| Abutilon theophrasti Medic.is the whole plant of Abutilon,which belongs to Abutilon genus of Malvaceae family,mainly found in tropical and subtropical regions,and cultivated by many countries all over the world.It has many medical effects including relieving rheumatic pains,dispelling the wind and detoxicating.The chemical constituents of the roots of Abutilon theophrasti Medic.,the quality control,pharmacological activity of five parts?roots,stems,leaves,seeds and exocarps?of Abutilon theophrasti Medic.,and the pharmacokinetics of the active compound,syriacusin A were investigated in this study.Seventeen chemical constituents were isolated from 70%ethanol extraction of the roots of Abutilon theophrasti Medic.,by using silica gel column chromatography,Sephadex-LH20 column chromatography.The structures of following 9 compounds were fully elucidated by chemical,physical,spectroscopic and nuclear magnetic resonance spectroscopy methods?MS,UV and NMR?.Among them,7-Hydroxycadalin?1?and Clemiscosin A?6?,which belong to naphthalene and coumarins respectively,were isolated from Malvaceae family for the first time;Syriacusin A?2?and Dibutyl phthalate?5?were isolated from Abutilon genus for the first time;?-Sitoterol?3?,Stigmasterol?4?,Daucosterol?7?,Glucose?8?and Uridine?9?were isolated from the roots of Abutilon theophrasti Medic.for the first time.The chemical compositions of five parts from Abutilon theophrasti Medic.,were studied systematically through the following methods:?1?High performance liquid chromatography-electrospray ionization-quadrupoles-time of flight-mass spectrometry?HPLC-ESI-Q-TOF-MS?was applied to study the phenols components of Abutilon theophrasti Medic..The analysis was performed on a Diamonsil C18 column using gradient elution with the mobile phase consisting of acetonitrile and 0.1%formic acid solution?pH 2,4?and the flow rate was 1.0 mL·min-1.The detection mode was performed under anion pattern detection.Standards,references and other methods were used to identify the 15 phenols and 1 naphthalene compounds in five parts of Abutilon theophrasti Medic.;By comparing with the references,37 components were identified in five parts of Abutilon theophrasti Medic.;Data Analysis 3.4 software was used to presume the molecular formulae of several unknown chemical compositions in five parts of Abutilon theophrasti Medic..?2?A simple and sensitive gas chromatography tandem mass spectrometry?GC-MS?method was developed to study the fatty oil,volatile oil and some weak polar constituents in five parts of Abutilon theophrasti Medic..The separation of the fatty oil,volatile oil and some weak polar constituents,was conducted on DB-1 quartz capillary column with programmed column temperature,and El source;with reference and NBS database of MS,several weak polar components were identified in five parts of Abutilon theophrasti Medic..The components of fatty oil were 38,51,78,55 and 26 in the roots,stems,leaves,seeds and exocarps of Abutilon theophrasti Medic.,respectively;the components of volatile oil were 66,46,19,31 and 41 respectively and the components in chloroform extraction from the roots of Abutilon theophrasti Medic.were 42.Research on the chemical constituents in five parts of Abutilon theophrasti Medic.by liquid chromatography and gas chromatography,systemically provided information of chemical components of Abutilon theophrasti Medic.,revealed the structures of the compounds,which was used for the recognization of the pharmacological effect and the development of new efficacy of Abutilon theophrasti Medic..By orthogonal test,the optimal extraction condition of gallic acid,protocatechuic acid,catechin,vanillic acid,caffeic acid,ferulic acid,rutin,quercetin,and syriacusin A is:A3B3C2D2?which is 70%alcohol with 20 times solvent,ultrasonic extracting twice for 30min each time;the HPLC method was developed to determine the contents of the nine components above by using Diamonsil C18 column and gradient elution with the mobile phase consisting of acetonitrile and 0.1%formic acid solution?pH 2.4?and the flow rate was 1.0 mL·min-1.The instrumental intra-day and inter-day precision,and method precision were calculated as RSDs,which were less than 1.98%,3.41%,and 1.98%,separately.Recoveries were within the range of 87.3%-1 00.8%.LODs and LOQs of the methods were 0.045-0.400?g·mL-1?S/N=3?and 0.090-1.115?g·mL-1?S/N=10?,respectively.The result showed that the analysis method in this research is of high specificity,accuracy and convenience,which provided the references and basis for quality control of Abutilon theophrasti Medic..In order to improve the quality control standard of Abutilon theophrasti Medic,the following researches were performed for the quality control of common pesticides and elements:?1?The 10 pesticides residues in TCM were determined by GC-ECD method,OV-1701 quartz capillary column with programmed column temperature.The pesticides residues in samples were handled by ultrasonic extraction and silicon magnesium adsorption purification,and the results showed that the method was rapid,accurate and easy to operate,thus providing the evident for TCM quality control.?2?The content of 18 elements was determined by inductively coupled plasma optical emission spectrometry?ICP-OES?.The samples needed to be digested in nitric acid-perchloric acid?4:1,v/v?on the heating pad before measured.The result showed that Na,Mg,K,Ca and some macroelements were widely existed in Abutilon theophrasti Medic.,while the content of V,Ni,As,Cd and Pb and other toxic elements were all lower than limit content.This method can be used to determine the elements in TCM,which was easy and rapid.Antioxidant capacity and antibacterial ability researches were performed for the evaluation pharmacological effects:?1?Orthogonal test optimized and confirmed that the optimal extraction condition of the total flavonoids and total polyphenols in Abutilon theophrasti Medic.were A2B1C3,which is 70%alcohol with 20 times solvent,ultrasonic extracting twice for 30min each time;and A1B2C3,which is 30%alcohol with 16 times solvent,ultrasonic extracting three times for 30min each time.The content of total flavonoids,total polyphenol,and total anthocyanidin,reducing power,the ability to scavenge O2-·and · OH,were determined by ultraviolet spectrophotometry,systematically evaluating the antioxidant capacity of Abutilon theophrasti Medic..?2?Disk diffusion and gradually tube dilution methods were used to investigate the antibacterial ability of total flavonoids and fatty oil.Through the determing the size of inhibition zone,MIC and MBC,the antibacterial ability of total flavonoids and fatty oil in five parts of Abutilon theophrasti Medic.,was evaluated systematically.The result showed that the antibacterial ability of total flavonoids was much larger than fatty oil;the total flavonoids and fatty oil in the five parts showed the antibacterial ability,exocarps,roots and leaves were comparatively better.HPLC analysis method was developed to determine the syriacusin A in rat plasma which was pretreated by liquid-liquid extraction.The analysis was carried out on a Hypersil-C18?250 mm × 4.6 mm i.d.,5 ?m?using the mobile phase methanol and 0.1%formic acid solution?79:21,v/v?.The flow rate was 1.0 mL·min-1.The validated method was successfully applied to a pharmacokinetic study in rats after vena caudalis administrations of syriacusin A(20 mg·kg-1).The pharmacokinetic parameters of syriacusin A in rats' were as following:t1/2:0.88±0.39h,Tmax:0.08h,Cmax:2910.4±164.2mg·L-1,AUC?0-??:1443.5±253.8 mg·L-1·h-1.In conclusion,the constituents in five parts?roots,stems,leaves,seeds and exocarps?of the Abutilon theophrasti Medic.,were investigated by HPLC-ESI-Q-TOF-MS,GC-MS,HPLC,GC,ICP-OES and other analytical techniques;the methods of quality control based on HPLC and GC were established,the antioxidant and antibacterial activity of the five parts were determined;the pharmacokinetic study on the active compound-syriacusin A was conducted.This study provided useful information for the first exploration and research of pharmacodynamic basis,quality control,pharmacological effect and pharmacokinetics of Abutilon theophrasti Medic.. |
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