Inhibitory Effects on NO Production and DPPH Radicals and NBT Superoxide Activities of Diarylheptanoid Isolated from Enzymatically Hydrolyzed Ehthanolic Extract of Alnus sibirica

Alnus sibirica (AS) is geographically distributed in Korea, Japan, Northeast China, and Russia. Various anti-oxidant, anti-inflammation, anti-atopic dermatitis and anti-cancer biological effects of AS have been reported. Enzymatic hydrolysis decomposes the sugar bond attached to glycoside into aglycone which, generally, has a superior biological activity, compared to glycoside. Enzymatic hydrolysis of the extract (EAS) from AS was processed and the isolated compounds were investigated—hirsutanonol (1), hirsutenone (2), rubranol (3), and muricarpon B (4). The structures of these compounds were elucidated, and the biological activities were assessed. The ability of EAS and the compounds (1–4) to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and Nitroblue tetrazolium (NBT) superoxide, and to inhibit NO production was evaluated in vitro. EAS showed more potent antioxidant and anti-inflammatory activity than AS. All investigated compounds showed excellent antioxidant and anti-inflammatory activities.

Enzymatic modifications are used to transform compounds isolated from natural sources [16][17][18]. Enzymes are used in various industrial applications where specific catalysts are required. For instance, amylases are used for splitting polysaccharides and proteins in malt, in the brewing industry, and for producing sugars from starch, in food processing industries [11,19]. Aglycones-glycosides in which the sugar molecules have been replaced by hydrogen atoms after enzymatic hydrolysis by intestinal or colonic microflora-are more easily absorbed from the small intestine than glycosides [20,21]. Glycosidation enhances water solubility but reduces chemical reactivity. Therefore, glycosidases play a major role in biological processes and are important in the biological, biomedical, and industrial fields [22].
Glycosidation enhances water solubility but reduces chemical reactivity. Therefore, glycosidases play a major role in biological processes and are important in the biological, biomedical, and industrial fields [22].
From our previous study, 17 compounds from fermented AS (FAS) were isolated and evaluated for their antioxidant, anti-inflammatory, and anti-atopic dermatitis activities, in vitro and in vivo, including the quantitative analysis of its components [23][24][25]. The present paper describes the evaluation of antioxidant and anti-inflammatory effects on the enzymatic hydrolysis (EAS). Through this study, diverse but expected chemical and biological changes, such as increased bioavailability or biological activities, were observed, after the glycosides were converted to aglycones.

Enzymatic Hydrolysis
Extracts from A. sibirica processed by enzymatic hydrolysis (EAS) was prepared by using Fungamyl Super AX (Novozymes, Bagsvaerd, Denmark). The differences between EAS and AS were observed by thin layer chromatography (TLC). (TLC results data not shown, reaction pathway shows in Figure 1)
Compound 2 was in the form of an amorphous brown oil. A navy-blue spot was observed after spraying the TLC strip with FeCl3 solution. A dark-blue/deep-violet spot was also observed after
Compound 2 was in the form of an amorphous brown oil. A navy-blue spot was observed after spraying the TLC strip with FeCl 3 solution. A dark-blue/deep-violet spot was also observed after spraying with 10% H 2 SO 4 solution and heating. The 1 H-NMR (300 MHz, Acetone-d 6 Table S2) Thus, 2 was identified as hirsutenone, 1,7-bis-(3,4-dihydroxyphenyl)-4-hepten-3-one, after comparison of this spectrum with the reported spectral data for hirsutenone [26].
Compound 4 was in the form of an amorphous brown oil. A navy-blue spot was observed after spraying the TLC strip with FeCl 3 solution. A dark-green spot was also observed after spraying with 10% H 2 SO 4 solution and heating. The 1 H-NMR (600 MHz, Acetone-d 6 Table S4) Thus, 4 was identified as muricarpon B, 1,7-bis(3,4-dihydroxyphenyl)-3-heptanone, after comparison of this spectrum with the reported spectral data for muricarpon B [28].

Plant Material
Barks of AS were collected from 'Kuksabong , Seoul, Republic of Korea, in January 2015 and authenticated by Professor Lee (College of Pharmacy, Chung-Ang University, Seoul, Korea). The voucher specimen (201501-AS) was placed at the Laboratory of Pharmacognosy and Natural Product-Derived Medicine at the Chung-Ang University.

Enzymatic Hydrolysis
We used Fungamyl Super AX ® (Novozymes) for the hydrolysis experiments. The purchased enzyme was mixed with AS extract and distilled water, in the ratio of 3:1:1. The mixture was allowed to react at room temperature for 3 days. After the enzymatic hydrolysis, the enzyme was removed via ethyl acetate fractionation. For this, centrifugation was performed, and the supernatant obtained was mixed with an equal volume of ethyl acetate; this process was repeated thrice. The ethyl acetate layer was then evaporated to obtain EAS.

Extraction and Isolation
The barks of AS (2.8 kg) were extracted with 80% ethanol (30 L) at room temperature. After removing ethanol, the mixture was concentrated to obtain 121 g of AS extract. A part (23.27 g) of this extract was subjected to enzymatic hydrolysis using Fungamyl (to obtain EAS), followed by liquid-liquid partition usingethyl acetate. The rest of the extract was stored in the freezer and the ethyl acetate layer was then subjected to Sephadex LH-20 column chromatography and then eluted with a solvent gradient system of MeOH:H 2 O (from 2:8 to 10:0), yielding eight sub-fractions (EAS-1 to 8). From fraction EAS-2, compound 1 (hirsutanonol, 283 mg) was isolated. When EAS-6 (203 mg) in the ODS gel was subjected to MPLC (flow rate: 5 mL/min) with a gradient solvent system of MeOH:H 2 O (from 0:10 to 10:0), 2 (hirsutenone, 76.9 mg) was obtained. EAS-7 (1.5 g), when subjected to MCI gel open-column chromatography with a solvent gradient system of MeOH:H 2 O (from 6:4 to 10:0) yielded 3 (rubranol, 504 mg) and 4 (muricarpon B, 125.8 mg).

Measurement of DPPH Radical Scavenging Activity
The antioxidant activity was evaluated on the basis of the scavenging activity of the stable DPPH free radical (Sigma, St. Louis, MO, USA). Each sample (20 µL), in anhydrous ethanol, was added to 180 µL of DPPH solution (0.2 mM, dissolved in anhydrous ethanol). After mixing gently and letting it stand for 30 min at 37 • C, in a dark environment, the absorbance was measured at 517 nm, using an enzyme-linked immunosorbent assay (ELISA) reader (TECAN, Salzburg, Austria). The free radical scavenging activity was calculated as the inhibition rate (%) = 100 − (sample O.D./control O.D.) × 100.
l-ascorbic acid was used as the positive control.

RAW264.7 Cell Culture
The murine macrophage RAW264.7 cells were purchased from the Korean Cell Line Bank. These cells were grown at 37 • C in a humidified atmosphere (5% CO 2 ) in Dulbecco s Modified Eagle Medium (Sigma, St. Louis, MO, USA), containing 10% fetal bovine serum, 100 IU/mL penicillin G, and 100 mg/mL streptomycin (Gibco BRL, Grand Island, NY, USA). The cells were used in the in vitro experiments, after counting with a hemocytometer.

Measurement of Inhibitory Activity on NO Production
RAW264.7 macrophage cells were cultured in a 96-well plate and incubated for 4 h at 37 • C, in a humidified atmosphere (5% CO 2 ). The cells were incubated in a medium containing 10 µg/mL lipopolysaccharide (Sigma) and the test samples. After incubating for an additional 20 h, the NO content was evaluated by the Griess assay. The Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in 5% H 3 PO 4 solution; Sigma) was added to the supernatant of the cells treated with the test samples. The absorbance at 540 nm, against a standard sodium nitrite curve, was used to determine the NO content. L-NMMA was used as the positive control. NO production inhibitory activity was and was defined as IC 50 , which was the concentration that could inhibit 50% of NO production.

Statistical Analysis
All data are expressed as the mean ± SD of three replicates. Values were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls test using the Statistical Package for the Social Sciences (SPSS) software pack; a statistical difference was considered to be significant when the p-value was less than 0.05. Values bearing different superscripts in the same column are significantly different.

Conclusions
In order to evaluate the anti-oxidative and anti-inflammatory effects of the EAS and its compounds (1-4), DPPH radical, NBT superoxide scavenging activities, and inhibitory activity on NO production were evaluated, in vitro. According to the results, the anti-oxidative and anti-inflammatory activities of the EAS were much better than the ethanolic crude extract of AS. Isolated compounds 1-4 showed significantly better anti-oxidative and anti-inflammatory activities, compared to their respective positive controls. The contents of 1-4 were increased and this appeared to be important in increasing the efficacy of EAS. These results suggest that EAS is a new source for the development of anti-oxidative and anti-inflammatory agents.