Antioxidant and Anti-Inflammatory Activities of Six Flavonoids from Smilax glabra Roxb

This study aimed to isolate, prepare and identify the main flavonoids from a standardized Smilax glabra flavonoids extract (SGF) using preparative HPLC, MS, 1H NMR and 13C NMR, determine the contents of these flavonoids using UPLC, then compare their pharmacological activities in vitro. We obtained six flavonoids from SGF: astilbin (18.10%), neoastilbin (11.04%), isoastilbin (5.03%), neoisoastilbin (4.09%), engeletin (2.58%) and (−)-epicatechin (1.77%). The antioxidant activity of six flavonoids were evaluated by determining the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-Azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS+) radical scavenging activity and ferric reducing antioxidant power (FRAP). In addition, the anti-inflammatory activity of six flavonoids were evaluated by determining the production of cytokines (IL-1β, IL-6), nitric oxide (NO) using enzyme linked immunosorbent assay and the NF-κB p65 expression using Western blotting in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that (−)-epicatechin, astilbin, neoastilbin, isoastilbin and neoisoastilbin had strong antioxidant activities, not only in DPPH and ABTS+ radicals scavenging capacities, but in FRAP system. Furthermore, all the six flavonoids could significantly inhibit the secretion of IL-1β, IL-6, NO (p < 0.01) and the protein expression of NF-κB p-p65 (p < 0.01) in LPS-stimulated RAW264.7 cells. This study preliminarily verified the antioxidant and anti-inflammatory activities of six flavonoids in S. glabra.


Introduction
Smilax glabra Roxb. is a dried rhizome of Liliaceae plant. Hundreds of years ago, it had been used as food and folk medicine in some Asian countries, such as China, Japan, Korea and Vietnam. S. glabra was widely used in traditional Chinese medicine (TCM) for the treatment of syphilis, hypertonia, nephritis, heavy metal poisoning and other diseases in China [1,2]. In addition, it could be added to medicated diet as a functional ingredient. Modern pharmacological studies have shown that S. glabra has a variety of biological activities, such as antioxidant, anti-inflammatory, antiviral, antibacterial, hypouricemic, anti-gout, hepatoprotection and cardiovascular protection [3][4][5][6].
According to TCM theory, the treatment of diseases by traditional Chinese materia medica based on the interaction of multiple components. Therefore, in our previous studies, a standardized S. glabra flavonoids extract (SGF) was prepared from the rhizomes of S. glabra [20], the flavonoids content was ~687 mg rutin equivalents/g dry extract [21]. In in vivo experiments, SGF has strong antioxidant, hepatoprotective, hypouricemic and nephroprotective effects [4,20], which has laid a foundation for this study.
In order to clarify the pharmacodynamic material basis and the potential pharmacological mechanisms of SGF, it is necessary to perform further isolation, preparation and identification of the main active compounds in SGF. Furthermore, as mentioned above, a wide variety of diseases are associated with oxidative stress caused by free radicals, as well as inflammation. Thus, this study also evaluated the antioxidant and anti-inflammatory activities of the main active compounds (six flavonoids) in SGF.

Isolation of Six Flavonoids in SGF by Preparative High-performance Liquid Chromatography (PHPLC)
After optimized the isolation conditions of the six main compounds in SGF by ultra-highpressure liquid chromatography (UPLC), the PHPLC method was used to isolate and purify these six compounds ( Figure 1). The six flavonoids in SGF were identified by the spectral data of Q-TOF MS, 1 H-NMR and 13    The six flavonoids in SGF were identified by the spectral data of Q-TOF MS, 1 H-NMR and 13 C-NMR (Supplementary Materials Figures S1-S4). The detailed data were as follows. .98 (C-5), 156.28 (C-7), 156.61 (C-9). MS, 1 H and 13 C NMR spectral data of the isolated compound agree well with the data reported [22]. From these data, it was identified as (−)-epicatechin ((−)-cis-3,3 ,4 ,5,7-pentahydroxyflavane)), the molecular formula was C 15 H 14 O 6 ( Figure 2).

Anti-Inflammatory Activity of Six Flavonoids in SGF
The anti-inflammatory activity of six flavonoids in SGF on LPS-stimulated RAW264.7 cells was investigated by measuring IL-1β, IL-6 and NO concentrations in cell culture supernatant. As shown in Figure 5, we found LPS increased the concentrations of IL-1β, IL-6 and NO significantly. (−)-Epicatechin, astilbin, neoastilbin, isoastilbin, neoisoastilbin and engeletin showed strong anti-inflammatory capacities compared to the LPS-stimulated RAW264.7 cells (p < 0.01). To further clarify the anti-inflammatory mechanism of six flavonoids, we determined the protein expression of NF-κB p-p65 in RAW 264.7 cells. As shown in Figure 6, LPS has induced NF-κB activation, which showed the protein expression of phosphorylated p65 (p-p65) in LPS-stimulated RAW264.7 cells increased significantly. (−)-Epicatechin, astilbin, neoastilbin, isoastilbin, neoisoastilbin and engeletin had obvious inhibitory effects on the protein expression of p-p65 (p < 0.01).

Discussion
Astilbin is a flavonoids compound. Previous studies have shown that astilbin was the main compound in S. glabra. However, the other stereoisomers of astilbin (isoastilbin, neoisoastilbin, Molecules 2020, 25, x; doi: FOR PEER REVIEW www.mdpi.com/journal/molecules the anti-inflammatory mechanism of six flavonoids, we determined the protein expression of NF-κB p-p65 in RAW 264.7 cells. As shown in Figure 6, LPS has induced NF-κB activation, which showed the protein expression of phosphorylated p65 (p-p65) in LPS-stimulated RAW264.7 cells increased significantly. (−)-Epicatechin, astilbin, neoastilbin, isoastilbin, neoisoastilbin and engeletin had obvious inhibitory effects on the protein expression of p-p65 (p < 0.01).

Discussion
Astilbin is a flavonoids compound. Previous studies have shown that astilbin was the main compound in S. glabra. However, the other stereoisomers of astilbin (isoastilbin, neoisoastilbin,

Discussion
Astilbin is a flavonoids compound. Previous studies have shown that astilbin was the main compound in S. glabra. However, the other stereoisomers of astilbin (isoastilbin, neoisoastilbin, neoastilbin) are naturally low in content [11]. Thus, this study aimed to isolate and prepare the flavonoids compounds from a standardized S. glabra flavonoids extract (SGF), instead of S. glabra raw material. We isolated and prepared six flavonoids using PHPLC from SGF: astilbin, neoastilbin, isoastilbin, neoisoastilbin, engeletin and (−)-epicatechin. The content of these six flavonoids was 18.10%, 11.04%, 5.03%, 4.09%, 2.58% and 1.77%, respectively. All the purities of these flavonoids were more than 95% which determined by UPLC. The results showed that astilbin was still the main chemical component in SGF. However, the percentage of the other three stereoisomers of astilbin in SGF was higher than in the raw material.
Some studies have shown that astilbin had good effects in the treatment of various diseases [6,10,27]. However, there were few studies on the pharmacological roles of the other chemical constituents in S. glabra. Excess radicals and oxidative stress caused a variety of diseases in the body. Previous investigations have indicated that reducing radicals could effectively prevent the occurrence of oxidative stress related diseases [28][29][30]. Thus, this study evaluated the antioxidant capacities of six flavonoids in SGF by determining the DPPH radical scavenging activity, the ABTS+ radical scavenging activity, as well as the ferric reducing antioxidant power (FRAP). The results showed that most of the isolated flavonoids had strong antioxidant activity. Among them, (−)-epicatechin, isoastilbin and neoisoastilbin showed stronger antioxidant activity than astilbin. The isomerism of natural compounds may affect their pharmacological activity to some extent [11]. The four stereoisomers of astilbin based on the C-2 and C-3 configuration, which is categorized in 4 stereoisomers: astilbin (2R,3R), neoisoastilbin (2S,3R), isoastilbin (2R,3S) and neoastilbin (2S,3S). The trans-stereoisomers of astilbin (neoisoastilbin, isoastilbin) showed higher antioxidant activities than cis-stereoisomers (astilbin, neoastilbin). Moreover, structure-activity studies of flavonoids indicated that the substitution patterns of B-ring could affect the antioxidant capacity [31]. The chemical structures of these flavonoids showed that the four stereoisomers of astilbin have 3 -OH and 4 -OH on B-ring, and the engeletin only has 4 -OH on B-ring. In addition, engeletin had no obvious antioxidant activity in DPPH radical system and showed low activities in ABTS + radical scavenging and the FRAP evaluation, which indicated that 3 -OH on B-ring of flavonoids is more important to the antioxidant capacity.
In some diseases, oxidative stress and inflammation often occur simultaneously [32]. IL-1β, IL-6 and NO are the major mediators of inflammation in most inflammatory diseases. NF-κB was an important transcription factor with pro-inflammatory response. Upon activation, NF-κB moved rapidly into the nucleus and subsequently activated transcription of target genes, leading to the release of pro-inflammatory cytokines [33]. S. glabra was often used in the treatment of inflammatory diseases. Therefore, we also assessed the anti-inflammatory activity of six flavonoids in SGF by determining the cytokines (IL-1β, IL-6) and NO, and the expression of NF-κB p-p65/p65 in LPS-stimulated RAW 264.7 cells. The results showed that RAW 264.7 cells could release a variety of pro-inflammatory cytokines when stimulating by LPS, including IL-1β, IL-6 and NO, which were consistent with several literature reports [34,35]. Moreover, the production of IL-1β, IL-6, NO and phosphorylation of NF-κB (p-p65) in LPS-stimulated RAW264.7 cells were significantly inhibited by six flavonoids. The anti-inflammatory activities of astilbin and (−)-epicatechin were also consistent with several literature reports [27,36,37].

Isolation of Six Flavonoids in SGF by Preparative High-Performance Liquid Chromatography (PHPLC)
In order to optimize the isolation conditions of the six flavonoids in SGF, ultra-high-pressure liquid chromatography (UPLC) with ACQUITY UPLC H-Class system (Waters, Milford, MA, USA) was performed. The SGF was dissolved with methanol and centrifuged to obtain the supernatant. The isolated samples were lyophilized for further identification of structure. In this experiment, the six flavonoids were identified by confrontation of the spectral characteristics (Waters UPLC Synapt G2-S mass spectrometer (Waters, Milford, MA, USA). MS conditions: electrospray ionization (ESI), negative ion mode, drying gas flow rate is 11 L/min, drying gas temperature is 300 • C, capillary voltage is 3500 V, the nebulizer pressure is 25 psig, the fragmentor voltage is 175 V, the skimmer voltage is 60 V. 1 H-NMR and 13 C-NMR, Bruker 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany)) with those depicted in a previous study. The quantitative analysis and purity of six flavonoids in SGF were determined by ACQUITY UPLC H-Class system mentioned above.

DPPH Radical Scavenging Activity
DPPH radical has the maximal absorbance at 517 nm, the absorption value could be decreased by the antioxidant compounds. The capacity of six flavonoids in SGF to remove DPPH radical was determined according to a previously described procedure with slight modifications [38]. DPPH radical (20 mg) was accurately weighed and dissolved in absolute alcohol to 200 µM. Different concentrations of sample solutions (2 mL) were mixed with DPPH radical solution (2 mL). The mixture was incubated in the dark for 30 min at room temperature after vortex mixing, followed by measurement at 517 nm using UV-VIS 3600 spectrophotometer (Shimadzu, Tokyo, Japan), the absorbance is A i . Moreover, the absorbance of the mixture of different concentrations of sample solutions (2 mL) and absolute alcohol (2 mL) were determined as A j . The absorbance of the mixture of DPPH radical solution (2 mL) and methanol (2 mL) were determined as A c . Ascorbic acid standard was used for comparison. ABTS + radical has the maximal absorbance at 734 nm, the absorption value could be decreased by the antioxidant compounds. The radical scavenging activity of six flavonoids in SGF for ABTS + radical was determined by a previously described procedure [38]. ABTS + radicle was dissolved in deionized water to 7 mM, and the stock solution were generated by reacting ABTS + radical stock solution with 2.45 mM potassium persulfate in the dark for 16 h at room temperature. The working solution was diluted in ethanol to an absorbance of 0.7 ± 0.02 at 734 nm. ABTS + radical solution (3.6 mL) was mixed with different concentrations of sample solutions. Then, the mixture was incubated in the dark for 10 min at room temperature after vortex mixing, and the absorbance of the mixture was determined as A i . Moreover, the absorbance of the mixture of ABTS + radical solution (3.6 mL) and methanol (0.4 mL) was determined as A j . Ascorbic acid standard was used for comparison. The FRAP value of six flavonoids in SGF was determined by a previously described procedure with minor modifications [39]. FRAP working solution: 300 mM acetate buffer, 10 mM TPTZ diluted in 40 mM HCl and 20 mM FeCl 3 ·6H 2 O solution were mixed at 10:1:1.

Inflammatory Cytokines Determination by Enzyme Linked Immunosorbent Assay (ELISA)
The contents of IL-1β, IL-6 and NO were determined according to the manufacturer's protocols [19,41]. In short, 50 µL of the cell supernatant was added to a new 96-well plate, then incubated at 37 • C for 0.5 h. After washing 5 times using washing buffer (each wash lasts 30 s), 50 µL of HRP-conjugate reagent was added to each well and incubate at 37 • C for 0.5 h. After washing 5 times, 50 µL of chromogen solution A and B were added to each well, then protected from light and incubated at 37 • C for 10 min. Finally, 50 µL of stop solution was added to each well. The absorbance was read at 450 nm within 15 min. 4.4.3. NF-κB p-p65 Expression Assay by Western Blotting RAW264.7 cells protein lysis was collected and the concentration of protein was measured after centrifugation at 12,000 rpm for 10 min at 4 • C. Then, the sample proteins (20 µg) were electrophoresed and separated in 10% polyacrylamide gel prior to being transferred onto PVDF membranes. After transferring the target proteins to PVDF membranes, the membranes were blocked with 5% BSA in TBST for 1 h, then incubated with anti-NF-κB p65 (dilution, 1:1000; CST), anti-NF-κB p-p65 (dilution, 1:1000; CST) and anti-actin (dilution, 1:5000; CST) at 4 • C for overnight [32]. Finally, the PVDF membranes were incubated with the anti-mouse or anti-rabbit IgG for 2 h at room temperature and detected by the Two-color Infrared Laser Imaging System (LI-COR Odyssey Clx).

Statistical Analysis
The results were analyzed by one-way analysis of variance (ANOVA), followed by a post hoc test using the SPSS 20.0 software (IBM Corp., Armonk, NY, USA). The data are the mean ± SD of three independent experiments. p < 0.05 was considered significant.
Supplementary Materials: The Supplementary Materials are available online. Figure S1: The mass spectra of six flavonoids in Smilax glabra flavonoids extract (SGF). Figure S2: The 1 H NMR spectra of six flavonoids in Smilax glabra flavonoids extract (SGF). Figure S3: The 13 C NMR spectra of six flavonoids in Smilax glabra flavonoids extract (SGF). Figure S4: The total ion current (TIC) of Smilax glabra flavonoids extract (SGF).
Author Contributions: X.Z. (Xinyu Zhao) collated data, designed study and wrote original draft. D.X. put forward a concept, supervised the experiment, reviewed and edited. R.C. designed study and experimentalized. X.Z. (Xiaoxi Zhang) provided technical support and provide technical support. Y.S. and C.T. experimentalized and designed study. All authors have read and agreed to the published version of the manuscript.