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

Liza Meutia Sari; Zaki Mubarak; Dina Keumala Sari

doi:10.12688/f1000research.54887.1

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Research Article

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

[version 1; peer review: 1 approved with reservations, 1 not approved]

Liza Meutia Sari ¹, Zaki Mubarak², Dina Keumala Sari³

PUBLISHED 21 Jul 2021

Author details Author details

¹ Oral Medicine, Syiah Kuala University, Banda Aceh, Indonesia, 23111, Indonesia
² Oral Microbiology, Syiah Kuala University, Banda Aceh, Indonesia, 23111, Indonesia
³ Nutrition, Sumatera Utara University, Medan, Indonesia, 20155, Indonesia

Liza Meutia Sari
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Zaki Mubarak
Roles: Project Administration, Supervision, Validation, Visualization

Dina Keumala Sari
Roles: Conceptualization, Supervision, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

Abstract

Background: Areca nut (Areca catechu Linn.) is the seed of the fruit of the oriental palm that is commonly used among Southeast Asian communities. Chrysanthemum (Dendrathema grandiflora) is a flowering plant originating from East Asia and dominantly grows in China. Both of these plants have strong antioxidant activities. To investigate the mechanism of their wound healing activities, we prepared areca nut and chrysanthemum polyethylene oral gel and performed several in vivo assays using Sprague–Dawley rats.
Methods: Sprague–Dawley rats were divided into five groups: Negative control group (rats with base gel treatment), positive control group (rats treated with triamcinolone acetonide), F1 (treatment with 20% areca nut:80% chrysanthemum), F2 (treatment with 50% areca nut:50% chrysanthemum), and F3 (treatment with 80% areca nut:20% chrysanthemum). Traumatic ulcers were performed on the buccal mucosa of all experimental animals that received topical oral gel and triamcinolone acetonide twice a day for seven days. The clinical and histological characteristics were analyzed and scored.
Results: During the six days, the ulcerated area receded linearly over time and was completely cicatrized in F2 and positive control group (Dependent t-test, p<0.05). There were significant increases in body weight in F2 and positive control groups. There were no significant differences between groups in histology examination (Kruskal Wallis test, p<0.05). The moderate score of TNF-α levels was seen in F2 and positive control groups (ANOVA/Tukey test). Similar results were seen in the collagenases assay.
Conclusions: A balanced combination of areca nut and chrysanthemum extract in the oral gel can optimize the healing of traumatic oral ulcers in rats through the increase of TNF-α and collagen deposition.

Keywords

Oral ulcer, Areca nut, Chrysanthemum, TNF-α, Collagen

Corresponding author: Liza Meutia Sari

Competing interests: No competing interests were disclosed.

Grant information: The study was supported by grant from the PNBP fund Syiah Kuala University No 319/UN11.2.1/PT.01.03/PNBP/2021.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Meutia Sari L et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Meutia Sari L, Mubarak Z and Keumala Sari D. Evaluation of clinical, histology, TNF-α, and collagen expressions on oral ulcer in rats after treatment with areca nut and chrysanthemum oral gel [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2021, 10:623 (https://doi.org/10.12688/f1000research.54887.1) First published: 21 Jul 2021, 10:623 (https://doi.org/10.12688/f1000research.54887.1) Latest published: 12 Sep 2025, 10:623 (https://doi.org/10.12688/f1000research.54887.7)

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.), 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 Chrysanthemum or chrysanth (Dendrathema grandiflora) 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. 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

Ethical approval

This study was approved by the Ethics Committee for Animal Research of Tropical Biopharmaceutical Research Center (Trop BRC), Bogor Agricultural University, West Java, Indonesia, with number 042-2020-KEH TROP BRC.

Extract preparation

Areca nuts were obtained from the Pinang plant from Aceh Besar, Indonesia. 2 kg of areca nuts (gross weight) were cleansed of dirt and dried in the open air and sunlight. Further drying was done using an oven set at a temperature of 50°C. The nuts were crushed using a blender and then strained with a 20-mesh sieve. The maceration process was conducted using 96% ethanol diluent solvent for 7 days before being subsequently filtered and evaporated using a vacuum rotary evaporator at 30–40°C and then reconcentrated using a water bath until a solid dry powder extract was obtained. Chrysanthemum polyethylene (P.E.) (Product No. 1237X17911) was provided by Javaplant Company, Indonesia.

Formulation of areca nut and chrysanthemum extract oral gel

The extract was formulated into four gel dosage forms with various concentrations of the extract combination, namely F1 (20% of areca nut:80% of chrysanthemum), F2 (50% of areca nut:50% of chrysanthemum), F3 (80% of areca nut:20% of chrysanthemum), and base gel (gel without areca nut and chrysanthemum). Preparation of the extract oral gel began with making two mixtures. The first mixture consisted of a carbamoyl and extract combination of areca nut and chrysanthemum and mixed with 10 mL of water at 70°C. The second mixture consisted of methylparaben dissolved in a little water and then mixed with a mixture of glycerin and propylene glycol. This mixture was then combined and stirred with water so that it is mixed homogeneously.

Experimental animals

A total of 25 adult male Sprague–Dawley rats weighing 200–240 g were provided by the animal laboratory at Tropical Biopharmaceutical Research Center (Trop BRC), Bogor Agricultural University. The rats were kept for adaptation at 25°C in the well-ventilated laboratory for 14 days under a 12/12 h light/dark cycle and fed with a standard pellet diet and tap water ad libitum before being entered into the experiment. All the animals were given an initial examination for systemic health conditions and stored in boxes with sawdust.

Experimental protocol to induce the ulcers

Ulcer induction was performed after the animals were anesthetized by intraperitoneal injection of 33 mg/kg of ketamine and 13 mg/kg of xylazine (2%). The left buccal mucosa was smeared with 10% povidone–iodine on cotton pellets. An abrasion was made with 5 mm diameter, 1 mm depth, and was limited to the mucosa without muscular involvement. The ulceration was performed by using a number 15 scalpel blade. The standardization area was marked with an 8-mm diameter demarcator. The surgical and technique was standardized for animals and performed by the same operator.¹³ The ulceration was not performed in the normal group. The formation of the ulcers could be observed after 24 hours.

Groups and treatment

The animals were randomly divided into five groups with 12 animals each: negative control group (rats with base gel treatment), positive control group (rats treated with triamcinolone acetonide), F1 (treatment with 20% areca nut:80% chrysanthemum), F2 (treatment with 50% areca nut:50% chrysanthemum), and F3 (treatment with 80% areca nut:20% chrysanthemum). A normal group (without ulcer and treatment) was added for the microscopic evaluation. All the groups were treated every 12 hours for seven days with topical application. The application of the oral gel was performed by using the individual sterile and disposable dental micro brush (SDent, USA). Each group was sacrificed gradually through the end of the seventh-day study period. After the animals were sacrificed, a section of buccal mucosa containing the ulcer of each rat was collected. Histopathology, immunohistochemistry, and collagenases evaluation were analyzed in Primate Research Center, Bogor Agricultural University, Indonesia.

Clinical evaluation

The animal body weights and diameter of the ulcers were measured before and after treatment on the second and seventh days. The diameter of the ulcers were measured with the naked eyes using a digital stainless vernier caliper which is capable of measuring diameter in 0.05 mm increments.¹⁴^,¹⁵ All measurements were performed by the same operator.

Histopathological analysis

The collected fragments of ulcers were identified and immersed in 10% formol for 24 hours. After fixation in formol, the specimens were macroscopically analyzed, subjected to dehydration in crescent alcoholic series, diaphanized in xylol, impregnated in paraffin, and melted at 60°C. The fragments were packed in paraffin-forming blocks at room temperature. The fragments were sectioned to 5 μm in thickness through the use of microtome and histology using routine coloration by hematoxylin-eosin (HE) was performed. The histopathological parameters were determined and scored from 0 to 4 according to previously published criteria (Table 1).¹⁶

Table 1. Histopathologic analysis of induced oral ulcers (histological scores).

Score	Epithelium	Conjunctive tissue
0	No ulcer	Remodeled
1	No ulcer	Fibrosis and slight chronic inflammation
2	With ulcer	Fibrosis and moderate chronic inflammation
3	With ulcer	Chronic inflammation process (granulation tissue)
4	With ulcer	Acute process (dilated vessels, mixed inflammatory infiltrate with neutrophils)

Table 2. 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 = 60).

Groups	Clinical evaluation
	Area of ulcer changes (mm²)			Body weight changes (g)
	2^nd day	7^th day	p	2^nd day	7^th day	p
Negative control	9.34 ± 0.69	5.24 ± 8.89	0.131	118.58 ± 5.29	117.25 ± 6.19	0.291
Positive control	10.10 ± 0.17	0.03 ± 0,01	0.000*	109.58 ± 6.89	117.17 ± 7.50	0.005*
F1	10.04 ± 0.18	2.84 ± 0.44	0.000*	119.17 ± 13.27	115.33 ± 14.46	0.000*
F2	10.08 ± 0.16	0.11 ± 0.07	0.000*	116.83 ± 4.41	121.58 ± 4.87	0.010*
F3	10.08 ± 0.18	3.49 ± 0.42	0.000*	120.50 ± 4.32	117.75 ± 6.82	0.028*

Immunohistochemistry analysis

The selected tissue sections of the ulcers (2.5 μm) were deparaffinized and rehydrated with distilled water. Blocking endogen peroxidase activity was done using 3% hydrogen peroxide solution for 15 minutes, followed by washing with phosphate-buffered saline (PBS) three times every 5 minutes. After protein blocking, the dripping of Biocare’s Background Sniper was performed and the specimens were incubated for 15 minutes at 37°C. The dripping of normal serum was performed and incubated for 60 minutes at 37°C. The specimens were washed with PBS and anti-TNF-α antibody (TNFA/1172) (ab220210Abcam), was diluted at 1:100, and then dripped and incubated at 4°C for two days. After washing with PBS, the secondary antibody goat anti-rabbit IgG H&L horseradish peroxidase (HRP) (RRID: AB_955447; ab6721-Abcam) was given for 30 minutes at 25°C and followed by Betazoid DAB chromogen solution (BDB2004H-Biocare Medicare LLC). It was used for increasing stability and staining intensity for HRP detection in the specimens. The specimens were washed with distilled water and checked with the microscope. After counterstaining with hematoxylin for 30 seconds, the specimens were rinsed in tap water, dehydrated, purified, and mounted. The percentage of nuclear and cytoplasmic expression in the connective tissue was divided into four scores, namely: 0: no positive cells; 1 (mild): 1–33% of positive cells; 2 (moderate): 34–66% of positive cells; 3 (intense): 67–100% of positive cells. Two observers analyzed the same scores until they were considered as the final scores.¹⁷

Collagenesis analysis

The same fragments were sectioned to a thickness of 3 μm, de-wax, hydrate paraffin section, and stained using picrosirius red (solution A) for one hour. After washing in acidified water (solution B), the specimens were dehydrated and mounted for observation under polarized light microscopy. The collagen bundles shown in red in the image were then calculated using Adobe Photoshop CC 2017 (RRID:SCR_014199; GNU Image Manipulation Program (RRID:SCR_003182) is an open-access alternative) and ImageJ (v1.50i) (RRID:SCR_003070) software. The percentage of collagen was derived from collagen area pixel divided by tissue pixel and multiply by 100%. The mean of three percentages was used as a sample unit.¹⁸

Statistical analysis

All experiments were carried out in triplicate and the data were expressed as mean ± standard deviation (SD). Statistical analysis was performed by using the SPSS 20 software (SPSS Inc., Chicago, USA) (RRID:SCR_019096); JASP (RRID:SCR_015823) is an open-access alternative. Clinical evaluation in changes of the area of ulcer and body weight of rats analyzed by the dependent t-test. Differences among samples in histopathological and immunohistochemistry analysis were evaluated by using the Kruskal–Wallis test. Collagenesis analysis was evaluated by using analysis of variance (ANOVA/Tukey test). A significant difference was assumed at p < 0.05.

Results

Clinical evaluation

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 2). 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) and F2 treatment groups. Notably, the average ulcer size in positive control and F2 treatment on the second day was 10.10 ± 0.17 and 10.08 ± 0.16 mm². On the seventh day, it was seen that the size of the ulcer had almost closed, namely 0.03 ± 0.01 and 0.11 ± 0.07 mm². These data analyses showed that treatment with areca nut and chrysanthemum oral gel caused significantly (p < 0.05) ulcer healing in a dose-dependent manner. Analysis of changes in body weight showed a significant increase in the F2 and positive control groups, while a significant decrease in body weight occurred in the F1 and F3 treatment groups (Table 2). Meanwhile, in the negative control group, there was a decrease in body weight, but it was not significant.

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).

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 3, 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).

Table 3. 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).

Parameter	Microscopic evaluation
	Groups
	Negative control	Positive control	F1	F2	F3	Normal	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 (%)	14.90 ± 0.95	17.73 ± 9.32	12.63 ± 0.92	25.4 ± 2.12	5.63 ± 2.28	9.40 ± 1.05	0.001*

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 group: 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 3). 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 group: without ulcer and treatment (TNF-α immunostaining, 40×)

Collagenesis evaluation

The results of red picrosirius staining on the last experimental day showed the highest collagen deposition in the F2 group with a mean of 25.4 ± 2.12, while the lowest collagen deposition while collagen deposition in low level was seen in the F3 group (5.63 ± 2.28) (Table 3). 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 3). 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. Collagenesis analysis of the ulcerations in the buccal mucosa on the seventh day.

Discussion

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 balanced areca nut and chrysanthemum (50%:50%). 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. This result has 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

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 E₂ (PGE₂) and arachidonic acid-induced inflammation, thereby helping to accelerate ulcer healing.²⁶ 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.

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.²⁷ 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.²⁸ 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.²⁹ 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. ³⁰ 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.³¹ 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.³² 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.

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.³³ 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 and F2 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).

Collagenesis analysis showed a lot of collagen deposition in the F2 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.³⁴ 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.³⁵

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 a balanced composition of areca nut and chrysanthemum and a positive control group. This shows the good work of collagen to repair wounds. What's interesting from this observation is that, although there were groups that received treatment with a higher composition of areca nut in oral gel, this was not able to induce collagen as in a balanced composition of areca nut and chrysanthemum. This explains that the ulcer healing rate in this study was not dose dependent but more dependent on the peak efficacy of each phytochemistry composition of an extract

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

Underlying data

Dryad: Underlying data for ‘Evaluation of clinical, histology, TNF-α, and collagen expressions on oral ulcer in rats after treatment with areca nut and chrysanthemum oral gel’. https://doi.org/10.5061/dryad.8w9ghx3mw.

The project contains the following underlying data: body weight, oral ulcer’s size, histology and tnf-α score, collagen percentage.

References

1. Seyyedi SA, Sanatkhani M, Pakfetrat A, et al.: The therapeutic effects of chamomilla tincture mouthwash on oral aphthae: A Randomized Clinical Trial. J Clin Exp Dent. 2014; 6(5): e535–8. PubMed Abstract | Publisher Full Text | Free Full Text
2. Fitzpatrick SG, Cohen DM, Clark AN: Ulcerated lesions of the oral mucosa: Clinical and histologic review. Head Neck Pathol. 2019; 13: 91–102. PubMed Abstract | Publisher Full Text | Free Full Text
3. Mortazavi H, Safi Y, Baharvand M, et al.: Diagnostic feature of common oral ulcerative lesions: An updated decision tree. Int J Dent. 2016; 2016: 1–14. PubMed Abstract | Publisher Full Text | Free Full Text
4. Eming SA, Martin P, Tomic-Canic M: Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014; 6(265): 265sr66. PubMed Abstract | Publisher Full Text | Free Full Text
5. Gonzalez ACO, Costa TF, Andrade ZA, et al.: Wound healing - A literature review. An Bras Dermatol. 2016; 91(5): 614–20. PubMed Abstract | Publisher Full Text | Free Full Text
6. Bartolome RI, Uchiyama A, Molinolo AA, et al.: Transcriptional signature primes human oral mucosa for rapid wound healing. Sci Transl Med. 2018 July 25; 10(451) : 1–24. PubMed Abstract | Publisher Full Text | Free Full Text
7. Kang MH, Kim BH: Oral wound healing effects of acai berry water extracts in rat oral mucosa. Toxicol Res. 2018; 34(2): 97–102. PubMed Abstract | Publisher Full Text | Free Full Text
8. Kobayashi S, Ogawa N, Fujimura Y, et al.: Water-soluble Component in Dried Chrysanthemum Flower Stimulates Tumor Necrosis Factor-a Production by Mouse Macrophage-like Cell Line RAW 264.7. Food Sci Technol Res. 2006; 12(2): 144–7.
9. Azeez S, Amudhan S, Adiga S, et al.: Wound Healing Profile of Areca Catechu Extracts on Different Wound Models in Wistar Rat. Kuwait Med J. 2007; 39(1): 48–52.
10. Verma DK, Bharat M, Nayak D, et al.: Areca catechu: Effect of topical ethanolic extract on burn wound healing in albino rats. Int J Pharmacol Clin Sci. 2012; 1(3): 74–8.
11. Kim DY, Won KJ, Yoon MS, et al.: Chrysanthemum boreale Makino essential oil induces keratinocyte proliferation and skin regeneration. Nat Prod Res. 2015; 29(6): 562–4. PubMed Abstract | Publisher Full Text
12. Syahrajabian MH, Sun W, Zandi P, et al.: A review of Chrysanthemum, the eastern queen in traditional Chinese medicine with healing power in modern pharmaceutical sciences. Appl Ecol Environ Res. 2019; 17(6): 13355–69. Publisher Full Text
13. Oliveira BV, Barros Silva PG, Nojosa JS, et al.: TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats. J Appl Oral Sci. 2016; 24(3): 278–90. PubMed Abstract | Publisher Full Text | Free Full Text
14. Cavalcante GM, Paula RJS, Souza LP, et al.: Experimental model of traumatic ulcer in the cheek mucosa of rats. Acta Cir Bras. 2011; 26(3): 227–34. PubMed Abstract | Publisher Full Text
15. Jeon JK, Park SK, Lee JH: Effects of high voltage pulsed current stimulation with a visible contraction intensity on expression of TGF-β1 and synthesis of type I collagen in wound-induced white rats. J. Phys. Ther. Sci. 2015; 27(5): 1485–90. PubMed Abstract | Publisher Full Text | Free Full Text
16. Cho SAPJ, Seok SH, Junh JH, et al.: Effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on 5-FU-induced ulcerative mucositis in hamster buccal pouches. Exp Toxicol Pathol. 2006; 57(4): 321–8. PubMed Abstract | Publisher Full Text
17. Etemad-Moghadam S, Khalili M, Tirgary F, et al.: Evaluation of myofibroblasts in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med. 2009; 38(8): 639–43. PubMed Abstract | Publisher Full Text
18. Andrade TA, Iyer A, Das PK, et al.: The inflammatory stimulus of a natural latex biomembrane improves healing in rats. Braz J Med Biol Res. 2011; 44(10): 1036–47. PubMed Abstract | Publisher Full Text
19. Sari LM, Subita GP, Auerkari EI: Areca nut extract demonstrated apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma. F1000 Res. 2019; 7(723): 1–34. PubMed Abstract | Publisher Full Text | Free Full Text
20. Rani R, Arora S, Kaur J, et al.: Phenolic compounds as antioxidants and chemopreventive drugs from Streptomyces cellulosae strain TES17 isolated from rhizosphere of Camellia sinensis. BMC Complement Altern Med. 2018; 18(82): 1–18. PubMed Abstract | Publisher Full Text | Free Full Text
21. Qazi BS, Tang K, Qazi A: Recent Advances in Underlying Pathologies Provide Insight into Interleukin-8 Expression-Mediated Inflammation and Angiogenesis. Int J Inflam. 2011; 2011: 1–13. PubMed Abstract | Publisher Full Text | Free Full Text
22. Feugate JE, Li Q, Wong L, et al.: The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure. J Cell Biol. 2002; 156: 161–72. PubMed Abstract | Publisher Full Text | Free Full Text
23. Nakanishi T, Mukai K, Yumoto H, et al.: Anti-inflammatory effect of catechin on cultured human dental pulp cells affected by bacteria-derived factors. Eur J Oral Sci. 2010; 118(2): 145–50. PubMed Abstract | Publisher Full Text
24. Faouzi M, Neupane RP, Yang J, et al.: Areca nut extracts mobilize calcium and release pro-infammatory cytokines from various immune cells. Sci Rep. 2018; 1075: 1–13. PubMed Abstract | Publisher Full Text | Free Full Text
25. Liu LL, Xiao ZB: Chemical constituents from flowers of Chrysanthemum indicum Chin. Tradit. Herb. Drugs. 2018; 49(22): 5254–8. PubMed Abstract | Publisher Full Text
26. Khan S, Mehmood MH, Ali ANA, et al.: Studies on anti-inflammatory and analgesic activities of betel nut in rodents. J Ethnopharmacol. 2011; 135(3): 654–61. PubMed Abstract | Publisher Full Text
27. Okabe S, Amagase K: An Overview of Acetic Acid Ulcer Models —The History and State of the Art of Peptic Ulcer Research. Biol. Pharm. Bull. 2005; 28(8): 1321–41. PubMed Abstract | Publisher Full Text
28. Sherratt JA, Dallon JC: Theoretical models of wound healing: past successes and future challenges. C. R. Biologies. 2002; 325: 557–64. PubMed Abstract | Publisher Full Text
29. Frenkel ES, Ribbeck K: Salivary mucins in host defense and disease prevention. J Oral Microbiol. 2015; 7(29759): 1–9. PubMed Abstract | Publisher Full Text | Free Full Text
30. Holbrook J, Reyna SL, Griffiths HJ, et al.: Tumour necrosis factor signalling in health and disease. F1000Res. 2019; 8(F1000 Faculty Rev)(111): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text
31. Liu C, Tang J: Expression levels of tumor necrosis factorα and the corresponding receptors are correlated with trauma severity. Oncol. Lett. 2014; 8(6): 2747–51. PubMed Abstract | Publisher Full Text | Free Full Text
32. Ashcroft GS, Jeong MJ, Ashworth JJ, et al.: TNFα is a therapeutic target for impaired cutaneous wound healing. Wound Repair Regen. 2012; 20(1): 38–49. PubMed Abstract | Publisher Full Text | Free Full Text
33. Imai S, Otsuka T, Naito A, et al.: Triamcinolone Acetonide Suppresses Inflammation and Facilitates Vascular Barrier Function in Human Retinal Microvascular Endothelial Cells. Curr Neurovasc Res. 2017; 14(3). PubMed Abstract | Publisher Full Text
34. Harvey W, Scutt A, Meghji S, et al.: Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids. Arch. Oral Biol. 1986; 31(1): 45–49. PubMed Abstract | Publisher Full Text
35. Steiner SSM, Roy S, Sen CK: Collagen in Wound Healing. Bioeng. 2021; 8(63): 1–15.

Comments on this article Comments (1)

Version 7

VERSION 7 PUBLISHED 12 Sep 2025

Revised

Comment

Version 1

VERSION 1 PUBLISHED 21 Jul 2021

Discussion is closed on this version, please comment on the latest version above.

Author Response 19 Oct 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

19 Oct 2021

Author Response
Reviewer 2 response:
Dear Prof,
- The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult
... Continue reading
Reviewer 2 response:
Dear Prof,

The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Response: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment. The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin. The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin. We have added this explanation in the “Discussion” section.

Among all inflammatory cytokines, why TNF-alpha was chosen?

Response: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers. The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases. Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral gel

The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

Response: The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b in this comment field, the section was taken from one of the photos of positive control). We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The university of Western Ontario, Canada). It was said that if we use the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

The author should briefly explain the histology findings in the figure legend.

Response: Thank you for the advice given. We have added the histology findings in the figure legend.

The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Response: All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. The visual data are the actual data based on our observations before seeing the histological results.
Reviewer 2 response:
Dear Prof,

The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Response: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment. The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin. The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin. We have added this explanation in the “Discussion” section.

Among all inflammatory cytokines, why TNF-alpha was chosen?

Response: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers. The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases. Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral gel

The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

Response: The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b in this comment field, the section was taken from one of the photos of positive control). We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The university of Western Ontario, Canada). It was said that if we use the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

The author should briefly explain the histology findings in the figure legend.

Response: Thank you for the advice given. We have added the histology findings in the figure legend.

The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Response: All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. The visual data are the actual data based on our observations before seeing the histological results.
Competing Interests: We declare no competing interests Close
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Author details Author details

Liza Meutia Sari
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Writing – Original Draft Preparation, Writing – Review & Editing

Zaki Mubarak
Roles: Project Administration, Supervision, Validation, Visualization

Dina Keumala Sari
Roles: Conceptualization, Supervision, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The study was supported by grant from the PNBP fund Syiah Kuala University No 319/UN11.2.1/PT.01.03/PNBP/2021.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Meutia Sari L, Mubarak Z and Keumala Sari D. Evaluation of clinical, histology, TNF-α, and collagen expressions on oral ulcer in rats after treatment with areca nut and chrysanthemum oral gel [version 1; peer review: 1 approved with reservations, 1 not approved]. F1000Research 2021, 10:623 (https://doi.org/10.12688/f1000research.54887.1)

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Reviewer Report 24 Sep 2021

Nurrul Shaqinah Nasaruddin, Department of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

Approved with Reservations

https://doi.org/10.5256/f1000research.58412.r90097

The major concern of this manuscript is the technique of treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe

The major concern of this manuscript is the technique of treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?
Among all inflammatory cytokines, why TNF-alpha was chosen?
The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures were lacked with the contrast and all the tissues appear homogenously red.
The author should briefly explain the histology findings in the figure legend.
The clinical evaluation results were contradicted with histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: pathology, histology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

CITE

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Author Response 08 Oct 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

08 Oct 2021

Author Response

Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are ... Continue reading Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Answer: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment.

The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin.

The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin.
We have added this explanation in the “Discussion” section.

2. Among all inflammatory cytokines, why TNF-alpha was chosen?

Answer: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers.
The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases.

Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral topical gel.

3. The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b, the section was taken from one of the photos of positive control).

We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The University of Western Ontario, Canada). It was said that if we used the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

4. The author should briefly explain the histology findings in the figure legend.

Thank you for the advice given. We have added the histology findings in the figure legend.

5. The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The clinical examination data are the actual data based on our observations before seeing the histological results. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. We decided to perform the first data just the way they were without adjusting with the histological assessment.
Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Answer: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment.

The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin.

The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin.
We have added this explanation in the “Discussion” section.

2. Among all inflammatory cytokines, why TNF-alpha was chosen?

Answer: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers.
The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases.

Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral topical gel.

3. The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b, the section was taken from one of the photos of positive control).

We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The University of Western Ontario, Canada). It was said that if we used the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

4. The author should briefly explain the histology findings in the figure legend.

Thank you for the advice given. We have added the histology findings in the figure legend.

5. The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The clinical examination data are the actual data based on our observations before seeing the histological results. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. We decided to perform the first data just the way they were without adjusting with the histological assessment.
Competing Interests: We do not have competing interests Close
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COMMENTS ON THIS REPORT

Author Response 08 Oct 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

08 Oct 2021

Author Response

Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are ... Continue reading Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Answer: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment.

The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin.

The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin.
We have added this explanation in the “Discussion” section.

2. Among all inflammatory cytokines, why TNF-alpha was chosen?

Answer: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers.
The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases.

Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral topical gel.

3. The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b, the section was taken from one of the photos of positive control).

We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The University of Western Ontario, Canada). It was said that if we used the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

4. The author should briefly explain the histology findings in the figure legend.

Thank you for the advice given. We have added the histology findings in the figure legend.

5. The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The clinical examination data are the actual data based on our observations before seeing the histological results. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. We decided to perform the first data just the way they were without adjusting with the histological assessment.
Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Answer: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment.

The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin.

The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin.
We have added this explanation in the “Discussion” section.

2. Among all inflammatory cytokines, why TNF-alpha was chosen?

Answer: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers.
The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases.

Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral topical gel.

3. The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b, the section was taken from one of the photos of positive control).

We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The University of Western Ontario, Canada). It was said that if we used the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

4. The author should briefly explain the histology findings in the figure legend.

Thank you for the advice given. We have added the histology findings in the figure legend.

5. The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The clinical examination data are the actual data based on our observations before seeing the histological results. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. We decided to perform the first data just the way they were without adjusting with the histological assessment.
Competing Interests: We do not have competing interests Close
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Reviewer Report 16 Aug 2021

Gunawan Indrayanto, Faculty of Pharmacy, Surabaya University, Surabaya, Indonesia

Not Approved

https://doi.org/10.5256/f1000research.58412.r91931

Some important methods and data were not reported:

Scientific identification of the plant was missing.
Stability evaluation of the extract was missing.
No validation data of

Some important methods and data were not reported:

Scientific identification of the plant was missing.
Stability evaluation of the extract was missing.
No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)
No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.
Add Gandevia et al. (2021¹) and related references.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

References

1. Gandevia S, Cumming G, Amrhein V, Butler A: Replication: Do not trust your p‐value, be it small or large. The Journal of Physiology. 2021; 599 (11): 2989-2990 Publisher Full Text

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Analytical Method Development and its Validation; Natural Product Chemistry, Herbal Drugs

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

CITE

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Author Response 10 Sep 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

10 Sep 2021

Author Response
- Scientific identification of the plant was missing.
- Response: This research is a series of research on the benefits of areca nut seeds that has been started in
... Continue reading
Scientific identification of the plant was missing.

Response: This research is a series of research on the benefits of areca nut seeds that has been started in 2012. We have published several publications since 2016 using areca nut extract from the same source (Aceh Besar). We apologize for the negligence or forgetting to re-enter the scientific identification that has been done. We have re-entered the plant identification in the “Extract preparation” section in “Methods”. Thank you for your accuracy in reviewing.

Stability evaluation of the extract was missing.

Response: We have added the stability evaluation in the manuscript.

No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)

Response: We have included the validation data in the manuscript especially the selectivity index of areca nut and chrysanthemum. The studies of areca nut on oral squamous cell carcinoma cell lines (HSC-2 dan HSC-3 cell lines) and human dermal keratinocytes (HaCat cell lines) have been published in 2 previous journals:

Sari LM, Subita GP, Auerkari EI. Potential antioxidant and cytotoxic activities of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res 2017;10(10):286-91.

Sari LM, Subita GP, Auerkari EI. (2019). Areca nut extract demonstrated the apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma F1000 Res, 7(723), 1-34.

No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.

Response: Data have been changed to “Mean ± SD, 95% CI, p-value”. Several statements in “Discussion” dan “Results” has undergone changes regarding data interpretations.

Add Gandevia et al. (2021) and related references.

Response: We have added: Gandevia S. Replication: Do not trust your p-value, be it small or large. J Physiol 2021;599(11):2989-90.
Scientific identification of the plant was missing.

Response: This research is a series of research on the benefits of areca nut seeds that has been started in 2012. We have published several publications since 2016 using areca nut extract from the same source (Aceh Besar). We apologize for the negligence or forgetting to re-enter the scientific identification that has been done. We have re-entered the plant identification in the “Extract preparation” section in “Methods”. Thank you for your accuracy in reviewing.

Stability evaluation of the extract was missing.

Response: We have added the stability evaluation in the manuscript.

No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)

Response: We have included the validation data in the manuscript especially the selectivity index of areca nut and chrysanthemum. The studies of areca nut on oral squamous cell carcinoma cell lines (HSC-2 dan HSC-3 cell lines) and human dermal keratinocytes (HaCat cell lines) have been published in 2 previous journals:

Sari LM, Subita GP, Auerkari EI. Potential antioxidant and cytotoxic activities of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res 2017;10(10):286-91.

Sari LM, Subita GP, Auerkari EI. (2019). Areca nut extract demonstrated the apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma F1000 Res, 7(723), 1-34.

No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.

Response: Data have been changed to “Mean ± SD, 95% CI, p-value”. Several statements in “Discussion” dan “Results” has undergone changes regarding data interpretations.

Add Gandevia et al. (2021) and related references.

Response: We have added: Gandevia S. Replication: Do not trust your p-value, be it small or large. J Physiol 2021;599(11):2989-90.
Competing Interests: The authors declare no conflict of interests Close
Report a concern
Respond or Comment

COMMENTS ON THIS REPORT

Author Response 10 Sep 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

10 Sep 2021

Author Response
- Scientific identification of the plant was missing.
- Response: This research is a series of research on the benefits of areca nut seeds that has been started in
... Continue reading
Scientific identification of the plant was missing.

Response: This research is a series of research on the benefits of areca nut seeds that has been started in 2012. We have published several publications since 2016 using areca nut extract from the same source (Aceh Besar). We apologize for the negligence or forgetting to re-enter the scientific identification that has been done. We have re-entered the plant identification in the “Extract preparation” section in “Methods”. Thank you for your accuracy in reviewing.

Stability evaluation of the extract was missing.

Response: We have added the stability evaluation in the manuscript.

No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)

Response: We have included the validation data in the manuscript especially the selectivity index of areca nut and chrysanthemum. The studies of areca nut on oral squamous cell carcinoma cell lines (HSC-2 dan HSC-3 cell lines) and human dermal keratinocytes (HaCat cell lines) have been published in 2 previous journals:

Sari LM, Subita GP, Auerkari EI. Potential antioxidant and cytotoxic activities of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res 2017;10(10):286-91.

Sari LM, Subita GP, Auerkari EI. (2019). Areca nut extract demonstrated the apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma F1000 Res, 7(723), 1-34.

No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.

Response: Data have been changed to “Mean ± SD, 95% CI, p-value”. Several statements in “Discussion” dan “Results” has undergone changes regarding data interpretations.

Add Gandevia et al. (2021) and related references.

Response: We have added: Gandevia S. Replication: Do not trust your p-value, be it small or large. J Physiol 2021;599(11):2989-90.
Scientific identification of the plant was missing.

Response: This research is a series of research on the benefits of areca nut seeds that has been started in 2012. We have published several publications since 2016 using areca nut extract from the same source (Aceh Besar). We apologize for the negligence or forgetting to re-enter the scientific identification that has been done. We have re-entered the plant identification in the “Extract preparation” section in “Methods”. Thank you for your accuracy in reviewing.

Stability evaluation of the extract was missing.

Response: We have added the stability evaluation in the manuscript.

No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)

Response: We have included the validation data in the manuscript especially the selectivity index of areca nut and chrysanthemum. The studies of areca nut on oral squamous cell carcinoma cell lines (HSC-2 dan HSC-3 cell lines) and human dermal keratinocytes (HaCat cell lines) have been published in 2 previous journals:

Sari LM, Subita GP, Auerkari EI. Potential antioxidant and cytotoxic activities of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res 2017;10(10):286-91.

Sari LM, Subita GP, Auerkari EI. (2019). Areca nut extract demonstrated the apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma F1000 Res, 7(723), 1-34.

No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.

Response: Data have been changed to “Mean ± SD, 95% CI, p-value”. Several statements in “Discussion” dan “Results” has undergone changes regarding data interpretations.

Add Gandevia et al. (2021) and related references.

Response: We have added: Gandevia S. Replication: Do not trust your p-value, be it small or large. J Physiol 2021;599(11):2989-90.
Competing Interests: The authors declare no conflict of interests Close
Report a concern

Comments on this article Comments (1)

Version 7

VERSION 7 PUBLISHED 12 Sep 2025

Revised

Comment

Version 1

VERSION 1 PUBLISHED 21 Jul 2021

Discussion is closed on this version, please comment on the latest version above.

Author Response 19 Oct 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

19 Oct 2021

Author Response
Reviewer 2 response:
Dear Prof,
- The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult
... Continue reading
Reviewer 2 response:
Dear Prof,

The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Response: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment. The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin. The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin. We have added this explanation in the “Discussion” section.

Among all inflammatory cytokines, why TNF-alpha was chosen?

Response: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers. The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases. Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral gel

The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

Response: The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b in this comment field, the section was taken from one of the photos of positive control). We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The university of Western Ontario, Canada). It was said that if we use the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

The author should briefly explain the histology findings in the figure legend.

Response: Thank you for the advice given. We have added the histology findings in the figure legend.

The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Response: All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. The visual data are the actual data based on our observations before seeing the histological results.
Reviewer 2 response:
Dear Prof,

The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Response: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment. The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin. The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin. We have added this explanation in the “Discussion” section.

Among all inflammatory cytokines, why TNF-alpha was chosen?

Response: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers. The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases. Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral gel

The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

Response: The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b in this comment field, the section was taken from one of the photos of positive control). We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The university of Western Ontario, Canada). It was said that if we use the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

The author should briefly explain the histology findings in the figure legend.

Response: Thank you for the advice given. We have added the histology findings in the figure legend.

The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Response: All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. The visual data are the actual data based on our observations before seeing the histological results.
Competing Interests: We declare no competing interests Close
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Gunawan Indrayanto, Surabaya University, Surabaya, Indonesia
Nurrul Shaqinah Nasaruddin, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
Ali Akbar Nekooeian, Shiraz University of Medical Sciences, Shiraz, Iran
Mingsan Miao, Henan University of Chinese Medicine, Zhengzhou, China

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18 Views

26 Jan 2023 | for Version 5

Mingsan Miao, Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou, China

18 Views Cite this report Responses(1)

Not Approved

1. For example, it is better to have a simple model group.

2. It is better to provide the appraiser and Latin of betel nut and chrysanthemum for research; There should also be the extraction method of chrysanthemum.

3. The weight range of rats is a little large, generally the same experiment does not exceed 20 grams.

4. The level and certificate number of rats shall be provided; Provide the license number of rat animal center; Provide the source and batch number of main reagents and drugs.

5. There are 12 in each group in the method, why is the result 18?

6. Pathology is best to have semi-quantitative results; The basis for sample selection, extraction process and sample quantity in the process of immunohistochemistry should be supplemented.

7. It is reported in the literature that long-term use of betel nut may cause oral cancer. Why should we choose betel nut to treat oral ulcer?

8. The discussion of the experimental results can be divided into categories and appropriately compressed.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pharmacology of Traditional Chinese Medicine

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Author Response

12 Sep 2025

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

1. For example, it is better to have a simple model group.

We divided the test animals into six simple groups: positive control, negative control, normal (no treatment), and three treatment groups.
These four groups are very important to determine whether the extract really works on the cells of the test animals. In this study, we also conducted tests to support each other's valid results.
• In the first stage, we tested the best gel dosage based on pH, viscosity or consistency, spreadability, organoleptic characteristics, and homogeneity. This dosage will be used for treating the test animals.
• In the second stage, we clinically observed the animals by weighing their body weight and measuring ulcer size before and after treatment.
• In the third stage, we performed three microscopic examinations: histopathological staining, TNF-α immunostaining, and collagenase analysis.
We strive to ensure that readers can understand the steps taken in this research to support the best possible results.

2. It is better to provide the appraiser and Latin of betel nut and chrysanthemum for research; There should also be the extraction method of chrysanthemum.

We have changed all the term betel nut into A. catechu, and chrysanthemum into D. Grandiflora.
We have added the maceration method in the “extract preparation section.”

3. The weight range of rats is a little large, generally the same experiment does not exceed 20 grams.
We have corrected. In this study, we used Sprague Dawley rats from the Indonesian veterinary laboratory in Bogor, West Java, which generally have an average weight of 110-128 grams.
Sprague Dawley rats were selected for this study because the ulcers that we need to form have a large diameter of 10 mm and a depth of ±2-3 mm. It will be created on their buccal mucosa. Ulcers formed in the buccal mucosa will require a wider buccal mucosa wall. Adult mice (weighing 20-40 grams) were not used in this study because we expected that using large rats, such as Sprague Dawley rats, would result in the formation of perfect ulcers and make their healing easier to observe.

4. The level and certificate number of rats shall be provided; Provide the license number of rat animal center; Provide the source and batch number of main reagents and drugs.
Specific license numbers for test animals at the IPB University School of Veterinary Medicine and Biomedical Sciences are not publicly available, but their use is regulated by the Ethical Use of Test Animals regulations that must be followed by every research facility in Indonesia. This research has passed ethical approval.
Test animal license numbers are part of an institution's internal data related to ethical and regulatory compliance. Specific information, such as license numbers, is not usually published for reasons of confidentiality and data security. All use of test animals in Indonesia must comply with the ethical guidelines and recommendations issued by the Test Animal Ethics Committee (KEPK).
We have added the brand and reagent numbers to the manuscript:
phosphate-buffered saline (PBS) (Sigm-Aldrich; P5493), anti-TNF-α antibody (RRID: AB_2892586; TNFA/1172) (ab220210Abcam), the secondary antibody goat anti-rabbit IgG H&L horseradish peroxidase (HRP) (RRID: AB_955447; ab6721-Abcam), Betazoid DAB chromogen solution (BDB2004H-Biocare Medicare LLC), Picro-Sirius red solution (Abcam; ab246832) (solution A), Adobe Photoshop CC 2017 (RRID:SCR_014199; GNU Image Manipulation Program (RRID:SCR_003182) is an open-access alternative) and ImageJ (v1.50i) (RRID:SCR_003070) software, SPSS 20 software (SPSS Inc., Chicago, USA) ( RRID:SCR_019096); JASP ( RRID:SCR_015823). ketamine HCl injection; OGB dexa, xylazine 20; Pantex Holland.

5. There are 12 in each group in the method, why is the result 18?
Thank you very much for the correction. Please let me explain the difference between the 12 rats in each group (materials) and 18 specimens (in the results).
“Effect of A. catechu and D. grandiflora oral gel on histological scores, number of connective tissue cells that expressed TNF-α, and percentage of collagen deposition area on the 7th experimental day (n = 18 specimens)”.

We indeed used 12 rats in each group (6 groups). After completing the study, we took ulcer tissue (biopsy) after the healing process. In each group (containing 12 rats), we took 3 biopsy tissues representing the 12 rats in 1 group for microscopic analysis (HE staining, picosirius staining, and immunohistochemistry). Taking 3 specimens in each group was considered to represent the overall healing process. This was also due to funding limitations in the study.
We hope reviewers can understand the limitations of this study.

6. Pathology is best to have semi-quantitative results; The basis for sample selection, extraction process, and sample quantity in the process of immunohistochemistry should be supplemented.
Thank you for the correction. We have corrected as instructed.

7. It is reported in the literature that long-term use of betel nut may cause oral cancer. Why should we choose betel nut to treat oral ulcers?

Thank you for the detailed correction.
Areca nut was chosen as a natural herbal ingredient because the quality of areca nuts in our region is very good. The percentage of patients with oral cancer is very low. Based on our survey of 87 cancer patients undergoing chemotherapy at our regional public hospital, only 4.87% suffered from oral squamous cell carcinoma with the risk factor of smoking tobacco with chewing betel nuts, not only areca nut. In addition, based on several references:
Areca nuts themselves do not contain carcinogens, but the carcinogenic effect is caused by nitrosamines produced by the nitrosation process of alkaloids (arecoline) from dried areca nuts when chewed and digested in the stomach under acidic conditions over a long period of time and without control.¹ Combination with nitric oxide produced by bacteria causes the formation of methylnitrosaminopropionitrile, which has been proven to cause carcinogenesis in animal tests.² This endogenous nitrosation is significantly higher, especially in people with poor oral hygiene.² When areca nuts are mixed with tobacco and chewed by people with poor oral hygiene, a large amount of nitrosamine products accumulate.³ This process occurs continuously over a long period of time because these nuts are addictive. Several studies have also reported an increase in reactive oxygen species, such as hydroxyl radicals in the oral cavity due to the combination of polyphenol autooxidation from areca nuts and high alkaline pH of lime.⁴ When areca nuts are chewed together with betel leaves and lime, these two ingredients cause erosion of the mucous membrane, making it easier for carcinogens to penetrate cells through the mucosa.⁵ Some communities in India and Pakistan use factory-packaged areca nuts called gutka and pan masala. Gutka consists of areca nuts, betel leaves, tobacco, and lime, while pan masala does not contain tobacco. Approximately 40% of gutka and pan masala packages are contaminated with carcinogenic aflatoxins from fungi of the species Aspergillus flavus, Aspergillus niger, and Rhizopus spp.⁶
References:

Bhisey RA, Boucher BJ, Hsi-Chen TH, Gajalakshmi V, Gupta PC, et al. Monographs on the Evaluation of Carcinogenic Risks to Humans.Betel-Quid and Areca-Nut Chewing and some Areca-Nut Related Nitrosamines. IARCS Monographs 2004;85:1-293.
Nagao T, Ikeda N, Warnakulasuriya S, Fukano H, Yuasa H, et al. Serum antioxidant micronutrients and the risk of oral leukoplakia among Japanese. Oral Oncol 2000;36:466-70.
Carossa S, Pera P, Doglio P, Lombardo S, Colagrande P, et al. Oral nitric oxide during plaque deposition. Eur J Clin Invest 2001;31:876-9.
Nair U, Bartsch H, Nair J. Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha and pan masala: a review of agents and causative mechanisms. Mutagenesis 2004;19:251-62.
Johnson NW, Jayasekara P, dan Amarasinghe HK. Squamous cell carcinoma and precursor lesions of the oral cavity: epidemiology and aetiology. Periodontology 2000. Singapura: John Wiley and Sons; 2011.
Anttila A, Bhat RV, Bhond JA, Bhorgoff SJ, Bosch FX, et al. Monographs on the Evaluation of Carcinogenic Risks to Humans. Some Traditional Herbal Medicines, Some Mycotoxins, Naphthalene and Styrene. IARCS Monographs 2002;82:171-4.

8. The discussion of the experimental results can be divided into categories and appropriately compressed.
Thank you. It is done.

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23 Views

09 Nov 2022 | for Version 4

Ali Akbar Nekooeian, Cardiovascular Pharmacology Research Lab, Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

23 Views Cite this report Responses(0)

Not Approved

Thank you for letting me to see this version of the manuscript.

In the method section in a paragraph titled by experimental animal mentions that 25 rats were used. However, in two paragraphs after this the manuscript mentions that animals were divided in 5 groups of 12 each. Moreover, as I mentioned earlier the study suffers from the lack of a control group not subjected to oral abrasion and not receiving medication. This is very vital that can tell us if the model was working. Therefore, regrettably, I cannot recommend the publication of the manuscript.

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35 Views

11 May 2022 | for Version 2

Ali Akbar Nekooeian, Cardiovascular Pharmacology Research Lab, Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

35 Views Cite this report Responses(1)

Not Approved

The manuscript examines the effects of areca and chrysanthemum oral gel on oral ulcers using clinical, histological and biomarkers criteria. Although nicely-written the manuscript suffers from a major problem and that is the lack of untreated group of rats with oral ulcer. The manuscript in the current form cannot answer the question that if untreated rats are left for the period used in this study, they would recover without treatment.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cardiovascular Pharmacology

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Author Response

04 Jan 2023

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

We understand that 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-alpha 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 the drug is swallowed. This pseudo equilibrium has an absorption half-life of about 0.87 minutes (52 seconds). It means that the oral mucosa absorbed the drug partition in the oral gel.

However, the effectiveness of the efficacy of the areca nut chrysanthemum gel can only be seen through clinical examination of the difference in cure rates between the treated groups with positive and negative controls. Although the results of the histology examination showed no significant difference, however, the results of the immunohistochemistry test showed an increase in TNF alpha expression which plays a role in the wound healing process as well as an increase in the work of collagen.

Several factors that influence the fast or slow absorption of drugs on the mucosal surface are permeability and blood supply. 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, facilitating the 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 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

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30 Views

26 Oct 2021 | for Version 2

Nurrul Shaqinah Nasaruddin, Department of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

30 Views Cite this report Responses(1)

Approved

The author has made the changes and amendments accordingly.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Pathology, histology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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34 Views

24 Sep 2021 | for Version 1

Nurrul Shaqinah Nasaruddin, Department of Craniofacial Diagnostics and Biosciences, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia

34 Views Cite this report Responses(1)

Approved With Reservations

The major concern of this manuscript is the technique of treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?
Among all inflammatory cytokines, why TNF-alpha was chosen?
The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures were lacked with the contrast and all the tissues appear homogenously red.
The author should briefly explain the histology findings in the figure legend.
The clinical evaluation results were contradicted with histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

pathology, histology

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Author Response

08 Oct 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Dear Reviewer,
I would like to respond to the comment's report:

1. The major concern of this manuscript is the technique of the treatment process. While the rats are fully awake, it is very difficult to apply the topical on the ulcerated buccal mucosa. The rats will chew the probe and it is very difficult to visualize the lesions for monitoring. Since the mucosal epithelium is highly dividing cells, how sure are you the healing occur due to treatment given?

Answer: In this study, we performed oral gel application on the buccal mucosa of the rat twice a day at 8 am and 8 pm. This application is by holding the rats, opening his mouth gently and slowly, then applying to the wound and holding it for about 20-30 seconds. After that, the mice were isolated briefly for 1 hour to avoid eating and drinking. We try our best to make the gel last well even if it's only for a few minutes, considering that this gel dissolves easily in saliva. We have acknowledged that this is one of the limitations of this study. We hope that with further research, we can make a formulation of topical oral gel in orabase so that it can last longer in the mouth. However, despite the brief application of the buccal mucosa, cellular changes can be seen differently on immunohistochemistry examination and collagenase assay after seven days of treatment.

The extent of absorption of materials through the skin or oral mucosa depends on the surface area over which the substance is applied, the concentration of the substance administered, the lipid solubility of the material or vehicle, whether the skin surface is intact, the skin thickness at the site of application, the length of time that the material is in contact with the skin surface, and the degree of skin hydration and surface occlusion, in that covered and well-hydrated skin absorbs substances faster than does uncover or dry skin.

The surface of the buccal mucosa is covered by non-keratinized stratified squamous epithelium. Keratinized mucosa is less permeable to the drugs as compared to the non-keratinized mucosa. This causes the buccal mucosa to more easily absorb the drug than the gingiva and palate. The turnover of the buccal epithelium which is due to differentiation and desquamation of the epithelium also accelerates the wound healing process happens faster. The turnover time for the buccal epithelium has been estimated at 14 days. Oral mucosa is well-hydrated skin that can absorb substances faster than dry skin.
We have added this explanation in the “Discussion” section.

2. Among all inflammatory cytokines, why TNF-alpha was chosen?

Answer: This research was initiated with lesions originating from simple injuries. We hope that the development of the results of this study can help the development of herbal extracts to accelerate the healing of oral diseases such as mucositis, RAS, and traumatic ulcers.
The reason why we choose TNF-α is that TNF-α is a central regulator of inflammation. TNF-α is also a crucial cytokine in the establishment and maintenance of inflammation in multiple autoimmune diseases.

Although its precise role in wound healing is not fully understood, however, Ritsu et al. (Ritsu M, Kawakami K, Kanno E, et al. Critical role of tumor necrosis factor-α in the early process of wound healing in skin. J. Dermatol. Dermatol. Surg. 2017. Vol 21(1). 14-19) reported that TNF-α is involved in the early process of wound healing. It is quickly released and initiates inflammation at wound tissues. The wound healing was accelerated on day 3 when mice were treated with bioactive TNF-α. Another previous study reported that impaired healing in animal models and age-related delayed healing of acute human wounds exhibit raised local and systemic levels of TNF-α that may parallel the pro-inflammatory phenotype. Despite previous studies showed varying results, there is an interest in observing the effect of TNF-α levels in ulcer healing after application of an oral topical gel.

3. The contrast color for special staining picrosirius red should be yellow and red, and the red indicates positive for collagen. However, the pictures lacked contrast and all the tissues appear homogenously red.

The resulting photos consist of two types; polarized and non-polarized light microscopy. The polarized colors of Picosirius red staining depend only on fiber thickness and packing, not on the composition of the specific collagen type within collagen bundles. Under polarized light, the oral mucosa sections seem to show collagen type 1 which showed thicker and denser fibers on the seventh day. We don't know why the red color was very generalized in the oral mucosa section although the Picosirius staining was done according to the standard procedure (as you can see in Figures 1a and b, the section was taken from one of the photos of positive control).

We searched the reason from another source (Kiernan JA. Sirius red for collagen staining protocol. The University of Western Ontario, Canada). It was said that if we used the polarized light, sometimes we will lose "the yellow background" of picric acid staining. The loss of the yellow cytoplasmic background is probably caused by the dehydrating process.

Figure 1.a. Polarized light microscopy (Positive control)
Figure 1. b. Non-polarized light microscopy (Positive control)

4. The author should briefly explain the histology findings in the figure legend.

Thank you for the advice given. We have added the histology findings in the figure legend.

5. The clinical evaluation results were contradicted histology evidence, whereby the author mentioned that the ulcer is almost healed. but histologically, it showed complete healing with thick stratified epithelium.

All measurements were performed by the same operator. We analyzed the ulcers directly and through photos. Histological examination results were analyzed several weeks after the visual examination was performed. At that time, some lesions showed the tip of the wound still have a light and thin ulcer while the other end had healed completely. The clinical examination data are the actual data based on our observations before seeing the histological results. The histological score showed that the group F1, F3, and positive control (Figure 2) had a score of 1. The normal, negative control and F2 showed a score of 0. We decided to perform the first data just the way they were without adjusting with the histological assessment.

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57 Views

16 Aug 2021 | for Version 1

Gunawan Indrayanto, Faculty of Pharmacy, Surabaya University, Surabaya, Indonesia

57 Views Cite this report Responses(1)

Not Approved

Some important methods and data were not reported:

Scientific identification of the plant was missing.
Stability evaluation of the extract was missing.
No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)
No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.
Add Gandevia et al. (2021¹) and related references.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

Partly
Are the conclusions drawn adequately supported by the results?

No

References

1. Gandevia S, Cumming G, Amrhein V, Butler A: Replication: Do not trust your p‐value, be it small or large. The Journal of Physiology. 2021; 599 (11): 2989-2990 Publisher Full Text

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Analytical Method Development and its Validation; Natural Product Chemistry, Herbal Drugs

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Author Response

10 Sep 2021

Liza Sari, Oral Medicine, Syiah Kuala University, Banda Aceh, 23111, Indonesia

Scientific identification of the plant was missing.
Response: This research is a series of research on the benefits of areca nut seeds that has been started in 2012. We have published several publications since 2016 using areca nut extract from the same source (Aceh Besar). We apologize for the negligence or forgetting to re-enter the scientific identification that has been done. We have re-entered the plant identification in the “Extract preparation” section in “Methods”. Thank you for your accuracy in reviewing.
Stability evaluation of the extract was missing.
Response: We have added the stability evaluation in the manuscript.
No validation data of the biological assays were reported, without any validation, the validity of the data cannot be confirmed (Z factors, Selective Index, etc.)
Response: We have included the validation data in the manuscript especially the selectivity index of areca nut and chrysanthemum. The studies of areca nut on oral squamous cell carcinoma cell lines (HSC-2 dan HSC-3 cell lines) and human dermal keratinocytes (HaCat cell lines) have been published in 2 previous journals:
- Sari LM, Subita GP, Auerkari EI. Potential antioxidant and cytotoxic activities of areca nut (Areca catechu Linn.) extract in human oral squamous cell carcinoma and keratinocyte cells. Asian J Pharm Clin Res 2017;10(10):286-91.
- Sari LM, Subita GP, Auerkari EI. (2019). Areca nut extract demonstrated the apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma F1000 Res, 7(723), 1-34.
No need to report the significance statistic evaluations, data should be presented ad Mean +/- Confidence interval; p values cannot be trusted.
Response: Data have been changed to “Mean ± SD, 95% CI, p-value”. Several statements in “Discussion” dan “Results” has undergone changes regarding data interpretations.
Add Gandevia et al. (2021) and related references.
Response: We have added: Gandevia S. Replication: Do not trust your p-value, be it small or large. J Physiol 2021;599(11):2989-90.

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Competing Interests

The authors declare no conflict of interests

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Seyyedi SA, Sanatkhani M, Pakfetrat A, et al.: The therapeutic effects of chamomilla tincture mouthwash on oral aphthae: A Randomized Clinical Trial. J Clin Exp Dent. 2014; 6(5): e535–8. PubMed Abstract | Publisher Full Text | Free Full Text

[2] 2. Fitzpatrick SG, Cohen DM, Clark AN: Ulcerated lesions of the oral mucosa: Clinical and histologic review. Head Neck Pathol. 2019; 13: 91–102. PubMed Abstract | Publisher Full Text | Free Full Text

[3] 3. Mortazavi H, Safi Y, Baharvand M, et al.: Diagnostic feature of common oral ulcerative lesions: An updated decision tree. Int J Dent. 2016; 2016: 1–14. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Eming SA, Martin P, Tomic-Canic M: Wound repair and regeneration: mechanisms, signaling, and translation. Sci Transl Med. 2014; 6(265): 265sr66. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Gonzalez ACO, Costa TF, Andrade ZA, et al.: Wound healing - A literature review. An Bras Dermatol. 2016; 91(5): 614–20. PubMed Abstract | Publisher Full Text | Free Full Text

[6] 6. Bartolome RI, Uchiyama A, Molinolo AA, et al.: Transcriptional signature primes human oral mucosa for rapid wound healing. Sci Transl Med. 2018 July 25; 10(451) : 1–24. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Kang MH, Kim BH: Oral wound healing effects of acai berry water extracts in rat oral mucosa. Toxicol Res. 2018; 34(2): 97–102. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Kobayashi S, Ogawa N, Fujimura Y, et al.: Water-soluble Component in Dried Chrysanthemum Flower Stimulates Tumor Necrosis Factor-a Production by Mouse Macrophage-like Cell Line RAW 264.7. Food Sci Technol Res. 2006; 12(2): 144–7.

[9] 9. Azeez S, Amudhan S, Adiga S, et al.: Wound Healing Profile of Areca Catechu Extracts on Different Wound Models in Wistar Rat. Kuwait Med J. 2007; 39(1): 48–52.

[10] 10. Verma DK, Bharat M, Nayak D, et al.: Areca catechu: Effect of topical ethanolic extract on burn wound healing in albino rats. Int J Pharmacol Clin Sci. 2012; 1(3): 74–8.

[11] 11. Kim DY, Won KJ, Yoon MS, et al.: Chrysanthemum boreale Makino essential oil induces keratinocyte proliferation and skin regeneration. Nat Prod Res. 2015; 29(6): 562–4. PubMed Abstract | Publisher Full Text

[12] 12. Syahrajabian MH, Sun W, Zandi P, et al.: A review of Chrysanthemum, the eastern queen in traditional Chinese medicine with healing power in modern pharmaceutical sciences. Appl Ecol Environ Res. 2019; 17(6): 13355–69. Publisher Full Text

[13] 13. Oliveira BV, Barros Silva PG, Nojosa JS, et al.: TNF-alpha expression, evaluation of collagen, and TUNEL of Matricaria recutita L. extract and triamcinolone on oral ulcer in diabetic rats. J Appl Oral Sci. 2016; 24(3): 278–90. PubMed Abstract | Publisher Full Text | Free Full Text

[14] 14. Cavalcante GM, Paula RJS, Souza LP, et al.: Experimental model of traumatic ulcer in the cheek mucosa of rats. Acta Cir Bras. 2011; 26(3): 227–34. PubMed Abstract | Publisher Full Text

[15] 15. Jeon JK, Park SK, Lee JH: Effects of high voltage pulsed current stimulation with a visible contraction intensity on expression of TGF-β1 and synthesis of type I collagen in wound-induced white rats. J. Phys. Ther. Sci. 2015; 27(5): 1485–90. PubMed Abstract | Publisher Full Text | Free Full Text

[16] 16. Cho SAPJ, Seok SH, Junh JH, et al.: Effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on 5-FU-induced ulcerative mucositis in hamster buccal pouches. Exp Toxicol Pathol. 2006; 57(4): 321–8. PubMed Abstract | Publisher Full Text

[17] 17. Etemad-Moghadam S, Khalili M, Tirgary F, et al.: Evaluation of myofibroblasts in oral epithelial dysplasia and squamous cell carcinoma. J Oral Pathol Med. 2009; 38(8): 639–43. PubMed Abstract | Publisher Full Text

[18] 18. Andrade TA, Iyer A, Das PK, et al.: The inflammatory stimulus of a natural latex biomembrane improves healing in rats. Braz J Med Biol Res. 2011; 44(10): 1036–47. PubMed Abstract | Publisher Full Text

[19] 19. Sari LM, Subita GP, Auerkari EI: Areca nut extract demonstrated apoptosis-inducing mechanism by increased caspase-3 activities on oral squamous cell carcinoma. F1000 Res. 2019; 7(723): 1–34. PubMed Abstract | Publisher Full Text | Free Full Text

[20] 20. Rani R, Arora S, Kaur J, et al.: Phenolic compounds as antioxidants and chemopreventive drugs from Streptomyces cellulosae strain TES17 isolated from rhizosphere of Camellia sinensis. BMC Complement Altern Med. 2018; 18(82): 1–18. PubMed Abstract | Publisher Full Text | Free Full Text

[21] 21. Qazi BS, Tang K, Qazi A: Recent Advances in Underlying Pathologies Provide Insight into Interleukin-8 Expression-Mediated Inflammation and Angiogenesis. Int J Inflam. 2011; 2011: 1–13. PubMed Abstract | Publisher Full Text | Free Full Text

[22] 22. Feugate JE, Li Q, Wong L, et al.: The cxc chemokine cCAF stimulates differentiation of fibroblasts into myofibroblasts and accelerates wound closure. J Cell Biol. 2002; 156: 161–72. PubMed Abstract | Publisher Full Text | Free Full Text

[23] 23. Nakanishi T, Mukai K, Yumoto H, et al.: Anti-inflammatory effect of catechin on cultured human dental pulp cells affected by bacteria-derived factors. Eur J Oral Sci. 2010; 118(2): 145–50. PubMed Abstract | Publisher Full Text

[24] 24. Faouzi M, Neupane RP, Yang J, et al.: Areca nut extracts mobilize calcium and release pro-infammatory cytokines from various immune cells. Sci Rep. 2018; 1075: 1–13. PubMed Abstract | Publisher Full Text | Free Full Text

[25] 25. Liu LL, Xiao ZB: Chemical constituents from flowers of Chrysanthemum indicum Chin. Tradit. Herb. Drugs. 2018; 49(22): 5254–8. PubMed Abstract | Publisher Full Text

[26] 26. Khan S, Mehmood MH, Ali ANA, et al.: Studies on anti-inflammatory and analgesic activities of betel nut in rodents. J Ethnopharmacol. 2011; 135(3): 654–61. PubMed Abstract | Publisher Full Text

[27] 27. Okabe S, Amagase K: An Overview of Acetic Acid Ulcer Models —The History and State of the Art of Peptic Ulcer Research. Biol. Pharm. Bull. 2005; 28(8): 1321–41. PubMed Abstract | Publisher Full Text

[28] 28. Sherratt JA, Dallon JC: Theoretical models of wound healing: past successes and future challenges. C. R. Biologies. 2002; 325: 557–64. PubMed Abstract | Publisher Full Text

[29] 29. Frenkel ES, Ribbeck K: Salivary mucins in host defense and disease prevention. J Oral Microbiol. 2015; 7(29759): 1–9. PubMed Abstract | Publisher Full Text | Free Full Text

[30] 30. Holbrook J, Reyna SL, Griffiths HJ, et al.: Tumour necrosis factor signalling in health and disease. F1000Res. 2019; 8(F1000 Faculty Rev)(111): 1–12. PubMed Abstract | Publisher Full Text | Free Full Text

[31] 31. Liu C, Tang J: Expression levels of tumor necrosis factorα and the corresponding receptors are correlated with trauma severity. Oncol. Lett. 2014; 8(6): 2747–51. PubMed Abstract | Publisher Full Text | Free Full Text

[32] 32. Ashcroft GS, Jeong MJ, Ashworth JJ, et al.: TNFα is a therapeutic target for impaired cutaneous wound healing. Wound Repair Regen. 2012; 20(1): 38–49. PubMed Abstract | Publisher Full Text | Free Full Text

[33] 33. Imai S, Otsuka T, Naito A, et al.: Triamcinolone Acetonide Suppresses Inflammation and Facilitates Vascular Barrier Function in Human Retinal Microvascular Endothelial Cells. Curr Neurovasc Res. 2017; 14(3). PubMed Abstract | Publisher Full Text

[34] 34. Harvey W, Scutt A, Meghji S, et al.: Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids. Arch. Oral Biol. 1986; 31(1): 45–49. PubMed Abstract | Publisher Full Text

[35] 35. Steiner SSM, Roy S, Sen CK: Collagen in Wound Healing. Bioeng. 2021; 8(63): 1–15.

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

Abstract

Keywords

Introduction

Methods

Ethical approval

Extract preparation

Formulation of areca nut and chrysanthemum extract oral gel

Experimental animals

Experimental protocol to induce the ulcers

Groups and treatment

Clinical evaluation

Histopathological analysis

Table 1. Histopathologic analysis of induced oral ulcers (histological scores).

Table 2. 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 = 60).

Immunohistochemistry analysis

Collagenesis analysis

Statistical analysis

Results

Clinical evaluation

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

Histological evaluation

Table 3. 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 7th experimental day (n = 18).

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

Immunohistochemical evaluation

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

Collagenesis evaluation

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

Discussion

Conclusion

Data availability

Underlying data

References

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Table 3. 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).