New phenolic glycosides from Anemone chinensis Bunge and their antioxidant activity

ABATRACT Nine compounds, five phenolic glycosides (1, 2, 4–6), three phenylpropanoids (7–9), and a furanone glycoside (3), were isolated from aqueous soluble extract of the dried roots of Anemone chinensis Bunge. The structures of new compounds (1–4) were elucidated by comprehensive spectroscopic data analysis as well as chemical evidence. Pulsatillanin A (1) demonstrated significant antioxidant effects through scavenging free radical in DPPH assay, and relieved the oxidative stress in LPS-induced RAW 264.7 cells by reducing ROS production, enhancing antioxidant enzyme SOD activity, replenishing depleted GSH in a dose-dependent manner. Western blot analysis revealed that 1 showed antioxidant activity via activating Nrf2 signaling pathway.


Introduction
Pulsatillae Radix, the dried roots of Anemone chinensis Bunge (synonym Pulsatilla chinensis (Bunge) Regel), is officially listed in the Chinese Pharmacopeia as a well-known Traditional Chinese Medicine. A. chinensis is traditionally used to treat intestinal amebiasis, dysentery, and enteritis (Muluye et al. 2014;. Modern pharmacological studies revealed that the chemical constituents of A. chinensis exhibited antitumor , antibacterial (Feng et al. 2018), anti-inflammatory, and immunomodulatory effects (Kang et al. 2019). Especially, the clinical efficacy of A. chinensis in ulcerative colitis treatment received significant attention Ma et al. 2020). Phytochemical investigations showed triterpenoid saponins are the main type of chemical component of A. chinensis and responsible for the bioactivities of this traditional medicine (Mimaki et al. 2001;Shu et al. 2013;Xu et al. 2013).
Phenolic compounds are well-known antioxidant active ingredients in vegetables, fruits, herb medicine, and other natural sources. They play a key role in the herb medicine to prevent oxidative damage under pathological conditions including cellular injury, aging, cancer, and cardiovascular disorders (Sytar 2015;Aryal et al. 2019). To investigate the potential of isolated phenolic compounds, the antioxidant activity of isolates 1-9 were evaluated by DPPH assay. Meanwhile, the effects of pulsatillanin A (1) scavenging ROS, increasing superoxide gasification enzyme (SOD) and non-enzyme antioxidant glutathione (GSH) levels by activating Nrf2 signaling pathway were investigated.
Compound 3 was obtained as a colorless solid and assigned a molecular formula of C 11 H 18 O 8 according to HRESIMS and 13 C NMR data. Its IR spectrum revealed the presence of hydroxy groups (3375 cm À1 ) and a carbonyl (1758 cm À1 ). The 1 H NMR spectrum presented signals for six oxygenated methines at d H 4.68-2.96, four methylenes at d H 3.96-2.02. The 13 C NMR and HSQC spectra demonstrated signals for all eleven carbons and assigned as one lactone carbonyl at d C 177.7, one oxygenated methine at d C 79.1, one oxygenated methylene at d C 70.6, two methylenes at d C 28.2, 23.4), and a group of carbon signals for a hexose moiety d C 103.4, 77.0, 76.8, 73.6, 70.1, 61.2. All the hydrogen signals were assigned to the corresponding carbons through carefully HSQC spectral analysis. The 1 H NMR, 13 C NMR, COSY, and HMBC spectra indicated 3 was a c-lactone alcohol glycoside. The aglycone was identified as in figure 1 based on acid hydrolysis product 3a, which showed nearly identical optical rotation value and NMR data with those of (S)-dihydro-5-(hydroxymethyl)furanone (Wrona et al. 2010).
The configuration of C-4 was further confirmed to be S based on the experimental ECD spectrum of 3a similar to the calculated ECD curve of S (Supplementary material Figure S2). The b-D-glucose was determined by the same chemical method as 1 and analysis of coupling constant of its anomeric proton. The connectivity of aglycone and D-glucopyranose was established from HMBC correlation observed between H-1 0 (d H 4.18) and C-5 (d C 70.6), between H-5 (d H 3.96, 3.57) and C-1 0 (d C 103.4). Based on the above evidence, the structure of 3 was determined as (S)-dihydro-5-(b-Dglucopyranosyloxymethyl)furanone.
Compound 4 was confirmed to have the molecular formula C 15 H 20 O 10 by its HRESIMS and 13 C NMR data. The 1 H and 13 C NMR data (Supplementary material Table S1) of 4 showed it was a phenolic glycoside. Acid hydrolysis of 4 afforded two products, aglycone (4a) and a sugar residue. 4a was elucidated as in Figure 1 by comparing its optical rotation value and NMR data with those of (R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid (tanshinol) (Kelley et al. 1976;Dai et al. 2010). Comparing the experimental and calculated ECD spectra (Supplementary material Figure S2) of 4a further determined the absolute configuration of C-8 was R. The sugar was identified as a D-glucose through using the same GC-MS analysis of the sugar derivative as those of compounds 1-3. HMBC cross-peaks found from H-1 0 (d H 4.59, d, J ¼ 7.2 Hz) to C-3 (d C 144.9) confirmed the sugar residue linkage with C-3. The b-configuration of glucose was determined according to the coupling constant of anomeric hydrogen ( 3 J H-1/H-2 ¼ 7.2 Hz). The structure of 4 was further confirmed by 1 H-1 H COSY and HMBC data analysis (Supplementary material Figure S1). As shown in Figure S1 (Supplementary material), the structure of 4 was elucidated and named as tanshinol-3-O-b-D-glucopyranoside.

Antioxidant activity determined by DPPH assay
To explore the antioxidant capacity of other isolates, the DPPH assay was carried out. As the result (Supplementary material Table S2), besides FA (8) and IFA (9), pulsatillanin A (PA, 1) showed effective antioxidant activity with IC 50 value at 177.17 ± 8.18 mM. Whereas, no significant antioxidant effect was observed among compounds 2-7 (IC 50 > 200 mM). Subsequently, new phenolic glycoside PA (1) was selected to evaluate the antioxidant effects in RAW 264.7 cells. FA (8) was used as the reference standard, which was reported to exhibit antioxidant ability by activating Nrf2 signaling pathway (Mahmoud et al. 2020).

Effects of PA on cell viability and oxidative stress in RAW 264.7 cells
Prior to investigate the antioxidant activity of PA (1), the cytotoxicity of PA on RAW 264.7 cells was evaluated by MTT assay. The result showed that there was no significant effects on cell viability after exposure to PA (1) at the concentration of 0 $ 100 mM for 24 h (Supplementary material Figure S3A).
The ROS level in LPS-stimulated RAW 264.7 cells increased significantly comparing to control group, which was in turn attenuated in a dose-dependent manner after treated with PA for 24 h (Supplementary material Figure S3B and C). The data further verified the protective effect of PA against oxidative stress in LPS-induced cells. Superoxide dismutase (SOD) and non-enzyme antioxidant glutathione (GSH) play the key role in the protection of cells against damage induced by oxidative stress . SOD is one of the key antioxidase involved in scavenging ROS and promoting the decomposition of hydrogen peroxide (G€ ur et al. 2020). The ratio of reduced glutathione and oxidized glutathione (GSH/GSSG) is widely used to determine the oxidative stress status. To investigate the effect of PA on the enzymatic and non-enzymatic antioxidant ability, the expression levels of SOD and GSH/GSSG were determined in LPSstimulated RAW 264.7 cells with PA treatment. According to the experimental result (Supplementary material Figure S3D), an obvious elevation in SOD level was observed in PA-treated group. Besides, PA also significantly reversed GSH level and GSH/GSSG ratio compared with LPS group (Supplementary material Figure S3E and F).

Effects of PA on Nrf2 signaling pathway in LPS-induced RAW264.7 cells
Under normal condition, Nrf2 is inactive in the cytoplasm and interacted with Kelchlike ECH-associated protein 1 (Keap1). Once exposed to oxidative stress, Nrf2 dissociated from Keap1 and translocated into the nucleus, subsequently combined with antioxidant response element (ARE) resulting in the expression of antioxidant/ detoxification enzymes, including NAD(P)H, quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), glutamate cysteine ligase catalytic subunit (GCLC) (Choi et al. 2020;Sun et al. 2020).
To explore the mechanism responsible for the antioxidant activity of PA, the protein expression levels of Nrf2 and its target genes GCLC, NQO-1 were detected by western blot. As shown in Figure S4 (Supplementary material), the protein expression level of Nrf2 was slightly elevated, but GCLC and NQO1was no significant changes in RAW 264.7 cells after LPS treatment. The observation indicate that oxidative stress occurred after LPS stimulation, and compensatory increase of Nrf2 was insufficient to induce antioxidant defense in cells. Nevertheless, compared with LPS group, the protein expression levels of Nrf2, GCLC and NQO1 significantly increased in a dose-dependent manner in LPS-induced cells with PA treatment. Hence, the antioxidant effect of PA might be attributed to activating Nrf2 signal pathway, which resulted in an enhancement of detoxification and antioxidant capacity in cells.