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Isolation and characterization of two new phenolic acids from cultured cells of Saussurea involucrata
Phytochemistry Letters 7 (2014) 133–136
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Isolation and characterization of two new phenolic acids from cultured cells of Saussurea involucrata Xiaowei Zou a,1, Dan Liu b,1, Yaping Liu c, Yanhun Fu b, Xiaozhe Zhang b, Zhilong Xiu a,**, Hongbin Xiao b,* a
Department of Bioscience and Biotechnology, School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, PR China Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China c Dalian Practical Biotechnology Co., Ltd., 96 Renmin Road, Dalian 116001, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2013 Received in revised form 7 November 2013 Accepted 11 November 2013 Available online 27 November 2013
Two new phenolic acids, 1, 5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (1) and 3, 5-di-O-caffeoyl-1-O-(2O-caffeoyl-4-maloyl)-quinic acid (2), were isolated from cultured cells of Saussurea involucrata. Their structures were elucidated using 2D NMR spectroscopy and MS. Further in vitro bioactive investigations demonstrated that 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4-maloyl)-quinic acid (2) had significant scavenging activities against radicals 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and 2, 20 -azino-bis-3ethylbenzothiazoline-6-sulphonic acid (ABTS). ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Cultured cells of Saussurea involucrata Phenolic acid Free radical scavenging activity
1. Introduction Saussurea involucrata Kar.et Kir., the herbal name ‘‘Tianshanxuelian’’, is a well-known Chinese herbal medicine plant distributed mainly in Xinjiang Uighur Autonomous Region of China, which is used for the treatment of rheumatic arthritis, gynopathy, headache, stomachache, several female disorders in Chinese folk medicine (Yi et al., 2010). This herb now becomes endangered due to the long period of its regeneration and over-harvesting. Therefore, it is highly desired to use cultured cells of S. involucrata (CCS) as an alternative material, which could theoretically generate comparable secondary metabolites and may thus serve as a new source of drug for clinical usage. Indeed, previous studies reported the CCS had anti-fatigue effects (Jia and Wu, 2008), radioprotective effect (Liu et al., 2011), anti-inflammatory, analgesic activities (Jia et al., 2005), and phytochemical investigations revealed CCS contained terpenes (Chen et al., 2009), flavones and lignans (Li et al., 2007), reflecting its potential values for replacing natural S. involucrata.
* Corresponding author. Tel.: +86 411 84379667; fax: +86 411 84379756. ** Corresponding author. Tel.: +86 411 84706369; fax: +86 411 84706369. E-mail addresses: [email protected] (Z. Xiu), [email protected] (H. Xiao). 1 These authors contributed equally to this work.
In this study, we reported the identification of two new caffeoyl maloyl quinic acid type phenolic acids, 1, 5-O-dicaffeoyl-3-O-(4maloyl)-quinic acid (1)and 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4maloyl)-quinic acid (2). They were further assayed for free radical scavenging activities using vitamin C as a positive control. The new compounds showed much lower IC50 during the radical scavenging activity tests. 2. Results and discussion 2.1. Structural elucidation of new compounds Compound 1 was obtained as white amorphous power with optical rotation [a]D20 + 28.0 (c 0.2, MeOH); mp 205–206 8C. In the UV spectrum, the absorption peaks were at 330 (4.31), 300 (sh, 4.18), 245 nm (4.02), which reflected the features of caffeic acid substituted compounds (Lee et al., 2010). Its IR (KBr disc) spectrum suggested the presence of hydroxy (3300 cm1), carbonyl (1680 cm1), arom double bond (1596, 1525 cm1), trans double bond (1260, 972 cm1). The pseudo molecular ion peak was at m/z 631.1294([MH+]), in correspondence with the molecular formula of C29H28O16 ([MH+] = 631.1305). Moreover, ESI-MS/ MS spectrum had fragment ions of m/z 469.0981 ([McaffeoylH+], M½caffeic acid ¼ 180:0423), 353.0875 (M½caffeoyl-quinic acidHþ ¼ 353:0878), 307.0667 ([M2caffeoylH+], 307.0671), 191.0563
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2013.11.002
X. Zou et al. / Phytochemistry Letters 7 (2014) 133–136
134
Fig. 1. 1H–1H COSY (bold) and HMBC correlations (arrows) of compound 1 and 2.
(M½quinic acidHþ ¼ 191:0561),suggestingtheexistenceofadicaffeoylquinic acid moiety. Interestingly, ion at m/z 307.0667 was also presented in the MS1 spectra, reflecting the thermal fragmentation of this compound in the ESI source. Tandem MS/MS spectrum of this ion presented product ions at m/z 191.0563 ([quinic acidH+]) and 133.0145 (M½maloyl ¼ 133:0145), thus suggesting the fragmentation of [maloyl-quinic acidH]. 1H NMR and 13C NMR spectra exhibited four doublets with coupling constants of 15.9 Hz characteristic for trans olefinic protons (dH 7.61, dH 6.30, dH 7.62, dH 6.35). The coupling patternoftwo1,3,4-trisubstitutedbenzenes[dH 6.79(d,8.2), dH 6.97(d, 8.2), dH 6.80(d,8.2), dH 6.99(d,8.2)]indicatedthepresenceoftwocaffeic acid moieties, which could be confirmed in 13C spectrum by compared with caffeic acid reported in a previous study (Lee et al., 2010). The carboxyl, dC 173.3 (C-10 ) and dC 171.7 (C-40 ) suggested that there was a 4-esterified-malic acid group, rather than a 1-esterified-malic acid group in previous studies (Dang et al., 2007; Yang et al., 2005). The rest signals of 1H NMR and 13C NMR were attributed to a quinic acid moiety (Pauli and Poetsch Nahrstedt, 1998). The low-field-shifted signals of dH Table 1 1 H NMR (500 MHz) and Position
13
C NMR (125 MHz) data of 1 and 2 (CD3OD, TMS). Compound 1
dH (mult, J in Hz) 1 2a 2b 3 4 5 6a 6b 7 1‘ 2‘ 3‘a 3‘b 4‘ 100 /1000 /10000 200 /2000 /20000 300 /3000 /30000 400 /4000 /40000 500 /5000 /50000 600 /6000 /60000 700 /7000 /70000 800 /8000 /80000 900 /9000 /90000
5.42 (1H, ddd, J = 4.2, 4.0, 4.2, H-5), and dH 5.48 (1H, m, H-3), indicated that 5-OHand 3-OHweresubstituted.The 1HNMRand 13CNMRdata of caffeoyl and quinic acid groups were highly similar with those of 1, 3, 5tri-O-caffeoyl-quinic acid (Cui et al., 2009), which had three acyl groups on C-1, 3, 5 of a quinic acid. In the HMBC spectrum, the presence of the correlation of dH 5.42 (1H, ddd, 4.2, 4.0, 4.2, H-5) to dC 168.7 (C-900 ) indicated there was a substitution of a caffeoyl on the C-5 of the quinic acid; The correlation of dH 5.48 (1H, m, H-3) to the carbonyl carbon signalat dC 171.7(C-40 )indicated asubstitutionofamaloylontheC-3 of the quinic acid (Fig. 1). It was worthy to note that the correlation of dH 2.75 (2H, m, H-30 ) to dC 171.7 (C-40 ) was stronger than the one from dH 4.45 (1H, m, H-20 ) to dC 171.7 (C-40 ), verifying the signal at dC 171.7 belonged to C-40 . Conclusively, the other caffeoyl group was unambiguously connected to 1-OH. Therefore, compound 1 was identified as 1, 5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (MDCQA). 1 H NMR and 13C NMR spectra data are shown in Table 1. Compound 2 was obtained as white amorphous power with optical rotation [a]D20 + 81.76 (c 0.17, MeOH); mp 233–234 8C;
2.84 2.46 5.48 3.95 5.42 2.03 2.64
(m) A (dd,15.4, 3.0) (m) (dd,3.5,9.1) (ddd,4.2,4.0,4.2) (dd, 13.4,10.3) (br. d,12.3)
4.45 (m) 2.75 (m) A
7.06 (d,1.9); 7.10(d,1.9)
6.79 6.80 6.97 6.99 7.61 6.30
(d, 8.2) (d, 8.2) (d, 8.2) (d, 8.2) (dd, 15.9); 7.62(dd, 15.9) (d,15.9); 6.35(d,15.9)
Note: the chemical shifts in the same letters were overlapped.
Compound 2
dC 80.8 33.1 73.3 71.7 71.3 40.7 174.4 173.3 68.2 37.7 171.7 127.7;127.8 115.0;115.2 146.8 149.7;149.8 116.55;116.6
dH (mult, J in Hz) 2.82 2.47 5.50 3.96 5.48 2.00 2.64
(d) B (d,14.4) (m) C (dd,3.2,9.4) (ddd) C (m) (br. d,12.6)
5.40 (m) 2.88 (dd,4.5,16.6) B 3.03 (dd,6.8,16.6)
dC 80.9 32.9 73.6 71.8 71.3 38.1 174.6 172.7 70.0 37.6
6.76-6.80 (m)
170.8 127.6;127.8 115.1;114.4;115.0 146.7;146.8 149.6;149.7 116.5
123.1;123.3
6.93-6.97 (m)
123.3;123.1;123.2
147.5;147.95 115.3;115.4 167.8;168.7
7.53-7.63 (m) 6.25-6.36 (m)
148.0;147.5;147.8 115.3;115.4 168.8;167.8;168.1
7.07 (m)
X. Zou et al. / Phytochemistry Letters 7 (2014) 133–136
UV and IR (KBr disc) spectra suggested 2 had a similar structure with compound 1. The ESI-MS analysis showed a pseudo molecular ion peak at m/z 793.1616 (calcd for C38H34O19, [MH], 793.1622). ESI-MS/MS fragment ions were found at m/z 631.1294 ([McaffeoylH]), 469.0981 ([Mcaffeoyl2H]), 353.0875 ([caffeoyl-quinic acidH]), 307.0667 ([maloyl-quinic acidH]), 277.0354 ([caffeoyl-maloylH2O]), and 191.0563 ([qunic acidH]). These data suggested 2 was a maloyl-caffeoyl-quinic acid type compound similar with 1. Compared with 1, the main differences in 1H NMR spectra were that there were one more set of caffeoyl signals, and the chemical shift of H-20 on malic acid group shifted to down-field with dH 5.40 (1H, m, H-20 ), which meant the OH located on C-20 acylated. The structure of 2 could be further assigned using 1H–1H COSY and HMBC spectra (Fig. 1). In the HMBC spectrum, the correlations of dC 170.8 (C-40 ) to dH 2.88 (1H, dd, J = 4.5, 16.6, H-30 a) and dH 3.03 (1H, dd, J = 6.8, 16.6, H-30 b) were stronger in comparison of dC 172.7 (C-10 ), which confirmed the assignment of C-40 . On other-cross signal of dH 5.40 (1H, m, H-20 ) to dC 170.8 (C-40 ) was weaker than that of H-20 to dC 172. 7 (C-10 ), which confirmed the assignment of C-10 . The HMBC correlations of dH 5.48 (1H, ddd, overlapped, H-5) to C-90 ’ (dC 168.8), and dH 5.50 (1H, m, H-3) to C-90000 (dC 168.1), indicated that two of caffeic acids were located at the C-3 and C-5, respectively; the correlation of dH 5.40 (1H, m, H-20 ) to C-9000 (dC 167.8) showed the substitution of the other caffeoyl moiety on C-20 of the malic acid group. Conclusively, the malic acid group was esterified on C-40 by 1-OH of the quinic acid. Therefore, compound 2 was determined as 3, 5-di-O-caffeoyl-1-O-(2-Ocaffeoyl-4-maloyl)-quinic acid (MTCQA). 2.2. Free radical-scavenging activity of the isolated compounds In this study, MDCQA (compound 1) and MTCQA (compound 2) were compared with vitamin C, to evaluate their free radicalscavenging activities. The values of IC50 for DPPH and ABTS radical scavenging are presented in Table 2, indicating that the capabilities of the two new compounds were much higher than the positive control. And the activities of compound 2 was higher than compound 1, which could be explained by the presence of the caffeoyl group (Go´ngora et al., 2003; Iwai et al., 2004; Kim et al., 2011).
3. Experimental 3.1. General methods Open column chromatography was performed with macroporous absorption resin HPD-100, NKA-9 (0.3–1.2 mm, Nankai resin technology limited company, Tianjin, China) or MCI GEL CHP 20P (75–150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semi-preparative HPLC was conducted on WatersTM 600 E separation module equipped with 996 photodiode array detector (205–450 nm) and a chromatocrex C18 column (20 mm i.d. 250 mm, 10 mm).
135
3.2. Materials and chemicals The CCS was regenerated using the method reported in a previous study (Chen et al., 2013). The herbs of S. involucrata were purchased from Xinjiang Uygur Autonomous Region, and the voucher specimens were deposited in the Herbarium of Institute of Botany, Chinese academy of sciences. 3.3. Extraction and isolation The dried CCS (1.5 kg) was extracted at 50 8C using 20 L boiling 70% ethanol (7 times) under reflux, with the assistance of vacuum pump and churn-dasher. The extract (702.8 g) was decentralized in 10 L water, then repartitioned using petroleum ether (Vwater: Vpetroleum ether = 1:1; 5 times), EtOAc (Vwater:VEtOAc = 1:1; 5 times), and n-BuOH (Vwater:Vn-BuOH = 1:1; 5 times) successively. The n-BuOH fraction (NBF, 171 g) was divided into 10% (128 g) and 50% (53 g) MeOH soluble parts, respectively. Six fractions (NBF-1 NBF-6) were obtained by separating the second MeOH soluble part on an open column chromatography (8 cm 200 cm, NKA-9 resin) using a step gradient of H2O-EtOH. Fraction NBF-3 (37 g) and NBF-5 (0.77 g) were passed through a column of MCI GEL CHP 20P (4 cm 30 cm) eluted using a step gradient of H2OEtOH to get NBF-3-a (21 mg) and NBF-5-a (61 mg), from which compound 1 (9 mg) and compound 2 (20 mg) were purified using preparative HPLC with the mobile phase of 40% methanol containing 0.5% formic acid. 3.4. Structural analysis NMR spectra were collected from AVANCE AV500 MHz spectrometer (Bruker Co., Ltd., Switzerland) with TMS as an internal standard. ESI-MS spectra of all samples were acquired on a Agilent G6200 Series Time-of-Flight with electrospray ionization (ESI) equipped with an Agilent 1290 infinity LC system and an Agilent ZORBAX poroshell 120 SB-C18 column (3 mm i.d 150 mm, 2.7 mm). The MS conditions were set as followed: negative ionization model, nebulizing gas (N2) press 30 psi, drying gas (N2) 8.0 L/min and gas temperature 350 8C. Fragmentation was set at 200 V, capillary voltage 3.5 kV, skimmer 65 V. Collision energy was set at 15 mV during MS/MS analysis. The mass scan rate was set to 100–1500 m/z, and the data was collected and analyzed using Agilent MassHunter Workstation Software. UV spectra were measured with a Hitachi JP/U-3010 spectrophotometer. IR spectra were recorded on Nicolet Is50 Adv FTIR Spectrometer (Thermo, the USA). Optical rotations were determined on a MCP200 modular circular polarimeter (Anton Paar, Austria). Melting points were measured on an X-4 micromelting point apparatus (Tech, Beijing, China). 3.5. DPPH and ABTS radical-scavenging activities of the isolated compounds The assays for DPPH and ABTS radical-scavenging were performed according to the method described by Yuan et al.
Table 2 Radical scavenging activities of compound 1and 2. Compounds
MDCQA(1) MTCQA(2) Vitamin C
DPPH radical scavenging
ABTS radical scavenging
IC50 (mmol/L)
95% Cl
IC50 (mmol/L)
95% Cl
1.053 0.748 5.725
(0.043, +0.049) (0.040, +0.040) (0.296, +0.332)
0.988 0.554 6.541
(0.056, +0.069) (0.024, +0.028) (0.301, +0.343)
Note: 95% Cl means Score of 95% confidence level.
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(2012). The data were measured with a multimode microplate spectrophotomter (Varioscan Flash, Thermo). Acknowledgements This work was financially supported by National Major New Drugs Innovation and Development Program (No. 2011ZX09307002-01) and China Postdoctoral Science Foundation (No. 2012M510078).
References Chen, R.D., Zou, J., Jia, J.M., 2009. Chemical constituents from the cell cultures of Saussurea involucrata. J. Asian Nat. Prod. Res. 12, 119–123. Chen, R., Liu, X., Zou, J., Yang, L., Dai, J., 2013. Qualitative and quantitative analysis of phenylpropanoids in cell culture, regenerated plantlets and herbs of Saussurea involucrata. J. Pharmaceut. Biomed. 74, 39–46. Cui, C.B., Jeong, S.K., Lee, Y.S., Lee, S.O., Kang, I.J., Lim, S.S., 2009. Inhibitory activity of caffeoylquinic acids from the aerial parts of Artemisia Princeps on rat rens aldose reductase and on the formation of advanced glycation end products. J. Korean Soc. Appl. Biomed. 52, 655–662. Dang, Q., Cao, J., Zhang, N., Lv, A.L., Wang, D., Pei, Y.H., 2007. Isolation and identification of chemical consistuents from fruits of Hippophae rhamnoides L. J. Shenyang Pharm. Univ. 29, 488–490. ˜ ez, S., Giner, R.M., Recio, M.C., Schinella, G., Rı´os, J.L., 2003. Go´ngora, L., Ma´n Inhibition of xanthine oxidase by phenolic conjugates of methylated quinic acid. Plata Med. 69 (5) 396–401.
Iwai, K., Kishimoto, N., Kakino, Y., Mochida, K., Fujita, T., 2004. In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans. J. Agric. Food Chem. 50 (13) 3718–3722. Jia, J.M., Wu, C.F., 2008. Antifatigue activity of tissue culture extracts of Saussurea involucrata. Pharm. Biol. 46, 433–436. Jia, J.M., Wu, C.F., Liu, W., Yu, H., Hao, Y., Zheng, J.H., Ji, Y.R., 2005. Antiinflammatory and analgesic activities of the tissue culture of Saussurea involucrata. Biol. Pharm. Bull. 28, 1612–1614. Kim, J.Y., Cho, J.Y., Ma, Y.K., Park, K.Y., Lee, S.H., Ham, K.S., Lee, H.J., Park, K.H., Moon, J.H., 2011. Dicaffeoylquinic acid derivatives and flavonoid glucosides from glasswort (Salicornia herbacea L.) and their antioxidative activity. Food Chem. 125, 55–62. Lee, E.J., Kim, S.J., Kim, H.P., Lee, J.H., Kang, S.S., 2010. Phenolic consistuents from the flower buds of Lonicera japonica and their 5-lipoxgenase inhibitory activities. Food Chem. 120, 134–139. Liu, Y.P., Chen, X.X., Liu, H.S., 2011. Effect of Saussurea involucrata culture on radiation injury. Chin. J. Basic Med. Trad. Chin. Med. 17, 968–970. Li, Y., Guo, S.X., Wang, C.L., Wu, L.Q., Xiao, P.G., 2007. Studies on chemical constituents in cell cultures of Saussurea involucrata. Chin. Pharm. Assoc. 42, 1768– 1769. Pauli, G.F., Poetsch Nahrstedt, A., 1998. Structure assignment of natural quinic acid derivatives using proton nuclear magnetic resonance techniques. Phytochem. Anal. 9, 177–185. Yang, J., Chen, S.Q., Ji, C.R., Liu, Y.Z., 2005. Isolation and characterization of chemical components of Cornus officinalis Sieb. et Zucc. Chin. Trad. Herbal Drugs 36, 1780–1782. Yuan, X.Y., Gao, M.Z., Xiao, H.B., Tan, C.Y., Du, Y.G., 2012. Free radical scavenging activities and bioactive substances of Jerusalemartichoke (Helianthus tuberosus L) leaves. Food Chem. 133, 10–14. Yi, T., Zhao, Z.Z., Yu, Z.L., Chen, H.B., 2010. Comparison of the anti-inflammatory and anti-nociceptive effects of three medicinal plants known as Snow Lotus herb in traditional Uighur and Tibetan medicines. J. Ethnopharmacol. 128, 405–411.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Isolation and characterization of two new phenolic acids from cultured cells of Saussurea involucrata Xiaowei Zou a,1, Dan Liu b,1, Yaping Liu c, Yanhun Fu b, Xiaozhe Zhang b, Zhilong Xiu a,**, Hongbin Xiao b,* a
Department of Bioscience and Biotechnology, School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, PR China Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China c Dalian Practical Biotechnology Co., Ltd., 96 Renmin Road, Dalian 116001, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 July 2013 Received in revised form 7 November 2013 Accepted 11 November 2013 Available online 27 November 2013
Two new phenolic acids, 1, 5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (1) and 3, 5-di-O-caffeoyl-1-O-(2O-caffeoyl-4-maloyl)-quinic acid (2), were isolated from cultured cells of Saussurea involucrata. Their structures were elucidated using 2D NMR spectroscopy and MS. Further in vitro bioactive investigations demonstrated that 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4-maloyl)-quinic acid (2) had significant scavenging activities against radicals 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and 2, 20 -azino-bis-3ethylbenzothiazoline-6-sulphonic acid (ABTS). ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Cultured cells of Saussurea involucrata Phenolic acid Free radical scavenging activity
1. Introduction Saussurea involucrata Kar.et Kir., the herbal name ‘‘Tianshanxuelian’’, is a well-known Chinese herbal medicine plant distributed mainly in Xinjiang Uighur Autonomous Region of China, which is used for the treatment of rheumatic arthritis, gynopathy, headache, stomachache, several female disorders in Chinese folk medicine (Yi et al., 2010). This herb now becomes endangered due to the long period of its regeneration and over-harvesting. Therefore, it is highly desired to use cultured cells of S. involucrata (CCS) as an alternative material, which could theoretically generate comparable secondary metabolites and may thus serve as a new source of drug for clinical usage. Indeed, previous studies reported the CCS had anti-fatigue effects (Jia and Wu, 2008), radioprotective effect (Liu et al., 2011), anti-inflammatory, analgesic activities (Jia et al., 2005), and phytochemical investigations revealed CCS contained terpenes (Chen et al., 2009), flavones and lignans (Li et al., 2007), reflecting its potential values for replacing natural S. involucrata.
* Corresponding author. Tel.: +86 411 84379667; fax: +86 411 84379756. ** Corresponding author. Tel.: +86 411 84706369; fax: +86 411 84706369. E-mail addresses: [email protected] (Z. Xiu), [email protected] (H. Xiao). 1 These authors contributed equally to this work.
In this study, we reported the identification of two new caffeoyl maloyl quinic acid type phenolic acids, 1, 5-O-dicaffeoyl-3-O-(4maloyl)-quinic acid (1)and 3, 5-di-O-caffeoyl-1-O-(2-O-caffeoyl-4maloyl)-quinic acid (2). They were further assayed for free radical scavenging activities using vitamin C as a positive control. The new compounds showed much lower IC50 during the radical scavenging activity tests. 2. Results and discussion 2.1. Structural elucidation of new compounds Compound 1 was obtained as white amorphous power with optical rotation [a]D20 + 28.0 (c 0.2, MeOH); mp 205–206 8C. In the UV spectrum, the absorption peaks were at 330 (4.31), 300 (sh, 4.18), 245 nm (4.02), which reflected the features of caffeic acid substituted compounds (Lee et al., 2010). Its IR (KBr disc) spectrum suggested the presence of hydroxy (3300 cm1), carbonyl (1680 cm1), arom double bond (1596, 1525 cm1), trans double bond (1260, 972 cm1). The pseudo molecular ion peak was at m/z 631.1294([MH+]), in correspondence with the molecular formula of C29H28O16 ([MH+] = 631.1305). Moreover, ESI-MS/ MS spectrum had fragment ions of m/z 469.0981 ([McaffeoylH+], M½caffeic acid ¼ 180:0423), 353.0875 (M½caffeoyl-quinic acidHþ ¼ 353:0878), 307.0667 ([M2caffeoylH+], 307.0671), 191.0563
1874-3900/$ – see front matter ß 2013 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe. http://dx.doi.org/10.1016/j.phytol.2013.11.002
X. Zou et al. / Phytochemistry Letters 7 (2014) 133–136
134
Fig. 1. 1H–1H COSY (bold) and HMBC correlations (arrows) of compound 1 and 2.
(M½quinic acidHþ ¼ 191:0561),suggestingtheexistenceofadicaffeoylquinic acid moiety. Interestingly, ion at m/z 307.0667 was also presented in the MS1 spectra, reflecting the thermal fragmentation of this compound in the ESI source. Tandem MS/MS spectrum of this ion presented product ions at m/z 191.0563 ([quinic acidH+]) and 133.0145 (M½maloyl ¼ 133:0145), thus suggesting the fragmentation of [maloyl-quinic acidH]. 1H NMR and 13C NMR spectra exhibited four doublets with coupling constants of 15.9 Hz characteristic for trans olefinic protons (dH 7.61, dH 6.30, dH 7.62, dH 6.35). The coupling patternoftwo1,3,4-trisubstitutedbenzenes[dH 6.79(d,8.2), dH 6.97(d, 8.2), dH 6.80(d,8.2), dH 6.99(d,8.2)]indicatedthepresenceoftwocaffeic acid moieties, which could be confirmed in 13C spectrum by compared with caffeic acid reported in a previous study (Lee et al., 2010). The carboxyl, dC 173.3 (C-10 ) and dC 171.7 (C-40 ) suggested that there was a 4-esterified-malic acid group, rather than a 1-esterified-malic acid group in previous studies (Dang et al., 2007; Yang et al., 2005). The rest signals of 1H NMR and 13C NMR were attributed to a quinic acid moiety (Pauli and Poetsch Nahrstedt, 1998). The low-field-shifted signals of dH Table 1 1 H NMR (500 MHz) and Position
13
C NMR (125 MHz) data of 1 and 2 (CD3OD, TMS). Compound 1
dH (mult, J in Hz) 1 2a 2b 3 4 5 6a 6b 7 1‘ 2‘ 3‘a 3‘b 4‘ 100 /1000 /10000 200 /2000 /20000 300 /3000 /30000 400 /4000 /40000 500 /5000 /50000 600 /6000 /60000 700 /7000 /70000 800 /8000 /80000 900 /9000 /90000
5.42 (1H, ddd, J = 4.2, 4.0, 4.2, H-5), and dH 5.48 (1H, m, H-3), indicated that 5-OHand 3-OHweresubstituted.The 1HNMRand 13CNMRdata of caffeoyl and quinic acid groups were highly similar with those of 1, 3, 5tri-O-caffeoyl-quinic acid (Cui et al., 2009), which had three acyl groups on C-1, 3, 5 of a quinic acid. In the HMBC spectrum, the presence of the correlation of dH 5.42 (1H, ddd, 4.2, 4.0, 4.2, H-5) to dC 168.7 (C-900 ) indicated there was a substitution of a caffeoyl on the C-5 of the quinic acid; The correlation of dH 5.48 (1H, m, H-3) to the carbonyl carbon signalat dC 171.7(C-40 )indicated asubstitutionofamaloylontheC-3 of the quinic acid (Fig. 1). It was worthy to note that the correlation of dH 2.75 (2H, m, H-30 ) to dC 171.7 (C-40 ) was stronger than the one from dH 4.45 (1H, m, H-20 ) to dC 171.7 (C-40 ), verifying the signal at dC 171.7 belonged to C-40 . Conclusively, the other caffeoyl group was unambiguously connected to 1-OH. Therefore, compound 1 was identified as 1, 5-O-dicaffeoyl-3-O-(4-maloyl)-quinic acid (MDCQA). 1 H NMR and 13C NMR spectra data are shown in Table 1. Compound 2 was obtained as white amorphous power with optical rotation [a]D20 + 81.76 (c 0.17, MeOH); mp 233–234 8C;
2.84 2.46 5.48 3.95 5.42 2.03 2.64
(m) A (dd,15.4, 3.0) (m) (dd,3.5,9.1) (ddd,4.2,4.0,4.2) (dd, 13.4,10.3) (br. d,12.3)
4.45 (m) 2.75 (m) A
7.06 (d,1.9); 7.10(d,1.9)
6.79 6.80 6.97 6.99 7.61 6.30
(d, 8.2) (d, 8.2) (d, 8.2) (d, 8.2) (dd, 15.9); 7.62(dd, 15.9) (d,15.9); 6.35(d,15.9)
Note: the chemical shifts in the same letters were overlapped.
Compound 2
dC 80.8 33.1 73.3 71.7 71.3 40.7 174.4 173.3 68.2 37.7 171.7 127.7;127.8 115.0;115.2 146.8 149.7;149.8 116.55;116.6
dH (mult, J in Hz) 2.82 2.47 5.50 3.96 5.48 2.00 2.64
(d) B (d,14.4) (m) C (dd,3.2,9.4) (ddd) C (m) (br. d,12.6)
5.40 (m) 2.88 (dd,4.5,16.6) B 3.03 (dd,6.8,16.6)
dC 80.9 32.9 73.6 71.8 71.3 38.1 174.6 172.7 70.0 37.6
6.76-6.80 (m)
170.8 127.6;127.8 115.1;114.4;115.0 146.7;146.8 149.6;149.7 116.5
123.1;123.3
6.93-6.97 (m)
123.3;123.1;123.2
147.5;147.95 115.3;115.4 167.8;168.7
7.53-7.63 (m) 6.25-6.36 (m)
148.0;147.5;147.8 115.3;115.4 168.8;167.8;168.1
7.07 (m)
X. Zou et al. / Phytochemistry Letters 7 (2014) 133–136
UV and IR (KBr disc) spectra suggested 2 had a similar structure with compound 1. The ESI-MS analysis showed a pseudo molecular ion peak at m/z 793.1616 (calcd for C38H34O19, [MH], 793.1622). ESI-MS/MS fragment ions were found at m/z 631.1294 ([McaffeoylH]), 469.0981 ([Mcaffeoyl2H]), 353.0875 ([caffeoyl-quinic acidH]), 307.0667 ([maloyl-quinic acidH]), 277.0354 ([caffeoyl-maloylH2O]), and 191.0563 ([qunic acidH]). These data suggested 2 was a maloyl-caffeoyl-quinic acid type compound similar with 1. Compared with 1, the main differences in 1H NMR spectra were that there were one more set of caffeoyl signals, and the chemical shift of H-20 on malic acid group shifted to down-field with dH 5.40 (1H, m, H-20 ), which meant the OH located on C-20 acylated. The structure of 2 could be further assigned using 1H–1H COSY and HMBC spectra (Fig. 1). In the HMBC spectrum, the correlations of dC 170.8 (C-40 ) to dH 2.88 (1H, dd, J = 4.5, 16.6, H-30 a) and dH 3.03 (1H, dd, J = 6.8, 16.6, H-30 b) were stronger in comparison of dC 172.7 (C-10 ), which confirmed the assignment of C-40 . On other-cross signal of dH 5.40 (1H, m, H-20 ) to dC 170.8 (C-40 ) was weaker than that of H-20 to dC 172. 7 (C-10 ), which confirmed the assignment of C-10 . The HMBC correlations of dH 5.48 (1H, ddd, overlapped, H-5) to C-90 ’ (dC 168.8), and dH 5.50 (1H, m, H-3) to C-90000 (dC 168.1), indicated that two of caffeic acids were located at the C-3 and C-5, respectively; the correlation of dH 5.40 (1H, m, H-20 ) to C-9000 (dC 167.8) showed the substitution of the other caffeoyl moiety on C-20 of the malic acid group. Conclusively, the malic acid group was esterified on C-40 by 1-OH of the quinic acid. Therefore, compound 2 was determined as 3, 5-di-O-caffeoyl-1-O-(2-Ocaffeoyl-4-maloyl)-quinic acid (MTCQA). 2.2. Free radical-scavenging activity of the isolated compounds In this study, MDCQA (compound 1) and MTCQA (compound 2) were compared with vitamin C, to evaluate their free radicalscavenging activities. The values of IC50 for DPPH and ABTS radical scavenging are presented in Table 2, indicating that the capabilities of the two new compounds were much higher than the positive control. And the activities of compound 2 was higher than compound 1, which could be explained by the presence of the caffeoyl group (Go´ngora et al., 2003; Iwai et al., 2004; Kim et al., 2011).
3. Experimental 3.1. General methods Open column chromatography was performed with macroporous absorption resin HPD-100, NKA-9 (0.3–1.2 mm, Nankai resin technology limited company, Tianjin, China) or MCI GEL CHP 20P (75–150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan). Semi-preparative HPLC was conducted on WatersTM 600 E separation module equipped with 996 photodiode array detector (205–450 nm) and a chromatocrex C18 column (20 mm i.d. 250 mm, 10 mm).
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3.2. Materials and chemicals The CCS was regenerated using the method reported in a previous study (Chen et al., 2013). The herbs of S. involucrata were purchased from Xinjiang Uygur Autonomous Region, and the voucher specimens were deposited in the Herbarium of Institute of Botany, Chinese academy of sciences. 3.3. Extraction and isolation The dried CCS (1.5 kg) was extracted at 50 8C using 20 L boiling 70% ethanol (7 times) under reflux, with the assistance of vacuum pump and churn-dasher. The extract (702.8 g) was decentralized in 10 L water, then repartitioned using petroleum ether (Vwater: Vpetroleum ether = 1:1; 5 times), EtOAc (Vwater:VEtOAc = 1:1; 5 times), and n-BuOH (Vwater:Vn-BuOH = 1:1; 5 times) successively. The n-BuOH fraction (NBF, 171 g) was divided into 10% (128 g) and 50% (53 g) MeOH soluble parts, respectively. Six fractions (NBF-1 NBF-6) were obtained by separating the second MeOH soluble part on an open column chromatography (8 cm 200 cm, NKA-9 resin) using a step gradient of H2O-EtOH. Fraction NBF-3 (37 g) and NBF-5 (0.77 g) were passed through a column of MCI GEL CHP 20P (4 cm 30 cm) eluted using a step gradient of H2OEtOH to get NBF-3-a (21 mg) and NBF-5-a (61 mg), from which compound 1 (9 mg) and compound 2 (20 mg) were purified using preparative HPLC with the mobile phase of 40% methanol containing 0.5% formic acid. 3.4. Structural analysis NMR spectra were collected from AVANCE AV500 MHz spectrometer (Bruker Co., Ltd., Switzerland) with TMS as an internal standard. ESI-MS spectra of all samples were acquired on a Agilent G6200 Series Time-of-Flight with electrospray ionization (ESI) equipped with an Agilent 1290 infinity LC system and an Agilent ZORBAX poroshell 120 SB-C18 column (3 mm i.d 150 mm, 2.7 mm). The MS conditions were set as followed: negative ionization model, nebulizing gas (N2) press 30 psi, drying gas (N2) 8.0 L/min and gas temperature 350 8C. Fragmentation was set at 200 V, capillary voltage 3.5 kV, skimmer 65 V. Collision energy was set at 15 mV during MS/MS analysis. The mass scan rate was set to 100–1500 m/z, and the data was collected and analyzed using Agilent MassHunter Workstation Software. UV spectra were measured with a Hitachi JP/U-3010 spectrophotometer. IR spectra were recorded on Nicolet Is50 Adv FTIR Spectrometer (Thermo, the USA). Optical rotations were determined on a MCP200 modular circular polarimeter (Anton Paar, Austria). Melting points were measured on an X-4 micromelting point apparatus (Tech, Beijing, China). 3.5. DPPH and ABTS radical-scavenging activities of the isolated compounds The assays for DPPH and ABTS radical-scavenging were performed according to the method described by Yuan et al.
Table 2 Radical scavenging activities of compound 1and 2. Compounds
MDCQA(1) MTCQA(2) Vitamin C
DPPH radical scavenging
ABTS radical scavenging
IC50 (mmol/L)
95% Cl
IC50 (mmol/L)
95% Cl
1.053 0.748 5.725
(0.043, +0.049) (0.040, +0.040) (0.296, +0.332)
0.988 0.554 6.541
(0.056, +0.069) (0.024, +0.028) (0.301, +0.343)
Note: 95% Cl means Score of 95% confidence level.
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(2012). The data were measured with a multimode microplate spectrophotomter (Varioscan Flash, Thermo). Acknowledgements This work was financially supported by National Major New Drugs Innovation and Development Program (No. 2011ZX09307002-01) and China Postdoctoral Science Foundation (No. 2012M510078).
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