the enzymatic treatment of rice bran improves the bran's bioactive compound profile but did not investigate its impact on chronic conditions such as inflammation. AIM OF THE STUDY: This study investigates the anti-inflammatory effects of endo-1,4-ß-xylanase (ERB) and Viscozyme (VRB) treated red rice bran extracts against lipopolysaccharide-induced inflammation in RAW264.7 macrophages in comparison with non-enzyme-treated bran (CRB). Further established their efficacy with known anti-inflammatory compounds-ferulic acid (FA), catechin (CAT), γ-tocopherol (GTP), and γ-oryzanol (ORZ). MATERIALS AND METHODS: The RAW 264.7 macrophage cells were pre-treated with non-toxic concentrations (10-200 µg/mL) of FA, CAT, GTP, ORZ, CRB, ERB, and VRB, followed by inflammatory stimulation with LPS for 24 h. Further, the cell supernatant and pellets were harvested to study the anti-inflammatory effects by evaluating and measuring their efficacy in inhibiting pro-inflammatory cytokines (TNF-α, IL-6, IL-10, IL-1ß) and mediators (ROS, NO, PGE2, COX2, iNOS) through biochemical, ELISA, and mRNA expression studies. RESULTS: The findings showed that both ERB and VRB effectively inhibited the production of pro-inflammatory markers (TNF-α, IL-6) and mediators (ROS, NO, PGE2) by downregulating mRNA expressions of inflammatory genes (TNF-α, IL-1ß, IL-6, IL-10, COX2, iNOS) and demonstrated anti-inflammatory efficacy higher than CRB. On comparison, ERB demonstrated exceptional efficacy by causing a reduction of 48% in ROS, 20% in TNF-α, and 23% in PGE2 at 10 µg/mL, surpassing the anti-inflammatory capabilities of all the bioactive compounds, FA and ORZ, respectively. At the same time, VRB exhibited remarkable efficacy by reducing NO production by 52% at 200 µg/mL and IL-6 by 66% at 10 µg/mL, surpassing FA, CAT, ORZ, and GTP. Further, ERB downregulated the mRNA expression of IL-10 and iNOS, while VRB downregulated TNF-α, IL-1ß, and COX2 expression. Both extracts equally downregulated IL-6 expression at 10 µg/mL, demonstrating the efficacy more remarkable/on par with established anti-inflammatory compounds. CONCLUSIONS: Overall, enzyme-treated rice bran/extract, particularly ERB, possesses excellent anti-inflammatory properties, making them promising agents for alternatives to contemporary nutraceuticals/functional food against inflammatory diseases.

Impact of extrusion on the content and bioaccessibility of fat soluble nutraceuticals, phenolics and antioxidants activity in whole maize.

The present study investigated the changes in fat soluble nutraceuticals, phenolics and antioxidant activity in extrudates of whole maize flour processed at 20 %, 25 % and 30 % feed moisture (M). Bioaccessibility of them in extrudates was also investigated at 20 % M. Extrusion resulted in a significant reduction in the content of components and antioxidant activity. Retention of phytosterols was more (77 %-100 %) followed by phenolics and flavonoids in extrudates. Reducing power decreased more than threefold, while DPPH radical scavenging activity and total antioxidant activity were half of its activity observed in raw. In bioaccessible fraction, the content of stigmasterol, ß-sitosterol and flavonoids was higher than in raw while phenolics and antioxidant activity were unchanged. Bioaccessibility of carotenoids, phytosterols, phenolics, and flavonoids, as well as antioxidant activity was high, except tocopherols and tocotrienols. Overall, extrusion of maize flour with 20 % M enhanced bioaccessibility most of fat solubles, phenolics and antioxidants activity.

Application of pulverization and thermal treatment to pigmented broken rice: insight into flour physical, functional and product forming properties.

The utilization of rice for food purposes involves pulverization and thermal processing which may affect its quality characteristics. Hence pigmented broken rice was processed in plate mill and hammer mill followed by thermal treatment by toasting to study the physical, and functional characteristics and their effect on rice noodle quality. Results showed that plate milled rice flour showed high concentration of particles with size below 148 µm particle (44%), increased redness (21%), bulk density (17%), sedimentation value (75%), damaged starch (72%), peak viscosity (17%), and caused microstructural changes compared to the hammer mill. The toasting of plate milled red and black rice flour caused an insignificant influence on particle size, color, and bulk density. However, it increased the sedimentation value to 134% and 94% and damaged starch by 44% and 19% in red and black rice flour respectively. Further, a reduction in peak viscosity (22%) in red, and increase (16%) in black, along with microstructural changes were also observed. The rice noodle prepared using plate milled, and toasted red rice flour was sensorily acceptable and exhibited excellent textural properties. The study showed that plate milling and thermal treatment significantly affect the quality characteristics of pigmented rice flour and end-product quality.

Influence of enzyme concentrations in enzymatic bioprocessing of red rice bran: A detailed study on nutraceutical compositions, antioxidant and human LDL oxidation inhibition properties.

Enzymes and their concentrations are crucial factors in improving the release of nutraceuticals bounded to rice bran's cell wall matrix. This study aims to investigate the optimal concentrations of Viscozyme and Fiberzyme at 3-30 beta-glucanase units/2 g in improving the release of phenolics, tocopherols, tocotrienols, and γ-oryzanol fractions and enhancing the bioactivities of red rice bran. At specific concentrations, Fiberzyme increased ferulic (301%) and caffeic acid (691%) in soluble phenolics, p-coumaric acid (98%), and catechin (161%) in bound phenolics as well as γ-oryzanol fractions(32%-134%) and increased ferric reducing power (90%), DPPH (41%), and hydroxyl (25%) radical scavenging activities. Viscozyme enhanced δ,γ,α-tocopherols (11%-164%) and tocotrienols (39%-271%) and scavenging activities against nitric oxide (144%), superoxide anion (120%), and inhibition of human LDL oxidation (40%). Cycloartenyl ferulate, ferulic acid, soluble phenolics, campesteryl ferulate, 24-methylenecycloartanyl ferulate, and α-tocotrienol showed a significant positive correlation with bioactivities. Thus, optimization of enzymatic processing will help process the red rice bran into a nutraceutical rich ingredient having higher biological activity.

Optimization of enzyme-assisted improvement of polyphenols and free radical scavenging activity in red rice bran: A statistical and neural network-based approach.

The current study is focused on optimizing the parameters involved in enzymatic processing of red rice bran for maximizing total polyphenol (TP) and free radical scavenging activity (FRSA). The sequential optimization strategies using central composite design (CCD) and artificial neural network (ANN) modeling linked with genetic algorithm (GA) was performed to study the effect of incubation time (60-90 min), xylanase concentration (5-10 mg/g), cellulase concentration (5-10 mg/g) on the response, i.e., total polyphenol and FRSA. The result showed that incubation time has a negative effect on the response, while the square effect of xylanase and cellulase showed positive effect on the response. A maximum TP of 2,761 mg ferulic acid Eq/100 g bran and FRSA of 778.4 mg Catechin Eq/100 g bran was achieved with incubation time (min) = 60.491; xylanase (mg/g) = 5.4633; cellulase (mg/g) = 11.5825. Furthermore, ANN-GA-based optimization showed better predicting capabilities as compared to CCD.

Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

Changes in the prostaglandin levels in alcohol toxicity: effect of curcumin and N-acetylcysteine.

The present study was undertaken to evaluate the potential role of curcumin, the antioxidant principal from Curcuma longa Linn., and the sulphur-containing amino acid N-acetylcysteine against ethanol-induced changes in the levels of prostanoids. Biochemical assessment of liver damage was done by measuring the activities of serum enzymes (i.e., aspartate transaminase and alkaline phosphatase), which were significantly increased in rats fed ethanol, whereas the elevated levels of these enzymes were decreased after curcumin and N-acetylcysteine treatment to rats fed ethanol. We observed a significant increase in the levels of prostaglandins E(1), E(2), F(2alpha), and D(2) in liver, kidney, and brain. Administration of curcumin and N-acetylcysteine was shown to decrease the level of these prostanoids in the tissue studied.

Serum levels of copper, zinc, iron and ceruplasmin in oral leukoplakia and squamous cell carcinoma.

Trace elements have been extensively studied in recent years to assess whether they have any modifying effects in the etiology of cancer. In the present study serum levels of copper, zinc, ceruloplasmin, total iron and total protein were estimated in 92 patients with oral leukoplakia and squamous cell carcinoma and 45 age and sex matched controls. Copper was significantly increased in oral leukoplakia and cancer in both sexes. The level of zinc decreased significantly only in male patients with leukoplakia and cancer. Copper zinc ratio was found significantly elevated in oral leukoplakia and cancer. A marked increase in serum ceruloplasmin was seen in oral leukoplakia and cancer in both sexes. A significant decrease of serum total iron and proteins were observed only in carcinomas. These findings suggest that reduction in the serum total iron and total protein may be due to malnutrition caused, on its turn, by tumour burden. Serum ceruloplasmin level and copper-zinc ratio demonstrated significant differences in the patients of both sexes from those of controls, while, comparing leukoplakia with cancer, a significant difference is only observed in male patients. Thus, the present study shows that these factors have diagnostic value only in differentiating malignancies from normal and a little value as biomarkers of disease progression. However, the exact mechanism responsible for the alteration of these factors in oral lesions is still not clear and a detailed study on large sample size is therefore needed.

Chemoprevention of oral leukoplakia with vitamin A and beta carotene: an assessment.

We conducted a double-blind placebo-controlled trial to evaluate the chemopreventive potential of either vitamin A alone or beta carotene alone in subjects with oral leukoplakia in Kerala, India. We randomised 160 fishermen and women with oral precancerous lesions to receive oral vitamin A (retinyl acetate 300,000 IU/week x 12 months, n = 50), or beta carotene (360 mg/week x 12 months, n = 55), or placebo (n = 55). Blood, saliva and urine samples were collected at baseline and at exit to study serum micronutrients and mutagenicity assays. Biopsies of the mucosal lesions at entry were performed for histopathological exclusion of malignancy. The subjects were examined once every 2 months to establish clinical response of lesions and toxicity, if any. The results are based on 43 complaint subjects on placebo, 42 on vitamin A and 46 on beta carotene. The complete regression rates were: 10% in the placebo arm, 52% with vitamin A and 33% with beta carotene (P < 0.0001). Homogeneous leukoplakias and smaller lesions responded better than non-homogeneous and larger lesions. No major toxicities were observed. Half of the responders with beta carotene and two thirds with vitamin A relapsed after stopping supplementation. Serum beta carotene concentration increased substantially with beta carotene administration while with vitamin A supplementation there was no change in serum retinol levels. In the vitamin A treated group there was a significant decrease in serum alpha tocopherol. Vitamin A administration resulted in a significant remission of oral leukoplakia without any side effects of prolonged vitamin A supplementation. The results of this study, as well as those from previous studies, appear to provide strong supporting evidence to justify long term trials with vitamin A in subjects with high-risk leukoplakias with oral cancer as an endpoint.

Effect of n-3 fatty acids on VLDL production by hepatocytes is mediated through prostaglandins.

The mechanism of the hypolipidemic effect of n-3 fatty acids was studied using isolated rat hepatocytes maintained in culture. EPA and DHA caused a significant reduction in the incorporation of 3[H]-leucine into apoB associated with the VLDL produced by hepatocytes in culture when compared to that in presence of palmitic acid. Presence of indomethacin, an inhibitor of cyclo-oxygenase reversed the effect of EPA on VLDL synthesis while diethyl carbamazine an inhibitor of lipoxygenase did not show any effect suggesting that the effect of EPA may be mediated through prostaglandins. This was further tested by invivo experiments where animals were fed fish oil containing diet with and without aspirin, which inhibits formation of prostaglandins. The incorporation of 3[H]-leucine into apo B and 14[C]-acetate into cholesterol of VLDL produced by hepatocytes from aspirin treated animals were significantly high. The reversal of the effect of n-3 fatty acids by agents which inhibit the formation of prostaglandin suggests that the n-3 fatty acids may exert their effect on VLDL production by liver cells through prostaglandins.

Prostaglandin metabolism during growth and differentiation of the regenerating vertebrate appendage.

House lizards are able to regenerate their tails. This is an ideal model to study the growth and differentiation of an organ. Prostaglandins (PGs) are local hormones having diverse and potent biological activities. In an effort to understand PG metabolism during the growth and differentiation of the regenerating lizard tail, we analysed the fatty acid (FA) composition of phospholipids are free FAs by GC, the activity of two rate-limiting enzymes (phospholipases A and C), the activity of the enzyme responsible for the oxygenation of polyunsaturated fatty acids to PGs (cyclooxygenase) and characterized the endogenous PGs by HPLC. It was observed that on the 20th day, i.e. the tissue differentiation period, there was an increase in phospholipase A activity, together with a sudden fall in the free arachidonic acid (AA) level, an increase in cyclooxygenase activity and the appearance of endogenous PGE2. PGE2 can stimulate cyclic adenosine monophosphate (cAMP) production and it may stimulate a cascade of events associated with tissue differentiation.

Effect of carnitine on malondialdehyde, taurine and glutathione levels in heart of rats subjected to myocardial stress by isoproterenol.

The effect of carnitine on free fatty acid, malondialdehyde, taurine and glutathione levels in myocardium was studied in rats administered isoproterenol to induce a stress in the myocardium resulting in myocardial ischaemia. Carnitine decreased the levels of free fatty acid and malondialdehyde (an index of lipid peroxidation) when compared to control rats given isoproterenol alone. Taurine and glutathione also registered a fall in the carnitine treated animals when compared to rats treated with isoproterenol alone. The results indicate that carnitine by decreasing the levels of these parameters helps the myocardium to survive from the stress induced by isoproterenol.