danio rerio embryo as a model for study abortifacient effects using ananas comosuss

www.wjpps.com Vol 3, Issue 7, 2014.

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Ramakrishna et al. World Journal of Pharmacy and Pharmaceutical Sciences

DANIO RERIO EMBRYO AS A MODEL FOR STUDY

ABORTIFACIENT EFFECTS USING ANANAS COMOSUS

Abhinava J V1, Srinivas Raju2, Guruprasad R*

Durga Femto Technologies and Research, #22,4th main, 4th cross, Chamarajpet, Bangalore-

560018, India.

ABSTRACT

Danio rerio is one of the best model organisms to study various kinds

of disorders. This study was conducted by treating Danio rerio

embryos with a well-known plant having abortifacient activity like

Ananas comosus. The ethanol and chloroform extract were subjected to

the embryos at different post fertilization periods and this includes

1cell stage, blastula, gastrula and organogenesis. The effects of the

different concentrations were studied at different time intervals. From

the statistical analysis for time with concentration, the blastula stage

embryos show the most significant effect P (F≥0.8753) = 0.05. As

embryos develop, they get resistance against both extracts and the

survival rate increases. Prolonged exposure to the embryos shows

death in all stages. This experiment demonstrates that Danio rerio is a suitable model

organism for the study of abortifacient activity.

Key Words: abortifacient, Danio rerio, Ananas comosus, model organism, embryogenesis.

INTRODUCTION

Increasing population is one of the critical things in developing countries like India. India is

one of the nations which adopted the family planning program during the year 1950. Today

controlling population becomes necessary, and for this the taking up of abortifacient

compound is common1. Thus the study of the abortifacient compounds from different sources

is very important; at present the screening of abortifacient activity is done by using different

model organisms.

Article Received on 07 May 2014, Revised on 27 May 2014, Accepted on 23 June 2014

*Correspondence for Author

Dr. Guruprasad

Ramakrishna

DurgaFemto Technologies and

Research, #22,4th main, 4th

cross, Chamarajpet, Bangalore,

India.

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The benzene extract of Achyranthesasperawas used to determine the abortifacient effect in

rabbits2. The unripe fruits of Carica papaya were used to determine the abortifacient activity

in rats3. In Guinea pigs the alkaloid called as vasicine shows the abortifacient effects4.

Ananas comosus is one of the known plants having abortifacien effect in India5. The steroids,

which are present in the Ananas comosus extract, show significant abortifacient in mice6, 7.

Throughout the study abortifacient effects of different plants were carried out on different

model organisms (mice, rat, rabbit, guineapigs, etc…), the maintenance of the organisms is

difficult when compared to aquatic organisms like zebrafish (Daniorerio).

Danio rerio is one of the best model organisms in current years8, 9. It is already proven that

80% of the Danio rerio genome matches the human genome10. Developments of all chordate

embryos are similar to human embryogenesis. The complete development of the embryo

from the single cell stage to larval stage requires up to 72hrs after post fertilization period.

The Danio rerio embryos are all transparent11.Due to the transparency of the embryos the

changes in the development of embryos can easily be studied and it not possible in higher

organism12, 13. The death of the embryos caused by the abortifacient compounds shows the

abortifacient activity of that compound. Thus adopting Danio rerio embryo for studying

abortifacient activity gives a faster and viable result than other organisms like tadpoles, mice,

rabbits, monkeys, etc…

MATERIALS AND METHODS

Preparation of extracts

The soxhelet apparatus was used to prepare Chloroform and Ethanol extracts of plant

material. The different concentrations (10, 20, 30, 40 and 50 mg/ml) were prepared using

double distilled water. The crude extract was prepared by homogenizing the plant materials.

Collection of embryos

The zebrafish (wild type) were maintained at standard lab condition. The embryos were

collected using natural spawning and they were incubated at 28.50c11.

Embryonic study

The crude Ananas comosus extract was added to petri-plates containing fertilized Danio rerio

embryo and observed at intervals of 1hr.


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The embryos with four different stages includes: 1cell stage (0-1hr post fertilization),

Blastula stage (2.25-5.25hr post fertilization), Gastrula stage (5.25-10hr post fertilization) and

Organogenesis stage (21-48hr post fertilization) were taken along with the different

concentrations of 0, 10, 20, 30, 40 & 50mg/ml of plant extract with distilled water and

incubated at 28.50c. Five embryos were taken in every plate for analysis. The readings were

noted every hour and the results were tabulated.

Statistical analysis

The death of the embryos against the concentration and time were taken for the analysis of

variance (ANOVA) and it was presented as mean ± SEM in different stages. From the data,

one way ANOVA analysis was done and F-value and P- values were calculated and analyzed

with the standard values. The comparative analysis of all stages with ethanol and chloroform

extract was presented as mean ± SEM.

RESULTS AND DISCUSSION

Embryonic study

Analysis of crude extract

The addition of crude extract shows the drastic change in the Danio rerio embryo

development and finally these extracts shows the death of the embryos within 1hr. The

microscopic image of the embryos with plant extracts along with the control was shown in

the figure 1.

Fig. 1: microscopic image of embryos with crude extract, showing the lyses of the

embryo (Right) and control having 5hr PF (Left).

Analysis of plant extract

The effect of Ananas comosus extracts was analyzed at four distinct stages and these include:


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1 Cell stage

The complete result of treated 1 cell stage (0.4hr PF) embryo with different time interval and

concentration was shown in Figure 2.

Fig. 2: Effect on the 1cell stage embryos of ethanol and chloroform extract along with

control. (A) Represent the survival rate with time; this indicates that survival of the

embryo decreases along with time. (B) Represent the Survival rate along with

concentration; this indicates that survival of embryo has a negative effect with

concentration. The values were presented as mean ± SEM and was assessed using

ANOVA. (C) Represent the microscopic view of 1cell embryo (0.4hpf), without plant

extract (above) and dead embryo of ethanol (1.5hpf) and chloroform extract (1.5hpf)

(below).

In this stage the death of the embryos were starting at the initial time (i.e. 1hr after

incubation). The variation in the concentration does not show much difference on the death

rate of the embryos.

Blastula Stage

The complete result of treated blastula stage (2.25hr PF) embryo with different time interval

and concentration was shown in Figure 3.


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Fig. 3: Effect on the blastula stage embryos of ethanol and chloroform extract along

with control. (A) Represent the Survival rate with time; this indicates that survival of

the embryo decreases along with time. (B) Represent the Survival rate along with

Concentration; this indicates that survival of embryo has a negative effect with

Concentration. The values were presented as mean ± SEM and were assessed using

ANOVA. (C) Represent the image of early blastula (2.25hpf) and late blastula (3.25hpf)

stage embryo without plant extract (above) and dead embryo of ethanol (4.5hpf) and

chloroform extract (3.5hpf) (below).

At this stage only the chloroform extract starts death at first hour and ethanol extract starts

death after second hour of plant extract addition. The concentration does not vary the embryo

death rate.

Gastrula stage

The complete result of treated gastrula stage (5.25hr PF) embryo with different time interval

and concentration was shown in Figure 4.


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Fig. 4: Effect on the Gastrula stage embryos of ethanol and chloroform extract along

with control. (A) Represent the Survival rate with time; this indicates that survival of

the embryo decreases along with time. (B) Represent the Survival rate along with

Concentration; this indicates that survival of embryo has a negative effect with

Concentration. The values were presented as mean ± SEM and was assessed using

ANOVA. (C) Represent the image of early gastrula (5.25hpf) and late gastrula (7.25hpf)

stage embryo without plant extract (above) and dead embryo of ethanol (7.25hpf) and

chloroform extract (7.25hpf) (below).

At this stage both the extractsshows death after second hour of plant extract addition. The

concentration does not vary the embryo death rate. The long exposure of the embryos shows

death of the embryos. This stage embryo shows more stable than the 1cell and blastula stage

embryos.

Organogenesis stage

The complete result of treated organogenesis stage (21hr PF) embryo with different time

interval and concentration was shown in Figure 5.


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Fig. 5: Effect on the Organogenesis stage embryos of ethanol and chloroform extract

along with control. (A) Represent the Survival rate with time; this indicates that

survival of the embryo decreases only at prolonged time intervals. (B) Represent the

Survival rate along with Concentration; this indicates that survival of the embryo not

shows much effect with Concentration. The values were presented as mean ± SEM and

were assessed using ANOVA. (C) Represent the image of early Organogenesis (21hpf)

and late Organogenesis (30hpf) stage embryo without plant extract (above) and dead

embryo of ethanol (26hpf) and chloroform extract (25hrpf) (below).

At this stage the long exposure of the plant extracts shows the death of the embryos. This

stage embryo develops more resistance than the 1hr, blastulaand gastrula stage embryos on

the plant extracts.

One way - ANOVA analysis

The one way ANOVA analysis is done by considering both time and concentration at a time

for different stages with different extracts. This data was subjected to a one way analysis and

the detailed results are shown in Figure 6.


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Fig. 6: (A) Represent the result for the P- level of all stages with two extracts. All P-

values are lesser than the table value, thus it shows the significance (p<0.05). (B) The P-

value decreased at the blastula stage and increased at gastrula and organogenesis stage.

The ethanol extract shows less significance than the chloroform extract at the gastrula

and organogenesis stage. The ethanol extract on blastula stage shows maximum

significant effect P (F≥0.8753) = 0.05.

The one way ANOVA analysis was carried out for every stage with different extract, the P-

value was compared with the table value. All the stages shows Significance (effect P≥0.05),

this indicates that both the extracts shows acceptable results. Among these ethanol extract on

blastula stage shows more acceptable result P (F≥0.8753) = 0.05.

Analysis of All stages

The compared analysis of all stages with time and concentration are shown in the figure 7.


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Fig. 7: (A) Represent the effect of time on different stages of embryos. (B) Showing the effect of concentration on different stages of embryos. In both cases the organogenesis stage embryo shows more tolerance to the extracts. The comparison studies were carried out between the different stages with time and

concentration. This study indicates that the development of embryo will increase the

resistance against the extract, thus organogenesis stage embryo has more resistance than the

gastrula stage, blastula stage and 1cell stage.Irrespective of the stages the long lime exposure

of the embryos shows death.

CONCLUSION

By using the Danio rerio embryo the abortifacient effects can be studied with different

parameters like time of exposure, concentration and stages. This experiment shows the better

efficacy in the blastula stage and this helps in designing herbal abortifacient drug. Thus the

Danio rerio is best model organism for studying abortifacient activity than the other higher

organisms like mice, rat, rabbit, etc.

ACKNOWLEDGEMENTS

The authors would grateful to the Mrs. Deepa, Department of Statistics and teaching staff,

Department of Biotechnology, Government Science College Bangalore, for there valuable

statistical assistance and there guidance.

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danio rerio embryo as a model for study abortifacient effects using ananas comosuss

Health & Medicine

significant abortifacient

abortifacient compounds

abortifacient effects4

danio rerio embryos

different model organisms

best model organisms

ananas comosus extract

abstract danio rerio