CURATELLA AMERICANA PDF

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Based on ethnopharmacological studies, a lot of plants, as well as its compounds, have been investigated for the potential use as wound healing agents. In Brazil, Curatella americana is traditionally used by local people to treat wounds, ulcers and inflammations.

However, to the best of our knowledge, its traditional use in the treatment of wounds has not been validated by a scientific study. Here, some compounds, many of them flavonoids, were identified in the hydroethanolic extract from the leaves of C. Besides that, solutions containing different concentrations of HECA and a gel produced with this extract were evaluated for its antimicrobial, coagulant and wound healing activities on an excision mouse wound model as well as its acute dermal safety.

A total of thirteen compounds were identified in HECA, mainly quercetin, kaempferol and glucoside derivatives of both, besides catechin and epicatechin known as wound healing agents. The present study provides scientific evidence of, this extract HECA possess remarkable wound healing activity, thereby, supporting the traditional use. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Many variables influence wound healing, such as age, nutritional status, infections and, especially, the simultaneous occurrence of diabetes and coagulation problems, generating a higher incidence and prevalence of chronic wounds.

Non-healing wounds remain a public health problem resulting in social disorders in patients requiring increasingly effective treatment and consequent high financial costs [ 1 , 2 ]. The developing of strategies for taking advantage of simpler, cheaper and safer technologies, as well as resources and raw materials found in less-developed regions, makes the treatment of wounds more accessible to a large number of people.

In this context, the research of natural products in the development of compounds to improve the healing process has been intensified in the last years [ 3 ]. A lot of plants, based on ethnopharmacological studies and its identified compounds, have been investigated for the potential use as wound healing agents, highlighting plants rich in antioxidant compounds and with anti-inflammatory activity [ 4 — 6 ].

The species Curatella americana Linn. This species is a woody perennial shrub that is characteristic of Neotropical Savanna, occurring from southern Mexico to Bolivia and in almost all savanna region of Brazil [ 7 — 9 ]. In the Amazonian savanna, C. In Brazilian folk medicine this species is used for inflammation, arthritis, bronchitis, high blood pressure [ 12 ], the leaf decoction is used as an antiseptic and astringent [ 13 ]; bark infusion is used for the treatment of cold, wounds healing and ulcers [ 14 ].

In Costa Rica, the cooked leaves are used to mitigate skin eruptions, for healing wounds, and the water for purifying blood [ 15 , 16 ]. The leaves and bark extracts of C. Although, popularly used as wound healing agent, this activity was not validated.

The purpose of this study was to evaluate the wound healing potential of hydroethanolic extract and gel formulation from the leaves of C.

Adult Swiss albino mice 30—40 g of either sex was used in all experiments. The microbiologic control was made before the analyses to ensure the quality of the results. The tests for identification of colony forming unities CFU were performed for bacterial and fungi. The bacterial pathogen identification Escherichia coli , Salmonella sp , Pseudomonas aeruginosa , and Staphylococcus aureus was performed in the presence of CFU, according to Brazilian Pharmacopoeia [ 22 ].

The analysis was performed using the broth microdilution method in the Mueller—Hinton broth as described by the Clinical and Laboratory Standards Institute [ 24 ]. The 3- 4,5-dimethylthiazol-2yl -2,5-diphenytetrazolium bromide MTT was used as a bacterial viability indicator, because it is reduced by metabolically active cells to a colorful water-soluble formazan derivative [ 25 ].

The MIC was read as the lowest concentration of the extracts at which a change in color occurred. The MBC was defined as the lowest concentration of extracts or fractions that resulted in either no growth or fewer than three colonies The fibrinogen coagulation FC assay was performed according to the methodology previously published by Moura and coworkers [ 27 ].

Coagulation time was recorded in seconds, and the assays were performed on a Multichannel Coagulometer CloTimer. The assay was developed in triplicate and calculations based on a calibration curve obtained with gallic acid. The results were expressed as milligram of gallic acid equivalents GAE per g of dry extract [ 29 ].

The acquisition parameters and chromatographic analysis were described in details previously, with some adjustments, for example, at the time of gradient elution to 20 minutes instead of 10 [ 29 ]. Before the experiments, two types of formulations, aqueous solution and aqueous gel, with different concentrations 0. All ingredients of the gel base were mixed until the required consistency, and then the lyophilized HECA were incorporated gently to obtain a uniform cream gel.

Each g of the gel prepared in the present study contained 0. It was also prepared a gel without extract for control use, CV. The samples of gel with HECA and the gel control were submitted to the physical preliminary and accelerate stability study described in the Cosmetic Products Stability Guide [ 31 ]. In the assays of preliminary stability were evaluated under centrifugations and the ice-thaw cycle for changes as volume loss, homogeneity, creaming and phase separation.

After the end of the stability test, the microbiological quality of the samples was carried out to investigate the presence of UFC above the recommended limits and pathogen bacterial as E.

A total of six animals 3 female and 3 male mice were used. The shaved region was covered with sterile gauze attached to the ends with tape to prevent the animal from withdrawing or ingesting the product for 24 h. After the exposure period, the test substance was removed, and the clinical signs and mortality were monitored at 30 minutes, 2, 4, 6 and 24 hours and once a day until day Individual body weights were measured just before administration day 0 , and on day 7 and 14 besides the volume of wather and food was measured every day.

The animals were randomly divided into six groups of ten animals each a total of 60 mice. One cutaneous full thickness, completely transdermal circular wound of 6 mm diameter was made on the pre-shaved, dorsal surface of the animal with the help of a biopsy punch Colgran M. Animals were allowed to recover and were housed individually in polypropylene cages containing autoclaved paper cuttings.

They received food and water ad libitum. The calculation of the contraction degree was expressed in percentage using the follow equations [ 33 ]. On the 7th and 14th day of the treatment, five animals from each group were euthanized in a CO 2 chamber for the collection of material for the morphometric analysis of the skin lesions. Next, the samples were processed for inclusion in paraffin by routine methods and cut using a microtome set to 2. The material obtained was placed on slides and coated with haematoxylin and eosin.

The microbiologic analysis of HECA was performed to ensure the safety of the animals during all experiments. The HECA was negative for bacteria and fungi contamination demonstrating the quality of the operational procedures in the extraction. Antibacterial screening revealed that the C. The prothrombin time test measures how long blood takes to clot and the fibrin coagulation test evaluated the interference of the extract on fibrin clot formation.

The extract did not exert any effect in the tested concentration over the fibrinogen neither in the prothrombin time. Nine compounds were identified at positive mode and four at negative mode. The gel was chosen because it has been used the dermatological basis for all skin types besides being a polymer of low cost and easy manipulation, it presents smooth spreading, low occlusion and can be used for incorporation of several active ingredients, including antimicrobial and anti-inflammatory agents [ 34 , 35 ].

The tests were conducted in triplicate and the samples stored under conditions that accelerate possible changes that can occur during the expiration date and show indications as to their stability.

In the preliminary stability, any changes in volume, homogeneity, creaming and phase separation were observed. The pH, organoleptic and microbiologic changes observed in the accelerated stability are shown in Table 3. In the analyzed formulations there was no growth of microorganisms above the recommended and no presence of pathogenic bacteria. In this test, no erythema, eschar, edema, symptoms of toxicity, mortality or any other reactions were observed in either animals and we classified the C.

There were no differences in the wound contraction between all formulations with the hydroalcoholic extract of C. The group C. Histopathological analysis of the seventh day showed that all groups treated with C. In the CaG 0. In the CV group, the cicatricial process was slowed by the presence of extensive seroma with neutrophil infiltration despite the presence of bulky fibroblasts and angiogenesis, see Fig 2.

Cr: crust with piocytes, FB: fibroblasts, N: neovasal, Sr: seroma, Ep: epithelization, In: Inflammatory cells, Fv: fibrovascular proliferation, Black arrow indicating granular tissue with initial remodeling.

On the 14th day, it was possible to observe in all groups treated with HECA complete epithelization and tissue remodelling at an advanced stage with denser collagen fibers like the FIB group.

While in the CV group, despite re-epithelialization and early stages of remodeling, impairment was observed in the cicatricial process due to the presence of microabscesses Fig 3. Ep: epithelization, Mc: Microabcesses, IRm: initial remodelling, Rm: advanced remodelling, Black arrow indicating collagenization. The wound healing is a complex physiologic process that involves blood clothing, inflammatory response, re-epithelization, and angiogenesis.

These processes are divided into three phases: inflammatory in 0—3 days, cellular proliferation between 2—14 days and remodelling initiated in 14 days and may persists months [ 36 , 37 ].

One of the leading causes of delayed healing is the persistence of inflammation, mainly for reactive oxygen species ROS , infectious processes or an inadequate angiogenic response [ 38 ]. Based on this, an excellent healing agent should combine the antimicrobial activity, antioxidant activity and mainly having enhanced mitogenic activity to increase cell proliferation, and consequently angiogenesis making possible the faster contraction.

The time that the blood takes to clot is clinically relevant for the homeostasis in the skin injury [ 39 ]. Although any concentration test of HECA presented coagulant effects, this did not inhibit the normal process of coagulation.

After clot formation, the cellular response is established 24 hours after injury, and neutrophils initiate debridement of the devitalized tissue and phagocytosis of infectious agents through the release of lysosomal enzymes and reactive oxygen species ROS. In 48 hours the macrophages migrate to the site of the lesion contributing to the end of the cleaning and disinfection initiated by neutrophils and producing ROS [ 36 , 37 ].

Inappropriate scavenging of these ROS generally results in a delay of wound healing, individuals with chronic leg ulcers and lesions due to ischemia present excessive production of oxygen radicals with significant xanthine oxidase activity [ 40 ]. In this context, plant extracts with healing activities have their effect justified by the presence of metabolites capable of capturing oxygen radicals [ 3 , 41 , 42 ].

The mass spectrum of HECA revealed the presence of flavonoids, terpenoids, phenolic derivatives confirming studies performed by El-Azizi et al. Other wise, in this study was possible identify catechin and epicatechin by comparison with injection of authentic standards. These compounds have been previously found in the bark [ 18 , 20 ], besides kaempferol and apigenin- 7-O- glucoside that were never identified in this specie before.

Among the identified phenolics, flavan- 3- ols as catechins and epicatechin has shown significant antioxidant activity including the potential inhibitory effect on xanthine oxidase [ 46 ]. Besides, the pretreatment of fibroblasts cells with catechin accelerated the proliferative response even after induction of oxidative stress [ 47 ]. Hiruma-Lima and coworkers, identified in C.

The critical role of PGE2 in the healing process and fibroblast proliferation is confirmed by the fact that immediately after the injury the cyclooxygenase 2, COX 2 , the primary enzyme producing PGE2, is induced in the epidermis and the assays in which the use of specific inhibitors of COX-2 led to delays in healing [ 50 ].

The intake of catechins from the green tea for 12 weeks induced the incorporation of catechin metabolites into human skin associated with abrogated UVR-induced pro-inflammatory mediators hydroxyeicosatetraenoic acid HETE but did not reduce PEG2 [ 51 ].

Plant extracts rich phenolics compounds including catechin and epicatechin stimulated the growth of ephitelial and fibroblast cell as well as enhance the vessels formation in vitro [ 52 , 53 ], catechin was able to increase the viability of human umbilical vein endothelial cells HUVEC , reducing apoptosis, but it contoled the angiogenesis in excess of VEGF [ 54 ].

The compounds kaempferol and apigenin- 7- O- glucoside have never been identified before in this specie. Regarding quercetin and its derivatives, anti-oxidant and anti-inflammatory activities have been attributed in the same way the kaempferol, apigenin and its derivatives [ 55 , 56 ].

While apigenin and kaempferol strongly inhibit COX-2 induction by inhibiting factor-kB nuclear transcription factor NF-kB via kB-inhibitor, on the other hand, the derivatives of these compounds as well as quercetin and catechins appear to slightly inhibit COX- 2 [ 55 ], this is important to modulate the inflammation. Topical application of quercetin appears to be effective in treating wounds by enhancing granular tissue formation, enhancing fibroblast proliferation, and collagen production with decreased inflammatory cytokines, including in diabetic animals [ 57 , 58 ].

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