Morelloflavone and Molecular Docking from Stembark of Chisocheton lasiocarpus and Its Cytotoxic Activity Against Breast Cancer Mcf-7 Cell Lines

. Chisocheton is a plant of the Meliaceae family which has been known as a source of limonoids, triterpenoids, steroids, alkaloids


INTRODUCTION
Meliaceae has approximately 53 genera and 650 species. This family has been known as plants that produce various secondary metabolites and have interesting activities Laino et al., 2021;Nurlelasari et al., 2021). One of the genera from this family plant is Chisocheton which is widely distributed mainly in the tropic such as Malaysia, Thailand, Filipina, India, China, Papua New Guinea, Nepal, Myanmar, and Indonesia (Shilpi et al., 2016). Chisocheton plants traditionally have been used as a traditional medicine to treat several ailments including stomachache, backache, kidney complaints, fever, rheumatism, and malaria (Chan et al., 2012;Wang et al., 2021;Zhang et al., 2012).
This study aims to obtain a new compound from C. lasiocarpus which then tests its potential cytotoxic activity against MCF-7 breast cancer cells in vitro and in silico. Anticancer research is urgently needed to date because cancer treatment has many shortcomings because it not only kills cancer/tumor cells but also normal cells, and chemotherapy treatment can make cancer cells resistant to chemotherapeutic agents (Chang & Singh, 2017;Deng et al., 2021;Thrane et al., 2013).
Meanwhile, the International Agency for Research on Cancer stated that the number of cancer prevalence in 2020 was 13,587,202 cases of cancer in all genders and all ages, with 9,958,133 deaths. One type of cancer, namely breast cancer, occupies the first position in the number of prevalence in women, namely 2,261,419 cases and is the first highest cause of death in women with 684,996 cases. These numbers are predicted to increase every year (Sung et al., 2021). So that the search for natural compounds that have cytotoxic properties against cancer cells, one of which is breast cancer is very necessary.
In this paper, the isolation, structure determination, and cytotoxic activity of the flavanone-(3→8")-flavone biflavonoid (morelloflavone) and their inhibition of the target protein ER-α (PDB code: 3ERD), ER-β (PDB code: 1QKM), and HER-2 (PDB code: 3PP0) were described. In this experiment, doxorubicin was used as a positive control. Morelloflavone were first discovered in this species. The chemical structure of morelloflavone can be shown in Figure 1. The stembark of C. lasiocarpus were collected from Bogor Botanical Garden, Indonesia. Plant identifications were made from Bogoriense Herbarium, Indonesia and voucher specimen (VII. G. 168). The material for in silico we used three target protein: ER-α (PDB code: 3ERD), ER-β (PDB code: 1QKM) and HER-2 (PDB code: 3PP0) was obtained from Protein Data Bank (https://www.rcsb.org/, accesed on 12 January 2022). The tasted ligand is morelloflavone from C. lasiocarpus stembark (Compound 1, ID: 5464454) and ligan as positive control are genistein (ID 5280961) and doxorubicin (ID 31703) was obtained from PubChem compound database (https://pubchem.ncbi.nlm.nih.gov/). Instrumentation NMR spectra of 1 H, 13 C, 1 H-1 H COSY and HMBC were obtained using a Bruker AM 500 spectrometer at 500 MHz ( 1 H) and 125 MHz ( 13 C). Separations and identification were conducted with liquid chromatograph by Merck Si gel 60 (230-400 mesh), and thin-layer chromatograph (TLC) on aluminum plates coated with Merck Si gel 60 F254 and thickness of 0.25 mm, stain was observed on UV light and heated on the hotplate after spraying with 10% H2SO4 in ethanol.

Cytotoxic Assay
The cytotoxic assay compound 1 against breast cancer MCF-7 cell lines was determined by the method of Haryanti and Widiyastuti (2017). MCF-7 cells was incubated using EMEM medium containing 10% FBS and 100 µg/mL penicillin-streptomycin in incubator with 5% CO2 condition at 37 ºC for 48 hours. The cell suspension was transferred into all wells on microplate with density of 8000 cells/well in EMEM medium then incubated for 48 hours. Furthermore, Samples with various concentration was measured by transferred into well on the microplate containing the incubated cell, then re-incubated for 48 hours. After that, 100 µL 0,5% MTT salt in EMEM medium was added to each well and incubated at 37 ºC for 3 hours until the purple crystal formazan was seen, thus the crystal was deluted for overnight by 10% SDS in 0.01 N HCl as a stopper reagent. Furthermore, the colored formazan that has been produced was measured its absorbance using ELISA reader at wavelength of 595 nm. The IC50 value were taken from plotted graph of the percentage of live cells compared to the control (%).

Methods for Docking
Prepared of receptor and ligan.
Three target protein (ER-α, ER-β and HER 2) had prepared to remove water molecule and prepared between receptor and native ligand by Biovia Discovery Studio 2016 Client and saved in PDB format (Aertgeerts et al., 2011;Pike et al., 1999;Shiau et al., 1998), this can be seen in the Table 1 .
Morelloflavone (1), doxorubicin A and genistein as a ligand test was obtained from PubChem (SDF format) and converted to PDB format using Chem3D Pro 12. Receptor has prepared with Autodock Tolls 4.2 by adding kollman charge and hydrogen polar only. Meanwhile, all ligand had prepared with Autodock Tolls 4.2 by adding Gasteiger charge, hydrogen and marge non-polar. Receptor and ligan was saved in pdbqt format (Desdiani et al., 2020;Kelutur & Mustarichie, 2020).

Validation method.
The docking process used Autodock Tools 4.2, grid box parameter (Table 1) obtained had used for ligand test and genetic algorithm parameter which had set only number of GA 100 (100x). Its aimed to find the best position or conformation when receptor and ligan bind. The validation result that must be obtained is the RMSD value of redocking ≤ 3Å compared to the crystallographic results (Desdiani et al., 2020;Kelutur & Mustarichie, 2020). Docking ligand test and receptor.
The docking process used Autodock Tools 4.2 and carried out like validation. The molecular docking results obtained free energy bond (G). Visualization of orientation ligand in 2D and 3D bond macromolecules using Biovia Studio 2016 Client (Desdiani et al., 2020;Kelutur & Mustarichie, 2020

RESULTS AND DISCUSSION
The compound obtained, compound 1 (Figure 1), is a pale-yellow solid, was found to have a molecular formula of C30H20O11. However, in the 1 H NMR spectrum recorded in acetone-d6 at room temperature, the major peaks were accompanied by corresponding less intense peaks with close chemical shifts, indicated that the compound presented as two conformers. In solution, the 1 H and 13 C NMR signals of the major conformer were unequivocally assigned and listed in Table 2 since the minor conformer signals were generally of low intensity. The 1 H NMR spectrum of compound 1 showed an oxymethine proton at δH 5.86 (H-2) and methine proton at 4.99 (H-3) with the same coupling constant of 12.06 Hz and correlation in 1 H-1 H COSY indicated the presence of flavanone unit. Also 1 H NMR spectrum revealed p-disubstituted phenyl moiety at δH 7.24 (2H, d, J= 8.3 Hz, H-2', H-6') and 6.53 (2H, d, J= 8.3 Hz, H-3', H-5') and there is a singlet signal at δH 6,02 for 2H (H-6 and H-8) on meta position of ring A flavanone. The data also indicate the presence of flavone unit by showed signals due to 1,3,4-trisubstituted phenyl moiety, as supported by the hydrogen signals at δH 7.50 (1H, s, H-2'''), 7,02 (1H, d, J=8.4 Hz, H-5''') and 7.52 (1H, d, J=8.4 Hz, H-6'''). Additionally, two unsaturated methane protons were determinded by signals at δH 6.47 (1H, H-3'') and 6.30 (1H, H-6'') were observed in the 1 H NMR spectrum.
In the HMBC spectrum, the correlation of H-3/C-9'' were observed, suggesting that the linkage position of unit I and II was at C-3 and quaternary carbons C-8''. Therefore, the planar structure of compound 1 was elucidated to be a flavanone-(3→8'')-flavone biflavonoid, and with those data and comparison literature Jamila et al., 2014) could identify that structure of compound 1 as morelloflavone (Table 3) In this study, compound 1 was also tested for its cytotoxicity against breast cancer MCF-7 sell lines. The results indicated compound 1 considered as weak category with IC50 >100 µM that is 610 µM (Kuete & Efferth, 2015). In summary, Compound 1 is first reported biflavonoid isolated from C. lasiocarpus.
Doxurubicin and genistein are commonly used in treatment breast cancer. Genistein can enchane chemotherapeutic afficacy and overcome chemoresistance in breast cancer (Xue et al., 2014). The long duration treatment of breast cancer with doxorubicin can causes cancer cell resistance and has serious adverse effect to heart damage (Lovitt et al., 2018). Therefore, the development of anti-breast cancer is needed.
All protein were docked with morelloflavone (1), genistein and doxorubicin. According to the docking result, Morelloflavone has a highest activity on HER-2 protein with binding activity of -7.58 Kcal/mol. In ERα and ER-β, which has the highest activity is genistein with binding activity are -9.32 and -10.25, respectively. But when compared with doxuribicin, morelloflavone compounds have higher activity (Table  3). Binding affinity is the energy of intermolecular interaction between receptor and ligand. If the binding affinity value was lower, it's meant that ligand had more significant potential to interact with receptor and indicates a good activity (Kelutur & Mustarichie, 2020;Riyana et al., 2020).
The interaction of amino acid residues between receptor and ligand was in hydrogen and Van der Waals bond (Table 4-6 and Figure 3-5). The similarity of residue amino acid showed that the ligand had the same binding pocket (Herdiyati et al., 2020

CONCLUSIONS
Morelloflavone has been isolated for the first time from stembark of C. lasiocaropus and the cytotoxic activity of this compound considered as inactivity against breast cancer MCF-7 cell lines with IC50 value of 610 µM. The results of molecular docking analysis showed that morelloflavone had a highest activity on HER-2 protein with binding activity of -7.58 Kcal/mol. Whereas in ER-α and ER-β proteins, morelloflavone activity was greater than doxuribicin with binding activity are -9.32 and -10.25, respectively. Therefore, morelloflavone have a potency to develop as new antibreast cancer agent.

ACKNOWLEDGMENTS
This investigation was financially founded by PDUPT grant (