α4-nicotinic acetylcholine receptor (α4-nAChR) [2]
- α7-nicotinic acetylcholine receptor (α7-nAChR) [2]

- Acetylcholinesterase (AChE) [2]

- Toll-like receptor 4 (TLR4) [3]

- NF-κB pathway components (IRAK1, TAK1, I-κBα, p65) [3]

- MAPK pathway components (ERK, JNK, p38) [3]

- Th17 cell differentiation [3]

- Treg cell differentiation [3]

In heLa cells, sarasapogenin (20–80 μM) causes apoptosis through a mitochondrial route that is dependent on caspase. Through the caspase-dependent mitochondrial apoptotic pathway, sarsasapogenin also causes apoptosis. The formation of ROS generated by sarsasapogenin (60 μM) causes mitochondrial malfunction and ER stress[1]. Sarsasapogenin significantly reduces the activation of NF-κB and MAPK in LPS-stimulated macrophages, as well as the phosphorylation of IRAK1, TAK1, and IκBα. Additionally, sarsasapogenin prevents M2 macrophage polarization toward M1 macrophages and LPS binding to macrophage Toll-like receptor 4. [3].

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In LPS-stimulated mouse peritoneal macrophages, sarsasapogenin (2 and 5 μM) inhibited NF-κB activation, decreased pro-inflammatory cytokine TNF-α, IL-1β, and IL-6 levels, and suppressed COX-2 and iNOS expressions. The inhibition was more potent than timosaponin AIII. Sarsasapogenin did not affect NF-κB activation or iNOS/COX-2 expression in peptidoglycan-induced macrophages. [3]
- Sarsasapogenin (2 and 5 μM) inhibited the phosphorylation of IRAK1, TAK1, I-κBα, and p65 in LPS-stimulated peritoneal macrophages, as shown by immunoblotting. [3]
- Sarsasapogenin (2 and 5 μM) suppressed LPS-induced nuclear translocation of NF-κB p65 subunit in peritoneal macrophages, detected by confocal microscopy. [3]
- Sarsasapogenin (2 and 5 μM) inhibited LPS-induced phosphorylation of ERK, JNK, and p38 MAPKs in peritoneal macrophages. [3]
- Sarsasapogenin (2 and 5 μM) inhibited the binding of Alexa Fluor 488-conjugated LPS to TLR4 on peritoneal macrophages in a dose-dependent manner, as measured by flow cytometry and confocal microscopy. [3]
- In splenocyte CD4+ T cells stimulated under Th17-polarizing conditions (anti-CD3, anti-CD28, IL-6, TGF-β), sarsasapogenin (2 and 5 μM) significantly suppressed the population of IL-17+ T cells (from 37.1% to lower percentages), indicating inhibition of Th17 cell differentiation. [3]
- In OB rat model, sarsasapogenin (20 and 40 mg/kg) treatment increased hippocampal α4-nAChR and α7-nAChR protein expression and decreased AChE activity and expression, as shown by immunohistochemistry and western blot. [2]

Sarsasapogenin (20 and 40 mg/kg) enhances locomotor activity and significantly corrects the sucrose preference deficit caused by olfactory bulbectomy (OB). Compared to the OB group, the sarsasapogenin groups (20 and 40 mg/kg) exhibited considerably shorter immobility durations and increased levels of AChE protein expression. Additionally, when compared to rats in the OB group, the Sarsasapogenin (20 and 40 mg/kg) groups exhibit considerably higher levels of α7-nAChR protein expression as well as higher levels of α4-nAChR protein expression[2]. Sarsasapogenin (5 or 10 mg/kg, po) decreases NF-κB activation and decreases interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 levels while concurrently raising IL-10 in mice by inhibiting TNBS-induced colon shortening and myeloperoxidase activity[3].

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In olfactory bulbectomized (OB) rats, sarsasapogenin (20 and 40 mg/kg, oral administration for 14 days) reversed depressive-like behaviors: significantly restored sucrose preference (66.99% and 80.12% vs OB 55.40%, p<0.05 and p<0.01), reduced hyperlocomotion (ambulation: 21.05 and 18.36 vs OB 47.10, p<0.05 and p<0.01; rearing: 3.07 and 2.32 vs OB 6.48, p<0.01; grooming: 6.03 and 4.87 vs OB 13.8, p<0.05 and p<0.01), and decreased immobility time in forced swimming test (103.67 and 82.19 s vs OB 142.15 s, p<0.01). [2]
- In OB rats, sarsasapogenin (20 and 40 mg/kg) significantly reversed OB-induced increase in hippocampal AChE activity (p<0.01) and AChE protein expression (p<0.01), and increased α4-nAChR and α7-nAChR protein expression in hippocampus (p<0.01). [2]
- In TNBS-induced colitis mice, oral administration of sarsasapogenin (5 and 10 mg/kg) for 3 days significantly inhibited colon shortening, reduced macroscopic colitis scores, suppressed myeloperoxidase activity (10 mg/kg inhibited by 52.1% vs TNBS group, p<0.05), and ameliorated edema and epithelial cell disruption. Sarsasapogenin also restored TNBS-suppressed expression of tight junction proteins ZO-1, occludin, and claudin-1. [3]
- In TNBS-induced colitis mice, sarsasapogenin (5 and 10 mg/kg) decreased colonic levels of TNF-α, IL-1β, IL-6, and IL-17, while increasing IL-10 expression. It inhibited M1 macrophage markers (TNF-α, IL-1β, arginase II) and induced M2 markers (IL-10, arginase I). Sarsasapogenin also suppressed TNBS-induced COX-2, iNOS, and phosphorylation of IRAK1, TAK1, I-κBα, and p65 in colon tissues. [3]
- In TNBS-induced colitis mice, sarsasapogenin (5 and 10 mg/kg) inhibited Th17 cell differentiation and promoted Treg cell differentiation in colonic lamina propria, as analyzed by flow cytometry. It also reduced IFN-γ and IL-17 expression and increased Foxp3 expression. [3]

AChE activity assay: Hippocampal tissue homogenates were prepared from rats. AChE activity was determined using a commercial AChE assay kit. Absorbance was measured at 412 nm, and AChE activity was expressed as U/mg protein. Sarsasapogenin (20 and 40 mg/kg) significantly reversed the OB-induced increase in AChE activity (p<0.01). [2]
- Immunoblotting for AChE, α4-nAChR, α7-nAChR: Protein extracts from hippocampal tissue were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 10% non-fat dry milk, then incubated overnight at 4°C with rabbit polyclonal anti-AChE (1:1,000), mouse monoclonal anti-α4-nAChR (1:100), goat polyclonal anti-α7-nAChR (1:100), and mouse monoclonal anti-GAPDH (1:500). After washing, membranes were incubated with HRP-labeled secondary antibodies for 1 hour. Densitometric measurements were performed using ECL detection system. [2]
- NF-κB and MAPK phosphorylation assay: Peritoneal macrophages were stimulated with LPS (100 ng/mL) for 90 min in the presence or absence of sarsasapogenin (2 and 5 μM). Cells were lysed and proteins were separated by 8-10% SDS-PAGE, transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dried milk in PBST, probed with antibodies against p-IRAK1, IRAK1, p-TAK1, TAK1, p-I-κBα, I-κBα, p-p65, p65, p-JNK, JNK, p-p38, p38, p-ERK, ERK, p-IKKβ, IKKβ, or β-actin, then incubated with HRP-conjugated secondary antibodies. Bands were visualized with ECL kit. [3]
- LPS-TLR4 binding assay by flow cytometry: Peritoneal macrophages were treated with Alexa Fluor 488-conjugated LPS (1 μg/mL) for 10, 20, or 30 min in the presence or absence of sarsasapogenin (2 and 5 μM). Cells were fixed in PBS containing 3% sucrose and 4% paraformaldehyde for 20 min, washed, incubated with propidium iodide (10 μg/mL) for 10 min, and analyzed by flow cytometer. [3]
- LPS-TLR4 binding assay by confocal microscopy: Peritoneal macrophages were stimulated with Alexa Fluor 488-conjugated LPS (1 μg/mL) for 20 min in the presence or absence of sarsasapogenin (2 and 5 μM). Cells were fixed with 4% paraformaldehyde and 3% sucrose for 20 min, then examined under a confocal microscope. [3]

Peritoneal macrophage isolation and culture: Mice were intraperitoneally injected with 4% sodium thioglycolate solution, and killed 4 days later. Peritoneal cavity was washed with RPMI 1640, centrifuged at 200×g for 10 min. Macrophages were isolated using biotin-labeled anti-mouse F4/80 antibody and streptavidin magnetic beads. Macrophages (1×10^6 cells/well) were cultured in 12-well plates at 37°C for 2 days in RPMI 1640 with 10% fetal bovine serum. To examine anti-inflammatory effects, macrophages were incubated with or without sarsasapogenin (2, 5, or 10 μM) together with 100 ng/mL LPS or peptidoglycan. [3]
- ELISA for cytokine measurement: Macrophages (0.5×10^6 cells) were stimulated with LPS (100 μg/mL) for 20 h in the presence or absence of sarsasapogenin (2 and 5 μM). Supernatants were collected and cytokine levels (TNF-α, IL-1β, IL-6) were determined using ELISA kits. [3]
- Immunoblotting for COX-2 and iNOS: Macrophages were treated as above, lysed, and proteins were analyzed by immunoblotting using antibodies against COX-2, iNOS, and β-actin. [3]
- NF-κB nuclear translocation assay by confocal microscopy: Peritoneal macrophages were stimulated with LPS (100 ng/mL) for 90 min in the presence or absence of sarsasapogenin (2 and 5 μM). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with goat polyclonal anti-p65 antibody at 4°C for 2 h, then incubated with Alexa 488-conjugated secondary antibodies and propidium iodide (10 μg/mL) for 1 h. Images were captured by confocal microscope. [3]
- Th17 cell differentiation assay in vitro: Splenocytes were prepared from mice, and CD4+ T cells were stimulated under Th17-polarizing conditions (immobilized anti-CD3 (1 μg/mL) and anti-CD28 (1 μg/mL) with IL-6 (20 ng/mL) and TGF-β (1 ng/mL)) for 4 days in the presence of sarsasapogenin (2 or 5 μM). Cells were then stained for surface CD4 and intracellular IL-17, and analyzed by flow cytometry. [3]
- Cytotoxicity assay: Peritoneal macrophages were treated with sarsasapogenin (2, 5, 10 μM) under experimental conditions, and cell viability was assessed. Sarsasapogenin showed no cytotoxicity at these concentrations. [3]

Olfactory bulbectomy (OB) rat model of depression: Male Sprague-Dawley rats (180-220 g) were anesthetized with chloral hydrate (10%, 3.3 mL/kg, intraperitoneal). The skull over the olfactory bulbs was exposed via a midline incision, and two holes (2 mm diameter) were drilled 8 mm anterior to bregma and 2 mm lateral to midline. Both olfactory bulbs were aspirated. Holes were filled with glass-ionomer cement, and scalp sutured. Sham-operated rats underwent same procedure without bulb aspiration. Rats received intramuscular penicillin (0.2 mL/300 g, 8×10^5 U) once daily for 3 days post-surgery. After 14 days recovery, drug treatment began. Sarsasapogenin (20 or 40 mg/kg) or amitriptyline (10 mg/kg) was administered orally once daily for 14 days (days 15-28). After behavioral tests, rats were sacrificed, and lesions assessed. Animals with <15% tissue removal were excluded. [2]
- Sucrose preference test: Rats were provided with 1% (w/v) sucrose solution for 24 h, then free access to both sucrose and water for 48 h. After 23 h food and water deprivation, rats were given access to both water and 1% sucrose for 1 h. Sucrose preference (%) = (sucrose intake / (sucrose intake + water intake)) × 100. [2]
- Open field test: Rats were placed individually in the center of a 100×100×40 cm metallic cage with floor divided into 16 sections. Number of line crossings and rearing frequency were scored manually over 5 min. [2]
- Forced swimming test (FST): Rats were forced to swim in a cylinder (40 cm height × 18 cm diameter) containing fresh water (25±1°C) to a height of 30 cm for 6 min. Total immobility time was recorded during the last 4 min. [2]
- TNBS-induced colitis mouse model: Male C57BL/6 mice (19-22 g, 7 weeks) were anesthetized and intrarectally injected with 2.5% (w/v) TNBS solution (100 μL, dissolved in 50% ethanol) using a thin round-tip needle. Normal control received vehicle alone. Sarsasapogenin (5 or 10 mg/kg) or sulfasalazine (50 mg/kg) dissolved in 2% Tween 80 was orally administered once daily for 3 days after TNBS treatment. Mice were killed 18 h after final administration. Colon was removed for macroscopic scoring (0-5 scale), MPO activity, histology, ELISA, and immunoblotting. [3]
- Myeloperoxidase (MPO) assay: Colon tissues were homogenized in 0.5% hexadecyl trimethyl ammonium bromide in 10 mM potassium phosphate buffer (pH 7.0), centrifuged at 20,000×g for 30 min at 4°C. Supernatant (50 μL) was added to reaction mixture containing 1.6 mM tetramethyl benzidine and 0.1 mM H2O2, incubated at 37°C for 3 min, and absorbance measured at 650 nm. MPO activity expressed as unit/mg protein. [3]

In mice, orally administered timosaponin AIII (a saponin precursor) is metabolized to sarsasapogenin by gut microbiota. In a study, timosaponin AIII (100 mg/kg) was orally administered to mice, and after 12 h, sarsasapogenin was detected in colon fluid, while both timosaponin AIII and sarsasapogenin were present, with sarsasapogenin being the main compound. Incubation of timosaponin AIII with mouse fecal suspension also resulted in its conversion to sarsasapogenin. This suggests that sarsasapogenin is an intestinal metabolite of timosaponin AIII. [3]
- Sarsasapogenin is more hydrophobic than its parent compound timosaponin AIII, which may affect its absorption and pharmacological activity. [3]

In vitro cytotoxicity assay: Sarsasapogenin (2, 5, and 10 μM) showed no cytotoxic effects on mouse peritoneal macrophages under the experimental conditions used. [3]

[1]. Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells. Biochem Biophys Res Commun. 2013 Oct 26.

[2]. Sarsasapogenin reverses depressive-like behaviors and nicotinic acetylcholine receptors induced by olfactory bulbectomy. Neurosci Lett. 2017 Feb 3;639:173-178.

[3]. Timosaponin AIII and its metabolite sarsasapogenin ameliorate colitis in mice by inhibiting NF-κB and MAPK activation and restoring Th17/Treg cell balance. Int Immunopharmacol. 2015 Apr;25(2):493-503.

(25S)-5β-spirostane-3β-ol is a saponin. It has been reported that diosgenin exists in diosgenin, fenugreek, and other organisms with relevant data.

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Sarsasapogenin is the major active component of Anemarrhena asphodeloides (Liliaceae) and has anti-oxidative properties. It has been shown to enhance neurogenesis and ameliorate cognitive impairment, and improve memory by increasing acetylcholine receptor density in the brain of memory-impaired rat models. It also has antidepressant-like effects in mice as assessed by behavioral despair test. [2]
- In the OB rat model, abnormal cholinergic signaling in the hippocampus contributes to the development of depression. Sarsasapogenin reverses depressive-like behaviors and modulates cholinergic system dysfunction by increasing α4- and α7-nAChR expression and normalizing AChE activity. [2]
- Sarsasapogenin is more potent than its parent compound timosaponin AIII in inhibiting inflammatory responses in vitro and in vivo. The anti-inflammatory mechanism involves inhibiting the binding of LPS to TLR4 on macrophages, blocking NF-κB and MAPK signaling pathways, and restoring the balance of Th17/Treg cells. [3]
- Orally administered timosaponin AIII is metabolized to sarsasapogenin by gut microbiota, and both compounds simultaneously ameliorate inflammatory diseases such as colitis. [3]

C27H44O3

416.6365

416.329

126-19-2

92095

White to off-white solid powder

1.1±0.1 g/cm3

516.6±20.0 °C at 760 mmHg

194°C

266.2±21.8 °C

0.0±3.0 mmHg at 25°C

1.552

6.21

1

3

0

30

694

12

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[C@H]6[C@@]5(CC[C@@H](C6)O)C)C)C)OC1

GMBQZIIUCVWOCD-WWASVFFGSA-N

InChI=1S/C27H44O3/c1-16-7-12-27(29-15-16)17(2)24-23(30-27)14-22-20-6-5-18-13-19(28)8-10-25(18,3)21(20)9-11-26(22,24)4/h16-24,28H,5-15H2,1-4H3/t16-,17-,18+,19-,20+,21-,22-,23-,24-,25-,26-,27+/m0/s1

(1R,2S,4S,5'S,6R,7S,8R,9S,12S,13S,16S,18R)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icosane-6,2'-oxane]-16-ol

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)

DMSO : ~3.57 mg/mL (~8.57 mM)
Ethanol : ~1 mg/mL (~2.40 mM)

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.

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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).

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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)

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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).

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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

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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.

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