Correlation of the free radical and antioxidant activities of Eriobotrya Japonica Lindl . with phenolic and flavonoid contents

Various external environmental factors such as environmental pollutants, ultraviolet rays, smoking, and alcohol intake in modern society are causing the occurrence of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), which causes health problems. Active oxygen species such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical etc. are used in the process of oxygen oxidation during respiratory process and are produced through various metabolic processes. It is regulated by an in vivo antioxidant defense system such as SOD, glutathione, catalase and the like present in the human body (Hwang & Thi, 2014). However, excessively produced ROS damage tissues and cells, inhibit protein breakdown, DNA synthesis, reduce skin elasticity, skin wrinkle formation and cause pigmentation such as spots, freckles, blotch, and skin aging (Halliwell et al., 1992). In addition, RNS refers to nitrogen compounds such as NO, NO2, and ONOO can easily diffuse into the cell membrane and react with other free radicals. In particular, NO combines with ∙Oto produce peroxynitrite (ONOO-), which is highly reactive (Carr et al., 2000) (Figure 1), which induces cytotoxicity by peroxidizing tissue proteins, nucleic acids, phospholipids, and other senile diseases. Oxidative stress of the ROS and RNS can induce a variety of diseases that not only contribute to the aging process, but also degenerative diseases, atherosclerosis, diabetes, hypertension, and cancer, etc. (Chung et al., 2000; Yu, 1996; Rittié & Fisher, 2002).

Therefore, this research was carried out on the protective effect of different ethanol extracts of E. japonica against free radicals under in vitro situations. In vitro assays were evaluated on TPC (total phenolic content), TFC (total flavonoid content), DPPH radical scavenging, ABTS radical scavenging, FRAP (ferric reducing antioxidant power), HP (Hydrogen peroxide scavenging), NO (nitric oxide). Further, the free radical scavenging activities were correlated with phytochemical contents of the E. japonica leaves extracts. This is the reason that there is considerable scientific and commercial interest in discovering new antioxidants from natural product sources.

Plant materials
The loquat leaves used in this study was purchased from Goheung, Korea's loquat orchard, in September-October 2018 to identify and use the loquat leaves from the Jeonnam Herbal Medicine Agricultural Cooperative. The loquat sample was washed clean and then dried in a dry oven at 80 °C for 5 h to be crushed to a diameter of about 1 cm or less.

Extraction
Extraction conditions according to the ethanol concentration were selected through preliminary tests of conditions for various solvent concentrations of E. japonica leaves. The E. japonica leaves were extracted with 10 vol (v/w) ethanol (0%, 20%, 40%, 60%, 80%, 100%) using a heating mantle at 100 °C for 4 h and concentrated. The concentrated sample was frozen in a deep freezer at -70 °C for 24 h, then lyophilized and stored at 4 °C for use in the experiment.

Determination of total polyphenol contents
Total polyphenol content analysis was measured by applying the modified method of Folin & Denis (1912). The sample solution (0.5 mL) was placed in the EP tube with Folin reagent (0.5 mL) and 10% sodium carbonate (0.5 mL). After incubation for 1h at 25 °C, absorbance was measured at 760 nm using a UV/VIS spectrophotometer (Neogen, Optizen 2120 UV, Sejong, Korea). After creating a standard curve using tannic acid, the polyphenol amount was calculated as the tannic acid equivalent amount, and the equation was calculated as y = 0.0366x-0.0033 (r 2 = 0.9933).

Determination of total flavonoid contents
Total flavonoid content analysis was measured by applying the modified method of Saleh & Hameed (2008). The sample (150 μL) was mixed with 80% ethanol (600 μL) and 5% sodium nitrite (45 μL) and reacted at room temperature for 5 min, after which 10% aluminium chloride (45 μL) was added and left at room temperature for 5 min. 1 N NaOH was mixed with 300 μL, and absorbance was measured at 510 nm using a UV/VIS spectrophotometer (Neogen, Optizen 2120 UV, Sejong, Korea). A standard curve was prepared using Catechin to calculate the amount of flavonoids in terms of catechin equivalent, and the equation was calculated as y = 0.0025x + 0.0142 (r 2 = 0.9992).

Assay for the estimation of free radical
DPPH radical scavenging activity DPPH radical scavenging ability is one of the methods to confirm the electron-donating ability and was measured by applying the modified method of Blois (1958). The sample solution (10 μL) was placed in 96 well plate with 0.2 mM DPPH (190 μL). After incubation for 0.5 h at 25 °C, absorbance was measured at 515 nm using an ELISA reader (Thermo Fisher SCIENTIFIC, Multiskan Sky, KOREA). L-ascorbic acid was used as a positive control. The antioxidant activity was expressed as a percentage in the following manner (Equation 1).
A: absorbance of control B: absorbance of test sample

ABTS radical scavenging activity
ABTS radical scavenging ability is one of the methods to confirm the electron-donating ability and was measured by applying the modified method of Re et al. (1999). 7 mM ABTS and 2.45 mM potassium persulfate were mixed at a ratio of 1: 1 (v /v) to react in a darkroom at 25 °C for 24 hours to generate radicals. Radical stock solution was diluted with distilled water so that the absorbance value at 734 nm was 0.70 ± 0.02. After incubation for 5 min in a dark room at 25 °C, absorbance was measured at 734 nm using a UV/VIS spectrophotometer (Neogen, Optizen 2120 UV, Sejong, Korea). L-ascorbic acid was used as a positive control. The antioxidant activity was expressed as a percentage in the following manner (Equation 2). (2) A: absorbance of control B: absorbance of test sample

Nitric oxide scavenging activity
The scavenging activity of nitric oxide forms a triazolo fluoresceun that emits green fluorescence at an excitation wavelength of 490 ~ 495 nm by the specific NO indicator DAF-2 trapping NO between its two amino groups. DAF-2 solution was prepared by dissolving 1 mg of DAF-2 in 0.55 ml of Dimethyl sulfoxide and diluting it again to 400 times (v/v) using 50 mM phosphate buffer (pH 7.4). 10 μL of the sample was mixed with 130 μL of 50 mM phosphate buffer (pH 7.4), after which 10 μL of 40 mM SIN-1 and 50 μL of DAF-2 solution were added. The fluorescence intensity of triazolofluorescein produced by the reaction of DAF-2 and NO for 10 min at room temperature was measured at excitation 485 nm and emission 525 nm using a fluorescence microplate reader (Molecular Devices, Gemini EM, U.S.A).

Statistical analysis
The experimental data of this study were expressed as the mean ± standard deviation after three repeated experiments. Significant difference tests were performed by one-way variance analysis and Tukey's multiple range test (TMRT) method using SPSS (statistical package for the social sciences, ver. 25) (p < 0.05). Correlation analysis was performed using the Pearson linear correlation method at a significance level of 0.05.

Extraction
The yield of the plant extract is considered an important factor in the measurement of antioxidant activity, and even if the physiological activity of the extract is excellent, the economic efficiency is insufficient when the yield of the extract is low. The extraction yield is an important part to be considered for various commercialization and industrialization purposes of functional extracts (Ham et al., 2015). Therefore, according to the results of the preliminary test from E. japonica, it was extracted for each ethanol concentration of 20%, 40%, 60%, 80%, and 100% suitable ingredient for food, medicine, and cosmetics. The ethanol extracts were concentrated under vacuum and the total extraction yields of ethanolic extracts from E. japonica are shown in Table 1. The yield of hot water (20.0%), 20% EtOH (14.5%), and 80% EtOH (14.4%) was higher than those of 40% EtOH (5.2%), 60% EtOH (9.37%), and 100% EtOH (9.16%), respectively. This is presumed to show the difference in extraction yield as a variable of the mixing ratio of water and ethanol for the sample. If the extraction yield is more than 10%, it is known to be economical, so the hot water, 20% EtOH, and 80% EtOH extract are considered as economically high-potential plant materials .

Total phenolic and total flavonoid contents
The phenolic compounds of the representative secondary metabolite from plants are an aromatic compound having a hydroxyl group and are known to be involved in various physiological activities. It is known that the effect on antioxidant activity varies depending on the type or content of the phenolic compound (Liu, 2004;Manach et al., 2005;Ryu et al., 2006). In addition, flavonoids, which are known to inhibit oxidative action in vivo, are important compounds in determining the antioxidant power of natural products and have the ability to scavenge free radicals as the most helpful substances for immune enhancement, and it have been reported to exhibit the ability to scavenge free radicals and inhibit the formation of lipid peroxide (Middleton & Kandaswami, 1994;Song et al., 2015).
In this study, the total polyphenol and flavonoid compound contents were measured by tannic acid and catechin, respectively ( Table 1). The total polyphenol content was highest in the hot water extract at 31.97 ± 1.29 mg TAE/g, followed by 60% ethanol (27.51 ± 0.71) and 20% ethanol extract (20.33 ± 0.02). The total flavonoid content showed the highest value as 96.10 ± 0.31 mg CE g of hot water extract, and it contained total flavonoids in the order of 40% ethanol (88.69 ± 0.20) and 60% ethanol extract (88.47 ± 0.26).

DPPH radical scavenging
The DPPH assay is a principle of a stable free radical measures such as vitamin C, tocophenol and aromatic compounds that has been widely used to evaluate the radical scavenging ability of various samples (Yoo et al., 2004). The fee radical scavenging activity evaluated by DPPH was expressed as the RC 50 value (the concentration of sample required for scavenging radical by 50%). All extract samples showed a high activity of 80% or higher at concentrations above 200 μg/mL (not show). RC 50 values of extracts by ethanol extracts of E. japonica leaves are 80% EtOH (61.96 ± 5.01 μg/mL, 60% EtOH (63.87 ± 0.59 μg/mL), 40% EtOH (72.92 ± 05.22 μg/mL), 20% EtOH (75.53 ± 1.14 μg/mL), hot water (102.49 ± 3.04 μg/mL), and 100% EtOH (117.96 ± 0.88 μg/mL), respectively (Figure 2A). This is thought to be a complex action of several compounds extracted according to the intrinsic color and ethanol concentration of E. japonica leaves extract (Kyeoung-Cheol & Ju-Sung, 2018).

ABTS radical scavenging
ABTS assay performs the antioxidant activity of decolorization when free radicals generated by reaction with potassium persulfate react with antioxidants to remove free radicals (Park et al., 2016). The RC 50 value of ethanolic extracts from E. japonica leaves was highest in 60% EtOH at 73.81 ± 0.16 μg/mL, hot water 103.12 ± 0.67 μg/mL, and 100% EtOH 169.78 ± 0.89 μg/mL, respectively (Figure 2A). These results did not show a certain tendency to activity. ABTS radical scavenging activity results showed relatively higher activity than DPPH radical scavenging activity. ABTS is capable of measuring both the hydrophilicity and hydrophobic materials of the radical scavenging activity from the extract sample and thus exhibiting high antioxidant activity (Re et al., 1999;Choi & Shin, 2015;Lee et al., 2016b).

FRAP Reducing power
The FRAP method is a principle that measures the reduction of trivalent iron to divalent iron by donating electrons directly rather than the radical scavenging assay. It is based on the principle that the ferric tripyridyltriazine (Fe (III) -TPTZ) complex agent is reduced to ferrous tripyridyltriazine (Fe (II) -TPTZ) by antioxidants capacity according to the reduction degree of the sample at a low pH and it can be said to have a high reducing power when the absorbance value increases as it turns blue (Benzie & Strain, 1996). FRAP activity of E. japonica leaves extract by ethanol concentration was highest in 60% ethanol extract (559.17 ± 2.69 mg TE/g) and did not show a significant difference from hot water extract (554.12 ± 1.78 mg TE/g). The lowest value of FRAP activity was obtained with 100% ethanol extract (317.18 ± 1.42 mg TE/g) ( Figure 2B). These FRAP results showed contrary outcomes to the Kyeoung-Cheol & Ju-Sung (2018) study that FRAP activity increased as the ethanol content increased.

Nitric oxide radical scavenging
Nitric oxide (NO) of the active nitrogen species is a highly reactive radical produced from arginine through catalysis of NO synthase (NOS) enzymes in vivo, and has physiological activities such as blood coagulation, blood pressure control, and immune function against cancer cells (Chung et al., 2001). As a result of measuring the nitric oxide radical scavenging activity value of ethanolic extracts from E. japonica leaves, it was confirmed that the scavenging activity increased in concentration-dependent from EtOH extracts. The NO scavenging activity of hot water showed at the concentration of a scavenging activity 31 -83% at 25 ~ 400 μg/mL, which was similar to positive control (BHA) of scavenging activity. The RC 50 value of ethanolic extracts was highest in hot water at 52.51 ± 8.54 μg/mL, followed by 60% EtOH (59.33 ± 9.88 μg/mL), 40% EtOH (5.66 ± 7.82 μg/mL), 20% EtOH (75.96 ± 9.31 μg/mL), 80% EtOH (106.48 ± 8.55 μg mL), 100% EtOH (154.79 ± 3.29 μg/mL), respectively (Figure 2A). This is a different result from that of 80% EtOH showing the best scavenging activity in nitric oxide scavenging ability  because it is considered to be the difference between extraction conditions and methods (Akowuah et al., 2005).

Correlation of antioxidant capacity with free radical scavenging
The correlation of the antioxidant activities with phenolic and flavonoid contents from E. japonica leaves is shown in Table 2. The value of the correlation coefficient (r) shows a positive correlation closer to 1 based on 0, and a negative correlation closer to -1 based on 0. The correlation between the polyphenol  ------1 1) Correlation is significantly different at *p < 0.05 and **p < 0.01 (Pearson). 2) FRAP (Ferric reducing antioxidant power) 3) DPPH (α, α-diphenyl-β-picrylhydrazyl) radical scavenging 4) ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging 5) HP (Hydrogen peroxide) scavenging 6) NO (Nitric oxide) radical scavenging and flavonoid contents of ethanolic extracts (0.955, p < 0.01) showed a significant correlation, and no correlation with nitric oxide. The correlation between DPPH and ABTS confirming the electron donating ability was 0.972 (p < 0.01), which showed a significant high correlation ( Table 2). The correlation of DPPH for TFC, FRAP, HP, and NO showed 0.670, 0.737, 0.801, and 0.905 (p < 0.01), respectively, and significant correlation was confirmed ( Figure 3A). In addition, the correlation of ABTS for TFC, FRAP, HP, and NO was measured 0.654 0.707, 0.874, and 0.933 (p < 0.01), respectively, and significant correlation was confirmed ( Figure 3B). Therefore, it is thought that the free radical activity in extracts by ethanol concentration from E. japonica leaves is involved in scavenging activity of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Also, the polyphenol and flavonoid contents of the extract showed a significant correlation with the free radical scavenging ability (DPPH, ABTS) and electron donating ability (FRAP), but does not appear to correlation with the NO scavenging ability.

Conclusions
The results of this study indicated that the hot water extract of E. japonica leaves owing to high levels of flavonoids and phenols content, showed reductions activity of FRAP, DPPH, ABTS, HP, NO assay. In the extract of E. japonica leaves, the correlation of the free radical and antioxidant activities with polyphenol and flavonoid contents was showed high significant. Therefore, the phytochemical content of hot water extract from E. japonica leaves revealed potential and economic value because of its use in antioxidant functional food and anti-ageing cosmetics raw materials, as well as in pharmaceuticals. In addition, correlations in the studied antioxidant activities will offer better understanding of post-harvest physiology of E. japonica leaves.