2.1. KCHO-1 preparation
KCHO-1 is consisted of nine herbs, Radix Paeoniae Alba (Paeonia lactiflora PALL.), Radix Glycyrrhizae (Glycyrrhiza uralensis FISCH.), Radix Aconiti Lateralis Preparata (Aconitum carmichaeli DEBX.), Radix Salviae Miltiorrhizae (Salvia miltiorrhiza BGE.), Rhizoma Gastrodiae (Gastrodia elata BL.), Rhizoma Curcumae Longae (Curcuma longa L.), Fructus Chaenomelis (Chaenomeles speciosa NAKAI), Rhizoma Atractylodis Japonicae (Atractylodes japonica KOIDZ. ex KITAM.), Radix Polygalae Tenuifoliae (Polygala tenuifolia WILLD.). KCHO-1 was provided by the Nervous & Muscular System Disease Clinical Research Center of Wonkwang University Gwang-ju Korean Medical Hospital. The test substance was used as it was without conversion into purification, and the concentrations were 50 mg/mL, 100 mg/mL, and 200 mg/mL. The completed extracts were stored at room temperature (1–30°C).
2.2.1. Animals and Husbandry
The animals used in oral dose toxicity test were 5-week old Sprague-Dawley (SD) rats. The reason SD rats were chosen is that they have been widely used on safety test in the field of medicine and they can be compared with a lot of other data bases easily. The mean weights of the rats were 112.9 ~ 139.3g and 93.0 ~ 111.5g for the male and the female rats at the time of oral dose toxicity test. For all animals, a visual inspection was conducted.
The temperature of the lab was 20.9~23.8°C, and the humidity was 47.6~62.8%. Enough food (Rodent Diet 20 5053) and UV-filtered water were provided. The temperature and humidity of the animal room were measured every 30 minutes by an automatic temperature and humidity meter. The environmental conditions such as illumination were measured according to standard work instructions. Variation that would affect the test results was not observed.
After acclimatization, healthy individuals were selected, and grouping was performed randomly so that the mean weight and standard deviation among the groups were equal. (Table 1) After grouping, the remaining animals were anesthetized with isoflurane and euthanized via exsanguination.
No toxicological changes related to the administration of the test substance were observed in the high-dose of 2000 mg/kg B.W. of the 4-week repeated dose determination test using this test substance. Therefore, the high-dose was set at 2000 mg/kg B.W., and the geometric ratio was set at 2, the mid-dose was set at 1000 mg/kg B.W., and the low-dose was set at 500 mg/kg B.W. The control group was administered with the same amount of excipient as the experimental group. In addition, in order to evaluate the reversibility of the toxicity change according to the test substance administration, the recovery group was set separately in the control group and the high-dose group.
During the administration of the test substance preparation, it was directly administered into the stomach using a syringe with a sonde for administration while shaking by hand. The test substance was administered to the SD rats 7 days/week, 1 time/day for 26 week. And the dose (mL/kg) was calculated based on the most recently measured body weight at the time of administration.
2.3.1. General symptoms
During the experimental period, all animals were observed for changes in general symptoms, toxic symptoms, and deaths when before and after the administration during administration period and once a day during the recovery period.
2.3.2. Weight measurements
Body weight was measured once a week at the time of introduction, separation of group, and up to 3 months after initiation of administration. After that, it was measured once every 4 weeks and once a week during the recovery period. In order to calculate the relative organ weights (%), fasting weight was measured on the day of the necropsy and measured at the time of death or moribund animal observation.
2.3.3. Feed intakes
Feed intake was measured once a week before the start of administration, up to three months after initiation of administration. After that, it was measured once every 4 weeks and once a week during the recovery period. The average daily intake (g/rat/day) was calculated by dividing the difference of the amount of daily dose and the remaining amount of the day after feeding into numbers per cage. The average daily intake (g/rat/day) was calculated by dividing the difference between the amount of daily dose and remaining amount of the next day after feeding into number of per cage. In the case of before administration, the daily intake from the day of grouping to the day of administration was measured.
For each group, 5 male and 5 female animals were examined for the following items in the final week of administration period and the final week of recovery period. Urine was subjected to urine chemistry test, urine sedimentation and urine color test for fresh urine obtained using metabolic cage. And urinary volume stored for about 24 hours was measured.
2.3.5. Ophthalmological examination
For all animals in the high-dose and control groups, the appearance of the animal eyes was made an ocular inspection before administration, at the final week of the administration period, and at the final week of the recovery period. After the ocular inspection, the pupil was dilated using a mydriatica [1 % Mydriacyl (Tropicamide 1%), Alcon, Belgium], and an ophthalmological examination was performed using an ophthalmoscope (Genesis, Kowa, Japan).
During the planned necropsy, surviving animals were anesthetized with isoflurane and exsanguinated and euthanized. Then, the external surface & all orifices, cranial cavity, thoracic and abdominal cavities & their contents were conducted macroscopy. The animals that died during the administration were also conducted macroscopy about the external surface & all orifices, cranial cavity, thoracic and abdominal cavities & their contents.
2.3.7. Organ weights
At the time of necropsy, the wet weight of the following organ was measured for all animals, and the relative organ weight ratio to the fasting weight was calculated: brain, pituitary, heart, lung, liver, spleen, kidneys, adrenals, tested, epididymides, ovaries, uterus, thymus, prostate, salivary gland. The bilateral organs were measured separately and the weights were summed up and calculated.
2.3.8. Hematologic examination
All animals were anesthetized with isoflurane and exsanguinated from the abdominal artery after fasting for more than 18 hours before the blood collection. 2 mL of the exsanguinated blood was placed in a CBC bottle (EDTA 2K, BD vacutainer) containing EDTA and the following items were measured using a Blood analysis apparatus (ADVIA 2120i, Siemens, Germany). Blood clotting time was measured by blood coagulation analyzer (ACL 7000, Instrumentation Laboratory, U.S.A.) using plasma obtained by refrigerated centrifugation (3000 rpm, 4°C 10minutes) in a vacutainer (9NC Sodium citrate, BD vacutainer).
2.3.9. Blood chemical examination
The remaining blood, except the blood used for the hematologic examination and the blood coagulation examination, was coagulated at room temperature and then subjected to refrigerated centrifugation (3000 rpm, 4°C, 10minutes). The obtained serum was measured using a biochemical analyzer (TBA-120FR, TOSHIBA, Japan) as follows.
2.3.10. Histopathologic examination
The following organs were isolated from each animal and fixed in 10% neutral buffered formalin: liver, kidneys, adrenals, pituitary gland, spleen, brain, heart, Lung and bronchus, testes, seminal vesicle, ovaries, epididymides, uterus, prostate gland, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, parathyroid gland, urinary bladder, small/large intestine, eyeball, submandibular gland, pancreas, sciatic nerve, sternum, mammary gland, femur, spinal cord, skin, mesenteric lymph node. Among them, the testes (epididymis) were fixed in Bouin fixative and the eye was fixed in Davidson solution. Among fixed organs tissues, all organs of control and high-dose groups were subjected to general tissue processing such as dehydration, paraffin infiltration, tissue paraffin embedding, and dissection. And sample slides were fabricated and Hematoxylin & Eosin (H & E) staining was performed. After sample fabrication, the remaining and fixed organ • tissues were stored in 10% neutral buffered formalin.
2.4. Statistical Analysis
The weight, feed intake, hematologic examination, Blood chemical examination and organ weight data from the experiments were analyzed by using SPSS program (Ver. 19.0). A Levene test was conducted to evaluate the homogeneity of the variance. The one-way ANOVA test was conducted to confirm the significance of the variance. One way ANOVA test was carried out and the significance between experimental groups was confirmed.
Therefore, the post-hoc test (Scheffe multiple test if variance is homogenous, Dunnett’s T3 if variance is heterogeneous) was conducted according to the presence or absence of homogeneity of variance. In the recovery group, statistical significance test between experimental groups was performed by the independent sample T-test.