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Clerodendrum Thomsoniae Balf.F. on DMBA-Induced Breast Cancer in Female Sprague-Dawley Rats

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Clerodendrum Thomsoniae Balf.F. on DMBA-Induced Breast Cancer in Female Sprague-Dawley Rats Original Article Antiproliferative Potential of Ethyl acetate Extract of Clerodendrum thomsoniae Balf.f. on DMBA-induced Breast Cancer in Female Sprague-dawley Rats VK Muhammed Ashraf1,*, VK Kalaichelvan1, R Ragunathan2, VV Venkatachalam1 1Department of Pharmacy, Annamalai University, Annamalai Nagar, Tamil Nadu, INDIA. 2Centre for Bioscience and Nanoscience Research, Coimbatore, Tamil Nadu, INDIA. ABSTRACT Background: Clerodendrum is a genus of around 500 species that has a place with the family Lamiaceae. Numerous types of this class have been demonstrated for the treatment of different maladies including cancer. This study aimed to investigate the anti-cancer activity of ethyl acetate fractions of Clerodendrum thomsoniae Balf.f. (EACT) in female Sprague-Dawley rats. Methods: 7, 12-dimethylbenz (a) anthracene (DMBA) was used to induce breast cancer. Two doses of (25 and 50 mg/kg) EACT were used for the study. Various biochemical, hematological and, antioxidant parameters were analyzed. Results: Reduction in mean tumor volume and tumor weight was observed in EACT treated groups. The blood parameters were seen as normal. In both DMBA and doxorubicin groups, malondialdehyde were increased and the level was significantly reduced in EACT-treated groups. The effect of catalase has been seen as reduced in the groups administered by DMBA and doxorubicin and normal in the EACT groups. In the DMBA control groups, nitrate and nitrite were found to increase, the levels in others were normal. There were less necrosis and infiltration in breast tissues treated with doxorubicin as well as in EACT. The curative effect was found to be dose-dependent in animals treated with EACT. Some of the damaged breast patterns were restored to normal by the treatments. GC-MS analysis confirmed 2, 4-bis(1-phenylethyl)-phenol (39%), which was found to be the major compound present in EACT. Conclusion: The study concludes that C. thomsoniae may be a potential anticancer candidate. Key words: Clerodendrum thomsoniae Balf.f., 2, 4-bis(1-phenylethyl)-phenol, Breast cancer, EACT, Antiproliferative potential. Submission Date: 03-09-2020; INTRODUCTION Revision Date: 27-10-2020; Accepted Date: 20-01-2021 Breast malignant growth is one of the most mammary glands of a few rodent strains, DOI: 10.5530/ijper.55.1.23 common and significant diseases among predominantly Sprague-Dawley and Wistar Correspondence: ladies, representing 25% (1.7 million) of rats are more sensitive chemical carcinogens Mr. Muhammed Ashraf VK recent cases and 15% of cancer mortality.1 and two most generally utilized breast cancer Department of Pharmacy, Annamalai University, Breast cancer is described by extreme cell inducers are 7,12-dimethylbenz(a)anthracene Chidambaram-608002, Tamil proliferation, wrong regulation of cellular (DMBA) and N-methylnitrosurea.6,7 Nadu, INDIA. Phone no: +91 9747313918 2 differentiation and inadequate apoptosis. The tumor incited by this model is Email id: ashrafvkclt@gmail. However, numerous studies in the last morphologically and histologically like com decade have shown that inflammation and that saw in human estrogen-subordinate angiogenesis are fundamental for primary dependent breast cancer.8 Several cancer, including breast cancer, development, markers for chemoprevention have been invasion and metastases.3-5 Chemically designed. These depend on the way that incited rat models of mammary malignancy chemopreventive drugs can apply their have been widely utilized for many years to anti-carcinogenic impacts by one or more imitate human breast carcinogenesis. The combination mechanism such as hindering www.ijper.org Indian Journal of Pharmaceutical Education and Research | Vol 55 | Issue 1 | Jan-Mar, 2021 205 Ashraf, et al.: Anticancer Potential of Clerodebdrum thomsoniae Balf.f. on Rats the arrangement of reactive carcinogenic metabolites, Collection and identification of plant material promoting enzymes that detoxify cancer-causing agents, Aerial parts of C. thomsoniae were obtained locally from scavenging reactive oxygen species, affecting apoptosis Calicut district (Kerala, India). Department of Botany, 9,10 and repressing cell proliferation. University of Calicut (Calicut, India) identified the plant The earliest chemotherapy approach involves single materials and authenticated them. Voucher specimens alkylating and antimetabolite therapies which have now as specimen No.148249 were deposited in the same been replaced by combination treatment since it has herbarium department. been shown that two or more drugs have been used together for improved outcomes.11 The free radicals Selection of experimental animals play a crucial function in the development of tumors Sprague–Dawley rats (Female, 125-175 g body weight) through direct chemical reactions or modifications of were used and kept in the animal house of the cellular metabolic pathways and their scavengers can pharmacology department at K.M. College of Pharmacy, inhibit cancer cells at different phases of carcinogenesis. Tamil Nadu (India). The experiments were planned and The toxicity of reactive oxygen (ROS), by the hindrance executed in compliance with ethical standards approved of ROS production, may be controlled by antioxidants.12 by the Institutional Animal Ethical Committee The modern management or treatment of breast cancer (IAEC) of the same college under reference number consists of surgery, chemotherapy and radiotherapy IAEC/MUHAMMED ASHRAF VK/Ph.D/AU/ also hormone and anti-Her-2 therapies.13,14 Even 1661130022/KMCP/91/2020. The selected rats were though the immense alternatives of treatment and the kept in plastic cages (34×47×18 cm3) at the institutional innovations in breast malignancy treatment the remedial animal house, in a cooled situation with five mice in methodology at present still produces many reactions, every cage and kept up at room temperature of (25 ± 2)°C which directly influence in the eminence of life and the with relative humidity (60% ± 10%) under 12 h light accomplishment of the treatment.15 As a result, the search and dark cycle. All the animals were given easy access for remedies that could ease the side effects, alone or in to standard food and water ad libitum and relocated to combination with drugs previously consumed in breast laboratory environments before the test. cancer management, is a necessary step to upgrade the control of the disease. Preparation and extraction of the plant material Clerodendrum thomsoniae Balf.f. is a twined, rambling, The aerial parts of C. thomsoniae were dried appropriately wine-like shrub native to the tropical part of West in the shade for approximately three weeks, segregated, Africa and the collective name is Bleeding-heart or powdered by a mechanical grinder and, then passed Bag-flower.16,17 They have beautiful white flowers and through a 40 mesh sieve. About 1 kg of air-dried plant are available in a different part of the world because of material was extracted with ethyl acetate in the soxhlet ornamental demand. These species are not extensively assembly. A rotary vacuum evaporator was used to studied for pharmacological studies, even though many concentrate the extract. Weighed the extract obtained plants listed under genus Clerodendrum are well studied and measured the percentage yield in terms of the for various pharmacological studies including cancer. dry weight of the plant material. The extract was also According to the data available, the species C. thomsoniae labeled with color and consistency. For this whole job, have phytoconstituents with excellent free radical all solvents were of analytical rank (Merck, Mumbai). scavenging properties and are useful in multiple Fractionation of extract conditions such as sores, cuts, bruises and skin rashes.18 The target of this study was to explore the anticancer Column chromatography using Silica Gel (60-120 activity of ethyl acetate fractions of C. thomsoniae mesh size) was employed to fractionate ethyl acetate (EACT) on female Sprague-Dawley rats using extracts. Fifty grams of the extract was submitted to chemically induced (DMBA) breast cancer model. flash chromatography utilizing silica gel (mesh size 60-120) as the stationary phase. The silica column was prepared using ethyl acetate by wet packing method and MATERIALS AND METHODS the column was washed using 100 ml of ethyl acetate. Chemicals For easy distribution of the sample, the ethyl acetate DMBA was supplied from Sigma Chemical Company extract had been blended and made of fine powder with St. Louis, MO, USA. All other chemical materials used silica gel. On top of the pre-packed silica column, the in this study are of analytical quality, brought from powdered sample mass was loaded. The elution was Himedia Private Laboratories Ltd., Mumbai, India. performed by utilizing solvent polarity at increasing 206 Indian Journal of Pharmaceutical Education and Research | Vol 55 | Issue 1 | Jan-Mar, 2021 Ashraf, et al.: Anticancer Potential of Clerodebdrum thomsoniae Balf.f. on Rats order made of hexane: ethyl acetate and methanol Drug administration began when tumors became mixtures. Every 10 ml of fractions were collected in vials stable and clearly palpable. and further analyzed by thin-layer chromatography. Seven General observations identical fractions (F1-F7) have been mixed, evaporated to dryness and kept in the dark for further analysis. All Throughout the experiment, the total changes in body fractions were tested for cytotoxicity by MTT assay by weight in the animals were recorded every week. Rats means of MCF-7
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