| Thymus quinquecostatus Celak belongs to the genus thymus of Lamiaceae family,which the dry aerial part used as the medicin.It was used in the treatment of gastric pain,chronic bronchitis,chronic stomach pain,arthritis and Kaschin-Beck disease in the folk.The members of this laboratory from the T.quinquecostatus Celak found several flavonoids and phenolic ingredients,such as scutellarin,danshensu,rosmarinic acid,caffeic acid and so on.The ethanol part having significantly reduce pathological model mouse infarction range.In order to fully study T.quinquecostatus played efficacy material basis on cerebral ischemic cardiovascular disease,research on the chemical composition,manufacture process and quality control of T.quinquecostatus effective fraction.The paper has divided into two parts:literature review and experimental research.The literature review has separately summarized the research progress of phenols and metal complexes in cardio-cerebrovascular diseases;the review was made on the progress in the research of cerebrovascular disease drugs,which is containing scutellarin and rosmarinic acid as the bioactive ingredients.Study one:Research on chemical constituents of T.quinquecostatus.Members of the research group first identified 18 compounds from ethanol extracts of T.quinquecostatus,including 9 flavonoids and 7 phenolic acids.UPLC-MS/MS was used to investigate the chemical composition of T.quinquecostatus.19 phenolic compounds were identified in both positive and negative ion patterns,including 12 flavonoids:scutellarin/luteolin-7-O-glucuronide,apigenin-7-O-glucuronide/baicalin,eridioctyol/aromadendrin,luteolin,thymusin,naringenin,methylated luteolin,cirsimaritin,cirsilineol,genkwanin,xanthomicrol,gardenin B.7 phenolic acids:danshensu/syringic acid,vanillic acid,gentisic acid/protocatechuic acid,chlorogenic acid,hydroxybenzoic acid,caffeic acid,rosmarinic acid.Most of the above compounds have good pharmacological activities for treating cardiovascular and cerebrovascular diseases.The study suggests that the above ingredients may also be the active ingredients for treatment of heart and brain vascular diseases.A HPLC method was established for the simultaneous determination of five components of danshensu,caffeic acid,scutellarin,rosmarinic acid,and 4’,5-dihydroxy-6,7,8-trimethoxyflavone in T.quinquecostatus.The method is quick and accurate,it can be used for the quality evaluation of T.quinquecostatus.Particularly worthy of late research is concerned that the contents of mesenterol and rosmarinic acid in the medicinal materials of T.quinquecostatus are all close to 1%.It is suggested that these two components may be the main medicinal ingredients in the treatment of cardio-cerebrovascular diseases.In view of the fact that the phenolic components are easy to coordinate with metal elements,and the complexes have more significant therapeutic activity for cardiovascular and cerebrovascular diseases.ICP-OES and ICP-MS were applied to determine the contents of K,Ca,Na,Mg,Mn,Fe,Zn,and Cu from T.quinquecostatus.The contents of them respectively were 3332,16059,48.75,3269,113.1,1795,27.25,and 12.02 μg/g.It is showed that the content of metal elements in T.quinquecostatus is relatively high,especially Ca and Mg elements.According to the coordination characteristics of phenolic components,the phenolic constituents of T.quinquecostatus may easily form metal complexes with Ca,Mg,Zn and Cu.Study two:Research on chemical composition and quality control of total phenols from T.quinquecostatusA method for the manufacture process of total phenols of T.quinquecostatus was established.First,a single factor investigation was performed on the extracting method of T.quinquecostatus,extraction solvent,extraction time,extraction times,and material-liquid ratio,and the preferred process was selected.Based on this study,L9(34)orthogonal experiments were further used to evaluate the extraction process of T.quinquecostatus,the content of total phenols,scutellarin and rosmarinic acid in T.quinquecostatus are as an indicator.Through comprehensive analysis,the best extract of the effective parts of T.quinquecostatus:D1%ethanol,reflux extraction B2 times,each A2 min,the solvent dosage is C3 g/mL.The macroporous adsorption resin was used to measure the adsorption amount of phenols and the indicator components of scutellarin and rosmarinic acid,the content of eluate,and the metastasis rate.After investigating the absorptive property and separate property of 7 kinds of macroporousresin,A resin was selected as the best resin,due to its has good adsorption and desorption of the total phenols,scutellarin and rosmarinic acid.The total phenols,scutellarin and rosmarinic acid content and metastasis rate were used as the main indices.The optimal enrichment process parameters was as following:C2 g/mL density(equal to the raw herbs),pH=B4,the flow rate of adsorption D2 BV/h,the ratio of loading capacity and to resin volume was E2,the ratio of diameter to height was H2,the volume of removing impurities was W2 BV of water,the elution solvent was P%ethanol,the elution flow rate was R2 BV/h,and the eluent volume was S BV.Under this condition,the content of total phenols was 47.16%,and the transferring ratio was 80.84%;the content of scutellarin was 8.36%,and the transferring ratio was 63.28%;the content of rosmarinic acid was 8.25%,and the transferring ratio was 67.40%.The results of preparation and content determination of three batches of samples showed that the extraction and purification technology of total phenols from T.quinquecostatus was reasonable and stable.UPLC-MS/MS was used to investigate the chemical composition of total phenols of T.quinquecostatus.15 phenolic compounds were identified in both positive and negative ion,including 8 flavonoids:scutellarin/luteolin-7-O-glucuronide,apigenin-7-O-glucuronide,cirsilineol,eridioctyol/aromadendrin,luteolin,thymusin,apigenin,scutellarein.7 phenolic acids:danshensu/syringic acid,vanillic acid,chlorogenic acid,salvianolic acid A,rosmarinic acid,ferulic acid,hydroxybenzoic acid.According to the results of mass spectrometry analysis.The visible spectrophotometric method was used to determine the total phenols content of T.quinquecostatus by using FeCl3-K3Fe(CN)6.The total phenols were quantitatively analyzed with rosmarinic acid as the reference substance.The calibration curve are linear at ranges of 1.10～2.05 μg/mL(r=0.9993).The regression equation was as following:Y=0.3439X-0.0318(r=0.9993),the total phenols content of T.quinquecostatus effective part was 47.75%,RSD was 1.50%.The experimental results showed that the method was simple,rapid and reliable,and could be used as a quality control method for phenolic fraction of T.quinquecostatus.A HPLC method was developed to determine the contents of scutellarin and rosmarinic acid,which are the target components in phenolic fraction of T.quinquecostatus.The calibration curve are linear at ranges of 0.019～0.592 mg/mL(r=0.9990).The regression equation was as following:Y=26449X-37.657,the scutellarin content of T.quinquecostatus effective part was 8.36%,RSD was 1.81%.The calibration curve are linear at ranges of 0.009～0.280 mg/mL(r=1).The regression equation was as following:Y=17000X-9.3631,the rosmarinic acid content of T.quinquecostatus effective part was 8.19%,RSD was 1.27%.The experimental results showed that the method was simple,rapid and reliable,it could be used as a quality control method for phenolic fraction of T.quinquecostatus.ICP-OES and ICP-MS were applied to determine the contents of K,Ca,Na,Mg,Mn,Fe,Zn,and Cu from T.quinquecostatus extract.The contents of them respectively were 20366,12232,326.3,7898,81.61,1262,19.27,38.83 μg/g.The content of K,Na,Mn,and Cu in the phenolic fraction was higher than that in T.quinquecostatus,which was 6.11,6.69,2.42,3.23 times that of K,Na,Mn and Cu in T.quinquecostatus,respectively,indicating that these four elements are relatively easy extracted from T.quinquecostatus.Innovation:1 For the first time,establish quality control methods of T.quinquecostatus and its phenolic fraction.2 Firstly,establish the manufacture process of phenolic fraction from T.quinquecostatus.3 It was the first time to study the chemical constituents of T.quinquecostatus and its phenolic fraction by UPLC-MS/MS method.4 ICP-OES and ICP-MS were used for the first time to determine the trace element contents of T.quinquecostatus and its phenolic fraction. |
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