Asian Journal of Biochemical and Pharmaceutical Research Embryo-Toxic and Teratogenic Effects of Banaba (Lagerstroemia speciosa L. Pers) Leaves Water Extract to the Zebrafish (Danio rerio) Embryo

Asian Journal of Biochemical and Pharmaceutical Research Issue 1(Vol. 8) 2018 ISSN: 2231-2560 10.24214/AJBPR/8/1/7886 CODEN (USA): AJBPAD Research Article

Asian Journal of Biochemical and Pharmaceutical Research Embryo-Toxic and Teratogenic Effects of Banaba (Lagerstroemia speciosa L. Pers) Leaves Water Extract to the Zebrafish (Danio rerio) Embryo Kevin Smith P. Cabuhat*, Lerma C. Batac, Princess May R. Mendoza, Alfredo P. Carpio Jr. Oliver R. Alaijos and Raymundo F. Javier Department of Biology, College of Science, Bulacan State University, City of Malolos, Bulacan, Philippines

Received: 18 March 2018; Revised: 29 March 2018; Accepted: 10 April 2018

Abstract: This work presents the embryo-toxic and teratogenic effects of Lagerstroemia speciosa

leaves water extract in Danio rerio embryos. Apparently, as early as 12 hours post treatment application (hpta), 100% mortality of embryos was observed in 3% and higher concentrations while mortality in lower concentrations increased as time of exposure prolongs. The effect was found to be as time and dose dependent. Coagulation was the toxic effect of the extract. On the other hand, heartbeat rate and hatchability rate of the zebrafish embryo in 0.05%, 0.1% and 0.5% decreases as treatment concentration increases. However, no hatchability and heartbeat perceived in 1% and higher concentrations due to early coagulation. Growth retardation was the most marked teratogenic effect. Moreover, head malformation, hook-tail, s-shaped tail, scoliosis, limited movement, stunted tail, bent tail, yolk deformity, light pigmentation, pericardial edema, abdominal edema and yolk edema were observed. Altogether, L. speciosa leaves contain bioactive components that exhibit toxic and teratogenic effects to the embryo of D. rerio. Keywords: Banaba, Danio rerio, Embryo-toxic, Lagerstroemia speciosa, Teratogenic, Zebrafish be associated by its active phytochemicals such as glycosides, sugars, tannins, proteins, steroids, anthraquinone glycosides, flavonoids and saponins [5].

INTRODUCTION: Plants are important to humankind. They are rich in bioactive phytochemicals and nutrients that play an important role in the physiology of human body systems [1]. However, in spite of its biological activities, some of those plants could pose adverse effects to living beings including human [2].

Despite of the profound medicinal advantages of L. speciosa, the embryo-toxic and teratogenic effects are not yet well established in detail. Toxicity is defined as the degree to which a substance can harm humans or animals [6]. On the other hand, teratogenicity is the occurrence of adverse effects on the developing organism caused by teratogens [7]. However, many anticancer drugs are teratogenic and teratogens can be used as anticancer drugs [8].

Lagerstroemia speciosa (L.) Pers., commonly known as Banaba, is a semideciduous belonging to family Lythraceae [3]. The dried leaves have been extensively studied for different bioactivities particularly its potency in curing diabetes and kidney diseases [4]. These functional activities could 78

Asian Journal of Biochemical and Pharmaceutical Research Issue 1(Vol. 8) 2018 ISSN: 2231-2560 CODEN (USA): AJBPAD

maintained at 26oC ± 1oC condition. The fishes were fed twice a day using dry flakes. To ensure the standard quality of water, excess feeds were released out of the aquarium [11].

Today, assessment of the toxic and teratogenic effects of any substances can be accomplished using Zebrafish Embryo Toxicity (ZET) test. It is considered as an attractive, cost-effective and scientifically sound model for assessing and understanding the teratogenic mechanisms of compounds from natural sources [9]. Due to its transparency, fast development of its embryo, high fecundity, embryonic membrane permeability, external fertilization and similarities to mammalian molecular mechanisms of developmental and cellular physiology [10], zebrafish suggested as a reliable model for teratology and toxicology.

Preparation of Embryo Medium : Following the latest protocol described by Thomas [12], embryo medium was prepared by combining 1 mL of Hank’s solution 1 (prepared by combining 8 g of Sodium chloride [NaCl], 0.4 g of Potassium chloride[KCl] and 100 mL of double distilled water); 0.1 mL of Hank’s solution 2 (prepared by adding 0.67 g of Sodium biphosphate heptahydrate [Na2HPO4 • 7 H2O] and 0.60 g of Potassium dihydrogen phosphate [KH2PO4] and 100 mL of double distilled water); 1 mL of Hank’s solution 4 (prepared by a combination of 1.92 g of Calcium chloride dehydrate [CaCl2 • 2 H2O] and 100 mL of double distilled water); 96.5 mL of double distilled water; 1 mL of Hank’s solution 5 (prepared using 2.46 g of Magnesium sulfate heptahydrate [MgSO4 • 7 H2O] and 100 mL of double distilled water); 0.35 mL of Hank’s solution 6 (prepared 0.35 g of Sodium bicarbonate [NaHCO3] and 10 mL of double distilled water). 1 M of Sodium Hydroxide [NaOH] was gradually added to the solution until a working solution was brought to the pH of 7.2. The pH of the solution was checked using the pH meter.

With the general intention establishing the biosafety and bio-potentialities of L. speciosa, the potential embryo-toxic and possible teratogenic effects of the plant were assessed using the embryo of D. rerio which could lead to the discovery of other promising bioactivities of this plant. MATERIALS AND METHODS: Research Specimen: The zebrafish were obtained from Cartimar, Pasay City, Philippines with the number of 10 female and 20 male. Banaba leaves sample was collected from Santol Balagtas, Bulacan, Philippines. The collected plant was submitted to the Botany Division of the National Museum of the Philippines for verification and authentication. The remaining leaves were washed and air-dried for 10 days. After which, the leaves were milled and prepared for hot water extraction process.

Preparation of Treatment Concentrations: The extract of Banaba (L. speciosa) leaves was obtained following the hot water extraction protocol of Eguchi et al., [13]. The powdered leaves sample (30g) was extracted in 300 mL hot water at 80-90oC in a water bath for 2 hours. Afterwards, the extract was filtered using Whatman Filter Paper No. 2. Ten mL of different concentrations (10%, 5%, 3%, 1%, 0.5%,

Acclimatization and Maintenance of D. rerio: Ten female and 20 male zebrafish were acclimatized in the aquarium with untreated tap water saturated with oxygen within one week prior to spawning. The set-up was 79

Asian Journal of Biochemical and Pharmaceutical Research Issue 1(Vol. 8) 2018 ISSN: 2231-2560 CODEN (USA): AJBPAD

0.1%, 0.05% and control) were prepared by diluting in embryo medium. Treatments were refrigerated prior the assay.

endpoint evaluation of zebrafish was based on parameters established by Nagel [16]: lethal (coagulation, tail not detached, no somites and no heart-beat), teratogenic (malformation of head and tail, light pigmentation, scoliosis, growth retardation, stunted tail, and limited movement) and normal. The images projected from the microscope were captured using an android phone with 8 megapixels.

Spawning of Zebrafish and Harvesting of Embryo: Following the procedure described by Jose et al., [14], with minor modification, 10 female and 20 male zebrafish were localized in an aquarium containing 30 L of dechlorinated tap water. Plastic mesh was used to prevent the released eggs from cannibalism. To induce spawning, the aquarium was covered by a black trash bag for 12 hours. Then, after 12 hours, the trash bag was uncovered and let the eggs be exposed in light condition to allow fertilization. Fertilization occurs 30 minutes after the light was turned on. Embryos were siphoned out using a hose and were transferred in a beaker. The collected embryos were rinsed thrice using distilled water and were transferred into the petri dish then check the uniformity and normal conditions of embryos using compound microscope. Deformed and unfertilized eggs were discarded while uniformed eggs were used in the assay.

Data Analysis: Using SPSS Program (Version 17.0), data were gathered and analyzed using One-Way ANOVA (Analysis of Variance) followed by a post-test, Duncan’s Multiple Range Test (DMRT). Difference between means was considered valid at 5% level of significance (P