Quality Evaluation Of Juniperus Rigida Sieb.et Zucc. Based On SSR Molecular Marker,Chemical Composition And Biological Activity

Juniperus rigida Sieb.et Zucc.(J.rigida),the evergreen coniferous arbor of the J.rigida,is endemic to East Asia.The J.rigida have been widely known as traditional Korean medicine for the treatment of rheumatoid arthritis and dropsy.The essential oils of J.rigida were also multiple applied in pharmacological industries.However,there are few studies onthe quality evaluation of J.rigida.Consequently,the SSR molecular marker,chemical composition,bioactivity and fingerprints combined with chemometrics were employed in order to establish a useful method for the quality evaluation of J.rigida,which may further provide theoretical basis for provenance resources protection,new variety breeding and the results were expressed in the follow:1.Genetic diversityof J.rigidabased on SSR molecular marker.(1)23 pairs of SSR molecular marker primer sequences were developed,polymorphism rate was 98.37%,specificity 24.35%;(2)the UPGMA tree was establishedand all the provenance can be divided into three categories: 1): S2(Fugu)and S3(Yijun),2)S8(Jinhua),S5(Chongli),S10(Fengcheng),S9(Changbai Mountain),S4(Datong),S1(Helan Mountain),S11(Qidaogou),S13(Xianghuangqi)and S12(Liulin);3)S6(Dabaozhen)andS7(Xiaosuangou);(3)PCA analysis verified the results of HCA analysis that the J.rigida comes from the same area tend to form a separate cluster subgroup andJ.rigidafrom similar genetic background also showed a close genetic relationship.2.Geographical differences of J.rigidabased on chemical composition and bioactivity:(1)GC-MS method determined 48 compounds in the essential oils and the S8 owned the highest yield(0.4%),themost varigied compositionswere?-pinene(11.81 ~ 26.60%),caryophyllene(5.75 ~ 16.60%),?-guaiacol(1.51 ~ 15.97%),?-farnesene(5.33 ~ 12.35%)and bornyl acetate(1.77 ~ 8.81%),limonene(3.63 ~ 7.57%)and?-myrcene(2.44 ~ 7.43%),in which S2 owned the highest contents;(2)the HPLC method detected 10 phenolic compounds in J.rigida phenolic extractsand themost varigied compoundswerechlorogenic acid(53.32 ±0.17 ~ 163.38± 0.17 mg/g),catechins(112.52 ± 0.08 ~ 288.88± 0.38 mg/g),podophyllotoxin(71.11 ± 0.06 ~ 189.18± 0.04 mg/g)and amentoflavone(57.68 ± 0.01 ~ 146.16± 0.05 mg/g),the S2 and S8 owned the highest contents.(3)Theklebsiella.pneumoniae,salmonella enteritidisand salmonella paratyphidisplayed most sensitive and the S2 and S10 exhibited the strongest antibacterial activity.In conclusion,the S2,S8 and S10 showed better quality.3.Establishment of GC-MS and HPLC fingerprint combined with chemometrics:(1)The GC-MS fingerprintanalysisselected41 common peaks and the similarities of chromatographsvaried from 0.638~0.995;HCAanalysis clustered the J.rigida into three major groups(G1:(S8,S9 andS10);G2: S3;G3:(S1,S4,S2,S4,S5,S6 and S7)(2)PCAanalysis results picked the(+)-4-carene,cymene,campholene,terpineol,?-bourbonene,ylangene,?-farnesene,neryl isobutyrate as indicating compounds;(3)14 common peaks were selected in HPLC fingerprint analysis and the similarities of chromatographsvaried from 0.855~0.991.HCAanalysisclustered the J.rigidainto three major groups(G1:(S8,S10);G2 :S2,S3;G3 :S1,S4,S5,S6,S7 and S14(Xiawulaha));(4)PCAanalysispicked the podophyllotoxinand amentoflavone as the indicating compounds;(5)DA analysis constructed discriminant functions for further discriminate and classify the unknown membership4.Genetic factors andchemical compositioncorrelation analysis showed the metabolites content differedfrom geneticdiversity.The ylangene,?-farnesene,neryl isobutyrate,?-guaiene,podophyllotoxin,amentoflavone and formononetin were selected as the indicating compounds.