2.1 Instruments

NMR spectra were recorded at 400 (1H) and 100 MHz (13C) on a Bruker NMR spectrometer, Japan. The NMR spectra were recorded in deuterated CD3OD using TMS as an internal standard.

2.2 Isolation of the compounds

Plant material: Hedera helix L. leaves were collected from a private garden in Giza, Egypt, and authenticated by Mrs Teresa Labib, Head of the Taxonomists at Orman Botanic Garden. A voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy, College of Pharmacy, Cairo University, Egypt.

Extraction and fractionation: Two kilograms of the air-dried powder were exhaustively extracted with 70% ethanol using a homogenizer-assisted extraction method. The ethanolic extract was combined and evaporated under reduced pressure to dryness. A portion of the extract (200 g) was dissolved in 500 mL methanol and allowed to precipitate for 1 day at 5 °C after the addition of anhydrous acetone (2 L), then filtered. Both filtrate and precipitate were monitored using precoated TLC plates (Fluka, Sigma-Aldrich Chemicals, Germany), where the filtrate was found to be enriched in flavonoids (70 g), and the precipitate was found to be enriched in saponins and further purified by precipitation by acetone to yield saponin-enriched residue (SRF, 130 g). Then, SRF (50 g) was subjected to flash column chromatography on silica gel (0.04–0.063 mm, Merck) and eluted with CHCl3–MeOH–H2O (26: 14: 3) to afford 5 fractions.

Isolation and identification: Fraction 4 (15 g) was further purified on a polyamide column (SC 6 0.07 mm, Merck) with 50% of MeOH then on another silica gel (0.04–0.063 mm, Merck) and eluted with CHCl3–MeOH–H2O (26: 14: 3) to obtain 210 mg of compound (1). Compound 1: Colorless solid; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD, TMS) (Supplementary Figs. S1 & S2). Additionally, Fraction 5 (7 g) was purified on a polyamide column (SC 6 0.07 mm, Merck) with 20% of MeOH, then on RP-18 silica gel (Fluka, Sigma-Aldrich Chemicals, Germany) and eluted with MeOH–H2O (50: 50) to obtain 140 mg of compound (2). Compound 2: Colorless solid; 1H NMR (400 MHz, CD3OD) and 13C NMR (100 MHz, CD3OD, TMS) (Supplementary Figs. S3 & S4).

The structure of saponins was elucidated by 1H and 13C NMR and compared with the literature to be α-hederin (1) and hederacoside C (2) [26].

1H NMR (1) (CD3OD, TMS) δ: 0.72 (3H, s), 0.84 (3H, s), 0.93 (3H, s), 0.96 (3H, s), 0.97 (3H, s), 0.99 (3H, s), 1.0 (1H, m), 1.17 (3H, s), 1.24 (3H, m), 1.28–1.94 (18H, m), 2.88 (1H dd, J = 13.86, 3.84 Hz), 3.29–3.93 (12H, m), 4.57 (1H, d, J = 9.0 Hz), 5.17 (1H, d, J = 1.35 Hz), 5.26 (1H, s).

13C NMR (1) (CD3OD, TMS) δ: 12.3, 14.9, 16.3, 16.5, 17.4, 22.5, 22.7, 23.1, 25.0, 25.0, 27.4, 30.2, 32.0, 32.4, 33.5, 36.2, 38.2, 39.1, 41.3, 41.5, 42.5, 45.8, 46.2, 46.7, 46.9, 63.3, 63.3, 67.7, 68.7, 70.7, 70.7, 72.2, 72.5, 72.5, 75.3, 80.9, 100.5, 102.9, 122.2, 143.8, 180.8.

1H NMR (2) (CD3OD, TMS) δ: 0.72 (3H, s), 0.82 (3H, s), 0.93 (3H, s), 0.97 (3H, s), 1.00 (3H, s), 1.16–2.06 (31H, m), 2.86 (1H dd, J = 14.71, 4 Hz), 3.26–4.09 (29H, m), 4.41 (1H, d, J = 10.62 Hz), 4.56 (1H, d, J = 8.05 Hz), 5.18 (1H, m), 5.27 (1H, m), 5.36 (1H, d, J = 8.05 Hz).

13C NMR (2) (CD3OD, TMS) δ: 12.4, 15.1, 15.1, 16.5, 16.6, 17.3, 22.7, 22.7, 24.9, 25.0, 27.4, 30.1, 32.1, 33.7, 36.2, 38.2, 39.2, 41.1, 41.7, 42.5, 45.8, 46.6, 46.7, 46.7, 47.0, 47.4, 47.7, 47.8, 48.0, 48.3, 48.3, 60.4, 63.2, 62.5, 65.2, 67.9, 68.2, 68.6, 69.2, 69.5, 70.8, 70.8, 71.1, 72.4, 72.4, 73.6, 75.2, 75.4, 76.2, 78.3, 81.2, 94.5, 100.2, 101.5, 102.7, 102.8, 122.4, 143.5, 176.7.

2.3 Formulation and characterization of the prepared nano delivery systems

Composite nanovesicle material: Epikuron 200 was provided by Cargill Texturizing Solutions, Germany. Transcutol was kindly supplied by Gattefosse Company, France. Phosphotungstic acid, low molecular weight chitosan (50 kDa) with 70% acetylation degree, and dialysis membrane with an average flat width of 33 mm were obtained from Sigma Aldrich Company, USA. Absolute ethanol was purchased from Al-Gomhorea pharmaceutical company, Cairo, Egypt. Nanosep centrifuge tubes equipped with an ultra-filter were obtained from Pall Life Sciences, USA.

Preparation of composite nanovesicles using the ethanol injection method: Composite nano-vesicular systems were formulated using the ethanol injection technique, according to Ang et al. [27]. In brief, 5 mL of absolute ethanol comprising 50 mg Epikuron 200 (phospholipid), 25 mg of α-hederin, or hederacoside C, or both were inserted dropwise into 10 mL of a warm 0.6% chitosan acetate buffer solution pH 4.6 encompassing 5% transcutol (acting as a penetration enhancer) using a plastic syringe at an injection rate of 2 mL/min. The dispersions were mechanically stirred at 100 r.p.m. (Lab Tech LMS -1003, Korea) maintained in ambient conditions (25 ℃) till complete evaporation of the employed absolute ethanol. Finally, the obtained dispersions were preserved overnight at refrigeration temperature for further investigations. Table 1 shows the composition of the three vesicular preparations.

Table 1 Composition and Characterization of composite nanovesicles

Determination of the particle size (P.S.), Polydispersity index (PDI), and zeta potential (ζ-potential): Following appropriate dilution, a Zetasizer (model ZS3600, Malvern Instruments Ltd., Worcestershire, UK) was implemented to assess the aforementioned colloidal properties of the composite vesicles [28].

Determination of entrapment efficiency (EE%): Nanosep centrifugal devices were employed to separate the unentrapped drug from loaded nanoparticles [29]. A volume equivalent to 0.5 mL was transferred to the Nanosep centrifugal devices, then centrifuged in a high-speed cooling centrifuge (SIGMA-3-30KS, Germany) at −4 °C and 3000 rpm for 50 min. After dilution with acetate buffer, pH 6.4, the free drug was measured in the filtrate via a reported HPLC method. Isocratic elution was applied using a mobile phase comprising ammonium acetate (pH 8.5) and acetonitrile 70:30 (v/v) at 1.2 mL min−1. Moreover, 1 μl sample volume was injected into a C18 column (Agilent 1290 infinity, Germany) with column effluent being monitored at 220 nm at 30 °C [30], using acetate buffer (pH 6.4) serving as the control solution. The following equation was implemented to compute EE% [31]:

EE% = \(\frac\) × 100. Where At represents the total quantity of saponin(s) being integrated into vesicular formulations, and Af represents the quantity of free saponin(s).

Storage stability study: The P.S., PDI, ζ-potential, and EE% of the three preparations were re-determined after 3 months of storage at 2–8 °C to reflect their physical stability [32].

In vitro release study: The present study was assessed utilizing Hanson Franz-type diffusion apparatus (model 60–301–106, CA, Los Angeles, USA) [33]. The cellulose membranes were loaded between the upper and lower chambers of Franz diffusion cells, and the lower chamber comprised 7.2 mL simulated tear fluid (STF), comprising 2 g NaHCO3, 0.08 g CaCl2, and 6.7 g NaCl in 1 L of deionized water, which was agitated at 600 rpm for a period of 24 h at 34 ± 0.5 °C [34]. A specified volume of the three nano vesicular formulations was introduced to the upper (donor) chamber, with the saponin(s) suspensions in acetate buffer (pH 6.4) serving as control. At specified time intervals (0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h), a volume equivalent to 2 mL was taken out from the release media and compensated with an equal volume of STF. The amount of released saponin(s) from each formulation was analyzed using a reported HPLC method and measured at ƛmax of 220 nm [35]. In addition, the release data were introduced into several kinetic models to evaluate the mechanism of release of the saponin(s) from the obtained dispersions.

Determination of the surface morphological features of the composite nanosystems employing transmission electron microscope (TEM): The surface morphological features of the formulae were assessed using TEM (JEM—100S, Joel, Tokyo, Japan). Samples were negatively stained imparting 1% phosphotungstic acid and examined under TEM [35].

Sterilization of composite nanosystems by Gamma Irradiation: The three preparations were exposed to gamma radiation at room temperature using a 60 °C radiation source at a dosage rate of 10 kGy/hour. After being subjected to gamma rays, the physicochemical properties of the formulae were assessed, and the variations from non-irradiated samples were compared statistically [36]. Furthermore, a sterility test was performed to assess the sterilization efficiency of the gamma radiation dose delivered to the formulae.

2.4 Antipseudomonal activity2.4.1 Bacterial strain

Three clinical keratitis P. aeruginosa isolates collected from tertiary care hospitals in the greater Cairo area were used in this study. Standard microbiological techniques such as Gram staining and oxidase testing were used as an initial identification. Further confirmation was performed using the Vitek® 2 automated system (bioMérieux, Marcyl’Etoile, France) [37]. The antibiotic susceptibility profile of P. aeruginosa isolates was evaluated using the agar diffusion method against the most clinically used antimicrobials [37, 38]. Results were construed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2024). Various antibiotic classes were used, such as penicillins (ampicillin, AMP), carbapenems (imipenem, IPM and meropenem, MRP), third-generation cephalosporins (ceftriaxone, CTR and cefotaxime, CTX), and penicillin-like inhibitors with beta-lactamase inhibitors (amoxicillin-clavulanic AMC). Tests were performed twice for confirmation. According to the World Health Organization (WHO), isolates that were resistant to three or more antibiotic classes were considered multidrug-resistant (MDR).

2.4.2 In vitro antimicrobial activity

Agar well diffusion: The antimicrobial activity of α-hederin and hederacoside C was evaluated using the agar well diffusion method. A concentration of 1 × 108 CFU/mL of P. aeruginosa suspension was streaked on Muller-Hinton agar plates (Oxoid, UK). Six concentrations (100%, 50%, 25%, 12.5%, 6.25%, 3%, 1.5%, and 0.75%) of the investigated compounds were examined. Then, 50 μl of each previous concentration was added to wells before overnight incubation at 37 °C. Diameters of a zone of inhibition were measured, and the experiments were performed in triplicate [39, 40].

2.4.3 In vitro anti-virulent activity

Pyocyanin production inhibition: In a broth containing MIC and another containing sub-MIC, the P. aeruginosa isolate was grown overnight at room temperature, then centrifuged, and the obtained supernatant was mixed with chloroform, followed by centrifugation. Finally, 0.2 M HCl was added, followed by centrifugation, and the absorbance of pyocyanin was measured at 520 nm [40].

Swarming and twitching motility inhibition: The medium was prepared by the addition of supplementing 0.5% (w/v) casamino acids to Luria–Bertani (LB), while the twitching medium consisted of 1.5% LB agar. Before agar solidification, MIC and sub-MIC concentrations of PEVIII were added, and then 2.5 μL of the inoculum was applied to the swarming and twitching agar plates before overnight incubation at room temperature. The migration zones appearing on the agar plates were monitored. All motility experiments were conducted in triplicate [41, 42].

2.4.4 In vivo evaluation of anti-pseudomonal activity

Antimicrobial resistance profiling of the obtained strains: An MDR carbapenem-resistant isolate, which showed resistance to four different classes of antibiotics, was used for the in vivo animal model (Fig. S5).

Animals: The study was approved by the Research Ethics Committee (REC) of the Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt, under permit number REC2524. The study adhered strictly to the Guidelines for the Care and Use of Laboratory Animals (National Research Council, 2011). A total of 35 healthy female Sprague–Dawley rats, weighing between 200 and 400 g, were used. All animals were acclimated for 7 days in controlled environmental conditions before the experiment commenced. The rats were randomly assigned to seven groups (n = 5 per group). Group 1 served as the negative control; Group 2 was the positive control (infected, non-treated); Groups 3, 4, and 5 were infected and treated with PEVI, PEVII, and PEVIII, respectively; Group 6 was infected and treated with the blank formula; and Group 7 received gentamicin sulphate ophthalmic ointment (GENTAWISE®). At the end of the study, the animals were humanely euthanized with ketamine, followed by cervical dislocation.

Induction of keratitis and grouping of animals: A corneal epithelial defect was created in all rats except for negative control groups (group 1) under anesthesia on the first day, and 0.05 mL per rat was inoculated from a solution containing 1 × 108 colony-forming units (CFU)/ mL of P. aeruginosa clinical isolate, and those with keratitis at the end of the third day were added to the group. On the 7th day, which was the end of the study, the rats were sacrificed, their globule structures were removed as a whole, and some were stored in 10% formaldehyde for histopathological examination, whereas the rest were frozen for biochemical parameters evaluation [43].

Gross Lesion Monitoring: The mice were examined at 1, 3, 5, and 7 days postinfection (p.i) to monitor disease progression, and digital images of the mice' corneas were taken.

Determination of bacterial bioburden: Ocular bacterial count was estimated after homogenization of samples in 2 mL of phosphate buffer saline (PBS) followed by serial dilution in PBS (1:10, 1:100, 1:1,000, and 1:10,000), then plating on cetrimide agar plates in triplicate and overnight incubation. To ensure reproducibility, the experiment was conducted in triplicate. All colony counts were expressed as log10 CFU per milliliter.

Histopathological examination and scoring: Sacrificed rats were examined microscopically by hematoxylin–eosin (H&E) staining by a pathologist who was unaware of the groups, and the results were evaluated and scored. The eye was placed in the solution immediately after enucleation and trimming. A gauze pad was used to keep the eye submerged. The globe remained in Davidson’s solution for 1–2 days. The eye was then taken out of the solution and placed in 10% paraformaldehyde. Tissue specimens were trimmed off, washed, and dehydrated in ascending grades of alcohol. The dehydrated specimens were then cleared in xylene, embedded in paraffin blocks, and sectioned at 4–6 µm thick. The obtained tissue sections were deparaffinized using xylol and stained using hematoxylin and eosin (H&E) for histopathological examination through the electric light microscope [44]. Davidson's Solution consisted of: glacial acetic acid, 100 mL, 95% ethyl alcohol, 300 mL, 10% neutral buffered formalin, 200 mL, and distilled water, 300 mL. Lesions were scored as follows: (−) for the absence of lesion, ( +) for Mild lesion (+ +) for Moderate lesion (+ + +) for Sever Lesion.

Enzyme-linked immunosorbent assays (ELISA): The eye content of tumor necrosis factor-α (TNF-α; SEA133Ra, Cloud-Clone Corp., USA), nuclear factor kappa B (NF-κB; MBS453975, MyBioSource, CA, USA), reactive oxygen species (ROS; MBS039665, MyBioSource, CA, USA), and AKT1 (AKT1; S-F49321, lifespan Biosciences) were measured in the eye homogenate using the corresponding ELISA kit according to the manufacturer’s protocol.

Quantitative real-time PCR (RT-PCR) analysis: The gene expression of PI3K and OPRL was assessed in eye tissues after the extraction of RNA. The Direct-zol™ RNA MiniPrep Plus kit (Cat # R2072, ZYMO Research Corp., CA, USA) was used in the extraction process. The Beckman dual spectrophotometer (USA) was used to assess the purity and concentrations of extracted RNA. As directed by the manufacturer, the SuperScript™ IV One-Step RT-PCR kit (Cat # 12594100; Thermo Scientific, MA, USA) was used.

The forward and reverse primers for PI3K were 5'-ACACCACGGTTTGGACTATGG-3' and 5'- GGCTACAGTAGTGGGCTTGG-3', respectively.

For OPRL, the forward primer was 5'-ATGGAAATGCTGAAATTCGGC-3' and the reverse primer was 5'-CTTCTTCAGCTCGACGCGACG-3'.

For GAPDH forward primer was 5'-TGGATTTGGACGCATTGGTC-3' and the reverse primer was 5'-TTTGCACTGGTACGTGTTGAT-3'.

Quantitative real-time PCR reactions were conducted using the Step One™ system (Applied Biosystems, CA, USA). The relative expression levels of PI3K and OPRL were determined according to the Livak and Schmittgen 2 − ΔΔCt method using GAPDH as an internal standard and presented as fold change from control.

Statistical analysis of data: One-way ANOVA followed by Tukey–Kramer post-test was utilized for data statistical analysis. Bonferroni correction and Cohen’s d were also applied to assess multiple comparisons and effect size, respectively. Data were expressed as mean ± SD, using Graphpad Instat software.