Natural flavonoid from green tea
(-)-Epigallocatechin-3-(3''-O-methyl) gallate targets multiple pathways involved in oxidative stress, inflammation, and cell proliferation. It scavenges DPPH-derived radicals (IC50: 36.54 µM) and ABTS radicals (IC50: 2.59 µM). It scavenges intracellular ROS production in RAW264.7 cells (12.5 µM, 30 min). It promotes cell proliferation in HaCaT cells (6.25 and 12 µM, 24-72 h). It increases HO-1 expression in HaCaT cells treated with 250 µM H2O2 (6.25 and 12 µM, 12 h), indicating a cytoprotective role against oxidative stress. It suppresses FcepsilonRI expression, negatively regulating basophil activation. Its strong cytotoxic activity against rat cancer cells suggests anticancer potential.

Antioxidative activity of (-)-epigallocatechin-3-(3''-O-methyl)gallate (catechin e) was examined. Catechin e showed a strong antioxidative activity. A preliminary test using rat cancer cells suggests that catechin e also has a strong cytotoxic activity. Among tested catechins, only catechin e has strong activity for both[1].
In vitro, (-)-Epigallocatechin-3-(3''-O-methyl) gallate scavenges DPPH-derived radicals with an IC50 of 36.54 µM, and scavenges ABTS radicals with an IC50 of 2.59 µM. At 12.5 µM for 30 minutes, it scavenges intracellular ROS production in RAW264.7 cells. At 6.25 and 12 µM for 24-72 hours, it promotes cell proliferation in HaCaT cells. At 6.25 and 12 µM for 12 hours, it increased HO-1 expression in HaCaT cells treated with 250 µM H2O2, indicating a cytoprotective role. It has strong cytotoxic activity for rat cancer cells. It exhibits antiallergic activity by negatively regulating basophil activation through suppression of FcepsilonRI expression.

In vivo activity data for (-)-Epigallocatechin-3-(3''-O-methyl) gallate is limited in publicly available literature. As a natural product from tea leaves with strong antioxidative and anti-inflammatory activities, it is hypothesized to exhibit similar effects in animal models of oxidative stress, inflammation, and cancer. Its strong cytotoxic activity against cancer cells suggests potential for in vivo antitumor studies. However, specific in vivo efficacy studies, including pharmacokinetic and pharmacodynamic parameters, have not been detailed in the available sources. Further research is needed to fully characterize its in vivo activity and therapeutic potential.

DPPH Assay[2]
DPPH decolorimetric assay was performed to examine the scavenging effect of 3″Me-EGCG as previously described. 3″Me-EGCG (0–12.5 μM) was mixed with 250 mM DPPH and incubated at 37 °C for 30 min. Ascorbic acid (500 μM) was used as a positive control. After incubation, the absorbance at 517 nm was measured by spectrophotometry. DPPH scavenging effect was expressed as percent inhibition as follows:
DPPH scavenging effect (%) = [(A0 − A1)/A0] ∗ 100 (1)
in which A0 indicates the absorbance of DPPH, and A1 is the absorbance of samples.

---

ABTS Assay[2]
ABTS scavenging assay was conducted as previously described with little modification. A mixture of 7.4 mM ABTS and 2.4 mM potassium persulfate at a 1:1 ratio was incubated overnight at room temperature to generate ABTS radical cation (ABTS•+). ABTS solution and 3″Me-EGCG (0–12.5 μM) were mixed at a 1:1 ratio in a 96-well plate. Ascorbic acid (50 μM) was used as a positive control. After 30 min of incubation at 37°C, the absorbance of each fraction was measured at 730 nm. ABTS scavenging effect was expressed as a percentage as follows:
ABTS scavenging effect (%) = [(A0 − A1)/A0] ∗ 100 (2)
in which A0 indicates the absorbance of ABTS, and A1 is the absorbance of samples.

---

ROS Generation[2]
The intracellular ROS level was determined by changes of fluorescence resulting from oxidation of the DHR123 fluorescent probe. Briefly, 1 × 106 RAW264.7 cells were incubated with 3″Me-EGCG for 30 min, and then SNP (0.25 mM) was added to induce ROS production. The cells were further incubated with 20 μM of the fluorescent probe DHR123 for 30 min at 37 °C, and cells were washed with PBS. The degree of fluorescence, which corresponds to the level of intracellular ROS, was determined using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA, USA) as reported previously
For in vitro cell-free assays, the antioxidant activity of (-)-Epigallocatechin-3-(3''-O-methyl) gallate can be measured using DPPH and ABTS radical scavenging assays. The compound is incubated with DPPH or ABTS radicals, and the reduction in absorbance is measured spectrophotometrically. IC50 values are calculated from dose-response curves. Its ability to scavenge intracellular ROS can be measured using fluorescent probes such as DCFH-DA in cell lysates or in cell-based assays. HO-1 expression can be measured by Western blot or ELISA.

Cell Viability Test[2]
The cytotoxicity of 3″Me-EGCG in RAW264.7 and HaCaT cells was evaluated as previously reported [6]. RAW264.7 cells (1 × 106 cells/mL) and HaCaT cells (4 × 106 cells/mL) were plated and cultured overnight, and 3″Me-EGCG (0–12.5 μM) was added for 24 h. Cell culture media (100 μL) were removed, and 10 μL MTT solution was added to each well. After 3 h of formazan formation, formazan dissolving solution was added. The absorbance at 570 nm was measured.

---

UVB Irradiation[2]
HaCaT cells were seeded at 7 × 105 cells per well in six-well plates and incubated for 24 h under starvation conditions using serum-free MEM. The media were changed to DMEM with 10% FBS and 1% penicillin-streptomycin, and cells were pre-treated with 3″Me-EGCG (0–12.5 μM) for 30 min. Cells were washed with DPBS to remove media, and 1 mL media was added to each well. Cells were irradiated with 30 mJ/cm2 UVB (UVB lamp, Bio-link crosslinker BLX-312; Vilber Lourmat, Collegien, France). Media was removed, and DMEM media with 3″Me-EGCG (0–12.5 μM) was added to cells; cells were incubated for 48 h.

---

Reactive oxygen species (ROS) are generated from diverse cellular processes or external sources such as chemicals, pollutants, or ultraviolet (UV) irradiation. Accumulation of radicals causes cell damage that can result in degenerative diseases. Antioxidants remove radicals by eliminating unpaired electrons from other molecules. In skin health, antioxidants are essential to protect cells from the environment and prevent skin aging. (-)-Epigallocatechin-3-(3″-O-methyl) gallate (3″Me-EGCG) has been found in limited oolong teas or green teas with distinctive methylated form, but its precise activities have not been fully elucidated. In this study, we examined the antioxidant roles of 3″Me-EGCG in keratinocytes (HaCaT cells). 3″Me-EGCG showed scavenging effects in cell and cell-free systems. Under H2O2 exposure, 3″Me-EGCG recovered cell viability and increased the expression of heme oxygenase 1 (HO-1). Under ultraviolet B (UVB) and sodium nitroprusside (SNP) exposure, 3″Me-EGCG protected keratinocytes and regulated the survival protein AKT1. By regulating the AKT1/NF-κB pathway, 3″Me-EGCG augmented cell survival and proliferation in HaCaT cells. These results indicate that 3″Me-EGCG exhibits antioxidant properties, resulting in cytoprotection against various external stimuli. In conclusion, our findings suggest that 3″Me-EGCG can be used as an ingredient of cosmetic products or health supplements[2].
For in vitro cellular assays, the cytoprotective and antioxidant activity of (-)-Epigallocatechin-3-(3''-O-methyl) gallate is assessed in various cell lines. RAW264.7 macrophage cells are treated with 12.5 µM of the compound for 30 minutes, and intracellular ROS production is measured using fluorescent probes. HaCaT keratinocyte cells are treated with 6.25 and 12 µM for 24-72 hours to assess cell proliferation using MTT or similar assays. HO-1 expression is measured by Western blot after 12 hours of treatment with 6.25 and 12 µM in cells treated with 250 µM H2O2. Cytotoxicity against rat cancer cells can be assessed using standard viability assays.

For in vivo studies, (-)-Epigallocatechin-3-(3''-O-methyl) gallate could be administered orally or intraperitoneally in rodent models of oxidative stress, inflammation, or cancer. In models of oxidative stress, endpoints include measurement of antioxidant enzyme levels, lipid peroxidation, and tissue damage. In models of inflammation, endpoints include inflammatory cytokine levels and histopathology. In cancer models, endpoints include tumor volume measurement and survival analysis.

(-)-Epigallocatechin-3-(3''-O-methyl) gallate (CAS 83104-87-4) has a molecular formula of C23H20O11 and a molecular weight of 472.4 g/mol. It is also known as (-)-EGCG-3''-O-ME. Appearance: typically a powder. Solubility: DMSO 50 mg/mL (105.84 mM). For in vivo formulation: 10% DMSO >> 40% PEG300 >> 5% Tween-80 >> 45% saline, solubility ≥ 1.25 mg/mL (2.65 mM). Storage: 4°C, protect from light; in solvent: -80°C for 6 months, -20°C for 1 month. Purity: ≥98%.

No detailed toxicity data is publicly available. As a natural product from tea leaves, it is generally considered to have low toxicity, but standard toxicological studies would be required for drug development. In vitro cytotoxicity assays in various cell lines are typically performed alongside efficacy studies to confirm that observed effects are not due to a general reduction in cell viability. Its strong cytotoxic activity against cancer cells suggests potential for therapeutic applications but also warrants careful toxicological evaluation.

[1]. Antioxidative activity of (-)-epigallocatechin-3-(3''-O-methyl)gallate isolated from fresh tea leaf and preliminary results on its biological activity. Biosci Biotechnol Biochem. 2000 Oct;64(10):2218-20.

[2]. Antioxidant and Cytoprotective Effects of (-)-Epigallocatechin-3-(3″-O-methyl) Gallate. Int J Mol Sci. 2019 Aug 16;20(16):3993.

(-)-Epigallocatechin 3-(3-methylgallate) is a catechin. Epigallocatechin 3-O-(3-O-methyl)gallate has been reported to exist in Limonium sinense, and relevant data are available for reference.
(-)-Epigallocatechin-3-(3''-O-methyl) gallate is a research-grade compound and is not approved for therapeutic use. It serves primarily as a pharmacological tool for studying oxidative stress, inflammation, and cancer. Its mechanism of action involves strong antioxidative activity through scavenging of DPPH and ABTS radicals and intracellular ROS. It exhibits cytoprotective effects by increasing HO-1 expression. It has strong cytotoxic activity against rat cancer cells. It negatively regulates basophil activation through suppression of FcepsilonRI expression. No clinical trials have been reported.

C₂₃H₂₀O₁₁

472.40

472.1

83104-87-4

9804842

Off-white to pink solid powder

1.77

856.1±65.0 °C at 760 mmHg

221-223 ºC(dec.)

298.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.796

2.03

7

11

5

34

694

2

COC1=CC(=CC(=C1O)O)C(=O)O[C@@H]2CC3=C(C=C(C=C3O[C@@H]2C4=CC(=C(C(=C4)O)O)O)O)O

WVRDOLPMKOCJRJ-DENIHFKCSA-N

InChI=1S/C23H20O11/c1-32-18-5-10(4-16(28)21(18)30)23(31)34-19-8-12-13(25)6-11(24)7-17(12)33-22(19)9-2-14(26)20(29)15(27)3-9/h2-7,19,22,24-30H,8H2,1H3/t19-,22-/m1/s1

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

83104-87-4; (-)-EGCG-3''-O-Me; Epigallocatechin 3-O-(3-O-methyl)gallate; (-)-Epigallocatechin-3-(3''-O-methyl) gallate; Egcg3''me; O97U9TPY8V; Epigallocatechin 3-O-(3-O-methylgallate); CHEMBL562716;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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