| Background:Rosa roxburghii is the fruit of silk flower reeling from deciduous perennial shrub of rosaceae,also known as anemone fruit,acanthopterygium,and spring return.It is widely distributed in warm temperate and subtropical regions,and mainly distributed in guizhou,hunan and other provinces in China.Rosa roxburghii has the function of nourishing the spleen,astringent and stopping diarrhea.Because it is rich in vitamin C and essential trace elements for the human body,it has the function of regulating the immunity of the body and delaying aging.Rosa roxburghii has been used as raw materials for medicine,fruit wine,fruit juice and other commodities.The root,fruit and seeds of Rosa roxburghii can be used as medicine,has the efficacy of tonifying stomach,nourishing and antidiarrheal.At present,it mainly develops roxburgh rose juice and roxburgh rose preserved fruit,while the seed of Rosa roxburghii are basically discarded due to their bitter taste,hard shell and small size,which makes seed of Rosa roxburghii a great waste of this green resource.Existing research reports clearly indicate that seed of Rosa roxburghii contain a variety of fatty acids,which have the functions of scavenging free radicals,anti-inflammatory,lowering blood sugar,protecting liver and fighting cancer.At present,the research on seed of Rosa roxburghii is limited to the extraction of low-polarity parts.Volatile esterified compounds were obtained by derivatization and analyzed qualitatively and quantitatively by Gas chromatography or Gas chromatography-Mass spectrometry.There are various side reactions in the process of esterification,and it is difficult to guarantee the complete esterification.The composition information of volatile and non-esterified components could not be obtained.The analysis time is long,make the analysis complicated and so on.Objective:Analyze and identify the chemical constituents of seed of Rosa roxburghii extract by UPLC-Q-TOF-MSE.The contents of trace elements in seed of Rosa roxburghii were measured by microwave digestion and inductively coupled plasma mass spectrometry.The purpose of this study was to evaluate the pharmacological activity of seed of Rosa roxburghii extract,and to study the mechanism of anti-hepatocellular carcinoma in order to lay a foundation for the further research and product development of seed of Rosa roxburghii extract.Methods:1.Chemical composition analysis of seed of Rosa roxburghii:The chemical composition of ethyl acetate part of methanol extract from seed of Rosa roxburghii was analyzed and identified by UPLC-Q-TOF-MSE and UNIFI screening platform.The laws of flavonoids and triterpenes were summarized and applied to the rapid identification of compounds in methanol extract of seed of Rosa roxburghii.To establish an analytical method for the determination of trace elements in roxburl seeds by inductively coupled plasma mass spectrometry.Microwave digestion method was used to pretreat sample powder of seed of Rosa roxburghii.Re,Y,Rh,In,Sc as internal standard,using external standard standard curve method to determine the content of trace elements In the seed of Rosa roxburghii.2.Pharmacological activity evaluation of seed of Rosa roxburghii:To evaluate the antioxidant activity of roxburgh rose seeds in vitro by DPPH method;The inhibition of seed of Rosa roxburghii on tyrosinase activity was determined by L-dopa.The inhibitory effect of seed of Rosa roxburghii on Chick chorioallantoic membrane was investigated by CAM model experiment.The inhibitory effect of seed of Rosa roxburghii on the proliferation of HepG2 was investigated by MTT method.3.Anticancer mechanism of seed of Rosa roxburghii:Inhibition of migration and invasion of seed of Rosa roxburghii on HepG2 was detected by cell scratch test.Annexin V-FITC/PI staining and flow cytometry were used to detect the effect of seed of Rosa roxburghii on the apoptosis rate of HepG2.The effects of seed of Rosa roxburghiion the expression of apoptosis-related proteins Bcl-2,Bax,Caspase-3 and Caspase-9 were measured by Western Blot.Results:1.Mass spectrometry data of methanol extract of seed of Rosa roxburghii were collected by using UPLC-Q-TOF-MSE.A total of 55 compounds,including 19 flavonoids and 36 triterpenes,were identified by UNIFI screening platform in combination with the relative retention time,precise molecular mass,molecular formula,characteristic fragment ion and relevant literature data.A method for the determination of trace elements in the seed of Rosa roxburghii by Inductively coupled plasma mass spectrometry was developed by observing the detection limit,precision and recovery of samples.In this study,ICP-MS method was used to measure the contents of 15 trace elements in seed of Rosa roxburghii,which laid a foundation for the study of the relationship between the element types and contents in seed of Rosa roxburghii and their therapeutic effect.It provides data support for the research on the material basis and mechanism of action of seed of Rosa roxburghii.It provides a basis for evaluating the quality of seed of Rosa roxburghii.And it can provide some reference for the better development and utilization of seed of Rosa roxburghii resources.2.The results of DPPH experiment showed that seed of Rosa roxburghii had a strong antioxidant capacity in vitro.By comparing the IC50 value,it can be seen that the antioxidant capacity of seed of Rosa roxburghii was stronger than that of ascorbic acid.The results of L-dopa test showed that seed of Rosa roxburghii had a good inhibitory effect on tyrosinase,suggesting that seed of Rosa roxburghii might inhibit the synthesis of melanin by inhibiting the activity of tyrosinase,thus playing a role in whitening and skin care.The results of chicken embryo villoallantoic membrane experiment showed that seed of Rosa roxburghii had the activity of inhibiting the growth of blood vessels,which suggested that seed of Rosa roxburghii might inhibit the growth and metastasis of tumor cells by inhibiting the formation of new blood vessels,thus effectively inhibiting the growth of tumor tissues and preventing and treating tumors.The inhibition rate of HepG2 proliferation of seed of Rosa roxburghii samples at different concentrations was dose-dependent and time-dependent,and the inhibitory effect increased with the increase of drug concentration and the prolongation of action time(p<0.05),the difference was statistically significant.3.Cell scratch test results showed that seed of Rosa roxburghii extract had the ability to inhibit HepG2 migration.The inhibitory effect was enhanced with the increase of concentration.The results of cell apoptosis experiments showed that seed of Rosa roxburghii extract could induce apoptosis of HepG2.With the increase of seed of Rosa roxburghii extract concentration,the sum of apoptotic rates of early and late HepG2 increased significantly(p<0.05),and the difference was statistically significant.Western Blot results showed that different concentrations of seed of Rosa roxburghii extract had an effect on the protein expression levels of Bax,Bcl-2,Caspase-3 and Caspase-9.With the increase of concentration,Bax protein expression level increased gradually.The expression levels of Bcl-2,Caspase-3 and Caspase-9 decreased gradually. |
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