- Email: [email protected]
Bioactive mexicanolide-type limonoids from the fruits of Trichilia connaroides
Phytochemistry Letters 20 (2017) 17–21
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Bioactive mexicanolide-type limonoids from the fruits of Trichilia connaroides A-Hong Chena,b,1, Qing Wena,b,1, Yan-Lei Maa,b , Zhi-Hua Jianga,b , Qing-Long Liua,b , Jin-Ying Tanga,b , Wei Xub , Yan-Ping Liua,** , Yan-Hui Fua,b,* a b
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR China College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, PR China
A R T I C L E I N F O
Article history: Received 26 October 2016 Received in revised form 10 February 2017 Accepted 9 March 2017 Available online xxx Keywords: Trichilia connaroides Mexicanolide-type limonoids Trichiconnarones A and B Nitric oxide production inhibition
A B S T R A C T
Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), along with six known analogues (3–8), were isolated from the fruits of Trichilia connaroides. The structures of 1 and 2 were elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literatures. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory effects with IC50 values comparable to that of hydrocortisone. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Limonoids isolated from the Meliaceae family have attracted continuous attentions due to their complex and diverse chemical structures and their significant biological activities. As a result, a large array of limonoids with various biological activities, such as anti-inflammatory (Yuan et al., 2013), anti-feedant (Cai et al., 2012), anti-microbial (Yuan et al., 2012), anti-HIV (Yu et al., 2015), anti-malarial (Bickii et al., 2000), and cytotoxic properties (Ning et al., 2010), had been isolated from the plants of the Meliaceae family in recent years. The genus Trichilia (Meliaceae) comprising of 419 species is mainly distributed in tropical regions of America, Africa, and Asia. There are about two species and one variety of this genus in China, namely T. connaroides, T. sinensis, and T. connaroides var. Microcarpa. Among them, T. connaroides is widely distributed throughout the southern part of China (Chen et al., 1997). As a traditional folk medicine, T. connaroides has been widely used as an anti-inflammatory agent for the treatment of arthritis, pharyngitis, tonsillitis, chronic osteomyelitis, and eczema in Asian area, such as China, India, Malaysia, and Nepal (Garima, 2011), and so on. Earlier
chemical and pharmacological studies on T. connaroides had showed that its limonoids account for its anti-inflammatory activities (Wang et al., 2013; Ji et al., 2015). Our preliminary experimental results showed that the 90% EtOH extract of the fruits of T. connaroides exhibited significant inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with an IC50 value of 6.8 mg/mL. As an ongoing program to explore structurally and biologically interesting natural products from tropical medicinal plants distributed in China's Hainan Island, two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), along with six known analogues (3–8), were isolated from the fruits of T. connaroides. The structures of 1 and 2 were elucidated by extensive spectroscopic methods. In addition, new compounds 1 and 2 were evaluated for their antiinflammatory activity via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory effects with IC50 values comparable to that of hydrocortisone. Herein, we describe the isolation, structural elucidation and anti-inflammatory activities of these new compounds. 2. Results and discussion
* Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR China ** Corresponding author. E-mail addresses: [email protected] (Y.-P. Liu), [email protected] (Y.-H. Fu). 1 A-Hong Chen and Qing Wen are co-first authors.
The 90% EtOH extract of the fruits of T. connaroides was suspended in water and extracted successively with petroleum ether and EtOAc. The EtOAc extract fraction was repeatedly
http://dx.doi.org/10.1016/j.phytol.2017.03.008 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
18
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
subjected to silica gel, reversed-phase C18 silica gel, Sephadex LH20 CC and semi-preparative HPLC, to yield a total of eight mexicanolide-type limonoids, including two new ones, as shown in Fig. 1. Trichiconnarone A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C31H38O9 by HRESI–MS with m/z 577.2425, [M+Na]+ (calcd: 577.2414), indicating 13 of unsaturation. The IR absorptions at 3448, 1729 and 1650 cm1 showed the presence of hydroxyl group, carbonyl group and double bond group, respectively. The 13C NMR and DEPT data revealed the presence of 31 carbon atoms, including 12 sp2 carbon atoms, four sp3 quaternary carbons, five sp3 methines, four sp3 methylenes and six methyl groups. The 12 sp2 carbon atoms were attributable to the functionalities of one di-substituted double bond, one tri-substituted double bond, one furan ring group, three ester carbonyl groups and one ketone carbonyl group, which accounted for nine out of 13 of unsaturation, the remaining four degrees of unsaturation were assumed for the presence of four rings system in 1. The above data revealed that 1 was a typical mexicanolide-type limonoid. Further analysis of 13C NMR and DEPT data suggested 1 was similar to that of methyl-2-hydroxy3b-tigloyloxy-1-oxomeliac-8(30)-enate (3) (Jimenez et al., 1998), except that the tigloyloxy group at C-3 was substituted by a 2-
Fig. 1. Structures of trichiconnarones A (1) and B (2).
butenoyl group, which was further supported by the HMBC correlations of H-3, H-20 and H-30 to C-10. The coupling constant between H-20 and H-30 (J = 15.6 Hz) permitted the assignment of orientation of the olefinic bond between C-20 and C-30 of the 2butenoate group as E, which was further supported by the ROESY correlation of H-20 with H3-40 Detailed analysis of 2D NMR (HSQC, HMBC, 1H-1H COSY and ROESY) spectra confirmed the planar structure of 1 as shown in Fig. 2. The relative configurations of 1 were assigned by analysis of the ROESY data (Fig. 2). The ROESY correlations of H-5/H3-29, H-5/H11b, H-5/H-17, and H-11b/H-17 suggested that they were cofacial and were arbitrarily assigned a b-orientation. Consequently, the ROESY correlations of 2-OH/H-3, 2-OH/H-9, 2-OH/H-30, H-3/H328, H-9/H-12a, H-9/H-14, H-9/H3-19, H-12a/H3-18, and H-14/H318 indicated that 2-OH, H-3, H-9, 10-CH3, 13-CH3, and H-14 were a-oriented. Thus, the relative configurations of 1 were arbitrarily established as depicted in Fig. 2. Trichiconnarone B (2) was obtained as a white amorphous powder. Its molecular formula was established as C31H38O9 by HRESIMS (m/z 577.2428, [M+Na]+; calcd: 577.2414), which was same with that of 1. The 1H and 13C NMR data (Table 1) suggested that 2 possessed 31 carbon signals which show similar structural features to those of 1, except that the 2-butenoyl group at C-3 was substituted by a methacryloyl group, which was further supported by the HMBC correlations of H-3, H-30 and H-40 to C-10. Detailed analysis of 2D NMR (HSQC, HMBC, 1H-1H COSY) spectra confirmed the planar structure of 2 as shown in Fig. 3. In addition, The relative configurations of 2 were the same with 1, as demonstrated by comparison of its ROESY spectrum with that of 1. In addition to new limonoids, trichiconnarones A (1) and B (2), six other known analogues were isolated and identified as methyl2-hydroxy-3b-tigloyloxy-1-oxomeliac-8(30)-enate (3) (Jimenez et al., 1998), ruageanin B (4) (Mootoo et al., 1996), heytrijunolide D (5) (Yang et al., 2012), trichinenlide D (6) (Xu et al., 2013), cipadesin N (7) (Ning et al., 2010), and 2-hydroxy-3-O-isobutyrylproceranolide (8) (Lin et al., 2009), by comparing their experimental and reported physical data. Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), were evaluated their anti-inflammatory activitis via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7
Fig. 2. Selected 2D NMR correlations for trichiconnarone A (1).
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
19
Table 1 1 H and 13C NMR data of trichiconnarones A (1) and B (2) in CDCl3. trichiconnarone A (1) Position 1 2 3 4 5 6a 6b 7 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16 17 18 19 20 21 22 23 28 29 30 2-OH 7-OCH3 10 20 30 a 30 b 40 a b
d
a H
trichiconnarone B (2)
dC
4.89 (1H, s) 3.30 (1H, dd, J = 8.3, 2.0 Hz) 2.32 (1H, dd, J = 16.5, 8.3 Hz) 2.33 (1H, dd, J = 16.5, 2.0 Hz)
2.21 (1H, overlapped) 1.63 (1H, m) 2.06 (1H, m) 1.39 (1H, m) 1.60 (1H, m)
b
215.2 s 77.2 s 84.5 d 39.3 s 41.6 d 32.7 t 174.0 s 136.6 s 56.5 d 49.5 s 20.4 t 34.3 t
2.21 (1H, overlapped) 2.80 (1H, dd, J = 18.2, 5.9 Hz) 2.83 (1H, dd, J = 18.2, 2.6 Hz)
36.8 s 44.8 d 29.7 t 168.9 s 76.8 d 21.8 q 15.7 q 120.7 s 141.8 d 109.7 d 143.0 d 19.7 q 21.9 q 129.0 d
5.64 (1H, s) 1.05 (3H, s) 1.19 (3H, s) 7.77 (1H, s) 6.44 (1H, s) 7.39 (1H, s) 0.78 (3H, s) 0.72 (3H, s) 5.33 (1H, s) 4.15 (1H, s) 3.68 (3H, s) 5.79 (1H, d, J = 15.6 Hz) 6.97 (1H, dq, J = 15.6, 6.8 Hz)
52.3 q 165.7 s 121.5 d 147.2 d
1.81 (3H, d, J = 6.8 Hz)
18.1 q
dH a
4.92 (1H, s) 3.37 (1H, dd, J = 8.6, 1.8 Hz) 2.36 (1H, dd, J = 16.7, 8.6 Hz) 2.38 (1H, dd, J = 16.7, 1.8 Hz)
2.26 (1H, overlapped) 1.69 (1H, m) 2.10 (1H, m) 1.43 (1H, m) 1.66 (1H, m) 2.26 (1H, overlapped) 2.83 (1H, dd, J = 18.7, 5.8 Hz) 2.85 (1H, dd, J = 18.7, 2.9 Hz) 5.62 (1H, s) 1.08 (3H, s) 1.23 (3H, s) 7.78 (1H, s) 6.44 (1H, s) 7.41 (1H, s) 0.82 (3H, s) 0.77 (3H, s) 5.36 (1H, s) 4.11 (1H, s) 3.71 (3H, s)
5.64 (1H, s) 6.17 (1H, s) 1.97 (3H, s)
Measured at 400 MHz. Measured at 100 MHz.
Fig. 3. Selected 2D NMR correlations for trichiconnarone B (2).
dCb 215.1 s 77.2 s 85.1 d 39.5 s 41.4 d 32.6 t 173.9 s 137.0 s 56.7 d 49.4 s 20.5 t 34.4 t 36.9 s 45.0 d 29.7 t 168.8 s 76.7 d 21.7 q 15.7 q 120.6 s 141.9 d 109.7 d 143.0 d 19.8 q 22.0 q 128.9 d 52.3 q 166.7 s 135.1 s 127.5 d 18.1 q
20
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
Fig. 4. Cell viability (A) and inhibitory activities on LPS-stimulated NO production (B) in RAW 264.7 cells of trichiconnarones A (1) and B (2).
cells in vitro. As a result, trichiconnarones A (1) and B (2) showed significant inhibitory activities with the IC50 values of 2.2 0.12 and 2.9 0.08 mM, respectively. While the positive control, hydrocortisone, showed an inhibitory activity with the IC50 value of at 3.4 0.18 mM. No cytotoxicities were observed in trichiconnarones A (1) and B (2) treated cells (cell viability >90%) (Fig. 4). 3. Experimental 3.1. General experiment procedure Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR spectra were recorded on Bruker 400 MHz spectrometers using TMS as an internal standard, with chemical shifts recorded as d values. HR-ESI–MS spectra were measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (250 9.4 mm, 5 mm). Silica gel (300–400 mesh, Qingdao Marine Chemical Inc., China), Silica gel H (10–40 mm, Qingdao Marine Chemical Inc., China), Lichroprep RP-18 gel (40–63 mm, Merck, Darmstadt, Germany), and Sephadex LH-20 (40–70 mm, Amersham Biosciences, Sweden) were used for column chromatography (CC). 3.2. Plant material The fruits of T. connaroides were collected from Bawangling Nature Reserve, Hainan Province China, in August 2015, and identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University. A voucher specimen (No. Fu20150802) has been deposited at the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University. 3.3. Extraction and isolation The dried powdered fruits of T. connaroides (2.8 kg) were extracted with 90% EtOH at room temperature for three times, each for 7 days. The solvent was evaporated in vacuum to obtain a crude extract. After suspended in water, the crude extract was extracted successively with petroleum ether and EtOAc. The EtOAc extract (68.6 g) was subjected to silica gel column chromatograph, eluted with petroleum ether/acetone (from 95:5 to 10:90) yielding eight fractions (Fr.1-Fr.8). Fr.2 (8.6 g) was subjected to RP-18 eluting with CH3OH/H2O (from 50% to 100%) to afford six fractions (Fr.2A-Fr.2F).
Fraction 2 B (268.6 mg) was purified using Sephadex LH-20 eluted with CHCl3/CH3OH (50:50, v/v), then separated by silica gel column chromatography eluted with petroleum ether/EtOAc 70:30 to yield 3 (23.6 mg) and 7 (42.8 mg). Fraction 2C (683.8 mg) was purified using Sephadex LH-20 eluted with CHCl3/CH3OH (50:50, v/v), then separated by semi-preparative HPLC (Agilent Eclipse XDB-C18 column, i.d. 250 9.4 mm, 5 mm, 73% CH3OH, 3.0 mL/min, tR 23.2, 25,7 and 28.9 min) to afford compound 1 (118.6 mg), 4 (65.2 mg) and 8 (36.9 mg). Fraction 2D (862.8 mg) was purified using Sephadex LH-20 eluted with CH3OH, then separated by semipreparative HPLC (Agilent Eclipse XDB-C18 column, i.d. 250 9.4 mm, 5 mm, 62% CH3CN, 3.0 mL/min, tR 20.8, 21.9 and 26.2 min) to afford compound 2 (62.4 mg), 5 (22.8 mg) and 6 (18.9 mg). 3.4. Trichiconnarone A (1) White amorphous powder; ½a20 D 58.2 (c 0.11, CH3OH); IR (KBr) nmax 3448, 2950, 1729, 1650, 1438, 1379, 1259, 1223, 1171, 1025 and 973 cm1; UV (CH3OH) lmax (log e) 212 (4.02); 1H and 13C NMR
data (see Table 1); ESIMS m/z 577 [M+Na]+; HRESIMS m/z 577.2425 (M + Na; calcd for C31H38O9Na, 577.2414). 3.5. Trichiconnarone B (2)
White amorphous powder; ½a20 D 46:8 (c 0.13, CH3OH); IR (KBr) nmax 3447, 2948, 1728, 1649, 1439, 1380, 1258, 1222, 1168, 1026 and 890 cm1; UV (CH3OH) lmax (log e) 213 (4.13); 1H and 13C NMR data (see Table 1); ESIMS m/z 577 [M + Na]+; HRESIMS m/z 577.2428 (M + Na; calcd for C31H38O9Na, 577.2414). 3.6. Anti-inflammatory bioassays The RAW 264.7 cells (Peking Union Medical College Cell Bank, Beijing, People’s Republic of China) were incubated in RPMI 1640 medium containing 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cell concentration was adjusted to 5 105 cells/mL, and 200 mL of cell suspension was seeded in each well of a 96-well plate. After 24 h incubation, cells were treated with LPS (1 mg/mL) and test samples dissolved in DMSO (final DMSO concentration 0.2%, v/v) for 24 h at 37 C. A 100 mL sample of the culture supernatant was determined by the Griess reaction (Wang et al., 2001), The Griess reagent (50 mL of 1% sulfanilamine in 5% H3PO4, and 50 mL of 0.1% N-1-naphthylethylenediamine dihydrochloride) was added to each well. After 10 min, the reaction products were colorimetrically quantitated at
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
540 nm using a microplate reader. The experiments were performed in triplicate. Hydrocortisone was used as a positive control; The cytotoxicity assay was performed using the MTT method in 96-well microplates (Mosmann, 1983). An MTT solution (200 mg/mL) was added after the 24 h treatment and then incubated for another 4 h at 37 C. The reduced MTT-formazan was solubilized with 150 mL of DMSO, and the absorbance of the MTT-formazan solution at 570 nm was measured by an immunoreader. The percentage of suppression was calculated by comparing the absorbance of sample treated cells with that of nontreated cells. 4. Concluding remarks Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), as well as six known analogues, were isolated from the fruits of T. connaroides. The discovery of 1 and 2 is not only a further addition to diverse and complex array of limonoids, but also, its presence as characteristic marker may be helpful in chemotaxonomical classifications. The inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells of trichiconnarones A (1) and B (2) were also investigated, and found to be quite potent. The significant inhibitory activities on nitric oxide (NO) production of trichiconnarones A (1) and B (2) may be used as an explanation of the folk use of T. connaroides, which was used as an anti-inflammatory drug in China. Acknowlegments This work was financially supported by the National Natural Science Foundation of China (Nos. 21302181, 21662011 and 31660097), the Applied Technology Research and Development and Demonstration Project of Hainan Province (No. ZDXM2015063), the Key Research and Development Project of Hainan Province (No. ZDYF2016221), the Program for Science and Technology plans to youth academic innovation of Hainan Association (No. HAST201636) and the Natural Science Foundation of Hainan Province (Nos. 20162021 and 20162024). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.008.
21
References Bickii, J., Njifutie, N., Foyere, J.A., Basco, L.K., Ringwald, P., 2000. In vitro antimalarial activity of limonoids from Khaya grandifoliola CDC (Meliaceae). J. Ethnopharmacol. 69, 27–33. Cai, J.Y., Zhang, Y., Luo, S.H., Chen, D.Z., Tang, G.H., Yuan, C.M., Di, Y.T., Li, S.H., Hao, X. J., He, H.P., 2012. Aphanamixoid A, a potent defensive limonoid, with a new carbon skeleton from Aphanamixis polystachya. Org. Lett. 14, 2524–2527. Chen, S.K., Li, H., Chen, B.Y., 1997. Flora of China, vol. 43. Science Press, Beijing, pp. 65–68 3. Garima, G., 2011. Trichilia connaroides Wight and Arnott: ethnobotany, phytochemistry and pharmacology. Chin. J. Nat. Med. 9, 241–248. Ji, K.L., Cao, D.H., Li, X.F., Guo, J., Zhang, P., Xu, Y.K., 2015. Two new limonoids from the roots of Trichilia connaroides with inhibitory activity against nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells. Phytochem. Lett. 14, 234–238. Jimenez, A., Villarreal, C., Toscano, R.A., Cook, M., Arnason, J.T., Bye, R., Mata, R., 1998. Limonoids from Swietenia humilis and Guarea grandiflora (Meliaceae) Taken in part from the PhD and MS theses of C. Villarreal and M.A. Jiménez respectively. Phytochemistry 49, 1981–1988. Lin, B.D., Yuan, T., Zhang, C.R., Dong, L., Zhang, B., Wu, Y., Yue, J.M., 2009. Structurally diverse limonoids from the fruits of Swietenia mahagoni. J. Nat. Prod. 72, 2084– 2090. Mootoo, B.S., Ramsewak, R., Khan, A., Tinto, W.F., Reynolds, W.F., McLean, S., Yu, M., 1996. Tetranortriterpenoids from Ruagea glabra. J. Nat. Prod. 59, 544–547. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Ning, J., Di, Y.T., Fang, X., He, H.P., Wang, Y.Y., Li, Y., Li, S.L., Hao, X.J., 2010. Limonoids from the leaves of Cipadessa baccifera. J. Nat. Prod. 73, 1327–1331. Wang, N.L., Yao, X.S., Ishii, R., Kitanaka, S., 2001. Antiallergic agents from natural sources. 3. Structures and inhibitory effects on nitric oxide production and histamine release of five novel polyacetylene glucosides from Bidens parviflora WILLD. Chem. Pharm. Bull. 49, 938–942. Wang, H.Y., Wang, J.S., Shan, S.M., Wang, X.B., Luo, J., Yang, M.H., Kong, L.Y., 2013. Chemical constituents from Trichilia connaroides and their nitric oxide production and alpha-glucosidase inhibitory activities. Planta Med. 79, 1767– 1774. Xu, J.B., Lin, Y., Dong, S.H., Wang, F., Yue, J.M., 2013. Trichinenlides A-T, mexicanolidetype limonoids from Trichilia sinensis. J. Nat. Prod. 76, 1872–1880. Yang, W., Kong, L.M., Li, S.F., Li, Y., Zhang, Y., He, H.P., Hao, X.J., 2012. Five new mexicanolide type limonoids from Heynea trijuga. Nat. Prod. Bioprospect. 2, 145–149. Yu, J.H., Wang, G.C., Han, Y.S., Wu, Y., Wainberg, M.A., Yue, J.M., 2015. Limonoids with anti-HIV activity from Cipadessa cinerascens. J. Nat. Prod. 78, 1243–1252. Yuan, C.M., Zhang, Y., Tang, G.H., Di, Y.T., Cao, M.M., Wang, X.Y., Zuo, G.Y., Li, S.L., Hua, H.M., He, H.P., Hao, X.J., 2012. Khayseneganins A-H, limonoids from Khaya senegalensis. J. Nat. Prod. 76, 327–333. Yuan, C.M., Tang, G.H., Zhang, Y., Wang, X.Y., Cao, M.M., Guo, F., Li, Y., Di, Y.T., Li, S.L., Hua, H.M., He, H.P., Hao, X.J., 2013. Bioactive limonoid and triterpenoid constituents of Turraea pubescens. J. Nat. Prod. 76, 1166–1174.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Bioactive mexicanolide-type limonoids from the fruits of Trichilia connaroides A-Hong Chena,b,1, Qing Wena,b,1, Yan-Lei Maa,b , Zhi-Hua Jianga,b , Qing-Long Liua,b , Jin-Ying Tanga,b , Wei Xub , Yan-Ping Liua,** , Yan-Hui Fua,b,* a b
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR China College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, PR China
A R T I C L E I N F O
Article history: Received 26 October 2016 Received in revised form 10 February 2017 Accepted 9 March 2017 Available online xxx Keywords: Trichilia connaroides Mexicanolide-type limonoids Trichiconnarones A and B Nitric oxide production inhibition
A B S T R A C T
Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), along with six known analogues (3–8), were isolated from the fruits of Trichilia connaroides. The structures of 1 and 2 were elucidated by extensive spectroscopic methods and the known compounds were identified by comparisons their data with those reported in the literatures. All new compounds were evaluated for their anti-inflammatory activities via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory effects with IC50 values comparable to that of hydrocortisone. © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Limonoids isolated from the Meliaceae family have attracted continuous attentions due to their complex and diverse chemical structures and their significant biological activities. As a result, a large array of limonoids with various biological activities, such as anti-inflammatory (Yuan et al., 2013), anti-feedant (Cai et al., 2012), anti-microbial (Yuan et al., 2012), anti-HIV (Yu et al., 2015), anti-malarial (Bickii et al., 2000), and cytotoxic properties (Ning et al., 2010), had been isolated from the plants of the Meliaceae family in recent years. The genus Trichilia (Meliaceae) comprising of 419 species is mainly distributed in tropical regions of America, Africa, and Asia. There are about two species and one variety of this genus in China, namely T. connaroides, T. sinensis, and T. connaroides var. Microcarpa. Among them, T. connaroides is widely distributed throughout the southern part of China (Chen et al., 1997). As a traditional folk medicine, T. connaroides has been widely used as an anti-inflammatory agent for the treatment of arthritis, pharyngitis, tonsillitis, chronic osteomyelitis, and eczema in Asian area, such as China, India, Malaysia, and Nepal (Garima, 2011), and so on. Earlier
chemical and pharmacological studies on T. connaroides had showed that its limonoids account for its anti-inflammatory activities (Wang et al., 2013; Ji et al., 2015). Our preliminary experimental results showed that the 90% EtOH extract of the fruits of T. connaroides exhibited significant inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells with an IC50 value of 6.8 mg/mL. As an ongoing program to explore structurally and biologically interesting natural products from tropical medicinal plants distributed in China's Hainan Island, two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), along with six known analogues (3–8), were isolated from the fruits of T. connaroides. The structures of 1 and 2 were elucidated by extensive spectroscopic methods. In addition, new compounds 1 and 2 were evaluated for their antiinflammatory activity via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells in vitro. Compounds 1 and 2 exhibited inhibitory effects with IC50 values comparable to that of hydrocortisone. Herein, we describe the isolation, structural elucidation and anti-inflammatory activities of these new compounds. 2. Results and discussion
* Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR China ** Corresponding author. E-mail addresses: [email protected] (Y.-P. Liu), [email protected] (Y.-H. Fu). 1 A-Hong Chen and Qing Wen are co-first authors.
The 90% EtOH extract of the fruits of T. connaroides was suspended in water and extracted successively with petroleum ether and EtOAc. The EtOAc extract fraction was repeatedly
http://dx.doi.org/10.1016/j.phytol.2017.03.008 1874-3900/© 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
18
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
subjected to silica gel, reversed-phase C18 silica gel, Sephadex LH20 CC and semi-preparative HPLC, to yield a total of eight mexicanolide-type limonoids, including two new ones, as shown in Fig. 1. Trichiconnarone A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C31H38O9 by HRESI–MS with m/z 577.2425, [M+Na]+ (calcd: 577.2414), indicating 13 of unsaturation. The IR absorptions at 3448, 1729 and 1650 cm1 showed the presence of hydroxyl group, carbonyl group and double bond group, respectively. The 13C NMR and DEPT data revealed the presence of 31 carbon atoms, including 12 sp2 carbon atoms, four sp3 quaternary carbons, five sp3 methines, four sp3 methylenes and six methyl groups. The 12 sp2 carbon atoms were attributable to the functionalities of one di-substituted double bond, one tri-substituted double bond, one furan ring group, three ester carbonyl groups and one ketone carbonyl group, which accounted for nine out of 13 of unsaturation, the remaining four degrees of unsaturation were assumed for the presence of four rings system in 1. The above data revealed that 1 was a typical mexicanolide-type limonoid. Further analysis of 13C NMR and DEPT data suggested 1 was similar to that of methyl-2-hydroxy3b-tigloyloxy-1-oxomeliac-8(30)-enate (3) (Jimenez et al., 1998), except that the tigloyloxy group at C-3 was substituted by a 2-
Fig. 1. Structures of trichiconnarones A (1) and B (2).
butenoyl group, which was further supported by the HMBC correlations of H-3, H-20 and H-30 to C-10. The coupling constant between H-20 and H-30 (J = 15.6 Hz) permitted the assignment of orientation of the olefinic bond between C-20 and C-30 of the 2butenoate group as E, which was further supported by the ROESY correlation of H-20 with H3-40 Detailed analysis of 2D NMR (HSQC, HMBC, 1H-1H COSY and ROESY) spectra confirmed the planar structure of 1 as shown in Fig. 2. The relative configurations of 1 were assigned by analysis of the ROESY data (Fig. 2). The ROESY correlations of H-5/H3-29, H-5/H11b, H-5/H-17, and H-11b/H-17 suggested that they were cofacial and were arbitrarily assigned a b-orientation. Consequently, the ROESY correlations of 2-OH/H-3, 2-OH/H-9, 2-OH/H-30, H-3/H328, H-9/H-12a, H-9/H-14, H-9/H3-19, H-12a/H3-18, and H-14/H318 indicated that 2-OH, H-3, H-9, 10-CH3, 13-CH3, and H-14 were a-oriented. Thus, the relative configurations of 1 were arbitrarily established as depicted in Fig. 2. Trichiconnarone B (2) was obtained as a white amorphous powder. Its molecular formula was established as C31H38O9 by HRESIMS (m/z 577.2428, [M+Na]+; calcd: 577.2414), which was same with that of 1. The 1H and 13C NMR data (Table 1) suggested that 2 possessed 31 carbon signals which show similar structural features to those of 1, except that the 2-butenoyl group at C-3 was substituted by a methacryloyl group, which was further supported by the HMBC correlations of H-3, H-30 and H-40 to C-10. Detailed analysis of 2D NMR (HSQC, HMBC, 1H-1H COSY) spectra confirmed the planar structure of 2 as shown in Fig. 3. In addition, The relative configurations of 2 were the same with 1, as demonstrated by comparison of its ROESY spectrum with that of 1. In addition to new limonoids, trichiconnarones A (1) and B (2), six other known analogues were isolated and identified as methyl2-hydroxy-3b-tigloyloxy-1-oxomeliac-8(30)-enate (3) (Jimenez et al., 1998), ruageanin B (4) (Mootoo et al., 1996), heytrijunolide D (5) (Yang et al., 2012), trichinenlide D (6) (Xu et al., 2013), cipadesin N (7) (Ning et al., 2010), and 2-hydroxy-3-O-isobutyrylproceranolide (8) (Lin et al., 2009), by comparing their experimental and reported physical data. Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), were evaluated their anti-inflammatory activitis via examining the inhibitory activity on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7
Fig. 2. Selected 2D NMR correlations for trichiconnarone A (1).
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
19
Table 1 1 H and 13C NMR data of trichiconnarones A (1) and B (2) in CDCl3. trichiconnarone A (1) Position 1 2 3 4 5 6a 6b 7 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16 17 18 19 20 21 22 23 28 29 30 2-OH 7-OCH3 10 20 30 a 30 b 40 a b
d
a H
trichiconnarone B (2)
dC
4.89 (1H, s) 3.30 (1H, dd, J = 8.3, 2.0 Hz) 2.32 (1H, dd, J = 16.5, 8.3 Hz) 2.33 (1H, dd, J = 16.5, 2.0 Hz)
2.21 (1H, overlapped) 1.63 (1H, m) 2.06 (1H, m) 1.39 (1H, m) 1.60 (1H, m)
b
215.2 s 77.2 s 84.5 d 39.3 s 41.6 d 32.7 t 174.0 s 136.6 s 56.5 d 49.5 s 20.4 t 34.3 t
2.21 (1H, overlapped) 2.80 (1H, dd, J = 18.2, 5.9 Hz) 2.83 (1H, dd, J = 18.2, 2.6 Hz)
36.8 s 44.8 d 29.7 t 168.9 s 76.8 d 21.8 q 15.7 q 120.7 s 141.8 d 109.7 d 143.0 d 19.7 q 21.9 q 129.0 d
5.64 (1H, s) 1.05 (3H, s) 1.19 (3H, s) 7.77 (1H, s) 6.44 (1H, s) 7.39 (1H, s) 0.78 (3H, s) 0.72 (3H, s) 5.33 (1H, s) 4.15 (1H, s) 3.68 (3H, s) 5.79 (1H, d, J = 15.6 Hz) 6.97 (1H, dq, J = 15.6, 6.8 Hz)
52.3 q 165.7 s 121.5 d 147.2 d
1.81 (3H, d, J = 6.8 Hz)
18.1 q
dH a
4.92 (1H, s) 3.37 (1H, dd, J = 8.6, 1.8 Hz) 2.36 (1H, dd, J = 16.7, 8.6 Hz) 2.38 (1H, dd, J = 16.7, 1.8 Hz)
2.26 (1H, overlapped) 1.69 (1H, m) 2.10 (1H, m) 1.43 (1H, m) 1.66 (1H, m) 2.26 (1H, overlapped) 2.83 (1H, dd, J = 18.7, 5.8 Hz) 2.85 (1H, dd, J = 18.7, 2.9 Hz) 5.62 (1H, s) 1.08 (3H, s) 1.23 (3H, s) 7.78 (1H, s) 6.44 (1H, s) 7.41 (1H, s) 0.82 (3H, s) 0.77 (3H, s) 5.36 (1H, s) 4.11 (1H, s) 3.71 (3H, s)
5.64 (1H, s) 6.17 (1H, s) 1.97 (3H, s)
Measured at 400 MHz. Measured at 100 MHz.
Fig. 3. Selected 2D NMR correlations for trichiconnarone B (2).
dCb 215.1 s 77.2 s 85.1 d 39.5 s 41.4 d 32.6 t 173.9 s 137.0 s 56.7 d 49.4 s 20.5 t 34.4 t 36.9 s 45.0 d 29.7 t 168.8 s 76.7 d 21.7 q 15.7 q 120.6 s 141.9 d 109.7 d 143.0 d 19.8 q 22.0 q 128.9 d 52.3 q 166.7 s 135.1 s 127.5 d 18.1 q
20
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
Fig. 4. Cell viability (A) and inhibitory activities on LPS-stimulated NO production (B) in RAW 264.7 cells of trichiconnarones A (1) and B (2).
cells in vitro. As a result, trichiconnarones A (1) and B (2) showed significant inhibitory activities with the IC50 values of 2.2 0.12 and 2.9 0.08 mM, respectively. While the positive control, hydrocortisone, showed an inhibitory activity with the IC50 value of at 3.4 0.18 mM. No cytotoxicities were observed in trichiconnarones A (1) and B (2) treated cells (cell viability >90%) (Fig. 4). 3. Experimental 3.1. General experiment procedure Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer in KBr discs. NMR spectra were recorded on Bruker 400 MHz spectrometers using TMS as an internal standard, with chemical shifts recorded as d values. HR-ESI–MS spectra were measured on a Micromass Q-TOF Ultima Global GAA076 LC mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1260 LC series with a DAD detector using an Agilent Eclipse XDB-C18 column (250 9.4 mm, 5 mm). Silica gel (300–400 mesh, Qingdao Marine Chemical Inc., China), Silica gel H (10–40 mm, Qingdao Marine Chemical Inc., China), Lichroprep RP-18 gel (40–63 mm, Merck, Darmstadt, Germany), and Sephadex LH-20 (40–70 mm, Amersham Biosciences, Sweden) were used for column chromatography (CC). 3.2. Plant material The fruits of T. connaroides were collected from Bawangling Nature Reserve, Hainan Province China, in August 2015, and identified by Prof. Qiong-Xin Zhong, College of Life Science, Hainan Normal University. A voucher specimen (No. Fu20150802) has been deposited at the Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University. 3.3. Extraction and isolation The dried powdered fruits of T. connaroides (2.8 kg) were extracted with 90% EtOH at room temperature for three times, each for 7 days. The solvent was evaporated in vacuum to obtain a crude extract. After suspended in water, the crude extract was extracted successively with petroleum ether and EtOAc. The EtOAc extract (68.6 g) was subjected to silica gel column chromatograph, eluted with petroleum ether/acetone (from 95:5 to 10:90) yielding eight fractions (Fr.1-Fr.8). Fr.2 (8.6 g) was subjected to RP-18 eluting with CH3OH/H2O (from 50% to 100%) to afford six fractions (Fr.2A-Fr.2F).
Fraction 2 B (268.6 mg) was purified using Sephadex LH-20 eluted with CHCl3/CH3OH (50:50, v/v), then separated by silica gel column chromatography eluted with petroleum ether/EtOAc 70:30 to yield 3 (23.6 mg) and 7 (42.8 mg). Fraction 2C (683.8 mg) was purified using Sephadex LH-20 eluted with CHCl3/CH3OH (50:50, v/v), then separated by semi-preparative HPLC (Agilent Eclipse XDB-C18 column, i.d. 250 9.4 mm, 5 mm, 73% CH3OH, 3.0 mL/min, tR 23.2, 25,7 and 28.9 min) to afford compound 1 (118.6 mg), 4 (65.2 mg) and 8 (36.9 mg). Fraction 2D (862.8 mg) was purified using Sephadex LH-20 eluted with CH3OH, then separated by semipreparative HPLC (Agilent Eclipse XDB-C18 column, i.d. 250 9.4 mm, 5 mm, 62% CH3CN, 3.0 mL/min, tR 20.8, 21.9 and 26.2 min) to afford compound 2 (62.4 mg), 5 (22.8 mg) and 6 (18.9 mg). 3.4. Trichiconnarone A (1) White amorphous powder; ½a20 D 58.2 (c 0.11, CH3OH); IR (KBr) nmax 3448, 2950, 1729, 1650, 1438, 1379, 1259, 1223, 1171, 1025 and 973 cm1; UV (CH3OH) lmax (log e) 212 (4.02); 1H and 13C NMR
data (see Table 1); ESIMS m/z 577 [M+Na]+; HRESIMS m/z 577.2425 (M + Na; calcd for C31H38O9Na, 577.2414). 3.5. Trichiconnarone B (2)
White amorphous powder; ½a20 D 46:8 (c 0.13, CH3OH); IR (KBr) nmax 3447, 2948, 1728, 1649, 1439, 1380, 1258, 1222, 1168, 1026 and 890 cm1; UV (CH3OH) lmax (log e) 213 (4.13); 1H and 13C NMR data (see Table 1); ESIMS m/z 577 [M + Na]+; HRESIMS m/z 577.2428 (M + Na; calcd for C31H38O9Na, 577.2414). 3.6. Anti-inflammatory bioassays The RAW 264.7 cells (Peking Union Medical College Cell Bank, Beijing, People’s Republic of China) were incubated in RPMI 1640 medium containing 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cell concentration was adjusted to 5 105 cells/mL, and 200 mL of cell suspension was seeded in each well of a 96-well plate. After 24 h incubation, cells were treated with LPS (1 mg/mL) and test samples dissolved in DMSO (final DMSO concentration 0.2%, v/v) for 24 h at 37 C. A 100 mL sample of the culture supernatant was determined by the Griess reaction (Wang et al., 2001), The Griess reagent (50 mL of 1% sulfanilamine in 5% H3PO4, and 50 mL of 0.1% N-1-naphthylethylenediamine dihydrochloride) was added to each well. After 10 min, the reaction products were colorimetrically quantitated at
A.-H. Chen et al. / Phytochemistry Letters 20 (2017) 17–21
540 nm using a microplate reader. The experiments were performed in triplicate. Hydrocortisone was used as a positive control; The cytotoxicity assay was performed using the MTT method in 96-well microplates (Mosmann, 1983). An MTT solution (200 mg/mL) was added after the 24 h treatment and then incubated for another 4 h at 37 C. The reduced MTT-formazan was solubilized with 150 mL of DMSO, and the absorbance of the MTT-formazan solution at 570 nm was measured by an immunoreader. The percentage of suppression was calculated by comparing the absorbance of sample treated cells with that of nontreated cells. 4. Concluding remarks Two new mexicanolide-type limonoids, trichiconnarones A (1) and B (2), as well as six known analogues, were isolated from the fruits of T. connaroides. The discovery of 1 and 2 is not only a further addition to diverse and complex array of limonoids, but also, its presence as characteristic marker may be helpful in chemotaxonomical classifications. The inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in mouse macrophage RAW 264.7 cells of trichiconnarones A (1) and B (2) were also investigated, and found to be quite potent. The significant inhibitory activities on nitric oxide (NO) production of trichiconnarones A (1) and B (2) may be used as an explanation of the folk use of T. connaroides, which was used as an anti-inflammatory drug in China. Acknowlegments This work was financially supported by the National Natural Science Foundation of China (Nos. 21302181, 21662011 and 31660097), the Applied Technology Research and Development and Demonstration Project of Hainan Province (No. ZDXM2015063), the Key Research and Development Project of Hainan Province (No. ZDYF2016221), the Program for Science and Technology plans to youth academic innovation of Hainan Association (No. HAST201636) and the Natural Science Foundation of Hainan Province (Nos. 20162021 and 20162024). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.008.
21
References Bickii, J., Njifutie, N., Foyere, J.A., Basco, L.K., Ringwald, P., 2000. In vitro antimalarial activity of limonoids from Khaya grandifoliola CDC (Meliaceae). J. Ethnopharmacol. 69, 27–33. Cai, J.Y., Zhang, Y., Luo, S.H., Chen, D.Z., Tang, G.H., Yuan, C.M., Di, Y.T., Li, S.H., Hao, X. J., He, H.P., 2012. Aphanamixoid A, a potent defensive limonoid, with a new carbon skeleton from Aphanamixis polystachya. Org. Lett. 14, 2524–2527. Chen, S.K., Li, H., Chen, B.Y., 1997. Flora of China, vol. 43. Science Press, Beijing, pp. 65–68 3. Garima, G., 2011. Trichilia connaroides Wight and Arnott: ethnobotany, phytochemistry and pharmacology. Chin. J. Nat. Med. 9, 241–248. Ji, K.L., Cao, D.H., Li, X.F., Guo, J., Zhang, P., Xu, Y.K., 2015. Two new limonoids from the roots of Trichilia connaroides with inhibitory activity against nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells. Phytochem. Lett. 14, 234–238. Jimenez, A., Villarreal, C., Toscano, R.A., Cook, M., Arnason, J.T., Bye, R., Mata, R., 1998. Limonoids from Swietenia humilis and Guarea grandiflora (Meliaceae) Taken in part from the PhD and MS theses of C. Villarreal and M.A. Jiménez respectively. Phytochemistry 49, 1981–1988. Lin, B.D., Yuan, T., Zhang, C.R., Dong, L., Zhang, B., Wu, Y., Yue, J.M., 2009. Structurally diverse limonoids from the fruits of Swietenia mahagoni. J. Nat. Prod. 72, 2084– 2090. Mootoo, B.S., Ramsewak, R., Khan, A., Tinto, W.F., Reynolds, W.F., McLean, S., Yu, M., 1996. Tetranortriterpenoids from Ruagea glabra. J. Nat. Prod. 59, 544–547. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63. Ning, J., Di, Y.T., Fang, X., He, H.P., Wang, Y.Y., Li, Y., Li, S.L., Hao, X.J., 2010. Limonoids from the leaves of Cipadessa baccifera. J. Nat. Prod. 73, 1327–1331. Wang, N.L., Yao, X.S., Ishii, R., Kitanaka, S., 2001. Antiallergic agents from natural sources. 3. Structures and inhibitory effects on nitric oxide production and histamine release of five novel polyacetylene glucosides from Bidens parviflora WILLD. Chem. Pharm. Bull. 49, 938–942. Wang, H.Y., Wang, J.S., Shan, S.M., Wang, X.B., Luo, J., Yang, M.H., Kong, L.Y., 2013. Chemical constituents from Trichilia connaroides and their nitric oxide production and alpha-glucosidase inhibitory activities. Planta Med. 79, 1767– 1774. Xu, J.B., Lin, Y., Dong, S.H., Wang, F., Yue, J.M., 2013. Trichinenlides A-T, mexicanolidetype limonoids from Trichilia sinensis. J. Nat. Prod. 76, 1872–1880. Yang, W., Kong, L.M., Li, S.F., Li, Y., Zhang, Y., He, H.P., Hao, X.J., 2012. Five new mexicanolide type limonoids from Heynea trijuga. Nat. Prod. Bioprospect. 2, 145–149. Yu, J.H., Wang, G.C., Han, Y.S., Wu, Y., Wainberg, M.A., Yue, J.M., 2015. Limonoids with anti-HIV activity from Cipadessa cinerascens. J. Nat. Prod. 78, 1243–1252. Yuan, C.M., Zhang, Y., Tang, G.H., Di, Y.T., Cao, M.M., Wang, X.Y., Zuo, G.Y., Li, S.L., Hua, H.M., He, H.P., Hao, X.J., 2012. Khayseneganins A-H, limonoids from Khaya senegalensis. J. Nat. Prod. 76, 327–333. Yuan, C.M., Tang, G.H., Zhang, Y., Wang, X.Y., Cao, M.M., Guo, F., Li, Y., Di, Y.T., Li, S.L., Hua, H.M., He, H.P., Hao, X.J., 2013. Bioactive limonoid and triterpenoid constituents of Turraea pubescens. J. Nat. Prod. 76, 1166–1174.