COX-2; β-Elemonic acid is a multi-target molecule, with a confirmed direct target of prostaglandin E synthase (IC₅₀ = 900 nM). Computational predictions suggest additional targets including cathepsin D (96.03%), cyclooxygenase-1 (95.32%), NF-κB p105 subunit (92.94%), MAP kinase ERK2 (86.90%), and glucocorticoid receptor (75.63%). Experimental studies demonstrate that β-elemonic acid inhibits phosphorylation of p42/44, MAPK/JNK, and p38, while activating the PERK/eIF2α/ATF4/CHOP endoplasmic reticulum stress pathway.

In human A549 lung cancer cells, β-elemonic acid (1, 3, 10, 20 µM; 24 hours) significantly promotes apoptosis in a time- and dose-dependent manner [1]. Human NSCLC A549 cells are subjected to strong cytotoxic effects from β-elemonic acid (1, 3, 10, 20 µM; 24 hours) that are dose-dependent. Following a 24-hour exposure to beta-citrate, the IC50 value is 6.92 µM[1]. G0/G1 phase cell percentage at 24 hours with 20 µM beta-citrate was 58.01%[1]. In A549 cells, β-elemonic acid (1, 3, 10, 20 µM; 24 hours) suppresses p42/44, MAPK/JNK, and p38 phosphorylation [1].

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In vitro studies demonstrate that β-elemonic acid exhibits significant anti-proliferative activity against various cancer cells. In human non-small cell lung cancer A549 cells, β-elemonic acid inhibits cell viability in a dose- and time-dependent manner, with a 24-hour IC₅₀ of 6.92 μM. This compound (20 μM for 24 hours) increases the percentage of A549 cells in the G0/G1 phase to 58.01%, induces phosphatidylserine externalization (Annexin V-positive), and elevates ROS levels. In osteosarcoma HOS and 143B cells, β-elemonic acid (1.0-50 μM) dose- and time-dependently inhibits cell viability, induces ER stress-mediated apoptosis, and suppresses the Wnt/β-catenin signaling pathway. The compound shows no significant cytotoxicity against normal WI-38 lung epithelial cells. Additionally, β-elemonic acid inhibits prolyl endopeptidase activity with an IC₅₀ of 39.74 μM.

EA significantly suppressed the growth of transplanted colorectal tumors in nude mice [3].
In vivo studies confirm the anti-tumor activity of β-elemonic acid in osteosarcoma xenograft models. In nude mice bearing osteosarcoma tumors, β-elemonic acid treatment significantly inhibits tumor growth, induces ER stress-mediated mitochondria-dependent apoptosis, and suppresses the Wnt/β-catenin signaling pathway. The compound distributes to both tumor and bone tissues, suggesting potential bone-targeting properties. Furthermore, β-elemonic acid exhibits rapid absorption, a short elimination half-life, and linear pharmacokinetic characteristics in vivo.

Xanthine Oxidase Inhibitory Assay [1].
Xanthine oxidase (XO) (EC 1.1.3.22) inhibition activity was assayed in phosphate buffer (0.1 M, pH 7.5, 250 μL) and XO (0.003 unit/well, 20 μL), and the test sample in 10 μL of DMSO was diluted to the desired range of concentrations, mixed in a 96-well microplate, and preincubated for 10 min at room temperature. The reaction was initiated by adding 20 μL of 0.1 mM xanthine. The uric acid formation was measured spectrophotometrically at 295 nm by using a microtiter plate reader (Molecular Devices, Spectramax 384).

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Phosphodiesterase I Inhibitory Assay[1].
Activity against phosphodiesterase I (Sigma P 4631) (EC 3.1.4.1) was assayed by using the reported method with the following modifications:  33 mM tris-HCl buffer pH 8.8, 30 mM Mg(C2H3O2)2·4H2O with 0.000742 U/well final concentration using microtiter plate assay, and 0.33 mM bis(p-nitrophenyl) phosphate (Sigma N-3002) as a substrate. Cystein and EDTA were used as positive controls (IC50 = 748 ± 15.00 and 274 ± 7.00 μM, respectively). After 30 min of incubation, the enzyme activity was monitored spectrophotometrically at 37 °C on a microtiter plate reader by following the release of p-nitrophenyl phosphate at 410 nm. Assays were conducted in triplicate.

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PEP Inhibitory Activity[1].
Prolyl endopeptidase (EC 3.4.21.26) was purchased from Seikagaku Corporation (Tokyo, Japan). N-Benzyloxycarbonyl-Gly-Pro-pNA and bacitracin were purchased from BACHEM Fine Chemicals Co. and Sigma Co., Ltd., respectively. PEP inhibitory activities were measured by a method developed by Yoshimoto 21 et al. and described in our previous publications.

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Enzyme Source Preparation: Use recombinantly expressed human prostaglandin E synthase. Substrate Preparation: Use prostaglandin H₂ as substrate. Inhibitor Incubation: Pre-incubate varying concentrations of β-elemonic acid (0.1-10 μM) with the enzyme in reaction buffer containing glutathione at 4°C. Reaction Initiation and Termination: Initiate the reaction by adding substrate and terminate after appropriate incubation. Product Detection: Quantify prostaglandin E₂ production by ELISA. Data Analysis: Calculate the IC₅₀ value of 900 nM.

Colony Formation
CRC cells were cultured in 12-well plates at 37°C with 5% CO2 for 12 h until the cells adhered to the wall. Next, they were treated with 0–20 μg/ml of EA for 10 days. The cells were stained using crystal violet solution according to the manufacturer’s instructions.

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Cell Migration Assay
Cell migration assay was performed to check the inhibitory effect of EA on migration of CRC cells. Cells were seeded at 40% confluence into 12-well plates around culture inserts and incubated at 37°C with 5% CO2. After 12 h, the inserts were removed, and the suspension cells were washed with phosphate-buffered saline (PBS). Fresh medium supplemented with 0–20 μg/ml EA was added. After 24 h incubation period, the width of cells scratches was observed under a microscope, and images were captured.

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DNA Synthesis Assay
DNA synthesis was examined using the EDU-594 Cell Proliferation Assay Kit, according to the manufacturer’s instructions, and staining results were recorded under a fluorescence microscope

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Cell Culture: Culture A549 human lung cancer cells or HOS/143B osteosarcoma cells in RPMI-1640/DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO₂ incubator. Drug Treatment: Dissolve β-elemonic acid in DMSO to prepare stock solution, dilute to working concentrations (e.g., 1.0-50 μM) with culture medium, and treat cells for 24-72 hours. Viability Assay: Measure cell viability by MTT assay; the 24-hour IC₅₀ in A549 cells is 6.92 μM. Apoptosis Detection: Detect apoptosis using Annexin V-FITC/PI double staining by flow cytometry. Cell Cycle Analysis: Analyze cell cycle distribution by PI staining and flow cytometry. ROS Detection: Measure reactive oxygen species levels using the DCFH-DA fluorescent probe. Western Blot Analysis: Detect expression of MAPK pathway proteins, COX-2, CHOP, Wnt/β-catenin, and other related proteins.

Xenografts
The mice experiment was approved by the ethics committee of Jining medical university, China. Female BALB/c nude mice were subcutaneously injected with 5 × 106 SW480 cells and randomly divided into two groups (n = 5 in each group). When the tumor reached a volume of 200 mm3, the mice were intraperitoneally injected with 10 mg/kg of EA (treatment group) or DMSO (vehicle) every 2 days for 24 days. All mice were euthanatized with CO2 at 24 days post-injection, and their tumors, kidneys, livers, and hearts were placed in 10% formalin for fixation.

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Hematoxylin and Eosin, Immunochemistry, and Immunofluorescence Staining
Mouse tissues were fixed with 10% formalin, dehydrated, and embedded into paraffin blocks. The paraffin-embedded specimens were sectioned at a thickness of 4 μm using a microtome. The sections were dewaxed and stained with H&E. After being dewaxed and antigen repaired, the sections were stained for IHC and IF staining. The primary antibodies used in IHC were Rabbit-anti Ki67 and Rabbit-anti FTL (1:100; Proteintech, China). The secondary antibodies and 3, 3′-diaminobenzidine were purchased from Boster Bio. In IF staining, Rabbit-LC3 (1:250, Proteintech) was used as the primary antibody, and Goat-anti rabbit IgG H&L (Alexa Fluor 488, 1:400; Abcam) was used as the secondary antibody.

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Animal Model: Use 4-6 week-old female BALB/c nude mice to establish xenograft tumor models via subcutaneous injection of osteosarcoma cells (HOS or 143B cells). Dosing Regimen: When tumor volumes reach approximately 100 mm³, randomize animals into control and β-elemonic acid treatment groups (e.g., 25 mg/kg or 50 mg/kg), administer by intraperitoneal injection once daily for 21 days. Tumor Growth Monitoring: Measure tumor volume and body weight every 3-4 days to calculate tumor growth inhibition rate. Pharmacokinetic Sampling: Collect plasma samples at various time points (0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) after administration and detect drug concentrations by LC-MS/MS. Tissue Distribution Study: Euthanize animals after administration, collect tissues including tumor, bone, liver, kidney, heart, spleen, lung, and brain to detect drug distribution. Histological Analysis: Collect tumor tissues for immunohistochemistry (Ki67, TUNEL, CHOP, β-catenin) and Western blot analysis. Data Analysis: Compare tumor volume, pharmacokinetic parameters, and tissue distribution between treatment and control groups.

β-Elemonic acid exhibits rapid absorption, a short elimination half-life, and linear pharmacokinetic characteristics in rats. Following oral administration, the compound is rapidly absorbed and distributes to both tumor and bone tissues. The calculated LogP values are 7.20 (XLogP) or 8.56 (ACD/LogP), with LogD values of 6.86 at pH 5.5 and 5.06 at pH 7.4. The topological polar surface area is 54.40 Ų, with 1 hydrogen bond donor and 2 hydrogen bond acceptors. Predictions indicate favorable human intestinal absorption (99.64%), Caco-2 permeability (63.55%), and blood-brain barrier penetration (65.00%). β-Elemonic acid is predicted to be a P-glycoprotein inhibitor (58.17% probability) and a CYP3A4 substrate (64.19% probability).

Predicted toxicological assessments based on available data indicate that β-elemonic acid is negative for Ames mutagenicity (58.54% probability), carcinogenicity (100% probability negative prediction), and hepatotoxicity (65.72% probability negative prediction). The compound shows no eye corrosion (99.54%), no eye irritation (92.97%), and no skin corrosion (95.48%). However, it may exhibit skin irritation (65.21%), respiratory toxicity (63.33%), and mitochondrial toxicity (81.25%). Predictions indicate reproductive toxicity risk (100%), suggesting caution during research handling. At the cellular level, β-elemonic acid exhibits cytotoxicity against cancer cells but shows no significant toxicity to normal WI-38 lung epithelial cells.

[1]. Bioactive constituents from Boswellia papyrifera. J Nat Prod. 2005 Feb;68(2):189-93.

[2]. β-Elemonic acid inhibits the cell proliferation of human lung adenocarcinoma A549 cells: The roleof MAPK, ROS activation and glutathione depletion. Oncol Rep. 2016 Jan;35(1):227-34.

β-elemic acid is a triterpenoid compound. (2S)-6-methyl-2-[(5S,10S,13S,14S,17R)-4,4,10,13,14-pentamethyl-3-oxo-1,2,5,6,7,11,12,15,16,17-decahydrocyclopenta[a]phenanthrene-17-yl]hept-5-enoic acid has been reported in Boswellia papyrifera, and relevant data are available.

C₃₀H₄₆O₃

454.68

454.344

28282-25-9

28282-25-9

24721570

White to off-white solid

1.07

565.2±50.0 °C at 760 mmHg

216-219 ºC

309.6±26.6 °C

0.0±3.3 mmHg at 25°C

1.542

Triterpene from Boswellia carterii

8.56

1

3

5

33

904

6

O=C1C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])C([H])(C1(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])([H])C1=C2C([H])([H])C([H])([H])[C@@]2(C([H])([H])[H])C([H])(C([H])(C(=O)O[H])C([H])([H])C([H])([H])/C(/[H])=C(\C([H])([H])[H])/C([H])([H])[H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H]

XLPAINGDLCDYQV-SDTWUMECSA-N

InChI=1S/C30H46O3/c1-19(2)9-8-10-20(26(32)33)21-13-17-30(7)23-11-12-24-27(3,4)25(31)15-16-28(24,5)22(23)14-18-29(21,30)6/h9,20-21,24H,8,10-18H2,1-7H3,(H,32,33)/t20-,21-,24-,28+,29-,30+/m0/s1

(2S)-6-methyl-2-[(5R,10S,13S,14S,17S)-4,4,10,13,14-pentamethyl-3-oxo-1,2,5,6,7,11,12,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]hept-5-enoic acid

βElemonic acid; Elemonic acid; DTXSID40590814; RefChem:590705; DTXCID101778811; 28282-25-9; β Elemonic 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: 25~35 mg/mL (55~77 mM)
H2O: < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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.)