Study On Content Determination Of Different Parts Of Lonicera Japonica And Fingerprint Combined With Chemical Pattern Recognition

Lonicera japonica is a traditional medicinal plant in China.The whole plant of Lonicera japonica is a treasure that can be used as medicine.It has many efficacities such as heat-clearing,detoxification,antibacteria,anti-inflammation and so forth.Before the song dynasty,the stems and leaves of Lonicera japonica were used as medicine.However,in the Ming dynasty,the stems,leaves and flowers were gradually used as medicine.Compendium of Materia Medica records:"the stems and leaves of Lonicera japonica have the same efficacy as the flowers".The Chinese Pharmacopoeia?2015 edition?has included the two medicinal materials of Lonicerae Japonicae Flos and Lonicerae Japonicae Caulis,and what's more,Lonicera japonica leaves are not included in the pharmacopoeia.Lonicera japonica leaves are usually regarded as non-medicinal parts of Lonicera japonica and without being fully utilized,resulting in the waste of resources.At present,the study of Lonicera japonica is mostly focused on Lonicerae Japonicae Flos and Lonicerae Japonicae Caulis,and the study of Lonicera japonica leaves is relatively few,and the comparative study of chemical constituents among them has not been reported.The content determination index of Lonicerae Japonicae Flos and Lonicerae Japonicae Caulis is less in Chinese Pharmacopoeia?2015 edition?,so the quality control of Lonicera japonica needs to be further improved.To solve these problems,in this study,up to 11 chemical components were chosen as index components and studied by ultra-high performance liquid chromatography?UPLC?in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.Wavelength switching method was used to establish respective fingerprints.Cluster analysis,principal component analysis and orthogonal partial least square discriminant analysis were used to analyze the results of content determination and fingerprint.This study provided the methods and basis for comprehensive control of the quality of different parts of Lonicera japonica and also supported data for the comprehensive development and utilization of Lonicera japonica.Objective:To establish a UPLC method for simultaneous determination of 11 chemical components in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves and to obtain fingerprints of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves;The chemical pattern recognition such as cluster analysis and principal component analysis were used to compare contents and chemical components of phenolic acids,flavonoids and iridoid glycosides in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.It could provide reference for the development and utilization of Lonicera japonica leaves and the quality control of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.Methods:1 The study of content determination:The content of 11 chemical components in Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves were simultaneously determined by UPLC.The UPLC method was carried out on an ACQUITY UPLC BEH C₁₈8 column?2.1×100 mm,1.7?m?by a gradient elution using acetonitrile and 0.1%phosphoric acid.The flow rate was 0.3 mL·min^-1;The temperature in column compartment was maintained at 30?;The temperature in autosampler was8?.The wavelength was set at 326 nm for new chlorogenic acid,chlorogenic acid,caffeic acid,cryptochlorogenic acid,artichoke,isochlorogenic acid B,isochlorogenic acid A and isochlorogenic acid C,352 nm for rutin and lignin,and 238 nm for loganin.The injection volume was 1?L.The chemical pattern recognition such as cluster analysis and principal component analysis were used to compare the contents of 11 chemical components of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.2 The study of fingerprint:The fingerprints of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves were determined by UPLC.The UPLC method was carried out on an ACQUITY UPLC BEH C₁₈8 column?2.1×100 mm,1.7?m?by a gradient elution using acetonitrile and 0.1%phosphoric acid.The flow rate was 0.3 mL·min^-1;The temperature in column compartment was maintained at 30?;The temperature in autosampler was 8?.The wavelength was set at 326 nm?0⁸min,16²1 min for phenolic acids?,238 nm?8¹4 min for iridoid glycosides?,and 350 nm?14¹6 min for flavonoids?.The injection volume was 1?L.The chemical pattern recognition such as cluster analysis and principal component analysis were used to compare the fingerprints of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.Results:1 The results of Content determination:The 11 chemical components was well separated,each component had a wide linear range and a good linear relationship?r?0.9996?,The precision,repeatability,stability and recovery rate of sample were accorded with the requirement of content determination.The results of cluster analysis showed that 28 batches of samples were mainly divided into two groups,of which the first was Lonicera japonica Caulis and the second were Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.The results showed that the contents of 11 chemical components in Lonicera japonica Caulis were different from those in Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.The contents of 11 chemical components in Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves were generally similar and consistent in quality.The results of orthogonal partial least squares discriminant analysis showed that the Luteoloside was the main differential compound between Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.Combined with the results of content determination,the content of Luteoloside in Lonicerae Japonicae Leaves was significantly higher than that in Lonicerae Japonicae Flos.2 The results of Fingerprint:28 batches of samples were analyzed and23 chromatographic peaks were detected,labeled F1^F23.Among which,10batches of Lonicerae Japonicae Flos and 9 batches of Lonicerae Japonicae Leaves were identified by 14 common peaks and 8 chromatographical peaks.20 common peaks were identified in 4 batches of Lonicera japonica Caulis collected in May,16 common peaks were identified in 5 batches of Lonicera japonica Caulis harvested in autumn and winter,and 10 chromatographical peaks were identified.The similarity of each Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves was more than 0.948,and the similarity of 9batches of Lonicera japonica Caulis was more than 0.629.The results of cluster analysis and principal component analysis showed that 28 batches of samples were mainly divided into two groups,of which the first was Lonicera japonica Caulis and the second were Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.The results showed that the chemical components of the Lonicera japonica Caulis was different from Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves,and the features of chemical components were generally similar between Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.The results of orthogonal partial least squares discriminant analysis showed that the chromatographic peaks of F18,F17?Luteoloside?,F21 and F10 were the main differential compounds,and which could be used as the Characteristically differenent indicators to distinguish between Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.Conclusions:The multi-component content determination method and fingerprints established in this study are accurate,stable and reproducible.It could provide reference for the quality control of Lonicerae Japonicae Flos,Lonicerae Japonicae Caulis and Lonicerae Japonicae Leaves.The chemical pattern recognition method was used to compare and distinguish Lonicerae Japonicae Caulis,Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.The results showed that Lonicerae Japonicae leaves and Lonicerae Japonicae Flos were similar in the compositions of phenolic acids,flavonoids and cyclic etheriterpenoid glycosides and the Lonicerae Japonicae leaves had strong development value.The chromatographic peaks of F18,F17?Luteoloside?,F21 and F10 were the main differential compounds of Lonicerae Japonicae Flos and Lonicerae Japonicae Leaves.