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

Seonu, Seo Yeon; Kim, Min Ji; Lee, Min Won

doi:https://doi.org/10.1155/2022/4428269

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Conflicts of Interest Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2022 | Article ID 4428269 | https://doi.org/10.1155/2022/4428269

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

Seo Yeon Seonu,¹Min Ji Kim,¹and Min Won Lee¹

Academic Editor: Talha Bin Emran

Received12 Oct 2021

Revised16 May 2022

Accepted19 May 2022

Published28 Dec 2022

Abstract

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.

1. Introduction

The 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. The species Q. serrata is widely distributed throughout Korea, Japan, and China and can reach up to 10–15 m in height. The bark is light grey in color with longitudinal furrows. The leaves are 6–12 cm long and 2.5–5 cm wide, with light grey elongated oval patterns on the underside [1]. The Quercus genus reportedly exhibits several types of bioactivity, including antioxidant [2, 3], anti-inflammatory [4–7], antifungal [8–10], 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 flavonoids [4, 5, 19], triterpenoids [20, 21], and tannins [22–24]. Natural products have historically contributed to the treatment of various diseases. Scientific developments in recent years have demonstrated the applicability of natural products in various fields, and this has led to increased scientific interest in such products [25–29]. Several natural products have been shown to exert inflammasome 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 [32–38]. In this respect, the inflammasome inhibitory profile 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) inflammasome 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 inflammatory diseases by evaluating nitric oxide (NO) production and proinflammatory cytokine levels [18] determined that such extracts can be applied for treating inflammatory diseases such as arthritis. Inflammasomes 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). Inflammasomes activate inflammatory responses and may trigger the maturation of proinflammatory cytokines such as interleukin (IL)-18 and IL-1β [39]. The NLRP3 inflammasome 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 inflammasome activation may also facilitate pyroptosis (caspase-1-mediated rapid cell death) [40, 41]. Inflammasomes can cause inflammatory disorders such as arthritis, multiple sclerosis, Alzheimer’s disease, atherosclerosis, type 2 diabetes, and systemic lupus erythematosus [42]. Arthritis is a chronic inflammatory disease that is caused by diverse triggers. Numerous studies have sought to identify the correlation between arthritis and inflammasomes. The 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 inflammasome proteins and show activity as antioxidants, anti-inflammatory agents, and collagenase inhibitors. Based on this bioactivity profile, we propose that this extract and its constituent compounds can potentially be applied for the treatment of inflammatory diseases such as arthritis.

2. Materials and Methods

2.1. Plant Materials

Q. serrata leaves were collected from Pocheon, Republic of Korea, in May 2020. The species was identified by Dr. Sung Sik Kim. A voucher specimen was stored at the Herbarium of the College of Pharmacy, Chung-Ang University.

2.2. General Experimental Procedure

Column chromatography was conducted using MCI gel CHP 20P (75–150 μm, Mitsubishi Chemical, Tokyo, Japan), ODS-B gel (40–60 μm, Daiso, Osaka, Japan), and Sephadex LH-20 (10–25 μm, GE Healthcare Bio-Science AB, Uppsala, Sweden).

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 (H₂SO₄) or anisaldehyde-H₂SO₄ was heated and sprayed with ferric chloride (FeCl₃) to determine the location of the stains on the chromatograph.

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

2.3. Extraction and Isolation

Extracts were obtained from 2.2 kg of Q. serrata leaves using 80% acetone at room temperature. Such extracts were obtained three times over the course of three days. The extracts were concentrated by removing the acetone under vacuum, and this procedure yielded 350.12 g of concentrated extract. The Q. serrata extract (300 g) was then filtered using Celite 545, and the remaining extract was stored at -20°C. The filtered Q. serrata extract was loaded onto a Sephadex LH-20 chromatography column with a methanol: water (MeOH : H₂O) gradient solvent system (ratio between 0 : 10 and 10 : 0) and yielded 12 subfractions (Q. serrata-1–12).

The fractionation of Q. serrata-8 (44.97 g) during repeated column chromatography using the MCI gel CHP 20P with a gradient solvent system of MeOH : H₂O (from 0 : 10 to 10 : 0) yielded 24 subfractions. The Q. serrata-8-12 subfractions (494.9 mg) were loaded onto octadecyl silica (ODS) gel with a gradient solvent system of MeOH : H₂O (from 0 : 10 to 10 : 0) and yielded compounds 1 (caffeic acid, 34.5 mg) and 2 (myricetin-3-O-cellobioside, 52.8 mg).

The fractionation of Q. serrata-7 (7.7 g) during repeated column chromatography using the MCI gel with a gradient solvent system of MeOH : H₂O (from 0 : 10 to 10 : 0) yielded compound 3 (phloroglucinol, 94.8 mg).

Q. serrata-9 (17.01 g) was loaded onto an MCI gel CHP 20P column with a gradient solvent system including MeOH : H₂O (from 0 : 10 to 10 : 0) and produced 22 subfractions. Q. serrata-9-5 yielded four subfractions when loaded onto an ODS column with a gradient solvent system including MeOH : H₂O (from 0 : 10 to 10 : 0). Column chromatography was repeated for Q. serrata-9-5-1 (1.55 g) using Sephadex LH-20 with a gradient solvent system including MeOH : H₂O (from 0 : 10 to 10 : 0), yielding compounds 4 ((S)-2,3-HHDP-D-glucopyranoside, 21.3 mg) and 5 (pedunculagin, 897.1 mg).

2.4. High-Performance Liquid Chromatography (HPLC) Analysis

The constituents of the Q. serrata extract were analyzed by HPLC. The mobile phase consisted of solvent A (0.2% acetic acid in H₂O) and solvent B (acetonitrile, ACN) (Table 1). The Q. serrata extract and the isolated constituent compounds were dissolved in 100% MeOH.

2.5. 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. The 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). The DPPH free radical scavenging activity was calculated in terms of percent inhibition (%). Half-maximal inhibitory concentration (IC₅₀) values were defined 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:

2.6. Cell Culture

RAW 264.7 cells were purchased from the Korean Cell Line Bank. The cells were grown at 37°C in a humidified atmosphere (with approximately 5% CO₂) in Dulbecco’s modified Eagle’s medium (DMEM) (Corning, NY, USA) supplemented with 10% fetal bovine serum (Welgene, Gyeongsangbuk-do, Korea), 100 IU/mL penicillin G, and 100 mg/mL streptomycin (Gibco BRL, Grand Island, NY, USA).

2.7. Cell Viability Assay

The cytotoxicity of the test extract/compounds was determined prior to the biological assay based on the mitochondria-dependent reduction of 3-(4,5-dimethylthiazol-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. The 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. The optical density of the formazan generated in the control cells was considered to represent 100% viability. The 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 humidified atmosphere (with approximately 5% CO₂). The 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. The absorbance of the samples was recorded at 540 nm [44]. NG-monomethyl-L-arginine monoacetate salt (L-NMMA) was used as a positive control. The IC50 value was defined as the concentration that could inhibit 50% of NO production. The inhibitory activity with reference to NO production was calculated as follows:

2.9. Western Blot Analysis

RAW 264.7 macrophage cells (1 × 106 cells/well) were pre-incubated for 16 h at 37°C and treated with 1 μg/mL of LPS for 24 h. The treated cells were harvested and washed twice with phosphate-buffered saline. Cell lysates were then prepared using radio-immunoprecipitation assay buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, and 1 mM ethylenediaminetetraacetic acid) (Thermo Fisher Scientific, MA, USA). The cell lysates were centrifuged at 13,000 rpm for 15 min at 4°C, and the supernatants (protein: 60 μg) were electrophoresed using SDS-polyacrylamide gels (10% and 12%). The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, CA, USA). The membrane was blocked using a blocking buffer (Thermo Fisher Scientific) for 30 min at room temperature. Then, the membrane was incubated overnight at 4°C with antibodies NLRP3 (1 : 1000; Cell Signaling, MA, USA), ASC (1 : 100; Santa Cruz, CA, USA), and caspase-1 (1 : 1000; Cell Signaling). After washing the membranes with Tris-buffered saline-Tween (TBS-T) (Bio-Rad, CA, USA) three times, they were incubated with horseradish peroxidase (HRP)-linked anti-rabbit IgG secondary antibody (1 : 3000; Cell Signaling) and m-IgGκ binding protein (BP)-HRP (1 : 1000; Santa Cruz) for 1 h at room temperature. The bands were visualized using a LAS-500 luminescent image analyzer (GE Healthcare Life Sciences, NJ, USA) after treating the membrane with ECL detection reagent (GE Healthcare Life Sciences).

2.10. Collagenase Assay

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 buffer (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. The inhibitory activity was calculated as follows:O.D. control: O.D. of control with collagenase - O.D. of control without collagenase O.D. sample: O.D. in the presence of test sample with collagenase - O.D. in the presence of test sample without collagenase.

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. The resulting values were considered significantly different at .

3. Results

3.1. Isolation and Structural Identification of the Compounds

Chromatographic analysis of the Q. serrata extract using Sephadex LH-20, MCI gel CHP–20P, and ODS-B gel yielded five compounds (1–5). These compounds were identified as caffeic acid (1), myricetin-3-O-cellobioside (2), phloroglucinol (3), (S)-2,3-HHDP-D-glucopyranoside (4), and pedunculagin (5) based on instrumental analysis and comparisons with references (Figure 1). This is the first time these compounds (1–5) have been isolated from Q. serrata. ¹H and ¹³C-NMR spectra of the compounds (1–5) are included in Supplementary Material as Figures S1–S5.

3.1.1. Compound 1

1 was a brown powder. A purple spot was detected by spraying the TLC plate with 10% H₂SO₄ followed by heating, and a black spot was detected using FeCl₃.

The 1H-NMR spectrum of 1 revealed ABX-type protons (δ 7.12 (1H, d, J = 1.8 Hz, H-2), 6.95 (1H, dd, J = 7.8, 1.8 Hz, H-6), 6.82 (1H, d, J = 7.8 Hz, H-5)) and two doublet signals with high J values, indicating trans-type protons (δ 7.48 (1H, d, J = 16.2 Hz, H-7), 6.21 (1H, d, J = 16.2 Hz, H-8)).

By comparing the spectral data with those from previous literature [46], the structure of 1 was identified as caffeic acid.

3.1.2. Compound 2

2 was a brown powder. A black spot was detected using FeCl₃, and a yellow spot was detected by spraying the TLC plate with 10% H₂SO₄ followed by heating.

The 1H-NMR spectrum of 2 revealed a myricetin moiety (δ 7.15 (2H, s, H-2′, 6′), 6.34 (1H, m, H-8), 6.16 (1H, m, H-6)) and two anomeric proton signals (δ5.43 (1H, d, J = 7.8 Hz, H-1″) and 5.31 (1H, d, J = 7.2 Hz, H-1‴)).

The 13C-NMR spectrum of 2 also showed myricetin moiety (δ 177.92 (C-4), 164.62 (C-7), 161.76 (C-5), 156.78 (C-9), 156.70 (C-2), 145.89 (C-3′, 5′), 137.16 (C-4′), 134.00 (C-3), 120.56 (C-1′), 109.05 (C-2′, 6′), 104.48 (C-10), 99.15 (C-6), and 93.88 (C-8)). The presence of a 3-O linkage was indicated by a 9.89 ppm downshift of C-2 (δ 156.70) and a 2.18 ppm upshift of C-3 (δ 134.00). A cellobiose moiety (δ 102.52 (C-1‴), 101.38 (C-1″), 78.13 (C-4″), 77.08 (C-5‴), 76.44 (C-3‴), 74.44 (C-2‴), 73.79 (C-3″), 71.72 (C-2″), 70.42 (C-5″), 68.52 (C-4‴), 61.59 (C-6‴), and 60.56 (C-6″)) was observed with a 9 ppm downshift of C-1’’; this indicated that C-1″ has a linkage with the 3-hydroxyl group of myricetin.

By comparing the results with data from previous literature [47, 48], the structure of 2 was identified as myricetin-3-O-cellobioside.

3.1.3. Compound 3

3 was a brown powder. A grey spot was detected by spraying the TLC plate with 10% H₂SO₄ followed by heating, and a dark blue spot was detected using FeCl₃.

The 1H-NMR spectrum of 3 showed only one signal at δ 6.87, and the 13C-NMR spectrum showed two signals (δ 145.90 (C-1, 3, 5) and 109.27 (C-2, 4, 6)).

By comparing our results with those from previous literature [49], the structure of 3 was identified 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% H₂SO₄ followed by heating, and a dark blue spot was detected using FeCl₃.

The 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 spectrum of 4 revealed one hexahydroxydiphenoyl (HHDP) signal (δ 6.65, 6.64, 6.56, 6.55 (4H in total, s, HHDP-H)) in the aromatic region and sugar protons existing as α and β forms in the sugar region. The 13C-NMR spectrum of 4 revealed four signals in the aromatic region indicating–COO (δ 169.97, 169.94, 169.40, 169.20) and included HHDP and two isomer sugar forms in the sugar region (δ 93.92, 90.58, 79.89, 77.58, 77.17, 77.07, 74.82, 71.96, 67.43, 67.21, 61.00, 60.85).

By comparing these results to NMR data from previous literature [50], the structure of 4 was identified 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% H₂SO₄ followed by heating, and a dark blue spot was detected using FeCl₃.

Anomeric proton signals below 5.45 ppm were observed on the 1H-NMR spectrum of 5, and all signals were duplicated. Thus, 5 is thought to be a mixture of two isomers caused by unacylated anomeric centers on a sugar. The 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 identified as pedunculagin.

3.2. HPLC Analysis

Contents analysis of the Q. serrata extract was conducted using HPLC. The 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%. The results showed that the major component was compound b, which comprised 16.56% of the QS extract (Figures 2 and 3, Table 2).

The antioxidant ability of the Q. serrata extract and its component compounds (1–5) was evaluated using the DPPH radical scavenging activity. The 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. The reaction is thus monitored by measuring the absorbance at 517 nm.

The IC₅₀ of the test compounds was calculated by plotting the inhibitory activity (%) in the DPPH free radical scavenging assay against the concentration. A low IC₅₀ value indicates a high antioxidant activity. The Q. serrata extract (IC₅₀ = 65.84 ± 5.79 μg/mL) demonstrated superior radical scavenging activity compared with ascorbic acid (IC₅₀ = 11.20 ± 5.97 μg/mL) (Figure 4, Table 3). Compounds 1, 4, and 5 also exhibited potent radical scavenging activity compared with ascorbic acid (IC₅₀ = 51.07 ± 1.47 μM). In particular, compound 5 (IC₅₀ = 24.18 ± 1.06 μM) exhibited the strongest activity compared with ascorbic acid (Figure 5, Table 4).

3.3. MTT Cell Viability Assay

The effect 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. The formation of formazan indicates a lack of cytotoxicity.

The 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. The cell viability was maintained at >80% at all concentrations of the extract. These results demonstrate that the inhibition of NO production and the production of inflammatory molecular products due to treatment with Q. serrata do not cause cytotoxicity (Figure 6).

3.4. Inhibition of NO Production

The anti-inflammatory 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. Thus, the inhibition of inflammatory response-related NO production might be a useful therapeutic and prophylactic method in arthritis.

The Q. serrata extract showed a potent inhibitory effect on NO production (IC₅₀ = 6.32 ± 0.19 μg/mL); the activity was comparable to that of the positive control L-NMMA (IC₅₀ = 5.10 ± 0.14 μg/mL) (Figure 7, Table 5). Compound 4 (117.66 ± 2.63 μM) and compound 5 (111.79 ± 2.52 μM) showed good inhibition of NO production compared with that of the other compounds (Figure 8, Table 6).

3.5. Inhibitory Effects on Inflammasome Protein Expression

The inhibitory effects of the Q. serrata extract and ellagitannin (pedunculagin, compound 5) on the expression of the components of the NLRP3 inflammasome (NLRP3, ASC, and caspase-1) in RAW 264.7 cells were evaluated using Western blot analysis. The NLRP3 complex is considered important as a treatment target for inflammatory diseases because it controls inflammatory cytokine levels.

The 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).

3.6. Inhibition of collagenase

The inhibitory effects of the Q. serrata extract and ellagitannin (pedunculagin, compound 5) on collagenase activity were evaluated. The 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.

Our results indicated that the Q. serrata extract (99.68%) and compound 5 (57.52%) showed potent inhibitory activity against collagenase compared with the positive control, EGCG (85.30%), at 25 μM (Tables 7, 8 and 9).

4. Discussion

We aim to evaluate an extract made from Q. serrata with reference to inflammasome inhibition, antioxidant, anti-inflammatory, and collagenase inhibition activities. Inflammasomes are multi-protein complexes that regulate the secretion of inflammatory cytokines such as IL-1 and IL-18. They are known to be key mediators of inflammation and immunity. When inflammatory cytokines are overexpressed due to inflammasome regulatory disorders, pyroptosis (a type of apoptosis) is induced, and various chronic inflammatory diseases such as diabetes, inflammatory bowel disease, arthritis, and prostate hypertrophy (please add a space between the concentration numeral and the “uM” in the X-axis labels) occur [39–42]. Recently, various types of inflammasomes have been studied, of which the NLRP3 complex is considered very important as a treatment target for inflammatory diseases. This is because this complex regulates inflammatory cytokine production and secretion. As mentioned earlier, the NLRP3 inflammasome consists of a sensor (NLRP3), an adaptor (ASC), and an effector (caspase-1). Upon stimulation, NLRP3 oligomerizes and recruits ASC. Natural plant products with antioxidant and anti-inflammatory activity are expected to contribute to the regulation of inflammasome activity, and inflammasome-related studies have been reported on natural products [52–56]. Therefore, we previously investigated the use of a natural product from Quercus spp. As a therapeutic agent for inflammatory diseases by evaluating the production of NO and proinflammatory cytokines [18], the results of our previous study demonstrated that Q. serrata extract can potentially be applied for the treatment of inflammatory 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 anti-inflammatory effects. 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 inflammasome. Both the extract and compound 5 suppressed the expression of the NLRP3 inflammasome 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. The extract and compound 5 also showed potent antioxidant and anti-inflammatory 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. There has been increased interest regarding the role of the inflammasome in arthritis. Increased secretion of NLRP3, ASC, and caspase-1 has been reported in arthritis patients, and 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 inflammasome will be beneficial in arthritis. Therefore, targeting the NLRP3 inflammasome 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 inflammasome component expression assay to comprehensively evaluate the bioactivity profile of the Q. serrata extract and its component compounds. Our results showed that the QS extract and its component compounds may have therapeutic benefits towards inflammatory diseases such as arthritis.

5. Conclusions

In this study, five compounds (1–5) were isolated from Q. serrata, namely caffeic 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. The antioxidant activity of the extract and the component compounds was evaluated by measuring DPPH radical scavenging activity. The Q. serrata extract and the isolated compounds (1–5) exhibited increased DPPH radical scavenging activity, and compound 5 performed the best in this assay. The anti-inflammatory activities were evaluated based on the inhibition of NO production. The Q. serrata extract and compounds 4 and 5 showed potential NO inhibitory activity. The Q. serrata extract and compound 5 potently inhibited the protein expression of the components of the NLRP3 inflammasome, including NLRP3, ASC, and caspase-1, further confirming their anti-inflammatory nature. The Q. serrata extract and compound 5 also showed potent collagenase inhibitory activity. The present results suggest that the Q. serrata extract and compound 5 are promising candidates for treating inflammatory diseases such as arthritis. However, further in vivo studies in arthritis animal models are required to confirm these findings.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this study.

Acknowledgments

This research was supported by the Chung-Ang University Research Grants in 2021. The authors would like to thank Editage (http://www.editage.co.kr) for English language editing. This work was supported by the National Research Foundation of Korean (NRF) grant funded by the Korean government (MSIT) (No. NRF-2020R1F1A1073407).

Supplementary Materials

Supplementary Material files include the NMR spectra of five compounds isolated from Q. serrata. Figure S1-1: ¹H-NMR spectrum of compound 1 (600 MHz, acetone- d₆+D2O); Figure S1-2: ¹³C-NMR spectrum of compound 1 (150 MHz, acetone- d₆+D₂O); Figure S2-1: ¹H-NMR spectrum of compound 2 (600 MHz, DMSO-d₆+D₂O); Figure S2-2: ¹³C-NMR spectrum of compound 2 (150 MHz, DMSO-d₆+D₂O); Figure S3-1: ¹H-NMR spectrum of compound 3 (600 MHz, DMSO-d₆+D₂O); Figure S3-2: ¹³C-NMR spectrum of compound 3 (150 MHz, DMSO-d₆+D₂O); Figure S4-1: ¹H-NMR spectrum of compound 4 (600 MHz, acetone- d₆+D₂O); Figure S4-2: ¹³C-NMR spectrum of compound 4 (150 MHz, MeOD); and Figure S5-1: ¹H-NMR spectrum of compound 5 (600 MHz, DMSO-d₆+D₂O). (Supplementary Materials)

References

K. Koh and E. Jeon, Ferns, Fern-Allies and Seed-Bearing Plants of Korea, 2003.
D. T. K. Tuyen, D. T. Khang, P. T. Thu Ha, T. Thu Ha, A. Hai, and T. D ElzaawelyXuan, “Antioxidant capacity and phenolic contents of three quercus species,” International Letters of Natural Sciences, vol. 54, pp. 85–99, 2016.
View at: Publisher Site | Google Scholar
A. Pandey and R. S. SekarTamtaRawal, “Assessment of phytochemicals, antioxidant and antimutagenic activity in micropropagated plants of Quercus serrata, a high value tree species of Himalaya,” Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, vol. 152, no. 5, pp. 929–936, 2018.
View at: Publisher Site | Google Scholar
M. Oh, K. H. Park, M. H. Kim et al., “Anti-oxidative and anti-inflammatory effects of phenolic compounds from the stems of Quercus acuta Thunberg,” Asian Journal of Chemistry, vol. 26, no. 15, pp. 4582–4586, 2014.
View at: Publisher Site | Google Scholar
J. Yin and M. W. KimHwangKimLee, “Anti-Inflammatory effects of phenolic compounds isolated from Quercus Mongolica Fisch. ex Ledeb. on UVB-Irradiated human skin cells,” Molecules, vol. 24, no. 17, p. 3094, 2019.
View at: Publisher Site | Google Scholar
H. H. Kim, On UVB-Irradiated Human Skin Cells, The Graduate School Chung-Ang University, Seoul, South Korea, 2017.
M. Kim, Anti-acne Vulgaris Effect of Pedunculagin Isolated from Quercus Mongolica Leaves by Inhibiting Inflammasome, The Graduate School Chung-Ang University, Seoul, South Korea, 2020.
N. Velmurugan, S. Han, and Y. Lee, “Antifungal activity of neutralized wood vinegar with water extracts of Pinus densiflora and Quercus serrata saw dusts,” International Journal of Environmental Research, vol. 3, no. 2, pp. 167–176, 2009.
View at: Google Scholar
S. Akroum, “Antifungal activity of acetone extracts from Punica granatum L. quercus suber L. and vicia faba L,” Journal de Mycologie Médicale, vol. 27, no. 1, pp. 83–89, 2017.
View at: Publisher Site | Google Scholar
H. D. Yeo, “Antifungal activity of the quercus mongolica extracts against botrytis cinerea,” Journal of the Korean Wood Science and Technology, vol. 36, no. 1, pp. 88–101, 2008.
View at: Google Scholar
A. Dogan, I. Celik, and M. S. Kaya, “Antidiabetic properties of lyophilized extract of acorn (Quercus brantii Lindl.) on experimentally STZ-induced diabetic rats,” Journal of Ethnopharmacology, vol. 176, pp. 243–251, 2015.
View at: Publisher Site | Google Scholar
A. W. Indrianingsih and K. TachibanaDewiItoh, “Antioxidant and α-glucosidase inhibitor activities of natural compounds isolated from Quercus gilva Blume leaves,” Asian Pacific Journal of Tropical Biomedicine, vol. 5, no. 9, pp. 748–755, 2015.
View at: Publisher Site | Google Scholar
M. Güllüce, A. Adıgüzel, H. Öğütçü, M. Şengül, I. Karaman, and F. Şahin, “Antimicrobial effects of Quercus ilex L. extract,” Phytotherapy Research, vol. 18, no. 3, pp. 208–211, 2004.
View at: Publisher Site | Google Scholar
F. W. WangWang and Y. C. JiaoChengTanSong, “Antimicrobial potentials of endophytic fungi residing in Quercus variabilis and Brefeldin A obtained from Cladosporium sp,” World Journal of Microbiology and Biotechnology, vol. 23, no. 1, pp. 79–83, 2007.
View at: Publisher Site | Google Scholar
N. Amessis-Ouchemoukh and J. OuchemoukhMeziantIdiriHernanzStincoRodríguez-PulidoHerediaMadaniLuis, “Bioactive metabolites involved in the antioxidant, anticancer and anticalpain activities of Ficus carica L., Ceratonia siliqua L. and Quercus ilex L. extracts,” Industrial Crops and Products, vol. 95, pp. 6–17, 2017.
View at: Publisher Site | Google Scholar
S. K. Panchal and L. Brown, “Cardioprotective and hepatoprotective effects of ellagitannins from European oak bark (Quercus petraea L.) extract in rats,” European Journal of Nutrition, vol. 52, no. 1, pp. 397–408, 2013.
View at: Publisher Site | Google Scholar
E. AkramAkram, K. Masoud, and N. Vahid, “Evaluation of the antibacterial and wound healing activity of Quercus persica,” Journal of Basic & Applied Sciences, vol. 8, no. 1, pp. 118–123, 2012.
View at: Publisher Site | Google Scholar
S. H. Youn, J. H. Kwon, J. Yin et al., “Anti-inflammatory and anti-urolithiasis effects of polyphenolic compounds from Quercus gilva blume,” Molecules, vol. 22, no. 7, p. 1121, 2017.
View at: Publisher Site | Google Scholar
K. M. McDougal and C. R. Parks, “Elevational variation in foliar flavonoids of quercus rubra L. (Fagaceae),” American Journal of Botany, vol. 71, no. 3, pp. 301–308, 1984.
View at: Publisher Site | Google Scholar
R.-K. Ko and N.-H. Lee, “Isolation of a new secolignan compound from Quercus glauca,” Bulletin of the Korean Chemical Society, vol. 29, no. 12, pp. 2531-2532, 2008.
View at: Google Scholar
J. Huang and X. WangLiWangHe, “Triterpenes isolated from acorns of Quercus serrata var. brevipetiolata exert anti-inflammatory activity,” Industrial Crops and Products, vol. 91, pp. 302–309, 2016.
View at: Publisher Site | Google Scholar
H. H. Kim, D. H. Kim, M. H. Oh, K. J. Park, J. H. Heo, and M. W. Lee, “Inhibition of matrix metalloproteinase-1 and type-I procollagen expression by phenolic compounds isolated from the leaves of Quercus mongolica in ultraviolet-irradiated human fibroblast cells,” Archives of Pharmacal Research, vol. 38, no. 1, pp. 11–17, 2015.
View at: Publisher Site | Google Scholar
G.-i. Nonaka, H. Nishimura, and I. Nishioka, “Tannins and related compounds. Part 26. Isolation and structures of stenophyllanins A, B, and C, novel tannins from Quercus stenophylla,” Journal of the Chemical Society, Perkin Transactions 1, vol. 1, p. 163, 1985.
View at: Publisher Site | Google Scholar
G.-i. Nonaka, “Tannins and related compounds. CIX. Isolation of alienanins A and B, novel C,C-linked ellagitannin dimers from Quercus aliena Blume,” Chemical and Pharmaceutical Bulletin, vol. 39, no. 4, pp. 884–888, 1991.
View at: Publisher Site | Google Scholar
P. Tagde, P. Tagde, F. Islam et al., “The multifaceted role of curcumin in advanced nanocurcumin form in the treatment and management of chronic disorders,” Molecules, vol. 26, no. 23, p. 7109, 2021.
View at: Publisher Site | Google Scholar
F. Islam, S Bibi, A. F. K. Meem et al., “Natural bioactive molecules: an alternative approach to the treatment and control of COVID-19,” International Journal of Molecular Sciences, vol. 22, no. 23, Article ID 12638, 2021.
View at: Publisher Site | Google Scholar
A. Akter, “CNS Depressant Activities of Averrhoa Carambola Leaves Extract in Thiopental-Sodium Model of Swiss Albino Mice: Implication for Neuro-Modulatory Properties,” 2022, https://www.researchgate.net/publication/359117990_CNS_depressant_activities_of_Averrhoa_carambola_leaves_extract_in_thiopental-sodium_model_of_Swiss_albino_mice_implication_for_neuro-modulatory_properties.
View at: Google Scholar
M. M. Rahman, M. R. Islam, S. Shohag et al., “The multifunctional role of herbal products in the management of diabetes and obesity: a comprehensive review,” Molecules, vol. 27, no. 5, p. 1713, 2022.
View at: Publisher Site | Google Scholar
M. M. RahmanRahman, F. Islam, A. Parvez et al., “Citrus limon L. (lemon) seed extract shows neuro-modulatory activity in an in vivo thiopental-sodium sleep model by reducing the sleep onset and enhancing the sleep duration,” Journal of Integrative Neuroscience, vol. 21, no. 1, p. 042, 2022.
View at: Publisher Site | Google Scholar
H. Wang, X. Guo, J. Liu, T. Li, X. Fu, and R. H. Liu, “Comparative suppression of NLRP3 inflammasome activation with LPS-induced inflammation by blueberry extracts (Vaccinium spp.),” RSC Advances, vol. 7, no. 46, pp. 28931–28939, 2017.
View at: Publisher Site | Google Scholar
S. B. Kwak, S. Koppula, E. J. In et al., “Artemisia extract suppresses NLRP3 and AIM2 inflammasome activation by inhibition of ASC phosphorylation,” Mediators of inflammation, vol. 2018, 2018.
View at: Publisher Site | Google Scholar
A. Shahbaz, B. A. Abbasi, J. Iqbal et al., “Chemical composition of Gastrocotyle hispida (Forssk.) bunge and Heliotropium crispum Desf. and evaluation of their multiple in vitro biological potentials,” Saudi Journal of Biological Sciences, vol. 28, no. 11, pp. 6086–6096, 2021.
View at: Publisher Site | Google Scholar
E. Küpeli Akkol, Y. Genç, B. Karpuz, E. Sobarzo-Sánchez, and R. Capasso, “Coumarins and coumarin-related compounds in pharmacotherapy of cancer,” Cancers, vol. 12, no. 7, p. 1959, 2020.
View at: Publisher Site | Google Scholar
V. Martínez, A. Iriondo De-Hond, F. Borrelli, R. Capasso, M. D. Del Castillo, and R. Abalo, “Cannabidiol and other non-psychoactive cannabinoids for prevention and treatment of gastrointestinal disorders: useful nutraceuticals?” International Journal of Molecular Sciences, vol. 21, no. 9, p. 3067, 2020.
View at: Publisher Site | Google Scholar
J. Iqbal, B. A. Abbasi, R. Ahmad et al., “Phytogenic synthesis of nickel oxide nanoparticles (NiO) using fresh leaves extract of Rhamnus triquetra (wall.) and investigation of its multiple in vitro biological potentials,” Biomedicines, vol. 8, no. 5, p. 117, 2020.
View at: Publisher Site | Google Scholar
M. A. Freitas, A. Vasconcelos, E. C. D. Gonçalves et al., “Involvement of opioid system and TRPM8/TRPA1 channels in the antinociceptive effect of spirulina platensis,” Biomolecules, vol. 11, no. 4, p. 592, 2021.
View at: Publisher Site | Google Scholar
T. A. Bristy, N. Barua, A. Montakim Tareq et al., “Deciphering the pharmacological properties of methanol extract of Psychotria calocarpa leaves by in vivo, in vitro and in silico approaches,” Pharmaceuticals, vol. 13, no. 8, p. 183, 2020.
View at: Publisher Site | Google Scholar
O. Goni, M. F. Khan, M. M. Rahman et al., “Pharmacological insights on the antidepressant, anxiolytic and aphrodisiac potentials of Aglaonema hookerianum Schott,” Journal of Ethnopharmacology, vol. 268, Article ID 113664, 2021.
View at: Publisher Site | Google Scholar
L. I. Bouwman, M. R. de Zoete, N. M. Bleumink-Pluym, R. A. Flavell, and J. P. van Putten, “Inflammasome activation byCampylobacterjejuni,” The Journal of Immunology, vol. 193, no. 9, pp. 4548–4557, 2014.
View at: Publisher Site | Google Scholar
M. Dagenais, A. Skeldon, M. Saleh, and A. Skeldon, “The inflammasome: in memory of Dr. Jurg Tschopp,” Cell Death & Differentiation, vol. 19, no. 1, pp. 5–12, 2012.
View at: Publisher Site | Google Scholar
J. Tőzsér and S. Benkő, “Natural compounds as regulators of NLRP3 inflammasome-mediated IL-1β production,” Mediators of Inflammation, vol. 2016, 2016.
View at: Google Scholar
H. Guo, J. B. Callaway, and J. P. Y. Ting, “Inflammasomes: mechanism of action, role in disease, and therapeutics,” Nature Medicine, vol. 21, no. 7, pp. 677–687, 2015.
View at: Publisher Site | Google Scholar
L. A. B. JoostenJoosten, M. G.Netea, G. Fantuzzi et al., “Inflammatory arthritis in caspase 1 geneâ “deficient mice: contribution of proteinase 3 to caspase 1â” independent production of bioactive interleukin-1Î²,” Arthritis & Rheumatism, vol. 60, no. 12, pp. 3651–3662, 2009.
View at: Publisher Site | Google Scholar
Y. Sawabe, K. Yamasaki, S. Iwagami, K. Kajimura, and K. Nakagomi, “Inhibitory effects of natural medicines on the enzymes related to the skin,” Yakugaku Zasshi, vol. 118, no. 9, pp. 423–429, 1998.
View at: Publisher Site | Google Scholar
M. H. Lee, J. M. Lee, S. H. Jun et al., “The anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO production,” Journal of Ethnopharmacology, vol. 112, no. 1, pp. 49–54, 2007.
View at: Publisher Site | Google Scholar
C. Andary, H. Ravnb, R. Wyldec, A. Heitzc, and E. M. Florac, “Crassifolioside, a caffeic acid glycoside ester from Plantago crassifolia,” Phytochemistry, vol. 28, no. 1, pp. 288–290, 1989.
View at: Publisher Site | Google Scholar
M. U. Roslund, P. Tähtinen, M. Niemitz, and R. Sjöholm, “Complete assignments of the 1H and 13C chemical shifts and JH,H coupling constants in NMR spectra of d-glucopyranose and all d-glucopyranosyl-d-glucopyranosides,” Carbohydrate Research, vol. 343, no. 1, pp. 101–112, 2008.
View at: Publisher Site | Google Scholar
P. K. Agrawal, Carbon-13 NMR of Flavonoids, Elsevier, Amsterdam, Europe, 2013.
K. Umehara, I. PalSingh, H. Etoh, M. Takasaki, and T. Konoshima, “Five phloroglucinol-monoterpene adducts from Eucalyptus grandis,” Phytochemistry, vol. 49, no. 6, pp. 1699–1704, 1998.
View at: Publisher Site | Google Scholar
D.-J. Kwon and Y.-S Bae, “Ellagitannins from bark of Juglans mandshurica,” Journal of the Korean Wood Science and Technology, vol. 37, no. 5, pp. 480–485, 2009.
View at: Google Scholar
Y.-M. Xu, T. TANAKA, G.-I. NONAKA, and I. NISHIOKA, “Tannins and related compounds. CVII. Structure elucidation of three new monomeric and dimeric ellagitannins, flosin B and reginins C and D, isolated from Lagerstroemia flos-reginae Retz,” Chemical and Pharmaceutical Bulletin, vol. 39, no. 3, pp. 647–650, 1991.
View at: Publisher Site | Google Scholar
S.-B. Kwak, “Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation,” Mediators of inflammation, vol. 2018, 2018.
View at: Google Scholar
G. Yang, H. E. Lee, S.-H. Han, T.-J. An, J.-K. Jang, and J. Y. Lee, “A Natural Product Inhibitor of the NLRP3 Inflammasome Derived from Glycyrrhiza Uralensis for the Treatment of Inflammatory Diseases,” The Journal of Immunology, vol. 198, 2017.
View at: Google Scholar
X. Sun, D. W. Shim, J. W. Han et al., “Anti-inflammatory effect of Impatiens textori Miq. extract via inhibition of NLRP3 inflammasome activation in in vitro and in vivo experimental models,” Journal of Ethnopharmacology, vol. 170, pp. 81–87, 2015.
View at: Publisher Site | Google Scholar
Y.-K. Kim, “Inhibitory effect and mechanism of arctium lappa extract on Nlrp3 inflammasome activation,” Evidence-Based Complementary and Alternative Medicine, vol. 2018, 2018.
View at: Google Scholar
K. Wang, Q. Lv, Y. M. Miao, S. M. Qiao, Y. Dai, and Z. F. Wei, “Cardamonin, a natural flavone, alleviates inflammatory bowel disease by the inhibition of NLRP3 inflammasome activation via an AhR/Nrf2/NQO1 pathway,” Biochemical Pharmacology, vol. 155, pp. 494–509, 2018.
View at: Publisher Site | Google Scholar
S. S. Chaurasia, R. R. Lim, B. H. Parikh et al., “The NLRP3 inflammasome may contribute to pathologic neovascularization in the advanced stages of diabetic retinopathy,” Scientific Reports, vol. 8, no. 1, p. 2847, 2018.
View at: Publisher Site | Google Scholar
F. G. Thankam, Z. K. Roesch, M. F. Dilisio et al., “Association of inflammatory responses and ECM disorganization with HMGB1 upregulation and NLRP3 inflammasome activation in the injured rotator cuff tendon,” Scientific Reports, vol. 8, no. 1, p. 8918, 2018.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2022 Seo Yeon Seonu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

486

Downloads

334

Citations