| ObjectiveAccording to our previous research,we compared the difference of the fingerprints of ethyl acetate extract from SPM and PSPM,what is more,we identified the components of the ethyl acetate extract.We separated and identified the chemical composition of PSPM in order to study the material basis of the change of the efficacy of the SPM before and after processing.In order to establish the fingerprint of the extract of ethyl acetate extract,the fingerprints of SPM and PSPM were studied.We compare the content of 3,7-dimethyl quercetin in SPM and PSPM,and compare the content of angiotensin I and calcitonin gene-related peptide levels in rats influenced by SPM and PSPM.Methods1.Silica gel,gel and semi-preparative liquid phase chromatography were used to isolate and purifie the chemical constituents of PSPM.The obtained monomer compounds were subjected to nuclear magnetic resonance spectroscopy to obtain a spectrum.The name and structure of the compound were determined by the physical and chemical properties and spectral data,thin layer chromatography and then combined with the literature.2.The HPLC method was used to compare the fingerprints of the ethyl acetate extract of SPM and PSPM,three common peaks were identified.3.HPLC-MS was used to study the composition of ethyl acetate extract from SPM and PSPM.The data were analyzed by Thermo Xcalibur software.4.The content of 3,7-dimethyl quercetin in SPM and PSPM was determined by HPLC,and the difference was found in SPM and PSPM.5.SELDI-TOF-MS was used to study the plasma content of plasma in rats.According to the different administration,the rats were divided into normal control group,product group and processed product group.The common proteins contained in the serum were detected after 30 days of intragastric administration.6.The effects of SPM and PSPM on the levels of angiotensin I and calcitonin gene-related peptides in rat plasma were measured by radioimmunoassay.Results1.Seven components were isolated and identified from PSPM,3,7-dimethyl quercetin,3-O-methyl quercetin,ent-16?,17-Dihydroxy-kauran-19-oic acid,Darutigenol,Darutoside,12-hydroxy-kirenol and caffeic acid.2.As the results show,there are 19 common peaks in SPM and 12 in PSPM.The number of common peaks decrease during the process of processing.By comparing the fingerprints of SPM PSPM,it was found that the peak area of the PSPM was reduced,indicating that the content of the products was reduced.Three components were successfully identified:kirenol,5"-methoxyepipinoresinol and Hythiemoside A.3.HPLC-MS results showed that in the positive/negative ion mode,10 compone nts were analysised in SPM,including(6S,9S)-roseoside,3,4-Dihydroxybenzaldehyde,5''-methoxyepipinoresinol,ent-16?,17,18-trihydroxy-kauran-19-oic acid,ent-2?,15,16-tr ihydroxypimar-8(14)-en-19-oic acid,12-hydroxy-kirenol,ent-16?,17-dihydroxykauran-19-oic acid,?-sitos-terol and 3,7-dimethyl quercetin.But only three components were analy sised,ent-16?,17,18-trihydroxy-kauran-19-oic acid,12-hydroxy-kirenol and 3,7-dimethyl quercetin included.4.The contents of 3,7-dimethyl quercetin in SPM and PSPM were 228.33 ?g/ml and 202.63 ?g/ml,respectively.The contents of 3,7-dimethyl quercetin in SPM is slightly higher than that in PSPM.5.8 proteins were found in rat serum,including Angiotensin-I,Rimorphin {ECO:0000250},VGF(375-407),Secretoneurin,Calcitonin gene-related peptide,Pancreatic hormone,Protein FAM229B and Unique cartilage matrix-associated protein,its content in rat plasma to be further studied.6.Angiotensin I was expressed in normal rats with renin activity,the results of radioimmunoassay showed that the level was 27.84 ± 1.70 ng/ml.hr in the normal group and 11.82 ± 0.76 ng/ml.hr in the positive control group,the level in the product group was 8.16 ±1.14 ng/ml.hr and the level in the processed product group was 16.20 ± 2.03 ng/ml.hr;The level of calcitonin gene-related peptide(CGRP)in the normal group was 80.18 ± 3.07 pg/ml,the level of the positive control group was 93.85 ± 1.92 pg/ml,the level in the product group was 119.81 ± 5.32 pg/ml,while 107.69 ± 2.71 pg/ml in the processed group.It is found that PSM has a stronger antihypertensive effect.ConclusionThe components isolated from PSPM are also contained in SPM,but the content is changed.Fingerprint studies have shown that the composition of the ethyl acetate extract after processing is reduced,it may be converted to other ingredients or lost in the process of processing,the identified three components in the processed products also reduced.The content of 3,7-dimethyl quercetin did not change largely,suggesting that the flavonoids were stable and did not change during the process.The HPLC-MS results showed that the composition of the ethyl acetate extract had reduced,and the common composition was also reduced.The results of the effects of SPM and PSPM on the level of angiotensin I and calcitonin gene-related peptideshowed that both can decrease the level of AI and increase the level of CGRP.Futher more,SPM showed a better effect than that of PSPM.It is incidated that PSM may cause antihypertensive effect by inhibiting AI and promote the level of CGRP. |
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