Peroxisome proliferator-activated receptor gamma (PPARγ) (suppresses transcriptional activity) [1]
CCAAT-enhancer-binding protein alpha (C/EBPα) (suppresses expression) [1]

In 3T3-L1 preadipocytes, Soyasaponin Ab (at concentrations of 25, 50, and 100 µM) dose-dependently inhibited lipid accumulation and triglyceride content during adipocyte differentiation over an 8-day period. Treatment with 25, 50, and 100 µM of Soyasaponin Ab reduced lipid accumulation by up to 45%, 50%, and 70%, respectively. [1]
Soyasaponin Ab (at 50 and 100 µM) significantly suppressed the mRNA expression of key adipogenic transcription factors PPARγ and C/EBPα in fully differentiated (8-day) 3T3-L1 adipocytes, as measured by quantitative real-time PCR. [1]
Western blot analysis showed that Soyasaponin Ab (at 50 and 100 µM) significantly suppressed the protein expression of PPARγ and C/EBPα in fully differentiated 3T3-L1 adipocytes. [1]
In a luciferase reporter assay using HEK 293T cells transfected with a PPRE-luciferase reporter and PPARγ plasmid, treatment with Soyasaponin Ab (at 25, 50, and 100 µM for 24 hours) significantly suppressed PPARγ transcriptional activity in a dose-dependent manner. [1]
Quantitative real-time PCR analysis in fully differentiated 3T3-L1 adipocytes showed that treatment with Soyasaponin Ab (at 50 and 100 µM) markedly reduced the mRNA expression of various adipogenic marker genes, including adiponectin, adipocyte determination and differentiation factor 1/sterol regulatory element binding protein 1c (ADD1/SREBP1c), adipocyte fatty acid-binding protein 2 (aP2), fatty acid synthase (Fas), and resistin. [1]

Cell Cytotoxicity Assay (MTT assay): 3T3-L1 cells were seeded at 5 x 10³ cells per well in 100 µL culture medium. After one day, cells were treated with various concentrations (12.5 to 200 µM) of Soyasaponin Ab for 24 hours in a humidified 5% CO₂ atmosphere at 37°C. MTT solution (5 mg/mL in PBS) was added to each well and incubated for 3 hours. The medium was aspirated, and 0.1 mL of buffered dimethyl sulfoxide was added to each well to dissolve the formazan crystals. Absorbance was measured at 540 nm using a microtiter plate reader. No significant cytotoxic effects were observed at concentrations up to 200 µM. [1]
Adipocyte Differentiation and Lipid Accumulation Assay (Oil Red O Staining): 3T3-L1 preadipocytes were cultured until fully confluent and then induced to differentiate using initiation medium (containing insulin, dexamethasone, and 3-isobutyl-1-methylxanthine) for 2 days, followed by progression medium (containing insulin) and maintenance medium. During the 8-day differentiation period, cells were treated with various concentrations (25, 50, 100 µM) of Soyasaponin Ab. On day 8, cells were washed, fixed with 3.7% formaldehyde, stained with 0.35% Oil Red O dye in isopropyl alcohol for 30 minutes, washed, and the stained lipid droplets were dissolved in isopropyl alcohol containing 4% Nonidet P-40. The triglyceride content was quantified by measuring absorbance at 510 nm. [1]
Luciferase Reporter Assay: HEK 293T cells were seeded in 24-well plates and cultured for 24 hours before transfection. Cells were transfected with 200 ng of PPRE-luciferase reporter plasmid, 50 ng of pcDNA3-hPPARγ plasmid, and 20 ng of pRLSV40 plasmid using a lipofection reagent. After 24 hours of transfection, cells were treated with Soyasaponin Ab (25, 50, 100 µM) for 24 hours. Luciferase activity was measured using a dual-luciferase reporter assay system, and relative luciferase activity was normalized to Renilla luciferase activity. [1]
Quantitative Real-Time PCR: Total RNA was extracted from treated 3T3-L1 cells using an RNA extraction kit. One microgram of RNA was used for cDNA synthesis via reverse transcription. Newly synthesized cDNA was amplified using gene-specific primers and a SYBR Premix Ex Taq system on a real-time PCR instrument. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The mRNA levels of target genes (PPARγ, C/EBPα, adiponectin, ADD1/SREBP1c, aP2, Fas, resistin) were quantified relative to GAPDH. [1]
Western Blot Analysis: Treated 3T3-L1 cells were washed with PBS and lysed on ice in RIPA buffer containing protease inhibitors for 30 minutes. Lysates were centrifuged, and supernatants were collected. Proteins (50 µg per sample) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were blocked and then incubated with primary antibodies against PPARγ, C/EBPα, or actin overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected using an enhanced chemiluminescence detection reagent. [1]

In the MTT cytotoxicity assay using 3T3-L1 cells, soyosaponin antibodies did not show significant cytotoxicity after treatment for 24 hours at concentrations ranging from 12.5 to 200 µM. [1]

[1]. Soyasaponins Aa and Ab exert an anti-obesity effect in 3T3-L1 adipocytes through downregulation of PPARγ. Phytother Res. 2015 Feb;29(2):281-7.

soyosaponin Ab has been reported to exist in soybeans, and relevant data are available. soyosaponin Ab is a group A soyosaponin, a disaccharide glycoside, with its two sugar chains linked to the C-3 and C-22 hydroxyl groups of the soyosaponin A, respectively. Its chemical structure differs from that of soyosaponin Aa in that the terminal sugar group at the C-22 position is different: soyosaponin Ab has 2,3,4,6-tetraacetyl glucosyl. [1] soyosaponin Ab was purified from the hypocotyl of the soybean variety Dayuanguang. The purity of the isolated compound was greater than 95% by liquid chromatography-photodiode array-tandem mass spectrometry. [1] This study shows that soyosaponin Ab exerts an anti-obesity effect in vitro by downregulating key adipogenic transcription factors PPARγ and C/EBPα, as well as several downstream adipogenic marker genes, thereby inhibiting adipocyte differentiation and lipid accumulation. [1]

C67H104O33

1437.5229

1436.645

118194-13-1

102004833

White to off-white solid powder

1.5±0.1 g/cm3

1.620

6.61

14

33

23

100

2960

35

CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O[C@H]2[C@H](CO[C@H]([C@@H]2O)O[C@@H]3[C@@H](C(C[C@@H]4[C@]3(CC[C@@]5(C4=CC[C@H]6[C@]5(CC[C@@H]7[C@@]6(CC[C@@H]([C@]7(C)CO)O[C@H]8[C@@H]([C@H]([C@@H]([C@H](O8)C(=O)O)O)O)O[C@H]9[C@@H]([C@H]([C@H]([C@H](O9)CO)O)O)O[C@H]1[C@@H]([C@H]([C@@H]([C@H](O1)CO)O)O)O)C)C)C)C)(C)C)O)O)OC(=O)C)OC(=O)C)OC(=O)C

YZNCIXVBVQRGQN-YUTHWCJWSA-N

InChI=1S/C67H104O33/c1-26(71)87-24-35-48(89-27(2)72)52(90-28(3)73)53(91-29(4)74)61(94-35)96-47-32(75)23-88-57(46(47)83)100-55-54(84)62(5,6)20-31-30-12-13-37-64(8)16-15-38(65(9,25-70)36(64)14-17-67(37,11)66(30,10)19-18-63(31,55)7)95-60-51(44(81)43(80)49(97-60)56(85)86)99-59-50(42(79)40(77)34(22-69)93-59)98-58-45(82)41(78)39(76)33(21-68)92-58/h12,31-55,57-61,68-70,75-84H,13-25H2,1-11H3,(H,85,86)/t31-,32-,33+,34+,35+,36+,37+,38-,39+,40-,41-,42-,43-,44-,45+,46+,47-,48+,49-,50+,51+,52-,53+,54-,55+,57-,58-,59-,60+,61-,63+,64-,65+,66+,67+/m0/s1

(2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8aR,9S,10R,12aS,14aR,14bR)-9-[(2S,3R,4S,5S)-3,5-dihydroxy-4-[(2S,3R,4S,5R,6R)-3,4,5-triacetyloxy-6-(acetyloxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-10-hydroxy-4-(hydroxymethyl)-4,6a,6b,8a,11,11,14b-heptamethyl-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-5-[(2S,3R,4S,5R,6R)-4,5-dihydroxy-6-(hydroxymethyl)-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,4-dihydroxyoxane-2-carboxylic acid

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 : ~100 mg/mL (~69.56 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (1.74 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (1.74 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (1.74 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 25.0 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.)