The water soluble ginseng pharmacopuncture was prepared in a sterile room at the Korean Pharmacopuncture Institute (Korea-Good Manufacturing Practice, K-GMP). After the mixing process with pure water had been completed, the pH was controlled to between 7.0 and 7.5; then, NaCl was added to make a 0.9% isotonic solution. The completed extract was stored in a refrigerator (2.1 ─ 6.6°C). A high performance liquid chromatography (HPLC) analysis was performed to determine the changes in the chemical constituents in ginseng saponin following the enzyme treatment. HPLC results showed the appearance of new peaks (Rh1, Rg3, protopanaxtriol (PPT), Compound K, and Rh2), indicative of ginseng saponin metabolites (Fig. 1, Table 1).
The animals used in this study were 6 week old Sprague-Dawley (SD) rats (Orientbio Inc., Korea). The rats were received at an age of 5 weeks, and they were kept for 1 week at room temperature. The mean weights of the rats were 189.5 ─ 209.2 g (male) and 145.1 ─ 167.6 g (female) at the time of injection. For all animals, a visual inspection was conducted; all animals were weighed using a CP3202S system (Sartorius, Germany). During the 7 days of acclimatization, the general symptoms of the rats were observed once a day. The weights of the rats were recorded on the last day of acclimatization. No abnormalities were found. The temperature of the laboratory was 21.0 ─ 23.2°C, and the humidity was 40.9% ─ 59.4%. Enough food (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C) and ultra violet (UV)-filtered water were provided. The lights were on for 12 hours/day (from 7 am to 7 pm). Groupings were done after 7 days of acclimatization. Animals were selected if their weights were close to the mean weight. In total, 20 male rats and 20 female rats were selected. The animals were randomly distributed into 4 groups (5 male and 5 female rats per group, Table 2).
The administration route was intramuscular because of clinical considerations, and the administered volume was 1.0 mL/animal of normal saline in the control group and high dose group, 0.1 mL/animal in low dose group, 0.5 mL/ animal in mid dose group. The syringe used in the experiment was 1 mL disposable 26G syringe. For the low dose group and mid dose group, the rats were administrated on the Lt. thigh, single injection. But for the control group and high dose group, the rats on the both thigh, 0.5 mL on each thigh.
The expected volume of GP administered in clinical use is 1.0 mL per treatment. No death occurred in a pilot test in which 1.0 mL of GP was injected into each male and female rat. In this study 1.0 mL/animal was set as a high dose, and 0.5 mL and 0.1 mL were set as the mid and the low doses, respectively. In the control group, 1.0 mL of normal saline solution was administered. This study was conducted under the approval of the Institutional Animal Ethic Committee of Biotoxtech.
From the 1st day to the 14th day of treatment, the general symptoms were examined once a day. On the day of injection (day 0), the general symptoms (toxicological effects, manifestation time, recovery time, etc.), as well as mortality, were examined at 30 minutes and 1, 2, 3, and 4 hours after injection. Body weights were measured immediately before treatment and at 3, 7 and 14 days after treatment.
After the rats had fasted for more than 18 hours, they were anesthetized by using isoflurane. A blood sample was taken from the abdominal aorta on necropsy day (15 days after injection) and was inserted into an ethylenediaminetetra acetic acid (EDTA) coated tube. The 1 mL of blood was analyzed by using an automatic hematology analyzer (ADVIA 120, SIEMEMS, Germany). The items measured were RBC (erythrocytes), hemoglobin, hematocrits, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leucocytes (WBC), WBC differential counting (neutrophils, lymphocytes, monocytes, eosinophils), and reticulocytes. A 2.0 mL blood sample underwent centrifugation for the blood coagulation test (3,000 rpm, 10 minutes), and serum was taken. The results were measured by using an automated coagulation analyzer (Coapresta 2000, SEKISUI, Japan). The items measured were the prothrombin time (PT) and the activated partial thromboplastin time (APTT).
Blood taken from the abdominal aorta was used in the blood biochemical test. The results were measured by using an automatic analyzer (7180, HITACHI, Japan) and an electrolyte analyzer (AVL9181, Roche, Germany). The items measured were alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), albumin/globulin (A/G) ratio, total cholesterol (T-Chol), triglyceride (TG), phosphate (P), glucose (Glu), calcium (Ca), chloride (Cl) and potassium (K).
After the termination of all observations, organs and tissues of all surviving animals were visually inspected and were examined under a microscope after they had been stabilized using 10% neutral buffered formalin. For the injection site, tissue slices were stained with hematoxylin & eosin (H&E).
The body weights and the results from the hematologic examinations and the blood biochemical tests were analyzed by using statistical analysis system (SAS) software (version 9.3, SAS Institute Inc., U.S.A.). The Bartlett test was conducted to evaluate the homogeneity of the variance and the significance. The significance level was 0.05. The one-way analysis of variance (ANOVA) test was conducted, and when homogeneity of the variance was recognized, Dunnett’s t test was conducted; if homogeneity was rejected, then the Kruskal-Wallis test was conducted post-hoc.