| Objective:To optimize the processing technology of Jinzhai Gastrodia elata;To establish HPLC fingerprint of Jinzhai Gastrodia elata before and after processing,and analyze the changes of chemical components of Gastrodia elata before and after processing;To evaluate the quality of processed products of Gastrodia elata in Jinzhai with different storage time;To explore the protective effect of Gastrodia elata before and after processing on CCl4induced acute liver injury in mice.Methods:（1）With the total content of gastrodin and p-hydroxybenzyl alcohol as the inspection index,the steaming process of extra-grade,first-grade,second-grade,third-grade and fourth-grade Jinzhai Wuhong gastrodia elata and red gastrodia elata was optimized.The processing technology of Jinzhai Gastrodia elata was optimized by using the multi-index orthogonal experiment method,taking the drying time,drying temperature and slice thickness as the factors,and taking the contents of gastrodin,p-hydroxybenzyl alcohol,Parishin,Parishin B,Parishin C and Parishin E as the evaluation indexes.（2）HPLC fingerprints of Wuhong Gastrodia elata and Red Gastrodia elata were established before and after 10 batches of processing in Jinzhai,Anhui Province.The differences of chemical components of Wuhong Gastrodia elata and Red Gastrodia elata before and after processing were analyzed by mathematical metrology,and the content changes of six effective components before and after processing were compared.The content of six active ingredients was used as the evaluation index to optimize the effect of different storage time（0,3,6,12 months）on the quality of Jinzhai Wuhong gastrodia elata and its processed products.（3）Sixty-three SPF grade male C58BL/6J mice were randomly divided into 9 groups,namely blank group,model group,positive control group（biphenyl double fat group）,high and low dose group of Wuhong gastrodia elata before processing,and high and low dose group of Wuhong gastrodia elata after processing,with 7 rats in each group.In the prophylactic administration stage,each administration group was continuously gavaged for 15 days,and the normal group and model group were gavaged with the same amount of normal saline,with the dose of 0.1m L/10g;At the modeling stage,one hour after the last administration,the mice in the model group and the prophylactic group were injected intraperitoneally with CCl4olive oil solution with a volume fraction of 2%,and the dose was 0.1m L/10g.After fasting for 24 hours,the eyeball was removed to take blood,and the liver was killed and separated.The content of aspartate Glutamic transaminase（AST）and Glutamic-pyruvic transaminase（ALT）in serum,the activity of Superoxide dismutase（SOD）and Catalase（CAT）in liver homogenate,and the content of Trace reduced glutathione（GSH）and Malondialdehyde（MDA）were measured.The concentration of Interleukin-6（IL-6）in serum was determined by ELISA,and the pathological changes of liver tissue were observed by Hematoxylin-eosin（HE）staining,To study the protective effect of Wuhong Gastrodia elata before and after processing on acute liver injury induced by CCl4in mice.Results:（1）The best steaming time of Jinzhai Wuhong gastrodia elata and red gastrodia elata super-grade is 4h,the best steaming time of the first,second,third and fourth grade gastrodia elata is 2h,1.5h,1h and 0.5h,respectively.The best drying process is drying temperature 60℃,drying time 8h,slice thickness 2mm.（2）The comparison fingerprint of 20 batches of Wuhong gastrodia elata samples showed that 9 common peaks were identified and 5 components were identified in the gastrodia elata samples before and after processing,of which the 7#peak was gastrodin,the 9#peak was Parishin E,the 11#peak was Parishin B,the 12#peak was Parishin C,and the 13#peak was Parishin.The relative peak area of gastrodia elata before and after processing is quite different.The principal component analysis clearly divides gastrodia elata before and after processing into two categories,indicating that there are differences in the internal quality of gastrodia elata before and after processing.Four different components of Gastrodia elata before and after processing were determined by least partial multiplication analysis,two of which were gastrodin and Parishin B.The content results showed that the content of six main components of Wuhong Gastrodia elata and Red Gastrodia elata changed significantly before and after processing,and the total content of gastrodin and p-hydroxybenzyl alcohol and total content of balisen glycosides increased significantly after processing,and the total content of each index component of Gastrodia elata in Jinzhai was the highest after 6 months of harvesting.（3）Compared with the blank group,the liver index of mice in the model group was significantly increased（p<0.01）,the contents of ALT and AST in serum were significantly increased（p<0.01）,the activities of SOD,CAT and GSH in liver tissue were significantly decreased（p<0.01）,the content of MDA was significantly increased（p<0.01）,and the expression of IL-6 in serum was significantly increased（p<0.01）.The HE staining results showed that the liver cells of mice were significantly denatured,the liver cells were swollen,the cytoplasm was transparent,and most cells were necrotic,The presence of inflammatory cell infiltration indicates that CCl4-induced acute liver injury model in mice has been successfully established.Compared with the model group,the positive control group and the low,medium and high dose groups before and after the processing of black red gastrodia elata can reduce the liver index of mice to varying degrees,reduce the content of ALT and AST in serum,increase the activity of SOD,CAT and GSH in liver tissue,reduce the content of MDA,and reduce the level of IL-6 expression in serum.The liver histopathological observation results show that the degree of liver tissue damage in the high,medium and low dose groups before and after the processing of black red gastrodia elata has improved,The liver cord gradually appears,the infiltration of inflammatory cells decreases,and the swelling of nucleus decreases.It showed that before and after processing,the black red gastrodia elata could reduce the acute liver injury induced by CCl4in mice.In addition,most of the indexes in the middle and high dose groups of processed Gastrodia elata are better than those before processing,indicating that the efficacy of processed Gastrodia elata is better than that before processing,and the degree of protection is dose dependent,that is,within the safe range,the higher the concentration of the decoction of Gastrodia elata,the stronger the protective effect on the liver of mice.Conclusion:Through this research,the best processing technology of Jinzhai Wuhong Gastrodia elata and Red Gastrodia elata has been confirmed,the quality of Jinzhai Wuhong Gastrodia elata and Red Gastrodia elata before and after processing has been comprehensively evaluated and the best storage time has been confirmed,and the HPLC analysis shows that there are significant differences in the chemical components of Jinzhai Wuhong Gastrodia elata and Red Gastrodia elata before and after processing,The results of animal experiments showed that the protective effect of Jinzhai Wuhong Gastrodia elata on liver injury before and after processing may be related to antioxidation and inhibition of inflammation. |
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