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ORIGINAL ARTICLE
Year : 2021  |  Volume : 24  |  Issue : 11  |  Page : 1694-1705

The investigation of biochemical and microbiological properties of four different honey types produced in turkey and the comparison of their effects with silver sulfadiazine on wound healing in a rat model of burn injury


1 Department of General Surgery, Vocational School of Health Services, University of Health Sciences and Esenler Obstetrics, Gynecology and Children's Hospital, Kayseri, Turkey
2 Department of Pathology, Haseki Training and Research Hospital, Kayseri, Turkey
3 Department of Clinical Microbiology and Infectious Diseases, Vocational School of Health Services, University of Health Sciences and Bakirkoy Dr. Sadi Konuk Training and Research Hospital, Kayseri, Turkey
4 Department of Medical Biochemistry, Hamidiye Medical Faculty, University of Health Sciences, Kayseri, Turkey
5 Department of Medical Microbiology, University of Health Sciences, Hamidiye Medical Faculty, İstanbul, Turkey
6 Department of Surgical Oncology, Erciyes University, Faculty of Medicine, Kayseri, Turkey

Date of Submission20-Sep-2020
Date of Acceptance02-Apr-2021
Date of Web Publication15-Nov-2021

Correspondence Address:
Dr. S Pamak Bulut
Assist Prof at University of Health Sciences, Surgeon. Address: Mekteb-i Tibbiye-i Sahane (Hamidiye) Kulliyesi Selimiye Mah. Tibbiye Cad. No:38 34668 Uskudar, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_582_20

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   Abstract 


Background: To determine and compare the effects of different honey types on wound healing in an animal model, with silver sulfadiazine as the standard treatment. Materials and Methods: Five different groups were created with eight rats in each group. Partial-depth burns were created, and different types of honey and silver sulfadiazine were applied to the respective groups. Rats were monitored for 21 days, and wound cultures were obtained. Histopathological evaluation and cytokine analysis of final tissue samples were performed. In addition, the biochemical and microbiological analyses of the four types of honey used in the study were performed. Results: Wound shrinkage comparisons showed that all four honey-treated groups (Bingöl, Konya, cotton, and citrus) performed better than the silver sulfadiazine group (honey groups, respectively, 86.86%, 84.72%, 89.61%, and 95.33% vs. control 82.90%). However, only citrus honey caused a significant difference in wound shrinkage rate when compared with other groups as well with control group (95.34% vs. 82.9%, P < 0.05). In tissues, all honey groups had higher cytokine (interleukin [IL]-6, IL-1B, tumor necrosis factor [TNF]-α) values compared with controls (P < 0.001). Honey analysis showed a significant inverse relationship between Fe (iron) and the number of diastases. Conclusions: The results of this study support the role of honey in wound healing, due to its antibacterial and immunomodulatory effects. More studies are needed to identify the role of honey composition in wound healing.

Keywords: Burn, cytokines, honey, phytochemicals, rat, wound healing


How to cite this article:
Bulut S P, Gurbuzel M, Karabela S N, Pence H H, Aksaray S, Topal U. The investigation of biochemical and microbiological properties of four different honey types produced in turkey and the comparison of their effects with silver sulfadiazine on wound healing in a rat model of burn injury. Niger J Clin Pract 2021;24:1694-705

How to cite this URL:
Bulut S P, Gurbuzel M, Karabela S N, Pence H H, Aksaray S, Topal U. The investigation of biochemical and microbiological properties of four different honey types produced in turkey and the comparison of their effects with silver sulfadiazine on wound healing in a rat model of burn injury. Niger J Clin Pract [serial online] 2021 [cited 2021 Nov 28];24:1694-705. Available from: https://www.njcponline.com/text.asp?2021/24/11/1694/330473




   Introduction/Background Top


Burns are one of the leading causes of traumatic injuries in homes and workplaces. In 2004, around 11 million people worldwide required medical care due to burns.[1] Although the incidence of injuries caused by burn have shown a decreasing trend throughout the years, it is still a health problem that causes significant morbidity.[1],[2] Burn wounds constitute an important social and financial burden due to the need for long-term care, loss of labor, and significant costs of treatment for all health systems around the world.[3] Although major burns have been studied in detail with regard to their outcome and costs, there is not much evidence for minor and moderate burns; however, it is possible to the estimate the treatment cost of minor and moderate cases by taking into account the growing wound care products market. A considerable number of options for treatment exist, but they remain somewhat insufficient in wounds that heal slowly, especially when infection or other factors that disrupt wound healing are present.[4] It is expected that an ideal agent used in wound treatment will not damage intact tissue while fighting against microorganisms and other conditions that impair wound healing. The search for new options in wound treatment is ongoing, with frequent studies on new methods and products.

Honey stands out as a natural product that provides wound healing and beneficial immunomodulation. Honey and sugar are sometimes used in the clinic as traditional treatment tools, especially in stubborn and problematic wounds that do not respond to standard treatment. The use of honey as a food supplement or topically for medical purposes dates back to ancient times.[5–7] Furthermore, a comprehensive examination of available literature seems to support that there is a fair amount of reliable evidence on the effectiveness of honey in burns, laceration, abrasion, and wound infections; however, it goes without saying that the lack of standardization is an important limitation.[5–8] In addition, it has been shown that there are only a few reports that establish the application of honey in such conditions (type of honey, amount to be applied, how to control application area, etc.).[9]

Even in the age of Aristotle and Dioscorides, honey composition was known to be influenced by the season of collection; thus, it was suggested that season had a considerable effect on the treatment spectrum of the honey. Furthermore, different floral sources were believed to cause different therapeutic properties.[10]

Today, we know that honey consists of more than 200 different substances. The major elements of honey are quite straightforward: sugar (about 76%) and water (about less than 20%), whereas proteins, enzymes, organic acids, vitamins, minerals, pigments, and other compounds are found in its composition, but they may be at significantly different concentrations. The variable content of honey also causes its effectiveness to vary. Previous studies show that the differences in effectiveness of honey on wound healing can result from the difference between the types of honey as well as the design of research. It is known that the composition of honey is influenced by many factors, particularly the geographical floral structure. On the other hand, the industrial and marketing processes used until honey reaches the end consumer are also important contributors to its composition and purity, which may cause important alterations from the hive to the shelf. Unfortunately, it is also well-known that many honey-labeled products sold on market shelves are neither natural nor do they come through a responsible industrial process. When it comes to using honey for medical purposes, the purity and quality of honey are even more important.

Turkey is ranked second after China in world honey production with 107,000 tons of yearly production,[11] and it has great potential, not only in terms of quantity but also in terms of having a rich floral variety.

Silver sulfadiazine is one of the most preferred drugs as a control group comparison treatment in animal experiments (similar to human studies) due to its easy application, low risk of toxicity and allergies, and antibacterial effectiveness.[12] In this study, we aimed to evaluate the effects of four honey types on wound healing in partial-depth burn and to compare results with standard silver sulfadiazine cream treatment.

Cytokines play a crucial role in wound healing process and anti-infective activity.[13] At the cellular level, cytokine activity is detected in the local region of wounds, even when an increase in systemic circulation cannot be detected.[14] We decided to examine cytokine changes in the tissue because the burn wounds we created were small; thus, systemic levels may not have been reliable or conclusive.


   Materials and Methods Top


Study design

When choosing honey, it was preferred that they should not to have any antibiotic and pesticide contamination, and we also aimed to represent a reliable group of the different types of honey produced in Turkey.

The different types of honey investigated in this study were collected directly from beekeepers from different regions of Turkey (during the course of 2019) [Table 1], and chemical analyses were performed to evaluate and confirm the discriminating flavors and odors that are specific to each of these honey types.
Table 1: Types of honey and harvested geographical area

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An animal experiment was designed to analyze the effect of four different honey types on wound healing through an in vivo burn model. We planned to compare clinical results between honey types and the control group as well as to determine the relationships between treatment results and the biochemical and microbiological properties of honey.

The selection of the four honey types and the microbiological and biochemical analysis of the honey were carried out by our project partner Altıparmak Food R&D Center. The known names of the selected honey types are as follows: Konya honey, Bingöl honey, cotton honey, and citrus honey. The honey analysis was carried out by Altıparmak Food R&D Center, and the honey samples were delivered to us after being coded numerically. To prevent researcher bias, honey types were packaged with numerical codes during all experiments. The analysis information was used to evaluate relationships only after animal studies were complete.

Approval for the study was obtained from the University of Health Sciences Local Ethics Committee on Animal Experimentation, decision number: 2018-08/02. All the procedures of the experiment were conducted in accordance with the best ethical practices and rules of animal experiments. The experiment was carried out in the animal laboratory at the University of Health Sciences Experimental Medicine Research and Application Center.

Preparation of rats

A total of 40 rats, 20 male and 20 female Sprague–Dawley rats (250–350 g, 8–10 weeks old) were used in the experiment. Although gender was reported to not be effective in burn wound healing,[15] we chose to include equal numbers of male and female rats because of the use of different types of honey that could have different allergic profiles and varying effects on treatment. All rats were kept in a temperature-controlled (25 ± 1°C) environment with a 12-h light/dark cycle in individual cages. They were fed ad libitum with standard chow and normal water. The anesthesia of all 40 rats was performed by intraperitoneal administration of 80 mg/kg ketamine + 10 mg/kg xylazine.

Creation of a burn wound

To minimize wound contact after burn damage, a region on the back closest to the head was selected and shaved. Then a culture sample was taken to analyze skin flora. A literature review of in vivo burn studies on animals showed that using a well-conductive brass rod to create the burns was the best overall method for burn application.[12],[16],[17] The wound size was planned as a 14 × 14 mm sized square; providing sufficient tissue sample for histological and cytokine analysis, while also minimizing systemic response.

Fluke 17B + digital multimeter and heat probe were supplied from the Biomedical Laboratory of the University of Health Sciences. A brass rod with a 14 × 14 mm square surface (18 cm length and 300 g of weight) was combined with a heat probe and connected to the multimeter. It was heated to 96°C in boiling water. Contact with the shaved tissue area was performed for 20 s with very gentle pressure [Figure 1]a.
Figure 1: (a) Creation of a burn wound with a brass rod. (b) Honey application after burn

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Formation of treatment groups and implementation of treatment

The rats were assigned to random groups with four males and four females in each group. After the burn was created, the first four groups received specific honey types that were applied with cotton swabs, whereas the control group was applied silver sulfadiazine in a similar manner [Figure 1]b. Meanwhile, the rats were taken into cages. They were kept in separate cages for the duration of the experiment as a precaution to prevent wound-area interactions between the rats and also to avoid the risk of cannibalism. For analgesia, 200 mg/kg paracetamol was added to their water for 3 days. If the burn width was less than 20% in partial or full-thickness burns, no postburn fluid treatment was given because parenteral fluid therapy is not recommended under these circumstances.[12] During the experiment, honey and silver sulfadiazine were applied topically to the wounds of the rats once a day.

Exclusion

One rat (numbered 29) was excluded from the study due to ophthalmitis that had been present from the first day of the experiment.

Microbiological examinations

Wound culture was performed on the third, seventh, and 14th days. The samples taken in sterile conditions were delivered to the laboratory immediately and were cultured in 5% sheep blood agar, MacConkey agar, and incubated at 37°C for 18 to 24 hours. Gram staining and other conventional methods were used in addition to the VITEK 2 (bioMérieux, France) fully automated identification system and MALDI-TOF MS (matrix-assisted laser desorption ionization–time-of-flight mass spectrometry) system (bioMérieux, France) for the identification of the bacteria detected in culture.

Ending the experiment

On the 21st day, the last wound photographs of the rats were taken under IP 80 mg/kg ketamine + 10 mg/kg xylazine anesthesia, and the wound tissue was excised. Half of the tissue sample was placed in formalin and was used for histopathological examination, whereas the other half was put in Eppendorf tubes in a box containing dry ice. The latter half was used for the analysis of immune cytokines. Finally, the rats were sacrificed by decapitation while under anesthesia.

Macroscopic evaluation

Wound photos were taken throughout the study on the third, seventh, 14th, and 21st days. The wound size and the macroscopic changes were observed. The following characteristics were observed and recorded: color, edema, crusting, contraction, discharge, and necrosis development.

The wound shrinkage rate (WSR) was calculated using the following formula:



Histopathological examinations

A total of 39 biopsy materials were identified and obtained with 10% formaldehyde from the separate numbered boxes. After 24 hours, each tissue was sampled. Paraffin blocks were prepared from tissues undergoing routine evaluations. Sections of 4 μm thickness were taken and stained with hematoxylin and eosin (H&E). The preparations were scanned under a light microscope (Nikon Eclipse Ni-U model) by a single pathologist with 400 × magnification area (40 × objective lens, 10 × ocular lens). According to Ehrlich and Hunt scoring system, the inflammatory cells (lymphocyte, neutrophil, eosinophil, and macrophage) were evaluated. Blood vessels and fibroblasts were scored from 0 to 4 (0: absent, 1: rare, 2: rare, 3: medium, and 4: intense/obvious).[18]

Cytokine analysis

Cytokines measured to evaluate inflammatory activity were quantified in the tissue by ELISA (enzyme-linked immunosorbent assay). After the skin area was excised, homogenization was first applied to tissue samples reserved for cytokine analysis. Tissue weighing 0.2 g was placed in a clean 1.5 mL of Eppendorf and 500 μL of PBS was added. The tissue shredding was performed on ice. After centrifuging at 5,000 xg for 5 minutes at 4°C, the supernatant was removed and used for quantification.

The biochemical and microbiological analysis of honey types

The color, moisture, acid and conductivity measurements, hydroxymethylfurfural, diastase count, proline levels, sugar profile, protein content, antioxidants, total phenolic content, minerals, heavy metal content, starch/pollen ratio, and the pollen content of honey were analyzed. In addition, antibiotic and pesticide contamination in honey was investigated.

The presence of total aerobic bacteria and mold in honey was examined. Antibacterial activity of honey was determined using the microdilution method according to ISO 20776-1 toward Staphylococcus aureus Scientific Name Search  (ATCC 25923) and  Escherichia More Details coli (ATCC 25922) reference strains. The concentration with 80% or more reduction compared with the positive control was recorded as minimum inhibitory concentration (MIC), and the lowest concentration (99% reduction in absorbance) without growth was recorded as minimum bactericidal concentration (MBC).

Statistical analysis

All statistical analyses were performed by using SPSS Version 23.0 (IBM, Armonk, NY, USA). Chi-square tests were used to compare categorical variables. To examine the differences between categorical and continuous variables, the Shapiro–Wilk test was used to determine whether data were suitable for normal distribution. The one-way ANOVA test was used to compare continuous variables between more than two groups. The categories of variables found to be different were compared in pairs using the Tukey test for post hoc correction. In the study, P values less than 0.05 were considered as statistically significant.


   Results Top


The average wound size was 13.72 mm × 14.32 mm. When the wound shrinkage rate was evaluated after 21 days, the group that received citrus honey was the best with an average of 95.34% closure (median 94.9%), whereas the control group treated with silver sulfadiazine was the worst with an average of 82.9% closure (median 90.39%) [Figure 2].
Figure 2: Comparison of wound shrinkage rates in experimental groups (Due to the number of samples, Mann–Whitney U test was used.)

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Each experimental group was compared with the control group in terms of wound shrinkage rate, and only citrus honey was found to demonstrate a statistically significant difference.

Wound shrinkage rate was found to be significantly higher in female rats than in male rats (P < 0.001) [Table 2].
Table 2: Comparison of gender in wound healing

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No edema or discharge was observed after the first 3 days. Crusting and contraction in the wounds was a common macroscopic finding. The contraction was evident from the 14th day and accelerated thereafter. Re-epithelialization was observed only during the third week.

A total of 43 flavonoids were investigated in the total phenolic content analysis. In the flavonoid group, 2-hydroxy cinnamic acid (o-Coumaric Acid), 3,4-dimethoxybenzaldehyde, apigenin, CAPE, catechin, chrysin, epicatechin, epigallocatechin, galangin, genistein, hesperetin, isorhamnetin, kaempferol, luteolin, accor myricetin, naringenin, pinocembrin, quercetin, resveratrol, rosmarinic acid, rutin, and taxifolin were not detected.

Furthermore, 3,4-dimethoxycinnamic acid, 4-hydroxy benzoic acid, caffeic acid, chlorogenic acid, cinnamyl aldehyde, ellagic acid, ferulic acid, gallic acid, homogentisic acid, methyl syringate, p-coumaric acid, phenyllactic acid, phloroglucinol, protocol quinic acid, shikimic acid, syringic acid, trans-cinnamic acid, and vanillic acid were detected in the analysis [Figure 3]. There was no significant difference between the four types of honey in terms of variety. In terms of quantity, the flavonoid level of Konya honey was the highest.
Figure 3: Flavonoid compositions of honey types

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Contaminants, such as multiple sulfate groups and pesticides, were below the detectable limit in all honey types.

In terms of mineral content, Bingöl honey was poor in Ca (calcium), K (potassium), and Cu (copper) compared with the others, Konya honey was rich in Mn (manganese), Ca, K, Sn (tin), Mg (magnesium), and Cu compared with the others, and cotton honey was rich in Zn (zinc), Na (sodium), and Mo (molybdenum) compared with the others. Citrus honey was poor in Mn, Na, Co, Mo, Mg, and Fe (iron) compared with the others, and was rich in Zn compared with the others – except for cotton honey.

In the microbiological analysis of the honey, the highest total aerobic living amount was determined in Konya honey (250 cfu [colony forming units]/g). The fermentum was available only in cotton honey (20 cfu/g). Mold, E. coli, coliform bacteria, and S. aureus were not detected in any honey types. When in vitro antibacterial efficacy was examined, S. aureus (ATCC 25923) MIC and MBC values were lower in cotton honey than in others. For E. coli (ATCC 25922), which was lower in the antibacterial activity of cotton honey, MIC and MBC values were the same for all four honey types.

In the residue analysis of the honey, we must note that the absence of pollutants and antibiotics reveals its purity and quality, whereas the diastasis coefficient shows its naturalness.

Correlation analysis was performed between the wound healing averages and the parameters of honey analysis. A significant inverse ratio between Fe and diastasis number was found. There was no significant relationship between the other data and wound healing [Table 3].
Table 3: Relationship between wound shrinkage rate (WSR) and biochemical composition

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In the statistical analysis of the histopathological examination results, no significant difference was observed in terms of inflammation, blood vessel, and fibroblast values compared with the control group [Figure 3].

When the results of cytokine analyses were compared with the control group, it was seen that all honey groups had higher cytokine (interleukin [IL]-6, IL-1B, tumor necrosis factor [TNF]-α) values compared with controls, and the differences were statistically significant (P < 0.001). There was no difference between the honey types [Figure 4].
Figure 4: (Top) Epithelial loss and presence of fibrinopurulent exudate; capillary proliferation in the dermis and active chronic inflammation around it; increased fibroblast activity (HE 100×) (Bottom) Pronounced inflammation cells and prominent capillary vessel proliferation

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With regard to the results of samples cultures,[19] we found the following results: Considering the suppression of reproductive bacteria in all groups, least suppression was in the fourth group (citrus honey), and the most was in the control group. In the first culture assessment of the first and second groups, three plaques remained sterile. However, in the next week, this suppression was only present in one plaque. The other types had less concentrated bacterial suppression.

The basic physicochemical properties of the honey species are summarized in [Table 4].
Table 4: Main physicochemical properties of honey species

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   Discussion Top


In a systematic review published in recent years, where the efficacy of silver sulfadiazine and honey were compared in the treatment of burns, the wound healing time and the ratio of infected wounds to sterile ones were evaluated. Based on the findings, it was concluded that honey is superior to silver sulfadiazine in the treatment of burns.[20] There are also several other studies showing the superiority of honey in terms of wound healing rate, infection control, and inflammation, especially in burns with partial depth.[7],[21]

Our study revealed results that were in support of the known positive roles of honey in wound healing. Citrus honey showed a statistically significant effect on wound healing compared with the other three honey types and the standard treatment silver sulfadiazine.

It seems the effect of gender on wound healing process provides an advantage in female rats.

In the biochemical content analysis, the inverse ratio between Fe and diastasis number was found to be statistically significant. There are many studies claiming that the phytochemicals in honey, which are known to consist of about 200 kinds of components, are related to the effectiveness of honey. In our study, 43 flavonoids were investigated, and only 20 were within detectable levels in honey. There was no significant relationship between flavonoids and wound shrinkage rate.

The reported effects of honey in wound healing can be summarized as follows: It provides better wound contraction, granulation tissue formation, provides epithelialization, increases collagen synthesis and tissue growth, stimulates new vascular formation, reduces edema, reduces inflammation, reduces bad smell, maintains the moisture of the wound, makes debridement easier, and reduces pain.[5] Epithelialization was not observed microscopically in the tissue samples. Granulation tissue and active chronic inflammation were dominant in the examination.

The antibiotic effect of honey is explained mainly by two mechanisms: peroxide and nonperoxide activity.[22] Hydrogen peroxide (H2O2), formed by the action of the glucose oxidase enzyme, inhibits bacterial growth. Nonperoxide activity depends mainly on the activity of complex phenols called flavonoids and organic acids.[6] In particular, the phytochemicals that contribute to nonperoxide activity are thought to change according to the floral source of honey.[23] However, peroxide activity was found to be different even when the honey was derived from the same type of floral source. This has been associated with different geographical features, changing environmental conditions, and even the health of bee colony.[10]

Honey, with its nonperoxide antioxidant activity, counteracts free radicals that destroy tissue and increase inflammation. This feature was found to be more evident in darker-colored honey types.[23],[24] In the current study, antioxidant activity was the highest in Konya honey, which has the darkest color of the four honey types in our study.

It is thought that the acidic pH and high osmolarity of honey also contribute to its antibacterial properties.[25] With its hygroscopic feature, honey attracts water from the environment, dehydrating bacteria and reducing wound moisture.[25] In addition, the acidic nature of honey facilitates the release of oxygen from hemoglobin into the tissue, promoting granulation tissue formation and wound healing.[26]

In the first culture assessment of the first and second groups, three plaques remained sterile, which was interpreted as an equal suppression with the fifth group.

In vivo animal studies have shown that the effectiveness of honey on bacterial growth was not significant compared with its in vitro antimicrobial efficacy.[27] This may be due to the difficulty in providing wound isolation in animal studies.

Honey is known to be effective on anaerobic, aerobic, gram-positive, and gram-negative bacteria and has bacteriostatic or bactericidal effects.[23],[25]

In the in vitro analysis of honey, antibacterial activities to E. coli and S. aureus strains were determined by measuring the MIC and MBC values. In the in vivo experiment, honey was applied to the wounds without dilution. Due to contamination and the mobility of the rats, reproduction of the intestinal and skin flora bacteria was observed in the culture results; this was less frequent in the control group. This could be due to the fact that the honey was not prepared as a treatment preparation (depending on its viscosity and density). It is noteworthy that the wound healing was better in the honey-treated groups despite the presence of intense contamination. This observation supports the suggestion that the effects of honey on wound healing are associated with other factors than only its antimicrobial properties.

In one study, it has been reported that the antimicrobial potency difference between the different geographic, seasonal, and botanical sources, and between harvest, processing, and storage conditions can be up to 100 times.[23],[25] In the medical literature, there is no guide that has put forth a method for the selection of honey type in infection treatment. The antibacterial properties and potencies of honey species are different, and storage conditions may change these features. It may be possible to utilize the antibacterial properties of honey more effectively by revealing factors influenced by storage conditions.[28] Although they came from the different geographical and floral sources, it was observed that the four types of honey used in this research (which were stored in the same conditions) had beneficial effects on wound healing, despite small differences between each other.

One of the most interesting features of honey is immunomodulation. Depending on the condition of the wound, it can inhibit or stimulate the release of cytokines such as TNF-α, IL-1β, and IL-6 from monocytes and macrophages.[6],[29] Although honey increases the release of inflammatory cytokines in cases with low levels of inflammation, it can suppress the release of cytokines such as TNF-α and IL-1β in the presence of infection.[29] Cytokines are considered important markers in the pathophysiology of burn injury.[30] TNF-α plays an important role in wound healing by directly or indirectly stimulating fibroblast proliferation, re-epithelialization, and neovascularization, and increasing wound fracture strength.[31]

In the tissue samples taken on the 21st day of the experiment, there were statistically significant higher values in all groups treated with TNF-α, IL-1β, and IL-6 compared with the control group treated with silver sulfadiazine as the standard treatment [Figure 5]. Despite the high microbiological load of the wounds, the immunomodulatory effect of honey may explain why wound healing is better than the control group.
Figure 5: Comparison of cytokine changes in honey groups with the control group

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Depending on the condition of the wound microenvironment, it can activate or suppress the release of reactive oxygen species (ROS) from neutrophils.[6] The immunomodulating feature of honey is quite complex in relation to the different compositions of honey compiled from different sources.[29] There are studies showing that honey achieves its anti-inflammatory effect by inhibiting COX-2 (cyclo-oxygenase-2) and iNOS (inducible nitric oxide synthase) expression, and with its anti-oxidant and free radical scavenging effects.[7]

In a study examining the molecular mechanisms of honey's antioxidant, antibacterial, antifungal, antiviral, anti-inflammatory, antidiabetic, antimutagenic, anticancer, cardiovascular protective, and immunomodulatory effects, it was found that the probiotic bacteria in honey repair the damaged immune system. In the same study, it was found that honey contributes to immune regulation by increasing the levels of immunoglobulin released from B lymphocytes, interferon, and phagocytic activity. In addition, antioxidants were found to contribute to immune regulation in different ways such as oxidative burst and H2O2 production due to sugar content, macrophage stimulation by providing glycolysis substrate, formation of short-chain fatty acids fermentation products, and cytokine release from monocytes.[8],[32] Honey increases lymphocytic and phagocytic activity in the wound,[26] and it ensures the removal of debris and bacteria. There are studies claiming that the proinflammatory activity of honey depends on intrinsic endotoxins.[33] TNF-α, IL-1β, and IL-6, also known as proinflammatory cytokines, are key elements in wound healing.[34] Moreover, honey has effects on keratinocyte and fibroblast proliferation, production and degradation of extracellular matrix proteins, stimulation of fibroblast chemotaxis, and regulation of immune response. In the in vitro studies, a minimal toxic effect of honey on keratinocytes and fibroblasts was observed.[8] Especially in the inflammatory phase of recovery, these cytokines usually peak.[34] In our study, significantly higher values of IL-1 β, IL-6, and TNF-α (high local cytokine levels) compared with the control group indicate the immunomodulatory effect of honey.

The variable composition of honey is a limiting factor for its direct use as a medicinal product, but it also offers a great potential for research.

In terms of phytochemicals analyzed in citrus honey, no specific increases were detected compared with the other honey types. In such a situation, it would have undoubtedly been easy to define a linear relationship and link this effect to a single component. The existence of a compositional effect that cannot be attributed to a single component should be considered. Indeed, Gheldof et al. found that the in vitro singular antioxidant activities of flavonoids in honey produce only a part of the total antioxidant capacity of honey.[35]

Today, the increasing microbial resistance to antibiotics is a serious problem.[36] Therefore, natural and traditional treatment methods such as honey have reemerged as candidate treatments.[7] There are in vitro findings showing that honey reverses the resistance to antibiotics and reduces microbial pathogenicity.[27] These are promising findings for further investigations in a translational context. There have been no reports of microbial resistance against honey.[37],[38] The fact that the antibacterial effect of honey is accomplished by multiple mechanisms with its multifactorial and complex composition is advocated as possible reasons for the absence of resistance.[26]

The antimicrobial effect of honey is mainly realized by direct antimicrobial effect and immune regulation, thereby indirectly increasing the body's resistance to microbes.[39] In this regard, it is different and unique from antibiotics.

H2O2 is one of the important components of the antimicrobial activity of honey. Depending on the glucose oxidase activity in honey, it is slowly released during the interaction with wound exudate. It is known that H2O2 concentration decreases when the concentration of honey increases.[40] For this reason, honey with low concentrations is more suitable for the purpose of wound treatment. In fact, the dilution of honey in the wound increases glucose oxidase activity and H2O2.[40] The H2O2 level in honey is 1,000 times lower than in 3% H2O2 solutions that are used as antiseptic.[5] Although it does not damage the tissue at this concentration, it can also show antibacterial effects.

On one hand, honey has antibacterial and inflammatory-stimulating effects as a source of H2O2, while it also has considerable antioxidant activity. H2O2 has also been shown to stimulate fibroblast activity and angiogenesis.[5] However, it is also known that excess H2O2 can have a toxic effect on human cells. Honey works as a curbing mechanism with the antioxidant substances it contains, and it clears superoxide radicals by preventing tissue damage through the control of peroxide activity. Perhaps it is this balanced composition that makes honey a more effective wound-friendly antiseptic than H2O2 solutions.

The level of iron in citrus honey was significantly lower than that of other honey types. It plays an important role together with copper in iron reduction reactions. In the presence of Fe, H2O2 leads to the production of toxic metabolites.[41] H2O2, which has an important role in wound healing, will be less to reactive in oxygen type formation with Fenton reaction when Fe amount is low. With the Fenton reaction, Fe2 + oxidizes to Fe3+, and turns H2O2 into the radical OH•. Thus, the use of H2O2 and the formation of ROS can also be reduced. This may explain why honey with a low Fe level had significantly better effect, because the great majority of bacteria need iron to reproduce. The low iron content of this specific honey type may prevent accelerated bacterial reproduction as well as lowering the peroxide activity of honey, thus inhibiting the production of ROS that damage healthy cells in the tissue.

Honey can be contaminated with microorganisms. However, because honey is not a fertile environment for microorganisms, only spore-producing bacteria and some yeasts can survive, but even these cannot proliferate. The limited and controlled presence of these microorganisms may be stimulating for the cells of the immune system.[42] The cellular components of microorganisms can cause the release of cytokines from immune cells through pattern-recognizing receptors (PRR) such as toll-like receptors (TLRs).[42]

The positive effects of honey on wound healing, its antimicrobial, anti-inflammatory, and immunomodulatory effects continue to arouse interest, and many studies have attempted to understand the mechanisms. However, it has not found enough place in clinical practice. For surgeons to prefer honey in burn and wound treatments, its efficacy should also be demonstrated by clinical prospective randomized studies. Concerns about the purity of honey in the market and its difficulty in application can cause reluctance. Knowing which honey or which component of honey provides benefit in the treatment may provide the basis for the development of standardized products for medical use.

In burn wound care in addition to the antimicrobial effect of silver sulfadiazine, honey can be safely preferred due to its antimicrobial, anti-inflammatory, and immune modulatory effects. Our study is the first novel research that investigated and compared the biochemical composition and microbiological effects of honey types specific to the flora of Turkey in terms of their influence on burn wounds in a rat model. It is unique and valuable in its attempt to reveal the possible relationships between honey composition and its known effects on wound healing.

Limitations

Although microbiological contamination of honey is possible in the process of harvesting, transporting, and processing, the contamination is limited because honey is not suitable for the growth and survival of microorganisms. It is recommended as a general precaution not to give honey to children up to 1 year of age, due to the presence of spore-producing species and the risk of botulism. Also, grayanotoxin-containing honey, which is known as “mad honey” should be distinguished. The hypersensitivity reactions to honey should be taken into account, especially in relation to pollen allergy.[7],[32]

One of the major limitations of the burn model in rats is the differences between rat skin and human skin. Although rats have a multilayered structure like epidermis and dermis similar to human skin, they have some differences. Rats are classified as loose-skin animals. Whereas the human skin is better attached to the underlying structures, the skin of the rat is more elastic and loose. It also has the l-gulonolactone enzyme that produces vitamin C, which is not found in humans, and is important for wound healing. Although re-epithelialization is more important in wound healing in humans, wound contraction is much more important in rats, due to the presence of the panniculus carnosus muscle, which is absent in humans. These features, which also provide faster recovery in rats, make rats attractive for the researchers as an economical option in burn and wound models.[43]

The first difficulty of applying drugs to the burn wound in rats is the difficulty of ensuring that the applied substance remains on the wound. Various behaviors of rats, such as wound licking, scratching with extremities, and attempts of cleaning the area by rubbing it to their bedding, can reduce long-term contact. They also tend to remove the dressing materials. For these reasons, the substances tested in burns and wound healing should also be tested in cell culture and clinical studies.


   Conclusions Top


This study aims to contribute to the literature by answering the previously unaddressed question in this field: which honey, and why? thereby providing a new baseline for future studies. The choice of honey type, its dosage, and form of application in wounds are still not clear for clinicians. However, some certain criteria must be determined, and standardization must be provided to use honey safely in critical wounds. Observing the differences in efficacy among honey types investigated in previous studies, performing comparative in vitro and in vivo studies, and the planning of clinical studies to identify distinctive factors are essential for the determination of the medicinal efficacy and safety of this natural product that provides important therapeutic benefits.

In this study, the positive effect of citrus honey, which is a multifloral honey produced in the Mediterranean region, was found to be significantly better than control treatment in partial-depth burn wound healing. Although Bingöl honey, Konya honey, and cotton honey also provided higher rates of wound shrinkage than the control group, the differences from the control group were not statistically significant. The cytokine levels in wound tissue were significantly higher in all groups treated with honey compared with the control group, which indicates the immunomodulation effect of honey in wound healing despite the presence of microbiological load in the wounds.

Turkey is among the most important countries in the world in terms of both honey production and floral diversity. More numerous and qualified research is needed to evaluate this potential in the most accurate way.

Declarations

The data sets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Pamak Bulut acknowledge the support of her advisor Prof. Dr. Nihat Bengisu who inspired her with his clinical work to investigate the healing effects of honey in this study.

We also acknowledge the assistance of our project partner Altıparmak Food Industry and Trade Inc. and Balparmak Research and Quality Control Laboratory for Honey and Other Bee Products (APILAB) for sharing their experiences in honey selection and analysis.

And, additionally, We thank Lecturer Bekir Erdoğan for his contribution in statistical analysis.

Financial support and sponsorship

This work was supported by the University of Health Sciences Scientific Research Projects Coordinatorship [Grant Number 2019/056].

Conflicts of interest

The authors whose names are listed above report the following details of affiliation or involvement in an organization or entity with a financial or nonfinancial interest in the subject matter or materials discussed in this manuscript. Balparmak Research and Quality Control Laboratory for Honey and Other Bee Products (APILAB) of Altıparmak Food Industry and Trade Inc. shared their experiences in honey selection and analysis. (It is not a producer company, but collects producers' goods.)



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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