A 14-Day Repeated Dose Toxicity of Epimedii Herba Aqueous Extract Administered by Oral Gavage in F344 Rats

Abstract

The objective of this study is to characterize a toxicity of Epimedii Herba (EH) in F344 rats and to find a dose levels for the 13 weeks toxicity study. EH is well known as medicinal herb in many Asian countries for traditional medicines of antibacterial and antiviral effects, estrogenic and antiestrogenic effects, and for treatment of osteoporosis, hypotensives, fatigue, kidney disorders, and related complications. However, the indispensable and basic information of toxicological evaluation of EH extract is insufficient to support its safe use. Therefore, we conducted toxicological evaluation of this drug in compliance with OECD and MFDS guideline in this study. The extract of EH was administered orally to F344 rats at dose levels of 0, 500, 1000, 2000, 3500, and 5000 mg/kg/day for 2 weeks. Each group was composed of 5 male and female rats. In this study, there were no treatment of EH-related adverse changes in clinical observations, mortality, body weights, food consumption, urinalysis, gross finding at necropsy, and organ weight examination. Total red blood cell count, hematocrit, mean corpuscular hemoglobin concentration, total cholesterol, and phospholipid were decreased in males and females at 5000 mg/kg/day compared to the control animals. Mean corpuscular volume and reticulocyte counts were increased in males and females at 5000 mg/kg/day compared to control animals. Therefore, we recommend that dose level of 5000 mg/kg/day is a highest treatment group in 13-week EH extract exposure study for further toxicity assessment.

Introduction

Herbal medicine has been traditionally used in Asian countries for a long time. Recently, they have been widely used as an alternative medicine even in western countries. Herbs used traditionally for long-time are considered to be safety in human (Jordan et al., 2010).1 Although herbal medicinal products are also considered to be lower risk compared with synthetic drugs, they are not completely free from the possibility of toxicity or other adverse effect (Smet, 2004).2 Recently, the safety issue for herbal products has been increased.

Epimedii Herba (EH; Epimedium koreanum, E. breviconum, E. sagittatum, E. pubescen or E. wushanense) is well known as medicinal herb in many Asian countries for treatment of osteoporosis, hypotensives, fatigue, and kidney disorders and has been traditionally used for antibacterial and antiviral effects, estrogenic and antiestrogenic effects (Kang et al., 2012; Fan et al., 2011).3,4 In addition, this herbal medicine was considered as hepatoprotective agent (Shindel et al., 2010).5 EH contains a lot of flavonoids such as icariin, icariside II, epimedin, epimedosides, hyperoside, quercetin, and chlorogenic acid (Cho et al., 2012).6 However, the indispensable and basic information of toxicological evaluation of EH extract is insufficient to support its safe use. Therefore, the objective of this study was to evaluate a toxicity of EH aqueous extract (EHAE) orally administered in male and female F344 rats and to find a dose level for the 13 weeks toxicity study. The present study was performed in compliance with the Good Laboratory Practice (GLP) of the Organization for Economic Cooperation and Development (OECD, 1997)7 and the Ministry of Food and Drug Safety (MFDS, Korea, 2014).

 

Experimental

Preparation of EHAE and HPLC analysis − Freezedried and powdered EHAE was extracted from leaves of Epimedium koreanum which were purchased from oriental market in Ulsan-si (Kwangmyungdang Medicinal Herbs Co.) and authenticated by Prof. Byung-Sun Min, College of Pharmacy, Catholic University of Daegu, Korea. Botanical identification and HPLC analysis were performed by Prof. Byung-Sun Min, and the voucher specimen CUD-1484-1 was deposited at the Herbarium of the College of Pharmacy, Catholic University of Daegu, Korea. The authentic EH was proved to be safe from typical contaminations such as pesticides (total dichlorodiphenyltrichloroethane, total hexachlorocyclohexane, aldrin, endrin and dieldrin), heavy metals (As, Cd, Hg and Pb), and aflatoxin B1 (JH Keum et al., 2014).8 EH was extracted according to a standard hot water extraction method of the Korea Pharmacopoeia and then freeze-dried. Components of EHAE were measured using a high performance liquid chromatography (HPLC) method. The content of icariin, the marker compound of EH, was 5.00 mg/g. EHAE was stable at 5 ℃ for 6 months (JH Keum et al., 2014).8 5 g of freeze-dried EHAE powder was suspended in 10 mL distilled water and the suspension of EHAE (highest dose group) was gradationally diluted to prepare lower dose groups.

Animals and maintenance − 60 male and female specific pathogen-free F344 rats were obtained from Orient Bio Co. (Seongnam-si, Republic of Korea) at 6 weeks of age. The animals were acclimated for 8 days and healthy animals were used on the study. 60 male and female rats were randomly assigned to 6 groups (one control group and 5 treatment groups) using Path/Tox system (Version 4.2.2, Xybion Medical Systems Corporation, USA). Each group consisted of 5 rats of each sex. The body weight range prior to the start of dosing ranged from 120.5 to 182.3 g for males and 96.4 to 115.7 g for females. The animals were housed in polycarbonate cage with bedding (Laboratory animal Aspen bedding, ABEDD BALTIC LTD., Latvia) throughout the study period. Sterilized tap water and pelleted food for experimental animals (PMI nutrition International, USA) were given to animals ad libitum. The animal room was maintained at a temperature of 23 ± 3 ℃, relative humidity of 50 ± 10%, air ventilation of 10 to 20 times/hour and light intensity of 150 to 300 Lux with 12 hour light/dark cycle. This study was approved by Institutional Animal Care and Use Committee in Korea Institute of Toxicology and performed in compliance with Testing Guidelines for Safety Evaluation of Drugs (Notification No. 2014-6 issued by the Ministry of Food and Drug Safety on 29 January, 2013).

Treatment and toxicity assessment − Oral administration was chosen for this study because it is the intended clinical route of EHAE administration in humans and has been used in previous non-clinical study. The dosing volume of 10 mL/kg was calculated using Path/Tox system based on the most recent body weight. In a previous study of similar crude drug in Korea Institute of Toxicology (not published), doses of 0 (vehicle), 500, 1000, 2000, 3500 and 5000mg/kg/day were well tolerated. The same doses with previous study were selected for this 2-week repeated-dose study. All record for the measurement and examination were performed using Path/Tox system. The condition and behavior of all animals was made once daily throughout the acclimation period. All animals were examined and clinical signs recorded twice daily (before and after dosing) during the treatment period and once before on the day of necropsy. Animals were weighed prior to randomization on the day of arrival, before dosing on the first day of treatment and once weekly thereafter. A final weighing was performed on the day of necropsy. Cage food consumption was recorded once during the acclimation period and once weekly during the treatment period. Individual food consumption was calculated as g/rat/day.

Urine samples were collected overnight (for approximately 16 hours) from animals housed in metabolism cages in the last week of treatment. Each animal was housed in an individual metabolism cage, food was withdrawn overnight during urine collection but water was available. Urinalysis was performed using urine automatic analyzer (Cobas U411, Roche, Germany) and urine stick (Multistix, 10 TM, Roche, Germany) to evaluate the following parameters; urine volume, color, specific gravity, pH, protein, ketone body, occult blood, glucose (GLU), bilirubin (BIL), nitrite, and urobilinogen. Also microscopic examination for urine cast, epithelial cell, red blood cell (RBC), and white blood cell (WBC) was performed.

All animals were fasted overnight before necropsy and blood sampling. Blood samples for hematology and clinical chemistry were collected from the vena cava of all animals at necropsy under isoflurane anesthesia. Blood samples for hematology analysis were collected into tubes containing EDTA-2K and analyzed to evaluate white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelet, differential leucocyte absolute counts (neutrophil, lymphocyte, monocyte, eosinophil, basophil and large unstained cell), and reticulocyte absolute and relative (%) count were analyzed using ADVIA 2120i Hematology analyzer (Siemens, USA). In addition, blood samples treated with 3.2% sodium citrate were analyzed for prothrombin time and activated partial thromboplastin time using ACL Elite Pro coagulation analyzer (Instrumental Laboratory, Italy).

Blood samples for clinical chemistry analysis were collected into tubes without anticoagulant at the same time as for hematology, placed at the room temperature (for at least 90minutes) and then centrifuged (approximately 3,000 rpm, 10 minutes, at room temperature) to obtain serum. The parameters including blood urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine (CREA), GLU, total cholesterol (TCHO), albumin/globulin ratio (A/G), total protein (TP), albumin (ALB), creatine kinase (CK), triglycerides (TG), total TBIL, and phospholipids (PL) were measured using an automatic analyzer (TBA 120FR NEO, Toshiba Co., Japan).

After blood sampling, the animals were killed by exsanguination from the vena cava and aorta under isoflurane anesthesia. Complete necropsy examination were performed in all animals. The absolute organ weight including brain, pituitary gland, adrenal gland, liver, spleen, kidneys, heart, thymus, lungs, salivary gland, thyroid gland, testes, epididymides, seminal vesicle, prostate, uterus, and ovaries were weighed and the relative organ weight (% of terminal body weight) were calculated.

Statistical analysis − The collected data were statistically analyzed by multiple comparison methods. When the Bartlett’s test indicated no significant deviations from variance homogeneity, the ANOVA test was conducted to determine if any of the group means differed at the P < 0.05 level. When found significant in ANOVA, Dunnett’s test was used to determine the difference between the control and treatment groups. In the case of significant deviations from variance homogeneity in the Bartlett’s test, a non-parametric comparison test, Kruskal-Wallace (H) test, was conducted to determine if any of the group means differed at the P < 0.05 level. When a significant difference was observed in the Kruskal-Wallace (H) test, the Dunn's Rank Sum test was conducted to quantify the specific pairs of group comparison, which are significantly different. The Fisher’s exact test was conducted to determine the pairs of group comparison (including prevalence or percentage). The level of probability was taken as 1 or 5%. Statistical analyses were performed by comparing the different dose groups with a vehicle control group using Path/Tox (version 4.2.2, Xybion Medical Systems Corporation).

 

Results and Discussion

Although the pharmacological effect or the analysis for extracts of Epimedii Herba (EH) have been revealed by a numerous reports, the information on its safety or toxicity could not be found anywhere. To obtain a toxicity data of EH aqueous extract (EHAE), F344 rats were orally treated with EHAE for 2 weeks. Rats consisted of six groups, which were treated with a vehicle control or 500 mg/kg/day, 1000 mg/kg/day, 2000 mg/kg/day, 3500 mg/kg/day, and 5000mg/kg/day EHAE. Mortality, clinical observations, body weight, food consumption, hematology, clinical chemistry, urinalysis, macroscopic findings, and organ weight were observed.

In 14-day repeated oral toxicity study, there were no treatment-related mortalities and clinical signs in any groups throughout the study period. In addition, none of groups treated with EHAE exhibited drug-related changes in body weight (Figs. 1 and 2) and food consumption (Table 1), compared with the vehicle control group. There were no significant changes found in urinalysis test for volume, specific gravity, pH, protein, ketone body, GLU, BIL, nitrite, urobilinogen, urine cast, epithelial cell, RBC, and WBC, which were compared with control group (data not shown).

Fig. 1.Changes in body weights after treatment with 500, 1000, 2000, 3500 and 5000 mg/kg/day in male of EHAE.

Fig. 2.Changes in body weights after treatment with 500, 1000, 2000, 3500 and 5000 mg/kg/day in female of EHAE.

Table 1.* Significant differences from control group (p < 0.05). + Significant differences from control group (p < 0.01).

In hematology shown as Table 2, RBC count (0.90X control), hemoglobin (HGB, 0.91X), hematocrit (HCT, 0.94X), and mean corpuscular hemoglobin concentration (MCHC, 0.97X) were decreased at 5000 mg/kg/day male group. Mean corpuscular volume (MCV, 1.04X), reticulocyte count (1.70X), and reticulocyte ratio (1.89X) were increased at 5000 mg/kg/day male group (Table 2). RBC count (0.93X control), HGB (0.94X), HCT (0.96X), and MCHC (0.98X) were decreased at 5000 mg/kg/day female group. MCV (1.04X), reticulocyte count (1.88X), and reticulocyte ratio (2.03X) were increased at 5000 mg/kg/day female group. In clinical chemistry shown as Table 3, TCHO (0.82X, 0.84X control) and PL (0.86X, 0.87X control) were decreased at 5000mg/kg/day male and female groups. These hematology or clinical chemistry values showed apparent difference from the control mean values, and/or were not normal limits (Charles River Laboratories Japan, Inc, Japan). Therefore, these changes of parameters in hematology or clinical chemistry are considered to be closely related to the treatment of EHAE, and require having further study for the long-term outcomes. Absolute organ weights are shown in Table 4. There were no treatment-related in either the absolute and relative organ weights changes in the treated groups compared to the vehicle control group (Table 5). In addition, no treatment-related macroscopic findings were observed in any of the treated animals.

Table 2.* Significant differences from control group (p < 0.05). + Significant differences from control group (p < 0.01).

Table 3.* Significant differences from control group (p < 0.05). + Significant differences from control group (p < 0.01).

Table 4.Absolute organ weights after treatment with 500, 1000, 2000, 3500 and 5000 mg/kg/day of EHAE

Table 5.Relative organ weights after treatment with 500, 1000, 2000, 3500 and 5000 mg/kg/day of EHAE

In conclusion, EH was administered to male and female F344 rats at doses of 0 (vehicle control), 500, 1000, 2000, 3500 and 5000 mg/kg/day for 2 weeks by oral gavage. There were no treatment-related adverse changes in clinical observations, mortality, body weights, food consumption, urinalysis, macroscopic finding at necropsy and organ weight examination. Total RBC count, HCT and MCHC, TCHO, PL were decreased in males and females at 5000 mg/kg/day compared to the control animals. MCV and reticulocyte counts were increased in males and females at 5000 mg/kg/day compared to the control animals. Therefore, we recommend that a dose level of 5000 mg/kg/day is a highest treatment group in 13-week EH extract exposure study for further toxicity assessment.