# Evaluation on Genotoxicity and Teratogenicity of Aqueous Extract fromCyclocarya paliurus Leaves

Authors: Lihong Deng; Jiandu Lei; Jing He; Jing Liu; Luying Wang; Rui Zhang; Xinhua Liu; Yun Liu
Journal: The Scientific World Journal
 (2014)

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## Abstract

Tremendous attentions have been attracted to the foods labeled with natural, green, organic, and nuisanceless conception of healthy diet. Therefore, it is of great significance to establish relative defining guidance for safe assessment of botanicals.Cyclocarya paliurus (Batal.) Iljinsk (family Cyclocaryaceae), called sweet tea tree, is a well-known edible and medicinal plant, which has been widely used in China as drug formulation for the treatment of hypertension and diabetes. Despite its benefits, no reports have been described on the safe assessment of C. paliurus leaves aqueous extract. In this study, we have conducted the genotoxicity assay (including Ames test, bone marrow polychromatic erythrocyte micronucleus test, and sperm abnormality test in mice) and traditional teratogenicity assay in rats (maternal toxicity, embryo toxicity, and teratogenicity test) to assess the genetic and teratogenic safety of aqueous extracts from C. paliurus leaves. Results of each assay show that the highest dose of C. paliurus leaves aqueous extract is considered relatively nonmutagenic and nonteratogenic, revealing that C. paliurus leaves possess safety and quality as a functional additional ingredient in food.


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## Body

## 1. Introduction

Cyclocarya paliurus, an endemic plant in China and the sole species in the genusCyclocarya Iljinskaja (Juglandaceae), is mainly found in the cloudy and foggy high-mountainous regions in southern China [1, 2], and possesses activities of anti-microorganisms, enhancement of mental efficiency and immunomodulation [3–9]. It has also been revealed that the chemical constituents of this plant contain protein, polysaccharides [3, 5, 10–12], triterpenoids, flavonoids, steroids, saponins, and phenolic compounds [13–16].The leaves ofC. paliurus have been used for drug formulations in traditional Chinese medicine, as well as an ingredient in functional foods in China [5] or dietary supplements [3]; they have even been a food resource for maritime people for a long time [13], which are processed as tea products, the first FDA-approved health tea in China in 1999 [17], and consumed as a beverage in Chinese daily life. The recommended human dose of this plant’s leaves is 0.133 g/kg·bw. With the developing concerns about healthy diet, the natural green plant and its derived products have turned to be new favorite products in food industry [18]. However, in modern society, because of the undesirable adverse health effects appearing due to the use of plant-based outcomes, it has been clearly realized that “natural” is not equal to “safe” [19]. Recently, there have been reports that some traditional medical plants cause genotoxicity or cancer. The botanicals and herbal food supplements, which are generally recognized as safe, need to be set up legislative frameworks and guidance for risk assessment forwardly [20]. Delightedly, several organizations have undertaken the work and achieved results [21, 22]. However, to our knowledge, little information has been critically given on the issue of safety, such as the toxicity ofC.paliurus.The acute and subacute toxicity test of aqueous extracts fromC. paliurus leaves in mice and rats had been conducted in our group. The result shows that the highest tested dosage of 40.0 g/kg·bw, equivalent to 300-fold of human daily intake (EDI), was not associated with any toxicity concern. Therefore, the aim of this study was focused on examining whether the aqueous extracts ofC.paliurus leaves cause genotoxicity and teratogenic toxicity or not. Ames test, mouse bone marrow polychromatic erythrocyte micronucleus test, and mouse sperm abnormality test were conducted to evaluate the genetic safety of aqueous extracts ofC.paliurus leaves. This study may give further information about the edible security ofC.paliurus leaves as a potential source for functional food ingredient with multibioactivities.


## 2. Materials and Methods

### 2.1. Materials and Tested Animals

Leaves ofC. paliurus were provided by HunanCyclocarya paliurus Technology Development Co. Ltd, Suining county, Hunan province, China, with recommended human dose of 0.133 g/kg·bw. The air-dried leaves are light brown and were milled into powder using 0.2 mm sieve. The milled leaves were extracted with distilled water for 30 min at 85°C at a ratio of water to leaves of 10 : 1 (mL : g). The extraction was performed duplicate and all extracts were combined and concentrated to a concentration equivalent to 2 g leaves per millimeter liquid.The SPF grade Kunming mice, SD rats, and the fodder were provided by Dongchuang Animals Experiment Technology Services, Kaifu district, Changsha (experimental animal production license number: SCXK (Xiang) 2009-0012, experimental animal permission license number: SYXK (Xiang) 2010-0010). The test animals were developed in the shielding environment of temperature of 22~24°C and humidity of 52~56%.


### 2.2. Bacterial Reverse Mutation Test (Ames Test)

Ames tests were carried out in histidine auxotrophicSalmonella typhimuriumstrains TA97, TA98, TA100, and TA102. Test groups received five doses of the aqueous extract ofC. paliurusleaves, equivalent to the doses of 5000 μg/plate, 1000 μg/plate, 200 μg/plate, 40 μg/plate, and 8 μg/plate, in the presence or absence of rat liver microsomal enzyme (S-9) as in vitro metabolic activation system. The following controls were conducted simultaneously: spontaneous reverse mutation test for all strains, solvent for all strains, positive for TA97+S9, TA98+S9, and TA100+S9 of 10 μg/plate 2-AF, positive for TA97-S9 and TA98-S9 of 0.2 μg/plate 9-fluorenone, positive for TA100-S9 of 1.5 μg/plate NaN3, positive for TA102+S9 of 50.0 μg/plate 1,8-mitoxanone, and positive for TA102-S9 of 0.3 μg/plate MMC, respectively. It was defined as positive when the number of reverse mutant colonies of the tested substance exceeded over twice as much as that of the spontaneous reverse mutation test or reflected a dose-response relationship. The whole test was repeatedly carried out twice under the same conditions.


### 2.3. Bone Marrow Micronucleus Test in Mice

The test was conducted by method of oral gavage twice interval of 24 h. 50 Kunming mice with weight of 25~30 g were divided randomly into 5 groups of 10 mice each, 5 males and 5 females. The test groups were administered 0.2 mL differently diluted aqueous extracts liquid of per 10 g body ofCyclocarya paliurus leaves, equivalent to the dose of 10.0 g/kg·bw (high dose level), 5.0 g/kg·bw (middle dose level), and 2.5 g/kg·bw (low dose level), respectively. 40 mg/kg·bw of cyclophosphamide was used as positive controls and distilled water as negative controls. Six hours after last oral gavage, the mice were sacrificed by cervical dislocation. The sternum marrow was taken to dilute with calf serum and smear. The slides were fixed with methanol, washed with distilled water, and stained with Giemsa. A total of 5000 polychromatic erythrocytes (PCE) calculated from 1000 PCEs per animal were analyzed to count the total number of micronucleated polychromatic erythrocyte (MNPCE) under microscope. The micronucleus incidence was counted by MNPCEs/PCEs (‰). A total of 1000 polychromatic erythrocytes (PCE) calculated from 200 PCEs per animal were used to determine the index of PCEs/NCEs (norm chromatic erythrocytes).


### 2.4. Sperm Deformity Test in Mice

25 male Kunming mice with weight of 25 g ~30 g were divided randomly into 5 groups, including three dosages test groups, one negative control group and one positive group, 5 mice each. At intervals of 24 h, the test groups received oral gavages of aqueous extracts ofCyclocarya paliurus leaves at three doses, 10.0 g/kg·bw (high dose level), 5.0 g/kg·bw (middle dose level), and 2.5 g/kg·bw (low dose level), respectively. 40 mg/kg·bw of Cytoxan was used as positive controls and distilled water as negative controls. The oral gavage was conducted continuously for 5 days. On the thirtieth day after the last gavage, the mice were sacrificed by cervical dislocation to get the bilateral epididymis. The sperms were spread on a slide glass and stained with eosin. A total of 1000 structurally completed sperms were scored for each animal under light microscope to evaluate the incidence of sperm deformity. A positive result for the sperm deformity in each test group was determined by a dose-dependent increase in the number of abnormal sperms or a reproducible positive reaction and statistical significance at one or more dosages.


### 2.5.Cyclocarya paliurus Teratogenicity Test

120 sexual maturity female SD rats and 60 male SD rats were employed to receive the test. One female and one male were kept in the same cage. Every morning the finding of vaginal plug or sperm in the female vagina identified the copulation of the rats and the day was regarded as the zero-day of fertilization. If the copulation did not occur within 5 days, the male rat would be replaced. The 60 detected pregnant rats were weighed, numbered, and divided randomly into 5 groups, including three dosages test groups, one negative control group and one positive group, 12 rats each group. In the gestation of 7 to 16 days, the three test groups received oral gavage of 1.0 mL differently diluted aqueous extracts ofCyclocarya paliurus leaves per 100 g body weight at interval of 24 h, equivalent to the dose of 20.0 g/kg·bw (high dose level), 10.0 g/kg·bw (middle dose level), and 5.0 g/kg·bw (low dose level), respectively. The oral gavage of 1.0 mL aspirin/100 g·bw was used as positive controls, equivalent to the dose of 0.27 g/kg·bw, and distilled water as negative controls. Each rat was provided enough diet. The oral gavage amount was adjusted based on the body weighing on the 0, 7th, 12th, 16th, and 20th day of gestation. On the 20th day, the pregnant rats were sacrificed and punched to obtain the ovaries and matrixes. The corpora lutea, implantation, absorbed fetus, dead fetus, and live fetus were counted. Ovaria, wombs linking to placenta, and placentas were weighed, respectively. The live fetus sexuality was identified one by one and the body weight, body length, and tail length were determined and checked whether appearance dysmorphia was occurred or not. After that, half of live fetuses (odd or even) of each pregnant rat were fixed in 95% (v/v) ethanol for 3 weeks, washed with running water, and immersed in the 2 g/100 mL potassium hydroxide for 72 hours. When the fetuses became hyaline, they were stained with alizarin red for 48 hours with shaking twice a day till the skulls became red. Then the fetuses were soaked in transparent liquid A for 2 days and transparent liquid B for 3 days in succession till the bones were incarnadined while the soft tissue faded. The overall examination and skeletal survey were conducted by the stereo-microscope. The other half live fetus was fixed by immersing in Bouin’s for 2 weeks for viscous examination.


### 2.6. Data Statistical Analysis

The data were analyzed by Spss11.0. The homogeneity of variance of the data was tested firstly. If it was homogeneous variance, single factor analysis of variance was used for the overall comparison. When diversity occurred, Dunnett method was adopted for pairwise comparisons among the different dosages, positive controls and negative controls. While the variance was heterogeneous, appropriate variable conversion was done for the original data to test the homogeneity of variance. If the variance was homogeneous, the converted data was used in statistics. Otherwise, rank-sum test was used. If there were diversities in the overall comparison, Tamhane’sT
2 test was used for pairwise comparisons, and x
2 test was used for statistics of rate.


## 2.1. Materials and Tested Animals

Leaves ofC. paliurus were provided by HunanCyclocarya paliurus Technology Development Co. Ltd, Suining county, Hunan province, China, with recommended human dose of 0.133 g/kg·bw. The air-dried leaves are light brown and were milled into powder using 0.2 mm sieve. The milled leaves were extracted with distilled water for 30 min at 85°C at a ratio of water to leaves of 10 : 1 (mL : g). The extraction was performed duplicate and all extracts were combined and concentrated to a concentration equivalent to 2 g leaves per millimeter liquid.The SPF grade Kunming mice, SD rats, and the fodder were provided by Dongchuang Animals Experiment Technology Services, Kaifu district, Changsha (experimental animal production license number: SCXK (Xiang) 2009-0012, experimental animal permission license number: SYXK (Xiang) 2010-0010). The test animals were developed in the shielding environment of temperature of 22~24°C and humidity of 52~56%.


## 2.2. Bacterial Reverse Mutation Test (Ames Test)

Ames tests were carried out in histidine auxotrophicSalmonella typhimuriumstrains TA97, TA98, TA100, and TA102. Test groups received five doses of the aqueous extract ofC. paliurusleaves, equivalent to the doses of 5000 μg/plate, 1000 μg/plate, 200 μg/plate, 40 μg/plate, and 8 μg/plate, in the presence or absence of rat liver microsomal enzyme (S-9) as in vitro metabolic activation system. The following controls were conducted simultaneously: spontaneous reverse mutation test for all strains, solvent for all strains, positive for TA97+S9, TA98+S9, and TA100+S9 of 10 μg/plate 2-AF, positive for TA97-S9 and TA98-S9 of 0.2 μg/plate 9-fluorenone, positive for TA100-S9 of 1.5 μg/plate NaN3, positive for TA102+S9 of 50.0 μg/plate 1,8-mitoxanone, and positive for TA102-S9 of 0.3 μg/plate MMC, respectively. It was defined as positive when the number of reverse mutant colonies of the tested substance exceeded over twice as much as that of the spontaneous reverse mutation test or reflected a dose-response relationship. The whole test was repeatedly carried out twice under the same conditions.


## 2.3. Bone Marrow Micronucleus Test in Mice

The test was conducted by method of oral gavage twice interval of 24 h. 50 Kunming mice with weight of 25~30 g were divided randomly into 5 groups of 10 mice each, 5 males and 5 females. The test groups were administered 0.2 mL differently diluted aqueous extracts liquid of per 10 g body ofCyclocarya paliurus leaves, equivalent to the dose of 10.0 g/kg·bw (high dose level), 5.0 g/kg·bw (middle dose level), and 2.5 g/kg·bw (low dose level), respectively. 40 mg/kg·bw of cyclophosphamide was used as positive controls and distilled water as negative controls. Six hours after last oral gavage, the mice were sacrificed by cervical dislocation. The sternum marrow was taken to dilute with calf serum and smear. The slides were fixed with methanol, washed with distilled water, and stained with Giemsa. A total of 5000 polychromatic erythrocytes (PCE) calculated from 1000 PCEs per animal were analyzed to count the total number of micronucleated polychromatic erythrocyte (MNPCE) under microscope. The micronucleus incidence was counted by MNPCEs/PCEs (‰). A total of 1000 polychromatic erythrocytes (PCE) calculated from 200 PCEs per animal were used to determine the index of PCEs/NCEs (norm chromatic erythrocytes).


## 2.4. Sperm Deformity Test in Mice

25 male Kunming mice with weight of 25 g ~30 g were divided randomly into 5 groups, including three dosages test groups, one negative control group and one positive group, 5 mice each. At intervals of 24 h, the test groups received oral gavages of aqueous extracts ofCyclocarya paliurus leaves at three doses, 10.0 g/kg·bw (high dose level), 5.0 g/kg·bw (middle dose level), and 2.5 g/kg·bw (low dose level), respectively. 40 mg/kg·bw of Cytoxan was used as positive controls and distilled water as negative controls. The oral gavage was conducted continuously for 5 days. On the thirtieth day after the last gavage, the mice were sacrificed by cervical dislocation to get the bilateral epididymis. The sperms were spread on a slide glass and stained with eosin. A total of 1000 structurally completed sperms were scored for each animal under light microscope to evaluate the incidence of sperm deformity. A positive result for the sperm deformity in each test group was determined by a dose-dependent increase in the number of abnormal sperms or a reproducible positive reaction and statistical significance at one or more dosages.


## 2.5.Cyclocarya paliurus Teratogenicity Test

120 sexual maturity female SD rats and 60 male SD rats were employed to receive the test. One female and one male were kept in the same cage. Every morning the finding of vaginal plug or sperm in the female vagina identified the copulation of the rats and the day was regarded as the zero-day of fertilization. If the copulation did not occur within 5 days, the male rat would be replaced. The 60 detected pregnant rats were weighed, numbered, and divided randomly into 5 groups, including three dosages test groups, one negative control group and one positive group, 12 rats each group. In the gestation of 7 to 16 days, the three test groups received oral gavage of 1.0 mL differently diluted aqueous extracts ofCyclocarya paliurus leaves per 100 g body weight at interval of 24 h, equivalent to the dose of 20.0 g/kg·bw (high dose level), 10.0 g/kg·bw (middle dose level), and 5.0 g/kg·bw (low dose level), respectively. The oral gavage of 1.0 mL aspirin/100 g·bw was used as positive controls, equivalent to the dose of 0.27 g/kg·bw, and distilled water as negative controls. Each rat was provided enough diet. The oral gavage amount was adjusted based on the body weighing on the 0, 7th, 12th, 16th, and 20th day of gestation. On the 20th day, the pregnant rats were sacrificed and punched to obtain the ovaries and matrixes. The corpora lutea, implantation, absorbed fetus, dead fetus, and live fetus were counted. Ovaria, wombs linking to placenta, and placentas were weighed, respectively. The live fetus sexuality was identified one by one and the body weight, body length, and tail length were determined and checked whether appearance dysmorphia was occurred or not. After that, half of live fetuses (odd or even) of each pregnant rat were fixed in 95% (v/v) ethanol for 3 weeks, washed with running water, and immersed in the 2 g/100 mL potassium hydroxide for 72 hours. When the fetuses became hyaline, they were stained with alizarin red for 48 hours with shaking twice a day till the skulls became red. Then the fetuses were soaked in transparent liquid A for 2 days and transparent liquid B for 3 days in succession till the bones were incarnadined while the soft tissue faded. The overall examination and skeletal survey were conducted by the stereo-microscope. The other half live fetus was fixed by immersing in Bouin’s for 2 weeks for viscous examination.


## 2.6. Data Statistical Analysis

The data were analyzed by Spss11.0. The homogeneity of variance of the data was tested firstly. If it was homogeneous variance, single factor analysis of variance was used for the overall comparison. When diversity occurred, Dunnett method was adopted for pairwise comparisons among the different dosages, positive controls and negative controls. While the variance was heterogeneous, appropriate variable conversion was done for the original data to test the homogeneity of variance. If the variance was homogeneous, the converted data was used in statistics. Otherwise, rank-sum test was used. If there were diversities in the overall comparison, Tamhane’sT
2 test was used for pairwise comparisons, and x
2 test was used for statistics of rate.


## 3. Results and Discussion

### 3.1. Mutagenicity Assay

#### 3.1.1. Ames Test

Ames test had been expensively used to help evaluate the mutagenic and carcinogenic risks for a many number of chemicals. McCann and Ames observed that a total of 5000 chemical compounds were revealed to present a mutagenic and carcinogenic risk, as determined by the Ames test [23]. In this study, the results of Ames test are summarized in Tables 1 and 2.Table 1
The results of Ames test onC. paliurus leaf aqueous extract (for the first time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
−S9
5000
150.0
±
13.5
147.0
±
7.5
42.7
±
7.5
35.3
±
4.0
179.3
±
17.1
162.3
±
18.8
285.0
±
15.1
261.3
±
9.9
1000
149.7
±
26.1
151.0
±
15.1
34.3
±
4.6
39.3
±
7.5
169.3
±
15.6
174.7
±
17.5
271.3
±
10.7
275.3
±
15.5
200
142.3
±
9.5
147.3
±
25.1
32.5
±
5.5
43.0
±
6.9
171.7
±
11.8
175.7
±
7.0
268.3
±
18.2
278.3
±
12.7
40
141.0
±
15.5
142.7
±
16.0
37.3
±
8.0
39.0
±
4.6
162.7
±
10.5
169.0
±
17.7
277.3
±
14.2
265.7
±
13.3
8
156.3
±
12.7
145.3
±
18.6
40.7
±
4.2
37.0
±
4.4
172.3
±
14.3
164.3
±
12.3
279.0
±
13.5
267.0
±
12.3
Spontaneous reverse
145.7
±
16.6
148.7
±
14.4
41.7
±
7.6
40.0
±
5.6
173.7
±
13.7
170.7
±
12.2
263.7
±
11.0
269.3
±
23.2
Solvent controls
154.3
±
14.8
145.7
±
12.9
34.7
±
5.0
37.0
±
8.5
172.3
±
15.0
179.7
±
10.8
273.7
±
22.5
267.3
±
11.2
Positive controls
1316.3
±
130.6
1232.0
±
134.2
2664.0
±
168.7
2133.3
±
107.7
2396.7
±
137.6
2426.3
±
123.6
1023.3
±
127.4
2235.3
±
162.0
All the above data were average value of three plates.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).Table 2
The results of Ames test onC. paliurus leaf aqueous extract (for the second time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
− S9
5000
139.3
±
7.8
144.7
±
10.1
39.7
±
7.5
36.7
±
8.1
164.7
±
15.3
175.7
±
18.5
266.3
±
26.0
268.0
±
22.6
1000
154.0
±
14.7
149.0
±
23.6
36.0
±
4.0
34.0
±
4.6
174.0
±
19.1
168.7
±
19.6
272.7
±
21.1
276.0
±
11.3
200
142.0
±
13.9
151.0
±
18.0
38.3
±
4.9
41.7
±
4.0
167.7
±
12.5
164.3
±
21.4
276.7
±
14.2
266.7
±
23.2
40
153.3
±
14.2
148.3
±
18.7
42.0
±
7.8
38.7
±
8.6
177.3
±
23.7
167.0
±
14.0
273.0
±
15.1
270.0
±
20.8
8
141.7
±
19.2
143.7
±
9.3
35.0
±
5.6
42.3
±
4.2
166.3
±
18.9
171.0
±
14.7
266.3
±
14.2
270.7
±
8.1
Spontaneous reverse
138.0
±
15.5
148.0
±
9.6
39.3
±
9.3
38.0
±
7.2
165.3
±
19.5
178.3
±
12.5
265.0
±
12.5
267.0
±
23.0
Solvent controls
152.0
±
26.0
141.7
±
12.5
43.7
±
5.9
40.7
±
4.5
173.0
±
12.1
166.0
±
26.1
268.3
±
8.0
277.7
±
18.1
Positive controls
1206.7
±
130.5
1186.3
±
162.2
2548.0
±
216.7
2283.0
±
157.4
2452.3
±
171.5
2522.7
±
160.1
984.0
±
73.4
2122.7
±
122.9
All the above data were average value of three plates± standard deviation.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).As can be seen, the numbers of colonies of each group at every dose of aqueous extract ofCyclocarya paliurus leaves in either presence or absence of S9 did not exceed twice of those of spontaneous reverse mutation group. Reversion mutation colonies did not grow with increasing dosages of aqueous extract compared to the solvent plates, indicating that no dose-response relationship was reflected. On the other hand, on all of positive plates, reversion mutation colonies grew greater than twice as frequently as on the negative plates. The results of the repeated test reflected the same pattern.


#### 3.1.2. Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test

Mouse bone marrow micronucleus assay is internationally recognized as the standard method of detecting mutagenicity of chemicals, which is originated by Schmid in the early 1970’s [24]. Generally micronucleus is derived from chromosome losing centromere or chromosome fragment after the chromosome was damaged by certain physical and chemical factors. Micronuclei incidence may reflect the extent of how chromosomes are damaged. The results of mouse bone marrow polychromatic erythrocyte micronucleus test of aqueous extract fromCyclocarya paliurus leaves are shown in Table 3.Table 3
Effect ofC. paliurus leaf aqueous extract on the rate of mouse bone marrow micronucleus. Data are expressed as mean ± SD.
Sex
Dose (g/kg·bw)
Number of animals
MNPCEs/PCEs (‰)
NCEs
PCEs/NCEs
Male
10.0
5
1.0
±
0.7
850
1.181
±
0.079
5.0
5
1.6
±
0.9
871
1.152
±
0.077
2.5
5
0.6
±
0.5
883
1.137
±
0.083
Negative
5
0.8
±
0.8
848
1.185
±
0.089
Positive
5
25.0
±
3.7
1004
0.999
±
0.055
Female
10.0
5
1.0
±
1.2
836
1.201
±
0.086
5.0
5
1.0
±
1.0
842
1.193
±
0.085
2.5
5
1.4
±
0.5
874
1.152
±
0.108
Negative
5
1.2
±
0.8
850
1.180
±
0.074
Positive
5
26.2
±
2.6
1010
0.992
±
0.046
PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; MNPCE: micronucleated polychromatic erythrocyte.
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.Whether male or female, the incidence of micronucleus of tested group for each dosage level had no significant statistical difference compared to the negative control, so as the index of PCE/NCE (P
>
0.05). There was no significant correlation between the micronucleus incidence and the dosage. Furthermore, each dose of the aqueous extract did not cause differences between sexes. However, there was significant difference between cyclophosphamide group and negative control group (P
<
0.01). So it could be considered that the aqueous extracts ofC. paliurus leaves had no marked effect on the incidence of mouse bone marrow micronucleus.


#### 3.1.3. Sperm Deformity Test in Mice

Sperm deformity test is one way to detect reproductive toxicity, including determination of the number of sperms, sperm motility, and sperm morphology. According to reports the results of three trials are consistent. Mammalian sperm abnormality test was to evaluate the reproductive toxicity of chemicals in a reliable and easy way. Singh et al. divided the sperm deformity into five kinds: acrosome defect, wedges, banana, bubble neck, and amorphous [25]. Xu et al. reported that sperm deformity through toxicity test could be divided into seven kinds: no hooks, bananas, fat head, double heads, twin tails, tail folded, and amorphous [26]. According to the experimental results, this paper reports the type of deformity with reference to Xu et al. [26].As can be seen in Table4, the kinds of abnormal sperm in any group were mainly no hooks, bananas, fat heads, and amorphous. The incidence of mouse sperm deformity of tested group at each dosage level had no significant difference from that of the negative control (P
>
0.05). Significant differences could be observed between the positive controls and negative controls (P
<
0.01). The summary of no hooks and amorphism in the three dosage groups and negative control group accounted for about 73% of the total, while in positive control group it decreased to about 60%.Table 4
Effect ofCyclocarya paliurus leaf aqueous extract on the rate of mouse sperm abnormality. Data are expressed as mean ± SD.
Dose/(g/kg·bw)
Mice number
Tested sperms
Abnormal sperms
Abnormality incidence
Constituent of different abnormal sperms/%
Lack hook
Banana-like
Fat-head
Morphism
Folding-tail
Double-headed
Twin-tailed
10.0
5
5000
114
2.28
±
0.41
30.7
14.9
15.8
38.6
0.0
0.0
0.0
5.0
5
5000
106
2.12
±
0.52
33.0
7.5
17.9
41.5
0.0
0.0
0.0
2.5
5
5000
111
2.22
±
0.31
32.4
10.8
13.5
43.2
0.0
0.0
0.0
Negativecontrols
5
5000
109
2.18
±
0.53
33.0
7.3
19.3
40.4
0.0
0.0
0.0
Positivecontrols
5
5000
462
9.24
±
0.90
28.6
19.5
21.0
30.3
0.0
0.0
0.6
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.


### 3.2. Teratogenicity Assays

During the gestation, no signs of miscarriage, no deaths, and no gross anatomical abnormalities were observed to any pregnant rat in all groups. Summary of the pregnant rat weight data was presented in Table5. As can be seen, the weight of pregnant rats in negative controls increased in a normal manner, so did the pregnant rats in the three dosage groups. On the 20th day, the weight gains of the three dosage groups had no significantly statistical diversity compared to the negative controls (P
>
0.05). However, the weight growth of the pregnant rats in positive control group was slower, especially in the mid- and late-pregnancy (from 12th to 20th day), which resulted in that the weight gain was significantly lower than the negative control group on the 20th day (P
<
0.05 or P
<
0.01).Table 5
Effect ofC. paliurus leaf aqueous extract on pregnant rat weight. Data are expressed as mean ± SD.
Pregnant rat weight/(g)
Weight gain (g)
Groups/(g/kg·bw)
Number of pregnant rats
0 day
7th day
12th day
16th day
20th day
x
¯  ±  s (g)
P
x
¯  ± s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s
P
Negative controls
12
260.8
±
17.1
1.000
287.9
±
18.7
0.820
312.5
±
21.1
0.768
355.3
.
5
±
20.5
0.120
390.2
±
20.7
/
133.4
±
16.7
/
5.0
12
260.6
±
17.1
283.0
±
17.7
304.5
±
18.7
347.7
±
19.2
386.7
±
18.9
0.935
126.0
±
20.1
1.000
10.0
12
261.7
±
17.6
286.2
±
16.0
309.8
±
18.6
358.1
±
19.7
392.1
±
20.2
0.999
130.3
±
16.4
1.000
20.0
12
261.3
±
17.3
288.8
±
20.2
306.8
±
18.3
354.2
±
20.0
388.4
±
24.4
0.995
127.1
±
15.2
1.000
Positive controls
12
261.1
±
18
280.7
±
21.6
302.4
±
25.8
336.5
±
2
7
.
4
359.7
±
27.9
0.006
98.6
±
26.3
0.029The effects ofC. paliurus leaves aqueous extract on indexes of embryo toxicity were presented in Table 6. The numbers of live embryos, the weight of uterus linking to placenta, and the weight of placenta of positive controls were significantly lower than those of negative controls (P
<
0.05 or P
<
0.01). The incidences of dead and adsorptive fetus of positive controls were significantly higher than those of negative controls (P
<
0.01). There were no distinguishable differences among the results of three dosages (5.0, 10.0, and 20.0 g/kg·bw) groups and negative controls (P
>
0.05) explaining the aqueous extract ofC. paliurus leaves had not produced obvious toxicity to the embryos of rats.Table 6
Effect ofC. paliurus leaf aqueous extract on the indexes of embryo toxicity in pregnant rat. Data are expressed as mean ± SD.
Groups/(g/kg·bw)
Numbers
Dead and adsorptive Incidence/(%)
Weight/(g)
Pregnant rat number
Corpora lutea
Nidation
Live embryo
Ovary
Uterus linking to Placenta
Placenta
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
Negative controls
12
13.3
±
2.3
0.905
11.8
±
2.1
0.690
11.6
±
2.1
/
1.42
/
0.186 ± 0.042
0.805
66.42
±
6.86
/
6.70
±
1.25
/
5.0
12
13.5
±
2.6
11.3
±
2.4
11.2
±
2.5
0.977
0.74
1.000
0.183 ± 0.039
63.34
±
9.73
0.789
6.95
±
0.92
0.970
10.0
12
13.6
±
1.8
11.7
±
2.3
11.4
±
2.3
0.999
2.14
0.684
0.194 ± 0.043
65.64
±
8.32
0.998
6.78
±
1.34
0.999
20.0
12
13.2
±
2.2
11.3
±
2.6
11.2
±
2.7
0.977
1.47
1.000
0.198 ± 0.046
64.80
±
10.25
0.973
6.56
±
1.66
0.997
Positive controls
12
12.8
±
2.1
10.5
±
2.0
7.8
±
1.9
0.001
25.4
0.000
0.179 ± 0.040
44.43
±
7.01
0.000
5.05
±
1.26
0.012The sexuality, fetal body weight, body length, and tail length of the live fetuses were presented in Table7. There were no distinguishable diversities of sex ratio of the fetuses among all of groups, illustrating that both aspirin and aqueous extract ofC. paliurushave no effect on sexuality of embryos. Compared with the negative controls, the fetus body weights of positive controls were obviously lower (P
<
0.01), and both body length and tail length were obviously shorter (P
<
0.01). The fetal body weight, body length, and tail length had no significant differences among the three dosage groups and the negative control group (P
>
0.05).Table 7
Effect ofC. paliurus leaf aqueous extract on sexuality, fetal body weight, body length, and tail length of fetal rat. Data are expressed as mean ± SD.
Groups/(g/kg·bw)
Live fetus
Male/female
Fetal body weight/(g)
Fetal body length/(mm)
Fetal tail length/(mm)
Ratio
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
Negative controls
139
67/72 (0.93)
/
3.79
±
0.40
/
37.38
±
1.31
/
13.80
±
0.65
/
5.0
134
64/70 (0.91)
0.942
3.74
±
0.39
0.727
37.15
±
1.28
0.456
13.66
±
0.62
0.511
10.0
137
63/74 (0.85)
0.712
3.72
±
0.45
0.473
37.14
±
1.34
0.409
13.72
±
0.87
0.995
20.0
134
69/65 (1.06)
0.587
3.81
±
0.42
0.983
37.35
±
1.37
0.999
13.63
±
0.70
0.314
Positive controls
94
45/49 (0.92)
0.961
3.18
±
0.46
0.000
35.32
±
1.70
0.000
13.00
±
0.66
0.000Stereoscope examination showed that no visible external abnormality had occurred to fetal rats of three dosage groups and negative control group, while encephalocele, exomphalocele, and anophthalmos had occurred to part of fetal rats in positive control group. Viscus examination showed that no obvious developmental deformity occurred to the fetal head, thorax, and abdominal viscera in three dosage groups and negative control group, while in positive controls, there was one fetal rat with anophthalmos, one with horseshoe kidney, one with absence of testis, and one with hydronephrosis.The information about skeletal development of fetal rats was summarized in Table8. The incidences of spine hypoplasia, sternum hypoplasia, pelvis hypoplasia, metacarpal hypoplasia, and phalanx hypoplasia of fetal rats in positive control group were significantly higher than those in negative control group (P
<
0.05 or P
<
0.01). Each kind of listed skeletal dysplasia proportion was from 18% to 40%, meaning multiple skeletal dysplasia appeared in the positive controls. The skeletal hypoplasia incidence showed no significant difference among the three dosage groups and the negative control group (P
>
0.05), and the main kinds of skeletal hypoplasia were sternum, metacarpal, and phalanx, congruously. No obvious abnormalities of libs, carpal, long bones, and occipital bones were observed in each dosage group.Table 8
Effect ofC. paliurus leaf aqueous extract on skeletal development of fetal rats.
Groups/(g/kg·bw)
Fetal rats number
Hypoplasia incidence/(%)
Spine/P
Sternum/P
Pelvis/P
Metacarpal/P
Phalanx/P
Negative controls
71
0.0/—
11.3/—
1.4/—
11.3/—
18.3/—
5.0
71
1.4/1.000
9.8/0.785
1.4/1.000
12.7/0.796
14.1/0.494
10.0
72
0.0/—
13.9/0.637
2.8/1.000
9.7/0.763
12.5/0.336
20.0
69
0.0/—
13.0/0.748
1.4/1.000
17.4/0.301
15.9/0.710
Positive controls
50
18.0/0.001
40.0/0.000
24.0/0.000
32.0/0.005
38.0/0.016All results summarized above gave detailed information about the developments of rat embryos in each group. The data about fetal body weight, body length, tail length, and skeletal development of fetal rats provided evidences about the weight gains of mother rats shown in Table5. Compared with the negative controls, all the data from the three dosages had no statistically significant differences. It can be considered that the aqueous extract ofC. paliurus leaves has not any teratogenic effect on the embryos of rats, even at the highest dose of 20 g/kg·bw.


## 3.1. Mutagenicity Assay

### 3.1.1. Ames Test

Ames test had been expensively used to help evaluate the mutagenic and carcinogenic risks for a many number of chemicals. McCann and Ames observed that a total of 5000 chemical compounds were revealed to present a mutagenic and carcinogenic risk, as determined by the Ames test [23]. In this study, the results of Ames test are summarized in Tables 1 and 2.Table 1
The results of Ames test onC. paliurus leaf aqueous extract (for the first time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
−S9
5000
150.0
±
13.5
147.0
±
7.5
42.7
±
7.5
35.3
±
4.0
179.3
±
17.1
162.3
±
18.8
285.0
±
15.1
261.3
±
9.9
1000
149.7
±
26.1
151.0
±
15.1
34.3
±
4.6
39.3
±
7.5
169.3
±
15.6
174.7
±
17.5
271.3
±
10.7
275.3
±
15.5
200
142.3
±
9.5
147.3
±
25.1
32.5
±
5.5
43.0
±
6.9
171.7
±
11.8
175.7
±
7.0
268.3
±
18.2
278.3
±
12.7
40
141.0
±
15.5
142.7
±
16.0
37.3
±
8.0
39.0
±
4.6
162.7
±
10.5
169.0
±
17.7
277.3
±
14.2
265.7
±
13.3
8
156.3
±
12.7
145.3
±
18.6
40.7
±
4.2
37.0
±
4.4
172.3
±
14.3
164.3
±
12.3
279.0
±
13.5
267.0
±
12.3
Spontaneous reverse
145.7
±
16.6
148.7
±
14.4
41.7
±
7.6
40.0
±
5.6
173.7
±
13.7
170.7
±
12.2
263.7
±
11.0
269.3
±
23.2
Solvent controls
154.3
±
14.8
145.7
±
12.9
34.7
±
5.0
37.0
±
8.5
172.3
±
15.0
179.7
±
10.8
273.7
±
22.5
267.3
±
11.2
Positive controls
1316.3
±
130.6
1232.0
±
134.2
2664.0
±
168.7
2133.3
±
107.7
2396.7
±
137.6
2426.3
±
123.6
1023.3
±
127.4
2235.3
±
162.0
All the above data were average value of three plates.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).Table 2
The results of Ames test onC. paliurus leaf aqueous extract (for the second time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
− S9
5000
139.3
±
7.8
144.7
±
10.1
39.7
±
7.5
36.7
±
8.1
164.7
±
15.3
175.7
±
18.5
266.3
±
26.0
268.0
±
22.6
1000
154.0
±
14.7
149.0
±
23.6
36.0
±
4.0
34.0
±
4.6
174.0
±
19.1
168.7
±
19.6
272.7
±
21.1
276.0
±
11.3
200
142.0
±
13.9
151.0
±
18.0
38.3
±
4.9
41.7
±
4.0
167.7
±
12.5
164.3
±
21.4
276.7
±
14.2
266.7
±
23.2
40
153.3
±
14.2
148.3
±
18.7
42.0
±
7.8
38.7
±
8.6
177.3
±
23.7
167.0
±
14.0
273.0
±
15.1
270.0
±
20.8
8
141.7
±
19.2
143.7
±
9.3
35.0
±
5.6
42.3
±
4.2
166.3
±
18.9
171.0
±
14.7
266.3
±
14.2
270.7
±
8.1
Spontaneous reverse
138.0
±
15.5
148.0
±
9.6
39.3
±
9.3
38.0
±
7.2
165.3
±
19.5
178.3
±
12.5
265.0
±
12.5
267.0
±
23.0
Solvent controls
152.0
±
26.0
141.7
±
12.5
43.7
±
5.9
40.7
±
4.5
173.0
±
12.1
166.0
±
26.1
268.3
±
8.0
277.7
±
18.1
Positive controls
1206.7
±
130.5
1186.3
±
162.2
2548.0
±
216.7
2283.0
±
157.4
2452.3
±
171.5
2522.7
±
160.1
984.0
±
73.4
2122.7
±
122.9
All the above data were average value of three plates± standard deviation.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).As can be seen, the numbers of colonies of each group at every dose of aqueous extract ofCyclocarya paliurus leaves in either presence or absence of S9 did not exceed twice of those of spontaneous reverse mutation group. Reversion mutation colonies did not grow with increasing dosages of aqueous extract compared to the solvent plates, indicating that no dose-response relationship was reflected. On the other hand, on all of positive plates, reversion mutation colonies grew greater than twice as frequently as on the negative plates. The results of the repeated test reflected the same pattern.


### 3.1.2. Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test

Mouse bone marrow micronucleus assay is internationally recognized as the standard method of detecting mutagenicity of chemicals, which is originated by Schmid in the early 1970’s [24]. Generally micronucleus is derived from chromosome losing centromere or chromosome fragment after the chromosome was damaged by certain physical and chemical factors. Micronuclei incidence may reflect the extent of how chromosomes are damaged. The results of mouse bone marrow polychromatic erythrocyte micronucleus test of aqueous extract fromCyclocarya paliurus leaves are shown in Table 3.Table 3
Effect ofC. paliurus leaf aqueous extract on the rate of mouse bone marrow micronucleus. Data are expressed as mean ± SD.
Sex
Dose (g/kg·bw)
Number of animals
MNPCEs/PCEs (‰)
NCEs
PCEs/NCEs
Male
10.0
5
1.0
±
0.7
850
1.181
±
0.079
5.0
5
1.6
±
0.9
871
1.152
±
0.077
2.5
5
0.6
±
0.5
883
1.137
±
0.083
Negative
5
0.8
±
0.8
848
1.185
±
0.089
Positive
5
25.0
±
3.7
1004
0.999
±
0.055
Female
10.0
5
1.0
±
1.2
836
1.201
±
0.086
5.0
5
1.0
±
1.0
842
1.193
±
0.085
2.5
5
1.4
±
0.5
874
1.152
±
0.108
Negative
5
1.2
±
0.8
850
1.180
±
0.074
Positive
5
26.2
±
2.6
1010
0.992
±
0.046
PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; MNPCE: micronucleated polychromatic erythrocyte.
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.Whether male or female, the incidence of micronucleus of tested group for each dosage level had no significant statistical difference compared to the negative control, so as the index of PCE/NCE (P
>
0.05). There was no significant correlation between the micronucleus incidence and the dosage. Furthermore, each dose of the aqueous extract did not cause differences between sexes. However, there was significant difference between cyclophosphamide group and negative control group (P
<
0.01). So it could be considered that the aqueous extracts ofC. paliurus leaves had no marked effect on the incidence of mouse bone marrow micronucleus.


### 3.1.3. Sperm Deformity Test in Mice

Sperm deformity test is one way to detect reproductive toxicity, including determination of the number of sperms, sperm motility, and sperm morphology. According to reports the results of three trials are consistent. Mammalian sperm abnormality test was to evaluate the reproductive toxicity of chemicals in a reliable and easy way. Singh et al. divided the sperm deformity into five kinds: acrosome defect, wedges, banana, bubble neck, and amorphous [25]. Xu et al. reported that sperm deformity through toxicity test could be divided into seven kinds: no hooks, bananas, fat head, double heads, twin tails, tail folded, and amorphous [26]. According to the experimental results, this paper reports the type of deformity with reference to Xu et al. [26].As can be seen in Table4, the kinds of abnormal sperm in any group were mainly no hooks, bananas, fat heads, and amorphous. The incidence of mouse sperm deformity of tested group at each dosage level had no significant difference from that of the negative control (P
>
0.05). Significant differences could be observed between the positive controls and negative controls (P
<
0.01). The summary of no hooks and amorphism in the three dosage groups and negative control group accounted for about 73% of the total, while in positive control group it decreased to about 60%.Table 4
Effect ofCyclocarya paliurus leaf aqueous extract on the rate of mouse sperm abnormality. Data are expressed as mean ± SD.
Dose/(g/kg·bw)
Mice number
Tested sperms
Abnormal sperms
Abnormality incidence
Constituent of different abnormal sperms/%
Lack hook
Banana-like
Fat-head
Morphism
Folding-tail
Double-headed
Twin-tailed
10.0
5
5000
114
2.28
±
0.41
30.7
14.9
15.8
38.6
0.0
0.0
0.0
5.0
5
5000
106
2.12
±
0.52
33.0
7.5
17.9
41.5
0.0
0.0
0.0
2.5
5
5000
111
2.22
±
0.31
32.4
10.8
13.5
43.2
0.0
0.0
0.0
Negativecontrols
5
5000
109
2.18
±
0.53
33.0
7.3
19.3
40.4
0.0
0.0
0.0
Positivecontrols
5
5000
462
9.24
±
0.90
28.6
19.5
21.0
30.3
0.0
0.0
0.6
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.


## 3.1.1. Ames Test

Ames test had been expensively used to help evaluate the mutagenic and carcinogenic risks for a many number of chemicals. McCann and Ames observed that a total of 5000 chemical compounds were revealed to present a mutagenic and carcinogenic risk, as determined by the Ames test [23]. In this study, the results of Ames test are summarized in Tables 1 and 2.Table 1
The results of Ames test onC. paliurus leaf aqueous extract (for the first time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
−S9
5000
150.0
±
13.5
147.0
±
7.5
42.7
±
7.5
35.3
±
4.0
179.3
±
17.1
162.3
±
18.8
285.0
±
15.1
261.3
±
9.9
1000
149.7
±
26.1
151.0
±
15.1
34.3
±
4.6
39.3
±
7.5
169.3
±
15.6
174.7
±
17.5
271.3
±
10.7
275.3
±
15.5
200
142.3
±
9.5
147.3
±
25.1
32.5
±
5.5
43.0
±
6.9
171.7
±
11.8
175.7
±
7.0
268.3
±
18.2
278.3
±
12.7
40
141.0
±
15.5
142.7
±
16.0
37.3
±
8.0
39.0
±
4.6
162.7
±
10.5
169.0
±
17.7
277.3
±
14.2
265.7
±
13.3
8
156.3
±
12.7
145.3
±
18.6
40.7
±
4.2
37.0
±
4.4
172.3
±
14.3
164.3
±
12.3
279.0
±
13.5
267.0
±
12.3
Spontaneous reverse
145.7
±
16.6
148.7
±
14.4
41.7
±
7.6
40.0
±
5.6
173.7
±
13.7
170.7
±
12.2
263.7
±
11.0
269.3
±
23.2
Solvent controls
154.3
±
14.8
145.7
±
12.9
34.7
±
5.0
37.0
±
8.5
172.3
±
15.0
179.7
±
10.8
273.7
±
22.5
267.3
±
11.2
Positive controls
1316.3
±
130.6
1232.0
±
134.2
2664.0
±
168.7
2133.3
±
107.7
2396.7
±
137.6
2426.3
±
123.6
1023.3
±
127.4
2235.3
±
162.0
All the above data were average value of three plates.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).Table 2
The results of Ames test onC. paliurus leaf aqueous extract (for the second time). Data are expressed as mean ± SD.
Dose (μg/plate)
TA97
TA98
TA100
TA102
+S9
−S9
+S9
−S9
+S9
−S9
+S9
− S9
5000
139.3
±
7.8
144.7
±
10.1
39.7
±
7.5
36.7
±
8.1
164.7
±
15.3
175.7
±
18.5
266.3
±
26.0
268.0
±
22.6
1000
154.0
±
14.7
149.0
±
23.6
36.0
±
4.0
34.0
±
4.6
174.0
±
19.1
168.7
±
19.6
272.7
±
21.1
276.0
±
11.3
200
142.0
±
13.9
151.0
±
18.0
38.3
±
4.9
41.7
±
4.0
167.7
±
12.5
164.3
±
21.4
276.7
±
14.2
266.7
±
23.2
40
153.3
±
14.2
148.3
±
18.7
42.0
±
7.8
38.7
±
8.6
177.3
±
23.7
167.0
±
14.0
273.0
±
15.1
270.0
±
20.8
8
141.7
±
19.2
143.7
±
9.3
35.0
±
5.6
42.3
±
4.2
166.3
±
18.9
171.0
±
14.7
266.3
±
14.2
270.7
±
8.1
Spontaneous reverse
138.0
±
15.5
148.0
±
9.6
39.3
±
9.3
38.0
±
7.2
165.3
±
19.5
178.3
±
12.5
265.0
±
12.5
267.0
±
23.0
Solvent controls
152.0
±
26.0
141.7
±
12.5
43.7
±
5.9
40.7
±
4.5
173.0
±
12.1
166.0
±
26.1
268.3
±
8.0
277.7
±
18.1
Positive controls
1206.7
±
130.5
1186.3
±
162.2
2548.0
±
216.7
2283.0
±
157.4
2452.3
±
171.5
2522.7
±
160.1
984.0
±
73.4
2122.7
±
122.9
All the above data were average value of three plates± standard deviation.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0μg/plate); TA97 − S9 and TA98 − S9 adopted 9-fluorenone (dose: 0.2 μg/plate); TA100 − S9 adopted NaN3 (dose: 1.5 μg/plate); TA102 + S9 adopted 1,8-mitoxanone (dose: 50.0 μg/plate); TA102 − S9 adopted MMC (dose: 0.5 μg/plate).As can be seen, the numbers of colonies of each group at every dose of aqueous extract ofCyclocarya paliurus leaves in either presence or absence of S9 did not exceed twice of those of spontaneous reverse mutation group. Reversion mutation colonies did not grow with increasing dosages of aqueous extract compared to the solvent plates, indicating that no dose-response relationship was reflected. On the other hand, on all of positive plates, reversion mutation colonies grew greater than twice as frequently as on the negative plates. The results of the repeated test reflected the same pattern.


## 3.1.2. Mouse Bone Marrow Polychromatic Erythrocyte Micronucleus Test

Mouse bone marrow micronucleus assay is internationally recognized as the standard method of detecting mutagenicity of chemicals, which is originated by Schmid in the early 1970’s [24]. Generally micronucleus is derived from chromosome losing centromere or chromosome fragment after the chromosome was damaged by certain physical and chemical factors. Micronuclei incidence may reflect the extent of how chromosomes are damaged. The results of mouse bone marrow polychromatic erythrocyte micronucleus test of aqueous extract fromCyclocarya paliurus leaves are shown in Table 3.Table 3
Effect ofC. paliurus leaf aqueous extract on the rate of mouse bone marrow micronucleus. Data are expressed as mean ± SD.
Sex
Dose (g/kg·bw)
Number of animals
MNPCEs/PCEs (‰)
NCEs
PCEs/NCEs
Male
10.0
5
1.0
±
0.7
850
1.181
±
0.079
5.0
5
1.6
±
0.9
871
1.152
±
0.077
2.5
5
0.6
±
0.5
883
1.137
±
0.083
Negative
5
0.8
±
0.8
848
1.185
±
0.089
Positive
5
25.0
±
3.7
1004
0.999
±
0.055
Female
10.0
5
1.0
±
1.2
836
1.201
±
0.086
5.0
5
1.0
±
1.0
842
1.193
±
0.085
2.5
5
1.4
±
0.5
874
1.152
±
0.108
Negative
5
1.2
±
0.8
850
1.180
±
0.074
Positive
5
26.2
±
2.6
1010
0.992
±
0.046
PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; MNPCE: micronucleated polychromatic erythrocyte.
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.Whether male or female, the incidence of micronucleus of tested group for each dosage level had no significant statistical difference compared to the negative control, so as the index of PCE/NCE (P
>
0.05). There was no significant correlation between the micronucleus incidence and the dosage. Furthermore, each dose of the aqueous extract did not cause differences between sexes. However, there was significant difference between cyclophosphamide group and negative control group (P
<
0.01). So it could be considered that the aqueous extracts ofC. paliurus leaves had no marked effect on the incidence of mouse bone marrow micronucleus.


## 3.1.3. Sperm Deformity Test in Mice

Sperm deformity test is one way to detect reproductive toxicity, including determination of the number of sperms, sperm motility, and sperm morphology. According to reports the results of three trials are consistent. Mammalian sperm abnormality test was to evaluate the reproductive toxicity of chemicals in a reliable and easy way. Singh et al. divided the sperm deformity into five kinds: acrosome defect, wedges, banana, bubble neck, and amorphous [25]. Xu et al. reported that sperm deformity through toxicity test could be divided into seven kinds: no hooks, bananas, fat head, double heads, twin tails, tail folded, and amorphous [26]. According to the experimental results, this paper reports the type of deformity with reference to Xu et al. [26].As can be seen in Table4, the kinds of abnormal sperm in any group were mainly no hooks, bananas, fat heads, and amorphous. The incidence of mouse sperm deformity of tested group at each dosage level had no significant difference from that of the negative control (P
>
0.05). Significant differences could be observed between the positive controls and negative controls (P
<
0.01). The summary of no hooks and amorphism in the three dosage groups and negative control group accounted for about 73% of the total, while in positive control group it decreased to about 60%.Table 4
Effect ofCyclocarya paliurus leaf aqueous extract on the rate of mouse sperm abnormality. Data are expressed as mean ± SD.
Dose/(g/kg·bw)
Mice number
Tested sperms
Abnormal sperms
Abnormality incidence
Constituent of different abnormal sperms/%
Lack hook
Banana-like
Fat-head
Morphism
Folding-tail
Double-headed
Twin-tailed
10.0
5
5000
114
2.28
±
0.41
30.7
14.9
15.8
38.6
0.0
0.0
0.0
5.0
5
5000
106
2.12
±
0.52
33.0
7.5
17.9
41.5
0.0
0.0
0.0
2.5
5
5000
111
2.22
±
0.31
32.4
10.8
13.5
43.2
0.0
0.0
0.0
Negativecontrols
5
5000
109
2.18
±
0.53
33.0
7.3
19.3
40.4
0.0
0.0
0.0
Positivecontrols
5
5000
462
9.24
±
0.90
28.6
19.5
21.0
30.3
0.0
0.0
0.6
Compared to negative controls;P
>
0.05. Compared to positive controls; P
<
0.01.


## 3.2. Teratogenicity Assays

During the gestation, no signs of miscarriage, no deaths, and no gross anatomical abnormalities were observed to any pregnant rat in all groups. Summary of the pregnant rat weight data was presented in Table5. As can be seen, the weight of pregnant rats in negative controls increased in a normal manner, so did the pregnant rats in the three dosage groups. On the 20th day, the weight gains of the three dosage groups had no significantly statistical diversity compared to the negative controls (P
>
0.05). However, the weight growth of the pregnant rats in positive control group was slower, especially in the mid- and late-pregnancy (from 12th to 20th day), which resulted in that the weight gain was significantly lower than the negative control group on the 20th day (P
<
0.05 or P
<
0.01).Table 5
Effect ofC. paliurus leaf aqueous extract on pregnant rat weight. Data are expressed as mean ± SD.
Pregnant rat weight/(g)
Weight gain (g)
Groups/(g/kg·bw)
Number of pregnant rats
0 day
7th day
12th day
16th day
20th day
x
¯  ±  s (g)
P
x
¯  ± s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s (g)
P
x
¯  ±  s
P
Negative controls
12
260.8
±
17.1
1.000
287.9
±
18.7
0.820
312.5
±
21.1
0.768
355.3
.
5
±
20.5
0.120
390.2
±
20.7
/
133.4
±
16.7
/
5.0
12
260.6
±
17.1
283.0
±
17.7
304.5
±
18.7
347.7
±
19.2
386.7
±
18.9
0.935
126.0
±
20.1
1.000
10.0
12
261.7
±
17.6
286.2
±
16.0
309.8
±
18.6
358.1
±
19.7
392.1
±
20.2
0.999
130.3
±
16.4
1.000
20.0
12
261.3
±
17.3
288.8
±
20.2
306.8
±
18.3
354.2
±
20.0
388.4
±
24.4
0.995
127.1
±
15.2
1.000
Positive controls
12
261.1
±
18
280.7
±
21.6
302.4
±
25.8
336.5
±
2
7
.
4
359.7
±
27.9
0.006
98.6
±
26.3
0.029The effects ofC. paliurus leaves aqueous extract on indexes of embryo toxicity were presented in Table 6. The numbers of live embryos, the weight of uterus linking to placenta, and the weight of placenta of positive controls were significantly lower than those of negative controls (P
<
0.05 or P
<
0.01). The incidences of dead and adsorptive fetus of positive controls were significantly higher than those of negative controls (P
<
0.01). There were no distinguishable differences among the results of three dosages (5.0, 10.0, and 20.0 g/kg·bw) groups and negative controls (P
>
0.05) explaining the aqueous extract ofC. paliurus leaves had not produced obvious toxicity to the embryos of rats.Table 6
Effect ofC. paliurus leaf aqueous extract on the indexes of embryo toxicity in pregnant rat. Data are expressed as mean ± SD.
Groups/(g/kg·bw)
Numbers
Dead and adsorptive Incidence/(%)
Weight/(g)
Pregnant rat number
Corpora lutea
Nidation
Live embryo
Ovary
Uterus linking to Placenta
Placenta
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
Negative controls
12
13.3
±
2.3
0.905
11.8
±
2.1
0.690
11.6
±
2.1
/
1.42
/
0.186 ± 0.042
0.805
66.42
±
6.86
/
6.70
±
1.25
/
5.0
12
13.5
±
2.6
11.3
±
2.4
11.2
±
2.5
0.977
0.74
1.000
0.183 ± 0.039
63.34
±
9.73
0.789
6.95
±
0.92
0.970
10.0
12
13.6
±
1.8
11.7
±
2.3
11.4
±
2.3
0.999
2.14
0.684
0.194 ± 0.043
65.64
±
8.32
0.998
6.78
±
1.34
0.999
20.0
12
13.2
±
2.2
11.3
±
2.6
11.2
±
2.7
0.977
1.47
1.000
0.198 ± 0.046
64.80
±
10.25
0.973
6.56
±
1.66
0.997
Positive controls
12
12.8
±
2.1
10.5
±
2.0
7.8
±
1.9
0.001
25.4
0.000
0.179 ± 0.040
44.43
±
7.01
0.000
5.05
±
1.26
0.012The sexuality, fetal body weight, body length, and tail length of the live fetuses were presented in Table7. There were no distinguishable diversities of sex ratio of the fetuses among all of groups, illustrating that both aspirin and aqueous extract ofC. paliurushave no effect on sexuality of embryos. Compared with the negative controls, the fetus body weights of positive controls were obviously lower (P
<
0.01), and both body length and tail length were obviously shorter (P
<
0.01). The fetal body weight, body length, and tail length had no significant differences among the three dosage groups and the negative control group (P
>
0.05).Table 7
Effect ofC. paliurus leaf aqueous extract on sexuality, fetal body weight, body length, and tail length of fetal rat. Data are expressed as mean ± SD.
Groups/(g/kg·bw)
Live fetus
Male/female
Fetal body weight/(g)
Fetal body length/(mm)
Fetal tail length/(mm)
Ratio
P
x
¯  ±  s
P
x
¯  ±  s
P
x
¯  ±  s
P
Negative controls
139
67/72 (0.93)
/
3.79
±
0.40
/
37.38
±
1.31
/
13.80
±
0.65
/
5.0
134
64/70 (0.91)
0.942
3.74
±
0.39
0.727
37.15
±
1.28
0.456
13.66
±
0.62
0.511
10.0
137
63/74 (0.85)
0.712
3.72
±
0.45
0.473
37.14
±
1.34
0.409
13.72
±
0.87
0.995
20.0
134
69/65 (1.06)
0.587
3.81
±
0.42
0.983
37.35
±
1.37
0.999
13.63
±
0.70
0.314
Positive controls
94
45/49 (0.92)
0.961
3.18
±
0.46
0.000
35.32
±
1.70
0.000
13.00
±
0.66
0.000Stereoscope examination showed that no visible external abnormality had occurred to fetal rats of three dosage groups and negative control group, while encephalocele, exomphalocele, and anophthalmos had occurred to part of fetal rats in positive control group. Viscus examination showed that no obvious developmental deformity occurred to the fetal head, thorax, and abdominal viscera in three dosage groups and negative control group, while in positive controls, there was one fetal rat with anophthalmos, one with horseshoe kidney, one with absence of testis, and one with hydronephrosis.The information about skeletal development of fetal rats was summarized in Table8. The incidences of spine hypoplasia, sternum hypoplasia, pelvis hypoplasia, metacarpal hypoplasia, and phalanx hypoplasia of fetal rats in positive control group were significantly higher than those in negative control group (P
<
0.05 or P
<
0.01). Each kind of listed skeletal dysplasia proportion was from 18% to 40%, meaning multiple skeletal dysplasia appeared in the positive controls. The skeletal hypoplasia incidence showed no significant difference among the three dosage groups and the negative control group (P
>
0.05), and the main kinds of skeletal hypoplasia were sternum, metacarpal, and phalanx, congruously. No obvious abnormalities of libs, carpal, long bones, and occipital bones were observed in each dosage group.Table 8
Effect ofC. paliurus leaf aqueous extract on skeletal development of fetal rats.
Groups/(g/kg·bw)
Fetal rats number
Hypoplasia incidence/(%)
Spine/P
Sternum/P
Pelvis/P
Metacarpal/P
Phalanx/P
Negative controls
71
0.0/—
11.3/—
1.4/—
11.3/—
18.3/—
5.0
71
1.4/1.000
9.8/0.785
1.4/1.000
12.7/0.796
14.1/0.494
10.0
72
0.0/—
13.9/0.637
2.8/1.000
9.7/0.763
12.5/0.336
20.0
69
0.0/—
13.0/0.748
1.4/1.000
17.4/0.301
15.9/0.710
Positive controls
50
18.0/0.001
40.0/0.000
24.0/0.000
32.0/0.005
38.0/0.016All results summarized above gave detailed information about the developments of rat embryos in each group. The data about fetal body weight, body length, tail length, and skeletal development of fetal rats provided evidences about the weight gains of mother rats shown in Table5. Compared with the negative controls, all the data from the three dosages had no statistically significant differences. It can be considered that the aqueous extract ofC. paliurus leaves has not any teratogenic effect on the embryos of rats, even at the highest dose of 20 g/kg·bw.


## 4. Conclusions

From genotoxicity assay (including Ames test, micronucleus test, and sperm abnormality test), it was demonstrated that aqueous extract ofC. paliurus leaves had no genotoxicity effects in the Kunming mice at the highest dosage of 5000 μg/plate, 10 g/kg·bw, and 10 g/kg·bw, respectively. While from the teratogenicity assay including the weights of pregnant rats, the indexes of embryo toxicity, the sexuality, fetal body weight, body length, and tail length of the live fetuses, the external deformities and deformity in viscera, and skeletal of the live fetuses, it was revealed that aqueous extract ofC. paliurus leaves showed negative reproductive toxicity on rats at the highest dosage of 20.0 g/kg·bw, equivalent to 150-fold of recommended human dosage. Therefore, it can be considered thatC. paliurus leaves are safe functional additive ingredient in food industries.



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*Source: 498134-2014-03-18.xml*