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Efficacy of mangiferin on serum and heart tissue lipids in rats subjected to isoproterenol induced cardiotoxicity
Toxicology 228 (2006) 135–139
Efficacy of mangiferin on serum and heart tissue lipids in rats subjected to isoproterenol induced cardiotoxicity Prabhu Sukumaran Nair ∗ , C.S. Shyamala Devi Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India Received 25 June 2006; received in revised form 13 August 2006; accepted 18 August 2006 Available online 1 September 2006
Abstract The efficacy of mangiferin, on metabolism of lipids was tested in experimental cardiotoxic rats. The cardiotoxicity was induced by myocardial infarction through subcutaneous administration of isoproterenol hydrochloride for 2 days using 0.1 ml saline. Mangiferin drug was given as pretreatment for 28 days through intraperitonial administration using 0.2 ml dimethyl sulphoxide. Mangiferin significantly reduced the cholesterol, triglycerol, free fatty acids levels in serum and heart of the cardiotoxic myocardial infarcted rats. Mangiferin also increased the level of heart tissue phospholipids significantly in isoproterenol induced cardio toxic rats. The experiment thus concludes that mangiferin possess cardioprotective and hypolipidemic effect on experimentally induced cardiotoxic myocardial infarcted rats. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Isoproterenol; Cardiotoxicity; Mangiferin; Hypolipidemic; Rats; Serum and heart lipids
1. Introduction The present study was undertaken to evaluate the efficacy of mangiferin with respect to lipid metabolism on isoproterenol (ISPH) induced experimental cardiotoxic rats. ISPH (Chagoya de Sanchez et al., 1997) has been found to cause a severe stress in the myocardium resulting in infarct like necrosis of the heart muscle. It also increases the levels of serum and myocardial lipids, which in turn leads to coronary heart disease (Prince and Rajadurai, 2005). It is well known to generate free radicals and to stimulate lipid peroxidation (LPO), which is ∗
Corresponding author at: No. 6/2, S.S. Bhavanam, Saraswathy Street, Guruswamy Nagar Extension, Gowrivakkam, PIN: 601302 Kanchi District, Tamil Nadu, India. Tel.: +91 9840180535; fax: +91 44 22781352. E-mail addresses: [email protected], [email protected] (P.S. Nair).
a causative factor for irreversible damage to the myocardial membrane (Sathish et al., 2003) and thus favors the deposition of myocardial lipids. The current study is a part of our research findings and that supports scientifically to the protection of heart by reducing the excess lipids through pretreatment with mangiferin. Mangiferin is a pharmacologically active phytochemical and a natural polyphenolic (C-glucoxyl xanthone) antioxidant derivative present in the bark, fruits, roots and leaves of Mangifera indica Linn. (Anacardiaceae) and is recommended in the Indian systems of medicine (Ghosal et al., 1996) for the treatment of immuno-deficiency diseases such as arthritis, diabetes, hepatitis, cardiac, mental disorders, cancer, autoimmune disorders, arteriosclerosis and coronary heart disease (Leiro et al., 2003). Some reports have showed that mangiferin has suppressive effect on blood lipids in diabetes (Miura et al., 2001). In our earlier studies we have reported that maniferin has cardioprotective (Prabhu et al., 2006a) and
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antioxidant property (Prabhu et al., 2006b). In view of this we have discovered that mangiferin possess hypolipidemic activity in isoproterenol induced cardiotoxic myocardial infarcted (MI) rats. 2. Experiments 2.1. Mangiferin The isolated mangiferin powder from M. indica L., was obtained from Herbo Organic Ltd., Chennai, Tamil Nadu. The purity of the compound was confirmed by a HPLC method as described by Geodakyan et al. (1992) using C18 column for separation. HPLC analysis of reference mangiferin purchased from Sigma–Aldrich Company showed a single peak (95.5%), which closely matched to its specified purity of isolated mangiferin (greater than 90%, w/w). 2.2. Dissolvation, toxicity study and dosage fixation of mangiferin For the experimental study, 100 mg of mangiferin was dissolved in 2 ml of DMSO and administered intraperitoneally (i.p.) to kg body weight of each rat (Muruganandan et al., 2002) Mangiferin showed no lethal effect at least up to a dose of 1000 mg/kg body weight indicating that LD50 if any should be higher than this dose (Prabhu et al., 2005). Assessment on the effective dose of mangiferin and duration of treatment against ISPH induced myocardial injury in rats was done based on the activities of serum lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes (Prabhu et al., 2006a). 2.3. Chemicals Choloesterol, uranyl acetate, digitonin, triglyceride, activated silicic acid, reference mangiferin and ISPH were purchased from Sigma Company, USA and all other chemicals were purchased from Loba Chemical Company, Bombay, India. 2.4. Animals Adult male albino rats of wistar strain weighing 150–200 g obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai were used for the present study. The animals were housed at 27 ± 2 ◦ C in temperature, 55% in humidity, and a 12 h-light/12 h-dark cycle. They were fed with standard laboratory chow (Hindustan Lever Foods, Bangalore, India) and provided with water ad libitum. All experiments were carried out according to the guidelines of Institutional Animal Ethics Committee (IAEC No.: 01/046/04). 2.5. Grouping of animals Animals were grouped into four, each group consisting of six animals. Group I, control rats received DMSO (2 ml/kg
body weight) as a vehicle i.p., for 28 days. Other than standard diet and water ad libitum, these groups of rats did not receive any other samples on 29th and 30th day. In Group II, the rats were administered with ISPH (200 mg/kg body weight suspended in 1 ml of 0.9% saline) s.c., (subcutaneously) once a day for 2 days at an interval of 24 h (Prabhu et al., 2005) for 29th and 30th day only. Here the Group II rats did not receive any other samples other than water ad libitum and standard laboratory diet initially for 28 days. Group III rats were treated with mangiferin alone (100 mg/kg body weight i.p., suspended in 2 ml of DMSO) for 28 days and did not receive any other samples for 29th and 30th day other than standard diet and water. Group IV rats pretreated with mangiferin (100 mg/kg body weight i.p., suspended in 2 ml of DMSO) for 28 days and then ISPH was administered s.c., once a day for 2 days at an interval of 24 h as the dose mentioned in Group II, for 29th and 30th day. Since DMSO was not administered with ISPH in Group II rats comparing to other groups, its interference with ISPH was tested using yet another group of rats, i.e. Group V rats. The Group V rats were received 0.2 ml of DMSO i.p., daily for 28 days and ISPH s.c., for 2 days like Group II rats. Since the serum myocardial marker enzymes of Group V rats did not show any deviated significant changes in their activity from Group II ISPH alone treated rats, the Group V rats were dropped from the study. 2.6. Collection of serum and preparation of tissue After the experimental period (i.e. after 30 days), the animals were anaesthetized with pentobarbital sodium (35 mg/kg, i.p.). Blood was drawn from the external jugular vein of the rat and serum was separated by centrifugation. The heart tissues were dissected out immediately and washed in ice-cold saline. Hundred milligrams of heart tissue was weighed accurately and homogenized in 5 ml of 0.1 M Tris–HCl buffer (pH 7.4) in icecold condition. The homogenate was centrifuged at 2500 × g and the clear supernatant solution was taken for the analytical procedures. 2.7. Extraction and estimation of serum and heart tissue lipids From the samples of serum and heart tissue homogenate the lipids were extracted by the method of Folch et al. (1957). To a known volume of serum or tissue homogenate, 10 ml of chloroform–methanol mixture was added and mixed well for 30 min using shaker and was filtered through What-man filter paper (No. 42) into a separating funnel. The filtrate was mixed with 0.2 ml of physiological saline and the mixture was kept overnight undisturbed. The lower phase containing the lipid was drained off into pre-weighed beakers. The upper phase was re-extracted with more of chloroform–methanol mixture and the extracts were pooled and evaporated under vacuum at room temperature. The lipid extract was re-dissolved in 3 ml of chloroform–methanol (2:1) mixture and the aliquots were collected. The aliquots were named as LEA (Lipid Extract
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Aliquots), which was taken further for the estimation of serum and heart tissue lipids. Cholesterol was estimated by the method of Parekh and Jung (1970). To 0.5 ml of LEA, 2.5 ml of ferric chloride-uranyl acetate reagent was added. Then, 2 ml of sulphuric acid, ferrous sulphate reagent was added and mixed well. After 20 min of incubation at room temperature the optical density was measured at 560 nm using green filter. Phospholipids were estimated as inorganic phosphorus by the method of Fiske and Subbarow (1925) after Barlette (1959) “acid digestion” process. To 0.5 ml of LEA, 1 ml of perchloric acid was added and digested on a sand bath until the mixture become colorless. It was then made up to a known volume (3 ml) and a suitable aliquot (0.5 ml) was taken and diluted with water to 4.5 ml. Then 0.5 ml of ammonium molybdate and 0.2 ml of ANSA was added. The contents were mixed well. The blue color developed was read after 10 min at 620 nm using red filter. The phosphorus content was multiplied by a factor 25 to get total phospholipid content. Triglycerides (TG) were estimated by the method of Rice (1970). To 0.5 ml of LEA, 3.5 ml of isopropanol was added followed by 50 mg of activated alumina. It was mixed well and left for 15 min. It was then centrifuged and 2 ml of the supernatant was taken for analysis. About 0.6 ml of alkaline potassium hydroxide was added to all the tubes. The tubes were incubated at 60 ◦ C for 10 min. The tubes were cooled and 1 ml of sodium metaperiodate reagent was added to the tubes followed by the addition of 0.5 ml of acetyl acetone reagent. The tubes were cooled and the color developed was read at 405 nm using blue filter. Free fatty acids (FFA) were measured by the method of Horn and Menahan (1981) with the color reagent based on the method of Itaya (1977). About 0.5 ml of LEA was added to 5.5 ml of CHM (chloroform heptane methanol) solvent. After adding 200 mg of activated silicic acid the contents were shaken well and centrifuged. Two milliliters of Cu-TEA (copper triethanolamine) reagent was added and mixed well. The tubes were centrifuged to separate the two phases and 2 ml of the upper phase from each tube was transferred to another set of tubes. To all these tubes 1 ml of 0.1% DDC (diethyl dithio carbamate) color reagent was added and shaken well. The color intensity was measured at 430 nm using blue filter.
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Blanks and standards were run along with the test samples when the estimation of an individual lipid component was carried out. Blanks comprised the respective reagents of the individual lipids to be analyzed but without any standards or test samples. Using blanks, zero optical density was set in a Shimadzu UV spectrophotometer at respective filters of lipids quantification and a calibration graph was prepared with each standard lipid. 2.8. Statistical analysis Results are presented as mean ± S.D. The significance of difference among the groups was assessed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) multiple comparison test. Significance was set at P < 0.05, P < 0.01 and P < 0.001 (Fisher, 1958).
3. Results The levels of serum, heart tissue total cholesterol, TG, and FFA in normal and experimental groups of rats are represented in Tables 1 and 2. Total cholesterol, TG and FFA level in serum and heart showed a significant increase (P < 0.001) in Group II ISPH induced MI rats when compared to Group I control rats. In mangiferin pretreated Group IV rats, the levels of serum and heart tissue cholesterol, FFA and TG (P < 0.001) showed a significant decrease as compared with Group II ISPH induced MI rats. Similarly in Group III drug control rats, it showed a non-significant (NS) change for the above parameters in serum when compared to Group I control rats. Fig. 1 represent the level of PL in the heart of normal and experimental group of rats. Group II ISPH induced MI rats showed a significant (P < 0.001) decrease in heart tissue PL level when compared to Group I control rats whereas a significant increase (P < 0.001) in heart tissue PL level was observed in Group IV mangiferin pretreated rats as compared with Group II ISPH induced MI rats. A non-significant (NS) change was observed in Group
Table 1 Levels of serum choloesterol, free fatty acids and triglyceride in control and experimental group of rats Parameters (mg/dl)
Group I
Group II
Group III
Group IV
Total cholesterol Free fatty acids Triglyceride
37.23 ± 2.63 30.60 ± 2.69 22.42 ± 1.53
55.75 ± 3.54a,*** 43.37 ± 3.06a,*** 31.14 ± 1.77a,***
35.25 ± 2.27a NS 31.53 ± 2.55a NS 21.16 ± 1.46a NS
41.58 ± 2.24b,*** 36.69 ± 2.94b,*** 24.61 ± 1.76b,***
Values are expressed as mean ± S.D. for six animals in each group. Statistical calculation: one-way ANOVA followed by posthoc test LSD. *** p < 0.001, ** p < 0.01, * p < 0.05, NS: non-significant. Group I: control rats, Group II: ISPH induced MI rats, Group III: mangiferin alone treated rats, and Group IV: mangiferin + ISPH treated rats. a Compared with Group 1. b Compared with Group 2.
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Table 2 Levels of heart tissue choloesterol, free fatty acids and triglyceride in control and experimental group of rats Parameters (mg/g)
Group I
Group II
Group III
Group IV
Total cholesterol tissue Free fatty acids tissue Triglyceride tissue
5.24 ± 0.12 0.49 ± 0.04 3.25 ± 0.14
5.78 ± 0.14a,*** 0.73 ± 0.05a,*** 4.10 ± 0.19a,***
5.18 ± 0.18a NS 0.50 ± 0.04a NS 3.16 ± 0.14a NS
5.30 ± 0.19b,*** 0.60 ± 0.05b,*** 3.50 ± 0.18b,***
Values are expressed as mean ± S.D. for six animals in each group. Statistical calculation: one-way ANOVA followed by posthoc test LSD. *** p < 0.001, ** p < 0.01, * p < 0.05, NS: non-significant. Group I: control rats, Group II: ISPH induced MI rats, Group III: mangiferin alone treated rats, and Group IV: mangiferin + ISPH treated rats. a Compared with Group 1. b Compared with Group 2.
Fig. 1. Effect of mangiferin on heart tissue phospholipid (PL) level in control and experimental group of rats. Group II ISPH induced MI rats showed a significant (P < 0.001) decrease in heart tissue PL level as compared to Group I control rats. Group IV mangiferin + ISPH treated rats showed a significant increase (P < 0.001) in heart tissue PL level as compared with Group II ISPH induced MI rats. A non-significant (NS) change was observed in Group III mangiferin alone treated rats as compared to Group I control rats.
III drug control rats when compared to Group I control rats. 4. Discussion The present study has showed an increase of serum and heart tissue lipids with a decrease in heart tissue phospholipids in ISPH induced cardiotoxic Group II MI rats. LPO can play an important role in lipoprotein modifications, which makes them susceptible to atherogenesis, which could be the reason for acute MI mediated cardiotoxicity by ISPH (Nirmala and Puvanakrishnan, 1996). Our earlier studies (Prabhu et al., 2006a) have also proved that ISPH induces LPO and accelerates myocardial necrosis. Increased level of cholesterol and TG (Manjula et al., 1992) is associated with cardiovascular disturbances and ISPH promotes lipolysis in myocardium (Sushamakumari et al., 1990). Enhancement in lipolysis and subsequent elevation of plasma FFA levels may lead to an increase in hepatic TG synthesis and secretion of elevated plasma TG concentration (Rayssiguier, 1990) and cholesterol. An increased synthesis of TG in heart tissue could be due to accumulation of acyl coA and an augmented production of glycerol
by increased glycolytic flux (Subramanian et al., 2003). The decrease in heart PL level may be explained due to increase in phospholipase activity and the activation of phospholipases is mediated through LPO with significant decrease in ATP levels during ISPH induced myocardial necrosis. Accelerated PL degradation could result in cell injury and ultimately cell death and is evidenced by the accumulation of thiobarbituric acid (TBA) reacting substances and the loss of both extractable PL and their polyunsaturated acyl groups (Sushamakumari et al., 1990). In Group IV mangiferin pretreated rats the level of serum and heart tissue lipids were reduced significantly with a significant increase in heart tissue PL. Polyphenols like mangiferin from different plant sources have been reported (Zern et al., 2003; Vinson and Jang, 2001) to alter the hepatic cholesterol metabolism which result in less accumulation of cholesterol in the aorta and able to significantly inhibit atherosclerosis and decrease the incidence of coronary heart disease. Mangiferin polyphenol possesses antioxidant property, which could have inhibited LPO due its scavenging lipid peroxy and alkoxy radicals and thereby preventing continued abstraction of hydrogen from cellular lipids (Ghosal et al., 1996). The higher rate of conversion of cholesterol to bile acids and elimination of faecal bile acids and neutral sterols could also be considered in the present study for the reduction of cholesterol in Group IV mangiferin pretreated rats. The improvement of hyperlipidemia may also be due to the significant reduction in lipolysis and hence decreased level of triacyl glycerol and FFA are observed. The increased level of PL in Group IV rats could be due its antioxidant and anti lipid peroxidative property, which could have protected tissue membrane PL from ISPH mediated peroxidation. Thus mangiferin has significantly reduced the serum and heart tissue lipids level except heart tissue PL. Hence i.p., administration of mangiferin has played a major role in lipid metabolism due to its free radical
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scavenging, antilipid peroxidive and cardiotonic activity and the present study strongly supports the efficacy of mangiferin in controlling lipid parameters in serum and heart of the experimental cardiotoxic rats. These observations highlight that mangiferin is one of the promising herbal drug for improving defense mechanisms in the physiological systems against hyperlipidemia and oxidative stress caused by ISPH mediated cardiotoxicity in experimental myocardial infarction. References Barlette, G.R., 1959. Phosphorous assay in column chromatography. J. Biol. Chem. 234, 466–468. Chagoya de Sanchez, V., Hernandez-Munoz, R., Lopez-Barrera, F., Yanez, L., Vidrio, S., Suarez, J., Cota-Garza, M.D., ArandaFraustro, A., Cruz, D., 1997. Sequential changes of energy metabolism and mitochondrial function in myocardial infarction induced by isoproterenol in rats: a long-term and integrative study. Can. J. Physiol. Pharmacol. 75 (12), 1300–1311. Fisher, R.A. Statistical Methods for Research Workers. Hafner (Ed.). Oxford, New York, 1958, pp. 356–78. Fiske, Subbarow, Y., 1925. The colorimetric determination of phosphorous. J. Biol. Chem. 66, 375–400. Folch, J., Lees, M., Stanley, G.S.H., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Geodakyan, S.V., Voskoboinikova, I.V., Kolesnik, J.A., Tjukavkina, N.A., Litvinanko, V.I., Glyzin, V.I., 1992. High-performance liquid chromatographic method for the determination of mangiferin, likviritin and dihydroquercetin in rat plasma and urine. J. Chromatogr. 577, 371–375. Ghosal, S., Rao, G., Saravanan, V., Misra, N., Rana, D., 1996. A pausible chemical mechanism of the bioactivities of mangiferin. Ind. J. Chem. 35B, 561–566. Horn, W.T., Menahan, L.A., 1981. A sensitive method for the determination of free fatty acids in plasma. J. Lipid Res. 22, 377–381. Itaya, K., 1977. A more sensitive and stable colorimetric determination of free fatty acids in blood. J. Lipid Res. 18, 663–665. Leiro, J.M., Alvarez, E., Arranz, J.A., Siso, I.G., Orallo, F., 2003. In vitro effects of mangiferin on superoxide concentrations and expression of the inducible nitric oxide synthase, tumour necrosis factor-alpha and transforming growth factor-beta genes. Biochem. Pharmacol. 65 (8), 1361–1371. Manjula, T.S., Geetha, A., Ramesh, T.G., Shyamala Devi, C.S., 1992. Reversal of Changes of myocardial lipids by chronic administration of aspirin in isoproterenol induced myocardial damage in rats. Ind. J. Physiol. Pharmacol. 36 (1), 47–50. Miura, T., Iwamoto, N., Kato, M., Ichiki, H., Kubo, M., Komatsu, Y., Ishida, T., Okada, M., Tanigawa, K., 2001. The suppressive effect
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of mangiferin with exercise on blood lipids in type 2 diabetes. Biol. Pharm. Bull. 24 (9), 1091–1092. Muruganandan, S., Gupta, S., Kataria, M., Lal, J., Gupta, P.K., 2002. Mangiferin protects the streptozotocin-induced oxidative damage to cardiac and renal tissues in rats. Toxicology 176 (3), 165–173. Nirmala, C., Puvanakrishnan, R., 1996. Protective role of curcumin against isoproterenol induced myocardial infarction in rats. Mol. Cell. Biochem. 159 (2), 85–93. Parekh, A.C., Jung, D.H., 1970. Determination of cholesterol with ferric acetate, uranium acetate and sulfuric acid, ferrous sulphate reagents. Anal. Chem. 42, 1423–1427. Prabhu, S., Jainu, M., Sabitha, K.E., Shyamala Devi, C.S., 2005. A study on the beneficial effect of mangiferin on isoproterenol induced myocardial infarction in rats—research article. Pharmacol. Mag. 1 (3), 92–96. Prabhu, S., Mallika, J., Sabitha, K.E., Devi, C.S.S., 2006a. Cardioprotective effect of mangiferin on isoproterenol induced myocardial infarction in rats. Ind. J. Exp. Biol. 44 (3), 209–215. Prabhu, S., Jainu, M., Sabitha, K.E., Shyamala Devi, C.S., 2006b. Role of mangiferin on biochemical alterations and antioxidant status in isoproterenol induced myocardial infarction in rats. J. Ethnopharmacol. 11; 107 (1), 126–133. Prince, P.S., Rajadurai, M., 2005. Preventive effect of Aegle marmelos leaf extract on isoprenaline-induced myocardial infarction in rats- biochemical evidence. J. Pharm. Pharmacol. 57 (10), 1353– 1357. Rayssiguier, Y., 1990. Metal ions in biological systems, magnesium and its role in biology, nutrition and physiology.Metal ions in biological systems, magnesium and its role in biology, nutrition and physiology. In: Sigel, H. (Ed.), Magnesium and lipid metabolism, 26. Marcel Dekker, New York, pp. 341–358. Rice, E.W., 1970. Triglycerides in serum.Triglycerides in serum. In: Roderick, R., Donald, M. (Eds.), Standard methods of clinical chemistry, vol. 6. Academic Press, New York, pp. 215–222. Sathish, V., Ebenezar, K.K., Devaki, T., 2003. Synergistic effect of Nicorandil and Amlodipine on tissue defense system during experimental myocardial infarction in rats. Mol. Cell. Biochem. 243 (1–2), 133–138. Subramanian, R., Ramaswamy, M., Wasan, K.M., 2003. Role of lipid and lipoprotein metabolizing enzymes in the development of atherosclerosis. Ind. J. Exp. Biol. 41 (1), 14–25. Sushamakumari, S., Varghese, A., Muraleedharan, D., Menon, V.P., 1990. Protective action of aspirin in experimental myocardial infarction induced by isoproterenol in rats and its effect on lipid peroxidation. Ind. J. Exp. Biol. 28 (5), 480–485. Vinson, J.A., Jang, J., 2001. In vitro and in vivo Lipoprotein antioxidant effect of a citrus extract and ascorbic acid on normal and hypercholesterolemic human subjects. J. Med. Food. Winter 4 (4), 187–192. Zern, T.L., West, K.L., Fernandez, M.L., 2003. Grape polyphenols decrease plasma triglycerides and cholesterol accumulation in the aorta of ovariectomized guinea pigs. J. Nutr. 133 (7), 2268–2272.
Efficacy of mangiferin on serum and heart tissue lipids in rats subjected to isoproterenol induced cardiotoxicity Prabhu Sukumaran Nair ∗ , C.S. Shyamala Devi Department of Biochemistry, University of Madras, Guindy Campus, Chennai, India Received 25 June 2006; received in revised form 13 August 2006; accepted 18 August 2006 Available online 1 September 2006
Abstract The efficacy of mangiferin, on metabolism of lipids was tested in experimental cardiotoxic rats. The cardiotoxicity was induced by myocardial infarction through subcutaneous administration of isoproterenol hydrochloride for 2 days using 0.1 ml saline. Mangiferin drug was given as pretreatment for 28 days through intraperitonial administration using 0.2 ml dimethyl sulphoxide. Mangiferin significantly reduced the cholesterol, triglycerol, free fatty acids levels in serum and heart of the cardiotoxic myocardial infarcted rats. Mangiferin also increased the level of heart tissue phospholipids significantly in isoproterenol induced cardio toxic rats. The experiment thus concludes that mangiferin possess cardioprotective and hypolipidemic effect on experimentally induced cardiotoxic myocardial infarcted rats. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Isoproterenol; Cardiotoxicity; Mangiferin; Hypolipidemic; Rats; Serum and heart lipids
1. Introduction The present study was undertaken to evaluate the efficacy of mangiferin with respect to lipid metabolism on isoproterenol (ISPH) induced experimental cardiotoxic rats. ISPH (Chagoya de Sanchez et al., 1997) has been found to cause a severe stress in the myocardium resulting in infarct like necrosis of the heart muscle. It also increases the levels of serum and myocardial lipids, which in turn leads to coronary heart disease (Prince and Rajadurai, 2005). It is well known to generate free radicals and to stimulate lipid peroxidation (LPO), which is ∗
Corresponding author at: No. 6/2, S.S. Bhavanam, Saraswathy Street, Guruswamy Nagar Extension, Gowrivakkam, PIN: 601302 Kanchi District, Tamil Nadu, India. Tel.: +91 9840180535; fax: +91 44 22781352. E-mail addresses: [email protected], [email protected] (P.S. Nair).
a causative factor for irreversible damage to the myocardial membrane (Sathish et al., 2003) and thus favors the deposition of myocardial lipids. The current study is a part of our research findings and that supports scientifically to the protection of heart by reducing the excess lipids through pretreatment with mangiferin. Mangiferin is a pharmacologically active phytochemical and a natural polyphenolic (C-glucoxyl xanthone) antioxidant derivative present in the bark, fruits, roots and leaves of Mangifera indica Linn. (Anacardiaceae) and is recommended in the Indian systems of medicine (Ghosal et al., 1996) for the treatment of immuno-deficiency diseases such as arthritis, diabetes, hepatitis, cardiac, mental disorders, cancer, autoimmune disorders, arteriosclerosis and coronary heart disease (Leiro et al., 2003). Some reports have showed that mangiferin has suppressive effect on blood lipids in diabetes (Miura et al., 2001). In our earlier studies we have reported that maniferin has cardioprotective (Prabhu et al., 2006a) and
0300-483X/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2006.08.030
136
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antioxidant property (Prabhu et al., 2006b). In view of this we have discovered that mangiferin possess hypolipidemic activity in isoproterenol induced cardiotoxic myocardial infarcted (MI) rats. 2. Experiments 2.1. Mangiferin The isolated mangiferin powder from M. indica L., was obtained from Herbo Organic Ltd., Chennai, Tamil Nadu. The purity of the compound was confirmed by a HPLC method as described by Geodakyan et al. (1992) using C18 column for separation. HPLC analysis of reference mangiferin purchased from Sigma–Aldrich Company showed a single peak (95.5%), which closely matched to its specified purity of isolated mangiferin (greater than 90%, w/w). 2.2. Dissolvation, toxicity study and dosage fixation of mangiferin For the experimental study, 100 mg of mangiferin was dissolved in 2 ml of DMSO and administered intraperitoneally (i.p.) to kg body weight of each rat (Muruganandan et al., 2002) Mangiferin showed no lethal effect at least up to a dose of 1000 mg/kg body weight indicating that LD50 if any should be higher than this dose (Prabhu et al., 2005). Assessment on the effective dose of mangiferin and duration of treatment against ISPH induced myocardial injury in rats was done based on the activities of serum lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes (Prabhu et al., 2006a). 2.3. Chemicals Choloesterol, uranyl acetate, digitonin, triglyceride, activated silicic acid, reference mangiferin and ISPH were purchased from Sigma Company, USA and all other chemicals were purchased from Loba Chemical Company, Bombay, India. 2.4. Animals Adult male albino rats of wistar strain weighing 150–200 g obtained from Tamil Nadu Veterinary and Animal Sciences University, Chennai were used for the present study. The animals were housed at 27 ± 2 ◦ C in temperature, 55% in humidity, and a 12 h-light/12 h-dark cycle. They were fed with standard laboratory chow (Hindustan Lever Foods, Bangalore, India) and provided with water ad libitum. All experiments were carried out according to the guidelines of Institutional Animal Ethics Committee (IAEC No.: 01/046/04). 2.5. Grouping of animals Animals were grouped into four, each group consisting of six animals. Group I, control rats received DMSO (2 ml/kg
body weight) as a vehicle i.p., for 28 days. Other than standard diet and water ad libitum, these groups of rats did not receive any other samples on 29th and 30th day. In Group II, the rats were administered with ISPH (200 mg/kg body weight suspended in 1 ml of 0.9% saline) s.c., (subcutaneously) once a day for 2 days at an interval of 24 h (Prabhu et al., 2005) for 29th and 30th day only. Here the Group II rats did not receive any other samples other than water ad libitum and standard laboratory diet initially for 28 days. Group III rats were treated with mangiferin alone (100 mg/kg body weight i.p., suspended in 2 ml of DMSO) for 28 days and did not receive any other samples for 29th and 30th day other than standard diet and water. Group IV rats pretreated with mangiferin (100 mg/kg body weight i.p., suspended in 2 ml of DMSO) for 28 days and then ISPH was administered s.c., once a day for 2 days at an interval of 24 h as the dose mentioned in Group II, for 29th and 30th day. Since DMSO was not administered with ISPH in Group II rats comparing to other groups, its interference with ISPH was tested using yet another group of rats, i.e. Group V rats. The Group V rats were received 0.2 ml of DMSO i.p., daily for 28 days and ISPH s.c., for 2 days like Group II rats. Since the serum myocardial marker enzymes of Group V rats did not show any deviated significant changes in their activity from Group II ISPH alone treated rats, the Group V rats were dropped from the study. 2.6. Collection of serum and preparation of tissue After the experimental period (i.e. after 30 days), the animals were anaesthetized with pentobarbital sodium (35 mg/kg, i.p.). Blood was drawn from the external jugular vein of the rat and serum was separated by centrifugation. The heart tissues were dissected out immediately and washed in ice-cold saline. Hundred milligrams of heart tissue was weighed accurately and homogenized in 5 ml of 0.1 M Tris–HCl buffer (pH 7.4) in icecold condition. The homogenate was centrifuged at 2500 × g and the clear supernatant solution was taken for the analytical procedures. 2.7. Extraction and estimation of serum and heart tissue lipids From the samples of serum and heart tissue homogenate the lipids were extracted by the method of Folch et al. (1957). To a known volume of serum or tissue homogenate, 10 ml of chloroform–methanol mixture was added and mixed well for 30 min using shaker and was filtered through What-man filter paper (No. 42) into a separating funnel. The filtrate was mixed with 0.2 ml of physiological saline and the mixture was kept overnight undisturbed. The lower phase containing the lipid was drained off into pre-weighed beakers. The upper phase was re-extracted with more of chloroform–methanol mixture and the extracts were pooled and evaporated under vacuum at room temperature. The lipid extract was re-dissolved in 3 ml of chloroform–methanol (2:1) mixture and the aliquots were collected. The aliquots were named as LEA (Lipid Extract
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Aliquots), which was taken further for the estimation of serum and heart tissue lipids. Cholesterol was estimated by the method of Parekh and Jung (1970). To 0.5 ml of LEA, 2.5 ml of ferric chloride-uranyl acetate reagent was added. Then, 2 ml of sulphuric acid, ferrous sulphate reagent was added and mixed well. After 20 min of incubation at room temperature the optical density was measured at 560 nm using green filter. Phospholipids were estimated as inorganic phosphorus by the method of Fiske and Subbarow (1925) after Barlette (1959) “acid digestion” process. To 0.5 ml of LEA, 1 ml of perchloric acid was added and digested on a sand bath until the mixture become colorless. It was then made up to a known volume (3 ml) and a suitable aliquot (0.5 ml) was taken and diluted with water to 4.5 ml. Then 0.5 ml of ammonium molybdate and 0.2 ml of ANSA was added. The contents were mixed well. The blue color developed was read after 10 min at 620 nm using red filter. The phosphorus content was multiplied by a factor 25 to get total phospholipid content. Triglycerides (TG) were estimated by the method of Rice (1970). To 0.5 ml of LEA, 3.5 ml of isopropanol was added followed by 50 mg of activated alumina. It was mixed well and left for 15 min. It was then centrifuged and 2 ml of the supernatant was taken for analysis. About 0.6 ml of alkaline potassium hydroxide was added to all the tubes. The tubes were incubated at 60 ◦ C for 10 min. The tubes were cooled and 1 ml of sodium metaperiodate reagent was added to the tubes followed by the addition of 0.5 ml of acetyl acetone reagent. The tubes were cooled and the color developed was read at 405 nm using blue filter. Free fatty acids (FFA) were measured by the method of Horn and Menahan (1981) with the color reagent based on the method of Itaya (1977). About 0.5 ml of LEA was added to 5.5 ml of CHM (chloroform heptane methanol) solvent. After adding 200 mg of activated silicic acid the contents were shaken well and centrifuged. Two milliliters of Cu-TEA (copper triethanolamine) reagent was added and mixed well. The tubes were centrifuged to separate the two phases and 2 ml of the upper phase from each tube was transferred to another set of tubes. To all these tubes 1 ml of 0.1% DDC (diethyl dithio carbamate) color reagent was added and shaken well. The color intensity was measured at 430 nm using blue filter.
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Blanks and standards were run along with the test samples when the estimation of an individual lipid component was carried out. Blanks comprised the respective reagents of the individual lipids to be analyzed but without any standards or test samples. Using blanks, zero optical density was set in a Shimadzu UV spectrophotometer at respective filters of lipids quantification and a calibration graph was prepared with each standard lipid. 2.8. Statistical analysis Results are presented as mean ± S.D. The significance of difference among the groups was assessed using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) multiple comparison test. Significance was set at P < 0.05, P < 0.01 and P < 0.001 (Fisher, 1958).
3. Results The levels of serum, heart tissue total cholesterol, TG, and FFA in normal and experimental groups of rats are represented in Tables 1 and 2. Total cholesterol, TG and FFA level in serum and heart showed a significant increase (P < 0.001) in Group II ISPH induced MI rats when compared to Group I control rats. In mangiferin pretreated Group IV rats, the levels of serum and heart tissue cholesterol, FFA and TG (P < 0.001) showed a significant decrease as compared with Group II ISPH induced MI rats. Similarly in Group III drug control rats, it showed a non-significant (NS) change for the above parameters in serum when compared to Group I control rats. Fig. 1 represent the level of PL in the heart of normal and experimental group of rats. Group II ISPH induced MI rats showed a significant (P < 0.001) decrease in heart tissue PL level when compared to Group I control rats whereas a significant increase (P < 0.001) in heart tissue PL level was observed in Group IV mangiferin pretreated rats as compared with Group II ISPH induced MI rats. A non-significant (NS) change was observed in Group
Table 1 Levels of serum choloesterol, free fatty acids and triglyceride in control and experimental group of rats Parameters (mg/dl)
Group I
Group II
Group III
Group IV
Total cholesterol Free fatty acids Triglyceride
37.23 ± 2.63 30.60 ± 2.69 22.42 ± 1.53
55.75 ± 3.54a,*** 43.37 ± 3.06a,*** 31.14 ± 1.77a,***
35.25 ± 2.27a NS 31.53 ± 2.55a NS 21.16 ± 1.46a NS
41.58 ± 2.24b,*** 36.69 ± 2.94b,*** 24.61 ± 1.76b,***
Values are expressed as mean ± S.D. for six animals in each group. Statistical calculation: one-way ANOVA followed by posthoc test LSD. *** p < 0.001, ** p < 0.01, * p < 0.05, NS: non-significant. Group I: control rats, Group II: ISPH induced MI rats, Group III: mangiferin alone treated rats, and Group IV: mangiferin + ISPH treated rats. a Compared with Group 1. b Compared with Group 2.
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Table 2 Levels of heart tissue choloesterol, free fatty acids and triglyceride in control and experimental group of rats Parameters (mg/g)
Group I
Group II
Group III
Group IV
Total cholesterol tissue Free fatty acids tissue Triglyceride tissue
5.24 ± 0.12 0.49 ± 0.04 3.25 ± 0.14
5.78 ± 0.14a,*** 0.73 ± 0.05a,*** 4.10 ± 0.19a,***
5.18 ± 0.18a NS 0.50 ± 0.04a NS 3.16 ± 0.14a NS
5.30 ± 0.19b,*** 0.60 ± 0.05b,*** 3.50 ± 0.18b,***
Values are expressed as mean ± S.D. for six animals in each group. Statistical calculation: one-way ANOVA followed by posthoc test LSD. *** p < 0.001, ** p < 0.01, * p < 0.05, NS: non-significant. Group I: control rats, Group II: ISPH induced MI rats, Group III: mangiferin alone treated rats, and Group IV: mangiferin + ISPH treated rats. a Compared with Group 1. b Compared with Group 2.
Fig. 1. Effect of mangiferin on heart tissue phospholipid (PL) level in control and experimental group of rats. Group II ISPH induced MI rats showed a significant (P < 0.001) decrease in heart tissue PL level as compared to Group I control rats. Group IV mangiferin + ISPH treated rats showed a significant increase (P < 0.001) in heart tissue PL level as compared with Group II ISPH induced MI rats. A non-significant (NS) change was observed in Group III mangiferin alone treated rats as compared to Group I control rats.
III drug control rats when compared to Group I control rats. 4. Discussion The present study has showed an increase of serum and heart tissue lipids with a decrease in heart tissue phospholipids in ISPH induced cardiotoxic Group II MI rats. LPO can play an important role in lipoprotein modifications, which makes them susceptible to atherogenesis, which could be the reason for acute MI mediated cardiotoxicity by ISPH (Nirmala and Puvanakrishnan, 1996). Our earlier studies (Prabhu et al., 2006a) have also proved that ISPH induces LPO and accelerates myocardial necrosis. Increased level of cholesterol and TG (Manjula et al., 1992) is associated with cardiovascular disturbances and ISPH promotes lipolysis in myocardium (Sushamakumari et al., 1990). Enhancement in lipolysis and subsequent elevation of plasma FFA levels may lead to an increase in hepatic TG synthesis and secretion of elevated plasma TG concentration (Rayssiguier, 1990) and cholesterol. An increased synthesis of TG in heart tissue could be due to accumulation of acyl coA and an augmented production of glycerol
by increased glycolytic flux (Subramanian et al., 2003). The decrease in heart PL level may be explained due to increase in phospholipase activity and the activation of phospholipases is mediated through LPO with significant decrease in ATP levels during ISPH induced myocardial necrosis. Accelerated PL degradation could result in cell injury and ultimately cell death and is evidenced by the accumulation of thiobarbituric acid (TBA) reacting substances and the loss of both extractable PL and their polyunsaturated acyl groups (Sushamakumari et al., 1990). In Group IV mangiferin pretreated rats the level of serum and heart tissue lipids were reduced significantly with a significant increase in heart tissue PL. Polyphenols like mangiferin from different plant sources have been reported (Zern et al., 2003; Vinson and Jang, 2001) to alter the hepatic cholesterol metabolism which result in less accumulation of cholesterol in the aorta and able to significantly inhibit atherosclerosis and decrease the incidence of coronary heart disease. Mangiferin polyphenol possesses antioxidant property, which could have inhibited LPO due its scavenging lipid peroxy and alkoxy radicals and thereby preventing continued abstraction of hydrogen from cellular lipids (Ghosal et al., 1996). The higher rate of conversion of cholesterol to bile acids and elimination of faecal bile acids and neutral sterols could also be considered in the present study for the reduction of cholesterol in Group IV mangiferin pretreated rats. The improvement of hyperlipidemia may also be due to the significant reduction in lipolysis and hence decreased level of triacyl glycerol and FFA are observed. The increased level of PL in Group IV rats could be due its antioxidant and anti lipid peroxidative property, which could have protected tissue membrane PL from ISPH mediated peroxidation. Thus mangiferin has significantly reduced the serum and heart tissue lipids level except heart tissue PL. Hence i.p., administration of mangiferin has played a major role in lipid metabolism due to its free radical
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