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2.1. Preparation of SDE

The rhizomes of SD were purchased as a dried herb from Hanherb Co. (Guri, Korea). The plant materials were confirmed taxonomically by Dr. Go-Ya Choi of the Korea Institute of Oriental Medicine (KIOM). A voucher specimen (number 2014 SDE-6) was deposited in the Korean Herbarium of Standard Herbal Resources. Dried rhizomes of SD (320 g) were extracted twice with 70% ethanol (with a 2 h reflux) and the extract was then concentrated under reduced pressure. The decoction was filtered, lyophilized, and stored at 4°C. The yield of dried extract from crude starting materials was 48.13% (w/w).

 

2.2. Quantitative High-Performance Liquid Chromatography (HPLC) Analysis

Chromatographic analysis was performed with a HPLC system (Waters Co., Milford, MA, USA) and a photodiode array detector. For the HPLC analysis of SDE, the prim-O-glucosylcimifugin standard was purchased from the Korea Promotion Institute for Traditional Medicine Industry (Gyeongsan, Korea), and sec-O-glucosylhamaudol and 4′-O-β-D-glucosyl-5-O-methylvisamminol were isolated within our laboratory and identified by spectral analyses, primarily by NMR and MS.

SDE samples (0.1 mg) were dissolved in 70% ethanol (10 mL). Chromatographic separation was performed with an XSelect HSS T3 C18 column (4.6 × 250 mm, 5 μm, Waters Co., Milford, MA, USA). The mobile phase consisted of acetonitrile (A) and 0.1% acetic acid in water (B) at a flow-rate of 1.0 mL/min. A multistep gradient program was used as follows: 5% A (0 min), 5–20% A (0–10 min), 20% A (10–23 min), and 20–65% A (23–40 min). The detection wavelength was scanned at 210–400 nm and recorded at 254 nm. The injection volume was 10.0 μL. Standard solutions for the determination of three chromones were prepared at a final concentration of 7.781 mg/mL (prim-O-glucosylcimifugin), 31.125 mg/mL (4′-O-β-D-glucosyl-5-O-methylvisamminol), and 31.125 mg/mL (sec-O-glucosylhamaudol) in methanol and kept at 4°C.

2.3. Evaluation of Anti-Inflammatory Activity In Vitro

2.3.1. Cell Culture and Sample Treatment

RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM medium containing 1% antibiotics and 5.5% FBS. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. To stimulate the cells, the medium was replaced with fresh DMEM medium, and lipopolysaccharide (LPS, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) at 1 μg/mL was added in the presence or absence of SDE (200 or 400 μg/mL) for an additional 24 h.

2.3.2. Determination of Nitric Oxide (NO), Prostaglandin E2 (PGE2), Tumor Necrosis Factor-α (TNF-α), and Interleukin-6 (IL-6) Production

Cells were treated with SDE and stimulated with LPS for 24 h. NO production was analyzed by measuring nitrite using the Griess reagent according to a previous study [12]. Secretion of the inflammatory cytokines PGE2, TNF-α, and IL-6 was determined using an ELISA kit (R&D systems) according to manufacturer instructions. The effects of SDE on NO and cytokine production were determined at 540 nm or 450 nm using a Wallac EnVision™ microplate reader (PerkinElmer).

2.4. Evaluation of Antiosteoarthritis Activity In Vivo

2.4.1. Animals

Male Sprague-Dawley rats (7 weeks old) were purchased from Samtako Inc. (Osan, Korea) and housed under controlled conditions with a 12-h light/dark cycle at °C and % humidity. Rats were provided with a laboratory diet and water ad libitum. All experimental procedures were performed in compliance with the National Institutes of Health (NIH) guidelines and approved by the Animal Care and Use Committee of the Daejeon university (Daejeon, republic of Korea).

2.4.2. Induction of OA with MIA in Rats

The animals were randomized and assigned to treatment groups before the initiation of the study ( per group). MIA solution (3 mg/50 μL of 0.9% saline) was directly injected into the intra-articular space of the right knee under anesthesia induced with a mixture of ketamine and xylazine. Rats were divided randomly into four groups: (1) the saline group with no MIA injection, (2) the MIA group with MIA injection, (3) the SDE-treated group (200 mg/kg) with MIA injection, and (4) the indomethacin- (IM-) treated group (2 mg/kg) with MIA injection. Rats were administered orally with SDE and IM 1 week before MIA injection for 4 weeks. The dosage of SDE and IM used in this study was based on those employed in previous studies [10, 13, 14].

2.4.3. Measurements of Hindpaw Weight-Bearing Distribution

After OA induction, the original balance in weight-bearing capability of hindpaws was disrupted. An incapacitance tester (Linton instrumentation, Norfolk, UK) was used to evaluate changes in the weight-bearing tolerance. Rats were carefully placed into the measuring chamber. The weight-bearing force exerted by the hind limb was averaged over a 3 s period. The weight distribution ratio was calculated by the following equation: [weight on right hind limb/(weight on right hind limb + weight on left hind limb)] × 100 [15].

2.4.4. Measurements of Serum Cytokine Levels

The blood samples were centrifuged at 1,500 g for 10 min at 4°C; then the serum was collected and stored at −70°C until use. The levels of IL-1β, IL-6, TNF-α, and PGE2 in the serum were measured using ELISA kits from R&D Systems (Minneapolis, MN, USA) according to manufacturer instructions.

2.4.5. Real-Time Quantitative RT-PCR Analysis

Total RNA was extracted from knee joint tissue using the TRI reagent® (Sigma-Aldrich, St. Louis, MO, USA), reverse-transcribed into cDNA and PCR-amplified using a TM One Step RT PCR kit with SYBR green (Applied Biosystems, Grand Island, NY, USA). Real-time quantitative PCR was performed using the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Grand Island, NY, USA). The primer sequences and the probe-sequence are shown in Table 1. Aliquots of sample cDNAs and an equal amount of GAPDH cDNA were amplified with the TaqMan® Universal PCR master mixture containing DNA polymerase according to manufacturer instructions (Applied Biosystems, Foster, CA, USA). PCR conditions were 2 min at 50°C, 10 min at 94°C, 15 s at 95°C, and 1 min at 60°C for 40 cycles. The concentration of target gene was determined using the comparative Ct (threshold cycle number at cross-point between amplification plot and threshold) method, according to manufacturer instructions.

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