15 February 2010, Volume 30 Issue 1

    

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  Study on Inhibiting Effects of Tea Catechins on the Formation of Age Pigment Fluorescence Products

  CAI Shu-xian, LIU Zhong-hua, HUANG Jian-an, LUO Guo-an, YU Xing-long, DONG Xin-rong, YANG Zhi-hui, YE Fei

  Journal of Tea Science. 2010, 30(1): 1-8. https://doi.org/10.13305/j.cnki.jts.2010.01.001

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  Malondialdehyde (MDA) is one of the active carbonyl compounds existing in human body, which can react with protein to form age pigment fluorescence products (APFs) by carbonyl-ammonia cross linking reaction. The purpose of this study is to determine whether tea catechins can inhibit the formation of the age pigment fluorescence products induced by MDA-modified human serum albumin (HSA). Results showed that tea catechins, especially the gallated-catechin, could inhibit the formation of APFs. The structure-activity relationship studies suggested that the inhibiting effects of tea catechins on the formation of APFs were correlated with their nucleophilicity, besides their trapping MDA directly. This research indicated that inhibiting carbonyl-ammonia cross linking reaction may be one of the mechanisms of tea catechins preventing and curing diseases resulting from carbonyl stress reaction.
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  Research on the Acute Toxicity of Formulated Puer Tea

  MA Wei-guang, HUANG Zhi-pu, YOU Wen-long, LI Jin-shan, ZENG Guang-yuan, LIU Yu-qing

  Journal of Tea Science. 2010, 30(1): 9-12. https://doi.org/10.13305/j.cnki.jts.2010.01.002

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  In order to verify the security of the formulating Puer tea products, the acute toxicity experiments were carried out on mice. The standard Puer tea extracts were taken as the control. LD₅₀ of the Pure tea, Ganodorma lucidum’s Puer tea, American ginseng’s Puer tea, Phyllanthus emblica’s Puer tea and the Fagopyrum tartaricum’s Puer tea were determined as 10.32 g/kg, 10.02 g/kg, 10.02 g/kg, 10.96 g/kg, 10.32 g/kg individually. The formulating Puer tea products and the Pure tea showed no significant difference in the aspects of the physical and chemistry comparison tests. The acute toxicity experimental results indicated that these four formulating puer tea products belong to the actual non-toxic grade according to the food toxicology standard.
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  Experiment on Safety of Ectropis grisescens Nucleopolyhedrovirus(EgNPV)

  GONG Zi-ming, LIU Ming-yan, TAN Rong-rong, MAO Ying-xin, LI Long, CHEN Guo-hua, ZHOU Zheng-min, LI Xiao-wen

  Journal of Tea Science. 2010, 30(1): 13-18. https://doi.org/10.13305/j.cnki.jts.2010.01.003

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  A series of toxicological researches conducted in this study. the acute toxicity test, the eyes and skin stimulative test on rabbit, the skin sensitivity test on Guinea pig and the acute pathogenic test on mice. According to the experimental results of acute oral toxicity (LD₅₀>5 000 mg/kg) and acute dermal toxicity(LD₅₀>2 000 mg/kg), the acute toxicity of EgNPV on mice belongs to the category of low toxicity. It is not only non-irritating to skin but also has no stimulation on eyes of rabbits. The sensitization rate was 0%, did not cause hypersensitic reaction on Guinea pig. The results of histopathological test showed that no pathological changes were found in internal organs of mice in each experimental groups.
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  Simplification of EcoTILLING Technique for Tea Plant

  JIN Ji-qiang, CHEN Liang, YAO Ming-zhe, WANG Xin-chao, MA Chun-lei

  Journal of Tea Science. 2010, 30(1): 19-26. https://doi.org/10.13305/j.cnki.jts.2010.01.004

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  EcoTILLING(Ecotype Targeting Induced Local Lesions In Genomes) had been used as a SNP(Single Nucleotide Polymorphism) discovery tool to examine DNA variation in natural populations. A simple EcoTILLING technique for tea plant was established. Firstly, specific PCR primers for the amplification of the target gene were screened out. Then through comparison, a fine CELⅠ enzyme digestion could be resulted using 20 min with 10× buffer containing 250 mmol/L Tris-HCl (pH7.4), 150 mmol/L KCl, 15 mmol/L MgSO₄, 4 μg/mL BSA and 0.04%Triton x-100, and 1.8 μL CJE(Celery Juice Extract)/10 μL reacting solution at 47.5℃. Moreover, a good picture could be obtained when using 5% polyacrylamide gels (PAGE), adding 4~5 μL digesting products in PAGE electrophoresis. The 870 bp fragment within the PPO (Polyphenol Oxidase) gene of 6 tea germplasms was distinguished from each other using the simplified EcoTILLING. The CELⅠdigested bands were ascertained that according to the position of SNP in or between the 6 accessions through sequencing verification.
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  Cloning and Expression Analysis of Leucoanthocyantin Reducase Gene of Tea Plant (Camellia sinensis)

  MA Chun-lei, QIAO Xiao-yan, CHEN Liang

  Journal of Tea Science. 2010, 30(1): 27-36. https://doi.org/10.13305/j.cnki.jts.2010.01.005

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  The leucoanthocyantin reducase gene (LAR), which was an important functional gene of catechins biosynthesis pathway, was cloned from tea plant by EST sequencing and rapid amplification of cDNA ends (RACE). The full-length cDNA of LAR is 1 301 bp (GenBank accession No. EF205148), containing a 1 029 bp ORF encoding a 342 amino acids protein, and its 3′ untranslated region has an obvious polyadenylation signal. The deduced protein molecular weight was 37.5 kD and its theoretical isoelectric point was 5.81. The gene was then constructed into expression vector pET-32a(+) for over expression in prokaryotic cells. The SDS-PAGE showed that induced by IPTG, the leucoanthocyantin reducase proteins was expressed in Escherichia coli BL21, and its molecular weigh was found to be about 60 kD by checking with SDS-PAGE, nearly equal to the predicted. The deduced amino acid sequence of LAR from tea plant showed high identity with that of other plants, for instance 71%, 70% and 68% with Vitis vinifera, Gossypium arboretum and Fragaria ananassa, respectively. Four different catechin content cultivars were selected from our tea germplasm appraisal database to assay the gene expression level of the seven genes, flavonol synthase, dihydroflavonol-4-reductase, leucoanthocyanidin reductase, anthocyanidin synthase, etc, which were involved in the flavonoids biosynthesis. The result showed that, the DFR and LAR transcripts were expressed increased with the increasing of tea catechin contents. Nevertheless, the others did not show this tendency clearly.
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  Cloning and Sequence Analysis of EoblGOBP in the Antennal of Ectropis obliqua hypulina

  CHEN Hua-cai, LIU Jun, ZHANG Xiao-yan, YIN Kun-shan

  Journal of Tea Science. 2010, 30(1): 37-44. https://doi.org/10.13305/j.cnki.jts.2010.01.006

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  The full-length sequences of two general odorant binding protein (GOBP) genes in Ectropis obliqua (named EoblGOBP1 and EoblGOBP2) were cloned with degenerated digonudeotide primers by RT-PCR and the rapid amplification of cDNA ends (RACE) using the total RNA isolated from the antenna of Ectropis obliqua as template. The length of EoblGOBP1 was 1 528 bp with a 501bp ORF which encoded a peptide of 166 amino acid residues. The predicted Mw and pI of the peptide were 18 835.63 D and 5.45 respectively. The NCBI/GenBank accession numbers of cDNA and amino acid of EoblGOBP1were FJ156732 and ACN29680.1, respectively. The length of EoblGOBP2 was 1 315 bp with a 405 bp ORF which encoded a peptide of 160 amino acid residues. The predicted Mw and pI of the peptide were 18 002.75 D and 5.32 respectively. The GenBank accession numbers of cDNA and amino acid of EoblGOBP2 were FJ 156733 and ACN29681.1, respectively. The amino acid sequences from the two gene sequences had 6 typical conservative Cys. Blast searching in NCBI showed that the amino acid sequences were highly similar to that of other GOBPs from Lepidoptera with sequence identities of 62%~82% and 76%~88%.
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  The Association Analysis of Phenotypic Traits with EST-SSR Markers in Tea Plants

  YAO Ming-zhe, QIAO Ting-ting, MA Chun-lei, JIN Ji-qiang, CHEN Liang

  Journal of Tea Science. 2010, 30(1): 45-51. https://doi.org/10.13305/j.cnki.jts.2010.01.007

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  Association analysis is an approach to identify the relationship of molecular markers or candidate genes with traits in a given population based on linkage disequilibrium (LD). In this study 55 EST-SSR markers developed independently were used for genotyping of 112 clonal tea cultivars. LD of pairwise loci and population structure were firstly analyzed, then association analysis between EST-SSR markers and 7 penotypic traits were performed using TASSEL GLM. The different degrees of LD were detected among 55 pairwise EST-SSR loci. Genetic structure analysis showed that the total of 112 tea cultivars can be classified into two sub-populations. Five EST-SSR markers associated with phenotypic traits were screened out at the level of P<0.01. Three Markers CS298, CS317 and CS84 were associated with the weight of 100 shoots with one bud and two leaves (BW), leaf length (LL) and content of tea polyphenol, and they could explain phenotypic variation with 0.11, 0.15 and 0.35, respectively. Two Markers CS330 and CS338 were both detected association with content of caffeine (CAF), and they could explain phenotypic variation with 0.14 and 0.15, respecitvely.
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  Determination of Phenylureas Herbicide Residues in Tea with Matrix Solid-phase Dispersion - RP-HPLC

  XIAO Liang, WANG Yuan-chao, CHENG Mei-rong

  Journal of Tea Science. 2010, 30(1): 52-56. https://doi.org/10.13305/j.cnki.jts.2010.01.008

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  A new method for the simultaneous determination of 4 herbicides (Monuron, Chlortoluron, Diuron and Linuron) was established with MSPD-RP-HPLC. The mixture of tea samples and C₁₈ stuffing were pulverized, then mixed with alumina packed to a syringe column, the extraction and purification of the samples were carried out with one-step using dichloromethane as eluting reagent. The effecting factors of MSPD and conditions of the chromatography were discussed. Under optimum conditions, the linear ranges of Monuron was 0.01~10.0 mg/L with 5 μg/L of the detection limits, and the linear ranges of Chlortoluron, Diuron and Linuron were 0.025~10.0 mg/L with 10 μg/L of the detection limits. The average recoveries of 4 herbicides ranged from 83.5% to100.4% with less than 7.1% of the relative standard deviations in tea samples. The method suggested was simple, rapid, and less interference with high recovery rate, and meet the analytical requirements of herbicide residues in tea.
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  Cellulase Enzymatic Property of Eurotium Cristatum from Brick Tea

  CAI Zheng-an, LIU Su-chun, LIU Zhong-hua, HU Zheng-rong, JIA Hong-xin, HU Bo-han, HUANG Jian-an

  Journal of Tea Science. 2010, 30(1): 57-62. https://doi.org/10.13305/j.cnki.jts.2010.01.009

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  Eurotium cristatum isolated from brick tea were enlarged to foster by liquid fermentation and then crude enzyme was prepared from the culture of Eurotium cristatum. Cellulase activity of the crude enzyme liquid was measured by 3,5-dinitrosalicylic acid method under different conditions. The results showed that the optimum temperature for enzymatic reaction was 50℃, and the optimum reactive pH value was at 4.8. The cellulase activity was stabile under conditions of 30~50℃ and pH 4.0~6.0. The cellulase activity was stimulated by Ca²⁺、Mg²⁺ ions and inhibited by Mn²⁺、Zn²⁺、Pb²⁺ ions. The research will be significance for production of brick tea.
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  Mini DPSA-1 Coupled to Electrochemical Sensor for the Simultaneous Rapid Determination of Lead, Cadmium and Copper in Tea by Differential Potentiometric Stripping

  ZHAO Guang-ying, SHEN Yi-han

  Journal of Tea Science. 2010, 30(1): 63-71. https://doi.org/10.13305/j.cnki.jts.2010.01.010

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  Mini DPSA-1 device, coupled to screen-printed carbon electrodes (SPCE), was used for simultaneous rapid detection of lead, cadmium and copper in tea by differential potentiometric stripping analysis (DPSA). The optimized experimental parameters were as follows, Hg²⁺ concentration — 3×10^-4 mol/L, supporting electrolyte — 1.5 mol/L pH4.0 NH₄Cl, deposition potential — -1.1 V, final potential —-0.2 V, deposition time — 200 s. The potentiometric stripping response for lead, cadmium and copper following 200 s deposition was linear over the concentration range examined (20~840 μg/L) with r values of 0.99749, 0.99740 and 0.99612 and detection limit of 1.12 μg/L, 1.09 μg/L and 1.23 μg/L (S/N=3), respectively. The interferences of various metal ions were also examined in the determination of lead, cadmium and copper. No significant difference existed after the rapid detection results were compared with GB method using t test. In comparing with conventional detection technique, this detection method for lead, cadmium and copper in tea, using mini DPSA-1 instrument, coupled to SPCE by DPSA, was a cheaper rapid detection technique. It could achieve a high percentage of sample screening and be applied to the simultaneous rapid detection of lead, cadmium and copper in other food and environmental samples.
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  Analysis on the Decline of Macau’s Tea Trade in Qing Dynasty

  LIU Xin-qiu, FENG Wei-ying, JIANG Ai-qin, FANG Wan-ping, ZHU Zi-zhen, LI Xing-hui

  Journal of Tea Science. 2010, 30(1): 72-78. https://doi.org/10.13305/j.cnki.jts.2010.01.011

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  Qing dynasty opened up a precedent of tea export trade in Chinese history, and Macau was the first tea export port. Studies on tea export trade in Qing dynasty were mostly focused on the “thirteen business” in Guangzhou, the business between Fujian (Quanzhou and Xiamen) and Southeast Asia. There are almost no data currently available about the Macau’s tea trade in Qing dynasty, so the course of prosperity and decline of Macau’s tea export trade in Qing dynasty were analyzed, the important position of Macau as the first tea export port in the history of tea trade development in China was indicated in the study. Furthermore, the historical and social reasons of the decline of Macau’s tea trade were also discussed.