Formulation and Characterization of Nanoemulsion Incorporating Chamomilla recutita L. Extract Stabilized with Hyaluronic Acid

Skin lesions are an important health concern, exposing the body to infection risks. Utilizing natural products containing chamomile (Chamomilla recutita L.) holds promise for curative purposes. Additionally, hyaluronic acid (HA), an active ingredient known for its tissue regeneration capacity, can expedite healing. In this study, we prepared and characterized an extract of C. recutita and integrated it into a nanoemulsion system stabilized with HA, aiming at harnessing its healing potential. We assessed the impact of alcoholic strength on flavonoid extraction and chemically characterized the extract using UHPLC/MS while quantifying its antioxidant and antimicrobial capacity. We developed a nanoemulsion loaded with C. recutita extract and evaluated the effect of HA stabilization on pH, droplet size, polydispersity index (PDI), zeta potential, and viscosity. Results indicated that 70% hydroalcoholic extraction yielded a higher flavonoid content. The extract exhibited antioxidant capacity in vitro, a desirable trait for skin regeneration, and demonstrated efficacy against key microbial strains (Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, and Pseudomonas aeruginosa) associated with skin colonization and infections. Flavonoids spireoside and apiin emerged as the most abundant bioactives. The addition of HA led to increased viscosity while maintaining a suitable pH for topical application. Zeta potential, droplet size, and PDI met acceptable criteria. Moreover, incorporating C. recutita extract into the nanoemulsion enhanced its antimicrobial effect. Hence, the nanoemulsion system loaded with C. recutita and HA stabilization exhibits favorable characteristics for topical application, showing promise in aiding the healing processes.


Introduction
Skin trauma, characterized by interruptions in skin-mucosal integrity, serves as a potential entry point for bacterial infections, significantly impacting individuals' lives and causing socioeconomic disruptions [1].A growing body of evidence suggests that natural products hold promise in treating some skin diseases.This is due to their diverse pharmacological effects, notably anti-inflammatory, antioxidant, and antimicrobial properties, alongside good tolerability and safety, which contribute synergistically to tissue repair [2].
For thousands of years, the floral heads of chamomile (Chamomilla recutita L., Matricaria recutita L., or Matricaria chamomilla) have been employed in traditional and folk medicine via infusion, attributed to their antioxidant, anti-inflammatory, and antibacterial properties, particularly associated with the flavonoids present in the extract.These properties have facilitated its use across pharmaceutical, cosmetic, and food industries [3].Moreover, chamomile oil extract has demonstrated efficacy in accelerating tissue repair post-skin injury [4] and burns [5] in in vivo models.
Additionally, the distinctive physicochemical properties and multifaceted physiological functions of hyaluronic acid (HA), a primary component of the skin's extracellular matrix, have rendered it invaluable in regenerative medicine and tissue engineering, showing promising applications in skin tissue repair [6].Recent advancements in the treatment and prevention of bacterial infections, leveraging nanomaterials, have underscored the efficacy of combining HA with antibacterial agents to overcome the limitations of traditional antibiotics in infection management and enhance the resolution of chronic wounds [7].Hence, this study undertook the preparation, characterization, and integration of C. recutita extract into a nanoemulsion system stabilized with HA, aiming to augment tissue repair processes.

Preparation of Chamomilla Recutita Extract
Floral heads of C. recutita sourced from Mandirituba (Paraná State, Brazil), harvested in August 2021, were procured from a supplier of botanical raw materials in Cascavel (Paraná State, Brazil), along with a quality assurance report.
Concurrently, conventional extraction was performed via infusion, maintaining a plant/solvent ratio of 1:10.Boiling water was added, and the system was allowed to rest for 30 min, followed by filtration, freezing, and freeze-drying.
To determine the optimal extraction conditions, the total flavonoid content (TFT) of all extracts was quantified using a spectrophotometer (Model IL-592, Kasuaki, São Paulo, SP, Brazil) at 425 nm, following the methodology outlined by Woisky and Salatino [8].Quercetin served as the standard, providing the equation for the calibration curve: y = 81.561x− 126.41 (R 2 = 0.9966).Analyses were performed in triplicate, and the results were expressed as µg quercetin equivalents per gram of extract (µg QUE mg ext −1 ).Chromatographic profiling and identification were conducted using an ultra-highpressure liquid chromatograph (UHPLC) equipped with a BEH C-18 water absorption column (150 mm × 2.1 mm × 1.7 µm), coupled to a mass spectrometer (MS) featuring a quadrupole-time-of-flight system (BRUKER, Q-TOFII ® , Billerica, MA, USA).Analyses were carried out in both positive and negative modes, following the conditions outlined by Dalmagro et al. [9].

Antioxidant Capacity
The extract, at a concentration of 1000 µg mL −1 , underwent triplicate evaluation for its capacity to eliminate the radicals DPPH and ABTS •+ , as well as its reducing capacity via the FRAP assay, using a spectrophotometer.

Nanoemulsion System Loaded with C. recutita and Stabilized with Hyaluronic Acid Development
The nanoemulsion preparation involved two steps.Firstly, a pre-emulsion was prepared by blending 0.1 M saline solution (22.0%, w/w), sodium dodecyl sulfate (2.5%, w/w), and chamomile extract (1.0%, w/w), followed by gradual addition of this mixture to a blend of corn oil (70%, w/w) and Span 80 (4.5%, w/w) through dripping, using a high-speed shearing apparatus at 9000 rpm for 300 s.The resulting pre-emulsion underwent sonication using an ultrasound device equipped with a 13 mm diameter ultrasonic tip (Eco-Sonics, Indaiatuba, SP, Brazil), operating at a frequency of 20 kHz and power of 80%, for 60 s [13].Concentrations were selected based on pre-formulation studies encompassing the lower and upper limits of each variable.
In the second stage, the pre-emulsion was dripped into an aqueous phase consisting of a surfactant agent (Cremophor RH 40) and 0.1 M saline solution, as outlined in Table 1, using a high-speed shearing device at 9000 rpm for 300 s.Hyaluronic acid was subsequently introduced, and stirring was continued for 2 h at room temperature (25 • C) employing a magnetic stirrer [14].The control formulation followed identical procedures but omitted the incorporation of C. recutita extract into the pre-emulsion.All preparations were conducted in triplicate and left to stand for 24 h at 25 ± 1 • C before characterization.The selection of surfactant was based on previous studies confirming its safety and non-toxicity [15,16].

Droplet Size and Polydispersity Index
For droplet size and polydispersity index (PDI) measurements, 20 µL of samples were dispersed in 20 mL of 0.1 M NaCl solution at the time of analysis.The refractive index of the oil phase was 1.420.Analyses were conducted using a laser diffraction particle size analyzer (Partica LA-960, HORIBA Scientific, Piscataway, NJ, USA), with an evaluation range from 10 nm at 5 mm, at 25 ± 1 • C, in triplicate.Results were expressed as the mean ± standard deviation.

Viscosity
Viscosity was measured at speeds ranging from 1 to 40 rpm using number 2 cylindrical spindles, with readings limited to the maximum speed, in a digital Brookfield viscometer (QUIMIS ® , model Q860M26, Diadema, SP, Brazil).The instrument provided precise readings of ±2.0% with a measurement range of 1 to 6,000,000 mPa s, at 25

Morphology
For morphological evaluation, the F2 + HA nanoemulsion was placed on a 300-mesh copper grid coated with carbon film and negatively stained with a 2% phosphotungstic acid solution.The grids were air-dried for 24 h at 25 ± 1 • C [19], and images were captured using transmission electron microscopy (TEM) (JEOL JEM 1400 TEM, Peabody, MA, USA).

In Vitro Antimicrobial Activity
The minimum inhibitory concentration (MIC) was determined using the serial microdilution method in 96-well plates, in triplicate, following a previously described methodology [9].The test was performed against the following microorganisms: Staphylococcus aureus (ATCC 12026), Streptococcus pyogenes (ATCC 19615), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 9027).The positive control comprised the first three lines containing broth, microorganisms, and commercial antimicrobial (neomycin at concentrations ranging from 1.22 × 10 −3 to 2.5 mg mL −1 ).The negative control consisted of the last line containing broth and microorganisms.The C. recutita extract, as well as the nanoemulsions control (C2 and C2 + HA) and those loaded with extract (F2 and F2 + HA), were added to microplates containing BHI at concentrations ranging from 0.24 to 500 mg mL −1 .The plates were then incubated at 36 ºC for 24 h, after which 2% TTC developer was added and the appearance of a pink color was observed, indicating bacterial growth.

Statistical Analysis
The results were subjected to analysis of variance (ANOVA) and compared using the Tukey test at a significance level of 5%, using the STATISTICA 13.0 program (Statsoft ® , Tulsa, OK, USA).

Results
This study aimed to prepare, characterize, and integrate C. recutita extract into a nanoemulsion system stabilized with HA, with the potential to combat bacterial infections and facilitate differential healing of skin lesions.Different alcoholic contents were employed to optimize the extraction of flavonoids (Table 2).The 70% ethanol extraction yielded a higher TFT, and the extract obtained under this condition underwent phytochemical characterization by UHPLC-MS (Table 3).This extract, at a concentration of 1000 µg mL −1 , exhibited scavenging capacities for DPPH and ABTS  Smaller droplet sizes and PDI values (Table 4) were attained with a 10.0% surfactant concentration (hydrophilic-lipophilic balance = 11.37).Incorporating the C. recutita extract did not cause a statistically significant difference in droplet size or PDI, compared to the corresponding control, indicating that the extract does not interfere with nanoemulsion formation.The systems exhibited pH levels compatible with topical application and satisfactory zeta potential.The incorporation of HA notably elevated the viscosity of the nanoemulsion systems compared to those not stabilized with HA.Moreover, higher surfactant concentrations in HA-stabilized systems resulted in a slight increase in resting viscosity.Rheogram analysis revealed pseudoplastic flow behavior (Figure 1), confirming the heightened viscosity in nanoemulsions stabilized with HA.Importantly, the addition of the extract did not exert a significant influence on viscosity parameters.
nanoemulsion system loaded with C. recutita extract.HA: hyaluronic acid for stabilization.* Viscosity at 5 rpm.** Only the most promising formulations were evaluated for zeta potential.
The incorporation of HA notably elevated the viscosity of the nanoemulsion systems compared to those not stabilized with HA.Moreover, higher surfactant concentrations in HA-stabilized systems resulted in a slight increase in resting viscosity.Rheogram analysis revealed pseudoplastic flow behavior (Figure 1), confirming the heightened viscosity in nanoemulsions stabilized with HA.Importantly, the addition of the extract did not exert a significant influence on viscosity parameters.To elucidate the morphology and validate the droplet size, the nanoemulsion exhibiting satisfactory characteristics (F2 + HA) was examined via TEM.As depicted in Figure 2, the nanoemulsion displays a spherical shape with a narrow distribution.Additionally, the presence of HA chains surrounding the droplets was observed.Importantly, the droplet size observed aligns with the data obtained from laser diffraction.To elucidate the morphology and validate the droplet size, the nanoemulsion exhibiting satisfactory characteristics (F2 + HA) was examined via TEM.As depicted in Figure 2, the nanoemulsion displays a spherical shape with a narrow distribution.Additionally, the presence of HA chains surrounding the droplets was observed.Importantly, the droplet size observed aligns with the data obtained from laser diffraction.In conclusion, the assessment of MIC affirmed the efficacy of C. recutita extract and nanoemulsions loaded (F2) and stabilized with HA (F2 + HA) in inhibiting bacterial multiplication (Table 5).It is noteworthy that the MIC for F2 denotes the concentration (mg mL −1 ) of nanoemulsion, where 250 mg is equivalent to 0.5 mg of C. recutita extract, indicating the augmentation of the antibacterial effect of the extract when incorporated into the nanoemulsion system.Additionally, the stabilization of the system with HA reduced the MIC against S. aureus and P. aeruginosa (MIC equivalent to 0.333 mg of extract).As anticipated, nanoemulsions (C2 and C2 + HA) lacking C. recutita extract demonstrated no inhibitory activity.In conclusion, the assessment of MIC affirmed the efficacy of C. recutita extract and nanoemulsions loaded (F2) and stabilized with HA (F2 + HA) in inhibiting bacterial multiplication (Table 5).It is noteworthy that the MIC for F2 denotes the concentration (mg mL −1 ) of nanoemulsion, where 250 mg is equivalent to 0.5 mg of C. recutita extract, indicating the augmentation of the antibacterial effect of the extract when incorporated into the nanoemulsion system.Additionally, the stabilization of the system with HA reduced the MIC against S. aureus and P. aeruginosa (MIC equivalent to 0.333 mg of extract).As anticipated, nanoemulsions (C2 and C2 + HA) lacking C. recutita extract demonstrated no inhibitory activity.* Values correspond to the amount (in mg mL −1 ) of nanoemulsion required for antimicrobial effect.(-) indicates no activity.C: nanoemulsion system without C. recutita extract.F: nanoemulsion system loaded with C. recutita extract.HA: hyaluronic acid for stabilization.

Discussion
The vortical extraction method demonstrated a significantly higher potential for flavonoid extraction when using 70% ethanol.This observation is consistent with findings by Weber et al. [20], who illustrated superior extraction outcomes for chamomile using hyphenation techniques at the same ethanol concentration.Additionally, Asadi et al. [21] reported that C. recutita extract in 70% ethanol affects macrophages, promoting the reduction of nitric oxide synthesis and displaying anti-inflammatory properties.This activity was attributed to apigenin, which was also identified during the chemical characterization of our extract.
Flavonoids comprise the majority class in the floral heads of C. recutita.These compounds are directly linked to antioxidant, cytotoxic, anti-allergic, analgesic, and bactericidal activities, as well as acceleration of the healing process [28][29][30].Studies on structure-activity relationships have identified several factors potentially responsible for the high antioxidant activity of flavonoids.These factors include the presence of hydroxyl groups at positions 3 and 5 of ring A, position and quantity of -OH in ring B, degree of methylation of 3-OH, the double bond between carbons 2-3 in conjugation, presence of the 4-oxo function in the C ring, and angulation of flavonoid skeleton [31,32].
Furthermore, after a skin injury, ROS over production during the inflammatory phase can induce damage and hinder wound healing [33].Hence, the application of C. recutita extract offers a promising alternative as a natural product in the development of topical delivery systems.Moreover, the extract has demonstrated potential antimicrobial activity, fostering a conducive environment for the healing process.
Flavonoids are well known for their antimicrobial effects through various mechanisms.For example, apigenin disorients the lipid components of the membrane, leading to cell disruption [34].Additionally, apigenin, naringenin, kaempferol, chrysin, and quercetin interfere with biofilm formation [35].Moreover, epicatechin and quercetin, along with their glycosylated derivatives such as spireoside, induce oxidative damage to the membrane, increasing cellular permeability [36].Furthermore, quercetin and luteolin inhibit bacterial DNA replication [34].
Notably, variations in surfactant concentration during the nanoemulsion system development cause changes in droplet size and PDI.As the concentration of surfactant agent increases up to 10.0%, particle size and PDI gradually decrease.This is because surfactants play a crucial role in maintaining the stability and resistance of nanoemulsion structures to variations [37].However, adding surfactants at concentrations above the ideal leads to particle entanglement, thereby destabilizing the system [38].This observation aligns with the data presented here, wherein a concentration of 12.5% surfactant increased in size and droplet PDI.
In general, most nanoemulsions without HA exhibited a narrower size distribution.The increase in droplet diameter and PDI in the presence of HA can be attributed to the presence of HA chains at the interface of the oil droplets and the formation of surfactant-HA aggregates on the interfacial surface of the droplet [14].Despite the increase in PDI, the values remained below 0.3, which is considered a narrow distribution and indicates homogeneous droplets [19,39].Although coalescence is an unfavorable phenomenon for emulsifier systems and occurs in formulations with high PDI, it is important to note that PDI is only one of the general factors that influence stability.Therefore, despite the increase in PDI, HA also increased viscosity, reducing Brownian movement and collision between droplets and consequently protecting the coalescence system [14].
The viscosity increase resulting from HA inclusion is linked to the hygroscopic nature of the molecule.This property attracts water to its polysaccharide structure, forming a threedimensional network capable of enhancing the viscoelasticity of emulsifying systems [40].The rheograms revealed that as shear force increased, viscosity tended to decrease, a characteristic of formulations with pseudoplastic flow [14].This phenomenon occurs because as shear stress rises, the polymer structure aligns along the shear direction, resulting in faster subsequent shear and a decrease in apparent viscosity [19].Notably, HA exhibits great potential as a carrier molecule for delivering active ingredients, both in topical and transdermal systems.Its viscoelasticity, biocompatibility, biodegradability, and nonallergenic characteristics make it pivotal for the development of emulsions [41].
The pH of a formulation must be compatible with biological tissues to ensure its stability and effectiveness.Additionally, extremely low pH values should be avoided in nanoemulsion systems because they diminish electrostatic repulsion between particles, leading to an increase in droplet size and coalescence [42].In this study, the pH of the nanoemulsions ranged between 5.80-6.55,which is suitable for topical application [43].Studies have indicated that pH values close to neutrality (≈6-7) promote an increase in zeta potential in emulsions containing HA.This occurs through the deprotonation of carboxyl (-COO-) and hydroxyl (OH-) groups in the molecular structure of HA, imparting a negative charge to the oil-water interface [14,44,45], consistent with the findings of this study.
High zeta values (>30 mV absolute value) have been proposed as an indicator of physical stability, as they ensure the creation of a high repulsive energy barrier between lipid droplets [46].Furthermore, in addition to the effect of HA at the droplet interface, the use of a non-ionic surfactant (Cremophor) on the surface also contributed to the adequate absolute zeta value.This is due to the presence of free fatty acids in the surfactant, which facilitate the adsorption of OH-ions from the water onto the surface of the droplets, resulting in a negative charge at pH close to neutrality [14,47].Based on the smallest droplet size and satisfactory PDI, zeta potential, pH, and viscosity, the nanoemulsions prepared with 10.0% surfactant were found to be promising for the intended purpose and were evaluated for antimicrobial effect.
In the early infection stage, S. aureus and S. pyogenes are the dominant pathogens involved, while E. coli and P. aeruginosa are more frequently found when a chronic wound develops [48].Notably, in addition to antimicrobial resistance, S. aureus and P. aeruginosa are strains that most express virulence factors affecting skin healing [49].Although the antimicrobial activity of chamomile is well-documented scientifically, this work found that the incorporation of the extract into the nanoemulsion system (F2) enhanced the antimicrobial effect compared to the isolated extract, against all bacteria tested.Furthermore, in addition to providing an adequate skin distribution system, the emulsification or encapsulation of antimicrobial agents can accelerate their absorption and increase the bioavailability of the active ingredients, thereby enhancing the therapeutic effect [50], justifying the findings of this study.
Additionally, against S. aureus and P. aeruginosa, the MIC significantly decreased after stabilization of the nanoemulsion with HA (F2 + HA), indicating a possible synergistic effect.According to Zamboni et al. [51], HA can exert a bacteriostatic effect.Therefore, incorporating products with antimicrobial action in systems with HA can potentially generate a synergistic action, inhibiting the multiplication of bacteria and promoting an effective approach to treating topical infections.Hyaluronic acid plays an integral role in wound healing, facilitating fibrin coagulation, modulating inflammatory response, promoting re-epithelialization, inducing migration and multiplication of dermal fibroblasts, and favoring angiogenesis [52].Hence, combining the fight against oxidative stress and the antimicrobial effect of C. recutita extract, linked to the inherent benefits of the presence of HA in a formulation, leads to the inference that the F2 + HA nanoemulsion has potential for topical application, with a possible auxiliary effect on the healing process.The scope of this study was limited to the development of the nanoemulsion system.In future studies, the physicochemical stability and in vivo wound healing activity should be evaluated.
system without C. recutita extract.F: nanoemulsion system loaded with C. recutita extract.HA: hyaluronic acid for stabilization.

Table 2 .
Total flavonoid content (µg QUE mg ext −1 ) in C. recutita extracts as a function of the extractive alcohol content.Method Alcohol Content (%) Total Flavonoid Content (µg QUE mg ext −1 ) d Mean value (n = 3) ± standard deviation.Averages showing the same letter within columns do not differ significantly from each other (p < 0.05) according to the ANOVA with Tukey's test.
* Percentages of phytochemical compounds were calculated based on the total amount of identified compounds.

Table 4 .
Influence of different surfactant concentrations on the size, PDI, pH, viscosity, and zeta potential of nanoemulsion systems loaded with C. recutita and stabilized with HA.

Table 5 .
Minimum inhibitory concentration (mg mL −1 ) of the pure C. recutita extract incorporated into a nanoemulsion system stabilized with HA.

Table 5 .
Minimum inhibitory concentration (mg mL −1 ) of the pure C. recutita extract incorporated into a nanoemulsion system stabilized with HA.