| Thymi herba(??)is the dried leaves and flowers of Thymus mongolicus(T.mongolicus)or Thymus przewalskii(T.przewalskii),which has been included in the Chinese Pharmacopoeia(1977)for the treatment of coughs,headaches,abdominal pains,and diarrhea.Modern pharmacological studies have found that Thymi herba has good anti-inflammatory,antibacterial,antioxidant,anti-tumor,antithrombotic,and antiallergic effects.The study on the chemical constituents of genus Thymus mainly focuses on the volatile constituents,among which Thymol and Carvacrol have good antibacterial,antioxidation,antitumor and other biological activities.None chemical composition of T.przewalskii has been reported.Up to now,there are no TLC identification and content determination items in quality standard of Thymi herba.To clarify the chemical substance basis of Thymi herba and improve its quality standard,this subject has studied its chemical composition and quality standard.The main research results are as follows:1.A Survey of Studies on ThymusIn this paper,the varieties and distribution,chemical components,pharmacological effects and quality standards of Thymus plants are systematically reviewed.Literature research shows that Thymus is about 400 species in the world,distributed in northern Africa,Europe and temperate Asia.There are 11 species and 2 varieties in China.The chemical structure types isolated and identified in Thymus include volatile oils,flavonoids,lignins,steroids,terpenoids,phenolic acids,long chain hydrocarbon and other components.Pharmacological activities mainly include antibacterial,anti-inflammatory,antioxidant,anti-tumor,inhibition of thrombosis,etc.2.Experimental Research2.1 Chemical Constituents of Thymi herba29 compounds were obtained from the ethanol extract of T.przewalskii by a combination of various chromatographies separation methods,including silica gel,reversed-phase silica gel C18,Sephadex LH-20,MCI and other chromatographic fillers.Using modern spectroscopy techniques(UV,IR,MS,NMR,2D-NMR,CD,etc.)combined with physical and chemical properties,a total of 24 compounds were identified,namely:5,6-dihydroxy-7,8,4'-trimethoxydihydroflavone(1),5,4'-dihydroxy-7,8-dimethoxydihydroflavone(2),5,4'-dihydroxy-6,7-dimethoxydihydroflavone(3),Sakuranetin(4),Diosmetin(5),Lysionotin(6),Methyl rosmarinate(7),Xanthomircrol(8),Salcolin A(9),Salcolin B(10),Quercetin(11),Tricin(12),Luteolin(13),Gardenin B(14),Caffeic Acid(15),Ursolic Acid(16),Oleanolic Acid(17),?-sitosterol(18),Cirsimaritin(19),Salvianolic acid A(20),Syringic acid(21),Amentoflavone(22),Apigenin-7-O-beta-D-glucuronide(23),Scutellarin(24).Compound 1 was a new compound and its configuration was determined by CD spectrum,compound 2 was isolated from natural products for the first time and its configuration was determined by CD spectrum,compounds 3-24 are the first separation from T.przewalskii,2-4,6,7,9-12,14,20-23 are the first separation from Thymus.2.2 Cytotoxicity Test ResearchThe CCK-8 was used to measure and evaluate the anti-tumor activities of four flavanones(compounds 1-4)isolated from T.przewalskii.The results showed that the four flavanones had weak antitumor activities inhibited the proliferation of human lung cancer cells A549 at the concentration ranging from 50?150 ?M,with the IC50 values 135.6 ?M,93.3?M,113.9?M and 74.5 ?M,respectively.It suggested that the four flavanones(1-4)may not be the main antitumor active components of Thymi herba.2.3 Volatile Components of Thymi herbaHS-SPME and GC-MS analysis were used to study the volatile components of Thymi herba from different habitats.L9(34)orthogonal design was used to optimize the sample volume(0.5,1.0,1.5 g),extraction time(20,40,60 min),extraction temperature(60,80,100?),desorption time(1,2,3 min)and other factors.The order of influence on the experimental results was determined as extraction temperature>desorption time>sample volume>extraction time.The optimum solid phase microextraction conditions were determined as follows:sample weight 1.0 g,extraction temperature 80? extraction time 60 min,desorption time 2 min.The dried leaves and flowers of 16 T.mongolicus and 4 T.przewalskii were analyzed under the optimum conditions.Through database comparison,102 volatile components were identified,inc luding 10 common components,namely:1,6-Octadien-3-ol,3,7-dimethyl-?3-Cycl ohexen-1-ol,4-methyl-1-(1-methylethyl)-?2,6-Octadien-1-ol,3,7-dimethyl-,(E)-?2,6-Octadienal,3,7-dimethyl-,(E)-?Thymol?Carvacrol?(-)-Spathulenol?Bicyclo[7.2.0]undec-4-ene,4,11,11-trimethyl-8-methylene-,[1R-(1R*,4Z,9S*)]-?5,9-Undecadien-2-one,6,10-dimethyl-,(Z)-?Eucalyptol.Among the common components,Thymol a nd Carvacrol were the highest,accounting for 9.11%?34.49%,15.06%?55.48%of the total volatile components,respectively,which indicated that Thymol an d Carvacrol were the main components of the volatile components of Thymi h erba.The above results show that there are some similarities between the volatile components from different sources and different places of origin.Thymol and Carvacrol are the main components of the volatile components,and the correlation between the content and the quality of the medicinal materials needs further study.2.4 Determination of Thymol and Carvacrol in Thymi herba by UPC2In this study,a rapid and sensitive ultraperformance convergence chromatography method was firstly established for quantification of Thymol and Carvacrol,two positional isomers of a major bioactive in the volatile oil of Thymi herba.Using a TrefoilTM CEL1 column,Thymol and Carvacrol were separated in less than 2.5 min and resolution was enhanced.The method was validated with respect to precision,accuracy,and linearity according to the Chinese Pharmacopoeia(2015 Version,Volume 1)and its appendix method.The optimized method exhibited good linear correlation(r=0.9998-0.9999),excellent precision(relative standard deviations(RSDs)<1.50%),and acceptable recoveries(87.29?102.89%).The limits of detection for Thymol and Carvacrol were 1.31 and 1.57 ng,respectively,while their corresponding limits of quantification were 2.63 and 3.14 ng.Finally,the quantities of the two compounds present in 16 T.mongolicus and 4 T.przewalskii samples were successfully evaluated by employing the developed method.The results of this study will serve as a guideline for the quality control of Thymi herba.2.5 Quality Standard of Thymi herbaQualitative identification of Thymi herba by TLC;Determination of rosmarinic acid in Thymi herba by HPLC;According to the Chinese Pharmacopoeia(2015 Version,Volume 1)and its appendix method,the water,total ash,acid insoluble ash and ethanol extractives were analyzed for Thymi herba.The results show that TLC identification of Thymi herba has strong characteristics,under the UV lamp(365 nm),fluorescent spots of the same color appeared in the corresponding position of the chromatogram of rosmarinic acid reference substance.The linear relationship of rosmarinic acid was good in the range of 3.44?3520.45 ng(r=0.9999),and the recovery is 97.41%?103.47%(RSD=1.76%).The average content of rosmarinic acid in the 20 batches of Thymi herba is 2.10?15.40 mg·g-1,the water content is 3.90%?8.54%,the total ash content is 5.80%?16.30%,the acid insoluble ash content is 0.90%?8.00%,the ethanol extractives content is 6.20%?11.20%.The results indicated that the content of rosmarinic acid should not be less than 0.20%,the ethanol extractives should not be less than 6.0%,the water,total ash,acid insoluble ash,were not more than 10.0%,17.0%,8.0%,respectively.The qualitative and quantitative evaluation methods of Thymi herba were established for the first time.The method is simple,accurate and reproducible,and can be used for the quality control of Thymi herba.Conclusively,this paper has conducted a systematic study on Thymi herba from the aspects of chemical composition and quality control,including 24 isolated and identified compounds,qualitative and quantitative analysis of Thymol,Carvacrol and rosmarinic acid.Herein,the study filled the chemical knowledge gap of Thymi herba from T.przewalskii source,improved the quality standard,and provided a theoretical basis and scientific evidence for the development and utilization. |
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