Investigation on key molecular targets responsible for antidiabetic properties of Symplocos cochinchinensis (Lour.) S. Moore

Diabetes mellitus is a heterogeneous metabolic disorder characterized by hyperglycemia with disturbances in carbohydrate, protein and lipid metabolism resulting from defects in insulin secretion, insulin action or both. Currently there are 387 million people with diabetes worldwide and is expected to affect 592 million people by 2035. Insulin resistance in peripheral tissues and pancreatic beta cell dysfunction are the major challenges in the pathophysiology of diabetes. Diabetic secondary complications (like liver cirrhosis, retinopathy, microvascular and macrovascular complications) arise from persistent hyperglycemia and dyslipidemia can be disabling or even life threatening. Current medications are effective for control and management of hyperglycemia but undesirable effects, inefficiency against secondary complications and high cost are still serious issues in the present prognosis of this disorder. Hence the search for more effective and safer therapeutic agents of natural origin has been found to be highly demanding and attract attention in the present drug discovery research. The data available from Ayurveda on various medicinal plants for treatment of diabetes can efficiently yield potential new lead as antidiabetic agents. For wider acceptability and popularity of herbal remedies available in Ayurveda scientific validation by the elucidation of mechanism of action is very much essential. Modern biological techniques are available now to elucidate the biochemical basis of the effectiveness of these medicinal plants. Keeping this idea the research programme under this thesis has been planned to evaluate the molecular mechanism responsible for the antidiabetic property of Symplocos cochinchinensis, the main ingredient of Nishakathakadi Kashayam, a wellknown Ayurvedic antidiabetic preparation. A general introduction of diabetes, its pathophysiology, secondary complications and current treatment options, innovative solutions based on phytomedicine etc has been described in Chapter 1. The effect of Symplocos cochinchinensis (SC), on various in vitro biochemical targets relevant to diabetes is depicted in Chapter 2 including the preparation of plant extract. Since diabetes is a multifactorial disease, ethanolic extract of the bark of SC (SCE) and its fractions (hexane, dichloromethane, ethyl acetate and 90 % ethanol) were evaluated by in vitro methods against multiple targets such as control of postprandial hyperglycemia, insulin resistance, oxidative stress, pancreatic beta cell proliferation, inhibition of protein glycation, protein tyrosine phosphatase-1B (PTP-1B) and dipeptidyl peptidase-IV (DPPxxi IV). Among the extracts, SCE exhibited comparatively better activity like alpha glucosidase inhibition, insulin dependent glucose uptake (3 fold increase) in L6 myotubes, pancreatic beta cell regeneration in RIN-m5F and reduced triglyceride accumulation in 3T3-L1 cells, protection from hyperglycemia induced generation of reactive oxygen species in HepG2 cells with moderate antiglycation and PTP-1B inhibition. Chemical characterization by HPLC revealed the superiority of SCE over other extracts due to presence of bioactives (beta-sitosterol, phloretin 2’glucoside, oleanolic acid) in addition to minerals like magnesium, calcium, potassium, sodium, zinc and manganese. So SCE has been subjected to oral sucrose tolerance test (OGTT) to evaluate its antihyperglycemic property in mild diabetic and diabetic animal models. SCE showed significant antihyperglycemic activity in in vivo diabetic models. Chapter 3 highlights the beneficial effects of hydroethanol extract of Symplocos cochinchinensis (SCE) against hyperglycemia associated secondary complications in streptozotocin (60 mg/kg body weight) induced diabetic rat model. Proper sanction had been obtained for all the animal experiments from CSIR-CDRI institutional animal ethics committee. The experimental groups consist of normal control (NC), N + SCE 500 mg/kg bwd, diabetic control (DC), D + metformin 100 mg/kg bwd, D + SCE 250 and D + SCE 500. SCEs and metformin were administered daily for 21 days and sacrificed on day 22. Oral glucose tolerance test, plasma insulin, % HbA1c, urea, creatinine, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein etc. were analysed. Aldose reductase (AR) activity in the eye lens was also checked. On day 21, DC rats showed significantly abnormal glucose response, HOMA-IR, % HbA1c, decreased activity of antioxidant enzymes and GSH, elevated AR activity, hepatic and renal oxidative stress markers compared to NC. DC rats also exhibited increased level of plasma urea and creatinine. Treatment with SCE protected from the deleterious alterations of biochemical parameters in a dose dependent manner including histopathological alterations in pancreas. SCE 500 exhibited significant glucose lowering effect and decreased HOMA-IR, % HbA1c, lens AR activity, and hepatic, renal oxidative stress and function markers compared to DC group. Considerable amount of liver and muscle glycogen was replenished by SCE treatment in diabetic animals. Although metformin showed better effect, the activity of SCE was very much comparable with this drug. xxii The possible molecular mechanism behind the protective property of S. cochinchinensis against the insulin resistance in peripheral tissue as well as dyslipidemia in in vivo high fructose saturated fat diet model is described in Chapter 4. Initially animal were fed a high fructose saturated fat (HFS) diet for a period of 8 weeks to develop insulin resistance and dyslipidemia. The normal diet control (ND), ND + SCE 500 mg/kg bwd, high fructose saturated fat diet control (HFS), HFS + metformin 100 mg/kg bwd, HFS + SCE 250 and HFS + SCE 500 were the experimental groups. SCEs and metformin were administered daily for the next 3 weeks and sacrificed at the end of 11th week. At the end of week 11, HFS rats showed significantly abnormal glucose and insulin tolerance, HOMA-IR, % HbA1c, adiponectin, lipid profile, liver glycolytic and gluconeogenic enzyme activities, liver and muscle triglyceride accumulation compared to ND. HFS rats also exhibited increased level of plasma inflammatory cytokines, upregulated mRNA level of gluconeogenic and lipogenic genes in liver. HFS exhibited the increased expression of GLUT-2 in liver and decreased expression of GLUT-4 in muscle and adipose. SCE treatment also preserved the architecture of pancreas, liver, and kidney tissues. Treatment with SCE reversed the alterations of biochemical parameters, improved insulin sensitivity by modifying gene expression in liver, muscle and adipose tissues. Overall results suggest that SC mediates the antidiabetic activity mainly via alpha glucosidase inhibition, improved insulin sensitivity, with antiglycation and antioxidant activities.

Synthesis and Characterisation of Thermoplastic Ionomers based on Natural Rubber

Chemically modified novel thermo-reversible zinc sulphonated ionomers based on natural rubber (NR), radiation induced styrene grafted natural rubber (RI-SGNR), and chemically induced styrene grafted natural rubber (CI-SGNR) were synthesized using acetyl sulphate/zinc acetate reagent system. Evidence for the attachment of sulphonate groups has been furnished by FTIR spectra. which was supplanted by FTNMR results. Estimation of the zinc sulphonate group was done using spectroscopic techniques such as XRFS and ICPAES. The TGA results prove improvement in the therrno-oxidative stability of the modified natural rubber. Both DSC and DMTA studies show that the incorporation of the ionic groups affect the thermal transition of the base polymer. Retention of the improved physical properties of the novel ionomers even after three repeated cycles of mastication and molding at 120 degree C may be considered as the evidence for the reprocessabiJity of the ionomer. Effect of both particulate (carbon black. silica & zinc stearate) and fibrous fillers (nylon & glass) on the properties of the radiation induced styrene grafted natural rubber ionomer has been evaluated. Incorporation of HAF carbon black results in maximum improvement in physical properties. Silica reinforces the backbone chain and weakens the ionic associations. Zinc stearate plays the dual role of reinforcement and ptasticization. The nylon and glass filled lonorner compounds show good improvement in the physical properties in comparison with the neat ionomer. Dispersion and adhesion of the fillers in the ionomer matrix has been amply supported by their SEM micrographs. Microwave probing of the electrical behavior of the 26.5 ZnSRISGNR ionomer reveals that the maximum relative complex conductivity and the complex permittivity appear at the frequency of 2.6 GHz. The complex conductivity of the base polymer increases from 1.8x 10.12 S/cm to 3.3xlO·4 S/cm. Influence of fillers on the dielectric constant and conductivity of the new ionic thermoplastic elastomer has been studied. The ionomer I nylon compound shows the highest microwave conductivity. Use of the 26.5 ZnS-RISGNR ionomer as a compatibilizer for obtaining the technologically compatible blends from the immiscible SBR/NBR system has been verified. The heat fugitive ionic cross-linked natural rubber may be, therefore, useful as an alternative to vulcanized rubber and thermoplastic elastomer

Chemical and Biochemical Studies on Natural Antioxidants from sesamum Species

In the present study on natural antioxidants, the focus has been kept mainly on oil seeds, especially sesame and its by-products. Sesame, which has been under cultivation in India for centuries is called the 'Queen of oil seed crops' because of the high yield of oil obtained and the nutritional qualities of the seed, oil, and meal. Though India is the largest producer of sesame in the world, research on the various health benefits of sesame has been carried out by Japanese Sesame has an important place in the foods and tradit..ional medicine of India from time immemorial. Foreseeing the potential of sesame and its byproducts as an important antioxidant source and its availability in bulk, the present study was focussed on Sesamum species. There are not many reports on the wild species of Sesamum in India, especially of the Kerala region. Hence, in the present study we also included antioxidants of Sesamurnrnalabaricumdistributed throughout the coastal region.The important characteristics of sesame are attributed to the presence of the umquc compounds lignans. Lignans arc a group of natural products of phenyl propanoid ongm, whieh are widely distributed in nature. They display important physiological functions in plants, in human nutrition and medicine, given their extensive health promotive and curative properties. Much interest has been focussed on their effectiveness as antineoplastic agents and research in this area has revealed several modes of action by which they can regulate the growth of mammalian cells. Sesame is an important source of furofuran lignans, of which sesamin and the rare oxygenated derivative sesamoIin are the most abundant. Others include sesamol and glucosides of lignans. Sesarnin and episesamin are reported to have hypocholesterolemic effect, suppressive effect on chemically induced cancer, alleviation of allergy symptoms etc. Sesamol, sesamolin and the lignan glycosides are reported to inhibit lipid peroxidation. Present investigation on sesame and its byproducts have been carried out to explore the possibility of developing a natural antioxidant extract from available resources to be used as a substitute to synthetic ones in vegetable oils and foods. Preliminary analysis showed that sesame cake, a byproduct could still be utilized as a major source of lignans. Sesame cake, which is now used only as a cattlefeed, can be better utilized in the form of a valuable antioxidant source. The present study explains the development of a feasible process for the extraction of antioxidant compounds from sesame cake. The antioxidant extract so prepared from sesame cake has been tested for vegetable oil protection and is found to be effective at low concentration. In addition, studies also include the antioxidant, radical scavenging, anticancer, mosquitocidal and pesticidal activities of extract and individual compounds.

Hypothalamic GABA receptor functional regulation and liver cell proliferation

GABAergic alterations in hypothalamus during compensatory hyperplasia after partial hepatectomy (PH), lead nitrate (LN) induced direct hyperplasia and N-nitrosodiethylamine (NDEA) induced neoplasia in liver were investigated. Serum GABA levels were increased in all 3 experimental groups compared with the control. GABA content decreased in hypothalamus of PH and NDEA treated rats, while it increased in LN treated rats. GABAA receptor number and affinity in hypothalamic membrane preparations of rats showed a significant decrease in PH and NDEA treated rats, while in LN treated rats the affinity increased without any change in the receptor number. The GABAB receptor number increased in PH and NDEA treated rats, while it decreased in LN treated rats. The affinity of the receptor also increased in NDEA treated rats. Plasma NE levels showed significant increase in PH and NDEA rats compared with the control while it decreased in LN treated rats. The results of the present study suggests that liver cell proliferation is influencing the hypothalamic GABAergic neurotransmission and these changes regulate the hepatic proliferation through the sympathetic stimulation.