Chemical Constituents from the Roots of Polygala arillata and Their Anti-Inflammatory Activities

A new compound, named arillatanoside E, which was elucidated as 3-O-β-D-glucopyranosyl presenegenin 28-O-β-Dxylopyranosyl-(1⟶ 3)-β-D-xylopyranosyl-(1⟶ 4)-α-L-rhamnopyranosyl-(1⟶ 2)-(4-O-acetyl)-β-D-fucopyranosyl ester, along with 11 known compounds was isolated from the ethanolic extract of the roots of Polygala arillata. 1e 11 known compounds were identified as oleanolic acid (2), 3′-E-3,4,5-trimethoxy cinnamoyl-6-benzoyl sucrose (3), trans-ferulic acid (4), trans-feruloyl-glucoside (5), feruloyl-glucoside (6), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), monopentadecanoin (9), sinapic acid (10), p-hydroxybenzaldehyde (11), and palmitic acid (12). Among them, seven isolated compounds 1, 2, 4, 5, 7, 8, and 10 exhibited little cytotoxic activity on macrophage RAW 264.7 cells. 1en, the inhibitory effects of 7 isolates on nitric oxide (NO) production in lipopolysaccharide-activated macrophages were evaluated. As a result, 3 compounds have significant anti-inflammatory activity, and they were arillatanoside E (1), oleanolic acid (2), and 2,4,6-trimethoxy-1-O-β-Dglycoside (7).


Introduction
Polygala arillata Buch.-Ham.ex D. Don belongs to the genus of Polygala (Polygalaceae), which is mainly distributed in Nepal, India (Sikkim), Myanmar, and the north of Vietnam.In China, it is mainly distributed in several provinces in the south of the Yangtze River; among them, Yunnan Province has the largest production.e roots and stems of this plant are commonly used to treat irregular menstruation, hepatitis, pneumonia, and so on [1].
During inflammation, activation of macrophages leads to the production of many proinflammatory cytokines, prostaglandin E 2 , and nitric oxide (NO).NO is a short-lived free radical and is synthesized by NO synthase (NOS).ree different isoforms of NOS exist and are referred to as neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) [13].Lipopolysaccharides (LPS) are constituents of the cell walls of Gram-negative bacteria and act as the activator of the immune system.LPS-stimulated macrophages induce the expression of iNOS, which stimulates NO production [13].
NO plays a key role in the regulation of cytokines especially related to inflammation and has anti-inflammatory with cytotoxic effects in inflammatory reaction [13,14].In this case, by measuring the amount of NO produced by mouse macrophages, we can screen out the compounds with anti-inflammatory activity in P. arillata [15].Here, the concentration of NO secreted by cells was detected by the Griess reagent method, and the potentially antiinflammatory activity of the compounds was screened [16].
We observed that with higher three compounds (1, 2, 7) concentration, the levels of nitrite concentration were decreased, which indicated that the three compounds have potential anti-inflammatory activity against the damage induced by LPS, and 1, 2 and 7 may be therapeutic agents for inflammation.However, among the three compounds, 1 and 7 with 12.5 μg/mL had no significant inhibitory effect for NO, maybe because the drug concentration was too low to reach the effective concentration.Our results will promote further application of the three compounds in pharmacology.
in-layer chromatography (TLC) was performed on glass-precoated silica gel GF 254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), detection under UV light or by heating after spraying with 10% sulfuric acid (H 2 SO 4 ) in 90% ethanol (EtOH).Column chromatography was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Inc., Qingdao, China), and Sephadex LH-20 (Pharmacia Biotech, Switzerland) were used for the chromatography column.Other chemicals and reagents of analytical grade were from Tianjin Concord Technology (Tianjin, China).

Plant Material.
e roots of P. arillata were collected from Yunnan province, China.
e plant material was identified by Associate Prof. Chun-hua Wang of the School of Pharmaceutical Engineering, Tianjin University of Traditional Chinese Medicine and a voucher specimen (No. 201612CH03) has been deposited in Tianjin International Joint Academy of Biotechnology and Medicine.

Extraction and Isolation.
e extraction process was as follows: 30 kg of air-dried samples were extracted three times with 75% aqueous ethanol solution (v/v) at room temperature by infusion each for a week.

Arillatanoside E (1)
. It is white powder; for 1 H-NMR, 13 C-NMR, and DEPT-135 spectroscopic data, see 2.5.Acid Hydrolysis and HPLC Analysis.4 mg of arillatanoside E was accurately weighed and dissolved in 2 M hydrogen chloride (HCl) (2 mL) for 1 h at 85 °C in the reaction bottle and then extracted twice with isovolumic EtOAc.After vacuum evaporation of water layer, the residue was dissolved in 2 mL pyridine containing L-cysteine methyl ester (4 mg) and O-tolyl isothiocyanate (4 mg) and reacted at 60 °C for 1 h, respectively, and successively [13].Derivatives were further detected with a steady flow (0.8 mL/min) and column temperature (35 °C) by HPLC using 25% acetonitrile-water (0.01% formic acid (HCOOH)) as the mobile phase, and the eluate was monitored at 250 nm.As in the above case, the standard sugar samples (1 mg) were subjected to the same reaction and HPLC conditions.en, the retention times of the derivatized sugars in HPLC were 17.464 (for D-glucose), 20.496 (for D-xylose), 24.340 (for D-fucose), and 30.146 min (for D-rhamnose), respectively.

Biological Activity Assays
2.6.1.Cell Culture.RAW 264.7 mouse macrophages were obtained from the Binhai lab (Bio-Swamp, MD, USA) and cultured in Dulbecco's modified eagle medium (DMEM) (high glucose), complete medium, which was refreshed every other day supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in an incubator (STIK (Shanghai) Co., Ltd., Shanghai, China) which is a 5% carbon dioxide-(CO 2 -) humidified atmosphere.e subculture was carried out at 2 to 3 day intervals.When the cells were approximately 80% confluent, cells were kept in the logarithmic phase by cell scraper and subcultured.

CCK-8 Cell Viability Assay.
e cells in the logarithmic phase were seeded in 96-well culture plates at 1 × 10 4 cells per well (100 μL) and incubated for 2 h for adhesion at 37 °C in a humidified atmosphere with 5% CO 2 .Meanwhile, blank control wells were added to 100 μL of culture medium and maintained under the same conditions.Next, 100 μL of the above culture medium containing three concentrations of 7 compounds (50, 25, and 12.5 μg/mL), dexamethasone sodium phosphate (5 μg/mL), and normal culture medium (untreated control group) were added, respectively, and incubated for 1 h.Moreover, in the dark environment, 10 μL of CCK-8 was added to each well and incubated for 1 h to 4 h.Last, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent reader (FlexStation 3, Molecular Devices, San Francisco, CA, USA).e survival rate of cell was calculated as survival rate (%) � (A sample − A blank )/ (A control − A blank ) × 100%.

Anti-Inflammatory Activity
Assay.500 μL of the above cells in the logarithmic phase were seeded in 24-well culture plates at 5 × 10 5 cells each well and incubated for 2 h for adhesion at 37 °C in a humidified atmosphere with 5% CO 2 .After the culture medium was replaced by the medium with different concentrations of 7 compounds (50, 25, 12.5 μg/ mL), dexamethasone sodium phosphate (5 μg/mL), and normal culture medium (untreated control group), the volume of them were 400 μL, respectively, and incubated for 1 h, and all of them except the blank control group were exposed to 0.1 μg/mL LPS (100 μL) for 16 h.After that, all of the supernatant was collected which were divided into three wells (50 μL), added to 96-well culture plates, and mixed with an equal volume (50 μL) of Griess reagents I (Sulfanilamide Solution) and II (NED).Absorbance values were measured at 548 nm using a microplate reader.Here, sodium nitrite was used to generate a standard reference curve (Figure S8), and the curve is obtained as follows: y � 0.0072x + 0.0557 (R 2 � 0.9995).

Statistical
Analysis.e t-test was used to analyze the differences between groups of data.Testing the significance of difference among groups by the statistical method with SPSS version 20.0 (International Business Machines Corporation, Armonk, NY, USA) and making statistical diagram by using GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA) were further made.p values less than 0.05 (p < 0.05) were considered statistically significant.p values less than 0.01 (p < 0.01) were considered of notable statistically significance.
e structure of the new compound 1 was elucidated on the basis of extensive NMR spectroscopic analysis, including a series of 2D-NMR experiments (Figure 2) (heteronuclear singular quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC)), and MS data.
What is noteworthy is that, in the HMBC spectrum, a correlation between the methyl proton at δ H 2.18 (CH 3 CO-) and δ C 172.8 (CH 3 CO-) and between δ H 5.10 (Fuc-4) and 172.8 (CH 3 CO-) indicated acetyl existed and methyl was attached by an ester linkage to the position of C-4 of Fuc.In addition, compared with arillatanoside A [17], the stereochemical structure of compound 1 had not changed.Synthesizing the above analysis of all the proton and carbon signals, we established the structure of compound 1, which was named as arillatanoside E, as 3

Biological Activity Assays.
e roots of P. arillata have anti-inflammatory effect, but its active components are not clear.Here, by measuring the amount of inhibition of LPSinduced NO production in RAW 264.7 mouse macrophages, 7 out of 12 compounds were selected for primary screening of anti-inflammatory components [15,18].

Anti-In ammatory Activity Assay.
As an important physiological messenger and e ector molecule, NO can regulate various physiological and pathological responses.However, it was rapidly metabolized into nitrite, which was relatively stable and could be detected by the Griess reagent kit (Promega, USA).For this, the Griess reagent kit was used to determine the level of NO which was secreted by macrophage cells, and 7 isolated compounds (1, 2, 4, 5, 7, 8, and 10) which did not exhibit cytotoxic activity on LPS-induced RAW 264.7 mouse macrophages were screened for anti-in ammatory activity with the LPS group as control.Meanwhile, dexamethasone sodium phosphate (C 22 H 28 FNa 2 O 8 P) was taken as the positive control group [18].Compared with the positive control group and negative control group, the results (Figure 5) indicated that three compounds (1, 2, and 7) had signi cant anti-in ammatory activity and 5 had anti-in ammatory activity at the highest concentration, while the other three compounds (4, 8, and 10) without anti-in ammatory activity.With higher three compounds (1, 2, and 7) concentration, the levels of nitrite concentration were decreased, which indicated that the three compounds have potential anti-in ammatory activity Journal of Chemistry against the damage induced by LPS and 1, 2, and 7 may be therapeutic agents for in ammation.However, among the three compounds, 1 and 7 with 12.5 μg/mL had no significant inhibitory e ect for NO, maybe the drugs concentration was too low to reach the e ective concentration.In addition, the compound 5 was e ective at the highest concentration, for the e ective concentration was too high, in-depth research on it is unnecessary either in theory or in reality.

Conclusion
In this research, a new compound, named as arillatanoside E (1), which was established as a derivative of the chemotaxonomic marker 3

Data Availability
e NMR and MS data used to support the ndings of this study are included within the supplementary information le.e other data used to support the ndings of this study are available from the corresponding author upon request.