Evaluation of Honey as a Topical Therapy for Intraoral Wound Healing in Rats

Objective. Honey is one of the oldest known medicines. Its medical and therapeutic importance has been recently rediscovered. Honey is an effective treatment for infected wounds and ulcers. The aim of this study was to determine the effect of honey as a topical therapy for intraoral wound healing in rats. Materials and Methods. Thirty-two male rats were divided into experimental and control groups (consisting of 16 rats, 4 animals in each group). A 2-mm mucosal defect was made to the depth of the periosteum using punch biopsy. Honey was applied to the wound every day, and the ulcer size was measured daily. On days 2, 4, 6, and 8, four rats were euthanized from each group (experimental and control groups), and tissues were histopathologically evaluated. Healing processes were studied as follows: the size of ulcer, inflammatory response, reepithelialization, and granulation tissue formation. Results. The mean rank of wound size was significantly reduced in the honey group (2.50), as compared to the control group (6.50). Reepithelialization and granulation tissue formation mean rank were significantly higher in the honey group (6.50) than in the control group (2.50). Inflammation mean rank was statistically lower in the honey group (2.63) compared with the control group (6.38). Conclusion. Honey was shown to have a beneficial effect on the healing of oral ulcers in rats in this model. Further research may shed light on the effects of honey on different types of ulcers in humans.

Honey has been valued for its nutritional and medicinal properties throughout history.¹  About 4500 years ago, almost as long as bacteria have been known, medicinal and antimicrobial activities of honey for wound healing were recognized.^2-4 The medicinal use of honey has reappeared in modern medicine.^5,6 Honey has antibacterial, anti-inflammatory, anti-allergic, antithrombotic, vasodilatory action, and wound healing properties without harmful effects on tissues because it contains antioxidants and flavonoids.⁷ It is also effective in healing wounds, burns, and gastrointestinal disease,^8,9 and it provides protection against gastric lesions.^10,11

Honey clears infection, removes malodors, reduces inflammation and pain, and subsides edema and exudation. In dentistry and oral medicine, honey is useful for periodontal therapy, prevention of infection in wounds following extraction or oral surgery, other oral infections, erosion of mucosa, radiotherapy-induced stomatitis, and oral ulcers, without any adverse reaction.^12-14 Researchers have demonstrated that many oral cariogenic bacteria and fungi including Streptococcus mutans and Lactobacillusare are sensitive to honey.^15-19 Honey has less demineralization depth and lower caries activity than the other sugars.²⁰ The use of honey mouth rinse effectively inhibits oral microorganisms,²¹ but very limited research exists on the healing properties of honey in oral mucosa.^5,22,23 Therefore, this study aims to evaluate the effect of honey as a topical therapy for intraoral wound healing in rats.

Setting. Thirty-two male rats (190–300 g) were used in this study and divided into 2 groups: experimental (honey) and control. The animals were fed a standard diet ad libitum. The experimental and control groups each consisted of 16 rats. Ethical approval of the study was given by the Animal Care Committee of Neuroscience Research Center of Kerman University of Medical Sciences. All procedures were conducted in accordance with the Helsinki Declaration guidelines of the Animal Care Ethics. 

Wound and treatment. The animals were anesthetized with ketamine 100 mg/kg (Alfasan Co, Netherlands) and xylazine (Rompun) 10 mg/kg, intraperit-oneally (Alfasan Co). A 2-mm palatal mucosal defect was made to the depth of the periosteum using a round stainless steel blade designed for punch biopsy (Figure 1). The honey used in this study consisted of nectar from the blossoms of wild flowers and was harvested from a natural honey farm in Malayer, Hojat Asgari Farm, Iran. The honey contains no artificial preservatives or diluents. 

Study design. A cotton swab was soaked in honey and packed into the wound (Figure 2). The wound size was measured with slide-calipers (Preco Machine Tool Co, China) (mm), and the soaked swab was reapplied at the same time (Figure 3). A single jar of honey from 1 source was used for all animal treatments. Ulcers were clinically assessed immediately prior to application of honey every day. The control group injuries were left untreated. On postinjury days 2, 4, 6, and 8, excisional biopsies were made from tissues for histologic evaluation.^24,25 Eight rats (4 rats from experimental and 4 from control groups) were euthanized on day 2, and the remaining were euthanized on days 4, 6, and 8, respectively, with an overdose of anesthetic.

Histopathological evaluation and statistical analysis. Each wound was excised using biopsy punch 5 mm, and approximately 3 mm of mucosa around the incision was maintained. The excised tissue was fixed in 10% formalin solution for histopathological examination. The specimen was oriented for maximum representation, embedded in paraffin, and stained with hematoxylin and eosin (H&E) (Figures 5, 6). A pathologist, who was unaware of the treatments applied to the animals, evaluated the slides.

The ulcer score was histopathologically graded on a 0–5 scale based on the inflammation, 1–5 scale for the level of reepithelialization, and 0–3 scale based on the granulation tissue formation of the tissue (Table 1). The scores were totaled to provide the condition scores of the rat palatal mucosa.^4,26 The results were analyzed using SPSS version 13 (IBM, Armonk, NY) by non-parametric Mann-Whitney U test. 

In this study, there were statistically significant differences in reepithelialization between the honey and control groups on days 4, 6, and 8. Reepithelialization occurred more rapidly in the honey group (P < .05). Granulation tissue formation was also statistically higher in the honey group on days 4 and 8 (P < .05). Inflammatory cells infiltrate decreased, and inflammation was lower in the honey group, and this difference was statistically significant (P < .05). The difference in wound size reduction between the honey and control groups was also significant on days 2, 4, 6, and 8. The wound size was smaller in the honey group in comparison with the control group (P < .05) (Table 2). 

Wound healing was not completed in this study, because the study was performed in 8 days. Normally, the healing process of the oral mucosa lasts about 8 days, but this study was performed on the attached mucosa of the palate in which the healing process is longer than usual (eTable 3).

Honey is created when bees collect nectar from a wide variety of plants and convert the nectar to honey in their crop in combination with various proteins and enzymes. Honey has been used as a medicine since ancient times.^2,27 The natural antioxidants and flavonoids of honey have a wide range of biological effects and wound healing properties.^12-14

An oral ulcer is one of the most common manifestations of oral diseases such as vesiculobullous disease, aphtha, herpes simplex, mucositis, drug eruption, allergic and contact stomatitis, chemical burns, and traumatic ulcer. Oral ulcerations can be painful and interfere with oral functions. Therefore, treatment and management of oral ulcers is very important. Although there are many medical and palliative therapies for oral ulcers, the application of a newer, cheaper, easier, and more available therapy is needed.²⁸

The findings of the present study indicate that honey promotes the healing process of oral ulcers. The results show wound size reduction, reepithelialization, and granulation tissue formation were significantly higher, and inflammatory cells infiltrations were significantly lower in the honey group. Thus, honey clinically and histologically was effective in the wound healing process of rats.

Honey was used as an antiseptic by ancient Egyptians, Assyrians, and Greeks during war and by the Germans as late as World War I.²⁹ Although research studies have been widely performed on the effect of honey on skin wounds, burn ulcers, and intestinal mucosa,²⁷ there is limited evidence about the effect of honey on oral ulcer healing.^5,22,23

This study showed granulation tissue formation was statistically higher after application of honey. Topical application of honey has been recognized to be effective in infection control and clean granulation tissue bed production.⁷ In general, honey acts as a hyperosmolar medium and prevents inflammation. Based on the results of this study, inflammatory cells infiltrate decreased in the honey group. Similarly, in another study²³ on the therapeutic effect of honey on oral mucosal ulcers, there was a statistically significant difference between the honey and control groups on day 7. 

Honey inhibits the growth of a wide range of bacterial species in vitro as well as 2 main cariogenic bacteria: Streptococcus mutans and Lactobacill.^6,18,21,30 The antibacterial activity of honey against cariogenic bacteria and its other beneficial properties may lead to lower caries activity, and it could be used instead of other cariogenic sugar in all sweet nutrients and items such as tooth paste, gum, candy, and chocolate.^20,21 Honey was effective in mucositis prevention, but in the long term, it may be cariogenic for the teeth.^23,31 However, according to a recent study,²⁰ honey had lower caries activity than glucose and fructose.

The authors herein used honey that was harvested from natural honey in Malayer, Iran. Antibacterial activity and composition of honey varies based on the source of honey.³² The results of this study showed natural honey of this region (Malayer) has healing properties.

The antibacterial properties of honey are produced by its high osmotic pressure, hydrogen peroxide effect, stimulating leukocytes activity, high acidity, glucose oxidation reaction, physical properties, low water activity, low pH, acidic environment, low protein content, high carbon-to-nitrogen ratio, low redox potential due to the high content of reducing sugars, a viscosity limiting dissolved oxygen and other chemical agents, phytochemicals, antioxidants, volatiles, flavonoid, beeswax, nectar pollen, and propolis.^{1,18,20,32,33}

A study showed Streptococcus mutans significantly decreased in the radiation-treated cancer patients with xerostomia after honey consumption.³⁴ In the present study, wound size significantly reduced in the honey group. High viscosity of honey causes a physical barrier and moist environment, which accelerates wound healing process. Honey also contains bee pollen enzymes and propolis, all of which can stimulate new tissue growth. The study results showed reepithelialization was faster in the honey group in comparison with the control group. The nutrient contents of honey such as laevulose and fructose improve local substance supply and may promote reepithelialization.^5,22,23,35

As previously mentioned, the findings of the present study indicate that honey increases the formation of granulation tissue, epithelialization, and fibroblast action. These results are in agreement with previous reports.^12,35 Moreover, in keeping with earlier reports, honey decreased inflammation, infection, and wound size in this study. ^36,37 Afshar and Ghaderi²⁹ also concluded that honey seems to be effective in fibroblast activation and granulation tissue formation.

Honey is an available, inexpensive, and acceptable herbal food. The study authors showed that honey works well on acute intraoral wounds, but this finding does not mean that it can heal an ulcer in the mouth caused by a disease or infection. Most previous studies have focused on the effect of honey on mucositis, so conducting more studies on other oral mouth ulcers including recurrent aphthous ulcers, traumatic, or other common oral ulcers is recommended.^5,23 Although honey has been used as mouthwash for prevention or treatment of mucositis, more research is required to evaluate the effects of other forms of remedies with honey including applying honey with an adhesive pad on oral ulcers. Normally, the healing process of the oral mucosa lasts about 8 days. In palatal-attached mucosa, the healing process is longer than usual. In this study, the wound healing was not completed. If the authors were to redo the same study, they would evaluate the healing process time of honey and take a larger sample size in human oral cavity.

This study had a few limitations. One of which was that only 1 type of natural honey was studied. It is better to design studies to compare honey from different sources — natural and synthetic honeys — and study their effect on the wound healing process. The other 2 limitations included a small sample size and the short duration of the study. 

Honey’s medicinal use is effective, safe, and practical. Honey was effective in the oral wound healing process of rats herein, and it may be used as an alternative material for oral ulcer treatment in the future. This study outlines what is known about the medical properties of honey and indicates the potential of honey in the management of acute traumatic oral ulcers in the mouth.

From the Department of Oral Medicine, Dental School, Kerman University of Medical Sciences, Kerman, Iran; Department of Pharmacognosy, School of Pharmacy, Kerman University of Medical Sciences; Department of Genetic and Molecular Medicine, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; Department of Persian Literature and English Language, School of Medicine, Hamadan University of Medical Sciences; and Dental Research Center and Research Center for Molecular Medicine, Department of Oral Medicine, Dental School, Hamadan University of Medical Sciences

Address correspondence to:
Fatemeh Ahmadi-Motamayel, DDS, MS
Associate Professor
Dental Research Center and Research Center for Molecular Medicine
Department of Oral Medicine 
Hamadan Dental School
Hamadan University of Medical Sciences
Hamadan, Iran
f.ahmadi@umsha.ac.ir 
fatahmadim@yahoo.com

Disclosure: The authors disclose no conflicts of interest. Funding was provided by Kerman University of Medical Sciences, Kerman, Iran.