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Regulatory Toxicology and Pharmacology 71 (2015) 365–370

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Genotoxicity studies on the root extract of Polygala tenuifolia Willdenow Ki Young Shin a,b,⇑, Beom Young Won a, Hyun Jee Ha a, Yeo Sang Yun a, Hyung Gun Lee a a b

Research & Development Center, Braintropia Co. Ltd., Anyang-si, Gyeonggi-do 431-716, Republic of Korea Department of Microbiology, College of Natural Science, Dankook University, Cheonan-si, Chungnam 330-714, Republic of Korea

a r t i c l e

i n f o

Article history: Received 5 December 2014 Available online 7 February 2015 Keywords: Polygala tenuifolia Willdenow Genotoxicity Reverse mutation Chromosomal aberration Micronucleus test

a b s t r a c t The root of Polygala tenuifolia Willdenow has been used for the treatment against insomnia, amnesia, depression, palpitations with anxiety, and memory improvement. However, there is no sufficient background information on toxicological evaluation of the root to given an assurance of safety for developing dietary supplements and functional foods. As part of a safety evaluation, the potential genotoxicity of the root extract of P. tenuifolia was evaluated using a standard battery of tests (bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay). In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, the extract did not increase the number of revertant colonies in any tester strain with or without metabolic activation by S9 mix, and did not cause chromosomal aberration in short-period test with the S9 mix or in the continuous (24 h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg/day showed no significant or dose dependent increase in the frequency of micronucleated polychromatic erythrocytes (PCE). These results indicate that ingesting the rot extract P. tenuifolia is not genotoxic at the proper dose. Ó 2015 Published by Elsevier Inc.

1. Introduction A number of medicinal plants have a long history of traditional use, and they have played many important roles for humans since the dawn of civilization (Pankaj et al., 2009), because medicinal herbs are used for the prevention and treatment of diseases (Firenzuoli and Gori, 2007; Wang et al., 2009). The use of herbal products as primary therapeutics or supplements for improving health-related conditions is popular worldwide (Seeff et al., 2001). Interestingly, medicinal plants have provided high opportunities for the development of herbal food products, dietary supplements, and functional foods (Chau and Wu, 2006). Recently, concerns have been raised over the lack of quality control and of scientific evidence for the efficacy and safety of these products (Firenzuoli and Gori, 2007; Rousseaux and Schachter, 2003). Especially, people become more interested in the food safety and well-being in recent years, so the demand for functional food from natural sources is increased (Lee et al., 2003), because medicinal plants have undesirable side effects (Chan and Cheung, 2000). In traditional oriental medicine, the root of Polygala tenuifolia Willdenow has been prescribed in Asia for thousands of years,

because of its expectorant, tonic, tranquilizer, and antipsychotic properties (Spelman et al., 2006; Wang et al., 2007; Nagai and Suzuki, 2001; Klein et al., 2012; Jin et al., 2012). Previously, we reported that the root extract of P. tenuifolia can enhance memory and cognitive function in two animal models or two human models (Park et al., 2002; Lee et al., 2009; Shin et al., 2009a,b) and the acute or subchronic toxicity of the root extract was not toxic to dogs and rats (Shin et al., 2014). Several researchers have demonstrated the effects of P. tenuifolia, but information on its safety is lacking. Therefore, systematic evaluation of the safety of the root extract of this herb is necessary for development of new foods or drugs. In this study, we evaluated the potential genotoxicity of the dried root extract of P. tenuifolia was conducted using the standard battery of tests recommended by OECD and the Korea Food and Drug Administration (KFDA). The test included the bacterial reverse mutation test (Ames test), the chromosomal aberrations test and the micronucleus tests to assure its safe use in dietary supplements or functional ingredients.

2. Material and methods 2.1. Extraction of Polygala tenuifolia

⇑ Corresponding author at: Department of Microbiology, College of Natural Science, Dankook University, Cheonan-si, Chungnam 330-714, Republic of Korea. Fax: +82 031 425 3719. E-mail address: newsky73@braintropia.com (K.Y. Shin). http://dx.doi.org/10.1016/j.yrtph.2015.01.016 0273-2300/Ó 2015 Published by Elsevier Inc.

The dried root extract of P. tenuifolia (500 g) was refluxed with 75% ethanol for 4 h in a boiling water bath. This procedure was repeated twice and the ethanol solution was concentrated under


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vacuum. The concentrated ethanol fraction (125 g) of the plant root, obtained as described above, was used for this study (Park et al., 2002). 2.2. Bacterial mutation assay (Ames test) The Ames test was carried out in according to the methods described in OECD (1997a) and the KFDA Notification No. 199961 (KFDA, 1999). Two strains of Salmonella typhimurium, TA 100 and TA 1535, and Escherichia coli WP2 uvrA were used for the detection of base-pair substitution mutations. Two additional S. typhimurium strains, TA 98 and TA 1537, were used for the detection of frameshift mutations. The tester strains were obtained from Molecular Toxicology, Inc. (111 Gibralter avenue, Annapolis, MD21401, USA) and Dr. M.H.L Green (University of Sussex, Falmer, Brighton, UK). The phenotypic properties of the S. typhimurium strains, including histidine requirement, presence of uvrB mutation, presence of R-factor, presence of rfa mutation, and number of spontaneous revertants were evaluated. For E. coli, the tryptophan requirement, presence of uvrA mutation, and the number of spontaneous revertants were checked according to the method of Maron and Ames (1983). These procedures were conducted at Preclinical Research Center (ChemOn, Inc., Gyeonggi-do, Korea) prior to the conduct of the study. The following chemicals were used as positive control: sodium azide (SA), 2-aminoanthracene (2-AA), 4-nitroquinoline-N-oxide (4-NQO), and 9-aminoacridine (9-AA) (all obtained from Sigma Aldrich Company). Sterile distilled water was included as a negative control and served as the solvent for sodium azide. All the other positive controls substances were dissolved in dimethylsulfoxide (Sigma–Aldrich Company). The metabolic activation system consisted of a commercial S9 fraction obtained from Molecular Toxicology, Inc. (Boone, NC, USA). The S9 fraction was prepared from the livers of male Sprague–Dawley rats pretreated with Aroclor 1254. Cofactor-1 (MgCl6H2O, KCl, glucose6-phosphate, NADPH, NADH, and sodium phosphate buffer) was supplied from Wako Pure Chemical, Ind., Ltd. (Japan). The S9 mix containing 50 lL of S9/mL solution was prepared according to standard methods. The study was conducted using the direct plate incorporation method. Briefly, this involved addition to a sterile tube of 2.0 mL top agar (45 °C), 0.1 mL test substance, 0.5 mL of the S9-mix or sodium phosphate buffer and 0.1 mL bacterial culture. Following vortexing the contents were poured onto 25 mL minimal glucose agar plates. Following this and the hardening of the top agar, plates were turned over and incubated at 37 °C for 48 h. At this time, the numbers of revertant colonies were counted. Two separate experiments were conducted, a dose-range finding test and a main test. The range-finding study was conducted in both the presence and absence of S9-mix and used 1 plate/dose level. The doses tested were 1.6, 8, 40, 200, 1000, 2500, 5000 lg/plate. Given the lack of growth inhibition of the colonies at any concentration, in the main study 5000 lg/plate was selected as the highest dose. Four lower doses of 62, 185, 556, 1667, and 5000 lg/plate were also included. 2.3. Mouse micronucleus test The micronucleus test was carried out in according to the methods described in OECD (1997b) and the KFDA Notification No. 199961 (KFDA, 1999). Total 30 healthy male ICR mice (8-weeks-of-age; Samtako, BIO KOREA) were used in the micronucleus assay to evaluate. The test substance was administered once a day for 2 days by gavage to male ICR mice at doses 0, 500, 1000 and 2000 mg/kg. The test substance was dissolved in DW and animals were orally administrated the test substance at the levels listed for two consecutive days. Cycliphosphamide (CPA) was administrated intraperitoneally at 70 mg/kg as the positive control. The clinical signs and body

weight were assessed once daily. Bone marrow preparations were made according to Schmid (1975) and two slides of the cell suspension per animal were made. Following the sacrifice with fetal bovine serum using a disposable syringe with 23G needle, the cell suspension was centrifuged and the supernatant was decanted. After removing the supernatant, a small drop of the viscous suspension was smeared onto clean microscope slides. Preparations were air-dried, and fixed by submerging in absolute methanol for 5 min. Fixed slides were stained with May-Grunwald and Giamsa. Stained slides were rinsed with distilled water, dried, and mounted with mountant (Depex, Fluka). Slides were examined under 1000 magnification. Small round or oval shaped bodies, with a size of about 1/5–1/20 of the diameter of a polychromatic erythrocyte (PCE), were counted as micronuclei. A total of 2000 PCEs were scored per animal by the same observer for determining the frequencies of micronucleated polychromatic erythrocytes (MNPCEs). PCE/(PCE + NCE (normochromatic erythrocyte)) ratio was calculated by counting 500 cells. 2.4. Chromosome aberration in Chinese hamster lung (CHL) cells The chromosome aberration study was carried out in accordance with the methods described in OECD (1997c) and the KFDA Notification NO. 1999-61 (KFDA, 1999). The experimental methods were based on the published reported by Ishidate et al. (1981) and Dean and Danford (1984). The Chinese hamster lung (CHL) fibroblast cell line used in the experiment was CHL/IU from American Type Culture Collection (ATCC #CRL-1935, CHL/IU). The cells were subcultured and maintained at Preclinical Research Center (ChemOn, Inc., Gyeonggi-do, Korea) prior to use in the study. The modal chromosome number of the CHL cells was 25 with a doubling time of 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 were cultured in reconstituted minimum essential medium (MEM, Gibco-BRL #41500-034) supplemented with 2200 mg of sodium bicarbonate, 292 mg of L-glutamine, antibiotics (Penicillin G and Streptomycin sulfate, GibcoBRL 15140-122) and 10% (v/v) fetal bovine serum (FBS, Gibco-BRL #16000-044) per liter. CHL cell was seeded at 6  104 cells/mL using a plastic plate and incubated for 3 days at 37 °C with 95% air and 5% CO2. Cells were subcultured every 2–3 days. Rapidly growing cultures were trypsinized, suspended in culture medium, and counted. Culture flasks (25 cm2) were seeded with 6  104 cells, each in 5 mL medium, and incubated for 3 days. The test substance and the positive controls, cyclophosphamide (CPA) and ethylmethanesulfonate (EMS), were dissolved in DW. A range finding study was performed to determine the highest concentration for the main study. The highest concentration was set as 5000 lg/ml according to the OECD guideline (OECD, 1997c). The test substance and positive controls were treated in the presence (+S9) and absence (S9) of the S-9 mix. A preliminary range-finding test (Study No. 04-VG-255P) was performed to select vehicle and dose levels of the present study. In the preliminary study cells were treated over at the doses of 0, 0.8, 2.3, 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 lg/mL in the presence and absence of metabolic activation system, with the same method as present study. After 24 h from the start of treatment, the cells of each flask were dissociated and counted to calculate relative cell count (RCC) which was regarded as the index of cytotoxicity. Based on the results of the preliminary study the doses which induced inhibition of cell growth 50% or greater were calculated. The treatment plan of main study is as shown in the Supplementary 1. The presence of aberrant metaphases was evaluated in each treatment group at two concentrations that met the acceptance criteria for viability and analyzable metaphases. At the end of treatment, cells were washed with Ca2+ and Mg2+ free Dulbecco’s phosphate buffered saline (CMFD-PBS), and fresh media was added for harvest.


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Approximately 22 h after the initial treatment, colchicine was added to each culture (final concentration, 1 lM) and incubated for an additional 2 h. After incubated, cells were collected count (RCC) was calculated. Cells were collected by centrifugation and resuspended in a 75 mM KCl solution. After 10 min at room temperature, cells were fixed in fixative (methanol:glacial acetic acid = 3:1, v/v) and then dropped onto slides. Slides were stained with 5% Giemsa solution (Sigma–Aldrich Co., St. Louis, MO, USA). Chromosomal aberrations were morphologically identified according to the principles described in the ‘Atlas of chromosome aberration by chemicals (JEMS-MMS, 1988)’. Any structural aberrations were recorded including type breaks and exchanges; and chromosome type breaks and exchanges. Each type of aberration was recorded; the number of aberrant metaphases (showing one or more aberration, including and excluding gaps) and the total aberrations (including and excluding gaps) were calculated (Supplementary 2).

nificant increase in the frequencies of micronucleated PCEs at any dose level of the test substance. The positive control substance, cyclophosphamideH2O, induced a statistically significant increase of MNPCE (75.17) when compared with that of the vehicle control (P < 0.01). The cytotoxicity indices, PCE/(PCE + NCE) ratios, were 0.38, 0.35, 0.45 and 0.34 for the vehicle control (0), 500, 1000 and 2000 mg/kg/day group, respectively. There was no statistically significant difference in the ratios at any dose level of the test substance when compared with that of the vehicle control. The positive control substance induced statistically significant decrease in the ratio, 0.21, as compared with that of the vehicle controls (P < 0.01). No mortality was observed at any of the dose levels and there were no abnormal signs attributable to the administration of the test substance. There was no statistically significant difference in the body weights of animals in all groups (Table 2 and Supplementary 3).

2.5. Statistical analysis

3.3. Chromosome aberration in Chinese hamster lung (CHL) cells

For incidences of micronucleated immature erythrocytes, due to the non-normality of the data, data were transformed by arcsin (sqrt(x)) and analyzed by the Kruskal and Wallis (1952). Kruskal–Wallis analysis of variance was followed by multiple comparisons using the Dunnett’s test (Dunnett, 1964). The distribution of PCE/(PCE + NCE) ratio satisfied normality, and the data were analyzed using one-way analysis of variance (ANOVA). The study were greater was accepted when all of the PCE/(PCE + NCE) ratio were greater than 0.1 (Heddle et al., 1984). The result was judged as positive when there was a statistically significant and dose-related increase or a reproducible increase in the frequency of MNPCEs (in the micronucleus test) or aberrant metaphases (chromosomal aberration test) at least at one dose level. Body weight values were also subject to ANOVA. Results were considered significant with P < 0.05. Statistical analyses were selected based on the methods used in published reports (Richardson et al., 1989) using Statistical Analysis System (SAS) software (Ver. 9.1.3, SAS Institute Inc., NC, USA). Metaphases were classified either as normal metaphase or as aberrant metaphase with one or more aberrations, and the frequency of aberrant metaphase was analyzed. Numerical aberrations were classified into diploid (DP), polyploidy (PP, P37 chromosomes) and endoreduplication (ER), and the frequency of PP + ER was analyzed. Differences were regarded as statistically significant if P < 0.05. Fisher’s exact test was used to determine differences between the vehicle and positive control groups (Fisher, 1970).

The frequencies of aberrant metaphase were 0.0 per 100 metaphases in the negative control and all test substance-treated groups, and there were no statistically significant increases in the frequencies of aberrant metaphases in any of the treatment groups when compared with the negative control group. The frequencies of metaphases with numerical aberration were 0.0 per 100 metaphases in the negative control and all test substance treated groups. On the other hand, there was a statistically significant (P < 0.01) increase in the frequency of aberrant metaphase (25.0) in the positive control group when compared with the negative control group. In 6 h treatment with the absence of S9 mix, the frequencies of aberrant metaphase were no more than 0.5 per 100 metaphases in the negative control and all test substance-treated groups, and there were no statistically significant increases in the frequencies of aberrant metaphases in any of the treatment groups when compared with the negative control group. The frequencies of metaphases with numerical aberration were no more than 0.5 per 100 metaphases in the negative control and all test substance-treated groups. There was a statistically significant (P < 0.01) increase in the frequency of aberrant metaphase (24.0) in the positive control group when compared with the negative control group (Table 3). In 24 h treatment with the absence of S9 mix, the frequencies of aberrant metaphase were 0.0 per 100 metaphases in the negative control and all test substance-treated groups, and there were no statistically significant increases in the frequencies of aberrant metaphases in any of the treatment groups when compared with the negative control group. The frequencies of metaphases with numerical aberration were 0.0 per 100 metaphases in the negative control and all test substance-treated groups. There was a statistically significant (P < 0.01) increase in the frequency of aberrant metaphase (19.5) in the positive control group when compared with the negative control group (Table 4).

3. Results 3.1. Bacterial mutation assay (Ames test) There was neither increase of colonies nor cytotoxicity in S. typhimurium TA100, TA1535, TA98 and TA1573 at any dose level of test substance both in the presence and absence of metabolic activation system. In WP2 uvrA, there was neither increase of colonies nor cytotoxicity at any dose level of the test substance both in the presence and absence of metabolic activation system. The background lawn was also normal in all strains. There was significant increase in the number of colonies in all positive control groups (Table 1). 3.2. Mouse micronucleus test The frequencies of micronucleated PCEs among 2000 PCEs were 0.17, 0.33, 0.00 and 0.50 for the vehicle control (0), 500, 1000 and 2000 mg/kg/day group, respectively. There was no statistically sig-

4. Discussion P. tenuifolia Willdenow is a perennial herbaceous plant distributed widely in China and Korea. In particular, the root of the herb is used against insomnia, neurasthenia, amnesia, depression, palpitations with anxiety, restlessness, and disorientation, dementia, and memory failure (Huang, 1993; Liu et al., 2010). The extract has been shown to contain C-glycosides, triterpene saponins, sucrose esters, and oligosaccharide esters (Ikeya et al., 2004; Ling et al., 2013). It also contains various substances like tenuigenin, tenuifolin, DISS (3,60 -disinapolyl sucrose), and TMCA (3,4,5-trimethoxycinnamic acid), which have been shown to have proliferative and protective effects


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Table 1 Bacterial reverse mutation assay of the root of Polygala tenuifolia. Tester strain

Chemical treated

Dose (lg/plate)

Revertant colonies/platea (mean) [factor] Without S9 mix

With S9 mix

TA 100

The root of P. tenuifolia

0 62 185 556 1667 5000

186 ± 12 171 ± 8 [0.9] 167 ± 14 [0.9] 180 ± 4 [1.0] 189 ± 7 [1.0] 183 ± 12 [1.0]

171 ± 7 168 ± 4 [1.0] 179 ± 9 [1.0] 179 ± 6 [1.0] 183 ± 13 [1.1] 182 ± 16 [1.1]

TA 1535

The root of P. tenuifolia

0 62 185 556 1667 5000

17 ± 4 16 ± 5 16 ± 2 19 ± 3 18 ± 2 16 ± 3

[0.9] [0.9] [1.1] [1.1] [0.9]

15 ± 5 18 ± 2 17 ± 1 17 ± 3 16 ± 5 17 ± 4

[1.2] [1.1] [1.1] [1.1] [1.1]

0 62 185 556 1667 5000

30 ± 3 29 ± 9 32 ± 5 32 ± 7 34 ± 5 31 ± 7

[1.0] [1.1] [1.1] [1.1] [1.0]

26 ± 4 28 ± 4 26 ± 5 26 ± 3 26 ± 3 27 ± 5

[1.1] [1.0] [1.0] [1.0] [1.0]

0 62 185 556 1667 5000

15 ± 3 14 ± 9 14 ± 0 14 ± 4 15 ± 3 17 ± 2

[0.9] [.9] [0.9] [1.0] [1.0]

15 ± 3 13 ± 6 13 ± 1 10 ± 3 10 ± 1 13 ± 4

[0.9] [0.9] [0.7] [0.7] [0.9]

0 62 185 556 1667 5000

27 ± 1 22 ± 1 27 ± 4 29 ± 4 26 ± 3 26 ± 4

[0.8] [1.0] [1.1] [1.0] [1.0]

18 ± 4 16 ± 3 20 ± 6 18 ± 3 21 ± 2 15 ± 6

[0.9] [1.1] [1.0] [1.2] [0.8]

0.5 2 0.5 1 2 0.5 0.5 0.5 50 0.5

1109 ± 47 [6.0] 228 ± 14 [13.4] 907 ± 23 [30.2] 412 ± 16 [27.5] 110 ± 12 [4.1]

TA 98

TA 1537

E. coli WP2 uvrA

Positive controls TA 100 TA1535 TA 98 TA 1537 WP2 uvr A TA 100 TA 1535 TA 98 TA 1537 WP2 uvr A

The root of P. tenuifolia

The root of P. tenuifolia

The root of P. tenuifolia

2-AA 2-AA 2-AA 2-AA 2-AA SA SA 4NQO 9-AA 4NQO

34 ± 3 [1.3]

370 ± 16 [2.2] 216 ± 12 [14.4] 267 ± 16 [10.3] 265 ± 55 [17.7] 95 ± 7 [5.3]

9-AA: 9-Aminoacridine. 4NQO: 4-Nitroquinoline N-oxide. 2-AA: 2-Aminoanthracene. SA: Sodium azide. a No. of colonies of treated plate/no. of colonies of negative control plate.

Table 2 Bone marrow micronucleus test in male ICR mice treated with the root of Polygala tenuifolia. Treatment

Dose (mg/kg)

Number of animals

MNPCE/2000PCE (%)

PCE/(PCE + NCE) (%)

Vehicle control The root of P. tenuifolia

02 500  2 1000  2 2000  2 70  1

6 6 6 6 6

0.17 ± 0.408 0.33 ± 0.817 0.00 ± 0.000 0.50 ± 0.837 75.17 ± 14.972**

0.38 ± 0.078 0.35 ± 0.109 0.45 ± 0.114 0.34 ± 0.097 0.21 ± 0.032**

CPA

Vehicle: sterilin distilluted water. CPA administered once on the day of 2nd admin. Abbreviations: PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; MNPCE: micronucleated polychromatic erythrocyte. CPA: cyclophosphamideH2O (positive control article). ** Significantly different from the vehicle control group at P < 0.01.

on hippocampal neurons (Chen et al., 2010; Chen et al., 2012). However evidence-based information on its safety is limited. Medicinal herbs and herbal formulas are generally considered to be safer than other medications (Lynch and Berry, 2007; Jordan

et al., 2010), there is limited safety data of gerbil formulations (Manu and Anurag, 2013). Many studies have reported the pharmacological efficacy and benefits of P. tenuifolia Willdenow, but little information has been reported about risk and safety. To


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S9 mix

Timec (h)

Mean aberrant metaphasesd

Mean total aberrationsd

Mean of PP + ER

Relative cell counts (%)

0 87.5 175 350 CPA 10

+ + + + +

6–18 6–18 6–18 6–18 6–18

0.5/0.0 0.0/0.0 0.0/0.0 0.0/0.0 26.5/25.0e,**

0.5/0.0 0.0/0.0 0.0/0.0 0.0/0.0 37.0/35.5

0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0

100 93 90 58 71

Test article: Polygala tenuifolia extracts powder. PP: polyploid. ER: endoreduplication. CPA: cyclophosphamide monohydrate. RCC: relative cell counts = cell counts of treated flask/cell counts of control flask  100 (%). ** Significantly different from the control at P < 0.01. a See the Supplementary 3 and 4 for individual data. b Nominal concentration of the test substance. c Treatment time–recovery time. d Gaps included/excluded, means of duplicate cultures. 100 metaphases were examined per culture. e Fisher’s exact test.

Table 4 Chromosome aberration test in the absence of S-9 mix.a Nominal conc. of test itemb (lg/ml)

S9 mix

Timec (h)

Mean aberrant metaphasesd

Mean total aberrationsd

Mean of PP + ER

Relative cell counts (%)

0 162.5 325 650 EMS 800 0 150 300 600 EMS 600

         

6–18 6–18 6–18 6–18 6–18 24–0 24–0 24–0 24–0 24–0

1.0/0.5 0.0/0.0 0.5/0.0 0.0/0.0 26.5/24.0e,** 0.5/0.0 0.0/0.0 0.0/0.0 0.0/0.0 23.0/19.5e,**

1.0/0.5 0.0/0.0 0.5/0.0 0.0/0.0 37.0/33.0 0.5/0.0 0.0/0.0 0.0/0.0 0.0/0.0 30.0/25.5

0.5 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0 0.0 + 0.0

100 79 75 49 78 100 108 106 52 86

Test article: Polygala tenuifolia extracts powder. PP: polyploid. ER: endoreduplication. CPA: cyclophosphamide monohydrate. RCC: relative cell counts = cell counts of treated flask/cell counts of control flask  100 (%). ** Significantly different from the control at P < 0.01. a See the Supplementary 3 and 5 for individual data. b Nominal concentration of the test substance. c Treatment time–recorvery time. d Gaps included/excluded, means of duplicate cultures. 100 metaphases were examined per culture. e Fisher’s exact test.

evaluate some potential genotoxicities of P. tenuifolia extracts, a bacterial reverse mutation test, a chromosomal aberration test and a micronuclei test were carried out in this study. First, the Ames test is a biological assay developed by Ames and coworkers in the early 1970s to assess the mutagenic potential of chemical compounds and complex environmental mixtures, and is a simple and quick assay to estimate the carcinogenic potential. The Ames test uses amino acid requiring strains of S. typhimurium and E. coli to detect point mutation involving substitution, addition, or deletions of one or more DNA base pairs (Maron and Ames, 1983). The Ames test is employed commonly as an initial screen of genotoxicity, particularly point mutation induction activity. Bacterial reverse mutation tests in four histidine auxotroph strain of S. typhimurium and one tryptophan auxotroph strain of E. coli were investigated. There were no increases in the number of revertant colonies at any concentration (5000, 1667, 556, 185 and 62 lg/plate) with and without metabolic system in S. typhimurium (TA 98, TA100, TA1535 and TA1537) and E. coli (WP2 uvrA). Under the conditions of this study, our results indicated that the root extract of P. tenuifolia did not show mutagenicity in bacterial reverse mutation test. Second, the in vitro chromosome aberration test is used to identify agents that induce structural chromosomal aberrations in cultured mammalian cells. Structural aberrations can affect chromosomes or chromatids. The majority of chemical

mutagens induce aberrations of the chromatid type, but chromosomal aberrations can also occur (Ishidate and Sofuni, 1985). In the chromosome aberration assay, there were no statistically significant increases in the number of metaphases with structural aberrations at any dose of P. tenuifolia extracts in the presence or absence of the metabolic activation system in CHL cells. However, positive control showed significant increase in the frequency of metaphases with aberrant chromosomes. Because of its simplicity and efficacy, the micronucleus test has become a popular and useful in vivo procedure for the detection of chemicals-induced chromosome damage. Therefore, P. tenuifolia showed a negative response in the chromosome aberration assay under the conditions of this study. Furthermore, these findings were in accordance with that reported by Manu and Anurag (2013). Third, the micronucleus test is a popular and useful in vitro procedure for the detection of chemically induced chromosome damage and the number of reports using micronucleus testing has increased dramatically in the scientific literature (Ashby, 1985). In the micronucleus test using ICR mice, no abnormal signs in general appearance and body weight were observed. Also, there was no significant increase in the frequency of micronuclei in any dose of P. tenuifolia compared with the vehicle control. The PCE/(PCE + NCE) ratio, an indicator of cytotoxicity, was no significantly decrease compared with negative control. Similar results were obtained in study of


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Genotoxicity studie on the root extract of polygala tenuifolia wildenow  

Genotoxicity studie on the root extract of polygala tenuifolia wildenow  

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