DMBA) Induced Breast Cancer in Wistar Rats Mohammad Kashif1, Avinash Kumar Singh2, Harmandeep Singh3, Arya Vidyadhari4, Kanchan Kohli4, Manju Sharma5 1

JNROnline Journal Journal of Natural Remedies ISSN: 2320-3358 (e) Vol. 21, No. 7(S3), (2020) ISSN: 0972-5547(p)

Protective effect of Diosgenin for treatment of Breast Cancer in 7,12 Dimethyl benz anthracene (DMBA) induced breast cancer in Wistar rats Mohammad Kashif1, Avinash Kumar Singh2, Harmandeep Singh3, Arya Vidyadhari4, Kanchan Kohli4, Manju Sharma5 1. Faculty of Pharmacy, Sachchidanand Sinha College, Aurangabad, Bihar-824102 2. Department of Pharmaceutical Medicine (Division of Pharmacology), School of Pharmaceutical Education And Research, Jamia Hamdard, New Delhi-110062 3. Senior Clinical Research Associate, Department of Clinical Research, Ankura, Gurugram, Haryana 4. Department of Pharmaceutics, School of Pharmaceutical Education And Research, Jamia Hamdard, New Delhi-110062 5. Department of Pharmacology, School of Pharmaceutical Education And Research, Jamia Hamdard, New Delhi-110062 Corresponding author: 1. Dr. Manju Sharma. 2. Prof. Kanchan Kohli. Abstract Breast cancer is considered a major health problem causing death in women around the world and is ranked second most common carcinoma. Millions of women are diagnosed with breast cancer every year, and more than half million women die annually due to this disease. So this work was designed to detect the effect of diosgenin on breast cancer. The present study aimed to unveil the pharmacological and medicinal effects of diosgenin in 7,12-dimethylbenzanthracene (DMBA)- induced mammary carcinoma in wistar rats. The diosgenin was subjected to in vivo antioxidant and anticancer studies in various concentrations in wistar rats. We have measured the changes in body weight, along with other tumor parameters such as tumor volume, tumor weight, and serum biochemical parameters of breast tissue. As the oxidative stress further enhances the development of cancer, the antioxidant property of the diosgenin against cancer treatment. Also, the normal liver and kidney functioning revealed the non-toxicity nature of the diosgenin. Diosgenin could be one of the effective naturally obtained anti-breast cancer medications. Keywords: diosgenin, 7,12-dimethylbenzanthracene, breast cancer, antioxidant, liver, kidney Introduction Breast cancer is one of the most common forms of female cancers and is currently considered the leading cause of death among women (accounting for 35% of all cancers and 20% of all cancer deaths) worldwide. Excluding cancers of the skin, breast cancer is the most frequently diagnosed cancer in women(1). In a high percentage of cases, it proves to be hormone-dependent because tumor progression is dependent on high levels of circulating estrogens, which play a critical role in cancer cell proliferation. It has been estimated that 5-10% of breast cancer cases result from inherited mutations or alterations in breast cancer antigen (BRCA-1 and BRCA-2). In many of the developed countries, notification of cancer cases is compulsory for every hospital. However, in most developing countries including India, the provision of information is voluntary. Cancer incidence rates in Indian women are about half the experience of the USA and European women. The Indian Council of Medical Research (ICMR) registry records 1, 00,000 every year as new cases in Indian women and 40% of them die either of late detection or shyness to get examined (ICMR report 2008)(2-4). Diosgenin is a steroidal saponin, which is found in a variety of plants including fenugreek (Trigonella foenum graecum), roots of wild yam (Dioscorea villosa), Solanum incaunm, and Solanum xanthocarpum(5). Diosgenin has been reported to arrest the growth of HER2 oncoprotein-overexpressing AU565 human breast adenocarcinoma cells at the sub-G1 phase. It also inhibits the growth of MCF-7 human breast cancer cells in vitro, and it provides the mechanism for the anticancer activity(6). Diosgenin caused a cell cycle arrest associated with strong apoptosis in vitro(7-9). These studies support the use of Diosgenin for breast cancer

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chemoprevention due to its influence in suppressing the growth of breast cells without significant toxicity. Based on these findings preclinical evaluation of diosgenin for treatment of breast cancer has been evaluated in the present study. Methodology Experimental animals Female Wistar rats of 43 days were obtained. The animals were housed in an air-conditioned room with a 12h light and dark cycle in the animal house, Poona College of Pharmacy, Pune. The animals were acclimatized to the laboratory conditions for a week before the experimentation and were randomly divided into six groups of eight animals each. They were kept in standard polycarbonate cages, with paddy husk as the bedding material. The animals were fed with standard rodent pellets and tap water was freely accessible ad libitum to the animal throughout the study except during fasting, before the study, the experimental protocol was submitted to the CPCSEA/ IAEC and got approved for 48 animals by the IAEC of Bharati Vidyapeeth University Poona College of Pharmacy, with registration No. 1703/PO/Re/S/01/CPCSEA. Source of carcinogen (DMBA) For the induction of mammary gland tumors in rats, 7, 12-Dimethylbenz anthracene (DMBA) was used. DMBA was procured from Sigma-Aldrich Corporation, Bangalore, India. (CAS Number: 57-97-6, C20H16, and Molecular Weight: 256.34). Preparation of DMBA 0.7 mg of DMBA was freshly dissolved in (0.1ml) of DMSO, and adjusted to pH 4.0 using 3% acetic acid, and maintained at 35- 40°C before administration. Administration of DMBA At the age of 50 days, mammary tumors were induced in the animals with DMBA following overnight fasting according to the methods of Thompson et al. and Rivera et al. with some modifications. DMBA was administered subcutaneously in four weekly doses of 7.5 mg/kg body weight each. Source of Treatment compound For the treatment of mammary gland tumors in rats, Diosgenin was used. Diosgenin was procured from Sigma-Aldrich Corporation Bangalore, India. (CAS Number: 512-04-9, C27H42O3, and Molecular Weight: 414.62. Diosgenin is a solid powder stored in dry, dark and at 0-4°C for short term (days to weeks) or -20°C for long term (months to years). It is soluble in DMSO. Preparation of Test compound Diosgenin was prepared by suspending in distilled water using DMSO & 0.5%v/v tween 80. Storage of Diosgenin Diosgenin was stored in the freezer and the fresh suspension was prepared every day for administration. Standard Drug (Vincristine) For the treatment of mammary gland tumors in rats of the standard group, vincristine was used. Vincristine was obtained from CIPLA LTD., B.NO. GE 70153. Vincristine was stored in the freezer and was administered at a dose of 0.5mg/kg i.p once a week for four weeks. Experimental design The animals were divided randomly into six groups with eight rats per group as follows:  Group I: Normal Control: Vehicle  Group II: DMBA Control: 7.5mg/kg of DMBA subcutaneously once a week for 4 weeks.  Group III: Standard (Vincristine), 7.5mg/kg of DMBA subcutaneously once a week for 4 weeks + (500μg/kg) of vincristine Weekly, ip from 8 weeks to 12 weeks.  Group IV: Diosgenin 10mg/kg, 7.5mg/kg of DMBA subcutaneously once a week for 4 weeks + (10mg/kg) of Diosgenin orally daily, from 8 weeks to 12 weeks.  Group V: Diosgenin 20mg/kg, 7.5mg/kg of DMBA subcutaneously once a week for 4 weeks + (20mg/kg) of Diosgenin orally, daily. from 8 weeks to 12 weeks.  Group VI: Diosgenin 40mg/kg, 7.5mg/kg of DMBA

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Journal of Natural Remedies Vol. 21, No.7(S3), (2020) subcutaneously once a week for 4 weeks + (40mg/kg) of Diosgenin orally daily, from 8 weeks to 12 weeks. Study Protocol Female Wistar rats of 43 days were obtained. The animals were housed in polycarbonate cages and acclimatized to standard animal house conditions for 1 week before the commencement of experimental procedures. After one week at the age of 50 days, rats were anesthetized with Thiopentone Sodium (35 mg/kg, i.p), and mammary gland tumor was induced by DMBA at a dose of 7.5 mg/kg subcutaneously. DMBA was administered once a week for four weeks. The animals were weighed weekly following DMBA administration. After three weeks of the first DMBA injection, their mammary glands were palpated twice per week for detection of tumors. The animals were examined with regular bodyweight for a further four weeks & the tumor was palpitated twice per week. After eight weeks the animals were randomly assigned into untreated and treated groups. The animals were treated with Diosgenin by oral gavage once daily for four weeks at a dose of 10,20,40mg/kg. Response to the treatment was assessed weekly by the measurement of animal body weight and physical tumor growth parameters (tumor number, size, and volume). Animals were euthanized after treatment. Blood was collected by cardiac puncture and aliquoted into dipotassium ethylenediamine tetra-acetic acid (K2EDTA) vials for assessment of full blood count (FBC) and into plane tube gels (SST II advance) for collection of serum used for the assessment of parameters for hepatic and kidney injury. Biochemical parameters for liver and kidney function impairment were analyzed using automated chemistry analyzer (Architect c8000; Abbott Park, USA) according to the manufacturer’s guidelines. The parameters measured were aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine. FBC parameters were assessed using automated hematology analyzer XE-5000 (Sysmex Asia Pacific, Pte Ltd, Singapore) according to the manufacturer’s guidelines. The parameters measured include total red blood cell (RBC) count, hemoglobin (Hb) content, packed cell volume (PCV) and red cell indices [i.e., mean corpuscular volume (MCV)] count were also measured. MDA, GSH, and SOD were measured using commercial spectrophotometric analysis kits (Bio- Diagnostic Company, Delhi, India). Statistical Analysis Statistical analysis was evaluated by using one-way analysis of variance (ANOVA) with posthoc test using Bonferroni multiple comparisons in the PASW program (version 18) for Windows (SPSS Inc. Chicago, IL, USA). The data were reported as the mean ± S.E.M.; a probability value less than 0.05 was considered significant. Results Effect of Diosgenin in the Bodyweight in DMBA induced breast cancer in rats At the end of the study, the body weight was found to be significantly p<0.001 decreased in the DMBA control group as compared to the normal control group. In the diosgenin-treated group (20 and 40mg/kg), the body weight was found to be significantly (p<0.001) increased as compared to the DMBA control group (Figure 1). Body wt 200 Normal cont. *** ### *** *** DMBA Cont. 150 Std.Vincri(0.5mg/kg) Diosgenin (10mg/kg) 100 Diosgenin (20mg/kg) Diosgenin (40mg/kg)

Body wt(gm) Body 50

Normal cont.DMBA Cont.

Std.Vincri(0.5mg/kg)DiosgeninDiosgenin (10mg/kg)Diosgenin (20mg/kg) (40mg/kg) Groups Figure 1: Effect of Diosgenin in the body weight in DMBA induced breast cancer in rats.

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Values are represented as mean± SEM (n=8). Data were analyzed by using Two Way ANOVA followed by Bonferonni post-test. ###p<0.001 as compared to a normal group, ***p<0.001 as compared to DMBA control group. Effect of Diosgenin in the Tumor size in DMBA induced breast cancer in rats At the end of the study, the tumor size was found to be significantly p<0.01 decreased in the diosgenin treated group (20 and 40mg/kg) as compared to the DMBA control group (Figure 2). Tumor size 8 DMBA Cont. Std. Vincri.(0.5mg/kg) 6 Diosgenin (10mg/kg) * * ** Diosgenin (20mg/kg) 4 Diosgenin (40mg/kg)

2 tumor size (mm) tumor size

DMBA Cont.

Std. Vincri.(0.5mg/kg)Diosgenin (10mg/kg)Diosgenin (20mg/kg)Diosgenin (40mg/kg) Groups Figure 2: Effect of Diosgenin in the tumor size in DMBA induced breast cancer in rats. Values are represented as mean± SEM (n=8). Data were analyzed by using Two Way ANOVA followed by Bonferroni post-test. **p<0.01 as compared to the DMBA control group. Effect of Diosgenin in serum AST, ALT, Urea and Creatinine level in DMBA induced breast cancer in the rat At the end of the study, the serum AST level was found to be significantly (p<0.001) increased in the DMBA control group as compared to the normal group. In the diosgenin-treated group (10, 20, and 40mg/kg), the AST level was found to be significantly (p<0.001) decreased as compared to the DMBA control group. At the end of the study, the serum ALT level was found to be significantly (p<0.001) increased in the DMBA control group as compared to normal control. In diosgenin treated group (20 and 40mg/kg), the ALT level was found to be significantly (p<0.001) decreased as compared to DMBA control rats. At the end of the study, the serum Urea was found to be significantly changed in the DMBA control group as compared to the normal control group. In the diosgenin-treated group (10,20 and 40mg/kg), the serum urea was significantly (p<0.001) decreased as compared to the DMBA control group. At the end of the study, the serum Creatinine was found to be significantly p<0.001 increased in the DMBA control group as compared to the normal control group. In the diosgenin-treated group (20 and 40mg/kg), the serum creatinine level was found to be significantly (p<0.001) decreased as compared to the DMBA control group (Table 1). Table 1: Effect of diosgenin in serum AST, ALT, Urea, and Creatinine level in DMBA induced breast cancer in rats.

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Effect of Diosgenin in Haemoglobin, R.B.C, total WBC count, PCV, MCV in DMBA induced breast cancer in rats At the end of the study, the hemoglobin was found to be significantly (p<0.001) decreased in the DMBA control group as compared to the normal control group. In the diosgenin treated group (10, 20, and 40 mg/kg), hemoglobin was found to be significantly (p<0.001) increased as compared to the DMBA control group. However, the hemoglobin in standard vincristine was found to be significantly decreased than DMBA control. The result indicates bone marrow suppression by vincristine. Furthermore, treatment with diosgenin at all the dose levels increased the hemoglobin, level which indicates there was no bone marrow suppression with diosgenin. At the end of the study, the serum R.B.C was found to be significantly (p<0.001) decreased in the DMBA control group as compared to a normal control group. In the diosgenin treated group (10, 20, and 40mg/kg), the serum R.B.C. was found to be significantly (p<0.001) increased as compared to the DMBA control group. However, the serum R.B.C in standard vincristine was found to be significantly decreased than DMBA control. The result indicates bone marrow suppression by vincristine. Furthermore, treatment with diosgenin at all the dose levels increased the serum R.B.C level which indicates there was no bone marrow suppression with diosgenin. At the end of the study, the total WBC count was found to be significantly (p<0.001) increased in the DMBA control group as compared to the normal control group. In the diosgenin treated group (20 and 40mg/kg), the total WBC count was found to be significantly (p<0.001) decreased as compared to the DMBA control group. At the end of the study, the PCV was found to be significantly (p<0.001) decreased in the DMBA control group, as compared to the normal control group. In the diosgenin treated group (10, 20, and 40mg/kg), PCV was found to be significantly (p<0.001) increased as compared to the DMBA control group. At the end of the study, the MCV was found to be significantly (p<0.001) increased in the DMBA control group as compared to the normal control group. In the diosgenin-treated group (10, 20, and 40mg/kg), the PCV was found to be significantly (p<0.001) decreased as compared to the DMBA control group (Table 2). Table 2: Effect of Diosgenin in Haemoglobin, R.B.C, total WBC count, PCV, MCV in DMBA induced breast cancer in rats

Values are represented as mean± SEM (n=8). Data were analyzed using One Way ANOVA followed by Dunnett’s test. ###p<0.001 as compared to the normal group, ***p<0.001 as compared to DMBA control group. Effect of Diosgenin in the MDA, SOD, GSH level in DMBA induced breast cancer in rats At the end of the study, the MDA level was found to be significantly (p<0.001) increased in the DMBA control group as compared to the normal control group. In the diosgenin treated group (10, 20, and 40mg/kg), the MDA was found to be significantly (p<0.001) decreased as compared to the DMBA control group. At the end of the study, the SOD level was found to be significantly(p<0.05) decreased in the DMBA control group as compared to the normal control group. In the diosgenin treated group (10, 20, and 40mg/kg), the SOD level was found to be nonsignificant as compared to the DMBA control group. At the end of the study, the GSH level

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Journal of Natural Remedies Vol. 21, No.7(S3), (2020) was found to be significantly p<0.001 decreased in the DMBA control group as compared to the normal control group. In the diosgenin treated group (10, 20, and 40mg/kg), the GSH level was found to be significantly (p<0.001) increased as compared to the DMBA control group (Table 3). Table 3: Effect of Diosgenin in the MDA, SOD, GSH level in DMBA induced breast cancer in rats

Values are represented as mean± SEM (n=8). Data were analyzed using One Way ANOVA followed by Dunnett’s test. ###p<0.001 as compared to the normal group, ***p<0.001 as compared to DMBA control group. Discussion Breast cancer is one of the most common forms of female cancer and is currently considered the leading cause of death among women (accounting for 35% of all cancers and 20% of all cancer deaths) worldwide. To study mammary carcinogenesis, DMBA (chemical carcinogens) induced rats are widely used as a model. In the present study, chemopreventive efficacy was evaluated using diosgenin against DMBA induced mammary gland tumorogenesis in female Wistar rats. Currently, there is a growing interest in the use of phytocompounds to develop safe and more effective therapeutic agents for cancer treatment throughout the world(10). This is due to the unfavorable side effects and resistance of many anticancer agents that have been developed are severe problems. The mechanism of action of many anticancer drugs is based on their ability to induce apoptosis(11, 12). Animal body weight has been observed on weekly basis in all the groups throughout the study at the end of the study the body weight was found to be significantly p<0.001 decreased in the DMBA control group as compared to the normal control group, due to the toxic effect of DMBA. In the diosgenin treated group the body weight was found to be significantly (p<0.001) increased as compared to the DMBA control group(13-16). For DMBA induced rats, the tumor size has been found to increase with time. The diosgenin- treated group showed a reduction in the tumor size during four weeks treatment period. A similar trend has been found in cell growth inhibition ability. The lifespan of the DMBA induced rats increased remarkably when treated with the test compound (diosgenin). The prolongation of the lifespan of cancer-bearing rats is a very important and reliable criterion for judging the potency of any drug as an anticancer agent(17). AST and ALT are the pathophysiological markers used to find tissue damage. Tissue damage or inflammation leads to leakage of these enzymes from liver cytosol to the bloodstream. The higher level of the liver enzymes present in the DMBA control group due to the toxic effect of DMBA in the liver. Whereas, the diosgenin-treated group showed a decreased level of AST and ALT compared to DMBA control rats(18-20). The serum creatinine is a specific indicator of normal kidney function. A higher level of creatinine in serum represents abnormalities. In the present study, the level of serum creatinine in DMBA control rats was higher due to the toxic effect of DMBA and these were significantly (p<0.001) reduced after treatment with diosgenin(21). Hematological parameters were found to be altered from normal values along with treatment of test compounds. Hemoglobin content, RBC counts were found to be increased and WBC counts

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Journal of Natural Remedies Vol. 21, No.7(S3), (2020) were found to be decreased during the treatment period. The decrease in RBC and hemoglobin content result from the hemolyzing power of the compounds. The increase in WBC count can be correlated with an increase in antibody production which helps in the survival and recovery of the animals. After the treatment period with a test compound, it was observed that the parameters were restored almost towards normal. The effects of the test compound on hematological parameters(22, 23). The low level of hemoglobin in the DMBA control group is due to the toxic effect of DMBA. Whereas, in the present study the diosgenin-treated group showed an increased level of hemoglobin as compared to DMBA control rats. The result indicates bone marrow suppression by vincristine. Furthermore, treatment with diosgenin at all the dose levels increased the hemoglobin level which indicates there was no bone marrow suppression with diosgenin. In lipid peroxidation, oxidative stress is known to be involved in carcinogenesis and higher levels of their products play a role in the early phases of tumor growth(24). Also in patients with breast cancer have increased MDA levels which in turn act as indicators of lipid peroxidation when compared to normal. In the present study, DMBA induced rats showed increased activity of lipid peroxidation level. The diosgenin treated rats significantly (p<0.001) altered the lipid peroxidation level and reversed near to normal level, thus indicating its anti-lipo peroxidative property(25, 26). GSH serves as a marker for evaluation of oxidative stress and it acts as an antioxidant at both extracellular and intracellular levels it was observed decreased activity of GSH in the DMBA control group(27). The diosgenin-treated group increased the GSH levels, which suggest its antioxidant property. Conclusion In conclusion, the present study demonstrates that Diosgenin exerts inhibitory effects on DMBA- induced Breast Carcinoma. In the diosgenin-treated group, the body weight was found to be significantly increased as compared to the DMBA control group. Tumor size was found to be decreased in the diosgenin treated group during four weeks of the treatment period. The biochemical alterations were observed in the DMBA control group in the present study. However, administration of diosgenin significantly reversed the alteration to near-normal levels in cancer- bearing animals. From the results it can be concluded that diosgenin positively modulated the antioxidant activity by detoxifying the free radicals induced by DMBA, considering the antioxidant property of diosgenin. References 1. Altun A, Saraydin SU, Soylu S, Inan DS, Yasti C, Ozdenkaya Y, et al. Chemopreventive effects of hydatid disease on experimental breast cancer. Asian Pacific journal of cancer prevention : APJCP. 2015;16(4):1391-5. 2. Alvarado A, Lopes AC, Faustino-Rocha AI, Cabrita AMS, Ferreira R, Oliveira PA, et al. Prognostic factors in MNU and DMBA-induced mammary tumors in female rats. Pathology, research and practice. 2017;213(5):441-6. 3. Gupta G, Kazmi I, Afzal M, Rahman M, Saleem S, Ashraf MS, et al. Sedative, antiepileptic and antipsychotic effects of Viscum album L.(Loranthaceae) in mice and rats. Journal of ethnopharmacology. 2012;141(3):810-6. 4. Gupta G, Sharma RK, Dahiya R, Mishra A, Tiwari J, Sharma GN, et al. Aphrodisiac activity of an aqueous extract of wood ear mushroom, Auricularia polytricha (Heterobasidiomycetes), in male rats. International journal of medicinal mushrooms. 2018;20(1). 5. Avtandilyan N, Javrushyan H, Mamikonyan A, Grigoryan A, Trchounian A. The potential therapeutic effect of N(G)-hydroxy-nor-L-arginine in 7,12-dimethylbenz(a)anthracene-induced breast cancer in rats. Experimental and molecular pathology. 2019;111:104316. 6. Baltaci SB, Mogulkoc R, Baltaci AK, Emsen A, Artac H. The effect of zinc and melatonin supplementation on immunity parameters in breast cancer induced by DMBA in rats. Archives of physiology and biochemistry. 2018;124(3):247-52. 7. Bishayee A, Mandal A, Thoppil RJ, Darvesh AS, Bhatia D. Chemopreventive effect of a novel oleanane triterpenoid in a chemically induced rodent model of breast cancer. International journal of cancer. 2013;133(5):1054-63.

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8. Gupta G, de Jesus Andreoli Pinto T, Chellappan DK, Mishra A, Malipeddi H, Dua K. A clinical update on metformin and lung cancer in diabetic patients. Panminerva medica. 2018;60(2):70-5. 9. Hatware KV, Sharma S, Patil K, Rajput H, Gupta G. Therapeutic role of natural agents in the management of coronary artery disease: a review. Journal of Environmental Pathology, Toxicology and Oncology. 2020;39(2). 10. Brennan MJ. Endocrinology in cancer of the breast. Status and prospects. American journal of clinical pathology. 1975;64(6):797-809. 11. Gupta G, Krishna G, Chellappan DK, Gubbiyappa KS, Candasamy M, Dua K. Protective effect of pioglitazone, a PPARγ agonist against acetaminophen-induced hepatotoxicity in rats. Molecular and cellular biochemistry. 2014;393(1-2):223-8. 12. Liu X, Sharma RK, Mishra A, Chinnaboina GK, Gupta G, Singh M. Role of aqueous extract of the wood ear mushroom, auricularia polytricha (agaricomycetes), in avoidance of haloperidol-lnduced catalepsy via oxidative stress in rats. International journal of medicinal mushrooms. 2019;21(4). 13. De Oliveira Andrade F, Yu W, Zhang X, Carney E, Hu R, Clarke R, et al. Effects of Jaeumkanghwa-tang on tamoxifen responsiveness in preclinical ER+ breast cancer model. Endocrine-related cancer. 2019;26(3):339-53. 14. Devlin KL, Sanford T, Harrison LM, LeBourgeois P, Lashinger LM, Mambo E, et al. Stage-Specific MicroRNAs and Their Role in the Anticancer Effects of Calorie Restriction in a Rat Model of ER-Positive Luminal Breast Cancer. PloS one. 2016;11(7):e0159686. 15. Gupta G, Singhvi G, Chellappan DK, Sharma S, Mishra A, Dahiya R, et al. Peroxisome proliferator-activated receptor gamma: promising target in glioblastoma. Panminerva medica. 2018;60(3):109-16. 16. Kazmi I, Afzal M, Rahman M, Gupta G, Anwar F. Aphrodisiac properties of Polygonatum verticillatum leaf extract. Asian Pacific Journal of Tropical Disease. 2012;2:S841-S5. 17. Hamdy SM, Latif AK, Drees EA, Soliman SM. Prevention of rat breast cancer by genistin and selenium. Toxicology and industrial health. 2012;28(8):746-57. 18. Hilakivi-Clarke L, Clarke R, Lippman ME. Perinatal factors increase breast cancer risk. Breast cancer research and treatment. 1994;31(2-3):273-84. 19. Madhu A, Gupta G, Arali B, K Chellappan D, Dua K. Anti-psychotic activity of aqueous root extract of Hemidesmus indicus: a time bound study in rats. Recent patents on drug delivery & formulation. 2017;11(1):36-41. 20. Rajeshkumar S, Menon S, Kumar SV, Tambuwala MM, Bakshi HA, Mehta M, et al. Antibacterial and antioxidant potential of biosynthesized copper nanoparticles mediated through Cissus arnotiana plant extract. Journal of Photochemistry and Photobiology B: Biology. 2019;197:111531. 21. Kabel AM, Elkhoely AA. Ameliorative potential of fluoxetine/raloxifene combination on experimentally induced breast cancer. Tissue & cell. 2016;48(2):89-95. 22. Prasher P, Sharma M, Mudila H, Gupta G, Sharma AK, Kumar D, et al. Emerging trends in clinical implications of bio-conjugated silver nanoparticles in drug delivery. Colloid and Interface Science Communications. 2020;35:100244. 23. Rawat S, Pathak S, Gupta G, Singh SK, Singh H, Mishra A, et al. Recent updates on daidzein against oxidative stress and cancer. EXCLI journal. 2019;18:950. 24. Karnam KC, Ellutla M, Bodduluru LN, Kasala ER, Uppulapu SK, Kalyankumarraju M, et al. Preventive effect of berberine against DMBA-induced breast cancer in female Sprague Dawley rats. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2017;92:207-14. 25. Sharma S, Pathak S, Gupta G, Sharma SK, Singh L, Sharma RK, et al. Pharmacological evaluation of aqueous extract of syzigium cumini for its antihyperglycemic and antidyslipidemic properties in diabetic rats fed a high cholesterol diet—Role of PPARγ and PPARα. Biomedicine & Pharmacotherapy. 2017;89:447-53.

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26. Singh Y, Samuel VP, Dahiya S, Gupta G, Gillhotra R, Mishra A, et al. Combinational effect of angiotensin receptor blocker and folic acid therapy on uric acid and creatinine level in hyperhomocysteinemia‐associated hypertension. Biotechnology and applied biochemistry. 2019;66(5):715-9. 27. Kilańczyk E, Gwoździński K, Wilczek E, Filipek A. Up-regulation of CacyBP/SIP during rat breast cancer development. Breast cancer (Tokyo, Japan). 2014;21(3):350-7.

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