Corneal tissue was obtained from a cadaver (a thirty-two years-old male) and used for corneal transplantation up to 48 h postmortem, the residual tissue after transplantation was transferred to the cell culture lab, under sterile conditions to be used for cell culture (23). Limbal biopsy (approximately 1 × 2 mm²) was extracted from the limbus and cultured in DMEM/F12 (Shell max, Iran) supplemented with 10% fetal bovine serum (FBS, Gibco, Germany) and 1% penicillin/streptomycin (Shell max, Iran). LSCs explants were cultured so that the epithelial layer was upward. After 15 min for the initial adhesion of tissue explants to the plate surface, the culture medium was gently added to the edge of the plate and gently tilted to spread throughout the culture dish. Approximately 7 days after the initial culture, LSCs showed growth from the bottom of the samples. Every 5 days, half of the culture medium was replaced with a fresh culture medium. When the cultures reached about 60–70% of confluence, the cells were characterized for the expression of LSCs markers including cytokeratin 19, vimentin (immunocytochemistry), CD44, P63, and ABCG2 (flow cytometry), and used for further analysis. In this study we intended to investigate the effect of 1% HSM on LSCs in their original cell architecture. As LSCs are susceptible to enzyme treatment and lose their cell–cell connections, all experiments were conducted using LSCs from primary cultures. This approach was specifically chosen to ensure the preservation of the cells’ intrinsic properties and to minimize any potential alterations in gene expression or functionality that might occur. Primary LSCs provide a more physiologically relevant model for assessing the effects of interventions.

Keratocyte cell culture was performed using the peripheral stroma of the same corneoscleral rime (a thirty-two years-old male) as described previously (24–26). Under sterile conditions, the corneal rim was relocated to the culture room. After rinsing the tissue with sterile phosphate buffer (PBS), the epithelial and endothelial layers were removed by knife scratching, and the rest of the tissue was cut into small segments, each roughly 1 mm in size. Stromal keratocyte explants were cultured in DMEM/F12 (Shell max, Iran) enriched with 10% FBS (Gibco, Germany) as well as 1% penicillin/streptomycin (Shell Max, Iran). Medium change and subculture were performed regularly every two to three days. Cells from passages 5–7 were checked for the expression of keratocytes markers (LUM and KERA) using polymerase chain reaction (PCR) and applied for further analysis.

Wax-free coriander honey from Koozeasal Co. (Product ID: hd-4, Esfahan, Iran), was analyzed and confirmed to be free from toxins or microorganisms.

Honey (at concentration of 1% w/v) was dissolved in DMEM/F12 (Shell Max, Iran) at 37°C, then filtered through a 0.22 μm filter, and kept at 4°C until the time of the experiment. Before usage, the pH of HSM was determined and verified in terms of neutrality (pH ~7.2–7.4). Then, HSM-treated cells underwent relative gene expression analysis and cell viability assays, and their results were contrasted to those of controls (culture media without honey).

Using an MTT test, the impact of HSM on LSCs and keratocyte proliferation was assessed. Cells were cultured in a 96-well microplate at a concentration of 10⁴ cells per well, and exposed to either 1% HSM or control media for 24 h at 37°C. Subsequently, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (5 mg/mL PBS; GoldBio, United States) was added to each well and kept at 37°C for approximately 4 h. Crystals of formazan are created by live cells using MTT. After that, 100 μL acidic SDS [10% SDS (Parstous, Iran) in 0.01 M HCL] was added to each well and incubated at 37°C overnight. SDS helps dissolve cell membrane and formazan crystals and produces a purple solution, which optical density (OD) reflects the number of living cells. The OD of each well was then assessed at 545 nm wavelength using a microplate reader (BMG Labtech, Germany) and was used to evaluate keratocyte and LSCs survival in HSM vs. control groups.

LSCs and keratocytes (10⁶ cells per 10 cm² culture dish) were exposed to 1% HSM medium for 24 h at 37°C. We used the RNeasy kit (Parstous Co, Iran) for total RNA extraction and the Easy cDNA Synthesis Kit (Parstous, Iran) for cDNA synthesis. AlleleID software (v.7.5, Premier Biosoft International, Palo Alto, CA, United States) was utilized for primer design of BAX, BCL-2, IL-1β, and ACTB (house-keeping gene) (Table 1). The StepOneTM Real-Time PCR machine (Applied Biosystems) was used to run real-time PCR using RealQ Plus Master Mix Green (Ampliqon). PCR reactions were comprised of 7.5 μL master mix, 1.5 μL F and R primers, and 6 μL of DNA (total volume of 15 μL). Every PCR run had a single-step temperature profile with 40 cycles of 95°C hold phase for 15 min, 95°C denaturation phase for 10 s, and 61°C annealing and extension phase for 45 s. According to the 2^−ΔΔCt formulation, relative RNA expression was quantified.

To assess the antimicrobial activity of 1% HSM on ocular pathogens, an antimicrobial susceptibility test was performed. Cultures of nine common human ocular pathogenic strains were obtained from the local hospital of the study. Species included Escherichia coli, Pseudomonas aeruginosa, Klebsiella, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Staphylococcus haemolyticus, Enterococcus faecalis, and Acinetobacter. Every sample was cultured, and if microbial growth materialized, differential cultures and assays were run to distinguish between various bacterial strains. Disc diffusion method was used for antibacterial activity tests. Mueller–Hinton agar, placed into either 100 mm or 150 mm Petri dishes, was the medium utilized in this experiment. The agar had a pH in the range of 7.2 to 7.4. The Mueller–Hinton agar culture medium (Merck, Germany) was streaked to create a bacterial lawn after being inoculated with a straight saline suspension of isolated colonies that had been corrected to 0.5 McFarland turbidity standard. The plates were dried and the HSM stock solution was diluted to concentration of 1%. A volume of 25 μL of each dilution was loaded into sterile, blank discs 6 mm in diameter (Padtanteb, Iran), and the disks were fully dried before placing on the plates. Then the disk was gently placed on top of the agar and was lightly pressed down with the pancetta. After 16–18 h of incubation at 35°C, the area of inhibited bacterial growth was measured. Antibiotic disks as positive control used for the tests included: chloramphenicol, gentamicin, ciprofloxacin, and ofloxacin. Distilled water-loaded discs were used as negative controls, also a vehicle medium (culture medium without honey) was used as control. The concentration of the solution would be at its peak right adjacent to the disc and would gradually diminish as the distance from the disk increase. No colonies would grow in the zone of inhibition if the drug was effective against bacteria at doses higher than critical concentration, that is, the region of the agar where the concentration of the antibacterial agent was greater than or equal to this minimum effective concentration. This-together with the rate of antibiotic diffusion-was utilized to determine how susceptible the bacteria were to that specific antibiotic.

The study protocol was approved by the ethics committee of Shiraz University of Medical Sciences (IR.SUMS.MED.REC.1400.548) and registered with the Iranian Registry of Clinical Trials (IRCT20220117053744N1). Patients diagnosed with dry eye disease based on clinical signs and symptoms were recruited for the study. Inclusion criteria included a Schirmer test result of less than 5 mm in 5 min and tear break time of less than 10 s. Patients with blepharitis, meibomian gland disorder, a history of taking tetracycline or oral corticosteroids within the past 3 months, ocular surface disorders, previous eye surgery, or a history of allergies were excluded from the study.

To minimize bias and ensure the integrity of the study, blinding procedures were implemented. Both the participants and the researchers involved in data collection and analysis were blinded to the treatment assignment. The honey and placebo eye drops were visually indistinguishable, as they had the same appearance and packaging. Only the pharmacist responsible for preparing the eye drops had access to the treatment allocation information. Allocation concealment was maintained to prevent selection bias. The treatment assignments were concealed from the researchers enrolling participants and assigning them to their respective groups. The allocation sequence was generated by a randomization procedure using computer-generated random numbers. The treatment assignments were then placed in opaque, sealed envelopes sequentially numbered. Each envelope was opened only at the time of participant enrollment, ensuring that the treatment allocation remained concealed until the participants were assigned to their respective groups.

The treatment group received honey eye drops, administered three times a day for 3 weeks. Follow-up evaluations were conducted 6 weeks after completing the treatment. The control group received placebo formulation following the same time and duration protocol.

Honey eye drops were prepared in a clean room according to Good Manufacturing Practice (GMP) protocols. The concentration of honey used was 1% (w/v) in artificial tear eye drops. The honey solution underwent sterilization through 0.45 μm and 0.22 μm filtration, followed by packaging and labeling. Benzalkonium chloride (BAK) (5 μg/mL), hydroxypropyl methyl cellulose (0.001 g/mL) and dextran 70 (0.001 g/mL) was used in both the honey and placebo eye drops.

The subjective improvement in symptoms of dry eye, tear break-up time and Schirmer’s test results were used as study outcome. The ocular surface disease index (OSDI) questionnaire was employed to assess the subjective improvement of dry eye symptoms. All patients were asked regarding any adverse events experienced during the study period.

Chemical analysis of coriander honey used in the ophthalmic formulation revealed notable characteristics, including a high percentage of reducing sugars (72.52% before and 73.56% after hydrolysis), low sucrose content (0.99 g/100 g), and balanced levels of fructose and glucose (36.3 g/100 g each, F/G ratio of 1.00). The presence of hydroxymethylfurfural (HMF) at 7.19 mg/kg, an indicator of honey quality, and a proline content of 734.7 mg/kg were also identified. These findings contribute valuable insights into the chemical composition of coriander honey, underscoring its significance as a key component in the ophthalmic formulation, potentially influencing the therapeutic efficacy and safety of the product. The summarized results are presented in Table 2.

The MTT assay results demonstrated that the 1% HSM did not adversely affect the survival of corneal LSCs and keratocytes, as evidenced by comparable viability to the control group. However, a non-significant reduction in cell proliferation was observed, with LSCs exhibiting 87.16 ± 10.03% viability compared to the control (100 ± 10.04%, p = 0.4167), and keratocytes showing 84.64 ± 1.37% viability compared to the control (100 ± 6.30%, p = 0.0758) (Figure 2).

The findings of our study indicate that the treatment of LSCs and keratocytes with 1% HSM did not result in a significant impact on the expression of BAX and BCL-2 genes, as illustrated in Figure 3. However, we observed a decreasing trend in the expression of the IL-1β gene in both LSCs and keratocytes treated with 1% HSM. Notably, this reduction reached statistical significance in keratocytes, indicating a potential anti-inflammatory effect of 1% HSM on these corneal cells. These results contribute to a better understanding of the molecular responses of LSCs and keratocytes to 1% HSM treatment, suggesting a specific influence on the expression of genes associated with inflammation in the keratocyte population.

The antibacterial activity of the 1% HSM solution was carefully evaluated by measuring the diameter of the zone of growth inhibition around the disks and comparing these measurements to those obtained with established antibiotics, including gentamicin, chloramphenicol, ciprofloxacin, and ofloxacin (Table 3). The length of the inhibitory zone is directly proportional to the inhibitory effect of the tested compound. Our analysis emphasizes that the notable antibacterial potential of 1% HSM was particularly evident against Staphylococcus aureus and Staphylococcus epidermidis, where it demonstrated comparable inhibitory effects in comparison to the reference antibiotics. Specifically, the zones of inhibition for these bacteria ranged from 10.00 mm to 15.44 mm, with Staphylococcus epidermidis showing the largest zone of inhibition at 15.44 mm. It is important to highlight that while 1% HSM showed promising results against these specific strains, it did not exhibit inhibitory effects against Acinetobacter and Pseudomonas aeruginosa. The vehicle medium (culture medium without honey) that was used as control showed growth for all the studied bacterial strains (see Table 3).

This outcome underscores the potential application of 1% HSM in targeting specific ocular pathogens and supports its consideration for inclusion in eye drop formulations, especially for infections known to be caused by susceptible bacterial strains.

In the present study, out of the initially assessed 112 participants, 80 were randomized into either the honey intervention group (40 participants) or the placebo intervention group (40 participants). Four participants in the honey group and 12 in the placebo group were lost to follow-up. Consequently, 36 participants in the honey group and 28 participants in the placebo group were included in the final analysis. The participants’ flow is summarized in the CONSORT flow diagram (Figure 4).