Thirteen-Week Oral Toxicity Study of HVC1 in Rats

Studies on the safety of herbal medicine are essential for the development of new drugs. The aim of this study was to evaluate the no-observed-adverse-effect-level (NOAEL) of HVC1 (Gamisamhwangsasim-tang, a 30% ethanol extract of a mixture of Pruni Cortex, Scutellariae Radix, Coptidis Rhizoma, and Rhei Rhizoma) and identify its target organs after oral administration to Sprague-Dawley (SD) rats repeatedly for 13 weeks. Three test groups were treated with HVC1 at a dose of either 500 (low-dose), 1,000 (middle-dose), or 2,000 (high-dose) mg/kg/day. Another group received high-dose HVC1 and was observed for 4 weeks following treatment to examine recovery from the effects of the extract. All treatment groups were compared to a vehicle control group. During the study, mortality, clinical signs, body weight changes, food consumption, abnormal lesions in the eye, urinary parameters, hematological parameters, blood coagulation time, blood biochemical parameters, changes in organ weight, gross findings, and histopathological changes were examined. No systemic toxicity related to HVC1 was observed in any group, and it was concluded that the NOAEL of HVC1 was 2,000 mg/kg/day. No target organ was identified.


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Evidence-Based Complementary and Alternative Medicine

Materials and Methods
. . Compliance with Ethical and Procedural Standards. This study was performed at Korea Conformity Laboratories (KCL, Incheon, Korea) with the approval of KCL's Institutional Animal Care and Use Committees (IA14-00150). The procedures used in this study were in compliance with KCL's ethical standards, in accordance with Good Laboratory Practice (GLP) of the Ministry of Food and Drug Safety (MFDS, No. 2013-40), and in line with the applicable guidelines for repeated-dose 90-day oral toxicity studies in rodents (OECD guideline No. 408) and the testing guidelines for safety evaluation of drug (MFDS guideline No. 2014-06).
. . Preparation of HVC . A decoction of HVC1 was produced by Kolmar Korea Co., Ltd. (Seojeong, Korea) from a mixture of chopped crude herbs (Table 1). Professor Hocheol Kim of Kyung Hee University identified the materials. The herbal mixture (20 kg) was extracted with 30% ethanol for 2 h in a reflux apparatus. After reflux and filtration, the solvent was removed under reduced pressure at 80-95 ∘ C to yield 4.6 kg of crude extract (1 g extract = about 4.35 g raw/dried herb).
. . Test Substance and Dosing Solution. HVC1 was dissolved in distilled water and dosing solutions were prepared daily. The dose volume was 10 mL/kg/day for all animals. To maintain a constant dose, the dose volume was adjusted to the latest recorded body weight for each individual rat.
. . High-Performance Liquid Chromatography (HPLC) Conditions. The HPLC analysis was performed at room temperature, at a flow rate of 0.5 mL/min, and for a duration of 100 min using the same equipment used in the previous study [16]. The injection volume was 10 L. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (HPLC grade, J.T. Baker5, Avantor Performance Materials, Inc., Center Valley, PA, USA) (B) in a ratio specified by the following binary gradient with linear interpolation: 0 min, 20% B; 60 min, 30% B; 70 min, 60% B; and 100 min, 70% B. The column eluent was monitored at 250 nm.
. . Animals. Male and female pathogen-free SD rats (approximately 5-week-old) were obtained from Orientbio Inc. (Sungnam, Gyeonggi province, Korea). On arrival, the animals were examined for signs of ill health and were allowed to acclimatize for 6 days prior to start of the study. One day before the start of oral administration, the animals were divided into 4 main groups and 2 recovery groups according to body weight. The rats were housed in stainless steel cages with a barrier system to control the light-dark cycle (08-20 h), air exchange (10-15 changes/h), temperature (23.1 ± 1.0 ∘ C), and relative humidity (54.2 ± 5.6 %) during the study. Animals were provided Teklad certified global 18% protein rodent diet (Harlan Co., Ltd., Madison, WI, USA) and filtered municipal tap water. Food and water were provided ad libitum.
. . Experimental Design . . . Experimental Groups and Administration. In previous studies using rats, the effective dose of HVC1 was 250 mg/kg/day. We calculated the pharmacologically active dose in humans according to guidelines on converting animal doses to human-equivalent doses [17]: 250 mg/kg/day/6.17 (conversion factor for rats)/10 (safety factor) × 60 (mean weight of a person in kg) = 243.1 mg/kg/day. Therefore, the recommended daily dose of HVC1 for humans is 250 mg/person/day. In a previous dose selection study (repeated dosing of 0, 500, 1000, or 2000 mg/kg/day HVC1 for 4 weeks), treatment-related toxicity was not observed. Therefore, in the present 13-week study, the dose range of 0, 500, 1,000, and 2000 mg/kg/day HVC1 was chosen.
Evidence-Based Complementary and Alternative Medicine The study included 4 main groups with 10 males and 10 females in each group and 2 recovery groups with 5 males and 5 females in each group. The main groups were a vehicle (distilled water) control group and 3 treatment groups receiving 500, 1,000, or 2,000 mg/kg/day HVC1 for 13 weeks. The recovery groups (a vehicle-treated control group and a group treated with high-dose (2,000 mg/kg/day) HVC1) received treatment for 13 weeks, followed by 4 weeks without further treatment. The test substances and vehicle were orally administered once daily into the stomach by gavage (using a stainless steel catheter attached to a disposable syringe). daily for clinical signs. Body weight and food consumption were measured weekly just before dosing and on the day of necropsy.
. . . Ophthalmoscopy and Urinalysis. Ophthalmoscopic examinations of all rats were conducted prior to the start of the treatment and prior to necropsy by using an ophthalmoscope (Vantage plus, Keeler Ltd., Windsor, UK) and a digital fundus camera (Genesis, Kowa Co., Ltd., Nagoya, Japan).
Urine was collected for urinalysis from 5 rats in each group prior to necropsy. Urinalysis was performed to assess urine volume, specific gravity, pH, glucose, bilirubin, ketone bodies, protein, urobilinogen, nitrates, leukocytes, cast, occult blood, red blood cells, white blood cells, and epithelial cells using a urine analyzer (CliniTek 50, Siemens, Munich, Germany).
. . . Hematology and Serum Biochemistry. Blood samples were collected from an abdominal artery and analyzed to determine white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, neutrophil count, lymphocyte count, monocyte count, eosinophil count, basophil count, large unstained cell count, reticulocyte count, platelet count, percentage of neutrophils, percentage of lymphocytes, percentage of monocytes, percentage of eosinophils, percentage of basophils, percentage of large unstained cells, mean corpuscular volume, mean corpuscular hemoglobin concentration, red cell width distribution, and mean platelet volume using an automatic hematological analyzer (ADVIA 2120, Siemens, Munich, Germany). In addition, blood samples were treated with 3.2% sodium citrate and analyzed for prothrombin time and activated partial thromboplastin time using an automatic coagulation time meter (ACL 7000, Instrumentation Laboratory, Bedford, MA, USA).

. . . Gross Observation and Measurement of Organ Weights.
After exsanguination from the abdominal aorta, macroscopic examination of the full external surface, cranial cavity, and organs of the thoracic and abdominal cavities was performed. The absolute weights of the spleen, liver, adrenal glands, kidney, heart, lung, brain, pituitary, thymus, testis, prostate, ovary, and uterus were measured, and their relative organ weights (organ-to-body weight ratios) were calculated.
. . Statistical Analysis. Statistical analyses were performed using SPSS v.12.0 statistical analysis software (IBM Corp., Evidence-Based Complementary and Alternative Medicine 5 Table 3: Urinalysis results of HVC1-treated male and female rats.

Results
. . Calibration Curve and Limits of Detection and Quantification. Calibration curves were calculated for the 9 reference compounds using linear regression derived from the peak 6 Evidence-Based Complementary and Alternative Medicine  area. The regression equations (correlation coefficient, 2 ) of the reference compounds are shown in Table 2. The calibration curves had good linearity.
. . Mortality, Clinical Signs, and Body Weight. Throughout the 13-week study period, no signs of ill health or mortality related to HVC1 treatment were observed. There were no significant differences in body weight between the control and HVC1-treated groups (Figure 2). There were no significant differences in feed intake between the control and HVC1treated groups (data not shown). There were no significant differences in clinical observations, body weight, and feed intake during the recovery period (data not shown).
. . Ophthalmoscopy and Urinalysis. Ophthalmoscopic examination did not reveal changes after HVC1 treatment (data not shown).
Urinalysis showed significant differences in glucose level, ketone body level, and leukocyte count between the control and HVC1-treated male rats. Significant differences in ketone body level were observed between the control and HVC1treated female rats. No other significant differences between the control and HVC1-treated male and female groups were observed (Table 3).
. . Hematology and Serum Biochemistry. In male rats, white blood cell levels significantly increased in the groups treated with 1,000 and 2,000 mg/kg/day HVC1 (p < 0.05), and the percentage of lymphocytes in the group treated with 2,000 mg/kg/day HVC1 also significantly increased (p < 0.01). The mean corpuscular hemoglobin level in the group treated with 500 mg/kg/day HVC1 and the percentage of neutrophils in the group treated with 2,000 mg/kg/day HVC1 significantly decreased (p < 0.05) in comparison with those of the control group. No other significant differences in hematological and 9.4 ± 0.6 9.2 ± 0.6 9.8 ± 0.6 10.7 * * ± 1.1 Chloride (mmol/L) 106 ± 1 1 0 7 ± 2 1 0 7 ± 2 1 0 7 ± 1 Magnesium (mg/dL) 2.6 ± 0.2 2.6 ± 0. plasma coagulation values were observed between the control and HVC1-treated male and female groups (Tables 4 and 5).
In male rats treated with 2,000 mg/kg/day HVC1, levels of uric acid (p < 0.05), inorganic phosphorus (p < 0.01), magnesium (p < 0.05), and potassium (p < 0.01) significantly increased. No other significant differences in serum biochemical values were observed between the control and HVC1treated male and female groups (Tables 6 and 7).

. . Gross Observations and Organ Weights.
No treatmentrelated changes in gross findings were observed in any of the treated animals. Absolute spleen weight in the male rats treated with 500 or 2,000 mg/kg/day HVC1 significantly decreased (p < 0.05), whereas absolute spleen weight significantly increased in the female rats treated with 1,000 mg/kg/day HVC1, and absolute kidney weight of both kidneys significantly increased in the female rats treated with 1,000 or 2,000 mg/kg/day HVC1 (p < 0.05) ( Table 8). The relative weight of the left kidney in the male rats treated with 1,000 or 2,000 mg/kg/day HVC1 significantly increased (p < 0.05). The relative weight of the spleen in the female rats treated with 1,000 mg/kg/day HVC1 (p < 0.05) and the relative weight of both the kidneys in the female rats treated with 1,000 or 2,000 mg/kg/day HVC1 significantly increased (p < 0.01) ( Table 9). No other significant differences in absolute or relative organ weights were observed between the control and HVC1-treated male and female groups (data not shown).
. . Histopathological Findings. No abnormal histopathological changes were observed in any animals (data not shown).

Discussion
In the present study, no signs of ill health or mortality were observed after oral administration of HVC1. Although salivation was sporadically observed in the male and female rats treated with 1,000 and 2,000 mg/kg/day, it was concluded that this symptom was temporarily stimulated by administration and did not have any toxicological significance. There were no significant differences in body weight and feed intake between the control and HVC1-treated groups.
Ophthalmoscopy revealed no abnormal lesions in any of the animals examined. The urinalysis showed significant differences in glucose, ketone body level, and leukocyte level in HVC1-treated male rats and in ketone body level in HVC1treated female rats, in comparison with the control group. The color of the urine changed in a dose-dependent manner in animals of both sexes in all dosing groups and tended to have a reddish color (amber, orange, or dark red). The change in urine color was thought to be caused by the test substance. No changes were observed in biochemical parameters related to the kidneys, such as blood urea nitrogen and creatinine levels, and histopathological examination revealed no kidneyrelated abnormalities. In addition, no differences in urine color were observed during the 4-week recovery period, and   Values are expressed as mean ± SD (n = 10). * < 0.05 compared with the control group. Values are expressed as mean ± SD (n = 10). * < 0.05, * * < 0.01 compared with the control group.
changes in urine color were thus not considered a toxic result of HVC1 treatment. According to hematological testing, the male rats in HVC1-treated groups showed increased white blood cell count and lymphocyte percentage, as well as decreased mean corpuscular hemoglobin level and neutrophil percentage. However, these changes were within the range of natural biological variation and were not observed during the recovery period. No other significant differences in hematological parameters and plasma coagulation values were observed.
Evidence-Based Complementary and Alternative Medicine 9 Analysis of serum biochemistry revealed increased levels of uric acid, inorganic phosphorus, magnesium, and potassium in the male rats treated with 2,000 mg/kg HVC1. However, these changes were within the range of natural biological variation. These differences were not observed during the recovery period.
The absolute spleen weights in male rats decreased, while the absolute and relative weights of the spleen and both kidneys in females, as well as the relative weight of the left kidney in males, increased. However, these changes in organ weight were not dose-dependent and were not observed during the recovery period, and no spleen-or kidneyrelated abnormalities were observed in the biochemical and histopathological analyses. No abnormal histopathological changes related to HVC1 oral administration were observed in any animals.

Conclusion
In this study, no systemic toxicity related to HVC1 was observed in any of the primary treatment or recovery groups after repeated dosing for 13 weeks. Therefore, the NOAEL (no-observed-adverse-effect-level) of HVC1 was established as 2,000 mg/kg/day, and no target organ of HVC1 toxicity was identified. This result suggested that there are no toxicity and side effects from repeated administration of HVC1. For the clinical trial, we have obtained IND approval from this result and previous hypolipidemia efficacy results and toxicity results in Korea. We are going to conduct phase II clinical trials. Like this, many studies are currently being carried out actively to discover new efficacies of traditional herbal prescriptions and to develop new prescriptions in Korea. If many clinical trials based on ethnopharmacological studies are carried out, it is expected that many new drugs using herbal medicine could be developed.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that there are no conflicts of interest.