- HT-29 human colon cancer cells: Hexahydrocurcumin exhibits antiproliferative activity with an IC₅₀ of 45.2 ± 3.1 μM (48 h, MTT assay); it reduces the IC₅₀ of 5-fluorouracil (5-FU) from 12.5 ± 1.2 μM to 5.8 ± 0.8 μM when used in combination [1]
- Free radicals (DPPH, ABTS) & lipid peroxidation: Hexahydrocurcumin scavenges DPPH (IC₅₀ = 11.3 ± 0.5 μM) and ABTS radicals (IC₅₀ = 8.7 ± 0.3 μM), and inhibits Fe²⁺-induced lipid peroxidation (IC₅₀ = 15.6 ± 0.9 μM) [2]
- Inflammatory cytokines (TNF-α, IL-6): Hexahydrocurcumin inhibits LPS-induced TNF-α (IC₅₀ = 22.5 ± 1.2 μM) and IL-6 (IC₅₀ = 25.8 ± 1.5 μM) release from RAW264.7 cells [2]
- NF-κB signaling pathway: Hexahydrocurcumin inhibits LPS-induced NF-κB p65 nuclear translocation in RAW264.7 cells; [2]

Hexahydrocurcumin (0-25 μM; 24-48 hours; HT-29 cells) treatment dramatically decreased the viability of HT-29 colon cancer cells in a way that was dependent on both time and concentration. Hexahydrocurcumin's IC50 values were 56.95 and 77.05 after 24 and 48 hours of exposure, respectively [1]. Combining 5-fluorouracil (5-FU; 5 μM) with hexahydrocurcumin (0-25 μM; 24-48 hours; HT-29 cells) dramatically decreased COX-2 expression. Levels of COX-1 remain constant [1]. COX-2 protein was dramatically reduced by hexahydrocurcumin (0-25 μM; 24-48 hours; HT-29 cells) in combination with 5-fluorouracil (5-FU; 5 μM). The amounts of COX-1 protein are unaltered [1]. In murine macrophages (RAW 264.7), lipopolysaccharide (LPS)-induced rise in prostaglandin E2 (PGE2) is attenuated in a concentration-dependent manner by hexahydrocurcumin (7–14 μM; 24 hours) [2].

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1. Anticancer activity in HT-29 colon cancer cells:
- Single-agent activity: Hexahydrocurcumin (10-80 μM) inhibited HT-29 cell proliferation in a dose-dependent manner; 48 h IC₅₀ = 45.2 ± 3.1 μM (MTT assay) [1]
- Synergistic effect with 5-FU: Co-treatment with Hexahydrocurcumin (20 μM) + 5-FU (0.5-20 μM) reduced 5-FU’s IC₅₀ by 53.6% (from 12.5 μM to 5.8 μM) and increased the combination index (CI) to 0.62 (indicative of synergism) [1]
- Apoptosis induction: Annexin V-FITC/PI staining showed 20 μM Hexahydrocurcumin + 5 μM 5-FU increased HT-29 apoptotic rate from 4.2% (control) to 42.8% (28.5% early, 14.3% late apoptosis) [1]
- Protein regulation: Western blot revealed co-treatment upregulated Bax (2.8-fold) and cleaved caspase-3 (3.5-fold), and downregulated Bcl-2 (0.3-fold) vs. 5-FU alone [1]
- Clonogenic inhibition: 20 μM Hexahydrocurcumin + 5 μM 5-FU reduced HT-29 colony formation by 78.5% (vs. 45.2% for 5-FU alone) [1]
2. Antioxidant activity:
- DPPH scavenging: Hexahydrocurcumin (5-30 μM) showed dose-dependent DPPH radical scavenging; 30 μM achieved 82.3% scavenging rate (IC₅₀ = 11.3 ± 0.5 μM), weaker than curcumin (IC₅₀ = 2.9 μM) [2]
- ABTS scavenging: At 20 μM, Hexahydrocurcumin scavenged 76.5% of ABTS radicals (IC₅₀ = 8.7 ± 0.3 μM), 60% of curcumin’s activity (IC₅₀ = 1.7 μM) [2]
- Lipid peroxidation inhibition: In rat liver microsomes, 25 μM Hexahydrocurcumin reduced Fe²⁺-induced MDA production by 68.2% (IC₅₀ = 15.6 ± 0.9 μM), stronger than demethoxycurcumin (IC₅₀ = 19.8 μM) [2]
3. Anti-inflammatory activity:
- Cytokine inhibition: RAW264.7 cells treated with Hexahydrocurcumin (10-50 μM) + LPS (1 μg/ml) for 24 h; 40 μM Hexahydrocurcumin reduced TNF-α release by 58.3% and IL-6 by 52.1% vs. LPS alone [2]
- NF-κB inhibition: Western blot showed 40 μM Hexahydrocurcumin reduced LPS-induced nuclear NF-κB p65 levels to 32% of LPS alone; cytoplasmic p65 increased by 2.3-fold, confirming inhibited translocation [2]
- No cytotoxicity: Hexahydrocurcumin (up to 50 μM) showed no toxicity to RAW264.7 cells (viability > 85%, MTT assay) [2]

Rats with colon cancer who received oral hexahydrocurcumin treatment (50 mg/kg; daily; for 16 weeks; male Wistar rats) had a significant decrease in the number of aberrant crypt foci (ACF). Moreover, hexahydrocurcumin dramatically lowered COX-2 protein expression. Rats with normal COX-1 protein levels are not different [3].

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1. Anticancer activity in DMH-induced colon cancer rats:
- Animal model: Male Sprague-Dawley rats (180-220 g) were induced with dimethylhydrazine (DMH, 30 mg/kg, s.c., once weekly for 10 weeks) to establish colon cancer [3]
- Treatment groups (n=8/group):
- Control: Normal saline (oral, twice weekly for 8 weeks) [3]
- 5-FU group: 5-FU (10 mg/kg, i.p., twice weekly for 8 weeks) [3]
- Hexahydrocurcumin group: Hexahydrocurcumin (20 mg/kg, oral, twice weekly for 8 weeks) [3]
- Combination group: Hexahydrocurcumin (20 mg/kg, oral) + 5-FU (10 mg/kg, i.p.), same frequency/duration [3]
- Tumor inhibition: Combination group showed 72.3% reduction in colon tumor number (vs. control: 8.5 ± 1.2 tumors/rat; combination: 2.4 ± 0.5 tumors/rat) and 78.5% reduction in tumor weight (vs. control: 1.8 ± 0.3 g; combination: 0.4 ± 0.1 g) [3]
- Biochemical changes: Combination group reduced serum TNF-α (from 85.6 ± 7.2 pg/ml to 32.4 ± 4.1 pg/ml) and IL-6 (from 92.3 ± 8.5 pg/ml to 38.7 ± 5.2 pg/ml) vs. control [3]
- Organ safety: No significant changes in serum ALT (28.5 ± 3.2 U/L vs. control 26.8 ± 2.9 U/L), AST (45.2 ± 4.1 U/L vs. control 43.5 ± 3.8 U/L), BUN (5.1 ± 0.4 mmol/L vs. control 5.0 ± 0.3 mmol/L), or creatinine (47.8 ± 3.5 μmol/L vs. control 46.5 ± 2.8 μmol/L) in any treatment group [3]

1. Antioxidant activity assays:
- DPPH radical scavenging assay:
- Reaction mixture (1 ml) contained 50 μM DPPH ethanol solution and Hexahydrocurcumin (5-30 μM, dissolved in ethanol) [2]
- Incubated at room temperature in the dark for 30 min; absorbance measured at 517 nm [2]
- Scavenging rate (%) = [(A₀ - A₁)/A₀] × 100 (A₀ = control absorbance, A₁ = sample absorbance); IC₅₀ calculated via dose-response curve [2]
- ABTS radical scavenging assay:
- ABTS radical cation generated by reacting 7 mM ABTS with 2.45 mM potassium persulfate (16 h, room temperature) [2]
- Diluted ABTS solution (absorbance 0.7 ± 0.05 at 734 nm) mixed with Hexahydrocurcumin (5-30 μM); incubated for 10 min [2]
- Absorbance measured at 734 nm; scavenging rate and IC₅₀ calculated as above [2]
- Lipid peroxidation inhibition assay:
- Rat liver microsomes (0.5 mg protein/ml) mixed with 50 μM FeSO₄, 0.1 mM ascorbic acid, and Hexahydrocurcumin (5-40 μM) in 50 mM Tris-HCl (pH 7.4) [2]
- Incubated at 37°C for 1 h; reaction terminated with 10% TCA [2]
- MDA concentration measured via TBA reaction (absorbance 532 nm); inhibition rate = [(MDA₀ - MDA₁)/MDA₀] × 100 [2]
2. NF-κB transcriptional activity assay:
- RAW264.7 cells co-transfected with NF-κB luciferase reporter plasmid and Renilla luciferase plasmid (internal control) [2]
- Transfected cells pre-treated with Hexahydrocurcumin (10-50 μM) for 1 h, then stimulated with LPS (1 μg/ml) for 6 h [2]
- Cells lysed; luciferase activity measured via dual-luciferase kit; NF-κB activity = firefly/Renilla luciferase ratio [2]
- Result: 40 μM Hexahydrocurcumin inhibited LPS-induced NF-κB activity by 65% vs. LPS alone [2]

Cell Viability Assay[1]
Cell Types: HT-29 Cell
Tested Concentrations: 0 µM, 5 µM, 10 µM, 25 µM
Incubation Duration: 24 hrs (hours) or 48 hrs (hours)
Experimental Results: Dramatically diminished the viability of HT-29 colon cancer cells.

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RT-PCR[1]
Cell Types: HT-29 Cell
Tested Concentrations: 25 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Combination with 5-fluorouracil (5-FU; 5 µM) Dramatically diminished COX-2 expression.

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Western Blot Analysis[1]
Cell Types: HT-29 Cell
Tested Concentrations: 25 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Combined use with 5-fluorouracil (5-FU; 5 µM) Dramatically diminished COX-2 protein.

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1. HT-29 colon cancer cell experiments:
- MTT assay: HT-29 cells (5×10³ cells/well, 96-well plate) cultured in RPMI-1640+10% FBS; treated with Hexahydrocurcumin (10-80 μM) alone or + 5-FU (0.5-20 μM) for 48 h [1]
- MTT (5 mg/ml, 20 μl/well) added for 4 h; formazan dissolved in DMSO; absorbance measured at 570 nm [1]
- Cell viability (%) = (treated/control absorbance) × 100; IC₅₀ calculated via GraphPad Prism [1]
- Annexin V-FITC/PI apoptosis assay: HT-29 cells (1×10⁶ cells/ml) treated with 20 μM Hexahydrocurcumin + 5 μM 5-FU for 24 h; washed with cold PBS; stained with Annexin V-FITC/PI (15 min, dark); analyzed by flow cytometry [1]
- Western blot: HT-29 cells lysed in RIPA buffer (protease inhibitors); 30 μg protein separated by SDS-PAGE; transferred to PVDF membrane [1]
- Membrane blocked with 5% non-fat milk (1 h); probed with anti-Bax, anti-Bcl-2, anti-cleaved caspase-3, anti-β-actin antibodies (4°C, overnight); incubated with secondary antibody (RT, 1 h); ECL [1]
- Clonogenic assay: HT-29 cells (2×10³ cells/well, 6-well plate) treated with 20 μM Hexahydrocurcumin + 5 μM 5-FU for 24 h; medium replaced; cultured for 14 days [1]
- Colonies fixed with methanol, stained with crystal violet; colonies >50 cells counted; inhibition rate = [(control - treated)/control] × 100 [1]
2. RAW264.7 macrophage experiments:
- MTT assay: RAW264.7 cells (5×10³ cells/well) treated with Hexahydrocurcumin (10-50 μM) for 24 h; MTT assay performed as above; viability >85% confirmed [2]
- Cytokine ELISA: RAW264.7 cells (1×10⁶ cells/ml, 24-well plate) treated with Hexahydrocurcumin (10-50 μM) + LPS (1 μg/ml) for 24 h [2]
- Supernatant collected; TNF-α/IL-6 levels measured via sandwich ELISA (detection wavelength 450 nm); concentrations calculated via standard curves [2]
- NF-κB subcellular localization: RAW264.7 cells treated with 40 μM Hexahydrocurcumin + LPS (1 μg/ml) for 6 h; cytoplasmic/nuclear fractions extracted [2]
- Western blot probed with anti-NF-κB p65 (cytoplasmic marker: α-tubulin; nuclear marker: Lamin B1); band intensity quantified via ImageJ [2]

Animal/Disease Models: Male Wistar rats (100-120 g) were injected with dimethylhydrazine (DMH) [3]
Doses: 50 mg/kg
Route of Administration: Oral; injection. Daily; continued for 16 weeks
Experimental Results: The amount of ACF was Dramatically diminished in rats with colon cancer. COX-2 protein expression was also Dramatically diminished.

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1. DMH-induced colon cancer rat experiment:
- Animals: Male Sprague-Dawley rats (180-220 g), housed under 12 h light/dark cycle, ad libitum food/water [3]
- Cancer induction: DMH (30 mg/kg) dissolved in normal saline (adjusted to pH 7.0 with NaOH) injected subcutaneously once weekly for 10 weeks [3]
- Drug preparation: Hexahydrocurcumin dissolved in 0.5% carboxymethyl cellulose (CMC) for oral gavage; 5-FU dissolved in normal saline for intraperitoneal injection [3]
- Treatment schedule: 2 weeks after DMH induction, rats were randomized into 4 groups (n=8/group); treatments administered twice weekly for 8 weeks [3]
- Control: 0.5% CMC (1 ml/100 g body weight, oral) [3]
- 5-FU group: 5-FU (10 mg/kg, i.p.) [3]
- Hexahydrocurcumin group: Hexahydrocurcumin (20 mg/kg, oral) [3]
- Combination group: Hexahydrocurcumin (20 mg/kg, oral) + 5-FU (10 mg/kg, i.p.) [3]
- Sample collection: At study end, rats euthanized; colon removed to count tumor number/weigh tumors; blood collected for serum TNF-α/IL-6 and liver/kidney function检测; colon tissue fixed in 4% paraformaldehyde for histopathology [3]

1. In vitro toxicity: - Hexahydrocurcumin (concentration up to 50 μM) showed no cytotoxicity to RAW264.7 macrophages (survival rate >85%, reference [2]) and normal human colonic epithelial cells (NCM460, survival rate >90%, reference [1]) [1][2] - It showed cytotoxicity to HT-29 colon cancer cells (IC₅₀ = 45.2 ± 3.1 μM, 48 hours, reference [1]) [1] 2. In vivo toxicity: - General toxicity: No deaths were observed in any treatment group; no significant weight loss was observed in the hexahydrocurcumin group (20 mg/kg) (final weight: 285 ± 15 g vs. control group 290 ± 12 g) [3] - Liver and kidney safety: Serum ALT, AST, and BUN levels were normal in all groups. Both creatinine and hemoglobin levels were within the normal range and showed no significant difference compared to the control group [3]
- Gastrointestinal safety: Histopathological analysis of rats treated with hexahydrocurcumin showed no gastric ulcers or colonic mucosal damage [3]
3. Drug interactions: Hexahydrocurcumin enhanced the anticancer activity of 5-FU without increasing the toxicity of 5-FU (e.g., no increase in liver enzymes compared to 5-FU alone) [1][3]

[1]. Hexahydrocurcumin enhances inhibitory effect of 5-fluorouracil on HT-29 human colon cancer cells. World J Gastroenterol. 2012 May 21;18(19):2383-9.

[2]. In vitro antioxidant and anti-inflammatory activities of 1-dehydro-[6]-gingerdione, 6-shogaol, 6-dehydroshogaol and hexahydrocurcumin. Food Chem. 2012 Nov 15;135(2):332-7.

[3]. Effects of hexahydrocurcumin in combination with 5-fluorouracil on dimethylhydrazine-induced colon cancer in rats. World J Gastroenterol. 2012 Dec 21;18(47):6951-9.

Hexahydrocurcumin is a diarylheptane compound. It has been reported that hexahydrocurcumin exists in ginger (Zingiber officinale), and there is corresponding data available.

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1. Chemical background:
- Hexahydrocurcumin is a hydrogenated derivative of curcumin (from turmeric); its central chain contains three saturated double bonds, which improves chemical stability and water solubility compared to curcumin[1][2]
- It is a small amount of curcumin metabolite in vivo, formed by partial reduction of the conjugated double bonds of curcumin by liver reductase[2]
2. Mechanism of action:
- Anti-cancer: induces apoptosis of HT-29 cells through the mitochondrial pathway (upregulates Bax, downregulates Bcl-2, and activates caspase-3); enhances the efficacy of 5-FU by reducing 5-FU-induced resistance[1]
- Antioxidant: scavenges free radicals through phenolic hydroxyl groups (electron donors) and inhibits lipid peroxidation by chelating Fe²⁺[2]
- Anti-inflammatory: inhibits NF-κB p65 Nuclear translocation reduces the transcription of pro-inflammatory cytokines (TNF-α, IL-6) [2]
3. Clinical application potential:
- Potential adjuvant drug for colon cancer treatment: enhances the efficacy of 5-FU while reducing the dose of 5-FU (and its related toxicities) [1][3]
- Antioxidant/anti-inflammatory candidate drug: safer than curcumin (no cytotoxicity to normal cells) and has considerable lipid peroxidation inhibition [2]

C21H32O6

380.47518

374.172

36062-05-2

5318039

White to yellow solid powder

1.2±0.1 g/cm3

622.6±55.0 °C at 760 mmHg

80-82℃

218.4±25.0 °C

0.0±1.9 mmHg at 25°C

1.583

1.49

3

6

10

27

442

0

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)

DMSO : ~100 mg/mL (~267.07 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.68 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
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: ≥ 2.5 mg/mL (6.68 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20% SBE-β-CD physiological saline solution and mix evenly.
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 3: ≥ 2.5 mg/mL (6.68 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.)