Materials
SAA (the chemical structure is presented in Fig. 1) was provided by Shandong Target Drug Research Co. Ltd (Shandong, China). The natural extracted SAA is low in content. Thus, SAA was synthetized through chemical conversion of raw material salvianolic acid B. This method could be scaled up for large-scale industrial production. The purity of SAA was determined to be higher than 98% by HPLC method with external standard. In this study, SAA was dissolved in 5% glucose solution.
Experimental animals
BALB/c mice (6–7 weeks old) were provided by Experimental Animal Center of Shandong University (Jinan, China). Beagle dogs (6–9 months old) were purchased from Guangzhou Institute of Pharmaceutical Industry. All animals were acclimated for at least 1 week at a temperature of 22 ± 2℃ and relative humidity of 42%~68%. All animals were housed in cages with food and tap water ad libitum. Room were well-lit and ventilated. All experiments were performed according to the guidelines specified in the Good Laboratory Practice Regulations by China Food and Drug Administration (CFDA) and recommendations of the National Institutes of Health Guide regarding the Care and Use of Laboratory Animals. The permission of animal use was approved by Office of Experimental Animal Management Committee of Shandong Province, China (License number: SYXK [Lu] 20,180,028). The Animal Ethics Committee of Yantai University gave consent to all animal protocols.
Acute toxicity studies in mice and dogs
A simple randomization was performed by Excel software. BALB/c mice (60 females, 60 males) were randomly assigned into the following groups (n = 20): Control, SAA (1600, 1340, 1130, 950, 800 mg/kg). After assigned to groups, mice were administered intravenously with different doses of SAA. For mice in control group, mice were administered with 5% glucose injection with an equal volume.
Six Beagle dogs (3 females, 3 males) were used in this study with approximate lethal dose method [15]. The effective dose of SAA was 10 mg/kg in rat [8, 16]. Thus, the equivalent dose of SAA in dog was 3.3 mg/kg according to the conversion of body surface area. We selected 60 mg/kg as initial dose, approximately 18 times higher than the effective dose. Successively, SAA was administered at doses of 90, 135, 202, 303, 455, 682, 1023, 1535 mg/kg, with an increasing proportion of 50%. After administration, animals were observed for changes in clinical signs and body weight. All animals were continuously observed for 14 days after administration. Finally, gross anatomy observation was carried out on dead and surviving animals at the end of the experiment. Organs with obvious pathological changes were examined histopathologically. During the experiment, the animal care staff and those who administer treatments were blind to the group allocation.
Subchronic toxicity study
Study design
This work was conducted according to the “Technical guidelines of chronic toxicity for traditional Chinese medicine and natural drugs” (SFDA, 2005) [17]. A simple randomization was performed by Excel software. In brief, twenty-four Beagle dogs (12 female, 12 males) were randomly divided into four groups: Control, SAA 20, 80 and 300 mg/kg, with 6 dogs in each group. Animals were infused with SAA, once-daily for a whole month. At the end of administration, 16 dogs (2 dogs/sex/group) were sacrificed, and after the 2-week recovery period, autopsy were conducted on the remaining dogs (1 dog/sex/group) to examine delayed occurrence, persistence, and whether the toxicities were reversible. During the experiment, animal care staff and those who administer treatments were blind to group allocation.
Clinical observations
Animals were observed for changes in clinical signs, toxic reaction and mortality. Body weight was measured once a week, while food intake was determined every day. In addition, the body temperature measurement, ophthalmic examinations, urinalysis and electrocardiographic examination were carried out before administration, days 15 and 29 after the administration and recovery period, respectively.
Laboratory testing
Blood was collected for hematology and serum biochemistry analyses from the forelimb veins of animals. Hematological parameters and biochemical parameters were consistent with previous reports [18, 19].
Necropsy and histopathology
On days 29 and following the 2-week recovery phase, a total of 4 dogs(2 dogs/sex/group)in each group and the remaining animals in recovery period were sacrificed and dissected. Firstly, visual observation was performed, and then, absolute and relative organ weights were determined for key organs including brain, heart, liver, lung, kidneys, adrenals, thymus, spleen, testes, epididymis, uterus and ovaries. Other organs of the following were collected: spinal cord(cervical, thoracic and lumbar), pituitary, thyroid, parathyroid glands, esophagus, salivary glands, stomach, Small and large intestine, gallbladder, pancreas, trachea, aorta, prostate, mammary gland, sciatic nerve, bladder, optic nerve, bone marrow, muscle, thyroid (including parathyroid gland), lymph gland, local tissues of administration and other organs with obviously abnormal lesions. All samples were fixed in 4% neutral buffered formalin, paraffin embedded, and were sliced into 5 μm sections. Sections were stained with H&E and followed microscopic examination.
Genotoxicity studies
Ames test
Salmonella typhimurium TA97, TA98, TA100, TA102 and TA1535 were used in this study. In a pilot study, we determined the bacterial toxicity of SAA (5000, 2500, 1250, 625, 312 µg/plate) in TA100 without a metabolic activation system. The result indicated that SAA did not show any toxicity against TA100. Thus, SAA 5000 µg/plate was selected as the maximum test concentration, other concentrations were set at 1000, 100, 10 and 1.0 µg/plate. This test was conducted by using the plate incorporation approach under the condition of presence or absence of S9 metabolic activation. We added 0.1 ml of bacterial culture solution, 0.1 ml of test article, and 0.5 ml of S9 mixture solution (metabolic activation group) into a 2 ml top culture medium in order, quickly mixed them on the oscillating mixer. Mixtures with or without S9 was plated onto basal culture medium and cultured for 48 and 72 h at 37 ℃. Positive controls included 9-aminoacridine, 2,7-diaminofluorene, sodium azide, mitomycin C, 2-aminofluorene, 1,8-dihydroxyanthraquinone. Each plate was counted for the number of revertant colonies.
In vivo bone marrow micronucleus assay
In vivo bone marrow micronucleus assay was conducted according to the previously described methods [18]. In brief, 60 BALB/C mice were randomly assigned into the following five groups: Vehicle control, positive control (cyclophosphamide, CP, 50 mg/kg, ip), SAA (200, 400, 800 mg/kg, iv). Mice were euthanized 24 and 48 h after administration. The sternum marrow of mice was flushed out and smears were prepared for Giemsa staining. A total of at least 2000 polychromatic erythrocytes (PCEs) were observed for the presence of micronucleus in each sample and the frequency of micronucleus in PCEs was calculated. In addition, a total of 200 erythrocytes [PCEs + normochromatic erythrocytes (NCEs)] were counted per animals. The ratio of PCEs to total erythrocytes was also calculated.
Statistical analysis
The statistical analyses were performed using SPSS 11.0. All results were expressed as mean ± standard deviation (SD). Quantitative data were tested for homogeneity of variance. If the variance was homogeneous, one-way ANOVA followed by Dunnett test was used. Comparisons between groups of nonparametric data were made using the Kruskal-Wallis test followed by the Mann-Whitney U test. P < 0.05 was considered significant.