Antioxidant Activities of Agrimonia pilosa Ledeb: In Vitro Comparative Activities of Its Different Fractions

Abstract

The antioxidant potentials of the methanol extract and its various fractions from Agrimonia pilosa Ledeb were measured as 1,1-diphenyl-2-picrylhydrazyl (DPPH), and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging abilities as well as the reducing power decreasing NO in vitro. The methanol extract of Agrimonia pilosa Ledeb showed significantly strong scavenging effects on free radicals of DPPH, ABTS, and NO. Hexane fraction (HF), ethyl acetate fraction (EF) and n-butyl alcohol fraction (BF) were prepared by solvent fractionation. By comparison of polyphenol contents among BF and HF, EF with high polyphenol contents showed the highest DPPH, ABTS, and NO scavenging effects with the value of 50.2%, 80.4%, and 65.7%, respectively, at the highest tested dose. HPLC analysis revealed the presence of various phenolic compounds in EF of Agrimonia pilosa Ledeb. These results suggest that EF could be considered as natural antioxidant sources and dietary nutritional supplements to prevent oxidation-related diseases.

Introduction

Free radicals can be generated in biological systems in the form of reactive oxygen species (ROS), such as superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, and the singlet oxygen (Halliwell et al., 1995). High levels of free radicals or active oxygen species create oxidative stress (Ordonez et al., 2006). Although normal cells possess antioxidant defense systems against ROS, the continuous accumulation of damage to the cells induces diseases such as gastric ulcers (Sakurai et al., 2005), colon cancer (Babbs, 1990), and hemolytic anemia (Jain and Hochstein, 1979). Human bodies are protected from oxidative stress by an efficient defense system, however, its capacity is affected by age, diet, and health status of the individual (Chun et al., 2003). Therefore, there is a growing interest in searching for natural antioxidants from medical herbs.

The species of Agrimonia pilosa, belonging to the Rosaceae, are listed in the oriental medicine as an astringent hemostatic to treat bleeding. Many pharmacological studies have reported that Agrimonia pilosa Ledeb showed broad biological properties, such as antioxidant activity (Zhu et al., 2009), anti-viral effect (Shin et al., 2010), and acetylcholinesterase inhibitory effect (Jung and Park, 2007). Agrimonia pilosa Ledeb contains abundant flavonoids, such as catechin, hyperoside, quercitrin, quercetin, and rutin. These compounds are demonstrated to be the major bioactive constituents having anti-inflammatory, anti-tumor, anti-virus, anti-bacteria, and anti-oxidation functions (Jian and Xiao, 1986). Considering that these functional effects might be correlated with antioxidant properties, we evaluated the antioxidant properties of the methanol extract from Agrimonia pilosa Ledeb and its fractions using diverse radical scavenging assays. This study included 2, 2'-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, 2, 2'-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, and NO radical scavenging activity. The total phenolic and total flavonoid contents were investigated. Flavonoids components of hexane fraction (HF), ethyl acetate fraction (EF) and n-butyl alcohol fraction (BF) were also compared by HPLC analysis.

 

Materials and Methods

Plant samples

The whole plant of Agrimonia pilosa Ledeb was purchased from Daegu yangnyeong Market (Daegu Korea). A voucher specimen (NP-116) was deposited at the herbarium of the sample shelf of Kyungpook National University (Daegu, Korea). Four kilogram of Agrimonia pilosa Ledeb were extracted at 85℃ reflux with 100% MeOH for 3 h (5 L×3 times). After evaporation of the solvent under vacuum (JP/N 1000S-W; Eyela, Japan) and water bath (Digital Water Bath SB-651; Rikakikai, Tokyo, Japan), the crude MeOH extract (ME) (304.4 g) was obtained and suspended in hot water and partitioned with n-hexane, EtOAc, and BuOH successively, to afford n-hexane-(HF) (26.0 g), EtOAc-(EF) (95.0 g), and BuOH-(BF) (83.4 g) soluble fractions, respectively. The final samples were dissolved in dimethyl sulfoxide (DMSO).

Total phenolic content

The amount of total phenolic content was determined according to the Folin–Ciocalteu colorimetric method (Maria et al., 2002). Gallic acid was used as the standard for calibration curve. The results were expressed as gallic acid equivalents per gram of sample in dry weight (㎎ GE / g DW). Eighty microliters of 0.2 N Folin-Ciocalteu reagent was added to samples at room temperature for 3 min, followed by the addition of sodium carbonate solution. After mixing for 20 min, absorption at 725 ㎚ was measured by using a Spectrophotometer (Power Wave XS, Bio-Tek Instrument, Winooski, VT). All tests were done in triplicate and the results were presented as means ± SD.

Total flavonoids content

Total flavonoids content was determined by colorimetric assay. At zero time, each sample was mixed with distilled water and 5% NaNO2. After incubation for 6 min, 10 μl 10% AlCl3 and 1 M NaOH solution were added. The absorbance was recorded at 415 ㎚ after 1 hour. Quercetin was used to make the calibration curve and the flavonoids content was expressed as milligram quercetin equivalent per g dry weight (㎎ QE/g DW.). All samples were analyzed in triplicate.

DPPH scavenging activity

DPPH radical scavenging effect was carried out with our previous method (Chen and Kang, 2014; Park et al., 2011). The tested sample solution was mixed with 0.4 mM DPPH solution. After 30 min of standing in darkness, the absorbance was measured at of 517 ㎚. Gallic acid was used as a positive control.

ABTS scavenging activity

The total antioxidant activity was measured by the ABTS radical cation decolourisation assay (Chen and Kang, 2013; Kim and Kang, 2010). 7 mM ABTS solution was mixed with 2.45 mM potassium persulfate solution. After 12 h, the absorbance was adjusted to 0.70 ± 0.05 at 734 ㎚. To determine the scavenging activity of the samples, ABTS reagent was mixed with sample. The absorbance was measured after 6 min at 734 ㎚. Trolox was used as a positive control.

Nitric oxide radical scavenging activity

NO generated from sodium nitroferricyanide dehydrate (SNP) was measured using the Griess reagent, as described previously (Chen and Kang, 2013). Each sample or quercetin was mixed with 25 μl of 10 mM SNP solution (pH 7.4), and incubated at 25℃ for 150 min. And then 50 μl of Griess reagent (2% sulfanilamide in distilled water and 0.2% naphthyletylene-diamine dihydrochloride in 4% phosphoric acid) was added. The absorbance of chromophore formed during the diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylene-diamine was read at 540 ㎚. Quercetin was used as the reference for NO radical scavengers.

Pattern analysis by HPLC

The phenolic compounds of the methanol extract and its solvent fractions were analyzed by HPLC (Jasco, Tokyo, Japan) on ZORBAX Eclipse XDB-C18 (4.6 ㎝ × 250 ㎜, 5 ㎛, Agilent, Santa Clara, CA, USA) column, associated with UV-Visible detector (Jasco, Tokyo, Japan). The solvent system was a gradient of mobile phase: soln. A, 0.5% formic acid in H2O; soln. B, MeOH. The following gradient was used: 0–2 min, 80% A; 2–5 min, 80% A to 70% A; 5-10 min, 50% A; 10-15 min, 50% A to 30% A, 15-20 min, 0% A, 20-25 min, 80% A. Operating conditions were as follows: flow rate, 1.0 ㎖/min; injection volume, 20 μl; UV detector at 280 ㎚. The column was operated at a temperature of 40℃.

Statistical analysis

Data were expressed as means ± standard deviations (SD) of three replicates. Determinations were then analyzed by SPSS V.13 (SPSS Inc., Chicago, USA). One way analysis of variance (ANOVA) and Duncan’s New Multiple-range test were used to determine the differences among the means. P values of < 0.05 were regarded as significant.

 

Results and Discussion

Total phenolics and flavonoid content

The contents of phenolics and flavonoids in the ME of Agrimonia pilosa Ledeb and its fractions, hexane, ethylacetate (EA), and BuOH frac. are shown in Fig. 1. The results revealed that total phenol contents were significantly higher than the flavonoid contents in each sample. The ME contained the highest amount of phenolics (21.5 ㎎ GE/g DW) among all the analyzed samples. The EF and BF had a comparatively lower presence of total phenol content at 20.3 and 18.6 ㎎ GE/g DW, respectively. Many phenolic compounds are known as potent antioxidants because of their ability to scavenge free radicals and active oxygen species such as singlet oxygen, superoxide anion radical and hydroxyl radicals (Chen et al., 2012). The highest flavonoid content was also found in ME representing 18.3 ㎎ QE/g DW. The contents of flavonoids and phenolics in tested samples were in the order of ME > EF > BF > HF. Significant correlation has been found between the total phenolics and flavonoids content in Agrimonia pilosa Ledeb (r = 0.78, p = 0.002). Total phenolic and fl avonoid contents of the hexane fraction were lowest, 7.9 ㎎ GE/g DW and 11.2 ㎎ QE/g DW, respectively. This is in agreement with previous reports that the hexane extract of Xylaria sp. contained higher total flavonoids than phenolic content (Liu et al. 2007). Previous studies on the aerial parts of Agrimonia pilosa showed the similar content of flavonoids (Tsai et al., 2007) compared with our results. Agrimonia pilosa Ledeb has a considerable amount of phenolic compounds, indicating that this plant may be a resource of efficient natural antioxidant.

Fig. 1Comparative analysis of total phenolic and flavonoid contents of the methanol extract and its fractions from Agrimonia pilosa Ledeb.

Antioxidant activities of Agrimonia pilosa Ledeb

Several solvent systems have been used to extract antioxidants for evaluations of different antioxidative activities (Apostolidis et al., 2011). To find out the antioxidant principle of Agrimonia pilosa Ledeb, the crude MeOH extract (ME) was obtained and fractionated to hexane, EtOAc, and BuOH fractions successively. The scavenging activities of the methanol extract and its various fractions from Agrimonia pilosa Ledeb on DPPH radical are shown in Fig. 2. All the tested samples showed DPPH radicals scavenging effects in a dose-dependent manner. Overall, ME showed the strongest scavenging effect on DPPH radical. The scavenging activity of ME reached to 55.6% at the concentration of 50 ㎍/㎖. No significant difference was found between ME and EF (50.2%). Due to the different biological potentials of compounds with different polarity (Julkunen-Tiito, 1985), solvent fractionation is frequently used for isolation of biological compounds. Zhu et al. (2009) reported that DPPH activity highly exhibited in aqueous extract of Agrimonia pilosa Ledeb with IC50 (50% inhibitory concentration) values of 13.0 ㎍/㎖. However, significantly less yields of extract were generally found in aqueous extract than organic solvent (Jayaprakasha et al., 2001). In the case of requirement for further separation, alcoholic solvent was used because of high extracting efficiency. Among organic solvents, methanol and ethanol are usually preferred for the recovery of phenolic compounds (Sultana et al., 2007). The DPPH scavenging activity of Agrimonia pilosa Ledeb is arranged in the following descending order: ME > EF > BF > HF. Generally, solvent fraction shows more potent activity than total alcoholic extract. But in the case of Agrimonia pilosa Ledeb, DPPH scavenging activity of the methanol extract is higher than EF or BF. This result might be attributed to the synergistic effects of diverse antioxidative components of EF and BF. Parejo et al. (2003) reported a similar result on DPPH scavenging activity. MeOH extract from Baccharis pentlandii DC was significantly higher than that of the other fractions including EF, BF, and HF. HF with the lowest total phenol content showed the lowest DPPH scavenging effect. ME, EF and BF showed DPPH radical scavenging effect depending total phenol content. Previous studies have also demonstrated that the significant correlation exists between the DPPH-scavenging ability and its total phenolic content (Katsube et al., 2004).

Fig. 2.DPPH radical scavenging activities of the methanol extract and its fractions from Agrimonia pilosa Ledeb at different concentrations. Different letters indicate a significant difference by Ducan’s multiple ranges at p < 0.05.

The blue/green chromophore ABTS was produced through the reaction between ABTS and potassium sulphate. Trolox, a vitamin-E analogue was used as a positive control in this assay. The ABTS radical scavenging activities were measured at three concentrations, ranging from 6.25-25 ㎍/㎖. Of all the tested samples, ME showed an outstanding scavenging activity with a scavenging value of 93.1% at 25 ㎍/㎖ concentration. Fraction EF showed 17% more potent activity than BF at 6.25 ㎍/㎖ concentration. Interestingly, HF containing non-polar compounds also showed significantly strong activity (74.3%) at the same dose (Fig. 3). These results suggested that Agrimonia pilosa Ledeb contains polar and non-polar substances with a strong H-donating capacity and major antioxidants are mainly soluble in polar solvents such as ME, EF and BF.

Fig. 3.ABTS radical scavenging activities of the methanol extract and its fractions from Agrimonia pilosa Ledeb at different concentrations. Different letters indicate a significant difference by Ducan’s ranges at p < 0.05.

Because of the complex nature of plants, the biological activity of the extract from plants cannot be evaluated by single method. In order to elucidate the active principles, the biological activity should be evaluated by diverse bioassay systems. In this study, we additionally adopted in vitro NO inhibitory methods to evaluate the antioxidative ability of Agrimonia pilosa Ledeb. NO can be viewed as a radical itself. Acker et al. (2014)> reported that NO molecules are directly scavenged by flavonoids. Therefore, NO scavenging effect of flavonoids has been speculated to play an important role in the therapeutic potentials of flavonoids. Fig. 4. showed the nitric oxide (NO) radical scavenging activity of the methanol extract and its fractions from Agrimonia pilosa Ledeb. The methanol extract and all fractions of Agrimonia pilosa Ledeb showed NO scavenging activity. ME exhibited the strongest NO scavenging activity, which was 20% greater than the HF, at the concentration of 250 ㎍/㎖. The scavenging activity of ME reached a plateau of 68.2%. EF (65.7%) showed higher NO scavenging activity than BF (57.5%). HF containing non-polar compounds showed higher activity on NO scavenging than BF at the concentration of 62.5 ㎍/㎖. This result was of very good agreement with previous studies who reported that n-hexane fraction of Hizikia fusiformis showed stronger NO radical scavenging activity than other fractions (Karawita et al., 2005).

Fig. 4.NO radical scavenging activities of the methanol extract and its fractions from Agrimonia pilosa Ledeb at different concentrations. Different letters indicate a significant difference by Ducan’s multiple ranges at p < 0.05.

Components analysis by HPLC

The phenolic compounds of ME, EF, BF, and HF of Agimonia pilosa Ledeb are monitored at 280 ㎚. Fig. 5. displays the composition profiles of all constituents, which was compared by plotting peak areas of all the constituents in each fraction. The ME has three major peaks at retention times of 13.3, 14.4, and 15.4 min with relative intensity of 10.0 12.7, and 8.9%, respectively. The crude extract was incompletely separated and three major constituents were overlapped in EF and BF. However, these major peaks were not found in the HF fraction. The antioxidant activities of Agrimonia pilosa Ledeb were directly proportional to the concentration of phenolic substances. The ABTS and DPPH radical scavenging effects of ME, and its solvent fractions decreased in the following order: ME > EF > BF > HF. HF has an inferior scavenging activity compared with that of other fractions. These results suggested that the antioxidant activities may directly proportional to the concentration of these three major constituents. HF with inhibition values of 39.6% at 125 ㎍/㎖, exhibited noticeable efficiencies on NO radicals scavenging, which were similar with BF. Accordingly, HF may contain some antioxidants scavenging NO radicals. In conclusion, these results suggest that the methanol extract of Agrimonia pilosa Ledeb and its fractions could be a potent scavenger against diverse radicals.

Fig. 5.HPLC chromatograms of ME (A), EF (B), BF (C) and HF (D) from Agrimonia pilosa Ledeb recorded at 280 ㎚.

Correlations between antioxidant capacity and functional compounds content

The total phenolics and flavonoids contents were measured and statistical correlations between these constituents and antioxidant activity were evaluated. The results are shown in Table 1. Significant correlation has been found between DPPH radical scavenging activity and the contents of phenolic (r = 0.89, < 0.001) and flavonoids (r = 0.87, < 0.001) from all the fractions from Agrimonia pilosa Ledeb. ABTS and DPPH radical scavenging activities were found to correlate strongly to the total phenolics (Table 1). The results reveal that the fractions of Agrimonia pilosa Ledeb containing high levels of phenolic and flavonids can contribute to antioxidant functions. There was a significant reverse correlation between NO radical scavenging activity and the total phenolics or flavonoids content. This result might be explained by the potential effects of solvent used in antioxidant extraction on the results of antioxidant activity tests and different antioxidants except phenolics or flavonoids.

Table 1.zTPC: total phenolics contents; TFC: total flavonoid contents

In conclusion, the fractions including the compounds with different polarity possess different magnitudes of antioxidant potencies. The methanol extract of Agrimonia pilosa Ledeb and its ethyl acetate fraction exhibit strong antioxidant activities. Especially, the ethyl acetate fraction (EF) of Agrimonia pilosa Ledeb was demonstrated to have the highest total phenolic and flavonoids contents. EF could be considered as natural antioxidant sources and dietary nutritional supplements to promote human health and prevent oxidation-related diseases. Further investigations to isolate and characterize the antioxidative constituents of Agrimonia pilosa Ledeb will be needed.