从铜耐性植物海州香薷中提取分离芹菜素糖苷

Apigenin-7- O -β- D -glycoside isolation from the highly copper-tolerant plant Elsholtzia splendens 从铜耐性植物海州香薷中提取分离芹菜素糖苷 1 corresp first-author ‡Corresponding Author ‡Corresponding Author †E-mail:penghongyun@zju.edu.cn †E-mail:penghongyun@zju.edu.cn penghongyun@zju.edu.cn 1 2 Key Laboratory of Environmental Remediation and Ecological Health, Ministry of Education, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China Key Laboratory of Environmental Remediation and Ecological Health, Ministry of Education, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China Institution of Marine Biology, Ocean College, Zhejiang University, Hangzhou 310058, China Institution of Marine Biology, Ocean College, Zhejiang University, Hangzhou 310058, China Elsholtzia splendens (Lamiaceae) is a copper-tolerant plant species growing on copper deposits in the south of China. Chromatographic separation of n -BuOH extracts from the flowering aerial biomass afforded apigenin-7- O -β- D -glycoside, using macroporous resin, Sephadex™ LH-20 gel, polyamide resin as well as preparative high-performance liquid chromatography (P-HPLC) columns. Chemical structure was elucidated using HPLC/ESI-MS (electrospray ionization-mass spectrometry), Fourier transform infrared (FTIR), and 1 D-and 2 D-nuclear magnetic resonance (NMR). Apigenin-7- O -β- D -glycoside could be the post-harvesting product from E. splendens biomass. 目的 2 建立从铜耐性植物海州香薷地上部提取分离芹菜素糖苷的工艺技术，探索铜耐性植物海州香薷资源化途径。 创新点 2 结合使用大孔树脂、凝胶树脂、聚酰胺树脂多种色谱柱技术，从铜耐性植物海州香薷中提取分离纯度达98%的黄酮苷类化合物芹菜素糖苷。 方法 2 以铜耐性植物海州香薷为原料，用60%乙醇溶液浸提，提取液依次使用石油醚、乙酸乙酯、饱和正丁醇萃取。实验中，对饱和正丁醇萃取层，使用D101型大孔吸附树脂粗分离提取，Sephadex™ LH-20型凝胶筛分分离，并借助半制备液相和聚酰胺树脂分离纯化，得到芹菜素糖苷单体（图1）。利用紫外光谱、液相色谱–质谱联用、红外光谱、一维和二维核磁图谱，鉴定芹菜素糖苷结构（图2~5）。 结论 2 芹菜素糖苷是铜耐性植物海州香薷体内的天然产物，利用色谱技术可以从海州香薷植物体中分离出芹菜素糖苷单体。 Chromatographic separation Copper-tolerant plant Elsholtzia splendens Flavonoid glycoside Structure elucidation 色谱分离 铜耐性植物 海州香薷 黄酮苷 结构解析 Introduction 1 The Chinese native herb Elsholtzia splendens Nakai (Lamiaceae) was first recognized as a copper indicator during copper ore exploration in the 1950s, as it was the dominant plant species colonizing the mining sites (Xie and Xu, 1952 1952 ). E. splendens has evolved to tolerate and acclimate to high copper toxicity, which affects photosynthesis metabolism (Peng et al., 2013 2013 ). Interestingly, ingredients from the aerial biomass of E. splendens have been used in North-East Asia in folk and conventional medicine for treating coughs, headaches, and inflammation, and also for anti-oxidative activities (Kim et al., 2003 2003 ; Choi et al., 2007 2007 ; 2008 2008 ; Choi and Kim, 2008a 2008a ; 2008b 2008b ; Chung and Kim, 2010 2010 ). Apigenin (5,7,4'-trihydroxyflavone) and luteolin (5,7,3',4'-tetrahydroxyflavone) were the flavone ingredients of interest in ethyl acetate extracts of flowering E. splendens biomass, owing to their high inhibitory properties against cancer cell lines (Peng et al., 2014 2014 ). Flavones are active due to the presence of phenolic hydroxyls and carbonyls. These phenolic hydroxyls are always methoxylated and transformed as their corresponding glycoside, resulting in the production of the flavone glycoside. For instance, apigenin-7- O -β- D -glycoside (cosmosiin) isolated from Lonicera gracilipes var. glandulosa can inhibit the growth of various human cancer cells (Kikuchi and Matsuda, 1996 1996 ). A study on flowering E. splendens biomass indicated that the total flavonoids from the n -BuOH extracts exhibited significant concentration-dependent vasorelaxation in endothelium-intact rings of rat, which were not abolished but were significantly reduced by the removal of endothelium (Wang et al., 2014 2014 ). Therefore, we focused on the n -BuOH extracts of the flowering aerial biomass of E. splendens , using macroporous resin (MR), Sephadex™ LH-20, polyamide (PA) resin, and preparative high-performance liquid chromatography (P-HPLC) columns during chromatographic column (CC) separation. This led to the isolation of apigenin-7- O -β- D -glycoside, and we present its isolation and structural elucidation here. Materials and methods 1 General 2 Column chromatography was performed on Macroporous resin D101 (Shanghai Mosu Chemicals Co., Ltd., China), Sephadex™ LH-20 (25–100 μm, Pharmacia Fine Chemical Co., Ltd., Sweden), and PA resin (100–200 mesh, Qingdao Haiyang Chemical Co., China). Thin layer chromatography (TLC) was carried out on PA pre-coated plates (Taizhou Chemical Co., China) with MeOH-H 2 O (3:1, v/v, the same below) and/or EtOAc-MeOH (5:1) as developing agents. The spots were detected by spraying with 3% (0.03 g/ml) FeCl 3 -ethanol solution. Ultraviolet (UV) spectra were obtained on a UV 210A spectrometer (Shimadzu, Kyoto, Japan). Structural elucidations were achieved using standard spectroscopic techniques, such as Fourier transform infrared (FTIR), 1 H nuclear magnetic resonance (NMR)/ 13 C NMR, and HPLC/ESI-MS (electrospray ionization-mass spectrometry). FTIR spectra were recorded on a Nicolet Nexus-670 spectrometer with a Ge/KBr beam splitter and a deuterated triglycine sulfate (DTGS) detector at room temperature. The wavenumber accuracy was 0.01 cm −1 ; 40 scans were recorded at a resolution of 1 cm −1 and averaged for the spectra. 1 H and 13 C NMR spectra were recorded with a Bruker AVIII 500 spectrometer (500 MHz for 1 H and 125 MHz for 13 C), using tetra-methylsilane (TMS) as an internal standard ( δ =0). The values of chemical shifts were given in δ (ppm, 10 −6 ) and coupling constants in J (Hz); 1 H spectra were referenced to the residual dimethyl sulfoxide (DMSO) peak at δ 2.5 and 13 C NMR at δ 39.5. Prior to analysis, samples were dissolved in DMSO- d 6 during the transfer to the NMR tube. Nuclear Overhauser effect (NOE) NMR was recorded with DRX-500 spectrometers (400 MHz for 1 H). ESI-MS spectra were recorded on an Agilent 6460 Triple Quad liquid chromatography (LC)/ESI-MS (VG, Manchester, UK) in both negative and positive modes. Further, purity of compound was assessed by an Agilent 6460 Triple Quad LC/ESI-MS, which was equipped with a Zorbax SB C18 column (250 mm×4.6 mm; 5 μm particle size); samples were detected at 230 nm in a G1314E 1290 VWD UV detector with acetonitrile/H 2 O (60%–0%, v/v) as mobile phase at a flow rate of 1 ml/min. Each fraction during separation was sampled and analyzed by HPLC on an Agilent Technology 1200 series, equipped with an eclipse XDB C18 column (250 mm×4.6 mm, 5 μm particle size) in a G1316C 1290 TCC thermostat-monitored column compartment and G1314B VWD UV detector (samples detected at 230 nm). Plant materials and extraction 2 The flowering aerial biomass of adult E. splendens plants was harvested from a copper-rich site located at Fuyang County, Zhejiang Province, East China, in October 2014. After being naturally air-dried, the leaves and flowers of plants were ground into powder. The powdered E. splendens (2 kg) biomass was extracted with 60% ethanol (3×30 L) at room temperature for one month. After filtration, the filtrate was vacuum-concentrated to yield a crude extract (520 g), which was successively partitioned between H 2 O (3×3000 ml) and petroleum ether (3×3000 ml), EtOAc (3×3000 ml) and H 2 O-saturated n -BuOH (3×3000 ml), yielding deep-brown n -BuOH extract (144 g). Isolation of the compound with MR and Sephadex™ columns 2 The n -BuOH extract (deep-brown) was chromatographed on an MR column of D101 type, and eluted with a gradient H 2 O-EtOH from 90:10 to 5:95, (v/v); each proportion used 4 to 8 L, and afforded yellow-brown extract (20.2 g) from the 70% (v/v, the same below) ethanol-eluted fractionation. Yellow-brown extract was dissolved in 70% MeOH-H 2 O, then chromatographed over a Sephadex™ LH-20 gel column (eluted with 70% and 95% MeOH-H 2 O), giving Fraction 1 (0.58 g, Pellet A). Purification of the compound with P-HPLC and PA 2 Pellet A (0.58 g) was isolated on an Agilent P-HPLC, which was equipped with a Sepax amethyst C18-H column (21.2 mm×250 mm, 5 μm particle size), with 40% MeOH-H 2 O as mobile phase. The partitions that peaked at around 10–15 min were collected (230 nm) and vacuum-concentrated, giving Subfraction 1.1 (0.10 g, Pellet A1) with a purity of 80%. Pellet A1 was subjected to a PA column, and eluted with EtOAc/MeOH (5:1, v/v), affording a compound (yellow powder) from the partitions through the PA column (TLC, R f =0.6, 254 nm) with a purity of 98%. Results and discussion 1 Column chromatography (MR, Sephadex™ LH-20, P-HPLC, and PA) was performed on the n -BuOH extracts of flowering E. splendens biomass, affording a compound (Fig. 1 1 ) with a purity of 98%, as determined by HPLC analyses (Fig. 2 2 ). The chemical structures were elucidated by HPLC/ESI-MS, FTIR, and NMR (Figs. 2 2 – 4 4 ). The compound was a light-yellow amorphous powder; its molecular formula was determined to be C 21 H 20 O 10 as derived from its positive ESI-MS ( m/z 433.2 [M-H] + , 887.3 [2M - Na] + ) and negative ESI-MS ( m/z 431.1 [M - H] − , 863.3 [2M - H] − ) (Fig. 2 2 ), and further confirmed by a 13 C NMR spectrum which has 19 efficient carbon signals (Fig. 4 4 ), indicating the presence of 12 degrees of unsaturation. The peak at m/z 271.1 [M - H] + from positive ESI-MS exhibited the presence of a flavone skeleton (She et al., 2009 2009 ). The UV maxima ( λ max ) (MeOH) were at 267 nm (Band II) and 337 nm (Band I), characteristics of a flavone skeleton (Malikov and Yuldashev, 2002 2002 ). (a) HPLC graph, purity of 98%. (b) ESI-MS spectra, m/z 433.2 [M-H] + and 431.1 [M-H] − , M=432. * Retention time An FTIR spectrum (KBr) (Fig. 3 3 ) exhibited the occurrence of hydroxyl (3423 cm −1 ), a weak peak attributed to a stretching vibration of aromatic C–H (3139 cm −1 ), saturated hydrocarbon (2925 cm −1 ), γ-pyrone carbonyl (1655 cm −1 ), aromatic C=C (1609, 1590, 1510 cm −1 ), aromatic hydroxyl (1371, 1276 cm −1 ), aromatic C–H (1499, 1452 cm −1 ), aromatic rings (1416, 910, 834, 771 cm −1 ), and glycoside C–O (1100, 1082, 1030 cm −1 ) (Berashvili et al., 2005 2005 ). An IR spectrum (KBr) at 3423, 1655, and 1590 cm −1 had the characteristic bands of apigenin. 1 H NMR spectra (Table 1 1 and Fig. 4a 4a ) further confirmed the presence of apigenin and glucose moieties in the structure. The occurrence of an apigenin skeleton was viewed from a hydroxyl δ H at 12.97 (s, OH-5), two doublets δ H at 6.43 (d, J =2.2 Hz, H-6) and δ H at 6.82 (d, J =2.2 Hz, H-8) on the A-ring; A 2 B 2 -type aromatic δ H at 7.95 (d, J =8.9 Hz, H-2', H-6') and δ H at 6.93 (d, J =8.9 Hz, H-3', H-5'), as well as a hydroxyl δ H at 10.51 (s, OH-4') on the B-ring; together with an olefinic δ H at 6.87 (s, H-3) on a flavone C-ring. 1 H NMR data fit exactly with previously reported data in the literature (Shen et al., 1993 1993 ; Berashvili et al., 2005 2005 ). Besides this, glycosidic δ H at 5.44 (d, J =7.4 Hz, H-1'') and δ C at 99.9 (C-1'') were evident in the 1 H and 13 C NMR spectra. The multiplet δ H at 3.27–3.47 (5H, m, H-3''–H-6'') was assignable to the coupling between protons and methylene protons of the glucosyl ring. Proton δ H at 3.71 (m, H-2''), hydroxyl δ H at 5.12 (s, OH-2''), δ H at 5.07 (s, OH-3''), δ H at 5.05 (s, OH-4''), and δ H at 4.65 (s, OH-6'') were assigned in the glycosidic ring. In DMSO-d 6 , δ in ppm, J in Hz. Measured at 125 MHz. Measured at 500 MHz Moity C atom δ C a a δ H b b Aglycone 2 163.4 3 103.5   6.87 (1H, s) 4 182.5 5 157.4 6 100.3   6.43 (1H, d, J =2.2 Hz) 7 164.7 8 95.3   6.82 (1H, d, J =2.2 Hz) 9 161.9 10 105.8 1' 121.4 2' 129.1   7.95 (1H, d, J =8.9 Hz) 3' 116.5   6.93 (1H, d, J =8.9 Hz) 4' 161.6 5' 116.5   6.93 (1H, d, J =8.9 Hz) 6' 129.1   7.95 (1H, d, J =8.9 Hz) 5-OH   12.97 (1H, s, OH-5) 4'-OH   10.51 (1H, s, OH-4') Glucose 1'' 99.9   5.44 (1H, d, J =7.4 Hz) 2'' 73.5   3.71 (1H, d, J =10.3 Hz, H-2'') 3'' 77.6   3.27–3.47 (5H, m, J =6.8 Hz, glucose H-3'', -4'', -5'', -6'') 4'' 69.9 3   5.12 (1H, s, OH-2''), 5.07 (1H, s, OH-3''), 5.05 (1H, s, OH-4''), 4.65 (1H, s, OH-6'') 5'' 76.9 6'' 63.5 Analyses of the 13 C NMR spectrum (Table 1 1 and Fig. 4b 4b ) revealed the existence of 21 carbons, including a hexose moiety at δ C (99.9, 73.5, 77.6, 69.9, 76.9, and 63.5). The 13 C NMR spectrum also exhibited the presence of δ C (157.4, 100.3, 164.7, and 95.3) for the A-ring, δ C (163.4, 103.5, 182.5, 161.9, and 105.8) for the C-ring, and δ C (121.4, 129.1, 116.5, and 161.6) for the B-ring of the flavone. The NOESY NMR spectra suggested an NOE association between the anomeric proton (H-1'') of the glucoside moiety and the H-8 singlet on the A-ring of the aglycone, confirming the attachment of the sugar unit to the 7-OH functional group (Fig. 5a 5a ). Also, the glycosidic linkage at C-7 of the aglycon was viewed from the upfield shift of δ C (C-7) as compared with apigenin, which can be further deduced from the existence of δ C at 99.9 (C-1'') (Table 1 1 and Fig. 4b 4b ). The large H-1'' coupling constant of 6.8 Hz between H-1'' and H-2'' indicated a β-coupled glucose. The glycoside stereochemistry was affirmed by positive NOEs between H-1'', -3'', and -5'' (Fig. 5b 5b ) and by the characteristic 1 H chemical shifts (Fig. 4a 4a ). Therefore, the structure of the purified compound was elucidated as apigenin-7- O -β- D -glycoside, in accordance with the reported data in the literature (Yang et al., 2007 2007 ; Liu et al., 2012 2012 ). Conclusions 1 Apigenin-7- O -β- D -glycoside could be the post-harvesting product from E. splendens biomass. Chromatographic column separation was set up for the preparation and purification of apigenin-7- O -β- D -glycoside in the n -BuOH extracts from the flowering aerial biomass of E. splendens . References DT Berashvili , MD Alaniya , AD Bakuridze , 等 . Apigenin glucuronide from Perilla nankinensis leaves . Chem Nat Compd , 2005 . 41 ( 1 ): 97 - 98 . . Apigenin glucuronide from Perilla nankinensis leaves EJ Choi , GH Kim . In vivo antioxidative characteristics of extracts from the aromatic herb Elsholtzia splendens . Food Sci Biotechnol , 2008 . 17 ( 5 ): 1128 - 1130 . . In vivo antioxidative characteristics of extracts from the aromatic herb Elsholtzia splendens EJ Choi , GH Kim . Effect of Elsholtzia splendens extracts on the blood lipid profile and hepatotoxicity of the mice . Food Sci Biotechnol , 2008 . 17 413 - 416 . . Effect of Elsholtzia splendens extracts on the blood lipid profile and hepatotoxicity of the mice EJ Choi , YS Lee , GH Kim . Antioxidative characteristics of extracts from aromatic herb Elsholtzia splendens . Food Sci Biotechnol , 2007 . 16 489 - 492 . . Antioxidative characteristics of extracts from aromatic herb Elsholtzia splendens EJ Choi , T Kim , GH Kim . Antioxidant effects of Elsholtzia splendens extract on DMBA-induced oxidative stress in mice . Food Sci Biotechnol , 2008 . 17 1341 - 1344 . . Antioxidant effects of Elsholtzia splendens extract on DMBA-induced oxidative stress in mice MS Chung , GH Kim . Effects of Elsholtzia splendens and Cirsium japonicum on premenstrual syndrome . Nutr Res Pract , 2010 . 4 ( 4 ): 290 - 294 . (Available from: http://dx.doi.org/10.4162/nrp.2010.4.4.290 http://dx.doi.org/10.4162/nrp.2010.4.4.290 ) . Effects of Elsholtzia splendens and Cirsium japonicum on premenstrual syndrome M Kikuchi , N Matsuda . Flavone glycosides from Lonicera gracilipes var . glandulosa J Nat Prod , 1996 . 59 ( 3 ): 314 - 315 . (Available from: http://dx.doi.org/10.1021/np960180j http://dx.doi.org/10.1021/np960180j ) . Flavone glycosides from Lonicera gracilipes var DW Kim , KH Son , HW Chang , 等 . Anti-inflammatory activity of Elsholtzia splendens . Arch Pharm Res , 2003 . 26 232 - 236 . . Anti-inflammatory activity of Elsholtzia splendens J Liu , L Chen , S Cai , 等 . Semisynthesis of apigenin and acacetin-7- O -β- D -glycosides from naringin and their cytotoxic activities . Carbohyd Res , 2012 . 357 41 - 46 . . Semisynthesis of apigenin and acacetin-7-O-β-D-glycosides from naringin and their cytotoxic activities VM Malikov , MP Yuldashev . Phenolic compounds of plants of the Scutellaria L. genus. Distribution, structure, and properties . Chem Nat Compd , 2002 . 38 ( 4 ): 358 - 406 . . Phenolic compounds of plants of the Scutellaria L. genus. Distribution, structure, and properties HY Peng , PMH Kroneck , H Kupper . Toxicity and deficiency of copper in Elsholtzia splendens affect photosynthesis biophysics, pigments and metal accumulation . Environ Sci Technol , 2013 . 47 ( 12 ): 6120 - 6128 . . Toxicity and deficiency of copper in Elsholtzia splendens affect photosynthesis biophysics, pigments and metal accumulation HY Peng , Y Xing , LL Gao , 等 . Simultaneous separation of apigenin, luteolin and rosmarinic acid from the aerial parts of the copper-tolerant plant Elsholtzia splendens . Environ Sci Pollut Res , 2014 . 21 ( 13 ): 8124 - 8132 . . Simultaneous separation of apigenin, luteolin and rosmarinic acid from the aerial parts of the copper-tolerant plant Elsholtzia splendens GM She , ZQ Guo , HN Lv , 等 . New flavonoid glycosides from Elsholtzia rugulosa Hemsl . Molecules , 2009 . 14 ( 10 ): 4190 - 4196 . . New flavonoid glycosides from Elsholtzia rugulosa Hemsl CC Shen , YS Chang , LK Ho . Nuclear magnetic resonance studies of 5,7-dihydroxy flavonoids . Phytochemistry , 1993 . 34 ( 3 ): 843 - 845 . . Nuclear magnetic resonance studies of 5,7-dihydroxy flavonoids HP Wang , JF Lu , GL Zhang , 等 . Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas . Environ Toxicol Phar , 2014 . 38 ( 2 ): 453 - 459 . (Available from: http://dx.doi.org/10.1016/j.etap.2014.07.019 http://dx.doi.org/10.1016/j.etap.2014.07.019 ) . Endothelium-dependent and -independent vasorelaxant actions and mechanisms induced by total flavonoids of Elsholtzia splendens in rat aortas XJ Xie , ZB Xu . Elsholtzia haichouensis –an indicator of copper mine . Geol Acta , 1952 . 32 360 - 368 . (in Chinese) . Elsholtzia haichouensis–an indicator of copper mine AM Yang , RH Lu , YP Shi . Study on flavonoids from Lagotis ramalana Batalin . Chin Pharm J , 2007 . 19 1459 - 1461 . (in Chinese) . Study on flavonoids from Lagotis ramalana Batalin Compliance with ethics guidelines Hong-yun PENG, Xue-hong ZHANG, and Jin-zhong XU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500242 Project supported by the National Natural Science Foundation of China (No. 21277121) 2015-10-08 2016-05-12 2015-12-18 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 06 17