The fresh berries of B. asiatica were collected from the Ghorakhal District in Nainital, Uttarakhand, India, a region situated in a forested area. After the harvest, the fruits were quickly taken to the laboratory and preserved at –20°C for subsequent analysis. Sample collection was guided by ethnopharmacological and ethnobotanical literature [9] and authenticated by taxonomist Dr. D. S. Rawat, Department of Biological Sciences, GBPUA&T, Pantnagar. Specimens of the fruits were archived in the institution’s herbarium for future reference.

All chemicals and reagents used in the study were of analytical grade and procured from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Wherever available, catalogue/item numbers have been provided.

Fresh fruits were thoroughly washed with distilled water and shade-dried in the dark at room temperature for 5–10 days. The dried fruits were powdered and used for extraction following previously reported methods [10, 11] with slight modifications. Briefly, 20 g of fruit powder was separately soaked in four solvents of increasing polarity, hexane, chloroform, 80% methanol (v/v), and 50% hydroalcohol (v/v), for 72 h. The mixtures were stirred intermittently every 24 h to enhance extraction efficiency and then filtered. Filtrates were concentrated at 30°C using a water bath, and the resulting extracts were stored at 4°C for further analysis.

We utilized an automated FT-IR spectrometer from PerkinElmer, Germany, for the identification of bioactive molecule groups. The FT-IR spectrometer analysis was carried out in the range of 4,000–400 cm^–1, employing the KBr pellet technique. Identification of bioactive molecules within the 80% crude methanol extract of B. asiatica fruit was achieved by comparing the spectral FT-IR analysis with published literature.

TAC in the methanol extract (80%) followed the procedure proposed by Aguila and McCann [12] with slight adjustments. The samples were diluted with a solution of ethanol:water:HCl (conc.) in a 70:30:1 ratio, and the absorbance was recorded at 540 nm. The assessment of TAC was executed, and the findings were expressed as malvidin-3-glucoside equivalents through the utilization of the following formula:

T A C   =   A 540   n m   ×   16.7   ×   d

Where d = dilution.

The identification of bioactive compounds, specifically anthocyanins, was conducted in the 80% crude methanol extracts of the sample fruit. This was achieved through the utilization of the LC-MS/MS analytical technique, provided by Agilent Technologies India Pvt. Ltd., Bangalore. The methodology employed a single quadrupole (SQ) LC/mass selective detector (MSD), and the LC-MS/MS system used in the analysis was equipped with an autosampler featuring a photo-diode array detector. For this investigation, a reversed-phase RP C18 column (150 × 3.0 mm, 2.5 µm) was selected, maintaining a constant temperature at 40°C. Mobile phase A: methanol, mobile phase B: acetonitrile. Chromatographic separation conditions: 0 min, 5% B; 1 min, 5% B; 15 min, 97% B; 20 min, 97% B; 21 min, 5% B; 25 min, 5% B. A consistent flow rate of 0.4 mL/min was consistently maintained during the entire analysis. The fruit extracts from B. asiatica in 80% v/v methanol were first dissolved in a water and methanol mixture (60:40 v/v) to create a stock solution with a concentration of 1 mg/mL. For liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) analysis, a 2 µL aliquot from the stock solution was utilized. The analysis utilized a Triple Quad mass spectrometer with an ESI source, specifically the Waters Quattro Premier XE from the USA.

The parameters were set as follows: electrospray capillary voltage at 3.5 kV, source temperature at 100°C, desolvation gas flow at 1,000 L/h, cone gas flow at 60 L/h, desolvation temperature at 350°C, nitrogen and cone voltage at 30 V, was employed in the ESI source, and the multiplier voltage was adjusted to 650 V. LC-MS analysis was performed in both positive and negative ionization modes using a full scan range of m/z 50–1,400. The recorded results included the total ion chromatogram along with the corresponding spectra.

Molecular docking simulations were performed via AutoDock 4.2 program package [18]. The crystal structures of (PDB: 4ewp) [19], (PDB: 1kzn) [20], (PDB: 7bly) [21], and (PDB: 3nrz) [22] were procured from the RCSB protein data bank (https://www.rcsb.org/). First, water molecules were removed, and then missing hydrogens and Gasteiger charges were added to the system during the preparation of the receptor input file. Then, AutoDock Tools was used to prepare the ligand and protein files (PDBQT). Pre-calculation of grid maps was performed with AutoGrid to save time during docking. Geometric optimization of all compounds was performed using ACD (3D viewer) software (http://www.filefacts.com/acd3d-viewer-freeware-info) and the visualization and analysis of interactions were performed using Discovery Studio 2017R2 (https://www.3ds.com/products/biovia/reference-center) and PyMOL 0.99rc6 [23].

All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD). Differences among solvent extracts and the standard reference (ascorbic acid) for antioxidant assays were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Statistical significance was considered at p < 0.05.

The functional group positions and characterization of molecules in the crude methanol extract of B. asiatica, determined by FT-IR spectroscopy, are shown in Figure 1 and Table 1. FT-IR spectroscopic analysis of the crude methanolic extract of B. asiatica revealed the presence of several functional groups associated with bioactive secondary metabolites. The broad band observed at 3,983.78 cm^–1, typical of O-H stretching, together with the signal at 1,384.26 cm^–1 (O-H deformation), indicates a high concentration of hydroxyl groups, characteristic of phenolic compounds and alcohols, commonly associated with antioxidant activity. The bands at 2,852.79 cm^–1 and 2,924.55 cm^–1, corresponding to C-H stretching of aliphatic chains, suggest the presence of lipophilic compounds such as fatty acids. The intense absorption at 1,721.07 cm^–1 is related to carbonyl stretching (C=O), frequently present in carboxylic acids, ketones, and esters, while the signals at 1,600.11 and 1,514.08 cm^–1 indicate the presence of conjugated aromatic rings, supporting the occurrence of flavonoids and berberine-type alkaloids.

In the region between 1,300 and 1,000 cm^–1, the bands at 1,266.85 cm^–1 and 1,071.53 cm^–1 are attributed to C-O vibrations, consistent with primary and secondary alcohols, and glycosidic bonds, correlating with glycosylated flavonoids and anthocyanins identified by LC-MS/MS (e.g., cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside). The signals at 873.75, 819.27, and 779.33 cm^–1, associated with out-of-plane aromatic C-H deformations, indicate tri- or tetra-substituted aromatic rings, common in complex phenolic compounds and isoquinoline alkaloids. The band at 619.69 cm^–1 is attributed to the vibration of the C-C skeleton, also typical of aromatic structures.

The quantification of the anthocyanin content in the 80% methanolic extract of B. asiatica using a spectrophotometric method revealed a significant value of 128.72 mg/g of plant material Table 2. However, when compared to studies of other species in the same genus, such as Berberis vulgaris, the results differ significantly.

The LC-MS/MS analysis of the methanolic extract of B. asiatica fruit revealed a phytochemical core consisting of eight glycosylated anthocyanins: cyanidin-3-O-glucoside (Cy3G), cyanidin-3,5-diglucoside (Cy3diG5G), malvidin-3-O-arabinoside (M3A), petunidin-3-O-glucoside, peonidin-3-O-glucoside (P3G), and pelargonidin-3-O-glucoside (Pg3G), catechin and epicatechin as shown in Table 3. Their retention times (2.53–9.65 min) and diagnostic fragments (m/z 271–331) confirm the dominance of 3- and 3,5-substituted anthocyanins. The co-elution of catechin and epicatechin at ~5.3–7.8 min indicates a secondary flavanol pool that can reinforce antioxidant molecules in the crude methanolic extract of B. asiatica. Figure 2 displays the LC-MS/MS chromatogram, illustrating how compounds are separated based on their retention time as they elute from the LC column. Each peak primarily represents an intact (parent) molecule detected during the chromatographic run. In contrast, Figure 3 showcases the MS/MS fragmentation spectra, where selected parent ions are further fragmented into product (fragment) ions. This process reveals the characteristic splitting pattern of each molecule. A greater number of compounds appear in Figure 3 because a single parent compound generates multiple fragment ions during MS/MS analysis. These fragments are detected as additional signals, providing structural information and confirming compound identity, rather than representing new or distinct compounds. Figure 4 represents the structure of compounds identified in the methanolic extract of B. asiatica.

The antioxidant characteristics of solvent extracts obtained from B. asiatica were assessed through traditional methods, including DPPH free radical scavenging activity, superoxide anion radical scavenging activity, H₂O₂ inhibition, and total antioxidant activity. The findings indicated that the 80% methanol extract exhibited noteworthy antioxidant activity, with subsequent rankings observed for the hydroalcoholic, hexane, and chloroform extracts. These findings held statistical significance when compared to the standard reference control, ascorbic acid at 1 mg/mL (Table 4).

The potential utilization of B. asiatica as a natural source of antioxidants opens avenues for developing products or supplements enriched with these beneficial compounds. This application holds the promise of offering health benefits to the public. By harnessing the antioxidant properties identified in B. asiatica, it becomes possible to create formulations that contribute to improving overall health and well-being. These natural antioxidants can act as a safeguard against oxidative stress, addressing health challenges associated with the harmful effects of free radicals in the body. Incorporating B. asiatica-derived antioxidants into various products or supplements aligns with the growing interest in holistic and natural approaches to health, providing individuals with a proactive and natural solution to support their well-being.

The antimicrobial efficacy of B. asiatica solvent extracts was assessed at a concentration of 200 µg/mL through the well diffusion method. The study revealed that the methanol extracts (80% v/v) and hydroalcoholic extracts (50% v/v) of B. asiatica demonstrated pronounced antimicrobial activity, surpassing the effectiveness of hexane and chloroform extracts, particularly against M. luteus, S. epidermidis, E. coli, A. niger, S. abony, and C. albicans. Interestingly, chloroform extracts exhibited no antimicrobial activity against S. abony, A. niger, and C. albicans. The observed inhibition zones indicate a clear trend of higher antimicrobial potency for polar solvent extracts when compared descriptively with the positive controls. The detailed antimicrobial response of all tested extracts and controls is presented in Table 5 and Figure 5.

Molecular docking is an established in silico structure-based technique widely used in drug discovery [24]. Motivated by this information, herein, molecular docking was used to identify a possible mechanism of action that correlated with the recorded antimicrobial and antioxidant potentials by modeling the identified phytocompounds (Cy3G, M3A, catechin, Cy3diG5G, epicatechin, petunidin-3-O-glucoside, P3G and Pg3G). In the current studies, among the strains used in the in vitro tests, the most sensitive strain against the phytomolecules was chosen to select the target receptors for carrying out the docking. ‘FabH from M. luteus’ (PDB: 4ewp) was taken as the target receptor to predict the sensitivity of a Gram-positive bacterium and ‘E. coli 24 kDa Domain’ (PDB: 1kzn) was used to conjecture the sensitivity of a Gram-negative bacterium against the selected phytocompounds in addition to ‘chitin deacetylase AngCDA from A. niger’ (PDB: 7bly) that used for the antifungal activity. Furthermore, ‘Xanthine Oxidase’ (PDB: 3nrz) was used as a receptor for the antioxidant potential.

Against M. luteus (PDB: 4ewp), the results presented in Table 6 show that the majority of docked phytocompounds have acceptable scores. Indeed, the most effective ligand is ‘Cy3diG5G’, which exhibited the best docking score (binding energy value = −7.3 kcal/mol). The 2D model in Figure 6 exhibits that this ligand fits well in the binding cavity of the targeted enzyme. The involved interactions depicted in Figure 6, which demonstrate the formation of five H-bonds with His96, Ala97, Trp197, and Ile200, reduced the binding energy score and consequently stabilized the docking complex. Further, other interactions are included, such as: Pi-donor hydrogen bond with Ser120, Pi-Pi T-shaped with Trp197.

Regarding the docking complex: ‘E. coli 24 kDa Domain’ (PDB: 1kzn), as Table 6 displays, all docked phytocompounds showed binding energy values ranging from –8 to –5 kcal/mol, have more interesting scores than the standard (azithromycin) except ‘Cy3diG5G’. For more details, ‘M3A’ was considered as the most bioactive ligand by exhibiting a conventional H-bond with Val43 and carbon hydrogen bond with Asp73, Pi-Cation with Arg76, Pi-Anion with Glu50, Pi-donor hydrogen bond with Asn46 and Pi-Alkyl with Ile78 and Pro79 (Figure 7). All other docked molecules exhibited different interactions, especially H-bonds.

Toward ‘chitin deacetylase AngCDA from A. niger’, Table 7 displays that all docked phytoligands have more interesting scores than the standard (fluconazole). Indeed, Figure 8 shows that the most effective ligand ‘Cy3G’ (–7.8 kcal/mol) is involved in three H-bonds with Asp47 and His97, Pi-donor hydrogen bond with Tyr166, Pi-Pi stacked with Tyr166, Pi-Pi T-shaped with Tyr138, Phe139, His195, Amide Pi-Stacked with Tyr138, as well as Pi-Alkyl with Leu193.

Against ‘Xanthine Oxidase’, it can be observed from the docking outcomes depicted in Table 8 that ‘catechin’, exhibiting the lowest binding energy (−7.3 kcal/mol) was found to be the most bioactive phytoligand compared to its analogues. Catechin was nicely bound to the target receptor via three H-bonds with Arg880, Thr1010, and Glu1261 through its hydroxy groups in addition to Pi-Sigma (Leu1014 and Ala1079), Pi-Pi stacked (Phe914), Pi-Pi T-shaped (Phe1009), and Pi-Alkyl (Leu873, Val1011, and Ala1078) (Figure 9).

All the above results show that these docked phytoconstituents of the B. asiatica fruit extract have a significant tendency to inhibit FabH from M. luteus, E. coli 24 kDa Domain, chitin deacetylase AngCDA from A. niger and Xanthine Oxidase.