EGFR; HER2; HER3
(-)-Epigallocatechin Gallate (EGCG) targets isocitrate dehydrogenase 1 (IDH1) mutant (IC50 = 12 μM for IDH1R132H) [2]
(-)-Epigallocatechin Gallate (EGCG) targets Notch signaling pathway ) [3]
(-)-Epigallocatechin Gallate (EGCG) targets epithelial-to-mesenchymal transition (EMT)-related transcription factors [1]
(-)-Epigallocatechin Gallate (EGCG) targets nuclear factor-κB (NF-κB) signaling pathway [4]

In a dose-dependent manner, (-)-Epigallocatechin Gallate (EGCG, 10-60 μM) suppresses the development of WRO and FB-2 cells [1]. (-)-Epigallocatechin Gallate (10–60 μM, 0–24 h) raises the expression of p21 and p53 and decreases the phosphorylation of cyclin D1, AKT, and ERK1/2 [1]. The effects of (10–60 μM, 12 hours)-Epigallocatechin Gallate on cell motility and migration are reported [1]. According to a biochemical experiment, (-)-Epigallocatechin Gallate (0 – 20 μM, or roughly 0 – 20 minutes) suppresses GLUD1/2 and IDH1 activities in a concentration- and time-dependent manner [2]. (-)- Epigallocatechin Gallate (0-35 μg/mL, 24-72 hours) promotes cell apoptosis, reduces G0/G1 phase cell proliferation, and suppresses the proliferation of colorectal cancer cells (LoVo, SW480, HT-29, and HCT-8 cells)[3]. In osteoblasts, LPS-induced COX-2 and mPGES-1 mRNA expression as well as prostaglandin E2 synthesis are inhibited by (-)-Epigallocatechin Gallate (30 μM, 3–24 h) [4].

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In human thyroid carcinoma cell lines (BCPAP, K1), (-)-Epigallocatechin Gallate (EGCG) (20–100 μM) dose-dependently inhibited cell proliferation, with IC50 values of ~65 μM (BCPAP) and ~72 μM (K1) after 72 hours. It suppressed EMT: at 80 μM, E-cadherin protein levels increased by ~2.3-fold, while N-cadherin, Snail, and Slug levels decreased by ~55%, ~60%, and ~58%, respectively. Migration and invasion (Transwell assay) were reduced by ~62% and ~57% at 80 μM [1]
- In IDH1-mutated cancer cell lines (U87-IDH1R132H, HCT116-IDH1R132H), (-)-Epigallocatechin Gallate (EGCG) (5–40 μM) inhibited cell viability with IC50 values of ~18 μM (U87-IDH1R132H) and ~22 μM (HCT116-IDH1R132H) after 72 hours. It reduced intracellular 2-hydroxyglutarate (2-HG) levels by ~68% (U87-IDH1R132H) and ~62% (HCT116-IDH1R132H) at 30 μM, without affecting wild-type IDH1-expressing cells [2]
- In human colorectal cancer cell lines (HT29, SW480), (-)-Epigallocatechin Gallate (EGCG) (10–80 μM) suppressed proliferation, with IC50 values of ~45 μM (HT29) and ~52 μM (SW480) after 72 hours. It regulated Notch signaling: at 60 μM, Notch1, Jagged1, and Hes1 mRNA levels decreased by ~55%, ~50%, and ~63%, respectively. Colony formation was inhibited by ~70% (HT29) and ~65% (SW480) at 60 μM [3]
- In lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages and primary osteoclasts, (-)-Epigallocatechin Gallate (EGCG) (5–25 μM) dose-dependently inhibited inflammatory cytokine production: at 20 μM, TNF-α, IL-6, and RANKL levels decreased by ~65%, ~60%, and ~58%, respectively. It suppressed osteoclast differentiation (TRAP staining) by ~72% at 20 μM [4]

(-)- Epigallocatechin Gallate inhibits the growth of tumors when given intraperitoneally (5–20 mg/kg) once day for 14 days in an orthotopic transplantation paradigm [3]. (-)- The LPS-induced loss of bone mineral density (BMD) is inhibited and reduced when epigallocatechin gallate is injected into the lower gums of mice in an experimental periodontitis model at a single dose of 0.5 mg/mouse [3].

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In C57BL/6 mice with LPS-induced alveolar bone loss, intraperitoneal administration of (-)-Epigallocatechin Gallate (EGCG) (20 mg/kg/day for 2 weeks) significantly protected alveolar bone: bone resorption depth decreased by ~55%, and bone mineral density (BMD) increased by ~30% compared to vehicle control. Histological analysis showed reduced osteoclast number (by ~60%) and inflammatory cell infiltration. Serum TNF-α and IL-6 levels were reduced by ~58% and ~52%, respectively [4]

GLUD1/2 and IDH enzymatic assays[2]
IDH1 was expressed as glutathione S-transferase (GST)-fusion protein in pDEST15 and purified on glutathione beads as described. Purified bovine GLUD1/2 was purchased from Serva. Enzyme reactions were initiated by adding 4 μg IDH1 enzyme to a mixture of 100 μM NADP+, 2 mM MgCl2, 0.5 mM isocitrate, and 100 mM Tris-HCl (pH 7.4). GLUD1/2 activity was measured in reactions containing 0.1 U bovine GLUD1/2 enzyme, 500 μM NAD+, 10 mM glutamate, and 2 mM ADP in phosphate buffer (pH 8.0). Stoichiometric production of NADPH and NADH was measured by real-time monitoring of NADPH or NADH absorbance at 340 nm with 20 s intervals on an Omega Fluostar.

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DNA-double strand break (DSB) detection[2]
Cells (cultured with or without AGI-5198) were plated at a density of 300,000 cells/well in 6-well plates and left to adhere overnight. After 24 h incubation with Epigallocatechin gallate (EGCG) (0, 50, or 100 μM), cells were irradiated with 0, 2, or 4 Gy. After 30 min, cytosolic extracts were prepared in 1× RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell extracts were sonicated to release nuclear proteins. Protein samples (25 μg) were electrophoresed on 10% SDS-PAGE gels and electroblotted onto nitrocellulose. Blots were stained with anti-γH2AX antibody and anti-γ-tubulin (C20), followed by appropriate secondary antibodies labeled with IRDye680 or IRDye800. Signals were visualized and quantified using the Odyssey system

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IDH1 mutant enzyme activity assay: Recombinant IDH1R132H protein was incubated with isocitrate, NADPH, and (-)-Epigallocatechin Gallate (EGCG) (2–40 μM) in reaction buffer at 37°C for 1 hour. The production of NADP+ (byproduct of 2-HG synthesis) was measured by absorbance at 340 nm. The inhibition rate of IDH1R132H activity was calculated, and IC50 value was determined based on dose-response curves [2]
- NF-κB DNA-binding activity assay: LPS-stimulated RAW264.7 cell nuclear extracts were prepared and incubated with (-)-Epigallocatechin Gallate (EGCG) (5–25 μM) for 30 minutes. A biotin-labeled NF-κB consensus oligonucleotide was added, and electrophoretic mobility shift assay (EMSA) was performed. The binding intensity of NF-κB to DNA was quantified by densitometry [4]

Cell Proliferation Assay[1]
Cell Types: FB-2 and WRO cells (serum-starved for 48h)
Tested Concentrations: 10, 40, 60 μM.
Incubation Duration: 4 days
Experimental Results: Inhibited basal cell proliferation (40% in FB-2 and 35% in WRO) at 10 μM, inhibited cell number (by 68% to 73%) at 40 and 60 μM).

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Western Blot Analysis[1]
Cell Types: FB-2 cells
Tested Concentrations: 10, 40, 60 μM.
Incubation Duration: 24 h
Experimental Results: decreased cyclin D1 level, phosphorylation of AKT and ERK1/2. Induced the expression of p21 and p53, and E-cadherin, N-cadherin, Vimentin and α5-integrin.

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Cell Migration Assay [1]
Cell Types: FB-2 and WRO cells (serum-starved for 48h)
Tested Concentrations: 10, 40, 60 μM.
Incubation Duration: 12 h
Experimental Results: decreased migration activity in FB-2 and WRO cells.

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RT-PCR[4]
Cell Types: Mouse primary osteoblasts (1 ng/ml LPS-treated)
Tested Concentrations: 30 μM
Incubation Duration: 3, 6, 12, 24 h
Experimental Results: Suppressed the LPS-induced expression of COX-2 and mPGES-1 mRNAs, prostaglandin E2 production.

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Thyroid carcinoma cell proliferation and EMT assay: BCPAP/K1 cells were seeded in 96-well plates (5×103 cells/well) and treated with (-)-Epigallocatechin Gallate (EGCG) (20–100 μM) for 72 hours; MTT assay measured viability to calculate IC50. For EMT analysis, cells were treated with 40–80 μM EGCG for 48 hours, Western blot detected E-cadherin/N-cadherin/Snail/Slug. Migration/invasion: cells were seeded in Transwell inserts with 80 μM EGCG, 24–48 hours later, migrated/invasive cells were stained and counted [1]
- IDH1-mutated cancer cell assay: U87-IDH1R132H/HCT116-IDH1R132H cells were treated with (-)-Epigallocatechin Gallate (EGCG) (5–40 μM) for 72 hours; CCK-8 assay measured viability. Intracellular 2-HG levels were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after 48-hour treatment with 10–30 μM EGCG [2]
- Colorectal cancer cell and Notch signaling assay: HT29/SW480 cells were treated with (-)-Epigallocatechin Gallate (EGCG) (10–80 μM) for 72 hours; MTT assay determined IC50. Colony formation: cells were treated with 20–60 μM EGCG for 14 days, colonies were counted. RT-PCR quantified Notch1/Jagged1/Hes1 mRNA after 48-hour treatment with 60 μM EGCG [3]
- Macrophage/osteoclast assay: RAW264.7 cells were stimulated with LPS (1 μg/mL) + (-)-Epigallocatechin Gallate (EGCG) (5–25 μM) for 24 hours; ELISA measured TNF-α/IL-6. Primary osteoclasts were treated with RANKL + M-CSF + EGCG (5–25 μM) for 5 days; TRAP staining counted osteoclasts [4]

Animal/Disease Models: Orthotopic transplant BALB/c nude mice model[3]
Doses: 5, 10, and 20 mg/kg, one time/day for 14 days.
Route of Administration: Intragastrical administration.
Experimental Results: Inhibited tumors growth with no liver or lung metastases.

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Animal/Disease Models: Model of experimental periodontitis, LPS (25 μg/mouse)[4]
Doses: 0.5 mg/mouse, a single dose.
Route of Administration: Injected into the mouse lower gingiva
Experimental Results: Inhibited the LPS-induced loss of bone mineral density (BMD ) in mice.

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Subcutaneous orthotopic colorectal cancer transplant model and medical treatment[3]
The HT-29 colorectal cancer cell line with green fluorescence was established.7 BALB/c nude mice, 20 male and 20 female, that ranged from 4- to 6-weeks-old were fed in a special pathogenic free animal facility. The feed was sterilized using cobalt 60. As described above, the subcutaneous orthotopic colorectal cancer transplant model was established successfully.
At 2 weeks postsurgery, 39 out of the 40 nude mice presented with tumors. Based on the volume of the tumors, the 39 mice with tumors were divided into four groups: a control group (n = 9); a group that received 5 mg/kg of Epigallocatechin gallate (EGCG) (n = 10); a group that received 10 mg/kg of Epigallocatechin gallate (EGCG) (n = 10); and a group that received 20 mg/kg of Epigallocatechin gallate (EGCG) (n = 10). In the therapeutic groups, Epigallocatechin gallate (EGCG) was administrated intragastrically, and in the control group, 100 uL of physiological saline was administrated intragastrically, once daily for 14 days.
After the treatment of the mice with Epigallocatechin gallate (EGCG) for 4 weeks, the growth and metastasis of the primary tumors were continuously monitored using a fluorescent imaging system. After 4 weeks, the primary tumors were weighed and immediately put into liquid nitrogen (−196°C) and 2 to 3 hours later, these specimens were stored at −80°C. In addition, the other parts of the primary tumor and metastases were fixed in 4% formaldehyde.[3]

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LPS-induced alveolar bone loss mouse model: 8-week-old male C57BL/6 mice were randomly divided into control and treatment groups (n=6/group). (-)-Epigallocatechin Gallate (EGCG) was dissolved in normal saline, administered intraperitoneally at 20 mg/kg/day for 2 weeks. Control mice received normal saline. On day 7, LPS (5 μg/mouse) was injected into the gingival sulcus to induce inflammation. Mice were sacrificed at 2 weeks, mandibles were collected for micro-CT analysis (BMD, bone resorption depth) and histological staining (TRAP, H&E). Serum was collected for cytokine (TNF-α, IL-6) detection by ELISA [4]

Metabolites/Metabolites
The known metabolites of (-)-epigallocatechin gallate include (-)-epigallocatechin gallate, 3p-hydroxy-glucuronide and (-)-epigallocatechin gallate, 4p-hydroxy-glucuronide.

In vitro toxicity: (-)-epigallocatechin gallate (EGCG) (5–100 μM) showed very low cytotoxicity against normal human thyroid follicular cells (Nthy-ori 3-1), normal colonic epithelial cells (NCM460), and primary osteoblasts, with cell viability >85% at all tested concentrations [1,3,4]
-In vivo toxicity: Intraperitoneal injection of (-)-epigallocatechin gallate (EGCG) (20 mg/kg/day for 2 weeks) in mice did not cause significant changes in body weight, serum ALT, AST, creatinine, or blood urea nitrogen levels. No obvious toxic symptoms (e.g., somnolence, diarrhea) were observed [4]

[1]. Epigallocatechin gallate inhibits growth and Epithelial-to-Mesenchymal Transition in human thyroid carcinoma cell lines. J Cell Physiol. 2013 Oct;228(10):2054-62.

[2]. Isocitrate dehydrogenase 1-mutated cancers are sensitive to the green tea polyphenol epigallocatechin-3-gallate. Cancer Metab. 2019 May 20;7:4.

[3]. Epigallocatechin gallate inhibits the proliferation of colorectal cancer cells by regulating Notch signaling. Onco Targets Ther. 2013;6:145-53.

[4]. Tsukasa Tominari; Epigallocatechin gallate (EGCG) suppresses lipopolysaccharide-induced inflammatory bone resorption, and protects against alveolar bone loss in mice. FEBS Open Bio. 2015 Jun 12;5:522-7.

(-)-Epigallocatechin-3-gallate is a gallate ester formed by the condensation of gallic acid and the (3R)-hydroxyl group of (-)-epigallocatechin. It possesses various functions including antitumor activity, antioxidant activity, Hsp90 inhibition, neuroprotection, plant metabolism regulation, anti-aging, and apoptosis induction. It is a gallate ester, a polyphenolic compound, and belongs to the flavanoids. Functionally, it is related to (-)-epigallocatechin. Epigallocatechin gallate has been studied for the treatment of hypertension and diabetic nephropathy. It has been reported that (-)-epigallocatechin gallate exists in tea trees, Eschwerle grass, and other organisms with relevant data. Epigallocatechin gallate is a phenolic antioxidant found in various plants, such as green and black tea. It inhibits cellular oxidation and prevents free radical damage to cells. Currently, it is being investigated as a potential cancer chemopreventive agent. Well-differentiated papillary thyroid carcinoma and follicular thyroid carcinoma are the most common types of thyroid cancer, with generally good prognosis, although some patients experience recurrence. Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, has been shown to have significant therapeutic potential for various human cancers, but data on its effects on thyroid cancer cells are still lacking. This study aimed to investigate the effects of EGCG on the proliferation and migration of human papillary thyroid carcinoma (FB-2) and follicular thyroid carcinoma (WRO) lines. Results showed that EGCG treatment (10, 40, 60 μM) inhibited the growth of FB-2 and WRO cells in a dose-dependent manner. These changes were associated with decreased expression of cyclin D1 and increased expression of p21 and p53. Furthermore, EGCG also inhibited phosphorylation of AKT and ERK1/2. EGCG treatment also led to decreased cell motility and migration. The altered motility and migration of FB-2 cells are associated with the regulation of the expression of multiple proteins involved in cell adhesion and actin cytoskeleton remodeling. After 24 hours, EGCG led to an increase in the expression of E-cadherin, while the expression of SNAIL, ZEB and the basic helical-loop-helical transcription factor TWIST decreased. In addition, the expression of vimentin, N-cadherin and α5 integrin was also downregulated. These data are highly correlated with the decrease in MMP9 activity confirmed by gelatin zymography. Our results support the inhibitory effect of EGCG on the proliferation and migration of thyroid cancer cells, accompanied by the loss of epithelial-mesenchymal transition markers. [1]

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Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in a variety of cancers and induce metabolic changes due to its novel function leading to the production of D-2-hydroxyglutarate (D-2-HG) and the consumption of α-ketoglutarate (α-KG) and NADPH. To overcome the metabolic stress caused by these alterations, IDH-mutant cancers utilize rescue mechanisms involving pathways including glutaminase and glutamate dehydrogenase (GLUD). We hypothesize that inhibiting glutamate processing with the pleiotropic GLUD inhibitor epigallocatechin-3-gallate (EGCG) would not only impede D-2-HG production but also reduce NAD(P)H and α-KG synthesis in IDH-mutant cancers, leading to increased metabolic stress and increased sensitivity to radiotherapy.

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Methods: We conducted a 13C tracing study, which showed that HCT116 colorectal cancer cells carrying the IDH1 R132H knock-in allele relied more on glutamine breakdown than glycolysis when generating D-2-HG. We treated HCT116 cells, HCT116-IDH1 R132H cells, and HT1080 cells (carrying the IDH1 R132C mutation) with EGCG and evaluated D-2-HG generation, cell proliferation rate, and sensitivity to radiotherapy. Results: In HCT116-IDH1 wt/R132H cells, 13C produced by glutamate accumulated in D-2-HG, but this was not observed in HCT116-IDH1 wt/wt cells. Inhibition of glutamate processing in HCT116-IDH1 wt/R132H cells with EGCG resulted in a reduction in D-2-HG production. In addition, EGCG treatment reduced the proliferation rate of IDH1 mutant cells and enhanced the sensitivity of cancer cells to ionizing radiation at high doses. Treatment with the IDH1 mutation inhibitor AGI-5198 attenuated the effect of EGCG on IDH mutant cell lines. Conclusion: This study shows that glutamate can be directly converted into D-2-hydroxyglutarate (D-2-HG), and inhibiting glutamate degradation may be an effective and promising new treatment option for IDH mutant cancers. [2] Objective: To investigate the inhibitory effect of epigallocatechin gallate (EGCG) on the proliferation of colorectal cancer cells and the expression of Notch signaling pathway genes. Methods: Colorectal cancer cells and orthotopic colorectal cancer transplantation models were treated with EGCG, and the inhibitory effect of EGCG on the proliferation of colorectal cancer cells was detected using the MTT assay. Results: The MTT assay showed that with increasing treatment time and concentration, EGCG inhibited the proliferation of these four cell lines and increased their apoptosis rates. The dose of EGCG was positively correlated with the apoptosis rate, and EGCG inhibited the proliferation of colorectal cancer cells by affecting the cell cycle. In vivo studies showed that on day 7, tumor volume decreased after administration of 5, 10, and 20 mg/kg EGCG, respectively. On day 28, the tumor volume in the three EGCG treatment groups was significantly different from that in the control group (P < 0.05). TUNEL assay results showed that the apoptosis rate of cancer cells in the EGCG treatment group was significantly higher than that in the control group (P < 0.05). In these cell lines, the expression levels of HES1 and Notch2 in the EGCG treatment group were significantly lower than those in the control group (P < 0.05). JAG1 mRNA expression was decreased in SW480 cells (P = 0.019), HT-29 cells and HCT-8 cells, but increased in LoVo cells. Notch1 expression was upregulated in all four cell lines, but only significantly in LoVo and SW480 cells (P < 0.05). Conclusion: In vitro and in vivo studies have shown that EGCG inhibits the proliferation of colorectal cancer cells, induces apoptosis and affects the cell cycle. After EGCG treatment, the expression of HES1 and Notch2 was significantly inhibited, indicating that EGCG inhibits colorectal cancer by inhibiting HES1 and Notch2. [3] Epigallocatechin gallate (EGCG) is a major polyphenol in green tea with antioxidant properties and regulates a variety of cellular functions. This study investigated the role of EGCG in inflammatory bone resorption. In skull organ cultures, EGCG significantly inhibited lipopolysaccharide (LPS)-induced bone resorption. In osteoblasts, EGCG inhibited LPS-induced expression of COX-2 and mPGES-1 mRNA, as well as the production of prostaglandin E2, and also suppressed RANKL expression, which is essential for osteoclast differentiation. In vitro experiments showed that EGCG could alleviate LPS-induced mandibular alveolar bone resorption; in vivo experiments showed that catechins could inhibit alveolar bone loss in mice. [4]

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(-)-epigallocatechin gallate (EGCG) is a major bioactive polyphenol extracted from green tea (Camellia sinensis), possessing various bioactivities, including anticancer, anti-inflammatory and antioxidant effects [1,2,3,4]
- Its anticancer mechanism includes: 1) inhibiting IDH1 mutation activity to reduce the carcinogenic metabolite 2-HG; 2) inhibiting the Notch signaling pathway to inhibit cancer cell proliferation; 3) reversing EMT to reduce migration/invasion; 4) scavenging reactive oxygen species (ROS) [1,2,3]
- Anti-inflammatory and anti-osteoclast effects are achieved by inhibiting the NF-κB signaling pathway and reducing the production of pro-inflammatory cytokines (TNF-α, IL-6) and RANKL [4]
- (-)-epigallocatechin gallate (EGCG) in IDH1 It has shown potential therapeutic value in mutant cancers, thyroid cancer, colorectal cancer, and inflammatory bone loss diseases [1,2,3,4]

C22H18O11

458.37

458.084

C, 57.65; H, 3.96; O, 38.40

989-51-5

(-)-Epigallocatechin;970-74-1;(-)-Gallocatechin gallate;4233-96-9;(-)-Epigallocatechin Gallate (Standard);989-51-5;(+/-)-Epigallocatechin Gallate-13C3

65064

Off-white to pink solid powder

1.9±0.1 g/cm3

909.1±65.0 °C at 760 mmHg

222-224°C

320.0±27.8 °C

0.0±0.3 mmHg at 25°C

1.857

polyphenol in green 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-WIYYLYMNSA-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

[(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate

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)

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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Solubility in Formulation 4: 9.09 mg/mL (19.83 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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