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| ln Vitro |
G's treatment when he's suspended. Significantly lowers radioactivity entering saponins and their precursors, but not sterols and steryl glucosides, when papiculata cells are treated with methyl gypsogenin-3-O-glucuronide (Gypsogenin-3-O-(6-O-methyl)-glucoside) 24 hours prior to [14C]acetate injection [1].
- In Gypsophila paniculata cell suspension cultures, pretreatment with gypsogenin 3,O-glucuronide (16 mg/L, added 24 hr before [2-14C]acetate feeding) resulted in a marked reduction in the incorporation of radioactivity into saponins (from 2778 dpm in control to 1027 dpm after 24 hr pretreatment) and their precursors, while phytosterol and steryl glucoside labelling was much less affected. Squalene accumulation increased under these conditions. [1] - Microsomal fractions from G. paniculata treated with gypsogenin 3,O-glucuronide (16 mg/L, added 8 or 24 hr before harvest) showed a greater inhibition of 2,3-oxidosqualene-amyrin cyclase (OSAC) activity than of 2,3-oxidosqualene-cycloartenol cyclase (OSCC). The OSCC/OSAC specific activity ratio increased from 51/49 in controls to 80/20 after 24 hr pretreatment, reflecting a marked reduction in OSAC activity. Prolonged treatment (24 hr) also reduced both OSCC and OSAC activities, leading to accumulation of uncyclized precursors such as squalene. [1] - In Saponaria officinalis cell suspension cultures, pretreatment with gypsogenin 3,O-glucuronide (16 mg/L, 24 hr) enhanced the cyclization of 2,3-oxidosqualene into both tetra- and pentacyclic triterpenoids, without a significant change in their ratio. The ratio of tetracyclic to pentacyclic triterpenoids in S. officinalis was approximately 1:3, which differs from that in G. paniculata. [1] |
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| Enzyme Assay |
- Microsomal fractions were prepared from cell suspension cultures harvested during exponential growth. Cells (50-70 g fresh weight) were rinsed with Tris-HCl buffer (0.1 M, pH 7.5) containing sucrose (0.5 M), EDTA (1 mM), and reduced glutathione (10 mM), then homogenized. The homogenate was filtered and centrifuged at 7000g for 10 min, followed by centrifugation of the supernatant at 10000g for 90 min. The microsomal pellet was resuspended in Tris-HCl buffer (0.1 M, pH 7.4) containing MgCl2 (4 mM) and reduced glutathione (2.5 mM). All procedures were performed at 4°C. [1]
- 2,3-Oxidosqualene-cycloartenol and -amyrin cyclase activities were assayed using 2,3-oxido[3-3H]squalene (100 μmol, 5×10^5 dpm) and Tween 80 (1 mg/mL final concentration). Substrates were added to test tubes as organic solutions, evaporated under nitrogen, and emulsified in a minimal volume of buffer. Microsomal fraction (1 mL, 3-6 mg protein) was added and incubated for 1.5 hr at 30°C. The reaction was stopped by addition of 6% ethanolic KOH (1 mL). Neutral lipids were extracted with hexane and analyzed by TLC on silica gel using CH2Cl2 (twice) as solvent. The 4,4-dimethyl sterols and amyrins (Rf 0.45) were separated from 4α-methyl sterols (Rf 0.40) and 4-demethyl sterols (Rf 0.30), eluted after radioactivity scanning. Authentic α/β-amyrins and cycloartenol + 24-methylenecycloartenol (300 μg each) were added as carriers, and the fraction was acetylated. Analytical argentation TLC (10% AgNO3) with cyclohexane-toluene (7:3, twice) was used to separate amyrin acetates (Rf 0.57) from cycloartenyl acetate (Rf 0.36), 24-methylene cycloartenyl acetate (Rf 0.17), cyclolaudenyl acetate (Rf 0.24), and cyclosadyl acetate (Rf 0.43). Radioactivity was determined by liquid-scintillation spectrometry. A boiled enzyme preparation was used as a control. [1] |
| Cell Assay |
- Cell suspension cultures of Gypsophila paniculata or Saponaria officinalis were transferred to fresh liquid medium (100 mL in 250 mL Erlenmeyer flasks) and shaken at 120 rpm for 15 hr. The pH was adjusted to 4.2-4.3 to enhance acetate incorporation. Sodium [2-14C]acetate (5.4 μCi, 48 Ci/mol) was then added to each flask. At each experimental time point, cells from one flask were harvested by filtration. The plant material was divided into two fractions. One fraction was lyophilized and used for sterol extraction after addition of unlabelled lyophilized tissue (200 mg). Sterols were extracted three times with refluxing cyclohexane for 30 min. The combined extracts were dried and dissolved in CH2Cl2 (1 mL). An aliquot (200 μL) was purified by preparative TLC on silica gel eluted with CHCl3 (twice), and the free sterol band was recovered for radioactivity assay. The second fraction was mixed with fresh unlabelled plant material and used for extraction of gypsogenin 3,O-glucuronide and steryl glucosides. Methanolic extraction was performed, concentrated, and suspended in water acidified with acetic acid (pH 2-3). Steryl glucosides were extracted with ethyl acetate (three times), dried, dissolved in methanol (2 mL), and purified by preparative TLC on silica gel with BuOH-HOAc-H2O (4:1:5). The aqueous phase was treated to extract gypsogenin 3,O-glucuronide in methanol (2 mL) and radioactivity was measured after HPLC purification. [1]
- For microsome preparation and cyclase assays, cells were harvested during exponential growth from untreated cultures or cultures treated with gypsogenin 3,O-glucuronide (16 mg/L, added 8 or 24 hr before harvest). The same compound was dissolved in 0.1% aqueous NaHCO3, sterilized by filtration (0.2 μm), and added aseptically to cell suspension flasks. Control flasks received the same volume of sterile 0.1% NaHCO3. [1] |
| References | |
| Additional Infomation |
Reports indicate that fly grass contains gypsy saponin-3-O-glucuronide, and relevant data is available for reference.
- Gypsogenin 3,O-glucuronide is a naturally occurring saponin precursor in Gypsophila paniculata and related species. In intact plants, these saponins are biosynthesized and stored only in roots and rhizomes, not in shoots. However, in in vitro cultures (calli, cell suspensions, or multiple shoots), they are produced independently of organ differentiation. [1] - The study suggests that 2,3-oxidosqualene cyclases (OSCC and OSAC) are rate-limiting steps in the isoprenoid pathway, directing the metabolic flux toward either tetracyclic phytosterols or pentacyclic triterpenoid saponins. Exogenous gypsogenin 3,O-glucuronide can inhibit (in G. paniculata) or stimulate (in S. officinalis) OSAC activity, while OSCC remains largely unaffected. The effect is time-dependent (greater after 24 hr than 8 hr pretreatment), leading to the hypothesis of a transcriptional or translational effect of the compound on the cyclases. [1] |
| Molecular Formula |
C37H56O10
|
|---|---|
| Molecular Weight |
660.8346
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| Exact Mass |
660.387
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| CAS # |
96553-02-5
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| PubChem CID |
21626375
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| Appearance |
White to off-white solid powder
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| Density |
1.3±0.1 g/cm3
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| Boiling Point |
743.1±60.0 °C at 760 mmHg
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| Flash Point |
223.3±26.4 °C
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| Vapour Pressure |
0.0±5.6 mmHg at 25°C
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| Index of Refraction |
1.582
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| LogP |
8.95
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
47
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| Complexity |
1330
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| Defined Atom Stereocenter Count |
14
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| SMILES |
C[C@]12CC[C@@H]([C@@]([C@@H]1CC[C@@]3([C@@H]2CC=C4[C@]3(CC[C@@]5([C@H]4CC(CC5)(C)C)C(=O)O)C)C)(C)C=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)C(=O)OC)O)O)O
|
| InChi Key |
LHZZULHOQSQSJN-UPGAAKEKSA-N
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| InChi Code |
InChI=1S/C37H56O10/c1-32(2)14-16-37(31(43)44)17-15-35(5)20(21(37)18-32)8-9-23-33(3)12-11-24(34(4,19-38)22(33)10-13-36(23,35)6)46-30-27(41)25(39)26(40)28(47-30)29(42)45-7/h8,19,21-28,30,39-41H,9-18H2,1-7H3,(H,43,44)/t21-,22+,23+,24-,25-,26-,27+,28-,30+,33-,34-,35+,36+,37-/m0/s1
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| Chemical Name |
(4aS,6aR,6aS,6bR,8aR,9S,10S,12aR,14bS)-9-formyl-2,2,6a,6b,9,12a-hexamethyl-10-[(2R,3R,4S,5S,6S)-3,4,5-trihydroxy-6-methoxycarbonyloxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~151.32 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.78 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.78 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.78 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.5132 mL | 7.5662 mL | 15.1325 mL | |
| 5 mM | 0.3026 mL | 1.5132 mL | 3.0265 mL | |
| 10 mM | 0.1513 mL | 0.7566 mL | 1.5132 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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