Evaluation of antioxidant, anti-inflammatory, and cytotoxic activities of Crotalaria pallida Aiton leaves

• Cite this article as: Chinnadhurai M^1*,
• Al-Otaibi F¹,
• Nelson K¹,
• Kandasamy G¹

1. ¹ Shanmugam M, Venkatnarayanan R. Protective Effect of Madhuca longifolia Leaves in 7, 12-Dimethylbenz(a)anthracene Induced Mammary Carcinoma in Sprague Dawley Rat model. Phcog Mag 2019;15:S396-401. MAHESWARI CHINNADHURAI, et al.: Protection of Madhuca longifolia in Mammary Carcinoma Pharmacognosy Magazine, Volume 15, Issue 66, October-December 2019 (Supplement 3) S397 accounting for almost one in four cancer cases among women.[1] Breast cancer develops in the lobules are called as lobular carcinoma and develops in cells lining of milk ducts are called as ductal carcinoma. Several studies have reported that 7, 12‑Dimethylbenz(a)anthracene (DMBA) is used to induce mammary carcinomas in rats. In the mammary gland, DMBA produces epoxides and active metabolites with a capacity for damaging the DNA molecule leading to carcinogenesis. With the higher cellular proliferative index of Types 1 and 2 lobules, there is higher metabolic activity and more epoxide formation.[2,3] Physicians and patients are in need of maximum therapeutic value with no or less side effects to improve the quality of the life of breast cancer patients. Several medicinal herbs constitute such a group. In recent years, many scientists have examined the effect of herbals used traditionally by herbalist and indigenous healers to treat different types of cancer. Several hundred plants have been studied for the treatment of different types of cancer. There have been only a handful of plants fairly well researched. After the extensive search of medicinal plant for the treatment of mammary carcinoma, the plant selected for the study is Madhuca longifolia, which comes under Sapotaceae family. It is a large‑sized tropical deciduous tree, grown up to 16–17 m height and distributed in India, and Nepal. Different parts of this plant were reported to contain sapogenins, steroids, saponins, flavonoids, tannins, β‑amyrin, betulinic acid, ethyl cinnamate, ursolic acid, stigmasterol, β‑carotene, xanthophylls, quercetin, dihyroquercetin, β‑sitosterol, sesquiterpene alcohol, triterpenoids, α‑terpineol, Mi‑saponin A and B, 3‑β‑monocaprylic ester of erythrodiol, myricetin, erythrodiol, and glycosides.[4,5] Leaves are used for chronic bronchitis, cancer, and Cushing’s disease.[6,7] Based on the literature review, there was no established work in the protective activity of Madhuca longifolia leaves in mammary carcinoma. Hence, the study was planned to evaluate the chemopreventive activity of Madhuca longifolia leaves in DMBA‑induced mammary carcinoma in rat. MATERIALS AND METHODS Drugs and chemicals DMBA and Vincristine were purchased from Sigma‑Aldrich Chemicals Pvt. Ltd., Bengaluru, India. All other chemicals used in the study were purchased from local sources and were of analytical grade. Collection, authentication, and extraction of plant The fresh leaves of Madhuca longifolia were collected from Sankarankovil, Tamil Nadu, India. The plant was authenticated by Botanical Survey of India, Coimbatore, and the preserved specimen of an identified plant has been kept in Pharmacognosy Department of R.V.S. College of Health Sciences, Coimbatore, India. Extraction After authentication, the fresh leaves of Madhuca longifolia were properly dried in shade for 2–3 weeks. It was reduced to fine particles in a blender, sieved, and used for the further experimental studies. About 2 kg of shade dried plant leaves of Madhuca longifolia was extracted in Soxhlet successively extracted with n‑hexane, chloroform, ethyl acetate, and methanol. Each extract was evaporated using rotary vacuum evaporator. The extracts collected from each solvent was weighed, and the percentage yield was calculated. The consistency and color of the plant leaf extracts were noted. Phytochemical analysis and free radical scavenging activity The n‑hexane, chloroform, ethyl acetate, and methanol extracts of the leaf powder of Madhuca longifolia were subjected to qualitative chemical analysis based on the method of Takeda et al.[8] Diphenylpicrylhydrazyl, superoxide and nitric oxide free radical scavenging activity were determined by the method of Sanchez‑Moreno et al.[9] Experimental animals and acute toxicity Adult female Sprague Dawley rats weighing 100–150 g were purchased from R.V.S. College of Health Sciences, Coimbatore, India. Animals were maintained in well‑ventilated housing conditions and fed with commercial rodent diet. They were provided with water ad libitum during the experiment. The Institutional Animals Ethics Committee (Register number 1012/c/06/CPCSEA) permitted the study. Acute toxicity study was done according to Organisation for Economic Co‑operation and Development guidelines 423.[10,11] Experimental design A total of thirty female Sprague Dawley rats were divided into five groups and each group consisting of six rats. Group I (vehicle‑treated control group) – administered with excipient (single dose of 1 mL of emulsion of sunflower oil and physiological saline) subcutaneously and 1 mL of 2% dimethyl sulfoxide per orally for 16 weeks. Group II (negative control group) – administered with single subcutaneous injection of 25 mg of DMBA in 1 mL of emulsion of sunflower oil and physiological saline. Group III (test group lower dose) – administered per orally with 100 mg/kg of MEML, dissolved in 2% dimethyl sulfoxide, started 7 days before the exposure of the DMBA. Group IV (test group higher dose) – administered per orally with 200 mg/kg of MEML, dissolved in 2% dimethyl sulfoxide, started 7 days before the exposure of the DMBA. Group V (positive control group) – administered intraperitoneally once per week with 0.5 mg/kg of standard drug Vincristine, started 7 days before the exposure of the DMBA. Groups III, IV, and V induced mammary carcinogenesis by giving single subcutaneous injection of 25 mg of DMBA in 1 mL of emulsion of sunflower oil and physiological saline and continued for 16 weeks. The experiment was terminated at 16th week to determine the protective activity of Madhuca longifolia during DMBA‑induced mammary carcinogenesis. The rats from all groups were sacrificed at the end of experiment by cervical dislocation method for biochemical analysis and histopathology. Biochemical analysis and histopathology Blood samples were collected from all groups and used for biochemical estimations. Half portion of tumor tissues from rats were fixed in formaldehyde for histopathological study and the remaining portion were used for biochemical analysis. Thiobarbituric acid reactive substances (TBARS) in plasma sample were determined using the method of Yagi[12] Tissue lipid peroxidation was estimated using the method of Ohkawa et al.[13] The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were determined using the method of Kakkar et al.,[14] Sinha,[15] and Rotruck et al.,[16] respectively. Glutathione (GSH) in mammary tissues and plasma was estimated using method of Beutler and Kelly[17] Chemiluminescent immunoassay was used for the estimation of serum 17 β‑estradiol (E2) using the method of Buscarlet et al.[18] Statistical analysis The results were given as mean ± standard error of the mean of six animals from all groups. The statistical analyses were done using, Sri Lanka.

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