Effects Of The Origin Processing On The Parishins Transformation And Quality Formation Of Gastrodiae Rhizoma

Objectives The origin processing method of Gastrodiae Rhizoma,which is steamed before drying,has been in use since the Song Dynasty.2020"Chinese Pharmacopoeia"stipulate that the origin processing method of Gastrodiae Rhizoma is"washed,moistened or steamed soft,thinly sliced and dried".However,the biochemical reactions and mechanisms during the origin processing of Gastrodiae Rhizoma are unclear,which leads to the lack of a set of standard processing methods in the actual production,resulting in uneven quality of Gastrodiae Rhizoma.The aim of this project is to study the influences of the origin processing method of Gastrodiae Rhizoma on its chemical composition and anti-inflammatory activity before and after processing,to investigate its potential medicinal substance and anti-inflammatory mechanism,and to provide theoretical support to reveal the scientific connotation of the origin processing and to standardize the processing method and improve the quality of Gastrodiae Rhizoma.Methods 1.A study of the effect of origin processing on the chemical composition of Gastrodiae Rhizoma.Using the controlled variable method,five processing methods were established.（1）Drying at 45 ℃ vs.steaming+drying at 45 ℃:to investigate the effect of high temperature on hydrolases and components during steaming;（2）drying at 45 ℃ vs.ultra-high-pressure enzyme deactivation+drying at 45 ℃:to investigate the effect of hydrolases on components;（3）ultra-high-pressure enzyme deactivation+drying at 45 ℃ vs.ultra-high-pressure enzyme deactivation+drying at 45 ℃:to investigate the effect of high temperature on components;（4）ultra-high-pressure enzyme deactivation+drying at 45 ℃ vs.ultra-high-pressure enzyme deactivation+freeze-drying:to investigate the effect of low temperature drying on components.The HPLC fingerprints of five processed Gastrodiae Rhizoma samples were established and common peaks were determined based on the“Similarity Evaluation System of TCM Chromatographic Fingerprints”.Then hierachical cluster analysis（HCA）,principal component analysis（PCA）,and orthogonal partial least squares-discriminant analysis（OPLS-DA）were used to cluster the five processed Gastrodiae Rhizoma samples and analyze the differential components.Finally,the content of the main components in five processed samples was determined.Elucidate the effects of processing on the quality of Gastrodiae Rhizoma from a chemical perspective.2.A study of the"spatial-temporal-content"changes of the chemical composition of Gastrodiae Rhizoma during the steaming process.Five Gastrodiae Rhizoma samples were steamed for 0 min,2 min,4 min,6 min and 8 min,and then placed at room temperature for24 h before slicing.The spraying matrix was then optimized to establish a high-coverage visualized matrix-assisted laser desorption/ionization mass spectrometry imaging（MALDI-MSI）method for analyzing phenolic components in Gastrodiae Rhizoma,and image,identify and analyze phenols by combining liquid-mass spectrometry to investigate the"spatial-temporal-content"changes of phenolic components during the steaming process.The activity of hydrolases related to the degradation of parishins in Gastrodiae Rhizoma was measured.The enzymatic hydrolysis of gastrodin（GAS）byβ-glucosidase（β-GC）was performed in vitro.3.A spectrum-effect relationship study of the anti-inflammatory activity of Gastrodiae Rhizoma.A model of LPS-induced inflammation in BV-2 was established,and the cytotoxicity of different extracted parts of Gastrodiae Rhizoma was investigated using the MTT method,and the anti-inflammatory active extracts were screened using the NO,TNF-αand IL-6 as indicators,and the anti-inflammatory activity before and after processing of Gastrodiae Rhizoma were compared.The HPLC fingerprint profiles of 10 batches of commercial Gastrodiae Rhizoma were established,the common peaks were identified by liquid-mass spectrometry（LC-MS）.Partial least square（PLS）and grey relational analysis（GRA）were used to establish the spectrum-effect relationship,and the correlation between its components and the secretion inhibition of NO,TNF-αand IL-6 was analyzed,to determine the potential medicinal substances basis of Gastrodiae Rhizoma’s anti-inflammatory effect.4.Studies on the identification of potent substances and mechanisms of the anti-inflammatory effects of Gastrodiae Rhizoma.The levels of NO,ROS,TNF-α,IL-6,i NOS and COX-2 in the LPS-induced inflammatory BV-2 cells under the treatment of potential pharmacodynamic substances were determined,and the relevant signal pathways and key target proteins of the anti-inflammatory effects of Gastrodiae Rhizoma were predicted by molecular docking.In addition,based on the LPS-induced inflammatory BV-2 cells and Western blot,the in vitro verification was conducted to investigate the influence of potential pharmacodynamic substances on the expression of major target proteins in related signal pathways.The components and its mechanism of the anti-inflammatory effect of Gastrodiae Rhizoma was demonstrated.Results 1.Systematic elucidation of the effects of origin processing on the chemical composition of Gastrodiae Rhizoma.The HPLC fingerprints identified gastrodin（GAS）,p-hydroxybenzyl alcohol（4-HBA）,parishin A（PA）,parishin B（PB）,parishin C（PC）,and parishin E（PE）as the common peaks of the five processed Gastrodiae Rhizoma samples.The results of HCA,PCA,and OPLS-DA showed that the direct-dried and steamed-dried samples could be clustered well individually,while the three groups of Gastrodiae Rhizoma samples treated with ultra-high pressure were clustered into one group,and the differential components affecting the clustering were 4-HBA and PA.The results of content determination showed that the content of 4-HBA in the direct-dried Gastrodiae Rhizoma was significantly higher than that in other groups（P<0.001）,and the contents of PA and PE were significantly lower than those of other groups（P<0.001）;the content of PA was lower in the steamed samples compared with that of samples with ultra-high-pressure following steaming treatment（P<0.01）;in addition,there was no significant difference in the content of chemical composition between the hot-air dried samples and freeze-dried samples.All the results indicates that steaming deactivates the hydrolases and inhibits the degradation of the parishins;while the high temperature during steaming also has the effect of promoting the degradation of PA;in addition,low temperature drying has no significant effect on the degradation of parishins.2.The"spatial-temporal-content"changes of phenols of Gastrodiae Rhizoma during the steaming process was presented.of t.By optimizing 1,5-DAN as the MALDI matrix,13phenols were imaged,including PA、PT/PU、PK、PB/PC、PW、PJ、PE/PG、PD、4-HBA and CA.With the increasing steaming time,the distribution of CA,4-HBA,PD and PW gradually decreased from the outside to the center,while macromolecule parishins such as PA,PT/PU,PK,PB/PC,PJ,and PE/PG spreading from the outside to the center.The small molecule metabolites in Gastrodiae Rhizoma were formed from the macromolecule parishins by breaking the ester andβ-glucosidic bonds in the presence of Car E andβ-GC,which were deactivated at the same time as the steaming.In vitro enzymatic hydrolysis experiments showed that steaming could inactivateβ-GC and inhibit the deglucose-degradation of GAS.All the results demonstrated that the steaming of Gastrodiae Rhizoma was to deactivate hydrolases and protect parishins from outside to inside.Moreover,the degradation pathways of parishins related to Car E andβ-GC in Gastrodiae Rhizoma were summarized.3.It was demonstrated that the anti-inflammatory activity of Gastrodiae Rhizoma was significantly enhanced by steaming,with GAS,4-HBA,PA,PB and PC as potential active substances.The MTT results showed that both the 70%ethanol extract and the CHCl₂ extract of Gastrodiae Rhizoma were cytotoxic to BV-2 cells.LPS-induced BV-2 inflammation model was established,and NO,TNF-αand IL-6 were used as indexes to screen the anti-inflammatory activity of n-Bu OH extract and H₂O extract of Gastrodiae Rhizoma.n-Bu OH extract was found to have better anti-inflammatory effect and was identified as the medicinal extract of Gastrodiae Rhizoma.The anti-inflammatory effect of direct drying Gastrodiae Rhizoma was further compared with that of origin processed Gastrodiae Rhizoma.It was found that the production of NO,TNF-αand IL-6 by BV-2 cells under the steaned Gastrodiae Rhizoma was lower than that under the direct drying Gastrodiae Rhizoma,indicating that the anti-inflammatory effect of Gastrodiae Rhizoma was improved after steaming.There were12 common peaks of active parts of Gastrodiae Rhizoma and 11 of them were identified by LC-MS.The 11 compounds were 4,4’-dihydroxybenzyl sulfoxide（peak 1）,CA（peak 2）,bis-（4-hydroxybenzyl）sulfide（peak 3）,GAS（peak 4）,methyl citric acid（peak 5）,4-HBA（peak6）,PE（peak 7）,PJ（peak 8）,PB（peak 9）,PC（peak 10）and PA（peak 11）.The peak area of the 11 common peaks combined with the inhibition rate of NO,TNF-αand IL-6 release in the LPS-induced BV-2 inflammatory model of the n-Bu OH extract of10 batches of Gastrodiae Rhizoma to established the spectrum-effect relationship.In PLS,peaks 4,6,10,11 were positively correlated with drug efficacy,and VIP value was greater than 1.In GRA,among the 12 common peaks closely related to NO inhibition,the top 5 were 6>4>7>12>10.The top 5 factors that were closely related to the secretion inhibition of TNF-αwere4>2>6>10>12.The top 5 most closely related to the inhibition of IL-6 secretion were6>4>7>12>10.In conclusion,peak 4（GAS）,peak 6（4-HBA）,peak 10（PB）,peak 11（PC）and peak 12（PA）were the potential material bases for n-Bu OH extract of Gastrodiae Rhizoma to exert anti-inflammatory activity.4.The anti-inflammatory activity and mechanism of PB were proved.PB inhibited the release of inflammatory mediators（NO,intracellular ROS,TNF-αand IL-6）in BV-2 cells stimulated by LPS,and decreased the activity of i NOS and the COX-2 level,indicating that PB could reduce the inflammation of BV-2 cells induced by LPS.Western Blot results showed that PB could exert anti-inflammatory effects by inhibiting LPS-induced phosphorylation of IKKβ,IκBαand p65,and to inhibit LPS-induced activation of NF-κB pathway.Molecular docking predicted that the binding ability of PB to AMPKαand SIRT1proteins were-12.1 k J·mol^-1 and-7.6 k J·mol^-1,respectively,which speculated that PB may act on AMPKα/SIRT1 proteins to regulate downstream inflammatory signaling pathways.Western Blot results showed that LPS inhibited AMPKαphosphorylation and SIRT1expression,while PB activated AMPKαand SIRT1 proteins and played an anti-inflammatory role by inhibiting AMPK/SIRT1 signaling.In conclusion,PB inhibits NF-κB pathway by activating AMPK/SIRT1 pathway,and plays an anti-LPS-induced neuroinflammatory role in BV-2.Conclusions In conclusion,the origin processing of Gastrodiae Rhizoma has the effect of inhibiting hydrolyses degradation of parishins and promoting chemical transformation,which can deactivate hydrolyses from the outside to the inside during the steaming process and inhibit the degradation process of parishins.The n-Bu OH extract is the active extract to exert anti-inflammatory effects of Gastrodiae Rhizoma,and the anti-inflammatory activity of Gastrodiae Rhizoma is significantly enhanced after steaming,and its main effective substances are GAS,4-HBA,PA,PB and PC.Among them,PB could inhibit NF-κB pathway by activating AMPK/SIRT1 pathway,and plays an anti-LPS-induced neuroinflammatory role in BV-2.In this study,the scientific connotation of processing for quality improvement of Gastrodiae Rhizoma was illustrated through the correlation of"processing-composition changes-pharmacological effects-quality improvement",which provides a theoretical basis for the establishment of a scientific and standardized processing method and quality evaluation system for asparagus.This study provides a theoretical basis for the establishment of a scientific and standardized origin processing method and quality evaluation system for Gastrodiae Rhizoma,and is of great significance in promoting the development of the discipline of traditional Chinese medicine and the high-quality development of Chinese medicine agriculture.