| ObjectiveBased on UPLC-Q-TOF/MS detection system,a sensitive and efficient method for the isolation and identification of various aristolochic acid components(AAs)and their related metabolites in vivo was established.Aristolochiae fructus was taken as a typical Chinese medicine containing aristolochic acid,and biological samples of serum,liver,kidney,urine and feces after subacute and long-term experimental administration were used.The levels of various AAs components and their metabolites were tested qualitatively and semi-quantitatively,and the possible in vivo metabolism of water extract of Aristolochiae fructus after short-term or long-term administration was comprehensively analyzed.The in vivo metabolism of multi-component AAs was preliminarily discussed,combined with the results of toxic effects studies,to provide a basis for clinical regulation of the potential toxicity of traditional Chinese medicine containing AAs and the study of AAs metabolism.Method1.Establish a qualitative analysis method for aristolochic acids and their metabolites in Aristolochiae fructusIn this study,the UPLC-Q-TOF/MS detection system was used to establish a qualitative detection method for five main aristolochic acids(AAs)in combination with existing literature reports and actual conditions.The Waters Acquity UPLC BEH C18 column(2.1*100mm,1.7 μ m)was used for gradient elution with 0.01%formate methanol-water(containing 5 mmol·L-1 ammonium acetate)as the mobile phase,and the flow rate was 0.3μL.min-1.Ten male SD rats were randomly divided into control group and AAI group(20mg kg-1·d-1).After continuous administration for 5 days,blood was taken under anesthesia,and kidney tissues were dissected after death.Use the serum and kidney of the blank control group to optimize the biological sample processing method,and calculate the recovery rate of AAs qualitative analysis method.Determine the biological sample treatment method and AAs qualitative analysis method.The metabolic product analysis method was verified with rat serum of AAI group.And the feasibility of the method was verified by qualitative analysis of AAs in serum of rats in AAI group.2.Study on subacute toxicity of Aristolochiae fructus in rats and qualitative analysis of aristolochic acid metabolites in vivoSD rats were selected and given water extract of Aristolochiae fructus(AFE)(110 g crude drug·kg-1·d-1)and AAI(5 mg · kg-1·d-1)by gavage for 5 days respectively.Serum,urine and feces were collected,liver and kidney tissues were extracted,and organ coefficient,hematology(RBC,PLT,WBC,etc.)and serum biochemical(ALT,AST,ALP,BUN,CRE,etc.)indexes were detected.The liver and kidney were examined by pathology.At the same time,UPLC-Q-TOF-MSE was used to qualitatively analyze the types of AAs prototype and related metabolites in biological samples(serum,urine,feces,liver,kidney),and parallelly compare the similarities and differences of metabolites in rats in the subacute toxicity test of AFE group and AAI group.3.Study on long-term toxicity of Aristolochiae fructus in rats and qualitative analysis of aristolochic acid metabolites in vivoIn this experiment,200 SD rats,half male and half female,were given three doses of Aristolochiae fructus extract by gavage(0.95 g crude drug ·kg-1·d-1,3.75 g crude drug·kg-1·d-1and 15g crude drug·kg-1 · d-1,respectively,equivalent to 6.35,25 and 100 times of human clinical dose).The drug was continuously administered for 6 months,and the observation was continued for 1 month after the drug was stopped.At the same time,a single aristolochic acid component AAI of the same dose was set as a positive control group to explore the toxicity of Aristolochiae fructus and single components and the similarities and differences of metabolites.Throughout the experiment real-time observation of animal behavior and recording of animal death,dynamic monitoring of animal weight and food intake.At the end of three months,six months and one month after the drug withdrawal period,some animals were killed,their urine and feces were collected,and hematology(RBC,PLT,WBC,etc.)and serum biochemistry(ALT,AST,ALP,BUN,CRE,etc.)were detected.The animals were dissected,by weighing the liver and kidney to calculatie the organ index and histopathological examination was performed.At the same time,UPLC-Q-TOF-MSE was used to qualitatively analyze the AAs prototype and related metabolites in biological samples(serum,urine,feces,liver,kidney).Result1.Establish a qualitative analysis method for aristolochic acids and their metabolites in Aristolochiae fructusIn this paper,an effective qualitative detection method of UPLC-Q-TOF/MS is selected.The specific chromatographic and mass spectrum conditions are as follows:the combined system of Waters UPLC-I class and Xevo G2-XS Q-TOF and the Waters Acquity UPLC BEH C18 chromatographic column(2.1*100mm,1.7 μ m)are used;Mobile phase 0.01%formic acid water(A)-0.01%formic acid methanol(B)(phase A and B contain 5 mmol·L-1 ammonium acetate),0-1.0 min,10%B;1.0～2.0 min,10%B～40%B;2.0～10.0 min,40%～85%B;From 10.0 to 10.2 min,85%B to 95%B;10.2～13.2 min,95%B;13.2 min to 15.0 min,95%B to 10%B.Gradient elution is performed.The flow rate was 0.3 mL·min-1,and the column temperature was 35℃.The parameters of mass spectrometer are ion source temperature 100℃,capillary voltage 3kV,desolvent gas temperature 250℃,desolvent gas flow 800L/Hr,MS/MSE mode,cone voltage 30 V,collision energy 6 eV.Under this condition,the separation effect of each AAs is better;The RSD of retention time and peak area of each AAs is less than 5%,and the instrument precision is good;In the range of 0.01～0.5 μg·mL-1,the linear relationship of AAs reference substances is good,and the correlation coefficient r ≥0.9980;In 48h,when the concentration of AAs is 0.05～0.5 μg·mL-1,the stability is good,and the RSD value is less than 10%except for a few.After the biological sample is treated by the treatment method,the recovery rate of the added sample at each concentration is less than 20%,which proves that this biological sample treatment method is feasible.After continuous administration of AAI(20 mg·kg-1)for 5 days,a total of 13 AAI-related metabolites were measured in rat serum according to the established AAs qualitative detection method according to the serum biological sample processing method.It is proved that the AAs metabolite detection method is feasible.Under the experimental conditions,the separation effect of each AAs is better.RSD of retention time and peak area of all AAs were less than 5%,indicating high precision of the instrument.In the range of 0.01～0.5 μg·mL-1,the linear relationship between the AAs was good,and the correlation coefficient r≥0.9980.In addition,the stability of AAs was good within 48h when the concentration of AAs was 0.05-0.5 μg·mL-1,and RSD were all less than 10%except some.After the biological samples were treated by the treatment method,the recovery rate was less than 20%at each concentration,indicating that the biological sample pretreatment method met the requirements.After 5 days of continuous administration of AAI(20 mg·kg-1),13 metabolites related to AAs were detected in serum by the established qualitative detection method according to the biological sample treatment method,which was basically consistent with the literature reports,confirming the feasibility of the method for detection of AAs metabolites.2.Study on subacute toxicity ofAristolochiae fructus in rats and qualitative analysis of aristolochic acid metabolites in vivoAfter continuous administration of AFE(110g crude drug·kg-1·d-1)for 5 days,although it had certain effects on the proportion of white blood cells in whole blood and the levels of BUN and CRE in serum,there were significant differences compared with the control group(P<0.05).However,the above changes were within the normal physiological and biochemical range of SD rats,and no changes related to drug toxicity were found.At the same time,no significant pathological changes were found in liver and kidney of AFE group.The results of organ coefficients showed that the liver and kidney coefficients of male rats in AFE group were significantly higher than those in control group(P<0.01),and were significantly different from those in equal dose AAI group(P<0.05 or P<0.01).However,AFE had no significant effect on hepato-renal coefficient of female rats.Correspondingly,AAI had little effect on the liver and kidney coefficients of rats,and the female liver coefficients were significantly decreased compared with the control group(P<0.05).AAI,AAII and ALI were detected in liver and kidney samples of AFE group.In contrast,AAI group only detected AAI and ALI in kidney tissue,and only ALI in liver tissue.In addition,the levels of AAI and ALI in liver and kidney tissues of animals in AFE group were significantly higher than those in AAI group(P<0.01 or P<0.001),which may be caused by the coexistence of other AAs components and the influence of AAI metabolism.In addition,no AAs metabolites were identified in the liver and kidney tissues of the AFE group and the AAI group,suggesting that no accumulation of AAs metabolites was found in the liver and kidney tissues of animals after short-term administration of AFE.AAs and their metabolites were mainly concentrated in serum,urine and feces in AFE group and AAI group.In the biological samples(serum,urine and feces)of AFE group,not only 6,10 and 13 common metabolic structures were identified,but also 14,20 and 30 unique metabolic structures were found,which were different from those of AAI group.Moreover,the main AAs components all follow the metabolic processes of demethylation,nitric acid reduction and binding.Parallel analysis with AAI group showed that AAI prototype components and most metabolic derivatives of AAI in biological samples of AFE group were significantly increased(P<0.05,P<0.01 or P<0.001).However,fecal metabolism of aristolocholic lactam I(ALI)in AFE group was significantly lower than that in AAI group(P<0.001).In addition,a variety of unique ALI efflux derivatives not found in Ai group were also identified in urine and feces of AFE group.It is speculated that the coexistence of various AAs components in AFE may affect the transformation of AAI into ALI by reducing the production of ALI and accelerating the efflux of ALI metabolism,so as to achieve a certain attenuated effect.3.Study on long-term toxicity of Aristolochiae fructus in rats and qualitative analysis of aristolochic acid metabolites in vivoFor the duration of the experiment,AFE was administered continuously for 6 months,although it had varying effects on some blood cytological markers(such as LYM%,NEUT%,PLT,WBC,and EOS%),coagulation markers(PT,PP,and APTT),and some serum biochemical markers(ALP,BUN,and ALT).However,the changes fluctuated within the normal range of SD rats,and no effects related to drug toxicity were found.After long-term administration of AFE and AAI,it was found that there were serious irreversible lesions in the stomach of the high-dose AFE group and the AAI group,while the pathological changes in the stomach of the medium-dose AFE group and the low-dose AFE group were produced and aggravated with the extension of administration time,suggesting that the stomach injuries caused by long-term and high-dose administration of Aristolochiae fructus should be avoided.In addition,the renal coefficient and liver coefficient of male rats in AFE high-dose group and AAI group were significantly changed compared with the control group(P<0.05 or P<0.01).Pathological examination of liver and kidney showed that part of renal tubular epithelial cells were degenerated and exfoliated in the renal tissues of rats in the AFE high-dose group and AAI group,and scattered exfoliated cells were observed in the lumen.Cell necrosis and tubular lesions were seen in some animals.The above lesions were basically consistent with those in the AAI group.In addition,only slight fibroblast hyperplasia was observed in the glomeruli in the high dose AFE and AAI groups.In addition,only focal inflammatory cell infiltration,mild eosinophilic lesion and occasional hepatocyte necrosis were observed in the high-dose AFE and AAI groups.It is suggested that AFE has the potential toxicity similar to AAI in high dose and long term administration.Metabolite analysis showed that 1,3 and 45 AAs prototypes or metabolites were detected in serum,urine and stool,respectively,3 months after AFE administration.Metabolite analysis showed that 1,3 and 45 AAs prototypes or metabolites were detected in serum,urine and stool,respectively,3 months after AFE administration.At the same time,1,2 and 51 AAs prototypes or metabolites could be detected 6 months after AFE administration.Moreover,the metabolic level of high dose AFE group was similar to that of AAI group.After 1 month of drug withdrawal,AAS-related metabolites were reduced in rats,and no metabolites were detected in serum,feces,liver or kidney tissue.It was suggested that AAs in AFE did not accumulate in liver and kidney tissue.In conclusion,after long-term administration of AFE,AAs-related chemical components can be metabolized and excreted rapidly in vivo,and the higher the dosage,the higher the amount of metabolites,and the rule of metabolites is consistent with the toxicity results.At the same time,1,2,51 AAs prototypes or metabolites could be detected 6 months after AFE administration.Moreover,the metabolic level of high dose AFE group was similar to that of AAI group.After 1 month of drug withdrawal,AAs-related metabolites were reduced in rats,and no metabolites were detected in serum,feces,liver or kidney tissue.It was suggested that AAs in AFE did not accumulate in liver and kidney tissue.In conclusion,after long-term administration of AFE,AAs-related chemical components can be metabolized and excreted rapidly in vivo,and the higher the dosage,the longer the administration time,the higher the amount of metabolites,and the rule of metabolites is consistent with the toxicity results.Conclusion1.This paper established a qualitative analysis method for aristolochic acids and their metabolites.The biological sample treatment method is fast and effective,and can be used for qualitative analysis of aristolochic acids and their metabolites.2.Under the conditions of this experiment,on the one hand,after continuous administration of AFE 110g crude drug·kg-1 for 5d,no significant hepatorenal toxicity related to AAs was found in the AAI group with the same dose.On the other hand,after continuous administration of AFE 15g crude drug·kg-1 for 6 months,the liver and kidney tissues showed certain pathological changes.Moreover,the disease worsened with the extension of administration time,and there was no significant improvement after withdrawal.The degree of liver and kidney damage was similar to that of the same dose of AAI.However,no significant toxic effects were found in 3.75g crude drugs·kg-1 and 0.95g crude drugs·kg-1 AFE during the whole experiment period.Comprehensive analysis showed that the dosage and duration of aristolochia should be strictly controlled in clinical application.Low dosage and short duration may have certain safety,but the potential risk of liver and kidney injury cannot be ruled out,and the liver and kidney function should be monitored at any time.3.In the subacute and long-term toxicity tests,the in vivo metabolism of various AAs in AFE basically followed the similar rule of AAI.The metabolites of AAs in AFE showed a certain correlation with the dosage and time of AFE administration.However,no matter the type or metabolic level of AAs-related metabolites,there were significant differences between the AFE group and the AAI group,including the in vivo metabolites related to AAI,suggesting that the coexistence of various AAs components in aristolochia may have certain effects on the metabolism of AAI contained in them. |
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