Study of antibacterial activity of Essential Oil components obtained from pericarp of Zanthoxylum rhetsa (Indian origin) using HS-GCMS

Significance of the topic

Essential oils from plant materials are widely valued for their potential as natural antimicrobial agents. Zanthoxylum rhetsa (Tirfal) pericarp oil has traditional culinary and medicinal uses in India but its precise antibacterial components and mechanisms remain underexplored. Elucidating the active constituents of this oil and their effects on common bacterial pathogens supports the development of alternative or complementary antimicrobial strategies in food safety, pharmaceuticals and industrial quality control.

Study objectives and overview

This study aimed to:

• Extract and analyze the chemical composition of essential oil from Z. rhetsa pericarp.
• Assess its antibacterial potency against Staphylococcus aureus MTCC 96 by minimum bactericidal concentration (MBC) assays.
• Identify which volatile components are depleted by bacterial exposure, suggesting their role in antibacterial activity, using headspace-GCMS.

Methodology and Used Instrumentation

The research was conducted in three stages:

• Oil extraction: Ground pericarp was hydro-distilled in water (20 g/200 mL) repeatedly to yield sufficient essential oil.
• Chemical profiling: Direct liquid injection GC–MS (Shimadzu GCMS-QP2010 Ultra) with FFNSC library matching and Kováts linear retention indices was used to identify components present at ≥ 0.3% of total area.
• Antibacterial assessment:
  • Tube assay to determine inhibition of S. aureus growth at oil concentrations of 1%, 2% and 4%.
  • MBC assay by spot inoculation from tubes onto nutrient agar.
  • Headspace-GCMS analysis (Shimadzu HS-20 headspace sampler coupled to GCMS-QP2010 Ultra) comparing 2% oil vials incubated with and without culture for 24 h to detect component depletion.

Results and Discussion

Chemical profiling revealed key constituents including sabinene (33.2%), β-pinene (11.4%), phellandrene isomers (total ~12%), terpinen-4-ol (5.7%), and decanal (5.3%).

The tube assay showed growth inhibition at 2% and 4% oil; MBC was confirmed at 2%.

Comparative headspace-GCMS of vials with and without bacterial exposure identified five compounds with significant area reduction, implicating them in antibacterial action:

• Dec-1-ene (~49% decrease)
• α-Terpinene (~71% decrease)
• γ-Terpinene (~74% decrease)
• n-Octanol (100% depletion)
• n-Decanal (~80% decrease)

These findings suggest that these volatiles are consumed or bound during interaction with S. aureus, correlating with bactericidal effects.

Benefits and Practical Applications

The study demonstrates a rapid workflow combining hydrodistillation, GC–MS profiling and headspace analysis to pinpoint active antimicrobial volatiles. The identified compounds offer candidates for natural preservatives, anti-infective formulations or synergistic blends in food, cosmetic and pharmaceutical industries. Moreover, the approach can be adapted to screen other botanical extracts for bioactive constituents.

Future Trends and Potential Uses

Opportunities for further research include:

• Assessing synergistic effects among the identified compounds and with standard antibiotics.
• Evaluating spectrum of activity against Gram-negative and fungal pathogens.
• Formulation development for controlled release in packaging or topical applications.
• Exploration of structural analogs to optimize potency and stability.

Conclusion

This study confirms the bactericidal efficacy of Z. rhetsa pericarp essential oil against S. aureus at 2% concentration and identifies five volatile constituents likely responsible for this activity. The combined use of GC–MS and headspace analysis proved effective in isolating target compounds, paving the way for natural antimicrobial development.

References

1. R.-X. Zhu, K. Zhong, W.-C. Zeng et al., Essential oil composition and antibacterial activity of Zanthoxylum bungeanum, African Journal of Microbiology Research, Vol. 5(26), 4631–4637 (2011).
2. FFNSC-MS Library, ver. 2, Chromaleont, Messina, Italy (2012).
3. E. Kovats, Advances in Chromatography 1, 229 (1965).