The Hominis placenta pharmacopuncture extract was prepared by adhering to Korea-Good Manufacturing Practice (K-GMP) in a clean room in a laboratory at the Korean Pharmacopuncture Institute. The pH was controlled to between 7.0 and 7.5. The completed extract was stored in a refrigerator (2.1 - 5.3°C).
The animals used in this study were 6 week old Sprague-Dawley (SD) rats. The reason SD rats were chosen is that they have generally been used in stability tests of medicine, so the data obtained in this study should be easily compared with many other databases. The mean weights of the rats were 185.1 - 205.9 g and 149.7 - 167.2 g for the male and the female rats, respectively. For all animals, a visual inspection was done, and all animals were weighed using a CP3202S system scale (Sartorius, Germany). After 7 days of acclimatization, the rats general symptoms and changes in weight were observed. The weights were recorded on the last day of acclimatization. No abnormalities were noted. The temperature of the lab was 21.1 - 24.1°C, and the humidity was 40.7% - 64.5%. Sufficient food (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C) and ultra violet (UV)-filtered water were provided. Group separations were done after 7 days of acclimatization. The animals were randomly distributed into 4 groups of 5 male and 5 female rats per group (Table 1): the control, low dose, mid dose and high dose groups.
The expected dose of Hominis placenta pharmacopuncture for clinical applications is 1.0 mL. In a pilot study (Biotoxtech Study No.: B13473P), 0.5 mL/animal of Hominis placenta pharmacopuncture was injected into each male and female rat; no deaths were observed. From this result, the doses for Hominis placenta pharmacopuncture were set as follows: animals in group 1 (G1, the control group) were injected with 0 mL/animal of pharmacopuncture and 0.5 mL/animal of normal saline solution (Choongwae Pharma Corp., Korea), animals in group 2 (G2, the low dose group) were injected with 0.125 mL/animal of pharmacopuncture, animals in group 3 (G3, the mid dose group) were injected with 0.25 mL/animal of pharmacopuncture, and animals in group 4 (G4, the high dose group) were injected with 0.5 mL/animal of pharmacopuncture. Using a disposable syringe (1 mL, 26 G), all rats were injected with a single dose in the left thigh muscle. This study was conducted under the approval of the Institutional Animal Ethics Committee of Biotoxtech.
The general symptoms (types of toxic symptoms, revealing times, recovery times, etc.) and the mortality were examined 30 minutes, and 1, 2, 4 and 6 hours after the injection on the day of dosing (day 0). From the 1st day to the 14th day of treatment, the general symptoms were examined once a day. The body weights were measured immediately before treatment and at 3, 7 and 14 days after injections.
All animals were fasted for more than 18 hours before the necropsy. The rats were anesthetized by using isoflurane, and blood samples were taken from the abdominal aorta (15 days after injection). An automatic hematology analyzer (ADVIA 2120i, SIEMENS, Germany) was used to analyze blood for the hematological examinations (Table 2). Two mL blood samples were centrifuged for the blood coagulation test (3,000 rpm, 10 minutes). Coagulation test results were measured by using an automated coagulation analyzer (Coapresta 2000, SEKISUI, Japan) (Table 3). For the biochemical tests, the blood remaining after carrying out the hematological tests was centrifuged at 3,000 rpm for 10 minutes, and the serum was collected. Biochemical test results were measured by using an automatic analyzer (7180, HITACHI, Japan) and an electrolyte analyzer (AVL9181, Roche, Germany) (Table 4).
After the observations, organs and tissues from the entire bodies of all surviving animals were visually inspected. Tissue samples of all the animals were fixed in 10% neutral buffered formalin. Routine histological methods, like trimming, dehydration, and paraffin embedding, were conducted on the fixed organs and tissues. Fixed samples were sliced and stained with hematoxylin & eosin (H&E).
All the results from the experiments were analyzed by using statistical analysis system (SAS) software (version 9.3, SAS Institute Inc., U.S.A.). A Bartlett test was conducted to evaluate the homogeneity of the variance and the significance . The one-way analysis of variation (ANOVA) test was conducted when homogeneity of the variance was recognized, and the Dunnett's t-test was conducted posthoc . The Kruskal-Wallis test was conducted when heterogeneity of the variance was recognized, and the steel test was conducted post-hoc.