Two Limonoids from The Seeds of Chisocheton Macrophyllus and Their Cytotoxic Activity Against Mcf-7 Breast Cancer Cells

Limonoids (tetranortriterpenoids) are triterpenoid compounds that lose four terminals in their structural framework. These compounds have a wide variety of structures and interesting activities including anti-inflammatory, anticancer, anti-tumor and anti-malarial properties. The purpose of this study was to find limonoid compounds from the Indonesian Chisocheton plant, and one of which is Chisocheton macrophyllus. The dried and powdered seeds of C. macrophyllus (4.5 kg) were extracted with methanol and partitioned successively with n-hexane, ethyl acetate and n-butanol. Evaporation of each extract resulted in the crude extracts of n-hexane (346.6 g), ethyl acetate (60.8 g) and n-butanol (14.6 g). The n-hexane fraction was subjected to a silica gel vacuum-liquid chromatography (VLC) column packed with silica gel 60 using gradient of n-hexane, ethyl acetate and methanol (10% stepwise) to afford thirteen fractions (A-M). Fraction F (4.4 g) was subjected to silica gel column chromatography using gradient of n-hexane and ethyl acetate (5% stepwise). Subfraction F5 (1.2 g) was chromatographed on a column of silica gel eluted with n-hexane: CH2Cl2: EtOAc (2:7.5:0.5) to give compound 1 (19.7 mg) and fraction H (1.8 g) was subjected to silica gel column chromatography using gradient of nhexane and ethyl acetate (5% stepwise) as eluting solvent to give 2 (12.0 mg). Chemical structures of 1 and 2 were elucidated by spectroscopic methods and determined as 6α-acetoxyepoxyazadiradione (1) and Dysobinin(2). Dysobinin (2) showed weak cytotoxic activity against MCF-7 breast cancer cells with an IC50 value of 228.15 μM


INTRODUCTION
Limonoids are a class of tetranortriterpenoids that are formed through the loss of four terminal carbons of side chain of euphane (20-S) or tirucallane (20-R) skeleton that are followed by a cyclization to form a 17β-furan ring (Tan, & Luo, 2011;Shi et al., 2020). Limonoids are classified into ten classes based on the differences on A, B, C, D, and furan ring of the limonoid skeleton and can be identified by their biosynthetic relationships (Fang, Di, & Hao, 2011;Tan & Luo, 2011;Shi et al., 2020). Ten classes of limonoid include protolimonoids, apoeuphol skeleton, D-ring seco, B, D-ring seco, A-ring seco, A,B-ring seco, Cring seco, A,D-ring seco and B-ring seco limonoids.
Limonoids occur mainly in the plant order of Rutales and most of them are found in Meliaceae and Rutaceae families (Li, Peng, & Zheng, 2016). Limonoids isolated from species of the family of Meliaceae have been of interest due to their diverse structures and their biological activities, including antifeedant, anticancer, antimicrobial, antimalarial, and antiviral properties (Tan, & Luo, 2011;Wong et al., 2011;Gualdani, Cavalluzzi, Lentini, & Habtemariam, 2016;Shilpi et al., 2016;Chong et al., 2019;Supratman et al., 2020). Nimbolide is a major limonoid isolated from the leaves of Azadirachta indica A. Juss or known as neem tree. Nimbolide as a neem limonoid is widely used for anti-malaria, antibacterial activity against S. aureus and S. coagulase, anti-feedant and insecticidal activity (Kumar & Navaratnam, 2013;Bodduluru, Kasala, Thota, Barua & Sistla, 2014;Wang et al., 2016;Sophia et al., 2018). Nimbolide was presumed to be a more potent anticancer. Nimbolide shows anticancer activity throughout selective modulation of signaling pathways linked to inflammation, survival, growth, invasion, angiogenesis and metastasis. Nimbolide was reported to induce apoptosis by disruption of Mitochondrial Outer Membrane Potential (MOMP) and inhibits tumor cell proliferation through alterations of cyclins, cdks, PCNA and p53 levels. In addition, nimbolide also reducing the nuclear translocation and DNA-binding activity of NF-κB in cancer cells (Kumar & Navaratnam, 2013;Bodduluru, Kasala, Thota, Barua, & Sistla, 2014;Wang et al., 2016;Sophia et al., 2018). Beside nimbolide, other limonoids, such azadirachtin, salannin, nimbin and nimbic acid, have been isolated from A. indica (Wang et al., 2016). Azadirachtin-A (AzaA) is a prominent limonoid known as strong antifeedant and has been exploited commercially. AzaA is present in seed, leaves and other parts of A. indica. Natural pesticide like AzaA is widely used to control the insect. AzaA can keep the insect engaged in defensive while reducing food consumption. In silico studies suggests that AzaA accommodated in the hydrophobic pocket of juvenile hormone esterase and interact with active site residues. AzaA generally targets more than one protein and was presumed to be a potent biopesticide (Dawkar et al., 2019). Other limonoids from Meliaceae family also have potential applications in the food and pharmaceutical industries and have been used as food additives and pesticides (Gualdani, Cavalluzzi, Lentini, & Habtemariam, 2016;Shi et al., 2020).
Chisocheton plant is a genus from Meliaceae that consists of more than 50 species. The genus is distributed mainly in India, Thailand, Malaysia, Indonesia and becomes the second largest genus of family Meliaceae (Katja et al., 2016;Supriatno et al., 2018). Previous phytochemical studies on Chisocheton have discovered several limonoid compounds, such as malayanines A and B, two novel limonoids, that were isolated from the bark of Malaysian C. erythrocarpus Hiern (Chong et al., 2012), chisomicines D and E, two new limonoids, that have been isolated from the bark of Malaysian C. ceramicus (Miq.) (Najmuldeen et al., 2012), chisotrijugin, a trijugin-type limonoid, from the bark of C. cumingianus (Katja et al., 2016), and pentandricine, a new vilacinine-type limonoid, that was isolated from the stembark of C. pentandrus, together with ceramicine B, 6-de(acetyloxy)-23oxochisocheton, and 6-de(acetyloxy)-23-oxo-7-Odeacetylchisocheton that have been (Supriatno et al., 2018). Chisocheton genus has also been known as the producers of limonoid compounds with interesting biological activities, for example, ceramicine G and I from C. ceramicus, which have cytotoxic activity against MCF-7 breast cancer cells (Wong et al., 2011) and erythrocarpine E from C. erythrocarpus, which has anticancer properties against HSC-4 human oral cancer cells (Nagoor et al., 2011).
In order to investigate cytotoxic limonoids from Indonesian Chisocheton plants (Katja et al., 2016;Nurlelasari et al., 2017), we continue to carry out a phytochemical investigation on Chisocheton macrophyllus seeds. C. macrophyllus species are distributed in Nicobar Islands, peninsular Thailand, Peninsular Malaysia, Singapore, Sumatra, Anambas Islands, Java and also Borneo. C. macrophyllus is a higher plant with the tree up to 35 m tall. The oil isolated from C. macrophyllus seed has been used for lighting in Indonesia. The wood of C. macrophyllus are used as timber because it is not durable and splits easily (Vossen, & Umali, 2002;Nurlelasari et al., 2017). Previous investigation on limonoids from C. macrophyllus seeds has showed that the plant resulted in bioactive limonoids including dysobinol, 7αhydroxyneotricilenone, dysobinin and nimonol with cytotoxic activity against P-388 murine leukemia cells (Nurlelasari et al., 2017). In this paper, we describe the isolation, structure elucidation and cytotoxic properties against MCF-7 breast cancer cells of 6αacetoxyepoxyazadiradione (1) and dysobinin (2), isolated from C. macrophyllus seeds.

EXPERIMENTAL SECTION Material and instrumentation
Seeds of C. macrophyllus were collected from Bogor Botanical Garden, Bogor, West Java Province, Indonesia with voucher specimen (No. Bo-1295453).
IR spectra and mass spectra were recorded on an One PerkinElmer spectrum-100 FT-IR in KBr and Waters Xevo QTOF MS respectively. NMR spectra were obtained with JEOL JNM-ECZ500R/S1 at 500 MHz for 1 H and 125 MHz for 13 C. Chemical shifts are given on a δ (ppm) scale with tetramethylsilane (TMS) as an internal standard. Chromatographic separations were carried out on the silica gel 60 (70-230 and 230-400 mesh, Merck). TLC analysis was carried out on 60 GF254 (Merck, 0.25 mm) using various solvent systems, and detection by irradiating under ultraviolet-visible light Vilber Lourmat (λ 254 nm dan λ 365 nm) followed by heating of silica gel plates, sprayed with 10% H2SO4 in ethanol and Ehrlich's reagent (pdimethylaminobenzaldehyde).

Extraction and isolation
The dried and powdered seeds of C. macrophyllus (4.5 kg) was extracted with methanol at room temperature for 3 x 4 L x 24 hours. After the solvent removal under vacuum, a total 560 g of methanol extract was obtained and partitioned with n-hexane, ethyl acetate and n-butanol. Evaporation on each extract resulted the crude extracts of n-hexane (346.6 g), ethyl acetate (60.8 g) and n-butanol (14.6 g).
The n-hexane soluble fraction was subjected to a silica gel vacuum-liquid chromatography (VLC) column packed with silica gel 60 using gradient of nhexane, ethyl acetate and methanol (10% stepwise) to afford thirteen fractions (A-M). Fraction F (4.4 g) was subjected to silica gel column chromatography using gradient of n-hexane and ethyl acetate (5% stepwise) as eluting solvent to afford twelve subfractions (F1-F12). Subfraction F5 (1.2 g) was chromatographed on a column of silica gel eluted with n-hexane: CH2Cl2: EtOAc (2:7.5:0.5) to give compound 1 (19.7 mg). Fraction H (1.8 g) was subjected to silica gel column chromatography using gradient of n-hexane and ethyl acetate (5% stepwise) as eluting solvent to give 2 (12.0 mg).

Bioassays for cytotoxic activity
The cytotoxicity assay was conducted according to the method previously described by Examinati, Wulandari, Harneti, & Poniah (2018) and Supriatno et al., (2018). MCF-7 cells plated in 96 multiwell culture plates at a density of 1.7 × 10 4 cells/well. After twentyfour hours, medium was discarded and fresh medium containing sample with different concentrations 7. 81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00 g/mL and control was added. After incubation with sample for 24h, prestoBlue ® reagent (resazurin dye) was added. The PrestoBlue ® assay results were read using multimode reader at 570 nm. The IC50 values were determined by linier regression method using Microsoft Excel software. The IC50 value corresponds to the concentration of compounds that decreases 50% number of viable cells and the absorbance in control corresponds to 100% viability.

RESULTS AND DISCUSSION
The n-hexane extract from the seeds of C. macrophyllus was subjected to a silica gel vacuumliquid chromatography (VLC) column packed with silica gel 60 by gradient elution. The VLC fractions were repeatedly subjected to normal phase column chromatography on silica gel to yield compounds 1 and 2 (Figure 1).
Correlations from oxygenated sp 3 methine protons at δ H 3.40 (H-15) to δ C 208.1 (C-16) and δ C 50.9 (C-17) and correlations from δ H 3.86 (H-17) to δ C 28.7 (C-12), δ C 45.3 (C-13), δ C 208.1 (C-16), δ C 116.5 (C-20), δ C 141.7 (C-21) and δ C 111.0 (C-22) were used to assign position of an epoxide between C-14 and C-15, a carbonyl located on C-16 and a furan ring attached at C-17. Chemical structure of 1 was presumed to be the same as 6 acetoxyepoxyazadiradione due to the high similarity of the NMR chemical shifts that was previously reported (Table 1.) (Pereira et al., 2014). The indicating the relative stereochemistry of epoxide between C-14/C-15 of 1 are -oriented and acetyl group at C-6 and C-7 is -oriented. Therefore, compound 1 was identified as a 6 -acetoxyepoxyazadiradione and showed in this plant for the first time.
Compounds 1 and 2 were evaluated for their cytotoxic activity against MCF-7 breast cancer cell and cisplatin as a positive control according to a method previously described (Examinati, Wulandari, Harneti, & Poniah, 2018;Supriatno et al., 2018). 6 α -Acetoxyepoxyazadiradione (1) was found to be inactive and dysobinin (2)    Tabel 1. NMR data for compounds 1 dan 6α-acetoxyepoxyazadiradione (Pereira et al., 2014)  6 -Acetoxyepoxyazadiradione (1) and dysobinin (2) have been isolated from the seeds of C. macrophyllus. The discovery of 1 and 2 supported the occurrence of limonoid in the Chisocheton genus. Compound 1 and 2 were evaluated for their cytotoxic activity against MCF-7 breast cancer cell line. Compound 1 was inactive and compound 2 demonstrated weak cytotoxic activity (228.15 M) against MCF-7 breast cancer cell line. The bioassay data suggested that the carbonyl at C-16 and epoxide at C-14/C-15 in 6 α -acetoxyepoxyazadiradione (1) decreased the cytotoxic properties.