The flowers of
The anti-inflammatory activity of
Most of the literatures that report the medicinal activity of
Dried and pulverized flowers of
To obtain the ethanolic extract (CEE), the pulverized flowers were extracted with 96° GL PA ethanol by cold maceration and concentrated by rotary evaporator at 40°C. To obtain of the hexanic and dichloromethane fractions, the CEE was soluble into a MeOH/H2O (7 : 1) mix followed by an extraction with hexane and afterwards with dichloromethane. The resulting extracts which were hexanic (HCF) and dichloromethane (DCF) were concentrated by rotary evaporator at 40°C.
As the ethanolic extract, the hexanic and dichloromethane fractions were insoluble in distilled water, solutions of 1% of the extract and fractions previously diluted in 70% ethanol were prepared and then used to test the angiogenic activity of the CAM. To test the angiogenic activity of the ethanolic extract on cutaneous wounds in rats, an aqueous solution of 1% of the extract was daily prepared.
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75 fertilized eggs from Cobb chickens, two days old, were used and obtained from matrixes with age varying from 34 to 35 weeks old. We followed the ethical principles in animal experimentation recommended by the National Council for Control of Animal for Experiments (CONCEA).
The experimental protocol of the animals used in this study was evaluated and approved by the Ethics in Research Committee of the Federal University of Goias (protocol number 019/2007).
The CAM was prepared aiming the angiogenic activity evaluation according to an adapted methodology [
According to previously established protocols [
After the euthanasia of the embryos, through medullar section of the atlantoocciptal region, fragments of the CAM from each egg were collected to morphometric analysis and quantification of the blood vessels.
To the morphometric evaluation of the CAM, a fresh fragment was collected from 10 eggs from each group and distended on a glass slide and microscopically analyzed.
The microscopical analysis was performed with a digital image analysis system. The percentage of the vascular area per field was calculated through the Image J 1.3.1 (NIH, USA) software. 30 random fields were photographed and analyzed per sample, and this number was determined by accumulated mean [
The membranes were fixed in tamponated formalin, processed and blocked into paraffin, and then sectioned into 5 micrometer sections and stained with hematoxylin and eosin (HE). 20 randomly chosen fields were photographed, number determined by accumulated mean. The inflammatory cells found in the CAM were classified in a qualitative form into absent (0/3), discrete (1/3), moderate (2/3), and accentuated (3/3).
The counting of the blood vessels in mesoderm from the CAM was performed through planimetry by counting of the points by using the GIM; P 2.4.3 software. A square reticulum composed by 25 points was overlapped on top of the histologic image [
The animals were weighted and randomly divided in two groups (
Control (C), animals were treated with distilled water. C1 was evaluated in the 4th PO day, C2 in the 7th PO day, and C3 in the 14th PO day.
CEE, animals were treated with aqueous solution of the ethanolic extract at 1%. CEE1 evaluated in the 4th PO day, CEE2 in the 7th PO day and CEE3 in the 14th PO day.
To the wound induction, a circular methalic punch of 1 cm of diameter was used on the dorsocervical region of each animal. The anesthetics consisted of the association between ketamin (70 mg/Kg, IM) and xylazine (10 mg/Kg, IM). Right after the surgery and daily afterwards on the same hour, 100
Each animal was daily examined as to their general aspect, and the macroscopic evaluation of the wound was performed. The animals were euthanized in a CO2 chamber at 4, 7, and 14 PO days accordingly to previous protocols by Lopes et al. [
The morphometric analysis of the wounds was performed through the photographed images of the wounds at days zero, 4, 7, and 14 PO aiming of the determination of the contraction of the wound area [
The histopathologic analysis was performed after the fragments of the wounds were fixed with 10% buffered formalin, processed, and blocked with paraffin, and then sectioned into 5 micrometer sections and stained with hematoxylin and eosin (HE). The sections were also stained with Picrossirius to the collagen quantification under polarized light [
The histopathologic analysis at 4, 7, and 14 PO days determined the healing phases of the wound through histologic variables such as the presence of fibrin, hemorrhage, hyperemia, inflammatory infiltration, reepithelialization, and epithelial hyperplasia [
Sections were incubated with bovine serum albumin (BSA), the blockage of endogenous peroxidase was performed, and the sections were incubated with the vascular endothelial growth factor (VEGF) primary antibody (147) (Santa Cruz Biotechnology-507), 1 : 500 dilution in a wet chamber, overnight at 4°C.
After this step, the streptavidin-biotin-peroxidase complex (kit LSAB-Dako K0690) was instilled over the sections for 20 minutes each reaction in a wet chamber at room temperature. The reaction was revealed with diaminobenzidine (DAB) solution, for 1 minute. A PBS solution was used for washing between the steps. The sections were counterstained with Mayer’s hematoxylin, for 30 seconds, and the slides were mounted with synthetic resin (Sigma Aldrich, USA) and histological coverslips.
The counting of the blood vessels was performed through planimetry, and the intensity of the VEGF expression into the endothelial cells was evaluated.
To determine the minimum inhibitory concentration (MIC), the cultures of
The MIC was determined through the plaque assay method when 1000 mg of the CEE, 500 mg of the HCF, and 350 mg of the DHC were incubated in essay tubes with 1.0 mL of DMSO.
The results were submitted to the statistical analysis through the GraphPad InStat programme (Version 3.05 for Windows). From the Kolmogorov-Smirnov normality test, the morphometric data from the CAM were evaluated through the Kruskal-Wallis test followed by the Dunn posttest. The counting of the blood vessels was analyzed through ANOVA and the Tukey test. The histopatologic and immunohistochemical variables were analyzed through the Mann-Whitney test. The significance level was of
The pulverized flowers of
The total content of the ashes was of 8.47%. The thin layer chromatography evidenced the presence of rutin (Rf-0.47), flavonoids (Rf-0.29), and aglycone flavonoids (Rf-0.92). The contents of total flavonoids from the pulverized flowers were of 0.77% and in the CEE of 1.29%. WHO monographs [
All data obtained from the pharmacognostic analysis are in accordance to what is described in the literature [
The evaluation of the angiogenic activity in the CAM showed through the morphometric analysis an increase in the vascular area in the positive control, ethanolic extract, dichloromethane fraction, and hexane fraction groups when compared to the solvent control 1 (Table
Morphometry (median, minimum/maximum) of the blood vessels from the fresh chorioallantoic membrane from fertilized chicken eggs.
Treatment | Marked area of the CAM |
---|---|
CEE | 10.47 (1.43/57.49)* |
HCF | 9.33 (1.61/71.22)* |
DCF | 9.11 (2.26/54.35)* |
PC | 8.11 (3.56/20.64)* |
SC1 | 5.10 (0.61/49.83) |
SC2 | 5.78 (1.20/40.37) |
CAM: chorioallantoic membrane, CEE: ethanolic extract of flowers from
The quantification of the blood vessels performed through planimetry of the CAM stained with H&E and treated with positive control, ethanolic extract, dichloromethane fraction, and hexane fraction evidenced an increase in the number of blood vessels when compared to the solvent control 1 group (Table
Influence of the
Treatment | Number of blood vessels |
---|---|
CEE | 1 (0/4)* |
HCF | 1 (0/4)* |
DCF | 2 (0/13)* |
PC | 2 (0/13)* |
SC1 | 1 (0/3) |
SC2 | 1 (0/3) |
CEE: ethanolic extract of flowers from
When substances are administered on the surface of the CAM, they may induce nonspecific inflammatory reactions leading to a secondary vessel proliferative response [
In the evaluation of the wound healing activity of
The macroscopic evaluation of the wounds did not show purulent exudate in any of the wounded animals, but it was possible to observe a serous exudation in the animals from the CEE group up until the 4th PO day and in the control group up until the 7th PO day. The crusts started to form from the 3rd day, and, in the CEE group, they presented thinner and wetter when compared to the control group which were thicker and drier. At the 7th PO day, the crusts began to detach showing signs of epithelialization of the wounds, and, in the 14th PO day, the complete healing was verified on both groups.
The microscopic evaluation of the CEE group at 4 and 7 PO days showed a significant decrease as to the presence of fibrin (median = 1.0 and 2.0, resp.)and hyperemia (median = 1.0 and 1.5, resp.) when compared to the control group at the same PO days (median = 3.0 and 2.5, resp.) (Figure
Photomicrograph of cutaneous wounds in rats at 4 (a1, a2) and 7 (b1, b2) PO days highlighting the presence of fibrin (Fb). (a1) and (b1) refer to the control group, treated with distilled water. (a2) and (b2) refer to CEE group. HE staining.
Previous studies evaluated and evidenced the anti-inflammatory activity of
It was possible to observe a significant increase in the collagen amount in the CEE group at 4 and 7 PO days (median = 8.27 and 6.33, resp.) when compared to the control group (median = 5.33 and 4.31, resp.) which indicates fibroplasia (Figure
Photomicrograph of the cutaneous wound in rats (
There are no reports found in the literature on the collagen mensuration through Picrossirius staining of cutaneous wounds treated with
The immunohistochemical evaluation showed an increase in the number of blood vessels in the dermis of the rats treated with CEE as it had a positive marking for VEGF (Figure
Photomicrograph from the dermis of rats at 7th PO days (
The immunohistochemistry technique is a viable tool to angiogenesis evaluation through VEGF marking in this experimental model. Our data indicated that the CEE angiogenic property is not directly related to the increase of the intensity of expression of this marker. Other pro-aniogenic factors such as fibroblast growth factor (FGF), angiogenic cytokines as interleukin 8 (IL-8), tumor necrosis factor-alfa (TNF-
The antibacterial activity of the CEE and the hexanic fraction was observed against Gram-positive bacteria, and this effect was not observed on the dichloromethane fraction. The lowest concentrations of the extracts that inhibit microorganism’s growth were CEE, MIC of 0.39 mg/mL against
In the case of wound contaminations, there is the formation of a great quantity of exudate and toxin productions which may retard the healing process [
This study was performed with the collaboration of the Animal Pathology and Preventive Veterinary Medicine Sectors of the Veterinary School of the Federal University of Goias; also of the Natural Products Research Laboratory from the Pharmacy Faculty and the Pathology Sector of the Tropical Pathology and Public Health Institute from the Federal University of Goias; also of the company Empresa Perdigão S/A (Rio Verde-GO). The authors would like to thank CAPES (Coordenação de aperfeiçoamento de pessoal de nível superior) for the financial support.