A.L. Gindri Et Al.Natural Product Research

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A.L. Gindri et al.Natural Product Research

SUPPLEMENTARY MATERIAL

Genotoxic evaluation, secondary metabolites and antioxidant capacity of leaves and roots of Urera baccifera Gaudich (Urticaceae)

A.L. Gindria, L.B. de Souzab, R.C. Cruza, A.A. Boligona, M.M Machadoc, M. L. Athaydea*

aPost Graduate Program in Pharmaceutical Sciences, Industrial Pharmacy Department, Federal University of Santa Maria, Santa Maria, Brazil.

bPost Graduate Program in Agrobiology, Biology Pharmacy Department, Federal University of Santa Maria, Santa Maria, Brazil.

cPost Graduate Program in Pharmaceutical Sciences, Federal University of Pampa, Uruguaiana, Brazil.

Genotoxic evaluation, secondary metabolites and antioxidant capacity of leaves and roots of Urera baccifera Gaudich (Urticaceae)

In addition to phenolics, flavonoids, flavonols, alkaloids, and condensed tannins, our tests identified the antioxidant and genotoxic properties in the crude extract and fractions of Urera baccifera (Urticaceae) roots and leaves. Oxalic acid contents were determined by HPLC-DAD and presented high values in the roots (1.82±0.21, 1.79±0.22, and 1.38±0.15 mg/g in butanolic, crude extract and ethyl acetate fraction, respectively). Oxalic acid caused a reduction of 30.7% in the leukocyte proliferation, followed by butanolic fractions of roots (24.15%) and leaves (23.28%). The mitotic index was lower in butanolic fractions of leaves (8.7%) and roots (8.3%), similarly to oxalic acid index, which was 6.0%. The DNA damage index in cultured leukocytes were observed for oxalic acid (19.33) and butanol fractions treatments (22.67 and 16, respectively, for leaves and roots). Antioxidant capacity (DPPH and TBARs) was moderated, confirmed by the low phenolic, flavonol and flavonoid contents in both parts of the plant.

Keywords: HPLC-DAD; DPPH; oxalic acid; phenolic compounds; TBARS; genotoxicity.

* Corresponding author:

Phytochemical Research Laboratory at Department of Industrial Pharmacy, Federal

University of Santa Maria, Build 26, room 1115, Santa Maria, RS, ZIP Code 91105-900, Brazil. Phone: 55 3220 8095

Experimental

Preparation of the sample extracts and fractions

The leaves and the roots of U. baccifera were collected in São Francisco de Assis, RS, Brazil (29°37.115'S, 054°53.970'W; height 150m) in May, 2010. The plant was identified by Dr. Renato Zachia, of Federal University of Santa Maria, and a voucher has been deposited at the University Herbarium with thevoucher specimen number 13.070. The material (1,000 grams of roots and 400 grams of leaves) were reduced, dried, milled and taken to maceration with 70% ethanol for 28 days, with daily agitation. Once every week, each extract were filtrated, giving the respective ethanol extract, and fresh extraction solvents were added. The extracts were combined and the ethanol was eliminated by a rotary evaporator thereby producing the aqueous extracts (roots and leaves separated). Parts of the aqueous extracts were completely dried, creating crude extracts (CE), and part were fractionated with the crescent polarity solvents chloroform (CHCl3), ethyl acetate (EtOAc) and butanol (BuOH). The fractions were equally dried.

Determination of total polyphenols

The total polyphenol contents of the crude extract and the fractions from the leaves and roots of U. baccifera were measured following the Chandra Mejia (2004) methodology. In this method, 1.0ml of Folin-Ciocalteau reagent was added to 1.0 ml of each sample at 150µg/ml. After 5 minutes 2.0 ml of calcium carbonate 20% were added. The test was performed in triplicate and all solutions were diluted in ethanol. The absorbance was read after 10 minutes in a spectrophotometer at 730 nm. The total polyphenol content was expressed in milligram equivalents of gallic acid per milliliter of each extract or fraction. Gallic acid was used as standard (0.001–0.03 mg/ml of gallic acid). The obtained equation was y=30.767x-0.0087, r=0.9992.

Determination of total flavonoids

The quantification of flavonoids followed the Woisky Salatino (1998) methodology with few modifications. Into 2.0 ml of the crude extract and the fractions from the leaves and roots U. baccifera, in a concentration of 150 µg/ml,1.0 ml of aluminum chloride 2% was added. After one hour the samples were read in spectrophotometer at 420 nm. All solutions were diluted in methanol and the test was made in triplicate. The equation obtained with the standard quercetin (0.002–0.018 mg/ml) was y=40.175x+0.001, r=0.9998 and the results were expressed in milligram equivalents of quercetin per milliliter of extract/fraction.

Determination of total flavonols

The total flavonols were estimated using the method of Jimoh, Adedapo, Aliero, Afolayan (2010), where into 2.0 ml of the samples in the concentration of 400µg/ml each,2.0 ml of aluminum chloride 2% and 3.0ml of sodium acetate 50 g/l were added. After 2.5 hours, the absorbance was read at 440nm. The test was made in triplicate and all solutions were diluted in ethanol. Standard quercetin (0.002–0.026 mg/ml) was used to obtain the calibration curve: y=24.478x - 0.0251, r=0.9926. Results were expressed in milligram equivalents of quercetin per milliliter of extract/fraction.

Determination of condensed tannins

Condensed tannins were determined using the methodology ofMorrison, Asiedu, Stuchbury & Powell (1995). Briefly, 0.1ml of the samples from the leaves and the roots in 25mg/ml in methanol, were added to 0.9ml of methanol and 5.0ml of vanillin reagent (equal volumes of: 8.0mlhydrochloric acid concentrated in 100.0ml methanol and 1.0g vanillin in 100.0ml methanol). After a 20-minutes water bath 40ºC, the absorbance was read at 500nm. The analysis was carried out in triplicate and the standard was catechin (0.0025–0.2 mg/ml): y=0.0015x-0.0005, r=0.9968. The results were expressed in milligram equivalents of catechin per milliliter of extract/fraction.

Determination of total alkaloids

The method of Oliveira et al. (2006) was used to determine the total alkaloids on the crude extract and the fractions. In this methodology, 5.0ml of the samples (60.0mg/ml in water, pH adjusted to 2.0 with hydrochloric acid 1%) were mixed with 2.0mlof Dragendorff. The tubes were subject to centrifugation for 30 minutes at 2400rpm and the supernatant was removed. The precipitate was washed with ethanol and the solvent was discarded. The residue was treated with dissodic sulfite 1% and centrifuged for 10 minutes at 2400 rpm, removing the supernatant one more time. The precipitate was dissolved in concentrated nitric acid and transferred to a 10.0ml volumetric flask filled with distillated water. An aliquot of 1.0ml of this solution was mixed with 5.0ml of thiourea, and read as 435nm on a spectrophotometer, using as a background a solution of 1.0ml nitric acid and 5.0ml of thiourea. The test was made in triplicate and the standard was bismuth nitrate. The equation obtained (0.01–0.09 mg/ml) was y=2.2783x+0.0361, r=0.9997. The results were expressed in milligram equivalents of alkaloids per milliliter of extract/fraction.

Antioxidant activity using the DPPH photometric assay

The antioxidant activity with the DPPH (2,2-diphenyl-1-picryl-hydrazyl radical) photometric assay was based on Choi et al. (2002). Six different ethanol dilutions of each fraction and crude extract consisting at 250, 125, 62.5, 31.25, 15.62 and 7.81 µg/ml were mixed with 1.0 ml of DPPH (12.0 mg DPPH + ethanol qsp 100.0 ml). Ethanol (1.0 ml) + plant extract/fraction (2.5 ml) was used as a blank for each concentration and the negative control was DPPH (1.0 ml) + ethanol (2.5 ml). The positive control was ascorbic acid in the same concentration as the samples. The entire test was performed in triplicate. The reaction occured in 30 minutes, and then the absorbance was read on the spectrophotometer at 518 nm. The values were converted into percentage using the following equation: scavenging capacity % = 100 – [(Abs of sample – Abs of blank) X 100/ abs control]. A graphic was plotted, with the results of scavenging capacity % (ordinate) related with the concentration of the samples (abscissa) while the IC50 (extract inhibitory concentration of 50% DPPH radical activity) was calculated by linear regression of plots.

Lipid peroxidation assay

Lipid peroxidation was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method ofOhkawa, Ohishi, & Yagi (1979). Peripheral human blood samples were collected after 12 hours of overnight fasting by vein puncture (protocol 23081.005770/2009-38) using top Vacutainer (BD Diagnostics,Plymouth, UK) tubes with heparin. Blood specimens (5.0 ml) were routinely centrifuged within one hour of collection for 15 min at 2500 g, and aliquots of plasma and erythrocyte samples were stored at -20ºC until biochemical analysis. Red blood cells were placed into contact with the varying concentrations of the plant for two hours. After this period, 10 mM of freshly prepared iron sulfate solution (FeSO4) was added. The measurement was expressed in nmol of malondialdeyde (MDA)/ ml erythrocyte.

Oxalic acid quantification by high performance liquid chromatography (HPLC)

Oxalic acid was quantified following the Fu et al. (2006) methodology with minor modifications. High performance liquid chromatography (HPLC-DAD) was performed with the HPLC system (Shimadzu, Kyoto, Japan), Prominence Auto-Sampler (SIL-20A), equipped with Shimadzu LC-20 AT reciprocating pumps connected to the degasser DGU 20A5 with integrator CBM 20A, UV–VIS detector DAD SPD-M20A and Software LC solution 1.22 SP1. C-18 column (4.6 mm x 250 mm) packed with 5 µm diameter particles. The mobile phase was 25 mM phosphate buffer (pH 2.5): acetonitrile at 95:5, at flow rate 0.8 ml/min. The peak was identified by comparison with the retention time of the standard solution, in a wavelength of 207 nm. The samples concentrations (5.0 mg/ml) were tested in triplicate. The calibration curve of oxalic acid in the concentration range of 1.0–12.0 mg/ml was made in triplicate and the equation obtained was: y= 10.548.869x + 6.236.233, r=0.9958.

Determination of the effect on the genetic material of leukocytes in culture

Leukocyte cultures were prepared using samples collected from human donors by vein puncture. Whole blood collected was immediately transferred to a culture medium containing 1ml of RPMI 1640, supplemented with 10% fetal bovine serum and 1% streptomycin / penicillin, as described in a previous study by Santos Montagner et al. (2010). The cells were placed in an incubator at 37°C in a microaerophilic environment for 72 hours. Each sample (extracts and fractions) and 0.8 mg/ml oxalic acid (Sigma standard), diluted in PBS buffer, was added to the blood to get a final concentration of 100 μg/ml. Each group was composed of three culture flasks. After growth for 72 hours, the genetic parameters were analyzed using the techniques described below.

Viability was assessed by the loss of membrane integrity, using the trypan blue (Burow et al. 1998).The differentiation of living/dead cells was observed by the blue coloration of dead cells. At least 300 cells were counted in this technique. Cell viability was expressed as a percentage of the control value. The amount of total leukocytes (proliferation) was determined by counting in a Neubauer chamber. In this technique, samples are mixed with Turk's solution (acetic acid 3% and 1% gentian violet in water) and placed in the chamber and counted in four quadrants (Santos Montagner et al. 2010). The genotoxicity test was evaluated by Comet Assay(Singh, McCoy, Tice & Schneider,1988). In this technique, the samples are dispersed in a slide containing agarose gel, which passes by electrophoresis and staining with silver nitrate, allowing the visualization of DNA residues. These residues have different electrophoretic mobility, forming a tail-to-tail resembling a comet.

Statistical analysis

The statistical analysis was performed using version 7.0 of the Statistica software. Differences among experimental data were analyzed by one-way ANOVA followed by Tukey test, and those at p< 0.05 were considered significant.

To the genotoxic evaluation ANOVA was used for statistical analysis followed by post hoc test of Bonferroni. Differences between groups were considered significant at p < 0.05.

Acknowledgement:

We thank Professor R.A. Zachia (Federal University of Santa Maria) for providing the botanical identification of Urera baccifera.

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Table S1.Results of polyphenols, flavonoids, flavonols, condensed tannins, and alkaloids obtained from the roots and leaves of U. baccifera.

SD=Standard deviation; CE=crude extract; CHCl3=Chloroform fraction; EtOAc=ethyl acetate fraction; BuOH=butanol fraction; Different letters in each treatment represent significant differences.

Table S2.Results of the roots and leaves of U. baccifera in the lipid peroxidation assay (nmol MDA/ ml) and the DPPH assay. IC50 and percentage of DPPH scavenging at 250 µg/ml.

CE=crude extract; CHCl3=Chloroform fraction; EtOAc =ethyl acetate fraction; BuOH =butanol fraction; IC50 (inhibition concentration of 50% oxidant activity); SD: Standard deviation. Different letters in each treatment represent significant differences.

Table S3. Levels of oxalic acid (mg/g) in roots and leaves of U. baccifera

SD=Standard deviation; CE=crude extract; CHCl3=Chloroform fraction; EtOAc=ethyl acetate fraction; BuOH =butanol fraction. Different letters in each treatment represent significant difference

Figure S1. A. Effects of U. baccifera on cell proliferation in cultured leukocytes. B. Effects of U. baccifera on cellular death in cultured leukocytes. Different letters represent statistically different results. Legend: NC: negative control; OA: Oxalic acid; EBR: Crude extract of Root; ECR: Root chloroformic fraction; EAR: Root ethyl acetate fraction; EBuR: Root butanol fraction; EBF: Crude leaf extract; ECF: Leaves’ chloroformic fraction; EAF: Leaves’ ethyl acetate fraction; EBuF: Leaves’ butanol fraction.

Figure S2. A. Effects of U. baccifera on mitotic index in cultured leukocytes. B. Effects of U. baccifera on the production of micronuclei in cultured leukocytes. C.Effects of U. baccifera in DNA damage index in cultured leukocytes.Different letters mean statistically different results. Legend: NC: negative control; OA: Oxalic acid; EBR: Crude extract of root; ECR: Root chloroformic fraction; EAR: Root ethyl acetate fraction; EBuR: Root butanol fraction; EBF: Crude leaf extract; ECF: Leaves’ chloroformic fraction; EAF: Leaves’ ethyl acetate fraction; EBuF: Leaves’ butanol fraction.