| Chinese medicine processing is a unique pharmaceutical technology. Rhubarb is a "typical example" it is used as the raw and the processed in clinical. This thesis focused on processing can change the property of rhubarb. Using UHPLC-Q-TOFMS technology combined with the spectrum effect relationship is to explore the changes of chemical components of rhubarb before and after processing and to determine the effective components related to pharmacodynamic differences. Effect of rhubarb has changed on the purgative activity. It was used as start point that the purgative activity has weakened after being processed. The hot junction constipation model was established in line with the root-and-branch theory of Chinese Traditional Medicine(TCM). Dachengqi decoction was used as a carrier to evaluate the effect of defecation, gastrointestinal hormones and neurotransmitters, water channel protein and H22 tumor growth on hot junction constipation model. From the viewpoint of prescription, it is processing that can change the property of rhubarb was rational and correct. ObjectiveThis thesis focused on the mechanism of rhubarb processing that the purgative activity has weakened after being processed and the raw rhubarb and the processed rhubarb can treat different disease. Using UPLC-Q-TOFMS technology combined with the spectrum effect relationship is to explore the changes of chemical components of rhubarb before and after being processed and to determine the effective components related to pharmacodynamic differences. Establishment of hot junction constipation model for evaluation of the difference of the purgative activity between the raw and the processed. Mechanism of rhubarb processing was researched by investigating the anti-tumor activity of HepG2, MG-63, B16, Hela cells in vitro and inhibitory effects of H22 on tumor bearing mice.MethodThe research on processing mechanism of rhubarb was carried out by using literature, pharmacology and chemical method.1 The changes of chemical components of rhubarb before and after processing was investigated by using UPLC-Q-TOFMS technology combined with the spectrum effect relationship to determine the effective components.2 By using the pharmacological methods and establishing hot junction constipation model, the efficacy differences between the raw and the processed rhubarb was carried out not only taking alone but also prescription.3 By using colormetric assay, the effect of the different concentrations(0,2.5,5, 10,20,40,80 mg/mL) of the raw and the processed rhubarb extract on the inhibitory effect on HepG2, MG-63, B16, Hela cell proliferation was investigated.4 By establishing the ascites hepatoma cell line of mice transplanted model, the effect of efficacy differences between the raw and the processed rhubarb on mice general state, thymus index, spleen index, inhibition rate, the survival rate of mice was investigated.5 By using hot junction constipation model, efficacy differences between the raw and the processed rhubarb was studied on fecal index, gastrointestinal hormones, neurotransmitters and other aspects.6 By using the channel protein AQP2, AQP3, AQP4 gene expression combined with the "VIP-PKA-CAMP-AQP3" signal pathway, the mechanisms of rhubarb processing that the purgative activity has weakened after being processed have been elucidated.7 To reveal the correlation between changes in the efficacy of rhubarb and intrinsic chemical composition, the spectrum-effect relationship was established by using the grey relational analysis between the UHPLC fingerprint and El, AQP3,ET-1.Results1 The defecation experiment and diarrhea index determination of normal mice and hot junction constipation mice model showed that the raw rhubarb and its prescription Dachengqi Decoction each dose group EI significantly increased, the processed rhubarb and its prescription Dachengqi Decoction each dose group El not changed significantly. Above experiments indicate that the purgative effect of the raw rhubarb is stronger than the processed rhubarb (P<0.05) and the mechanisms of rhubarb processing that the purgative activity has weakened after being processed have been proved from single medicine to prescription.2 Gastrointestinal hormones-Neurotransmitter-Ghrelin system:Compared with the raw group, the contents of GAS, MTL, VIP, NT, SS, AchE, SP and ET-1 in serum and MTL, VIP, GAS and NT in colon of the processed group were significant differences(P<0.01, P<0.05). By inhibiting the release of VIP in the colon, the raw rhubarb can increased the levels of MTL, NT, SS, SP, MTL and AchE in serum and NT in colon. In a word, the raw rhubarb is able to increase colonic motility and promote defecation.3 Gastrointestinal hormones-Neurotransmitter-Ghrelin system:Compared with Dachengqi Decoction using the raw group, the levels of GAS, MTL, VIP, NT, SS, AchE, SP, and ET-1 of the processed were significant differences(.P<0.01, P<0.05).4 The expression of water channel protein in the colon:The expression of AQP2, AQP3 and AQP4 were increased in colon of hot junction constipation mice model, as the mean while, the expression of AQP2, AQP3 and AQP4 were decreased in colon of the raw, Dachengqi Decoction using the raw, and Dachengqi Decoction using the processed respectively. Compared with the model group, the raw andDachengqi Decoction using the raw group were differences significantly(P<0.05), the processed and Dachengqi Decoction using the processed group have gradual downward trend.5 VIP-CAMP-PKA-AQP3 signal pathway:The levels of CAMP and PKA of the raw group were decreased significantly(P<0.05, P<0.01), the levels of PKA of the processed group were decreased significantly(P<0.01), the levels of CAMP and PKA of Dachengqi Decoction using the raw group were decreased significantly(P<0.01), the levels of PKA of Dachengqi Decoction using the processed were decreased significantly(P<0.01), compared with the model group. The raw group were differences significantly compared with the processed group(P<0.05). Dachengqi Decoction using the raw group were differences significantly compared with Dachengqi Decoction using the processed group(P<0.05). The expression of AQP3 in colon decreased significantly, the other groups have gradual downward trend compared with the model group.6 ENS-ICC-SMC: Compared with model group, the expression of ICC both Dachengqi Decoction using the raw and the processed group were increased significantly(P<0.01), the expression of ICC of the raw group were increased significantly(P<0.05).7 Inhibition of HEPG2, MG-63, B16, HELA:The water extracts of the raw and the processed rhubarb had no inhibitory effect on all the four tumor cells. The ethanol extract of the raw rhubarb had a little inhibitory effect on HEPG2 and HELA, had no effect on MG-63 and B16. The ethanol extract of the processed rhubarb had obvious inhibitory effect on HEPG2 and HELA.Rhein-8-O-p-D-glucopyranoside (RHG) had a little inhibitory effect on HELA, emodin-8-O-p-D-glucopyranoside(EMG)and hyscion-8-O-β-D-glucopyranoside(PHG) had no effect. The RHG had obvious inhibitory effect on HEPG2. The EMG had a little inhibitory effect on HEPG2. The PHG had no effect. RHG and EMG had a little inhibitory effect on MG-63. The PHG had no effect on MG-63. RHG, EMG and PHG had no obvious effect on B16.8 H22 tumor bearing mice:Compared with the model group, all of the processed groups, high dose of the raw group and the tegafur group had obvious differences of average tumor weight(P<0.01, P<0.05), M and L dose group had no significant differences.Compared with the model group, all of the processed groups and the low dose of the raw group had obvious differences of the thymus index(P<0.01,P<0.05). Compared with the normal group, all of the processed groups had no significant differences of the spleen index.Compared with the model group, all of the processed groups had obvious differences of the survival time(P<0.01, P<0.05), all of the raw groups and tegafur group had no differences of the survival time. Compared with the tegafur group, all of the processed groups had obvious differences of the survival time(P<0.05). Compared with the raw group, all of the processed groups had obvious differences of the survival time(P<0.05).9 The chemical composition of raw, processed rhubarb were identified by using UPLC/Q-TOF MS technology, this experiment detected more than 50 kinds of ingredients in rhubarb,46 compounds were preliminary identified, including 28 kinds of anthraquinone compounds,2 kinds of stilbene glucoside compounds,3 kinds of tannin compounds,2 kinds of flavonoids, the others 9 compounds.10 The fingerprints of the raw and processed rhubarb were established by using UHPLC/Q-TOF MS Technology.11 Spectrum-effect relationshipEI correlation coefficient is greater than 0.70: physcion-8-O-β-D-glucopyranoside>sennosideB>emodin-1-O-β-D-glucopyranoside> torachrysone-8-O-β-D-glucopyranoside>emodin>(-)epicatechin-3-O-gallateAQP3 correlation coefficient is greater than 0.70:gallicacid>aloe-emodin-3-CH2-O-β-D-glucopyranoside>Torachrysone>rhein>chrysophano 1>sucrose>physcion>catechin>aloe-emodin>gallic acid-4-O-β-D-glucopyranosideET-1 correlation coefficient is greater than 0.70:physcion-8-O-β-D-glucopyranoside>emodin-1-O-β-D-glucopyranoside>sennoside B>torachrysone-8-O-β-D-glucopyranoside>emodin>(-)epicatechin-3-O-gallateConclusion1 Purgative activity difference between the raw and processed rhubarb was investigated by establishing and using the model combining disease with syndrome.The results indicated that El of the raw rhubarb is bigger than the processed rhubarb. All the same results were obtained from the Dachengqi Decoction using the raw or the processed rhubarb. The purgative activity has weakened after being processed.2 Taking the gastrointestinal hormone, neurotransmitter, expression of water channel protein, VIP-CAMP-PKA-AQP3 pathway, and ENS-ICC-SMC as the starting point, the mechanism of rhubarb processing that the purgative activity has weakened after being processed was investigated intensively.3 The anti-tumor experiments was carried out in vitro and in vivo, from the whole to the components. The results indicated the anti tumor activity of the raw is stronger than that of the processed. Processing can enhance promoting blood circulation and removing blood stasis effect of the rhubarb.4 Spectrum-effect relationship research was carried out to find out the relationship between the differences in efficacy and differences in components.The results are as follows:physcion-8-O-β-D-glucopyranoside, sennosideB. gallic acid, catechin, physcionInnovation1 The hot junction constipation model was established in line with the root-and-branch theory of Chinese Traditional Medicine(TCM). The prescription was used as a carrier to study the mechanism of rhubarb processing.2 To study the mechanism of rhubarb processing that the purgative activity has weakened after being processed, many experiments were carried out By using multi index and multi channel.3 By carrying out anti-tumor experiments in vitro and in vivo, from the whole to the components, the mechanisms of rhubarb processing that the purgative action has changed into the tonic effect after being processed and the raw rhubarb and the processed rhubarb can treat different disease were elucidated intensively.4 Spectrum-effect relationship research was carried out to find out the relationship between the changes of the material basis and the effect of processing, which provides a new method for the research of processing principle of Chinese herbal medicine.
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