Effect of 4% and 15% moringa leaf extract gel on gingival wound healing in rats

Traumatic dental procedures such as incisions and gingival flaps are the leading causes of injury to the gingival structure. One of natural ingredients that can potentially accelerate the wound healing process is Moringa leaves ( Moringa oleifera ). Moringa leaf has several active compounds, one of which is flavonoids, which can be an anti-inflammatory and antibacterial agent, and increase collagen synthesis. A randomized posttest-only control-group design was used in this study. 48 wistar rats were categorized into four groups: CMC-Na gel, hyaluronic acid, 4% Moringa leaf extract gel, and 15% Moringa leaf extract gel. The samples were then euthanized on days 1, 3, 5, and 7. The two-way ANOVA test described significant differences (p < 0.05) for all the components of the observation (neutrophils, fibroblasts, angiogenesis, and epithelial thickness). The lowest mean number of neutrophils, the highest mean number of fibroblasts, and the highest mean of angiogenesis were found in the 15% Moringa extract group on the 7 th day. The highest mean epithelial thickness was found in the use of 4% Moringa extract on the 5 th day. The microscopic images showed that the treatment group gained more effective wound healing processes than the control group. The microscopic image showed that, in terms of neutrophils, fibroblasts, and angiogenesis, 15% Moringa extract was more effective for wound healing compared to 4% Moringa extract.


INTRODUCTION
The function of the gingiva is to protect the underlying structures against the influence of the oral environment.The function of the gingiva as a protective tissue causes it to be susceptible to injury. 1 Wounds can occur as a result of trauma and surgery. 2 Wound healing is divided into several stages, namely hemostasis, inflammation, proliferation, and remodeling. 3The wound healing process in the oral cavity passes through the same fundamental pathway as that in other regions. 4However, the abundance of pathogenic and opportunistic bacteria in the oral cavity can trigger acute and chronic infections.Acceleration of the healing process in the oral cavity is highly recommended to reduce discomfort and pain in dental patients. 5Topical application is used to accelerate the wound healing process.Topical (transdermal) drugs have several advantages, such as eliminating fluctuations in gastrointestinal absorption and providing constant and controlled drug input. 6One of the topical drug dosage forms that can be applied to the oral cavity is a gel.The advantage of gel preparations is better penetration, which maximizes the local effect and minimizes the systemic effect. 7ne of natural ingredients that have the potential to accelerate the healing process of gingival wounds are Moringa leaves (Moringa oleifera).Moringa leaves have several active ingredients, one of them is flavonoids.Flavonoids function as antibacterial and anti-inflammatory agents, and play a role in increasing collagen synthesis by fibroblasts during a wound healing process. 8,9Previous research showed that the use of Moringa leaf extract gel was proven to accelerate the wound closure process on the palate of rats. 8Another study also stated that the administration of gel preparations from Moringa leaf extract could shorten the bleeding time and increase the collagen density of the incision wound on the gingiva of Cavia porcellus. 10This study aimed to determine the effect of Moringa oleifera extract gel on the gingival wound healing process in wistar rats (Rattus norvegicus) seen from the number of neutrophils, the number of fibroblasts, angiogenesis, and epithelial thickness.

MATERIALS AND METHODS
This study was a laboratory experiment with a randomized posttest-only control group design.This research was approved by the research ethics committee of the Faculty of Medicine, Udayana University with clearance number 2303/UN14.2.2.VII.14/LT/2021.The making of Moringa Leaf Extract Gel started by sterilizing, drying, and refining it into powder.The powder was then extracted using a Soxhlet extractor with 95% ethanol as solvent.The process took three cycles per hour.The extract was put into a flash evaporator.Gels containing 4% and 15% Moringa leaf extract were obtained by dissolving 2 g and 7.5 g of Moringa leaf extract in 5 ml of distilled water.A total of 50 g of CMC-Na gel base was added to the mixture gradually until the mixture was homogeneous.Moringa Leaf Extract Gel was prepared in the Laboratory of Phytochemistry and Experimental Animals, Faculty of Mathematics and Natural Sciences, Udayana University.
Fourty-eight (n = 48) male wistar rats (Rattus norvegicus) aged 2-3 months whose body weight was 150-200 grams were categorized into four groups: CMC-Na gel, hyaluronic acid, and 4% Moringa oleifera extract gel, and 15% Moringa oleifera extract gel.The research samples were given standard laboratory pellet and tap water ad libtum.They were first adapted for seven days under normal laboratory condition with sufficient light and ventilation in the Laboratory of Phytochemistry and Experimental Animals, Faculty of Mathematics and Natural Sciences, Udayana University.
Intramuscular injection was carried out with the administration of anesthetic drugs.The labial gingiva of the rats' mandibular incisor was injured using a 2.5 mm punch biopsy.0.5 mL of Moringa leaf extract was applied with the help of cotton buds every morning and evening.Hyaluronic Acid (Gingigel) and CMC-Na gel were applied in the control group with the same procedure.Gingival samples were taken using surgical scissors and a scalpel.The tissue was stored in a 10% formalin buffer solution and histological preparations were made.The sacrificed rats were then buried properly.All histological microscopic features were measured on days 1, 3, 5, and 7 and stained with H&E, hematoxylin-eosin in the control and treatment groups.Fibroblasts, neutrophils, and angiogenesis were counted using a binocular light microscope at 400× magnification and Image Raster 3.0 (United States) software.In five fields of view, observations were made on randomly selected as the regions of interest in gingival wound area.The epithelial thickness was measured by microruler with a scale of 1:1,000 at 400x magnification.Observations were made with the help of an Olympus light microscope with an Optilab digital camera.The magnification was 400x in five fields of view.The analysis was carried out using Saphiro-Wilk Test of normality, followed by Levene's test.The statistical test was done with Two-Way ANOVA as a comparative test, followed by post hoc LSD.

RESULTS
The lowest mean number of neutrophils was found in the 15% Moringa leaf extract gel group on the 7 th day of euthanasia, while the highest mean number was found in the 2% CMC Na gel group on the 1 st day of euthanasia.The lowest mean number of fibroblasts was found in the 2% CMC-Na Gel group on the 1 st day of euthanasia, while the highest mean was found in the 15% Moringa leaf group on the 7 th day.
The lowest mean number of angiogenesis was found in the 2% CMC Na Gel group on the 1 st day of euthanasia, while the highest mean was found in the 15% Moringa leaf group on the 7 th day.The lowest mean epithelial thickness was found in the 2% CMC Na group on the 3 rd day of euthanasia, while the highest mean was found in the 4% Moringa leaf extract group on the 5 th day.
The normality and homogeneity test resulted in a p-value of > 0.05 in all the observed cells.It can be concluded that the research data were normal and homogeneous.The two-way ANOVA results indicated a significant difference (p < 0.05) in all the observed components (neutrophils, fibroblasts, angiogenesis, and epithelial thickness).The data were then analyzed using the Post-hoc test LSD (Least Significant Different).             1. Histological microscopic feature of: neutrophil (A) 15% Moringa leaf extract gel group on the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibroblast (C) 2% CMC-Na Gel group on the 1 st day (D) 15% Moringa leaf group on the 7 th day; angiogenesis (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa leaf group on the 7 th day; epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4% Moringa leaf extract group on the 5 th day of euthanasia.All of histological microscopic feature stained with H&E, hematoxylin-eosin, and 400× magnification (indicated by black arrow).

DISCUSSION
The descriptive analysis of neutrophils showed that the total mean was lower in the treatment group (4% and 15% Moringa leaf gel) than the control group (2% Na CMC gel and hyaluronic acid).The observations of fibroblasts, angiogenesis, and epithelial thickness showed a higher total mean in the 4% and 15% Moringa leaf gel groups than the 2% Na and hyaluronic acid CMC gel groups.The twoway ANOVA test indicated significant differences in all the observed wound healing factors (neutrophils, fibroblasts, angiogenesis, and epithelial thickness).
The observation of the neutrophils in the 4% Moringa leaf group showed a significant difference on the 1 st day compared to the 3 rd , 5 th , and 7 th day.This significant difference was caused by the active compounds in the Moringa leaf extract gel.Flavonoids play a role in accelerating wound healing.In the inflammatory phase, flavonoids help the wound healing process by inhibiting the activity of the COX-2 enzyme. 11The observation of the fibroblasts in the 4% Moringa leaf group showed significant differences on the 1 st day compared to the 3 rd , 5 th , and 7 th days and the 3 rd compared to the 7 th day.This significant difference was caused by the bioactive compounds in the gel, one of which is flavonoids.Flavonoids have antibacterial and anti-inflammatory properties, which cause the proteins in the bacteria around the wound to become denatured so that the bacterial cells break down and make the proper wound-healing process. 8,11,12The results of the observation of the epithelial thickness in the 4% Moringa leaf extract gel group showed a significant difference on the 1 st euthanasia day compared to the 5 th euthanasia day, the 3 rd euthanasia day compared to the 5 th euthanasia day, and the 5 th euthanasia day compared to the 7 th euthanasia day.It is because Moringa leaf extract contains flavonoids.The flavonoids in Moringa leaf extract were proven to increase the expression of TGF-ß1, CD68, and VEGF.Increased VEGF stimulates endothelial cell proliferation in wound healing mechanisms.Saponin compounds in leaf extract activate the functions of VEGF, TGF-ß, FGF, and epidermal growth factor (EGF). 13,14 The results of the observation of the neutrophils in the 15% Moringa leaf group showed a significant difference on the 1 st euthanasia day compared to the 5 th and 7 th euthanasia day, and the 3 rd euthanasia day compared to the 7 th euthanasia day.This significant difference was caused by several bioactive compounds in the Moringa leaf extract gel, which have antiinflammatory properties, including flavonoids and alkaloids. 11,15The observation of the fibroblasts in the 15% Moringa leaf extract gel group showed a significant difference between all the euthanasia days.This significant difference was due to several bioactive compounds in Moringa leaf extract, one of which is flavonoids.Flavonoids in Moringa leaf extract can accelerate the wound healing process by increasing collagen synthesis, decreasing the number of macrophages, and increasing the number of fibroblasts. 11,16This is directly proportional to research conducted by Poernomo and Setiawan, 2019, showing that 15% Moringa leaf extract gel can shorten bleeding time and increase collagen density.The increase in collagen density occurs because fibroblasts play a role in the formation of collagen fibers.In a normal wound healing process, collagen provides tissue integrity and strength in the injured area. 16,17,18he observation of the angiogenesis in the 15% Moringa leaf extract gel group found a significant difference on the 1 st euthanasia day compared to the 5 th and 7 th euthanasia day and the 3 rd euthanasia day compared to the 7 th euthanasia day.This is because flavonoids can stimulate angiogenesis during the wound healing process. 11n the other hand, the post hoc analysis of the groups using 4% and 15% Moringa leaf extract gel showed no significant differences, presumably because of some confounding factors that could interfere with the wound healing process. 19owever, this study has some limitations.This study involved a limited number of samples and a limited variation of extract concentrations in the gel.There is still a possibility that other concentrations might have a significant impact on healing process.It is recommended for further research to use other variations of extract concentrations in the gel and more samples.In addition, the application of the extract with other gel bases in the healing process of gingival wounds is still needed to identify its impact on wound healing.It is also necessary to carry out a quantitative phytochemical testing to determine which compounds play an important role in the healing process.

CONCLUSION
The study results concluded that 15% Moringa leaf extract gel had a better anti-inflammatory effect in reducing the number of neutrophils than 4% Moringa leaf gel, especially on the 3 rd day.In addition, 15% Moringa leaf extract gel was also better in increasing the number of fibroblasts compared to 4% Moringa leaf gel, especially on the 7 th day of wound healing.The study results also concluded that 15% moringa leaf extract gel was better in increasing angiogenesis than 4% Moringa leaf gel, especially on the 7 th day of wound healing.However, 4% moringa leaf gel was better in increasing epithelial thickness than 15% Moringa leaf gel, primarily on the 5 th day of wound healing.

ACKNOWLEDGMENT
This study was carried out with the help of Udayana University PNPB research program with letter number B/4969-5/UN14.2.2.VII.1/PT.01.03/2021.The authors express gratitude to the Pharmacy Laboratory and Histology Laboratory of Udayana University for the completion of this research.

CONFILCT OF INTEREST
There is no potential conflict of interest in this study.

Figure 1 .
Histological microscopic feature of: neutrophil (A) 15% Moringa leaf extract gel group on the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibroblast (C) 2% CMC-Na Gel group on the 1 st day (D) 15% Moringa leaf group on the 7 th day; angiogenesis (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa leaf group on the 7 th day; epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4% Moringa leaf extract group on the 5 th day of euthanasia.All of histological microscopic feature stained with H&E, hematoxylin-eosin, and 400× magnification (indicated by black arrow).

Figure 1 .
Histological microscopic feature of: neutrophil (A) 15% Moringa gel group on the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibrobl CMC-Na Gel group on the 1 st day (D) 15% Moringa leaf group on th angiogenesis (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa lea the 7 th day; epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4 leaf extract group on the 5 th day of euthanasia.All of histological microsco stained with H&E, hematoxylin-eosin, and 400× magnification (indicated arrow).

Figure 1 .
Histological microscopic feature of: neutrophil (A) 15% Moringa gel group on the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibrobl CMC-Na Gel group on the 1 st day (D) 15% Moringa leaf group on th angiogenesis (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa lea the 7 th day; epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4 leaf extract group on the 5 th day of euthanasia.All of histological microsco stained with H&E, hematoxylin-eosin, and 400× magnification (indicated arrow).

Figure 1 .
Histological microscopic feature of: neutrophil gel group on the 7 th day (B) 2% CMC Na gel group on CMC-Na Gel group on the 1 st day (D) 15% Moringa angiogenesis (E) 2% CMC Na Gel group on the 1 st day ( the 7 th day; epithelial thickness (G) 2% CMC Na group o leaf extract group on the 5 th day of euthanasia.All of hi stained with H&E, hematoxylin-eosin, and 400× mag arrow).
H) stological microscopic feature of: neutrophil (A) 15% Moringa leaf extract the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibroblast (C) 2% l group on the 1 st day (D) 15% Moringa leaf group on the 7 th day; (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa leaf group on epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4% Moringa roup on the 5 th day of euthanasia.All of histological microscopic feature H&E, hematoxylin-eosin, and 400× magnification (indicated by black eutrophil

Figure 1 .
Figure 1.Histological microscopic feature of: neutrophil (A) 15% Moringa leaf extract gel group on the 7 th day (B) 2% CMC Na gel group on the 1 st day; fibroblast (C) 2% CMC-Na Gel group on the 1 st day (D) 15% Moringa leaf group on the 7 th day; angiogenesis (E) 2% CMC Na Gel group on the 1 st day (F) 15% Moringa leaf group on the 7 th day; epithelial thickness (G) 2% CMC Na group on the 3 rd day (H) 4% Moringa leaf extract group on the 5 th day of euthanasia.All of histological microscopic feature stained with H&E, hematoxylin-eosin, and 400× magnification (indicated by black arrow).

Table 2 .
Descriptive analysis of fibroblast

Table 4 .
Descriptive analysis of epithelial thickness

Table 6 .
Post hoc neutrophil test of 15% Mori

Table 5 .
Post hoc neutrophil test of 4% Moringa leaf extract gel group

Table 9 .
Post hoc angiogenesis test for 4% Moringa leaf extract gel group

Table 10 .
Post hoc angiogen leaf extract gel group

Table 5 .
Post hoc neutrophil test of 4% Moringa leaf extract gel group

Table 9 .
Post hoc angiogenesis test for 4% Moringa leaf extract gel group st day (D) 15% Moringa leaf group on the 7 th day; el group on the 1 st day (F) 15% Moringa leaf group on (G) 2% CMC Na group on the 3 rd day (H) 4% Moringa y of euthanasia.All of histological microscopic feature n-eosin, and 400× magnification (indicated by black 3): 151 -......
th day (B) 2% CMC Na gel group on the 1 st day; fibroblast (C)

Table 7 .
Post hoc fibroblast test of 4% Moringa leaf extract gel group

Table 8 .
Post hoc fibroblast test of 15% Moringa leaf extract gel group

Table 9 .
Post hoc angiogenesis test for 4% Moringa leaf extract gel group

Table 10 .
Post hoc angiogenesis test of 15% Moringa leaf extract gel group

Table 11 .
Post hoc test of the epithelial thickness of 4% Moringa leaf extract gel group

Table 12 .
Post hoc test of the epithelial thickness of 15% Moringa leaf extract gel group