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Effect of Saururus chinensis leaves extract on type II collagen-induced arthritis mouse model

  • Jong-Hyun Nho
  • Hyeun-Joo Lee
  • Ho-Kyung Jung
  • Ji-Hun Jang
  • Ki-Ho Lee
  • A-Hyeon Kim
  • Tae-Kyoung Sung
  • Hyun-Woo ChoEmail author
Open Access
Research article
Part of the following topical collections:
  1. Basic Research

Abstract

Background

Saururus chinensis leaves have been used as traditional medicine in Korea for pain, intoxication, edema, and furuncle. According to previous reports, these leaves exert renoprotective, neuroprotective, and antioxidant effects by attenuating inflammatory responses. However, the beneficial effect of Saururus chinensis leaves on arthritis has not been elucidated. Thus, we evaluated the water extract of Saururus chinensis leaves (SHW) using type II collagen-induced arthritis (CIA) mice models.

Methods

Quantitative analysis of major components from SHW was performed by HPLC. Arthritis was induced by injection of type II collagen. Each group was orally administered SHW (100 mg/kg and 500 mg/kg). Methotrexate (MTX) was used as a positive control. Serum levels of interleukin-6, TNF-alpha, and type II collagen IgG in the animal models were measured using ELISA. Histological features were observed by H&E staining.

Results

Quantitative analysis of SHW showed the contents as 56.4 ± 0.52 mg/g of miquelianin, 7.75 ± 0.08 mg/g of quercetin 3-O-(2”-O-β -glucopyranosyl)-α-rhamnopyranoside, and 3.17 ± 0.02 mg/g of quercitrin. Treatment with 500 mg/kg SHW decreased the serum level of Interleukin-6 (IL-6), TNF-alpha, and collagen IgG in the CIA model. Moreover, SHW treatment diminished the swelling of hind limbs and monocyte infiltration in blood vessels in CIA animal models. The results indicate that SHW could decrease CIA-induced arthritis in vivo.

Conclusions

The results indicate that SHW could be used to improving arthritis by reducing inflammatory factors (IL-6 and TNF-alpha). However, further experiments are required to determine how SHW influences signal transduction in animal models.

Keywords

Saururus chinensis leaves Arthritis Inflammation 

Abbreviations

ALT

Alanine amino transferase

AST

Aspartate serum transferase

BUN

Blood urine nitrogen

CFA

Complete Freund’s adjuvant

CIA

Type II collagen-induced arthritis

Cre

Creatinine

ELISA

Enzyme-linked immunosorbent assay

IACUC

Institutional animal care and use committee

IFA

Incomplete Freund’s adjuvant

IL-6

Interleukin-6,

LPS

Lipopolysaccharide

MeOH

Metahnol

MTX

Methotrexate

NIKOM

National development institute of Korean medicine

NO

Nitric oxide

NSAIDs

Non-steroidal anti-inflammatory drugs

PGE2

Prostaglandin E2,

RA

Rheumatoid arthritis

SHW

The water extract of Saururus chinensis leaves

TNF-alpha

Tumor necrosis factor-alpha

Background

Saururus chinensis is a perennial herbaceous plant belonging to the family Saururaceae. It is commonly found in South Korea, Japan, and China. As a traditional medicine in South Korea and China, Saururus chinensis leaves have been used for the treatment of pneumonia, edema, urination, and jaundice [1]. According to previous reports, the extract of Saururus chinensis (leaves, stems and flowers) shows renoprotective and antioxidant effects in rats fed a high-fructose diet [2], and inflammation-mediated neurotoxicity was found to be attenuated by treatment with the ethanol extract of Saururi herba in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells [3]. These leaves also possess anti-asthmatic, anti-atopic, and anti-angiogenic activities [3, 4, 5, 6]. In Korean traditional medicine, Saururi herba is used for the treatment of inflammatory diseases including fever, edema, and jaundice [7]. Sauchinone isolated from Saururus chinensis was found to reduce SREBP-1c-mediated hepatic steatosis and oxidative stress, as well as iron-induced liver injury [8, 9]. However, the beneficial effect of Saururus chinensis leaves on type II collagen-induced arthritis has not been elucidated.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, affecting about 2% of the world population [10]. It is characterized by destruction of the cartilage and tendons, and inflammation in synovial joints. The pathophysiological mechanisms and causes of RA remain unclear, it is known that various immune cells including T- and B-lymphocytes, osteoclasts, fibroblast-like synoviocytes, and chondrocyte are involved in auto-immunity and chronic inflammation during RA pathogenesis [11, 12]. The type II collagen-induced arthritis (CIA) model is an animal model of rheumatoid arthritis that has been commonly used to validate therapeutic drugs based on the clinical and immunological features of RA [13]. Therefore, most drugs used for the treatment of RA have anti-inflammatory and anti-oxidative stress effects [14].

Inflammation is one of the defense mechanisms against body injury caused by infection. Inflammation disease presents symptoms such as fever and pain, and supports regeneration of damaged tissues [15, 16]. However, long-lasting inflammation responses induce neurodegenerative disease or inflammatory disease [17]. Inflammatory factors including intereukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) are secreted upon inflammation responses in the body, and these factors are used as indicators of inflammatory responses [18].

According to existing literature, current treatment strategies for RA are focused on improvement of joint damage and inflammatory response including swelling and fever. Thus, therapeutic agents for RA include glucocorticoids, specific inhibitors of inflammatory cytokines, and non-steroidal anti-inflammatory drugs (NSAIDs) [19]. However, most of these therapeutic agents induce adverse effects that influence the cardiovascular and gastrointestinal system, kidneys, and liver. Thus, searching for natural products with safety and efficacy in treating RA has become indispensable [20, 21]. In this study, we performed quantitative analysis of major components from Saururus chinensis leaves and examined the effect of SHW on RA using the type II collagen-induced arthritis (CIA) animal model.

Methods

Sample preparation and extraction

Thirteen dried Saururus chinensis leaf samples were purchased from Kyungdong market (Seoul, Korea). These samples were sourced from various regions and were identified by Professor Jong Gil Jeong (Plant taxologist, Dongshin University, Korea). A voucher specimen (TKM-II-7-1~13) of this plant was deposited in the Herbarium at the National Development Institute of Korea Medicine. We performed fingerprint and reproducibility analysis (Additional file 1:Supplementary Figure S1) for major components and selected seven samples. The same amount of Saururus chinensis leaves (3.1 kg, 443 g × 7) were extracted with water (30 L, 3 times) under reflux conditions for 3 h and filtered. The extracts were lyophilized using a freeze dryer (LYOPH-PRIDE 20R, IlShinBioBase, Dongducheon, Korea) to obtain SHW powder (652 g, yield: 21%). The lyophilized powder was dissolved in 0.5% carboxymethyl cellulose (CMC, Sigma-Aldrich, USA) before being used in experiments.

LC-IT-TOF-MS conditions

Electrospray ionization-mass spectra were obtained on a LC-IT-TOF mass spectrometer (Shimadzu, Japan). All solvents used for analyses were of HPLC grade and were purchased from J.T. Baker (PA, USA). LC analysis was performed on a Shimadzu analytical UFLC (Kyoto, Japan) system comprising two LC-30 AD XR pumps, a CTO-20A column oven, a DGU-20A3 degasser, an SPD-20A detector, and an SIL-20A XR auto-injector. For full scan MS analysis, spectra were recorded in the range m/z 100–1000. Data were later processed using LC/MS solution software (version 3, Shimadzu, Kyoto, Japan), which includes a formula predictor. Detailed analytical conditions are listed in Table 1. SHW (50 mg) was dissolved in 5 mL of 70% methanol and filtered through a 0.2 μm syringe filter (Adventec, Canada) for the analysis.
Table 1

LC-IT-TOF MS conditions for S. chinensis extract

HPLC condition

 Column

BEH C18 (1.7 μm, 2.1 × 150 mm)

 Flow rate

0.21 mL/min

 Injection volume

1 μL

 Column temperature

40 °C

Mobile phase

A: 0.1% formic acid in water

B: acetonitrile

Time

A (%)

B (%)

0

100

0

25

55

45

MS condition

 Ionization mode

ESI, positive

 Capillary voltage (kV)

4.5

 CDL voltage (V)

10

 Detector voltage (kV)

1.67

 CDL temperature

200 °C

 Heat block temperature

200 °C

 Nebulizing gas

N2, 1.5 L/min

 Collision gas

Ar

 Collision Energy

30%

Isolation and identification

Dried leaves of Saururus chinensis (300 g) were suspended in 3 L water containing 2% acetic acid and then partitioned with n- hexane, methylene chloride and n-butanol, yielding 2 g, 3.5 g, 65 g, 205 g, respectively. The BuOH fraction (1 g) was dissolved in 10 mL of methanol and purified using a Luna C18-100A column (Phenomenex, USA; 25 cm × 3 mm, 5 μm particle size) on an LC20AP series high-performance liquid chromatography (HPLC) system equipped with SPD-M20A (Shimadzu, Tokyo, Japan). The mobile phase consisted of 0.02% formic acid in water (A) and acetonitrile (B), starting with 10% B increasing to 30% B for 45 min. The flow rate was 35.0 mL/min and the wavelength was 254 nm. The fraction was purified as an eluent to yield pure compound 2 (18.5 min, 70 mg) and 3 (21.1 min, 200 mg). NMR spectra were obtained using a Varian UNITY INOVA 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C) using CD3OD (Sigma-Aldrich, USA); chemical shifts are given in ppm (δ).

Qualitative analysis

SHW powder (200.0 mg) was extracted respectively with 45 mL of 0, 30, 50, 70, and 100% methanol under sonication for 60 min at room temperature. The extract was filtered, adjusted to a final volume of 50 mL in a volumetric flask, and then filtered through 0.2 μm syringe filter. Quercitrin purchased was Sigma-Aldrich (St. Louis, MO, USA). Miquelianin and quercetin 3-O-(2”-O-β-glucopyranosyl)-α-rhamnopyranoside [Q-3-(2″-glu)-rham] were isolated from Saururus chinensis leaves. A mixture of miquelianin, Q-3-(2″-glu)-rham and quercitrin was prepared in 70% methanol and serially diluted (concentration: 420–26.25 μg/mL, 105–6.25 μg/mL, 58.75–3.67 μg/mL) to obtain calibration curves. The chromatographic system used was an Agilent 1200 series HPLC system (Agilent, USA) with an MG II column (C18, 4.6 × 250 mm, Shiseido, Japan). The mobile phase was a gradient of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The linear gradient elution was 15% of B to 25% of B for 0–30 min at a flow rate of 1.0 mL/min. The column oven temperature was 40 °C, and the wavelength was 254 nm.

Animals and euthanasia

Male DBA/1 mice (6 weeks of age) purchased from Samtako (Osan, Korea) were separated into 5 groups (Control; n = 7, CIA; n = 7, CIA + MTX; n = 7, CIA + 100 mg/kg SHW; n = 7, CIA + 500 mg/kg SHW; n = 7). Immunization grade bovine type II collagen (Chondrex, Redmond, WA, USA) was dissolved in complete Freund’s adjuvant (CFA, Sigma Aldrich, St. Louis, MO, USA) or incomplete Freund’s adjuvant (IFA, Sigma Aldrich, St. Louis, MO, USA). The first immunization was performed using bovine type II collagen dissolved in CFA (1:1 ratio), mice were injected at the base of the tail (100 μl). After one week, bovine type II collagen dissolved in IFA (1:1 ratio) was injected at the base of the tail (100 μl) [22]. After 1 week of the second immunization, the CIA + MTX group was administered with methotrexate (0.2 mg/kg, p.o., once a day, 3 weeks). The CIA + 100 mg/kg SHW group was administered with 100 mg/kg SHW (p.o., once a day, 3 weeks). The CIA + 500 mg/kg SHW group was administered with 500 mg/kg SHW (p.o., once a day, 3 weeks). At the end of the experiment, mice were euthanized by carbon dioxide (CO2) inhalation according to the standard laboratory operating procedures of the IACUC.

Assessment of arthritis and histologic score

Clinical arthritis was assessed weekly beginning from 21 days of the second immunization, and arthritic scoring was performed by three independent examiners, three times per week. Arthritis scoring was performed as described by Endale et al. [23]. The clinical assessment was as follows: 0 = symptomless, 2 = erythema, 4 = mild swelling and erythema, 6 = mild swelling, erythema from the tarsals to the ankle, 8 = moderate swelling, erythema from the metatarsal joints to the ankle, 10 = severe swelling and erythema from the foot to the ankle. Histological sections were stained with hematoxylin and eosin, and analyzed microscopically by three observers for the degree of inflammation and bone erosion according to the method reported previously [24, 25]. The following scale was used: 0 = normal, 1 = cell infiltration in synovial membrane, 2 = cartilage erosion, 3 = erosion of subchondral bone, and 4 = loss of joint integrity and ankylosis.

Measurement of type II collagen IgG

Measurement of type II collagen IgG in serum was performed using Mouse anti-mouse type II collagen IgG antibody assay (2036, Chondrex, WA, USA). Blood was collected in BD Vacutainer™ SST tubes (Thermo, MA, USA), incubated at room temperature for 10 min, and centrifuged for 10 min at 4000 rpm at 4 °C. Separated serum samples were used for measuring the type II collagen IgG in serum according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was conducted for the measurement of IL-6 and TNF-alpha levels in the serum. After euthanasia, blood was collected in BD Vacutainer™ SST tubes (Thermo, MA, USA), incubated at room temperature for 10 min, and centrifuged for 10 min at 4000 rpm at 4 °C. Separated serum samples were used for measuring the IL-6 and TNF-alpha levels. The ELISA kits used were Mouse IL-6 DuoSet ELISA (DY406–05, R&D systems, MN, USA) and Mouse TNF-alpha DuoSet ELISA (DY410–05, R&D systems, MN, USA). All experiments were conducted according to the manufacturer’s instructions.

Hematoxylin & Eosin staining

Hind limbs were harvested from DBA/1 mice, fixed overnight in 10% NBF (Sigma Aldrich, St. Louis, MO, USA) and then paraffinized with paraplast (39,603,002, LEICA biosystems, Wetzlar, Germany), xylene (Sigma Aldrich, 534,056, St. Louis, MO, USA), and diluted ethanol. Paraffinized hind limb samples were cut into 5 μm sections using a microtome (LEICA biosystems, Wetzlar, Germany). The paraffin embedded sections were deparaffinized with xylene and hydrated with water and diluted ethanol, and then subjected to hematoxylin & eosin staining.

Blood chemistry analysis

Blood chemistry analysis was conducted using the FUJI DRI-CHEM 4000i analyzer (Fujifilm, Tokyo, Japan), according to the manufacturer’s instructions. Blood collected in BD Vacutainer™ SST tube (Thermo, MA, USA), was incubated at room temperature for 20 min and then centrifuged for 10 min at 4000 rpm at 4 °C. The separated serum was used for blood chemistry analysis (BUN, blood urine nitrogen; Cre, creatinine; AST, aspartate serum transferase; ALT, alanine amino transferase).

Statistical analysis

Results were expressed as the mean ± SEM. Between group comparisons were conducted using one-way ANOVA by SPSS (SPSS Inc., IL, USA), followed by Tukey’s post hoc test. A value of p < 0.05 was considered significant.

One-way ANOVA with SPSS was performed to compare the groups based on rheumatoid arthritis score, followed by Tukey-Kramer’s multiple comparison test. P values < 0.05 were considered statistically significant.

Results

Isolation and structural elucidation

The HPLC chromatogram and total ion chromatogram of Saururus chinensis leaves is shown in Fig. 1. Compound 1 was detected at 14.5 min, m/z 479.08 [M + H]+, MS/MS fragment ion occurred at m/z 303.05 [M-glucuronic acid+H]+. Compound 2 was detected at 15.1 min, m/z 611.16 [M + H]+, fragment ions occurred at m/z 449.10 [M-glu + H]+, m/z 303.05 [M-glu-rham+H]+. Compound 3 was found at 15.3 min, m/z 479.08 [M + H]+, MS/MS fragment ion at m/z 303.05 [M-rham+H]+. These compounds have been reported in a previous study on Saururus chinensis leaves but a standard preparation of compound 2 is not commercially available. To obtain the standard compound we performed isolation and identification. Compound 1 was identified as miquelianin and compound 2 was identified as quercetin 3-O-(2”-O-β-glucopyranosyl)-α-rhamnopyranoside upon comparison of spectroscopic data with literature; the purity was confirmed via LC-IT-TOF-MS. Compound 3 was identified as quercitrin based on comparison of its mass spectroscopic data with that of the purchased standard compound [26, 27].
Fig. 1

LC-IT-TOF MS chromatograms of Saururus chinensis leaves. (a) HPLC chromatogram at 254 nm; (b) Total ion chromatogram in the positive ion mode

Qualitative analysis of Saururus chinensis leaves

HPLC chromatograms are shown in Fig. 2. Three compounds were detected at tR 16.3 min, 18.0 min, and 21.1 min without interference. All of the regressions coefficients were r2 > 0.999 and the calibration curve showed good linearity of the detector over the range, respectively (Table 2). Several extraction solvents were examined in order to obtain satisfactory extraction efficiency. Most of the flavonoid glycosides were more soluble in a mixture of organic solvent:water rather than in a single solvent [28]. In this study, 30–50% methanol showed similar results for the three compounds, but the best proportion of the extracting solvent was chosen as 70% methanol (Table 3) with sonication for 60 min. As a result, the content of miquelianin was 56.4 ± 0.52 mg/g, that of Q-3-(2″-glu)-rham was 7.75 ± 0.08 mg/g, and the of quercitrin was 3.17 ± 0.02 mg/g.
Fig. 2

HPLC chromatograms of SHW (a) and standard solution (b). 1, Miquelianin; 2, Q-3-(2″-glu)-rham; 3, Quercitrin

Table 2

Linearities, regression equation of compound 1–3

 

Regression equation

R2

Linear range (ug/mL)

Miquelianin

y = 8.3173x-50.936

0.9999

420–26.25

Q-3-(2″-glu)-rham

y = 18.359x-14.669

0.9999

105–6.563

Quercitrin

y = 31.668x + 4.057

1

58.75–3.672

Table 3

Concentration of compounds from extraction solvent

Analytes

Extraction solvent

Contents (mg/g)

Mean

SD

%RSD

1

2

3

Miquelianin

H2O

53.478

53.142

53.216

53.279

0.176

0.331

30% MeOH

54.802

54.614

54.369

54.595

0.217

0.398

50% MeOH

56.722

55.673

55.474

55.959

0.670

1.198

70% MeOH

56.448

56.897

55.853

56.399

0.524

0.928

MeOH

35.938

35.414

35.179

35.510

0.388

1.093

Q-3-(2″-glu)-rham

H2O

7.340

7.285

7.307

7.311

0.027

0.373

30% MeOH

7.506

7.483

7.441

7.477

0.033

0.439

50% MeOH

7.779

7.682

7.609

7.690

0.086

1.114

70% MeOH

7.763

7.831

7.669

7.754

0.081

1.050

MeOH

5.532

5.502

5.468

5.501

0.032

0.584

Quercitrin

H2O

2.698

2.726

2.751

2.725

0.027

0.979

30% MeOH

2.850

2.805

2.951

2.869

0.075

2.608

50% MeOH

3.155

3.183

3.110

3.149

0.037

1.181

70% MeOH

3.168

3.189

3.158

3.172

0.016

0.4926

MeOH

2.312

2.354

2.349

2.338

0.023

0.988

SHW administration decreased the serum level of IL-6 and TNF-alpha and did not influence toxicity markers of the liver and kidneys in CIA animal models

To confirm the toxicity of SHW on the liver and kidneys, we evaluated the commonly used toxicity markers including BUN (blood urine nitrogen), Cre (creatinine), AST (aspartate serum transferase), and ALT (alanine amino transferase). The experimental schedule is indicated in a diagram (Fig. 3A). BUN and Cre are typically used as indicators of kidney function, whereas AST and ALT are used as biochemical indicators of liver function [29]. In this study, SHW administration slightly increases BUN in DBA/1 mice, but did not affect Cre, ALT, and AST (data not shown).
Fig. 3

SHW administration decreased the serum level of IL-6 and TNF-alpha and did not influence the liver and kidney toxicity markers in the CIA animal models. (a) Experimental schedule. (b-c) Mice were separated into 5 groups (Control; n = 7, CIA; n = 7, CIA + MTX; n = 7, CIA + 100 mg/kg SHW; n = 7, CIA + 500 mg/kg SHW; n = 7). IL-6 and TNF-alpha levels in serum were analyzed by ELISA. Data are presented as the mean ± SEM (n = 7). #p < 0.05, versus normal control group; *p < 0.05, versus CIA group. Between groups comparisons were conducted using one-way ANOVA with Tukey’s post hoc test. CIA, collagen induced arthritis; MTX, methotrexate; SHW, water extract of Saururus chinensis leaves

To confirm the anti-inflammatory effect of SHW administration, SHW was orally administered to CIA animal models. Administration of 500 mg/kg SHW reduced the serum level of IL-6 (Fig. 3B), but it was not more efficient than the CIA + MTX groups. As shown in Fig. 3C, the serum level of TNF-alpha was decreased by administration of 500 mg/kg SHW. However, administration of 500 mg/kg SHW was not more efficient than the administration of 200 μg/kg MTX (Fig. 3C).

Effect of SHW administration on CIA-induced swelling and erythema in the hind limbs

To confirm the protective effect of SHW administration, morphological analysis of the hind limbs was performed. Treatment with SHW at 500 mg/kg significantly reduced the swelling and erythema scores compared to those in the CIA groups. However, administration of 500 mg/kg SHW in CIA animal models was not more efficient than the administration of 200 μg/kg MTX (Fig. 4A). As shown Fig. 4B, the swollen hind limbs induced by CIA were diminished by both 200 μg/kg MTX and 500 mg/kg SHW administration (Fig. 4B).
Fig. 4

Effect of SHW administration on CIA-induced swelling and erythema of hind limbs. (a-b) Mice were separated into 5 groups (Control; n = 7, CIA; n = 7, CIA + MTX; n = 7, CIA + 100 mg/kg SHW; n = 7, CIA + 500 mg/kg SHW; n = 7). Morphological analysis was carried out on hind limbs. Rheumatoid arthritis score was assessed weekly beginning from 21 days of the second immunization, by examiners over three times per week. Clinical assessment was scored as follows: 0 = symptomless, 2 = erythema, 4 = mild swelling and erythema, 6 = mild swelling, erythema from the tarsals to the ankle, 8 = moderate swelling, erythema from the metatarsal joints to the ankle, 10 = severe swelling and erythema from the foot to the ankle. The X axis indicates Weeks. Data are presented as the mean ± SEM (n = 7). *p < 0.05, versus the CIA group by one-way ANOVA with Tukey-Kramer’s multiple comparison test. CIA, collagen induced arthritis; MTX, methotrexate; SHW, water extract of Saururus chinensis leaves

Effect of SHW administration on type II collagen IgG levels and infiltration of inflammatory cells in the synovial membrane

Finally, to elucidate the effect of SHW on CIA animal models, we confirmed the histological features and the serum levels of type II collagen IgG. As shown Fig. 5A, the serum level of type II collagen IgG was increased in the CIA groups. However, it was decreased by 200 μg/kg MTX, 100 mg/kg SHW, and 500 mg/kg SHW administration. Moreover, the inflammatory response was diminished by SHW administration in the synovial membrane and knee joints (Fig. 5B). These data suggest that SHW administration may reduce the inflammatory response in CIA animal models.
Fig. 5

Effect of SHW administration on type II collagen IgG levels and infiltration of inflammatory cells in the synovial membrane. Mice were separated into 5 groups (Control; n = 7, CIA; n = 7, CIA + MTX; n = 7, CIA + 100 mg/kg SHW; n = 7, CIA + 500 mg/kg SHW; n = 7). (a) Type II collagen IgG in serum was analyzed using ELISA. (b) Representative images were stained with hematoxylin and eosin (H&E). Infiltration of inflammatory cells is indicated with black arrowheads. (c) Histological scores were determined on H&E stained sections in different groups of mice. Histologic scoring of inflammation and bone erosion was performed by three independent observers. Data are presented as the mean ± SEM (n = 7). #p < 0.05, versus normal control group; *p < 0.05, versus CIA group. Between group comparisons were conducted using one-way ANOVA with Tukey’s post hoc test. CIA, collagen induced arthritis; MTX, methotrexate; SHW, water extract of Saururus chinensis leaves

Discussion

This study demonstrated the anti-inflammatory effect of SHW administration in CIA animal models through improved inflammatory responses such as elevated IL-6, TNF-alpha, and type II collagen IgG in serum, as well as swelling of the hind limbs. SHW administration could inhibit the development of arthritis. In addition, safety evaluation of medicinal herbs used as traditional medicine in Korea, China and Japan [30, 31, 32]. We evaluated the safety of SHW using toxicity marker such as BUN, Cre, AST, and ALT. As previously mentioned, BUN and Cre are commonly used as indicators of renal function [29]. BUN and Cre are nitrogenous end products, BUN is the metabolite derived from dietary and tissue protein. Similarly, Cre is a product of muscle creatinine metabolism. Both are distributed throughout the total body fluids, and are increased during kidney dysfunction such as nephrotoxicity and diabetic nephropathy [33].

IL-6 (Interleukin-6) is pivotal cytokine that mediates RA pathogenesis, and is found in the synovial fluid and serum of RA patients. Moreover, IL-6 promotes joint destruction by stimulating neutrophil infiltration, osteoclast maturation, and pannus formation [34]. TNF-alpha (Tumor necrosis factor alpha) is a pleiotropic cytokine in RA. It is significantly increased in RA synovial tissue, but not in osteoarthritis synovial tissue [35]. Thus, we confirmed the beneficial effect of SHW administration on the IL-6 and TNF alpha. Based on our results, the serum levels of IL-6 and TNF alpha in the CIA animal models were statistically increased upon collagen administration, but this increase was diminished by SHW administration at 500 mg/kg (Fig. 3B and C). Recently, Meng et al. reported that anti-inflammatory effects of Saururus chinensis Baill. in murine macrophages regulating heme oxygenase-1 [36]. Moreover, various reports revealed that Saururus chinensis Baill. has anti-inflammatory effects [37, 38, 39]. This evidence supports the current results and suggests that SHW administration may suppress the pathophysiological features of RA.

The pathological and immunological characteristics induced by CIA administration are similar to those observed in RA in humans [40]. Our results demonstrate that treatment with 500 mg/mg SHW decreased the swelling in hind limbs (Fig. 4A and B). RA is an autoimmune disease, wherein the immune system mistakenly attacks the own body tissue, resulting in pain and swelling [35]. Our results demonstrate that SHW may affect inflammatory responses such as arteriole dilation, neutrophil migration, and expansion of capillary beds. Recent studies have revealed that type II collagen IgG is present in the synovial fluid and serum of RA patients [41]. Our results show that type II collagen IgG levels in serum are decreased upon SHW treatment (Fig. 5A). Moreover, SHW administration decreased the infiltration of inflammatory cells in the synovial membrane (Fig. 5B).

Prior to treatment, qualitative analysis was performed. Miquelianin, Q-3-(2″-glu)-rham, and quercitrin were identified in the water extract of Saururus chinensis leaves. Quantitative analysis of each compound performed by HPLC indicated their individual content as follows: 56.4 ± 0.52 mg/g (miquelianin), 7.75 ± 0.08 mg/g (Q-3-(2″-glu)-rham), 3.17 ± 0.02 mg/g (quercitrin).

Quercetin derivatives are the most common group of flavonoids and are associated various health benefits. Miquelianin, the major compound of SHW, is more easily and rapidly absorbed compared to quercetin and is the major bioactive component in plasma; it inhibits peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein [42, 43]. Moreover, miquelianin interferes with the protein-protein interaction of Aβ (1–40) and Aβ (1–42) [44], and inhibits the noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cell by regulating the β2-adrenergic signaling pathway through matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) [45]. These results could be useful for further pharmacokinetic studies on SHW and suggest that SHW treatment can be used to manage RA. However, further experiments are required to explore how SHW administration influences inflammatory signaling pathways including the NF-κB, integrin, and TNF signaling pathways.

Conclusions

SHW administration improved the various features of arthritis and rheumatoid arthritis including elevated serum levels of IL-6, TNF-alpha, type II collagen IgG, swelling of hind limbs, and infiltration of inflammatory cells in the synovial membrane. Thus, SHW acts as therapeutic agent against arthritis and rheumatoid arthritis. However, further experiments are required to explore how SHW influences inflammatory signaling pathways such as the NF-κB and TNF signaling pathways.

Notes

Acknowledgements

This study was supported by the Ministry of Health and Welfare (MHW).

Funding

This work was supported by the Korean Medicinal Herb-based Business of the Korean Traditional Resource subsidized by the Ministry of Health and Welfare (Republic of Korea).

Availability of data and materials

The materials can be obtained upon request via email to the corresponding author.

Authors’ contributions

JHN and HJL contributed to the data analysis, manuscript writing, laboratory experiments, and design of experiments. HKJ, JHJ, KHL, AHK, and TKS contributed to the extraction of samples and participated in the animal experiment. HWC proposed the idea and finalized the manuscript. All the authors have read and approved the final manuscript.

Ethics approval and consent to participate

All researchers on the animal experiment complied with the standards for the care of experimental animals. The animal laboratory was examined by the Institutional animal care and use committee (IACUC) for animal management, feeding environment, and visitor’s safety in accordance with the laboratory animal law. This study was approved by the National Development Institute of Korean Medicine-division of traditional Korean medicine resource-IACUC (NIKOM-DTKMR-IACUC, deliberation number; 2017–1-03, approval number; NIKOM-2017-003).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary material

12906_2018_2418_MOESM1_ESM.docx (38 kb)
Additional file 1: Figure S1. HPLC chromatograms of several samples used on this study. A~F, the water extracts of Saururus chinensis leaves. 1, miquelianin; 2, Q-3-(2″-glu)-rham; 3, quercitrin. (DOCX 38 kb)

References

  1. 1.
    Gao X, He J, Wu XD, Peng LY, Dong LB, Deng X, Li Y, Cheng X, Zhao QS. Further lignans from Saururus chinensis. Planta Med. 2013;79:1720–3.CrossRefGoogle Scholar
  2. 2.
    Choi HN, Park YH, Kim JH, Kang MJ, Jeong SM, Kim HH, Kim JI. Renoprotective and antioxidant effects of Saururus chinensis Baill in rats fed a high-fructose diet. Nutr Res Pract. 2011;5(4):365–9.CrossRefGoogle Scholar
  3. 3.
    Kim BW, Koppula S, Park SY, Hwang JW, Park PJ, Lim JH, Choi DK. Attenuating of inflammatory-mediated neurotoxicity by Saururus chinensis extract in LPS-induced BV-2 microglia cells via regulation of NF-κB signaling and anti-oxidant properties. BMC Complement Altern Med. 2014;14:502–12.CrossRefGoogle Scholar
  4. 4.
    Quan Z, Lee YJ, Yang JH, Lu Y, Li Y, Lee YK, Jin M, Kim JY, Choi JH, Son JK, Chang HW. Ethanol extracts of Saururus chinensis suppress ovalbumin-sensitization airway inflammation. J Ethnopharmacol. 2010;132(1):143–9.CrossRefGoogle Scholar
  5. 5.
    Choi MS, Kim EC, Lee HS, Kim SK, Choi HM, Park JH, Han JB, An HJ, Um JY, Kim HM, Han AR, Hong MC, Bae H, Min BI. Inhibitory effect of Saururus chinensis (LOUR.) Baill on the development of atopic dermatitis-like skin lesions in NC/Nga mice. Biol Pharm Bull. 2008;31(1):51–6.CrossRefGoogle Scholar
  6. 6.
    Yoo HJ, Kang HJ, Jung HJ, Kim K, Lim CJ, Park EH. Anti-inflammatory, anti-angiogenic and anti-nociceptive activities of Saururus chinensis extract. J Ethnopharmacol. 2008;120(2):282–6.CrossRefGoogle Scholar
  7. 7.
    Chung B, Shin MG. Dictionary of Korean folk medicine. Seoul: Young Lim Sa; 1990. p. 813–4.Google Scholar
  8. 8.
    Kim YW, Lee SM, Shin SM, Hwang SJ, Brooks JS, Kang HE, Lee MG, Kim SC, Kim SG. Efficacy of sauchinone as a novel AMPK-activating lignin for preventing iron-induced oxidative stress and liver injury. Free Radic Biol Med. 2009;47:1082–92.CrossRefGoogle Scholar
  9. 9.
    Kim YW, Kim YM, Yang YM, Kim TH, Hwang SJ, Lee JR, Kim SC, Kim SG. Inhibition of SREBP-1c-medicated hepatic steatosis and oxidative stress by sauchinone, an AMPK-activating lignin in Saururus chinensis. Free Radic Biol Med. 2010;48:567–78.CrossRefGoogle Scholar
  10. 10.
    Kaur A, Nain P, Nain J. Herbal plants used in treatment of rheumatoid arthritis: a review. Int J Pharm Pharm Sci. 2012;4:44–57.Google Scholar
  11. 11.
    Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rhematology. 2012;51:44–57.Google Scholar
  12. 12.
    Scrivo R, Di Franco M, Spadaro A, Valesini G. The immunology of rheumatoid arthritis. Ann N Y Acad Sci. 2007;1108:312–22.CrossRefGoogle Scholar
  13. 13.
    Williams RO. Collagen-induced arthritis in mice: a major role for tumor necrosis factor-alpha. Methods Mol Biol. 2007;361:265–84.PubMedGoogle Scholar
  14. 14.
    Khanna G, Sethi KS, Ahn MK, Pandey AB, Kunnumakkara B, Sung A, Aggarwal BB. Aggarwal, natural products as a gold mine for arthritis treatment. Curr Opin Pharmacol. 2007;10:77–88.Google Scholar
  15. 15.
    Nathan C. Points of control on inflammation. Nature. 2002;420(6917):846–52.CrossRefGoogle Scholar
  16. 16.
    Zamora R, Vodovotz Y, Billiar TR. Inducible nitric oxide synthase and inflammatory disease. Mol Med. 2000;6(5):347–73.CrossRefGoogle Scholar
  17. 17.
    Simons RK, Junger WG, Loomis WH, Hoyt DB. Acute lung injury in endotoxemic rats is associated with sustained circulating IL-6 levels and intrapulmonary CINC activity and neutrophil recruitment role of circulating TNF-alpha and IL-beta? Shock. 1996;6:39–45.CrossRefGoogle Scholar
  18. 18.
    Kim KH, Kwun MJ, Han CW, Ha KT, Choi JY, Joo MS. Suppression of lung inflammation in an LPS-induced acute lung injury model by the fruit hull of Gleditsia sinensis. BMC Complement Altern Med. 2014;14:402.CrossRefGoogle Scholar
  19. 19.
    Quan LD, Thiele GM, Tian J, Wang D. The development of novel therapies for rheumatoid arthritis. Expert Opin Ther Pat. 2008;18:723–38.CrossRefGoogle Scholar
  20. 20.
    Li X, Yuan FL, Lu WG, Zhao YQ, Li CW, Li JP, Xu RS. The role of interleukin-17 in mediating joint destruction in rheumatoid arthritis. Biochem Biophys Res Commun. 2010;397(2):131–5.CrossRefGoogle Scholar
  21. 21.
    Yuan FL, Li X, Lu WG, Li CW, Xu RS, Dong J. IL-33: a promising therapeutic target for rheumatoid arthritis? Expert Opin Ther Targets. 2011;15(5):529–34.CrossRefGoogle Scholar
  22. 22.
    Kim HO, Lee SI. Experimental animal models for rheumatoid arthritis: methods and applications. J Rheum Dis. 2012;19(4):189–95.CrossRefGoogle Scholar
  23. 23.
    Endale M, Lee WM, Kwak YS, Kim NM, Kim BK, Kim SH, Cho JY, Kim S, Park SC, Yun BS, Ko D, Rhee MH. Torilin ameliorates type II collagen-induced arthritis in mouse model of rheumatoid arthritis. Int Immunophamacol. 2013;16:232–42.CrossRefGoogle Scholar
  24. 24.
    Sun J, Jia Y, Li R, Guo J, Sun X, Liu Y, Li Y, Yao H, Liu X, Zhao J, Li Z. Altered influenza virus haemagglutinin (HA)-derived peptide is potent therapy for CIA by inducing Th1 to Th2 shift. Cell Mol Immunol. 2011;8:348–58.CrossRefGoogle Scholar
  25. 25.
    Nishikawa M, Myoui A, Tomita T, Takahi K, Nampei A, Yoshikawa H. Prevention of the onset and progression of collagen-induced arthritis in rats by the potent p38 mitogen-activated protein kinase inhibitor FR167653. Arthritis Rheum. 2003;48:2670–81.CrossRefGoogle Scholar
  26. 26.
    Nugroho A, Song BM, Lee KT, Park HJ. Quantification of antidepressant in mature and immature fruits of Korean rubus species. Nat Prod Sci. 2014;20:258–61.Google Scholar
  27. 27.
    Hasler A, Gross GA, Meier B, Sticher O. Complex flavonol glycosides from the leaves of ginkgo biloba. Phytochemistry. 1992;31:1391–4.CrossRefGoogle Scholar
  28. 28.
    Ferreira O, Pinho SP. Solubility of flavonoids in pure solvents. Ind Eng Chem Res. 2012;51:6586–90.CrossRefGoogle Scholar
  29. 29.
    Zhao B, Fei J, Chen Y, Ying YL, Ma L, Song XQ, Huang J, Chen EZ, Mao EQ. Vitamin C treatment attenuates hemorrhagic shock related multi-organ injuries through the induction of heme oxygenase-1. BMC Complement Altern Med. 2014;14:442.CrossRefGoogle Scholar
  30. 30.
    Zhang JH, Onakpoya IJ, Posadzki P, Eddouks M. The safety of herbal medicine: from prejudice to evidence. J Evid Based Complementary Altern Med. 2015;316709.Google Scholar
  31. 31.
    Yoon WH. Genotoxicological safety evaluation of the solvent extracts for medicinal herbs that are of highly domestic spendings. Korean J food Nutr. 2013;26(4):814–423.CrossRefGoogle Scholar
  32. 32.
    Kobayashi H, Ishii M, Takeuchi S, Tanaka Y, Shintani T, Yamatodani A, Kusunoki T, Furue M. Efficacy and safety of a traditional herbal medicine, Hochu-ekki-to in the long-term management of Kikyo (delicate constitution) patients with atopic dermatitis: a 6-month, multicenter, double-blind, randomized, placebo-controlled study. Evid Based Complement Alternat Med. 2010;7(3):367–73.CrossRefGoogle Scholar
  33. 33.
    Lopez-Giacoman S, Madero M. Biomarkers in chronic kidney disease, from kidney function to kidney damage. World J Nephrol. 2015;4(1):57–73.CrossRefGoogle Scholar
  34. 34.
    Srirangan S, Choy EH. The role of interleukin 6 in the pathophysiology of rheumatoid arthritis. Ther Adv Musculoskelet Dis. 2010;2(5):247–56.CrossRefGoogle Scholar
  35. 35.
    Matsuno H, Yudoh K, Katayama R, Nakazawa F, Uzuki M, Sawai T, Yonezawa T, Saeki Y, Panayi GS, Pitzalis C, Kimura T. The role of TNF-α in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera. 2002;41:329–37.Google Scholar
  36. 36.
    Meng X, Kim I, Jeong YJ, Cho YM, Kang SC. Anti-inflammatory effects of Saururus chinenesis aerial parts in murine macrophages via induction of heme oxygenase-1. Exp Biol Med. 2016;241(4):396–408.CrossRefGoogle Scholar
  37. 37.
    Lee JJ, Kim DH, Lim JJ, Kim GS, Min W, Lee HJ, Chang HH, Kim S. Antibacterial effect of Saururus chinenesis Baill. Ethanol extract against Salmonella typhimurium infection in RAW 264.7 macrophage. Kor J Vet Publ Hlth. 2010;34(4):285–91.Google Scholar
  38. 38.
    Kim BW, Koppula S, Park SY, Hwang JW, Park PJ, Lim JH, Choi DK. Attenuation of inflammatory-mediated neurotoxicity by Saururus chinensis extract in LPS-induced BV-2 microglia cells via regulation of NF-κB signaling and anti-oxidant properties. BMC Complement Altern Med. 2014;14:502.CrossRefGoogle Scholar
  39. 39.
    Cho HY, Cho CW, Song YS. Antioxidative and anti-inflammatory effects of Saururus chinensis methanol extract in RAW 264.7 macrophage. J Med Food. 2005;8(2):190–7.CrossRefGoogle Scholar
  40. 40.
    MCNamee K, Williams R, Seed M. Animal models of rheumatoid arthritis: how informative are they? Eur J Pharmacol. 2015;759:278–86.CrossRefGoogle Scholar
  41. 41.
    Clague RB, Morgan K, Reynolds I, Williams HJ. The prevalence of serum IgG antibodies to type II collagen in American patients with rheumatoid arthritis. Br J Clague. 1994;33:336–8.Google Scholar
  42. 42.
    Yang LL, Xiao N, Li XW, Fan Y, Alolga RN, Sun XY, Wang SL, Li P, Qi LW. Pharmacokinetic comparison between quercetin and quercetin 3-O-β-glucuronide in rats by UHPLC-MS/MS. Sci Rep. 2016;6:35460.CrossRefGoogle Scholar
  43. 43.
    Terao J, Yamaguchi S, Shirai M, Miyoshi M, Moon JH, Oshima S, Inakuma T, Tsushida T, Kato Y. Protection by quercetin and quercetin 3-O-Bata-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein. Free Radic Res. 2001;35:925–31.CrossRefGoogle Scholar
  44. 44.
    Ho L, Ferruzzi MG, Janle EM, Wang J, Gong B, Chen TY, Lobo J, Cooper B, Wu QL, Talcott ST, Percival SS, Simon JE, Pasinetti GM. Identification of bratin-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer’s disease. FASEB J. 2013;27:769–81.CrossRefGoogle Scholar
  45. 45.
    Yamazaki S, Miyoshi N, Kawabata K, Yasuda M, Shimoi K. Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking β2-adrenergic signaling. Arch Biochem Biophys. 2014;557:18–27.CrossRefGoogle Scholar

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  1. 1.Tradition Korean Medicine Research TeamNational Development Institute of Korean MedicineSeoulSouth Korea

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