lipid regulating agent； β-Sitosterol is a multi-target natural product with multiple identified direct or indirect molecular targets. Molecular docking studies have shown that β-sitosterol can bind to estrogen receptor beta (binding energy of -6.14 kcal/mol) and caspase-3 (binding energy of -7.98 kcal/mol). Recent studies have identified catalase as a direct target of β-sitosterol, with the compound forming hydrogen bonds in the catalase pocket and enhancing its enzymatic activity. Furthermore, β-sitosterol exerts its effects by inhibiting the activation of the PI3K/Akt/mTOR signaling pathway. Other predicted targets include inflammation-related proteins such as cyclooxygenase-2 and NF-κB.

Beta-Sitosterol is one of the most abundant dietary phytosterols. According to one study, beta-sitosterol has the ability to prevent leukemia, ovarian cancer, breast, prostate, colon, lung, stomach, and colon cancer.

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In vitro studies confirm that β-sitosterol exhibits extensive biological activities. Regarding anti-proliferative activity, β-sitosterol shows IC₅₀ values of 187.61 µg/mL against MCF7 breast cancer cells and 874.156 µg/mL against MDA-MB-231 triple-negative breast cancer cells. Morphological observations show that β-sitosterol treatment induces apoptotic bodies (cell membrane blebbing) in MCF7 cells. Regarding anti-inflammatory activity, β-sitosterol concentration-dependently scavenges DPPH free radicals, achieving a clearance rate of 78.12% at 250 µg/mL. In atherosclerosis studies, β-sitosterol (5-20 µM) inhibits oxLDL-induced lipid deposition and phenotypic transformation in vascular smooth muscle cells. Mechanistic studies reveal that β-sitosterol treatment upregulates caspase-9 and caspase-3 mRNA expression, inducing apoptosis in cancer cells.

The total cells and eosinophils in the bronchoalveolar lavage (BAL) fluid markedly decreased (p<0.05) after L-BS or beta-sitosterol (BS) administration (1 mg/kg; i.p.), and the ROS production also decreased in comparison to the asthma control. Histopathological features were detected by performing histochemistry, including H&E and alcian blue & P.A.S staining. Both L-BS and beta-sitosterol (BS) mitigated the inflammation by eosinophil infiltration and mucus hypersecretion by goblet hyperplasia. These effects of L-BS were superior to those of BS. L-BS and BS inhibited the increased mRNA and protein expression of IL-4 and IL-5 in the lung tissue and BAL fluid, respectively. The IgE concentration in the BAL fluid and serum was measured by performing ELISA and the ovalbumin-specific IgE in the BAL fluid was uniquely inhibited by L-BS (p<0.05). The splenocytes were isolated from the normal and asthmatic mice and incubated in the absence and presence of 100 microg/ml ovalbumin, respectively. L-BS blocked the survival rate of the splenocytes of the mice (p<0.01). This finding indicates the possibility of L-BS and BS as potential therapeutic molecules in asthma and may contribute to the need to improve current therapeutic drugs. [2]

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In vivo studies confirm that β-sitosterol exhibits significant activity in various animal models. In ApoE⁻/⁻ mouse models of atherosclerosis, β-sitosterol (25, 50, 100 mg/kg) dose-dependently reduces aortic sinus lesion areas and intraplaque lipid deposition, while decreasing serum total cholesterol and triglyceride levels. Anti-inflammatory activity studies show that β-sitosterol exhibits 50-70% inhibition in rat paw edema assays, reduces pleural exudate volume by 46% in rat pleurisy assays, and decreases neutrophil counts by 20%. In mouse ear edema assays, β-sitosterol achieves an average inflammatory inhibition of 75%. In xenograft tumor models, dietary β-sitosterol reduces tumor volume by one-third and decreases lymph node and lung metastases by 20%. In prostate cancer xenograft models, β-sitosterol (100 mg/kg twice daily by oral gavage) significantly inhibits tumor growth.

Protocol for determining the effect of β-sitosterol on catalase activity: Compound Preparation: Dissolve β-sitosterol in DMSO to prepare stock solution, dilute to working concentrations (e.g., 5, 10, 20 µM) with reaction buffer. Enzyme Source Preparation: Use recombinantly expressed or purified catalase protein. Reaction System: Mix varying concentrations of β-sitosterol with catalase and hydrogen peroxide substrate in reaction buffer, incubate at 37°C. Product Detection: Measure hydrogen peroxide consumption spectrophotometrically (decrease in absorbance at 240 nm). Data Analysis: Calculate enzyme activity inhibition or enhancement rates. Results show that β-sitosterol concentration-dependently enhances catalase activity.

Viability of mouse splenocytes [2]
We performed MTT assay and apoptosis assay to determine the cell viability with using the MTT assay kit and annexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit. The splenocytes were isolated from the spleens of normal and asthmatic control mice by syringe pumping. After three washings with 10 ml of DMEM supplemented with antibiotics–antimycotics, the splenocytes were incubated with 3 ml of RBC lysis buffer for 10 min at room temperature, and then this was washed twice with 10 ml of the wash medium. 2 × 105 splenocytes in 100 μl of DMEM medium containing 10% FBS were seeded onto a 96-well culture plate. L-BS, BS or dexamethasone (1 μg/ml) in the absence or presence of 100 μg/ml ovalbumin was added to the individual well and then the plate was incubated for 48 h at 37 °C in a CO2 incubator. After the addition of 10 μl of MTT solution in each well, the plate was incubated at 37 °C for 4 h in a CO2 incubator and 100 μl of solubilization solution was added to each well for MTT assay. After 24 h incubation, the absorbance was measured by using an ELISA reader at 550 nm. For the apoptosis assay, the cells were harvested and resuspended in binding buffer. Annexin V-FITC and PI were added and incubated for 15 min at room temperature. The cells were analyzed by FACSort cytofluorimeter using CellQuest software. Negative cells against annexin V and PI staining were considered as viable or non-apoptotic cells.[2]

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Cell Culture: Culture MCF7 breast cancer cells, MDA-MB-231 cells, or vascular smooth muscle cells (VSMCs) in DMEM/RPMI-1640 medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. Drug Treatment: Dissolve β-sitosterol in DMSO to prepare stock solution, dilute to working concentrations (e.g., 5-20 µM for VSMCs; 187-874 µg/mL for cancer cells) with culture medium, and treat cells for 24-72 hours. Keep final DMSO concentration below 0.1%. Viability Assay: Assess cell viability using PrestoBlue or MTT assays to calculate IC₅₀ values. Lipid Deposition Detection: Evaluate lipid deposition in VSMCs by Oil Red O staining. Apoptosis Detection: Detect caspase-9 and caspase-3 expression by Western blot; verify binding to target proteins by molecular docking. Data Analysis: Compare cell viability, apoptosis markers, and lipid deposition levels between treatment and control groups.

Induction of asthma in mice[2]
Six to eight-week-old female BALB/c mice were obtained from Daehan Biolink Co. LTD. They were maintained in an air-conditioned room. The room temperature (about 22 ± 1 °C) and humidity (about 55 ± 10%) were automatically controlled. The mice were divided into five groups (n = 5), and airway inflammation was induced in four groups. Each mouse was immunized through intraperitoneal (i.p.) injection with 20 μg of chicken OVA and 1 mg of aluminum hydroxide on days 1 and 14, as shown in Fig. 2. The mice were exposed to a 5% ovalbumin solution aerosolized using an ultrasonic nebulizer for 1 h per day from days 21 to 27 after the second sensitization. The mice were placed in a Plexiglass chamber (30 × 30 × 15 cm3) that contained small ventilation holes on one side during the inhalation challenge. The aerosol was generated with a nebulization rate of 1 ml/min. Three groups of asthma-induced mice were treated through i.p. injection with 1 mg/kg of L-BS, BS or dexamethasone between days 14 and 27. Both L-BS and BS dissolved in DMSO diluted less than 1/100 by phosphate-buffered saline (PBS). The negative control group was sensitized and challenged with PBS without drug administration.

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Animal Models: Establish atherosclerosis models using ApoE⁻/⁻ mice (male, 6-8 weeks old) fed a high-fat diet for 12 weeks; establish xenograft tumor models using immunodeficient nude mice (e.g., BALB/c nude mice) via subcutaneous injection of tumor cells. Dosing Regimen: Administer β-sitosterol by oral gavage. In atherosclerosis models, doses are 25, 50, 100 mg/kg once daily for 4 weeks. In xenograft models, the dose is 100 mg/kg twice daily (3 days on/4 days off cycle). Efficacy Assessment: Atherosclerosis: Assess aortic sinus lesion area by H&E and Oil Red O staining; measure serum TC and TG levels by biochemical assays; detect platelet activation by flow cytometry. Anti-inflammatory Activity: Measure paw edema volume, pleural exudate volume, and myeloperoxidase activity. Antitumor: Measure tumor volume every 2-3 days using calipers with the formula (length × width²) × 0.52; excise and weigh tumors at endpoint. Data Analysis: Compare lesion areas, tumor volumes, and biochemical parameters between treatment and control groups.

The oral bioavailability of β-sitosterol is relatively low, typically ranging from 0.5% to 5%. Pharmacokinetic studies in Beagle dogs show that following intravenous administration, the concentration-time profile of β-sitosterol fits a two-compartment open model, with a distribution half-life (t₁/₂α) of approximately 3 hours and a terminal elimination half-life (t₁/₂β) of approximately 129 hours. The volume of distribution of the central compartment is 0.56 L/kg (corresponding to total body fluid), and the apparent volume of distribution is approximately 0.92 L/kg (corresponding to total body weight). The mean residence time is approximately 185 hours. The absolute bioavailability following oral administration is approximately 9%, with PEG embedding significantly increasing the absorption rate but not the extent of absorption. Nanoformulations can significantly improve bioavailability: following oral administration of β-sitosterol nanosuspension in dogs, Cmax increases from 152.3 ng/mL to 546.8 ng/mL, with relative bioavailability reaching 239%.

The oral LD50 in mice was >25 gm/kg. (Cancer Letters, 127(135), 1998 [PMID:9619869])
β-Sitosterol exhibits a favorable safety profile with no significant toxicity observed in extensively studied animal models and clinical trials. Acute toxicity data: The oral LD₅₀ in mice has not been clearly reported, but no severe toxic reactions have been observed at conventional experimental doses. In ApoE⁻/⁻ mouse models of atherosclerosis, β-sitosterol (up to 100 mg/kg) administered daily for 4 weeks showed no abnormal body weight loss or behavioral changes. In xenograft tumor models, β-sitosterol treatment caused no obvious toxicity. However, plasma concentrations of β-sitosterol are in the micromolar range, and the effects of long-term high-dose use require further investigation. This compound is for research use only; standard laboratory safety practices and appropriate personal protective equipment should be used when handling.

[1]. Beta-Sitosterol: A Promising but Orphan Nutraceutical to Fight Against Cancer. Nutr Cancer. 2015;67(8):1214-20.
[2]. Int Immunopharmacol. 2007 Dec 5;7(12):1517-27. doi: 10.1016/j.intimp.2007.07.026.

Sitosterol is a phytosterol compound with a structure of stigmaster-5-ene substituted with a β-hydroxyl group at the 3-position. It has multiple functions, including as a sterol methyltransferase inhibitor, a cholesterol-lowering drug, an antioxidant, a plant metabolite, and a rodent metabolite. It is a 3β-sterol, stigmasterol, 3β-hydroxy-Δ(5)-steroid, C29-steroid, belonging to the phytosterol class of compounds. It is derived from the hydrogenation of stigmasterane. The active ingredient in Solanum trilobatum can alleviate the toxic side effects of radiation. It has been reported that β-sitosterol is also found in elderberry (Sambucus chinensis), Rhodiola rosea (Erythrophleum fordii), and several other organisms with relevant data. Currently, all available cancer treatments are expensive, and none are safe. However, traditional plant-derived drugs or compounds are relatively safe. β-sitosterol (BS) is a well-known compound that is a plant-derived nutrient with anticancer properties and can fight breast cancer, prostate cancer, colon cancer, lung cancer, gastric cancer, ovarian cancer and leukemia. Studies have shown that BS can interfere with a variety of cell signaling pathways, including cell cycle, apoptosis, proliferation, survival, invasion, angiogenesis, metastasis and inflammation. Most studies are incomplete, partly because BS is relatively low in efficacy. However, almost all research communities have overlooked the fact that it is generally considered non-toxic, which is the opposite of all currently available anticancer chemotherapy drugs. To compensate for the low efficacy of BS, there is great potential to design BS delivery systems that are "cancer cell specific". Liposome delivery of BS is one of the most promising approaches. However, no further research progress has been made in either the field of β-sitosterol (BS) drug delivery or in how to improve BS-mediated anticancer activity, so BS is still regarded as an orphan nutritional supplement. Therefore, it is strongly recommended that BS be extensively studied as a potent anticancer nutritional supplement. [1] Asthma is a disease characterized by chronic lung inflammation, and the number of asthma patients is increasing year by year. β-Sitosterol (BS) and β-sitosterol glucoside are found in various plants and possess antitumor, antibacterial, and immunomodulatory activities. However, the exact roles of BS and β-sitosterol glucoside in asthma remain unclear. This study aimed to investigate the inhibitory effects of BS and lactose-β-sitosterol (L-BS) on ovalbumin-induced pathophysiological processes in asthmatic mice. Administration of L-BS or BS (1 mg/kg; intraperitoneal injection) significantly reduced the total cell count and eosinophil count in bronchoalveolar lavage fluid (BALF) (p<0.05), and also reduced reactive oxygen species (ROS) production compared to the asthma control group. Histopathological features were examined by histochemical staining (including hematoxylin-eosin staining and alicin blue-PAS staining). Both L-BS and BS alleviated inflammation caused by eosinophil infiltration and excessive mucus secretion caused by goblet cell hyperplasia. These effects of L-BS were superior to those of BS. L-BS and BS inhibited the increase of IL-4 and IL-5 mRNA and protein expression in lung tissue and BALF, respectively. The concentration of IgE in BALF and serum was detected by ELISA. The results showed that L-BS specifically inhibited the level of ovalbumin-specific IgE in BALF (p<0.05). Spleen cells were isolated from normal mice and asthmatic mice and incubated with or without 100 μg/ml ovalbumin. L-BS inhibited the survival rate of mouse spleen cells (p<0.01). This finding suggests that L-BS and BS may be potential therapeutic molecules for asthma and may help improve existing therapeutic drugs. [2]

C29H50O

414.71

414.386

C, 83.99; H, 12.15; O, 3.86

83-46-5

222284

White to off-white solid powder

1.0±0.1 g/cm3

501.9±19.0 °C at 760 mmHg

139-142 ºC

220.4±13.7 °C

0.0±2.9 mmHg at 25°C

1.521

10.73

1

1

6

30

634

9

C[C@@]12[C@@H]([C@H](C)CC[C@@H](CC)C(C)C)CC[C@H]1[C@@H]1CC=C3C[C@H](CC[C@]3(C)[C@H]1CC2)O

KZJWDPNRJALLNS-VJSFXXLFSA-N

InChI=1S/C29H50O/c1-7-21(19(2)3)9-8-20(4)25-12-13-26-24-11-10-22-18-23(30)14-16-28(22,5)27(24)15-17-29(25,26)6/h10,19-21,23-27,30H,7-9,11-18H2,1-6H3/t20-,21-,23+,24+,25-,26+,27+,28+,29-/m1/s1

(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5-ethyl-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

(-)-beta-Sitosterol; 22,23-Dihydrostigmasterol; 24-alpha-Ethylcholesterol; AI3-26020; alpha-Dihydrofucosterol; Rhamnol; Angelicin; beta-Sitosterol; CCRIS 5529; Azuprostat; Cinchol; Cupreol; Harzol; Nimbosterol; Prostasal; Quebrachol; Triastonal

2934.99.03.00

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)

Solubility in Formulation 1: 20 mg/mL (48.23 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: ≥ 1 mg/mL (2.4 mM) (saturation unknown) in 10% EtOH + + 40% PEG300 + 5% Tween80 + + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 25 mg/mL EtOH + stock solution and add to 400 μL of PEG300, mix well; Then add 50 μL of Tween 80 to the above solution, mix well; Finally, add 450 μL of saline to the above solution, mix well.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (2.4 mM) (saturation unknown) in 10% EtOH + + 90% (20% SBE-β-CD in saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can take 100 μL of 25 mg/mL EtOH + stock solution and add to 900 μL of 20% SBE-β-CD in saline, mix well.
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 4: ≥ 1 mg/mL (2.4 mM) (saturation unknown) in 10% EtOH + + 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 take 100 μL of 25 mg/mL EtOH + stock solution and add to 900 μL of corn oil, mix well (clear solution).

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Solubility in Formulation 5: ~5 mg/mL (12.1 mM) in 15% Cremophor EL + + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution.

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Solubility in Formulation 6: ~10 mg/mL (24.1 mM) in Corn Oil , clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)