Phytochemical composition and antimicrobial potential of Stevia rebaudiana Bertoni extract and its topical spray formulation against animal skin pathogens
Authors: Singh Ranee , Borlace Glenn Neville , Sringam Patchanee , Thongkham Eakachai , Aiemsaard Jareerat 
Published: December 2024

Background and Aim: The rise of antimicrobial resistance in veterinary medicine is a significant concern, particularly for pathogens responsible for skin infections. Although Stevia rebaudiana Bertoni (stevia) has demonstrated effective antimicrobial properties, there is limited research on its efficacy against animal skin pathogens. This study aimed to identify natural compounds in stevia extract, develop a topical spray formulation, and assess its effectiveness against six common bacterial and fungal pathogens associated with animal skin infections.

Materials and Methods: The aerial parts of stevia plants were extracted using hexane in a Soxhlet apparatus. Total phenolic and flavonoid contents were quantified using colorimetric assays. The volatile oil content was analyzed using gas chromatography-mass spectrometry (GC-MS). The antimicrobial activity of stevia extract against Staphylococcus pseudintermedius, Malassezia pachydermatis, Microsporum canis, Microsporum gypseum, Microsporum gallinae, and Trichophyton mentagrophytes was evaluated using broth microdilution and time-kill tests. Environmental scanning electron microscopy (E-SEM) and leakage studies were conducted to assess the extract’s impact on microbial morphology and cell membrane integrity. The antimicrobial efficacy and stability of a topical spray formulation containing stevia extract were evaluated using time-kill and freeze-thaw testing.

Results: The stevia extract yield was 3.59% of the dry plant weight with 259.96 ± 23.66 mg gallic acid equivalent (GAE)/g extract of total phenolics and 247.41 ± 19.92 mg quercetin equivalent (QE)/g extract of total flavonoids. GC-MS analysis identified major volatile components, including N-acetyl-14, 15, 16-trinorlabd-8(17)-en-13-amine (37.70% of peak area), phytol (11.02% of peak area), (-)-spathulenol (9.46% of peak area), n-hexadecanoic acid (8.01% of peak area), and (diphenylphosphinoyloxymethyl) dimethylsilane (7.59% of peak area). The minimum inhibitory concentration of the extract against the tested microorganisms ranged from 0.25 to 128.00 mg/mL. Time-kill kinetics exhibited time- and concentration-dependent germicidal effects. E-SEM and cell leakage analyses indicated that stevia extract compromised microbial cell membrane integrity. A spray formulation containing 10% w/w stevia extract displayed excellent eradication efficacy, achieving a 99.9999% reduction of S. pseudintermedius and a 99.999% reduction of M. pachydermatis and dermatophytes, with good stability after six freeze-thaw cycles.

Conclusion: Stevia extract is an effective antimicrobial against S. pseudintermedius, M. pachydermatis, Mi. canis, Mi. gypseum, Mi. gallinae, and T. mentagrophytes in vitro. Future research will investigate the pharmaceutical properties and toxicity profiles of purified compounds and determine appropriate dosages and clinical efficacy.

Keywords: animal skin infection, antimicrobial activity, Stevia rebaudiana, topical formulation.