Research Article |
Corresponding author: Warsi Warsi ( warsi@pharm.uad.ac.id ) Corresponding author: Nurkhasanah Mahfudh ( nurkhasanah@pharm.uad.ac.id ) Academic editor: Paraskev Nedialkov
© 2024 Warsi Warsi, Irwandi Jaswir, Qamar Uddin Ahmed, Nurkhasanah Mahfudh, Mohamed Sufian bin Mohd. Nawi, Abdul Rohman, Alfi Khatib.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Warsi W, Jaswir I, Ahmed QU, Mahfudh N, bin Mohd. Nawi MS, Rohman A, Khatib A (2024) Morphological, teratogenic and behavioral evaluations of Gelidium spinosum methanol extract on zebrafish embryos. Pharmacia 71: 1-10. https://doi.org/10.3897/pharmacia.71.e109918
|
Gelidium spinosum is an edible red seaweed from the family Gelidiaceae with possibility to be developed. The potential medical benefits of G. spinosum have been established, yet adequate empirical research on its toxicity is still lacking. Hence, the present work was aimed to examine the toxicity of G. spinosum methanol extract (GsME) on zebrafish (Danio rerio) embryos and to identify the phytoconstituents using gas chromatography-mass spectrometry (GC-MS) analysis. Results of this study showed that GsME induced morphological defects in zebrafish embryos, including reduction in eye size and body length. Moreover, yolk sac size and mortality were increased in zebrafish embryos exposed to GsME in a dose-dependent manner. GsME at high concentrations triggered teratogenic effects in zebrafish embryos such as decrease in heartbeat/minute, lack of pigmentation, lack of somite, structural deformity, pericardial oedema, and yolk oedema. The LC50 and EC50 of GsME were < 100 mg/L, which classified as a harmful category. The teratogenic index of GsME was found to be > 1, indicating its teratogenic attribute. Additionally, GsME exerted behavioral effects i.e. significantly lower total distance of movement and slower swimming speed of zebrafish embryos. GC-MS analysis of GsME was confirmed the presence of amino acid, phenolics, carboxylic acids, reducing sugars, saturated fatty acids and brominated saturated fatty acid in the extract. It suggesting that compounds of 13-bromotetradecanoic acid, palmitic acid, and stearic acid containing in GsME were contributed to the toxic effects.
Behavior, Gelidium spinosum, GC-MS, Red seaweed, Toxicity
Gelidium sp. is frequently recognized as red seaweed from division Rhodophyta (
The zebrafish (Danio rerio) model is widely used in preliminary toxicology evaluations to verify teratogenic potential of both pharmaceutical products (
The purpose of the current study was to assess the acute toxicity of G. spinosum methanol extract (GsME), by focusing on the morphological, teratogenic and behavioral effects on zebrafish embryos. Besides, this work also aimed to identify the phytoconstituents in GsME using GC-MS analysis.
Red seaweed (G. spinosum) was collected in January 2020 from Drini beach, Gunungkidul, Special Region of Yogyakarta, Indonesia. The sample was authenticated by Dr. Abdul Razaq Chasani (Ph.D.), Plant Systematics Laboratory, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia (document number 014893/S.Tb./IX/2020).
Red seaweed was initially cleaned with distilled water and then dried at 28 ± 1 °C. The dried red seaweed was ground using a simplicia grinder. The resultant dried powder (25.0 g) was extracted with 150 mL of methanol (1:6 w/v) and stirred for 8 h at 800 rpm and then macerated at room temperature for 1 day. The crude extract was filtered using Whatman filter paper no. 1. Residue was again macerated with methanol (1:6 w/v) then filtered. The supernatant was concentrated using an evaporator at 65 ± 5 °C and then freeze-dried using a freeze dryer to dry the extract. The freeze dried red seaweed extract was maintained at -78 ± 2 °C for further experiments (
Albino adult zebrafish were cared for at The Central Research and Animal Facility of International Islamic University Malaysia, Kuantan, Malaysia (authorized ethical registration number #IIUM/504/14/2/IACUC). The zebrafish were kept in an aquarium with a 12–16 h photoperiod and a 1 L water loading capacity per fish. Artemia dry granules of food were given three times each day. All the zebrafish were not given any medication for two months prior to spawning. The method used for spawning zebrafish corresponded with guidelines of standard literature (
Zebrafish embryos were processed with the sample in accordance with standard procedures (
The morphological observations of zebrafish embryos were conducted at 1, 2, 3 and 4 dpf. Lethal concentration (LC50) was determined using parameter endpoints of mortality (Equation 1), while effective concentration (EC50) was calculated using endpoints of mortality and malformation (Equation 2) in accordance with The Organisation for Economic Co-operation and Development (OECD) guideline (
(1)
(2)
Behavioral analysis was carried out following procedures previously reported by some researchers (
Phytoconstituents of G. spinosum methanol extract (GsME) were identified based on Gas Chromatography-Mass Spectrometry (GC-MS) profile by following a technique reported by
The samples were analyzed by the gas chromatography mass spectrometry-electron ionization (GCMS-EI) system which was designed up of an Agilent 6890 GC-MS (Agilent Technologies, Santa Clara, CA, USA) and a HP 5973 selective mass detector (Agilent Technologies). A DB-5MS 5% phenyl methyl siloxane column with diameter of 250 μm, a film thickness of 0.25 μm and length of 30.0 m was chosen for the analysis. A 2 μL of the sample filtrate was injected into the GC-MS machine in splitless mode with helium supplied at a speed of 0.8 mL/min. The oven’s first temperature was 85 °C. The starting temperature was consistently maintained to the intended temperature of 315 °C at a rate of 2 °C per minute, and then stabilized for 5 minutes. The temperatures of ion source and injector were established at 200 and 250 °C, respectively. The solvent barrier was set to 6 minute. Data were obtained in a mass scan ranging from 50 to 550 m/z, correlation between % abundance and retention time (minute). ACD/Spec Manager v. 12.00 (Advanced Chemistry Development, Inc., ACD/Labs Ontario, Toronto, ON, Canada) was used for converting the GC-MS raw data to the CDF file. The spectra were further analyzed using the Windows tool MZmine 2.53 (Institute of Organic Chemistry and Biochemistry, Prague, CZE). The National Institute of Standards and Technology’s (NIST) 14 dataset library was used to compare the spectra for each peak of GC-MS, as well as compound name, m/z, retention time (RT), percentage of area, and similarity index (SI) of common primary and secondary metabolites. The SI for each of selected compounds was approximately ≥ 90.
All data were analyzed statistically using the IBM SPSS 23 (IBM Corporation, New Orchard Road Armonk, United States) statistics software package for Windows 10. Kolmogorov-Smirnov was used to verify normal distribution of the data, whereas Levene’s test was used to confirm homogeneity of the data. The analysis then was followed by One-way ANOVA and Post-Hoc Tukey HSD tests to determine significant differences within the data groups. Body length, eye size, yolk size, total distance moved, and total swimming speed were parameters used to generate the data.
A toxicity examination is necessary to determine the potential risk from consumption of G. spinosum extract by human. The zebrafish as an animal model is assessed at different stages of its life: embryonic, larval and adult (
Mortality rate (%) in zebrafish embryos exposed to GsME is displayed in Fig.
The dosage required to kill 50% or cause malformation and mortality in 50% of the investigated zebrafish embryos was used to determine the lethal concentration (LC50) and effective concentration (EC50), respectively. The linear regression curve relating log concentration and mortality rate (%) and relationship between log concentration and percentage malformation (%) were used to calculate the LC50 and EC50) values, respectively (Fig.
The results of this research show that LC50 and EC50 values of GsME in zebrafish embryos were < 100 mg/L (Table
Timepoints (Day) | LC50 (mg/L) | EC50 (mg/L) | TI (LC50/ EC50) |
---|---|---|---|
1 dpf | 76.20 | 61.49 | 1.24 |
2 dpf | 50.22 | 32.83 | 1.53 |
3 dpf | 37.82 | 25.10 | 1.51 |
4 dpf | 28.49 | 16.53 | 1.72 |
Zebrafish embryo LC50 and EC50 values were dosage and time-dependent manner. Longer exposures were associated with lower LC50 and EC50 values (
The heart rate of zebrafish embryo was recorded at 3 dpf (Fig.
Body length is a morphological parameter that is used as an indicator to assess for malfunction in zebrafish embryos toxicity tests. Body length was used to determine the effect of GsME on the growth of the zebrafish embryos. It was measured at 5 dpf. The body length measurements of zebrafish embryos treated with GsME is presented in Table
Extract dosage (mg/L) | Hatching rate (%) | Eye size ×104 (µm²) | Body length (mm) | Yolk size ×105 (µm²) |
---|---|---|---|---|
250 | 0 | NA | NA | NA |
125 | 10 | NA | NA | 6.85 ± 0.03a |
62.5 | 25 | NA | NA | 6.03 ± 0.05b |
30 | 100 | 7.88 ± 0.06a | 2.29 ± 0.06a | 5.66 ± 0.03c |
15 | 100 | 9.15 ± 0.10b | 2.37 ± 0.05b | NA |
5 | 100 | 10.19 ± 0.13c | 2.42 ± 0.05b | 5.16 ± 0.02d |
SC | 100 | 10.74 ± 0.06d | 2.75 ± 0.05c | 4.99 ± 0.03e |
NC | 100 | 10.67 ± 0.05d | 2.76 ± 0.05c | 5.01 ± 0.02e |
PC | 10 | NA | NA | NA |
Zebrafish embryos have the capacity to hatch while they are still in the early stages of development between 2 and 3 dpf. Following exposure to GsME at concentrations between 5 and 30 mg/L, all zebrafish embryos were able to hatch. This was similar to the normal control and solvent control at 3 dpf. Meanwhile, a hatching defect was discovered in embryos treated with GsME at doses ranging from 62.6 to 125 mg/L. Because 100% of the embryos were coagulated at 1 dpf, the hatching rate of zebrafish embryos could not be measured at maximum concentration (250 mg/L).
Another appropriate endpoint indicator for assessing a compound’s toxicity is the size of the eyes in zebrafish embryos. This is due to the fact that zebrafish eyes and human eyes have several similarities, such as the predominance of cylinders in zebrafish vision and the way that zebrafish cells absorb light (
Yolk sac could be used to identify organ malfunction in zebrafish. The yolk sac provides the zebrafish embryo’s main food supply for the first week of development. Zebrafish eggs have yolk sacs that are 70% neutral lipids, mainly liver-metabolized triacylglycerol and cholesterol ester. Thus, hepatic dysfunction causes a delay in the digestion and absorption of yolks as well as an increased fat deposition (
The embryo tail-bud usually begins to detach at 1 dpf, and a non-detaching tail indicates the embryo is dead (
Extract dosage (mg/L) | Teratogenic parameters | |||||
---|---|---|---|---|---|---|
LP | LS | SD | PO | UH | YO | |
250 | NA | NA | NA | NA | NA | NA |
125 | + | + | + | + | + | + |
62.5 | + | + | + | + | + | + |
30 | - | - | + | + | - | + |
15 | - | - | + | - | - | - |
5 | - | - | - | - | - | - |
NC | - | - | - | - | - | - |
SC | - | - | - | - | - | - |
PC | NA | NA | + | + | + | + |
At 4 dpf, embryos exposed to GsME at concentrations of 62.5 and 125 mg/L had abnormal spines as well as reduced pigmentation (Fig.
Several variables can be used in behavior analysis, such as spontaneous movement, which starts at 17 hours post fertilization (hpf), touch-evoked tail coiling which starts at 21 hpf, and swimming which starts at 27 hpf (
Extract doses (mg/L) | Total distance moved × 103 (mm) | Total swimming speed × 104 (mm/s) | ||
---|---|---|---|---|
Light | Dark | Light | Dark | |
30 | 2.37 + 0.05a | 2.81 + 0.06a | 7.92 + 0.07a | 8.19 + 0.13a |
15 | 2.65 + 0.05b | 3.22 + 0.06b | 9.41 + 0.09b | 9.86 + 0.09b |
5 | 3.09 + 0.05c | 3.45 + 0.05c | 10.27 + 0.11c | 10.52 + 0.10c |
2.5 | 3.39 + 0.05d | 4.00 + 0.03d | 10.83 + 0.09d | 11.16 + 0.13d |
SC | 3.63 + 0.04e | 4.44 + 0.06e | 11.29 + 1.85e | 11.94 + 0.10e |
NC | 3.62 + 0.04e | 4.45 + 0.05e | 11.21 + 1.41e | 11.90 + 0.14e |
Identified metabolites in derivatized GsME of GC-MS analysis are presented in Fig.
Peak No | Compounds | m/z | RT (min) | Area (%) | SI |
---|---|---|---|---|---|
1 | 1-Isoleucine | 86.05 | 5.06 | 0.77 | 90 |
2 | Butanedioic acid (Succinic acid) | 73.05 | 6.81 | 1.91 | 95 |
3 | D-Arabinonic acid | 73.05 | 8.68 | 1.33 | 91 |
4 | Protocatechuic acid | 193.00 | 11.11 | 0.53 | 91 |
5 | 1,2,3-Propanetricarboxylic acid (Citric acid) | 73.05 | 11.39 | 8.63 | 90 |
6 | D-(-)-Fructose | 73.05 | 12.19 | 4.02 | 94 |
7 | D-Psicose (D-Allulose, Pseudofructose) | 73.05 | 12.32 | 3.32 | 93 |
8 | D-(+)-Talose (D-Talopyranoside) | 73.05 | 12.62 | 5.97 | 93 |
9 | D-Galactose | 73.05 | 12.91 | 1.53 | 91 |
10 | Hexadecanoic acid (Palmitic acid) | 117.05 | 14.57 | 7.84 | 94 |
11 | Myo-inositol (Vitamin B8) | 73.05 | 15.78 | 1.04 | 93 |
12 | Ribonic acid | 73.05 | 17.40 | 1.17 | 94 |
13 | Glyceryl glycoside | 204.05 | 19.62 | 0.79 | 91 |
14 | D-(+)-Galacturonic acid | 73.05 | 20.60 | 0.86 | 91 |
15 | O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside (Sucrose) | 73.05 | 22.48 | 3.14 | 90 |
16 | Octadecanoic acid (Stearic acid) | 73.05 | 23.02 | 1.12 | 94 |
17 | 13-Bromotetradecanoic acid | 227.15 | 23.26 | 0.05 | 90 |
18 | Catechin | 73.05 | 23.97 | 0.70 | 90 |
GsME was contained mainly reducing sugars compounds, including D-arabinonic acid, D-fructose, D-(+)-galacturonic acid, D-galactose, glyceryl glycoside, myo-inositol, D-psicose, sucrose and D-(+)-talose. Based on evidence, it was reported that these sugars were not found to be toxic at low concentration and short time (
The findings of this toxicology investigation, show that G. spinosum methanol extract (GsME) had LC50 and EC50 of < 100 mg/L in a dose and time-dependent manner. A teratogenic index of > 1 suggested that GsME had teratogenic effects. GsME was significantly reduced eye size, body length, total distance of movement and swimming speed, meanwhile the amount of yolk in zebrafish embryos was increased in a dose-dependent manner. Low doses of GsME did not cause any developmental defects in zebrafish embryos, whereas high concentrations led to malformations including lack of pigmentation, lack of somite, structural deformities, pericardial oedema, and yolk oedema. GC-MS analysis of GsME was identified several important phytoconstituents belonging to different classes of biologically active compounds namely amino acid, phenolics, carboxylic acids, reducing sugars, saturated fatty acids and brominated saturated fatty acid.
The authors are grateful to Universitas Ahmad Dahlan, Yogyakarta, Indonesia for their support under the Program of 100 Doctoral Graduates within Year 2019–2023.