Natural Product Research Formerly Natural Product Letters ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20 A new limonoid from stem bark of Chisocheton pentandrus (Meliaceae) Supriatno, Nurlelasari, Tati Herlina, Desi Harneti, Rani Maharani, Ace Tatang Hidayat, Tri Mayanti, Unang Supratman, Mohamad Nurul Azmi & Yoshihito Shiono To cite this article: Supriatno, Nurlelasari, Tati Herlina, Desi Harneti, Rani Maharani, Ace Tatang Hidayat, Tri Mayanti, Unang Supratman, Mohamad Nurul Azmi & Yoshihito Shiono (2018): A new limonoid from stem bark of Chisocheton pentandrus (Meliaceae), Natural Product Research, DOI: 10.1080/14786419.2018.1428600 To link to this article: https://doi.org/10.1080/14786419.2018.1428600 View supplementary material Published online: 25 Jan 2018. Submit your article to this journal Article views: 5 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Natural Product research, 2018 https://doi.org/10.1080/14786419.2018.1428600 A new limonoid from stem bark of Chisocheton pentandrus (Meliaceae) Supriatnoa Nurlelasaria, Tati Herlinaa, Desi Harnetia, Rani Maharania,b, Ace Tatang Hidayata,b, Tri Mayantia, Unang Supratmana,b, Mohamad Nurul Azmic and Yoshihito Shionod a Faculty of Mathematics and Natural sciences, department of chemistry, universitas Padjadjaran, Jatinangor, Indonesia; bcentral laboratory, universitas Padjadjaran, Jatinangor, Indonesia; cschool of chemical sciences, universiti sains Malaysia, Minden, Malaysia; dFaculty of agriculture, department of Food, life and environmental science, Yamagata university, tsuruoka, Japan ABSTRACT ARTICLE HISTORY A new limonoid, pentandricine (1), along with three known limonoids, ceramicine B (2), 6-de(acetyloxy)-23-oxochisocheton (3), 6-de(acetyloxy)-23-oxo-7-O-deacetylchisocheton (4), have been isolated from the stembark of Chisocheton pentandrus. The chemical structures of the new compound were elucidated on the basis of spectroscopic evidence. All of the compounds were tested for their cytotoxic effects against MCF-7 breast cancer cells. Compounds 1–4 showed weak and no cytotoxicity against MCF-7 breast cancer cells with IC50 values of 369.84, 150.86, 208.93 and 120.09 μM, respectively. received 6 december 2017 accepted 9 January 2018 KEYWORDS Chisocheton pentandrus; limonoid; pentandricine; Meliaceae; McF-7 breast cancer 1. Introduction Chisocheton is genus belong to Meliaceae family, consist more than 50 plant species that are distributed mainly in India, Thailand, Malaysia and Indonesia (Heyne 1982; Yang et al. 2009). The genus Chisocheton was distributed in the tropical regions and widely known for CONTACT unang supratman unang.supratman@unpad.ac.id supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1428600. © 2018 Informa uK limited, trading as taylor & Francis Group 2 SUPRIATNO ET AL. its insecticidal limonoid constituents (Jain and Tripathi 1993; Roy and Saraf 2006). Previous phytochemical studies on Chisocheton species have yielded a number of interesting compounds, including limonoids (Connolly et al. 1979; Gunning et al. 1994), antifungal meliacin-type compounds (Bordoloi et al. 1993), dammarane-type triterpenoids, with an inhibitory effect on Epstein-Barr virus activation (Inada et al. 1993) and spermidine alkaloids (Tzouros et al. 2004). In our continous search for novel cytotoxic constituents from Indonesian Chisocheton plants, we isolated a new limonoid, dysobinol, from the seed of C. macrophyllus (Nurlelasari et al. 2017), and a lanostane-type triterpenoid from the bark of C. cumingianus (Katja et al. 2016). In the further screening for novel cytotoxic compounds from Indonesia Chisocheton plants, we found that the methanol extract of Chisocheton pentandrus showed a significant cytotoxic activity against MCF-7 breast cancer cells. In this paper, we report the isolation and structural elucidation of a new limonoids, pentandricine (1) and known limonoid compounds 2–4, along with their cytotoxic activity against MCF-7 breast cancer cells. 2. Results and discussion The n-hexane extract of the bark of C. pentandrus was chromatographed over a vacuum-liquid chromatographed (VLC) column packed with silica gel 60 by gradient elution. The VLC fraction were repeatedly subjected to normal and reverse phase column chromatography and preparative TLC on silica gel GF254 to afford compounds 1–4 (Figure 1). Pentandricine (1) was obtained as a colourless amorphous solid. Its molecular composition was established to be C26H32O6 from a combined analysis of the HR-ESI-TOFMS spectra (m/z 439.2025 [M + H]− and NMR data, thus requiring 11 degree of unsaturations. The UV spectrum showed an absorption maximun at 250 nm (log ε 4.2), indicating the presence of a conjugated carbonyl group. The IR spectrum showed bands which were ascribed to hydroxyl (νmax 3540 and 3450 cm−1), an α,β-unsaturated carbonyl (νmax 1690 cm−1), a conjugated ester (νmax 1710 cm−1), and an ether group (νmax 1130 and 1108 cm−1). The 1H NMR spectrum showed four tertiary methyls (δH 1.25, 1.17, 1.13 and 0.82, each 3H), four sp2 methine protons at δH 6.01 (1H, d, J = 5.6 Hz), 5.43 (1H, br.s), 5.73 (1H, d, J = 9.4 Hz) and 7.13 (1H, d, J = 9.4 Hz), one oxygenated methylene proton at δH 3.62 (1H, d, J = 7.2 Hz) and 3.43 (1H, d, J = 7.2 Hz), three oxygenated methine protons at δH 4.20 (1H, d, J = 3.6 Hz), 4.46 (1H, dd, J = 12.3, 3.6 Hz) and a hemiacetal protons at δH 6.01 (1H, d, J = 5.6 Hz). In addition, three sp3 methylenes at δH 2.50 (1H, m), 2.20 (1H, m), 1.80 (1H, m), 1.62 (1H, m), 2.41 (1H, m) and 1.70 (1H, m) as weel as three sp3 methines at δH 3.16 (1H, m), 2.22 (1H, m) and 2.59 (1H, d, J = 12.3 Hz) also were observed in 1H NMR spectrum. The 13C NMR together with the DEPT spectra revealed 26 carbon signals, including a carbonyl [δC 203.7 (s)], a carbonyl ester [δC 171.6 (s)], four sp2 methines [δC 153.4 (d), 129.2 (d), 119.5 (d) and 118.7 (d)], two sp2 quaternary carbon [δC 158.8 (s) and 132.7 (s)], three sp3 oxygenated methine carbons [δC 99.4 (d), 73.9 (d) and 72.6 (t)], one oxygenated methylene carbon at δC 79.2 (d), three sp3 methylenes, three sp3 methines, four sp3 quaternary and four methyls carbons. These functionalities accounted for five out of the total eleven degrees of unsaturation. The remaining six degrees of unsaturation were consistent with the limonoid containing six rings (Mohamad et al. 2009). A comparison of the NMR data of 1 with those of ceramicine D isolated from Chisocheton ceramicus (Mohamad et al. 2009) revealed that the structures of the two compounds are closely related, the main difference were the absence of an oxygenated NATURAL PRODUCT RESEARCH 3 Figure 1. structures of compounds 1–4. methylene at [δC 70.3, δH 4.77 (br. s)] and appeareance of an oxygenated methine in downfield region at [δC 99.4, δH 6.01 (d, J = 6.8 Hz), suggesting that 1 was a hydroxyl derivative of ceramicine D to form a hemiacetal group at C-23. In order to clarify the position of a newly hydroxyl group, the H–H COSY and HMBC experimets were carried, and the results was shown in Figure S2. The 1H–1H COSY spectrum of 1 showed correlations in H1-H2, H6-H7-H8, H9-H10-H11, H15-H16-H17 and H22-H23, supporting the presence of limonoid structure in 1. In the HMBC spectrum, the correlations arising from the tertiary methyl protons to their neighbouring carbons enabled the assignment of the four singlet methyls. Furthermore, an olefinic protons at δH 5.73 and 7.13 are couple each other and were correlated to carbonyl at δC 203.7 (C-1) indicated that an α,β-unsaturated carbonyl was located at C-1, C-2 and C-3, respectively. Correlation from an olefinic proton at H-22 (δH 6.01) to oxygenated carbon C-23 (δC 99.4) and an oxygenated proton at δH 6.01 to C-20 (δC 132.7) and C-21 (δC 171.6) were used to assign a new hydroxyl group was located at C-23. The relative configuration of 1 was determined by NOESY experiment (Figure S3) and by comparison with those similar compound previously reported, ceramicines D (Mohamad et al. 2009) and walsogyne A (Mohamad et al. 2008). NOESY correlations of H-6/CH3-19, H-7/CH3-30 indicated that H-6 and H-7 were each β-configuration. The α-configuration of H-9 and H-5 were assigned by the NOESY cross peaks of H-9/CH3-18 and H-5/H-9. The NOESY correlations between CH3-18/H-23 and CH318/H-9, supported that the hydroxyl group at C-23 was β-oriented. The stereochemistry at hemiacetal C-23 was assigened not to be epimerised because only single signal at NMR 4 SUPRIATNO ET AL. spectra (C-20, δC 132.7; C-22, δC 118.7; C-23, δC 99.4) and quite different to those of walsogyne A, isolated from Walsura chrysogyne (Mohamad et al. 2008). The NOESY cross peak also observed between CH3-30/H-17 indicated that the α-pyrone ring at C-17 was α-oriented. Other correlations in the NOE spectra supported that the relative configuration of 1 was similar to those of ceramicines D (Mohamad et al. 2009), therefore, the structure of 1 was elucidated as the new limonoid and, namely pentandricine. The known compounds ceramicines B (2) (Mohamad et al. 2009), 6-de(acetyloxy)-23oxochisocheton (3) (Gunning et al. 1994) and 6-de(acetyloxy)-23-oxo-7-O-deacetylchisocheton (4) (Gunning et al. 1994) were identified by comparison of their spectroscopic data with reported values. The cytotoxic activity of the isolated compounds 1–4 was evaluated against the against MCF-7 breast cancer cells according to a method described (Skehan et al. 1990) and Cisplatin (IC50 27.0 μM) was used as a positive control (Hadisaputri et al. 2012). Based on the IC50 value of compounds 1, 3 and 4, compound 4 showed stronger activity, suggesting that the presence of an acetyl, hydroxyl in lactone ring and ether ring seems to increase the cytotoxic activity. 3. Experimental 3.1. General experimental procedures Optical rotations were recorded on an ATAGO AP-300 automatic polarimeter. UV spectra were measured using a TECAN Infinite M200 pro, with MeOH. The IR spectra were recorded on SHIMADZU IRPrestige-21 in KBr. The mass spectra were recorded with a Waters Xevo QTOF MS. NMR data were recorded on a Bruker Topspin spectrometer at 500 MHz for 1H and 125 MHz for 13C using TMS as internal standard. Column chromatography was conducted on silica gel 60. TLC plates were precoated with silica gel GF254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% H2SO4 in EtOH, followed by heating and irridiation under ultraviolet–visible light at a wavelength of 257 and 364 nm. 3.2. Plant material The stem bark of C. pentandrus was collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia in June 2016. The plant was identified by the staff of the Bogoriense Herbarium, Bogor, Indonesia and a voucher specimen (No. Bo-104) was deposited at the Herbarium. 3.3. Extraction and isolation The dried ground stembark (1.8 kg) of C. pentandrus was extracted with methanol exhaustively (14 L) at room temperature for 7 days. After removal of the solvent under vacuum, the viscous concentrate of MeOH extract (340.01 g) was first suspended in H2O and then partitioned with n-hexane, EtOAc, and n-butanol, successively. Evaporation resulted in the crude extracts of n-hexane (10.90 g), EtOAc (25.18 g), and n-butanol (228.63 g), respectively. The n-hexane soluble fraction (10.90 g) was fractionated by column chromatography on silica gel using a gradient n-hexane, EtOAc and MeOH to give fractions A–H, combined according NATURAL PRODUCT RESEARCH 5 to TLC results. Fraction B (5.39 g) was subjected to column chromatography over silica gel using a gradient mixture of n-hexane-CH2Cl2-EtOAc (5% stepwise) as eluting solvents to afford thirteen subfractions (B1-B13). Subfraction B9 (912.2 mg) was chromatographed on a column of silica gel, eluted with n-hexane:CH2Cl2:EtOAc (5:4:1), to give 6 subfractions (B9A– B9F), subfraction B9C to give (2) (62.2 mg). Subfraction B9E was chromatographed on a column of silica gel, eluted with n-hexane:EtOAc (6:4), to give (3) (2.2 mg). Fraction C (698.8 mg) was subjected to column chromatography over silica gel using a gradient mixture of n-hexane-CH2Cl2-EtOAc (5% stepwise) as eluting solvents to afford nine subfraction (C1-C9). Subfraction C2 (126.1 mg) was chromatographed on a column of silica gel, eluted with n-hexane:CH2Cl2:EtOAc (5:1.5:3.5), to give six subfractions (C2A–C2F), Subfraction C2B (16.8 mg) was chromatographed on preparative TLC, eluted with n-hexane:CH2Cl2:EtOAc (4:2.5:5), to give (3) (3.4 mg) and (4) (4.6 mg). Fraction E (819.8 mg) was chromatographed on a column of silica gel, eluted with n-hexane:CH2Cl2:EtOAc (5:1.5:3.5), Subfraction E4 (61.2 mg) was chromatographed on a column of silica gel, eluted with CH2Cl2:EtOAc (6.5:3.5), to give (1) (23.4 mg). ◦ Pentandricine-A (1), colourless amorphous solid, [𝛼]20 D + 39 (c 0.1, DMSO), UV (MeOH) λmax 230 nm (log ε 4.2); IR (KBr) vmax 3535, 3457, 2862, 1720, 1457, 1387, 1247 cm−1; HR-TOFMS m/z 439.2025 [M + H]-, (Calcd C26H32O6 m/z 440.2121). 1H NMR (DMSO, 500 MHz): δH 7.13 (1H, d, J = 9.4 Hz, H-3), 6.06 (1H, d, J = 5.6 Hz, H-22), 6.01 (1H, d, J = 5.6 Hz, H-23), 5.73 (1H, d, J = 9.4 Hz, H-2), 5.43 (1H, br.s, H-15), 4.46 (1H, dd, J = 12.3, 3.6 Hz, H-6), 4.20 (1H, d, J = 3.6 Hz, H-7), 3.62 (1H, d, J = 7.2 Hz, H-28a), 3.43 (1H, d, J = 7.2 Hz, H-28b), 3.16 (1H, m, H-17), 2.59 (1H, d, J = 12.3 Hz, H-5), 2.50 (1H, m, H-16a), 2.41 (1H, m, H-11a), 2.22 (1H, m, H-9), 2.20 (1H, m, H-16b), 1.70 (1H, m, H-11b), 1.80 (1H, m, H-12a), 1.62 (1H, m, H-12b), 1.25 (3H, s, CH3-29), 1.17 (3H, s, CH3-19), 1.13 (3H, s, CH3-30), 0.82 (3H, s, CH3-18); 13C NMR (DMSO, 125 MHz): 203.7 (C-1), 171.6 (C-21), 158.8 (C-14), 153.4 (C-3), 132.7 (C-20), 129.2 (C-2), 119.5 (C-15), 118.7 (C-22), 99.4 (C-23), 79.2 (C-28), 73.9 (C-6), 72.6 (C-7), 53.4 (C-17), 41.9 (C-4), 47.7 (C-5), 47.1 (C-13), 46.9 (C-8), 46.9 (C-11), 35.8 (C-10), 34.3 (C-12), 33.2 (C-16), 26.1 (C-30), 22.4 (C-18), 18.1 (C-12), 14.3 (C-19), 14.3 (C-29). 3.4. Bioassays for cytotoxic activity (Skehan et al. 1990) The MCF-7 cells were seeded into 96-well plates at an initial cell density of approximately 3 × 104 cells cm−3. After 24 h of incubation for cell attachment and growth, varying concentrations of samples were added. The compounds added were first dissolved in DMSO at the required concentration. Subsequent six desirable concentrations were prepared using PBS (phosphoric buffer solution, pH 7.30–7.65). Control wells received only DMSO. The assay was terminated after a 48 h incubation period by adding MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; also named as thiazol blue] and the incubation was continued for another 4 h, in which the MTT-stop solution containing SDS (sodium dodecyl sulphate) was added and another 24 h incubation was conducted. Optical density was read using a micro plate reader at 550 nm. IC50 values were taken from the plotted graph of percentage live cells compared to control (%), receiving only PBS and DMSO, vs. the tested concentration of compounds (μM). The IC50 value is the concentration required for 50% growth inhibition. Each assay and analysis was run in triplicate and averaged. 6 SUPRIATNO ET AL. 4. Conslusions A new limonoid compound, namely pentandricine (1), along with three known limonoidcompounds, 2–4, were isolated from the stembark of C. pentandrus. Compounds 1–4 showed weak and no cytotoxic activity against MCF-7 breast cancer cells with IC50 values of 369.84, 150.86, 208.93 and 120.09 μM, respectively, indicating the presence of an acetyl group, hydroxyl group in lactone ring and ether ring in compounds 1, 3 and 4, seems to increase the cytotoxic activity. Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by the Lembaga Pengelola Dana Pendidikan (LPDP); the Directorate General of Higher Education, Ministry of Research, Technology and Higher Education, Indonesia (under LPDP post graduate grand, 2016–2017, by Supriatno) [grant number 718/UN6.3.1/PL/2017]. References Bordoloi M, Saikia B, Mathur RK, Goswami BN. 1993. A meliacin from Chisocheton panniculatus. Phytochemistry. 34:583–584. Connolly JD, Labbe C, Rycroft DS, Taylor DAH. 1979. Tetranortriterpenoids and related compounds. Part 22. New apotirucallol derivatives and tetranortriterpenoids from the wood and seeds of Chisocheton paniculatus (Meliaceae). J Chem Soc Perkin Trans I. 12:2959–2964. Gunning PJ, Jeffs LB, Isman MB, Towers GHN, Ibrahim KS. 1994. Two limonoids from Chisocheton microcarpus. Phytochemistry. 5:1245–1248. Hadisaputri YE, Pharm D, Miyazaki T, Suzuki S, Yokobori T, Kobayashi T, Tanaka N, Inose T, Sohda M, Kuwano H. 2012. 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