Cytoprotective Effect of Eugenia Uniflora L. Against the Waste

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ORIGINAL ARTICLE Cytoprotective eﬀect of Eugenia uniﬂora L. against the waste contaminant mercury chloride

Francisco A.B. Cunha a,b, Antonio I. Pinho a,b, Joycy F.S. Santos a, Celestina E. Sobral-Souza c, Rosimeire S. Albuquerque c, Edinardo F.F. Matias c, Nadghia F. Leite c, Saulo R. Tintino c, Jose´G.M. Costa c, Aline A. Boligon b, Emily P. Waczuk b, Joa˜ o B.T. Rocha b, Thais Posser b, Henrique D.M. Coutinho c,*, Lucindo J. Quintans-Ju´nior d, Jeferson L. Franco b

a Departamento de Cieˆncias Biolo´gicas da Universidade Regional do Cariri – URCA, Crato, CE, Brazil b Programa de Po´s Graduac¸a˜o em Bioquı´mica Toxicolo´gica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil c Departamento de Quı´mica Biolo´gica da Universidade Regional do Cariri – URCA, Crato, CE, Brazil d Departamento de Fisiologia da Universidade Federal de Sergipe – UFS, Aracaju, SE, Brazil

Received 22 January 2016; accepted 27 April 2016

KEYWORDS Abstract Environmental stressors such as mercury are important for the search of substances to Mercurium chloride; lower induced oxidative damage. Accordingly, secondary metabolites from plants with antioxidant Negative geotactic effect; activity represent an interesting alternative. The aim of this work was to evaluate the cytoprotective Chelating effect; effect of the ethanol extract of Eugenia uniflora against mercury chloride. The main antioxidant Cytoprotection; phytochemicals were quantified by HPLC. The chelating effect was evaluated using the Heavy metal O-phenanthroline method. The cytoprotective effect was assayed using bacterial and plant models. The cytotoxic and the negative geotaxis effect was determined using Drosophila melanogaster. The ethanol extract of E. uniflora demonstrated chelating effect against iron, and these results can be related to the total phenols (1079 mg/g) and flavonoids (946.9 mg/g), detected by HPLC and quantified. The same extract demonstrated a cytoprotective effect in both models and a non-toxic effect against D. melanogaster, with low mortality and low inhibition of geotaxis, demonstrating that the extract can reduce the toxicity of this heavy metal against prokaryotic and Eukaryotic organisms. Due the results, we can conclude that the phytocompounds in the ethanol extract of E. uniflora, possibly phenols and flavonoids, could be interesting agents to protect different organisms against the heavy metal damages by a chelating or an antioxidative mechanism. Other studies are still

* Corresponding author at: Universidade Regional do Cariri, Departamento de Quı´ mica Biolo´ gica, Av. Cel. Antoˆnio Luiz, 1161, CEP: 63105-000 Crato, CE, Brazil. E-mail address: [email protected] (H.D.M. Coutinho). Peer review under responsibility of King Saud University.

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http://dx.doi.org/10.1016/j.arabjc.2016.04.018 1878-5352 Ó 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

necessary; however, these ﬁndings can open a new perspective to the recuperation of environments contaminated by heavy metals. Ó 2016 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction 2.2. Plant material

Brazil has a large biodiversity, so prospecting for biologically active Leaves of E. uniflora L. were collected in the Horto Botaˆnico molecules represents an excellent opportunity to enhance our knowl- of Crato, Brazil, with the following coordinates: 7°14028.000S edge about this diversity, preserve these natural resources and discover ° 0 00 and/or develop technological novelties. The search for natural prod- and 39 24 56.7 W. The plant was identified by Dr. Maria ucts with antioxidant activities such as flavonoids and phenols to min- Arlene Pessoa, and a sample was deposited in the Herbarium imize or reverse toxic effects from environmental stressors is a growing Da´ rdano de Andrade Lima, at the Universidade Regional do research area, with interesting perspectives for environmental conser- Cariri- URCA, under voucher number #3106. vation and industrial development (Cacciola et al., 2007; Dartora et al., 2011). 2.3. Preparation of ethanol extract of E. uniflora leaves(EEEU) Eugenia uniflora (Myrtaceae), known as ‘‘pitanga,” is a tree found all over Brazil. The family Myrtaceae comprises 144 genera with 5774 species (The Plant List, 2013). Several biological activities have been The extract was prepared by immersing 650 g leaves in ethanol reported for this plant, including antimicrobial (Oliveira et al., 2006; for 72 h at room temperature, which was filtered and concentrated Simo˜es et al., 2010), antioxidant (Velazquez et al., 2003) and hypoten- using a vacuum rotary evaporator (model Q-344B- Quimis, sive (Consolini and Sarubbio, 2002). Brazil) and warm water bath (model Q214M2- Quimis Brazil). The leaves of this plant contain several biologically active sub- The yield of crude extract was 1.07% (Matos, 2009). stances such as mono- and triterpenes, flavonoids, tannins and others (Amorim, 2009). Among the flavonoids, several compounds with 2.4. Apparatus and general procedures known antioxidant activity have been reported including myricetin, quercetin and rhamnosides (Rattmann, 2012). The leaves of this species have been found to contain several phytochemicals with chelating High performance liquid chromatography (HPLC-DAD) was and antioxidant activities, which suggest that this has interesting performed with a Shimadzu Prominence Auto Sampler (SIL- potential to protect against or reverse the toxic effect of heavy metals 20A) HPLC system (Shimadzu, Kyoto, Japan), equipped with such as mercury. The main models for the study of neurological dam- Shimadzu LC-20AT reciprocating pumps connected to a DGU age induced by mercury use rats or mice, but Balamurugan et al. 20A5 degasser with a CBM 20A integrator, SPD-M20A diode (2009) and Posgai et al. (2011) reported that invertebrate models, array detector and LC solution 1.22 SP1 software. although rarely used, represent good models with compatible results in several cases. One of the main invertebrate models involves 2.5. Quantification of compounds by HPLC-DAD Drosophila melanogaster (Paula, 2012). This fly species is a model widely used in genetic research, but a few years ago, this model began being utilized in research of neurodegenerative diseases such as Reverse phase chromatographic analyses were carried out Parkinson´s disease. The advantage of using adult flies is the synchro- under gradient conditions using C18 column nized aging of their cells, except the gonads and intestinal cells, and (4.6 mm 250 mm) packed with 5-lm diameter particles. the similarity with mammals with regard to dopaminergic neurons The mobile phase was water containing 1% formic acid (A) (Jimenes-del-Rio, 2010). Other models to investigate mercury toxicity and acetonitrile (B), and the gradient composition was as fol- make use of bacteria and plants, but these models are much more lows: 13% B until 10 min and changed to 20%, 30%, 50%, uncommon. Sobral-Souza et al. (2014) used these models and demon- 60%, 70%, 20% and 10% B at 20, 30, 40, 50, 60, 70 and strated the cytoprotective effect of Eugenia janbolana Lam. against the toxic damage induced by mercury. In this same work, the extract 80 min, respectively, according to Kamdem et al. (2013) with exhibited a cytoprotective effect, while the essential oil of the same slight modifications. E. uniflora extracts and mobile phase were plant demonstrated a cytotoxic potential, making it an interesting filtered through a 0.45-lm membrane filter (Millipore) and bioinsecticide. then degassed using an ultrasonic bath prior to use, and the The aim of the present work was to evaluate the cytoprotective samples from E. uniflora extracts and decoction were analyzed effect of the ethanol extract of E. uniflora L. using bacterial and plant at a concentration of 5 mg/mL. The flow rate was 0.6 mL/min, models and to determine the in vivo toxicity using the D. melanogaster and injection volume 50 lL The detection wavelength was model. 254 nm for gallic acid, 280 nm for catechin and epicatechin, 324 nm for caffeic, chlorogenic and ellagic acids, and 366 nm for quercitrin, quercetin, rutin and kaempferol. All samples 2. Materials and methods and mobile phase were filtered through a 0.45-lm membrane filter (Millipore) and then degassed using an ultrasonic bath 2.1. Chemical reagents prior to use. Stock solutions of reference standards were prepared in HPLC mobile phase in a concentration range of All chemicals were of analytical grade. Acetonitrile, formic 0.030–0.250 mg/mL for catechin, epicatechin, quercitrin, quer- acid, gallic acid, chlorogenic acid, caffeic acid and ellagic acid cetin, kaempferol and rutin, and 0.025–0.300 mg/mL for gallic, were purchased from Merck (Darmstadt, Germany). Catechin, chlorogenic, caffeic and ellagic acids. The chromatography epicatechin, quercitrin, quercetin, rutin and kaempferol were peaks were confirmed by comparing their retention time with acquired from Sigma Chemical Co. (St. Louis, MO, USA). those of reference standards and by DAD spectra (200 to

Please cite this article in press as: Cunha, F.A.B. et al., Cytoprotective effect of Eugenia uniflora L. against Eugenia uniflora L. –>the waste contaminant mercury chloride. Arabian Journal of Chemistry (2016), http://dx.doi.org/10.1016/j.arabjc.2016.04.018 Cytoprotective effect of Eugenia uniflora L. 3

600 nm). The calibration curves were as follows: gallic acid, concentrations (400, 300, 200, 100, 50, 25 and 10 mg/mL) pre- Y = 12674x + 1283.6 (r = 0.9999); catechin, Y = 13185x + pared in 99.6% ethanol. 1196.5 (r = 0.9993); chlorogenic acid, Y = 12984x + 1271.4 (r = 0.9999); caffeic acid, Y = 11972x + 1370.3 (r = 2.9. Chelating effect 0.9996); epicatechin, Y = 13256x + 1371.2 (r = 0.9993); ellagic acid, Y = 12679x + 1277.1 (r = 0.9995); quercitrin. The method of Benzie and Strain (1996) and Benzie and Szeto Y = 13183x + 1274.6 (r = 0.9995); quercitrin, Y = 12547x + (1999) with modiﬁcations was adopted for the assay. The prin- 1163.5 (r = 0.9997); rutin. Y = 12983x + 1321.6 (r = 0.9998); ciple is based on the formation of O-phenanthroline–Fe2+ and kaempferol, Y = 13048x + 1256.7 (r = 0.9994). All chro- complex and its disruption in the presence of chelating agents. matography operations were carried out at ambient temperature A volume of 100 lL of extract, from 64 to 2048 lg, was added in triplicate. The limit of detection (LOD) and limit of quantiﬁ- to 50 lL of 2.0 mM aqueous FeSO4. The controls contained all cation (LOQ) were calculated on the basis of the standard devi- the reaction reagents except the extract or positive control sub- ation of the responses and the slope using three independent stance. After 10 min incubation, the reaction was initiated by analytical curves. LOD and LOQ were calculated as 3.3 and 200 lL of 6.0 mM O-phenanthroline. After a 10-min equilibra- r r 10 /S, respectively, where is the standard deviation of the tion period, the absorbance was read at 510 nm. Iron chelation response and S is the slope of the calibration curve (Boligon activity was calculated from the absorbance of the control (Ac) et al., 2013). and of the sample (As) and expressed as Na2EDTA equivalents (mg Na2EDTA/g extract). The values are presented as 2.6. Microorganisms the means of triplicate analyses.

The bacterial strain utilized was Escherichia coli 11105, 2.10. Evaluation of cytoprotective potential of EEEU against donated by the Mycology Laboratory of Universidade Federal mercury chloride da Paraı´ ba (UFPB). The minimum inhibitory concentration (MIC) of EEEU was 2.7. Drosophila stock and culture determined by the microdilution assay according to Coutinho et al. (2008), with modifications, using suspensions D. melanogaster (Harwich strain) was obtained from the of E. coli ATTC 11105 in saline at 105 CFU/mL and extracts National Species Stock Center, Bowling Green, OH. The flies starting at a concentration of 1024 lg/mL. MIC was defined were reared in 2.5 6.5 cm2 glass bottles containing 5 mL of as the lowest concentration at which no growth was observed. standard medium (1% w/v brewer’s yeast; 2% w/v sucrose; The protective effect of EEEU against heavy metals was eval- 1% w/v powdered milk; 1% w/v agar; 0.08% v/w nepagin) uated by using subinhibitory concentrations of extracts, bacte- at constant temperature and humidity (25 ± 1 °C; 60% rela- rial suspensions of 105 CFU/mL in M9 Tris medium with 2% tive humidity, respectively). All experiments were performed glucose and dilutions of mercury chloride from 10 to with the same strain. 0.004883 mM. The microdilution plates were incubated for 48 h at 37 °C. The minimum bactericidal concentration 2.8. Total phenols and flavonoids (MBC) was determined as the lowest concentration capable of inhibiting the growth of the microorganisms, utilizing Petri The quantity of total phenols was determined by adding plates with heart infusion agar (HIA) for transfer of solutions 200 lL of extract dilution (300, 100, 50 and 25 l g/mL in incubated in microdilution plates. 99.6% ethanol) to 1 mL of Folin–Ciocalteau reagent (10%, v/v) and mixing for 1 min. Next, 800 lL of 7.5% sodium car- 2.11. Test for cytoprotective effect in lettuce seeds bonate was added, and the sample was homogenized for 30 s. (germination) After 1 h, absorbance was read in a spectrophotometer with the wavelength set at 765 nm. The blank was determined with The experiments were performed in clean, dry, sterile Petri all reagents, but the extract was replaced with distilled water. plates lined with two filter paper disks, on which lettuce seeds The test was done in triplicate. The mean of three readings were placed. In each plate, 3 mL of solution was added. The was used to determine the total phenols, expressed as mg gallic concentration of extract was 256 lg/mL, and mercury chloride acid equivalents/g extract, by extrapolating this value on the solutions were at concentrations of 1.25, 0.5, 0.1 and 0.05 mM. calibration curve constructed with gallic acid standards. The A control plate was moistened with 3 mL of distilled water. calibration curve for gallic acid was determined using different The experiments were conducted in a BOD germination cham- concentrations (300, 100, 75, 25 and 10 mg/mL). Flavonoids ber at a temperature of approximately 25 °C with a 12-h pho- were quantified by preparing solutions of extract (300, 200, toperiod for seven days. The treatments were arranged in a 100 and 50 mg/mL) and adding 1 mL of these to 1 mL of randomized complete block design (RCBD), with three repeti- 2% (w/v) aluminum chloride (AlCl3). Aluminum chloride tions of 20 seeds per plate. The parameters analyzed at the end was replaced with distilled water in a blank tube. After of seven days were as follows: number of seeds germinated, 30 min incubation at room temperature, absorbance was read germination speed index (GSI), biometry of plumule and radi- using a 415-nm filter. The test was done in triplicate, and the cle, and number of necrotic radicles and seedling abnormali- mean was used for determination of the quantity of total flavo- ties, according to the guidelines for analysis of seeds (Brasil, noids and expressed as mg quercetin equivalents/g extract. The 2009). Seeds were considered germinated if their radicle calibration curve for quercetin was determined using different reached 1 mm in length. GSI was evaluated every 24 h, which

2.12. Mortality tests

Adult flies (males and females) were placed in 330-mL flasks containing at the bottom 1 g filter paper impregnated with 20% sucrose in distilled water. A counter-lid of polyethylene terephthalate (PET) was placed in the screw cap of the flask, to which parchment paper was taped for application of the different concentrations of essential oil. The flasks received the following treatments: 20% sucrose (control) and 2.5, 5 and 7.5 mg essential oil, with a 12-h light/dark cycle and controlled temperature at 25 ± 5 °C. Readings were taken at 6, 12, 24 and 48 h. Figure 1 Representative high performance liquid chromatogra- 2.13. Negative geotaxis assay phy profile of Eugenia uniflora. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), ellagic acid (peak 5), epicatechin (peak 6), rutin (peak 7), quercitrin (peak 8), Locomotor ability was determined using the negative geotaxis quercetin (peak 9) and kaempferol (peak 10). assay as described by Coulom and Birman (2004). Briefly, for each assay, 20 flies (7 days old; both genders) were immobi- lized on ice for 1–2 min and placed separately in vertical glass columns (length, 25 cm; diameter, 1.5 cm). After 30 min recov- Table 1 Phenolics and flavonoid composition of Eugenia ery, flies were gently tapped to the bottom of the column and uniflora. the time required for flies to climb up to 8 cm in the columns Compounds E. uniflora LOD LOQ was recorded. The assays were repeated five times at 1-min mg/L % lg/mL intervals. Results are presented as mean time (s) ± SE obtained in three independent experiments. In parallel with Gallic acid 4.93 ± 0.02 a 0.49 0.027 0.091 Catechin 1.08 ± 0.01 b 0.10 0.011 0.036 individual performance, in a second experiment, a group of Chlorogenic acid 8.75 ± 0.01 c 0.87 0.009 0.034 20 flies were gently tapped to the bottom of a glass column Caffeic acid 1.13 ± 0.02 b 0.11 0.036 0.119 and after 1 min, the number of flies that reach 8 cm of the col- Ellagic acid 11.65 ± 0.01 d 1.16 0.015 0.049 umn (top) and flies that remained below this mark (bottom) Epicatechin 2.47 ± 0.02 e 0.24 0.031 0.102 were counted. Rutin 5.19 ± 0.01 a 0.51 0.008 0.027 Quercitrin 11.73 ± 0.03 d 1.17 0.034 0.112 2.14. Statistical analysis Quercetin 13.82 ± 0.01 f 1.38 0.045 0.149 Kaempferol 4.74 ± 0.03 a 0.47 0.018 0.057 Statistical differences between groups were analyzed by one- Results are expressed as mean ± standard deviations (SD) of three way ANOVA followed by Duncan’s post hoc test. Differences determinations. LOD limit of detection (lg/ml), LOQ limit of l were considered statistically significant when P < 0.05. quantification ( g/ml). Averages followed by different letters differ by Tukey test at P < 0.05. 3. Results and discussion

3.1. HPLC analysis Table 2 Total of phenols, flavono´ ides and chelating effect of HPLC fingerprinting of E. uniflora extracts revealed the pres- EEEU. ence of gallic acid (RT = 10.32 min; peak 1), catechin Gallic acid Quercetine EEEU EDTA (RT = 15.26 min; peak 2), chlorogenic acid (RT = 20.15 min; Total phenols 1079.1 mg/g – 75.6 mg/g – peak 3), caffeic acid (RT = 24.91 min; peak 4), ellagic acid Flavono´ ides – 946.9 mg/g 42.5 mg/g – (RT = 30.04 min; peak 5), ellagic acid (RT = 34.98 min; peak IC50 – – 2430.9 lg/ml – 6), rutin (RT = 38.81 min; peak 7), quercitrin (RT = IC50 – – – 136 lg/ml 43.59 min; peak 8), quercetin (RT = 47.25 min; peak 9) and EEEU – Ethanol extract of leaves from Eugenia uniflora Lam.; IC50 kaempferol (RT = 58.16 min; peak 10) (Fig. 1 and Table 1). – Chelating concentration of 50% of Hg. HPLC analysis indicated the following phytochemicals in the ethanol extract of the leaves of E. uniflora: quercetin (peak 9), quercitrin (peak 8), ellagic acid (peak 5) and chlorogenic acid (peak 3). Rattmann et al. (2012), in studying leaves of these compounds are associated with the antioxidant activity ‘‘pitanga tree” identified similar compounds, including rham- observed. Using the fruits, Celli (2011) found the phytochem- nosides, myricetin and quercetin. Analyzing the seeds of icals myricetin 3 – O- hexoside, myricetin 3 – O- pentoside, E. uniflora, Oliveira (2014) observed the presence of ellagic myricetin 3 – O- rhamnoside, quercetin 3 – O- hexoside, acid, quercitrin and kaempferol, indicating in this study that quercetin 3 – O- pentoside, quercetin 3 – O- rhamnoside and

1000 *** 900 100 Sucrose 800 10 mg/mL **

M) 25 mg/mL

µ 700 50 mg/mL 600 95 75 mg/mL 300 100 mg/mL

200 Surveillance (%)

90 Concentration ( 100 3h 6h 12h 24h 48h 0 Time EEEU Control Figure 5 Biocide effect of EEEU (mg/mL) against D. melano- Figure 2 Cytoprotective effect of Eugenia uniflora against gaster. *** value statistically significant with P < 0.05. Escherichia coli. EEEU – Ethanol extract of leaves from Eugenia uniflora. *** Value statistically significant with P < 0.001. concentration of phenolic compounds (Okuda et al., 1992), mainly due to the oxidoreduction potential of various phyto- 4 chemicals on metal ions or due to the inhibition of lipid perox- Caulicles idation (Okuda et al., 1992). Radicles 3 *** 3.3. Chelating effect 2 ** Length (cm) 1 Table 2 shows the chelating effect of EEEU when compared with the control with EDTA. The EEEU demonstrated a sig- 0 nificant chelating effect, with EC50 = 2430.86 lg/mL. Accord- 1 H O EEEU ing to Cobbet et al. (2002), cellular sequestration of metals is 2 an efficient defense mechanism, where it is accomplished Figure 3 Germination and extension of caulicles and radicules of through metal chelators such as organic acids and amino acids. Lactuca sativa. Concentration of natural product used was 256 lg/ mL. EEEU – Ethanol extract of the leaves of Eugenia uniflora. 3.4. Evaluation of the cytoprotective potential of EEEU against *** value statistically significant with P < 0.0001. Mercury chloride using a bacterial model (E. coli)

According to the results in Fig. 2, EEEU exerted a significant myricetin deoxyhexoside-galate, and reported the relationship cytoprotective effect, because a higher concentration of mer- between these compound and the antioxidant activity. On the cury needed to kill the bacteria in the presence of this extract. basis of the HPLC analysis, the ethanol extract of E. uniflora Zerin (2012), in studying eukaryotic cells, observed that quer- appeared to have antioxidant activity, which indicated the cetin reduced the oxidative damage induced by paraquat due cytoprotective potential of this extract. to the reduction in reactive oxygen species production, demonstrating the cytoprotective effect of this compound. The same 3.2. Total phenols and flavonoids mechanism could be observed using the bacterial model with E. coli, because of quercetin being the main phytochemical in The results presented in Table 2 show the concentration of the the extract as observed in HPLC analysis. This is the first phenols and flavonoids present in EEEU. The antioxidant report of the cytoprotective effect of E. uniflora using this potential of a natural product is frequently related to the model.

4 *** Caulicles + HgCl 2 *** Radicles + HgCl 2 Caulicles + HgCl + EEEU 3 2 Radicles + HgCl 2+ EEEU

2 *** Lenght (cm) 1 *** 0

0,05mM 0,1mM 0,5mM 1,25mM

Concentration of HgCl 2

*** Figure 4 Cytoprotective effect of Eugenia uniﬂora (128 lg/mL) associated with HgCl2. Value statistically signiﬁcant with P < 0.0001.

Geotaxia Negative

20 * ** ** ** 15

10 Drosophilas

5 Nº of

10 25 50 75 10 25 50 75 10 25 50 75 10 25 50 75 10 25 50 75 Sac. 100 Sac. 100 Sac. 100 Sac. 100 Sac. 100

3 Hours 6 Hours 12 Hours 24 Hours 48 Hours

Figure 6 Negative Geotactic effect of EEEU (mg/mL) against D. melanogaster. Value *** statistically signiﬁcant at P < 0.05. Suc.: Sucrose.

3.5. Cytoprotective assay with seeds of lettuce (Lactuca sativa) References

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