Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats

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Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats Jun-Won Yun a,1, Jeong-Hwan Che a,b,1, Euna Kwon a, Yun-Soon Kim a, Seung-Hyun Kim a, Ji-Ran You a, Woo Ho Kim c, Hyeon Hoe Kim d, Byeong-Cheol Kang a,b,e,f,⇑ a

Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Biomedical Center for Animal Resource and Development, N-BIO, Seoul National University, Seoul, Republic of Korea Department of Pathology, Seoul National University College of Medicine, Seoul, Republic of Korea d Department of Urology, Seoul National University College of Medicine, Seoul, Republic of Korea e Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea f Designed Animal and Transplantation Research Institute, Institute of GreenBio Science Technology, Seoul National University, Pyeongchang-gun, Gangwon-do, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 30 April 2015 Available online xxxx Keywords: Angelica gigas Traditional medicine Toxicity Subchronic Genotoxicity

a b s t r a c t As a well-known traditional medicine, Angelica gigas (AG) and its active constituents, including decursin and decursinol, have been shown to possess several health beneficial properties such as anti-bacterial, immunostimulating, anti-tumor, neuroprotective, anti-nociceptive and anti-amnestic activities. However, there is lack of toxicity studies to assess potential toxicological concerns, especially long-term toxicity and genotoxicity, regarding the AG extract. Therefore, the safety of AG extract was assessed in subchronic toxicity and genotoxicity assays in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a subchronic toxicity study for 13 weeks (125, 250, 500, 1000 and 2000 mg/kg body weight, delivered by gavage), data revealed no significant adverse effects of the AG extract in food consumption, body weight, mortality, hematology, biochemistry, necropsy, organ weight and histopathology throughout the study in male and female rats. These results suggest that no observed adverse effect level of the AG extract administered orally was determined to be greater than 2000 mg/kg/day, the highest dose tested. In addition, a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay suggested that the AG extract was not genotoxic. In conclusion, the AG extract appears to be safe as a traditional medicine for oral consumption. Ó 2015 Published by Elsevier Inc.

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1. Introduction

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Angelica gigas (AG) belongs to the Umbelliferae family. Based on its area of distribution, three common species of Angelica roots can

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Abbreviations: AG, Angelica gigas; OECD, Organization for Economic Co-operation and Development; WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; PLT, platelet; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; BUN, blood urea nitrogen; TC, total cholesterol; TP, total protein; TB, total bilirubin; ALP, alkaline phosphatase; AST, aspartate transaminase; ALT, alanine transaminase; cGT, c-glutamyl transferase; TG, triglyceride; CHL, Chinese hamster lung; MNPCEs, micronucleated polychromatic erythrocytes; NCEs, normochromatic erythrocytes; HPLC, high-performance liquid chromatography. ⇑ Corresponding author at: Graduate School of Translational Medicine, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul 110-744, Republic of Korea. Fax: +82 2 741 7620. E-mail address: [email protected] (B.-C. Kang). 1 Contributed equally to this work.

be found in Asia: A. gigas from Korea, A. sinensis from China, and A. acutiloba from Japan (Lv et al., 2007). In particular, AG, known by the Korean name ‘Cham-dang-gui’, grows naturally and is cultivated in the alpine region of Korea (Hwang and Yang, 1997). The dried roots of AG have long been used as a traditional folk medicine for various pharmacologic effects including anti-bacterial (Lee et al., 2003b), immunostimulating (Han et al., 1998), anti-tumor (Lee et al., 2003a), neuroprotective (Kang et al., 2005), anti-nociceptive (Choi et al., 2003a) and anti-amnestic (Kang et al., 2003) activities. AG is also known for the improvement of hypercholesterolemia and prevention of atherosclerosis (Jang et al., 2014). In addition, AG exerted anti-inflammatory activities against carrageenan-induced inflammation (Shin et al., 2009), croton oil-induced ear inflammation (Shin et al., 2010), dinitrofluorobenzene-induced allergic dermatitis models (Joo et al., 2010), and thermal burn (Shin et al., 2008).

http://dx.doi.org/10.1016/j.yrtph.2015.05.025 0273-2300/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Yun, J.-W., et al. Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats. Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.05.025

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As there is considerable concern about the adverse effects of synthetic chemical drugs, there is increasing interest in natural products since traditional medicines are widely perceived as natural, safe, and free from side effects (Markman, 2002; Shin et al., 2013). In particular, traditional medicines can be less toxic when prescribed strictly with attention to plant origin, method of preparation, dose, and treatment duration (Liu et al., 2011). In other words, the popularity of traditional medicines demands a comprehensive analysis of safety issues. In fact, we recently found that various beneficial herbs have toxic side effects (Che et al., 2014, 2015; Yun et al., 2015). Although AG is currently available on the market as an herbal medicine in East Asian countries, there is lack of toxicity studies that have been carried out for the toxicity of the AG extract, and their safety is not guaranteed. In this study, we report the results from subchronic toxicity studies of orally administered AG extract and from genotoxicity studies including a bacterial reverse mutation assay (Ames test), in vitro chromosome aberration assay and a micronucleus assay in mice.

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2. Materials and methods

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2.1. Test substance and animals

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A hot water AG extract was provided by the National Institute of Food and Drug Safety Evaluation (Osong, Korea). AG roots were purchased from an Oriental medicine market in Korea, and an extract of AG was obtained according to a method described previously (Yun et al., 2015). In brief, dried AG roots were ground by a mixer, and incubated with distilled water (DW) at 110 °C. After filtration through filter paper, the filtrate was freeze-dried and dissolved in DW for oral administration. The extraction yield of the hot water AG extract was 0.216 g of freeze-dried AG extract/g of dried AG root. Analysis of decursin and decursinol obtained from the AG extract was performed using the HPLC equipment Shimadzu SCL-10AVP (Kyoto, Japan). The analysis was carried out using a YMC-Pack ODS-A column (150  6 mm, 5 lm particle size) and methanol-based mobile phase composed of methanol and 1% acetic acid water with gradient elution as follows: 0–10 min, methanol 55%; 10–11 min, methanol 55–80%; 11–25 min, methanol 80%; 25–26 min, methanol 80–100%; 26–40 min, methanol 100%. The detection was carried out using a diode array detector. F344 rats (SLC, Hamamatsu, Japan) and ICR mice (Orient Bio, Seongnam, Korea) were used after a week of quarantine and acclimatization. During the studies, the animal facility was maintained under standard conditions (22 ± 2 °C, 40–60% humidity, and 12 h light/dark cycle). The animals were fed a rodent diet (LabDiet 5002 Certified Rodent Diet, PMI Nutrition International, St. Louis, MO, USA) and tap water ad libitum. All of the animal experiments were approved by the Institutional Animal Care and Use Committee of the Biomedical Research Institute at the Seoul National University Hospital, and this study was performed in compliance with the guidelines published by the Organization for Economic Cooperation and Development (OECD) as well as the guidance for Good Laboratory Practices for toxicity tests issued by the Ministry of Food and Drug Safety (MFDS, 2005).

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2.2. Experimental design for the oral toxicity study

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For the 14-day repeat-dose toxicity study, the hot water AG extract was administered to F344 rats (5/sex/group) by oral gavage at doses of 125, 250, 500, 1000, and 2000 mg/kg of body weight/10 ml DW once daily for 14 days. For the 13-week repeat-dose toxicity study, the hot water AG extract was administered to F344 rats (10/sex/group) by oral gavage at doses of 125, 250, 500, 1000, and 2000 mg/kg of body weight/10 ml DW once

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daily for 13 weeks in accordance with OECD guideline 408 (OECD, 1998) and the US National Toxicology Program (NTP) protocol (https://ntp.niehs.nih.gov/testing/types/cartox/protocols/ 13 week/index.html). During the administration period, the rats were observed for general appearance daily, and body weights, food intake, and water consumption were recorded weekly. The rats were anesthetized with isoflurane one day after the final gavage.

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2.3. Hematology and serum biochemistry

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Blood samples were collected via the posterior vena cava. The hematology parameters were measured using an automatic hematology analyzer MS9-5 Hematology Counter (Melet Schloesing Laboratories, Osny, France) for the following parameters: total white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelet (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and differential WBC. And, the standard serum biochemistry parameters were analyzed with an automatic chemistry analyzer 7070 (Hitachi, Tokyo, Japan) to evaluate the following serum biochemistry parameters: blood urea nitrogen (BUN), total cholesterol (TC), total protein (TP), albumin, total bilirubin (TB), alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), c-glutamyl transferase (cGT), creatinine, creatinine kinase, triglyceride (TG), and glucose.

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2.4. Gross findings, organ weights, and histopathological assessments

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At the end of the treatment period, animals were exsanguinated, and organs and tissues were observed macroscopically. Organ weights were obtained for the liver, kidney, testis, thymus, heart, and lung. The eyes with the Harderian glands were fixed in Davidson solution (30 ml 95% ethyl alcohol + 20 ml formalin + 10 ml glacial acetic acid + 30 ml DW). The testis and epididymis were fixed in Bouin’s solution. Other organs including the liver, kidney, adrenal gland, urinary bladder, spleen, pancreas, thymus, thyroid gland, parathyroid gland, trachea, esophagus, lung, heart, salivary gland, lymph node, stomach, duodenum, jejunum, ileum, colon, rectum, preputial gland, clitoral gland, skin, brain, pituitary gland, bone marrow, prostate, seminal vesicle, ovary, uterus, and vagina were fixed in 10% neutral buffered formalin. The nasal cavity and femora were treated with a decalcification solution for up to 3 weeks. Tissue samples were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin (H&E). After staining, the histological preparations from animals in the control, 1000, and 2000 mg/kg groups were initially examined via light microscopy. With respect to the organs and tissues showing significant histological changes, preparations of all rats in the other groups were examined microscopically.

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2.5. Genotoxicity study

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Five characterized histidine-dependent strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535, TA1537; MFDS, Osong, Korea) were utilized for bacterial reverse mutation assay (Ames test) in accordance with OECD guideline 471 (OECD, 1997a). S. typhimurium strains were incubated with the AG extract with or without an S9 mix in the dark at 37 °C for 48 h. The standard mutagens (2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine, and 2-aminoanthracene; Sigma–Aldrich, St. Louis, MO, USA) were used as positive controls. The extract was considered to be positive if there was a twofold increase relative to negative control or a dose-dependent increase in the number of revertant colonies.

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An in vitro chromosomal aberration test using Chinese hamster lung (CHL) fibroblast cells was conducted in accordance with OECD guideline 473 (OECD, 1997b). The cells were incubated in a CO2 incubator (5% CO2, 37 °C, high humidity) with the AG extract in the presence or absence of an S9 mix for 6 h or 24 h. Mitomycin C and cyclophosphamide (Sigma–Aldrich) were used as positive controls. After colcemid (0.2 lg/ml, GIBCO, Carlsbad, CA, USA) was added for 2 h, the cells were treated with hypotonic solution, fixed in 3:1 methanol/glacial acetic acid, and stained with 4% Giemsa. An in vivo bone marrow micronucleus test was conducted in accordance with OECD guideline 474 (OECD, 1997c). 8-week-old male ICR mice were orally treated with the AG extract at 0, 500, 1000, and 2000 mg/kg body weight once daily for 4 d. Mitomycin C (2 mg/kg) served as a positive control and was intraperitoneally injected. The mice were euthanized at 24 h after the last dose. The femoral bone marrow cells were isolated, centrifuged, smeared on the slides, and dried. After fixation with methanol and 5% Giemsa staining, the number of micronucleated polychromatic erythrocytes (MNPCEs) was counted from 2000 PCEs. In addition, the PCE/(PCE + NCE) ratio, where the NCEs indicate the normochromatic erythrocytes, was calculated to detect the possibility of cytotoxicity (Heddle et al., 1984).

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2.6. Statistical analysis

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All of the values are expressed as mean ± SD. The statistical analysis was performed using a one-way ANOVA, followed by a multiple comparison procedure with a Tukey/Duncan test using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). P values of less than 0.05 were considered to be statistically significant.

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3. Results

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3.1. 14-day repeat-dose oral toxicity study

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Several coumarin derivatives, including decursin, decursinol, decursinol angelate, nodakenin, nodakenetin and umbelliferone, have been identified previously as major components in the AG extract (Chi and Kim, 1970; Kang et al., 2003; Shin et al., 2010). The presence of decursinol (1.7 mg/g of freeze-dried AG extract) and decursin (0.7 mg/g of freeze-dried AG extract) in the AG extract used in this study were confirmed by high performance liquid chromatography (HPLC). In the 14-day repeat-dose oral toxicity study, no animal deaths, abnormal clinical signs, body weight changes (Supplementary Fig. 1) related to the administration of the AG extract were observed throughout the duration of the experiment at any dose used. Likewise, no test substance-related effects were evident in macroscopic lesions at all dosages at necropsy after the 14-day observation period. There were no significant differences in absolute and relative organ weights among the groups (Supplementary Table 1). Based on the results from the current study, we selected 2000 mg/kg as a high-dose for the 13-week repeat-dose oral toxicity study.

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3.2. 13-week repeat-dose oral toxicity study

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3.2.1. General observation, body weight, feed intake, and water consumption No mortalities were noted in all groups. Neither abnormal signs nor symptoms attributable to the AG extract administration were observed in males and females in any group. Similarly, the body weight showed a normal increase in the AG extract-treated groups compared with the control group throughout the study (Fig. 1).

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Although statistically significant changes in the mean daily food (Supplementary Fig. 2A) and water consumption (Supplementary Fig. 2B) were often observed among the groups, these changes was transient and showed no dose-dependency.

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3.2.2. Hematology and clinical chemistry In the 250 (18.2 ± 0.4) and 2000 mg/kg (18.1 ± 0.4) male group, slight, yet statistically significant, decreases in MCH were noted relative to the control group (18.6 ± 0.3). However, values were within historical control ranges (Table 1). No significant changes were observed in other hematological parameters measured. The biochemical parameters are presented in Table 2. The TC levels in males treated with 250 (72.5 ± 4.0), 500 (73.3 ± 6.0), 1000 (78.7 ± 6.6) and 2000 mg/kg (74.9 ± 4.2) of the AG extract were significantly higher than that in the control group (66.9 ± 3.6). The TP levels significantly increased in males treated with 250 (6.9 ± 0.2), 500 (6.9 ± 0.2), 1000 (7.0 ± 0.2) and 2000 mg/kg (6.9 ± 0.1) of the AG extract relative to the control males (6.6 ± 0.2). The albumin levels in males treated with the AG extract with doses of 250 (3.1 ± 0.1), 500 (3.2 ± 0.1), 1000 (3.2 ± 0.1) and 2000 mg/kg (3.2 ± 0.1) were significantly greater than those in the control group (3.0 ± 0.1). Serum AST and ALT levels in males exhibited significant decreases as a result of the treatment with the AG extract at a dose of 2000 mg/kg (AST, 78.1 ± 12.0; ALT, 52.3 ± 110.8), relative to the respective control groups (AST, 100.1 ± 24.5; ALT, 68.6 ± 20.3). However, these changes were considered spurious and were not considered treatment-related because they were very slight in degree and were within physiologically acceptable ranges.

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3.2.3. Organ weights and histopathological changes Table 3 shows the results for the absolute and relative organ weights in the male and female rats administered the AG extract. Significant increases in the absolute liver weights were noted in females treated with 1000 (5.54 ± 0.50) and 2000 mg/kg (5.71 ± 0.41) of the AG extract compared with the control group (4.98 ± 0.37). The relative liver weight significantly increased in females treated with 1000 mg/kg AG extract (3.01 ± 0.33) compared with the control group (2.70 ± 0.20). And, a significant increase in the absolute kidney weight was noted in females treated with 2000 mg/kg AG extract (0.64 ± 0.03) in comparison to the control group (0.58 ± 0.04). However, the changes were very minimal and not toxicologically meaningful. No obvious differences were found in other organs between the control group and the AG extract-treated groups of either sex. Gross pathological examination of the organs of animals treated with the AG extract did not show any difference when compared with the untreated groups in both sexes. The histopathological examinations of the sampled organs revealed that all the features were within the normal parameters and no dose-related abnormal findings were attributed to the AG extract administration (data not shown).

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3.3. Genotoxicity

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3.3.1. Ames test An Ames test was performed to assay the mutagenic potential of the AG extract using histidine requiring strains of S. typhimurium TA98, TA100, TA102, TA1535 and TA1537. The AG extract treatments at all concentration levels (312.5, 625, 1250, 2500 and 5000 lg/plate) did not significantly increase the revertant colonies in plates of five strains in the absence and presence of S9 activation when compared to the negative control (Table 4). In contrast, the various mutagens induced a twofold or greater increase in the number of His+ mutants with or without of S9 mix relative to the negative control. Therefore, no mutagenic effect of the AG extract was observed in the bacterial strains tested under the current conditions.

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Fig. 1. Effects of the AG extract on the body weight changes after oral administration in male and female rats for 13 weeks. Data expressed as means ± SD.

Table 1 Hematological data for male and female F344 rats orally administered with Angelica gigas extract for 13 weeks. Dose of Angelica gigas (mg/kg)

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0

125

250

500

1000

2000

Males WBC (103/mm3) RBC (106/mm3) HGB (g/dl) HCT (%) PLT (103/mm3) MCV (fl) MCH (pg) MCHC (g/dl) Neutrophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%)

8.5 ± 0.9 9.0 ± 0.3 16.5 ± 0.4 43.2 ± 1.9 553.2 ± 52.6 48.6 ± 1.2 18.6 ± 0.3 38.2 ± 1.3 16.2 ± 2.8 0.6 ± 1.0 0.8 ± 0.1 76.7 ± 2.4 4.4 ± 0.4

8.1 ± 1.1 8.8 ± 0.3 16.4 ± 0.5 43.0 ± 1.3 544.2 ± 36.2 48.6 ± 0.5 18.5 ± 9.5 38.1 ± 0.6 16.0 ± 2.7 0.5 ± 0.6 0.7 ± 0.2 77.2 ± 3.8 4.3 ± 0.6

8.1 ± 1.2 9.0 ± 0.2 16.4 ± 0.3 43.7 ± 1.6 542.3 ± 29.2 51.7 ± 9.5 18.2 ± 0.4* 37.4 ± 1.4 17.2 ± 1.3 0.4 ± 0.4 0.6 ± 0.1 76.2 ± 1.5 4.4 ± 0.5

8.2 ± 1.5 8.8 ± 0.2 16.3 ± 0.5 42.3 ± 1.0 574.9 ± 24.4 48.2 ± 1.0 18.5 ± 0.3 38.4 ± 0.9 16.2 ± 1.8 0.4 ± 0.3 0.6 ± 0.1 77.3 ± 2.2 4.1 ± 0.5

8.1 ± 1.2 8.9 ± 0.2 16.3 ± 0.4 43.1 ± 1.0 545.8 ± 38.5 48.3 ± 0.8 18.2 ± 0.4 37.8 ± 0.8 28.2 ± 35.8 0.7 ± 1.3 0.7 ± 0.2 76.5 ± 3.8 4.4 ± 0.6

8.0 ± 1.6 8.8 ± 0.7 16.3 ± 0.4 43.1 ± 1.3 565.4 ± 27.2 47.7 ± 0.9 18.1 ± 0.4* 37.9 ± 1.2 16.1 ± 2.2 0.3 ± 0.1 0.6 ± 0.1 77.8 ± 2.4 3.9 ± 0.4

Females WBC (103/mm3) RBC (106/mm3) HGB (g/dl) HCT (%) PLT (103/mm3) MCV (fl) MCH (pg) MCHC (g/dl) Neutrophils (%) Eosinophils (%) Basophils (%) Lymphocytes (%) Monocytes (%)

6.3 ± 1.0 8.3 ± 0.2 16.4 ± 0.6 43.0 ± 1.5 568.8 ± 39.9 51.9 ± 1.0 19.7 ± 0.4 38.0 ± 0.9 13.9 ± 4.0 0.2 ± 0.2 0.7 ± 0.2 79.6 ± 4.7 4.1 ± 0.7

6.3 ± 1.1 8.4 ± 0.4 16.7 ± 0.7 43.5 ± 2.5 590.5 ± 52.9 51.8 ± 0.6 19.8 ± 0.3 38.3 ± 0.8 13.7 ± 3.0 0.2 ± 0.1 0.6 ± 0.1 79.8 ± 3.6 4.3 ± 0.8

6.0 ± 0.7 8.3 ± 0.2 16.4 ± 0.5 43.1 ± 1.7 558.4 ± 42.1 52.3 ± 2.1 19.8 ± 0.4 37.9 ± 1.6 13.8 ± 2.7 0.2 ± 0.2 0.7 ± 0.1 80.0 ± 3.2 3.9 ± 0.5

5.8 ± 0.6 8.2 ± 0.2 16.3 ± 0.4 42.4 ± 0.8 577.7 ± 46.7 51.7 ± 0.8 19.9 ± 0.5 38.4 ± 1.3 13.5 ± 2.4 0.3 ± 0.2 0.6 ± 0.2 80.2 ± 2.8 4.0 ± 0.5

5.8 ± 1.2 9.1 ± 2.4 16.4 ± 0.5 51.2 ± 24.7 859.9 ± 908.4 54.1 ± 7.8 18.7 ± 3.1 35.5 ± 8.1 13.6 ± 2.7 0.2 ± 0.1 0.7 ± 0.1 79.9 ± 3.7 4.0 ± 0.9

6.2 ± 1.0 8.5 ± 0.3 16.5 ± 0.7 43.7 ± 2.0 574.1 ± 46.1 51.7 ± 0.9 19.5 ± 0.5 37.8 ± 1.4 13.5 ± 2.5 0.3 ± 0.3 0.8 ± 0.3 79.8 ± 3.6 4.0 ± 0.7

Significantly different from control group (p < 0.05).

3.3.2. In vitro chromosomal aberration assay From the results obtained with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay (Supplementary Table 2), 5000 lg/ml of the AG extract (CHL cell survival rate, 93.25%) was selected as the highest exposure level for in vitro chromosomal aberration assay. Cultures treated with the positive controls showed significant increases in the incidence of structural chromosome aberrations with or without the S9 mix (Table 5). In contrast, no significant increase in the incidence of chromosomal aberrations, including breaks, fragments, and exchanges, could be detected in the presence or absence of S9 mix at any of the concentration of the AG extract.

49.4%, 57.0%, and 46.7% for the negative control, at 500, 1000, 2000 mg/kg of the AG extract, and the positive control, respectively (Table 6). No decrease in this ratio reflects a lack of toxic effects of the AG extract (Heddle et al., 1984). As for the number of micronuclei, the animals of the mitomycin C-treated group showed a significant increase when compared with the negative control. In contrast, no significant induction of micronuclei was observed by the AG extract treatments at 500, 1000, or 2000 mg/kg of body weight. Although the micronuclei number in high-dose group (2000 mg/kg) was a 3.5 times increase relative to the negative control, this number was within historical control ranges (0–4/2000 PCEs, 0.9 ± 1.1).

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3.3.3. In vivo bone marrow micronucleus assay During the study, no abnormality occurred and no abnormal signs in general appearance were noted in any of the groups. The mean ratio of PCEs to total erythrocytes were 52.2%, 62.1%,

4. Discussion

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The dried root of AG is an important herbal medicine that is traditionally used for the treatments of anemia and some circulatory

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J.-W. Yun et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx Table 2 Serum biochemistry data for male and female F344 rats orally administered with Angelica gigas extract for 13 weeks. Dose of Angelica gigas (mg/kg)

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250

500

1000

2000

Males BUN (mg/dL) TC (mg/dL) TP (g/dL) Albumin (g/dL) TB (mg/dL) ALP (IU/L) AST (IU/L) ALT (IU/L) cGT (IU/L) Creatinine kinase (IU/L) Creatinine (mg/dL) TG (mg/dL) Glucose (mg/L)

19.2 ± 1.8 66.9 ± 3.6 6.6 ± 0.2 3.0 ± 0.1 0.1 ± 0.0 173.5 ± 26.8 100.1 ± 24.5 68.6 ± 20.3 0.0 ± 0.0 387.9 ± 160.4 0.6 ± 0.0 180.5 ± 45.3 177.9 ± 21.9

18.9 ± 3.2 71.4 ± 5.4 6.7 ± 0.2 3.0 ± 0.1 0.1 ± 0.0 178.9 ± 27.0 103.0 ± 13.2 65.8 ± 6.5 0.1 ± 0.3 390.3 ± 239.9 0.6 ± 0.0 181.0 ± 48.7 173.1 ± 15.2

19.8 ± 2.3 72.5 ± 4.0** 6.9 ± 0.2** 3.1 ± 0.1** 0.1 ± 0.0 185.8 ± 37.6 98.0 ± 11.4 66.6 ± 9.7 0.0 ± 0.0 300.7 ± 79.6 0.6 ± 0.0 219.7 ± 62.5 187.2 ± 14.2

19.8 ± 3.1 73.3 ± 6.0** 6.9 ± 0.2** 3.2 ± 0.1** 0.1 ± 0.0 172.2 ± 23.9 91.0 ± 20.8 62.0 ± 9.0 0.0 ± 0.0 242.1 ± 102.3 0.6 ± 0.0 175.7 ± 52.2 192.1 ± 22.9

19.3 ± 3.2 78.7 ± 6.6** 7.0 ± 0.2** 3.2 ± 0.1** 0.1 ± 0.0 174.4 ± 35.7 90.7 ± 16.5 59.2 ± 11.0 0.0 ± 0.0 349.8 ± 150.2 0.6 ± 0.0 218.2 ± 61.1 189.1 ± 24.0

17.7 ± 3.3 74.9 ± 4.2** 6.9 ± 0.1** 3.2 ± 0.1** 0.1 ± 0.0 165.8 ± 23.6 78.1 ± 12.0* 52.3 ± 10.8* 0.0 ± 0.0 268.1 ± 113.5 0.6 ± 0.0 228.3 ± 40.4 179.7 ± 16.3

Females BUN (mg/dL) TC (mg/dL) TP (g/dL) Albumin (g/dL) TB (mg/dL) ALP (IU/L) AST (IU/L) ALT (IU/L) cGT (IU/L) Creatinine kinase (IU/L) Creatinine (mg/dL) TG (mg/dL) Glucose (mg/L)

18.9 ± 3.2 89.8 ± 8.7 6.5 ± 0.3 3.0 ± 0.2 0.1 ± 0.0 180.4 ± 40.6 90.9 ± 12.1 60.9 ± 7.3 0.4 ± 0.7 695.1 ± 561.9 0.6 ± 0.1 91.5 ± 34.4 165.1 ± 16.9

18.4 ± 2.4 93.2 ± 5.2 6.7 ± 0.4 3.2 ± 0.2 0.1 ± 0.0 163.8 ± 42.5 97.5 ± 17.0 61.0 ± 8.3 0.4 ± 0.7 561.6 ± 329.0 0.6 ± 0.0 79.9 ± 24.9 157.9 ± 17.7

18.4 ± 3.3 89.6 ± 5.2 6.7 ± 0.3 3.1 ± 0.2 0.1 ± 0.0 159.1 ± 45.1 89.5 ± 14.9 61.1 ± 10.3 0.3 ± 0.7 445.7 ± 330.6 0.6 ± 0.0 90.7 ± 33.9 165.4 ± 19.8

17.5 ± 3.9 94.0 ± 5.7 6.8 ± 0.2 3.2 ± 0.1 0.1 ± 0.0 142.5 ± 31.7 90.9 ± 17.4 58.9 ± 9.8 0.6 ± 0.7 432.1 ± 201.3 0.6 ± 0.0 95.2 ± 24.4 167.4 ± 16.2

15.3 ± 5.1 82.8 ± 28.2 5.9 ± 2.0 2.8 ± 1.0 0.1 ± 0.0 145.2 ± 53.3 77.7 ± 23.3 51.8 ± 17.2 0.5 ± 0.7 429.3 ± 225.6 0.5 ± 0.2 93.0 ± 33.1 139.7 ± 44.2

17.8 ± 5.6 98.1 ± 8.2 6.8 ± 0.4 3.2 ± 0.2 0.1 ± 0.0 158.4 ± 24.1 78.1 ± 11.1 58.2 ± 10.1 0.4 ± 0.7 492.2 ± 308.9 0.6 ± 0.0 120.2 ± 37.3 159.6 ± 14.7

Significantly different from control group (p < 0.05). Significantly different from control group (p < 0.01).

Table 3 Organ weights for male and female F344 rats orally administered with Angelica gigas extract for 13 weeks. Dose of Angelica gigas (mg/kg)

Males Liver Kidney Testis Thymus Heart Lung Females Liver Kidney Thymus Heart Lung

* **

339 340 341 342 343

0

125

250

500

1000

2000

(g) (%BW) (g) (%BW) (g) (%BW) (g) (%BW) (g) (%BW) (g) (%BW)

10.82 ± 1.21 3.05 ± 0.30 1.09 ± 0.06 0.31 ± 0.02 1.54 ± 0.05 0.43 ± 0.01 0.24 ± 0.02 0.07 ± 0.01 0.98 ± 0.05 0.28 ± 0.01 1.25 ± 0.06 0.35 ± 0.02

10.60 ± 0.66 3.05 ± 0.27 1.07 ± 0.05 0.31 ± 0.01 1.49 ± 0.03 0.43 ± 0.02 0.25 ± 0.02 0.07 ± 0.01 0.95 ± 0.04 0.27 ± 0.01 1.22 ± 0.05 0.35 ± 0.01

10.72 ± 0.81 3.10 ± 0.23 1.07 ± 0.06 0.31 ± 0.01 1.51 ± 0.05 0.44 ± 0.01 0.25 ± 0.02 0.07 ± 0.01 0.96 ± 0.03 0.28 ± 0.01 1.22 ± 0.07 0.35 ± 0.02

10.77 ± 0.80 3.12 ± 0.23 1.09 ± 0.04 0.31 ± 0.01 1.50 ± 0.06 0.43 ± 0.02 0.25 ± 0.03 0.07 ± 0.01 0.96 ± 0.05 0.28 ± 0.01 1.20 ± 0.07 0.35 ± 0.02

10.89 ± 0.83 3.14 ± 0.19 1.08 ± 0.04 0.31 ± 0.01 1.49 ± 0.05 0.43 ± 0.02 0.24 ± 0.02 0.07 ± 0.01 0.95 ± 0.04 0.27 ± 0.00 1.21 ± 0.08 0.35 ± 0.01

11.50 ± 1.06 3.34 ± 0.25 1.09 ± 0.06 0.32 ± 0.01 1.52 ± 0.06 0.44 ± 0.02 0.23 ± 0.02 0.07 ± 0.01 0.95 ± 0.03 0.28 ± 0.01 1.25 ± 0.07 0.36 ± 0.01

(g) (%BW) (g) (%BW) (g) (%BW) (g) (%BW) (g) (%BW)

4.98 ± 0.37 2.70 ± 0.20 0.58 ± 0.04 0.31 ± 0.02 0.19 ± 0.02 0.10 ± 0.01 0.60 ± 0.02 0.32 ± 0.01 0.84 ± 0.04 0.45 ± 0.02

5.32 ± 0.43 2.79 ± 0.23 0.61 ± 0.02 0.32 ± 0.02 0.20 ± 0.01 0.10 ± 0.01 0.67 ± 0.20 0.35 ± 0.10 0.88 ± 0.04 0.46 ± 0.03

5.12 ± 0.22 2.72 ± 0.19 0.59 ± 0.03 0.31 ± 0.02 0.18 ± 0.02 0.10 ± 0.01 0.60 ± 0.03 0.32 ± 0.01 0.83 ± 0.04 0.44 ± 0.02

5.13 ± 0.28 2.74 ± 0.18 0.58 ± 0.04 0.31 ± 0.02 0.19 ± 0.02 0.10 ± 0.01 0.61 ± 0.03 0.33 ± 0.01 0.83 ± 0.05 0.44 ± 0.03

5.54 ± 0.50** 3.01 ± 0.33* 0.62 ± 0.06 0.33 ± 0.02 0.20 ± 0.05 0.10 ± 0.02 0.61 ± 0.03 0.33 ± 0.04 0.86 ± 0.06 0.47 ± 0.04

5.71 ± 0.41** 2.98 ± 0.17 0.64 ± 0.03* 0.33 ± 0.02 0.20 ± 0.01 0.10 ± 0.00 0.62 ± 0.03 0.32 ± 0.01 0.86 ± 0.04 0.45 ± 0.02

Significantly different from control group (p < 0.05). Significantly different from control group (p < 0.01).

disorders (Jung et al., 1991), and as a sedative, an analgesic (Sarker and Nahar, 2004; Kim et al., 2006b), an anti-inflammatory (Shin et al., 2009), an anti-cancer agent (Lee et al., 2003a). The roots of this plant are composed of various active components including decursin, decursinol, decursinol angelate, nodakenin, nodakenetin,

and umbelliferone (Chi and Kim, 1970; Kang et al., 2003; Shin et al., 2010). Among these, decursin, the most abundant component of the AG roots (Ahn et al., 2008), inhibited the induction of inflammatory mediators from lipopolysaccharide-stimulated macrophages (Kim et al., 2006a). Decursinol was shown to possess

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Table 4 Results of S. typhimurium reversion assay with Angelica gigas extract. S9

+

Chemical

Dose (lg/plate)

His+ revertanat colonys/plate TA98

TA100

TA102

TA1535

TA1537

Distilled watera 2-Nitrofluoreneb Sodium azideb Mitomycin Cb Sodium azideb 9-Aminoacridineb Angelica gigas

– 10 5 0.5 0.5 80 312.5 625 1250 2500 5000

26 ± 5 307 ± 23* – – – – 17 ± 7 25 ± 7 30 ± 6 24 ± 6 28 ± 4

193 ± 16 – 1442 ± 90* – – – 206 ± 4 232 ± 26 249 ± 18 249 ± 32 280 ± 11

368 ± 9 – – 2221 ± 530* – – 411 ± 18 374 ± 86 467 ± 21 409 ± 37 531 ± 22

12 ± 5 – – – 423 ± 43* – 12 ± 3 14 ± 4 22 ± 4 19 ± 2 19 ± 4

72 ± 11 – – – – 1132 ± 142* 72 ± 5 96 ± 6 77 ± 9 79 ± 12 96 ± 12

Distilled watera 2-Aminoanthraceneb

– 2 5 312.5 625 1250 2500 5000

47 ± 2 286 ± 23* – 34 ± 8 45 ± 1 39 ± 7 38 ± 7 38 ± 5

198 ± 16 791 ± 69* – 173 ± 27 203 ± 6 178 ± 17 192 ± 24 223 ± 4

458 ± 63 – 1029 ± 122* 452 ± 24 429 ± 74 501 ± 32 557 ± 33 419 ± 72

10 ± 4 – 245 ± 49* 11 ± 6 13 ± 3 11 ± 3 15 ± 4 16 ± 5

74 ± 14 – 736 ± 87* 67 ± 3 81 ± 18 75 ± 10 81 ± 11 86 ± 3

Angelica gigas

a b *

Negative control. Positive control. Significantly different from negative control group (p < 0.05).

Table 5 Results of chromosomal aberration induced by Angelica gigas extract. Dose (lg/ml)

Substance

Number of cells scored

No. of cells with aberrations S9

MEMa Mitomycin Cb Cyclophosphoamideb Distilled water Angelica gigas

a b *

– 0.1 5 – 625 1250 2500 5000

200 200 200 200 200 200 200 200

Substance Distilled water Angelica gigas

Mitomycin Cb

a

Dosage (mg/kg BW)

Number of mice

MNPCEc

PCE/ (PCE + NCE)d

0.0 500 1000 2000 2

5 5 5 5 5

0.4 ± 0.6 0.0 ± 0.0 0.4 ± 0.9 1.4 ± 1.1 66.0 ± 8.3*

52.2 ± 11.2 62.1 ± 8.4 49.4 ± 6.9 57.0 ± 11.6 46.7 ± 2.7

a

Negative control. b Positive control. c Polychromatic erythrocyte with micronuclei was calculated from 2000 polychromatic erythrocytes (%). d The ratio of polychromatic erythrocytes to all erythrocytes (polychromatic + normochromatic) (%). * Significantly different from negative control group (p < 0.05).

350 351 352 353

24 h

6h

3.5 ± 0.7 34.0 ± 7.1* – 3.0 ± 1.4 5.0 ± 1.4 4.0 ± 2.8 2.0 ± 2.8 5.0 ± 4.2

0.5 ± 0.7 55.0 ± 5.7* – 0.5 ± 0.7 0.0 ± 0.0 0.5 ± 0.7 2.0 ± 2.8 0.5 ± 0.7

3.0 ± 0.0 – 95.0 ± 1.4* 3.0 ± 1.4 0.5 ± 0.7 2.5 ± 0.7 1.5 ± 2.1 1.5 ± 0.7

Minimum essential medium (negative control). Positive control. Significantly different from negative control group (p < 0.05).

Table 6 Micronucleated polychromatic erythrocytes (MNPCEs) in mice bone marrow following treatment with Angelica gigas extract.

349

+S9

6h

various pharmacological activities, including antinociceptive, analgesic, antiangiogenic, and anticancer effects (Choi et al., 2003b; Lee et al., 2009; Seo et al., 2009; Son et al., 2009). Also, decursinol angelate from the root of the AG showed in vitro cytotoxicity and protein kinase C activating activities (Ahn et al., 1997). Nodakenin

has been known to ameliorate scopolamine-induced memory disruption via enhancement of cholinergic signaling (Kim et al., 2007). Umbelliferone has been found to modulate reactive oxygen species generation in human blood lymphocytes (Kanimozhi et al., 2011), and have antitumor activity as well as immunomodulatory activity (Marshall et al., 1994). Despite many findings of the beneficial activities of the AG extract and its active components, there are limited data available to assess potential toxicological concerns, especially long-term toxicity, regarding the AG extract. Therefore, we have performed genotoxicity and subchronic oral toxicity study in rats using the AG extract, which was confirmed on the presence of considerable decursin and decursinol by HPLC analysis. In a 14-day repeat-dose oral toxicity study, the parameters of body weight, clinical observations, and organ weights showed no differences between the control and the AG extract treatment groups up to 2000 mg/kg, which is the highest recommended dose to be tested in the 13-week toxicity study. We have previously demonstrated the subchronic toxicities of various well-known traditional medicines in liver and kidney. Paecilomyces tenuipes, which has long been widely used for allergic diseases, asthma, cancer and tuberculosis (Zhu et al., 1998a,b), showed renal toxicity potential at the concentration higher than 500 mg/kg body weight. And, we

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also found that the hepatotoxic potentials of Sophorae radix, which has been observed to have anti-inflammatory (Kim et al., 2002) and free radical scavenging (Jung et al., 2005) activities, and vinegar-processed Genkwa flos, which has been used to treat edema, ascites, sudden cough, asthma and cancer (Zhan et al., 2005; Hong et al., 2011), in 13-week repeat-dose toxicity study. Therefore, various hematological and biochemical parameters were assessed to evaluate the toxic symptoms in main organs, including the kidney and liver, which are major organs that have several important functions (Worasuttayangkurn et al., 2012), in the 13-week repeat-dose toxicity study. The hematological analysis revealed that all of the observed changes were likely toxicologically irrelevant because they were within normal physiological ranges. Along with histopathological analysis, the detections of serum biochemical markers such as creatinine and BUN are the primary options for monitoring kidney dysfunction (Vaidya et al., 2010; Chen et al., 2013). We found no significant changes in the levels of serum BUN and creatinine between the control and the AG extract treatment groups. AST, ALT, and ALP are primarily used as sensitive markers for screening for liver disease (Mukinda and Syce, 2007). Serum ALT and AST are considered to be indicators for hepatocellular damage (Han et al., 2011). And, elevation of ALP is the most sensitive indicator of biliary obstruction (Ozdil et al., 2010). The analysis of these serum liver biomarker enzymes (AST, ALT, and ALP) showed that no test substance-related effects were evident. In addition, the AG extract did not produce any severe toxicity symptoms as seen from histopathological examinations of major organs (i.e. liver, kidney, lung, spleen, pancreas, and brain) that could be affected by long-term administration. Decursin, one of major components of the AG extract, was found to be metabolized rapidly (initial half-life of decursin, 0.05 h) to decursinol via an extensive hepatic first-pass metabolism after oral administration of decursin (Park et al., 2012) although decursinol exhibited high oral bioavailability following oral administration (Song et al., 2011). Thus, further study is needed for pharmacokinetic study to analyze the bioavailability of the AG extract in vivo. The battery of regulatory genotoxicity studies, including an Ames test, a chromosome aberration assay, and a micronucleus assay, were conducted to investigate the clastogenic and mutagenic potentials of the AG extract. The Ames test is the most commonly used in vitro method for detection of the mutagenic effects of genotoxicants (Hakura et al., 1999; Maron and Ames, 1983). In the Ames study, the S. typhimurium tester strains were employed to detect the induction of frameshift (TA98 and TA1537), base-pair substitution (TA100 and TA1535), or oxidative and cross-linking (TA102) mutation (Kaleeswaran et al., 2009). The results of this assay showed that no significant increases in revertant colonies of each test strains were noted in the AG extract-treated groups relative to the negative control group, indicating that the AG extract was not detectably mutagenic. The chromosomal aberration assay and micronucleus assay have been widely used in primary screening for detecting DNA damage at the chromosomal level (Fenech and Morley, 1985; Perry and Evens, 1975; Tucker et al., 1993). The current data for chromosome aberration in vitro and micronucleus formation in vivo indicated that the AG extract had no clastogenic activity. In conclusion, the 13-week repeat-dose oral toxicity result together with the negative genotoxicity results as seen from the Ames assay, in vitro chromosome aberration assay, and in vivo micronucleus assay clearly support that administration of the AG extract did not promote toxic effects. These results are consistent with the previous findings that evaluated the effects of decursin, the major component of the AG extract, in both genotoxicity (Kim et al., 2009a) and acute toxicity (Kim et al., 2009b). The NOAEL of the AG extract was considered to be greater than 2000 mg/kg in both male and female rats. According to the

7

surface-area-guided dosing adjustment of the US Food and Drug Administration (Reagan-Shaw et al., 2008), the human dose 324 mg/kg converted from the rat dose 2000 mg/kg could be considered for further clinical study. To our knowledge, this study is the first to demonstrate long-term toxicity and genotoxicity of the AG extract.

443

Conflict of Interest

449

The authors declare that they have no conflict of interest.

444 445 446 447 448

450

Transparency Document

451

The Transparency document associated with this article can be found in the online version.

452 456 455 453 454

Acknowledgments

457

This research was supported with a grant (05122MFDS471) from the Ministry of Food and Drug Safety. We thank Dr. Hyeong-Kyu Lee at Korea Research Institute of Bioscience and Biotechnology for kindly providing HPLC data for the AG extract.

458

Appendix A. Supplementary data

462

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.yrtph.2015.05. 025.

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Please cite this article in press as: Yun, J.-W., et al. Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats. Regul. Toxicol. Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.05.025