Phytochemical profile , antibacterial , antioxidant and cytotoxicity activities of Euphorbia cotinifolia

The aim of this study was to determine the content of phenolic compounds and flavonoids, the antibacterial, antioxidant and cytotoxicity activities of hydroethanolic extracts of root, stem, leaf and fruit of Euphorbia cotinifolia. Phytochemical screening was performed using spectrophotometric methods (phenolic and flavonoid content) and liquid cromatography. The antibacterial activity was determined by agar diffusion and broth microdilution technique. Additionally, antioxidant activity was determined by diphenylpicrylhydrazyl (DPPH) radical scavenging method and cytotoxicity by the MTT method using BHK-21 (newborn hamster’s kidney) cells. All extracts presented notable content of phenolic compounds, flavonoids and tannins. The high-performance liquid chromatography with photodiode array detection (HPLC-DAD) analysis showed higher concentration of phenolic compounds in dried leaves than in fresh leaves and it indicated the presence of caffeic acid. The extracts of leaf, stem, root and fruit showed activity against five gram-positive bacteria, six gram-negative bacteria and two yeasts, but not for mycobacterial. The highest antioxidant activity was exhibited in the extract of dried leaf (EC50 = 7.32 μg/ml). Extracts showed no cytotoxicity at the concentrations tested. All extracts showed antibacterial, antifungal and antioxidant activities, phenolic compounds, tannins and flavonoids. The results provided evidence that the studied plant might indeed be potential sources of natural antioxidant and antimicrobial agents.


INTRODUCTION
The use of medicinal plants to treat diseases is virtually an old strategy used by all populations in the world.Between 25 to 30% of all medicines available in therapeutics are derived from natural products (plants, microorganisms and animals) (Ramos et al., 2008).The increased resistance to the available antimicrobials has attracted the attention of the scientific community to investigate new effective drugs of natural origin (Bitu et al., 2012).Natural products are an important source of bioactive compounds that can be used as an alternative to the treatment of diseases and also as a natural preservative in the food industry.Studies report that many biological activities, as well as antioxidant and antimicrobial, are due to the content of total phenols, tannins and flavonoids (Einbond et al., 2004;Banerjee and Dasgupta, 2005;Choi et al., 2006).The largest genus in the euphorbiaceae family is the Euphorbia.Plants from this genus have a preference for dried areas and they are found in South America and also in African and Asian continents.They are found as weeds in gardens or as ornamental elements at homes.The plants of the Euphorbia genus are traditionally used as medicinal plants, since they have several bioactive compounds, such as flavonoids, alkaloids, tannins and terpenes (Wang et al., 2006;Pusztai et al., 2007;Zhang et al., 2008;Shlamovitz et al., 2009).E. cotinifolia is used in folk medicine to cauterize wounds and also as a laxative (Mortan, 1962).The E. cotinifolia presents molluscicide (Pereira et al., 1978), antiviral (Betancur-Galvis et al., 2002) and antimicrobial activity (Jayalakshmi et al., 2011).In this context, this study aimed to determine the content of phenolic compounds and flavonoids and also determine the presence of antimicrobial, antioxidant and cytotoxicity activities of hydroethanolic extracts of root, stem, leaf and flower of dried and fresh E. cotinifolia.

Plant identification and extract production
E. cotinifolia root, stem, leaf and flower were obtained in the city of  at an elevation of 908 m, on November, 2012.The plant was identified, registered and filed in the Herbarium of the Federal University of Alfenas, thus, getting the specimen voucher number 2337.Fresh plant parts (root, stem, leaf and fruit) were washed under non-sterile water and cut into small pieces using non-sterile scissors.To prepare extracts from fresh plant parts, 200 grams of the fragments were weighed and added to a flask containing 800 ml of ethanol at 70%.To prepare dry extracts from plant parts, the samples were dehydrated at 37°C for seven days to reach constant weight.The samples were grounded into a powder and particle size was determined according to the Brazilian Pharmacopoeia 5th ed.using the Sieve Shaker -Electromagnetic method (Ertel ®) (Brazilian Pharmacopoeia, 2010).The obtained dried powder was used to prepare the extracts as described previsiously.The extracts were macerated for seven days in dark and after maceration, the extracts were filtered using filterpaper.Subsequently, the extracts were concentrated by rotaevaporator apparatus using negative pressure of 500 mmHg at a temperature of 60°C and lyophilized.All extracts were ressuspendend at final concentration of 50 mg/ml using dimethylsulfoxide (DMSO) and sterilized using 0.22 µm filters.

Evaluation of the phytochemical profile of the extracts
The extracts of root, stem, leaf and flower of E. cotinifolia were subjected to phytochemical tests based on colorimetry and precipitation for detection of the major bioactive constituents: anthraquinones, flavonoids, tannins, steroids, saponins and alkaloids (Costa, 1994).For the determination of phenolic compounds, an aliquot of each extract (0.5 ml) at 0.1 mg/ml was mixed with 2.5 ml of Folin-Ciocalteu reagent (diluted 1:10 in distilled water) and 2.0 ml of Na 2 CO 3 4% (w/v) in distilled water.After two hours incubation in the dark at room temperature, the absorbance was measured at 750 nm in a spectrophotometer.The results were expressed as gallic acid equivalents (mg GAE/g) calculated by a curve constructed with concentrations ranging from 5 to 100 mg/ml (Singleton, 1999).The flavonoid content was determined according to Kalia et al. (2008).An aliquot of 0.5 ml of the extracts (at a concentration of 1.5 mg/ml) was mixed with 1.5 ml of ethanol, 0.1 ml of aluminum chloride (AlCl 3 .6H2O) 10% (w/v), 0.1 ml 1 M potassium acetate, 2.8 ml of distilled water and 5 ml of total reaction.After 30 minutes, the absorbance of the mixture was measured at 425 nm.The total flavonoid standard curve was made using quercetin.The total flavonoids was expressed as quercetin equivalents (mg QE/g), and the values were presented by mean of a triple analysis.High Performance Liquid Chromatography (HPLC) analyses were performed on Shimadzu chromatograph matched with a diode array detector (DAD) and Shimadzu C18 ODS column (250 x 4.5 mm, 5 mm in particle size).The eluents used were acetic acid solution 5% v/v (eluent A) and methanol (eluent B), the injection volume was 25 µl and a flow rate of 1.0 ml/min.The analysis started at 10% B, and the linear gradient followed by 100% B in 35 min.The concentration of B was maintained until 50 min.Analysed extracts were dissolved in the mobile phase.Ascorbic acid, caffeic acid, quercetin, benzoic acid, and gallic acid were used as standards in the HPLC analysis.

Evaluation of antibacterial activity of the extracts
The antimicrobial activity was evaluated by agar diffusion according to the methodology proposed in document M7-A6 (CLSI, 2003)  For the agar diffusion test, chlorhexidine 0.12% (v/v) was used as positive control and distilled water as negative control for gram positive, gram negative and yeasts.The antibiotic Rifamycin was used as positive control for antimicobacterial test.The minimum inhibitory concentration (MIC) was performed by broth microdilution according to the methodology proposed in document M27A3 (CLSI, 2008a) using 96 well plates.The extracts were diluted in Mueller Hinton broth at concentrations of 25 mg/ml to 0.05 mg/ml.It was used Mueller Hinton broth inoculated with the tested organism as positive control, Mueller Hinton broth uninoculated as a negative control and Mueller Hinton broth with only the extract as sterility control of the extract.To determinate the minimum microbicidal concentration (MMC), 10 µL of each well was inoculated into nutrient agar plates.After an incubation of 24 h at 37°C, the MMC was considered as the lowest concentration where no visible growth and was detected on nutrient agar.All experiments were performed in triplicate.

Antioxidant activity of extracts
Different concentrations of the extracts (from 400 to 1.56 mg/ml) in an ethanolic solution (2 ml) were mixed with 0.5 ml of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (0.5 mM, diluted in ethanol).After incubation for 30 min in the dark, the absorbance was measured at 517 nm.The blank test was composed of all reagents except extracts.Ascorbic acid, butylated hydroxytoluene (BHT) and quercetin were used as positive controls.The abduction property was calculated as the percentage of abducted DPPH radical by using the following equation: Seizure of DPPH (%) = [(absorbance of blank -absorbance sample)/(absorbance blank)] x 100 and the EC50 was determined for each extract.All experiments were performed in triplicate (Yen, 2005).

Evaluation of the cytotoxic activity of extracts on cell culture
Cytotoxicity was assessed by 3 -(4,5-dimethylthiazol-2YL) -2,5diphenyltetrazolium bromide (MTT) method.In this test, 1 x 10 4 BHK-21 cells (baby hamster's kidney cells) were seeded per well in 96-well plates containing the medium Eagle's Minimum Essential (MEM) with 10% fetal bovine serum and antibiotics.After 24 h, the medium was discarded and then 0.1 ml of MEM containing 1% fetal bovine serum with decreasing dilutions of the extracts (5 to 0.039 mg/ml) was added to the cultures for 48 h.After incubation, 10 μL of MTT at a concentration of 5 mg/ml was added and incubated for 4 h at 37°C for the MTT incorporation and for the formation of formazan crystals.To solubilize the formazan crystals, the medium was discarded and 0.1 ml of DMSO was added to wells.Spectrophotometric analysis was performed on a microplate reader at 570 nm.The percentage of cytotoxicity was calculated by using the formula [(A-B)/ Ax100], where A and B are values of the optical densities of the control and treated cells, respectively.All experiments were performed in triplicates (Araújo, 2008).

Statistical analysis
Statistical analysis of the results was performed by the SISVAR 5.3 software, using the analysis of variance (ANOVA) and the Scott-Knott test to observe significant differences between average values (p <0.05 ) (Scott and Knott, 1964)

RESULTS AND DISCUSSION
The granulometric assessment of the ground material is impostant, as it has direct influence on the efficiency of the extraction process.The powders were classified as moderately thick according to the Brazilian Pharmacopeia (2010).The average partical size for leaf, stem, root and fruit were 204.43, 218.04, 216.39 and 200.20 mm, respectively.Particles with homogeneous dimensions increase the contact area between the solid material and extractor liquid, making the extraction more efficient (Migliato et al., 2007).Phytochemical screening revealed the presence of flavonoids and tannins for all extracts.
The phenolic content ranged from 71.66 to 335.64 mg GAE/g.The dried stem extract showed the highest content of phenolic compounds (335.64 mg GAE/g) and dried fruit showed the lowest (71.66 mg GAE/g) (Table 1).Regarding the content of flavonoids, the dried leaf extract showed the highest content (18.52 mg QE/g) and the fresh root showed the lowest (1.01 mg QE/g) (Table 1).The dried extracts showed statistically higher values than fresh extracts.In studies conducted by Jayalakshmi et al. (2011) and Jayalakshmi et al. (2012), leaf extract of E. cotinifolia also presented flavonoids and tannins, corroborating our results.Taking the higher biological activity into account, the extracts of fresh and dried plant were selected for  chemical analysis by high performance liquid chromatography (HPLC).It can be observed in the chromatograms that the first eluted compounds exhibit more polarity characteristics than later compounds.Thus, a higher methanol concentration for the drag of these substances were required.
Considering the polar characteristics of phenolic compounds, it is suggested that first eluted peaks correspond to phenolic compounds, since they have spectra with absorption in the ultraviolet region.Comparing the peaks of around retention time 18 min of fresh leaf (peak 2; Figure 1A) and the dried leaf (peak 6; Figure 1B,), the area of the peak in the dried leaf is about 10 times larger than in the fresh leaf.According to the used standards, it was possible to identificated the presence of caffeic acid in leaf extracts (for fresh peak 2, Figure 1A, for dry peak 6, Figure 1B).The result obtained by liquid chromatography confirms the results expressed in the analysis of phenolic compounds by the Folin-Ciocalteau method and flavonoids by the aluminum chloride method, where it was also observed a highest concentration of phenolic and flavonoid compounds in the dried leaf extract when compared to fresh leaf extract.
In previous studies, 17 polyphenols were isolated using spectrometry, mass spectrometry (ESI-MS) and nuclear magnetic resonance (1H NMR) and 1D/2D NMR from the leaf of E. cotinifolia (Marzouk et al., 2012).Highlighting the presence of phenolic compounds in E. cotinifolia.Results of agar diffusion test showed an antibacterial activity against B.subtilis, B.cereus, M. luteus, S. aureus and P. mirabilis.M. luteus was the most sensitive bacteria and presented
areas of inhibition between 15 and 20 mm (Table 2).The P. mirabilis was inhibited by all extracts and inhibition areas ranged between 10 to 13 mm.No inhibition was observed for E. faecalis, E. aerogenes, E. coli, P. aeruginosa, S. typhimurium, S. marcescens, M. bovis, M. tuberculosis, C. albicans and S. cerevisiae.The leaf extracts (fresh or dry) showed greater spectrum of activity followed by the stem, root and fruit extracts, respectively.The MIC values were below 6.25 mg/ml and the MMC between 3.12 and 25 mg/ml (Table 3).The leaf extracts presented the lowest MICs, followed by stem, root and fruit extracts, respectively.The dried extracts showed lower MICs and MMCs when compared to fresh extracts and the highest concentration of phenolics and flavonoids for leaf extract may be closely related to the antimicrobial activity (Choi et al., 2006).Phenolic compounds act on the cytoplasmic membrane, changing its structure and function, change the active transport and coagulate the cellular content (Burt, 2004) (MTCC 7443).The MIC ranged from 0.312 to 1.25 mg/ml (Jayalakshmi et al., 2014).Although the same bacteria had been used, they showed lower MIC, but they are different strains, which may lead to these variations.Differences arising from the soil, climate and seasonality can influence the chemical composition of the plant, resulting in a higher concentration of compounds, thus influencing the biological activity.For the DPPH free radical scavenging activity, the highest antioxidant potential was shown on the extract of dried leaf, which had the lowest concentration capable of sequestering 50% of DPPH radicals, with EC 50 of 7.32 μg/ml (Table 4).Correlation between phenolic compounds and antioxidant activity was positive (r 2 = 0.32).Extracts showed the highest concentration of phenolic compounds showed higher antioxidant activity.
Several biological activities such as the antioxidant and antimicrobial may be related to phenol contents such as tannins and flavonoids.The dried extracts showed higher concentration of the total phenolics and flavonoids and this could explain the fact that there is greater antioxidant activity in the dry extracts, since the phenolic compounds are closely linked to antioxidant activity (Einbond et al., 2004;Banerjee and Dasgupta, 2008;Choi et al., 2006).
Although the dried leaf extract did not present the highest concentration of phenolic compounds, this extract has the highest concentration of flavonoids among all tested extracts.This differential flavonoid concentration could justify the high antioxidant activity observed.Marzouk et al. (2012) isolate polyphenols, including two new ellagitannins and one trigaloil glucosilcampferol from E.cotinifolia, and they showed high activity in the DPPH assay, with EC 50 values lower than the ascorbic acid.In a study by Jayalakshmi et al. (2014) the E.cotinifolia chloroform extract was the most active with an EC 50 of 15 µg/ml followed by petroleum ether, ethyl acetate and methanol, with EC 50 values of 17, 18 and 19 mg/ml, respectively.These scavenging activities of the extracts were considered higher when compared with the standard.These studies corroborate with our results, since the leaf extracts leaf (dry and fresh), dry root, dry fruit, fresh stem, presented better results for the BHT, emphasizing the potential antioxidant activity of E. cotinifolia.The extracts showed no citoxicity against BHK-21 cells in all concentrations tested.Betancur-Galvis et al. (2002) valuated the cytotoxic activity using the MTT method of several species of Euhorbia genus, among them, the E. cotinifolia.The hydromethanolic extracts of leaf and stem also showed no cytotoxicity, corroborating our results.

Conclusion
All extracts of E. cotinifolia showed antibacterial, antifungal and antioxidant activities, high levels of phenolic compounds and the presence of secondary metabolites.The HPLC-DAD analysis suggests the presence of caffeic acid in leaf extracts.The extract of dried leaves showed lower MIC and MMC for antimicrobial activity and lower EC 50 values for antioxidant activity.Extracts showed no activity against mycobacterial.Extracts showed no significant citoxicity.The results provided evidence that the studied plant might indeed be potential sources of natural antioxidant and antimicrobial agents.

Table 1 .
Content of total phenolics and flavonoids in the extracts of Euphorbia cotinifolia.
c *Results expressed as mean ± standard deviation (n = 3).Means with different letters are statistically different in the same column or compound (Scott -Knott p < 0.05).** Milligrams of gallic acid (GA) per gram of sample.*** Milligrams of quercetin per gram of sample.

Table 3 .
Minimum inhibitory concentration (MIC) and minimum concentration microbicide (MMC) in mg/ml extracts of Euphorbia cotinifolia.