Semecarpus anacardium L.f. leaf extract exhibits activities against breast cancer and prolongs the survival of tumor-bearing mice

Abstract Semecarpus anacardium L.f. has been commonly used in various traditional medicines from ancient times. The nuts have been described in Ayurveda medication systems to treat numerous clinical ailments. However, isolating phytochemical constituents from nuts remain challenging and exhibits cytotoxic effects on other cells. In this study, we have standardized procedures for isolating phytochemicals from the leaf extract. The ethyl acetate leaf extract selectively affects cancer cells in a dose-dependent manner (IC50: 0.57 µg/ml in MCF-7 cells) in various cancer cell lines and induces apoptosis in cancer cells. However, the non-malignant cells were relatively insensitive to the extract. Next, the incubation of the leaf extract induces cell cycle arrest and suppresses cancer cell migration in the cell culture model. Moreover, oral administration of extract significantly restored tumor growth in mice. Together, these observations suggest the anti-cancer activities of S. anacardium L.f. leaf potential for both in vitro and in vivo models. Graphical Abstract


Introduction
Breast cancer is one of the leading diseases among women and creates a significant global disruption in the healthcare system.though much progress has been made in designing therapeutics, however; the treatment of breast cancer remains at its infant stage.moreover, chemotherapy remains the common pharmacological approach for its treatment with many limitations, including severe toxic side effects leading to death in cancer patients (arya et al. 2015).nevertheless, natural products have been used to treat many clinical ailments as described in different traditional systems of medicine since antiquity (Kim et al. 2018).among these, ayurveda is one of the traditional systems that describes the holistic approach to treating various human clinical ailments, including processed medicinal plants, animal parts, and minerals (Joshi et al. 2010).Semecarpus anacardium L.f. (Family: anacardiaceae) is described in ayurveda for treating several clinical ailments such as inflammation, neurological disorders, microbial infection, diabetes etc. (mathivadhani et al. 2007).however, recent studies have indicated it as a potential source of antioxidant, antimicrobial, anti-inflammatory, anti-cancerous and stimulant for the central nervous system (Semalty et al. 2010).the nut exhibits cytotoxicity activity against breast, liver, lung, cervical, and blood cancer cells (Sharma et al. 1995;Selvam et al. 2004;Semalty et al. 2010).however, isolating phytochemicals in nuts remains challenging and the active constituents include bhilawanols, cardol, anacardic acid, semecarpol and anacardol (nair et al. 2009; adhami et al. 2012).due to the high toxicity and challenges in isolating essential bioactive compounds from nuts, we have investigated the other parts of the plant for potential anti-cancer activity.While assessing the plants, we identified leaves that could be used as an alternative source for isolating potential anti-cancer constituents against cancer cells. in this study, we showed that the ethyl acetate extract of S. anacardium L.f. extract induces apoptosis, thus inhibiting breast cancer progression and enhancing survival in tumor-bearing mice.therefore, our results show the potency of S. anacardium L.f. leaves extract as a raw material for future anti-cancer drug discovery for breast cancer.

Results and discussion
our analysis of plant extract from different parts (root, bark, leaf and fruit) revealed that the leaf is an excellent source for isolating potential phytochemicals that showed higher cytotoxic activity against breast cancer cells (Figure S1).

Semecarpus anacardium L.f. leaf extract exhibits anti-cancer activity and inhibits tumor progression in mice
the evaluate anti-cancer activity, we treated either petroleum ether (BLPe), ethyl acetate (BLea) or methanol (BLm) derived leaf extract in six different cell lines, including mCF7, mda-mB-231, hCt-15, min6, eaC and L929. the concentrations of the extracts ranging from 0 to 200 µg/ml were used in our study.among these, BLea-derived leaf extract induced the highest cytotoxicity among all cancer cell lines, including mCF-7 and eaC cells, with an iC50 of 0.57 and 1.71 µg/ml, whereas hCt-15, min-6 and hepG2 cells showed most minor sensitivity with an iC50 of 6.66, 15.77 and 44.93 µg/ml towards the extracts (Figure 1 and table S1; p value: *≤0.05, **≤0.025,***≤0.01.) moreover, BLea showed very low cytotoxicity in normal L929 cells (iC50 value 29.62 µg/ml), indicating that the extracts were less toxic for normal proliferating cells.therefore, BLea was selected against mCF-7 cells for further studies (table S1).additionally, we compared the cytotoxicity and iC50 values of BLea with paclitaxel in mCF-7 cells.however, paclitaxel exhibits higher iC50 values than BLea (Figure S2).these data indicate that BLea-derived leaf extract could be a potential alternative source for cancer treatment.next, we examined the anti-cancer potency of BLea using eaC (ehrlich ascites carcinoma) cells induced tumor model in mice.Based on a pilot study, a 100 mg/kg body weight dose of BLea was selected to evaluate anti-tumor activity in mice (Figure S4). the treatment of tumor-bearing mice started from the 8th day of eaC cell injection, when tumors became measurable, with a daily dose of 100 mg/kg body weight of BLea till the 28th day.next, we monitored the tumor progression on alternative days.interestingly, the oral administration of BLea resulted in a significant decrease in tumor volume (58.67, 224.00 and 298.67 mm 3 ) compared to the control groups (Figure S7(b-d)).in contrast, the mice that did not receive any treatment for BLea, the mean tumor volume was significantly increased (58.67, 388.98 and 780.44 mm 3 ) on the day 8th, 18th and 28th compared to the treated groups (Figure S7(a); p value: *≤0.05, **≤0.025,***≤0.01.).additionally, the histological sections of thigh from tumor-bearing mice revealed alterations in cellular morphology compared to the treated and control mice (Figure S7(f-h)).altogether, these data suggest that BLea has the potency to reduce tumor progression significantly in mice.therefore, BLea can be used in vivo and in vitro models to study the nature of cancer progression.
the development of chemoresistance among cancer cells is a major concern in the treatment, leading to a high mortality rate in cancer patients.the inactivation, alteration and efflux of drug targets influence the chemoresistance of cancer (arya et al. 2015).therefore, traditionally used medicinal plants or a mixture of multi-targeted compounds with anti-tumor activity are potent tools to overcome drug resistance with minimal side effects.Semecarpus anacardium L.f. is a widely studied medicinal plant, which has been described in ayurveda for the preparation of several poly-herbal formulations to treat clinical ailments.the nut extract inhibits the activity of a critical enzyme involved in carbohydrate metabolism, which is elevated in cancer cells during tumor growth to enhance the synthesis of nucleic acid and lipids (Sharma et al. 1995;Semalty et al. 2010).Besides, S. anacardium L.f. extract has been studied in hypoxia-associated factors and extracellular matrix-associated enzymes in tumors, inhibiting tumor progression and metastasis in dmBa-induced tumor-bearing rats (Sharma et al. 1995;arya et al. 2015).

Conclusion
in this study, we evaluated the anti-cancer properties of S. anacardium L.f. as an alternative source of phytochemicals for therapeutic use.moreover, the leaf extracts were prepared and evaluated for anti-cancer activity using various cell lines and tumor models.the ethyl acetate extract showed the most potent cytotoxic activity against different cancer cell lines and induced apoptosis in mCF-7 cells.Besides, BLea exhibited cell cycle arrest in the G1 phase and apoptotic cell death in mCF-7 cells.additionally, the oral administration of BLea induces significant tumor growth suppression and enhances the longevity of tumor-bearing mice.moreover, BLea inhibited tumor growth, delayed death and significantly enhanced survival in the tumor-bearing mice.therefore, BLea could be a potential extract for developing anti-cancer chemotherapy.however, additional studies are necessary to develop and elucidate molecular pathways involved in developing an efficient anti-cancer drug for cancer prevention.

Figure 1 .
Figure 1.cytotoxicity assay of semecarpus anacardium l.f.leaf extract in different cell lines.(a-f ) histogram showing cytotoxic assay of leaf extract in various cell lines treated with BlPe, Blea & BlM and cytotoxicity was evaluated by Mtt assay after 48 hours of treatment as indicated in the figure.p value: *≤0.05, **≤0.025,***≤0.01.