Identification of antibacterial component from extract of Garcinia indica fruit rinds using LC/MS/MS

Importance of the topic

Medicinal plants remain a critical source for new antibacterial agents amid rising resistance to conventional antibiotics. Garcinia indica fruit rinds have a history of culinary and medicinal use and contain bioactive prenylated chalcones. Rapid detection and identification of such compounds are essential for drug discovery and quality control of natural products.

Aims and overview of the study

The study aimed to isolate and identify the antibacterial constituent of Garcinia indica rind extract. Direct bioautography on thin layer chromatography was used to locate active fractions against the plant pathogen Xanthomonas axonopodis pv punicae. The bioactive zone was then characterized by liquid chromatography tandem mass spectrometry with multiple reaction monitoring.

Methodology and instrumentation

Preparation of extract

• Shade-dried Garcinia indica rinds were ground and subjected to Soxhlet extraction with ethyl acetate for 18–24 hours.
• Extracts were evaporated and dissolved in methanol for analysis.

Bioautography

• TLC plates developed in toluene–ethyl acetate–formic acid (4 : 1 : 0.5) were sprayed with Xanthomonas axonopodis culture and incubated at 28 C.
• Plates were stained with p-iodonitrotetrazolium dye; clear zones indicated bacterial inhibition.

LC/MS/MS analysis

• Shimadzu LCMS-8040 Triple Quadrupole with electrospray ionization was used.
• Chromatography was performed on a C18 column at 40 C, 0.6 mL/min flow rate, with a gradient of 10 mM ammonium acetate and methanol and UV detection at 238 nm.
• Optimisation of collision energies for the precursor ion m/z 603 allowed identification of characteristic product ions.

Main results and discussion

Bioautography revealed a single inhibition band. LC/MS full scan in positive mode detected [M + H]+ at m/z 603 and in negative mode [M − H]− at m/z 601. Collision induced dissociation at incremental energies produced fragment ions at m/z 585, 535, 467, 411, 343, 287, 233, 177, 137, 81 and 57. The fragmentation pattern matched that of garcinol, confirming the active compound as the prenylated chalcone garcinol.

Benefits and practical applications

• The combined bioautography and LC/MS/MS workflow enables rapid screening and precise identification of antibacterial compounds in complex plant extracts.
• Identification of garcinol supports its potential development as an antibacterial agent in agriculture and clinical settings.
• The approach is applicable for quality control of herbal preparations and for discovery of new antimicrobial leads.

Future trends and opportunities

• Integration of high resolution mass spectrometry and nuclear magnetic resonance will allow definitive structural confirmation and stereochemical analysis.
• Comprehensive metabolomic profiling could uncover additional synergistic bioactives in Garcinia indica.
• Automated TLC bioautography combined with mass spectrometry imaging offers high throughput screening capabilities.
• Further work on formulation and standardisation of extracts could advance translation into commercial antibacterial products.

Conclusion

This study demonstrates an efficient workflow combining direct bioautography and LC/MS/MS to isolate and identify garcinol as the antibacterial principle in Garcinia indica rind extract. The method offers a model for rapid natural product screening and bioactive compound discovery.

References

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• Choma IM Grezelak EM Bioautography detection in Thin layer chromatography J Chromatogr A 2010
• Quality Standards of Indian Medicinal Plants Medicinal plants unit ICMR New Delhi 2008 Vol 7 123–132