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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2017, Vol. 11 Issue (2) : 253-265     DOI: 10.1007/s11684-017-0508-9
RESEARCH ARTICLE |
Screening for main components associated with the idiosyncratic hepatotoxicity of a tonic herb, Polygonum multiflorum
Chunyu Li1,2, Ming Niu1, Zhaofang Bai1, Congen Zhang1, Yanling Zhao1, Ruiyu Li1, Can Tu1, Huifang Li7, Jing Jing3, Yakun Meng1, Zhijie Ma1,4, Wuwen Feng1, Jinfa Tang1, Yun Zhu3, Jinjie Li6, Xiaoya Shang6, Zhengsheng Zou5, Xiaohe Xiao3(), Jiabo Wang1()
1. China Military Institute of Chinese Medicine, 302 Military Hospital, Beijing 100039, China
2. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, China
3. Integrative Medical Center, 302 Military Hospital, Beijing 100039, China
4. Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China
5. Diagnosis and Treatment Center for Non-infectious Diseases, 302 Military Hospital, Beijing 100039, China
6. Beijing Union University, Beijing 100101, China
7. Shanxi University of Traditional Chinese Medicine, Taiyuan 030619, China
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Abstract  

The main constituents of a typical medicinal herb, Polygonum multiflorum (Heshouwu in Chinese), that induces idiosyncratic liver injury remain unclear. Our previous work has shown that cotreatment with a nontoxic dose of lipopolysaccharide (LPS) and therapeutic dose of Heshouwu can induce liver injury in rats, whereas the solo treatment cannot induce observable injury. In the present work, using the constituent “knock-out” and “knock-in” strategy, we found that the ethyl acetate (EA) extract of Heshouwu displayed comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats. Results indicated a significant elevation of plasma alanine aminotransferase, aspartate aminotransferase, and liver histologic changes, whereas other separated fractions failed to induce liver injury. The mixture of EA extract with other separated fractions induced comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats. Chemical analysis further revealed that 2,3,5,4'-tetrahydroxy trans-stilbene-2-O-β-glucoside (trans-SG) and its cis-isomer were the two major compounds in EA extract. Furthermore, the isolated cis-, and not its trans-isomer, displayed comparable idiosyncratic hepatotoxicity to EA extract in LPS-treated rats. Higher contents of cis-SG were detected in Heshouwu liquor or preparations from actual liver intoxication patients associated with Heshouwu compared with general collected samples. In addition, plasma metabolomics analysis showed that cis-SG-disturbing enriched pathways remarkably differed from trans-SG ones in LPS-treated rats. All these results suggested that cis-SG was closely associated with the idiosyncratic hepatotoxicity of Heshouwu. Considering that the cis-trans isomerization of trans-SG was mediated by ultraviolet light or sunlight, our findings serve as reference for controlling photoisomerization in drug discovery and for the clinical use of Heshouwu and stilbene-related medications.

Keywords Polygonum multiflorum      idiosyncratic hepatotoxicity      metabolomics      stilbene      cis-transisomerization     
Corresponding Authors: Xiaohe Xiao,Jiabo Wang   
Just Accepted Date: 22 January 2017   Online First Date: 17 March 2017    Issue Date: 01 June 2017
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0508-9     OR     http://academic.hep.com.cn/fmd/EN/Y2017/V11/I2/253
Fig.1  Ultraviolet light mediated cis-isomerization of trans-stilbenes. Ultraviolet light mediated cis-isomerization of trans-SG. (A) 2,3,5,4'-tetrahydroxy trans-stilbene-2-O-β-glucoside (trans-SG) can be transformed into its cis-isomer (cis-SG) by ultraviolet light or sunlight. (B) Reference substances oftrans-SG and cis-SG. (C) Evident transformation of cis-SG was found in the Heshouwu herbal liquor used by an actual DILI patient. (D) Low content of cis-SG was found in the herb liquor of a regular raw material of Heshouwu; however, it could be induced to produce high content of cis-SG by sunlight (E). Exposure to ultraviolet light (365 nm) marks of cis-isomerization of pure trans-SG compound in solution was found in quartz, transparent glass or transparent polyethylene bottles (F, G and H) whereas no transformation was found in light shielding or brown glass bottles (I and J).
Fig.2  Stilbene-containing ethyl acetate (EA) extract was demonstrated as the major hepatotoxic fraction in Heshouwu. (A) Framework of constituent “knock-out” strategy. (B) Liquid chromatograms showing nearly complete separation of compounds with different polarities in the different extracting fractions from the whole extract of Heshouwu (PM). CH, the chloroform extract; EA, the ethyl acetate extract; and RE, the residue. (C) Liver dysfunction was indicated by significantly elevated serum ALT activities in either LPS+/PM+ or LPS+/EA+ rat groups (n = 9). (D) Framework of constituent “knock-in” strategy. (E) Liquid chromatograms showing stepwise increased contents of the EA extract in the mixture of different fractions. (F) Liver dysfunction indicated by significantly elevated serum ALT activities was observed only in LPS/RE+ CH+ 100%EA rat group (n = 9). *, P<0.05, compared with the normal control (LPS/drug) group; #, P<0.05, compared with the model control (LPS+/drug) group.
Fig.3  cis-SG was demonstrated as the major hepatotoxic compound in the EA extract. (A) Hepatocyte injury is indicated by elevated serum ALT and AST activities only in response to cis-SG and the EA extract in LPS-treated rats (n = 9). The dosages of cis-SG,trans-SG, and emodin glycoside (EG) were 30, 200, and 20 mg/kg, respectively, and were set comparable with their contents in the EA extract. (B) LPS/cis-SG treatment induced histological liver lesions whereas no visible histological changes were observed in other groups. (C) Significant increases of the serum TNF-α, IL-6, and IFN-g were observed in the LPS/cis-SG group, compared with the model control group. *, P<0.05, compared with the normal control (LPS/drug) group; #, P<0.05, compared with the model control (LPS+/drug) group.
Fig.4  Results of multivariate statistical analysis. PCA score plot derived from the LC-MS analysis of rats from control (Con), LPS, LPS/trans-SG, and LPS/cis-SG groups in ESI+ mode (A) and ESI mode (B). (C)–(F) are the results of multivariate statistical analysis derived from LC-MS metabolite profiles of LPS/trans-SG and LPS/cis-SG in ESI+ mode. (C) Score plot of LPS/trans-SG and LPS/cis-SG from PCA in ESI+ mode, PC1 versus PC2. (D) PLS-DA score plot of LPS/trans-SG and LPS/cis-SG displayed with the first two components. (E) Loading plot. Differentially expressed metabolites were cycled in a red square. (F) 100-permutation test of PLS-DA model (LPS/trans-SG group in blue and the LPS/cis-SG group in green).
No.R.T. (min)Potential biomarkersMassFormulaContent variancea
ESI+
10.90D-arginine174.1124C6H14N4O2
21.05Pantothenic acid219.1108C9H17NO5
31.383-hydroxyanthranilic acid153.0451C7H7NO3
43.57Kynurenic acid189.0443C10H7NO3
54.06Phenylacetylglycine193.0733C10H11NO3
68.024-imidazolone-5-propionic acidb156.0532C6H8N2O3
78.25Prostaglandin D3340.1891C20H30O5
813.37D-alanyl-D-alanine160.0893C6H12N2O3
913.37Diaminopimelic acid190.0985C7H14N2O4
1013.89Fexofenadineb501.2817C32H39NO4
1117.38Cortol368.2514C21H36O5
ESI
11.02D-ornithine132.0853C5H12N2O2
21.064-pyridoxic acid183.0502C8H9NO4
31.08Isocitric acid192.0224C6H8O7
41.33Heptanoic acid130.0968C7H14O2
51.55L-aspartyl-4-phosphate213.0031C4H8NO7P
64.32Pyridoxine 5′-phosphate249.0415C8H12NO6P
75.75Uridine122.0321C9H12N2O6
87.954-imidazolone-5-propionic acidb156.0535C6H8N2O3
911.83Uridine 5′-diphosphate464.0267C9H14N2O12P2
1013.89Fexofenadineb501.2769C32H39NO4
Tab.1  Identification and trends of change for potential biomarkers
Fig.5  Schematic of the disturbed enriched pathway related to LPS/cis-SG treatment. The notations are as follows: (↑) in red, metabolite higher in LPS/cis-SG treated group than in LPS/trans-SG group; (↓) in blue, metabolite lower in LPS/cis-SG treated group than in LPS/trans-SG group. The related enriched pathways are cycled in a black box [39–41]; ALT, glutamate pyruvate transaminase; AST, aspartate aminotransferase; Arg & Orn metabolism, arginine and ornithine metabolism.
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