Pharmacodynamic Effect Of "Laxative Followed By Astringent" Of Raw Rhubarb And Mechanism Studies Based On Metabolomics

Rhubarb,originally listed in《Shen Nong Ben Cao Jing》，is one of the most commonly used traditional Chinese medicines in clinical practiceand has a wide range of pharmacodynamic effects,which can be used for laxative,invigorating blood circulation and eliminating stasis,diuresis,anti-inflammation,and antitumor effects.In traditional Chinese medicines that affect the world,rhubarb is included.The medicinal effects of rhubarb for the treatment of constipation have been documented in the Pharmacopoeia of nineteen countries.There are currently more than 120 types of standard compound Chinese patent medicines containing rhubarb with laxative effects.Thus,as a kind of clinical medication of diarrhea effect,rhubarb has precise efficacy and remarkable effects.However,recent clinical studies have found that raw rhubarb not only has a "diarrhea"effect but also a"convergence"effect.Many patients with habitual constipation and young women,who blindly pursue weight loss,take long-term,high-dose laxative medicines containing raw rhubarb,producing good laxative effects at the initial stage,but more severe side effects such as constipation and even colonic melanosis appear in the later stage,seriously harming their own health.The pharmacodynamic effects or side effects originate from the pharmacodynamic substance basis in medicines.The "diarrhea followed by convergence" effect that occurs clinically with the long-term use of raw rhubarb is related to the active ingredients it contains.Our group has systematically studiedthe pattern of changes in the chemical composition of different processed products of rhubarb and their correlation with changes in drug property for many years.Based on the previous study,we took the phenomenon of "diarrhea followed by convergence"of raw rhubarb as an entry point and took the steamed rhubarb,which has moderate drug property and weak laxative effect for contrast to reveal the material basis of the pharmacodynamic effect of "diarrhea followed by convergence" of raw rhubarb by systematically examining the correlation between its chemical composition and efficacy.Based on UPLC-MS/MS metabolomics technology,we systematically compared the blood obtained from normal animals and animal models of rhubarb intervention at different time nodes,dissected biomarkers with monitored efficacy during the period from diarrhea to constipation at the molecular level,and explored the "diarrhea followed by convergence" effect mechanisms of raw rhubarb to provide a scientific basis for reasonably guiding the clinical use of rhubarb.1.Quantitative characterization of chemical constituents in raw rhubarbUsing UPLC-DAD analysis,12 chemical components in the raw rhubarb extractwere quantified in this study.These 12 components were aloe-emodin,rhein,emodin,chrysophanol,physcion,aloe emodin-8-o-β-D-glucoside,rhein-8-o-β-D-glucoside,emodin-8-o-β-D-glucoside,ethyl gallate,lindleyin,isolindleyin,4-(4’hydroxyphenyl-2-butanone-4’-O-β-D-(2"-O-galloyl-6"-O-p-coumaroyl-glucophyranos ide.The concentrations of each component in raw rhubarb were 0.05%,0.66%,0.19%,0.12%,0.36%,0.23%,1.81%,0.29%,0.99%,5.46%,7.73%,1.69%,respectively.For the subsequent study,steamed rhubarb was used as a control sample,the determination of each component in steamed rhubarb was performed at the same time and showed that 0.13%,1.29%,0.23%,0.21%,0.55%,0.18%,1.76%,0.14%,0.03%,2.49%,2.89%,0.12%,respectively.Compared with raw rhubarb,the content of anthraquinone glycosides and phenylbutanone glycosides in steamed rhubarb such as:aloe-emodin-8-O-β-D-glucoside,rhein-8-o-β-D-glucoside,emodin-8-o-βD-glucoside,4-(4’-hydroxyphenyl-2-butanone-4’-O-β-D-(2"-O-galloyl-6"-O-p-coumaroyl-glucoph yranoside,ethyl gallate,lindleyin,isolindleyin and phenylbutanone glycosides decreased,whereas the content of aloe-emodin,rhein,emodin,rhein,and physcionin raw rhubarb increased.The study of quantitative characterization of each component in raw rhubarb provides a research basis for further elucidating the material basis associated with "laxative followed by astringent" of raw rhubarb.2.Pharmacodynamic study of "laxative followed by astringent" of total extracts and active fractions in raw rhubarbSteamed rhubarb was used as a control to investigate the pharmacodynamic effects of raw rhubarb that "laxative followed by astringent" effect.Raw rhubarb extracts were administered to mice by gavage for 7 consecutive days,and samples were taken on Day 1,3and 7 of dosing.The diarrheal index(EI),changes in the level of three gut hormones as motilin(MTL),vasoactive intestinal peptide(VIP),epinephrine(EPI)in serum were measured and pathological changes in the colonic tissues of mice to clarify the "diarrhea followed by convergence" pharmacodynamic effects of raw rhubarb.Meanwhile,to explore the relevant medicinal components responsible for the pharmacodynamic effects of "laxative followed by astringent" of raw rhubarb,further pharmacodynamic study was performed on the anthraquinone and tannin fractions of raw rhubarb.The EI and the levelchanges of three gut hormones in serum were measured.El results showed that the laxative effect was quite significant on Day 1 administration of raw rhubarb,the highest on Day 3,and the decrease in diarrhea on Day 4,while the phenomenon of constipation appeared on Day 5 and gradually aggravated on Day 6 and Day 7.The anthraquinone component of raw rhubarb did not exert a significant laxative effect during the early period and showed constipation on Day 5,which was maintained thereafter.The tannin component of raw rhubarb exhibited a gradually increased laxative effect in the early stage.It showed the strongest laxative effect on Day 3,and showed a gradually decreased laxative effect in the later stage,and the phenomenon of constipation appeared on Day 7.Raw rhubarb had obvious pharmacodynamic effect of "laxative followed by astringent",in which anthraquinones and tannins components are the relevant components for this effect.The ratio of anthraquinone glycosides to aglycones in raw rhubarb was 4:1,speculating that the high concentration of anthraquinone glycosides inhibited the activity of intestinal flora,so that anthraquinone glycosides could not be hydrolyzed to aglycones and the laxative effect disappeared.The condensed tannins in raw rhubarb have a strong laxative effect during the pre-administration period,but a"astringent" effect because condensed tannins would be hydrolyzed to monomeric tannin gallic acid in the intestine.The presence of anthraquinone and tannin components in raw rhubarb made raw rhubarb with the "misplaced effect" of"laxative followed by astringent".In vivo hormone level assay results showed that the increased MTL level and decreased VIP and EPI level on Day 1 of raw rhubarb administration and it played a role in diarrhea.Although MTL secretion inhibited,raw rhubarb simultaneously reduced EPI concentration to exert a laxative effect.However,raw rhubarb inhibited the MTL secretion effect and promoted a large increase in the concentrations of VIP and EPI,exerting a convergent effect,and the phenomenon of constipation appeared on Day 7.In vivo hormone level measurements showed that raw rhubarb may exert its pharmacodynamic effects of "laxative followed by astringent" by affecting the changes of hormone levels in vivo,such as MTL,VIP,and EPI.It may be that different hormones exert their primary effects during different medication periods,and the common effects of these hormones promote raw rhubarb to exert its Pharmacodynamics effects.Anthraquinones and tannins components of raw rhubarb would affect the levels of hormones such as MTL,VIP,and EPI in the body,thus exhibiting different pharmacodynamic effects in the early and later stages.The result of pathological sections showed that long-term administration of raw rhubarb produced inflammation in the colon of mice.Therefore,it is speculated that the side effect of constipation in the later stage of raw rhubarb may be associated with inflammation.3.A metabolomics study on the mechanism of the "laxative followed by astringent"effect in raw rhubarbThis chapter utilized UHPLC/MS methods with high resolution and sensitivity,and untargeted metabolomics profiling was performed using normal phase and reverse chromatographic separationto investigate the effects of long-term administration of raw rhubarb on serum endogenous metabolites in mice,and to explore the mechanism of effect of "laxative followed by astringent" in raw rhubarb.The PCA plot showed that there was a clear separation trend between the raw rhubarb group and the blank group,which indicated that the administration of raw rhubarb could change the metabolism in mice and there were obvious differences in endogenous metabolites.The raw rhubarb and steamed rhubarb groups showed aggregation trends in different directions,indicating that raw rhubarb and steamed rhubarb have different regulatory effects on metabolism in mice.Metabolites with VIP>1 and P<0.05 were identified as characteristic metabolites by OPLS-DA model and T-test.A total of 63 and 55 differential metabolites were screened in the early phase(Day 1)and late phase(Day 7)of drug administration,respectively.Most of the selected metabolites were phosphatidylcholine(PC),lysophosphatidylcholine(LysoPC),amino acidand carnitine classes.Allopurinol,hypoxanthine,oleamide,sphingomyelin(SM),and creatine were added to the list.Compared with the blank control,most of the PC and LysoPC changed from up-regulation on Day 1 to down-regulation on Day 7.This reverse regulation may be associated with the "diarrhea followed by convergence" effect of raw rhubarb.PC and lysoPC are able to inhibit the up-regulation of inflammatory factors.Therefore,down-regulation of these metabolites may trigger inflammation,which in turn leads to constipation.Compared with the blank control,L-arginine changed from down-regulation on Day 1 to up-regulation on Day 7.L-proline could not be detected on Day 1,but showed an up-regulation trendon Day 7.L-histidine was significantly down-regulated on Day 1 and was not detected onDay 7.L-valine levels were down-regulated on Day 1 but became up-regulated on Day 7.The reversed regulation seen on Day 1 and Day 7 of these amino acid metabolites may affect the pharmacodynamic effects of raw rhubarb through their respective mechanisms of action,which further produces the phenomenon of "laxative followed by astringent".For carnitine components;,the content of L-carnitine,L-acetylcarnitine as well as other acylcamitines was down-regulated on Day 1,and most carnitines were undetectable on Day 7.Deficiency of carnitine may be associated with constipation as well as inflammation.Compared with the blank control,both of allopurinol and hypoxanthine were up-regulated on Day 1 but down-regulated on Day 7.These metabolites are associated with colonic inflammation and may cause the"convergence" effect of raw rhubarb by regulating inflammatory pathways.Oleamide was not detected on Day 1,but was detected on Day 7,speculating that the production of oleamide may be one of the causes of secondary constipation.Metabolomics results showed that the differential metabolites screened mainly included:phospholipids such as PC,LysoPC,amino acids such as L-arginine,L-proline,L-histidine,L-valine,carnitines such as L-carnitine,acylcarnitines,and other small molecules such as allopurinol,hypoxanthine,and oleamide.The results of metabolomics studies showed that the reason of the "laxative followed by astringent"pharmacodynamic effect of raw rhubarb after long-term administration may be closely related to the metabolisms of these differential metabolites.The analysis of metabolic pathways suggested that glycerophospholipid metabolism pathway,arginine metabolism pathway,proline metabolism pathway and histidine metabolism pathway may be associated with the side effects of secondary constipation of raw rhubarb.The "laxative followed by astringent" effect of raw rhubarb may be related to inflammation,and the differential metabolites may produce side effects of constipation by regulating inflammatory signaling and molecules.Therefore,raw rhubarb should not be used for a long time when it is used clinically as a laxative medicine.Long term administration triggers intestinal inflammation,impaired intestinal function,and subsequently side effects of constipation.Especially in patients with spleen and stomach of qi deficiency or yang deficiency,use caution.However,steamed rhubarb consistently exerted a stable,moderating laxative effect during the administration period,and no inflammation appeared.Meanwhile,raw rhubarb processed as steamed rhubarb,which can be applied for clinical treatment of constipation for a long time,and it also reveals the significance of TCM processing.Therefore,it can be used to treat constipation in patients with weak body.The research in this thesis is of great importance to reasonably guide the clinical use of rhubarb and avoid its secondary constipation side effects.