Teratogenic effect of Ethanolic extract of Solanum xanthocarpum berries in Zebrafish embryo

 

Sabarinath C¹*, Nandhu T¹, Sudhakar P¹, Gayathiri N.M¹, Shanmuganath C²

¹Department of Pharmacology, Swamy Vivekanandha College of Pharmacy,

Tiruchengode – 637205, Namakkal DT, Tamilnadu, India.

²Veterinary Assistant Surgeon, Tamilnadu Animal Husbandry Department,

Kovilpatti – 628502, Thoothukudi DT, Tamilnadu, India.

 

ABSTRACT:

Zebrafish model produce hundreds of offspring at weekly intervals providing scientists with an ample supply of embryos to study. As zebrafish eggs are fertilised and develop outside the mother’s body it is an ideal model organism for studying early development. In our present investigation we carried out the teratogenic effect of ethanolic extract of Solanum xanthocarpum berries in zebrafish embryo. Teratology is the study of abnormal development in embryo and causes congenital malformation or birth defects. Solanum xanthocarpum berries were extracted and prepared the five different concentrations of the extract 10%, 5%, 1%, 0.5%, and 0.1% respectively. The embryo-toxic effects, hatchability, and morphological abnormalities of extract treated embryos were determined. Zebrafish embryos exposed to different concentrations of Solanum xanthocorpum extract was proven to have embryotoxic (lethal) and teratogenic effects.

 

 

 

INTRODUCTION:

The medicinal plants consider as a rich resource of ingredients which can be used in drug development and synthesis. Medicinal plants have a promising future because there are about half million plants around the world, and most of them their medical activities have not investigate yet, and their medical activities could be decisive in the treatment of present or future studies. Medicinal plants are major source of new drugs.

 

Medicine poisonings associated with certain species of Solanum are not uncommon and may be fatal. However, several species are locally used in folk medicine, particularly by native peoples who have long employed them. Based on the teratogenicity of Solanum species should be due to their alkaloidal content, particularly the glycosidal alkaloids.

 

Several Solanum species are teratogenic to several lab animals, the active agent appears to be solanidane and spirosalane steroidal alkaloids, as there glycosides⁽²⁾. The presence of saponins in the drug may promote gastrointestinal absorption of alkaloids.

 

Studies designed to test the teratogenic potential of environmental agents use animal model systems. Early teratologists exposed pregnant animals to environmental agents and observed the fetuses for gross visceral and skeletal abnormalities. While this is still part of the teratological evaluation procedures today, the field of teratology is moving to a more molecular level, seeking the mechanism (s) of action by which these agents act. In addition, pregnancy registries are large, prospective studies that monitor exposures women receive during their pregnancies and record the outcome of their births. These studies provide information about possible risks of medications or other exposures in human pregnancies. Understanding how a teratogen causes its effect is not only important in preventing congenital abnormalities but also has the potential for developing new therapeutic drugs safe for use with pregnant women⁽³⁾.

 

Danio rerio (zebrafish) are seen as the latest ‘model’ for embryological development studies. These embryos have great advantage that they develop as “see through” embryos. That is all internal development can be clearly observed from the outside in the living embryo. Genetic similarity with humans, eases to house and care, than rodents, impact of genetic mutation with drug treatment is easy to seen (transparent), lots of offspring, ease to introduce genetic change, easily increase value to in genetic and developmental biological research and can test number of toxicities⁽⁴⁾.

 

According to the literature review, there is a paucity of scientific data for anti-inflammatory activity of the whole plant. The present research work, therefore, was initiated to investigate the teratogenicity activity of ethanolic extract of S.xanthocarpum berries in zebrafish. Despite the enormous medicinal properties of Solanum xanthocarpum, its teratogenic effect is not yet well established in zebrafish.

 

MATERIALS AND METHODS:

ANIMAL MODEL:

The zebrafish as a ‘model’- past and present use of zebrafish in research begins to increase as the field of molecular biology progressed in recent years⁽⁵⁾.

 

Egg Quality:

Obtaining good quality eggs from animals is important, not only for helping to realise research objectives, but also to keep number of animals used to a minimum. For most of the academic areas in which zebra fish eggs are used, it is particularly important that higher percentage of eggs are fertilised successfully, that the eggs undergo a clean and even first cleavage, and that they remain normally developed at gestation. Healthy eggs have a translucent, yellowish appearance. Numerous husbandry parameters are often manipulated in an attempt to improve egg quality. Such factors include diet, lightning, water salinity, water flow, frequency of cleaning, tank size, type of hormonal stimulations, frequency of egg collections, and age of females.

 

By natural mating, zebrafish are normally kept under laboratory conditions to replicate perpetual summer. Depending upon food availability and temperature they can breed all year round with females generally producing eggs once every 1 to 3 days. Darkness allows the fish to rest and the return of light will trigger the fish to breed. Females consistently spawn more frequently and produce larger clutches of eggs with some males than others. However, this effect does not appear to be related to male dominance rank. A good clutch consists of between 70 and 300 eggs, of which at least 80% are fertilised. It has been reported that keeping females together for four days prior to being separated and mated with a single male significantly supresses the number of eggs produced.

 

Frequency of egg collection:

Though zebrafish females are capable of spawning on a near daily basis, a female which lay eggs daily is very unlikely to produce a good quantity or quality of eggs. The full impact on the fish of maintaining such a rigorous egg production schedule for more than two to three weeks has yet to be evaluated. It is likely that frequency at which eggs of good quality can be collected form females, and the impact of this processes on this animal, is heavily influenced by parameters such as water quality and nutrients.

 

Spawning of Mature Zebrafish:

A glass aquarium filled with untreated, clean tap water with continuous supply of oxygen was provided to house adult male and female zebra fishes in 1:2 ratio. To investigate spawning of zebra fishes, the glass aquarium was covered with a black plastic sheet (dark condition) for 12 hours. Once spawning was finished, the eggs were subjected to a fluorescent bulb (lighted condition) for 12 hours as well. Fertilization of eggs takes place within 20 minutes after the introduction of light. The fertilized eggs were siphoned out of the aquarium after 12 hours of fertilization using a hose. The embryos were then rinsed three times and placed in a clean watch glass containing embryo medium for microscopic observations⁽⁶⁾.

 

Preparation of embryo water:

The preparation of the embryo water was done following the formulation of Hank’s solution for teratogenic assays. The pH of the solution was checked using the p^H meter⁽⁷⁾.

 

Extraction:

The crushed material of berries of Solanum xanthocapum was subjected to hot continuous percolation extraction by Soxhlet apparatus. The extracted material weighed about 30gms⁽⁸⁾.

 

Teratogenicity and Embryo toxicity of Solanum xanthocarpum berries Extract:

Three ml of each treatment concentration of Solanum xanthocarpum extract prepared using embryo water as diluent (10%, 5%, 1%, 0.5%, and 0.1%) and control (embryo water) were placed into each well of the 12-well ELISA plate. Six embryos at segmentation phase were transferred into each well containing the different treatments. The plates were incubated at 26°C±1°C. Teratogenic activity was examined under 40X magnification using a compound microscope after 12, 24, 36 and 48h of incubation. Morphological endpoint evaluation of zebra fish was based on the parameters established by lethal (coagulation, tail not detached, no somites, and no heart-beat), teratogenic (malformation of head, tail and heart, scoliosis, deformity of yolk, and growth retardation), and normal. Hatchability, malformation and mortality rates were recorded, and death was defined as coagulated embryos⁽⁹⁾. All tests were repeated three times and conducted in accord with national guidelines for animal welfare.

 

Data and evaluation of mortality and hatchability rates of zebrafish:

The data recorded were used to

(a) Assess gross morphological endpoints of zebrafishes;

(b) Determine Percent mortality (characterized by the number of coagulated zebrafish embryos after 12, 24, 36 and 48 hours post-treatment application (HPTA) computed using the formula:

 

                                No. of dead embryos

% Mortality =    –––––––––––––––––––––     × 100

                              Initial No. of embryos

 

(c)  Percent hatchability (characterized by hatched zebrafish embryo after 48 HPTA)

 

Computed using the formula:

                                  No. of hatched embryos

% Hatchability =    –––––––––––––––––––––    × 100

                                   Initial No. of embryos

 

Statistical Analysis:

To check which treatments are significantly different from each other, the data were subjected to One-Way Analysis of Variance, Duncan Multiple Range Test and Tukey’s Test⁽¹⁰⁾.

 

RESULTS AND DISCUSSION:

In this study, the spawning and fertilization were successful with approximately 95% rate of fertilization. The successful embryonic development was observed in three distinct periods, namely segmentation phase (12 hours post fertilization), pharyngula period (24-36 hours post fertilization) and hatching period (48-72 hours post fertilization). The effects of the different concentrations of Solanum xanthocarpum berries extract on the developmental processes of the embryos were evaluated.

 

Embryo-toxic Effect:

Mortality was defined as coagulation of embryos. The percentage mortality of D. Rerio embryos after 12, 24, 36, and 48 hours of exposure in varying concentrations of Solanum xanthocarpum berries extract are shown in Table 1.

 

Table 1: Effect of ethanolic extract of Solanum xanthocarpum on Mortality of zebrafish

The embryo-toxic effects of the extracts were found dependent on dose and time of exposure. The results of the present study indicate that S. xanthocarpum berries extract higher concentration is more toxic and producing embryo-toxic effect.

 

Hatchability of Zebrafish Embryo:

Hatching is an indicative of the successful developmental processes of the embryos. The percentage hatchability of embryos treated with the different concentrations of S. xanthocarpum extract at 48 HPTA is depicted in Table 2.

 

Table 2: Effect of ethanolic extract of Solanum xanthocarpum on Hatchability of zebrafish

S. No.	Extract	Concentration (%)	Hatchability (%)
1.	Berries of Solanum xanthocarpum	10.00	0.00
		5.00	44.44
		1.00	55.56
		0.50	77.78
		0.10	100.00

 

Hatching was completed at 48 hours post treatment exposure in control embryos, 0.10% of concentration extract-treated embryos. However, hatching of some embryos at 0.5% to 5% of extract was also observed. No hatched embryo was recorded at 10% concentration extract of S. xanthocarpum. Apparently, hatching of embryos was affected by the varying concentrations of the different extracts: as the extract concentration increased the percent hatchability decreased. This delayed hatching process strongly dictates growth retardation and possibly explained by the morphological abnormalities observed in embryos that limit hatching.

 

The varying concentrations of extract of Solanum xanthocorpum affected the hatchability of embryos: as the extract concentration increased the percent hatchability decreased. The low hatchability could be attributed to the delayed development of embryos as one of the important sublethal effects of the extract.

 

Teratogenic activity of S. xanthocarpum:

Morphological abnormalities or malformations are the most important teratogenic parameters in D. Rerio teratogenicity assay. Apparently, embryos exposed at 5% and some embryos at 1% of both extracts showed different malformations (Figure 1). Larvae at 5% of extract showed twisted tail and body whereas the extract at 1% caused bent tail tip and hook-like tail, respectively. These malformations of larvae could be associated as effects of the delayed development of embryos at these two concentrations. Embryos at 10% extract concentration coagulated after 12 hours. All embryos at 0.1% extract concentration and at embryo water were found normal. In this study, morphological malformations such as perverted and hook-like tail were the most common teratogenic effect of Solanum xanthocorpum extract. In the present work, the delayed development and morphological abnormalities were the most distinct teratogenic effects of Solanum xanthocorpum extracts in zebrafish. The failure of organogenesis leading to the underdevelopment of the head and tail morphology was also noticed in the treated embryos. This might be attributed to the inhibition or disturbance of essential substances for growth and developmental processes of embryos.

 

Figure 1: Effect of ethanolic extract of Solanum xanthocarpum on morphological characters of zebrafish

 

CONCLUSION:

This study has been used in an introductory toxicology course, allowing gaining hands-on experience with zebrafish as a toxicological model.

 

Zebrafish embryos exposed to different concentrations of Solanum xanthocorpum extract was proven to have embryotoxic (lethal) and teratogenic effects. It was observed that the concentrations of Solanum xanthocorpum extract 10% concentration caused lethality to zebrafish embryos after 12 HPTA. On the other hand, at concentrations ranging from 1% to 5% resulted to abnormalities to the growth of zebrafish embryos, however, prolonged exposure of the embryos also led to death.

 

Altogether, it can be concluded that Solanum xanthocorpum has toxic and teratogenic activities against zebrafish embryo model. The molecular mechanism of its toxic and teratogenic effect and other multifunctional activities of Solanum xanthocorpum should be further investigated in the future studies.

 

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Received on 09.11.2019           Modified on 02.02.2020

Accepted on 24.03.2020         © RJPT All right reserved