Biocompatibility of Membranes Based on a Mixture of Chitosan and Lythri herba Aqueous Extract

Abstract

In the current context of concern for the improvement and protection of environmental conditions, emphasis is placed on the provision of non-toxic, eco-friendly, renewable biomaterials to replace established chemical substances. Lythri herba is the aerial part of the plant species Lythrum salicaria L., known in the scientific literature especially for its content of tannins and total polyphenols, which highlight its antioxidant, hemostatic, antibacterial and antidiarrheal properties. Chitosan is a biopolymer widely used in industry and medicine due to its abundance in nature, its biodegradability, lack of toxicity and the ease with which it can be transformed into several basic forms (hydrogel, membrane, sponge). The aqueous solutions and membranes obtained in this study by merging these two natural resources were biologically tested in terms of genotoxicity (SOS-Chromo assay), hemolytic activity, thrombin generation activity and bacterial adhesion to reveal outwardly the lack of these properties and their use for medical purposes. The results of the current study attest to the absence of mutagenic and slight hemolyzing properties, thus supporting the possibility of using this extract and membrane in medical and pharmaceutical therapeutic practice. The surface parameters of membranes were examined and important influences at thrombin activity were found. Also, bacterial adhesion results showed a correlation between Lythri herba and chitosan concentrations and membranes’ appearances (swelling, stability). The results show that the membranes could be a promising material for biomedical applications.

1. Introduction

Biomaterials have gained an important role in medical fields, but biocompatibility testing of many materials remains challenging [1,2]. The assessment of biomaterials’ biocompatibility for various medical applications considers several factors, such as the physical characteristics and chemical nature of the materials used [1,3], the specific tissue with which they come into contact [4] and the contact duration [5].

Biopolymeric materials offer a wide spectrum of applications in the manufacture of medical devices [6]. They are used in both conventional and innovative drug delivery systems, and have superior adhesion, drug release and anti-inflammatory properties [7,8].

New biopolymer research trends focus mostly on the development of multifunctional [9] and intelligent films [10,11] with antibacterial, antioxidant and pH-responsive properties intended for the food packaging industry [12] and on advanced biomaterials for pharmaceutical or biomedical fields [9,13,14,15]. Consequently, emphasis is placed on the mass production of natural, non-toxic, biodegradable, eco-friendly, easy to procure and relatively cheap materials, such as gelatin [16,17], chitosan [18,19,20], chitin [21,22], collagen [23,24] and cellulose [25,26], in order to achieve diverse biomedical, environmental and food applications.

One such material, chitosan, is a cationic biopolymer indispensable in its use as a carrier of therapeutic substances [27,28] due to its non-toxic, biodegradable, biocompatible, antibacterial and antioxidant nature [29].

Chitosan, a biopolymeric macromolecule, possesses a multitude of technical and biological properties that have gained significant attention in diverse global research, in particular concerning biomedical healing applications that require anti-microbial and anti-inflammatory capacities [30,31,32,33,34,35,36]. At the same time, attractive aspects were analyzed for various fields in the food, cosmetic, medical and pharmaceutical industries, such as the abundant availability and low cost of chitosan, which falls within the general desideratum of obtaining materials through renewable green resources [37,38].

In addition, an important property of chitosan is its mucoadhesiveness as a result of its ability to interact with negatively charged mucins through electrostatic attraction [39], since this biopolymer is the only positively charged polysaccharide in the world due to the presence of amino groups in its chemical structure [38]. Moreover, chitosan is known as a hemostatic agent in the pharmaceutical industry for the formulation of wound dressings due to its affinity for red blood cells and its ability to induce clotting.

Although chitosan is widely used in the fields of biotechnology, wastewater treatment, agriculture, the food industry, cosmetics, medicine and pharmacy for its unique properties, it has some limitations in the biomedical field caused by its poor solubility and inferior mechanical properties [40,41]. The solubility issue can be improved through structural modifications, thereby creating novel derivatives with superior physical properties, broadening its application range [41].

Some of the most recent studies evaluate chitosan-based membranes in which plant extracts [42] or their secondary metabolites [43,44] have been incorporated, precisely due to the superior ability of chitosan to form membranes [45,46,47]. An underestimated traditional medicinal plant rich in bioactive compounds (polyphenols, tannins) is the Lythrum salicaria L. species.

Lythrum salicaria L. is a widely known plant for its beneficial astringent and hemostatic properties, being used in cases of diarrhea, hemorrhoidal diseases and hemorrhages [37]. Previous research conducted on Lythri herba plant material has demonstrated various beneficial properties. These include antioxidant, anti-inflammatory, anti-nociceptive and hemostatic activity [48,49]. Additionally, in vitro experiments have revealed antibacterial and fungistatic activities [50,51,52]. Furthermore, animal models have highlighted that Lythri herba exhibits hypoglycemic effects [53] and modulatory effects on the intestinal microbiota’s composition through its postbiotic metabolites, known as urolithins [54].

Additionally, our previous study on Lythrum salicaria L. confirmed the existence of polyphenols (16.39%), tannins (10.53%) and anthocyanosides (0.3598%) and underlined the very low cytotoxic activity of the aqueous extract using Artemia salina as a biotester [55].

The strategy of this study was to blend, for the first time, the chitosan biopolymer and Lythrum salicaria L. aqueous extract to obtain a new membrane combination allowing the correlated use of both the properties of the plant extract and the multifunctionality of the polymer. This study aims to enhance the characteristics of chitosan by incorporating a natural extract, resulting in the development of a stable membrane form. This membrane form has the potential to be utilized as a biomaterial for surface or implant devices, benefiting from the combined effects of both components. The obtained membranes were qualitatively assessed for surface characteristics, swelling, stability and biocompatibility. In this study, our objective was to assess the suitability of the extract and chitosan as potential initial components for the formulation of biomedical devices by analyzing the necessary endpoint components for biological safety evaluation.

2. Materials and Methods

2.1. Plant Material and Chemicals

The plant materials used in this stage were represented by the dried floral tips of the species Lythrum salicaria L. collected in August 2019 and 2021 from Constanta, Romania. A voucher specimen of L. salicaria L. plant was registered at the Pharmacognosy Department of the Pharmacy Faculty, Ovidius University of Constanța, under an individual number.

The aqueous extract, obtained by refluxing for 2 h and filtering with millipore filter paper, was concentrated using a Buchi R-215 rotavapor and dried via cryo-drying using an Christ Alpha 1–2 B lyophilizer. At the end of the lyophilization process, approximately 12 g of freeze-dried Lythri herba aqueous extract (Ly) was obtained, which was transferred to a glass bottle with a tight stopper and stored in a desiccator. To prepare the concentrations of herbal aqueous extract solutions that would be incorporated into the studied membranes, a stock solution of 10 g/L aqueous extract was prepared.

The chitosan powder (CS) with medium molar mass and a deacetylation degree (DDA) ≥ 75% (product number 448877) and glacial acetic acid ReagentPlus^®, ≥99% (product number A6283), were purchased from Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.

2.2. Plant–Chitosan Membranes

Development of membranes based on chitosan (1% and 0.5%) with aqueous extracts of Lythri herba (0.5, 1 and 2 g/L) was carried out in the first step by mixing the two types of solutions in a volumetric ratio of 1:1 (v/v) under medium magnetic stirring, on the heating plate at 50 °C for 3 h, according to an improved and adapted study by Ahmadi et al. (2022) [56]. During the second step, the mixtures were poured into PTFE dishes, and the solvent was evaporated at 40 °C temperature for 6 h in a drying oven. Similarly, membranes based on 0.5% chitosan with aqueous herbal extract concentration levels of 0.5, 1 and 2 g/L were prepared. The colors of the obtained membranes ranged from light brownish to a darker shade of brown, depending on the concentration of the aqueous plant extract (the higher the concentration, the darker the color). The 1% and 0.5% CS membranes chosen as a reference were obtained by dissolving the chitosan powder in a dilute solution of acetic acid. Diluted acetic acid (1%) solutions were used to prepare the membranes, according to a slightly improved method by Cui et al. (2018) [57]. The 1% chitosan solution in 1% acetic acid was poured into PTFE dishes and left in a drying oven at 40 °C for 6 h.

2.3. Membrane Evaluation

The membranes were qualitatively evaluated according to the degree of swelling (%), FT-IR spectroscopy analysis and surface microscopic characterization.

2.3.1. Membranes’ Swelling Percentage (%)

To determine the degree of swelling (%) of the previously obtained membranes, the working method of Al-Dhubiab et al. (2016) [58] was adapted, by which the hydration properties of the membranes were measured. The surfaces of 1 cm² with an initial weight (m_i) were cut from the membranes, immersed in phosphate-buffered solution (PBS) and kept in an incubator at 37 °C for 10, 20 and 30 min. After each time interval, the membranes were removed from the solution, lightly dried and weighed again (m_f). The swelling percentage of the membranes was determined using the equation of Nair et al. (2013) [59]:

$$\mathrm{Swelling} (\%)=\frac{m_f-m_i}{m_f}·100$$

(1)

where m_i is the initial weight of the membrane, and m_f is the final weight of the membrane after immersion in PBS.

2.3.2. Microscopic Evaluation of Membranes’ Surfaces

In order to carry out this evaluation, fragments measuring a few millimeters from each membrane were sectioned, making preparations, that were subsequently observed and used for a topographic analysis of the membranes’ surfaces. The microscopic images were achieved using a VWR microscope VisiScope 300D (VWR International, Radnor, PA, USA) and processed with VisiCam Image Analyzer 2.13. All observations were carried-out in triplicate. Fluorescent images (FM) were obtained using an OPTIKA B-350 microscope (Ponteranica, Italy) with a blue filter (λ_ex = 450–490 nm; λ_em = 515–520 nm) and green filter (λ_ex = 510–550 nm; λ_em = 590 nm). The FM images were processed with Optikam Pro 3 Software (OPTIKA S.R.L., Ponteranica, Italy).

2.3.3. FT-IR Spectroscopy Analysis of CS-Ly-Based Membranes

The Fourier-transform infrared (FT-IR) spectroscopy analysis of chitosan (CS) membranes with Lythri herba aqueous extracts (Ly) was carried out with a Perkin-Elmer Spectrum BX FT-IR spectrophotometer equipped with ATR. IR spectra of solid samples (membranes) were recorded in the 4000 cm⁻¹ and 900 cm⁻¹ ranges, with a resolution of 8 cm⁻¹ and accumulation of 64 scans, at room temperature and low humidity.

2.4. Biological Analysis

2.4.1. The Genotoxic Assay of the Aqueous Solution of Chitosan and Lythri herba Extract

To evaluate the genotoxicity, we used an SOS-Chromo assay kit developed by EBPI (Environmental Bio-Detection Products Inc., Mississauga, ON, Canada), solutions of chitosan, Lythri herba plant extract (2 g/L, 1 g/L) and combinations of chitosan and extract of differing concentrations. It should be mentioned that only the high concentrations of the test solutions where an effect was assumed were used. The bacterium used in the SOS-Chromo test was a PQ37 mutant strain of Escherichia coli developed as a genotoxicity test indicator organism and included a gene encoding the β-galactosidase enzyme linked to an SOS promoter. When DNA damage occurred, the SOS system was activated, and β-galactosidase was transcribed proportionally to the level of SOS induction. The production of β-galactosidase was measured through its reaction with a blue chromogen as highlighted in the work protocol (SOS-ChromoTestTM kit, for rapid detection of genotoxicity or DNA damage, Product Number 5031).

EBPI uses traditional metabolic bioactivation methods for indirect genotoxins by using S9 liver homogenate from Sprague Dawley rats induced by Aroclor 1254 in the presence of the necessary cofactors. Many carcinogens are known to require metabolic conversion to a reactive metabolite before interacting with DNA. Therefore, both direct and indirect genotoxins can be detected if S9 is included in the assay design.

To carry out the quantitative evaluation, the following calculation formulas were used:

• growth factor for bacteria by measuring alkaline phosphatase (G),

$$\mathrm{G}=\frac{A_{420S}-A_{420B}}{A_{420N}-A_{420B}}$$

(2)

A_420S = sample absorbance, A_420B = blank absorbance and A_420N = negative control absorbance read at 420 nm.

• β-galactosidase activity (β-gal),

$$\mathit{\beta -gal}=\frac{A_{600S}-A_{600B}}{A_{600N }-A_{600B}}$$

(3)

A_600S = sample absorbance, A_600B = blank absorbance and A_600N = negative control absorbance read at 600 nm.

• Induction factor (IF),

$$\mathrm{IF}=\frac{\mathit{\beta -gal}}{G}$$

(4)

β-gal = β-galactosidase activity, G = growth factor for bacteria by measuring alkaline phosphatase.

• Survival rate (%),

$$\mathrm{Survival} \mathrm{rate} (\%)=\frac{{OD}_{420 sample}}{{OD}_{420 negative control}}·100$$

(5)

OD_{420, sample} = sample absorbance, OD_{420, control negative} = negative control absorbance, read at 420 nm.

The effect of Lythri herba (Ly) concentrations tested between 19.23 µg/mL and 153.85 µg/mL were compared with those of 1% CS solution, as well as with genotoxic solutions of 2-AA (2-aminoanthracene) and 4-NQO (4-hydroxyquinoline 1-oxide). This last two compounds were recognized and validated as positive controls, mutagens (according to the kit procedure).

The microplate with the samples to be analyzed was kept in the incubator at 37 °C for 2 h, during which the potentially genotoxic solutions interacted with the DNA of the SOS-Chromo assay bacteria and induced the de novo synthesis of β-galactosidase. In the last step of the SOS-Chromo test, the relative amount of enzyme produced because of the induction of the SOS system is measured by adding a blue chromogenic substrate. The blue chromogen produces a blue color that allows for quantitative evaluation (spectrophotometry). Since the success of the β-galactosidase assay and production depend on bacterial viability during the assay, cell survival (ATP activity) was assessed using alkaline phosphatase. In this way, both the acute bacterial toxicity and the genotoxicity of the samples could be measured simultaneously. Absorbances at 600 nm to determine β-galactosidase production (genotoxicity) and at 420 nm to determine bacterial viability were read immediately.

2.4.2. Hemolytic Activity

The hemolytic activity was evaluated with a Biomaterial Hemolytic Assay kit developed by HaemoScan (Company with Quality Management System Certified by DNV GL, ISO 9001). The absorbance (415 nm/450 nm/380 nm) of the samples was read at 1 h and 2 h intervals. To calculate the absorbance (OD), the Harboe method (6) was used:

$$\mathrm{OD}=\left(2\times A_{415\mathrm{n}\mathrm{m}})-(A_{450\mathrm{n}\mathrm{m}}+A_{380\mathrm{n}\mathrm{m}} \right)$$

(6)

A_{415 nm} = sample absorbances at 415 nm, A_{450 nm} = sample absorbances at 450 nm, A_{380 nm} = sample absorbances at 380 nm.

A correction was made against the negative control using the following formula:

A_sample − A_{negative control}

(7)

A_sample = absorbance of the test sample, A_{negative control} = negative control absorbance.

The previously obtained values represent hemoglobin in mg/0.5 cm² (area of the membranes tested), which were then reported for an area of 1 cm². Then, the hemoglobin percentage (%/cm²) was calculated considering the total hemoglobin value of 100% (9).

$$\mathrm{Ht} (\%)=1.2505\times \mathrm{10,000}=\mathrm{12,505}$$

(8)

$${\mathrm{H}}_{\mathrm{sample}} (\%)=\frac{V_{sample}}{\mathrm{12,505}}·100$$

(9)

V_sample = value of the tested sample.

2.4.3. Thrombin Activity

The examination relied on the methodology of determining the thrombogenicity induced by the exposure of biomaterials to plasma [60,61]. A HaemoScan kit, Thrombin Generation Assay, is a suitable method for evaluating the hemocompatibility of biomaterials and medical devices in accordance with the international standard ISO 10993-4:2002. Only the samples containing CS 1% and extract were tested. Medical steel (0.6 cm²) was used as a reference material. All the parts of the membranes tested and analyzed had dimensions of 0.5–0.6 cm². All measurements were performed in duplicate. The results obtained were related to the surfaces of the parts analyzed and to the exposure time (ΔmU/mL/min/cm²).

2.4.4. Bacterial Adhesion Inhibitions Assay of CS-Ly-Based Membranes

Sample parts of 5–6 mm were washed in 70% alcohol at 37 °C for 30 min. The tested strains were Staphyloccocus aureus ATCC 23235 (Gram-positive), Enterococcus faecalis ATCC 29212 (Gram-positive) and Klebsiella pneumoniae ATCC 13883 (Gram-negative). Bacterial suspensions of 1 × 10⁷ CFU/mL with Mueller–Hinton Broth, according to the 0.5 McFarland standard, measured with a densitometer (Biomerieux, Marcy-l’Étoile, France), were prepared. The dilution was made with physiological serum. The membranes were placed in the sterile cell culture plates (24 wells). Bacterial suspensions (1 mL, 10 % Mueller–Hinton Broth) were added and incubated for 24 h in static conditions at 37 °C in a My Temp mini Z763322 digital incubator (Benchmark Scientific Inc., Sayreville, NJ, USA). After incubation, the membranes were washed with distilled water 3 times to remove the suspended bacteria, which did not attach to the biofilm. For control, standardized 0.5 mm type samples, such as silicone elastomer, medical steel and chitosan membranes without the plant extract, were used. At the end, the samples (pieces of membranes) were placed on 1 mL buffer solution (PBS) and inserted for 5 min in sterile Eppendorf tubes in a MiniLabRoller Agitator to dislodge the bacteria adhering to the substrate. Then, a series of 5 dilutions were made (1:2, 1:4, 1:8, 1:16, 1:100), and from each microvolume (5 µL) was placed on the TSA culture medium, in spots.

The plates with TSA were incubated for 12 h at 37 °C. The number of colonies/spot and CFU were reported to the surface of the samples (mm²) and then compared with the control samples. All successive steps were performed in a microbiological protection niche with laminar flow (Asal Srl, Cernusco (MI) Italy model Aslair Vertical 700). The membranes were also analyzed under a microscope to confirm the previous observations. To highlight the bacteria, staining with 0.01 % acridine orange (AO) was performed for 2–4 min. The membranes were analyzed under a white light and epifluorescence microscope. Fluorescent images (FM) were processed with Optikam Pro 3 Software (OPTIKA S.R.L., Ponteranica, Italy).

2.5. Statistical Analysis

The presented data correspond to the mean ± standard deviation of three experimental values. The statistical differences between data were evaluated through one-way analysis of variance (ANOVA), and p < 0.05 was statistically significant (StatPlus:mac, AnalystSoft Inc. (Alexandria, VA, USA)—statistical analysis program for macOS, Version v8. See https://www.analystsoft.com/en/, accessed on 11 August 2022).

3. Results

3.1. Plant–Chitosan Membranes

The obtained membranes were thin, transparent, malleable and elastic, similar to a plastic material and with visible color differences when the plant extract (Ly) was added (Figure 1).

3.2. Membrane Evaluation

3.2.1. Membrane Percentage of Swelling (%)

When membranes come into contact with an alkaline buffer solution, they suffer a loss of rigidity and become more flexible, making them easier to fold. Furthermore, they do not undergo distortion, and upon drying, they regain their original characteristics. Following the first 10 min of immersion in PBS solution, membranes combined with 0.5 g/L and 1 g/L aqueous extracts of Lythri herba (Figure 2A) reached approximately 60% degree of swelling (Figure 2A) and up to 80%, respectively (Figure 2B), and afterwards the uptake level stabilized in the next 20–30 min. At CS 1, without extract, the membranes had a swelling rate close to that of membranes with Ly, in the first 10 min. Following 30 min of immersion in PBS solution, the process changed, with a decrease in the swelling percentage, possibly induced by the hydrophobicity of some poly-GlcNAc groups [33]. At CS 0.5%, membranes’ swelling percentage varied from 120% to 140% (Figure 2B). The swelling measurements of CS 0.5–Ly were around 80% for all concentrations, and remained stable after the first 10 min (Figure 2B). This study suggested the significance of chitosan concentrations employed and the extract role in maintaining the stability of the chitosan matrix. Another significant aspect related to the preservation of the original color and the absence of increased deformity, indicating that the plant extract engaged in chemical bonding with the chitosan matrix, thereby providing notable stability.

In other studies in which chitosan is associated with plant extracts, this swelling process is correlated with the replacement of the extract in the spaces created by the chitosan matrix. The extract leaves the gaps in the polymer matrix passing into the aqueous solutions, consequently leading to an escalation in the rate of swelling [13,14].

3.2.2. Microscopic Evaluation of Membranes Surfaces

The surface analysis of membranes (Figure 3) revealed similarities between the studied materials. The micro-elevations and waves were remarkable among all topographies. Epifluorescence microscopy clearly highlights details of the multi-lamellar arrangement in parallel layers and the oblique arrangement of filaments specific to chitosan structures (Figure 3e–h). The microstructure of the membrane shows a uniform appearance with no porosity (Figure 3a–h).

The general uniformity of the chitosan and extract membranes suggests polymer rearrangement. This detail is important in correlation with other processes such as bacterial adhesion and cellular interactions. CS–Ly mixed membranes could be an alternative to chemical modification with the aim of modulating the natural polymer properties, which is essential for contact applications and frequently used in the last few years [62].

3.2.3. FT-IR Spectroscopy Analysis of CS-Ly-Based Membranes

FT-IR analysis was carried out to determine the main functional groups of chitosan (CS) and of the Lythri herba lyophilized aqueous extract (Ly), as well as the interaction between certain groups of these two components of the CS–Ly mixed membranes.

FT-IR transmittance spectra were recorded and analyzed for the membranes obtained from mixing the solution of CS in diluted acetic acid solution (1%) with solutions of aqueous extract of Lythi herba, so that, in the end, the analyzed samples had the following final composition: CS 1% with 0.5 g/L, 1 g/L and 2 g/L Lythri herba, respectively (Figure 4). As a reference, a simple CS membrane in 1% acetic acid was chosen. Samples from all membranes were cut to small sizes and placed in the appropriate crystal holder of a Perkin-Elmer Spectrum BX spectrophotometer.

As shown in Figure 4, the CS–Ly membranes showed absorption bands common to those of the simple CS membrane at wavenumbers 3736 cm⁻¹, 2918 cm⁻¹, 2849 cm⁻¹ and 2358 cm⁻¹. Based on the literature data, the results can be interpreted as follows: the common transmittance band of all four samples at 3736 cm⁻¹ can be attributed to the O–H stretching vibrations of the -CH₂-OH group or the glycosidic ring involved in intramolecular hydrogen bonds [63] that overlap with the N-H stretching vibrations of the primary amines in the samples subjected to infrared (IR) analysis. The corresponding common peaks at 2918 cm⁻¹ and 2849 cm⁻¹ are characteristic of the C-H vibrations of the methine groups in the pyranose nuclei of CS, CS–Ly membranes and polyholozides from the lyophilized extract. The common peak recorded at 1650 cm⁻¹ is attributed to the ν_(C=O) stretching vibrations of the C=O from the NH–CO–CH₃ group (amide I band) of CS and its mixtures [64], and the one at 1398 cm⁻¹ corresponds to the C-N (amide III) stretching vibration. The C³–OH and C⁶–OH groups present in the CS structure were characterized by the peaks recorded at 1044 cm⁻¹ and 900 cm⁻¹ [65], but after the formation of the membrane by mixing with lyophilized extract, these peaks were not found in the spectra of CS–Ly membranes (Figure 4). Additionally, the spectra of CS–Ly membranes exhibited two peaks of low intensity at 1536 cm⁻¹ and 1618 cm⁻¹, which can be attributed to aromatic rings. These peaks are suggestive of the presence of polyphenolic acids, phenolic acids or tannins, which are specific to the chemical composition of the lyophilized aqueous extract (Ly).

3.3. Biological Analysis

3.3.1. The Genotoxic Assay of the Aqueous Solution of Chitosan and Lythri herba Extract

The genotoxicity of the aqueous extract and of the mixtures of chitosan and aqueous extract was detected via SOS-Chromo test. The test consists of colorimetric assays of enzymatic activities (β-galactosidase and alkaline phosphatase) after incubating the tester strain in the presence of various amounts of compound [66]. All samples showed a non-genotoxic effect compared with 4-NQO (4-hydroxyquinoline 1-oxide) and 2-AA (2-aminoanthracene), which were used as positive controls (

Table 1. Results of SOS-Chromo assay samples (mean ± SE, n = 3). 4-NQO (4-hydroxyquinoline 1-oxide) and 2-AA (2-aminoanthracene) were used at positive control (C+).

Samples	Biological Effect	Induction Factor (IF) *
		without microsomal activation (-S9)	microsomal activation (+S9)
Negative control	Non effect	-	-
C+ (4-NQO)	High genotoxic	>2	>2
C+ (2-AA)	Moderate genotoxic	1.5–2	1.5–2
CS 1	Non-genotoxic	<0.01	<0.1
CS 0.5	Non-genotoxic	<0.01	<0.1
Ly 1	Non-genotoxic	0.78 ± 0.0005	0.42 ± 0.001
Ly 2	Non-genotoxic	0.26 ± 0.0001	0.21 ± 0.001
CS–Ly 2	Non-genotoxic	0.46 ± 0.0030	0.04 ± 0.001
CS–Ly 1	Non-genotoxic	0.36 ± 0.0047	0.04 ± 0.01

* Evaluating criteria: IF < 1.5: non-genotoxic; 1.5 < IF > 2: moderate genotoxic; IF > 2: high genotoxic; the mean ± SE.

).

Additionally, the potential indirect mutagenic effect was evaluated through metabolic products, where all the samples analyzed with the microsomal enzyme activator (+S9) showed a lack of genotoxicity due to the values of the induction factor (IF) being lower than 1.5 (Table 1). A significant decrease in genotoxicity was observed especially in combination with CS–Ly with microsomal activation (+S9) compared with the same non-activated combinations (-S9). In the presence of microsomal enzymes, mixtures of CS–Ly were ten times less genotoxic. Consequently, indirect mutagenesis was non-existent, and this will allow safe applications without systemic effects.

The results of testing both genotoxic activity (with and without microsomal activation) evaluated from a quantitative point of view (IF) were adapted after our previous work, Iancu et al. [67].

3.3.2. Hemolysis Activity

Blood compatibility is a gold standard for all new materials developed specifically for biomedical applications. Hemolytic activity is a requirement to be tested for any medical device that encounters blood and is calculated as the percentage of red blood cell lysis produced as a result of the interaction with the biomaterial.

The hemolysis effects were between 0.2 and 0.4%/cm². Also, no significant differences were recorded between 1 h and 2 h. It was noted that with hemolysis, the amount of Ht (%) increased when the CS concentration was smaller (0.5%) in the mixture’s biofilm (Figure 5) as a result of the weakening of the bonds between chitosan and the plant extract compounds. The biofilm formed by the CS–Ly mixture allowed the amine groups to bind to the polyphenols in the extract [38]. Chitosan interacts not only with red blood cells, but also with platelets through amine groups that are attracted to the negative charge of blood elements [32]. In the case of the analyzed membranes, the hemolysis effect was low and perhaps determined by the groups left free after the interaction with the extract.

The percentage values of the hemolysis from the tested samples were graphically interpreted according to Figure 5.

3.3.3. Thrombin Activity

Thrombin, an essential enzyme within the coagulation cascade, provides valuable insights into the clotting potential of a biomaterial when measured. The assessment of novel biomaterials commonly includes the measurement of thrombin activity as a standardized parameter. Our results showed a significant activity of all samples compared with the reference control after 3 and 4 min of membrane exposure. The highest thrombin synthesis was noted at the mixed CS–Ly extract membranes, while the chitosan membrane had a thrombin activity comparable with that of medical steel, a highly reactive reference material (Figure 6).

The results show that the plant extract added to the membranes significantly amplified the thrombin effect of the membranes, suggesting the synergistic value of the extract.

3.3.4. Bacterial Adhesion Inhibitions Assay of CS–Ly-Based Membranes

The adhesion of Staphylococcus aureus, Enterococcus faecalis and Klebsiella pneumoniae bacteria on the tested membranes was compared with the materials used as standards (medical steel and silicone elastomer) (Figure 7).

These results show that all mixed CS–Ly membranes showed higher adhesiveness compared with the chitosan membranes (CS 1 and CS 0.5) without the Lythri herba extract. The chitosan membranes recorded several CFU/mm² colonies of approximately 4 × 10³, a value very close to that of the standard materials tested (medical steel, silicone elastomer).

The inhibition of the adhesiveness of the microorganisms on the membranes with an Ly content was not noticed. Correlations between the different concentrations of the extract and the recorded effects were also not observed. For the CS 1–Ly membranes, the adhesiveness of the K. pneumoniae bacterial species was two times lower compared with the other bacteria, which suggests the importance of chitosan concentration in fixing these Gram-negative bacteria. In the case of Gram-positive bacteria, a lower attachment of S. aureus species compared with E. faecalis could be noted on the surface of CS 0.5–Ly membranes. Therefore, the observations indicate that the adhesiveness of the microorganisms under investigation was influenced by two key factors: the concentration of the polymer and the specific type of bacteria. The presence of the plant extract did not align with the initial assumptions regarding its potential to reduce bacterial adhesiveness. The enhancement of the biopolymer-induced phenomenon was not found in the presence of the extract.

4. Discussion

The therapeutic plant extract L. salicaria L. is known in the specialized literature for its high quantity of tannins [37], and the formulation of chitosan membranes is the first attempt to capitalize on these plant metabolites with as diverse applications as possible, such as obtaining mucoadhesive patches [45] or dressings with a fast local effect in a general context for the reduction of synthetic materials and the development of eco-friendly technologies.

The use of water as the main solvent for the extraction of active compounds from the plant L. salicaria L. followed by the choice of a natural, biodegradable and non-toxic material as a support in the development of a pharmaceutical or medical application were decisions made in accordance with the “green technology” principle to protect the environment by limiting its contamination with aggressive solvents or toxic materials [68].

The accumulation of the extract within the chitosan membranes can be attributed to the chemical interactions that occur between the biopolymer matrix and the components of the extract. These interactions involved the amino groups from chitosan interacting with tannic acids as well as the OH groups of C₆ from the glycosidic ring of chitosan interacting with tannic acids. The formation of a homogeneous film indicates that the ingredients used were properly incorporated into the polymer matrix. The firm and flexible appearance is suitable for formulations with medical applications [69,70,71], ensuring better ease of administration [72].

Previous studies in the current collection of literature have focused on the process of functionalizing chitosan with polyphenols, which are derived from plants as secondary metabolites. This particular research direction has been pursued due to the observed enhancement of antioxidant activity associated with these polyphenols [73]. Among the various options available, tannic acids (TA) emerge as the most suitable choice due to their notable antioxidant properties and excellent water solubility [74,75]. Furthermore, the coalescence of chitosan and TA has been observed to enhance the bioavailability of TA while also providing protection against their degradation [76]. In relation to the hydration level of the membranes, it was observed that the use of the aqueous freeze-dried extract of Lythri herba resulted in membranes that exhibited enhanced stability compared with the 1% and 0.5% chitosan membranes. Furthermore, these Lythri herba-treated membranes did not undergo deformation upon exposure to an alkaline solution. Instead, they displayed desirable properties of flexibility and elasticity, making them suitable for applications in mucoadhesive systems. Due to the presence of hydroxyl and amino groups in its chemical structure, CS chitosan is easily hydrated in water, because water molecules interact strongly with these hydrophilic groups, leading to the weakening of intermolecular hydrogen bonds [77].

Several particular investigations have been conducted on membranes and biofilms made from chitosan, and these studies consistently found that these materials have low water resistance [78,79]. This aspect was observed at the 0.5% CS membrane tested, whose fragility was the highest. Ferreira et al. (2022) drew attention to a phenomenon analogous to that observed in the present study. Specifically, they found that membranes containing chitosan as a constituent, along with green banana peel extract, exhibited a lower degree of swelling in both water and phosphate-buffered saline (PBS) solutions compared with membranes composed solely of chitosan. This finding suggests that the robust interactions between chitosan and the extract compounds result in a reduced ability to absorb water [80]. The water content has a considerable influence on the bioadhesive behavior of the membranes, which is modified by disrupting the polymer bonds with the active ingredients [81].

The utilization of extracts derived from medicinal plants has experienced a notable rise in recent decades. It has been observed that several of these plants harbor chemical compounds that are recognized for their mutagenic and/or carcinogenic properties [82,83]. In our study, it was observed that neither the aqueous extract of Lythri herba nor the combined solutions of chitosan and extract exhibited any mutagenic processes.

One may underscore the observation that the non-activated and metabolically activated samples exhibited the most elevated induction factor (IF) values at a concentration of 1 g/L of Lythri herba extract. Specifically, values of 0.78 (-S9) and 0.46 (+S9) were recorded. However, it is noteworthy that these values fall below the threshold of 1.5, thereby indicating that the Lythri herba extract, sourced from Romania, lacks genotoxic properties at the aforementioned concentration.

The plant species studied in vivo has a very low cytotoxic action [55] and a great potential for exploitation. More investigations are needed to decide whether this specific extract or extracts from other medicinal plants can be used without hazards to human health [84,85].

The hemocompatibility of the new blended membranes (chitosan and Lythri herba aqueous extract) was evaluated using a hemolytic assay, which is a common method for evaluating the blood compatibility of materials [86].

The membranes obtained from the combination of the biopolymer chitosan (CS) with different concentrations of Lythri herba aqueous extract had the weakest hemolyzing effect, comparable with the standard material (medical steel). A possible explanation would be that the presence of the extract induces chemical bonds with the chitosan biopolymer, thus blocking the ability of these positively charged groups to interact with the negatively charged erythrocyte membranes. This evaluation highlighted hemolysis ratio values of all tested samples that were lower than the 5% recommended by the general standard for hemocompatibility materials. According to the ASTM standard F756, a hemoglobin release of 0–2%, 2–5% or >5% of the total release is classified as non-hemolytic, slightly hemolytic or hemolytic, respectively [87]. Chitosan, in blood contact processes, involves erythrocyte aggregation, platelet activation, the contact activation pathway and the formation of the space net structure. Also, the hemostatic effect of chitosan is influenced by factors such as the quantity of protonated amine groups, molecular weight and degree of deacetylation [88].

A slow hemolysis effect was recorded after 1 h and 2 h between the membranes and blood contact; the effect was similar to other chitosan derivates [89] and chitosan oligosaccharides tested [90]. Berteanu et al. (2016) compared the hemolytic activity of the variants of membranes based on collagen, chitosan and alkaline phosphatase cross-linked with glutaraldehyde and concluded that simple collagen membranes do not show hemolytic activity, but chitosan ones are slightly hemolyzing. When collagen and alkaline phosphatase are added to chitosan membranes, there is a significant decrease in the hemolyzing property, which suggests that the combination of chitosan with other non-interacting red cells’ biomaterials reduces the hemolytic effect [91].

An additional significant aspect to consider in the evaluation of biocompatibility for biomaterials [92,93] pertains to their capacity to engage with specific blood constituents, thereby initiating a series of reactions that promote the process of coagulation. Surfaces that come in contact with plasma have been found to play a significant role in accelerating the activation of FXII through the prekallikrein and high molecular weight kininogen systems [93]. The existence of a significant activity of thrombin generation in the studied CS–Ly membranes compared with medical steel is a result that confirms the information from the local traditional practice of using the plant as an anticoagulant. The presence of a prolonged effect of thrombin synthesis in membranes with a higher Ly concentration can be correlated with the level of bioactive compounds of the extract included in CS–Ly bonds. However, the prothrombinic effect requires additional evaluations to identify the action mechanisms of the extract on the coagulation cascade. A series of studies revealed the importance of knowing thrombin activity both in pathologies and in the use of plant extracts [94].

Chitosan is a biopolymer known for its hemostatic properties, which can be attributed to its ability to create artificial clots when it comes into contact with blood. Chitosan induces platelet adhesion, aggregation and activates intrinsic blood coagulation [95]. The hemostatic mechanism of chitosan involves the agglutination of erythrocytes of the classical coagulation system; in other words, the positively charged structure of chitosan attracts the negatively charged red cells, leading to the appearance of the phenomenon of agglutination and, therefore, coagulation [96].

The ability of bacteria to develop biofilm on various surfaces is a crucial characteristic associated with pathogenicity, particularly in the context of biomaterial manufacturing technologies. The Ly extract composition comprises compounds that engage in interactions with the chitosan polymer, resulting in alterations to the surface of the membranes in conjunction with variations in chitosan concentration. In contrast to findings from previous studies, the results indicate a reduced level of bacterial adhesion on the CS-Ly membranes examined, in comparison with other materials commonly employed in the production of dental implants, after a 24 h period [97]. These topographic changes of the membrane surfaces also change the contact with the bacteria and make the adhesion different [98].

The results obtained are encouraging and suggest that adjusting the concentrations of the two components in the mixture may serve as a modulating factor for the effects of bacterial anti-adhesiveness and biocompatibility, as well as correlation with the studies about standardization of the extract [99]. Nevertheless, the subsequent studies endeavor to consolidate the information pertaining to prolonging the duration of exposure beyond 24 h while also diversifying the control experiments.

5. Conclusions

The CS–Ly membranes were produced by combining chitosan with an aqueous extract of Lythri herba. This resulted in enhanced properties and qualities of the biopolymer while also combining the synergistic activities of both compounds.

The examination of these membranes using FT-IR spectroscopy analysis demonstrated that there is a stable bond between the components of the plant extract and the biopolymer. Moreover, these links elucidate specific characteristics that, from the perspective of the biocompatibility assessment, offer initial insights into the feasibility of employing the membranes in biomedical applications with minimal risk upon contact. The absence of hemolytic activity and the enhanced capacity for thrombin generation exhibited by the membranes in comparison with a conventional medical material (medical steel) are encouraging findings. In future studies, an increase in the concentration of the Ly extract is considered and an observed effect induced by the release of the important chemical compounds of the extract (tannins) is necessary, favoring both anti-adherence and bactericidal activity.

In conclusion, these new membranes represent the first stage in the development of a natural material with pharmaceutical and biomedical field applications.