Chitosan Xylotrupes gideon encapsulated lemongrass leaf ethanol extract reduce H 2 O 2 ‐induced oxidative stress in human dermal fibroblast

During phagocytosis, phagocyte cells discharge reactive oxygen species referred to as respiratory bursts, inducing a rise in pro‐oxidants and subjecting the cell to oxidative stress. Such stress is a biological mechanism related to an imbalance in pro‐oxidant/antioxidant homeostasis, which generates toxic reactive oxygen. Encapsulation is a coating process to improve the stability of bioactive compounds from lemongrass extract. Therefore, this study aims to determine the encapsulation activity of lemongrass leaf extract with chitosan X. gideon (LEChXg) to reduce the oxidative stress of fibroblasts. The research used the human dermal fibroblast (HDF) cell line, comprising negative and positive controls and use of LEChXg 100, 200, 300, 400, and 500 µg/mL. HDF cell migration was evaluated by employing the scratch wound healing method and the wound closure was oberseved at 0, 2, 4, 6, and 24 h intervals. The cell proliferation was observed at 24, 48, and 72 h using CCK‐8 at a 450 nm wavelength. The results showed that the observations at 0, 2, and 4 h did not demonstrate any significant difference on the cell migration ( p > 0.05) among the groups. However, the wound closure at 4 and 6 h showed a significant difference ( p < 0.05) with LEChXg 300 µg/mL. Despite the lack of any significant variation observed up to 24 h, fibroblast subjected to the stressor did not achieve complete closure. The groups treated with LEChXg were more stable in maintaining fibroblast proliferation up to the end of the observation than those with stressors at 24, 48, and 72 h. Fibroblast induced with a stressor was also more stable in maintaining migration and proliferation in groups receiving LEChXg 300 µg/mL.


Introduction
Mouth or oral ulcers are generally known as discontinu ities of oral mucosa characterized by epithelial tissue dam age and connective tissue in lamina propria of the mucosa in oral cavity (Zakiawati et al. 2020).About 40% of peo ple have been estimated to suffer from oral ulcers disease (Zakiawati et al. 2020) due to trauma during medical treat ment.Fibroblast is a crucial cell for mouth ulcers heal ing, which undertakes essential functions like synthesis and replenishment of the connective fibers and the amor phous substance during tissue repair (Lendahl et al. 2022).By the primary defence mechanism, the ulcer disappears through the healing process, which is divided into three phases, namely inflammation, proliferation, and remod elling (Toma et al. 2021).In inflammation, neutrophils and macrophages migrate to the ulcer area, resolve respi ratory bursts using high oxygen during phagocytosis, and increase reactive oxygen species (ROS), such as superox ide and hydrogen peroxide (Arief and Widodo 2018).Fur thermore, high ROS production can cause a prooxidant increase and oxidative stress (Bhattacharyya et al. 2014; Phaniendra et al. 2015).This condition interferes the cells communication and causes damage to influence the ulcer healing process (Pisoschi and Pop 2015), such as length ening the inflammation phase as well as hindering migra tion process and fibroblast proliferation (Buranasin et al. 2018).
Herbal medicine for the ulcer healing process has been widely used, such as lemongrass (Cymbopogon cit ratus DC) (Veronica et al. 2021).Lemongrass is one of the spices growing in the tropics and is widely used in Southeast Asia, including Indonesia (Maria et al. 2021).Lemongrass leaf is often discarded without being utilized, whereas its stem serves as a highly valued spice in culi nary applications.The leaf contains valuable active com pounds, including alkaloids, flavonoids, tannins, steroids, triterpenoids, and saponins, which possess notable antiox idant properties (CominoSanz et al. 2021).Natural in gredient with antioxidant content is proven to accelerate the ulcer healing process (Ozougwu 2016) by reducing fi broblast oxidative due to respiratory burst from phagocytic cells (Deng et al. 2021) and to accelerate migration and proliferation (Grgić et al. 2020).
The pharmacological activity of the active compound has bioavailability and absorption limitations in the body that can be controlled with encapsulation technology (Rahim et al. 2022), i.e. by protecting the active compound from oxidation to improve its therapeutic potential (Negi and Kesari 2022).The common polymer material used as a trapping matrix for encapsulation is chitosan (Andikop utri et al. 2021), which is a natural polymer compound ob tained from insect exoskeleton (Baharlouei and Rahman 2022), such as horn beetle (Xylotrupes gideon) (Veronica et al. 2021).Thus, it can be developed to facilitate a drug delivery system due to its biocompatible, biodegradable, low toxicity level (Agarwal et al. 2018), and simple prepa ration method (Mohammed et al. 2017).Chitosan physical modification also increases the absorption, diffusion, and penetration to the mucosal layer better than its normal size (Detsi et al. 2020).
Chitosan is a polymer widely used as an active com pound trap of a natural ingredient.Previous studies show that Prunus avium L. extract encapsulation using nanochi tosan and gallic acid can decrease oxidative stress on en dothelium cells (Beconcini et al. 2018) and 3T3 fibrob last cells (de Paiva et al. 2021), respectively.Further more, the active compound encapsulation of lemongrass with chitosan polymer reduces ROS production of fibrob last by inducing hydrogen peroxide stressor (Fitria et al. 2022).The observation with 2'7' dichlorodihydrofluo rescein diacetate (H2DCFDA) staining using a fluores cent microscope also shows the intensity of green fluores cent cells, indicating reduced ROS production (Andikop utri et al. 2021; Veronica et al. 2021.
This study assesses the activity of chitosan encapsulated active compounds of lemongrass leaf ethanol exctract.The chitosan was derived from X. gideon through the ionic gelation method.It mitigates fibroblast oxidative stress induced by hydrogen peroxide to promote a decrease in oxidative stress.This reduc tion in oxidative stress can be demonstrated through enhanced migration and proliferation of fibroblast during the healing process in mouth ulcers.Encapsulation of the lemongrass bioactive compounds is an innovative approach allowing protection against oxidation, thermal degradation and increasing bioavailability.Encapsulation is promising to improve the performance of medicines in oral health, such as mouthwashes.

Polymeric materials
The source of chitosan in this study was X. gideon obtained from Cangkurawok, Damaga, and Balumbang Jaya, Bo gor, East Java.All parts of X. gideon body were detached, followed by drying for five days, and then continied to the processes of demineralization (3N HCl), deproteinization (3N NaOH), discoloration (4% H 2 O 2 ) and deacetylation (50% NaOH) (Komariah et al. 2019).

Lemongrass extract (LE) preparation
Lemongrass (Cymbopogon citratus) was collected from Balai Penelitian Tanaman Rempah dan Obat (BALITRO), Indonesian Medicinal and Aromatic Crops Research Insti tute (IMACRI), West Java, Indonesia.The determination was carried out at Pusat Riset Biologi, Badan Riset dan Or ganisasi Nasional (BRIN), Cibinong, West Java, Indone sia.The leaves were dried in an oven at a temperature of 45 °C for one week and extracted using the maceration method (Felicia et al. 2022).They were soaked in 70% ethanol at a 1:10 (w/v) ratio for 24 h at room temperature.The macerated mixture was filtered using a filter paper and evaporated with a rotary vacuum evaporator at a tempera ture of 40 °C at 100 rpm for 2 h (Fitria et al. 2022).

The preparation of LE-loaded on chitosan X. gideon (LEChXg)
Chitosan with 83% degrees of deacetylation and a weight value of 0.5 g was dissolved in 1% acetic acid (Merck, Germany).Subsequently, 2 mL of 10% lemongrass leaf extract (LE) and 100 mL of distilled water were added (Veronica et al. 2021).Stirring was conducted using a magnetic stirrer (IKA RH basic 2, Germany) and heating at 40 °C with a speed of 2,500 rpm for 20 min and was subsequently carried out without heating for 100 min.Fol lowing the previous step, 40 mL of 0.1% tripolyphosphate (SigmaAldrich, USA) was added dropwise while stir ring for one hour.Subsequently, 0.1 mL of 0.1% Tween 80 (Merck, France) was introduced, and the mixture was stirred again at a speed of 2,500 rpm for 30 min (Andikop utri et al. 2021; Veronica et al. 2021.The particle size of lemongrass extract was determined using a particle size analyzer (PSA) (Horiba Scientific, Nano Particle Analyzer SZ100, UK) (Budi et al. 2020).

Culture of fibroblast
Human dermal fibroblast (HDF) was obtained from the Biorepository of Stem Cell Research Center, Yarsi Univer sity, Indonesia (Fitria et al. 2022).Fibroblast was planted in a cell culture dish and incubated at 37 °C with 5% CO 2 for 24 h.(Fitria et al. 2022).

Proliferation assay
Fibroblasts planted at a density of 1 × 10 3 cells/well in a 96well plate were incubated at 37 ºC for 24 h.After 24, 48, and 72 h of treatment with a various concentration of LEChXg, the cells were washed by PBS 1×, and 100 μL of CCK8 solution was added.The absorbance was measured at 450 nm using a microplate reader (Tecan Group Ltd. Mannedorf, Switzerland) and the percentage of fibroblast proliferation was calculated as shown below (Felicia et al. 2022).

Migration assay
The cells were planted in a 24well plate (2.9 × 10 3 cells/well) (Kauanova et al. 2021) and incubated until con fluent.After reaching confluency, the monolayer was scratched gently using a white tip perpendicularly to the bottom of the monolayer.After the first scratch was con ducted, a second scratch was made by crossing the first one.Thus, a cross pattern would be formed.Next, the cells were washed in PBS once and subsequently were treated (Felicia et al. 2022).The cell migration was observed af ter 0, 2, 4, 6, 24, and 48 h after the that and photographed using a microscope (EVOS FLc Cell Imaging System).At the end of the experiment, the wound closure was analyzed using ImageJ.

Data Analysis
Statistical quantification was conducted using SPSS ver sion 2.3 and the data were presented as mean ± standard deviation (SD).Meanwhile, MANOVA was used to com pare the groups against several dependent variables (times) in migration and proliferation assays.For a significant dif ference (p < 0.05), Post Hoc's Tukey test was performed.

Characteristics of LEChXg particle
Characteristic of LEChXg particle was used to estimate and determine the particle size and distribution of parti cle size.Meanwhile, particle size was measured using a PSA with a repetition of three times.The result of particle measurement and polydispersity index (PDI) LEChXg is shown in Table 1.The LEChXg particle measurement indicated that the average size was 489.57nm.Therefore, LEChXg fulfilled the requirements as a nanoparticle with a size range be tween 50-500 nm particle (Ismail and Harun 2019).Ac cording to Idacahyati et al. (2021), a nanosize particle should range from 1-1000 nm.PDI is a value that shows particle size distribution with a range of 0-1.A sample with a bigger and smaller size range has higher PDI values (Karmakar 2019).The result of particle distribution with PDI values 0.035 to 0.05 is considered to have monodis perse particle distribution (Clayton et al. 2016).Poly dispersity is a macromolecule with various good weights, sizes, and mass distribution (Kim et al. 2019).The zeta po tential value of LEChXg was 31.2 mV.Nanoparticles with zeta potential values smaller than 31.2mV and greater than +30 mV also indicated good stability (Prakash et al. 2014).A dispersion system with a small zeta potential value was easier to form, such as the Van der Waals style in particle interaction (Juliantoni et al. 2020).

Migration of fibroblast
The migration test was conducted using scratch by mak ing an artificial gap (scratch) in confluent monolayer cells.The gap allowed cells to communicate with each other.It also showed the ability of fibroblasts to move toward the ulcer, carry out proliferation, and form an extracellu lar matrix.The results of fibroblast migration showed that migration in 0 and 2 h did not indicate a significant differ ence (p = 0.222).However, at the observations of 4 and 6 h, there was a significant difference (p < 0.05) between the group with a stressor and the LEChXg group at a con centration of 300 µg/mL.The group treated with LEChXg showed a good migration activity by closing the most sig nificant gap compared to those treated with a H 2 O 2 stres sor only.At 24 h, all study groups had no significant dif ference (p > 0.05).However, the fibroblast group treated with stressor showed less optimal migration due to an open gap of 0.51 ± 0.88 µm 2 .The average fibroblast migration ability is shown in Table 2. Microscopic observation of the cell migration can be seen in Figure 1.
The observation of fibroblast migration when closing the gap is related to the ulcer healing process.The inflam mation cells are stimulated to release various mediators (Chen et al. 2018) and growth factors such as the trans forming growth factorbeta 1 (TGFβ) factor of fibroblast when oxidative stress is reduced (Jimi et al. 2020).H 2 O 2 is one of the most critical compounds in ROS signaling studies due to its physicochemical properties, relatively low reactivity, and ability to diffuse through membranes.The addition at low concentrations increases intracellular ROS levels without causing oxidative stress.It also in creases the migration of mesenchymal cells through extra cellular signalregulated kinases (ERK) 1/2 and focal ad hesion kinase (FAK) pathways.Cellular abnormalities are increased when cells experience oxidative stress (Waheed Indonesian Journal of Biotechnology 28(4), 2023, 191-199  2018) stated that gingival fibroblast exposed to high glucose concentrations causes oxidative stress by increasing ROS production and inhibit ing the migration process associated with inhibiting basic fibroblast growth factor (bFGF) signaling.

Fibroblast proliferation
The results of fibroblast proliferation in 24 h showed that the group treated with stressor was significantly differ ent (p < 0.05) from those without stressor, as well group treated with ascorbic acid as a nonenzymatic antioxidant and LEChXg.The group treated with stressors indicated low cell proliferation compared to the others.Hydrogen peroxide is a molecule with low reactivity but can easily penetrate the cell membrane, generating the most reactive type of oxygen, hydroxyl radical, and converting Fe 2+ atau Cu + to OH (Nita and Grzybowski 2016).
Fibroblast proliferation without stressors had the high est proliferative compared to the other groups.The av erage proliferation is shown in Figure 2. Fibroblast can counteract an elevation in free radicals by augmenting the synthesis of endogenous antioxidants, thereby prevent ing any adverse impact on the proliferation of stressor unexposed cells (Tsuneda 2020) The group treated with ascorbic acid showed good proliferation after exposing the cells to stressors H 2 O 2 .Ascorbic acid is an antioxidant that can neutralize oxidative stress by donating an electron to prevent other oxidized compounds and scavenging su peroxide anion, hydroxyl radical, and lipid hydroperoxide (Pehlivan 2017).
Fibroblast proliferation in 48 h of observation showed that the group treated with H 2 O 2 is significantly differ ent (p < 0.05) from those without stressor, as well as the group treated with ascorbic acid, and LEChXg concentra tions at 200, 300, and 400 µg/mL.Meanwhile, the group with stressors did not report a significant difference (p > 0.05) from LEChXg 100 and 500 µg/mL.The group treated with H 2 O 2 at 48 h showed a decrease in proliferation compared to the 24h observation.It indi cates that the cells experienced oxidative stress could not detoxify or repair the damage resulting from free radicals (Phaniendra et al. 2015).Therefore, it caused cell damage and affected proliferation.The ascorbic acid group and those treated with LEChXg at all the the tested concentra  tions could detoxify or repair the damages resulting from increased free radicals to enable a relatively stable prolif eration.
The observation at 72 h showed that the group treated with stressor was significantly different (p < 0.05) from LEChXg concentrations 100, 200, 300, and 400 µg/mL.The difference showed increased proliferation higher than the group treated with a stressor.In contrast, the non stressor, ascorbic acid, and LEChXg 500 µg/mL groups showed no significant difference (p = 1.000).Even though there was no difference, these groups exhibited higher pro liferation cells than those treated with stressors.The pro liferation of fibroblast in 24, 48, and 72 h observation is shown in Figure 3.
The results of the proliferation of the nonstressor, stressor, and ascorbic acid groups experienced decreased proliferation.However, a proliferation of the group treated with LEChXg was relatively stable.LEChXg maintained or stabilized proliferation until 72 h observation of fibrob last, which experienced oxidative stress.Pan et al. (2022) showed that active compounds of lemongrass increase cell proliferation power by reducing oxidative stress resulting from high ROS.Similarly, Roriz et al. (2014) indicated that lemongrass showed an antioxidant effect by improv ing the superoxide dismutase enzyme (SOD) activity and reducing the production of ROS in macrophages.Chitosan plays a role in scavenging free radicals and inhibiting ox idative damage (Pellis et al. 2022).The leading functional group, such as hydroxyl and an amino groups can reduce free radicals after a reaction at C2, C3, and C6 positions of the pyranose ring to produce a stable macromolecule (Muthu et al. 2021).At the end of our observation, there was an aggregate formed in the LEChXg group due to polydisperse particle distribution with various molecular weights, sizes, and mass distributions, as well as zeta po tential value which was relatively greater than +30 mV (Dipahayu and Kusumo 2021).
Our results were consistent with Beconcini et al. (2018), where the encapsulation of Prunus avium extract with chitosan and its derivatives reduces oxidative stress in human umbilical vein endothelial cells (HUVEC).An in vivo study on rat liver cells showed that encapsulated Pinus merkusii extract with chitosan reduces malondialde hyde (MDA) levels.An increase in MDA levels indicates hepatocyte cell membrane damage after exposure to ROS.Meanwhile, the decrease showed that the encapsulation of P. merkusii extract inhibits ROS production (Di Santo et al. 2021).
Damage in the epithelium and lamina propria as con nective tissue with a predominance of fibroblast in mouth ulcers accelerated the healing process by encapsulating lemongrass leaf extract ethanol with chitosan X. gideon.The acceleration of the process was determined through increased migration and proliferation of fibroblasts after experiencing oxidative stress during the inflammatory pro cess.

FIGURE 2
FIGURE 2 Fibroblast proliferation.a-c in different hours shows a significant difference (p < 0.05) The superscript 'a' indicates higher proliferation compared to groups 'b' and 'c,' while 'b' indicates higher proliferation than group 'c.' Superscript 'ab' implies no significant difference between groups 'a' and 'b,' and superscript 'bc' no significant difference between groups 'b' and 'c'.

FIGURE 3
FIGURE 3 Fibroblast proliferation at 24, 48, and 72-h observation as indicated.The yellow arrow shows the fibroblast, which is living with a clearly visible cell nucleus, while the red arrow shows a dead fibroblast by shrinking in the cells.A: negative control, B: H 2 O 2 only.C-H received H 2 O 2 with treatment as follows: C: ascorbic acid as positive control, D: LEChXg 100 µg/mL, E: LEChXg 200 µg/mL, F. LEChXg 300 µg/mL, G. LEChXg 400 µg/mL, and H LEChXg 500 µg/mL.The observation was at 1,000× magnification.