Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

高效液相色谱法分析元宝枫叶中儿茶素类物质

本文建立了元宝枫树叶中儿茶素种类及其含量的高效液相色谱(HPLC)测定方法。采用反相C18色谱柱,以甲醇/水(含0.5%乙酸)=25/75(V/V)为流动相,对没食子儿茶素(GC)、表没食子儿茶素(EGC)、儿茶素(C)、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素(EC)和没食子儿茶素没食子酸酯(GCG)进行定性、定量分析;以甲醇/水(含0.5%乙酸)=35/65(V/V)为流动相,对表儿茶素没食子酸酯(ECG)和儿茶素没食子酸酯(CG)进行定性分析,柱温均为35℃,检测波长为278 nm,流速为1.0mL/min。结果表明:元宝枫叶中有EGC、EC和GCG,其它五种则无。EGC平均含量为0.0389 mg/g,方法精密度(RSD)为0.42%(n=6);EC平均含量为0.0289 mg/g,方法RSD为1.5%(n=6);GCG平均含量为0.284 mg/g,方法RSD为0.32%(n=6)。该方法简便、准确、分离效果好,为元宝枫叶开发成茶叶、饮料以及医疗保健品提供重要依据。     

Determination of exogenous epigallocatechin gallate peracetate in mouse plasma using liquid chromatography with quadrupole time‐of‐flight mass spectrometry

A robust method for the quantitation of epigallocatechin gallate peracetate in plasma for pharmacokinetic studies is lacking. We have developed a validated method to quantify this compound using liquid chromatography with quadrupole time‐of‐flight mass spectrometry with isopropanol and tert‐butyl methyl ether (3:10) extraction and thin‐layer chromatography purification. The epigallocatechin gallate peracetate‐1‐¹³C₈ isotope was used as an internal standard. The linear range (r² > 0.9950) was from 0.05 to 100.00 μg/mL. The lower limit of quantification of the method was 0.05 μg/mL. Reproducibility, coefficient of variation, was between 0.7 and 12.6% (n = 6), accuracy between 83.7 and 104.6% (n = 5), and recovery ranged from 82.4 to 109.0% (n = 4). Ion suppression was approximately 40%. No mass spectral peaks were found to interfere between the standard and internal standard or the blank plasma extracts. Epigallocatechin gallate peracetate in plasma was stably stored at –80°C over three months even after three freeze–thaw cycles. Extracts were stable in the sampler at 4°C for over 48 h. Plasma levels were maintained at 1.36 μg/mL for 360 min after intraorbital intravenous injection at 50 mg/kg in mice. This method can be used to reliably measure epigallocatechin gallate peracetate in plasma for pharmacokinetic studies.     

茶中茶多酚的高效液相色谱法分离分析

用改进的Ａｇａｒｗａｌ方法萃取不同种类茶叶和茶饮料中的茶多酚,建立了用高效液相色谱（ＨＰＬＣ）法对茶多酚进行分离分析方法。ＨＰＬＣ可有效分离ＧＴＰｓ主要组成成分ＥＣ、ＥＧＣ、ＥＣＧ和ＥＧＣＧ并精确定量,相对标准偏差小于５％。茶叶加工过程对ＧＴＰｓ含量有很大影响,绿茶总ＧＴＰｓ含量在６￣１５ｇ／１００ｇ干茶叶、乌龙茶总ＧＴＰｓ含量在５￣７ｇ／１００ｇ干茶叶,红茶总ＧＴＰｓ含量低于２ｇ／１００ｇ干茶叶     

Rapid, direct determination of polyphenols in tea by reversed-phase column liquid chromatography.

Column liquid chromatography on a C18-bonded silica column with water-methanol-acetic acid as eluent was used to determine polyphenols and caffeine in tea. Without any pretreatment, catechin, epicatechin gallate, epigallocatechin gallate, epigallocatechin, epicatechin and caffeine were separated successfully within 15 min. The detection limits (S/N = 3) of polyphenols studied were 1.8-24 mg/l at a detection wavelength 270 nm. The linear range of the peak area calibration curves for the analytes were over two orders of magnitude with a correlation coefficient of 0.996-0.999. Using this method, some Chinese tea samples were analyzed with a good reproducibility (RSD are below 5%).     

Dodecyl gallate as a pro-ecological antioxidant for food packing materials

Electrochemical oxidation of dodecyl gallate (lauryl gallate), the main monomer flavanol found in green tea, was investigated on platinum electrodes using cyclic voltammetry (CV) and differential pulse (DPV) methods. The rate constant, electron transfer coefficient and diffusion coefficients were determined for dodecyl gallate electrochemical oxidation. The oxidation mechanism proceeds in sequential steps related to the hydroxyl groups in the aromatic ring of dodecyl gallate. The confirmed antioxidant activity of lauryl gallate justified its use in polymers, for instance, in an environment-friendly stabiliser aimed at improving the resistance to aging of elastomeric materials. Based on energy change during deformation, cross-linking density and oxygen induction time in the TG method, we confirmed the high antioxidant activity of lauryl gallate in polymers. Moreover, the research on biodegradation confirmed the environmentally friendly interest of the antioxidant by the fact that it increases the susceptibility of the elastomeric materials to disintegration by mildew mushrooms.     

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2'-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (-)-epicatechin and (-)-epigallocatechin gallate solutions, were linear over the range of 0.1-50 microM. The detection limits (signal-to-noise ratio=3) were 0.8 pmol for (-)-epigallocatechin gallate and 1.2 pmol for (-)-epicatechin. The high-performance liquid chromatography-chemiluminescence (HPLC-CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.     

Speciation of chromium by selective separation and preconcentration of Cr(III) on an immobilized nanometer titanium dioxide microcolumn

Nanometer titanium dioxide immobilized on silica gel (immobilized nanometer-scale TiO2 particles) was prepared by a sol-gel method and characterized by X-ray diffraction and scanning electron microscopy. The adsorptive behavior of Cr(III) and Cr(VI) on immobilized nanometer TiO2 was assessed. Cr(III) was selectively sorbed on immobilized nanometer TiO2 in the pH range of 7-9, while Cr(VI) was found to remain in solution. A sensitive and selective method has been developed for the speciation of chromium in water samples using an immobilized nanometer TiO2 microcolumn and inductively coupled plasma atomic emission spectrometry. Under optimized conditions (pH 7.0, flow rate 2.0 mL/min), Cr(III) was retained on the column, then eluted with 0.5 mol/L HNO3 and determined by ICP-AES. Total chromium was determined after the reduction of Cr(VI) to Cr(III) by ascorbic acid. The adsorption capacity of immobilized nanometer TiO2 for Cr(III) was found to be 7.04 mg/g. The detection limit for Cr(III) was 0.22 ng/mL and the RSD was 3.5% (n = 11, c = 100 ng/ mL) with an enrichment factor of 50. The proposed method has been applied to the speciation of chromium in water samples with satisfactory results.     

Synthesis and preliminary anticancer activity studies of C4 and C8-modified derivatives of catechin gallate (CG) and epicatechin gallate (ECG)

We have developed an improved and reliable method for stereoselective functionalization at C4 of naturally occurring (+)-catechin. Our method utilizes DDQ oxidation followed by trapping of the quinonemethide intermediate with allyl alcohol. The quinonemethide intermediate can be regenerated from the allyl ether by exposure to boron trifluoride diethyl etherate. This reactive intermediate can be trapped with a wide range of external nucleophiles. NBS bromination, lithium halogen exchange, and alkylation gave access to C8-allyl derivatives of (+)-catechin, and this allyl group was used in a series of cross-metathesis experiments to prepare novel dimeric catechin-derived products. Gallate ester derivatives of the novel C4- and C8-substituted catechins were prepared, and these materials were screened for potential anticancer activity in a range of human cancer cell lines. From these preliminary cytotoxicity assays (MTT) we found that C8-propyl-catechin gallate was more active (IC50 = 31 microM) than catechin gallate (CG, IC50 = 53 microM) or epicatechin gallate (ECG, IC50 = 76 microM) against the colorectal adenocarcinoma cell line HCT116. Differential sensitivity in pancreas (Pan1), bladder (RT112), stomach (MGLVA1), liver (HepG2), and fibroblasts (46Br.1G1) cell lines was also observed.     

Extraction of Catechin Compounds from Green Tea with a New Green Solvent

An emerging green solvent called a deep eutectic solvent（DES） was applied to the extraction and determination of catechin（C）,（＋）epicatechin gailate（ECG） and （-）epigallocatechin gallate（EGCG） from Chinese green tea.After evaluating different combinations of them by extraction methods and DESs,a DES-based extraction method was established and optimized by a systematic investigation of the influencing factors.As a result,a total of 3.629,35.25 or 114.2 mg/g catechin,（＋）epicatechin gallate or （-）epigaliocatechin gallate were extracted respectively under optimal conditions with extraction efficiencies of 82.7％,92.3％ and 97.0％,respectively.By comparing with other common used solvents for extracting catechin compounds,DESs were proved to be potential extraction solvents for bioactive ingredients.     

Attomole catechins determination by capillary liquid chromatography with electrochemical detection.

Attomole quantities of catechins were determined by a capillary liquid chromatography system with electrochemical detection (CLC-ECD) and the system is applied to the determination of catechins in human plasma. The eight catechins: catechin (C), epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), catechin gallate (Cg), epicatechin gallate (ECg), gallocatechin gallate (GCg), and epigallocatechin gallate (EGCg), were separated within 10 min using a capillary column (0.2 mm i.d.) and a mobile phase of phosphoric acid (85%)-methanol-water (0.5:27.5:72.5, v/v/v), and were detected at +0.85 V vs. Ag/AgCl. Peak heights were found to be linearly related to the amount of catechins injected, from 200 amol to 500 fmol (r > 0.998). The detection limits of the catechins were 61 amol for EGC, 75 amol for EC, 54 amol for GC, 61 amol for C, 67 amol for GCg, 75 amol for EGCg, 75 amol for ECg and 89 amol for Cg (S/N = 3). Because the present method is highly sensitive and allows facile pretreatment for plasma sample, the time courses of concentrations of catechins (GCg, EC, EGCg, ECg, and Cg) and their conjugates in human plasma obtained from a 10 microl plasma sample after ingestion of green tea could be determined.     

In vitro evaluation of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB

Although some polyphenols are known to possess anticancer activity against different cancer cell lines through induction of apoptosis, the mode of antiproliferative effect of ethyl gallate against human oral squamous carcinoma cell line KB was not studied until now. Therefore, the antiproliferative effect of ethyl gallate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in comparison with the reference drug paclitaxel. Generation of reactive oxygen species, mitochondrial membrane potential loss, DNA damage and apoptosis were determined using 2,7-diacetyldichlorofluorescein fluorescence, uptake of rhodamine-123 by mitochondria, comet assay and acridine orange/ethidium bromide dual-dye staining method. Both ethyl gallate and paclitaxel exhibited cytotoxicity in a dose-dependent manner. The 50% inhibitory concentration for ethyl gallate was 30 and 20 μg/mL for paclitaxel. A volume of 50 μg/mL of ethyl gallate was found to be significantly effective (P < 0.05) in controlling the cancer cell proliferation leading to acute apoptosis.     

Screening of Binding Properties of Con-A Immobilized on Bead Cellulose by Flow Microcalorimetry Using Invertase and Anti-Con-A Antibody as Reporting Systems

SUMMARY

Flow micro-calorimeter so-called enzyme thermistor (ET) is nowadays increasingly used for rapid screening of the sorbents with immobilized protein ligands. In the present paper, chlorotriazine-, dialdehyde-, formylmethyl-, oxirane- and diazotized derivatives of bead cellulose were applied as matrices for immobilization of Concanavalin-A (Con-A). The amounts of free-accessible Con-A in each of the above sorbents were evaluated: i.) by adsorption of invertase; ii.) by means of ELISA using both, mouse anti-Con-A antibody and pig anti-mouse antibody linked with peroxidase. Reactions sub i and ii were monitored in dual way; thermometrically, with the aid of an ET as well as using common spectophotometric methods. The results obtained by spectrophotometry and thermometry intercorrelated significantly (p=0.0028 and p=0.0001, for ELISA and the invertase, respectively). The amounts of immobilized Con-A were directly proportional with the ELISA signal of peroxidase reaction whether this was detected by means of ET (r=0.975, p=0.001) or by spectrophotometry (r=0.983, p=0.0005). Slightly less tight correlation was found between the amounts of immobilized Con-A and the activity of invertase in conjugates when they were assayed by means of thermometry (r=0.933, p=0.0065) and spectrophotometry (r=0.920, p=0.0094). The above data indicate, that thermometry may be considered as a method suitable for rapid evaluation of the amount and accessibility of immobilized Con-A.     

Preparation,characterization, and luminescence of (SBA-15) immobilized pepsin

SBA-15 mesoporous silica was synthesized by hydrothermal method and its surface was methylated by treatment with methyltrimethoxysilane. Pepsin was immobilized on the obtained materials giving host-guest composite materials (SBA-15)-pepsin and (methylated SBA-15)-pepsin. The optimum conditions for preparation of these materials were established. Methylated SBA-15 (M-SBA-15) has improved immobilization efficiency of enzyme compared to initial SBA-15 silica. It was shown that with the gradual increase of NaCl solution ionic strength the immobilized amount of enzyme was reduced. Powder X-ray diffraction and Fourier transform infrared spectroscopy showed that the host frameworks in the prepared host-guest composite materials are intact and the ordered structure was retained. Scanning electron microscopic studies revealed fibrous morphologic characteristics of the SBA-15 and the immobilized pepsin composite materials. The average particle diameter of (SBA-15)-pepsin composite was 338 ± 10 and 343 ± 10 nm for (M-SBA-15)-pepsin. The low temperature N₂ adsorption-desorption study at 77 K showed that the pore sizes and specific surface areas of the host-guest composite materials were smaller than those before the introduction of the enzyme, suggesting that the immobilized enzyme occupied a definite position in the host material pore channels. The UV-vis solid diffuse reflectance and luminescence studies showed that the enzyme was successfully immobilized on to the host material and that after the immobilization of enzyme on SBA-15 the conformation of pepsin macromolecule has not been changed.     

吸附-纤维素覆膜联合固定化酶

以食品工业中常用的木瓜蛋白酶为模式酶, 建立了吸附-纤维素覆膜联合固定化酶方法. 通过对吸附载体类别、 纤维素种类及溶剂、 保护剂种类及其浓度、 干燥方式及时间等的优化, 得到最佳的吸附-纤维素覆膜联合固定化酶工艺. 以硅藻土或HPD-417(大孔树脂)作为吸附载体, 甲基纤维素(分子量40000~50000)丙酮溶液作为覆膜溶液, 加入6%(质量分数)的聚乙二醇或麦芽糖作为覆膜保护剂, 于4 ℃干燥9 h, 制得固定化木瓜蛋白酶, 硅藻土吸附-纤维素覆膜固定化酶酶活回收率达到96.50%, HPD-417吸附-纤维素覆膜固定化酶酶活回收率达到93.92%. 对吸附-纤维素覆膜固定化酶的性质进行了研究, 发现纤维素覆膜后固定化酶具有良好的热稳定性, 于80 ℃下保存12 h后, 固定化酶活残余率仍然能保持90%左右; 在pH=4.5~9.5的范围内, 固定化酶的稳定性较好; 连续使用9次后, 固定化酶活残余率仍能保持95%左右.     

Determination of proanthocyanidins in fresh grapes and grape products using liquid chromatography with mass spectrometric detection

Fresh grapes and grape products, such as grape wine and grape juice, were analyzed for proanthocyanidins (PACs) using liquid chromatography with electrospray ionization mass spectrometric (MS) detection. PACs were successfully separated and analyzed on the basis of their protonated molecules, allowing the identification of PACs in different degrees of polymerization from monomers to oligomers (up to 7 units), and in various isomeric forms. Using reversed-phase high-performance liquid chromatography (HPLC) combined with MS detection, the PAC monomers, (+)-catechin (C), (-)-epicatechin (EC), (-)-catechin gallate (CG), and (-)-epicatechin gallate (ECG), were successfully quantified using selected ion monitoring (SIM) mode. Standard curves were fitted for each PAC ranging from 43.8 to 5600 ng/mL for C, from 42.2 to 5400 ng/mL for EC, from 36.7 to 4700 ng/mL for CG, and from 39.8 to 5100 ng/mL for ECG. Good linearity (r2>0.999) was achieved for each analyte. The accuracy and precision (RSD) were within 10% (n=8) at the limit of detection. This method allows direct quantification of monomeric PACs in fresh grapes and grape-derived products. Additionally, flow injection analysis (FIA) was applied to estimate the concentration levels of PAC oligomers by comparing their FIA-MS peak areas, which were well correlated (r2=0.936) to the total concentrations of PAC monomers.     

Detailed investigations of phase transitions and magnetic structure in Fe(III), Mn(II), Co(II) and Ni(II) 3,4,5-trihydroxybenzoate (gallate) dihydrates by neutron and X-ray diffraction

The effect of cation valency on the complex structures of divalent and trivalent transition metal gallates has been examined using a combination of neutron and synchrotron X-ray powder diffraction, single-crystal X-ray diffraction and XANES spectroscopy. In the divalent frameworks, M(C(7)H(4)O(5))·2H(2)O (M = Mn, Co and Ni), it was found that charge balance was achieved via the presence of protons on the meta-hydroxyl groups. It was also established that these compounds undergo a discontinuous phase transition at lower temperatures, which is driven by the position of the extra-framework water molecules in these materials. By contrast, in the trivalent Fe gallate, Fe(C(7)H(3)O(5))·2H(2)O, it was found that the stronger bonding between the meta-hydroxy oxygen and the cations leads to a weakening of the bond between this oxygen and its proton. This is turn is thought to lead to stronger hydrogen bonding with the extra-framework water. The lattice water is disordered in the Fe(III) case, which prevents the phase transition found in the M(II) gallates. Refinement against the neutron diffraction patterns also revealed that the relatively mild microwave synthesis of gallate frameworks in D(2)O led to an extensive deuteration of the ortho-hydrogen sites on the aromatic ring, which may suggest a more versatile method of deuterating aromatic organics. The antiferromagnetic structure of Co gallate has also been determined.     

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.