Drug preparation and administration

A. leucostomum Worosch. (~30 kg) was obtained from Nilka County (Yili, Xinjiang, China) and identified by pharmacist Professor Yonghe Li at the Traditional Chinese Medicine Hospital Affiliated to Xinjiang Medical University (Urumqi, China). Monomer components were isolated and purified according to our previously published procedures, and identified to be delvestidine (WF-1) and anthranoyllycoctonine (WF-4), according to spectral analysis (22). Three kinds of processed products were prepared using water-boiling, high-pressure steaming and excipient co-boiling methods, respectively (23).

Decoction solution of the crude drug and processed products was prepared as previously reported (24). Briefly, 200 g crude drug or processed product was immersed in water (w/v=1/8) for 1 h, then boiled for 30 min. Following filtration with 4 layers of bandage, the solution was collected and subjected to another decoction. Three decoctions were performed in total. Then, 40% (v/v) ethanol (95%) was added to the decoction solution, which was kept at 4°C for 2 days. Following centrifugation 700 × g at room temperature for 10 min, the supernatant was concentrated under reduced pressure in a 65°C water bath and dry extract was obtained.

For drug administration, HFLS-RA cells were divided into the following groups: i) Control group, in which the cells received no treatment; five treatment groups, in which the cells were treated with ii) 2 mg/ml crude drug, iii) 1.8 mg/ml water-boiled processed products, iv) 1.5 mg/ml high-pressure steamed processed products, v) 1.2 mg/ml excipient co-boiled processed products, vi) 40 µg/ml monomer component WF-1 or vii) 100 µg/ml monomer component WF-4; and viii) a positive control group, in which cells were treated with 150 µg/ml leflunomide, which is used for the treatment of RA (25) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For the drug treatment, WF-1, WF-4, and leflunomide were dissolved in DMSO, and dry drug extracts were dissolved in the DMEM medium, which were then used to incubate the cell cultures, respectively. The inhibition rates were calculated based on the data from the control group.