Steroidal saponin

Researchers investigated the effect of Tigogenin on adipocytic and osteoblastic differentiation in mouse bone marrow stromal cells (BMSCs). Tigogenin enhanced the proliferation of BMSCs significantly. Tigogenin treatment reduced the adipogenic induction of lipid accumulation, visfatin secretion, and the expressions of peroxisome proliferation-activated receptor (PPAR)gamma2 and adipocyte fatty acid-binding protein (ap)2. Moreover, Tigogenin had no effect on the mitotic clonal expansion. On the other hand, tigogenin significantly elevated alkaline phosphatase (ALP) activity and the expressions of Cbfa1, collagen type I (COL I) and osteocalcin (OCN), as well as the content of matrix calcium in BMSCs. Further, SB-203580 antagonized the tigogenin-promoted osteogenesis. These observations suggested that tigogenin may modulate differentiation of BMSCs to cause a lineage shift away from the adipocytes and toward the osteoblasts, which is at least mediated by inhibition of PPARgamma and via p38 MAPK pathway, and is a potential drug preventing the development of osteoporosis and the related disorders [1].

Assessment of proliferation and mitotic clonal expansion of primary BMSCs [1]
BMSCs were plated in 96-well culture plates and cultured for 6 days. Tigogenin [(25R)-5α-spirostane-3β-ol] diluted with DMSO to prepare the stock solution (10 mmol/L) was then added to the wells at final concentrations of 10, 30 or 90 μmol/L. Cells were incubated for 72 h. Upon completion of the incubation, MTT dye solution (20 μL, 5 g/L, Sangon, China) was added to each well. After 4 h incubation, the supernatant was removed and 100 μL DMSO was added to solubilize the MTT. The optical density of each well was measured on a microplate spectrophotometer at a wavelength of 570 nm. For the assay of mitotic clonal expansion in adipogenesis, MTT test was conducted after BMSCs were treated with tigogenin (10, 30 or 90 μmol/L) from day 0 to 3 of adipocytic differentiation.

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Oil red O staining and measurement [1]
BMSCs were cultured in the presence of adipogenic inducers and Tigogenin at the concentration of 10, 30 or 90 μmol/L for 18 days. Fat droplets within differentiated adipocytes from BMSCs were observed using the oil red O staining method. Cell monolayers were fixed in 4% formaldehyde for 20 min, washed in water and stained with a 0.6% (w/v) oil red O solution (60% isopropanol, 40% water) for 45 min at 37 °C. For quantification, cell monolayers were then washed extensively with water to remove unbound dye, then 1 ml of isopropyl alcohol was added to the stained culture plate. After 5 min, the absorbance of the extract was assayed by a spectrophotometer at 510 nm. Quantification of oil red O staining is normalized to cell number.

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ELISA for secreted visfatin protein [1]
After being treated with adipogenic supplement and Tigogenin (10, 30 or 90 μmol/L) for 18 days, the culture medium of BMSCs was collected. The levels of extracellular visfatin protein in culture medium were assayed by using EIA kit according to the user's manual of the kit.

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Alkaline phosphatase (ALP) activity assay [1]
BMSCs were propagated to confluence, and 2 days later, cells were treated with 10, 30 and 90 μmol/L Tigogenin respectively for 5 days, and then the cells were washed twice with ice-cold PBS and lysed by two cycles of freezing and thaw. Aliquots of supernatants were subjected to ALP activity and protein content measurement using an ALP assay kit and a micro-Bradford assay kit respectively. In the experiment for probing the signaling pathway, cells were pretreated with 50 μmol/L ERK1/2 pathway inhibitor PD-98059 or 10 μmol/L p38 MAPK pathway inhibitor SB-203580 for 60 min, and then stimulated with 30 μmol/L tigogenin for 5 days. Cells were collected and ALP activity was determined. Western blotting analysis [1]
BMSCs treated with Tigogenin (30 μmol/L) were lysed with NETN buffer (20 mM Tris–HCl, pH 7.8, 1 mM EDTA, 50 mM sodium chloride, and 0.5% NP-40) at day 5 of adipogenic induction and lysates were centrifuged at 12,000 rpm at 4 °C for 10 min. Supernatants were collected and protein concentration was determined by BCA assay kit. Western blotting analysis was carried out according to the manufactuers’ protocol. Antibodies against PPARγ, COL I, β-actin, phospho-p38, and non-phospho-p38 were used. Protein expression was visualized with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence.

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Quantification of matrix calcium deposition [1]
BMSCs were treated with varied concentrations of Tigogenin, and the culture continued up to 18 days. Calcification was assessed by the method previously described (Wada et al., 1999) with modification. Briefly, BMSCs were decalcified for 24 h with 0.6 mol/L HCl and the calcium content in supernatant was determined with the use of the calcium C-test Wako Kit. After decalcification, the cells were washed three times with PBS and solubilized with 0.1 mol/L NaOH/0.1% SDS. The total protein concentration was determined by BCA assay kit. The calcium content of the cells was normalized to protein content.

rat LDLo intraperitoneal 10 mg/kg Toxins of Animal and Plant Origin, Proceedings International Symposium, 2nd, 1970, de Vries A., and E. Kochva, eds., 3 vols., New York, Gordon and Breach Science Pub., 1971-73, 2(597), 1972

[1]. Tigogenin inhibits adipocytic differentiation and induces osteoblastic differentiation in mouse bone marrow stromal cells. Mol Cell Endocrinol. 2007 May 30;270(1-2):17-22.

Tigogenin is a widely used steroidal sapogenin isolated from several plant species and used for synthesizing steroid drugs. It has a role as a gout suppressant and a plant metabolite.
Tigogenin has been reported in Ipheion uniflorum, Trigonella foenum-graecum, and other organisms with data available.

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To explore whether tigogenin exerted the corresponding influences on osteogenesis of BMSCs, ALP activity, expression levels of Cbfa1, COL I and OCN, and the content of matrix calcium in BMSCs exposed to tigogenin were assayed. We have ever compared the capacity for osteogenesis in BMSCs induced by osteogenic supplements with or without tigogenin in previous studies, and found that tigogenin treatment did not significantly promoted the osteogenesis of BMSCs induced by osteogenic supplements, whereas the osteogenesis could be slightly but significantly elevated when BMSCs were treated with tigogenin only. We speculated that the strong effect of osteogenic inducers on the osteogenesis of BMSCs might make it inapparent the weak effect of tigogenin when both were added to the cells. Thus, in the current study, we employed BMSCs treated without osteogenic inducers. It was found that ALP activity was significantly elevated in the presence of tigogenin. Similarly, the mRNA expression of other osteoblastic phenotypes, such as Cbfa1, COL I and OCN, also increased following the addition of tigogenin. These results in osteoblastic differentiation suggest that tigogenin is able to differentiate BMSCs into an osteoblastic lineage without adding any osteogenic factors, although the effect appears modest. This conclusion is further strengthened by the evidence that tigogenin increased the matrix calcium deposition in BMSCs slightly but significantly. To make it clear the mechanism involved in this stimulatory effect of tigogenin, the role of MAPK in mediating the cell response to tigogenin was assessed. It has been reported that two key MAPK pathways, ERK1/2 and p38, are essential for osteoblastic differentiation of bone cells (Lai et al., 2001, Lai and Cheng, 2002, Chen et al., 2004). In the current study, SB-203580, a specific inhibitor of p38 pathway, remarkably blocked the stimulatory effect of tigogenin on ALP activity of BMSCs, whereas, PD-98059, inhibitor of ERK1/2, barely changed this promotion. Furthermore, we found that the amount of phospo-p38 protein was increased in BMSCs treated with tigogenin. Thus, it is suggested that this promotion of osteoblastic differentiation by tigogenin is at least partially mediated via p38 MAPK pathway. The above results indicate that tigogenin exerts dual influences on BMSCs by inhibiting adipocytic differentiation and promoting osteoblastic differentiation. These results got further substantiated by the fact that tigogenin promoted the proliferation of BMSCs. Taken together, we deduced from our results that tigogenin might have protective effect on bone and be helpful in preventing the development of osteoporosis by inhibiting adipocyte formation and stimulating osteoblast formation from BMSCs. [1]

C27H44O3

416.6365

416.329

77-60-1

99516

White to off-white solid

1.1±0.1 g/cm3

516.6±20.0 °C at 760 mmHg

202-204ºC

266.2±21.8 °C

0.0±3.0 mmHg at 25°C

1.552

6.21

1

3

0

30

694

12

O1[C@]2(C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])C([H])([H])O2)[C@@]([H])(C([H])([H])[H])[C@@]2([H])[C@]1([H])C([H])([H])[C@@]1([H])[C@]3([H])C([H])([H])C([H])([H])[C@@]4([H])C([H])([H])[C@]([H])(C([H])([H])C([H])([H])[C@]4(C([H])([H])[H])[C@@]3([H])C([H])([H])C([H])([H])[C@@]12C([H])([H])[H])O[H]

GMBQZIIUCVWOCD-MFRNJXNGSA-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,18S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icosane-6,2'-oxane]-16-ol

Tigogenin; 77-60-1; (25R)-5alpha-Spirostan-3beta-ol; 5-alpha-Spirostan-3-beta-ol; (5alpha,25R)-Spirostan-3beta-ol; UNII-4SMU15RR44; 5-epi-sarsasapogenin; TICOGENIN;

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)

Ethanol : ~14.29 mg/mL (~34.30 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.)