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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
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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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:     OR
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
21.05Pantothenic acid219.1108C9H17NO5
31.383-hydroxyanthranilic acid153.0451C7H7NO3
43.57Kynurenic acid189.0443C10H7NO3
68.024-imidazolone-5-propionic acidb156.0532C6H8N2O3
78.25Prostaglandin D3340.1891C20H30O5
913.37Diaminopimelic acid190.0985C7H14N2O4
21.064-pyridoxic acid183.0502C8H9NO4
31.08Isocitric acid192.0224C6H8O7
41.33Heptanoic acid130.0968C7H14O2
64.32Pyridoxine 5′-phosphate249.0415C8H12NO6P
87.954-imidazolone-5-propionic acidb156.0535C6H8N2O3
911.83Uridine 5′-diphosphate464.0267C9H14N2O12P2
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.
1 Stickel F, Patsenker E, Schuppan D. Herbal hepatotoxicity. J Hepatol 2005; 43(5): 901–910 
doi: 10.1016/j.jhep.2005.08.002 pmid: 16171893
2 Stedman C. Herbal hepatotoxicity. Semin Liver Dis 2002; 22(2): 195–206 
doi: 10.1055/s-2002-30104 pmid: 12016550
3 Teschke R, Eickhoff A. Herbal hepatotoxicity in traditional and modern medicine: actual key issues and new encouraging steps. Front Pharmacol 2015; 6: 72 
doi: 10.3389/fphar.2015.00072 pmid: 25954198
4 Dong Q, Li N, Li Q, Zhang CE, Feng WW, Li GQ, Li RY, Tu C, Han X, Bai ZF, Zhang YM, Niu M, Ma ZJ, Xiao XH, Wang JB. Screening for biomarkers of liver injury induced by Polygonum multiflorum: a targeted metabolomic study. Front Pharmacol 2015; 6: 217 
doi: 10.3389/fphar.2015.00217 pmid: 26483689
5 But PP, Tomlinson B, Lee KL. Hepatitis related to the Chinese medicine Shou-wu-pian manufactured from Polygonum multiflorum. Vet Hum Toxicol 1996; 38(4): 280–282
pmid: 8829347
6 Wang J, Ma Z, Niu M, Zhu Y, Liang Q, Zhao Y, Song J, Bai Z, Zhang Y, Zhang P, Li N, Meng Y, Li Q, Qin L, Teng G, Cao J, Li B, Chen S, Li Y, Zou Z, Zhou H, Xiao X. Evidence chain-based causality identification in herb-induced liver injury: exemplification of a well-known liver-restorative herb Polygonum multiflorum. Front Med 2015; 9(4): 457–467
doi: 10.1007/s11684-015-0417-8 pmid: 26459430
7 Wang JB, Li CY, Zhu Y, Song HB, Bai ZF, Xiao XX. Integrated evidence chain-based identification of Chinese herbal medicine- induced hepatotoxicity and rational usage: exemplification by Polygonum multiflorum (He shou wu). Chin Sci Bull 2016; 61(09): 971–980 (in Chinese)
8 Shaw PJ, Ganey PE, Roth RA. Idiosyncratic drug-induced liver injury and the role of inflammatory stress with an emphasis on an animal model of trovafloxacin hepatotoxicity. Toxicol Sci 2010; 118(1):7–18
doi: 10.1093/toxsci/kfq168 pmid: 20538741
9 Björnsson ES. Drug-induced liver injury: an overview over the most critical compounds. Arch Toxicol 2015; 89(3): 327–334
doi: 10.1007/s00204-015-1456-2 pmid: 25618544
10 Poulsen KL, Olivero-Verbel J, Beggs KM, Ganey PE, Roth RA. Trovafloxacin enhances lipopolysaccharide-stimulated production of tumor necrosis factor-α by macrophages: role of the DNA damage response. J Pharmacol Exp Ther 2014; 350(1): 164–170
doi: 10.1124/jpet.114.214189  pmid: 24817034
11 Roth RA, Ganey PE. Animal models of idiosyncratic drug-induced liver  injury—current  status. Crit  Rev  Toxicol  2011; 41(9): 723–739 
doi: 10.3109/10408444.2011.575765 pmid: 21726137
12 Li CY, Li XF, Tu C, Li N, Ma ZJ, Pang JY, Jia GL, Cui HR, You Y, Song HB, Du XX, Zhao YL, Wang JB, Xiao XH. The idiosyncratic hepatotoxicity of Polygonum multiflorum based on endotoxin model. Acta Pharmaceutica Sinica (Yao Xue Xue Bao) 2015; 50(1):28–33 (in Chinese)
13 Wang JB, Xiao XH, Du XX, Zou ZS, Song HB, Guo XX. Identification and early diagnosis for traditional Chinese medicine-induced liver injury based on translational toxicology. China J Chin Materia Medica (Zhongguo Zhong Yao Za Zhi) 2014; 39(1): 5–9 (in Chinese)
pmid: 24754159
14 Wu X, Chen X, Huang Q, Fang D, Li G, Zhang G. Toxicity of raw and processed roots of Polygonum multiflorum. Fitoterapia 2012; 83(3): 469–475
doi: 10.1016/j.fitote.2011.12.012 pmid: 22210538
15 Lin CM, Singh SB, Chu PS, Dempcy RO, Schmidt JM, Pettit GR, Hamel E. Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin: a structure-activity study. Mol Pharmacol 1988; 34(2): 200–208
pmid: 3412321
16 Siles R, Ackley JF, Hadimani MB, Hall JJ, Mugabe BE, Guddneppanavar R, Monk KA, Chapuis JC, Pettit GR, Chaplin DJ, Edvardsen K, Trawick ML, Garner CM, Pinney KG. Combretastatin dinitrogen-substituted stilbene analogues as tubulin-binding and vascular-disrupting agents. J Nat Prod 2008; 71(3): 313–320 
doi: 10.1021/np070377j pmid: 18303849
17 Sun JL, Huang XL, Wu HQ, Huang F. Determination of content and light stability of cis- and trans-2,3,5,4′-tetrahydroxystilbene-2-O-β-D- glucoside in Radix Polygoni multiflori by HPLC/DAD/MS. Chin Pharm J (Zhongguo Yao Xue Za Zhi) 2009(7):541–544 (in Chinese)
18 Chalasani NP, Hayashi PH, Bonkovsky HL, Navarro VJ, Lee WM, Fontana RJ; Practice Parameters Committee of the American College of Gastroenterology. ACG Clinical Guideline: the diagnosis and management of idiosyncratic drug-induced liver injury. Am J Gastroenterol 2014; 109(7): 950–966, quiz 967 
doi: 10.1038/ajg.2014.131 pmid: 24935270
19 Xiao XH, Li XH, Zhu Y, Wang JB, Li L, Zhang T, Liu CH, Sun KW, Yang HS, Guo YM. Guideline for diagnosis and treatment of herb-induced liver injury. China J Chin Materia Medica (Zhongguo Zhongyao Zazhi) 2016; (7): 1165–1172
20 Luyendyk JP, Lehman-McKeeman LD, Nelson DM, Bhaskaran VM, Reilly TP, Car BD, Cantor GH, Deng X, Maddox JF, Ganey PE, Roth RA. Coagulation-dependent gene expression and liver injury in rats given lipopolysaccharide with ranitidine but not with famotidine. J Pharmacol Exp Ther 2006; 317(2): 635–643
doi: 10.1124/jpet.105.096305  pmid: 16401727
21 Sajish M, Schimmel P. A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol. Nature 2015; 519(7543): 370–373
doi: 10.1038/nature14028 pmid: 25533949
22 Langcake P, Pryce RJ. Production of resveratrol by Vitis vinifera and other members of Vitaceae as a response to infection or injury. Physiol Plant Pathol 1976; 9(1): 77–86 
doi: 10.1016/0048-4059(76)90077-1
23 Crowell JA, Korytko PJ, Morrissey RL, Booth TD, Levine BS. Resveratrol-associated renal toxicity. Toxicol Sci 2004; 82(2):614–619
doi: 10.1093/toxsci/kfh263 pmid: 15329443
24 Minezawa N, Gordon MS. Photoisomerization of stilbene: a spin-flip density functional theory approach. J Phys Chem A 2011; 115(27): 7901–7911
doi: 10.1021/jp203803a  pmid: 21639100
25 Zaki MA, Balachandran P, Khan S, Wang M, Mohammed R, Hetta MH, Pasco DS, Muhammad I. Cytotoxicity and modulation of cancer-related signaling by (Z)- and (E)-3,4,3′,5′-tetramethoxystilbene isolated from Eugenia rigida. J Nat Prod 2013; 76(4): 679–684
doi: 10.1021/np300893n  pmid: 23547843
26 Woods JA, Hadfield JA, Pettit GR, Fox BW, McGown AT. The interaction with tubulin of a series of stilbenes based on combretastatin A-4. Br J Cancer 1995; 71(4): 705–711
doi: 10.1038/bjc.1995.138  pmid: 7710932
27 Manis JP. Knock out, knock in, knock down—genetically manipulated mice and the Nobel Prize. N Engl J Med 2007; 357(24): 2426–2429
doi: 10.1056/NEJMp0707712 pmid: 18077807
28 Tong C, Li P, Wu NL, Yan Y, Ying QL. Production of p53 gene knock-out rats by homologous recombination in embryonic stem cells. Nature 2010; 467(7312): 211–213
doi: 10.1038/nature09368 pmid: 20703227
29 Zhu Y, Li YG, Wang Y, Wang LP, Wang JB, Wang RL, Wang LF, Meng YK, Wang ZX, Xiao XH. Analysis of clinical characteristics in 595 patients with herb-induced liver injury. Chin J Integr Tradit Western Med (Zhongguo Zhong Xi Yi Jie He Za Zhi) 2016; 36(1):44–48 (in Chinese)
30 El Kasmi KC, Anderson AL, Devereaux MW, Vue PM, Zhang W, Setchell KD, Karpen SJ, Sokol RJ. Phytosterols promote liver injury and Kupffer cell activation in parenteral nutrition-associated liver disease. Sci Transl Med 2013; 5(206): 206ra137
doi: 10.1126/scitranslmed.3006898  pmid: 24107776
31 Mitchell D, Wagner C, Stone WJ, Wilkinson GR, Schenker S. Abnormal regulation of plasma pyridoxal 5′-phosphate in patients with liver disease. Gastroenterology 1976; 71(6): 1043–1049
pmid: 992265
32 Myers BA, Dubick MA, Reynolds RD, Rucker RB. Effect of vitamin B-6 (pyridoxine) deficiency on lung elastin cross-linking in perinatal and weanling rat pups. Biochem J 1985; 229(1): 153–160
doi: 10.1042/bj2290153 pmid: 2864042
33 Canellakis ES, Jaffe JJ, Mantsavinos R, Krakow JS. Pyrimidine metabolism. IV. A comparison of normal and regenerating rat liver. J Biol Chem 1959; 234(8): 2096–2099
pmid: 13673019
34 Fausto N, Brandt JT, Kesner L. Possible interactions between the urea cycle and synthesis of pyrimidines and polyamines in regenerating liver. Cancer Res 1975; 35(2): 397–404
pmid: 1109804
35 Barbul A. Arginine: biochemistry, physiology, and therapeutic implications. J Parenter Enteral Nutr 1986; 10(2): 227–238
doi: 10.1177/0148607186010002227 pmid: 3514981
36 Satriano J. Arginine pathways and the inflammatory response: interregulation of nitric oxide and polyamines. Amino Acids 2004; 26(4): 321–329
doi: 10.1007/s00726-004-0078-4 pmid: 15290337
37 Corraliza IM, Soler G, Eichmann K, Modolell M. Arginase induction by suppressors of nitric oxide synthesis (IL-4, IL-10 and PGE2) in murine bone-marrow-derived macrophages. Biochem Biophys Res Commun 1995; 206(2): 667–673
doi: 10.1006/bbrc.1995.1094 pmid: 7530004
38 Bronte V, Zanovello P. Regulation of immune responses by L-arginine metabolism. Nat Rev Immunol 2005; 5(8): 641–654 
doi: 10.1038/nri1668 pmid: 16056256
39 Block  WD, Westhoff  MH, Steele  BF. Histidine metabolism in the human adult: histidine blood tolerance, and the effect of continued free L-histidine ingestion on the concentration of imidazole compounds in blood and urine. J Nutr 1967; 91(2):189–194
pmid: 6021220
40 Machado  MV, Kruger  L, Jewell  ML, Michelotti  GA, Pereira Tde  A, Xie G, Moylan CA, Diehl AM. Vitamin B5 and N-acetylcysteine in nonalcoholic steatohepatitis: a preclinical study in a dietary mouse model. Dig Dis Sci 2016; 61(1):137–148 
doi: 10.1007/s10620-015-3871-x pmid: 26403427
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