The Effect of Oral Consumption of Propolis Alone and in Combination With Silver Nanoparticles on Wound Healing in Male Wistar Rats

Research to identify and develop compounds that facilitate wound healing is important, especially for hard-to-heal chronic wounds. Purpose: This study was conducted to investigate the effects of orally administered propolis (a resinous substance found in beehives), alone and in combination with silver nanoparticles (SNPs), on the wound healing process in male rats. Methods: Forty (40) male Wistar rats were randomly divided into 4 groups of 10 each: 1 control group received no treatment, and 3 study groups received a daily dose of 1) propolis (100 mg/kg), 2) propolis + 30 ppm SNPs, or 3) propolis + 60 ppm SNPs. Healing rate was determined by wound surface area reduction on days 4, 6, 8, and 10 post-surgery. On day 12 after wound creation, histological changes of wound healing, including number of new vessels, inflammatory cells (neutrophils, eosinophils, and mast cells) and fibroblasts were counted based on morphology using a 400x objective lens, and collagen deposition density was determined using hematoxylin and eosin and trichrome staining, respectively. The histological scores were based on a 0 to 4 scale from lowest to highest amount of improving tissue status and were analyzed using one-way analysis of variance, Tukey test, Kruskal-Wallis test, t test, and Mann-Whitney U test to examine differences among the groups. Significance was set at P <.05. Results: The rate of wound healing was significantly different between the control and the treated groups on days 4, 6, 8, and 10 (percent change was not assessed on day 12) post-surgery, especially in the propolis + 30 ppm SNPs group compared to the control group. This difference was more significant on days 6 (wound healing percentage [WHP]: 75% and 45%) and 8 (WHP: 88% and 65% ) post-surgery (P <.001). Mean neutrophil count on day 12 was highest in the control (34.8 ± 2.97) and lowest in the propolis + 30 ppm SNPs group (16.55 ± 2.12). The number of eosinophils on day 12 was considerably higher in the control group (1.05 ± 4) compared to those in the propolis group (3 ± 0.70), propolis + 30 ppm SNPs group (60/0 ± 1/1), and propolis + 60 ppm SNPs group (0.5 ± 0.52) (P <.001). Mean propolis + 30 ppm SNPs scores for epithelialization and granulation tissue formation were 3 and 4, respectively; in the propolis + 60 ppm SNPs, scores  were 2 and 3, respectively; in the propolis alone group scores were 2 and 3, respectively (statistical significance not computed for semiquantitative values). The highest fibroblast count was in the propolis + 30 ppm SNPs group (114.44 ± 3.90) compared to control group (73.2 ± 2.8); P <.001). The difference in collagen fiber density scores was also significant: 1.2 ± 0.42 in the control  and 3.66 ± 0.50 in the propolis + 30 ppm SNPs group; (P <.001). The mean of collagen fiber density in the propolis + 60 ppm SNPs group was 2.63 ±  0.51. Conclusion: Oral propolis alone and in combination with 30 ppm SNPs appears to provide anti-inflammatory effects and increase fibroblast proliferation and collagen deposition in experimental wounds, which may explain the observed differences in healing. Propolis + 60 ppm SNPs appears to have a cytotoxic effect. Research confirming these results and that examines toxicity levels in animals and humans is needed.

A comprehensive systematic literature review by El-Nour et al¹ has shown silver provided by various routes of administration has been widely used for health and medical purposes for thousands of years, including for the treatment of wounds. Silver nitrate 1% solution as an eye drop was introduced in 1884 to prevent conjunctivitis in newborns, silver compounds were broadly applied topically for the treatment of infected wounds during the First World War, and silver sulfadiazine topical ointment is commonly prescribed for antibacterial applications and widely used to treat burn wounds.^1,2 A systematic review by Mathur et al³ showed silver nanoparticles (SNPs; 1 nm to 100 nm) exhibit particular physicochemical properties, as well as biological activity, and have various routes of application and uses in the health care field, such as biomedical applications, infertility management, antibacterial effects, and to treat skin damage, burns, and cancer. The systematic review by Völker et al⁴ showed the benefits of nanosilver products of various technologies were that they were  nontoxic, eco-friendly, nonstimulating and nonallergenic, and heat-resistant; they have wide availability and are highly and rapidly effective in numerous medical applications, even at low concentrations. Moreover, SNPs in topical or oral application do not degrade and induce long-lasting disinfectant effects at the surfaces where they are consumed. Hence, utilization of nanosilver particle technology does not create or increase resistance and compatibility in microorganisms. However, more research regarding the safety of topical or oral application of SNPs is necessary to consider the possible toxic effects of SNPs.⁴

Propolis, a resinous substance found in beehives, is rich in minerals, micronutrients, and vitamins; it also has a wide variety of flavonoids and antioxidants that boost the immune system, as well as other materials with beneficial bioactive properties that have anti-angiogenic, antiulcer, anti-inflammatory, antiviral, antiprotozoal, anesthetic, antitumoral, anticancer, antifungal, antiseptic, and antimutagenic effects.^5,6 The substances present in propolis vary with the season, the time of its collection, geographical region, and temperature zone. Specific important bioactive components of propolis include phenolic ingredients comprising flavonoids; experimental and review studies^5,6 have demonstrated antimicrobial properties against both gram-positive and gram-negative microorganisms, along with aromatic acids and their esters. Furthermore, flavonoids, such as kaempferol, quercetin, hesperetin, and tangeretin, have been shown in experimental and review studies^7-9 to accelerate wound healing by increasing the amount of prostaglandin in the mucosa and preventing lipid peroxidation.

Skin wound healing consists of 3 closely related phases: hemostasis, inflammation, and structural repair. Experimental and review studies¹⁰ have shown any factor, product, or substance that accelerates reepithelialization, granulation tissue formation, and angiogenesis and reduces infection potentially increases wound healing rate. Current conventional treatments for wounds often include using moisture-retentive dressings that have very few side effects. In certain conditions and chronic ulcers, biological dressings, such as skin autograft, are applied. Despite their benefits, conventional wound treatments may not be sufficient.¹⁰ Especially when wounds become chronic (ie, remain unhealed more than 3 months) and/or occur in patients with concomitant comorbidities, it is important to identify interventions that can facilitate the wound healing process without harmful side effects.¹¹

he results of systematic review and meta-analysis, respectively, by Öhnstedt et al¹¹ and Sung et al,¹² have shown propolis has the ability to boost the immune system and reduce inflammation and infection risk. In a multicenter randomized trial, Pina et al¹³ tested the efficacy of an oral gel form of Brazilian propolis extract on denture stomatitis among 40 older adults. The clinical cure rate of denture stomatitis using propolis extract was equal with miconazole; therefore, propolis extract could be suggested as an alternative and supplementary therapy for elderly persons with denture stomatitis.

The results of a systematic review by Boateng et al¹⁴ showed SNPs can be used to treat various types of injuries and burn wounds due to their significant antimicrobial effects. However, despite broad use of nanoparticles, the biomechanisms in different biological systems still remain ambiguous,¹⁵ underscored by the fact that research on the oral administration of SNPs is limited, the topic is not adequately investigated, and most studies have been conducted to investigate the effects of inhaling, not ingesting, SNPs.^16,17 Additionally, many studies affirm silver’s negative effects. For example, an experimental study by Guo et al¹⁶ evaluated the adverse pulmonary effects of SNP inhalation on the rat small airway epithelium as a target. The results showed damage of respiratory epithelium and modification of cellular cycle decreased proliferation rate and activation of apoptotic signaling. In addition, an experimental study by Jia et al¹⁷ evaluated the effect of oral exposure to SNPs or silver ions (Ag+) in the livers of overweight mice compared with normal weight mice (fed a high-fat diet and normal diet, respectively). The findings showed SNPs and Ag+ at a nontoxic dose initiated development of steatohepatitis from liver fatty changes only in overweight mice.  
In their experimental study, Kim et al¹⁸ evaluated the 28-day oral toxicity, genotoxicity, and gender-related tissue distribution of SNPs in Sprague-Dawley rats. According to study results, no significant differences were noted in feeding and weight changes between the experimental and control groups. Among animals in treated groups given a high dose of SNPs, serum alkaline phosphatase and cholesterol were raised, findings that suggested hepatotoxicity. In their experimental study, Kim et al¹⁹ tested the subchronic oral toxicity of SNPs in 40 rats over 13 weeks and noted a significant decline in body weight among male rats after 4 weeks of SNP consumption, as well as a dose-related rise in serum alkaline phosphatase and cholesterol. These findings demonstrated that high doses of SNPs may be lead to mild hepatotoxicity.

The oral use of propolis and SNPs is not well researched and currently rarely used in North America,^20,21 but the authors believed propolis, as a natural substance, provided orally along with SNPs, might produce synergistic effects in the process of wound healing. The present study aimed to investigate the effects of oral consumption of propolis alone and in combination with SNPs on skin wound healing rates in male rats.

Experimental groups. This experimental study, conducted in the Biophysical and Pathobiology departments of the Qazvin University of Medical Sciences between March 2017 and February 2018, involved 40 male Wistar rats weighing 230 to 250 g. All animals were kept in clean, standard cages with a 12:12 hour light-dark cycle, controlled temperature (22˚ C ± 2˚ C), and free access to food and water. A randomized sampling method was applied. Mechanical wounds were created on the animals and animal care was provided according to all the relevant provisions of the Helsinki Accords. All experiments were carried out in accordance with the approved guidelines and the ethical code of IR.QUMS.REC.1395.154 from the Ethics Committee of Qazvin University of Medical Sciences.

The rats were randomly divided into 4 groups — 1 control and 3 treatment groups. Rats in the control group received oral gavage with a vehicle control. Rats in the propolis and propolis + SNPs groups received oral treatment with propolis (100 mg/kg), propolis + 30 ppm SNPs, and propolis + 60 ppm SNPs over 10 days (daily oral gavage).
Wound creation. The rats were provided general anesthesia by injection (ketamine xylazine, 50.5 mg/kg; Merck) before surgery. The dorsum of the rats was shaved using a sterile blade and disinfected with alcohol (70%). After demarcating, a full-thickness wound approximately 2 cm in diameter and 0.2 cm in thickness was created using a sterile scissor blade on the back along the longitudinal axis animal body without damaging the muscle layer. The wound was created without fixating the skin (ie, nonsplinted). To prevent the animal from biting or manipulating the wounded area, the nape area was utilized.²² Post-surgery, routine care included daily washing of the wound with normal saline solution for all 40 animals. The  wounds were left uncovered/open to the air.

Propolis preparation. The propolis, obtained from a beehive, was stored at -4˚ C. Before extraction from the raw state, 30 g of propolis were vacuum-dehydrated and homogenized, so that 30 g of pure, dry propolis was added to 100 mL of a serial dilution of ethanol (30%, 50%, 70%, and 99% pure ethanol/distilled water solution). The solutions were stored at room temperature for 10 days and shaken once a day. The ethanol extracts then were filtered using Whatman 42 filters, and the solutions were left to dry under freezing conditions. A freeze-dry device was used for this purpose. The obtained propolis then was diluted in 20% propylene and 80% distilled water for oral administration.²³

Preparation of SNPs; combination of propolis and SNPs. Spherical SNPs with an average diameter of 10 nm and stock concentration of 500 ppm were purchased from the Nano Danesh Caspian Co. SNPs were processed using organic and inorganic reducing agents and citrate oxidation. The intended dilutions of the nanoparticles were prepared from the stock solution using the serial dilution method; the prepared dilutions then were mixed in ethylene glycol24 and stored in dark plastic containers with a shelf life of approximately 1 year.

Wound healing rate. The wound healing rate was determined on days 0, 4, 6, 8, and 10 post-surgery by measuring the area of the wound in mm2. To measure the wound surface area, the animals were first held in a standard crouching position; the shape of the wound then was drawn on transparent paper and the wound surface area computed using AutoCAD software. Trained staff performed daily examinations at specific hours.
The following formula was applied to determine the percentage of wound healing:

Sampling and histological evaluation. To perform histological examinations on day 12 post-surgery (ie, in the active wound healing phase), biopsies of the wound and the healthy skin of the adjacent area were obtained after inducing deep sedation with peritoneal injection of ketamine and xylazine (in each of the control and experimental groups) in a closed environment. The obtained samples then were fixed in 10% formaldehyde without being washed. Paraffin was used for blocking, and serial sections of 5-µm thickness tissue from both the wound and healthy tissues were obtained using a microtome. The tissue sections then were stained with hematoxylin and eosin in order to examine the rates (by number of cells) of angiogenesis, inflammatory cells (neutrophils, eosinophils, and mast cells), and fibroblasts. Trichrome stain was used to evaluate collagen deposition density. An Iwf-Iox-Holland eyepiece was applied to the microscope in order to count the cells.

Histological changes scoring of wound healing. The number of neutrophils, eosinophils, mast cells, and fibroblasts was measured using a 400x objective lens. In order to evaluate angiogenesis, an object 100x lens was first used to identify the areas with high vessel density, and then the vessel count was performed in 3 separate fields of these areas using a 400x objective lens. The method proposed by Talas²⁵ was applied to determine and score collagen fiber density in the tissue sections stained with trichrome. The different inflammatory cell types were distinguished based on histomorphology by an expert pathologist, and the cellular element count performed using a 400x objective lens. The cellular number indicated capacity of inflammatory response. A neutrophil was a round cell with a segmented nucleus (2–5 lobes), obvious pink cytoplasm, and abundant fine specific granules. A mast cell was oval shaped with dense granular cytoplasm and centrally located nucleus. Eosinophils were round cells with large eosinophilic cytoplasmic granules and bilobed nucleus.²⁶

Once sampling was accomplished, the rats were euthanized after being anesthetized by ether inhalation.

After histopathological examination, the indices of granulation tissue formation as well as reepithelialization were evaluated semiquantitatively. Histopathological examinations were conducted on the coded tissue slides to perform semiquantitative evaluation,²⁷  where 0 = inflammatory exudate in at least 70% of the tissue; 1 = inflammatory exudate and granulation tissue in at least 60% of the tissue; 2 = granulation tissue in at least 40% of the tissue; 3 = presence of large granulation tissue, together with the formation of collagen fibrils and vessels vertically disposed toward collagen fibrils; and 4 = complete tissue formation in at least 80% of the tissue. For reepithelialization, scoring was as follows: 0 = absence of the thickening of the edges and proliferation of spinous cells; 1 = presence of the proliferation of spinous cells and epithelial projections at the edges of the wound; 2 = migration of spinous cells across the wound; 3 = complete bridging of spinous cells across the wound; and 4 = presence of keratin on the wound (see Table 1).

Data collection and analysis. Data were entered into researcher-developed checklists and analyzed using SPSS V.20. software. One-way analysis of variance (ANOVA), Tukey test and, if necessary, Kruskal-Wallis test were applied for statistical analyses. In addition, a t test and Mann-Whitney U test were used to examine differences between the control and experimental groups.  P values <.05 were considered as significant.

Wound healing. The average percentage of wound healing at days 4, 6, 8, and 10 post-wounding were 26%, 45%, 65%, and 82%, respectively, in the control group; 39%, 66%, 79%, and 91%, respectively, in the propolis group; 44%, 75%, 88%, and 96%, respectively, in the propolis + 30 ppm SNPs group; and 37%, 64%, 79% , and 88%, respectively, in the propolis + 60 ppm SNPs group. The propolis + 30 ppm SNPs group was the only experimental group to achieve 100% wound healing, (exhibited the highest reepithelialization rate). Surface area was not assessed on day 12;  the measure assessed was complete and mature epithelialization with presence of epidermal appendages (see Figure 1 and Figure 2F).
The wound healing percentage (WHP) was significantly higher in the treatment groups, especially in the propolis + 30 ppm SNPs group, compared with the control group. This difference was more significant on day 6 (WHP: 75% and 45% ) and day 8 (WHP: 88% and 65% ) post-surgery (P <.001) (see Figure 1).

Histopathological findings. The control group had the highest (34.8 ± 2.97) and the propolis + 30 ppm SNPs group had the lowest (16.55 ± 2.12) mean neutrophil count. Mean tissue neutrophil counts were almost equal in the propolis alone and propolis + 60 ppm SNPs groups. The control group had the highest mean tissue eosinophil content (4 ± 1.05), and the propolis + 60 ppm SNPs had the lowest (0.5 ± 0.52) (P <.001).

Results regarding the number of tissue mast cells were similar to those of the tissue eosinophil — the control group had the highest mean tissue mast cell count (4.7 ± 0.94) and the propolis +  60 ppm SNPs group had the lowest (2 ± 0.81) (P <.05). The control group tissue had the lowest fibroblasts count (73.2 ± 2.22) and the propolis + 30 ppm SNPs group had the highest (114.44 ± 3.90) (P <.05) (see Table 2 , Table 3, and Figure 2B-J).

Hence, the difference in the numbers of inflammatory cells and fibroblasts were significant between the propolis + 30 ppm SNPs and the propolis + 60 ppm SNPs groups. The increased inflammation and decreased number of fibroblasts suggested a cytotoxic effect of SNPs at the concentration of 60 ppm (see Figure 2A-J).

The control group had the lowest angiogenesis count, and the propolis + 30 ppm SNP had the highest, while the angiogenesis rates were approximately equal in the propolis alone and propolis + 60 ppm SNPs groups (see Table 2). The highest reepithelialization rate was detected in the propolis + 30 ppm SNPs group), while this parameter was equal in the other 3 experimental (see Table 2).

The lowest level of collagen density was noted in the control group (1.2 ± 0.42) and the highest level was noted in the propolis + 30 ppm SNPs group (3.66 ± 0.50) (P <.001). Levels of collagen density were almost equal in the propolis alone and propolis + 60 ppm SNPs groups (see Table 2, Table 3, and Figure 2B-J).

No side effects of treatment were noted during the study; the animals appeared to be otherwise healthy.

The aim of the present study was to compare the effect of oral consumption of propolis alone and in combination with SNPs to control on wound healing in male Wistar rats.

Wound healing occurs in a series of sequential, overlapping phases after injury. Immediately after tissue damage, hemostatic platelet plugs form as framework for inflammatory cell infiltration. The next phase involves inflammatory reaction and includes many cellular elements and chemokine. In the third phase, epithelial, endothelial, and fibroblast cells proliferate. Ultimately, remodeling or healing occurs. The authors used day 12 post-surgery to examine tissue for healing, seeking to assess the active wound healing phase.²⁸

On day 12 post-surgery,  the lowest mean tissue neutrophil count was observed in the propolis + 30 ppm SNPs group, the lowest amounts of eosinophil and mast cells were noted in the propolis + 60 ppm SNPs, and the highest levels of both eosinophil and mast cells were seen in the control group. Furthermore, the control group exhibited the lowest mean level scores for rate and amount of reepithelialization, granulation tissue formation, tissue collagen density, and tissue fibroblast count, while the highest levels of these measures occurred in the propolis + 30 ppm SNPs group. These differences were statistically significant in cases of collagen density, mast cell, fibroblast, eosinophil, and neutrophil counts. The combined treatment with propolis + 30 ppm SNPs induced significant differences in terms of collagen density and fibroblast, neutrophil, and eosinophil counts compared to the propolis alone treatment. Moreover, significant differences were detected in mast cell, fibroblast, and eosinophilic variables in the propolis + 60 ppm SNPs group compared to the propolis alone group. The granulation and reepithelialization scores were the highest in the propolis + 30 ppm SNPs group.  

To the authors’ knowledge, no comprehensive study has yet been conducted to compare the efficacy of oral administration of propolis and SNPs with topical administration of propolis and SNPs in wound healing process. Generally, topical application of active substances such as silver sulfadiazine cream is recommended to obtain adequate effect and with minimal side effects; inactive materials such as propolis that may play a role appear to be activated during many complex processes after oral consumption. In addition, the results of Pina et al,¹³ Boateng et al,¹⁴ Jia et al,¹⁷ and Kim et al¹⁸ demonstrated oral provision at a nontoxic dose of these materials had beneficial effects on healing and therefore were safe to use in normal weight mice. Therefore, topical application of these materials is discussed as follows.

In an experimental study, Jafari et al²⁹ tested the effect of topical yellow plant aloe vera and alcoholic extract of propolis on improving diabetic rat ulcers over 21 days in 50 rats and found the average wound healing rate in the treated group was lower than those of the other experimental groups (P ≤.01). These findings were similar to the results of the current study.

The meta-analysis results of the beneficial effects of topical propolis on wound healing conducted by Oryan et al³⁰ also are consistent with the findings of the present study. Additionally, the authors³⁰ recommended more research on dosing, effectiveness, and side effects of propolis in wound healing.

The beneficial effects of SNPs also have been reported. Fatemi et al³¹ conducted an experimental study and found the use of topical modified chitosan gel (containing SNPs) accelerated burn wound healing and increased the number of fibroblasts compared to the treatment group with the conventional chitosan gel (without SNPs), findings consistent with the current study results. In an experimental study, Jameii et al³² demonstrated that treating Leishmaniasis ulcers in mice with half-wave rectified sine electricity plus SNPs significantly reduced the diameter of the lesion compared to the untreated group.

Results of the experimental study by Rahnema et al³³ that investigated the macroscopic and microscopic effects of  topical application of SNPs supported accelerated wound healing using topical SNPs, but use was associated with some toxic effects. The results of the Rahnema et al study³³ were similar to the current study in terms of healing.

An experimental study by Heidarnejad et al³⁴ that explored the effects of  topical SNPs on hematological parameters during wound healing in mice noted treatment of the wound with SNPs significantly reduced the mean surface area of the wound with immune system stimulation 14 days post-wounding compared to a control group without side effects on certain blood-related parameters. The results of this study were consistent with the findings of the present study regarding healing process.

he experimental study by Seyyedmir et al³⁵ evaluated the effects of nanosilver dressing (ie, a cloth filled with SNPs) on wounds created in rats. Results showed the dressing can reduce wound surface area and wound healing duration in rats and with fewer complications compared with a nonsilver dressing, findings similar to  the current study.

Although the research is limited, it is consistent with the current study findings in terms of beneficial effects of medical application of SNPs confirmed by experimental study.³⁶ SNPs can be applied as medication in the treatment of certain diseases and conditions, such as acne, various types of injuries, and burns, as well as bacterial and fungal infections. However, SNPs can produce reactive oxygen species; therefore, they can induce dose-dependent toxic effects. For instance, an animal study³⁶ has shown long-term oral consumption of high doses of SNPs not only damages liver tissue, but it also decreases the number of white blood cells and may downregulate  the immune system of the treated animal. This negative characteristic of SNPs may result in their limited therapeutic applications. However, not all experimental research has demonstrated the toxic effects of SNPs.³⁷

Overall, SNPs appear to accelerate the wound healing process due to their antimicrobial effects and their ability to prevent the spread of infection. In an experimental study, Adomavičiūtė  et al³⁸ investigated a wound dressing comprised of an electrospun, fast-dissolving nano/microfiber mat formulation containing propolis and SNPs. The authors demonstrated  the antimicrobial effects of combining propolis and SNPs in electrospun mats on many gram-positive, gram-negative bacteria, and Candida albicans strains, finding that this combination induced inhibitory effects on microorganisms such as Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Bacillus subtilis, B cereus, and C albicans.

Just as the antimicrobial effects of SNPs have been proven by several experimental studies,³⁹ the antimicrobial properties of propolis also have been widely reported. Caffeic acid phenethyl ester, a lipophilic compound, acts as a major propolis activator. In agreement with the findings of the present study, a review of the literature⁴⁰ supported that the positive effects of propolis on wound healing are boosted when used in combination with SNPs.

Silver ions can replace the phosphate groups in the nucleotides of the DNA strands; therefore, they can reduce DNA replication. It initially was assumed that SNPs release silver ions in order to induce their antimicrobial effects. However, later experimental studies⁴¹ have indicated that SNPs exert their antimicrobial effects without releasing silver ions. SNPs can penetrate the cell membrane. This study utilized SNPs, not silver ions.

A review⁴² suggested that SNPs induce a pleiotropic effect on bacterial cells. SNPs bind to the surface of the membrane and disrupt its permeability by altering membrane potential and inhibiting cellular respiration. SNPs cations bind to the thiol groups of the bacterial proteins and disrupt their activity, which leads to cell death. Hence, the combination of SNPs with the lipophilic molecules of propolis is a desirable complex, and an experimental study⁴³ suggested that this combination accelerates the burn wound healing. 

Because the effects of SNPs are not cell-specific and all cell types are affected by them, it might be possible to reduce their side effects and enhance their beneficial effects by changing their formulation, combining them with other substances such as plant extracts and designing plant nanocomposites. Additional research with propolis and other compatible substances is warranted.

Preclinical studies have inherent limitations, and additional research is needed to evaluate the effect of these substances on human wounds. In addition, the biodistribution of SNPs in various organs differs between rats and humans. Research is required to investigate the safety of oral SNPs in humans. The beneficial effects of propolis have been long-approved, but few previous studies described the effects of oral SNPs on wound healing, leading the current authors to evaluate the effect of propolis + SNPs in more than 1 strength/dose.

An experimental study that investigated the effects of oral administration of propolis alone and in combination with SNPs on the wound healing process in male rats found that oral consumption of propolis in combination with SNPs accelerated the healing process. The highest beneficial effects, including wound healing rate, were noted when propolis + 30 ppm SNPs was administered. However, increased inflammation and reduced fibroblasts counts indicated SNPs at a higher dose (60 ppm) produced cytotoxic effects. As such, the safety of oral consumption of SNPs requires further research.

The authors thank the Clinical Research Center of Kosar Hospital and Mrs. Simindokht Molaverdikhani for helping to us in manuscript submission in this journal.

Dr. Gheibi is a professor, Biophysics, Biochemistry & Genetics Department, Cellular and Molecular Research Center, Research Institute for Non-communicable Diseases; Dr. Farzam is an assistant professor, Pathology, Pathobiology Department, School of Medicine; Dr. Habibian is a general physician, Pathobiology Department; and Dr. Samiee-Rad is an associate professor of Pathology, Metabolic Disease Research Center, Research Institute for Non-communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran. Please address correspondence to: fsamieerad@gmail.com.