| Flos Lonicerae Japonicae(FLJ)is the dry flower bud or open flower of Lonicera japonica with high medicinal value and wide application in traditional Chinese medicine.However,the raw material plant sources of FLJ are complex and frequently mixed raw materials harvested from different sources and different stages are used.In addition,the lack of effective quality control standards causes irregularity of the raw material quality,which seriously affects the effective utilization of FLJ in clinics.In order to resolve these problems,we examined the accumulation dynamics of chlorogenic acid(CGA)and luteoloside,two major medicinal components of FLJ,at six different growth stages of L.japonica,including the young alabastrum stage(S1),the green alabastrum stage(S2),the slightly white alabastrum stage(S3),the whole white alabastrum stage(S4),the silvery flower stage(S5)and golden flower stage(S6)],using a combination of phytochemistry,physiology and molecular methods.Whist,the medicinal materials quality of FLJ from different sources was systematic researched based on the methods of ATR-FTIR,HPLC-DAD and chemometrics.The main results are summarized below.1.The accumulation dynamics of the CGA and luteoloside are correlated with the expression patterns of their key biosynthetic genesWe found that the accumulation of both CGA and luteoloside first increased and then decreased throughout the growth of L.japonica.The highest content of CGA was observed in S3,the amount of luteoloside was relatively higher in S3 and S4 than other growth stages.In addition,we found that HQT and CHI activity levels were directly correlated with changes in CGA and luteoloside contents,respectively.Moreover,the accumulation of CGA and luteoloside during S1-S4 was also correlated with variations in the activities of PAL,C4 H and 4CL enzymes of the phenylalanine metabolic pathway.Further,gene expression analysis revealed a correlation between the HQT transcript levels with HQT activities and CGA contents.Based on these results,S3-S4 are recommended as the optimal harvesting stage for high-quality Flos Lonicerae Japonicae.2.CGA is mainly synthesized in the chloroplast and cytoplasm and finally stored in the vacuoles in L.japonica corolla tubeThe in situ hybridization and histochemical localization results indicated that the tissue specific expression of HQT in tissue or cellular specificity significantly influenced the distribution of CGA in L.japonica corolla tube cells at different development stages.For example,in S1-S3,the HQT was presented in the inner and outer epidermal cells,and parenchyma cells and vascular bundle.CGA was also found in these tissues.HQT and CGA were only distributed around in vascular bundle and inner epidermal cells at S5-S6.The results of the rabbit anti-HQT Immunogold(lgG)and the laccase colloidal gold demonstrated that anti-HQT-lgG and laccase-lgG particles were mainly located in chloroplasts and cytoplasm at S1-S2.At S3-S4,accompanying the weakening of the structure and function of chloroplast,the structure of starchs was gradually destroryed and degraded,the anti-HQT-lgG particles were reduced in chloroplast and mainly distributed in the cytoplasm.During the process of vacuolization,we observed that the laccase-lgG particles were reduced in the plastid and cytoplasm,but increased in the vacuole.After the buds fully opened,the large central vacuole almost occupied the whole cell,very few anti-HQT-lgG particles were observed in cytoplasm.The anti-HQT-lgG particles were not found in vacuole and cell wall throughout the different growth stages.Whereas the laccase-lgG particles were mainly located in the vacuole in S5 and S6.Therefore,we concluded that chlorogenic acid was mainly synthesized in the chloroplasts,plasmids and cytoplasm,and these sites could be used as the temporary storage sites for chlorogenic acid in the early stage.In middle and later stages of L.japonica,chlorogenic acid was mainly synthesized in the cytoplasm,and stored in the vacuole.3.Quality evaluation of diploid and tetraploid FLJ as well as related medicinal plants3.1 Comparison of the medicinal materials quality between diploid and tetraploid FLJThe chemical fingerprints based on HPLC-DAD determination results indicated that although the accumulation dynamics of some polyphenols compounds exhibited significant differences in different growth stages of two ploidy L.japonica,multiple compounds accumulated at significantly higher levels in the tetraploid than in the diploid plants.The results of antioxidant activity combined with principal component and similarity analyses showed that the tetraploid L.japonica displayed better antioxidant activity than the diploid during most of the growth stages,and the levels of antioxidant activity were significantly correlated with the variations of chlorogenic acid,rutin,hyperoside,luteoloside in the two ploidy L.japonica(P < 0.05).Notably,the antioxidant activities were more closely associated with the amounts of chlorogenic acid and luteoloside in tetraploid L.japonica.The highest effective component yields such as chlorogenic acid,luteoloside,hyperoside and total flavonoid in tetraploid L.japonica were observed during S3-S5.Therefore,S3-S5 are recommended as the optimal stage for harvesting high-yield,high-quality tetraploid Flos Lonicerae Japonicae.3.2.A comparative study on the chemical fingerprint chromatography between L.japonica and its relative plants.The chemical fingerprint chromatography combined with chemometrics methods such as principal component analysis(PCA)and clustering analysis(HCA)were used to identify the differences of chemical components between L.japonica and that relative plants.Results from both methods revealed that the chemistry composition of L.japonica was similar to L.confuse but significantly different from that of L.macranthoides.and L.fulvotomentosa.The loading analysis results indicated that the differences of chemical components were mainly associated with the constituent and content of organic acid and flavonoids between L.japonica and its relative plants.Ten different chemical component contents were simultaneously determined by using HPLC and mixed standard methods between L.japonica and its relative plants.We found that the highest content of CGA was observed in sample D,whereas the highest contents of 3,4-dio-caffeoylquinic acid and 3,5-dio-caffeoylquinic acid were observed in sample C,respectively.The highest content of luteoloside was observed in sample A,and lowest content was observed in sample B.We show that the identification model of SIMCA could be successfully applied to predict unknown samples from L.japonica and the relative plants,and the recognition rate could reach 100 %.These results will provide a guiding reference for rapid identification and quality control of medicinal materials and improvement the clinical curative effects of L.japonica. |
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