Study On The Dynamic Changes Of Chemical Compounds And Antioxidant Activity Of Green Tea

Tea, an infusion prepared with the leaves of Camellia sinensis (L.) O. Ktze, is the most widely consumed drink in the world, besides water. In this paper, dynamic changes of chemical compounds and antioxidant activities, pharmacokinetics and the antioxidant mechanism of green tea were studied.A ultra flow liquid chromatographic mass spectrometric (UFLC/MS-IT-TOF)method combined with Formula predictor soft ware were employed for screening and identifying the main constituents of green tea. Together 18 compounds were identified including theanine; theophylline and caffeine; epigallocatechin, gallocatechin,epicatechin gallate, catechin gallate, epicatechin, catechin, epigallocatechin-3-gallate,gallocatechin-3-gallate, 1-galloyl-glucose; rutin, epigallocatechin3-O-(3"-O-methyl)gallate, epigallocatechin-3,5-digallate; procyanidin dimmer; gallic acid, 5-galloylquinic acid and the fragmentation pathways of EGC/GC, ECG/CG, EC/C and EGCG/GCG were also studied.The dynamic antioxidant activity changes of green tea was measured by different analytical methods including total phenols Folin-Ciocalteu method, DPPH-free radical scavenging activity assay, ABTS+ free radical scavenging activity assay and Ferric reducing antioxidant capacity assay. These methods were used to evaluate the changes of antioxidant activity of green tea samples with the different collection periods. The total phenols contents and the antioxidant activity of Shifeng Longjing and Meijiawu Longjing green tea displayed the same dynamic trends, first increased then decreased gradually, furthermore, the collection period of the potentest antioxidant activity were June and July, respectively; and September was the lowest. There was a positive correlation between the polyphenols contents and the antioxidant activity, indicating polyphenols was the key antioxidant components in green tea.A ultra flow liquid chromatographic mass spectrometric (UFLC/MS-IT-TOF)method has been developed and validated for the determination of 13 compounds in green tea including: theanine; theophylline and caffeine; epigallocatechin, gallocatechin,epicatechin gallate, catechin gallate, epicatechin, catechin, epigallocatechin-3-gallate,gallic acid, rutin and gallocatechin-3-gallate. Separation was carried out with a Gemini C18 (250 mm × 4.6 mm id, 5 ?m). A gradient program was used with the mobile phase consisting of 0.1 % formic acid water and 0.1% formic acid acetonitrile. Analysis was performed in selected ion monitoring (SIM) mode with positive and negative electrospray ionization (ESI) interface. All the analytes showed good linearity (r0.9991, n = 5) in the concentration ranges. The average percentage recoveries were evaluated by calculating the ratio of detected amount versus added amount. The recovery of the method was in the range of 93.9 % ?107.3 %,with RSD less than 5.5 %.The method was validated for good accuracy, repeatability and precision, and was used as a valid analytical method to evaluate the variations of 13 compounds in green tea with different production regions and collection periods. The results indicated that the production regions had little effect on the dynamic changes of chemical compounds,while the collection periods had significant effect on them. Caffeine, epigallocatechin,gallocatechin, epicatechin, catechin, and gallocatechin-3-gallate showed the overall first increased and then decreased trends, while theanine,theophylline, gallic acid, rutin, epicatechin gal late, catechin gallate and epigallocatechin-3-gallate displayed a downward trends gradually. Total alkaloids and polyphenols presented a first increased and then decreased trends.A ultra fast liquid chromatographic mass spectrometric (UFLC/MS-IT-TOF)method was developed and validated for the simultaneous determination of EGC, EC,EGCG and ECG in rat plasma. After addition of the internal standard protocatechuic acid, plasma samples were extracted by liquid-liquid extraction method with ethyl acetate and separated on an ShimpackXR ODS column (75mm × 2.0 mm id, 2.2 ?m). A gradient program was used with mobile phase composed of 0.1 % formic acid water and 0.1 % formic acid acetonitrile within a runtime of 5.0 min. The analytes were detected without interference in the selected ion monitoring (SIM) mode with positive electrospray ionization. The linear range was 2.1 ? 839.2 ng·mL-1, 3.3 ? 1324 ng·mL-1,8.7 ? 3460 ng·mL-1,11 ?4388 ng·mL-1,for EGC,EC,EGCG and ECG, respectively.The recovery of analytes and internal standard both were more than 75.0 %. The intra-day and inter-day precisions (RSD %) were both within 7.0 % and 11.0 % for the four analytes, while the accuracy (relative error, RE %) were all less than 7.0 %. The validated method was successfully applied to the pharmacokinetic study of EGC, EC,EGCG and ECG in rat plasma after tail intravenous injection of the mixture of EGC, EC,EGCG and ECG. And the Cmax of EGC,EC,EGCG and ECG was 582.2 ± 83.8, 617.1± 117.8, 2659 ± 427.8 and 1201 ± 234.2 ng·ml-1; AUC(0-?) was 1.016×104 ± 9.60×102, 2.397×104 ± 1.736×104, 5.783×104 ± 2.690×104, 4.369×104 ± 1.715×104?g·min·L-1; t1/2z was 25.9 ± 5.5,26.7 ± 3.91,38.6 ± 11.3 and 32.8 ± 12.5 min,respectively. The results indicated they were eliminated rapidly within 6 hours.Compared with EGC, EC and EGCG, ECG had the much higher absorption.Rats chronically injected with D-gal had been used as an animal aging model for anti-aging study. The results of Morris water maze test, biochemistry and histopathology showed that the aging animal model of D-gal treated rats was successfully reproduced and the GTE (green tea extract) was demonstrated to intervene its effects positively, and the action of HG (hight dosage group) was mostly strong,which could lay the foundation for antioxidant mechanism of green tea by metabonamic methods.The metabonomics was introduced to study the influence of green tea polyphenols on aging rats induced by D-gal. An UFLC/MS-IT-TOF method combined with Profiling solution soft ware were developed and applied to analyze the serum, brain and urine samples from control group, model group and GTE treated group. The analytical data were converted to the Profiling solution data format and processed via the principle component analysis (PCA). PCA score plots of analytical data indicated the GTP treated group located between the model group and control group, and much closer to the control group, exhibiting a tendency recovering to healthy control group after taking GTE. The dominant metabolites obtained from loading plot were the differential metabolites among different groups, 28 of which were identified. They were LPC(16:0),LPC(18:2), LPC(18:0),LPC(20:5),LPC(20:4), LPC(22:6),C16DHS,C18DHS,C20DHS,C18PHS, C20 PHS and Tryptophan from serum samples; Lactate, Cysteine, Taurine,Creatine,Adenosine,C16DHS,C18DHS,C20DHS,C18PHS and C20 PHS from brain samples; Creatinine, Creatine, Tryptophan, Phenylalanine and Hippuric acid from urine samples. The mechanism of pathological changes might be elucidated with the biomarks metabolic pathways.