New secoiridoids from the fruits of Ligustrum lucidum

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1 Journal of Asian Natural Products Research ISSN: (Print) (nline) Journal homepage: New secoiridoids from the fruits of Ligustrum lucidum Zuo-Cheng Qiu, Xiao-Xiao Zhao, Qing-Chang Wu, Jian-Wu Fu, Yi Dai, Man-Sau Wong & Xin-Sheng Yao To cite this article: Zuo-Cheng Qiu, Xiao-Xiao Zhao, Qing-Chang Wu, Jian-Wu Fu, Yi Dai, Man- Sau Wong & Xin-Sheng Yao (2018): New secoiridoids from the fruits of Ligustrum lucidum, Journal of Asian Natural Products Research, DI: / To link to this article: Published online: 28 Mar Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at

2 Journal of Asian Natural Products Research, New secoiridoids from the fruits of Ligustrum lucidum Zuo-Cheng Qiu a#, Xiao-Xiao Zhao a#, Qing-Chang Wu a, Jian-Wu Fu b, Yi Dai a,b, Man- Sau Wong c and Xin-Sheng Yao a,b,d a Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, College of Pharmacy, Jinan University, Guangzhou , China; b Key Laboratory of Standard Material in Natural Medicine of Guangdong Province, Guangzhou , China; c Shenzhen Research Institute of the Hong Kong Polytechnic University, State Key Laboratory of Chinese Medicine and Molecular Pharmacology, Shenzhen , China; d College of Traditional Chinese Medicine, Shenyang Pharmaceutical University, Shenyang , China ABSTRACT Three new secoiridoids, nuezhenelenoliciside (1), isojaslanceoside B (2), 6 --trans-cinnamoyl-secologanoside (3), were isolated from the dried fruits of Ligustrum lucidum. Their structures were elucidated by comprehensive spectroscopic analysis. Among them, 1 featured a rare rearrangement product of secoiridoid, which underwent the cleavage of chemical bond between C-1 and -2, and the reformation of a new iridoid ring between C-8 and -2. In addition, all compounds were tested for their osteogenic activity on pre-osteoblastic MC3T3-E1 cells. As a result, 1 and 3 exhibited potent effects on promoting cell proliferation of pre-osteoblast cells. ARTICLE HISTRY Received 8 January 2018 Accepted 15 March 2018 KEYWRDS leaceae; secoiridoids; ligustri lucidi fructus; MC3T3-E1 cells; osteogenic activity 1. Introduction Ligustri Lucidi Fructus (LLF) is the dried fruit of Ligustrum lucidum Ait., which belongs to the leaceae family [1]. It is widely distributed in eastern Asia and India [2]. According to the records of traditional Chinese medicine, LLF was effective in nourishing the liver, kidney, and strengthening the bone and muscle [3]. Modern pharmacological study indicated that CNTACT Yi Dai daiyi1004@163.com; Xin-Sheng Yao tyaoxs@jnu.edu.cn # These authors contributed equally to this work Informa UK Limited, trading as Taylor & Francis Group

3 2 Z.-C. QIUA ET AL. H H 1'' 1' H H H H CH HC CCH H HC 3 3 CCH '' H ''' H H H 1' H 1' H 1 9 H 8 H H Figure 1. Chemical structures of compounds 1 3. LLF possesses antitumor, immune regulation, anti-inflammatory, and anti-osteoporosis activities [4 7]. Moreover, LLF was also approved as a dietary supplement by the Ministry of Health of China [8]. Chemical investigations on LLF indicated that it mainly contains various triterpenoids, iridoids, flavones, and phenolic glucosides [9]. Some compounds such as ursolic acid [10], salidroside, and nuzhenide were demonstrated to exert osteogenic effects [8]. However, the active chemical components responsible for anti-osteoporosis activity of LLF are far from clear, which seriously hindered the quality control and the application of LLF as a medicinal herb or dietary supplement. In our continuous chemical investigation on LLF, three new secoiridoids with osteogenic activity were discovered. In this paper, we mainly reported the isolation and structural elucidation of these three new secoiridoids (see Figure 1) from LLF, as well as the evaluation of their effects on promoting proliferation and bone nodule calcified formation of pre-osteoblastic MC3T3-E1 cells. 2. Results and discussion Compound 1 was obtained as yellow solid, and the molecular formula was established as C 31 H based on the molecular ion [M H] observed at m/z (C 31 H 41 17, calcd ) in the high-resolution electrospray ionization mass spectrum (HRESIMS). The 1 H-NMR spectrum of compound 1 (600 MHz, in CD 3 D) revealed a methyl signal at δ H 1.41 (3H, d, J = 6.9 Hz), a methoxy group at δ H 3.67 (3H, s), two olefinic proton signals at δ H 7.52 (1H, s) and δ H 6.58 (1H, s), as well as the presence of a para-substituted benzene ring [δ H 7.07 (2H, d, J = 8.5 Hz) and δ H 6.70 (2H, d, J = 8.5 Hz)]. The anomeric proton signals observed at δ H 4.32 (1H, d, J = 7.8 Hz) and δ H 4.49 (1H, d, J = 7.2 Hz) indicated the existence of two monosaccharide residues. Acid hydrolysis of 1 was performed to determine the types of monosaccharide residues and their absolute configuration, which were both identified as D-glucosyl residues. In addition, the anomeric configurations were determined to be β-configuration due to the large coupling constants. A total of 31 carbons signals were observed in 13 C-NMR spectrum of 1. It displayed six aromatic carbons [δ C 156.9, ( 2), 130.7, ( 2)] and carbons for D-glucosyl residues [δ C 104.5, 75.0, 78.0, 71.8, 75.3, 65.6 and 104.3, 74.5, 78.6, 71.2, 77.8, 62.6]. Besides, two carbonyl groups [δ C and δ C 169.3], four olefinic carbons [δ C 155.9, 143.4, 114.1, 107.3], and four remaining carbon signals at δ C 22.6, 28.7, 41.0, and 75.3 indicated the presence of an iridoid moiety in 1. The 1 H 1 H CSY spectrum showed obvious correlations between δ H 4.62 (1H, q, J = 6.9 Hz, H-8) and δ H 1.41 (3H, d, J = 6.9 Hz, H 3-10); δ H 4.05 (1H, dd, J = 9.7, 4.9 Hz, H-5)

4 JURNAL F ASIAN NATURAL PRDUCTS RESEARCH 3 H H H H CCH3 H H H H H H 1 H- 1 H CSY HMBC NESY Figure 2. Key 2D NMR correlations of compound 1. and δ H 2.45 (1H, dd, J = 12.7, 9.7 Hz, H-6b); δ H 2.84 (2H, dd, J = 15.5, 8.9 Hz, H-7 ) and δ H 3.97 (2H, dd, J = 15.5, 8.9 Hz, H-8 ), which demonstrated that a conjunction between δ C 75.3 (C-8) and δ C 22.6 (C-10); δ C 41.0 (C-6) and δ C 28.7 (C-5); δ C 36.4 (C-7 ) and δ C 72.4 (C-8 ). The HMBC spectrum exhibited correlations of 1 (Figure 2) from δ H 7.07 (H-2 ) to δ C (C-4 ), (C-6 ), 36.4 (C-7 ); δ H 3.97 (H-8 ) to δ C (C-1 ), (C-1 ) which indicated the existence of a salidroside residue. HMBC correlations from δ H 7.52 (H-3) to δ C (C-11), 75.3 (C-8), 28.7 (C-5); δ H 4.05 (H-5) to δ C (C-7), (C-11), (C-1), 75.3 (C-8); δ H 4.62 (H-8) to δ C (C-1) inferred the gross structure of secoiridoid moiety of 1. The HMBC correlations of δ H 4.37 (H-6 )/δ C 174.1(C-7) and δ H 6.58 (H-1)/δ C (C-1 ) indicated that the salidroside residue was attached to C-7, and the other glucose moiety was linked to C-1 of the aglycone. The relative configuration of 1 was elucidated by NESY experiment. The correlation between H-1 and H-8 observed in the NESY spectrum revealed that the double bond of C-1 and C-9 was E configuration. And the other significant NESY correlation between H 3-10 and H-6b indicated that they both situated on the same side. Therefore, H-5, H-8, and H-1 were situated on the other side. Up till now, all secoiridoids isolated from Ligustrum Linn. featured a β configuration of H-5 [2,11,12]. Thus, the structure of 1 was determined and shown in Figure 2, namely nuezhenelenoliciside (1). 1 featured a rare rearrangement product of secoiridoid, which underwent the cleavage of chemical bond between C-1 and -2, and the reformation of a new iridoid ring between C-8 and -2. Compound 2 was obtained as yellow solid. The molecular formula was established as C 26 H according to the molecular ion [M H] at m/z (C 26 H 29 14, calcd ). The 1 H NMR spectrum of 2 (600 MHz, in CD 3 D), showed two typical proton signals of iridoids glycosides at δ 6.01 (1H, s) and 7.54 (1H, s). Two signals at δ 7.45 (2 H, d, J = 8.6 Hz) and 6.80 (2H, d, J = 8.6 Hz) indicated the presence of a para-substituted benzene ring. The signals at δ 7.61 (1H, d, J = 15.9 Hz) and 6.31 (1H, d, J = 15.9 Hz) were assigned to coupled trans-alkene protons, while the anomeric proton signal at δ 4.83 (1H, d, J = 7.8 Hz) indicated the presence of a monosaccharide moiety. Signals for the methoxy groups were also observed at δ 3.73 (3H, s, CMe). The signal patterns of the 1 H and 13 C NMR spectra were similar to those of jaslanceoside B [13], except that compound 2 had a hydroxyl group at C-7 and a methoxy group at C-11 instead of the methoxy group and hydroxyl group in janslanceoside B. The geometry of the double bonds of 8 in 2 was also

5 4 Z.-C. QIUA ET AL. assigned as E configuration by the NESY correlation between H-1 and H-8. The relative configuration of 2 was elucidated by NESY correlations between H-1 and H-6b, which indicated the same side situation of the two protons. As described in 1, up till now, all secoiridoids isolated from Ligustrum Linn. were featured a β configuration of H-5 [2,11,12]. Thus, the structure of 2 was determined, namely isojaslanceoside B. Compound 3 was isolated as yellow solid and the molecular formula was established as C 25 H based on the molecular ion [M + H] + determined at m/z (C 25 H 29 12, calcd ). The signal patterns of the 1 H and 13 C NMR spectra were similar to those of 6 -trans-p-coumaroyl-secologanoside [14], except that 3 had a trans-cinnamoyl moiety [δ H (2H, m, H-2, 6 ), (3H, m, H-3, 4, 5 ), 7.72 (1H, d, J = 16.0 HZ, H-7 ), 6.57 (1H, d, J = 16.0 Hz, H-8 ); δ C (C-1 ), (C-3, 5 ), (C-2, 6 ), (C-4 ), (C-7 ), (C-8 ), (C-9 )], instead of the trans-p-coumaroyl moiety in 6 -E-p-coumaroyl-secologanoside. The relative configuration of 3 was also elucidated by NESY experiment. The correlation between H-1 and H-6b in the NESY spectrum suggested that H-1 and H-5 were in trans-configuration. In addition, the NESY correlation of H-6b/H-9 combined with coupling constants of the vicinal protons J 1,9 (4.1 Hz) indicated that these protons were cofacial on the cyclohexane ring. Taking biosynthetic pathway into account, the structure of 3 was determined as 6 --trans-cinnamoyl-secologanoside. As extracts of LLF and its components were widely reported to exert stimulatory effect on osteoblast-related bone formation [3,8,9], the osteogenic activity of 1 3 on murine pre-osteoblastic MC3T3-E1 cells was evaluated in vitro. As a result, 1 and 3 increased the proliferation of pre-osteoblast MC3T3-E1 cells. Effects of 1 3 on mineralization of osteoblasts were also investigated by Alizarin Red S staining and further quantified by measurement of the absorbance of dissolved calcium nodule. As shown in Figure 3, 1 (10 6, 10 5 M), 2 (10 5 M), and 3 (10 6 M) significantly promoted mineralization of MC3T3-E1 cells (p < vs. control). It was the first report that secoiridoids in the fruits of Ligustrum lucidum possessed anti-osteoporosis activity. 3. Experimental 3.1. General experimental procedures ptical rotations were measured on a JASC P1020 digital polarimeter (Jasco International Co. Ltd, Tokyo, Japan). The UV spectra were recorded on a JASCV-550. IR data were recorded on a JASC FT/IR-480 plus spectrometer (Jasco International Co. Ltd, Tokyo, Japan). The HR-ESI-MS were obtained on a Micromass Q-TF mass spectrometer (Waters Corporation, Milford, USA). The NMR spectra were recorded on Bruker Avance III 600 spectrometer (Bruker BioSpin Group, Faellanden, Switzerland) using solvent signals (CD 3 D: δ H 3.31/δ C 49.0) as internal standard. The analytical HPLC was performed on a Waters 2695 system equipped with a 2998 PDA (Waters, Manchester, U.K.) and Alltech 3300 ELSD (Alltech 64 Inc., Deerfield, Illinois, U.S.A.), using a CSMSIL 5C18-MS-II ( mm, 5 μm, Nacalai 66 Tesque, Kyoto, Japan). The preparative HPLC was performed on Waters 1515 system with a 2489 PDA (Waters, Manchester, U.K.), using a CSMSIL 5C18-MS-II ( mm, 5 μm, Nacalai 66 Tesque, Kyoto, Japan). Silica gel ( mesh, Haiyang Chemical Co. Ltd, Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) were employed in open column chromatography operation.

6 JURNAL F ASIAN NATURAL PRDUCTS RESEARCH 5 Figure 3. Effects of 1 3 on osteogenic activity of pre-osteoblastic MC3T3-E1 cells. Notes: (A) Cell proliferation assay. (B) Bone calcified nodule formation assay. Left: Representative morphological pictures of bone calcified nodule formation. Right: Quantification of bone nodules formation by the measurement of absorbance of the released calcified nodules. The values represent mean ± S. E. M of three independent experiments, each with more than triplicate wells. ***p < vs. control Plant material The plant materials were purchased from Bozhou Huqiao Pharmaceutical company, Henan province, which was identified as fruits of Ligustrum lucidum by Prof. G. X. Zhou, Jinan University. A voucher specimen ( ) was deposited in XiangXue Pharmaceutical Co., LTD, Guangzhou, China Extraction and isolation The dried fruits of Ligustrum lucidum (20.0 kg) were refluxed 2 times with 280 L of 70% EtH H2 for 2 h each time. After filtration, the EtH was removed under reduced pressure to yield a crude extract (6.8 kg). Then the crude extract was suspended in water, and partitioned with equal volume of ethyl acetate twice to obtain water fraction and ethyl acetate fraction (EAF) The EAF (300 g) was subjected to open silica gel column chromatography, which was eluted with CHCl3 MeH H2 (100:0:0 8:2:0.2, V/V/V) in gradient to yield 15 fractions (Fr.1 Fr.15). Fr.15 (15.8 g) was subjected to open DS column chromatography eluting

7 6 Z.-C. QIUA ET AL. Table 1. 1 H and 13 C NMR spectral data for 1 3 (δ in ppm, in CD 3 D, 600 and 125 MHz) Pos. δ C δ H (multi., J, Hz) δ C δ H (multi., J, Hz) δ C δ H (multi., J, Hz) s br s d (4.1) s s d (1.8) dd (9.7, 4.9) dd (10.0, 3.6) m a 2.89 dd (12.7, 4.9) a 2.81 dd (15.3, 3.6) a 2.97 dd (16.6, 4.6) 6b 2.45 dd (12.7, 9.7) 6b 2.45 dd (15.3, 10.0) 6b 2.22 dd (16.6, 9.4) q (6.9) m dt (17.2, 10.4) m d (6.9) dd (13.4, 8.0) a 5.21 d (17.2) 4.84 overlap 10b 5.15 dd (10.4, 1.7) CH s s d (7.8) d (7.8) d (7.9) dd (9.7, 7.8) m m m m m m m m m m m dd (11.8, 1.9) dd (12.0, 1.7) dd (11.9, 2.2) 4.24 dd (11.8, 6.8) 3.67dd (12.0, 5.6) 4.40 dd (11.9, 6.1) d (8.5) d (8.6) m d (8.5) d (8.6) m m dd (15.5, 8.9) d (15.9) d (16.0) dd (15.5, 8.9) d (15.9) d (16.0) d (7.2) m m m m d (11.2) 3.68 overlap with MeH/H 2 gradients to afford six fractions (Fr.15.1 Fr.15.6). Fr.15.5 (1.1 g) was purified by Sephadex LH-20 column chromatography with MeH H 2 9:20 to afford sub-fractions A D, and then semi-preparative HPLC (45% MeH/H 2, flow rate 4 ml/ min) to give 1 (2.8 mg, with retention time 36.5 min) from sub-fraction C. Fr.13 (7.0 g) was subjected to open DS column chromatography eluting with MeH/H 2 gradients to afford eight fractions (Fr.13.1 Fr.13.8). Fr.13.3 (3.9 g) was purified by Sephadex LH-20 column chromatography with MeH H 2 (2:3) to afford sub-fractions A I, and then semi-preparative HPLC (20% MeCN/H 2, flow rate 4 ml/min) to give 2 (64.3 mg, with retention time 36.0 min) from sub-fraction I. Fr.13.7 (381.4 mg) was purified by Sephadex LH-20 column chromatography with MeH H 2 11:20 to afford sub-fractions A I. 3 (81.7 mg) was obtained from sub-fraction G Nuezhenelenoliciside (1) Yellow solid; [α] 25 D 5.06 (c 0.65, MeH); UV (MeH) λ (log ε): (3.50), (4.00) max nm; IR (KBr): υ max 3450, 1698, 1621, 1514, 1444, 1381, 1278, 1186, 1079 cm 1 ; The 1 H (600 MHz, CD 3 D) and 13 C (150 MHz, CD 3 D) NMR spectral data, see Table 1. HRESIMS: m/z [M H] (calcd for C 31 H 41 17, ).

8 JURNAL F ASIAN NATURAL PRDUCTS RESEARCH Isojaslanceoside B (2) Yellow solid; [α] 25 D (c 0.67, MeH); UV (MeH) λ (log ε): (3.86), max (3.79) nm; IR (KBr): υ max 3316, 1698, 1633, 1511, 1444, 1373, 1272, 1165, 1038, 985 cm 1 ; The 1 H (600 MHz, CD 3 D) and 13 C (150 MHz, CD 3 D) NMR spectral data, see Table 1. HRESIMS: m/z [M H] (calcd for C 26 H 29 14, ) trans-cinnamoyl-secologanoside (3) Yellow solid; [α] 25 D 3.10 (c 0.55, MeH); UV (MeH) λ (log ε): (3.78), (3.62) max nm; IR (KBr): υ max 3393, 1701, 1633, 1408, 1275, 1189, 1065, 861 cm 1 ; The 1 H (600 MHz, CD 3 D) and 13 C NMR spectral data (150 MHz, CD 3 D), see Table 1. HRESIMS: m/z [M + H] + (calcd for C 25 H 29 12, ) Acid hydrolysis and derivatization of 1 Acid hydrolysis and derivatization of 1 (1.5 mg) were performed according to the reference [15]. Then, the reaction mixture was directly analyzed by HPLC (column: CSMSIL 5C18-MS-II column, mobile phase: 25% MeCN; flow rate; 0.8 ml/min; detective wavelength: 250 nm). The standard monosaccarides of D-Glc, L-Glc, D-Gal, L-Gal, D-Man, L-Man were subjected to the same method. The absolute configuration of the sugar residue was determined by comparing the retention times of the sample with each standard monosaccharide In vitro osteogenic activity assay Cell proliferation assay The effects of 1 3 on proliferation of MC3T3-E1 cells were evaluated by MTS assay according to the procedure described previously [16] Bone calcified nodule formation assay The effects of 1 3 on bone calcified nodule formation of MC3T3-E1 were detected by Alizarin Red S. After being cultured for 21 days, the cells were stained with 1% Alizarin Red S to detect the bone nodules. Each well was captured for visual comparison. The staining bone nodules were further dissolved in 0.5 M HCl and 5% SDS, and quantified by measuring the absorbance at 415 nm by microplate reader (BioTek Synergy H1, USA). Disclosure statement The authors declare no competing financial interest. Funding This research was funded by the National Natural Science Foundation of China [grant numbers , ]; and the National Major Scientific and Program of Introducing Talents of Discipline to Universities [grant number B13038].

9 8 Z.-C. QIUA ET AL. References [1] Pharmacopoeia Commission of the Ministry of Public Health, Chinese pharmacopoeia, Part I, (Chemical Industrial Press, Beijing, 2010), p. 43. [2] X.J. Huang, Y. Wang, Z.Q. Yin, and W.C. Ye, J. Asian Nat. Prod. Res. 12, 685 (2010). [3] Q. Li, Y.S. Fan, Z.Q. Gao, K. Fan, and Z.J. Liu, J. Ethnopharmacol. 170, 88 (2015). [4] J. Wang, A. Shan, T. Liu, C. Zhang, and Z. Zhang, Int. Immunopharmacol. 14, 758 (2012). [5] A.S. Ravipati, L. Zhang, S.R. Koyyalamudi, S.C. Jeong, N. Reddy, J. Bartlett, P.T. Smith, K. Shanmugam, G. Münch, M.J. Wu, M. Satyanarayanan, and B. Vysetti, BMC Complement Altern. Med. 12, 1472 (2012). [6] H.J. An, H.J. Jeong, J.Y. Um, Y.J. Park, R.K. Park, E.C. Kim, H.J. Na, T.Y. Shin, H.M. Kim, and S.H. Hong, J. Pharm. Pharmacol. 59, 1279 (2007). [7] X.L. Dong, M. Zhao, K.K. Wong, C.T. Che, and M.S. Wong, Br. J. Nutr. 108, 92 (2012). [8] Q. Chen, L. Yang, G. Zhang, and F. Wang, Phytother. Res. 27, 973 (2013). [9] L. Gao, C. Li, Z. Wang, X. Liu, Y. You, H. Wei, and T. Guo, Nat. Prod. Res. 29, 493 (2015). [10] S. Lee, S. Park, H. Kwak, J. h, Y. Min, and S. Kim, Pharmacol. Res. 58, 290 (2008). [11] S. Aoki, Y. Honda, T. Kikuchi, T. Miura, R. Sugawara, Y. Yaoita, M. Kikuchi, and K. Machida, Chem. Pharm. Bull. 60, 251 (2012). [12] Y. Zhang, L. Liu, J. Gao, C. Wu, L. Han, E. Liu, P. Shi, X. Gao, and T. Wang, Fitoterapia 91, 107 (2013). [13] Y.C. Shen, S.L. Lin, and C.C. Chyh-Chung Chein, Phytochemistry 44, 891 (1997). [14] H.K. bied, P. Karuso, P.D. Prenzler, and K. Robards, J. Agric. Food Chem. 55, 2848 (2007). [15] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, and I. Kouno, Chem. Pharm. Bull. 55, 899 (2007). [16] H.H. Xiao, C.Y. Fung, S.K. Mok, K.C. Wong, M.X. Ho, X.L. Wang, X.S. Yao, and M.S. Wong, J. Steroid Biochem. Mol. Biol. 143, 141 (2014).

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