Semecarpus Anacardium Linn. Leaf Extract Exhibits Activities Against Breast Cancer And Prolongs The Survival of Tumor Bearing Mice

Semecarpus anacardium Linn. is a commonly used Ayurvedic medicinal plant which nuts have been described in Ayurveda and Sidda system of medicine to treat clinical ailments such as vitiligo, inamation, microbial infection, geriatric problem, baldness and neuro related problems. In this study, anti-cancer activity of the leaves of Semecarpus anacardium Linn was evaluated for future drug development. The phytochemical screening was done by GC-MS analysis, cytotoxicity was examined using MTT assay, mode of cell death was evaluated by uorescence microscopy and nally antitumor activity was determined in EAC cell induced tumor bearing mice. The ethyl acetate extract from the leaves of the plant induced cytotoxicity in cancer cells in a dose dependent manner (IC 50 : 0.57 µg/ml in MCF-7 cells) in different cancer cell lines. The non-malignant cells were relatively insensitive to the extract. The staining with acridine orange, ethidium bromide and DAPI conrmed that the extract induced apoptosis in cancer cells. Furthermore, the extract induced cell cycle arrest at G1 phase and suppressed cancer cell migration. An oral administration of the extract suppressed the tumor growth in mice model bearing ehrlich ascites carcinoma cells. The ethyl acetate extract was also found to prolong the survival of tumor bearing mice. Overall, these observations suggest the anticancer activities of the ethyl acetate extract of the leaves of S. anacardium. The study opens a new window to examine the phytochemical constituents from the leaves of the plant responsible for the anticancer activities. property of the leaves. Here, it is important to focus that leaves are alternative and ecofriendly source of a plant, may be used without altering any biodiversity. Our study showed that the ethyl acetate extract of S. anacardium Linn. leaves induces apoptosis and arrests the cell cycle progression in G1 phase and more importantly, the oral administration of the extract shows inhibition of tumor growth, delays the tumor associated death and enhances the survival in tumor bearing mice signicantly. The results show that the potency of S. anacardium leaves extract for being used as raw material for future anticancer drug discovery to treat the breast cancer. Thus, this study explores the anticancer property of leaves for the rst time with less toxicity and more ecacy as compare to the nuts.


Introduction
Breast Cancer is the most common cancer amongst the women and a major health issue globally. Its incidence in 2018 was 2.09 million new cases diagnosed and 0.63 million mortality with a projection of 3.06 million new cases and 0.99 million death in 2040 [1]. Chemotherapy remains the common pharmacological approach for its treatment with many drawbacks including severe toxic side effects, leading to death in cancer patients [2]. These drugs are often cytotoxic and induce cancer cell death in solid tumors either by cell cycle arrest, DNA synthesis inhibition, necrotic cell death, or by inducing apoptosis. There are several known adverse effects of these drugs which cause severe damage to the vital organs and normal proliferating cells such as heart, liver, kidneys, hair follicles, stem cells, etc [3,4].
New e cient and non toxic chemotherapeutic agents are the need of time to overcome the problem.
Natural products have been used to treat many clinical ailments since antiquity [5] as described in different traditional system of medicine across the world. Ayurveda, one of the traditional system of medicine, specially popular in Indian subcontinent, which describes the holistic approach for the treatment of human clinical ailments using processed medicinal plants, some animal parts and minerals [6]. The medicinal plants described in Ayurveda may serve as a prime source for rational drug designing for many diseases including cancer. A large number of phytoconstituents are isolated from Ayurvedic medicinal plants which are promising lead molecules such as alkaloids from Rauwol a serpentina, The nuts of the plant are described for several ethnopharmacological properties in different Ayurvedic formulations including anticancer property, but there are no studies have been carried out to investigate the anticancer activity of other parts of the plant except the fruit. Therefore, in this study we carried out to explore the anticancer property of the leaves. Here, it is important to focus that leaves are alternative and ecofriendly source of a plant, may be used without altering any biodiversity.
Our study showed that the ethyl acetate extract of S. anacardium Linn. leaves induces apoptosis and arrests the cell cycle progression in G1 phase and more importantly, the oral administration of the extract shows inhibition of tumor growth, delays the tumor associated death and enhances the survival in tumor bearing mice signi cantly. The results show that the potency of S. anacardium leaves extract for being used as raw material for future anticancer drug discovery to treat the breast cancer. Thus, this study explores the anticancer property of leaves for the rst time with less toxicity and more e cacy as compare to the nuts.

Collection of plant materials and phytochemical analysis
The leaves of Semecarpus anacardium Linn. were collected in the spring season of 2017 from Banaras Hindu University, Varanasi, India and identi ed by the Department of Dravyaguna (Ayurvedic The plant materials were dried at room temperature for two weeks and grinded into a coarse powder through electrical domestic grinder. The 1 kg of leaves powder was macerated up to 24 h with continuous stirring in 5 litres of petroleum ether, ethyl acetate and methanol respectively three times. The supernatant was recovered by ltration through Whatman lter paper of 11µm pore size. Further, each ltrate was completely dried by rotary vacuum evaporator in reduced pressure [18]. The compounds of ethyl acetate extract of S. anacardium leaves (SLE) were assessed by Gas chromatography-mass spectrometry (GC-MS), the JEOL GCMATE II GC-MS at SAIF, IIT Madras using standard procedure [19]. The data were matched with the standards [20,21].

Cytotoxicity assay
MTT assay was performed for evaluation of the cytotoxicity of the leaves extracts in which tetrazolium rings of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide is cleaved by mitochondrial enzyme succinate dehydrogenase of live cells. The yellowish MTT solution is turned into water-insoluble violet colored formazan crystals depending on the number of viable cells. This assay is also sensitive, reliable, quantitative and colorimetric method to measure cell viability. The 100 µl of cell solution in fresh media at a cell density of 1 X 10 4 cells/ml was seeded in each well of 96-well plate and incubated overnight. Then, the old culture media was replaced with 200 µl fresh media and treated with the extracts by performing serial dilution which was further incubated for 48 hours. On completion of treatment duration, the treated culture media was replaced with 100 µl of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) solution at a concentration of 0.5 mg/ml incomplete culture media, i.e., the culture media without fetal bovine serum was kept for 4 hours at 37°C. The treated cells were centrifuged at 750 rpm for 5 minutes (Remi R8C) and the supernatant was replaced with 100 µl DMSO to solubilize water-insoluble purple colored-formazan crystal and absorbance was measured at 570 nm using microtitre plate reader (Biorad, India) [18]. The percentage of cell viability was calculated as Percentage of cell viability = (OD of the treated cells /OD of the untreated cells as control) × 100, then IC 50 was calculated.

Analysis of Cell morphology
The MCF-7 cells were seeded into 96 wells plate and then old media were replaced with fresh media after 24 h, the extract was administred and further washed with 10 mM PBS (pH 7.4) aftr incubation of 24 h and observed under the inverted light microscope (Dewinter, India) at 40X objective lense [18,22]. Further, Acridine orange (AO) and Ethidium bromide (EtBr) double staining was used for detection of the mode of cell death. Acridine orange is a cell permeable dye which binds with the nucleic acid to emit green uorescence while ethidium bromide can enter the cell membrane and binds to the nucleic acid to emit red uorescence. MCF-7 cells were seeded into 96 wells plate (1X10 4 cells per well) for 24 h at 37°C with fresh culture medium and the extract to test the drug, Paclitaxel was used as control positive group while 10 mM PBS was taken as control negative. Then the culture media were removed and cells were washed with 10 mM PBS (pH 7.4) and incubated for further 30 minutes in 100 µg/ml AO and 100 µg/ml EtBr in dark. It was washed with 10 mM PBS (pH 7.4) and nally cells were examined underthe uorescence inverted microscope (Dewinter, India) using 40× objective lense [18,22].

Analysis of nuclear morphology
Nuclear Morphology was studied by DAPI (4′-6-diamidino-2-phenylindole) stain (Genetix, India) which forms uorescent complexes with natural double-stranded DNA [22]. Human breast cancer (MCF-7) cells were seeded at a density of 1 X 104 cells per well and incubated for 24 hours. Then, the treatments were given as per previous experiment followed by washing of cells with PBS and stained with DAPI solution. After 30 minute incubation, the cells were again washed with 10 mM PBS (pH 7.4) and cells were studied by uorescence inverted microscope using the blue lter [18].

Cell cycle analysis
The cell cycle analysis was perfomed with slight modi edi cation of previously described method [8]. In brief, the cells were washed with PBS at the end of the treatment time, then stained with 70% chilled methanol, treated with RNaseA and stained with PI. The population of cells in different phases of the cell cycle was determined by FACScan and the analysis was performed by Cell Quest software (Becton Dickinson).
Scratch wound healing assay MCF-7 cells were grown upto 80% con uency and trypsinized for seeding into 6-well plate (1 × 10 5 cells/ml) at 37 ˚C for 24 h in DMEM medium. Further, media were replaced with fetal bovine serum free media further 24 h and scratch wound was created in the middle of the well with a sterile 20 µl pipette tip and washed with 10 mM PBS thrice. 1 ml of complete medium was given to the wound containing cells and extract was administrated in test group while medium in control group. The cells were examined at an interval of 24 h, 48 h and 72 h. Images were analyzed for cellular migration by inverted microscope (Dewinter, India) using 10X objective lense [18].

Animal model and Ethical statement
For in vivo studies, Swiss albino mice of either sex weighing 18-22 grams of about 8-10 weeks old were procured from the central animal facility, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India with approval of the institutional ethical committee (Dean/2016/CAEC/337). The animals were kept in polypropylene cages and provided standard pellet diet (Agro Corporation Pvt. Ltd., Bengaluru, India) and water ad libitum. The mice were accommodated under controlled temperature (27 ± 2 ˚C) and humidity with a 12 hours light and dark cycle by following CPCSEA guidelines for experimental study.

Antitumor evaluation
To evaluate antitumor activity, a total of 18 tumor-bearing mice and 6 normal mice were divided into four groups. Mice of group I, group II and group III were administered 20 mg/kg body weight Paclitaxel, 100 mg/kg bodyweight BLEA and equal volume of water (as BLEA suspended in it) respectively, while group IV was normal mice without tumor. All mice of group I and Group II were given respective drugs regularly throughout the experimental period of 28 days. The mice of group IV were given no treatment to served as control negative group. Tumor size was measured using vernier calipers on alternative days for tumor animals and tumor volume was calculated using the formula V = 0.5 × a × b2, where 'a' and 'b' indicates the major and minor diameter, respectively. At the end of 28th day of experimental period, animals from each group were sacri ced to collect kidney, liver, thigh and spleen were collected from Paclitaxel, BLEA treated, tumor and normal animals and were evaluated for morphological changes using hematoxylineosin stained histological slides [23].

Animal survival study
The effect of BLEA on life span of the tumor-bearing mice was assessed. For this study, total 12 mice have received EAC cells in their left thigh. They were further divided into two groups (6 mice in each).
Group I was served as tumor control and group II received oral administration of BLEA 100 mg/kg body weight daily till the survival of last animal, starting after 7 days of tumor (EAC) cell injection.
The life span of BLEA treated mice was calculated and compared with untreated tumor-bearing animals. The life span of experimental animals was monitored and calculated by using the following formula [(T − C)/C] × 100, where 'T' indicates the number of days the treated animals survived and 'C' indicates that number of days that tumor animals survived [2].

Histology
The tissues were collected from control, BLEA treated animals, and further processed for histological evaluations [24]. The excised tissues were xed in 4% neutral saline formalin, dehydrated in alcohol and embedded in para n wax. The section (5 µm) of tissues was cut using Microtome (Leica Biosystems, Germany) and then stained with haematoxylin and eosin. Images of all tissue sections were captured using light microscope (Dwinter).
In silico studies ADME Screening: The parameters of pharmacokinetics such as absorption, distribution, metabolism and elimination (ADME) play an important role in drug discovery and development [25]. The ADME properties of compounds were analyzed using SwissADME (http://www.swissadme.ch) and the data were recorded to analyze the possibility of identi ed compounds to be the drug candidates [26].
Molecular docking studies: All identi ed phytochemicals were used as ligands and their structures were drawn using Chembiodraw Ultra 14.0 (Cambridge Soft) and saved as pdb le. Crystal structures of receptor proteins CDK2 (protein ID: 1W0X) was downloaded from Protein Data Bank (https://www.rcsb.org/structure/2B53), then water molecules and ligand were removed. Patchdock (Tel Aviv University, Israel) was used for molecular docking studies using CDK2 receptor protein and ligands considering settings as clustering RMSD 4.0 and complex type default [27]. The resultant complex of receptor-ligand with solution number, docking score, area and six-dimensional transformation space was recorded. The docking solutions obtained in Patchdock were further re ned with FireDock (Tel Aviv University, Israel) and top 10 solutions among 1000 rescored solution were downloaded with global energy, atomic contact energy, attractive Van der Waals and considered for further analysis. Each complex of ligand-receptor obtained through FireDock was ranked based on their minimum global energy and Chimera 1.12 used for surface visualization of the complex. Discovery Studio 2017 R2 Client was used for analysis of interaction of the receptor-ligand complex.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 6.0. The analysis of variance was performed on three independent cytotoxicity results by one way ANOVA analysis using tukey as post test. The results were considered signi cant at p value < 0.05. The data were expressed as mean ± SD.

Results
Based on the selectivity of different extracts [28], the plant leaf extract was selected for this study, as it showed higher cytotoxicity in cancer (MCF-7) cells and less toxicity for normal (L929) cells (selectivity 7.84) among all four extracts i.e. root, bark, leaf and fruit (supplement Fig. 1).
The extracts induce cytotoxicity in cancer cells.
In this study, total six cell lines MCF7, MDA-MB-231, HCT-15, MIN6, EAC and L929 have been used to evaluate the anticancer activity of leaf extracts of Semecarpus anacardium Linn. in different organic solvents, petroleum ether, ethyl acetate and methanol which were coded as BLPE, BLEA and BLM respectively. The cells were treated with different concentration of the extracts, ranging from 0 to 200 µg/ml, which induced cytotoxicity signi cantly in dose-depended manner as evaluated by MTT assay (Fig. 1).
BLEA showed the most active extract and induced cytotoxicity among all cancer cell lines (supplementary  table 1 BLEA induces apoptosis BLEA induced cytotoxicity through altering cellular morphology and intracellular vacuolizations. So, to know the cell death mode, stained with DAPI staining and the cells were examined under uorescence microscopy which revealed a signi cantly alteration in the morphology of nucleus in treated group of cells, such as chromatin condensation, nuclear fragmentation and shrunken nuclear morphology, were observed ( g. D-F). The untreated cells showed intact nuclear morphology. This result also supports apoptosis as a mode of cell death. Along with the above results, the cells were treated with BLEA at same concentration and apoptosis assay was performed using Ethidium bromide-Acridine orange double staining medoth. MCF-7 cells were treated with BLEA at 0.57 µg/ml concentrations (equivalence IC 50 ) for 48 h, then stained with Ethidium bromide and Acridine orange which showed that the death induced in MCF-7 cells by BLEA was apoptosis, as the nuclear fragmentation, nuclear contraction, cytoplasmic membrane blebbing, entry of ethidium bromide into the cells, leading orange to red color uorescence in cells were observed (Fig. 2G-I).
BLEA induces cell cycle arrest As cells were died through apoptosis when treated with BLEA, the effect of BLEA on cell cycle alteration was studied. BLEA was found to modestly increase the population of cells at the G1 phase. Furthermore, the sub G1 population of cells was also modestly increased at the higher concentration of BLEA (500 ng/ml). Conversely, the percentage of cells in the S and G2/M phase was decreased with an increase of BLEA concentration (Fig. 2J-L). Overall, these observations suggest thatBLEA induces cell cycle arrest at the sub-G1 and G1 phases.

BLEA inhibits cellular migration
Cellular migration is one of the important characteristics of cancer cells which enables them to metastasis. To examine the effect of BLEA on cellular migration, the scratched wound created and treated with BLEA. The cells were examined on the day 1st, 3rd and 5th for the healing of wound and found that BLEA decreases the cellular migration signi cantly, resulting delayed in wound healing (Fig. 3).

BLEA safe for oral administration in mice
The toxicity of BLEA for oral administration was evaluated by oral acute toxicity with the standard protocol (test number 425 of OECD) in mice. The BLEA did not show any sign of toxicity even at high dose in mice. The tissues of vital organs were further analyzed for any cellular architectural alteration due to toxicity which did also not show any sign of toxicity. Interestingly, there was no any body weight loss was recorded in the animal treated with BLEA, which indicates that BLEA is non-toxic to the animals for oral administration (supplementary table 2 and supplement Fig. 4).

BLEA inhibits of tumor progression in mice
The anticancer potency of BLEA was evaluated using EAC (Ehrlich ascites carcinoma) cells induced mice tumor model. Based on a pilot study ( Supplementary Fig. 5), 100 mg/kg b.wt. dose of BLEA was selected for evaluation of anti-tumor activity. The treatment of tumor-bearing mice was started from 8th day of EAC cells injection, when tumors became measurable, with a daily dose of 100 mg/kg b.wt of BLEA with feeding needle till 28th day. The volume of tumors was measured on alternative days. The oral administration of BLEA resulted in a signi cant (p ≤ 0.05) decrease in tumor volume upon the treatment with BLEA, whereas the tumor volume was continuously increasing in control group of tumor-bearing mice (Fig. 4A).
The mean volume of tumor observed on the day 8th, 18th and 28th were 58.67, 388.98 and 780.44 mm 3 respectively in untreated tumor-bering control group of mice, whereas BLEA treatment reduced the tumor volume and found 58.67, 224.00 and 298.67 mm 3 respectively as compare to the control (Fig. 4B-D). The histological examination of tissues from thigh, liver and spleen from tumor-bearing control group of mice showed alterations in cellular morphology compared to the treated and normal mice (Fig. 4F-H). There were dead cells seen in tumor tissue of treated mice. Therefore, the study suggests that BLEA has potency to reduce tumor progression signi cantly in mice models.
BLEA delays tumor associated death and enhances the survival of tumor bearing mice Importantly, this study showed that the tumor associated death in mice was delayed and rst death in BLEA treated mice was recorded on the day 49th whereas the rst death in control tumor bearing mice was on the day 22nd. It was observed that about 50% survival in tumor bearing mice treated with BLEA was increased as compare to the control group of tumor bearing mice. The control untreated tumor bearing mice survived only upto 73 days after EAC injection, whereas the BLEA treated tumor bearing mice were survived more than 110 days. These results show the anticancer potential of BLEA which caused tumor growth regression as well as increased survival with the delay in tumor associated death in tumor bearing mice model. Further, the histological examination of dissected tumor tissues was done using haematoxylin-eosin double staining to explore the mechanism lying behind the tumor growth in tumor bearing mice models. The study showed a signi cant decrease in number of EAC cells in the tissue of thigh tumor of the mice received BLEA orally in comparison of untreated controls tumor bearing mice ( Fig. 4E).

Compounds of BLEA inhibit CDK2
All 17 compounds of BLEA extract were found to have zero violation of Lipinski rule, which was suggested the possibility of the compound to be drug candidate with good bioavailability (data not shown). Compound 14 was bound to CDK2 with lowest Gibbs free energy, forming hydrogen bond with amino acid residues of CDK2 side chain such as Ile10, Thr14, Val18, Leu134, Ala144 and Asp145, while other closely interacting residues were Gly11, Glu12, Gly13, Tyr15, Lys33, His84, Gln85, Asp86, Lys89, Asp127, Lys129, Gln131 and Asn132 with a pocket area of 760.10 Å 2 . The binding a nity of compound 13 was highest after compound 14, followed by compound 12 and 10 with − 45.27, -41.94 and − 40.75 kcal/mol respectively. Compound 13 was found to interact with amino acid residues Ile10, Tyr15, Val18, Lys89, Leu134 and Ala144 through hydrogen bond directly, while closely interacting residues were Gly11, Glu12, Gly13, Thr14, Lys33, Gln85, Asp86, Asp127, Lys129, Gln131, Asn132 and Asp145 with a pocket area of 693.30 Å 2 . Similarly, other compounds were found to have inhibitory action against CDK2 protein with different a nities and pocket areas, as showed in the table 2 and Fig. 5.
Overall, our results showed that BLEA induced cytotoxicity by inducing apoptosis, inhibited tumor growth, delayed the tumor associated death leading to signi cant increase in the survival of tumor bearing mice model. Thus, this study suggests that BLEA could be served as a prime source for development of chemotherapeutic agent for human cancer prevention.

Discussion
The development of chemoresistance among cancer cells is major obstacle in its treatment, leading to high mortality rate in cancer patients. The drug inactivation, drug target alteration and drug e ux are in uencing the chemoresistance development for existing target speci c single molecule drug [2]. Therefore, plant extract or fractions which are mixture of multi-targeted compounds, having antitumor activity are potent tools to overcome the drug resistance. Traditionally used medicinal plants serve as the prime source for development of chemotherapeutic drugs with minimal side effect which also reduce the time and cost to develop potential chemotherapeutic agents. shows synergistic effect with conventional drugs such as doxorobocin and induce apoptosis via arresting cells in G2/M phase of cell cycle in cancer cells [15,36].
In this study, anticancer properties of S. anacardium leaves was investigated, which is an alternative source of phytochemicals and could be used for therapeutic purposes. The medicinal application of leaves remain unknown till the date. Here, the leaf extracts in three different organic solvents were prepared and evaluated rst time for anticancer activity using various cell lines and animal tumor model. The ethyl acetate extract was found more potent cytotoxic against different cancer cell lines and induces apoptosis in MCF-7 cells. However, it was found fairly safe for normal (L929) cells. Moreover, the treatment of MCF-7 cells with BLEA exhibited cell cycle arrested in G1 phase and apoptotic cell death. The in vivo study using mice as animal model also exhibited that BLEA is safe for oral administration and potent for tumor growth inhibition. The extract delays the tumor related death and enhance the survival of tumor bearing mice. ether, ethyl acetate and methanol respectively which are also higher than the previous studies [15]. The selectivity index is an important tool in bioassay guided fractionation for selection of the best extract/fraction which indicate cytotoxicity to cancer cells and safety for normal cells [28]. The selectivity index is range from 0.40 to 52.17 for all three extract in different cancer cells. Among these, the selectivity index of ethyl acetate extract of the leaf is higher in human breast carcinoma (MCF-7) cells, resulting the selection of BLEA and MCF-7 cells for further studies.
The discovery and development of anticancer chemotherapeutic agents focuses on mode of cell death induced and their effect on cell cycle [37]. As apoptotic cell death is highly organized and programmed process. The drugs with apoptosis inducing property and inhibiting the tumor progression in cancer, are considered as novel anticancer chemotherapeutic drugs [38]. There are several cytoplasmic vacuoles induced after treatment with BLEA in breast cancer (MCF-7) cells which indicating the induction of apoptosis like cell death [39,40]. Double staing with AO-EtBr, followed by uorescence microscopy for detection mode of cell death and differentiation of untreated cells, early apoptotic cells, late apoptotic cells and necrotic cells are indicating BLEA induced apoptosis in MCF-7 cells. As Acridine orange permeable to the unbroken plasma membrane and has ability to bind DNA, resulting green uorescence, whereas Ethidium bromide enters inside the cells only broken plasma membrane, binds the nucleic acid ladder and emits red uorescence. The uorescing color green, yellowish orange, orange and red represent normal, early apoptotic, apoptotic and late apoptotic/necrotic cell death respectively [41]. The altered morphology was seen in the cells treated with BLEA and stained with DAPI stain, which further con rmed the apoptosis mode of cell death induction [41]. The cell cycle is series of events, regulated by various molecular signals at different checkpoints, resulting synthesis of DNA and division of cell into daughter cells. BLEA alters the cell cycle and arrests the MCF-7 cells at G1 phase. Interestingly, the nut extract has reported for arresting the cells at G2/M phase [35]. The above ndings suggest that the molecular mode of action of nut extract and leaf extract are different in cancer cells [42]. The scratched wound assay is an important tool for study of cell migration as well as metastasis. Numerous phytochemicals and herbal extract have been reported for inhibition of cellular migration which may play an important role in prevention of metastasis in cancer patients [43][44][45]. The leaf extract, BLEA has also showed inhibition of cellular migration of MCF-7 cells in this study.
The progression of cell cycle through G1 phase depends on CDK2-Cyclin E complex [46]. The genotoxic stress induces cell cycle arrest with the help of CDK inhibitors such as P 21 , P 27 , P 57 , etc. The CDK inhibitors bind to CDK, as a result the function of CDK-Cyclin complex is ceased. Similarly, the binding of P 21 to CDK2 causes cells arrest in G1 phase of cell cycle [46]. Therefore, CDK2 inhibitor phytochemicals may be implemented as an e cient G1 phase arrest in treatment of cancer cells. Numerous phytochemicals are reported to have CDK2 inhibitory activity such as Artocarpin, Curcumin, Piperine, Urosolic Acid, etc [47]. All identi ed compounds were showed binding a nity to the target protein (CDK2), whereas compound 10, 12, 13, 14 were showed strong binding a nity to the active site of CDK2 protein.
The compounds of the potent extract BLEA, which have higher a nity to the target protein CDK2, also showed strong hydrogen bonding with the conserved amino acid residues such as Val18 and Leu134 as shown in table X and gure Y. The molecular docking studies shows that the compounds of BLEA are capable to bind to the active site of CDK2, inhibit the binding of CDK2 to cyclin E, resulting arrest of cell cycle progression in G1 phase. The interaction of compounds with CDK2 shows possible cause of cell cycle arrest in G1 phase of cancer cells.
The different extracts of Semecarpus anacardium nuts are well known for their toxicity in human and animals [17,48,49]. The puri cation processes are described in Ayurveda to reduce its toxicity of nuts before clinical use [50]. However, in this study it has found that the ethyl acetate extract of leaf is safe for oral administration. There was no any morphological, physiological or behavioral sign of toxicity sign as well as death occurred in mice even received high dose (5000 mg/kg b. wt.) of the extract (BLEA). Interestingly, haematoxylin and eosin stained histological examination of stomach, liver, kidney and lung tissues did not show any alteration in cellular architecture in treated group of animals.
The study has further extended to evaluate in vivo anti-tumor activity in EAC cells induced mice tumor model. The EAC cells are mouse derived ascetic carcinoma cells which easily accepted in mice body with high transplantable e cacy, rapid growth and con rmed malignancy. The subcutaneous injection of EAC cells, results the development of solid tumor model in mice, which is selected in this study [51,52]. Availability of data and material (data transparency): The data will be available on demand.
Code availability (software application or custom code): Not applicable.     Visualization of the interaction of receptor protein, CDK2 with ligands molecules in which A represents 3D surface view of binding site of compounds, B represents 3D spatial interactions and C represents 2D interaction of the compounds respectively. 1-17 represents the compounds.

Supplementary Files
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