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

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.

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.

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).

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.

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.

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 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.

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.

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 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).

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].

HPLC chromatograms are shown in Fig. 2. Three compounds were detected at t_R 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.

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).

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).

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).

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.

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.