SUMMARY AND CONCLUSION

Transcription

1 CHAPTER VI SUMMARY AND CONCLUSION

2 CHAPTER 6. SUMMARY AND CONCLUSION "The doctor of the future will no longer treat the human frame with drugs, but rather will cure and prevent disease with nutrition." -Thomas Alva Edison Chapter 6: Summary and Conclusion Toxicological research on arsenic induced toxicity is of increased importance world-wide, in recent years, and recent publications cover all aspects of toxicology. Over centuries exposures to arsenic continue to be a major public health problem in many countries. The high priority of this issue is now recognized politically in a number of countries. Also experimental research on toxicokinetics and toxicodynamics and on modes of toxic action is moving very rapidly. The matter is of high regulatory concern, and effective preventive measures are required in a number of countries. Camellia sinensis (Green tea) has been consumed for centuries as a hot beverage and has got immense medicinal properties [Sherwani et al., 2013]. It is a popular herbal ingredient that has been manufactured into more than 100 supplements. Green tea has been shown to have a wide range of beneficial physiological and pharmacological effects [Sherwani et al., 2013]. Green tea s most touted benefits are its antioxidant and weight-loss properties [Shreena S Patel et al., 2013]. Likewise, cocoa and cocoa based products like chocolate are widely consumed by all age groups in many countries and cultures. It is well known that plants which possess antioxidative and pharmacological properties are related to the presence of many phytoconstituents [Ilhami Gulcin, 2012]. Therefore, cocoa and chocolate may be considered as functional foods [Rusconi and Conti, 2010]. Increasing interest in the health benefits of green tea (Camellia sinensis) and cocoa (Theobroma cacao) has led to the inclusion of green tea and cocoa in the group of beverages with functional properties. The research interest based on tea and cocoa components may provide an approach to decrease the incidence of and mortality from various diseases. Hence, the present study was taken up to check the role of these beverages (Green tea and Cocoa) on Arsenic Toxicity. In order to check the quality of both the plant products the physico-chemical analysis such as total ash, acid insoluble ash, water soluble ash, alcohol soluble, water soluble extractive and crude fiber content were determined in C. sinensis and T. cacao as per the methods referred in Ayurvedic pharmacopoeia. All physico-chemical 178

3 values were found to be well within the permissible limit approving the quality of the raw material to be considered for use as a nutraceutical supplement. Further to the quality assessment by physiochemical standards, both the C. sinensis and T. cacao extracts and its fractions were screened for the phytochemicals in them qualitatively using standard methods. Subsequently, quantitative analysis of primary metabolites such as carbohydrates, lipid and proteins, followed by secondary metabolites like total phenols, tannins, flavonoids, Vitamin E and Vitamin C were carried out in C. sinensis and T. cacao extracts and fractions. The qualitative phytochemical screening showed the presence of tannins, phenol, flavonoids, alkaloids, reducing sugar, proteins, steroids and quinones except triterpenoids. Phenolic compounds were found to be much higher in C. sinensis and T. cacao crude extracts than its fraction. Likewise, the various macro and micro elements like Na, K, Ca, Zn, Fe, Cu, Mn, Mg, and toxic elements such as As, Hg and Pb were estimated in both the raw material using a flame photometer and an Atomic absorption spectrometer (AAS) (Perkin Elmer). The nutrient elements were found to be present in the raw material which probably mediates the antioxidant potential of our plant products. However, the toxic elements are well within the permissible limit as per WHO and AYUSH (Govt of India) guidelines. HPTLC fingerprinting of the crude extracts, tannified fraction and detannified fraction of both the plants C. sinensis and T. cacao and its fractions were performed using Toluene: Ethyl acetate: Formic acid: Methanol (30:30:8:2) mobile phase. The mobile phase was suitable for the separation of tannins. The HPTLC fingerprinting of the tannin rich crude extract was carried out and a total of 5-6 peaks were observed. The results showed the presence of phytoconstituents which was quantitatively estimated by the catechin, and epicatechin content by the corresponding peaks in UV light at 254 and 366 nm. The antioxidant potential of the extracts of C. sinensis and T. cacao and its fractions were tested using different standard in vitro antioxidant assay methods viz. DPPH stable radical scavenging, Nitric oxide scavenging, Lipid peroxidation inhibition, and they were compared with standard Vitamin E and Epicatechin. Crude extracts were found to be better than its fractions which were confirmed through its IC 50 values. ABTS radical cation decolourisation assay were also performed and 179

4 compared with standards. The ABTS result showed that crude extracts C. sinensis and T. cacao were found to be better than its detannified fractions showing better antioxidant potential. Altogether, the results confirm that the tannin rich green tea and cocoa exhibited better in vitro antioxidant activity and potent free radical scavenging potential which might be due to the presence of high tannin and polyphenol content. However, the crude tannin rich extract of T. cacao when compared to C. sinensis showed higher amount of polyphenols and exhibited better in vitro antioxidant activity and potent free radical scavenging potential. MTT cytoxicity was carried out using Chang liver cell lines. In MTT assay, crude drugs (GTC and CC) increase the percentage of viability of cells when compared to the number of viable cells in the detannified treated groups. The degree of apoptosis was confirmed by staining techniques. Compared to the percentage of cells in the control, the percentage of cells proliferation was significantly increased after a 24 h of arsenic treatment. Among the cells treated with NaAsO 2, the percentages of cell viability in the cells grown in tannin rich crude drug treatment were significantly higher than those in the cells grown in the detannified drug treated groups. Based on the results of the in vitro studies on the biological activity, in vivo studies using experimental animals were carried out after due ethical clearance. Hence, an attempt was made to study the extent of cellular and oxidative damage caused by arsenic and its protection by both crude extract and detannified fraction of C. sinensis and T. cacao. Arsenic intoxication was evaluated at three dose levels (30, 100, 300 ppm) in drinking water. Changes in body weight, arsenic content, biochemical markers and histopathological sections were analyzed in liver and kidneys to support the dose and duration of the arsenic intoxication. The dose of 30 ppm showed less alteration compared to other doses and a dose of 300 ppm showed very high damage in both liver and kidney tissues. However, the dose of 100 ppm produced severity of arsenic toxicity but it was in a dose relevant manner with very less severity and not very high damage. Therefore, based on the dose and duration study,100 ppm of arsenic was found to be better as it showed lower signs of toxicity and no mortality as that of 300 ppm, where 50% mortality had occurred. Based on the above validation a dose of 100 ppm for a period of 28 days was used in female rats to 180

5 determine the efficacy of green tea and cocoa in ameliorating the toxic effects of Arsenic. Chapter 6: Summary and Conclusion The crude (GTC) and detannified (GTDT) fractions of C. sinensis and the crude (CC) and detannified (CDT) fractions of T. cacao were subsequently administered orally to the rats exposed to arsenic toxicity via drinking water for a period of 28 days. In efficacy treatment study, no significant changes in body weight of animals in different groups were noted. However, the growth was comparatively poor in arsenic exposed untreated rats (group II) as evidenced by lower mean body weights as compared to the normal control rats. Weight of the brain, heart, liver, and kidney was taken at the end of experimental period. Arsenic intoxicated (groups II) animals showed difference in organ weight when compared to the normal. Likewise, hematological parameters showed significant changes when compared to the group II arsenic poisoned group animals. Biochemical parameters such as glucose, cholesterol, triglycerides, urea, creatinine, Uric acid, Total protein, Lactate dehydrogenase (LDH), Gamma glutamyl transferase (GGT), Bilirubin, Alanine transferase (ALT), Aspartate transferase (AST), Alkaline phosphatase (ALP), Acid phosphatase (ACP), Albumin, Globulin were measured in plasma using Accurex kit in a semi autoanalyzer. Elevation of these plasma markers in group II confirms the severity of Arsenic toxicity in rats. Crude extract treatment of GTC and CC prevented the elevation of these marker enzymes when compared to the detannified fraction treated groups of GTDT and CDT. Arsenic toxicity marker enzyme (ALAD) was measured in the hemolysate and all major organs using spectrophotometric method. The level of marker enzyme was decreased significantly in group II animals. The treatment groups of GTC and CC showed significantly enhanced levels of ALAD when compared to the detannified treated groups. Enzymic antioxidants (GPx, SOD), non enzymic antioxidants (GSH and nitrite) and malondialdehyde were measured in hemolysate and all major organs using spectrophotometric method. In group II treated rat, administration of arsenic enhances the stress through the elevation of malondialdehyde thereby reducing the levels of enzymic and nonenzymic antioxidants which was significantly reversed by the GTC (300 mg/kg) and CC treatment (30, 100 and 300 mg/kg) when compared to the GTDT and CDT groups. Tissue losses its membrane integrity by the arsenic administration via inhibiting the membrane bound enzymes Na + /K + ATPase, and Ca 2+ ATPase which were confirmed 181

6 by the animals in group II. This severity was protected by the admistration of tannin rich crude extract treated groups than the detannified treated groups. Total sulphydryl content, protein bound sulphydryl and non protein bound sulphydryl was measured because of its important role in the detoxification process via facilitating removal of arsenic from the cellular sites and stimulating excretion of methylated arsenic. In the present study, administration of tannin rich crude extract significantly restores the depleted thiol levels as seen in Group II than that of the detannified treated groups. Histopathological examinations of all major organs were carried out using H and E staining. Histopathological examinations of all major tissue of group 2 animal confirms the severity of toxicity whereas the GTC and CC treatment protected the damage and thereby confirm its protectivity similar to the results of biochemical assays. TEM examinations of brain, liver and kidney tissues were carried out. These major organ tissues of group II animal confirm the severity of damage whereas the tannin rich crude extract drug treatment protected these damages when compared to the detannified fraction. Green tea tannin rich administration mostly at 300 mg/kg bw shows effectiveness as a good inhibitor against arsenic-induced oxidative injury as observed by decrease in lipid peroxidation and increase in ALAD activity, oxidative stress markers and also maintained the structural form of cellular structure. It is interesting to note that the cocoa tannin rich crude (CC) extract almost at all doses (30 mg/kg, 100mg/kg and 300 mg/kg) play a protective role against sodium arsenite induced damage than that of cocoa detannified fraction (CDT). Thereby, issues related to arsenic poisoning can be ameliorated by common beverages such as Camellia sinensis and Theobroma cacao which are rich in tannins. However, Theobroma cacao seems to be more effective in ameliorating the damage caused by arsenic even at low dose (30 mg/kg). Hence, this study has brought in new insights in the possible role of both the beverages Green Tea and Cocoa in Arsenic toxicity mediated by modulating the oxidative stress. The new insights of its mechanism of action in the toxic metal intoxication would be a potential area in the development of nutraceuticals/functional foods specifically for men/children living in areas of arsenic burden. Overall tea and cocoa is an affordable beverage of natural origin compared to modern beverages such as soft drinks hence can be recommended to people of all ages. 182

7 The mechanistic pathways that the tannin rich Camellia sinensis and Theobroma cacao used to exhibit its protective action against arsenic induced toxicity are not fully clear except for its antioxidant property. Further study ought to be conducted for the exploration of isolated molecules of Camellia sinensis and Theobroma cacao to find out the exact mechanistic pathways that can be effective, safer, cheaper, non toxic enough for ameliorating the arsenic induced toxicities for the benefit of mankind. 183