Study On The Material Basis,Process And Hepatotoxicity Reduction Of Braised Polygoni Multiflori Radix

ObjectivePolygoni Multiflori Radix（PMR）,first published in the Kaibao Ben Cao,is a traditional Chinese medicine that is used in both raw and prepared forms.It is often used to treat sores and carbuncles,scrofula,itchy rubella,chronic malaria,and constipation.After prepared,Polygoni Multiflori Radix Praeparata（PMRP）is used to nourish the liver and kidney,benefit the essence and blood,darken the hair,strengthen the tendons and bones,remove turbidity,lower lipids,and is often used to treat blood deficiency and yellowing,dizziness and tinnitus,premature greying of the hair,soreness of the waist and knees,numbness of the limbs,collapse and deficiency of the belt,and hyperlipidemia^[1].The difference in efficacy and application between raw and prepared Radix et Rhizoma Polygonatum is evident in the significant effect of a concoction on it.However,studies have shown that different concoction methods result in significant differences in chemical composition changes,potency and hepatotoxicity of He Shou Wu.Braised Polygoni Multiflori Radix Praeparata（BPMR）is one of the specialties of the Jiangxi Jianchang Gang.Braising and steaming can eliminate the diarrhea-causing effect and enhance the tonic power,while wine can enhance the warming power.However,the chemical composition of BPMR has not yet been systematically studied,and the preparation process still needs to be optimized,and the research on the reduction of hepatotoxicity is still not in-depth.In this study,the chemical composition of BPMR from different origins before and after concoction was studied systematically and compared with that of PMRP recorded in the Chinese Pharmacopoeia（2022 edition）.Finally,a zebrafish model was used to compare the hepatotoxicity of BPMR with that of the other two processed products.The aim of this study is to provide a material basis for the elucidation of the concoction mechanism of BPMR and a scientific basis for the formulation of quality standards and the clinical application of BPMR.Methods and results（1）The color parameters of different processed products of PMR were determined using a color difference meter.The results showed that the L*and E*ab of PMR,PMRP,and BPMR decreased in order,indicating that the apparent brightness of the samples gradually deepened from bright to dark.This is consistent with the color trends of the sample powders:the PMR is yellowish-brown,the PMRP is brownish-yellow,and the BPMR is brownish-brown.The microscopic differences between the different processed products of PMR were characterized by scanning electron microscopy（SEM）.At a low magnification of 800×,the surfaces of both BPMR and PMRP were relatively smooth and not significantly different,while closely arranged cells were visible on the surface of PMR.With increasing magnification,the structure of the surface of the concoction became clearer.At a low magnification of 6400×,the surface of PMR was visible as undamaged intact single cells with compactness between adjacent cells;some small cells remained on the surface of the PMRP,while the surface of BPMR was still smooth and no residual cells were found.This indicates that the color and microstructure of PMR changed significantly after concoction,and that the changes in BPMR were more pronounced than those in PMRP.（2）The odour of different prepared products of PMR was identified by electronic nose,and the volatile components were analysed by HS-GC-MS technique for a total of 30 batches of PMR,BPMR,and PMRP from three main origins.A total of 59volatile components were identified,with 11 components common to all three.The relative percentage of 2-methyl-2-butenal was much higher in BPMR and PMRP than in the other three batches,and PCA and OPLS-DA analyses revealed that the volatile components of the three batches differed significantly and could be grouped into three distinct categories.The method of processing had a greater influence on the volatile components than the origin.According to the principle of VIP≥1.5,12 and 11different volatile components were obtained from PMR and BPMR,BPMR and PMRP,respectively;among them,aldehydes accounted for the major part.The relative percentages of the three 11 common components and the associated colour value parameters were correlated,and significant correlations（P<0.05）were obtained between L^*and three volatile components,including 2-methyl-2-butenal,2-methyltetrahydrofuran-3-oneand2,3-dihydro-3,5-dihydroxy-6-methyl-4（H）-pyran-4-one.（3）The contents of total polysaccharides,total polyphenols,total saponins,and total flavonoids in different processed products of PMR were determined by the phenol-sulfuric acid method,Folin-Ciocalteu method,vanillin-perchloric acid colorimetric method,and Na NO₂-Al（NO₃）₃-Na OH colorimetric method,respectively.The content of total polysaccharides in PMR after braising increased significantly,while the content of total polysaccharides in PMRP by pharmacopoeia decreased significantly;the total polyphenols,total saponins,and total flavonoids in PMR decreased after processing,and the content of BPMR was significantly lower than that of the other two concoctions.（4）UPLC-Q-TOF-MS/MS was used to analyze nonvolatile components in 30batches of PMR,BPMR,and PMRP from three different origins.65 components were identified,including 16 stilbenes,21 anthraquinones,22 flavonoids,and 6 others.The non-volatile components of PMR could be clustered into three distinct categories,and the effect of the concoction method on the non-volatile components of PMR was greater than the factor of origin.According to the principles of VIP≥1 and P≤0.5,36different non-volatile components were obtained from the three species.Among them,33components,includingemodin-8-O-（6′-O-malonyl）-glucoside,physcion-8-O-（6′-O-acetyl）-glucoside,proantho-cyanindins,and caspianin-8-O-glucoside,decreased or disappeared in response intensity after braising.（5）By HPLC fingerprint graph,effective component content determination,and chemical measurement model identification methods such as HCA,PCA,and OPLS-DA for the three main sites,a total of 30 batches of PMR and its different processed products were used for quality analysis studies.In the similarity of fingerprint graphs,PMR,BPMR,and PMRP,each of the 10 batches’similarity is>0.99,indicating the difference in the component group between the different batches of the same drink is not significant and the overall quality of the same cannon product is more stable.In the total of 30 batches,the similarity of PMR,BPMR,and PMRP is 0.893 to 0.925,0.799 to 0.892,and 0.976 to 0.993,respectively,and the similarities of different types of cannon products can be seen decrease.Compared with PMR and PMRP,the content of gallic acid,emodin,and physcion is significantly increased,while the contents of diphenyl ethylene glycoside,emodin-8-O-（6′-O-malonyl）-glucoside,and physcion-8-O-（6′-O-acetyl）-glucoside are significantly reduced.（6）This study carried out two orthogonal experiments and three single-factor experiments on the process of BPMR.by means of multiple factors such as the ratio of excipients,the amount of excipients,the state of the excipients,the process of braising temperature,temperature preservation,temperature reduction,wine steaming time,drying temperature,drying time,and so on,and used stilbene glycoside,resveratrol,emodin-8-O-glucoside,physcion-8-O-glucoside,emodin,physcion,and ethanol-soluble extract as the inspection indicators.Through the combination of the coefficient of variation method and the AHP composite weighted calculation weight and comprehensive score,the mathematical statistical analysis of multiple indicators was carried out.The best process for BPMR was obtained as follows:Take the pure PMR block,soak it with black beans overnight until there was no dry heart,put it in the stew tank,add 40–50°C warm water,cover it,and move it to the surrounding area.Place charcoal and dried bran（5 kg charcoal and 80 kg dried bran for every 100 kg of PMR）around the pot,ignite,and simmer for 21 hours（5 hours of heating,6 hours of heat preservation,and 10 hours of cooling）,until the bran is completely gray and cold,and take it out;Dry in a 75°C oven to 70%dry;Then mix the remaining medicine juice with the yellow rice wine and steam it for 6 hours after it has been sucked out.After ceasing fire for a night,take it out and put it in an oven to dry.For every 100 kg of PMR,10 kg of black beans and 20 kg of yellow wine are used.Three batches of medicinal materials were taken for process validation,and the results showed that the processing technology of BPMR was stable and feasible.（7）Finally,the zebrafish model was used to investigate the hepatotoxicity of different processed products of PMR.The mortality of zebrafish in each experimental group was calculated.The maximum non-lethal concentration（MNLC）and 10%lethal concentration（LC10）of the acute toxicity of BPMR,PMR,and PMRP to zebrafish were simulated by Origin Pro 2021 software.The results showed that the MNLC and LC10 of BPMR were 53.6 and 43.1 times those of PMR,or 18.2 and 12.6times,respectively.The PMRP(900 mg·L^-1)concentration group could significantly cause liver enlargement,which was statistically different from the normal control group（p<0.05）;however,PMR(60,180 mg·L^-1)and BPMR(60,180,540,900,2700mg·L^-1)had no significant effect on liver area.Both concentrations of PMR(60 and180 mg·L^-1)can cause liver degeneration,which is statistically different from the normal control group（p<0.05,p<0.01）;the PMRP(900 mg·L^-1)concentration group can cause obvious liver degeneration（p<0.01）;BPMR(60,180,540,900,and 2700mg·L^-1)did not cause liver degeneration and showed high safety.It can be seen from the results of the liver histopathological experiment that both the 60 and 180 mg·L^-1concentration groups of PMR have liver toxicity,the PMRP 180 mg·L^-1concentration is the safe concentration,and the BPMR 1350 mg·L^-1concentration is the safe concentration.The safe concentration of BPMR is 7.5 times that of PMRP and 22.5times that of PMR.ConclusionIn this paper,the appearance color and microstructure of different processed products of PMR were compared and analyzed based on color difference instruments and SEM.The results showed that the color of processed products from PMR deepened and the microstructure changed.Compared with the PMRP,the BPMR is darker in color and smoother in microstructure,which indicates that the stewing method is more suitable for the preparation of the PMR.The results of the electronic nose test showed significant differences in the odour of BPMR,PMR,and PMRP.Further HS-GC-MS was used to analyse the substance basis that formed the different odours of the three.The results of the correlation between color and composition showed that three volatile components were significantly correlated with L^*.The unique flavor of BPMR may be related to aldehydes in different components.The total polysaccharides,total polyphenols,total saponins,and total flavonoids in different processed products of PMR were determined by the principle of spectrophotometry.Compared with PMR,the total polysaccharides content of BPMR increased significantly,but the total polysaccharides content of PMRP decreased significantly;After two methods of processing,the content of the other three functional substances in PMR decreased,and the content of BPMR decreased more.The whole-component analysis of BPMR,PMR,and PMRP was then performed using the UPLC-Q-TOF-MS/MS technique,and 36 differential components were obtained.Among them,physcion-8-O-glucoside,gallocatechin gallate,and catechin were strongly associated components of the hepatotoxicity of PMR,and Polygonumnolides B1,Polygonumnolides C1 and Polygonumnolides A1 were also found to have weak hepatotoxic effects.These components decreased or disappeared in response intensity after braising.Multi-component analysis of different processed products of PMR was carried out by HPLC fingerprinting combined with the stoichiometric method.The similarity showed that the chemical components of PMR and PMRP were similar to those of BPMR.The results of HCA,PCA,and OPLS-DA could confirm this conclusion.After processing,the content of stilbene glycoside and bound anthraquinone in Polygonum multiflorum decreased,while the content of gallic acid and free anthraquinone increased.Compared with PMR and PMRP,the content of BPMR had significant changes.Furthermore,two orthogonal experiments and three single-factor experiments were designed based on important factors such as excipients,braising,winemaking,and drying to optimize the process of BPMR.Finally,in order to verify the hepatotoxicity advantages of BPMR,this study used a zebrafish model to evaluate the hepatotoxicity of BPMR,PMR,and PMRP from the aspects of lethal rate,delayed area of yolk sac absorption,liver pathological section,and other indicators,and concluded that the safe concentration of BPMR was 7.5times that of PMRP and 22.5 times that of PMR.This study will provide a reliable reference for the formulation of the quality standard for BPMR,provide a material basis for the clarification of its processing mechanism,and make a certain contribution to the exploration and inheritance of traditional characteristic stewing methods.