Antioxidative potential of Chenopodium botrys L . ( Amaranthaceae )

Evaluation of the antioxidant potential of methanol extract of Chenopodium botrys L. (Amaranthaceae) collected from six different locations in Republic of Macedonia was performed. Several methods were used for testing the antioxidative activity: 1) 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging assay, 2) ferric reduction power assay (FRAP), 3) inhibition of H2O2 activity, 4) non-sitespecific hydroxyl radical-catalyzed 2-deoxy-D-ribose degradation (NSSOH) and 5) site-specific hydroxyl radical-catalyzed 2-deoxy-D-ribose degradation (SSOH). The IC50 values ranged from 0.26-3.10 mg/mL, 3.01-12.71 mg/mL and 2.60-12.29 mg/mL, for DPPH, NSSOH and SSOH assays, respectively. The H2O2 inhibition activity and the ferric reducing power capacity were from 28.84-46.56% and 26.1443.40%, respectively. The obtained data establish the antioxidant potency in concentration-dependent manner. Additionally, total phenols (TPC) and total flavonoid content (TFC) were determined. The estimated values ranged from 27.77-71.25 mg GAE/g DW and from 7.3516.33 mg QE/g DW, respectively.


Introduction
Oxidative stress plays an important role in the etiology of chronic diseases such as cancer, atherosclerosis and diabetes as well as age-related neurological degenerative disorders.In the last couple of decades, the misperception of safety chronic consumption of synthetic antioxidants has increased the interest in phytochemicals as biological antioxidants which are defined as substances present in low concentrations that can reduce or prevent oxida-tion of the easily oxidisable substrate (Shikov et al., 2006;Benzie and Strain, 1996).Among enormous variety of secondary metabolites, polyphenols have a wide range of biological activities: anti-inflammatory, antiallergenic, antioxidant and antitumor, and have an impact on improving the immune system, as well (Pasko et al., 2008).Numerous studies have demonstrated the importance of naturally occurring dietary polyphenols in promoting of cardiovascular health and emphasized the significant role these compounds play in limiting the effects of cellular aging (Khura et al., 2013).Also, they pointed to the impact of Ljubica Adji Andov, Marija Karapandzova, Blagica Jovanova, Gjose Stefkov, Ivana Cvetkovikj Karanfilova, Tatjana Kadifkova Panovska and Svetlana Kulevanova phenolic compounds on carcinogenic cells which is crucial for their antitumor potential (Boivin et al., 2009).Anticancer activity is partly attributed to the antioxidative effects of phenolic compounds and polyphenols as they are defined as reducing agents, free radical scavengers, hydrogen donors and inhibitors of pro-oxidative enzymes (Gawlik-Dziki, 2012;Cai et al., 2004).Since, recent studies are increasingly directed towards finding new antioxidant compounds, that requires a lot of information and additional research related to antioxidative potential of natural components (Nikolova et al., 2011).
The genus Chenopodium comprises numerous members of perennial and annual species, which occur everywhere in the world.They could grow as aromatic or non-aromatic herbaceous plants or as shrubs and small trees (Fuentes-Bazan et al., 2012).C. botrys L. (syn.Dysphania botrys (L.) Mosyakin and Clemants), known as Jerusalem Oak Goosefoot or Feathered Geranium, commonly grows in Europe and is native to Mediterranean region, but it also could be found in Asia, India, Himalayas, Turkey, Cyprus, Africa, Australia and North and South America (Seidemann, 2005).The plant is known as traditional medicine for different ailments in respiratory tract, especially for the treatment of catarrh and humoral asthma, when it is used as a substitute for C. ambrosioides (Yadav et al., 2007).Concerning literature data, C. botrys contains flavonoids, alkaloids, terpenes and essential oil (Kokanova-Nedialkova et al., 2009).Regarding this, pharmacological analyses confirm medical potential of C. botrys in the development of new drugs.Various isomers of specific monoterpene compound, ascaridole, were identified in C. botrys oil which was associated with the activity against various tumor cell lines in vitro (Morteza-Semnani, 2015).Additionally, in the aerial parts of C. botrys, the flavon chrysoeriol as well as flavonoids: salvigenin, sinensetin, hispidulin, quercetin and their derivatives were identified, which were related to a potential antioxidant activity (Kokanova-Nedialkova et al., 2009).
In the flora of the Republic of Macedonia (RM), 15 species of the genus Chenopodium occur naturally, including C. botrys (Micevski, 1995).Dried over ground flowering parts of the plant (herba) are utilized by local people for preparing infusions or liquid extracts with diuret-ic, antispasmodic, carminative and antidiarrheal properties (Maksimovic et al., 2005).Despite its usage, chemical composition and biological activity of this plant are poorly known.Therefore, the aim of this study was determination of the total phenolic and total flavonoid content as well as evaluation of the antioxidant capacity of C. botrys collected from different localities of the Republic of Macedonia (RM).

Plant material
The aerial parts of C. botrys were collected in the period from July to September in 2012, 2013 and 2014 at six localities in RM (Table 1).Plant identity was verified and a herbarium voucher specimen (NoCB1/12; NoCB1/13 and NoCB1/14) was deposited at the Department of Pharmaceutical Botany, Institute of Pharmacognosy, Faculty of Pharmacy, University 'Ss.Cyril and Methodius', Skopje, R. Macedonia.Collected plant material was air-dried and preserved in paper bags until performing the analysis, when it was properly milled and homogenised.

Extract preparation
2.5 g of dried and fine grounded plant material was extracted in ultrasonic bath (50/60 Hz, 720 W) at room temperature, twice with 15 mL of 100% methanol (HPLC grade), each time for 15 min.After filtration, the volume was made up to 25 mL with methanol.Obtained methanol extracts (0.1 g/mL) were used for determination of total phenols and total flavonoids and for evaluation of the

Determination of total phenolic content (TPC)
TPC was determined with the Folin-Ciocalteu reagent according to a procedure described by Singleton et al., (1999) with slight modifications.Folin-Ciocalteu reagent (2.5 mL, 1:10 v/v diluted with distilled water) was added to the test sample (0.1 mL methanol extract), and stirred for 5 min at room temperature.Subsequently, 3 mL of 7% sodium carbonate was added and made up to 10 mL with distilled water.This mixture was incubated at room temperature in dark place for 1 h.After incubation, absorbance of blue color was measured at 765 nm using a Agilent 8453 UV-Vis spectrophotometer.The total phenolic content was estimated as mg of gallic acid equivalents (GAE) per gram of dried weight of plant material (mg GAE/g DW) using an equation obtained from standard gallic acid calibration curve.

Determination of total flavonoid content (TFC)
TFC was determined using the Aluminum chloride assay described by Lallianrawna et al., (2013).An aliquot of the test sample (0.1 mL methanol extract) was mixed with 0.1 mL of 5.0% sodium nitrite and allowed to stand for 5 min.Then, 0.15 mL of 10.0% aluminum chloride were added to the mixture, kept at room temperature for six more minutes and followed by the addition of 0.5 mL of 1 M sodium hydroxide.The total volume was made up to 2.5 mL with distilled water.The resulting solution was mixed well and the absorbance was measured immediately at 510 nm using an Agilent 8453 UV-Vis spectrophotometer.The blank was prepared by replacing the extracts with an equal volume of deionized water.The TFC was expressed as mg of quercetin equivalents (QE) per gram of dried weight of plant material (mg QE/g DW) using an equation obtained from standard quercetin calibration curve.

Free radical scavenging activity (DPPH assay)
The scavenging activity of DPPH free radicals of C. botrys extracts was done according the method reported by Kurin et al.,(2012) with minor modifications.200 µL of different concentrations of tested samples (100, 50 and 20 mg/mL) were transferred in a cuvette and 4 mL of 100 µM ethanol solution of DPPH radical were added.Mixtures were shaken vigorously for one min and left to stand 10 min in dark place at room temperature.The reduction of the DPPH free radicals was measured by reading the absorbance at 517 nm on final concentrations of extracts in the reaction mixture (1.0, 2.5 and 5.0 mg/mL).Methanol was used as control blank.Quercetin and BHA were used as standards, with final concentrations in the reaction mixture of 0.0005, 0.001 and 0.0025 mg/mL.The antioxidant capacity of methanol extracts was expressed as IC 50 val-ues (extract concentration providing 50% of radical scavenging activity) that were calculated by interpolation from the graph obtained by plotting the percentage of inhibition against extract concentration.

Ferric reducing antioxidant power (FRAP)
The ferric reducing power of C. botrys methanol extracts was determined by using the Potassium ferricyanide/ferric chloride assay reported by Shahat et al. with minor modifications [23].Different concentrations of extracts (100, 50 and 20 mg/mL) were added to 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide.The mixtures were incubated at 50 °C for 20 min, after which 2.5 mL of 10% trichloroacetic acid was added.An aliquot of the mixture (2.5 mL) was taken and mixed with 2.5 mL water and 0.5 mL 1% FeCl 3 .After 30 min, the absorbance was measured at 700 nm on the final concentrations of extracts in the reaction mixture (2.8 mg/mL).Quercetin and ascorbic acid were used as standards with final concentrations in the reaction mixture of 0.007 mg/mL.The ferric reducing antioxidant power was expressed in percentage (%) after construction of FeSO 4 x 7H 2 O calibration curve.Higher absorbance of the reaction mixture indicated greater reducing power.

H 2 O 2 inhibition effect
The H 2 O 2 inhibition effect of C. botrys extracts was determined according to the method reported by Serteser et al., (2009).One mL of extract (100 mg/mL), 3.4 mL of 0.1 M phosphate buffer (pH 7.4) and 0.6 mL of 43 mM H 2 O 2 were mixed and after 60 minutes the absorbance of the mixture with final concentrations of extracts (20 mg/ mL) in the reaction mixture was measured at 230 nm.Controls without H 2 O 2 were prepared for each sample concentration.To determine the H 2 O 2 mM concentration that did not involve the reaction, a linear regression equation was used.3.4 mL of phosphate buffer were added to 0.6 mL of 10, 15, 25, 43 and 50 mM H 2 O 2 and the absorbance was measured at 230 nm.Linear regression equations were obtained by the diagram of concentration against absorbance.Quercetin was used as standard solution.The percentage of inhibition was calculated from the H 2 O 2 concentration of control and H 2 O 2 concentration of sample using the following equation:

Nonsite-Specific Hydroxyl Radical-Mediated 2-Deoxy-D-Ribose Degradation (NSSOH)
Inhibition of nonsite-specific hydroxyl radical-mediated degradation was carried out according to Halliwell et After vortexing and 30 min incubation at 50 °C, trichloroacetic acid (1 mL, 2.8%) and thiobarbituric acid (1 mL, 1.0%) were added.The mixture was vortexed and heated at 50 °C for 30 more minutes.The extent of oxidation was estimated from the solution absorbance of the final concentrations of extracts in the reaction mixture (0.6, 1.6 and 3.3 mg/mL) at 532 nm.Quercetin and BHA were used as standards with final concentrations in the reaction mixture of 0.6, 1.6 and 3.3 μg/mL.IC 50 values were estimated using linear regression of the linear part of the curve.

Statistical analysis
Data obtained from determination of TPC and TFC were expressed as mean values ± SD as all experiments were carried out in triplicate.Statistical analyses were performed by statistical package STATGRAPH 21.0.The p value less than 0.05 was considered to be statistically significant.

Total phenolic and total flavonoids content
The C. botrys samples analysed in this study showed presence of total phenolic content in range of 27.77 to 71.25 mg of gallic acid equivalents per gram of dried weight of plant material (mg GAE/g DW), for samples from Stip (CB6/14) and Pretor (CB2/13), respectively (Table 2).The quantitative analysis of total flavonoid content in different samples of C. botrys revealed values of TFC from 7.35 to 16.33 mg quercetin equivalents per gram of dried weight of plant material (mg QE/g DW) in samples from Radovis (CB5/13) and Kozuf (CB1/12), respectively (Table 2).
In the available literature, there is no data about the C. botrys TPC and TFC, thus only few articles could be found related to the total phenolic and total flavonoid content of other Chenopodium species.Few authors reported that C. album herb extracts contained 3.36 mg/g DW of free polyphenols (Nowak et a., 2015), while leaves of C. album comprised 44.2 µg GAE/mg of total phenolics and 9.53 µg CE/mg of flavonoids (catechins) (Dasgupta and De, 2007).

DPPH radical scavenging activity
The methanol extract of C. botrys showed DPPH radical scavenging activity with IC 50 values from 0.26-3.10mg/mL (Table 3).The radical scavenging ability of methanol extracts was compared to the scavenging activity of two reference substances, quercetin and BHA.The estimated IC 50 values were incomparably lower (1.0 mg/mL and 6.0 mg/mL, respectively).The highest DPPH scavenging activity showed the methanol extract prepared from samples from Strumica (CB3/14).

Non-site-specific hydroxyl radical-catalyzed 2-deoxy-Dribose degradation (NSSOH)
In the method with 2-deoxy-D-ribose as a substrate, hydroxyl radicals (OH•) are generated in conditions when EDTA-chelated iron (III) ions are incubated at physiological pH with reducing agent and H 2 O 2 .These hydroxyl radicals are capable to transfer on substrate and to fragment it into thiobarbituric acid reactive substances (TBARS).An extract that inhibits the development of TBARS in this system can be considered capable for scavenging hydroxyl radicals, protecting the carbohydrates from oxidative degradation (Shikov et al., 2006).C. botrys methanol extract has shown capability of inhibiting the formation of TBARS in a concentration-dependent manner with IC 50 values from 3.01 to 12.71 mg/mL.According the estimated values, extracts from samples collected from Strumica (CB3/14) and Antioxidative potential of Chenopodium botrys L. (Amaranthaceae) Pretor (CB2/13) have shown better radical scavenging activity compared to samples from other localities (Table 3).The antioxidant activity of quercetin and BHA was significantly higher in comparison to the activity of the tested samples (IC 50 values were 0.29 mg/mL for both standards).

Site-specific hydroxyl radical-catalyzed 2-deoxy-D-ribose degradation (SSOH)
In the site-specific hydroxyl radical-mediated 2-deoxy-D-ribose assay, an extract that inhibits the formation of TBARS in this system does not scavenge hydroxyl radicals but prevents their formation by chelating and deactivating iron (III) ions.Thus, the experiment is performed without EDTA (Shikov et al., 2006).The methanol extract of C. botrys was capable for preventing hydroxyl radical mediated oxidative degradation of 2-deoxy-D-ribose at physiological pH, with IC 50 values from 2.60 to 12.29 mg/ mL.The extracts from the samples of C. botrys collected from Strumica (CB3/14) and Pretor (CB2/13) have shown the most prominent activity (Table 3).The antioxidant activity of quercetin and BHA was significantly higher, with IC 50 values 6.39 μg/mL and 7.10 μg/mL, respectively.

FRAP assay
Ferric reduction is often used as an indicator of electron-donating activity, which is an important mechanism of phenolic antioxidant action.The initial concentration of C. botrys methanol extract (100 mg/mL) showed ferric reducing power from 26.14-43.40%(Table 3).The extract of C. botrys collected from Pretor (CB2/13) and Strumica (CB3/14) have shown better activity than other samples.The reducing power of methanol extracts was compared to the activity of quercetin and ascorbic acid.These substances with initial concentration of 0.25 mg/mL showed 21.37% and 27.71% of reducing potential of ferric ions, respectively.

H 2 O 2 -inhibition activity
Reactive oxygen hydroxyl radical can be formed by Fenton reaction, in the presence of metal ions and H 2 O 2 .Indirect neutralization of resulting hydroxyl radical by H 2 O 2 -inhibition activity is a significant method for the determination of antioxidant activity.Scavenging of H 2 O 2 by extracts may be attributed to their phenolic compounds, which can donate electrons to H 2 O 2 , thus neutralizing it to water (Ebrahimzadeh et al., 2010).The estimated percentages of inhibition showed that methanol extract of C. botrys was capable of scavenging hydrogen peroxide in a concentration dependent manner.H 2 O 2 -inhibition activity ranged from 28.84 to 46.56%.The extracts with initial concentration of 100 mg/mL prepared from C. botrys samples from Strumica (CB3/14) and Pretor (CB2/13) showed the highest H 2 O 2 inhibition (36.73% and 46.56%, respectively) but lower potential than quercetin (64.89 %) with the initial concentration of 0.25 mg/mL (Table 3).
In the present study, the evaluation of the antioxidant activity of methanol extracts of C. botrys was tested by methods that involve different biochemical mechanisms: DPPH radical scavenging activity, ferric reducing power determination (FRAP), H 2 O 2 -inhibition activity and inhibition of non-site-specific and site-specific hydroxyl radical-catalyzed 2-deoxy-D-ribose degradation.All examined extracts showed activity in each performed assay in concentration-dependent manner, with better antioxidant activity in higher concentrations.The methanol extracts of C. botrys from Strumica (CB3/14) and Pretor (CB2/13) have shown better activity in all assays, as they have lower IC 50 values in DPPH, NSSOH and SSOH assays, and higher inhibition percentage in FRAP and H 2 O 2 -inhibition activity assays (Fig. 1).
Comparing the obtained results for the antioxidant activity to additionally determined TPC and TFC, positive correlation can be found for the most samples of C. botrys, especially for samples collected from Pretor and Strumica, as they contained the highest TPC (71.25 and 51.28 mg GAE/g DW, in CB2/13 and CB3/14, respectively).However, the content of TFC cannot be correlated, as the sam- Positive correlation of antioxidant activity with higher TPC was previously reported.Thus, for Marrubium peregrinum L. (Lamiaceae), high content of phenolic compounds as well as significant linear correlation between their concentration values and antioxidant activity indicated that these compounds contributed to the strong antioxidant activity (Stankovic, 2011).Total phenolic and flavonoid contents showed positive correlations with the DPPH radical scavenging activity (p<0.01) and negative correlations with IC 50 (p<0.01)for methanol extracts of four Ulva species (Farasat et al., 2014).The extracts of Inula viscose showed significant antioxidant activity as the plant contained considerable levels of phenols.The ethanol extract (0.3 mg/mL) showed a remarkable antioxidant activity in three systems of assays (against 2,2-diphenyl-1-pic-rylhydrazyl, total antioxidant capacity and iron-reducing capacity), which was probably due to its high content of polyphenols (274.39±6.94mg GAE/g dry extract) (Chahmi et al., 2015).
Since phenols are generally responsible for antioxidant activity of plant extracts, in almost all cases, the antioxidant activity followed the total phenolic content.That was proven by estimation of the correlation coefficient between the total phenolic content and antioxidant activity by Kopjar et al., (2009).The correlation coefficients between the parameters of interest ranged from 0.5749 to 0.9604.A positive relationship between TEAC (Trolox equivalent antioxidant capacity), ABTS + (2,2'-azinobis [3-ethylbenzthiazoline]-6-sulfonic acid radical) and FRAP values and total phenolic content, measured by HPLC, was found in many representative herbs within Lamiaceae and Asteraceae families, as well (Wojdylo et al., 2007).Finally, con- tribution of the phenolics (catechin gallate, catechin, vanillic acid, kaempferol, ferulic acid, quercetin, resorcinol and 4-methylresorcinol) to the TAC (total antioxidant capacity) value indicated that phenolics (resorcinol) remarkably forwarded to the antioxidant capacity of the water-soluble extract of Chenopodium pallidicaule.This plant is considered as a potential source of natural antioxidants and other bioactive compounds which can be important for human health (Penarrieta et al., 2008).

Conclusion
Methanol extract of several specimens of Chenopodium botrys (Amaranthaceae), collected from different regions of the territory of Republic of Macedonia, showed scavenging activity against DPPH radical as well as antioxidant activity in non-site specific and site-specific hydroxyl radical catalyzed 2-deoxy-D-ribose degradation.The extracts exhibited inhibition of ferric reduction power (FRAP assay) and in H 2 O 2 activity as well.In comparison to the antioxidant activity of the reference substances (quercetin, BHA and ascorbic acid) the antioxidant activity of C. botrys methanol extracts was consider as very poor, despite the relatively high content of total phenols and total flavonoids.

Fig. 1 .
Fig. 1.Comparison of antioxidative activity of methanol extract of C. botrys obtained by different antioxidative assays.

Table 1 :
Plant material of C. botrys

Table 2 .
Total phenolic and flavonoid content in C. botrys

Table 3 :
Antioxidant activity of methanol extracts of C. botrys * initial concentration of ascorbic acid and quercetin 0.25 mg/mL ples that contained the highest amounts of TFC did not act as the best antioxidant agents. *