Original Article FORMULATION AND CHARACTERIZATION OF SAMBILOTO EXTRACT ORALLY DISSOLVING FILMS: A DUAL MECHANISM FOR PROBIOTIC GROWTH AND ANTIBACTERIAL ACTION MIKSUSANTI MIKSUSANTI * , ELSA FITRIA APRIANI , ADIK AHMADI , SHAUM SHIYAN , DINA PERMATA WIJAYA , VIO AGISTER RISANLI Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Sriwijaya, Indralaya, South Sumatra, Indonesia * Corresponding author: Miksusanti Miksusanti; * Email: miksusanti@unsri.ac.id Received: 09 Oct 2024, Revised and Accepted: 26 Nov 2024 ABSTRACT Objective: This study aimed to develop Orally Dissolving Films (ODFs) containing Sambiloto leaf extract and evaluate their effects on the growth of the probiotic bacterium Bifidobacterium longum (B. longum) and their antibacterial activity against Escherichia coli (E. coli). Methods: The ODFs were prepared using the solvent casting method with three concentrations: F1 (0.4%), F2 (0.6%), and F3 (0.8%). The growth of B. longum was assessed through the Total Plate Count method, while antibacterial activity was determined using the disc diffusion method. Results: F2 was chosen as the optimal formulation, characterized by a smooth texture, a pH of 6.240±0.026, thickness of 0.102±0.008 mm, weight of 0.059±0.002 mg, disintegration time of 16.633±0.822 seconds, folding endurance of 433.00±2.000 folds, and elongation of 22.250±1.372%. F2 significantly enhanced the growth of B. longum, yielding 2.43×10¹⁰ CFU/ml and a prebiotic index of 1.056 (p<0.05). Additionally, it demonstrated antibacterial activity with an inhibition zone diameter of 7.500±0.408 mm (p<0.05). Conclusion: This research highlights F2's potential as a nutraceutical product with both probiotic growth-enhancing and antibacterial properties. Keywords: Antibacterial activity, Bifidobacterium longum, Nutraceutical, Orally dissolving films (ODF), Sambiloto extract © 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2025v17i2.52902 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Escherichia coli (E. coli) infection is a major cause of infectious diarrheal diseases. According to the latest report from the World Health Organization (WHO), there are approximately 1.7 billion cases of diarrhea caused by E. coli infection each year, with significant mortality rates, particularly among children under the age of five in developing countries. The increasing antibiotic resistance presents an essential challenge in managing E. coli infections. Recent studies have demonstrated that E. coli strains resist to several antibiotics, including ampicillin, sulfonamides, tetracycline, streptomycin, and gentamicin [1]. The urgency to find new solutions is heightened by the substantial clinical impact and the rising incidence of antibiotic resistance, necessitating the development of more effective and sustainable alternative treatments. Probiotics are a promising approach for managing infections caused by E. coli. Probiotics are beneficial intestinal microbiota that play a crucial role in maintaining the balance of the intestinal ecosystem and overall health. Specifically, probiotics such as Bifidobacterium longum (B. longum) are essential for intestinal health by inhibiting pathogen growth through nutrient competition and enhancing the immune system. Previous studies have shown that B. longum can increase lactic acid production, lowering intestinal pH and creating an inhospitable environment for pathogens like E. coli [2]. Additionally, research by Sharma et al. [3] revealed that B. longum strains could inhibit E. coli growth by producing bacteriocins, natural antimicrobials such as bifidocin. Bifidocin kills E. coli by breaking down its cell structure in several ways. It damages the cell membrane, leading to leaks of essential molecules like potassium, phosphate, and ATP. Additionally, bifidocin disrupts the cell’s energy balance by breaking the electric and pH gradients across the membrane, which are crucial for cell survival [4, 5]. These findings confirm the potential of probiotics in controlling E. coli infections, emphasizing their importance as a preventive and therapeutic strategy for maintaining intestinal health and combating pathogenic infections. The existence of probiotic bacteria is greatly influenced by the availability of nutrients that can support their activity and proliferation. Prebiotics are the primary nutrients probiotic bacteria need, including dietary fiber, oligosaccharides, and polysaccharides that function as fermentation substrates, as well as vitamins and minerals needed for bacterial metabolism. Flavonoids, bioactive compounds in many plants, also play an important role in increasing probiotic bacteria population. Flavonoids can function as prebiotic agents by providing additional substrates beneficial for the growth of probiotic bacteria [6]. Sambiloto (Andrographis paniculata L.) is a traditional medicinal plant renowned for its diverse health benefits. Extensive research has highlighted that this plant contains bioactive compounds, including flavonoids like quercetin. In addition to its prebiotic activity, flavonoids in sambiloto leaves have been demonstrated to inhibit the growth of pathogenic bacteria, including E. coli. This inhibition occurs through mechanisms such as the suppression of protein synthesis, disruption of bacterial cell membranes, and the creation of unfavorable environmental conditions for pathogens [7, 8]. Administering nutraceuticals, such as probiotics, through an Orally Dissolving Film (ODFs) can enhance bioavailability and provide effective antibacterial action. Due to the fast disintegration and resorption characteristics of ODFs from oral mucosa, the active principles can easily enter systemic circulation, bypassing gastrointestinal degradation phenomena compared to conventional dosage forms, hence the strong antibacterial action. For instance, a study on ODFs containing extracts from Vaccinium oxycoccos and Plectranthusamboinicus demonstrated effective inhibition of Streptococcus mutans at minimal inhibitory concentrations of 25 μg/ml and 50 μg/ml, respectively[9]. Also, because the extract usually used from the sambiloto leaf is highly bitter, a special formulation will be required to mask the bitter taste to enhance patient compliance. The ODF dosage form, flexible pharmaceutical films that rapidly dissolve in the mouth without water, effectively mask bitterness and enhance the ease of drug administration, increasing patient adherence. Thus, ODF formulations might offer a more efficient, patient-friendly alternative for managing E. coli infections. The primary components of ODFs are film formers and plasticizers. In this study, pullulan and maltodextrin were used as film formers. International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 17, Issue 2, 2025