A New Flavonol From Saussurea involucrata With Anti-Inflammatory Activity

Abstract

A new flavonol named 6-(2'',3''-epoxy-3''-methylbutyl)-resokaempferol (1), together with five known compounds (2-6) were isolated from the EtOAc-soluble extract of the aerial part of Saussurea involucrata. Their structures were elucidated on the basis of spectroscopic methods. All compounds were evaluated for their anti-inflammatory effects by measuring the production of nitric oxide (NO) and TNF-α in vitro. Among them, compound 1 showed potential inhibitory activity on the production of NO and TNF-α in LPS-induced RAW 264.7 cells with IC₅₀ values of 48.0 ± 1.5 and 41.4 ± 1.7 µM, respectively.

Saussurea involucrata (Kar. et Kir.) Sch. -Bip (Asteraceae), called “Tianshan Snow Lotus” or“Xinjiang Xuelian”in China, is one of the precious medicinal herbs that is widely distributed in Central Asia. ¹ As an important Chinese traditional medicine, S. involucrata is used to treat cough, ² rheumatoid arthritis, ^3,4 dysmenorrhea, ⁵ altitude sickness and stomachache. ⁶ Published studies have showed that S. involucrata possesses a number of bioactive compounds including lignans, ⁷ flavonoids, ⁷ coumarins, ⁸ sesquiterpene lactones, ⁹ steroids and phenylpropanoids. ¹⁰ Some of these compounds displayed a wide range of biological activities such as anti-inflammatory, ¹¹ anti-aging, ¹¹ anti-oxidative, anti-fatigue, ¹² and anti-tumor effects. ² During the course of our search for novel lead compounds with anti-inflammatory effect from Chinese traditional medicines, a new flavonol named 6-(2'',3''-epoxy-3''-methylbutyl)-resokaempferol (1), together with five known compounds (2-6), were isolated from the whole plant of S. involucrata. Herein we report the isolation, structural elucidation and anti-inflammatory activities of these compounds.

Results and Discussion

Compound 1 was isolated as a yellow amorphous powder, and its molecular formula was determined to be C₂₀H₁₈O₆ by HREIMS (Supplemental Figure S4) from the ion at m/z 354.1023. The IR (KBr) spectrum showed absorptions attributable to hydroxyl (3357 cm^-1) and carbonyl (1743 cm^-1) groups. The ¹H NMR spectroscopic data of 1 (Table 1, Supplemental Figure S1) showed six aromatic protons at δ_H 7.67 (s, H-5), 6.57 (s, H-8), 7.76 (d, J = 8.5 Hz, H-2'/6') and 6.90 (d, J = 8.5 Hz, H-3'/5'), an oxygenated methine at δ _H 2.90 (t, J = 6.0 Hz, H-2''), a methylene at δ _H 2.74‐2.83 (m, H-1'') and two methyls at δ _H 1.26 (s, H-4'') and 1.35 (s, H-5''). The ¹³C NMR (Table 1, Supplemental Figure S2) displayed twenty carbons, including a carbonyl carbon at δ _C 182.1 (C-4), twelve aromatic carbons at δ _C 164.9‐100.1, two olefinic carbons at δ _C 145.7 (C-2) and 133.8 (C-3), and two methyl carbons at δ _C 25.1 (C-4'') and 19.2 (C-5''). When combined with the corresponding downfield region (δ_C 182.1‐100.1) in the ¹³C NMR spectrum of 1 and the comparison of NMR data of 1 with those of kaempferol, ¹³ the flavonol skeleton of 1 was deduced. The structure of the 2'',3''-epoxy-3''-methylbutyl moiety at C-6 could be deduced by the key HMBC correlations of δ _H 2.74‐2.83 (H-1'') to δ _C 128.5 (C-5), 121.9 (C-6) and 63.5 (C-2''), δ _H 2.90 (H-2'') to δ _C 121.9 (C-6), 28.9 (C-1'') and 58.7 (C-3''), and δ _H 1.26/1.35 (H-4''/5'') to δ _C 63.5 (C-2'') and 58.7 (C-3'') (Figure 1 and Supplemental Figure S3). In addition, the comparison of NMR data between 1 and (2S)−7,4'-hydroxyl-6-(2'',3''-epoxy-3''-methylbutyl) flavanone further confirmed the structure of 1. ¹⁴ Because of the small amount of 1 available, the absolute configuration of C-2'' was not determined. Thus, the structure of compound 1 was determined as 6-(2'',3''-epoxy-3''-methylbutyl)-resokaempferol.

Table 1

¹H and ¹³C NMR Spectral Data of Compound 1 in Acetone-D ₆ (¹H: 500 MHz, ¹³C: 125 MHz).

No.	1
	δ C	δ H
2	145.7
3	133.8
4	182.1
5	128.5	7.67, s
6	121.9
7	164.9
8	100.1	6.57, s
9	163.7
10	115.1
1'	122.8
2'	129.0	7.76, d, (8.5)
3'	116.2	6.90, d, (8.5)
4'	158.7
5'	116.2	6.90, d, (8.5)
6'	129.0	7.76, d, (8.5)
1''	28.9	2.74‐2.83, m
2''	63.5	2.90, t (6.0)
3''	58.7
4''	25.1	1.26, s
5''	19.2	1.35, s

Figure 1

Key HMBC correlations of compound 1.

Five known compounds (2-6) were also obtained from S. involucrata, and identified as quercetin (2), ¹⁵ quercetin 3-O-rhamnoside (3), ¹⁵ isorhamnetin 3-O-rutinoside (4), ¹⁵ kaempferol 7-O-glucopyranoside (5), ² and chrysoeriol 7-O-glycoside (6) ² by comparing their NMR data with those in the literature (Figure 2).

Figure 2

The structures of compounds 1-6.

S. involucrata possesses a significant anti-inflammatory effect. Thus, in this study, all isolates were evaluated for their anti-inflammatory activities in vitro. Nitric oxide (NO) and TNF-α are two key signals to reflect the occurrence of inflammatory diseases in the body such as tissue injury, ulcerative colitis and osteoarthritis. Therefore, the anti-inflammatory activities of compounds 1-6 were tested by evaluating the production of NO and TNF-α in LPS-induced RAW 264.7 cells (Table 2); dexamethasone and silybin were used as positive control. Compounds 1, 3-5 and 6 displayed moderate anti-inflammatory activities by inhibiting the production of NO and TNF-α in RAW264.7 cells. Compound 1 showed potential inhibitory activity on the production of NO and TNF-α with IC₅₀ values of 48.0 ± 1.5 and 41.4 ± 1.7 µM, respectively. Furthermore, compound 4 also showed inhibitory activity on NO production with an IC₅₀ value of 20.1 ± 1.3 µM.

Table 2

Anti-inflammatory Activities of Compounds 1-6.

Compound	IC50(μM)
	NO	TNF-α
1	12.0 ± 1.1	41.4 ± 1.7
2	>100	>100
3	41.6 ± 0.9	>100
4	20.1 ± 1.3	50.7 ± 1.4
5	67.0 ± 1.0	>100
6	>100	>100
Dexamethasone a	8.2 ± 0.4	-
Silybin a	-	62.7 ± 1.1

IC50 values represent the means ± SEM of three parallel measurements.

^aPositive control.

Investigations on S. involucrata have resulted in the isolation of various flavonoid derivatives, including flavonoid glycosides and flavonols; these compounds exhibited moderate to high anti-inflammatory effect. In this article, we report a novel flavonol with potent anti-inflammatory activity from the dried aerial part of S. involucrata. Further investigation of the chemical constituents of S. involucrata will be undertaken.

Experimental

General

Optical rotations were measured with a Perkin-Elmer 241 polarimeter, UV spectra with a JASCO V-650 spectrophotometer (JASCO, Tokyo, Japan), IR spectra with a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer, and nuclear magnetic resonance (NMR) spectra with a Varian Unity Inova 500 MHz spectrometer (Varian Unity Inova, Phoenix, USA) using TMS as the internal standard. Mass spectra were obtained on a QTOF2 high resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography (CC) was performed on silica gel 200‐300 mesh (Yantai Xinde Chemical Co., Ltd, Yantai, China) and RP-18 (Merck, Darmstadt, Germany). TLC was performed with silica gel GF₂₅₄ glass plates (Qingdao Marine Chemical Co. Ltd., Qingdao, China). HPLC was carried out using a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu, Kyoto, Japan), and an ODS column (250 mm × 10 mm i.d., 5 µm, YMC-ODS-A).

Plant Material

The dried aerial parts of S. involucrata were collected in tianshan mountains, Xinjiang, China, and authenticated by Professor Qiang Zhao (College of Pharmacy, Hangzhou Medical College). A voucher specimen of the plant (No. 20190315) was deposited at Hangzhou Medical College, Hangzhou, China.

Extraction and Isolation

The dried aerial parts of S. involucrata (5.0 kg) were extracted 3 times with 95% EtOH under reflux and concentrated under vacuum to obtain a crude extract (2.1 kg). This was suspended in H₂O and partitioned successively with light petroleum (PE), CH₂Cl₂, EtOAc and n-BuOH. The anti-inflammatory effects of these four fractions were further evaluated through measuring the inhibition of NO production in LPS-induced RAW 264.7 cells. The EtOAc-soluble fraction (97.5 g) was subjected to silica gel column chromatography using a gradient of CH₂Cl₂-MeOH, and was separated into 12 fractions (Fr.1-Fr.12). Fr.3 (4.4 g) was chromatographed over silica gel, eluted with a gradient of PE-EtOAc, and was separated into 15 fractions (Fr.3.1-Fr.3.15). Fr.3.4 (215.3 mg) was chromatographed on a Sephadex LH-20 column (MeOH) to give compounds 2 (10.3 mg) and 4 (6.4 mg). Fr.3.5 (312.6 mg) was further purified by HPLC using a gradient solvent system of 45 to 55% MeOH in H₂O over 70 minutes to yield compounds 1 (2.8 mg, t _R = 33.4 minutes), 3 (6.2 mg, t _R = 42.5 minutes), 5 (5.8 mg, t _R = 50.3 minutes) and 6 (7.1 mg, t _R = 61.4 minutes).

6-(2'',3''-Epoxy-3''-methylbutyl)-resokaempferol (1 ). A yellow amorphous powder; [α] −10.5 (c 0.1, MeOH); UV (MeOH) λ_max (log ε): 230 (3.89), 351 (3.22) nm; IR (KBr disc) ν _max 3357, 2930, 1743, 1610, 1447 cm^{-1; 1}H (500 MHz) and ¹³C NMR (125 MHz) spectral data in acetone-d ₆, see Table 1; HREIMS: m/z 354.1023 [M]⁺ (calcd for C₂₀H₁₈O₆, 354.1103).

Anti-inflammatory Assays

In vitro anti-inflammatory activities of compounds 1-6 were evaluated by monitoring the production of NO and TNF-α in RAW264.7 cells. ¹⁶ Briefly, RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% antibiotics and _L-glutamine, and seeded in a 96-well chamber (3 × 10⁴ cells/well). Next, cells were treated with different concentrations of compounds 1-6 after 24 hours incubation. Then RAW264.7 cells were exposed to LPS (1 µg/mL) for another 24 hours. The concentration of NO was measured using a Griess kit (Beyotime Biotechnology, Shanghai, China), with dexamethasone as the positive control. The concentrations of TNF-α were determined by an ELISA kit (R&D company, Minnesota, USA) according to the manufacturer’s instructions. Finally, the level of TNF-α was determined from a standard curve with silybin used as a positive control.

Supplemental Material

Supplementary Material 1 - Supplemental material for A New Flavonol From Saussurea involucrata With Anti-Inflammatory Activity

Supplemental material, Supplementary Material 1, for A New Flavonol From Saussurea involucrata With Anti-Inflammatory Activity by Sheng Pan and Zi-Guan Zhu in Natural Product Communications