15 December 2012, Volume 32 Issue 6

    

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  Application of Caco-2 Cell Model in the Research of Tea Bioactive Components

  XIE Yan-lan, GUO Hao-wei, ZHENG Xian-mu, XU Hai-rong, Wilfried Andlauer, Agnieszka Kosińska

  Journal of Tea Science. 2012, 32(6): 471-479. https://doi.org/10.13305/j.cnki.jts.2012.06.003

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  Caco-2 cell monolayer possesses the similar morphological and functional properties to the human intestinal epithelial cells, is one of the important in vitro cell models to study nutrients or drugs absorption, transport and metabolism. In recent years the Caco-2 cell model also widely used in the research of tea bioactive components. The absorption and transport mechanisms of catechins, theanine, gallic acid and caffeine, the metabolism of catechins and the pharmacodynamic mechanism of tea bioactive components in Caco-2 cells were reviewed. What’s more, the rapid assessment of tea bioactive components’ absorption features using Caco-2 cell model in order to promote the development of tea-care products and preparations were prospected.
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  Contents of Caffeine and Catechin in Guizhou Green Tea

  LI Jun, GUO Xiao-guan, PANG Hong-yu, ZHU Fu-jian, WANG Zhen, LAI Fei

  Journal of Tea Science. 2012, 32(6): 480-484. https://doi.org/10.13305/j.cnki.jts.2012.06.010

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  Contents of caffeine and catechin of 243 green tea samples from Guizhou province are detected, using high performance liquid chromatographic (HPLC). The results show that, the catechin contents range 9.14%~27.28% in green tea of Guizhou province, the average content is 15.71%. The caffeine contents range 1.08%~3.33%, the average content is 2.24%. The catechin quality indexes of samples from 9 regions mainly are 1000~2000, and the highest one (the sample is from Qiannan) is 1975.51. The catechin bitter taste index of samples from 9 regions mainly are 8.00~17.04．According to the results, there is high value for making use of effective components of Guizhou green tea, and developing high quality spring tea is strongly recommended, and relatively the autumn tea is suitable for deep processing or extracting active principles.
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  Hydroxyapatite/SDBS Modified Screen-printed Carbon Electrodes for the Rapid Determination of Trace Lead in Tea

  YANG Fang, ZHAO Guang-ying, DOU Wen-chao

  Journal of Tea Science. 2012, 32(6): 485-493. https://doi.org/10.13305/j.cnki.jts.2012.06.002

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  In order to improve the stability, sensitivity and safety of the electrochemical sensor for rapid determination of lead, the usual toxic mercury with screen-printed carbon electrode was replaced by the hydroxyapatite dispersed by SDBS, with added Nafion membrane to increase stability, so to establish a new electrochemical sensor for rapid determination of trace lead. The analysis was performed by square-wave stripping voltammetry, Sodium acetate buffer (0.1mol/L) was employed as the supporting electrolyte. Under the optimized working conditions, including amount of hydroxyapatite, pH of solution, accumulation time, a linear response range from 2μg/L to 300μg/L was obtained, the detection limit for lead determination was 0.460µg/L(S/N) =3. The sensor presented good repeatability, evaluated in terms of relative standard deviation (R.S.D.=4.59%). The result determined by this method was similar to ICP-MS method. The novel electrochemical sensor for rapid detection of lead effectively improved the sensitivity, stability and security. In addition, it had accurate results and possessed a certain application potential.
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  Preparation of Gallic Acid Derivatives by Solid-phase Extraction Cartridge Coupled with Preparative High Performance Liquid Chromatography from Tea Plant

  LU Zhong-wei, JIANG Xiao-lan, LIU Ya-jun, LIU Li, GAO Li-ping, XIA Tao

  Journal of Tea Science. 2012, 32(6): 494-499. https://doi.org/10.13305/j.cnki.jts.2012.06.001

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  Gallic acid derivatives are the chief compounds of phenolic acid in tea. They are important umami-enhancing compounds in tea beverage, and possess the effects of inhibiting IgE producton, enhancing cognition-anting depressant. A reversed-phase PHPLC coupled with SPE was developed to purify and to prepare high purity 3-O-galloyquinic acid and β-D-glucogalline from fresh tea leaves. The obtained fractions were successfully identified by LC/TOF-ESI-MS, ¹H-NMR and ¹³C-NMR. This method is simple, effective for the preparation of gallic acid derivatives.
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  Cloning and Sequencing of UBA1 Gene Full-length cDNA from Tea Plant

  DENG Ting-ting, WU Yang, LI Juan, LI Yin-hua, HUANG Jian-an, LIU Zhong-hua

  Journal of Tea Science. 2012, 32(6): 500-508. https://doi.org/10.13305/j.cnki.jts.2012.06.011

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  The cDNA-AFLP technology was applied to analyze gene expression during periodic albinism process of Anji Baicha. Some transcript-derived fragments (TDFs) were isolated occurring in both the albinistic and re-greening stage leaves. One of them showed a high similarity to ubiquitin-activating enzyme 1 (UBA1) gene. Based on the fragment, the full length of UBA1 gene with 3764bp (GenBank Accession No. JN180299) cDNA was obtained via rapid amplification of cDNA ends (RACE), named Camellia Sinensis UBA1 gene. It contained an open reading frame (ORF) encoding a polypeptide of 1094 amino acid residues with a predicable molecular mass of 121kD. Analysis of the nucleotide sequence and deduced amino acid sequence showed 82%, 81%, 79%, 79%, 77% homology with UBA1 genes from Nicotiana tabacum, Ricinus communis, Oryza sativa subsp. japonica, Triticum aestivum, Arabidopsis thaliana, respectively. Analysis by qRT-PCR showed that the transcript of UBA1 was significantly up-regulated at the albinistic stage to 2.49-fold higher than that at the re-greening stage. This is a key enzyme in the ubiquitin-proteasome mediated protein degradation system. The clone and analysis of the tea plant UBA1 gene establishes a good foundation for further study on the molecular mechanism of periodic albinism in Anji Baicha.
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  Expression Analysis of Auxin-related Genes at Different Winter Dormant Stages of Axillary Buds in Tea Plant (Camellia sinensis)

  WANG Xin-chao, MA Chun-lei, YANG Ya-jun, CAO Hong-li, HAO Xin-yuan, JIN Ji-qiang

  Journal of Tea Science. 2012, 32(6): 509-516. https://doi.org/10.13305/j.cnki.jts.2012.06.012

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  The expression patterns of some auxin-related genes, including auxin response factors (ARFs), primary auxin responsive genes, auxin-regulated genes, auxin influx carrier gene and auxin-binding protein (ABP) gene, were studied in different stages of tea plant axillary bud dormancy and its release. The results showed that (1) the expression levels of five in six ARF homologous EST fragments were up-regulated in the deep dormancy and down-regulated after sprouted, the level of the last one showed the opposite expression levels. (2) There were significant differences among the expression patterns of 3 ESTs which are homologous with primary auxin responsive genes. EST which is homologous with SAURs was up-regulated in the dormancy stage but down-regulated after dormancy release. On the contrary, the 2 ESTs which are homologous with GH3 and Aux/IAAs were up-regulated in sprouting stage while down-regulated in dormant stages and after sprouting stages. (3) The CsGLP1 gene which was homogenous with ABPs has a lower expression level in the dormancy stage but maintain rising trend after germination.(4) The EST which is homogenous with auxin influx carrier gene showed the highest expression levels during the deep dormant stage but maintain lower levels in other stages. (5) Two auxin-regulated ESTs showed different expression patterns. The expression levels of auxin-induced EST were higher in the dormant stages than those in the sprouting stages, while the levels of the other auxin-repressed EST were higher in the sprouting stage than that in the dormant stages. These results indicated that auxin promote the dormancy of tea axillary buds in winter.
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  Study of SNP and Relative dCAPS Markers in Tea Plant (Camellia sinensis)

  ZHANG Cheng-cai, WANG Li-yuan, WEI Kang, CHENG Hao, BAO Yun-xiu, LIU Ben-ying, WANG Yun-gang

  Journal of Tea Science. 2012, 32(6): 517-522. https://doi.org/10.13305/j.cnki.jts.2012.06.013

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  SNPs were mined from tea-derived EST sequences. Primers were designed to evaluate the predicted SNPs in genomic DNA from tea plant. The PCR products were sequenced from both directions to confirm the valid SNPs. Meanwhile, the dCAPS primers were designed based on the valid SNPs and the genomic DNA was used for amplification. The amplicons were digested by restrict enzymes to convert SNPs into dCAPS markers. Furthermore, we also detected the separation of the new markers in a F1 population. The results showed that seventeen SNPs (54.8%) were confirmed in 11 amplycons, eight SNPs were successfully converted to dCAPS markers (72.7%), two of them which have polymorphisms in parents exhibited a separate ratio of 1︰1 in the F1 population. The new markers would be useful in tea genetic research. It is also believed that dCAPS technology will promote the use of SNP technology in genetics and breeding research in tea plant.
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  Molecular Cloning and Quantitative Analysis of GDH, GS and GOGAT Genes from Leave of Tea Plant

  LIN Zheng-he, ZHONG Qiu-sheng, CHEN Chang-song

  Journal of Tea Science. 2012, 32(6): 523-529. https://doi.org/10.13305/j.cnki.jts.2012.06.014

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  This research has separated gene conservative regions of glutamate dehydrogenase (GDH), Glutamine synthetase (GS) and Glutamine oxoglutarate aminotransferase (GOGAT), which were key enzymes involving in the metabolism of amino acid in tea plant, from four tea germplasms. Sequences of GDH, GS and GOGAT conservative regions have been submitted to GeneBank and their accession number were JN602371, JN602372 and JN602373, respectively. Real-time quantitative PCR analysis revealed that GDH gene has higher transcription in 0314C (germplasm with relatively high amino acid content) than the other three germplasms, while lower transcription in 0212-15 (germplasm with relatively low amino acid content) comparing to the others. The transcription of GS and GOGAT gene were in apparently low level in 0314C and high level in 0212-15 and 0318D, respectively. Regression analysis indicated that the expression of GS gene was negatively related to the contents of theanine, lysine and lactamine, whereas the expression of GDH gene was positively related to the content of theanine.
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  Evaluation on the Alleviating Physical Fatigue Function of Theanine Compound Preparation

  TAN Jun-feng, LIN Zhi, PENG Qun-hua, GUO Li, LV Hai-peng

  Journal of Tea Science. 2012, 32(6): 530-534. https://doi.org/10.13305/j.cnki.jts.2012.06.006

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  According to “The Standard of Health Food Test and Assesment”, the alleviating physical fatigue function of theanine compound preparation was evaluated. The recommended daily dose of human is 1.4g/60kg body weight. The low, middle and high dosage groups of Kunming mice were respectively 0.12, 0.23 and 0.70g/kg, equivalent to 5, 10 and 30 times of human recommended doses. The control group with equal distilled water by gavage, on 30 consecutive days. The results showed that 0.70g/kg dose group could significantly prolong the loaded swimming time of mice, reduce the serum urea content and increase hepatic glycogen content, indicating the theanine compound preparation possessed the function of alleviating physical fatigue.
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  Study on Remission of Drunk Symptomsby the Tea Complex

  YIN Ai-wu, TIAN Run, LI Tan-fang

  Journal of Tea Science. 2012, 32(6): 535-538. https://doi.org/10.13305/j.cnki.jts.2012.06.007

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  ：To study anti-inebriation effects of a kind of tea complex and its effects were evaluated by observing the time loss and the recovery time of righting reflex, the death rate induced by acute alcoholism, alcohol-induced spontaneous activity and alcohol concentration in mice plasma. The results showed low, middle and high dose of tea complex prolonged the loss time of righting reflex and shortened the recovery time of righting reflex, reduced the death rate induced by acute alcoholism, inhibited ethanol-induced spontaneous activity of mice and obviously decreased alcohol concentration in mice plasma. The alcohol concentration at 30, 60, 90, 120, 150min after oral administration high dose of tea complex were (523±129), (671±175), (954±203), (1012±326), (634±139)mg/L respectively which showed significant differences compared with that of control group(P<0.05 or P<0.01). This research would provide the groundwork for the development of tea.
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  Protective Effect of Fuzhuan Brick Tea on H₂O₂-Induced Cellular Oxidative Damage in LLC-PK1 Cells

  SONG Jia-le

  Journal of Tea Science. 2012, 32(6): 539-542. https://doi.org/10.13305/j.cnki.jts.2012.06.004

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  The aim of this study is to investigate the protective effect of ethanolic extract from Fuzhuan brick tea (FTE) on H₂O₂-induced oxidative damage in LLC-PK1 cells. Various concentration (10~200μg/mL) of FTE were pre-incubated with LLC-PK1 cells for 24h, and then exposed to H₂O₂ (500μmol/L) for 4h. The cell viability was determined by MTT assay, contents of malondialdehyde (MDA) were determined by thiobarbituric acid reactive substances (TBARS) assay. Intracellular antioxidant enzyme activities, including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) were determined by colorimetric assay according to the protocols of the commercial kit. The cell viabilities of H₂O₂ treated LLC-PK1 cells were increased, and the contents of MDA were decreased after 24h pre-incubated with different concentrations of FTE. In addition, the antioxidant enzymes (CAT, SOD and GSH-px) levels were also increased in FTE compared with H₂O₂ treated group. There was statistical significant difference between the FTE treated groups and untreated H₂O₂ treated groups (all P<0.05). Those results suggested that pre-incubated with FTE showed a protective effect against H₂O₂-induced oxidative damage in LLC-PK1 cells.
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  Comparative Study on Lipid-lowering and Anti-obesity Effect of Different Pu-erh Tea Fractions

  XIONG Chang-yun, PENG Yuan-ju, WANG Xing-hua, CUI Wen-rui, LI Feng-juan, HE Pu-ming

  Journal of Tea Science. 2012, 32(6): 543-551. https://doi.org/10.13305/j.cnki.jts.2012.06.008

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  In this study, Pu-erh tea was extracted with acetone, water, chloroform, ethyl acetate, and n-butanol. then, the extraction was concentrated with rotary evaporation and dried with lyophilizer into chloroform layer, ethyl acetate layer, n-butanol layer and surplus water layer extracts, which were used to administer ICR obese mouse for analyzing the lipid-lowering and anti-obesity effects. The experimental results demonstrated that the pu-erh tea total extract, ethyl acetate extract and surplus water extract are all effective on lipid-lowering and anti-obesity of ICR obese mouse, and the effects of ethyl acetate extract and surplus water extract on inhibiting fat accumulation（inhibition ratio was 24.7% and 22.5%）was slightly less than those in the L-carnitine （38.8%）, but the effect on improving HDL-C level(61.7% and 73.4%) significantly superior to that in the L-carnitine (9.4%), the similar results were obtained in the mice liver, which suggest that the key lipid-lowering and anti-obesity function components were existed in the ethyl acetate layer and (or) surplus water layer extracts of pu-erh tea.
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  Study on a Remote Controlling Machine for Electrostatic Spraying and Fertilizing in Tea Garden

  LI Ming, LIU Zhong-hua

  Journal of Tea Science. 2012, 32(6): 552-558. https://doi.org/10.13305/j.cnki.jts.2012.06.009

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  In order to decrease the more influence on tea production development for the shortage of labor, and increase the utilization of pesticide and fertilization to reduce the environmental pollution, a remote controlling machine for electrostatic spraying and fertilizing in tea garden was investigated. The machine was mainly made of frame, power, spraying system, fertilizing system and controlling system, which held the manual and remote controlling operations. The machine’s driving and fertilizing function adopted 48 Voltage direct current brushless motor and the spraying 12 Voltage direct current vane pump. It used the capacity adjustment shallow fertilization and can be soil covered. The tea plant’s profile modeling spraying pole and high voltage electrostatic electricity were utilized to realize spraying. Experimental results showed that the machine could complete the fertilization and spraying preferably, and improved the fogdrop deposition on the leaf obviously. The machine is prosperous in the future.
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  A Comparative Study of the Chan Tea Culture between the Lingyin Temple and the Shaolin Temple

  MA Xiao-li

  Journal of Tea Science. 2012, 32(6): 559-564. https://doi.org/10.13305/j.cnki.jts.2012.06.005

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  Nowadays, there is great interest to culture and tea academics in studying Chan, Chan tea and its culture. The paper is based on the knowledge of tea culture and economy of Henan and Zhejiang province and the great interest in studying Buddhism for years. By surveying the tea culture activities and its spreading, the believers and protection of knowledge and so on, the author does a comparative study of the contribution to Chan tea culture in the two temples.