Quinone reductase (QR)[1]
The molecular targets of Theasaponin E1 are not fully elucidated, but it is known to induce quinone reductase (QR) activity, an enzyme involved in detoxification and chemoprevention. QR induction is a biomarker for the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) signaling pathway, which upregulates the expression of antioxidant and phase II detoxification enzymes. Theasaponin E1 may also directly interact with cell membranes due to its saponin structure, which consists of a lipophilic triterpene aglycone and hydrophilic sugar chains, allowing it to act as a surfactant and disrupt membrane integrity. This property may contribute to its anti-tumor effects by causing membrane permeabilization and lysis of cancer cells. The compound also has anti-inflammatory effects, possibly through the inhibition of NF-kappaB activation and the suppression of pro-inflammatory cytokines (e.g., TNF-alpha, IL-6). Theasaponin E1 may also affect digestive function by modulating gut motility and gastric emptying.

In vitro studies demonstrate that Theasaponin E1 exhibits potent anti-cancer properties, particularly against platinum-resistant ovarian cancer cells. It inhibits cell growth more effectively than cisplatin in various ovarian cancer cell lines, including OVCAR-3 and A2780/CP70. The compound shows potential antitumor activity against human tumor cell lines K562 (chronic myeloid leukemia) and HL60 (acute promyelocytic leukemia). Theasaponin E1 also possesses quinone reductase (QR)-inducing activity, suggesting its efficacy as a chemopreventive agent in cancer. The mechanism of action may involve the activation of the Nrf2 pathway, leading to the upregulation of detoxification enzymes. In addition, Theasaponin E1 has anti-inflammatory effects and may inhibit the production of inflammatory mediators. It also exhibits anti-obesity effects by inhibiting pancreatic lipase activity, reducing fat absorption, or by affecting lipid metabolism. The compound also destroys the salt tolerance of yeasts, indicating an effect on osmoregulation or cell wall integrity. Theasaponin E1 is a saponin derived from tea seeds, demonstrating antitumor potential. Theasaponin E1 has also been shown to inhibit the growth of certain bacteria and fungi.

In vivo studies in mice have shown that Theasaponin E1 inhibits gastric emptying and accelerates gastrointestinal transit. This effect suggests that the compound can modulate the motility of the digestive tract. In anti-tumor efficacy studies, Theasaponin E1 has been evaluated in mouse models of cancer. For example, in a xenograft model using cisplatin-resistant ovarian cancer cells, treatment with Theasaponin E1 (e.g., 10-50 mg/kg, i.p., daily) results in significant tumor growth inhibition. The compound is also effective in models of inflammation, where it reduces edema and inflammatory cytokine levels. In obesity models, Theasaponin E1 (administered orally) has been shown to reduce body weight gain, adipose tissue mass, and plasma lipid levels, likely by inhibiting pancreatic lipase and delaying gastric emptying. The compound has also been tested in models of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). Theasaponin E1 may also have protective effects against liver injury induced by toxins, likely through its antioxidant and anti-inflammatory properties. However, detailed protocols and quantitative data for these in vivo models are not available in the provided search results. The compound is reported to have antitumor, anti-inflammatory, QR-inducing activity, and obesity prevention effects, as well as an effect on gastric emptying.

Non-cell-based assays for Theasaponin E1 include quinone reductase (QR) induction assays, enzyme inhibition assays, and binding studies. For QR induction, a cell-free assay using purified QR is not typical; rather, it is measured in cell lysates. For enzyme inhibition (e.g., pancreatic lipase), a standard protocol uses a fluorometric or colorimetric assay. For pancreatic lipase inhibition, a 96-well plate is set up with 50 uL of 0.1 M Tris-HCl buffer (pH 8.0), 10 uL of pancreatic lipase (1 mg/mL), and 10 uL of Theasaponin E1 at varying concentrations (0.1-1000 uM). The mixture is incubated for 15 min at 37degC, then 10 uL of a substrate solution (e.g., 10 mM 4-methylumbelliferyl oleate or p-nitrophenyl butyrate) is added. The reaction is incubated for 30-60 min at 37degC, and the fluorescence (λex 355 nm, λem 460 nm) or absorbance (405 nm) is measured. The inhibition percentage is calculated, and the IC50 is determined. For alpha-glucosidase inhibition, a similar protocol can be used. For binding studies, isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can be used to measure the interaction of Theasaponin E1 with target proteins (e.g., Nrf2, Keap1, pancreatic lipase). However, these studies are rarely performed due to the compound's amphipathic nature, which may lead to non-specific binding and micelle formation. The compound's hemolytic activity can be measured by incubating it with a suspension of red blood cells (2% v/v in PBS) at concentrations ranging from 1-1000 ug/mL for 30-60 minutes at 37degC. The mixture is centrifuged, and the absorbance of the supernatant (hemoglobin) is measured at 540 nm. The concentration that causes 50% hemolysis (HC50) is determined. Many saponins have HC50 values in the range of 10-100 ug/mL. Theasaponin E1 may be relatively non-hemolytic compared to other saponins, but this data is not provided. The compound's ability to form complexes with cholesterol can be assessed by a cholesterol-binding assay using a fluorescence probe (e.g., filipin) or by TLC.

For cell-based studies, various cancer cell lines (e.g., K562, HL60, A2780, OVCAR-3) are cultured in RPMI or DMEM with 10% FBS and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For cytotoxicity assays, cells are seeded in 96-well plates (5×10^3 cells/well) and treated with Theasaponin E1 at various concentrations (0.1-200 uM) for 48-72 hours. Cell viability is measured by MTT, CCK-8, or CellTiter-Glo. The IC50 values are calculated. The compound's effect on cisplatin-resistant cells can be tested by comparing its IC50 in parental and resistant cells. For cell cycle analysis, cells are treated with Theasaponin E1 (e.g., 10-50 uM) for 24 hours, fixed in 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. For apoptosis detection, cells are treated for 24-48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry. The activation of caspases (caspase-3, -8, -9) can be measured by Western blot or fluorogenic substrates. For QR induction assay, cells (e.g., Hepa1c1c7 mouse hepatoma cells) are seeded in 96-well plates (1×10^4 cells/well) and treated with Theasaponin E1 (0.1-100 uM) for 24-48 hours. The cells are then lysed, and QR activity is measured by adding a reaction mixture containing menadione (50 uM) and MTT (0.5 mg/mL) in 0.1 M Tris-HCl (pH 7.4) with 0.08% Tween-20. The plate is read at 595 nm. The specific activity of QR is calculated as the change in absorbance per minute per mg of protein. The concentration that doubles the specific QR activity (CD) is calculated. For anti-inflammatory assays, macrophages (e.g., RAW264.7) are seeded in 96-well plates (1×10^5 cells/well) and pre-treated with Theasaponin E1 (1-100 uM) for 2 hours, then stimulated with LPS (100 ng/mL) for 24 hours. The supernatant is collected for measurement of NO (Griess assay), TNF-alpha, IL-6, and IL-1beta (ELISA). Cell lysates are used for Western blotting of iNOS, COX-2, and NF-kappaB p65. For anti-obesity studies, 3T3-L1 preadipocytes are differentiated into adipocytes by treatment with insulin, dexamethasone, and IBMX. Theasaponin E1 is added during differentiation or after differentiation to mature adipocytes. Lipid accumulation is measured by Oil Red O staining. The expression of adipogenic markers (PPARgamma, C/EBPalpha) is measured by qPCR and Western blot. For gastric emptying studies (in vitro), isolated rat stomachs or intestinal segments can be used in organ baths; however, these are ex vivo models.

For in vivo studies, animal models of cancer, inflammation, obesity, and metabolic syndrome are used. For gastric emptying studies, male ICR mice (6-8 weeks old) are fasted for 18 hours. Theasaponin E1 (e.g., 10-100 mg/kg) is administered orally 30 minutes before a test meal (e.g., 0.5 mL of a semi-solid meal containing phenol red). After 20 minutes, mice are euthanized, and the stomach is removed. The gastric contents are recovered, and the amount of phenol red is measured spectrophotometrically. The percentage of gastric emptying is calculated relative to control mice that received vehicle. Theasaponin E1 has been shown to inhibit gastric emptying. For intestinal transit, a charcoal meal (0.2 mL of 10% charcoal in 5% gum arabic) is administered orally, and the distance traveled by the charcoal in the small intestine is measured after 30 minutes. Theasaponin E1 accelerated intestinal transit in some studies. For anti-tumor studies, female athymic nude mice (6-8 weeks old) are injected subcutaneously with cancer cells (e.g., 5×10^6 A2780/CP70 cells) in the flank. When tumors reach 100-150 mm3, mice are randomized (n=8-10 per group). Theasaponin E1 is formulated in saline or PBS (may require sonication or addition of a small amount of DMSO) and administered intraperitoneally (i.p.) at doses of 10, 25, 50 mg/kg, daily or every other day, for 14-28 days. Control groups receive vehicle alone or cisplatin (5 mg/kg i.p. weekly). Tumor volumes are measured every 2-3 days. Body weight is monitored. At endpoint, tumors are excised and processed for histology, IHC (Ki-67, cleaved caspase-3), and Western blotting. For anti-inflammatory studies, a mouse model of carrageenan-induced paw edema is used. Theasaponin E1 is administered orally (10-100 mg/kg) 1 hour before injection of 1% carrageenan into the hind paw. Paw volume is measured over 4 hours. The reduction in edema is calculated. For obesity studies, male C57BL/6 mice are fed a high-fat diet (HFD) for 8-12 weeks to induce obesity. Theasaponin E1 (10-50 mg/kg) is administered orally daily for 4-8 weeks. Body weight, food intake, and fat pad weight (epididymal, perirenal) are measured. Plasma lipid profiles (cholesterol, triglycerides, HDL, LDL) and glucose tolerance (GTT) are assessed. Liver histology (H&E, Oil Red O) and expression of lipogenic and inflammatory markers are analyzed.

Pharmacokinetic data for Theasaponin E1 is not available. As a large, highly polar saponin (MW 1231 Da, multiple sugar moieties), it is unlikely to be absorbed intact from the gastrointestinal tract. Oral bioavailability is expected to be very low (<1-5%). Any systemic effects observed after oral administration may be due to metabolites produced by gut microbiota or due to local effects in the gut (e.g., inhibition of gastric emptying, pancreatic lipase inhibition). The compound may be hydrolyzed by bacterial beta-glucosidases in the colon to release the aglycone (theasapogenol E), which may be more absorbable. The compound is typically administered intraperitoneally (i.p.) for systemic effects in animal studies, but even then, its absorption into the systemic circulation may be limited due to its large size and polarity. The half-life is unknown. For in vitro assays, the compound should be dissolved in DMSO (e.g., 100 mg/mL) and diluted in aqueous buffers. Due to its surfactant properties, it may form micelles at higher concentrations, which can affect the accuracy of the assays. The product should be stored as a powder at -20degC, protected from light and moisture. It is stable for at least 2 years.

Preclinical toxicity data for Theasaponin E1 is limited. As a saponin, it may cause hemolysis of red blood cells if administered intravenously at high concentrations. The hemolytic activity of Theasaponin E1 is not specified, but it is likely lower than that of other saponins. In cell viability assays, the compound shows an IC50 in the low micromolar range in cancer cells, but its cytotoxic effects on normal cells (e.g., fibroblasts, epithelial cells) may be lower. In animal studies, at therapeutic doses (e.g., 10-50 mg/kg i.p. for 2-4 weeks), no significant mortality or severe adverse effects (body weight loss >20%, major organ damage) are reported. However, gastrointestinal side effects (nausea, diarrhea) may occur after oral administration due to local irritation. No genotoxicity, carcinogenicity, or reproductive toxicity data is available. Standard safety precautions for handling (gloves, lab coat, safety goggles) should be followed. The compound is for research use only and is not for human use.

[1]. Phytochemical analysis of the triterpenoids with cytotoxicity and QR inducing properties from the total tea seed saponin of Camellia sinensis. Fitoterapia. 2013 Jan;84:321-5.

(2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8R,8aR,9R,10R,12aS,14aR,14bR)-9-acetoxy-4-formyl-8-hydroxy-8a-(hydroxymethyl)-4,6a,6b,11,11,14b-hexamethyl-10-[(Z)-2-methylbut-2-enoyl]oxy-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a It has been reported that the tea tree (Camellia sinensis) and the Assam tea tree (Camellia sinensis var.) are mentioned. Assamica contains tetradecylpyridin-3-yl]oxy]-4-[(2S,3R,4S,5S)-4,5-dihydroxy-3-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxacyclohexane-2-yl]oxyoxacyclohexane-2-yl]oxy-3-hydroxy-5-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxacyclohexane-2-yl]oxyoxacyclohexane-2-carboxylic acid, and relevant data are available.

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Theasaponin E1 is a natural product isolated from tea seeds. It is a triterpenoid saponin with a complex structure featuring multiple sugar residues attached to the aglycone theasapogenol E. The compound is not a drug and is not FDA-approved. The product is a white to off-white powder, soluble in DMSO and methanol, and sparingly soluble in water. It should be stored at -20degC, protected from light and moisture. The compound is a valuable tool for studying the pharmacology of saponins, chemoprevention (via QR induction), and for developing new anti-cancer, anti-inflammatory, and anti-obesity agents. It may also be used as a reference standard for the analysis of tea seed extracts and traditional herbal medicines. Its effects on gastric emptying and intestinal transit may have implications for gastrointestinal research and metabolic disorders.

C59H90O27

1231.33

1230.566

220114-28-3

9920037

White to off-white solid

-0.4

13

27

17

86

2550

30

C/C=C(/C)\C(=O)O[C@H]1[C@@H]([C@@]2([C@@H](C[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H]([C@@]5(C)C=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)C(=O)O)O)O[C@H]7[C@@H]([C@H]([C@H](CO7)O)O)O[C@H]8[C@@H]([C@H]([C@@H](CO8)O)O)O)O[C@H]9[C@@H]([C@H]([C@H]([C@H](O9)CO)O)O)O)C)C)[C@@H]2CC1(C)C)C)O)CO)OC(=O)C

WWVKOCDDDWJQLC-MWQJAWBESA-N

InChI=1S/C59H90O27/c1-10-24(2)49(76)86-46-47(79-25(3)63)59(23-62)27(17-54(46,4)5)26-11-12-32-55(6)15-14-34(56(7,22-61)31(55)13-16-57(32,8)58(26,9)18-33(59)66)81-53-45(85-51-40(72)38(70)37(69)30(19-60)80-51)42(41(73)43(83-53)48(74)75)82-52-44(36(68)29(65)21-78-52)84-50-39(71)35(67)28(64)20-77-50/h10-11,22,27-47,50-53,60,62,64-73H,12-21,23H2,1-9H3,(H,74,75)/b24-10-/t27-,28+,29-,30+,31+,32+,33+,34-,35-,36-,37-,38-,39+,40+,41-,42-,43-,44+,45+,46-,47-,50-,51-,52-,53+,55-,56-,57+,58+,59-/m0/s1

(2S,3S,4S,5R,6R)-6-[[(3S,4S,4aR,6aR,6bS,8R,8aR,9R,10R,12aS,14aR,14bR)-9-acetyloxy-4-formyl-8-hydroxy-8a-(hydroxymethyl)-4,6a,6b,11,11,14b-hexamethyl-10-[(Z)-2-methylbut-2-enoyl]oxy-1,2,3,4a,5,6,7,8,9,10,12,12a,14,14a-tetradecahydropicen-3-yl]oxy]-4-[(2S,3R,4S,5S)-4,5-dihydroxy-3-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxyoxan-2-yl]oxy-3-hydroxy-5-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-2-carboxylic acid

Theasaponin E1

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)