Accepted Manuscript Title: Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae Author: Zhao-Guang Lu Ming-Hui Li Jun-Song Wang Dan-Dan Wei Qing-Wang Liu Ling-Yi Kong PII: DOI: Reference:
S0890-6238(14)00100-2 http://dx.doi.org/doi:10.1016/j.reprotox.2014.05.015 RTX 6970
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
7-12-2013 11-4-2014 24-5-2014
Please cite this article as: Lu Z-G, Li M-H, Wang J-S, Wei D-D, Liu Q-W, Kong LY, Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae, Reproductive Toxicology (2014), http://dx.doi.org/10.1016/j.reprotox.2014.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine
2
and sophocarpine, in zebrafish (Danio rerio) embryos/larvae
3
Zhao-Guang Lua, †, Ming-Hui Lia, †, Jun-Song Wangb, *, Dan-Dan Weia, Qing-Wang
5
Liua, Ling-Yi Konga, *
ip t
4
6 a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
cr
7
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR
9
China
10
b
us
8
Center for Molecular Metabolism, School of Environmental & Biological
Engineering, Nanjing University of Science & Technology, 200 Xiao Ling Wei Street,
12
Nanjing 210094, PR China
an
11
14
*
M
13
Corresponding author: Prof. Ling-Yi Kong and Prof. Jun-Song Wang
Prof. Ling-Yi Kong:
16
E-mail: [email protected]
17
Address: Department of Natural Medicinal Chemistry, China Pharmaceutical
18
University, 24 Tongjia Xiang, Nanjing 210009, Jiangsu, PR China
24
Ac ce p
te
d
15
25
Phone/Fax: +86 25 8327 1402
19 20 21 22 23
Phone/Fax: +86 25 8327 1405
Prof. Jun-Song Wang:
E-mail: [email protected] Address: School of Environmental & Biological Engineering, Nanjing University of Science & Technology, 200 Xiao Ling Wei Street, Nanjing 210094, PR China
26 27
†
These authors contributed equally to this work
28 29
Page 1 of 39
29
ABSTRACT: Matrine and sophocarpine are two major matrine-type alkaloids
31
included in the traditional Chinese medicine (TCM) Kushen (the root of Sophora
32
flavescens Ait.). They have been widely used clinically in China, however with few
33
reports concerning their potential toxicities. This study investigated the developmental
34
toxicity and neurotoxicity of matrine and sophocarpine on zebrafish embryos/larvae
35
from 0 to 96/120 hours post fertilization (hpf). Both drugs displayed teratogenic and
36
lethal effects with the EC50 and LC50 values at 145 and 240 mg/L for matrine and 87.1
37
and 166 mg/L for sophocarpine, respectively. Exposure of matrine and sophocarpine
38
significantly altered spontaneous movement and inhibited swimming performance at
39
concentrations below those causing lethality and malformations, indicating a
40
neurotoxic potential of both drugs. The results are in agreement with most
41
mammalian studies and clinical observations.
M
an
us
cr
ip t
30
42
KEYWORDS:
Matrine;
Sophocarpine;
Zebrafish;
Developmental
44
Spontaneous movement; Swimming behavior; Neurotoxicity
toxicity;
te
d
43
Ac ce p
45
Page 2 of 39
45 46
1. Introduction
47
Kushen (the root of Sophora flavescens Ait.) is a well-known traditional Chinese
49
medicine (TCM) used in the treatment of cancer, psoriasis, as well as other diseases
50
[1-4]. Xiaoyin Recipe (Xiaoyin Tablet), a Chinese herbal formulation containing
51
Kushen, has been used successfully to treat psoriasis patients [3, 5, 6]. Matrine and
52
sophocarpine (Fig. 1), two matrine-type alkaloids, are its main active components.
53
Possessing a variety of pharmacological effects such as anti-inflammation, antivirus,
54
anti-tumor and antiarrhythmic activities [7-9], both drugs have high frequency of
55
clinical use in China. There are many dosage forms of matrine (injection, capsule and
56
suppositories, etc.) in China for the treatment of hepatitis B, enteritis, gynecological
57
infections, cancer and cardiac arrhythmia [10]. The two alkaloids are also widely used
58
in agriculture and forestry production to prevent or control pests in China [11, 12].
M
an
us
cr
ip t
48
Despite their frequent use, studies on their toxicities and side effects are still sparse.
60
Several studies in rodents suggested the nervous system as the main target organ of
61
the induced toxicity by both alkaloids. Liu et al. [13] reported that matrine and
62
sophocarpine
te
d
59
increased
dopamine
metabolites
68
Ac ce p
significantly
69
including abnormal gait, convulsions, dysphasia, mental changes, dystonia and other
70
nervous system symptoms. Noteworthily, most of these poisoning cases were found in
71
juvenile and infant patients taking these drugs [15-18], which combined with the fact
72
that they were also used by pregnant women, necessitate the study on the potential
73
risk of early developmental exposure to matrine and sophocarpine.
63 64 65 66 67
3,4-dihydroxyphenylacetic acid and homovanillic acid in rat striatum and limbic area, and that the high dose of sophocarpine lowered the content of dopamine in rat striatum. Intraperitoneal injection of matrine to mice led to degenerative changes of the nerve cells in the brain tissue [14]. Several clinical cases reported that oral administration of high dose of prescriptions containing matrine and/or sophocarpine resulted in severe poisoning to patients, with complex neurological manifestations
Page 3 of 39
In order to determine developmental toxicity, pregnant animals, usually rats or
75
rabbits, are exposed to test compounds and the toxic effects are evaluated in fetuses
76
[19, 20]. These conventional developmental toxicity tests not only require the use of
77
high numbers of experimental animals, but are also time- and resource-consuming due
78
to the long reproductive cycle. As a novel experimental model organism for
79
developmental biology, zebrafish offers many benefits over other mammals such as
80
economic husbandry requirements, small size, high fecundity and rapid extracorporeal
81
development [21-24]. Another major advantage of using zebrafish embryo for
82
teratogenicity assay is its transparency. In mammalian assays, the mothers have to be
83
sacrificed to allow the inspection of embryonic development, which are of ethical
84
concern. However, embryonic development could be easily and directly observed in
85
embryos of zebrafish or other fish species. Many aspects of neurodevelopmental
86
process and function in zebrafish are similar to those of mammals, thus providing a
87
simple and accessible model system for investigation [25].
M
an
us
cr
ip t
74
In this study, we investigated the developmental toxicities and neurotoxicities of
89
matrine and sophocarpine in the zebrafish embryos to estimate their toxicological
90
potential and potential risk. The early developmental toxicities on zebrafish embryos
91
induced by the two drugs, were detected by analysis of lethality, malformations and
93 94 95 96 97
te
Ac ce p
92
d
88
heart beat frequency. Spontaneous movement change and behavior alteration were used to evaluate the potential neurotoxicity of both drugs.
2. Materials and methods
2.1. Materials
98 99
Matrine (CAS-number: 519-02-8; HPLC purity: ≥98%; molecular weight: 248.36;
100
molecular formula: C15H24N2O) and sophocarpine (CAS-number: 145572-44-7;
101
HPLC purity: ≥98%; molecular weight: 246.17; molecular formula: C15H22N2O) were
102
purchased from Ningxia Bauhinia Pharmaceutical Co., Ltd. (Ningxia, China). Sodium
Page 4 of 39
chloride (NaCl), potassium chloride (KCl), sodium hydrogen carbonate (NaHCO3),
104
calcium chloride (CaCl2) and potassium dihydrogen phosphate (KH2PO4) were of
105
analytical grade and obtained from Nanjing Chemical Reagent Co., Ltd. (Nanjing,
106
China). Acetonitrile and diethylamine used for HPLC were of HPLC grade and
107
purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).
108
Ultra-pure water (resistance ≥18.2 MΩ cm-1) for Holtfreter's solution (60 mM NaCl,
109
2.4 mM NaHCO3, 0.8 mM CaCl2, 0.67 mM KCl) [26, 27] preparation and HPLC was
110
produced using a Barnstead Easypure II ultrapure water system (Thermo Fisher
111
Scientific, Hudson, NH, USA). Superior non-residue shrimp flake food (product
112
ingredients: crude protein ≥48%, crude fat ≥8%, crude fibre ≤4%, crude ash ≤18%
113
and moisture ≤10%) was bought from Guangdong Yuequn Ocean Biological
114
Research and Development Co., Ltd. (Jieyang, China).
an
us
cr
ip t
103
Matrine and sophocarpine were dissolved in Holtfreter's solution to obtain two
116
stock solutions with concentration of 1 mg/L. Within 1 day, the stock solutions were
117
diluted into a series of test solutions with Holtfreter's solution for exposure
118
experiment. The stock solutions of both drugs were stable during storage for 48 hours
119
at 4 °C with little change in concentrations by HPLC analysis.
te
d
M
115
126
Ac ce p
120
127
commercially available non-residue shrimp flake food twice a day. The water used for
128
housing and breeding was maintained under the following conditions based on OECD
129
guidelines [28]: temperature (26±1 °C), conductivity (200 μS/cm), hardness (116
130
mg/L CaCO3), dissolved oxygen (O2 ≥80% saturation 7.75±0.23 mg/L O2), pH
131
(7.5±0.5), nitrites (
S0890-6238(14)00100-2 http://dx.doi.org/doi:10.1016/j.reprotox.2014.05.015 RTX 6970
To appear in:
Reproductive Toxicology
Received date: Revised date: Accepted date:
7-12-2013 11-4-2014 24-5-2014
Please cite this article as: Lu Z-G, Li M-H, Wang J-S, Wei D-D, Liu Q-W, Kong LY, Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae, Reproductive Toxicology (2014), http://dx.doi.org/10.1016/j.reprotox.2014.05.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine
2
and sophocarpine, in zebrafish (Danio rerio) embryos/larvae
3
Zhao-Guang Lua, †, Ming-Hui Lia, †, Jun-Song Wangb, *, Dan-Dan Weia, Qing-Wang
5
Liua, Ling-Yi Konga, *
ip t
4
6 a
State Key Laboratory of Natural Medicines, Department of Natural Medicinal
cr
7
Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR
9
China
10
b
us
8
Center for Molecular Metabolism, School of Environmental & Biological
Engineering, Nanjing University of Science & Technology, 200 Xiao Ling Wei Street,
12
Nanjing 210094, PR China
an
11
14
*
M
13
Corresponding author: Prof. Ling-Yi Kong and Prof. Jun-Song Wang
Prof. Ling-Yi Kong:
16
E-mail: [email protected]
17
Address: Department of Natural Medicinal Chemistry, China Pharmaceutical
18
University, 24 Tongjia Xiang, Nanjing 210009, Jiangsu, PR China
24
Ac ce p
te
d
15
25
Phone/Fax: +86 25 8327 1402
19 20 21 22 23
Phone/Fax: +86 25 8327 1405
Prof. Jun-Song Wang:
E-mail: [email protected] Address: School of Environmental & Biological Engineering, Nanjing University of Science & Technology, 200 Xiao Ling Wei Street, Nanjing 210094, PR China
26 27
†
These authors contributed equally to this work
28 29
Page 1 of 39
29
ABSTRACT: Matrine and sophocarpine are two major matrine-type alkaloids
31
included in the traditional Chinese medicine (TCM) Kushen (the root of Sophora
32
flavescens Ait.). They have been widely used clinically in China, however with few
33
reports concerning their potential toxicities. This study investigated the developmental
34
toxicity and neurotoxicity of matrine and sophocarpine on zebrafish embryos/larvae
35
from 0 to 96/120 hours post fertilization (hpf). Both drugs displayed teratogenic and
36
lethal effects with the EC50 and LC50 values at 145 and 240 mg/L for matrine and 87.1
37
and 166 mg/L for sophocarpine, respectively. Exposure of matrine and sophocarpine
38
significantly altered spontaneous movement and inhibited swimming performance at
39
concentrations below those causing lethality and malformations, indicating a
40
neurotoxic potential of both drugs. The results are in agreement with most
41
mammalian studies and clinical observations.
M
an
us
cr
ip t
30
42
KEYWORDS:
Matrine;
Sophocarpine;
Zebrafish;
Developmental
44
Spontaneous movement; Swimming behavior; Neurotoxicity
toxicity;
te
d
43
Ac ce p
45
Page 2 of 39
45 46
1. Introduction
47
Kushen (the root of Sophora flavescens Ait.) is a well-known traditional Chinese
49
medicine (TCM) used in the treatment of cancer, psoriasis, as well as other diseases
50
[1-4]. Xiaoyin Recipe (Xiaoyin Tablet), a Chinese herbal formulation containing
51
Kushen, has been used successfully to treat psoriasis patients [3, 5, 6]. Matrine and
52
sophocarpine (Fig. 1), two matrine-type alkaloids, are its main active components.
53
Possessing a variety of pharmacological effects such as anti-inflammation, antivirus,
54
anti-tumor and antiarrhythmic activities [7-9], both drugs have high frequency of
55
clinical use in China. There are many dosage forms of matrine (injection, capsule and
56
suppositories, etc.) in China for the treatment of hepatitis B, enteritis, gynecological
57
infections, cancer and cardiac arrhythmia [10]. The two alkaloids are also widely used
58
in agriculture and forestry production to prevent or control pests in China [11, 12].
M
an
us
cr
ip t
48
Despite their frequent use, studies on their toxicities and side effects are still sparse.
60
Several studies in rodents suggested the nervous system as the main target organ of
61
the induced toxicity by both alkaloids. Liu et al. [13] reported that matrine and
62
sophocarpine
te
d
59
increased
dopamine
metabolites
68
Ac ce p
significantly
69
including abnormal gait, convulsions, dysphasia, mental changes, dystonia and other
70
nervous system symptoms. Noteworthily, most of these poisoning cases were found in
71
juvenile and infant patients taking these drugs [15-18], which combined with the fact
72
that they were also used by pregnant women, necessitate the study on the potential
73
risk of early developmental exposure to matrine and sophocarpine.
63 64 65 66 67
3,4-dihydroxyphenylacetic acid and homovanillic acid in rat striatum and limbic area, and that the high dose of sophocarpine lowered the content of dopamine in rat striatum. Intraperitoneal injection of matrine to mice led to degenerative changes of the nerve cells in the brain tissue [14]. Several clinical cases reported that oral administration of high dose of prescriptions containing matrine and/or sophocarpine resulted in severe poisoning to patients, with complex neurological manifestations
Page 3 of 39
In order to determine developmental toxicity, pregnant animals, usually rats or
75
rabbits, are exposed to test compounds and the toxic effects are evaluated in fetuses
76
[19, 20]. These conventional developmental toxicity tests not only require the use of
77
high numbers of experimental animals, but are also time- and resource-consuming due
78
to the long reproductive cycle. As a novel experimental model organism for
79
developmental biology, zebrafish offers many benefits over other mammals such as
80
economic husbandry requirements, small size, high fecundity and rapid extracorporeal
81
development [21-24]. Another major advantage of using zebrafish embryo for
82
teratogenicity assay is its transparency. In mammalian assays, the mothers have to be
83
sacrificed to allow the inspection of embryonic development, which are of ethical
84
concern. However, embryonic development could be easily and directly observed in
85
embryos of zebrafish or other fish species. Many aspects of neurodevelopmental
86
process and function in zebrafish are similar to those of mammals, thus providing a
87
simple and accessible model system for investigation [25].
M
an
us
cr
ip t
74
In this study, we investigated the developmental toxicities and neurotoxicities of
89
matrine and sophocarpine in the zebrafish embryos to estimate their toxicological
90
potential and potential risk. The early developmental toxicities on zebrafish embryos
91
induced by the two drugs, were detected by analysis of lethality, malformations and
93 94 95 96 97
te
Ac ce p
92
d
88
heart beat frequency. Spontaneous movement change and behavior alteration were used to evaluate the potential neurotoxicity of both drugs.
2. Materials and methods
2.1. Materials
98 99
Matrine (CAS-number: 519-02-8; HPLC purity: ≥98%; molecular weight: 248.36;
100
molecular formula: C15H24N2O) and sophocarpine (CAS-number: 145572-44-7;
101
HPLC purity: ≥98%; molecular weight: 246.17; molecular formula: C15H22N2O) were
102
purchased from Ningxia Bauhinia Pharmaceutical Co., Ltd. (Ningxia, China). Sodium
Page 4 of 39
chloride (NaCl), potassium chloride (KCl), sodium hydrogen carbonate (NaHCO3),
104
calcium chloride (CaCl2) and potassium dihydrogen phosphate (KH2PO4) were of
105
analytical grade and obtained from Nanjing Chemical Reagent Co., Ltd. (Nanjing,
106
China). Acetonitrile and diethylamine used for HPLC were of HPLC grade and
107
purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China).
108
Ultra-pure water (resistance ≥18.2 MΩ cm-1) for Holtfreter's solution (60 mM NaCl,
109
2.4 mM NaHCO3, 0.8 mM CaCl2, 0.67 mM KCl) [26, 27] preparation and HPLC was
110
produced using a Barnstead Easypure II ultrapure water system (Thermo Fisher
111
Scientific, Hudson, NH, USA). Superior non-residue shrimp flake food (product
112
ingredients: crude protein ≥48%, crude fat ≥8%, crude fibre ≤4%, crude ash ≤18%
113
and moisture ≤10%) was bought from Guangdong Yuequn Ocean Biological
114
Research and Development Co., Ltd. (Jieyang, China).
an
us
cr
ip t
103
Matrine and sophocarpine were dissolved in Holtfreter's solution to obtain two
116
stock solutions with concentration of 1 mg/L. Within 1 day, the stock solutions were
117
diluted into a series of test solutions with Holtfreter's solution for exposure
118
experiment. The stock solutions of both drugs were stable during storage for 48 hours
119
at 4 °C with little change in concentrations by HPLC analysis.
te
d
M
115
126
Ac ce p
120
127
commercially available non-residue shrimp flake food twice a day. The water used for
128
housing and breeding was maintained under the following conditions based on OECD
129
guidelines [28]: temperature (26±1 °C), conductivity (200 μS/cm), hardness (116
130
mg/L CaCO3), dissolved oxygen (O2 ≥80% saturation 7.75±0.23 mg/L O2), pH
131
(7.5±0.5), nitrites (
