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Evaluation of the subacute toxicity of Yongdamsagan-tang, a traditional herbal formula, in Crl:CD Sprague Dawley rats
Journal of Ethnopharmacology 238 (2019) 111852
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Evaluation of the subacute toxicity of Yongdamsagan-tang, a traditional herbal formula, in Crl:CD Sprague Dawley rats
T
Eunsook Parka, Mee-Young Leeb, Chang-Seob Seoa, Hyeun-Kyoo Shina, Su-Cheol Hanc, Hyekyung Haa,∗ a
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, Republic of Korea Clinical Medicine Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, Republic of Korea c General Toxicology Research Center, Korea Institute of Toxicology, 30 Baekhak 1-gil, Jeongeup, Jeonbuk, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Yongdamsagan-tang water extract Subacute toxicity Crl:CD sprague Dawley rats
Ethnopharmacological relevance: Yongdamsagan-tang, a traditional herbal formula, is used widely for the treatment of inflammatory and viral diseases. However, the safety of Yongdamsagan-tang has not been established. Aim of the study: To evaluate the subacute toxicity of Yongdamsagan-tang water extract (YSTE) in Crl:CD Sprague Dawley rats. Materials and methods: We evaluated the subacute toxicity of YSTE in male and female Crl:CD Sprague Dawley rats (n = 5 per group). Rats were treated with YSTE at doses of 0, 1000, 2000 and 5000 mg/kg administered once a day by oral gavage for 4 weeks. Results: There were no significant changes in mortality, body weight, food intake, serum biochemistry, or results of hematology and urinalysis after YSTE administration. However, all rats treated with 5000 mg/kg/day YSTE exhibited excessive salivation and discolored urine. Necropsy findings showed discoloration in the liver of both male (n = 1) and female (n = 3) rats treated with 5000 mg/kg/day YSTE, and an increase in the relative weights of kidney and liver was also found in male rats treated with 5000 mg/kg/day. In addition, decreases in serum creatinine, total bilirubin, alanine transaminase, and alkaline phosphatase were observed in male rats treated with 2000 or 5000 mg/kg/day YSTE. Conclusions: Abnormalities in some rats are considered to be independent of YSTE toxicity. Therefore, the results suggest that oral administration of YSTE in rats for 4 weeks is safe at doses of up to 5000 mg/kg/day.
1. Introduction Herbal formulas composed of several herbs are a traditional medicine of Asian type and are widely used in China, Korea, and Japan (Grayson, 2011). As interest in health and natural products increases, the popularity of traditional herbal formulas as complementary or alternative medicines is growing in both Western and Asian countries (Lu and Lu, 2014). However, there are still many limitations on the use of herbal formulas as treatment for various diseases or as dietary supplements because of the lack of scientific evidence for their safety and efficacy (Xiong et al., 2017; Xu et al., 2012). Therefore, systematic toxicology studies are required to demonstrate their safety. Yongdamsagan-tang, also known as Long Dan Xie Gan Tang in Chinese and Ryutanshakan-to in Japanese, is a traditional herbal
formula widely used in Korea for treating inflammation and viral diseases (YUZhou, 2001). Recently, it has been reported that Yongdamsagan-tang has therapeutic effects in bacterial infection, benign prostatic hyperplasia, and polycystic ovary syndrome via its antioxidant, antiproliferative, and anti-inflammatory activities (Jin, 2004; Lee and Chang, 2010; Lim et al., 2007; Park et al., 2016a, 2017). Despite of scientific trials to establish the efficacy of Yongdamsagan-tang, toxicological studies to determine any adverse effects of Yongdamsagantang in vivo are scarce. Therefore, to investigate the safety profile of Yongdamsagan-tang, we evaluated the subacute toxicity of Yongdamsagan-tang water extract (YSTE) in male and female Crl:CD Sprague Dawley rats. This scientific information about the subacute toxicity of YSTE may assist in the development of new treatments based on herbal formulas including
∗
Corresponding author. Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. E-mail addresses: [email protected] (E. Park), [email protected] (M.-Y. Lee), [email protected] (C.-S. Seo), [email protected] (H.-K. Shin), [email protected] (S.-C. Han), [email protected] (H. Ha). https://doi.org/10.1016/j.jep.2019.111852 Received 22 June 2018; Received in revised form 24 March 2019; Accepted 31 March 2019 Available online 04 April 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 238 (2019) 111852
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Fig. 1. Three-dimensional HPLC chromatogram of YSTE using HPLC–PDA. Table 1 Amounts of eight marker components in YSTE at 0, 1 and 4 weeks by HPLC (n = 3). Compound
0 week
Chlorogenic acid Gentiopicroside Liquiritin apioside Liquiritin Nodakenin Baicalin Wogonoside Glycyrrhizin
1 week
4 week
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
0.981 16.692 2.365 2.043 1.876 6.937 2.344 3.000
0.016 0.032 0.028 0.006 0.016 0.073 0.031 0.007
1.626 0.189 1.174 0.302 0.829 1.055 1.321 0.242
0.976 16.964 2.323 2.108 1.967 7.212 2.522 3.024
0.005 0.207 0.032 0.039 0.029 0.169 0.061 0.088
0.464 1.222 1.392 1.872 1.483 2.350 2.424 2.907
1.021 17.853 2.404 2.132 1.954 7.694 2.645 3.111
0.007 0.123 0.025 0.023 0.012 0.060 0.021 0.059
0.660 0.686 1.020 1.101 0.633 0.778 0.794 1.904
Table 2 Mortality in rat administered orally with YSTE for 4 weeks. Group
Dosing phase 1 day
Male rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day Female rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day a
≤1 week
≤2 week
Table 3 Clinical signs in rat administered orally with YSTE for 4 weeks. Final mortalitya
Group Male rats 0 mg/kg 1000 mg/kg 2000 mg/kg 5000 mg/kg Female rats 0 mg/kg 1000 mg/kg 2000 mg/kg 5000 mg/kg
≤4 week
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 1
0/5 0/5 0/5 1/5
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 1
0/5 0/5 0/5 1/5
a
Number of animals with unscheduled death/Total animal number.
2
Salivationa
Discolored urinea
Irregular respirationa
Palenessa
0/5 0/5 1/5 5/5
0/5 0/5 0/5 4/5
0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5
0/5 1/5 0/5 5/5
0/5 0/5 0/5 4/5
0/5 0/5 0/5 1/5
0/5 0/5 0/5 1/5
Number of animals with sign/Total number of animals observed.
Journal of Ethnopharmacology 238 (2019) 111852
E. Park, et al.
Fig. 2. Body weights (g) of male (A) and female (B) rats treated orally with YSTE at doses of 0 (○), 1000 (■), 2000 (▲), and 5000 (●) mg/kg/day for 4 weeks. Values are presented as mean ± SD.
Fig. 3. Food intake (g/animal/day) of male (A) and female (B) rats treated orally with YSTE at doses of 0 (○), 1000 (■), 2000 (▲), and 5000 (●) mg/kg/day for 4 weeks. Values are presented as mean ± SD. Table 4 Urinalysis of rats administered orally with YSTE for 4 weeks. Group Male rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day Female rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day
Table 5 Gross necropsy observations of rats administered orally with YSTE for 4 weeks.
Volume (mL)
Glucosea
Specific gravity
pH
22 20 27 22
± ± ± ±
7.4 6.1 15.4 10.7
0/5 0/5 0/5 0/4
1.008 1.012 1.011 1.014
± ± ± ±
0.0027 0.0045 0.0042 0.0025
7.0 7.0 6.7 6.6
± ± ± ±
0.00 0.00 0.27 0.25
10 12 14 17
± ± ± ±
3.6 5.5 5.3 4.8
0/5 0/5 0/5 0/4
1.016 1.015 1.014 1.014
± ± ± ±
0.0042 0.0035 0.0042 0.0025
6.5 6.8 6.7 6.6
± ± ± ±
0.35 0.27 0.27 0.25
Dose (mg/kg/day) Male rats No remarkable findinga Liver discolorationa Female rats No remarkable findinga Liver discolorationa a
0
1000
2000
5000
5/5 0/5
5/5 0/5
5/5 0/5
1/4 3/4
5/5 0/5
5/5 0/5
5/5 0/5
3/4 1/4
Number of animals with sign/Total number of animals observed.
Yongdamsagan-tang.
Values are presented as mean ± SD. a Number of animals with sign/Total number of animals observed.
2. Materials and methods 2.1. Preparation of YSTE Yongdamsagan-tang extract was manufactured by Sungil Bioex Co. Ltd. (Hwaseong, Korea). Briefly, the eleven component herbs of 3
Journal of Ethnopharmacology 238 (2019) 111852
E. Park, et al.
Table 6 Relative organ weights (% of body weight) in rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats Brain Pituitary gland Liver Spleen Heart Thymus Salivary glands Seminal vesicle Prostate Kidneys Adrenal glands Testes Epididymides Lung Thyroid/parathyroid Female rats Brain Pituitary gland Liver Spleen Heart Thymus Salivary glands Kidneys Adrenal glands Ovaries Lung Thyroid/parathyroid Uterus/cervix
0
1000
2000
5000
0.522 0.033 3.180 0.186 0.323 0.141 0.167 0.303 0.112 0.797 0.015 0.848 0.300 0.379 0.004
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0387 0.0001 0.1215 0.0360 0.0090 0.0346 0.0216 0.0404 0.0139 0.0323 0.0017 0.0326 0.0143 0.0362 0.0004
0.520 0.003 3.164 0.182 0.324 0.127 0.170 0.326 0.125 0.823 0.016 0.840 0.285 0.380 0.005
± 0.0277 ± 0.0004 ± 0.0790 ± 0.0277 ± 0.0218 ± 0.0114 ± 0.0172 ± 0.0432 ± 0.0249 ± 0.0778 ± 0.0016 ± 0.0409 ± 0.0226 ± 0.0181 ± 0.0006**
0.510 0.003 3.273 0.192 0.325 0.145 0.169 0.304 0.104 0.858 0.015 0.785 0.266 0.372 0.005
± 0.0110 ± 0.0005 ± 0.3032 ± 0.0373 ± 0.0094 ± 0.0356 ± 0.0021 ± 0.0271 ± 0.0198 ± 0.0271 ± 0.0011 ± 0.0821 ± 0.0133* ± 0.0163 ± 0.0003
0.533 0.003 3.779 0.206 0.336 0.120 0.174 0.337 0.113 0.968 0.017 0.902 0.301 0.391 0.005
± 0.0186 ± 0.0004 ± 0.2381** ± 0.0293 ± 0.0271 ± 0.0099 ± 0.0139 ± 0.0731 ± 0.0308 ± 0.0297** ± 0.0016 ± 0.0375 ± 0.0120 ± 0.0203 ± 0.0004
0.760 0.006 3.245 0.207 0.352 0.185 0.180 0.831 0.032 0.037 0.484 0.006 0.211
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0577 0.0005 0.2771 0.0203 0.0200 0.0315 0.0145 0.0501 0.0069 0.0043 0.0257 0.0004 0.0247
0.807 0.005 3.256 0.226 0.358 0.211 0.171 0.832 0.029 0.040 0.526 0.005 0.220
± ± ± ± ± ± ± ± ± ± ± ± ±
0.783 0.005 3.445 0.210 0.359 0.205 0.173 0.860 0.034 0.037 0.498 0.006 0.208
± ± ± ± ± ± ± ± ± ± ± ± ±
0.825 0.005 3.520 0.240 0.354 0.198 0.169 0.892 0.027 0.040 0.515 0.006 0.232
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0826 0.0003 0.2254 0.0273 0.0263 0.0579 0.0142 0.0459 0.0019 0.0028 0.0452 0.0007 0.0620
0.0644 0.0008 0.2254 0.0169 0.0206 0.0108 0.0070 0.0487 0.0072 0.0055 0.0321 0.0009 0.0343
0.0588 0.0008 0.1016 0.0321 0.0180 0.0199 0.0091 0.0647 0.0028 0.0055 0.0269 0.0014 0.0940
Values are presented as the mean ± SD. * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, when compared with the vehicle control group.
column used was Gemini C18 (250 mm × 4.6 mm; 5 μm, Phenomenex, Torrance, CA, USA) maintained at 40 °C, and 0.1% (v/v) trifluoroacetic acid in distilled water and acetonitrile were used as the mobile phase.
Yongdamsagan-tang, i.e. Gentianae Scabrae Radix (Gentiana scabra Bunge; 22 kg), Bupleuri Radix (Bupleurum falcatum Linne; 22 kg), Alismatis Rhizoma (Alisma orientale Juzepczuk; 22 kg), Akebiae Caulis (Akebia quinata Decaisne; 11 kg), Plantaginis Semen (Plantago asiatica Linne; 11 kg), Poria Sclerotium (Poria cocos Wolf; 11 kg), Rehmanniae Radix Crudus (Rehmannia glutinosa Liboschitz ex Steudel; 11 kg), Angelicae Gigantis Radix (Angelica gigas Nakai; 11 kg), Gardeniae Fructus (Gardenia jasminoides Ellis; 11 kg), Scutellariae Radix (Scutellaria baicalensis Georgi; 11 kg), and Glycyrrhizae Radix et Rhizoma (Glycyrrhiza uralensis Fischer; 11 kg) were purchased from Kwangmyungdang Medicinal herbs (Ulsan, Korea), mixed, and extracted in a 10-fold mass (1540 L) of water at 80 °C for 2 h using the reflux method. The extracted water solution was freeze-dried to give a powder (33 kg, yield: 21.4%).
2.3. Experimental design The experimental design was based on that of a previous study (Park et al., 2016b). All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Institute of Toxicology (approval number: 1505-0116) and performed under the current Good Laboratory Practice (GLP) regulations for nonclinical laboratory studies. Five-week-old specific pathogen-free Crl:CD Sprague Dawley rats (n = 20 of each sex) were obtained from Orient Bio Co. (Seoul, Korea) and acclimatized for one week to laboratory conditions before the start of experiments. The rats were maintained in an animal facility under a controlled temperature of 23 ± 3 °C and a relative humidity of 50 ± 20% with 12 h/12 h light cycle. Healthy rats were randomly assigned to four weight-matched groups (n = 5 per group) using a Path/Tox System (version 4.2.2; Xybion Medical Systems Corporation, Cedar Knolls, NJ). Three different doses of YSTE (1000, 2000 and 5000 mg/kg/day) dissolved in distilled water were administered to rats daily by oral gavage for 4 weeks. The vehicle control group (0 mg/kg YSTE) were daily administered an equal volume of distilled water. YSTE was prepared fresh once a week and kept at 4 °C. The daily dose (in 10 mL/kg body weight) of YSTE was individually calculated based on the most recent body weight of each animal.
2.2. High-performance liquid chromatography (HPLC) analysis of YSTE Liquiritin (PubChem CID: 503737, purity 99.6%), baicalin (PubChem CID: 64982, purity 98.0%), and glycyrrhizin (PubChem CID: 14982, purity 99.0%) were purchased from Wako Chemicals (Osaka, Japan). Chlorogenic acid (PubChem CID: 1794427, purity 99.6%), gentiopicroside (PubChem CID: 88708, purity 98.3%), liquiritin apioside (PubChem CID: 10076238, purity 98.0%), nodakenin (PubChem CID: 73191, purity 98.0%), and wogonoside (PubChem CID: 3084961, purity 98.2%) were purchased from Acros Organics (Pittsburgh, PA, USA), Biopurify Phytochemicals (Chengdu, China), Shanghai Sunny Biotech (Shanghai, China), NPC Bio Technology (Yeongi, Korea) and Tauto Biotech (Shanghai, China), respectively. Standard stock solutions of these eight components were prepared at a concentration of 1.0 mg/ mL in methanol and stored at 4 °C until use. The detailed conditions for simultaneous analysis of YSTE were described previously (Park et al., 2016a). Briefly, a Prominence LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) coupled with a photodiode detector array was used. The
2.4. General observations During the dosing period, general observations including mortality, clinical signs, body weights, and food intake were recorded as described 4
Journal of Ethnopharmacology 238 (2019) 111852
E. Park, et al.
Table 7 Hematological parameters of rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats WBC (103/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) Large unstained cells (%) RBC (106/μL) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/dL) Reticulocytes (%) Platelet (103/μL) PT (sec) APTT (sec) Female rats WBC (103/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) Large unstained cells (%) RBC (106/μL) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/dL) Reticulocytes (%) Platelet (103/μL) PT (sec) APTT (sec)
0
1000
2000
5000
8.87 ± 1.70 11.2 ± 3.63 85.6 ± 3.89 0.9 ± 0.25 1.3 ± 0.33 0.3 ± 0.11 0.7 ± 0.15 8.0 ± 0.35 15.4 ± 0.61 49.0 ± 1.91 61.7 ± 0.75 19.4 ± 0.52 31.5 ± 0.48 2.6 ± 0.31 917.2 ± 104.79 14.3 ± 0.67 16.4 ± 1.85
8.3 ± 1.11 11.4 ± 2.77 84.9 ± 3.48 1.0 ± 0.38 1.4 ± 0.51 0.4 ± 0.13 0.9 ± 0.24 8.2 ± 0.35 15.7 ± 0.26 49.9 ± 0.71 61.2 ± 1.80 19.2 ± 0.67 31.5 ± 0.32 2.82 ± 0.39 952.0 ± 98.84 13.9 ± 0.41 15.9 ± 0.77
9.4 ± 2.38 10.4 ± 4.00 86.4 ± 4.24 0.6 ± 0.25 1.3 ± 0.18 0.4 ± 0.09 0.8 ± 0.22 8.2 ± 0.32 15.6 ± 0.39 49.4 ± 1.58 60.1 ± 2.46 18.9 ± 0.68 31.5 ± 0.30 2.7 ± 0.45 918.4 ± 176.86 14.1 ± 0.59 15.7 ± 0.60
8.8 ± 2.59 15.3 ± 2.76 81.1 ± 2.65 0.9 ± 0.15 1.6 ± 0.39 0.4 ± 0.22 0.7 ± 0.26 8.0 ± 0.40 15.0 ± 0.37 47.5 ± 2.00 59.1 ± 0.99 18.7 ± 0.51 31.7 ± 0.60 2.9 ± 0.34 1076.0 ± 91.80 14.9 ± 0.47 14.3 ± 1.85
8.9 ± 1.20 8.3 ± 2.98 87.4 ± 3.53 1.2 ± 0.37 1.8 ± 0.69 0.3 ± 0.05 1.0 ± 0.27 8.2 ± 0.60 15.7 ± 0.84 48.2 ± 2.90 59.0 ± 1.38 19.3 ± 0.46 32.7 ± 0.54 2.7 ± 0.36 919.0 ± 90.78 13.7 ± 0.58 13.9 ± 2.01
12.86 ± 3.22 12.1 ± 5.87 83.5 ± 6.60 0.9 ± 0.31 2.0 ± 0.66 0.4 ± 0.11 1.1 ± 0.44 8.5 ± 0.19 16.1 ± 0.09 48.9 ± 0.73 57.7 ± 1.61 19.0 ± 0.36 33.0 ± 0.41 2.4 ± 0.39 999.2 ± 69.76 13.9 ± 0.90 12.4 ± 3.10
10.9 ± 1.30 8.0 ± 1.63 88.0 ± 1.94 0.9 ± 0.30 1.8 ± 0.33 0.4 ± 0.04 0.9 ± 0.29 8.2 ± 0.16 16.0 ± 0.50 48.4 ± 1.46 58.9 ± 1.02 19.5 ± 0.41 33.1 ± 0.81 3.2 ± 0.33 941.4 ± 64.71 13.7 ± 0.35 12.0 ± 2.54
12.5 ± 3.66 7.9 ± 3.17 87.6 ± 4.09 0.9 ± 0.23 1.9 ± 0.61 0.4 ± 0.00 1.3 ± 0.62 8.3 ± 0.64 15.8 ± 0.68 47.3 ± 2.13 57.1 ± 2.21 19.1 ± 0.82 33.4 ± 0.41 2.6 ± 0.57 1000.3 ± 118.22 14.0 ± 0.41 12.2 ± 1.16
WBC, white blood cell count; RBC, red blood cell count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PT, prothrombin; APTT, activated partial thromboplastin time. Values are presented as the mean ± SD.
2.7. Hematology and serum biochemistry
previously (Park et al., 2016b). Briefly, rats of all groups were observed twice a day for mortality or any other clinical signs. The body weight of each rat was recorded prior to dosing on day 1 and once a week during the dosing period and finally on the day of necropsy. The daily food intake of each rat (g/animal/day) was determined by dividing the weekly food intake by the total weight of the rats in each cage.
All analyses were performed as described previously (Park et al., 2016b). Prior to necropsy, blood samples (3 mL) from each rat were collected from the posterior vena cava, and 0.5 mL was placed in a tube containing dipotassium ethylenediamine tetraacetic acid for hematological analysis. The following hematological parameters were analyzed using an ADVIA2120i hematology analyzer (Siemens, USA): total red blood cell count (RBC), total leukocyte count (WBC), hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count, activated partial thromboplastin time (APTT), and white cell differential including neutrophils, lymphocytes, eosinophils, monocytes, and basophils. Plasma was obtained from 1.0 mL of the blood sample, which was transferred into a tube containing 3.2% sodium citrate and then centrifuged at 3000 rpm for 10 min. Prothrombin time and activated partial thromboplastin time of the plasma were measured using an ACL Elite pro coagulation analyzer (Instrumentation Laboratory, Italy). For serum biochemistry analysis, 1.5 mL of blood was placed in a collection tube and kept at room temperature for 90 min. Serum was separated from the clotted blood by centrifugation at 3000 rpm for 10 min. The following biochemistry parameters were analyzed using a Toshiba 120 FR chemistry analyzer (Toshiba Co., Japan): glucose, blood urea nitrogen (BUN), creatinine, total protein, albumin, albumin/ globulin ratio, total cholesterol, triglyceride, phospholipids, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total
2.5. Urinalysis 16 h prior to necropsy, urine samples were collected from rats housed in metabolic cages. Urine glucose, specific gravity, and pH were measured using a Cobas U411 urine analyzer (Roche, Germany) and Combur 10 TM urine sticks (Roche). 2.6. Necropsy After 28 days of YSTE administration, all rats were transferred to metabolic cages and fasted overnight (approximately 16 h) before necropsy. On day 29, rats were euthanized by inhaled anesthetics with overdose of isoflurane and blood samples were collected. Complete gross postmortem by examination was performed as described previously (Park et al., 2016b). Different organs like brain, pituitary gland, liver, spleen, heart, thymus, salivary gland, seminal vesicles, prostate, kidneys, adrenal glands, testes, epididymides, lungs, thyroid, ovaries, and uterus were removed from each rat, trimmed free of fat, and weighed. Relative organ weights were calculated based on the organ-tobody weight ratio. 5
Journal of Ethnopharmacology 238 (2019) 111852
E. Park, et al.
Table 8 Biochemical parameters of rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats Glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) Total protein (g/dL) Albumin (g/dL) Albumin/globulin ratio Total cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipid (mg/dL) AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL) ALP (IU/L) Creatine kinase (IU/L) Ca++ (mg/dL) IP (mg/dL) Na+ (mmol/L) K+ (mmol/L) Cle (mmol/L) GGT (IU/L) Female rats Glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) Total protein (g/dL) Albumin (g/dL) Albumin/globulin ratio Total cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipid (mg/dL) AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL) ALP (IU/L) Creatine kinase (IU/L) Ca++ (mg/dL) IP (mg/dL) Na+ (mmol/L) K+ (mmol/L) Cle (mmol/L) GGT (IU/L)
0
1000
2000
5000
95.20 ± 10.73 14.60 ± 1.08 0.43 ± 0.02 6.32 ± 0.27 4.12 ± 0.17 1.87 ± 0.01 48.60 ± 10.04 15.40 ± 2.46 74.60 ± 9.37 134.50 ± 20.66 33.70 ± 5.44 0.10 ± 0.02 621.90 ± 98.01 711.20 ± 303.45 10.59 ± 0.14 10.54 ± 0.46 145.80 ± 1.64 8.21 ± 0.85 101.20 ± 1.64 0.29 ± 0.23
99.10 ± 18.33 15.60 ± 1.06 0.41 ± 0.02 6.35 ± 0.25 4.19 ± 0.11 1.95 ± 0.08 62.60 ± 10.36 14.90 ± 3.87 90.60 ± 7.02 135.60 ± 2.77 29.70 ± 3.53 0.09 ± 0.02 569.60 ± 48.65 707.00 ± 165.48 10.50 ± 0.33 10.56 ± 1.04 145.80 ± 2.05 7.80 ± 1.44 100.80 ± 0.45 0.27 ± 0.08
103.00 ± 14.17 14.70 ± 1.53 0.41 ± 0.02 6.37 ± 0.10 4.16 ± 0.06 1.89 ± 0.07 66.40 ± 11.13 20.50 ± 5.60 96.60 ± 10.78 141.00 ± 19.75 26.30 ± 3.80* 0.10 ± 0.03 513.50 ± 58.16 730.20 ± 257.00 10.77 ± 0.19 10.25 ± 1.25 146.40 ± 2.07 7.50 ± 1.23 99.80 ± 1.30 0.26 ± 0.11
93.40 ± 17.46 15.80 ± 1.83 0.36 ± 0.02** 6.23 ± 0.41 4.10 ± 0.30 1.93 ± 0.12 54.80 ± 16.32 19.00 ± 12.20 86.30 ± 17.97 120.10 ± 7.38 25.10 ± 2.63* 0.05 ± 0.03* 445.20 ± 64.85** 608.50 ± 134.05 10.49 ± 0.56 10.19 ± 1.00 146.30 ± 0.96 8.48 ± 1.52 100.50 ± 0.58 0.26 ± 0.12
84.30 ± 18.28 25.40 ± 5.05 0.54 ± 0.09 6.55 ± 0.56 4.35 ± 0.36 1.98 ± 0.03 59.00 ± 5.61 8.70 ± 1.95 105.00 ± 7.35 155.10 ± 45.73 29.10 ± 7.74 0.15 ± 0.03 400.60 ± 45.76 830.20 ± 481.37 10.99 ± 0.34 9.83 ± 0.72 145.20 ± 1.64 7.74 ± 0.64 102.40 ± 0.89 0.95 ± 0.51
77.30 ± 19.63 19.10 ± 1.67* 0.45 ± 0.03 6.48 ± 0.22 4.30 ± 0.07 1.98 ± 0.17 60.40 ± 20.78 15.00 ± 10.43 113.00 ± 33.11 156.10 ± 5.24 29.20 ± 7.65 0.15 ± 0.04 394.90 ± 109.88 773.60 ± 195.94 11.08 ± 0.41 10.14 ± 0.64 145.00 ± 0.71 7.88 ± 0.61 102.40 ± 2.70 1.08 ± 0.39
82.80 ± 28.00 19.60 ± 2.07* 0.46 ± 0.03 6.61 ± 0.22 4.42 ± 0.10 2.03 ± 0.08 50.80 ± 11.19 10.00 ± 4.17 96.40 ± 18.06 141.70 ± 8.16 24.20 ± 3.44 0.15 ± 0.04 407.20 ± 71.59 628.00 ± 109.17 11.27 ± 0.26 10.65 ± 0.75 146.20 ± 1.92 7.98 ± 0.70 102.40 ± 1.95 1.51 ± 0.60
87.7 ± 19.95 20.00 ± 2.61 0.44 ± 0.09 6.60 ± 0.19 4.36 ± 0.10 1.96 ± 0.19 68.3 ± 21.69 11.60 ± 3.91 116.30 ± 29.35 135.00 ± 18.27 22.90 ± 1.83 0.11 ± 0.04 319.30 ± 62.84 797.30 ± 85.18 11.09 ± 0.10 9.71 ± 0.44 146.80 ± 0.50 7.06 ± 0.45 103.00 ± 0.82 1.05 ± 0.36
BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; Ca++, calcium; IP, inorganic phosphorus; Na++, sodium; K+, potassium; Cle, chloride; GGT, gamma glutamyl transpeptidase. Values are presented as the mean ± SD. * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, when compared with the vehicle control group.
bilirubin, alkaline phosphatase (ALP), creatine kinase, calcium (Ca++), inorganic phosphorus (IP), sodium (Na+), potassium (K+), chloride (Cl−), and gamma glutamyl transpeptidase (GGT).
3. Results 3.1. HPLC analysis of YSTE In this study, HPLC analysis of eight marker components of YSTE was performed using an established HPLC analytical method. All the marker compounds, i.e., chlorogenic acid, gentiopicroside, liquiritin apioside, liquiritin, nodakenin, baicalin, wogonoside, and glycyrrhizin, were detected at 9.95, 11.26, 14.49, 14.85, 15.57, 19.13, 22.12, and 26.98 min, respectively (Fig. 1), and the amounts of these components in YSTE at 0, 1, and 4 weeks were 0.981–16.692, 0.976–16.964, and 1.021–17.853 mg/g, respectively (Table 1). These data indicate that the components of YSTE were stable during the period of administration to rats.
2.8. Statistical analyses Statistical analyses were performed essentially as described previously (Park et al., 2016b). Briefly, analyses were performed using the Path/Tox System (version 4.2.2 and 6.4.0, Xybion Medical System Co., USA). All data were analyzed using Bartlett's test for homogeneity of variance. When Bartlett's test indicated that there was no significant deviation from homogeneity, a one-way analysis of variance was performed and the significance of difference between pairs of groups was determined using Dunnett's test. When Bartlett's test indicated a significant deviation from homogeneity, the Kruskal–Wallis test was performed and the significance of differences between specific pairs of groups was determined using Dunn's Rank Sum test. P < 0.05 was considered significant. Values are presented as mean ± standard deviation (SD).
3.2. Effects of YSTE on mortality and clinical signs in rats During oral treatment with YSTE, one female and one male rat treated with 5000 mg/kg/day were found dead at day 24 (Table 2). The clinical signs, however, were not observed in the dead animals. Salivation was observed in one male rat treated with 2000 mg/kg/day at day 26 and in both male and female rats treated with 5000 mg/kg/day 6
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stress (Arantes-Rodrigues et al., 2012; Brown et al., 2000). Therefore, we consider that death from these injuries resulted from the physical process of intubation with the gavage needle catheter and was independent of YSTE toxicity. Excessive salivation and discolored urine were observed in all rats of both sexes treated with 5000 mg/kg/day YSTE. Salivation and gaping are behavioral responses associated with ingestion of a bitter substance (Matsuo et al., 2001; Yarmolinsky et al., 2009) and Gentianae Scabrae Radix, one of the major components of Yongdamsagan-tang, has a bitter taste (Han et al., 2018; Hayashi, 1976). Therefore, the salivation seen after the highest dose of YSTE was considered to be caused by its bitter taste. With regard to the discolored urine, the absolute and relative weights of the kidneys were also increased in rats treated with 5000 mg/kg/day YSTE but there were no associated changes in urinalysis, which are used as a diagnostic tool to detect urinary tract diseases, such as renal dysfunction or urinary tract infections (Rigby and Gray, 2005; Simerville et al., 2005). Because of discoloration of urine occurred in all rats treated with 5000 mg/kg/day YSTE and the characteristics of YSTE, we consider that the discolored urine was likely related to the pigment content of YSTE, not toxicologically significant change. Furthermore, studies on the subchronic toxicity against the administration of YSTE over 13 weeks should be performed to clarify the cause of these symptoms. On gross necropsy, abnormal lesions of liver tissue in which all lobes were darkened in color were observed in both male and female rats treated with 5000 mg/kg/day YSTE. Considering the frequency of these changes in both sexes, we suggest that this discoloration of the liver was also related to the deposition of YSTE pigments in hepatocytes. Nutrients and foreign bodies, as well as oxygen, are transported to the cells of the whole body through the blood. Therefore, hematological and serum biochemical parameters are important indicators of the toxicity of a substrate for target organs (Khoo et al., 2010; Larrey, 2002; Milner et al., 2003). Serum biochemistry analysis showed some changes in parameters including total bilirubin, creatinine, ALT, and ALP in male rats. Bilirubin, which is secreted in bile and urine, acts as an indicator of liver damage (Fevery, 2008). An elevated concentration of total bilirubin, the combination of direct and indirect bilirubin, indicates a high probability of a hepatic or extrahepatic disorder, whereas decreased levels of bilirubin are often harmless (Giannini et al., 2005). In this study, the decrease in average total bilirubin in male rats treated with 5000 mg/kg/day YSTE was the result of an excessive inhibition in one rat (to 0.01 mg/dL) but not the others (0.056–0.085 mg/dL) and showed little dose dependency. Creatinine, which is filtered through the kidney glomeruli and then excreted in urine, is an index used commonly to measure renal toxicity (Gowda et al., 2010; Perrone et al., 1992). Although we observed a reduction in serum creatinine in male rats treated with 5000 mg/kg/day YSTE, the results of urinalysis showed no difference in any other parameter despite the urine discoloration. Serum ALT and ALP values are used as indicators of hepatocellular injury and hepatobiliary disease, respectively (Limdi and Hyde, 2003). Although the reductions in creatinine, ALT and ALP of male rats treated with 2000 or 5000 mg/kg/day YSTE were significant, these changes were small and were not consistent between male and female rats. All these changes were in the normal ranges. Therefore, these biochemical changes were considered to be unrelated to toxicity from YSTE. The daily dose of YST is known to be 26.25 g based on dried raw herbal medicines (Hŏ, 2012) and is equivalent dose of YSTE 5.62 g when the extraction yield is 21.4%. In the present study, the human equivalent dose (HED) of YSTE applied to rats (Van Miert, 1986) is calculated to be 580 mg/kg, which is equivalent to 29% of the highest dose 2000 mg/kg without adverse event, regardless of toxicity in rats.
at all time points (Table 3). Discolored urine was observed in both male (n = 4) and female (n = 4) rats treated with 5000 mg/kg/day YSTE (Table 3). Irregular respiration and paleness were also observed in one female rat treated with 5000 mg/kg/day YSTE (Table 3). 3.3. Effects of YSTE on body weight and food intake changes in rats No significant change in body weight (Fig. 2) or food intake (Fig. 3) showed in rats of either sex treated orally with YSTE compared with the control vehicle-treated group. 3.4. Effects of YSTE on urinalysis Urinalysis of rats of both sexes showed that there were no significant differences in urine volume, glucose, specific gravity or pH in rats treated with 1000, 2000 or 5000 mg/kg/day YSTE compared with the vehicle-treated control group (Table 4). 3.5. Effects of YSTE on gross necropsy and organ-to-body weights in rats After YSTE treatment, all rats except for those that had died were subjected to gross necropsy. Rats treated with 1000 or 2000 mg/kg/day YSTE exhibited no notable changes at gross necropsy (Table 5). However, discoloration of the liver was observed in both male (n = 3) and female (n = 1) rats treated with 5000 mg/kg/day YSTE. The effects of YSTE on organ weight relative to total body weight are shown in Table 6. In male rats treated with 5000 mg/kg/day YSTE, significant increases in the relative weights of liver (1.19-fold) and kidney (1.21fold) were observed. In female rats, no significant change was observed in the relative organ weights related to oral administration of YSTE. 3.6. Effects of YSTE on hematological and biochemical parameters in rats No significant change in hematological parameters related to YSTE administration was observed in rats of either sex (Table 7). However, slight changes in some biochemical parameters were observed in YSTEtreated male but not female rats (Table 8). Compared with the vehicle control group, significant decreases in creatinine (0.84-fold; P < 0.01), total bilirubin (0.54-fold; P < 0.05) and ALP (0.72-fold; P < 0.01) were observed in rats treated with 5000 mg/kg, and ALT levels reduced 0.72-fold (P < 0.05) and 0.75-fold (P < 0.05) in rats treated with 2000 mg/kg/day and 5000 mg/kg/day YSTE, respectively. All these changes in biochemical parameters were in the normal ranges. There was no clinically significant change in the biochemical parameters of YSTE-treated rats compared with vehicle-treated controls. 4. Discussion A preliminary acute toxicity study previously conducted by our group showed that YSTE administration to rats at doses of up to 5000 mg/kg/day caused no detectable effects on the parameters tested (data not shown). Based on these results, the present study evaluated the toxicity of YSTE in rats over 4 weeks. YSTE administration to rats at doses of up to 5000 mg/kg/day for 4 weeks resulted in no major changes in mortality, body weight, food intake, or the results of serum biochemistry, hematology, or urinalysis. However, abnormalities in some rats were noted as follows. During the dosing period, one male and one female rat treated with 5000 mg/kg/day YSTE died. In the female rat, irregular respiration and paleness were observed prior to death. In the male rat, no clinical signs were observed except for excessive salivation, and there was no change in body weight or food intake. However, the autopsy findings revealed esophageal perforation and a fluid-filled thoracic cavity in both rats. It has been reported that oral gavage for substance administration involves many complications that can increase mortality, including esophageal perforation, esophageal rupture, aspiration pneumonia, and
5. Conclusion YSTE administration at doses of up to 5000 mg/kg/day for 28 days 7
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References
resulted in no notable adverse effects on mortality, body weight, food intake, urinalysis, gross necropsy, relative organ weights, or hematological and biochemical analysis. However, discolored liver and urine, and increased absolute and relative kidney weights were observed in rats treated with 5000 mg/kg/day YSTE, suggesting that these symptoms are associated with the pigments contained in YSTE. Taken together, these findings suggest that doses of up to 5000 mg/kg/day YSTE are nontoxic for rats. Moreover, further testing including the assessment of the subchronic toxicity of YSTE should be required.
Arantes-Rodrigues, R., Henriques, A., Pinto-Leite, R., Faustino-Rocha, A., Pinho-Oliveira, J., Teixeira-Guedes, C., Seixas, F., Gama, A., Colaco, B., Colaco, A., Oliveira, P.A., 2012. The effects of repeated oral gavage on the health of male cd-1 mice. Lab. Anim 41 (5), 129–134. Brown, A.P., Dinger, N., Levine, B.S., 2000. Stress produced by gavage administration in the rat. Contemp. Top. Lab. Anim. Sci. 39 (1), 17–21. Fevery, J., 2008. Bilirubin in clinical practice: a review. Liver Int. 28 (5), 592–605. Giannini, E.G., Testa, R., Savarino, V., 2005. Liver enzyme alteration: a guide for clinicians. CMAJ (Can. Med. Assoc. J.) 172 (3), 367–379. Gowda, S., Desai, P.B., Kulkarni, S.S., Hull, V.V., Math, A.A., Vernekar, S.N., 2010. Markers of renal function tests. N. Am. J. Med. Sci. 2 (4), 170–173. Grayson, M., 2011. Traditional asian medicine. Nature 480 (7378), S81. Han, X., Jiang, H., Han, L., Xiong, X., He, Y., Fu, C., Xu, R., Zhang, D., Lin, J., Yang, M., 2018. A novel quantified bitterness evaluation model for traditional Chinese herbs based on an animal ethology principle. Acta Pharm. Sin. B 8 (2), 209–217. Hayashi, T., 1976. [studies on crude drugs originated from gentianaceous plants. I. Determination of gentiopicroside, the bitter principle of gentianae radix and gentianae scabrae radix (author's transl)]. Yakugaku Zasshi 96 (3), 356–361. Hŏ, C., 2012. Donguibogam: Principles and Practice of Eastern Medicine. Ministry for Health, Welfare and Family Affairs. Jin, Y., 2004. A combined use of acupuncture, moxibustion and long dan xie gan tang for treatment of 36 cases of chronic pelvic inflammation. J. Tradit. Chin. Med. 24 (4), 256–258. Khoo, Z.Y., Teh, C.C., Rao, N.K., Chin, J.H., 2010. Evaluation of the toxic effect of star fruit on serum biochemical parameters in rats. Phcog. Mag. 6 (22), 120–124. Larrey, D., 2002. Epidemiology and individual susceptibility to adverse drug reactions affecting the liver. Semin. Liver Dis. 22 (2), 145–155. Lee, T.Y., Chang, H.H., 2010. Longdan xiegan tang has immunomodulatory effects on cd4+cd25+ t cells and attenuates pathological signs in mrl/lpr mice. Int J Mol 25 (5), 677–685. Lim, J.-H., Lee, J.-R., Kim, S.-C., Jee, S.-Y., 2007. Inhibitory effect of yongdamsagantang water extract on il-6 and nitric oxide production in lipopolysaccharide-activated raw 264.7 cells. Orient Pharm Exp Med 7 (3), 321–329. Limdi, J.K., Hyde, G.M., 2003. Evaluation of abnormal liver function tests. Postgrad. Med. 79 (932), 307–312. Lu, W.I., Lu, D.P., 2014. Impact of Chinese herbal medicine on american society and health care system: perspective and concern. Evid Based Complement Alternat Med 251891 2014. Matsuo, R., Yamauchi, Y., Kobashi, M., Funahashi, M., Mitoh, Y., Adachi, A., 2001. Role of parabrachial nucleus in submandibular salivary secretion induced by bitter taste stimulation in rats. Auton. Neurosci. 88 (1–2), 61–73. Milner, J.M., Stien, A., Irvine, R.J., Albon, S.D., Langvatn, R., Ropstad, E., 2003. Body condition in svalbard reindeer and the use of blood parameters as indicators of condition and fitness. Can. J. Zool. 81 (9), 1566–1578. Park, E., Lee, M.Y., Jeon, W.Y., Lee, N., Seo, C.S., Shin, H.K., 2016a. Inhibitory effect of yongdamsagan-tang water extract, a traditional herbal formula, on testosterone-induced benign prostatic hyperplasia in rats. Evid Based Complement Alternat Med 1428923 2016. Park, E., Lee, M.Y., Seo, C.S., Jeon, W.Y., Shin, H.K., 2017. Yongdamsagan-tang, a traditional herbal formula, inhibits cell growth through the suppression of proliferation and inflammation in benign prostatic hyperplasia epithelial-1 cells. J. Ethnopharmacol. 209, 230–235. Park, E., Lee, M.Y., Seo, C.S., Yoo, S.R., Jeon, W.Y., Shin, H.K., 2016b. Acute and subacute toxicity of an ethanolic extract of melandrii herba in crl:Cd sprague dawley rats and cytotoxicity of the extract in vitro. BMC Complement Altern. Med. 16 (1), 370. Perrone, R.D., Madias, N.E., Levey, A.S., 1992. Serum creatinine as an index of renal function: new insights into old concepts. Clin. Chem. 38 (10), 1933–1953. Rigby, D., Gray, K., 2005. Understanding urine testing. Nurs. Times 101 (12), 60–62. Simerville, J.A., Maxted, W.C., Pahira, J.J., 2005. Urinalysis: a comprehensive review. Am. Fam. Physician 71 (6), 1153–1162. Van Miert, A., 1986. The Use in Animals of Drugs Licensed for Human Use Only. Comparative Veterinary Pharmacology, Toxicology and Theraphy. Springer, pp. 489–500. Xiong, X., Che, C.T., Borrelli, F., Moudgil, K.D., Caminiti, G., 2017. Evidence-based tam classic herbal formula: from myth to science. Evid Based Complement Alternat Med 9493076 2017. Xu, J., Liu, M., Xia, Z., 2012. Asian medicine: call for more safety data. Nature 482 (7383), 35. Yarmolinsky, D.A., Zuker, C.S., Ryba, N.J., 2009. Common sense about taste: from mammals to insects. Cell 139 (2), 234–244. YUZhou, Y., 2001. Chines Medical Formulae. People'medical publishing house, Beking, pp. 142–143.
Conflicts of interest The authors declare that they have no competing interests. Authors’ contributions MYL and HH designed and conceived the study. EP and MYL participated in the data analyses and manuscript preparation. CSS carried out the preparation of YSTE, HPLC analysis, and manuscript preparation. HKS edited the draft manuscript. SCH carried out the toxicity assay. All authors have read and approved the final manuscript. Acknowledgements This research was supported through the grants ‘Construction of Scientific Evidences for Herbal Medicine Formulas (K17251)’ and ‘Construction of safety and efficacy for traditional herbal prescriptions of medicinal institution (K18241)’ from the Korea Institute of Oriental Medicine (KIOM) in South Korea. List of abbreviations ALP ALT APTT AST BUN Ca++ Cl− GGT HCT HPLC K+ IP MCH MCHC MCV Na+ SD RBC WBC YSTE
alkaline phosphatase alanine aminotransferase activated partial thromboplastin time aspartate aminotransferase blood urea nitrogen calcium chloride gamma glutamyl transpeptidase hematocrit High-performance liquid chromatography potassium inorganic phosphorus mean corpuscular hemoglobin mean corpuscular hemoglobin concentration mean corpuscular volume sodium standard deviation red blood cell white blood cell Yongdamsagan-tang water extract
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.111852.
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Evaluation of the subacute toxicity of Yongdamsagan-tang, a traditional herbal formula, in Crl:CD Sprague Dawley rats
T
Eunsook Parka, Mee-Young Leeb, Chang-Seob Seoa, Hyeun-Kyoo Shina, Su-Cheol Hanc, Hyekyung Haa,∗ a
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, Republic of Korea Clinical Medicine Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, Republic of Korea c General Toxicology Research Center, Korea Institute of Toxicology, 30 Baekhak 1-gil, Jeongeup, Jeonbuk, Republic of Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Yongdamsagan-tang water extract Subacute toxicity Crl:CD sprague Dawley rats
Ethnopharmacological relevance: Yongdamsagan-tang, a traditional herbal formula, is used widely for the treatment of inflammatory and viral diseases. However, the safety of Yongdamsagan-tang has not been established. Aim of the study: To evaluate the subacute toxicity of Yongdamsagan-tang water extract (YSTE) in Crl:CD Sprague Dawley rats. Materials and methods: We evaluated the subacute toxicity of YSTE in male and female Crl:CD Sprague Dawley rats (n = 5 per group). Rats were treated with YSTE at doses of 0, 1000, 2000 and 5000 mg/kg administered once a day by oral gavage for 4 weeks. Results: There were no significant changes in mortality, body weight, food intake, serum biochemistry, or results of hematology and urinalysis after YSTE administration. However, all rats treated with 5000 mg/kg/day YSTE exhibited excessive salivation and discolored urine. Necropsy findings showed discoloration in the liver of both male (n = 1) and female (n = 3) rats treated with 5000 mg/kg/day YSTE, and an increase in the relative weights of kidney and liver was also found in male rats treated with 5000 mg/kg/day. In addition, decreases in serum creatinine, total bilirubin, alanine transaminase, and alkaline phosphatase were observed in male rats treated with 2000 or 5000 mg/kg/day YSTE. Conclusions: Abnormalities in some rats are considered to be independent of YSTE toxicity. Therefore, the results suggest that oral administration of YSTE in rats for 4 weeks is safe at doses of up to 5000 mg/kg/day.
1. Introduction Herbal formulas composed of several herbs are a traditional medicine of Asian type and are widely used in China, Korea, and Japan (Grayson, 2011). As interest in health and natural products increases, the popularity of traditional herbal formulas as complementary or alternative medicines is growing in both Western and Asian countries (Lu and Lu, 2014). However, there are still many limitations on the use of herbal formulas as treatment for various diseases or as dietary supplements because of the lack of scientific evidence for their safety and efficacy (Xiong et al., 2017; Xu et al., 2012). Therefore, systematic toxicology studies are required to demonstrate their safety. Yongdamsagan-tang, also known as Long Dan Xie Gan Tang in Chinese and Ryutanshakan-to in Japanese, is a traditional herbal
formula widely used in Korea for treating inflammation and viral diseases (YUZhou, 2001). Recently, it has been reported that Yongdamsagan-tang has therapeutic effects in bacterial infection, benign prostatic hyperplasia, and polycystic ovary syndrome via its antioxidant, antiproliferative, and anti-inflammatory activities (Jin, 2004; Lee and Chang, 2010; Lim et al., 2007; Park et al., 2016a, 2017). Despite of scientific trials to establish the efficacy of Yongdamsagan-tang, toxicological studies to determine any adverse effects of Yongdamsagantang in vivo are scarce. Therefore, to investigate the safety profile of Yongdamsagan-tang, we evaluated the subacute toxicity of Yongdamsagan-tang water extract (YSTE) in male and female Crl:CD Sprague Dawley rats. This scientific information about the subacute toxicity of YSTE may assist in the development of new treatments based on herbal formulas including
∗
Corresponding author. Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea. E-mail addresses: [email protected] (E. Park), [email protected] (M.-Y. Lee), [email protected] (C.-S. Seo), [email protected] (H.-K. Shin), [email protected] (S.-C. Han), [email protected] (H. Ha). https://doi.org/10.1016/j.jep.2019.111852 Received 22 June 2018; Received in revised form 24 March 2019; Accepted 31 March 2019 Available online 04 April 2019 0378-8741/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Three-dimensional HPLC chromatogram of YSTE using HPLC–PDA. Table 1 Amounts of eight marker components in YSTE at 0, 1 and 4 weeks by HPLC (n = 3). Compound
0 week
Chlorogenic acid Gentiopicroside Liquiritin apioside Liquiritin Nodakenin Baicalin Wogonoside Glycyrrhizin
1 week
4 week
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
0.981 16.692 2.365 2.043 1.876 6.937 2.344 3.000
0.016 0.032 0.028 0.006 0.016 0.073 0.031 0.007
1.626 0.189 1.174 0.302 0.829 1.055 1.321 0.242
0.976 16.964 2.323 2.108 1.967 7.212 2.522 3.024
0.005 0.207 0.032 0.039 0.029 0.169 0.061 0.088
0.464 1.222 1.392 1.872 1.483 2.350 2.424 2.907
1.021 17.853 2.404 2.132 1.954 7.694 2.645 3.111
0.007 0.123 0.025 0.023 0.012 0.060 0.021 0.059
0.660 0.686 1.020 1.101 0.633 0.778 0.794 1.904
Table 2 Mortality in rat administered orally with YSTE for 4 weeks. Group
Dosing phase 1 day
Male rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day Female rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day a
≤1 week
≤2 week
Table 3 Clinical signs in rat administered orally with YSTE for 4 weeks. Final mortalitya
Group Male rats 0 mg/kg 1000 mg/kg 2000 mg/kg 5000 mg/kg Female rats 0 mg/kg 1000 mg/kg 2000 mg/kg 5000 mg/kg
≤4 week
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 1
0/5 0/5 0/5 1/5
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 1
0/5 0/5 0/5 1/5
a
Number of animals with unscheduled death/Total animal number.
2
Salivationa
Discolored urinea
Irregular respirationa
Palenessa
0/5 0/5 1/5 5/5
0/5 0/5 0/5 4/5
0/5 0/5 0/5 0/5
0/5 0/5 0/5 0/5
0/5 1/5 0/5 5/5
0/5 0/5 0/5 4/5
0/5 0/5 0/5 1/5
0/5 0/5 0/5 1/5
Number of animals with sign/Total number of animals observed.
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Fig. 2. Body weights (g) of male (A) and female (B) rats treated orally with YSTE at doses of 0 (○), 1000 (■), 2000 (▲), and 5000 (●) mg/kg/day for 4 weeks. Values are presented as mean ± SD.
Fig. 3. Food intake (g/animal/day) of male (A) and female (B) rats treated orally with YSTE at doses of 0 (○), 1000 (■), 2000 (▲), and 5000 (●) mg/kg/day for 4 weeks. Values are presented as mean ± SD. Table 4 Urinalysis of rats administered orally with YSTE for 4 weeks. Group Male rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day Female rats 0 mg/kg/day 1000 mg/kg/day 2000 mg/kg/day 5000 mg/kg/day
Table 5 Gross necropsy observations of rats administered orally with YSTE for 4 weeks.
Volume (mL)
Glucosea
Specific gravity
pH
22 20 27 22
± ± ± ±
7.4 6.1 15.4 10.7
0/5 0/5 0/5 0/4
1.008 1.012 1.011 1.014
± ± ± ±
0.0027 0.0045 0.0042 0.0025
7.0 7.0 6.7 6.6
± ± ± ±
0.00 0.00 0.27 0.25
10 12 14 17
± ± ± ±
3.6 5.5 5.3 4.8
0/5 0/5 0/5 0/4
1.016 1.015 1.014 1.014
± ± ± ±
0.0042 0.0035 0.0042 0.0025
6.5 6.8 6.7 6.6
± ± ± ±
0.35 0.27 0.27 0.25
Dose (mg/kg/day) Male rats No remarkable findinga Liver discolorationa Female rats No remarkable findinga Liver discolorationa a
0
1000
2000
5000
5/5 0/5
5/5 0/5
5/5 0/5
1/4 3/4
5/5 0/5
5/5 0/5
5/5 0/5
3/4 1/4
Number of animals with sign/Total number of animals observed.
Yongdamsagan-tang.
Values are presented as mean ± SD. a Number of animals with sign/Total number of animals observed.
2. Materials and methods 2.1. Preparation of YSTE Yongdamsagan-tang extract was manufactured by Sungil Bioex Co. Ltd. (Hwaseong, Korea). Briefly, the eleven component herbs of 3
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Table 6 Relative organ weights (% of body weight) in rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats Brain Pituitary gland Liver Spleen Heart Thymus Salivary glands Seminal vesicle Prostate Kidneys Adrenal glands Testes Epididymides Lung Thyroid/parathyroid Female rats Brain Pituitary gland Liver Spleen Heart Thymus Salivary glands Kidneys Adrenal glands Ovaries Lung Thyroid/parathyroid Uterus/cervix
0
1000
2000
5000
0.522 0.033 3.180 0.186 0.323 0.141 0.167 0.303 0.112 0.797 0.015 0.848 0.300 0.379 0.004
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0387 0.0001 0.1215 0.0360 0.0090 0.0346 0.0216 0.0404 0.0139 0.0323 0.0017 0.0326 0.0143 0.0362 0.0004
0.520 0.003 3.164 0.182 0.324 0.127 0.170 0.326 0.125 0.823 0.016 0.840 0.285 0.380 0.005
± 0.0277 ± 0.0004 ± 0.0790 ± 0.0277 ± 0.0218 ± 0.0114 ± 0.0172 ± 0.0432 ± 0.0249 ± 0.0778 ± 0.0016 ± 0.0409 ± 0.0226 ± 0.0181 ± 0.0006**
0.510 0.003 3.273 0.192 0.325 0.145 0.169 0.304 0.104 0.858 0.015 0.785 0.266 0.372 0.005
± 0.0110 ± 0.0005 ± 0.3032 ± 0.0373 ± 0.0094 ± 0.0356 ± 0.0021 ± 0.0271 ± 0.0198 ± 0.0271 ± 0.0011 ± 0.0821 ± 0.0133* ± 0.0163 ± 0.0003
0.533 0.003 3.779 0.206 0.336 0.120 0.174 0.337 0.113 0.968 0.017 0.902 0.301 0.391 0.005
± 0.0186 ± 0.0004 ± 0.2381** ± 0.0293 ± 0.0271 ± 0.0099 ± 0.0139 ± 0.0731 ± 0.0308 ± 0.0297** ± 0.0016 ± 0.0375 ± 0.0120 ± 0.0203 ± 0.0004
0.760 0.006 3.245 0.207 0.352 0.185 0.180 0.831 0.032 0.037 0.484 0.006 0.211
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0577 0.0005 0.2771 0.0203 0.0200 0.0315 0.0145 0.0501 0.0069 0.0043 0.0257 0.0004 0.0247
0.807 0.005 3.256 0.226 0.358 0.211 0.171 0.832 0.029 0.040 0.526 0.005 0.220
± ± ± ± ± ± ± ± ± ± ± ± ±
0.783 0.005 3.445 0.210 0.359 0.205 0.173 0.860 0.034 0.037 0.498 0.006 0.208
± ± ± ± ± ± ± ± ± ± ± ± ±
0.825 0.005 3.520 0.240 0.354 0.198 0.169 0.892 0.027 0.040 0.515 0.006 0.232
± ± ± ± ± ± ± ± ± ± ± ± ±
0.0826 0.0003 0.2254 0.0273 0.0263 0.0579 0.0142 0.0459 0.0019 0.0028 0.0452 0.0007 0.0620
0.0644 0.0008 0.2254 0.0169 0.0206 0.0108 0.0070 0.0487 0.0072 0.0055 0.0321 0.0009 0.0343
0.0588 0.0008 0.1016 0.0321 0.0180 0.0199 0.0091 0.0647 0.0028 0.0055 0.0269 0.0014 0.0940
Values are presented as the mean ± SD. * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, when compared with the vehicle control group.
column used was Gemini C18 (250 mm × 4.6 mm; 5 μm, Phenomenex, Torrance, CA, USA) maintained at 40 °C, and 0.1% (v/v) trifluoroacetic acid in distilled water and acetonitrile were used as the mobile phase.
Yongdamsagan-tang, i.e. Gentianae Scabrae Radix (Gentiana scabra Bunge; 22 kg), Bupleuri Radix (Bupleurum falcatum Linne; 22 kg), Alismatis Rhizoma (Alisma orientale Juzepczuk; 22 kg), Akebiae Caulis (Akebia quinata Decaisne; 11 kg), Plantaginis Semen (Plantago asiatica Linne; 11 kg), Poria Sclerotium (Poria cocos Wolf; 11 kg), Rehmanniae Radix Crudus (Rehmannia glutinosa Liboschitz ex Steudel; 11 kg), Angelicae Gigantis Radix (Angelica gigas Nakai; 11 kg), Gardeniae Fructus (Gardenia jasminoides Ellis; 11 kg), Scutellariae Radix (Scutellaria baicalensis Georgi; 11 kg), and Glycyrrhizae Radix et Rhizoma (Glycyrrhiza uralensis Fischer; 11 kg) were purchased from Kwangmyungdang Medicinal herbs (Ulsan, Korea), mixed, and extracted in a 10-fold mass (1540 L) of water at 80 °C for 2 h using the reflux method. The extracted water solution was freeze-dried to give a powder (33 kg, yield: 21.4%).
2.3. Experimental design The experimental design was based on that of a previous study (Park et al., 2016b). All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Korea Institute of Toxicology (approval number: 1505-0116) and performed under the current Good Laboratory Practice (GLP) regulations for nonclinical laboratory studies. Five-week-old specific pathogen-free Crl:CD Sprague Dawley rats (n = 20 of each sex) were obtained from Orient Bio Co. (Seoul, Korea) and acclimatized for one week to laboratory conditions before the start of experiments. The rats were maintained in an animal facility under a controlled temperature of 23 ± 3 °C and a relative humidity of 50 ± 20% with 12 h/12 h light cycle. Healthy rats were randomly assigned to four weight-matched groups (n = 5 per group) using a Path/Tox System (version 4.2.2; Xybion Medical Systems Corporation, Cedar Knolls, NJ). Three different doses of YSTE (1000, 2000 and 5000 mg/kg/day) dissolved in distilled water were administered to rats daily by oral gavage for 4 weeks. The vehicle control group (0 mg/kg YSTE) were daily administered an equal volume of distilled water. YSTE was prepared fresh once a week and kept at 4 °C. The daily dose (in 10 mL/kg body weight) of YSTE was individually calculated based on the most recent body weight of each animal.
2.2. High-performance liquid chromatography (HPLC) analysis of YSTE Liquiritin (PubChem CID: 503737, purity 99.6%), baicalin (PubChem CID: 64982, purity 98.0%), and glycyrrhizin (PubChem CID: 14982, purity 99.0%) were purchased from Wako Chemicals (Osaka, Japan). Chlorogenic acid (PubChem CID: 1794427, purity 99.6%), gentiopicroside (PubChem CID: 88708, purity 98.3%), liquiritin apioside (PubChem CID: 10076238, purity 98.0%), nodakenin (PubChem CID: 73191, purity 98.0%), and wogonoside (PubChem CID: 3084961, purity 98.2%) were purchased from Acros Organics (Pittsburgh, PA, USA), Biopurify Phytochemicals (Chengdu, China), Shanghai Sunny Biotech (Shanghai, China), NPC Bio Technology (Yeongi, Korea) and Tauto Biotech (Shanghai, China), respectively. Standard stock solutions of these eight components were prepared at a concentration of 1.0 mg/ mL in methanol and stored at 4 °C until use. The detailed conditions for simultaneous analysis of YSTE were described previously (Park et al., 2016a). Briefly, a Prominence LC-20A HPLC system (Shimadzu Co., Kyoto, Japan) coupled with a photodiode detector array was used. The
2.4. General observations During the dosing period, general observations including mortality, clinical signs, body weights, and food intake were recorded as described 4
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Table 7 Hematological parameters of rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats WBC (103/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) Large unstained cells (%) RBC (106/μL) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/dL) Reticulocytes (%) Platelet (103/μL) PT (sec) APTT (sec) Female rats WBC (103/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) Large unstained cells (%) RBC (106/μL) Hemoglobin (g/dL) Hematocrit (%) MCV (fL) MCH (pg) MCHC (g/dL) Reticulocytes (%) Platelet (103/μL) PT (sec) APTT (sec)
0
1000
2000
5000
8.87 ± 1.70 11.2 ± 3.63 85.6 ± 3.89 0.9 ± 0.25 1.3 ± 0.33 0.3 ± 0.11 0.7 ± 0.15 8.0 ± 0.35 15.4 ± 0.61 49.0 ± 1.91 61.7 ± 0.75 19.4 ± 0.52 31.5 ± 0.48 2.6 ± 0.31 917.2 ± 104.79 14.3 ± 0.67 16.4 ± 1.85
8.3 ± 1.11 11.4 ± 2.77 84.9 ± 3.48 1.0 ± 0.38 1.4 ± 0.51 0.4 ± 0.13 0.9 ± 0.24 8.2 ± 0.35 15.7 ± 0.26 49.9 ± 0.71 61.2 ± 1.80 19.2 ± 0.67 31.5 ± 0.32 2.82 ± 0.39 952.0 ± 98.84 13.9 ± 0.41 15.9 ± 0.77
9.4 ± 2.38 10.4 ± 4.00 86.4 ± 4.24 0.6 ± 0.25 1.3 ± 0.18 0.4 ± 0.09 0.8 ± 0.22 8.2 ± 0.32 15.6 ± 0.39 49.4 ± 1.58 60.1 ± 2.46 18.9 ± 0.68 31.5 ± 0.30 2.7 ± 0.45 918.4 ± 176.86 14.1 ± 0.59 15.7 ± 0.60
8.8 ± 2.59 15.3 ± 2.76 81.1 ± 2.65 0.9 ± 0.15 1.6 ± 0.39 0.4 ± 0.22 0.7 ± 0.26 8.0 ± 0.40 15.0 ± 0.37 47.5 ± 2.00 59.1 ± 0.99 18.7 ± 0.51 31.7 ± 0.60 2.9 ± 0.34 1076.0 ± 91.80 14.9 ± 0.47 14.3 ± 1.85
8.9 ± 1.20 8.3 ± 2.98 87.4 ± 3.53 1.2 ± 0.37 1.8 ± 0.69 0.3 ± 0.05 1.0 ± 0.27 8.2 ± 0.60 15.7 ± 0.84 48.2 ± 2.90 59.0 ± 1.38 19.3 ± 0.46 32.7 ± 0.54 2.7 ± 0.36 919.0 ± 90.78 13.7 ± 0.58 13.9 ± 2.01
12.86 ± 3.22 12.1 ± 5.87 83.5 ± 6.60 0.9 ± 0.31 2.0 ± 0.66 0.4 ± 0.11 1.1 ± 0.44 8.5 ± 0.19 16.1 ± 0.09 48.9 ± 0.73 57.7 ± 1.61 19.0 ± 0.36 33.0 ± 0.41 2.4 ± 0.39 999.2 ± 69.76 13.9 ± 0.90 12.4 ± 3.10
10.9 ± 1.30 8.0 ± 1.63 88.0 ± 1.94 0.9 ± 0.30 1.8 ± 0.33 0.4 ± 0.04 0.9 ± 0.29 8.2 ± 0.16 16.0 ± 0.50 48.4 ± 1.46 58.9 ± 1.02 19.5 ± 0.41 33.1 ± 0.81 3.2 ± 0.33 941.4 ± 64.71 13.7 ± 0.35 12.0 ± 2.54
12.5 ± 3.66 7.9 ± 3.17 87.6 ± 4.09 0.9 ± 0.23 1.9 ± 0.61 0.4 ± 0.00 1.3 ± 0.62 8.3 ± 0.64 15.8 ± 0.68 47.3 ± 2.13 57.1 ± 2.21 19.1 ± 0.82 33.4 ± 0.41 2.6 ± 0.57 1000.3 ± 118.22 14.0 ± 0.41 12.2 ± 1.16
WBC, white blood cell count; RBC, red blood cell count; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PT, prothrombin; APTT, activated partial thromboplastin time. Values are presented as the mean ± SD.
2.7. Hematology and serum biochemistry
previously (Park et al., 2016b). Briefly, rats of all groups were observed twice a day for mortality or any other clinical signs. The body weight of each rat was recorded prior to dosing on day 1 and once a week during the dosing period and finally on the day of necropsy. The daily food intake of each rat (g/animal/day) was determined by dividing the weekly food intake by the total weight of the rats in each cage.
All analyses were performed as described previously (Park et al., 2016b). Prior to necropsy, blood samples (3 mL) from each rat were collected from the posterior vena cava, and 0.5 mL was placed in a tube containing dipotassium ethylenediamine tetraacetic acid for hematological analysis. The following hematological parameters were analyzed using an ADVIA2120i hematology analyzer (Siemens, USA): total red blood cell count (RBC), total leukocyte count (WBC), hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, reticulocyte count, activated partial thromboplastin time (APTT), and white cell differential including neutrophils, lymphocytes, eosinophils, monocytes, and basophils. Plasma was obtained from 1.0 mL of the blood sample, which was transferred into a tube containing 3.2% sodium citrate and then centrifuged at 3000 rpm for 10 min. Prothrombin time and activated partial thromboplastin time of the plasma were measured using an ACL Elite pro coagulation analyzer (Instrumentation Laboratory, Italy). For serum biochemistry analysis, 1.5 mL of blood was placed in a collection tube and kept at room temperature for 90 min. Serum was separated from the clotted blood by centrifugation at 3000 rpm for 10 min. The following biochemistry parameters were analyzed using a Toshiba 120 FR chemistry analyzer (Toshiba Co., Japan): glucose, blood urea nitrogen (BUN), creatinine, total protein, albumin, albumin/ globulin ratio, total cholesterol, triglyceride, phospholipids, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total
2.5. Urinalysis 16 h prior to necropsy, urine samples were collected from rats housed in metabolic cages. Urine glucose, specific gravity, and pH were measured using a Cobas U411 urine analyzer (Roche, Germany) and Combur 10 TM urine sticks (Roche). 2.6. Necropsy After 28 days of YSTE administration, all rats were transferred to metabolic cages and fasted overnight (approximately 16 h) before necropsy. On day 29, rats were euthanized by inhaled anesthetics with overdose of isoflurane and blood samples were collected. Complete gross postmortem by examination was performed as described previously (Park et al., 2016b). Different organs like brain, pituitary gland, liver, spleen, heart, thymus, salivary gland, seminal vesicles, prostate, kidneys, adrenal glands, testes, epididymides, lungs, thyroid, ovaries, and uterus were removed from each rat, trimmed free of fat, and weighed. Relative organ weights were calculated based on the organ-tobody weight ratio. 5
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Table 8 Biochemical parameters of rats administered orally with YSTE for 4 weeks. Dose (mg/kg/day) Male rats Glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) Total protein (g/dL) Albumin (g/dL) Albumin/globulin ratio Total cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipid (mg/dL) AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL) ALP (IU/L) Creatine kinase (IU/L) Ca++ (mg/dL) IP (mg/dL) Na+ (mmol/L) K+ (mmol/L) Cle (mmol/L) GGT (IU/L) Female rats Glucose (mg/dL) BUN (mg/dL) Creatinine (mg/dL) Total protein (g/dL) Albumin (g/dL) Albumin/globulin ratio Total cholesterol (mg/dL) Triglycerides (mg/dL) Phospholipid (mg/dL) AST (IU/L) ALT (IU/L) Total bilirubin (mg/dL) ALP (IU/L) Creatine kinase (IU/L) Ca++ (mg/dL) IP (mg/dL) Na+ (mmol/L) K+ (mmol/L) Cle (mmol/L) GGT (IU/L)
0
1000
2000
5000
95.20 ± 10.73 14.60 ± 1.08 0.43 ± 0.02 6.32 ± 0.27 4.12 ± 0.17 1.87 ± 0.01 48.60 ± 10.04 15.40 ± 2.46 74.60 ± 9.37 134.50 ± 20.66 33.70 ± 5.44 0.10 ± 0.02 621.90 ± 98.01 711.20 ± 303.45 10.59 ± 0.14 10.54 ± 0.46 145.80 ± 1.64 8.21 ± 0.85 101.20 ± 1.64 0.29 ± 0.23
99.10 ± 18.33 15.60 ± 1.06 0.41 ± 0.02 6.35 ± 0.25 4.19 ± 0.11 1.95 ± 0.08 62.60 ± 10.36 14.90 ± 3.87 90.60 ± 7.02 135.60 ± 2.77 29.70 ± 3.53 0.09 ± 0.02 569.60 ± 48.65 707.00 ± 165.48 10.50 ± 0.33 10.56 ± 1.04 145.80 ± 2.05 7.80 ± 1.44 100.80 ± 0.45 0.27 ± 0.08
103.00 ± 14.17 14.70 ± 1.53 0.41 ± 0.02 6.37 ± 0.10 4.16 ± 0.06 1.89 ± 0.07 66.40 ± 11.13 20.50 ± 5.60 96.60 ± 10.78 141.00 ± 19.75 26.30 ± 3.80* 0.10 ± 0.03 513.50 ± 58.16 730.20 ± 257.00 10.77 ± 0.19 10.25 ± 1.25 146.40 ± 2.07 7.50 ± 1.23 99.80 ± 1.30 0.26 ± 0.11
93.40 ± 17.46 15.80 ± 1.83 0.36 ± 0.02** 6.23 ± 0.41 4.10 ± 0.30 1.93 ± 0.12 54.80 ± 16.32 19.00 ± 12.20 86.30 ± 17.97 120.10 ± 7.38 25.10 ± 2.63* 0.05 ± 0.03* 445.20 ± 64.85** 608.50 ± 134.05 10.49 ± 0.56 10.19 ± 1.00 146.30 ± 0.96 8.48 ± 1.52 100.50 ± 0.58 0.26 ± 0.12
84.30 ± 18.28 25.40 ± 5.05 0.54 ± 0.09 6.55 ± 0.56 4.35 ± 0.36 1.98 ± 0.03 59.00 ± 5.61 8.70 ± 1.95 105.00 ± 7.35 155.10 ± 45.73 29.10 ± 7.74 0.15 ± 0.03 400.60 ± 45.76 830.20 ± 481.37 10.99 ± 0.34 9.83 ± 0.72 145.20 ± 1.64 7.74 ± 0.64 102.40 ± 0.89 0.95 ± 0.51
77.30 ± 19.63 19.10 ± 1.67* 0.45 ± 0.03 6.48 ± 0.22 4.30 ± 0.07 1.98 ± 0.17 60.40 ± 20.78 15.00 ± 10.43 113.00 ± 33.11 156.10 ± 5.24 29.20 ± 7.65 0.15 ± 0.04 394.90 ± 109.88 773.60 ± 195.94 11.08 ± 0.41 10.14 ± 0.64 145.00 ± 0.71 7.88 ± 0.61 102.40 ± 2.70 1.08 ± 0.39
82.80 ± 28.00 19.60 ± 2.07* 0.46 ± 0.03 6.61 ± 0.22 4.42 ± 0.10 2.03 ± 0.08 50.80 ± 11.19 10.00 ± 4.17 96.40 ± 18.06 141.70 ± 8.16 24.20 ± 3.44 0.15 ± 0.04 407.20 ± 71.59 628.00 ± 109.17 11.27 ± 0.26 10.65 ± 0.75 146.20 ± 1.92 7.98 ± 0.70 102.40 ± 1.95 1.51 ± 0.60
87.7 ± 19.95 20.00 ± 2.61 0.44 ± 0.09 6.60 ± 0.19 4.36 ± 0.10 1.96 ± 0.19 68.3 ± 21.69 11.60 ± 3.91 116.30 ± 29.35 135.00 ± 18.27 22.90 ± 1.83 0.11 ± 0.04 319.30 ± 62.84 797.30 ± 85.18 11.09 ± 0.10 9.71 ± 0.44 146.80 ± 0.50 7.06 ± 0.45 103.00 ± 0.82 1.05 ± 0.36
BUN, blood urea nitrogen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; Ca++, calcium; IP, inorganic phosphorus; Na++, sodium; K+, potassium; Cle, chloride; GGT, gamma glutamyl transpeptidase. Values are presented as the mean ± SD. * and ** indicate a significant difference at P < 0.05 and P < 0.01, respectively, when compared with the vehicle control group.
bilirubin, alkaline phosphatase (ALP), creatine kinase, calcium (Ca++), inorganic phosphorus (IP), sodium (Na+), potassium (K+), chloride (Cl−), and gamma glutamyl transpeptidase (GGT).
3. Results 3.1. HPLC analysis of YSTE In this study, HPLC analysis of eight marker components of YSTE was performed using an established HPLC analytical method. All the marker compounds, i.e., chlorogenic acid, gentiopicroside, liquiritin apioside, liquiritin, nodakenin, baicalin, wogonoside, and glycyrrhizin, were detected at 9.95, 11.26, 14.49, 14.85, 15.57, 19.13, 22.12, and 26.98 min, respectively (Fig. 1), and the amounts of these components in YSTE at 0, 1, and 4 weeks were 0.981–16.692, 0.976–16.964, and 1.021–17.853 mg/g, respectively (Table 1). These data indicate that the components of YSTE were stable during the period of administration to rats.
2.8. Statistical analyses Statistical analyses were performed essentially as described previously (Park et al., 2016b). Briefly, analyses were performed using the Path/Tox System (version 4.2.2 and 6.4.0, Xybion Medical System Co., USA). All data were analyzed using Bartlett's test for homogeneity of variance. When Bartlett's test indicated that there was no significant deviation from homogeneity, a one-way analysis of variance was performed and the significance of difference between pairs of groups was determined using Dunnett's test. When Bartlett's test indicated a significant deviation from homogeneity, the Kruskal–Wallis test was performed and the significance of differences between specific pairs of groups was determined using Dunn's Rank Sum test. P < 0.05 was considered significant. Values are presented as mean ± standard deviation (SD).
3.2. Effects of YSTE on mortality and clinical signs in rats During oral treatment with YSTE, one female and one male rat treated with 5000 mg/kg/day were found dead at day 24 (Table 2). The clinical signs, however, were not observed in the dead animals. Salivation was observed in one male rat treated with 2000 mg/kg/day at day 26 and in both male and female rats treated with 5000 mg/kg/day 6
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stress (Arantes-Rodrigues et al., 2012; Brown et al., 2000). Therefore, we consider that death from these injuries resulted from the physical process of intubation with the gavage needle catheter and was independent of YSTE toxicity. Excessive salivation and discolored urine were observed in all rats of both sexes treated with 5000 mg/kg/day YSTE. Salivation and gaping are behavioral responses associated with ingestion of a bitter substance (Matsuo et al., 2001; Yarmolinsky et al., 2009) and Gentianae Scabrae Radix, one of the major components of Yongdamsagan-tang, has a bitter taste (Han et al., 2018; Hayashi, 1976). Therefore, the salivation seen after the highest dose of YSTE was considered to be caused by its bitter taste. With regard to the discolored urine, the absolute and relative weights of the kidneys were also increased in rats treated with 5000 mg/kg/day YSTE but there were no associated changes in urinalysis, which are used as a diagnostic tool to detect urinary tract diseases, such as renal dysfunction or urinary tract infections (Rigby and Gray, 2005; Simerville et al., 2005). Because of discoloration of urine occurred in all rats treated with 5000 mg/kg/day YSTE and the characteristics of YSTE, we consider that the discolored urine was likely related to the pigment content of YSTE, not toxicologically significant change. Furthermore, studies on the subchronic toxicity against the administration of YSTE over 13 weeks should be performed to clarify the cause of these symptoms. On gross necropsy, abnormal lesions of liver tissue in which all lobes were darkened in color were observed in both male and female rats treated with 5000 mg/kg/day YSTE. Considering the frequency of these changes in both sexes, we suggest that this discoloration of the liver was also related to the deposition of YSTE pigments in hepatocytes. Nutrients and foreign bodies, as well as oxygen, are transported to the cells of the whole body through the blood. Therefore, hematological and serum biochemical parameters are important indicators of the toxicity of a substrate for target organs (Khoo et al., 2010; Larrey, 2002; Milner et al., 2003). Serum biochemistry analysis showed some changes in parameters including total bilirubin, creatinine, ALT, and ALP in male rats. Bilirubin, which is secreted in bile and urine, acts as an indicator of liver damage (Fevery, 2008). An elevated concentration of total bilirubin, the combination of direct and indirect bilirubin, indicates a high probability of a hepatic or extrahepatic disorder, whereas decreased levels of bilirubin are often harmless (Giannini et al., 2005). In this study, the decrease in average total bilirubin in male rats treated with 5000 mg/kg/day YSTE was the result of an excessive inhibition in one rat (to 0.01 mg/dL) but not the others (0.056–0.085 mg/dL) and showed little dose dependency. Creatinine, which is filtered through the kidney glomeruli and then excreted in urine, is an index used commonly to measure renal toxicity (Gowda et al., 2010; Perrone et al., 1992). Although we observed a reduction in serum creatinine in male rats treated with 5000 mg/kg/day YSTE, the results of urinalysis showed no difference in any other parameter despite the urine discoloration. Serum ALT and ALP values are used as indicators of hepatocellular injury and hepatobiliary disease, respectively (Limdi and Hyde, 2003). Although the reductions in creatinine, ALT and ALP of male rats treated with 2000 or 5000 mg/kg/day YSTE were significant, these changes were small and were not consistent between male and female rats. All these changes were in the normal ranges. Therefore, these biochemical changes were considered to be unrelated to toxicity from YSTE. The daily dose of YST is known to be 26.25 g based on dried raw herbal medicines (Hŏ, 2012) and is equivalent dose of YSTE 5.62 g when the extraction yield is 21.4%. In the present study, the human equivalent dose (HED) of YSTE applied to rats (Van Miert, 1986) is calculated to be 580 mg/kg, which is equivalent to 29% of the highest dose 2000 mg/kg without adverse event, regardless of toxicity in rats.
at all time points (Table 3). Discolored urine was observed in both male (n = 4) and female (n = 4) rats treated with 5000 mg/kg/day YSTE (Table 3). Irregular respiration and paleness were also observed in one female rat treated with 5000 mg/kg/day YSTE (Table 3). 3.3. Effects of YSTE on body weight and food intake changes in rats No significant change in body weight (Fig. 2) or food intake (Fig. 3) showed in rats of either sex treated orally with YSTE compared with the control vehicle-treated group. 3.4. Effects of YSTE on urinalysis Urinalysis of rats of both sexes showed that there were no significant differences in urine volume, glucose, specific gravity or pH in rats treated with 1000, 2000 or 5000 mg/kg/day YSTE compared with the vehicle-treated control group (Table 4). 3.5. Effects of YSTE on gross necropsy and organ-to-body weights in rats After YSTE treatment, all rats except for those that had died were subjected to gross necropsy. Rats treated with 1000 or 2000 mg/kg/day YSTE exhibited no notable changes at gross necropsy (Table 5). However, discoloration of the liver was observed in both male (n = 3) and female (n = 1) rats treated with 5000 mg/kg/day YSTE. The effects of YSTE on organ weight relative to total body weight are shown in Table 6. In male rats treated with 5000 mg/kg/day YSTE, significant increases in the relative weights of liver (1.19-fold) and kidney (1.21fold) were observed. In female rats, no significant change was observed in the relative organ weights related to oral administration of YSTE. 3.6. Effects of YSTE on hematological and biochemical parameters in rats No significant change in hematological parameters related to YSTE administration was observed in rats of either sex (Table 7). However, slight changes in some biochemical parameters were observed in YSTEtreated male but not female rats (Table 8). Compared with the vehicle control group, significant decreases in creatinine (0.84-fold; P < 0.01), total bilirubin (0.54-fold; P < 0.05) and ALP (0.72-fold; P < 0.01) were observed in rats treated with 5000 mg/kg, and ALT levels reduced 0.72-fold (P < 0.05) and 0.75-fold (P < 0.05) in rats treated with 2000 mg/kg/day and 5000 mg/kg/day YSTE, respectively. All these changes in biochemical parameters were in the normal ranges. There was no clinically significant change in the biochemical parameters of YSTE-treated rats compared with vehicle-treated controls. 4. Discussion A preliminary acute toxicity study previously conducted by our group showed that YSTE administration to rats at doses of up to 5000 mg/kg/day caused no detectable effects on the parameters tested (data not shown). Based on these results, the present study evaluated the toxicity of YSTE in rats over 4 weeks. YSTE administration to rats at doses of up to 5000 mg/kg/day for 4 weeks resulted in no major changes in mortality, body weight, food intake, or the results of serum biochemistry, hematology, or urinalysis. However, abnormalities in some rats were noted as follows. During the dosing period, one male and one female rat treated with 5000 mg/kg/day YSTE died. In the female rat, irregular respiration and paleness were observed prior to death. In the male rat, no clinical signs were observed except for excessive salivation, and there was no change in body weight or food intake. However, the autopsy findings revealed esophageal perforation and a fluid-filled thoracic cavity in both rats. It has been reported that oral gavage for substance administration involves many complications that can increase mortality, including esophageal perforation, esophageal rupture, aspiration pneumonia, and
5. Conclusion YSTE administration at doses of up to 5000 mg/kg/day for 28 days 7
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References
resulted in no notable adverse effects on mortality, body weight, food intake, urinalysis, gross necropsy, relative organ weights, or hematological and biochemical analysis. However, discolored liver and urine, and increased absolute and relative kidney weights were observed in rats treated with 5000 mg/kg/day YSTE, suggesting that these symptoms are associated with the pigments contained in YSTE. Taken together, these findings suggest that doses of up to 5000 mg/kg/day YSTE are nontoxic for rats. Moreover, further testing including the assessment of the subchronic toxicity of YSTE should be required.
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Conflicts of interest The authors declare that they have no competing interests. Authors’ contributions MYL and HH designed and conceived the study. EP and MYL participated in the data analyses and manuscript preparation. CSS carried out the preparation of YSTE, HPLC analysis, and manuscript preparation. HKS edited the draft manuscript. SCH carried out the toxicity assay. All authors have read and approved the final manuscript. Acknowledgements This research was supported through the grants ‘Construction of Scientific Evidences for Herbal Medicine Formulas (K17251)’ and ‘Construction of safety and efficacy for traditional herbal prescriptions of medicinal institution (K18241)’ from the Korea Institute of Oriental Medicine (KIOM) in South Korea. List of abbreviations ALP ALT APTT AST BUN Ca++ Cl− GGT HCT HPLC K+ IP MCH MCHC MCV Na+ SD RBC WBC YSTE
alkaline phosphatase alanine aminotransferase activated partial thromboplastin time aspartate aminotransferase blood urea nitrogen calcium chloride gamma glutamyl transpeptidase hematocrit High-performance liquid chromatography potassium inorganic phosphorus mean corpuscular hemoglobin mean corpuscular hemoglobin concentration mean corpuscular volume sodium standard deviation red blood cell white blood cell Yongdamsagan-tang water extract
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jep.2019.111852.
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