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.

Oral LD50 in mice >1 g/kg, Japanese Patent Publication No. 93-944 (Tokyo Publication).

[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 formed by the condensation of the carboxyl group of gallic acid with the (3R)-hydroxyl group of (-)-gallocatechin. It is a natural product found in green tea. It has multiple functions, including as an inhibitor of EC 3.4.22.69 (SARS coronavirus main protease), a human xenobiotic metabolite, an antitumor agent, and a plant metabolite. It is a gallate, polyphenol, and catechin. Its functions are related to (-)-gallocatechin and gallic acid. It is the enantiomer of (+)-gallocatechin gallate. (-)-Gallocatechin gallate has been reported in tea (Camellia sinensis), Potentilla erecta, and other organisms with relevant data. Background: Catechins are the main polyphenolic compounds in tea (Camellia sinensis). To understand the relationship between gene expression and product accumulation, this study analyzed the content of catechins and the relative expression levels of key genes in tea leaves at different developmental stages. Results: Except for gallocatechin gallate, the content of catechins in tea leaves at different developmental stages showed significant differences. The study found that the expression of catechin synthesis genes was closely related to catechin accumulation. Correlation analysis showed that the expression of chalcone synthase 1, chalcone synthase 3, anthocyanin reductase 1, anthocyanin reductase 2, and colorless anthocyanin reductase genes was significantly positively correlated with total catechin content, suggesting that the expression of these genes may have a significant impact on total catechin accumulation. The expression of anthocyanin synthase was significantly correlated with catechin content. Furthermore, the expression of anthocyanin reductase 1, anthocyanin reductase 2, and colorless anthocyanin reductase was significantly positively correlated with the content of (-)-epigallocatechin gallate and (-)-epigallocatechin gallate. Conclusion: Our findings suggest that there is a synergistic effect between the expression of synthetic genes and the accumulation of catechins. Based on our findings, anthocyanin synthase may regulate the early steps of catechin conversion, while anthocyanin reductase and colorless anthocyanin reductase genes may both play important roles in the biosynthesis of galloyl catechins. [1] Yingde black tea is made from the leaves of Camellia sinensis var. assamica. In this study, five new flavanones, namely amilia ketone AE (1-5), and seven known compounds 6-12 were isolated and identified from the ethanol extract of Yingde black tea. The structures of these five new phenolic compounds were determined using a large number of 1D and 2D nuclear magnetic resonance spectroscopy experiments. In addition, the antioxidant activity, α-glucosidase inhibitory activity and cytotoxic activity of these compounds were evaluated. Compound 1 exhibited higher α-glucosidase inhibitory activity, with a half-maximal inhibitory concentration (IC50) of 10.2 µM, compared to acarbose (18.2 µM). [2]

C22H18O11

458.375

458.084

C, 57.65; H, 3.96; O, 38.40

4233-96-9

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

199472

White to off-white solid powder

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.)