Assessment of the antidiabetic potential of extract and novel phytoniosomes formulation of Tradescantia pallida leaves in the alloxan-induced diabetic mouse model.

Diabetes inflicts health and economic burdens on communities and the present antidiabetic therapies have several drawbacks. Tradescantia pallida leaves have been used as a food colorant and food preservative; however, to our knowledge antidiabetic potential of the leaves of T. pallida has not been explored yet. The current study aimed to investigate the antidiabetic potential of T. pallida leaves extract and its comparison with the novel nisosome formulation of the extract. The leaves extract and phytoniosomes of T. pallida in doses of 15, 25 and 50 mg/kg were used to assess the oral glucose loaded, and alloxan-induced diabetic mice models. The biological parameters evaluated were; change in body weight, blood biochemistry, relative organ to body weight ratio and histopathology of the liver, pancreas and kidney. Results revealed that the extract 50 mg/kg and phytoniosomes 25 and 50 mg/kg remarkably reduced the blood glucose level in all hyperglycemic mice by possibly inhibiting α-amylase and α-glucosidase production. Body weight and blood biochemical parameters were considerably improved in phytoniosomes 50 mg/kg treated group. The relative body weight was similar to those of healthy mice in extract 50 mg/kg, phytoniosomes 25 mg/kg, and phytoniosomes 50 mg/kg treated groups. Histopathology showed the regeneration of cells in the CHN50 treated group. Hyphenated chromatographic analysis revealed potent metabolites, which confirmed the antidiabetic potential of the extract by inhibiting α-amylase and α-glucosidase using in silico analysis. The present data suggested that phytoniosomes have shown better antidiabetic potential than crude extract of these leaves.

Fructooligosaccharides from Cynoglossum tubiflorus: Effect of the molecular size on their antidiabetic activity in high-fat diet and alloxan induced diabetic rats.

The use of acetylation followed by silica gel column purification allowed the isolation of eight fructooligosaccharides (FOS) from the ethanol extract of Cynoglossum tubiflorus roots. Each FOS was identified by analyzing its FT-IR, HRMS/MS and NMR data, including 1H, 13C and 2D NMR HH COSY, HMBC and NOESY. In diabetic rats treated with a series of FOS from Glc-(Fru)3 to Glc-(Fru)7, a significant inhibition of intestinal α-amylase was observed. This activity increases proportionally with the FOS molecular size. It was found that they delay the absorption of total cholesterol (TC), ldl-cholesterol (LDL-C) and increase HDL-cholesterol (HDL-C) in a molecular size-dependent manner. This inhibitory effect on the activity of the digestive enzyme causes a significant (p < 0.05) reduction in the level of glucose in the blood as an anti-diabetic action. The ethanolic extract (E.E) exerts a significant effect against α-amylase as well as antihyperglycemic and antihyperlipidemic actions, while its acetylation suppresses these effects. Therefore, this study demonstrates for the first time that pure FOS act as an efficient agent in preventing hyperglycemia and hyperlipidemia and that this action evolves in the same manner with their molecular size.

Chrysin-loaded PEGylated liposomes protect against alloxan-induced diabetic neuropathy in rats: the interplay between endoplasmic reticulum stress and autophagy.

BACKGROUND: Diabetic neuropathy (DN) is recognized as a significant complication arising from diabetes mellitus (DM). Pathogenesis of DN is accelerated by endoplasmic reticulum (ER) stress, which inhibits autophagy and contributes to disease progression. Autophagy is a highly conserved mechanism crucial in mitigating cell death induced by ER stress. Chrysin, a naturally occurring flavonoid, can be found abundantly in honey, propolis, and various plant extracts. Despite possessing advantageous attributes such as being an antioxidant, anti-allergic, anti-inflammatory, anti-fibrotic, and anticancer agent, chrysin exhibits limited bioavailability. The current study aimed to produce a more bioavailable form of chrysin and discover how administering chrysin could alter the neuropathy induced by Alloxan in male rats. METHODS: Chrysin was formulated using PEGylated liposomes to boost its bioavailability and formulation. Chrysin PEGylated liposomes (Chr-PLs) were characterized for particle size diameter, zeta potential, polydispersity index, transmission electron microscopy, and in vitro drug release. Rats were divided into four groups: control, Alloxan, metformin, and Chr-PLs. In order to determine Chr- PLs' antidiabetic activity and, by extension, its capacity to ameliorate DN, several experiments were carried out. These included measuring acetylcholinesterase, fasting blood glucose, insulin, genes dependent on autophagy or stress in the endoplasmic reticulum, and histopathological analysis. RESULTS: According to the results, the prepared Chr-PLs exhibited an average particle size of approximately 134 nm. They displayed even distribution of particle sizes. The maximum entrapment efficiency of 90.48 ± 7.75% was achieved. Chr-PLs effectively decreased blood glucose levels by 67.7% and elevated serum acetylcholinesterase levels by 40% compared to diabetic rats. Additionally, Chr-PLs suppressed the expression of ER stress-related genes (ATF-6, CHOP, XBP-1, BiP, JNK, PI3K, Akt, and mTOR by 33%, 39.5%, 32.2%, 44.4%, 40.4%, 39.2%, 39%, and 35.9%, respectively). They also upregulated the miR-301a-5p expression levels by 513% and downregulated miR-301a-5p expression levels by 65%. They also boosted the expression of autophagic markers (AMPK, ULK1, Beclin 1, and LC3-II by 90.3%, 181%, 109%, and 78%, respectively) in the sciatic nerve. The histopathological analysis also showed that Chr-PLs inhibited sciatic nerve degeneration. CONCLUSION: The findings suggest that Chr-PLs may be helpful in the protection against DN via regulation of ER stress and autophagy.

Symbiotic Antidiabetic Effect of Lactobacillus casei and the Bioactive Extract of Cleome droserifolia (Forssk.) Del. on Mice with Type 2 Diabetes Induced by Alloxan.

The consumption of probiotics protects pancreatic ß-cells from oxidative damage, delaying the onset of type 2 diabetes mellitus (T2DM) and preventing microvascular and macrovascular complications. This study aimed to evaluate the antidiabetic activity of CDE fermented by Lactobacillus casei (ATCC 39539) (LC) in alloxan-induced diabetic rats. The oxidative stress identified by catalase (CAT), serum AST, ALT, ALP, creatinine, urea, and uric acid were measured. The chemical profiles of the plant extract and the fermented extract were studied using HPLC/MS. The potential of the compounds towards the binding pockets of aldose reductase and PPAR was discovered by molecular docking. A significant reduction in fasting blood glucose in alloxan-treated rats. The CAT showed a significant decrease in diabetic rats. Also, serum AST, ALT, ALP, creatinine, urea, and uric acid were significantly decreased in the mixture group. Mild histological changes of pancreatic and kidney tissues suggested that the mixture of probiotics and cleome possesses a marked anti-diabetic effect. Overall, the study suggests that the combination of Cleome droserifolia fermented by Lactobacillus casei exhibits significant antidiabetic activity (p-value=0.05), reduces oxidative stress, improves lipid profiles, and shows potential for the treatment of diabetes.

Mentha piperita silver nanoparticle-loaded hydrocolloid film for enhanced diabetic wound healing in rats.

OBJECTIVE: To investigate the role of Mentha piperita silver nanoparticle-loaded carbopol gel for enhanced wound healing in a diabetic rat model. This research further aims to explore bioactive compounds derived from Mentha piperita obtained from high altitude. METHOD: Methanolic extracts of Mentha piperita (MP), Mentha spicata (MS) and Mentha longifolia (ML) were used to synthesise silver nanoparticles (AgNP). AgNP synthesis was confirmed by ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antioxidant activity was assessed by 2, 2-diphenyl-1-picrylhydrazyl (DDPH) assay. Antiglycation potential was determined by measuring the fluorescent advanced glycation end products. The bioactive compound identified in the Mentha piperita methanolic (MPM) fraction through electrospray ionisation tandem mass spectrometric analysis (ESI-MS) was responsible for the highest antiglycation. The effects of MPM and MPM.AgNP-loaded Carbopol (Sanare Lab, India) on wound healing were compared in male, alloxan-induced, diabetic albino rats (200-250g), divided into control and treated groups. Effects on wound healing were assessed via histopathology. RESULTS: UV-Vis and FTIR confirmed NP synthesis with peaks for flavonoids and polyphenols. SEM and XRD explored the cubical, 30-63nm crystalline NP. The maximum antioxidant and antiglycation potential was observed in order of; MP.AgNP>MS.AgNP>ML.AgNP. The highest antioxidant activity was observed by methanolic and aqueous MP.AgNPs (88.55% and 83.63%, respectively) at 2mg.ml-1, and (75.16% and 69.73%, respectively) at 1mg.ml-1, compared to ascorbic acid (acting as a positive control, 90.01%). MPM.AgNPs demonstrated the best antiglycation potential of 75.2% and 83.3% at 1mg.ml-1 and 2mg.ml-1, respectively, comparable to positive control (rutin: 88.1%) at 14 days post-incubation. A similar trend was observed for antimicrobial activity against Bacillus subtilis, Micrococcus luteus and Escherichia coli with an inhibition zone of 21mm, 21.6mm and 24.6mm. Rosmarinic acid was the active compound present in Mentha piperita, as identified by ESI-MS. MPM.AgNP-loaded Carbopol resulted in 100% wound closure compared with control at 20 days post-wounding. In the treatment group, re-epithelialisation was achieved by day 18, compared with 25 days for the positive control group. CONCLUSION: MPM.AgNP-loaded Carbopol demonstrated safer and more effective biological properties, hence accelerating the diabetic excision wound healing process in alloxan-induced diabetic rats.

In-vitro and in-vivo antidiabetic activity of aerial parts of Aitchisonia rosea supported by phytochemical and GC-MS analysis.

Medicinal plants contain a wide variety of bioactive phytoconstituents which can serve as new therapeutic agents for several diseases. This study examines the antidiabetic potential of Aitchisonia rosea in alloxan-induced diabetic rats and identifies its bioactive phytoconstituents using GC-MS. In vitro, antidiabetic potential was established using the α-amylase inhibition assay. In vivo, antidiabetic potential was investigated by employing the oral glucose tolerance test (OGTT). GC-MS analysis was used to identify the bioactive phytoconstituents. The in vitro and in vivo tests showed that the aqueous extract of A. rosea possesses better antidiabetic potential. The α-amylase inhibition assay highlighted an IC50 value of 134.87µg/ml. In an oral glucose tolerance test, rats given an aqueous A. rosea extract significantly lowered their blood sugar levels significant reduction in the blood glucose concentration was observed in the oral glucose tolerance test in rats treated with the aqueous A. rosea extract. GC-MS investigation revealed many phytoconstituents, with serverogenin acetate and cycloheptasiloxane tetradecamethyl being important antidiabetic agents. This study found anti-diabetic properties in A. rosea extract. The phytochemical and GC-MS investigation also found serverogenin acetate and cycloheptasiloxane tetradecamethyl, which could be used to develop new antidiabetic drugs.

CARDIOPROTECTIVE EFFECT OF GLYCYRRHIZA GLABRA EXTRACT AND GLYCYRRHIZA GLABRA SILVER NANOPARTICLE AGAINST ALLOXAN AND NICOTINAMIDE INDUCED DIABETIC CARDIAC INJURY IN RATS.

Objective - to study the Cardioprotective effect of Glycyrriza glabra ethanolic extract and Glycyrrhiza glabra Silver nanoparticle against alloxan and nicotinamide-induced diabetic cardiac injury in adult female Rats. The current study was performed on 36 days in which the G. glabra extract and G. glabra extract loaded on Silver nanoparticles were given to alloxan and nicotinamide-induced diabetic cardiac injured rats. The Cardioprotective effect has been evaluated biochemically. The results of induction of diabetic cardiac injury for 36 days showed a significantly increased (P˂0.05) serum Cardiac Troponin I (cTn-I) and Creatine Kinase (CK-MB) concentration in the diabetic cardiac injury induced (G2) group when compared with the control group (G1), and showed a significant decrease (P˂0.05) in the serum cTn-I and CK-MB concentration in (G3) group (received G. glabra extract) and (G4) group (G.glabra loaded on silver nanoparticle) in comparison with G2. This study concluded that Glycyrriza glabra extract and Glycyrrhiza glabra Silver nanoparticle have a significant Cardioprotective effect induced by alloxan and nicotinamide.

Effect of heterologous platelet-rich plasma on liver and modulation of glucose metabolism and Wnt signalling pathways in diabetic mice.

BACKGROUND: The current study was designed to highlight the effects of heterologous platelet-rich plasma (PRP) on deteriorated hepatic tissues and impaired glucose metabolism of alloxan-induced diabetic mice. METHODS: 30 male mice were divided into a control (CG), PRP (PG), diabetic (DG), and two treated groups (T1G and T2G). PG was given PRP treatment (0.5 ml/kg body weight) twice a week for four weeks. DG, T1G and T2G were given alloxan (150 mg/kg) to induce diabetes. After confirmation, PRP treatment was given to T1G and T2G for two and four weeks respectively while DG was left untreated. Upon completion of the said experimental period, liver samples were taken for histological and gene expression analyses. RESULTS: The study found that the liver tissue of the DG group showed signs of damage, including hepatocyte ballooning, sinusoid dilatation, and collagen deposition. However, these changes were significantly reduced in both T1G and T2G groups. The expression of several genes related to liver function was also affected, with upregulation of Fbp1 and Pklr, and downregulation of Pck1 in the DG group. PRP treatment restored Fbp1 expression and also increased the expression of glycolytic pathway genes Hk1 and Gck, as well as Wnt signalling pathway genes Wnt2, Wnt4, and Wnt9a in both treated groups. CONCLUSION: Current study revealed that heterologous PRP may partly alleviate high glucose levels in diabetics possibly by mediating glucose metabolism via inhibition of Wnt signalling pathway.

Fucoidan from Sargassum wightii reduces oxidative stress through upregulating Nrf2/HO-1 signaling pathway in alloxan-induced diabetic cardiomyopathy rats.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a form of cardiac dysfunction caused by diabetes, increasing heart failure and death. Studies shown that hyperglycemia-induced oxidative stress significantly affects heart structure and functional changes during diabetic cardiomyopathy. Fucoidans are sulfated polysaccharide derived from naturally available seaweeds and reported for various biological functions such as antioxidant, anti-diabetic, and anti-inflammatory. However, the therapeutic potential of Indian seaweeds against DCM remains largely unexplored. Therefore, the current study aimed to work on the cardioprotective effect of extracted fucoidan from Sargassum wightii (SwF) in alloxan-induced DCM. METHODS AND RESULTS: Diabetes (DM) was induced with alloxan monohydrate (150 mg/kg-1) dissolved in Nacl (0.9%) overnight-fasted rats. Group III, IV rats were DM induced, followed by treated with SwF (150 mg/kg-1) and (300 mg/kg-1). Group V and VI were non-diabetic rats and received SwF (150 mg/kg-1) and (300 mg/kg-1). SwF reduced classical progressive DM complications such as hyperglycemia, polydipsia, polyphagia, and polyurea in alloxan-induced diabetic rats. Biochemical analysis showed that SwF decreased blood glucose, cardiac markers enzymes, and lipid peroxidation levels compared to diabetic rats. SwF administration significantly increased Nrf2, HO-1, SOD, Catalase, and NQO1 gene expression. In addition, SwF-treated rats showed reduced heart tissue damage with increased Nrf2 and HO-1 protein expression. CONCLUSION: The current research concludes that targeting oxidative stress with SwF provided an effective role in the prevention of DCM. Thus, fucoidan could be used to develop functional food ingredients for DCM.

Fortification of Fermented Camel Milk with Salvia officinalis L. or Mentha piperita Leaves Powder and Its Biological Effects on Diabetic Rats.

The incorporation of fermented camel milk with natural additives possesses numerous benefits for the treatment of various pathological and metabolic conditions. The present study investigated the impact of fortification of fermented camel milk with sage or mint leaves powder (1 and 1.5%, respectively) on glucose and insulin levels, lipid profile, and liver and kidney functions in alloxan-induced diabetic rats. The gross chemical composition of sage and peppermint leaves powder was studied. The chemical composition of sage and mint extracts was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS) of sage and mint extracts. Furthermore, a total of forty-two adult normal male albino rats were included in this study, whereas one group was kept as the healthy control group (n = 6 rats) and diabetes was induced in the remaining animals (n = 36 rats) using alloxan injection (150 mg/kg of body weight). Among diabetic rats groups, a control group (n = 6 rats) was kept as the diabetic control group whereas the other 5 groups (6 rats per group) of diabetic rats were fed fermented camel milk (FCM) or fermented camel milk fortified with 1 and 1.5% of sage or mint leaves powder. Interestingly, the oral administration of fermented camel milk fortified with sage or mint leaves powder, at both concentrations, caused a significant decrease in blood glucose level and lipid profile, and an increase in insulin level compared to the diabetic control and FCM groups. Among others, the best results were observed in the group of animals that received fermented camel milk fortified with 1.5% sage powder. In addition, the results revealed that the fermented camel milk fortified with sage or mint leaves powder improved the liver and kidney functions of diabetic rats. Our study concluded that the use of sage and mint leaves powder (at a ratio of 1.5%) with fermented camel milk produces functional food products with anti-diabetic activity.

Determination of Hypoglycemic, Hypolipidemic and Nephroprotective Effects of Berberis Calliobotrys in Alloxan-Induced Diabetic Rats.

Many plants of the Berberis genus have been reported pharmacologically to possess anti-diabetic potential, and Berberis calliobotrys has been found to be an inhibitor of α-glucosidase, α-amylase and tyrosinase. Thus, this study investigated the hypoglycemic effects of Berberis calliobotrys methanol extract/fractions using in vitro and In vivo methods. Bovine serum albumin (BSA), BSA-methylglyoxal and BSA-glucose methods were used to assess anti-glycation activity in vitro, while in vivo hypoglycemic effects were determined by oral glucose tolerance test (OGTT). Moreover, the hypolipidemic and nephroprotective effects were studied and phenolics were detected using high performance liquid chromatography (HPLC). In vitro anti-glycation showed a significant reduction in glycated end-products formation at 1, 0.25 and 0.5 mg/mL. In vivo hypoglycemic effects were tested at 200, 400 and 600 mg/kg by measuring blood glucose, insulin, hemoglobin (Hb) and HbA1c. The synergistic effect of extract/fractions (600 mg/kg) with insulin exhibited a pronounced glucose reduction in alloxan diabetic rats. The oral glucose tolerance test (OGTT) demonstrated a decline in glucose concentration. Moreover, extract/fractions (600 mg/kg) exhibited an improved lipid profile, increased Hb, HbA1c levels and body weight for 30 days. Furthermore, diabetic animals significantly exhibited an upsurge in total protein, albumin and globulin levels, along with a significant improvement in urea and creatinine after extract/fractions administration for 42 days. Phytochemistry revealed alkaloids, tannins, glycosides, flavonoids, phenols, terpenoids and saponins. HPLC showed the presence of phenolics in ethyl acetate fraction that could be accountable for pharmacological actions. Therefore, it can be concluded that Berberis calliobotrys possesses strong hypoglycemic, hypolipidemic and nephroprotective effects, and could be a potential therapeutic agent for diabetes treatment.

Phenolics Extracted from Jasminum sambac Mitigates Diabetic Cardiomyopathy by Modulating Oxidative Stress, Apoptotic Mediators and the Nfr-2/HO-1 Pathway in Alloxan-Induced Diabetic Rats.

Diabetes mellitus is a chronic metabolic disorder defined as hyperglycemia and pancreatic ß-cell deterioration, leading to other complications such as cardiomyopathy. The current study assessed the therapeutic effects of phenolic acids extracted from Jasminum sambac phenols of leaves (JSP) against diabetes-induced cardiomyopathy in rats. The rats were divided into four groups, with each group consisting of 20 rats. The rats were given intraperitoneal injections of alloxan monohydrate (150 mg/kg) to induce diabetes. The diabetes-induced groups (III and IV) received treatment for six weeks that included 250 and 500 mg/kg of JSP extract, respectively. In the treated rats, the results demonstrated that JSP extract restored fasting glucose, serum glucose, and hyperlipidemia. Alloxan induced cardiomyopathy, promoted oxidative stress, and altered cardiac function biomarkers, including cardiac troponin I, proBNP, CK-MB, LDH, and IMA. The JSP extract-treated rats showed improved cardiac function indicators, apoptosis, and oxidative stress. In diabetic rats, the mRNA expression of caspase-3, BAX, and Bcl-2 was significantly higher, while Bcl-2, Nrf-2, and HO-,1 was significantly lower. In the treated groups, the expression levels of the BAX, Nrf-2, HO-1, Caspase-3, and Bcl-2 genes were dramatically returned to normal level. According to our findings, the JSP extract prevented cardiomyopathy and heart failure in the hyperglycemic rats by improving cardiac biomarkers and lowering the levels of hyperlipidemia, oxidative stress, apoptosis, hyperglycemia, and hyperlipidemia.

Bi-layered living cellular construct resulted in greater healing in an alloxan-induced diabetic porcine model.

Tissue-engineered skin constructs, including bi-layered living cellular constructs (BLCC) used in the treatment of chronic wounds, are structurally/functionally complex. While some work has been performed to understand their mechanisms, the totality of how BLCC may function in wound healing remains unknown. To this end, we have developed a delayed wound healing model to test BLCC cellular and molecular mechanisms of action. Diabetes was chemically-induced using alloxan in Yucatan miniature pigs, and full-thickness wounds were generated on their dorsum. These wounds were either allowed to heal by secondary intention alone (control) or treated with a single or multiple treatments of a porcine autologous BLCC. Results indicated a single treatment with porcine BLCC resulted in statistically significant wound healing at day 17, while four treatments resulted in statistically significant healing on days 10, 13, and 17 compared to control. Statistically accelerated wound closure was driven by re-epithelialisation rather than contraction or granulation. This porcine diabetic model and the use of a porcine BLCC allowed evaluation of healing responses in vivo without the complications typically seen with either xenogenic responses of human/animal systems or the use of immune compromised animals, expanding the knowledge base around how BLCC may impact chronic wounds.

High-fat diet-induced nonalcoholic steatohepatitis is accelerated by low carnitine and impaired glucose tolerance in novel murine models.

Carnitine deficiency and impaired glucose tolerance (IGT) exacerbate liver steatosis. Given the current lack of ideal murine nonalcoholic steatohepatitis (NASH) models, we investigated new NASH models using jvs/+ mice with low carnitine and wild-type mice with low-dose alloxan-induced IGT. The jvs/+ and wild-type mice were divided into jvs/+ mice fed a high-fat diet (HFD) from 3 weeks of age (HF hetero group), wild-type mice with low-dose alloxan treatment fed HFD (AL + HF wild group), wild-type mice fed HFD (HF wild group), and two types of mice fed a normal diet-jvs/+ and wild-type (intact group). All mice were sacrificed at 20 or 40 weeks of age. All male HFD-fed mice showed obesity, IGT, high blood insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR), high liver enzyme levels, and high cholesterol levels. The degree of IGT was the worst in the AL + HF wild group, and blood insulin levels and HOMA-IR score were remarkably increased from 20 to 40 weeks of age. Almost all HFD-fed mice showed steatosis, fibrosis, and lobular inflammation in the centrilobular zone. These changes were accompanied by hepatocyte ballooning and were enhanced at 40 weeks of age. Furthermore, the incidence rate of nodular hyperplasia and adenoma in both the HF hetero and AL + HF wild groups was nearly 30%. We successfully established two novel murine models of NASH using male jvs/+ mice with low carnitine and male wild-type mice with IGT that eventually developed obesity, fatty liver, insulin resistance, liver fibrosis, and tumorigenesis. These results suggest that low carnitine levels and early-stage induction of IGT are important factors in the progression of NASH to tumorigenesis, similar to human NASH.

Deacetylated nimbin analog N2 fortifies alloxan-induced pancreatic ß-cell damage in insulin-resistant zebrafish larvae by upregulating phosphoenolpyruvate carboxykinase (PEPCK) and insulin levels.

This study aims to evaluate the protective behaviour of N2, a semi-natural analog of nimbin, for its anti-diabetic efficacy against alloxan-induced oxidative damage and ß-cell dysfunction in in-vivo zebrafish larvae. A 500 µM of alloxan was exposed to zebrafish larvae for 24 h to induce oxidative stress in the pancreatic ß-cells and co-exposed with N2 to study the protection of N2 by inhibiting ROS by DCFH-DA, DHE and NDA staining along with Cellular damage, apoptosis and lipid peroxidation. The zebrafish was further exposed to 500 µM alloxan for 72 h to induce ß-cell destruction along with depleted glucose uptake and co-exposed to N2 to study the protective mechanism. Glucose levels were estimated, and PCR was used to verify the mRNA expression of phosphoenolpyruvate carboxykinase (PEPCK) and insulin. Alloxan induced (24 h) oxidative stress in the pancreatic ß-cells in which N2's co-exposure inhibited ROS by eliminating O-2 radicals and restoring the glutathione levels, thus preventing cellular damage and lipid peroxidation. The zebrafish exposed to 500 µM alloxan for 72 h was observed with ß-cell destruction along with depleted glucose uptake when stained with 2NBDG, wherein N2 was able to protect the pancreatic ß-cells from oxidative damage, promoted high glucose uptake and reduced glucose levels. N2 stimulated insulin production and downregulated PEPCK by inhibiting gluconeogenesis, attenuating post-prandial hyperglycemia. N2 may contribute to anti-oxidant protection against alloxan-induced ß-cell damage and anti-hyperglycemic activity, restoring insulin function and suppressing PEPCK expression.

Reprint of: Intracellular Production of Superoxide Radical and of Hydrogen Peroxide by Redox Active Compounds.

Several compounds have been found capable of diverting the electron flow in Escherichia coli and thus causing increased intracellular production of O2- and H2O2. One indication of this electron-shunting action was increased cyanide-resistant respiration and one cellular response was increased biosynthesis of the manganese-containing superoxide dismutase and of catalase. Blocking cytochrome oxidase with cyanide or azide increased the electron flow available for reduction of paraquat and presumably of the other exogenous compounds tested and thus increased their biological effects. Paraquat, pyocyanine, phenazine methosulfate, streptonigrin, juglone, menadione, plumbagin, methylene blue, and azure C were all effective in elevating intracellular production of O2- and H2O2. The effect of alloxan appeared paradoxical in that it increased cyanide-resistant respiration without significantly increasing the cell content of the manganese-superoxide dismutase and with only a small effect on the level of catalase. The alloxan effect on cyanide-resistant respiration was artifactual and was due to an oxygen-consuming reaction between alloxan and cyanide, rather than to a diversion of the intracellular electron flow. With paraquat as a representative electron-shunting compound, the increase in biosynthesis of the manganese-superoxide dismutase was prevented by inhibitors of transcription or of translation, but not by an inhibitor of replication. The increase in this enzyme activity, caused by paraquat and presumably by the other compounds, was thus due to de novo enzyme synthesis activated or derepressed at the level of transcription.

A novel in vitro approach to test the effectiveness of fish oil in ameliorating type 1 diabetes.

Diabetes type 1 (T1D) characterized by destruction of pancreatic ß-cells results in inadequate insulin production and hyperglycaemia. Generation of reactive oxygen species and glycosylation end-products stimulates toxic impacts on T1D. Dietary w-3 fatty acids present in Fish oil (FO) might be helpful in the prevention of oxidative stress and lipid peroxidation, thus, beneficial against T1D. But how the cellular secretion from ß-cells under influence of FO affects the glucose homeostasis of peri-pancreatic cells is poorly understood. In the current study, we aimed to introduce an in vitro model for T1D and evaluate its effectiveness in respect of alloxan treatment to pancreatic Min6 cells. We use alloxan in the Min6 pancreatic ß-cell line to induce cellular damage related to T1D. Further treatment with FO was seen to prevent cell death by alloxan and induce mRNA expression of both insulin 1 and insulin 2 isoforms under low-glucose conditions. From the first part of the study, it is clear that FO is effective to recover Min6 cells from the destructive effect of alloxan, and it worked best when given along with alloxan or given after alloxan treatment regime. FO-induced secretion of molecules from Min6 was clearly shown to regulate mRNA expression of key enzymes of carbohydrate metabolism in peri-pancreatic cell types. This is a pilot study showing that an improved in vitro approach of using Min6 along with muscle cells (C2C12) and adipose tissue cells (3T3-L1) together to understand the crosstalk of molecules could be used to check the efficacy of an anti-diabetic drug.

ß-cells regeneration by WL15 of cysteine and glycine-rich protein 2 which reduces alloxan induced ß-cell dysfunction and oxidative stress through phosphoenolpyruvate carboxykinase and insulin pathway in zebrafish in-vivo larval model.

BACKGROUND: Pancreatic ß-cells are susceptible to oxidative stress, leading to ß-cell death and dysfunction due to enhanced ROS levels and type 2 diabetes. To inhibit the ß-cells damages induced by the oxidative stress, the present study investigates the beneficial effect of various peptides (WL15, RF13, RW20, IW13 and MF18) of immune related proteins (cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively). Also, the molecular mechanism of WL15 from cysteine and glycine-rich protein 2 on ß-cell regeneration was identified through PEPCK and insulin pathway. MATERIALS AND METHODS: In this study, a total of five peptides including WL15, RF13, RW20, IW13, and MF18 were derived from immune-related proteins such as cysteine and glycine-rich protein 2, histone acetyltransferase, vacuolar protein sorting associated protein 26B, serine threonine-protein kinase and CxxC zinc finger protein, respectively. These protein sequences were obtained from an earlier constructed transcriptome database of a teleost Channa striatus. The identified peptides were evaluated for their antioxidant as well as antidiabetic activity. Based on the in silico analysis and in-vitro screening experiments, WL15 was predicted to have better antioxidant and antidiabetic activity among the five different peptides. Therefore, WL15 alone was further analyzed for apoptosis, antioxidant capacity, glucose metabolism, and gene expression performance, which was investigated on the alloxan (500 µM) induced zebrafish in vivo larval model. RESULTS: The results showed alloxan exposure to zebrafish larvae for a day, the ROS was generated in the ß-cells. Interestingly, WL15 treatment showed a protective effect by reducing the toxicity of alloxan exposed zebrafish larvae by increasing their survival and heart rate. Moreover, WL15 reduced the intracellular ROS level and apoptosis in alloxan-induced larvae. The superoxide anion and lipid peroxidation levels are also reduced by improving the glutathione content after the WL15 treatment. Besides, WL15 treatment increased the proliferation rate of ß-cells and decreased the glucose level. Further, the gene expression studies revealed that WL15 treatment normalized the PEPCK expression while upregulating the insulin expression in alloxan exposed larvae. CONCLUSION: Overall, the findings indicate that WL15 of cysteine and glycine-rich protein 2 can act as a potential antioxidant for type 2 diabetes patients in respect of improving ß-cell regeneration.

Antidiabetic and pancreas-protective potential of Parthenium Hysterophorous (Carrot grass) in Alloxan induced diabetic rabbits.

Parthenium hysterophorus has been used to cure cancer, fever, malaria, diarrhea, dysentery, and neurologic disorders. This study evaluates the anti-diabetic effects of methanolic extract of P. hysterophorus (MEPH) in alloxan-induced diabetic rabbits. Twenty-five rabbits were divided into 5groups (N=5). Group-I served as a negative control. Groups II to V were injected with freshly prepared alloxan solution 150 mg/kg intraperitoneally to induce diabetes. Group II till V received following treatments orally: Group II: Alloxan 150 mg/kg alone; group III: Alloxan + MEPH (50 mg/kg); group IV: Alloxan + MEPH (100 mg/kg); group V: Alloxan +Glucophage (62.5 mg/kg), respectively for 10 days. The body weight of all animals was recorded on the 1st, 4th, 7th and 10th days. Short-term (1st, 3rd, 5th and 7th hour) and long-term (4th, 7th and 10th day) hypoglycemic effects were also recorded. All animals were sacrificed on the 10th day to isolate the pancreas for histopathological examination. The results showed that MPEH reduced the blood glucose levels in all the groups of alloxan-induced diabetic rabbits. The histopathological studies depicted that 100 mg/kg of MEPH most effectively repaired alloxan-induced pancreatic damage. The study showed that the MPEH is useful for developing effective phytomedicine to treat diabetes mellitus.

Superoxide-producing thermostable associate from the small intestines of control and alloxan-induced diabetic rats: quantitative and qualitative changes.

BACKGROUND: NADPH oxidase 1 (Nox1), which is highly expressed in the colon, is thought to play a potential role in host defense as a physical and innate immune barrier against commensal or pathogenic microbes in the gastrointestinal epithelium. Diabetes can be caused by several biological factors, including insulin resistance is one of them. Alloxan is widely used to induce insulin-dependent diabetes in experimental animals. Alloxan increases the generation of reactive oxygen species as a result of metabolic reactions in the body, along with a massive increase in cytosolic calcium concentration. METHODS: Using a universal method, a superoxide radical (О2-)-thermostable associate between NADPH-containing lipoprotein (NLP) and NADPH oxidase (Nox)- NLP-Nox was isolated and purified from the small intestine (SI) of control (C) and alloxan-induced diabetic (AD) albino rats. RESULTS: In comparison to the C indices, in AD in the SI, an increase in the specific content of NLP-Nox associate and a decrease in the stationary concentration of produced О2- in liquid phase (in solution) and gas phase (during blowing by oxygen of the NLP-Nox solution) were observed. The NLP-Nox of SI associate in C and AD rats produced О2- by an immediate mechanism, using NLP as a substrate. The phenomenon of the hiding of the optical absorption maxima of the Nox in oxidized states at pH10,5 was observed in the composition of these SI associates of the C and AD rat groups. The characteristic absorption maxima of the «hidden¼ Nox were observed under these conditions after reduction by potassium dithionite. CONCLUSION: Thus, at AD, the decrease in the stationary concentration of produced О2- in the solution and gas phase was compensated for by an increase in the specific amount of associate. In addition,  the decrease in the stationary concentration of produced О2- by NLP-Nox associates at AD can be linked to a decrease in the level of NADPH in NLP-Nox composition. This could be used as a new mechanism of AD pathogenesis.