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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection a
a
ab
Sheng-Yuan Zhang , Chun-Lin Fan , Xiao-Jun Huang , Wen-Hua c
c
ab
Huang , Xue-Hua Liu , Lei Wang
ab
& Wen-Cai Ye
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou510632, China b
Click for updates
J, NU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou510632, China c
Limin Pharmaceutical Factory, Livzon Pharmaceutical Group, Shaoguan512028, China Published online: 08 Oct 2014.
To cite this article: Sheng-Yuan Zhang, Chun-Lin Fan, Xiao-Jun Huang, Wen-Hua Huang, Xue-Hua Liu, Lei Wang & Wen-Cai Ye (2014): A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.965161 To link to this article: http://dx.doi.org/10.1080/10286020.2014.965161
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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.965161
A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection Sheng-Yuan Zhanga1, Chun-Lin Fana1, Xiao-Jun Huangab, Wen-Hua Huangc, Xue-Hua Liuc, Lei Wangab* and Wen-Cai Yeab* a
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Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China; bJNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou 510632, China; cLimin Pharmaceutical Factory, Livzon Pharmaceutical Group, Shaoguan 512028, China (Received 26 February 2014; final version received 9 September 2014) A new phenylpropanoid and a new isoflavone glycoside were isolated from Shenqi Fuzheng Injection. Their structures were elucidated as (aS)-a-ethenyl-4-hydroxy-3methoxy-benzenemethanol (1) and calycosin 7-O-[a-D -glucopyranosyl (1 ! 4)]-b-D glucopyranoside (2) by means of spectroscopic methods including UV, IR, HR-ESIMS, and NMR. The absolute configurations of 1 and 2 were confirmed by quantum chemical calculation and acid hydrolysis. Keywords: Shenqi Fuzheng Injection; isoflavone glycoside; phenylpropanoid
1. Introduction Shenqi Fuzheng Injection (SFI) is a clinical drug, which was widely used as an adjuvant to chemotherapy for the treatment of cancer in China. SFI composed of two herbal medicines, Radix Codonopsis and Radix Astragali, which was approved by the State Food and Drug Administration (SFDA) of China since 1999 [1]. In clinical application, SFI exhibited anti-tumor synergy effects in cooperation with chemotherapy [1 – 3]. Previous phytochemical investigations on SFI had reported the presence of several flavonoids, phenolics, saponins, and carbohydrates by our group and other research teams [4 – 6]. Our further investigation has now led to the isolation of two new compounds, (aS)-aethenyl-4-hydroxy-3-methoxy-benzenemethanol (1) and calycosin 7-O-[a-D glucopyranosyl(1 ! 4)]-b-D -glucopyranoside (2) (Figure 1). Herein, the isolation and structural elucidation of the new compounds are described.
2. Results and discussion Compound 1 was obtained as an amorphous powder. The molecular formula of 1 was determined as C10H12O3 by its HRESI-MS at m/z 359.1503 [2M 2 H]2. The UV spectrum displayed absorption maxima at 204, 230, 277, and 302 nm. The IR spectrum of 1 suggested the presence of hydroxyl group (3388 cm21) and aromatic ring (1600, 1509, 1444 cm21). The 1H NMR spectrum of 1 showed signals for an 1,3,4-trisubstituent benzene ring [dH 7.08 (1H, d, J ¼ 1.9 Hz), 6.86 (1H, dd, J ¼ 8.1, 1.9 Hz), and 6.73 (1H, d, J ¼ 8.1 Hz)], a terminal double bond [dH 6.35 (1H, ddd, J ¼ 17.1, 10.2, 8.3 Hz), 5.23 (1H), 5.19 (1H)], an oxygenated methine [dH 4.48 (1H, d, J ¼ 8.2 Hz)], and a methoxy group [dH 3.85 (3H, s)] (Table 1). The 13C NMR spectrum of 1 displayed 10 signals including a methyl, a methylene, 5 methines, and 3 quaternary carbons. These data indicated the presence of a phenylpropanoid skeleton. Comparison of
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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S.-Y. Zhang et al.
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Figure 1. Chemical structures of 1 and 2.
the NMR data of 1 with those of the known compound eugenol revealed their considerable structural similarity [7]. The main difference was that 1 was substituted by a hydroxyl group at the C-a position. The 1H – 1H COSY correlations between H-a (dH 4.48) and H-b (dH 6.35), as well as the HMBC correlations between H-a (dH 4.48) and C-1 (dC 130.0)/C-b (dC 136.5)/C-g (dC 118.9) further confirmed that a hydroxyl group was attached to C-a (Figure 2). In addition, the methoxy group was located at C-3 position according to the HMBC correlation between the methoxyl protons (dH 3.85) and C-3 (dC 148.6), as well as the ROESY correlation between Table 1. 1H (400 MHz) and 13C NMR (100 MHz) spectral data for compound 1 (CD3OD, d in ppm, J in Hz).a No. 1 2 3 4 5 6 a b g 3OCH3 a
dH
dC
– 7.08 (1H, d, 1.9) – – 6.73 (1H, d, 8.1) 6.86 (1H, dd, 8.1, 1.9) 4.48 (1H, d, 8.2) 6.35 (1H, ddd, 17.1, 10.2, 8.3) a: 5.23 (1H) b: 5.19 (1H) 3.85 (3H, s)
130.0 114.1 148.6 146.9 115.8 123.3 71.6 136.5 118.9 56.4
Overlapped signals were reported without designating multiplicity.
the methoxyl (dH 3.85) and H-2 (dH 7.08) (Figure 2). Based on the above evidence, the planar structure of 1 was determined. The absolute configuration of 1 was determined to be aS by comparison of the optical rotation with the literature data [8], which was further confirmed by computational methods via calculation of the optical rotation values at three different levels (theory-B3LYP, B3P86, and PBE1PBE) using the 6-311 þ þ G(2d, p) and aug-cc-pVDZ basis set in the Gaussian 09 software [9,10] (Table 2). The calculated specific rotation values and the experimental data for 1 were summarized in Table 2. These results indicated that the absolute configuration of 1 corresponded to aS. Therefore, the structure of 1 was elucidated as (aS)-a-ethenyl4-hydroxy-3-methoxy-benzenemethanol. Compound 2 was obtained as an amorphous powder. The HR-ESI-MS of 2 exhibited a quasi-molecular-ion at m/z 631.1637 [M þ Na]þ, consistent with the molecular formula C28H32O15. The IR spectrum revealed the presence of hydroxyl group (3389 cm 21), carbonyl group (1624 cm21), and aromatic ring (1595, 1512, and 1443 cm21). The UV characteristic absorption maxima at 260 and 286 nm, as well as the proton singlet at dH 8.39 (H-2) in the 1H NMR spectrum suggested 2 to be an isoflavone. Furthermore, the 1H NMR spectrum of 2 showed signals for six
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Figure 2. Key 1H– 1H COSY and HMBC correlations of 1 and 2.
aromatic protons [d H 8.06 (1H, d, J ¼ 8.9 Hz), 7.25 (1H, d, J ¼ 2.2 Hz), 7.15 (1H, dd, J ¼ 8.9, 2.2 Hz), 7.07 (1H, s), 6.97 (2H, overlapped)], two anomeric protons [dH 5.18 (1H, d, J ¼ 7.8 Hz), 5.08 (1H, d, J ¼ 3.7 Hz)], and one methoxy group [dH 3.80 (3H, s)]. The 13C NMR spectrum of 2 displayed 15 aromatic carbon signals due to a C6 –C3 – C6 system and 12 aliphatic carbon signals for two sugar moieties, suggesting that 2 was an isoflavone glycoside with two sugar units. Detailed analysis of 1D and 2D NMR spectra of 2 and comparison with those of the known compound calycosin 7-O-b-D glucopyranoside revealed that their signals were similar except for the appearance of the signals for an additional sugar unit in 2 [5]. The methoxy group was located at C-40 position according to the HMBC correlation between the methoxyl (dH 3.80) and C-40 (dC 147.6), as well as the ROESY correlation between the methoxyl (dH 3.80) and H-50 (dH 6.97) (Figure 2). Acid hydrolysis of 2 afforded D -glucose, which
was identified by high-performance liquid chromatography (HPLC) analysis with authentic sample [11]. The relative configurations of two sugar units were determined as b and a based on the 3JH1, H2 coupling constants of the anomeric protons [dH 5.18 (1H, d, J ¼ 7.8 Hz), 5.08 (1H, d, J ¼ 3.7 Hz)], respectively. The linkage position of the two glucopyranosyl moieties could be determined by the HMBC correlations between H-1 of glc0 at dH 5.08 and C-4 of glc at dC 79.0, as well as between H-1 of glc at dH 5.18 and C-7 at dC 161.3 (Figure 2). Based on the above results, the structure of 2 was characterized as calycosin 7-O-[a-D -glucopyranosyl (1 ! 4)]-b-D -glucopyranoside. Previous phytochemical studies had revealed that phenylpropanoids are a type of characteristic constituents of Radix Codonopsis [12], while isoflavone glycosides typically exist in Radix Astragali [13]. Recently, an ultra-fast liquid chromatography coupled with ESI-Q-TOFMS/MS analysis of SFI and its individual
Table 2. Calculated and experimental specific rotation values of 1 [in 8 (dm g/cm – 3) – 1]. Calcd Methods PBE1BPE/aug-cc-pVDZ B3LYP/6-311 þ þ G(2d, p) B3P86/6-311 þ þ G(2d, p)
R
S
Exptl
þ11.6 þ31.0 þ32.6
211.6 231.0 232.6
210.8
4
S.-Y. Zhang et al.
raw material also indicated that phenylpropanoids and isoflavonoids could be assigned into Radix Codonopsis and Radix Astragali, respectively [14]. Based on the above evidences, the new phenylpropanoid (1) could be originated from Radix Codonopsis, and the new isoflavonoid (2) could come from Radix Astragali. 3.
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3.1
Experimental General experimental procedures
Optical rotations were measured by a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan) at room temperature. UV spectra were recorded on a Jasco V-550 UV–vis spectrophotometer (Jasco). IR spectra were conducted on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer (Jasco). HR-ESI-MS were performed on an Agilent 6210 ESI/TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1D (1H, 13C, and DEPT) and 2D (1H– 1H COSY, HSQC, HMBC, and ROESY) NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker, Faellanden, Switzerland) with TMS as the internal standard, and chemical shifts were expressed in d values (ppm). Silica gel (200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China), macroporous resin AB-8 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), Sephadex LH-20 (25 – 100 mm, Fluka, Buchs, Switzerland), and RP-18 (50 mm, YMC, Kyoto, Japan) were used for column chromatographies (CCs). Thin-layer chromatography was performed using precoated silica gel plates (GF254, Yantai Chemical Industry Research Institute, Yantai, China). Analytical HPLC was performed on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1316A photodiode array detector (Agilent Technologies), and a C18 reversed-phase column (Cosmosil, 5 mm, 4.6 mm £ 250 mm, Nacalai Tesque, Kyoto, Japan). Preparative HPLC was performed on a Varian Prostar system equipped with UV detectors (Varian, Santa Clara, CA, USA) and a preparative Cosmosil
C18 column (20 mm £ 250 mm, Nacalai Tesque). All solvents used in CC were analytical grade (Tianjin Damao Chemical Plant, Tianjin, China). 3.2 Plant material The active pharmaceutical ingredients of SFI (batch no. 20090116) were provided by the Livzon Pharmaceutical Group Inc. The plant Codonopsis pilosula (Franch.) Nannf (Longxi GAP base, Gansu province, China) is used for Radix Codonopsis, while Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Hunyuan GAP base, Shanxi province, China) is used for Radix Astragali. 3.3 Extraction and isolation The extract (1 kg) was suspended in H2O and then subjected to macroporous resin AB-8 column eluting with H2O–EtOH (100:0, 60:40, 30:70, 5:95, each 30 l) to give four fractions (fractions A – D). Fraction B (100.0 g) was subjected to silica gel column and eluted with CHCl3 – MeOH (100:0 ! 0:100, v/v) to afford six subfractions (fractions B1–B6). Fraction B2 (4.0 g) was further separated on silica gel column eluting with a gradient of CHCl3 –MeOH (100:0 ! 0:100, v/v) to afford five subfractions (B2A–B2E). Fraction B2C (200 mg) was subjected to Sephadex LH-20 column with the eluent of CHCl3 –MeOH (1:1) to yield compound 1 (10.1 mg). Fraction B2D (1.2 g) was subjected on ODS column eluting with CH3OH – H2O (10:90 ! 100:0, v/v) to afford six subfractions (B2D1–B2D6). Fraction B2D4 (188 mg) was purified by Sephadex LH-20 (CHCl3 – MeOH, 1:1) and preparative HPLC to yield compound 2 (MeOH–H2O, 35:70, 8 ml/ min, 254 nm, tR 28.8 min, 8.7 mg). 3.3.1 (aS)-a-Ethenyl-4-hydroxyl-3methoxyl-benzenemethanol (1) Amorphous powder. ½a25 D 2 10.8 (c 0.81, MeOH); UV (MeOH) lmax (log 1): 204
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(4.20), 230 (3.76), 277 (3.20), 302 (3.08) nm; IR (KBr) nmax: 3388, 2927, 1624, 1600, 1509, 1444, 1280, 1267, 1077 cm21; for 1H and 13C NMR spectral data (CD3OD), see Table 1. HR-ESI-MS m/z: 359.1503 [2M 2 H]2 (calcd for C20H23O6, 359.1500). 3.3.2 Calycosin 7-O-[a-D glucopyranosyl (1 ! 4)]-b-Dglucopyranoside (2) Amorphous powder. ½a25 D þ 22.7 (c 0.62, CH3OH); UV (CH3OH) lmax (log 1): 204 (4.10), 219 (3.06), 248 (3.28), 260 (4.08), 286 (3.81) nm; IR (KBr) nmax: 3389, 2925, 1624, 1512, 1443, 1267, 1074 cm21; for 1 H and 13C NMR spectral data (DMSOd6), see Table 3. HR-ESI-MS m/z: 631.1637 [M þ Na] þ (calcd for C28H32O15Na, 631.1633). 3.4 Acid hydrolysis of compound 2 Compound 2 (3.5 mg) in 4 mol/l HCl (10 ml) was refluxed at 908C for 6 h in a water bath. The reaction mixture was dissolved in H2O after evaporation, which was then extracted with EtOAc for three Table 3. 1H (400 MHz) and ppm, J in Hz).a
13
times. The aqueous layer was concentrated and dried by N2, and treated with dry pyridine (1 ml) and L -cysteine methyl ester hydrochloride (2 mg), followed by heating at 608C for 2 h, and then concentrated to dryness with N2. The residue was added with isothiocyanate (2 mg) and heated at 608C for another 1 h. The final reaction mixture was analyzed by HPLC under the following conditions: an Agilent 1200 chromatograph equipped with a Cosmosil 5C18-MS-II column (4.6 mm £ 250 mm i. d., Nacalai Tesque Inc.); column temperature: 358C; mobile phase: isocratic elution of 41% CH3CN –H2O (v/v) in 50 mmol/l HCl; flow rate: 0.8 ml/min; injection volume: 12 ml; UV detection wavelength: 254 nm. The standard D -glucose was subjected under the same condition. After the comparison of the retention times of monosaccharide derivative, the sample was confirmed to comprise D -glucose (tR ¼ 19.1 min) [11]. Acknowledgments Financial support of this research was provided by Program for National Natural Science Foundation of China (No. 81273391), the Ministry of Science and Technology of
C NMR (100 MHz) spectral data for compound 2 (DMSO-d6, d in
No.
dH
dC
No.
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60
– 8.39 (1H, s) – – 8.06 (1H, d, 8.9) 7.15 (1H, dd, 8.9, 2.2) – 7.25 (1H, d, 2.2) – – – 7.07 (1H, br s) – – 6.97 (1H) 6.97 (1H)
– 153.6 124.4 174.6 127.0 115.5 161.3 103.4 156.9 118.5 123.6 116.4 146.0 147.6 112.0 119.7
100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 30 -OH 40 -OCH3
a
5
dH 5.18 (1H, d, 7.8) 3.38 (1H) 3.62 (1H) 3.47 (1H) 3.67 (1H) a: 3.64 (1H) b: 3.48 (1H) 5.08 (1H, d, 3.7) 3.40 (1H) 3.51 (1H) 3.08 (1H) 3.42 (1H) a: 3.75 (1H, dd, 10.3, 4.9) b: 3.60 (1H) 9.01 (1H, s) 3.80 (3H, s)
Overlapped signals were reported without designating multiplicity.
dC 99.6 73.5 76.1 79.0 75.4 60.8 100.7 72.4 73.3 69.9 72.7 60.2 – 55.7
6
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China (Nos 2013DFM30080, 2013BAI11B05, 2012ZX09103201-056), and the Program of Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2200058).
Note 1.
These authors contributed equally to this work.
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References [1] J. Dong, S.Y. Su, M.Y. Wang, and Z. Zhan, J. Exp. Clin. Cancer Res. 29, 137 (2010). [2] X.R. Dong, J.N. Wang, L. Liu, X. Chen, M.S. Chen, J.H. Ren, Q. Li, and J. Han, Mol. Med. Rep. 3, 621 (2010). [3] J.X. Wang, X. Tong, P.B. Li, H. Cao, and W.W. Su, J. Ethnopharmacol. 139, 788 (2012). [4] D. Wu, X.G. Li, L.J. Wu, and H.Y. Gao, Mod. Chin. Med. 11, 19 (2009). [5] S.Y. Zhang, C.L. Fan, L. Wang, X.H. Liu, X.W. Sun, and W.C. Ye, Chin. Tradit. Pat. Med. 33, 1743 (2011).
[6] H.Y. Gu, S.Y. Zhang, W.H. Huang, X. H. Liu, Y. Wang, C.L. Fan, and W.C. Ye, Chin. Tradit. Pat. Med. 35, 1494 (2013). [7] C. Chen, H.M. Wang, S.H. Chung, W. L. Lo, W.L. Yang, and S.C. Yang, Chem. Nat. Compd. 3, 474 (2010). [8] J. Stambasky, A.V. Malkov, and P. Kocovsky, J. Org. Chem. 73, 9148 (2008). [9] F.P. Miao, X.R. Liang, X.L. Yin, G. Wang, and N.Y. Ji, Org. Lett. 14, 3815 (2012). [10] F. Wang, Y. Gao, L. Zhang, B. Bai, Y. N. Hu, Z.J. Dong, Q.W. Zhai, H.J. Zhu, and J.K. Liu, Org. Lett. 12, 3196 (2010). [11] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, and I. Kouno, Chem. Pharm. Bull. 55, 899 (2007). [12] K. Mizutani, M. Yuda, O. Tanaka, Y. I. Saruwatari, M.R. Jia, Y.K. Ling, and X. F. Pu, Chem. Pharm. Bull. 36, 2726 (1998). [13] X.F. Ma, T.Y. Zhang, Y. Wei, P.F. Tu, Y. J. Chen, and Y. Ito, J. Chromatogr. A 962, 243 (2002). [14] M.H. Liu, X. Tong, J.X. Wang, W. Zou, H. Cao, and W.W. Su, J. Pharm. Biomed. 74, 141 (2013).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection a
a
ab
Sheng-Yuan Zhang , Chun-Lin Fan , Xiao-Jun Huang , Wen-Hua c
c
ab
Huang , Xue-Hua Liu , Lei Wang
ab
& Wen-Cai Ye
a
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou510632, China b
Click for updates
J, NU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou510632, China c
Limin Pharmaceutical Factory, Livzon Pharmaceutical Group, Shaoguan512028, China Published online: 08 Oct 2014.
To cite this article: Sheng-Yuan Zhang, Chun-Lin Fan, Xiao-Jun Huang, Wen-Hua Huang, Xue-Hua Liu, Lei Wang & Wen-Cai Ye (2014): A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.965161 To link to this article: http://dx.doi.org/10.1080/10286020.2014.965161
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
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systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.965161
A new phenylpropanoid and a new isoflavone glycoside from Shenqi Fuzheng Injection Sheng-Yuan Zhanga1, Chun-Lin Fana1, Xiao-Jun Huangab, Wen-Hua Huangc, Xue-Hua Liuc, Lei Wangab* and Wen-Cai Yeab* a
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Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China; bJNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou 510632, China; cLimin Pharmaceutical Factory, Livzon Pharmaceutical Group, Shaoguan 512028, China (Received 26 February 2014; final version received 9 September 2014) A new phenylpropanoid and a new isoflavone glycoside were isolated from Shenqi Fuzheng Injection. Their structures were elucidated as (aS)-a-ethenyl-4-hydroxy-3methoxy-benzenemethanol (1) and calycosin 7-O-[a-D -glucopyranosyl (1 ! 4)]-b-D glucopyranoside (2) by means of spectroscopic methods including UV, IR, HR-ESIMS, and NMR. The absolute configurations of 1 and 2 were confirmed by quantum chemical calculation and acid hydrolysis. Keywords: Shenqi Fuzheng Injection; isoflavone glycoside; phenylpropanoid
1. Introduction Shenqi Fuzheng Injection (SFI) is a clinical drug, which was widely used as an adjuvant to chemotherapy for the treatment of cancer in China. SFI composed of two herbal medicines, Radix Codonopsis and Radix Astragali, which was approved by the State Food and Drug Administration (SFDA) of China since 1999 [1]. In clinical application, SFI exhibited anti-tumor synergy effects in cooperation with chemotherapy [1 – 3]. Previous phytochemical investigations on SFI had reported the presence of several flavonoids, phenolics, saponins, and carbohydrates by our group and other research teams [4 – 6]. Our further investigation has now led to the isolation of two new compounds, (aS)-aethenyl-4-hydroxy-3-methoxy-benzenemethanol (1) and calycosin 7-O-[a-D glucopyranosyl(1 ! 4)]-b-D -glucopyranoside (2) (Figure 1). Herein, the isolation and structural elucidation of the new compounds are described.
2. Results and discussion Compound 1 was obtained as an amorphous powder. The molecular formula of 1 was determined as C10H12O3 by its HRESI-MS at m/z 359.1503 [2M 2 H]2. The UV spectrum displayed absorption maxima at 204, 230, 277, and 302 nm. The IR spectrum of 1 suggested the presence of hydroxyl group (3388 cm21) and aromatic ring (1600, 1509, 1444 cm21). The 1H NMR spectrum of 1 showed signals for an 1,3,4-trisubstituent benzene ring [dH 7.08 (1H, d, J ¼ 1.9 Hz), 6.86 (1H, dd, J ¼ 8.1, 1.9 Hz), and 6.73 (1H, d, J ¼ 8.1 Hz)], a terminal double bond [dH 6.35 (1H, ddd, J ¼ 17.1, 10.2, 8.3 Hz), 5.23 (1H), 5.19 (1H)], an oxygenated methine [dH 4.48 (1H, d, J ¼ 8.2 Hz)], and a methoxy group [dH 3.85 (3H, s)] (Table 1). The 13C NMR spectrum of 1 displayed 10 signals including a methyl, a methylene, 5 methines, and 3 quaternary carbons. These data indicated the presence of a phenylpropanoid skeleton. Comparison of
*Corresponding authors. Email: [email protected], [email protected] q 2014 Taylor & Francis
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Figure 1. Chemical structures of 1 and 2.
the NMR data of 1 with those of the known compound eugenol revealed their considerable structural similarity [7]. The main difference was that 1 was substituted by a hydroxyl group at the C-a position. The 1H – 1H COSY correlations between H-a (dH 4.48) and H-b (dH 6.35), as well as the HMBC correlations between H-a (dH 4.48) and C-1 (dC 130.0)/C-b (dC 136.5)/C-g (dC 118.9) further confirmed that a hydroxyl group was attached to C-a (Figure 2). In addition, the methoxy group was located at C-3 position according to the HMBC correlation between the methoxyl protons (dH 3.85) and C-3 (dC 148.6), as well as the ROESY correlation between Table 1. 1H (400 MHz) and 13C NMR (100 MHz) spectral data for compound 1 (CD3OD, d in ppm, J in Hz).a No. 1 2 3 4 5 6 a b g 3OCH3 a
dH
dC
– 7.08 (1H, d, 1.9) – – 6.73 (1H, d, 8.1) 6.86 (1H, dd, 8.1, 1.9) 4.48 (1H, d, 8.2) 6.35 (1H, ddd, 17.1, 10.2, 8.3) a: 5.23 (1H) b: 5.19 (1H) 3.85 (3H, s)
130.0 114.1 148.6 146.9 115.8 123.3 71.6 136.5 118.9 56.4
Overlapped signals were reported without designating multiplicity.
the methoxyl (dH 3.85) and H-2 (dH 7.08) (Figure 2). Based on the above evidence, the planar structure of 1 was determined. The absolute configuration of 1 was determined to be aS by comparison of the optical rotation with the literature data [8], which was further confirmed by computational methods via calculation of the optical rotation values at three different levels (theory-B3LYP, B3P86, and PBE1PBE) using the 6-311 þ þ G(2d, p) and aug-cc-pVDZ basis set in the Gaussian 09 software [9,10] (Table 2). The calculated specific rotation values and the experimental data for 1 were summarized in Table 2. These results indicated that the absolute configuration of 1 corresponded to aS. Therefore, the structure of 1 was elucidated as (aS)-a-ethenyl4-hydroxy-3-methoxy-benzenemethanol. Compound 2 was obtained as an amorphous powder. The HR-ESI-MS of 2 exhibited a quasi-molecular-ion at m/z 631.1637 [M þ Na]þ, consistent with the molecular formula C28H32O15. The IR spectrum revealed the presence of hydroxyl group (3389 cm 21), carbonyl group (1624 cm21), and aromatic ring (1595, 1512, and 1443 cm21). The UV characteristic absorption maxima at 260 and 286 nm, as well as the proton singlet at dH 8.39 (H-2) in the 1H NMR spectrum suggested 2 to be an isoflavone. Furthermore, the 1H NMR spectrum of 2 showed signals for six
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Figure 2. Key 1H– 1H COSY and HMBC correlations of 1 and 2.
aromatic protons [d H 8.06 (1H, d, J ¼ 8.9 Hz), 7.25 (1H, d, J ¼ 2.2 Hz), 7.15 (1H, dd, J ¼ 8.9, 2.2 Hz), 7.07 (1H, s), 6.97 (2H, overlapped)], two anomeric protons [dH 5.18 (1H, d, J ¼ 7.8 Hz), 5.08 (1H, d, J ¼ 3.7 Hz)], and one methoxy group [dH 3.80 (3H, s)]. The 13C NMR spectrum of 2 displayed 15 aromatic carbon signals due to a C6 –C3 – C6 system and 12 aliphatic carbon signals for two sugar moieties, suggesting that 2 was an isoflavone glycoside with two sugar units. Detailed analysis of 1D and 2D NMR spectra of 2 and comparison with those of the known compound calycosin 7-O-b-D glucopyranoside revealed that their signals were similar except for the appearance of the signals for an additional sugar unit in 2 [5]. The methoxy group was located at C-40 position according to the HMBC correlation between the methoxyl (dH 3.80) and C-40 (dC 147.6), as well as the ROESY correlation between the methoxyl (dH 3.80) and H-50 (dH 6.97) (Figure 2). Acid hydrolysis of 2 afforded D -glucose, which
was identified by high-performance liquid chromatography (HPLC) analysis with authentic sample [11]. The relative configurations of two sugar units were determined as b and a based on the 3JH1, H2 coupling constants of the anomeric protons [dH 5.18 (1H, d, J ¼ 7.8 Hz), 5.08 (1H, d, J ¼ 3.7 Hz)], respectively. The linkage position of the two glucopyranosyl moieties could be determined by the HMBC correlations between H-1 of glc0 at dH 5.08 and C-4 of glc at dC 79.0, as well as between H-1 of glc at dH 5.18 and C-7 at dC 161.3 (Figure 2). Based on the above results, the structure of 2 was characterized as calycosin 7-O-[a-D -glucopyranosyl (1 ! 4)]-b-D -glucopyranoside. Previous phytochemical studies had revealed that phenylpropanoids are a type of characteristic constituents of Radix Codonopsis [12], while isoflavone glycosides typically exist in Radix Astragali [13]. Recently, an ultra-fast liquid chromatography coupled with ESI-Q-TOFMS/MS analysis of SFI and its individual
Table 2. Calculated and experimental specific rotation values of 1 [in 8 (dm g/cm – 3) – 1]. Calcd Methods PBE1BPE/aug-cc-pVDZ B3LYP/6-311 þ þ G(2d, p) B3P86/6-311 þ þ G(2d, p)
R
S
Exptl
þ11.6 þ31.0 þ32.6
211.6 231.0 232.6
210.8
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S.-Y. Zhang et al.
raw material also indicated that phenylpropanoids and isoflavonoids could be assigned into Radix Codonopsis and Radix Astragali, respectively [14]. Based on the above evidences, the new phenylpropanoid (1) could be originated from Radix Codonopsis, and the new isoflavonoid (2) could come from Radix Astragali. 3.
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3.1
Experimental General experimental procedures
Optical rotations were measured by a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan) at room temperature. UV spectra were recorded on a Jasco V-550 UV–vis spectrophotometer (Jasco). IR spectra were conducted on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer (Jasco). HR-ESI-MS were performed on an Agilent 6210 ESI/TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). 1D (1H, 13C, and DEPT) and 2D (1H– 1H COSY, HSQC, HMBC, and ROESY) NMR spectra were recorded on a Bruker AV-400 spectrometer (Bruker, Faellanden, Switzerland) with TMS as the internal standard, and chemical shifts were expressed in d values (ppm). Silica gel (200–300 mesh, Qingdao Marine Chemical Plant, Qingdao, China), macroporous resin AB-8 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), Sephadex LH-20 (25 – 100 mm, Fluka, Buchs, Switzerland), and RP-18 (50 mm, YMC, Kyoto, Japan) were used for column chromatographies (CCs). Thin-layer chromatography was performed using precoated silica gel plates (GF254, Yantai Chemical Industry Research Institute, Yantai, China). Analytical HPLC was performed on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1316A photodiode array detector (Agilent Technologies), and a C18 reversed-phase column (Cosmosil, 5 mm, 4.6 mm £ 250 mm, Nacalai Tesque, Kyoto, Japan). Preparative HPLC was performed on a Varian Prostar system equipped with UV detectors (Varian, Santa Clara, CA, USA) and a preparative Cosmosil
C18 column (20 mm £ 250 mm, Nacalai Tesque). All solvents used in CC were analytical grade (Tianjin Damao Chemical Plant, Tianjin, China). 3.2 Plant material The active pharmaceutical ingredients of SFI (batch no. 20090116) were provided by the Livzon Pharmaceutical Group Inc. The plant Codonopsis pilosula (Franch.) Nannf (Longxi GAP base, Gansu province, China) is used for Radix Codonopsis, while Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao (Hunyuan GAP base, Shanxi province, China) is used for Radix Astragali. 3.3 Extraction and isolation The extract (1 kg) was suspended in H2O and then subjected to macroporous resin AB-8 column eluting with H2O–EtOH (100:0, 60:40, 30:70, 5:95, each 30 l) to give four fractions (fractions A – D). Fraction B (100.0 g) was subjected to silica gel column and eluted with CHCl3 – MeOH (100:0 ! 0:100, v/v) to afford six subfractions (fractions B1–B6). Fraction B2 (4.0 g) was further separated on silica gel column eluting with a gradient of CHCl3 –MeOH (100:0 ! 0:100, v/v) to afford five subfractions (B2A–B2E). Fraction B2C (200 mg) was subjected to Sephadex LH-20 column with the eluent of CHCl3 –MeOH (1:1) to yield compound 1 (10.1 mg). Fraction B2D (1.2 g) was subjected on ODS column eluting with CH3OH – H2O (10:90 ! 100:0, v/v) to afford six subfractions (B2D1–B2D6). Fraction B2D4 (188 mg) was purified by Sephadex LH-20 (CHCl3 – MeOH, 1:1) and preparative HPLC to yield compound 2 (MeOH–H2O, 35:70, 8 ml/ min, 254 nm, tR 28.8 min, 8.7 mg). 3.3.1 (aS)-a-Ethenyl-4-hydroxyl-3methoxyl-benzenemethanol (1) Amorphous powder. ½a25 D 2 10.8 (c 0.81, MeOH); UV (MeOH) lmax (log 1): 204
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(4.20), 230 (3.76), 277 (3.20), 302 (3.08) nm; IR (KBr) nmax: 3388, 2927, 1624, 1600, 1509, 1444, 1280, 1267, 1077 cm21; for 1H and 13C NMR spectral data (CD3OD), see Table 1. HR-ESI-MS m/z: 359.1503 [2M 2 H]2 (calcd for C20H23O6, 359.1500). 3.3.2 Calycosin 7-O-[a-D glucopyranosyl (1 ! 4)]-b-Dglucopyranoside (2) Amorphous powder. ½a25 D þ 22.7 (c 0.62, CH3OH); UV (CH3OH) lmax (log 1): 204 (4.10), 219 (3.06), 248 (3.28), 260 (4.08), 286 (3.81) nm; IR (KBr) nmax: 3389, 2925, 1624, 1512, 1443, 1267, 1074 cm21; for 1 H and 13C NMR spectral data (DMSOd6), see Table 3. HR-ESI-MS m/z: 631.1637 [M þ Na] þ (calcd for C28H32O15Na, 631.1633). 3.4 Acid hydrolysis of compound 2 Compound 2 (3.5 mg) in 4 mol/l HCl (10 ml) was refluxed at 908C for 6 h in a water bath. The reaction mixture was dissolved in H2O after evaporation, which was then extracted with EtOAc for three Table 3. 1H (400 MHz) and ppm, J in Hz).a
13
times. The aqueous layer was concentrated and dried by N2, and treated with dry pyridine (1 ml) and L -cysteine methyl ester hydrochloride (2 mg), followed by heating at 608C for 2 h, and then concentrated to dryness with N2. The residue was added with isothiocyanate (2 mg) and heated at 608C for another 1 h. The final reaction mixture was analyzed by HPLC under the following conditions: an Agilent 1200 chromatograph equipped with a Cosmosil 5C18-MS-II column (4.6 mm £ 250 mm i. d., Nacalai Tesque Inc.); column temperature: 358C; mobile phase: isocratic elution of 41% CH3CN –H2O (v/v) in 50 mmol/l HCl; flow rate: 0.8 ml/min; injection volume: 12 ml; UV detection wavelength: 254 nm. The standard D -glucose was subjected under the same condition. After the comparison of the retention times of monosaccharide derivative, the sample was confirmed to comprise D -glucose (tR ¼ 19.1 min) [11]. Acknowledgments Financial support of this research was provided by Program for National Natural Science Foundation of China (No. 81273391), the Ministry of Science and Technology of
C NMR (100 MHz) spectral data for compound 2 (DMSO-d6, d in
No.
dH
dC
No.
1 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60
– 8.39 (1H, s) – – 8.06 (1H, d, 8.9) 7.15 (1H, dd, 8.9, 2.2) – 7.25 (1H, d, 2.2) – – – 7.07 (1H, br s) – – 6.97 (1H) 6.97 (1H)
– 153.6 124.4 174.6 127.0 115.5 161.3 103.4 156.9 118.5 123.6 116.4 146.0 147.6 112.0 119.7
100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 30 -OH 40 -OCH3
a
5
dH 5.18 (1H, d, 7.8) 3.38 (1H) 3.62 (1H) 3.47 (1H) 3.67 (1H) a: 3.64 (1H) b: 3.48 (1H) 5.08 (1H, d, 3.7) 3.40 (1H) 3.51 (1H) 3.08 (1H) 3.42 (1H) a: 3.75 (1H, dd, 10.3, 4.9) b: 3.60 (1H) 9.01 (1H, s) 3.80 (3H, s)
Overlapped signals were reported without designating multiplicity.
dC 99.6 73.5 76.1 79.0 75.4 60.8 100.7 72.4 73.3 69.9 72.7 60.2 – 55.7
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China (Nos 2013DFM30080, 2013BAI11B05, 2012ZX09103201-056), and the Program of Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2200058).
Note 1.
These authors contributed equally to this work.
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[6] H.Y. Gu, S.Y. Zhang, W.H. Huang, X. H. Liu, Y. Wang, C.L. Fan, and W.C. Ye, Chin. Tradit. Pat. Med. 35, 1494 (2013). [7] C. Chen, H.M. Wang, S.H. Chung, W. L. Lo, W.L. Yang, and S.C. Yang, Chem. Nat. Compd. 3, 474 (2010). [8] J. Stambasky, A.V. Malkov, and P. Kocovsky, J. Org. Chem. 73, 9148 (2008). [9] F.P. Miao, X.R. Liang, X.L. Yin, G. Wang, and N.Y. Ji, Org. Lett. 14, 3815 (2012). [10] F. Wang, Y. Gao, L. Zhang, B. Bai, Y. N. Hu, Z.J. Dong, Q.W. Zhai, H.J. Zhu, and J.K. Liu, Org. Lett. 12, 3196 (2010). [11] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, and I. Kouno, Chem. Pharm. Bull. 55, 899 (2007). [12] K. Mizutani, M. Yuda, O. Tanaka, Y. I. Saruwatari, M.R. Jia, Y.K. Ling, and X. F. Pu, Chem. Pharm. Bull. 36, 2726 (1998). [13] X.F. Ma, T.Y. Zhang, Y. Wei, P.F. Tu, Y. J. Chen, and Y. Ito, J. Chromatogr. A 962, 243 (2002). [14] M.H. Liu, X. Tong, J.X. Wang, W. Zou, H. Cao, and W.W. Su, J. Pharm. Biomed. 74, 141 (2013).
