| Xihuangcao is a special Chinese medicinal materials in Lingnan area.The dried aerial part of Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara and Rabdosia.serra(Maxim.)H.Hara are used as Xihuangcao in《Guangdong Chinese Materia Medicine Criterion》.It has a long history of use in Guangdong,Guangxi,Jiangxi,Fujian and other southern regions,and is commonly used as clinical and folk medicine,as well as the main raw material of a variety of health products and proprietary Chinese medicine.It has a relatively big market potentials.The germplasm resources of Xihuangcao are in chaos,such as the plants included in the standards of local medicinal materials are not the same and scholars have different views on the classification of the two varieties of Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara.Based on literature and market investigation and germplasm collection,the team members found that in addition to the plants included in the standards of local medicinal materials are used as Xihuangcao herbs,Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara and Rabdosia.nervosa(Hems1.)C.Y.Wu et H.W.Li are also used as Xihuangcao herbs among the people.The confusion of the germplasm resources of Xihuangcao and the good germplasm is not clear are the problems that restrict the Xihuangcao industry development.Therefore,it is necessary to sort out,identify and evaluate the quality of the germplasm resources of Xihuangcao herbs,which is of great significance to ensure the effectiveness and controllability of its clinical application.ObjectiveTo study and establish a method for the identification and quality evaluation of Xihuangcao herbs,and the quality evaluation of them were carried out systematically,which provide scientific basis for the construction of chemical identification system of Xihuangcao herbs and select the good germplasm of them.Methods1.Study on UPLC fingerprint and UPLC-ESI-QTOF-MS/MS analysis of Xihuangcao herbsBased on the analysis of extract solvent,mobile phase system,elution conditions,column temperature,flow rate and detection wavelengths,the UPLC fingerprint of Rabdosia.serra(Maxim.)H.Hara and Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara were established separately.The fingerprints were compared for similarity using "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System"(2012.130723)and cluster analysis,principal component analysis were carried out by SPSS 22.0.UPLC-ESI-QTOF-MS/MS analysis were conducted to identify related chemical components of Rabdosia.serra(Maxim.)H.Hara,Rabdosia.nervosa(Hemsl.)C.Y.Wu et H.W.Li,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara.2.Determination of 5 index components contents in Xihuangcao herbsBased on the analysis of extract solvent,mobile phase system,elution conditions,column temperature,flow rate and detection wavelengths,the method to determine the contents of caffeic acid,rosmarinic acid,schaftoside,isoschaftoside and oridonin in Xihuangcao herbs were established.The methods were used to determine 5 index components contents of 31 batches of different kinds of Xihuangcao herbs.3.Determination of total flavonoids,total diterpenes and total phenolic acid contents in Xihuangcao herbsThe methods for the determination of total flavonoids,total diterpenes and total phenolic acid in Xihuangcao herbs were investigated and optimized,and orthogonal test was used to investigate the extraction conditions of the samples.The method were used to determine total flavonoids,total diterpenes and total phenolic acid contents of different kinds of Xihuangcao herbs.4.Determination of antioxidant activity of Xihuangcao herbsThe detection wavelength and reaction time were investigated respectively.The extraction condition of the samples was the same as the method under the content determination of each total component.DPPH method was used to determine the antioxidant activity of different kinds of Xihuangcao herbs.5.Comprehensive evaluation on the quality of Xihuangcao herbsCaffeic acid,rosmarinic acid,total flavonoids,total diterpenes and total phenolic acid were selected as evaluation index and TOPSIS method was used to make the comprehensive evaluation on the quality of Xihuangcao herbs.Term weight were obtained by principal component analysis and the processes of comprehensive evaluation were based on SPSS 22.0 and Excel software.Results1.Study on UPLC fingerprint and UPLC-ESI-QTOF-MS/MS analysis of Xihuangcao herbsThe results of UPLC fingerprint showed that the UPLC fingerprints of Rabdosia.serra(Maxim.)H.Hara were significantly different from Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara.16 batches of Rabdosia.serra(Maxim.)H.Hara herbs were significantly different,with the similarity between 0.680-0.983.Among them,the similarity between commercial herbs were relatively high,while the similarity between field-collected herbs were lower.It meant that the quality of the germplasm of different population were different;The similarity between 11 batches of Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara herbs were high,which were basically above 0.9.It meant that the quality of them were similar;The similarity between Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas were between 0.846 and 0.999.It meant that their chemical compositions were relatively similar and provides a scientific basis to some extent for the use of Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara as a medicinal material of Xihuangcao herbs among the people.UPLC-ESI-QTOF-MS/MS chemical analysis results showed that there were 8 common chemical components of Rabdosia.serra(Maxim.)H.Hara and Rabdosia.nervosa(Hemsl.)C.Y.Wu et H.W.Li,they were peaks 1,11,16,17,18,20,21 and 22,while peaks 2,3,5,13,15,19,23 and 24 were only detected in Rabdosia.serra(Maxim.)H.Hara and peak 14(nodosin)was only detected in Rabdosia.nervosa(Hemsl.)C.Y.Wu et H.W.Li;There were 7 common chemical components of Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara,they were peaks 1,12,26,27,28,29 and 31,and there were 10 common chemical components of Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara and Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara,they were peaks 1,7,8,12,26,27,28,29,31and 32,while peak 25 was only detected in Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,peaks 6,9 and 10 were only detected in Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara.2.Determination of 5 index components contents in Xihuangcao herbsThe contents of caffeic acid,rosmarinic acid,schaftoside,isoschaftoside and oridonin in 31 batches of Xihuangcao herbs were different.Caffeic acid,rosmarinic acid,isoschaftoside and oridonin can be detected in Rabdosia.serra(Maxim.)H.Hara,while schaftoside was difficult to be detected;caffeic acid,rosmarinic acid,schaftoside can be detected in Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas,while isoschaftoside and oridonin were difficult to be detected;caffeic acid,rosmarinic acid can be detected in Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara,while schaftoside,isoschaftoside and oridonin were hard to be detected.The contents of caffeic acid in different populations of Rabdosia.serra(Maxim.)H.Hara,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardi anus(Benth.)Hara,Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara herbs were between 0.17-0.32 mg·g-1,0.09-0.20 mg·g-1,0.15-0.46 mg·g-1,0.18-0.24 mg·g-1,0.22-0.76 mg·g-1,respectively,and the contents of rosmarinic acid were between 3.83-16.14 mg·g-1,3.20-17.42 mg·g-1,6.57-28.25 mg·g-1,13.96-33.25 mg·g-1,9.46-34.70 mg·g-1,respectively;The contents of schaftoside in different populations of Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara and Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara herbs were between 0.14-0.17 mg·g-1,0.10-0.91 mg·g-1,0.29-0.48 mg·g-1 respectively,but schaftoside can be only detected in sample RStr-02 among Rabdosia.Stracheyi(Benth.Ex Hook.f.)Hara herbs,with the content of 0.26 mg · g-1;The contents of isoschaftoside and oridonin in different populations of Rabdosia.serra(Maxim.)H.Hara herbs were between 1.20-6.93 mg·g-1,0.12-3.13 mg·g-1 respectively.The results showed that the chemical compositions and their contents in different kinds of Xihuangcao herbs were different.Rabdosia.serra(Maxim.)H.Hara was rich in oridonin which has antitumor pharmacological activity.The contents of rosmarinic acid in Rabdosia.lophanthoides(Buch.Ham.ex D.Don)H.Hara and its varietas or Rabdosia.stracheyi.Ex Hook.f.)Hara were higher than Rabdosia.serra(Maxim.)H.Hara.Caffeic acid and rosmarinic acid were the common chemical components of different kinds of Xihuangcao herbs.3.Determination of total flavonoids,total diterpenes and total phenolic acid contents in Xihuangcao herbsThe results of the determination of total flavonoids,total diterpenes and total phenolic acid showed that the contents of different kinds of Xihuangcao herbs were differet.The contents of total flavonoids in Rabdosia.serra(Maxim.)H.Hara,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graeiliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara herbs were between 1.65-2.65%,2.51-3.13%,1.99-3.60%,2.36-2.90%,2.80-3.93%,respectively,the contents of total diterpenes were between 9.12-15.58%,9.10-14.37%,9.62-17.81%,12.43-15.99%,13.40-18.43%,respectively,the contents of total phenolic acid were between 7.22-11.95%,7.09-13.07%,7.15-13.57%,11.58-15.39%,10.13-16.49%,respectively.The contents of the total components in the sample RS-02,RS-05 were relative higher among the Rabdosia.sarra(Maxim.)H.Hara herbs.The contents of the total components in the sample RL-01,RLGra-06,RLGer-01,RStr-04 were relative higher among the Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara war.gerardianus(Benth.)Hara,Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara herbs respectively.However,the quality of the Xihuangcao herbs can not be judged objectively and completely according to the content of a certain total component,it is necessary to establish a comprehensive evaluation method to evaluate the quality of different kinds of Xihuangcao herbs.4.Determination of antioxidant activity of Xihuangcao herbsThe results of IC50 in different kinds of Xihuangcao herbs showed that the IC50 of Rabdosia.serra(Maxim.)H.Hara,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graeiliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara,Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara herbs were between 24.60-57.00 ug·mL-1,28.70-79.80 ug·mL-1,20.00-41.91 ug·mL-1,29.29-34.75 ug·mL-1,18.30-42.00 ug·mL-1,respectively.The results showed that the antioxidant activity of Rabdosia.Lophanthoidesvar.graciliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara herbs were better than Rabdosia.serra(Maxim.)H.Hara.Multiple linear regression analysis results showed that the correlation coefficient between antioxidant capacity and the contents of total flavonoids,total diterpenes,total phenolic acids were 0.6240,0.7287,0.7315,respectively.And the difference was statistically significant(P≤0.05).The effect of antioxidant capacity were as fellows:total phenolic acid>total diterpene>total flavonoids.5.Comprehensive evaluation on the quality of Xihuangcao herbsThe ranking results of TOPSIS comprehensive evaluation method showed that sample RS-02 had better comprehensive quality in Rabdosia.serra(Maxim.)H.Hara herbs,sample RL-01 had better comprehensive quality in Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara herbs,sample RLGra-06 and RLGra-03 had better comprehensive quality in Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara herbs,sample RLGer-01 had better comprehensive quality in Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara herbs,sample RStr-02 and RStr-04 had better comprehensive quality in Rabdosia.stracheyi.Ex Hook.f.)Hara herbs;sample RStr-02,RLGra-06,RStr-04 and RLGra-03 had better comprehensive quality in different kinds of Xihuangcao herbs.ConclusionIn the study,the UPLC fingerprint of Rabdosia.serra(Maxim.)H.Hara and Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara were optimized and established and can be used for their quality control.UPLC-ESI-QTOF-MS/MS analysis can be used to identify related chemical components of Rabdosia.serra(Maxim.)H.Hara,Rabdosia.nervosa(Hemsl.)C.Y.Wu et H.W.Li,Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara,Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara,Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara and Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara.The method is fast,accurate and reliable.Relevant experimental results also provided a scientific basis for the use of Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara as a medicinal material of Xihuangcao and Isodon lophanthoides(Buch-Ham.ex D.Don)Hara var.gerardianus(Benth.)Hara can be used as Rabdosia.Lophanthoides var.graciliflora(Benth.)H.Hara herbs.The methods for the determination of 5 index components contents in Xihuangcao herbs and determination of total flavonoids,total diterpenes and total phenolic acid in Xihuangcao herbs,as well as the method for determining the antioxidant activity of Xihuangcao herbs had feasible operation and reliable result.Caffeic acid and rosmarinic acid as the common chemical components of different kinds of Xihuangcao herbs,which can be considered as the index components to evaluate different kinds of Xihuangcao herbs.TOPS IS method can be used to the comprehensive evaluation on the quality of Xihuangcao herbs and there are 4 germplasm types with good comprehensive quality were selected which can be used as breeding materials for new variety breeding in the future.The evaluation system can be used to evaluate the quality of the germplasm resources of Xihuangcao.The results of this study showed that the chemical composition and its contents of Rabdosia.serra(Maxim.)H.Hara and Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas are different and should not be used as the same.It should be selected according to their characteristics in the clinic prescription.Rabdosia.serra(Maxim.)H.Hara was rich in oridonin which has anti-tumor pharmacological activity and when its anti-tumor effect is given priority,it is suggested to select Rabdosia.serra(Maxim.)H.Hara;The contents of rosmarinic acid in Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas or Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara were higher than Rabdosia.serra(Maxim.)H.Hara.Rosmarinic acid has anti-inflammatory and antioxidant pharmacological activities and when its anti-inflammatory and anti-oxidant effect is given priority,it is suggested to select Rabdosia.lophanthoides(Buch.-Ham.ex D.Don)H.Hara and its varietas or Rabdosia.stracheyi(Benth.Ex Hook.f.)Hara.How to use the Xihuangcao herbs reasonably,relevant pharmacological studies and clinical trials should be carry on in the future to further verify.This study laid the foundation for the quality evaluation and good germplasm screening of Xihuangcao herb. |
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