Formulation, Antimicrobial Evaluation and Stability Kinetics of a Polyherbal Leather Insole Containing Bryophyllum pinnatum, Calendula officinalis and Althaea officinalis Extracts

Abstract

Infected diabetic foot ulcers (DFUs) are a leading cause of non-traumatic lower-limb amputation, and rising antimicrobial resistance is limiting conventional antibiotic therapy. A leather insole that carries plant-derived antimicrobials and sits in continuous contact with the plantar surface could complement standard wound care. We extracted leaves of Bryophyllum pinnatum, Calendula officinalis, and Althaea officinalis sequentially with n-hexane, dichloromethane, ethyl acetate, and ethanol, and screened the 12 extracts for phytochemicals. We profiled the ethanol extracts by GC-MS. The antimicrobial activity of the extracts, and of vegetable-tanned leather (VTL) loaded with them, was tested by agar disc diffusion, MIC and MBC/MFC against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniae and Candida albicans isolated from DFU patients. A polyherbal insole (1:1:1, 25 mg/mL) was characterised by FTIR, and its antimicrobial potency was monitored for 96 days and fitted to first-order decay kinetics. Steroids, cardiac glycosides and terpenoids were present in all 12 extracts, and the more polar extracts had the richest profiles. Fatty acid methyl esters dominated the GC-MS profiles; oleic acid methyl ester made up 42.3% of the C. officinalis extract and 20.8% of the B. pinnatum extract. The extracts produced inhibition zones of 18–42 mm and MICs of 0.10–1.25 mg/mL, with an MBC/MIC ratio of 2 in every case, which indicates cidal activity. C. officinalis was the most active extract against S. aureus (42 mm; MIC 0.10 mg/mL), outperforming ciprofloxacin (28 mm), and all three extracts exceeded fluconazole against C. albicans (33-37 mm vs 20 mm). Extract-loaded leather retained on average 77% of the free-extract zone of inhibition. FTIR band shifts (O-H 3360 → 3297 cm⁻¹; C=O 1709 → 1638 cm⁻¹) indicated hydrogen bonding between the extract and the collagen matrix. The inhibition zone of the polyherbal insole fell from 42 mm to 16 mm by Day 84 and then plateaued. Regression across Days 0–84 gave k = 0.0116 day⁻¹ (R² = 0.981), t_½ ≈ 60 days and t₉₀ ≈ 9 days. The three extracts have broad antibacterial and anticandidal activity that largely survives incorporation into leather. The polyherbal insole retains useful activity for about two months, but its short t90 suggests replacing the insole at defined intervals or improving storage conditions.