Bitter gourd (Momordica charantia) possess developmental toxicity as revealed by screening the seeds and fruit extracts in zebrafish embryos

Abstract

Background: Bitter gourd (Momordica charantia) has attracted the focus of researchers owing to its excellent anti-diabetic action. The beneficial effect of Momordica charantia on heart has been reported by in vitro and in vivo studies. However the developmental toxicity or potential risk of M. charantia on fetus heart development is largely unknown. Hence this study was designed to find out the developmental toxicity of M. charantia using zebrafish (Danio rerio) embryos.

Methods: The crude extracts were prepared from fruit and seeds of M. charantia. The Zebrafish embryos were exposed to serial dilution of each of the crude extract. The biologically active fractions were fractionated by C18 column using high pressure liquid chromatography. Fourier-transform infrared spectroscopy and gas chromatography coupled with mass spectrophotometry was done to identify chemical constituents in fruit and seed extract of M. charantia.

Results: The seed extract of M. charantia was lethal with LD₅₀ values of 50 μg/ml to zebrafish embryos and multiple anomalies were observed in zebrafish embryos at sub-lethal concentration. However, the fruit extract was much safe and exposing the zebrafish embryos even to 200 μg/ml did not result any lethality. The fruit extract induced severe cardiac hypertrophy in treated embryos. The time window treatment showed that M. charantia perturbed the cardiac myoblast specification process in treated zebrafish embryos. The Fourier-transform infrared spectroscopy analyses revealed diverse chemical group in the active fruit fraction and five new type of compounds were identified in the crude seeds extract of M. charantia by gas chromatography and mass spectrophotometry.

Conclusion: The teratogenicity of seeds extract and cardiac toxicity by the fruit extract of M. charantia warned that the supplementation made from the fruit and seeds of M. charantia should be used with much care in pregnant diabetic patients to avoid possible damage to developing fetus.

Keywords: Cardiac hypertrophy; Danio rerio; Developmental toxicity; Momordica charantia.

Fig. 1

Seed extract of Momordica charantia induced multiple teratological defects in developing zebrafish embryos. Representative photomicrograph of live zebrafish embryos at 3dpf control (a) and treated with various concentration of seeds extract of M. charantia (B-F). The highest concentration in which treated embryos could survive was 50 μg/ml and the representative image of treated embryos are presented in b. The embryos treated with 50 μg/ml of seeds extracts were severely developmentally delayed as compared to mock treated embryos as shown in a. The control embryos developed to 72hpf, after two days but treated embryos were at 15 somite stage after two days of exposure. c The zebrafish embryos which were treated with 25 μg/ml of the seeds extract exhibited multiple teratogenic effect. The treated embryos were developmentally delayed as compared to control, had small head, enlarged yolk (yolk), severe cardiac hypertrophy (arrow), enlarged cardiac cavity (arrow head) absence of tail extension (shown in small inset) absence of circulation (shown in Additional file 1 as supplementary data d). The zebrafish embryos treated with 15 μg/ml of seeds extract of M. charantia also had mild level of developmental delay, enlarged cardiac cavity (arrow head). The heart was severely affected as both atrium and ventricle were fused representing as string (black arrow). The treated embryos had curved trunk as shown in small at lower magnification. e. the zebrafish embryos treated with 10 μg/ml of the seeds extract had mild level of developmental delay but the treated embryos ad severe cardiac hypertrophy (arrow) and also enlarged cardiac cavity (arrow head). f). zebrafish embryos treated with 5 μg/ml of the seeds extract did not show development delay however, the cardiac hypertrophy (arrow) was evident in treated embryos. All embryos anterior to the left

Fig. 2

GC-MS analysis of phytochemical compounds in BG seed methanol extract. The highest peak with least retention time was identified as 1,2- CYCLOPENTANEDIONE which is newly identified compound from the seeds of M.charantia

Fig. 3

Crude fruit extract of M. charantia induced cardiac hypertrophy in treated zebrafish embryos. Representative photomicrograph of live zebrafish embryos at 3dpf. a and b Mock (methanol 0.5% V/V) exposed embryos showed normal embryonic development and heart growth (black arrow). c and d the Zebrafish embryos treated with methanol fruit extract of M. charantia (100 μg/ml). The treated embryos are not developmentally delayed as the protruding mouth structure which is prominent feature of zebrafish embryos at 72 hpf stage can also be seen clearly in treated embryos (arrow head). However, the treated embryos exhibited enlarged cardiac chamber (edema) and cardiac hypertrophy (arrow head). B and D are the higher magnification showing the heart of the same embryos presented in images A and C. The red encircled areas shows the areas which has been shown in μm at the bottom of the image. e and f) Zebrafish embryos which were exposed to the fraction 2 (10 μg/ml) obtained from the C18 column fractionation of ethyl acetate fraction of fruit extract of M. charantia. The treated embryos did not show developmental delay but had particular cardiac hypertrophy represented by black arrow. The area of the heart is almost 3X times bigger as compared to mock treated control embryos. Scale bar: 50 μm

Fig. 4

IR fingerprint of the rich-phenolic extract isolated from ethyl acetate fraction of M. charantia fruit extract

Fig. 5

The fruit extract of M .Charantia effected the heart formation not the heart growth in treated zebrafish embryos. Representative live photomicrograph of zebrafish embryos at 72 hpf. a: Untreated control embryos at 72 hpf showing the normal formation and development of heart (red dots area represented by arrow). b: Zebrafish embryos which were treated with fruit extract of M. charantia at one cells stage exhibited the cardiac hypertrophy (red dots area and black arrow). c: The mock (methanol 0.5% v/v) treated embryos which were treated with solvent at one cell stage had normally heart development. d: Zebrafish embryos which were exposed to fruit extract of M. charantia at 48 hpf also had normal heart formation and development (red dot area). Abbreviation hpf: hours post fertilization. Scale bar 50 μm

Fig. 6

The co exposure of fruit and seeds extracts of M. charantia resulted lethality and severe cardiac toxicity in zebrafish embryos. Representative photomicrograph showing live zebrafish embryos at 3dpf control or treated with fruit, seeds or combination of seeds and fruit extract of M. charantia. a The mock (methanol 0.5%V/V) exposed embryos did develop normally and had normal heart growth and development. b The zebrafish embryos treated with fruit extract (100 μg/ml) had severe cardiac hypertrophy, c the embryos treated with 15 μg/ml of seeds extract had cardiac hypertrophy and also enlarged cardiac cavity. d The embryos which were treated with combination of both extract showed severe effect on heart formation as fused ventricle and atrium represented as string and also enlarged heart cavity