Summary and Conclusions 125
Summary The thesis entitled Studies on Anti-hyperlipidemic and Anti-atherosclerotic activities of selected Indian Medicinal Plants incorporated the study of antioxidant, antihyperlipidemic and anti-atherosclerotic activities of selected plants, Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana. Hyperlipidemia is one of the greatest risk factor contributing to prevalence and severity of cardiovascular complications like coronary heart diseases including atherosclerosis. Many allopathic anti-hyperlipidemic drugs are available in the market but the side effects like hyperuricemia, myositis and hepatotoxicity were reported. Therefore attention is now paid to search natural hypolipidemic agents from plant sources. A number of plants have been found to be useful in treatment of hyperlipidemia mentioned in Ayurveda such as Allium sativum, Commiphora mukul, Boswellia serrata, Emblica officinalis, Garcinia cambogia, Terminalia arjuna, Trigonella foenum-graecum, Ocimum sanctum, Withania somnifera and Zingiber officinale. In view of the importance of the selected medicinal plants, Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana, the author has taken up the study on antioxidant, anti-hyperlipidemic and antiatherosclerotic activities and developed a suitable formulation. The above work was presented in seven chapters appended with a brief summary. Chapter I In the first chapter, the importance of medicinal plants in the treatment of various diseases was described with special emphasis on hyperlipidemia and atherosclerosis. Some of the medicinal plants and phytoconstituents which were reported to possess anti- 126
hyperlipidemic activity were listed and the need for natural anti-hyperlipidemic agents from plants was described. The scope and objectives of the present study was also discussed. Chapter II In this chapter a review of the selected plant species were recorded, with details about phytoconstituents so far reported, their structures and therapeutic applications. Based on the earlier reports, the leaves and gum resin of Boswellia ovalifoliolata (BOL, BOG), leaves and stem bark of Commiphora caudata (CCL, CCB), whole plant of Saccharum spontaneum (SSW) and pericarp of Garcinia mangostana fruits (GMP) were selected for the study. All the selected plants were reported to contain therapeutically important phytoconstituents such as phenols, flavonoids, sterols and terpenoids and were traditionally used in the treatment of several disorders including diabetes, cardiovascular diseases and obesity. But the therapeutic claims lack scientific documentation. Chapter III The third chapter includes the physico-chemical analysis of the selected plant materials and preparation of alcoholic extracts. The dried and powdered plant materials were defatted with hexane and extracted with alcohol in soxhlet apparatus. The physicochemical analysis was carried out to determine the extractive values, ash values and moisture content. The quantification of total phenols in the plant materials showed that high content of phenols were present in Garcinia mangostana pericarp and Commiphora caudata leaves. The phenolic content of the selected plants was in the order GMP (44.24 mg/g) > CCL (18.67 mg/g) >BOL (10.56 mg/g) > SSW (9.82 mg/g) > BOG (7.45 mg/g) > CCB (5.56 mg/g). The alcoholic extracts were subjected to preliminary phytochemical 127
analysis which revealed the presence of important phytoconstituents like terpenoids, phenols, steroids, proteins and saponins. The alcoholic extracts of the selected plants, B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana were initially tested for acute oral toxicity as per the OECD guidelines. Healthy young albino rats of either sex weighing between 150 to 200 gms were used for acute toxicity study and tested upto 2000 mg/kg body weight. After dosing, animals were observed periodically during the first 24 hours for any chances of toxic symptoms or mortality. The acute toxicity studies of the selected plant extracts showed no symptoms of toxicity or behavioral changes at the maximum dose (2000 mg/kg). The results indicate that the herbal extracts are safe upto a dose of 2000 mg/kg. Chapter IV In the fourth chapter the free radical scavenging activity of the alcoholic extracts of the selected plants was determined by DPPH, nitric oxide and inhibition of lipid peroxidation methods. The effective concentration of sample required to scavenge the free radicals by 50% (IC 50 value) was obtained by linear regression analysis of doseresponse curve plotting between percentage inhibition and concentrations. All the selected plant extracts showed dose dependant percentage inhibition of free radicals and lower IC 50 values indicate better protection. In the DPPH free radical scavenging activity, the ethanolic extracts of Commiphora caudata leaves and bark (ECCL, ECCB), leaves and gum of Boswellia ovalifoliolata (EBOL, EBOG), Saccharum spontaneum whole plant (ESSW) and Garcinia mangostana pericarp (EGMP) were evaluated for their free radical scavenging activity with ascorbic acid as standard compound. The best free radical scavenging 128
activity was obtained with ECCL (26.71 µg/ml) while the other extracts showed comparable levels of free radical scavenging activity. The activity was found to be in the following order: ECCL (26.71) >EGMP (44.99) >EBOG (49.16) > EBOL (69.59) > ESSW (88.71) > ECCB (134.83). Alcoholic extract of Commipora caudata leaves showed better activity than reference standard Ascorbic acid (32.05). The nitric oxide scavenging activity was determined by the method of Sreejayan et al 2009 and the percentage inhibition was calculated. The IC 50 values of the alcoholic extracts of selected plants and the order of efficacy were as follows: EGMP (69.34)> ECCL (94.33)> ESSW (122.33)>EBOG (151.13) > ECCB (216.63) >EBOL (379.37). The results showed that ECCL and EGMP showed potent anti oxidant activity which is similar or better than that of standard α-tocopherol (101.63). The selected plant extracts not only exhibited excellent ability for various radical scavenging activities, but also suppressed lipid peroxidation. The test extracts significantly inhibited the oxidation of linoleic acid which was determined by a ferric thiocyanate (FTC) assay. The result demonstrated that the extracts inhibited lipid peroxidation in a concentration dependent manner. The IC 50 (µg/ml) values were found to be, EGMP (28.48)> ECCL (74.23)> ESSW (112.27)>EBOG (91.67) > ECCB (423.97) >EBOL (388.27), while α-tocopherol showed IC50 value at 39.64. The results indicated that EGMP showed potent antioxidant activity than the standard. Chapter V The fifth chapter describes the studies carried out on evaluation of in-vitro antiadipogenic activity in 3T3-L1 cell line. To examine the effect of plant extracts on cell viability of 3T3-L1 preadipocytes, an MTT assay was performed, which assess cell 129
viability by measuring mitochondrial activity in 3T3-L1 cells, treated with different concentrations of the alcoholic extracts. The alcoholic extracts of the selected plants showed cytotoxicity with CTC 50 ranging from 100±0.31 (EBOG) to 720±0.18 µg/ml (ECCL) against 3T3-L1 cell line. The effect of plant extracts on fat droplet formation in 3T3-L1 cells, and inhibition through the quantification of Oil Red O staining was studied. The 3T3-L1 adipocytes were cultured and differentiated in a Dulbecco s Modified Eagle s Medium containing 10% fetal bovine serum for 6 to 8 days in the absence and presence of plant extracts. Adipogenesis was substantially inhibited by the test extracts and among the six extracts screened for anti adipogenic activity, test extracts ECCL (51.5), EBOG (46.1), ESSW (26) and EGMP (56.3 %) significantly reduced the lipid accumulation over control at 100, 700,250 and 250µg/mL respectively through the quantification method of Oil Red O staining. Other two extracts namely ECCB and EBOL did not show significant inhibition of fat accumulation at the tested concentration. Chapter VI The sixth chapter includes, studies carried out on the evaluation of antihyperlipidemic activity of the alcoholic extracts that showed significant activity in invitro studies. The methods available for the evaluation of antihyperlipidemic activity were reviewed and presented. The activity was tested in atherogenic diet induced rat model. Administration of atherogenic diet (2% cholesterol, 1% choline chloride and 2% Lard) to male Sprauge-Dawley rats for 21 days increased total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) and atherogenic index (AI) significantly in blood compared to control animals. The serum lipid parametes were estimated by using commercially available kits.in addition significant 130
increase in lipid peroxidation (LPO) levels and hepatic enzymes in liver was seen in AD fed rats. Treatment with alcoholic extracts ECCL, EBOG, ESSW and EGMP at 100 and 200 mg/kg and Atorvastatin for 21 days to atherogenic diet fed rats showed significant decrease in serum lipid profile in a dose dependent manner. Oxidative stress caused by atherogenic diet is associated with peroxidation of cellular lipids, which is determined by measurement of thiobarbituric acid reacting substance (TBARS). Feeding atherogenic diet increased LPO levels in diet control group whereas the extract treated groups showed reduction in LPO levels in graded manner with effects shown similar to that of standard drug. HDL has a protective role in hyperlipidemia and it was observed that the alcoholic extracts showed a trend towards increase in HDL levels in rats. Among all the extracts tested, G. mangostana was found to possess highest lipid lowering activity followed by C. caudata and B. ovalifoliolata. S. spontaneum recorded lowest activity among all the extracts tested. Hyperlipidemia induces liver damage and changes in serum AST, ALT and ALP levels are markers of hepatotoxicity. Animals in group A (control) showed no change in the AST, ALT, ALP levels and lies in the normal range. Significant increase of serum AST (96.47 U/L), ALT (75.61 U/L) and ALP (507.76 U/L) levels were detected in AD group. However treatment with alcoholic extracts of the selected plants decreased the levels of hepatic enzymes which indicate the hepatoprotective potential of the extracts. Chapter VII The seventh chapter presents the development of a Herbal formulation (FBCG) using alcoholic extracts of C. caudata, B. ovalifoliolata and G.mangostana as they have 131
shown significant anti-hyperlipidemic activity in-vivo. The suspension prepared was evaluated for sedimentation ratio and also showed good stability. Anti-hyperlipidemic and anti-atherosclerotic activities of the polyherbal formulation were evaluated by atherogenic diet induced hyperlipidemic rat model at doses of 100 and 200 mg/kg bw. The herbal formulation showed significant reduction in the TC, TG, LDL levels, body weight and AI with a significant increase in HDL levels. The activity of the formulation was comparable with that of the standard. Treatment with the herbal formulation prevented the peroxidation of lipids by reducing the concentration of TBARS. The increased levels of hepatic enzymes caused by atherogenic diet were significantly decreased by treatment with the polyherbal formulation which indicates the hepatoprotective potential of the formulation. When compared to the activity of individual extracts at the same dose, the activity of the formulation was relatively high. Histopathological changes in liver and aorta showed hyperlipidemia and atherogenesis in animals and administration of the herbal formulation restored normal architecture of the aorta. The histopathology of liver indicated that AD feeding to animals caused fatty liver, where nucleus was peripherally located and several fat globules were seen in cytoplasm of hepatocytes, whereas no histopathological abnormalities were seen in Atorvastatin and FBCG at 200 mg/kg treated groups. FBCG at 100 mg/kg showed few fat globules. Histopathology of coronary arteries from AD fed animals showed swollen endothelial cells in the intima. Animals treated with FBCG at 100 mg/kg showed few swollen endothelial cells. Atorvastatin and FBCG at 200 mg/kg treated rats showed normal histology of coronary arteries. 132
Thus the results of the present investigation clearly indicated that the selected medicinal plants possess good antihyperlipidemic activity in atherogenic diet induced hyperlipidemic rats and led to the development of new Herbal formulation possessing antihyperlipidemic and antiatherosclerotic activities. This is the first study which investigates the hypolipidemic activity of Commiphora caudata, Boswellia ovalifoliolata, Saccharum spontaneum and Garcinia mangostana. The results found are encouraging for further studies on the selected plants and to identify the bioactive compounds. An exhaustive bibliography was appended at the end of the thesis. 133
Conclusion: 1. Preliminary phytochemical investigations showed the presence of bioactive compounds like glycosides, sterols, terpenoids and phenolic compounds in selected plants namely, Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana. 2. The phenolic content of the selected plants were determined and were found to be in the order EGMP (Ethanolic extract of G.mangostana pericarp)> ECCL (Ethanolic extract of C.caudata leaves) >EBOL (Ethanolic extract of B.ovalifoliolata leaves) > ESSW (Ethanolic extract of S.spontaneum whole plant) > EBOG (Ethanolic extract of B.ovalifoliolata gum) > ECCB (Ethanolic extract of C.caudata bark). 3. The plant extracts showed no toxicity at a maximum dose of 2000 mg/kg. 4. All the extracts tested showed significant antioxidant activities in DPPH, Nitric oxide and lipid peroxidation methods in a dose dependant manner. 5. Among the six extracts tested for antiadiogenesis activity in-vitro, four extracts showed significant activity in the order EGMP> ECCL> EBOG>ESSW except ECCB and EBOL. 6. All the four extracts tested in-vivo showed decrease in lipid profiles and atherogenic index in a dose dependent manner. The efficacy was in the order, EGMP> ECCL> EBOG. However ESSW did not show good activity. The decrease in the levels of hepatic enzymes ALT, AST, ALP and lipid peroxidation indicate the hepatoprotective nature of the extracts. 7. The alcoholic extracts of selected herbal drugs could be formulated into effective hypolipidemic dosage form. The formulation possessed significant activity than the 134
individual extracts. This may be due to the synergistic effect of the combined extracts. Thus the results of the present investigation clearly indicated that the selected medicinal plants possess good antihyerlipidemic activity in atherogenic diet induced hyperlipidemic rats and led to the development of new Herbal formulation possessing antihyperlipidemic and antiatherosclerotic activities. This is the first study which investigates the hypolipidemic activity of Commiphora caudata, Boswellia ovalifoliolata, Saccharum spontaneum and Garcinia mangostana. The results found are encouraging for further studies on the selected plants and to identify the bioactive compounds. 135