Study On The Changes Of The Chemical Constituents And In Vivo Absorption And Metabolism Process Of Polygonum Multiflorum Radix Via Processing

ObjectivePolygonum multiflorum Radix（PM）has obvious different pharmacodynamic and toxic effects characteristics of raw and processed products.In recent years,PM-induced liver toxicity has been attracted wide attention.Up to now,many researchers have focused on the toxic components analysis of PM,but still has been inconclusive.That is mainly due to the complex chemical constitution of the herbal medicine,which effect the organism together and form a multiple target network system.Only one single compound or such a few of them can not represent the global effect of the herbal medicine.In addition,the toxic effects are directly related to the chemical components which absorbed or metabolism in vivo.Previous researches had clearly defined that the toxicity of PM is significantly reduced after processing,and compatibility with Sojae Seman Nigrum showed a significant effect of reducing toxicity and increasing efficiency of PM.Therefore,it is of great significance to clarify the effects of processing on the chemical composition and in vivo disposal of PM.In this study,UPLC-LTQ-Orbitrap MSn system was were carried out to provide clear chemical profiles of raw and processed PM.Normally.For only the exogenous chemicals with high enough exposure in target organs are considered as the potential effective and/or toxicity components,RPM and its processed products were respectively oral administrated in rats and xenobiotics in rat biosamples were detected and characterized.Then a UPLC-Exactive-Series MS system was employed to detect the dynamic changes of the compounds in vivo.A systematic analysis was performed to compare the changes of the compounds in normal or toxic states of rats after oral administration of raw and processed PM extracts,and the compatibility effect of Sojae Semen Nigrum was also considered.Based on chemical material basis in vivo,the toxic components were explored for improving the quality control and clinical safety of PM.Methods（1）spm（PM preparation only steamed with water）and bpm（PM preparation steamed with Sojae Semen Nigrum extract）were processed from the same batch of RPM（raw PM）in our laboratory.For the preparation of spm,the dry roots of 1 kg rpm were infiltrated by water and steaming at 100℃ for 24 h.The processed products were then dried under the drying oven at 55℃.For the preparation of bpm,100 g of Sojae Semen Nigrum were extracted twice with water（2 X600 mL,4 h per extraction）and the combined extract was condensed to 200 mL.After infiltrated by the black bean extract,the roots of rpm were then steaming at 100℃ for 24 h and then dried.The dry roots of rpm,spm,and bpm were extracted using following method:1 kg dry roots were extracted thrice with 70%ethanol（3 X 800 mL,60 min per extraction）,and the combined extracts were condensed to 500 mL under reduced pressure,respectively.（2）The qualitative analysis of the compounds was performed by the Thermo-fisher LTQ-Orbitrap XL hybrid mass spectrometer（Thermo Fisher Scientific,Bremen,Germany）coupled with Accela UHPLC instrument via an ESI interface.The software of Mass Frontier 6.0（Thermo Fisher Scientific,MA,USA）and Xcalibur 2.1（Thermo Fisher Scientific）was employed for data analysis.（3）The male specific pathogen free Sprague-Dawley（SD）rats were randomly assigned to five groups and orally administrated with different drugs.Rpm group:the rats only received rpm extract（10 mL/kg,2 g/mL）;spm group:the rats only received spm extract（10 mL/kg,2 g/mL）;bpm group:the rats only received bpm extract（10 mL/kg,2 g/mL）;rpmb group:the rats received rpm extract（10 ml/kg）and Sojae Semen Nigrum extract（5 mL/kg,1 g/mL）;and spm group:the rats received spm extract（10 mL/kg,2 g/mL）and Sojae Semen Nigrum extract（5 mL/kg,1 g/mL）.Each group was further divided into three sub-groups for plasma,urine,and bile sample collections.All rats were oral administration of the same and equal drugs once more time after 2 hours.Blood samples（400 μL）from orbital veins were collected at 0.5,0.75,1.0,2.0,and 4.0 h in heparinized 1.5 mL polythene tubes after the second administration and the plasma samples from the same rat were mixed.For bile sampling,a cannular was surgically implanted into the bile duct which allowed continuous collection of bile for 4 h after the second administration.For urine sampling,the rats were held in metabolism cages,and urine samples were collected for 6 h after the first administration.The qualitative analysis of the compounds also was performed by the Thermo-fisher LTQ-Orbitrap XL hybrid mass spectrometer（Thermo Fisher Scientific,Bremen,Germany）coupled with UHPLC instrument via an ESI interface.The MS data of the samples were acquired in full scan mode and data-dependent MSn scan was performed by dynamic exclusion setting.Comparing the total ion chromatograms（TIC）of treatment and blank samples,the probable prototype components and metabolites were screened out with endogenous components excluded.（4）The SD rats were randomly assigned to eight groups,including rpm group,spm group,bpm group,rpm-10-1 group,rpm-5-1 group,spm-10-1 group,spm-5-1 group and control group.Rpm,spm and bpm groups were oral administrated with rpm,spm and bpm extracts for 20g/kg,respectively.Rpm10-1 and rpm-5-1 groups were oral administrated with the same dose of rpm extracts and combined with low（4g/kg）or high（8g/kg）dose of Sojae Semen Nigrum extract.Spm-10-1 and Spm-5-1 groups were oral administrated with the same dose of spm extracts and combined with low（4g/kg）or high（8g/kg）dose of Sojae Semen Nigrum extract.All rats were successive administration of the extracts for 1,7 or 14 days.The control group were administration of saline solution for 10 mL/kg every day.The general state of the rats were observed and blood samples were collected at 1 h after administration on the 3rd,7th and 14th days.liver function indexs were measured with a fully automatic biochemical analyser.Blood samples（300 μL）from orbital veins were collected at 5 min,10 min,20 min,30 min,45 min,60 min,90 min,2h,4h,6h,8h and 24h in heparinized 1.5 mL polythene tubes after administration on the 1st,7th and 14th days.The plasma samples were then analyzed by the UPLC-ExactiveSeries MS system,6 prototypes and 28 metabolites were simultaneous determination.Since most analytes have no reference substances,the ratios of analyte peak area（Ad）to internal standard peak area（Ai）were employed as ordinate and the time as the abscissa,the values of area under the curves were calculated for AUC’.Clustering analysis and PCA analysis were used to assign similar and dissimilar groups by AUC’ values and clarify the main influencing factors for PM components disposition in vivo.Comprehensive analysis the detoxification mechanism of processing from the material basis In vitro and in vivo.Results（1）A total of 147 compounds were detected in PM extracts,and most of them were the derivatives of emodin,tetrahydroxystilbene,catechin,gallic acid,and torachrysone.Most compounds were identified or tentatively identified by using of characteristic diagnostic fragmentions or references.38 compounds were only detected in the processed products,which were probably new components produced during the steaming process.（2）In vivo study,7 prototype components and 66 metabolites were detected or tentatively identified in rat plasma,urine and bile.Among them,24 metabolites were reported for the first time and the novel metabolic pathways of emodin,THSH,gallic acid,catechin,and torachrysone were revealed.The results indicated that the phase II metabolic processes were the main pathways of all the compounds and the majority of metabolites were emodin-or THSH-related.（3）In the process of long-time administration,the rats showed different response of toxic effect.On the 3rd day,the ATL values of nonprocessed groups were significant increased and had statistic difference which compared with the control group（P<0.05）,while the TBA values of processed groups were statistic decreased（P<0.05）.On the 7th day,the DBLL values of all the groups were significant increased,and had statistic difference which compared with the control group.On the 14th day,the IBLL values of all the groups were statistic decreased（P<0.05）.The clustering analysis results showed that the processed groups and the non-processed groups were grouped into two categories.The results of PCA analysis showed that the processed group and the non-processed group were well differentiated at 1d,7d,and 14d.There was a tendency to differentiate between the rpm group and the groups compatibility with Sojae Semen Nigrum extract on the 7th day,which with an obvious differentiate at the 14th day.The total exposure of compounds in the unprocessed groups was significantly higher than those in the processed groups.The compounds with the most multiples are M2（catechin-O-glucuronide）,M3₁（methoxyepicatechin-O-glucuronide）,M3₂（emodic acidglucopyranoside·H2O）,M6（THSG-O-glucuronide）,and M16（torachrysone-Oglucuronide）,M20（torachrysone-O-sulfate）.The AUC’ values of the six metabolites at Id in the rpm group were 11.66,10.01,54.78,7.56,87.28,and 449.06 times for the spm group,respectively.In addition,compounds M4₁（emodin-O-di glucuronide）,M4₂（emodin-O-di glucuronide）,M5（THSGO-glucuronide sulfate）,M7（3,5,4’-tetrahydroxystilbene-glucuronide sulfate）,M9（3,5,4’-tetrahydroxystilbene-glucuronide）,M10（hydroxyemodin-O-glucuronide）,M11（3,5,4’-tetrahydroxystilbene-sulfate）,M12（emodic acid-3-O-glucuronide）,M13₁（emodin-O-glucuronide）,M14₃（emodin-O-sulfate）,M17（emodic acid-3-O-sulfate）,P3（polydatin）and other compounds can be more than doubled in the non-processed group during the administration process.With the extension of the administration time,the exposure of most compounds in the rpm group decreased,while the exposure of the spm and bpm groups increased.The compounds M7（3,5,4’tetrahydroxystilbene-glucuronide sulfate）,M9（3,5,4’tetrahydroxystilbene-glucuronide）and M11（3,5,4’-tetrahydroxystilbenesulfate）have a certain accumulation effect.The long-term compatibility of Sojae Semen Nigrum had a greater effect on the in vivo exposure of compounds in the rpm group,which can significantly increase the exposure of the metabolites.Conclusion（1）The processing has a greater impact on the material base composition of PM,mainly including the following three points:1)Hydrolyzation of the derivatives.There are a large number of glycosides and derivatives in PM.These components are prone to hydrolysis under long-term high temperature conditions.2)Formation of new isomers.There are many isomers in PM.In the processing process,isomerization reaction occurs,and new isomers are formed.3)New derivatives are formed.There are a large number of derivatives in PM.In the process of decomposition,it was easier to undergo substitution reactions with small organic compounds and generate new derivatives.The processes of compound changes during processing were very complicated mainly due to the influence of high temperature,few by Sojae Semen Nigrum.（2）At the same administration dosage,the toxic process of processed groups was slower than that of unprocessed groups.Processing and compatibility of Sojae Semen Nigrum had almost no effect on the types of absorbed components and metabolites,but most of them had higher exposure in the non-processed groups.Processing,duration of administration,and compatibility of Sojae Semen Nigrum all have effects on the process of PM in vivo disposal,of which processing is the primary influencing factor.（3）The reduction of the total exposure of exogenous compounds in vivo might be the main reason for the reduced toxicity of Polygonum multiflorum Radix after processing.With the extension of the administration time,various functions of the were body changed.The effect of compatibility of Sojae Semen Nigrum was not produced through direct interaction between the compounds,but involved through the regulation of the body’s functions in the long-time process of administration.The toxic mechanism of Polygonum multiflorum Radix may be related to the content of stilbene glycosides and its related derivatives.During the continuous administration process,large amount accumulation of the stilbene glycoside related metabolites including 3,5,4’-tetrahydroxystilbene-glucuronide sulfate,3,5,4’tetrahydroxystilbene-glucuronide and 3,5,4’-tetrahydroxystilbene-sulfate in vivo might lead to liver cell damage by causing biliary excretion obstacles.