The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell

The number of breast cancer patients is increasing high in the world. This study aims to determine the chemopreventive effect of a combination of ethanolic extracts of tea leaves and mandarin peel in silico and in vitro on T47D breast cancer cells. Extraction by the maceration method used ethanol solvent 70%. The research is an in silico molecular docking utilized software of Autodock Vina to determine the binding affinity of tangeretin compounds and Epigallocatechin gallate (EGCG) on HER-2 protein. The antioxidant test used the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method to determine the antioxidant activity of the combination of tea leaf and mandarine peel (CTM) extract. The in vitro test used the method of 3-(4,5-dimethyltiazol-2-il) -2,5-diphenyltetrazolium bromide (MTT) to determine the value of IC50 from CTM on T47D breast cancer cells. The result of this study showed that CTM had a vigorous antioxidant activity with an IC50 value of 83 μg/ml. CTM had a weak cytotoxic activity with an IC50 value of 1889 μg/ml. Molecular tethering results of tangeretin and EGCG compounds produced a docking score of -6.6 and -5.0 kcal/mol with docking score proportion consisting of -4.9 kcal/mol of original ligand, -6.1 kcal/mol of doxorubicin and -4.5 kcal/mol of 5-fluorouracil. CTM had potential as a chemopreventive agent based on the robust antioxidant activity data on T47D breast cancer cells and molecular docking on the HER-2 protein.


INTRODUCTION
Breast cancer is a deadly cancer attacking women with a high prevalence rate. In 2012, there were 26 cases per 100,000 women. 1 One of the proteins that mainly causes breast cancer cell growth is HER-2. In the majority of breast cancer patients, HER-2 protein experiences uncontrolled expression through gene amplification that increases HER-2 protein expression up to 40-100 times. [2][3][4][5] Breast cancer can grow and spread into its surrounding tissue, such as lymph nodes, and then enter the blood vessels and even to other organs (lungs, bones, liver, and brain). 6 Common treatments for breast cancer patients are such as chemotherapy, surgery, and radiation. 7 However, these treatments are expensive and also often cause adverse side effects, which can kill the healthy surrounding tissue and even the effects of immunosuppression. it is necessary to innovate in defeating cancer, for example, using safer alternative treatments, such as plant extracts. 8 Natural ingredients that have the potential as a chemopreventive agent are tea leaves and mandarin (mandarin orange) peels. Tea leaves contain a flavonoid-polyphenol compound, epigallocatechin gallate (EGCG), which is proven to have a substantial antioxidant property. Mandarin (Citrus reticulata) peel contains a compound called 'tangeretin' that can potentially inhibit tumor cell growth, which is proven to be able to induce cell-cycle G1 arrest processes. 9 Considering all those benefits, this study aims to determine the chemopreventive effect of a combination of ethanolic extracts of tea leaves and mandarine peels in silico and in vitro. The combination of both extracts is expected to have a good synergy effect that can inhibit the development of cancer cells from being more significant.

Plant Determination
Tea leaves (Camellia sinensis) were obtained from the Kaliurang area, Yogyakarta, and mandarin peel (Citrus reticulata) were obtained from the Bantul region, Yogyakarta. The determination of both plants was carried out in the Laboratory of Pharmacy Biology, Faculty of Pharmacy, UGM Yogyakarta.

Macerating Extraction
The selection of the extraction method was based on the natures and compositions to be isolated. 10 Extraction was conducted using ethanol solvent 70% and stirred routinely every day for five days. After that, the pulp was remacerated for two days. After two days, the maceration was filtered again and was then evaporated using a Rotary Evaporator until a thick extract was obtained. 11

Antioxidant Test of DPPH
The DPPH method was based on the freeradical reduction of DPPH with maximum absorption at a wavelength of 517 nm, which produced a purple color in ethanol. 12 After that, 2 mL of vitamin C solution was taken, which was the sample solution at various concentration series made into a 10-mL volumetric flask. The solution was added with 2 mL of DPPH and a methanol solution into the limit mark. The solution was vortexed for 30 seconds and left alone within the operating time. Uptake was read at  maximum and replicated three times.

Cytotoxic Test of MTT Assay
This method was based on the breakdown of tetrazolium salt or yellow Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 3 MTT (3-(4,5-dimethylthyazol-2-yl) -2,5diphenyl tetrazolium bromide) by dehydrogenase succinate enzyme contained in the cell mitochondria to turn into purplish blue formazan crystals. 13 Cells with a density of 70-80% (confluent) were distributed into 96 well plates, observed under an inverted microscope to identify the cells' distribution, and then incubated for 48 hours so that the cells could adapt and attach to the bottom of the wells. On the following day, the media in the wells were dumped by turning the well plates over the dump, then drained with a tissue. The wells were washed using 100 µL PBS which was added to all wells filled with cells, and then they were discarded. Furthermore, a series of concentrations of the test sample and culture media containing 0.2% DMSO (control) was added and then re-incubated for 24 hours. At the end of the incubation, culture media containing the sample were removed and washed with 100 µL PBS, then added with the MTT reagent. In this case, living cells would react with MTT to form purple formazan crystals. After 4 hours of incubation, the condition of the cells was examined under an inverted microscope. When formazan was created, a 200 μL SDS stopper solution was added into 0.1% HCl to dissolve the formazan crystals. The well plates were then covered with aluminum foil and incubated in a dark place at room temperature for 3 hours. Upon that process, the aluminum foil was opened and shaken for 10 minutes. The absorption was read with an ELISA reader at a wavelength of 595 nm. Based on the absorbance data, the percentage of living cells and IC50 values could be calculated.

Molecular Docking Test
Molecular docking is a computational method used to predict the interaction between ligands and receptors or proteins to obtain the value which describes their total bond energy. The protein structure selected as the target of molecular docking in this study was HER-2 (PDB ID: 3PP0). The structure was downloaded from the Protein Data Bank (PDB) through www.rcsb.org; thus, the protein's PDB ID could be identified. The application used for protein and ligand preparation in this study was DS Visualizer. RMSD value was determined by filling in the Windows Command Prompt according to the code; therefore, some conformations would appear. Each conformation showed its RMSD affinity value, then the conformation, which had an RMSD value of less than 2 Å, was selected. The visualization process of docking results used the DS Visualizer app. The visualization was to determine the position and the image of the bond between proteins and ligands in 3D.

Plant Determination
The results of the determination showed that the simplisia used in this study was tea leaf (Camellia sinensis) and mandarin orange peel (Citrus reticulata).

Macerating Extraction
The maceration method in this study used ethanol solvent 70%, 10 L for 1 kg of dried tea leaf powder, and 1 kg of dried mandarin peel powder. Based on the filtering result, 2,450 ml of liquid extract was obtained. It was then dried using a rotary evaporator and produced 27.3 grams of thick tea leaf extract (TLE) with a dark green color and 26.4 grams of a concentrated mandarin peel extract (MPE) with a dark yellowish-green color. Both tea leaf extract and mandarin peel extract were mixed to get a combined extract.  Tables 1 and 2, and the graphic is shown in Figures 1 and  2. Also, the IC50 value of the antioxidant test is shown in Table 3.

Molecular Docking with Autodock Vina
Molecular docking was conducted using the Autodock Vina application. Open babel, and the shape of the structure were then visualized in 2D and 3D with the DS Visualizer. The data on the molecular docking results can be seen in Table 4.

Cytotoxic Test of MTT Assay
The cytotoxic test was performed to determine MTT Assay activity towards T47D breast cancer cells by giving CTM extract treatment shown in Table 5.

Cytotoxic Test of MTT Assay
The cytotoxic test was performed to determine MTT Assay activity towards T47D breast cancer cells by giving CTM extract treatment, as shown in Table 6.   Antioxidant test results showed an IC50 value of vitamin C compounds was 14.87 μg/ml. Whereas, the IC50 value of TLE and MPE combination was 83.00 μg/ml, which was weaker in capturing radicals compared to vitamin C. It occurred as the combination sample (CTM) was still in the form of crude extracts; thus, the number of active compounds that could inhibit free radical activity was likely to be smaller. However, IC50 < 100 μg/ml was obtained and classified as antioxidants with vigorous activity. Therefore, the combination of CTM extract had strong potential in the activity of capturing free radical compounds.
Referring to a similar study, an antioxidant test of Kemuning extract (Murraya paniculata (L) Jack) produced an IC50 value of 126.17 μg/ml, and antioxidant analysis of mangosteen peel extract (Garcinia mangostana L.) resulted in an IC50 value of 44.49 μg/ml. 15 Compared to this, CTM extract had more substantial antioxidant potential than Kemuning extract. However, it was weaker compared to mangosteen peel extract. 16 The results of molecular docking showed that the EGCG compound had the most vigorous inhibitory activity on the HER-2 protein with a docking score of -6.6 kcal/mol, compared to other comparative compounds. Visualization of the docking results showed that EGCG compounds were comparable to doxorubicin which had 7 bounds interacting with amino acid Based on the cytotoxic test results, CTM extract had a weak potential in inhibiting the viability of T47D breast cancer cells with IC50 values of 1889 μg/ml. It was probably due to the small amount of tangeretin compound content in CTM extracts, so the cytotoxic effect was not reliable. The tangeretin flavonoid compound, which was suspected of having an effect as a chemopreventive agent, had a mechanism to induce cell cycle G1 arrest when the cell was in the restriction point phase. 22-24 A restriction point is a point where hyperphosphorylation occurs that causes the p27 inhibitor, a p53 expression gene (an apoptosis gene), to be degraded. At this point, the reparation of damaged DNA occurs. If DNA damage is severe and cannot be repaired, it will be immediately eliminated to the G0 phase. The mechanism of the cell cycle starts with the entry of it from the G0 phase to the G1 phase due to the growth factor stimuli. It then causes retinoblastoma protein (pRb) hyperphosphorylation and DNA to become loose. Furthermore, it later enters the restriction point and the S