Leaf Homogenates Research Articles

Measurement of partition coefficients for selected polycyclic aromatic hydrocarbons between isolated plant cuticles and water

Partition coefficients between plant cuticles and water (Kcutw) were measured for 10 selected polycyclic aromatic hydrocarbons (PAHs) to evaluate the sorption capacity of plant cuticular layers for hydrophobic organic chemicals. The partitioning properties of PAHs between cuticles and water were evaluated by using (1) isolated cuticular layers and (2) leaf homogenate. The abaxial and adaxial cuticular layers of Euonymus japonicus were isolated by enzymatic digestion. A third-phase partitioning method using poly(dimethylsiloxane) (PDMS) was used to obtain Kcutw. The Kcutw values for the selected PAHs showed no significant differences between the abaxial and adaxial cuticular layers and ranged between 104.1 and 107.6. These values are close to or slightly higher than their 1-octanol/water partition coefficient (log Kow), indicating high sorption capacity of plant cuticles. On the contrary, partition coefficients between the lipid tissues of homogenized leaves and water were lower than those obtained using isolated cuticular layers by factors of 3.7–190, which is likely due to the breakdown of lipid layers. This indicates that the sorption of hydrophobic organic chemicals by plant leaves is better evaluated using isolated cuticles and that the sorption potential of plant leaves may be underestimated when leaf homogenates are used.

Cometabolic degradation of trichloroethylene by Burkholderia cepacia G4 with poplar leaf homogenate.

Trichloroethylene (TCE), a chlorinated organic solvent, is one of the most common and widespread groundwater contaminants worldwide. Among the group of TCE-degrading aerobic bacteria, Burkholderia cepacia G4 is the best-known representative. This strain requires the addition of specific substrates, including toluene, phenol, and benzene, to induce the enzymes to degrade TCE. However, the substrates are toxic and introducing them into the soil can result in secondary contamination. In this study, poplar leaf homogenate containing natural phenolic compounds was tested for the ability to induce the growth of and TCE degradation by B. cepacia G4. The results showed that the G4 strain could grow and degrade TCE well with the addition of phytochemicals. The poplar leaf homogenate also functioned as an inducer of the toluene-ortho-monooxygenase (TOM) gene in B. cepacia G4.

Mechanisms of phototransformation of protochlorophyllide into chlorophyllide.

The purpose of this review is to summarize and discuss data obtained in studies on the mechanisms of the primary photophysical and photochemical reactions of protochlorophyllide photoreduction in plant materials (etiolated leaves and leaf homogenates) and in model systems. Based on the results of numerous studies, it can be stated that the reduction of active forms of the chlorophyll precursor is a multistep process comprising two or three short-lived intermediates characterized by a singlet ESR signal. The first intermediate is probably a complex with charge transfer between protochlorophyllide and the hydride ion donor NADPH. The conserved tyrosine residue Tyr193 of protochlorophyllide oxidoreductase is the donor of the second proton.

Head‐group acylation of monogalactosyldiacylglycerol is a common stress response, and the acyl‐galactose acyl composition varies with the plant species and applied stress

Formation of galactose-acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap-freezing. Here, lipidomic analysis using mass spectrometry showed that galactose-acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl-galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl-galactose components in response to the same stress. Additionally, the composition of the acyl-galactose component of Arabidopsis acMGDG (galactose-acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub-lethal freezing treatment, acMGDG contained mainly non-oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid-containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose-acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.

Effect of lead accumulation in maize leaves on their chemical images created by a flow-through electronic tongue

A flow-through electronic tongue based on miniaturized ion-selective electrode array was used for the classification of the maize leaf samples (Zea mays) exposed to media containing Pb(II) ions. The system provided a good recognition of the extracts from the plant leaves treated with solutions of varying concentrations of Pb(NO3)2. Additionally, samples derived from specific segments of the maize leaf, representing different developmental stages of cells, were also discriminated. The presented results showed that the developed sensor array combined with Partial Least Squares-Discriminant Analysis (PLS-DA) technique allowed to recognize the plant samples on the basis of the changes in the ionic composition of the leaf homogenates.

A highly sensitive nitric oxide biosensor based on hemoglobin–chitosan/graphene–hexadecyltrimethylammonium bromide nanomatrix

Nitric oxide (NO) is a key signaling molecule in different physiological processes of plants. It is therefore very important to establish a simple, fast, sensitive and accurate detection method for NO detection. In this study, a novel amperometric biosensor based on immobilization of hemoglobin (Hb), chitosan (CS), graphene (GR) and the surfactant hexadecyltrimethylammonium bromide (CTAB) on a glassy carbon electrode was constructed to quantitatively measure NO. Atomic force microscopy, scanning electron microscopy and electrochemical methods were used to characterize the Hb–CS/GR–CTAB nanocomposite film. The biosensor fabricated from the nanocomposite film showed direct electrochemistry with a fast electron-transfer rate (60.3s−1) and high electrocatalytic activity towards the reduction of NO. Under optimal conditions, the resulting biosensor exhibited a high sensitivity of 615μAmM−1, a low detection limit of 6.75×10−9M (S/N=3) and a small apparent Michaelis–Menten constant of 0.315μM. These results were superior to those for biosensors based on the graphite, GR/ionic liquid and carbon nanotubes. The biosensor was also used successfully for detection of additional NO in oilseed rape leaf homogenate samples. Furthermore, the biosensor had excellent long-term storage stability, as well as reproducibility and selectivity, and should have a potential application in monitoring NO in plant samples.

Isolation of intact and pure chloroplasts from leaves of Arabidopsis thaliana plants acclimated to low irradiance for studies on Rubisco regulation

<p>A protocol is presented for low-cost and fast isolation of intact and pure chloroplasts from leaves of plants acclimated to low irradiance. The protocol is based on a differential centrifugation of cleared leaf homogenate and omits a centrifugation on Percoll gradient step. The intactness and purity of the chloroplasts isolated from leaves of low irradiance-acclimated plants by using this protocol (confirmed by phase contrast microscopy as well as enzymatic and immunological approaches) allows plausible studies on low irradiance-dependent Rubisco regulation.</p>

깻잎 첨가 국수 대량 생산 공정 개발 및 이의 항산화 효과

This study was evaluated mass production processes for flour noodles containing perilla leaf (FNPL) and to examine the antioxidant properties of FNPL. The processes for perilla leaf preparation before dough making were the main focus. The sensory evaluation was used as a tool to determine the optimal conditions for each step in the process. The appearance, taste, flavor, texture and overall acceptability were evaluated using a 9 point scale sensory evaluation. Eight minutes blanching of perilla leaf in boiling water (1:30, w/v) was found to be a suitable time to remove the unpleasant leaf flavor remaining in the final noodle product when fresh perilla leaf was used. The appearance, taste, texture and overall acceptability of FNPL were significantly different from FNPLs prepared with other blanching times. The appearance of FNPL containing 40% (w/v) perilla leaf homogenates was the best. In the dough making process, additional water was not required when 6 portions of 40% perilla leaf homogenate were added to 10 portions of flour, suggesting that the water adding step in the dough preparation process can be skipped. The antioxidant activity of FNPL was expressed as the radical scavenging activity. The DPPH (; 0.56 mg/mL), super oxide radical (; 9.53 mg/mL) and hydroxy radical scavenging activities (; 169.2 /mL) of FNPL were increased 19.6 (p

Sensory Characteristics and Volatile Profiles of Parsley (Petroselinum crispum [Mill.] Nym.) in Correlation to Resistance Properties against Septoria Blight (Septoria petroselini)

Sixteen different genotypes of parsley, including two cultivars, six populations, and eight inbred lines, were investigated regarding their sensory characteristics in relation to the volatile patterns and resistance to Septoria petroselini . The sensory quality was determined by a combination of profile analysis and preference test, whereas the volatile patterns were analyzed by headspace-SPME-GC of leaf homogenates with subsequent nontargeted data processing to prevent a possible overlooking of volatile compounds. The more resistant genotypes are characterized by several negative sensory characteristics such as bitter, grassy, herbaceous, pungent, chemical, and harsh. In contrast, the contents of some volatile compounds correlate highly and significantly either with resistance (e.g., hexanal and α-copaene) or with susceptibility (e.g., p-menthenol). Some of these compounds with very strong correlation to resistance are still unidentified and are presumed to act as resistance markers.

First Report of Tomato spotted wilt virus on Tobacco in Campania, Italy.

During the spring and summer of 2010, a survey for viruses was conducted in two tobacco field trials at the Agricultural Research and Experimentation Council in Scafati, Campania, Italy. A total of 1,392 symptomatic and asymptomatic tobacco plants (cv. Burley) were sampled, and leaves were analyzed by double-antibody sandwich-ELISA using polyclonal antisera against five tobacco-infecting viruses: Tobacco mosaic virus, Potato virus Y, Cucumber mosaic virus, Alfalfa mosaic virus, and Tomato spotted wilt virus (TSWV) (Loewe, Munich, Germany). Only one plant was positive to TSWV. Symptoms on this plant were severe necroses on the stem with a few chlorotic/necrotic leaflets on the top of the plant. This result was subsequently confirmed by reverse transcription (RT)-PCR. The primers (5'-ATGTCTAAGGTTAAGCTC-3' forward and 5'-TTAAGCAAGTTCTGTGAG-3' reverse) targeted the nucleocapsid gene of TSWV and amplified the expected product of approximately 800 bp (2). The resultant sequence (GenBank Accession No. JF290419) was aligned and edited using BlastN, displaying 99.9% identity with deposited TSWV nucleocapsid gene sequences in GenBank, with no similarity to any other targets, which confirmed the presence of TSWV in tobacco. Leaf homogenate from the tobacco symptomatic plants was inoculated onto three plants of Nicotiana benthamiana, N. glutinosa, and Datura metel. All plants developed a systemic necrosis after 7 days. Inoculation from N. glutinosa back to cv. White Burley tobacco produced symptoms similar to those observed in the field. Two plants from each species were used as noninoculated controls, which remained asymptomatic. TSWV infection has been responsible for severe epidemics on tobacco throughout the United States and Greece, with losses estimated as much as 85% (1,3). The presence of TSWV in Italy could therefore represent a serious threat for tobacco in the region, especially considering that it is prevalent in other crops in the area and vectors are widespread.

Advances in understanding of the primary reactions of protochlorophyll(ide) photoreduction in cells and model systems

The key step in chlorophyll biosynthesis is photoreduction of its immediate precursor, protochlorophyllide. This reaction is catalyzed by a photoenzyme, protochlorophyllide oxidoreductase (POR) and consists in the attachment of two hydrogen atoms in positions C17 and C18 of the tetrapyrrole molecule of protochlorophyllide; the double bond is replaced with the single bond. Two hydrogen donors involved in protochloro-phyllide photoreduction are NADPH [1,2] and a conserved tyrosine residue Tyr193 of the photoenzyme POR [3]. The structure of active pigment-enzyme complex (Pchlide-POR-NADPH) ensures a favorable steric conditions for the transfer of hydride ion and proton. This review does not examine the ternary complex structure, but concentrates upon the mechanisms of primary photophysical and photochemical reactions during formation of chlorophyllide from protochlorophyllide in living objects (etiolated leaves and leaf homogenates) and model systems.

Quantitating carbon monoxide production from heme by vascular plant preparations in vitro

Heme in animals is mainly degraded enzymatically, producing a predictable amount of carbon monoxide (CO). Under some conditions, alternative sources of CO production are important, such as lipid peroxidation and photo-oxidation. Less is known about CO production in plants as a reflection of enzymatic activity or coupled oxidation, but a sensitive assay for CO production in plants would be a valuable tool to explore the various sources in plants as the conditions of the reactions and mechanisms are defined. Using gas chromatography, we determined the requirements for heme-supported in vitro CO generation by exogenous reactants (NADPH, tissue supernatant, oxygen), optimum reaction conditions (time, temperature, pH, light), and effects of various cofactors and substrates using supernatants from Spinacia oleracea (spinach) leaf and Solanum tuberosa (potato) tuber homogenates. We then determined the CO production rate distribution between organ (root, stem, leaf, flower, fruit) supernatants in a number of commercially available plant species. CO production ranged from 4-65 nmol CO/h/g fresh weight and occurred in all vascular plant tissues examined, with the highest rates in chloroplast-containing tissues. In spinach leaves, CO production was concentrated (>2-fold) in the particulate fraction, whereas in potato tubers, the particulate fraction accounted for <50% of the rates in homogenates. We conclude that gas chromatography is uniquely suited for the determination of CO production in pigmented, heterogeneous plant tissue preparations.

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which L: -dopa is formed from the substrate L-tyrosine. L-Dopa concentration and activity of L-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum L-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH(4), and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K (m) value of 808.63 microM for L-tyrosine at 37 degrees C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 microM L-tyrosine. Higher concentrations of the cofactor 6-MPH(4), however, completely inhibited the synthesis of L-dopa. Tyrosine hydroxylase converted specific monophenols such as L-tyrosine (808.63 microM) and tyramine (K (m) 1.1 mM) to diphenols L-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.

Chronic oral administration of Ocimum sanctum Linn. augments cardiac endogenous antioxidants and prevents isoproterenol-induced myocardial necrosis in rats

Wistar rats (200-250 g) of either sex were fed with fresh leaf homogenate of Ocimum sanctum by oral gavage in two different doses, 50 mg kg-1(Os 50) and 100 mg kg-1 (Os 100), daily for 30 days. This was followed by isoproterenol administration (85 mg kg-1 s.c. two doses at 24h intervals) in both control and 0. sanctum-fed rats to induce myocardial necrosis. Hearts were isolated for estimation of endogenous myocardial antioxidants (superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx) and myocardial lipid peroxidation) and light microscopic study. Increased basal myocardial antioxidant SOD (9.3 +/- 1.2 vs 3.7 +/- 0.7 units mg-1 protein; P<0.05) and catalase activities (34.3 +/- 5.4 vs 17.9 +/- 5.1 units mg-1 protein; P< 0.05) were observed in the Os 50 group only without any evidence of cellular injury in both the groups. In control rats, isoproterenol administration caused significant depletion of myocardial SOD (1.7 +/- 0.2 units mg-1 protein) and GPx (104 +/- 2mU mg-1 protein) activities and increase in GSH (551.7 +/- 30.9, microg g-1 wet weight of tissue) level, with evidence of myocardial necrosis. Isoproterenol-induced changes in myocardial SOD, GPx and GSH were prevented by both the doses of 0. sanctum, however cellular injury was minimal only with 50mg kg-1. The results indicate that long-term feeding of 0. sanctum offered significant protection against isoproterenol-induced myocardial necrosis through a unique property of enhancement of endogenous antioxidants.

Activation of the Chloroplast Monogalactosyldiacylglycerol Synthase MGD1 by Phosphatidic Acid and Phosphatidylglycerol

One of the major characteristics of chloroplast membranes is their enrichment in galactoglycerolipids, monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG), whereas phospholipids are poorly represented, mainly as phosphatidylglycerol (PG). All these lipids are synthesized in the chloroplast envelope, but galactolipid synthesis is also partially dependent on phospholipid synthesis localized in non-plastidial membranes. MGDG synthesis was previously shown essential for chloroplast development. In this report, we analyze the regulation of MGDG synthesis by phosphatidic acid (PA), which is a general precursor in the synthesis of all glycerolipids and is also a signaling molecule in plants. We demonstrate that under physiological conditions, MGDG synthesis is not active when the MGDG synthase enzyme is supplied with its substrates only, i.e. diacylglycerol and UDP-gal. In contrast, PA activates the enzyme when supplied. This is shown in leaf homogenates, in the chloroplast envelope, as well as on the recombinant MGDG synthase, MGD1. PG can also activate the enzyme, but comparison of PA and PG effects on MGD1 activity indicates that PA and PG proceed through different mechanisms, which are further differentiated by enzymatic analysis of point-mutated recombinant MGD1s. Activation of MGD1 by PA and PG is proposed as an important mechanism coupling phospholipid and galactolipid syntheses in plants.

Isozyme-Specific Modes of Activation of CTP:Phosphorylcholine Cytidylyltransferase in Arabidopsis thaliana at Low Temperature

Arabidopsis thaliana increases cellular phosphatidylcholine (PC) content during cold acclimation by up-regulating PC biosynthesis. The A. thaliana genes CCT1 and CCT2 encode CTP:phosphorylcholine cytidylyltransferases (CCTs; EC 2.7.7.15), which regulate PC biosynthesis via the CDP-choline pathway. We isolated the T-DNA-tagged knockout mutants cct1 and cct2 of A. thaliana (Wassilevskaja; WS). CCT activity in cct1 and cct2 plants accounted for 29 and 79% to the cellular CCT activity of WS plants, respectively. When plants were exposed to 2 degrees C for 7 d, CCT activity increased in both cct1 and cct2 plants, and immunoblot analyses revealed that cct1 contained an increased level of CCT2 protein whereas cct2 exhibited little increase in CCT1 level. For each mutant grown at 23 degrees C, CCT activity was mainly enriched in the particulate (15,000 x g pellet) and microsomal (150,000 x g pellet) fractions from rosette leaf homogenates. After exposure to cold, the particulate and microsomal fractions of cct1 plants had higher total CCT activity due to increased levels of CCT2; in contrast, the levels of CCT1 in cct2 plants remained unchanged in particulate and microsomal fractions despite a significant increase in the total CCT activity. We conclude that the CDP-choline pathway of A. thaliana is up-regulated at low temperature via isogene-specific modes: enhanced expression of CCT2 and post-translational activation/inactivation of CCT1 in membranes. PC levels were similarly maintained in both mutants and WS plants after 14 d at 2 degrees C, suggesting that either of the CCT genes is sufficient for PC biosynthesis at low temperature.

First Report of Cucumber mosaic virus in Anemone sp. in the United States

In the spring of 2008, more than a dozen, aphid-infested, anemone plants (Anemone sp.) grown at the campus of the University of Arkansas in Fayetteville showed stunting and mosaic, whereas only two were asymptomatic. Leaf homogenates from four symptomatic plants were inoculated onto Nicotiana benthamiana that became stunted and developed severe mosaic approximately 7 days postinoculation, whereas buffer-inoculated plants remained asymptomatic. Double-stranded RNA (dsRNA) extraction (4) from symptomatic anemone revealed the presence of four predominant bands of approximately 3.2, 2.9, 2.2, and 0.9 kbp, a pattern indicative of cucumovirus infection. Cucumber mosaic virus (CMV) is the only cucumovirus reported in anemone in Europe (2) and Israel (3), and for this reason, anemone and N. benthamiana plants were tested by Protein A ELISA with antisera against CMV developed by H. A. Scott. ELISA verified the presence of CMV in symptomatic anemone and inoculated N. benthamiana, while asymptomatic plants were free of the virus. Using cucumovirus degenerate primers, essentially as described by Choi et al. (1), a region of approximately 940 bases that includes the complete coat protein gene of the virus was amplified from symptomatic anemone and N. benthamiana but not asymptomatic plants of either species. This anemone isolate (GenBank Accession No. FJ375723) belongs to the IA subgroup of CMV because it shares 99% nucleotide and 100% amino acid sequence identities with the Fny isolates of the virus. To my knowledge, this is the first report of CMV infecting anemone in the United States and an important discovery for the ornamental industry since anemone is commonly grown together with several ornamental hosts of CMV in nursery and garden settings.

Aphrodisiac Property of the Elephant Creeper Argyreia nervosa

Based on ethno-medical clues, the aphrodisiac property of Argeria nervosa was studied in male mice. The root, flower and, to some extent, leaf (homogenate in 2% gum acacia) of the plant showed aphrodisiac activity as evidenced by an increase in mounting behavior of mice. When different extracts of the root were tested, the activity was found in the alcohol extract (200 mg/kg; p.o, single dose). The extract, 1 hr after administration, stimulated mounting behavior of male mice in a concentration-dependent manner. The root- or flower-treated male mice also exhibited a remarkable increase in mating performance. Further, the number of males was found to be more among the pups fathered by the herbal drug-treated mice compared to those by the control mice. Thus, the plant has promising potential to be developed into an effective medicine for stimulating male sexual activity with an influence on sex ratio favoring males.

The Mobile Thylakoid Phosphoprotein TSP9 Interacts with the Light-harvesting Complex II and the Peripheries of Both Photosystems

The localization of the plant-specific thylakoid-soluble phosphoprotein of 9 kDa, TSP9, within the chloroplast thylakoid membrane of spinach has been established by the combined use of fractionation, immunoblotting, cross-linking, and mass spectrometry. TSP9 was found to be exclusively confined to the thylakoid membranes, where it is enriched in the stacked grana membrane domains. After mild solubilization of the membranes, TSP9 migrated together with the major light-harvesting antenna (LHCII) of photosystem II (PSII) and with PSII-LHCII supercomplexes upon separation of the protein complexes by either native gel electrophoresis or sucrose gradient centrifugation. Studies with a cleavable cross-linking agent revealed the interaction of TSP9 with both major and minor LHCII proteins as identified by mass spectrometric sequencing. Cross-linked complexes that in addition to TSP9 contain the peripheral PSII subunits CP29, CP26, and PsbS, which form the interface between LHCII and the PSII core, were found. Our observations also clearly suggest an interaction of TSP9 with photosystem I (PSI) as shown by both immunodetection and mass spectrometry. Sequencing identified the peripheral PSI subunits PsaL, PsaF, and PsaE, originating from cross-linked protein complexes of around 30 kDa that also contained TSP9. The distribution of TSP9 among the cross-linked forms was found to be sensitive to conditions such as light exposure. An association of TSP9 with LHCII as well as the peripheries of the photosystems suggests its involvement in regulation of photosynthetic light harvesting.

Epigallocatechin gallate content change of the fresh tea leaf homogenates extracted by different methods in extraction and preservation

The fresh leaves of China green tea, Camellia sinensis, were collected from Fuyang, Zhejiang Province, China, in April. The tea polyphenols was extracted by four different methods (homogenized with distilled water at room temperature, homogenized with 0.3% citric acid (w/v) at room temperature, 5-min boiling and homogenized with distilled water at room temperature, homogenized with 85°C distilled water), and after preserving at room temperature, the change of the Epigallocatechin gallate (EGCG) contents of the extracts was investigated. Results indicated that the EGCG content of homogenate extracted with 85°C distilled water was the highest before the extract was preserved, followed by that of the extract homogenized with 0.3% citric acid at room temperature. During preservation, EGCG content changed obviously. The EGCG contents of homogenates extracted with distilled water at room temperature and 85°C distilled water declined quickly and separately reduced to 21.52% and 54.6% of their initial contents after preservation for 12 h. The EGCG contents extracted by 0.3% citric acid (w/v) solvent at room temperature and 5-min boiling/homogenized with distilled water at room temperature declined relatively slowly, and separately reduced to 76.9% and 85.16% of their initial contents after preservation for 12 h. It was also found that the citric acid can prevent the degradation of EGCG and the extract solution color is light green