Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 ALCHEMY Jurnal Penelitian Kimia Laman resmi: https://jurnal.uns.ac.id/alchemy A New Limonoid from the Seeds of Chisocheton lasiocarpus (Meliaceae) Ronauli Fitrianaa, Nurlelasari Nurlelasaria, Darwati Darwatia, Desi Harnetia, Rani Maharania,b, Tri Mayantia, Unang Supratmana,b a Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor 45363, Indonesia b Central Laboratory of Universitas Padjadjaran, Jatinangor 45363, Indonesia. Corresponding author: nurlelasari@unpad.ac.id * DOI: 10.20961/alchemy.17.2.44782.219-226 Received 11 October 2020, Accepted 08 June 2021, Published 09 September 2021 Keywords: limonoids; chisocheton; lasiocarpines; 14β,15βepoxynimonol. ABSTRACT. A New Limonoid from the Seeds of Chisocheton lasiocarpus (Meliaceae). Chisocheton is one of Meliaceae genus, which has about 53 species spreading in subtropical and tropical regions. One of the species is Chisocheton lasiocarpus. Chisocheton is rich in limonoids that have various biological activities such as anticancer, antimalarial, anti-inflammatory, antifeedant, antiviral, neuroprotective, and antimicrobial properties. This study aims to isolate limonoids from the seeds of C. lasiocarpus, and determine the structures. The dry powder of seeds of C. lasiocarpus (203.75 g) was macerated with subsequent n-hexane, ethyl acetate, and methanol. Extract of ethyl acetate was separated and purified using chromatography methods until a new limonoid, lasiocarpines (1) and one known limonoid, 14β,15β-epoxynimonol (2) were obtained. The purification process was guided by Ehrlich reagent. The chemical structures were identified by UV, IR, 1H-NMR, 13C-NMR, 2D NMR, and mass spectrometry. INTRODUCTION Limonoids are triterpenoid compounds which degrade on their four carbon side chains. This group of compounds has various biological properties including insecticidal, antibacterial, antimalarial, antiviral, antifungal, and anticancer activities (Mohamad et al., 2009). This group of compounds is found in the Meliaceae family, including in the Chisocheton genus (Shilpi et al., 2016). Chisocheton is a genus of the Meliaceae family which contains approximately 53 species spreading in India, Thailand, Malaysia, and Indonesia (Nurlelasari et al., 2017). Several limonoids were found in C. siamensis, C. paniculatus, C. microcarpus, C. ceramicus, C. erythrocarpus, C. cumingianus subsp. balansae, C. macrophyllus, and C. pentandrus (Shilpi et al., 2016; Nurlelasari et al., 2017 and Supriatno et al., 2018). Limonoid types that have been isolated from the Chisocheton genus include havanensin (Nurlelasari et al., 2017; Laphookhieo et al., 2008; Supriatno et al., 2018; Hoai et al., 2018), phragmalin (Najmuldeen et al., 2011; Chong et al., 2012) 30nor trijugine (Katja et al., 2016), mexicanolide (Awang et al., 2007), and vokensin (Yang et al., 2009). One of the Chisocheton species that has not been widely explored for its secondary metabolite content is C. lasiocarpus. Until now, there are only two phytochemicals of C. lasiocarpus isolated from this plant, namely stigmast-5 (6) -en-3β-ol (Nurlelasari et al., 2018) and lasiocarpon that showed moderate cytotoxic activity against MCF-7 breast cancer cell with an IC50 value of 42.5 mM (Hidayat et al., 2018). In this study we successfully isolated one new limonoid compound from C. lasiocarpus seeds, having unusual side chains and one known havanensin-type limonoid. EXPERIMENTAL In this study we used the seeds of C. lasiocarpus, which were collected in Bogor Botanical Garden, Bogor, West Java Province, Indonesia. General experimental procedures UV (ultraviolet) spectra were measured on a TECAN Infinite M200 pro, with MeOH. The IR spectra were recorded on a Perkin Elmer spectrum-100 FT-IR (fourier transform infrared) in KBr. Mass spectra were Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 219 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 obtained with a Water Xevo QTOF mass spectrometer instrument. 1H and 13C NMR (nuclear magnetic resonance) spectra were obtained with a JEOL JNM A-500 spectrometer using TMS (tetramethylsilane) as internal standard. Chromatographic separations were carried out on silica gel 60 (Merck). TLC plates were precoated with silica gel GF254 (Merck, 0.25 mm). The detection of the compounds on TLC plates was achieved under UV light at 254 and 367 nm and by spraying the plates with dimethylaminobenzaldehyde in 10% H2SO4 in ethanol (Ehrlich’s reagent) followed by heating. Extraction and Isolation C. lasiocarpus seed powder (203.5 g) were extracted successively with n-hexane, ethyl acetate, and methanol. The ethyl acetate extract (20 g) was separated on a silica gel vacuum-liquid chromatography (VLC) column eluted with an n-hexane-ethyl acetate stepwise gradient to afford ten fractions (A-J). Fraction H (1.65 g) was chromatographed using the same composition of solvents to give five subfractions (H1-H5). Then, subfraction H5 (301.8 mg) was purified using n-hexane:dichloromethane:ethyl acetate (5:4:1) until a new limonoid 1 was obtained. Fraction E (82.4 mg) was fractionated on silica gel column using n-hexane:ethyl acetate (8:2) to give 2. RESULTS AND DISCUSSION Figure 1. 1H-NMR spectrum of 1(500 MHz in acetone-d). Compound 1 (36.5 mg) was identified as white amorphous powder that can be dissolved in acetone. Its molecular formula, C30H39O6N, was established from the quasi-molecular ion peak at m/z 510.2312 [M + H]+ (calcd. m/z for C30H40O6N, 510.2401) in the HRTOFMS (high-resolution time-of-flight mass spectrometry), suggesting that 1 has twelve degrees of unsaturation. The UV spectrum showed two absorption maximum at 228 nm dan 254 nm (π→π* electronic transition). IR peaks revealed the presence of hydroxyl group at 3447 cm-1, α, β-unsaturated carbonyl at 1670 cm-1, an ester at 1735, and gem dimethyl at 1385 cm-1 as twin bands. 220 Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 Figure 2. 13C-NMR and DEPT 135o spectrum 1 (125 MHz in acetone-d). H-NMR spectrum of compound 1 (Figure 1, Table 1) revealed five tertiary methyls (δH 1.02, 1.19, 1.29, each 3H and 1.10 6H ), two methyls of the ester group at δH 1.94 and 2.10 ppm (each 3H), three oxygenated methine protons at δH 3.03 (1H, bs), 4.34 (1H; t); and 5.34 (1H, d) and five sp2 methines at δH 7.25 (1H, s), 7.27 (1H, s), 5.74 (1H; d); and 5.85 (2H, d)]. The combination of 13C-NMR and DEPT (distortionless enhancement by polarization transfer) (Figure 2) spectra showed 30 carbon signals including carbonyl at δC 204.4 ppm, five methyls at δC 17.5, 20.7, 21.4, 27.8, and 32.3 ppm, two acetyl groups at δC 21.9 (C-1'), 21.8 (C-1''), 171.5 (C-2') and 170.2 (C-2") ppm, three methylenes at δC 24.7, 26.3, and 43.1 ppm, three sp3 methines at δC 47.3, 62.0 and 81.7 ppm, five sp2 methines at δC 125.9, 126.1, 126.3, 159.3, and 159.6 ppm, three oxygenated methines at δC 70.9, 76.7, and 81.7 ppm, and seven quaternary carbons at δC 41.3, 45.5, 47.8, 58.6, 160.0, 162.0, and 167 ppm. 1 Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 221 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 Figure 3. 1H-NMR spectrum (500 MHz in CDCl3) of 2. The skeleton type and position of each functional group were determined using 2D NMR. 1H-1H COSY (correlation spectroscopy 1H-1H) spectrum indicated by the correlations between H-1/H-2, H-6/H-7, H-11/H9/H-12, H-16/H-17, and H-22/H-23 shows that 1 has an intact limonoid skeleton, the havanensin type. The presence of two acetyl groups and the determination of their positions were suggested through the correlations between H-1'/C-1"/C-12 and H-2'/C-2"/C-11. Then, the correlations between H-16/C-20, H-17/C-22, H-21/C23 (δH 81,7 ppm) indicate that the limonoid side chain forms a dihydrolactam ring. The orientation of two acetyls was deduced by the NOESY (nuclear overhauser enhancement spectroscopy) correlations of H-11/H29/H-30 and H-12/H-29. 222 Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 Figure 4. 13C-NMR and DEPT 135o spectrum (125 MHz in CDCl3) of 2. The chemical shift of 1 has similarities with the chemical shift of the 6α, 7α-diacetoxy-3-oxo-24,25,26,27tetranorapotirucalla-1,14,20(22)-trien-21,23-lactam (3) (Figure 6), which was isolated from C. paniculatus fruit, obtained from Vietnam (Hoai et al., 2018). The difference between 1 and 3 is in the acetyl groups position. Compound 3 contains two acetyl groups at C-6 and C-7, while compound 1 has two acetyl groups at C-11 and C-12 positions. There is also an olefinic group between C-5 and C-6 in the structure. In addition to that, the side chain of compound 3 is a lactam ring, while compound 1 has a dihydrolactam ring. This causes the chemical shift value of C-20 of compound 3 lower than the chemical shift of C-20 of compound 1. A comparison of the spectroscopic data for compounds 1 and 3 is shown in Table 1. H N O O 1' 1'' O 19 1 2 3 4 O 28 10 5 11 9 6 O 2'' 18 12 30 13 14 21 20 2' 23 O OH 22 17 16 15 8 7 29 Lasiocarpines (1) O O O OH O 14β,15β-epoxynimonol (2) Figure 5. Selected 1H-1H COSY and HMBC correlations for 1 and 2. Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 223 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 Figure 6. Structure of 6α,7α-diacetoxy-3-oxo-24,25,26,27-tetranorapotirucalla-1,14,20(22)-trien-21,23lactam (3) (Hoai et al., 2018). Compound 2 (6 mg) was identified as a white amorphous powder that can be dissolved in chloroform. Its molecular formula, C30H39O6N, was established from the quasi-molecular ion peak at m/z 469.2383 [M+H]+, (calcd. m/z for C28H37O6 469.2390) in the HRTOFMS suggesting that 2 has eleven degrees of unsaturation. 1HNMR spectrum of compound 2 (Table 2) indicated five tertiary methyls (δH 0.95, 1.22, 1.24 (3H, s), 1.05 (6H, s), one methyl of ester at δH 2.05 ppm (each 3H), two oxygenated methine protons at δH 3.42 (1H, s) dan 4.75 (1H, t, J= 2.7) and five sp2 methines at δH 5.84 (1H, d, J = 10.3 Hz), 6.15 (1H, s); 7.09 (1H, s), 7.18 (1H, s), and 7.35 (1H, s). The combination of 13C-NMR and DEPT spectra revealed 28 carbon signals including carbonyl at δC 206.1 ppm, five methyls at δC 19.3, 20.1, 21.8, 22.0, and 31.1 ppm, one acetyl groups at δC 21.6 (C-1'), and 171.9 (C-2') ppm, three methylenes at δ C 16.6, 29.6, and 32.2 ppm, two sp3 methines at δC 39.0, 50.4, three oxygenated methines at δC 57.5, 68.8 and 77.8 ppm, five sp2 methines at δC 111.0, 126.3, 139.7, 143.0, and 157.8 ppm, and six quaternary carbons at δC 40.3, 41.9, 43.0, 45.7, 73.1, and 123.7 ppm. Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) of 1 and 3 in acetone-d (Hoai et al., 2018). Compound 1 6α,7α-diacetoxy-3-oxo-24,25,26,27-tetranorapotirucalla-1,14,20(22)dCNM trien-21,23-lactam (3) dHNMR R 159.3 7.25 (s) 157.4 7.14 (d, 10.0) 125.9 5.85 (d, 10.4) 126.1 5.91 (d,10.0) 204.4 204.6 45.5 45 162 48.1 2.50a(d, 11.0) 126.1 5.74 (d, 1.8) 70 5.43 (dd, 11.0, 2.0) 26.3 1.01 (m) 74.4 5.45 (d; 2.0) 47.8 43.2 47.3 2.64 (d, 12,4) 37.2 2.24a(dd, 11.0; 7.0) 41.3 40.9 5.34 (dd, 2.3, 70.9 16.5 1.90 (m), 1.72 (m) 12.6) 76.7 6.08 (d, 2.3) 32.7 2.05 (m), 1.81 (m) 58.6 47.1 167 158.1 159.6 7.27 (s) 119.4 5.36 (d, 2.0) 3.54 (dd, 1.9, 24.7 34.1 2.50a(m), 2.24 13.2) 62 2.79 (m) 50.9 2.85 (dd, 6.0, 2.5) 17.5 1.02 (s) 21 0.86 (s) 32.2 1.19 (s) 20.8 1.18 (s) 160 139.4 43.1 2.23, 2.29 174.6 126.3 5.85 (d, 10.4) 139.9 6.5 (t, 1.0) 81.7 4.34 (d, 8.6) 46.4 3.98 (brs) 27.8 1.29 31.7 1.26 (s) 21.4 1.1 20.5 1.18 (s) 20.7 1.1 26.9 1.33 (s) 21.9 2.1 170.2 171.5 21.4 2.04 21.8 1.94 170.1 170.2 21.2 2.01 224 Copyright © 2021, ALCHEMY Jurnal Penelitian Kimia, ISSN 1412-4092, e ISSN 2443-4183 Fitriana et al., ALCHEMY Jurnal Penelitian Kimia, Vol. 17(2), 2021, 219-226 6.28 The skeleton type and position of each functional group were determined using 2D NMR (Figure 5). 1HH COSY spectrum indicates the correlations between H-1/H-2, H-5/H-6/H-7, H-11/H-12, H-16/H-17, and H22/H-23 showing that 2 has an intact limonoid skeleton, the havanensin type. The presence of one acetyl groups, one hydroxyl group, and their positions were suggested by correlations between H-1'/C-2'/C-7 H-7/C5/C-6/C-9 and H-6/C-5. The orientation of the hydroxyl group was deduced by the NOESY correlation of H6/H-18. The chemical shift comparison with the chemical shifts of 14β,15β-epoxynimonol [12], 2 was identified as the known 14β,15β-epoxynimonol (Govindachari et al., 1999). 1 Table 2. 1H NMR and 13C NMR of 2 and 14β,15β-epoxynimonol (Govindachari et al., 1999). Compound 2 14β,15β-epoxynimonol 1 13 1 H -NMR C-NMR H -NMR δH ppm (ΣH; mult; J=Hz) δC ppm δH ppm (ΣH; mult; J=Hz) 7.10 (1H, d, 10.1) 157.8 7.12 (1H, d, 9,9) 5.90 (1H, d, 10.1) 126.3 5.92(1H, d, 9.9) 206.1 40.3 2.22 (1H, d, 11.7) 50.4 2.22 (1H, d, 11.69) 4.27 (1H, dd, 2.8, 11.7) 68.8 5.40 (1H, dd, 11.61, 3.04) 4.91 (1H, d, 2.8) 77.8 4.93 (1H, d, 2.63) 43 2.58 (1H, m) 39 2.62 (1H, m) 41.9 1.23 (1H, m), 1.30 (1H, m) 29.9 1.86 (2H, m) 45.7 73.1 3.46 (1H, s) 57.5 3.49 (1H, s) 1.28 (1H, s), 1.30 (1H, m) 32.2 1.60 (1H. m), 2.13 (1H.m) 2.18 (1H, m) 40.3 2.62 (1H, m) 0.95 (3H, s) 22 0.97 (3H, s) 2.16 (3H, s) 31.1 1.38 (3H, s) 123.7 7.10 (1H, s) 139.7 7.38 (1H, t, 1.6) 6.16 (1H, s) 111 6.17 (1H, br, s) 7.36 (1H, t, 1.8) 143 7.12 (1H, br, s) 1.12 (3H, s) 21.8 1.13 (3H, s) 1.15 (3H, s) 19.3 1.17 (3H, s) 1.36 (3H, s) 20.1 1.31 (3H, s) 2.14 (3H, s) 21.6 2.15 (3H, s) 171.9 - C-NMR δC ppm 157.6 126,26 205.84 40.28 50.41 68.73 77.74 42.92 39.04 41.87 29.31 45.68 73.03 57.38 32.13 39.41 21.86 31.9 123.63 142.96 110.92 139.56 21.65 19.31 19.97 21.4 171.74 13 CONCLUSION One novel lasiocarpines (1) and one known 14β,15β-epoxynimonol (2), two havanensin-type limonoids, have been isolated from the seeds of Chisocheton lasiocarpus. 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