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MScPROJECT

  1. 1. The cardio protective effect of Tinospora crispaTo determine if Tinospora crispa has a cardio protective effect on the oxidative stress induced impairment of vascular tone in DiabetesMaulik Thakkar9/28/2009University of HertfordshireDepartment of Life Sciences<br />DECLARATION<br />I declare that:<br />(a)all the work described in this report has been carried out by me – and all the results (including any survey findings, etc.) given herein were first obtained by me – except where I may have given due acknowledgement to others;<br />(b)all the prose in this report has been written by me in my own words, except where I may have given due acknowledgement to others and used quotation marks, and except also for occasional brief phrases of no special significance which may be taken from other people’s work without such acknowledgement and use of quotation marks;<br />(c)all the figures and diagrams in this report have been devised and produced by me, except where I may have given due acknowledgement to others.<br />(d)the project has been spell and grammar checked.<br />I understand that if I have not complied with the above statements, I may be deemed to have failed the project assessment, and/or I may have some other penalty imposed upon me by the Board of Examiners.<br />Signed ……………………………Date …………………………...<br />ACKNOWLEDGEMENTS:<br />The following project carried out required a lot of hard work and motivation. Pursuing a career in the field of one’s choice is always a hard decision and requires crossing a large number of hurdles. Therefore I would like to take this opportunity to extend my gratitude to the people who have helped me and always motivated me in achieving my goals. <br />Foremost I would like to thank my parents for always believing in what I was doing and supporting in every possible manner, the work I was doing. Without their support this would not have been possible. Secondly I would like to thank my supervisor Dr. Anwar Baydoun, for giving me guidance and helping me figure out new ways in which I could approach the project. <br />I would also like to give a note of thanks to all the friends who have always stood by me and given me their unconditional support in what I was doing. <br />Last, but not the least, I would like to give a big hug to all the laboratory staff Komal Patel, Jayna Patel, Yugal Kalaskar and Leena Pye, for making this work seem like fun. It was an amazing time working on the project with you all. <br />MAULIK THAKKAR<br /><ul><li>INDEX</li></ul>List of Figrues…………………………………………………………………………………………5<br />List of Graphs…................................................................................................................................... 6<br />List of Tables………………………………………………………………………………………… 7<br />Abstract………………………………………………………………………………………………8<br /><ul><li>Introduction..............................................................................................................................9
  2. 2. Oxidative stress and the Reactive species.................................................................................9
  3. 3. Oxidative stress and diabetes...................................................................................................14
  4. 4. Sources of oxidative stress in Diabetes....................................................................................16
  5. 5. Antioxidants.............................................................................................................................17
  6. 6. Current Treatments..................................................................................................................18
  7. 7. Tinospora crispa......................................................................................................................18
  8. 8. Aims.........................................................................................................................................22
  9. 9. Objectives................................................................................................................................22
  10. 10. Methods..................................................................................................................................23
  11. 11. 2.1 Tissue Preparation..............................................................................................................23
  12. 12. 2.2 Experimental Protocol.......................................................................................................23
  13. 13. 2.3 Procedure...........................................................................................................................23
  14. 14. 2.4 Chemicals...........................................................................................................................24</li></ul>3. Results………………………………………………………………………………...............25<br /> 3.1 Contractions obtained with Phenylephrine…………………….......................................25<br /> 3.2 Relaxations obtained with Acetylcholine……………………………………….............35<br /> 3.3 Relaxations obtained with Sodium Nitroprusside………………………………............40<br />4. Discussion, Conclusion and Future Studies……………………………………………….. 44<br /> 4.1 Discussion……………………………………………………………………………… 44<br /> 4.2 Conclusion………………………………………………………………………………48<br /> 4.3 Drawbacks and Future Studies…………………………………………………... …….48<br />5. Bibliography………………………………………………………………………………….50<br /><ul><li>LIST OF FIGURES
  15. 15. FIGURE NO.TITLEPAGE NO.1Mechanism of Nitric Oxide production in endothelium122The various ways In which oxidative stress is induced in diabetes153Structure of Apigenein20Structure of cycloeucalenol and cycoeucalenone204Trace of the contraction responses obtained with Phenylephrine266Trace of the contraction response obtained with PE in the presence of 1 µM Antimycin A28Trace of contraction response obtained with PE in the presence of 0.5 µM Antimycin A297 (a)Trace of response obtained with PE in the presence of T. crispa (75 mg/ml) and Antimycin A (0.5 µM)318 (a)Trace of response as observed in the presence of T. crispa alone3310 (a)Trace of relaxations obtained with Acetylcholine in control rat aorta3611Trace showing partial relaxation with Acetylcholine in control rat aorta38Trace showing relaxation with SNP in cases of partial relaxations with acetylcholine in control rat aorta3812 (a)Trace of the relaxation obtained with ACh in the presence of 0.5 µM Antimycin A3913Trace of relaxation obtained with Ach in the presence of T. crispa (75mg/ml) and Antimycin A (0.5 µM)4014 (a)Trace of relaxation as observed with SNP in control rat aorta4115 (a)Trace of relaxation observed with SNP in 0.5 µM Antimycin A42</li></ul>LIST OF GRAPHS<br /><ul><li>GRAPH NO.TITLEPAGE NO.5Dose response curve of PE in control rat aorta27Graph showing % Response vs concentration of PE276 (c)Dose response curve of PE in the presence of 0.5 µM Antimycin A297 (b)Dose response curve of PE in the presence of T. crispa (75mg/ml) and Antimycin A (0.5 µM)318 (b)Dose response curve of PE in the presence of T. crispa only339Comparison between dose responses of PE in control, Antimycin A, T. crispa and Antimycin A and in T. crispa only34b) Graph of relaxation vs concentration of ACh in control rat aorta36Percentage relaxation observed with ACh in control rat aorta3712 (b)Relaxation dose response curve obtained with SNP when complete relaxations were not obtained with ACh in case of oxidative stress.3914 (b) Relaxation dose response curve obtained with SNP in control rat aorta4115 (b)Relaxation dose response curve obtained with SNP in Antimycin A (0.5 µM) induced oxidative stress4316Bar Graph showing the comparison between the maximum response obtained with PE in control, Antimycin A, T. crispa and Antimycin A and in T. crispa alone45
  16. 16. LIST OF TABLES
  17. 17. TABLE NO.TITLEPAGE NO.1Response in grams and percentage response obtained with PE in control rat aorta262Contractions obtained with PE in the presence of Antimycin A (0.5 µM)303Contractions obtained with PE in the presence of T. crispa (75mg/ml) and Antimycin A (0.5 µM)324Contractions obtained in the presence of T. crispa only345Relaxations obtained with ACh in control aorta376Relaxations obtained with SNP in control aorta42</li></ul>ABSTRACT:<br />Tinospora crispa is used in several Far East Asian countries in the treatment of diabetes. Studies show that it exhibits its anti- hyperglycemic and insulinotropic effect by β – cell Ca2+ modulation. Since diabetic patients sometimes show the presence of oxidative stress impaired vascular tone, this experiment was carried out to determine if the plant had any effect on the oxidative stress. Impairment of vascular tone in several diseases such as hypertension, atherosclerosis and other vascular complications has proved to be fatal. In the given experiment, contraction responses in rat aorta were taken with phenylephrine (PE). These responses were compared to the contraction responses in the presence of Antimycin A (0.5 µM) and also in the presence of T. crispa (75mg/ml) and Antimycin, both. Comparing the responses, Antimycin A significantly decreased PE induced contractions (p = 0.019). Prior administration of T. crispa did not bring about a change in the contractile response of PE in the presence of oxidative stress. In the absence of oxidative stress, no change in the contractile response with PE was observed with prior administration of T. crispa. This showed that the plant was unable to reverse the oxidative stress induced decrease in contraction response. When the relaxations were taken with endothelium dependent vaso relaxant acetylcholine (ACh), control tissue showed a 100% relaxation at a concentration of 1* 10-6M. The relaxation was a result of nitric acid (NO) production by endothelium. In some tissues, complete relaxations were not obtained, which meant there was damage to the endothelium. Relaxation with ACh in the presence of Antimycin A could not be achieved, which showed that oxidative stress was blocking the endothelium mediated vaso relaxation. When, T. crispa was administered prior to inducing oxidative stress, partial relaxation was observed with ACh. This meant that the plant was able to bring relaxation even in the presence of oxidative stress. This could be because of increased NO formation by the endothelium in the presence of the plant. Antimycin however did not bring a change in the concentration of endothelium independent vaso relaxant sodium nitro prusside (SNP) required to obtain relaxation with PE contractions in control and Antimycin A administered tissue. Relaxations with SNP, in T. crispa administered tissue were not obtained because of time constraints. These need to be performed to see if the plant has any effect on external NO donator like SNP. To conclude, the experiment performed, showed that T. crispa is able to decrease the impairment in oxidative stress induced vascular tone by stimulating the endothelium to produce more NO. However this needs to be verified by studying the levels of antioxidants as well as the reactive species in the presence and absence of drug.<br /><ul><li>INTRODUCTION:</li></ul>Millions of people suffer from diabetes all over the world. Insufficiency of secretion or receptor sensitivity to insulin leads to diabetes mellitus (Rolo & Palmeira, 2006). Diabetes mellitus is characterised by hyperglycemia. Diabetes mellitus can be classified into 2 types; Type 1: Insulin dependent diabetes which is caused as a result of destruction of pancreatic β- cells causing insulin deficiency (Newsholme, et al., 2007), Type 2: insulin independent diabetes caused as a result of insulin resistance (Rolo & Palmeira, 2006, Creager, et al., 2003). Approximately 5- 10% suffer from Type 1 diabetes while the remaining 90-95 % suffer from type 2 caused as a result of lifestyle patterns. In diabetes mellitus, the vascular endothelium has a reduced capacity for synthesizing vasodilators (Poston, 2007).The regulation and the functioning of the vascular tone is mediated through the control of locally produced agents (Rang, et al., 2003). The vascular tone in homeostasis is regulated by the balance between the vasoconstrictor agents such as endothelin and the vasodilator agents, amongst which nitric oxide (NO), plays a very important role (Mather, et al., 2001). The major complications arising as a result of diabetes are vascular diseases which are the principle cause of death and disability in patients. The macro vascular manifestations include atherosclerosis and medial calcification while the micro vascular complications include retinopathy and nephropathy leading to blindness and end stage renal failure (Creager, et al., 2003, King, 1996). Thus it is necessary for the physicians to be aware of the salient features of diabetic vascular diseases in order to treat the patients most effectively. Emphasis has therefore been laid upon exploring the mechanisms of endothelial dysfunction and thereby developing new drug therapies to either prevent or reverse endothelial damage.<br /><ul><li>Oxidative Stress and the reactive species:</li></ul>In mammalian cells, during aerobic respiration, molecular oxygen is converted into water and energy is produced. A number of intermediates are produced during this entire process. These are known as reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Johansen, et al., 2005, Moncada & Higgs, 2006). The reactive species generated under homeostatic conditions are involved to an extent in various signalling pathways and body defence mechanism like phagocytosis, neutrophil function and shear-stress induced vasorelaxation. However, in case of oxidative stress, there is an imbalance between the free reactive species produced and the antioxidant defence mechanisms (Nedeljkovic, et al., 2003). The over production of reactive species may lead to severe complications such as damage to proteins, lipids and DNA (Johansen, et al., 2005). The free radical formation is now known to play a very crucial role in a number of diseases as well as in normal ageing and therefore targeting the free radical formation seems to be a novel way for improving drug treatment of various pathological conditions (Cavin, et al., 1998).<br /> <br />The ROS includes free radicals such as superoxide (O2-), hydroxyl (OH-), peroxyl (RO2), hydroperoxyl (HRO2-), hydrogen peroxide (H2O2) and hyperchlorous acid (HClO) while the RNS consist of nitrogen dioxide (NO2-) and peroxynitrite (ONOO-) (Johansen, et al., 2005). Peroxynitrite is formed as a result of the reaction between the NO and the superoxide. As a result of this there is decreased availability of nitric oxide (NO) in the endothelium and therefore the inherent property of the endothelium to relax is lost leading to various vascular complications such as atherosclerosis, hypertension and heart failure. Amongst the above mentioned reactive species, O2- and ONOO- a play a very important role in pathogenesis of oxidative stress in diabetes and will therefore be discussed in detail. <br />Oxidative stress seen in diabetes, hypertension and atherosclerosis can cause a variety of complications. The major complications arising because of diabetes induced oxidative stress are of vascular nature. The endothelial dysfunction observed as a result of oxidative stress in each of the above causes a loss of the inherent property of endothelium to produce vaso relaxation. This could be a result of loss of endothelial nitric oxide synthase (eNOS) activity or degradation of NO because of the free radicals generated. In the absence of NO and increased oxidative species, the low density lipoprotein (LDL) is subjected to oxidation forming ox-LDL (Mehta). The endothelial cells, vascular smooth muscle cells and the monocytes are collectively able to oxidise LDL. Once oxidised, the macrophages within the vessel wall internalise the LDL via scavenger receptors (Nedeljkovic, et al., 2003). This subsequently causes the release of inflammatory cytokines and growth factors (Mehta, et al., 2006). All these lead to a fatty streak formation in the intima of the vessel wall. The vicious cycle is carried on and the plaque grows larger and larger ultimately narrowing the lumen of the vessel. This oxidative modification hypothesis of LDL, provides a link between the hypercholesterolemia and vascular disease (Nedeljkovic, et al., 2003).<br />There are a number of pathways in which the reactive species may be formed. Amongst these, the important ones are the NAD(P)H pathway, the xanthine oxidase pathway, the nitric oxide pathway and the PKC pathway.<br />Amongst the enzymatic sources contributing towards the formation of reactive species are the endothelial nitric oxide synthase (eNOS), NAD(P)H oxidase and the xanthine oxidase system. <br />The NAD(P)H oxidase pathway: <br />The NADH/NAD(P)H complex system generates the superoxide ion by reduction of molecular oxygen (,Nedeljkovic, et al., 2003, Newsholme, et al., 2007, Pacher, et al., 2007). The system may be activated in response to inflammatory cytokines (like TNF – α) and vaso active factors such as angiotensin II thereby causing an increased superoxide formation (Mehta, et al., 2006). There is also increased formation of NAD(P)H in the presence of protein kinase C. <br />Xanthine oxidase pathway:<br />During the purine metabolism, the conversion of hypoxanthine to xanthine is catalysed by the enzyme xanthine dehydrogenase (XD) (Mehta, et al., 2006, Cai & Harrison, 2009). This enzyme requires NAD+ as an active electron acceptor. Its other isoform, xanthine oxidase (XO), however reduces molecular oxygen generating superoxide ion. Besides the formation of the O2-, xanthine oxidase, may donate two electrons directly to O2 to produce H2O2 (Lubous, et al., 2008). The inhibition of XO activity, with oxypurinol in hypercholesterolemic patients, improved endothelium dependent vasodilation (Lubous, et al., 2008). This shows that the decrease in bioavailability of NO is a result of the superoxide formation by the enzyme XO (Pacher, et al., 2007).<br />Nitric oxide and the Nitric oxide synthase pathway:<br />The endothelium in the vasculature plays an important role in maintaining the vascular tone, inhibiting platelet and inflammatory cell adhesion, promote fibrinolysis and limit vascular proliferation (Nedeljkovic, et al., 2003, Papaharalambus & Griendling, 2007). This it does by the release of nitric oxide (Pacher, et al., 2007).The release of NO is mediated by acetylcholine (ACh). ACh stimulates the influx of calcium which binds to calmodulin to form the calcium calmodulin complex thereby activating eNOS. The enzyme then in the presence of arginine forms NO and citrulline. NO activates the enzyme soluble guanylyl cyclase (sGC) which in turn converts GTP to cGMP bringing about smooth muscle relaxation (Rang, et al., 2003, Brailoiu, et al., 1999). Figure 1 shows the pharmacological activation of NO and how it mediates the smooth muscle relaxation. A central feature of endothelium dysfunction is impairment in the bioavailibility of NO (Nedeljkovic, et al., 2003). The enzyme synthesizing NO is nitric oxide synthase. There are three isoforms of the enzyme namely neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (nNOS). While the first two isoforms are Ca2+/calmodulin dependent the activity of the latter is Ca2+/calmodulin independent (Mehta, et al., 2006, Moncado & Higgs, 2007). <br />1295400-60325<br />Figure 1: The mechanismof NO production in endothelium cells and the way in which it mediates it effects. ACh causes an increase in intracellular calcium influx. This calcium binds with the calcium calmodulin complex and activates the eNOS. In the presence of L- arginine, eNOS produces citrulline and NO. The NO activates the enzyme sGC which converts GTP to cGMP causing vaso relaxation. (eNOS: endothelial nitric oxide synthase, NO: nitric oxide, GC: guanylyl cyclase, GTP: guanosine tri phosphate, cGMP: cyclic guanosine mono phosphate) (Adapted from Rang)<br />Clinical experiments carried out on knock out eNOS isoform in animal models led to hypertensive phenotypes while over expression of eNOS in endothelial cells reduced blood pressure (Huang, 2000) which showed the relation between eNOS and vascular complications. The eNOS regulates the dilator action by activating the sGC and consequently increasing the intracellular cyclic 3̍̍ -5̍ guanosine monophosphate (cGMP) (Nedeljkovic, et al., 2003). High concentrations of glucose have shown endothelium dysfunction in both in vitro and in vivo experiments (Tesfamariam, et al., 2000). <br />The impairment in the endothelium function may be result of the decreased activity of eNOS, and/or decreased expression of NO downstream target sGC, and/or increased degradation of nitric oxide as a result of increased superoxide production. Nitric oxide is produced by eNOS in the presence of the substrate L- arginine (Moncado & Higgs, 2007). It also requires co factors flavin adenine dinucleotide (FAD), flavin adenine mononucleotide (FMN), heme, tetrahydrobiopterin (BH4) and Ca2+/calmodulin (Johansen,et al., 2005). If there is an absence or a decrease in any one of the co factors or the substrate, eNOS produces O2- instead of NO. This phenomenon is called uncoupling of NOS (Sydow& Munzel, 2003). To understand the mechanism of eNOS uncoupling, endothelial cells were incubated with glucose. The eNOS expression was then studied at RNA level and protein level. It was seen that the eNOS expression was up regulated. However at the same time, NOS mediated NO production increased only by 30-40% while the superoxide production rose by 300% (Sydow& Munzel, 2003). This showed that inspite of increased production of NO as a result of an increased transcription activity in the presence of hyperglycemia, the levels of superoxide formation were much higher, which could lead to impairment in the endothelial dependent vaso relaxation. The effect of BH4 on the uncoupling of NOS was studied by infusion of BH4 in diabetic patients (Heitzer, et al., 2009). The infusion improved endothelium dependent relaxation in response to ACh, but had no effect on healthy subjects. Further, the use of L-N- monomethyl arginine (L-NMMA), eNOS inhibitor, blocked the improvement by BH4 indicating that the endothelium dysfunction improvement was largely due to restoration of eNOS function (Heitzer, et al., 2009). Another concept suggesting endothelial dysfunction revolves around NO degradation. Recent studies showed that, treatment of vessels from diabetic animals with superoxide dismutase (SOD), and antioxidant, improved endothelial dependent relaxation (Sydow& Munzel, 2003). This suggested that the antioxidant prevented the degradation of the NO by scavenging the free radicals.<br />Role of protein kinase C (PKC):<br />The importance of PKC in generation of free radicals and impairment of NO mediated vaso relaxation is highlighted because of its ability to down regulate the messenger system of NO, i.e. sGC. The secondary messenger protein kinase C (PKC) is also known to increase oxidative stress. Incubation of endothelium cells and smooth muscle cells with glucose leads to an increase in the diacyl glycerol (DAG) levels intracellularly (Sydow& Munzel, 2003). Increase in DAG levels cause an increase in the PKC (Ishii, et al., 1998).The activity of eNOS, in vitro, was decreased as a result of PKC mediated phosphorylation (Hirata). This could be prevented by PKC inhibition. PKC is also known to increase the levels of NAD(P)H which as described above plays a major role in the formation of free radicals. Experiments carried out using PKC inhibitor, N- benzoyl- taurosporine, showed an improvement in the endothelial function as well as prevented down regulation of sGC and normalised the vascular suproxide levels (Sydow& Munzel, 2003).<br /> Besides these sources, ROS may also be generated due to decrease in the natural antioxidants present within the vasculature.<br /><ul><li>Oxidative Stress and Diabetes: </li></ul>Evidence of oxidative stress in diabetes is based on the increased levels of nitro tyrosine in plasma and the tissue of diabetic patients. The plasma levels of nitro tyrosine in normal healthy patients is undetectable but are present in the plasma of patients suffering from rheumatoid arthritis, chronic renal failure, septic shock and coeliac disease (Kaur & Halliwell, 1994, ter Steege, et al., 1998). It has also been previously reported in the placenta and kidney of diabetic patients (Thuraisingham, et al., 2000). Reaction of NO with the superoxide radical leads to the formation of the free radical peroxynitrite which can decompose into products that can nitrate aromatic amino acids (Ischiropoulous, 1998). Peroxynitrite thus nitrates tyrosine present in the proteins forming nitro tyrosine, which is a stable product of peroxynitrite oxidation, and can therefore be used as a useful marker in the assessment of the oxidative stress induced damage (Beckman & Koppenol, 1996). Cellular and experimental models of diabetes have shown the presence of endothelial dysfunction (Pieper, et al., 1995). A number of studies, though not all, show that endothelium dependent vasodilation is impaired in patients with type 1 and type 2 diabetes (Johnstone, et al., 1993, McVeigh, et al., 1992). Endothelium dependent relaxation is decreased in aortic rings incubated with glucose (Creager, et al., 2003).Several other metabolic dearrangements besides hyperglycemia may be seen in diabetes such as excess free fatty acid secretion(King, 1996) and insulin resistance (Creager, et al., 2003). Increase in the levels of fatty acids in diabetes also reduced endothelium dependent relaxation in animal models and in humans in vivo (Steinberg, et al., 1997). The role of fatty acid mediated abnormality was confirmed when co infusion of antioxidant ascorbic acid improved endothelium dependent vasodilation in humans treated with free fatty acid. Figure 2 shows the various ways in which oxidative stress can be induced in diabetes and the aftermath of oxidative stress. <br />Figure 2: Figure showing the various ways in which oxidative stress can be induced in diabetes mellitus. Since most of the complications arising from diabetes are of vascular nature such as atherosclerosis or thrombosis, it is of utmost importance that the physician understands the pathophysiology of diabetes so as to treat the patient effectively (adapted from creager).<br />There are multiple sources of oxidative stress in diabetes (johansen). These include non-enzymatic, enzymatic and mitochondrial pathways. <br /><ul><li>Sources of oxidative stress in diabetes:</li></ul>The non- enzymatic sources arise as a result of oxidative biochemistry of glucose. The non –enzymatic sources include auto-oxidation of glucose, formations of advanced glycation end-products, pylol pathway and mitochondrial respiratory transport chain. In cases of diabetes, there is excess glucose present in the blood. In diabetes, this glucose is either not metabolised by insulin or is resistant to insulin. As a result, the glucose undergoes auto- oxidation generating OH- (Johansen,et al., 2005). Hydroxyl ion is the most potent oxidising reactive species (Lubous, et al., 2008). In some instances, glucose reacts with proteins (Rolo & Palmeira, 2006). This reaction yields advanced glycation end products (AGEs). During this process, reactive oxygen species are produced at various steps. Also, once formed AGEs react with their receptors which increase the formation of proinflammatory cytokines, chemokines and other adhesion molecules such as VCAMs (Naka, et al., 2004). The pylol pathway also plays a role in increasing the reactive species formation in diabetes. The pylol pathway generally plays an important role in the detoxification of aldehydes and alcohols. However in hyperglycemia, the pathway converts glucose to sorbitol and finally to fructose (Mehta, et al., 2006). This reaction consumes large amounts of nicotinamide adenosine dinucleotide (phosphate) (NAD(P)H) which is a major factor for GSH regeneration (Mehta, et al., 2006). GSH, as mentioned above plays an important role as an antioxidant (Lubous, et al., 2008). Besides the above mentioned non enzymatic sources, the mitochondrial respiration transport chain also contributes to the formation of reactive species (Foster, et al., 2006). Mitochondrion plays a very important role in the production of ATP (Mehta, et al., 2006). Of the total ATP production, majority (>95%) is produced by oxidative phosphorylation in mitochondria, while the remaining is produced because of glycolysis in the cytoplasm(Foster, et al., 2006) The energy is generated via the transport of electrons through mitochondrial complexes I, II, III and IV. Complex -1 (NADH –ubiquinone oxidoreductase), Complex-II (succinate- ubiquinone oxidative reductase), Complex- III (ubiquinol-cytochrome c reductase) and Complex –IV (cytochrome c reductase) constitute the electron transport chain (Mehta, et al., 2006). Besides the production of ATP, the mitochondrion is also involved in the regulation of Ca2+ levels, intracellular pH and apoptosis and in the formation of ROS. Approximately, 85% of the total superoxide formation is generated by the mitochondrion (Foster, et al., 2006). In the process of electron transport, 2-5 % of the electrons escapes at complexes I and III and react with readily diffusible O2 to form the superoxide radical. The generation of superoxide radicals may increases significantly during enhanced mitochondrial activity as well in the presence of the complex inhibitors, for example, inhibition of complex I by rotenone causes an increase in H2O2 production. Similar results were seen with Antimycin A, a mitochondrial complex III inhibitor (Foster, et al., 2006r). In diabetes, the increased generation O2- in the endothelial cells occurs as a result of incomplete reduction of O2, mainly at complex I and complex III (Lubous, et al., 2008).<br /><ul><li>Antioxidants</li></ul>Under normal physiological conditions the damage arising due to production of oxidative reactive species is prevented by the antioxidants present within the body (Lubous, et al., 2008). These antioxidants either prevent the formation of the reactive species or they remove the reactive species before they can cause any damage to tissues. The non-enzymatic antioxidants present in endothelial cells are uric acid, ascorbic acid (Vitamin C), tocopherol (Vitamin E) and glutathione (GSH) (Sies). Amongst these GSH is the major antioxidant. In hypercholesterolemia, depletion of GSH has shown to alter the vascular walls ability to detoxify peroxynitrite. The antioxidant property of glutathione is attributed to the presence of thiol group in its cysteinyl moiety. Glutathione oxidises to glutathione disulfide (GSSG) and thereby serves as a substrate for glutathione peroxidises in the endothelial cells to eliminate lipid hydroperoxidases and H2O2 (Meister, 1994). Thus depletion of GSH may lead to decrease in the ability of the vascular cells to scavenge free radicals. Other important antioxidants present in the endothelial cells include superoxide dismutases (SOD), catalase, the thioredoxin system, peroxiredoxins, glutathione peroxidises and heme oxygenases. SODs convert O2- to H2O2 which is then reduced to H2O by peroxiredoxins, catalase, or glutathione peroxidises (Lubous, et al., 2008). The activity of SOD is under the influence of metal ion co factors copper, zinc, manganese or iron. Any change in the binding capacity of the metal ion, such as the one observed in the case of H2O2 mediated oxidative stress, lead to enzyme instability and thus reduces the enzyme activity (Sampson & Beckman, 2001). <br />Catalase on the other hand, which are expressed more in the peroxisomes in smooth muscles, reduce H2O2 to water. The reduction of the H2O2, organic hyroperoxides and peroxynitrite is under the control of peroxisomes (Lubous, et al., 2008). <br /><ul><li>Current treatments:</li></ul>Successful treatment of diabetes revolves around making a long difference in terms of health of the patient. However, the current treatments have not solved the problem completely. Treatment to type 1 diabetes consists of a carefully calculated diet, physical excercise and multiple doses of insulin. Type 2 diabetes may require oral medication or insulin injection. (Rang, et al., 2003). Hyperglycemia can be controlled using oral hypoglycaemic agents. The only successful medication that has shown to improve insulin sensitivity is group of compounds called biguanides. Amongst these metformin has shown to improve endothelium dependent vasodilation (Mather, et al., 2001). Another group of compounds, Thiazolidinediones act as endogenous agonist whereby they increase lipogenesis and enhance uptake of fatty acids and glucose (Rang, et al., 2003). Since oxidative stress is known in diabetes, several approaches have been made to treat diabetes by reducing oxidative stress and thereby improving endothelium function. Agents such as angiotensin converting enzyme inhibitors (ACE) have shown improvement in endothelium function by decreasing the NAD(P)H mediated oxidative stress (Munzel, & Keaney, 2001). Though pre treatment with antioxidants Vitamin C and Vitamin E have shown improvement in endothelium dysfunction, they are not able to improve the prognosis of the patients (Sydow& Munzel, 2003). Given the conflicting data of use of antioxidant therapies, newer strategies need to be designed in combating oxidative stress.<br />Since a cure for the oxidative stress induced impairment of vascular tone in diabetes has not been obtained, various novel approaches have been tried. Use of some plants such as Tinospora crispa, Musa sapienta, Piper sarmentosum etc, in the Far East countries of Asia has shown promising results in improving the diabetic symptoms. A number of studies have been carried out to identify the mechanisms by which they exert the anti diabetic activity. However the success has been limited and more studies need to be carried out.<br /><ul><li>Tinospora crispa: </li></ul>Tinospora crispa (Family: Menispermaceae) is a climber plant that is found in the tropical and sub-tropical India and parts of Far East particularly in the rainforest and the mixed deciduous forests of the Philippines. The climber is also widely distributed in Thailand, Vietnam, Indonesia, Malaysia and in other parts of tropical Asia at an altitude of upto 1000 metres.<br />left0 The widely accepted name is Tinospora crispa, but it also has a numerous synonyms Menispermum rimosum Blanco, Minospermum crispum, Cocculous cordifolious, Cocculous villosus, Tinospora tuberculata , Tinospora rumphii, Cocculous crispum, Menispermum tuberculatum, Menispurmam verrocosum. Also known as bitter wine, T. crispa has been used traditionally in many South Asian countries as a herbal medicine.<br />It is one of the key ingredients in Thai folk remedies for maintaining good health. A decoction of the stem, leaves, and roots is used in the treatment of fever, cholera, diabetes, rheumatism and snake bites. The stem infusion is used as a vermifuge while the decoction of stem is used for washing sore eyes and syphilitic sores. The crushed leaves can also be applied on wounds and made into a poultice for itching. Besides these, it is also used to treat internal inflammation, reduces thirst and increases appetite. The stem of the plant is a registered drug in Thailand pharmacopoeia and is used in hospitals in the treatment of diabetes. In Malaysia, it has been used to treat the diseases common to the tropic like malaria (Rahman, et al., 1999). The drug is considered as a universal medicine in Philippines and Malaysia. Commonly prescribed as an aqueous extract, it is used in curing of stomach disorders, indigestion and diarrhoea. Several cocktails contain bitter wine. The dichloro methane extract of the plan has also shown free radical scavenging activity against DPPH (2, 2- diphenyl-1-picrylhydrazyl) radical (Cavin, et al., 1998).<br />Chemical Constituents: Since the plant has been known for its medicinal effects, attempts have been made to identify the chemical constituents of the T. crispa. The leaves of the plant contain a bitter amorphous principle called picrotine. The whole plant contains traces of alkaloid and berberine, a glucoside. There is however a controversy over the presence of alkaloids. The CH2Cl2 extract of the stem showed the presence of N-cis-feruloyltyramine, N-trans-feruloyltyramine and secoisolariciresinol, all of which had anti oxidant and radical scavenging property against β- carotene. Besides these, the stem also contains two triterpenes namely cycloeucalenol and cycloeucalenone (kongkathip, et al., 2004). <br />Figure 3-a: The chemical constituents present in T. crispa: Apigenin <br />(b)(a) <br />Figure 3-b : The triterpenes present in T. crispa: a) cycloeucalenol, b) cycloeucalenone<br />The stem also contains apigenin (flavonoid), picroretoside, berberine, palmatine, picroretine and resin. <br /><ul><li>Anti diabetic and insulinotropic effect of Tinospora crispa:</li></ul>Several animal studies were carried out to observe the anti diabetic effect of T. crispa. Amongst the very first studies was the one carried out by Noor and colleagues where they studied the anti- hyperglycaemic effect and the insulinotropic effect of the drug in human and rat islets and the HIT-T15 B cells (Noor, et al., 1989). Diabetes was induced in albino rats using alloxan (40mg/ml) and the rats with fasting blood glucose levels of 14-20 mmol/l one week after alloxan administration were selected. Three weeks after alloxan administration, T. crispa extract (4g/l) was dissolved in drinking water and the extract administration lasted for 2 weeks. The glucose levels, insulin levels and the weight gain between the normal and the alloxan-diabetic rats were compared. The results showed that the blood glucose levels of treated diabetic rats reduced significantly which was maintained throughout the course of treatment. Besides decreasing the blood glucose levels, the serum insulin levels rose significantly in treated diabetic rats as compared to untreated diabetic rats. When compared to normal animals, T. crispa had no effect on the same parameters. Further, the treated diabetic rats showed signs of recovery of body weight as compared to diabetic controls. Experiments on rat islets showed that T. crispa extract produced a dose dependent increase in the basal (2mmol/l) insulin secretion as well as potentiated glucose-stimulated (10mmol/l) insulin secretion. The same results were seen in HIT-T15 B cells, where T. crispa induced both dose dependent and potentiation of glucose-stimulated insulin release. These were the first studies that provided biochemical evidence which substantiated the claims for an oral hypoglycaemic effect of Tinospora crispa (noor). The hypoglycaemic effect may be a direct result of increased insulin secretion .These experiments however did not characterize the exact mechanism of how T. crispa induced insulin release. The hypoglycaemic effect could be a result of inhibition of intestinal glucose absorption or stimulation of peripheral glucose uptake.Further experiments were needed, to determine in detail, the exact mechanism by which the plant showed it’s hypoglycaemic and insulin secretion effect. <br />In 1997, Noor and his colleagues carried out experiments to determine the pharmacological properties of T. crispa extract (Noor, et al., 1997). The severe post-prandial hyperglycaemia, commonly observed in diabetic patients, can be prevented if the rate of glucose uptake into the blood circulation from intestine could be prevented. Scientists showed that T. crispa had no effect on the uptake of glucose from the intestine. This showed that the hypoglycaemic property of the plant was not due to its interference with the intestinal glucose absorption. Similar to the entry of glucose in the blood circulation, its exit from the circulation could also affect the blood glucose levels. Therefore drugs like metformin which improve insulin activity can decrease the blood glucose levels. However, the extract of T. crispa showed no effect on the uptake of methyl-glucose in adipocytes, which meant that the hypoglycaemic effect of the plant was not because of its ability to mimic or improve insulin activity. As mentioned above, since the extract caused an increased secretion of insulin, it was challenged against insulin secretory antagonist adrenaline, verapamil and nifedipine. Adrenaline exerts its inhibitory effect by binding to α- adrenergic receptors and interferes with the cAMP system or alters CA2+. Since it was seen that the insulinotropic effect of T. crispa was inhibited by adrenaline, it suggested that the action of T. crispa could me mediated by the two mentioned secondary messenger systems namely cAMP or CA2+. However there was no significant increase in the cAMP concentration when the drug extract was added in an incubation medium containing HIT- T15 B cells. This ruled out the possibility of T. crispa mediated cAMP involvement. Further calcium channel blockers such as verapamil and nifedipine, caused a significant inhibition of T. crispa mediated insulin secretion. This led to the hypothesis that the hypoglycaemic activity of the plant could be due to modulation of calcium handling by β cells. Thus it was concluded that the insulinotropic and ultimately the antidiabetic effect of T. crispa is due to stimulation of insulin release via modulation of β- cell Ca2+ modulation (Noor, et al., 1997).<br />Other studies on the cardiac contractility of rat atria showed that cycloeucalenone produced a slight change in the right atria as compared to control (80%alcohol) while cycloeucalenol showed an initial reduction and then a slight increase in the contractility of right atria as compared to noradrenaline which has positive inotropic and chronotropic effect. The two compounds produced a slight increase in the right atrial rate. However both of them produced an initial and then a sustained reduction in the left atrial contraction while noradrenaline gave marked contraction. The mechanism of how cycloeucalenone and cycloeucalenol act on the atria is unknown. Further studies need to be undertaken to study the cardiotonic effects of these two terpenes. <br />Since the precise pharmacological properties of T. crispa in diabetes is not known, further studies need to be carried out to see if it could be useful in diabetes by acting in some other way.<br /><ul><li>AIMS:</li></ul>The given experiment was carried out to study the effect of Tinospora crispa on the oxidative stress induced impairment of vascular tone which may have serious consequences in Diabetes.<br /><ul><li>OBJECTIVES</li></ul>The following objectives would be achieved during the project.<br />Develop in vitro model to monitor change in vascular tone<br />Background understanding of diabetes and vascular physiology<br />Develop in vitro pharmacological techniques<br />Study effects of known pharmacological agents phenylephrine (vasoconstriction) and acetylcholine/sodium nitro prusside (vaso relaxants)<br />Effect of oxidative stress on vascular tone<br />Effect of Tinospora crispa on vascular tone in presence and absence of oxidative stress<br /><ul><li>METHODS:
  18. 18. Tissue preparation:</li></ul> The rats were dissected and the aorta was isolated. The connective tissue surrounding the aorta was removed carefully so as not to damage the tissue. The blood was removed from the aorta and it was then cut into rings of approximately 2mm length. The aortic rings thus isolated were kept in a buffered Krebs- Hanseleit solution prepared with the following composition (per litre) : NaCl,6.9 gms; KCl, 0.36 gms; MgSO4, 0.29gms; KH2PO4, 0.16 gms; NaHCO3; Glucose, 2gms; CaCl2, 0.37gms. The solution was kept at 37º C and aerated continuously with 95%O2 and 5% CO2.<br /><ul><li>Experimental protocol: </li></ul>The Lab-scribe was calibrated. The isolated aortic ring was then cut open taking care so as not to damage the aorta. The tissue was set up in the organ bath. The tissues were kept to relax at a tension of 2 grams for 2 hours, which ensured maximum contractile responses. <br /><ul><li>Procedure:</li></ul> Once the tissue was equilibrated in the organ bath for 2 hours, responses to phenylephrine (PE) were taken. Based on the contractile responses, the EC80 of PE was calculated. Thereafter, relaxations with acetylcholine (ACh) were recorded. For this, the tissue was contracted using the EC80 dose of PE and then ACh was added starting from the lowest concentration to higher concentrations till complete relaxation was observed. If relaxations were not obtained, sodium nitro prusside (SNP) was added to see if the tissue was alive. Similar to relaxation to EC80 induced contractions with ACh, relaxations with SNP were also recorded. To record the contraction and relaxation responses in present of oxidative stress, Antimycin A (0.5µM) was added after one hour of tissue stabilization. The responses to PE were recorded an hour later. The EC80 of PE in the presence of Antimycin was obtained. Relaxations in oxidative stress induced tissue were also obtained with ACh and SNP. Also, the contraction and relaxation responses to Tinospora crispa were noted down in presence of Antimycin and in absence of Antimycin. The crude extract was added immediately to the organ bath after equilibration. After an hour of stabilization, Antimycin A was added. The tissue was then left to stabilize for another hour and the contraction and relaxation response were recorded as per the same protocol performed for the control tissue.<br /><ul><li>Chemicals: </li></ul>Phenylephrine, sodium nitroprusside and acetylcholine were obtained from Sigma Aldrich, while the salts needed for preparation of Krebs- Hanseleit solution were obtained from Fisher chemicals. All other compounds used were of analytical grade. Ethanolic extract of the stem of T. crispa was used.<br /><ul><li>RESULTS:
  19. 19. The organ bath was set up and the responses to various drugs were noted down.
  20. 20. CONTRACTIONS WITH PHENYLEPHRINE:
  21. 21. Control: </li></ul>Once the tissue was stabilised for 2 hours in the organ bath, phenylephrine (PE) was added and the contraction responses were recorded using labscribe. The procedure was repeated three times. Figure 4 shows a trace of the responses obtained with PE. PE induced a dose dependent increase in contraction of the smooth muscles of the rat aorta. Initially it produced a gradual increase in contraction, but at a concentration of 1 * 10-8M produces a sharp increase in the response. At a bath concentration of 1*10-9 M PE marginal contraction of around 0.005 grams was observed which increased with the increase in concentration and reached a maximum of 0.69 grams at a concentration of 3* 10-5 M PE. Concentrations of PE beyond this did not produce any contractions in the tissue and a plateau was obtained indicating the maximum response. Table 1 shows the response obtained with various concentrations of PE (n = 3). Figure 5a indicates the concentration versus response curve of PE while figure 5b shows the graph of concentration of PE versus % response. <br /> <br /> Figure 4: Trace of the responses obtained with phenylephrine in a control rat aorta using the Labscribe. The tissue was kept in the organ bath under 2 grams relaxing tension. PE was added in a dose dependent manner. Each dose was added, and the contraction was noted. Once the maximum response with dose was obtained, the next dose was added and in this was the responses were noted till a maximum response was obtained. <br />Table 1: The response (in grams) and the % Response obtained with various concentrations of Phenylephrine in control rat aorta. The highest response obtained was considered as 100% and the responses at the other doses were calculated as a percentage of the highest response.<br />Figure 5- (a): Dose response curve of Phenylephrine in control Rat aorta. Phenylephrine produces a dose dependent increase in the contraction of rat aorta. a final bath concentration of 1*10-9 M PE marginal contraction of around 0.005 grams was observed which increased wit with the increase in contraction and reached a maximum of 0.69 grams at a concentration of 3* 10-5 M PE.<br />Figure 5- (b): Graph showing the % response obtained with increase in dose of Phenylephrine in control rat aorta. The highest response obtained was considered as 100% and the contractions at the other doses were calculated as a percentage of this highest dose.<br /><ul><li>In Presence of Antimycin A:</li></ul>Similar to the dose response curves obtained in the control tissues, contractions were obtained in tissues having oxidative stress. This oxidative stress was induced using various concentrations of Antimycin A. The highest concentration of Antimycin A used was 25 µM. However, at this concentration, PE did not show any contractions. Various concentrations of Antimycin A were therefore used. PE still did not evoke any responses at 20 µM, 15 µM, 10 µM and 5 µM concentrations of Antimycin A. A response of 0.28grams was seen with PE at a concentration of 1 µM concentration (figure 6 a). The dose of Antimycin was still lowered to 0.5 µM. PE gave an average response (n = 3) of 0.515 grams at this level of oxidative stress. Figure 6 b shows a trace of response obtained with PE at 0.5 µM Antimycin A. A graph (Figure 6 c) of response versus concentration of PE was plotted to calculate the EC80 of PE in the presence of Antimycin A (0.5 µM). Table 2 shows the values of contractions obtained with PE. Similar to the control, PE did not evoke a steep contraction at 1* 10-9M in the presence of Antimycin A. The contractions however increased on increasing the doses of PE and a maximum response (n=3) was obtained (0.515grams) at a concentration of 1 * 10-4M PE. <br /> <br /> Figure 6- a:A response of 0.28 grams obtained with PE as observed in the presence of 1 µM Antimycin A. The tissue was left to relax for an hour before oxidative stress was induced using 1 µM Antimycin A. After administering it, the tissue was further kept to relax for another hour and then the contractions were noted down.<br /> <br /> Figure 6 -b: Trace of Response to PE in presence of 0.5 µM Antimycin A. <br />Figure 6- c) - Dose response curve of Phenylephrine in the presence of Antimycin A (0.5 µM). The maximum response of 0.515 grams was observed at 1 * 10-4 M PE after which a plateau was reached.<br />Table 2: Contractions obtained with PE in the presence of Antimycin A. The response at 1* 10-4 was considered as 100%. The responses at other concentrations were expresses as a percentage of this response.<br /><ul><li>Phenylephrine response to Tinospora crispa in the presence of Antimycin A: </li></ul>The drug extract (75mg/ml) of the plant was administered immediately after setting the tissue under 2 gms tension. After an hour, Antimycin A (0.5µM) was added and the tissue was allowed to stabilise for another hour. Dose response curves (Table 3) were obtained for PE contractions in the presence of drug (T. Crispa-75 mg/ml) and Antimycin A(0.5 µM) induced oxidative stress. Figure 7 a shows a trace of the response obtained while figure 7 b shows the dose response curve. A maximum response of 0.44 grams (n=3) was observed in this case (table 3)<br /> <br /> Figure 7- a: Trace of the response obtained with PE in the presence of T. Crispa(75mg/ml) and Antimycin A (0.5µM). The drug extract was added immediately after setting the tissue up in organ bath. After an hour of relaxation, Antimycin A (0.5µM) was added. The tissue was kept to stabilize for another hour before taking contractions responses with PE.<br />Figure 7-b) Dose response curve of PE in the presence of T. crispa(75mg/ml) and antimycin A (0.5 µM)A maximum response of 0.44 grams was obtained with the highest concentration of PE.<br />Table 3: Responses obtained with PE in the presence of T. crispa(75 mg/ml) and Antimycin A (0.5µM)<br /><ul><li>PHENYLEPHRINE IN THE PRESENCE OF T. crispa ONLY</li></ul>Responses to PE in the presence of T. crispa only were also obtained without inducing oxidative stress. Figure 8 a shows the trace of the response obtained. These responses were almost of the same nature as compared to the control tissue. The responses obtained at various PE concentrations are as shown in table 4. Figure 8 b shows the dose response curve of the same, indicating that the maximum response obtained was 0.639 grams at a concentration of 3 * 10-4M PE. <br />Figure 8-a: Trace of the response obtained with PE in the presence of T. crispa alone. The procedure followed was the same as control. However the drug was added here. <br />Figure 8-b: Dose response curve of PE in the presence of T. crispa (75 mg/ml) only. The contractions obtained were almost of the similar type as the control tissue. The maximum response obtained was 0.639 grams<br />Table 4: Responses obtained with PE in the presence of T. crispa only (75mg/ml)<br />Figure 9: Comparison between the PE contractions on control, Antimycin A(AA)- 0.5µM, Antimycin A & T. crispa (75mg/ml) and T. crispa alone. The contractions were maximum in control aorta (0.697 gms). The lowest contractions were seen with Antimycin (0.515 gms) and with T. crispa in the presence of Antimycin A (0.44 gms). However, there was a marginal decrease in contractions in the presence of T. crispa alone (0.642 gms) as compared to control. There was a significant decrease in the response obtained between control and Antimycin A induced oxidative stress.<br />Comparing the responses of PE in each of the above cases, it was seen that control tissue showed the maximum response of 0.697 grams. Almost the same response (0.642grams) was observed with the presence of T. crispa (75 mg/ml). However the response decreased in the presence of Antimycin A induced oxidative stress. The response decreased to 0.516 grams in the presence of 0.5 µM Antimycin A and 0.44 grams in the presence of T. crispa and Antimycin A. Figure 9 shows a comparison between the various PE responses.<br />Applying two way anova to the above results, it was seen that there was not a significant difference (p= 0.394, p>0.05) between the control (PE alone) and T. crispa only. However there was a significant difference in the contraction obtained between control and Antimycin A (p= 0.019) and also between control and T. crispa and Antimycin A induced oxidative stress (p= 0.03).<br /><ul><li>RELAXATION WITH ACETYLCHOLINE:
  22. 22. In Control:</li></ul>The EC80 values for PE in the control were calculated from the dose response curve. Using PE EC80 (3 * 10-6M PE) to produce contractions, relaxations with ACh were obtained. Figure 10 a shows a trace of one such relaxation observed with ACh. The experiment was repeated three times. A graph was plotted to measure the ACh induced relaxations (figure 10 b). Table 5 shows the readings for Ach relaxations. Initial addition of ACh (1 * 10-9M) brought a small change in the relaxation of the rat aorta. On further additions of ACh, relaxations were observed (Figure 10 a) with a total relaxation being observed at approximately 1 * 10-6 M ACh. A sharp fall was seen at 3 * 10-7 M ACh.<br />Figure 10- a: Trace showing the relaxations obtained with ACh in PE EC80 induced contractions in rat aorta. Once the EC80 of PE was obtained, the tissue was given a wash. The EC80 concentration of PE was added again and the relaxations with ACh were recorded. On adding each dose of ACh, the tissue showed relaxation. It was allowed to relax to the maximum. <br />Figure 10 –b) relaxation brought about with ACh in rat aorta. Initial addition of ACh relaxed the tissue by approximately 0.005 grams. Further concentrations of ACh relaxed the aorta with a complete relaxation being observed at 1 * 10-6 M Ach<br />Figure 10- c) Percentage Relaxations observed with Ach in control rat aorta.<br />Table 5: Data showing the relaxation obtained with Ach in control rat aorta. Initial doses of ACh brought about a slight relaxation in the PE EC80 induced contractions. The relaxation increased on adding higher concentrations of ACh and reached complete relaxation at 1 * 10-6 M ACh.<br />In some cases a partial relaxation was observed with the highest concentration of ACh, as seen in figure 11-a. Since the relaxation was not 100% (but was greater than 70%) it was not considered in calculating the average results. Cases where a partial relaxation was observed with ACh, sodium nitro prusside (SNP) was added to bring about complete relaxation (Figure 11 b).<br />Figure 11 -a: Trace showing approximately 76% relaxation with acetylcholine in control tissue (The results were not considered in calculating the average relaxations). <br />Figure 11- b: Partial relaxation as seen with ACh in control tissue. In the above case relaxations were observed till a concentration of 3 * 10-5 M ACh. Endothelium independent vaso relaxant SNP was then added to bring about complete relaxation.<br /><ul><li>In Antimycin A induced Oxidative Stress </li></ul>The EC80 of PE was calculated in the presence of Antimycin A (0.5µM). It was approximately 3 * 10-6M. Relaxations with ACh were measured to see what changes oxidative stress brings about on the tissue. Figure 12 a shows the relaxation graph obtained with ACh in the presence of oxidative stress. Acetylcholine did not show any relaxations with endothelium under oxidative stress and therefore sodium nitro prusside was added to induce complete relaxations. <br />Figure 12- a: Trace of the relaxation obtained with Ach in the presence of 0.5 µM Antimycin A. Since ACh did not show any relaxations, SNP (3* 10-8 M) was used to bring relaxations.<br />Figure 12-b :Demonstrates the relaxation brought about with SNP when Ach did not relax the contractions of PE in the presence of Antimycin A. SNP immediately showed relaxations even with the lowest concentration of 1 * 10-9M (n=3).<br /><ul><li>Relaxation observed in the presence of T. crispa and Antimycin A:</li></ul>The EC80 for PE in the presence of T. crispa and antimycin A was calculated and was found out to be 3 * 10-6 M. ACh produced a dose dependent relaxation. However the contraction obtained was partial. The maximum relaxation obtained was 0.133 grams. Complete relaxation was therefore brought about using SNP as seen in figure 13.<br />Figure 13: Trace of relaxations as observed with the plant extract and oxidative stress. ACh did not relax the tissue completely. The maximum relaxation obtained was 0.133 grams. Further relaxations were not obtained. Complete relaxation was brought about using SNP. <br />Relaxations with Ach in the presence of T. crispa only were not recorded because of time constraints.<br /><ul><li>RELAXATION WITH SNP
  23. 23. Control</li></ul>Similar to the relaxations obtained with ACh, relaxation were also obtained with endothelium independent vaso dilator sodium nitro prusside (SNP). Figure 14-a shows the trace of the SNP induced relaxation in EC80 induced PE contractions. Figure 14 b shows the graphs plotted to calculate the relaxation observed with SNP. As compared to ACh, SNP at a concentration of 1 * 10-9 M, produced a significant relaxation of 0.105 grams. Further increasing the concentrations of SNP showed an increase in the relaxation of the rat aorta with a 0.55 grams relaxation observed at 1 * 10-7M SNP.<br /> <br /> Figure 14-a: Relaxation trace observed with Sodium Nitroprusside in control rat aorta. The tissue was contracted using the PE EC80 concentration. Once the tissue contracted to PE, SNP was added to cause relaxation. Complete relaxation was observed at a concentration of 1 * 10-7M SNP.<br />Figure 14-b SNP induced relaxations in control rat aorta. Significant Relaxations were observed at a dose of 1 * 10-9M SNP. Complete relaxation was seen at a concentration of 1 * 10-7M SNP.<br />Table 6: Relaxation as observed with endothelium independent vaso relaxant SNP<br /><ul><li>Relaxation with SNP in the presence of Antimycin A</li></ul>Figure 15 –a shows the graph of the relaxations observed with SNP in oxidative stress induced tissue. Relaxations were measured against maximum contractions that were induced using EC80 of PE in the presence of Antimycin A. Figure 15-b shows the graph of response versus relaxations.<br />Figure 15-a: Trace of relaxations observed with SNP in the presence of Antimycin A (75mg/ml). The tissue was relaxed for an hour under 2 grams tension. Oxidative stress was induced after an hour using 0.5 µM Antimycin A. After another hour of relaxation, PE EC80 contraction was obtained. SNP was added and the relaxations were noted down. <br />Figure 15- b: Figure showing the relaxation responses with SNP in the presence of Antimycin A. In the presence of Antimycin A, a concentration of 1 * 10-7M SNP gave a 100% relaxation.SNP showed a 100 % relaxation at a concentration of 0.45 grams at a concentration of 1 * 10-7M SNP.<br />Relaxations with SNP in T. crispa alone and also in presence of both T. crispa and Antimycin A were not obtained because of time constraints.<br /><ul><li>DISCUSSION AND CONCLUSION:
  24. 24. Discussion:</li></ul>The main objective of the study was to see if Tinospora crispa had any effect on the oxidative stress induced in diabetes (Creager, et al., 2003). Previous experiments carried out by Noor and colleagues have shown that the plant is able to decrease the glucose level and it also shows insulinotropic effect (Noor, et al., 1989, Noor, et al., 1997). To study the effect of T. crispa on oxidative stress, experimental model was designed whereby oxidative stress was induced in rat aorta using Antimycin A. Antimycin A is a known inhibitor of complex III in the mitochondrial respiratory transport chain (Raha, et al., 2000, Foster, et al., 2006). Thus it would mimic the oxidative stress seen in diabetes.<br />To compare whether the plant extract had any effect on the Antimycin A induced oxidative stress, control study was performed using the PE induced contractions in the normal rat aorta. PE brings about contraction by acting on the α1 – receptors located on the smooth muscles cells of the aorta (Rang, et al., 2003). Therefore agonists like PE may cause an increase in the systolic and diastolic arterial pressure by constricting the large arteries and veins. <br /><ul><li>Contractions with Phenylephrine</li></ul> In the experiment, PE was given to the tissue starting from the lowest concentration of 1 * 10-9M. The dose response curve was obtained till a maximum response was reached. Responses greater than 0.6 grams were considered good. PE initially caused a gradual increase in the contractions which gradually increased as the dose was increased. The maximum response was reached at a concentration of 3 * 10-5M. The average contraction of about 0.69 grams was obtained from three different tissues. Similar to response in controls, responses were also taken in the presence of oxidative stress. Oxidative stress was induced using Antimycin A. Since experiments involving formation of oxidative stress were not performed in organ bath, experiments performed on tissues were used to estimate the concentration of Antimycin A to be used. Based on a study performed by Raha and colleagues, it was seen that significant amount of reactive species was formed at a concentration of 20µM Antimycin A in rat mitochondrion (Raha, et al., 2000). A similar concentration was used to induce oxidative stress in the organ bath. However, at this concentration, PE did not induce any contractions. Thus it was inferred that the oxidative stress produced by Antimycin at this concentration was blocking the responses with PE. Therefore it was suggested to lower down the concentration of Antimycin. Responses were therefore noted at concentrations of 20µM, 15 µM, 10 µM, 5 µM, 1 µM and 0.5 µM. However the first response observed was at a 1 µM concentration ().28grams). The contraction obtained at this concentration was small and would have given a wrong result when relaxations were taken with ACh, because the contractions were not comparable to the control. Finally a contraction response of 0.515 grams was obtained with a concentration of 0.5µM concentration of Antimycin A. Once the concentration of Antimycin was decided to induce the oxidative stress, the responses were measured in the presence of the plant extract (75mg/ml). At the same level of oxidative stress, T. crispa evoked a response of 0.44 grams, which is a significant decrease as compared to the control tissue (p = 0.03, p < 0.05). When the results were compared in the presence of T. crispa alone, a response of 0.642 gms was obtained which is almost the same as that in the controls. This shows that oxidative stress causes a decrease in the PE induced contractions of the rat aorta.<br />Figure 16: Graph showing the contraction responses with PE under various conditions. There was a significant difference between the contractions between the control and the tissue in the presence of Oxidative stress (indicated by the p values). There was no significant difference between the contractions obtained with Antimycin A and T. crispa plus Antimycin A (p > 0.05). Similarly there was no difference in the contractions obtained in the control and T. crispa alone (p= 0.394, p> 0.05). This suggests that Antimycin A decreases the PE induced contractions by either damaging the tissue, or by blocking the receptors. (PE- phenylephrine, AA- Antimycin A, TC- T. crispa.) <br /> Figure 16 shows the contraction responses obtained with various conditions. The decrease in the contractions could be a result of excessive oxidative stress that could damage the tissues. This was confirmed by giving the tissue a wash with the Krebs – Hanseleit solution and then trying to obtain responses with PE. However when the tissue was given a complete wash, no responses were obtained which could mean that the tissue was being damaged by Antimycin A. The other reason could be that it could be blocking the α1 receptors with Antimycin A or the oxidative species as a result of which the tissue does not induce contractions. Since T. crispa was not able to reverse the decrease in the response with PE in the presence of Antimycin A, induced oxidative stress, it could be inferred that it is not able to prevent the formation of free radicals.<br /><ul><li>Relaxations with Acetylcholine</li></ul>Acetylcholine is an endothelium dependent vaso relaxant (Brailoiu, et al., 1999, Rang, et al., 2003). ACh induces relaxations by stimulating the endothelium to release NO (Nedeljkovic, et al., 2003). The mechanism of NO release as a result of ACh is discussed above. The nitric oxide has inherent pharmacological properties of vaso relaxation, inhibition of platelet aggregation, inhibition of vascular smooth muscle cell proliferation and inhibition of inflammatory adhesion molecules (Papaharlambus and Griendling, 20070. When relaxations were performed on the control tissue, ACh initially produced a marginal relaxation, which was followed by a steep relaxation with further increasing the concentration of ACh. Relaxations were obtained till the initial base line was reached. The highest response was considered as baseline to consider the relaxation with successive doses of ACh. Cases where a 100% relaxation was not obtained were not considered, because it meant the endothelium was damaged leading to decreased NO and therefore less relaxation. However the tissue having a relaxation of around 70% was deemed as having endothelium intact. In such cases, complete relaxation was obtained using external NO donor sodium nitro prusside (SNP) (Brailoiu, et al., 1999, Rang, et al., 2003). Relaxations were also checked at level of 0.5µM Antimycin A induced oxidative stress. However in the presence of oxidative stress no relaxations were observed with ACh, indicating that Antimycin was inhibiting the responses of ACh. This could be a result of excessive oxidative stress, whereby the amount of NO produced by the ACh is scavenged by the free radical ions. To check the viability of the tissue for relaxation, SNP was administered. This showed relaxations, indicating that the oxidative stress was inhibiting the NO produced by the endothelium and that the NO thus produced was therefore insufficient to bring about a relaxation which is as expected in diabetic oxidative stress conditions with vascular complications (Creager, et al., 2003). The concentration of SNP required to bring about complete relaxation in oxidative stress induced tissue, in the presence of ACh, was approximately 3 * 10-8M. When relaxations were taken in the presence of T. crispa (75mg/ml) and oxidative stress, partial relaxations were observed with ACh initially. Since no further relaxations were observed with ACh, relaxations were obtained using SNP. This showed T. crispa was able to negate the effects of Antimycin A induced oxidative stress. This effect could be because T. crispa was preventing Antimycin A from producing oxidative stress by stimulating the endothelium to produce more NO in the presence of ACh as a result of which the free radicals were scavenged. The other reason for the relaxant activity could be increased release of enzymes such as SOD, in the presence of the drug. However since complete relaxations were not seen, meant that some of the effects of oxidative stress were still present. Also, as mentioned earlier, it was not able to reverse the decrease in the contraction of PE in the presence of oxidative stress. This means that it could be having the vaso relaxant activity by stimulating the endothelium to produce more NO and not by mimicking the activity of antioxidant enzymes. Results also need to be verified at higher concentrations of T. crispa to see if complete relaxations can be obtained with ACh. Thus the levels of the antioxidant enzymes need to be studied in the presence and absence of drug. Also the effect of plant on the oxidative stress could be checked by measuring the amount of free radical species produced using luminiscence measurement (Moncado & Higgs, 2006). If T. crispa was able to decrease the formation of free radicals, there would less oxidative species obtained as compared to the tissue with Antimycin A only. <br /><ul><li>Relaxations with SNP</li></ul>SNP is an external NO donor (Brailoiu, et al., 1999, Rang, et al., 2003) and is thus responsible for relaxation of blood vessels. In the control tissue, when relaxation were obtained to EC80 PE induced contractions, a concentration of 1 * 10-7M SNP was requires. In the presence of oxidative stress the concentration of SNP required was 1 * 10-7M, which is the same as the control. Thus it could be said that oxidative stress had no effect on relaxation induced by external NO donor. However, in case of oxidative stress, where relaxations were not obtained with ACh, a lower concentration of 3 * 10-8M SNP was required. This shows that ACh induced NO production from the endothelium already scavenged a few free radicals as a result of which a lower concentration of SNP gave complete relaxation as compared to when SNP was used directly to induce relaxation in oxidative stress induced tissue. Because of time constraints, relaxations with SNP were not obtained in the presence of T. crispa oxidative stress and with T. crispa alone. This study was necessary to see the effect of the drug on external NO donor.<br /><ul><li>CONCLUSION</li></ul>Tinospora crispa has known anti hyperglycemic and insulinotropic effect. Here in the experiment, it was demonstrated that the crude plant product may also have some activity against diabetes induced oxidative stress. In the experiment it was seen that T. crispa was able to restore some of the acetylcholine induced relaxant activity in the presence of Antimycin A induced oxidative stress. This activity was not seen with acetylcholine in the presence of Antimycin A alone. However T. crispa was unable to reverse the decrease in contraction response seen with phenylephrine in the presence of oxidative stress, which meant that it was unable to prevent the Antimycin A induced free radical species generation. The other reason could be the increase or mimicking the activity of antioxidant enzymes such as SOD in the presence of drug. However these results need to further verified upon. Thus the vaso relaxant activity of T. crispa in the presence of oxidative stress seems to be mediated through increased NO production by the endothelium. Also since the drug shows antioxidant properties, its activity in other vascular diseases such as atherosclerosis where oxidative stress is present needs to be studied.<br /><ul><li>DRAWBACKS AND FUTURE STUDIES:</li></ul>In the above experiment it was demonstrated that the plant was able to induce acetylcholine mediated relaxations even in the presence of oxidative stress. Therefore it could be suggested that the plant was able to restore some of the inherent properties of the endothelium. However, further studies need to be carried out to verify the anti oxidant activity of the plant extract. This could be done using chemi luminiscence to measure the formation of oxidative species produced in the presence of the drug and comparing it with to the free radical formation in the absence of drug. Also the formation of NO could be studied and the mRNA expression of eNOS could be studied. The results could also be compared with the anti oxidant activity of enzymes such as super oxide dismutase or catalase which are known to decrease the formation of superoxide radicals. The experiments carried out were in a normal rat aorta. The same experiments need to be repeated in a diabetic rat aorta to verify the antioxidant activity of the drug in diabetes. The concentration of T. crispa used was 75mg per ml. At this concentration it evoked a partial relaxation was obtained with endothelium dependent vasodilator ACh. A higher concentration of the plant extract could be used to see if complete relaxation was observed with ACh. Also the concentration of Antimycin A required to produce oxidative stress needs to be verified by measuring the amount of reactive species so as to check if the amount of reactive species produced in diabetes are the same as that produced with Antimycin A. Relaxations with ACh at lower levels of Antimycin A need to be studied to determine the amount of oxidative stress that does not affect the inherent property of endothelium to relax the tissue. Beyond this concentration, oxidative stress could be having an adverse effect on the inherent properties of smooth muscle and the endothelium. 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