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Acute and genetic toxicity of GS-E3D, a new pectin lyase-modified red ginseng extract
Regulatory Toxicology and Pharmacology 104 (2019) 157–162
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Acute and genetic toxicity of GS-E3D, a new pectin lyase-modified red ginseng extract
T
Cho Rong Parka, Mi Kyung Pyob, Hwan Leeb, Seung Young Honga, Su Hwan Kima, Cheol Beom Parka, Seung Min Ohc,∗ a
Biotoxtech. Co. Ltd, Cheongju, 13000, South Korea International Ginseng and Herb Research Institute, Geumsan, 312-804, South Korea c Department of Nanofusion Technology, Hoseo University, Asan, 31499, South Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute toxicity Genotoxic effects Pectin lyase-modified red ginseng extract GS-E3D
Korean red ginseng and its extract have been used as traditional medicines and functional foods in countries worldwide. Pectin lyase-modified red ginseng extract (GS-E3D) was newly developed as a dietary supplement for obesity, diabetes-related renal dysfunction, etc. In this study, the safety of GS-E3D on acute toxicity and genotoxicity was evaluated. For acute study, Sprague-Dawley rats were administrated by oral gavage at a dose of 5000 mg/kg GS-E3D. To evaluate genotoxicity of GS-E3D, we conducted three-battery tests, which are Ames test using Escherichia coli (WP2uvrA pKM101) and Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), chromosomal aberration test -using Chinese hamster lung cells, and micronucleus test using ICR mice. In acute toxicity studies, there were no dead animals or abnormal necropsy findings in the control group and GSE3D (5000 mg/kg) treated group. GS-E3D did not induce mutagenicity in the bacterial test, chromosomal aberrations in Chinese hamster lung cells and micronuclei in bone marrow cells of mice. Conclusively, the approximate lethal dose of GS-E3D was greater than 5000 mg/kg bw and GS-E3D has no genotoxic potential in the three-battery tests on genotoxicity.
1. Introduction Ginseng, the root of panax ginseng Meyer, and its preparation, red ginseng, have been broadly used as traditional herb medicines in East Asia (Lee et al., 2012; Lu et al., 2009). One of the steroid saponins of Panax ginseng, ginsenoside contains over 150 identified substances (Shibata, 2001), which are generally divided into 2 groups: protopanaxadiols (Rb1, Rb2, Rb3, 20(S)-Rg3, 20R-Rg3 and Rd) and protopanaxatriols (Rg1, Re, Rf, Rg5, and Rk1). Ginseng and its major component, ginsenoside, have been studied on various beneficial effects including anticarcinogenic effects (Shibata, 2001), allergic reactions (Park et al., 2011), Alzheimer's disease (Chen et al., 2006; Shieh et al., 2008), central nerve system (Seong et al., 1995; Joo et al., 2008), cardiovascular disease (Buettner et al., 2006), and diabetes (Reeds et al., 2011; Attele et al., 2002). Korean red ginseng containing many functional chemicals and complexes is made by repetitive steaming and drying treatment (Tanaka and Kasai, 1984). The Korean red ginseng exhibits potent pharmacological and therapeutic effects on high blood pressure, atherosclerosis, and hyperlipidemia (Kim and Rhee, 2009;
Nocerino et al., 2000). In addition, pectin lyase-modified red ginseng extract (GS-E3D) treated with microbial pectin lyase was developed as a dietary supplement, which has beneficial effects on obesity (Lee et al., 2014), inflammation (Hong et al., 2015), and diabetes-related renal dysfunction (Kim et al., 2017). Korean red ginseng showed no toxic effects in subacute (Park et al., 2013) and subchronic oral toxicity (Park et al., 2018), and genotoxicity (Jeong et al., 2016). In addition, National Toxicology Program (NTP, 2011) reported that ginseng has no evidence of carcinogenic activity in 2-year study of F344/N Rats and B6C3F1 mice (gavage studies, 1250–5000 mg/kg) (NTP, 2011). Although safety results of various ginseng products have been reported, different preparations and manufacturing may exhibit different toxic effects. Therefore, in this study, we performed acute toxicity and genotoxicity tests to confirm toxic effects of pectin lyase-modified red ginseng extracts. To identify acute toxic effects of GS-E3D, we conducted the acute oral toxicity study and evaluated the approximated lethal dose in rats. For genotoxicity of drug and chemicals, the three-battery tests with the bacteria reverse mutation assay, the in vitro chromosomal
∗ Corresponding author. Department of Nanofusion Technology, Hoseo University, 20, Hoseo-ro 79beon-gil, Baebang-eup, Asan-si, Chungcheongnam-do, 31499, South Korea. E-mail address: [email protected] (S.M. Oh).
https://doi.org/10.1016/j.yrtph.2019.03.010 Received 16 August 2018; Received in revised form 15 March 2019; Accepted 16 March 2019 Available online 21 March 2019 0273-2300/ © 2019 Published by Elsevier Inc.
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Table 1 Clinical signs, necropsy findings, and mortality of Sprague-Dawley (SD) rats orally treated with GS-E3D in acute toxicity test. Sex
Group
Dose (mg/kg)
Clinical signs No. of animals (NOA/total)
Necropsy findings No. of animals (URF/total)
Mortality % (dead/total)
Male
G1: G2: G1: G2:
0 5000 0 5000
5/5 5/5 5/5 5/5
5/5 5/5 5/5 5/5
0/5 0/5 0/5 0/5
Female
Control GS-E3D Control GS-E3D
*NOA: No observable Abnormality. *URF: Unremarkable findings.
Fig. 1. The changes of body weights in the male (A) and female rats (B) orally treated with GS-E3D.
Table 2 Mutagenicity assays for GS-E3D in the absence of metabolic activation using salmonella typhimurium and Escherichia coli strains. Dose (μg/plate)
Number of revertant colonies/plate (Mean ± SD) Base-pair substitution type
0 39.1 78.1 156 313 625 1250 2500 5000 Positive Control (μg/plate)
Table 3 Mutagenicity assays for GS-E3D in the presence of metabolic activation using salmonella typhimurium and Escherichia coli strains. Dose (μg/plate)
Frameshift type
Base-pair substitution type TA100
TA1535
WP2uvrA
TA98
TA1537
113 ± 3 102 ± 9 100 ± 0 105 ± 9 106 ± 5 104 ± 4 93 ± 7 2-AA 2.0 847 ± 19
8±2 – 10 ± 1 10 ± 2 7±1 9±2 9±2 2-AA 3.0 157 ± 6
194 ± – 169 ± 172 ± 180 ± 194 ± 197 ± 2-AA 2.0 551 ±
29 ± 5 – 29 ± 6 23 ± 3 26 ± 5 31 ± 2 34 ± 1 2-AA 1.0 348 ± 9
8±2 – 10 ± 1 10 ± 2 7±1 9±2 9±2 2-AA 3.0 194 ± 7
TA100
TA1535
WP2uvrA
TA98
TA1537
80 ± 9 97 ± 3 91 ± 1 91 ± 5 97 ± 3 67 ± 10 57 ± 8 – – SA 1.5 615 ± 36
11 ± 1 – – 12 ± 2 10 ± 3 11 ± 3 10 ± 1 10 ± 2 10 ± 1 SA 1.5 437 ± 31
154 ± – – – 177 ± 177 ± 153 ± 187 ± 202 ± 4NQO 0.1 819 ±
21 ± 3 – – – 20 ± 2 23 ± 4 23 ± 3 21 ± 3 22 ± 5 2-NF 5.0 674 ± 31
7±1 7±2 8±2 7±1 7±2 6±2 3±2 – – 9-AA 80.0 542 ± 16
13
6 11 5 6 15
5
Number of revertant colonies/plate (Mean ± SD)
0 156 313 625 1250 2500 5000 Positive Control (μg/plate)
Frameshift type
5 15 7 11 16 19
13
2-AA: 2-aminoanthracene.
SA, sodium azide; 4NQO, 4-nitroquinoline-1-oxide; 2-NF, 2-nitrofluorene; 9AA, 9-aminoacridine.
(Maron and Ames, 1983; Mortelmans and Riccio, 2000). The formation of revertant colonies by frameshift and a base-pair substitution defects were easily identified at low levels of tryptophan or histidine. Second, the chromosomal aberration (CA) test using cultured mammalian cells was performed to evaluate structural chromosomal aberrations of GSE3D. The in vitro chromosome aberration assay has been performed as one of the sensitive methods to predict mutagens and/or carcinogens (Ishidate and Odashima, 1977). Finally, the rodent haematopoietic cell micronucleus assay is most widely used as an in vivo test to evaluate structural and numerical chromosomal aberrations (Hayashi, 2016). These toxicity tests were conducted according to the Good Laboratory Practice (GLP) regulations.
aberration test, and the in vivo micronucleus test have been frequently used by recommendation of regulatory agencies for determining genetic risk (Korea Food & Drug Administration, Food & Drug Administration, and OECD). These three-battery tests on genotoxicity have a high screening value to predict carcinogenicity in rodents (Mortelmans and Zeiger, 2000). Therefore, the potential genotoxicity of GS-E3D was examined using three-battery tests. First, we conducted bacterial reverse mutation test using the S. typhimurium strains TA98, TA100, TA1535 and TA1537 or the E. coli strain WP2uvrA in the presence or absence of metabolic activation. Four salmonella strains and the E. coli strain are histidine-dependent and tryptophan-dependent, respectively
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Array (PDA) detector (Waters 1525). A Gemnini C18 column (250 ⅹ 4.6 mm, 5 μm, Phenomenex, USA) was used for analysis. A binary gradient system was employed for elution, which consisted of water and acetonitrile (ACN), at a flow rate of 1.0 ml/min and a temperature of 40 °C. The program was as follows: 0–42 min, 18% ACN; 42–46 min, 24% ACN; 46–79 min, 29% ACN; 79–115 min, 40% ACN; 115–135 min, 65% ACN; 135–150 min, 85% ACN; 150–151 min, 18% ACN. The sample injection volume and detection wavelength were set at 20 μl and 203 nm, respectively.
Table 4 The Dose Range Finding of GS-E3D in CHL cells. Dose (μg/ plate)
0 19.5 39.1 78.1 156 313 625 1250 2500b 5000b a b
Relative Population Doubling (%) Treatment – Recovery time (6–18 h)
Treatment – Recovery time (24 - 0 h)
S9 mix (−)
S9 mix (+)
S9 mix (−)
100.0 95.7 90.6 82.8 78.9 77.6 78.3 70.0 55.2 −25.6
100.0 96.4 93.3 93.9 92.1 83.4 80.6 79.2 77.0 44.1
100.0 99.0 99.5 93.1 91.5 91.5 69.6 56.0 41.1 n/ca
2.2. Animal husbandry and maintenance Young adult SD rats (five-week old) of both sexes and ICR male mice were purchased from the Orient Bio Co., Ltd. (Gyeonggi-do, Korea) and acclimated for six days. The animals (5 rats per group for acute toxicity test, 5 mice per group for in vivo micronucleus test) were kept in a room with a 12 h/12 h light/dark cycle at a temperature of 23 ± 3 °C and a relative humidity of 55 ± 5%. Rodent chow (2.0 Mrad γ-ray sterilized EP pellet) and sterilized tap water were provided ad libitum for all animals. All animals were maintained in the facility following the guidelines for management and use of laboratory animals (Biotoxtech. Co. Ltd., Cheongju, Korea) approved by the Association for assessment and Accreditation of Laboratory Animal Care International (AAALAC). The protocol for the animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Biotoxtech Co., Ltd (Cheongju, Korea) (Approval No. 160409 for acute toxicity; Approval No. 160805 for micronucleus test).
n/c: RPD was not calculated because of cytotoxicity. Precipitation.
2. Materials and methods 2.1. Test substance and preparation The Panax (P.) ginseng used in this experiment was a 4-year-old dried ginseng purchased from a local market (Wooshin Industrial Co., Ltd., Geumsan, Korea) and was deposited in the International Ginseng and Herb Research Institute (No. GS201104). GS-E3D was prepared according to our early report (Kim et al., 2017). Briefly, red ginseng extract adjusted to 5 Brix, which is 5 g of saccharide in 100 g of solution, were incubated with 10% pectin lyase (EC 4.2.2.10, Novozyme, #33095, Bagsvaerd, Denmark) at 50 °C for 5 days in a shaking incubator (150 rpm). The process of extraction was terminated by heating at 95 °C for 10 min, and then freeze-dried for further experiment. GSE3D used in this study consists of 60% dried red ginseng extract, 39.5% water, etc. The ginsenoside in GS-E3D was analyzed by HPLC (high performance liquid chromatography) with 2998 Photodiode
2.3. Bacterial strains, CHL cells and S9 WP2uvrA (the Escherichia coli strain, pKM101), TA98, TA100, TA1535, and TA1537 (the Salmonella typhimurium strains) were purchased from Molecular Toxicology Inc. (Boone, NC, USA). Each strain was added in 2.5% nutrient broth number 2 solution and incubated for about 12 h at 150 rpm at 37 °C in a shaking incubator. When bacteria counts exceeded 1 ⅹ 109 cells/ml, they were used in the test. CHL cells purchased from American type culture collection (Manasass, VA, USA)
Table 5 Structural and numerical chromosome aberration of GS-E3D in the presence or absence of metabolic activation (S9) in CHL cells. Time (h)
With/without S9mix
Chemicals
Conc. (μg/ml)
Number of cells showing Structural chromosome aberrations Cell No.
ctb
csb
cte
cse
frg
Gap ctg
6h
24 h
S9mix (−)
GS-E3D
S9mix (+)
MMC GS-E3D
S9mix (−)
B [a]P GS-E3D
MMC
0 313 625 1250 2500a 0.1 0 625 1250 2500a 20 0 313 625 1250 2500a 0.1
200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200
1 0 0 0 0 10 0 0 0 0 15 0 0 0 0 0 21
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2
MMC: Mitomycin C, ctb: chromatid break, csb: chromosome break, cte: chromatid exchange. cse: chromosome exchange, frg: fragmentation, ctg: chromatid gap, csg: chromosome gap. gap-: total number of cells with structural aberrations excluding gap. gap+: total number of cells with structural aberrations including gap. Significant difference from negative control by Fisher's exact test: **p < 0.01. a Precipitation. 159
0 0 0 0 0 29 0 0 0 0 57 0 0 0 0 0 80
0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 1 1 0 2 0 0 0 0 2 1 0 0 0 0 1
Total csg
Gap1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 0 0 0 39** 0 0 0 0 66** 0 0 0 0 0 97**
Gap+ 4 0 1 1 0 41 0 0 0 0 68 1 0 0 0 0 98
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Table 6 Micronucleus assay of GS-E3D on male mice bone marrow cells. Groups a
Negative control (water treatment) GS-E3Da
Positive controlb MMC
Dose (mg/kg)
PCE/(PCE + NCE) % (Mean ± S.D.)
MNPCE/PCE % (Mean ± S.D.)
0 1250 2500 5000 2
31.5 32.0 33.5 34.9 29.1
0.05 0.01 0.07 0.07 8.46
± ± ± ± ±
1.93 2.42 1.55 1.74 1.66
± 0.05 ± 0.02 ± 0.08 ± 0.06 ± 0.80**
MNPCE: micronucleated polychromatic erythrocytes. PCE: Polychromatic erythrocytes. NCE: Normochromatic erythrocytes. S.D.: standard Deviation. MMC: Mytomycin C. Significant difference from negative control by Kastendaum & Bowman: **p < 0.01. a Peroral Injection. b Intraperitoneal injection.
using Chinese hamster lung (CHL) cells. To choose the exposure dose of GS-E3D, cytotoxicity (> 50%) of GS-E3D was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT assay) and the relative population doubling (RPD) was calculated as follows: RPD = 100 ⅹ no. of population doublings in treated cultures/ no. of population doublings in control cultures; where population doublings = [log (post-treatment cell number/initial cell number)]/log 2. Based on the cytotoxicity results, the tests for the short time treatment (6 h) in the presence or absence of S9 mix and the continuous treatment (24 h) without S9 mix were conducted at concentrations of 313, 625, 1250 and 2500 μg/ml. Negative control group was treated with sterilized water, while positive control groups were treated with mitomycin C (MMC, 0.1 μg/ml) or benzo [a]pyrene (B [a]P, 20 μg/ml). CHL cells (5 ⅹ 104 cells/5 ml) were seeded and incubated with 10% FBSEMEM in a 60 mm plastic culture dish. And then, the cells were exposed to GS-E3D, MMC or B [a]P for 6 h or 24 h. To collect metaphase cells, colcemid (0.2 μg/ml) was added to the culture medium for 2 h before cell harvesting. Chromosomes prepared by the air-drying method were stained with 3% Giemsa. The frequency of structural and numerical chromosomal aberrations (polyploidy and endoreduplication) in 200 metaphase cells was recorded. The structural aberrations include chromosome exchange, chromatid exchange, chromosome break, chromatid break and others (fragmentations, except for pulverization).
were grown in Eagle's Minimum Essential Medium (EMEM, Lonza Walkersville Inc., USA) with 10% Fetal bovine serum (Gibco, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml), and were incubated under humidified conditions of 5% CO2/95% air and 37 °C. A rat liver S9 fraction (Oriental Yeast Co., Ltd, Tokyo, Japan) was induced by phenobarbital and 5,6-benzoflavone in male Sprague-Dawley rats. 2.4. Acute oral toxicity test The study was conducted in accordance with the Ministry of Food and Drug Safety (MFDS) test guideline (MFDS TG no 2015–82) followed by GLP (MFDS no. 2014–67). Six-week-old male and female rats (5 rats/group) were assigned randomly. The GS-E3D suspended in distilled water as a 200 mg/ml was orally administered to one animal at a dose of 5000 mg/kg body weight (bw) (1st step). After 3 days, another four animals were treated by the same procedure (2nd step). Each animal was observed for clinical signs of toxicity at 1 and 3 h after GS-E3D administration, and then once a day for 14 days. Mortality and morbidity were checked daily. Body weights were measured before administration, and on Days 1 and 7, and at termination. On Day 15, rats were euthanized, and gross observation was recorded after necropsy. 2.5. Bacterial reverse mutation (Ames) test The bacterial reverse mutation test (Ames test) was performed according to MFDS TG (no. 2015–82) based on OECD TG 471 (1997 version). Simply, 0.1 ml of dosing formulations of GS-E3D was preincubated with the test strains (0.1 ml) and either 0.5 ml of sodium phosphate buffer (PB, 0.1 mol/l) or S9 mix (0.1 ml S9/ml, 8 μmol MgCl2/ml, 33 μmol KCl/ml, 5 μmol Glucose-6-phosphate/ml, 4 μmol NADPH, 4 μmol NADH, 100 μmol PB buffer/mL, pH 7.4, 0.275 ml purified water/ml) as metabolic activation system for 20 min at 37 °C prior to mixing with top agar. During pre-incubation, tubes were aerated using a shaking water bath. After pre-incubation, the strain exposed to GS-E3D was mixed with 2 ml of top agar and was poured onto minimal agar plate. Triplicate plating was used in each group to assess an adequate estimate of variation. After all plates in each test were incubated at 37 °C for 48 h, the number of revertant colonies per plate was counted.
2.7. In vivo micronucleus test
2.6. In vitro chromosomal aberration test
2.8. Statistical method
The in vitro chromosomal aberration test was performed according to MFDS TG no. 2015-82 based on OECD TG 473 (1997 revised version). CA test was performed as follows: a short-term treatment (6 h) in the absence or presence of S9 mixture (0.3 ml S9/ml, 5 μmol MgCl2/ml, 33 μmol KCl/ml, 5 μmol Glucose-6-phosphate/ml, 4 μmol NADP, 4 μmol HEPES buffer (pH 7.2)/ml, 0.1 ml purified water/ml), and a continuous treatment for 24 h to determine the mutagenic potential of GS-E3D
The data were expressed as the mean ± standard deviation. Statistical differences between the groups in the acute toxicity test were determined by Dunnett's or Duncan's multiple range test followed by the standard two-way analysis of variance (ANOVA) using SAS version 9.3 (SAS Institute Inc., USA). Fisher exact test was used in the chromosomal aberration test, and Mann-Whitney test and Student t-test were used in the micronuclei test. P values of < 0.05 and 0.01 were
The in vivo micronucleus test was performed according to MFDS TG no. 2015-82 based on OECD TG 474 (1997 revised version). To evaluate the in vivo micronucleus test, ICR male mice (Orientbio INC, Gyeongi, Korea) were used. The animals were sacrificed at 24 h, after either the GS-E3D (from 1 mg/kg-bw to 5000 mg/kg-bw) had been administered via gavage or MMC (2 mg/kg) had been given intraperitoneally. The treatment concentration of GS-E3D and the harvest time of the bone marrow were chosen based on a preliminary dose-range finding test. The air-dried slides with micronuclei of bone marrow were stained with 3% Giemsa. The ratio of polychromatic erythrocytes (PCEs) in 500 erythrocytes (PCE + normochromatic erythrocytes, NCE) and the frequency of micronucleated PCE (MNPCE) in 2000 PCEs per animal were determined under a light microscope (BX51, Olympus, Japan).
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medicine and functional food in the world. Main pharmacological component of Ginseng is known as ginsenosides, which are a group of steroidal saponins and identified as more than 150 ginsenosides (Shibata, 2001). In addition, Korean red ginseng, which is manufactured by repetitive steaming and drying of fresh Panax ginseng (Hong et al., 2011), contains high concentrations of bioactive ginsenosides (Lee et al., 2015). Recently, biotransformation methods including enzymatic conversion (Ko et al., 2003) and fermentation (Park et al., 2010) have been applied to increase the pharmacological potency of Korean red ginseng (Cheon et al., 2015; Lee et al., 2012). These process techniques are known to play an important role in enhancing intestinal absorption and bioactivity, and in reducing toxicity (Wang et al., 2011). In this study, GS-E3D is a newly developed pectin lyase-modified red ginseng extract and contains higher amount of ginsenosides than Korea red ginseng reported in Park et al. (2013). Especially, it contains an enhanced level of the ginsenoside Rd, which has been known to exhibit potent anti-inflammatory, anti-obesity, and anti-ischemic effects (Yoon et al., 2012; Wang et al., 2012; Liu et al., 2012; Yang et al., 2012). LD50 of various ginseng products (Panax Ginseng, Ginseng root extract, Ginsenoside No. 3, Ginseng, Saponin extract) is listed in the Registry of Toxic Effects of Chemical Substances (RTECS, 1998) and ranges from 54 to 910 mg/kg/day (NLM, 1998a; NLM, 1998b; Chen and Fu, 2007). In the single oral administration of red ginseng oil and ethanol/water extract of ginseng to SD rats, LD50 was more than 5000 mg/kg bw (Bak et al., 2014; NTP TR567 report, 2011). In this study of single oral administration to male and female SD rats, LD50 value of GS-E3D may be greater than 5000 mg/kg. When LD50 values of substances are higher than 5000 mg/kg-bw by oral administration, they are regarded as being safe or practically non-toxic (Kennedy et al., 1986). Therefore, we suggest that GS-E3D could be considered safe and not acutely toxic. Genotoxicity studies should be conducted for commercial approval of food and dietary supplement. The three-battery tests on genotoxicity showed a high screening value to predict carcinogenicity in rodents (Mortelmans and Zeiger, 2000). Ginseng root extract (Morimoto et al., 1981) and red ginseng oil (Seo et al., 2017) did not induce mutagenic effects in bacterial reverse mutation assay. Leaf extract of Ginseng did not show toxic potentials in three battery tests on genotoxicity (Kim et al., 2014). In this study, GS-E3D did not show any genotoxic potential in three battery tests. In the subchronic oral toxicity study of ginseng extract for 25 weeks, no effect level in rats was 105–210 mg/kg (Popov and Goldwag, 1973). In recent studies, subacute (4 weeks, Park et al., 2013) and subchronic effects (8 weeks; Park et al., 2018) of Korean red ginseng extract were not shown for oral exposure, and no observed adverse effect level (NOAEL) was greater than 2000 mg/kg bw/day for both sexes. In the case of red ginseng oil, NOAEL was greater than 2000 mg/kg bw/day (Seo et al., 2017). Although safety for subacute and subchronic exposure to various ginseng products has been reported, different preparations and manufacturing could exhibit different toxic effects. In this study, we focused on acute toxicity and genotoxic effects of GS-E3D. Therefore, further studies on subchronic oral toxicity should be conducted to assess a risk for human use as functional food or dietary supplement of GS-E3D. In conclusion, GS-E3D, a pectin lyase-modified red ginseng, did not induce any apparent acute toxicity in the single oral exposure study and showed a higher LD50 value than 5000 mg/kg. In addition, GS-E3D did not show any genotoxicity in three battery tests. GS-E3D was extracted with hot water, which is the method used clinically. Therefore, we suggest that the clinical use of GS-E3D could be safe and not acutely toxic on human health. This study is one of a series of studies to evaluate the safety of GS-E3D as functional food or dietary supplement. To obtain approval of GS-E3D as a functional food or dietary supplement in Korea, we will conduct a long-term animal study (4 weeks) based on the results of this acute study.
considered as statistical significance. 3. Results 3.1. Acute oral toxicity study The dried GS-E3D is composed of 62.39 mg/g crude saponin containing the following ginsenosides: 3.33 mg/g Rg1, 6.41 mg/g Re, 2.24 mg/g Rf, 15.45 mg/g Rb1, 9.92 mg/g Rc, 9.48 mg/g Rb2, and 15.58 mg/g Rd. To evaluate acute toxicity of GS-E3D, male and female rats were orally exposed to 5000 mg/kg of GS-E3D. A dose of 5000 mg/ kg GS-E3D did not cause any signs of toxicity, changes in behavior, mortality, clinical signs, and necropsy findings in male and female SD rats up to 14 days of observation (Table 1). As shown in Fig. 1, the body weights of treated male and female rats showed no significant change compared to the control groups, respectively. The results indicated that LD50 value of GS-E3D may be greater than 5000 mg/kg in male and female SD rats. 3.2. Bacterial reverse mutation (Ames) test Before performing the Ames test, the growth inhibition of GS-E3D was assessed in all bacterial strains used for the tests. The results showed that GS-E3D had no inhibitory growth at the doses used in the test (data not shown). As shown in Table 2 and Table 3, positive controls [sodium azide (SA), 4-nitroquinoline-1-oxide (4NQO), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AA)] showed a significant increase of revertant colonies in comparison with the negative control (0 μg/ plate) in four S. typhimurium strains (TA100, TA1535, TA98, and TA100) and one E. coli. strain (WP2uvrA) with/without S9 metabolic activation system. However, GS-E3D (from 39.1 to 5000 μg/plate) did not show any significant increase of revertant colonies in all 5 strains. These results indicated that GS-E3D has no mutagenic effect in bacterial reverse mutation test. 3.3. Chromosomal aberration test To choose the exposure concentration of GS-E3D, RPD (%) based on cytotoxicity was determined in CHL cells. As shown in Table 4, RPD in CHL cells exposed to GS-E3D (19.5–5000 μg/ml) showed a significant decrease in a dose-dependent manner. According to RPD results (> 50%) of GS-E3D, the highest dose was selected as 2500 μg/ml. As shown in Table 5, CHL cells exposed to GS-E3D (313–2500 μg/ml) did not show numerical or structural chromosome aberrations (ctb: chromatid break, csb: chromosome break, cte: chromatid exchange, cse: chromosome exchange, frg: fragmentation, ctg: chromatid gap, csg: chromosome gap) even after the application of the S9 metabolic activation system compared to the negative control group. However, the positive control (MMC or B(a)P) showed a significant increase of structural chromosome aberrations in comparison to the negative control group. 3.4. In vivo micronucleus test In Table 6, the PCE/(PCE + NCE) ratio revealed no significant difference in the GS-E3D treatment groups in comparison with the negative control group, indicating that the rate of cell division in bone marrow was not altered in all groups. Positive control group exposed to MMC (2 mg/kg) showed a significant increase of MNPCE/PCE in comparison to the negative control group. However, GS-E3D showed no significant increase of MNPCE/PCE (Table 6) indicating that GS-E3D has no genotoxic potential in in vivo micronucleus test. 4. Discussion Ginseng (Panax ginseng C.A. Meyer) is used as a popular herbal 161
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Conflicts of interest
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All authors declare no conflicts of interest. Acknowledgement This research was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries through Export Promotion Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (315049-05-2SB010) of South Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yrtph.2019.03.010. References Attele, A.S., Zhou, Y.P., Xie, J.T., Wu, J.A., Zhang, L., Dey, L., Pugh, W., Rue, P.A., Polonsky, K.S., Yuan, C.S., 2002. Antidiabetic effects of panax ginseng berry extract and the identification of an effective component. Diabetes 51 (6), 1851–1858. Bak, M.J., Kim, K.B., Jun, M., Jeong, W.S., 2014. Safety of red ginseng oil for single oral administration in Sprague-Dawley rats. J. Ginseng Res. 38 (1), 78–81. Buettner, C., Yeh, G.Y., Phillips, R.S., Mittleman, M.A., Kaptchuk, T.J., 2006. Systematic review of the effects of ginseng on cardiovascular risk factors. Am. Pharmacother. 40, 83–95. Chen, F., Eckman, E.A., Eckman, C.B., 2006. Reductions in levels of the Alzheimer's amyloid beta peptide after oral administration of ginsenosides. FASEB J. 20 (8), 1269–1271. Chen, P.C., Fu, P.P., 2007. Toxicity of Panax Ginseng – an herbal medicine and dietary supplement. J. Food Drug Anal. 15 (4), 416–427. Cheon, J.M., Kim, D.I., Kim, K.S., 2015. Insulin sensitivity improvement of fermented Korean Red Ginseng (Panax ginseng) mediated by insulin resistance hallmarks in oldaged ob/ob mice. J. Ginseng. Res. 39, 331–337. Hayashi, M., 2016. The micronucleus test – most widely used in vivo genotoxicity test. Genes Environ. 38, 18. Hong, S.C., Oh, M.H., Lee, H., Park, Y.S., Kim, N.Y., Park, S.H., Park, J.D., Jang, J.D., Kim, S.H., Kim, E.J., Pyo, M.K., 2015. Pectinase-modified red ginseng (GS-E3D) inhibit NFkB translocation and nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells. Int. J. Pharm. Pharm. Sci. 7 (9), 322–326. Hong, S.Y., Oh, J.H., Lee, I., 2011. Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus. Biosci. Biotechnol. Biochem. 75 (8), 1490–1495. Ishidate Jr., M., Odashima, S., 1977. Chromosome tests with 134 compounds on Chinese hamster cells in vitro a screening for chemical carcinogens. Mutat. Res. 48 (3–4), 337–353. Jeong, M.K., Cho, C.K., Yoo, H.S., 2016. General and genetic toxicology of enzyme-treated ginseng extract: Toxicology of Ginseng Rh2. J. Pharmacopunture 19 (3), 213–224. Joo, S.S., Yoo, Y.M., Ahn, B.W., Nam, S.Y., Kim, Y.B., Hwang, K.W., Lee, D.I., 2008. Prevention of inammation-mediated neurotoxicity by Rg3 and its role in microglial activation. Biol. Pharm. Bull. 31, 1392–1396. Kennedy, G.L., Ferenz, R.L.J., Burgess, B.A., 1986. Estimation of acute toxicity in rats by determination of the appromate lethal dose rather than LD50. J. Appl. Toxicol. 6, 145–148. Kim, C.S., Jo, K., Kim, J.S., Pyo, M.K., Kim, J., 2017. GS-E3D, a new pectin lyase-modified red ginseng extract, inhibited diabetes-related renal dysfunction in streptozotocininduced diabetic rats. BMC Complement Altern. Med. 17 (1), 430. Kim, E.H., Rhee, D.K., 2009. Anti-oxidative properties of ginseng. J. Ginseng Res. 33, 1–7. Kim, J.Y., Ri, Y., Do, S.G., Lee, Y.C., Park, S.J., 2014. Evaluation of the genotoxicity of ginseng leaf extract UG0712. Lab. Anim. Res. 30 (3), 104–111. Ko, S.R., Choi, K.J., Uchida, K., Suzuki, Y., 2003. Enzymatic preparation of ginsenosides Rg2, Rh1, and F1 from protopanaxatriol-type ginseng saponin mixture. Planta Med. 69, 285–286. Lee, E.J., Song, M.J., Kwon, H.S., Ji, G.E., Sung, M.K., 2012. Oral administration of fermented red ginseng suppressed ovalbumin-induced allergic responses in female BALB/c mice. Phytomedicine 19 (10), 896–903. Lee, H.Y., Park, K.H., Park, Y.M., Moon, D.I., Oh, H.G., Kwon, D.Y., Yang, H.J., Kim, O., Kim, D.W., Yoo, J.H., Hong, S.C., Lee, K.H., Seoul, S.Y., Park, Y.S., Park, J.D., Pyo, M.K., 2014. Effects of pectin lyase-modified red ginseng extracts in high-fat diet fed
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Acute and genetic toxicity of GS-E3D, a new pectin lyase-modified red ginseng extract
T
Cho Rong Parka, Mi Kyung Pyob, Hwan Leeb, Seung Young Honga, Su Hwan Kima, Cheol Beom Parka, Seung Min Ohc,∗ a
Biotoxtech. Co. Ltd, Cheongju, 13000, South Korea International Ginseng and Herb Research Institute, Geumsan, 312-804, South Korea c Department of Nanofusion Technology, Hoseo University, Asan, 31499, South Korea b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute toxicity Genotoxic effects Pectin lyase-modified red ginseng extract GS-E3D
Korean red ginseng and its extract have been used as traditional medicines and functional foods in countries worldwide. Pectin lyase-modified red ginseng extract (GS-E3D) was newly developed as a dietary supplement for obesity, diabetes-related renal dysfunction, etc. In this study, the safety of GS-E3D on acute toxicity and genotoxicity was evaluated. For acute study, Sprague-Dawley rats were administrated by oral gavage at a dose of 5000 mg/kg GS-E3D. To evaluate genotoxicity of GS-E3D, we conducted three-battery tests, which are Ames test using Escherichia coli (WP2uvrA pKM101) and Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537), chromosomal aberration test -using Chinese hamster lung cells, and micronucleus test using ICR mice. In acute toxicity studies, there were no dead animals or abnormal necropsy findings in the control group and GSE3D (5000 mg/kg) treated group. GS-E3D did not induce mutagenicity in the bacterial test, chromosomal aberrations in Chinese hamster lung cells and micronuclei in bone marrow cells of mice. Conclusively, the approximate lethal dose of GS-E3D was greater than 5000 mg/kg bw and GS-E3D has no genotoxic potential in the three-battery tests on genotoxicity.
1. Introduction Ginseng, the root of panax ginseng Meyer, and its preparation, red ginseng, have been broadly used as traditional herb medicines in East Asia (Lee et al., 2012; Lu et al., 2009). One of the steroid saponins of Panax ginseng, ginsenoside contains over 150 identified substances (Shibata, 2001), which are generally divided into 2 groups: protopanaxadiols (Rb1, Rb2, Rb3, 20(S)-Rg3, 20R-Rg3 and Rd) and protopanaxatriols (Rg1, Re, Rf, Rg5, and Rk1). Ginseng and its major component, ginsenoside, have been studied on various beneficial effects including anticarcinogenic effects (Shibata, 2001), allergic reactions (Park et al., 2011), Alzheimer's disease (Chen et al., 2006; Shieh et al., 2008), central nerve system (Seong et al., 1995; Joo et al., 2008), cardiovascular disease (Buettner et al., 2006), and diabetes (Reeds et al., 2011; Attele et al., 2002). Korean red ginseng containing many functional chemicals and complexes is made by repetitive steaming and drying treatment (Tanaka and Kasai, 1984). The Korean red ginseng exhibits potent pharmacological and therapeutic effects on high blood pressure, atherosclerosis, and hyperlipidemia (Kim and Rhee, 2009;
Nocerino et al., 2000). In addition, pectin lyase-modified red ginseng extract (GS-E3D) treated with microbial pectin lyase was developed as a dietary supplement, which has beneficial effects on obesity (Lee et al., 2014), inflammation (Hong et al., 2015), and diabetes-related renal dysfunction (Kim et al., 2017). Korean red ginseng showed no toxic effects in subacute (Park et al., 2013) and subchronic oral toxicity (Park et al., 2018), and genotoxicity (Jeong et al., 2016). In addition, National Toxicology Program (NTP, 2011) reported that ginseng has no evidence of carcinogenic activity in 2-year study of F344/N Rats and B6C3F1 mice (gavage studies, 1250–5000 mg/kg) (NTP, 2011). Although safety results of various ginseng products have been reported, different preparations and manufacturing may exhibit different toxic effects. Therefore, in this study, we performed acute toxicity and genotoxicity tests to confirm toxic effects of pectin lyase-modified red ginseng extracts. To identify acute toxic effects of GS-E3D, we conducted the acute oral toxicity study and evaluated the approximated lethal dose in rats. For genotoxicity of drug and chemicals, the three-battery tests with the bacteria reverse mutation assay, the in vitro chromosomal
∗ Corresponding author. Department of Nanofusion Technology, Hoseo University, 20, Hoseo-ro 79beon-gil, Baebang-eup, Asan-si, Chungcheongnam-do, 31499, South Korea. E-mail address: [email protected] (S.M. Oh).
https://doi.org/10.1016/j.yrtph.2019.03.010 Received 16 August 2018; Received in revised form 15 March 2019; Accepted 16 March 2019 Available online 21 March 2019 0273-2300/ © 2019 Published by Elsevier Inc.
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Table 1 Clinical signs, necropsy findings, and mortality of Sprague-Dawley (SD) rats orally treated with GS-E3D in acute toxicity test. Sex
Group
Dose (mg/kg)
Clinical signs No. of animals (NOA/total)
Necropsy findings No. of animals (URF/total)
Mortality % (dead/total)
Male
G1: G2: G1: G2:
0 5000 0 5000
5/5 5/5 5/5 5/5
5/5 5/5 5/5 5/5
0/5 0/5 0/5 0/5
Female
Control GS-E3D Control GS-E3D
*NOA: No observable Abnormality. *URF: Unremarkable findings.
Fig. 1. The changes of body weights in the male (A) and female rats (B) orally treated with GS-E3D.
Table 2 Mutagenicity assays for GS-E3D in the absence of metabolic activation using salmonella typhimurium and Escherichia coli strains. Dose (μg/plate)
Number of revertant colonies/plate (Mean ± SD) Base-pair substitution type
0 39.1 78.1 156 313 625 1250 2500 5000 Positive Control (μg/plate)
Table 3 Mutagenicity assays for GS-E3D in the presence of metabolic activation using salmonella typhimurium and Escherichia coli strains. Dose (μg/plate)
Frameshift type
Base-pair substitution type TA100
TA1535
WP2uvrA
TA98
TA1537
113 ± 3 102 ± 9 100 ± 0 105 ± 9 106 ± 5 104 ± 4 93 ± 7 2-AA 2.0 847 ± 19
8±2 – 10 ± 1 10 ± 2 7±1 9±2 9±2 2-AA 3.0 157 ± 6
194 ± – 169 ± 172 ± 180 ± 194 ± 197 ± 2-AA 2.0 551 ±
29 ± 5 – 29 ± 6 23 ± 3 26 ± 5 31 ± 2 34 ± 1 2-AA 1.0 348 ± 9
8±2 – 10 ± 1 10 ± 2 7±1 9±2 9±2 2-AA 3.0 194 ± 7
TA100
TA1535
WP2uvrA
TA98
TA1537
80 ± 9 97 ± 3 91 ± 1 91 ± 5 97 ± 3 67 ± 10 57 ± 8 – – SA 1.5 615 ± 36
11 ± 1 – – 12 ± 2 10 ± 3 11 ± 3 10 ± 1 10 ± 2 10 ± 1 SA 1.5 437 ± 31
154 ± – – – 177 ± 177 ± 153 ± 187 ± 202 ± 4NQO 0.1 819 ±
21 ± 3 – – – 20 ± 2 23 ± 4 23 ± 3 21 ± 3 22 ± 5 2-NF 5.0 674 ± 31
7±1 7±2 8±2 7±1 7±2 6±2 3±2 – – 9-AA 80.0 542 ± 16
13
6 11 5 6 15
5
Number of revertant colonies/plate (Mean ± SD)
0 156 313 625 1250 2500 5000 Positive Control (μg/plate)
Frameshift type
5 15 7 11 16 19
13
2-AA: 2-aminoanthracene.
SA, sodium azide; 4NQO, 4-nitroquinoline-1-oxide; 2-NF, 2-nitrofluorene; 9AA, 9-aminoacridine.
(Maron and Ames, 1983; Mortelmans and Riccio, 2000). The formation of revertant colonies by frameshift and a base-pair substitution defects were easily identified at low levels of tryptophan or histidine. Second, the chromosomal aberration (CA) test using cultured mammalian cells was performed to evaluate structural chromosomal aberrations of GSE3D. The in vitro chromosome aberration assay has been performed as one of the sensitive methods to predict mutagens and/or carcinogens (Ishidate and Odashima, 1977). Finally, the rodent haematopoietic cell micronucleus assay is most widely used as an in vivo test to evaluate structural and numerical chromosomal aberrations (Hayashi, 2016). These toxicity tests were conducted according to the Good Laboratory Practice (GLP) regulations.
aberration test, and the in vivo micronucleus test have been frequently used by recommendation of regulatory agencies for determining genetic risk (Korea Food & Drug Administration, Food & Drug Administration, and OECD). These three-battery tests on genotoxicity have a high screening value to predict carcinogenicity in rodents (Mortelmans and Zeiger, 2000). Therefore, the potential genotoxicity of GS-E3D was examined using three-battery tests. First, we conducted bacterial reverse mutation test using the S. typhimurium strains TA98, TA100, TA1535 and TA1537 or the E. coli strain WP2uvrA in the presence or absence of metabolic activation. Four salmonella strains and the E. coli strain are histidine-dependent and tryptophan-dependent, respectively
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Array (PDA) detector (Waters 1525). A Gemnini C18 column (250 ⅹ 4.6 mm, 5 μm, Phenomenex, USA) was used for analysis. A binary gradient system was employed for elution, which consisted of water and acetonitrile (ACN), at a flow rate of 1.0 ml/min and a temperature of 40 °C. The program was as follows: 0–42 min, 18% ACN; 42–46 min, 24% ACN; 46–79 min, 29% ACN; 79–115 min, 40% ACN; 115–135 min, 65% ACN; 135–150 min, 85% ACN; 150–151 min, 18% ACN. The sample injection volume and detection wavelength were set at 20 μl and 203 nm, respectively.
Table 4 The Dose Range Finding of GS-E3D in CHL cells. Dose (μg/ plate)
0 19.5 39.1 78.1 156 313 625 1250 2500b 5000b a b
Relative Population Doubling (%) Treatment – Recovery time (6–18 h)
Treatment – Recovery time (24 - 0 h)
S9 mix (−)
S9 mix (+)
S9 mix (−)
100.0 95.7 90.6 82.8 78.9 77.6 78.3 70.0 55.2 −25.6
100.0 96.4 93.3 93.9 92.1 83.4 80.6 79.2 77.0 44.1
100.0 99.0 99.5 93.1 91.5 91.5 69.6 56.0 41.1 n/ca
2.2. Animal husbandry and maintenance Young adult SD rats (five-week old) of both sexes and ICR male mice were purchased from the Orient Bio Co., Ltd. (Gyeonggi-do, Korea) and acclimated for six days. The animals (5 rats per group for acute toxicity test, 5 mice per group for in vivo micronucleus test) were kept in a room with a 12 h/12 h light/dark cycle at a temperature of 23 ± 3 °C and a relative humidity of 55 ± 5%. Rodent chow (2.0 Mrad γ-ray sterilized EP pellet) and sterilized tap water were provided ad libitum for all animals. All animals were maintained in the facility following the guidelines for management and use of laboratory animals (Biotoxtech. Co. Ltd., Cheongju, Korea) approved by the Association for assessment and Accreditation of Laboratory Animal Care International (AAALAC). The protocol for the animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Biotoxtech Co., Ltd (Cheongju, Korea) (Approval No. 160409 for acute toxicity; Approval No. 160805 for micronucleus test).
n/c: RPD was not calculated because of cytotoxicity. Precipitation.
2. Materials and methods 2.1. Test substance and preparation The Panax (P.) ginseng used in this experiment was a 4-year-old dried ginseng purchased from a local market (Wooshin Industrial Co., Ltd., Geumsan, Korea) and was deposited in the International Ginseng and Herb Research Institute (No. GS201104). GS-E3D was prepared according to our early report (Kim et al., 2017). Briefly, red ginseng extract adjusted to 5 Brix, which is 5 g of saccharide in 100 g of solution, were incubated with 10% pectin lyase (EC 4.2.2.10, Novozyme, #33095, Bagsvaerd, Denmark) at 50 °C for 5 days in a shaking incubator (150 rpm). The process of extraction was terminated by heating at 95 °C for 10 min, and then freeze-dried for further experiment. GSE3D used in this study consists of 60% dried red ginseng extract, 39.5% water, etc. The ginsenoside in GS-E3D was analyzed by HPLC (high performance liquid chromatography) with 2998 Photodiode
2.3. Bacterial strains, CHL cells and S9 WP2uvrA (the Escherichia coli strain, pKM101), TA98, TA100, TA1535, and TA1537 (the Salmonella typhimurium strains) were purchased from Molecular Toxicology Inc. (Boone, NC, USA). Each strain was added in 2.5% nutrient broth number 2 solution and incubated for about 12 h at 150 rpm at 37 °C in a shaking incubator. When bacteria counts exceeded 1 ⅹ 109 cells/ml, they were used in the test. CHL cells purchased from American type culture collection (Manasass, VA, USA)
Table 5 Structural and numerical chromosome aberration of GS-E3D in the presence or absence of metabolic activation (S9) in CHL cells. Time (h)
With/without S9mix
Chemicals
Conc. (μg/ml)
Number of cells showing Structural chromosome aberrations Cell No.
ctb
csb
cte
cse
frg
Gap ctg
6h
24 h
S9mix (−)
GS-E3D
S9mix (+)
MMC GS-E3D
S9mix (−)
B [a]P GS-E3D
MMC
0 313 625 1250 2500a 0.1 0 625 1250 2500a 20 0 313 625 1250 2500a 0.1
200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200 200
1 0 0 0 0 10 0 0 0 0 15 0 0 0 0 0 21
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2
MMC: Mitomycin C, ctb: chromatid break, csb: chromosome break, cte: chromatid exchange. cse: chromosome exchange, frg: fragmentation, ctg: chromatid gap, csg: chromosome gap. gap-: total number of cells with structural aberrations excluding gap. gap+: total number of cells with structural aberrations including gap. Significant difference from negative control by Fisher's exact test: **p < 0.01. a Precipitation. 159
0 0 0 0 0 29 0 0 0 0 57 0 0 0 0 0 80
0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 1 1 0 2 0 0 0 0 2 1 0 0 0 0 1
Total csg
Gap1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
1 0 0 0 0 39** 0 0 0 0 66** 0 0 0 0 0 97**
Gap+ 4 0 1 1 0 41 0 0 0 0 68 1 0 0 0 0 98
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Table 6 Micronucleus assay of GS-E3D on male mice bone marrow cells. Groups a
Negative control (water treatment) GS-E3Da
Positive controlb MMC
Dose (mg/kg)
PCE/(PCE + NCE) % (Mean ± S.D.)
MNPCE/PCE % (Mean ± S.D.)
0 1250 2500 5000 2
31.5 32.0 33.5 34.9 29.1
0.05 0.01 0.07 0.07 8.46
± ± ± ± ±
1.93 2.42 1.55 1.74 1.66
± 0.05 ± 0.02 ± 0.08 ± 0.06 ± 0.80**
MNPCE: micronucleated polychromatic erythrocytes. PCE: Polychromatic erythrocytes. NCE: Normochromatic erythrocytes. S.D.: standard Deviation. MMC: Mytomycin C. Significant difference from negative control by Kastendaum & Bowman: **p < 0.01. a Peroral Injection. b Intraperitoneal injection.
using Chinese hamster lung (CHL) cells. To choose the exposure dose of GS-E3D, cytotoxicity (> 50%) of GS-E3D was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT assay) and the relative population doubling (RPD) was calculated as follows: RPD = 100 ⅹ no. of population doublings in treated cultures/ no. of population doublings in control cultures; where population doublings = [log (post-treatment cell number/initial cell number)]/log 2. Based on the cytotoxicity results, the tests for the short time treatment (6 h) in the presence or absence of S9 mix and the continuous treatment (24 h) without S9 mix were conducted at concentrations of 313, 625, 1250 and 2500 μg/ml. Negative control group was treated with sterilized water, while positive control groups were treated with mitomycin C (MMC, 0.1 μg/ml) or benzo [a]pyrene (B [a]P, 20 μg/ml). CHL cells (5 ⅹ 104 cells/5 ml) were seeded and incubated with 10% FBSEMEM in a 60 mm plastic culture dish. And then, the cells were exposed to GS-E3D, MMC or B [a]P for 6 h or 24 h. To collect metaphase cells, colcemid (0.2 μg/ml) was added to the culture medium for 2 h before cell harvesting. Chromosomes prepared by the air-drying method were stained with 3% Giemsa. The frequency of structural and numerical chromosomal aberrations (polyploidy and endoreduplication) in 200 metaphase cells was recorded. The structural aberrations include chromosome exchange, chromatid exchange, chromosome break, chromatid break and others (fragmentations, except for pulverization).
were grown in Eagle's Minimum Essential Medium (EMEM, Lonza Walkersville Inc., USA) with 10% Fetal bovine serum (Gibco, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml), and were incubated under humidified conditions of 5% CO2/95% air and 37 °C. A rat liver S9 fraction (Oriental Yeast Co., Ltd, Tokyo, Japan) was induced by phenobarbital and 5,6-benzoflavone in male Sprague-Dawley rats. 2.4. Acute oral toxicity test The study was conducted in accordance with the Ministry of Food and Drug Safety (MFDS) test guideline (MFDS TG no 2015–82) followed by GLP (MFDS no. 2014–67). Six-week-old male and female rats (5 rats/group) were assigned randomly. The GS-E3D suspended in distilled water as a 200 mg/ml was orally administered to one animal at a dose of 5000 mg/kg body weight (bw) (1st step). After 3 days, another four animals were treated by the same procedure (2nd step). Each animal was observed for clinical signs of toxicity at 1 and 3 h after GS-E3D administration, and then once a day for 14 days. Mortality and morbidity were checked daily. Body weights were measured before administration, and on Days 1 and 7, and at termination. On Day 15, rats were euthanized, and gross observation was recorded after necropsy. 2.5. Bacterial reverse mutation (Ames) test The bacterial reverse mutation test (Ames test) was performed according to MFDS TG (no. 2015–82) based on OECD TG 471 (1997 version). Simply, 0.1 ml of dosing formulations of GS-E3D was preincubated with the test strains (0.1 ml) and either 0.5 ml of sodium phosphate buffer (PB, 0.1 mol/l) or S9 mix (0.1 ml S9/ml, 8 μmol MgCl2/ml, 33 μmol KCl/ml, 5 μmol Glucose-6-phosphate/ml, 4 μmol NADPH, 4 μmol NADH, 100 μmol PB buffer/mL, pH 7.4, 0.275 ml purified water/ml) as metabolic activation system for 20 min at 37 °C prior to mixing with top agar. During pre-incubation, tubes were aerated using a shaking water bath. After pre-incubation, the strain exposed to GS-E3D was mixed with 2 ml of top agar and was poured onto minimal agar plate. Triplicate plating was used in each group to assess an adequate estimate of variation. After all plates in each test were incubated at 37 °C for 48 h, the number of revertant colonies per plate was counted.
2.7. In vivo micronucleus test
2.6. In vitro chromosomal aberration test
2.8. Statistical method
The in vitro chromosomal aberration test was performed according to MFDS TG no. 2015-82 based on OECD TG 473 (1997 revised version). CA test was performed as follows: a short-term treatment (6 h) in the absence or presence of S9 mixture (0.3 ml S9/ml, 5 μmol MgCl2/ml, 33 μmol KCl/ml, 5 μmol Glucose-6-phosphate/ml, 4 μmol NADP, 4 μmol HEPES buffer (pH 7.2)/ml, 0.1 ml purified water/ml), and a continuous treatment for 24 h to determine the mutagenic potential of GS-E3D
The data were expressed as the mean ± standard deviation. Statistical differences between the groups in the acute toxicity test were determined by Dunnett's or Duncan's multiple range test followed by the standard two-way analysis of variance (ANOVA) using SAS version 9.3 (SAS Institute Inc., USA). Fisher exact test was used in the chromosomal aberration test, and Mann-Whitney test and Student t-test were used in the micronuclei test. P values of < 0.05 and 0.01 were
The in vivo micronucleus test was performed according to MFDS TG no. 2015-82 based on OECD TG 474 (1997 revised version). To evaluate the in vivo micronucleus test, ICR male mice (Orientbio INC, Gyeongi, Korea) were used. The animals were sacrificed at 24 h, after either the GS-E3D (from 1 mg/kg-bw to 5000 mg/kg-bw) had been administered via gavage or MMC (2 mg/kg) had been given intraperitoneally. The treatment concentration of GS-E3D and the harvest time of the bone marrow were chosen based on a preliminary dose-range finding test. The air-dried slides with micronuclei of bone marrow were stained with 3% Giemsa. The ratio of polychromatic erythrocytes (PCEs) in 500 erythrocytes (PCE + normochromatic erythrocytes, NCE) and the frequency of micronucleated PCE (MNPCE) in 2000 PCEs per animal were determined under a light microscope (BX51, Olympus, Japan).
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medicine and functional food in the world. Main pharmacological component of Ginseng is known as ginsenosides, which are a group of steroidal saponins and identified as more than 150 ginsenosides (Shibata, 2001). In addition, Korean red ginseng, which is manufactured by repetitive steaming and drying of fresh Panax ginseng (Hong et al., 2011), contains high concentrations of bioactive ginsenosides (Lee et al., 2015). Recently, biotransformation methods including enzymatic conversion (Ko et al., 2003) and fermentation (Park et al., 2010) have been applied to increase the pharmacological potency of Korean red ginseng (Cheon et al., 2015; Lee et al., 2012). These process techniques are known to play an important role in enhancing intestinal absorption and bioactivity, and in reducing toxicity (Wang et al., 2011). In this study, GS-E3D is a newly developed pectin lyase-modified red ginseng extract and contains higher amount of ginsenosides than Korea red ginseng reported in Park et al. (2013). Especially, it contains an enhanced level of the ginsenoside Rd, which has been known to exhibit potent anti-inflammatory, anti-obesity, and anti-ischemic effects (Yoon et al., 2012; Wang et al., 2012; Liu et al., 2012; Yang et al., 2012). LD50 of various ginseng products (Panax Ginseng, Ginseng root extract, Ginsenoside No. 3, Ginseng, Saponin extract) is listed in the Registry of Toxic Effects of Chemical Substances (RTECS, 1998) and ranges from 54 to 910 mg/kg/day (NLM, 1998a; NLM, 1998b; Chen and Fu, 2007). In the single oral administration of red ginseng oil and ethanol/water extract of ginseng to SD rats, LD50 was more than 5000 mg/kg bw (Bak et al., 2014; NTP TR567 report, 2011). In this study of single oral administration to male and female SD rats, LD50 value of GS-E3D may be greater than 5000 mg/kg. When LD50 values of substances are higher than 5000 mg/kg-bw by oral administration, they are regarded as being safe or practically non-toxic (Kennedy et al., 1986). Therefore, we suggest that GS-E3D could be considered safe and not acutely toxic. Genotoxicity studies should be conducted for commercial approval of food and dietary supplement. The three-battery tests on genotoxicity showed a high screening value to predict carcinogenicity in rodents (Mortelmans and Zeiger, 2000). Ginseng root extract (Morimoto et al., 1981) and red ginseng oil (Seo et al., 2017) did not induce mutagenic effects in bacterial reverse mutation assay. Leaf extract of Ginseng did not show toxic potentials in three battery tests on genotoxicity (Kim et al., 2014). In this study, GS-E3D did not show any genotoxic potential in three battery tests. In the subchronic oral toxicity study of ginseng extract for 25 weeks, no effect level in rats was 105–210 mg/kg (Popov and Goldwag, 1973). In recent studies, subacute (4 weeks, Park et al., 2013) and subchronic effects (8 weeks; Park et al., 2018) of Korean red ginseng extract were not shown for oral exposure, and no observed adverse effect level (NOAEL) was greater than 2000 mg/kg bw/day for both sexes. In the case of red ginseng oil, NOAEL was greater than 2000 mg/kg bw/day (Seo et al., 2017). Although safety for subacute and subchronic exposure to various ginseng products has been reported, different preparations and manufacturing could exhibit different toxic effects. In this study, we focused on acute toxicity and genotoxic effects of GS-E3D. Therefore, further studies on subchronic oral toxicity should be conducted to assess a risk for human use as functional food or dietary supplement of GS-E3D. In conclusion, GS-E3D, a pectin lyase-modified red ginseng, did not induce any apparent acute toxicity in the single oral exposure study and showed a higher LD50 value than 5000 mg/kg. In addition, GS-E3D did not show any genotoxicity in three battery tests. GS-E3D was extracted with hot water, which is the method used clinically. Therefore, we suggest that the clinical use of GS-E3D could be safe and not acutely toxic on human health. This study is one of a series of studies to evaluate the safety of GS-E3D as functional food or dietary supplement. To obtain approval of GS-E3D as a functional food or dietary supplement in Korea, we will conduct a long-term animal study (4 weeks) based on the results of this acute study.
considered as statistical significance. 3. Results 3.1. Acute oral toxicity study The dried GS-E3D is composed of 62.39 mg/g crude saponin containing the following ginsenosides: 3.33 mg/g Rg1, 6.41 mg/g Re, 2.24 mg/g Rf, 15.45 mg/g Rb1, 9.92 mg/g Rc, 9.48 mg/g Rb2, and 15.58 mg/g Rd. To evaluate acute toxicity of GS-E3D, male and female rats were orally exposed to 5000 mg/kg of GS-E3D. A dose of 5000 mg/ kg GS-E3D did not cause any signs of toxicity, changes in behavior, mortality, clinical signs, and necropsy findings in male and female SD rats up to 14 days of observation (Table 1). As shown in Fig. 1, the body weights of treated male and female rats showed no significant change compared to the control groups, respectively. The results indicated that LD50 value of GS-E3D may be greater than 5000 mg/kg in male and female SD rats. 3.2. Bacterial reverse mutation (Ames) test Before performing the Ames test, the growth inhibition of GS-E3D was assessed in all bacterial strains used for the tests. The results showed that GS-E3D had no inhibitory growth at the doses used in the test (data not shown). As shown in Table 2 and Table 3, positive controls [sodium azide (SA), 4-nitroquinoline-1-oxide (4NQO), 2-nitrofluorene (2-NF), 9-aminoacridine (9-AA)] showed a significant increase of revertant colonies in comparison with the negative control (0 μg/ plate) in four S. typhimurium strains (TA100, TA1535, TA98, and TA100) and one E. coli. strain (WP2uvrA) with/without S9 metabolic activation system. However, GS-E3D (from 39.1 to 5000 μg/plate) did not show any significant increase of revertant colonies in all 5 strains. These results indicated that GS-E3D has no mutagenic effect in bacterial reverse mutation test. 3.3. Chromosomal aberration test To choose the exposure concentration of GS-E3D, RPD (%) based on cytotoxicity was determined in CHL cells. As shown in Table 4, RPD in CHL cells exposed to GS-E3D (19.5–5000 μg/ml) showed a significant decrease in a dose-dependent manner. According to RPD results (> 50%) of GS-E3D, the highest dose was selected as 2500 μg/ml. As shown in Table 5, CHL cells exposed to GS-E3D (313–2500 μg/ml) did not show numerical or structural chromosome aberrations (ctb: chromatid break, csb: chromosome break, cte: chromatid exchange, cse: chromosome exchange, frg: fragmentation, ctg: chromatid gap, csg: chromosome gap) even after the application of the S9 metabolic activation system compared to the negative control group. However, the positive control (MMC or B(a)P) showed a significant increase of structural chromosome aberrations in comparison to the negative control group. 3.4. In vivo micronucleus test In Table 6, the PCE/(PCE + NCE) ratio revealed no significant difference in the GS-E3D treatment groups in comparison with the negative control group, indicating that the rate of cell division in bone marrow was not altered in all groups. Positive control group exposed to MMC (2 mg/kg) showed a significant increase of MNPCE/PCE in comparison to the negative control group. However, GS-E3D showed no significant increase of MNPCE/PCE (Table 6) indicating that GS-E3D has no genotoxic potential in in vivo micronucleus test. 4. Discussion Ginseng (Panax ginseng C.A. Meyer) is used as a popular herbal 161
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Conflicts of interest
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