Senkyunolide A and Z-ligustilide were obtained from Corescience Inc. (Seoul, Korea). The purities of the standards were more than 98.0%. HPLC system (Agilent Technologies Mfg GmbH&Co.KG, Waldbronn, Germany) consisted of a 1260 quaternary pump, autosampler and multiple wavelength detector carried out the qualitative and quantitative analyses of two compound in C. officinale. Chromatographic separation was performed on a Hector-M C18 column (250 mm × 4.6 mm I.d.; 5 µm). UV detection was set at 290 nm. The mobile phase was the gradient solvent system consisting of solvent A (0.1% formic acid in water) and solvent B (ACN) as follows: linear gradient 70 – 20 – 100% A (0 – 30–34 min) and isocratic 70% A (34 – 40 min). The flow rate was 1.0 mL min⁻¹ and 10 µL of the pulverized 70% ethanolic extract of C. officinale (10 mg/mL) and two standards (500 µg/mL), which were filtered through a 0.45-µm PVDF membrane, were injected using the autosampler for the analyses.

C. officinale extract (1 kg) was extracted in 70% ethanol (1 l) in the Soxhlet apparatus for 3 hr at 65~70 ℃, filtered and freeze-dried to 100 g of powder. It was packaged in the sterilized pouch until the further study. Briefly, for preparing the cream formulation of C. officinale extract (COE), white petrolatum (94.45 g) and sorbitan sesquioleate (0.5 g) were mixed by mild agitation with a magnetic stirring bar (2500 rpm) for 40 min at 80 ℃ and they were cooled at 75 ℃. The pulverized 70% ethanolic extract of C. officinale (0.05 g) and propylene glycol (4 g) were added and mixed by homogenizing at 2500 rpm for 30 min. After adding of propylene glycol (0.5 g), the mixture was blended by stirring at 250 rpm for 10 min at 75 ℃, followed by cooling and stirring at 2000 rpm and 30 ℃ for 10 min. Vehicle cream was prepared by the same procedure except for the addition of the 70% ethanolic extract of C. officinale. COE was stored in the refrigerator (−4 ℃) before the in vivo experiments. A commercially available imiquimod (IMQ) cream (Aldara®, iNova Pharmaceuticals Pty LTD., Chatswood, Australia), clobetasol (CLO) (Dermovate®, Glaxo Operation, UK) and tacrolimus (TAC) (Protopic®, Astellas Pharma Inc., Japan) were gifted from Hankook Korus Pharm, Co., LTD. (Chuncheon, Korea).

BALB/c mice were purchased from Orient Bio Inc. (Sungnam, Korea). Mice were kept under specific pathogen-free conditions and provided with water and food ad libitum. All mice used in the experiments were 8 weeks of age and bred for 7 days in animal care center (temperature 23 ± 2C, humidity 50 ± 5%, 12 h light-dark cycle), Hongcheon Institute of Medicinal Herb (Hongcheon, Korea) before the beginning of the experiments. All experiments were approved by the Institutional Animal Care and Use Committee of Hongcheon Institute of Medicinal Herb (No. 15–04). They were separated into four groups: 1) Vehicle group was the control group treated with control cream; 2) IMQ group was the model group received a daily dose of 62.5mg/day of IMQ mixed with control cream; 3) COE group was the model group treated with only COE; 4) IMQ + CLO group was the model group received CLO cream (17-propionate 0.5 mg/1 g) 6 h later after IMQ treatment; 5) IMQ + TAC group was the model group received TAC cream (tacrolimus 0.1%) 6 h later after IMQ treatment; 6) IMQ + COE group was the model group received COE 6 h later after IMQ treatment. All the experiments were treated for every 2 days, during 6 days.

The samples were rubbed 1 day after the removal of ear and back hairs for 0, 2, 4 and 6 days. The ear and back thickness were measured using Digimatic Thickness Gage (model no. 547-500S, Mitutoyo, Japan) using the hair removal products (Veet®, Oxy Reckitt Benckiser, Slough, UK). The erythema severity was expressed using Dermacatch® (Colorix, Swiss).

The severity of psoriasis was measured by the Psoriasis Area and Severity Index (PASI). Briefly, the ear and back thickness, and erythema were scored independently and calculated according to PASI index (http://pasi.corti.li, ver. 1.7.1).

Ear and back tissues were washed with PBS and fixed in 10% neutral buffered formalin (NBF) for 24 hours at room temperature. The tissues were dehydrated by gradually soaking them in alcohol and xylene and then embedded in paraffin. The paraffin-embedded specimens were cut into 5 µm sections, stained with hematoxylin and eosin (H&E) and observed with a digital light microscope (Axio Scan. Z1, Zeiss, Germany).

For evaluation of the changes of myeloperoxidase (MPO) and keratin 6A, sections were deparaffinized and incubated with mouse anti-MPO (Abcam, Cambridge, UK) and mouse anti-keratin 6A (Abcam, Cambridge, UK) in humidified chamber at 4 ℃ for 24 hours, followed by biotinylated goat anti-mouse IgG-B (SantaCruz Biotechnology, Santa Cruz, USA) at 4 ℃ for 24 hours. Immunostaining was performed by tris-HCl (pH 7.4) buffered with 0.05% 3,3′-diaminobenzidin (Sigma-Aldrich, St. Louis, USA) and 0.01% HCl 1 hour after the incubation with an avidin-biotin-HRP complex (Dako, Carpinteria, USA), and observed with a digital light microscope (Axio Scan. Z1, Zeiss, Germany).

Total mRNA was extracted from 50~100 mg of back skin with 1 ml of Trizol reagent (Invitrogen, USA) by homogenization. The mRNA was transcribed to cDNA with cDNA reverse transcription kit (Qiagen, Japan). IFN-γ, c-fos and IκB-α mRNA levels were measured by a SYBR Green (Applied Biosystems, USA) and 7500 Real-time Thermal Cycler (Applied Biosystems, USA). The specific primers were: IFN-γ: forward primer, 5′-TCA AGT GGC ATA GAT GTG GAA GAA-3′, reverse primer, 5′-TGG CTC TGC AGG ATT TTC ATG-3′; c-fos: forward primer, 5′-CCT TCG GAT TCT CCG TTT CTC-3′, reverse primer, 5′-TGG TGA AGA CCG TGT CAG GA-3′; IκB-α: forward primer, 5′-TGT CTG CAC CTA GCC TCT ATC CA-3′, reverse primer, 5′-ATC AGC ACC CAA AGT CAC CAA-3′; β-actin: forward primer, 5′-GGC TGT ATT CCC CTC CAT CG-3′, reverse primer, 5′-CCA GTT GGT AAC AAT GCC ATG T-3′. The results were analyzed by One step system software ver. 2.1 (Applied Biosystems, USA).

Data were expressed as the mean ± SD unless indicated otherwise. One-way ANOVA with the Tukey-HSD post hoc test was performed to compare the differences between two groups using GraphPad Prism 6.0 version (GraphPad Software, Inc., San Diego, CA, USA). A value of P < 0.05 was considered significant at the 95% confidence level (^*p < 0.05).

The LC chromatogram of the 70% ethanolic extract of C. officinale used in the present study was shown in Fig. 1, and senkyunolide A and Z-ligutilide were detected as the major components of C. officinale.7 The thickness of back and ear was unequivocally increased 2–3 days after IMQ application (62.5 mg/day) with the characteristic changes of psoriasis lesions, such as hyperkeratosis and desquamation compared to the positive controls, clobetasol and tacrolimus (Fig. 2a).12 COE ameliorated the thickness and scaling of back and ear, while back and ear erythema was not changed compared to IMQ group (Fig. 2a). This result related to erythema may be due to the colorization of COE on back and ear. It was consistent of the results of the independent scores in a representative experiment (Fig. 2b). After 6 days, the relative changes of the thickness of back and ear, erythema and scaling were scored together on a scale from 0 to 4 and applied to the PASI index (Fig. 2b). The PASI score of only IMQ-induced group (PASI score = 8.87) was significantly increased decreased compared to vehicle group (0.15). However, it was reversed psoriasis-like symptoms after the treatment of COE (5.56).

The effect of COE on the psoriasislike symptom induced by IMQ symptom was confirmed by the histological (H&E staining) and immunohistochemical analyses on back and ear. The treatment of IMQ locally induced and exacerbated the symptoms of psoriasislike skin lesions, such as epidermal acanthosis, the presence of nuclei in the stratum corneum (parakeratosis) in the back and ear skin, while COE significantly ameliorated these symptoms (Fig. 3a and 3b). Keratin 6A which is one of the psoriasis-typical markers was overexpressed in the IMQ-treated group (Fig. 4a and 4b).13 In the COE-treated group, the abnormal proliferation induced by IMQ was significantly ameliorated. Also, the overexpressed MPO level, which is another marker for the psoriasis-like skin lesion, was reversed after the treatment of COE (Fig. 4c and 4d).

We next examined the protective mechanism of COE against psoriasis-like lesion. It has been previously reported that IMQ has its immunomodulatory activity via the induction of the release of IFN-γ.14 Though IMQ may be effective in the infected disease, such as leishmaniasis, but the prolonged exposure may cause psoriasis-like symptoms. In the present study, IMQ induced the expression of IFN-γ, c-fos and IκB-α, which are highly secreted from immune cells, such as dendritic cells, macrophages, and T cells, and related to the stimulation of the keratinocytes proliferation.1516 However, COE significantly reduced the expression of IFN-γ, c-fos and IκB-α (Fig. 5).