Two new hemiterpene glycosides and one new phenolic glycoside from the roots of Securidaca inappendiculata Hassk

Two new hemiterpene glycosides and one new phenolic glycoside from the roots of Securidaca inappendiculata Hassk

Phytochemistry Letters 21 (2017) 74–77 Contents lists available at ScienceDirect Phytochemistry Letters journal homepage: www.elsevier.com/locate/ph...

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Phytochemistry Letters 21 (2017) 74–77

Contents lists available at ScienceDirect

Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol

Short communication

Two new hemiterpene glycosides and one new phenolic glycoside from the roots of Securidaca inappendiculata Hassk

MARK



Zhi Wanga, Haiyan Zhaa, Xuedong Yanga, , Licui Hua, Wenfeng Zhenga, Lizhen Xub a b

School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100094, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: Securidaca inappendiculata Hassk Polygalaceae Hemiterpene Phenolic glycoside

Two new hemiterpene glycosides, named as securiterpenoside B (1), and securiterpenoside C (2), and one new phenolic glycoside, named as securiphenoside A (3) were isolated from the roots of Securidaca inappendiculata Hassk. Their structures were elucidated on the basis of 1D and 2D NMR data and HRESIMS and comparisons with published data. Moreover, compounds 1–3 were evaluated for cytotoxicities against A549 (Lewis lung cancer), Hela (human cervical cancer) and MCF-7 (human breast cancer) cell lines.

1. Introduction Securidaca inappendiculata Hassk, a climbing shrub of the Polygalaceae family belonging to the tribe Polygaleae, is mainly distributed in Yunnan, Guangxi, Guangdong, and Hainan provinces of southern China and the tropical regions of Asia (Delecti Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae, 1997). As a traditional Chinese herbal medicine, the roots of S. inappendiculata are used as an anti-inflammatory, antibacterial, and antirheumatism agent (Jiangsu Institute of Botany, 1988). Our previous antitumor constituents investigations of the aqueous portion of ethanol extract from the roots of S. inappendiculata have shown that the plant is a rich source for acylated triterpene saponins (Zha et al., 2015). As a part of continuing studies on the same portion, we now report the isolation and elucidation of two new hemiterpene glycosides (1–2), and one new phenolic glycoside (3) (Fig. 1). Cytotoxicities of compounds 1–3 were evaluated against A549, Hela, and MCF-7 cell lines, respectively. 2. Results and discussion Compound 1, obtained as a white amorphous powder, was assigned a molecular formula of C12H20O8 which was deduced from the HRESIMS (m/z 315.1045 [M + Na]+). IR absorption at 3392 cm−1 and 1716 cm−1 supported the presence of hydroxyl and carbonyl groups. Combined analysis of 13C NMR and DEPT spectrum showed that there were two methylene carbon signals at δc 31.6 (C-3) and δc 66.9 (C-4), a carboxylic carbon at δc 166.7 (C-1), a quaternary carbon at δc 136.6 (C2), an exomethylene carbon at δc 126.9 (C-5), and a methoxy carbon at δc 51.7. In 1H NMR spectrum, signals at δH 6.13 and 5.80 (each 1H, d, ⁎

J = 1.1 Hz, H-5) can be assigned to a terminal double bond, signals at δH 2.52 (2H, t, J = 7.0 Hz, H-3), 3.85 (1H, m, H-4a) and δH 3.56 (1H, q, J = 11.9, 7.0 Hz, H-4b) can be ascribed to two methylenes, and signal at δH 3.68 (3H, s) indicated the presence of a methoxy group. HMBC correlations from H-5 to C-1, C-2 and C-3, from H-3 to C-1, C-2, C-4 and C-5, and from methoxy protons to C-1 provided the position of the aglycone (Fig. 2). These data led to the elucidation of the hemiterpene structure as 4-hydroxy-2-methylene-butyric acid methyl ester. The anomeric glucose proton signal at δH 4.13 (1H, d, J = 7.8 Hz) in 1H NMR spectra and six carbon signals at δc 102.8 (C-1′), δc 73.4 (C-2′), δc 76.7 (C-3′), δc 70.1 (C-4′), δc 76.8 (C-5′), δc 61.1 (C-6′) in 13C NMR spectra indicated the presence of a β-D glucopyranosyl moiety. The Dconfiguration of the glucose residue was assumed from biogenetic ground. Furthermore, the 1H NMR and 13C NMR spectural data of 1 was quite similar to those of securitepenoside (Yang et al., 2002) except for one additional signal due to a methoxy group. HMBC correlations of C-4 with H-1′ and of C-1′ with H-4 revealed a linkage between the aglycone and a glucopyranosyl moiety (Fig. 2). Therefore, the structure of 1, named as securiterpenoside B, was determined as 2-methene-butyric acid methyl ester-4-O-β-D-glucopyranoside. Compound 2 was obtained as a pale yellow amorphous powder. It’s molecular formula was determined to be C27H36O15 on the basis of the HRESIMS (m/z 623.1959 [M + Na]+). The IR spectrum showed the presence of the hydroxyl at 3419 cm−1 and carbonyl group at 1714 cm−1. Comparison of the 1H NMR and 13C NMR data with that of 1 manifested that structure of 2 was inclusive of one glucopyranosyl moiety and hemiterpene residue as 1, along with additional signals due to a 1,5-anhydroglucitol residue (Cheng et al., 2006; Ikeya et al., 1991) and a trans-feruloyl moiety. The presence of one 1,5-anhydroglucitol

Corresponding author at: School of Pharmaceutical Science and Technology Tianjin University No. 92 Weijin Road, Nankai District Tianjin 300072, PR China. E-mail address: [email protected] (X. Yang).

http://dx.doi.org/10.1016/j.phytol.2017.05.019 Received 24 January 2017; Received in revised form 19 May 2017; Accepted 30 May 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

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Fig. 1. Structures of compounds 1–3.

11) along with signals at δH 5.36 and 5.25 (each 1H, d, J = 12.6 Hz, H14) could be assigned to benzyl alcohol. The HMBC correlations of H-14 with C-7, C-8, and C-9/C-13, of H-9/H-13 with C-10/C-12, C-11, and C14, and of H-3, H-5 with C-2, C-6, and C-7 provided the position of the benzoyl residue. These data led to the elucidation of the aglycone structure as benzyl 2-hydroxy-6-methoxybenzoate. The two anomeric proton signals at δH 4.89 (1H, d, J = 7.8 Hz, H1′), 4.18 (1H, d, J = 6.5 Hz, H-1′′) in 1H NMR spectrum and two carbon signals at δc 100.3 (C-1′), 103.3 (C-1″) in 13C NMR were indicative of the presence of one glucose moiety and one arabinose/xylose moiety. Compared with the reference data, the signals of H-5 of xylose moiety [δH 3.81 dd (11.0, 6.0), 3.16 m] (Wang et al., 2010) and arabinose moiety [δH 3.79 dd (11.5, 3.5), 3.43 dd (11.5, 2.0)] (Zheng et al., 2008), signals at δH 3.67 (1H, dd, J = 11.9, 3.4), 3.30 (1H, dd, J = 11.9, 2.3) (H-5″) were assigned to one α-L-arabinose moiety which were further verified by homonuclear J-resolved spectroscopy. HMBC correlation from H-1′ to C-2 revealed a linkage between the aglycone and a glucopyranosyl moiety (Fig. 2). Correlation in the HMBC spectrum between H-1″ and C-6′ disclosed the (1 → 6) linkage between glucose and arabinose units (Fig. 2). Based on above evidence, compound 3, named as securiphenoside A, was elucidated as benzyl 2-O-[α-L-arahinopyranosyl-(1″ → 6′)-β-D-glucopyranosyl]-6-methoxybenzoate. The naturally occurring hemiterpene glycosides from Plygalaceae family have been rarely reported. Only six of this kind of compounds were isolated from the family including securitepenoside isolated from S. inappendiculata (Yang et al., 2002) and another five compounds obtained from Monnina obtusifolia (Lepore et al., 2011). The isolation of compounds 1 and 2 may reveals a close relationship between the two species, which could have chemotaxonomic significance to some extent. Compounds 1–3 were evaluated for their cytotoxicities in vitro against A549 (Lewis lung cancer), Hela (human cervical cancer) and MCF-7 (human breast cancer) cell lines using MTT assay method. Cisplatin was used as a positive control and IC50 values were 16.74, 16.74, 19.83 μM, respectively. All compounds did not show significant cytotoxicities against A549, Hela, and MCF-7 cells (IC50 > 100 μM).

residue was supported by two methylenes [δH 3.88 (H-1″a), δH 3.21 (H1″b), δH 4.41 (H-6″a), δH 4.14 (H-6″b)], four methines [δH 4.65 (H-2″), δH 3.47 (H-3″), δH 3.25 (H-4″), δH 3.42 (H-5″)], and six carbons [δC 66.1 (C-1″), δC 71.4 (C-2″), δC 74.8 (C-3″), δC 70.4 (C-4″), δC 78.2 (C-5″), δC 64.1 (C-6″)]. In 1H NMR spectrum, the E-olefinic protons of feruloyl moiety can be identified by the signals which appeared as two doublets at δH 7.57 (1H, d, J = 15.8 Hz) and 6.46 (1H, d, J = 15.8 Hz) and signals at δH 3.81 (3H, s) and 9.62 (1H, brs) were assigned to a methoxy group and a hydroxyl group substituted in benzene ring. Combined analysis of HSQC, HMBC and NOESY spectra afforded the assignments of protons and carbons of the feruloyl moiety which were in good agreements with literature data (Kobayashi et al., 2000; Yu et al., 2007). In HMBC spectra, correlation between δC 67.0 (C-4) and δH 4.15 (H-1′) revealed a linkage between the hemiterpene and a glucopyranosyl moiety (Fig. 2). The HMBC correlation of δC 166.2 (C-1) with δH 4.65 (H-2″) suggested the linkage between the hemiterpene and 1,5anhydroglucitol moiety (Fig. 2). In addition, the HMBC spectrum of 2 showed a correlation between δC 166.3 (C-9‴) and δH 4.41 (H-6″a), 4.14 (H-6″b) (Fig. 2). Full assignments of the 1H NMR and 13C NMR signals were secured by COSY, HSQC, HMBC and NOESY spectra. Analysis of all the available data led us to conclude that the structure of 2, named as securiterpenoside C, was 1-O-[6-O-feruloyl-1,5-anhydroglucitol]-2-methene-butyric acid methyl ester-4-O-β-D-glucopyranoside. Compound 3 was isolated as a colorless amorphous powder. It’s molecular formula was determined as C26H32O13 deduced from the HRESIMS (m/z 575.1702 [M + Na]+). The IR absorption of 3 showed absorption at 3413 cm−1 and 1724 cm−1, ascribed to hydroxyl and carbonyl moieties. The 1H NMR and 13C NMR spectral data of aglycone of 3 were similar to those of the known compound 2-hydroxy-4-O-[βxylopyranosyl(1″ → 6′)-β-glucopyranosyl]-benzoate (Wang et al., 2010). In 1H NMR spectrum, the presence of an aromatic ABC-spin system with three aromatic signals [δH 7.37 (1H, t, J = 8.6 Hz, H-4), 6.93 (1H, d, J = 8.5 Hz, H-3), 6.75 (1H, d, J = 8.5 Hz, H-5)] indicated the existence of 2, 6-disubstituted phenyl. In aromatic region of 1H NMR spectrum also showed five aromatic protons, including two pairs of symmetrical ones, which appeared at δH 7.46 (2H, d, J = 7.0 Hz, H9/13), 7.39 (2H, t, J = 7.1 Hz, H-10/12), 7.33 (1H, t, J = 7.2 Hz, H-

Fig. 2. Key HMBC and NOESY correlations for compounds 1-3.

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3. Experimental

Table 1 1 H (400 MHz), 13C NMR (150 MHz) spectroscopic data of compound 1 (DMSO-d6) and compound 2 (DMSO-d6).

3.1. General

Position

Optical rotations were measured on a Rudolph Research Analytical AutopoL II automation polarimeter. UV spectra were measured using an Agilent Cary 60 UV–vis spectrophotometer. IR spectra were recorded on Bruker TENSOR 27 spectrometer with KBr pellets. The 1D and 2D NMR spectra were recorded on Bruker Avance III 600 MHz and 400 MHz instruments with TMS as internal standard. HRESIMS spectra were performed on a Bruker MicroTOF-Q II spectrometer. Vacuum liquid chromatography (VLC) was carried out using silica gel (100–200 mesh, Qingdao Marine Chemical Co., Ltd). Medium pressure liquid chromatography (MPLC) was performed on Dr Flash II (Suzhou Lisui Science Co., Ltd) equipped with a UV3000 UV–vis Detector and Cosmosil glass columns (500 × 50 mm, RP-18, 50 μm) or (500 × 75 mm, RP-18, 50 μm). Preparative HPLC were carried out on a LC3000P instrument (Beijing Chuangxin Tongheng Science and Technology Co., Ltd) equipped with a P3000 pump, UV3000 UV–vis Detector, and a Cosmosil MS II C18 column (250 × 20 mm, 5 μm). All solvents used were of analytical grade (Tianjin Benchmark Chemical Reagent Co., Ltd).

1 δH (J in Hz)

1 2 3 4 5 1′ 2′ 3′ 4′ 5′ 6′

2.52 t (7.0) 3.85 m 3.56 q (11.9, 7.0) 6.13 d (1.1) 5.80 d (1.1) 4.13 d (7.8) 2.92 td (8.0, 4.9) 3.12 m 3.03 m 3.07 dd (5.6, 1.7) 3.65 m 3.42 m

2 δC 166.7 136.6 31.6 66.9 126.9 102.8 73.4 76.7 70.1 76.8 61.1

1″ 2″ 3″ 4″ 5″ 6″

3.2. Plant material The roots of S. inappendiculata were collected from Yunnan Province in the People’s Republic of China in 2010 and authenticated by Hong Wang, plant taxonomist of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. A voucher specimen (Y201011) was deposited in School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, People’s Republic of China.

1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴ OH OCH3

3.3. Extraction and isolation The dried and powdered roots of S. inappendiculata (9.85 kg) were extracted with 95% EtOH (3 × 13.0 L) under reflux for 3 × 2 h. Removal of the solvent in vacuo afforded a residue (1.25 kg), which was suspended in H2O (5.0 L) followed by successive partition with dichloromethane and ethyl acetate (each 3 × 3.0 L). The aqueous portion was subjected to D101 macroporous resin column (10.6 × 94.0 cm) and then eluted with H2O, 30% EtOH, 60% EtOH, and 95% EtOH, respectively. The 60% EtOH elution (180 g) was performed on a vacuum liquid chromatography column (VLC, 40 × 14 cm) using silica-gel (100–200 mesh) and eluted with a step gradient of CHCl3/MeOH/H2O (85: 15: 2, 75: 20: 5, 68: 24: 8, 60: 32: 8, 50: 42: 8, low phase) to obtain 30 fractions (C1–C30). Then, fractions C13–15 (14.0 g) was separated by MPLC (RP-18, 500 × 50 mm, 35 mL/min) eluted with MeOH/H2O (30: 70, 32: 6, 34: 66) to yield 6 subfractions. Subfraction 1 (600 mg) were further separated and purified by preparative HPLC (RP-18, 250 × 10 mm, 5 μm, 12 mL/min) to afford compound 1 (20 mg). Fractions C10–C12 (4.5 g) was separated by MPLC (RP-18, 500 × 75 mm, 55 mL/min) eluted with MeOH/H2O (31: 69, 32: 68, 33: 66) to yield 15 subfractions. Subfraction 13 (350 mg) were further separated and purified by preparative HPLC (RP-18, 250 × 10 mm, 5 μm, 12 mL/min) to afford compound 3 (80 mg). Subfraction 14 (600 mg) were further separated and purified by preparative HPLC (RP-18, 250 × 10 mm, 5 μm, 12 mL/min) to afford compound 2 (12 mg).

δH (J in Hz)

2.54 t (7.0) 3.87 m 3.59 q (11.8, 6.9) 6.16 s 5.81 s 4.15 d (7.8) 2.94 t (8.1) 3.12 m 3.04 m 3.08 dd (5.6, 1.8) 3.67 m 3.44 m 3.88 dd (10.8, 5.4) 3.21 t (10.8) 4.65 m 3.47 m 3.25 m 3.42 m 4.41 dd (11.9, 1.5) 4.14 dd (11.3, 5.9) 7.32 d (1.8)

6.79 d (8.1) 7.11 dd (8.1, 1.8) 7.57 d (15.8) 6.46 d (15.8)

3.68 s

51.7

9.62 brs 3.81 s

δC 166.2 136.7 31.6 67.0 127.2 102.9 73.4 76.8 70.1 76.9 61.1 66.1 71.4 74.8 70.4 78.2 64.1 125.6 111.1 148.0 149.5 115.5 123.4 145.6 114.2 166.3 55.7

50% and cisplatin was used as a positive control. All experiments were performed in three independent replicates. 3.5. Securiterpenoside B (1) White amorphous powder, [α]21.1 D : −13.6 (c 0.07, MeOH); UV (MeOH) λmax (log ε): 205 (3.43), 208 (3.40), 210 (3.36) nm; IR νmax 3392, 1716,1516, 1271, 1161 cm−1; HRESIMS m/z 315.1045 [M + Na]+ (calcd. for C12H20O8Na 315.1056); 1H NMR and 13C NMR data, see Table 1. 3.6. Securiterpenoside C (2) Pale yellow amorphous powder, [α]17.5 D : +14.1 (c 0.21, MeOH); UV (MeOH) λmax (log ε): 205 (1.38), 234 (0.62), 327 (0.95) nm; IR νmax 3419, 2921, 2353, 2309, 1714, 1632, 1599, 1516, 1271,1161, 1099, 1026 cm−1; HRESIMS at m/z 623.1959 [M + Na]+ (calcd. for C27H36O15Na 623.1952); 1H NMR and 13C NMR data, see Table 1. 3.7. Securiphenoside A (3)

3.4. MTT cytotoxicity bioassays Colorless amorphous powder, [α]18.7 D : −13.1 (c 0.15, MeOH); UV (MeOH) λmax (log ε): 206 (5.86), 226 (5.91), 308 (6.04) nm; IR νmax 3413, 2890, 1725, 1601, 1456, 1293, 1254, 1070, 1006 cm−1; HRESIMS m/z 575.1702 [M + Na]+ (calcd. for C26H32O13Na 575.1741); 1H NMR and 13C NMR data, see Table 2.

Cytotoxicities of compounds 1-3 against A549, Hela and MCF-7 cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann, 1983). IC50 values were determined as concentration of a compound resulting in cell growth inhibition by

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Table 2 1 H (400 MHz), Position 1 2 3 4 5 6 7 8 9, 13 10, 12 11 14

References 13

C (150 MHz) NMR spectroscopic data of compound 3 (DMSO-d6).

δH (J in Hz)

6.93 d (8.5) 7.37 t (8.6) 6.75 d (8.5)

7.46 d (7.0) 7.39 t (7.1) 7.33 t (7.2) 5.36 d (12.6) 5.25 d (12.6)

δC

Position

δH (J in Hz)

δC

113.3 154.5 107.9 131.4 105.0 156.4 165.1 136.1 127.7 128.2 127.8 66.1

1′ 2′ 3′ 4′ 5′ 6′

4.89 d (7.8) 3.25 td (7.5,5.6) 3.31 m 3.18 td (8.7,5.1) 3.56 m 3.96 dd (14.1, 4.5) 3.55 dd (9.0, 2.6) 4.18 d (6.5) 3.38 td (8.3,4.4) 3.33 m 3.62 m 3.67 dd (11.9, 3.4) 3.30 dd (11.9,2.3) 3.76 s

100.3 73.2 76.6 69.8 75.9 67.9

1” 2” 3” 4” 5” OCH3

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103.3 70.6 72.4 67.3 64.9 55.9

Acknowledgments This work was financially supported by the grant of the National Scientific and Technological Major Special Project for Significant New Drug Creation, People’s Republic of China (No. 2011ZX09201). 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.2017.05.019.

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