| Background:At present,osteoporosis has become a serious social and public health problem in China.However,the commonly used clinical treatment drugs such as bisphosphonates,calcitonin and fluoride can cause side effects including irritating esophageal inflammation and antigenic reactions.Plant medicine has broad application prospects because of its mild effect and small toxic and side effects.Therefore,the search for safe and effective anti-osteoporosis drugs from plant drugs has become a hot topic in the field of global medical research.The previous experimental research of our research group showed that the leaves of Dalbergia odorifera had good anti-osteoporosis effect.In addition,Dalbergia odorifera leaves have been included in the’Hainan food local safety standards’.Based on this,this paper deeply discusses the anti-osteoporosis mechanism of Dalbergia odorifera leaves and carries out quality analysis and research,in order to provide reference for the development of forestry waste-Dalbergia odorifera leaves resources,and also contribute to the protection of precious traditional Chinese medicine-Dalbergia odorifera plant resources.Objective:To explore the anti-osteoporosis mechanism of Dalbergia odorifera leaves,and to carry out the quality analysis of Dalbergia odorifera leaves.Methods:The chemical information of Dalbergia odorifera leaves was collected by UPLC-Q-TOF-MS/MS analysis technology.After that,combined with the mass spectrometry database and previous literature comparison,the chemical constituents in the leaves of Dalbergia odorifera were analyzed and determined.Combining the above research results with the previous literature reports on the chemical constituents of Dalbergia odorifera leaves,Swiss Target Prediction was used to obtain the target of the chemical constituents of Dalbergia odorifera leaves.At the same time,the disease targets of osteoporosis were collected by searching NCBI and Gene Cards disease target database.After that,the disease targets and component targets were intersected through the Venny platform to obtain potential targets.Then,the protein interaction network was constructed to screen out the core targets.Finally,the mechanism of anti-osteoporosis of Dalbergia odorifera leaves was discussed by GO biological function and KEGG pathway enrichment analysis.Aiming at Biochanin A and Genistein in Dalbergia odorifera leaves,MC3T3-E1cells were used as the activity evaluation model,and the concentration of Biochanin A and Genistein treated MC3T3-E1 cells was detected by CCK-8 method.Then,enzyme-linked immunosorbent assay（ELISA）,alkaline phosphatase（ALP）staining and alizarin red staining were used to study the osteogenic activity of the two with ALP,staining degree and mineralized nodules as activity evaluation indexes.Finally,the effects of different harvest time and different origin factors on the contents of Biochanin A,Genistein and Total phenolic acid in Dalbergia odorifera leaves were investigated by HPLC and UV-visible spectrophotometry,and the quality analysis of Dalbergia odorifera leaves was carried out.Results:Through mass spectrometry analysis,18 components in the leaves of Dalbergia odorifera were identified,including 16 flavonoids（Kaempferol,Luteolin,6-Methoxyluteolin,Chrysoeriol,Genistein,Tectorigenin,4′-Hydroxywogonin,Crisiliol,Diosmetin,Eupatilin,Genkwanin,Biochanin A,5,7-Dihydroxy-3′,4′-dimethoxyflavone,Tricin,Formononetin,and Prunetin）,one phenolic acid compound（Salvianolic acid F）,and one steroid compound（Dimethisterone）.Among them,Kaempferol,Luteolin,6-Methoxyluteolin,Chrysoeriol,4′-Hydroxywogonin,Crisiliol,Diosmetin,Eupatilin,Genkwanin,5,7-Dihydroxy-3’,4’-dimethoxyflavone,Tricin,Formononetin,Salvianolic acid F and Dimethisterone were first analyzed from the leaves of Dalbergia odorifera.According to the network pharmacology prediction analysis,there were 461intersection targets between compounds and diseases,of which 40 core targets with Degree value≧90.On this basis,477 biological processes（P<0.05）were obtained by GO enrichment analysis and 154 biological processes（P<0.05）were obtained by KEGG enrichment analysis.Lipid and atherosclerosis,AGE-RAGE signaling pathway in diabetic complications,HIF-1 signaling pathway and EGFR tyrosine kinase inhibitor resistance were speculated to be related to this study.At the same time,29 active components including Biochanin A and Genistein were predicted.Compared with the induction group,the corresponding ALP activity values of Biochanin A at 1×10-6and 1×10-5M doses were significantly increased（P<0.05,P<0.05）.At the same time,the corresponding ALP activity values of Biochanin A at1×10-7M dose and Genistein at 1×10-7,1×10-6and 1×10-5M doses showed an increasing trend,but there was no significant difference（P>0.05）.In addition,ALP staining results showed that the positive staining cells of Biochanin A and Genistein were more than those in the induction group.Moreover,compared with the induction group,the area of mineralized nodules increased significantly（P<0.01）at the dose of1×10-5M of Biochanin A,indicating that Biochanin A could promote the production of mineralized nodules.At the same time,Biochanin A at the dose of 1×10-7and1×10-6M and Genistein at the doses of 1×10-7,1×10-6and 1×10-5M,respectively,promoted the growth of mineralized nodule area,but there was no significant difference（P>0.05）.The above results show that Biochanin A and Genistein have a certain role in promoting bone formation.The effects of different harvest time on the contents of Biochanin A,Genistein and Total phenolic acids in the leaves of Dalbergia odorifera were investigated.The results showed that the contents of Biochanin A,Genistein and Total phenolic acids in the leaves of Dalbergia odorifera were 0.09±0.01～1.87±0.02 mg·g-1（dry leaf）,0.26±0.01～1.14±0.02 mg·g-1（dry leaf）and 6.20±0.19～9.02±0.04 mg·g-1（dry leaf）,respectively.The effects of different origin factors on the contents of Biochanin A,Genistein and Total phenolic acids in the leaves of Dalbergia odorifera were investigated.The results showed that the contents of Biochanin A,Genistein and Total phenolic acids were 0.64±0.02～37.12±0.25μg·g-1（dry leaf）,19.57±0.37～79.76±1.55μg·g-1（dry leaf）,and5.45±0.02～8.74±0.02 mg·g-1（dry leaf）,respectively.There were 6 common peaks in the fingerprint of Dalbergia odorifera leaves from different producing areas.The similarity of HPLC chemical fingerprint was 0.775～0.962.The 10 batches of Dalbergia odorifera leaves were divided into 4 categories by cluster analysis.The medicinal materials collected from 2 producing areas in Guangdong Province and 3producing areas in Guangxi Province were clustered into one category,the medicinal materials collected from Nanchang City in Jiangxi Province were clustered into one category,the medicinal materials collected from 2 producing areas in Hainan Province were clustered into one category,and the medicinal materials collected from Fujian and Zhejiang provinces were clustered into one category.Conclusion:In this paper,the anti-osteoporosis mechanism and quality analysis of Dalbergia odorifera leaves can lay a foundation for the development of anti-osteoporosis innovative drugs and the protection of precious traditional Chinese medicine-Dalbergia odorifera plant resources. |
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