Inhibition of LPS-induced phosphorylation of IκB-α, p38 MAPK, and ERK MAPK in microglial cells, without affecting JNK phosphorylation [1]

Diosgenin glucoside (1, 5, 10 µM) alone had no cytotoxic effect on the viability of rat primary microglia and BV-2 microglial cells after 24-hour incubation in serum-free DMEM [1]
Diosgenin glucoside (1, 5, 10 µM) inhibited the activity-induced cell death (AICD) of rat primary microglia and BV-2 cells incubated with LPS [1]
Diosgenin glucoside (1, 5, 10 µM) suppressed the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) (M1 markers) in LPS-activated rat primary microglia and BV-2 cells in a dose-dependent manner [1]
Diosgenin glucoside (10 µM) suppressed the LPS-induced mRNA expression of inducible nitric oxide synthase (iNOS), TNF-α, IL-6, interleukin-1β (IL-1β), interleukin-17 (IL-17), and interleukin-23 (IL-23) in BV-2 cells [1]
Diosgenin glucoside did not affect the production of interleukin-10 (IL-10) and interleukin-1 receptor antagonist (IL-1Ra) (M2 markers) in LPS-activated primary microglia and BV-2 cells, nor did it reverse the LPS-induced suppression of CD206 protein expression [1]
Diosgenin glucoside had no effect on the LPS-induced mRNA expression of IL-1Ra, IL-10, granulocyte-colony stimulating factor (G-CSF), suppressor of cytokine signaling 3 (SOCS3), CD206, resistin-like α (Fizz1), and chitinase 3-like 3 (Ym1) in BV-2 cells. It inhibited LPS-induced cyclooxygenase-2 (COX-2) mRNA expression but did not affect cyclooxygenase-1 (COX-1) and arginase-1 (Arg-1) mRNA levels [1]
Diosgenin glucoside had no effect on the IL-4-induced mRNA expression of IL-10, CD206, Arg-1, and Ym1 in BV-2 cells [1]
Diosgenin glucoside (10 µM) markedly inhibited LPS-induced phosphorylation of IκB-α, p38 MAPK, and ERK MAPK, but not JNK phosphorylation, in BV-2 cells [1]
Conditioned medium from BV-2 cells treated with LPS and Diosgenin glucoside (10 µM) was significantly less toxic to Neuro-2a neuronal cells compared to conditioned medium from cells treated with LPS alone, indicating indirect neuroprotection via modulation of microglial secretions [1]

For cell viability testing (CCK-8 assay), primary microglia and BV-2 cells were incubated with Diosgenin glucoside (1, 5, or 10 µM) for 24 hours in serum-free Dulbecco's Modified Eagle Medium (DMEM). A 10% CCK-8 solution was then added, and after 0.5 hours of incubation, optical absorbance at 450 nm was measured with a plate reader [1]
To assess activity-induced cell death (AICD), cells were pre-treated with Diosgenin glucoside (1, 5, or 10 µM) for 30 minutes, then stimulated with lipopolysaccharide (LPS) (0.1 µg/mL for BV-2 cells, 1 µg/mL for primary microglia) for 24 hours in serum-free DMEM, followed by the CCK-8 assay [1]
For nitric oxide (NO) and cytokine level determination, cells pre-treated with Diosgenin glucoside (1, 5, or 10 µM) for 30 minutes were stimulated with LPS for 24 hours. Supernatant nitrite levels were measured using Griess reagents, and absorbance was read at 550 nm. Levels of IL-6 and TNF-α were measured using enzyme-linked immunosorbent assay (ELISA) kits [1]
For quantitative real-time polymerase chain reaction (qRT-PCR), BV-2 cells were treated with LPS (0.1 µg/mL) and/or Diosgenin glucoside for 8 hours, or with interleukin-4 (IL-4, 40 ng/mL) and/or Diosgenin glucoside for 8 hours. Total RNA was extracted, reverse transcribed into cDNA, and mRNA levels were quantified using specific primers and SYBR Green PCR Master Mix [1]
For ELISA measurement of cytokines, cells were pre-treated with Diosgenin glucoside for 30 minutes, then stimulated with LPS for 24 hours. Supernatant concentrations of TNF-α, IL-6, IL-10, and IL-1Ra were determined using ELISA kits [1]
For Western blot analysis, BV-2 cells were treated with LPS and/or Diosgenin glucoside (10 µM) for 1 hour. Total proteins were extracted, quantified, separated by electrophoresis, transferred to membranes, and probed with primary antibodies against IκB-α, phospho-JNK, JNK, phospho-p38, p38, phospho-ERK, ERK, and β-actin, followed by appropriate secondary antibodies and detection [1]
For immunofluorescence, BV-2 cells treated with LPS and/or Diosgenin glucoside (10 µM) for 24 hours were fixed, blocked, and stained with an anti-CD206 antibody and a fluorescent secondary antibody. Nuclei were stained with DAPI, and samples were observed under a confocal microscope [1]
For microglia-conditioned medium toxicity assay, BV-2 cells were stimulated with LPS (0.1 µg/mL) and/or Diosgenin glucoside (1, 5, or 10 µM) for 24 hours. The conditioned media were then applied to Neuro-2a cells for 24 hours, after which Neuro-2a cell viability was assessed using the CCK-8 assay [1]

Diosgenin glucoside at concentrations of 1, 5, and 10 µM did not show cytotoxic effects on rat primary microglia and BV-2 microglia after 24 hours of incubation [1].

[1]. Int Immunopharmacol. 2017 Sep;50:22-29. doi: 10.1016/j.intimp.2017.06.008. Epub 2017 Jun 14.

[2]. Diosgenin Glucoside Protects against Spinal Cord Injury by Regulating Autophagy and Alleviating Apoptosis. Int J Mol Sci. 2018 Aug 2;19(8). pii: E2274.

Diosgenin-3-O-β-D-glucoside is a sterol-3-β-D-glucoside, its sterol component being diosgenin. It is a metabolite. It is a sterol-3-β-D-glucoside, a monosaccharide derivative, a hexagonal triterpenoid, and a spiroacetate. Functionally, it is related to diosgenin. It is derived from the hydrogenation of spirostane. The disaccharide has been reported to exist in Dioscorea panthaica, Polygonatum zanlanscianense, and other organisms with relevant data. Diosgenin is a saponin compound extracted from Tribulus terrestris L. [1] Studies have shown that diosgenin selectively inhibits the production/expression of pro-inflammatory M1 markers (e.g., IL-1β, NO, IL-6, TNF-α, IL-17, IL-23) in activated microglia while retaining several anti-inflammatory M2 markers (e.g., IL-10, IL-1Ra, CD206, Arg-1), thereby modulating microglia polarization. [1] The neuroprotective effect of diosgenin appears to be indirect, achieved through… By reducing the release of neurotoxic pro-inflammatory factors from activated microglia, rather than directly protecting neurons, diosgenin has the potential to treat microglia-mediated neuroinflammatory diseases [1].

C33H52O8

576.7612

576.366

14144-06-0

Diosgenin;512-04-9

11827970

White to off-white solid powder

1.3±0.1 g/cm3

705.1±60.0 °C at 760 mmHg

380.2±32.9 °C

0.0±5.1 mmHg at 25°C

1.594

3.89

4

8

3

41

1030

16

C[C@@H]1CC[C@@]2([C@H]([C@H]3[C@@H](O2)C[C@@H]4[C@@]3(CC[C@H]5[C@H]4CC=C6[C@@]5(CC[C@@H](C6)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O)O)O)C)C)C)OC1

WXMARHKAXWRNDM-GAMIEDRGSA-N

InChI=1S/C33H52O8/c1-17-7-12-33(38-16-17)18(2)26-24(41-33)14-23-21-6-5-19-13-20(8-10-31(19,3)22(21)9-11-32(23,26)4)39-30-29(37)28(36)27(35)25(15-34)40-30/h5,17-18,20-30,34-37H,6-16H2,1-4H3/t17-,18+,20+,21-,22+,23+,24+,25-,26+,27-,28+,29-,30-,31+,32+,33-/m1/s1

(2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(1S,2S,4S,5'R,6R,7S,8R,9S,12S,13R,16S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-ene-6,2'-oxane]-16-yl]oxyoxane-3,4,5-triol

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 (~216.73 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.61 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.)