Evaluation of clinical, histology, TNF-α, and collagen expressions on oral ulcer in rats after treatment with areca nut and chrysanthemum oral gel

Introduction

Oral ulceration is the most common presentation in the oral cavity caused by many etiologic factors.¹ Most of the etiologies of ulcerative lesions on the oral mucosa are divided into four categories; namely, infectious (bacterial and virus infection), immune-related (autoimmune and allergic), traumatic (mechanical trauma), and neoplastic (oral squamous cell carcinoma).² Traumatic ulcers are injuries to the oral mucosa caused by mechanical or physical trauma such as sharp food, accidental biting during mastication, biting while speaking, punctured by sharp objects, fractured, malformed, carious, or malposed teeth on the superficial epithelial layer or underlying connective tissue or may involve both.³

Currently, increasing the resolution of wound healing is one of the main priorities in the medical field to accelerate the healing of chronic wounds and traumatic injuries.⁴ The process of tissue regeneration and repair occurs immediately after lesion onset. The linear tissue repair and regeneration involves growth factors that induce cell proliferation, especially parenchymal cells, followed by dynamic changes in soluble mediators, blood cells, and the extracellular matrix.⁵ The unique oral cavity environment shows advantages in accelerating wound healing compared with skin repair. This is due to differences in the response to inflammation, differentiation and proliferative programs, modulation of stem cells, collagen synthesis, the role of macrophages, and epithelial remodeling.^6,7 Collagen synthesis and tumor necrosis factor-α (TNF-α) are important components during acute inflammation and responsible for a diverse range of signaling events within cells in the wound-healing process. TNF-α is one of inflammatory cytokine and regulates the immune system.⁸

Areca nut is the seed of the areca palm (Areca catechu Linn.; areca, Palmaceae), which grows thrive in tropical Pacific region, South and South East Asia, including Indonesia where it is known as “Pinang”. It has been consumed by people worldwide as part of ancient tradition, custom, or ritual for a long time. Several studies have been conducted to prove the healing ability of areca nut, including that the alkaloid and polyphenols content in areca nut could enhance the healing of burn wounds, leg ulcers, and skin graft surgery.^9,10 Areca nut contains alkaloid, phenolic, tannin, and flavonoid compounds. The presence of catechin, quercetin, and jacareubin contributed antioxidant and cytotoxic activities of areca nut extract.¹ Our previous study showed that areca nut extract had cytotoxic activity againts human oral cancer cell lines such as HSC-2 and HSC-3 with IC₅₀ values of 629.50 and 164.06 μg/mL, respectively.² The selectivity of the areca nut extract was verified by using normal cell line. Areca nut had no cytotoxic effect on human keratinocyte (HaCaT) cell line. Instead, it induced high rate of proliferation in HaCaT cells. Chrysanthemum or chrysanth (Dendrathema grandiflora; Asteracea) is a flowering plant originating from East Asia and dominantly grows in China. The flowers are generally consumed in herbal teas and as a supplement. This genus comprises 40 species and contains flavonoids (flavanone), terpenoids, anthocyanins, and steroids.^3,4 The selectivity index of Chrysanthemum indicum and morifilium oils were calculated 1.8 and 1.5, respectively, indicating low selectivity against trypanosomes compared to their toxicity versus HL-60 cells.⁵ Previous studies reported the effect of increasing keratinocyte proliferation and skin regeneration derived from Chrysanthemum boreale.¹¹ Chrysanthemums are usually used for treating allergies, anxiety, hypertension, inflammation, headache, cold, sore throat, and tinnitus by certain communities.¹²

However, scientific research has not been conducted related to the healing process on the oral mucosa by oral application of these herbal plants. This study aimed to evaluate the potential of the combination of areca nut and chrysanthemum oral gel on oral mucosa using the Sprague–Dawley rat model.

Methods

Results

Oral ulcer and body weight evaluations

The change comparison of ulcer size analysis in the buccal mucosa of rats showed a significant decrease in ulcer size in F1, F2, F3, and positive control groups (Figure 1A–J, Table 4). This observation was carried out on the second day after the ulcer was formed and at the end of the experiment. The most significant decrease in ulcer area was seen in positive control (triamcinolone acetonide orabase), F2, and F3 treatment groups. Notably, the average ulcer size in positive control, F2, and F3 treatment on the second day was 10.10 ± 0.17, 10.08 ± 0.16, and 10.08 ± 0.18 mm², respectively. On the seventh day, it was seen that the size of the ulcer had almost closed, namely 0.03 ± 0.01, 0.11 ± 0.07, and 3.49 ± 0.42 mm², respectively. In the end of experimental period, the clinical evaluation showed that although ulcers in the F2 group has smaller size, but the ulcers in the F3 group have tighter healing wound closure and more cicatricial tissue formation than other treatment groups. These data analysis showed that the ulcer healing in a dose-dependent manner in clinical analysis. Analysis of changes in bodyweight showed a significant increase in the normal, positive control, and F2 groups.

Table 4. Effect of areca nut and chrysanthemum oral gel on the area of ulcer and body weight changes in rats on the 2nd and 7th experimental days (n = 72).

Groups	Clinical evaluation
	Area of ulcer changes (mm 2)				Body weight changes (g)
	2 nd day	7 th day	95% CI	p	2 nd day	7 th day	95% CI	p
Negative control	9.34 ± 0.69	5.24 ± 8.89	6.34, 6.84	0.000	118.58 ± 5.29	117.25 ± 6.19	−1.31, 3.97	0.291
Positive control	10.10 ± 0.17	0.03 ± 0,01	9.96, 10.19	0.000	109.58 ± 6.89	117.17 ± 7.50	−12.31, −2.84	0.005
F1	10.04 ± 0.18	2.84 ± 0.44	6.89, 7.52	0.000	119.17 ± 13.27	115.33 ± 14.46	2.12, 5.54	0.000
F2	10.08 ± 0.16	0.11 ± 0.07	9.86, 10.08	0.000	116.83 ± 4.41	121.58 ± 4.87	−8.10, −1.39	0.010
F3	10.08 ± 0.18	1.81 ± 0.71	6.25, 7.59	0.000	120.50 ± 4.32	117.75 ± 6.82	0.35, 5.14	0.028
Normal	0	0	0		120.33 ± 4.77	125.67 ± 8.78	1.05, 9.61	0.019

Table 5. Organoleptics characteristics and physical stability tests of F III kept at 40°C/75% RH.

Evaluation	Weeks of storage
	0	2	4	6	8	95% CI	p
Organoleptic characteristics
Color	Dark chocolate	Dark chocolate	Dark chocolate	Dark chocolate	Dark chocolate
Aroma	Typical aromatic	Typical aromatic	Typical aromatic	Typical aromatic	Typical aromatic
Taste	Bitter	Bitter	Bitter	Bitter	Bitter
Shape	Condensed	Condensed	Liquid	Liquid	Liquid
Homogeneity	Homogeneous	Homogeneous	Homogeneous	Homogeneous	Homogeneous
pH	5.50 ± 0.03	5.52 ± 0.02	5.50 ± 0.03	5.45 ± 0.02	4.49 ± 0.01
Spreadability (cm)	5.53a ± 0.09	5.72a ± 0.08	6.21b ± 0.18	6.73c ± 0.15	6.79c ± 0.09	−1.37, −7.36d	0.000
Viscosity (m.Pas)	202.24e ± 1.12	184.52e ± 4.67	181.03e ± 0.98	161.96e ± 2.73	155.82e ± 0.45	−4.54, 35.42d	0.129

a Significantly different from the week 4, 6, and 8;

b Significantly different from the week 0, 2, 6, and 8;

c Significantly different from the week 0, 2, and 4;

d 95% CI week 0 vs week 8,

e No significant differences of viscosity among weeks of storage time (p > 0.05).

Figure 1. Clinical analysis of the traumatic ulcers in the buccal mucosa on the second and seventh day.

Negative control: base gel treatment group (A and B); Positive control: triamcinolone acetonide treatment (C and D); F1 treatment: 20% areca nut:80% chrysanthemum (E and F); F2 treatment: 50% areca nut:50% chrysanthemum (G and H); F3 treatment: 80% areca nut:20% chrysanthemum (I and J); Normal: without ulcer and treatment (K and L).

Table 6.

Effect of areca nut and chrysanthemum oral gel on histological scores, number of connective tissue cells that expressed TNF-α, and percentage of collagen deposition area on the 7 ^th experimental day (n = 18 specimens).

Parameter	Microscopic evaluation
	Groups
	Negative control	Positive control	F1	F2	F3	Normal	95% CI	p
Histological scores	0	1.34 ± 1.53	1.67 ± 1.53	0	1.34 ± 0.58	0		0.107
TNF-α immunostaining scores	1.33 ± 0.58	2.67 ± 0.58	1.00 ± 1.53	2.33 ± 0.58	1.33 ± 0.58	1.00 ± 0		0.025
Collagen deposition area (%)	5.63a ± 2.28	14.49 ± 0.95	17.73b ± 9.32	12.63c ± 0.92	25.4d ± 2.12	9.40 ± 1.05	1.61, 23.93e	0.001

a Significantly different from F1 and F3 groups.

b Significantly different from negative control.

c Significantly different from F3.

d Significantly different from F2, negative control, and normal groups.

e 95% CI of F2 compared with F3. Negative control: base gel treatment group; Positive control: triamcinolone acetonide treatment; F1: 20% areca nut:80% chrysanthemum; F2: 50% areca nut:50% chrysanthemum; F3: 80% areca nut:20% chrysanthemum; Normal: without ulcer and treatment.

Histological evaluation

To verify the mechanism of ulcer healing by areca nut and chrysanthemum oral gel, the ulcers were subjected to histological examination. As seen in Table 6, the mean of histological scores on the seventh day showed that positive control, F1, and F3 groups indicated that there were no ulcers in the epithelial layer, presence of fibrosis, and slight chronic inflammation in the conjunctive tissue. The score 0 indicating no ulcers and remodeling in the conjunctive tissue was seen in the negative control, F2, and normal groups, but significant differences between groups were not detected in histology examination. Analysis of photomicrograph of ulceration showed loss of ulcer with remodeled connective tissue and accompanied by a slight inflammatory infiltrate in Figure 2B, 2C, and 2E (score 1). There were no visible inflammations in Figure 2A, 2D, and 2F (score 0).

Figure 2. Histopathological analysis of the traumatic ulcers in the buccal mucosa at second and seventh day.

(A) Negative control: base gel; (B) Positive control: triamcinolone acetonide in orabase; (C) F1: 20% areca nut:80% chrysanthemum; (D) F2: 50% areca nut:50% chrysanthemum; (E) F3: 80% areca nut:20% chrysanthemum; (F) Normal: without ulcer and treatment (Hematoxylin and eosin, 40×).

Immunohistochemical evaluation

The immunostaining for TNF-α aims to show changes in TNF-α expression released by connective tissue cells in the cytoplasm during the healing process ( Figure 3). The increase in the expressions with a mean score of 2 (moderate) was showed by positive control (2.67 ± 0.58) and F2 groups (2.33 ± 0.58) (Table 6). The lower scores (1-mild) were seen in the negative control, F1, F3, and normal groups. This study showed significant differences between the six groups with changes in TNF-α expression. These results were in line with the pictures that revealed brown diffuse granular-like staining pattern of the connective tissue and epidermis in the Figure 3B and 3D, while the other groups did not show dominant TNF-α expressions except in the F3 group, the immunostaining of TNF-α was prominently found in the basal cell layer (Figure 3E).

Figure 3. Immunohistochemistry analysis of the traumatic ulcers in the buccal mucosa on the second and seventh day.

(A) Negative control: base gel; (B) Positive control: triamcinolone acetonide in orabase; (C) F1: 20% areca nut:80% and chrysanthemum; (D) F2: 50% areca nut:50% chrysanthemum; (E) F3: 80% areca nut:20% chrysanthemum; (F) Normal: without ulcer and treatment (TNF-α immunostaining, 40×).

Collagenase evaluation

The results of red picrosirius staining on the last experimental day showed the highest collagen deposition in the F3 group with a mean of 25.4 ± 2.12, while collagen deposition in low level was seen in the F2, normal, and negative control group (12.63 ± 2.28, 9.04 ± 1.05, 5.63 ± 2.28, respectively) ( Table 6). Figure 4B (Positive control) and 4D (F2 group) showed regular, dense, and thick collagen, whereas, in the negative control (Figure 4F), collagen deposition showed thin, irregular, and many empty gaps. The ANOVA analysis showed significant differences (p < 0.05) in collagen expression between groups (Table 6). The Tukey test demonstrated significant differences between F2 and F3 groups. Other significant differences were evaluated between F1, F2, F3, and negative control groups.

Figure 4. Collagenase analysis of the ulcerations in the buccal mucosa on the seventh day.

(A) Negative control: base gel; (B) Positive control: triamcinolone acetonide in orabase; (C) F1: 20% areca nut:80% chrysanthemum; (D) F2: 50% areca nut:50% chrysanthemum; (E) F3: 80% areca nut:20% chrysanthemum; (F) Normal: without ulcer and treatment (Picosirius red staining, 40×). Yellow arrow: Disintact collagen bundles. Blue arrow: Intact collagen bundles.

Discussion

The preparation of the oral topical gel in this study using a CMC-Na-based gel. The gel form was chosen because it is easy to dry and provides a cool feeling on the mucosal surface. The gel formulation was carried out by trial-error by modifying the gelling agent. The aim of designing a stable oral topical gel is to deliver antioxidant properties effectively over its shelf life. Gel stability can be affected by environmental stress such as temperature and humidity. The decrease in the effect of the active gel ingredients can be observed from physical and chemical changes such as color, shape, texture, odor, and pH. The stability test in our research is a step in optimizing the preparation based on the selection of the appropriate gelling agent level so that the oral gel can be stable in various storage settings. The organoleptic characteristics of the optimum gel (F III) showed promising stability because there were no significant changes in color, odor, texture, and shape. The gel is said to be homogeneous if there is an even color equation and no different particles are found. The consistency, acidity, and spreadability of the gel are related to the comfort of use on the surface of the oral mucosa. The disadvantages of the gel are that it cannot adhere tightly to the mucosa surface and is easily soluble in saliva. This research showed that viscosity stability is an important consideration in the flow and texture properties of the gel. The stability of the constituents extract in the gel is also important because the pharmacological properties depend on the chemical viability of the extract. As pointed out above, the catechins are pharmacologically active constituents of the areca nut; however they are more stable at high concentrations around pH 4 and low temperature.⁹ Their stability is affected by several factors such as pH, temperature, and concentration in solution state.¹⁰

In the present study, we demonstrated that application of the oral gel to ulcerated lesions for 6 days showed significant lesion healing in the composition of the oral gel containing 50% areca nut:50% chrysanthemum and 80% areca nut:20% chrysanthemum. This significant wound healing was based on the clinical appearance such as ulcer size reduction, reepithelization over the ulcer base, and formulation of granulation tissue around the ulceration. These results have the same clinical appearance as the positive control group that using triamcinolone acetonide. Areca nut, as one of the main ingredients, contains phenolic and flavonoids which function as antioxidants.¹⁹ Catechin is a component of flavonoids present in areca nut that acts as an antioxidant by chelating the ions and scavenging the free radicals particularly, superoxide (O ^2–), peroxyl, hydroxyl radicals (·OH), and hence inhibit both DNA damage and lipid peroxidation, which can cause membrane damage.²⁰ Catechin can reduce the expression of interleukin-6 (IL-6) and IL-8 which function to overcome inflammation and increase the wound healing process by chemotactic for fibroblasts, accelerates their migration and stimulates deposition of tenascin, fibronectin, and collagen I during wound healing in vivo.^21–24 The success of using areca nut in oral gel to heal the ulcers is in line with a previous study using ointment with 2% ethanolic extract on burns on the skin.¹⁰ This study also used a combination with chrysanthemum. Previous studies have shown that chrysanthemum has antioxidant, antiproliferative, antimicrobial, acetylcholine esterase inhibition, antitimelanogenic, and antiviral activities, especially Chrysanthemum morifolium, Chrysanthemum indicum, and Chrysanthemum coronarium. The active phytochemistry content of chrysanthemum is apigenin-7-O-glucoside.²⁵

The wound in untreated rats would recover without treatment for a certain period. The wound healing process normally begins immediately after the damage occurs, but the mechanism and speed of repair of damaged tissue depend on the type of wound and the body’s defense system. A normal wound healing progresses through inflammatory, proliferative, and remodeling phases in response to tissue injury. Impairments of any of these phases can cause the wound to be in a chronic, non-healing state. Although the body has its wound healing mechanism without the help of external medicinal materials, the acceleration of wound healing can be assisted by intervention in the form of a supply of materials and nutrients that can be obtained from outside to guide the process back to completion. In this study, medicinal plants have roles in accelerating the process of wound healing. Collagen is an important extracellular matrix component in the regulation of the phases of wound healing in its native. The administration of a combination of areca nut and chrysanthemum extract gel on wounds of the oral mucosa can produce better-wound healing with the ability to stimulate collagen and TNF-α activity so that it can increase the average wound contraction and increase re-epithelialization. This condition is expected to significantly reduce wound healing time.

The wounds on the buccal mucosa of rats can heal on their own, but the results of our study prove that there is a difference in wound healing in the group that received treatment and the group without treatment. In addition to the results from clinical examinations, differences in healing speed were also supported by differences in test results for TNF-α levels and collagenase activity. The technique of applying the oral gel to the oral mucosa of rats is important in this research process because at the time of application the rats are not anesthetized, so there is a possibility that the gel is not maximally absorbed in the oral mucosa and most of it dissolves in saliva and is swallowed by rats. In this study, the oral gel was applied over the wound and held for 1 minute. The drug partitions dissolved into the mucous membrane and within a few minutes will reach equilibrium with the drug in solution in the oral cavity. One of the previous studies revealed the existence of the term pseudo-equilibrium which refers to the balance of a drug in solution and a drug in tissue in a relatively short contact time in the oral cavity before a dose of a drug is swallowed. This pseudo equilibrium has an absorption half-life of about 0.87 minutes (52 seconds).²⁶ It means that the drug partition in the oral gel was absorbed by the oral mucosa.

Several factors that influence the fast or slow absorption of drugs on the mucosal surface are permeability and blood supply.²⁷ The buccal mucosa is the part that has a high permeability making it easier for drugs to enter. The buccal mucosa has a thin layer of nonkeratinized epithelium consisting of the stratum basale, filamentous, and distendum which facilitates absorption of the drug substance into the epithelium. In addition, the buccal mucosa lacks the intercellular lamellar bilayer structure found in the stratum corneum and hence is more permeable. Another factor that increases tissue permeability is the rich blood supply in the oral mucosa, which causes the absorption of drugs from the oral cavity into the mucosal tissue to be faster.

This present study observed the body weight of rats before and after application. There are similarities between ulcer repair and weight gain. A significant increase in body weight in the F2 and positive control showed faster healing of ulcers, making it easier for rats to consume more food. The use of healthy rats aims to evaluate the effect of ulcers in the oral cavity purely based on the ability of rats to gain healing process without being affected by systemic diseases that can affect rat’s body weight. Areca nut is capable of repressing prostaglandin E ₂ (PGE₂) and arachidonic acid-induced inflammation, thereby helping to accelerate ulcer healing.²⁸ The analgesic or antinociceptive activity of the areca nut is originated by inhibition of prostaglandin synthesis. The oral gel form was chosen to facilitate absorption in the oral mucosa and it has a rapid cool soothing effect, although the drawback is that the gel is easily soluble in saliva. Application twice a day in the morning and evening regularly showed a fairly good cicatrization process in both groups (F2 and positive control). The increase in body weight in the rat showed that in the last few days the diet was not disturbed by the size of the ulcer. In normal group, we also reported that there was a significant increase of body weight after feeding with areca nut. This is probably due to the areca nut extract’s ability to increase body weight. Its mechanism of action is that the main component of areca alkaloids is an inhibitor of the gamma-aminobutyric acid (GABA) receptor.²⁹ The inhibitory effects of GABA receptor may increase appetite with eventual fat accumulation in general and central area of the body. Another mechanism that might play role in gain weight is that the areca nitrosamines derived from alkaloids may induce insulin resistance which can cause hyperglycemic conditions. Another study has reported that areca nut chewing has closely associated with metabolic syndrome, general obesity, and type-2 diabetes mellitus.³⁰

Histological examination with HE staining on day seven showed no significant difference between all treatment groups, normal, positive, and negative control groups. The oral gel containing a balanced composition of areca nut and chrysanthemum, negative control, and normal groups did not show any ulcers and the epithelial tissue had been completely remodeled. Meanwhile, the other group still showed fibrosis and inflammation without ulcers. Fibrosis, persistent neutrophil infiltration, and poor angiogenesis in the ulcer base are factors involved in the underlying mechanism of delayed healing ulcers.³¹ The ulcer healing mechanism begins with ulcer base contraction which includes contraction of the ulcerated area, re-epithelization over the ulcer base, and formation of granulation tissue. The next step is epithelial regeneration and mucus secretion. This epidermal repair is regulated by a key growth factor (KGF) that is expressed at very high levels within 24 h of injury on dermal skin.³² The buccal mucosa is a non-keratinocyte layer, so it is probably the most involved in the re-epithelization process in the oral mucosa is salivary mucin. Mucin is the primary gel-forming component of mucus that provide a critical layer of protection on the wet epithelial surface including gastrointestinal, female genital, and respiratory tracts.³³ Angiogenesis is the next step of ulcer healing. It allows the supply of nutrients and oxygen to the ulcerated area thereby giving the collagen in connective tissue and epithelium an opportunity to recover. Matrix formation is the last step of ulcer repair. This remodeling formation is regulated by the temporospatial expression of the fibrillar and basement membrane collagen (types I, III, IV), and matrix metalloproteinase-2 (MMP-2). Fibronectin is important for cell proliferation, adhesion, differentiation, and matrix formation.

In the present study, the analysis of TNF-α showed that the increase in the moderate expressions was showed by positive control and F2 groups. This is in line with research by Kobayashi et al. which showed that the water-soluble component in dried chrysanthemum has antioxidant ability to increase TNF-α. This activity was observed within 3 hours after ulcer formation.⁸ TNF-α is a cytokine produced primarily by immune cells such as monocytes/macrophages, but several cell types, such as T and B lymphocytes, natural killer cells, neutrophils, fibroblasts, and osteoclasts can also secrete TNF-α in smaller quantities.³⁴ TNF-α levels which had the same score between F2 and positive control on the seventh day showed that the amount of this cytokine was still high detected in the local connective tissue. Prolonged or remaining elevated expression of TNF-α may occur for up to the third to the fifth day following the traumatic injury.³⁵ Prolonged increase in TNF-α especially in delayed healing reflects their augmented blood TNF-α, suggesting an association between impaired healing and hard work TNF-α in the inflammatory response so that wound healing is achieved.³⁶ In this study, we did not examine the level of TNF-α in the blood so that the role of areca nut-chrysanthemum oral gel to accelerate wound healing systemically cannot be explored.

TNF-α is a cytokine that has pleiotropic effects on various cell types. The pleiotropic effects mean that TNF-α can affect multiple systems or exert multiple pharmacological activities. It has been identified as a major regulator of inflammatory responses and is known to be involved in the pathogenesis of some inflammatory and autoimmune diseases. Although the precise role of TNF-α is not fully understood, among the pro-inflammatory cytokines, TNF-α is an important cytokine because it is quickly released and initiates inflammation in wound tissues. Ritsu et al. reported that TNF-α is involved in the early process of wound healing.³⁷ TNF-α is released rapidly by vascular endothelial cells, keratinocytes, and fibroblasts in the lesion area and initiates the inflammatory phase by promoting inflammatory leukocyte recruitment into the wounded tissues. The major cellular sources of TNF-α are changed into recruited neutrophils and macrophages, and this process yields a positive amplification circuit for extending the inflammatory responses. An addition of inflammatory phase, TNF-α also plays a role during the proliferation phase in the wound healing process. TNF-α secreted from macrophages promotes the formation of the extracellular matrix in the wounded tissues by inducing the generation of proteoglycan and fibronectin by fibroblasts. This process is further enhanced by collagen synthesis from the proliferating fibroblasts, which are promoted by tumor growth factor (TGF)-β.

Triamcinolone acetonide was chosen as a positive control because this preparation has efficacy as a synthetic corticosteroid that has anti-inflammatory, antipruritic, and anti-allergic effects. This preparation has the advantage of being an emollient dental paste that can attach the drug to the oral mucosal tissue so that it is not easily soluble in saliva. An important role of steroids in triamcinolone acetonide is to alter the localization of high junction proteins thereby increasing the adhesion density between epithelial cells.³⁸ A tight epithelial protective barrier can be achieved through tight cell adhesion. Contacts between adjacent cells are made up of tight junctions, adherens junctions, and desmosomes which help in preserving tissue homeostasis. This study showed that with the use of triamcinolone acetonide, F2, and F3 group on the seventh day, the ulcers had closed tightly without any inflammation. These results prove that there might be a possibility of areca nut–chrysanthemum oral gel also can close the adhesion bonds between epithelial cells so that the level of wound closure density which is similar to that of the group positive control (triamcinolone acetonide).

Collagenase analysis showed a lot of collagen deposition in the F3 group. The results of this study are in line with the fact that the alkaloids in areca nut are potent inducers to increase the amount of collagen. Previous research has shown that a series of synthetic arecaidine esters by oral mucosa fibroblast was closely correlated with the extent of collagen synthesis.³⁹ Alkaloids in the areca nut in a long term play a major role in the accumulation of collagen in oral submucous fibrosis (OSF). Collagen is a key component of the extracellular matrix that plays a critical role in the regulation of the phases of wound healing. Collagen exposure due to injury in the inflammatory phase will activate the clotting cascade, resulting in a fibrin clot that stops the initial bleeding. It also acts as potent chemoattractants for neutrophils, enhancing phagocytosis and immune response and modulating gene expressions. Collagen promotes anti-inflammatory, proangiogenic wound macrophage phenotype via microRNA signaling. In the angiogenesis phase, collagen stimulates angiogenesis in vitro and in vivo through the engagement of specific integrin receptors. The C-propeptide fragment of collagen I recruit endothelial cells, potentially triggering angiogenesis in the healing wound.⁴⁰

The results of the evaluation of collagen deposition after treatment, with picrosirius red staining, showed a picture of collagen with thicker density, large fiber size, and lots of collagen fibrils in the oral gel with higher composition of areca nut which consisted 80% of areca nut. This shows the good work of collagen to repair ulcers. Although the result of the collagenase assay has a significant p value, the range in the confidence interval (CI) is very wide. It indicates that these results have great uncertainty about the effect of the extract content in the gel for ulcer healing.¹¹ Further research is needed by using a larger sample size so that it can produce narrower estimated CIs and the estimated effect could become more reliable.

Conclusion

The present study provides the activity of the healing effect of combination areca nut and chrysanthemum. This oral gel can improve ulcer wound healing through the role of TNF-α and collagen with the same results as topical steroid therapy.

Data availability