Study On Quality Markers Of Zanthoxyli Radix Based On Pharmacokinetics And In Vitro/in Vivo Toxicity Evaluation Of Effective Components

1.Background and objectives:Zanthoxyli Radix is the dry root of plant belonged to Zanthoxylum in Rutaceae（Zanthoxylum nitidum（Roxb.）DC.;ZN）.In the field of Traditional Chinese Medicine（TCM）,ZN has various pharmacological activities of activating blood circulation to dissipate blood stasis,activating vital energy to stop pain,dispelling wind and removing obstruction in the meridians and detumescence by detoxification.It mainly used for traumatic injury,stomachache,toothache,rheumatic arthralgia,snake bite;external treatment of burns.At present,there are many articles about the chemical constituents of ZN.More than two hundard compounds were isolated from ZN.The chemical structure type mainly included alkaloids,coumarins,lignans,flavonoids and their glycosides,steroids and their glycosides,quinones,terpenoids and some organic acids.Modern pharmacological research about ZN mainly focused on anti-tumor,analgesic,anti-inflammatory and antibacterial activities,and most of them are about the pharmacological activities of alkaloid compounds in ZN.Many researches had shown that many alkaloid compounds in ZN have obvious anti-tumor activities,such as nitidine chloride,chelerythrine,magnoflorine and so on.Although there are many articles about some active ingredients in ZN,the pharmacodynamic material basis of ZN is still unclear.In addition,previous studies had shown that the medicinal sources of ZN are confused,Toddalia asiatica（L.）Lam.（TA）is widely used in the market instead of ZN.This study mainly attempts to use the theory and technology of analytical chemistry,pharmacokinetics,pharmacodynamics and toxicology to elaborate the pharmacodynamic material basis of ZN.At the same time,it attempts to critically analyze the current quality control patterns of TCM and take ZN as an example to analyze and discuss the new quality control mode of TCM.2.Methods:（1）Qualitative analysis of chemical constituents of ZN extracts and serum pharmacochemistry research.The chemical constituents of 75%ethanol extract from ZN and its common adulterants TA were qualitatively analyzed using UPLC-Q-TOF-MS.The UPLC-Q-TOF-MS information of the main active ingredients from the above-mentioned extracts were collected and compared with the literature.Based on it,the specific structure type of the main active ingredients could be analyzed and identified.At the same time,the blank and drug-containing plasma were collected from ophthalmic venous plexus before and after oral administration of 75%ethanol extract from ZN and its common adulterants TA（The administration dosages were 2g dry extract/kg weight and 1g dry extract/kg weight,respectively）.Based on the qualitative analysis of chemical constituents of ZN extracts,the prototypes and metabolites migrating to blood could be analyzed and identified.（2）The biotransformation pathway of main active ingredients in ZN and pharmacokinetic studyThe metabolits of main active alkaloids（chelerythrine,dihydrochelerythrine,skimmianine,and dictamnine）in rat liver microsomes（RLMs）in vitro and in vivo were analyzed and identified.The blank and substrate-containing RLMs（NADPH generating system and RLMs）incubation system were prepared after 90 min incubation in water bath（37℃）and injected into UPLC-Q-TOF-MS system for elaborating the biotransformation pathway of above-mentioned alkaloids in vitro.Blank and drug-containing urine,feces,bile and plasma samples were prepared and injected into UPLC-Q-TOF-MS system for elaborating the biotransformation patterns of above-mentioned alkaloids in vivo.An UPLC-MS-MS method for simultaneous determination of the above-mentioned alkaloids（dihydrochelerythrine,nitidine chloride,chelerythrine,sanguinarine,liriodenine,skimmianine,fagarine,dictamnine and toddalolactone）in rat plasma was developed and validated.The blank and drug-containing plasma samples were collected at different time points befor and after the oral administration of ZN and TA75%ethanol extract.The plasma samples were pepared according to the above-mentioned method,and injected into UPLC-MS-MS system for elaboraring the pharmacokinetic behavior of the main active ingredients in ZN and its common adulterants TA.（3）The relative molecular mechanism of skimmianine and dictamnine induced acute liver injury in mice and toxicological evaluation in vitroThe histopathological sections of liver and the levels of ALT,AST,and ALP in serum were analyzed for evaluating dictamnine-induced acute liver injury in mice.Trapping assay（nucleophilic reagents such as cysteine,homocysteine,glutathione and methoxyamine hydrochloride were added intoâ… phase incubation system of DIC）was carried out for identifying the structure and reactivity of epoxide intermediate derived from dictamnine and skimmianine.At the same time,the activity and expression of MPO,the content of MDA and the activity of GSH-Px in the liver and the content of TNF-α,IL-6 and IFN-γin the serum were determined for evaluating oxidative stress injury and inflammation were involved in the pathological process of dictamnine and skimmianine-induced acute liver injury in mice.In vitro and in vivo detection experiment of the conjugation between DIC metabolic reactive epoxide intermediate and cysteine residue have been confirmed the covalent binding between the electrophilic reactive intermediate and the protein containing cysteine residue might be the most important cause of dictamnine and skimmianine-induced acute liver injury in mice.L02 cell line was used to evaluate cytotoxicity in vitro of the above-mentioned active ingredients（nitidine chloride,chelerythrine,dihydrochelerythrine,magnoflorine,skimmianine,fagarine,dictamnine,sanguinarine and toddalolactone）.The IC₅₀ values of above-mentioned ingredients in L02 cell line were determinated.（4）Study on quality markers of ZN and the new strategy of safety and quality control of TCMA HPLC-DAD method for simultaneous determination of magnoflorine,nitidine chloride and chelerythrine in ZN was established and validated.The content of magnoflorine,nitidine chloride and chelerythrine in eighteen batches of ZN were determinated using the above-mentioned method.A TLC method was developed for simultaneous determination of nitidine chloride and ethoxychelerythrine in ZN and toddalolactone in its common adulterant TA.An UPLC-MS-MS method for simultaneous determination of dihydrochelerythrine,nitidine chloride,chelerythrine,sanguinarine,liriodenine,skimmianine,fagarine,dictamnine and toddalolactone in ZN and its common adulterant TA were developed and validated.The content of above-mentioned active ingredients in eighteen batches herbs were determinated using the above-mentioned method.The integration parameters Ii=Ci*Ai/Ei（Ci:the content of each active ingredients in ZN;Ai:the normalized value of the area under the plasma drug concentration time curve of each active ingredients in ZN;Ei:the normalized value of the half inhibition rate of each active ingredient in ZN）are calculated by integrating the above-mentioned content determination data（Content value）,pharmacokinetic data（AUC value）and toxicological data(IC₅₀ value).Based on it,the integration parameters can be used for evaluating the pharmacodynamic material basis（quality marker;Q-Marker）of ZN.3 Results（1）Qualitative analysis of chemical constituents of ZN extracts and serum pharmacochemistry research.An UPLC-Q-TOF-MS method was successfully established for the qualitative analysis of the chemical composition of 75%ethanol extract of ZN and its common adulterant TA.The result showed that there are many kinds of alkaloids in ZN,such as benzophenanthridine,furoquinoline,aporphine,quinoline,isoquinoline and benzylisoquinoline alkaloids.Nitidine chloride,chelerythrine,dihydrochelerythrine,sanguinarine,liriodendrine,skimmianine,fagarine and dictamnine were all detected in ZN.The result of TA showed that the active alkaloids in ZN also could be detected in TA.The chemical constituent differences between ZN and TA were mainly reflected in the coumarins in TA,such as toddalolactone,toddaculine and toddanin.Study on the serum pharmacochemistry of ZN and its common adulterant TA showed that various alkaloids in ZN were absorbed into blood in vivo,such as nitidine chloride,chelerythrine,dihydrochelerythrine,sanguinarine,liriodendrine,skimmianine,fagarine and dictamnine.The response of skimmianine in plasma was significantly higher than that of nitidine chloride and chelerythrine.The difference in the serum pharmacochemistry between ZN and TA mainly reflected in the high plasma drug concentration of toddalolactone after oral administration of TA 75%ethanol extract.（2）The biotransformation pathway of main active ingredients in ZN and pharmacokinetic studyThe main biotransformation patterns of the main active alkaloids in ZN in rats were studied.The metabolic pathways of benzophenanthridine alkaloids,such as chelerythrine,mainly included demethylation,hydroxylation,methylene dioxy cycle opening reaction andâ…¡phase metabolic reaction of glucuronization after exposure of active hydroxyl groups.The metabolic pathways of dictamnine and other furoquinoline alkaloids in rats mainly included epoxidation of furan ring,hydroxylation of aromatic rings,demethylation of methoxyl group,the conjugation between epoxide intermediate and various nucleophilic reagent in vivo（including glutathione binding products）,andâ…¡phase metabolic reaction of glucuronization after exposure of active hydroxyl groups.An UPLC-MS-MS method for simultaneous determination of dihydrochelerythrine,nitidine chloride,chelerythrine,sanguinarine,liriodenine,skimmianine,fagarine,dictamnine and toddalolactone in rat plasma was developed and validated.The established UPLC-MS-MS method was successfully applied to the pharmacokinetic study of eight active ingredients after oral administration of ZN and its common adulterant TA extract.In the pharmacokinetic study of ZN,skimmianine and liriodendrine have good oral absorption effect with maximum plasma drug concentrations of 233.56 and 377.90 ng/m L,respectively.By contrast,nitidine chloride,chelerythrine and dihydrochelerythrine have poor oral absorption effect with maximum plasma drug concentrations of 120.16,40.15,and 83.09 ng/m L,respectively.Considering the content difference of each component in the extract,AUC_0-∞/dose was used as an evaluation index for the absorption effect of each component.When the dose difference was deducted,the poor absorption effects of nitidine chloride,chelerythrine,and dihydrochelerythrine became evident.In the pharmacokinetic study of TA,the pharmacokinetic characteristics of nitidine chloride,chelerythrine,dihydrochelerythrine,skimmianine,fagarine and dictamnine were similar to those in the extract of ZN.In addition,the most significant characteristics of the pharmacokinetic behavior in TA reflected in the high plasma drug concentration of toddalolactone.（3）The relative molecular mechanism of skimmianine and dictamnine induced acute liver injury in mice and toxicological evaluation in vitroThe acute liver injury in mice induced by dictamnine and skimmianine was elaborated in detail.The structure and reactivity of furan epoxide intermediate of dictamnine and skimmianine were confirmed by trapping assay（adding nucleophilic reagent to theâ… phase incubation system of dictamnine and skimmianine）.At the same time,it was confirmed that the oxidative stress injury and inflammation induced by glutathione depletion were involved in the pathological process of acute liver injury induced by dictamnine and skimmianine.The covalent binding between the electrophilic reactive intermediate and the protein containing cysteine residue might be the most important cause of dictamnine and skimmianine-induced acute liver injury in mice.L02 cell line was used to evaluate the cytotoxicity of several active alkaloids（nitidine chloride,chelerythrine,dihydrochelerythrine,magnoflorine,skimmianine,fagarine,dictamnine,sanguinarine and toddalolactone）in vitro.The results showed that the IC₅₀ values of nitidine chloride and chelerythrine were significantly lower than that of skimmianine and dictamnine.（4）Study on quality markers of ZN and the new strategy of safety and quality control of TCMA HPLC-DAD method was established for the simultaneous determination of magnoflorine,nitidine chloride and chelerythrine in ZN.The contents of the above-mentioned three active components in 18 batches of ZN were determined.The results showed that there are four batches of medicinal materials do not conform with the"Chinese Pharmacopoeia"2020 edition standard in 11 batches of Zanthoxylum nitidum（Roxb.）DC.,the content of nitidine chloride is lower than the required standard.However,the content of nitidine chloride in No.14-16 conform with the required standard,but toddalolactone was detected（according to the standard of Chinese Pharmacopoeia 2020 edition,toddalolactone should not be detected）.Three above-mentioned active ingredients could not be detected in No.18,which indicated that the market of ZN was in chaos,and there was potential risk in the safety of clinical medication.An UPLC-MS-MS method was successfully established for the determination of nitidine chloride,chelerythrine,dihydrochelerythrine,sanguinarine,magnoflorine,skimmianine,fagarine,dictamnine and toddalolactone in ZN and its common adulterants TA.The results showed that the content of each active ingredient in different batches of medicinal materials was quite different,but the overall trend was consistent.The contents of nitidine chloride,chelerythrine and magnoflorine in the medicinal materials are significantly higher than that of skimmianine,fagarine and dictamnine.So far,the integration parameters Ii=Ci*Ai/Ei（Ci:the content of each active ingredient in ZN;Ai:the normalized value of the area under the plasma drug concentration time curve of each active ingredient in ZN;Ei:the normalized value of the half inhibition rate of each active ingredient in ZN）are calculated by integrating the above-mentioned content determination data（Content value）,pharmacokinetic data（AUC value）and toxicological data(IC₅₀ value).Theoretically,the components with higher integration parameters are likely to be the pharmacodynamic material basis of the herb,and it is easy to carry out quality control due to their high content.The ingredients with higher integrated parameters（quality marker;Q-Marker）were selected as the quality control indexes of the herb.4 ConclusionThrough the content analysis of various active ingredients in ZN,the study of pharmacokinetics in vivo,the study of toxicology in vitro and in vivo,and the analysis of the current quality control method of TCM.We propose to integrate the content of various active components（Content value）,in vivo pharmacokinetic parameters（AUC value）and in vitro toxicological parameters(IC₅₀ value)of ZN to form the integrated parameter Ii=Ci*Ai/Ei.The ingredients with higher integrated parameter most likely to be the material basis of the herb,the quality control of active ingredients will have great significance.For ZN and TA,it is more appropriate to select nitidine chloride and chelerythrine as the quality control index of ZN,while it is more reasonable to select toddalolactone as the quality control index of TA.Based on the above analysis,the safety,effectiveness,and quality control of TCM will be combined.This study also provides a reference for the identification of pharmacodynamic material basis（quality marker;Q-Marker）of TCM.