The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell Ega Hida Prabowo, Rifki Febriansah* School of Pharmacy, Faculty of Medicine and Health Science, Universitas Muhammadiyah Yogyakarta, Jl Brawijaya, Tamantirto, Kasihan, Bantul, Yogyakarta 55183. Abstract 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. Keywords: Antioxidant test; Camellia sinensis; Citrus reticulate; MTT Assay; Molecular docking; T47D breast cancer cells Data of article Received Reviewed Accepted : : : 18 Feb 2020 12 May 2020 23 Jul 2020 DOI 10.18196/jfaps.010101 Type of article: Research * Corresponding author, e-mail: rifki.febriansah@umy.ac.id Ega Hida Prabowo, Rifki Febriansah | The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell 2 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.2-5 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. METHODS 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 re- macerated 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 free- radical 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) -2,5- diphenyl 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. RESULTS AND DISCUSSION 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. Ega Hida Prabowo, Rifki Febriansah | The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell 4 Antioxidant Test of DPPH Absorbance data of DPPH antioxidant test results can be seen in 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. Table 1. Data of comparative inhibition percentage for vitamin C Concentration (μg/ml) Average absorbance Blank absorbance % Inhibition 0.5 0.641 0.702 8.73 1.0 0.638 0.702 9.11 2.0 0.625 0.702 11.01 5.0 0.557 0.702 20.76 10.0 0.402 0.702 42.76 20.0 0.189 0.702 73.04 30.0 0.055 0.702 92.22 Table 2. Data of inhibition percentage for CTM extract Concentration (μg/ml) Average absorbance Blank absorbance % Inhibition 5.0 0.721 0.702 -2.61 7.5 0.703 0.702 -0.09 10.0 0.664 0.702 5.46 15.0 0.633 0.702 9.87 20.0 0.472 0.702 32.87 30.0 0.381 0.702 45.80 40.0 0.346 0.702 50.78 Table 3. IC50 value of antioxidant test for vitamin C and CTM extract Compounds Linear regression equation IC50 value (μg/ml) Description Vitamin C y=2.9913x+7.5319 14.19 Very strong R2= 0.9859 CTM y=0.6427x-3.3458 83.00 Strong R2=0.9698 Table 4. The result of the molecular docking between ligands and HER-2 receptors Compounds RMSD value Docking Score Conformation Native ligand 1.957 -4.9 7 EGCG 1.343 -6.6 2 Tangeretin 1.197 -5.0 4 Doxorubicin 1.542 -6.1 4 5-Fluorouracil 1.576 -4.5 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. Table 5. Data of living cell after CTM extract treatment (%) Content (μg/ml) Average Sample Absorbance Standard Deviation % Living Cells 100 0.130 0.014 4.308 400 0.128 0.003 3.798 800 0.124 0.002 3.122 1000 0.118 0.001 2.009 Average Absorbance Cell Control 0.663 Media 0.103 Equation y = -0.0024x + 4.6713 R2 = 0.9233 IC50=1889 μg/ml 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. Table 6. Data of living cell after CTM extract treatment (%) Content (μg/ml) Average Sample Absorbance Standard Deviation % Living Cells 100 0.130 0.014 4.308 400 0.128 0.003 3.798 800 0.124 0.002 3.122 1000 0.118 0.001 2.009 Average Absorbance Cell Control 0.663 Media 0.103 Equation y = -0.0024x + 4.6713 R2 = 0.9233 IC50=1889 μg/ml Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 5 Figure 1. Chart of Vitamin C Inhibition Figure 2. Chart of CTM Extract Inhibition Figure 3. 3D structure of geometry optimization results: (A) Native ligand (B) EGCG (C)Tangeretin (D) Doxorubicin (E) 5-Fluorouracil y = 2,9913x + 7,5319 R² = 0,9859 0,00 20,00 40,00 60,00 80,00 100,00 120,00 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 % I n h ib it io n Concentration (μg/ml) y = 0,6427x - 3,3458 R² = 0,9698 (10,00) 0,00 10,00 20,00 30,00 40,00 50,00 60,00 0,0 20,0 40,0 60,0 80,0 100,0 % I n h ib it io n Concentration (μg/ml) (A) (B) (C) (D) (E) Ega Hida Prabowo, Rifki Febriansah | The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell 6 Figure 4. 2D structure of interaction of Test Compounds on HER-2 in atomic pockets: (A) Native Ligand (B) EGCG (C) Tangeretin (D) Doxorubicin (E) 5-Fluorouracil Figure 5. Graphic of Living Cells T47D with CTM Extract Treatment (%) Figure 6. Changes in T47D cell morphology (A) Before the extract treatment (B) Shortly after the extract treatment (C) After the extract treatment (sign ‘’ indicates damaged or dead cells) y = -0,0024x + 4,6713 R² = 0,9233 0,000 0,500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 5,000 0 200 400 600 800 1000 1200 % L iv in g C e ll s Concentration (μg/ml) (D) (E) (B) (C) (A) (C) (A) (B) Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 7 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 glutamine at position 407 (GLN 407), glutamic acid at position 299 (GLU 299), glutamic acid at position 383 (GLU 383), threonine at position 301 (THR 301), glutamic acid at position 382 (GLU 382), lysine at position 347 (LYS 347), and phenylalanine at position 349 (PHE 349). It was higher than the original ligand, which only had five bonds. The tangeretin compound interacted by binding to 6 bonds interacting with the amino acid threonine at position 301 (THR 301), lysine at position 346 (LYS 346), lysine at position 347 (LYS 347), glutamine at position 383 (GLU 383), and valine at position 300 (VAL 300).17-20 The 5-FU ligand and tangeretin interacted with five bonds of amino acids, as many as the original ligand. The more the bonds are formed between the ligand and the target protein, the stronger the potential activity of a compound will be. It indicates that the EGCG compound in silico has a strong potential to inhibit HER-2 protein.21 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 Ega Hida Prabowo, Rifki Febriansah | The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell 8 phase to replicate DNA. If there is damage during the synthesis, it can be repaired before it finally enters the M phase. In cancer cells, the p53 gene is mutated. CONCLUSION It can be concluded that the combination of ethanolic extract of tea leaves and mandarin orange peel (CTM) had strong potential as an antioxidant agent in chemopreventive activity based on DPPH and molecular docking tests. However, the results of the cytotoxic test showed a weak potential to inhibit the development of T47D breast cancer cells. Some compounds that were considered to play an essential role in the chemopreventive mechanism were tangeretin and EGCG compounds. ACKNOWLEDGMENT The authors would like to thank the Ministry of Research, Technology, and Higher Education Republic of Indonesia (RISTEKDIKTI) for funding this research through the Beginner Lecturer Research grant in 2019. CONFLICT OF INTEREST There is no potential for conflict of interest. REFERENCES 1. Waks, A. G., & Winer, E. P. (2019). Breast cancer treatment: a review. Jama, 321(3), pp. 288-300. 2. Chatterjee, S., Mukherjee, M. M., Nath, N. C., Samanta, S., & Saha, A. K. (2017). An observational study on the incidence of HER-2/neu receptor over-expression and comparison of clinical presentation between HER- 2/neu positive and HER-2/neu negative breast cancer. International Surgery Journal, 4(2), pp. 506-513. 3. Miedema, J., & Andea, A. A. (2020). Through the looking glass and what you find there: making sense of comparative genomic hybridization and fluorescence in situ hybridization for melanoma diagnosis. Mod Pathol 33, pp. 1318–1330. 4. Amirifard, N., Sadeghi, E., Payandeh, M., Mohebbi, H., Sadeghi, M., & Choubsaz, M. (2016). Relationship between HER2 proto-oncogene status and prognostic factors of breast cancer in the west of Iran. Asian Pac J Cancer Prev, 17(1), pp. 295-298. 5. Márquez-Garbán, D. C., Gorrín-Rivas, M., Chen, H. W., Sterling Jr, C., Elashoff, D., Hamilton, N., & Pietras, R. J. (2019). Squalamine blocks tumor-associated angiogenesis and growth of human breast cancer cells with or without HER-2/neu overexpression. Cancer letters, 449, pp. 66-75. 6. Anastasiadi, Z., Lianos, G. D., Ignatiadou, E., Harissis, H. V., & Mitsis, M. (2017). Breast cancer in young women: an overview. Updates in surgery, 69(3), pp. 313-317. 7. Mulder, R. L., Van Der Pal, H. J. H., Levitt, G. A., Skinner, R., Kremer, L. C. M., Brown, M. C., ... & Frey, E. (2016). Transition guidelines: an important step in the future care for childhood cancer survivors. A comprehensive definition as Journal of Fundamental and Applied Pharmaceutical Science, 1(1), August 2020 9 groundwork. European Journal of Cancer, 54, pp. 64-68. 8. Rodríguez-García, C., Sánchez- Quesada, C., & Gaforio, J. J. (2019). Dietary flavonoids as cancer chemopreventive agents: An updated review of human studies. Antioxidants, 8(5), 137. pp. 1- 23. 9. Surichan, S., Arroo, R. R., Tsatsakis, A. M., & Androutsopoulos, V. P. (2018). Tangeretin inhibits the proliferation of human breast cancer cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1– mediated metabolism to the product 4′ hydroxy tangeretin. Toxicology in Vitro, 50, pp. 274-284. 10. Khopkar, S. M. (2014). Konsep Dasar Kimia Analitik. Penerjemah: Saptorahardjo, A. Jakarta: Universitas Indonesia. 11. Azwanida, N. N. (2015). A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med Aromat Plants, 4(196), pp. 2167-0412. 12. Ialongo, C. (2017). Preanalytic of total antioxidant capacity assays performed in serum, plasma, urine and saliva. Clinical Biochemistry, 50(6), pp. 356-363. 13. Mittal, L., Raman, V., Camarillo, I. G., & Sundararajan, R. (2017). Electrical pulse-mediated veliparib for effective treatment of triple negative breast cancer: an in vitro model study. Int. J. of Curr. Res. and Academic Review, 5, pp. 53-64. 14. Forli, S., Huey, R., Pique, M. E., Sanner, M. F., Goodsell, D. S., & Olson, A. J. (2016). Computational protein–ligand docking and virtual drug screening with the AutoDock suite. Nature protocols, 11(5), pp. 905- 919. 15. Dungir, S. G., Katja, D. G., & Kamu, V. S. (2012). Aktivitas antioksidan ekstrak fenolik dari kulit buah manggis (Garcinia mangostana L.). Jurnal MIPA, 1(1), pp. 11-15. 16. Rahardjo, T. B. W., & Purwaningsih, E. H. (2017). Life Cycle Concept. The Power Of Jamu (English Version), 85. 17. Cheemanapalli, S., Chinthakunta, N., Shaikh, N. M., Shivaranjani, V., Pamuru, R. R., & Chitta, S. K. (2019). Comparative binding studies of curcumin and tangeretin on up- stream elements of NF-kB cascade: a combined molecular docking approach. Network Modeling Analysis in Health Informatics and Bioinformatics, 8(1), p. 15. 18. Shi, X., Zhang, T., Li, X., Feng, Y., Tan, X., & Jin, Y. (2016). Interaction between tangeretin and ovalbumin to reduce the allergic effects of ovalbumin. Chemical Research in Chinese Universities, 32(4), pp. 556- 560. 19. Surichan, S., Arroo, R. R., Tsatsakis, A. M., & Androutsopoulos, V. P. (2018). Tangeretin inhibits the proliferation of human breast cancer cells via CYP1A1/CYP1B1 enzyme induction and CYP1A1/CYP1B1– mediated metabolism to the product 4′ hydroxy tangeretin. Toxicology in Vitro, 50, pp. 274-284. 20. Briguglio, M., Hrelia, S., Malaguti, M., Serpe, L., Canaparo, R., Dell’Osso, B., Ega Hida Prabowo, Rifki Febriansah | The Chemopreventive Effects of The Combination Between Tea Leaf and Mandarine Peel Extract on Breast Cancer Cell 10 ... & Banfi, G. (2018). Food bioactive compounds and their interference in drug pharmacokinetic/pharmacodynamic profiles. Pharmaceutics, 10(4), p. 277. 21. Peter, B., Bosze, S., & Horvath, R. (2017). Biophysical characteristics of proteins and living cells exposed to the green tea polyphenol epigallocatechin-3-gallate (EGCg): review of recent advances from molecular mechanisms to nanomedicine and clinical trials. European Biophysics Journal, 46(1), pp. 1-24. 22. Meiyanto, E., & Hermawan, A. (2012). Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents. Asian Pacific journal of cancer prevention: APJCP, 13(2), p. 427. 23. Chahar, M. K., Sharma, N., Dobhal, M. P., & Joshi, Y. C. (2011). Flavonoids: A versatile source of anticancer drugs. Pharmacognosy reviews, 5(9), p. 1. 24. Tung, Y. C., Chou, Y. C., Hung, W. L., Cheng, A. C., Yu, R. C., Ho, C. T., & Pan, M. H. (2019). Polymethoxyflavones: Chemistry and Molecular Mechanisms for Cancer Prevention and Treatment. Current Pharmacology Reports, 5(2), pp. 98- 113.