- NF-κB (nuclear factor-κB) [1]
- PI3K/Akt signaling pathway [1]
- MAPK signaling pathway (including ERK1/2, JNK1/2, p38 MAPK) [1,2,3]
- IKK/IκBα [3]
- TLR4 [3]
- Nrf2/Keap1/HO-1 axis [3]
- ER stress-related proteins (PERK, IRE1, ATF6, GRP78, CHOP) [3]
- Cyclin B1, cdc2, cdc25c [4]
- Caspase-3, caspase-8, caspase-9, PARP [1,2,4]

Hispolon (at 25 and 50 μM, for 24 to 72 hours) reduces U87MG cell viability [2]. In U87MG cells, hispolon (25 and 50 μM, 24 and 48 hours) can cause apoptosis and G2/M cell cycle arrest [2]. Hispolon (25 and 50 μM, 2-8 hours) boosts the expression of the CDK inhibitor p21 but decreases the expression of the G1-S transition-related protein cyclin D4 [2]. The migration of U87MG cells is inhibited by hispolon (at 25 and 50 μM, for 24 hours) [2].

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- Hispolon demonstrated antiproliferative activity against multiple cancer cell lines including U87MG (glioblastoma), HeLa, SiHa (cervical cancer), MCF-7, MDA-MB-231 (breast carcinoma), NPC-39, HONE-1, NPC-BM, NPC-039 (nasopharyngeal cancer), A549, H661 (lung cancer), DU145, LNCaP, PC3 (prostate cancer), MV4-11, HL-60, U937, THP-1 (leukemia), SGC-7901, MKN-45, MGC-803 (gastric cancer), T24, J82 (bladder cancer), Hep3B, SK-Hep1 (hepatocellular carcinoma), TCMK-1 (renal cancer) and KB (human epidermoid) cancer cells. [1]
- In U87MG glioblastoma cells, hispolon (25 and 50 μM) decreased cell viability in a dose- and time-dependent manner as measured by MTT assay. At 24 h, only 50 μM hispolon decreased viability; at 48 and 72 h, both concentrations markedly suppressed viability (p<0.001). [2]
- In U87MG cells, hispolon (25 and 50 μM) induced G2/M cell cycle arrest. After 48 h treatment, the percentage of cells in G2/M phase was 11.7±1.7% in control, 25.9±1.1% with 25 μM hispolon, and 37.9±1.1% with 50 μM hispolon. [2]
- In U87MG cells, hispolon decreased cyclin D4 expression and increased p21 and p53 expression in a dose-dependent manner. Hispolon (50 μM) reduced pERK1/2 at 15 and 30 min, and reduced pAkt at 2 and 4 h. [2]
- In U87MG cells, hispolon (50 μM for 72 h) induced apoptosis with increased percentage of necrotic and apoptotic cells, increased caspase-3 expression, and increased cleaved PARP (89 kD) with decreased full-length PARP (116 kD). [2]
- In U87MG cells, hispolon (50 μM for 24 h) significantly inhibited cell migration as measured by Boyden chamber assay. [2]
- In DBTRG and C6 GBM cells, hispolon reduced cell viability in a dose- and time-dependent manner. IC50 values: for C6 cells, 68.1 μM (24 h) and 51.7 μM (48 h); for DBTRG cells, 55.7 μM (24 h) and 46.6 μM (48 h). [4]
- In DBTRG and C6 cells, hispolon (75 μM for 24 h) increased apoptosis ratio to 27% (C6) and 11.3% (DBTRG) compared to control (C6: 3.6%, DBTRG: 0.9%) as measured by Annexin V-FITC staining. [4]
- In DBTRG and C6 cells, hispolon (0-75 μM) induced cleavage of caspase-9, caspase-3, and PARP in a dose-dependent manner by Western blot. [4]
- In DBTRG and C6 cells, hispolon induced G2/M phase arrest. After 75 μM hispolon for 24 h, G2/M phase cells increased to 37.9% (C6) and 23.2% (DBTRG) compared to control (C6: 23.3%, DBTRG: 21.8%). Hispolon decreased cyclin B1, cdc2, and cdc25c expression in a dose- and time-dependent manner. [4]
- In DBTRG and C6 cells, hispolon (75 μM) decreased cell migration by wound-healing assay (C6: 56% reduction; DBTRG: 57% reduction) and inhibited migration (C6: 71.9%; DBTRG: 66%) and invasion (C6: 50%; DBTRG: 67.6%) by transwell assay. Hispolon upregulated E-cadherin mRNA and downregulated N-cadherin, vimentin, Snail1, Snail2, and Twist mRNA by qRT-PCR. [4]
- In BV-2 microglial cells, hispolon (0-20 μM) induced HO-1 protein expression and inhibited iNOS/NO production under LPS or LTA stimulation with no cytotoxicity. [3]
- In LPS-challenged mice lung tissues, hispolon decreased iNOS and COX-2 protein expression, inhibited IKK/IκBα/NF-κB signaling, and suppressed MAPK phosphorylation (ERK, JNK, p38). [3]
- In LPS-challenged mice, hispolon increased antioxidant enzyme expression (catalase, SOD, GPx), increased Nrf2 and HO-1 expression, decreased Keap1 expression, and increased PPARγ expression. Hispolon also inhibited TLR4/PI3K/Akt/mTOR signaling. [3]
- In LPS-challenged mice, hispolon decreased ER stress markers (CHOP, PERK, IRE1, ATF6, GRP78), decreased autophagy markers (LC3-II/I, Beclin-1), decreased LKB1/CaMKK-AMPK signaling, and decreased apoptosis (increased Bcl-2, decreased Bax and caspase-3). [3]

Hispolon (2.5-10 mg/kg, intraperitoneal injection) can decrease LPS-induced acute lung damage in mice [3]. Hispolon (5 and 10 mg/kg, subcutaneous injection) inhibits tumor growth in DBTRG xenograft mice [4].

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- In U87MG glioblastoma xenograft mice (not detailed in this paper; cited from other studies). [1]
- In acute myeloid leukemia (AML) xenograft mice, hispolon (10 mg/kg) inhibited tumor growth in vivo. [1,3]
- In LPS-induced acute lung injury (ALI) mouse model, hispolon (2.5, 5, and 10 mg/kg, intraperitoneal injection) significantly reduced histopathological lung injury scores, pulmonary edema (W/D ratio), MPO activity, total cell counts, and total protein in BALF compared to LPS-only group. [3]
- In LPS-induced ALI mice, hispolon (2.5, 5, 10 mg/kg, ip) decreased NO, TNF-α, IL-1β, and IL-6 levels in BALF as measured by ELISA. [3]
- In LPS-induced ALI mice, hispolon (10 mg/kg, ip) decreased ROS production in BALF. Co-treatment with NAC (ROS inhibitor) further enhanced hispolon's effects on p-AMPK, HO-1, and nuclear Nrf2 expression. Co-treatment with LY294002 (AKT inhibitor) or 4-PBA (ER stress inhibitor) further decreased inflammatory and ER stress-related proteins. [3]
- In DBTRG xenograft NOD-SCID mice, hispolon (5 and 10 mg/kg, subcutaneous injection every 2 days) significantly reduced relative tumor volume on day 21 (5 mg/kg: 61% of control, p=0.011; 10 mg/kg: 55% of control, p=0.015). Tumors were harvested, photographed, and analyzed. [4]
- In DBTRG xenograft mice, hispolon (10 mg/kg) increased cleaved caspase-3 expression in tumor tissues by Western blot and immunohistochemistry. Hispolon also decreased ki-67 expression and increased caspase-3 positive cells by IHC staining. [4]

- MTT assay for cell viability: U87MG cells (1×10⁴ cells/well in 96-well plates) were treated with hispolon (25 and 50 μM) or 0.1% DMSO for 24, 48, or 72 h. MTT (5 mg/mL) was added for 2 h, cells were lysed with DMSO, and absorbance was measured at 570 nm. [2]
- Cell cycle analysis by flow cytometry: U87MG cells (25×10⁴ cells) were treated with hispolon (25 and 50 μM) for 24, 48, or 72 h, fixed in 70% ethanol, stained with propidium iodide (50 μg/mL) for 30 min, and analyzed by flow cytometry. [2]
- Apoptosis assay by Annexin V-FITC: U87MG cells (5×10⁵ cells) were treated with hispolon (25 and 50 μM) or 0.1% DMSO for 24, 48, or 72 h, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. [2]
- Western blot analysis: Proteins (40 μg) from hispolon-treated U87MG cells were separated by SDS-PAGE (12.5% gel), transferred to nitrocellulose membranes, incubated with primary antibodies (cyclin D4, p21, p53, PARP, pAKT, pERK1/2) overnight at 4°C, then with secondary antibody, and detected by chemiluminescence. [2]
- Migration and invasion assays: U87MG cells were treated with hispolon (50 μM for 24 h), harvested, seeded into Boyden chambers (15×10⁴ cells/well) in serum-free medium, incubated for 24 h, then fixed, stained with Giemsa, and counted. For invasion, membranes were coated with Matrigel. [2]
- MTT assay for GBM cells: DBTRG and C6 cells were treated with hispolon (0-100 μM) for 24 or 48 h, and cell viability was measured by MTT assay. [4]
- Annexin V-FITC apoptosis assay: GBM cells were treated with hispolon (75 μM for 24 h), stained with Annexin V-FITC and PI, and analyzed by flow cytometry. [4]
- Cell cycle analysis: GBM cells were treated with hispolon (0-75 μM for 24 h), fixed with 70% ethanol, stained with PI (20 μg/mL) with RNase A (0.2 mg/mL) and Triton X-100 (0.1%) for 30 min, and analyzed by flow cytometry. [4]
- Western blot for cell cycle proteins: GBM cells were treated with increasing concentrations of hispolon for 48 h or with 75 μM hispolon for 12, 24, 48 h. Proteins were analyzed using antibodies against cyclin B1, cdc2, and cdc25c. [4]
- Wound-healing assay: GBM cells were seeded in 24-well plates with culture inserts for 24 h, then treated with hispolon (0-75 μM) for 24 h, and wound closure was photographed at 0, 4, 8, and 24 h. [4]
- Transwell migration and invasion assay: GBM cells were pretreated with hispolon (75 μM for 24 h), reseeded into upper chambers with or without Matrigel coating (2 mg/mL), incubated for 24 h with 10% FBS as chemoattractant, then fixed with 10% formalin, stained with 0.2% crystal violet, and counted at 200× magnification. [4]
- qRT-PCR: Total RNA was extracted using RNeasy Mini kit, reverse transcribed at 37°C for 60 min, and real-time PCR was performed to measure E-cadherin, N-cadherin, vimentin, Snail1, Snail2, and Twist mRNA expression. [4]

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Cell Viability Assay[2]
Cell Types: U87MG Cell
Tested Concentrations: 25 and 50 μM
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: Inhibition of cell viability in a dose- and time-dependent manner.

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Western Blot Analysis [2]
Cell Types: U87MG Cell
Tested Concentrations: 25 and 50 μM
Incubation Duration: 2, 4, 8 h
Experimental Results: cyclin D4 levels diminished and p21 levels increased.

- LPS-induced acute lung injury (ALI) model: Six-week-old male ICR mice (25-28 g) were randomly divided into 6 groups (n=6): control, LPS (5 mg/kg, intratracheal instillation), LPS + Dex (10 mg/kg, ip), and LPS + hispolon (2.5, 5, 10 mg/kg suspended in 0.5% CMC solution, ip). Hispolon and Dex were injected intraperitoneally 1 hour before LPS administration. After 6 hours, animals were sacrificed and samples collected. Lung tissues were collected for histopathology (H&E staining), W/D ratio, MPO activity, and Western blot. BALF was collected for total cell count, protein concentration, nitrite assay, ROS measurement, and cytokine ELISA. [3]
- DBTRG xenograft model: NOD-SCID mice were subcutaneously implanted with 1×10⁶ DBTRG cells into the dorsal subcutaneous tissue. When tumors reached 80-150 mm³, mice were randomly divided into control and treatment groups (n=6 per group). Hispolon (5 or 10 mg/kg) was administered by subcutaneous injection (contralateral flank to tumor site) every 2 days until the end of the experiment. Tumor volume was calculated as TV (mm³) = (L × W²)/2. Relative tumor volume (RTV) was expressed as RTVn = TVn/TV0. On day 21, xenograft tumors were harvested, photographed, fixed in 4% formalin, embedded in paraffin for histological analysis (HE staining, ki-67 and caspase-3 immunohistochemistry), and also analyzed by Western blot for cleaved caspase-3. [4]

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Animal/Disease Models: LPS-induced acute lung injury in mice [3]
Doses: 2.5, 5 and 10 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Reduce the pathological effects of LPS-challenged mice. Decreases W/D ratio and MPO activity in the lungs. The production of pro-inflammatory cytokines is diminished.

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Animal/Disease Models: DBTRG xenograft mice [4]
Doses: 5 and 10 mg/kg
Route of Administration: subcutaneous injection
Experimental Results: tumor volume reduction (RTV). HE and ki-67 staining inhibited GBM cell proliferation in vivo.

- Hispolon showed no significant toxicity for phorbol ester (TPA)-treated and untreated MDA-MB-231 cells between 0 and 40 μM (8.8 μg/mL) for 24 h. [1,3]
- Hispolon (0-20 μM) induced HO-1 protein expression in BV-2 cells under LPS or LTA stimulation with no cytotoxicity. [1,3]
- Hispolon was shown to be less cytotoxic to normal cells. [1,3]

[1]. Hispolon: A natural polyphenol and emerging cancer killer by multiple cellular signaling pathways. Environ Res. 2020 Nov;190:110017.

[2]. Effects of hispolon on glioblastoma cell growth. Environ Toxicol. 2017 Sep;32(9):2113-2123.

[3]. Attenuation of Lipopolysaccharide-Induced Acute Lung Injury by Hispolon in Mice, Through Regulating the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 Pathways, and Suppressing Oxidative Stress-Mediated ER Stress-Induced Apoptosis and Autophagy. Nutrients. 2020 Jun 10;12(6):1742.

[4]. Hispolon Induces Apoptosis, Suppresses Migration and Invasion of Glioblastoma Cells and Inhibits GBM Xenograft Tumor Growth In Vivo. Molecules. 2021 Jul 26;26(15):4497.

Hispolon belongs to the class of catechol compounds. It has been reported to exist in Tropicoporus linteus, Phellinus igniarius, and other organisms with relevant data.

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- Hispolon is a natural bioactive polyphenol isolated from Phellinus linteus, Inonotus hispidus, Phellinus igniarius, Phellinus merrillii, and Phellinus loncerinus. It was first isolated in 1996 from Inonotus hispidus. Its chemical structure is similar to cinnamic acid derivative (replacement of H with -OH groups at meta and para positions in aromatic ring and -OH by alkyl groups at the end of the chain). The major factor responsible for greater activity of hispolon compared to cinnamic acid is the -COCH₃ group. [1]
- Hispolon exhibits anticancer, antidiabetic, antioxidant, antiviral, anti-inflammatory, hepatoprotective, immunomodulatory, and neuroprotective activities. [1]
- Hispolon fights against cancer via induction of apoptosis, halting cell cycle, and inhibition of metastasis by targeting PI3K/Akt, MAPK, and NF-κB signaling pathways. [1]
- Structure-activity relationship (SAR) studies showed that methoxy (-OCH₃) substitution increases lipophilicity and cytotoxicity compared to hydroxyl (-OH) groups. [1]
- Hispolon acts as an anti-inflammatory agent by suppressing TNF-α, decreasing MMP-9 via inhibition of NF-κB pathway, and inhibiting JNK phosphorylation. [1]
- Hispolon exhibits antidiabetic activity with IC₅₀ values of 12.38 μg/mL against α-glucosidase and 9.47 μg/mL against aldose reductase. [1]
- Hispolon exhibits antiviral activity against influenza virus A and B. [1]

C12H12O4

220.221

220.073

C, 65.45; H, 5.49; O, 29.06

173933-40-9

10082188

Light yellow to yellow solid powder

1.3±0.1 g/cm3

477.2±45.0 °C at 760 mmHg

256.5±25.2 °C

0.0±1.3 mmHg at 25°C

1.666

0.7

3

4

3

16

306

0

CC(=O)/C=C(/C=C/C1=CC(=C(C=C1)O)O)\O

QDVIEIMMEUCFMW-QXYPORFMSA-N

InChI=1S/C12H12O4/c1-8(13)6-10(14)4-2-9-3-5-11(15)12(16)7-9/h2-7,14-16H,1H3/b4-2+,10-6-

(3Z,5E)-6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one

Hispolon; 173933-40-9; (3Z,5E)-6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one; DTXSID701045719; RefChem:146573;

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 : ~125 mg/mL (~567.61 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.)