A total of 60 male Wistar rats (weight, 230–280 g; age, 7 weeks) were obtained from the Animal Experimental Center of Southern Medical University (Guangzhou, China) and were maintained at a constant temperature (24±2°C) with a relative humidity of 55±15% in a 12-h light/dark cycle. All rats were administered standard laboratory rodent feed and water ad libitum. A total of 50 rats were randomly assigned into 5 groups: A spontaneous group (n =10), a control (n=10), 100 mg/kg of Artemisia extract-treated group (n=10), 200 mg/kg of Artemisia extract-treated group (n=10), 400 mg/kg of Artemisia extract-treated group (n=10). This study was in accordance with the Southern Medical University Guidelines and Regulations on the Use and Care of Lab Animals. Ethical approval was obtained for this study from the Southern Medical University.

The dried flowering tips of A. apiacea were obtained from Kunming Institute of Medicine (Kunming, China) and were ground into fine powder using an electric blender (Thermo Fisher Scientific Inc., Waltham, MA, USA). Next, 20 g powder was ground and refluxed with 80% ethanol (600 ml) using a Soxhlet extractor (Thermo Fisher Scientific Inc.) for 8 h. The extraction process was repeated twice. The extract was separated by passing the mixture through filter paper. and the resulting Artemisia extract was freshly dissolved in a 5% gum arabic (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) solution prior to use.

All rats received an intraperitoneal (i.p.) injection of compound 48/80 (8 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) to induce a systemic anaphylactic reaction. The systemic anaphylactic reaction model was conducted according to the method previously described by Shin et al (9). Artemisia extract was administered 1 h prior to the injection of compound 48/80. Following the induction of anaphylactic shock, mortality rate was recorded over a 1-h period.

The rats were cervically dislocated under anesthesia with 40 mg/kg pentobarbital sodium). Peritoneal mast cells were harvested from male Wistar strain rats using decollation under 350 mg/kg chloral hydrate. A total of 5 ml of medium was injected into enterocoelia of every mouse and the abdomen was gently massaged for 5–10 min. The cells were purified using Percoll density centrifugation (Thermo Fisher Scientific, Inc.) at 8,000xg at 4°C for 10 min, as described previously (10). All cells (3×104 cells/tube) were incubated in physiological buffer solution (Beyotime Institute of Biotechnology, Haimen, China) for 20 min at 37°C. Artemisia extract, 0.1 ml phosphate-buffered saline (Wuhan Procell Science and Technology Co., Ltd., Wuhan, China) and 0.5 mg/ml compound 48/80 were blended altogether. The miscible liquids were incubated for 20 min on ice. The histamine content was measured by means of a fluorometric assay (ELISTA kit; E-EL-0032c, Elabscience, Wuhan, China) by an ELx800 microplate reader (Biotek Instruments, Inc., Winooski, VT, USA). Briefly, the miscible liquids were incubated for 20 min on ice and 80 µl of affinity chain enzyme-HRP at 37°C was added for 1 h. Next, every well was washed with scrubbing solution. The histamine content was measured by means of a fluorometric assay (Synergy 2 Microplate Reader, Bio-Tek, USA) at 450 nm.

Scratching behavior was surveyed using a previously described method (11). The Artemisia extract was administered orally and scratching behavior was observed for 1 h. Next, 10 µg/0.02 ml compound 48/80, 100 nmol/0.02 ml histamine and 100 nmol/0.02 ml serotonin (both from Sigma-Aldrich) were injected intradermally into the rostral part of the back of the rats. Scratching behavior of rat was observed for a further 1 h, and evaluated using a HF-200 camera (Canon Legria, Canon Inc., Tokyo, Japan).

Artemisia extract was administered orally and 1 h passed before subsequent experiments. Next, 0.5 mg/0.02 ml compound 48/80 (i.p.), 10 nmol/0.02 ml histamine (i.p.) or 10 nmol/0.02 ml serotonin (i.p.) was administered into the rostral part of the back. In addition, 2% Evans blue solution (Beyotime Institute of Biotechnology) was intravenously injected into each animal. After a pentobarbital sodium injection (1.5%; Sigma-Aldrich) the rats were sacrificed using cervical dislocation and after 30 min, then the ‘bluing’ reaction diameter was measured at the injection site.

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was tested using one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference.

To determine the effect of artemisia extract on the systemic anaphylactic shock induced by compound 48/80, the mortality rate within 1 h after compound 48/80 injection was recorded. The administration of 8 mg/kg compound 48/80 (i.p.) increased the mortality rate in the allergic model rats (Table I). However, the Artemisia extract pretreatment reduced the mortality rate of allergic model rats in a concentration- and time-dependent manner (Table I).

A fluorometric assay was performed to investigate the effect of Artemisia extract on histamine release in the allergic model rats. Treatment with 0.5 mg/ml compound 48/80 significantly increased the histamine release in the allergic model rats (Fig. 1). Artemisia extract pretreatment (100, 200 and 400 mg/kg) significantly reduced the histamine release in the allergic model rats, compared with the untreated control (Fig. 1).

To elucidate the effect of Artemisia extract on the scratching behavior of allergic model rats, the scratching behavior was induced by treatment with compound 48/80. Furthermore, the Artemisia extract pretreatment (200 and 400 mg/kg) significantly reduced the scratching behavior of the allergic model rats (Fig. 2).

To assess the effect of Artemisia extract on the vascular permeability of allergic model rats, the vascular permeability was evaluated following 0.5 mg/0.02 ml compound 48/80 injection. Compound 48/80 effectually augmented the vascular permeability of allergic model rats (Fig. 3). The Artemisia extract pretreatment (200 and 400 mg/kg) significantly reduced the vascular permeability of allergic model rats (Fig. 3).

To examine the effect of Artemisia extract on the scratching behavior of allergic model rats, scratching behavior was induced with 100 nmol/0.02 ml histamine or serotonin injection. Histamine or serotonin significanlty increased the scratching behavior of allergic model rats (Fig. 4A and B). The Artemisia extract pretreatment (400 mg/kg) significantly reduced the scratching behavior induced by histamine (Fig. 4A). Furthermore, the Artemisia extract pretreatment (200 and 400 mg/kg) significantly reduced the scratching behavior induced by serotonin (Fig. 4B).

To analyze the effect of Artemisia extract on the vascular permeability of allergic model rats, the vascular permeability was investigated after 10 nmol/0.02 ml histamine or serotonin injection. Histamine or serotonin markedly increased the vascular permeability of allergic model rats (Fig. 5A and B). The Artemisia extract pretreatment (400 mg/kg) significantly reduced the vascular permeability induced by histamine (Fig. 5A). Furthermore, the Artemisia extract pretreatment (200 and 400 mg/kg) significantly reduced the vascular permeability induced by serotonin (Fig. 5B).