Inhibition of the NLRP3 Inflammasome by a Quercus Serrata Extract and Isolation of the Component Compounds for the Treatment of Arthritis

Quercus serrata belongs to the Fagaceae family. There are 600 known species of Quercus worldwide. Q. serrata is distributed nationally in Korea, Japan, and China and grows to a height of 10–15 m. It exhibits a light grey bark with longitudinal furrows; the leaves are 6–12 cm long and 2.5–5 cm wide. The Quercus genus reportedly exhibits several types of bioactivity, including antioxidant, anti-inflammatory, antifungal, antimicrobial, and anticancer activity. Additionally, it has been reported that Quercus produces diverse phytochemicals, including tannins, flavonoids, and triterpenoids. Herein, we describe the column chromatographic isolation of five compounds from a Q. serrata extract. The compounds included caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5). We assessed the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity, antioxidant activity, NLR family pyrin domain-containing 3 (NLRP3) inflammasome (including NLRP3, ASC, and caspase-1) inhibitory effects, and collagenase inhibition activity of the Q. serrata extract and its constituent compounds. Our results indicated that the Q. serrata extract and the isolated constituent compounds showed inhibitory activity with reference to nitric oxide production, inflammasome component expression, and collagenase activity. Our findings imply that the Q. serrata extract and the isolated constituent compounds are potential candidates for the treatment of inflammatory diseases such as arthritis.


Introduction
Te Quercus genus is part of the Fagaceae family, with 600 known species worldwide. Several variants of this species exist in Korea, including Q. acutissima, Q. variabilis, Q. dentata, Q. aliena, Q. mongolica, Q. serrata, Q. acuta, Q. glauca, Q. myrsinifolia, Q. salicina, and Q. gilva. Te species Q. serrata is widely distributed throughout Korea, Japan, and China and can reach up to 10-15 m in height. Te bark is light grey in color with longitudinal furrows. Te leaves are 6-12 cm long and 2.5-5 cm wide, with light grey elongated oval patterns on the underside [1]. Te Quercus genus reportedly exhibits several types of bioactivity, including antioxidant [2,3], anti-infammatory [4][5][6][7], antifungal [8][9][10], antidiabetic [11,12], antimicrobial [13,14], anticancer [15], anti-hepatoprotective [16], antibacterial [17], and anti-urolithiatic [18] activities. Phytochemical studies of Quercus spp. revealed that it produces favonoids [4,5,19], triterpenoids [20,21], and tannins [22][23][24]. Natural products have historically contributed to the treatment of various diseases. Scientifc developments in recent years have demonstrated the applicability of natural products in various felds, and this has led to increased scientifc interest in such products [25][26][27][28][29]. Several natural products have been shown to exert infammasome inhibitory activity [30,31]. Consequently, there has been an increasing amount of research into the use of natural products as pharmacotherapeutics and nutraceuticals for cancer, gastrointestinal disorders, nociceptive pain, and anxiety disorders; they have also been explored for application such as aphrodisiacs [32][33][34][35][36][37][38]. In this respect, the infammasome inhibitory profle of natural products is an interesting property that can be applied to develop phytochemical treatments for arthritis. However, studies investigating the application of natural products for arthritis treatment via the modulation of the NLR family pyrin domain-containing 3 (NLRP3) infammasome have not yielded convincing evidence, and studies on Q. serrata in this context are especially rare. We previously investigated the use of a Quercus spp. Extract as a therapeutic agent for infammatory diseases by evaluating nitric oxide (NO) production and proinfammatory cytokine levels [18] determined that such extracts can be applied for treating infammatory diseases such as arthritis. Infammasomes are cytosolic multi-protein complexes comprising a nucleotide-binding domain and leucine-rich repeat-containing proteins (NLRs), caspase-1, and the adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Infammasomes activate infammatory responses and may trigger the maturation of proinfammatory cytokines such as interleukin (IL)-18 and IL-1β [39]. Te NLRP3 infammasome contains the NLRP3 sensor, an ASC adaptor, and caspase-1 protease. Following its assembly into large cytoplasmic complexes and caspase-1 activation, NLRP3 catalyzes the maturation and secretion of IL-18 and IL-1β. In addition to cytokine production, NLRP3 infammasome activation may also facilitate pyroptosis (caspase-1-mediated rapid cell death) [40,41]. Infammasomes can cause infammatory disorders such as arthritis, multiple sclerosis, Alzheimer's disease, atherosclerosis, type 2 diabetes, and systemic lupus erythematosus [42]. Arthritis is a chronic infammatory disease that is caused by diverse triggers. Numerous studies have sought to identify the correlation between arthritis and infammasomes. Te levels of NLRP3, ASC, and caspase-1 increased in both arthritic rats and patients with arthritis [43]. In this study, we demonstrate that a Q. serrata extract and its constituent compounds can downregulate the expression of the NLRP3 infammasome proteins and show activity as antioxidants, anti-infammatory agents, and collagenase inhibitors. Based on this bioactivity profle, we propose that this extract and its constituent compounds can potentially be applied for the treatment of infammatory diseases such as arthritis. A pre-coated silica gel 60 F254 plate (Merck, Darmstadt, Germany) was used to perform thin-layer chromatography (TLC) with a mixture of chloroform, methanol, and distilled water (6 : 4:1, volume ratio) and a mixture of benzene, ethyl formate, and formic acid (1 : 7:2, volume ratio). A 10% solution of sulfuric acid (H 2 SO 4 ) or anisaldehyde-H 2 SO 4 was heated and sprayed with ferric chloride (FeCl 3 ) to determine the location of the stains on the chromatograph.

Materials and Methods
To clarify the structure of the isolated compounds, 1Dnuclear magnetic resonance (NMR) analyses such as 1H-(600 MHz) and 13 C-(150 MHz) NMR were performed and recorded using JEOL (JEOL, Massachusetts, USA) at Chung-Ang University.

High-Performance Liquid Chromatography (HPLC)
Analysis. Te constituents of the Q. serrata extract were analyzed by HPLC. Te mobile phase consisted of solvent A (0.2% acetic acid in H 2 O) and solvent B (acetonitrile, ACN) ( Table 1). Te Q. serrata extract and the isolated constituent compounds were dissolved in 100% MeOH.

DPPH Radical Scavenging Assay.
A DPPH radical scavenging assay was used to evaluate antioxidant activity. A 20 μL aliquot of each sample was dissolved in anhydrous ethanol and added to 180 μL of 0.2 mM DPPH (Sigma-Aldrich, St. Louis, MO, USA) solution. Te mixture was incubated for 37°C, 30 min, and the absorbance was measured at 517 nm using an enzyme-linked immunosorbent assay reader (Tecan Co., Ltd., Salzburg, Austria). Te DPPH free radical scavenging activity was calculated in terms of percent inhibition (%). Half-maximal inhibitory concentration (IC 50 ) values were defned as the concentrations that could scavenge 50% of the DPPH free radicals. L-ascorbic acid was used as the positive control. Inhibitory activity was calculated as follows:

Cell Viability Assay.
Te cytotoxicity of the test extract/ compounds was determined prior to the biological assay based on the mitochondria-dependent reduction of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) (Sigma-Aldrich) to formazan. RAW 264.7 cells (2 × 105 cells/well) were seeded in a 96-well plate and incubated at 37°C for 16 h, treated with 20 μL of serum-free DMEM, and incubated for 24 h at 37°C. Te medium was then removed, and 100 μL of MTT solution (0.5 mg/mL) was added to each well. After 4 h of incubation, the supernatant was aspirated. Finally, 100 μL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorbance at 540 nm was measured using an ELISA reader (Tecan Co., Ltd.). Relative cell viability was evaluated based on the quantity of MTT converted to the insoluble formazan salt. Distilled water served as the control sample.
Te optical density of the formazan generated in the control cells was considered to represent 100% viability. Te data are expressed as the mean percentage of viable cells versus the control.
2.8. NO Production Assay. RAW 264.7 murine macrophage cells (2 × 105 cells/well) were seeded in 96-well plates and pre-incubated for 16 h at 37°C in a humidifed atmosphere (with approximately 5% CO 2 ). Te cells were then incubated in serum-free medium containing the test compound/extract and 1 μg/mL of lipopolysaccharide (LPS) (Sigma-Aldrich). After incubating for an additional 24 h, the NO content was evaluated using a Griess assay. For the Griess assay, 100 μL of Griess reagent (Sigma-Aldrich) was added to 100 μL of supernatant from the treated cells. Te absorbance of the samples was recorded at 540 nm [44]. NG-monomethyl-L-arginine monoacetate salt (L-NMMA) was used as a positive control. Te IC50 value was defned as the concentration that could inhibit 50% of NO production. Te inhibitory activity with reference to NO production was calculated as follows: Collagenase assay was conducted using a previously described procedure [45]. Collagenase (5 μg) and 4-phenylazobenzyloxycarbonyl-L-Pro-L-Les-Gly-L-Pro-D-Arg (PZ-peptide, 0.5 mg) (a substrate of collagenase) were incubated with or without samples in 0.1 M Tris bufer (pH 7.4) at 37°C for 30 min (total volume 1.7 mL).
To terminate the enzyme reaction, 1 mL of 25 mM citric acid solution was added. After mixing with 5 mL of ethyl acetate, the absorbance of the organic layer was measured at 320 nm. Epigallocatechin gallate (EGCG) was used as the positive control. Te inhibitory activity was calculated as follows: 2.11. Statistical Analysis. All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) and the Student-Newman-Keuls test were performed using the Statistical Package for Social Sciences (SPSS 24) software. Te resulting values were considered signifcantly diferent at p < 0.05.

Compound 2.
2 was a brown powder. A black spot was detected using FeCl 3 , and a yellow spot was detected by spraying the TLC plate with 10% H 2 SO 4 followed by heating. By comparing the results with data from previous literature [47,48], the structure of 2 was identifed as myricetin-3-O-cellobioside.

Compound 3.
3 was a brown powder. A grey spot was detected by spraying the TLC plate with 10% H 2 SO 4 followed by heating, and a dark blue spot was detected using FeCl 3 .
By comparing our results with those from previous literature [49], the structure of 3 was identifed as phloroglucinol.
3.1.4. Compound 4. 4 was a brown powder. A dark pink spot was detected by spraying the TLC plate with 10% H 2 SO 4 followed by heating, and a dark blue spot was detected using FeCl 3 .
Te 1H-NMR of 4 showed duplicated signals below 5.34 ppm; thus, it is possible that 4 may have two isomers on an anomeric center on the sugar. In addition, the 1H-NMR By comparing these results to NMR data from previous literature [50], the structure of 4 was identifed as (S)-2,3-HHDP-D-glucopyranoside.
3.1.5. Compound 5. 5 was a brown amorphous powder. A brown spot was detected by spraying the TLC plate with 10% H 2 SO 4 followed by heating, and a dark blue spot was detected using FeCl 3 .
Anomeric proton signals below 5.45 ppm were observed on the 1H-NMR spectrum of 5, and all signals were duplicated. Tus, 5 is thought to be a mixture of two isomers caused by unacylated anomeric centers on a sugar. Te 1H-NMR spectrum also revealed two HHDP moieties (δ 6.64, 6.63, 6.57, 6.57, 6.53, 6.48, 6.30, 6.29 (8H in total, each s, HHDP-H)) in the aromatic region and a glucose core with 4C1 conformation (δ 3.74-5.45 (large coupling constants)) in the sugar region.
By comparing these results with data from previous literature [51], the structure of 5 was identifed as pedunculagin.

HPLC Analysis.
Contents analysis of the Q. serrata extract was conducted using HPLC. Te composition of the extract was found to be as follows: compound 1 0.04%, compound 2 0.32%, compound 3 0.22%, compound 4 6.22%, and compound 5 16.56%. Te results showed that the major component was compound b, which comprised 16.56% of the QS extract (Figures 2 and 3, Table 2).
Te antioxidant ability of the Q. serrata extract and its component compounds (1)(2)(3)(4)(5) was evaluated using the DPPH radical scavenging activity. Te DPPH free radical is deep purple in color and absorbs light at 517 nm. It loses electrons upon reacting with antioxidants, resulting in a reduced form, which is yellow in color. Te reaction is thus monitored by measuring the absorbance at 517 nm.

MTT Cell Viability Assay.
Te efect of the Q. serrata extract on cell viability and its cytotoxic activity were evaluated by the mitochondria-dependent reduction in MTT. When MTT is processed in living cells, it is reduced by the reductase in the mitochondria, forming formazan crystals. Te formation of formazan indicates a lack of cytotoxicity.

Evidence-Based Complementary and Alternative Medicine
Te MTT assay was performed with the Q. serrata extract at concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 μg/mL in RAW 264.7 cells. Te cell viability was maintained at >80% at all concentrations of the extract. Tese results demonstrate that the inhibition of NO production and the production of infammatory molecular products due to treatment with Q. serrata do not cause cytotoxicity ( Figure 6).

Inhibition of NO Production.
Te anti-infammatory activity of the Q. serrata extract and the isolated component compounds 1-5 was evaluated based on the inhibition of NO production in RAW 246.7 macrophage cells.
NO is biosynthesized from L-arginine by three nitric oxide synthase (NOS) enzyme isoforms (nNOS, eNOS, and iNOS). NO regulates the cellular and toxic responses. However, excessive NO production is considered to enhance tumor development and DNA methylation. Tus, the inhibition of infammatory response-related NO production might be a useful therapeutic and prophylactic method in arthritis.

Inhibitory Efects on Infammasome Protein Expression.
Te inhibitory efects of the Q. serrata extract and ellagitannin (pedunculagin, compound 5) on the expression of the components of the NLRP3 infammasome (NLRP3, ASC, and caspase-1) in RAW 264.7 cells were evaluated using Western blot analysis. Te NLRP3 complex is considered important as a treatment target for infammatory diseases because it controls infammatory cytokine levels.
Te expression of NLRP3 in LPS-treated cells incubated with the Q. serrata extract was lower than that in the control group, indicating that the extract inhibited NLRP3 protein expression. Similarly, compound 5 also inhibited the expression of NLRP3, caspase-1, and ASC (level compared with that in the control group) (Figures 9-12).

Inhibition of collagenase.
Te inhibitory efects of the Q. serrata extract and ellagitannin (pedunculagin, compound 5) on collagenase activity were evaluated. Te cartilage tissue associated with arthritis includes collagen and various proteins. Collagen plays an important role in joints, and collagenase is the key enzyme that cleaves and breaks down collagen.

Discussion
We aim to evaluate an extract made from Q. serrata with reference to infammasome inhibition, antioxidant, antiinfammatory, and collagenase inhibition activities.
Infammasomes are multi-protein complexes that regulate the secretion of infammatory cytokines such as IL-1 and IL-18. Tey are known to be key mediators of infammation and immunity. When infammatory cytokines are overexpressed due to infammasome regulatory disorders, pyroptosis (a type of apoptosis) is induced, and various chronic infammatory diseases such as diabetes, infammatory bowel disease, arthritis, and prostate hypertrophy (please add a space between the concentration numeral and the "uM" in the X-axis labels) occur [39][40][41][42]. Recently, various types of infammasomes have been studied, of which the NLRP3 complex is considered very important as a treatment target for infammatory diseases. Tis is because this complex regulates infammatory cytokine production and secretion. As mentioned earlier, the NLRP3 infammasome consists of a sensor (NLRP3), an adaptor (ASC), and an efector (caspase-1). Upon stimulation, NLRP3 oligomerizes and recruits ASC. Natural plant products with antioxidant and anti-infammatory activity are expected to contribute to the regulation of infammasome activity, and infammasomerelated studies have been reported on natural products [52][53][54][55][56]. Terefore, we previously investigated the use of a natural product from Quercus spp. As a therapeutic agent for infammatory diseases by evaluating the production of NO and proinfammatory cytokines [18], the results of our previous study demonstrated that Q. serrata extract can potentially be applied for the treatment of infammatory diseases such as arthritis treatment.
Our results in this study demonstrated that the Q. serrata extract evaluated here, as well as compounds 1, 4, and 5 exhibited good antioxidant ability; the major component, compound 5, was most potent in this regard. In addition, the Q. serrata extract and compounds 4 and 5 exerted antiinfammatory efects. Based on these results, we evaluated whether the Q. serrata extract and compound 5 were able to inhibit the expression of the components of the NLRP3 infammasome. Both the extract and compound 5 suppressed the expression of the NLRP3 infammasome components. Western blot analysis of the NLRP3 components revealed that the suppression of the protein expression of all the three components (NLRP3, caspase-1, and ASC) by the extract and compound 5 was clearly concentration-dependent. Te extract and compound 5 also showed potent antioxidant and anti-infammatory abilities. Inhibition of collagenase activity is assessed to evaluate whether the test compound can potentially be applied for arthritis treatment. In our study, both the Q. serrata extract and compound 5 showed a potent collagenase inhibitory activity. Tere has been increased interest regarding the role of the infammasome in arthritis. Increased secretion of NLRP3, ASC, and caspase-1 has been reported in arthritis patients, and  Evidence-Based Complementary and Alternative Medicine 7 suppression of NLRP3 activity was reported to suppress arthritis in rat models [43]. In addition, higher levels of NLRP3 and caspase-1 were observed in patients with rheumatoid arthritis than in patients with degenerative arthritis, and it was found that the administration of selective NLRP3 inhibitors reduced redness and cartilage degradation [57]. On the other hand, it has been reported that controlling the activity of NLRP3 is a good strategy for the treatment of wrist ligament injury [58]. Based on the above literature reports, it is clear that a drug that regulates the infammasome will be benefcial in arthritis. Terefore, targeting the NLRP3 infammasome may provide new therapeutic strategies for treating arthritis. In this study, we employed the DPPH radical scavenging activity, NO production inhibitory activity assay, and NLRP3 infammasome component expression assay to comprehensively evaluate the

Conclusions
In this study, fve compounds (1-5) were isolated from Q. serrata, namely cafeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5). Content analysis showed that pedunculagin (5) was the main constituent of this extract. Te antioxidant activity of the extract and the component compounds was evaluated by measuring DPPH radical scavenging activity. Te Q. serrata extract and the isolated compounds (1-5) exhibited increased DPPH radical scavenging activity, and compound 5 performed the best in this assay. Te anti-infammatory activities were evaluated based on the inhibition of NO production. Te Q. serrata extract and compounds 4 and 5 showed potential NO inhibitory activity. Te Q. serrata extract and compound 5 potently inhibited the protein expression of the components of the NLRP3 infammasome, including NLRP3, ASC, and caspase-1, further confrming their anti-infammatory nature. Te Q. serrata extract and compound 5 also showed potent collagenase inhibitory activity. Te present results suggest that the Q. serrata extract and compound 5 are promising candidates for treating infammatory diseases such as arthritis. However, further in vivo studies in arthritis animal models are required to confrm these fndings.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interest regarding the publication of this study.