Elucidation of phytochemicals and antioxidants properties of Sasa quelpaertensis

ABSTRACT Sasa quelpaertensis Nakai extract (SQE) has long been used as a traditional medicine and health drink. The present study aims to examine the properties and antioxidant activity of the phytochemicals isolated from S. quelpaertensis. Therefore, the SQE was fractionated with n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc), and n-butanol (BuOH). Phytochemicals present in the solvent fraction were also isolated using a high-performance liquid chromatography (HPLC) system. In addition, their DPPH radical scavenging ability and NO production inhibitory effect were compared. The results showed that phytochemicals p-hydroxybenzaldehyde (1), salicylic acid (2), syringaldehyde (3), methyl cis-p-hydroxycinnamate (4), methyl trans-p-hydroxycinnamate (5), p-coumaric acid (6), 2,3-dihydrozypropyl 9Z,12Z-octadecadienoate (7), (+)-(6S,7aS)-epilolide (8), (-)-(6 R,7aS)-loliolide (9), naringenin (10), 3-O-p-coumaroyl-1-(4-hydroxy-3,5-dimethoxyphenyl)-1-O-β-glucopyranosylpropanol (11), tricin (12), and tricin 7-O-b-D-glucopyranoside (13) were isolated from CHCl3, EtOAc, and BuOH fractions and then identified using nuclear magnetic resonance (NMR) spectrometry. This is an initial study on compounds 2, 3, 4, 5, 7, 8, and 9 obtained from S. quelpaertensis. Compounds 11 (IC50 120.3 μM) and 7 (IC50 43.62 μM) showed significant DPPH radical scavenging activity and anti-inflammatory activity, respectively. We believe these results may provide the basic data for developing effective antioxidants using the SQE.


Introduction
Reactive oxygen and nitrogen species (ROS and RNS, respectively) are derived from oxygen and nitrogen molecules through the biological system in the body and include free radicals such as superoxide anion radical (O 2 − ·), hydroxyl radical (·OH), nitric oxide (NO·), and nitric dioxide (NO 2 ·), as well as nonradical molecules such as hydrogen peroxide (H 2 O 2 ), singlet oxygen (O 2 ), nitrous acid (HNO 2 ), and dinitrogen tetroxide (N 2 O 4 ). [1] ROS and RNS are important intermediaries in the synthesis of critical signaling molecules that respond to abiotic stress and play an important role in the immune response against pathogens. [2] However, when their concentration in a cell exceeds the required levels, they begin to adversely affect critical cellular structures such as proteins, lipids, and nucleic acids and can lead to oxidative and nitrosative stress. [3,4] Oxidative and nitrosative stress is associated with various diseases such as cancer, inflammation, Alzheimer's, atherosclerosis, and arteriosclerosis. [5,6] Therefore, research to isolate antioxidant compounds from natural resources 1.0 L distilled water, followed by the addition of 1.0 L n-hexane, chloroform, ethyl acetate, and n-butanol, three times. Each partitioned fraction was filtered through a filter paper and then concentrated using a rotary evaporator (Rotavapor R-100, Büchi, Labortechnik AG, Flawil, Switzerland) and a freeze dryer (IlshinBioBase Co., Ltd., Gyeonggi, Korea) at 40°C. Each sample was stored in a refrigerator at −70°C until use.

Isolation and identification of phenolic compounds
An automated HPLC system (2695 Alliance system; Waters, Milford, MA, USA) equipped with a photodiode array (PDA) detector and a fraction collector was used to purify the phenolic compounds. Each phenolic compound was isolated using a Symmetryprep™ C18 column (7.8 × 300 mm ID. 7 μm; Waters, Milford, MA, USA). The column oven was maintained at 40°C. The mobile phase consisted of MeOH (A) and water (B). The gradient elution program was as follows: 0.0-60.0 min linear from 20% to 100% of A, quick decrease to 20% A for 2 min, and isocratic for 10 min. The isolated compound was monitored by recording the UV spectra of the irradiated samples between 200 and 600 nm. The CHCl 3 , EtOAc, and BuOH fractions were repeatedly injected in 100 μL at a concentration of 100 mg/mL. Their repeatedly injected fractions were obtained phytochemicals through a fraction collector. After monitoring the HPLC chromatogram, the fractions of the same compound were combined. The isolated compounds were analyzed using a JEOL FT/NMR 400 spectrometer (JEOL Ltd., Tokyo, Japan). Each isolated compound was recorded at 400 and 100 MHz by referring to the solvent signals (CD 3 OD) and was identified by comparing with the reference data.

Total phenolic (TP) and total flavonoid (TF) content
The TP contents were measured using the Folin-Ciocalteu method with some modifications. [19] Briefly, 0.5 mL of the extract (1 mg/mL in distilled water) was mixed thoroughly with 0.5 mL of 2 N Folin-Ciocalteu reagent for 5 min followed by the addition of 2 mL of 7% (w/v) sodium carbonate. The mixture was left to react at room temperature for 90 min and then its absorbance was measured at 750 nm. The TP contents were calculated using the calibration curve and were expressed as mg of GA equivalent (GAE) per gram dry weight. The TF contents were measured using the Davis method. [20] The extract (0.2 mL), 0.5 mL of 1 N NaOH, and 4 mL of diethylene glycol were mixed in a 15-mL conical tube and allowed to stand at 37°C for 1 h. The absorbance values were measured at 420 nm. Standard curves were prepared using NG standard solutions (5, 10, 50, 100, and 500 μg/mL). The TF contents were expressed as the mg of NG equivalent (NGE) per gram dry weight.

DPPH radical scavenging assay
DPPH radical scavenging activities of the extract, solvent fraction, and isolated compounds were investigated using the method of Floegel et al. [21] with some modifications. First, 100-μL samples of appropriate concentration (1.0 mg/mL) were dispensed in 96-well plates and mixed with 100 μL of 0.4 mM DPPH solution. The plates were then left to react in the dark for 10 min, and their absorbance values were measured at 517 nm. The radical scavenging activity was calculated as follows: DPPH radical scavenging rate (%) = (A control -A sample )/A control × 100, where A control is the absorbance of the control (DPPH + methanol) and A sample is the absorbance of the sample (DPPH + sample). The antioxidant activity was expressed as the IC 50 value, where IC 50 refers to the amount of antioxidant needed to reduce the initial radical concentration by 50%. BHA and ascorbic acid were used as reference compounds.

Cell culture
The Raw264.7 murine macrophage cell line was obtained from the Korea Cell Line Bank (Seoul, South Korea). The cells were cultured in 1% penicillin/streptomycin (PS)/DMEM containing 10% FBS at 37° C in a 5% CO 2 incubator.

MTT assay
Cell viability and cytotoxicity were determined by the MTT cell viability assay. The cells were seeded at a density of 3 × 10 4 /well into a 96-well flat-bottom cell culture plate. After 24 h of incubation, the extract was added to the cell culture plate and then after 1 h, it was cultured with LPS for 24 h. Mitochondrial enzyme activity, which is an indirect measure of the number of viable respiring cells, was determined using the MTT reagent after 24 h of treatment with the extracts. The absorbance was read using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA) at 595 nm. The effect of extracts on the cell viability was evaluated as the relative absorbance compared with that of control cultures.

Nitrite production assay
The amount of nitrite produced was determined by a colorimetric assay. Briefly, 100 μL of the cell culture medium were mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylene diamine in 5% phosphoric acid) and incubated for 10 min. The absorbance at 540 nm was recorded using a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The nitrite concentration was determined by extrapolating the sodium nitrite standard curve.

Statistical analysis
All experiments were conducted in triplicate. The results are expressed as the mean±standard deviation (SD). Values in the same column were measured using one-way ANOVA with Duncan's Post-Hoc test. Differences were considered statistically significant at p < .05. Associations between phenolic contents and radical scavenging activities were assessed using the Spearman rank correlation coefficient. All statistical analyses were performed using the SPSS software (ver. 18.0; SPSS Inc., Chicago, IL, USA).

Antioxidant activities of the fractions and phytochemicals
Both SQE and all solvent fractions were evaluated for antioxidant activities using DPPH free radical scavenging assay ( Table 1). The EtOAc fraction showed the strongest DPPH free radical scavenging activity (IC 50 = 42.9 μg/mL), followed by BuOH (IC 50 = 60.5 μg/mL), CHCl 3 (IC 50 = 152.6 μg/mL), SQE (IC 50 = 246.8 μg/mL), and n-hexane (IC 50 = 613.6 μg/mL). Previous studies have shown a high correlation between the phenolic compounds and the antioxidant capacity. [29,30] In this study, the relationship between the phenolic compounds of the extract and radical scavenging reactions (IC 50 ) is evaluated through Spearman rank correlation analysis, and a correlation coefficient value of −0.916 was obtained, which confirmed the significance at the 0.05 level. These results showed that phenolic compounds had a strong and close correlation with the DPPH free radical scavenging activity and that phenolic compounds are major contributors to the antioxidant capacity of S. quelpaertensis. Thus, the antioxidant activities of the isolated 13 phenols can also be used to investigate the DPPH free radical scavenging assay. Compounds 11 and 13 showed DPPH free radical scavenging activity in a concentration-dependent manner. BHA, which was used as a control group to evaluate the antioxidant effects of the SQE, showed 66.3% radical scavenging activity at 100 μM, while compounds 11 and 13 showed 43.7% and 16.6% radical scavenging activity, respectively ( Figure 2). These results show that compound 11 is a potential antioxidant for BHA.
Nitrogen oxides can induce an oxidative reaction in the human body and may be responsible for diseases such as cancer, arthritis, and neurodegenerative disorders. [31,32] Therefore, NO-productioninhibitory activity of the fractions was evaluated in LPS-stimulated RAW 264.7 macrophage cells. The SQE significantly inhibited NO production at concentrations above 62.5 mg/mL. No cytotoxicity was observed at any concentration level (Figure 3a). All fractions also inhibited NO production in a concentration-dependent manner (Figure 3b-e). The CHCl 3 fraction showed the most potent NOinhibition activity, followed by ethyl acetate, n-hexane, SQE, and n-butanol fractions. However, n-hexane and chloroform fractions showed cytotoxicity at a concentration of 250 ng/mL. Correlation analysis between each sample and inhibition of NO production was performed by Spearman rank correlation analysis. Unlike the high correlation between the antioxidant activity and the phenolic compounds, a low correlation of −0.641 (p < .05) was observed for the inhibitory activity of NO production on LPS-stimulated RAW 264.7 cells. For an in-depth evaluation of these results, more studies should be performed on the complex mechanism between phenol components and NO production in cells. The inhibitory effect of NO production in LPS-stimulated cells was confirmed for the 13 compounds isolated from S. quelpaertensis (Figure 4). Compounds 4, 7, 11, and 13 showed NO-production-inhibitory effect under experimental conditions. Compound 7 not only reduced NO production in a concentration-dependent manner but also showed the strongest NOproduction-inhibitory effect in LPS-stimulated RAW 264.7 macrophages. The isolated compounds did not show any cytotoxicity at all concentrations tested in the MTT assay.

Conclusion
This study provides good evidence that extracts and fractions, as well as the phenolic compounds isolated from S. quelpaertensis, can be used as antioxidants, and further suggested two available DPPH free radical inhibitors (compound 11 and 13) and four NO-production inhibitors (compound 4, 7, 11, 13). This is the first report on the NO-inhibition activity of 2,3-dihydroxypropyl 9Z,12Z- octadecadienoate derived from S. quelpaertensis. These results can be used as the basic data for developing effective antioxidant components from S. quelpaertensis. However, further studies are needed to investigate the chemical mechanism and synergistic effects of the relation between the antioxidant activity and phenolic compounds.