Chinese Journal of Natural Medicines  2019, Vol. 17Issue (2): 145-148  
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Cite this article as: 

CAO Duo, WANG Li-Qing, HAN Xiao-Min, GUAN Hui-Rui, LEI Meng, WEI Ya-Hui, CHENG Liang, YANG Pei-Ming, SUN Zheng-Liang, GAO Wen, DAI Jia-Kun. Two symmetrical unsaturated acids isolated from Viscum album[J]. Chinese Journal of Natural Medicines, 2019, 17(2): 145-148.
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Research funding

This work was supported by the National Science and Technology Development Fund (No. 2009ZX09301-007)

Corresponding author

peimingy@163.com (YANG Pei-Ming)
E-mails: sunzl6@126.com (SUN ZhengLiang)

Article history

Received on: 20-Oct-2018
Available online: 20 February, 2019
Two symmetrical unsaturated acids isolated from Viscum album
CAO Duo1,2 , WANG Li-Qing1 , HAN Xiao-Min1 , GUAN Hui-Rui1 , LEI Meng1 , WEI Ya-Hui1 , CHENG Liang2 , YANG Pei-Ming2 , SUN Zheng-Liang3 , GAO Wen4 , DAI Jia-Kun5     
1 College of Life Science, Northwest University, Xi'an 710069, China;
2 State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai 201203, China;
3 Shanghai University of Medicine & Health Sciences Affiliated Sixth People's Hospital South Campus, Shanghai 201499, China;
4 Shineway Pharmaceutical Group Co., Ltd., Shijiazhuang 051430, China;
5 Bio-Agricultue Institute of Shaanxi, Shaanxi 710043, China
[Abstract]: In the present study, two new acetylene conjugate compounds, dibutyl (2Z, 6Z)-octa-2, 6-dien-4-yne dioate (1), and dibutyl (2E, 6E)- octa-2, 6-dien-4-yne dioate (2), were isolated from the dry stem leaves of Viscum album, along with nine known compounds (3-11). Their structures were confirmed on the basis of spectroscopic data. Compounds 1 and 8 showed antioxidant activity against xanthine oxidase (XOD) and 1, 1-diphenyl-2-picrylhydrazyl radical 2, 2-diphenyl-1-(2, 4, 6-trinitrophenyl) hydroxyl (DPPH), with the IC50 of 1.22 and 1.33 μmol·L-1, and the SC50 of 4.34 and 8.22 μmol·L-1, respectively.
[Key words]: Viscum album     Symmetrical unsaturated acid     Antioxidant activity    
Introduction

As a traditional Chinese medicine, Viscum album (Loran thaceae), commonly named mistletoe, is widely used for the treatment of cardiovascular diseases. Intravenous injection of ethanol extract of mistletoe in dogs and rabbits can maintain antihypertensive effect for 3 min, which could be extended to 1 h with intraperitoneal injection, indicating that the ethanol extract of mistletoe has a significant antihypertensive effect [1]. A crude ethanolic extract of mistletoe has been found to have vasomotor reactivity of superfused rat aortic rings (with or without a functional endothelium) [2]. These results indicate that the ethanolic extract of mistletoe induces predominantly an endothelium-dependent relaxation, which seems to be mediated by the synthesis/release of nitric oxide [2]. However, the active compounds are not clearly identified. In the present study, we carried out a detailed phytochemical investigation on the aerial part of Viscum album, leading to the isolation of two new acetylene conjugate compounds, dibutyl (2Z, 6Z)-octa-2, 6-dien-4-yne dioate (1), dibutyl (2E, 6E)-octa-2, 6-dien-4-yne dioate (2), as well as nine known compounds 311. The antioxidant activities of these compounds were also evaluated to explore the pharmacological mechanism of action of these compounds.

Results and Discussion

Compound 1 had a molecular formula of C16H22O4 from the quasimolecular ion peak in positive HR-ESI-MS at m/z 279.0937 [M + H] + (Calcd. for C16H23O4, 279.15). Its IR spectrum showed absorption band for alkynyl (2300 cm–1) and carbonyl (1728.4 cm–1) groups. The 1H NMR spectrum revealed one methyl group at δH 0.91 (3H, t), three methylene groups at 1.35 (2H, m), 1.65 (2H, m) and 4.23 (2H, t), and two protons of cis alkene at 7.68 (1H, d, 10 Hz) and 7.70 (1H, d, 10 Hz). The 13C NMR spectrum DEPT revealed eight carbon signals for C-atoms from, one methyl (δC 13.6), three methylene (δC 18.7, 30.0 and 65.1), two alkenyls (128.7 and 131.6), one alkynyl (δC 91.7) and one carbonyl (δC 167.0), indicating the existence of symmetric skeleton. The assignments of the 13C NMR data were confirmed by 2D NMR experiments. The HMBC spectrum showed correlations of H-2 to C-1, C-3 and C-4; of H-3 to C-2 and C-4; of H-6 to C-5 and C-7; and of H-7 to C-5, C-6 and C-8 (Fig. 1). Based on the above evidences, the structure of compound 1 was elu ci dated as (2Z, 6Z)-dibutyl octa-2, 6-dien-4-yne dioate (Fig. 3).

Fig. 1 Key 1H-1H COSY and HMBC correlations of 1 and 2
Fig. 2 ORTEP view of compounds 1 and 2
Fig. 3 The chemical structures of compounds 1–11 obtained from Viscum album

Compound 2 was isolated as a colorless amorphous solid, the HR-ESI-MS spectrum (m/z 279.1402 [M –H]), suggested the molecular formula C16H22O4. The 1H and 13C NMR spectrum (Table 1) of Compound 2 showed that its structure was similar to that of 1, except for the relative configuration of the vinyl group. In the 1H NMR spectrum of 2, two hydrogen groups of alkenes at 7.66 (1H, d, 15.6 Hz) and 7.69 (1H, d, 15.6 Hz) were shown, indicating the presence of trans alkene. Thus, the structure of compound 2 was determined to be (2E, 6E)-dibutyl octa-2, 6-dien-4-yne dioate (Fig. 3).

Table 1 1H NMR(600 MHz) and 13C NMR(150 MHz) spectro scopic data of compound 1 and 2 in DMSO-d6 (J in Hz)

According to the reported data, the nine known compounds (Fig. 3) were identified as rhamnazine-3-O-β-D- apiofuranosyl(1→2)-[6''-(3-oxhydryl-3-methylgluaryl)]-O-β- D-glucopyranoside(3) [5], liquidamboside (4) [3-4], homerio dictyol-7-O-β-D-glucopyranoside-4′-O-β-D-apioside (5) [5], home riodictyol-7-O-β-D-apiofuranosyl (1→2)-O-β-D- glu copy rano side (6) [6-7], rhamnazin-3-O-β-D-glucopyranoside (7) [8-9], rhamnazine (8) [7-8], rhamnazin-3-O-β-D-(6″-acetyl)- O-β-D-glucopyranoside (9) [8-9] homeriodictyol-7-O-β-D-gluco pyrano side (10) [5, 8], and eriodictyol-7-O-β-D-glucopyrano side (11) [5].

Compounds 111 were evaluated for their antioxidant activity against xanthine oxidase (XOD) and 1, 1-Diphenyl-2- picrylhydrazyl radical2, 2-Diphenyl-1-(2, 4, 6- trinitrophenols) hydroxyl (DPPH). Compounds 1 and 8 demonstrated significant inhibitory activities against DPPH, with the SC50 of 4.34 and 8.22 μmol·L–1 respectively. They also displayed moderated inhibitory effects on XOD, with the IC50 of 1.22 and 1.33 μmol·L–1, respectively (Table 2). All these compounds showed antioxidant activity against XOD and DPPH at 12 μmol·L–1.

Table 2 Antioxidant activity against XOD and DPPH of com pounds 1–11
Experimental General

IR spectra were recorded on a Bruker Vector 22 spectrometer (Bruker Beijing Technology Co., Ltd., Beijing, China) with KBr disks. NMR spectra were measured on a Bruker AV-600 spectrometer with TMS as the internal standard. UV spectra were recorded on a Shimadzu UV-2550 spectrophotometer. ESI-MS were acquired on an Agilent LC-MSD Trap XCT mass spectrometer, whereas HR-ESI-MS were measured using a Waters Q-TOF micro mass spectrometer. Analytical HPLC was carried out with a Waters 515/2487 instrument and a Chiralpak Semi-preparative HPLC was conducted on a Waters 510/484 instrument with a YMC-Pack ODS-A column (5 μm, 10.0 mm × 250 mm). Materials for column chromatography were silica gel (200–300 and 300–400 mesh; Huiyou Silical Gel Development Co.), Sephadex LH-20 (40–70 μm; Amersham Pharmacia Biotech), and RP-18 silica gel (Greenherbs Sci & Tech Development Co.). All other chemicals used in the present study were of analytical grade.

Plant materials

The dry stem and leaves of Viscum album were collected in Anhui Province, China, in June 2014, and were identified by Dr. WU Tong, Department of Traditional Chinese Medicine, Shanghai Institute of Pharmaceutical Industry, Shanghai, China. A voucher specimen has been deposited in the Department of Traditional Chinese Medicine, Shanghai Institute of Pharmaceutical Industry (#14031901).

Extraction and isolation

The dry stem and leaves of Viscum album were extracted with 95% ethanol under refluxing four times, 2 h each. After evaporation of the solvent, the extract was diluted with water (4.0 L) and then partitioned three times with CHCl3 (1.8 L). The fluid extract of water diluted by 3.8 L of water, was subjected to column chromatography (CC) over macroporous resin (D101) and eluted with a mixture of water–ethanol (0%, 10%, 30%, 50%, 70%, and 95% ethanol) to give 6 major fractions on the basis of TLC (water, 10%, 30%, 50%, 70%, 95% ethanol). The fraction of 50% ethanol eluted was subjected to column chromatography (CC) over silica gel (200–300 mesh, 2.0 kg, 10 cm × 120 cm) eluted with a mixture of ethyl acetate–MeOH (100: 0 to 0: 100) to afford 5 subfractions on the basis of TLC (Frs. B1–B5). Fr. B3 (23.0 g) was re-chromato graphed on silica gel CC (200–300 mesh, 600 g, 5 cm × 80 cm) eluted with a mixture of EAOAc–MeOH (50: 1 to 1: 1) to afford 7 subfractions on the basis of TLC (Frs. B 3-1–B3-7). Fr. B3-4 (7 g) was further separated by repeated silica gel (300–400 mesh, 120 g, 2.5 cm × 60 cm) chromatogry (CHCl3–MeOH, 18: 1 to 1: 1) and purified by Sephadex LH-20 (100 g, 3.0 cm × 100 cm, MeOH, 800 ml) to give 5 (18.3 mg), 6 (23.7 mg), 8 (20.6 mg), and 11(13.5 mg). Fr. B3-5 (5.8 g) was subjected to an RP-18 column (180 g, 5.0 cm × 60 cm) eluted with H2O–MeOH (100% to 30%, 1800 mL) to obtain 3 (8.9 mg, 200–300 mL), 4 (14.3 mg, 600–900 mL), and 7 (17.4 mg, 1200–1400 mL). Fr. B3-7 (8.2 g) was subjected to silica gel CC (300–400 mesh, 200 g, 2.5 cm × 120 cm) eluted with acetate–MeOH (30 : 1 to 1 : 1), and finally purified by semi-preparative HPLC (MeOH–H2O, 40 : 60, 3.0 mL·min–1) to give 1 (6.0 mg, tR = 12.5 min), 2 (8.2 mg, tR = 18.9 min), 9 (7.9 mg, tR = 26.7 min), and 10 (6.3 mg, tR = 33.5 min).

Antioxidant activity

The antioxidant activity of compounds 111 (> 90% purity) were measured in vitro on XOD and DPPH. In the xanthine oxidase (XOD) inhibitory activity assay, added 100 μL of the samples to be tested, 50 μL of 0.08 U·mL−1 XOD solution to the 96-well plate, and used PB as a blank control, incubated for 3 min at 37 ℃, and added 50 μL of 0.48 mmol·L−1 XA solution to start the reaction at 295 nm. The readings were taken every 15 s and the absorbance values were recorded for a total of 10 min. Four sets of holes were arranged in parallel for each group. The XOD inhibition rate of the compound was calculated according to the following formula: XOD inhibitory activity (%) = [Ablank-Asample] /Ablank×100% Ablank is the absorbance value of the control group; Asample is the absorbance value of the sample group

In the DPPH free radical scavenging activity assay, 100 μL of the samples to be tested, DPPH (0.1 mmol·L−1) 100 μL were added to the 96-well plate, and the absorbance was measured at 517 nm after 30 min in the dark. Three replicate wells were set in parallel for each group. Methanol was used as a blank control. The DPPH free radical scavenging rate of the compound was calculated according to the following formula:

DPPH clearance (%)= [Ablank-Asample] /Ablank×100%

Ablank is the absorbance value of the control group; Asample is the absorbance value of the sample group

According to the concentration and the correspond-ding inhibition and clearance rate, half of the inhibitory concentration (IC50) and clear concentration (SC50) were calculated by GraphPad Prism 6.0.2. All the assays were performed in duplicate. Quercetin and allopurinol (purity > 95%) are used as positive controls.

Acknowledgements

We thank Prof. WU Jian-Jun (Shanghai Institute of Metrology and Testing, SIMT) for NMR spectral measurements.

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