Plant materials and extract
A. capillaris was purchased from Kwangmyungdang Medicinal herbs (Ulsan, Korea) in September 2009. These materials were confirmed taxonomically by Professor Je-Hyun Lee of Dongguk University, Korea. A voucher specimen (AC-2009-EBM30) has been deposited at the Herbal Medicine Formulation Research Group at the Korea Institute of Oriental Medicine.
The 300 g sample of dried A. capillaris was extracted with 70% EtOH (3 L × 3) by sonication for 60 min. The extract solution was filtered through Whatman No. 2 filter paper (150 mm diameter, Buckinghamshire, UK) and evaporated to dryness using a rotary evaporator. The yield of 70% EtOH extract was 8.30% (24.89 g).
Chemicals and reagents
Chlorogenic acid and caffeic acid were purchased from Acros Organics (Pittsburgh, PA, USA). Hyperoside and scoparone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isoquercitrin and isochlorogenic acid A were purchased from Biopurify Phytochemicals Ltd. (Chengdu, China). The purity of the six compounds was determined to be ≥97% by HPLC analysis. HPLC-grade reagents, methanol, acetonitrile, and water were obtained from J.T.Baker (Phillipsburg, NJ, USA). Glacial acetic acid was of analytical reagent grade and was procured from Junsei (Tokyo, Japan).
Chromatographic conditions of HPLC analysis
The HPLC analysis was performed using a Shimadzu LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) consisting of a solvent delivery unit, an on-line degasser, a column oven, an autosampler and a PDA detector. The data processor used LC Solution software (version 1.24, Shimadzu Co., Kyoto, Japan). The analytical column used was a Gemini C18 (250 × 4.6 mm; particle size 5 μm; Phenomenex, Torrance, CA, USA) maintained at 40°C. The mobile phases were composed of 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The gradient flow rate was as follows: 0–5 min, 0–10% B; 5–30 min, 10–50% B; 30–35 min, 50–50% B; 35–40 min, 50–10% B. The flow rate and injection volume were 1.0 mL/min and 10 μL, respectively. The detection wavelength was set at 254 nm for hyperoside and isoquercitrin, at 320 nm for chlorogenic acid, caffeic acid, and isochlorogenic acid A and at 340 nm for scoparone.
Preparations of standard and sample solutions
Standard stock solutions of three phenolic acids (chlorogenic acid, caffeic acid and isochlorogenic acid A), two flavonoids (hyperoside and isoquercitrin) and one coumarin (scoparone) (Figure 1A) were dissolved in methanol at concentrations of 1.0 mg/mL and kept at 4°C. The working standard solutions were diluted to calibration curves in the concentration range of 0.78–50.00 μg/mL for chlorogenic acid, hyperoside and scoparone, 0.16–10.00 μg/mL for caffeic acid and isoquercitrin and 2.34–150.00 μg/mL for isochlorogenic acid A.
The 70% EtOH extract (20 mg) was dissolved in 70% EtOH (10 mL) and then filtered through a 0.2 μm membrane filter (Woongki Science, Seoul, Korea) before injection into the HPLC for simultaneous analysis.
Measurement of nitric oxide (NO) production in LPS-treated RAW264.7 cells
RAW 264.7 murine macrophage cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, U.S.A.) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL., NY, U.S.A.) supplemented with 5.5% (v/v) fetal bovine serum (FBS, Gibco BRL., NY, U.S.A.), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco BRL, NY, U.S.A.). The cells were seeded at densities of 2.5×103 cells/well in 96-well plates for the cytotoxicity assay. The cells were then incubated with different concentrations of herbal extracts (10, 50 and 100 μg/mL) for 24 hr. The vehicle control was 1% DMSO. After treatment, 10 μL of Cell Counting Kit-8 reagent (CCK-8, Dojindo, Japan) was added to each well and the plates were incubated for 4 hr. The absorbance was measured at 450 nm using a microplate reader (BenchmarkPlus, Bio-Rad Laboratories Inc., U.S.A.) and the percentages of viable cells were calculated. AC extracts in the range of 20–200 μg/mL did not cause cytotoxicity in RAW 264.7 cells and non-cytotoxic concentrations of herbal extracts were used for the subsequent experiments.
RAW 264.7 cells were seeded at a density of 5×105 cells in a 48-well plate for the NO assay. After culturing for 16 hr, the cells were stimulated with 1 μg/ml of LPS in the presence or absence of AC extracts (20–200 μg/mL) for 18 hr. N
G-methyl-L-arginine (NMMA; Sigma-Aldrich, Inc., MO, U.S.A.) and indomethacin (Sigma-Aldrich, Inc., MO, U.S.A.) were used as positive controls. A Griess reagent system (Promega., WI, U.S.A.) was used to measure the production of NO in the culture supernatants. Briefly, samples (50 μL/well) were incubated at room temperature with 1% sulfanilamide for 10 min and with 1% α-naphthylamine for 10 min. The absorbance was then evaluated at 535 nm using a calibration curve generated using the standards.
Measurement of histamine in PMA/A23187-treated MC/9 cells
The murine mast cell line MC/9 was maintained in DMEM media containing 10% (v/v) FBS, 0.05 mM 2-mercaptoethanol (Sigma Chemical Co., MO, U.S.A.), 10% (v/v) Rat T-STIM (BD Biosciences, MA, U.S.A.), 100 U/mL of penicillin and 100 μg/mL of streptomycin in a humidified 5% CO2 atmosphere. The MC/9 cells were plated in 48-well plates at a concentration of 2×105 cells per well. The cells were either untreated or treated with either phorbol 12-myristate 13-acetate (50 nM; PMA, Sigma-Aldrich, Inc., MO, U.S.A.) and A23187 (1 μM; Sigma-Aldrich, Inc., MO, U.S.A.) alone or PMA/A23187 (PA) + AC (50–200 μg/mL) for 24 hr. The histamine levels in the MC/9 cell supernatants were measured by ELISA in accordance with the manufacturer’s instructions (Oxford Biomedical Research, U.S.A.).
Animals and sensitization
Male Nc/Nga mice (8 weeks old) were obtained from Central Laboratory Animal Inc. (Seoul, Korea) and housed individually in an air-conditioned room maintained at 24 ± 2°C with 55 ± 15% relative humidity. The animals were allowed to acclimatize for 2 weeks before the experiments were initiated. All experimental procedures were carried out in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals and were approved by Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee. The approval number for the animal study was #10-052. The animals were cared for in accordance with the dictates of the National Animal Welfare Law of Korea.
AD-like skin lesions were induced in male Nc/Nga mice using Dermatophagoides farinae extract ointment (Biostir-AD, Biostir Co., Ltd., Kobe, Japan) [12]. At ten weeks of age the mice were grouped randomly into four groups with seven mice per group. The mice were divided into untreated (normal; 200 μL of 70% EtOH/mouse/day), D. farinae-sensitized (control; 70% EtOH), D. farinae-sensitized plus Protopic® ointment-treated (Protopic; 50 mg/mouse/day) and D. farinae-sensitized plus AC extract-treated (AC; 10 mg/mouse/day) groups. For sensitization, 50 mg Biostir-AD was topically applied on the upper dorsal skin and the back of the ears twice weekly for 4 weeks. The AC extract was dissolved 70% ethanol and applied every day for 4 weeks.
Dermatitis score
The dermatitis scores were assessed by evaluating the dorsal skin and the ears once a week for 4 weeks. The Eczema Area and Severity Index (EASI) scoring system was employed to evaluate the severity of dermatitis. The dermatitis score was defined as the sum of the scores for erythema/hemorrhage, edema, excoriation/erosion and scaling/dryness and scored as follows: no symptoms, 0; mild, 1; moderate, 2; and severe, 3 [4].
Histological analysis
Mice were anesthetized by pentobarbital sodium (Entobar inj., Hanlim Pharm. Co., Ltd., Korea) injection (i.p.). Blood samples were taken and the animals were sacrificed by exsanguination from the aorta. A complete gross observation was performed on all terminated animals. The blood samples were collected in a microtainer (Becton, Dickinson and Company, NJ, USA) containing K2-EDTA. Plasma samples were collected after centrifugation at 10000 rpm and stored at -80°C until they were further assayed. The dorsal skin and one ear of each mouse were removed and fixed in 10% (v/v) natural buffered formalin for 24 hr. The tissues were embedded in paraffin and then sectioned at 4 μm thickness. The tissue sections were then stained with hematoxylin & eosin (H&E) or toluidine blue to estimate epidermal inflammation (hypertrophy and infiltration by inflammatory cells) and mast cell counts, respectively. The dermal mast cell content was quantified by counting the numbers of toluidine-blue positive cells in randomly selected high power fields for each specimen.
Plasma levels of IgE and histamine
The plasma levels of IgE (Bethyl Laboratories Inc., U.S.A.) and histamine (Oxford Biomedical Research, U.S.A.) were measured by ELISA accordance with the manufacturer’s instructions.
Statistical analysis
The data are expressed as the mean ± SEM and were analyzed using a one-way ANOVA followed by the Bonferroni multiple comparison test. A P-value <0.05 was defined as statistically significant. All statistical analyses were performed using the SYSTAT® 8.0 program (SYSTAT Inc., Evanston, IL, U.S.A.).
