Peptides from frog skin for wound repair: a systematic review

Abstract

Skin wounds are common injuries, and many treatments have been proposed, especially membranes. Some studies have been exploring the potential of the skin of Amphibians as a source of peptides for wound treatment, which has been an extremely promising source of bioactives to be used, although there is a lack of effects on pre-clinical animal models, which makes it difficult to understand the results and the choice of the best protocol to use. In this context, the purpose of this study was to perform a systematic review of the literature to examine the effects of membranes manufactured with peptides from frogs on the process of healing skin wounds using in vivo experimental models. This systematic review was carried out based in the orientations of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) utilizing PubMed, Scopus and Web of Science databases. This review confirms the significant benefits of using these peptides for injury repair and regeneration. Moreover, a faster healing and a full wound closure, with well-organized dermal and epidermal layers were found in the wounds treated peptides from frog skin.

Key words
Membranes; Tissue Engineering; Skin wound; systematic review

INTRODUCTION

Skin wounds are very common injuries, caused by many different reasons, including physicochemical or thermal damage and metabolic diseases such as diabetes resulting in functional imbalance, increased levels of pain, disability, or even death (Borena et al. 2015). It is estimated that approximately 5.7 million people in the US are affected by wound lesions, with the cost exceeding 50 billion dollars annually (Childs & Murtihy 2017).

Skin wounds can be divided into acute and chronic wounds. Acute wounds, in general, heal by themselves, in a smooth and efficient way (Childs & Murtihy 2017). Acute wounds normally heal in an orderly and efficient manner. They progress smoothly through the four distinct, but overlapping phases of wound healing: hemostasis, inflammation, proliferation, and remodeling. Conversely, in the presence of some specific situations such as bad nutrition, associated diseases, age, or extensive wound extension, the process of skin regeneration may be impaired, resulting in chronic or non-healed wounds (Diegelmann & Evans 2004, Monika et al. 2022). Many treatments for chronic skin injuries have been proposed such as surgical interventions, pharmacological treatments, growth factors, stem cells, and wound dressing or membranes (Tottoli et al. 2020).

Membranes are one of the most efficient treatments for skin injuries, serving as protection from infection, reducing pain levels, and stimulating and supporting cell migration and proliferation (Chilakamarthi et al. 2014, Rahman et al. 2019). It is important to emphasize that many different biomaterials serve as raw materials to be used for membrane manufacturing such as auto-grafts, synthetic materials (artificial polymers), and natural materials like chitosan and collagen (Col) (Chilakamarthi et al. 2014, Chen & Liu 2016, Silva et al. 2014). Col is the most abundant protein in vertebrates and it is involved in the structural integrity of many tissues (Shoulders & Raines 2009, Papaiordanou 2022). Based on their biocompatibility, bioactive, low immunogenicity, and mechanical properties, Col-derived grafts or membranes are extremely suitable for tissue engineering and regenerative medicine (TERM) strategies in human health issues, including skin wounds (Lui et al. 2023, Bonferoni et al. 2021, Camponogara et al. 2022, Riha et al. 2021). Col membranes also possess the ability to support cell growth, are easy to manipulate, and have adequate porosity and absorption rate (Takayama et al. 2023). Moreover, many different sources of Col for membrane manufacturing have been explored, including bovine and porcine bone and skin, marine species such as fish skin, marine sponges, and jellyfish (Furtado et al. 2022, Jafari et al. 2020, Liu et al. 2022). For example, Kaasi et al. (2018) observed a stimulatory effect on skin tissue healing in dogs and cats after the treatment with Col membranes, highlighting their safety and efficacy.

More recently, some studies have been exploring the potential of the skin of Amphibians as a source of Col for membrane manufacturing, which has been an extremely promising source of bioactive to be used for the treatment of human health problems (Indriani et al. 2023, Qin et al. 2018). It is known that the skin of amphibians is a favorable organic system for the development of biotechnological products. Amphibians of the order Anura, such as toads, frogs, and tree frogs have components in their skin that have attracted the attention of the community for decades scientific, mainly because they exhibit pharmacological properties (Childs & Murtihy 2017, Azevedo et al. 2007, de Brito-Gitirana & Azevedo 2005, Pelli et al. 2010). The interest in these animals of the Amphibia class lies primarily in the fact that components of the extracellular matrix of the dermis, such as collagen and glycosaminoglycans, as well as peptides that have properties like those derived from the skin of vertebrate (de Brito-Gitirana & Azevedo 2005, Pelli et al. 2010).

In that regard, some studies have been proving the promising potential of using frog skin for use with the purpose of tissue engineering (Indriani et al. 2023). The literature shows that frog skin has antimicrobial (Rollins-Smith 2023, Chen et al. 2022), antioxidant and anti-inflammatory properties (Cao et al. 2018), essential factors for successful skin repair. Studies, in vitro, demonstrated a positive effect on the proliferation and differentiation of epidermal cells and dermal cells cultivated in the presence of frog skin (Raghavan et al. 2010). Rezazade et al. (2015) used the skin of frogs of the species Rana ridibunda for use in dressings. The authors observed that they presented properties regenerative and antimicrobial properties, indicated through in vivo studies in an experimental of cutaneous wounds and in vitro in Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Another biologically important feature for the use of frog skin as a dressing biological function, more specifically of the bullfrog, is its selective permeability that would allow the concomitant administration of drugs and/or the use of other therapeutic resources to aid in wound repair without the need for frequent dressing changes (Willens et al. 2006).

Although all the evidence about the positive effects of Col from frogs is used as a raw material for manufacturing membranes, there is a lack of studies investigating its effects on pre-clinical animal models, which makes it difficult to understand the results and the choice of the best protocol to use. In this context, the purpose of this study was to perform a systematic review of the literature to examine the effects of membranes manufactured from different Col from frogs on the process of healing skin wounds using in vivo experimental models. Consequently, this work discusses the results with the intention of promoting a better understanding of the effects of these membranes in this type of injury.

MATERIALS AND METHODS

Review protocol

This systematic review was carried out based in the orientations of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guideline as outlined by de Vries et al. (2015). The search was performed between July and August of 2023, utilizing PubMed, Scopus and Web of Science databases. To initiate the review process, specific Medical Subject Headings (MeSH) terms were defined, including “frog”, “wound skin” and “in vivo”.

Study selection

Two independent reviewers (AS and IRA) conducted a comprehensive analysis of the titles and abstracts of the chosen literature, identifying prospective studies based on predefined inclusion and exclusion criteria. Additionally, these reviewers had access to the selected studies to confirm their suitability. Any differences in opinions were resolved through discussion. Subsequently, a thorough examination was undertaken during the full text screening phase, resulting in the exclusion of studies that did not align with the established eligibility criteria.

Eligibility criteria

Inclusion criteria

• 1) Animal experiments.

• 2) Animal studies employing the following experimental model: skin wounds treated with peptides and frog skin.

• 3) Manuscripts should be written in English and published in the last 10 years.

Exclusion criteria

• 1) Studies of characterization of the membrane, in vitro studies, in situ studies, reviews, case reports;

• 2) Studies without skin wounds, burns or injuries;

• 3) Lack of description of the skin wounds, burns or injuries, methodology or outcomes;

• 4) Animal models or clinical trials with other systemic diseases (such as diabetic rats).

Data extraction

The analyzed variable was based on the macroscopic analysis. In addition, the remaining variables were also extracted: studies, as author, species/strain, animal sex, age, weight, marine species and biomaterial, skin wound type, skin wound size, implantation period (days), way of treatment, peptide purification and synthesis, analysis performed in the in vivo studies and outcomes measures.

Types of Reported Results

The authors (JRP) utilized the GRADE (Grading of Recommendations, Assessment, Development, and Evaluations) tool, accessed at https://gdt.gradepro.org/app/, to evaluate the evidence quality of the selected studies in the review. This tool assesses the evidence quality across a set of studies rather than an individual study. The levels of evidence quality are categorized as follows: high (sufficient evidence in the effect estimate), moderate (true effect closely aligns with the estimate), low (limited confidence in the effect), and very low (minimal confidence in the effect estimate) (Balshem et al. 2011).

RESULTS

Description of the included studies

The search method applied in the current investigation is illustrated by the flow diagram (Figure 1). A total of 39 papers were obtained from PubMed, Web of Science, and Scopus databases. Then, the duplicated records were excluded, and 23 abstracts were assessed for eligibility. After the second qualitative analysis, 13 full-text articles were assessed for eligibility and 10 papers were excluded. Finally, 6 papers were included and examined in this systematic review after 7 research were excluded based on the inclusion criteria.

Figure 1
PRISMA flow diagram of search strategy used in the present study.

Table I shows the general characteristics of all selected studies. From these studies, three works used the specie Odorrana frogs and their respective peptides extracted from their skins: OA-FF10 (Liu et al. 2019), OM-LV20 (Li et al. 2018) and AH90 (Liu et al. 2014). Two works used the specie Rana frog and extracted the peptides RCSPs (Ji et al. 2020) and RP-1 (Zhang et al. 2013), and only Wu et al. (2018) used the specie Nanorana to extract cathelicidin-NV peptides.

Table I
Characteristics of the studies.

Authors	Frog species/peptides	Species/ Strain	Animal sex / age/ weight	Wound type and size	Method of treatment	Implantation period
Ji et al. (2020)	Rana chensinensis / RCSPs	Wistar rats	Male/ N.D. /200-250 g	Full-thickness wound on the back Ø = 1 cm	Hydrogel loaded with frog egg-like microspheres	4, 8 and 12 days
Liu et al. (2019)	Odorrana andersonii / OA-FF10	Kunming mice	Male / N.D. / 20-25 g	Two full-thickness wounds on the back size = 8 mm x 8 mm	Group 1: The right-sided wounds were treated with 20 µl (100 µM) of OA-FF10 and for left-sided were used 20 µl of Kangfuxin. Group 2: The right-sided wounds were treated with 20 µl (10 µM) of OA-FF10 and for left-sided were used 20 µl of saline solution. Group 3: The right-sided wounds were treated with 20 µl (1 µM) of OA-FF10 and for left-sided were used 20 µl of saline solution.	2, 4, 6 and 8 days
Wu et al. (2018)	Nanorana entripunctata / cathelicidin-NV	Kunming mice	Male / 6-7 weeks / N.D.	Two full-thickness wounds on the back Ø = 6 mm	Cathelicidin-NV (20 μl, 200 mg/ml) or EGF (20 μl, 100 mg/ml) applied directly to the wound area twice daily.	2, 4, 6, 8 and 10 days
Li et al. (2018)	Odorrana margaretae / OM-LV20	N.D. mice	Male / Adult / 22-25 g	Two full-thickness wounds on the back Size = 8 mm x 8 mm	Left-sided wound treated with 20 µl of OM-LV20 and right-sided wound treated with 20 µl of saline solution or EGF.	4, 7 and 10 days
Liu et al. (2014)	Odorrana grahami / AH90	Balb/c mice	Male / N.D. / N.D.	A single wound on the back skin Ø = 7 mm	AH90 or sAH90 dissolved in DPBS was applied to the wound 2 times per day.	2, 4, 6, 8 and 10 days
Zhang et al. (2013)	Rana pleurade / RP-1	Sprague-Dawley	Male / 4 weeks / N.D.	Irradiation buttock skin Size = 3 cm x 4 cm	50 µg de RP-1 or TAT-RP1 was applied two times per week.	48 days

• Abbreviations: AH90: a potential wound healing-promoting peptide; sAH90: a scrambled version of AH90; EGF: epidermal growth factor; DPBS: dulbecco’s phosphatebuffered saline; N.D: not described; RCSPs: Rana chensinensis skin peptides; RP-1: small peptide; TAT-RP1: OM-LV20: Novel peptide; cathelicidin-NV: a novel 24-residue peptide; OA-FF10: a Novel Amphibian Peptide; PVA: polyvinyl alcohol.

Furthermore, two works used rats Sprague-Dawley (Zhang et al. 2013) and Wistar (Liu et al. 2019). On the other hand, four works used mice, including Balb/c mice (Liu et al. 2014) and Kunming mice (Liu et al. 2019, Wu et al. 2018) and one work has not described which species was used (Liu et al. 2014). Also, all studies used male animals (Ji et al. 2020, Liu et al. 2014, 2019, Wu et al. 2018, Li et al. 2018, Zhang et al. 2013). Furthermore, one study informed the age of the mice ranged from 6 to 7 weeks (Wu et al. 2018), and another one informed that the mice used were “adult” (Li et al. 2018), but two other works have not described the age of the mice (Liu et al. 2019). About the age of the rats, one study used 4 weeks old animals (Zhang et al. 2013), and another one has not described the age of the rats used (Ji et al. 2020). In addition, the weight of the mice used ranged from 20 to 25 g (Liu et al. 2019, Li et al. 2018), and the weight of the rats ranged from 200 to 250 g (Ji et al. 2020). Three works have not described this variable (Wu et al. 2018, Liu et al. 2014, Zhang et al. 2013).

The size of the wounds in rats ranged from 78,5 mm2 to 1,200 mm2 (Ji et al. 2020, Zhang et al. 2013) and in mice ranged from 28.26 mm2 to 64 mm2 (Liu et al. 2014, 2019, Wu et al. 2018, Li et al. 2018, Zhang et al. 2013). Five works used the experimental model back wound (Ji et al. 2020, Liu et al. 2014, 2019, Li et al. 2018, Wu et al. 2018), and 3 of them performed 2 wounds in the same animal (Liu et al. 2019, Li et al. 2018, Wu et al. 2018). Only one study used the irradiation buttock skin model to create a wound (Zhang et al. 2013).

Different treatments were used by the different authors. Liu et al. (2014) used AH90 or sAH90 dissolved in DPBS on the wound. Zhang et al. (2013) treated the wounds with 50 µg of RP-1 or TAT-RP1 twice per week. Li et al. (2018) compared the effects of 2 treatments: 20 µl of OM-LV20 on the wound of the on the left-sided and 20 µl of saline solution or EGF on the wound of the right. Wu et al. (2018) used Cathelicidin-NV or EGF twice daily. Liu et al. (2019) used on the right-sided wound 20 µl of OA-FF10 with different concentrations and for the left-sided wound were used 20 µl of Kangfuxin or saline solution. Ji et al. (2020) used on the wound a hydrogel loaded with frog egg-like microspheres. The implantation period ranged from 2 to 48 days (Ji et al. 2020, Liu et al. 2014, 2019, Li et al. 2018, Wu et al. 2018, Zhang et al 2013).

Table II describes the synthesis and purification of the peptides, the methodologies for characterization, as well as the physical and morphological characteristics of the studies. One author synthesized the peptides commercially (Zhang et al. 2013), one author synthesized using solid phase (Liu et al. 2019), and four authors described their synthesis and purification processes (Ji et al. 2020, Wu et al. 2018, Liu et al. 2014, 2018). With this, Ji et al. (2020) dissolved the SA in deionized water and left it to stir overnight. Afterwards, the anhydrous CaCl₂ was dissolved in deionized water, then the RCSPs were dissolved in deionized water, followed by the addition of 10% glycerin. Wu et al. (2018) purified the secretions in a 0.1 M phosphate buffer solution with 1% protease inhibitors, then the collection was centrifuged and the supernatants were lyophilized. Li et al. (2018), the frogs were stimulated using an electronic massager in a 0.01% NaCl solution, then the secretions were collected and washed in 25 mm Tris-HCl. They were then centrifuged and freeze-dried. After the desalting step by reverse-phase HPLC, the fractions were pooled and purified on a C18 RP-HPLC column. Finally, according to Liu et al. (2014), the secretions were stimulated in an electronic massager and then collected using Tris-HCl buffer, after which the solution was centrifuged and the supernatant freeze-dried. The fractions were submitted to a C18 RP-HPCL column pre-equilibrated with 0.1% TFA.

Furthermore, the purification processes were described by three authors, who used HPLC (Li et al. 2018, Liu et al. 2014, Zhang et al. 2013). Among the authors, one author carried out the characterizations using SEM, FTIR, EE, mechanical properties, water absorption and kinetic release of peptides (Ji et al. 2020), two authors performed the MALDI-TOF analysis 3 performed the cDNA and 3 authors determined the primary structure of the peptide (Liu et al. 2014, 2019, Wu et al. 2018, Li et al. 2018).

In the morphological and physical characterizations, only four authors described their findings (Ji et al. 2020, Liu et al. 2019, Li et al. 2018, Wu et al. 2018), while two did not present their results (Liu et al. 2014, Zhang et al. 2013). Ji et al. (2020) carried out SEM analyses on the H-FMS and H-MS samples and found the presence of evenly distributed micropores when compared to the H0 group. In the FTIR analysis, characteristic peaks of SA, RCSPs, PVA and gelatine were observed. They also described how increasing the flow of the external and internal axes improved the EE of the RCSPs in the FMS. They also compared the mechanical properties of the H0, H-MS and H-FMS groups, which showed values of 290.83 ± 3.39, 242.95 ± 3.26 and 266.72 ± 3.64 KPa, respectively. Finally, in the water absorption test, the H-TMS group absorbed 1.105%, HMS 1.156% and H0 absorbed 937%. In contrast, Liu et al. (2019) revealed that OA-FF10 exhibited a molecular mass of 1105.25 Da along with a purity exceeding 95%. The pre-peptide sequence of OA-FF10 revealed an intramolecular heptapeptide arrangement featuring a disulfide bridge located at the C-terminus. This structural confirmation was further supported by the results, which demonstrated the presence of the disulfide bridge in both the annular and native forms of OA-FF10, established through the process of coelution. However, in Liu et al. (2019), it was revealed that OA-FF10 exhibited a purity exceeding 95%, boasting a molecular mass of 1105.25 Da. This peptide displayed resemblances to peptides found in the dermal secretions of amphibians, showcasing an intramolecular heptapeptide sequence. Moreover, a disulfide bridge located at the C-terminus was a notable structural feature, accompanied by paired cysteine residues. The investigation conclusively verified the presence of the disulfide bridge in both the annular and native conformations. In contrast, Wu et al. (2018) through their analysis, identified an amino acid sequence of ARGKKECKDDRCRLLMKRGSFSYV following purification. This sequence, comprising 24 amino acid residues, exhibited a potential intramolecular disulfide bridge. The peptide’s molecular mass was determined as 2845.9 Da, and its purity surpassed 98%. Additionally, the cathelicidin-NV precursor encompassed a total of 146 amino acid residues. This consisted of a designed 20-amino acid signaling peptide, a conserved domain spanning 102 amino acids, and a mature 24-amino acid peptide segment. Conversely, Li et al. (2018) revealed an amino acid sequence that spans from the N-terminal to the C-terminal as LVGKLLKGAVGDVCGLLPIC. This peptide has a purity exceeding 95% and a molecular mass of 1966.39 Da.

Table III presents the in vivo analysis and overall findings. Out of all the studies, all of them conducted a macroscopic evaluation of the wound (Ji et al. 2020, Liu et al. 2014, 2019, Li et al. 2018, Wu et al. 2018, Zhang et al. 2013). Moreover, 5 studies assessed the rate of wound closure or skin injury score (Ji et al. 2020, Liu et al. 2014, 2019, Wu et al. 2018, Li et al. 2018). Moreover, histological analysis was included in 3 works (Ji et al. 2020, Wu et al. 2018, Liu et al. 2014). Furthermore, only one study utilized immunohistochemistry (Wu et al. 2018).

Table III
In vivo analysis and overall results.

Author	In vivo analyses	Results	Outcomes
Ji et al. (2020)	Macroscopic analysis Wound closure rate Histological analysis	The wound closure rate of the H-FMS group ranged from 53.98% (4 days) to 97.58% (12 days) while the control, H0 and H-MS groups ranged from 40.28, 42.91, and 47.09% to 83.26, 89.71, and 92.51%, respectively. At 4 and 8 days, the H-MS and H-FMS groups showed decreased inflammatory process, with an increase in fibroblasts concentration reepithelialization at the site of the wound. On day 12, H-FMS demonstrated a well-developed structure of regenerated skin tissue compared to the other groups.	+
Liu et al. (2019)	Macroscopic analysis Wound closure rate	OA-FF10 (100 µM) treated animals presented a smaller wound area (¶5% of repair) compared to saline and KFX groups (・0% of repair). Also, the OA-FF10 has the highest number of closed wounds after 8 days post-surgery.	+
Wu et al. (2018)	Macroscopic analysis Wound closure rate Histological analysis Immunohistochemistry analysis Cytokine analysis	Cathelicidin-NV displayed a 22% lower wound area than control mice on the second day post-injury. By day 8, Cathelicidin-NV and EGF treatments led to wound size reductions of about 88% and 90% respectively, compared to controls. By day 10, Cathelicidin-NV and EGF-treated wounds were almost entirely closed, while control wounds remained about 6% open. After 4 days of Cathelicidin-NV treatment, re-epithelialization and increased epithelial cells were observed. Granulation tissue formation was slower in control wounds compared to the Cathelicidin-NV-treated groups at 4 and 10 days. Cathelicidin-NV displayed positive reactions to alpha-smooth muscle actin (α-SMA) and collagen, in contrast to the vehicle-treated group. Also, Cathelicidin-NV-treated wounds consistently showed higher expression levels of tested proteins (MCP-1, TNF-α, VEGF, and TGF-β1) compared to vehicle-treated wounds.	+
Li et al. (2018)	Macroscopic analysis Wound closure rate	The OM-LV20 group showed a lower wound area than CG on days 4, 7, and 10. Moreover, similarly healing process was observed between OM-LV20 and EGF groups. Moreover, the residual area of wound of all groups at the end of 10 experimental days were below 20 mm2.	+
Liu et al. (2014)	Macroscopic analysis Wound closure rate Histological analysis	The reduction in wound size compared to the initial area in the AH90 group varied from 72% after 2 days to 2% after 10 days compared to the initial area, while the control and EGF groups showed reductions ranging from 92% to 12% and 58% to 5%, respectively. The AH90 group exhibited results comparable to the CG. Starting from day 5, the AH90 group displayed noticeable improvements in re-epithelialization and the formation of granulation tissue. By day 9, the skin regeneration and remodeling in the AH90 group resembled that of normal mice.	+
Zhang et al. (2013)	Macroscopic analysis Skin injury score	The wound was significantly smaller in the TAT-RP1 group compared to the control group (PBS) from the 15th day after the application of radiation. The RP-1 group maintained tissue damage similar to the control group.	+

• Abbreviations: H-FMS: hydrogel containing frog egglike microspheres; H-MS: hydrogel containing solid microspheres; H0: hydrogel without microspheres; OA-FF10: a Novel Amphibian Peptide; KFX: Kangfuxin; cathelicidin-NV: a novel 24-residue peptide; EGF: epidermal growth factor; OM-LV20: Novel peptide; AH90: a potential wound healing-promoting peptide; TAT-RP1: HIV-TAT, Protein transduction domains-coupled RP-1 fusion protein.

Table II
Purification process of frog skin, peptide synthesis or manufacturing technique and physical and morphological characteristics.

Authors	Peptide purification and synthesis	Characterization methods	Physical and morphological characteristics
Ji et al. (2020)	SA was dissolved in deionized water (1.5% w/v) and stirred overnight at room temperature. Anhydrous CaCl2 was dissolved in deionized water (3% w/v). RCSPs were dissolved in deionized water (2% w/v), followed by the addition of 10% glycerine to protect the RCSPs during lyophilization.	SEM FTIR EE Mechanical properties Water absorption Release kinetics of RCSPs	In SEM, H-FMS and H-MS had an increased presence of micropores and uniform distribution within their microstructure when compared to H0. FTIR showed peaks at 1.460 cm−1 and 1.610 cm−1 characteristic of SA and peaks 3.410 cm−1, 1.670 cm−1, and 1.260 cm−1 characteristic of RCSP. The PVA characteristic bands were 3.407 cm−1, 1.936 cm−1 and 1.596 cm−1. The characteristic gelatin peaks corresponded to 3.413 cm−1, 1.652 cm−1, 1.554 cm−1, 3.505 cm−1, 1.681 cm−1, and 1.544 cm−1. Flow increasing rate of the inner and outer axes of EE of RCSPs in FMS was (46 ± 0.8%). compared to that of MS. The mechanical properties of H0 were 290.83 ± 3.39 KPa,H-MS and H-FMS groups were 242.95 ± 3.26 KPa and 266.72 ± 3.64 KPa. H-TMS and HMS absorbed 1.105 and 1.156% of water, respectively, while that of H0 was 937%.
Liu et al. (2019)	Solid-phase synthesis was employed for their creation using an Applied Biosystems model 433A.	MALDI-TOF MS analysis sequence specificity	Molecular mass 1105.25 Da and a purity of > 95% for OA-FF10. Pre-peptide OA-FF10 sequence and structure showed an intramolecular heptapeptide with a disulfide bridge at the C-terminus. The results confirm the presence of the disulfide bridge in the annular and native forms of OA-FF10 through coelution.
Wu et al. (2018)	The secretions were purified using 0.1 M phosphate buffer, pH 6.0, with a 1% protease inhibitor cocktail. After collection, the solutions were rapidly centrifuged (10,000 g for 10 minutes) and the supernatants were lyophilized.	Primary structural analysis cDNA cloning	The amino acid sequence of purified NV-catelicidin was ARGKKECKDDRCRLLMKRGSFSYV. Cathelicidin-NV comprises 24 amino acid residues: two cysteines, forming a possible intramolecular disulfide bridge. The molecular mass of cathelicidin-NV was s 2845.9 Da, with a purity level of over 98%. The precursor of cathelicidin-NV consists of a total of 146 amino acid residues, a designed signal peptide of 20 amino acids, a conserved cathelin domain spanning 102 amino acids and a mature peptide of 24 amino acids.
Li et al. (2018)	The frogs were submitted to a 6-second stimulation using an electronic massager (6 V) in a 0.01% NaCl solution. Skin secretions were collected by washing the frogs with 25 mm Tris-HCl buffer, followed by centrifugation (2,880 g for 15 minutes at 4°C). The supernatants were then lyophilized and stored at -80°C. After desalting by reverse-phase HPLC on a C4 column and lyophilization, the fractions were pooled and purified by a C18 RP-HPLC column, collecting peaks with wound healing activity.	Determination of peptide primary structure Cloning of cDNA	Molecular mass of OM-LV20 was 1966.39 Da with a purity of >95%. The amino acid sequence from the N-terminus to C-terminus was “LVGKLLKGAVGDVCGLLPIC”.
Liu et al. (2014)	Cutaneous secretions were acquired by washing the frogs with deionized water, stimulating them in 0.01% NaCl deionized water with an electronic massager (6 V, 6 s) and collecting secretions using 25 mM Tris-HCl buffer. The collected solutions were centrifuged (4000 rpm, 15 min, 4°C), producing supernatants that were lyophilized and stored at -80°C. The fractions underwent C18 RP-HPLC on a column pre-equilibrated with 0.1% (v/v) trifluoroacetic acid (TFA) in water. Elution utilized a linear 0.1% (v/v) TFA in acetonitrile gradient, 0.7 ml/min, monitored at 215 nm. Peaks were collected and lyophilized, followed by a second HPLC purification under the same conditions.	Primary structure	N.D.
Zhang et al. (2013)	Shanghai Mocellbio Scientific Inc. (Shanghai, China). Purified by HPLC.	MALDI-TOF mass spectrometry	N.D.

• Abbreviations: ACN: acetonitrile; AH90: a potential wound healing-promoting peptide; sAH90: a scrambled version of AH90; DTT: dithiothreitol; FMS: frog egglike microspheres; H-FMS: hydrogel containing frog egglike microspheres; H-MS: hydrogel containing solid microspheres; H0: hydrogel without microspher_es; dH2O: deionized water; MS: solid microspheres; N.D: not described; OMM: Observed Molecular Mass; OM-LV20: Novel peptide; cathelicidin-NV: a novel 24-residue peptide; OA-FF10: a Novel Amphibian Peptide; RCSPs: Rana chensinensis skin peptides solution; SA: sodium alginate; TFA: trifluoroacetic acid; TMM: Theoretical Molecular Mass.

Ji et al. (2020) and Liu et al. (2019) demonstrated the highest number of closed wounds after 12- and 8-days post-treatment with H-FMS and OA-FF10, respectively. Wu et al. (2018), Liu et al. (2014) and Li et al. (2018) presented wound healing after 10 days after treatment with Cathelicidin-NV, OM-LV20, and AH90, respectively. However, Zhang et al. (2013) achieved results only on the 15th-day post-treatment.

The authors reported an initial wound recovery rate ranging from approximately 30% to 98% within a timeframe of 2 to 15 days, based on the wound closure or contraction rate. After 12, 10, and 8 days Ji et al. (2020), Liu et al. (2014, 2019), Wu et al. (2018) and Li et al. (2018) obtained a rate close to 100% for H-FMS, OA-FF10, Cathelicidin-NV, OM-LV20, AH90, respectively, while Zhang et al. (2013) apply a score from 1 to 5 to quantify the wound damage and this score was close to 2 for TAT-RP1.

In the study performed by Ji et al. (2020), between day 4 and day 8, a noticeable reduction in the count of inflammatory cells and an increase in both fibroblast numbers and re-epithelialization tissue were observed in the H-MS and H-FMS groups. Similarly, in the study carried out by Wu et al. (2018), significant re-epithelialization was evident in the Cathelicidin-NV group on day 4, along with heightened cell migration, while the control group exhibited comparatively lower granulation tissue formation.

In contrast, findings from Liu et al. (2014) indicated that the AH90 group displayed enhanced re-epithelialization and more substantial granulation tissue formation starting from day 5, with regeneration comparable to the control observed by day 9.

In their immunohistochemical assessment, Wu et al. (2018) revealed that from day 4 to day 10, the Cathelicidin-NV-treated tissue displayed notably elevated levels of alpha-smooth muscle actin (α-SMA) and collagen expression, in contrast to the control group. Throughout all the treatment days, the Cathelicidin-NV group consistently demonstrated heightened levels of protein expression (MCP-1, TNF-α, VEGF, and TGF-β1) when compared to the control group.

Table IV displays the evidence quality as per the GRADE methodology concerning the impact of frog skin-derived membranes on the skin wound healing process. The evidence summary within the table indicates a moderate quality for experimental studies focused on Macroscopic analysis. These studies demonstrate favorable effects of frog skin in facilitating wound repair.

Table IV
Quality of evidence according to the GRADE approach for studies that applied the membranes manufactured from different Col from frogs on the process of healing skin wounds, in macroscopic analysis.

		Frog skin for wound repair
Outcome	Limitations	Inconsistency	Indirectness	Imprecision	Publications Bias	Trials	Intervention (n)	Comparison (n)	Significant difference or Statistics	GRADE level of evidence
Intervention: Frog skin x control
Variable: Macroscopic analysis
						Ji et al. (2020)	7	7	Yes
						Liu et al. (2019)	12	12	Yes
						Wu et al. (2018)	10	10	Yes
						Li et al. (2018)	10	10	Yes
						Liu et al. (2014)	#	#	Yes
						Zhang et al. (2013)	5	5	Yes
	Serious	No	No	No	Undetected					Moderate

• #: not described.

DISCUSSION

The present study evaluated the effects of different kinds of peptides extracted from frog skin on the process of healing in skin wounds in animal models. From the studies included in this review, it was possible to observe that many different kinds of peptides obtained (using different protocols of extraction) from the skin of frogs were used to treat skin wounds in the rats and mice. Additionally, for the in vivo experiments, many different skin wound sizes and types were used (since 78,5 mm2 to 1,200 mm2), as well as different timepoints of evaluation (2 days to 48 days). Peptides were purified and synthesized using different methodologies, but mainly through the lyophilization of the skin secretions of the frogs and some of them was commercially purchased. The characterization of the peptides was performed mainly using mass spectrometer for determination of primary structure of the peptide and the amino acid sequence was also determined. Although the use of different peptides, all the authors have found positive results in the process of skin healing in the animal models used. In general, the treatment produced a significant decrease in the wound, histological findings point out that a higher index of re-epitalization at the site of the injury, a higher amount of collagen deposition.

Some articles have been demonstrating that bioactive peptides extracted from amphibians have showed antimicrobial, antiseptic, antioxidant peptides and wound healing peptides (Liu et al. 2019). Of note, damaged amphibian skin has been found to heal quickly and often without scaring, indicating the existence of molecules in amphibian skin secretions (Yokoyama et al. 2011, Tang et al. 2014, Di Grazia et al. 2015). The works included in the present review used many different experimental conditions, varying the size of the wounds and the experimental period of treatment. However, all the studies performed the treatment placing the peptides directly at the site of the injury (and one associated with hydrogel).

Although the evidence about the positive effects of frog skins on the process of wound healing-promoting ability, on molecular level, almost no biomolecules or peptides derived from this source was identified and investigated for promoting wound re-epithelialization in frog and human skin (Meier et al. 2013). Interestingly, the authors included in this paper purified different peptides from the different specimens of frogs. Liu et al. (2010) used the peptide named AH90 (from Odorrana graham) (Meier et al. 2013), Ji et al. (2020) extracted skin peptides (RCSPs) of Rana chensinensis from the skin (Liu et al. 2019), Zhang et al. (2013) used small peptide isolated from the skin secretions of Rana pleurade, which show multifunctional properties, such as combined antioxidant, anti-inflammatory and antimicrobial activities, (Liu et al. 2010, Yang et al. 2009). Li et al. (2018) a novel peptide named OM-LV20, was identified from the skin secretions of Odorrana margaretae and promoted potent wound healing activity in both cellular and animal models, Li et al. (2018) and Wu et al. (2018) used the cathelicidin peptide named cathelicidin-NV 36 and Liu et al. (2023) investigated the new peptide, OA-FF10, from the skin secretions of O. andersonii, which was found to be highly effective in promoting wound healing both in vitro and in vivo (Li et al. 2018).

For the extraction of the peptides, most of the authors used methods of purification such as to lyophilize skin secretions and apply it to a gel filtration column. It is worthwhile to point out that a wide range of techniques for evaluating the physical and morphological membrane characteristics was used by the different authors. SEM, FTIR, mechanical properties, water absorption test, among others were performed with the aim of analyzing the morphology of the peptides, identifying their structure and molecules (Ji et al. 2020, Liu et al. 2014, 2019, Wu et al. 2018, Li et al. 2018, Zhang et al. 2013). These techniques are complementary to each other, making possible a screening and the characterization of peptides. Most of the studies demonstrated that the methods for purification and synthesis of the skin frog peptides extracted samples with a high purity and a structure resembling the substance present in the secretions of skin amphibians.

It is well known that skin from frogs is a rich source of collagen and peptides with a huge potential to be used as treatments for accelerating tissue healing in skin wounds (Mashregui et al. 2013). Rezazade et al. (2015) demonstrated that a compound of powder extracted from frog skin was found to contain considerable healing and antibacterial effects on wounds. Frog skin secretions of Rana ridibunda were also demonstrated to be significantly effective in promoting wound healing process (Mashregui et al. 2013). The in vivo results demonstrated that positive outcomes were found for all the studies included, with an improved process of healing in the skin wounds. Most of the authors used macroscopic analysis, wound closure rate and histological analysis to evaluate the effects of peptides from frog skin in the process of wound healing. Accordingly, to Ji et al. (2020) peptides from frog skin can promote cell adhesion and proliferation in the region of wounds, which maybe an effect of the formation of mature vascular structures in damaged tissues.

This systematic review has some limitations. The use of skin frog and their derived peptides as a treatment for accelerating skin wound healing is still undergoing animal studies. Further studies are needed, especially for investigating the mechanisms of action of the peptides from frog skin on the wounds to determine the efficiency and safety of this therapy. Moreover, the observation time and measurements vary among studies which cause high heterogeneity in the results. Hence, it was not possible to perform a meta-analysis.

The development of innovative multifunctional treatments for skin wounds is of great need in the clinical setting, as some wounds pass a threshold diameter such that they may not heal by themselves. Furthermore, in some patients with systemic diseases, wounds can become chronic causing a decrease in the quality of life and even, lead to mortality. In this context, skin frogs (and their biocompounds including peptides) present biological characteristics that are able of mimicking skin tissue, with an adequate porosity to support cell growth and releasing growth factors, consequently stimulating healing. Based on these statements, the use of this therapeutical intervention constitutes a promising treatment for chronic wounds.

CONCLUSIONS

In conclusion, this systematic review analyzed studies investigating the effects of peptides extracted from frog skin for wound healing in in vivo animal studies. First, this review confirms the significant benefits of using these peptides for injury repair and regeneration. A faster healing and a full wound closure, with well-organized dermal and epidermal layers were found in the wounds treated peptides from frog skin. Although all the positive biological effects demonstrated by the works, further studies are needed, especially, in the clinical setting in order to determine if frog skin and its peptides are safe and relevant before these devices can become part of standard clinical practice.

Acknowledgements

We gratefully acknowledge Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant No. 2023/10998-0, 2019/102285, 2021/02974-9 and 2021/13056-0) for funding support.

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Publication Dates

• Publication in this collection
  15 Sept 2025
• Date of issue
  2025

History

• Received
  18 Sept 2024
• Accepted
  01 Apr 2025