Chemicals and reagents
Anti-DNP IgE, DNP-human serum albumin (HSA), Evans blue, histamine phosphate, hyaluronidase, p-dimethylaminobenzaldehyde, fluocinonide ointment, disodium cromoglycate (DSCG), A23187, and PMA were purchased from Sigma (St. Louis, MO, USA). KU812 cells were purchased from the Shanghai Institute cell library. Iscove’s Modified Dulbecco’s Medium (IMDM), penicillin, and streptomycin were purchased from Gibco (Invitrogen Corporation, Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). Cytokine-specific enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems (USA). DMY was purchased from Shanghai Tauto Biotech. Co., Ltd. (Shanghai, China), and was determined to be greater than 98% pure by high-performance liquid chromatography (HPLC) analysis. Other reagents used were of analytical grade.
A. grossedentata, S. divaricata, S. flavescens, A. sinensis, O. japonicus, and C. officinalis were purchased from the Beijing Tongrentang Co., Ltd. (Beijing, China). All herbs were authenticated by Professor Y. Peng, a medical botanist at the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Science (CAMS), Beijing, China. The optimized ratio of the herbs was determined to be 3:2:2:1.7:1.2:1.2, using a Design-Expert regression model directed by hyaluronidase inhibitory activity. The formulation (100 g) was extracted by thermal recycling with 70% ethanol (1:13) for 140 min at 68 °C and filtered. The AEF was concentrated in a rotary vacuum evaporator, lyophilized, and stored at − 20 °C until use.
The AEF was standardized based on its DMY content. Chromatographic separation was carried out on an Agilent 1260 LC series system (Agilent Technologies, Palo Alto, CA, USA) equipped with online vacuum degasser, quaternary pump, autosampler, temperature-controlled column compartment, and a diode array detector. Agilent Technologies ChemStation software for LC (B.02.01) was used. Chromatographic separation was achieved on an Agilent RP C18 (150 mm × 4.6 mm, 5 μm) using a mobile phase consisting of water to acetic acid (99.8:0.1, v/v) (A) and methanol (B). The gradient program consisted of 60% (B) for 0–30 min. The flow rate was 0.5 mL/min, and the column temperature was set to 25 °C. The detection wavelength was 290 nm. DMY was detected at around 5.6 min in this system. The DMY content of the AEF was 10.25 ± 0.83 mg/g (n = 3).
KU812 cells were grown in IMDM culture medium supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin at 37 °C in 5% CO2. KU812 cells were pre-treated with the AEF (50, 200, or 300 μg/mL) for 2 h prior to stimulation with 40 nM PMA and 1 μM A23187 for different periods of time. The AEF was diluted in nuclease-free double-filtered distilled water, whereas PMA and A23187 were dissolved in DMSO.
Hartley guinea pigs (250 ± 10 g) and Sprague-Dawley rats (200–250 g) were obtained from the Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). These animals were housed under standard conditions with a 12/12-h light/dark cycle at a temperature of 22 ± 1 °C with 55 ± 10% humidity and were given standard laboratory feed (Beijing Jinmuyang Laboratory Animals, Inc., Beijing, China) and water ad libitum. The animal certification number was SCXK (Jing) 2010-0001. Animal protocols were developed in accordance with the institution’s guidelines for the care and use of laboratory animals and were approved by the local Animal Care and Use Committee.
Inhibition of hyaluronidase activity
Hyaluronidase inhibition was determined by measuring the amount of β-N-acetylglucosamine formed from sodium hyaluronate, using a spectrophotometer (Kakegawa 1992). Bovine hyaluronidase (500 μL of 7420 units/mL in 0.1 M acetate buffer; pH 5.6) was mixed with 100 μL calcium chloride (0.25 mM) and then incubated in a water bath at 37 °C for 20 min. The indicated test sample was added in a volume of 500 μL, and the mixture was incubated in a water bath at 37 °C for 20 min, after which sodium hyaluronate (500 μL of 0.5 mg/mL in 0.1 M acetate buffer; pH 5.6) was added. After a 30-min incubation in a water bath at 37 °C, 100 μL of 0.4 M sodium hydroxide was added to stop the reaction, and the mixture was placed in an ice-water bath for 5 min. Next, 500 μL acetylacetone was added, and the mixture was incubated in boiling water for 30 min to produce a chromogenic reaction. After cooling to 25 °C, 1.0 mL of p-dimethylaminobenzaldehyde solution was added to the reaction mixture for 20 min at 25 °C. The optical density of the reaction mixture was measured at 555 nm using a microplate reader (RT-6000; Leidu, Shenzhen, China). All determinations were performed in triplicate.
Measurement of pruritus
Hartley guinea pigs (250 ± 10 g) were randomly divided into four groups with eight animals/group; these groups consist of model control group (physiological saline, 100 mg/cm2), positive control group (DSCG, 100 mg/cm2), low dosage experimental group (AEF, 50 mg/cm2), middle dosage experimental group (AEF, 100 mg/cm2) and high dosage experimental group (AEF, 150 mg/cm2) applied to shaved dorsal skin sites (2 cm2) on their back right feet for 2 days. On the third day, the samples were applied to the shaved sites for 10 min, followed by 0.05 mL of increasing concentrations of histamine phosphate (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, or 0.1%), each of which was dripped onto the test site for 3 min. The scratching behavior induced by histamine phosphate was recorded, and the itch threshold was the level required to produce itching (Hu and Zhong 2013).
Induction of the PCA reaction
Sprague-Dawley rats (200–250 g) were divided into six groups with eight rats/group: the untreated control group, the model group (distilled water, 100 mg/cm2), the positive control group (fluocinonide ointment, 50 mg/cm2), and the high AEF (100 mg/cm2), middle AEF (50 mg/cm2), and low AEF (25 mg/cm2) dose groups. Each group had the appropriate treatment applied to the skin at three dorsal skin injection sites, which were outlined with a water-insoluble red marker. One hour later, the PCA reaction was generated by sensitizing the skin with an intradermal injection of 0.5 μg anti-DNP IgE into each of the sites. After 48 h, this was followed by a tail vein injection of 100 μg DNP-HSA in phosphate-buffered saline containing 4% Evans blue. The rats were sacrificed 30 min after the administration of DNP-HSA. The skin at the injection site was removed for measurement of the pigment area. The amount of dye was determined by colorimetry after extraction using a 1:1 mixture of acetone and physiological saline (Shin et al. 2004). The absorbance of the skin extract was measured at 620 nm in a microplate reader (RT-6000; Leidu), and the amount of dye was calculated using an Evans blue calibration curve.
Evaluation of skin repair activity
Hartley guinea pigs (250 ± 10 g) were randomly divided into five groups with eight animals/group: a blank control group (physiological saline), a model control group, and groups exposed to different dosages of AEF. The hair on the back of the neck of each guinea pig was shaved the day before the experiment to expose about 2 cm2 skin. Each group (except for the blank control group) had 150 μL acetone to ether (1:1) solution dripped onto the shaved skin. Test samples (0.1 mL/cm2) were smeared onto the shaved skin; 0.1 mL/cm2 distilled water was applied to the model control group. Treatments were administered twice daily for five consecutive days. On the fifth day, the skin moisture loss of the shaved skin was tested 20 min after sample administration. Protection rates (%) were calculated for each study group.
Levels of IL-6 and IL-8 in the culture medium were quantified by specific sandwich ELISAs. Briefly, KU812 cells were stimulated with PMA (40 nM) plus A23187 (1 μM) for 12 h, with or without pre-treatment with AEF (Rasheed et al. 2009). The ELISAs were performed using the culture supernatants, in accordance with the manufacturer’s instructions (R&D Systems). Plates were read at 450 nm using the RT-6000 microplate reader (Leidu).
The results were expressed as mean ± standard error of the mean (S.E.M.). The statistical significance of the differences between the treated and control groups were calculated using Student’s t test. Results with P < 0.05 were considered statistically significant.