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.