Use of propolis for skin wound healing: systematic review and meta-analysis

Propolis is a natural resin that is produced by bees. It has anti-inflammatory and antibiotic properties, promotes reepithelization, and stimulates skin regeneration. Propolis has great potential for the development of new therapeutic approaches to treat skin ulcers. The present study performed a systematic review and meta-analysis of published studies of the use of propolis for the regeneration of cutaneous wounds and its efficacy as a therapeutic agent. Data were collected from articles in the PubMed, SCOPUS, and Web of Science databases that were published since 1900 by searching the terms “propolis” AND “wound healing.” This search yielded 633 articles, of which 43 were included in this systematic review and meta-analysis. The results showed that interest in the therapeutic efficacy of propolis has increased over the years. The studies reported that the propolis was effective for the treatment of skin ulcers by promoting a higher percentage of healing than classically employed interventions. The mode of propolis application has also evolved. An increasing number of studies combined it with other substances and materials to achieve additive or synergistic effects on the skin regeneration process. Propolis appears to be an effective therapeutic alternative for the treatment of skin ulcers.


Introduction
The skin acts as a physical and chemical barrier that protects internal organs and the body as a whole against pathogens and dehydration [1,2]. The skin is most often exposed to injury and trauma, which represents a potential risk to the body's integrity. Skin regeneration is characterized by four stages: hemostasis, inflammation, proliferation, and remodeling. It acts to stop bleeding, prevent infection by removing debris and microorganisms from the wound bed, proliferate epithelial cells and fibroblasts, stimulate angiogenesis, granulation tissue formation, collagen deposition, epithelialization, and wound retraction, and remodel new tissue [3].
Skin lesions are a serious public health problem on a global scale and "are a health quality indicator that generates substantial morbidity and considerable health care costs" [4]. An estimated 2% of hospitalized patients worldwide are affected by chronic skin ulcers [4]. In 2017/2018, the United Kingdom's National Health Service estimated that 3.8 million people were affected by some type of wounds, at a cost of ~ £8.3 billion [5]. In the United States, skin injuries affected the quality of life of 2.5% of the population and affected ~ 8.2 million healthcare beneficiaries in 2014, with estimated expenditures between USD$28.1 and USD$96.8 billion for all types of wounds [6].
Sparse data on the occurrence and expenditures of skin wounds have been reported in Brazil. Brito, de Vasconcelos Generoso, and Correia [7] assessed the prevalence 1 3 and characteristics of pressure ulcers in hospitals in different regions of Brazil from March 2009 to February 2011. These authors found that 16.9% of patients had pressure ulcers. Becker et al. [8] evaluated the incidence and risk factors of pressure ulcers in adult patients who were admitted to intensive care units and reported an incidence of 13.6% of patients with pressure ulcers.
Self-regeneration of the skin is generally a considerably fast and efficient process. However, extensive injuries, deep burns, and such diseases as diabetes and peripheral neuropathy can compromise the normal process of skin healing and regeneration, often resulting in chronic ulcers [9]. Classic procedures that are routinely used therapeutically for skin ulcers have employed such materials as gauze and cotton. However, these dressings have low permeability and allow the leakage of exudates, which can facilitate bacterial infection and cause bleeding and damage to newly formed tissue when these dressings are removed [10].
Researchers have sought to develop new therapeutic alternatives for cutaneous ulcers, such as dressings that interact with the wound through the release of bioactive molecules and the development of materials that are able to maintain appropriate conditions for optimal skin regeneration [2,[11][12][13][14][15].
Besides that, therapeutic alternatives that are based on natural components, such as Aloe vera (vegetable based), silver (mineral based), and honey and propolis (animal based), have been proposed, based on practices and knowledge from different countries and cultures. Egyptians and Persians, for example, have used propolis and honey to treat wounds since the beginning of human civilization [16]. In recent decades, researchers have revealed more information about the mechanisms of action and biological activities of these compounds by characterizing their active constituents and evaluating their anti-inflammatory, antibiotic, antifungal, and antioxidant effects [9,[17][18][19].
Propolis is an animal product that is produced by bees. Together with honey, propolis is one of the best known antibacterial and anti-inflammatory agents that has been used by humans since ancient times [20]. In recent years, experimental research has shown that honey and propolis can accelerate and improve the percentage of wound repair than classically used procedures [21][22][23][24][25][26].
Propolis is a strongly adhesive natural resin that consists of plant exudates, wax, and salivary secretions from bees. It is known as a bee hive protector. Propolis is largely composed of flavonoids, phenolic acids, fatty acids, vitamins, ketones, and sugars, whose composition varies according to the locality where it is produced [27,28]. In Brazilian territory, 13 different types of propolis have been identified based on the locality and plant species by which it is produced, including brown, green, and red propolis [29].
In addition to having documented anti-inflammatory and antibiotic effects [30,31], propolis has been shown to have antitumor [32,33] and antioxidant [20,31] effects. With regard to skin wound regeneration, propolis can lead to an increase in reepithelialization, fibroblast growth factor-2 expression, and collagen deposition and stimulate the cellular proliferation of keratinocytes and fibroblasts, thereby contributing to faster and more effective skin regeneration [34][35][36].
Considering the epidemiological impact of skin lesions and potential of propolis as a new therapeutic alternative for skin ulcers, we conducted a systematic review and metaanalysis of the administration of propolis for the regeneration of skin ulcers. We evaluated its efficacy in the treatment of these wounds.

Data search
The present systematic review and meta-analysis followed the criteria of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [37]. Data were obtained from the SCOPUS, Web of Science, and PubMed databases by searching the terms "propolis" AND "wound healing" in the title, abstract, and keywords or as "Topics" in Web of Science for all years. The databases were last consulted on August 24, 2021. In addition to the articles surveyed, the reference lists in these articles were also searched. We sought to identify the largest possible number of studies that are related to the theme of this systematic review.

Inclusion criteria
We included studies that (i) evaluated the application of propolis as a therapeutic agent for the repair of skin ulcers and (ii) were available as full text in English or Portuguese.

Exclusion criteria
We excluded studies that (i) evaluated only one case report, (ii) did not address in vivo testing of wounds in animals or humans, (iii) did not assess wound healing percentage/ rate or wound area/size, (iv) did not report their results as means and standard deviations (which makes it impossible to perform a meta-analysis), and (v) after performing the analyses had results that differed considerably from the rest (i.e., outliers), possibly leading to statistically incoherent final results in this meta-analysis.

Data extraction
Information on wound area and the percentage of healing was extracted from the articles. Results that were presented by the articles as units of measurement area (cm 2 or mm 2 ) or length/diameter (cm or mm) were transformed into percentages of remaining area, according Eq. (1). Numerical data that were not presented in the text of the studies or in tables and that were presented only in graphs were extracted as proportions according to Eq. (2).
where Lx is the measurement of the result in the graph, Ly is the measurement of the y-axis for a given value, y is the determined value of y, and x is the result of the experiment, leading to an approximate result.

Meta-analysis
To perform the meta-analysis, a period of approximately 2 weeks of treatment was standardized for all studies to avoid potentially biased results. The parameters that were used to perform the meta-analysis were the percentage of healing and percentage of remaining area by comparing the most complete propolis-containing treatment with the positive control or reference treatment. A third analysis was also conducted to evaluate the effect of propolis that was combined with biomaterials or other compounds or biomolecules (1) Percentage of remaining area = (Final area)∕(Initial area) × 100 .y compared with the effect of propolis alone on the percentage of healing. The meta-analysis was performed using a random effect model for all parameters because of the high heterogeneity of the data (I 2 = 89.5 for percentage of wound healing, I 2 = 88.75 for percentage of remaining area, and I 2 = 93.15 for propolis combination treatments, p < 0.0001). The metafor package [38] of R software was used, using as inputs the sample sizes of the control and treatment groups and the means and standard deviations for each of the groups.

Results
A total of 633 articles were found in all databases, of which 43 were included in the present systematic review and metaanalysis after excluding duplicates (n = 260) and selecting studies according to the inclusion and exclusion criteria ( Fig. 1).
The first published study among the articles included was from 2008 [39] and was the only one from this year ( Table 1 More than two-thirds of the articles were published in the last 7 years ( Fig. 2A). For the distribution of articles according to the country of publication, Brazil stood out with 33% of the published articles, followed by Iran (18%), Egypt (16%), and Iraq (9%). Other countries like India, Poland, Australia, the United States, and Nigeria were   responsible for the publication of one or two articles among those that were analyzed herein, comprising 24% as "Others" in Fig. 2B.

Effect of propolis administration on cutaneous wound healing
The effect of propolis administration on skin ulcers was evaluated as the percentage of wound healing (wound closure) and percentage of remaining wound area. The effect size of propolis on the percentage of remaining area was significant (random effect model = −2.9474, 95% confidence interval [CI]: −3.6970 to −2.1978, p < 0.0001; Fig. 3).
Similarly, for the effect of propolis on the percentage of wound healing, the effect size was also significant (random effect model = 2.0995, 95% CI: 1.0972-3.1017, p < 0.0001; Fig. 4).
The effect size of propolis combined with biomaterials, substances, or other treatments on the percentage of wound healing was also evaluated using a random effect model (I 2 = 93.15, p < 0.0001), which was also significant (random effect model = 5.1886, 95% CI: 2.8759-7.5013, p < 0.0001; Fig. 5).

Bias analysis
Publication bias was assessed using a funnel plot of the relation between standard deviation and the difference of standardized means and using Egger's regression test. The funnel plot distribution was asymmetric for percent area remaining (Egger's regression: z = −9.0549, p < 0.0001), percent healing (Egger's regression: z = 4.2981, p < 0.0001), and wound healing of propolis combinations (Egger's regression: z = 7.2747, p < 0.0001; Fig. 6A-C).  The results of this meta-analysis showed that propolis was significantly more effective than controls in reducing the wound area and consequently achieving a higher percentage of regeneration. These findings justify research interest and the increase in the number of publications on applications of propolis for the treatment of cutaneous ulcers. According to the data that were collected in the present systematic review, Brazil, Iran, Egypt, and Iraq stand out as leaders in research on the subject. There may be a direct relationship between this finding and the fact that propolis preparations are popularly known in these countries, especially green and red propolis in Brazil and Iranian propolis.
Al-Waili [48] compared the efficacy of two samples of propolis that were collected in different regions of Iraq at different times of the year. Consequently, these samples were produced from different flora because vegetation in this geographic area varies according to climate and time of the year. The results showed higher efficacy of one of the samples. When these two types of propolis were mixed, a higher rate of wound regeneration was observed compared with either of the two samples alone, thus revealing their synergistic action.
Propolis composition includes several types of molecules with potential regenerative and differentiation effects, such as flavonoids, phenolic acids, fatty acids, vitamins, ketones, and sugars. Being produced from plant sap, the composition of propolis varies according to several aspects, such as, the botanical composition, geographical origin, time of collection and environmental conditions [49]. This variation in composition, and therefore in the presence and concentration of secondary metabolites, may result in different results for treatments developed with different types of propolis. Besides, the mechanisms of action in the regenerative process promoted by propolis are not well defined. This can be a very complex issue with natural products, whose activity is not due to a single chemical entity, but to a mixture of constituents, some of which are not yet identified. Some natural products contain many active compounds that act through different mechanisms and unique interactions collectively have very high efficacy [50].
Propolis appears to be effective in regenerating skin ulcers overall, however, this efficacy does not appear to be related solely to only one type of propolis that is tested. The analysis of results from studies that used propolis of different origins suggested that other aspects influence efficacy with regard to regeneration more than the type of propolis per se. Studies that used Iranian [28,[51][52][53][54], Egyptian [46], Brazilian red [43], or Iraqi [48] propolis all reported similar results, achieving more than 90% regeneration of wounds after 14 days of treatment.
Interestingly, studies of propolis of the same origin [46,55] reported quite different results. Sarhan and Azzazy [46] achieved 100% wound regeneration on day 12 of treatment, whereas Ahmed et al. [55] reported regeneration of only 30% after 14 days of treatment. The reason for this discrepancy may have more than one explanation.
One aspect to be considered is individual variations of wounds in each experimental design. Sarhan and Azzazy [46] evaluated the effect of Egyptian propolis on an acute "punch" wound, whereas Ahmed et al. [55] applied propolis to treat burns in animals with induced diabetes, a pathological condition that makes the skin regeneration process more complex. Additionally, Sarhan and Azzazy [46] applied propolis combined with nanofibers that were made of honey, polyvinyl acid, and chitosan, whereas Ahmed et al. [55] applied propolis combined with a commercial ointment with healing properties (1% silver sulfadiazine, Dermazine®). Bayrami et al. [56] tested Iranian propolis combined with zinc oxide (ZnO), copper oxide (CuO), and silver (Ag) in several different combinations. They found both positive and negative outcomes with these combinations. Thus, although the same type of propolis was used in these studies, the propolis/ZnO and propolis/Ag/ZnO combinations were more effective in promoting wound regeneration than the other combinations.
The great majority of the studies that were evaluated in this systematic review applied the therapeutic compounds topically or in the form of dressings [47,[57][58][59]. Only Corrêa et al. [30] administered an ethanolic extract of red propolis orally to animals. Until 2014, most of the published studies administered propolis in the form of an alcoholic or aqueous extract [39,60] or as a cream or powder [34,61,62]. Exceptions were Barud et al. [41], who evaluated the healing potential of a biocellulose membrane that was associated with propolis, and Sulaiman et al. [63], who combined an ethanolic extract of propolis with honey.
The results of this meta-analysis show that these combinations are successful treatments, in which the administration of propolis together with another compound or material was significantly more effective in the healing process than propolis alone. These other compounds or material may generally have properties that are favorable to the regeneration of epithelial tissue, resulting in possible additive or synergistic effects. Takzaree et al. [23] showed that the administration of a combination of honey and propolis was more effective than either of them applied alone.
These therapeutic components were evaluated in different types of wounds, including acute wounds [65][66][67][68] burns [40,45], and chronic wounds [69]. Eyarefe [25] administered an ethanolic extract of propolis on acute wounds that were infected with bacteria. Four studies evaluated wound regeneration in animals with induced diabetes [24,56,70,71]. An acute wound in a healthy animal will likely undergo a much more efficient and faster healing process than a wound in a diabetic animal or a lesion that is exposed to microorganisms, such as bacteria. Therefore, comparisons of the results of these studies that were obtained within the same treatment time without considering these other circumstances may not reflect clinical reality and thus may represent an analytical bias of the results.
However, these circumstances emphasize the treatment effect, as was presented by Voss et al. [42]. In this study, the effect of cellulosic films that were combined with vitamin C and propolis was compared between diabetic and nondiabetic rats. No significant difference in effects on ulcer regeneration was found between the treatment and control. However, when the process of wound repair was evaluated in diabetic animals, the results showed that the treatment had a significantly greater effect than the control, reaching a regeneration rate that was very close to wounds in nondiabetic animals. A similar result was reported by Mclennan et al. [39].
The procedures currently used to treat skin ulcers use dressings that have low permeability and allow the leakage of exudates, facilitating bacterial infection, besides causing damage to the newly formed tissue when removed. This process can hinder skin regeneration, especially when the wound is difficult to heal, such as infected wounds or chronic wounds. In contrast, the antibiotic effect of propolis enables the elimination and prevention of bacteria in the wound. In addition, its anti-inflammatory and antioxidant effect may be responsible for stimulating the regeneration process of chronic ulcers, such as diabetic ulcers. Thus, propolis could promote a faster and more effective regenerative process, even for wounds that are difficult to heal.
Another notable aspect that varied among studies was the different species that were used to evaluate ulcer repair. Most of the articles evaluated the efficacy of treatments in rats or mice, but some studies used dogs [72][73][74], rabbits [48,75], horses [76], and sheep [77]. We also identified three clinical studies, in which the therapeutic compound was applied to patients with chronic wounds [60,62,69].
This variety of species may partially explain the heterogeneity of results of the present meta-analysis. Nonetheless, the findings strongly suggest the high potential of propolis for veterinary use to resolve wounds that are otherwise difficult to manage clinically, especially in species that are more susceptible to injuries, such as horses and sheep, because of their specific physiological and behavioral characteristics [78].
In summary, the present systematic review and metaanalysis found strong evidence that propolis has therapeutic promise for the treatment of skin ulcers, including difficultto-heal ulcers that are found in patients with diabetes and in bedridden patients with pressure ulcers. The findings justify the growing interest in propolis in regenerative medicine, especially over the last few years. New studies are seeking to optimize the therapeutic effects of propolis by combining it with other substances and biomaterials to achieve additive or synergistic effects to improve the regeneration process of cutaneous ulcers of different etiologies.

Limitations
Although the present systematic review attempted to include all published articles that meet the inclusion criteria, the bias analysis indicated that there may be publication bias for all parameters evaluated. Another limitation was the presence of different controls in each study. We initially sought to compare propolis with a positive control or reference treatment. However, some studies used only negative controls (e.g., gauze or saline solution application) and compared 1 3 propolis to no intervention. This difference in controls may have considerably affected the final results of the metaanalysis because propolis likely has much more pronounced effects statistically compared with a negative control relative to a positive control or reference treatment. The efficacy of propolis that is equal to, or slightly better than, a reference treatment would be a much more clinically relevant finding.
Another limitation was the analysis of only one aspect of wounds (i.e., wound area), which may limit a more thorough understanding of the effects of propolis on all aspects of tissue regeneration. Future studies should include data on histological analyses of tissues, in addition to evaluating the presence or absence of indicators of the healing process, such as reepithelialization or granulation. Besides that, it would be of great importance to produce studies that try to determine more precisely which are the mechanisms of action involved in the efficacy of propolis in regenerating skin ulcers.