An Alternative Approach Wound Healing Field with Polypodium Vulgare.

OBJECTIVE: In this study, we examined the effects of Polypodium vulgare L. (Polypodiaceae) as a candidate to be used for wound healing scarred area. We investigated the antibacterial, and antioxidant activity of P. Vulgare on both in vivo, and in vitro wound healing using an excisional wound model in mice.
METHOD: We used 32 Balb-c mice equally divided into four groups: Group 1 control, Group 2 vehicle, Group 3 Polypodium vulgare, and Group 4 Centella asiatica extract (CAE). All treatments were applied topically once in a day. The scar area, percentage wound closure and epithelization time were measured. PDGF, VEGF, and collagen immunohistochemical staining were used for evaluation.
RESULTS: CAE and P. vulgare extract groups were observed to be more effective than the control and vehicle groups in terms of new vascular, epidermal and granulation tissue organization. PDGF, VEGF, and collagen immunohistochemical staining was stronger in the P.vulgare extract and CAE groups compared to the control and vehicle groups. In the P. vulgare and CAE groups, PDGF staining intensity was stronger than the control and vehicle groups, but VEGF and collagen staining in P. vulgare group was not different from the control group.
CONCLUSION: P. vulgare had an effect on the injured area by regenerating the epidermis and increasing vascularization. P. vulgare extract with known antioxidant, and antimicrobial activities may be helpful as a supportive treatment in wound healing. © Copyright Istanbul Medeniyet University Faculty of Medicine.

Introduction

Wound healing is a complicated period both for skin and tissues especially after an injury. Epidermis and dermis in the undamaged tissue is a protective barrier against external influences. When the barrier breaks, some repair processes are activated1. Wound healing is of great clinical importance since untreated wounds can be fatal2,3. It was reported that wounds, especially chronic ones can be quickly colonized by both Gram-positive or negative bacteria, fungus and even viruses2,4. Therefore, doctors recommend topical use of antibiotics to prevent unexpected effects such as sepsis and bacteremia. For wound healing and management, various pharmaceutical products can be used4. Traditional medicine is full of knowledge that has been developed through generations in various societies. Traditional medicine approaches are based upon natural products from plants, animals, and minerals. African and Asian communities have used plants for primary health care5. Herbal medicines include sections of plants, different plant parts, their combinations, or plant extracts. Plants are used frequently by medical practitioners in wound healing6. From past to present, various plants have been used as wound healing agents such as Laurocerasus sp., Capparis sp., and Prunus spinosa4,7.

In this study, Polypodium vulgare L. (Polypodace-ae) is evaluated for its potential as a wound healing candidate. Polypodium vulgare L. (P. vulgare) is a plant known and have been used by people since ancient times. Its rhizomes are more well-known and are included in some foods as sweetening agents8,9. In ethnobotanical medicine the plant is used for its antitussive and diuretic prop-erties10. Infusion prepared from its roots is used in abdominal pain and inflammatory conditions. In addition, the pharmacological activities of the plant on the liver and lung are also known. It has also antioxidant, anticholinesterase, antimicrobial, antiviral, antiepileptic, antipyretic, and analgesic properties11-14. P. vulgare is used in traditional medicine as an expectorant to treat cough and pertussis15. Centella asiatica extract (CAE) is a molecule that facilitates the repair process of the wound tissue and has been shown to exhibit antimicrobial activity16. For this reason, it has been preferred as a reference molecule in many studies including this one. We investigated the effects of P.vulgare on excisional wound healing in mice.

Materials and Methods

Preparation of Extract

P. vulgare aerial parts were collected from Belgrade forests, Istanbul, in 2018. Herbal ingredient was identified (specimen no. IMEF: 1068) and stored. The airdried herb was soft powdered in alcohol for 24 hours. Following permeation and vaporization (Heidolph, Germany), gel formulations were prepared by the methanol extract

Antioxidant Activity in Vitro

• 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging

DPPH• was used to determine the total antioxidant capacity17. The reaction mix contained 100 μM DPPH• in methanol and P. vulgare extract. After 30 min, UV spectrophotometer (UV-1800, Shimadzu, Japan) read absorbance value at 517 nm. The scavenging activity was calculated as the percent radical reduction as follows:

DPPH• RSA % = [(Absorbance control - Absor-bance test sample) / Absorbance control)] x 100

• 2,2-Azino-bis 3-ethylbenzthiazoline-6-sulfo-nic acid (ABTS) scavenging

Re and co-workers’ protocol was used for obtaining P. vulgare methanol extract18. Potassium per-sulfate and ABTS• blend was kept at room temperature in a dark room for 16 h prior to its use. The 734 nm was its absorbance measure. The study was repeated three times. For negative controls: ethanol, and positive controls: Trolox were used4. The absorbance of the extract obtained was measured as follows:

ABTS• RSA % = [(Absorbance control - Absor-bance test sample) / Absorbance control)] x 100

Antioxidant reading outcomes were studied comparatively with standard reference substances such as ascorbic acid and Trolox.

Antimicrobial Activity in Vitro

Broth microdilution assay was used for extract specification defined by the Clinical and Laboratory Standards Institute (2006) for minimum inhibitory concentrations (MIC). MIC is the lowest concentration of an antimicrobial agent that prevents growth of an organism.

Staphylococcus aureus ATCC 6538, Enterococ-cus faecalis ATCC 29212, Escherichia coli NRLL B-3008, and Pseudomonas aeruginosa ATCC 10145 strains were grown in Mueller Hinton Broth (MHB, Merck, Germany) at 37°C in aerobic conditions for 24 h. All microorganisms were standardized to 1 × 108 CFU/mL using McFarland No: 0.5 in sterile saline (0.85%). A modified microdilution assay was used to establish the antimicrobial action of the sample. Dimethylsulfoxide (DMSO) was used to prepare the stock solution.

Preparation of Polypodium Vulgare Extract Gel Formulation

The gel of the extract was formulated with hy-droxypropyl cellulose gel. For preparing the gel, hydroxypropyl cellulose (2%) was dissolved in distilled water for 24 hours, and then the P. vul-gare extract (5%) was added, and the blend was stirred slowly.

In Vivo Experiments

• Experimental Animals

Balb-c mice (25-28 g) were used in the study. The mice were hosted in regular cages with food and water intake ad libitum, at room temperature (24°C), with artificial light from 7.00 am to 7.00 pm. Before experiments, ethical clearance approval was obtained from the Local Ethics Committee (No: E.65386-89). During the experiments, the animals were cared for according to the ethical rules.

• Wound formation protocols and animal groups

The experimental animals were divided into four groups within 8 animals per group;

Group 1: Control group (with sterile saline).

Group 2: Vehicle group (no drug content)

Group 3: Polypodium vulgare extract gel group.

Group 4: Centella asiatica extract (CAE) reference molecule.

Mice were anesthetized intraperitoneally with a combination of 80-100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally. Two excisional wound tissues were formed using a 5 mm biopsy apparatus on the left side of the midline in the shaved back region, one cm apart from each other and 1.5 cm away from the midline. Treatments were applied to the wounds for ten days.

• Macroscopic Assessment

For score healing of the wound, photographs were taken at days 0 and 10. The surface areas were measured with the Image J program. After then, wound healing rates were calculated by the formula given in the literature19.

• Histological Assessment

On the 10th day, the animals were sacrificed and the scar tissue was removed. A 10% neutral formalin was used for fixation. After that, wound samples were dehydrated in ethanol series, cleared in toluene, and embedded in paraffin. Five μm thick sections were taken on glass slides and stained with hematoxylin-eosin (HE), immunohistochem-ical stains (vascular endothelial growth factor (VEGF) (Santa Cruz sc-7269), Collagen (COL1A1) (Santa Cruz sc-293182), and Platelet Derived Growth Factor (PDGF-A) (Santa Cruz sc-9974)).

The scoring system defined by Galeano et al. was used for the histological evaluation of wound healing20. For epidermal organization; Score 1: weak epidermal tissue organization ≥ 20% of the tissue, Score 2: incomplete epidermal tissue organization ≥ 40% of the tissue, Score 3: moderate epithelial tissue organization ≥ 60% of the tissue, Score 4: complete epidermal tissue organization ≥ 80% of the tissue. For the thickness of the granulation tissue; Score 1: weak granulation layer, Score 2: moderately dense granulation layer, Score 3: dense granulation layer, Score 4: very dense granulation layer.

For angiogenesis; Score 1: one to two vessels/ site, Score 2: few neovascularization (3-4/site), Score 3: newly formed capillary vessels (5-6/site) and Score 4 describes newly formed and normal appearing capillary vessels (>7/site).

The semiquantitative method was used for the immunoreactivity of immunohistochemical stain. Five randomly selected fields were evaluated and averaged. Score 0: No staining, Score 1: Poor staining, Score 2: Moderate staining, and Score 3: Strong staining21.

• Statistical analysis

For statistical analysis, a standard software package (SPSS 20 for Windows, SPSS Inc., Chicago, IL, USA) was used. Differences between groups were analyzed by one-way ANOVA test, followed by the least significant differences tests. All values were given as mean ± S.E.M. P values values less than 0.05 were considered significant.

Results

Antioxidant Activity

Antioxidant activity of P. vulgare extract is summarized in Table 1. It can be said that the extract has a moderate antioxidant capacity compared to the ones used as standard substances. As a result of the experiments, the antioxidant effect of the extract was revealed, and the antioxidant capacity is thought to be effective in the wound healing.

Table 1

ABTS and DPPH radical scavenging activities of P. vulgare methanol extract (IC50±SD (mg/mL).

	Extract IC50 (mg/mL)	Standards IC50 (mg/mL)
ABTS	3.25±0.03	0.034±0.001 (Trolox)
DPPH	2.13±0.04	0.009±0.002 (Ascorbic acid)

*Statistically significant compared to control, p<0.05.

Antimicrobial Activity

The antimicrobial activity of P. vulgare extracts is showed in Table 2. While the P. vulgare extracts were found to be most sensitive against S. au-reus; P. aeruginosa and E. coli were found to be resistant at tested concentrations (10 mg/mL). MIC value of P. vulgare methanol extract of 0.625 mg/mL against S. aureus was determined.

Table 2

MIC values of P. vulgare MeOH extract on tested concentration (MICs in mg/mL).

	E. coli	S. aureus	E. faecalis	P. aeruginosa
P. vulgare extract	-	0.625	1.25	-
Tetracycline	0.016	0.025	0.016	0.016

Macroscopic wound healing

Figure 1 exhibited the percentage of macroscopic wound healing rates. On day 10, statistically insignificant difference in wound contraction compared to the controls in the P.vulgare group was observed (p<0.2) (Figure 1). No local or systematic side effects were observed macroscopically during the application of the extract.

Figure 1

Macroscopic wound healing and healing percentage of wound area in each group (Control, vehicle, CAE, P. vul-gare groups).

Histology of wound healing

Histological evaluation (H&E evaluation and immunohistochemical (VEGF, PDGF, and collagen) were appraised separately. Histopathological-ly, H&E (Figure 2 A, B, C, D) and immunohistochemical staining were shown in Figure 3 D. CAE (p<0.05) and P. vulgare extract (p<0.05) groups were observed to be more effective than the control and vehicle groups in terms of new vascular organization, epidermal and granulation tissue organization (Figure 2 E, F, G). PDGF, VEGF, and collagen immunohistochemical staining was stronger in the P.vulgare extract and CAE groups compared to the control and vehicle groups (Figure 3 D). In the P.vulgare and CAE group, PDGF staining intensity was stronger than the control and vehicle groups, although not statistically significant (Figure 3A), but VEGF and collagen staining in the P.vulgare group were not different from the control group (Figure 3 B,C).

Figure 2

Histopathological view of injured tissues of the control (A), vehicle gel (B), CAE (C) and P.vulgare (D) extract gel on 10th day after wound incision (Original magnification X10). E, F, G; Histological scores of epidermal-dermal regeneration, granulation tissue thickness and angiogenesis of control, vehicle gel, CAE, P. vulgare groups. Statistically significant as compared to control; P<0.05. Values are presented as the mean ±SEM. The scale bars represent 100 μm for figure. *: Epidermal regeneration, →: Angiogenesis, blood vessels, ↔: Granulation formation.

Figure 3

A, B, C, Comparison of immunohistochemistry collagen, PDGF and VEGF wound healing scores among groups. Statistically significant as compared to control; P<0.05(*). Values are presented as the mean±SEM. D, Histopathological view of injured tissues of the untreated (control), vehicle, P.vulgare and CAE groups on 10th day after wound incision (Original magnification X10).

Discussion

Plants rich in antioxidant compounds are used for their wound-healing and anti-aging properties. Also, free oxygen radicals play an important role in apoptosis and cell proliferation mechanisms. Studies have shown that topical application of antioxidant-containing compounds will be beneficial for wound healing and protecting tissues from oxidative damage7,22. In our experiments, the antioxidant effect of the extract was revealed, and the antioxidant capacity was thought to be effective in wound healing. In our study, the antimicrobial activity of P. vulgare extract was shown. Prevention of microorganisms from multiplying in wound care and the healing process are an important parameters23. Therefore, the antibacterial effect of the extract against various microorganisms found in the human skin flora was investigated for this purpose in our study.

The skin is the largest organ in human body, which acts as the defensive set across physical damage, pathogens, fluid loss, and maintain the body ho-meostasis24. Thus, even a small breakage can impact the health of the individuals. Wound healing is a complex process, although some parts are explained in detail, the rest still need to be explained. The wound healing process is comprised of three different phases. The first is the inflammatory stage that leukocytes migrate to the wound area, followed by the proliferation phase, which includes reepithelization, angiogenesis, and granulation tissue formation. The proliferation phase is important and it starts after three days. The proliferation phase consists different stages to protect the barrier function of the tissue and provide protection against fluid loss and bacterial entry. The last stage is the restructuring phase when the wound is finally contracted7. Multiple studies have shown that essential oils of various medicinal plants increase the wound healing with active ingredients from aromatic plants and fruits7,25. In this study, we observed that P. vulgare increased wound healing, especially by increasing PDGF. In general, angiogenesis impacts embryonic development and wound healing. In our study, it was determined that CAE and P. vulgare extracts significantly effect neovascularization, epidermal organization, and granulation tissue formation compared to the control and vehicle groups. Besides, neovascularization is essential for wound healing4,7. Epidermal organization, granulation tissue formation, and revascularization are the critical agents of wound healing. Formation of organized granulation tissue, with the formation of capillary vessels are important criteria in wound healing. During healing process of the wounds, the granulation tissue acts to protect against infections and epithelial cells migrate to this area26. In our study, significant epidermal organization was observed in CAE and P.vulgare groups compared to the control and vehicle groups. The TGF-beta, PDGF, fibroblast growth factor (FGF), epidermal growth factor (EGF), and VEGF decreased in chronic wounds. In addition, grade of tumor necrosis factor-alfa (TNF-alfa), interleukins (IL) 1 and 6 decreased in chronic wounds27. it was shown that the expression of PDGF in CAE and P. vulgare groups was increased relative to that of control and vehicle groups. Angiogenesis improves fibroblast activity and feeding in the wound area. Fibroblast activity contributes to the formation of granulation tissue. Reactive oxygen specimen (ROS) in wounds decreases fibroblast proliferation and migration; and accordingly, collagen synthesis decreases28. In our study collagen staining of P. vulgare was not different from the control group probably for this reason. Angiogenesis scores of P. vulgare group were not different from the control group.

We observed that P.vulgare enhances skin wound healing. There were marked increases in epidermal regeneration and granulation tissue in the P.vulgare and CAE groups compared to the control group. In light of all this information, P. vulgare extract may be a new option in wound healing because of having similar properties of CAE.