THE DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR EVALUATING GALLIC ACID IN ETHYL ACETATE FRACTION (EAF) OF SNEDDS FORMULATION: QUANTITATIVE ANALYSIS WITH IN VITRO ASSAY

Objective: This study aimed to develop a simple, accurate, precise, sensitive, robust, and stable analytical method for the evaluation of gallic acid in Self-Nanoemulsifying Drug Delivery System (SNEDDS) incorporating ethyl acetate fraction (EAF) of Melastoma malabathricum leaves in combination with Gentamicin. Methods: Validation process followed ICH guidelines and applied a reverse phase HPLC method with a mobile phase of acetonitrile-phosphoric buffer at pH 3.03 (20:80 v/v). The stationary phase consisted of a VP-ODS shim-pack C-18 column (250x4.6 mm) with a flow rate of 0.2 ml/min and detection at 263 nm using an Ultraviolet detector. Additionally, antioxidant activity was assessed through the DPPH and FRAP methods, and SPF value was determined with a UV/Vis spectrophotometer in the 290-390 nm wavelength range. Results: The results showed that the retention time of quercetin was 16.648 min with a tailing factor of 1.623. The regression equation (y=224689x-989000) had a concentration range of 10-55 µg/ml and a correlation value of 0.9920. Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 2.394±0.086 and 7.254±0.260 µg/ml, respectively. Method accuracy, determined by recovery values at concentrations of 50%, 100%, and 150%, ranged from 91.18% to 109.49%. Repeatability inter-day variations were expressed as %RSD values of 1.027-1.963% for AUC and 0.150-0.145 for RT. Moreover, the applied method showed stability within a temperature range of 14 °C–35 °C. Analysis showed gallic acid content of 1.773±0.049 mg/g in SNEDDS EAF formulation. Antioxidant activity measured through the DPPH and FRAP methods yielded IC 50 values of 4.167±0.552 µg/ml and 20.253±0.619 µg/ml, respectively, while SPF value at SNEDDS concentration of 150 µg/ml was 36.993±0.183. Conclusion: This study successfully developed a precise, accurate, specific, and stable method for quantifying gallic acid levels in SNEDDS EAF of Melastoma malabathricum leaves in combination with Gentamicin. Therefore, SNEDDS EAF formulation exhibited an effective wound-healing potential, supported by a robust quality control process.


INTRODUCTION
Gallic acid (GA), chemically referred to as 3,4,5-trihydroxybenzoic acid, is one of the phenolic compounds in existence.Furthermore, its phenolic structure serves as a source of hydrogen atoms capable of generating free radicals with delocalized electrons within the phenol group [1].The results reported by Nayeem et al., 2016 showed that gallic acid could exhibit various pharmacological activities, including anticancer, antioxidant, and neuroprotective effects, while Agrawal et al., 2021 discovered its anti-inflammatory and analgesic properties.Yang et al., 2016 investigated the potential of this compound as a wound healer, with good antioxidant activity, found to stimulate fibroblast cell migration and activate kinase enzymes.Gallic acid can promote wound healing in animal models of both type 1 and type 2 diabetes, particularly in chronic cases [1][2][3].
The potential of gallic acid in wound healing has been explored through various formulations.Gan and Chin., 2021 incorporated this compound into a hydrocolloid film for wound dressing.Stefanov et al., 2018 showed the effectiveness of a hydrogel containing gallic acid and chitosan as a means to control oxidative stress and bacterial contamination in chronic wounds and foot ulcers.Seulgi et al., 2021 developed a chitosan and gallic acid-based synthetic tissue adhesive, which significantly improved wound closure and tissue regeneration through enhanced fibroblast cell production compared to the control group [10][11][12].
Pratiwi et al., 2021 created a nanoemulsion formulation within a Self-Nanoemulsifying Drug Delivery System (SNEDDS), combining EAF of Melastoma malabathricum leaves with the antibiotic gentamicin for the treatment of diabetic foot ulcers (DFU).This study reinforced the use of plants containing gallic acid in formulation development, specifically focusing Melastoma malabathricum [13].Sari et al., 2022 showed the synergistic effects of EAF and Gentamicin combination, which acted as an effective antibiotic against Bacillus cereus, Escherichia coli, and Staphylococcus aureus.Therefore, this current study aims to develop and validate an analytical method for quantifying gallic acid levels within SNEDDS formulation, a combination of Melastoma malabathricum leaves EAF and Gentamicin, which is promising for the effective treatment of chronic wounds such as DFU.The identification of active compounds (markers), particularly gallic acid in SNEDDS preparations, contributes scientifically to understanding pharmacological activity of the developed formulation.In this study, antioxidant activity tests through the DPPH and FRAP methods and the measurement of the Sun Protection Factor (SPF) value of formulation will be conducted.The development and validation of analytical method are expected to follow the ICH guidelines, using a reverse phase High-Performance Liquid Chromatography (HPLC) instrument to obtain reproducible and consistent measurement results.

Preparation of mobile phase
The mobile phase used in this study comprised a 20:80 ratio of acetonitrile and phosphate buffer (pH 3.08), which was prepared by diluting a 0.008% H3PO4 solution to a final volume of 0.5L with double-distilled water.

Sample preparation
The sample used was SNEDDS loaded with EAF of Melastoma malabathricum, dissolved in acetonitrile.The botanical identity of Melastoma malabathricum has voucer specimen number is 021/AL/lB/FMIPA/UNTAN/2023.EAF was obtained by constituting the maceration of Melastoma malabathricum leaves with ethanol 80%.Subsequently, the fractionation process applied n-hexane, chloroform, ethyl acetate, methanol, and water as solvents.Sample in this study, namely EAF (Ethyl Acetate Fraction), it was incorporated into SNEDDS formulation.

Preparation of SNEDDS EAF combination with gentamicin
Up to 10 mg of Melastoma malabathricum EAF and 10 mg of gentamicin were added to 10 ml soybean oil, VCO, olive oil, sunflower oil, Tween 20, and Cremophor EL.SNEDDS was prepared using a combination of Tween 80, propylene glycol, and soybean oil, to which EAF was added.The mixture was conditioned in a water bath at 40 °C for 10 min, then the lipid and water phases were blended at 70 °C and homogenized with a homogenizer at 750 rpm for 10 min [13].

RP-HPLC instrumentation for gallic acid analysis
The sample analyzed in this study was an SNEDDS-loaded EAF of Melastoma malabathricum with gentamicin (SNEDDS EAF), dissolved in acetonitrile.Furthermore, it was filtered through a 0.2 µm membrane filter (Whatman) before injection into the HPLC system.A standard solution of gallic acid was prepared by dissolving 25 mg of the compound in 25 ml of acetonitrile, and different concentrations, ranging from µg/ml 10-55 µg/ml, were produced as a calibration curve.All the standard solutions were filtered through a 0.2 µm membrane filter (Whatman) before being transferred into the HPLC system [9].

Chromatography condition in RP-HPLC
The chromatography system applied was a modification of the method described by Apridamayanti et al., 2023.This used a VP-ODS shim-pack C-18 column (250x4.6 mm), with a reverse phase column packed with 4.5 µm diameter particles.The mobile phase consisted of acetonitrile-phosphoric acid with pH 3.03 (20:80, v/v), filtered through a 0.2 µm membrane filter (Whatman) and de-aerated ultrasonically before use.The temperature was maintained at 30 °C, with an injection volume of 20 µl and a flow rate of 0.2 ml/min.The wavelength for gallic acid detection was set at 263 nm using a UV detector [9].

Assay validation
The RP-HPLC method for quantifying gallic acid was validated according to the International Council for Harmonisation (ICH) guidelines.Validation procedure included assessing linearity, range, precision, accuracy, sensitivity, and robustness [9,15].

Stability of analytical solution
The strength of analytical solutions was determined by examining the standard preparations at-14 °C (in a refrigerator), room temperature (30 °C), and 35 °C under 24 h.Five injections drawn from each solution were analyzed, and the peak and the RSD average were calculated.

Linearity and range
A calibration curve was prepared by diluting 25 mg gallic acid with acetonitrile as a standard stock solution to produce concentrations ranging from 10 µg/ml-55 µg/ml.Three injections from each concentration were analyzed under the same condition.Furthermore, linear regression analysis was used to evaluate the curve Linearity through the least square linear regression method (R 2 ).

Sensitivity
The determination of sensitivity included calculating the limit of Detection (LOD)/limit of Quantitation (LOQ) of gallic acid solution by analyzing different solutions and measuring the signal-to-noise ratio.LOD was the concentration that produced a signal-to-noise ratio of approximately 3:1, while LOQ generated a signal-to-noise ratio of 10:1.

Accuracy
The accuracy of the analytical method was assessed through recovery studies at three concentration levels of gallic acid (50%, 100%, and 150%, i.e., 0.7 mg, 1.5 mg, and 2.2 mg).Three samples from each concentration were injected into the HPLC system, and the percentage recovery of added gallic acid and their RSD was calculated.

Precision
The system and method precision (repeatability) was determined by several measurements of sample solution (n=5) and standard solution (n=9) through intraday tests.

Robustness
The robustness test was carried out by introducing minor and deliberate changes into the chromatography system to evaluate their influences.This included using a column temperature of±3 °C, flow rate of±0.05ml/min, wavelength of±5 nm, and mobile phase and organic composition (Buffer phosphate: Acetonitrile) at±5%.Moreover, five injections from each solution were analyzed, and the peak and the RSD average were calculated.In this study, column efficiency (N) and tailing factor (TF) parameters were found to be within the acceptable limit.

Quantitative analysis of gallic acid in SNEDDS EAF (Marker study in formulation)
A total of 1 ml of SNEDDS EAF preparation was dissolved in acetonitrile and then homogenized.The sample solution was passed through a 0.2 µm membrane filter (Whatman) before injection into the HPLC system.

Antioxidant activities
Antioxidant activity was assessed using the 1,1-Diphenyl-2picrylhydrazyl (DPPH) radical assay to determine free-radicalscavenging activity (DPPH and FRAP assay).The method followed the protocol described by Apridamayanti et al., 2022, with minor modifications.Up to 3 ml of each sample, with concentrations ranging from 1 µg/ml-6 µg/ml, was added to 3 ml of 1 mmol DPPH and incubated in the dark at room temperature for 30 min.Absorbance was measured at 515.5 nm with a Spectrophotometer UV/Vis for the DPPH assay.This study was conducted with experiments in triplicate measurement for each test [5].The Ferric Reduction Antioxidant Power (FRAP) assay was also conducted according to [5] with slight modifications.A total of 30 µl of each sample at different concentrations (5 µg/ml to 30 µg/ml) was added to 30 µl FeCl3 solution (3 mmol in 5 mmol citric acid) and 240 µl TPTZ reagent (1 mmol in 0.05 M HCL) in a 96-well microplate.After incubation at room temperature for 15-30 min, absorbance was measured at 615 nm using an Elisa Reader.

Identification of SPF
SNEDDS EAF formulation was dissolved in ethanol, and different concentrations were prepared at 50 µg/ml, 100 µg/ml, and 150 µg/ml.The solution was scanned in the UV range of 290 to 320 nm (at 5 nm intervals) using a spectrophotometer (n=3).The identification of sun protection activity was determined based on the equation proposed by Mansur [9].
Note: CF represents the correction factor, EE is the Eritema effect in the spectrum (nm), I denotes light intensity (nm), and Abs signifies the absorbance sample.During the experimental process, the absorbance sample was measured three times and used for SPF calculation.

Statistical analysis
Statistical analysis for validation and in vitro studies was conducted using Microsoft Office Excel 365 for Windows and SPSS software, respectively.Parameters such as average, sum, Standard Deviation (SD), Regression (RSQ), and scattered charts were used for linearity assessment.Moreover, ANOVA was applied for the in vitro studies, with a significance level of p<0.05 considered significant.

RESULTS AND DISCUSSION
The objective of this study was to develop and validate analytical method for quantifying gallic acid content in SNEDDS EAF formulation containing EAF of Melastoma malabathricum leaves.Analysis of marker compounds could provide scientific information related to the activity of formulation.

Method validation
The determination of gallic acid levels was conducted through spectroscopic and chromatographic techniques.In this study, an HPLC method was developed for quantifying gallic acid, a marker compound present in EAF of nanoemulsion preparations.The development of RP-HPLC aimed to ensure precision, thoroughness, sensitivity, and specificity in quantifying this marker compound in natural sources, following ICH guidelines for method validation.The results obtained were documented in the form of Peak Area, Retention Time (TR), Asymmetry Factor (TF), and Column Efficiency (N) within predetermined limits.Moreover, the asymmetry factor is often applied to characterize a chromatographic system, with increased asymmetry leading to decreased separation and measurement result repeatability.Column efficiency evaluates the performance and effectiveness of column in relation to the length used in the system [20].

Chromatographic system
In this study, the chromatographic system was used to assess the solubility of the marker compound, gallic acid, and the suitability of the mobile phase composition under several design variations to achieve effective compound separation and column efficiency.The details of the chromatography system are presented in table 1.

Stability testing of SNEDDS EAF formulation and gallic acid
Stability testing aims to assess the stability of the compound under test temperature conditions and during storage.The strength of the standard solution was evaluated under temperature variations of 30 °C and 35 °C, with storage times of 7 d at 30 °C and 24 h at 35 °C and 14 °C.Meanwhile, the sample solution was stored for 24 h at 35 °C.The test parameters examined included RSD values for Peak area (AUC) and retention time, as well as the TF and N values, which were compared against regulatory limits, and the results can be seen in table 2.

Linearity and range
The linearity test of the calibration curve for the standard gallic acid solutions was conducted by repeating the experiment three times (n=3), resulting in slope, intercept, and regression values (R 2 ).The range in this study was found to be 10-55 µg/ml, as shown in Tables 3 and 4.  Information: σ = SD The measured LOD and LOQ values are presented in table 4.

Precision
Precision testing constituted repeatability assessments of SNEDDS samples and gallic acid standard solution on days 1, 2, and 3.
The %RSD value was examined, with a criterion of <2% and the test results can be seen in tables 5 and 6.

Accuracy
The accuracy test assessed the performance of the analytical method using the accuracy value expressed as a percentage of recovery.This study included adding the standard solutions at concentrations of 50%, 100%, and 150%, and the results are presented in table Strength testing for analysis method (Robustness) The strength of the developed HPLC system was evaluated by analyzing changes made to various parameters, including column temperature, maximum wavelength, mobile phase composition, and flow rate within the HPLC system.Additionally, this condition was observed from the %RSD value<2%, TF, and N for each parameter change made to the AUC and RT.The results can be seen in table 8.   Quantification of Gallic Acid in SNEDDS EAFAnalysis of gallic acid, serving as a marker compound in SNEDDS EAF formulation, was conducted using a validated method.RP-HPLC was the instrumental method used for this purpose, and gallic acid content determined was 1.773±0.049mg/g, with an RT of 16.648 min.

Antioxidant activity
The assessment of antioxidant activity in the preparation aimed to elucidate the in vitro pharmacological activity associated with the benefits provided by SNEDDS EAF formulation.Antioxidant activity was determined by reacting SNEDDS EAF formulation with DPPH solution and FRAP reagent.Therefore, the ability of the preparation to stabilize oxidant compounds, often indicated by the IC50 value, signifies its potential pharmacological activity.The results of this assay are presented in Tables 10(A) and 10(B).

SPF test results
SPF test aimed to evaluate the effectiveness of SNEDDS EAF of Melastoma malabathricum formulation.Three different concentration levels were tested, and the test results are presented in table 11.SPF value obtained showed the effectiveness of the sample concentration in protecting against UV rays.The maximum SPF value observed was 36.993±0.183at a concentration level of 150 µg/ml.

DISCUSSION
The developed analytical method was designed as a preliminary step in the pharmaceutical formulation process [16], specifically for evaluating SNEDDS EAF formulation derived from Melastoma malabathricum leaves extract combined with Gentamicin.During the initial stages of method development, significant optimization efforts were made to ensure the solubility of the marker compound and the sample solution within the mobile phase of the applied RP-HPLC system used.The details of the developed chromatography system are provided in table 1.In this study, tests were carried out on SNEDDS formulation to determine the content of marker compounds present in the sample.
Arbianto et al., 2019 discovered that HPLC analysis at 44%, as recommended by the US Pharmacopeia, offered selective and accurate results for quantifying chemical compounds in formulated products.The presence of excipients in formulated products often disrupts the selectivity of analytical method.Therefore, the development of a particular analytical method tailored to the formulated products is needed to separate the analytes from excipients and ensure their accurate, specific, and sensitive quantification using HPLC [16].
To ensure the selectivity of the chromatography system, tests were performed on both standard gallic acid and sample solutions, as represented in fig. 1 a, b, and c.The development of an analytical method for measuring gallic acid present in SNEDDS EAF formulation is instrumental for quality control and assurance of compound content in pharmaceutical production.Therefore, validation of the analytical method was conducted following the guidelines set by ICH.
Linearity, expressed through a line based on a mathematical equation, establishes the relationship between analyte response and concentration variations, often referred to as the correlation value (R 2 ) in the linear regression equation.Additionally, Range is expressed in the interval of the largest and smallest concentration values in the test performed.In this study, an R-value of 0.9920 was obtained in the standard gallic acid solution concentration range of 10-55 (µg/ml), as presented in table 4.
LOD and LOQ indicate the minimum detectable and quantifiable levels of analytes measured statistically, precisely, and accurately by HPLC, and are often obtained through a linear regression line on a standard curve.Their values in this study were determined to be 2.394±0.086and 7.254±0.260,respectively as shown in table 4.
According to Patil, 2017, LOQ value determination is valuable for detecting low levels of analytes contained in matrix samples and evaluating impurities and degradation products [18].
Precision is the estimate of the variation in measurement results from individual tests on the sample collected from a homogeneous mixture, expressed as the relative standard deviation (% RSD) value [21,22].Repeatability tests were conducted five times on the sample solution and nine times on the standard solution over three days.The %RSD values for the peak area and retention time are presented in Tables 5 and 6.These were found to be 2.289±0.828 in the sample solution, while in gallic acid solution, the values ranged from 1.027±0.106-1.963±0.105.In accordance with Patil, 2017 and Snyder et al., 2010, the acceptable %RSD value for 100% analyte content (standard solution) is<2%, and for 1% analyte content (sample solution), it is stipulated as<2.7%.
Accuracy reflects the closeness of measured analyte levels to the actual values and is expressed as %Recovery.The accuracy test was conducted using the addition method, where predetermined quantities of gallic acid were mixed with SNEDDS (placebo) formulation, and the resulting amounts of gallic acid were compared with the actual levels.The %Recovery values obtained in this study were compared with the applicable regulatory value and ranged from 91.18% to 109.49%, as presented in table 7.
Robustness is a validation parameter used to assess the resilience and responses of analytical method to small changes in various HPLC system parameters, such as maximum wavelength, column temperature, flow rate, and mobile phase composition (ratio).This is often evaluated by observing the peak area, retention time, asymmetry factor (TF), and column efficiency (N).The test results are presented in table 8, and according to [18][19][20], TF value is <2% and N is>2000 (plate count).
The quantification of gallic acid levels contained in the samples using the RP-HPLC system is presented in table 1. Measurements were conducted on the models and repeated three times (n=3).Gallic acid levels in the samples were found to be 1.773±0.049mg/g with a retention time of 16.648 min.Antioxidants are chemical compounds capable of donating electrons to other molecules, such as Reactive Oxygen Species (ROS), one of the protective mechanisms within cells for neutralizing oxidants [24,25].Similarly to Reactive Nitrogen Species (RNS), ROS induced by ultraviolet radiation participate in wound healing [26].According to Gulumian et al., 2018, during the inflammatory phase, ROS plays a crucial role in facilitating the movement of neutrophils and monocytes in the blood vessels surrounding the wound [24].Dunnil et al., 2015 also stated that ROS contributes to infection control, as well as the proliferation of keratinocytes, endothelial cells, and fibroblasts, increasing angiogenesis and collagen deposition.However, uncontrolled ROS production can elevate oxidative stress, leading to wound severity and delayed healing.To maintain ROS levels within the range required by the body, antioxidants are used to stabilize ROS by donating electrons [24,27,28].
Regulating the amount of ROS content in the body during inflammation can serve as therapy, hence, stabilizing these oxidants within the inflammatory phase in wound tissue improves the healing process [25,27] [32,30].
SNEDDS is an isotropic mixture consisting of oil, surfactant, solvent, and co-solvent, with the addition of active pharmaceutical ingredients.SNEDDS can form stable solutions and emulsions with droplet sizes ranging from 10 to 200 nm [33].This lipid-based system helps protect drug compounds, slow down degradation, and enhance drug delivery to target tissues, minimizing side effects.
Moreover, the lipids in nanoemulsion preparations are suitable carriers for increasing the bioavailability of drugs at their target points [34].SNEDDS have been shown to significantly boost drug release by 89% compared to 24% for traditional formulation [35].
Literature studies conducted on nano-sized lipid systems have shown their potential to effectively encapsulate antioxidant compounds.For instance, nanoencapsulation using green tea and peppermint oil phases resulted in an IC50 value of 116.07 mg/ml for the antioxidant.Additionally, SNES formulation from olive, pomegranate, and grape seed oil had IC50 values of 0.49%, 0.18%, and 0.8%, respectively.The development of SNEDDS from spirulina powder and combination of spirulina powder, fish oil, and Tribulus terrestis yielded IC50 values of 1606 and 1122 mg/ml [36][37][38].
In this study, the protective effect against oxidative stress was assessed using the DPPH and FRAP methods.The results in table 10 showed that the optimal protective concentration against free radicals was 50%, corresponding to IC50 values of 4.167±0.552µg/ml and 20.253±0.619µg/ml.The photoprotective effect against UV radiation, as indicated in table 11, was most potent at a concentration of 150 µg/ml, with SPF value of 36.993±0.183.Other investigations have reported some similar results; for example, [39] discovered that SNEDDS from ginger extract (Zingiber officinale) and eel fish bone oil (Anguilla spp) exhibited antioxidant activity with an IC50 value of 428.4 mg/ml.According to Kholieqoh et al., SNEDDS formulation of Pandanus tectorius fruit extract showed an antioxidant activity 1.6 times higher than the non-formulated extract.Additionally, Lestari et al., 2023 documented an antioxidant activity of 56.74±1.04%from SNEDDS formulation containing Allium sativum extract [40].
Phenolic compounds derived from natural sources have the potential to protect against the negative impacts of UV radiation.According to Hettihewa et al., 2020, flavonoids and polyphenols present in fruit, flower, and vegetable samples have shown significant UV protection and antioxidant activity [26].This observation was supported by the study of Frei et al., 2023, which indicated SNEDDS containing curcumin extract to be capable of improving carrageenan and UV-induced inflammation in Mice [42].
The mechanism of action of antioxidant activity inhibition and UV protection constitutes the disruption of ROS activity in cells.Moreover, in a test conducted on human fibroblast cells, Liu et al., 2018 stated that the concentration of curcumin needed for UV protection was 5 µg/ml [43].To mitigate the inflammatory process associated with wounds and generally accelerate healing, designing a formulation with antioxidant activity and UV protection is essential.

CONCLUSION
In conclusion, this study successfully developed a precise, accurate, specific,and stable analytical method for quantifying gallic acid levels contained in SNEDDS EAF of Melastoma malabathricum leaves combined with Gentamicin.The prepared SNEDDS formulation exhibited promising pharmacological activity, including potent antioxidant properties with IC50 values of 4.167±0.552µg/ml and 20.253±0.619µg/ml, assessed using the DPPH and FRAP methods, respectively.Moreover, it showed significant protection against UV radiation, as indicated by SPF value of 36.993±0.183at a concentration of 150 µg/ml.These results showed the potential of SNEDDS formulation as an effective wound-healing agent, supported by a robust quality control process.

Table 2 : Stability test of SNEDDS EAF formulation and gallic acid
* Number of experiments, n=3

Table 4 : Linearity, LOD, and LOQ test results of gallic acid solution
* Values are expressed as mean±SD, Number of experiments, n=3

Table 9 : Quantification of gallic acid in SNEDDS EAF of Melastoma malabathricum Name of sample Retention time Gallic acid (mg/g)
* Values are expressed as mean±SD, Number of experiments, n=3

Table 10 : Antioxidant activity test of SNEDDS EAF of Melastoma malabathricum using DPPH method and FRAP method
*IC50 Values was expressed as mean±SD, Number of experiments, n=3

Table 11 : Measurement of photoprotector activity on SNEDDS EAF of Melastoma malabathricum
a,b,c have a significant difference value of P<0.05, *Values are expressed as mean±SD, Number of experiments, n=3 * . Gallic acid, a phenolic compound, is known to have good antioxidant activity.Studies by Monteiro et al., 2017; Yang et al., 2016 and Pressi et al., 2022 showed that gallic acid could inhibit ROS