A specimen of A. senticosus was foraged by Dr Qinglong Meng from the Liaodong planting base of Chinese Medicinal Materials (Qingyuan, China) and Professor Yueying Ren identified the specimen. The specimen was stored in the Cultivation and Breeding of Medicinal Plants Laboratory in State Administration of Traditional Chinese Medicine (Changchun, China) for experimental applications. The specimen was crushed to powder then passed through mesh sieves, the pass rate of the 20 mesh pharmacopoeia sieve was ≥90% and the 80 mesh pharmacopoeia sieve was ≤10%. The pharmacopoeia sieves were provided by Xinxiang Zhuohang Precision Mesh Filter Co., Ltd. (Henan, China). The specimen then underwent 60°C thermostatic drying for 6 h by blowing air in a thermostatic oven (model, DHG-92438-2; Shanghai Fuma Experimental Equipment Co., Ltd., Shanghai, China). The dried powder was extracted with supercritical CO₂ and placed in distilled water with 0.01–0.05% of mixed enzymes (protease: Cellulose: pectinase in the ratio 3:1.5:0.5). Continuous upstream extraction was performed using distilled water (material: Liquid, 1:18; extraction temperature, 75°C; extraction time, 2 h). The extracted liquid was filtered, adsorbed with non-polar macroporous resin, washed with 11.2 ml distilled water and filtered through a 0.22 µm membrane. The solid content was concentrated to 20% by 45°C thermostatic drying for 4 h in a thermostatic oven. The ASPS extraction yield was 10.76%.

Sephadex-G75 gel filtration (column size, 16×500 mm; internal diameter, 15 mm) was performed using a high performance liquid chromatography (HPLC) system (ÄKTAFPLC with Fraction Collector Frac-920; all GE Healthcare, Chicago, IL, USA) to purify active molecules from the ASPS extract. The molecules were separated using an Ultrahydrogel^™ 500 column (7.8×300 mm), which was provided by Waters GmbH (Eschborn, Germany), on the HPLC system using a 0.9% NaCl water solution and distilled water. The column was operated at 35°C. The samples were of 2 mg ASPS were diluted in 1 ml 0.9% NaCl water solution and a total of 20 µl was injected onto the column. The column was operated at a maximum of 1.6 MPa with a flow rate of 0.5 ml/min. Gas chromatography was then used to analyse the composition of ASPS. Firstly, the samples were hydrated with ethanol (Nanjing Chemical Reagent Co., Ltd., Nanjing, China) for 12 h at 22°C and silylanised with silylate (Shandong Baiqian Chemical Co., Ltd., Shangdong, China) for 30 min at 22°C. Secondly, the samples were analysed with aVarian 7890 gas chromatography spectrometer. The gasification temperature was 300°C. The samples were then separated in high purity nitrogen in a SE-54 column (15 m ×0.2 mm ×0.25 µm) at 120°C for 2 min, increasing by 8°C/min until the column reached 250°C, then 250°C for 30 min. An infrared spectrometer (Perkin Elmer, Inc., Waltham, MA, USA) was used to record the infrared spectrum of ASPS.

The tumour cell lines Crocker sarcoma S₁₈₀, hepatic carcinoma H₂₂ and uterine cervical carcinoma U₁₄ were provided by a drug clinical trial from Jilin Province Cancer Hospital (Changchun, China) and cultured fro 7 days at 37°C in the Cultivation and Breeding of Medicinal Plants Laboratory.

A total of 360 female Kunming mice, 6–7 weeks old and weighing 18–22 g, were provided by the Changchun Institute of Biological Products (Changchun, China; license number: SCXKJi 2013-0001). The mice were maintained in clean plastic cages in the laboratory of the College of Chinese Medicinal Materials at Jilin Agricultural University (Changchun, China). The temperature was controlled at 22±2°C with a 12 h light/dark cycle and relative humidity of 50–60%. Standard rodent chow and water were freely accessible. All mice were maintained for an acclimatization period of ~7 days under normal laboratory conditions. All mice were treated according to the National Regulations on the Usage and Welfare of Animals (29) and study protocols were approved by the Animal Ethical and Welfare Committee of the College of Chinese Medicinal Materials, Jilin Agricultural University prior to the experiments (approval no. 2013-006).

On day 0, each mouse was injected subcutaneously with 1×10⁸ tumour cells in 0.2 ml normal saline. A total of 50 Kunming mice used for each cell line and the same number of tumour cells were injected regardless of the cell line used. The inoculated mice were subsequently randomly divided into five groups (10 mice/group) as follows: Model group, treated with normal saline; positive control group, treated with cyclophosphamide (CTX; 30 mg/kg); and treatment groups, administered with 200, 100 or 50 mg/kg ASPS. Non-inoculated mice were divided into the normal group, treated with normal saline, and the negative control group, treated with 200 mg/kg ASPS (n=10/group). CTX and ASPS were administered intragastrically once daily for 10 days. All mice were sacrificed on day 11. Mice were weighed every 2 days throughout the treatment period and, following sacrifice, the tumours, spleens and thymuses were harvested and weighed. The tumour growth inhibition rates and immune organ indices were calculated using the following equations: Inhibition rate (%) = [(Mean tumour weight in the model group-mean tumour weight in the treatment group) / mean tumour weight in the model group] ×100. Immune organ index = Organ weight (mg) / body weight (g) (30,31). Blood was harvested from mice in each group and centrifuged at 1,409 × g at room temperature for 10–15 min. The serum levels of tumour necrosis factor (TNF)-α (cat. no. MTA00B), interleukin (IL)-2 (cat. no. M2000), IL-12 (cat. no. M1270) and interferon (INF)-γ (cat. no. MIF00) were determined using commercial ELISA kits according to the manufacturer's protocols (R&D Systems, Inc., Minneapolis, MN, USA).

A total of 50 female Kunming mice were inoculated, divided into groups and treated as above. For ascites tumour-bearing mice, there were no non-inoculated mice ILS was calculated using the following equation: ILS %=[(Mean survival days of treated group-mean survival days of model group)/mean survival days of model group] ×100 (31).

A total of 40 Kunming mice (20 male and 20 female; 6–7 weeks old; 20–22 g) were randomly divided into two groups: Administration and blank (n=20 in each). All mice were fasted for l2 h prior to experimentation, but were not deprived of water. Mice in the administration group were treated with 10 g/kg ASPS once daily for 14 days. Mice in the blank group were administered with 0.2 ml normal saline daily. Observations of the limb flexibility, feeding, diarrhoea, death, hair finish (whether the hair is dry and falling out), and stool and urine secretions were recorded. All mice were sacrificed on day 15. The major organs, including the heart, liver, spleen, lungs and kidneys of the mice, were tested for toxicity following the sacrifice of the mice.

Data are presented as the mean ± standard deviation. Differences between groups were analysed using the Least-Significant Difference post-test. Statistical analyses were conducted using SPSS (version 17.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The single, narrow, symmetrical peak of the analysed ASPS sample revealed its homogeneous composition (Fig. 1). The molecular weight of ASPS was 10 kDa and its monosaccharide composition was demonstrated to be (Arabinose: Xylose: Glucose: Mannose)=7.1:22.3:7.6:1.0 (Fig. 2).

Treatment with high, medium and low dose ASPS had a significant inhibitory effect on tumour growth in mice inoculated with S₁₈₀ (Table I), H₂₂ (Table II) and U₁₄ (Table III) compared with the model group (all P<0.01). The greatest inhibitory effect in S₁₈₀ and U₁₄ tumour-bearing mice was achieved with the medium dose (100 mg/kg; Tables I and III), whereas the high dose (200 mg/kg) had the greatest effect in H₂₂ tumour-bearing mice (Table II).

Treatment with high, medium and low doses of ASPS increased the immune organ indices in S₁₈₀, H₂₂ and U₁₄ solid tumour-bearing mice. Spleen and thymus indices in the CTX group were significantly decreased compared with the normal, control and model groups for all cell lines (P<0.01; Figs. 3–5). Spleen and thymus indices in the high, medium and low ASPS treatment groups were significantly increased compared with the positive control group for all cell lines (P<0.01; Figs. 3–5).

Treatment with high, medium and low doses of ASPS significantly increased serum IL-2 levels in S₁₈₀, H₂₂ and U₁₄ solid tumour-bearing mice compared with the normal, control, CTX and model groups (S₁₈₀, P<0.05, P<0.05 and P<0.01, respectively; H₂₂ and U₁₄, all P<0.01; Fig. 6). IL-2 levels were significantly decreased in the CTX group compared with the normal, control and model groups (S₁₈₀, P<0.01, P<0.05 and P<0.05, respectively; H₂₂ and U₁₄, all P<0.01; Fig. 6). High and medium doses of ASPS significantly increased serum IL-12 levels in H₂₂ and U₁₄ solid tumour-bearing mice compared with the normal, control, CTX and model groups (P<0.01; Fig. 7); furthermore, IL-12 levels were significantly increased with high or medium-dose ASPS treatment in S₁₈₀ tumour-bearing mice compared with the control, CTX and normal groups (P<0.01; Fig. 7). No significant differences in serum TNF-α levels were observed between groups, irrespective of treatment (Fig. 8). No significant differences in serum INF-γ levels were observed in S₁₈₀ and U₁₄ solid tumour-bearing mice in different treatment groups (Fig. 9). However, treatment with high (P<0.05), medium (P<0.01) and low (P<0.05) doses of ASPS significantly increased INF-γ expression in H₂₂ solid tumour-bearing mice compared with the positive control group (Fig. 9).

Treatment with medium (P<0.01) and low (P<0.05) doses of ASPS had inhibitory effects on the growth of S₁₈₀ in ascites tumour-bearing mice compared with the model group (Table IV). Treatment with high (P<0.01), medium (P<0.01) and low (H₂₂, P<0.05 and U₁₄, P<0.01) doses of ASPS had an inhibitory effect on the growth of H₂₂ and U₁₄in ascites tumour-bearing mice (Tables V and VI). The greatest inhibitory effect in S₁₈₀ tumour-bearing mice was observed with the ASPS medium dose (100 mg/kg; Table IV). The greatest inhibitory effect in H₂₂ and U₁₄ tumour-bearing mice was observed with the ASPS high dose (200 mg/kg; Tables V and VI).

All mice in the oral administration groups survived until the end of the 14-day observation period. Mice were sacrificed on day 15 and no visible lesions were observed in the vital organs. These results suggest that mice are able to tolerate >10 g/kg ASPS without adverse effects. According to the standard classification of acute toxicity of chemical substances (32), ASPS is a non-toxic material. No abnormalities in eating, drinking, stool, urine, disposition, mobility (data not shown) or body weight (Table VII) were observed in any groups.