| Pinelliae Rhizoma(PR),is the dried tuber of Pinellia ternata(Thunb.)Breit belongs to Araceae family,is widely used to reduce phlegm,eliminate dampness and disperse phlegm,etc.in clinical application.The raw products of PR has strong irritant toxicity.If eaten accidentally,it can cause symptoms such as tingling in the lips and tongue,salivation,and swelling of the mouth and tongue.Its toxicity was mainly manifested as a strong irritation to the mucous membrane,so its processed products were generally used as a medicine,because its toxicity had been significantly reduced after processing.The main processed products including:Pinelliae Rhizoma Praeparatum Cum Alumine(PRPCA),Pinelliae Rhizoma Praeparatum Cum Zingibere Alumine(PRPCZ),and Pinelliae Rhizoma Praeparatum(PRP).From our team preliminary perspective,the toxic needle crystals and lectin protein were considered as the main toxic substance for PR.Toxic needle crystals had significant inflammatory effects,and the toxic lectin protein contained in needle crystals,which could significantly increase the inflammatory toxicity caused by toxic needle crystals.Preliminary studies had clarified the processing and detoxification mechanism of PRPCA and PRPCZ.That was,the excipient(alum)and the heating process could destroy the structure of toxic needle crystals and lectin protein,and gingerol could also antagonize the inflammatory response caused by its toxic components,resulting in a significant reduction in toxicity.However,PRP,as another processing product of PR,had completely different processing excipients from PRPCZ and PRPCZ.The detoxification mechanism of PRP processed with licorice juice and lime water was unknown.Clinically,PRP had been proved to dry dampness and resolve phlegm,and it was widely used for dispelling cold phlegm.However,the material basis related to the effect of "dampness-dampening and phlegmresolving" of PRP is still inconclusive,and the mechanism of action is unknown.Based on this fact,we aimed to investigate the detoxification mechanism of PRP by processing and its material basis and action mechanism of phlegm-resolving efficacy.The content of this thesis was divided into two parts,and the first part of the content:the detoxification mechanism by processing of PRP.This paper established the mouse model of celiac inflammation and the rabbit model of eyes irritation.Based on the inflammatory toxicity before and after the co-processing of PR with licorice juice and lime water,the effects of processing excipients(licorice juice and lime water)immersion on the toxic needle crystals and lectin protein of PR were investigated,and the key detoxification component in licorice juice was also further screened during the processing and detoxification of PRP.Meanwhile,the impact of soaking lime water of different pH on the toxic needle crystals and lectin protein,in order to reveal the detoxification mechanism of PRP.The second part of the content:the phlegm-resolving effect and material basis related to PRP.The paper established a model of cold asthmatic mouse,which examined the role of PRP intervention treatment,and the system solvent method was used to separate the effective part of PRP decoction.High secretion with sputum formation indicator(MUC5AC)and fluid transfer indicator(AQP5)were used to evaluate the phlegm-resolving effect of PRP.Furthermore,network pharmacology and transcriptomics were used to investigate the possible action mechanism of PRP decoction.GO function,and KEGG signaling pathway enrichment analysis were performed to clarify the target and action mechanism related to the role of PRP.Meanwhile,UPLC-Q-TOF MS/MS was used to detect the ingredients of PRP derived from mouse serum,and they were compared with the effective parts obtained from the separation from PRP.Through the above research,we hoped to reveal the detoxification mechanism of PRP by processing and the material basis of its phlegm-resolving effect,and provided scientific basis and theoretical support for the quality control,clinical safety application,processing technology reform and innovation of PRP.It also provided demonstration and reference for the research of other types of traditional Chinese medicine in Araceae.The specific research contents were as follows:The first part of the content:the detoxification mechanism by processing of PRP.1.Effects of the processing process of PRP on the inflammatory toxicity and toxic componentsPR was soaked with licorice juice alone,lime water,and licorice juice and lime water,respectively.The mouse model of peritoneal inflammation and the rabbit model of eyes irritation were established to investigate the inflammatory toxicity of the processed products of PR,which were soaked with licorice juice alone,lime water alone,and licorice juice and lime water together.The results showed that the suspension of crude PR could significant cause strong inflammatory toxicity.However,the inflammatory toxicity of the processed products of PR had been significantly reduced,which were soaked with licorice juice alone,lime water alone,and licorice juice and lime water together.Among them,the inflammatory toxicity of processed products of PR was the weakest,especially after soaking with licorice juice with lime water together.The lectin protein and toxic needle crystals were soaked in lime water with different pH(pH=10,pH=11,pH=12.4)and licorice juice(supernatant and precipitate of licorice juice alcohol precipitation).Western blot and scanning electron microscopy were used to examine the content of lectin protein and the crystal structure of toxic needle crystal.Western blot was used to investigate the effect of immersion in different pH lime water(pH=10,pH=11,pH=12.4)and licorice juice(supernatant and precipitate of licorice juice alcohol precipitation)on the content of lectin protein.The effect of immersion of toxic needle crystals in different pH lime water and licorice juice on its crystal structure was observed by Scanning electron microscope(SEM).The results showed that immersion in lime water with pH>12 could significantly reduce the content of lectin protein and destroy the crystal structure of toxic needle crystals.The content of lectin protein when soaked with licorice juice and alcoholic precipitation supernatant of licorice juice was significantly reduced,but it had no effect on the crystal structure of toxic needles.These results suggested that PH>12 may be the key condition for the detoxification of lime water by processing.licorice juice and lime water might simultaneously play a detoxification role,and the "small molecules" compounds in licorice juice were related to detoxification by processing.The above results suggested that licorice juice and lime water might play a synergistic effect during the processing process of PR.2.Study on the detoxification mechanism of licorice juiceUPLC-Q-TOF MS/MS technology was used to detect the dynamic changes of components in licorice juice during the processing of PRP,and the contents of glycyrrhizic acid,liquiritin and liquiritigenin in licorice juice were determined by HPLC.Western blot was used to investigate the effect of glycyrrhizin,liquiritin and liquiritigenin contained in licorice juice on the content of lectin protein.Western blot was used to investigate the contents of lectin protein which was soaked in the glycyrrhizic acid,liquiritin,and liquiritigenin contained in licorice juice.The influences of glycyrrhizin,liquiritin and liquiritigenin solution immersed toxic needles on its crystal structure was observed by SEM.Simultaneously,SDS-PAGE combined with silver staining was used to determine the protein composition of supernatant and precipitate after soaking with lectin protein in licorice juice and glycyrrhizic acid solution.MALDI-TOF MS/MS technology was used to detect the molecular weight distribution of peptides in the supernatant and precipitate after soaking with lectin protein in licorice juice and glycyrrhizic acid solution.The isoelectric point of lectin protein was determined by isoelectric point precipitation method,and the influence of licorice juice and glycyrrhizic acid solution immersion on the isoelectric point was investigated;In addition,transmission electron microscopy was used to observe the effects of licorice juice and glycyrrhizic acid solution immersion on the polymerization of lectin proteins.The results showed that the content of lectin protein was significantly decreased by soaking with glycyrrhizic acid solution,while the content of lectin was not significantly affected by soaking with liquiritin and liquiritigenin solution,and the crystal structure of toxic needle crystals soaked with glycyrrhizic acid,liquiritigenin and liquiritin solution had no significant effect,which was compared with lectin proteins and toxic needle crystals soaked with water.Glycyrrhizic acid was detected in the precipitate after co-soaking lectin protein in licorice juice with lime water.The lectin protein soaked with licorice juice and glycyrrhizic acid solution had no significant effect on the molecular weight distribution of its constituent peptides.The pH of the isoelectric point of lectin protein was about 5～6,and the pH of licorice juice and glycyrrhizic acid solution were 5.25 and 5.62,respectively.After soaking with licorice juice and glycyrrhizic acid solution,the Zeta potential of lectin protein was close to 0,reached the isoelectric point and precipitation.After the lectin protein was soaked with licorice juice,glycyrrhizic acid solution,and licorice juice lime water,we observed that the lectin protein formed a large number of aggregates,and accelerated the precipitation by transmission electron microscope(TEM).The above results showed that glycyrrhizic acid was a component of licorice juice with detoxification during the processing of PRP.The lectin protein was precipitated by soaking in licorice juice alone and glycyrrhizic acid solution,which was caused by the fact that the lectin protein reached the isoelectric point and changed the solubility during the process of soaking.The lectin protein was soaked by licorice juice,licorice solution,and licorice juice and lime water together to form macromolecular complexes,which caused precipitation.In particular,lectin protein was soaked by licorice juice and lime water together,which would form a complex of the macromolecular structure.The above results showed that glycyrrhizic acid in licorice juice was one of the ingredients that had detoxification effect during the process of soaking.The above results suggested that the glycyrrhizic acid in licorice juice was one of the ingredients that had the effects of detoxification in the process of soaking.3.Study on the detoxification mechanism of lime water by processingThe lectin protein and toxic needle crystals were soaked in lime water with different pH and saturated strong alkali,strong alkali and weak acid salt solution,and the protein composition in the supernatant and precipitate after lime water soaked with lectin protein was determined by SDSPAGE method combined with silver staining technique.The effect of lectin protein immersed in saturated strong alkali,strong alkali and weak salt solution on its content was investigated by western blot,and the effect of toxic needle crystals immersed in saturated strong alkali,strong alkali and weak salt solution on its crystal structure was observed by SEM;Meanwhile,MALDITOF MS/MS was used to detect the molecular weight distribution of peptide fragments in the supernatant and precipitate after lime water soaked with lectin protein,and circular dichroism(CD)was used to detect the change of α-helix and β-sheet structure ratio of lectin protein during the soaking process.Toxic needle crystal matrix protein was isolated and extracted,and the composition of the poison needle crystal matrix protein and the changes of protein composition after enzymatic hydrolysis were analyzed by SDS-PAGE method combined with silver staining.The influence of its matrix protein composition was further used Q Exactive mass spectrometry technology to identify the differential matrix proteins before and after protease digestion.In addition,the rabbit model of eye irritation was used to investigate the irritating effects of lectin protein and poison needle crystals soaked in lime water on the rabbit eye conjunctiva.The results of the study showed that the supernatant after soaking with the lectin protein in lime water with pH<12(pH=10,pH=11)showed a band corresponding to the lectin protein at the position of 12 KDa,and there was no corresponding agglutination appeared in the precipitate.However,the corresponding lectin protein bands did not appear in the supernatant and precipitate after soaking the lectin protein in lime water with pH>12(pH=12.4).Soaking with saturated sodium hydroxide and lime aqueous solution could significantly reduce the content of lectin protein,but soaking with saturated sodium bicarbonate solution had no significant effect on the content of lectin protein,which was compared with the lectin protein soaked in water.Compared with the toxic needle crystals soaked in water,the toxic needle crystals soaked in saturated lime water and sodium hydroxide solution could destroy its crystal structure.However,the toxic needle crystals soaked in saturated sodium bicarbonate solution had no significant effect on its crystal structure.Compared with the lectin protein soaked in water,the lectin protein soaked in lime water with pH>12 could change the molecular weight distribution and α-helix and β-sheet structure ratio of lectin protein structure,while the lectin protein soaked in lime water with pH<12 could not change its structural ratio.There was no significant effect on molecular weight distribution and protein secondary structure.Compared with the toxic needle crystals soaked in water,after soaking in the compound protease solution,the needle tip fracture of the toxic needles crystals was aggravated;the toxic needle crystals soaked in lime water with pH>12 could denature their matrix proteins,and the crystal structure of the poisonous needles was destroyed.Toxic needle crystals soaked in lime water(pH>12)could be destroyed,lectin protein denatured,and the inflammatory toxicity was significantly reduced in conjunctival edema of rabbits.The above results showed that pH>12 was the key condition for the hydrolysis of lime during the processing of PRP,and immersion in lime water with pH>12 could irreversibly denature the lectin protein in PR,and the toxic needle crystals were broken.This was caused by the denaturation of the water-soluble matrix protein of the venomous crystals by lime water with pH>12,which led to the destruction of the crystalline morphology of the venomous needles and the change of the crystal structure.Soaking in lime water with pH>12 could destroy the poisonous needle crystals and denature the lectin protein,resulting in a significant decrease in the inflammatory toxicity of PR,which played a key role in processing and detoxification.The results of the above study stated that lime water was selected and kept in pH>12 during the process of the processing of PRP.Meanwhile,toxic needle crystals were soaked in licorice juice and lime water,which could destroy its matrix protein structure,thereby destroyed the toxic needle crystal structure.In addition,the proportion of the original α-spiral and β-folding structures of lectin protein had changed significantly,resulting in destruction of lectin protein structure and degradation.Glycyrrhizic acid,which came from licorice juice,could promote the formation of macromolecular polymers to cause precipitation.Licorice juice and lime water were synergistic to detoxify,which significantly reduced the inflammatory toxicity of PR.The second part of the content:the phlegm-resolving effect and material basis related to PRP.1.Phlegm-resolving effect of PRP decoction and preliminary analysis of its active components.A mouse model of cold asthma was established with ovalbumin(OVA)and cold-water bath stimulation,and then these mice were treated with PR and PRP decoction.We observed the general condition of mice,and determined their airway hyperresponsiveness,expectorant effect,bronchoalveolar lavage fluid(BALF)cell count,BALF and serum inflammatory cytokines,pathological changes,and the protein and mRNA of MUC5AC and AQP5 in lung tissue.Meanwhile,the bronchial epithelial cell(16HBE)model induced by lipopolysaccharide(LPS)was treated with PR and PRP decoction,the protein and mRNA expression levels of MUC5AC and AQP5 were detected.In addition,the serum components of PRP were also identified by UPLC-QTOF MS/MS.The results of the study showed that,compared with the model group,the general condition,airway hyperresponsiveness,expectorant effect,BALF,serum inflammatory cells,and pathological changes of the cold asthmatic model mice were significantly improved after treated with high-and medium-dose of PR and PRP decoction.After the intervention of PR and PRP decoction,the expression levels of MUC5AC protein and mRNA were significantly downregulated,and the expression levels of AQP5 protein and mRNA were significantly up-regulated in vitro and in vivo models.A total of 59 prototype components of PRP in the serum componentswere detected by UPLC-Q-TOF MS/MS,which mainly including organic acids,alkaloids,flavonoids and amino acids.Among them,54 ingredients were from PR and 5 ingredients were derived from licorice.The above results showed that the decoction of PR and PRP had a significant therapeutic effect on the cold asthmatic mice model,which may be related to the organic acids,alkaloids,amino acids,flavonoids,etc.The phlegm-resolving effect of PRP decoction was related to the high secretion with sputum formation indicator(MUC5AC)and fluid transfer indicator(AQP5)expression level of protein and mRNA.2.Study on the mechanism of action of phlegm-resolving effect of PRP decoctionNetwork pharmacology and transcriptomic analysis were used to predict the target and mechanism of action of PRP entered into blood.Meanwhile,western blot was used to investigate the effect of PRP by the participation of PKC-α/EGFR/MAPK/PI3K-Akt signaling pathway in the lung tissue of the cold asthmatic mice.The results of the study showed that the active components of the PRP decoction into the blood acted on a total of 276 targets in the cold asthmatic mice.The analysis of network topological characteristics showed that pentadecanoic acid,licochalcone A,βsitosterol and other components may be the main pharmacodynamic material basis for the treatment of cold phlegm by PRP decoction.In addition,such as SRC,MAPK1,MAPK3,STAT3,HSP90AA1,AKT1,etc.,were considered as hub genes,which may be the key targets of PRP for treating with cold phlegm.Molecular docking demonstrated that the above-mentioned main pharmacodynamic material basis and key targets existed strong affinity effect.GO function and KEGG pathway enrichment results of network pharmacology and transcriptomics were consistent.Western blot results showed that PRP decoction could significantly inhibit the expression of PKCα,SRC protein,the phosphorylation of EGFR,and the activation of MAPK and PI3K-Akt signaling pathways.The above results showed that the decoction of PRP could inhibit the activation of PKC/EGFR/MAPK/PI3K-Akt signaling pathway induced by OVA and cold-water bath,and the downregulation of MUC5AC,and the upregulation of AQP5,which played a key role in phlegmresolving effect.3.Study on the material basis of phlegm-resolving effect of PRP decoctionIn order to further screen the pharmacodynamic material basis of PRP for resolving phlegm,16HBE cells model induced by LPS was established,and the effect of MUC5AC and AQP5 protein expression of LPS-induced 16HBE were treated with ethanol-precipitated supernatant(it was separated into different polar sites)and ethanol-precipitated precipitation of PRP decoction by western blotting.The effective parts of PRP decoction were qualitatively analyze by UPLC-QTOF MS/MS.The results showed that the down-regulation trend of MUC5AC protein expression and the up-regulation trend of AQP5 expression were significantly better than those of alcohol precipitation after treated with alcohol precipitation of PRP decoction within a certain dose range,compared with the model group.The results showed that,compared with the model group,the down-regulation trend of MUC5AC protein expression and the up-regulation trend of AQP5 expression after alcohol precipitation of Pinellia decoction were significantly better than that of alcohol precipitation within a certain dose range.It indicated that the "small molecule" class components had stronger phlegm-resolving effect in the decoction of PPR".The expression of MUC5AC protein was significantly decreased and the expression of AQP5 protein was significantly increased after the intervention of methylene chloride,ethyl acetate,alkaloids and non-alkaloids parts of PRP decoction,while the expression of MUC5AC and AQP5 proteins had no significant changes after the intervention of n-butanol and the water part.A total of 62 compounds were identified in the dichloromethane part of PRP decoction,and 54 compounds were identified in the ethyl acetate part,mainly including organic acids,alkaloids,amino acids,flavonoids,etc.There are 38 kinds of components in the two parts.Compared with the serum components of the decoction,the blood components accounted for about 60.53%of the total components.Among them,the blood components derived from PR were mainly hydroxycinnamic acid,azelaic acid,anisic acid,palmitic acid amide,linoleic acid,glyceryl palmitate,protocatechuic aldehyde,etc.The main blood components derived from licorice are liquiritin,licorice chalcone A,chrysophanol and so on.These prototypical components of PR absorbed into the blood may be the relevant material basis for the phlegm-relieving effect of PRP.In this paper,the detoxification mechanism of PRP by processing and its material basis and action mechanism in phlegm-resolving effect were systematically studied,which basically clarified the mechanism of "detoxification-existing effect" during the processing of PRP.Lime water immersion with pH>12 could destroy the structure of lectin protein,the proportion of αhelix structure changed from the original 12.6%±0.1%to 0.1%±0.1%,and the proportion of βsheet structure changed from the original 43.9%±0.2%to 80.4%±0.1%,resulting in irreversible denaturation of lectin protein.Meanwhile,the matrix protein of the toxic needle crystal was destroyed,which led to the destruction of the crystal structure of the toxic needle crystal.After PR was processed into PRP,the toxicity was significantly reduced,and its phlegm-resolving effect was still retained.Various components derived from PR,including organic acids,such as oleic acid,linoleic acid,linolenic acid,etc.;alkaloids,such as palmitamide,matrine,soyline,etc.;amino acids,etc.Ingredients,such as tryptophan,phenylalanine,tyrosine,etc.And it also included liquiritin,licorice chalcone A,etc.in licorice,which were absorbed into the blood to play a role in reducing phlegm.The possible mechanism of action was as follows:PRP inhibited the activation of PKC/EGFR/MAPK/PI3K-Akt signaling pathway,the downregulation of the expression of MUC5AC,and the upregulation of the expression of AQP5,thereby exerting a phlegm-relieving effect. |
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