A bioactive compound isolated from Duku ( Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line

Background: Breast cancer is a major health problem for women globally. Many attempts have been promoted to cure cancer by finding new anticancer medicines from natural resources. Despite the richness of biodiversity discovered, there are some natural resources that remain unexplored. Fruit peels of Duku ( Lansium domesticum Corr.) are rich with compounds that may have the potential to be developed as anticancer drugs. This study aimed to isolate cytotoxic compounds from the fruit peels of L. domesticum and assess their cytotoxic nature against T47D cells. Methods: Powdered peels were macerated with ethyl acetate and the filtrate was evaporated to give EtOAc extract A. Dried extract A was triturated with n-hexane to give n-hexane soluble fraction B and insoluble fraction C. The cytotoxic nature of these three samples were assessed using MTT assay using T47D cells and doxorubicin as a control. Results: Fraction C that showed the smallest IC50 (25.56 ± 0.64μg/mL) value compared to extract A and fraction B. Fraction C was further fractionated by vacuum liquid chromatography to give 6 subfractions. Subfraction 2 showed a single compound based on thin layer chromatography, and this compound was identified as Lamesticumin A on the basis of its spectroscopic data. Lamesticumin A demonstrated cytotoxic activity against T47D cell lines with an IC 50 value of 15.68 ± 0.30µg/mL. Conclusions: Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.


Introduction
The most frequent cancer in women and that which causes the highest mortality is breast cancer.In Indonesia, it was reported that approximately 21% of cancer deaths among women were due to breast cancer 1 .Therefore, new medicines to eradicate this type of cancer is required.Duku (Lansium domesticum Correa) widely grows in Indonesia.Traditionally, L. domesticum bark and seeds have been used to treat dysentery and fever 2 .Based on previous studies, chloroform and methanol extracts of L. domesticum displayed cytotoxic activity on murine melanoma (B 16 F 10 ) and colon cancer (HT29) cells 3 .In addition, it has been shown that ethanol and ethyl acetate fractions of the peel have a deterrent activity on DNA damage in lymphoblast cells induced by H 2 O 2 exposure 4 .Onoceranoidtype of triterpenoids have been isolated from twigs and leaves of L. domesticum, and these compounds showed antibacterial and antimutagenic activities 5,6 .In this study, the cytotoxic effects of compound extracted from the peels of L. domesticum are assayed against breast cancer T47D cells.

Plant material
The fruits of L. domesticum were collected on March 2018 from Bantul, Yogyakarta (GPS : -7.871098, 110.394854) and identified at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada.

Extraction and fractionation
The peel was separated from the fruit, dried in oven 50°C for 24 hours and powdered using a blender.Powdered L. domesticum fruit peel (200 g) was macerated with ethyl acetate (EtOAc; 2 L) overnight.This solution was filtrated (0.45μm) and the filtrate was evaporated to dryness with a rotary evaporator set at 50°C, to give dried EtOAc extract (A, 50.13 g).In order to separate the extract into non-polar and polar fractions, 6 g of extract A was diluted 5 times using n-hexane (20 mL) to give soluble n-hexane fraction B (supernatant; 3.03 g) and insoluble n-hexane fraction C (residue; 2.83 g).
Fraction C was the most active among other fractions (see Results), and was therefore further fractionated using vacuum liquid chromatography as described by Mae Sri Hartati et al. 8 .In brief, using silica gel preparation grade (15 g) as stationary phase this was eluted with n-hexane and increasing amounts of ethyl acetate.Six subfractions were obtained and subfraction 2 contained a major compound which appeared as white crystals (referred to as compound 1).Compound 1 was obtained as a single compound from subfraction 2, while the other subfractions still contained various compounds.Compound 1 (142.6 mg), had a melting point at 140-150 o C (Figure 1).Compound 1 was identified using spectroscopy data such as ultraviolet (UV), infrared (IR), 13 C-NMR and 1 H-NMR (see section Chemicals and equipment).

Cytotoxic assay
The bioassay followed the methodology described by Bahuguna et al. 9 with modifications.In brief, 100 μl T47D and Vero cell line (in RPMI mediaFaculty of Medicine, Universitas Gadjah Mada) were placed in each well of a 96 well micr oplates, resulting in 1 × 10 4 cells/well.The cells were incubated for 24 hours at 37°C in a CO 2 incubator.
Absorbance was measured by microplate reader (Bio Rad) at 595 nm.positive control The data generated were used to plot a dose-response curve and IC 50 of the samples was determined.

Statistical analysis
The IC 50 values were analyzed by one-way ANOVA with statistical significance P < 0.05 using IBM SPSS ver.23.

Compound 1 characterization
Identified as Lamesticumin A.

Amendments from Version 1
In this version, we present cytotoxic data on normal cells (Vero cell line) in Table 2 and Figure 3 has been revised as recommended by the reviewer for major point revision.Besides, we also present the minor points revision such as grammar and typological errors.The infrared spectroscopy (KBr) spectrum of 1 showed a broad band at 3400-2800 cm -1 , which indicated the presence of -OH group, specifically -COOH due to intermolecular bonding.This data is supported by the appearance of -C=O at 1712 cm -1 .Compound 1 displayed UV absorption at 236,5 nm.The 13 C-NMR spectrum (500 MHz, CDCl 3 ) of compound 1 showed 30 carbons (Table 1).The IC 50 of the isolated compound from fraction C, compound 1/Lamesticumin A was 15.68 μg/mL.All samples inhibited T47D cell growth in a dose dependent behavior (Figure 3).

Discussion
In this study, the cytotoxic activity of Lamesticumin A, derived from the peel of L. domesticum, was demonstrated in the T47D cell line with IC 50 15.68 (μg/ml).The T47D cell line is an epithelial breast cancer cell subtype luminal A cell line that express estrogen and progesterone receptors 10 .Based on National Cancer Institute guidelines, a natural compound has potent anticancer activity if it has IC 50 <4 μg/ml or 10 μM 11 .
Many triterpenoid compounds have been previously isolated from L. domesticum.Most of these compounds are UV inactive or have no strong UV absorbance because triterpenoid's lack of a conjugated functional group 12 .Lansiosida A and Dukunolida A has been isolated from n-hexane extract of L. domesticum fruit peel 13,14 .Lamesticumin A is an onoceranoid-type triterpenoid, isolated previously from L. domesticum twigs, that has antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, Micrococcus pyogenes and Bacillus cereus with minimum inhibitory concentration of <15 μg/ml 5 .Another onoceranoid-type triterpenoid  Lansium acid I-IX were isolated from L. domesticum leaves, which was reported to have antimutagenic activity 6 .
Based on several in vitro tests, some terpenoid compounds had anticancer activity.Sesquiterpene lactone compounds are known to inhibit Nf-kB, thereby inducing apoptosis 15 .Celastrol has anticancer properties by regulating various transcription factors, angiogenesis processes, cell cycle arrest and induction of apoptosis 16 .Betulinic acid can induce apoptosis in HT-29 colon cancer cells and acts as a chemosensitizer for chemotherapeutic agents in wildtype adenocarcinoma cancer cells (SNU-C5/WT) 17 .Clematangoticosides D and F from Clematis tangutica are known to have cytotoxic activity against human gastric cancer cell line (SGC-7901) with IC 50 24.22 and 21.35 μM, respectively 18 .Cycloartane-type and oleanane-type triterpenoids from Ligularia przewalskii show cytotoxicity in Hela, HEPG2, SGC7901, MDA-MB-231, HL-60, and Lewis cell lines with IC 50 8.40-24.39μM 19 .
It has been reported that natural compounds combined with low doses of antineoplastics can increase effectiveness and reduce toxic effects 20 .Betulinic acid can induce apoptosis when combined with 5-fluorouracil, irinotecan and oxaliplatin 4 .Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anticancer activity through interfering with multiple signaling pathways.Furthermore, UA has been shown to act as a chemosensitizing agent to increase the effect of conventional anticancer drugs 21 , and to increase the effect of doxorubicin by increasing the cellular amount of the drug in the MCF-7 cell line 22 .Further study is needed to investigate the possibility of Lamesticumin A to be combined with doxorubicin for its potential to have synergistic effect.

Conclusions
Extract, fractions and Lamesticumin A derived from the peel of L. domesticum showed cytotoxic activity against the T47D breast cancer cell line.Further research is needed to investigate the potential of the natural compound Lamesticumin A derived from L. domesticum fruit peel as an anticancer therapy.
This project contains the following underlying data: -UV, infrared, 13 C-NMR and 1 H-NMR spectra of compound 1. -

Ratana Banjerdpongchai
Department of Biochemistry, Chiang Mai University, Chiang Mai, Thailand Major points: The extracts and purified bioactive compound, Lamesticumin A, from this plant, is not novel, since it was isolated from the twigs from this same herb in the previous report.The anticancer activity of the active compound is not promising since it is more than 4 microgram/mL or 10 micromolar, the level of which the National Cancer Institution defines is 4 microgram/mL or less than this.However, the authors discussed of using this compound as a chemosensitizing agent, which various steps still are needed before launching it as a therapeutic anti-cancer drug.The authors should explore the cytotoxic effects of the extracts, EtOAc extract (A), N-hexane fraction (B), Nhexane insoluble fraction (C) and compound 1 (Lamesticumin A) on normal cells.
In Figure 3, the percent cell viabilities of EtOAc extract (A), N-hexane fraction (B), N-hexane insoluble fraction (C) and compound 1 (Lamesticumin A) should be presented with the statistical significance by using asterisks, such as *p<0.05,**p<0.01,and so on, above the dots.

Minor points:
The sources of chemicals and instruments should indicate the city and country that they are produced from.Sometimes the authors used gr and sometimes g, it should be consistent with each other.In subtopic "Cytotoxic assay", in 3 rd -4 th lines, the authors should rewrite the sentences.There are many grammar and various typological errors, such as the number of g it should be 43.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? No If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests:
Cell viability and IC 50 values of extract A, fractions B and C, compound 1 and doxorubicin in T47D cell line.Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes
4 rather than 43,4, etc.For the previous findings, the sentences should be in present tense, but for the authors' research data in the Results and Discussion parts, the sentences would be in past tense.For MDA231, it should be MDA-MB-231 breast cancer cells.For MCF, it should be clarified as MCF-7 or not?In Ref. No. 12, there is no information of company, city, country of publication, is it a book?Ref. No. 21, the name of journal is full name, whereas others are in abbreviated forms.It should be consistent with each other.References 1. Manosroi A, Jantrawut P, Sainakham M, Manosroi W, et al.: Anticancer activities of the extract from Longkong (Lansium domesticum) young fruits.Pharm Biol.2012; 50 (11): 1397-407 PubMed Abstract | Publisher Full Text