# Chemical Constituents from the Roots ofPolygala arillata and Their Anti-Inflammatory Activities

Authors: Wei Xiang; Guo-Dong Zhang; Fang-Yi Li; Teng-long Wang; Tong-Chuan Suo; Chun-Hua Wang; Zheng Li; Yan Zhu
Journal: Journal of Chemistry
 (2019)

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## Abstract

A new compound, named arillatanoside E, which was elucidated as 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(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. The 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. Then, 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-D-glycoside (7).


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## Body

## 1. 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. The roots and stems of this plant are commonly used to treat irregular menstruation, hepatitis, pneumonia, and so on [1].Up to now, few compounds have been isolated from the plant ofP. arillata. It is reported that it mainly contains saponins, oligosaccharides, and ketones [2], which has various biological activities, such as immunoenhancement, sedation and hypnotism, antibacterial activity, and dispelling cold and relieving pain. [2]. To further study the chemical constituents of the roots of P. arillata, several kinds of chromatographic methods such as silica gel column chromatographic technology, octadecylsilyl (ODS) column chromatographic technique, Sephadex LH-20 column, and preparative high performance liquid chromatography (Pr-HPLC) were used. As a result, a new compound, named arillatanoside E, which was elucidated as 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(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 P. arillata. The other 11 known compounds were identified as oleanolic acid (2) [3], 3′-E-3,4,5-trimethoxy cinnamoyl-6-benzoyl sucrose (3) [4], trans-ferulic acid (4) [5], trans-feruloyl-glucoside (5) [6], feruloyl-glucoside (6) [6], 2,4,6-trimethoxy-1-O-β-D-glycoside (7) [7], 3-methoxy-4-hydroxybenzoic acid (8) [8], monopentadecanoin (9) [9], sinapic acid (10) [10], p-hydroxybenzaldehyde (11) [11], and palmitic acid (12) [12] by means of mass spectrometry (MS), ultraviolet spectrophotometry (UV), and nuclear magnetic resonance (NMR) spectroscopy, including 1D-NMR and 2D-NMR, such as 1H-NMR, 13C-NMR, HSQC, HMBC, and 1H-1H COSY.During inflammation, activation of macrophages leads to the production of many proinflammatory cytokines, prostaglandin E2, and nitric oxide (NO). NO is a short-lived free radical and is synthesized by NO synthase (NOS). Three 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 anti-inflammatory 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.


## 2. Experimental

### 2.1. General

HPLC was run on Agilent 1260 HPLC (Agilent, Palo Alto, CA, USA). Semipreparative HPLC was performed on Waters 2489 equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 μm; Waters, Maple St. Milford, MA, USA). NMR spectra were measured on an AV-400 spectrometer (Bruker Corporation, Faellanden, Switzerland) using methanol-d4 (CD3OD), chloroform-d (CDCl3), and dimethyl sulfoxide-d6 (DMSO-d6) (Sigma-Aldrich, St. Louis, MO, USA) as solvent. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an electrostatic field orbital trap mass spectrometer (Thermo Scientific, Bremen, Germany) using an ESI source. Thin-layer chromatography (TLC) was performed on glass-precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), detection under UV light or by heating after spraying with 10% sulfuric acid (H2SO4) 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).


### 2.2. Plant Material

The roots ofP. arillata were collected from Yunnan province, China. The 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.


### 2.3. Extraction and Isolation

The 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. The isolation process was as follows: (1) The extracting solution was merged for rotatory evaporation till no alcohol taste. (2) The liquid remaining in the still was suspended in water (H2O) and then partitioned successively with petroleum ether, ethyl acetate (EtOAc), and n-butyl alcohol to give petroleum ether, EtOAc (A, 100.0 g), and n-butyl alcohol (B, 300.0 g) fractions, respectively. (3) Fractions A and B were chromatographed over silica gel, eluting with methanol (CH3OH) in dichloromethane (0–100%, stepwise), yielding ten fractions (Fr.1–Fr.10), respectively. (4) The crude fraction Fr.5 (13.6 g) (fraction A) was further purified by semipreparative HPLC using CH3OH and 0.1% formic acid solution in water as the mobile phase, to yield compounds 4 (16 mg), 5 (32 mg), 6 (7 mg), 7 (12 mg), 8 (12 mg), 9 (8 mg), 10 (17 mg), 11 (6 mg), and 12 (9 mg). (5) The total fractions Fr.5 (11.3 g) and Fr.8 (17.6 g) (fraction B) were chromatographed over a silica gel column eluting with dichloromethane-methanol (CH2Cl2-CH3OH) (0–100%, stepwise), to afford compound 1 (8 mg), 2 (11 mg), and 3 (4 mg).


### 2.4. Arillatanoside E (1)

It is white powder; for1H-NMR, 13C-NMR, and DEPT-135 spectroscopic data, see Table 1 and Figures S1–S3 (Supplementary Materials); 1H-1H COSY, HSQC, and HMBC, see Figures S4–S6 (Supplementary Materials); HR-ESI-MS: m/z 1277.5839 [M-H]− (Calc. for C60H94O29: 1277.5803), see Figure S7 (Supplementary Materials).Table 1
The spectral data of1H-NMR (400 MHz) and 13C-NMR (100 MHz) of compound 1 in CD3OD.
Position
δ
H
δ
C
Position
δ
H
δ
C
1
2.12 (1H, m), 1.21 (1H, m)
44.6
3-O-Glc-1
4.55 (1H, d,J = 7.8 Hz)
105.6
2
3.55 (1H, m)
70.9
Glc-2
3.95 (m)
75.1
3
3.50 (1H, m)
87.7
Glc-3
3.26 (m)
77.7
4
—
53.2
Glc-4
3.92 (m)
71.7
5
1.70 (1H, m)
52.9
Glc-5
3.26 (m)
77.7
6
1.67 (1H, m), 1.21 (1H, m)
21.6
Glc-6
3.82 (m), 3.72 (1H, dd,J = 6.6, 4.8 Hz)
62.3
7
1.67 (1H, m), 1.27 (1H, m)
34.3
28-O-Fuc-1
5.44 (1H, d,J = 8.4 Hz)
95.0
8
—
41.7
Fuc-2
3.85 (m)
72.3
9
1.89 (1H, m)
50.1
Fuc-3
3.38 (m)
75.5
10
—
37.5
Fuc-4
5.10 (1H, d,J = 3.6 Hz)
75.3
11
1.99 (1H, m), 1.64 (1H, m)
23.9
Fuc-5
4.32 (m)
71.2
12
5.67 (1H, t)
128.8
Fuc-6
1.10 (3H, d,J = 6.6 Hz)
16.5
13
—
139.3
Ac at 4
2.19 (3H, s)
20.8
14
—
48.6
—
—
172.8
15
1.64 (1H, m), 1.44 (1H, m)
24.9
Rha-1
5.46 (1H, brs)
101.1
16
1.99 (1H, m), 1.91 (1H, m)
24.7
Rha-2
3.55 (m)
71.1
17
—
47.9
Rha-3
3.84 (m)
73.7
18
2.92 (1H, dd,J = 10.2, 3.6 Hz)
42.8
Rha-4
3.54 (m)
85.0
19
1.62 (1H, m), 1.23(1H, m)
46.2
Rha-5
3.87 (m)
68.5
20
—
31.6
Rha-6
1.32 (3H, d,J = 6.6 Hz)
18.2
21
1.39 (1H, s), 1.27 (1H, m)
34.7
Xyl-1 (inner)
4.51 (1H, d,J = 7.8 Hz)
106.8
22
1.75 (1H, m), 1.61 (1H, m)
33.0
Xyl-2
3.95 (m)
75.1
23
—
181.9
Xyl-3
4.13 (1H, d,J = 3.6 Hz)
85.9
24
1.40 (3H, s)
13.7
Xyl-4
3.54 (m)
69.4
25
1.29 (3H, s)
17.7
Xyl-5
3.77 (m), 3.56 (m)
66.5
26
0.76 (3H, s)
18.9
Xyl-1 (outer)
4.36 (1H, d,J = 7.8 Hz)
104.9
27
3.77 (1H, m), 3.55(1H, m)
64.7
Xyl-2
3.95 (m)
75.0
28
—
177.8
Xyl-3
3.37 (m)
77.6
29
0.94 (3H, s)
33.4
Xyl-4
3.27 (m)
71.1
30
0.97 (3H, s)
24.1
Xyl-5
3.92 (m), 3.24 (m)
67.2


### 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) andO-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. Then, 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.


### 2.6. 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 100μg/mL streptomycin at 37°C in an incubator (STIK (Shanghai) Co., Ltd., Shanghai, China) which is a 5% carbon dioxide-(CO2-) humidified atmosphere. The 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.


#### 2.6.2. CCK-8 Cell Viability Assay

The cells in the logarithmic phase were seeded in 96-well culture plates at 1 × 104 cells per well (100 μL) and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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). The survival rate of cell was calculated as survival rate (%) = (Asample − Ablank)/(Acontrol − Ablank) × 100%.


#### 2.6.3. Anti-Inflammatory Activity Assay

500μL of the above cells in the logarithmic phase were seeded in 24-well culture plates at 5 × 105 cells each well and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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 (R2 = 0.9995).


### 2.7. Statistical Analysis

Thet-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.


## 2.1. General

HPLC was run on Agilent 1260 HPLC (Agilent, Palo Alto, CA, USA). Semipreparative HPLC was performed on Waters 2489 equipped with a diode array detector and a C18 column (250 mm × 10 mm, 5 μm; Waters, Maple St. Milford, MA, USA). NMR spectra were measured on an AV-400 spectrometer (Bruker Corporation, Faellanden, Switzerland) using methanol-d4 (CD3OD), chloroform-d (CDCl3), and dimethyl sulfoxide-d6 (DMSO-d6) (Sigma-Aldrich, St. Louis, MO, USA) as solvent. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was performed on an electrostatic field orbital trap mass spectrometer (Thermo Scientific, Bremen, Germany) using an ESI source. Thin-layer chromatography (TLC) was performed on glass-precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), detection under UV light or by heating after spraying with 10% sulfuric acid (H2SO4) 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).


## 2.2. Plant Material

The roots ofP. arillata were collected from Yunnan province, China. The 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.


## 2.3. Extraction and Isolation

The 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. The isolation process was as follows: (1) The extracting solution was merged for rotatory evaporation till no alcohol taste. (2) The liquid remaining in the still was suspended in water (H2O) and then partitioned successively with petroleum ether, ethyl acetate (EtOAc), and n-butyl alcohol to give petroleum ether, EtOAc (A, 100.0 g), and n-butyl alcohol (B, 300.0 g) fractions, respectively. (3) Fractions A and B were chromatographed over silica gel, eluting with methanol (CH3OH) in dichloromethane (0–100%, stepwise), yielding ten fractions (Fr.1–Fr.10), respectively. (4) The crude fraction Fr.5 (13.6 g) (fraction A) was further purified by semipreparative HPLC using CH3OH and 0.1% formic acid solution in water as the mobile phase, to yield compounds 4 (16 mg), 5 (32 mg), 6 (7 mg), 7 (12 mg), 8 (12 mg), 9 (8 mg), 10 (17 mg), 11 (6 mg), and 12 (9 mg). (5) The total fractions Fr.5 (11.3 g) and Fr.8 (17.6 g) (fraction B) were chromatographed over a silica gel column eluting with dichloromethane-methanol (CH2Cl2-CH3OH) (0–100%, stepwise), to afford compound 1 (8 mg), 2 (11 mg), and 3 (4 mg).


## 2.4. Arillatanoside E (1)

It is white powder; for1H-NMR, 13C-NMR, and DEPT-135 spectroscopic data, see Table 1 and Figures S1–S3 (Supplementary Materials); 1H-1H COSY, HSQC, and HMBC, see Figures S4–S6 (Supplementary Materials); HR-ESI-MS: m/z 1277.5839 [M-H]− (Calc. for C60H94O29: 1277.5803), see Figure S7 (Supplementary Materials).Table 1
The spectral data of1H-NMR (400 MHz) and 13C-NMR (100 MHz) of compound 1 in CD3OD.
Position
δ
H
δ
C
Position
δ
H
δ
C
1
2.12 (1H, m), 1.21 (1H, m)
44.6
3-O-Glc-1
4.55 (1H, d,J = 7.8 Hz)
105.6
2
3.55 (1H, m)
70.9
Glc-2
3.95 (m)
75.1
3
3.50 (1H, m)
87.7
Glc-3
3.26 (m)
77.7
4
—
53.2
Glc-4
3.92 (m)
71.7
5
1.70 (1H, m)
52.9
Glc-5
3.26 (m)
77.7
6
1.67 (1H, m), 1.21 (1H, m)
21.6
Glc-6
3.82 (m), 3.72 (1H, dd,J = 6.6, 4.8 Hz)
62.3
7
1.67 (1H, m), 1.27 (1H, m)
34.3
28-O-Fuc-1
5.44 (1H, d,J = 8.4 Hz)
95.0
8
—
41.7
Fuc-2
3.85 (m)
72.3
9
1.89 (1H, m)
50.1
Fuc-3
3.38 (m)
75.5
10
—
37.5
Fuc-4
5.10 (1H, d,J = 3.6 Hz)
75.3
11
1.99 (1H, m), 1.64 (1H, m)
23.9
Fuc-5
4.32 (m)
71.2
12
5.67 (1H, t)
128.8
Fuc-6
1.10 (3H, d,J = 6.6 Hz)
16.5
13
—
139.3
Ac at 4
2.19 (3H, s)
20.8
14
—
48.6
—
—
172.8
15
1.64 (1H, m), 1.44 (1H, m)
24.9
Rha-1
5.46 (1H, brs)
101.1
16
1.99 (1H, m), 1.91 (1H, m)
24.7
Rha-2
3.55 (m)
71.1
17
—
47.9
Rha-3
3.84 (m)
73.7
18
2.92 (1H, dd,J = 10.2, 3.6 Hz)
42.8
Rha-4
3.54 (m)
85.0
19
1.62 (1H, m), 1.23(1H, m)
46.2
Rha-5
3.87 (m)
68.5
20
—
31.6
Rha-6
1.32 (3H, d,J = 6.6 Hz)
18.2
21
1.39 (1H, s), 1.27 (1H, m)
34.7
Xyl-1 (inner)
4.51 (1H, d,J = 7.8 Hz)
106.8
22
1.75 (1H, m), 1.61 (1H, m)
33.0
Xyl-2
3.95 (m)
75.1
23
—
181.9
Xyl-3
4.13 (1H, d,J = 3.6 Hz)
85.9
24
1.40 (3H, s)
13.7
Xyl-4
3.54 (m)
69.4
25
1.29 (3H, s)
17.7
Xyl-5
3.77 (m), 3.56 (m)
66.5
26
0.76 (3H, s)
18.9
Xyl-1 (outer)
4.36 (1H, d,J = 7.8 Hz)
104.9
27
3.77 (1H, m), 3.55(1H, m)
64.7
Xyl-2
3.95 (m)
75.0
28
—
177.8
Xyl-3
3.37 (m)
77.6
29
0.94 (3H, s)
33.4
Xyl-4
3.27 (m)
71.1
30
0.97 (3H, s)
24.1
Xyl-5
3.92 (m), 3.24 (m)
67.2


## 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) andO-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. Then, 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.


## 2.6. 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 100μg/mL streptomycin at 37°C in an incubator (STIK (Shanghai) Co., Ltd., Shanghai, China) which is a 5% carbon dioxide-(CO2-) humidified atmosphere. The 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.


### 2.6.2. CCK-8 Cell Viability Assay

The cells in the logarithmic phase were seeded in 96-well culture plates at 1 × 104 cells per well (100 μL) and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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). The survival rate of cell was calculated as survival rate (%) = (Asample − Ablank)/(Acontrol − Ablank) × 100%.


### 2.6.3. Anti-Inflammatory Activity Assay

500μL of the above cells in the logarithmic phase were seeded in 24-well culture plates at 5 × 105 cells each well and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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 (R2 = 0.9995).


## 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 100μg/mL streptomycin at 37°C in an incubator (STIK (Shanghai) Co., Ltd., Shanghai, China) which is a 5% carbon dioxide-(CO2-) humidified atmosphere. The 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.


## 2.6.2. CCK-8 Cell Viability Assay

The cells in the logarithmic phase were seeded in 96-well culture plates at 1 × 104 cells per well (100 μL) and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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). The survival rate of cell was calculated as survival rate (%) = (Asample − Ablank)/(Acontrol − Ablank) × 100%.


## 2.6.3. Anti-Inflammatory Activity Assay

500μL of the above cells in the logarithmic phase were seeded in 24-well culture plates at 5 × 105 cells each well and incubated for 2 h for adhesion at 37°C in a humidified atmosphere with 5% CO2. 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 (R2 = 0.9995).


## 2.7. Statistical Analysis

Thet-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.


## 3. Results and Discussion

### 3.1. Structure Elucidation

The known compounds (2–12) (Figure 1) were identified by comparison of their experimental spectral data with literature data [3–12].Figure 1
Chemical structures of compounds1–12.The structure of the new compound1 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.Figure 2
Key HMBC correlations of compound1 (arrows point from proton to carbon).Compound1 was obtained as a white powder with the molecular formula of C60H94O29 based on the negative ion peak at m/z 1277.5839 [M-H]− (calculated 1277.5803), implying 14 degrees of unsaturation. The 1H-NMR spectrum revealed the presence of two tertiary methyl protons at δH 1.32 (3H, d, J = 6.6 Hz) and 1.10 (3H, d, J = 6.6 Hz), five quaternary methyl protons at δH 1.40 (3H, s), 1.28 (3H, s), 0.97 (3H, s), 0.94 (3H, s), and 0.76 (3H, s), an olefinic proton at δH 5.67 (1H, t), and five anomeric protons at δH 5.46 (1H, brs), 5.44 (1H, d, J = 8.4 Hz), 4.55 (1H, d, J = 7.8 Hz), 4.51 (1H, d, J = 7.8 Hz), and 4.36 (1H, d, J = 7.8 Hz). The 13C-NMR spectrum displayed the presence of six quaternary carbons at δC 31.6, 37.5, 41.7, 47.9, 48.6, and 53.2, an oxygen-bearing methylene carbon at δC 64.7, two oxygen-bearing methine carbon at δC 70.9 and 87.7, five anomeric carbons at δC 95.0, 101.1, 104.9, 105.6, and 106.8, a set of olefinic carbons at δC 128.8 and 139.3, an ester carbonyl carbon at δC 177.8, and a carboxyl carbon at δC 182.9. All the above spectral information showed that the aglycone moiety of 1 was presenegenin [17] which belonged to oleanane triterpenoidal saponin, meanwhile combined with the chemical shift of the deshielded carbon C-3 (δC 87.7) and the carbonyl C-28 (δC 177.8) of presenegenin indicated that compound 1 was the 3,28-bisdesmoside of presenegenin, having five monosaccharide units.On acid hydrolysis, compound1 afforded an aglycone which was identified as presenegenin on the basis of the 1H-NMR and 13C-NMR data [17]. The sugars obtained from the saponin hydrolysate were identified as D-glucose, D-fucose, D-xylose, and L-rhamnose based on HPLC analysis (Figure 3). The NMR (Table 1) data of compound 1, along with the HSQC spectrum, showed the five anomeric proton signals at δH 5.46 (1H, brs), 5.44 (1H, d, J = 8.4 Hz), 4.55 (1H, d, J = 7.8 Hz), 4.51 (1H, d, J = 7.8 Hz), and 4.36 (1H, d, J = 7.8 Hz) and were correlated with anomeric carbon signals at δC 101.1, 95.0, 105.6, 106.8, and 104.9, respectively. Combined 13C-NMR with distortionless enhancement by polarization transfer (DEPT) data, the carbon signals of five monosaccharide units had been listed as follows: Glc’s carbon signals at δC 105.6 (Glc-1), 75.1 (Glc-2), 77.7(Glc-3), 71.7 (Glc-4), 77.7 (Glc-5), and 62.3 (Glc-6); Fuc’s carbon signals at δC 95.0 (Fuc-1), 72.3 (Fuc-2), 75.4 (Fuc-3), 75.3 (Fuc-4), 71.2 (Fuc-5), and 16.5(Fuc-6); Rha’s carbon signals at δC 101.1 (Rha-1), 71.1 (Rha-2), 73.7 (Rha-3), 85.0 (Rha-4), 68.5 (Rha-5), and 18.2 (Rha-6); Xyl’s (inner) carbon signals at δC 106.8 (Xyl-1), 75.1 (Xyl-2), 85.9 (Xyl-3), 69.4 (Xyl-4), 65.5 and (Xyl-5); Xyl’s (outer) carbon signals at δC 104.9 (Xyl-1), 75.0 (Xyl-2), 78.1 (Xyl-3), 71.1 (Xyl-4), and 67.2 (Xyl-5). With the analyzing results of HSQC, HMBC, DEPT, and NMR, five monosaccharide units of 1 were identified as one β-D-glucopyranosyl (Glc), one β-D-fucopyranosyl (Fuc), one α-L-rhamnopyranosyl (Rha) and two β-D-xylopyranosyls (Xyl). The positions of the sugar components in 1 were clarified by an HMBC experiment (Figure 2), which showed a correlation between δH 4.55 (Glc-1) and δC 87.7 (C-3) revealing that Glc was located at the position of C-3 of presenegenin, δH 5.44 (Fuc-1) and δC 177.8 (C-28) revealing that Fuc was located at the position of C-28 of presenegenin, δH 4.51 (Xyl-1, inner) and δC 85.0 (Rha-4) revealing that Xyl (inner) was located at the position of C-4 of Rha, δH 4.36 (Xyl-1, outer) and δC 85.9 (Xyl-3, inner) revealing that Xyl (outer) was located at the position of C-3 of Xyl (inner), and δH 5.48 (Rha-1) and δC 72.3 (Fuc-2) revealing that Rha was located at the position of C-2 of Fuc.Figure 3
The hydrolyzed sugar’s identification chromatogram of compound1.What is noteworthy is that, in the HMBC spectrum, a correlation between the methyl proton atδH 2.18 (CH3CO-) and δC 172.8 (CH3CO-) and between δH 5.10 (Fuc-4) and 172.8 (CH3CO-) 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-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1 ⟶ 3)-β-D-xylopyranosyl-(1 ⟶ 4)-α-L-rhamnopyranosyl-(1 ⟶ 2)-(4-O-acetyl)-β-D-fucopyranosyl ester.


### 3.2. Biological Activity Assays

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


#### 3.2.1. Cytotoxic Activity Assay

NO participates in the regulation of cytokines which is especially associated with inflammation and has anti-inflammatory protective effect and cytotoxicity in inflammatory reaction [15]. Meanwhile, NO is an effector in cytotoxicity of activated macrophages [15]. To further confirm, cytotoxicity screenings on the isolated compounds were measured by cell counting kit-8 (CCK-8) assay in LPS-induced RAW 264.7 macrophage cells. 7 compounds including arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10) were tested against RAW 264.7 macrophage cells using cell counting kit-8 (CCK-8) (Dojindo, Japan). The results (Figure 4) showed that there was no significant cytotoxicity for the compounds at the corresponding concentrations, which also means its cytotoxicity had no impact on anti-inflammatory activity.Figure 4
The effect of the isolated compounds on the survival rate of RAW 264.7 macrophage cells. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h. The data show the mean ± SD of three independent experiments performed in triplicates. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


#### 3.2.2. Anti-Inflammatory Activity Assay

As an important physiological messenger and effector 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-inflammatory activity with the LPS group as control. Meanwhile, dexamethasone sodium phosphate (C22H28FNa2O8P) 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 significant anti-inflammatory activity and 5 had anti-inflammatory activity at the highest concentration, while the other three compounds (4, 8, and 10) without anti-inflammatory 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-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 the drugs concentration was too low to reach the effective concentration. In addition, the compound 5 was effective at the highest concentration, for the effective concentration was too high, in-depth research on it is unnecessary either in theory or in reality.Figure 5
Anti-inflammatory effects of the isolated compounds and inhibition of LPS-induced NO production in RAW 264.7 mouse macrophages. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h and then stimulated with LPS (0.1 μg/mL) for 16 h. The data show the mean ± SD of three independent experiments performed in triplicates. ∗p<0.05 and ∗∗p<0.01 compared to the LPS-treated values. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


## 3.1. Structure Elucidation

The known compounds (2–12) (Figure 1) were identified by comparison of their experimental spectral data with literature data [3–12].Figure 1
Chemical structures of compounds1–12.The structure of the new compound1 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.Figure 2
Key HMBC correlations of compound1 (arrows point from proton to carbon).Compound1 was obtained as a white powder with the molecular formula of C60H94O29 based on the negative ion peak at m/z 1277.5839 [M-H]− (calculated 1277.5803), implying 14 degrees of unsaturation. The 1H-NMR spectrum revealed the presence of two tertiary methyl protons at δH 1.32 (3H, d, J = 6.6 Hz) and 1.10 (3H, d, J = 6.6 Hz), five quaternary methyl protons at δH 1.40 (3H, s), 1.28 (3H, s), 0.97 (3H, s), 0.94 (3H, s), and 0.76 (3H, s), an olefinic proton at δH 5.67 (1H, t), and five anomeric protons at δH 5.46 (1H, brs), 5.44 (1H, d, J = 8.4 Hz), 4.55 (1H, d, J = 7.8 Hz), 4.51 (1H, d, J = 7.8 Hz), and 4.36 (1H, d, J = 7.8 Hz). The 13C-NMR spectrum displayed the presence of six quaternary carbons at δC 31.6, 37.5, 41.7, 47.9, 48.6, and 53.2, an oxygen-bearing methylene carbon at δC 64.7, two oxygen-bearing methine carbon at δC 70.9 and 87.7, five anomeric carbons at δC 95.0, 101.1, 104.9, 105.6, and 106.8, a set of olefinic carbons at δC 128.8 and 139.3, an ester carbonyl carbon at δC 177.8, and a carboxyl carbon at δC 182.9. All the above spectral information showed that the aglycone moiety of 1 was presenegenin [17] which belonged to oleanane triterpenoidal saponin, meanwhile combined with the chemical shift of the deshielded carbon C-3 (δC 87.7) and the carbonyl C-28 (δC 177.8) of presenegenin indicated that compound 1 was the 3,28-bisdesmoside of presenegenin, having five monosaccharide units.On acid hydrolysis, compound1 afforded an aglycone which was identified as presenegenin on the basis of the 1H-NMR and 13C-NMR data [17]. The sugars obtained from the saponin hydrolysate were identified as D-glucose, D-fucose, D-xylose, and L-rhamnose based on HPLC analysis (Figure 3). The NMR (Table 1) data of compound 1, along with the HSQC spectrum, showed the five anomeric proton signals at δH 5.46 (1H, brs), 5.44 (1H, d, J = 8.4 Hz), 4.55 (1H, d, J = 7.8 Hz), 4.51 (1H, d, J = 7.8 Hz), and 4.36 (1H, d, J = 7.8 Hz) and were correlated with anomeric carbon signals at δC 101.1, 95.0, 105.6, 106.8, and 104.9, respectively. Combined 13C-NMR with distortionless enhancement by polarization transfer (DEPT) data, the carbon signals of five monosaccharide units had been listed as follows: Glc’s carbon signals at δC 105.6 (Glc-1), 75.1 (Glc-2), 77.7(Glc-3), 71.7 (Glc-4), 77.7 (Glc-5), and 62.3 (Glc-6); Fuc’s carbon signals at δC 95.0 (Fuc-1), 72.3 (Fuc-2), 75.4 (Fuc-3), 75.3 (Fuc-4), 71.2 (Fuc-5), and 16.5(Fuc-6); Rha’s carbon signals at δC 101.1 (Rha-1), 71.1 (Rha-2), 73.7 (Rha-3), 85.0 (Rha-4), 68.5 (Rha-5), and 18.2 (Rha-6); Xyl’s (inner) carbon signals at δC 106.8 (Xyl-1), 75.1 (Xyl-2), 85.9 (Xyl-3), 69.4 (Xyl-4), 65.5 and (Xyl-5); Xyl’s (outer) carbon signals at δC 104.9 (Xyl-1), 75.0 (Xyl-2), 78.1 (Xyl-3), 71.1 (Xyl-4), and 67.2 (Xyl-5). With the analyzing results of HSQC, HMBC, DEPT, and NMR, five monosaccharide units of 1 were identified as one β-D-glucopyranosyl (Glc), one β-D-fucopyranosyl (Fuc), one α-L-rhamnopyranosyl (Rha) and two β-D-xylopyranosyls (Xyl). The positions of the sugar components in 1 were clarified by an HMBC experiment (Figure 2), which showed a correlation between δH 4.55 (Glc-1) and δC 87.7 (C-3) revealing that Glc was located at the position of C-3 of presenegenin, δH 5.44 (Fuc-1) and δC 177.8 (C-28) revealing that Fuc was located at the position of C-28 of presenegenin, δH 4.51 (Xyl-1, inner) and δC 85.0 (Rha-4) revealing that Xyl (inner) was located at the position of C-4 of Rha, δH 4.36 (Xyl-1, outer) and δC 85.9 (Xyl-3, inner) revealing that Xyl (outer) was located at the position of C-3 of Xyl (inner), and δH 5.48 (Rha-1) and δC 72.3 (Fuc-2) revealing that Rha was located at the position of C-2 of Fuc.Figure 3
The hydrolyzed sugar’s identification chromatogram of compound1.What is noteworthy is that, in the HMBC spectrum, a correlation between the methyl proton atδH 2.18 (CH3CO-) and δC 172.8 (CH3CO-) and between δH 5.10 (Fuc-4) and 172.8 (CH3CO-) 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-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1 ⟶ 3)-β-D-xylopyranosyl-(1 ⟶ 4)-α-L-rhamnopyranosyl-(1 ⟶ 2)-(4-O-acetyl)-β-D-fucopyranosyl ester.


## 3.2. Biological Activity Assays

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


### 3.2.1. Cytotoxic Activity Assay

NO participates in the regulation of cytokines which is especially associated with inflammation and has anti-inflammatory protective effect and cytotoxicity in inflammatory reaction [15]. Meanwhile, NO is an effector in cytotoxicity of activated macrophages [15]. To further confirm, cytotoxicity screenings on the isolated compounds were measured by cell counting kit-8 (CCK-8) assay in LPS-induced RAW 264.7 macrophage cells. 7 compounds including arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10) were tested against RAW 264.7 macrophage cells using cell counting kit-8 (CCK-8) (Dojindo, Japan). The results (Figure 4) showed that there was no significant cytotoxicity for the compounds at the corresponding concentrations, which also means its cytotoxicity had no impact on anti-inflammatory activity.Figure 4
The effect of the isolated compounds on the survival rate of RAW 264.7 macrophage cells. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h. The data show the mean ± SD of three independent experiments performed in triplicates. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


### 3.2.2. Anti-Inflammatory Activity Assay

As an important physiological messenger and effector 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-inflammatory activity with the LPS group as control. Meanwhile, dexamethasone sodium phosphate (C22H28FNa2O8P) 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 significant anti-inflammatory activity and 5 had anti-inflammatory activity at the highest concentration, while the other three compounds (4, 8, and 10) without anti-inflammatory 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-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 the drugs concentration was too low to reach the effective concentration. In addition, the compound 5 was effective at the highest concentration, for the effective concentration was too high, in-depth research on it is unnecessary either in theory or in reality.Figure 5
Anti-inflammatory effects of the isolated compounds and inhibition of LPS-induced NO production in RAW 264.7 mouse macrophages. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h and then stimulated with LPS (0.1 μg/mL) for 16 h. The data show the mean ± SD of three independent experiments performed in triplicates. ∗p<0.05 and ∗∗p<0.01 compared to the LPS-treated values. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


## 3.2.1. Cytotoxic Activity Assay

NO participates in the regulation of cytokines which is especially associated with inflammation and has anti-inflammatory protective effect and cytotoxicity in inflammatory reaction [15]. Meanwhile, NO is an effector in cytotoxicity of activated macrophages [15]. To further confirm, cytotoxicity screenings on the isolated compounds were measured by cell counting kit-8 (CCK-8) assay in LPS-induced RAW 264.7 macrophage cells. 7 compounds including arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10) were tested against RAW 264.7 macrophage cells using cell counting kit-8 (CCK-8) (Dojindo, Japan). The results (Figure 4) showed that there was no significant cytotoxicity for the compounds at the corresponding concentrations, which also means its cytotoxicity had no impact on anti-inflammatory activity.Figure 4
The effect of the isolated compounds on the survival rate of RAW 264.7 macrophage cells. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h. The data show the mean ± SD of three independent experiments performed in triplicates. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


## 3.2.2. Anti-Inflammatory Activity Assay

As an important physiological messenger and effector 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-inflammatory activity with the LPS group as control. Meanwhile, dexamethasone sodium phosphate (C22H28FNa2O8P) 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 significant anti-inflammatory activity and 5 had anti-inflammatory activity at the highest concentration, while the other three compounds (4, 8, and 10) without anti-inflammatory 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-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 the drugs concentration was too low to reach the effective concentration. In addition, the compound 5 was effective at the highest concentration, for the effective concentration was too high, in-depth research on it is unnecessary either in theory or in reality.Figure 5
Anti-inflammatory effects of the isolated compounds and inhibition of LPS-induced NO production in RAW 264.7 mouse macrophages. The cells were pretreated with concentrations (12.5, 25, and 50μg/mL) of compounds (1, 2, 4, 5, 7, 8, and 10) or C22H28FNa2O8P (5 μg/mL) for 1 h and then stimulated with LPS (0.1 μg/mL) for 16 h. The data show the mean ± SD of three independent experiments performed in triplicates. ∗p<0.05 and ∗∗p<0.01 compared to the LPS-treated values. Arillatanoside E (1), oleanolic acid (2), trans-ferulic acid (4), trans-feruloyl-glucoside (5), 2,4,6-trimethoxy-1-O-β-D-glycoside (7), 3-methoxy-4-hydroxybenzoic acid (8), and sinapic acid (10).


## 4. Conclusion

In this research, a new compound, named as arillatanoside E (1), which was established as a derivative of the chemotaxonomic marker 3-O-(β-D-glucopyranosyl) presenegenin 28-[O-β-D-xylopyranosyl-(1 ⟶ 4)-O-α-L-rhamnopyranosyl-(1 ⟶ 2)-β-D-fucopyranosyl] ester, 3-O-β-D-glucopyranosyl presenegenin 28-O-β-D-xylopyranosyl-(1 ⟶ 3)-β-D-xylopyranosyl-(1 ⟶ 4)-α-L-rhamnopyranosyl-(1 ⟶ 2)-(4-O-acetyl)-β-D-fucopyranosyl ester, was found in P. arillata. During the past ten years’ literatures, we have also found that two compounds, 3-O-(β-D-glucopyranosyl) presenegenin 28-{O-β-D-galactopyranosyl-(1 ⟶ 3)-O-β-D-xylopyranosyl-(1 ⟶ 4)-O-α-L-rhamnopyranosyl-(1 ⟶ 2)-β-D-fucopyranosyl} ester [19] and arillatanoside A [17], have the same structure character and were found in this genus, whose type may be the chemotaxonomic marker of Polygala genus. On the other hand, more than 100 saponins have been isolated from Polygala genus, and their aglycones are almost oleanane triterpenoids. Thus, in our opinion, oleanane triterpenoids with diverse biological activities [20] are the chemotaxonomic markers of this genus. Furthermore, seven isolated compounds (1, 2, 4, 5, 7, 8, and 10) exhibited little cytotoxic activity on RAW 264.7 mouse macrophages; then, the inhibitory effects of 7 isolates on nitric oxide (NO) production in lipopolysaccharide-activated macrophages were evaluated. As a result, arillatanoside E (1), oleanolic acid (2), and 2,4,6-trimethoxy-1-O-β-D-glycoside (7) possessed potential anti-inflammatory activity.



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*Source: 8079619-2019-01-02.xml*