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Safety evaluation of Elsholtzia splendens extracts: Assessment of acute toxicity and mutagenicity
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Food and Chemical Toxicology 46 (2008) 1042–1047 www.elsevier.com/locate/foodchemtox
Safety evaluation of Elsholtzia splendens extracts: Assessment of acute toxicity and mutagenicity Soon-Mi Shim, Mi-Hee Choi, Gun-Hee Kim * Department of Food and Nutrition, Duksung Women’s University, Ssangmun-Dong, Dobong-Gu, Seoul 132-714, South Korea Received 2 March 2007; accepted 27 October 2007
Abstract Much attention is recently gained for Elsholtzia splendens extracts and issue on their usage is raised due to their biological properties. However, there is no sufficient background information on toxicological evaluation of E. splendens extracts to give an assurance of safety for developing dietary supplements and functional foods. The objective of this study was to evaluate safety on E. splendens extracts using acute oral toxicity, bacterial reverse mutation, and chromosome aberration test. Total flavonoids within E. splendens were extracted with 80% of methanol by a reflux condenser. Both female and male mice were orally administrated E. splendens extracts at the dose of 0, 500, 1000, and 2000 mg/kg body weight/day. Mutagenicity of the extracts was evaluated in a bacterial reverse mutation assay using histidine requiring Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and tryptophan-requiring Escherichia coli (WP2uvrA). In vitro chromosome aberration assay in Chinese Hamster Lung (CHL) was conducted to evaluate genotoxicity. Single administration of dose levels of 500, 1000, and 2000 mg/kg body weight/day to mice for 15 days did not produce any significant mortality, clinical signs, body weight loss, and gross findings. E. splendens extracts in the range of 156.3–5000 lg/plate did not induce mutagenicity in S. typhimurium and E. coli with and without metabolic activation system. Any significant chromosomal aberration was not observed in CHL cells 6 h after treating with the extract at the concentrations of 1250, 2500, and 5000 lg/mL in absence and presence of metabolic activation system. However, frequency of chromosomal aberration in 22 h after treatment without metabolic activation system was increased with showing a pattern of dose–response relationship. The highest concentration of 5000 lg/mL significantly induced chromosomal aberration. E. splendens extracts may induce chromosomal structure abnormality in CHL cells. This study suggests that further study is needed to assess the potential genotoxic effects of E. splendens extracts. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Elsholtzia splendens; Safety; Acute toxicity; Bacterial reverse mutation; CHL chromosomal aberration
1. Introduction Many species of genus Elsholtzia wild are predominantly distributed over the east of Asia and some of them are used as an ingredient of traditional medicine (Ling et al., 2004). Among them, Elsholtzia splendens is spontaneously growing in South Korea (Chung and Lee, 2002). It is perennial herb of aromatic plant and contains refined oil composed of elsholtzidiol, sterol, and phenol (WHO, 1998; Youn, 1992; Song and Chae, 2004). It has been used for perfume, aroma therapy, edible and medicinal plants (Lee et al., *
Corresponding author. Tel.: +82 2 901 8496; fax: +82 2 901 8372. E-mail address: [email protected] (G.-H. Kim).
0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.10.036
2005). Many studies have been reported that E. splendens provide effects on reducing pain, inflammation, fever and contamination of microorganism compared to other species of Elsholtzia wild (Youn, 1992). Due to its functional properties, it is currently used as a traditional medicine for the treatment of headaches, fevers, diarrhea, and edema in Asia (WHO, 1998). E. splendens also has abundant flavonoids which is well-known to have anti-cancer and anti-oxidative components (Middleton and Kandawami, 1993; Fiala et al., 1985; Chen et al., 2002). Several studies have been reported protective effect of flavonoids extracted from E. splendens on inhibiting inflammation (Kim and Kim, 2003), myocardial ischemia (Ling et al., 2004), and myocardial apoptosis (Ling and Lou, 2005). Since
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E. splendens are known to have medicinal properties mainly owing to flavonoids, interest by both consumer and industry is increasing for the development of food products with health benefits. However, there is insufficient background information on toxicological evaluation of E. splendens extracts to give an assurance of safety in developing dietary supplements and functional foods. Therefore, the objective of this study was to evaluate safety on extract of E. splendens using acute toxicity of oral acute toxicity, bacterial reverse mutation test, and chromosome aberration test. 2. Materials and methods 2.1. Sample preparation E. splendens harvested from Hwacheon around October, 2005 were obtained. E. splendens was freezing dried and then macerated to powder by mixer (Hanil Electrical Co., FM-681, South Korea). Total flavonoids within E. splendens were extracted according to the method of Kim and Kim (2003): The freeze dried E. splendens (1 g) was mixed with 60 mL of 80% methanol solution (containing 0.05% BHT). This mixture was sonicated for 10 min (Branson 3200 sonicator), then refluxed for 1 h. Following cooling to room temperature, the extract was filled up to 100 mL with 50% methanol. The extract was centrifuged gently (at 850g, Sorvall T 6000D) for 15 min. The supernatant was collected and nitrogen gas was gently applied to evaporate organic solvents. The extract was stored at 4 °C until analysis. Further analysis showed that the content of total flavonoids in the extract was 90.1%, which was detected by colorimetric method using apigenin (Fig. 1) as the control. We conducted sample preparation procedure coupled with Cirsium japonicum to validate the analysis, showing the similar result on total flavonoid in C. japonicum to previous study.
2.2. Acute oral toxicity study in mice Both 24 female and 24 male ICR mice (specific pathogen free, 5 weeks of age) were purchased from Orient Bio (Orient Bio Inc., South Korea). Mice were assigned to each group (n = 5) and acclimated for a week in the housing: Temperature was maintained at 23 ± 3 °C and relative humidity was 50 ± 10%. A 12-h light/dark cycle was observed and air was changed 10–20 times per day. Mice were freely accessed to stock rodent pellet (PMI Nutrition International, Richmond, In, USA) and tap water. The study protocol was reviewed by IACUC (Korea Institute of Toxicology under the Korea Research Institute of Chemical Technology, South Korea) and conducted in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) international recognition (2006). Body weight of the mice at study beginning was 29.60 ± 1.07 for males and 21.47 ± 1.26 g for females. Mice were fasted for 4 h prior to oral administration. E. splendens extract was dissolved in CMC (carboxymethyl cellulose). Constant volume (10 mL/kg body weight) containing different concentration of E. splendens extracts (50, 100, and 200 mg/mL) were orally administered to each five mice per sex to achieve the dose of 500, 1000, and 2000 mg/kg body weight/day. CMC was used as negative control. Feeds and water were provided after 4 h of
Fig. 1. The structure of apigenin in Elsholtzia splendens extracts.
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oral administration. Clinical symptoms including mortality, clinical sign, and gross findings were observed once a day for 15 days. Body weight was measured right before oral administration and 2, 4, 8, 15 days after administration. Histopathology evaluations were performed on organs and tissue after mice were sacrificed.
2.3. Bacterial reverse mutation assay Potential mutagenicity of E. splendens extracts was evaluated according to the standard Ames protocol (Maron and Ames, 1983). Histidine requiring S. typhimurium strains (TA100, TA1535, TA98, and TA1537) and tryptophan-requiring Escherichia coli (WP2uvrA) used in this study were obtained from Molecular Toxicology Inc. (Boone, NC, USA). E. splendens extracts were dissolved in DMSO (Dimethylsulfoxide, 99.99% of purity, Sigma–Aldrich Chemical Co.) to be a final concentration of 5 mg/ mL. This concentrate solution was assigned for the highest test concentration (5000 lg/mL) and serially diluted with DMSO for lower test concentrations (2500, 1250, 625, 312.5, 156.3 lg/mL) according to OECD guideline (1997a). The mutagenicity assay was carried out in both the presence and absence of metabolic activation system (S9 mix) (Rat liver S9, Molecular Toxicology Inc. Boone, NC, USA), along with the negative and positive controls as described in OECD Guidelines for the testing of chemicals TG No. 471 ‘Bacterial reverse mutation test’ (1997) (Table 3). Each treatment was made in triplicate. The sources and grades of positive control materials were as follows: Sodium azide (100.1% purity, Sigma– Aldrich Chemicals Co.), 2-nitrofluorene (98.1% purity, Aldrich Chemical Co., Milwaukee, WI, USA), 9-aminoacridine (97.7% purity, Merck Chemical Co.), 4-nitroquinoline 1-oxide (99% purity, Sigma Chemical Co.), 2-Aminoanthracene (99.8% purity, Aldrich Chemical Co.), Benzo(a)pyrene (99.8% purity, Sigma–Aldrich Chemical Co.).
2.4. In vitro mammalian chromosome aberration test In vitro mammalian chromosome aberration test was conducted according to OECD Guidelines for the testing of chemicals TG No. 473 ‘In vitro mammalian chromosome aberration test’ (OECD, 1997). Detail experiment procedure was performed as described in both Ishidate et al. (1981) and Dean and Danford (1984). Chinese Hamster Lung (CHL) cell derived from the female newborn Chinese hamster was obtained from American Type Culture Collection (Manassas, VA). Modal chromosome number of CHL was 25 and the cycle of fission was approximately 15 h. CHL cell kept in liquid nitrogen was thawed and cultured more than 7 days. Microbial contamination was tested before starting experiment. Cell medium was composed of Minimum Essential Medium (GIBCOInvitrogen, Carlsbad, California, USA), 10% Fetal Bovine Serum (FBS, GIBCO-Invitrogen), 2.2 g of sodium bicarbonate, 292 mg of L-glutamine, 100 lg/ml of streptomycin sulfate, and 100 units of penicillin GNa. CHL cell was seeded at 4 104 cells/mL using a plastic plate and incubated for 3 days at 37 °C with 95% air and 5% CO2. E. splendens extracts was solublized in DMSO. Three dose levels were finally chosen due to their cytotoxicities. Three experiment sets were performed with the concentration in the level of 1250, 2500, 5000 lg/mL both in presence and absence of metabolic activation system (S9 mix). Test concentration was selected according to OECD guideline (1997b). The cells were incubated for 6 h with/without metabolic activation system or 22 h without metabolic activation system. Two plates per concentration were used. Cells were washed with 5 mL of CMFD-PBS (Ca++ and Mg++ free Dulbecco’s phosphate buffered saline) after 6 h or 22 h-treatment. Colchicine (Sigma– Aldrich Chemical Co.) was added to accumulate metaphase cells 2 h before the end of culture. After incubation, cells were collected. DMSO, CPA (Cyclophosphamide monohydrate, 99.7% of purity, Sigma Chemical Co.), and EMS (Ethylmethanesulfonate, Sigma Chemical Co.) were used for negative control, positive control with metabolic activation, and positive control without metabolic activation, respectively. Cells were separated by Trypsin–EDTA for counting relative cell count. The cells were separated by centrifugation, and then resuspended in dilute (hypotonic) potassium chloride solution (75 mM KCl). The cells were fixed and
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washed in a mixture of methanol and acetic acid (3:1, v:v), then dropped onto glass microscope slides. Slides were stained with 3% of Giemsa (Sigma–Aldrich Co.), then mounted with cover slips and examined by light microscopy by trained personal.
exposed at 1000 or 2000 mg E. splendens extracts/kg body weight but there was no significant difference from control group.
2.5. Statistical analysis
3.2. Bacterial reverse mutation assay
The changes of body weights and clinical signs for 15 days after oral administration were analyzed by Path/Tox system (Version 4.2.2). All data were evaluated using the SAS program (version 8.2, SAS Institute Inc., Cary, NC). The average of the number of bacterial reverse mutation colonies was represented with standard deviation. Difference from the control group was tested statistically using a t-test. The significant difference (p < 0.05) in the frequency of chromosome aberration between negative control and treatment was evaluated by X2-test and Fisher’s exact test. Cochran–Armitage trend test was applied to estimate dose–response relationship. Difference in means between negative and positive control group was conducted by Fisher’s exact test.
Mutagenicity of the E. splendens extracts was evaluated in a bacterial reverse mutation assay using histidine requiring S. typhimurium (TA100, TA1535, TA98, and TA 1537) and tryptophan-requiring E. coli (WP2uvrA) (Table 2). Regardless of presence of S9 mix, the number of reverting colonies in all the level of treatment was not significantly higher than that in negative control (Table 2). There was no cytotoxicity in all bacterial systems used in mutation assay. Remarkable increase in the number of revertant colonies was founded in all positive control compared to negative control. Mutagenic activity of the E. splendens extracts at all tested concentration (156.3, 312.5, 625, 1250, 2500, and 5000 lg/plate) was not detected (Table 3).
3. Results 3.1. Acute oral toxicity study Mortality, clinical signs, gross findings, and body weights of mice were measured for 15 days after the oral single gavage administration of E. splendens extracts (0, 500, 1000, 2000 mg/kg body weight). All of the female and male mice after administration at the level of 0, 500, 1000, 2000 mg/kg body weight/day did not show any mortality (Table 1). There were no clinical signs of toxicity in any extract-treated mice at any dose level throughout the 15 day observation period (data not shown). Following the administration of 2000 mg E. splendens extracts/kg body weight/day to female mice, soft stools was observed on day 1. However, this symptom was recovered within 24 h. Slight body weight loss was found from female mice
3.3. In vitro mammalian chromosome aberration test The frequencies of metaphases with aberrant chromosomes and statistical analysis are presented in Table 3. In 6 h treatment with presence of metabolic activation (S9 mix), the frequency of metaphases with aberrant chromosomes in the cultures treated with none, 1250, 2500, and 5000 lg/mL were 1.0, 1.5, 2.0, and 4.5, respectively. Increases in the frequency of metaphases with aberrant chromosomes in those cultures were not either observed or significantly different from negative control (Table 3). Positive control showed significant increases in the frequency of metaphases with aberrant chromosomes
Table 1 Mortality of mice for 15 days after oral administration of Elsholtzia splendens extracts Dose (mg/kg)
Male 0 500 1000 2000 Female 0 500 1000 2000 A B
Days on test 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
aA bB a b a b a b
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
a b a b a b a b
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
a = Number animals alive at the start of each study day. b = Number of mortalities during each study day.
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Table 2 Bacterial reverse mutation assay with Elsholtzia splendens extracts Revertant colonies/plate (Mean) [factor]a
Tester strain
Chemical treated
Dose (lg/plate)
Without S9 mix
With S9 mix
TA 100
Test itemb
0 156.3 312.5 625 1250 2500 5000
104 ± 8 105 ± 8 [1.0] 118 ± 4 [1.1] 112 ± 3 [1.1] 103 ± 2 [1.0] 124 ± 14 [1.2] 122 ± 5 [1.2]
133 ± 10 113 ± 10 [0.8] 129 ± 11 [1.0] 121 ± 19 [0.9] 127 ± 19 [1.0] 121 ± 16 [0.9] 139 ± 4 [1.0]
TA 1535
Test item
0 156.3 312.5 625 1250 2500 5000
15 ± 1 18 ± 6 14 ± 5 16 ± 2 16 ± 2 17 ± 3 18 ± 2
[1.2] [0.9] [1.1] [1.1] [1.1] [1.2]
12 ± 1 10 ± 5 [0.8] 9 ± 4 [0.8] 14 ± 2 [1.2] 11 ± 1 [0.9] 12 ± 1 [1.0] 11 ± 3 [0.9]
0 156.3 312.5 625 1250 2500 5000
18 ± 2 25 ± 4 19 ± 2 21 ± 6 20 ± 4 16 ± 5 21 ± 6
[1.4] [1.1] [1.2] [1.1] [0.9] [1.2]
30 ± 6 35 ± 4 [1.2] 36 ± 11 [1.2] 37 ± 7 [1.2] 35 ± 10 [1.2] 39 ± 6 [1.3] 34 ± 2 [1.1]
0 156.3 312.5 625 1250 2500 5000
10 ± 3 9 ± 2 [0.9] 8 ± 2 [0.8] 9 ± 2 [0.9] 8 ± 3 [0.8] 7 ± 1 [0.7] 9 ± 2 [0.9]
21 ± 3 22 ± 5 19 ± 5 19 ± 3 23 ± 3 23 ± 2 29 ± 5
[1.0] [0.9] [0.9] [1.1] [1.1] [1.4]
0 156.3 312.5 625 1250 2500 5000
16 ± 4 21 ± 4 17 ± 3 13 ± 1 14 ± 5 15 ± 3 15 ± 4
25 ± 6 20 ± 2 20 ± 5 23 ± 2 26 ± 6 26 ± 6 28 ± 6
[0.8] [0.8] [0.9] [1.0] [1.0] [1.1]
0.5 0.5 2 50 0.5 2 2 2 2 4
620 ± 10 [6.0] 469 ± 39 [31.3] 574 ± 90 [31.9] 355 ± 88 [35.5] 103 ± 5 [6.4]
TA 98
TA1537
E. coli WP2uvrA
Positive controls TA 100 TA 1535 TA 98 TA 1537 WP2uvrA TA100 TA 1535 TA 98 TA 1537 WP2uvrA
Test item
Test item
Test item
SA SA 2-NF 9-AA 4NQO BP 2-AA BP BP 2-AA
[1.3] [1.1] [0.8] [0.9] [0.9] [0.9]
15 ± 4 [1.0] 19 ± 4 [1.1]
926 ± 39 [7.0] 463 ± 2 [38.6] 390 ± 57 [13.0] 155 ± 19 [7.4] 185 ± 9 [7.4]
Abbreviations: SA, sodium azide; 2-NF, 2-nitrofluorene; 9-AA, 9-aminoacridine; 4NQO, 4-nitroquinoline 1-oxide; 2-AA, 2-aminoanthracene; BP, benzo(a)pyrene. a Number of revertant colonies of treated plate/number of revertant colonies of vehicle control plate. b Test item: Elsholtzia splendens extracts.
(p < 0.01). In the absence of S9 mix, no biological or significant increases in the frequency of metaphases with aberrant chromosomes were found in cultures 6 h after treatment of E. splendens extracts (0, 1250, 2500, 5000 lg/mL) (Table 3). However, the cultures incubated for 22 h without S9 mix showed increases in the frequency of metaphases with aberrant chromosomes with the dose–
response relationship. Statistically significant increases was observed in culture treated with E. splendens extracts of 5000 lg/mL (p < 0.01). On the contrary, positive control in both 6 h and 22 h-treatment cultures without S9 mix presented 26.5 and 44.5 of increases in the frequency of metaphases with aberrant chromosomes, respectively, and it was statistically significant increases (p < 0.01). In the
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Table 3 Chromosome aberration test with Elsholtzia splendens extracts and relative cell count Nominal concentration of test itema (lg/mL)
Timesb (h)
Mean aberrant metaphases
Mean total aberrations
Mean of PP + ER
Relative cell countsf (%)
6 h treatment ( S9) 0 1250 2500 5000 EMS 800
6–18 6–18 6–18 6–18 6–18
2.5/2.0c 2.0/2.0 3.5/3.0 3.5/3.0 27.0/26.5**,d
2.5/2.0 3.0/3.0 5.0/4.5 4.5/4.0 37.5/37.0
0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0
100 123 101 86 119
22 h treatment ( S9) 0 1250 2500 5000 EMS 600
22–2 22–2 22–2 22–2 22–2
1.0/0.5 3.0/2.5 3.5/3.0 8.5/8.0**,d 45.5/44.5**,e
1.0/0.5 3.5/3.0 3.5/3.0 11.5/10.5 67.5/65.0
0.0 + 0.0 0.5 + 0.0 0.5 + 0.0 0.0 + 0.0 0.0 + 0.0
100 139 118 102 99
6–18 6–18 6–18 6–18 6–18
2.0/1.0 2.0/1.5 2.0/2.0 5.0/4.5 39.0/38.5**,d
3.5/2.5 2.0/1.5 3.0/3.0 5.5/4.5 58.5/57.5
0.0 + 0.0 0.5 + 0.0 0.0 + 0.0 1.0 + 0.0 0.5 + 0.0
100 97 91 82 58
6 h treatment (+S9) 0 1250 2500 5000 CPA 6
S9 mix
+ + + + +
**
Significantly different from the control at p < 0.01. Visible turbidity was observed when treated and at the end of the treatment. Abbreviations: PP, Polyploid; ER, Endoreduplication; EMS, Ethylmethanesulfonate; CPA, Cyclohosphamide monohydrate. a Elsholtzia splendens extracts. b Treatment time-recovery time. c Gaps included/excluded, means of duplicated cultures; 100 metaphases were examined per culture. d The vehicle and test-item treated group; Chi-square test and Fisher’s exact test. e The vehicle and positive control groups; Fisher’s exact test. f Relative cell counts = (cell counts of treated flask/cell counts of control flask) 100 (%).
#
experiment to examine if there was as reproducible effect or unaccounted experimental error in +S9 and S9, we obtained similar results to previous experiments. 4. Discussion The major flavonoid isolated from E. splendens extracts were apigenin, apigenin-7-O-glucoside, luteolin-7-Oglucoside, and linarin (Kim and Kim, 2003). Current study found that the total flavonoid extracted from E. splendens was apigenin, corresponding with 90.1%. Apigenin is widely found in dietary fruit, vegetables, and plant-derived beverages such as tea and wine (Noel et al., 2006). It has shown to possess antioxidant activity, anti-inflammatory, hypothermic, and antipyretic effect and protective effect on radiation-induced chromosomal damage (Youn, 1992; Bae et al., 2007; Kim and Kim, 2003; Rithidech et al., 2005). Those pharmacological effects have increased the public’s interest in the use of Elsholtzia splendens, which are containing significant of apigenin for its potential health benefits. However, the adverse effects of excessive apigenin intake remain highly uncertain. In order to provide safety information on E. splendens extracts, oral acute toxicity and genotoxicity using bacterial reverse mutation test and chromosomal aberration test are carried out in this study. Single dose of E. splendens extracts up to level of 2000 mg/kg body weight did not show adverse effect on clinical sign for 15 days, implying
that E. splendens extracts do not cause any acute toxicity. This result support the LD50 value of E. splendens extracts could be greater than 2000 mg/kg body weight/day. In bacterial reverse mutation test conducted in a range of 156.3, 312.5, 625, 1250, 2500, 5000 lg/plate for each S. typhimurium (TA100, TA1535, TA98, and TA 1537) and Escherichia coli (WP2uvrA), gene mutation was not induced. Similar to our result, genotoxic effect was not introduced in test with S. typhimurium strains TA 1538 and TA 1978 (Czeczot and Kusztelak, 1993). On the contrary, earlier study reported that apigenin showed weak mutagenic effects with S. typhimurium strains TA 100 and TA 98 compared to other flavonoids structurally related (Nagao et al., 1981). These observations imply that difference in bacterial reverse mutation depends on bacterial strain tested. A chromosomal aberration test using cultured CHL cells was performed with or without metabolic activation system. At the highest concentration of 5000 lg/mL in 22 h treatment without S9 mix showed significant increases in the incidence of chromosomal aberrations (p < 0.01). In addition, the frequency of chromosomal aberrations in treatment group was found to have a pattern with dose–response relationship. Results from our study illustrate that E. splendens extracts possibly have potential effect on inducing the chromosomal aberrations. This finding is similar to that previously reported in micronucleus assay (Noel et al., 2006). They found apigenin induced micronuclei in a dose-dependent manner at high dose levels between 600
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and 5400 mg/mL. On the other hand, lower dose of apigenin such as ranged from 2.5 to 25 lg/mL showed a radioprotective effect on radiation-induced chromosomal damage in human lymphocytes (Rithidech et al., 2005). As with other flavonoids, it is appeared that apigenin may show mutagenic or antimutagenic effects depending upon the intake level. Our results suggest that E. splendens extracts may not induce gene mutation but structural aberration of chromosome. Therefore, our study suggests that further studies such as in vivo micronucleus assay can quantitate activity of chromosomal aberrations should be performed. More research on the toxicological properties of E. splendens extracts containing apigenin that will be consumed as functional food or medicine are required for understanding of adverse health effects upon routine intake. In addition, bioavailability of E. splendens extracts including digestive stability, factors affecting intestinal absorption, and metabolism have to be investigated to clarify its health benefit or adverse health effects from oral intake. Acknowledgements The authors wish to thank Institute of toxicology for technical assistance to conduct animal and in vitro study for this research. This Study was supported by Agriculture R&D Promotion Center, Ministry of Agriculture and Forestry, Republic of Korea (204024-03-3-SB010) and in part from the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-005J13001). References AAALAC international (Association for Assessment and Accreditation of Laboratory Animal Care), 1998. Available from: (accessed July 2006). Bae, K., Jin, W., Thuong, P.T., Min, B.S., Na, M., Lee, Y.M., Kang, S.S., 2007. A new flavonoid glycoside from the leaf of Cephalotaxus koreana. Fitoterapia 78 (6), 409–413. Chen, J.W., Zhu, Z.Q., Hu, T.X., Zhu, D.Y., 2002. Structure–activity relationship of natural flavonoids in hydroxyl radical scavenging effects. Acta Pharmacol. Sinica 23, 667–672. Chung, M.S., Lee, M.S., 2002. Development of Elsholtzia splendensFlavored oils and analysis of flavor pattern using electronic nose. Korean J. Soc. Food Cookery Sci. 18 (4), 455–460.
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Czeczot, H., Kusztelak, J., 1993. A study of the genotoxic potential of flavonoids using short-term bacterial assays. Acta Biochim. Pol. 40 (4), 549–554. Dean, B.J., Danford, N., 1984. Assays for the detection of chemicallyinduced chromosome damage in cultured mammalian cells. In: Venitt, S., Parry, J.M., (Eds.), Mutagenicity Testing – A Practical Approach. IRL Press Limited, P.O. Box 1, Eynsham, Oxford OX8 1JJ, England, pp. 187–232. Fiala, E.S., Reddy, B.S., Weisburger, J.H., 1985. Naturally occurring anticarcinogenic substances in foodstuffs. Ann. Rev. Nutr. 5, 295–321. Ishidate, M., Sofuni, T., Yoshikawa, K., 1981. Chromosal aberration tests in vitro as a primary screening tool for environmental mutagens and/or carcinogens. GANN Monogr. Cancer Res. 27, 95–107. Kim, S.J., Kim, G.H., 2003. Identification for flavones in different parts of Cirsium japonicum. J. Food Sci. Nutr. 8, 330–335. Lee, S.Y., Chung, M.S., Kim, M.K., Baek, H.H., Lee, M.S., 2005. Volatile compounds of Elsholtzia splendens. Korean J. Food Sci. Technol. 37 (3), 339–344. Ling, H., Lou, Y., 2005. Total flavones from Elsholtzia reduce infarct size during acute myocardial ischemia by inhibiting myocardial apoptosis in rats. J. Ethnopharmacol. 101, 169–175. Ling, H., Lou, Y., Lou, H., Wu, H., 2004. Protective effect of total flavones from Elsholtzia blanda (TFEB) on myocardial ischemia induced by coronary occlusion in canines. J. Ethnopharmacol. 94, 101–107. Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173–215. Middleton, E., Kandawami, J.C., 1993. The impact of plant flavonoids on mammalian biology. Implications for immunity, inflammation and cancer. In: Harborne, J.B. (Ed.), The Flavonoids. Advances in Research Since 1986. Chapman and Hall, London, UK, pp. 619–652. Nagao, M., Morita, N., Yahagi, T., Shimizu, M., Kuroyanagi, M., Fukuoka, M., Yoshhira, K., Natori, S., Fujino, T., Sugimura, T., 1981. Mutagenicities of 61 flavonoids and 11 related compounds. Environ. Mutagen. 3 (4), 401–419. Noel, S., Kasinathan, M., Rath, S.K., 2006. Evaluation of apigenin using in vitro cytochalasion blocked micronucleus assay. Toxicol. In Vitro 20, 1168–1172. OECD, 1997a. Guideline for the testing of chemicals. TG No. 471 ‘Bacterial Reverse Mutation Test’. OECD, 1997b. Guidelines for the testing of chemicals. TG No. 473 ‘In vitro Mammalian Chromosome Aberration Test’. Rithidech, K.N., Tungjai, M., Whorton, E.B., 2005. Protective effect of apigenin on radiation-induced chromosomal damage in human lymphocytes. Mutat. Res. 585, 96–104. Song, S.E., Chae, Y.A., 2004. Characteristics of volatile oil components in Elsholtzia splendens Nakai collected in Korea. Korean J. Medicinal Crop Sci. 12 (6), 459–462. WHO (World Health Organization), 1998, Medicinal Plants in the Republic of Korea World Health Organization, Manila. Available from: (accessed May 2006). Youn, J.S., 1992. Comparison study on the beneficial effect of Elsholtzia Splendens and Elsholtzia Ciliata. J. Herbol. 7 (1), 33–45.
Food and Chemical Toxicology 46 (2008) 1042–1047 www.elsevier.com/locate/foodchemtox
Safety evaluation of Elsholtzia splendens extracts: Assessment of acute toxicity and mutagenicity Soon-Mi Shim, Mi-Hee Choi, Gun-Hee Kim * Department of Food and Nutrition, Duksung Women’s University, Ssangmun-Dong, Dobong-Gu, Seoul 132-714, South Korea Received 2 March 2007; accepted 27 October 2007
Abstract Much attention is recently gained for Elsholtzia splendens extracts and issue on their usage is raised due to their biological properties. However, there is no sufficient background information on toxicological evaluation of E. splendens extracts to give an assurance of safety for developing dietary supplements and functional foods. The objective of this study was to evaluate safety on E. splendens extracts using acute oral toxicity, bacterial reverse mutation, and chromosome aberration test. Total flavonoids within E. splendens were extracted with 80% of methanol by a reflux condenser. Both female and male mice were orally administrated E. splendens extracts at the dose of 0, 500, 1000, and 2000 mg/kg body weight/day. Mutagenicity of the extracts was evaluated in a bacterial reverse mutation assay using histidine requiring Salmonella typhimurium (TA 98, TA 100, TA 1535, and TA 1537) and tryptophan-requiring Escherichia coli (WP2uvrA). In vitro chromosome aberration assay in Chinese Hamster Lung (CHL) was conducted to evaluate genotoxicity. Single administration of dose levels of 500, 1000, and 2000 mg/kg body weight/day to mice for 15 days did not produce any significant mortality, clinical signs, body weight loss, and gross findings. E. splendens extracts in the range of 156.3–5000 lg/plate did not induce mutagenicity in S. typhimurium and E. coli with and without metabolic activation system. Any significant chromosomal aberration was not observed in CHL cells 6 h after treating with the extract at the concentrations of 1250, 2500, and 5000 lg/mL in absence and presence of metabolic activation system. However, frequency of chromosomal aberration in 22 h after treatment without metabolic activation system was increased with showing a pattern of dose–response relationship. The highest concentration of 5000 lg/mL significantly induced chromosomal aberration. E. splendens extracts may induce chromosomal structure abnormality in CHL cells. This study suggests that further study is needed to assess the potential genotoxic effects of E. splendens extracts. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Elsholtzia splendens; Safety; Acute toxicity; Bacterial reverse mutation; CHL chromosomal aberration
1. Introduction Many species of genus Elsholtzia wild are predominantly distributed over the east of Asia and some of them are used as an ingredient of traditional medicine (Ling et al., 2004). Among them, Elsholtzia splendens is spontaneously growing in South Korea (Chung and Lee, 2002). It is perennial herb of aromatic plant and contains refined oil composed of elsholtzidiol, sterol, and phenol (WHO, 1998; Youn, 1992; Song and Chae, 2004). It has been used for perfume, aroma therapy, edible and medicinal plants (Lee et al., *
Corresponding author. Tel.: +82 2 901 8496; fax: +82 2 901 8372. E-mail address: [email protected] (G.-H. Kim).
0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.10.036
2005). Many studies have been reported that E. splendens provide effects on reducing pain, inflammation, fever and contamination of microorganism compared to other species of Elsholtzia wild (Youn, 1992). Due to its functional properties, it is currently used as a traditional medicine for the treatment of headaches, fevers, diarrhea, and edema in Asia (WHO, 1998). E. splendens also has abundant flavonoids which is well-known to have anti-cancer and anti-oxidative components (Middleton and Kandawami, 1993; Fiala et al., 1985; Chen et al., 2002). Several studies have been reported protective effect of flavonoids extracted from E. splendens on inhibiting inflammation (Kim and Kim, 2003), myocardial ischemia (Ling et al., 2004), and myocardial apoptosis (Ling and Lou, 2005). Since
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E. splendens are known to have medicinal properties mainly owing to flavonoids, interest by both consumer and industry is increasing for the development of food products with health benefits. However, there is insufficient background information on toxicological evaluation of E. splendens extracts to give an assurance of safety in developing dietary supplements and functional foods. Therefore, the objective of this study was to evaluate safety on extract of E. splendens using acute toxicity of oral acute toxicity, bacterial reverse mutation test, and chromosome aberration test. 2. Materials and methods 2.1. Sample preparation E. splendens harvested from Hwacheon around October, 2005 were obtained. E. splendens was freezing dried and then macerated to powder by mixer (Hanil Electrical Co., FM-681, South Korea). Total flavonoids within E. splendens were extracted according to the method of Kim and Kim (2003): The freeze dried E. splendens (1 g) was mixed with 60 mL of 80% methanol solution (containing 0.05% BHT). This mixture was sonicated for 10 min (Branson 3200 sonicator), then refluxed for 1 h. Following cooling to room temperature, the extract was filled up to 100 mL with 50% methanol. The extract was centrifuged gently (at 850g, Sorvall T 6000D) for 15 min. The supernatant was collected and nitrogen gas was gently applied to evaporate organic solvents. The extract was stored at 4 °C until analysis. Further analysis showed that the content of total flavonoids in the extract was 90.1%, which was detected by colorimetric method using apigenin (Fig. 1) as the control. We conducted sample preparation procedure coupled with Cirsium japonicum to validate the analysis, showing the similar result on total flavonoid in C. japonicum to previous study.
2.2. Acute oral toxicity study in mice Both 24 female and 24 male ICR mice (specific pathogen free, 5 weeks of age) were purchased from Orient Bio (Orient Bio Inc., South Korea). Mice were assigned to each group (n = 5) and acclimated for a week in the housing: Temperature was maintained at 23 ± 3 °C and relative humidity was 50 ± 10%. A 12-h light/dark cycle was observed and air was changed 10–20 times per day. Mice were freely accessed to stock rodent pellet (PMI Nutrition International, Richmond, In, USA) and tap water. The study protocol was reviewed by IACUC (Korea Institute of Toxicology under the Korea Research Institute of Chemical Technology, South Korea) and conducted in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) international recognition (2006). Body weight of the mice at study beginning was 29.60 ± 1.07 for males and 21.47 ± 1.26 g for females. Mice were fasted for 4 h prior to oral administration. E. splendens extract was dissolved in CMC (carboxymethyl cellulose). Constant volume (10 mL/kg body weight) containing different concentration of E. splendens extracts (50, 100, and 200 mg/mL) were orally administered to each five mice per sex to achieve the dose of 500, 1000, and 2000 mg/kg body weight/day. CMC was used as negative control. Feeds and water were provided after 4 h of
Fig. 1. The structure of apigenin in Elsholtzia splendens extracts.
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oral administration. Clinical symptoms including mortality, clinical sign, and gross findings were observed once a day for 15 days. Body weight was measured right before oral administration and 2, 4, 8, 15 days after administration. Histopathology evaluations were performed on organs and tissue after mice were sacrificed.
2.3. Bacterial reverse mutation assay Potential mutagenicity of E. splendens extracts was evaluated according to the standard Ames protocol (Maron and Ames, 1983). Histidine requiring S. typhimurium strains (TA100, TA1535, TA98, and TA1537) and tryptophan-requiring Escherichia coli (WP2uvrA) used in this study were obtained from Molecular Toxicology Inc. (Boone, NC, USA). E. splendens extracts were dissolved in DMSO (Dimethylsulfoxide, 99.99% of purity, Sigma–Aldrich Chemical Co.) to be a final concentration of 5 mg/ mL. This concentrate solution was assigned for the highest test concentration (5000 lg/mL) and serially diluted with DMSO for lower test concentrations (2500, 1250, 625, 312.5, 156.3 lg/mL) according to OECD guideline (1997a). The mutagenicity assay was carried out in both the presence and absence of metabolic activation system (S9 mix) (Rat liver S9, Molecular Toxicology Inc. Boone, NC, USA), along with the negative and positive controls as described in OECD Guidelines for the testing of chemicals TG No. 471 ‘Bacterial reverse mutation test’ (1997) (Table 3). Each treatment was made in triplicate. The sources and grades of positive control materials were as follows: Sodium azide (100.1% purity, Sigma– Aldrich Chemicals Co.), 2-nitrofluorene (98.1% purity, Aldrich Chemical Co., Milwaukee, WI, USA), 9-aminoacridine (97.7% purity, Merck Chemical Co.), 4-nitroquinoline 1-oxide (99% purity, Sigma Chemical Co.), 2-Aminoanthracene (99.8% purity, Aldrich Chemical Co.), Benzo(a)pyrene (99.8% purity, Sigma–Aldrich Chemical Co.).
2.4. In vitro mammalian chromosome aberration test In vitro mammalian chromosome aberration test was conducted according to OECD Guidelines for the testing of chemicals TG No. 473 ‘In vitro mammalian chromosome aberration test’ (OECD, 1997). Detail experiment procedure was performed as described in both Ishidate et al. (1981) and Dean and Danford (1984). Chinese Hamster Lung (CHL) cell derived from the female newborn Chinese hamster was obtained from American Type Culture Collection (Manassas, VA). Modal chromosome number of CHL was 25 and the cycle of fission was approximately 15 h. CHL cell kept in liquid nitrogen was thawed and cultured more than 7 days. Microbial contamination was tested before starting experiment. Cell medium was composed of Minimum Essential Medium (GIBCOInvitrogen, Carlsbad, California, USA), 10% Fetal Bovine Serum (FBS, GIBCO-Invitrogen), 2.2 g of sodium bicarbonate, 292 mg of L-glutamine, 100 lg/ml of streptomycin sulfate, and 100 units of penicillin GNa. CHL cell was seeded at 4 104 cells/mL using a plastic plate and incubated for 3 days at 37 °C with 95% air and 5% CO2. E. splendens extracts was solublized in DMSO. Three dose levels were finally chosen due to their cytotoxicities. Three experiment sets were performed with the concentration in the level of 1250, 2500, 5000 lg/mL both in presence and absence of metabolic activation system (S9 mix). Test concentration was selected according to OECD guideline (1997b). The cells were incubated for 6 h with/without metabolic activation system or 22 h without metabolic activation system. Two plates per concentration were used. Cells were washed with 5 mL of CMFD-PBS (Ca++ and Mg++ free Dulbecco’s phosphate buffered saline) after 6 h or 22 h-treatment. Colchicine (Sigma– Aldrich Chemical Co.) was added to accumulate metaphase cells 2 h before the end of culture. After incubation, cells were collected. DMSO, CPA (Cyclophosphamide monohydrate, 99.7% of purity, Sigma Chemical Co.), and EMS (Ethylmethanesulfonate, Sigma Chemical Co.) were used for negative control, positive control with metabolic activation, and positive control without metabolic activation, respectively. Cells were separated by Trypsin–EDTA for counting relative cell count. The cells were separated by centrifugation, and then resuspended in dilute (hypotonic) potassium chloride solution (75 mM KCl). The cells were fixed and
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washed in a mixture of methanol and acetic acid (3:1, v:v), then dropped onto glass microscope slides. Slides were stained with 3% of Giemsa (Sigma–Aldrich Co.), then mounted with cover slips and examined by light microscopy by trained personal.
exposed at 1000 or 2000 mg E. splendens extracts/kg body weight but there was no significant difference from control group.
2.5. Statistical analysis
3.2. Bacterial reverse mutation assay
The changes of body weights and clinical signs for 15 days after oral administration were analyzed by Path/Tox system (Version 4.2.2). All data were evaluated using the SAS program (version 8.2, SAS Institute Inc., Cary, NC). The average of the number of bacterial reverse mutation colonies was represented with standard deviation. Difference from the control group was tested statistically using a t-test. The significant difference (p < 0.05) in the frequency of chromosome aberration between negative control and treatment was evaluated by X2-test and Fisher’s exact test. Cochran–Armitage trend test was applied to estimate dose–response relationship. Difference in means between negative and positive control group was conducted by Fisher’s exact test.
Mutagenicity of the E. splendens extracts was evaluated in a bacterial reverse mutation assay using histidine requiring S. typhimurium (TA100, TA1535, TA98, and TA 1537) and tryptophan-requiring E. coli (WP2uvrA) (Table 2). Regardless of presence of S9 mix, the number of reverting colonies in all the level of treatment was not significantly higher than that in negative control (Table 2). There was no cytotoxicity in all bacterial systems used in mutation assay. Remarkable increase in the number of revertant colonies was founded in all positive control compared to negative control. Mutagenic activity of the E. splendens extracts at all tested concentration (156.3, 312.5, 625, 1250, 2500, and 5000 lg/plate) was not detected (Table 3).
3. Results 3.1. Acute oral toxicity study Mortality, clinical signs, gross findings, and body weights of mice were measured for 15 days after the oral single gavage administration of E. splendens extracts (0, 500, 1000, 2000 mg/kg body weight). All of the female and male mice after administration at the level of 0, 500, 1000, 2000 mg/kg body weight/day did not show any mortality (Table 1). There were no clinical signs of toxicity in any extract-treated mice at any dose level throughout the 15 day observation period (data not shown). Following the administration of 2000 mg E. splendens extracts/kg body weight/day to female mice, soft stools was observed on day 1. However, this symptom was recovered within 24 h. Slight body weight loss was found from female mice
3.3. In vitro mammalian chromosome aberration test The frequencies of metaphases with aberrant chromosomes and statistical analysis are presented in Table 3. In 6 h treatment with presence of metabolic activation (S9 mix), the frequency of metaphases with aberrant chromosomes in the cultures treated with none, 1250, 2500, and 5000 lg/mL were 1.0, 1.5, 2.0, and 4.5, respectively. Increases in the frequency of metaphases with aberrant chromosomes in those cultures were not either observed or significantly different from negative control (Table 3). Positive control showed significant increases in the frequency of metaphases with aberrant chromosomes
Table 1 Mortality of mice for 15 days after oral administration of Elsholtzia splendens extracts Dose (mg/kg)
Male 0 500 1000 2000 Female 0 500 1000 2000 A B
Days on test 1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
aA bB a b a b a b
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
a b a b a b a b
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
5 0 5 0 5 0 5 0
a = Number animals alive at the start of each study day. b = Number of mortalities during each study day.
S.-M. Shim et al. / Food and Chemical Toxicology 46 (2008) 1042–1047
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Table 2 Bacterial reverse mutation assay with Elsholtzia splendens extracts Revertant colonies/plate (Mean) [factor]a
Tester strain
Chemical treated
Dose (lg/plate)
Without S9 mix
With S9 mix
TA 100
Test itemb
0 156.3 312.5 625 1250 2500 5000
104 ± 8 105 ± 8 [1.0] 118 ± 4 [1.1] 112 ± 3 [1.1] 103 ± 2 [1.0] 124 ± 14 [1.2] 122 ± 5 [1.2]
133 ± 10 113 ± 10 [0.8] 129 ± 11 [1.0] 121 ± 19 [0.9] 127 ± 19 [1.0] 121 ± 16 [0.9] 139 ± 4 [1.0]
TA 1535
Test item
0 156.3 312.5 625 1250 2500 5000
15 ± 1 18 ± 6 14 ± 5 16 ± 2 16 ± 2 17 ± 3 18 ± 2
[1.2] [0.9] [1.1] [1.1] [1.1] [1.2]
12 ± 1 10 ± 5 [0.8] 9 ± 4 [0.8] 14 ± 2 [1.2] 11 ± 1 [0.9] 12 ± 1 [1.0] 11 ± 3 [0.9]
0 156.3 312.5 625 1250 2500 5000
18 ± 2 25 ± 4 19 ± 2 21 ± 6 20 ± 4 16 ± 5 21 ± 6
[1.4] [1.1] [1.2] [1.1] [0.9] [1.2]
30 ± 6 35 ± 4 [1.2] 36 ± 11 [1.2] 37 ± 7 [1.2] 35 ± 10 [1.2] 39 ± 6 [1.3] 34 ± 2 [1.1]
0 156.3 312.5 625 1250 2500 5000
10 ± 3 9 ± 2 [0.9] 8 ± 2 [0.8] 9 ± 2 [0.9] 8 ± 3 [0.8] 7 ± 1 [0.7] 9 ± 2 [0.9]
21 ± 3 22 ± 5 19 ± 5 19 ± 3 23 ± 3 23 ± 2 29 ± 5
[1.0] [0.9] [0.9] [1.1] [1.1] [1.4]
0 156.3 312.5 625 1250 2500 5000
16 ± 4 21 ± 4 17 ± 3 13 ± 1 14 ± 5 15 ± 3 15 ± 4
25 ± 6 20 ± 2 20 ± 5 23 ± 2 26 ± 6 26 ± 6 28 ± 6
[0.8] [0.8] [0.9] [1.0] [1.0] [1.1]
0.5 0.5 2 50 0.5 2 2 2 2 4
620 ± 10 [6.0] 469 ± 39 [31.3] 574 ± 90 [31.9] 355 ± 88 [35.5] 103 ± 5 [6.4]
TA 98
TA1537
E. coli WP2uvrA
Positive controls TA 100 TA 1535 TA 98 TA 1537 WP2uvrA TA100 TA 1535 TA 98 TA 1537 WP2uvrA
Test item
Test item
Test item
SA SA 2-NF 9-AA 4NQO BP 2-AA BP BP 2-AA
[1.3] [1.1] [0.8] [0.9] [0.9] [0.9]
15 ± 4 [1.0] 19 ± 4 [1.1]
926 ± 39 [7.0] 463 ± 2 [38.6] 390 ± 57 [13.0] 155 ± 19 [7.4] 185 ± 9 [7.4]
Abbreviations: SA, sodium azide; 2-NF, 2-nitrofluorene; 9-AA, 9-aminoacridine; 4NQO, 4-nitroquinoline 1-oxide; 2-AA, 2-aminoanthracene; BP, benzo(a)pyrene. a Number of revertant colonies of treated plate/number of revertant colonies of vehicle control plate. b Test item: Elsholtzia splendens extracts.
(p < 0.01). In the absence of S9 mix, no biological or significant increases in the frequency of metaphases with aberrant chromosomes were found in cultures 6 h after treatment of E. splendens extracts (0, 1250, 2500, 5000 lg/mL) (Table 3). However, the cultures incubated for 22 h without S9 mix showed increases in the frequency of metaphases with aberrant chromosomes with the dose–
response relationship. Statistically significant increases was observed in culture treated with E. splendens extracts of 5000 lg/mL (p < 0.01). On the contrary, positive control in both 6 h and 22 h-treatment cultures without S9 mix presented 26.5 and 44.5 of increases in the frequency of metaphases with aberrant chromosomes, respectively, and it was statistically significant increases (p < 0.01). In the
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Table 3 Chromosome aberration test with Elsholtzia splendens extracts and relative cell count Nominal concentration of test itema (lg/mL)
Timesb (h)
Mean aberrant metaphases
Mean total aberrations
Mean of PP + ER
Relative cell countsf (%)
6 h treatment ( S9) 0 1250 2500 5000 EMS 800
6–18 6–18 6–18 6–18 6–18
2.5/2.0c 2.0/2.0 3.5/3.0 3.5/3.0 27.0/26.5**,d
2.5/2.0 3.0/3.0 5.0/4.5 4.5/4.0 37.5/37.0
0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0
100 123 101 86 119
22 h treatment ( S9) 0 1250 2500 5000 EMS 600
22–2 22–2 22–2 22–2 22–2
1.0/0.5 3.0/2.5 3.5/3.0 8.5/8.0**,d 45.5/44.5**,e
1.0/0.5 3.5/3.0 3.5/3.0 11.5/10.5 67.5/65.0
0.0 + 0.0 0.5 + 0.0 0.5 + 0.0 0.0 + 0.0 0.0 + 0.0
100 139 118 102 99
6–18 6–18 6–18 6–18 6–18
2.0/1.0 2.0/1.5 2.0/2.0 5.0/4.5 39.0/38.5**,d
3.5/2.5 2.0/1.5 3.0/3.0 5.5/4.5 58.5/57.5
0.0 + 0.0 0.5 + 0.0 0.0 + 0.0 1.0 + 0.0 0.5 + 0.0
100 97 91 82 58
6 h treatment (+S9) 0 1250 2500 5000 CPA 6
S9 mix
+ + + + +
**
Significantly different from the control at p < 0.01. Visible turbidity was observed when treated and at the end of the treatment. Abbreviations: PP, Polyploid; ER, Endoreduplication; EMS, Ethylmethanesulfonate; CPA, Cyclohosphamide monohydrate. a Elsholtzia splendens extracts. b Treatment time-recovery time. c Gaps included/excluded, means of duplicated cultures; 100 metaphases were examined per culture. d The vehicle and test-item treated group; Chi-square test and Fisher’s exact test. e The vehicle and positive control groups; Fisher’s exact test. f Relative cell counts = (cell counts of treated flask/cell counts of control flask) 100 (%).
#
experiment to examine if there was as reproducible effect or unaccounted experimental error in +S9 and S9, we obtained similar results to previous experiments. 4. Discussion The major flavonoid isolated from E. splendens extracts were apigenin, apigenin-7-O-glucoside, luteolin-7-Oglucoside, and linarin (Kim and Kim, 2003). Current study found that the total flavonoid extracted from E. splendens was apigenin, corresponding with 90.1%. Apigenin is widely found in dietary fruit, vegetables, and plant-derived beverages such as tea and wine (Noel et al., 2006). It has shown to possess antioxidant activity, anti-inflammatory, hypothermic, and antipyretic effect and protective effect on radiation-induced chromosomal damage (Youn, 1992; Bae et al., 2007; Kim and Kim, 2003; Rithidech et al., 2005). Those pharmacological effects have increased the public’s interest in the use of Elsholtzia splendens, which are containing significant of apigenin for its potential health benefits. However, the adverse effects of excessive apigenin intake remain highly uncertain. In order to provide safety information on E. splendens extracts, oral acute toxicity and genotoxicity using bacterial reverse mutation test and chromosomal aberration test are carried out in this study. Single dose of E. splendens extracts up to level of 2000 mg/kg body weight did not show adverse effect on clinical sign for 15 days, implying
that E. splendens extracts do not cause any acute toxicity. This result support the LD50 value of E. splendens extracts could be greater than 2000 mg/kg body weight/day. In bacterial reverse mutation test conducted in a range of 156.3, 312.5, 625, 1250, 2500, 5000 lg/plate for each S. typhimurium (TA100, TA1535, TA98, and TA 1537) and Escherichia coli (WP2uvrA), gene mutation was not induced. Similar to our result, genotoxic effect was not introduced in test with S. typhimurium strains TA 1538 and TA 1978 (Czeczot and Kusztelak, 1993). On the contrary, earlier study reported that apigenin showed weak mutagenic effects with S. typhimurium strains TA 100 and TA 98 compared to other flavonoids structurally related (Nagao et al., 1981). These observations imply that difference in bacterial reverse mutation depends on bacterial strain tested. A chromosomal aberration test using cultured CHL cells was performed with or without metabolic activation system. At the highest concentration of 5000 lg/mL in 22 h treatment without S9 mix showed significant increases in the incidence of chromosomal aberrations (p < 0.01). In addition, the frequency of chromosomal aberrations in treatment group was found to have a pattern with dose–response relationship. Results from our study illustrate that E. splendens extracts possibly have potential effect on inducing the chromosomal aberrations. This finding is similar to that previously reported in micronucleus assay (Noel et al., 2006). They found apigenin induced micronuclei in a dose-dependent manner at high dose levels between 600
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and 5400 mg/mL. On the other hand, lower dose of apigenin such as ranged from 2.5 to 25 lg/mL showed a radioprotective effect on radiation-induced chromosomal damage in human lymphocytes (Rithidech et al., 2005). As with other flavonoids, it is appeared that apigenin may show mutagenic or antimutagenic effects depending upon the intake level. Our results suggest that E. splendens extracts may not induce gene mutation but structural aberration of chromosome. Therefore, our study suggests that further studies such as in vivo micronucleus assay can quantitate activity of chromosomal aberrations should be performed. More research on the toxicological properties of E. splendens extracts containing apigenin that will be consumed as functional food or medicine are required for understanding of adverse health effects upon routine intake. In addition, bioavailability of E. splendens extracts including digestive stability, factors affecting intestinal absorption, and metabolism have to be investigated to clarify its health benefit or adverse health effects from oral intake. Acknowledgements The authors wish to thank Institute of toxicology for technical assistance to conduct animal and in vitro study for this research. This Study was supported by Agriculture R&D Promotion Center, Ministry of Agriculture and Forestry, Republic of Korea (204024-03-3-SB010) and in part from the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-005J13001). References AAALAC international (Association for Assessment and Accreditation of Laboratory Animal Care), 1998. Available from:
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