Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity and genotoxicity studies

Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity and genotoxicity studies

Journal Pre-proof Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity in ICR mice and genotoxicity studies Sarah Shin, Jin-Mu Yi, No ...

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Journal Pre-proof Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity in ICR mice and genotoxicity studies Sarah Shin, Jin-Mu Yi, No Soo Kim, Chan-Sung Park, Su-Hwan Kim, Ok-Sun Bang PII:

S0378-8741(19)33530-5

DOI:

https://doi.org/10.1016/j.jep.2019.112381

Reference:

JEP 112381

To appear in:

Journal of Ethnopharmacology

Received Date: 6 September 2019 Revised Date:

31 October 2019

Accepted Date: 7 November 2019

Please cite this article as: Shin, S., Yi, J.-M., Kim, N.S., Chan-Sung Park, , Kim, S.-H., Ok-Sun Bang, , Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity in ICR mice and genotoxicity studies, Journal of Ethnopharmacology (2019), doi: https://doi.org/10.1016/j.jep.2019.112381. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Forsythia viridissima Lindl.

safety

Safety

mAU

EFVF

Material

QC

Phytochemical profile by UHPLC

Dried fruits



Arctiin Arctigenin Matairesinol



Acute oral toxicity

Genotoxic assessment - In vitro Ames

Aqueous Extract

- In vitro chromosomal aberration - In vivo micronucleus

EFVF

1

Aqueous extract of Forsythia viridissima fruits: Acute oral toxicity

2

in ICR mice and genotoxicity studies

3 4 5

Sarah Shina, Jin-Mu Yia, No Soo Kima, Chan-Sung Parkb, Su-Hwan Kimb and Ok-Sun

6

Banga*

7 8 9

a

Clinical Medicine Division, Korea Institute of Oriental Medicine, 1672 Yuseong-daero,

10

Yuseong-gu, Daejeon 34054, Republic of Korea

11

b

12

Cheongju-si, Cheungcheongbuk-do 28115, Republic of Korea

Nonclinical Research Institute, Biotoxtech Co., Ltd., 53 Yeongudanji-ro, Cheongwon-gu,

13 14

Sarah Shin

15

Jin-Mu Yi

16

No Soo Kim

[email protected]

17

Chan-Sung Park

[email protected]

18

Su-Hwan Kim

[email protected]

19

Ok-Sun Bang

First author

[email protected] [email protected]

Corresponding author

[email protected]

20 21 22

*

23

Clinical Medicine Division, Korea Institute of Oriental Medicine

24

1672 Yuseong-daero, Yuseong-gu, Daejeon 34054, Republic of Korea

Corresponding author: Ok-Sun Bang, Ph.D.

1

25

Tel: +82-42-868-9353, Fax: +82-42-868-9370, E-mail: [email protected]

26 27

Abstract

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Ethnopharmacological relevance: Forsythiae Fructus (FF) is widely used in traditional

29

medicine to treat diverse diseases-related clinical symptoms, including fever, pain, vomiting,

30

nausea, and abscess. However, the safety of FF has not yet been fully assessed.

31

Aim of the study: In this study, we evaluated the acute oral toxicity and genotoxic potential

32

of an aqueous extract of Forsythia viridissima fruits (EFVF).

33

Materials and methods: For an acute oral toxicity test, male and female SD rats (n=5) orally

34

received a single dose of 5000 mg/kg EFVF. The genotoxic potential of EFVF was evaluated

35

with a battery of tests, including an in vitro bacterial reverse mutation test using five mutant

36

strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli

37

(WP2 uvrA), an in vitro chromosomal aberration test using Chinese hamster lung (CHL/IU)

38

cells, and an in vivo micronucleus test using bone marrow cells in male ICR mice that were

39

orally administered EFVF. All tests were completed in compliance with Organization for

40

Economic Cooperation and Development guidelines and/or regional regulatory standards for

41

toxicity tests.

42

Results: In the acute oral toxicity test, the animals did not show any significant mortality and

43

body weight changes for 14 days following a single dose of EFVF at 5000 mg/kg. There was

44

no evidence of genotoxicity of EFVF based on the results of the in vitro bacterial reverse

45

mutation test (up to 5000 µg/plate), the in vivo micronucleus test (up to 5000 mg/kg), and the

46

in vitro chromosomal aberration test (1100-2500 µg/mL). 2

47

Conclusions: We found that EFVF is safe with regard to acute toxicity in rats as well as

48

genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions.

49

These results might support the safety of EFVF as a potential therapeutic material for the

50

traditional use or pharmaceutical development.

51 52

Keywords: Forsythia viridissima, acute oral toxicity, Ames, chromosomal aberration,

53

micronucleus test

54

55

56

1. Introduction Herbal medicine has traditionally been used in many countries for thousands of years.

57

Recent years, the number of patients with geriatric or chronic diseases such as Alzheimer's,

58

dementia, cancer, and metabolic diseases are increasing as aging populations rapidly increase

59

worldwide (Mori et al., 2019; Prasad et al., 2012). In addition, disease treatment strategies

60

have focused more on patient quality of life than on the mere relief of clinical symptoms

61

(Adams and Jewell, 2007). Accordingly, the use of herbal medicine, including botanical drugs

62

and health care supplements has substantially grown worldwide and the global market for

63

these material is increasingly expanding (Pelkonen et al., 2014). However, despite the belief

64

that herbal medicines will be effective and safe due to their long-term use, the concerns

65

regarding their safety have been raised by both national health authorities and the general

66

public, due to a lack of scientific evidence. Therefore, the evaluation of the toxicity of herbal

67

medicines in a nonclinical assessment of herbal extracts is necessary.

68

Forsythiae Fructus (FF), the dried fruit of Forsythia suspensa Vahl and Forsythia 3

69

viridissima Lindl. (Oleaceae), is listed in the pharmacopoeias of China, Japan, and Korea

70

(IPC, 2015; JDECA, 2011; MFDS, 2015). According to a number of ancient medical studies,

71

FF has been prescribed to reduce fever, abscesses, and the swelling of wounds, as well as to

72

detoxify certain poisons as an antidote (Chen et al., 2015; Dong et al., 2017). Indeed, various

73

recent pharmacological studies have confirmed that fruits of F. suspensa and their

74

phytochemicals have anti-oxidant (Zhao et al., 2017), anti-bacterial (Guo et al., 2016), anti-

75

inflammatory (Hwang et al., 2017; Kuo et al., 2017), anti-tumor (Bao et al., 2016; Lee et al.,

76

2017), and neuroprotective effects (Zhang et al., 2016). Recently, the fruits of F. viridissima

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have also been found to exert anti-inflammatory (Lee et al., 2010), anti-oxidant (Kim et al.,

78

2006), anti-asthmatic (Lee et al., 2010) and neuroprotective effects (Yi et al., 2019). Although

79

FF has a popular history of use and clear therapeutic advantages (Chen et al., 2015; Dong et

80

al., 2017; Hwang et al., 2017; Kuo et al., 2017; Yi et al., 2019; Zhao et al., 2017), there is

81

little information regarding its safety. Therefore, it is necessary to evaluate its safety using a

82

standardized battery of tests to ensure a safer use of herbal medicines and for the further

83

development of novel pharmaceutical drugs. In a previous study, we showed that aqueous

84

extracts of F. viridissima fruits (EFVF) alleviates the pain of peripheral neuropathy caused by

85

the side effects of anticancer drugs including oxaliplatin, in both in vitro and in vivo

86

neuropathic animal models (Yi et al., 2019). As a follow-up study, in this study, we evaluated

87

the acute oral toxicity and genotoxic potential of EFVF in order to provide information on its

88

nonclinical safety.

89 90

2. Materials and methods

91

2.1. Chemicals and reagents 4

92

For ultra-high performance liquid chromatography (UHPLC) analysis, authentic

93

standard chemicals (STDs), including arctiin (AT), arctigenin (ATG), and matairesinol (MTR)

94

were purchased from ChemFaces (Wuhan, Hubei, China). UHPLC-grade water, acetonitrile,

95

and methanol were supplied from Fisher Scientific Ltd. (Loughborough, UK). Positive

96

control drugs for genotoxicity tests, including 2-aminoanthracene (2-AA), 9-aminoacridine

97

(9-AA), benzo[a]pyrene (B[a]P), mitomycin C (MMC), 2-nitrofluorene (2-NF), 2-

98

nitroquinoline N-oxide (4-NQO), and sodium azide (SA) were supplied from Sigma-Aldrich

99

Co (St. Louis, MO, USA). Sterilized distilled water was obtained from JW Pharma (Dangjin,

100

Chungnam, Republic of Korea). Colcemide, Oxoid Nutrient Broth No. 2 and bactoagar were

101

purchased from BD (Franklin Lakes, NJ, USA). Dulbecco’s phosphate-buffered saline (D-

102

PBS) was obtained from Lonza Walkersville Inc., (Basel, Switzerland). The metabolic

103

activator S9 mix was prepared using a phenobarbital/5,6-benzoflavon-pretreated rat liver S9

104

(Oriental Yeast Co., Ltd., Itabashi, Tokyo, Japan), and a cofactor-A for the in vitro bacterial

105

reverse mutation (Ames) test or cofactor-C for the in vitro chromosomal aberration test. The

106

S9 mix was freshly prepared prior to use and kept on ice during the experiment. All

107

chemicals and reagents except those described elsewhere were purchased from the Sigma-

108

Aldrich.

109

110

2.2. Preparation and stability analysis of EFVF

111

A voucher specimen (KIOM-CRC#518) of F. viridissima fruits was deposited in the

112

Clinical Medicine Division of KIOM. EFVF was prepared as previously reported (Yi et al.,

113

2019). The stability of EFVF was assessed by chromatographic analysis of the three major

114

constituents, AT, ATG, and MTR, in EFVF for 2 weeks. The analysis was conducted using an 5

115

UHPLC-diode array detection (DAD) system (1290 infinity, Agilent Technologies, Santa

116

Clara, CA, USA) equipped with a Lunaomega C18 column (2.1×50 mm, 1.6 µm,

117

Phenomenex, Torrance, CA, USA). The UHPLC profile was obtained by using a sequential

118

gradient mobile phase system; 0.1% formic acid: acetonitrile (v/v), 95:5 to 40:60, for 40 min

119

with a flow rate at 0.2 mL/min. The signal of each peak in the chromatograms was detected

120

with DAD at the wavelength range of 200-500 nm.

121

122

2.3. Animals

123

Protocols for the acute oral toxicity and the in vivo micronucleus test were approved

124

by the Institutional Animal Care and Use Committee (IACUC) of Biotoxtech (Protocol

125

#170725 and Protocol #170733, respectively). Sprague–Dawley (SD) rats (6-week-old

126

female and male rats weighing 110–125 g) and CrljOri:CD1(ICR) mice (7-week-old male

127

mice weighing 29.2–32.6 g) were obtained from Orient Bio (Sungnam, Gyeonggi, Republic

128

of Korea). During the entire period of the experiments, animals were maintained under

129

specific pathogen-free (SPF) laboratory conditions at a temperature of 22 ± 3 °C, relative

130

humidity of 45-60%, a light/dark cycle of 12 h/12 h (150-300 Lux), and ventilation at 10-15

131

times/h. All animals were quarantined and allowed to acclimate for 1 week before toxicity

132

studies.

133

134

2.4. Acute oral toxicity test

135

Acute oral toxicity test was conducted according to the Good Laboratory Practice

136

regulations for nonclinical laboratory studies and the Ministry of Food and Drug Safety 6

137

(MFDS) Standards Guidelines for the Nonclinical Studies and Toxicity Test of

138

Pharmaceuticals (MFDS Notification NO. 2017-32 and 2017-71, Republic of Korea) (MFDS,

139

2017a, b). Prior to treatment, animals were weighed, marked, and allowed for overnight

140

fasting with free access to water. Rats were randomly assigned to two groups of each sex

141

(n=5) on the basis of their individual body weights. Since the preliminary acute oral toxicity

142

test did not show any remarkable toxic effects up to 5000 mg/kg, rats of the treated group

143

received a single oral dose of 5000 mg/kg EFVF (10 mg/mL), whereas the control group

144

received distilled water. Clinical signs related with a drug-toxicity and the changes in the

145

general behaviors of the animals were monitored and recorded every 1 h for the first 6 h after

146

EFVF treatment, and then once daily over 14 days. Individual body weights were checked

147

immediately before drug treatment and then at day 1, 3, 7, and 14 thereafter. On day 14, all

148

animals were anesthetized with CO2 inhalation and exsanguinated from the abdominal aorta.

149

Complete gross postmortem examinations were performed on all animals in the test.

150

151

2.5. In vitro Ames test

152

The in vitro Ames test were conducted in accordance with the Organization for

153

Economic Cooperation and Development (OECD) guidelines for the Testing of Chemicals,

154

TG 471 Bacterial Reverse Mutation Test (OECD, 1997), and the MFDS Standards Guidelines

155

for Toxicity Test of Pharmaceuticals (MFDS Notification 2017-71) (MFDS, 2017b). The

156

experiment was performed using a pre-incubation procedure according to a slightly modified

157

method as described previously (Ames et al., 1975; Maron and Ames, 1983).

158

strains of the histidine auxotrophic mutants Salmonella typhimurium (S. typhimurium),

159

including TA98, TA100, TA1535, and TA1537, and one strain of tryptophan auxotrophic 7

Briefly, four

160

mutant, Escherichia coli (E. coli), WP2 uvrA were obtained from Molecular Toxicology Inc

161

(Boone, NC, USA). These strains have been proven for the sensitive detection of the

162

mutagenicity of diverse chemicals, and therefore were considered appropriate for this test

163

(Mortelmans et al, 2000). Bacterial cells were maintained in 2.5% oxoid nutrient broth No.2.

164

A minimal medium composed of 1.5% Bacto agar, Vogel–Bonner medium E, and 2% glucose

165

was prepared before the test. A preliminary range-finding test was first performed to

166

determine the solubility and toxicity of EFVF in bacterial growth, and 5000 µg/plate was

167

chosen as a maximum test dose. Bacterial strains were exposed to serially increasing doses of

168

EFVF (5-5000 µg/plate) in the presence and absence of a metabolic activator, which

169

consisted of a rat liver S9 mix (10%, v/v). In the presence of S9 metabolic activation, 2-AA

170

(1.0-3.0 µg/plate) was used as a positive control for all strains. In the absence of S9 metabolic

171

activation, SA (1.5 µg/plate) for TA100 and TA1535, 2-NF (5.0 µg/plate) for TA98, 9-AA

172

(80.0 µg/plate) for TA1537, and 4-NQO (0.1 µg/plate) for WP2 uvrA were used as positive

173

controls. Sterilized distilled water was used as a negative control. Mutant bacterial strains (>

174

1×109 cells/mL) were incubated with EFVF (5-5000 µg/plate) or the control drugs at 37 °C

175

for 20 min, in the presence and absence of the S9 metabolic activation, and then mixed with

176

the top agar containing 2.5% oxoid nutrient broth. The mixture was poured onto minimal

177

glucose agar plates; hardened plates were inverted and incubated at 37 °C for 48 h. To verify

178

sterility, 0.1 mL of EFVF (5000 µg/plate), 0.5 mL of S9 mix and 0.5 mL of 0.1 M sodium

179

phosphate buffer (pH 7.4) were incubated at 37 °C for 20 min without bacterial strain cell and

180

mixed with the top agar, and then cultured for 48 h. The numbers of revertant bacterial

181

colonies were counted visually using an automatic colony counter (ProtoCOL3 SYNBIOSIS,

182

Cambridge, UK). It was determined as a positive when the average numbers of revertant 8

183

colonies in one or more bacterial strains were increased by more than two fold compared to

184

those of the negative control irrespective of S9 metabolic activation.

185

186

2.6. In vitro mammalian chromosomal aberration test

187

The in vitro chromosomal aberration test was conducted in accordance with the

188

OECD guidelines for the Testing of Chemicals, TG 473 In Vitro Mammalian Chromosome

189

Aberration Test (OECD, 2014a), and the MFDS Standards Guidelines for Toxicity Test of

190

Pharmaceuticals (MFDS Notification 2017-71) (MFDS, 2017b). The chromosomal aberration

191

test was basically followed the methods described by Ishidate (Ishidate, 1985). A preliminary

192

dose range-finding test was performed to determine the toxicity of EFVF in Chinese hamster

193

lung (CHL/IU) cells by calculating the relative population of doubling (RPD) in substance-

194

treated cultures. The RPD value was determined using the following formula.

195

RPD (%) =

196

Population doubling =

x 100

*+

(

/ +

)-

.

197

According to the OECD guidelines for the Testing of Chemicals, TG 473 In Vitro

198

Mammalian Chromosome Aberration Test, a limit of about 50% growth reduction is

199

considered appropriate to determine the dose range (OECD, 2014a). Therefore, doses of 1650,

200

2500 and 1100 µg/mL of EFVF were chosen as maximum doses for 6 h treatment in the

201

absence and presence of the S9 mix, and 24 h treatment, respectively, at which the RPD

202

values were > 55%. B[a]P (20 µg/mL) and MMC (0.1 µg/mL) were used as positive control

203

drugs, respectively, in the presence and absence of S9 metabolic activation. Sterile water was 9

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used for the negative control. CHL/IU cells were obtained from the American Type Culture

205

Collection® (Manassas, VA, USA). They were inoculated as 1 x 105 cells/2 mL/6 well plate

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in growth medium and were maintained in a CO2 incubator at 37 °C for 3 days. CHL/IU cells

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were treated with serial doses of EFVF (413-1650 and 625-2500 µg/mL) or the

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positive/negative control drugs for 6 h followed by a brief washing with D-PBS and 18 h

209

recovery in fresh media in the presence and absence of the cofactor C-activated rat liver S9

210

mix (30%, v/v). In parallel, cells were also subjected to EFVF (275-1100 µg/mL) or

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positive/negative control drugs for a 24 h treatment followed by 0 h recovery in the absence

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of S9 metabolic activation. Two hours before the completion of the final culture, cells were

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incubated with 0.2 µg /mL colcemide solution for 2 h. Cells that were arrested at metaphase,

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were then collected and treated with 5 mL pre-warmed 75 mM KCl at 37 °C for 20 min, and

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finally fixed in a methanol: acetic acid (3:1) mixture. Two smears per slide were then

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prepared and stained with 3% (v/v) Giemsa solution. More than 100 metaphases per smear

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were analyzed under the microscope at 600x magnification (BX51, Olympus, Shinjuku,

218

Japan). Cells showing one or more chromosome aberrations were counted as one abnormal

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cell and the type of aberration was characterized in accordance with the Atlas of

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Chromosome Aberration by Chemicals (JEMS-MMS, 1988). Structural aberrations in

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metaphasic chromosomes were categorized into chromosome types of breaks (csb),

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exchanges (cse) or gaps (csg), chromatid types of breaks (ctb), exchanges (cte), or gaps (ctg),

223

and total cells with structural aberrations including (gap+) or excluding (gap-) multiple

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aberration. The numerical aberration in metaphasic chromosomes was then categorized into

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polyploidy (pol) and endo-reduplication (end). This method was regarded as valid in that the

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number of total cells with structural aberrations excluding gap (gap-) below 5%, was

10

227

considered negative, 5-10% was partial positive, and 10% or higher was considered positive

228

in accordance with the criteria of Sofuni (Sofuni, 1999).

229

230

2.7. In vivo micronucleus test

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The in vivo micronucleus test was conducted in accordance with the OECD

232

guidelines for the Testing of Chemicals, TG 474 Mammalian Erythrocyte Micronucleus Test

233

(OECD, 2014b), and the MFDS Standards Guidelines for Toxicity Test of Pharmaceuticals

234

(MFDS Notification 2017-71) (MFDS, 2017b). The micronucleus test basically followed the

235

methods described previously (Erexson, 2003) with slight modification. Briefly, based on the

236

data of the dose-range finding study, a dose of 5000 mg/kg/day was chosen as a maximum

237

dose for the in vivo micronucleus test. Male CrljOri:CD1(ICR) SPF mice were divided into

238

seven groups (n=3) on the basis of their body weights. Mice were orally administered EFVF

239

at 313–5000 mg/kg/day, or sterile water for 2 days as a negative control. The mice received

240

one intraperitoneal administration of MMC at 2 mg/kg 24 h before sacrifice. Morphological

241

observations of all experimental animals were conducted for 4 days, before and after each

242

drug administration. EFVF treatment did not cause any toxicity-related clinical signs in

243

experimental animals. Compound-colored stools that were shown on day 2 and 3 in all

244

EFVF-treated animals reverted to normal on day 4. Twenty-four hours after the second

245

treatment, mice were sacrificed by cervical dislocation, and then the femurs were procured.

246

Bone marrows were collected by flushing the femurs with 2 mL FBS using a 23 gauged

247

needle. Bone marrow cells were then centrifuged at 4 °C and 1000 rpm for 5 min and

248

smeared on a clean slide glass. Smeared slides were air-dried, fixed in methanol for 5 min,

249

and then stained with 3% Giemsa solution for 30 min. Stained slides were observed under the 11

250

fluorescence microscope at 600x magnification (BX51, Olympus). The numbers of

251

micronucleated polychromatic erythrocytes (MNPCEs), polychromatic erythrocytes (PCEs),

252

and normochromatic erythrocytes (NCEs) among the red blood cells (RBCs, PCE+NCE)

253

were counted in bone marrow. A genotoxic index was expressed as the average number of

254

MNPCEs in 1000 PCEs per mouse. A cytotoxic index was expressed as the average ratio of

255

PCE to RBCs by counting a total 500 RBCs.

256

2.8. Statistical analyses

257

Statistical analyses were performed using the Statistical Analysis System (SAS)

258

program (version 9.3, SAS Institute Inc., USA). For the acute oral toxicity test, the

259

homogeneity of variance on body weights was determined by the Fold-F test (α=0.05), and

260

the Student’s t-test was conducted to confirm its statistical significance (α=0.05 and 0.01,

261

two-tailed). No statistical analysis was performed for the in vitro Ames test and the in vitro

262

chromosomal aberration test. For the in vivo micronucleus test, the incidence of MNPCE was

263

verified by the Kastenbaum & Bowman method (α= 0.01, two-tailed). The Barlett’s test was

264

performed to compare the homogeneity of the variance between the vehicle control and the

265

EFVF treatment groups (α=0.05). One-way analysis of variance (ANOVA) was applied to

266

confirm its significance on body weights (α=0.05). The Fold-F test was conducted to

267

compare the homogeneity of the variance between the vehicle control and the positive control

268

group (α=0.05). The homogeneity of variance on the incidence of PCE among the total

269

erythrocytes was determined by applying the Fold-F test (α=0.05), and the Student t-test was

270

conducted to confirm its statistical significance (α=0.05 and 0.01, two-tailed). The difference

271

of means was considered to be statistically significant at p<0.05. 12

272

273

3. Results

274

3.1. Phytochemical analysis and stability

275

As shown in the three dimensional UHPLC profile of EFVF, AT, MTR, and ATG

276

were confirmed as major constituents of EFVF (Fig. 1), with their contents in EFVF

277

determined to be 22.86 ± 0.05 mg/g of AT, 59.35 ± 0.11 mg/g of MTR, and 23.16 ± 0.05

278

mg/g of ATG. The stability of EFVF was verified by analyzing the contents of the three

279

constituents in the extract for 2 weeks; their relative standard deviations (RSD) of the

280

contents were maintained less than 1.0% during this period (Table 1). Our results

281

demonstrated that the quality of EFVF was consistent with that of our previous extract (Yi et

282

al., 2019) and its constituents were stable for at least 2 weeks.

13

283 284 285

Fig. 1. Three-dimensional UHPLC profile of the aqueous extract of F. viridissima fruits (EFVF).

286

287

Table 1. Stability of the three major constituents in the aqueous extract of F. viridissima fruits

288

(n=3). Week 0

Week 1

Week 2

Constituents

Mean ± SD (mg/g)

RSD (%)

Mean ± SD (mg/g)

RSD (%)

Mean ± SD (mg/g)

RSD (%)

Arctiin

22.86 ± 0.05

0.22

22.77 ± 0.05

0.25

22.82 ± 0.02

0.11

Matairesinol

59.35 ± 0.11

0.19

59.23 ± 0.10

0.16

59.30 ± 0.01

0.01

Arctigenin

23.16 ± 0.05

0.22

23.05 ± 0.02

0.10

23.14 ± 0.03

0.12

289

14

290

291

3.2. Acute oral toxicity test

292

For 14 days after the single oral dose of EFVF (5000 mg/kg), no abnormal clinical

293

symptoms or mortalities were observed in both male and female rats. However, on day 1 after

294

oral administration of EFVF, compound-colored stools were observed in both sexes, which

295

were reverted to normal on the following day (data not shown). There were no significant

296

changes in body weight gains by EFVF treatment (Fig. 2). At necropsy, no remarkable

297

findings were noted in both sexes in the control and EFVF treatment groups. A single oral

298

dose of 5000 mg/kg EFVF did not cause any mortalities or toxic effects, indicating that the

299

approximate lethal dose (LD) of single oral dose toxicity of EFVF in rats was over 5000

300

mg/kg.

301 302

Fig. 2. Body weight changes in male (M) and female (FM) SD rats following a single oral

303

dose of aqueous extracts of F. viridissima fruits (EFVF). Body weights were monitored for 14

304

days and presented as the means ± SD (n=5).

305 15

306

3.3. In vitro Ames test

307

To evaluate pro-mutagenic potential, increasing doses of EFVF (0–5000 µg/plate)

308

were applied to mutant S. typhimurium (TA100, TA1535, TA98, TA1537) and E. coli (WP2

309

uvrA) strains. Turbidity or precipitation due to the low solubility of the test substance was not

310

found in top agars containing bacterial strains. Bacterial colonies due to microbial

311

contamination were not found confirming the sterility of the experiment conditions that

312

included EFVF and the S9 mix. As expected, all positive control drugs showed a remarkable

313

increase in the number of revertant colonies in all tested mutant bacterial strains regardless of

314

the presence of S9 metabolic activation, which confirmed the validity of the in vitro Ames

315

test system. Mean numbers of revertant bacterial colonies were comparable to the negative

316

control at all doses of EFVF. No dose-dependent increase in the number of revertant colonies

317

was observed up to 5000 µg/plate EFVF, regardless of the presence of the S9 metabolic

318

activator (Table 2). Collectively, these results demonstrated that the mutagenic potential of

319

EFVF is negative.

320

Table 2. Summary of the Ames test of the aqueous extracts of F. viridissima fruits (EFVF) in

321

the absence and presence of S9 metabolic activation. No. of revertant colonies/plate Strains

TA100

Drugs

EFVF

Doses (µg/plate) S9 mix (+)

S9 mix (-)

0

101.0 ± 1.4

83.5 ± 2.1

5

102.0 ± 4.2

82.0 ± 1.4

10

96.0 ± 1.4

86.0 ± 1.4

50

94.0 ± 5.7

86.0 ± 2.8

100

101.0 ± 7.1

82.5 ± 4.9

500 1000

103.0 ± 2.8 111.5 ± 2.1

81.0 ± 1.4 85.0 ± 4.2

16

TA1535

WP2 uvrA

TA98

TA1537

2500 5000

114.5 ± 6.4 111.0 ± 1.4

85.0 ± 2.8 90.5 ± 0.7

2-AA SA

2 1.5

774.0 ± 8.5 -

685.0 ± 1.4

EFVF

0 5 10 50 100 500 1000 2500 5000

13.5 ± 0.7 13.5 ± 0.7 12.5 ± 0.7 13.0 ± 1.4 13.5 ± 2.1 14.5 ± 0.7 14.0 ± 0.0 16.5 ± 0.7 14.5 ± 0.7

12.0 ± 1.4 12.5 ± 0.7 12.0 ± 1.4 12.5 ± 0.7 12.0 ± 1.4 12.5 ± 2.1 13.5 ± 0.7 13.0 ± 1.4 14.0 ± 1.4

2-AA SA

3 1.5

158.0 ± 4.2 -

524.5 ± 10.8

EFVF

0 5 10 50 100 500 1000 2500 5000

144.0 ± 4.2 140.5 ± 3.5 151.5 ± 0.7 141.5 ± 2.1 145.5 ± 2.1 154.5 ± 0.7 155.0 ± 4.2 151.5 ± 3.5 183.5 ± 20.5

82.0 ± 0.0 83.0 ± 4.2 87.5 ± 4.9 92.5 ± 6.4 91.5 ± 2.1 103.5 ± 3.5 96.0 ± 1.4 105.5 ± 2.1 95.0 ± 1.4

2-AA 4-NQO

2 0.1

546.0 ± 18.4 -

1014.5 ± 4.9

EFVF

0 5 10 50 100 500 1000 2500 5000

33.0 ± 1.4 32.5 ± 0.7 33.5 ± 2.1 30.5 ± 0.7 31.0 ± 1.4 30.5 ± 0.7 35.0 ± 1.4 31.0 ± 1.4 34.0 ± 2.8

17.5 ± 0.7 19.5 ± 0.7 18.5 ± 2.1 18.5 ± 2.1 17.0 ± 1.4 17.5 ± 0.7 17.5 ± 0.7 17.5 ± 0.7 19.5 ± 0.7

2-AA 2-NF

1 5

387.5 ± 10.6 -

723.0 ± 21.2

EFVF

0 5

18.0 ± 1.4 16.0 ± 0.0

8.5 ± 0.7 8.0 ± 0.0

17

2-AA 9-AA

10 50 100 500 1000 2500 5000

16.5 ± 0.7 17.0 ± 0.0 17.5 ± 2.1 19.0 ± 0.0 18.5 ± 0.7 19.0 ± 1.4 19.0 ± 1.4

8.0 ± 1.4 10.0 ± 1.4 9.5 ± 2.1 10.0 ± 1.4 8.5 ± 0.7 8.5 ± 0.7 9.5 ± 0.7

3 80

192.5 ± 6.4 -

601.0 ± 4.2

322

2-AA, 2-aminoanthracene; 9-AA, 9-nitroacridine; B[a]P, benzo[a]pyrene; SA, sodium azide;

323

2-NF, 2-nitrofluorene and 4-NQO, 4-nitroquinoline-1-oxide were used as bacterial strain

324

specific-positive control drugs.

325 326

3.4. In vitro mammalian chromosomal aberration test

327

The genotoxic potential of EFVF to induce chromosomal aberration was determined

328

in mammalian CHL/IU cells. Cells were treated with serial doses of EFVF (275-2500 µg/mL)

329

in the presence and absence of the metabolic activator S9 fraction for 6 or 24 h. No turbidity

330

or precipitation of the test substances was observed in all test doses during the treatment

331

periods. The frequency of metaphasic chromosomes showing structural aberrations was 0%

332

in the negative control. The positive control drugs, B[a]P and MMC in the presence and

333

absence of the S9 fraction, respectively, remarkably increased the frequencies of CHL/IU

334

cells showing chromosomal abnormalities up to more than 20%, which confirmed the validity

335

of the test (Table 3). However, the frequencies of the CHL/IU cells showing structural or

336

numerical aberrations were less than 3% and were not significantly increased compared with

337

negative control at all doses of EFVF in the tested conditions.

338

Table 3. Results of chromosomal aberration test of the aqueous extract of F. viridissima fruits

339

(EFVF) in the absence or presence of S9 metabolic activation. 18

No. of numerical aberration

No. of structural aberrations Drugs

Doses (µg/mL)

RPD (%)

ctb

cte

ctg

csb

cse

csg

Total (%) gap-

gap+

end

pol

Total (%)

6 h Trt-18 h Rec (-S9) EFVF

MMC

0

100

0

0

0

0

0

0

0

0

0

1

1

413

100

0

0

0

0

0

0

0

0

0

2

2

825

94

0

0

0

0

0

0

0

0

0

1

1

1650

57

1

2

0

0

0

0

3

3

0

0

0

0.1

60

6

17

0

0

0

0

21

21

0

0

0

6 h Trt-18 h Rec (+S9) EFVF

B[a]P

0

100

0

0

0

0

0

0

0

0

0

0

0

625

99

0

0

0

0

0

0

0

0

0

0

0

1250

96

2

0

0

0

0

0

2

2

0

0

0

2500

70

0

0

0

0

0

1

0

1

0

0

0

20

55

4

25

0

3

0

0

28

28

0

0

0

24 h Trt-0 h Rec (-S9) EFVF

MMC

0

100

0

0

0

0

0

0

0

0

0

0

0

275

77

0

0

0

0

0

0

0

0

0

1

1

550

71

1

0

0

0

0

0

1

1

0

0

0

1100

68

3

1

0

0

0

0

3

3

0

1

1

0.1

55

6

24

0

0

0

0

26

26

0

1

1

340

ctb, chromatid break; cte, chromatid exchange; ctg, chromatid gap; csb, chromosome break;

341

cse, chromosome exchange; csg, chromosome gap; end, endo-reduplication; gap-, total

342

number of cells with structural aberrations excluding gap; gap+, total number of cells with

343

structural aberrations including gap; pol, polyploidy; RPD, relative population doubling; Trt-

344

Rec, treatment-recovery. Mitomycin C (MMC) and benzo[a]pyrene (B[a]P) were used as

345

positive control drugs in the absence and presence of S9 metabolic activation, respectively.

346

347

3.5. In vivo micronucleus test 19

348

To examine the in vivo micronucleus test, first the oral toxicity of EFVF was

349

monitored for 4 days in male ICR mice. No toxicity-related clinical signs such as loss of body

350

weight or mortality (Table 4), were observed in experimental animals receiving an oral

351

administration of EFVF at 313–5000 mg/kg/day for 2 days.

352

Table 4. Body weight changes and the mortalities of male ICR mice following treatment of

353

with the oral aqueous extract of F. viridissima fruits (EFVF).

Drugs

Doses (mg/kg/day)

EFVF

MMC 354

Body weight (g, Mean ± SD)

Mortality

Day 0

Day 3

0

36.03 ± 1.21

35.30 ± 0.78

0/3

313

35.77 ± 1.26

35.20 ± 1.00

0/3

625

35.80 ± 1.20

35.13 ± 0.91

0/3

1250

35.83 ± 1.16

35.33 ± 1.17

0/3

2500

35.70 ± 1.35

34.43 ± 1.00

0/3

5000

35.67 ± 1.45

35.43 ± 1.26

0/3

2

35.47 ± 0.67

34.43 ± 1.59

0/3

Mitomycin C (MMC) was used as a positive control drug.

355

To determine the mutagenic potential of EFVF in bone marrow cells derived from

356

ICR mice, micronucleus test was done by determining the frequency of MNPCEs in 1000

357

PCEs per animal. As shown in Table 5, there was no significant increase in the frequencies of

358

MNPCEs at all test doses of EFVF when compared with the negative control (0.10 ± 0.00).

359

MMC, a positive control, significantly increased the frequency of MNPCEs (7.77 ± 0.86),

360

which was expected. The cytotoxic index [PCE/(PCE+NCE)] of EFVF was approximately

361

0.30 at all test doses, which was comparable with those of the negative and positive control

362

treatments.

363

Table 5. Summary of the micronucleus test of the aqueous extract of F. viridissima fruits

364

(EFVF) in male ICR mice. 20

Drugs

Doses (mg/kg/day)

MNPCE/1000 PCEs (%, Mean ± SD)

PCE/(PCE+NCE) (Mean ± SD)

EFVF

0

0.10 ± 0.00

0.29 ± 0.01

313

0.07 ± 0.06

0.30 ± 0.05

625

0.10 ± 0.00

0.29 ± 0.01

1250

0.07 ± 0.12

0.29 ± 0.04

2500

0.03 ± 0.06

0.28 ± 0.01

5000

0.13 ± 0.06

0.30 ± 0.02

2

7.77 ± 0.86*

0.28 ± 0.03

MMC 365

MMC, mitomycin C; PCE, polychromatic erythrocyte; NCE, normochromatic erythrocyte;

366

MNPCE, micronucleated polychromatic erythrocyte. *p<0.05.

367

368

4. Discussions

369

The use of herbal medicines has been increasing worldwide due in part to growing

370

elderly population and the general desire to avoid side effects or the inefficacy of modern

371

conventional drugs. Since herbal medicines are comprised of multiple components with

372

multiple efficacies, they can be useful for treating complex diseases, such as cancer and

373

various chronic diseases (Fu et al., 2018). In addition, herbal preparations are not only used

374

as a resource for the development of new drugs, they are still used in traditional or

375

complementary medicine. However, the safety of herbal medicines in general has not been

376

scientifically confirmed. Unlike synthetic drugs, most consumers have blind faith in the

377

safety of herbal medicines merely because they originated from nature and have been used

378

for a long time (Jordan et al., 2010). However, several studies have reported that some plants

379

frequently used in traditional medicine, including Ocotea duckei, Synadenium umbellatum

380

Pax Latex, Pterocaulon polystachyum, Cynara scolymus L, and Cryptolepis sanguinolenta 21

381

are potentially genotoxic on the basis of the Ames, chromosomal aberration, or commet tests

382

(Ansah et al., 2005; Marques et al., 2003; Melo-Reis et al., 2011; Regner et al., 2011; Zan et

383

al., 2013). Accordingly, the potential toxicity of herbal extracts/substances in nonclinical

384

studies should be evaluated (Di Stasi et al., 2002; Melo-Reis et al., 2011; Sponchiado et al.,

385

2016).

386

Despite of the popular use and pharmacological advantages of FF, little is known

387

regarding its toxicity. In the one study that did examine the toxicity of FF, the water extract

388

of F. suspensa was shown to have genotoxic potential in both chromosomal aberration and

389

micronucleus assays (Yin et al., 1991). In the present study, we first evaluated the acute oral

390

toxicity of the water extract of F. viridissima fruits in SD rats. A single oral dose of EFVF at

391

5000 mg/kg did not show any abnormal body weight changes, animal death, or gross lesions

392

in both male and female SD rats for 14 days. Therefore, the approximate LD50 of EFVF was

393

considered to be higher than 5000 mg/kg in both sexes indicating that EFVF is safe in terms

394

of acute oral toxicity at up to 5000 mg/kg in SD rats and safe in humans at up to 810 mg/kg.

395

Genotoxicity tests are routinely performed on drugs, and various herbal medicines in

396

order to evaluate the potential of these extracts/substances to interact with nucleic acids and

397

induce irreversible damage or mutations at relatively low concentrations. The most frequently

398

used methods to evaluate the genotoxicity of herbal extracts include the in vivo rodent bone

399

marrow micronucleus test for clastogenicity and aneuploidy, the in vitro Ames test for

400

detecting gene mutations in auxotrophic bacteria, and the in vitro chromosomal aberration

401

test for detecting clastogenicity in mammalian cells (Sponchiado et al., 2016). In the present

402

study, we evaluated the genotoxic potential of EFVF using a battery of genotoxic tests,

403

including the Ames, chromosomal aberration and micronucleus tests. 22

404

The Ames test was developed to assess the mutagenic potential of bacteria and to

405

estimate the carcinogenic potential of environmental mixtures (Ames et al., 1975). This test

406

is commonly employed as an initial screening to determine the mutagenic potential of new

407

chemicals and drugs as well as herbal medicines, particularly with activity for induced point

408

mutations, involving substitution, addition, or the deletion of one or more DNA base pairs.

409

In the present test, the histidine auxotrophic strains of S. typhimurium TA100, TA98,

410

TA1535, and TA1537 and the tryptophan auxotrophic strain E. coli WP2 uvrA were used as

411

described previously (Maron and Ames, 1983). The mean number of revertant colonies was

412

not at up to 5000 µg/plate of EFVF treatment for all test bacterial strains, regardless of

413

metabolic activation. Therefore, our results demonstrated that EFVF does not have

414

mutagenic potential toward any genes regarding point mutations under the present

415

experimental conditions.

416

The micronucleus test is useful to detect chemically induced chromosomal damages

417

(Ashby, 1985), as well as accumulated genotoxic damage in response to complex mixture of

418

contaminants risk (Lando et al., 2007; Murgia et al., 2008). Recently, it was also reported

419

that the increased frequency of micronucleus formation in peripheral blood lymphocytes is

420

related to cancer risk (Lando et al., 2007; Murgia et al., 2008). Prior to the micronucleus test,

421

we confirmed that EFVF-administered ICR mice did not show abnormal or toxicity-related

422

signs in general appearance and body weights. In the bone marrow cells derived from the

423

femur of the ICR mice, there was no significant or dose-related increase in the number of

424

MNPCE at any EFVF treatment dose level. In addition, the PCE/RBC (PCE + NCE) ratio,

425

an indicator of cytotoxicity, was not significantly decreased compared with the negative

426

control. Collectively, these data indicate that EFVF does not have the potential to induce 23

427

clastogenicity or the cytotoxicity of mouse bone marrow cells under the present experimental

428

conditions.

429

The chromosomal aberration test can be used to verify whether a test substance causes

430

the clastogenicity. That is abnormal changes in the structure or number of chromosomes,

431

which may play an important role in tumor progression (Emerit, 2007; Savage, 1991). There

432

are two types of chromosomal aberrations, involving chromatid and chromosome. A

433

chromatid is one of the two identical copies of DNA making up a replicated chromosome.

434

The majority of chemically induced aberrations are of the chromatid type, however,

435

chromosome type aberration also may occur (OECD, 2014a). In our chromosomal aberration

436

test of EFVF, the frequencies of structural aberrations in both the 6 h- and 24-treatment

437

groups were less than 5% at all test concentrations of EFVF, irrespective of metabolic

438

activation, and there was no increase in the frequencies of numerical aberrations. Accordingly,

439

EFVF is not inducible the chromosomal aberrations in cultured CHL/IU cells under the tested

440

dose conditions.

441

Collectively, our novel findings indicate that EFVF has no oral acute toxicity at up to

442

5000 mg/kg. In the Ames test, EFVF did not result in any detectable gene mutations in

443

bacterial strains at up to 5000 µg/plate. In addition, EFVF did not show the potential to

444

induce clastogenicity in the in vivo micronucleus test and the in vitro chromosomal aberration

445

test. Therefore, these results suggest that EFVF can be safely used as a source material for

446

traditional herbal medicine and EFVF-based drug development, at least within the dose range

447

used in the present study.

448

24

449

Acknowledgments

450 451

This work was supported by a grant from the Korea Institute of Oriental Medicine (grant number KSN1515294).

452

453

Authors’ contributions

454

SS wrote the manuscript. NSK and OSB contributed to the conception and design of

455

the study, and editing the original draft. SS performed and discussed the UHPLC analysis.

456

JMY, CSP and SHK performed the experiments and data validation. OSB contributed to the

457

discussion of the data, funding acquisition and supervision. All authors read and approved

458

final version of the manuscript.

459 460

Conflicts of interest

461

The authors declare no conflict of interests related to this work.

462

463 464

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