Antimicrobial synergism and cytotoxic properties of Citrus limon L., Piper nigrum L. and Melaleuca alternifolia (Maiden and Betche) Cheel essential oils

J Pharm Pharmacol. 2017 Nov;69(11):1606-1614. doi: 10.1111/jphp.12792. Epub 2017 Aug 17.


Objectives: The chemical composition, antimicrobial and synergistic effect, and cytotoxic activity of Citrus limon (lemon), Piper nigrum (green pepper) and Melaleuca alternifoila (tea tree) essential oils (EOs) were investigated.

Methods: Chemical analyses of essential oils were tested by GC-FID and GC-MS spectroscopy. The antimicrobial activity assay was conducted using microdilution method against several oral bacteria and Candida spp. originating from the humans with oral disorders. The synergistic antimicrobial activity was evaluated using checkerboard method. The cytotoxicity evaluation of EOs was assessed using MTT test.

Key findings: Limonene (37.5%) and β-pinene (17.9%) were the major compounds in C. limon oil, β-pinene (34.4%), δ-3-carene (19.7%), limonene (18.7%) and α-pinene (10.4%) in P. nigrum oil and terpinen-4-ol (38.6%) and γ-terpinene (21.7%) in M. alternifolia oil. The broad-spectrum antimicrobial activity was achieved by tested three EOs, with C. limon oil being the strongest against bacteria and M. alternifolia oil strongest against fungi. The EOs demonstrated synergism; their combined application revealed an increase in antimicrobial activity. All tested essential oils showed lower cytotoxic activity in comparison with the positive control, and the obtained results confirmed a dose-dependent activity.

Conclusions: The results of this study encourage use of tested EOs in development of a novel agent intended for prevention or therapy of corresponding oral disorders.

Keywords: Citrus limon; Melaleuca alternifolia; Piper nigrum; antimicrobial synergism; cytotoxic activity; essential oil.

Publication types

  • Comparative Study

MeSH terms

  • Anti-Infective Agents / administration & dosage
  • Anti-Infective Agents / isolation & purification
  • Anti-Infective Agents / pharmacology*
  • Antineoplastic Agents, Phytogenic / administration & dosage
  • Antineoplastic Agents, Phytogenic / isolation & purification
  • Antineoplastic Agents, Phytogenic / pharmacology*
  • Bacteria / drug effects
  • Bacteria / isolation & purification
  • Candida / drug effects
  • Candida / isolation & purification
  • Cell Line, Tumor
  • Citrus / chemistry
  • Dose-Response Relationship, Drug
  • Drug Synergism
  • Gas Chromatography-Mass Spectrometry
  • Humans
  • Melaleuca / chemistry
  • Oils, Volatile / administration & dosage
  • Oils, Volatile / isolation & purification
  • Oils, Volatile / pharmacology*
  • Piper nigrum / chemistry


  • Anti-Infective Agents
  • Antineoplastic Agents, Phytogenic
  • Oils, Volatile