Smilagenin targets the muscarinic receptor subtype 1 (M1 receptor) and the brain-derived neurotrophic factor (BDNF) pathway. The compound improves memory of aged rats by increasing M1 receptor density. Smilagenin attenuates Aβ(25-35)-induced neurodegeneration by stimulating the gene expression of BDNF, a key neurotrophic factor involved in neuronal survival, synaptic plasticity, and cognitive function. The compound counteracts oxidative dopaminergic neurotoxicity and helps restore neuronal growth factor and dopamine receptor deficits in neurodegeneration-related models. Smilagenin prevents and reverses MPTP-induced neuronal damage in Parkinson's disease models.

Sarsaparogenin (10 μM; 24 hours) boosted the survival rate of SH-SY5Y cells in comparison to Aβ(25–35) poisoned cells [3]. After a 24-hour administration of sarsaparilla (10 μM), GDNF and neurotrophic factors are increased in 1-methyl-4-phenylpyridinium (MPP+)-treated SH-SY5Y cells (BDNF) mRNA levels through the promotion of CREB phosphorylation[2].
In vitro studies demonstrate that Smilagenin (10 μM; 24 hours) increases SH-SY5Y cell survival compared to Aβ(25-35)-intoxicated cells. The compound attenuates Aβ(25-35)-induced neurodegeneration by stimulating BDNF gene expression. Smilagenin increases M1 receptor density. The compound acts as a non-peptide neurotrophic factor inducer, promoting neuronal survival and function. In neuronal cell cultures, Smilagenin protects against oxidative and neurotoxic insults. The compound's effects on BDNF expression and M1 receptor density are key to its neuroprotective activity.

In a chronic MPTP/probenecid-induced animal model, sarsaparilla (intragastric injection; 10 or 26 mg/kg once day; 60 days) protects damage to dopaminergic neurons [2].
In vivo studies show that Smilagenin improves memory of aged rats by increasing M1 receptor density. The compound attenuates Aβ(25-35)-induced neurodegeneration in animal models of Alzheimer's disease. Smilagenin prevents and reverses neuronal damage induced by MPTP in a mouse model of Parkinson's disease. The compound is orally active and induces neurotrophic factor expression in vivo. In aged rats, Smilagenin improves cognitive function and memory. The compound's ability to increase BDNF expression and M1 receptor density contributes to its neuroprotective and cognitive-enhancing effects.

The in vitro receptor binding assay for Smilagenin involves measuring M1 muscarinic receptor binding affinity. Membrane preparations from brain tissue or cells expressing M1 receptors are incubated with varying concentrations of Smilagenin (1 nM-100 μM) and a fixed concentration of radiolabeled M1 ligand (e.g., [3H]-pirenzepine). Non-specific binding is determined using excess atropine. IC50 values are calculated from competition binding curves. BDNF gene expression is measured by qPCR or Northern blot in cells treated with Smilagenin (0.1-100 μM) for 1-24 hours. Neuroprotection is assessed in neuronal cell cultures exposed to Aβ(25-35) or MPP+ in the presence of the compound.

Cell viability assay [3]
Cell Types: SH-SY5Y Cell
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: SH-SY5Y cell viability increased.

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RT-PCR[2]
Cell Types: SH-SY5Y Cell
Tested Concentrations: 10 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Increased GDNF and BDNF transcription.
In vitro cellular assays for Smilagenin typically use SH-SY5Y neuroblastoma cells or primary neuronal cultures. Cells are cultured in appropriate media and treated with the compound at concentrations of 1-100 μM for 1-72 hours. For neuroprotection studies, cells are exposed to Aβ(25-35) or MPP+ in the presence of Smilagenin. Cell viability is assessed using MTT or LDH release assays. BDNF expression is measured by ELISA or qPCR. M1 receptor expression is measured by Western blot or receptor binding assays. Neurite outgrowth is assessed by microscopy. Apoptosis is evaluated using caspase-3/7 activity assays and Annexin V staining.

Animal/Disease Models: MPTP/probenecid induced mouse model [2]
Doses: 10 or 26 mg/kg
Route of Administration: intragastric (po) (po)administration; 10 or 26 mg/kg; one time/day; 60-day
Experimental Results: MPTP/probenecid The exercise capacity of Shu-lesioned mice was improved.
In vivo animal studies for Smilagenin involve aged rat models for memory studies and mouse models of Alzheimer's and Parkinson's diseases. Aged rats are treated with the compound via oral gavage at doses of 1-50 mg/kg daily for 2-8 weeks. Memory and cognitive function are assessed using Morris water maze, novel object recognition, or passive avoidance tests. M1 receptor density is measured by receptor binding assays in brain tissues. BDNF levels are measured by ELISA or Western blot. For Parkinson's disease studies, MPTP-treated mice are used. Behavioral tests and dopaminergic neuron survival are assessed. Neurodegeneration is evaluated by histology.

Pharmacokinetic properties of Smilagenin indicate good oral bioavailability. The compound has a molecular weight of 416.64 and molecular formula of C27H44O3. It is a lipid-soluble steroidal sapogenin. The compound should be stored at -20°C for long-term preservation. After oral administration, the compound reaches systemic circulation and distributes to tissues including the brain. The duration of neuroprotective activity is consistent with the compound's half-life. Further studies on plasma half-life, clearance, and protein binding are needed.

Toxicological data for Smilagenin indicates a favorable safety profile as a natural product. The compound is isolated from plants used in traditional Chinese medicine for treating chronic neurodegeneration. The compound is for research use only and not for human therapeutic applications. Standard safety precautions should be followed when handling. For detailed toxicity information, specialized toxicological studies would need to be performed.

[1]. Smilagenin Protects Dopaminergic Neurons in Chronic MPTP/Probenecid-Lesioned Parkinson's Disease Models. Front Cell Neurosci. 2019 Feb 5;13:18.

[2]. Regulation of M1-receptor mRNA stability by smilagenin and its significance in improving memory of aged rats. Neurobiol Aging. 2010 Jun;31(6):1010-9.

[3]. Smilagenin attenuates beta amyloid (25-35)-induced degeneration of neuronal cells via stimulating the gene expression of brain-derived neurotrophic factor. Neuroscience. 2012 May 17;210:275-85.

(25R)-5β-spirostane-3β-ol is an oxaspirocyclic compound with the structure (5β,25R)-spirostane substituted with a β-hydroxyl group at the 3-position. It possesses antitumor activity and is also a metabolite. It is an oxaspirocyclic compound, a 3β-hydroxy steroid, an organic heterohexacyclic compound, and a saponin. It is derived from the hydride of (25R)-5β-spirostane. Smilagenin is a novel, non-peptide, orally bioavailable neurotrophic factor inducer that effectively reverses the free radical neurotoxicity produced by 1-ethyl-4-phenylpyridine (MPP+) in dopaminergic neurons and reverses the reduction of neurotrophic factor and dopamine receptors in the brain. Preclinical studies have shown that smilagenin has neuroprotective effects, counteracting β-amyloid and glutamate damage (one of the causes of Alzheimer's disease) and reversing lesions in brain regions associated with Parkinson's disease. P58 is a protein synthesis stimulant whose mechanism of action is to restore the levels of abnormal proteins in the aging brain, reverse the loss of neural receptors in the aging brain, and potentially promote the regeneration of neural connections. Therefore, P58 provides a novel mechanism of action with potentially significant implications for diseases related to brain aging. P58 is a class of phytochemicals isolated from traditional geriatric therapies, and previous studies have shown that these substances have significant efficacy in treating Alzheimer's disease. Diosgenin has been reported to be found in dioscorea collettii, fenugreek (Trigonella foenum-graecum), and other organisms with relevant data. Drug indications: It has been investigated for the treatment of Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases. Mechanism of Action Diosgenin is a novel, non-peptide, orally bioavailable neurotrophic factor inducer that rapidly reverses the free radical neurotoxicity produced by 1-ethyl-4-phenylpyridine (MPP+) in dopaminergic neurons and the reduction of nerve growth factor and dopamine receptors in the brain. Pharmacodynamics Diosgenin is a novel P58, a non-peptide, orally bioavailable neurotrophic factor inducer that rapidly reverses the free radical neurotoxicity produced by 1-ethyl-4-phenylpyridine (MPP+) in dopaminergic neurons and the reduction of nerve growth factor and dopamine receptors in the brain. P58 is a protein synthesis stimulator whose mechanism of action is to restore altered protein levels in the aging brain, reverse the loss of neural receptors in the aging brain, and potentially promote the regeneration of neural connections. Therefore, P58 provides a novel mechanism of action and has potentially important implications for diseases related to brain aging. P58 belongs to a class of phytochemicals isolated from traditional geriatric therapies, and previous studies have shown that these substances have significant therapeutic effects in treating Alzheimer's disease.
Smilagenin (SMI) is a natural product with CAS number 126-18-1. Its molecular formula is C27H44O3 and molecular weight is 416.64. The compound is a steroidal sapogenin isolated from various plant species. It is an orally active non-peptide neurotrophic factor inducer that improves memory and protects against neurodegeneration. The compound increases M1 receptor density and stimulates BDNF expression. It has been studied for Alzheimer's and Parkinson's diseases. This compound has not been approved by the FDA and is strictly for research purposes only.

C27H44O3

416.646

416.329

126-18-1

91439

White to off-white solid powder

1.11g/cm3

516.6ºC at 760mmHg

-185ºC

266.2ºC

7.83E-13mmHg at 25°C

1.552

5.793

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-UQHLGXRBSA-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-/m1/s1

(1R,2S,4S,5'R,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

PYM 50028; PYM50028; Smilagenin

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)

Ethanol :≥ 10 mg/mL (~24.00 mM)
DMSO : ~5 mg/mL (~12.00 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.)