| Treatment based on syndrome differentiation is one of the major features in traditional Chinese medicines?TCMs?,it emphasizes that dosage should be adapted to patients with changing disease condition.At the same time,the clinical experience in the syndrome differentiation shows that dosage of traditional Chinese medicines?TCMs?affects much of clinical efficacy and safety.So goes the proverb"Dosage is the mystery of TCM".But how can we make this mystery scientific and valid?In this research we put forward the thought of syndrome?disease?-dosage relationship for the first time to explore the scientific evidences of dosage of TCMs with multi-efficacy.Rhubarb was selected as model drug due to its representative multi-efficacy in clinical treatments of constipation,cholestatic hepatitis and acute pancreatitis.Classical pharmacology and efficacy-associated biomarkers were jointly applied to elucidate the syndrome?disease?-dosage relationship as well as the therapeutic window of rhubarb.The results would provide reference basis for clinical rational use of rhubarb.The scientific mechanism of disease-dosage relationship of rhubarb would also be investigated by metabolic biomarkers and the network pharmacology.Through this project,it would provide new strategy and exploratory methodology to reconstruct and improve the scientific theory of dosimetry and therapeutics of TCMs.The major contents and results of the study are as following:1 Sample collection,sample preparation and quality control of rhubarbIn the present studty,raw rhubarb?Rheum tanguticum Maxim.ex Balf.?was collected and the decoction pieces of rhubarb were prepared.To ensure the quality of rhubarb used for animal studies.The content of five anthraquinones was determined by high performance liquid chromatography?HPLC?,and the chemical fingerprint of rhubarb was established.What's more,the chemical information of water extract was established by ultra-high performance liquid chromatography-mass spectrometry?UHPLC-MS?system.The results showed that total content of the five anthraquinone compounds were 2.14%,which was in accord with the quality standard of Chinese Pharmacopoeia?CP??2010?.Further,the similarity values of all the samples was higher than 0.95,the stability of sample was proved.At last,altogether 37 compounds,including aloeemodin,rhein and gallic acid were tentatively identified by UHPLC-MS system.2 The disease-dosage relationship study of rhubarb based on classic pharmacodynamics and metabolomics2.1 The dose response study of rhubarb in rat model of constipationThe anti-consitipation effect of five doses of rhubarb?0.21,0.66,2.10,6.60,and21.0 g/kg?in a loperamide-induced rat model of constipation was examined,acute hepatotoxicity and cause of diarrhea were also examined.LC-MS-based untargeted metabolomics together with pathway analysis were further applied to characterize the metabolic alterations induced by the different rhubarb doses,and the dose-response curve was also studied by efficacy-related biomarkers.As a result,an obvious dose-effect-toxicity relationship was observed.The optimal dose of rhubarb on constipated rats was 0.66g-2.10g/kg,and overdose of 21.0 g/kg may led to acute hepatotoxicity and severe diarrhea.Altogether,14 biomarkers were identified.The dose-response curve indicated that doses in the 0.94g±0.11g/kg range?Corresponding to 8.56-10.92g in the clinic?were optimal effective for constipation treatment.The pathway analysis showed that amino acid metabolism and glycometabolism pathway were altered by rhubarb in a dose-dependent manner.2.2 The dose response study of rhubarb in rat model of cholestatic hepatitisThe anti-cholestatic effect of five doses of rhubarb?0.21,0.66,2.10,6.60,and 21.0g/kg?in an alpha-naphthylisothiocyanate?ANIT?-induced rat model of cholestatic hepatitis was examined by histopathology and serum biochemistry.A dose-dependent anti-cholestatic effect of rhubarb?0.21-6.60 g/kg?was observed,and an overdose of21.0 g/kg showed a poor effect.LC-MS-based untargeted metabolomics together with pathway analysis were further applied to characterize the metabolic alterations induced by the different rhubarb doses.Altogether,13 biomarkers were identified.The dose-response curve based on 9 important biomarkers indicated that doses in the 0.42-6.61g/kg range(EC20-EC80 range,corresponding to 4.00-62.95 g in the clinic)were effective for cholestasis treatment.The pathway analysis showed that bile acid metabolism and excretion,inflammation and amino acid metabolism were altered by rhubarb in a dose-dependent manner and might be involved in the dose-response relationship and therapeutic mechanism of rhubarb for cholestasis treatment.2.3 The dose response study of rhubarb in rat model of acute pancreatitisThe anti-pancreatitis effect of five doses of rhubarb in a cerulein-induced rat model of acute pancreatitis was examined.Untargeted metabolomics were further applied to characterize the metabolic alterations induced by the different rhubarb doses.As a result,a dose-dependent anti-pancreatitis effect of rhubarb?0.21-2.10 g/kg?was observed,and dose of 2.10 g/kg showed an optimal effect.16 biomarkers such as creatinine,tryptophan were identified in metabolomics study.The dose-response curve based on12 efficacy-related biomarkers indicated that doses in the 0.22–3.26 g/kg range(EC20-EC80 range,corresponding to 2.35–37.56 g in the clinic)were effective for pancreatitis treatment.The pathway analysis showed that amino acid metabolism and arachidonic acid metabolism were altered by rhubarb in a dose-dependent manner.3.The summary and potential mechanism exploration of disease-dosage relationship based on analysis of biomarkers and network pharmacologyOur results preliminarily revealed the objective reality of disease-dosage relationship of rhubarbAt first,the optimal dosage of rhubarb for treating constipation,cholestatic hepatitis and acute pancreatitis were summarized.Analysis of classic pharmacodynamics indexes showed that the optimal therapeutic dosage of rhubarb on constipation,cholestatic hepatitis and pancreatitis were quite different.Rhubarb can used at low dose of about 0.66g-2.10g/kg for treating constipation,and this dosage can generate optimal efficacy without significant toxic and side effects.And we can get that rhubarb has limited effect on cholestatic hepatitis,large dose of rhubarb at about6.60g/kg can generate moderate efficacy on cholestatic hepatitis.Moderate dose of rhubarb at about 2.10-6.60g/kg can be used for treaing mild pancreatitis.Then,the mathematical model of dose-response curves based on biomarkers was used for the evaluation of disease-dosage relationship of rhubarb.Based on the parameter of dose-response curves,the different pharmacodynamic characteristics of rhubarb on the three diseases were obtained.The optimal dosage of rhubarb were 0.94g±0.11g/kg for constipation,6.61±0.13 g/kg for cholestatic hepatitis and 3.26±0.17g/kg for pancreatitis,corresponding to 8.56-10.92 g?9.74±1.18 g?,58.60g-61.41g?60±1.41g?and 35.78-39.34g?37.56±1.78 g?in the clinic,respectively.The possible material basis and potential targets that help to elucidate disease-dosage relationship of rhubarb were preliminarily studiedBy the analysis of network pharmacology based on biomarkers,we found rhubarb owns unique and same target networks for treating constipation,cholestatic hepatitis and pancreatitis.And we also found that rhubarb can dose-dependently affect the target networks.Through the comparison we can get that the chemical components,the potential targets the the level of biomarkers were quite different in the model of constipation,cholestatic hepatitis and pancreatitis.The possible material basis and potential targets in the three models and the response of biomarkers to rhubarb at different dose can also help to uncover the disease-dosage relationship of rhubarb.The main innovations of this paper could be summarized as follows:?1?The concept and research methods of syndrome?disease?-dosage of TCMs were firstly proposed.And in the present study rhubarb was selected as model drug.The optimal therapeutic window of rhubarb on constipation,cholestatic hepatitis and pancreatitis were revealed.The findings is conducive to improving therapeutic effects and rational use of rhubarb,and it would provide the new strategy and exploratory methodology to reconstruct and improve the scientific theory of dosimetry and therapeutics of TCMs.?2?The potential mechanism of disease-dosage relationship of rhubarb was preliminarily revealed based on the components-targets network.And the present study can present a new perspective on the research of material basis and potential targets for functional directions of TCMs. |
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