2.1 Preparation of herbal extracts for Yinxieling.
According to the normal clinical dosage, A: RADIX REHMANNIAE (Rehmannia glutinosa Libosch. ), B: RHIZOMA SMILACIS GLABRAE: (Smilax glabra Roxb.), C: FRUCTUS MUME (Prunus mume(Sieb.) Sieb.etZucc.), D: HERBA SARCANDRAE (Sarcandra glabra(Thunb.) Nakai), E: RADIX ARNEBIAE/RADIX LITHOSPERMI (Lithospermum erythrorhizon Sieb. et Zucc. ), F: RADIX PAEONIAE RUBRA (Paeoniae Radix Rubra ), G: RHIZOMA CURCUMAE (Curcuma phaeocaulis Val. ), H: RHIZOMA CHUANXIONG (Ligusticum chuanxiong Hort. ), I: RADIX ANGELICAE SINENSIS (Angelica sinensis(Oliv.)Diels ) , J: RADIX GLYCYRRHIZAE (Glycyrrhiza uralensis Fisch. ) with a weight ratio of 20:15:15:15:15:9:9:9:6:6. All these herbs were afforded by Guangdong provincial hospital of Chinese Medicine (Guangzhou, China) and kept in 4℃ refrigeration storage. The above herbs were soaked in 10 times the amount of water for 30min, and extracted for 1h, then filtered. The extraction process was repeated twice and filtrate was incorporated. The extract was concentrated to 200 mL by vacuum rotary evaporation at 65℃, anhydrous ethanol was added to adjust the alcohol concentration to 65%, and stood overnight at 4℃. The supernatant was concentrated to extract form, freeze-dried into powder and sealed at 4℃.
UPLC qualitative analysis.
UPLC analysis were performed to control the quality of the extration of YXL, using a Waters Acquity Ultra performance LC system (Waters, MA, USA), including quaternary solvent manager, sampler manager, column compartment and PDA detector, equipped with Waters Empower software. The separation was carried out on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d, 1.7 μm, Waters Corp., Milford, MA, USA). The column temperature was set at 40 ºC. The mobile phase consisted of 0.1 % formic acid (A) and acetonitrile (B). The gradient condition was as follows: 0-5 min, 4% B; 5-6 min, 4-8 % B; 6-9 min, 8-9 % B; 9-14 min, 9-10 % B; 14-21 min, 10-19 % B; 21-24 min, 19%B; 24-26 min, 19-21 % B; 26-27 min, 21-70 % B; 27-31min, 70%B, and finally reconditioning the column with 4 % B isocratic for 10 min after washing column with 100 % B for 4min. The flow rate was set at 0.35 mL min-1 and the injection volume was set at 5 μL. The detection wavelength was set at 290nm. FIGURE S1.
Mice and treatment
Male C57BL/6 mice with SPF grade of 7-9 weeks of age were purchased from Guangdong Experimental Animal Center (Guangzhou, China). During the experiment, the animal room and feed were provided by the Guangdong Academy of Traditional Chinese Medicine Sciences. The animal room was dry and ventilated, the ambient temperature was controlled between 22-26℃, the humidity was 50%-70%, drinking water and eating were free, and the light and darkness cycle was carried out for 12h, which matched the requirements of the animals to be tested.
Mice were randomly divided into 6 groups (n = 3 per group, repeat 3 times): control group, Imiquimod (IMQ) group, IMQ + Cyclosporin (CsA) group, YXL high dose group (IMQ+YXL-H), YXL medium dose group (IMQ+YXL-M) medium and low dose group (IMQ+YXL-L). All the mice had been oral treated for 7 days. Among them, oral treated group were given 25 mg/kg CSA, 3.0 g/kg(equal to about 17g raw herbs), 2.0 g/kg and 1.0 g/kg YXL, which were dissolved in distilled water, while control group and IMQ group were only given distilled water.
From day 3rd, All the IMQ treated mice were topically treated daily with 50 mg 5 % Imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd. Sichuan, China) on shaved back skin for five consecutive days. And the control group was given the base material.
Assessment of psoriasis-like dermatitis
IMQ was applied on the 3rd day after intragastric administration, and the psoriasis lesions and disease severity were scored on the 4th day. PASI score was modified for evaluating the mouse psoriasis-like skin lesions from human psoriasis area and severity index clinical scoring system. The modified PASI had three parameters, including skin erythema, scaling and thickness, which were used to assess the severity of the IMQ-induced psoriasis-like dermatitis. These indexes were evaluated independently using a defined rating system (0: none, 1: mild, 2: moderate, 3: marked, 4: severe)[20]. PASI score was the sum of these three numerical values.
Body weights were recorded daily at the first day when IMQ was treated on the mice. Weight gain or reduction was calculated, comparing to the first day body weight.