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Section 3. Pharmaceutical Sciences
Agzamkhujaeva Khilola Tojimurod Qizi, Mirzaakhmedov Sharafitdin Yashinovich, Yakubova Rana Abdrimovna, Tashmukhamedov Mugrajitdin Salakhovich, Institute of Bioorganic Chemistry, Academy of Sciences Republic of Uzbekistan E-mail: t_meroj@mail.ru
STUDY OF EMBRYOTOXIC, TERATOGEN ACTIONS OF THE DRUG "BAKAGIN" FROM THE PAROTID SECRETES CENTRAL ASIAN GREEN TOAD BUFO VIRIDIS LAUR AND ITS EFFECT TO THE REPRODUCTIVE FUNCTION OF RATS
Abstract. The sum of bufadienolides, named as "Bakagin" obtained from the venom of the Central Asian green toad Bufo viridis Laur. The effect of the preparate to the embryotoxic, teratogenic activities and influence on the reproductive function was studied in laboratory rats. Obtained results show that, "Bakagin" when injected by intraperitoneally, in doses of0.046 and 0.093 mg/kg, has not cause embryotoxic and teratogenic activities. A study of "Bakagin" on to reproductive function in white rats showed that in the studied doses it has not negative affect to the reproductive function of white outbred rats.
Keywords: Embryotoxicity, teratogenicity, bufadienolides, bakagin, reproductive function, persecuted females, pre-implantation, post-implantation, prenatal, fecundity of females, embryos.
In previous studies we identified bufadieno- bufarenogin (3(,12p,14(- trihydroxybufa-20,22-
lide composition of "Bakagin", from the Central dienolide) and bufalin (3(,14(-dihydroxy-20,22-
Asian green toad Bufo viridis Laur venom. In that dienolide) [1; 2].
work by chromatographic methods six individual It is known that when using medicines it is neces-
bufadienolides were isolated, and their chemi- sary to take in to account about its possible impact on
cal and conformational structures determined the mother's organism, on the natural physiological
using NMR and X-ray analysis. In a result bufadi- barrier - placenta and direct impact to the embryo.
enolides identified as: arenobufagin (3(,11a,14- Embryotoxic action can be appear in increased levels
trihydroxy-12-oxo-5p,14(-bufa-20,22-dienolide), of fetal mortality, delayed ossification of the skeleton,
gamabufotalin (3 (, 11 a, 14-trihydroxy-5 (3,14(- changing in body mass and cranyo caudal size of the
bufa-20,22-dienolide), telocinobufagin (3(,5p,14(- fetus, an increasing in perinatal pathology and etc. [3].
trihydroxy-20,22-dienolid) marinobufagin (3(,5(- Approximately 1% of fetal developmental ab-
dihydroxy-14,15(-epoxybufa-20,22-dienolid) normalities in pregnant women cause uncontrolled
and not correct use of drugs. Therefore in modern medicine more attention is paid to study the effects of drugs on the intrauterine development of a child and on a pregnant woman herself at different stages of pregnancy. In the pre-clinic evaluation on safety of new drugs, it is necessary to know their effect to the embryogenesis of laboratory animals [4].
Purpose of this work is study of embryotoxic, teratogen effects of "Bakagin" and its influence on the reproductive function of laboratory rats.
Experimental
Investigation of embryotoxic, teratogen effects of "Bakagin"
Experiments were carried out on white outbred rats (180 ± 20g), animals were divided into 3 groups of10 male and 20 female. Experimental animals were kept and feeding by standard conditions [5]. During 48 days, the males were intra-peritoneal injected with Bakagin at a daily therapeutic dose: 1-group -0.046 mg/kg; 2-group - 0.093 mg/kg; 3-group -physiological solution, as a control. At the end of this period, the female rats were placed in a ratio of 2:1 to the males. During the week, pregnant females were isolated (the presence of sperms in a vaginal smear) and they were daily injected with drug in the same doses.
At 7, 17 and 20 days after pregnancy, females were killed by dislocation of cervical vertebrae and dissected. As indicators of embryotoxic action, against to control were determined pre - and postimplantation embryonic mortality, morphological (anatomical) malformations, a general retardation of fetal development.
Pre-implantation mortality was determined by difference between numbers ofyellow bodies in ovaries and implantation sites in uterus. Post-implantation mortality is determined by difference between numbers of implantation and live fetuses.
Teratogenic effect was determined by proportion of anomalous fetuses at external observation. For that 20-day yields of each litter weighted and their cranyo caudal dimensions were measured. After ex-
ternal examination, yields of each litter were fixed in Buen's solution and used to study of internal organs. To study of skeleton state other yields were fixed in 96% ethanol.
Due to the fact that abnormalities in fetuses development can appear in a later period, part of pregnant females from each group were kept in separate cells for natural childbirth, followed by monitoring of offspring development in the postnatal period (generation was investigated during 1 month).
Obtained results were statically processed by standard methods, using Microsoft Excel program: determination of average value (M) and average error (m).
Investigation of "Bakagin" on to reproductive function of white rats
Investigation of "Bakagin" on to reproductive function of white rats was carried out on 24 male and 60 female of white outbred rats (160-180g).
"Bakagin" was intra peritoneal injected into females for 15 days at doses of0.046 and 0.093 mg/kg of body weight (the group of "hounded females"). Then the animals were mated with intact males. Females were planted to males in a 2:1 ratio for 10 days period. During the study general physical condition and behaviour of animals were monitored. Fertilization was recorded using vaginal smears. In the third week, part of the pregnant females were killed by dislocation of the cervical vertebrae and dissected. State of the reproductive organs of females was studied, in the uterus number of yellow bodies, in the ovaries implants live and dead fetuses were counted. Based on the data obtained, indicators of pre-implant and post-implantation mortality were determined and the fertility index - ratio of the number of pregnant females to the seated females was calculated. In each group of females, a part ofpregnant rats were left before the birth and during one month offspring development - the general physical condition, behaviour and rats death was observed.
In males ("group of hounded males"), "Bakagin" was injected by intra peritoneal in a dose of 0.046
and 0.093 mg/kg of body weight for 48 days. After end of injection of the drug to the males, intact females were planted in a ratio of 1:2 for 10 days. A status of reproductive function of males was estimated by the results by study of reproductive organs of females paired with males who received the drug of "Bakagin".
Results and discussion
Results of investigation embryotoxic, teratogen actions of the drug "Bakagin"
Results obtained showed that in female white rats who received "Bakagin" in doses of 0.046 and 0.093 mg/kg, all parameters of embryo-lethality do not have significant differences with those in the females in control group (see Table 1).
Indicators Group of animals
Control 0.046 mg/kg 0.093 mg/kg
Number of females in the experience 20 20 20
Fecundity of females 16 17 16
Number of yellow bodies per 1 female 8.63 ± 0.37 8.53 ± 0.36 8.25 ± 0.40
Number of dead and resorbed yields 4.0 5.0 4.0
Death of embryos,%
Pre-implantation 2.90 3.44 3.03
Post-implantation 3.62 4.13 3.78
Overall embryo survival,% 93.5 92.4 93.2
Mass of embryos, g 2.53 ± 0.12 2.51 ± 0.17 2.67 ± 0.50
Size of embryos, mm 25.8 ± 0.27 26.8 ± 0.29 26.3 ± 0.14
Indicators of pre-implantation mortality in control and studied variants are 2.90, 3.44 and 3.03%, and post-implantation mortality - 3.62, 4.13 and 3.78%, respectively. The mass ofyields in control and studied groups after injection of Bakagin in doses of 0.046 mg/kg, 0.093 mg/kg is 2.53 ± 0.12, 2.51 ± 0.17 and 2.67 ± 0.50 g, respectively. They do not have significant differences and cranyo-caudal dimensions: 25.8 ± 0.27, 26.8 ± 2.94 and 26.3 ± 0.14 mm, respectively. All indicators of embryo toxicity remain at the control level. During external examination of yields of each litter development of visible external anomalies are not observed.
Statistical difference in survival and death of fetuses before implantation and after implantation in the studied groups were not found.
A daily examination ofpregnant females after inj ec-tion of Bakagin did not reveal a significant difference in their common condition, neatness and revealed no significant differences by increase of body mass, compared to control group ofpregnant female rats.
In the study of the teratogenic effect of "Bakagin" on the basis of macro anatomical analysis of internal organs and embryo skeleton anomalies, no significant deviations compared to control were detected
(see Tables 2 and 3). Table 2.- Results of macroscopic study of embryos by influence of "Bakagin"
Indicators Group of animals
Control 0.046 mg/kg 0.093 mg/kg
1 2 3 4
Number of investigated embryos 60 60 60
Hemorrhage,%
Subcutaneous — — —
Facial skull 2 2 2
Table 1. - Effect of Bakagin on pregnancy and fetal development
1 2 3 4
In the chest cavity - - -
Into the abdominal cavity 3 3 3
Anomalies of brain development,%
Hemorrhage - - -
Expansion of brain ventricles - - -
Spinal hemorrhage - - -
Table 3.- State skeleton of rats yields by action of Bakagin
Indicators Group of animals
Control 0,046 Mr/Kr 0,093 Mr/Kr
Number of yields, investigated by Dauson 50 50 50
Number of ribs
Right 13.00 13.00 13.00
Left 13.00 13.00 13.00
Number of embryos with anomaly development
External anomalies - - -
Internal anomalies - - -
Thus, based on obtained results, it is possible to say that "Bakagin" at intra peritoneal injection in doses of0.046 and 0.093 mg/kg, has not embryotoxic and teratogenic properties.
Results of studies of "Bakagin" on the reproductive function of white rats
Studies have shown that injection of "Bakagin" and did not affect to the accouchement time. So, in rats receiving the drug, and control, this period was 20-21 days. During drug injection, the state and behaviour of experimental rats did not difference
against to control. In all experimental groups, the body weight of pregnant rats injected with Bakagin increased by an average of 34.8 and 35.5% relative to the outcome. The same increase in body weight of pregnant rats was observed in the control of 37.4%.
Indicators of the reproductive function of white female rats after intra peritoneal injection of Bakagin in doses of0.046 and 0.093 mg/kg did not difference against to control animals. The data obtained are shown in the (Table 4).
Table 4.- Effects of "Bakagin" on the fertility of rats in the group of "baited females"
Indicators Groups
Control Ç - intact S - intact 0.046 mg/kg Ç - hunted S - intact 0.093 mg/kg Ç - hunted S - intact
Number of females hooked with males 20 20 20
Number of fertilized females 17 18 17
Number of pregnant females 17 16 17
Fertility Index,% 85.0 90.0 85.0
The results are shown in Table 4 shows that remained at the level of the fertility index of the the fertility index of the females of this group control group. Thus, analysis of obtained results
allows concluding, that fecundity of females in ex- implantation sites and their number per 1 female,
perimental groups is equivalent against to control number of live fetuses and their number per 1 female
values. were determined. These indicators of female repro-
In the third week of pregnancy number of yel- duction did not statistically significantly different
low bodies, their number per 1 female, number of against to control (see Table 5).
Table 5.- Action of "Bakagin" on the reproductive function of female rats (group "baited females")
Indicators Groups
Control $ - intact S - intact 0.046 mg/kg $ - hunted S - intact 0.093 mg/kg $ - hunted S - intact
Number of pregnant females 8.0 8.0 8.0
Number of yellow bodies per 1 female 70/8.75 68/8.50 69/8.6
Number of implantation sites per 1 female 67/8.37 66/8.25 65/8.12
Number of live yields per 1 female 65/8.12 64/8.0 65/8.12
Thus, the analysis of obtained data allows con- Results of the experimental group of "hounded
cluding that reproductive indicators of females in the males" by fertility show that the fertility index of
experimental group of "hounded females" remain at females in these experimental groups does not dif-
the level, against to control group. This result indi- ference against to control. Results on the fertility of
cates that "Bakagin" in used does has not negative female rats in the "hounded males" groups are given
affect to the reproductivity of females. in (Table 6).
Table 6.- Effects of "Bakagin" on the fertility of male rats in the group "baited males"
Indicators Groups
Control $ - intact S - intact 0.046 mg/kg $ - hunted S - intact 0.093 mg/kg $ - hunted S - intact
Number of females hooked with males 20 20 20
Number of fertilized females 18 17 18
Number of pregnant females 8 8 8
Fertility Index,% 90.0 85.0 90.0
The obtained experimental data show that the fertility index of animals - females of these experimental groups, does not statistically significantly difference against to control. This data indicates that Bakagin in used doses does not negative affect to the fertility of male rats.
For investigation the effect of "Bakagin" to other reproductive indicators of rats, males from the group of "hounded males" took pregnant rats in the third week, the part of pregnant animals was opened and the number of yellow bodies was counted, their
number per 1 female, the number of implantation sites and their number per 1 female, the number of live fruits and their number per 1 female.
The results of the reproductive performance of female rats in the "hounded males" groups are given in the (Table 7).
As can be seen from the obtained data, reproductive performance of female rats in the "baited males" groups did not difference against to control.
Left to give birth to females of all experimental groups, the process of childbirth and care for the
offspring were unremarkable. The average number of eyes, lowering of testes and opening of vagina
of fetuses per female, the ratio of rats by sex, the in the litters of both the experimental and control
level of death of new born rats did not change in groups were similar. The results of physiological
the litters of all experimental groups. The timing of developments of the offspring of rats by influence
the out-off the auricle, the appearance of the pri- of "Bakagin" in the early postnatal period are given
mary hairline, the incision of the incisors, opening in (Table 8).
Table 7.- Effects of Bakagin on the reproductive ability of rats of the "persecuted males" group
Indicators Groups
Control Ç - intact S - intact 0.046 mg/kg Ç - hunted S - intact 0.093 mg/kg Ç - hunted S - intact
Number of pregnant females 8.0 8.0 8.0
Number of yellow bodies 68/8.5 67/8.4 68/ 8.6
Number of implantation sites 66/8.2 65/8.1 65/8.1
Number of live yields 65 64 64
Таблица 8.- Results of physiological developments of the offspring of rats by influence of "Bakagin" in early postnatal period
Indicators Groups
Control Ç - intact S - intact 0.046 mg/kg Ç - hunted S - intact 0.093 mg/kg Ç- hunted S - intact
Number of females in experiment 10 10 10
Amount of yields 60 60 60
Death of rats in the period of feeding (number) 3 2 2
Auricle Day 3.3 ± 0.08 3.5 ± 0.01 3.6 ± 0.05
Primary Hair Day 5.6 ± 0.14 5.5 ± 0.11 5.4 ± 0.12
Teething Day 8.7 ± 0.12 8.8 ± 0.10 8.7 ± 0.10
Eye opening day 14.6 ± 0.13 15.2 ± 0.13 14.7 ± 0.17
Day of the lowering of the testes 26.0 ± 0.32 26.1 ± 0.15 26.4 ± 0.14
Vagina opening day 31.0 ± 0.21 31.5 ± 0.14 31.5 ± 0.16
Mass of rats, g
4 day 8.49 ± 0.20 8.84 ± 0.50 8.80 ± 0.11
7 day 12.2 ± 0.10 11.6 ± 0.15 11.4 ± 0.32
14 day 21.4 ± 0.09 21.7 ± 0.14 21.9 ± 0.23
21 day 30.4 ± 0.23 30.2 ± 0.16 30.5 ± 0.16
Death of females during the feeding period 0 ± 0 0 ± 0 0 ± 0
Thus, based on obtained results, it can be as- negative affect to the reproductive function of white sumed that "Bakagin" in the studied doses does not outbred rats.
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