Methanolic leaf extract of Punica granatum attenuates ischemia-reperfusion brain injury in Wistar rats: Potential antioxidant and anti-inflammatory mechanisms

Document Type : Original Article

Authors

1 Research Scholar, JNT University Anantapur, Ananthapuramu 515002, India

2 Connexios Life Sciences Pvt Ltd, Basavanagudi, Bangalore 560 004, India

3 Oil Technological and Pharmaceutical Research Institute (OTPRI), JNT University Anantapur, Ananthapuramu 515 002, India

Abstract

Objective(s): This study was conducted to evaluate the cerebroprotective effect of methanolic leaf extract of Punica granatum (MePG) in Wistar rats.
Materials and Methods: The MePG was initially assessed for in vitro antioxidant activity, and later evaluated on LPS-induced RAW 264.7 cell line assay. Finally, the MePG was evaluated against ischemia-reperfusion (I/R) induced brain injury in Wistar rats.
Results: In DPPH, FRAP and ORAC assays, the MePG has exhibited potent antioxidant activity. Further, the MePG has significantly inhibited the generation of nitrite, ROS and TNF-α in LPS-induced RAW 264.7 cell lines. Besides, global ischemia followed by reperfusion caused significant changes in the neurological and behavioral functions in I/R control animals compared to sham control. Additionally, in the I/R control group there was a substantial decrease in the catalase and superoxide dismutase activities; Likewise, reduced glutathione levels reduced and lipid peroxidation levels enhanced significantly. Also, pro-inflammatory cytokines such as TNF-α, IL-6, and ICAM-I were increased and the levels of IL-10 was decreased significantly. Furthermore, the I/R insult caused increase in brain volume and cerebral infarct formation. Similarly, histopathology of the brain tissue revealed hallmarks like necrosis, leukocyte infiltration, cerebral edema and vascular congestion in I/R control. Notably, MePG (200 and 400 mg/kg) pretreatment for 7 days, has attenuated all the I/R-persuaded pathological changes compared to I/R control. In addition, the LC-MS/MS analysis showed presence of acteoside, apigenin, gallic acid, gossypin, pentagalloyl glucose, quercetin, and rutin as major ingredients in the MePG.
Conclusion: These findings suggest that the MePG possesses significant cerebroprotective activity.

Keywords

Main Subjects


1. Ringelstein EB, Nabavi DG. Cerebral small vessel diseases: cerebral microangiopathies. Curr Opin Neurol 2005;18: 179-188. 
2. Guzik A, Bushnell C. Stroke Epidemiology and Risk Factor Management. Continuum 2017; 23:15-39.
3. Mahajan SK, Kashyap R, Sood BR, Jaret P, Mokta J, Kaushik NK, Prashar BS. Stroke at moderate altitude. JAPI 2004;52: 699–702.
4. Allen C, Bayraktutan U. Oxidative stress and its role in the pathogenesis of ischaemic stroke. Int J Stroke 2009;4:461–470.
5. Albarracin SL, Stab B, Casas Z. Effects of natural antioxidants in neurodegenerative disease. Nutr Neurosci 2012;15: 1–9.
6. Tsai PJ, Tsai TH, Yu CH, Ho SC. Evaluation of the NO-suppressing activity of several Mediterranean culinary spices. Food Chem Toxicol 2007;45:440-447.
7. Rodrigues FTS, de Sousa CNS, Ximenes NC, Almeida AB, Cabral LM, Patrocinio CFV, et al. Effects of standard ethanolic extract from Erythrina velutina in acute cerebral ischemia in mice. Biomed Pharmacother 2017;96:1230-1239.
8. Rao A, Balachandran B. Role of oxidative stress and antioxidants in neurodegenerative diseases. Nutr Neurosci 2002; 5:291–309.
9. Durukan A, Tatlisumak T. Acute ischemic stroke: overview of major experimental rodent models, pathophysiology, and therapy of focal cerebral ischemia. Pharmacol Biochem Behav 2007;87:179-197.
10. Fisher M. New approaches to neuroprotective drug development. Stroke 2011;42:S24-S27.
11. Kritikar K, Basu B, 2007. Indian Medicinal Plants. International Book Publishers, Bombay.
12. Nadkarni K. 1976. Indian Material Medica. Bombay Popular Prakashan, Bombay.
13. Basiri S. Evaluation of antioxidant and antiradical properties of Pomegranate (Punica granatum L.) seed and defatted seed extracts. J Food Sci Technol 2015; 52:1117-23.
14. Viswanatha GL, Venkataranganna MV, Prasad NBL. Evaluation of anti-epileptic activity of leaf extracts of Punica granatum on experimental models of epilepsy in mice. J Intercult Ethnopharmacol 2016;5:415-421.
15. Braidy N, Selvaraju S, Essa MM, Vaishnav R, Al-Adawi S, Al-Asmi A, et al. Neuroprotective effects of a variety of pomegranate juice extracts against MPTP-induced cytotoxicity and oxidative stress in human primary neurons. Oxid Med Cell Longev 2013;2013:685909.
16. Tapias  V,  Cannon  JR,  Greenamyre JT. Pomegranate juice exacerbates oxidative stress and nigrostriatal degeneration in Parkinson’s disease. Neurobiol Aging 2014 35:1162-76.
17. DaSilva NA, Nahar PP, Ma H, Eid A, Wei Z, Meschwitz S, et al. Pomegranate ellagitannin-gut microbial-derived metabolites, urolithins, inhibit neuroinflammation in vitro. Nutr Neurosci 2017;  7:1-11.
18. MM Essa, S Subash, M Akbar, S Al-Adawi, GJ Guillemin. Long term dietary supplementation of pomegranates, figs and dates alleviate neuroinflammation in a transgenic mouse model of Alzheimer’s disease. PLoS One 2015;10:e0120964.
19. Morzelle MC, Salgado JM, Telles M, Mourelle D, Bachiega P, Buck HS, et al. Neuroprotective effects of Pomegranate Peel Extract after Chronic Infusion with Amyloid–β Peptide in Mice. PLoS One 2016;11:e0166123.
20. Adiga S, Trivedi P, Ravichandra V, Deb D, Mehta F. Effect of Punica granatum peel extract on learning and memory in rats. Asian Pac J Trop Med 2010;3:687-90.
21. West T, Atzeva M, Holtzman DM. Pomegranate polyphenols and resveratrol protect the neonatal brain against hypoxic-ischemic injury. Dev Neurosci 2007;29:363-72.
22. Loren DJ, Seeram NP, Schulman RN, Holtzman DM. Maternal dietary supplementation with pomegranate juice is neuroprotective in an animal model of neonatal hypoxic-ischemic brain injury. Pediatr Res 200557:858-64.
23. Anturlikar S, Rafiq M, Azeemuddin M, Viswanatha G, Jagadeesh M, Rao KS, et al. Free radical scavenging and hepatoprotective activity of HD-03/ES in experimental models. J Exp Integr Med2012; 2:161-166.
24. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem1996; 239: 70-76.
25. Rafiq M, Azeemuddin M, Anturlikar SD,Viswanatha GL, Patki PS. Application of oxygen radical absorbance capacity (ORAC) assay in the estimation of antioxidant value of botanicals. Oxid Antioxid Med Sci 2012;1: 87-90.
26. Viswanatha GL, Venkataranganna MV, Prasad NBL, Hanumanthappa S. Chemical characterization and cerebroprotective effect of methanolic root extract of Colebrookea oppositifolia in rats. J Ethnopharmacol 2018; 223: 63-75.
27. Mathew, G, Jacob A, Durgashivaprasad E, Reddy ND, Unnikrishnan MK. 6b,11b-Dihydroxy-6b,11b-dihydro-7H-indeno[1,2-b]naphtho[2,1-d]furan-7-one (DHFO), a small molecule targeting NF-kappaB, demonstrates therapeutic potential in immunopathogenic chronic inflammatory conditions. Int Immunopharmacol 2013;15:182-189.
28. Viswanatha GL, Kumar LM, Rafiq M, Kavya KJ, Thippeswamy AH, Yuvaraj HC, et al. LC-MS/MS profiling and neuroprotective effects of Mentat(R) against transient global ischemia and reperfusion-induced brain injury in rats. Nutrition 2015; 31:1008-1017.
29. Viswanatha GL, Shylaja H, Mohan CG. Alleviation of transient global ischemia/reperfusion-induced brain injury in rats with 1, 2, 3, 4, 6-penta-O-galloyl-beta-d-glucopyranose isolated from Mangifera indica. Eur J Pharmacol 2013; 720: 286-293.
30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ.Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275.
31. Luck H. Catalase, in: H.O., B. (Ed.), Methods of Enzymatic analysis. Academic Press, New York, 1971, pp. 885-893.
32. Wills ED.Mechanisms of lipid peroxide formation in animal tissues. Biochem J 1966; 99:667-676.
33. Sedlak J. Lindsay RH.Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 1968; 25:192-205.
34. Moukdar F. Robidoux J. Lyght O. Pi J. Daniel KW. Collins S.Reduced antioxidant capacity and diet-induced atherosclerosis in uncoupling protein-2-deficient mice. J Lipid Res 2009; 50:59-70.
35. Ainsworth EA, Gillespie KM.Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat Protoc 2007; 2: 875-877.
36. Choi SY, Ko HC, Ko SY, Hwang JH, Park JG, Kang SH, et al. Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits. Biol Pharm Bull 2007; 30:772-778.
37. Conforti F. Menichini F.Phenolic compounds from plants as nitric oxide production inhibitors. Curr Med Chem 2011;18:1137-1145.
38. Tsuchiya M, Sako K, Yura S, Yonemasu Y. Local cerebral glucose utilisation following acute and chronic bilateral carotid artery ligation in Wistar rats: relation to changes in local cerebral blood flow. Exp Brain Res 1993;95:1-7.
39. Hillard CJ. Role of cannabinoids and endocannabinoids in cerebral ischemia. Curr Pharm Des 2008;14:2347-2361.
40. Soliman ML, Puig KL, Combs CK, Rosenberger TA.Acetate reduces microglia inflammatory signaling in vitro. J Neurochem 2012;123:555-567.
41. Liu C, Tu FX, Chen X. Neuroprotective effects of apigenin on acute transient focal cerebral ischemia-reperfusion injury in rats. Zhong Yao Cai 2008;31:870–873.
42. Sun J, Li YZ, Ding YH, Wang J, Geng J, Yang H, et al. Neuroprotective effects of gallic acid against hypoxia/reoxygenation-induced mitochondrial dysfunctions in vitro and cerebral ischemia/reperfusion injury in vivo. Brain Res 2014;1589:126–139.
43. Chandrashekhar VM, Ganapathy S, Ramkishan A, Narsu ML.Neuroprotective activity of gossypin from Hibiscus vitifolius against global cerebral ischemia model in rats. Ind J Pharmacol 2013; 45: 575-580.
44. Rivera F, Costa G, Abin A, Urbanavicius J, Arruti C, Casanova G, et al. Reduction of ischemic brain damage and increase of glutathione by a liposomal preparation of quercetin in permanent focal ischemia in rats. Neurotox Res 2008;13:105-114.
45. Khan MM, Ahmad A, Ishrat T, Khuwaja G, Srivastava P, Khan MB, et al. Rutin protects the neural damage induced by transient focal ischemia in rats. Brain Res 2009;1292:123–135.
46. Jang JW, Lee JK, Hur H, Kim TW, Joo SP, Piao MS. Rutin improves functional outcome via reducing the elevated matrix metalloproteinase-9 level in a photothrombotic focal ischemic model of rats. J Neurol Sci 2014; 15;339:75-80.