QBD-based optimization of sequential extraction of anthocyanins from Krishna Tulsi (Ocimum tenuiflorum): investigation of its bioactivities for biomedical applications.

QBD-based optimization of sequential extraction of anthocyanins from Krishna Tulsi (Ocimum tenuiflorum): investigation of its bioactivities for biomedical applications - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice 3 Biotech . 2025 May 12;15(6):162. doi: 10.1007/s13205-025-04334-1 QBD-based optimization of sequential extraction of anthocyanins from Krishna Tulsi ( Ocimum tenuiflorum ): investigation of its bioactivities for biomedical applications Amrita Chatterjee 1 , Rajdeep Saha 1 , Ravi Adinarayan Somabattini 2 , Satish Kumar 1 , Riya Ghosh 1 , Anupam Roy 3 , Uma Ranjan Lal 4 , Kunal Mukhopadhyay 5 , Satheesh Kumar Nanjappan 2 , Biswatrish Sarkar 1, ✉ Author information Article notes Copyright and License information 1 Group Polyphenol-BIT, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215 India 2 Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, India 3 Department of Chemical Engineering, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215 India 4 Department of Natural Products, National Institute of Pharmaceutical Education and Research, S.A.S. Nagar, Mohali, Punjab 160062 India 5 Department of Bio-Engineering and Biotechnology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand 835215 India ✉ Corresponding author. Received 2024 Nov 9; Accepted 2025 Apr 28; Issue date 2025 Jun. © King Abdulaziz City for Science and Technology 2025. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. PMC Copyright notice PMCID: PMC12069208  PMID: 40370430 Abstract Krishna Tulsi ( Ocimum tenuiflorum L.), is known for its rich polyphenolic composition, including anthocyanins, which contribute to its bioactivity. Efficient extraction methods are critical to maximizing the yield of these bioactive compounds. This research focuses on optimizing the parameters for the extraction of anthocyanins from Ocimum tenuiflorum L. using response surface methodology. Extraction conditions were designed with the Box–Behnken model varying solvent concentration [ethanol (20–80%), HCl (0.1–2.2%), and temperatures (10–55 °C)]. Further responses such as total anthocyanin content and antioxidant activity were validated. Anthocyanins such as peonidin, cyanidin, delphinidin, and their derivatives were identified by UHPLC–QTOF–MS. Bioactivities such as DNA protection, antibacterial properties, and in-silico binding affinity with biofilm-forming proteins were assessed. Optimal conditions (79.396% ethanol and 1.288% HCl at 21.102 °C) yielded 188.16 mg/g anthocyanins, with IC 50 values of 12.28 and 20.74 µg/mL in DPPH and ABTS assays, respectively. The extract reversed Fenton’s reaction of oxidative damage to calf thymus DNA. It exhibited significant antibacterial activity against gram-negative ( Escherichia coli and Pseudomonas aeruginosa ) and gram-positive ( Staphylococcus aureus and Bacillus subtilis ) bacteria. In addition, considerable biofilm inhibition (ranging from 91 ± 6.110 at 4*MIC to 30.667 ± 3.055% at MIC/2 for S. aureus ) and destruction of bacterial surface morphology were observed in FESEM. Docking studies revealed strong binding affinities of cyanidin and peonidin derivatives with biofilm-forming proteins (PDB id—3TIP and 4KH3), highlighting their potential as biofilm inhibitors. Therefore, by integrating computational and experimental strategies, these findings support the development of phytopharmaceutical formulations leveraging anthocyanins from Krishna Tulsi as potential therapeutic agents in the future. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-025-04334-1. Keywords: Anthocyanin, Solvent optimization, Response surface methodology, DNA protection, In-silico , Antimutagenic, Antimicrobial, Biofilm Introduction Water-soluble anthocyanins are widely distributed secondary metabolites of plants commonly found in flowers, leaves, stems fruits, and vegetables. Distinct anthocyanin types, i.e., peonidin, cyanidin, and delphinidin, bear potential functional effects, such as antioxidant, anti-cancer, anti-aging, and anti-inflammation properties (Zhang et al. 2020 ; Ghareaghajlou et al. 2021 ). Based upon consumer awareness of natural anthocyanin, an adult in the United States, consumes around 12.5 mg of anthocyanins per day from their diet (Wu et al. 2006 ). The selective antimicrobial action of anthocyanin against human pathogens (Ma et al. 2019 ) and its protective function during oxidative stress and inflammation hold a functional value in the food and nutraceutical industry (Speer et al. 2020 ). Oxidative stress can alter the structure and functions of DNA bases leading to cellular injury and death. The antioxidant effects of anthocyanins supported by their free radical scavenging properties also protect against oxidative DNA damage and serve as the major physiological arsenal in combating cellular damage induced by oxidative stress. The food industry suffers recurrent loss from degradation due to food-borne bacteria which tend to form biofilm over food surfaces. Biofilm formation is also part of microbial defense under stress from hostile surroundings, host immunological responses, or the presence of antimicrobials, which enhances their immune responses and allows them to stay within the biofilm matrix (Tamfu et al. 2020 ). These biofilms are highly protected by a self-secreted exopolymeric matrix made up of proteins, carbohydrates, lipids, nucleic acids, little signaling molecules, etc. Bacterial resistance against antimicrobial agents increases when it enters into biofilm formation (Mohammadi et al. 2019 ). Despite having synthetic biofilm eradicators, consumers prefer natural compounds. Since anthocyanins are already in use as food colorants, their antimicrobial and antibiofilm properties have increasingly been studied (Jeyaraj et al. 2022 ). This demands a search for sustainable sources of anthocyanins— Ocimum species commonly known as Tulsi is a rich reservoir of bioactive compounds. Ocimum tenuiflorum L. (Lamiaceae) or Krishna Tulsi is unique in the Basil community due to its variety of ethnomedicinal applications. Sometimes, the presence of anthocyanins in this species is also a unique feature in comparison with other common Basil species (Sankhalkar and Vernekar 2016 ; Sarkar et al. 2018 ; Chatterjee and Sarkar 2024 ). Different parts of this plant are being used for ceremonial, religious, and medicinal values. Leaves of the plant are mostly valued and used in herbal teas or as green tea additives that have antimicrobial, antioxidant, anti-inflammatory, and antipyretic activities (Rabeta and Lai 2013 ). Thus, extracting, concentrating, anthocyanin profiling, and exploring the biological activity is important for exploring the functional uses of Ocimum tenuiflorum. Since extraction of anthocyanins depends on several variable factors, optimization of the process is a cumbersome technique. Various solvents (such as absolute organic solvents or their aqueous mixtures) with varying pH, have been employed for the extraction of anthocyanins from plant parts. Sametime different plant parts require different compositions of solvent system for optimal anthocyanin extraction. In this regard, Response Surface Methodology (RSM) has been effectively utilized to determine optimal extraction conditions across various domains, notably in extracting valuable phytochemicals. RS investigates the relationship between process variables and responses using minimal experimental runs. The Box–Behnken Design (BBD), a spherical and rotatable design within RSM, consists of points situated at the midpoint of each edge and replicated center points of a multidimensional cube (Jeganathan et al. 2014 ). The extraction of anthocyanins from Krishna Tulsi also presents scientific significance given its ethnopharmacological importance in India. Therefore, in this study, Box–Behnken design for optimization was employed to evaluate the individual and interactive effects of parameters—specifically solvent system (ethanol:hydrochloric acid:water) and temperature for extraction—on the extraction yield of anthocyanin compounds from Krishna Tulsi. It was hypothesized that optimizing the solvent system to extract the highest content of anthocyanin from O. tenuiflorum can be correlated with its antioxidant potential. Apart from the anthocyanin profiling elucidating other bioactivities (such as DNA damage protection, antimicrobial, and biofilm inhibition) would help to emphasize functional uses of Ocimum tenuiflorum anthocyanin extract (OTAE). Thus, this study explored the optimization of solvent systems for extracting anthocyanins from Ocimum tenuiflorum L. , employed UHPLC–QTOF–MS, to profile the anthocyanins and same time investigated the anti-genotoxicity, antimicrobial, and biofilm inhibition properties In addition, an in-silico study was performed to understand the interaction of anthocyanins with the biofilm-forming proteins of Staphylococcus aureus and Escherichia coli . Materials and methods Chemicals and antibiotics All the chemicals and solvents used for the study were of analytical grade with 99% purity, while double distilled water was used for extraction purposes unless otherwise mentioned. The respective suppliers of the chemicals and reagents are listed in the supplementary file. Plant material and test microorganisms Fresh Ocimum tenuiflorum L . (Lamiaceae) plant parts grown in the medicinal plant garden of BIT, Mesra, Ranchi campus were collected and shade dried (moisture content 9% dry basis). The plant had been identified and authenticated by the Central National Herbarium, Botanical Survey of India (CNH/30/2012/Tech II). Bacterial strains used for antimicrobial activity study were obtained from the Institute of Microbial Technology Chandigarh, India, and maintained according to the specifications provided. Extraction of anthocyanins 125 mg powdered aerial part (#40 mesh or less than 420 µM particle size instead) were macerated with 5 mL of solvent while stirring with a magnetic stirrer at 800 rpm for 4 h in the dark. The mixture was then centrifuged at 4000 rpm for 15 min at 4 °C. Samples were stored at − 20 °C in amber bottles and kept until required for analysis (Sarkar et al. 2018 ). Determination of total anthocyanin content and antioxidant activity of anthocyanin extract The total anthocyanin content (TAC) determination The pH differential method measured the TAC and reported as mg cyanidin-3-glucoside/100 gm. dry weight. Briefly, the sample was diluted with distilled water, and two aliquots were prepared. The first aliquot was mixed with a potassium chloride buffer (pH 1.0) and the second with a sodium acetate buffer (pH 4.5). After equilibrating for 15 min at room temperature, the absorbance of each aliquot was measured at 520 and 700 nm using a UV–Vis spectrophotometer. The absorbance values were corrected for haze by subtracting the reading at 700 nm from that at 520 nm. The difference in absorbance (ΔA) between the two pH conditions was used to calculate the monomeric anthocyanin pigment concentration, expressed as cyanidin-3-glucoside equivalents (mg/gm), using the formula: C (mg/gm) = (ΔA × MW × DF × 1000)/(ε × 1 × m), where MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF is the dilution factor, and ε is the molar extinction coefficient (26,900 L·cm⁻ 1 ·mol⁻ 1 ), m is the mass of sample (Giusti and Wrolstad 2001 ). Antioxidant activity DPPH radical scavenging assay The scavenging activity was checked by measuring the quenching of DPPH free radicals, according to the earlier reported methods with some modification (Re et al. 1999 ; Sharma and Bhat 2009 ; Chatterjee et al. 2021 ). Briefly, 100 µL of freshly prepared 0.2 mM DPPH solution in methanol was added to different concentrations (5–80 µg/mL) of the extract. After incubation in the dark for 30 min at room temperature (20 ± 5 °C), the reduction in absorbance of DPPH free radicals was estimated using a 96-well plate reader (Thermo Scientific Multiskan GO) at 517 nm and the percentage inhibition was calculated using a standardized formula. Ascorbic acid at a concentration (5–80 µg/mL) was taken as a standard and antioxidant activity was determined by calculating IC 50 (inhibitory concentration 50% value). ABTS •+ scavenging assay Another antioxidant potential was evaluated by measuring the quenching of ABTS •+ free radicals, according to the earlier reported methods with some modifications (Re et al. 1999 ; Sharma and Bhat 2009 ; Chatterjee et al. 2021 ). Briefly, the stock solution was prepared by mixing 7.4 mM ABTS ·+ and 2.6 mM potassium persulfate in methanol and kept in the dark for 12–16 h. at room temperature. Before beginning the assay, the ABTS solution was diluted with methanol to an absorbance of 0.700 at 734 nm. Various concentrations (5–80 µg/mL) of the extract were mixed with 100 µL of stock solution and incubated in the dark for 30 min at room temperature (20 ± 5 °C) and absorbance was taken at 734 nm. Quercetin was used as positive control for this assay and antioxidant activity was determined by calculating IC 50 . Box–Behnken-based optimization of the anthocyanin extraction process A Box–Behnken statistical screening design was employed for the systematic development of a predictive model and the comprehensive evaluation of the main, interaction, and quadratic effects of key process parameters on the anthocyanin content and antioxidant activity, as measured by DPPH and ABTS radical scavenging assays. The Box–Behnken design (BBD) was utilized to optimize the number of experimental runs and to identify the most significant factors influencing the extraction process. The effects of extraction parameters, including ethanol concentration (20–80%), hydrochloric acid concentration (0.1–2.2%), and extraction temperature (10–55˚C), were assessed by varying these factors at three levels across 15 experimental runs, including three central points. Model predictions were generated using the Design Expert software, version 13.0.3.0 (Stat-Ease, Inc., Minneapolis, MN), and demonstrated a strong correlation with the observed experimental results. UHPLC–ESI–QTOF–MS analysis The separation and structural characterization of anthocyanins present in OTAE obtained from an optimized solvent system was inferred using UHPLC–QTOF–MS ( Agilent 1290 HPLC that was coupled to an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (MS) with a Dual Jet Stream electrospray ionization) through untargeted metabolomics approaches. The UHPLC (Agilent Corp., Santa Clara, CA, USA) consisted of a binary pump, an autosampler, a column compartment, and a diode array detector. The elution was performed using a binary gradient system with eluent 0.1% formic acid (solvent A) and Acetonitrile (solvent B) at a gradient program is as follows: 0–8 min, 2–10% B; 8–12 min, 10–13% B; 12–15 min, 13–20% B; 15–19 min, 20–25% B; 19–21 min, 25–30% B; 21–23 min, 30–30% B; 23–30 min, 30–2% B; 30–35 min, 2–2% B. Agilent Eclipse Plus C18 (100 mm × 2.1 mm, 1.8 µm) column was used and oven temperature was set to 40 °C. The flow rate of the mobile phase was 0.6 mL/min and the injection volume used was 5 μL. The separated components were passed through a Q-TOF mass spectrometer (Agilent Corp., Santa Clara, CA, USA), equipped with a Dual AJS ESI interface and supported by MassHunter Workstation. The instrument parameters are as follows: Gas Temp. 320 °C, Gas Flow 10 l/min, Nebulizer 35 psig, 350 Sheath Gas Temp. with 10 Sheath Gas Flow. Scan Source Parameters set to VCap 3500 V, Nozzle Voltage 1000 V, Fragmentor 175, Skimmer1 65, Octopole RF Peak 750 V, and Collision Energy 10 V. Samples were analyzed by setting the scanning range from 50 to 1700 m/z for MS/MS in the negative mode of ionization. Antimutagenic properties in DNA A Fenton-induced DNA nicking assay of OTAE was performed following the protocol described by Wu and Dhanasekaran ( 2020 ), with minor adjustments. In summary, a reaction mixture totaling 20 µL was prepared, containing 600 ng of calf thymus DNA and 20 mM phosphate-buffered saline (PBS). This mixture was treated with 200 µg of OTAE in a microcentrifuge tube and incubated for 10 min at room temperature. Subsequently, a Fenton-reaction mixture, composed of 15 mM ethylenediaminetetraacetic acid (EDTA), 15 mM hydrogen peroxide (H 2 O 2 ), and 8 mM ferrous sulfate (FeSO 4 ), was added and the incubation continued for 30 min. Controls included calf thymus DNA alone and calf thymus DNA treated with 200 µg of OTAE. Following the treatments, the samples were subjected to electrophoretic analysis, and DNA mobility was visualized by photographing the gels under UV light at 312 nm using a transilluminator. In vitro antimicrobial activity Disc diffusion assay The antimicrobial properties of OTAE were assessed by disc diffusion assay. Briefly, 150 µL of the bacterial culture (gram-positive Staphylococcus aureus , Bacillus subtilis, and gram-negative Pseudomonas aeruginosa , Escherichia coli) grown at mid-log phase (5 × 10 6 CFU/mL) in LB broth. The cultures were subsequently spread over a sterilized LB agar medium plate to achieve confluent growth. Filter paper discs (6 mm) impregnated with 15 µL of anthocyanin extracts at three different concentrations (1, 0.5, 0.25 mg/mL) were placed on the agar plates inoculated with the specific bacteria (Sarkar et al. 2022 ). Streptomycin was taken as a positive control, whereas disc without any drugs was considered a negative control (Sati et al. 2019 ). The impact of OTAE was checked, and the zone of inhibition diameter in the plate was measured. Determination of MIC and MBC Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of OTAE against both gram-positive and gram-negative bacteria were measured through the micro-plate-dilution method by the National Committee for Clinical Laboratory Standards (NCLS original reference). The anthocyanin extracts were serially diluted (4000 µg to 1.95 µg) in 96 well plates. The lowest concentration of the test sample that showed prevention against microbial growth (clear lane as indicative of no growth) was referred to as MIC value (Tong et al. 2021 ). To determine MBC, 100 μL of the previously grown bacterial culture, up to MIC concentration, were plated on LB agar and incubated for 24 h at 37 °C. After 24 h, colonies grown on plates were counted and compared against positive control. MBC of the extract is the lowest concentration at which no microbe could survive was observed. All the MIC and MBC analyses were carried out in triplicate (Roy et al. 2020 ). Anti-biofilm screening The anti-biofilm activity of OTAE was studied against two biofilm-forming foodborne pathogens ( S. aureus , and E. coli ) using the 96-well plate technique (Barabadi et al. 2021 ). In brief, 100 μL of liquid culture (10. 6 CFU/mL) of each bacterium was put into 96 well plates and incubated for an initial period of 24 h. It is further co-supplemented with 100 μL of fresh Nutrient broth for subsequent 2 days with incubation followed by discarding the broth and washing the cells with 0.1% normal saline (w/v). 100 µL of anthocyanin extract with varying concentrations (a final concentration of 0.5, 1, 2, and 4*MIC) was further added over the grown biofilm in the well, incubated for 24 h at 37 °C, stained with 0.4% crystal violet absorbance was taken at 570 nm (SpectraMax i3x Multi-Mode Microplate Reader). The biofilm inhibition percentage was estimated using the method below (Ni et al. 2022 ; Gharaei et al. 2022 ) Biofilm inhibition (\% ) = OD control - OD treated / OD control × 100 FESEM analysis of the biofilm inhibitory potential The effect of OTAE on the morphology of S. aureus bacterial biofilm was seen under a field emission scanning electron microscope (FESEM) (accelerating voltage: 20 kV) (Carl Zeiss Microscope Ltd., Germany; Sigma 300). The cells were fixed with glutaraldehyde (2.5%) and dehydrated ethanol solutions (30%, 50%, 70%, 90%, 95%, and 100%) (Roy et al. 2020 ; El-Maati et al. 2022 ) and seen under field emission scanning electron microscopy. Protein structure preparation and molecular docking The molecular docking study was carried out through several key steps, starting with the retrieval of the film-forming protein structures of Staphylococcus aureus (PDB ID: 3 TIP) and Escherichia coli (PDB ID: 4 KH3) from the Protein Data Bank. These protein structures were prepared using AutoDock Tools and Biovia Discovery Studio. Ligand structures in SDF format were obtained from the PubChem database. The active sites of the 3 TIP and 4 KH3 receptors were identified using PyMOL software, and grid boxes were generated at these sites for docking. Molecular docking was then conducted using AutoDock Vina with default settings, enabling the evaluation of binding affinities and interactions between the ligands and the receptors. The docking outcomes were analyzed in Biovia Discovery Studio, which provided summaries of the best-docked complexes and their binding energies (Ni et al. 2022 ; Mousavi et al. 2023 ). Finally, the binding modes and affinities of compounds from Ocimum tenuiflorum to the selected receptors were compared to identify the most promising candidates for further investigation. Statistical analysis All data were expressed as mean ± standard deviation (SD) with n = 3. One-way ANOVA followed by Tukey's multiple comparison test was performed using GraphPad Prism software version 5.0 to determine the significant difference between control and test and standard groups, whereby p < 0.05 was considered significant. Results Effect of extraction conditions on anthocyanin yield and antioxidant properties of Krishna Tulsi During the optimization procedure, fifteen trials were conducted. Anthocyanins from Ocimum tenuiflorum were extracted using ethanol (20–80%) and hydrochloric acid (0.1–2.2%) under controlled temperatures (10–55 °C). The Box–Behnken design (BBD) (three factors, three levels) assessed extraction parameters on anthocyanin yield and antioxidant activity. Effects of ethanol concentration (A), hydrochloric acid concentration (B), and temperature (C), on the total anthocyanin content, can be described by the following equation. Total anthocyanin content TAC = 150.11666666667 + 31.88375 A + 3.90875 B - 5.7925 C - 1.4975 AB + 0.535 AC - 1.625 BC + 2.2804166666667 A 2 - 10.654583333333 B 2 - 4.9320833333333 C 2 DPPH = 33.4017 - 27.8415 A - 4.15875 B + 4.97925 C + 1.8005 AB - 2.208 AC - 1.384 BC + 6.62142 A 2 + 9.01742 B 2 + 2 . 91742 C 2 ABTS = 41.4407 - 26.0899 A - 4.35462 B + 5.14975 C + 2.22225 AB - 1.7475 AC - 0.906 BC + 5.3670 4 A 2 + 9.18054 B 2 + 2.99529 C 2 The factors (A, B, and C) were studied for optimization with the help of BBD in all possible combinations (Tables S1 and S2). The observed values of the responses are presented in Table S2. The quadratic model significantly influenced outcomes. At optimal conditions (79.644% ethanol, 1.231% HCl, 26.699 °C) predicted yield was 185.034 mg/g anthocyanins with IC 50 values of 11.726 µg/mL (DPPH) and 20.124 µg/mL (ABTS). The desirability function reached 1.000. The results demonstrated a statistically significant influence (p < 0.05) on ethanol concentration and temperature on anthocyanin content within the extract (Table S1 ). These findings were graphically depicted using 3D surface plots (Fig. 1 ), illustrating the relationships derived from the associated equation. The extraction efficiency of anthocyanins increased with rising temperature, reaching an optimal point before declining as temperatures increased. The DPPH radical scavenging activity of the 15 tested samples, expressed as IC 50 values, ranged from 13.743 to 86.548 µg/mL, while the reference compound ascorbic acid exhibited an IC 50 of 23.21 ± 2.73 µg/mL. Similarly, quercetin, used as a standard in the ABTS assay, presented an IC 50 of 10.450 ± 0.99 µg/mL, with the test samples showing IC 50 values ranging from 21.855 µg/mL to 91.568 µg/mL. Ethanol concentration, hydrochloric acid concentration, and temperature significantly impacted antioxidant activity, which increased with higher ethanol concentrations, corroborating the correlation between elevated anthocyanin yield and enhanced antioxidant capacity. The fitting curves (Fig. 1 ) reveal that antioxidant activity peaked at approximately 32.5 °C before declining, with a similar pattern observed at an acid concentration around 1.15%, followed by a subsequent decrease. All relationships demonstrated statistical significance ( p < 0.05). Fig. 1. Open in a new tab 3D response surface plots to illustrate the influence of extraction parameters. Effects of the three independent variables, i.e., ethanol (20–80%), hydrochloric acid (HCl) (0.1–2.2%), and temperatures (10–55 °C), on three dependent or response variables, i.e., total anthocyanin content (TAC) estimation, DPPH, and ABTS radical scavenging activity, were evaluated. a The anthocyanin content as a variable of ethanol concentration and HCl concentration, b the anthocyanin content as a variable of HCl concentration and temperature, and c the anthocyanin content as a variable of temperature and ethanol concentration. The extraction efficiency of anthocyanins increased with rising temperature, reaching an optimal point before declining as temperatures increased. A similar trend was observed for the influence of hydrochloric acid concentration, where anthocyanin yield increased up to a mid-range concentration of 1.15% but declined beyond a certain acid–alcohol threshold. d The inhibitory concentration of 50% (IC 50 ) of DPPH response as a variable of ethanol concentration and HCl concentration, e IC 50 of DPPH response as a variable of HCl concentration and temperature, f IC 50 of DPPH response as a variable of temperature and ethanol concentration, g IC 50 of ABTS response as a variable of ethanol concentration, and HCl concentration, h IC 50 of ABTS response as a variable of HCl concentration and temperature, i IC 50 of ABTS response as a variable of temperature and ethanol concentration. The 3D fitting curves reveal that antioxidant activity peaked at approximately 32.5 °C before declining, with a similar pattern observed at an acid concentration of around 1.15%, followed by a subsequent decrease Model fitting The experimental design was structured using the Box–Behnken model, with the corresponding results detailed in Table S2. A strong correlation was observed between the experimental and predicted values, indicating high model accuracy. The coefficient of determination ( R 2 ) and adjusted R 2 values for the proposed models were substantial (Table S3), reflecting strong predictive capability and model reliability. The minimal difference (< 0.2) between the predicted R 2 and adjusted R 2 further underscores the robustness and applicability of the models employed. Validation of the model Model validation was performed by comparing experimental data with the predicted values under optimized extraction conditions (Table 1 ). The results indicated a high degree of concordance between the experimental and predicted values, thereby confirming the robustness of the model fit. This validation reinforces the reliability of the model and underscores its applicability in optimizing anthocyanin extraction from OT. Table 1. Validation of the model Factors Responses Desirability Ethanol (%) Hydrochloric acid (%) Temperature (°C) TAC (mg/gm) DPPH (IC 50 ) ABTS (IC 50 ) Predicted value 79.396 1.288 21.102 185.196 11.734 19.990 1.000 Experimental value 79.396 1.288 21.102 188.16 ± 2.43 12.28 ± 0.589 20.74 ± 1.19 – Error (%) – − 1.602 − 4.621 − 3.745 – Open in a new tab From the data that was obtained through the software and the experiments, differences found within the observed and the predicted values were − 1.602%, − 4.621, and − 3.745, respectively, for TAC, DPPH, and ABTS responses (Table 1 ). The acceptable range of the difference must be within ± 5%, so, the model that was used can be identified as valid and accurate (Choudhary and Pramanik 2021 ). UHPLC–Q-ToF–ESI–MS analysis for identification of anthocyanin compounds A comprehensive chemical profiling applying the UHPLC–Q-ToF–MS technology was conducted to investigate the anthocyanins in the optimized anthocyanin extract (OTAE), with the highest anthocyanin yield and lowest IC 50 or highest antioxidant potential. The Q-ToF–MS-based untargeted metabolomics approach was used in this investigation because of its excellent accuracy and sensitivity for detecting secondary metabolites. Figure 2 illustrates the total ion chromatogram of the sample. The LC–MS raw data sets were further matched with the PCDL libraries in (Agilent, USA) for compound identification, as tabulated in Table 2 . Once the large compound splits into little compounds, producing distinctive peaks at varied m/z ratios, diverse compounds eluted at varying retention durations were identified. Few of the compounds with bioactive properties that have been found have the potential to become future therapeutic candidates. As a result, 10 compounds were identified in the negative mode (Fig. 2 ) and their mass spectrum is mentioned in Figs. S1 – S10 . The compounds were identified in the negative mode with good scores, mass, retention time, and area are represented in Table 2 . Structures of anthocyanin compounds detected in Krishna Tulsi are illustrated in Fig. 3 . Fig. 2. Open in a new tab UHPLC–PDA chromatogram of the crude anthocyanin-rich extract of O. tenuiflorum L. Chromatogram captured at 360 nm wavelength. (Peak assignment: 1—Cyanidin-3,5-diglucoside; 2—Peonidin-3-O-alpha-arabinopyranoside; 3—Peonidin-3-(6″-acetoyl) galactoside; 4—Cyanidin-3-glucoside; 5—Cyanidin; 6—Peonidin-3-O-beta-galactopyranoside; 7—Peonidin; 8—Cyanidin 3-O-(6-O-p-coumaroyl) glucoside; 9—Cyanidin3-(6″-acetoyl) galactoside; 10—Delphinidin) Table 2. List of anthocyanins identified in O. tenuiflorum Linn. by UHPLC–PDA–ESI–MS analysis (Sarkar et al. 2014 , 2018 ; Flanigan and Niemeyer 2014 ; Olawale et al. 2022 ) Sr. no. Proposed compound name Formula Molecular weight RT (min) Width Height Area [M–H] − (m/z) Error (ppm) Score 1 Cyanidin-3,5-diglucoside C 27 H 31 O 16 611.1656 14.169 0.443 7538 204,983 610.1587 7.25 74.4 2 Peonidin-3-O-alpha-arabinopyranoside C 21 H 20 O 10 432.1124 17.806 0.379 3507 64,221 431.1054 15.68 52.49 3 Peonidin-3-(6″-acetoyl) galactoside C 24 H 25 O 12 505.1311 22.007 0.254 21,553 368,223 504.123 −7 52.59 4 Cyanidin-3-glucoside C 21 H 21 O 11 449.1117 23.601 0.115 6432 49,659 448.1039 7.28 62.7 5 Cyanidin C 15 H 11 O 6 287.0562 23.8 0.559 73,494 2,578,898 286.0486 2.16 92.82 6 Peonidin-3-O-beta-galactopyranoside C 22 H 23 O 11 463.1266 26.341 0.254 4771 101,864 462.1193 5.52 71.57 7 Peonidin C 16 H 13 O 6 301.072 27.371 0.365 1907 29,183 300.0643 2.73 50.52 8 Cyanidin 3-O-(6-O-p-coumaroyl) glucoside C 30 H 27 O 13 595.1509 29.148 0.362 259,172 6,161,214 594.144 9.68 47.28 9 Cyanidin3-(6″-acetoyl) galactoside C 23 H 23 O 12 491.1206 29.978 0.421 25,305 641,795 490.1151 3.45 70.64 10 Delphinidin C 15 H 11 O 7 303.0514 31.356 1.093 4522 234,884 302.044 2.98 74.21 Open in a new tab RT: Retention time; ppm: parts per million Fig. 3. Open in a new tab Structures of anthocyanin compounds detected in Krishna Tulsi. The antioxidant capacities of anthocyanins are attributed to the reducing power of the ortho-dihydroxy groups and the number of hydroxyl groups (–OH) in the B ring of the anthocyanin structure, which confers a stable radical form and facilitates electron delocalization. Glycosylated anthocyanins (Cyanidin-3-glucoside, Cyanidin-3,5-diglucoside, Cyanidin 3-O-(6-O-p-coumaroyl) glucoside) are more easily soluble in aqueous environments and transport in the bloodstream than their aglycone forms Evaluation of antimutagenic properties of anthocyanins Figure 4 shows the protection imparted by OTAE to Fenton (hydroxyl radical) induced damage to calf thymus DNA. Control calf thymus DNA (Lane 1) showed proper structural integrity, whereas the Fenton reaction mixture (containing hydroxyl radical) induced damage to calf thymus DNA (Lane 2) showed significant damage to DNA. The DNA integrity is not compromised when treated with OTAE in Lane 4–9. DNA damage protection followed a concentration-dependent manner from 25 to 800 µg/mL, the protection increased along with the concentration. Fig. 4. Open in a new tab Agarose gel electrophoretic pattern of calf thymus DNA with and without treating anthocyanin extract. Anthocyanins imparted protection against Fenton (hydroxyl radical) induced damage to calf thymus DNA. Control calf thymus DNA (Lane 1) showed proper structural integrity, whereas the Fenton reaction mixture (containing hydroxyl radical) induced damage to calf thymus DNA (Lane 2) showed significant damage to DNA. The DNA integrity is not compromised when treated with OTAE in Lane 4–9. DNA damage protection followed a concentration-dependent manner from 25 to 800 µg/mL, the protection increased along with the concentration. Lane 1: Control Calf thymus DNA; Lane 2: Calf thymus DNA + Fenton reaction mixture; Lane 3: Calf thymus DNA + OTAE without Fenton reaction mixture; and Lane 4: Calf thymus DNA + Fenton reaction mixture + OTAE 25 µg/mL; Lane 5: Calf thymus DNA + Fenton reaction mixture + OTAE 50 µg/mL; Lane 6: Calf thymus DNA + Fenton reaction mixture + OTAE 100 µg/mL; Lane 7: Calf thymus DNA + Fenton reaction mixture + OTAE 200 µg/mL; Lane 8: Calf thymus DNA + Fenton reaction mixture + OTAE 400 µg/mL; Lane 9: Calf thymus DNA + Fenton reaction mixture + OTAE 800 µg/mL Evaluation of antibacterial properties of anthocyanins The results of the antimicrobial activity in terms of ZOI for different concentrations of OTAE are shown in Table 3 and MIC, and MBC values are presented in Table 4 . In disc diffusion analysis (Fig. 5 ) OTAE had the highest inhibitory effect against P. aeruginosa with an inhibition zone of 24.4 ± 0.4 mm. In the microdilution assay, the extract offered antimicrobial action at varying concentration ranges. It offered excellent antimicrobial action against both bacterial gram-positive and gram-negative bacteria. MBC was within a range of 6.25–25 µg/mL for OTAE and for the standard antibiotic streptomycin it was in a range between 12.5 and 100 µg/mL; which was quite higher than the MBC range of OTAE. Table 3. Inhibition zones of anthocyanin-rich extracts against four bacteria Bacterial strains Diameter of inhibition zone (mm) [including 6 mm disc diameter] 0.25 mg/mL 0.5 mg/mL 1 mg/mL STR E. coli 9.6 ± 0.4 a 16.6 ± 0.4 b 19.6 ± 0.8 c 14.4 ± 0.2 d P. aeruginosa 10.4 ± 0.2 a 13.4 ± 0.8 b 24.4 ± 0.4 c 19.8 ± 0.6 d S. aureus 11.8 ± 0.4 a 14.4 ± 0.8 b 17.4 ± 0.6 c 13.4 ± 0.4 ab B. subtilis 14.8 ± 0.4 a 17.4 ± 0.6 b 22.4 ± 1.4 c 15.6 ± 0.8 a Open in a new tab Data presented as mean ± SD, n = 3; values showing the different letters are significantly different ( p > 0.05) from one other in the same row OTAE, Ocimum tenuiflorum anthocyanin extract; STR, streptomycin Table 4. Antimicrobial activity of anthocyanin-rich extracts Bacterial strains OTAE STR MIC (µg/mL) MBC (µg/mL) MIC (µg/mL) MBC (µg/mL) E. coli 6.25 12.5 25 50 P. aeruginosa 12.5 25 25 50 S. aureus 6.25 12.5 6.25 12.5 B. subtilis 3.125 6.25 50 100 Open in a new tab MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; OTAE, Ocimum tenuiflorum anthocyanin extract; STR, streptomycin Fig. 5. Open in a new tab Visible clear zones produced by different concentrations of OTAE and streptomycin against four pathogens P. aeruginosa MTCC 741, E. coli MTCC 443, S. aureus MTCC 96, and B. subtilis MTCC 441. Anthocyanins from O. tenuiflorum exhibited antimicrobial activity against both gram-positive and gram-negative bacteria in a concentration-dependent manner Estimation of anti-biofilm potential Two common foodborne pathogens ( Staphylococcus aureus and Escherichia coli ) were tested for the antibiofilm activity of anthocyanins at MIC, sub, and super MIC concentrations, and the results are shown in Fig. 6 . The gram-positive bacteria S. aureus showed a wide range of biofilm formation inhibition percentages, ranging from 91 ± 6.110 at 4*MIC to 30.667 ± 3.055% at MIC/2. Antibiofilm activity on gram-negative bacteria ranged from 86.667 ± 4.163% at 4*MIC to 26.333 ± 2.082% at MIC/2 for E. coli . Fig. 6. Open in a new tab Antibiofilm activities of anthocyanins from O. tenuiflorum extract (% inhibition). Anthocyanins from O. tenuiflorum inhibited biofilm formation of both S. aureus and E. coli over a broad range of concentrations from MIC/2 to 4× MIC, indicating that the anti-biofilm efficacy of anthocyanins is significantly enhanced by high anthocyanin concentrations. The results are shown as the Mean ± SD ( n = 3) analyzed by one-way ANOVA Tukey's multiple comparison test. Different superscripted letters indicate significant differences ( p < 0.05) with each other for individual bacteria. Small letters were used to signify S. aureus and capital letters signify E. coli Evaluation of morphological changes in the biofilm Based upon the preliminary antibiofilm data, S. aureus was chosen for the model system and the morphological changes of S. aureus bacterial biofilm cells before and after exposure to the OTAE were investigated by FESEM (Fig. 7 ) . Fig. 7. Open in a new tab Observation of changes in cell architecture and membrane function using a field emission scanning electron microscope. A Control cells showing round shape and smooth cell wall; B cells treated with anthocyanin extract at MIC showing dents and blebbing; C cells treated with anthocyanin extract at 2× MIC showing a loss of membrane integrity and subsequent cell death Docking result analysis of biofilm-forming proteins PDB id-3 TIP for S. aureus and 4 KH3 for E. coli The bioactive compounds identified from the UHPLC–Q-ToF–ESI–MS analysis from Ocimum tenuiflorum were investigated for their binding interactions with the biofilm-forming proteins of S. aureus and Escherichia coli through molecular docking studies. This in-silico docking analysis facilitated the prediction of binding sites and key interacting amino acid residues for the phytocompounds and their binding interaction energies. Our result provides insights into the best-docked poses, characterized by the lowest binding energies, and highlighted potential interactions such as hydrogen bonds, electrostatic interactions, and π–π interactions between the amino acids and the bioactive compounds, as depicted in Table 5 . These findings are crucial for identifying potential lead compounds for future drug development. Our docking results of bioactive compounds against biofilm-forming proteins (3 TIP and 4 KH3) revealed that cyanidin and peonidin derivatives exhibit notable binding affinities, with Cyanidin 3-O-(6-O-p-coumaroyl) glucoside showing the strongest interaction (− 6.1 kcal/mol with 3 TIP and − 6.3 kcal/mol with 4 KH3). Key hydrogen bonds were formed with residues, such as TYR625, GLU628, and ARG567, indicating potential inhibition of biofilm formation. Peonidin derivatives also displayed significant binding, though slightly lower than cyanidin derivatives. Table 5. Molecular docking study of biofilm-forming proteins PDB id-3 TIP for S. aureus and 4 KH3 for E. coli Bioactive anthocyanins Biofilm forming proteins Auto dock results Docking score (Kcal/mol) Molecular interaction Cyanidin 3 TIP − 5.9 Hydrogen Bond: TYR625; GLY626; GLU628, THR629 Hydrophobic Bond: PHE569; PRO627 4 KH3 − 4.8 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; THR27; SER25; GLY26; ALA54; GLY56; THR57 Hydrophobic Bond: GLY55 Cyanidin 3-(6''-acetoyl-galactoside) 3 TIP − 5.8 Hydrogen Bond: ARG567; THR577; PRO627; GLY626 Hydrophobic Bond: GLU628; GLU578; TYR625 4 KH3 − 5.1 Hydrogen bond: PRO7; GLY8; GLU9; THR10; SER25; GLY26; THR27; GLY55; GLY56; THR57 Hydrophobic bond: ARG59 Cyanidin 3-glucoside 3 TIP − 5.7 Hydrogen Bond: GLU578; GLY576; TYR625; GLU628; PRO627; PHE569 4 KH3 − 5.8 Hydrogen Bond: HIS6; PRO7; GLU9; THR10; SER25; GLY26; THR27; GLY55; THR57; ARG59; THR76 Hydrophobic Bond: GLY8 Cyanidin 3-O-(6-O-p-coumaroyl) glucoside 3 TIP − 6.1 Hydrogen Bond: TYR625; GLU628; ARG567; THR629; GLU566; PRO627 Hydrophobic Bond: PHE569 4 KH3 − 6.3 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; ASN12; SER25; THR27; ASN29; ALA54; GLY55; THR57; ARG59 Cyanidin-3,5-diglucoside 3 TIP − 5.9 Hydrogen Bond: THR269; PRO627; GLU628; TYR625; ARG567; GLY576; THR577 Hydrophobic Bond: GLU578 4 KH3 − 5.7 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; ASN12; ASN29; THR27; ALA54; THR57; ARG59 Delphinidin 3 TIP − 5.8 Hydrogen Bond: TYR625; GLU628; GLY626; THR629 Hydrophobic Bond: PRO627; PHE569 4 KH3 − 4.8 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; ASN12; ASN29; THR27; ALA54; THR57; ARG59 Peonidin 3 TIP − 6.0 Hydrogen Bond: TYR625; GLY626; GLU628; THR629 Hydrophobic Bond: PHE569; PRO627 4 KH3 − 4.8 Hydrogen Bond: GLY8; GLU9; THR10; SER25; GLY26; THR27; ALA54; GLY56; THR57 Hydrophobic Bond: PRO7 Peonidin 3-(6''-acetyl-galactoside) 3 TIP − 5.6 Hydrogen Bond: PRO267; GLU628; GLY626; GLU578; ARG567 Hydrophobic Bond: PHE569; TYR625 4 KH3 − 5.8 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; GLY26; THR27; GLY55; GLY56; THR57; GLY74; THR76 Peonidin-3-o-alpha-arbinopyranoside 3 TIP − 5.6 Hydrogen Bond: TYR625; GLU628; THR629 Hydrophobic Bond: PRO627; PRO571; PHE569 4 KH3 − 5.5 Hydrogen Bond: PRO7; GLY8; THR10; SER25; GLY26; THR27; GLY55; THR57; ARG59 Peonidin-3-O-beta-galactopyranoside 3 TIP − 5.4 Hydrogen Bond: TYR625; ARG567; PRO627; PHE569; GLY626; GLU578 4 KH3 − 5.1 Hydrogen Bond: PRO7; GLY8; GLU9; THR10; GLY26; THR27; GLY56; THR57 Hydrophobic Bond: GLY55; ARG59 Open in a new tab Discussion In this study, 3D response surface plots were generated to illustrate the influence of extraction parameters. Effects of the three independent variables, i.e., ethanol, hydrochloric acid, and temperatures, on three dependent or response variables, i.e., total anthocyanin content (TAC), IC 50 values of DPPH, and ABTS radical scavenging activity were evaluated. The extraction efficiency of anthocyanins was found to reach an optimal point at 32.5 °C and then decreased as temperature was further increased to 55 °C (Fig. 1 ). A similar trend was observed for the influence of hydrochloric acid concentration, where anthocyanin yield increased up to a mid-range concentration of 1.15% but declined beyond a certain acid–alcohol threshold. Sametime this increased yield of anthocyanins was found to have a direct effect on antioxidant activity as revealed from the fitting curves. The minimum TAC, quantified as 102.94 mg/g cyanidin-3-glucoside (C3G) equivalent, was observed under extraction conditions comprising 20% ethanol and 0.1% hydrochloric acid at 32.5 °C. This has an immediate bearing on the antioxidant activity indicated by the higher IC₅₀ values obtained in DPPH (86.548 µg/mL) and ABTS assays (91.568 µg/mL). Elevated conditions, i.e., ethanol (80%), acid (1.15%), and 55 °C, demonstrated a substantial increase in TAC, yielding 172.55 mg/g C3G equivalent, and thereby an improved antioxidant effect evident from lower the IC₅₀ values in DPPH (19.596 µg/mL) and ABTS (27.658 µg/mL) assays. Interestingly, under identical concentrations of ethanol (i.e., 80%) and acid (1.15%) with a reduced extraction temperature of 10 °C, TAC further increased to 184.81 mg/g C3G equivalent, which could be further correlated with the antioxidant efficiency of the extract (DPPH, IC₅₀ = 13.743 µg/mL; and ABTS, IC₅₀ = 21.855) µg/mL. These findings indicate a strong correlation between ethanol and acid concentration with anthocyanin yield and antioxidant efficacy. The observed trends were statistically significant ( p < 0.05), highlighting the critical role of optimization of extraction parameters in enhancing phytochemical bioactivity. Response surface methodology-based optimization revealed that the ideal extraction parameters for maximizing anthocyanin yield were 79.396% ethanol, 1.288% hydrochloric acid with an optimal temperature of 21.102 °C. Under these conditions, the predicted anthocyanin yield was 185.196 mg/g C3G equivalent, and IC₅₀ values for the DPPH and ABTS radical scavenging assays were determined to be 11.734 µg/mL and 19.99 µg/mL, respectively. While experimental validation of the predicted values at the said optimized conditions, TAC was estimated to be 188.16 ± 2.43 mg/g C3G equivalent, with the IC₅₀ values 12.28 ± 0.589 (DPPH) and 20.74 ± 1.19 (ABTS), yielding a desirability index of 1.0. These findings conclude that anthocyanins extraction from O. tenuiflorum is temperature and acid-dependent, as TAC peaked at optimal conditions before declining due to thermal degradation and excessive acidity. Ruenroengklin et al. reported a comparable pattern in TAC with thermal modulation, wherein both TAC and antioxidant potential of litchi anthocyanins exhibited a significant enhancement with increasing temperature, reaching an optimum at 45 °C. However, prolonged incubation at 45 °C led to a marked decline in antioxidant activity which indicated that maintaining an optimal thermal condition is critical for preserving the bioactivity and thermodynamic stability of anthocyanins (Ruenroengklin et al. 2008 ). Similarly, hydrochloric acid improves anthocyanin yield up to a mid-range concentration but diminishes beyond a certain acid–alcohol threshold, likely due to pigment degradation or structural alterations (Suganya Devi et al. 2012 ). The stability of anthocyanin pigments is markedly influenced by pH, with acidic conditions favoring greater stability. This is primarily due to the predominance of the flavylium cation at lower pH levels, which is the most stable and colored form of anthocyanins. The stabilization of the chromophore species, particularly the quinoidal base, can be further enhanced through co-pigmentation mechanisms. These include intermolecular interactions with other phenolic compounds, such as flavonoids and polyphenols, as well as intramolecular associations, for instance, via acylation of sugar moieties within the anthocyanin structure (Jackman et al. 1987 ; Brouillard and Dangles 1994 ; Ghareaghajlou et al. 2021 ). Suganya Devi et al. demonstrated that anthocyanins extracted from red sorghum exhibited maximum stability at pH 1.1 in acidified methanolic extracts. Under such conditions, the flavylium cation predominates, thereby conferring enhanced pigment stability (Suganya Devi et al. 2012 ). Another study demonstrated that using a solvent system acidified with 0.01% hydrochloric acid significantly improved anthocyanin extraction from blackberry residues, resulting in higher pigment retention and antioxidant activity (Machado et al. 2015 ). These critical relationships of choice of solvent systems and temperature with the extraction of anthocyanins establish importance of the optimization tools such as Box–Behnken experimental design (BBD). Several studies have employed BBD to obtain efficient anthocyanin extraction. Mugwagwa and Chimphango employed a BBD to optimize the extraction of anthocyanins from mango ( Mangifera indica ) peels using independent variables, such as ethanol and, acetic acid concentration, and extraction time. Similarly, another study showed that acidified ethanol (0.05% HCl) enhanced the extraction efficiency of anthocyanins from purple sweet potatoes, leading to greater color intensity and stability than neutral solvents (Pramitasari and Ekaputra 2020 ). Anthocyanin extraction from pomegranate flowers with varying methanol concentration significantly influenced antioxidant activity, peaking at 34.56%, whereas temperature and extraction time showed minor, non-significant effects ( p > 0.05), with antioxidant activity increasing until 50 °C before declining and slightly rising with time (Gościniak et al. 2022 ). BBD helped to deduce the effects of aqueous-alcoholic solvents on anthocyanin extraction from raspberry, blueberry, cherry, and red pear peel, (Rocha et al. 2020 ), whereby anthocyanin content increased with changes in solvent polarity (Fig. 1 ). It is long known that the inclusion of water enhances the extraction of hydrophilic anthocyanins, rendering acidified ethanol alone less effective as an extraction solvent (Fuleki and Francis 1968 ). Most monomeric anthocyanins, such as delphinidin, cyanidin, peonidin, and malvidin derivatives, were identified using different polar solvents (Wang et al. 2016 ). In this study, acidification of ethanol (≈80%) proved most effective in stabilizing the anthocyanins and enhancing antioxidant properties. Therefore, these insights support the development and optimization of efficient extraction techniques for enhancing the bioactivity of anthocyanin-rich functional foods. The UHPLC–PDA–ESI–MS analysis of the optimized anthocyanin extract of Krishna Tulsi (OTAE) (Table 2 ) showed the presence of ten bioactive anthocyanins, namely, presence of Cyanidin-3,5-diglucoside, Peonidin-3-O-alpha-arabinopyranoside, Peonidin-3-(6″-acetoyl) galactoside, Cyanidin-3-glucoside, Cyanidin, Peonidin-3-O-beta-galactopyranoside, Peonidin, Cyanidin 3-O-(6-O-p-coumaroyl) glucoside, Cyanidin-3-(6″-acetoyl) galactoside, and Delphinidin (Figs. 2 and 3 ). The efficient antioxidant effects of OTAE are justified by the presence of these varied classes of anthocyanin. The antioxidant mechanism of these compounds is attributed to the reducing power of the ortho-dihydroxy groups and the number of hydroxyl groups (-OH) in the B ring of the anthocyanin structure, which confers a stable radical form and facilitates electron delocalization (Kalita et al. 2022 ; Chatterjee et al. 2024 ). Glycosylated anthocyanins [Cyanidin-3-glucoside, Cyanidin-3,5-diglucoside, and Cyanidin 3-O-(6-O-p-coumaroyl) glucoside] are more easily soluble in aqueous environments and transport in the bloodstream than their aglycone forms, due to the presence of sugar moiety. All these compounds have been reported for different biological and pharmacological activities aligning to their antioxidant activities. Cyanidin-3-O-glucoside exhibits bioactivity as a DNA radical scavenger, gastroprotective, anti-inflammatory, anti-thrombotic, and chemopreventive agent, functioning as an epigenetic modulator. Its protective effects extend to Helicobacter pylori infection, age-associated pathologies, type 2 diabetes mellitus, cardiovascular disorders, metabolic syndrome, and oral carcinogenesis (Olivas-Aguirre et al. 2016 ). Another glycosylated anthocyanin, Cyanidin-3,5-diglucoside administration resulted in an upregulation of active glucagon-like peptide-1 (GLP-1) levels in the serum of KKAy mice. In addition, cyanidin 3,5-diglucoside supplementation led to a significant reduction in blood glucose and glycated hemoglobin (HbA1c) levels, indicating improved glycaemic regulation. The most prominent molecular mechanisms underlying its therapeutic potential include radical scavenging activity, epigenetic regulation, competitive protein–ligand interactions, and enzymatic inhibition (Yamane et al. 2019 ). The magenta or purple color of O. tenuiflorum is conferred by its rich presence of Delphinidin. This anthocyanin is highly stable under acidic conditions but unstable in alkaline and neutral pH. It has been found to possess anticancer, anti-inflammatory, neuroprotective, cardioprotective, antihypertensive, antidiabetic, antiosteoporotic, anti-psoriatic, hepatoprotective, antiviral, and nasal polyp protective potential (Husain et al. 2022 ). Peonidin-3-galactoside found in OTAE is also another potent suppressor of oxidative stress (Guo et al. 2024 ). Among its antioxidant activity anthocyanins demonstrate efficient DNA protection. Similarly, in this study, pretreatment with OTAE at concentrations ranging from 25 to 800 µg/mL demonstrated a dose-dependent protection of genomic DNA integrity (Fig. 4 ) against Fenton-induced genotoxicity. The Fenton reaction, which involves hydrogen peroxide (H₂O₂) and ferrous iron (Fe 2 ⁺), produces highly reactive hydroxyl radicals (•OH) that can cause extensive single and double-strand breaks in DNA molecules due to the abstraction of hydrogen atoms from its deoxyribose sugar backbone. Moreover, the Fenton reaction can induce crosslinking between DNA and proteins, particularly histones, which are essential for chromatin structure (Wu and Dhanasekaran 2020 ). Anthocyanins protect DNA integrity through a synergistic combination of antioxidant, metal-chelating, enzyme-modulating, and DNA repair-enhancing mechanisms. Their natural origin, low toxicity, and multi-targeted actions make them promising candidates for preventing mutagenicity, oxidative damage, and chemical genotoxicity (Devi et al. 2012 ; Liu et al. 2013 ). Lazzé et al. investigated the protective effects of anthocyanin pre-treatment with delphinidin, cyanidin, and their glycosylated (glycoside and rutinoside) derivatives against tert-butyl hydroperoxide (t-BHP)-induced genotoxicity in rat smooth muscle and hepatoma cell lines. The findings which demonstrated that anthocyanins effectively mitigated t-BHP-induced cytotoxicity, DNA single-strand breaks (SSBs), oxidative base modifications, and lipid peroxidation (Lazzé et al. 2003 ) also corroborates with our results. OTAE also exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. Compared to streptomycin, S. aureus was more sensitive to anthocyanins among the four tested bacterial pathogens (Fig. 5 ). The inhibitory effects of OTAE were observed in both solid and liquid media (Tables 3 and 4 ). We have found OTAE to be rich in cyanidin derivatives. A recent report suggests the intense antimicrobial action of cyanidin-3-O-glucoside ester derivatives against Pseudomonas aeruginosa , Escherichia coli , and Staphylococcus aureus . The lipopolysaccharide layer in gram-negative bacteria impedes the diffusion of hydrophobic compounds through the outer cell membrane, rendering the outer layer relatively resistant to larger molecules. Therefore, the ability of anthocyanin derivatives to penetrate bacterial membranes is influenced by the side chain length, enhancing their antibacterial effect (Oliveira et al. 2021 ). Zhang et al. investigated the anti-biofilm activity of an anthocyanin-rich aqueous extract from purple highland barley bran, which contains Cyanidin malonyl glucoside, Cyanidin-3-galactoside, Cyanidin acetyl galactoside, Cyanidin di-glucoside, and Peonidin glucoside, against two biofilm-forming bacteria, P. aeruginosa and S. enterica (Zhang et al. 2020 ). Similarly, pomegranate peel extract, containing delphinidin, cyanidin, and pelargonidin glycosides demonstrated the strongest inhibitory activity, reducing biofilm development of S. aureus by more than 65% at a sub-MIC (1/16) concentration (Salim et al. 2023 ). OTAE inhibited biofilm formation of both S. aureus and E. coli over a broad range of concentrations from MIC/2 to 4 × MIC, indicating that the anti-biofilm efficacy of anthocyanins is significantly enhanced by high anthocyanin concentrations (Fig. 6 ). FESEM surface morphology analysis revealed that untreated S. aureus cells were round-shaped with smooth and intact cell walls (Fig. 7 ). However, cells treated with MIC and 2 × MIC concentrations of OTAE exhibited significant perturbations on the cell walls, including numerous blebs and pores, leading to a loss of membrane integrity and subsequent cell death (Xu et al. 2017 ). The antimicrobial and anti-biofilm properties observed could be attributed to OTAE's ability to attach to the peptidoglycan layer of the cell wall, disrupting membrane permeability and the function of respiratory chain enzymes within bacterial cells (Cushnie and Lamb 2005 ). The docking analysis reveals that cyanidin and peonidin derivatives, particularly Cyanidin 3-O-(6-O-p-coumaroyl) glucoside, are promising candidates for inhibiting biofilm formation due to their strong binding affinities with key biofilm-forming proteins. The significant interactions, especially the hydrogen bonds with key residues, such as TYR625 and GLU628, highlight the ability of these compounds to disrupt protein functions essential for biofilm formation (Gruszka et al. 2012 ; Mousavi et al. 2023 ). The slight variation in binding affinities among the derivatives indicates that structural modifications could further optimize their efficacy. These results suggest that these compounds could be effective in targeting biofilm-associated proteins, offering an alternative approach to combat biofilm-associated infections (Table 5 ). Biofilm-forming and colonizing bacteria are responsible for over half of all infections. Quorum sensing (QS), a cell-density-dependent communication method regulating the expression of various genes, including virulence genes, is closely associated with biofilm formation (Zhang et al. 2020 ; Dawan et al. 2022 ; Khan et al. 2023 ). The results of this study suggest that the anthocyanin-rich extract of Krishna Tulsi could be developed as a novel QS inhibitor and anti-biofilm agent, potentially extending food shelf life and improving food safety. These findings indicate that bioactive anthocyanins in OTAE also possess strong antioxidant and anti-mutagenic properties, which could be beneficial in managing health issues related to degenerative diseases. Conclusion This study demonstrates the relevance of optimized extraction conditions in maximizing the therapeutic potential of natural compounds for biomedical applications. The high anthocyanin content in Krishna Tulsi is unique, and the extracted anthocyanins demonstrated strong radical scavenging activity, antimutagenic properties, and potent antimicrobial and biofilm disruption abilities, validating their biomedical application in the nutraceutical and functional food industries. The Box–Behnken optimized extraction enhances bioavailability, supporting the use of these compounds in treating oxidative stress-related diseases. In addition, these extracts could be considered safer food additives to impart color to various food materials while providing health benefits. A limitation of the study is that UV light modulates anthocyanin biosynthesis in O. tenuiflorum , which may cause seasonal fluctuations in bioactivity due to changes in anthocyanin content. Further research is needed to evaluate the impact of using anthocyanins extracted with this optimized system as a food colorant on food taste (Vyas et al. 2014 ). Supplementary Information Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 625 KB) (625.5KB, docx) Author contributions Amrita Chatterjee: literature search, experimental work, data analysis, writing—original draft, and writing—review and editing; Rajdeep Saha: literature search, data analysis, and writing—review; Ravi Adinarayan Somabattini: literature search and experimental work; Satish Kumar: experimental work, data analysis, and writing—original draft; Riya Ghosh: literature search and experimental work; Anupam Roy: supervision and writing—review and editing; Uma Ranjan Lal: supervision and writing—review and editing; Kunal Mukhopadhyay: supervision and writing—review and editing; Satheesh Kumar Nanjappan: supervision and writing—review and editing; Biswatrish Sarkar: conceptualization, supervision, and editing. Funding The work was financially supported by an Extra Mural Research Grant of DST–SERB, Govt. of. India (Grant No. EMR/2016/005695). Amrita Chatterjee was working as a Junior Research Fellow, supported by the above grant. Availability of data and materials The data sets used or analyzed during the current study are available from the corresponding author upon reasonable request. Declarations Conflict of interest No conflict of interest was reported by the authors. Research involving human participants and/or animals To accomplish the goals of this study, no approval of research ethics committees was required, because experimental work did not involve the use of living matter. Informed consent Not applicable. Consent to publish All authors have approved the manuscript for publication. References Barabadi H, Mojab F, Vahidi H et al (2021) Green synthesis, characterization, antibacterial and biofilm inhibitory activity of silver nanoparticles compared to commercial silver nanoparticles. 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