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Toonamicronoids A–D, four new B-seco-limonoids from Toona microcarpa
Phytochemistry Letters 31 (2019) 225–228
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Toonamicronoids A–D, four new B-seco-limonoids from Toona microcarpa a,b,1
a,1
a
a
a
b
a
Ling Zhang , Jianjun Xia , Jianyun Yang , Long Zhu , Danyu Tang , Ji Ma , Yingliang Zhao , ⁎ Xiaoying Zenga, Minghua Qiub, a b
T
R&D Center of China Tabacco Yunnan Industrial Co., Ltd. Kunming, 650202, China Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China
ARTICLE INFO
ABSTRACT
Keywords: Toona microcarpa Toonamicronoids A–D Limonoid Identification Cytotoxicity
Four new B-seco-limonoids, toonamicronoids A–D (1–4), together with four known ones, toonaciliatin H–J (5–7), and toonacilianin A (8), were isolated from Toona microcarpa. Their structures were identified on the basis of ESIMS, HREIMS and 1D/2D NMR analysis. Compounds 1 and 4 showed moderate cytotoxicities against HL-60 with the IC50values of 17.3 ± 1.5 and 22.7 ± 1.9 μM, respectively.
1. Introduction
2. Experimental Section
The genus Toona, belonging to the family of Meliaceae, comprises five species including Toona ciliate, Cedrela toona, Toona sinensis, Toona microcarpa, Toona febrifuga, and Toona calantas (Flora of China Editorial Committee, 2004). Their barks, leaves, and fruits are frequently used as the food additives and herbal medicine for treating various disease, such as anabrosis, metrorrhagia, dysentery, enteritis, etc. Previous phytochemical investigations on the genus Toona showed the major components of phenols (Kakumu et al., 2014) and terpenoids (De Leo et al., 2018) which possess various pharmacological effects (Chen et al., 2014; Hseu et al., 2011; Huang et al., 2012; Zhang et al., 2011). T. microcarpa was a macrophanerophyte of Toona (Meliaceae). Phytochemical research revealed only three phenols and one terpenoid reported, i.e. (+)-catechin, (6R,7S,8S)-7a-[(β-D-glucopyranosyl)oxy] lyoniresinol, (6R,7R,8R)-7a-[(β-D- glucopyranosyl)oxy]lyoniresinol, and 20-hydroxyecdysone (Fang et al., 2010). To explore new components of T. microcarpa, in the present study, an investigation on chemical constituents of T. microcarpa was carried out. Four new B-secolimonoids, toonamicronoids A–D (1–4), along with four known ones toonaciliatin H–J (5–7) (Chen et al., 2009), and toonacilianin A (8) (Liu et al., 2011) (Fig. 1), were isolated from T. microcarpa. Their structures were identified on the basis of ESIMS, HREIMS and 1D/2D NMR analysis. The cytotoxic activities of the new compounds isolated were also evaluated.
2.1. Plant material The barks of Toona microcarpa (C. DC.) C. Y. Wu were collected by Mr. Qing-Yong Wu in Ning’er County of Yunnan Province, China in August 2017, and was identified by Prof. Zong-Yu Wang, a botanic taxonomist at the Kunming Institute of Botany, Chinese Academy of Sciences. The voucher specimen (TC-2017-01) had been deposited in Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. 2.2. Extraction and isolation The air-dried and powdered barks of T. ciliata (25 Kg) were extracted with MeOH under reflux for three times. The methanol extract was concentrated under reduced pressure to afford a black residue. The residue was suspended in water (10 L) and then partitioned with petroleum ether (10 L × 3) and EtOAc (10 L × 3), successively. The EtOAc extract was evaporated in a vacuum to obtain a brownish black residue (600 g). The EtOAc soluble portion was subjected to silica gel chromatography column (CC) and eluted successively with CHCl3, CHCl3MeOH (100:1), CHCl3-MeOH (50:1), and CHCl3-MeOH (15:1). The CHCl3-MeOH (50:1) portion of the EtOAc extract was subjected to silica gel CC and eluted with PE/acetone (10:1) to afford four fractions (Fr. I–V). Fraction III was subjected to RP-18 silica gel CC (MeOH/H2O, 50:50 → 80:20, v/v) to afford eight subfractions III1-5. Subfraction III2
Corresponding author. E-mail address: [email protected] (M. Qiu). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.04.010 Received 16 December 2018; Received in revised form 3 April 2019; Accepted 5 April 2019 1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
Phytochemistry Letters 31 (2019) 225–228
L. Zhang, et al.
Fig. 1. The structures of 1–4.
[M]+ (calcd. for C25H30O9 474.1890); 1H and data, see Table 1.
was rechromatographed over an RP-18 silica gel CC eluting with MeOH/H2O (60:40) to yield compound 1 (5 mg), 3 (11 mg), 5 (28 mg), and 6 (7.5 mg). Subfraction III3 was separated by an RP-18 silica gel (MeOH/H2O, 70:30) to yield compound 2 (8 mg) and 7 (10 mg). Subfraction III4 was further chromatographed on an RP-18 silica gel CC eluting with MeOH/H2O (65:35) to yield compound 4 (4 mg) and 8 (5 mg).
13
C NMR spectroscopic
2.2.3. Toonaciliatone C (3) White amorphous powder; m.p. 215 C; [α]20 D = + 40.4 (c 0.40, MeOH); m/z 567 [M+Na]+, 1111 [2M+Na]+; HREIMS m/z 544.2313 [M]+ (calcd. for C29H36O10 544.2308); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.1. Toonaciliatone A (1) White amorphous powder; m.p. 250 ○C; [α]20 D = + 108.9 (c 0.10, pyridine); m/z 523 [M+Cl]−, 1011 [2M+Cl]−; HREIMS m/z 488.1716 [M]+ (calcd. for C25H28O10 488.1682); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.4. Toonaciliatone D (4) White amorphous powder; m.p. 168 C; [α]20 D = + 42.2 (c 0.41, CHCl3); m/z 535 [M+Na]+, 1047 [2M+Na]+; HREIMS m/z 512.2374 [M]+ (calcd. for C29H36O8 512.2410); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.2. Toonaciliatone B (2) White amorphous powder; m.p. 210 C; [α]20 D = + 56.9 (c 0.50, MeOH); m/z 497 [M+Na]+, 971 [2M+Na]+; HREIMS m/z 474.1879 Table 1 The NMR spectroscopic data of toonamicronoids A–D (1–4). Position
toonamicronoid A (1) in C5D5N
toonamicronoid B (2) in C5D5N
toonamicronoid C (3) in C5D5N
toonamicronoid D (4) in CDCl3
δC (mult.)
δC (mult.)
δC (mult.)
δC (mult.)
δH (mult. J in Hz)
153.0 d 125.9 d 203.1 s 45.9 s 43.9 d 31.8 t
7.18 d (10.4) 6.03 d (10.4)
1 2 3 4 5 6
106.2 s 126.9 d 132.9 d 86.7 s 82.0 s 77.7 d
7 8 9 10 11 12
174.0 s 72.1 s 54.4 d 55.1 s 79.7 d 210.3 s
13 14 15 16
53.9 75.5 57.3 31.5
17 18 19 20 21 22 23 28 29 30 7-OCH3 29- OCOCH3 29-OCOCH3
35.8 d 19.2 q 12.9 q 124.0 s 141.6 d 113.2 d 143.2 d 24.3 q
s s d t
23.7 q
δH (mult. J in Hz) 6.16 d (10.4) 5.89 d (10.4) 4.69 s
3.75 s 5.29 d (13.6)
3.80 2.06 2.26 3.16 1.46 2.00
m m m m s s
7.55 6.90 7.62 1.92
s s s s
1.78 s
105.4 s 126.1 d 133.9 d 87.1 s 82.2 s 77.9 d 174.3 s 75.7 s 55.6 d 54.9 s 74.1 d 44.1 t 41.5 73.3 56.1 31.5
s s d t
35.8 d 23.3 q 13.3 q 123.8 s 140.2 d 111.4 d 143.6 d 24.4 q 24.0 q
δH (mult. J in Hz) 6.16 d (10.4) 5.94 d (10.4) 4.69 s
3.42 d (13.6) 4.85 m 2.65 dd (12.4, 2.8) 1.74 m 3.57 1.86 2.12 2.89 1.22 1.96
m m m m s s
7.56 7.20 7.64 1.97
s s s s
188.1 s 97.9 d 206.1 s 48.8 s 49.0 d 71.2 d 173.8 s 74.2 s 54.8 d 50.1 s 82.7 d 31.6 t 47.5 74.8 56.4 33.7
s s d t
41.7 d 20.4 q 16.5 q 123.5 s 140.1 d 111.8 d 143.3 d 25.9 q 66.6 t 24.2 q 51.5 q 170.4 s 20.8 q
1.66 s
226
δH (mult. J in Hz) 5.60 s 3.11 m 4.69 s
3.62 m 5.11 m 1.88 m 2.22 m 3.52 3.04 3.86 3.08 1.51 1.58
m m m m s s
7.47 6.48 7.55 1.24 4.50 1.59 3.50
s s s s m s s
1.99 s
173.9 s 135.7 s 56.7 d 41.8 s 73.3 d 75.0 d 45.3 72.8 60.3 31.8
s s d t
39.3 d 13.1 q 20.0 q 123.6 s 140.1 d 112.3 d 142.1 d 22.5 q 22.8 q 120.5 t 51.9 q 170.7 s 21.0 q
2.62 m 2.35 m 2.38 m 2.49 s 5.25 d (3.9) 4.08 d (3.9)
3.88 1.86 2.24 2.90 0.74 0.94
s m m m s s
7.16 6.40 7.26 1.04 1.04 5.12 3.61
s s s s s s s
1.98 s
Phytochemistry Letters 31 (2019) 225–228
L. Zhang, et al.
toonamicronoid A. The 1H and 13C NMR spectroscopic data were assigned based on its 1H-1H COSY, HSQC, HMBC, and ROESY spectra (Table 1). Compound 2 was a white power and its molecular formula of C25H30O9 was deduced on the basis of its ESIMS ion peaks at m/z 497 [M+Na]+ and 971 [2M +Na]+, HREIMS ion peak at m/z 474.1879 [M]+ (calcd. for C25H30O9 474.1890), and the 13C NMR data. Comparing the NMR data with the reported ciliatonoid C (Liu et al., 2016) revealed that 2 is a B-seco limonoid. Analysis of its 2D NMR data, 1 H-1H COSY correlations of H-2/H-3, H-9/H-11, H-11/H-12, H-15/H16 and H-16/H-17 and the HMBC correlations from H-2 to C-1, H-3 to C-4 and C-28, H-6 to C-7, C-4, and C-5, H-9 to C-5 and C-30, H-11 to C1, C-12 and C-13, H-15 to C-14 and C-16, and H-17 to C-13, C-18, and C-20, suggested a same planar structure with the known ciliatonoid C. The relative configuration of 2 was estimated by ROESY experiment. As shown in Fig. 1, the ROESY correlations of H-30/H-17 and H15 showed the configurations of OH-8α, H-15β, and H-15β. The presence of the NOE correlations of H-19/ H-11 and H-30, H-9/H-18, combing with the absence of NOE correlation of H-17/H-18 suggested the relative configurations of H-9, H-11, CH3-19, and CH3-18 are α, β, β, and α, respectively. Because there is no NOE correlation of H-6/H-9 and H-30, the relative configurations of both OH-5 and H-6 are estimated as α. Furthermore, the ROESY correlation of H-6/H-28 showed an α position of CH3-28. Thus, the relative configuration of 2 was evaluated as shown, which is different from ciliatonoid C (Liu et al., 2016). Therefore, the structure of 2 was identified and named toonamicronoid B. Compound 3 was obtained as a white amorphous powder. Its molecular formula was deduced to be C29H36O10 by ESIMS at m/z 567 [M +Na]+ and 1111 [2M +Na]+, HREIMS at m/z 544.2313 [M]+ (calcd. for C29H36O10 544.2308) and 13C NMR spectrum. Comparison of the NMR data with toonamicronoid B (2) and the reported toonaciliatone F (Yang et al., 2015) suggested 3 possesses a same skeleton as toonaciliatone F. This was supported by the 1H-1H COSY correlations of H-5/H6, H-11/H-9 and H-12, and H-16/H-15 and H-17 and the HMBC correlations from H-2 to C-1, H-29/30 to C-4, and C-3, H-5 to C-4 and C-6, H-19 to C-1 and C-10, H-30 to C-8 and C-9, H-11 to C-1, and H-18 to C17, C-12, and C-13, and H-17 to C-20. The major differences are the C-
2.3. Assay for cytotoxic activity The cytotoxic activities of compounds 1 against five human cancer cell lines were evaluated by the reported method (Alley et al., 1988; Fang et al., 2018; Zhang et al., 2019). HL-60, SMMC-7721, A-549, MCF7, and SW480 (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 or DMEM medium (Hy-clone, Logan, UT, USA) by means of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4- sulfopheny)- 2Htetrazolium (MTS) assay. Cis- dichlorodiamineplatinum (DDP) and taxol were included as a positive control. 3. Results and discussion Compound 1 was isolated as a white power and the molecular formula of C25H28O10 was deduced on the basis of its ESIMS ion peaks at m/z 523 [M+Cl]− and 1011 [2M +Cl]− and HREIMS ion peak at m/z 488.1716 [M]+ (calcd. For C25H28O10 488.1682) alongside the 13C NMR and DEPT data. Comparison of the 13C NMR data with that of the known toonaciliatone C reported in the literature (Yang et al., 2015) suggested that 1 is a B-seco-29-nor-limonoid triterpenoid. The presence of 1H-1H COSY correlations of H-2/H-3, H-9/H-11, H-15/H-16, H-16/H17, and H-22/H-23 and HMBC correlations from H-2 to C-4 and C-10, H-3 to C-28 and C-5, H-6 to C-4 C-5, and C-7, H-19 to C-1, C-9 and C-10, H-11 to C-1, C-10, C-12 and C-13, H-30 to C-8 and C-9, H-15 to C-14, C16, and C-17, H-17 to C-13, C-18, and C-20, H-21 to C-20 and C-23, and H-22 to C-20 (Fig. 2) revealed the planar structure of 1 is the same as toonaciliatone C. The relative configuration of 1 was estimated by ROESY experiment. The presence of ROESY correlations of H-19/H-11, H-11/H-17, H-9/H-18, and H-18/H-15 and the absence of NOE correlations of H18/H-17 suggested the configurations of C-10, 9, 11, 13, 17, 14, 15 shown in Fig. 1. Furthermore, the relative configuration of C-4, 5, 6 were estimated according to the presence of NOE correlation of H-19/ H-6 and the absence of NOE correlation of H-28/H-6. The absence of NOE correlation of H-9/H-30 showed a configuration of OH-8α. The configuration of OH-6 is α which is different from that of the reported toonaciliatone C. Hence, compound 1 was identified and named as
Fig. 2. Key 1H-1H COSY, HMBC and ROESY correlations of 1–4. 227
Phytochemistry Letters 31 (2019) 225–228
L. Zhang, et al.
12 and C-29 with toonaciliatone F. C-12 is non-submitted and C-19 is substituted by CH3COO according to the presence of HMBC correlation from H-29 to 29eCOO. Therefore, the planar structure of 3 was estimated as shown. ROESY experiment was employed to identify the relative configuration. The presence of NOE correlations of H-18/H-9, and H-17, H15/H-30, H-30/H-11 and the absence of NOE correlation of H-30/H-9 and H-18/H-15 estimate the relative configurations of C-9, C-8, C-11, C14, C-15, C-13, and C-17 as shown. The ROESY correlations of H-19/H5, H-11, and H-29 showed the configuration at C-4, C-5, and C-10. Hence, 3 was identified and named toonamicronoid C. Compound 4 was isolated as a white amorphous powder and its molecular formula was deduced to be C29H36O10 by ESIMS at m/z 535 [M+Na]+ and 1047 [2M+Na]+, HREIMS at m/z 512.2374 [M]+ (calcd. for C29H36O8 512.2410) and 13C NMR spectrum. Analyzing its NMR data, 4 is a limonoid by comparison of the NMR data with the reported compound toonacilin (Luo et al., 2017). The presence of 1H-1H COSY correlations of H-1/H-2, H-5/H-6, H-11/H-9 and H-12, and H-16/ H-15 and H-17 and the HMBC correlations from H-1 to C-3, H-2 to C-3, H-29/30 to C-3, C-4, and C-5, H-5 to C-10, H-6 to C-7, H-19 to C-1, C10, and C-9, H-11 to C-9, C-12, and C-8, H-18 to C-12, C-13, and C-17, H-15 to C-14 and C-16, and H-17 to C-20 suggested a skeleton same as toonacilin. The HMBC correlations from H-30 to C-8, C-9, and C-14 showed a double bond between C-8 and C-30. A CH3COO group was linked to C-11 according to the HMBC correlation from H-11 to 11eCOO. The presence of the HMBC correlation from OCH3 to C-7 revealed an OCH3 located at C-7. Hence, the planar structure was estimated. The relative configuration of 4 was evaluated by REOSY experiment. The NOE correlations of H-17/H-11 and H-12, and H-11/H-15, combing with the absence of NOE from H-9/H-12 and H-17/H-18, estimate the relative configurations of C-9, C-11, C-12, C-13, C-14, C-15, and C-17 as shown. The ROESY correlation of H-19/H-6 identified the cis-configuration between CH3-19 and CH2-6. Therefore, 4 was identified and named toonamicronoid D. The cytotoxic activities of toonamicronoids A–D (1-4) against HL60, SMMC-7721, A-549, MCF-7, and SW480 were evaluated. Compounds 1 and 4 showed moderate cytotoxicity against HL-60 with the IC50 values of 17.3 ± 1.5 and 22.7 ± 1.9 μM, respectively.
Acknowledgments This work was financially supported by a grant from the Science and Technology Project of Yunnan Province (Nos. 2018BA084, 2018BA035) and a grant from Foundational Project of China Tabacco Yunnan Industrial Co., Ltd. (No. 2015CP02). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.04.010. References Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, B.J., Mayo, J.G., Shoemaker, R.H., Boyd, M.R., 1988. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48, 589–601. Chen, H.-D., Yang, S.-P., Wu, Y., Dong, L., Yue, J.-M., 2009. Terpenoids from Toona ciliata. J. Nat. Prod. 72, 685–689. Chen, H., Jin, M., Wang, Y.-F., Wang, Y.-Q., Meng, L., Li, R., Wang, J.-P., Gao, L., Kong, Y., Wei, J.-F., 2014. Effect of Toona microcarpa harms leaf extract on the coagulation system. Biomed Res. Int. 2014, 1–7. De Leo, M., Milella, L., Braca, A., De Tommasi, N., 2018. Cedrela and Toona genera: a rich source of bioactive limonoids and triterpenoids. Phytochem. Rev. 17, 751–783. Fang, X., Di, Y.-T., He, H.-P., Hu, G.-W., Li, S.-L., Hao, X.-J., 2010. Chemical constituents of Toona microcarpa (C. DC.) Harms in Engl. (Meliaceae). Biochem. Syst. Ecol. 38, 128–130. Fang, X., Zeng, R.-T., Zhuo, Z.-G., Shen, Y.-H., Zhang, W.-D., 2018. Sesquiterpenoids from Ainsliaea yunnanensis and their cytotoxic activities. Phytochem. Lett. 26, 25–29. Flora of China Editorial Committee, 2004. Flora Reipublicae Popularis Sinicae. Science Press, Beijing. Hseu, Y.-C., Chen, S.-C., Lin, W.-H., Hung, D.-Z., Lin, M.-K., Kuo, Y.-H., Wang, M.-T., Cho, H.-J., Wang, L., Yang, H.-L., 2011. Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells. J. Ethnopharmacol. 134, 111–121. Huang, P.-J., Hseu, Y.-C., Lee, M.-S., Kumar, K.J.S., Wu, C.-R., Hsu, L.-S., Liao, J.-W., Cheng, I.S., Kuo, Y.-T., Huang, S.-Y., Yang, H.-L., 2012. In vitro and in vivo activity of gallic acid and Toona sinensis leaf extracts against HL-60 human premyelocytic leukemia. Food Chem. Toxicol. 50, 3489–3497. Kakumu, A., Ninomiya, M., Efdi, M., Adfa, M., Hayashi, M., Tanaka, K., Koketsu, M., 2014. Phytochemical analysis and antileukemic activity of polyphenolic constituents of Toona sinensis. Bioorg. Med. Chem. Lett. 24, 4286–4290. Liu, J., Yang, S.-P., Su, Z.-S., Lin, B.-D., Wu, Y., Yue, J.-M., 2011. Limonoids from the stems of Toona ciliata var. henryi (Meliaceae). Phytochemistry 72, 2189–2196. Liu, C.-P., Wang, G.-C., Gan, L.-S., Xu, C.-H., Liu, Q.-F., Ding, J., Yue, J.-M., 2016. Ciliatonoids A and B, two limonoids from Toona ciliata. Org. Lett. 18, 2894–2897. Luo, J., Huang, W.-S., Hu, S.-M., Zhang, P.-P., Zhou, X.-W., Wang, X.-B., Yang, M.-H., Luo, J.-G., Wang, C., Liu, C., Yao, H.-Q., Zhang, C., Sun, H.-B., Chen, Y.-J., Kong, L.-Y., 2017. Rearranged limonoids with unique 6/5/6/5 tetracarbocyclic skeletons from Toona ciliata and biomimetic structure divergence. Org. Chem. Front. 4, 2417–2421. Yang, M.-S., Hu, S.-M., Kong, L.-Y., Luo, J., 2015. B-seco-29-nor-Limonoids from the stem barks of Toona ciliate var. yunnanensis. Tetrahedron 71, 8472–8477. Zhang, F., Liao, S.-G., Zhang, C.-R., He, X.-F., Chen, W.-S., Yue, J.-M., 2011. Limonoids and Diterpenoids from Toona ciliata Roem. var. yunnanensis. Planta Med. 77, 1617–1622. Zhang, Q.-Q., Sun, Z.-Y., Feng, X.-Y., Chen, R.-J., Deng, W., Tang, Y.-L., Guo, Z.-Y., Liu, C.X., Chen, J.-F., Zou, K., 2019. Thymol derivatives from the roots of Eupatorium chinense and their cytotoxic activities. Phytochem. Lett. 29, 165–168.
4. Conclusion This paper presents that four new B-seco-limonoids, toonamicronoids A–D (1-4), along with four known ones, toonaciliatin H–J (5-7), and toonacilianin A (8), were isolated from T. microcarpa. Compounds 1 and 4 showed moderate cytotoxicity against HL-60. Conflict of interest The authors have declared no conflict of interest.
228
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Toonamicronoids A–D, four new B-seco-limonoids from Toona microcarpa a,b,1
a,1
a
a
a
b
a
Ling Zhang , Jianjun Xia , Jianyun Yang , Long Zhu , Danyu Tang , Ji Ma , Yingliang Zhao , ⁎ Xiaoying Zenga, Minghua Qiub, a b
T
R&D Center of China Tabacco Yunnan Industrial Co., Ltd. Kunming, 650202, China Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China
ARTICLE INFO
ABSTRACT
Keywords: Toona microcarpa Toonamicronoids A–D Limonoid Identification Cytotoxicity
Four new B-seco-limonoids, toonamicronoids A–D (1–4), together with four known ones, toonaciliatin H–J (5–7), and toonacilianin A (8), were isolated from Toona microcarpa. Their structures were identified on the basis of ESIMS, HREIMS and 1D/2D NMR analysis. Compounds 1 and 4 showed moderate cytotoxicities against HL-60 with the IC50values of 17.3 ± 1.5 and 22.7 ± 1.9 μM, respectively.
1. Introduction
2. Experimental Section
The genus Toona, belonging to the family of Meliaceae, comprises five species including Toona ciliate, Cedrela toona, Toona sinensis, Toona microcarpa, Toona febrifuga, and Toona calantas (Flora of China Editorial Committee, 2004). Their barks, leaves, and fruits are frequently used as the food additives and herbal medicine for treating various disease, such as anabrosis, metrorrhagia, dysentery, enteritis, etc. Previous phytochemical investigations on the genus Toona showed the major components of phenols (Kakumu et al., 2014) and terpenoids (De Leo et al., 2018) which possess various pharmacological effects (Chen et al., 2014; Hseu et al., 2011; Huang et al., 2012; Zhang et al., 2011). T. microcarpa was a macrophanerophyte of Toona (Meliaceae). Phytochemical research revealed only three phenols and one terpenoid reported, i.e. (+)-catechin, (6R,7S,8S)-7a-[(β-D-glucopyranosyl)oxy] lyoniresinol, (6R,7R,8R)-7a-[(β-D- glucopyranosyl)oxy]lyoniresinol, and 20-hydroxyecdysone (Fang et al., 2010). To explore new components of T. microcarpa, in the present study, an investigation on chemical constituents of T. microcarpa was carried out. Four new B-secolimonoids, toonamicronoids A–D (1–4), along with four known ones toonaciliatin H–J (5–7) (Chen et al., 2009), and toonacilianin A (8) (Liu et al., 2011) (Fig. 1), were isolated from T. microcarpa. Their structures were identified on the basis of ESIMS, HREIMS and 1D/2D NMR analysis. The cytotoxic activities of the new compounds isolated were also evaluated.
2.1. Plant material The barks of Toona microcarpa (C. DC.) C. Y. Wu were collected by Mr. Qing-Yong Wu in Ning’er County of Yunnan Province, China in August 2017, and was identified by Prof. Zong-Yu Wang, a botanic taxonomist at the Kunming Institute of Botany, Chinese Academy of Sciences. The voucher specimen (TC-2017-01) had been deposited in Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China. 2.2. Extraction and isolation The air-dried and powdered barks of T. ciliata (25 Kg) were extracted with MeOH under reflux for three times. The methanol extract was concentrated under reduced pressure to afford a black residue. The residue was suspended in water (10 L) and then partitioned with petroleum ether (10 L × 3) and EtOAc (10 L × 3), successively. The EtOAc extract was evaporated in a vacuum to obtain a brownish black residue (600 g). The EtOAc soluble portion was subjected to silica gel chromatography column (CC) and eluted successively with CHCl3, CHCl3MeOH (100:1), CHCl3-MeOH (50:1), and CHCl3-MeOH (15:1). The CHCl3-MeOH (50:1) portion of the EtOAc extract was subjected to silica gel CC and eluted with PE/acetone (10:1) to afford four fractions (Fr. I–V). Fraction III was subjected to RP-18 silica gel CC (MeOH/H2O, 50:50 → 80:20, v/v) to afford eight subfractions III1-5. Subfraction III2
Corresponding author. E-mail address: [email protected] (M. Qiu). 1 These authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.phytol.2019.04.010 Received 16 December 2018; Received in revised form 3 April 2019; Accepted 5 April 2019 1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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Fig. 1. The structures of 1–4.
[M]+ (calcd. for C25H30O9 474.1890); 1H and data, see Table 1.
was rechromatographed over an RP-18 silica gel CC eluting with MeOH/H2O (60:40) to yield compound 1 (5 mg), 3 (11 mg), 5 (28 mg), and 6 (7.5 mg). Subfraction III3 was separated by an RP-18 silica gel (MeOH/H2O, 70:30) to yield compound 2 (8 mg) and 7 (10 mg). Subfraction III4 was further chromatographed on an RP-18 silica gel CC eluting with MeOH/H2O (65:35) to yield compound 4 (4 mg) and 8 (5 mg).
13
C NMR spectroscopic
2.2.3. Toonaciliatone C (3) White amorphous powder; m.p. 215 C; [α]20 D = + 40.4 (c 0.40, MeOH); m/z 567 [M+Na]+, 1111 [2M+Na]+; HREIMS m/z 544.2313 [M]+ (calcd. for C29H36O10 544.2308); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.1. Toonaciliatone A (1) White amorphous powder; m.p. 250 ○C; [α]20 D = + 108.9 (c 0.10, pyridine); m/z 523 [M+Cl]−, 1011 [2M+Cl]−; HREIMS m/z 488.1716 [M]+ (calcd. for C25H28O10 488.1682); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.4. Toonaciliatone D (4) White amorphous powder; m.p. 168 C; [α]20 D = + 42.2 (c 0.41, CHCl3); m/z 535 [M+Na]+, 1047 [2M+Na]+; HREIMS m/z 512.2374 [M]+ (calcd. for C29H36O8 512.2410); 1H and 13C NMR spectroscopic data, see Table 1.
2.2.2. Toonaciliatone B (2) White amorphous powder; m.p. 210 C; [α]20 D = + 56.9 (c 0.50, MeOH); m/z 497 [M+Na]+, 971 [2M+Na]+; HREIMS m/z 474.1879 Table 1 The NMR spectroscopic data of toonamicronoids A–D (1–4). Position
toonamicronoid A (1) in C5D5N
toonamicronoid B (2) in C5D5N
toonamicronoid C (3) in C5D5N
toonamicronoid D (4) in CDCl3
δC (mult.)
δC (mult.)
δC (mult.)
δC (mult.)
δH (mult. J in Hz)
153.0 d 125.9 d 203.1 s 45.9 s 43.9 d 31.8 t
7.18 d (10.4) 6.03 d (10.4)
1 2 3 4 5 6
106.2 s 126.9 d 132.9 d 86.7 s 82.0 s 77.7 d
7 8 9 10 11 12
174.0 s 72.1 s 54.4 d 55.1 s 79.7 d 210.3 s
13 14 15 16
53.9 75.5 57.3 31.5
17 18 19 20 21 22 23 28 29 30 7-OCH3 29- OCOCH3 29-OCOCH3
35.8 d 19.2 q 12.9 q 124.0 s 141.6 d 113.2 d 143.2 d 24.3 q
s s d t
23.7 q
δH (mult. J in Hz) 6.16 d (10.4) 5.89 d (10.4) 4.69 s
3.75 s 5.29 d (13.6)
3.80 2.06 2.26 3.16 1.46 2.00
m m m m s s
7.55 6.90 7.62 1.92
s s s s
1.78 s
105.4 s 126.1 d 133.9 d 87.1 s 82.2 s 77.9 d 174.3 s 75.7 s 55.6 d 54.9 s 74.1 d 44.1 t 41.5 73.3 56.1 31.5
s s d t
35.8 d 23.3 q 13.3 q 123.8 s 140.2 d 111.4 d 143.6 d 24.4 q 24.0 q
δH (mult. J in Hz) 6.16 d (10.4) 5.94 d (10.4) 4.69 s
3.42 d (13.6) 4.85 m 2.65 dd (12.4, 2.8) 1.74 m 3.57 1.86 2.12 2.89 1.22 1.96
m m m m s s
7.56 7.20 7.64 1.97
s s s s
188.1 s 97.9 d 206.1 s 48.8 s 49.0 d 71.2 d 173.8 s 74.2 s 54.8 d 50.1 s 82.7 d 31.6 t 47.5 74.8 56.4 33.7
s s d t
41.7 d 20.4 q 16.5 q 123.5 s 140.1 d 111.8 d 143.3 d 25.9 q 66.6 t 24.2 q 51.5 q 170.4 s 20.8 q
1.66 s
226
δH (mult. J in Hz) 5.60 s 3.11 m 4.69 s
3.62 m 5.11 m 1.88 m 2.22 m 3.52 3.04 3.86 3.08 1.51 1.58
m m m m s s
7.47 6.48 7.55 1.24 4.50 1.59 3.50
s s s s m s s
1.99 s
173.9 s 135.7 s 56.7 d 41.8 s 73.3 d 75.0 d 45.3 72.8 60.3 31.8
s s d t
39.3 d 13.1 q 20.0 q 123.6 s 140.1 d 112.3 d 142.1 d 22.5 q 22.8 q 120.5 t 51.9 q 170.7 s 21.0 q
2.62 m 2.35 m 2.38 m 2.49 s 5.25 d (3.9) 4.08 d (3.9)
3.88 1.86 2.24 2.90 0.74 0.94
s m m m s s
7.16 6.40 7.26 1.04 1.04 5.12 3.61
s s s s s s s
1.98 s
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toonamicronoid A. The 1H and 13C NMR spectroscopic data were assigned based on its 1H-1H COSY, HSQC, HMBC, and ROESY spectra (Table 1). Compound 2 was a white power and its molecular formula of C25H30O9 was deduced on the basis of its ESIMS ion peaks at m/z 497 [M+Na]+ and 971 [2M +Na]+, HREIMS ion peak at m/z 474.1879 [M]+ (calcd. for C25H30O9 474.1890), and the 13C NMR data. Comparing the NMR data with the reported ciliatonoid C (Liu et al., 2016) revealed that 2 is a B-seco limonoid. Analysis of its 2D NMR data, 1 H-1H COSY correlations of H-2/H-3, H-9/H-11, H-11/H-12, H-15/H16 and H-16/H-17 and the HMBC correlations from H-2 to C-1, H-3 to C-4 and C-28, H-6 to C-7, C-4, and C-5, H-9 to C-5 and C-30, H-11 to C1, C-12 and C-13, H-15 to C-14 and C-16, and H-17 to C-13, C-18, and C-20, suggested a same planar structure with the known ciliatonoid C. The relative configuration of 2 was estimated by ROESY experiment. As shown in Fig. 1, the ROESY correlations of H-30/H-17 and H15 showed the configurations of OH-8α, H-15β, and H-15β. The presence of the NOE correlations of H-19/ H-11 and H-30, H-9/H-18, combing with the absence of NOE correlation of H-17/H-18 suggested the relative configurations of H-9, H-11, CH3-19, and CH3-18 are α, β, β, and α, respectively. Because there is no NOE correlation of H-6/H-9 and H-30, the relative configurations of both OH-5 and H-6 are estimated as α. Furthermore, the ROESY correlation of H-6/H-28 showed an α position of CH3-28. Thus, the relative configuration of 2 was evaluated as shown, which is different from ciliatonoid C (Liu et al., 2016). Therefore, the structure of 2 was identified and named toonamicronoid B. Compound 3 was obtained as a white amorphous powder. Its molecular formula was deduced to be C29H36O10 by ESIMS at m/z 567 [M +Na]+ and 1111 [2M +Na]+, HREIMS at m/z 544.2313 [M]+ (calcd. for C29H36O10 544.2308) and 13C NMR spectrum. Comparison of the NMR data with toonamicronoid B (2) and the reported toonaciliatone F (Yang et al., 2015) suggested 3 possesses a same skeleton as toonaciliatone F. This was supported by the 1H-1H COSY correlations of H-5/H6, H-11/H-9 and H-12, and H-16/H-15 and H-17 and the HMBC correlations from H-2 to C-1, H-29/30 to C-4, and C-3, H-5 to C-4 and C-6, H-19 to C-1 and C-10, H-30 to C-8 and C-9, H-11 to C-1, and H-18 to C17, C-12, and C-13, and H-17 to C-20. The major differences are the C-
2.3. Assay for cytotoxic activity The cytotoxic activities of compounds 1 against five human cancer cell lines were evaluated by the reported method (Alley et al., 1988; Fang et al., 2018; Zhang et al., 2019). HL-60, SMMC-7721, A-549, MCF7, and SW480 (ATCC, Manassas, VA, USA) were cultured in RPMI-1640 or DMEM medium (Hy-clone, Logan, UT, USA) by means of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4- sulfopheny)- 2Htetrazolium (MTS) assay. Cis- dichlorodiamineplatinum (DDP) and taxol were included as a positive control. 3. Results and discussion Compound 1 was isolated as a white power and the molecular formula of C25H28O10 was deduced on the basis of its ESIMS ion peaks at m/z 523 [M+Cl]− and 1011 [2M +Cl]− and HREIMS ion peak at m/z 488.1716 [M]+ (calcd. For C25H28O10 488.1682) alongside the 13C NMR and DEPT data. Comparison of the 13C NMR data with that of the known toonaciliatone C reported in the literature (Yang et al., 2015) suggested that 1 is a B-seco-29-nor-limonoid triterpenoid. The presence of 1H-1H COSY correlations of H-2/H-3, H-9/H-11, H-15/H-16, H-16/H17, and H-22/H-23 and HMBC correlations from H-2 to C-4 and C-10, H-3 to C-28 and C-5, H-6 to C-4 C-5, and C-7, H-19 to C-1, C-9 and C-10, H-11 to C-1, C-10, C-12 and C-13, H-30 to C-8 and C-9, H-15 to C-14, C16, and C-17, H-17 to C-13, C-18, and C-20, H-21 to C-20 and C-23, and H-22 to C-20 (Fig. 2) revealed the planar structure of 1 is the same as toonaciliatone C. The relative configuration of 1 was estimated by ROESY experiment. The presence of ROESY correlations of H-19/H-11, H-11/H-17, H-9/H-18, and H-18/H-15 and the absence of NOE correlations of H18/H-17 suggested the configurations of C-10, 9, 11, 13, 17, 14, 15 shown in Fig. 1. Furthermore, the relative configuration of C-4, 5, 6 were estimated according to the presence of NOE correlation of H-19/ H-6 and the absence of NOE correlation of H-28/H-6. The absence of NOE correlation of H-9/H-30 showed a configuration of OH-8α. The configuration of OH-6 is α which is different from that of the reported toonaciliatone C. Hence, compound 1 was identified and named as
Fig. 2. Key 1H-1H COSY, HMBC and ROESY correlations of 1–4. 227
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12 and C-29 with toonaciliatone F. C-12 is non-submitted and C-19 is substituted by CH3COO according to the presence of HMBC correlation from H-29 to 29eCOO. Therefore, the planar structure of 3 was estimated as shown. ROESY experiment was employed to identify the relative configuration. The presence of NOE correlations of H-18/H-9, and H-17, H15/H-30, H-30/H-11 and the absence of NOE correlation of H-30/H-9 and H-18/H-15 estimate the relative configurations of C-9, C-8, C-11, C14, C-15, C-13, and C-17 as shown. The ROESY correlations of H-19/H5, H-11, and H-29 showed the configuration at C-4, C-5, and C-10. Hence, 3 was identified and named toonamicronoid C. Compound 4 was isolated as a white amorphous powder and its molecular formula was deduced to be C29H36O10 by ESIMS at m/z 535 [M+Na]+ and 1047 [2M+Na]+, HREIMS at m/z 512.2374 [M]+ (calcd. for C29H36O8 512.2410) and 13C NMR spectrum. Analyzing its NMR data, 4 is a limonoid by comparison of the NMR data with the reported compound toonacilin (Luo et al., 2017). The presence of 1H-1H COSY correlations of H-1/H-2, H-5/H-6, H-11/H-9 and H-12, and H-16/ H-15 and H-17 and the HMBC correlations from H-1 to C-3, H-2 to C-3, H-29/30 to C-3, C-4, and C-5, H-5 to C-10, H-6 to C-7, H-19 to C-1, C10, and C-9, H-11 to C-9, C-12, and C-8, H-18 to C-12, C-13, and C-17, H-15 to C-14 and C-16, and H-17 to C-20 suggested a skeleton same as toonacilin. The HMBC correlations from H-30 to C-8, C-9, and C-14 showed a double bond between C-8 and C-30. A CH3COO group was linked to C-11 according to the HMBC correlation from H-11 to 11eCOO. The presence of the HMBC correlation from OCH3 to C-7 revealed an OCH3 located at C-7. Hence, the planar structure was estimated. The relative configuration of 4 was evaluated by REOSY experiment. The NOE correlations of H-17/H-11 and H-12, and H-11/H-15, combing with the absence of NOE from H-9/H-12 and H-17/H-18, estimate the relative configurations of C-9, C-11, C-12, C-13, C-14, C-15, and C-17 as shown. The ROESY correlation of H-19/H-6 identified the cis-configuration between CH3-19 and CH2-6. Therefore, 4 was identified and named toonamicronoid D. The cytotoxic activities of toonamicronoids A–D (1-4) against HL60, SMMC-7721, A-549, MCF-7, and SW480 were evaluated. Compounds 1 and 4 showed moderate cytotoxicity against HL-60 with the IC50 values of 17.3 ± 1.5 and 22.7 ± 1.9 μM, respectively.
Acknowledgments This work was financially supported by a grant from the Science and Technology Project of Yunnan Province (Nos. 2018BA084, 2018BA035) and a grant from Foundational Project of China Tabacco Yunnan Industrial Co., Ltd. (No. 2015CP02). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.04.010. References Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, B.J., Mayo, J.G., Shoemaker, R.H., Boyd, M.R., 1988. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48, 589–601. Chen, H.-D., Yang, S.-P., Wu, Y., Dong, L., Yue, J.-M., 2009. Terpenoids from Toona ciliata. J. Nat. Prod. 72, 685–689. Chen, H., Jin, M., Wang, Y.-F., Wang, Y.-Q., Meng, L., Li, R., Wang, J.-P., Gao, L., Kong, Y., Wei, J.-F., 2014. Effect of Toona microcarpa harms leaf extract on the coagulation system. Biomed Res. Int. 2014, 1–7. De Leo, M., Milella, L., Braca, A., De Tommasi, N., 2018. Cedrela and Toona genera: a rich source of bioactive limonoids and triterpenoids. Phytochem. Rev. 17, 751–783. Fang, X., Di, Y.-T., He, H.-P., Hu, G.-W., Li, S.-L., Hao, X.-J., 2010. Chemical constituents of Toona microcarpa (C. DC.) Harms in Engl. (Meliaceae). Biochem. Syst. Ecol. 38, 128–130. Fang, X., Zeng, R.-T., Zhuo, Z.-G., Shen, Y.-H., Zhang, W.-D., 2018. Sesquiterpenoids from Ainsliaea yunnanensis and their cytotoxic activities. Phytochem. Lett. 26, 25–29. Flora of China Editorial Committee, 2004. Flora Reipublicae Popularis Sinicae. Science Press, Beijing. Hseu, Y.-C., Chen, S.-C., Lin, W.-H., Hung, D.-Z., Lin, M.-K., Kuo, Y.-H., Wang, M.-T., Cho, H.-J., Wang, L., Yang, H.-L., 2011. Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells. J. Ethnopharmacol. 134, 111–121. Huang, P.-J., Hseu, Y.-C., Lee, M.-S., Kumar, K.J.S., Wu, C.-R., Hsu, L.-S., Liao, J.-W., Cheng, I.S., Kuo, Y.-T., Huang, S.-Y., Yang, H.-L., 2012. In vitro and in vivo activity of gallic acid and Toona sinensis leaf extracts against HL-60 human premyelocytic leukemia. Food Chem. Toxicol. 50, 3489–3497. Kakumu, A., Ninomiya, M., Efdi, M., Adfa, M., Hayashi, M., Tanaka, K., Koketsu, M., 2014. Phytochemical analysis and antileukemic activity of polyphenolic constituents of Toona sinensis. Bioorg. Med. Chem. Lett. 24, 4286–4290. Liu, J., Yang, S.-P., Su, Z.-S., Lin, B.-D., Wu, Y., Yue, J.-M., 2011. Limonoids from the stems of Toona ciliata var. henryi (Meliaceae). Phytochemistry 72, 2189–2196. Liu, C.-P., Wang, G.-C., Gan, L.-S., Xu, C.-H., Liu, Q.-F., Ding, J., Yue, J.-M., 2016. Ciliatonoids A and B, two limonoids from Toona ciliata. Org. Lett. 18, 2894–2897. Luo, J., Huang, W.-S., Hu, S.-M., Zhang, P.-P., Zhou, X.-W., Wang, X.-B., Yang, M.-H., Luo, J.-G., Wang, C., Liu, C., Yao, H.-Q., Zhang, C., Sun, H.-B., Chen, Y.-J., Kong, L.-Y., 2017. Rearranged limonoids with unique 6/5/6/5 tetracarbocyclic skeletons from Toona ciliata and biomimetic structure divergence. Org. Chem. Front. 4, 2417–2421. Yang, M.-S., Hu, S.-M., Kong, L.-Y., Luo, J., 2015. B-seco-29-nor-Limonoids from the stem barks of Toona ciliate var. yunnanensis. Tetrahedron 71, 8472–8477. Zhang, F., Liao, S.-G., Zhang, C.-R., He, X.-F., Chen, W.-S., Yue, J.-M., 2011. Limonoids and Diterpenoids from Toona ciliata Roem. var. yunnanensis. Planta Med. 77, 1617–1622. Zhang, Q.-Q., Sun, Z.-Y., Feng, X.-Y., Chen, R.-J., Deng, W., Tang, Y.-L., Guo, Z.-Y., Liu, C.X., Chen, J.-F., Zou, K., 2019. Thymol derivatives from the roots of Eupatorium chinense and their cytotoxic activities. Phytochem. Lett. 29, 165–168.
4. Conclusion This paper presents that four new B-seco-limonoids, toonamicronoids A–D (1-4), along with four known ones, toonaciliatin H–J (5-7), and toonacilianin A (8), were isolated from T. microcarpa. Compounds 1 and 4 showed moderate cytotoxicity against HL-60. Conflict of interest The authors have declared no conflict of interest.
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