Anti-Inflammatory and Anti-Oxidative Activities of Phenolic Compounds from Alnus sibirica Stems Fermented by Lactobacillus plantarum subsp. argentoratensis

Fermentation of Alnus sibirica (AS) stems using Lactobacillus plantarum subsp. argentoratensis was conducted and three compounds isolated from the Alnus species were identified for the first time, 7-(3,4-dihydroxyphenyl)-1-(4-hydroxyphenyl)-heptan-3-one, 1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptan-3-one and 4-(3,4-dihydroxyphenyl)-butan-2-one, along with 14 known compounds. The anti-oxidative and anti-inflammatory abilities of AS and fermented AS (FAS) as well as the isolated phenolic compounds from FAS were investigated. FAS showed stronger anti-oxidative and anti-inflammatory activities than non-fermented AS.


Introduction
The discovery of new compounds plays a crucial role in drug development. Although there is an extensive diversity of natural compounds, the discovery of novel compounds has become increasingly difficult. Many approaches have been proposed to overcome these challenges. They include exploring unused natural resources from the ocean as well as their secondary metabolites. One of these approaches that has shown promise is fermentation. Along with increasing the overall yield of chemical constituents, fermentation helps to improve pharmacological effects, while also decreasing toxicity [1][2][3][4][5].
The fermentation of traditional medine has been used for a long time in China, Korea and India, etc., and the fermentation of Chinese herbal medicine has been the subject of many investigations because of its undeniable benefits in terms of increasing pharmacological effects with fewer adverse effects [2]. Fermentation is a metabolic process which converts sugars to acids or alcogol, and degrades the organic components through an oxidation-reduction process. The microbial fermentation process is extensively used in the production of food and pharmaceuticals.
Alnus sibirica (AS), a member of the Alnus species, is geographically distributed throughout Korea, Japan, China and Russia [6]. In Korean traditional medicine, the bark of AS has been used as an antipyretic, an expectorant, antiphlogistils, cough lozenges, an antiasthmatic and a health tea for alcoholism [6]. Together with investigations of the Alnus species [7,8], studies on the chemical constituents of AS have led to the isolation of various pharmacogically important compounds. Moreover, we isolated various components from AS including diarylheptanoids, tannins, flavonoids, and triterpenoids [9,10]. In addition, biological activities including anti-oxidative, anti-inflammatory, anti-atopic, melanogenesis inhibitory, anti-adipogenic, anti-tumor, and cytotoxic effects have been studied with the extracts of the plants and the compounds from those plants [10][11][12][13][14][15][16][17][18].

Evaluation of Anti-Oxidative and Anti-Inflammatory Activities
AS, FAS and all the isolated phenolic compounds from FAS were measured to assess their antioxidative activities. Both AS and FAS exhibited potent anti-oxidative activities and FAS showed a more potent activity than AS ( Table 2). All compounds exhibited potent anti-oxidative activities except 8 (Table 2). Moreover, compounds 3-6, 9-13 exhibited more potent radical scavenging activity than ascorbic acid (the positive control) . Compounds 3, 6, and 13 showed a strong anti-oxidative activity, followed by 1, 2 and 10, which is similar to the results in Reference [27]. Moreover, 4 and 5 also showed potent anti-oxidative activity, especially 5, which was newly produced, and fermentation

Evaluation of Anti-Oxidative and Anti-Inflammatory Activities
AS, FAS and all the isolated phenolic compounds from FAS were measured to assess their anti-oxidative activities. Both AS and FAS exhibited potent anti-oxidative activities and FAS showed a more potent activity than AS ( Table 2). All compounds exhibited potent anti-oxidative activities except 8 (Table 2). Moreover, compounds 3-6, 9-13 exhibited more potent radical scavenging activity than ascorbic acid (the positive control). Compounds 3, 6, and 13 showed a strong anti-oxidative activity, followed by 1, 2 and 10, which is similar to the results in Reference [27]. Moreover, 4 and 5 also showed potent anti-oxidative activity, especially 5, which was newly produced, and fermentation led to the obviously increased content of 13 [31], and this it had the most potent anti-oxidative activity ability. This may be the key of the reason that FAS showed stronger anti-oxidative activity.
Furthermore, the nitric oxide (NO) production inhibitory effects of AS, FAS and all of the isolated phenolic compounds from FAS in LPS-stimulated RAW 264.7 cells were evaluated. While all samples including AS, FAS and all the isolated compounds showed more potent nitric oxide production inhibitory effects than L-NMMA (positive control), FAS exhibited stronger activity than AS, and 3, 5, 9, 13 showed potent NO production inhibitory activities (Table 3) [32,33].

Plant Material
The stems of AS were collected from Kuksabong, Seoul, Republic of Korea, in January 2015, certificated by Prof. Min Won Lee (Pharmacognosy Laboratory, College of Pharmacy, Chung-Ang University). A voucher specimen was deposited at the herbarium of the College of Pharmacy, Chung-Ang University (Seoul, Korea).

Extraction and Fermentation
The stems of AS (2.7 kg) were extracted with 80% prethanol A (30 L) at room temperature followed by removal of the prethanol A under vacuum, to yield 312 g of the extract. One hundred grams of the extract was fermented with Lactobacillus plantarum subsp. argentoratensis.
AS extract was sterilized for 20 min at 121 • C after adding 9 L of water. Sterilized AS was then inoculated in a liquid medium, mixed at room temperature, and fermented for seven days. After the fermentation, it was evaporated into the final fermented FAS.

Measurement of DPPH Radical Scavenging Activity
First 20 µL of each sample was added into 180 µL 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution 0.2 mM in absolute ethanol. An ELISA reader (TECAN, Salzburg, Austria) was used to measure the optical density of the solution at 540 nm after mixing and incubating for 30 min at room temperature. DPPH radical scavenging activity was calculated as inhibition rate (%) = [1 − (sample optical density/control optical density)] × 100. The IC 50 value was defined as the concentration at which 50% of the DPPH free radicals were scavenged. Ascorbic acid was used as a positive control.

Cell Culture
Mouse monocyte-macrophage RAW 264.7 cells (Korean Cell Line Bank) were maintained in DMEM media supplemented with penicillin (100 U/mL) and 10% fetal bovine serum at 37 • C in a humidified incubator with 5% CO 2 and 95% air, the medium was changed every three days.

Measurement of Inhibition of NO Production
RAW 264.7 macrophages were seeded at density of 1 × 10 5 cells per well in 200 µL in a 96-well plate. The cells were incubated at 37 • C in 5% CO 2 for 3 h to ensure adherence. Afterwards, the medium was removed and the cells were treated with AS, FAS (100, 50, 25, 12.5, 6.125 µg/mL, DMEM medium) and isolated phenolic 1-17 (100, 50, 25, 12.5, 6.125 µM, DMEM medium) for 1 h. The cells were stimulated with 10 µg/mL lipopolysaccharide (LPS) for 24 h. The spent media were collected and analyzed for nitric oxide (NO) production measurement with Griess reagent by mixing 100 µL of cell culture supernatant with the same volume of reagent. NO production inhibitory activity (I) was calculated as inhibition rate (%) calculated from the following formula. L-N G -monomethyl arginine citrate (L-NMMA) was used as the positive control.
where: A sample: Absorbance of compound-treated cell sample; A control: Absorbance of only LPS-treated cell sample; A blank: Absorbance of non-treated cell sample.

Statistical Analysis
All data are expressed as mean±standard deviation (SD). Values were performed by one-way analysis of variance (ANOVA) followed by post hoc Turkey test p < 0.05 value was used to determine statistical significance. All analyses were performed at least three independent experiments and each experiment was analyzed at least triplicate. In all experiments, untreated cells were considered as blank (B), treated cells with only LPS were considered as control (C).