Eyes are the most important sensory organ, through which we get 80% of all information [1
]. Globally, the number of persons who have moderate and severe eye disorders is constantly growing. Inflammatory eye diseases are among the most common diseases in contemporary societies [2
]. The causative agents of these diseases are viruses, bacteria, fungi, and protozoa [3
]. With the increasing popularity of natural aids, the demand for natural ophthalmic preparations is growing as well, but the use of natural materials in ophthalmology should be based on scientific research [3
]. The data from scientific research show that rational nutrition and consumption of fruit and vegetables help to preserve vision and even reverse vision disorders [4
]. That positive effect is associated with the presence of some phytochemicals, which have bioactive properties, such as polyphenols and carotenoids [5
]. Polyphenols can diminish the frequency of diseases—neurodegenerative diseases included—associated with oxidative stress [6
]. Eye drops with polyphenols are used in glaucoma treatment in order to suppress neuroinflammation, which is responsible for neurodegeneration and retinal ganglion cell (RGC) death [9
]. For patients who have dry eye syndrome (DES), symptomatic treatment is applied using eye drops, which are called artificial tears [12
]. Polyphenolic additives in artificial tears have antioxidative, antibacterial, and anti-inflammatory properties, which are important in seeking a positive treatment effect [13
]. Polyphenol-enriched eye drops are effective in protecting trabecular meshwork cells from oxidative stress [15
Eye drop compositions usually contain plant and propolis extracts [16
]. The use of plant extracts and propolis in eye drops is associated with their antibacterial [18
], anti-inflammatory [19
], and antioxidative activity [20
]. These medicinal properties are mostly associated with the biologically active compounds, such as anthraquinones, flavonoids, and phenolic acids [23
]. Propolis collected in Europe is often defined as poplar-type propolis, because poplar buds are one of the primary sources of propolis [24
]. Since ancient times, poplar buds extracts and decoctions have been used for the alleviation of dermatitis symptoms, treatment of rheumatism or infections of the upper respiratory tract, and wound healing. Balsam poplar buds, as with propolis, are a source of phenolic acids and flavonoids. Researchers have found that p
-coumaric acid predominates in both ethanolic propolis and balsam poplar buds extracts collected in Lithuania [25
]. Poplar buds, due to their antibacterial [26
], antioxidant [27
], and anti-inflammatory [28
] action, are a potential active material in the production of eye drops. Italian scientists determined that the use of eye drops with p
–coumaric acid protects eye tissues and lessens the harmful effect of UVB rays, due to its antioxidative properties. p
-Coumaric acid is useful in protecting the eyes from free radical damage, which can be caused by solar rays and UV lamps. Scientific research has proven that eye nanogel with ferulic acid positively impacts the growth of fibroblasts, as well as wound healing [29
]. The results of scientific research show that eye drops with propolis decrease the inflammation of the cornea [31
] and, due to their antibacterial properties, can serve as an auxiliary measure in cases of keratitis [32
]. On the basis of the scientific research data published in existing scientific literature, it is possible to declare that the search for new natural polyphenolic compounds suitable for use in ophthalmology is still ongoing. For our research we chose balsam poplar buds (Populus balsamifera
L.), which are one of the main plant-based precursors of propolis. Therefore, this study presents new data on the application of poplar buds extract in the modelling of ophthalmological preparations. The use of balsam poplar extract in eye drops would expand the group of users, because propolis preparations are not acceptable for vegans. One of the important stages is the selection of a pharmaceutical form.
Eye drops comprise the biggest part of ophthalmic preparations. The major problems in conventional liquid ophthalmic formulations are the washing out of the drug from the precorneal area immediately upon instillation because of constant lachrymal secretion, nasolacrimal drainage, and the short precorneal residence time of the solution [33
]. This problem can be overcome by using in situ gels. In situ gels are conveniently dropped as a solution into the conjunctival sac, where they undergo a transition into a gel, with favorable residence time [34
]. Ocular drug delivery systems based on the concept of in situ gel formation are aimed at longer precorneal residence time, improved ocular bioavailability, and improved patient acceptability. In situ gels are a suitable alternative for common eye drops. After dropping aqueous solution—which contains temperature-sensitive polymers—into the conjunctival sac, viscous and mucoadhesive gels are formed on the surface of the eye [35
]. This leads to the improvement of precorneal residence time and ocular bioavailability. Poloxamer 407 was chosen as a gelling substance [36
], as it is widely used in biomedicine because of its low toxicity and compatibility with many excipients.
Poloxamer is widely used in pharmaceutical formulations as the carrier for most routes of administration, including the rectal, vaginal, ocular, intranasal, topical, and oral routes [37
]. Hydroxypropyl methylcellulose (HPMC) was chosen for its ability to prolong drug release and as a viscosity enhancer [38
]. HPMC has good biological compatibility, and is nontoxic to humans. The quality of gels produced in situ is evaluated by determining various parameters, such as their physical appearance, drug content, clarity, pH value, viscosity, drug release, rheological properties, behavior, and sol–gel transition temperature. It is necessary to evaluate the modeled eye drops for their possible irritant effect on the eyes, because eyes are a sensitive organ. Scientists perform these tests using animal models and ocular cell models [39
]. In order to avoid harmful effects on animals, a short time exposure (STE) in vitro test using a rabbit corneal cell line (SIRC) is recommended as an alternative method for assessing eye irritation [40
]. The aim of this research is to adapt the balsam poplar buds extract for use in the production of ophthalmic gels in situ, and to evaluate their quality by conducting tests of their chemical composition, rheological properties, and biological activity in vitro. Promising results of these tests can serve as a basis for further research.
2. Materials and Methods
Reagents, standards, and solvents of analytical grade were used. Purified deionized water was prepared with the Milli-Q® (Millipore, Arlington, Massachusetts, USA) water purification system. For food purposes, 96.3% rectified ethanol (JSC “Vilniaus Degtine”, Vilnius, Lithuania) was used, along with Folin–Ciocalteu reagent (Sigma-Aldrich, Buchs, Switzerland); acetonitrile (Sigma-Aldrich, Steinheim, Germany); reference standards p-coumaric acid (Sigma-Aldrich, Steinheim, Germany), caffeic acid (Sigma-Aldrich, Steinheim, Germany), ferulic acid (Sigma-Aldrich, Buchs, Switzerland), chlorogenic acid (Sigma-Aldrich, Steinheim, Germany), vanillic acid (Sigma-Aldrich, Buchs, Switzerland), cinnamic acid (Sigma-Aldrich, Germany), apigenin (Sigma-Aldrich, Buchs, Switzerland), pinobanksin (Sigma-Aldrich, Buchs, Switzerland), pinocembrin (Sigma-Aldrich, Buchs, Switzerland), galangin (Sigma-Aldrich, Buchs, Switzerland), and salicin (Sigma-Aldrich, Buchs, Switzerland); sodium carbonate (Sigma-Aldrich, Saint-Quentin-Fallavier, France), and aluminum trichloride hexahydrate (Sigma-Aldrich, Steinheim, Germany). An ultrasonic bath (Bandelin electronic GmbH & Co.KG, Germany) and a lyophilizer (LyoQuest Telstar, Wertheim, Germany) were used for preparation of the extracts.
2.2. Populus Balsamifera Extraction
Balsam poplar buds were collected in Lithuania in March 2020 from the supplier Jadvyga Balvočiūtė’s organic herb farm; fresh material was dried by the supplier. Purified water was chosen as the extractant for the extraction of the balsam poplar buds. Extraction was performed in an ultrasonic bath [41
] for 60 min at a temperature of 25 °C, with a 1:10 ratio of raw material to extractant. After receiving the aqueous balsam poplar buds extract (L2), the extract was freeze-dried (lyophilized) [42
]. Next, 100 mL of aqueous balsam poplar buds extract was frozen, the frozen extract was placed in a lyophilizer, and freeze-drying was carried out at −50 °C for 24 h. Then, 1% aqueous solution (L1) was prepared from the freeze-dried balsam poplar buds extract powder, which was then used in experimental ophthalmic formulations. The extracts were stored in a refrigerator at 5 °C.
2.3. Total Phenolic Compounds
The total content of phenolic compounds was determined in an aqueous extract of balsam poplar buds, and in a 1% aqueous solution of balsam poplar buds’ lyophilized form. The reaction was performed according to the method of Singleton et al., with some modifications [43
]. The phenolic compounds content was determined using the Folin–Ciocalteu reagent, with the results expressed as the p
-coumaric acid equivalent/g of dry weight (mg CAE/g, DW). The extracts were prepared in 25 mL volumetric flasks; 1 mL of extract, 9 mL of purified water, and 1 mL of Folin–Ciocalteu reagent were added; after 3 min, 1.5 mL of Na2
was also added. The reaction mixture was then diluted to the 25 mL mark with purified water. Samples were incubated for 40 min at room temperature (RT) in the dark. The absorbance was measured using a spectrophotometer (Agilent Technologies 8453 UV-Vis, Santa Clara, California, USA) at a wavelength of 760 nm.
2.4. Total Flavonoids
Solutions of extracts were prepared in 25 mL volumetric flasks, with 5 mL of the extracts added to a volumetric flask and diluted up to the mark with 96% ethanol (v
). A further reaction with diluted extracts for the identification of total flavonoids was performed in a new 25 mL flask, according to Woisky and Salatino’s methodology, with some modifications [44
]. Then, 1 mL of the extracts’ solution was added to the flask, followed by 10 mL of 96% ETOH (v
) and 2 mL of AlCl3
(10%) added to the volumetric flask, and the reaction was carried out in an acid medium (33% acetic acid). The reaction mixture was stirred, left in the dark at RT for 20 min and, after incubation, the reaction mixture was diluted with 96% ETOH (v
) to the 25 mL mark. The results are expressed as the mg rutin equivalent/g of dry weight (mg RE/g, DW), and the absorbance was measured with spectrophotometer on a 407 nm wavelength.
2.5. HPLC Analysis
The identification of the predominant active compounds was performed via high-performance liquid chromatography (HPLC) [45
]. A Waters 2695 chromatographic system with a Waters 996 diode array detector and an ACE 5C18 chromatography column (250 × 4.6 mm) was used. The data were processed using Empower 2 Chromatography Data Software. The eluent system consisted of 100% acetonitrile and 1% trifluoroacetic acid. The elution program was used as presented in Table 1
, with an injection volume of 10 µL, a mobile phase flow rate of 1 mL/min, a flow time of 81 min, and a column temperature of 25 °C. The active polyphenols in testing samples were identified, evaluating the retention time of the analytes and reference substances present, as well as the UV absorption from 300 to 360 nm. The reference compounds were salicin (R2
= 0.9999), p
-coumaric acid (R2
= 0.9999), caffeic acid (R2
= 0.9999), vanillic acid (R2
= 0.9999), cinnamic acid (R2
= 0.9999), ferulic acid (R2
= 0.9999), chlorogenic acid (R2
= 0.9999), apigenin (R2
= 0.9999), galangin (R2
= 0.9998), pinobanksin (R2
= 0.9999), and pinocembrin (R2
= 0.9998). The extracts were diluted 10 times with 70% ethanol (v
). The results are presented as the mean of three measurements, p
2.6. Ophthalmic Gel Formulation
Ophthalmic gels were formulated using different concentrations of polymers. Poloxamer 407 (Fagron, St. Paul, MN, USA), hydroxypropyl methylcellulose (HPMC) (Sigma-Aldrich, Steinheim, Germany), propane-1,2-diol (AppliChem GmbH, Darmstadt, Germany), purified water, and a 1% solution of lyophilized balsam poplar buds extract (L1) were used to form the experimental in situ gels. We chose to add 10% (w
) of 1% solution of balsam poplar extract (L1) to each formulation. Poloxamer 407 and HPMC gels were prepared separately. The appropriate amount of poloxamer was weighed (10%, 12%, or 15% (w
)) and mixed with the appropriate amount of purified water, and the mixtures left in a refrigerator (5 °C) for 24 h. The HPMC mixture was prepared by weighing an appropriate amount (0.5% or 0.75% (w
)) of polymer and adding an appropriate amount of water, with the mixture placed on a magnetic stirrer at 50 °C until a homogeneous gel form was obtained. In the preparation of the in situ gels, the poloxamer 407 and HPMC gels were mixed with a magnetic stirrer to form a homogeneous structure. Next, 10% (w
) propylene glycol and 10% (w
] L1 were added dropwise to the formulations, and the gels were mixed to form a homogeneous structure. All experimental formulations were stored in the refrigerator (5 °C).
2.7. In Vitro Release Test Determining Total Phenolic Compounds
Release of phenolic compounds from formulations was performed using Franz-type diffusion cells with natural cellulose dialysis membranes (Medicell International Ltd., London, UK). Phosphate-buffered saline of pH 7.4, which is most commonly used in ophthalmic release tests, was used as the acceptor medium, with a volume of 15 mL. During the release test, the temperature was maintained at 34 ± 0.5 °C, and the medium was stirred continuously [47
]. Samples of 1 mL of acceptor medium were taken every hour; the last samples were taken after 8 h. The total amount of phenolic compounds in the acceptor medium is expressed as the p
-coumaric acid equivalent/g of dry weight (mg CAE/g, DW).
2.8. Physical Characterization (pH, Viscosity)
The pH of the experimental formulations was evaluated at room temperature with a pH meter (766 with a Knick SE 104N electrode). The pH meter was calibrated with buffer solutions at pH 4.0–7.0. The viscosity of the gels was assessed with a vibrating viscometer (Vibro viscometer SV-10, A&D Company ltd, Tokyo, Japan) at 5 °C (immediately after removal from refrigerator), at room temperature, and at 37 °C (the experimental in situ gel samples were kept in a thermostat).
2.9. Rheological Tests
The rheological properties of the in situ formulations were evaluated with a rheometer (Physica MCR, Anton Paar GmbH, Austria) using a system of parallel steel plates and a standard-size concentric cylinder geometry, taking the formulation composition system into account. Storage modulus G′ and loss modulus G″ were measured using a system of parallel plates, the samples were carefully placed on the lower rheometer plate, and the measurements were carried out at a temperature from 20 °C to 50 °C, at an angular frequency omega of 1 rad/s, amplitude gamma of 0.5%, and temperature change rate of 2 °C per min. Flow properties were assessed using a concentric cylinder system, containing 10 g of the experimental formulation substance in a concentric cylinder; measurements were made at 22 °C, 32 °C, and 37 °C, with the shear rate from 1 to 200 1/s [36
]. Data were processed using RheoPlus software (Anton Paar GmbH, German). The analysis was performed at least three times for each composition.
2.10. Cell Viability: The Short Time Exposure (STE) Test
A rabbit corneal cell line (SIRC, American Type Culture Collection (ATCC)) was used for the study. The cell line was cultivated in the flask (75 cm2
) according to the protocol provided by the ATCC [50
]. Eagle’s Minimum Essential Medium (American Type Culture Collection (ATCC)) was used, with 10% fetal bovine serum (FBS) (v
), 1% penicillin/streptomycin solution, and 1% L
-glutamine (all reagents were purchased from Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Cells were grown in a thermostat at 37 °C with a CO2
level of 5%.
The STE assay was performed with an SIRC cell line based on the MTT method protocol, with some modifications [51
]. Cells from the SIRC culture line were seeded in 96-well plates (1 × 104 cells/well) and incubated in a 37 °C thermostat for 24 h. The experimental in situ formulations—aqueous balsam poplar buds extract (L2), and 1% lyophilized extract aqueous solution (L1)—were incubated on the cells for a short time: 5 min and 30 min, respectively. Empty gel G0 was used as a control. Extracts with a cell viability of 70% or less were considered to have an irritant effect, while those with a cell viability of 70% or more were considered to have a non-irritant effect.
2.11. Antioxidant Activity by ABTS and FRAP Methods
ABTS: The antiradical activity of the extracts was determined using the ABTS assay method, with certain modifications according to Yim et al.’s methodology [52
]. A stock solution of ABTS (0.0548g ABTS (Sigma-Aldrich, Oakville, ON, Canada), 50 mL purified water, and 0.0095g K2
(2 mmol/L) (Riedel–de Haën, Seelze, Germany) was prepared. The stock solution was kept in the dark for 16 h. The ABTS working solution was prepared by diluting a stock solution with purified water until the absorption at a wavelength of 734 nm reached 0.8 +/−0.03. 3 µL of balsam poplar buds extracts and propolis extracts were mixed with 3000 µL of ABTS working solution. All reaction mixtures were incubated at room temperature for 30 min. The absorbance of the reaction mixtures was measured spectrophotometrically at 734 nm.
FRAP: Reducing activity was assessed based on Raudonės et al.’s methodology, with some modifications [53
]. A working FRAP solution was prepared from 300 mmol/L sodium acetate buffer solution (0.775g CH3
COONa (Scharlau, Sentmenat, Spain); 4 mL glacial acetic acid, diluted to 250 mL with purified water), 10 mmol/L TPTZ solution (0.0781g TPTZ (Carl Roth, Karlsruhe, Germany); 40 mmol/L HCl-acidified purified water, in a 25 mL volumetric flask), and 20 mmol/L FeCl3
O aqueous solution (0.1352 g FeCl3
O (Vaseline-Fabrik Rhenania, Bonn, Germany), purified water in a 25 mL volumetric flask) with ratio of 10:1:1. Next, 10 µL of balsam poplar buds extract was mixed with 3000 µL of FRAP working solution. The samples were incubated at room temperature for 30 min in the dark. The absorbance of the reaction mixtures was measured spectrophotometrically at 593 nm.
Calibration curves were obtained from Trolox standard solutions of different concentrations. The results were expressed as the µmol Trolox equivalent per gram of tested raw material (µmol TE/g).
2.12. Statistical Analysis
Results are expressed as the mean and standard deviation of three measurements. For variables where normal conditions were not satisfied, a correlation was calculated based on Spearman’s correlation coefficient. Independent measurements were evaluated by a non-parametric Kruskal–Wallis test. Data were evaluated and plotted using IBM SPSS Statistics 27 (SPSS Inc., Chicago, IL, USA) and OriginPro®2021 (OriginLab, Northampton, MA, USA). Results were considered to be statistically significant at p < 0.05.