| Eucommia ulmoides is a unique Tertiary relict plant in my country,and it is a national second-class precious protected tree species.The dry bark of Eucommia ulmoides is a traditional medicinal part.Due to the huge demand of domestic and foreign markets,the bark of Eucommia ulmoides has been exploited in large quantities,which seriously affects the sustainable utilization of Eucommiae resources.In recent years,the comprehensive utilization value of Eucommia ulmoides resources has become increasingly prominent with the deepening of the research on leaves,male flowers and seeds of Eucommia ulmoides.Eucommia ulmoides leaves have been included in the Chinese Pharmacopoeia,and also listed in the homologous catalogue of medicine and food.In order to explore the resource substitutability of the bark and leaf of Eucommia ulmoides,the material basis,antiosteoporosis effect,quality control and other aspects were systematically compared.Research purposes:To clarify the material basis of the bark and leaves of Eucommia ulmoides,and to explore their anti-osteoporosis pharmacodynamics and mechanism,and on this basis,to compare the quality of the bark and leaves of Eucommia ulmoides.Research method.Methods of study:(1)Chemical Constituents of Bark and Leaf of Eucommia ulmoides and Prediction of Their Difference.Firstly,the characteristic chromatograms of cortex and leaves of Eucommia ulmoides were established by high performance liquid chromatography.Then,the chemical constituents of cortex and leaves of Eucommia ulmoides were identified by HPLC/ESI-QTOFMS combined with molecular ion peaks,primary and secondary fragment ion information and relevant literature.At the same time,on the basis of characteristic chromatogram and LC-MS,PCA and OPLS-da analysis were carried out to preliminarily explain the difference of material basis of Eucommia ulmoides bark and leaf from different producing areas.(2)Study on the mechanism of anti-osteoporosis effect and the prediction of pharmacodynamic substances based on network pharmacology.The chemical components of Eucommia ulmoides were screened by network pharmacology,and the relationship between chemical components and targets and pathways for the treatment of osteoporosis was analyzed.To predict the possible targets and pathways of anti-osteoporosis effect of Eucommia ulmoides,the key targets and corresponding compounds were screened and verified by molecular docking.(3)Study on anti-osteoporosis effect of bark and leaf of Eucommia ulmoides Oliv.Based on the differences of physical properties of different chemical components,different solvents were selected for extraction and enrichment,and mouse skull bone subclone 14(MC3T3-E1Subclone14)cells were used to investigate the effects of different solvent extracts of Eucommia bark and Eucommia leaf on the proliferation and ALP activity of pre-osteogenic index components.(4)Comparative study on quality of bark and leaf of Eucommia ulmoides based on pharmacodynamic substances.Through the above research,the pharmacodynamic material basis of cortex eucommiae and Folium eucommiae for treating osteoporosis was initially determined.The contents of active components and total flavonoids were determined by HPLC and UV,and the quality differences between Eucommia bark and Eucommia ulmoides leaves were analyzed.Findings:(1)HPLC specific chromatogram of cortex Eucommiae and Folium Eucommiae were established.Twenty-four characteristic peaks were identified,including 2 lignans,3 iridoids,7phenolic acids,11 flavonoids and 1 terpene.OPLS-da and PCA analysis showed that there were great differences in the chemical components of the bark and leaves of Eucommia ulmoides from different areas.Geniposidic acid,pinoresinol diglucoside,chlorogenic acid,quercitrin and geniposide were the main differential components in cortex eucommiae,while geniposide,catechin,chlorogenic acid and geniposidic acid were the main differential components in Folium eucommiae.(2)Network pharmacological studies have shown that active ingredients such as Kaempferol,Quercetin,Dehydrodieugenol,beta-sitosterol,8-Hydroxypinoresinol,betacarotene,Epiquinidine,Dehydrodiconiferyl alcohol,Hirsutrin may interfere with tumor necrosis factor,rheumatoid arthritis,MAPK signaling pathway and other related pathways by acting on PTGS2AKT1,MAPK1,JUN,PTGS1,FOS,TNF,IL6,VEGFA,DPP4 and other target proteins,and then play an anti-osteoporosis role in Eucommia ulmoides.Molecular docking results showed that Kaempferol,Quercetin,Syringetin and other compounds had high docking scores with target proteins,and could be well docked to the active cavity of potential target proteins.(3)The proliferation of MC3T3-E1Subclone14 cells treated with the extracts from the bark and leaves of Eucommia ulmoides Oliv.was investigated.The results showed that the extracts had no toxic effect on MC3T3-E1Subclone14 cells after 24 H treatment.Compared with the blank group,the ethyl acetate and n-butanol fraction of Eucommia bark 10 μg/m L group had no significant proliferation with osteoblasts after 24 H of administration,the other groups could promote the proliferation of osteoblasts.The effect of n-butanol extract on the proliferation of MC3T3-E1 cells was better than that of ethyl acetate extract.The results of MC3T3-E1Subclone14 cell ALP activity experiment showed that the ALP activity value of cells treated with n-butanol fraction was higher than that of ethyl acetate at the same concentration,and the ability of n-butanol fraction to promote MC3T3-E1Subclone14 cells to secrete ALP activity was higher.The chemical constituents of the extracts were analyzed,and the results showed that hyperin,pinoresinol diglucoside,chlorogenic acid,kaempferol and geniposidic acid were identified as the anti-osteoporosis substances in the bark and leaf of Eucommia ulmoides.(4)The results showed that the contents of geniposidic acid and pinoresinol diglucoside in the bark were significantly higher than those in the leaves.The contents of chlorogenic acid and hyperoside in Eucommia ulmoides leaves were significantly higher than those in bark.There were also significant differences in the content of the same part of the drug among different producing areas.The contents of geniposidic acid and chlorogenic acid in DZPSN003 were higher than those in the other two areas,but the contents of hyperoside and kaempferol were lower than those in the other two areas.The content of total flavonoids in Eucommia ulmoides leaves was significantly higher than that in Eucommia ulmoides bark,especially the content of total flavonoids in DZYSN003 was nearly 10 times higher.Conclusion:Through the above research work,the pharmacodynamic material basis of Eucommia ulmoides bark and leaves was preliminarily determined,the anti-osteoporosis effect and its mechanism were explained,and the difference law of pharmacodynamic substances in different parts and producing areas was obtained,which provided experimental basis for the quality control and rational utilization of Eucommia ulmoides bark and leaves. |
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