Secondary Metabolites from the Barks of Eucommia ulmoides and Their Bioactivities.
Summary: Eucommia ulmoides Oliv., exhibiting diverse bioactivities including antimicrobial, antioxidant, neuroprotective, anti-tumor and anti-inflammatory properties, has long been known as an active ingredient commonly used in antihypertensive herbal prescriptions in China. The 95% ethanol extract of the barks of E. ulmoides showed antimicrobial and antioxidant activities and then was isolated by bioactivity-guided fractionation to obtain a new cinnamyl glucoside, 1-O-trans-cinnamoyl-[beta]-D-apiofuranosyl-(1a6)-[beta]-D-glucopyranose (1), together with twenty known secondary metabolites (2-21). And their structures were identified by extensive spectroscopic analyses, including 1D and 2D NMR, UV, IR and HRESIMS, and chemical methods. All secondary metabolites were evaluated for their antimicrobial and antioxida nt activities in vitro.
Compounds 7, 8, 11, and 21 exhibited moderately antimicrobial activity. In the antioxidant activity assay, compounds 3, 7, 11, and 14 showed intense activity in ferric reducing antioxidant power (FRAP) with the values of 9.82 mmol/g, 19.25 mmol/g, 14.52 mmol/g, and 12.58 mmol/g in comparison to Trolox (9.02 mmol/g). And compounds 2, 11, and 14-16 showed more potent activity with IC50 values ranging from 18.34 to 24.36 uM than Trolox (26.35 uM) in ABTS+* scavenging assay.
Key Words: Eucommia ulmoides Oliver, Secondary metabolites, Structural identification, Antimicrobial activity, Antioxidative activity.
Eucommia ulmoides Oliver (Eucommiaceae), an indigenously important economic tree in central and southern China, is the sole species of the sole genus Eucommia. It has been considered as a living fossil and introduced in other countries, such as Japan, Korea, and America. The dried leaves have been commonly consumed as a health drink (Du-Zhong tea) in China and Japan. The barks (Eucommiae Cortex) are well-known and valued traditional Chinese medicine. Eucommiae Cortex has been used extensively for the treatment of varieties ailments including hypertension, impotence, lumbago and ischialgia . Especially, it is considered to be the best natural antihypertensive medicine without side effects . Previous phytochemical investigations on the genus Eucommia have shown the presence of lignans, phenolics, iridoids and flavonoids, which exhibit a wide range of bioactivities including anti-hypertensive, neuroprotective, anti-tumor, and anti-inflammatory [3, 4].
To our knowledge, minimal studies were focused on antimicrobial and antioxidant activities of Eucommiae Cortex.
Here, we describe the isolation and structure identification of a new cinnamyl glucoside, 1-O-trans-cinnamoyl-[beta]-D-apiofuranosyl-(1a6)-[beta]-D-glucopyranose (1), and twenty known compounds, salidroside (2) , 3,4-dihydroxyphenethyl alcohol-3-O-[beta]-D-glucopyranoside (3) , vanillactic acid (4) , 3'-(4-hydroxy-3-methoxyphenyl)propane-1,2-diol (5) , arbutin (6) , protocatechuic acid (7) , 3,5-dimethoxy-benzyl alcohol 4-O-[beta]-D-glucopyranoside (8) , 2,6-dimethoxy-4-hydroxyphenol-1-O-[beta]-D-glucopyranoside (9) , isosalicin (10) , kaempferol (11) , geniposide (12) , eucomoside B methyl ester (13) , cyclo-olivil (14) , cyclo-olivil 9-O-[beta]-D-glucopyranoside (15) , cyclo-olivil 4'-O-[beta]-D-glucopyranoside (16) , olivil 4-O-[beta]-D-glucopyranoside (17) , olivil 4'-O-[beta]-D-glucopyranoside (18) , arctigenin (19) , pinoresinol 4'-O-[beta]-D-glucopyranoside (20) , and pinoresinol 4,4'-di-O-[beta]-D-glucopyranoside (21)  (Fig. 1).
Among them, compounds 3-5, 8-10, 13, 15, 16, and 19 were obtained from this genus for the first time. In addition, antimicrobial and antioxidant activities of all compounds were evaluated by bioassays in vitro, respectively.
NMR spectra were performed on a Bruker AVANCE-600 spectrometer (Bruker, Rheinstetten, Germany) with tetramethylsilane (TMS) as an internal standard at 600 MHz for 1H-NMR and 150 MHz for 13C-NMR. IR spectra were carried out on a Varian Cary 610/670 FTIR microspectrometer (Varian, Palo Alto, CA, USA) with KBr disk. UV spectra were recorded on a Varian Cary 5000 UV-vis-NIR spectrophotometer (Varian, Palo Alto, CA, USA). HRESIMS spectra were obtained by electrospray ionization (ESI) on a Bruker MaXis ultra-high resolution (UHR) TOF apparatus (Bruker, Bremen, Germany).
TLC was carried out on silica gel plates (Yantai Institute of Chemical Technology, China). Column chromatography (CC) was performed on silica gel (200-300 mesh; Qingdao Marine Chemical Factory) and Sephadex LH-20 (40-70 um, Amersham Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on a LC3000 instrument (Chuangxintongheng, China) with an Alltima (250 mmx10 mm, YMC, Japan) preparative column packed with C18 (5 um).
The barks of E. ulmoides were collected in Jiangxi Province, China, in September 2011. A herbarium sample (No. 20111201) has been deposited in the College of Bioscience and Biotechnology, Yangzhou University, China.
Extraction and Isolation
The air-dried and powdered barks of E. ulmoides (50 Kg) were refluxed with ethanol (95% v/v) three times, 2 h for each. After the removal of the solvent under reduced pressure, the ethanol extract (3.5 kg) was partitioned between petroleum ether and H2O. The H2O layer was passed through a macroporous resin HPD 100 column. After the column washed with H2O, the adsorbed materials were eluted with 95% ethanol. By bioactivity-guided fractionation procedure, the 95% ethanol eluate (1.4 Kg) displayed antimicrobial and antioxidant activities and was chromatographed on silica gel CC, eluting with a gradient of CHCl3-MeOH (100:0a100:5a100:10a100:15a100:20), to give five fractions (F1-F5).
F5 was further divided into four fractions (F5-1-F5-4) through MCI gel CC (MeOH-H2O, 5:95a35:65a55:45a70:30). F5-3 was applied to ODS CC (MeOH-H2O, 15:85a25:75a40:60a60:40) to obtain four fractions F5-3-1-F5-3-4. compound 1 (12 mg, tR 35.7 min) was obtained by preparative HPLC (MeOH-H2O, 35:65, v/v, flow rate of 1.5 mL/min) after being subjected to Sephadex LH-20 with MeOH from F5-3-3. Twenty known compounds 2 (10 mg), 3 (4 mg), 4 (23 mg), 5 (3 mg), 6 (12 mg), 7 (14 mg), 8 (8 mg), 9 (5 mg), 10 (6 mg), 11 (35 mg), 12 (351 mg), 13 (75 mg), 14 (53 mg), 15 (32 mg), 16 (32 mg), 17 (124 mg), 18 (131 mg), 19 (25 mg), 20 (121 mg), and 21 (15 g) were obtained by MCI CC (MeOH-H2O), Sephadex LH-20 CC (MeOH), ODS CC (MeOH-H2O), and PHPLC (MeOH-H2O or MeCN-H2O) form F2-F4, respectively.
White amorphous powder (12 mg), IR (KBr) cm-1: 3501 (OH), 1724 (C=O), 1617 and 1584 for aromatic system; UV I>>max (EtOH) nm (log Iu): 206 (3.46), 216 (3.72), 275 (3.54);The 1H-NMR and 13C-NMR data, Table-1; HRESIMS: m/z 465.1376 [M+Na]+ (calcd. for C20H26O11, 465.1373).
Table-1: 1H- (600 MHz) and 13C- (150 MHz) NMR spectral data of 1 in DMSO-d6 (in I' ppm).
###Position###I' H###I' C###Position###I' H###I' C
###2###7.63 (m)###129.4###3'###3.46 (m)###78.0
###3###7.42 (m)###130.1###4'###3.36 (m)###71.4
###4###7.42 (m)###131.8###5'###3.57 (m)###77.7
###3.99 (dd, J = 2.1, 11.4 Hz)
###3.62 (dd, J = 5.9, 11.4 Hz)
###6###7.63 (m)###129.4###1"###4.97 (d, J = 2.3 Hz)###111.0
###7###6.58 (d, J = 16.0 Hz)###147.7###2"###3.90 (d, J = 2.3 Hz)###78.0
###8###7.81 (d, J = 16.0 Hz)###118.6###3"###-###80.6
###3.97 (d, J = 9.7 Hz)
###3.75 (d, J = 9.7 Hz)
###1'###5.58 (d, J = 7.7 Hz)###95.9###5"###3.58 (br.s)###65.7
Acid hydrolysis of 1
The acid hydrolysis reaction was performed according to the method described by Jiang et al. with some modifications . Briefly, A solution of compound 1 in H2O (4 mL) was hydrolyzed with HCl (2 N, 0.5 mL) at 80AdegC for 2 h. The reaction mixture was extracted with EtOAc (4 x 4 mL) to yield the EtOAc extract and H2O phase after removing the solvents. The aqueous phase of the hydrolysate was dissolved in pyridine (0.15 mL), and then L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.2 mL) was added to this solution. The mixtures were heated at 70AdegC for 2 h, and 0.15 mL of TMS was added, followed by heating at 70AdegC for 2 h. The dried products were partitioned with petroleum ether and H2O (0.2 mL each), and the organic layer was analyzed by GC. By comparing of retention times, sugars in compound 1 were identified as D-glucose and D-apiose.
Evaluation of Antimicrobial and Antioxidant Activities
The antimicrobial activities of the compounds were assayed according to the procedures as previously described . The samples were dissolved in DMSO at a concentration of 4.5 mg/mL (95% ethanol eluate) and 0.2 mg/mL (compounds). Strains including four species of bacteria [Bacillus subtilis (1790), Staphylococcus aureus (3103), Escherichia coli (1672) and Salmonella typhi (733)] and three species of fungi [Alternaria alternate (DSM 2006), Aspergillus niger (ATCC 6275), Fusarium oxysporum (ITCC 6246)] were used for test. Rifampicin and Nystatin were used as positive controls for antibacterial and antifungal activities, respectively.
The ABTS radical cation scavenging activity of the compounds was determined according to the procedures as previously described . Trolox was used as the positive control. Free radical scavenging rate (%) = [(OD control - OD sample) / (OD control - OD blank)]x100 %.
The FRAP assay was performed referring to the procedures as previously described . Trolox was used as the positive control. A calibration curve was established using FeSO4 at different concentrations from 0.15 to 5.00 mM. FRAP values were calculated based on the standard curve and expressed as millimoles of FeSO4 per gram of samples (mmol/g).
Each concentration of test compounds was performed in triplicate. The results of antimicrobial activity were presented as the mean values of the diameters of the inhibitory zones from three runs. All values of antioxidant activity tests were expressed as mean +- SD of three parallel measurements. Analysis of variance was performed by ANOVA procedure in SPSS 20.0 software package, and p 100###9.82+-0.01###15###20.13+-0.08###6.31+-0.01
Compounds 2-21 were identified and confirmed by comparison with the reported data.
The 95% ethanol eluate and isolated compounds 1-21 (purity >95%) were screened for their antioxidant and antimicrobial activities in vitro. Among them, some exhibited apparently biological activity as shown in Table-2 and Table-3.
The ethanol eluate showed antioxidant and antimicrobial activities, which indicated the potential presence of bioactive secondary metabolites in the E. ulmoides. Compounds 7, 8, 11, and 21 exhibited antimicrobial activity (Table-2). Noticeably, compound 21 may be developed as a potential antimicrobial agent due to its antibacterial and antifungal activities. Compounds 2, 7, 11, and 14-16 showed ABTS+* scavenging activity with IC50 values ranging from 20.13 to 86.41 uM (Table-3). In particular, the activity of compounds 2, 11, and 14-16 were more potent than that of the positive control Trolox (26.35 uM). In the FRAP assay, compounds 2, 3, 7, 9, 11, 14-16, and 20 had antioxidant activity in the range of 2.49 to 19.25 mmol/g (Table-3), among them, 3, 7, 11, and 14 showed stronger antioxidant activity than that of the positive control Trolox (9.02 mmol/g). These results indicated that polyphenols were involved in the antimicrobial and antioxidant effects of E. ulmoides Oliver.
This work was supported by Students Science and Technology Innovation Fund of Yangzhou University (x20160738), Jiangsu Students' Project for innovation and entrepreneurship training program (201711117085X) and open foundation of Green Pesticides and Biotechnology Key Laboratory of Guizhou University of Ministry of Education (2017GDGP0102/2017GDGP0103). The research work was partially supported by program for the Analytical Detective Center, Yangzhou University.
1. X. He, J. Wang, M. Li, D. Hao, Y. Yang, C. Zhang, R. He and R. Tao, Eucommia ulmoides Oliv.: Ethnopharmacology, Phytochemistry and Pharmacology of an Important Traditional Chinese Medicine, J. Ethnopharmcol., 151, 78 (2014).
2. S. Y. Hu, A Contribution to Our Knowledge of Tu-chung-Eucommia ulmoides, Am. J. Chin. Med., 7, 5 (1979).
3. X. Chai, Y. Wang, Y. Su, A. J. Bah, L. Hu, Y. Gao and X. Gao, A Rapid Ultra Performance Liquid Chromatography-Tandem Mass Spectrometric Method for the Qualitative and Quantitative Analysis of Ten Compounds in Eucommia ulmodies Oliv., J. Pharm. Biomed. Anal., 57, 52 (2012).
4. S. H. Kwon, H. K. Lee, J. A. Kim, S. I. Hong, S. Y. Kim, T. H. Jo, Y. I. Park, C. K. Lee, Y. B. Kim, S. Y. Lee and C. G. Jang, Neuroprotective Effects of Eucommia ulmoides Oliv. Bark on Amyloid Beta25-35-Induced Learning and Memory Impairments in Mice, Neurosci. Lett., 487, 123 (2011).
5. Y. Yang, N. J. Yu, Y. Zhang, F. X. Ren, R. Xu, J. L. Xu and Y. M. Zhao, Chemical Constituents from Leaves of Loropetalum chinense, Chin. Pharm., J. 50, 205 (2015).
6. W. K. Whang, Aromatic Compounds and Triterpenoidal Saponins from Clematis koreana var. umbrosa, Arch. Pharm. Res., 17, 5 (1994).
7. L. Tang, X. F. Li, S. X. Yang, Y. Qiu and K. Yuan, Chemical Constituents of Hyptis rhomboidea and Their Antifungal Activity, Zhongguo Zhong Yao Za Zhi, 39, 2284 (2014).
8. H. Kikuzaki, S. Hara, Y. Kawai and N. Nakatani, Antioxidative Phenylpropanoids from Berries of Pimenta dioica, Phytochemistry, 52, 1307 (1999).
9. W. Wang and W. Y. Kang, Chemical Constituents from Ainsliaea bonatii, Chin. Pharm., J. 48, 174 (2013).
10. W. G. Jing, Z. M. Wang, Y. Zhao, J. Fu, X. L. Zhao and A. Liu, Chemical Constituents from Seeds of Brassica Campestris, Zhongguo Zhong Yao Za Zhi, 39, 2521 (2014).
11. K. M. Park, M. C. Yang, K. H. Lee, K. R. Kim, S. U. Choi and K. R. Lee, Cytotoxic Phenolic Constituents of Acer tegmentosum Maxim, Arch. Pharm. Res., 29, 1086 (2006).
12. S. K. Liu, S. Que, W. Cheng, Q. Y. Zhang and H. Liang, Chemical Constituents from Whole Plants of Carduus acanthoides, Zhongguo Zhong Yao Za Zhi, 38, 2334 (2013).
13. K. Kurashima, M. Fujii, Y. Ida and H. Akita, Simple Synthesis of [beta]-D-Glycopyranosides Using [beta]-Glycosidase from Almonds, Chem. Pharm. Bull., 52, 270 (2004).
14. X. Zhang, Y. M. Ma, D. M. Wang, J. J. Liu and Y. X. Kang, Chemical Constituents from Geum aleppicum (I), J. Northwest Forestry Univ., 27, 124 (2012).
15. C. J. Cai, Z. L. Zhang, Y. M. Zuo, Y. Y. Zhu, G. M. Luo and J. Zhang, Studies on the Chemical Components of Iridoids of Gardenia jasminoides Ellis., Lishizhen Med. Mater. Med. Res., 24, 342 (2013).
16. C. Takamura, T. Hirata, T. Ueda, M. Ono, H. Miyashita, T. Ikeda and T. Nohara, Iridoids from the Green Leaves of Eucommia ulmoides, J. Nat. Prod., 70, 1312 (2007).
17. F. Abe, T. Yamauchi and A. S. C. Wan, Lignans Related to Olivil from Genus Cerbera (Cerbera. VI), Chem. Pharm. Bull., 36, 795 (1988).
18. X. G. Yan, J. M. Jia, L. Tang, L. Y. Shi, Y. Q. Wang and B. M. Feng, New Chemical Constituents of Roots of Urtica triangularis Hand-Mass, Chem. Pharm. Bull., 56, 1463 (2008).
19. M. Suglyama, E. Nagayama and M. Kikuchi, Lignan and Phenylpropanoid Glycosides from Osmanthus asiaticus, Phytochemistry, 33, 1215 (1993).
20. M. M. A. Rahman, P. M. Dewick, D. E. Jackson and J. A. Lucas, Lignans of Forsythia intermedia, Phytochemistry, 29,1971 (1990).
21. W. W. Huang, D. Y. Kong and P. M. Yang, Studies on Lignan Constituents of Clematis armandii Franch., Chin. J. Nat. Med., 1, 199 (2003).
22. N. X. Nhiem, P. V. Kiem, C. V. Minh, N. Kim, S. Park, H. Y. Lee, E. S. Kim, Y. H. Kim, S. Kim, Y. S. Koh and S. H. Kim, Diarylheptanoids and Flavonoids from Viscum album Inhibit LPS-Stimulated Production of Pro-inflammatory Cytokines in Bone Marrow-Derived Dendritic Cells, J. Nat. Prod., 76, 495 (2013).
23. Q. Ma, H. Xie, S. Li, R. Zhang, M. Zhang and X. Wei, Flavonoids from the Pericarps of Litchi chinensis, J. Agric. Food Chem., 62, 1073 (2014).
24. I. F. Palici, E. Liktor-Busa, I. Zupko, B. Touzard, M. Chaieb, E. Urban and J. Hohmann, Study of in Vitro Antimicrobial and Antiproliferative Activities of Selected Saharan Plants, Acta Biol. Hung., 66, 385 (2015).
25. T. Fukunaga, I. Kajikawa, K. Nishiya, K. Takeya and H. Itokawa, Studies on the Constituents of the Japanese Mistletoe, Viscum album L. var. coloratum Ohwi Grown on Different Host Trees, Chem. Pharm. Bull., 37, 1300 (1989).
|Printer friendly Cite/link Email Feedback|
|Author:||Liu, Wen-Yan; Dong, Zi-Ling; Li, Ke-Huan; Shao, Jian-Hua; Shen, Jie; Zhao, Chun-Chao; Kang, Xing-Don|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Feb 28, 2019|
|Previous Article:||Angiotensin-I Converting Enzyme Inhibitory Peptides from Sweet Sorghum Grain Protein: Optimisation of Hydrolysis Conditions and Hydrolysate...|
|Next Article:||Synthesis and Evaluation of Some Novel 6-Substituted Quinazoline Derivatives as Antitumor Agents.|