Anti-Inflammatory Activity of Fern Pteridium aquilinum Ethanol Extract Gel on Mice (Mus musculus) Incision Wounds

The utilization of natural resources around the community is one of the alternative anti-inflammatory medications. One of the several studies on this area reported that commonly people use Pteridium aquilinum leaves to heal wound instead of commercial gel medications. The research aimed to determine the activity and it effective dose of Pteridium aquilinum ethanol extract gel as an anti-inflammatory. The ethanol extract of Pteridium aquilinum was formulated in a gel with varying dosage concentrations of 1%, 1.5%, and 3% for 2 times applied a day in 14 days, with the incision wounds without fern ethanol extract gel as negative control and incision wound was treated with commercially as positive control. Observation on the wound length and the injured tissue histology of mice incision wound carried out on 3, 5, 7, and 14 days of treatment. The results prove that fern ethanol extract gel formula has anti-inflammatory activity on mice incision wound. Wound healing indicated by the closure of the wound on the skin. Based on the results can be concluded that Pteridium aquilinum ethanol extract has anti-inflammatory and heals mice incision wound affectively in 14 days with the best dosage concentration of 3%.


Introduction
Inflammation is a normal protective response on tissue injury caused by physical trauma, damaging chemicals, or microbiological agents [1].Inflammation is the body's mechanism to damage and deactivate invading organisms accompanied by inflammation through a complete healing process [2].Generally, inflammation treatment includes two aspects, namely relieving pain which is often a symptom, and efforts to stop the process of tissue damage.Reducing inflammation using steroids and non-steroidal antiinflammatory drugs (NSAIDs) can reduce inflammatory reactions well, but long-term use can have side effects [3].Therefore, using natural remedy found around the community place have been chosen to avoid the side effect anti-inflammatory medicines.
Various studies have been reported on the use of plants to treat various diseases, among them the fern plant known by the scientific name Pteridium aquilinum.P. aquilinum is used as a vegetable ingredient [4].This plant is known contain anti-thiamine which is useful for exterminating insects, as an ornamental plant, and fertilizing peatlands [5].P. aquilinum can also be used as a source of potassium or fertilizer [6].In Bangka Belitung, Pteridium aquilinum leaves are used as a medicine for infected wounds [7].This is under empirical data where the people of Ambon also use P. aquilinum leaves as medicine to treat wounds.
Plants can be used as medicine for disease because of the secondary metabolites they produce.Secondary metabolites are organic molecules that do not have a direct role in growth and development [8].Secondary metabolites in plants have several functions: 1) defense against viruse, bacterial, and fungi; competitor plants; and most importantly against herbivores, 2) attractants (odor, color, taste) for pollinators and seed-dispersing animals, 3) protection from UV rays and N-storage [9].Studies report that several ferns have biological activities including anti-inflammatory and antinociceptive [10].
A group of compounds derived from plants such as tannins can help the wound healing process because their function as antioxidants and antimicrobials which is affecting wound healing and also accelerate epithelialization.Tannins and saponins also act as antiseptics on surface wounds and bacteriostatic.Steroids act as anti-inflammatory.Flavonoids have antioxidant properties by reducing excessive ROS, antibacterial, and can increase wound contraction with their antimicrobial and astringent properties [11].
Topical medicinal preparations for wound healing on the market can be found in the form of creams, ointments and gels.Preparations in gel form are more widely used because they are clear, dry easily, form a film layer that is easy to wash off and provide a cool feeling on the skin [12].Based on the description above, this study aimed to determine the activity and effective dose of Pteridium aquilinum ethanol extract gel as an anti-inflammatory.

Research Procedures 2.2.1. Ethanol extract of fern (Pteridium aquilinum) preparation
A total of 250 grams of the dried simplicia ferns were soaked in 1000 mL of 96% ethanol, covered with aluminum foil and left at room temperature for 5 days, stirred occasionally.After 5 days, the filtrate was set aside, the precipitate was added with 750 mL of 96% Ethanol, covered with aluminum foil and left for 2 days then filtered.The resulting filtrate is mixed with the filtrate from the previous filtering.The mixture was evaporated in a water bath at a temperature of 40°C to obtain a thick fern extract.[13].In this study, gel preparations were made with modifications of fern ethanol extract concentration of 1%, 1.5% and 3% and amounting 25 grams applied twice a day for 14 days of observation [14].The ethanol extract of the fern (Pteridium aquilinum) is dissolved in some heated water, then add Na-CMC, stirred until homogen.Addes glycerin, propylene glycol, and water, then stirred until a homogeneous gel is formed and packaged in a gel container [15].

Preparing test animals and making incisions
The test animals used were 15 male mice.Before treatment, mice were acclimatized to the research environment for 7 days.The day before the incision was made, the fur on the back of the test animals were shaved.Then anesthetized with lidocaine.Next, an incision was made with a length of 2 cm and a wound depth of 1.5 mm with a scalpel.The incision will be treated twice a day.

Testing the effectiveness of fern ethanol extract gel on incision wounds
Observation was carried out for 14 days to see the wound healing process by measuring the diameter of the wound [16].
The treatment given to the test samples is as follows: Treatment 1 : Incision wounds without fern ethanol extract gel (negative control) Treatment 2 : The incision wound was treated with commercially used wound healing gel (positive control) Treatment 3 : The incision wound was treated with 1% fern ethanol extract gel.Treatment 4 : The incision wound was treated with 1.5% fern ethanol extract gel.Treatment 5 : The incision wound was treated with 3% fern ethanol extract gel.
The gel applied by spread it evenly on the back of the mouse where the wound has been made, approximately ± 0.2 g twice a day.

Histopathological observations
Histopathological observation was carried out on day 3, 5, 7, and 14 in each treatment group to see the collagen formed after treatment.Histopathological observations were carried out according to [17].

Data analysis
Data measuring the length of the incision wound after treatment was expressed in dx (cm), then analyzed using SPSS One-Way ANOVA method with a 0.05 with the formula [2]: Where: P = percentage of wound healing d0 = initial length of incision wound dx = length of incision after treatment This animal study was approved by Pattimura University ethics committee.This research was carried out following animal welfare standards and policies according to the Government Regulation Concerning Animal Welfare, Chapter III Animal Welfare, Part 1; Article 83.

Wound Length Measurement Data
The data of the observation for 14 days on wound healing and measuring it diameter shown on Table 2.

Observation
Days to- Wound healing is a long recovery process for the skin due to damage or dis-integrity of skin tissue.A wound is a break in the continuity of a tissue due to injury or surgery [18].The observation results showed that the wound had closed well on day 14 th with the both treatments given of the positive control and the 3% concentration.In contrary to the negative control, there were changes on wound because the mice were not given any medication on the incision wounds.The treatments concentrations of 1% and 1.5% also showed no different significantly results with the negative control.This concentration only had a little ability to close wound.The administration of extract gel with various concentrations shows different effects on wound healing.This is also supported by the homogeneity test analysis using SPSS in Table 3.The results of the homogeneity test analysis using SPSS showed that the relationship between fern ethanol extract gel and wound length in wounds healing had a significant value greater than 0.05 (0.102 > 0.05), so the data distribution was homogeneous (the data variance was the same).Closure of wound healing on each mice treatment group means that ethanol extract of the Pteridium aquilinum treatment has an effect on wound healing.1329 (2024) 012002 IOP Publishing doi:10.1088/1755-1315/1329/1/0120025

Re-epithelialization Presentation
Re-epithelialization is a stage of wound repair including mobilization, migration, mitosis and differentiation of epithelial cells.These stages will restore lost skin integrity [19].There are striking differences between treatment groups as shown on Table 4. On the 14th day of observation, the average re-epithelialization on the positive control group given commercial drugs and 3% gel concentration treatment had reached around 97%, whereas on the negative control group without Pteridium aquilinum gel treatment only 3%.The average reepithelialization on 1% and 1.5% gel treatment showed a small presentation value.These results showed that Pteridium aquilinum ethanol extract gel has relatively the same ability as commercial drugs to increase epidermal re-epithelialization, so the wounds close more quickly.From the results shown in Figure 1, it can be seen that on groups (a), (b) and (e), the epidermal re-epithelialization process has not occurred completely, scabs/fibrin are still visible.On groups (c) and (d), the re-epithelialization process has begun to complete, the scab has fallen off, the wound has narrowed, and fibrosis is at a high density.On the 14th day, the density of fibroblasts was completed on the Pteridium aquilinum ethanol extract gel group as well as on the commercial gel group, but both of them were different with the control group where the density was dense but there were still cavities.All of that showed that the re-epithelialization process or closure wounds process on the 3% fern ethanol extract group more quickly than the other groups.
The group of tannin compounds contained in Pteridium aquilinum extract can help the wound healing process due to its function as an antioxidant and antimicrobial affecting wound healing and also accelerates epithelialization.Tannins and saponins also act as antiseptics on surface wounds due to bacteriostatics.Steroids act as anti-inflammatory and flavonoids have antioxidant properties by reducing excessive ROS due to antibacterial and can increase wound related to antimicrobial and astringent properties [11].Tannins have a role in increasing collagen, epithelium, and new blood vessels for wound closure [20].

Conclusion and Recommendations
Pteridium aquilinum ethanol extract gel has the anti-inflammatory activity as well as commercial gel medicine.Pteridium aquilinum ethanol extract gel has been proven to be able to heal wounds in mice by accelerating the process of re-epithelialization, neocapillaryization, and increasing the formation of skin connective tissue so it can be used as an alternative for wound healing.The best Pteridium aquilinum ethanol extract gel concentration of 3% effective in healing incision mice within 14 days.

Figure 1 .
Figure 1.Microscopic view of the wound re-epithelialization process on the 14th day