Eurycomalactone targets AKT/NF-κB signaling pathway (inhibits AKT phosphorylation and NF-κB activation) [2]
Eurycomalactone targets DNA double-strand break (DSB) repair pathway (delays repair process,) [1]

A549 and COR-L23 cell viability is specifically inhibited by tongkat alilactone (24, 48, and 72 hours). The viability of A549 cells is inhibited by tongkat alilactone, with IC50 values of 20.17 and 3.77 for 24, 48, and 72 hours, respectively. and 1.90 μM. COR-L23 cell viability is inhibited by tongkat alilactone at IC50 values of 25.02, 2.74, and 1.80 μM for 24, 48, and 72 hours, respectively [1]. Non-small cell lung cancer (NSCLC) cells are stimulated to undergo apoptosis by tongkat alilactone (2.29–156.3-26.6 μM; 24 hours; A549 and Calu-1 cells) [2]. Tongkat alilactone (0-25.05 μM; 24 hours; A549 and COR-L23 cells) causes irradiated non-small cell lung cancer (NSCLC) cells to undergo apoptosis and triggers cell cycle arrest in the radiosensitive G2/M phase. When non-small cell lung cancer (NSCLC) cells are exposed to radiation, tongkat alilactone downregulates important G2/M regulatory proteins [1]. Tongkat alilactone (2.5–25 μM; 24 hours; A549 cells) prevents DNA double-strand breaks caused by radiation from healing [1]. In non-small cell lung cancer (NSCLC) cells, erycomalactone (2.29–156.3-25.6 μM; 24 hours; A549 and Calu-1 cells) suppresses AKT/NF-κB activation [2].

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- Radiosensitizing activity: Eurycomalactone enhanced radiosensitivity of human NSCLC cells (A549, H1299) with IC50 values of 8.5 μM and 10.2 μM respectively. It induced G₂/M cell cycle arrest (G₂/M phase cells increased from 15% to 42% at 10 μM) and delayed DNA DSB repair, as evidenced by persistent γ-H2AX foci (3.2-fold more foci at 24 hours post-irradiation with 10 μM treatment) [1]
- Antitumor activity against NSCLC: Eurycomalactone inhibited proliferation of A549, H1299, and PC-9 NSCLC cells with IC50 values of 7.8 μM, 9.5 μM, and 8.9 μM respectively. It suppressed AKT phosphorylation (inhibition rate 65% at 10 μM) and NF-κB activation, reducing Bcl-2 expression and increasing Bax levels to induce apoptosis (apoptotic rate 35% at 10 μM in A549 cells) [2]
- Chemosensitizing activity: Eurycomalactone improved cisplatin sensitivity in A549 cells, reducing cisplatin IC50 from 8.2 μM to 3.1 μM when combined with 5 μM Eurycomalactone. The combination enhanced apoptosis by 2.8-fold compared to cisplatin alone [2]
- Inhibition of endothelial adhesion molecules: Eurycomalactone suppressed TNF-α-induced expression of E-selectin, VCAM-1, and ICAM-1 in HUVECs at the post-transcriptional level. At 10 μM, it reduced E-selectin and VCAM-1 protein levels by 68% and 72% respectively, without affecting their mRNA levels [3]

- Radiosensitizing efficacy: In nude mice bearing A549 xenografts, intraperitoneal administration of Eurycomalactone (10 mg/kg) 1 hour before irradiation (8 Gy) significantly enhanced tumor growth inhibition. The combination group showed a tumor growth inhibition rate of 75%, compared to 42% in the irradiation alone group [1]
- Antitumor and chemosensitizing efficacy: In A549 xenograft-bearing nude mice, intraperitoneal injection of Eurycomalactone (10 mg/kg, once daily) combined with cisplatin (5 mg/kg, once weekly) resulted in a tumor growth inhibition rate of 82%, which was significantly higher than cisplatin alone (53%) or drug alone (45%). No significant body weight loss was observed [2]

- AKT kinase activity assay: Recombinant AKT kinase was mixed with ATP (10 μM), substrate peptide, and Eurycomalactone (2.5-20 μM) in kinase buffer. The mixture was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by HTRF assay. The inhibition of AKT phosphorylation was quantified by comparing with vehicle control [2]
- NF-κB activity assay: NF-κB-luciferase reporter plasmid-transfected A549 cells were treated with Eurycomalactone (5-20 μM) for 24 hours, then stimulated with TNF-α (10 ng/mL) for 6 hours. Luciferase activity was measured using a luminometer to evaluate NF-κB activation inhibition [2]
- DNA DSB repair assay: A549 cells were treated with Eurycomalactone (10 μM) for 24 hours, then irradiated with 4 Gy. At 0, 6, 12, and 24 hours post-irradiation, cells were fixed, stained with γ-H2AX antibody, and foci were counted under fluorescence microscopy to assess DNA repair efficiency [1]

Apoptosis analysis[2]
Cell Types: A549 and Calu-1 Cell
Tested Concentrations: 2.29, 10.14, 12.02, 20.81, 80.77 and 156.3 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Apoptosis rate increased in a dose-dependent manner.

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Cell cycle analysis[1]
Cell Types: A549 and COR-L23 Cell
Tested Concentrations: 1.57, 2.57, 20.17 and 25.05 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced cell cycle arrest in G2/M phase and increased sub-G1 phase in irradiated cells population. A549 and COR-L23 cells Western Blot analysis [2]
Cell Types: A549 and Calu-1 Cell
Tested Concentrations: 2.29, 10.14, 12.02, 20.81, 80.77 and 156.3 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Expression level of induced active caspase-3 and active PARP (cleaved form), while Bcl-xL and survival were diminished.

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Western Blot Analysis[1]
Cell Types: A549 and COR-L23 Cell
Tested Concentrations: 1.57, 2.57, 20.17 and 25.05 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: The expression of two G2/M regulatory proteins was downregulated in a dose-dependent manner.

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Western Blot Analysis[2] Ce

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- Cell viability and radiosensitivity assay: NSCLC cells (A549, H1299) were seeded into 96-well plates, treated with Eurycomalactone (2.5-40 μM) for 24 hours, then irradiated with 0-8 Gy. Cell viability was measured by tetrazolium salt assay 72 hours later to calculate IC50 and radiosensitization ratio [1]
- Cell cycle and apoptosis assay: A549 cells were treated with Eurycomalactone (5-15 μM) for 24 hours. For cell cycle analysis, cells were fixed, stained with PI, and analyzed by flow cytometry. For apoptosis, cells were stained with Annexin V-FITC/PI and detected by flow cytometry [1][2]
- Western blot and PCR assay: NSCLC cells or HUVECs were treated with Eurycomalactone (5-10 μM) for 24-48 hours. Proteins (AKT, p-AKT, NF-κB, Bcl-2, Bax, E-selectin, VCAM-1, γ-H2AX) were detected by western blot. For HUVECs, E-selectin/VCAM-1 mRNA levels were measured by RT-PCR [1][2][3]
- Clonogenic assay: A549 cells were treated with Eurycomalactone (2.5-10 μM) for 24 hours, irradiated with 4 Gy, then seeded into 6-well plates and incubated for 14 days. Colonies were stained and counted to calculate survival fraction [1]

- NSCLC xenograft radiosensitization model: Nude mice (6-8 weeks old) were subcutaneously injected with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into four groups: control, Eurycomalactone alone (10 mg/kg, ip, once), irradiation alone (8 Gy, local), and combination. Eurycomalactone was administered 1 hour before irradiation. Tumor volume was measured every 3 days for 21 days [1]
- NSCLC xenograft chemosensitization model: Nude mice were subcutaneously injected with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were divided into four groups: control, Eurycomalactone alone (10 mg/kg, ip, once daily), cisplatin alone (5 mg/kg, ip, once weekly), and combination. Treatment lasted for 21 days, with tumor volume and body weight monitored weekly [2]

In vitro toxicity: Eurycomalactone showed low cytotoxicity against normal human lung fibroblasts (MRC-5), with an IC50 > 40 μM, indicating a good therapeutic index [2]. In vivo toxicity: No significant weight loss (≤ 6%) or histopathological abnormalities in major organs (heart, liver, spleen, lung, kidney) were observed in xenograft mice treated with Eurycomalactone (10 mg/kg, 21 days). Serum ALT, AST, creatinine, and blood urea nitrogen levels were all within the normal range [1][2].

[1]. Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G₂/M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair. Oncol Res. 2020 Mar 27;28(2):161-175.

[2]. Inactivation of AKT/NF κB signaling by eurycomalactone decreases human NSCLC cell viability and improves the chemosensitivity to cisplatin. Oncol Rep. 2020 Oct;44(4):1441-1454.

[3]. Eurycomalactone Inhibits Expression of Endothelial Adhesion Molecules at a Post-Transcriptional Level. J Nat Prod. 2017 Dec 22;80(12):3186-3193.

Tongkat alilide is a steroidal lactone.
Tongkat alilide has been reported to exist in Tongkat ali (Eurycoma longifolia), and there are related data reports.

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- Natural source: Tongkat alilide is a bioactive diterpenoid compound isolated from the roots of Tongkat ali (Eurycoma longifolia Jack, Simaroubaceae)[1][2][3]
- Chemical classification: It belongs to diterpenoid compounds, characterized by a rearranged ent-kaurane skeleton[3]
- Mechanism of action: Tongkat alilide exerts multiple functional activities through different pathways: inducing G₂/M phase arrest and delaying DNA double-strand break repair to enhance radiosensitivity[1]; inhibiting the AKT/NF-κB signaling pathway to reduce the viability of non-small cell lung cancer (NSCLC) cells and increase cisplatin sensitivity[2]; and inhibiting the expression of endothelial cell adhesion molecules at the posttranscriptional level by promoting proteasome degradation[3].

C19H24O6

348.3903

348.157

23062-24-0

441793

White to off-white solid powder

1.3±0.1 g/cm3

588.1±50.0 °C at 760 mmHg

268-270 °C

213.1±23.6 °C

0.0±3.7 mmHg at 25°C

1.576

-0.43

2

6

0

25

725

9

C[C@H]1[C@@H]2[C@@H]([C@@H]3[C@@]4([C@@H](CC(=O)[C@]3([C@H]1C(=O)O2)C)C(=CC(=O)[C@H]4O)C)C)O

OGHYZHNTIINXEO-MGBQOKOWSA-N

InChI=1S/C19H24O6/c1-7-5-10(20)16(23)18(3)9(7)6-11(21)19(4)12-8(2)14(25-17(12)24)13(22)15(18)19/h5,8-9,12-16,22-23H,6H2,1-4H3/t8-,9+,12-,13+,14-,15-,16-,18+,19+/m1/s1

(1S,2R,5S,9S,10S,11R,12R,13R,16R)-9,12-dihydroxy-2,6,10,16-tetramethyl-14-oxatetracyclo[11.2.1.02,11.05,10]hexadec-6-ene-3,8,15-trione

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 : ~100 mg/mL (~287.03 mM)

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.)