COX-1 and COX-2 (IC50 for COX-1: 116.3 ± 0.03 μM; IC50 for COX-2: 94.7 ± 0.02 μM) [1]

With IC50 values of 34.2 and 28.4 μM at 24 and 48 hours, respectively, taraxerolacetate (10-150 μM; 24 or 48 hours) causes dose- and time-dependent cytotoxicity in U87 cells [2]. The proportion of apoptotic cells increased from 7.3% in control cells to 10, 50, and 150 µM dandelion alcohol acetate-treated cells after dandelion alcohol acetate (10, 50, and 150 µM) was applied for 24 hours. 16.1, 44.1, and so forth. Moreover, sub-G1 cell cycle stoppage and a related decrease in S-phase cell count were seen upon dandelion alcohol acetate administration [2].

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Taraxerol acetate showed potent antiproliferative effects against U87 human glioblastoma cells in a dose- and time-dependent manner. The half maximal inhibitory concentration (IC50) was 34.2 μM at 24 h and 28.4 μM at 48 h as determined by MTT assay [2].
Morphological changes observed under phase contrast microscopy: untreated U87 cells appeared as tightly packed multilayers, while cells treated with 10, 50, or 150 μM Taraxerol acetate became rounded, shrunken, disconnected, or floating [2].
Acridine orange/ethidium bromide double staining showed that living control cells had large green nuclei, whereas treatment with 10, 50, or 150 μM Taraxerol acetate markedly reduced the number of cells with intact nuclei and induced nuclear condensation and apoptotic body formation at 150 μM [2].
Annexin V-FITC/PI flow cytometry revealed that Taraxerol acetate induced early and late apoptosis in a dose-dependent manner. The percentage of apoptotic cells increased from 7.3% in control to 16.1% (10 μM), 44.1% (50 μM), and 76.7% (150 μM) after 48 h treatment [2].
Cell cycle analysis by flow cytometry showed that Taraxerol acetate treatment (10, 50, 150 μM for 48 h) increased the sub-G1 phase population from 2.4% (control) to 18.6%, 33.21%, and 48.6%, respectively, while decreasing the S-phase population from 33.15% (control) to 27.21%, 16.21%, and 12.9%, respectively [2].
Taraxerol acetate induced dose-dependent DNA fragmentation (a hallmark of apoptosis) in U87 cells as visualized by agarose gel electrophoresis after treatment with 10, 50, or 150 μM for 48 h [2].
Western blot analysis showed that Taraxerol acetate increased the protein expression of the CDK inhibitor p21 in a dose-dependent manner, and decreased the expression of cyclin B, cyclin D, CDK2, CDK4, and CDK6 compared to control [2].
Taraxerol acetate induced autophagy in U87 cells as evidenced by increased protein expression of LC3B-II in a dose-dependent manner (0, 10, 50, 150 μM) and time-dependent manner (3, 6, 12, 24 h) [2].
Wound healing assay demonstrated that Taraxerol acetate (50 and 150 μM for 48 h) significantly reduced the number of U87 cells migrating into the scratched area in a dose-dependent manner [2].

In a female BALB/c nude mouse xenograft model bearing U87 human glioblastoma cells, intraperitoneal injection of Taraxerol acetate at 0.25 and 0.75 μg/g once orally (as described in the protocol, though injection route was intraperitoneal) significantly reduced tumor weight from 1.2 g in PBS-treated control to 0.81 g and 0.42 g, respectively, and reduced tumor volume from 1.3 cm³ in control to 0.67 cm³ and 0.25 cm³, respectively, after 24 days [2].

The COX inhibition assay was performed using Tris buffer (50 mM; pH 7.5) to dissolve COX-1 and COX-2 enzymes, co-factors adrenaline (5 mM) and hematin (1 μM). Hematin was first dissolved in NaOH. The reaction mixture was added to 96-well plates at a final volume of 190 μL per well. Different concentrations of Taraxerol acetate were diluted in Tris buffer and placed into a 96-well plate, then cofactor and enzyme (10 U/mL; 180 μL) were added. The plates were incubated at 37°C for 30 min before adding 10 μL of 1 M HCl to stop the reaction, followed by 10 μL of 1 M NaOH to adjust the pH to 7.5. The formation of PGE₂ (a metabolite of PGH₂) was measured by radioimmunoassay to determine enzyme activity and the inhibitory effect of different concentrations of Taraxerol acetate. Data were presented as percentage of control PGE₂ production. All experiments were performed in triplicate, and IC₅₀ values were determined using the EZFit Enzyme Kinetics program. Results were expressed as Mean ± SEM [1].

Cell proliferation was assessed by MTT assay: U87 cells (1×10⁵ cells/well) were seeded into 96-well plates and incubated for 5-7 h at 37°C for attachment. After treatment with Taraxerol acetate (0, 5, 10, 25, 50, 100, 150 μM) for 48 h, MTT solution (10 μL; 5 mg/mL in PBS) was added for 4 h at 37°C. Formazan crystals were dissolved in 150 μL DMSO, and absorbance was measured at 490 nm using a microplate reader. Cell viability inhibition ratio (%) was calculated as [OD₄₉₀(control) - OD₄₉₀(treated)] / OD₄₉₀(control) × 100 [2].
Phase contrast and fluorescence microscopy: U87 cells (1×10⁵ cells/mL) were plated in 6-well plates for 24 h, then treated with Taraxerol acetate (0, 10, 50, 150 μM) for 48 h. Cells were examined under phase contrast microscope, and images were captured. For fluorescence staining, cells were washed twice with PBS, then AO/EB solution (10 μg/mL) was added for 30 min at 37°C, and images were captured using a fluorescence microscope [2].
Annexin V-FITC/PI apoptosis assay: U87 cells treated with Taraxerol acetate (0, 10, 50, 150 μM) for 48 h were stained with PI and Annexin V-FITC according to manufacturer's instructions. The percentages of viable, apoptotic, and necrotic cells were analyzed by flow cytometry using CellQuest software [2].
Cell cycle analysis: U87 cells (1×10⁵) treated with Taraxerol acetate (0, 10, 50, 150 μM) for 48 h were collected, washed with ice-cold PBS twice, fixed with 70% alcohol at 4°C for 12 h, stained with PI in the presence of 3% RNase A at 37°C for 20 min, and analyzed by flow cytometry. The percentage of cells in each cell cycle phase was determined using ModFit LT software [2].
DNA fragmentation analysis: U87 cells in 100-mm dishes were treated with Taraxerol acetate (0, 10, 50, 150 μM) for 48 h. Cells were harvested, lysed with DNA lysis buffer (2% NP-40, 20 mM EDTA, 40 mM Tris-HCl) for 30 min, centrifuged at 6,600 × g for 5 min. Supernatants were mixed with equal volume of 1.5% SDS, incubated with 2.5 mg/mL RNase A at 60°C for 2 h, then with 2.5 mg/mL proteinase K for 2 h at 20°C. After adding 0.5 volumes of 10 M ammonium acetate, DNA was precipitated with cold ethanol, collected by centrifugation at 6,600 × g for 20 min, dissolved in gel loading buffer, separated by electrophoresis in 1.5% agarose gel (1 h at 100 V), and visualized under UV light after ethidium bromide staining [2].
Wound healing (cell migration) assay: U87 cells (1×10⁵ cells/mL) were seeded into 6-well plates and incubated at 37°C for 24 h until 95% confluent. After 12 h starvation, a straight cell-free wound was created using a pipette tip. Each well was washed three times with PBS to remove debris, then treated with Taraxerol acetate (0, 10, 50, 150 μM) in DMEM. After 48 h incubation at 27°C, cells were fixed and stained with 5% ethanol containing 0.3% crystal violet for 30 min. Images were captured under an inverted research microscope, and the number of cells that migrated into the scratched area was counted; wound lengths were determined by ImageJ software [2].
Western blot assay: Total protein was extracted from U87 cells and concentration measured using a bicinchoninic acid protein assay kit. Protein samples (100 μg) were run on SDS-polyacrylamide gel (3 h at 70 V) and transferred to nitrocellulose membranes. Membranes were blocked with 5% bovine serum albumin for 2 h at room temperature, incubated with primary antibodies (anti-p21, anti-cyclin B, anti-cyclin D, anti-CDK2, anti-CDK4, anti-CDK6, anti-LC3B, anti-α-tubulin, anti-GAPDH) overnight at 4°C, then with relevant secondary antibodies for 1 h at room temperature. Bands were visualized using a ChemiDoc MP Imaging System [2].

Female BALB/c nude mice (age 8 weeks; weight 20 g) were housed in a pathogen-free environment with a 12-h light/dark cycle, provided water and food ad libitum. U87 cells (1×10⁵ cells/mouse) were subcutaneously injected into the right rear flank of each mouse to generate tumors. After tumor generation, mice were divided into three groups (n=5) and treated once orally (though the compound was injected intraperitoneally per the text) with 1X PBS (control), 0.25 μg/g Taraxerol acetate, or 0.75 μg/g Taraxerol acetate (injected intraperitoneally). Mice were sacrificed after 24 days by cervical dislocation. Tumors were removed, and tumor weight and volume (calculated as length × width × 0.5 width) were measured [2].

[1]. Molecular docking of taraxerol acetate as a new COX inhibitor. Journal of the Bangladesh Pharmacological Society.

[2]. Anticancer activity of taraxerol acetate in human glioblastoma cells and a mouse xenograft model via induction of autophagy and apoptotic cell death, cell cycle arrest and inhibition of cell migration.Mol Med Rep. 2016 Jun;13(6):4541-8.

According to reports, taraxacin acetate has been found in Codonopsis pilosula, Codonopsis pilosula var. pubescens, and other organisms with available data.

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Taraxerol acetate was isolated from the ethyl acetate fraction of Artemisia roxburghiana methanolic extract via silica gel column chromatography using hexane-ethyl acetate mixtures. The structure was confirmed by NMR and mass spectral data compared with previously reported data [1].
The compound has ethnomedicinal background: A. roxburghiana is used in Pakistan to treat fever, rheumatism, malaria, dysentery, hepatitis, and diabetes. The current investigation supports its anti-inflammatory use via COX inhibition [1].
Molecular docking simulations using AutoDock 4.2 (with Lamarckian genetic algorithm, grid 60×60×60 points centered on the catalytic pocket, grid spacing 0.375 Å) on COX-2 (PDB ID: 3BGP) revealed that Taraxerol acetate interacts with the active site via hydrophobic interactions: methyl groups at C-29 and C-30 interact with Val116 and Leu531; Val349 interacts with C-11 and C-12 of ring C; Leu359 interacts with ring E; the acetyl group shows electrostatic interactions with Trp387, Met522, and the amide group of Leu384. Flurbiprofen was used as a reference (binding energy RMSD 0.453) [1].
In the glioblastoma study, Taraxerol acetate induced both apoptotic and autophagic cell death, increased sub-G1 arrest, decreased S-phase cells, upregulated p21, downregulated cyclins and CDKs, and inhibited cell migration. These effects suggest its potential as a therapeutic agent against glioblastoma [2].

C32H52O2

468.7541

468.396

C, 81.99; H, 11.18; O, 6.83

2189-80-2

94225

Solid powder

1.0±0.1 g/cm3

505.1±49.0 °C at 760 mmHg

303-305ºC

256.2±17.4 °C

0.0±1.3 mmHg at 25°C

1.529

11.95

0

2

2

34

897

8

O(C(C([H])([H])[H])=O)[C@@]1([H])C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@@]([H])(C([H])([H])C([H])([H])[C@@]3(C([H])([H])[H])C4=C([H])C([H])([H])[C@]5(C([H])([H])[H])C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[C@@]5([H])[C@]4(C([H])([H])[H])C([H])([H])C([H])([H])[C@]23[H])C1(C([H])([H])[H])C([H])([H])[H]

YWJGYBXHXATAQY-BOTWUFHUSA-N

InChI=1S/C32H52O2/c1-21(33)34-26-13-17-30(7)22(28(26,4)5)11-15-31(8)23-10-14-29(6)19-18-27(2,3)20-25(29)32(23,9)16-12-24(30)31/h10,22,24-26H,11-20H2,1-9H3/t22-,24+,25+,26-,29-,30-,31-,32+/m0/s1

[(3S,4aR,6aR,6aS,8aR,12aR,14aR,14bR)-4,4,6a,6a,8a,11,11,14b-octamethyl-1,2,3,4a,5,6,8,9,10,12,12a,13,14,14a-tetradecahydropicen-3-yl] acetate

Taraxerol acetate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)