Natural polyphenol; anticancer

The content of (-)-gallatechin gallate in leaves is minimal and does not vary depending on the stage [1]. When (−)-epigallocatechin gallate and active catechin ((−)-epigallocatechin gallate) are combined, they have a synergistic effect that stops PC-9 cell development and induces apoptosis. α-Glucosidase and DPPH are inhibited by (−)-gallocatechin gallate, with IC50 values of 30.2 μM and 12.2 μg/mL, respectively [2].

α-Glucosidase inhibition assay[2]
This assay was conducted in 96-well microtiter plates following the procedures described in the literature (Yang et al., 2016a, Yang et al., 2016b). In brief, test compounds were dissolved in dimethyl sulfoxide (DMSO) to six serial concentrations. α-Glucosidase and p-NPG were dissolved in 60 mM sodium phosphate buffer with pH 6.8 at 0.5 U/mL and 5 mM, respectively. Four kinds of solutions were made. Test solution contained 112 μL buffer, 20 μL enzymes, and 8 μL compounds. Test bank solution contained 112 μL buffer and 8 μL test compounds. Negative control solution contained 112 μL buffer, 20 μL enzyme, and 8 μL DMSO. Negative blank solution contained 132 μL buffer and 8 μL DMSO. Acarbose was used as positive control. The plates were carefully shaken to thoroughly mix the solutions and kept at 37 °C for 15 min. A total of 20 μL p-NPG was added to quench the reaction. The amount of p-nitrophenol hydrolyzed from p-NPG by α-glucosidase was quantified by measuring its OD value at 405 nm.

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DPPH free radical scavenging assay[2]
DPPH free radical scavenging activities of compounds were measured according to the method previously described. Briefly, 20 μL of test samples at different concentrations was mixed with 180 μL DPPH solution for 30 min in the dark. Then, the change in absorbance at 517 nm for DPPH was measured on a microplate reader. DMSO was used as a negative control. Results were expressed as percentage of inhibition, relative to a control containing DMSO in place of the sample, and as half maximal inhibitory concentration (IC50 values, μg/mL).

Cytotoxicity assay[2]
Cell viability was examined by measuring the capability of cells to metabolize MTT to a purple formazan dye (Zhou et al., 2015). Human liver cancer HepG2 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, 100 units mL−1 penicillin, and 50 units mL−1 streptomycin at 37 °C in a humidified incubator with 5% CO2 atmosphere. Cells were seeded in 96-well tissue culture plates for 24 h and then incubated with the tested compounds at different concentrations for 72 h. After incubation, 25 μL MTT in 5 mg/mL PBS was added and incubated for 4 h. The medium was aspirated and replaced with 150 μL dimethyl sulfoxide (DMSO) to dissolve the formazan salt. The color intensity of the formazan solution, which reflects the cell growth condition, was measured at 570 nm using a microplate spectrophotometer.

mouse LD50 oral >1 gm/kg Japanese Kokai Tokyo Koho Patents., #93-944

[1]. Accumulation of catechins and expression of catechin synthetic genes in Camellia sinensis at different developmental stages. Bot Stud. 2016 Dec;57(1):31.

[2]. C-geranylated flavanones from YingDe black tea and their antioxidant and α-glucosidase inhibition activities. Food Chem. 2017 Nov 15;235:227-233.

(-)-gallocatechin gallate is a gallate ester obtained by formal condensation of the carboxy group of gallic acid with the (3R)-hydroxy group of (-)-gallocatechin. A natural product found in found in green tea. It has a role as an EC 3.4.22.69 (SARS coronavirus main proteinase) inhibitor, a human xenobiotic metabolite, an antineoplastic agent and a plant metabolite. It is a gallate ester, a polyphenol and a catechin. It is functionally related to a (-)-gallocatechin and a gallic acid. It is an enantiomer of a (+)-gallocatechin gallate.
(-)-Gallocatechin gallate has been reported in Camellia sinensis, Potentilla erecta, and other organisms with data available.

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Background: Catechins are the main polyphenol compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, the levels of catechins and relative expressions of key genes in tea leaves of different developmental stages were analyzed.
Results: The amounts of catechins differed significantly in leaves of different stages, except for gallocatechin gallate. Close correlations between the expression of synthesis genes and the accumulation of catechins were identified. Correlation analysis showed that the expressions of chalcone synthase 1, chalcone synthase 3, anthocyanidin reductase 1, anthocyanidin reductase 2 and leucoanthocyanidin reductase genes were significantly and positively correlated with total catechin contents, suggesting their expression may largely affect total catechin accumulation. Anthocyanidin synthase was significantly correlated with catechin. While both ANRs and LAR were significantly and positively correlated with the contents of (-)-epigallocatechin gallate and (-)-epicatechin gallate.
Conclusion: Our results suggest synergistic changes between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanidin synthase may regulate earlier steps in the conversion of catechin, while the anthocyanidin reductase and leucoanthocyanidin reductase genes may both play important roles in the biosynthesis of galloylated catechins.[1]

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YingDe black tea is produced from crude tea prepared from leaves of Camellia sinensis var. assamica. In this work, we isolated and identified five novel flavanones, namely, amelliaone A-E (1-5), along with seven known compounds 6-12 from the ethanol extract of YingDe black tea. The structures of these five novel phenolic compounds were determined using extensive 1D and 2D nuclear magnetic resonance spectroscopy experiments. The compounds were further evaluated for antioxidant, α-glucosidase inhibitory, and cytotoxic activities. Compound 1 exhibited higher α-glucosidase inhibitory activity with a half-maximum inhibitory concentration value (IC50) of 10.2µM compared with acarbose (18.2µM).[2]

C22H18O11

458.375

458.084

C, 57.65; H, 3.96; O, 38.40

4233-96-9

(-)-Epigallocatechin Gallate;989-51-5

199472

Typically exists as solid at room temperature

1.9±0.1 g/cm3

909.1±65.0 °C at 760 mmHg

320.0±27.8 °C

0.0±0.3 mmHg at 25°C

1.857

Tea

2.08

8

11

4

33

667

2

O1C2=C([H])C(=C([H])C(=C2C([H])([H])[C@]([H])([C@]1([H])C1C([H])=C(C(=C(C=1[H])O[H])O[H])O[H])OC(C1C([H])=C(C(=C(C=1[H])O[H])O[H])O[H])=O)O[H])O[H]

WMBWREPUVVBILR-NQIIRXRSSA-N

InChI=1S/C22H18O11/c23-10-5-12(24)11-7-18(33-22(31)9-3-15(27)20(30)16(28)4-9)21(32-17(11)6-10)8-1-13(25)19(29)14(26)2-8/h1-6,18,21,23-30H,7H2/t18-,21+/m1/s1

[(2S,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-3-yl] 3,4,5-trihydroxybenzoate

CCRIS-9286; CCRIS 9286; (-)-Gallocatechin gallate; 4233-96-9; (-)-Gallocatechol gallate; (2S,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl 3,4,5-trihydroxybenzoate; [(2S,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-chromen-3-yl] 3,4,5-trihydroxybenzoate; Gallocatechin gallate, (-)-; (2S,3R)-2-(3,4,5-Trihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol 3-(3,4,5-trihydroxybenzoate); MFCD00214298; CCRIS9286; (-)-gallocatechin-3-O-gallate; (-)-Gallocatechol gallate; Gallocatechin gallate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~218.16 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.54 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 20.8 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.54 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.54 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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