| Objective:Polygoni multiflori radix is traditional Chinese tonic herbs.However, because of liver injury reports caused by Polygoni multiflori radix and its medical preparations wherever at home or abroad caused are increasing, which turned out to aggravate people's fears of Polygoni multiflori radix even the entire Chinese herbal medicine. In this thesis, the research was focused on the diagnosis method of Polygoni multiflori radix-induced liver injury and its possible mechanism. In the last,the potencial factors which might be related to liver injury were studied.Methods:(1) Polygoni multiflori radix-induced liver damage markers screeningA total of 58 clinical plasma samples were analysed by LC-Q-TOF/MS in combination with multivariate statistical analyses. Using DILIs induced by Polygonum multiflorum as an example, the metabolic characteristics of the DILI were screened based on comparisons of patients with autoimmune hepatitis (AIH) and hepatitis B (HBV) and healthy controls.(2) Metabolomics research of different parts of Polygoni multiflori radix based on immunological stress ratThe sample analysis was performed by Agilent ZORBAX SB C18 column, and the mobile phase were acetonitrile and water. Both positive and negative ion mode were conducted. Metabolic fingerprints of different parts of the Polygoni multiflori radix of liver injury model in rats plasma were set up. After all, the metabolites and related metabolic pathways differences between groups was analysed.(3) Investigation on the influence of metal ions and light to the 2,3,5,4'-tetrahydroxy stilbene-2-O-?-D-glucoside contained in Polygoni Multiflori RadixBy using ultra high performance liquid chromatography coupled to mass spectrometry, the main ingredient in Polygoni multiflori radix 2,3,5,4'-tetrahydroxy stilbene-2-O-beta-D-glucoside, THSG was studied, Water containing different metal ions were used to investigate and compare the influence on THSG; The influence of light was preliminary conducted.Results:(1) Multivariate data analysis showed the disturbances in glucose, amino acid and sphingolipid metabolism varied between the diseases. Among all 24 selected metabolites, melatonin and valine exhibited stepwise increases and phytosphingosine exhibited stepwise reductions from AIH to DILI to HBV, which were selected as biomarkers to construct a classification model in the discrimination of DILI and yielded a good sensitivity (92.3%) and specificity (95.6%).(2) Based on metabonomics,48 endogenous biomarkers were selected under the positive ion mode while 7 endogenous biomarkers were selected under the negative ion mode. Totally 6 metabolism pathways were caculated based on KEGG database path analysis (www.genome.jp/kegg/).(3) The content of THSG could be decreased by different kinds of metal ions, and the effect was concentration-dependent. Moreover, Fe3+ and Fe2+ showed the greatest influence at the same concentration; and our study has shown that THSG decreased more than 98% in Fe3+ and Fe2+ solutions at 500ppm concentration. In the same time we found out p-hydroxybenzaldehyde (molecular weight:122.0367) and 2,3,5-trihydroxybenzaldehyde-2-O-glycoside (molecular weight:316.0794) were the main degradation products of THSG in tap water and water containing Cu2+, Ca2+, Zn2+, Mg2+, and Al3+. The product of THSG dimer with a water molecule was found in water containing Fe3+and Fe2+.(4) Cis-THSG also have photosensitiveness which is much weaker than its Trans-form. It indicates that there is a equilibrium between the transformation of Cis and Trans-forms.Conclusion:(1) This research presents a holistic view of the metabolic features of DILI, and the combination of melatonin, valine and phytosphingosine exhibited great potential to serve as biomarkers of DILI in clinical diagnosis.(2) The possible toxicity mechanism might associated with bile acid secretion and amino acid metabolic pathways, which is need to be further confirmed.(3) Objective authenticity of "Iron forbidden" of Polygoni multiflori radix was preliminary confirmed, At the same time, decoction or medicinal preparation containing Polygoni multiflori radix should be kept away from light. |
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