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.

Intraperitoneal injection of 10 mg/kg LDLo animal and plant-derived toxins in rats, Proceedings of the Second International Symposium, 1970, edited by de Vries A. and E. Kochva, Vol. 3, 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 saponin, isolated from various plants, used in the synthesis of steroidal drugs. It has anti-gout effects and is also a plant metabolite. Tigogenin has been reported to exist in Ipheion uniflorum, Trigonella foenum-graecum, and other organisms with relevant data. To investigate whether Tigogenin affects osteogenic activity in bone marrow mesenchymal stem cells (BMSCs), we examined the alkaline phosphatase (ALP) activity, Cbfa1, type I collagen (COL I), and osteocalcin (OCN) expression levels, as well as matrix calcium content, in BMSCs exposed to Tigogenin. In a previous study, we compared the effects of osteogenic inducers on the osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) with and without tegronine. We found that tegronine treatment did not significantly promote osteogenic inducer-induced osteogenic activity in BMSCs, while tegronine alone slightly but significantly enhanced osteogenic activity. We hypothesized that the potent effect of osteogenic inducers on BMSC osteogenic activity might mask the weak effect of tegronine. Therefore, in this study, we used BMSCs without osteogenic inducers. The results showed that alkaline phosphatase (ALP) activity was significantly increased in the presence of tegronine. Similarly, the mRNA expression levels of other osteoblast phenotypes (such as Cbfa1, COL I, and OCN) also increased after the addition of tegronine. These results of osteoblast differentiation indicate that tegronine can induce BMSCs to differentiate into osteoblast lineages without the addition of any osteogenic factors, although the effect appears to be relatively mild. Tegonine slightly but significantly increased matrix calcium deposition in BMSCs, further reinforcing this conclusion. To elucidate the mechanism of tegonine stimulation, we evaluated the role of MAPK in mediating the cellular response to tegonine. It has been reported that the ERK1/2 and p38 MAPK pathways are crucial for osteogenic differentiation of osteoblasts (Lai et al., 2001; Lai and Cheng, 2002; Chen et al., 2004). In this study, the p38 pathway-specific inhibitor SB-203580 significantly blocked the stimulatory effect of tegonine on alkaline phosphatase (ALP) activity in bone marrow mesenchymal stem cells (BMSCs), while the ERK1/2 inhibitor PD-98059 had little effect on this promoting effect. Furthermore, we found an increase in phosphorylated p38 protein in tegonine-treated BMSCs. Therefore, this suggests that the osteoblast-promoting effect of tegonine is at least partially mediated through the p38 MAPK pathway. The above results indicate that tegonine has a dual effect on BMSCs, both inhibiting adipocyte differentiation and promoting osteoblast differentiation. The fact that tegonine promotes BMSC proliferation further confirms the above results. In summary, our research results suggest that tegonine may have a protective effect on bone, and may help prevent osteoporosis by inhibiting adipocyte formation and stimulating bone marrow mesenchymal stem cells to differentiate into osteoblasts. [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.)