Toll-like receptor 4 (TLR4) / Myeloid differentiation primary response 88 (MyD88) signaling pathway, and Dynamin-related protein 1 (Drp1)-mediated mitochondrial fission. [1]

Schaftoside modulates the actions of lipoxygenase (LOX), cyclooxygenase 2 (COX-2), and apoptotic phospholipase A2 (sPLA2). Schaftoside also controls the expression of pro-inflammatory factors (IL-1β, TNF-α, and IL-6) both on the mRNA and the protein level [1].

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In BV2 microglial cells subjected to oxygen-glucose deprivation (OGD), treatment with Schaftoside (0.01, 0.1, 1 µM) for 4 hours dose-dependently suppressed the mRNA and protein expression of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), as measured by qRT-PCR, ELISA, and western blot analysis. The effect of Schaftoside (1 µM) was comparable to the TLR4 inhibitor TAK-242 (1 µM). [1]
Schaftoside treatment (0.01, 1 µM) also significantly reduced the OGD-induced increase in both protein and mRNA expression of TLR4 and its downstream adapter protein MyD88 in BV2 cells, as determined by western blot, qRT-PCR, and immunofluorescence. [1]
Furthermore, Schaftoside (0.01, 0.1, 1 µM) decreased the total protein expression of Drp1, its phosphorylation at Ser616 (p-Drp1), and the translocation of Drp1 from the cytosol to mitochondria in OGD-stimulated BV2 cells. Mitochondrial fission, assessed by Mito-Tracker staining and confocal microscopy, was significantly reduced by Schaftoside (1 µM) treatment, resulting in more elongated, rod-shaped mitochondria compared to the fragmented mitochondria observed in the OGD-only group. [1]

Oxygen-Glucose Deprivation (OGD) Model: BV2 microglial cells were cultured. To induce OGD, the culture medium was replaced with glucose-free DMEM containing Schaftoside (0.01, 0.1, or 1 µM) or TAK-242 (1 µM). The cells were then placed in a hypoxic incubator (94% N₂, 5% CO₂, 1% O₂) at 37°C for 4 hours. Control cells were incubated in normal complete medium under normoxic conditions (95% air, 5% CO₂) for the same duration. [1]
ELISA: After 4 hours of OGD, the culture media from BV2 cells were collected. The protein levels of IL-1β, IL-6, and TNF-α in the media were quantified using commercial ELISA kits according to the manufacturer's protocols. [1]
qRT-PCR: After 4 hours of OGD, total RNA was extracted from BV2 cells. mRNA expression levels of IL-1β, IL-6, TNF-α, TLR4, and MyD88 were measured by quantitative real-time PCR using specific primers. [1]
Immunofluorescence: Cells were fixed, permeabilized, and blocked. They were then incubated overnight at 4°C with primary antibodies against Drp1, TLR4, or MyD88. After washing, cells were incubated with fluorescently labeled secondary antibodies. Nuclei were stained with DAPI. Fluorescent images were captured using a confocal microscope. For mitochondrial localization of Drp1, cells were co-stained with Mito-Tracker Deep Red FM and an anti-Drp1 antibody followed by a fluorescent secondary antibody. [1]
Mitochondrial Fission Analysis: BV2 cells were incubated with Mito-Tracker Deep Red FM to label mitochondria. After fixation and staining, mitochondrial morphology was visualized by confocal microscopy. The degree of mitochondrial fission (fragmented vs. elongated structures) was assessed. [1]
Western Blot: BV2 cells were lysed, and protein concentration was determined. Equal amounts of protein were separated by SDS-PAGE, transferred to membranes, and probed with primary antibodies against IL-1β, IL-6, TNF-α, TLR4, MyD88, Drp1, phospho-Drp1 (Ser616), COX4 (mitochondrial loading control), and β-actin (total protein loading control). Signals were detected using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. For mitochondrial protein analysis, mitochondrial fractions were isolated from BV2 cells using a mitochondria isolation kit before western blot analysis for Drp1 and p-Drp1. [1]

[1]. Schaftoside ameliorates oxygen glucose deprivation-induced inflammation associated with the TLR4/Myd88/Drp1-related mitochondrial fission in BV2 microglia cells. J Pharmacol Sci. 2019 Jan;139(1):15-22.

5,7-Dihydroxy-2-(4-hydroxyphenyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl]-8-(3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)-4H-chromen-4-one belongs to the flavonoid class and is a C-glycoside compound. It has been reported that 5,7-dihydroxy-2-(4-hydroxyphenyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxecyclohexane-2-yl]-8-(3,4,5-trihydroxyoxecyclohexane-2-yl)chromen-4-one is found in Acanthus ebracteatus, Trigonella foenum-graecum, and other organisms with relevant data.

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Schaftoside is a natural flavonoid compound found in various Chinese herbal medicines. This study demonstrates that Schaftoside ameliorates OGD-induced inflammation in BV2 microglial cells by inhibiting the TLR4/MyD88 signaling pathway and subsequently suppressing Drp1-mediated mitochondrial fission. The proposed mechanism involves Schaftoside downregulating TLR4 and MyD88 expression, which leads to reduced Drp1 activation (phosphorylation at Ser616) and translocation to mitochondria, thereby preventing excessive mitochondrial fragmentation and ultimately attenuating the production of pro-inflammatory cytokines. [1]
The study suggests that regulation of mitochondrial fission in microglia could be a potential therapeutic target for reducing neuroinflammation after stroke. [1]

C26H28O14

564.49

596.137

51938-32-0

3550102

White to yellow solid powder

1.8±0.1 g/cm3

1028.2±65.0 °C at 760 mmHg

228ºC

342.7±27.8 °C

0.0±0.3 mmHg at 25°C

1.754

-3.06

10

14

4

40

938

0

MMDUKUSNQNWVET-UHFFFAOYSA-N

InChI=1S/C26H28O14/c27-6-13-18(32)21(35)23(37)26(40-13)15-19(33)14-10(29)5-12(8-1-3-9(28)4-2-8)39-24(14)16(20(15)34)25-22(36)17(31)11(30)7-38-25/h1-5,11,13,17-18,21-23,25-28,30-37H,6-7H2

5,7-dihydroxy-2-(4-hydroxyphenyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-8-(3,4,5-trihydroxyoxan-2-yl)chromen-4-one

Apigenin 6-C-glucoside-8-C-arabinoside Schaftoside

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 : ~125 mg/mL (~221.44 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.68 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 (3.68 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 (3.68 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.)