Plants are important sources of bioactive compounds, and are a major potential source of new therapeutic agents against several diseases, including those of interest to public health, in particular neglected diseases such as leishmaniasis.
The bioactive compounds produced by plant cells may vary depending on soil, climate and the season that the plant, fruit or leaves were collected. Soil and climate conditions, along with the plant’s need for adaptation and defense, bring about this change in chemical constituents . Phytochemical analysis of A. chica revealed the presence of flavonoids, tannins, anthocyanidins, chalcones and phenolic compounds, as previously described for this species [26, 27].
The first chemical study with the leaves of A. chica was performed by Chapman et al. and reported the isolation of the substance 3-deoxyanthocyanidin, more specifically 1,6,7-dihydroxy-5,4′-dimethoxy-flavylium (carajurin) and 6,7,4′-trihydroxy-5′, methoxy-flavyluim (carajurone), responsible for the reddish color of the extract . In other studies, other compounds, such as flavone acacetin, oleanolic triterpene acid and two deoxiantocyanidins (6, 7, 3′-4′-tetrahydroxy-5-methoxy-flavylium and 6, 7, 3′-trihydroxy-5, 4′ dimethoxy-flavyliume) were isolated [29-31].
Anthocyanins are plant pigments belonging to the family of flavonoids, included in the group of phenolic compounds [32, 33]. More than 50 new anthocyanins have been isolated from the flowers, fruits, seeds and leaves of plants. They play a pivotal role in flower pollination, seed dispersal by insects and defense against predatory insects .
Pharmacological studies with anthocyanins reveal their high antioxidant effect  on metabolic diseases . The antioxidant, cytotoxic, antimicrobial and diuretic potential of the ethanol extract and fractions of A. chica was evaluated by Amaral et al. that confirmed this antioxidant and diuretic action, and the absence of an antimicrobial and cytotoxic response . The therapeutic activity of the phenolic compounds is mainly attributed to their antioxidant capacity, the chelation of metal ions, modulation of gene expression and interaction with cell signaling pathways .
Phenolic composition and antioxidant activity of the leaves of A. chica was evaluated by Siraichi et al. by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and verified the presence of two important flavonoids, apigenin and scutellarein, which are most responsible for the significant antioxidant action .
In pharmacological tests, Alves et al. determined the chemical potential of A. chica dye, which is widely used by indigenous tribes, and verified the presence of alkaloids, anthocyanidins, anthocyanins, anthraquinone, steroids, triterpenoids, phenols, flavanonois, flavanols, flavanones, saponins and catechin tannin . This study described some compounds undetected in the present study, differing in the presence of anthraquinones, steroids, triterpenoids and saponins. This fact is probably due to the climate differences between the two places where the plant was collected, the states of Maranhão and Pará, as well as the method by which the extract was obtained. Both were obtained using ethanol as a solvent; however, in the present study this was submitted to 72 h rotoevaporation at 40 °C, while in the aforementioned study the extract was macerated for 10 days in a capped percolator.
In vitro pharmacological studies have demonstrated the antineoplasic and antioxidant potential of A. chica, and this action was directly related to the high content of flavonoids, especially anthocyanins . These and other benefits made the search for new sources of anthocyanins essential .
The secondary metabolites in A. chica, such as flavonoids, the antimicrobial, antiviral, anti-inflammatory, antineoplastic, antifungal and anti-protozoan action of which has already been reported in literature [17, 38–40], encourages many researchers to study this plant.
In the present study, the leishmanicidal potential of the crude extract and fractions of A. chica, which displayed satisfactory results at concentrations between 60 and 155.9 μg/ml, depending on the type of fraction. The chemical constituents of the plant were more selective for the protozoan than the host cell, with a cytotoxicity effect at concentrations higher than those with leishmanicidal action.
Several studies have aimed to identify and characterize crude extracts, fractions and essential oils [41-48], as well as the isolated compounds of the plant [49, 50], with leishmanicidal activity. The screening of these natural compounds is performed with cultures of Leishmania spp., as it is easy to maintain and produce .
According to the World Health Organization, a candidate drug must undergo in vitro cytotoxicity and antiparasitic activity testing. After this initial screening, the results are evaluated to define the continuity of studies and the possibility of in vivo assays .
Studies performed by Rodrigues et al.  investigated the leishmanicidal effect of five fractions obtained from the crude hexane extract of A. chica, on L. amazonensis and L. infantum promastigote forms, by determining the minimum inhibitory concentration (MIC), which was found to be 37.2 and 18.6 μg/ml, respectively. This result is related to one of the fractions used, the main chemical components of which were linoleic acid methyl ester (25.38 %), n-hexadecanoic acid (19.61 %), octadecanoic acid (14.10 %) and gamma-sitosterol (12.85 %). The study also evaluated ultrastructural alterations of the parasite under the influence of this fraction, and observed mitochondrial swelling, disruption of mitochondrial membrane, presence of vesicles in the cytoplasm, mitochondria and flagellar pocket and modifications in the Golgi complex.
The significant increase in studies of plant compounds with leishmanicidal potential is due to of the major importance of this disease, of which there are large numbers of new cases annually. According to the World Health Organization 1.3 million new cases are recorded per year, being 300,000 cases of visceral leishmaniasis and 1 million cutaneous leishmaniasis .
Leishmaniasis are characterized as neglected diseases, as they are associated with low social indicators and are more prevalent in developing countries, whose resources in the areas of control, treatment and research are insufficient .
Additionally, plant extracts or plant compounds can represent a valuable source of new medicinal agents and alternative, effective and less toxic treatments, compared to conventional drugs (pentavalent antimoniates, amphotericin B, pentamidine), leading to a search for natural products with a potential leishmanicidal action. Furthermore, the benefits obtained from the search for natural products have encouraged interest in valuable synthetic compounds .
The crude extract from the leaves of A. chica had a cytotoxic effect at a concentration of 189.9 μg/mL. Maliofete et al. , also testing the ethanol extract from the leaves of A. chica, observed low oral toxicity, absence of cytotoxicity and antibacterial activity against Helicobacter pylori. In vivo experimental studies did not show any clinical or histopathological signs of extract toxicity in the pleura , open or sutured wounds or burns . Studies by Barbosa et al., assessing the antifungal, antimicrobial and antiprotozoal potential of the ethanolic extract and fractions of A. chica, observed growth inhibition of Trichophyton mentagrophytes and a significant trypanocidal effect against Trypanosoma cruzi, as well as the absence of relevant acute toxicity, even at the highest dose tested (1000 mg/kg) .
Several factors must be taken into consideration when evaluating the toxic effects of a plant extract, such as seasonal and environmental factors, the genotypic variations of the species, the part of the plant used to produce the extract, the time of collection and the age of the plant .
In traditional medicine in the Amazon region, A. chica is widely used as an anti-inflammatory, healing and anti-anemic medicine, and is prepared from the leaves as a tea, for oral or vaginal administration [16, 17, 56]. However, despite its wide popular use, several studies are still necessary to evaluate the medical potential of this plant.
In the present study, the healing potential of the crude extract of the leaves of A. chica in skin lesions was evaluated. A satisfactory response was initially observed, with angiogenesis and collagen deposition, but over the 21-day observation period it was found that the extract did not accelerate healing, presenting a similar profile to the control group.
Several local and systemic factors can cause variations in the healing process, leading to a delay or even prolongation of healing. Among these are the local of the lesion, existence of infection, surgical technique, tissue ischemia and bandages, as well as malnutrition and/or deficiency of trace minerals and vitamins [57, 58].
As cited above the importance of bandages in the healing process should be noted. Several studies have demonstrated that there the healing process is more successful when bandages are placed on the location of the wound after the application of the test product [16, 59] to protect the injury area. This step, which was not performed in the present study, may have contributed to the retardation of healing. The application of a suitable compress enables an increase of the natural debridement and simplifies healing, as it keeps the exudates rich on mediators on the wound surface .
Wound healing is a complex and dynamic process of the replacement of a devitalized tissue by a new tissue, which occurs through a series of biochemical cascade events; usually divided into three phases: the inflammatory, proliferative, and remodeling phases . Wound healing treatment has improved considerably over time, notably in order to achieve the best scarring results in the shortest possible time.
In the present study, a strong inflammatory response was observed at the beginning of the healing process, with an increased lesion area until the 3rd day after surgical incision and the presence of inflammatory cells in histological evaluation. The inflammatory phase is characterized by increased vascular permeability and immune cells chemotaxis to the site of injury, due to the release of mediators (histamine, serotonin, bradykinin, prostaglandins, thromboxanes) .
The most pronounced reduction period in the wound area was between the third and the 10th day, with retraction of the wound area, representing the proliferative phase. During this phase fibroplasia, granulation tissue formation, and removal of cellular debris were observed, with the formation of a temporary repair tissue during inflammation, and development of tissue substitutes .
In the healing process, fibroplasia occurs together with neovascularization . As the wound healing process advances, fibroblasts undergo phenotypic changes, with an abundance of rough endoplasmic reticulum, due to intense protein synthesis; and subsequent transformation into myofibroblasts, which help in the retraction of the lesion [64, 65].
Studies performed by Jorge et al. , when evaluating the healing, anti-inflammatory, antiulcerogenic and antioxidant effect of crude methanolic extract of A. chica, found that there was no reduction of paw edema induced by carrageenan or ear edema induced by croton oil in rats. However, in vitro fibroblast proliferation induction, stimulation of collagen synthesis in vivo and in vitro, anti-ulcer activity and moderate antioxidant capacity were observed.
After 10 days, it was verified that 96 % of wounds in the experimental group had healed, with a prominent collagen deposition, while in the control group, treated with saline solution, only 36 % of the wounds had healed, with a mild collagen deposition .
The topic healing effect of A. chica extract in the tendons of Wistar rats was investigated by Aro et al., that found better collagen organization in the treated groups than in the control group, as well as an increase in sulfate, on the 14th day treatment .
Compounds exhibiting antioxidant activity can indicate good therapeutic agents in the healing process, according to Houghton et al. . Non-enzymatic antioxidant activity is promoted by flavonoids, carotenoids, ascorbic acid, glutathione, and vitamin E, among others [58, 66, 67]. Therefore, the healing activity of A.chica is attributed to the antioxidant activity of anthocyanins and to fibroblast proliferation stimuli and consequent collagen synthesis [16, 68].
Fibroblasts are connective tissue cells, essential for dermis formation, due to collagen production, and responsible for structural firmness. After tissue injury, fibroblasts near the lesion site proliferate, migrate to the wound and produce a large amount of matrix material rich in collagen (type I and III), which helps to isolate and repair the injured tissue .
The proliferation of fibroblasts is directly related to the presence of macrophages in the healing process, as these release growth factors and cytokines essential for the maturation of the inflammatory phase and the beginning of the healing. Among the growth factors responsible for the proliferation, insulin-like growth factor 1 (IGF-I) and transforming growth factor beta (TGF-β) play an important role. Studies have demonstrated that IGF-I increases the procollagen chain expression in cultured dermal fibroblasts .
In the present study, on day 14 the control and experimental groups presented a smaller healing area than on day 07. At the end of the experiment, after 21 days, both groups were in the final stages of the healing process, in which the remodeling phase predominates, characterized by the reduction of inflammatory cells  and blood vessels at the lesion site , being the stage in which the scar acquires maximum resistance .
Several plants are used in popular medicine for the treatment of skin disorders. A number of these, such as Aloe vera (“babosa”), Schinus terebenthifolius (“aroeira”), Stryphnodendrom barbatiman (“barbatimão”), Calendula officinalis (“calendula”) and Triticum vulgare (“trigo”) are especially worthy of note due to the number of Brazilian studies involving the phytopharmaceutical use of these species [71–74].