Antitumor activity of Neem leaf Extract and Nimbolide on Ehrlich Ascites Carcinoma Cells in Mice
Abstract
Background: Azadirachta indica (Neem) has been used traditionally for many centuries. Some impressive therapeutic qualities have been discovered. Aim: Our study aims to investigate the in vivo antitumor and antioxidant activities of Ethanolic Neem Leaf Extract (ENLE) and its fraction called Nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica). Also, to study the side effects of the ethanolic Neem leaf extract and Nimbolide fraction on the liver and kidney. Materials & Methods: We assessed the effect of nimbolide and ethanolic Neem leaves extract (ENLE) on replicative lifespan prolongation in vitro and on the levels of malondialdehyde (MDA), nitric oxide (NO), catalase, glutathione peroxidase (GPx), Caspase-3, and Cytochrome c. Also, our study estimated their effect on Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Total Protein (TP), Albumin (Alb), bilirubin, urea and creatinine. Results: Ethanolic neem leaves extract and nimbolide resulted in increases in replicative lifespan. Also, they showed a significant decrease in malondialdehyde and nitric oxide and an increase in catalase, glutathione peroxidase, caspase-3 activities, and cytochrome c concentration. Hence, it may be possible that Nimbolide and ENLE decrease lipid peroxidation level due to their antioxidant effect and enhance apoptosis. The Neem leaves extract and nimbolide showed no side effects on liver and kidney. Also, they showed a significant protection for both liver and kidney histopathologically.
Keywords
References
Aebi H 1984 Catalase in vitro. Methods Enzymol 105 121-126
Ahmed E, Mohamed S & Ahmed M 2014 Azadirachta indica Attenuates Cisplatin-Induced Nephrotoxicity and Oxidative Stress. Biomed Res Int 2014 647131
Anima T, Tulsidas G Shrivastav & Chaube SK 2011 Aqueous extract of Azadirachta indica (neem) leaf induces generation of reactive oxygen species and mitochondria-mediated apoptosis in rat oocytes. J Assist Reprod Genet 29 15-23
Anitha G, Raj JJ, Krishnan VR, Narasimhan S, Solomon KA & Rajan SS 2007 Semi-synthetic modification of nimbolide to 6-homodesacetylnimbin and 6-desacetylnimbin and their cytotoxic studies. J Asian Nat Prod Res 9 73-78
Azman NA, Gallego MG, Juliá L, Fajari L & Almajano M. 2015 The Effect of Convolvulus arvensis Dried Extract as a Potential Antioxidant in Food Models. Antioxidants (Basel) 4(1) 170-184
Babykutty S, S PP, J NR, Kumar MA, Nair MS, Srinivas P & Gopala S 2011 Nimbolide retards tumor cell migration, invasion, and angiogenesis by downregulating MMP-2/9 expression via inhibiting ERK1/2 and reducing DNA-binding activity of NF-κB in colon cancer cells. Mol Carcinog 51 475-490
Baligar NS, Aladakatti RH, Ahmed M & Hiremath MB 2014 Evaluation of acute toxicity of neem active constituent, Nimbolide and its hepatoprotective activity against acute dose of carbon tetrachloride treated albino rats. IJPSR 50 3455-3466
Basu AK 2018 DNA Damage, Mutagenesis and Cancer. Int J Mol Sci 19 970 doi:103390/ijms19040970
Bhanwra S, Singh J & Khosla P 2000 Effect of Azadirachta indica (Neem) leaf aqueous extract on paracetamol-induced liver damage in rats. Indian J Physiol Pharmacol 44 64-68
Cai J, Yang J & Jones DP 1998 Mitochondrial control of apoptosis: the role of cytochrome c. Biochem Biophys Acta 1366 139-149
Casarez E 2001 Basic Principles of Toxicology. BIOC 597c
Casciola-Rosen L, Nicholson DW, Chong T, Rowan KR, Thornberry NA, Miller DK & Rosen A 1996 Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med 183 1957-1964
Chaney AL & Marbach CP 1962 Modified reagents for determination of urea and ammonia. Clin Chem 8 130-132
Chattopadhyay RR 1998 Possible biochemical mode of anti-inflammatory action of Azadirachta indica AJuss in rats. Indian J Exp Biol 36 418-420
Chhavi Sh, Andrea J Vas PG, Taher M Gh, Tahir AR & Arif H 2014 Ethanolic Neem (Azadirachta indica) Leaf Extract Prevents Growth of MCF-7 and HeLa Cells and Potentiates the Therapeutic Index of Cisplatin. Journal of Oncology 2014 321754
Crump KS, Hoel DG, Langley CH & Peto R 1976 Fundamental Carcinogenic Processes and Their Implications for Low Dose Risk Assessment. Cancer Res 36 2973–2979
Dasgupta T, Banerjee S, Yadava PK & Rao AR 2004 Chemopreventive potential of Azadirachta indica (Neem) leaf extract in murine carcinogenesis model systems. J Ethnopharmacol 92 23-36
Doaa E-D, Mohamed S G, Abdel Razik F & Ahmed AM 2011 Physiological and histological impact of Azadirachta indica (neem) leaves extract in a rat model of cisplatin-induced hepato and nephrotoxicity. J Med Plant Res 5 5499-5506
Doumas, BT, Bayse, DD, Carter RJ, Elevitch F, Garber CC, Graby RA, Hause LL, Mather A, Peters T Jr, Rand RN, Reeder DJ, Russell SM, Schaffer R & Westgard JO 1981 Candidate reference method for determination of total proteins in serum I Development and validation, II Tests for transferability. Clin Chem 27 1642 – 1654
Doumas, BT, Watson, WA & Biggs HG 1971 Albumin standards and the measurement of serum albumin with bromocresol green. Clin Chim Acta 31 87 – 96
Dubey NK 2015 Plants as a Source of Natural Antioxidants. CABI, 1st edition
Dutta M, Raychaudhuri U, Chakraborty R & Maji D 2011 Role of diet and plants on diabetic patients- A critical appraisal. Sci Cultur 77
Elumalai P, Gunadharini DN, Senthilkumar K, Banudevi S, Arunkumar R, Benson CS, Sharmila G & Arunakaran J 2012 Ethanolic neem (Azadirachta indica A Juss) leaf extract induces apoptosis and inhibits the IGF signaling pathway in breast cancer cell lines. Biomed Prevent Nutr 2 59–68
Elumalai P, Gunadharini DN, Senthilkumar K, Banudevi S, Arunkumar R, Benson CS, Sharmila G & Arunakaran J 2012 Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway. Toxicol Lett 215 131– 142
Fahim FA, Esmat AY, Mady EA & Amin MA 1997 Serum LDH and ALP isozyme activities in mice bearing solid Ehrlich carcinoma and/or treated with maximum tolerated dose (MTD) of aloin. Dis Markers 13 183-193
Gangar SC, Sandhir R, Rai DV & Koul A 2006 Modulatory effects of Azadirachta indica on benzo(a)pyrene induced forestomach tumorigenesis in mice. World J Gastroenterol 12 2749-2755
Gerhauser C 2008 Cancer chemopreventive potential of apples, apple juice, and apple components. Planta Med 74 1608-1624
Ghimeray AK, Jin C, Ghimire BK & Cho DH 2009 Antioxidant activity and quantitative estimation of azadirachtin and nimbin in Azadirachta IndicaA Juss grown in foothills of Nepal. Afr J Biotechnol 8 3084-3091
Glinsukon T, Somjaree R, Piyachaturawat P & Thebtaranonth Y 1986 Acute toxicity of nimbolide and nimbic acid in mice, rats and hamsters. Toxicol Lett 30 159-166
Gunadharini DN, Elumalai P, Arunkumar R, Senthilkumar K & Arunakaran J 2011 Induction of apoptosis and inhibition of PI3K/Akt pathway in PC-3 and LNCaP prostate cancer cells by ethanolic neem leaf extract. J Ethnopharmacol 134 644- 650
Gupta SC, Prasad S, Sethumadhavan DR, Nair MS, Mo YY & Aggarwal BB 2013 Nimbolide, a limonoid triterpene, inhibits growth of human colorectalcancerxenograftsby suppressing the proinflammatory microenvironment. Clin Cancer Res 19 4465-4476
Gupta SC, Reuter S, Phromnoi K, Park B, Hema PS, Nair M & Aggarwal BB 2011 Nimbolide Sensitizes Human Colon Cancer Cells to TRAIL through Reactive Oxygen Species- and ERK-dependent Up-regulation of Death Receptors, p53, and Bax. J Biol Chem 286 1134–1146
Haque E, Mandal I, Pal S & Baral R 2006 Prophylactic dose of neem (Azadirachta indica) leaf preparation restricting murine tumor growth is nontoxic, hematostimulatory and immunostimulatory. Immunopharmacol Immunotoxicol 28 33-50
Jendrassik L & Gróf P (1938) Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins. Biochemische Zeitschrift 297 82-89 Kale BP, Kothekar MA, Tayade HP, Jaju JB & Mateenuddin M 2003 Effect of aqueous extract of Azadirachta Indica leaves on hepatotoxicity induced by antitubercular drugs in rats. Indian Journal of Pharmacology 35 177-180
Kikuchi T, Ishii K, Noto T, Takahashi A, Tabata K, Suzuki T & Akihisa T. 2011 Cytotoxic and Apoptosis-Inducing Activities of Limonoids from the Seeds of Azadirachta indica (Neem). J Nat Prod 74 866–870
Kumar S, Suresh PK, Vijayababu MR, Arunkumar A & Arunakaran J 2006 Anticancer effects of ethanolic neem leaf extract on prostate cancer cell line (PC-3). J Ethnopharmacol 105 246-250
Kumaraguruparan R, Subapriya R, Viswanathan P & Nagini S 2002 Tissue lipid peroxidation and antioxidant status in patients with adenocarcinoma of the breast. Clin Chim Acta 325 165-170
Levesque R 2007 SPSS Programming and Data Management: A Guide for SPSS and SAS Users. Fourth Edition, SPSS Inc, Chicago, III
Lillie RD 1976 Histopathologic technique. Practical histochemistry 95 851-859
Maha G, Bhuvaneshwari V, Amsaven R, Ragavendran P & Kalaiselvi M 2015 Antioxidant and antibacterial activity from whole plant of Eclipta Alba (l)- an in vitro model. Int J Biosci Nanosci 2 1-8
Mallick A, Ghosh S, Banerjee S, Majumder S, Das A, Mondal B, Barik S, Goswami KK, Pal S, Laskar S, Sarkar K, Bose A & Baral R. 2013 Neem leaf glycoprotein is nontoxic to physiological functions of Swiss mice and Sprague Dawley rats: histological, biochemical and immunological perspectives. Int Immunopharmacol 15 73-83
Markmee S & Saiin C 2003 Isolation and Structure Modification of Biologically Active Compound Nimbolide from Azadiracthta indica A Juss Var siamensis Valeton. Naresuan University Journal 11 45-49
Mazumdar UK, Gupta M, Maiti S & Mukherjee D 1997 Antitumor activity of Hygrophila spinosa on Ehrlich ascites carcinoma and sarcoma-180 induced mice. Indian J Exp Biol 35 473-437
McLimans WF, Davis EV, Glover FL & Rake GW 1957 The submerged culture of mammalian cells. The Spinner Culture. J Immunol 79 428-433
Meier J & Theakston RD 1986 Approximate LD50 determination of snake venuoms using eight to ten experimental animals. Toxicon 24 395-401
Montgomery HC & Dymock JF 1961 The determination of nitrite in water. Analyst 86 414-416
More YW, Raut RR & Taware AS 2015 Review on Biological Activities of Azadirachta. IJIFR 2 1327- 1334
Murray RL & Kaplan A 1984 Creatinine. Clinical Chemistry, St Louis Toronto Princeton, 1261-1266, 418
Naundri S, Thunuguntla SSR, Nyavanandi VK, Kasu S, Kumar PM, Ram PS, Rajagopal S, Kumar RA, Deevi DS, Rajagopalan R & Venkateswarlu A 2003 Biological investigation and structure-activity relationship studies on azadirone from Azadirachta indica A Juss. Bioorg Med Chem Lett 13 4111-4115
Neto CC 2007 Cranberry and its phytochemicals: a review in vitro anticancer studies. J Nutr 137 186S–193S
Nguyen TT, Tran E, Nguyen TH, Do PT, Huynh TH & Huynh H. 2004 The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis 25 647-659
Nnenna OP & Okey AA 2013 Toxicity and nutritional assessment of aqueous Azadirachta indica (neem) leaf extract in broiler chicks. IJB 3 172-180
Paglia DE & Valentine WN 1967 Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70 158-169
Pati HN, Das U, Kawase M, Sakagami H, Balzarini J, De Clercq E & Dimmocka JR 2008 1-Aryl-2-dimethylaminomethyl-2-propen-1-one hydrochlorides and related adducts: A quest for selective cytotoxicity for malignant cells. Bioorg Med Chem 16 5747-5753
Phillips DR, Rasbery JM, Bartel B & Matsuda SP 2006 Biosynthetic diversity in plant triterpene cyclization. Curr Opin Plant Biol 9 305-314
Porter AG & Jänicke RU 1999 Emerging roles of caspase-3 in apoptosis. Cell Death Differ 6 99-104
Reitman S & Frankel S 1957 A colorimetric method for the determination of serum glutamic oxalacetic and glutamic-pyruvic transaminases. Am J Clin Pathol 28 56-63
Rojanapo W, Suwanno S, Somjaree R, Glinsukon T & Thebtaranont Y 1985 Mutagenic and antibacterial activity testing of nimbolide and nimbic acid. J Sci Soc Thailand 11 177-181
Roy MK, Kobori M, Takenaka M, Nakahara K, Shinmoto H, Isobe S & Tsushida T 2007 Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica). Phytother Res 21 245–250
Sánchez-Alcázar JA, Ault JG, Khodjakov A & Schneider E 2000 Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells. Cell Death Differ 7 1090-1100
Satoh K 1978 Serum Lipid Peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 90 37-43
Schluster VL & Seldin DW 2004 Renal clearance. Seldin DW, Giebisch G, Eds The Kidney: Physiology and Pathology. Raven Press; 1985, pp 365–395
Schottenfeld D 2015 Causes of cancer. Ann Epidemiol 25 145-216
Schumacker PT 2015 Reactive Oxygen Species in Cancer: A Dance with the Devil. Cancer Cell 27 156-157
Subapriya R, Bhuvaneswari V, Ramesh V & Nagini S 2005 Ethanolic leaf extract of neem (Azadirachta indica) inhibits buccal pouch carcinogenesis in hamsters. Cell Biochem Funct 23 229-238
Subapriya R & Nagini S 2003 Ethanolic neem leaf extract protects against N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in Wistar rats. Asian Pac J Cancer Prev 4 215-223
Subapriya R & Nagini S 2005 Medicinal properties of neem leaves: a review. Curr Med Chem Anticancer Agents 5 149-156
Tafani M, Karpinich NO, Hurster KA, Pastorino JG, Schneider T, Russo MA & Farber JL 2002 Cytochrome c release upon Fas receptor activation depends on translocation of full-length bid and the induction of the mitochondrial permeability transition. J Biol Chem 277 10073-10082
Wong RS 2011 Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 30 87
Yoshikawa T & Naito Y 2002 What Is Oxidative Stress? JMAJ 45 271-276
Refbacks
- There are currently no refbacks.
Copyright © 2021 Journal of Molecular Biochemistry