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Subchronic toxicological evaluation of Bojungikki-tang water extract: 13-Week oral repeated-dose toxicity study in Crl:CD (SD) rats
Journal of Ethnopharmacology 252 (2020) 112551
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Subchronic toxicological evaluation of Bojungikki-tang water extract: 13Week oral repeated-dose toxicity study in Crl:CD (SD) rats
T
Woo-Young Jeona, Chang-Seob Seoa, Hyekyung Haa, Hyeun-Kyoo Shina, Jae-Woo Chob, Da Hee Kimb, Mee-Young Leea,∗ a b
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea Korea Institute of Toxicology, 30 Baekhak 1-Gil, Jeongeup-si, Jeollabuk-do, 56212, Republic of Korea
ARTICLE INFO Keywords: Traditional herbal medicines Bojungikki-tang Safety evaluation Subchronic toxicity NOAEL
Ethnopharmacological relevance: Bojungikki-tang is a traditional herbal medicine used to boost immunity and reduce fatigue. However, there is not enough scientific evidence about its toxicological safety profile to support its continued clinical application. Aim of the study: The objective of this study was to investigate the subchronic toxicity profile of Bojungikki-tang water extract (BITW) in Sprague Dawley rats who were exposed to it in multiple doses and various concentrations. Materials and methods: BITW was administered to rats orally, once daily at doses of 0, 500, 1000, or 2000 mg/ kg/day for 13 weeks. We checked toxicological parameters including general observations, organ/body weights, food consumption, ophthalmological signs, hematological and serum biochemical values, urinalysis values and histopathological findings. Results: The 13 week repeated oral administration of BITW to rats at doses at doses levels of less than or equal to 2000 mg/kg/day caused no significant toxicological changes and only minor nonsignificant changes. Conclusions: Our findings indicate that administration of BITW for up to 13 weeks may be safe and nontoxic, with a no-observed-adverse-effect-level of > 2000 mg/kg/day for both male and female rats.
1. Introduction The safety of drugs is defined by their potential to cause side effects related to their administration. A preclinical evaluation of safety is a meaningful constituent of the overall testing of biological products and new drug candidates (Gautier, 2010). An essential part of the drug development process is evaluation of the organ-, dosage-, and speciesspecific toxic influences of an investigational drug. This process is principally used to calculate the no-observed-adverse-effect level (NOAEL) of the drug for clinical research and to check for specific adverse events (Parasuraman, 2011; Setzer and Kimmel, 2003). The NOAEL is usually based on in vivo studies using various protocols that differ in their use of animal model (rodent or nonrodent), exposure route (oral, inhalation, intradermal, subcutaneous, intramuscular, intravascular, or intraperitoneal), and exposure range (short or long period) (SCCS , 2012). The present study focused on investigating the toxicity, dose–response relationships, and effects on major target organs of rats during a 13-week period of repeated oral dosing with Bojungikki-tang.
Traditional herbal medicines, known as herbal remedies have been widely used for the treatment and prevention of various diseases, and to improved healthcare or give enhanced quality of life. Although herbal medicines are believed to be safe and are frequently used, there is a lack of scientific evidence concerning their safety profiles (Benzie and Wachtel-Galor, 2011). Therefore, for approval of an herbal prescription that includes pharmacologically active ingredients for clinical use, it is necessary to establish its safety profile by evaluating its toxicity through experimental data analysis (Mythilypriya et al., 2007). Bojungikki-tang (known as Bu-zhong-yi-qi-tang in China or Hochuekkito in Japan) is one of the most frequently prescribed traditional herbal medicines used in Asian countries to alleviate severe fatigue (Lee et al., 2014). Recent studies have demonstrated that Bojungikki-tang water extract (BITW) exerts a variety of pharmacological effects, including anticancer (Yu et al., 2017), immunomodulatory (Yang et al., 2015), and gastroprotective effects (Lee et al., 2012a). However, there is limited scientific evidence concerning its safety profile to support its continued therapeutic application. Our laboratory
Corresponding author. E-mail addresses: [email protected] (W.-Y. Jeon), [email protected] (C.-S. Seo), [email protected] (H. Ha), [email protected] (H.-K. Shin), [email protected] (J.-W. Cho), [email protected] (D.H. Kim), [email protected] (M.-Y. Lee). ∗
https://doi.org/10.1016/j.jep.2020.112551 Received 7 August 2019; Accepted 4 January 2020 Available online 07 January 2020 0378-8741/ © 2020 Elsevier B.V. All rights reserved.
Journal of Ethnopharmacology 252 (2020) 112551
W.-Y. Jeon, et al.
Fig. 1. Chemical structures of reference standards (A) and HPLC chromatograms of the standard mixture (B), 1 week (C), and 13 weeks (D) of Bojungikki-tang water extract at 254 nm (I), 275 nm (II), and 325 nm (III).
previously conducted an evaluation of the subacute toxicity (Yoo et al., 2017) of BITW in vivo under conditions of Good Laboratory Practice (GLP) and Organization for Economic Cooperation and Development (OECD) guidelines. The present study evaluated the potential toxicity of a 13-week repeated-dose schedule of BITW in rats, to provide additional toxicological information about BITW.
2.2. High-performance liquid chromatography (HPLC) analysis of BITW The reference standard compounds, liquiritin and glycyrrhizin, were purchased from Wako Pure Chemical Co. (Osaka, Japan). Nodakenin and hesperidin were purchased from NPC Bio Technology Inc. (Yeongi, Republic of Korea) and Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), respectively. The purity of these compounds was greater than 98.0% and their chemical structures are shown in Fig. 1A. A standard stock solution of each reference standard compound was prepared in methanol at a concentration of 1000 μg/mL and stored in the refrigerator. The conditions for HPLC analysis of BITW were described previously (Lee et al., 2012a).
2. Materials and methods 2.1. Preparation of BITW BITW was produced by Sungil Bioex Co. Ltd. (Hwaseong, Republic of Korea). Briefly, the 8 component herbal medicines of Bojungikki-tang, Astragalus membranaceus Bunge (36.885 kg), Glycyrrhiza uralensis Fischer (24.590 kg), Panax ginseng C. A. Meyer (24.590 kg), Atractylodes japonica Koidzumi (24.590 kg), Citrus unshiu Markovich (12.295 kg), Angelica gigas Nakai (12.295 kg), Cimicifuga heracleifolia Komarov (7.377 kg), and Bupleurum falcatum Linne (7.377 kg) were purchased from Kwangmyungdang Medicinal herbs Co. (Ulsan, Republic of Korea), mixed (total 150 kg), and extracted in a 10-fold mass (1500 L) of water at 100 °C for 2 h using the reflux method. The water extraction was lyophilized to obtain 47.0 kg of powder (yield: 31.3%).
2.3. Experimental animals Studies of subchronic toxicity were performed in animals according to the guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (accredited by The Association for Assessment and Accreditation of Laboratory Animal Care International, 1998) under the GLP Regulations for Non-Clinical Laboratory Studies (approval numbers: 1605–0159). Five-week-old specific pathogen-free Crl:CD Sprague Dawley rats were obtained from Orient Bio Inc. (Seongnam, Republic of Korea) and used after they were acclimatized
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Journal of Ethnopharmacology 252 (2020) 112551 101.70 98.60 96.20 101.00 0.74 0.88 0.82 1.78
2.4. Grouping and dose selection
101.60 99.00 96.70 100.90
93.71 61.63 80.31 230.50
A previous four-week repeated-dose oral toxicity study of BITW administration at a range of doses observed no toxicological effects (Yoo et al., 2017). Therefore, in the present study, the highest dose of BITW was set at 2000 mg/kg/day, the same dose used in the previous study. Animals were treated with doses of BITW ranging from a low dose of 500 mg/kg/day, a medium dose of 1000 mg/kg/day, and a high dose of 2000 mg/kg/day. The rats were divided into 4 groups (10 males and 10 females per group). BITW was administered once daily by oral gavage at 0, 500, 1000, or 2000 mg/kg for 91 days. The daily application volume (10 mL/kg) of BITW was applied based on the most recently measured body weights of the individual rats. 2.5. General observations
102.30 99.20 97.50 102.30 94.28 61.96 81.32 233.58
0.89 0.61 0.47 0.74
2.7. Necropsy
94.41 62.92 83.22 229.33 100.00 100.00 100.00 100.00
The animals were fasted overnight before blood and urine collection or necropsy. All rats were anesthetized with isoflurane inhalation and then sacrificed by exsanguination. Complete gross postmortem examinations were carried out on all rats. Absolute organ weights were measured and relative organ weights (organ weight/body weight ratios) were calculated for the following organs: brain, pituitary gland, liver, spleen, heart, thymus, salivary glands (submandibular and sublingual), seminal vesicles (with coagulation gland), prostate, kidneys, adrenal glands, testes, ovaries, epididymides, uterus (with cervix), lung and thyroids (with parathyroids) as described previously (Shin et al., 2012). 2.8. Hematology, serum biochemistry, and urinalysis Hematological analyses, serum biochemistry analyses and urinalysis were conducted as described previously (Shin et al., 2012; Jeong et al., 2015; Jeon et al., 2019). 2.9. Histopathology
102.40 100.70 99.70 100.50 1.17 0.53 1.47 0.09 0.39 0.47 0.90 0.74
% RSD (%)
95.13 63.09 82.01 228.58
Conc. (μg/mL) Conc. (μg/mL) %
92.19 62.48 83.44 228.26
Histopathological analyses were performed as described previously (Lee et al., 2012b). 2.10. Statistical analyses
Liquiritin Nodakenin Hesperidin Glycyrrhizin
RSD (%) Conc. (μg/mL)
An external eye examination with a slit lamp (XL-1, Ohira Co. Ltd., Japan) and indirect binocular ophthalmoscope (Vantage Plus Digital, Keeler Ltd., England) was performed during the last week of BITW administration, and the appearance of the conjunctiva, sclera, cornea, lens, and iris of each eye was recorded as described previously (Lee et al., 2012b; Jeong et al., 2015).
0.15 0.87 0.44 0.38
103.20 101.00 98.30 100.10
% RSD (%) 2 day
RSD (%)
Conc. (μg/mL)
2.6. Ophthalmic examination
%
The mortality, clinical signs, body weight, and food intake of rats were monitored for 91 days. The general observations were recorded as described previously (Lee et al., 2012b; Jeong et al., 2015; Jeon et al., 2019).
1 day 0 day Compound
Table 1 Stability of four compounds for 10 days in the BITW (n = 3).
for one week. The animal housing conditions were as described previously (Jeon et al., 2019).
93.65 61.86 80.69 230.31
0.85 0.45 0.74 1.62
Conc. (μg/mL) % Conc. (μg/mL)
RSD (%)
10 day 4 day
7 day
RSD (%)
%
W.-Y. Jeon, et al.
All statistical analyses were based on those described previously (Dunnett, 1964; Shin et al., 2012; Jeong et al., 2015) and were carried out using the Path/Tox System. Differences with P values < 0.05 and < 0.01 were considered significant. 3
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Table 2 Amounts of four marker components in the BITW at 0, 1 and 13 weeks by HPLC (n = 3). Compound
Liquiritin Nodakenin Hesperidin Glycyrrhizin
0 week
1 week
13 week
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
1.88 1.27 1.72 4.68
0.04 0.01 0.04 0.05
2.02 0.47 2.46 1.15
1.78 1.25 1.58 4.57
0.03 0.01 0.02 0.03
1.81 1.07 1.04 0.65
1.87 1.27 1.66 4.60
0.04 0.02 0.04 0.08
2.30 1.63 2.13 1.75
Fig. 2. Changes in body weight of rats treated with Bojungikki-tang water extract (BITW) by oral gavage at 0 (○), 500 (●), 1000 (▲), and 2000 (■) mg/kg/day for 91 days. (A) Change in body weight of male rats and (B) change in body weight of female rats. Values are expressed as means ± SD (n = 10 per group).
3. Results
hesperidin, and glycyrrhizin were detected at 17.35, 18.22, 19.14, and 34.07 min, respectively (Fig. 1B–D), and the amounts of these components at 0, 1, and 13 weeks in BITW were 1.27–4.68 mg/g, 1.25–4.57 mg/g, and 1.27–4.60 mg/g, respectively (Table 2).
3.1. HPLC analysis of BITW The stability of four marker components (liquiritin, nodakenin, hesperidin, and glycyrrhizin) of BITW was measured for 10 days (at 0, 1, 2, 4, 7, and 10 days, respectively) using the prepared sample solution. As a result, stability of all components was maintained 96.2–103.2% as compared with the initial content at 0 day (Table 1). Simultaneous analysis of four major marker components was conducted using optimized HPLC analytical conditions. Liquiritin, nodakenin,
3.2. General observations No treatment-related mortality or abnormal symptoms were observed in any of the rats treated with BITW during the study period (data not shown). Other symptoms unrelated to the administration of BITW, such as hair loss, scratch wounds, and swelling, were
Fig. 3. Accumulated food intake of rats treated with Bojungikki-tang water extract (BITW) by oral gavage at 0 (○), 500 (●), 1000 (▲), and 2000 (■) mg/kg/day for 91 days. (A) Accumulated food intake of male rats and (B) accumulated food intake of female rats. Values are expressed as means ± SD (n = 10 per group). Significant differences at #P < 0.05 and ##P < 0.01 (Dunn's rank-sum test) compared with the normal control group (0 mg/kg/day).
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Journal of Ethnopharmacology 252 (2020) 112551
3.3. Changes in body weight and cumulative food intake There was no significant BITW administration-related difference between the BITW-treated groups (up to 2000 mg/kg/day) and the normal control group for body weight (Fig. 2) or food intake (Fig. 3) of rats of either sex, except that male rats treated with BITW at 500 and 2000 mg/kg/day significantly decreased their food intake. This reduction was slight and temporary, and was considered to be an accidental change. 3.4. Ophthalmological examination There were no BITW administration-related abnormal ophthalmologic findings in rats of either sex in any group (data not shown). 3.5. Changes in relative organ weights Table 3 lists the relative organ weights calculated as % body weight (% body weight = (each organ weight (g)/fasted body weight (g) × 100)). There was no significant BITW administration-related difference between the BITW-treated and the normal control groups in the relative organ weights for rats of either sex. 3.6. Hematology and serum biochemistry The observed values of hematological parameters are presented in Table 4. There was no significant BITW administration-related difference between the BITW-treated and normal control groups for any hematological parameter in rats of either sex. As shown as in Table 5, which indicates serum biochemistry parameters, there was no significant BITW administration-related difference between rats of either sex from the BITW-treated and normal control groups for any toxicologically relevant parameter except for Na and Cl. The significant changes in Na and Cl values are slight or accidental change (not observed changes of histopathologic findings in both gender), which is considered to be toxicologically meaningless.
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; M, male; F, Female.
0.3172 ± 0.04832 0.0046 ± 0.00090 0.3304 ± 0.05435 0.0042 ± 0.00111 0.3256 ± 0.03436 0.0045 ± 0.00087
0.4276 ± 0.05377 0.0064 ± 0.00128
0.4429 ± 0.03168 0.0071 ± 0.00090 0.4605 ± 0.04030 0.0066 ± 0.00173 0.3196 ± 0.01962 0.0042 ± 0.00107
± ± ± ± 0.7830 0.0285 0.0318 0.2873 0.08969 0.00401 0.00258 0.04172 ± ± ± ± 0.7034 0.0246 0.0311 0.1833 0.07634 0.00458 0.00358 0.04667
549.3 ± 75.27 0.3880 ± 0.04990 0.0024 ± 0.00051 2.8374 ± 0.21251 0.1601 ± 0.02631 0.3005 ± 0.03296 0.0759 ± 0.01353 0.1430 ± 0.01835 0.3389 ± 0.09456 0.1126 ± 0.04053 0.7017 ± 0.09011 0.0108 ± 0.00139 0.6616 ± 0.11710 0.2997 ± 0.03568 539.8 ± 54.79 0.3988 ± 0.03501 0.0024 ± 0.00033 2.8404 ± 0.29447 0.1479 ± 0.01579 0.2960 ± 0.03385 0.0752 ± 0.01226 0.1421 ± 0.02123 0.3264 ± 0.08623 0.1151 ± 0.03153 0.7035 ± 0.09446 0.0112 ± 0.00128 0.6358 ± 0.09191 0.3028 ± 0.03306
Total body weight (g) Brain (% body weight) Pituitary gland (% body weight) Liver (% body weight) Spleen (% body weight) Heart (% body weight) Thymus (% body weight) Salivary glands (% body weight) Seminal vesicles + Coagulation gland (% body weight) Prostate (% body weight) Kidneys (% body weight) Adrenal glands (% body weight) M: Testes (% body weight), F: Ovaries (% body weight) M: Epididymides (% body weight), F: Uterus + Cervix (% body weight) Lung (% body weight) Thyroids + Parathyroids (% body weight)
570.2 ± 52.52 0.3893 ± 0.04249 0.0022 ± 0.00026 2.7842 ± 0.15407 0.1549 ± 0.01477 0.3002 ± 0.02984 0.0825 ± 0.01511 0.1348 ± 0.02031 0.3201 ± 0.05871 0.1226 ± 0.02756 0.7084 ± 0.07520 0.0108 ± 0.00200 0.6589 ± 0.06463 0.2995 ± 0.02969
± ± ± ±
observed in specific individuals, but did not correlate with the dose of BITW.
0.4538 ± 0.05941 0.0068 ± 0.00184
0.05356 0.00284 0.00728 0.11351 ± ± ± ± 0.7560 0.0278 0.0333 0.2716 0.7575 0.0282 0.0323 0.2298
0.07067 0.00405 0.00566 0.12365
290.0 ± 30.46 0.6819 ± 0.06239 0.0072 ± 0.00104 2.9647 ± 0.18403 0.1897 ± 0.03378 0.3461 ± 0.02219 0.0975 ± 0.01114 0.1733 ± 0.01591 306.7 ± 47.12 0.6546 ± 0.08681 0.0064 ± 0.00142 2.7302 ± 0.22234 0.1730 ± 0.05660 0.3406 ± 0.03965 0.1116 ± 0.03236 0.1605 ± 0.00768 281.5 ± 16.20 0.7033 ± 0.02561 0.0073 ± 0.00101 2.9303 ± 0.16470 0.1834 ± 0.03438 0.3584 ± 0.02139 0.1081 ± 0.01861 0.1657 ± 0.01295
545.2 ± 39.17 0.3986 ± 0.02879 0.0024 ± 0.00032 2.8325 ± 0.24427 0.1497 ± 0.02372 0.3050 ± 0.01964 0.0783 ± 0.00660 0.1519 ± 0.01660 0.2963 ± 0.03809 0.1002 ± 0.02486 0.6879 ± 0.05557 0.0108 ± 0.00196 0.6497 ± 0.04699 0.2910 ± 0.01998
299.3 ± 32.07 0.6479 ± 0.06621 0.0077 ± 0.00155 2.9160 ± 0.16341 0.1688 ± 0.01683 0.3513 ± 0.04178 0.1043 ± 0.02337 0.1636 ± 0.02142
1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day 2000 mg/kg/day 1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day
Female Male
Parameters BITW (mg/kg)
Table 3 Relative organ weights (% body weight) of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks.
2000 mg/kg/day
W.-Y. Jeon, et al.
3.7. Urinalysis There were no significant BITW administration-related differences between the BITW-treated and normal control groups for any urinalysis parameter in rats of either sex (Table 6). 3.8. Histopathology There were no abnormal histopathology findings for the liver (Fig. 4A–D) or kidney (Fig. 4E–H) of rats of either sex from the BITWtreated and control groups. The observed minor histopathologic changes, including anterior pituitary hyperplasia (Fig. 4I– L), were considered to be toxicologically insignificant. Considering the frequency and degree of their occurrence, they were considered to be unrelated to the administration of BITW and regarded as spontaneous findings often seen in rats of similar age. 4. Discussion The present study was performed to investigate the potential toxicity in experimental rats of 13 weeks of repeated oral administration of BITW at concentrations of 0, 500, 1000, or 2000 mg/kg/day. Our findings showed that treatment with BITW at up to 2000 mg/kg/day for 13 weeks was not associated with adverse effects on toxicologically relevant parameters in rats of either sex. The safety of long-term use and intake of herbal medicines is 5
Journal of Ethnopharmacology 252 (2020) 112551
W.-Y. Jeon, et al.
Table 4 Hematological values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks. Parameters BITW (mg/kg) 3
Male
WBC ( × 10 /μL) RBC ( × 106/μL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT ( × 103/μL) RET% (%) RETA ( × 109/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) LUC (%) NEUA ( × 103/μL) LYMA ( × 103/μL) EOSA ( × 103/μL) MONA ( × 103/μL) BASA ( × 103/μL) LUCA ( × 103/μL) PT (sec) APTT (sec)
Female
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
10.65 ± 1.941 9.27 ± 0.484 15.9 ± 0.64 50.7 ± 2.35 54.7 ± 1.03 17.2 ± 0.46 31.3 ± 0.52 976.5 ± 98.30 2.03 ± 0.283 187.4 ± 24.61 16.0 ± 7.59 78.7 ± 7.56 1.0 ± 0.32 2.7 ± 0.71 0.4 ± 0.15 1.2 ± 0.36 1.76 ± 1.041 8.33 ± 1.499 0.11 ± 0.036 0.29 ± 0.094 0.04 ± 0.016 0.13 ± 0.041 14.6 ± 0.80 16.9 ± 1.51
10.03 ± 3.132 9.44 ± 0.550 15.8 ± 0.84 50.6 ± 2.49 53.6 ± 1.69 16.8 ± 0.80 31.2 ± 0.57 973.7 ± 119.89 1.91 ± 0.375 178.7 ± 29.06 14.2 ± 3.77 80.4 ± 3.31 1.2 ± 0.42 2.8 ± 0.95 0.3 ± 0.12 1.2 ± 0.36 1.37 ± 0.412 8.11 ± 2.714 0.11 ± 0.042 0.29 ± 0.116 0.03 ± 0.013 0.12 ± 0.059 14.7 ± 1.38 17.2 ± 0.83
11.62 ± 4.197 9.31 ± 0.343 16.1 ± 0.79 51.2 ± 2.43 55.0 ± 1.34 17.3 ± 0.56 31.4 ± 0.61 1058.2 ± 117.32 2.05 ± 0.411 190.9 ± 39.01 11.4 ± 3.06 83.5 ± 3.70 1.0 ± 0.20 2.5 ± 0.89 0.3 ± 0.11 1.2 ± 0.31 1.35 ± 0.689 9.69 ± 3.485 0.12 ± 0.048 0.28 ± 0.118 0.04 ± 0.020 0.14 ± 0.047 14.7 ± 0.93 17.5 ± 1.13
11.98 ± 3.372 9.47 ± 0.500 16.3 ± 0.80 51.7 ± 2.35 54.7 ± 2.09 17.2 ± 0.70 31.4 ± 0.35 987.3 ± 97.82 1.79 ± 0.277 169.3 ± 23.70 16.0 ± 6.14 79.0 ± 6.07 1.1 ± 0.46 2.6 ± 0.92 0.3 ± 0.07 1.0 ± 0.24 1.89 ± 0.806 9.48 ± 2.847 0.13 ± 0.068 0.33 ± 0.173 0.03 ± 0.009 0.12 ± 0.039 14.8 ± 0.75 17.4 ± 0.94
7.54 ± 2.452 8.52 ± 0.161 15.5 ± 0.41 47.9 ± 1.15 56.2 ± 1.38 18.2 ± 0.41 32.4 ± 0.43 927.7 ± 100.29 1.99 ± 0.364 169.3 ± 30.01 10.5 ± 3.69 84.5 ± 3.80 1.2 ± 0.55 2.5 ± 0.48 0.3 ± 0.11 1.0 ± 0.31 0.78 ± 0.331 6.40 ± 2.242 0.08 ± 0.029 0.18 ± 0.043 0.02 ± 0.007 0.07 ± 0.021 13.7 ± 0.61 16.1 ± 1.63
7.69 ± 3.653 8.93 ± 0.620 15.6 ± 0.69 48.9 ± 2.27 54.8 ± 2.08 17.5 ± 0.90 31.9 ± 0.69 955.1 ± 125.36 1.93 ± 0.340 170.9 ± 24.79 11.0 ± 4.06 82.8 ± 4.12 1.2 ± 0.45 3.4 ± 1.35 0.3 ± 0.20 1.2 ± 0.41 0.91 ± 0.652 6.29 ± 2.730 0.09 ± 0.037 0.29 ± 0.288 0.03 ± 0.033 0.09 ± 0.041 13.2 ± 0.37 14.9 ± 1.15
7.46 ± 3.045 8.62 ± 0.329 15.5 ± 0.60 47.9 ± 2.16 55.6 ± 1.01 18.0 ± 0.36 32.4 ± 0.38 920.3 ± 116.91 2.13 ± 0.391 183.4 ± 36.78 12.0 ± 4.34 82.4 ± 4.54 1.3 ± 0.46 3.0 ± 1.04 0.3 ± 0.12 1.2 ± 0.47 0.84 ± 0.351 6.21 ± 2.866 0.09 ± 0.041 0.22 ± 0.088 0.02 ± 0.008 0.09 ± 0.039 13.4 ± 0.63 16.2 ± 0.42
7.33 ± 2.099 8.65 ± 0.384 15.6 ± 0.55 48.2 ± 1.83 55.8 ± 1.48 18.1 ± 0.48 32.4 ± 0.59 944.4 ± 89.08 2.06 ± 0.471 177.3 ± 38.88 11.6 ± 3.85 83.4 ± 3.91 1.1 ± 0.17 2.4 ± 0.71 0.4 ± 0.30 1.1 ± 0.26 0.84 ± 0.315 6.12 ± 1.818 0.08 ± 0.024 0.18 ± 0.088 0.02 ± 0.010 0.08 ± 0.037 13.7 ± 0.58 16.5 ± 1.39
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; WBC, white blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; RET%, reticulocyte count (relative) and RETA, reticulocyte count (absolute); leucocytes, differential leucocytes count (relative) and NEUA, LYMA, EOSA, MONA, BASA and LUCA, differential leucocytes count (absolute); LUC, large unstained cells; PT, Prothrombin time; APTT, activated partial thromboplastin time.
Table 5 Serum biochemical values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks. Parameters BITW (mg/kg)
Male
Female
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
GLU (mg/dL) BUN (mg/dL) CREA (mg/dL) TP (g/dL) ALB (g/dL) A/G (ratio) AST (IU/L) ALT (IU/L) TBIL (mg/dL) GGT (IU/L) ALP (IU/L) TCHO (mg/dL) TG (mg/dL) Ca (mg/dL) IP (mg/dL) K (mmol/L) CK (IU/L) PL (mg/dL) Na (mmol/L) Cl (mmol/L)
122.6 ± 26.16 16.6 ± 1.51 0.51 ± 0.042 6.80 ± 0.296 4.25 ± 0.153 1.67 ± 0.103 108.5 ± 12.79 29.6 ± 4.40 0.096 ± 0.0150 0.41 ± 0.263 272.1 ± 46.65 77.2 ± 15.35 36.0 ± 14.53 10.91 ± 0.494 9.02 ± 1.257 7.81 ± 1.064 499.5 ± 95.80 108.0 ± 17.22 142.0 ± 1.05 99.2 ± 1.55
122.7 ± 24.27 15.6 ± 2.02 0.52 ± 0.054 6.71 ± 0.341 4.24 ± 0.184 1.72 ± 0.076 112.6 ± 20.01 32.7 ± 6.26 0.111 ± 0.0119 0.47 ± 0.142 285.4 ± 43.93 77.5 ± 10.10 35.0 ± 17.94 10.94 ± 0.349 9.04 ± 0.793 7.74 ± 1.292 471.2 ± 112.19 110.7 ± 9.38 142.4 ± 1.51 99.7 ± 1.06
135.8 ± 23.43 15.6 ± 1.94 0.55 ± 0.056 7.02 ± 0.373 4.40 ± 0.175 1.68 ± 0.080 116.8 ± 14.15 31.4 ± 4.59 0.103 ± 0.0190 0.35 ± 0.159 292.7 ± 78.13 78.5 ± 14.15 38.5 ± 19.36 11.29 ± 0.429 9.22 ± 1.034 6.95 ± 1.020 520.9 ± 141.61 111.2 ± 20.35 145.3 ± 3.33# 101.0 ± 1.94*
126.3 ± 32.17 16.6 ± 1.43 0.54 ± 0.043 7.03 ± 0.342 4.38 ± 0.160 1.66 ± 0.106 116.2 ± 13.76 31.7 ± 8.07 0.094 ± 0.0110 0.24 ± 0.160 261.2 ± 48.71 85.9 ± 16.88 40.7 ± 29.76 11.12 ± 0.630 8.45 ± 1.058 7.27 ± 0.923 531.5 ± 212.45 116.5 ± 20.64 148.0 ± 1.25## 102.6 ± 1.17**
122.4 ± 23.76 20.3 ± 4.48 0.68 ± 0.106 7.66 ± 0.474 4.88 ± 0.270 1.77 ± 0.164 118.1 ± 18.04 31.2 ± 14.14 0.137 ± 0.0202 0.91 ± 0.377 169.2 ± 49.48 92.7 ± 12.20 25.8 ± 8.27 11.47 ± 0.592 7.25 ± 1.113 6.69 ± 0.883 583.3 ± 165.18 175.7 ± 21.11 145.5 ± 1.65 103.2 ± 1.32
121.9 ± 12.69 19.1 ± 3.11 0.63 ± 0.041 7.72 ± 0.555 4.97 ± 0.361 1.81 ± 0.099 127.8 ± 43.43 41.1 ± 33.44 0.164 ± 0.0407 1.21 ± 0.392 172.1 ± 42.75 79.5 ± 15.83 26.2 ± 8.45 11.58 ± 0.447 7.88 ± 1.068 6.94 ± 1.242 476.0 ± 107.87 147.9 ± 21.50 146.3 ± 1.57 102.9 ± 1.60
145.9 ± 29.50 20.3 ± 5.49 0.61 ± 0.080 7.65 ± 0.383 4.93 ± 0.228 1.82 ± 0.086 119.9 ± 19.20 31.2 ± 9.74 0.132 ± 0.0335 1.24 ± 0.403 175.3 ± 52.88 83.2 ± 16.69 23.5 ± 11.39 11.35 ± 0.277 7.16 ± 0.727 6.05 ± 1.002 501.0 ± 152.46 156.8 ± 27.06 148.0 ± 1.33** 102.6 ± 1.43
134.1 ± 23.88 17.9 ± 1.66 0.61 ± 0.049 7.70 ± 0.550 4.93 ± 0.392 1.78 ± 0.081 131.0 ± 22.84 42.1 ± 17.46 0.136 ± 0.0185 0.92 ± 0.335 148.0 ± 34.29 92.5 ± 18.53 32.0 ± 18.14 11.63 ± 0.473 7.85 ± 1.282 6.92 ± 0.697 491.6 ± 143.50 166.9 ± 28.02 148.1 ± 0.74** 103.6 ± 1.26
Values are expressed as means ± SD (n = 10 per group). Significant differences at *P < 0.05 and **P < 0.01 (Dunnett LSD test) compared with the normal control group (0 mg/kg/day), respectively. Significant differences at #P < 0.05 and ##P < 0.01 (Dunn Rank Sum Test) compared with the normal control group (0 mg/kg/day), respectively. BITW, Bojungikki-tang water extract; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine; TP, total protein; ALB, albumin; A/G, albumin/globulin ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin; GGT, gamma glutamyl transpeptidase; ALP, alkaline phosphatase; TCHO, total cholesterol; TG, triglyceride; Ca, calcium; IP, inorganic phosphorus; K, potassium; CK, creatine phosphokinase; PL, phospholipid; Na, sodium; Cl, chloride.
6
Journal of Ethnopharmacology 252 (2020) 112551
important. Most of these medicines are used for general healthcare purposes, such as enhancing immune function, and are likely to be used long-term. Therefore, it is necessary to perform appropriate preclinical toxicity assessments to select a safe dose for human health (Ismail et al., 2014). Bojungikki-tang is a traditional herb prescription that is composed of a mixture of crude extracts from eight medicinal herbs (Astragalus membranaceus Bunge, Glycyrrhiza uralensis Fischer, Panax ginseng C. A. Meyer, Atractylodes japonica Koidzumi, Citrus unshiu Markovich, Angelica gigas Nakai, Cimicifuga heracleifolia Komarov, and Bupleurum falcatum Linne). The combination of these different herbs is considered to improve their therapeutic effects by allowing herb–herb interactions while preventing potential side effects and toxic influence (Bensky and Barolet, 1990). Therefore, we performed a subchronic toxicity study of BITW in rats to determine the toxicity of BITW administration over a long period of 13 weeks and, ultimately, to establish its safety profile. General toxicity studies are classified as acute (14 days), subacute (28 days), subchronic (90 days), and chronic (6–12 months), depending on the specific experimental period (Prieto et al., 2005). Subchronic studies evaluate the potential adverse events of repeated or continuous exposure to drugs over a portion of the average life span of laboratory animals (e.g. mice, rats, and rabbits). Specifically, they provide valuable information about the toxicity of these drug preparations for the target organ and are designed to identify the NOAEL. This can provide the necessary supporting evidence to determine suitable dose regimens for long-term studies (NRC, 2006). An important requirement for toxicological studies in vivo is the ability to evaluate the therapeutic effects of medications on specific organs. Sensitive indicators are a change in body and organ weight related to the change in food intake responsible for weight gain or loss, hematological and serum biochemistry, and urinary and histopathological parameters (Fatchiyah et al., 2017). Our findings show that administration of BITW at doses up to 2000 mg/kg/ day for 13 weeks did not produce any apparent adverse effects in rats of either sex. Some minor changes (including serum Na and Cl values and histological anterior pituitary hyperplasia) were considered to be toxicologically insignificant because they appeared to be accidental changes or spontaneous changes unrelated to the administration of BITW. The NOAEL is used to classify noxious substances, and is obtained from subchronic or chronic repeated-dose toxicity studies. The NOAEL is defined as the maximum level of exposure at which there are no biologically meaningful increases in the intensity, severity or frequency of side effects between the exposed group and the appropriate control group (Lewis et al., 2002). In the present subchronic toxicity study, we determined a best-estimate NOAEL range of a dietary dose of > 2000 mg/kg/day BITW in both male and female rats.
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; SG, specific gravity; pH, hydrogen exponent; U–K, urine potassium; U–Cl, urine chloride; U–Na, urine sodium.
11.4 ± 4.01 1.016 ± 0.0039 6.7 ± 0.34 127.42 ± 42.551 41.4 ± 16.47 44.7 ± 16.45 13.2 ± 5.75 1.015 ± 0.0044 6.7 ± 0.35 104.88 ± 39.251 44.9 ± 19.27 48.9 ± 17.53 12.6 ± 4.12 1.013 ± 0.0035 6.8 ± 0.42 93.32 ± 23.580 41.5 ± 15.20 46.0 ± 18.47 13.8 ± 5.53 1.013 ± 0.0035 7.0 ± 0.16 98.88 ± 34.429 42.9 ± 10.70 49.9 ± 14.65 15.6 ± 5.72 1.010 ± 0.0037 7.0 ± 0.16 152.47 ± 66.789 48.5 ± 20.30 45.8 ± 19.66 14.8 ± 5.09 1.012 ± 0.0024 7.2 ± 0.47 152.05 ± 41.932 48.4 ± 15.06 50.9 ± 20.80 Urine volume (mL) SG pH U–K (mmol/L) U–Cl (mmol/L) U–Na (mmol/L)
16.0 ± 4.71 1.011 ± 0.0032 7.0 ± 0.16 134.43 ± 37.780 42.9 ± 14.43 42.7 ± 24.12
14.4 ± 5.40 1.013 ± 0.0035 6.9 ± 0.21 145.03 ± 37.481 42.4 ± 15.57 41.6 ± 20.04
0 mg/kg/day 1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day
2000 mg/kg/day
Female Male
Parameters BITW (mg/kg)
Table 6 Urinalysis values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks.
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
W.-Y. Jeon, et al.
5. Conclusions In conclusion, the findings of our subchronic toxicity study in rats suggested that oral administration of BITW at doses ≤2000 mg/kg/day for 13 weeks does not cause any adverse effects. Under these experimental conditions, the NOAEL of BITW was considered to be > 2000 mg/kg/day for rats of both sexes. Authors’ contributions Woo-Young Jeon, Chang-Seob Seo, Hyekyung Ha, Hyeun-Kyoo Shin and Mee-Young Lee participated in the design of the data analyses and manuscript preparation. Woo-Young Jeon, Chang-Seob Seo, Jae-Woo Cho and Da Hee Kim performed the experiments and analyzed the relevant data. All authors were involved in revising the manuscript and approved the final manuscript. 7
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Fig. 4. Representative histopathological findings in the liver, kidney, and anterior pituitary ( × 200). BITW, Bojungikki-tang water extract; M, male; F, female. (A) Male normal control group liver, (B) male 2000 mg/kg/day BITW treatment group liver, (C) female normal control group liver, (D) female 2000 mg/kg/day BITW treatment group liver, (E) male normal control group kidney, (F) male 2000 mg/kg/day BITW treatment group kidney, (G) female normal control group kidney, (H) female 2000 mg/kg/day BITW treatment group kidney, (I) male normal control group anterior pituitary, (J) male 2000 mg/kg/day BITW treatment group anterior pituitary, (K) female normal control group anterior pituitary, (L) female 2000 mg/kg/day BITW treatment group anterior pituitary. Liver, kidney, and anterior pituitary tissues were fixed, sectioned at 4 μm thickness, and stained with hematoxylin and eosin solution.
Declaration of competing interest
Altern. Med. 2014, 207613. https://doi.org/10.1155/2014/207613. Lee, M.Y., Shin, I.S., Jeon, W.Y., Seo, C.S., Ha, H., Huh, J.I., Shin, H.K., 2012a. Protective effect of Bojungikki-tang, a traditional herbal formula, against alcohol-induced gastric injury in rats. J. Ethnopharmacol. 142, 346–353. https://doi.org/10.1016/j.jep. 2012.04.043. Lee, M.Y., Shin, I.S., Seo, C.S., Kim, J.H., Han, S.R., Shin, H.K., 2012b. Subchronic oral toxicity studies of the traditional herbal formula Bangpungtongseong-san in Crl: CD (SD) rats. J. Ethnopharmacol. 144, 720–725. https://doi.org/10.1016/j.jep.2012.10. 019. Lewis, R.W., Billington, R., Debryune, E., Gamer, A., Lang, B., Carpanini, F., 2002. Recognition of adverse and nonadverse effects in toxicity studies. Toxicol. Pathol. 30, 66–74. https://doi.org/10.1080/01926230252824725. Mythilypriya, R., Shanthi, P., Sachdanandam, P., 2007. Oral acute and subacute toxicity studies with Kalpaamruthaa, a modified indigenous preparation, on rats. J. Health Sci. 53, 351–358. https://doi.org/10.1248/jhs.53.351. NRC (National Research Council), 2006. Toxicity Testing for Assessing Environmental Agents: Interim Report. The National Academies Press, Washington, DC, USA. Parasuraman, S., 2011. Toxicological screening. J. Pharmacol. Pharmacother. 2, 74–79. https://doi.org/10.4103/0976-500X.81895. Prieto, P., Clemedson, C., Meneguz, A., Pfaller, W., Sauer, U.G., Westmoreland, C., 2005. Subacute and subchronic toxicity. Alternatives to laboratory animals 33 (1), 109–116. SCCS (Scientific Committee on Consumer Safety), 2012. The SCCS's notes of guidance for the testing of cosmetics substances and their safety evaluation 8th revision. http://ec. europa.eu/health/scientific_committees/consumer_safety/docs/sccs_s_006.pdf. Setzer, R.W., Kimmel, C.A., 2003. Use of NOAEL, benchmark dose, and other models for human risk assessment of hormonally active substances. Pure Appl. Chem. 75, 2151–2158. https://doi.org/10.1351/pac200375112151. Shin, I.S., Lee, M.Y., Kim, Y., Seo, C.S., Kim, J.H., Shin, H.K., 2012. Subacute toxicity and stability of Soshiho-tang, a traditional herbal formula. In: Sprague-dawley Rats. BMC Complementary and Alternative Medicine, vol 12. pp. 266. https://doi.org/10.1186/ 1472-6882-12-266. Yang, S.H., Kao, T.I., Chiang, B.L., Chen, H.Y., Chen, K.H., Chen, J.L., 2015. Immunemodulatory effects of bu-zhong-yi-qi-tang in ovalbumin-induced murine model of allergic asthma. PLoS One 10, e0127636. https://doi.org/10.1371/journal.pone. 0127636. Yoo, S.R., Ha, H., Lee, M.Y., Shin, H.K., Han, S.C., Seo, C.S., 2017. A 4-week repeateddose oral toxicity study of bojungikgi-tang in Crl:CD Sprague Dawley rats. Evid. Based Complement Altern. Med. 2017, 4748904. https://doi.org/10.1155/2017/4748904. Yu, N., Xiong, Y., Wang, C., 2017. Bu-zhong-yi-qi decoction, the water extract of Chinese traditional herbal medicine, enhances cisplatin cytotoxicity in A549/DDP cells through induction of apoptosis and autophagy. BioMed Res. Int. 2017, 3692797. https://doi.org/10.1155/2017/3692797.
The authors declare no competing financial interests. Acknowledgments This research was supported by a Grant for “Construction of scientific evidences for herbal medicine formulas [grant number K16251]” and “Construction of safety and efficacy for traditional herbal prescriptions of medicinal institution [grant number KSN1812240]” from the Korea Institute of Oriental Medicine (KIOM). References Bensky, D., Barolet, R., 1990. Chinese Herbal Medicine: Formulas and Strategies. Eastland Press, Seattle WA. Benzie, I.F.F., Wachtel-Galor, S., 2011. Herbal Medicine: Biomolecular and Clinical Aspects, second ed. CRC Press/Taylor and Francis, Boca Raton (FL). Dunnett, C.W., 1964. New tables for multiple comparisons with control. Biometrics 20, 482–491. Fatchiyah, F., Setiawan, B., Kania, N., Ohta, T., Agustina, V., Suharjono, S., 2017. Safe dosage of caprine CSN1S2 protein from fresh local goat milk for rats evaluated by acute and sub-chronic toxicity testing. Journal of Mathematical and Fundamental Sciences 49, 90–103. https://doi.org/10.5614/j.math.fund.sci.2017.49.1.8. Gautier, J.C., 2010. Drug safety evaluation: methods and protocols. J. Pharm. Pharm. Sci. 427 Humana Press, New York, NY, ISBN: 978-1-60327-186-8. Ismail, Z., Halim, S.Z., Abdullah, N.R., Afzan, A., Abdul Rashid, B.A., Jantan, I., 2014. Safety evaluation of oral toxicity of carica papaya linn. Leaves: a subchronic toxicity study in Sprague Dawley rats. Evid. Based Complement Altern. Med. 2014, 741470. https://doi.org/10.1155/2014/741470. Jeon, W.Y., Jin, S.E., Seo, C.S., Lee, M.Y., Shin, H.K., Han, S.C., Ha, H., 2019. Safety assessment of Gyejibokryeong-hwan water extract: study of acute and subacute toxicity, and influence on drug metabolizing enzymes. J. Ethnopharmacol. 240, 111913. https://doi.org/10.1016/j.jep.2019.111913. Jeong, S.J., Huh, J.I., Shin, H.K., 2015. Cytotoxicity and sub-acute toxicity in Crl:CD (SD) rats of traditional herbal formula Ojeok-san. BMC Complement Altern. Med. 15, 38. https://doi.org/10.1186/s12906-015-0582-y. Lee, A.J., Lee, H.J., Kim, J.D., Jung, H.J., Bae, S.H., Ryoo, H.M., Kim, S.G., 2014. Changes of peripheral blood lymphocyte subtypes in patients with end stage cancer administered localized radiotherapy and Bojungikki-tang. Evid. Based Complement
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Subchronic toxicological evaluation of Bojungikki-tang water extract: 13Week oral repeated-dose toxicity study in Crl:CD (SD) rats
T
Woo-Young Jeona, Chang-Seob Seoa, Hyekyung Haa, Hyeun-Kyoo Shina, Jae-Woo Chob, Da Hee Kimb, Mee-Young Leea,∗ a b
Herbal Medicine Research Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, Yuseong-gu, Daejeon, 34054, Republic of Korea Korea Institute of Toxicology, 30 Baekhak 1-Gil, Jeongeup-si, Jeollabuk-do, 56212, Republic of Korea
ARTICLE INFO Keywords: Traditional herbal medicines Bojungikki-tang Safety evaluation Subchronic toxicity NOAEL
Ethnopharmacological relevance: Bojungikki-tang is a traditional herbal medicine used to boost immunity and reduce fatigue. However, there is not enough scientific evidence about its toxicological safety profile to support its continued clinical application. Aim of the study: The objective of this study was to investigate the subchronic toxicity profile of Bojungikki-tang water extract (BITW) in Sprague Dawley rats who were exposed to it in multiple doses and various concentrations. Materials and methods: BITW was administered to rats orally, once daily at doses of 0, 500, 1000, or 2000 mg/ kg/day for 13 weeks. We checked toxicological parameters including general observations, organ/body weights, food consumption, ophthalmological signs, hematological and serum biochemical values, urinalysis values and histopathological findings. Results: The 13 week repeated oral administration of BITW to rats at doses at doses levels of less than or equal to 2000 mg/kg/day caused no significant toxicological changes and only minor nonsignificant changes. Conclusions: Our findings indicate that administration of BITW for up to 13 weeks may be safe and nontoxic, with a no-observed-adverse-effect-level of > 2000 mg/kg/day for both male and female rats.
1. Introduction The safety of drugs is defined by their potential to cause side effects related to their administration. A preclinical evaluation of safety is a meaningful constituent of the overall testing of biological products and new drug candidates (Gautier, 2010). An essential part of the drug development process is evaluation of the organ-, dosage-, and speciesspecific toxic influences of an investigational drug. This process is principally used to calculate the no-observed-adverse-effect level (NOAEL) of the drug for clinical research and to check for specific adverse events (Parasuraman, 2011; Setzer and Kimmel, 2003). The NOAEL is usually based on in vivo studies using various protocols that differ in their use of animal model (rodent or nonrodent), exposure route (oral, inhalation, intradermal, subcutaneous, intramuscular, intravascular, or intraperitoneal), and exposure range (short or long period) (SCCS , 2012). The present study focused on investigating the toxicity, dose–response relationships, and effects on major target organs of rats during a 13-week period of repeated oral dosing with Bojungikki-tang.
Traditional herbal medicines, known as herbal remedies have been widely used for the treatment and prevention of various diseases, and to improved healthcare or give enhanced quality of life. Although herbal medicines are believed to be safe and are frequently used, there is a lack of scientific evidence concerning their safety profiles (Benzie and Wachtel-Galor, 2011). Therefore, for approval of an herbal prescription that includes pharmacologically active ingredients for clinical use, it is necessary to establish its safety profile by evaluating its toxicity through experimental data analysis (Mythilypriya et al., 2007). Bojungikki-tang (known as Bu-zhong-yi-qi-tang in China or Hochuekkito in Japan) is one of the most frequently prescribed traditional herbal medicines used in Asian countries to alleviate severe fatigue (Lee et al., 2014). Recent studies have demonstrated that Bojungikki-tang water extract (BITW) exerts a variety of pharmacological effects, including anticancer (Yu et al., 2017), immunomodulatory (Yang et al., 2015), and gastroprotective effects (Lee et al., 2012a). However, there is limited scientific evidence concerning its safety profile to support its continued therapeutic application. Our laboratory
Corresponding author. E-mail addresses: [email protected] (W.-Y. Jeon), [email protected] (C.-S. Seo), [email protected] (H. Ha), [email protected] (H.-K. Shin), [email protected] (J.-W. Cho), [email protected] (D.H. Kim), [email protected] (M.-Y. Lee). ∗
https://doi.org/10.1016/j.jep.2020.112551 Received 7 August 2019; Accepted 4 January 2020 Available online 07 January 2020 0378-8741/ © 2020 Elsevier B.V. All rights reserved.
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Fig. 1. Chemical structures of reference standards (A) and HPLC chromatograms of the standard mixture (B), 1 week (C), and 13 weeks (D) of Bojungikki-tang water extract at 254 nm (I), 275 nm (II), and 325 nm (III).
previously conducted an evaluation of the subacute toxicity (Yoo et al., 2017) of BITW in vivo under conditions of Good Laboratory Practice (GLP) and Organization for Economic Cooperation and Development (OECD) guidelines. The present study evaluated the potential toxicity of a 13-week repeated-dose schedule of BITW in rats, to provide additional toxicological information about BITW.
2.2. High-performance liquid chromatography (HPLC) analysis of BITW The reference standard compounds, liquiritin and glycyrrhizin, were purchased from Wako Pure Chemical Co. (Osaka, Japan). Nodakenin and hesperidin were purchased from NPC Bio Technology Inc. (Yeongi, Republic of Korea) and Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), respectively. The purity of these compounds was greater than 98.0% and their chemical structures are shown in Fig. 1A. A standard stock solution of each reference standard compound was prepared in methanol at a concentration of 1000 μg/mL and stored in the refrigerator. The conditions for HPLC analysis of BITW were described previously (Lee et al., 2012a).
2. Materials and methods 2.1. Preparation of BITW BITW was produced by Sungil Bioex Co. Ltd. (Hwaseong, Republic of Korea). Briefly, the 8 component herbal medicines of Bojungikki-tang, Astragalus membranaceus Bunge (36.885 kg), Glycyrrhiza uralensis Fischer (24.590 kg), Panax ginseng C. A. Meyer (24.590 kg), Atractylodes japonica Koidzumi (24.590 kg), Citrus unshiu Markovich (12.295 kg), Angelica gigas Nakai (12.295 kg), Cimicifuga heracleifolia Komarov (7.377 kg), and Bupleurum falcatum Linne (7.377 kg) were purchased from Kwangmyungdang Medicinal herbs Co. (Ulsan, Republic of Korea), mixed (total 150 kg), and extracted in a 10-fold mass (1500 L) of water at 100 °C for 2 h using the reflux method. The water extraction was lyophilized to obtain 47.0 kg of powder (yield: 31.3%).
2.3. Experimental animals Studies of subchronic toxicity were performed in animals according to the guidelines of the Institutional Animal Care and Use Committee of the Korea Institute of Toxicology (accredited by The Association for Assessment and Accreditation of Laboratory Animal Care International, 1998) under the GLP Regulations for Non-Clinical Laboratory Studies (approval numbers: 1605–0159). Five-week-old specific pathogen-free Crl:CD Sprague Dawley rats were obtained from Orient Bio Inc. (Seongnam, Republic of Korea) and used after they were acclimatized
2
Journal of Ethnopharmacology 252 (2020) 112551 101.70 98.60 96.20 101.00 0.74 0.88 0.82 1.78
2.4. Grouping and dose selection
101.60 99.00 96.70 100.90
93.71 61.63 80.31 230.50
A previous four-week repeated-dose oral toxicity study of BITW administration at a range of doses observed no toxicological effects (Yoo et al., 2017). Therefore, in the present study, the highest dose of BITW was set at 2000 mg/kg/day, the same dose used in the previous study. Animals were treated with doses of BITW ranging from a low dose of 500 mg/kg/day, a medium dose of 1000 mg/kg/day, and a high dose of 2000 mg/kg/day. The rats were divided into 4 groups (10 males and 10 females per group). BITW was administered once daily by oral gavage at 0, 500, 1000, or 2000 mg/kg for 91 days. The daily application volume (10 mL/kg) of BITW was applied based on the most recently measured body weights of the individual rats. 2.5. General observations
102.30 99.20 97.50 102.30 94.28 61.96 81.32 233.58
0.89 0.61 0.47 0.74
2.7. Necropsy
94.41 62.92 83.22 229.33 100.00 100.00 100.00 100.00
The animals were fasted overnight before blood and urine collection or necropsy. All rats were anesthetized with isoflurane inhalation and then sacrificed by exsanguination. Complete gross postmortem examinations were carried out on all rats. Absolute organ weights were measured and relative organ weights (organ weight/body weight ratios) were calculated for the following organs: brain, pituitary gland, liver, spleen, heart, thymus, salivary glands (submandibular and sublingual), seminal vesicles (with coagulation gland), prostate, kidneys, adrenal glands, testes, ovaries, epididymides, uterus (with cervix), lung and thyroids (with parathyroids) as described previously (Shin et al., 2012). 2.8. Hematology, serum biochemistry, and urinalysis Hematological analyses, serum biochemistry analyses and urinalysis were conducted as described previously (Shin et al., 2012; Jeong et al., 2015; Jeon et al., 2019). 2.9. Histopathology
102.40 100.70 99.70 100.50 1.17 0.53 1.47 0.09 0.39 0.47 0.90 0.74
% RSD (%)
95.13 63.09 82.01 228.58
Conc. (μg/mL) Conc. (μg/mL) %
92.19 62.48 83.44 228.26
Histopathological analyses were performed as described previously (Lee et al., 2012b). 2.10. Statistical analyses
Liquiritin Nodakenin Hesperidin Glycyrrhizin
RSD (%) Conc. (μg/mL)
An external eye examination with a slit lamp (XL-1, Ohira Co. Ltd., Japan) and indirect binocular ophthalmoscope (Vantage Plus Digital, Keeler Ltd., England) was performed during the last week of BITW administration, and the appearance of the conjunctiva, sclera, cornea, lens, and iris of each eye was recorded as described previously (Lee et al., 2012b; Jeong et al., 2015).
0.15 0.87 0.44 0.38
103.20 101.00 98.30 100.10
% RSD (%) 2 day
RSD (%)
Conc. (μg/mL)
2.6. Ophthalmic examination
%
The mortality, clinical signs, body weight, and food intake of rats were monitored for 91 days. The general observations were recorded as described previously (Lee et al., 2012b; Jeong et al., 2015; Jeon et al., 2019).
1 day 0 day Compound
Table 1 Stability of four compounds for 10 days in the BITW (n = 3).
for one week. The animal housing conditions were as described previously (Jeon et al., 2019).
93.65 61.86 80.69 230.31
0.85 0.45 0.74 1.62
Conc. (μg/mL) % Conc. (μg/mL)
RSD (%)
10 day 4 day
7 day
RSD (%)
%
W.-Y. Jeon, et al.
All statistical analyses were based on those described previously (Dunnett, 1964; Shin et al., 2012; Jeong et al., 2015) and were carried out using the Path/Tox System. Differences with P values < 0.05 and < 0.01 were considered significant. 3
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Table 2 Amounts of four marker components in the BITW at 0, 1 and 13 weeks by HPLC (n = 3). Compound
Liquiritin Nodakenin Hesperidin Glycyrrhizin
0 week
1 week
13 week
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
Mean (mg/g)
SD
RSD (%)
1.88 1.27 1.72 4.68
0.04 0.01 0.04 0.05
2.02 0.47 2.46 1.15
1.78 1.25 1.58 4.57
0.03 0.01 0.02 0.03
1.81 1.07 1.04 0.65
1.87 1.27 1.66 4.60
0.04 0.02 0.04 0.08
2.30 1.63 2.13 1.75
Fig. 2. Changes in body weight of rats treated with Bojungikki-tang water extract (BITW) by oral gavage at 0 (○), 500 (●), 1000 (▲), and 2000 (■) mg/kg/day for 91 days. (A) Change in body weight of male rats and (B) change in body weight of female rats. Values are expressed as means ± SD (n = 10 per group).
3. Results
hesperidin, and glycyrrhizin were detected at 17.35, 18.22, 19.14, and 34.07 min, respectively (Fig. 1B–D), and the amounts of these components at 0, 1, and 13 weeks in BITW were 1.27–4.68 mg/g, 1.25–4.57 mg/g, and 1.27–4.60 mg/g, respectively (Table 2).
3.1. HPLC analysis of BITW The stability of four marker components (liquiritin, nodakenin, hesperidin, and glycyrrhizin) of BITW was measured for 10 days (at 0, 1, 2, 4, 7, and 10 days, respectively) using the prepared sample solution. As a result, stability of all components was maintained 96.2–103.2% as compared with the initial content at 0 day (Table 1). Simultaneous analysis of four major marker components was conducted using optimized HPLC analytical conditions. Liquiritin, nodakenin,
3.2. General observations No treatment-related mortality or abnormal symptoms were observed in any of the rats treated with BITW during the study period (data not shown). Other symptoms unrelated to the administration of BITW, such as hair loss, scratch wounds, and swelling, were
Fig. 3. Accumulated food intake of rats treated with Bojungikki-tang water extract (BITW) by oral gavage at 0 (○), 500 (●), 1000 (▲), and 2000 (■) mg/kg/day for 91 days. (A) Accumulated food intake of male rats and (B) accumulated food intake of female rats. Values are expressed as means ± SD (n = 10 per group). Significant differences at #P < 0.05 and ##P < 0.01 (Dunn's rank-sum test) compared with the normal control group (0 mg/kg/day).
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Journal of Ethnopharmacology 252 (2020) 112551
3.3. Changes in body weight and cumulative food intake There was no significant BITW administration-related difference between the BITW-treated groups (up to 2000 mg/kg/day) and the normal control group for body weight (Fig. 2) or food intake (Fig. 3) of rats of either sex, except that male rats treated with BITW at 500 and 2000 mg/kg/day significantly decreased their food intake. This reduction was slight and temporary, and was considered to be an accidental change. 3.4. Ophthalmological examination There were no BITW administration-related abnormal ophthalmologic findings in rats of either sex in any group (data not shown). 3.5. Changes in relative organ weights Table 3 lists the relative organ weights calculated as % body weight (% body weight = (each organ weight (g)/fasted body weight (g) × 100)). There was no significant BITW administration-related difference between the BITW-treated and the normal control groups in the relative organ weights for rats of either sex. 3.6. Hematology and serum biochemistry The observed values of hematological parameters are presented in Table 4. There was no significant BITW administration-related difference between the BITW-treated and normal control groups for any hematological parameter in rats of either sex. As shown as in Table 5, which indicates serum biochemistry parameters, there was no significant BITW administration-related difference between rats of either sex from the BITW-treated and normal control groups for any toxicologically relevant parameter except for Na and Cl. The significant changes in Na and Cl values are slight or accidental change (not observed changes of histopathologic findings in both gender), which is considered to be toxicologically meaningless.
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; M, male; F, Female.
0.3172 ± 0.04832 0.0046 ± 0.00090 0.3304 ± 0.05435 0.0042 ± 0.00111 0.3256 ± 0.03436 0.0045 ± 0.00087
0.4276 ± 0.05377 0.0064 ± 0.00128
0.4429 ± 0.03168 0.0071 ± 0.00090 0.4605 ± 0.04030 0.0066 ± 0.00173 0.3196 ± 0.01962 0.0042 ± 0.00107
± ± ± ± 0.7830 0.0285 0.0318 0.2873 0.08969 0.00401 0.00258 0.04172 ± ± ± ± 0.7034 0.0246 0.0311 0.1833 0.07634 0.00458 0.00358 0.04667
549.3 ± 75.27 0.3880 ± 0.04990 0.0024 ± 0.00051 2.8374 ± 0.21251 0.1601 ± 0.02631 0.3005 ± 0.03296 0.0759 ± 0.01353 0.1430 ± 0.01835 0.3389 ± 0.09456 0.1126 ± 0.04053 0.7017 ± 0.09011 0.0108 ± 0.00139 0.6616 ± 0.11710 0.2997 ± 0.03568 539.8 ± 54.79 0.3988 ± 0.03501 0.0024 ± 0.00033 2.8404 ± 0.29447 0.1479 ± 0.01579 0.2960 ± 0.03385 0.0752 ± 0.01226 0.1421 ± 0.02123 0.3264 ± 0.08623 0.1151 ± 0.03153 0.7035 ± 0.09446 0.0112 ± 0.00128 0.6358 ± 0.09191 0.3028 ± 0.03306
Total body weight (g) Brain (% body weight) Pituitary gland (% body weight) Liver (% body weight) Spleen (% body weight) Heart (% body weight) Thymus (% body weight) Salivary glands (% body weight) Seminal vesicles + Coagulation gland (% body weight) Prostate (% body weight) Kidneys (% body weight) Adrenal glands (% body weight) M: Testes (% body weight), F: Ovaries (% body weight) M: Epididymides (% body weight), F: Uterus + Cervix (% body weight) Lung (% body weight) Thyroids + Parathyroids (% body weight)
570.2 ± 52.52 0.3893 ± 0.04249 0.0022 ± 0.00026 2.7842 ± 0.15407 0.1549 ± 0.01477 0.3002 ± 0.02984 0.0825 ± 0.01511 0.1348 ± 0.02031 0.3201 ± 0.05871 0.1226 ± 0.02756 0.7084 ± 0.07520 0.0108 ± 0.00200 0.6589 ± 0.06463 0.2995 ± 0.02969
± ± ± ±
observed in specific individuals, but did not correlate with the dose of BITW.
0.4538 ± 0.05941 0.0068 ± 0.00184
0.05356 0.00284 0.00728 0.11351 ± ± ± ± 0.7560 0.0278 0.0333 0.2716 0.7575 0.0282 0.0323 0.2298
0.07067 0.00405 0.00566 0.12365
290.0 ± 30.46 0.6819 ± 0.06239 0.0072 ± 0.00104 2.9647 ± 0.18403 0.1897 ± 0.03378 0.3461 ± 0.02219 0.0975 ± 0.01114 0.1733 ± 0.01591 306.7 ± 47.12 0.6546 ± 0.08681 0.0064 ± 0.00142 2.7302 ± 0.22234 0.1730 ± 0.05660 0.3406 ± 0.03965 0.1116 ± 0.03236 0.1605 ± 0.00768 281.5 ± 16.20 0.7033 ± 0.02561 0.0073 ± 0.00101 2.9303 ± 0.16470 0.1834 ± 0.03438 0.3584 ± 0.02139 0.1081 ± 0.01861 0.1657 ± 0.01295
545.2 ± 39.17 0.3986 ± 0.02879 0.0024 ± 0.00032 2.8325 ± 0.24427 0.1497 ± 0.02372 0.3050 ± 0.01964 0.0783 ± 0.00660 0.1519 ± 0.01660 0.2963 ± 0.03809 0.1002 ± 0.02486 0.6879 ± 0.05557 0.0108 ± 0.00196 0.6497 ± 0.04699 0.2910 ± 0.01998
299.3 ± 32.07 0.6479 ± 0.06621 0.0077 ± 0.00155 2.9160 ± 0.16341 0.1688 ± 0.01683 0.3513 ± 0.04178 0.1043 ± 0.02337 0.1636 ± 0.02142
1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day 2000 mg/kg/day 1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day
Female Male
Parameters BITW (mg/kg)
Table 3 Relative organ weights (% body weight) of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks.
2000 mg/kg/day
W.-Y. Jeon, et al.
3.7. Urinalysis There were no significant BITW administration-related differences between the BITW-treated and normal control groups for any urinalysis parameter in rats of either sex (Table 6). 3.8. Histopathology There were no abnormal histopathology findings for the liver (Fig. 4A–D) or kidney (Fig. 4E–H) of rats of either sex from the BITWtreated and control groups. The observed minor histopathologic changes, including anterior pituitary hyperplasia (Fig. 4I– L), were considered to be toxicologically insignificant. Considering the frequency and degree of their occurrence, they were considered to be unrelated to the administration of BITW and regarded as spontaneous findings often seen in rats of similar age. 4. Discussion The present study was performed to investigate the potential toxicity in experimental rats of 13 weeks of repeated oral administration of BITW at concentrations of 0, 500, 1000, or 2000 mg/kg/day. Our findings showed that treatment with BITW at up to 2000 mg/kg/day for 13 weeks was not associated with adverse effects on toxicologically relevant parameters in rats of either sex. The safety of long-term use and intake of herbal medicines is 5
Journal of Ethnopharmacology 252 (2020) 112551
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Table 4 Hematological values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks. Parameters BITW (mg/kg) 3
Male
WBC ( × 10 /μL) RBC ( × 106/μL) HGB (g/dL) HCT (%) MCV (fL) MCH (pg) MCHC (g/dL) PLT ( × 103/μL) RET% (%) RETA ( × 109/μL) Neutrophils (%) Lymphocytes (%) Eosinophils (%) Monocytes (%) Basophils (%) LUC (%) NEUA ( × 103/μL) LYMA ( × 103/μL) EOSA ( × 103/μL) MONA ( × 103/μL) BASA ( × 103/μL) LUCA ( × 103/μL) PT (sec) APTT (sec)
Female
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
10.65 ± 1.941 9.27 ± 0.484 15.9 ± 0.64 50.7 ± 2.35 54.7 ± 1.03 17.2 ± 0.46 31.3 ± 0.52 976.5 ± 98.30 2.03 ± 0.283 187.4 ± 24.61 16.0 ± 7.59 78.7 ± 7.56 1.0 ± 0.32 2.7 ± 0.71 0.4 ± 0.15 1.2 ± 0.36 1.76 ± 1.041 8.33 ± 1.499 0.11 ± 0.036 0.29 ± 0.094 0.04 ± 0.016 0.13 ± 0.041 14.6 ± 0.80 16.9 ± 1.51
10.03 ± 3.132 9.44 ± 0.550 15.8 ± 0.84 50.6 ± 2.49 53.6 ± 1.69 16.8 ± 0.80 31.2 ± 0.57 973.7 ± 119.89 1.91 ± 0.375 178.7 ± 29.06 14.2 ± 3.77 80.4 ± 3.31 1.2 ± 0.42 2.8 ± 0.95 0.3 ± 0.12 1.2 ± 0.36 1.37 ± 0.412 8.11 ± 2.714 0.11 ± 0.042 0.29 ± 0.116 0.03 ± 0.013 0.12 ± 0.059 14.7 ± 1.38 17.2 ± 0.83
11.62 ± 4.197 9.31 ± 0.343 16.1 ± 0.79 51.2 ± 2.43 55.0 ± 1.34 17.3 ± 0.56 31.4 ± 0.61 1058.2 ± 117.32 2.05 ± 0.411 190.9 ± 39.01 11.4 ± 3.06 83.5 ± 3.70 1.0 ± 0.20 2.5 ± 0.89 0.3 ± 0.11 1.2 ± 0.31 1.35 ± 0.689 9.69 ± 3.485 0.12 ± 0.048 0.28 ± 0.118 0.04 ± 0.020 0.14 ± 0.047 14.7 ± 0.93 17.5 ± 1.13
11.98 ± 3.372 9.47 ± 0.500 16.3 ± 0.80 51.7 ± 2.35 54.7 ± 2.09 17.2 ± 0.70 31.4 ± 0.35 987.3 ± 97.82 1.79 ± 0.277 169.3 ± 23.70 16.0 ± 6.14 79.0 ± 6.07 1.1 ± 0.46 2.6 ± 0.92 0.3 ± 0.07 1.0 ± 0.24 1.89 ± 0.806 9.48 ± 2.847 0.13 ± 0.068 0.33 ± 0.173 0.03 ± 0.009 0.12 ± 0.039 14.8 ± 0.75 17.4 ± 0.94
7.54 ± 2.452 8.52 ± 0.161 15.5 ± 0.41 47.9 ± 1.15 56.2 ± 1.38 18.2 ± 0.41 32.4 ± 0.43 927.7 ± 100.29 1.99 ± 0.364 169.3 ± 30.01 10.5 ± 3.69 84.5 ± 3.80 1.2 ± 0.55 2.5 ± 0.48 0.3 ± 0.11 1.0 ± 0.31 0.78 ± 0.331 6.40 ± 2.242 0.08 ± 0.029 0.18 ± 0.043 0.02 ± 0.007 0.07 ± 0.021 13.7 ± 0.61 16.1 ± 1.63
7.69 ± 3.653 8.93 ± 0.620 15.6 ± 0.69 48.9 ± 2.27 54.8 ± 2.08 17.5 ± 0.90 31.9 ± 0.69 955.1 ± 125.36 1.93 ± 0.340 170.9 ± 24.79 11.0 ± 4.06 82.8 ± 4.12 1.2 ± 0.45 3.4 ± 1.35 0.3 ± 0.20 1.2 ± 0.41 0.91 ± 0.652 6.29 ± 2.730 0.09 ± 0.037 0.29 ± 0.288 0.03 ± 0.033 0.09 ± 0.041 13.2 ± 0.37 14.9 ± 1.15
7.46 ± 3.045 8.62 ± 0.329 15.5 ± 0.60 47.9 ± 2.16 55.6 ± 1.01 18.0 ± 0.36 32.4 ± 0.38 920.3 ± 116.91 2.13 ± 0.391 183.4 ± 36.78 12.0 ± 4.34 82.4 ± 4.54 1.3 ± 0.46 3.0 ± 1.04 0.3 ± 0.12 1.2 ± 0.47 0.84 ± 0.351 6.21 ± 2.866 0.09 ± 0.041 0.22 ± 0.088 0.02 ± 0.008 0.09 ± 0.039 13.4 ± 0.63 16.2 ± 0.42
7.33 ± 2.099 8.65 ± 0.384 15.6 ± 0.55 48.2 ± 1.83 55.8 ± 1.48 18.1 ± 0.48 32.4 ± 0.59 944.4 ± 89.08 2.06 ± 0.471 177.3 ± 38.88 11.6 ± 3.85 83.4 ± 3.91 1.1 ± 0.17 2.4 ± 0.71 0.4 ± 0.30 1.1 ± 0.26 0.84 ± 0.315 6.12 ± 1.818 0.08 ± 0.024 0.18 ± 0.088 0.02 ± 0.010 0.08 ± 0.037 13.7 ± 0.58 16.5 ± 1.39
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; WBC, white blood cell count; RBC, red blood cell count; HGB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; RET%, reticulocyte count (relative) and RETA, reticulocyte count (absolute); leucocytes, differential leucocytes count (relative) and NEUA, LYMA, EOSA, MONA, BASA and LUCA, differential leucocytes count (absolute); LUC, large unstained cells; PT, Prothrombin time; APTT, activated partial thromboplastin time.
Table 5 Serum biochemical values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks. Parameters BITW (mg/kg)
Male
Female
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
0 mg/kg/day
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
GLU (mg/dL) BUN (mg/dL) CREA (mg/dL) TP (g/dL) ALB (g/dL) A/G (ratio) AST (IU/L) ALT (IU/L) TBIL (mg/dL) GGT (IU/L) ALP (IU/L) TCHO (mg/dL) TG (mg/dL) Ca (mg/dL) IP (mg/dL) K (mmol/L) CK (IU/L) PL (mg/dL) Na (mmol/L) Cl (mmol/L)
122.6 ± 26.16 16.6 ± 1.51 0.51 ± 0.042 6.80 ± 0.296 4.25 ± 0.153 1.67 ± 0.103 108.5 ± 12.79 29.6 ± 4.40 0.096 ± 0.0150 0.41 ± 0.263 272.1 ± 46.65 77.2 ± 15.35 36.0 ± 14.53 10.91 ± 0.494 9.02 ± 1.257 7.81 ± 1.064 499.5 ± 95.80 108.0 ± 17.22 142.0 ± 1.05 99.2 ± 1.55
122.7 ± 24.27 15.6 ± 2.02 0.52 ± 0.054 6.71 ± 0.341 4.24 ± 0.184 1.72 ± 0.076 112.6 ± 20.01 32.7 ± 6.26 0.111 ± 0.0119 0.47 ± 0.142 285.4 ± 43.93 77.5 ± 10.10 35.0 ± 17.94 10.94 ± 0.349 9.04 ± 0.793 7.74 ± 1.292 471.2 ± 112.19 110.7 ± 9.38 142.4 ± 1.51 99.7 ± 1.06
135.8 ± 23.43 15.6 ± 1.94 0.55 ± 0.056 7.02 ± 0.373 4.40 ± 0.175 1.68 ± 0.080 116.8 ± 14.15 31.4 ± 4.59 0.103 ± 0.0190 0.35 ± 0.159 292.7 ± 78.13 78.5 ± 14.15 38.5 ± 19.36 11.29 ± 0.429 9.22 ± 1.034 6.95 ± 1.020 520.9 ± 141.61 111.2 ± 20.35 145.3 ± 3.33# 101.0 ± 1.94*
126.3 ± 32.17 16.6 ± 1.43 0.54 ± 0.043 7.03 ± 0.342 4.38 ± 0.160 1.66 ± 0.106 116.2 ± 13.76 31.7 ± 8.07 0.094 ± 0.0110 0.24 ± 0.160 261.2 ± 48.71 85.9 ± 16.88 40.7 ± 29.76 11.12 ± 0.630 8.45 ± 1.058 7.27 ± 0.923 531.5 ± 212.45 116.5 ± 20.64 148.0 ± 1.25## 102.6 ± 1.17**
122.4 ± 23.76 20.3 ± 4.48 0.68 ± 0.106 7.66 ± 0.474 4.88 ± 0.270 1.77 ± 0.164 118.1 ± 18.04 31.2 ± 14.14 0.137 ± 0.0202 0.91 ± 0.377 169.2 ± 49.48 92.7 ± 12.20 25.8 ± 8.27 11.47 ± 0.592 7.25 ± 1.113 6.69 ± 0.883 583.3 ± 165.18 175.7 ± 21.11 145.5 ± 1.65 103.2 ± 1.32
121.9 ± 12.69 19.1 ± 3.11 0.63 ± 0.041 7.72 ± 0.555 4.97 ± 0.361 1.81 ± 0.099 127.8 ± 43.43 41.1 ± 33.44 0.164 ± 0.0407 1.21 ± 0.392 172.1 ± 42.75 79.5 ± 15.83 26.2 ± 8.45 11.58 ± 0.447 7.88 ± 1.068 6.94 ± 1.242 476.0 ± 107.87 147.9 ± 21.50 146.3 ± 1.57 102.9 ± 1.60
145.9 ± 29.50 20.3 ± 5.49 0.61 ± 0.080 7.65 ± 0.383 4.93 ± 0.228 1.82 ± 0.086 119.9 ± 19.20 31.2 ± 9.74 0.132 ± 0.0335 1.24 ± 0.403 175.3 ± 52.88 83.2 ± 16.69 23.5 ± 11.39 11.35 ± 0.277 7.16 ± 0.727 6.05 ± 1.002 501.0 ± 152.46 156.8 ± 27.06 148.0 ± 1.33** 102.6 ± 1.43
134.1 ± 23.88 17.9 ± 1.66 0.61 ± 0.049 7.70 ± 0.550 4.93 ± 0.392 1.78 ± 0.081 131.0 ± 22.84 42.1 ± 17.46 0.136 ± 0.0185 0.92 ± 0.335 148.0 ± 34.29 92.5 ± 18.53 32.0 ± 18.14 11.63 ± 0.473 7.85 ± 1.282 6.92 ± 0.697 491.6 ± 143.50 166.9 ± 28.02 148.1 ± 0.74** 103.6 ± 1.26
Values are expressed as means ± SD (n = 10 per group). Significant differences at *P < 0.05 and **P < 0.01 (Dunnett LSD test) compared with the normal control group (0 mg/kg/day), respectively. Significant differences at #P < 0.05 and ##P < 0.01 (Dunn Rank Sum Test) compared with the normal control group (0 mg/kg/day), respectively. BITW, Bojungikki-tang water extract; GLU, glucose; BUN, blood urea nitrogen; CREA, creatinine; TP, total protein; ALB, albumin; A/G, albumin/globulin ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin; GGT, gamma glutamyl transpeptidase; ALP, alkaline phosphatase; TCHO, total cholesterol; TG, triglyceride; Ca, calcium; IP, inorganic phosphorus; K, potassium; CK, creatine phosphokinase; PL, phospholipid; Na, sodium; Cl, chloride.
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Journal of Ethnopharmacology 252 (2020) 112551
important. Most of these medicines are used for general healthcare purposes, such as enhancing immune function, and are likely to be used long-term. Therefore, it is necessary to perform appropriate preclinical toxicity assessments to select a safe dose for human health (Ismail et al., 2014). Bojungikki-tang is a traditional herb prescription that is composed of a mixture of crude extracts from eight medicinal herbs (Astragalus membranaceus Bunge, Glycyrrhiza uralensis Fischer, Panax ginseng C. A. Meyer, Atractylodes japonica Koidzumi, Citrus unshiu Markovich, Angelica gigas Nakai, Cimicifuga heracleifolia Komarov, and Bupleurum falcatum Linne). The combination of these different herbs is considered to improve their therapeutic effects by allowing herb–herb interactions while preventing potential side effects and toxic influence (Bensky and Barolet, 1990). Therefore, we performed a subchronic toxicity study of BITW in rats to determine the toxicity of BITW administration over a long period of 13 weeks and, ultimately, to establish its safety profile. General toxicity studies are classified as acute (14 days), subacute (28 days), subchronic (90 days), and chronic (6–12 months), depending on the specific experimental period (Prieto et al., 2005). Subchronic studies evaluate the potential adverse events of repeated or continuous exposure to drugs over a portion of the average life span of laboratory animals (e.g. mice, rats, and rabbits). Specifically, they provide valuable information about the toxicity of these drug preparations for the target organ and are designed to identify the NOAEL. This can provide the necessary supporting evidence to determine suitable dose regimens for long-term studies (NRC, 2006). An important requirement for toxicological studies in vivo is the ability to evaluate the therapeutic effects of medications on specific organs. Sensitive indicators are a change in body and organ weight related to the change in food intake responsible for weight gain or loss, hematological and serum biochemistry, and urinary and histopathological parameters (Fatchiyah et al., 2017). Our findings show that administration of BITW at doses up to 2000 mg/kg/ day for 13 weeks did not produce any apparent adverse effects in rats of either sex. Some minor changes (including serum Na and Cl values and histological anterior pituitary hyperplasia) were considered to be toxicologically insignificant because they appeared to be accidental changes or spontaneous changes unrelated to the administration of BITW. The NOAEL is used to classify noxious substances, and is obtained from subchronic or chronic repeated-dose toxicity studies. The NOAEL is defined as the maximum level of exposure at which there are no biologically meaningful increases in the intensity, severity or frequency of side effects between the exposed group and the appropriate control group (Lewis et al., 2002). In the present subchronic toxicity study, we determined a best-estimate NOAEL range of a dietary dose of > 2000 mg/kg/day BITW in both male and female rats.
Values are expressed as means ± SD (n = 10 per group). BITW, Bojungikki-tang water extract; SG, specific gravity; pH, hydrogen exponent; U–K, urine potassium; U–Cl, urine chloride; U–Na, urine sodium.
11.4 ± 4.01 1.016 ± 0.0039 6.7 ± 0.34 127.42 ± 42.551 41.4 ± 16.47 44.7 ± 16.45 13.2 ± 5.75 1.015 ± 0.0044 6.7 ± 0.35 104.88 ± 39.251 44.9 ± 19.27 48.9 ± 17.53 12.6 ± 4.12 1.013 ± 0.0035 6.8 ± 0.42 93.32 ± 23.580 41.5 ± 15.20 46.0 ± 18.47 13.8 ± 5.53 1.013 ± 0.0035 7.0 ± 0.16 98.88 ± 34.429 42.9 ± 10.70 49.9 ± 14.65 15.6 ± 5.72 1.010 ± 0.0037 7.0 ± 0.16 152.47 ± 66.789 48.5 ± 20.30 45.8 ± 19.66 14.8 ± 5.09 1.012 ± 0.0024 7.2 ± 0.47 152.05 ± 41.932 48.4 ± 15.06 50.9 ± 20.80 Urine volume (mL) SG pH U–K (mmol/L) U–Cl (mmol/L) U–Na (mmol/L)
16.0 ± 4.71 1.011 ± 0.0032 7.0 ± 0.16 134.43 ± 37.780 42.9 ± 14.43 42.7 ± 24.12
14.4 ± 5.40 1.013 ± 0.0035 6.9 ± 0.21 145.03 ± 37.481 42.4 ± 15.57 41.6 ± 20.04
0 mg/kg/day 1000 mg/kg/day 500 mg/kg/day 0 mg/kg/day
2000 mg/kg/day
Female Male
Parameters BITW (mg/kg)
Table 6 Urinalysis values of male and female rats oral-administrated with BITW at doses of 0, 500, 1000, and 2000 mg/kg/day for 13 weeks.
500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
W.-Y. Jeon, et al.
5. Conclusions In conclusion, the findings of our subchronic toxicity study in rats suggested that oral administration of BITW at doses ≤2000 mg/kg/day for 13 weeks does not cause any adverse effects. Under these experimental conditions, the NOAEL of BITW was considered to be > 2000 mg/kg/day for rats of both sexes. Authors’ contributions Woo-Young Jeon, Chang-Seob Seo, Hyekyung Ha, Hyeun-Kyoo Shin and Mee-Young Lee participated in the design of the data analyses and manuscript preparation. Woo-Young Jeon, Chang-Seob Seo, Jae-Woo Cho and Da Hee Kim performed the experiments and analyzed the relevant data. All authors were involved in revising the manuscript and approved the final manuscript. 7
Journal of Ethnopharmacology 252 (2020) 112551
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Fig. 4. Representative histopathological findings in the liver, kidney, and anterior pituitary ( × 200). BITW, Bojungikki-tang water extract; M, male; F, female. (A) Male normal control group liver, (B) male 2000 mg/kg/day BITW treatment group liver, (C) female normal control group liver, (D) female 2000 mg/kg/day BITW treatment group liver, (E) male normal control group kidney, (F) male 2000 mg/kg/day BITW treatment group kidney, (G) female normal control group kidney, (H) female 2000 mg/kg/day BITW treatment group kidney, (I) male normal control group anterior pituitary, (J) male 2000 mg/kg/day BITW treatment group anterior pituitary, (K) female normal control group anterior pituitary, (L) female 2000 mg/kg/day BITW treatment group anterior pituitary. Liver, kidney, and anterior pituitary tissues were fixed, sectioned at 4 μm thickness, and stained with hematoxylin and eosin solution.
Declaration of competing interest
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