Development and characterization of stabilized omega-3 fatty acid and micronutrient emulsion formulation for food fortification.

We report a novel stabilized emulsified formulation containing omega-3 fatty acid (ω-3 FA) and micronutrient that can be readily used for food fortification. The emulsification methodology for producing a stabilized formulation containing both ω-3 FA and micronutrients is described. The formulation was developed considering the human requirement of ω-3 FA and recommended daily allowance of important micronutrients. This formulation was characterized for physical appearance, pH, specific gravity, color measurement etc. Fatty acid analysis revealed formulations (2, 4 and 5 g/serve) were able to provide 500-1300 mg of alpha linoleic acid. Oxidative stability assessment (peroxide value, free fatty acid content) of the formulations showed, stability of the formulation as non-significant alterations were noted in these parameters when the formulations were compared with raw flaxseed oil. Rheological evaluation indicated formulation followed Non-Newtonian system with shear thickening behavior. Particle size was found to be between 673.83 to 798.76 nm and poly-dispersity index was between 0.438 to 0.681. Microscopic analysis by Cryo-SEM analysis of the formulation showed stable homogeneous nature of formulation. Stability of the formulations was confirmed by freeze-thawing, dilution test and emulsion stability index. Acute oral toxicity study as per OECD guideline showed safety of the formulations. Most importantly, in vivo bioavailability study of ω-3 FA confirmed better bioavailability of the metabolites of ω-3 FA i.e. eicosapentaenoic and docosahexaenoic acids in formulation treated group as compared to flax oil and comparable bioavailability to that of fish oil.

Anticancer and antimetastatic potential of enterolactone: Clinical, preclinical and mechanistic perspectives.

Currently cancer is the second leading cause of death globally and worldwide incidence and mortality rates of all cancers of males and females are rising tremendously. In spite of advances in chemotherapy and radiation, metastasis and recurrence are considered as the major causes of cancer related deaths. Hence there is a mounting need to develop new therapeutic modalities to treat metastasis and recurrence in cancers. A significant amount of substantiation from epidemiological, clinical and laboratory research highlights the importance of diet and nutrition in cancer chemoprevention. Enterolactone (EL) is a bioactive phenolic metabolite known as a mammalian lignan derived from dietary lignans. Here in we review the reported anti-cancer properties of EL at preclinical as well as clinical level. Several in-vivo and in-vitro studies have provided strong evidence that EL exhibits potent anti-cancer and/or protective properties against different cancers including breast, prostate, colo-rectal, lung, ovarian, endometrial, cervical cancers and hepatocellular carcinoma. Reported laboratory studies indicate a clear role for EL in preventing cancer progression at various stages including cancer cell proliferation, survival, angiogenesis, inflammation and metastasis. In clinical settings, EL has been reported to reduce risk, decrease mortality rate and improve overall survival particularly in breast, prostate, colon, gastric and lung cancer. Further, the in-vitro human cell culture studies provide strong evidence of the anticancer and antimetastatic mechanisms of EL in several cancers. This comprehensive review supports an idea of projecting EL as a promising candidate for developing anticancer drug or adjunct dietary supplements and nutraceuticals.

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-ß-induced epithelial-mesenchymal transition.

OBJECTIVE: Triple-negative breast cancer (TNBC) is highly metastatic, and there is an urgent unmet need to develop novel therapeutic strategies leading to the new drug discoveries against metastasis. The transforming growth factor-ß (TGF-ß) is known to promote the invasive and migratory potential of breast cancer cells through induction of epithelial-mesenchymal transition (EMT) via the ERK/NF-κB/Snail signaling pathway, leading to breast cancer metastasis. Targeting this pathway to revert the EMT would be an attractive, novel therapeutic strategy to halt breast cancer metastasis. METHODS: Effects of enterolactone (EL) on the cell cycle and apoptosis were investigated using flow cytometry and a cleaved caspase-3 enzyme-linked immunosorbent assay (ELISA), respectively. Effects of TGF-ß induction and EL treatment on the functional malignancy of MDA-MB-231 breast cancer cells were investigated using migration and chemo-invasion assays. The effects of EL on EMT markers and the ERK/NF-κB/Snail signaling pathway after TGF-ß induction were studied using confocal microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and flow cytometry. RESULTS: Herein, we report that EL exhibits a significant antimetastatic effect on MDA-MB-231 cells by almost reverting the TGF-ß-induced EMT in vitro. EL downregulates the mesenchymal markers N-cadherin and vimentin, and upregulates the epithelial markers E-cadherin and occludin. It represses actin stress fiber formation via inhibition of mitogen-activated protein kinase p-38 (MAPK-p38) and cluster of differentiation 44 (CD44). EL also suppresses ERK-1/2, NF-κB, and Snail at the mRNA and protein levels. CONCLUSIONS: Briefly, EL was found to inhibit TGF-ß-induced EMT by blocking the ERK/NF-κB/Snail signaling pathway, which is a promising target for breast cancer metastasis therapy.

Altered Kinetics Properties of Erythrocyte Lactate Dehydrogenase in Type II Diabetic Patients and Its Implications for Lactic Acidosis.

Recent studies have been noted that the erythrocytes from Type II diabetic patients show significantly altered structural and functional characteristics along with the changed intracellular concentrations of glycolytic intermediates. More recent studies from our laboratory have shown that the activities of enzymes of glycolytic pathway changed significantly in RBCs from Type II diabetic patients. In particular the levels of lactate dehydrogenase (LDH) increased significantly. Lactic acidosis is an established feature of diabetes and LDH plays a crucial role in conversion of pyruvate to lactate and reportedly, the levels of lactate are significantly high which is consistent with our observation on increased levels of LDH. Owing to this background, we examined the role of erythrocyte LDH in lactic acidosis by studying its kinetics properties in Type II diabetic patients. Km, Vmax and apparent catalytic efficiency were determined using pyruvate and NADH as the substrates. With pyruvate as the substrate the Km values were comparable but Vmax increased significantly in the diabetic group. With NADH as the substrate the enzyme activity of the diabetic group resolved in two components as against a single component in the controls. The Apparent Kcat and Kcat/Km values for pyruvate increased in the diabetic group. The Ki for pyruvate increased by two fold for the enzyme from diabetic group with a marginal decrease in Ki for NADH. The observed changes in catalytic attributes are conducive to enable the enzyme to carry the reaction in forward direction towards conversion of pyruvate to lactate leading to lactic acidosis.

Enterolactone Suppresses Proliferation, Migration and Metastasis of MDA-MB-231 Breast Cancer Cells Through Inhibition of uPA Induced Plasmin Activation and MMPs-Mediated ECM Remodeling

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the 'uPA-plasmin-MMPs system'.

Altered Erythrocyte Glycolytic Enzyme Activities in Type-II Diabetes.

The activity of enzymes of glycolysis has been studied in erythrocytes from type-II diabetic patients in comparison with control. RBC lysate was the source of enzymes. In the diabetics the hexokinase (HK) activity increased 50 % while activities of phosphoglucoisomerase (PGI), phosphofructokinase (PFK) and aldolase (ALD) decreased by 37, 75 and 64 % respectively but were still several folds higher than that of HK. Hence, it is possible that in the diabetic erythrocytes the process of glycolysis could proceed in an unimpaired or in fact may be augmented due to increased levels of G6P. The lactate dehydrogenase (LDH) activity was comparatively high in both the groups; the diabetic group showed 85 % increase. In control group the HK, PFK and ALD activities showed strong positive correlation with blood sugar level while PGI activity did not show any correlation. In the diabetic group only PFK activity showed positive correlation. The LDH activity only in the control group showed positive correlation with marginal increase with increasing concentrations of glucose.

Hepatoprotective effect of withanolide-rich fraction in acetaminophen-intoxicated rat: decisive role of TNF-α, IL-1ß, COX-II and iNOS.

CONTEXT: Overdose of acetaminophen (APAP) is common in humans and is often associated with hepatic damage. Withania somnifera (L.) Dunal (Solanaceae) shows multiple pharmacological activities including antioxidant and anti-inflammatory potential. OBJECTIVE: To evaluate the possible mechanism of hepatoprotective activity of withanolide-rich fraction (WRF) isolated from a methanolic extract of Withania somnifera roots. MATERIALS AND METHODS: Hepatotoxicity was induced by oral administration of APAP (750 mg/kg, p.o.) for 14 d. The control group received the vehicle. APAP-treated animals were given either silymarin (25 mg/kg) or graded doses of WRF (50, 100 and 200mg/kg) 2 h prior to APAP administration. Animals were killed on 15th day and blood and liver tissue samples were collected for the further analysis. RESULTS: In WRF-treated group, there was significant and dose-dependent (p < 0.01 and p < 0.001) decrease in serum bilirubin, ALP, AST and ALT levels with significant and dose-dependent (p < 0.01 and p < 0.001) increase in hepatic SOD, GSH and total antioxidant capacity. The level of MDA and NO decreased significantly (p < 0.01) by WRF treatment. Up-regulated mRNA expression of TNF-α, IL-1ß, COX-II and iNOS was significantly down-regulated (p < 0.001) by WRF. Histological alternations induced by APAP in liver were restored to near normality by WRF pretreatment. CONCLUSION: WRF may exert its hepatoprotective action by alleviating inflammatory and oxido-nitrosative stress via inhibition of TNF-α, IL-1ß, COX-II and iNOS.

Oral L-glutamine administration attenuated cutaneous wound healing in Wistar rats.

The objective of this study was to evaluate the wound healing potential of L-glutamine in laboratory rats using excision and incision wound models. Excision wounds of size 500 mm(2) and depth 2 mm were made on the dorsal portion of male Wistar rats (230-250 g) and were used for the study of oral L-glutamine (1 g/kg) treatment on the rate of contraction of wound and epithelisation. Histological evaluation of wound tissue was also performed. Six-centimetre-long two linear-paravertebral incisions in male Wistar rats (230-250 g) were used to study the effect of L-glutamine (1 g/kg, p.o.) treatment on tensile strength, total protein and hydroxyproline content in the incision model. Oral administration of L-glutamine (1 g/kg) significantly decreased wound area, epithelisation period and wound index, whereas the rate of wound contraction significantly increased (P < 0·001) when compared with vehicle control rats in the excision wound model. Tensile strength, hydroxyproline content and protein level were significantly increased (P < 0·001) in L-glutamine (1 g/kg, p.o.)-treated rats when compared with vehicle control rats in the incision wound model. Histological evaluation of wound tissue from L-glutamine (1 g/kg, p.o.)-treated rats showed complete epithelialisation with new blood vessel formation and high fibrous tissues in the excision wound model. In conclusion, oral administration of l-glutamine (1 g/kg) promotes wound healing by acting on various stages of wound healing such as collagen synthesis, wound contraction and epithelialisation.

Evaluation of the bioavailability of major withanolides of Withania somnifera using an in vitro absorption model system.

Withania somnifera (L.) Dunal, shows several pharmacological properties which are attributed mainly to the withanolides present in the root. The efficacy of medicinally active withanolides constituents depends on the absorption and transportation through the intestinal epithelium. We examined these characteristics by employing the Sino-Veda Madin-Darby canine kidney cells culture system, which under in vitro condition shows the absorption characteristics similar to the human intestinal epithelium. Thus, the aim of the present investigation was to assess the bioavailability of individual withanolides. Withanolides were diluted in Hank's buffered saline at a concentration of 2 µg/ml were tested for permeability studies carried out for 1 h duration. Permeability was measured in terms of efflux pump (P eff) in cm/s. P eff values of withanolide A (WN A), withanone (WNN), 1,2-deoxywithastramonolide (1,2 DWM), withanolide B (WN B), withanoside IV-V (WS IV-V), and withaferin A were 4.05 × 10(-5), 2.06 × 10(-5), 1.97 × 10(-5), 1.80 × 10(-5), 3.19 × 10(-6), 3.03 × 10(-6) and 3.30 × 10(-7) respectively. In conclusion, the nonpolar and low molecular weight compounds (WN A, WNN, 1,2 DWM, and WN B) were highly permeable. As against this, the glycosylated and polar WS IV and WS V showed low permeability. Surprisingly and paradoxically, the highly biologically active withaferin A was completely impermeable, suggesting that further studies possibly using human epithelial colorectal adenocarcinoma (Caco-2) cells may be needed to delineate the absorption characteristics of withanolides, especially withaferin A.

Significance of antioxidant potential of plants and its relevance to therapeutic applications.

Oxidative stress has been identified as the root cause of the development and progression of several diseases. Supplementation of exogenous antioxidants or boosting endogenous antioxidant defenses of the body is a promising way of combating the undesirable effects of reactive oxygen species (ROS) induced oxidative damage. Plants have an innate ability to biosynthesize a wide range of non-enzymatic antioxidants capable of attenuating ROS- induced oxidative damage. Several in vitro methods have been used to screen plants for their antioxidant potential, and in most of these assays they revealed potent antioxidant activity. However, prior to confirming their in vivo therapeutic efficacy, plant antioxidants have to pass through several physiopharmacological processes. Consequently, the findings of in vitro and in vivo antioxidant potential assessment studies are not always the same. Nevertheless, the results of in vitro assays have been irrelevantly extrapolated to the therapeutic application of plant antioxidants without undertaking sufficient in vivo studies. Therefore, we have briefly reviewed the physiology and redox biology of both plants and humans to improve our understanding of plant antioxidants as therapeutic entities. The applications and limitations of antioxidant activity measurement assays were also highlighted to identify the precise path to be followed for future research in the area of plant antioxidants.

Effect of macronutrient deficiency on withanolides content in the roots of Withania somnifera and its correlationship with molybdenum content.

CONTEXT: The content of withanolides in the roots of Withania somnifera (L.) Dunal (Solanaceae) is important for therapeutic application. Earlier studies have shown that the deficiency of macro- and micronutrients affects the growth of W. somnifera. Therefore, we examined the effect of these deficiencies on the withanolides content of the roots. OBJECTIVE: To examine the effect of molybdenum accretion in nitrogen-, phosphorus-, calcium- and potassium-deficient soils on the accumulation of withanolides in the roots of W. somnifera. Different withanolides have different therapeutic applications hence major bioactive withanolides assume importance. MATERIALS AND METHODS: Methanol extracts of the roots were subjected to HPTLC and individual withanolides were identified by comparing their Rf values with those of the authentic samples. Molybdenum was quantified by atomic absorption spectroscopy. Free radical scavenging activity was monitored by the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay. RESULTS: Molybdenum content in roots of nitrogen-, phosphorus-, calcium-, potassium-deficient, and control plants were 7.02 ± 2.1, 13.1 ± 1.6, 17.1 ± 0.9, 33.5 ± 3.3, and 33.9 ± 1.6 ppm, respectively. Levels of withaferine A increased with the increase in the Mo content in roots from 7.79 ± 2.2 mg/g to 12.57 ± 3.4 mg/g. Antioxidant activity of nitrogen-deficient plants was the lowest (24.7 ± 2.2%) compared to other groups. DISCUSSION AND CONCLUSION: It was observed that nitrogen metabolism-dependent molybdenum uptake influences the withanolides accumulation in the roots.

Isolation, characterization and antihyperlipidemic activity of secoisolariciresinol diglucoside in poloxamer-407-induced experimental hyperlipidemia.

CONTEXT: Linum usitatissimum L. (Linaceae), commonly known as flaxseed, is a good source of dietary fiber and lignans. Earlier we reported cardioprotective, antihyperlipidemic, and in vitro antioxidant activity of flax lignan concentrate (FLC) obtained from flaxseed. OBJECTIVES: To isolate secoisolariciresinol diglucoside (SDG) from FLC and to evaluate the antihyperlipidemic activity of SDG in poloxamer-407 (P-407)-induced hyperlipidaemic mice. MATERIAL AND METHODS: FLC was subjected to column chromatography and further subjected to preparative HPTLC to isolate SDG. The chemical structure of the isolated compound was elucidated by UV, IR, (1)H NMR, (13)C NMR, DEPT, COSY, HSQC, HMBC, ROESY, MS, and specific optical rotation was recorded. Further, we have investigated the antihyperlipidaemic effect of SDG (20 mg/kg) in P-407-induced hyperlipidaemic rats. Hyperlipidaemia was induced by intraperitoneal administration of P-407 (30% w/v). Serum lipid parameters such as total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) levels were measured. RESULTS AND DISCUSSION: The structure and stereochemistry of the isolated compound were confirmed on the basis of 1D and 2D spectral data and characterized as SDG. Finally, isolated pure SDG was screened using a P-407-induced mice model for its antihyperlipidemic action using serum lipid parameters. The isolated SDG (20 mg/kg) significantly reduced serum cholesterol, triglyceride (p < 0.001), very low-density lipoprotein (p < 0.05), and non-significantly increased HDL-C. CONCLUSION: Finally, it was concluded unequivocally that SDG showed antihyperlipidaemic effects in P-407-induced hyperlipidaemic mice. Isolated pure SDG confirms that SDG is beneficial in the prevention of experimental hyperlipidemia in laboratory animals.

Mitochondrial dysfunction in psychiatric and neurological diseases: cause(s), consequence(s), and implications of antioxidant therapy.

Mitochondrial dysfunction is at the base of development and progression of several psychiatric and neurologic diseases with different etiologies. MtDNA/nDNA mutational damage, failure of endogenous antioxidant defenses, hormonal malfunction, altered membrane permeability, metabolic dysregulation, disruption of calcium buffering capacity and ageing have been found to be the root causes of mitochondrial dysfunction in psychatric and neurodegenerative diseases. However, the overall consequences of mitochondrial dysfunction are only limited to increase in oxidative/nitrosative stress and cellular energy crises. Thus far, extensive efforts have been made to improve mitochondrial function through specific cause-dependent antioxidant therapy. However, owing to complex genetic and interlinked causes of mitochondrial dysfunction, it has not been possible to achieve any common, unique supportive antioxidant therapeutic strategy for the treatment of psychiatric and neurologic diseases. Hence, we propose an antioxidant therapeutic strategy for management of consequences of mitochondrial dysfunction in psychiatric and neurologic diseases. It is expected that this will not only reduces oxidative stress, but also promote anaerobic energy production.

Cardioprotective activity of flax lignan concentrate extracted from seeds of Linum usitatissimum in isoprenalin induced myocardial necrosis in rats.

The objective of the study was to evaluate the cardioprotective activity of flax lignan concentrate (FLC) in isoprenalin (ISO) induced cardiotoxicity in rats. Male Wistar rats (200-230 g) were divided into three groups. Group I: control, Group II: isoprenalin, Group III: FLC (500 mg/kg, p.o.) orally for 8 days and in group II and III isoprenalin 5.25 mg/kg, s.c. on day 9 and 8.5 mg/kg on day 10. On day 10 estimation of marker enzymes in serum and haemodynamic parameters were recorded. Animals were sacrificed, histology of heart was performed. Isoprenalin showed cardiotoxicity, manifested by increased levels of marker enzymes and increased heart rate. FLC treatment reversed these biochemical changes significantly compared with ISO group. The cardiotoxic effect of isoprenalin was less in FLC pretreated animals, which was confirmed in histopathological alterations. Haemodynamic, biochemical alteration and histopathological results suggest a cardioprotective protective effect of FLC in isoprenalin induced cardiotoxicity.

Unstable genes unstable mind: beyond the central dogma of molecular biology.

Schizophrenia has a polygenic mode of inheritance and an estimated heritability of over 80%, but success in understanding its genetic underpinnings to date has been modest. Unlike in trinucleotide neurodegenerative disorders, the phenomenon of genetic anticipation observed in schizophrenia or bipolar disorder has not been explained. For the first time, we provide a plausible molecular explanation of genetic anticipation and pathophysiology of schizophrenia, at least in part, with supporting evidence. We postulate that abnormally increased numbers of CAG repeats in many genes being expressed in the brain, coding for glutamine, cumulatively press for higher demand of glutamine in the respective brain cells, resulting in a metabolic crisis and dysregulation of the glutamate-glutamine cycle. This can adversely affect the functioning of both glutamate and GABA receptors, which are known to be involved in psychosis, and may also affect glutathione levels, increasing oxidative stress. The resulting psychosis (gain in function), originating from unstable genes, is described as an effect "beyond the central dogma of molecular biology". The hypothesis explains genetic anticipation, as further expansions in subsequent generations may result in increased severity and earlier occurrence. Many other well described findings provide proof of concept. This is a testable hypothesis, does not deny any known facts and opens up new avenues of research.

Decreased antioxidant enzymes and membrane essential polyunsaturated fatty acids in schizophrenic and bipolar mood disorder patients.

Oxidative stress-mediated cell damage has been considered in the pathophysiology of schizophrenia. Abnormal findings have often been considered related to differences in ethnicity, life style, dietary patterns and medications, all of which influence indices of oxidative stress and oxidative cell damage. To minimize these confounds, schizophrenic patients were compared with age-matched control subjects with the same ethnic background and similar lifestyle, as well as with bipolar mood disorder (BMD) patients. Levels of antioxidant defense enzymes (i.e. superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx) were lower in schizophrenic patients than in controls, indicating conditions for increased oxidative stress. The contents of plasma thiobarbituric acid reactive substances (TBARS) were only marginally higher in schizophrenic patients, who had normal levels of arachidonic acid (AA), a major source of TBARS, indicating no significant oxidative membrane lipid peroxidation. Levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), however, were significantly lower in schizophrenic patients. When the same indices in BMD patients were compared with findings in matched controls, levels of only SOD and CAT were lower in the patients, whereas GPx was not. Again, as in schizophrenia, the contents of TBARS were marginally higher in BMD patients with no change in levels of AA. Levels of alpha-linolenic acid and EPA were significantly lower and levels of DHA were slightly lower in BMD patients. These data indicate that certain biochemical characteristics may be common to a spectrum of psychiatric disorders, and suggest supplementation of antioxidants and essential fatty acids might affect clinical outcome.