Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells

J Ethnopharmacol. 2016 Aug 2;189:139-47. doi: 10.1016/j.jep.2016.05.020. Epub 2016 May 10.


Ethnopharmacological relevance: Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation.

Aims of the study: The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity.

Methods: The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice.

Results: Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice.

Conclusion: The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.

Keywords: Antitumor activity; Apoptosis; Cell cycle arrest; DNA damage; Doxorubicin (PubChem CID: 31703); Piper nigrum; Piperine (PubChem CID: 638024); Piperyline (PubChem CID: 636537); ROS overproduction.

MeSH terms

  • Animals
  • Antineoplastic Agents, Phytogenic / isolation & purification
  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Apoptosis / drug effects*
  • Apoptosis Regulatory Proteins / metabolism
  • Biomarkers, Tumor / metabolism
  • Breast Neoplasms / drug therapy*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / metabolism
  • Breast Neoplasms / pathology
  • Carcinoma, Ehrlich Tumor / drug therapy*
  • Carcinoma, Ehrlich Tumor / genetics
  • Carcinoma, Ehrlich Tumor / metabolism
  • Carcinoma, Ehrlich Tumor / pathology
  • Cell Cycle Checkpoints / drug effects*
  • Cell Cycle Proteins / metabolism
  • DNA Damage*
  • Dose-Response Relationship, Drug
  • Ethanol / chemistry*
  • Female
  • HT29 Cells
  • Humans
  • Lipid Peroxidation / drug effects
  • MCF-7 Cells
  • Male
  • Mice, Inbred BALB C
  • Oxidants / isolation & purification
  • Oxidants / pharmacology*
  • Oxidative Stress / drug effects*
  • Phytotherapy
  • Piper nigrum / chemistry*
  • Piperidines / isolation & purification
  • Piperidines / pharmacology*
  • Plant Extracts / isolation & purification
  • Plant Extracts / pharmacology*
  • Plants, Medicinal
  • Protein Carbonylation / drug effects
  • Reactive Oxygen Species / metabolism*
  • Solvents / chemistry*
  • Time Factors
  • Tumor Burden / drug effects
  • Up-Regulation


  • Antineoplastic Agents, Phytogenic
  • Apoptosis Regulatory Proteins
  • Biomarkers, Tumor
  • Cell Cycle Proteins
  • Oxidants
  • Piperidines
  • Plant Extracts
  • Reactive Oxygen Species
  • Solvents
  • Ethanol