Gentiana Asclepiadea L. Roots Extract

EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

Original article:

CHEMICAL COMPOSITION, ANTIOXIDANT AND ANTIGENOTOXIC ACTIVITIES OF DIFFERENT FRACTIONS OF GENTIANA ASCLEPIADEA L. ROOTS EXTRACT

Vladimir Mihailović1*, Sanja Matić2, Danijela Mišić3, Slavica Solujić1, Snežana Stanić2, 1 1 1 Jelena Katanić , Milan Mladenović , Nevena Stanković

1 Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia 2 Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia 3 Institute for Biological Research "Siniša Stanković", University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia * Corresponding author: V. Mihailović Tel.: +381 34 336 223; Fax: +381 34 335 040; E-mail address: [email protected]

ABSTRACT The aim of this study was to evaluate the antioxidant and antigenotoxic activities of chloroform, ethyl acetate and n-butanol fractions obtained from Gentiana asclepiadea L. roots methanolic extract. The main secondary metabolites sweroside, swertiamarin and gentiopicrine were quantified in G. asclepiadea root extracts using HPLC-DAD analysis. Amount of total phenols, flavonoids, flavonols and gallotannins was also determined. The antigenotoxic potential of extracts from roots of G. asclepiadea was assessed using the standard in vivo procedure for the detection of sex linked recessive lethal mutations in Drosophila melanogaster males treated with ethyl methanesulfonate (EMS). The results showed that the most abundant secoiridoid in G. asclepiadea roots was gentiopicrine and its content in the n- butanol fraction (442.89 mg/g) was the highest. Among all extracts, ethyl acetate fraction showed the highest antioxidant activity, as well as total phenolics (146.64 GAE/g), flavonoids (44.62 RUE/g), flavonols (22.71 RUE/g) and gallotannins (0.99 mg GAE/g) content. All the fractions showed antioxidant activity using in vitro model systems and the results have been correlated with total phenolics, flavonoids, flavonols and gallotannins content. In addition to antioxidant activity, G. asclepiadea root extract fractions possess an antigenotoxic effect against DNA damage induced by alkylation with EMS. The antioxidant activity exhibited by G. asclepiadea depended on the phenolic compounds content of the tested extracts, while there was no significant difference in the antigenotoxic potential between fractions.

Keywords: antioxidant activity, antigenotoxicity, gentiopicrine, Gentiana asclepiadea L., phenolic contents, secoiridoids

INTRODUCTION 2007). Many herbs and spices are the sub- ject of ongoing scientific investigations re- With recent advances in medical and lated to antioxidant properties and health nutrition sciences, natural products and (Kaefer and Milner, 2008). Nowadays, health-promoting foods have received ex- there is a growing interest in bioprospecting tensive attention from both health profes- and the analysis of novel natural antioxi- sionals and the common population (Zhao,

807 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

dants for use in foods. Namely, some of poor appetite, digestive problems and as synthetic antioxidants, such as butylated hepatoprotective agents worldwide. As nat- hydroxytoluene (BHT) and butylated hy- ural sources of food flavoring they are uti- droxyanisole (BHA), which have been lized in alcoholic and nonalcoholic bever- widely used in foods and beverages, ages (Jensen and Schripseme, 2002; Szücs showed potential health hazards, because of et al., 2002; Jiang et al., 2005; Aberham et the formation of possible toxic or carci- al., 2011). The major bioactive constituents nogenic components during their degrada- of Gentiana plants are iridoids, secoir- tion (Dastmalchi et al., 2007; Hsouna et al., idoides, xanthones, and flavonoids (Krstić 2011). The protective effects of plant pro- et al., 2004; Zhao et al., 2010). Swertiama- ducts are ascribed to several components rine, gentiopicrine, sweroside and ama- which have distinct mechanisms of action, rogentin are the main secoiridoids of Genti- including low molecular weight com- ana plants. Because its bitter taste, secoir- pounds, e.g. vitamins E and C, carotenoids idoids are used in preparation different bit- and so-called non-nutrient antioxidants, e.g. ter tonics (Wolfender et al., 1993). phenolic and polyphenolic compounds Gentiana asclepiadea L. is medicinal (Fürst, 2009; Kim et al., 2011). Most anti- plant which used in Serbia as traditional oxidants isolated from higher plants are medicine for hepatitis infections and the lo- phenolic compounds (e.g. phenolic acids, cal name of this plant is a "grass of jaun- flavonoids, flavonols, catechins, lignans dice". Also, herb and roots of this plant are and tannins), which have diverse biological widely used for improving digestion (Sarić, activities, such as anti-inflammatory, anti- 1989; Menković et al., 2011). Secoiridoid- carcinogenic and anti-atherosclerotic ef- glycosides: gentiopicrine, its 6'-O-glycosi- fects, as a result of their antioxidant activity de, swertiamarine and sweroside (Mpondo (Kaisoon et al., 2011; Krishnaiah et al., and Chulia, 1988; Szücs et al., 2002); fla- 2011). vone- and xanthone-C-glycosydes and phe- In the past few years, there has been nolic acids (Kitanov and Spassov, 1992) are significant interest in natural products with the secondary metabolites identified in G. antigenotoxic properties. Plants used in tra- asclepiadea. However, no correlation has ditional medicine, as well as their second- been proved between the biochemical com- ary metabolites, have been proposed for position of this plant and its biological ac- their antigenotoxic activities (De Flora, tivity or therapeutic uses. Potential antioxi- 1998; Alekperov, 2002; El Hamss et al., dant activity of some Gentiana species, 2003). Previous studies on the genotoxicity such as G. lutea (Kušar et al., 2006) and G. and antigenotoxicity of plant extracts can arisanensis (Ko et al., 1998) have been con- help evaluate the safety of these herbal firmed. In addition, our previous studies al- products (Romero-Jiménez et al., 2005; so showed that methanol extract of aerial Alpsoy et al., 2011). Therefore, it is neces- parts of G. asclepiadea had a good antioxi- sary to assess the antigenotoxic potential of dant activity in vitro (Nićiforović et al., plant extracts. 2010) and aerial parts and roots extracts had Gentiana asclepiadea L. belongs to the hepatoprotective activities against carbon genus Gentiana that comprises about 400 tetrachloride induced liver injury in rats species. Of the 29 Gentiana species recor- (Mihailović et al., 2013). Previous study ded in Europe, eleven of them are distri- showed that G. asclepiadea extracts are buted in Serbia (Josifović, 1973). In ge- non-genotoxic to rat liver cells (Mihailović neral, the medicinal part of Gentiana spp. is et al., 2013). Therefore, the objectives of the root. The underground parts of various the present study were to evaluate the anti- Gentiana species have been included in oxidant, radical-scavenging and antigeno- many herbal formulations as remedies for toxic activities of chloroform, ethyl acetate

808 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

and n-butanol fractions obtained from the Preparation of the extracts root extract of G. asclepiadea, as an alter- The roots (90 g) of G. asclepiadea were native natural source of antioxidants which dried and fine powdered. The obtained can potentially be used as functional food powder was extracted with methanol (3 × ingredients. In addition to antioxidant activ- 500 ml, 24 h each) at room temperature. ity, the antigenotoxic potential of fractions After filtration through Whatman No. 1 fil- was assessed in Drosophila melanogaster ter paper, the extract was concentrated un- treated with the well-established mutagenic der vacuum at 40 °C by using a rotary eva- agent ethyl methanesulfonate (EMS) using porator, yielding brown residues (32.49 g). the standard in vivo sex-linked recessive le- The resulted residue was suspended in thal (SLRL) test. The amounts of genti- 500 ml of distilled water and sequentially opicrine, swertiamarin and sweroside were partitioned into chloroform (3 × 100 ml), determined by HPLC-DAD analyses. At the ethyl acetate (3 × 100 ml) and then n- same time, phenolic content of the same butanol (3 × 100 ml). The resulting three plant material was determined in order to solutions were concentrated in vacuo to evaluate their amount, as well as their con- dryness, to give chloroform (0.621 g), ethyl tribution to the antioxidant capacity. acetate (0.217 g) and n-butanol (2.846 g) soluble fractions. The extracts were stored MATERIALS AND METHODS in darkness at 4 °C until used.

Chemicals and apparatus All chemicals used in experiments were Determination of total phenolics content of analytical grade and were purchased The total phenolics content was deter- from Sigma Chemical Co. (St. Louis, MO, mined by the method as described by Sin- USA), Aldrich Chemical Co. (Steinheim, gleton et al. (1999). A reaction mixture Germany) and Alfa Aesar (Karlsruhe, Ger- consisting of 0.5 ml of extracts (1 mg/ml), many). Standard compounds gentiopicrine 2.5 ml of Folin–Ciocalteu reagent (diluted (≥ 98 % purity) was obtained from Roth, 10-fold) and 2 ml of NaHCO3 (7.5 %) was Karlsruhe, Germany. Swertiamarin and prepared. After incubation for 15 min at sweroside (both 98 % purity) were obtained 45 °C, the absorbance was measured at 765 from Oskar Tropitzsch, Marktredwitz, nm. The mean of three independent anal- Germany and mangiferin (≥ 98 % purity) yses were used and the total phenol content was purchased from Sigma-Aldrich, Stein- was expressed in milligrams of gallic acid heim, Germany. The spectrophotometric equivalents/g extract (mg GAE/g extract). measurements were performed on UV–VIS spectrophotometer MA9523-SPEKOL 211 Determination of flavonoids content (ISKRA, Horjul, Slovenia). The total flavonoid content determination was based on the method reported Brighente et al. (2007). 0.5 ml of 2 % AlCl Plant material 3 G. asclepiadea was collected from Ja- and the same volume of methanol solution dovnik Mountain (Gostun-Kumanica, at the of plant extracts were incubated for 1 h at Serbia–Montenegro border) in the month of room temperature, and the absorbance was October during its flowering time. The col- measured at 415 nm. The mean of three in- lected plant was authenticated and voucher dependent analyses were used and the total specimen (No. 16337) was deposited in the flavonoids content was expressed in milli- Herbarium of the Department of Botany, grams of rutin equivalents/g extract (mg Faculty of Biology, University of Belgrade, RUE/g extract). Belgrade, Serbia.

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Determination of flavonols content order to determine total antioxidant capaci- The content of flavonols was deter- ty of extracts the method of Prieto et al. mined by Yermakov et al. (1987) method. (1999) was used. The total antioxidant ac- The rutin calibration curve was prepared by tivity of extracts was monitored by the for- mixing 2 ml of various concentrations of mation of a green phosphate/Mo (V) com- ethanolic solutions of rutin with 2 ml plex at acid pH. Briefly, 0.3 ml of extracts (20 mg/ml) aluminium trichloride and 6 ml in methanol (1 mg/ml) was added to 3 ml of (50 mg/ml) sodium acetate. The absorbance reagent solution (0.6 M sulfuric acid, at 440 nm was read after 2.5 h. The same 28 mM sodium phosphate and 4 mM am- procedure was used for 2 ml of plant extract monium molybdate). Obtained mixtures (10 mg/ml) instead of rutin solution. All de- were incubated at 95 °C for 90 min. The terminations were carried out in three inde- absorbance was recorded at 695 nm after pendent experiments. The content of flavo- cooling to room temperature. The results nols was calculated as rutin equivalents were evaluated through the standard curve (mg RUE/g dry extract). of ascorbic acid (AA) obtained by the same procedure. The total antioxidant capacity is Determination of gallotannins content expressed as milligrams of AA per gram of The content of gallotannins was deter- the dry extract. mined by Haslam method (1965). The assay is based on the reaction of potassium iodate Determination of DPPH free-radical scav- (KIO3) with galloyl esters, which will form enging activity a red intermediate and ultimately a yellow In order to determine scavenging DPPH compound. The concentration of the red in- radical activity of the extracts, the method termediate can be measured spectrophoto- proposed by Takao et al. (1994) was used metrically at 550 nm. The reaction was ini- with slight modification (Kumarasamy et tially performed by adding 1.5 ml of a satu- al., 2007). Different concentracions of rated potassium iodate solution to 3.5 ml of extracts in methanol (2 ml each) were methanol solution of plant extracts, fol- mixed with DPPH (2 ml, 80 g/ml). After lowed by incubation at 15 °C until a maxi- 30 min of incubation at room temperature, mum absorbance was reached (regardless of the absorbance was measured at 517 nm. the time) (Bate-Smith, 1977). Gallotannins Each sample was analyzed in three inde- content was determined as gallic acid pendent experiments. Ascorbic acid (AA), equivalents (mg GAE/g extract) and the gallic acid (GA) and butylated hydroxytol- values are presented as means of three in- uene (BHT) were used as reference stand- dependent experiments. ards. The precentage of DPPH free radical scavenging activity was calculated by HPLC analysis of secoiridoids and xan- following equation: thones % inhibition = [(AC – AS)/AC] x 100 The amount of compounds swertia- where A is the absorbance of the control marin, gentiopicrine, sweroside, and man- C solution and A is the absorbance for the giferin in fractions obtained from the root S fraction in the presence the DPPH solution. extract of G. asclepiadea was determined The concentration of extracts providing by the HPLC-DAD method as described 50 % scaviening (IC ) was calculated from previously (Mihailović et al., 2013). 50 the sigmoidal dose-response curve plotted

scavening percentage against extract Determination of total antioxidant capacity Determination of total antioxidant ca- concentration (g/ml). pacity is based on the reduction of Mo (VI) – Mo (V) by the antioxidant compounds. In

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Determination of the inhibitory activity ed X-chromosomes, were used in the exper- toward lipid peroxidation iment. The fruit flies were cultivated on The lipid peroxidation inhibition activi- corn flour medium with yeast, agar, sugar ty of extract fractions was determined using and nipagin (Cas No. H5501, Sigma- the ferric thiocyanate method (Hsu et al., Aldrich, St. Louis, MO, USA) at 25 °C and 2008). The linoleic acid emulsion was pre- 60 % relative humidity in a 12:12 h pared by homogenizing 0.2804 g of linoleic light/dark regime. acid, 0.2804 g of Tween-20 as emulsifier Chloroform, ethyl acetate and n-butanol and 50 ml 40 mM phosphate buffer (pH fractions obtained from G. asclepiadea 7.0). Reaction mixture was composed of 2.5 roots methanolic extract were dissolved in ml linoleic acid emulsion and 0.5 ml extract 1 % sucrose and 5 % solutions were used solution prepared in different concentra- immediately in treatments. EMS (Cas No. tions. The final volume was adjusted to 5 62-50-0, Sigma-Aldrich, St. Louis, MO, ml with 40 mM phosphate buffer (pH 7.0) USA) in concentration of 0.75 ppm was and samples were incubated at 37 oC in the used as a positive control and as the DNA dark. After 72 h of incubation, 4.7 ml of damage-inducing agent. ethanol (75 %) was mixed with 0.1 ml of In order to evaluate the antigenotoxicity the reaction mixture and 0.1 ml FeCl2 of chloroform, ethyl acetate and n-butanol (20 mM). Finally, 0.1 ml ammonium thio- fractions obtained from G. asclepiadea cyanate (30 %) was added to this solution roots methanolic extract, Canton S males and the absorbance was measured at were divided into five groups, with fifteen 500 nm, after it was stirred for 3 min. Inhi- males in each and starved for 5 h prior to bition percent of linoleic acid peroxidation treatments. The first group of males, re- was calculated using following formula: ferred as the negative control, was treated

% inhibition = [(AC – AS)/AC] x 100 by acute feeding exposure to 1 % sucrose for 24 h (Lewis and Bacher, 1968). The Measurement of reducing power second group, referred as the positive con- In order to determine reducing power of trol group, was treated for 24 h with EMS. extracts, the method of Oyaizu (1986) was The third, fourth and fifth groups were used. Different concentrations of extracts treated with EMS for 24 hours before to (2.5 ml) prepared in distilled water were treatment with single dose of 5 % chloro- mixed with same volume of 0.2 M sodium form, ethyl acetate and n-butanol fractions, phosphate buffer (pH 6.6) and 1 % potassi- respectively. um ferricyanide. The mixture was incubat- The SLRL test on D. melanogaster was ed at 50 °C and after 20 min of incubation, carried out according to the standard proce- reaction mixture acidified with 2.5 ml of dure originally described by Würgler and 10 % trichloroacetic acid (w/v). Reaction Graf (1985). The frequencies of sex linked mixture then centrifuged at 1000 rpm for 8 recessive lethal mutations are detected by min and the upper layer (5 ml) was mixed examining males’ eye color and shape in with 1 ml of FeCl3 (0.1 %). The absorbance the second generation (F2). By taking into was measured at 700 nm. Increased absorb- account the fact that recessive lethal muta- ance indicates a higher reducing activity. tion is expressed before the adult stage, the absence of wild type males in the F2 indi- Antigenotoxic potential of chloroform, cates the occurrence of recessive lethal mu- ethyl acetate and n-butanol fractions tation. The SLRL test was applied on D. mela- According to test procedure, cells were nogaster available from Bloomington Stock exposed in successive spermatogenetic Centre (Indiana, USA). Canton S males and stages (three broods), so the effect of chlo- marked balancer Basc females, with invert- roform, ethyl acetate and n-butanol frac-

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tions on frequency of mutations is assayed each fraction varied between 55.57 and at each of the germ-line stages: the first and 146.64 mg/g of extract and ethyl acetate the second broods (I and II) gives the effect fraction showed the highest total phenolics of treated postmeiotic stages, spermatozoids content. As for total phenolics, flavonoids and spermatids, respectively, while the third and flavonols contents also varied depend- brood (III) reveals the effect on the premei- ing on the solvent extraction with maxi- otic stage (spermatocytes). mum values of 44.62 and 22.71 mg RUE/g of extract, respectively, in ethyl acetate Statistical evaluations fraction (Table 1). Results showed that all The data are expressed as mean ± fractions contain low amount of gallotan- standard deviation (SD). The IC50 for in nins (0.34 to 0.99 mg GAE/g) compared vitro antioxidant potential was calculated with other groups of examined phenolic using nonlinear regression analysis from the compounds. The effect of solvent on gallo- sigmoidal dose-response inhibition curve. tannins solubility showed approximately Statistical analyses on the data were ana- the same classification as phenolics. The to- lyzed using analysis of variance (ANOVA) tal phenolics, flavonoids, flavonols and gal- and the group means were compared by the lotannins contents were found to be signifi- least signiﬁcant difference test (LSD). The cantly higher (p < 0.05) in the ethyl acetate results were considered statistically signiﬁ- than in the chloroform and n-butanol frac- cant if the p < 0.05, p < 0.01 and p < 0.001. tions. The total flavonoids and flavonols For SLRL test, by testing for large inde- contents were significantly higher pendent samples, the statistically signifi- (p < 0.05) in the n-butanol than in the chlo- cance of percentage differences in lethal roform fraction, while the amounts of total cultures was calculated, and results are ana- phenolics and gallotannins did not show lyzed for difference in proportions (Petz, significant variations (p > 0.05) among n- 1985). butanol and chloroform fractions. The vari- ability in the phenolic contents in different RESULTS AND DISCUSSION extracts could be the result of the varying solubility of the phenolic compounds; this Chemical composition of extract fractions variation in solubility may be driven by the The crude methanolic extract of G. as- solvent polarity (Marinova and Yanishlieva, clepiadea roots was fractionated by se- 1997). In the present study, chloroform was quential extraction with solvents of in- less effective for extracting phenolic com- creasing polarity: chloroform, ethyl acetate pounds than the n-butanol, while ethyl ace- and n-butanol. The total phenolics, flavo- tate extraction is able to extract phenolics, noids, flavonols and gallotannins contents flavonoids, flavonols and gallotannins in of various solvents fractions are presented relative good amounts. in Table 1. The total phenolics content of

Table 1: Total phenolics, flavonoids, flavonols and gallotannins content of chloroform, ethyl acetate and n-butanol fractions from Gentiana asclepiadea roots

total phenolics flavonoids flavonols gallotannins Fractions (mg GAE/g) (mg RUE/g) (mg RUE/g) (mg GAE/g) Chloroform 55.57 ± 2.64a 17.54 ± 1.67a 4.50 ± 0.68a 0.38 ± 0.03a Ethyl acetate 146.64 ± 7.49b 44.62 ± 0.19b 22.71± 1.37b 0.99 ± 0,05b n-Butanol 73.51 ± 1.51a 34.07 ± 0.19c 15.33 ± 1.01c 0.34 ± 0,02a Each value represents the mean ± SD of three independent experiments; RUE, rutin equivalents; GAE, gallic acid equivalents; means in the same column with superscript with different letters are significantly different at p < 0.05.

812 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

Table 2: HPLC analysis of secoiridoid compounds contents in chloroform, ethyl acetate and n-butanol fractions from Gentiana asclepiadea root extract

Swertiamarin Gentiopicrine Sweroside Fractions (mg/g of extract) (mg/g of extract) (mg/g of extract) Chloroform 7.12  3.01a 14.39  1.70a 1.52  0.03a Ethyl acetate 8.47  2.99a 287.02  40.02b 10.95  0.57b n-Butanol 16.62  6.07a 442.89  21.94b 27.85  4.80c Each value represents the mean ± SD of three independent experiments; means in the same column with superscript with different letters are significantly different at p < 0.05

HPLC-DAD analyses of extracts show- idoids than phenolic compounds while ed that secoiridoid compounds; sweroside, chloroform fraction contained significant swertiamarin and gentiopicrine, exist in G. higher (p < 0.001) concentration of total asclepiadea root extracts at different phenolics than secoiridoid compounds. The amounts, depending on the solvent used for highest amount of secoiridoids was present the extraction (Table 2). The results showed in n-butanol fraction (48.74 %) while the that the most abundant secoiridoid com- chloroform fraction contained all examined pound in the extracts was gentiopicrine, and compounds in significantly lower concen- its amount varied within the range of 14.39 tration compared with n-butanol and ethyl to 442.89 mg/g, depending on the extrac- acetate fractions. That was also confirmed tion procedure. The sweroside contents in in other studies where the highest amount the extracts were 1.52-27.85 mg/g, while of secoiridoids was extracted with n-bu- the swertiamarin contents ranged from 7.12 tanol (Krstić et al, 2004; Janković et al., to 16.62 mg/g. The n-butanol fraction pos- 2005). sessed the highest amount of secoiridoid compounds and its swertiamarin, sweroside and gentiopicrine contents were found to be 16.62, 27.85, and 442.89 mg/g of extract, respectively. The lowest amounts of sweroside and gentiopicrine were found in the chloroform fraction, while no significant difference (p > 0.05) in concentrations of swertiamarin between extracts. In root fractions of G. asclepiadea, mangiferin was not present in detectable amounts. Quantitative data obtained for G. asclepiadea root in the present study are in good agreement with Figure 1: The content of total secoiridoid glyco- previously published data suggesting that sides, phenolics, flavonoids, flavonols and gallotannins in roots of G. asclepiadea expressed gentiopicrine is the main secoiridoid com- in % of dry extracts (mean values of three inde- pound in this plant (Dević et al., 2006; pendent measurements with error bars repre- Szücs et al., 2002). senting SD); Means for same solvent fraction Amounts of the total secoiridoid gly- with different letters are significantly different at cosides and total phenolics, flavonoids, fla- p < 0.001 vonols, and gallotannins investigated in the underground parts of G. asclepiadea, ex- Total antioxidant capacity pressed in percents of fractions are shown The results for total antioxidant capacity in Figure 1. It is obvious that n-butanol and are given in Table 3. Ethyl acetate fraction ethyl acetate fractions contained significant had a significantly higher (p < 0.05) ca- higher (p < 0.001) concentration of secoir-

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pacity (401.33 mg AA/g) than the other the chloroform, ethyl acetate and n-butanol fractions. The total antioxidant activity of fractions obtained from the G. asclepiadea the plant extracts are in the order: ethyl ace- root extract, along with the reference stand- tate fraction > n-buthanol fraction > chloro- ards, are shown in Table 3. All examined form fraction. The dependence of total anti- extracts exhibited considerable levels of oxidant capacity in relation to the total phe- free radical scavenging activity in the nolics, flavonoids, flavonols and gallotan- DPPH assay, indicated by IC50, i.e., the nins content, was also evaluated. The strong concentration at which the compound re- linear relationship of the total antioxidant duces 50 % of the DPPH. capacity results and total phenolics and gal- The most active radical scavengers were lotannins contents were observed ethyl acetate fraction, with IC50 of (R2 = 0.9322 and R2 = 0.9996), suggesting 107.28 µg/ml, while chloroform fraction that total phenolics and gallotannins were was considerably less effective radical sca- likely to be contributing to the total antioxi- vengers. Gallic acid showed the greatest ra- dant capacity of these extracts. The total dical scavenging activity (IC50 = 3.79 µg/ flavonoids and flavonols contents of the ex- ml) among examined referent antioxidants, tracts had a less linear correlation with their its IC50 value was approximately 30 times total antioxidant capacity (R2 = 0.5573 and lower than the concentration of ethyl ace- R2 = 0.5750, respectively), while no corre- tate fraction to reduce 50 % of DPPH start- lation or insignificant relationship between ing concentration. Based on the results of the secoiridoids and total antioxidant capac- DPPH assay, the extract performing the ity of the extracts (R2 < 0.5). highest antioxidant activity had the highest concentration of total phenolics, flavonoids, DPPH radical scavenging activity flavonols and gallotannins. Excellent corre- DPPH assay was employed in our study lation of IC50 values of DPPH scavenging to determine the proton–radical-scavenging activity to the total flavonols (R2 = 0.9830) activity based on the reaction that the pur- and flavonoids (R2 = 0.9781) content was ple color of DPPH solution fades quickly noticed, while IC50 values correlated to the when it reacts with proton–radical scaven- total phenolics and gallotannins content gers. BHT, ascorbic acid and gallic acid are with R2 = 0.9035 and R2 = 0.7189, respec- the reagents used as standards. The results tively. of DPPH radical scavenging activities of

Table 3: Antioxidative activity of chloroform, ethyl acetate and n-butanol fractions from Gentiana asclepiadea root extract

IC (µg/ml) Total antioxidant 50 Fractions/ Inhibitory activity capacity (mg AA/g DPPH scavenging standards toward lipid of dry extract) activity peroxidation Chloroform 325.80 ± 16.02a 361.55 ± 21.97a 183.16 ± 11.55a Ethyl acetate 401.33 ± 25.60b 107.28 ± 15.60b 40.96 ± 5.11b n-Butanol 318.70 ± 20.05a 239.23 ± 18.55c 116.90 ± 8.69c Gallic acid - 3.79 ± 0.69d 255.43 ± 11.68d Ascorbic acid - 6.05 ± 0.34d >1000 BHT - 15.61 ± 1.26d 1.00 ± 0.23e α-Tocopherol - - 0.48 ± 0.05e

IC50 values were determined by nonlinear regression analysis. Results are mean values ± SD from three independent experiments; AA, ascorbic acid; -, Not tested. Means in the same column with superscript with different letters are significantly different at p < 0.05

814 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

Inhibitory activity toward lipid peroxida- vate oxidants (Gülçin, 2009). The reducing tion capacity of extracts and referent compounds The inhibitory activity of the extracts was measured by the direct reduction of 3+ 3- 2+ 4- against lipid peroxidation was determined [Fe (CN)6] to [Fe (CN)6] . Reduced by the thiocyanate method, and the results product reacts with FeCl3 and forms the are given in Table 3. The ferric thiocyanate intense Perl’s Prussian blue complex, method measures the amount of peroxide Fe4[Fe(CN)6]3, which has a strong absorb- produced during the initial stages of oxida- ance at 700 nm. tion, which is the primary product of lipid An increase in absorbance of the reac- oxidation. In this assay, indirect measure- tion mixture would indicate an increase in ment was performed for the amount of hy- the reducing capacity due to an increase in droperoxides produced from linoleic acid the formation of the complex. Figure 2 emulsion by auto-oxidation during the ex- shows the reductive capability of fractions periment period (Inatani et al., 1983). All obtained from methanol root extract of G. tested extracts added to linoleic acid emul- asclepiadea, compared to ascorbic acid, sion were able to reduce the formation of gallic acid and BHT. The results indicate hydroperoxide. The IC50 values of the G. that ethyl acetate extract exhibited a greater asclepiadea extracts ranged from 40.96 to reduction power than the chloroform and n- 183.16 μg/ml. Ethyl acetate fraction has butanol extract. As in previous described been found as the highest effective antioxidant studies, in this work, strong re- (p < 0.05) among the G. asclepiadea ex- ducing power of the ethyl acetate extract tracts, with IC50 value of 40.96 μg/ml. In was expected, since it had a high content of the linoleic acid system (Table 3), referent phenolic compound. compounds α-tocopherol (IC50 = In the present study, a nonsignificant 0.48 µg/ml) and BHT (IC50 = 1 µg/ml) ex- correlation was found between antioxidant hibited much lower IC50 values than exa- activities and sweroside, swertiamarin and mined plant extracts. In comparison, ethyl gentiopicrine contents in G. asclepiadea acetate, n-butanol and chloroform fractions root extracts, R2 values were less than 0.5. showed higher activity than referent com- Results showed that the polyphenol- pounds gallic acid and ascorbic acid.These enriched fraction (ethyl acetate fraction) results together confirm that the polar anti- possessed higher antioxidant activities than oxidants are less effective in protecting the any other fractions. Therefore, total phenol- emulsiﬁed linoleic acid, whereas lipophilic ics, flavonoids, flavonols and gallotannins, antioxidants due to higher partition into the appear to be the major contributors to the lipid phase reveal greater activity in the high antioxidant activities. emulsion (Terpinc et al., 2012). The results show that the amounts of total phenols, fla- Antigenotoxic potential of chloroform, vonoids and flavonols correlated strongly ethyl acetate and n-butanol fractions IC50 values obtained in this method, with The use of plant extracts to cure various linear correlation coefficients R2 = 0.9126, diseases is very common in traditional med- 0.9732, and 0.9787, respectively, which icine. Although a large number of plant somewhat better than correlation with gal- species is a potential source of biologically lotannins content (R2 = 0.7330). active compounds, the effect on genetic material is often unknown. In this study, the Reducing power efficacy of three different fractions of G. Reducing power reflects the electron asclepiadea root extract against DNA dam- donating capacity of bioactive compounds age induced by EMS was tested. The aim and is associated with antioxidant activity. was to investigate new potential antigeno- Antioxidants can be reductants and inacti- toxic agents from natural sources, for pos-

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Figure 2: Reducing power of extract fractions obtained from G. asclepiadea roots compared to reducing power of standards at different concentrations. Each value is the average of three independent measurements with error bars representing SD.

sible use to protect the integrity of DNA tigenotoxic effect, as they significantly re- from various genotoxic agents. duces the rate of mutation compared to As can be seen from Table 4, EMS cau- EMS. Results analyzed by simultaneous sed statistically signiﬁcant increase in fre- comparison between three fractions from G. quency of lethal mutations in comparison to asclepiadea revealed no statistically signif- the sucrose, observed as an increase in the icant difference in the protective potential percentage of lethal (33.63 %). Although of the extract fractions (Table 7). EMS increased the frequency of mutation, According to Hung et al. (2009) and chloroform fraction from G. asclepiadea Kuroda et al. (2001) there are two main roots decreased the genotoxicity of EMS groups of protective mechanisms for DNA: (Table 5). Ethyl acetate fraction at concen- desmutagenesis, in which tested substances tration of 5 % statistically significantly re- act on mutagens or inactivate them, and the duced the frequency of mutations induced other is bio-antimutagenesis in which tested by EMS in all three broods. Post-treatment substances act on the processes of muta- with n-butanol fraction from G. asclepiadea genesis or repair DNA damages that result drastically reduced the frequency of muta- in a decrease in the frequency of mutation. tions induced by EMS with high signifi- Desmutagens could be detected with pre- cance in all broods (p < 0.001***). treatment, while bio-antimutagenicity could Compared to the negative control all be better detected with post-treatment (De fractions decreased the genotoxicity of Flora, 1998). Considering the genotoxin EMS in postmeiotic germinative cell lines - administered with tested fractions, there is a at spermatozoids and spermatids and in possibility that the antimutagens in frac- premeiotic line - spermatocytes (Table 6). tions exert protective effect in bio- The average values in frequencies of muta- antimutagenic manner, which suppresses tions for I, II and III broods indicate that all mutation fixation after DNA is damaged by fractions in concentration of 5 % exhibit an- EMS.

816 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

Table 4: Frequencies of sex linked recessive lethal mutations in the Drosophila melanogaster test after post-treatments with EMS and G. asclepiadea roots extract fractions

Broods Treatments No of crosses No of lethal % of lethal Sa 150 3 2.0 EMSb 110 37 33.63 c I EMS+GAch 69 4 5.80 d EMS+GAea 76 2 2.63 e EMS+GAnb 85 4 4.71 S 135 3 2.22 EMS 81 27 33.33

II EMS+GAch 62 2 3.23

EMS+GAea 65 2 3.10

EMS+GAnb 68 4 5.88 S 126 3 2.38 EMS 59 15 25.42

III EMS+GAch 74 2 2.70

EMS+GAea 80 2 2.50

EMS+GAnb 65 2 3.10 S 411 9 2.19 EMS 250 79 31.6

Σ EMS+GAch 205 8 3.90

EMS+GAea 221 6 2.71

EMS+GAnb 218 10 4.60

a b c S; sucrose, negative control, 1 %; EMS; ethyl methanesulphonate, positive control, 0.75 ppm; GAch; d chloroform fraction from G. asclepiadea roots, 5 %; GAea; ethyl acetate fraction from G. asclepiadea e roots, 5 %; GAnb; n-butanol fraction from G. asclepiadea roots, 5 %.

Table 5: The significance of differences in the frequency of recessive lethal X-linked mutations in Drosophila melanogaster males after post-treatments with G. asclepiadea roots extract fractions compared to the positive control

b a c d e Broods EMS /S EMS/EMS+GAch EMS/EMS+GAea EMS/EMS+GAnb t 5.33 5.32 6.41 5.86 I p 0.001*** 0.001*** 0.001*** 0.001*** t 5.74 5.36 5.36 4.59 II p 0.001*** 0.001*** 0.001*** 0.001*** t 4.0 3.84 3.80 3.70 III p 0.001*** 0.001*** 0.001*** 0.001*** t 9.90 9.23 9.10 8.30 I+II+III p 0.001*** 0.001*** 0.001*** 0.001***

a b c S; sucrose; negative control, 1 %; EMS; ethyl methanesulphonate, positive control, 0.75 ppm; GAch; chloro- d form fraction from G. asclepiadea roots, 5 %; GAea; ethyl acetate fraction from G. asclepiadea roots, 5 %; e GAnb; n-butanol fraction from G. asclepiadea roots, 5 %; The results are analyzed by the test for difference in proportions by simultaneous comparison with the positive control (t values); Asterix indicates significantly higher frequency compared to EMS as positive control.

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Table 6: The significance of differences in the frequency of sex-linked recessive lethal mutations of Drosophila melanogaster males after post-treatments with G. asclepiadea roots extract fractions compared to the negative control

a b c d e Broods S /EMS S/EMS+GAch S/EMS+GAea S/EMS+GAnb t 5.33 1.23 0.30 1.05 I p 0.001*** 0.05 0.05 0.05 t 5.74 0.50 0.5 1.27 II p 0.001*** 0.05 0.05 0.05 t 4.0 0.25 0.05 0.24 III p 0.001*** 0.05 0.05 0.05 t 9.90 1.27 0.62 1.50 I+II+III p 0.001*** 0.05 0.05 0.05

a b c S; sucrose, negative control, 1 %; EMS; ethyl methanesulphonate, positive control, 0.75 ppm; GAch; chloroform d e fraction from G. asclepiadea roots, 5 %; GAea; ethyl acetate fraction from G.asclepiadea roots, 5 %; GAnb; n- butanol fraction from G. asclepiadea roots, 5 %; The results are analyzed by the test for difference in proportions by simultaneous comparison with the negative control (t values); Asterix indicates significantly higher frequency compared to sucrose as negative control.

Table 7: A comparison in the significance of differences in the frequency of sex-linked recessive lethal mutations of Drosophila melanogaster males after post-treatments with three G. asclepiadea roots extract fractions

a b c Broods EMS+GAch /EMS+GAea EMS+GAch/EMS+GAnb EMS+GAea/EMS+GAnb t 0.97 0.31 0.75 I p 0.05 0.05 0.05 t 0.07 0.81 0.83 II p 0.05 0.05 0.05 t 0.08 0.40 0.21 III p 0.05 0.05 0.05 t 0.71 0.37 0.95 I+II+III p 0.05 0.05 0.05

a b GAch; chloroform fraction from G. asclepiadea roots, 5 %; GAea; ethyl acetate fraction from G. asclepiadea roots, 5 %; c GAnb; n-butanol fraction from G. asclepiadea roots, 5 %; The results are analyzed by the test for difference in proportions by simultaneous comparison of three G. asclepiadea L. roots extract fractions (t values)

Mutagenic action of alkylating agents sion is supported by De Flora et al. (2005) may be related to their ability to form O6- who reported that the bioantimutagenic methylguanine (O6MeG) (Stephanou et al., agents reverted the mutagenic effects and 1996). O6MeG is repaired by protein preventing the fixation of mutations acting termed O6-methylguanine DNA methyl- on the physiological mechanisms of DNA transferase (MGMT) (Margison and San- protection and repair. tibáñez-Koref, 2002), by transferring ethyl Potential genotoxicity and/or antigeno- group from a base lesion to a cysteine resi- toxicity of the methanolic and aqueous ex- due, thus restoring the integrity of the DNA tracts from the haulm and flower of G. as- without creating additional DNA damage clepiadea were investigated previously in (Kaina and Christmann, 2002). This means human lymphocytes, human embryonic kid- that fractions from G. asclepiadea have the ney (HEK 293), and monkey kidney (COS ability of anti-alkylation damage and im- 1) cell lines using the comet assay (Hude- proves the activity of MGMT. This conclu- cová et al., 2010; Hudecová et al., 2012a,

818 EXCLI Journal 2013;12:807-823 – ISSN 1611-2156 Received: June 12, 2013, accepted: September 04, 2013, published: September 12, 2013

2012b). In those studies, extracts of G. as- tions. It seems the reason for this difference clepiadea had neither genotoxic nor cytoto- just is that the phenolic content in ethyl ace- xic effect in a wide range of concentrations. tate fraction is higher than in other frac- Likewise, our previous study showed that in tions. Regarding the fact that n-butanol vivo exposure to the methanol extracts of fraction contains the highest amount of se- aerial parts and roots of G. asclepiadea did coiridoids compounds in relation to the oth- not induce DNA damage in liver cells of er two fractions and that the ethyl acetate Wistar rats (Mihailović et al., 2013). fraction exhibit stronger antioxidant activi- The antigenotoxic effect can be attribut- ty, it can be concluded that phenolics are ed to a wide range of phytochemicals in- main compounds responsible for the antiox- cluding simple phenols, flavonoids and fla- idant effects. Experiments performed in D. vonols (Aaronson, 2000; Siriwardhana et melanogaster showed an antigenotoxic ef- al., 2003; Karawita et al., 2005), detected in fect of three different extract fractions of G. extracts of G. asclepiadea. Previous studies asclepiadea by post-treatment. These re- prove the ability of phenolics and flavo- sults suggested that extracts act intracellu- noids in scavenging mutagens or free radi- lary by affect the DNA repair damage in- cals (Rice-Evans et al., 1996; Yao et al., duced by EMS in a bio-antimutagenic man- 2004). Also, the antigenotoxic activity of ner. some flavonoids was induced by radical scavenging activity (Edenharder and ACKNOWLEDGMENTS Grunhage, 2003). Fedeli et al. (2004) re- This work was supported by the Minis- ported that tannins are able to protect DNA try of Education, Science and Technologi- against damage in low concentrations, cal Development of the Republic of Serbia while at high concentrations they could be (projects No. III 43004 and 41010). genotoxic. To the best of our knowledge, there are no available literature data about the antimutagenic effect of the secoiridoids. REFERENCES Regarding the fact that chloroform fraction contains lower amounts of phenolic com- Aaronson S. Important vegetable supple- pounds and secoiridoids in relation to the ments. In: Kiple KF, Ornelas KC (ed): The other two fractions (Figure 1), and that they Cambridge world history food (pp 231-49). exhibit the same level of antigenotoxicity New York: Cambridge University Press, (Table 7), it cannot be conclude with cer- 2000. tainty which of the above mentioned compounds are responsible for the protective ef- Aberham A, Pieri V, Croom JrEM, Ellmer- fect. It can be assumed that the presence of er E, Stuppner H. Analysis of iridoids, se- all these components in the extracts cumu- coiridoids and xanthones in Centaurium er- latively may have been responsible for re- ythraea, Frasera caroliniensis and Gentia- duction of DNA binding and ethylation na lutea using LC–MS and RP-HPLC. J with EMS. Pharm Biomed Anal 2011;54:517-25.

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