SIRT1 [1]
- Nrf2 [1]
- NF-κB [1]
- MAPK pathways (ERK1/2, p38, JNK) [1]

2'-O-Galloylhyperin (25, 50, 100 μM) did not show significant cytotoxicity to RAW 264.7 cells in the absence or presence of LPS (100 ng/mL) for 24 h [1].
- Pretreatment with 2'-O-Galloylhyperin attenuated LPS-induced release of pro-inflammatory molecules, including NO, TNF-α, and IL-6, in a concentration-dependent manner (25, 50, 100 μM) in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin blocked the LPS-induced increase of TLR4 protein expression in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin inhibited LPS-induced IκBα phosphorylation and degradation, and inhibited IKKα/β phosphorylation in a concentration-dependent manner in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin reversed LPS-induced increase in nuclear p65 level and blocked LPS-induced nuclear translocation of p65 in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin suppressed LPS-induced upregulation of NF-κB luciferase activity in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin significantly suppressed LPS-induced phosphorylation of ERK1/2, p38, and JNK in a concentration-dependent manner in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin reversed the LPS-induced decrease in SIRT1 protein and mRNA expression in RAW 264.7 cells [1].
- The inhibitory effects of 2'-O-Galloylhyperin on TNF-α, IL-6, and NO production were markedly abolished by the SIRT1 inhibitor Ex527 in LPS-stimulated RAW 264.7 cells [1].
- The suppressive effects of 2'-O-Galloylhyperin on IKK phosphorylation, IκBα phosphorylation, and IκBα degradation were reduced by EX527 in LPS-stimulated RAW 264.7 cells [1].
- Blockage of SIRT1 by EX527 significantly elevated LPS-induced p65 protein levels in the nuclear fractions in the presence of 2'-O-Galloylhyperin [1].
- 2'-O-Galloylhyperin inhibited LPS-induced intracellular accumulation of ROS in RAW 264.7 cells [1].
- The antioxidant NAC, similar to 2'-O-Galloylhyperin, inhibited LPS-induced secretion of NO [1].
- NAC significantly blocked LPS-induced SIRT1 down-regulation in RAW 264.7 cells [1].
- Treatment with 2'-O-Galloylhyperin showed a concentration-dependent increase in nuclear Nrf2 translocation in the presence or absence of LPS in RAW 264.7 cells [1].
- 2'-O-Galloylhyperin up-regulated HO-1 expression regardless of LPS presence in RAW 264.7 cells [1].
- The specific HO-1 inhibitor ZnPP significantly reversed the 2'-O-Galloylhyperin-mediated suppression of NO, IL-6, and TNF-α production in LPS-stimulated RAW 264.7 cells [1].
- 2'-O-Galloylhyperin-induced upregulation of HO-1 expression was significantly abolished by the SIRT1 inhibitor EX527 in the presence of LPS [1].

In a mouse model of LPS-induced septic shock, administration of 2'-O-Galloylhyperin (10 or 50 mg/kg, i.p.) 12 h and 1 h before LPS injection (30 mg/kg, i.p.) resulted in survival rates of 30% and 60% respectively, 72 h post-injection, compared to 10% survival in the LPS-only group [1].
- Treatment with 2'-O-Galloylhyperin (10 or 50 mg/kg, i.p.) significantly inhibited the LPS-induced increase of TNF-α and IL-6 in mouse serum [1].
- In LPS-challenged mice, 2'-O-Galloylhyperin treatment (10 or 50 mg/kg, i.p.) significantly reduced the activity of the liver damage markers ALT and AST in a dose-dependent manner (ALT: 44.2 ± 1.78 U/L for 10 mg/kg, 34.8 ± 3.29 U/L for 50 mg/kg vs. 54.4 ± 6.15 U/L for LPS control; AST: 96.9 ± 4.71 U/L for 10 mg/kg, 82.5 ± 3.85 U/L for 50 mg/kg vs. 117.4 ± 6.74 U/L for LPS control) [1].
- 2'-O-Galloylhyperin treatment suppressed LPS-induced hepatic cell necrosis, hemorrhage, and infiltration of inflammatory cells in liver tissue of mice [1].
- In livers of endotoxic shock mice, 2'-O-Galloylhyperin treatment significantly increased the expression of SIRT1, nuclear Nrf2, and the antioxidant enzymes HO-1, SOD (67.4 ± 1.54 U/mg protein for 10 mg/kg, 78.1 ± 1.17 U/mg protein for 50 mg/kg vs. 61.9 ± 1.29 U/mg protein for LPS control), and GSH-Px (79.8 ± 3.68 U/mg protein for 10 mg/kg, 91.3 ± 6.02 U/mg protein for 50 mg/kg vs. 61.4 ± 5.88 U/mg protein for LPS control) [1].
- 2'-O-Galloylhyperin treatment obviously inhibited NF-κB p65 protein expression in the liver of LPS-challenged mice [1].

Luciferase Reporter Assay for NF-κB Activity: RAW 264.7 cells were co-transfected with a pNF-κB-Luc plasmid and a pRL-TK vector. After 24 hours of transfection, cells were pretreated with 2'-O-Galloylhyperin for 2 hours and then stimulated with LPS (100 ng/mL) for an additional 12 hours. Cells were then lysed, and luciferase activities were measured using a dual-luciferase reporter assay kit. The results showed that 2'-O-Galloylhyperin suppressed LPS-induced upregulation of NF-κB luciferase activity [1].

Cell Viability Assay: RAW 264.7 cells were treated with different concentrations of 2'-O-Galloylhyperin for 24 hours, with or without LPS (100 ng/mL). Cell viability was measured using the MTT assay. 2'-O-Galloylhyperin did not show significant cytotoxicity [1].
- Nitric Oxide (NO) Measurement: RAW 264.7 cells were pretreated with 2'-O-Galloylhyperin for 2 hours and then stimulated with LPS (100 ng/mL) for 24 hours. Nitric oxide production was measured by detecting its stable oxidative metabolite, nitrite. 100 μL of culture media was mixed with 50 μL of Griess reagents I and II for 10 minutes at room temperature. Absorbance was measured at 540 nm. The amount of nitrite was evaluated based on a sodium nitrite standard curve. 2'-O-Galloylhyperin attenuated LPS-induced NO release [1].
- Cytokine Measurement: RAW 264.7 cells were pretreated with 2'-O-Galloylhyperin for 2 hours and then stimulated with LPS (100 ng/mL) for 24 hours. The concentrations of TNF-α and IL-6 in the culture medium were measured using corresponding ELISA kits. 2'-O-Galloylhyperin attenuated LPS-induced release of TNF-α and IL-6 [1].
- Western Blotting: RAW 264.7 cells were pretreated with 2'-O-Galloylhyperin for 2 hours and then stimulated with LPS (100 ng/mL) for various times (15 min, 1 h, 18 h, 24 h). For nuclear and cytoplasmic extraction, cells were lysed using a kit, and protein concentration was determined. Equal amounts of total or nuclear protein were separated by SDS-PAGE, electroblotted to PVDF membranes, blocked with BSA, and incubated with primary antibodies (1:500) followed by secondary antibodies (1:1000). Protein abundance was densitometrically determined. This method was used to assess iNOS, TNF-α, IL-6, TLR4, p-IκBα, IκBα, p-IKKα/β, p65, MAPKs (p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK), SIRT1, Nrf2, and HO-1 [1].
- Immunofluorescence: RAW 264.7 cells cultured on coverslips were pretreated with 2'-O-Galloylhyperin for 2 hours prior to LPS (100 ng/mL) stimulation. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, and blocked. They were then incubated with a primary NF-κB p65 antibody overnight at 4°C, followed by an Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained with DAPI. Images were captured using fluorescence microscopy. This showed that 2'-O-Galloylhyperin blocked LPS-induced nuclear translocation of p65 [1].
- ROS Detection: RAW 264.7 cells were treated with 2'-O-Galloylhyperin (25 μM) in the presence or absence of NAC for 1 hour, followed by stimulation with LPS (100 ng/mL) for 24 hours. Intracellular ROS accumulation was monitored using the fluorescent probe DCFH-DA. Cells were collected, washed with PBS, and incubated with 10 μM DCFH-DA for 30 minutes at 37°C in the dark. Fluorescence was measured by flow cytometry and fluorescence microscopy. 2'-O-Galloylhyperin inhibited LPS-induced intracellular ROS accumulation [1].
- Reverse Transcriptase Polymerase Chain Reaction (RT-PCR): Total RNA was isolated from RAW 264.7 cells using Trizol reagent. 1.0 μg of total RNA was converted to cDNA. The cDNA was amplified using specific primers for SIRT1 and Actin. The PCR products were electrophoresed on an agarose gel. This showed that 2'-O-Galloylhyperin reversed the LPS-induced decrease in SIRT1 mRNA expression [1].

Mouse Septic Shock Model and Survival Study: Male ICR mice (25-30 g) were used. For the septic mortality experiment, mice were intraperitoneally (i.p.) injected with 2'-O-Galloylhyperin (10 or 50 mg/kg) or vehicle 12 hours and 1 hour before LPS injection (30 mg/kg, i.p.). Mice were monitored every 12 hours for 72 hours. Survival rates were recorded [1].
- Sample Collection and Analysis: Serum and liver tissues were collected 12 hours after LPS injection (30 mg/kg, i.p.). Mice were pre-treated with 2'-O-Galloylhyperin (10 or 50 mg/kg, i.p.) 12 hours and 1 hour before LPS challenge [1].
- Histopathology: Harvested livers from mice (treated as described above) were fixed in paraformaldehyde, dehydrated with ethanol, embedded in paraffin, and cut into 4 μm sections. The sections were stained with H&E. Pathological changes were evaluated under a light microscope [1].
- Biochemistry Analysis: Levels of TNF-α and IL-6 in mouse serum (from the LPS-challenged model) were measured using assay kits. The biochemical parameters AST and ALT in serum, and SOD and GSH in liver tissues, were measured with enzymatic assays [1].

2'-O-Galloylhyperin (25, 50, 100 μM) did not have significant cytotoxicity to RAW 264.7 cells in the absence or presence of LPS (100 ng/mL) as measured by MTT assay [1].
- In vivo, treatment with 2'-O-Galloylhyperin (10 or 50 mg/kg, i.p.) significantly reduced LPS-induced liver damage markers (ALT and AST) and suppressed hepatic necrosis and hemorrhage, indicating a protective, not toxic, effect in this model [1].

[1]. 2'-O-Galloylhyperin Isolated From Pyrola incarnata Fisch. Attenuates LPS-Induced Inflammatory Response by Activation of SIRT1/Nrf2 and Inhibition of the NF-κB Pathways in Vitro and Vivo. Front Pharmacol. 2018 Jun 27;9:679.

[2]. Hepatoprotective effect of 2'-O-galloylhyperin against oxidative stress-induced liver damage through induction of Nrf2/ARE-mediated antioxidant pathway. Food Chem Toxicol. 2017 Apr;102:129-142.

2''-Galloylkaolin belongs to the flavonoid class and is a glycoside. It has been reported that 2'-O-galloylkaolin exists in Euphorbia crescentis, Euphorbia esula, and other organisms with relevant data.

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2'-O-Galloylhyperin is a major compound isolated from Pyrola incarnata Fisch. [1].
- The plant Pyrola incarnata Fisch. has been reported for hypertension, cardiovascular disease, gastric, and pulmonary hemorrhage, but its anti-inflammatory effect was unclear prior to this study [1].
- This study was the first to demonstrate the anti-inflammatory activities of 2'-O-Galloylhyperin both in vitro and in vivo [1].
- The anti-inflammatory effect of 2'-O-Galloylhyperin is mediated by activation of the SIRT1/Nrf2 pathway and inhibition of the NF-κB pathway [1].
- The findings suggest that 2'-O-Galloylhyperin could potentially be a novel functional food candidate or a potential therapeutic agent for the treatment of sepsis and other inflammatory diseases [1].

C28H24O16

616.48

616.106

53209-27-1

6453359

Light yellow to yellow solid powder

1.9±0.1 g/cm3

1106.9±65.0 °C at 760 mmHg

365.6±27.8 °C

0.0±0.3 mmHg at 25°C

1.838

3.61

10

16

7

44

1070

5

C1=CC(=C(C=C1C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O[C@H]4[C@@H]([C@H]([C@H]([C@H](O4)CO)O)O)OC(=O)C5=CC(=C(C(=C5)O)O)O)O)O

PXGWEUQZDRUMRE-UNZYZCBSSA-N

InChI=1S/C28H24O16/c29-8-18-21(37)23(39)26(43-27(40)10-4-15(34)20(36)16(35)5-10)28(42-18)44-25-22(38)19-14(33)6-11(30)7-17(19)41-24(25)9-1-2-12(31)13(32)3-9/h1-7,18,21,23,26,28-37,39H,8H2/t18-,21+,23+,26-,28+/m1/s1

(2S,3R,4S,5R,6R)-2-((2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl)oxy)-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl 3,4,5-trihydroxybenzoate

FT-0689359 FT 0689359FT0689359 2''-GalloylhyperinQ 100605Q-100605 Q100605

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 (~162.21 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (1.35 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (1.35 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 8.3 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: ≥ 0.83 mg/mL (1.35 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 8.3 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.)