Natural flavonoid in green tea; antioxidant

Four isoflavone-metabolizing bacteria were tested for their abilities to degrade (-)-epigallocatechin (EGC) and its isomer (-)-gallocatechin (GC). Biotransformation of both EGC and GC was observed with Adlercreutzia equolifaciens JCM 14793, Asaccharobacter celatus JCM 14811, and Slackia equolifaciens JCM 16059, but not Slackia isoflavoniconvertens JCM 16137. With respect to the degradation of EGC, strain JCM 14793 only catalyzed 4'-dehydroxylation to produce 4'-dehydroxylated EGC (7). Strain JCM 14811 mainly produced 7, along with a slight formation of the C ring-cleaving product 1-(3,4,5-trihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (1). Strain JCM 16059 catalyzed only C ring cleavage to form 1. Interestingly, the presence of hydrogen promoted the bioconversion of EGC by these bacteria. In addition, strain JCM 14811 revealed the ability to catalyze 4'-dehydroxylation of 1 to yield 1-(3,5-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (2) in the presence of hydrogen. In the case of GC, strain JCM 14793 mainly produced C ring-cleaving product (1) with only a very small amount of 4'-dehydroxylated GC (8), while Strain JCM 14811 only catalyzed 4'-dehydroxylation to form 8. Strain JCM 16059 formed 1. The bioconversion of GC by the three strains was stimulated by hydrogen. Strain JCM 14793 showed the ability to convert 1 into 2 in the presence of hydrogen as did strain JCM 14811. Furthermore, strains JCM 14793 and JCM 14811 were found to have the ability to catalyze p-dehydroxylation of the pyrogallol moiety in the EGC metabolites 4-hydroxy-5-(3,4,5-trihydroxyphenyl)valeric acid (3) and 5-(3,4,5-trihydroxyphenyl)-γ-valerolactone (4), and this ability was enhanced by the presence of hydrogen[1].

It has been known that tea catechins, (-)-epicatechin (1), (-)-epigallocatechin (2), (-)-epicatechin gallate (3), and (-)-epigallocatechin gallate (4) are epimerized to(-)-catechin (5), (-)-gallocatechin (6), (-)-catechin gallate (7), and (-)-gallocatechin gallate (8), respectively, during retort pasteurization. We previously reported that tea catechins, mainly composed of 3 and 4, effectively inhibit cholesterol absorption in rats. In this study, the effect of heat-epimerized catechins on cholesterol absorption was compared with tea catechins. Both tea catechins and heat-epimerized catechins lowered lymphatic recovery of cholesterol in rats cannulated in the thoracic duct and epimerized catechins were more effective than tea catechins. The effect of purified catechins on micellar solubility of cholesterol was examined in an in vitro study. The addition of gallate esters of catechins reduced micellar solubility of cholesterol by precipitating cholesterol from bile salt micelles. Compounds 7 and 8 were more effective to precipitate cholesterol than 3 and 4, respectively. These observations strongly suggest that heat-epimerized catechins may be more hypocholesterolemic than tea catechins[3].

---

Aging leads to cognitive impairments characterized by reduced hippocampal functions that are associated with impairment of long-term potentiation of CA1 synapses. Here, we assessed the safety and efficacy of modified (-)-gallocatechin gallate (GCG)-enriched green tea extract (HTP-GTE) in ameliorating the cognitive dysfunctions in late middle-aged murine model. We developed a novel HTP-GTE that was enriched with GCG via epimerization that involved heating. We compared the effects of oral administrations of conventional green tea and HTP-GTE in young and aged male C57/BL6 mice, and examined the changes in the hippocampal functions related to aging process. The functional outcome was assessed by the electrophysiological experiments to measure the long-term potentiation (LTP). HTP-GTE improved the age-related cognitive impairments via restoring long-term synaptic plasticity. We also identified that GCG was the main active component responsible for the HTP-GTE effect. The main molecular pathway in ameliorating the age-related cognitive dysfunctions involved protein kinase A (PKA) which was shown to be modulated by HTP-GTE. Thus, HTP-GTE has a therapeutic potential as a dietary supplement which may aid to rescue the impaired cognitive functions at the early phase of aging process through the modulation of LTP threshold[4].

Canned and bottled tea drinks contain not only green tea epicatechins (GTE), namely (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC), but also four GTE epimers, namely (-)-gallocatechin gallate (GCG), (-)-catechin gallate (CG), (-)-gallocatechin (GC) and (-)-catechin (C). In the present study we examined the antioxidant activity and bioavailability of these epimers compared with their corresponding precursors. The epimerisation reaction was induced by autoclaving GTE extract derived from longjing green tea at 120 degrees C for 20 min. Isolation and purification of each GTE and epimer were accomplished by various column chromatographic and semi-preparative HPLC techniques. The antioxidant activity of each epimer with its corresponding GTE precursor was conducted in the three in vitro systems, namely human LDL oxidation, ferric reducing-antioxidant power (FRAP), and anti-2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assays. The results of all three assays demonstrated that CG had similar antioxidant activity with its precursor ECG, while GC was less potent as an antioxidant than its precursor EGC. Regarding EGCG and GCG, the antioxidant potency was similar for both LDL oxidation and DPPH free radical assays, but GCG was statistically less effective than EGCG in the FRAP assay. For EC and C, the latter had less anti-free radical activity in the DPPH assay, but in LDL oxidation and FRAP assays the antioxidant activity was similar. Oral and intravenous dosing of GTE-epimer mixture led to increase in total plasma antioxidant capacity in rats. In general, both epicatechins and epimers had low bioavailability (0.08-0.31) and most of the observed differences between epicatechins and their corresponding epimers were small, even if they were statistically significant in some cases. It was concluded that the epimerisation reaction occurring in manufacturing canned and bottled tea drinks would not significantly affect antioxidant activity and bioavailability of total tea polyphenols[2].

Cell viability test with methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay[4]
Hippocampal neurons were cultured in the 24-well plate with the confluence of 2 × 10~6 cells per well for 14 days at 37 °C in the incubator with mixed gas (95% O2 and 5% CO2). After the full maturation, the neurons were treated with GTE or HTP-GTE for 2 h, then, the medium was changed with fresh Neurobasal medium containing diluted methylthiazolyldiphenyl-tetrazolium bromide (MTT) (10% MTT) for further 2 h at 37 °C. After the reaction, the media was removed and 200 μL dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. MTT results were measured via the light absorbance at 570 nm using a microplate reader, and cell viability data was acquired by the ratio of optical density of the treated group over the control group.

Studies on Lymphatic Recovery of 14C-Cholesterol in Rats Cannulated in the Thoracic Duct. [3]
Eight-week old male SD rats were fed a commercial chow for 1 week until the operation. The left thoracic lymphatic duct cephalad to the cisterna chyli of these rats was cannulated as described previously. A second indwelling catheter was placed in the stomach for administration of a test emulsion. After surgery, the animals were placed in restraining cages and intragastrically given a continuous infusion of a solution containing 139 mM glucose and 85 mM NaCl at a rate of 3.4 mL/h until the end of the experiment. The same solution was given as drinking water. The next morning, animals with a constant lymph flow rate were administered 3 mL of a test emulsion containing 14C-cholesterol with or without catechin preparations. The test emulsion (3 mL) contained 200 mg of sodium taurocholate, 50 mg of fatty-acid-free bovine albumin fraction V, 200 mg of triolein, 37 kBq of 14C-cholesterol and 25 mg of cholesterol. The mixture was emulsified by sonication. When tea catechins and heat-epimerized catechins were administered, these catechins were added in the emulsion at 100 mg and 120 mg in 3 mL, respectively. Because the content of catechins (3, 4, 7, and 8) was decreased in heat-epimerized catechins, the amount of catechins was adjusted to the same levels between tea catechins and epimerized catechins. The molar ratios of total catechins to cholesterol in lipid emulsions were about 2.5. Lymph was collected in ice-chilled tubes containing EDTA, and the radioactivity was measured.

[1]. Biotransformation of (-)-epigallocatechin and (-)-gallocatechin by intestinal bacteria involved in isoflavone metabolism. Biol Pharm Bull. 2015;38(2):325-30.

[2]. Comparison of antioxidant activity and bioavailability of tea epicatechins with their epimers. Br J Nutr. 2004 Jun;91(6):873-81.

[3]. Heat-epimerized tea catechins rich in gallocatechin gallate and catechin gallate are more effective to inhibit cholesterol absorption than tea catechins rich in epigallocatechin gallate and epicatechin gallate. J Agric Food Chem. 2003 Dec 3;51(25):7303-7.

[4]. Ahn JW, Kim S, Ko S, Kim YH, Jeong JH, Chung S. Modified (-)-gallocatechin gallate-enriched green tea extract rescues age-related cognitive deficits by restoring hippocampal synaptic plasticity. Biochem Biophys Rep. 2022;29:101201.

(-)-gallocatechin is a a gallocatechin that has (2S,3R)-configuration. It has a role as an antioxidant, a radical scavenger and a metabolite. It is an enantiomer of a (+)-gallocatechin.
(-)-Gallocatechin has been reported in Camellia sinensis, Berchemia formosana, and other organisms with data available.

C15H14O7

306.26746

306.073

C, 58.83; H, 4.61; O, 36.57

3371-27-5

(-)-Epigallocatechin;970-74-1

9882981

White to off-white solid powder

1.7±0.1 g/cm3

685.6±55.0 °C at 760 mmHg

368.5±31.5 °C

0.0±2.2 mmHg at 25°C

1.776

-0.1

6

7

1

22

380

2

C1[C@H]([C@@H](OC2=CC(=CC(=C21)O)O)C3=CC(=C(C(=C3)O)O)O)O

XMOCLSLCDHWDHP-DOMZBBRYSA-N

InChI=1S/C15H14O7/c16-7-3-9(17)8-5-12(20)15(22-13(8)4-7)6-1-10(18)14(21)11(19)2-6/h1-4,12,15-21H,5H2/t12-,15+/m1/s1

(2S,3R)-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol

(-)-Gallocatechin; 3371-27-5; ent-gallocatechin; (2S,3R)-gallocatechin; (2S,3R)-2-(3,4,5-Trihydroxyphenyl)chroman-3,5,7-triol; CHEBI:71225; MFCD01632616; (2S,3R)-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~326.51 mM)
H2O : ~1.67 mg/mL (~5.45 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (8.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

 (Please use freshly prepared in vivo formulations for optimal results.)