The study used the biofilm-forming clinical keratitis isolate C. hoffmannii (GenBankaccession number: MN453262.1), previously identified (Figure 1A) and isolated from a 70-year-old patient with an agrarian background by our team. The organism was used to evaluate the MIC, MFC, and biofilm inhibitory effects of floral extracts from C. ternatea. The growth colony morphology of this strain was examined on Congo red agar. To confirm its biofilm-forming nature, the isolate was streaked on Congo red agar supplemented with Congo red stain, sucrose, and BHI agar (HiMedia Laboratories, India). Afterwards, the inoculated plates were incubated at 27°C for 24–48 h by the method outlined (Khadija et al., 2019). The growth characteristics of the formed colony were observed and documented.

The blue-colored flowers of the Asian pigeon wings (Figure 1B), locally known as Sangu Pushpam, were collected in and around Tirunelveli, Tamil Nadu, India, at GPS coordinates 8° 44′ 28.3992” N; 77° 41′ 40.6536″ E, between September and December 2022. The plant voucher specimen has been identified as C. ternatea L. using the website http://www.worldfloraonline.org. The initial identification was confirmed by the Southern Regional Office of the Botanical Survey of India in Coimbatore. The flowers were collected, dried in the shade, and ground into a coarse powder. The Soxhlet extraction method was used to extract the powdered flower using a sequential gradient of solvents that ranged from nonpolar to polar, including petroleum ether, ethyl acetate, acetone (SRL, Mumbai, India), and water in the ratio 1:10. The solvents were filtered through Whatman™ filter paper and condensed with a help of using a rotary evaporator (RV10 IKA^®). The resulting solvent-free extracts were stored at 4°C for later use (Rajput et al., 2021).

Floral extracts from C. ternatea were evaluated for their antifungal activity on potato dextrose agar (PDA) plates following the well diffusion method. C. hoffmannii inoculum was cultured in a PDB medium for 48 h at 100 rpm and 27°C. The yeast cell suspension density was adjusted to 0.5 McFarland units using a spectrophotometer (Jasco V-770, Japan). The PDA plates were evenly coated with the prepared inoculum. The solvent-free plant extracts were mixed with DMSO (SD Fine Chemicals Ltd., Mumbai, India) at concentrations (µg/mL) of 1,000, 1,500, 2000, and 2,500. Agar plates were prepared with wells for adding the extracts. Fluconazole (HiMedia Laboratories, Mumbai, India) served as a positive control, whereas the negative control was DMSO. The inhibitory zone’s diameter was measured in millimetres after 48 h of incubation at 27°C (Parveen et al., 2018).

The MIC and MFC values of C. ternatea flower extracts were evaluated using broth dilution and plate methods against C. hoffmannii. Four extracts were obtained by diluting concentrations from 0.5 to 2.5 mg/mL in DMSO using two-fold serial dilutions. A culture mixture of 48-h-old C. hoffmannii culture at 1.5 × 10⁸ CFU/mL mixed with 100 mL of PDB was prepared. The MIC was determined by adding different amounts of plant extracts (0.25–2.5 mg/mL) to various test tubes. The MIC is the minimum concentration of the extract required to inhibit the growth of the inoculum after a 48-h incubation period at 27°C. To examine MFC, 10 μL of culture from tubes showing no visible growth was added onto a PDA plate. The MFC was calculated as the lowest concentration of extract which allows no fungal growth during incubation on PDA agar plates (Sahal et al., 2020). The floral extract of C. ternatea with the highest inhibition rate at the lowest concentration was chosen for further investigation in subsequent tests.

The DPPH method was utilized to evaluate the CFEA sample’s antioxidant capacity using ascorbic acid as the standard. Varied concentrations of the extract (200, 100, 50, 30, and 10 mg/mL) were mixed with DMSO and an equal volume of DPPH in ethanol (0.004%), and the mixture was kept in the dark for another 30 min. The extract’s free radical scavenging capabilities were assessed by monitoring the purple DPPH color change to yellow and measuring its OD at 517 nm. The IC₅₀ value, which represents the amount of extract needed to eliminate 50% of the DPPH, was then calculated using the described method (Rajput et al., 2021).

The plant CFEA sample’s bioactive compound profiling was carried out using a Shimadzu QP2020 NX GC–MS. The instrument had a single quadrupole, a 190 L/s / 170 L/s (He) differential exhaust turbomolecular pump, a 1,035 kPa pressure range flow controller, and a flame ionization sensor. A 1 mg/mL plant sample was prepared and introduced into the instrument at a split ratio of 1:20 after a 3-min preheating phase at 50°C. The gas chromatograph separation was performed using an Rxi-5sil MS column. The column’s initial temperature was set at 280°C for 2 min, then gradually rose to 330°C at a rate of 12°C every 40 min. The carrier helium gas flow rate was set at 1 mL/min through the injection port maintained at 280°C. For mass spectrometry analysis, an ionization potential of 70 eV was used, along with electron ionization (EI) and transmission line temperatures of 260°C and 280°C, respectively. The sample was fully scanned with a cut-off time of 3 min, from 25 to 500 amu. The gas chromatograph ran for a total of 39 min, with the mass spectrometry analysis taking place between 5 and 40 min. Unidentified compounds within the extract were identified using the Shimadzu GC–MS solution™ Ver.4 software, which utilized the “NIST20R-library” (Syeda and Riazunnisa, 2020).

The major functional groups and structural features of the phytoactive chemicals in the plant sample were determined by FTIR spectral analysis. The infrared (IR) spectral details were acquired employing a Nicolet iS5 FT-IR spectrometer, scanning the mid-IR band 32 times at a resolution of 2 cm⁻¹. The plant samples were placed in an IR chamber with iD3 ATR attachments before analysis. The resulting spectral details were collected, examined, and processed using Thermo Fischer Scientific OMNIC software, which compared the output spectrum with a reference spectrum from a library to identify the functional groups.

The mutagenic potential, carcinogenicity, and eye irritation ability of the phytocompounds were predicted using the STopTox web portal (Pokharkar et al., 2022). The Swiss-ADME program and ADMET lab 2.0 were used to predict the ADME pharmacokinetic properties. Additionally, the physicochemical properties that influence corneal permeability, such as molecular weight (MW), molecular volume (MV), hydrogen-bond donor (HBD), hydrogen-bond acceptor (HBA), total hydrogen bonds (HBtot), octanol–water partition coefficient (logP), and distribution coefficient logD7.0, were recorded using ADMET lab 2.0 software (Jha et al., 2022).

The CFEA phytochemical’s toxicity on SIRC (NCCS, Pune, India) cells was assessed using MTT colorimetric assay. DMEM media (Gibco, United States) with FBS (10%) and antibiotic solution (1%) supplementation was used growing the cells until reaching 1 × 10⁵ cells/mL concentration in a 96-well culture plate. After incubation at 37°C for 24–48 h, the wells were rinsed with sterile PBS and treated with various amounts of plant samples in a serum-free DMEM medium. The plate was then incubated for 24 h at 37°C with 5% CO₂. Following that, 10 μL of MTT (5 mg/mL) was added to each well and incubated until a purple precipitate appeared under an inverted microscope. The well plates were washed with 1X PBS after the spent culture and MTT were removed. The formed formazan crystals were dissolved by adding 100 μL of DMSO and shaking the plate for 5 min. Cell viability percentages were calculated by quantifying the MTT reduction to formazan crystal through measuring the OD values at 570 nm using the microplate reader (Thermo Fisher Scientific, United States) as described by Chainumnim et al. (2022).

The in vitro hen’s egg chorioallantoic membrane-embryo irritation (CAE-EI) experiment, as described by Rangel et al. (2020) was utilized to assess the CFEA sample’s potential for causing ocular irritation. Fertilized White Leghorn chicken eggs were obtained from the Veterinary College and Research Institute in Tirunelveli after being incubated for 9 days. The eggshells were sterilized with ethanol, and a rotary dentist saw blade was used to carefully remove them without harming the chorioallantoic membrane (CAM). To test for eye irritation, a solution containing 0.3 mL of the extracted sample in a 0.5% DMSO solution was applied to the CAM. The positive control used was 0.1 N NaOH, and the negative control was 0.9% NaCl. Following the ICCVAM methodology, the CAM reactions such as vascular lysis or coagulation were observed after 5 min. The resulting irritation scores, ranging from 0 to 21, were assigned to categorize irritants as none (0–0.09), weak (1–4.9), medium (5–9.9), and strong (10–21), and recorded for analysis (Luepke and Kemper, 1986).

The data from the experiments performed thrice were reported as mean with ± standard deviation. SPSS 22.0 was used to carry out a one-way ANOVA analysis and the p-values less than 0.05 were considered as statistically significant.

Ongoing research is being conducted on the potential therapeutic benefits of the blue flowers of C. ternatea, which have traditionally been used to treat eye ailments. The study found that C. ternatea flower extracts had significant antifungal activity against C. hoffmannii, a clinical isolate fungus. CFEA showed the strongest inhibitory effect at the lowest concentration. The water extract had no inhibitory effects, while petroleum ether and acetone extracts had reduced inhibitory effects. The average inhibition zone of the active extracts ranged from 9.83 ± 0.28 to 23 ± 1.0 mm. The CFEA extract showed the most potent antifungal activity, ranging from 11.50 ± 0.50 to 23 ± 1.0 mm (Figure 2A). The DMSO used as a negative control had no inhibitory effects. In contrast, the positive control, fluconazole, resulted in an inhibition zone of 23 ± 0.5 mm. The results demonstrate that C. hoffmanni is more susceptible to CFEA extract than other extracts, having MIC and MFC values of 0.5 and 1 mg mL⁻¹, respectively.

Under an optical microscope, living cells stained with crystal violet were observed as part of the antibiofilm assay (Figure 3). The ability of CFEA to effectively inhibit biofilms was demonstrated by exposing C. hoffmannii to varying doses of the compound (Figure 2B). The intensity of biofilm formation decreased as the concentration of CFEA increased, indicating a dose-dependent relationship for inhibiting biofilm formation. The least effective MIC doses for inhibiting biofilm growth were 1/4 and 1/2.

At concentrations (mg/mL) of 10, 50, 100, 200, and 300, the antioxidant levels of CFEA were observed to increase by 72%, 78%, 83%, 91%, and 92%, respectively. These results closely matched those of the control group, with ascorbic acid indicating the strong antioxidant capacity of CFEA (Figure 2C). The IC₅₀ of CFEA was found to be 1 mg, demonstrating its ability to reduce DPPH radical scavenging activity by 50% at this concentration. This study highlights the impressive antioxidant effects of CFEA and its effectiveness in eliminating DPPH radicals.

Upon comparing the CFEA extract to the “NIST20R library,” the GCMS spectral data analysis revealed the presence of 23 phytochemicals in different quantities. The GC–MS chromatogram (Figure 4) indicates 23 peaks in the CFEA. These include compounds such as Hexanoic acid, 2-ethyl-, anhydride (49.18%); n-Hexadecanoic acid (11.86%); 1,2,3-Propanetriol, 1-acetate (11.71%); Glycerol 1,2-diacetate (5.58%); Hexadecanoic acid, octyl ester (4.03%); Phthalic acid, diethyl ester (3.89%); 1-Hexacosene (3.70%); 9-Tricosene, (Z)- (1.26%); D-Glucitol, 1,4-anhydro- (1.04%); Phenol, 2,4-bis (1,1-dimethyl ethyl)- (0.99%); Sorbitol (0.87%); Benzofuran, 2,3-dihydro- (0.77%); 2,5-O-Methylene-D-mannitol (0.73%); Isopropyl hexacosyl ether (0.66%); Phthalic acid, bis(2-ethylhexyl) ester (0.65%); Hexadecanoic acid, 3-hydroxy-, methyl ester (0.57%); 5-Hydroxymethyl-2-furaldehyde (0.47%); Propanoic acid, 2-methyl-, nonyl ester (0.47%); 2-Methylhexacosane (0.45%); 1-Docosanol, methyl ether (0.44); Dotriacontyl isopropyl ether (0.43%); 9,9-dimethoxy bicyclo [3.3.1] nonane-2,4-dione- (0.27%) and Sulfurous acid, dodecyl 2-propyl ester (0.23%).

The CFEA FTIR spectrum revealed several functional groups of CFEA phytocompounds. Major absorption peaks were found at specific wave numbers (cm⁻¹): 851, 1,047, 1,238, 1,371, 1,438, 1739, 2,987, and 3,328, which confirms the presence of significant chemical bonding groups. The peak at 1,047 cm⁻¹ corresponds to the CO-O-CO bond stretch in the anhydride group, while the peak at 851 cm⁻¹ represents the C=C alkene group bend. Peaks at 1,371 cm⁻¹, 1,438 cm⁻¹, and 3,328 cm⁻¹ indicate the O-H groups of phenols, carboxylic acids, and alcohols, respectively. Additionally, peak values at 1,238 cm⁻¹, 1,739 cm⁻¹, and 2,987 cm⁻¹ represent the C-O, C=O, and C-H stretches of alkyl aryl ethers, carbonyl esters, and alkane groups (Figure 5).

The corneal permeability and other pharmacological parameters of compounds are crucial for formulating topical ophthalmic medications. This data includes MW, MV, HBD, HBA, total hydrogen bonds (HBtot), Log P, and Log D. Some CFEA phytocompounds adhere to the Lipinski rule of drug-likeness, showing potential for conversion into orally bioavailable medications and topical treatments for FK. However, some CFEA compounds violate Lipinski’s rule, and some are predicted to have ocular irritation, carcinogenic, mutagenic, or mutagenic potential. However, some compounds of CFEA violate Lipinski’s rule. They include Sorbitol, 9-tricosene, (Z)-, 1-hexacosene, 2-methylhexacosane, Hexadecanoic acid, octyl ester, bis(2-ethylhexyl) phthalate, and Isopropyl hexacosyl ether, each with one violation and Dotriacontyl isopropyl ether with two violations. The compounds Glycerol 1,2-diacetate, D-Glucitol, 1,4-anhydro-, 1,2,3-Propanetriol, 1-acetate, 9,9-dimethoxybicyclo[3.3.1]nonane-2,4-dione-, and 2,5-O Methylene-D-mannitol are designated as corneal-permeable, druggable phytocompounds based on their properties. Table 1 provides each compound’s physiochemical and pharmacokinetic properties.

The antioxidant, antifungal, and antibiofilm properties of CFEA, along with the abundance of identified bioactive phytocompounds and their high-affinity interaction with fungal virulence target enzymes and human inflammatory proteins, have led us to propose the following mechanism of action for CFEA:

Figure 10 depicts the CFEA phytocomponents’ proposed general mechanism, qualifying them as constituents in a topical treatment to be produced to combat FK. When rich CFEA phytocompounds come into contact with the surface of the fungal cell wall, their interaction may cause a variety of damaging events at the cell wall, resulting in cell integrity breakdown and the release of internal components. They also inhibit biosynthesis as well as the development of new cell walls and biofilm. Furthermore, their in-silico druggable corneal permeability and other pharmacological features, as well as their high-affinity association with pathogenic and anti-inflammatory-linked proteins, may help to minimize post-infection inflammation in the eye.