Studies On The Quality Evaluation Method And Pharmacokinetics Of Longjing Green Tea

Longjing green tea(also called Dragon Well tea)is a kind of unfermented dried leaves of Camellia sinensis(L.)O.Kuntze,and has become one of the most widely consumed beverages all over the world.In this paper,chemical components,extraction method,quality evaluation method and pharmacokinetics of Longjing green tea were studied systematically.The chemical components were separated and analyzed using various separation methods.11 compounds were achieved from Longjing green tea after extracted with 70%alcohol-water.They were identified on the basis of physi-chemical and spectral methods,including 6 catechins:catechin,epicatechin,gallocatechin,epigallocatechin,epigallocatechin gallate and epicatechin gallate;2 alkaloids:caffeine and theobromine;2 flavonoids:kaempferol and quereetin;1 sterol:a-spinasterol.The extraction method was optimized in the study.Contents of all the catechins were selected as observing index.Extraction temperature?extraction durations?the ratio of material to liquid and extraction times were optimized by using the single factor test.The experiment proved that optimized method was using 600 folds amount of boiling water for three times and extracted 10 min each time.The optimized process was stable,feasible and suitable for the extraction of Longjing green tea in our daily life.A high performance liquid chromatographic(HPLC)method with diode array detection was developed and validated to simultaneously determine eleven compounds in Longjing green tea including gallic acid,theobromine,gallocatechin,epigallocatechin,caffeine,catechin,epicatechin,epigallocatechin gallate,gallocatechin gallate,rutin and epicatechin gallate.The determination was performed on a Waters Symmetry C1g column,with the mobile phase consisting of acetonitrile(A)and 0.5%phosphoric acid in water(B)with gradient elution mode at a flow rate of 1.0 ml·min-1.The detection wavelength was 203 nm.The method showed good linear relationship for the 11 analytes within the tested ranges(n=6,r>0.9999).The average recoveries were between 89.6%and 97.6%,the RSD was less than 3.8%.The established method was applied to determine the amounts of the eleven compouds in thirteen samples from different regions.The results indicated that significant difference existed in the contents of the polyphenols,while almost no difference exsited in the contents of alkaloids.RP-HPLC fingerprint chromatogram of 13 batches of Longjing green tea from different regions was established.The separation was performed on an Agilent ZORBAX SB-C18 column,with the mobile phase consisting of acetonitrile(A)and 0.5%phosphoric acid in water(B)with gradient elution mode at a flow rate of 1.0 ml·min-1.The detection wavelength was 203 nm.20 common peaks were acquired in the fingerprint and 11 of them were identified including the internal reference peak-caffeine.Similarity tests were performed using the professional software named Similarity Evaluation System for Chromatographic Fingerprint(2010).The similarity was more than 0.95 for the plant samples coming from the traditional plant areas and less than 0.95 for those coming from new areas.The method was simple and accuracy with good reproducibility and can be used as a quality control method for Longjing green tea.A liquid chromatography hybrid ion trap time-of-flight mass spectrometric(LC-IT-TOF-MS)method was developed and validated for identification and simultaneous determination of the potential bioactive components from green tea in rat plasma.Two prototype components and one metabolite were successfully identified as caffeine,theobromine and theophylline.After addition of two internal standards,hypoxanthine and paracetamol,plasma samples were extracted by liquid-liquid extraction(LLE)method with ethyl acetate and separated on a Shim-pack XR-ODS ? column,with the mobile phase composed of 0.1%formic acid in acetonitrile(A)and 0.1%formic acid in water(B)with gradient elution mode.The linear range was 10.0-10,000 ng-ml-1 for caffeine,1.0-1,000 ng·ml-1 for theobromine and 2.0-2,000 ng·ml-1 for theophylline with the lower limit of quantitation of 10.0,1.0 and 2.0 ng·ml-1,respectively.The recoveries of the analytes and the internal standards were both higher than 71.22%.The intra-day and inter-day precision were within 6.0%and 10.9%,and accuracy(relative error,RE%)were less than 4.8%and 6.5%,respectively.The validated method was successfully applied to investigate the dynamic change rules of caffeine,theobromine and theophylline in rat plasma after oral administration of caffeine,theobromine and Longjing green tea extract.After caffeine or aqueous extract of Longjing green tea were orally treated,the Cmax of caffeine was 9.18 ×103±1.39 ×103and 5.500×103±1.655 ×103 ng·ml-1,AUC(0-?)was 9.90×respectively.After caffeine,theobromine or aqueous extract of Longjing green tea were orally treated,the Cmsx of theobromine was 74.25±8.57,33.86±11.23 and 82.1± 10.4 ng·ml-1 AU(0-?)was 305.7±24.2,1474± 1021 and 2752±984 ngh·ml-1,T1/2 was 2.4±0.6,11.3±6.3 and 13.0±8.0 h,respectively.After caffeine or aqueous extract of Longjing green tea were orally treated,the Cmax of theophylline was 108.7±18.0 and 121.2±64.6 ng·ml-1,ACUC(0-?)was 1695±426 and 2668±1125 ngh·ml-1,T1/2 was 4.8±0.6 and 10.2±7.2 h,respectively.The results indicated that there were obvious differences in the pharmacokinetic behaviors of caffeine and theobromine after oral administration of the mono substance and the aqueous extract of Longjing green tea,while there was no difference in the pharmacokinetic behaviours of theophylline.