Journal of Ethnopharmacology 151 (2014) 1072–1078

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Research Paper

Subchronic oral toxicity of evodia fruit powder in rats Duyeol Kim a, Yong-Hoon Lee a, Sun Hee Park a, Mi Ju Lee a, Myoung Jun Kim a, Ho-Song Jang a, Jung-Min Lee a,e, Hye-Yeong Lee a, Beom Seok Han b, Woo-Chan Son c, Ji Hyeon Seok d, Jong Kwon Lee d, Jayoung Jeong d, Jin Seok Kang e,n, Jong-Koo Kang f,nn a

Department of Pathology, Biotoxtech Co., Ltd., 686-2 Yangcheong-ri, Ochang-eup, Cheongwon-gun, Chungbuk 363-883, Republic of Korea Hoseo Toxicity Research Center, Hoseo University Biomedical Laboratory Science, 79 Hoseo-ro BaeBang-Myeon, Asan, Chungnam 336-795, Republic of Korea c Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, 88 Olympic-ro-43-gil, Songpa-gu, Seoul 138-736, Republic of Korea d Toxicological Research Divison, National Institute of Food and Drug Safety Evaluation, Korea Food and Drug Administration, Osong Health Technology Administration Complex, 187 Osongsaengmyeong2-ro, Osong, Cheongwon, Chungbuk 363-700, Republic of Korea e Department of Biomedical Laboratory Science, Namseoul University, 21 Maeju-ri, Seonghwan-eup, Cheonan, Chungnam 331-707, Republic of Korea f Department of Laboratory Animal medicine, College of Veterinary medicine, Chungbuk National University, 410 Naesudong-ro, Heungdeok-gu, Cheongju, Chungbuk 361-763, Republic of Korea b

art ic l e i nf o

a b s t r a c t

Article history: Received 7 June 2013 Received in revised form 23 October 2013 Accepted 3 December 2013 Available online 30 December 2013

Ethnopharmacological relevance: Evodia, a fruit from Evodia rutaecarpa, has been used in oriental medicine, and since its various pharmaceutical actions, including anti-cancer activity, have become known, evodia has been widely used as a dietary supplement. However, information regarding its toxicity is limited. Materials and methods: Evodia fruit from Evodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (0, 25, 74, 222, 667, and 2000 mg/kg) was administered orally five times per week for 13 weeks. Clinical signs, body weight, food consumption, hematology, serum chemistry, urinalysis, vaginal cytology, sperm morphology, organ weight, and gross and histopathological findings were evaluated. Results: Urinary ketone body excretion was detected in males at 667 and 2000 mg/kg and in females at 2000 mg/kg. An increase in absolute/relative liver weight was observed in both sexes at 2000 mg/kg. Although levels of serum alanine aminotransferase, glucose, total cholesterol, and triglycerides were significantly reduced in males and/or females at 200 and/or 667 and 2000 mg/kg, all values were within normal ranges and were considered non-adverse. In addition, no treatment-related differences in body weight, food consumption, hematology, vaginal cytology, sperm morphology, or gross and histopathological examination were detected. Conclusions: The subchronic no-observable-adverse-effect level for evodia fruit powder following oral administration in rats is greater than 2000 mg/kg. & 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: Evodia fruit Rat Subchronic toxicity

1. Introduction Evodia, named “Wu-Chu-Yu” in China, is an immature fruit from Evodia rutaecarpa. Evodia has been used in oriental medicine to relieve headaches, stomachaches induced by gastrointestinal disorders, and postpartum bleeding (Sheu, 1999). Currently, evodia and its alkaloids has been shown to improves testosterone secretion (Lin et al., 1999) and exhibits anti-inflammatory (Chiou et al., 1997), anti-dementia (Park et al., 1996), anti-obesity (Kobayashi et al., 2001), and thermoregulatory (Tsai et al., 1995) effects. Many

n

Corresponding author. Tel.: þ 82 41 580 2721; fax: þ 82 41 580 2932. Corresponding author. Tel.: þ 82 43 261 2607; fax: þ 82 43 267 2595. E-mail addresses: [email protected] (J.S. Kang), [email protected] (J.-K. Kang).

nn

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.006

recent studies have focused on the pharmaceutical potential of the alkaloids in evodia, such as the major alkaloid evodiamine, which inhibits the growth of human colon carcinoma cells (Ogasawara et al., 2001) and hepatoblastoma cell lines (Xu et al., 2006). These studies demonstrated that the anti-tumor actions of evodiamine are associated with the inhibition of proliferation, invasion, and metastasis, as well as with the induction of apoptosis. Rutaecarpin, another alkaloid in evodia, also exhibits prospective pharmaceutical activity, such as vasodilation for treatment in cardiovascular disorders (Sheu, 1999). A recent study investigating the effects of the total alkaloids of evodia found that their antioxidant activity is related to the inhibition of 2,20 -diphenyl-1-picrylhydrazyl free radical formation and lipid peroxidation (Tan et al., 2012). Dietary supplements containing evodia are becoming more popular as news of their pharmaceutical potential spreads (Haller

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et al., 2008). In addition, there is growing interest in the possible medical uses of the alkaloids in evodia. However, very little is known about the safety of evodia. The results from one previous study suggested that evodia might terminate pregnancy in female mice (Kong et al., 1986). Another study conducted in humans reported palpitations, trembling, dizziness, nervousness, and gastrointestinal symptoms after administration of an evodia extract (Kim et al., 2008). The results from several studies indicate that evodia affects drug metabolism or detoxication of environmental pollutants through the inhibition of cytochrome P450 in liver, and evodia can intensify these effects or side effects (Engels et al., 2004; Iwata et al., 2005; Jan et al., 2005). With the exception of acute oral toxicity studies in mice and Drosophila melanogaster, and repeated oral toxicity study in mice for 30 days (Miyazawa et al., 2002; Yang et al., 2006; Yang, 2008; Zhou et al., 2011), no toxicological reports on evodia have been published. Therefore, the aim of this study was to evaluate the safety of the powder of evodia in rats by examining subchronic toxicity following a 13-week repeated oral dosing schedule.

2. Materials and methods 2.1. Test substance Evodia powder from Evodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang was provided by the National Institute of Toxicological Research (NITR), Korea Food and Drug Administration. The plant material was identified by Professor Kihwan Bae, Chungnam National University, Korea and voucher specimens was deposited at the Herbarium of College of Pharmacy, Catholic University of Daegu. The evodia fruits were cleaned and extracted with water (80 kg evodia fruits add 800 kg of distilled water) at temperatures of 100 1C for 4 h. After cooling the extract was filtered twice and was condensed into 30 L at 40–50 1C. The extract was then lyophilized at  70 1C, at pressure of 0.06 mbar for 72 h. The whole lyophilized powder provided for the study was produced as the result of single operation procedure. Identity and purity analyses were conducted by HPLC followed by electrochemical detection using an Alliance 2695 (Waters, Milford, MA) located in the Department of Herbal Medicine, Catholic University of Daegu (Li et al., 2012). Evodia powder was stored at 3.3–7.1 1C. Evodiamine (C19H17N8O, 99% purity) was used as the standard reference (Sigma Aldrich Co., Ltd., St. Louis, MO). The peak retention time for the test compound (20.905 min) was identical to that for the standard reference (20.904 min) (Fig. 1). Compound specificity was 91.20% and 90.98% at 1 and 400 mg/ml, respectively. 2.2. Experimental animals Specific pathogen-free F344 rats (age 6 weeks) of both sexes were purchased from Japan SLC, Inc. (Shizuoka, Japan), and acclimated for 2 weeks prior to drug administration. The animals were housed in a good laboratory practice (GLP) facility with controlled temperature (2271.5 1C), humidity (52712.5%), ventilation (10–15 times/h), light (12:12 h, light:dark cycle), and illumination (150–300 lx). Food (certified rodent diet 5002; Labdiet, USA) and sterilized tap water were provided ad libitum. The animals were randomly allocated to one control group and five treatment groups, each consisting of 10 males and 10 females. The ethical committee at Biotoxtech Co., Ltd. approved our use of the animals and our study design (Approval no. 09102). 2.3. Administration All procedures were carried out in accordance with the National Toxicology Program (NTP)0 s guidelines for the “Descriptions of NTP

Fig. 1. HPLC of standard reference and test substance. Chromatograms of standard reference and test substance. (A) Evodiamine (retention time: 20.905 min) and (B) test substance (retention time: 20.904 min).

Study Types, Toxicology/Carcinogenicity, 13-Week Toxicity Study” (as updated on January 18, 2006) and the study was conducted in compliance with the Good Laboratory Practice Regulations (No. 2005-79) issued by the Korea Food and Drug Administration, and the Principles of Good Laboratory Practice issued by the Organization for Economic Cooperation and Development. Evodia powder was obtained from the National Institute of Food and Drug Safety Evaluation (Osong, Korea). The test compound was suspended in water for injection (WFI: Choonwae Pharma Corp., Seoul, Korea) and was prepared once a week in accordance with the results from stability analyses assessed previously. The test substance was suspended in 5 ml of WFI and one dose (25, 74, 222, 667, or 2000 mg/kg body weight) per group per day was orally administered by gavage 5 days per week for 13 weeks; WFI (5 ml/kg/day) was administered to the controls. The treatment dose was determined by the previous 2-week repeated oral toxicity study. There were no adverse effects in 2000 mg/kg/day during the 2-week treated period (data not shown). 2.4. Body weight, food consumption, and toxicological signs The animals were observed throughout the course of the study. On each day of dosing, animals were observed once before compound administration and once 6 h after administration. Body weight and food consumption were measured on day 0 and then weekly after treatments began. 2.5. Urinalysis At the end of treatment, urinalyses with 3-h urine specimens were performed using either an autoanalyzer (Miditron Junior II; Roche Diagnostics, Mannheim, Germany, and 7080; Hitachi, Japan) or test kit (Combur 10 Test M stick; Roche Diagnostics, Mannheim, Germany). The 3-h urine specimens were analyzed for pH, protein,

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glucose, ketone bodies, bilirubin, erythrocyte, color, transparency, and urinary sediment. The 24-h urine specimens were analyzed for volume, specific gravity, N-acetyl-β-glucosaminidase (NAG), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST). Analyses of enzymes in 24-h urine specimens were performed using an automated analyzer (7080; Hitachi, Tokyo, Japan). 2.6. Biochemical and hematological analysis After 13 weeks of treatment, all animals were fasted for approximately 18 h. Blood was collected from the abdominal aorta under isoflurane inhalation and used for routine hematological and serum biochemistry analysis. The hematological analysis was performed using an automated hematology analyzer (Advia 120; Siemens Healthcare Diagnostics Inc., Erlangen, Germany, and ACL 7000; Instrumentation Laboratory, Bedford, Massachusetts, USA). The hematological parameters examined included total erythrocyte count (red blood cells, RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelet (PLT), total white blood cell count (WBC), WBC differential counting (neutrophil, lymphocyte, monocyte, eosinophil, and basophil ratio), reticulocyte (Reti), prothrombin time (PT), and activated partial thromboplastin time (APTT). The collected plasma was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), sorbitol dehydrogenase (SDH), creatine kinase (CK), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), albumin/globulin ratio (A/G), total cholesterol (T-Chol), triglyceride (TG), phosphorus (P), glucose (Glu), calcium (Ca), chloride (Cl), sodium (Na), and potassium (K) using an automated analyzer (7080; Hitachi, Tokyo, Japan, and AVL9181; Roche, Mannheim, Germany).

2.10. Necropsy and histopathology After urine and blood were collected, animals were euthanized by exsanguination from the abdominal aorta and any gross abnormal appearances were recorded. The following organs were excised and fixed in 10% neutral buffered formalin: brain, pituitary, harderian gland, zymbal gland, tongue, thyroid with parathyroid gland, trachea, esophagus, salivary gland, submandibular and mesenteric lymph nodes, sternum, femur including bone marrow, thymus, heart, lung, aorta, liver, adrenal gland, kidney, stomach, small and large intestine, pancreas, urinary bladder, ovary, uterus, vagina, prostate, seminal vesicles, epididymis, preputial/clitorial gland, mammary gland, skin, nasal turbinates, and any gross lesions, if present. The eyes and testis were fixed in Davidson0 s solution. After fixing in formalin, the organs and tissues from the all groups were prepared for microscopic examination by routine tissue processing, embedding in paraffin, sectioning, and staining with hematoxylin and eosin. 2.11. Statistical analysis Statistical analysis was carried out using the SAS program (version 9.1.3, SAS Institute Inc.; Cary, NC, USA). Body weight, food consumption, urine volume, clinical pathology data, vaginal cytology, sperm motility, and organ weights from the control and the evodia-treated groups were analyzed by one-way analysis of variance (ANOVA) after first using Bartlett0 s test to determine the homogeneity of variance (p 40.05, two-sided). If variances were not homogeneous, the data were analyzed by using the nonparametric Kruskal–Wallis test. If statistical significance was observed (po 0.05), comparison between controls and treated groups were made using Dunnett0 s multiple comparisons test (homogeneous data, p o0.05, two-sided) or Dunn0 s multiple comparisons test (heterogeneous data, p o0.05, two-sided).

2.7. Vaginal cytology Five rats in each group were evaluated in weeks 11 and 12 of compound administration. Vaginal smears were collected every morning for 14 days, stained with Diff-Quick solution and observed microscopically. The estrous cycle was determined by the features of each cell type in the vaginal smear and characterized as proestrus, estrus, metestrus, or diestrus. 2.8. Sperm motility and deformity test Five rats in each group were evaluated at necropsy. Sperm from the left cauda epididymis was collected by dissecting the tissue into 2 ml of 1% DPBS containing 0.5% bovine serum albumin and Dulbecco0 s phosphate-buffered saline. After 10–20 min incubation at 37 1C (with 5% CO2), the motility of sperm reserves was evaluated using the standard hemocytometric method (Sperm analysis system, HTM-TOX IVOS; Hamilton Thorne Biosciences, USA). To determine sperm deformity rate, incubated sperm fluid was smeared, stained with Diff-quick, and evaluated microscopically. Two hundred sperm cells from each rat were examined for shape and deformity, and the sperm deformity rate (%) was calculated as follows: Sperm deformity rateð%Þ ¼ ðnumber of sperm deformed=total number of spermÞ  100:

ð1Þ

3. Results 3.1. Clinical signs, body weight, and food consumption No mortality or adverse clinical signs were observed in animals treated with evodia following the course of study, and there were no statistically significant differences in body weight of evodiatreated groups as compared to the control group (Fig. 2). Food consumption was significantly decreased (  6.8%; po 0.05) for females at an evodia dose of 2000 mg/kg at week 11 as compared to food consumption in the control group at this same time point. 3.2. Urinalysis Ketone body concentration was judged for color intensity from 7 (5 mg/dL) to 3 þ (150 mg/dL), and was detected in males at a dose of evodia of 666 mg/kg (1/5, 7) and 2000 mg/kg (2/5 and 1/5 for the 7 and 1 þ, respectively) and in females at a dose of evodia of 2000 mg/kg (2/5, 7 ). The increase in AST was statistically significant (p o0.05) in males at an evodia dose of 25 mg/kg (37.9713.6) as compared with controls (22.2 72.2). No additional significant changes were detected. 3.3. Hematology and serum chemistry

2.9. Organ weight Absolute and relative weights were determined during necropsy for the following organs: liver, kidney, heart, thymus, lung, and testes.

Significant differences in hematology were observed between the treated and control groups at the end of the treatment period. Increases in PLT and Reti were observed in males at an evodia dose of 2000 mg/kg, and the eosinophil ratio of the WBC differential

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3.5. Organ weights The absolute and relative (organ-to-body weight ratio) weights in liver were significantly increased in males at a dose of evodia of 2000 mg/kg (p o0.01) and in females at a dose of 2000 mg/kg (p o0.01 and p o0.05, respectively) as compared to controls. The weights of the other organs were not different between control and treated animals (Tables 3 and 4). 3.6. Necropsy and histopathological examination During necropsy, one male gave evodia at a dose of 222 mg/kg and two females in the control group showed opacity in either the left or right eye. Yellow nodules in the abdominal cavity were noted in one female given evodia at a dose of 25 mg/kg, and a hepatodiaphragmatic nodule was observed in one female at an evodia dose of 2000 mg/kg. Microscopic examination indicated that these findings were in concordance with cataracts in the eye and fat necrosis in the abdominal cavity. All microscopic findings appeared sporadic, unrelated to dose, and consistent with F344 rats at this age.

4. Discussion

Fig. 2. Body weight for rats in 13-week oral toxicity of evodia fruit powder. (A) Males and (B) females. Each point presented as mean 7 S.D. (n ¼10).

increased in males at an evodia dose of 25 mg/kg. Decreases in APTT and MCV were observed in males at an evodia dose of 2000 mg/kg and in females at 222 mg/kg and 2000 mg/kg, respectively (Table 1). In the serum chemistry, ALT was decreased significantly in males at evodia doses of 222, 667, and 2000 mg/kg and in females at 667 and 2000 mg/kg as compared to controls (p o0.01). Decreases in AST were observed in males at evodia doses of 222 and 2000 mg/kg (p o0.05). ALP levels were decreased in males at a dose of evodia of 2000 mg/kg (p o0.05) and SDH levels decreased in females at a dose of evodia of 2000 mg/kg (p o0.01). Serum glucose levels were decreased in females at evodia doses of 222 (p o0.05), 667 (p o0.01), and 2000 mg/kg (p o0.05). T-Chol was decreased in males at evodia doses of 667 and 2000 mg/kg (p o0.01). Triglycerides were decreased in males at evodia doses of 667 (p o0.01) and 2000 mg/kg (p o0.05). Alb and BUN were increased in males at an evodia dose of 2000 mg/kg (p o0.01). The A/G ratio was elevated in males and females at an evodia dose of 2000 mg/kg (p o0.05 and p o0.01, respectively). In contrast, this parameter was reduced in males at an evodia dose of 25 mg/kg (po 0.05) (Table 2).

3.4. Vaginal cytology, sperm motility, and deformity test No statistically significant changes were observed (data not shown).

A 13-week, repeated-dose toxicity study in rats was performed to establish the safety profile of evodia fruit powder for traditional medical use and for drug development. Although we observed some treatment-related differences in the clinical chemistry and organ weights, evodia did not significantly alter clinical signs, body weight, food consumption, urinalysis, hematology, or cause any gross histopathological changes. In addition, there were no relevant changes in the appearance or behavior for any treatment group. Recorded clinical signs, including soft stool, chromodacryorrhea, crust and lens opacity, were sporadic and did not show a dose-response relationship, and were, therefore, considered unrelated to evodia treatment. Similarly, the slight decrease in food consumption observed in females only at the highest evodia dose (2000 mg/kg) and only at week 11 with no significant changes for any group of treated animals as compared to controls in body weight, was also considered unrelated to treatment. Decreased food consumption could be attributed to the physical stress induced by handling and gavage. Urinary ketone bodies were detected in males (1/5) given evodia at a dose of 667 mg/kg and in both males (2/5) and females (1/5) at a dose of 2000 mg/kg. However, this was not considered toxicologically relevant because the color intensities depicting urinary ketone body concentrations were low and similar to those observed in fasting F344 rats ( 7 to þ; Okamura et al., 2011). Moreover, evidence from observed clinical signs, serum chemistry, and histopathological examination did not indicate ketoacidosis. The AST levels in urine were minimally increased in males and only at a low dose of evodia, 25 mg/kg. Although an increase in urinary AST is generally correlated with damage to the proximal tubules in kidney (Stonard et al., 1987), it was not associated with histological finding. Thus, this urinary AST alteration was not regarded as a treatment-related change. Some of the hematological parameters in the treatment groups were statistically different to those in the control group. For example, differences were observed in PLT, APTT, Reti, and EOS in males and in MCV in females. However, because all of these differences occur within normal ranges (Okamura et al., 2011), they are not considered toxicologically relevant. Thus, evodia does not affect erythropoiesis, myelopoiesis, or blood coagulation.

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Table 1 Hematologic results for rats treated with evodia fruit powder for 13 weeks. Treatment Control

Male RBC (  106 cells/μL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (  103 cells/μL) Reti (%) WBC (  103 cells/μL) NEU (%) LYM (%) MONO (%) EOS (%) BASO (%) PT (s) APTT (s) Female RBC (  106 cells/μL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT (103 cells/μL) Reti (%) WBC (  103 cells/μL) NEU (%) LYM (%) MONO (%) EOS (%) BASO (%) PT (s) APTT (s)

Evodia fruit powder (mg/kg) 25

74a

222

667

2000

9.45 70.45 15.4 70.3 45.0 70.7 47.7 72.1 16.3 70.6 34.1 70.4 762 745 1.9 70.2 4.88 70.77 29.1 75.9 66.9 76.4 1.8 70.6 1.3 70.3 0.1 70.1 14.3 70.2 21.0 70.9

9.247 1.00 15.2 7 0.5 43.7 7 3.8 47.4 7 1.5 16.6 7 2.0 35.07 3.0 7757 57 1.9 7 0.2 4.69 7 1.11 29.17 8.2 66.37 8.7 2.0 7 0.5 1.6 7 0.4n 0.2 7 0.1 14.4 7 0.4 20.4 7 1.9

9.39 70.62 15.3 70.5 44.6 71.7 47.6 71.7 16.3 71.2 34.3 71.2 751 7110 2.0 70.2 4.6771.17 28.7 76.8 67.0 76.7 1.9 70.2 1.5 70.2 0.2 70.1 14.1 70.7 19.9 72.5

9.43 7 0.45 15.3 7 0.3 45.5 7 1.1 48.47 2.8 16.3 7 0.6 33.77 0.8 789 7 43 1.9 7 0.3 5.177 0.80 25.2 7 4.7 70.77 4.7 1.9 7 0.4 1.3 7 0.2 0.2 7 0.1 14.5 7 0.3 20.7 7 0.6

9.25 7 0.59 15.17 0.5 44.6 7 1.3 48.37 2.7 16.4 7 0.9 33.97 0.2 8017 36 2.2 7 0.3 5.03 7 0.74 28.2 7 7.5 67.9 7 7.7 1.7 7 0.3 1.3 7 0.2 0.17 0.1 14.5 7 0.2 20.4 7 0.5

9.25 7 0.23 15.0 7 0.4 44.2 7 1.1 47.8 7 0.4 16.3 7 0.2 34.0 7 0.4 843 7 31nn 2.2 7 0.2nn 5.26 7 0.74 26.8 7 4.7 69.7 7 4.7 1.6 7 0.5 1.0 7 0.2 0.17 0.1 14.3 7 0.4 19.4 7 0.5nn

9.45 70.45 15.4 70.3 45.0 70.7 47.7 72.1 16.3 70.6 34.1 70.4 762 745 1.9 70.2 4.88 70.77 29.1 75.9 66.9 76.4 1.8 70.6 1.3 70.3 0.1 70.1 14.3 70.2 21.0 70.9

9.247 1.00 15.2 7 0.5 43.7 7 3.8 47.4 7 1.5 16.6 7 2.0 35.07 3.0 7757 57 1.9 7 0.2 4.69 7 1.11 29.17 8.2 66.37 8.7 2.0 7 0.5 1.6 7 0.4 0.2 7 0.1 14.4 7 0.4 20.4 7 1.9

9.39 70.62 15.3 70.5 44.6 71.7 47.6 71.7 16.3 71.2 34.3 71.2 751 7110 2.0 70.2 4.6771.17 28.7 76.8 67.0 76.7 1.9 70.2 1.5 70.2 0.2 70.1 14.1 70.7 19.9 72.5

9.43 7 0.45 15.3 7 0.3 45.5 7 1.1 48.47 2.8nn 16.3 7 0.6 33.77 0.8 789 7 43 1.9 7 0.3 5.177 0.80 25.2 7 4.7 70.77 4.7 1.9 7 0.4 1.3 7 0.2 0.2 7 0.1 14.5 7 0.3 20.7 7 0.6

9.25 7 0.59 15.17 0.5 44.6 7 1.3 48.37 2.7 16.4 7 0.9 33.97 0.2 8017 36 2.2 7 0.3 5.03 7 0.74 28.2 7 7.5 67.9 7 7.7 1.7 7 0.3 1.3 7 0.2 0.17 0.1 14.5 7 0.2 20.4 7 0.5

9.25 7 0.23 15.0 7 0.4 44.2 7 1.1 47.8 7 0.4n 16.3 7 0.2 34.0 7 0.4 843 7 31 2.2 7 0.2nn 5.26 7 0.74 26.8 7 4.7 69.7 7 4.7 1.6 7 0.5 1.0 7 0.2 0.17 0.1 14.3 7 0.4 19.4 7 0.5nn

RBC: red blood cell, HGB: hemoglobin, HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, PLT: platelet, Reti: reticulocyte, WBC: total white blood cell count, NEU: neutrophil, LYM: lymphocyte, MONO: monocyte, EOS: eosinophil, BASO: basophil, PT: prothrombin time, and APTT: activated partial thromboplastin time. n

Data presented as mean7 S.D. Significantly different from control: p o 0.05. Data presented as mean7 S.D. Significantly different from control: p o 0.01. For N ¼ 9 in females at 74 mg/kg.

nn

a

For the biochemical parameters, we observed a significant dose-dependent reduction in ALT levels. In addition, statistically significant decreases in other liver enzymes, AST, ALP, SDH, and Alb, were found in the serum of both males and females at the highest evodia dose of 2000 mg/kg. Pyridoxine deficiency has been suggested as a typical cause of large reduction in serum ALT and AST (Ennulat et al., 2010), and chronic liver damage might be associated with reduction of hepatic transaminase, as well as decrease in serum protein (Rasekh et al., 2008). In this study, however, the reduction in ALT levels was modest, remaining within normal background levels (Okamura et al., 2011), and was not accompanied by histopathological alterations in the liver. The decreases observed in the other liver enzymes were also probably unrelated to treatment because they were small in magnitude, lacked a dose-response relationship, and/or showed no correspondence between the sexes. Glucose, total cholesterol, and triglyceride levels were significantly altered in males and/or females at evodia doses of 667 and 2000 mg/kg. These results parallel those from previous studies, which reported that evodiamine contributes to reduced serum glucose levels as well as lower levels of hepatic cholesterol and triglycerides (Kobayashi et al., 2001; Wang et al., 2008). However, the small decrease in glucose, total cholesterol, and triglyceride

levels observed in the present study is not toxicologically relevant, because these were within normal background ranges (Okamura et al., 2011), and no concurrent changes were recorded during the histopathological examination of the liver or pancreas. Slight increases were observed in absolute and relative liver weights at the highest evodia dose of 2000 mg/kg in both sexes. However, this was of no toxicological relevance because the liver weight changes were within one standard deviation from mean background levels (Okamura et al., 2011), and not associated with the morphological alterations in liver. All macroscopic and microscopic findings in the examined organs appeared sporadic, uncorrelated with dose-response relationships, and could be attributed to spontaneous lesions generally occurring at this age in F344 rats. Comparing our results with previous study, there was some differences as well as some similarities. Zhou et al. (2011) were reported that oral gavaging of water decoction at dose of 50,000 mg/kg for 8, 15, 20, 25, and 30 days induced hepatotoxicity including increase in ALT and pathological lesions in mice, and liver index were increased with course of time. On the contrary to this, the liver enzymes level in the present study was decreased. In the case of acute oral toxicity study, no toxicologic signs in mice were observed at 10,000 mg/kg of water extracts of evodia fruits

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Table 2 Biochemical results for rats treated with evodia fruit powder for 13 weeks. Treatment Control

Male ALT (U/L) AST (U/L) ALP (U/L) SDH (U/L) CK (U/L) Glu (mg/dL) BUN (mg/dL) Crea (mg/dL) T-Bili (mg/dL) T-Chol (mg/dL) TG (mg/dL) TP (g/dL) Alb (g/dL) A/G ratio Ca (mg/dL) Na (mmol/L) K (mmol/L) Cl (mmol/L) Female ALT (U/L) AST (U/L) ALP (U/L) SDH (U/L) CK (U/L) Glu (mg/dL) BUN (mg/dL) Crea (mg/dL) T-Bili (mg/dL) T-Chol (mg/dL) TG (mg/dL) TP (g/dL) Alb (g/dL) A/G ratio Ca (mg/dL) Na (mmol/L) K (mmol/L) Cl (mmol/L)

Evodia fruit powder (mg/kg) 25

74a

222

667

2000

57.17 8.8 95.7 7 8.0 396.6 7 36.3 9.5 7 2.5 1067 18 1527 14 15.5 7 1.3 0.477 0.05 0.017 0.01 797 5 867 41 6.4 7 0.1 2.9 7 0.1 0.82 7 0.03 10.8 7 0.1 1407 1 4.17 0.2 1037 1

56.6 7 13.5 94.7 7 11.4 399.6 7 53.8 9.2 7 3.1 1037 15 1487 30 16.0 7 1.0 0.497 0.06 0.017 0.01 797 7 637 35 6.5 7 0.2 2.8 7 0.1 0.777 0.05n 10.8 7 0.3 1407 1 4.3 7 0.2 1037 1

55.4 7 7.3 93.17 10.8 397.2 7 32.4 8.2 7 2.1 987 14 1517 16 16.2 7 1.1 0.477 0.04 0.017 0.01 777 8 747 48 6.4 7 0.2 2.8 7 0.1 0.80 7 0.01 10.8 7 0.2 1407 1 4.17 0.3 1037 1

45.7 7 4.7nn 84.9 7 6.7n 400.6 7 37.6 7.2 7 2.1 957 10 1407 12 16.5 7 0.6 0.487 0.07 0.017 0.01 737 7 637 20 6.3 7 0.1 2.9 7 0.1 0.82 7 0.03 10.9 7 0.2 1407 1 4.0 7 0.2 1037 1

45.0 7 4.6nn 87.5 7 8.0 402.9 7 43.0 6.7 7 1.7 1037 11 1377 15 16.4 7 1.5 0.487 0.05 0.02 7 0.01 64 7 5nn 447 21nn 6.2 7 0.1 2.9 7 0.1 0.84 7 0.05 10.8 7 0.2 1407 1 4.2 7 0.1 1037 1

43.17 4.4nn 83.3 7 9.2n 397.2 7 37.2 5.8 7 2.5nn 106 7 15 1387 11 18.2 7 0.7nn 0.487 0.03 0.02 7 0.01 66 7 9nn 49 7 13n 6.4 7 0.2 3.0 7 0.1nn 0.90 7 0.05n 11.0 7 0.2 1407 2 4.17 0.3 104 7 1

42.2 7 5.9 90.9 7 15.0 374.9 7 41.1 4.7 7 2.7 1037 21 1347 13 17.0 7 1.4 0.46 7 0.06 0.02 7 0.01 1057 8 267 10 6.3 7 0.2 2.9 7 0.1 0.86 7 0.03 10.6 7 0.2 1407 1 4.4 7 0.3 1057 1

39.9 7 7.8 92.2 7 17.9 376.7 7 55.1 3.8 7 1.6 1067 15 1277 14 16.9 7 1.6 0.477 0.04 0.03 7 0.02 977 12 177 4 6.2 7 0.1 2.9 7 0.1 0.86 7 0.06 10.5 7 0.3 1397 1 4.5 7 0.1 1057 1

37.6 7 8.8 88.4 7 12.4 333.3 7 64.2 2.4 7 0.9 1007 14 1227 12 17.7 7 1.2 0.447 0.06 0.03 7 0.02 997 7 187 3 6.17 0.2 2.9 7 0.1 0.89 7 0.03 10.4 7 0.2 1407 1 4.5 7 0.3 1057 1

39.8 7 5.9 92.2 7 10.2 356.07 36.5 3.4 7 1.7 997 21 1217 10n 17.2 7 1.2 0.477 0.03 0.03 7 0.02 957 8n 227 13 6.17 0.1 2.9 7 0.1 0.89 7 0.03 10.5 7 0.2 1397 2 4.5 7 0.2 106 7 1

31.9 7 2.9nn 81.8 7 5.0 327.4 7 66.5 2.6 7 2.3 1167 29 1167 6nn 17.5 7 1.8 0.477 0.08 0.03 7 0.01 847 8nn 197 6 6.2 7 0.2 3.0 7 0.1 0.917 0.05 10.7 7 0.2 1397 1 4.5 7 0.2 1057 1

29.5 7 3.5nn 82.3 7 6.3 302.3 7 28.1n 3.0 7 2.2 1007 18 1197 7n 18.0 7 1.8 0.45 7 0.06 0.02 7 0.01 827 6nn 197 7 6.2 7 0.3 3.17 0.1 0.977 0.06nn 10.7 7 0.3 1397 2 4.3 7 0.2 1057 1

ALT: alanine aminotransferase, AST: aspartate aminotransferase, ALP: alkaline phosphatase, SDH: sorbitol dehydrogenase, CK: creatine kinase, BUN: blood urea nitrogen, Crea: creatinine, T-Bili: total bilirubin, TP: total protein, Alb: albumin, A/G ratio: albumin/globulin ratio, T-Chol: total cholesterol, TG: triglyceride, P: phosphorus, Glu: glucose, Ca: calcium, CL: chloride, Na: sodium, and K: potassium. n

Data presented as mean7 S.D. Significantly different from control: p o 0.05. Data presented as mean 7S.D. Significantly different from control: po 0.01. For N ¼ 9 females at 74 mg/kg.

nn

a

Table 3 Absolute organ weights for rats treated with evodia fruit powder for 13 weeks. Treatment Control

Male Heart (g) Lung (g) Liver (g) Thymus (g) Kidney (right) (g) Testis (right) (g) Female Heart (g) Lung (g) Liver (g) Thymus (g) Kidney (right) (g) n

Evodia fruit powder (mg/kg) 25

74

222

667

2000

0.90 7 0.04 1.147 0.05 9.167 0.50 0.2047 0.021 1.03 7 0.05 1.487 0.05

0.917 0.03 1.147 0.04 9.067 0.56 0.2137 0.038 1.02 7 0.05 1.46 7 0.05

0.92 7 0.03 1.18 7 0.06 9.16 7 0.49 0.223 7 0.033 1.03 7 0.05 1.44 7 0.05

0.89 70.05 1.13 70.06 8.80 70.58 0.217 70.023 0.99 70.07 1.46 70.08

0.89 7 0.04 1.127 0.06 9.02 7 0.66 0.2157 0.018 1.017 0.07 1.43 7 0.04

0.89 70.04 1.15 70.04 10.11 70.75nn 0.219 70.023 1.03 70.06 1.44 70.08

0.617 0.03 0.84 7 0.05 4.95 7 0.29 0.1947 0.012 0.647 0.03

0.59 7 0.03 0.86 7 0.04 4.78 7 0.33 0.1847 0.025 0.62 7 0.04

0.58 7 0.03 0.82 7 0.03 4.61 7 0.30 0.185 7 0.020 0.60 7 0.04

0.61 70.04 0.84 70.03 4.76 70.28 0.196 70.018 0.62 70.03

0.59 7 0.03 0.84 7 0.03 4.85 7 0.23 0.1877 0.019 0.62 7 0.03

0.60 70.03 0.83 70.03 5.31 70.33n 0.193 70.016 0.63 70.04

Data presented as mean7 S.D. Significantly different from control: p o 0.05. Data presented as mean 7S.D. Significantly different from control p o 0.01.

nn

1078

D. Kim et al. / Journal of Ethnopharmacology 151 (2014) 1072–1078

Table 4 Relative organ weights for rats treated with evodia fruit powder for 13 weeks. Treatment Control

Male Heart (g% B.W.) Lung (g% B.W.) Liver (g% B.W.) Thymus (g% B.W.) Kidney (right) (g% B.W.) Testis (right) (g% B.W.) Female Heart (g% B.W.) Lung (g% B.W.) Liver (g% B.W.) Thymus (g% B.W.) Kidney (right) (g% B.W.) nn

Evodia fruit powder (mg/kg) 25

74

222

667

2000

0.277 0.01 0.357 0.02 2.777 0.14 0.062 7 0.007 0.317 0.01 0.45 7 0.01

0.28 70.01 0.3570.01 2.74 70.14 0.065 70.012 0.31 70.01 0.44 70.01

0.28 7 0.01 0.357 0.02 2.75 7 0.09 0.0677 0.011 0.317 0.02 0.43 7 0.02

0.28 7 0.01 0.357 0.01 2.737 0.10 0.068 7 0.008 0.317 0.01 0.45 7 0.02

0.28 7 0.01 0.357 0.01 2.777 0.13 0.066 7 0.007 0.317 0.01 0.447 0.02

0.277 0.01 0.36 7 0.02 3.127 0.12nn 0.068 7 0.007 0.32 7 0.01 0.45 7 0.04

0.32 7 0.01 0.447 0.02 2.63 7 0.12 0.1037 0.003 0.34 7 0.02

0.32 70.01 0.46 70.03 2.58 70.09 0.099 70.009 0.3370.02

0.32 7 0.01 0.45 7 0.01 2.52 7 0.10 0.1017 0.008 0.337 0.02

0.337 0.02 0.46 7 0.02 2.577 0.13 0.106 7 0.007 0.337 0.02

0.32 7 0.01 0.46 7 0.01 2.63 7 0.05 0.1027 0.007 0.34 7 0.02

0.32 7 0.01 0.447 0.01 2.81 7 0.13nn 0.1027 0.007 0.34 7 0.03

Data presented as mean 7S.D. Significantly different from control: po 0.01.

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