| Scutellaria baicalensis is a perennial herb in the Lamiaceae family and its dried root is used as a famous traditional Chinese medicine（TCM）.Its main medicinal active ingredients are flavonoids,which have good effects in anti-oxidation,anti-virus,anti-tumor and other aspects.With the large-scale development and utilization of related products of S.baicalensis and its extracts,wild resources have declined sharply.Some related species and cultivated S.baicalensis resources have become an important way to solve the pressure on wild resources of S.baicalensis.Among them,Scutellaria viscidula,a related species of the same genus,is used as a substitute for medicinal purposes or as a raw material for extracting active ingredients in some regions.However,there are currently few studies on the molecular mechanism of the accumulation of medicinal active ingredients between the two species and the differences in their formation.And it is urgent to reveal the formation mechanism of the quality of S.baicalensis in terms of the origin,harvest period,processing and other aspects of cultivated S.baicalensis,so as to lay a foundation for further solving the instability of the quality of S.baicalensis.In this study,S.baicalensis and S.viscidula were used as experimental materials.High performance liquid chromatography（HPLC）was used to determine the content of baicalin,baicalein,wogonoside,wogonin,scutellarin,scutellarein,apigenin and chrysin in the roots,stems and leaves of the two species from May to October.Real-time fluorescent quantitative PCR was used to determine the expression levels of the key enzyme genes F6H,F8H,UBGAT and GUS in the downstream of the flavonoid synthesis pathway,and combined with the changes in the content of medicinal active ingredients to preliminarily reveal the molecular mechanism of the accumulation of active ingredients of the two species.The contents of 8 active ingredients in different germplasm,different growth years and different parts of the root of S.baicalensis were compared and analyzed to explore the factors affecting the quality of S.baicalensis.The content changes of six different post-harvest processing technology combinations and fresh roots under different treatments at room temperature were compared and analysis to explore the effect of S.baicalensis post-harvest processing on medicinal active ingredients.The main findings are as follows:1.Eight medicinal active ingredients of S.baicalensis and S.viscidula showed different accumulation trends in different tissues at different growth stages,and their contents in roots,stems and leaves showed significant differences and tissue specificity.The contents of baicalin,wogonoside,baicalein,wogonin in the roots of the two species were the highest,and except for wogonoside and scutellarein,the contents of the other six active ingredients in the roots of S.baicalensis were significantly higher than those in S.viscidula,（P<0.01）.The content of scutellarin was the highest in the stems and leaves of two species.Among them,the contents of baicalein,wogonoside,wogonin and apigenin in the stem of S.viscidula were significantly higher than those in S.baicalensis（P<0.01）,and except for wogonin,scutellarein and chrysin,the contents of the other five active ingredients in the leaves of S.baicalensis were significantly higher than those of S.viscidula（P<0.01）.2.The four key enzyme genes of S.baicalensis and S.viscidula showed different expression level in different tissues at different growth stages.（1）The four key enzyme genes are both expressed in the roots,stems and leaves of the two species,and there are tissue differences.Except for GUS gene,the relative expression levels of F6H,F8H and UGBAT genes in the roots of the two species were significantly higher than those in stems and leaves（P<0.05）.（2）The expression of F6H gene in the stem and leaf of S.viscidula was significantly higher than that of S.baicalensis in the early and middle growth stages（P<0.01）.The expression levels of F8H and UBGAT genes in root and UBGAT gene in stem of S.baicalensis were significantly higher than those of S.viscidula from June to October（P<0.01）.GUS gene expression in root of S.viscidula from June to July was significantly higher than that of S.baicalensis（P<0.01）,but the expression in stem and leaf was significantly lower than those of S.baicalensis（P<0.01）.3.The expression of key enzyme genes in S.baicalensis and S.viscidula was significantly correlated with the accumulation of medicinal active ingredients.Correlation analysis between the expression of key enzyme genes and the content of medicinal active ingredients of the two species showed that the contents of baicalein,wogonin and scutellarein in the root of S.baicalensis were extremely significantly positively correlated with the expression of F6H gene（P<0.01）;the expression of F8H gene was extremely significantly positively correlated with the content of wogonoside in the leaf of S.baicalensis（P<0.01）;the expression of UBGAT gene was significantly positively correlated with the content of baicalein and wogonin in the roots of S.viscidula（P<0.05）;the expression of GUS gene was significantly positively correlated with the content of baicalin in the roots of the two species（P<0.05）.4.There are significant differences in the content of some medicinal active ingredients in S.baicalensis from different germplasm sources.The content of baicalin in 13 different sources of S.baicalensis was higher than the minimum standard（9%）stipulated in the Pharmacopoeia.Germplasm with relatively high content of medicinal active ingredients is mainly concentrated in the three main producing areas of Inner Mongolia,Shaanxi and Hebei,while the content of S.baicalensis in Shanxi,Shandong and Henan is relatively low.5.Growth years and different parts of root have significant effects on the content of active ingredients of S.baicalensis.The total content of the four main medicinal active ingredients of S.baicalensis in different growth years is as follows:15 years old>9 years old>2 years old>7 years old>12 years>3 years old>5 years old>4 years old>4 years old=1 year old.In addition,the content of medicinal active ingredients in the cortex of "Kuqin" was significantly higher than that of "Ziqin"（P<0.01）,but the stele was significantly lower than that of "Ziqin"（P<0.01）.6.Different processing treatments after harvest have a significant impact on the active content and quality of S.baicalensis.The total content of the four main medicinal active ingredients under different processing technology combinations is shown as follows:drying（D）>slicing+drying（CD）>slicing+steaming+drying（CSD）>drying+steaming+slicing+drying（DSCD）>steaming+slicing+drying（SCD）>drying+steaming+slicing（DSC）.7.The storage time of fresh roots of S.baicalensis under different treatment at room temperature（20℃）had a significant effect on the medicinal active components.The contents of baicalin,baicalein,wogonoside,wogonin in the whole fresh root of S.baicalensis at room temperature were slightly higher than those at the moment of harvest.The contents of baicalin,wogonoside and scutellarin in fresh roots of S.baicalensis after being sliced at room temperature were significantly decreased,while the contents of baicalein and wogonin were significantly increased（P<0.05）.The contents of chrysin,baicalein,wogonin,baicalin and scutellarein in fresh roots of S.baicalensis after being crushed at room temperature were significantly increased,while the contents of baicalin and wogonoside were significantly decreased（P<0.05）.Correlation analysis of the contents of medicinal active components in fresh roots of S.baicalensis under different treatments showed that there were correlations between baicalin and baicalein,wogonoside and wogonin,and scutellarin and scutellarein.The above research results will lay a theoretical foundation for further revealing the regulation mechanism of the medicinal active ingredients of S.baicalensis and S.viscidula during the growth period and the molecular mechanism of the differences in content between species,and provides a theoretical basis for determining the harvesting period of S.baicalensis and the processing and technology of the production area. |
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