This study was conducted at Biotoxtech under the regulations of Good Laboratory Practice. Mahwangcheonoh pharmacopuncture extracts were prepared at the Korean Pharmacopuncture Institute (Seoul, Korea) in a clean room, were stored in a refrigerator at 2.1 - 5.3°C, and remained stable for 6 months following production (Lot No. KPI-2013-03). The normal saline used as a control material was manufactured by Choongwae Pharma Corp. (Seoul, Korea, Lot No. 12133) and stored at room temperature.
The animals used in this study were 6-week-old specific-pathogen-free (SPF) Sprague-Dawley (SD) male (184.7 - 204.0 g) and female (156.1 - 179.5 g) rats purchased from Orientbio Inc. (Gyeonggi-do, Korea). This study was approved by the Institutional Animal Care and Use Committees of Biotoxtech (No. 130364). When the animals were received, a visual inspection was done, and weights were recorded using electronic scales (CP3202S, Sartorius, Göttingen, Germany). The general symptoms and changes were checked once daily during the seven days of acclimatization, after which all animals were checked and found to be normal.
All animals were bred in stainless-steel wire breeding boxes (260 mm (W) × 350 mm (D) × 210 mm (H)) kept at a temperature from 22.1 to 24.1°C, a humidity from 47.7% to 70.8%, and an illuminance from 150 to 300 Lux, with a 12-hour light (7:00 am to 7:00 pm)/dark cycle. Each breeding group was divided by three animals during acclimatization periods and was divided by one animal during observation periods. Feed (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C, Harlan Laboratories Inc., USA) and ultraviolet (UV) sterilizer running water were provided freely.
Groupings were done at end of acclimatization. Twenty male and twenty female rats with weights closest to the mean weight were selected. The selected animals were distributed into four groups (five rats of each sex per group). The Mahwangcheonoh pharmacopuncture extracts were injected into the animals via a muscular route, and the high dose was set at 1.0 mL per animal because no mortalities had occurred in a preliminary test (Biotoxtech Study No.: B13474P) in which a single dose of 1.0 mL of Mahwangcheonoh pharmacopuncture extract had been injected into the muscles of male and female rats. Based on this high dose, we set 0.5 mL per animal as the medium dose and 0.25 mL per animal as the low dose. For the control group, 1.0 mL of normal saline was injected. In the low-dose group (G2) and the medium-dose group (G3), the Mahwangcheonoh pharmacopuncture extracts were injected into the left femoral muscle. For the control group (G1) and the high-dose group (G4), 0.5 mL of normal saline and 0.5 mL of Mahwangcheonoh pharmacopuncture extract, respectively, were injected into the left and the right femoral muscles, for a total of 1.0 mL per animal. All injections were made using disposable syringes (1.0 mL, 26 G).
On the day of injection (day 0), general symptoms (types of toxic indications, times of toxic expression, recovery times) and any deaths were recorded at 30 minutes and at 1, 2, 4 and 6 hours after injection. From day 1 until day 14 after injection, general symptoms were observed once daily. Body weights were measured on the day of injection (before injection) and on days 3, 7 and 14 after injection.
All animals were denied access to food and water (fasted) for at least 18 hours prior to necropsy. On the day of necropsy (15 days after injection), the animals were anesthetized with isoflurane, and blood was collected from the abdominal aorta. One mL of the collected blood was put into a tube containing ethylene diamine tetraacetic-acid (EDTA), and the hematology parameters, the prothrombin time (PT) and the activated partial thromboplastin time (APTT) were measured using a hematology analyzer (ADVIA 2120i, SIEMENS, Germany). Two mL of the collected blood were put into a tube containing 3.2% sodium citrate; then, the plasma was collected after centrifugation at 3,000 rpm for 10 minutes. A coagulation examination was performed using coagulation time analyzers (Coapresta 2000, SEKISUI, Japan) to measure the PT and the APTT.
The blood biochemistry was determined by using serum taken from blood that had been collected from the abdominal aorta and had been centrifuged at 3,000 rpm for 10 minutes. A blood chemistry analyzer (7180, HITACHI, Japan) and an electrolyte analyzer (AVL 9181, Roche, Germany) were used for the measurements.
For the histopathology, the organs and the tissues collected from all animals after necropsy were inspected. After necropsy, tissues surrounding the injection regions were excised and fixed in 10% neutral buffered formalin solution, after which local resistance tests were performed on those tissues. Fixed organs and tissues sections were produced using a common process: cutting, dehydrating and embedding in paraffin. The tissue sections were stained using hematoxylin & eosin (H&E). The weights, as well as the hematological and the blood biochemical results, were analyzed by using SAS (version 9.2, 9.3, SAS Institute Inc., USA). A Bartlett test was conducted to evaluate equal variance between the test and the control groups (P < 0.05). The one-way analysis of variation (ANOVA) test was conducted when equal variance was recognized on the Bartlett test (P < 0.05), and the Dunnett’s t-test was conducted when equal variance was recognized on the ANOVA test (both P < 0.05, P < 0.01). Also, the Kruskal-Wallis test was conducted when equal variance was not recognized on the Bartlett test (P < 0.05), and the Steel-test was conducted when equal variance was recognized on the Kruskal-Wallis test (both P < 0.05, P < 0.01).