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Enhanced Cutaneous Wound Healing In Vivo by Standardized Rough Excerpt of Poincianella pluviosa

• Fernanda Giacomini Bueno,
• Eduarda Antunes Moreira,
• Gutierrez Rodrigues de Morais,
• Isabela Almeida Pacheco,
• Mauro Luciano Baesso,
• Eneri Vieira de Souza Leite-Mello,
• João Carlos Palazzo de Mello

x

• Published: March 3, 2016
• https://doi.org/10.1371/journal.pone.0149223

Figures

Abstruse

Wound healing is a complex process that involves several biological events, and a filibuster in this process may cause economic and social problems for the patient. The search continues for new alternative treatments to aid healing, including the use of herbal medicines. Members of the genus Caesalpinia are used in traditional medicine to treat wounds. The related species Poincianella pluviosa (DC.) Fifty.P. Queiroz increases the cell viability of keratinocytes and fibroblasts and stimulates the proliferation of keratinocytes in vitro. The crude excerpt (CE) from bark of P. pluviosa was evaluated in the wound-healing process in vivo, to validate the traditional use and the in vitro activity. Standardized CE was incorporated into a gel and practical on cutaneous wounds (TCEG) and compared with the formulation without CE (Control) for iv, seven, ten, or 14 days of treatment. The effects of the CE on wound re-epithelialization; cell proliferation; permeation, using photoacoustic spectroscopy (PAS); and proteins, including vascular endothelial growth factor (VEGF), superoxide dismutase ii (SOD-2) and cyclooxygenase 2 (COX-two) were evaluated. The TCEG stimulated the migration of keratinocytes at solar day iv and proliferation on the following days, with a high concentration of cells in metaphase at vii days. Type I collagen formed more than apace in the TCEG. PAS showed that the CE had permeated through the pare. TCEG stimulated VEGF at mean solar day iv and SOD-2 and COX-two at mean solar day 7. The results suggest that the CE promoted the regulation of proteins and helped to accelerate the processes involved in healing, promoting early angiogenesis. This led to an increase in the re-epithelialized surface, with significant mitotic activity. Maturation of collagen fibers was also enhanced, which may impact the resistance of the extracellular matrix. PAS indicated a correlation between the rate of diffusion and biological events during the healing process. The CE from P. pluviosa appears promising as an assistance in healing.

Citation: Bueno FG, Moreira EA, Morais GRd, Pacheco IA, Baesso ML, Leite-Mello EVdS, et al. (2016) Enhanced Cutaneous Wound Healing In Vivo by Standardized Rough Extract of Poincianella pluviosa. PLoS I 11(3): e0149223. https://doi.org/10.1371/periodical.pone.0149223

Editor: Joseph P. R. O. Orgel, Illinois Institute of Engineering science, United states of america

Received: September 6, 2015; Accustomed: January five, 2016; Published: March 3, 2016

Copyright: © 2016 Bueno et al. This is an open access article distributed nether the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in whatever medium, provided the original author and source are credited.

Data Availability: All relevant data are within the newspaper and its Supporting Data files.

Funding: We give thanks CAPES, CNPq, and Fundação Araucária for financial support. Additionally, we would like to thank Conselho Nacional de Desenvolvimento Científico e Tecnológico – JCPM #303391/2009-0. The funders had no function in report design, data collection and analysis, determination to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The skin forms a bulwark that protects the body against intentional or adventitious damage such as burns, cuts, abrasions or cutaneous ulcers, which tin can compromise its function [i,two]. These types of impairment are repaired in the wound-healing process, which is very complex and involves several biological events, including vascular and cellular changes, epithelial proliferation, collagen synthesis and deposition, fibroblast proliferation, and wound contraction. However, the time required to complete these stages can change when wound healing is impaired or fails. Almost six million people worldwide are estimated to suffer from unhealed wounds [3].

The cells produce pro-inflammatory cytokines and reactive oxygen species (ROS) such as the superoxide anion and hydrogen peroxide [4,v]. ROS are essential to protect the tissue against microorganisms [5] and stimulate immune cells to release loftier levels of vascular endothelial growth gene (VEGF) [6]. VEGF induces migration and proliferation of endothelial cells [7]. A "respiratory outburst" is caused by an excessive increase in ROS release [viii]. Extensive tissue impairment including inhibition of cell migration and proliferation can occur if ROS are non detoxified [9]. Superoxide dismutase (SOD), catalase, and some peroxidases are scavengers of these reactive species. Hydrogen peroxide (H₂O₂) can be produced by the activeness of SOD on the superoxide anion. In the endothelial cells, H_iiO_ii can stimulate the expression of cyclooxygenase ii (COX-2) and metalloproteinases [10].

A delay in wound repair causes economic and social problems for the patient, raising concerns regarding the reduction in quality of life, mental and concrete health, and complications that tin can cause morbidity and mortality [11]. Due to the high toll of treatment associated with poor wound healing, the search for new drugs to advance the healing process has get a priority. For this procedure to exist effective, the wound must close rapidly. A normal healing process should produce a resistant and esthetically satisfying scar [12]. In mod medicine, herbal compounds are assuming an important role in tissue repair. Some reports have described the furnishings of herbal drugs on wound healing [13,14]. In Brazil, several traditional medicinal plants have been used and studied for their acceleration of wound healing [14,15], although many species remain to exist evaluated.

A member of the family Fabaceae, Poincianella pluviosa (DC.) L.P. Queiroz is popularly known equally "sibipiruna" or "false Brazilwood", and is also reported under its synonyms Caesalpinia peltophoroides (Benth.), Caesalpinia pluviosa DC., and Caesalpinia pluviosa var. peltophoroides (Benth.) Grand.P. Lewis [three,16]. The bark has been investigated as an antimalarial [17] and for its healing activeness. Bueno et al. showed that a crude extract of P. pluviosa increased the in vitro prison cell viability of keratinocytes (HaCaT), and fibroblasts (pNHDF), stimulated the proliferation of keratinocytes and demonstrated the presence of hydrolyzable tannins in the active fraction [sixteen]. In traditional Indian medicine, members of the related genus Caesalpinia are used to treat wounds and other injuries [18]. Caesalpinia contains nigh 500 species, and their compounds have diverse biological activities [19]. Several species are used and/or have been evaluated for their healing potential [20,21,22,23].

The nowadays study evaluated the in vivo healing effect of a crude extract from the bark of P. pluviosa on the process of wound re-epithelialization, antioxidant effects, angiogenesis, cell proliferation, and permeation.

Materials and Methods

Plant material and rough extract (CE) training

Bark of P. pluviosa was collected on the campus of the Universidade Estadual de Maringá (UEM), Maringá, Paraná, Brazil (23°24'10''S; 51°56'28''W, 564 m a.due south.l.). A voucher specimen was deposited in the UEM Herbarium nether number HUEM-12492. The bark samples were dried under forced-air apportionment (40°C) and then milled in a Tigre ASN-5 stainless-steel hammer manufacturing plant. Milled bark of P. pluviosa (ten% due west/v) was extracted using fifty% ethanol (v/v) by turbo-extraction (Ultra-Turrax UTC 115KT, IKA, U.s.; 15 min; t <40°C). The crude excerpt (CE) was concentrated in a rotary evaporator under reduced pressure and and then lyophilized.

Antioxidant capacity and total polyphenol content

Antioxidant capacity was estimated based on the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging action, according to the method described by Amarowicz et al., and the results are presented as IC_l (μg/mL) [24]. Vitamin C was used as a reference (W.P., China, 100.0%). The CE was standardized co-ordinate to Bueno et al. [xvi,25], and the full polyphenol content (TP) was determined using a modified Folin-Ciocalteu method [25].

Animal experimentation

Gel conception.

Two carbopol gel formulations with hydrophilic characteristics, a base gel without CE (BG) and a CE gel containing i% CE (CEG), were prepared as described by Silva-Corazza et al. and stored at iv–8°C [26]. The formulations were prepared before the beginning of the experiments, and were used during the entire treatment period.

Ethics statement and experimental animals.

The study was canonical by the Beast Ideals Committee of the Universidade Estadual de Maringá (Permit number: 141/2010). Male Wistar rats (Rattus norvegicus) weighing 220 to 240 thou were kept in private cages, on a 12-h light/night cycle, temperature 22°C, with water and chow (Nuvital) advert libitum. The animals (northward = 34) were divided into four groups, corresponding to 4, 7, 10, and 14 days of treatment. Each group was evaluated by means of histological tests (n = 5), photoacoustic spectroscopy measurements (northward = 3), and the Western Blot test (n = 3; except at twenty-four hour period 14).

The animals were anesthetized with 2% Rompum (Bayer, São Paulo, Brazil)/10% Ketamine Agener (Agener União, São Paulo, Brazil) (1:i; 0.1 mL/100 g), positioned for a cervical epilation, and two wounds (1 cm² each) were made side by side, past removing the epidermis and dermis. Starting time on the next twenty-four hour period, each cutaneous wound was treated daily with base gel on the correct side (Control) and CEG on the left side (TCEG). After 4, 7, ten, or 14 days of handling, the animals were euthanized with an overdose of anesthetic (120 mg/kg Thiopentax, Cristália, São Paulo, Brazil). The cutaneous wounds were examined visually and then pare samples were removed. In the histological examination, 2 h before the animals were euthanized, vincristine sulfate (0.5 mg/kg; Tecnocris one mg/mL, Zodiac, São Paulo, Brazil) was administered to cake mitosis in the epithelial cells.

Histological study.

The pare samples taken at days 4 and 7 were cut in half. All samples were Bouin-fixed, methane series-embedded, and cut in semi-series 6 μm-thick sections. The slides were stained with hematoxylin-eosin (HE) and Sirius red [27]. The re-epithelialization (length and thickness) and number of metaphases were evaluated under an Olympus BX41 light microscope with a 3.2 Megapixel Olympus Q-Color-three Imaging Arrangement coupled to an epitome capture system (Q-Capture Pro). Types I and Iii collagen fibers were quantified by the Picro-Sirius technique nether an optical microscope coupled to a polarizer (Zipper Nszh-KPO). All slides were analyzed using Image Pro-Plus (v. 4.v).

Analyses of re-epithelialization.

At days 4 and 7, the upper re-epithelialized surface was measured on each side of the wound. The thickness was evaluated at days 10 and 14, by measuring the re-epithelialized surface at three different points, starting from the center of the wound. Three sections of each slide were analyzed, using a 10X objective. The number of cells in mitosis was determined higher up the basal and supra-basal surface layers. Five sections of each slide, with a total length of 10,000 μm, were analyzed under a 40X objective. The results were expressed in number of cells in metaphase/mm [28].

Collagen fibers.

The natural birefringence of collagen, revealed with Picro-Sirius staining and polarized light, allows the types of collagen to exist differentiated by their density [27]. The collagen-stained surface area was calculated by the density of the fibers. Light-green-stained fibers stand for blazon Iii collagen, and reddish-, orange- or yellow-stained fibers represent type I. Three fields were analyzed on each slide, observed using a 20X objective. The results were expressed as pct of fibers.

Analysis of protein past Western Absorb.

At days 4, 7, or 10, the wounds were removed, fragmented, and homogenized with Tris buffer (50 mmol/L, pH 6.eight) containing the protease inhibitors PMSF (10 mg/mL) and aprotinin (2 mg/mL) [29]. The samples were placed in an ultrasonic bath (3 10 15 s) and centrifuged at 4°C. Full protein (μg/mL) present in the supernatant of each sample was measured past the Bradford method [30], then diluted in a solution containing i% SDS, ii% 2-mercaptanol, and x% glycerol, and placed in boiling h2o for five min. Separation and packaging gels containing 10% and 4% polyacrylamide, respectively, and molecular weight standards from half-dozen.9 to 200 kDa (Mark12 Unstained Standard) were used. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) is a constitutive protein and was used every bit a loading control. Later separation by electrophoresis in SDS-PAGE, the proteins were transferred to a nitrocellulose membrane and blocked with Tris buffer solution containing 0.2% Tween-twenty (TBST, pH 7.v), and 10% milk protein for 1 h. The membrane was incubated overnight with rabbit monoclonal VEGF, COX-2, SOD-2, and GAPDH (1:250) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and washed with TBST. The membrane was revealed using a secondary antibiotic F(ab') 2 fragment of goat anti-mouse IgG (Santa Cruz Biotechnology) conjugated to peroxidase (1:1000) for 1 h. The blot was incubated in a chemiluminescence solution (Novex Chemiluminescent Substrates, Invitrogen) at room temperature, and was autoradiographed with a ChemiDoc XRS System (Bio-Rad). Poly peptide levels were analyzed past densitometry (ImageJ 1.47) and normalized against the GAPDH response (100%).

Photoacoustic spectroscopy (PAS) measurements.

PAS measurements were carried out according to Rocha et al. [31]. The photoacoustic optical assimilation spectra were measured using the calorie-free modulation frequency at 22 Hz and scanned the wavelength between 200 and 800 nm. The gel was applied 30 min earlier the samples were collected and analyzed on the epidermal and dermal surfaces. The absorptions of the CE, BG, and CEG were measured. Spectra obtained on the dermal and epidermal surfaces were subtracted to ameliorate assess the skin permeation.

Statistical assay

The software Statistica eight.0 (StatSoft, Inc. 1984–2007) was used for the statistical analyses. Data are expressed as hateful±standard deviation (SD) using the Mann-Whitney test, a nonparametric analysis for Western Blot; and the Tukey test, a unilateral analysis of variance (one-fashion ANOVA) for multiple comparisons. Meaning differences were adamant using p<0.05 as the significance benchmark.

Results

Antioxidant activity estimated past the DPPH method showed that the inhibitory concentration (IC_fifty) was vii.40±0.10 μg/mL for the CE and 4.36±0.08 μg/mL for vitamin C. The total polyphenol content of the CE was 22.7%. The gel formulation was therefore standardized to 22.7 mg% of full polyphenols. The amount of CE in the carbopol gel was optimized from previous studies with Stryphnodendron adstringens (barbatimão) [15]. Visual observation of TCEG showed no exudate, inflammation or bleeding on all days of treatment. However, on the second solar day a rapid browning (darker ruddy-ruddy color) and drying crust were observed in the TCEG. In the Control, the chaff was less consistent and colored bright red (Fig 1A). These different colors were observed until twenty-four hours 5.

Fig i. (A) Photographs of excisional wounds on day 2 of treatment: TCEG and Control; (B) Photomicrographs of histological sections stained in hematoxylin-eosin: skin at days 4, seven, 10, and fourteen of TCEG and Control.

The original magnification was 2x. (●) Representative re-epithelialized surface. (^▬) 500 μm. TCEG: wound treated with gel containing one% crude extract; Control: wound treated with base gel.

https://doi.org/10.1371/periodical.pone.0149223.g001

Histological study

Fig 1B shows the re-epithelialized surface on the days of treatment. Fig 2A and 2B shows the length (at days four and seven) and thickness (at days 10 and 14) of the re-epithelialization surface, respectively. At 24-hour interval iv, the length of the re-epithelialized surface at the wound heart was greater in the TCEG. At days ten and 14, the epidermal layer was thicker than that of the Command. At day 7, the re-epithelialized surface peaked in the Command but was thinner than in the TCEG. Statistical analysis showed significant differences (p<0.05) on all days in both treatments. In the evaluation of mitotic activity (Fig 2C), at twenty-four hour period 4 the TCEG and Control showed 25.0±0.46 and 12.0±0.22 cells in metaphase/ten mm, respectively. At day seven this number doubled for the treatments, resulting in 45% more than cells in metaphase in the TCEG. At day 10, more than cells in metaphase were present in the Command, and at day fourteen in the TCEG. The results of the treatments were statistically different (p<0.05).

Fig 2.

(A) Length at days 4 and 7; (B) thickness at days 10 and 14 of the re-epithelialization surface. (C) Number of cells in metaphase/mm in basal and supra-basal re-epithelialized surface at days 4, 7, ten, and 14. The results are expressed equally mean±standard divergence. Values are ways of three independent experiments. Statistical data were used to compare the days of treatment between TCEG and Control (*p<0.05). (█) TCEG and (█) Command. TCEG: wound treated with gel containing ane% crude excerpt; Control: wound treated with base gel.

https://doi.org/10.1371/periodical.pone.0149223.g002

The percentage of type Three (immature) collagen was higher at days four and vii for the Control (l.02±i.99 and 56.32±5.61, respectively) likewise as for the TCEG (53.14±ix.56 and 52.23±three.69, respectively). The percentage decreased on the succeeding days, due to replacement by type I collagen (mature). However, at day 10 at that place was a significant difference (p<0.05) between treatments, with 67% more than type I collagen in the TCEG. At mean solar day xiv there was re-institution of the type I collagen in the Control. Fig 3A and 3B shows the collagen on all days of treatment. At days 4, seven, and 10, type I collagen fibers were present in higher percentages (p<0.05) in the TCEG compared to the Control (S1 Fig).

Fig three. Percentages of (A) blazon Three collagen (young) and (B) blazon I collagen (mature) at days 4, seven, 10, and 14.

The results are expressed as mean±standard deviation. Values are means of iii independent experiments. Statistical data were used to compare the days of treatment between TCEG and Control (*p<0.05). (█) TCEG and (█) Control. TCEG: wound treated with gel containing i% crude excerpt; Control: wound treated with base gel.

https://doi.org/10.1371/journal.pone.0149223.g003

Analysis of protein by Western blot

The levels of SOD-2, VEGF, and COX-2 protein was detected at 24, 45, and 70 kDa (Fig 4A), respectively. At mean solar day 4, no changes in the protein levels of SOD-2 and COX-2 were observed. In TCEG, VEGF showed a significant (p<0.05) increase at 4 twenty-four hours and SOD-2 and COX-2 showed significant (p<0.05) upward-regulation at day 7. The proteins returned to basal levels afterward the maximum peak (Fig 4B).

Fig 4.

(A) Western absorb analyses of COX-2, SOD-2, VEGF, and GAPDH poly peptide at days four, seven, and 10 of TCEG (T) and Command (C). (B) Measurement obtained from the ratio betwixt optical densities of protein bands from treatments and loading control (GAPDH). GAPDH was fix to 100%. The results are expressed as mean±standard deviation. Values are means of 3 contained experiments. Statistical data were used to compare the days of treatment between TCEG and Control (*p<0.05). TCEG: wound treated with gel containing 1% rough extract; Control: wound treated with base gel.

https://doi.org/x.1371/journal.pone.0149223.g004

Photoacoustic spectroscopy (PAS) measurements

The results obtained from photoacoustic spectroscopy showed the spectra of BG and CEG (Fig 5A), where BG has no absorption in the spectral range of 250–450 nm. In Fig 5A-inset, from the determination of Gaussian fit, assimilation bands of P. pluviosa are shown considering the main band at 290 nm. Fig 5B shows the spectra of the dermis treated with CEG, where all spectra have assimilation betwixt 250 and 450 nm. Fig 5C shows the subtraction of TCEG dermal spectra from the spectra for the Control dermis, and the presence of bands around 290 nm compared to the band of the CEG. Fig 5C-inset shows the Gaussian fit adjustment of the CEG contribution to the subtraction spectra. The area of this band at 290 nm provides an approximate of the permeation during the healing process (Fig 5D).

Fig five. (A) photoacoustic spectra of BG, CEG, and in the inset the Gauss fit of absorption bands of CE; (B) photoacoustic spectra of TCEG over 14 days of treatment; (C) difference between TCEG and control dermal spectra, and in the inset the Gauss fit of absorption bands of CEG; (D) the area of permeation behavior of the absorption ring at 290 nm.

The results are expressed as mean±standard deviation. Values are ways of iii independent experiments. BG: base of operations gel; CE: crude excerpt; CEG: gel containing 1% CE; Control: wound treated with BG; TCEG: wound treated with CEG.

https://doi.org/ten.1371/journal.pone.0149223.g005

Discussion

Seconds after an injury, hemostasis is triggered with hemorrhage into the wound. Instantly, a claret clot is formed, serving as a physical bulwark and producing chemotactic signals [eleven,32]. The fibrin jell acts every bit a temporary matrix for cell migration in the adjacent wound-repair stages [1,11]. In our experiment, during the starting time five days of TCEG, the jell was darker and more than consistent than in the Command. This feature can be explained past the presence of polyphenol compounds (22.seven%) in CE. Tannins, belonging to the polyphenol group, are able to precipitate with proteins and form a dark crust that covers the wound. They have astringent, antioxidant, and antimicrobial properties [33].

The proliferative phase begins with re-epithelialization involving the extracellular matrix and collagen production [34]. Re-epithelialization closes the wound, reorganizing the cytoskeleton through the migration and proliferation of keratinocytes from the wound edges [35]. As wound closure progresses, epidermal re-epithelialization can be adamant past the thickness (days 10 and 14) and length (days 4 and 7) of the re-epithelialization layer. At day 4, the TCEG showed intense migration and proliferation of keratinocytes, and at day seven the migration was inhibited and proliferation was stimulated. At days 10 and 14. keratinocyte proliferation and differentiation were visible. Cellular proliferation is essential to form a dense hyperproliferative epithelium and restore pare integrity [35]. In the Command, the keratinocyte migration was observed at days four and 7, and keratinocyte proliferation at days 10 and 14. Interruption of mitosis at metaphase by the assistants of vincristine sulfate, which binds to tubulin and prevents microtubule formation [36], tin demonstrate cell proliferation in the TCEG. Mitosis in the TCEG remained loftier up to day 7, while in the Control, mitosis peaked later day 10, virtually iii days afterwards. The results showed that TCEG accelerated re-epithelialization compared to the Control. Values in the re-epithelialization surface area (afterwards 4 days) and for epidermal thickness (days 10 and xiv) were e'er higher in the TCEG.

While re-epithelialization is proceeding, the extracellular matrix is being laid down. Granulation tissue is the new stroma and consists of fibroblasts, collagen fibers, and new vessels. Fibroblasts are responsible for producing collagen in the temporary extracellular matrix, which is beginning to be replaced by a resistant elastic tissue [34]. At this stage of healing (days 4 and 7), larger amounts of type III (young) collagen were quickly replaced by blazon I (mature) collagen fibers in the TCEG, with subsequent formation of stronger and less-vascularized tissue [37]. The fibroblast response is related to the increased proliferation of this cell type in this phase of healing [38]. The dermis at days 10 and 14 showed better-organized type I collagen fibers than in the Control. During the fibroblast attachment and maturation, the process of wound contraction reaches its maximum efficiency and the tensile strength may exist increased. The ratio of the re-epithelialized area, number of metaphases, and accelerated progression to mature collagen fibers indicate a higher proliferative potential and resistance in wounds treated with the gel containing the CE of P. pluviosa.

Each stage of wound healing can be regulated by many bioactive compounds including growth factors, cytokines, and eicosanoids. Prostaglandins (PGs), which are inflammatory mediators, vest to the eicosanoid class. Arachidonic acid is converted past cyclooxygenase 1 and ii (COX) into PGs [39]. The major PG is PGE_two, which is formed past COX-ii, and is involved in keratinocyte proliferation [40], angiogenesis [41], and mediation of the inflammatory response. [42] observed large amounts of COX-2 in the basal layer of wound epidermis and likewise expressed in inflammatory cells. In this written report, COX-ii was stimulated (p<0.05) past TCEG, indicating a possible effect on inflammatory cells and keratinocyte proliferation. Nevertheless, VEGF was stimulated by TCEG at day 4 and returned to basal levels, indicating that P. pluviosa promoted angiogenesis. VEGF is the nearly important proangiogenic cistron that increases with initial hypoxia [43,44]. The epicatechin gallate, a phenolic compound, stimulated COX-two and VEGF, improving wound healing in a rat model [45].

Another procedure that influences wound healing is the presence of big amounts of reactive oxygen species (ROS), which are formed from oxide-reduction reactions during the processes of energy production, phagocytosis, regulation of jail cell growth, synthesis of substances, and intercellular signaling [46,47]. Free radicals produce numerous disorders, only can exist eliminated past antioxidants that are present in the crude extract, which facilitates the healing procedure [48]. The CE IC₅₀ was similar to that of vitamin C; this activity is probably due to phenolic compounds. Another possibility is the production of SOD-ii (day vii), the most common scavenger of ROS found in the mitochondrial matrix [49].

The gel formulation for topical application was likewise evaluated for permeation of the CE of P. pluviosa from the dermis to the bloodstream [50]. The PAS spectra were used to determine the penetration profile of the substances through the pare. The resulting bands were evaluated by Gaussian analysis on each twenty-four hour period of treatment. The permeation profile was obtained past subtracting the absorption spectrum obtained for the control dermis from the spectrum for the treated dermis, giving the absorption profile for the CE only. As with sunscreens, the gel used to treat skin wounds should have a minimum permeation to the bloodstream, performing its effects in the peel layers [51]. The PAS technique allowed the states to determine that the crude extract was absorbed in the wound, showing that the formulation was advisable to evaluate the wound-healing treatment. The rates of drug improvidence in wounds are affected by the morphological evolution during the repair process, where the nigh important effect for the re-establishment of skin integrity is re-epithelialization. At solar day 4, the increase in vascularity favored the CE transport into the systemic apportionment, every bit was observed with a propolis extract after 7 days of handling [fifteen]. Even with the remodeling of the number of blood vessels and the proliferation and differentiation of keratinocytes, the corporeality of CEG increased until ten days of treatment. At 14 days, the wound wrinkle, epidermal reconstitution, and germination of collagen fibers reduce the penetration charge per unit in the skin to a low level [15].

Decision

Employ of the formulation containing the rough excerpt of P. pluviosa stimulated the formation of collagen fibers and re-epithelialization, indicating that it promotes the formation of more-organized tissue and accelerates wound healing. The up-regulation of proteins by the cells helped to accelerate the processes involved in healing. Phenolic compounds with antioxidant activity present in the CE had a positive effect, providing greater protection for the injured tissue past inhibiting the oxidant agents produced in backlog. Photoacoustic spectroscopy allowed us to determine the spread of the formulation during the skin healing, showing a correlation between the rate of diffusion and the biological events of the healing procedure. Therefore, the CE of P. pluviosa, applied topically, proved to exist a potent promoter of wound repair.

Supporting Information

S1 Fig. Photomicrographs of histological sections stained with Sirius Crimson: type I collagen (yellow/orange/ruby) and type Iii collagen (greenish) at days 10 and xiv, for the TCEG and Command. The original magnification was 2x. (^▬) 500 μm. TCEG: wound treated with gel containing 1% crude extract; Control: wound treated with base gel.

https://doi.org/10.1371/journal.pone.0149223.s001

(TIF)

Acknowledgments

We thank CAPES, CNPq, and Fundação Araucária for financial support. Nosotros are very grateful to M.East.C. Cancino, A. Arantes, and C.R. Novello for technical assist. Thanks are due to Dr. Janet W. Reid, JWR Associates, Trumansburg, New York, for English revision.

Author Contributions

Conceived and designed the experiments: FGB MLB EVSL-M JCPM. Performed the experiments: FGB EAM GRM IAP. Analyzed the data: FGB MLB EVSL-M JCPM. Contributed reagents/materials/analysis tools: MLB EVSL-Yard JCPM. Wrote the paper: FGB GRM MLB EVSL-M JCPM.

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