Chemical Characterization and Wound Healing Property of Jacaranda decurrens Cham. (Bignoniaceae): An Experimental Study Based on Molecular Mechanisms

Background Jacaranda decurrens Cham., known as carobinha, is prevalent in the Cerrado biome and presents popular use in treatment of dermatological diseases. The present study aimed to investigate the healing action of topical formulation of Jacaranda decurrens Cham. (FtEHJ) in mice cutaneous lesions. Methods Phytochemical analysis of J. decurrens hydroalcoholic extract was carried out by using HPLC-PDA-ESI-MS and FIA-ESI-IT-MSn. Swiss mice were treated topically with formulation base (FtB) or Fibrinase® or ointment FtEHJ (15 mg/g; 50 mg/Kg). At the end of treatment periods, the inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the lesions were measured by using ELISA and gene expression of TGF-β, Collagen I, and Collagen III was demonstrated by RTqPCR method and histological evaluation. Results Ten compounds were identified in the extract, distributed among the classes of flavonoids and triterpenes. Treatment with FtEHJ increased the wound contraction in 24 hours, such as reduction of TNF-α, IL-1β, and IL-6 (pg/mL) cytokines in the lesion. The TGF-β and collagen gene expression was increased and the wound closure accelerated to nine days, with discrete inflammation, collagenization, and accented reepithelialization. Conclusions. The results obtained suggest chemical compounds present in the FtEHJ accelerates wound healing by being a gene expression modulator, and protein content of different molecules are involved in tissue repair.


Introduction
Wound healing is a physiological, multicellular phenomenon and aimed at restoring the epithelium after injury. is process depends on multiplicity of growth factors and cytokines, which contribute to regular signaling in order to alter the growth, differentiation, and metabolism of target cells. During acute wound healing, active polypeptides are present in wound bed and perform functions at all stages: inflammation, proliferation, and remodeling [1]. It has been found that disorders in wound healing are more frequent in inflammation and/or proliferation stages, which depend on interactions between different cell types and the extracellular matrix, predominantly synthesized by fibroblasts [2,3]. Acute healing occurs along a coordinated biochemical cascade. However, a wound may become chronic if the inflammatory and proliferative phases of the cascade suffer some imbalance. Chronic wounds cause substantial morbidity, mortality, and increased health costs [4].
ere are countless curative options available in the market, which are distributed from simplest coverage as solutions for hygiene and antisepsis, ointments, gels, growth factors, and even most complex types of dressings, called "smart dressings" or "bioactive dressings" [5]. However, in view of tissue repair response complexity, it is perceived that treatment with a single factor or cellular component reaches limited effectiveness in chronic wound healing. e challenge is in combined therapeutic approaches development or preferably in products development that having more than one biologically active compound, which stimulate both angiogenesis and matrix deposition and epithelial migration [6]. ere has been growing increase in plants study recommended by traditional medicine due to the access ease and cultural compatibility with popular traditions, seeking to validate its use as a safe and effective herbal medicine [7,8].
e species Jacaranda decurrens Cham. has a scientific synonym Jacaranda pteroides Silva Manso, receiving popular names such as carobinha and caroba [9]. In Brazil, it is a frequent plant in the Brazilian Cerrado of states of Goiás, Mato Grosso, Maranhão, and São Paulo [10]. e literature describes that Brazilian population uses J. decurrens in hypertension treatment, infectious processes, and hepatic disorders and as blood depurative [11,12]. e infusion and decoction of leaves and stem bark have also been employed for the cutaneous healing treatment and to cure pruritus. A preparation called "garrafada" in Brazil, made from wine with roots and leaves of J. decurrens, is used in treatment of syphilis, rheumatism, inflammation, and dermatological diseases [9]. It has also been reported the use of teas and "garrafadas" of J. decurrens subsp. symmetrifoliolata for uterine and ovarian wound healing, treatment of gynecological infections, giardiasis, and amebiasis [13].
Pharmacological studies revealed that species of Jacaranda genus have properties such as chemopreventive, antioxidant, [14] and antimicrobial [12]. Chemical studies with Jacaranda decurrens revealed the presence of ursolic and oleanolic acids (triterpenes) [14]. Oleanolic acid is a metabolite that has significant antioxidant activity and ursolic acid showed antimicrobial, antiviral, hepatoprotective, immunoregulatory, and inhibitory activity of human cancer cells [15][16][17]. Santos et al. [18] demonstrated that the hydroalcoholic extract of Jacaranda decurrens leaves significantly reduced the formation of paw edema and myeloperoxidase activity in rats. Some studies on the potential efficacy of phenolic compounds in prevention or treatment of skin disorders and reduction of healing time have been published [19][20][21].
Although secondary metabolites with healing activity have been identified in plants of Jacaranda genus [14][15][16][17][18], there are still few studies on chemical and pharmacological activity of Jacaranda decurrens Cham. [11,12,14]. erefore, it is necessary to study the chemical composition and cicatrizant action of it, once the population reports the use of the plant for such activity. us, the objectives of this work were to investigate the hydroalcoholic extract chemical composition of J. decurrens Cham. and evaluate the wound healing effect of a topical formulation containing the plant extract.

Botanical Material.
e leaves of Jacaranda decurrens Cham. were collected in September 2013, in the municipality of São Raimundo das Mangabeiras, Maranhão, Brazil. A species sample was cataloged byÁtico Seabra Herbarium of Federal University of Maranhão (UFMA) under number 1140/SLS017213.

Extraction and Phytochemical Analysis.
e leaves were dried in air circulation in a greenhouse with a temperature of 40-45°C and blended in a DeLeo ® electric mill. e powder, moderately thick (710-250 µm), of the leaves was submitted to hydroalcoholic extraction by maceration with 70% ethanol in the ratio 1 : 10 (weight/volume) during 15 days, under manual shaking for 10 min once daily. e hydroalcoholic extract of J. decurrens (EHJ) was vacuum filtered, concentrated in a rotaevaporator (60°C), and lyophilized, obtaining a yieldof 16.19%. A part of lyophilized material was used in preparation of topical pharmaceutical formulation (FtEHJ) [22,23].

LC-ESI-IT-MS/MS and FIA-ESI-IT-MSn from the Hydroalcoholic Leaves Extract of Jacaranda decurrens Cham.
For the HPLC-ESI-IT-MS/MS and FIA-ESI-IT-MS n a cleanup step was performed to remove any contaminants; the solution was purified by solid phase extraction (SPE) using Phenomenex Strata C18 cartridges (500 mg of stationary phase) that were previously activated with 5 mL of MeOH and equilibrated with 5 mL of MeOH : H 2 O (1 : 1, v/v). e compounds were eluted from cartridges using 1 mL of MeOH : H 2 O (1 : 1, v/v) with a final volume of 5 mL. e samples were then filtered through a 0.22 µm PTFE filter and dried. e extract was diluted to 10 µg/mL in HPLC solvent. Aliquots of 20 µL were injected directly into the LC-ESI-IT-MS. e analysis was performed on an online LC-ESI-IT-MS in a mass spectrometer LCQ Fleet, ermo Scientific ® . e analytical column used for LC separation was a Kinetex ® C18 (2.1 × 100 mm, 100Å and 5 µm). e analysis was carried out with water containing 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B) added to 0.1% formic acid in exploratory gradient starting with 10% to 100% B in 6 minutes at a flow rate of 0.4 ml/min. e sample was infused into the mass spectrometer from the HPLC system, where the sample was analyzed online by ESI-MS in negative mode and UV detector associated. e mass spectra data were obtained in the same Fleet LCQ mass spectrometer from ermo Scientific ® .  Evidence-Based Complementary and Alternative Medicine CA, USA). A stainless-steel capillary tube at 280°C was used, with a spray voltage of 5.00 kV, capillary voltage of −90 V, tube lens of −100 V, and a 5 µL min −1 flow. Full-scan analysis was recorded in m/z ranging from 100-1000. Multiple-stage fragmentations (ESI-MS n ) were performed using the collision-induced dissociation (CID) method against helium for ion activation. e first event was a full-scan mass spectrum to acquire data on ions in that m/z range. e second scan event was an MS/MS experiment performed by using a datadependent scan on the (M-H) − molecules from the compounds of interest at a collision energy of 30% and an activation time of 30 ms. e product ions were then submitted to further fragmentation in the same conditions until no more fragments were observed. e identification of the different compounds in the chromatographic profile of the hydroalcoholic extract was done by comparing their retention times and UV spectra with literature data.

Topical Formulation Development (FtEHJ).
In preparing the FtEHJ, lyophilized extract was resuspended in propylene glycol and added to molten polyethylene glycol 4000 (PEG 4000), forming a homogeneous mixture, moss green, with pasty consistency and characteristic odor. e topical formulation final concentration was 15 mg/g. After raw materials homogenization, it was stored in containers protected from light [23]. 2.6. Healing Activity. Mice (n � 60) were divided into the following groups: formulation base (BFt; n � 20); Fibrinase ® (fibrinolysin 3.33 U/kg; deoxyribonuclease 2.220 U/kg; chloramphenicol 33.3 U/kg; n � 20) and FtEHJ (1.5 g/100 g; n � 20). e animals were anesthetized (8 mg/kg xylazine, 10 mg/kg ketamine). e lesions were made in the dorsal region with the aid of 1 cm diameter surgical punch. All groups received approximately 0.1 g topically from respective treatments with daily application, at the same time. During treatment the following parameters were observed: bleeding; hyperemic halo; secretion, and extension of crusts. e lesions in animals were photographed daily by digital camera coupled to a tripod at a distance of 12 cm. e lesion area analysis was done through the Image J ® program, and the percentage contraction calculation was given by the following equation [24]: x � [(initial area − average area of the day/initial area) × 100].
(1) e animals were euthanized by anesthetic overdose of xylazine and ketamine at four different times: 1st, 7th, 9th, and 12th (n � 5/group/treatment time). At the end of the 1st and 7th day, part of tissue was removed and stored at −20°C to perform the ELISA technique. Part of cutaneous tissue removed on the 7th day was stored with RNAlater ® solution (Ambion ® ) at −20°C for further extraction of mRNA and tissue repair gene dosage [24,25]. At the end of the 9th day, the removed tissue was used to make histological slides.

ELISA Assay.
For total protein extraction, the skin tissue (Approximately 250 mg) was sprayed using a stainless steel bowl cooled for a long time in liquid nitrogen which keeps frozen samples. e powder was resuspended in 1 mL of protein lysis buffer (NP40-NP40 10%; glycerol 10%; NaCl 135 mM; Tris 20 mM; PMSF 1 mM). e samples were homogenized on a seesaw shaker for 30 minutes on ice and centrifuged (1400 rpm/30 min/4°C). e supernatant was collected, and total protein content was determined as described by Bradford, 1976 (Bio Agency ® ) [26]. e cytokines TNF-α, IL-1β, and IL-6 were measured in the animal's cutaneous tissue on the 1st and 7th day after treatment by quantitative ELISA analysis, using a microtiter plate reader (photometry at 505 nm), and cytokine concentrations were calculated from standard curves and are expressed as picograms per milligram of total protein, following the manufacturer's protocol R&D Systems, Minneapolis, MN, USA, based assay range: TNF-α 10.9-700 pg/mL; IL-1β 12.5-800 pg/mL; IL-6 3.1 300 pg/mL.

Histology.
e tissue samples removed on the 9th day after lesion treatment were embedded in 10% formaldehyde buffered solution for 24 hours, mounted in paraffin blocks, cut in 5 µm sections, and stained with Hematoxylin and Eosin (HE) and Masson's Trichrome to evaluate the following parameters: inflammatory infiltrate; granulation tissue; angiogenesis; fibroblast migration; reepithelialization; Evidence-Based Complementary and Alternative Medicine and collagenization. Specimens were examined under a light microscope (100x and 400x) and scored accordingly: absent; discrete (up to 25% of the parameter); moderate (25-50%); and marked (50-100%) [25,27].

Statistical Analysis.
e results were expressed as mean ± standard error of the mean (SEM) using GraphPad Prism Software Inc, version 5.00 (GraphPad Software Inc., San Diego, CA, USA). Differences among groups were assessed by one-way analysis of variance (one-way ANOVA) used for multiple comparisons. A Newman-Keuls posttest was used to compare individual groups. Differences were considered statistically significant when p < 0.05.

Chemical Constituents of EHJ.
e total ion chromatogram from the hydroalcoholic extract of Jacaranda decurrens acquired by LC-ESI-MS (m/z: 100-1000 Da) and Compound 7, which produced a (M-H) − at m/z 301, was tentatively established to quercetin by comparing the mass spectrum data and chromatography with an authentic standard. Peak 8 was identified as luteolin, also the MS/MS spectrum obtained in negative ionization mode presented the expected fragmentation patterns for luteolin, and the precursor ion at m/z 285 (M-H) − is in agreement. In the other hand, kaempferol (peak 9) has the same molecular formula as luteolin, but the product ions formed in MS/MS spectrum in the positive ionization mode allowed distinction between them. en, luteolin had m/z 151 as the bp, whereas kaempferol had m/z 211 as the bp and other characteristic product ion at m/z 163. Also, during the MS/MS in the negative ionization mode, luteolin showed a characteristic ion at m/z 197.Compound 10, which produced a (M-H) − at m/z 487, was tentatively established to arjunolic acid by comparing the mass spectrum data and chromatography with an authentic standard.  Figure 2). e FtEHJ group showed a percentage of 80% contraction, higher than BFt (33%) and Fibrinase ® (50%) groups (Figure 3(b)). On the 9th day, the 5 (five) animals treated with FtEHJ had no crusts and the skin tissue was already restored (Figure 2), indicating a regression of 100% of the wound mean area, being a superior evolution when compared to BFt (65%) and Fibrinase ® (85%) control groups who completely closed the lesions on the 12th day ( Figures 2  and 3(b)).

Effect of FtEHJ on Cytokine Concentration (ELISA).
e proinflammatory cytokines TNF-α, IL-1β, and IL-6 dosed in the tissue after the 1st and 7th day after treatment were significantly reduced in FtEHJ when compared to the other groups (Figure 4). is reduction was also seen in IL-6, but only on the first day. Figure 5 shows the effects of FtEHJ on gene expression of the molecules involved in healing process on the 7th day after injury induction. Lesions treatment with FtEHJ and Fibrinase significantly increased the gene expression of TGF-β. Type I collagen gene expression increased after FtEHJ treatment. ere was no difference between the groups regarding the gene expression of type III collagen.

Histological Evaluation.
Microscopic analysis of tissues removed on the 9th and 12th day after treatment is described in Table 2 and shown in Figure 6.

Discussion
Several studies have shown that plant extracts from polar solvents contribute to better healing, mainly by reducing inflammation, stimulating the proliferation of tissue repair cells, and collagenization [1,3,24,[28][29][30]. In this work, we observed that topical formulation of Jacaranda decurrens Cham. (FtEHJ) promoted a wound healing evolution homogeneously, with controlled inflammatory process and in a shorter time compared to other groups. On the first day after lesion induction, FtEHJ     Evidence-Based Complementary and Alternative Medicine had promoted 18% wound regression and reduced the presence of important proinflammatory cytokines such as TNF-α, IL-1β, and IL-6. In the healing first days, these cytokines contributed to the signaling of phagocytes with the function of removing debris from the wound, but at the same time this inflammatory process can generate damage to the tissue. Modarresi et al. [1] found evidence that the topical administration of M. piperita ointment is able to reduce infiltration of immune cells into the wound region, and control inflammation of the tissue to promote downregulation of inflammatory cytokines and has provided acceptable efficiency in both pathological and molecular phases.
us, interventions capable of modulating the injury inflammatory phase can be an effective strategy for wound management [31].
e chemical characterization of plant showed that the extract of J. decurrens Cham. presents compounds of the class of flavonoids, tannins, and triterpenes, which have been reported with anti-inflammatory, antioxidant, and healing activities [32][33][34][35][36]. e inflammation modulation is important to reduce the damage resulting from proteases release by neutrophils, such as excessive degradation of extracellular matrix and proteins important for tissue repair, besides the free radicals production [37]. Among these proteases the matrix metalloproteinases-(MMP-) 2 and MMP-9, which together with their inhibitors, are important for maintenance of balance between extracellular matrix degradation and collagen remodeling, and when super expressed impair tissue repair [38]. e suppressor effect of quercetin [39] and quinic acid [40] (which are also present in EHJ) on these proteases has been demonstrated.
e FtEHJ modulating effect contributed to the transition process from the inflammatory phase to proliferative phase, favoring a greater wound contraction, as observed.
On the 7th day after lesion treatment, the FtEHJ already presented 80% wound retraction. is result may be associated with the lower amount of proinflammatory cytokines (TNF-α and IL-1β), characteristic of a successful proliferative phase. In contrast, IL-6 concentration was high in the group treated with FtEHJ. A study published by Zi-Qing Lin et al. [41] found that knockout mice for IL-6 showed delayed healing when compared to wild-type mice, indicating that this cytokine plays an important role in healing by leukocyte infiltration regulation, angiogenesis, and collagen accumulation. Gallucci et al. [42] observed that the delay in mice healing, after immunosuppression induction by glucocorticoids, was reversed when administered recombinant murine IL-6 (rMuIL-6). e results of this study were evidenced by the presence of epithelialization, granulation tissue formation, and wound closure. Such characteristics could be seen in the histological analysis ( Figure 6), corroborating with the author's findings. It was also shown that there was greater stimulation of TGF-β gene expression in both the FtEHJ and Fibrinase ® groups compared to the CTL group. e main component of Fibrinase ® ointment is fibrinolysin, a debriding enzyme that removes fibrin and nonviable cells, facilitating the migration of fibroblasts to scar formation and tissue remodeling [43]. e increase in TGF-β gene expression in animals of these groups may be related to the migration and differentiation of fibroblasts in lesions, since this growth factor acts by inhibiting the matrix proteins degradation, decreasing the synthesis of metalloproteinases and increasing the production of their inhibitors as well as inducing the differentiation of fibroblasts into myofibroblasts, favoring cell migration and wound closure [44]. Different flavonoids do not play the same role in TGF-β expression. Kaempferol has been reported to promote suppressive activity of this molecule in a lung cancer model [45]. However, quercetin acts positively on modulation of the TGF-β signaling pathway, favoring healing [46]. ese flavonoids are also present in propolis [47], which has healing and anti-inflammatory activity, as in other plants of Jacaranda genus [48,49]. ere was an increase in type I collagen gene expression in the FtEHJ group compared to the others, although type III collagen gene expression was not different between the groups. Type I collagen fiber is described in literature as more resistant to tensile strength because it has a denser characteristic than type III fiber. erefore, the more mature the scar tissue, the greater the type I collagen fibers deposit, since during remodeling there is a renewal of collagen that changes from type III to type I [31]. e greater gene expression of collagen I in the treated group with FtEHJ may indicate more stimulus to collagen fibers organization to be produced and deposited in bigger quantity, contributing to the remodeling phase [50].
Histological evaluation made with tissues on the 9th day after lesion treatment validates the accelerating healing effects of the extract, showing characteristics of inflammatory and proliferation phases in a more discreet way and with marked collagenization and reepithelialization, indicating that the FtEHJ group was in the remodeling phase while the other groups were in the proliferative phase. At the same time, the animals treated with FtEHJ showed slight angiogenesis in the scar tissue, while in the control animals there was still moderate formation of these vessels. Since this event is related to increase of cellular metabolism, characteristic of the proliferation phase, it is possible that the animals treated with FtEHJ were already experiencing a more mature phase of healing, in transition to remodeling phase [51]. Masson's trichrome staining revealed the characterization of denser collagen fibers, organized and in greater quantity in tissues treated with FtEHJ. Collagen high levels can lead to rapid healing by increasing the tensile strength of wounds and improving the collagen fibers stabilization [52]. It was also observed that the FTEHJ group no longer presented crusts in the wounds, criterion used in the indication of experimental cicatricial process completion. e other groups remained with the crust until the 12th day after injury. ese results evidenced the healing action of Jacaranda decurrens hydroalcoholic extract.
Considering the secondary metabolites detected in the leaf extract of this plant, the observed pharmacological effects can be attributed to the presence mainly of phenolic compounds, highlighting the flavonoids revealed in phytochemical screening and analysis by HPLC-DAD-ESI-MS and FIA-ESI-IT-MSn.
is activity is mainly related to modulating effect that these compounds have on inflammatory molecules, controlling inflammation, preventing microorganism's proliferation, and contributing to rapid 10 Evidence-Based Complementary and Alternative Medicine lesion retraction and evolution of other cicatrization stages [3,[53][54][55].

Conclusion
e results of this study reveal that the hydroalcoholic extract of Jacaranda decurrens Cham. has important phytochemical constituents of the classes of flavonoids, tannins, and triterpenes that may be involved in cutaneous wound healing.
us, FtEHJ proved to be effective in lesions treatment by modulating the action of proinflammatory cytokines, contributing to inflammatory control, stimulating the growth factor synthesis and collagen maturation essential in cicatricial process.

Data Availability
e data used to support the findings of this study are included within the supplementary information file(s).