Jatropha gossypiifolia L . CYTOTOXIC , ANTIMICROBIAL AND HEALING ACTIVITY OF THE Jatropha gossypiifolia L EXTRACT

Objective: to investigate the cytotoxic, antimicrobial and cicatrizant potential of extracts of leaves, branches and stem of J. gossypiifolia L. Method: quantitative, experimental study. The extracts were obtained by maceration in ethanol, concentrated in a rotary evaporator and dried in a vacuum desiccator. In the analysis, phytochemical prospecting; cytotoxicity; microdilution in broth and Scratch assay tests were performed. Among the detected metabolites, were: tannins, steroids, flavonoids, flavones, xanthones. Results: the stem extract presented cell viability above 80%. The leaves were moderately cytotoxic and the branches showed no cell viability. The extracts inhibited the growth of S. aureus, S. epidermidis and P. aeruginosa at different concentrations. Scratch assay showed that the methanolic fraction of the leaves allowed the cellular migration in 45% more than the control. Conclusion: studies with this plant species should be continued for isolation of the active principle, aiming at the production of a wound healing phytotherapic. Descriptors: Healing; Nursing; Citotoxicity; Medicinal Plants. RESUMEN Objetivo: investigar el potencial citotóxico, antimicrobiano y cicatrizante de los extractos de las hojas, ramas y el tallo de J. gossypiifolia L. Método: estudio cuantitativo, experimental. Los extractos fueron obtenidos por maceración en etanol, concentrados en evaporador rotatorio y seco en desecador al vacuo. En el análisis, se realizaron pruebas de prospección fitoquímica; citotoxicidad; microdilución en caldo y Scratch asay. Entre los metabolitos detectados, estuvieron: taninos, esteroides, flavonoides, flavonas, xantonas. Resultados: el extracto del tallo presentó viabilidad celular por encima del 80%. Las hojas fueron moderadamente citotóxicas y las ramas presentaron ausencia de viabilidad celular. Los extractos inhibieron el crecimiento de S. aureus, S. epidermidis y P. aeruginosa en diferentes concentraciones. El Scratch assay evidenció que la fracción metanólica de las hojas propició la migración celular en un 45% más que el control. Conclusión: estudios con esta especie vegetal deben ser continuados para aislamiento del principio activo, visando la producción de un fitoterápico cicatrizante de heridas. Descriptores: La Curación; Enfermería; Citotoxicidad; Plantas Medicinales. Enfermeiro do HUPAA-AL, Mestrando, Programa de Pós-Graduação em Enfermagem, Universidade Federal de Alagoas/PPGEnf/UFAL. Maceió (AL), Brasil. E-mail: enfermagemheha@gmail.com; ORCID iD: : https://orcid.org/0000-0002-572o-6638; Enfermeira, Especialista em Docência e Gestão do Ensino Superior. Mestranda, Programa de Pós-Graduação em Enfermagem, Universidade Federal de Alagoas. Maceió (AL), Brasil. E-mail: raquelloppes@gmail.com; ORCID iD: http://orcid.org/0000-0002-2061-7038; Enfermeiro, Mestrando pelo Programa de Pós-Graduação em Enfermagem, Universidade Federal de Alagoas. Maceió (AL), Brasil. E-mail: jefer_caetano@hotmail.com; ORCID iD: https://orcid.org/0000-0003-2556-4817; Enfermeiro, Mestrando pelo Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Alagoas. Maceió (AL), Brasil. E-mail: wanderley89@live.com; ORCID iD: https://orcid.org/0000-0001-9813-8857; Enfermeira, Professora Doutora em Biotecnologia, Universidade Federal de Alagoas/UFAL. Maceió (AL), Brasil. E-mail: salesregina@hotmail.com; ORCID iD: http://orcid.org/0000-0002-8636-5152; Enfermeira, Professora Doutora em Ciências, Universidade Federal de Alagoas/UFAL. Maceió (AL), Brasil. E-mail: lysetebastos@gmail.com ORCID iD: https://orcid.org/0000-0003-1752-7645 ARTIGO ORIGINAL Silva PSG da, Lopes RF, Silva JC da et al. Atividade citotóxica, antimicrobiana e cicatrizante...

Throughout the ages, medicinal plants have been used to treat numerous diseases and also to heal wounds.This knowledge has passed from generation to generation, through empirical knowledge and, has now, increased the number of publications with the purpose of scientifically proving this use, since they represent an important source of metabolites that act as inhibitors of various biological activities through the action of their molecules bioactive. 1e indiscriminate and inadequate use of antibiotics has contributed to the emergence of a microorganism resistant to several drugs, contributing to an increase in the number of colonized and/or infected wounds, thereby, delaying the healing process.According to data from the World Health Organization, 25% of the world's deaths are due to infectious diseases. 2Wound care is part of the Nursing care attributions, with a large number of studies published in this area, demonstrating the important role and responsibility of nurses in this process and in the search for new ways of care, attentive to the innovations of care in this area, to improve the quality of care. 3 this way, the use of therapies, other than conventional therapies, such as the use of herbal medicines, extracts or, even medicinal plants in natura, for wound healing, has been increased, in recent years, by the search for active principles isolated from plants, that have an effective role in the healing process. 4e species chosen, Jatropha gossypiifolia L., is popularly known as a purple top, teutonpotato, purgative herb, jalapon, mamoninha, teuton-root, purple peon and purple pinion.Several parts of the plant have been used, in folk medicine for the treatment of peptic ulcers, diabetes, neoplasias, diarrhea and, as a healer and diuretic.It is notable for its analgesic and anti-inflammatory potential, as well as being used for the treatment of eczema, abscesses, wound healing, dysentery, leprosy, arthritis, otitis, alopecia, venereal diseases, stomach pains, obstructions of the abdominal tract, rheumatism and bite of venomous animals [5][6] , becoming pertinent in the practice of folk medicine based on the specific knowledge of this plant.
It is a cut of a basic, experimental research that sought to generate new knowledge for the advancement of science.This type of study forms the foundation for clinical research and is recognized as a fundamental tool for the development of new therapies. 7is study aimed to investigate the cytotoxic, antimicrobial and healing potential of leaf, branch and stem extracts of J. gossypiifolia L.

 Plant material
The plant material was collected in the Cidade Universitária -Maceió-Alagoas complex, whose coordinates 9° 32'47.3"S 35° 47'07.6"W, in the month of January 2016, and was identified at the Institute of the Environment (IMA) of State of Alagoas, the exsicata being deposited with the MAC registry 241.

 Obtaining Ethanolic Extract
The plant material of the leaves, branches and stem, of the plant species J. gossypiifolia L., underwent drying, at room temperature, for five days.Then, the material was minced and was extracted by steeping with 98% ethanol.Subsequently, the extracts obtained were rotated at 40 ° C to concentrate them.

 Fractioning of the crude ethanolic extract
The fractionation of the extracts of the species in question was carried out with an aliquot of 9.9 grams of leaves and 9.6 grams of stem of the ethanolic extracts, in which they were partitioned in a vacuum filtering column, using, as a stationary phase, silica gel and, as mobile phase, hexane, chloroform (CHCl3), ethyl acetate (EtOAc) and methanol (MeOH), following that order of polarity. 8e solutions obtained were concentrated in a rotary evaporator, resulting in four phases: hexane, chloroform, ethyl acetate and methanol.Thereafter, the wet materials were placed in suitable vials and dried at room temperature.

 Phytochemical Analysis
Phytochemical prospecting of J. gossypiifolia L. was performed according to a methodology already used. 8

INTRODUCTION METHOD  Evaluation of cell viability by the MTT technique
Cytotoxicity was evaluated by the use of the colorimetric method by the methyltetrazolium salt (MTT), in which macrophages of the J774 lineage were used. The ethanolic extracts from leaves, stems and branches of J. gossypiifolia, previously dissolved in Ethanol, were serially diluted in the RPMI medium to obtain the final concentrations and added in a 96 well plate (100 μL/well).Cells (1.5 x 10 5 cells/well) were cultured in a 96-well plate with culture medium (RPMI-1640 supplemented with 10% fetal bovine serum, 1% glutamine and 40 μg/mL gentamicin).Therefore, the cells were treated with extracts at concentrations ranging from 62.5; 125 or 250 μg/mL in culture medium.They were then incubated, for 24 hours, in a 5% CO2 incubator at 37° C and, then 25 μl of the MTT solution were added, incubating the plates for a further three hours.After this time the absorbance reading was carried out in spectrophotometer at 595 nm. 11

 Microdilution in broth for determination of Minimum Inhibitory Concentration (MIC)
CIM was performed according to the protocol based on the Clinical Laboratory Standards Institute, in which the Mueller-Hinton Broth (CMH) was distributed in the 96 well plates and 100 μL/well was placed. 12The solubilization of the samples occurred by the association of 2000 μg of the sample in question, added to 20 μL of DMSO solution, being diluted in 980 μL of sterile saline, resulting in a stock concentration of 2000 μg/mL.In columns one to nine of line A, 100 μL of the solubilized samples were added, and columns ten, 11 and 12 were destined for the Growth Control, Negative Control and Plate Sterility Control, respectively.
After this process, 100 μL of the contents of each hole in line A were transferred to line B and, after homogenization, this process was repeated until line H, in which excess was discarded.Microbial inoculums were prepared based on an already used protocol, which used the McFarland 0.5 scale (108 CFU/mL) . 12acteria were diluted in three mL of sterile saline and one mL of this was rediluted in the ratio of 1:10 sterile saline solution, then, five μL (104 CFU/mL) was deposited in the wells of columns one to 11.The plates were then incubated in a bacteriological oven at 35° C, for 16 to 20 hours.After this time, 20 μL of Triphenyl Tetrazolium Chloride (TTC) was added to each well and the plates were reincubated for another three hours.Then, the wells that changed from colorless to red indicated the presence of a living microorganism.However, absence of staining means positive result for inhibition of the samples on the evaluated bacteria.

 In vitro healing potential using Scratch assay
Fibroblast proliferation was studied in vitro using a 3T3 fibroblast assay of mouse embryo tissue. 13Initially, fibroblasts were cultured in DMEM culture medium supplemented with 10% fetal bovine serum (FBS) and incubated in a greenhouse at 5% CO2 at 37° C, for 24 hours.Subcultures were performed using the trypsin-EDTA solution to peel off the adhered cells.After growing and being trypsinized, the fibroblasts were seeded in 12-well plates and kept in a 5% CO2 oven, at 37°C.The culture medium was then removed and the Scratch assay was performed, in which a straight line was made in the median region of the plate with the tip of the 200 μL pipette, causing a rupture between the cells and causing a mechanical lesion.For the removal of debridis resulting from the lesion, the wells were washed with phosphate buffered saline (PBS).
Then, the plates were given the extracts of J. gossypiifolia, in concentration of 125 μg / mL.Established this concentration, after results of the MTT, with the fractions of leaves and stems of said plant, whose values in the concentration 125 μg/mL evidenced cell viability above 90% of the viability, using the migration times in hours, that were zero, 12 and 24 hours. 13e images were captured with a digital camera coupled to the inverted phase microscope using NIS Elements F 3.2 software, and obtained from the same field of view of the wound, creating reference points on the outside of the plate and on the microscope stage with fine-point markers.Thus, the migration of fibroblasts was evaluated by means of these photographs, in which the lesional area was measured at times zero, 12, 24 hours.

 Phytochemical prospecting
In the phytochemical prospection of the crude extracts of the leaves, stems and branches of the plant species J. gossypiifolia, the presence of the tannin, steroid and flavonoid secondary compounds was evidenced in the three samples tested, and in the leaf extracts the constituents flavones and Xanthones were also found, as evidenced in Table 1.In relation to the chloroform and hexane fractions of the stem, the presence of tannins, flavonoids and steroids was verified, whereas in the methanolic fraction, the presence of flavonoids and steroids.In hexane only, flavones were found.For the leaf fractions, tannins and steroids were found in the four fractions samples, while in the methanolic and ethyl acetate fractions flavonoids, were identified.In the same manner as in the ethyl acetate fraction, the flavones were evidenced.In none of the extracts and fractions were found saponins nor alkaloids.

 Cell Viability Assay by MTT Method
This assay was performed by the MTT method against J774 macrophages cells to evaluate possible cytotoxicity of the ethanolic extracts of leaves, stems and branches of J. gossypiifolia (Figure 1).The stem extracts, at the concentration of 62.5 μg/mL, had a percentage of cell viability of 77.09%, whereas, in the concentration of 125 μg/mL and 250 μg / mL, the percentage of viable cells was higher, with values of 87.00% and 82.00%, respectively.
In the MTT fractions, 3T3 fibroblasts were used, since, for the MTT test of the fractions of the leaves and stems, the choice of 3T3

RESULTS
fibroblasts rested on the need to perform Scracth assay, which is done with fibroblasts of this lineage.Figures 2 and 3 show these results.As shown in figure 2, only fractions of ethyl acetate and hexane in the concentration, of 250 μg / mL, showed lower percentages of cell viability, both with 67.02% and 67% 85%, respectively.
At the concentration of 125 μg / mL, the methanolic and chloroform fractions induced cell proliferation, presenting percentages of 117.08% and 110.02%, respectively.While at the concentration of 62.5 μg / mL, the methanolic fractions (108%) and ethyl acetate (115.3%) were those that induced cell proliferation.

 Determination of Minimal Inhibitory Concentration (MIC)
The MIC was initially performed with the crude extracts and was repeated with the leaves and stem fractions from these extracts.All the ethanol extracts showed inhibitory activity against three microorganisms.The ethanolic extract of the leaves inhibited the S. aureus, S. epidermidis and P. aeruginosa microorganisms, at the concentration of 500 μg/mL.The stem was resistant to S. aureus and P. Aeruginosa, at 1000 μg/mL.At the concentration of 500 μg / mL inhibited S. epidermidis growth.The ethanolic extract from the leaves inhibited S. aureus at the concentration of 1000 μg/mL, P. aeruginosa at 500 μg/mL and S. epidermidis, at a concentration of 250 μg/mL.
It is important to stress that the ethanolic extracts did not present antimicrobial activity for E. coli and reaffirm that the fractions English/Portuguese J Nurs UFPE on line., Recife, 12(2):465-74, Feb., 2018 470 showed no inhibitory activity for the tested microorganisms.

 In vitro healing potential using Scratch assay
In order to evaluate the cicatrization activity in vitro, the methanolic fraction from the leaves of J. gossypiifolia was chosen because it obtained the best results against MTT with 3T3 fibroblasts.In figure 4, fibroblasts treated with this fraction at 125 μg/mL, exhibited an increase in migration at 12 h and 24 h times, as compared to cells treated with culture media.The other fractions did not promote this migration.It was verified that the treatments with the fractions led to changes in these cells, inhibiting the migration process.The methanolic fraction may be a viable alternative to invest in further research that provides the necessary safety for the use of this plant species in the treatment of wounds.
The effect of the methanolic fraction of the leaves on the migration of the fibroblasts during the experiment at zero time, did not distinguish the control and the fraction.It was noticed that, in the time of 12h and 24h, the methanolic fraction surpassed in 45%, more than the control.
The following secondary metabolites: tannins, flavonoids, steroids and coumarins, were evidenced in the species J. gossypiigolia of this study. Another study found secondary metabolites such as tannins, flavonoids, steroids and saponins in the different parts of J. gossypiifolia, which corroborates this study. 6owever, metabolites such as terpenoids, quinones, lactones and lignans reported by other studies were not found in the extracts studied in this study. 6,15e Jatropha genus has several secondary metabolites such as organic acids, alkaloids, carotenes, phenoesteroids, phytosteroids, flavonoids, glycosides, lactones (coumarins), lignans, mucilages, pectins, polysaccharides, quinones, saponins, tannins and terpenes, which characterize it as a genre that presents different biological activities, among them antimicrobian, antiinflamatory and adstringent. 15 The presence of the secondary metabolites, found in the plant species in question, serves as a possible basis, for the biological activities found in the tests performed with the J. gossypiifolia samples.
As for cytotoxicity, the crude extracts were initially tested against macrophages, which presented slightly cytotoxic results, for extracts of the stems at concentrations 62.5; 125 and 250 μg/ml.For leaves, at the same concentrations, the results obtained were moderately cytotoxic, while the result of the branches, also in the same concentrations, showed severe cytotoxicity, with more than 50% of nonviability, which made it impossible to continue the study with this part of the plant.
In the in vitro cytotoxicity assay against HEK-293 cells, at concentrations of 62.5; 125; 250 and 500 μg/mL, from the crude extract of the leaves of J. gossypiifolia obtained by decoction, no cytotoxic effect, was evidenced, therefore, being considered 100% cell viability. 18 cytotoxicity test of the crude extracts of the stem to the macrophages presented slightly cytotoxic results, the leaves were moderately cytotoxic; and the branches presented cell viability of less than 50%.
However, when cell viability tests were carried out, with fractions of leaf and stem extracts of J. gossypiifolia, the methanolic and ethyl acetate fractions, in the three concentrations studied were not cytotoxic, with viability greater than 90%, while the chloroform fraction, in the concentration of 250 μg/mL, presented viability less than 50%.Virtually all the stem fractions did not present cytotoxicity except ethyl acetate and hexane at concentrations of 250 μg/mL considered to be moderately cytotoxic with a percentage of 50-79% viability.13Increased chloroform fraction concentration induced a decline in number of viable cells, leading to a reduction of 50% (p <0.001) in cell viability, at the concentration of 250 μg/mL.The hexane fraction decreased the viability of 3T3 fibroblasts at all concentrations tested.In parallel, preclinical toxicological studies, in rats treated with the leaf and stem ethanolic extract of J. gossypiifolia demonstrated low acute oral toxicity.However, their prolonged use in an animal model of chronic toxicity revealed severe liver, pulmonary and renal damage.Therefore, further studies should be performed to elucidate the cytotoxic potential of this plant. 14 relation to the minimal inhibitory concentration, the extracts of J. gossypiifolia showed inhibition against the bacteria tested, while the ethanolic extract of the leaves presented a MIC of 500 μg / mL for S. aureus, S. epidermidis and P. while the ethanolic extract of the branches exhibited an inhibition of 1000, 500 and 250 μg/mL, and the ethanolic extract of the stem showed antimicrobial activity at 1000, 500 and 1000 μg/mL, respectively.It was also found that the extracts tested were not able to promote the inhibition of the E. coli microorganism.
J. gossypiifolia had antimicrobial action against eight microorganisms tested (Bacillus cereus var.Mycoides, B. pumilus, B. subtilis, Bordetella bronchiseptica, S. epidermidis, Klebsiella pneumoniae, Streptococcus faecalis and Candida albicans). 19As well as extracts of J. gossypiifolia presented activity against P. aeruginosa and Bacillus subtilis. 15These results corroborate with another study that states that extracts of the genus Jatropha have antibacterial action. 17other study about the antimicrobial activity of the root, stem and leaf extracts of Croton pulegioides Baill, belonging to the same Jatropha family, concluded that the methanolic extract of the stem showed antimicrobial activity against the strains of S. aureus and S. epidermidis, while the methanolic extract of the leaves showed no activity.On the other hand, all the extracts were inactive in the concentrations evaluated against the Gram-negative bacteria used in the study (Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae), a result that was also observed in the in vitro antimicrobial assays in this study. 20 is important to note that, the inhibition of the fractions in the extracts is probably due to the synergism of the secondary metabolites, that, when the fractions were separated, the antimicrobial action was inactivated.The actual efficacy of a medicinal plant may not be due to a major active component, but to the combined action of different compounds, which in many cases the synergism of the secondary metabolites potentiates the biological action of numerous plant species. 21study carried out confirmed the antibacterial activity of extracts of leaves extracted in chloroform and methanol against Salmonella typhi, P. aeruginosa, S. aureus and C. albicans. 6A study carried out with methanolic and ethyl acetate extracts of Jatropha multifidia latex active against the following microorganisms: Bacillus subtilis, S. aureus, P. aeruginosa, E. coli, K. pneumoniae and Candida sp. 20This result, different from the one found in this study, when fractionating leaf and stem extracts, no antibacterial activity was found.
[24] In this study the evaluation of cicatrization was performed using an in vitro bioassay using Scratch assay, which evaluates cell migration in the time intervals of zero, 12 and 24 hours.This technique is based on the creation of an interruption of continuity of a cellular monolayer, or wound, in which the follow-up of the closure of this lesion is performed by observation under an inverted phase microscope. 25This method was used in order to observe the cicatrizing action in vitro of extracts fractions of leaves and stem, measured by the cellular migration of 3T3 fibroblasts.
Time zero is the time when the wound is performed and there is no presence of cells, fibroblasts.For stem fractions, the migration of these cells was not observed at any of the times.For the leaf fractions, a significant migration occurred in the methanolic fraction (45%), observing this increase in the migration rate when used in the 125 μg / mL concentration, which demonstrates the possibility of this fraction being an alternative for the production of a phytotherapic for the wound care.
MTT results indicated that the stem extract presented cell viability from mildly cytotoxic to non-cytotoxic, while leaf extract was moderately cytotoxic.As for the branches, the result was severely cytotoxic, preventing the continuation of the study with this sample.
The CIM of the leaf extract was 500 μg/mL for the S. aureus, S. epidermidis and P. aeruginosa bacteria, while the ethanolic extract of the branches exposed an inhibition of 1000, 500 and 250 μg/mL and the ethanolic extract of the stem exhibited antimicrobial activity in 1000, 500 and 1000 μg/mL, respectively.It was also found that, the extracts tested were not able to promote inhibition of the E. coli microorganism.The CIM, performed with the fractions from the extracts in leaves and stem ethanol, resulted in the absence of inhibitory activity for these bacteria.
The healing evaluation, by the Scratch Assay method showed that the methanolic fraction of the leaves allowed a 45% increase in fibroblast migration, signaling the possibility of this fraction being an alternative for the production of a herbal medicine for the treatment of wounds.Studies with this plant species should be continued for isolation of the active principle aiming at the production of a wound healing phytotherapic.

Figure 3
Figure 3 shows that the methanolic fraction induced cell proliferation at all concentrations, especially the concentration of 250 μg/mL with a percentage of cell viability of 225.08%.