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
28
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
77
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
204
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
206
in growth medium and were maintained in a CO2 incubator at 37 °C for 3 days. CHL/IU cells
207
were treated with serial doses of EFVF (413-1650 and 625-2500 µg/mL) or the
208
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
211
positive/negative control drugs for a 24 h treatment followed by 0 h recovery in the absence
212
of S9 metabolic activation. Two hours before the completion of the final culture, cells were
213
incubated with 0.2 µg /mL colcemide solution for 2 h. Cells that were arrested at metaphase,
214
were then collected and treated with 5 mL pre-warmed 75 mM KCl at 37 °C for 20 min, and
215
finally fixed in a methanol: acetic acid (3:1) mixture. Two smears per slide were then
216
prepared and stained with 3% (v/v) Giemsa solution. More than 100 metaphases per smear
217
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
219
cell and the type of aberration was characterized in accordance with the Atlas of
220
Chromosome Aberration by Chemicals (JEMS-MMS, 1988). Structural aberrations in
221
metaphasic chromosomes were categorized into chromosome types of breaks (csb),
222
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
224
aberration. The numerical aberration in metaphasic chromosomes was then categorized into
225
polyploidy (pol) and endo-reduplication (end). This method was regarded as valid in that the
226
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
231
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
References
465
Adams, M., Jewell, A.P., 2007. The use of Complementary and Alternative Medicine by
466
cancer
467
patients. Int. J. Surg. Oncol. 4, 10. https://doi.org/10.1186/1477-7800-4-10.
468
Ames, B.N., McCann, J., Yamasaki, E., 1975. Methods for detecting carcinogens and
469
mutagens 25
470
with the salmonella/mammalian-microsome mutagenicity test. Mutat. Res. Envir. Muta.
471
31, 347–363. https://doi.org/10.1016/0165-1161(75)90046-1.
472
Ansah, C., Khan, A., Gooderham, N.J., 2005. In vitro genotoxicity of the West African anti-
473
malarial herbal Cryptolepis sanguinolenta and its major alkaloid cryptolepine. Toxicology
474
208, 141–147. https://doi.org/10.1016/j.tox.2004.11.026.
475
Ashby, J., 1985. Is there a continuing role for the intraperitoneal injection route of exposure
476
in short-term rodent genotoxicity assays?. Mutat. Res. Genet. Toxicol. 156, 239–243.
477
https://doi.org/10.1016/0165-1218(85)90069-2.
478
Bao, J., Ding, R., Zou, L., Zhang, C., Wang, K., Liu, F., Li, P., Chen, M., Wan, J.B., Su, H.,
479
Wang, Y., He, C., 2016. Forsythiae Fructus Inhibits B16 Melanoma Growth Involving
480
MAPKs/Nrf2/HO-1 Mediated Anti-Oxidation and Anti-Inflammation. Am. J. Chin. Med.
481
44, 1043–1061. http://doi.org/10.1142/S0192415X16500580.
482
Chen, H.Y., Lin, Y.H., Huang, J.W., Chen, Y.C., 2015. Chinese herbal medicine network and
483
core treatments for allergic skin diseases: Implications from a nationwide database. J.
484
Ethnopharmacol. 168, 260–267. https://doi.org/10.1016/j.jep.2015.04.002.
485
IPC, 2015. In Pharmacopoeia of People’s Republic of China; Chemical Industry Press.
486
International Pharmacopoeia Commission; Pharmacopoeia Commission of the People's
487
Republic of China, Beijing, China, pp. 1–1750.
488
Di Stasi, L.C., Oliveira, G.P., Carvalhaes, M.A., Queiroz-Junior, M., Tien, O.S., Kakinami,
489
S.H., Reis, M.S., 2002. Medicinal plants popularly used in the Brazilian Tropical Atlantic
490
Forest. Fitoterapia 73, 69–91. https://doi.org/10.1016/S0367-326X(01)00362-8.
491
Dong, Z., Lu, X., Tong, X., Dong, Y., Tang, L., Liu, M., 2017. Forsythiae Fructus: A Review
492
on its Phytochemistry, Quality Control, Pharmacology and Pharmacokinetics. Molecules
493
22. https://doi.org/10.3390/molecules22091466. 26
494 495
Emerit, L., 2007. Clastogenic factors as potential biomarkers of increased superoxide production. Biomark insights 2, 429–438.
496
Erexson, G.L., 2003. Lack of in vivo clastogenic activity of grape seed and grape skin
497
extracts in a mouse micronucleus assay. Food. Chem. Toxicol. 41, 347–350.
498
https://doi.org/10.1016/S0278-6915(02)00236-3.
499
Fu, B., Wang, N., Tan, H.-Y., Li, S., Cheung, F., Feng, Y., 2018. Multi-Component Herbal
500
Products in the Prevention and Treatment of Chemotherapy-Associated Toxicity and Side
501
Effects: A Review on Experimental and Clinical Evidences. Front. Pharmacol. 9, 1394–
502
1394. https://doi.org/10.3389/fphar.2018.01394.
503
Guo, N., Gai, Q.-Y., Jiao, J., Wang, W., Zu, Y.-G., Fu, Y.-J., 2016. Antibacterial Activity of
504
Fructus forsythia Essential Oil and the Application of EO-Loaded Nanoparticles to Food-
505
Borne Pathogens. Foods 5, 73. https://doi.org/10.3390/foods5040073.
506
Hwang, Y.H., Kim, D.G., Li, W., Yang, H.J., Yim, N.H., Ma, J.Y., 2017. Anti-inflammatory
507
effects
508
Ethnopharmacol. 206, 73–77. https://doi.org/10.1016/j.jep.2017.05.011.
509 510 511 512 513 514
of
Forsythia
suspensa
in
dextran
sulfate
sodium-induced
colitis.
J.
Ishidate, M., Jr., Sofuni, T., 1985. The in vitro chromosomal aberration test using chinese hamster lung (CHL) fibroblast cells in culture. Pro. Mutat. Res. 5, 427–432. JDECA, 2011. Japanese Pharmacopoeia, in: Ministry of Health, L.W. (Ed.). The Japan Drug Editional Commission of Administration, Tokyo, Japan, pp. 1–2131. JEMS–MMS, 1988. Atlas of Chromosome Aberration by Chemicals. Japanese Enviromental Mutagen Society-Mammalian Mutagenicity Study Group.
515
Jordan, S.A., Cunningham, D.G., Marles, R.J., 2010. Assessment of herbal medicinal
516
products: challenges, and opportunities to increase the knowledge base for safety
517
assessment.
Toxicol.
Appl.
Pharmacol. 27
243,
198–216.
518 519 520
https://doi.org/10.1016/j.taap.2009.12.005. Kim, M.J., Kim, J.Y., Jung, T.K., Choi, S.W., Yoon, K.-S., 2006. Skin anti-aging effect of Forsythia viridissima L. extract. Korean J. Biotechnol. Bioeng. 6, 444–450.
521
Kuo, P.C., Hung, H.Y., Nian, C.W., Hwang, T.L., Cheng, J.C., Kuo, D.H., Lee, E.J., Tai, S.H.,
522
Wu, T.S., 2017. Chemical Constituents and Anti-inflammatory Principles from the Fruits
523
of
524
https://doi.org/10.1021/acs.jnatprod.6b01141.
Forsythia
suspensa.
J.
Nat.
Prod.
80,
1055–1064.
525
Lando, C., Ceppi, M., Bonassi, S., Bigatti, M.P., Cebulska-Wasilewska, A., Fabianova, E.,
526
Fucic, A., Hagmar, L., Joksic, G., Martelli, A., 2007. An increased micronucleus
527
frequency in peripheral blood lymphocytes predicts the risk of cancer in humans.
528
Carcinogenesis 28, 625–631. https://doi.org/10.1093/carcin/bgl177.
529
Lee, J.Y., Moon, H., Kim, C.J., 2010. Effects of hydroxy pentacyclic triterpene acids from
530
Forsythia viridissima on asthmatic responses to ovalbumin challenge in conscious guinea
531
pigs. Biol. Pharm. Bull. 33, 230–237.
532
Lee, S.E., Lim, C., Ahn, S.C., Cho, S., 2017. A Study of the Anti-Cancer Effects of the
533
Hexane Fraction of the Methanol Extract of Forsythiae Fructus. Pharmacogn. Mag. 13,
534
719–724. https://doi.org/10.4103/0973-1296.211079.
535 536
Maron, D.M., Ames, B.N., 1983. Revised methods for the Salmonella mutagenicity test. Mutat. Res 113, 173–215. https://doi.org/10.1016/0165-1161(83)90010-9.
537
Marques, R.C.P., De Medeiros, L.R.B., Da Silva Dias, C., Barbosa-Filho, J.M., Agnez-Lima,
538
L.F., 2003. Evaluation of the mutagenic potential of yangambin and of the hydroalcoholic
539
extract of Ocotea duckei by the Ames test. Mutat. Res. Gen.Tox. En. 536, 117–120.
540
https://doi.org/10.1016/S1383-5718(03)00040-8.
541
MFDS, 2015. South Korean Pharmacopoeia; Monographs Part II, Ministry of Food and Drug 28
542 543 544 545 546
Safety, Sejong, Republic of Korea, 1004–1241. MFDS, 2017a. Guidelines for the Nonclinical Laboratory Test. No. 2017–32. Ministry of Food and Drug Safety, Sejong, Republic of Korea. MFDS, 2017b. Guidelines for Toxicity Tests of Pharmaceuticals. No. 2017–71. Ministry of Food and Drug Safety, Sejong, Republic of Korea.
547
Melo-Reis, P., Bezerra, L., Vale, M., Canhête, R., Chen-Chen, L., 2011. Assessment of the
548
mutagenic and antimutagenic activity of Synadenium umbellatum Pax latex by
549
micronucleus test in mice. Braz. J. Biol. 71, 169–174. https://doi.org/10.1590/S1519-
550
69842011000100024.
551
Mori, T., Hamada, S., Yoshie, S., Jeon, B., Jin, X., Takahashi, H., Iijima, K., Ishizaki, T.,
552
Tamiya, N., 2019. The associations of multimorbidity with the sum of annual medical and
553
long-term
554
https://doi.org/10.1186/s12877-019-1057-7.
555
care
expenditures
in
Japan.
BMC
Geriatrics
19,
69.
Mortelmans, K., Zeiger, E., 2000. The Ames Salmonella/microsome mutagenicity assay.
556
Mutat.
557
5107(00)00064-6.
Res.
Envir.
Muta.
455
(1-2),
29–60.
https://doi.org/10.1016/S0027-
558
Murgia, E., Ballardin, M., Bonassi, S., Rossi, A.M., Barale, R., 2008. Validation of
559
micronuclei frequency in peripheral blood lymphocytes as early cancer risk biomarker in
560
a
561
https://doi.org/10.1016/j.mrfmmm. 2007.10.010.
nested
case–control
study.
Mutat.
Res.
Fund.
Mol.
M.
639,
27–34.
562
OECD, 1997. Guideline for the Testing of Chemical. No. 471: Bacterial Reverse Mutation
563
Test. Organization for Economic Cooperation and Development. https://doi.org/10.1787/
564
9789264071247-en.
565
OECD, 2014a. Guideline for the Testing of Chemical. No. 473: In vitro Mammalian 29
566
Chromosome Aberration Test. Organization for Economic Cooperation and Development.
567
https://doi.org/10.1787/9789264224223-en.
568
OECD, 2014b. Guideline for the Testing of Chemical. No. 474: Mammalian Erythrocyte
569
Micronucleus Test. Organization for Economic Cooperation and Development.
570
https://doi.org/10.1787/9789264224292-en.
571
Pelkonen, O., Xu, Q., Fan, T.-P., 2014. Why is Research on Herbal Medicinal Products
572
Important and How Can We Improve Its Quality? J. Tradit. Complement. Med. 4, 1–7.
573
https://doi.org/10.4103/2225-4110.124323.
574
Prasad, S., Sung, B., Aggarwal, B.B., 2012. Age-associated chronic diseases require age-old
575
medicine:
576
https://doi.org/10.1016/j.ypmed.2011.11.011.
role
of
chronic
inflammation.
Prev.
Med.
54,
S29–S37.
577
Regner, G.G., Gianesini, J., Von Borowski, R.G., Silveira, F., Semedo, J.G., Ferraz, A.d.B.F.,
578
Wiilland, E., Von Poser, G., Allgayer, M., Picada, J.N., Pereira, P., 2011. Toxicological
579
evaluation of Pterocaulon polystachyum extract: A medicinal plant with antifungal
580
activity.
581
https://doi.org/10.1016/j.etap.2010.11.003
Environ.
Toxicol.
Pharmacol.
31,
242–249.
582
Savage, J.R.K., 1991. Chromosomal Aberrations: Basic and Applied Aspects. Edited by G.
583
Obe and A. T. Natarajan. Berlin, Heidelberg: Springer-Verlag. 1990. Genet. Res. 57, 201–
584
201. https://doi.org/10.1017/S001667230002927X.
585
Sofuni, T., 1999. Data book of of chromosomal aberration test in vitro. Tokyo: LIC Ltd.
586
Sponchiado, G., Adam, M.L., Silva, C.D., Silva Soley, B., De Mello-Sampayo, C., Cabrini,
587
D.A., Correr, C.J., Otuki, M.F., 2016. Quantitative genotoxicity assays for analysis of
588
medicinal
589
https://doi.org/10.1016/j.jep.2015.10.026.
plants:
A
systematic
review.
30
J.
Ethnopharmacol.
178,
289–296.
590
Yi, J.-M., Shin, S., Kim, N.S., Bang, O.-S., 2019. Neuroprotective Effects of an Aqueous
591
Extract of Forsythia viridissima and Its Major Constituents on Oxaliplatin-Induced
592
Peripheral
593
https://doi.org/doi:10.3390/molecules24061177.
Neuropathy.
Molecules
24,
1177.
594
Yin, X.Y., Liu, D.X., Wang, H.C., Zhou, Y., 1991. A study on the mutagenicity of 102 raw
595
pharmaceuticals used in Chinese traditional medicine. Mutat. Res. Genet. Toxicol. 260,
596
73–82. https://doi.org/10.1016/0165-1218(91)90082-W.
597
Zan, M.A., Ferraz, A.B.F., Richter, M.F., Picada, J.N., de Andrade, H.H.R., Lehmann, M.,
598
Dihl, R.R., Nunes, E., Semedo, J., Da Silva, J., 2013. In Vivo Genotoxicity Evaluation of
599
an Artichoke (Cynara scolymus L.) Aqueous Extract. J. Food Sci. 78, T367–T371.
600
https://doi.org/10.1111/1750-3841.12034.
601
Zhang, S., Shao, S.Y., Song, X.Y., Xia, C.Y., Yang, Y.N., Zhang, P.C., Chen, N.H., 2016.
602
Protective effects of Forsythia suspense extract with antioxidant and anti-inflammatory
603
properties in a model of rotenone induced neurotoxicity. Neurotoxicology 52, 72–83.
604
https://doi.org/10.1016/j.neuro.2015.09.009.
605
Zhao, P., Piao, X., Pan, L., Zeng, Z., Li, Q., Xu, X., Wang, H., 2017. Forsythia suspensa
606
extract attenuates lipopolysaccharide-induced inflammatory liver injury in rats via
607
promoting
608
https://doi.org/10.1111/asj.12717.
antioxidant
defense
mechanisms.
609
31
Anim.
Sci.
J.
88,
873–881.