论文部分内容阅读
Abstract In this study， Paulownia flower extract of different concentrations was administered to pregnant rats by gavage to investigate its teratogenic effect. It was found that Paulownia has neither teratogenic effect nor growth inhibitory effect on rats， and it can be used in animal production as new feed additive.
Key words Paulownia flower extract； Rat； Concentration； Test
Paulownia flower contains various chemical components such as flavonoids， terpenoids and volatile oils[1-5]. It has antibacterial， antiasthmatic and antiallergic inflammation[6]functions， can enhance immune function[7]and promote the development of immune organs， and has antitumor， antioxidation， antiviral， antihyperglycemia， antihyperlipidemia and other extensive pharmacological activities[8]. In clinics， it is used for the treatment of respiratory infections， bronchial pneumonia， acute tonsillitis， dysentery， acute enteritis， acute conjunctivitis and mumps， varying degrees of burns， foot and hand tinea， etc. It is a biological active resource with development and utilization value and has become an important traditional Chinese herbal medicine in China.
In this study， the teratogenicity test of Paulownia flower extract on pregnant rats was conducted with reference to the traditional teratogenicity test guidelines of the Ministry of Agriculture for new veterinary drugs， aiming to accumulate data for the toxicity study of the product.
Materials
Experimental animal
About 80 10weekold Wistar rats （half male and half female， clean grade） were provided as experimental animal by the Institute of Comparative Medicine， College of Veterinary Medicine， Yangzhou University with animal production license number of SCXK （Su） 20120004 and animal use license number of SYXK （Su） 20120029. The rats were reared in cages， with free access to drinking water， room temperature of （22 ± 3）℃， natural light， and relatively humidity of 55%-75%. They were transferred to the test environment 3 d before the test and starved for 12 h before administration. Drinking water was not restricted.
Drug
Paulownia flower extract （lot number 20141022） was produced by a biotechnology company. Just before use， it was prepared into suspension （0.25 g/ml） with 2% sodium carboxymethyl cellulose solution.
Methods
Preparation of pregnant rats
The female and male rats were caged according to the ratio of 1∶1， and the vaginal plugs were examined the following morning. The day when the vaginal plug was detected was recorded as day 0 of pregnancy. The detected fertilized rats were randomly grouped. On day 7-17 of pregnancy， the drug was given orally every day. The dose of drug was adjusted according to the body weight during the test. Grouping
The test was divided into 4 groups with 12-15 pregnant rats in each group. Different concentrations （2.00， 0.65 and 0.20 g/kg， equivalent to 1/10 LD501/100 LD50； LD50 for acute toxicity test of rats greater than 20 mg/kg） of Paulownia flower extract was administered to the rats by gavage according to the dose of 0.008 ml/g. The rats in the normal control group were given normal saline （0.008 ml/g）.
Examination of fetal rats
On day 20 of pregnancy， the experimental rats were weighed and sacrificed by cervical dislocation， and the uteri were removed. Then， the litter （uterus and fetal rat） weight， placenta weight， number of live births， number of stillbirths and number of resorbed fetuses， as well as the gender， body length， body weight and tail length of the fetuses were recorded. The anomalies in the appearance， internal organs and skeletons of the fetal rats were examined. The rates of malformed fetuses， maternal malformed fetuses， stillborn fetuses， and resorbed fetuses were calculated according to the following formulas：
Malformation rate （%） = Total number of malformed fetuses/Total number of fetuses × 100%；
Average malformation rate of live fetuses （%） = Total number of malformed fetuses/Total number of live fetuses × 100%；
Maternal malformation rate （%） = Number of mothers with malformed fetuses/Total number of pregnancies × 100%；
Stillbirth rate （%） = Total number of stillbirths/Total number of fetuses × 100%；
Resorption rate （%） = Number of resorbed fetuses/Total number of fetuses × 100%.
Result judgement
If the malformation indicators （malformation rate， average malformation rate of live fetuses， maternal malformation rate， stillbirth rate and resorption rate， etc.） in the test group are significantly higher than those in the normal control group and there is a dosedependent relationship， it can be determined that the test substance has teratogenesis.
Data statistics and analysis
Data processing and analysis was performed using SPSS 12.0. The data was expressed as mean ± standard deviation. The significance test was performed using ttest or chisquare test.
Results
Effect of Paulownia flower extract on fetal rats
The measurement results of litter size， placental weight， and body weight， body length and tail length of live births were shown in Table 1.
As shown in Table 1， no significant differences were found in the litter size， fetal average body weight， fetal average body length， fetal average tail length， placental weight， and gender ratio between the treatment and control groups （P>0.05）. The results of live birth rate， stillbirth rate and resorption rate were shown in Table 2.
As shown in Table 2， there were no significant differences in the total number of fetuses， live birth rate， stillbirth rate and resorption rate between the treatment and control groups （P>0.05）.
Teratogenic effect of Paulownia flower extract on fetal rats
In the malformation examination， it was found that the distance between two ribs increased on one side or on both sides （the number of ribs did not change）. This phenomenon appeared in one fetus of the normal control group and each treatment group， and no any other abnormalities were observed. The results were shown in Table 3.
As shown in Table 3， no significant differences were found in the common malformation indicators such as malformation rate， average malformation rate of live fetuses and maternal malformation rate between different doses of Paulownia flower extract treatment groups and the normal control group （P>0.05）.
Discussion
Teratogenicity is the most important manifestation of development toxicity and an important indicator of the development toxicity of foreign compounds. If the mother is exposed to harmful substances that can pass through the placenta barrier during pregnancy， the differentiation and development of the embryos organs will be influenced， leading to structural and functional defects， that is， fetal malformations. Therefore， after the implantation of the embryo of the pregnant animal， injecting the test substance during the organ formation can help to detect its teratogenic effect on the fetus. The body weight of pregnant rats and reproductive indicators such as the number of corpus lutea， number of implantations， number of live births， number of resorbed fetuses， and number of stillbirths can reflect the influence of the test substance on rat reproductive function. Examination of the appearance， visceral and skeletal deformity of the fetus can reflect the teratogenic effect of the test substance on the test animal. In this study， it was foundthat there were no significant differences in the litter size， fetal average body weight， fetal average body length， fetal average tail length， placental weight and gender ratio， as well as live birth rate， stillbirth rate and resorption rate between different doses of Paulownia flower extract treatment groups and the normal control groups （P>0.05）. The malformation examination showed that the spacing between two ribs increased on one side or on both sides， but the number of ribs did not change. This phenomenon appeared in one fetus of the normal control group and each treatment group， and no other abnormalities were observed. The results showed that Paulownia flower extract has neither teratogenic effect nor inhibitory effect on animal development， and it can used as a new feed additive for animal production. References
[1]DUAN WD， ZHANG J， XIE G， et al. Chemical constituents from the flower of Paulownia fortunei （Seem.） Hemsl[J]. Journal of Chinese Medicinal Materials， 2007， 30（2）： 168-170.
[2]LIANG FT. Studies on chemical constituents of the extractives by petroleum from the leaves of Paulownia fortunei （Seem.） Hemsl[D]. Lanzhou： Lanzhou University， 2007.
[3]ZHANG PF， LI C. Flavones from flowers of Paulownia fortunei[J]. Chinese Journal of Chinese Materia Medica， 2008， 33（22）： 2629-2632.
[4]CHEN LY， ZHAO L， YU XH， et al. Content determination of ursolic acid in different parts of Paulownia fortunei[J]. Journal of Chinese Medicinal Materials， 2007， 30（8）： 914-915.
[5]WANG X， CHENG CG， LIU JH， et al. Chemical composition of the essential oil from Paulownia tomentosa flowers[J]. Chemistry and Industry of Forest Products， 2005（2）： 99-102.
[6]WEI XY， HE Y， JIANG LF， et al. The study of the antibacterial activity in vitro and determination of flavones of flos paulowniae[J]. Natural Product Research and Development， 2006（3）： 401-404.
[7]LI YC， ZHAO YH， CHEN ZY， et al. Study on the allergic inflammation of bronchial asthma treated with volatile oil of Paulownia flower[J]. Journal of Medical Forum， 2006， 27（16）： 71-72.
[8]FU MZ， YUAN FH， LU XM， et al. Antibacterial effect of flavonoids from Paulownia flower and its effect on immune function[J]. Chinese Journal of Veterinary Medicine， 1999， 25（5）： 46-47.
Editor： Tingting XU Proofreader： Xinxiu ZHU
Key words Paulownia flower extract； Rat； Concentration； Test
Paulownia flower contains various chemical components such as flavonoids， terpenoids and volatile oils[1-5]. It has antibacterial， antiasthmatic and antiallergic inflammation[6]functions， can enhance immune function[7]and promote the development of immune organs， and has antitumor， antioxidation， antiviral， antihyperglycemia， antihyperlipidemia and other extensive pharmacological activities[8]. In clinics， it is used for the treatment of respiratory infections， bronchial pneumonia， acute tonsillitis， dysentery， acute enteritis， acute conjunctivitis and mumps， varying degrees of burns， foot and hand tinea， etc. It is a biological active resource with development and utilization value and has become an important traditional Chinese herbal medicine in China.
In this study， the teratogenicity test of Paulownia flower extract on pregnant rats was conducted with reference to the traditional teratogenicity test guidelines of the Ministry of Agriculture for new veterinary drugs， aiming to accumulate data for the toxicity study of the product.
Materials
Experimental animal
About 80 10weekold Wistar rats （half male and half female， clean grade） were provided as experimental animal by the Institute of Comparative Medicine， College of Veterinary Medicine， Yangzhou University with animal production license number of SCXK （Su） 20120004 and animal use license number of SYXK （Su） 20120029. The rats were reared in cages， with free access to drinking water， room temperature of （22 ± 3）℃， natural light， and relatively humidity of 55%-75%. They were transferred to the test environment 3 d before the test and starved for 12 h before administration. Drinking water was not restricted.
Drug
Paulownia flower extract （lot number 20141022） was produced by a biotechnology company. Just before use， it was prepared into suspension （0.25 g/ml） with 2% sodium carboxymethyl cellulose solution.
Methods
Preparation of pregnant rats
The female and male rats were caged according to the ratio of 1∶1， and the vaginal plugs were examined the following morning. The day when the vaginal plug was detected was recorded as day 0 of pregnancy. The detected fertilized rats were randomly grouped. On day 7-17 of pregnancy， the drug was given orally every day. The dose of drug was adjusted according to the body weight during the test. Grouping
The test was divided into 4 groups with 12-15 pregnant rats in each group. Different concentrations （2.00， 0.65 and 0.20 g/kg， equivalent to 1/10 LD501/100 LD50； LD50 for acute toxicity test of rats greater than 20 mg/kg） of Paulownia flower extract was administered to the rats by gavage according to the dose of 0.008 ml/g. The rats in the normal control group were given normal saline （0.008 ml/g）.
Examination of fetal rats
On day 20 of pregnancy， the experimental rats were weighed and sacrificed by cervical dislocation， and the uteri were removed. Then， the litter （uterus and fetal rat） weight， placenta weight， number of live births， number of stillbirths and number of resorbed fetuses， as well as the gender， body length， body weight and tail length of the fetuses were recorded. The anomalies in the appearance， internal organs and skeletons of the fetal rats were examined. The rates of malformed fetuses， maternal malformed fetuses， stillborn fetuses， and resorbed fetuses were calculated according to the following formulas：
Malformation rate （%） = Total number of malformed fetuses/Total number of fetuses × 100%；
Average malformation rate of live fetuses （%） = Total number of malformed fetuses/Total number of live fetuses × 100%；
Maternal malformation rate （%） = Number of mothers with malformed fetuses/Total number of pregnancies × 100%；
Stillbirth rate （%） = Total number of stillbirths/Total number of fetuses × 100%；
Resorption rate （%） = Number of resorbed fetuses/Total number of fetuses × 100%.
Result judgement
If the malformation indicators （malformation rate， average malformation rate of live fetuses， maternal malformation rate， stillbirth rate and resorption rate， etc.） in the test group are significantly higher than those in the normal control group and there is a dosedependent relationship， it can be determined that the test substance has teratogenesis.
Data statistics and analysis
Data processing and analysis was performed using SPSS 12.0. The data was expressed as mean ± standard deviation. The significance test was performed using ttest or chisquare test.
Results
Effect of Paulownia flower extract on fetal rats
The measurement results of litter size， placental weight， and body weight， body length and tail length of live births were shown in Table 1.
As shown in Table 1， no significant differences were found in the litter size， fetal average body weight， fetal average body length， fetal average tail length， placental weight， and gender ratio between the treatment and control groups （P>0.05）. The results of live birth rate， stillbirth rate and resorption rate were shown in Table 2.
As shown in Table 2， there were no significant differences in the total number of fetuses， live birth rate， stillbirth rate and resorption rate between the treatment and control groups （P>0.05）.
Teratogenic effect of Paulownia flower extract on fetal rats
In the malformation examination， it was found that the distance between two ribs increased on one side or on both sides （the number of ribs did not change）. This phenomenon appeared in one fetus of the normal control group and each treatment group， and no any other abnormalities were observed. The results were shown in Table 3.
As shown in Table 3， no significant differences were found in the common malformation indicators such as malformation rate， average malformation rate of live fetuses and maternal malformation rate between different doses of Paulownia flower extract treatment groups and the normal control group （P>0.05）.
Discussion
Teratogenicity is the most important manifestation of development toxicity and an important indicator of the development toxicity of foreign compounds. If the mother is exposed to harmful substances that can pass through the placenta barrier during pregnancy， the differentiation and development of the embryos organs will be influenced， leading to structural and functional defects， that is， fetal malformations. Therefore， after the implantation of the embryo of the pregnant animal， injecting the test substance during the organ formation can help to detect its teratogenic effect on the fetus. The body weight of pregnant rats and reproductive indicators such as the number of corpus lutea， number of implantations， number of live births， number of resorbed fetuses， and number of stillbirths can reflect the influence of the test substance on rat reproductive function. Examination of the appearance， visceral and skeletal deformity of the fetus can reflect the teratogenic effect of the test substance on the test animal. In this study， it was foundthat there were no significant differences in the litter size， fetal average body weight， fetal average body length， fetal average tail length， placental weight and gender ratio， as well as live birth rate， stillbirth rate and resorption rate between different doses of Paulownia flower extract treatment groups and the normal control groups （P>0.05）. The malformation examination showed that the spacing between two ribs increased on one side or on both sides， but the number of ribs did not change. This phenomenon appeared in one fetus of the normal control group and each treatment group， and no other abnormalities were observed. The results showed that Paulownia flower extract has neither teratogenic effect nor inhibitory effect on animal development， and it can used as a new feed additive for animal production. References
[1]DUAN WD， ZHANG J， XIE G， et al. Chemical constituents from the flower of Paulownia fortunei （Seem.） Hemsl[J]. Journal of Chinese Medicinal Materials， 2007， 30（2）： 168-170.
[2]LIANG FT. Studies on chemical constituents of the extractives by petroleum from the leaves of Paulownia fortunei （Seem.） Hemsl[D]. Lanzhou： Lanzhou University， 2007.
[3]ZHANG PF， LI C. Flavones from flowers of Paulownia fortunei[J]. Chinese Journal of Chinese Materia Medica， 2008， 33（22）： 2629-2632.
[4]CHEN LY， ZHAO L， YU XH， et al. Content determination of ursolic acid in different parts of Paulownia fortunei[J]. Journal of Chinese Medicinal Materials， 2007， 30（8）： 914-915.
[5]WANG X， CHENG CG， LIU JH， et al. Chemical composition of the essential oil from Paulownia tomentosa flowers[J]. Chemistry and Industry of Forest Products， 2005（2）： 99-102.
[6]WEI XY， HE Y， JIANG LF， et al. The study of the antibacterial activity in vitro and determination of flavones of flos paulowniae[J]. Natural Product Research and Development， 2006（3）： 401-404.
[7]LI YC， ZHAO YH， CHEN ZY， et al. Study on the allergic inflammation of bronchial asthma treated with volatile oil of Paulownia flower[J]. Journal of Medical Forum， 2006， 27（16）： 71-72.
[8]FU MZ， YUAN FH， LU XM， et al. Antibacterial effect of flavonoids from Paulownia flower and its effect on immune function[J]. Chinese Journal of Veterinary Medicine， 1999， 25（5）： 46-47.
Editor： Tingting XU Proofreader： Xinxiu ZHU