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.
The leaves of
The acute and subacute toxicity test of aqueous extracts from
Leaves of
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%.
Ames tests were carried out in histidine auxotrophic
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 of
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 of
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 of
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’s
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 [
The results of Ames test on
Dose ( |
TA97 | TA98 | TA100 | TA102 | ||||
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+S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | −S9 | |
5000 |
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1000 |
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200 |
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40 |
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8 |
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Spontaneous reverse |
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Solvent controls |
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Positive controls |
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All the above data were average value of three plates.
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0
The results of Ames test on
Dose ( |
TA97 | TA98 | TA100 | TA102 | ||||
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+S9 | −S9 | +S9 | −S9 | +S9 | −S9 | +S9 | − S9 | |
5000 |
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1000 |
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200 |
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40 |
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Spontaneous reverse |
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Solvent controls |
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Positive controls |
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All the above data were average value of three plates
Positive controls: TA97 + S9, TA98 + S9, and TA100 + S9 adopted 2-AF (dose: 10.0
As can be seen, the numbers of colonies of each group at every dose of aqueous extract of
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 [
Effect of
Sex | Dose (g/kg |
Number of animals | MNPCEs/PCEs ( |
NCEs | PCEs/NCEs |
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Male | 10.0 | 5 |
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850 |
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5.0 | 5 |
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871 |
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2.5 | 5 |
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883 |
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Negative | 5 |
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848 |
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Positive | 5 |
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1004 |
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Female | 10.0 | 5 |
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836 |
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5.0 | 5 |
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842 |
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2.5 | 5 |
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874 |
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Negative | 5 |
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850 |
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Positive | 5 |
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1010 |
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PCE: polychromatic erythrocyte; NCE: normochromatic erythrocyte; MNPCE: micronucleated polychromatic erythrocyte.
Compared to negative controls;
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 (
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 [
As can be seen in Table
Effect of
Dose/(g/kg·bw) | Mice number | Tested sperms | Abnormal sperms | Abnormality incidence | Constituent of different abnormal sperms/% | ||||||
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Lack hook | Banana-like | Fat-head | Morphism | Folding-tail | Double-headed | Twin-tailed | |||||
10.0 | 5 | 5000 | 114 |
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30.7 | 14.9 | 15.8 | 38.6 | 0.0 | 0.0 | 0.0 |
5.0 | 5 | 5000 | 106 |
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33.0 | 7.5 | 17.9 | 41.5 | 0.0 | 0.0 | 0.0 |
2.5 | 5 | 5000 | 111 |
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32.4 | 10.8 | 13.5 | 43.2 | 0.0 | 0.0 | 0.0 |
Negative |
5 | 5000 | 109 |
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33.0 | 7.3 | 19.3 | 40.4 | 0.0 | 0.0 | 0.0 |
Positive |
5 | 5000 | 462 |
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28.6 | 19.5 | 21.0 | 30.3 | 0.0 | 0.0 | 0.6 |
Compared to negative controls;
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 Table
Effect of
Pregnant rat weight/(g) | Weight gain (g) | ||||||||||||
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Groups/(g/kg·bw) | Number of pregnant rats | 0 day | 7th day | 12th day | 16th day | 20th day | |||||||
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Negative controls | 12 |
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1.000 |
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0.820 |
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0.768 |
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0.120 |
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/ |
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/ |
5.0 | 12 |
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0.935 |
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1.000 | ||||
10.0 | 12 |
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0.999 |
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1.000 | ||||
20.0 | 12 |
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0.995 |
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1.000 | ||||
Positive controls | 12 |
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0.006 |
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0.029 |
The effects of
Effect of
Groups/(g/kg·bw) | Numbers | Dead and adsorptive Incidence/(%) | Weight/(g) | ||||||||||||
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Pregnant rat number | Corpora lutea | Nidation | Live embryo | Ovary | Uterus linking to Placenta | Placenta | |||||||||
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Negative controls | 12 |
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0.905 |
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0.690 |
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/ | 1.42 | / | 0.186 ± 0.042 | 0.805 |
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/ |
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/ |
5.0 | 12 |
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0.977 | 0.74 | 1.000 | 0.183 ± 0.039 |
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0.789 |
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0.970 | |||
10.0 | 12 |
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0.999 | 2.14 | 0.684 | 0.194 ± 0.043 |
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0.998 |
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0.999 | |||
20.0 | 12 |
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0.977 | 1.47 | 1.000 | 0.198 ± 0.046 |
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0.973 |
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0.997 | |||
Positive controls | 12 |
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0.001 | 25.4 | 0.000 | 0.179 ± 0.040 |
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0.000 |
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0.012 |
The sexuality, fetal body weight, body length, and tail length of the live fetuses were presented in Table
Effect of
Groups/(g/kg·bw) | Live fetus | Male/female | Fetal body weight/(g) | Fetal body length/(mm) | Fetal tail length/(mm) | ||||
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Ratio |
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Negative controls | 139 | 67/72 (0.93) | / |
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/ |
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/ |
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5.0 | 134 | 64/70 (0.91) | 0.942 |
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0.727 |
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0.456 |
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0.511 |
10.0 | 137 | 63/74 (0.85) | 0.712 |
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0.473 |
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0.409 |
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0.995 |
20.0 | 134 | 69/65 (1.06) | 0.587 |
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0.983 |
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0.999 |
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0.314 |
Positive controls | 94 | 45/49 (0.92) | 0.961 |
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0.000 |
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0.000 |
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0.000 |
Stereoscope 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 Table
Effect of
Groups/(g/kg·bw) | Fetal rats number | Hypoplasia incidence/(%) | ||||
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Spine/ |
Sternum/ |
Pelvis/ |
Metacarpal/ |
Phalanx/ | ||
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 | 38.0/0.016 |
All 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 Table
From genotoxicity assay (including Ames test, micronucleus test, and sperm abnormality test), it was demonstrated that aqueous extract of
There is no conflict of interests regarding the publication of this paper.
Lihong Deng, Rui Zhang, and Yun Liu contributed equally to this paper.
The authors greatly acknowledge the Hunan Provincial Disease Prevention and Control Center for doing experiments and the financial support from Fundamental Research Funds of the Central Universities (Grant no. TD2011-10).