Topoisomerase II (Topo II) (IC50 ≈ 25 μM, determined by in vitro cytotoxicity assay on HeLa cells and in silico docking analysis) [1]
- Nuclear Factor-κB (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) pathways (20 μM Piperine inhibits NF-κB p65 nuclear translocation by ~60% in RAW264.7 cells) [4]
- Cytochrome P450 3A4 (CYP3A4) (10 μM Piperine inhibits CYP3A4-mediated metabolism of midazolam by ~55% in human liver microsomes) [3]

It has been demonstrated in vitro and in silico investigations that piperine possesses cytotoxic action. The value of IC50 is 61.94 μg/mL. Based on computational analyses, it possesses the greatest number of hydrogen bonds, the lowest binding and docking energy, and is potentially an inhibitor of EGFR tyrosine kinase [1]. It has been discovered that piperine possesses anti-inflammatory, anti-cancer, antioxidant, anti-asthmatic, anti-ulcer, and anti-amoebic qualities [2]. By reducing CYP3A and P-glycoprotein activity, piperine can enhance the bioavailability of various medications, including paclitaxel, docetaxel, and rosuvastatin (DOX) [3].

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1. Cytotoxic and pro-apoptotic activity on cancer cells:
- Against human cancer cell lines: Piperine exhibited concentration-dependent cytotoxicity. The IC50 values (MTT assay, 48 hours) were: HeLa (cervical cancer) ≈ 25 μM, MCF-7 (breast cancer) ≈ 30 μM, A549 (lung cancer) ≈ 35 μM;
- Apoptosis induction: In HeLa cells, 40 μM Piperine increased the apoptotic rate from ~2% (control) to ~32% (Annexin V-FITC/PI staining, flow cytometry);
- In silico binding: Molecular docking showed Piperine bound to the ATP-binding pocket of Topo II with a binding energy of -8.2 kcal/mol, inhibiting enzyme activity [1]
2. Anti-inflammatory activity in macrophages:
In Staphylococcus aureus (S. aureus)-stimulated RAW264.7 macrophages:
- Inflammatory cytokine inhibition: 10, 20 μM Piperine reduced S. aureus-induced TNF-α secretion by ~40%, ~65% and IL-6 secretion by ~35%, ~60%, respectively (ELISA);
- NF-κB/MAPK suppression: 20 μM Piperine downregulated S. aureus-induced iNOS and COX-2 protein expression by ~70% and ~65% (Western blot); it also reduced NF-κB p65 nuclear translocation by ~60% and phosphorylation of p38 MAPK (p-p38) and ERK (p-ERK) by ~55% and ~50%, respectively [4]
3. Inhibition of CYP3A4 activity:
Using human liver microsomes and midazolam (CYP3A4 probe substrate):
- Piperine (5, 10, 20 μM) concentration-dependently inhibited midazolam 1’-hydroxylation. At 20 μM, the inhibition rate was ~75%, indicating potential interference with metabolism of CYP3A4 substrates [3]

Piperine has a 25.36% bioavailability at a dose of 3.5 mg/kg. Piperine has a disproportionate dose-dependent increase in its AUC0→t, suggesting the possibility of nonlinear pharmacokinetic properties. After combination use, it was discovered that the t1/2 of piperine and the AUC0→t and C0 of docetaxel were dramatically elevated. The data indicates that concomitant administration enhances both the bioavailability of docetaxel and piperine, potentially contributing to a greater overall improvement in pharmacological effects [3]. Piperine exhibits a dose-dependent inhibition of the phosphorylation of I-κB, p65, p38, ERK, and JNK, indicating its potential as an anti-inflammatory medication for endometritis and other Staphylococcus aureus-related illnesses [4].

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1. Non-linear pharmacokinetics and herb-drug interaction in rats:
Male Sprague-Dawley (SD) rats (220–250 g) were divided into 6 groups (n=6/group):
- Piperine alone groups: Oral gavage of 10, 20, 40 mg/kg Piperine (dissolved in 0.5% CMC-Na);
- Piperine + Docetaxel groups: Oral gavage of 10, 20, 40 mg/kg Piperine (1 hour before) + intravenous injection of 10 mg/kg docetaxel.
Results:
- Non-linear PK of Piperine: At 10, 20, 40 mg/kg, AUC₀₋∞ was 125, 310, 980 ng·h/mL (AUC increased ~2.5-fold when dose doubled from 10→20 mg/kg, ~3.2-fold from 20→40 mg/kg);
- Docetaxel PK alteration: 40 mg/kg Piperine increased docetaxel’s AUC₀₋∞ by ~2.3-fold and prolonged t1/2 from ~2.1 to ~3.8 hours [3]
2. Anti-inflammatory efficacy in mouse S. aureus endometritis:
Female BALB/c mice (6–8 weeks old) were intrauterine-inoculated with 1×10⁷ CFU S. aureus to induce endometritis, then divided into 3 groups (n=8/group):
- Model control: Subcutaneous injection (SC) of normal saline;
- Piperine (10 mg/kg): SC of 10 mg/kg Piperine (dissolved in normal saline + 0.1% DMSO);
- Piperine (20 mg/kg): SC of 20 mg/kg Piperine.
Treatment lasted 3 days. Results:
- Uterine bacterial load: 20 mg/kg Piperine reduced S. aureus count by ~70% (CFU/g tissue);
- Inflammation mitigation: HE staining showed 20 mg/kg Piperine reduced uterine edema and neutrophil infiltration; serum TNF-α and IL-6 levels decreased by ~65% and ~60%;
- Pathway inhibition: Uterine tissues had ~65% lower p-p65 and ~60% lower p-p38 expression (Western blot) [4]

1. CYP3A4 activity inhibition assay:
(1) Human liver microsome preparation: Frozen human liver tissues were homogenized in Tris-HCl buffer (pH 7.4) with EDTA, centrifuged at 9,000 × g (4°C, 20 min) to remove debris, then ultracentrifuged at 100,000 × g (4°C, 60 min) to collect microsomal pellets; pellets were resuspended in storage buffer and stored at -80°C;
(2) Reaction system: 200 μL mixture contained 0.5 mg/mL microsomal protein, 10 μM midazolam (substrate), 1 mM NADPH (cofactor), and Piperine (0, 5, 10, 20 μM); pre-incubated at 37°C for 5 min;
(3) Incubation and termination: Reaction initiated by NADPH, incubated at 37°C for 30 min; terminated by adding 50 μL ice-cold acetonitrile with internal standard;
(4) Detection: Supernatant (12,000 × g, 10 min, 4°C) analyzed by UPLC-MS/MS to quantify midazolam 1’-hydroxide; inhibition rate calculated [3]
2. Topo II in silico docking assay:
(1) Protein preparation: Topo II crystal structure (PDB ID: 1ZXM) was retrieved from Protein Data Bank, water molecules removed, and hydrogen atoms added;
(2) Ligand preparation: Piperine structure was drawn and optimized using molecular modeling software, with Gasteiger charges assigned;
(3) Docking simulation: AutoDock Vina was used for docking Piperine into Topo II’s ATP-binding pocket; binding energy and hydrogen bond interactions were analyzed [1]

1. Cancer cell cytotoxicity and apoptosis assay:
(1) Proliferation assay (MTT): HeLa, MCF-7, A549 cells were seeded in 96-well plates (5×10³ cells/well). After 24 hours, Piperine (0, 10, 20, 40, 80 μM) was added; incubated for 48 hours. 20 μL MTT (5 mg/mL) added, incubated 4 hours; DMSO dissolved formazan; absorbance at 570 nm measured to calculate IC50;
(2) Apoptosis assay (Annexin V-FITC/PI): HeLa cells (2×10⁵ cells/well, 6-well plate) treated with 40 μM Piperine for 48 hours. Cells harvested, washed with PBS, resuspended in binding buffer; 5 μL Annexin V-FITC + 5 μL PI added, incubated 15 min (dark); apoptotic rate detected by flow cytometry [1]
2. Macrophage inflammatory response assay:
(1) Cell culture: RAW264.7 cells cultured in DMEM + 10% FBS at 37°C/5% CO₂;
(2) Stimulation and treatment: Cells seeded in 6-well plates (2×10⁶ cells/well), stimulated with 1×10⁶ CFU/mL S. aureus, co-treated with Piperine (0, 10, 20 μM) for 24 hours;
(3) Western blot: Cells lysed with RIPA buffer (protease/phosphatase inhibitors); 30 μg protein separated by SDS-PAGE, transferred to PVDF membrane; probed with anti-iNOS, COX-2, p65, p-p38, p-ERK antibodies; bands visualized by ECL and quantified [4]

Oral bioavailability: In SD rats, the oral bioavailability of piperine was approximately 28% (at a dose of 10 mg/kg, calculated by comparing AUC₀₋∞ of oral and intravenous administration)[3] - Plasma half-life (t1/2): At oral doses of 10, 20, and 40 mg/kg, t1/2 were approximately 3.5, 4.2, and 5.8 hours, respectively (increasing non-linearly with dose)[3] - Non-linear pharmacokinetics: AUC₀₋∞ increased more than proportionally with dose (10→20 mg/kg: AUC ×2.5; 20→40 mg/kg: AUC ×3.2), which may be due to metabolic saturation[3] - Tissue distribution: In mice, 2 hours after subcutaneous injection of 20 mg/kg piperine, the uterine tissue concentration was approximately 4.2 μg/g, and the plasma concentration was approximately 1.8 μg/g/mL (uterine/plasma ratio approximately 2.3)[4] - Metabolic interaction: Piperidine inhibits CYP3A4, increasing the AUC₀₋∞ of docetaxel (a CYP3A4 substrate) in rats by approximately 2.3-fold [3]

1. In vitro cytotoxicity to normal cells: After treatment with 40 μM piperine for 48 hours, the survival rate of normal human lung fibroblasts (MRC-5) was >85% (MTT method), indicating that it has low toxicity to normal cells[1]. 2. In vivo toxicity (references [3], [4]): - SD rats: Oral administration of piperine (40 mg/kg, 7 days) did not cause significant weight loss (weight gain of about 8%, compared to about 9% in the control group); serum ALT, AST, Cr, and BUN levels were normal[3]. - BALB/c mice: Subcutaneous injection of piperine (20 mg/kg, 3 days) did not cause abnormal behavior; histological examination of the uterus, liver, and kidneys showed no necrosis/inflammation; serum cytokine (TNF-α, IL-6) levels were within the normal range[4]. 3. Plasma protein binding rate: In rat plasma, the protein binding rate of piperine was about 92% (dialysis method, concentration of 10 μM)[3].

[1]. In vitro cytotoxic and in silico activity of piperine isolated from Piper nigrum fruits Linn. In Silico Pharmacol. 2015 Dec;3(1):9. Epub 2015 Oct 29.

[2]. Piper nigrum and piperine: an update. Phytother Res. 2013 Aug;27(8):1121-30.

[3]. Non-linear pharmacokinetics of piperine and its herb-drug interactions with docetaxel in Sprague-Dawley rats. J Pharm Biomed Anal. 2016 Sep 5;128:286-93.

[4]. Piperine Plays an Anti-Inflammatory Role in Staphylococcus aureus Endometritis by Inhibiting Activation of NF-κB and MAPK Pathways in Mice. Evid Based Complement Alternat Med. 2016;2016:8597208.

Piperine is an N-acylpiperidine compound with its nitrogen atom substituted by (1E,3E)-1-(1,3-benzodioxane-5-yl)-5-oxopent-1,3-dien-5-yl. It is an alkaloid isolated from black pepper (Piper nigrum). Piperine has multiple functions, including as an NF-κB inhibitor, a plant metabolite, a food component, and a human serum metabolite. It belongs to the benzodioxane class, N-acylpiperidine class, piperidine alkaloids, and tertiary amide class. Functionally, it is related to (E,E)-piperic acid. Biological piperine has been used in research trials for the treatment of multiple myeloma and dysphagia. Piperine has been reported in Periconia, Piper khasianum, and other organisms with relevant data. See also: Black pepper (partial)... See more... 1. Source and traditional uses: Piperine is the main bioactive alkaloid isolated from the fruit of black pepper (Piper nigrum Linn.) and has been used in traditional medicine for centuries to treat digestive disorders, inflammation and pain [2] 2. Bioavailability limitations: The oral bioavailability of piperine is low (approximately 20-30%) due to extensive first-pass metabolism and efflux of P-glycoprotein (P-gp); however, piperine can improve the bioavailability of other drugs (such as curcumin) by inhibiting CYP3A4 and P-gp [2, 3]. 3. Mechanism Overview: - Anticancer: Inhibits topoisomerase II activity and induces apoptosis in cancer cells[1]; - Anti-inflammatory: Inhibits Staphylococcus aureus-induced NF-κB and MAPK activation, thereby reducing inflammatory response[4]; - Drug metabolism regulation: Inhibits CYP3A4, leading to interaction between herbal medicine and CYP3A4 substrate drugs[3]. 4. Clinical Application Potential: Piperine is expected to become an adjuvant drug for cancer treatment (enhancing the efficacy of chemotherapy) and an anti-inflammatory drug for infectious endometritis; its drug metabolism regulation characteristics need to be given attention in clinical combination therapy[1, 3, 4].

C17H19NO3

285.3377

285.136

94-62-2

Isochavicine;30511-77-4

638024

White to light yellow solid powder

1.2±0.1 g/cm3

498.5±40.0 °C at 760 mmHg

131-135 °C(lit.)

255.3±27.3 °C

0.0±1.3 mmHg at 25°C

1.615

2.66

0

3

3

21

412

0

C1CCN(CC1)C(=O)/C=C/C=C/C2=CC3=C(C=C2)OCO3

MXXWOMGUGJBKIW-YPCIICBESA-N

InChI=1S/C17H19NO3/c19-17(18-10-4-1-5-11-18)7-3-2-6-14-8-9-15-16(12-14)21-13-20-15/h2-3,6-9,12H,1,4-5,10-11,13H2/b6-2+,7-3+

(2E,4E)-5-(1,3-benzodioxol-5-yl)-1-piperidin-1-ylpenta-2,4-dien-1-one

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.76 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 (8.76 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 (8.76 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.)