Protective Effects of Combined Therapy of Rutin with Silymarin on Experimentally-Induced Diabetic Neuropathy in Rats

DOI: 10.4236/pp.2014.59098   PDF   HTML     3,405 Downloads   4,395 Views   Citations


The management of diabetic neuropathy (DN) is still a challenge for physicians. Hyperglycemia induced oxidative stress involves in the development of diabetic neuropathy, which could be reversed by supplementation of antioxidants. In the present study, it has targeted the oxidative stress mediated nerve damage in DN by using combined therapy of rutin (RT) and silymarin (SM). Diabetes was induced by single streptozotocin (STZ, 65 mg/kg i.p.) injection. The diabetic rats were treated daily with RT (100 mg/kg), SM (60 mg/kg) and RT (50 mg/kg) + SM (30 mg/kg) for 6 consecutive weeks. Pain-related behavior tests were performed including tail flick, paw-pressure analgesia and Rota-rod treadmill performance. Serum glucose, insulin, tumor necrosis factor-α (TNF-α), interleukine-6 (IL-6) and interleukine-1β (IL-β) levels were estimated. Thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) levels and enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) were measured. Diabetic rats that developed neuropathy were revealed by decreased tail-flick latency, paw-withdrawal latency and motor coordination. RT (100 mg/kg/day) and SM (60 mg/kg/day) dosed to diabetic rats, ameliorated hyperalgesia, analgesia and led to improved motor coordination. However, the combined therapy of RT (50 mg/kg/day) with SM (30 mg/kg/day) showed more significant effects in these parameters. STZ significantly increased TBARS and decreased GSH levels in sciatic nerve whereas combined therapy of RT and SM produced higher significant protection compared to individual. Similarly, combined therapy showed more significant amelioration in decreased levels of SOD, CAT, GST, GS and GPx activities in sciatic nerve of diabetic rats. Present results concluded that the combined therapy of phenolic compounds such as RT and SM had higher protective effects than their individual supplementations against DM.

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Al-Enazi, M. (2014) Protective Effects of Combined Therapy of Rutin with Silymarin on Experimentally-Induced Diabetic Neuropathy in Rats. Pharmacology & Pharmacy, 5, 876-889. doi: 10.4236/pp.2014.59098.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Wild, S., Roglic, G., Green, A., et al. (2004) Global Prevalence of Diabetes: Estimates for the Year 2000 and Projections for 2030. Diabetes Care, 27, 1047-1053.
[2] Roa, W.I. (2006) Definition and Diagnosis of Diabetes Mellitus and Intermediate Hyperglycemia. WHO Document Production Services, 1-46.
[3] Callaghan, B.C., Little, A.A., Feldman, E.L., et al. (2012) Enhanced Glucose Control for Preventing and Treating Diabetic Neuropathy. Cochrane Database Systematic Review, 6, Article ID: CD007543.
[4] Abdi, S., Haruo, A. and Bloomstone, J. (2004) Electroconvulsive Therapy for Neuropathic Pain: A Case Report and Literature Review. Pain Physician, 7, 261-263.
[5] Woolf, C.J. (2004) Dissecting out Mechanisms Responsible for Peripheral Neuropathic Pain: Implications for Diagnosis and Therapy. Life Sciences, 74, 2605-2610.
[6] Galer, B.S., Gianas, A. and Jensen, M.P. (2000) Painful Diabetic Polyneuropathy: Epidemiology, Pain Description, and Quality of Life. Diabetes Research and Clinical Practice, 47, 123-128.
[7] Cameron, N.E., Jack, A.M. and Cotter, M.A. (2001) Effect of Alpha-Lipoic Acid on Vascular Responses and Nociception in Diabetic rats. Free Radical Biology and Medicine, 31, 125-135.
[8] Stevens, M.J., Obrosova, I., Cao, X., et al. (2000) Effects of DL-Alpha-Lipoic Acid on Peripheral Nerve Conduction, Blood Flow, Energy Metabolism, and Oxidative Stress in Experimental Diabetic Neuropathy. Diabetes, 49, 1006-1015.
[9] Calcutt, N.A. (2004) Experimental Models of Painful Diabetic Neuropathy. Journal Neurological Sciences, 220, 137-139.
[10] Sima, A.A., Zhang, W., Xu, G., et al. (2000) A Comparison of Diabetic Polyneuropathy in Type II Diabetic BBZDR/Wor Rats and in Type I Diabetic BB/Wor Rats. Diabetologia, 43, 786-793.
[11] Edwards, J.L., Vincent, A.M., Cheng, H.T. and Feldman, E.L. (2008) Diabetic Neuropathy: Mechanisms to Management. Pharmacology & Therapeutics, 120, 1-34.
[12] Figueroa-Romero, C., Sadidi, M. and Feldman, E.L. (2008) Mechanisms of Disease: The Oxidative Stress Theory of Diabetic Neuropathy. Reviews in Endocrine and Metabolic Disorders, 9, 301-314.
[13] Cameron, N.E. and Cotter, M.A. (1999) Effects of Antioxidants on Nerve and Vascular Dysfunction in Experimental Diabetes. Diabetes Research and Clinical Practice, 45, 137-146.
[14] Yu, J., Zhang, Y., Sun, S., Shen, J.P., Qiu, J., Yin, X.X., Yin, H.L. and Jiang, S.J. (2006) Inhibitory Effects of Astragaloside IV on Diabetic Peripheral Neuropathy in Rats. Canadian Journal of Physiology and Pharmacology, 84, 579-587.
[15] Arora, M., Kumar, A., Kaundal, R.K. and Sharma, S.S. (2008) Amelioration of Neurological and Biochemical Deficits by Peroxynitrite Decomposition Catalysts in Experimental Diabetic Neuropathy. European Journal of Pharmacology, 596, 77-83.
[16] Cui, X.P., Li, B.Y., Gao, H.Q., Wei, N., Wang, W.L. and Lu, M. (2008) Effects of Grape Seed Proanthocyanidin Extracts on Peripheral Nerves in Streptozocin-Induced Diabetic Rats. Journal of Nutritional Science and Vitaminology (Tokyo), 54, 321-328.
[17] Cunha, J.M., Jolivalt, C.G., Ramos, K.M., Gregory, J.A., Calcutt, N.A. and Mizisin, A.P. (2008) Elevated Lipid Peroxidation and DNA Oxidation in Nerve from Diabetic Rats: Effects of Aldose Reductase Inhibition, Insulin, and Neurotrophic Factors. Metabolism-Clinical and Experimental, 57, 873-881.
[18] Bierhaus, A., Ziegler, R. and Nawroth, P.P. (1998) Molecular Mechanisms of Diabetic Angiopathy-Clues for Innovative Therapeutic Interventions. Hormone Research, 50, 1-5.
[19] Tuttle, K.R. (2005) Linking Metabolism and Immunology: Diabetic Nephropathy Is an Inflammatory Disease. Journal of the American Society of Nephrology, 16, 1537-1538.
[20] Navarro-Gonzalez, J.F. and Mora-Fernandez, C. (2008) The Role of Inflammatory Cytokines in Diabetic Nephropathy. Journal of the American Society of Nephrology, 19, 433-442.
[21] Goldberg, R.B. (2009) Cytokine and Cytokine-Like Inflammation Markers, Endothelial Dysfunction, and Imbalanced Coagulation in Development of Diabetes and Its Complications. The Endocrine Society. Journal of Clinical Endocrinology and Metabolism, 94, 3171-3182.
[22] Rangkadilok, N., Sitthimonchai, S., Worasuttayangkurn, L., Mahidol, C., Ruchirawat, M. and Satayavivad, J. (2007) Evaluation of Free Radical Scavenging and Antityrosinase Activities of Standardized Longan Fruit Extract. Food and Chemical Toxicology, 45, 328-336.
[23] Lopez-Revuelta, A., Sanchez-Gallego, J.I., Hernandez-Hernandez, A., Sánchez-Yagüe, J. and Llanillo, M. (2006) Membrane Cholesterol Contents Influence the Protective Effects of Quercetin and Rutin in Erythrocytes Damaged by Oxidative Stress. Chemico-Biological Interactions, 161, 79-91.
[24] ChoiI, P.Y., Choi, H. and Lee, E.H. (2006) Anti-Adipogenic Activity of Rutin in 3T3-L1 Cells and Mice Fed with High-Fat Diet. BioFactors, 26, 273-281.
[25] Nencini, C., Giorgi, G. and Micheli, L. (2007) Protective Effect of Si-lymarin on Oxidative Stress in Rat Brain. Phytomedicine, 14, 129-135.
[26] Soto, C., Recoba, R., Barrón, H., Alvarez, C. and Favari, L. (2003) Silymarin Increases Antioxidant Enzymes in Alloxan-Induced Diabetes in Rat Pancreas. Comparative Biochemistry and Physiology. Toxicology & Pharmacology, 136, 205-212.
[27] Ihme, N., Kiesewetter, H., Jung, F., Hoffmann, K.H., Birk, A., Müller, A. and Grützner, K.I. (1996) Leg Oedema Protection from a Buckwheat Herb Tea in Patients with Chronic Venous Insufficiency: A Single-Centre, Randomised, Double-Blind, Placebo-Controlled Clinical Trial. European Journal of Clinical Pharmacology, 50, 443-447.
[28] Lindahl, M. and Tagesson, C. (1997) Flavonoids as Phospholipase A2 Inhibitors: Importance of Their Structure for Selective Inhibition of Group II Phospholipase A2. Inflammation, 21, 347-356.
[29] Agarwal, R., Agarwal, C., Ichikawa, H., Singh, R.P. and Aggarwal, B.B. (2006) Anticancer Potential of Silymarin: From Bench to Bed Side. Anticancer Research, 26, 4457-4498.
[30] Polyak, S.J., Morishima, C., Shuhart, M.C., Wang, C.C., Liu, Y. and Lee, D.Y.W. (2007) Inhibition of T-Cell Inflammatory Cytokines, Hepatocyte NF-κB Signaling and HCV Infection by Standardized Silymarin. Gastroenterology, 132, 1925-1936.
[31] Jacobs, B.P., Dennehy, C., Ramirez, G., Sapp, J. and Lawrence, V.A. (2002) Milk Thistle for the Treatment of Liver Disease: A Systematic Review and Meta-Analysis. American Journal of Medicine, 113, 506-515.
[32] Aleisa, A.M., Abouhashish, H.M., Ahmed, M.M., Al-Rejaie, S.S., Alkhamees, O.A. and Alroujayee, A.S. (2013) Ameliorative Effects of Rutin and Ascorbic Acid Combination on Hypercholesterolemia-Induced Hepatotoxicity in Female Rats. African Journal of Pharmacy and Pharmacology, 7, 280-288.
[33] Sugimoto, K., Rashid, I.B., Shoji, M., Suda, T. and Yasujima, M. (2008) Early Changes in Insulin Receptor Signaling and Pain Sensation in Streptozotocin-Induced Diabetic Neuropathy in Rats. Journal of Pain, 9, 237-245.
[34] Kamboj, S.S., Vasishta, R.K. and Sandhir, R. (2010) N-Acetylcysteine Inhibits Hyperglycemia-Induced Oxidative Stress and Apoptosis Markers in Diabetic Neuropathy. Journal of Neurochemistry, 112, 77-91.
[35] Sedlak, J. and Lindsay, R.H. (1968) Estimation of Total, Protein-Bound, and Nonprotein Sulfhydryl Groups in Tissue with Ellman’s Reagent. Analytical Biochemistry, 25, 192-205.
[36] Kono, Y. (1978) Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase. Archives of Biochemistry and Biophysics, 186, 189-195.
[37] Aebi, H. (1974) Catalase. In: Bergmeyer, H.U., Ed., Methods in Enzymatic Analysis, New York, 674-684.
[38] Apfel, S.C., Asbury, A.K., Bril, V., et al. (2001) Positive Neuropathic Sensory Symptoms as Endpoints in Diabetic Neuropathy Trials. Journal of the Neurological Sciences, 189, 3-5.
[39] Li, F., Drel, V.R., Szabo, C., Stevens, M.J. and Obrosova, I.G. (2005) Low-Dose Poly (ADP-Ribose) Polymerase Inhibitor-Containing Combination Therapies Reverse Early Peripheral Diabetic Neuropathy. Diabetes, 54, 1514-1522.
[40] Obrosova, I.G. (2009) Diabetic Painful and Insensate Neuropathy: Pathogenesis and Potential Treatments. Neurotherapeutics, 6, 638-647.
[41] Romanovsky, D., Hastings, S.L., Stimers, J.R. and Dobretsov, M. (2004) Relevance of Hyperglycemia to Early Mechanical Hyperalgesia in Streptozotocin-Induced Diabetes. Journal of the Peripheral Nervous System, 9, 62-69.
[42] Fuchs, D., Birklein, F., Reeh, P.W. and Sauer, S.K. (2010) Sensitized Peripheral Nociception in Experimental Diabetes of the Rat. Pain, 151, 496-505.
[43] Ramirez, M.R., Guterres, L., Dickel, O.E., de Castro, M.R., Henriques, A.T., de Souza, M.M. and Barros, D.M. (2010) Preliminary Studies on the Antinociceptive Activity of Vaccinium ashei Berry in Experimental Animal Models. Journal of Medicinal Food, 13, 336-342.
[44] Lee, J.Y., Jang, Y.W., Kang, H.S., Moon, H., Sim, S.S. and Kim, C.J. (2006) Anti-Inflammatory Action of Phenolic Compounds from Gastrodia elata Root. Archives of Pharmacal Research, 29, 849-858.
[45] Al-Enazi, M.M. (2013) Combined Therapy of Rutin and Silymarin Has More Protective Effects on Streptozotocin-Induced Oxidative Stress in Rats. Journal of Applied Pharmaceutical Science, 4, 21-28.
[46] Prabu, S.M., Shagirtha, K. and Renugadevi, J. (2011) Naringenin in Combination with Vitamins C and E Potentially Protects Oxidative Stress-Mediated Hepatic Injury in Cadmium-Intoxicated Rats. Journal of Nutritional Science and Vitaminology, 57, 177-185.
[47] Li, J., Wei, G.H., Huang, H., Lan, Y.P., Liu, B., Liu, H., Zhang, W. and Zuo, Y.X. (2013) Nerve Injury-Related Autoimmunity Activation Leads to Chronic Inflammation and Chronic Neuropathic Pain. Anesthesiology, 118, 416-429.
[48] Djordjevic, A., Bursac, B., Velickovic, N., Vasiljevic, A. and Matic, G. (2013) The Impact of Different Fructose Loads on Insulin Sensitivity, Inflammation, and PSA-NCAM-Mediated Plasticity in the Hippocampus of Fructose-Fed Male Rats. Nutritional Neuroscience, in press.
[49] Locksley, R.M., Killeen, N. and Lenardo, M.J. (2001) The TNF and TNF Receptor Superfamilies: Integrating Mammalian Biology. Cell, 104, 487-501.
[50] Brownlee, M. (2005) The Pathobiology of Diabetic Complica-tions: A Unifying Mechanism. Diabetes, 54, 1615-1625.
[51] Abu-El-Fattah, A.A., El-Sawalhi, M.M., Rashed, E.R. and El-Ghazaly, M.A. (2010) Possible Role of Vitamin E, Coenzyme Q10 and Rutin in Protection against Cerebral Ischemia/Reperfusion Injury in Irradiated Rats. International Journal of Radiation Biology, 86, 1070-1078.
[52] Alsaif, M.A. (2009) Beneficial Effects of Rutin and Vitamin C Co-Administration in a Streptozotocin-Induced Diabetes Rat Model of Kidney Nephrotoxicity. Pakistan Journal of Nutrition, 8, 745-754.
[53] Kwon, K.H., Murakami, A., Tanaka, T. and Ohigashi, H. (2005) Dietary Rutin, but Not Its Aglycone Quercetin, Ameliorates Dextran Sulfate Sodium-Induced Experimental Colitis in Mice: Attenuation of Pro-Inflammatory Gene Expression. Biochemical Pharmacology, 69, 395-406.
[54] Ashkavand, Z., Malekinejad, H., Amniattalab, A., Rezaei-Golmisheh, A. and Vishwanath, B.S. (2012) Silymarin Potentiates the Anti-Inflammatory Effects of Celecoxib on Chemically Induced Osteoarthritis in Rats. Phytomedicine, 19, 1200-1205.
[55] Gharagozloo, M., Velardi, E., Bruscoli, S., Agostini, M., Di Sante, M., Donato, V., Amirghofran, Z. and Riccardi, C. (2010) Silymarin Suppress CD4+ T Cell Activation and Proliferation: Effects on NF-κB Activity and IL-2 Production. Pharmacological Research, 61, 405-409.
[56] Bonnefont-Rousselot, D. (2002) Glucose and Reactive Oxygen Species. Current Opinion in Clinical Nutrition & Metabolic Care, 5, 561-568.
[57] Viggiano, A., Monda, M., Viggiano, A., Viggiano, D., Viggiano, E., Chiefari, M., Aurilio, C. and De Luca, B. (2005) Trigeminal Pain Transmission Requires Reactive Oxygen Species Production. Brain Research, 1050, 72-78.
[58] Kuzumoto, Y., Kusunoki, S., Kato, N., Kihara, M. and Low, P.A. (2006) Effect of the Aldose Reductase Inhibitor Fidarestat on Experimental Diabetic Neuropathy in the Rat. Diabetologia, 49, 3085-3093.
[59] Arai, K., Maguchi, S., Fujii, S., Ishibashi, H., Oikawa, K. and Taniguchi, N. (1987) Glycation and Inactivation of Human Cu-Zn-Superoxide Dismutase. Identification of the in Vitro Glycated Sites. Journal of Biological Chemistry, 262, 16969-16972.
[60] Kamalakkannan, N. and Prince, P.S.M. (2006) Antihyperglycaemic and Antioxidant Effect of Rutin, a Polyphenolic Flavonoid, in Streptozotocin-Induced Diabetic Wistar Rats. Basic & Clinical Pharmacology & Toxicology, 98, 97-103.
[61] Cecen, E., Dost, T., Culhaci, N., Karul, A., Ergur, B. and Birincioglu, M. (2011) Protective Effects of Silymarin against Doxorubicin-Induced Toxicity. Asian Pacific Journal of Cancer Prevention, 12, 2697-2704.
[62] Jain, A., Yadav, A., Bozhkov, A.I., Padalko, V.I. and Flora, S.J.S. (2011) Therapeutic Efficacy of Silymarin and Naringenin in Reducing Arsenic-Induced Hepatic Damage in Young Rats. Ecotoxicology and Environmental Safety, 74, 607-614.
[63] Soto, C., Pérez, J., García, V., Uría, E., Vadillo, M. and Raya, L. (2010) Effect of Silymarin on Kidneys of Rats Suffering from Alloxan-Induced Diabetes Mellitus. Phytomedicine, 17, 1090-1094.
[64] Carlberg, I. and Mannervik, B. (1985) Glutathione Reductase. Methods in Enzymology, 113, 484-490.
[65] Ulusu, N.N., Sahilli, M., Avci, A., Canbolat, O., Ozansoy, G., Ari, N., et al. (2003) Pentose Phosphate Pathway, Glutathione-Dependent Enzymes and Antioxidant Defense during Oxidative Stress in Diabetic Rodent Brain and Peripheral Organs: Effects of Stobadine and Vitamin E. Neurochemical Research, 28, 815-823.
[66] Sanders, R.A., Rauscher, F.M. and Watkins, J.B. (2001) Effects of Quercetin on Antioxidant Defense in Streptozotocin-Induced Diabetic Rats. Journal of Biochemical and Molecular Toxicology, 15, 143-149.
[67] Ahmad, I., Shukla, S., Kumar, A., et al. (2012) Biochemical and Molecular Mechanisms of N-Acetyl Cysteine and Silymarin-Mediated Protection against Maneb- and Paraquat-Induced Hepatotoxicity in Rats. Chemico-Biological Interactions, 201, 9-18.
[68] Mak, D.H., Ip, S.P., Li, P.C., Poon, M.K.T. and Ko, K.M. (1996) Alterations in Tissue Glutathione Antioxidant System in Streptozotocin-Induced Diabetic Rats. Molecular and Cellular Biochemistry, 162, 153-158.

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