Saline extract from Malpighia emarginata DC leaves showed higher polyphenol presence, antioxidant and antifungal activity and promoted cell proliferation in mice splenocytes

Currently, the research of new natural compounds with biological potential demonstrates great ethnopharmacological importance. In this study, we evaluated the biological properties promoted by saline extract from Malpighia emarginata DC leaves, whose objective is to evaluate the antioxidant, antimicrobial and cytotoxicity potential. Phytochemical characterization was performed by UPlC-MS chromatography to identify the chemical compounds. For the antioxidant potential, DPPH, ATT and FRAP methods were used. The antibacterial and antifungal tests were performed evaluating the MIC50, MIC90, CMB and CMF parameters. Moreover, antibiofilm action was evaluated. Cytotoxicity and proliferation were performed using splenocytes from Balb/c mice and were evaluated by cytometry. We found a list of phenolic compounds among other bioactive compounds in the M. emarginata saline extract. In addition, higher antioxidant profile and antifungal activity against different strains of Candida spp. was promoted by the saline extract. Splenocytes showed greater cell viability (more than 90%) and showed higher proliferate index in 24 and 48 hours of incubation with the extract. Saline extract from Malpighia emarginata DC has potential action like antioxidant and antifungal agent without promote animal cell damage.

study of phytochemical constituents, therapeutic action mechanism and identify active principles responsible for many biological activities observed in medicinal plants (lin et al. 2018). Moreover, many authors have been publishing evidences about the efficacy of medicinal plants and their constituents on the improvement of diseases as cancer (Shirzad et al. 2011), atherosclerosis (Asgari et al. 2012) and diabetes (Asgary et al. 2011).
Malpighia emarginata DC. is a plant found throughout Central and South America. It is a tropical specie originating in the Caribbean region, being known as the Antillean cherry (Oliveira et al. 2012, Moreira et al. 2016. In Brazil the fruit is known as acerola and possess high content of ascorbic acid (vitamin C), carotenoids, anthocyanins, thiamine, riboflavin, niacin, proteins and mineral salts, mainly iron, calcium and phosphorous, being considered a functional food (Mezquita and Vigoa 2000, Rosso et al. 2008, Alvarez-Suarez et al. 2017. This plant also has other phytochemical constituents such as flavonoids, phenolic acids and polyphenols (Bataglion et al. 2015, Malegori et al. 2017). All these compounds are able to minimize oxidative damage caused by the reactive oxygen and nitrogen species in animal organism (Oliveira et al. 2012) and prevent some chronic diseases such as cancer (Mezadri et al. 2008, Leffa et al. 2014. Moreover, these molecules also exhibit potential therapeutic effect as anti-inflammatory, radiation-protective, chemoprotective, vasoprotective, inhibition of lDl oxidation and decrease the risks of cardiovascular diseases (Wang et al. 1997, Seeram and Nair 2002, Rosso et al. 2008.
local communities, especially in northeast of Brazil, use the juice of acerola mainly for respiratory diseases as flu and bronchitis. However, some accounts showed that leaves of plant were used to make tea associated with garlic and honey to flu symptoms. In addition, recent biological studies have shown that acerola consuming is not dangerous to human cells.
Many researches show important biological actions promoted by different organic extracts (like phenolic and ethanolic fractions) made using leaves of M. emarginata (Silva 2014, Oliveira 2015, Rezende et al. 2018. However, is known that the true use of M. emarginata in local communities is like teas or infusions using water as the solvent.
Although the studies about M. emarginata fruit are extensive, is very important to study the phytochemical composition and biological activities promoted by other organs of this plant. Thus, the present study aimed investigates the phytochemical composition of the saline extract from Malpighia emarginata DC leaves and shows the initial biological activities promoted by this compound.

VegeTABle MATeRIAl AND eXTRACT PRePARATION
The leaves of Malpighia emarginata were collected in the city of Recife, state of Pernambuco, Brazil. The botanical identification was performed by the Professor Marlene Barbosa, on May 30, 2017, at the Herbarium geraldo Mariz, located at the Center of Biosciences of the Federal University of Pernambuco through exsiccate deposited under registration number 82.615. After collection, the leaves were kept at room temperature for drying during 4 days. The dried leaves were crushed in Reverse Black Oster and the pulverized material (20g) was diluted in 0.15 M NaCl (200 ml) in the proportion of 10% (w/v). Therefore, using an orbital and alternative incubator shaker (lab Companion™ IS-971) the material was kept under agitation (400 rpm) in constant temperature (28 ºC) for 16 hours. After the stirring time, the material was filtered using paper filter and centrifuged (15 min, 10,000 x g). The obtained supernatant was collected and called saline extract from Malpighia emarginata leaves. After this, the extract was lyophilized and BÁRBARA R.S. BARROS et al.

Malpighia emarginata PReSeNT BIOlOgICAl ACTIVITIeS
An Acad Bras Cienc (2019)  Ultra-performance liquid chromatography (UPlC) was performed with an Acquity H-Class (Waters) employing a 2.1 x 100 mm BeH column with a particle size of 1.7 μm. The column was maintained under a constant temperature of 40 ºC and the auto injector at 10 ºC. We use an aqueous solution (eluent A) containing 2% methanol (MeOH), 5Mm ammonium formate and 0.1% formic acid and a methanol solution (eluent B) containing 0.1% formic acid, which were pumped at a flow rate of 0.3 ml/min. Ten microliters of the M. emarginata extract was injected. elution was performed in gradient mode and the initial condition (98% A / 2% B) was maintained for 0.25 minutes. The B ratio increased linearly to 99% in 8.5 minutes, remaining at 99% B for one minute, followed by immediate decreased to 2% B, where it was maintained for up to 11 minutes. The UPlC system was coupled to a single quadrupole mass spectrometer SQ Detector 2 (Waters®). The data were obtained in fullscan mode, analyzing masses between 100 and 1000 Da, in negative ionization. The acquisition of the chromatograms and mass spectra was obtained through Masslynx ™ software (Waters®).

Total Antioxidant Activity
The total antioxidant activity promoted by saline extract from Malpighia emarginata leaves was determined as a function of ascorbic acid, according with (Pietro et al. 1999). Saline extract (100µl at 500 µg/ml) was mixture with ascorbic acid (1 mg/ ml) and 1 ml of phosphomolybdenum solution (600 mM sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). This mixture was incubated in water at 95 °C for 90 minutes.
After returning to room temperature, the absorbance are measured at 695 nm against a blank (1 ml of solution and 0.1 ml of water). A standard curve with ascorbic acid (0 -500 μg/mL) was performed to obtain the equation Y = 0.019x + 0.0723, R² = 0.9937. Assays were performed in quintuplicate. Total antioxidant activity was calculated by the formula ATT (%) = [(As -Ac) / (Aaa -Ac)] 100, where: Ac = Control Absorbance, As = Sample Absorbance and Aaa = Ascorbic Acid Absorbance.

Reduction of Ferric Ion (FRAP assay)
The stock solution of the FRAP assay was prepared with 300 mM acetate buffer (3.1 g CH 3 COONa and 16 ml CH 3 COOH) at pH 3.6, 10 mM TPTZ (2.4, 6-tripyridyl-s-triazine) solubilized in 40 mM HCl, and 20 mM FeCl 3 solution. The working solution was prepared by mixing the acetate buffer, TPTZ and FeCl 3 in a ratio of 10:1:1 (v/v/v) and incubated for 5 min at 37 ºC. Saline extract (25µl at 1 mg/ ml of concentration) was added in 180 µl of FRAP reagent and this solution was incubated for 30 minutes at 37 °C in the dark. Subsequently the absorbance was measured at 593 nm. A standard curve with FeSO 4 (0 -1000 μg / mL) was performed to obtain the equation Y = 0.0024 x + 0.0019, R² = 0.9953. The results were expressed as mg eFeSO4 (II)/g of extract.
ANTIMICROBIAl ASSAYS

Preparation of splenocytes
This procedure was performed in accordance with Melo et al. 2011. After the inoculation of anesthetics (10mg/Kg of xylazine and 115mg/Kg of ketamine i.p. injection) followed of cervical displacement, spleen of each mouse was removed aseptically and placed in a Falcon tube containing RPMI 1640 with fetal calf serum (complete medium). In a vertical flow, each spleen was transferred to a petri dish where they were soaked. The cell suspensions obtained from each spleen were transferred to Falcon tubes containing approximately 10 ml of incomplete medium. Spleen homogenates were overlaid onto Ficoll-Paque™ PlUS layer, with the density adjusted to 1.076 g/ml, and centrifuged at 1000 x g at room temperature for 25 min. The interface cell layer containing immune cells was recovered by Pasteur pipette, washed twice in phosphate-buffered saline (PBS) and centrifuged twice at 500 x g for 10 min. Cells were counted in a Neubauer chamber, and cell viability was determined by the trypan blue exclusion method. Cells were only used when viability was> 98%.

Analysis of cell viability by Annexin V-FITC and Propidium Iodide Staining
Mice splenocytes (10 6 cells) were treated with saline extract from Malpighia emarginata leaves at 50, 25, 12.5, 6 and 3 μg/mL and were maintained in 24-well plates for 24 hours to analyze their cell viability. Untreated cells (control) were used as a negative control. Following this, splenocytes were centrifuged at 4 °C, 450 x g for 10 min. After discarding the supernatant, 1 ml of PBS 1X was added to the precipitate and centrifuged at 4 °C, 450 x g for 10 min. After discarding the supernatant, the pellet was resuspended in a binding buffer of cell viability kit (Becton Dickinson Biosciences) and Annexin V conjugated with fluorescein isothiocyanate (FITC): 50 μg/mL) and propidium iodide (PI, 20 μg/mL) were added to each labeled cytometer tube. Flow cytometry was performed in a FACS Calibur (Becton Dickinson Biosciences) and analyzed using Cell Quest Pro software (Becton Dickinson). Result of the analysis was performed in graphs by dot plot. Annexin-FITC negative/PI positive cells were considered necrotic cells and Annexin-FITC positive/PI negative represented splenocytes in the early stage of apoptosis. Double negatives were considered viable cells.

Cell proliferation analysis using CFSE staining
Same protocol to obtained splenocytes was used for proliferation assay. After splenocytes obtainment, cell solution was centrifuged at 300 x g at room temperature for 5 min with sterile PBS 1X added with SFB 5% (pH 7.2). After this, the cell solution was adjusted to10x10 6 cells/ml and received 5mM of 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSe). Cells were incubated for 10 min at room temperature in the dark. Cells were centrifuged twice at 300 x g / 5 min with sterile PBS 1X. Cells stained were cultured for 24 and 48 hours with 12.5 µg/ml of saline extract from M. emarginata leaves and only culture complete RPMI 1640 medium (negative control). After culture time cells were centrifuged (300 x g / 5 min), were carry to acquisition on FACSCalibur platform (Becton Dickinson Biosciences) and results were analyzed using Cell Quest Pro software (Becton Dickinson).

STATISTICAl ANAlYSIS
To test the normal hypothesis on the variable involved in this study the Shapiro-Wilke test was applied. Means of samples were analyzed using non-parametric tests. The statistical difference between two groups was analyzed by Wilcoxon test and that among more than three groups by one-way analysis of variance (ANOVA). All the conclusions BÁRBARA R.S. BARROS et al.
For statistical analysis was used graphPad Prim 5.01 software.  (Table I).

PReVAleNCe
In addition, we found 18 compounds in the saline extract in accordance with the area, retention time, molecular weight, molecular formula and negative mass, identifying other phenolic compounds, like Protocatechuic acid, Apigenin-7-O-glucoside flavonoid and terpenoids, like Isotriptophenolide (see Table II).
SAlINe eXTRACT FROM M. emarginata leAVeS SHOWeD SIgNIFICANT ANTIOXIDANT POWeR Saline extract from M. emarginata leaves showed higher antioxidant power being five times superior to butylated hydroxytoluene (BHT) standard (20.32±0.89 / 4.12±0.10 to saline extract and BHT, respectively). Saline extract also was able to  increase of free radical sequestration (38,59 %) and promoted higher ferric ion reduction, also like BHT standard (see Table I).  concentrations (Table III). In addition, was also possible observe that saline extract presented more significant results as antifungal against C. albicans and C. parapsilosis, comparing to the other species.
Only the C. glabrata species was resistant to saline extract.
Antibiofilm activity was evaluated with the saline extract following the concentrations of MICs: 64 MIC, 16 MIC, 4 MIC, MIC, 1/4 MIC, 1/16 MIC and 1/64 MIC; according to the graphs in figure 2 and Table IV.   The results of the antibiofilm activity confirm the antimicrobial tests performed. The saline extract of Malpighia emarginata presented a considerable reduction in the biofilm formation of C. albicans, C. krusei and C. parapsilosis species, as described in table IV. Mice Balb/c spleen cells treated with saline extract in different concentrations (50, 25, 2.5, 6 and 3 μg/ mL), did not suffer significant cell death, for both apoptosis and necrosis, in none concentrations evaluated (Figure 3). In fact, saline extract from M. emarginata leaves promoted a higher cellular viability, superior to 90% of surviving. For measuring if saline extract was able to activate immune spleen cells, we performed other assay to investigate the proliferation index of these cells challenged with only one concentration of extract (12.5 μg/mL) during two days of cell culture. Results showed that saline extract from M. emarginata leaves induced higher proliferation indices in both times evaluated (Figure 4).

DISCUSSION
Popular culture collaborates with the indiscriminate use of medicinal plants within the context of selfmedication, requiring the study of its constituents, the mechanism of therapeutic action and the identification of active principles responsible for the various biological activities reported by the population (Ford et al. 2014). Here, we used a saline extract made from M. emarginata leaves. The proposal was investigated if an extract of plant, made with standard physiological solution (NaCl 0.15M), is cytotoxic to cells and microorganisms and if this extract is also able to be antioxidant. In similarity to the results found by Nowotarska et al. (2017), the saline extract from leaves of M. emarginata, as described in Table II prepared in its phytochemicals 18 characterized compounds, being a larger number of a class of phenolic and terpenoid compounds.
As described in Table I, saline extract from M. emarginata leaves showed a potential antioxidant capacity, especially when compared to butylated hydroxytoluene (BHT) a known antioxidant compound used as standard (Pasa et al. 2016. We suggest that this antioxidant potential is due to higher amounts of phenolic compounds, previously described in several studies as antioxidants molecules (Zengin et al. 2018). Similar to our results, other studies also showed that plants rich in phenolic compounds have antioxidant potential as well as the extracts of Organic extracts from various organs of M. emarginata presented potential antimicrobial activity against different pathogenic bacteria species as described by Silva (2014) and Motohashi et al. (2004). Although such solvents (hexane and ethyl acetate) have high toxicity to microorganisms (Yung et al. 2016), the indiscriminate use of these solvents is also associated with intoxication, either by ingestion or inhalation, causing serious damage to the individual, such as severe lung injury and hepatotoxicity (Ford et al. 2014, Tormohelen et al. 2014, Connellan, 2017.
The results obtained in this study did not show action against the species of bacteria used. However, the saline extract of M. emarginata leaves presented a potential fungicidal, fungistatic and antibiofilm action, suggesting this capacity, once again, to the presence of phenolic compounds (Haghdoost et al. 2016, Barral et al. 2017, Jara et al. Wang et al. 2018. As described by Khan et al. (2014) andglorybai et al. (2015) plants are commonly used by traditional medicine as a source of cure for various diseases. However, the irrational use or the conception that a natural product does not cause harm to the health can be harmful to the individual, Here we also investigate the cytotoxicity profile promoted by the saline extract from M. emarginata leaves in mice splenocytes and the results showed that, same in higher concentrations, this extract is not toxic. Moreover, M. emarginata extract also promoted lymphocyte proliferation in both 24 and 48 hours of incubation, suggesting immunostimulatory properties. Recent study showed that extract of several species of Byrsonima spp. (Malpighiaceae) in different concentrations (12.5 and 50 μg/mL) were not cytotoxic against RAW 264-7 cells (Fraige et al. 2018). In 2016, Düsman et al. evaluated the cytotoxicity of the aqueous extract from Malpighia glabra linnaeus fruits in vivo and in vitro in Wistar rats, and against bone marrow derived cells from these animals. Their results demonstrated that the extract also was not cytotoxic.
The activation and cellular proliferation are directly associated with healing processes and with the replacement of dead cells of the tissues that are in constant development in the organism. For this, is very important investigate if a compound can promote cell proliferation, especially if these cells are immunological because the major activation mechanism of lymphocytes is the clonal expansion after it activation.
Plants extracts belonging to the Malpighiaceae family also present other potential pharmacological properties, such as antimicrobial, wound healing and antitumor (Düsman et al. 2016, Specian et al. 2016.
Recent studies with different species belonging to the genus Byrsonima (family Malpighiaceae) showed no cytotoxic action against different human cell lines (monocytes and cells of primary gastric epithelium) but demonstrated a potential cytotoxic capacity against colon cancer cell lines (HT29) and hepatocellular carcinoma (Hepg2) (Specian et al. 2016, Verdam et al. 2014. Our results associated with these findings suggest the possibility of future investigations about antitumor, wound healing and immunological responses promoted by saline extract from Malpighia emarginata leaves.

CONCLUSIONS
Saline extract from Malpighia emarginata leaves showed the presence of a considerable concentration of total phenolic compounds, among them some flavonoids and phenolic acids. Moreover, showed antioxidant and antifungal properties and was not cytotoxicity against mice splenocytes, inducing high proliferation in these cells. These results are promisors, demonstrate the safe use of this plant against normal cells and stimulate new investigations to use this extract like antifungal and immunostimulant compound in future assays.