Thanigavelan, (2016) Cardioprotective Activity of Linga Mathirai in Rodents. Doctoral thesis, The Tamilnadu Dr.M.G.R. Medical University, Chennai.
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Abstract
Ischemic heart disease (IHD), otherwise known as Coronary artery disease (CAD) is a term for a group of closely related syndromes caused mainly due to atherosclerosis of Coronary artery causing ischemia of myocardium. Among IHD, Myocardial Infarction (MI) commonly known as heart attack occurs due to the blockage in any one of the coronary arteries and cause ischemic necrosis on myocardium. The most common triggering event is the disruption of an atherosclerotic plaque in an epicardial coronary artery leading to clotting cascade that result in occlusion of the lumen. Inflammation is known to be an important step in plaque formation. In Siddha medicine, purified Lingam (Cinnabar) based medicines have known to be a potent anti-inflammatory drug and which cures the diseases that occurs in Naadi (Blood Vessel). In the literature Anuboga Navaneetham, Linga Mathirai (a compound pill) was indicated for treating Uruthira Vayvu (Cardiac pain) at the dosage of 60 mg/dose with the vehicle Ginger decoction. With the above background, the research work was carried out on Linga Mathirai (LM) to validate its safety and efficacy in the management of Ischemic Heart disease in both preventive and curative aspect in animal model. The test drug LM was prepared as per the traditional method cited in Anuboga Navaneetham with modification in the purification process of Lingam. LM was prepared by triturating equal proportion of purified powdered Lingam and purified powdered Vengaaram (Borax) in breast milk for 3 h. 1. The test samples were analyzed for qualitative and quantitative estimation. Preliminary physical parameters such as total ash, moisture content and extractive values were analyzed. The functional groups were analyzed by FT-Raman Spectroscopic study. The content of lead and cadmium were analyzed using Atomic Absorption Spectroscopic study. The concentration of elements in oxide form was analyzed through Energy dispersive X-Ray Fluorescence. The concentration of trace and heavy metals were analyzed using Inductively Coupled Plasma – Optical Emission Spectrometer. From the result of above studies, we inferred that LM has long stability period having better quality but the concentrations of mercury and sulphur was beyond the WHO permissible limit. WHO framed the permissible limits of heavy metals in herbal raw drugs only but not for traditional compound drugs particularly herbo-metallic formulations. Linga Mathirai is a purely mineral formulation processed under continuous trituration with breast milk which has the properties of detoxifying as well as antagonist against the mercurial compounds. Even though LM contains high concentrations of mercury and sulphur, to validate its safety, the toxicity studies in animal model were carried out. 2. An acute oral toxicity study was conducted on Sprague Dawley (SD) rats to determine the safety for Linga Mathirai (LM) to produce toxicity from a single dose of 2000 mg/kg via oral route following OECD guidelines 423. Based on the results of this study, the single dose acute oral median lethal dose (LD50) of the LM is greater than 2000 mg/kg of body weight in SD rat. 2000 mg/kg of body weight was administered to six healthy SD rats by oral gavage. No mortality, signs of toxicity and behavioural changes were observed at least once daily for 14 days. Body weights were recorded on initial day before LM administration and again on 7 day and 14 day. At the terminal period, since all rats appeared healthy, the rats were not sacrificed and necropsy study was not performed. 3. To determine the target organ toxicity, no observed effect level (NOEL) and reversibility of signs and toxicity after recovery period for the test drug LM, the sub chronic toxicity study of LM administered orally to SD rats for 90 days was conducted. For 90 days study, OECD guidelines No. 407 was followed with some modification in dose fixation of test drug using AYUSH protocol. SD rats of male and female in controlled age and body weight were selected and randomized and grouped into five groups. LM was administered 11, 55 and 110 mg/kg body weight as suspension prepared using ginger decoction to the three groups respectively. Control and LM 110 mg/kg treated satellite groups were allotted. End points were body weight, functional observational battery including motor activity and neurological examination, haematogical and biochemical analysis. The results were recorded on day 91 and for satellite group on day 119. All rats participated in the study treated up to 110 mg/kg survived throughout the dosing period of 90 days and the recovery period of 28 days. No intoxication signs were observed in male and female rats from different dose groups during the dosing and recovery period. Male and female rats from all the treated dose groups had comparable gain in their body weight with that of control group throughout the dosing and recovery period. Comparable food consumption was found between control and treated rats. Functional observation tests done before the scarification revealed no abnormalities. Haemotological, biochemical and urine analysis revealed no abnormalities attributable to the LM treatment. Organs weight of treated groups recorded on sacrificed day were found to be comparable with control. Gross study on the internal organs revealed no abnormalities. Histopathological examination of H&E sliced vital organs except cerebrum, kidney and small intestine showed normal architecture. The accumulation of mercury in kidney was studied and revealed that reduced amount of mercury was found in the LM treated groups when compared with control. So, the changes found in the cerebrum, kidney and small intestine in the high dose and high satellite groups were not specific to the treatment of LM. 4. In vitro clot lysis and platelet aggregation assays were employed for the study on blood samples of rat. Whole blood and platelets obtained by centrifugation was used for clot lysis and aggregation assay. Platelet aggregation was induced by agonists Thrombin and Adenosine diphosphate (ADP). The different concentrations of LM (100, 200, 500, 1000 μL) was prepared with the adjuvant Ginger decoction for test samples and 100 μL low molecular weight heparin was used for treating standard group. Against clot and platelet aggregation, LM did not exhibit significant efficacy on compared with normal saline (control) and heparin. However, LM at its higher concentration 1000 μL exhibited significant inhibitory activity on platelet aggregation induced by both Thrombin and ADP when compared with distilled water (control). 5. The cardio-protective effect of LM at 11 mg/kg body weight, orally, daily for 8 days as pre and post treatment against myocardial infarction in SD rat’s myocardium induced by Isoproterenol (ISO) injection subcutaneously at the dosage of 5.25 and 8.5 mg/kg twice at an interval of two days was investigated on comparison with standard drug Clopidogrel. For the study, five groups of rats were allotted viz., normal control, ISO control, pretreated standard and test drug, and post treated standard and test drug groups. The biochemical cardiac markers such as Aspartate Amino transferase (AST), Alanine amino transferase (ALT), Creatine phosphokinase (CPK), Acid phosphatase (ACP), Alkaline phosphatase (ALP), Lactate dehydrogenase (LDH), cardiac Troponin T (cTnT) and Homocysteine (Hcy) in plasma were determined. The histo-morphometric studies were done on the myocardium to estimate the infarction area, inter myocardial fiber distance and microscopic features in Hematoxylin and Eosin stained ventricle slices. The antioxidant status on heart tissues were also evaluated by analyzing the activities of antioxidant enzymes such as Lipid peroxides (LPO), Reduced Glutathione (GSH), Glutathione peroxidase (Gpx), Glutathione-S-transferase (GST), Catalase (CAT) and Superoxide dismutase (SOD).In the positive control rats, ISO induced myocardial infarction was manifested by a significant increase in the levels of diagnostic markers such as AST, ALT, LDH, CPK, ACP and ALP troponin T and homocysteine in plasma. The histo-morphological observation of myocardium also confirmed the necrotic damage induced by isoproterenol. No mortality and morbidity was observed in all groups during and after experiment.AST, ALT, CPK, ACP, ALP, LDH, Troponin T and Homocysteine levels were reduced significantly in group C (Standard Pretreated), group D (LM pre-treated), group E (Standard Post-treated) and group F (LM post-treated) as compared to group B (Iso control).LPO level were reduced significantly in group C (Standard Pretreated), group D (LM pre-treated), group E (Standard Post-treated) and group F (LM post-treated) on compared with group B (Iso control).GSH, GST, CAT and SOD levels were increased significantly in group C (Standard Pretreated), group D (LM pre-treated), group E (Standard Post-treated) and group F (LM post-treated) on compared with group B (Iso control).GPx level was increased significantly in group C (Standard Pretreated), group E (Standard Post-treated) and group F (LM post-treated) on compared with group B (Iso control).The percentage of infarction was reduced significantly in group C (Standard Pretreated), group D (LM pre-treated), group E (Standard Post-treated) and group F (LM post-treated) when compared with group B (Iso control).There was significant decrease of the distance of inter-myocardial fiber in group E (Standard Post-treated) and group F (LM post-treated) when compared with group B (Iso control).There was significant decrease of histological score in group C (Standard Pretreated), group D (LM pre-treated), group E (Standard Post-treated) and group F (LM post-treated) when compared with group B (Iso control).There was significant increase of fibrosis score in group E (Standard Post-treated) and group F (LM post-treated) when compared with group B (Iso control). 139 6. Anti-atherogenic effect of LM at 11 mg/kg body weight was studied in SD rat‟s atherosclerotic aorta induced by 5 days treatment of Vitamin D3 based Atherogenic diet. For the study, five groups of rats were allotted viz., normal control, atherosclerotic control, standard drug Atorvastatin and test drug LM groups treated for 14 days, and another test drug group treated for 7 days. At the end of treatment, blood samples of rats of different groups were analyzed for serum lipid profile estimation. The histo-morphometric studies were done on the aorta to measure the atherosclerotic lesions. Percentage of atheroma formation, microscopic changes in Hematoxylin and Eosin stained aortic slices and grading of atherosclerotic lesions were done.No mortality and morbidity was observed in all groups during and after experiment. Total cholesterol, TG, VLDL, LDL levels and Atherogenic index were reduced significantly in group III (Standard 14d), group IV (LM 14d) and group V (LM 7d) when compared with group II (Athero control).HDL level was increased significantly in group III (Standard 14d), group IV (LM 14d) and group V (LM 7d) when compared with group II (Athero control).The percentage of atheroma formation was reduced significantly in group III (Standard 14d) and group IV (LM 14d) when compared with group II (Athero control).There was no significant difference of total wall area of aorta and thickness of intima, media and adventitia of group III (Standard 14d), group IV (LM 14d) and group V (LM 7d) when compared with group II (Athero control).There was no significant difference in aortic lumen size of group III (Standard 14d), group IV (LM 14d) and group V (LM 7d) when compared with group II (Athero control). 140 From the above studies, it can be concluded as follows 1. Linga Mathirai has long shelf life period and free from toxins, and it contains metallic group specifically mercury attached with sulphur. 2. Median lethal dose for Linga Mathirai was calculated as more than 2000 mg/kg body weight. 3. No observed effect level (NOEL) of Linga Mathirai prepared by us to Sprague Dawley Rats through oral route over a period of 90 days was found to be 110 mg/kg body weight for both male and female rats. 4. Linga Mathirai demonstrated no significant clot lytic property in different rat blood samples on compared with low molecular weight heparin but the study revealed that LM have antiplatelet aggregation property. It can be concluded that Linga Mathirai show better thrombolytic activity but not so better efficacy as low molecular weight heparin. 5. Administration of Linga Mathirai is effective in minimizing all the deleterious effects induced by isoproterenol, thereby justifying its use as a potent cardio-protective agent. 6. Treatment of Linga Mathirai lowers the atherosclerotic formation in aorta and grade of atherosclerotic lesions and serum lipids. The overall cardioprotective effect of Linga Mathirai is probably related to its vasodilator effect and thrombolytic effect and antioxidant property evidenced by its ability to reduce lipid peroxidation and to maintain the activities of free radical enzymes and nonenzymatic antioxidants, its membrane stabilizing action and to its hypolipidemic property. CONCLUSION: IschemicHeart Disease remains the major cause of mortality in both developed and developing countries. Myocardial infarction is the rapid development of ischemic necrosis of myocardium. Atherosclerosis in coronary artery accounts for myocardial ischemia in more than 90% cases. The present research work is an attempt to characterize a Siddha Sastric formulation – Linga Mathirai and to validate the Cardioprotective effect of Linga Mathirai against Isoproterenol induced myocardial infarction and Vitamin B3 based atherogenic diet induced atherosclerosis in Sprague dawley rats by estimating the biochemical markers and histopathological studies. The data of the research showed that Linga Mathirai at the dosage of 11 mg/kg body weight in rats modulated most of the biochemical parameters and histopathological lesions to near to the normal status and assuring its cardioprotective role. Linga Mathirai has been scientifically studied and validated to use as a drug in the management of Ischemic Heart Disease at the intended therapeutic dose 130 mg/kg twice daily cited in the literature along with the adjuvant dried ginger decoction.
| Item Type: | Thesis (Doctoral) |
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| Additional Information: | Ref.No.00068/2012 |
| Uncontrolled Keywords: | Cardioprotective activity, Linga Mathirai, Rodents. |
| Subjects: | AYUSH > Gunapadam |
| Depositing User: | Subramani R |
| Date Deposited: | 01 Jan 2019 14:52 |
| Last Modified: | 14 Oct 2022 16:38 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/10297 |
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