Histopathological and Toxicological Studies on Zebra Fish Using White-Fruited and Green-Fruited Varieties of Momordica charantia

Momordica charantia is well known for its medicinal properties. It has exhibited various pharmacological activities, such as antidiabetic, anti-inflammatory, and antimicrobial activities. Although this plant is used worldwide as a vegetable and medicinal ingredient in herbal medicines, its toxicity studies have not been conducted to date. This study attempts to understand its toxicity. The present study examined the activity of two enzymes, acetylcholinesterase and succinate dehydrogenase, as well as histopathological variations in the liver, intestine, and gills of zebrafish. The results of the acetylcholinesterase assay showed that the concentrations of 40 mg/L and 60 mg/L of the four extracts (leaf and fruit extracts of both varieties) exhibited increased enzyme activity. Interestingly, the leaves of the green fruit variety at a concentration of 60 mg/L showed the highest activity, with a value of 2.824 ± 0.0682 micromoles/min compared to the control value of 1.8347 ± 0.0046 micromoles/min. On the other hand, the succinate dehydrogenase assay revealed that the concentrations of 40 mg/L and 60 mg/L of the extracts decreased the enzyme activity. The highest inhibition was observed in the concentration of 60 mg/L of the leaves of the white-fruited variety and the green-fruited variety, with values of 1.884 ± 0.0482 micromoles/min compared to the control value of 2.747 ± 0.0046 micromoles/min. The studies on histopathological changes also demonstrated abnormalities in the brain, liver, intestine, and gills of zebrafish after the exposure to the extracts of M. charantia. The severity of the damage varied from low to high concentraions. In general, this study sheds light on the safety profile of Momordica charantia and highlights its potential toxicity in animal models. The findings suggest that more research is needed to fully understand the toxicity of this plant and its implications for human use.


Introduction
Momordica charantia, commonly known as bitter gourd, has been extensively researched due to its medicinal potential.It belongs to the Cucurbitaceae family and has various pharmacological activities, such as anti-infammatory, antidiabetic, antimicrobial, and anticancer efects [1].Two of its powerful ingredients, momordicin and charantin, which are cucurbitane-type triterpenoids, have been linked to its biological actions [2].Bitter gourd is widely used in South America, Africa, and Asia and is considered safe for human consumption.M. charantia has garnered signifcant attention due to its pharmacobotanical aspects and its efects on various biological systems.In particular, research has spotlighted its pharmacological properties and potential therapeutic benefts, including its impact on glucose metabolism, infammation, and cancer.However, its safety profle has not been fully evaluated in animal models.
Zebrafsh (Danio rerio) have been widely used in toxicological and histopathological investigations by the scientifc community.Zebrafsh are a model organism that reacts to chemicals in a way similar to that of humans and have been used in numerous toxicological and pharmacological research [3].Te safety of natural substances has become a growing concern, and it is crucial to assess how therapeutic herbs impact living things.Terefore, the objective of this study was to assess the toxicity of the white and green Momordica charantia varieties in zebrafsh and any potential histopathological alterations.Danio rerio has been used in numerous investigations as a model organism for behavioral, toxicological, and cancer-related research.Justifcations for using this fsh as a model organism are its short generation time, external fertilization methods, and reduced maintenance costs for the husbandry.Due to the external nature of fertilization, genetic and developmental modifcations are feasible.Since mature zebrafsh are small in size, it is possible to assess the cytotoxic efects on the organism and the organ system, which may lead to the development of new metabolites [4].
Te main objective of this investigation was to compare the toxicity of the white-fruited bitter gourd (BGW) and green-fruited bitter gourd (BGG) varieties of M. charantia in larvae of zebrafsh and investigate their histopathological changes.Te results of this study will help to understand the safety profle of bitter gourd (M.charantia) and develop a better understanding of its toxicity in animal models.
Furthermore, histopathological analysis provides important information on the microscopic modifcations caused by these types of bitter gourds.Carefully examining and analyzing these changes can be helpful in identifying possible side efects and understanding the mechanisms underlying the reported toxicological consequences.
Te fndings of this study will advance our knowledge of Momordica charantia toxicity and pave the way for future studies in this feld.Toroughly evaluating the safety profle of this plant using animal models will create a solid foundation for the use of bitter gourd in the development of prospective treatment interventions or dietary guidelines.Te results of this study will contribute signifcantly to a better understanding of Momordica charantia and its potential toxicity.Tis research will provide adequate understanding and guidance for future investigations in the feld, ultimately ensuring the safe and efective use of M. charantia.

Preparation of Plant Extract.
Te fruits and leaves of the green and white varieties of M. charantia (Authentication/SMPU/RARIMD/BNG/2018-19/288) collected from Bangalore, Karnataka were dried and powdered.Te powdered plant materials were extracted with methanol.Te sample extracts were prepared by adding 50 g of dried powder in 250 mL of methanol using Soxhlet extraction methods.Te extract was then subjected to evaporation, and the resulting extract was used for further studies.Serial dilution of M. charantia extracts: all four diferent extracts were diluted to 3 diferent concentrations (20 mg/mL, 40 mg/L, and 60 mg/mL) [1].Te experiments were carried out using four diferent extract samples.

Maintenance of Danio rerio.
Te fsh were quarantined and stored in a 20 L aquarium and the fsh were fed brine shrimp cultured under laboratory conditions once every 36 hours.In total, 13 same-size aquariums were used with one liter of dechlorinated and reverse osmosis water, 12 aquariums were used for testing, and one aquarium for control.Each aquarium was maintained with 3 adult Danio rerio.
Administration of plant extracts in fsh: the plant extracts were injected into experimental Danio rerio using six replicates, including a control following the protocol of OECD [5].

Isolation of Acetylcholinesterase (AChE) from the Brain.
Te brains of Danio rerio were dissected and homogenized with 3 mL of phosphate bufer (PBS) (0.05 M, pH 7.2) using a prechilled mortar and pestle.Te homogenate was diluted with 17 mL of cold PB and then centrifuged at 5000 rpm for 15 minutes at 4 °C.Te supernatant was collected, which served as the source of the enzyme.Te enzyme source was diluted at 20x concentration [6].
2.1.4.Acetylcholine Esterase Assay.In this assay [6], to 0.5 mL of enzyme, 5 mL of acetylcholine (100 mM) was added and incubated for 5 minutes at room temperature and then the enzyme was deactivated by placing it in a boiling water bath for 2-3 minutes.Te terminated mixture was then titrated against NaOH (0.05 M) with phenolphthalein as an indicator until a pale pink colour was obtained.Te titer value was noted, and the enzyme activity was calculated.

Isolation of Mitochondria and Assay of the Succinate Dehydrogenase (SDH) Enzyme. A diferential centrifugation
technique is adopted to separate mitochondria from a supernatant free of cell debris in brain cells.Osmoprotectants (sucrose or mannitol) are used to help mitochondria stay free of osmotic stress and intact.To prevent heavy metal toxicity, EDTA was used to chelate metal ions and prevent mitochondrial damage.Bufers were used at pH 7.4 or pH 7.2 to maintain the biological pH proximities.
Te following reagents were used for the assay of SDH: 1 M sucrose was prepared by adding 342.8 g of sucrose to 1000 mL of distilled water.Tris/HCl bufer was prepared by adding 12.1 g of tris HCl in 500 mL of distilled water, the pH was set at 7.4 and the solution was made up to 1000 mL, 0.1 M EDTA was prepared, and the pH was set at 7.4 using Tris powder.Te isolation bufer was prepared by adding 10 mL of 0.1 M Tris/ HCl 1 mL of EDTA and 20 mL of 1 M sucrose.Volume was made up to 100 mL and pH was set at 7.4.All prepared reagents were autoclaved at 121 °C at 15 PSI for a period of 15−20 min and stored at 4 °C.For the extraction of mitochondria, it was essential that the microcentrifuge tube would be fresh and autoclaved.For the performance of the assay, clean test tubes were soaked in 1% H 2 SO 4 .Te test tubes were then rinsed in water and dried to remove any water [7].

Isolation of Mitochondria.
Te tissue was rinsed multiple times using an ice-cold isolation bufer to prevent blood clotting and soaked in the isolation bufer at 4 °C and 2 Scientifca then fnely cut the tissue into small pieces.Tese samples were transferred to a chilled pestle and mortar.Te pestle and mortar overnight were prechilled in the freezer and used only while performing the experiment and the tissue was homogenized (powder-like or amorphous) until it became a fne powder (powder-like or amorphous).5 mL of ice-cold isolation bufer was added for every 1 g of tissue and homogenized until a paste was formed.Te homogenate was transferred to a sterile Eppendorf tube and centrifuged at 1600 rpm for 10 min at 4 °C.Te supernatant was transferred to a sterile Eppendorf tube and centrifuged at 3000 rpm for 15 min at 4 °C.Te pellet was discarded and the supernatant was transferred to an Eppendorf tube and centrifuged at 7000 rpm for 15 mins at 4 °C.Te supernatant was discarded and the pellet was resuspended in a fresh isolation bufer, and continued to centrifuge at 10,000 rpm for 15 mins at 4 °C.Te pellet was stored at 4 °C for the enzymatic assay (the pellet was suspended in 0.5 mL of 0.1 M phosphate bufer to use it in the SDH assay).Te morphology of the mitochondria was studied by smearing the pellet and staining the smear with Janus green stain.Te assay was performed within 24-36 h of isolation [7].

Histological Variations of the Liver, Intestine, and Gills of
Danio rerio.Histological studies were carried out following the McManus and Mowry protocol [8] and Xavier and Kripasana [9].After further treatment with M. charantia fruit and leaf extracts, intestine, gills, and liver samples were dissected and stored in Bouins fuid for 24 hours.Tissue samples from the control and treatment groups were dehydrated and cleaned in xylene prior to embedding in parafn wax.Sections of 5 μm thickness were taken and stained with hematoxylin and eosin stains.Te sections were mounted in DPX (distyrene plasticizer xylene) after deparafnization.Sections were observed under a light microscope for structural variations [9].

Statistical Analysis.
Statistical analysis of the present study was conducted using SPSS statistical software version 22. Te signifcance of the data was analyzed using Tukey's test at a probability of less than p ≤ 0.05.

Acetycholinesterase Assay.
Te in vivo assay showed that 40 mg/L and 60 mg/L of the four extracts (leaf and fruit extracts of the green and white fruits of M. charantia) showed increased enzyme activity.60 mg/L of leaf extracts from the green-fruited variety of M. charantia has shown the highest activity of 2.824 ± 0.0682 micromoles/min when compared to the control of 1.8347 ± 0.0046 micromoles/min.Tere were no signifcant diferences in the other concentrations of 20 mg/L in the four extracts (Table 1).2) During the comparison, it was found that the addition of 40 and 60 mg of leaf extracts of both varieties (green and white fruits of M. charantia) had a higher rate of inhibition than the fruits.

Histopathological Variations.
Te general histological evaluation indicated a low to high incidence of tissue damage to Danio rerio, after exposure to M. charantia extracts.Histopathological changes were observed in the brain, liver, intestine, and gill tissues.

Histopathology of the Brain.
To determine the histopathological variations of the brain, the fshes were exposed to 40 mg/mL and 60 mg/mL of the plant extracts of M. charantia for 4 days.Te alteration of the brain tissue was observed under a light microscope (Figures 1, 2,  3).Diferent concentrations of plant extract treated with fsh showed a signifcant exponential increase in histological modifcations over time.In terms of the histomorphology of the brain, obvious tissue chaos was noticed.Te brain of Danio rerio exposed to a higher concentration of plant extract at 60 mg/mL showed necrosis in the brain.Purkinje cell degeneration was observed in the brain region of zebrafsh in the M. charantia treatment groups.Disturbed tissue anarchy was observed in the leaf extract-treated samples.Intracellular spaces in the treatment group samples were diferent from those in the control samples.Te presence of serious lesions was observed in the treatment groups, suggesting the toxicity of M. charantia extracts.

Histopathology of the Liver Treated with BGG Green
Variety Leaf Extract.M. charantia samples at higher concentrations of 200 and 400 mg/L have induced nuclear pyknosis and cytoplasmic vacuolization (Figures 4,5,6,7,8).Hepatic granulation in liver cells seems to be normal in the 4 Scientifca control samples.Te intervals in the hepatic chords were regular.Te cytoplasm of the hepatocytes was normal.Erythrocytic infltration in hepatocytes was found to increase in the treatment groups.Samples at lower concentrations of 50 and 100 mg/L had induced severe cytoplasmic vacuolization.fgures.As the gills have a higher chance of reacting to changes in water quality due to M. charantia extracts, histopathological studies were carried out in the gill regions to analyze the possible role of M. charantia extracts in the regulation of respiration and osmoregulation.However, the severity, but not the type of lesions observed in exposed fsh was concentration dependent.Histopathological lesions were observed in the gills of the exposed fsh (Figures 14, 15,16,17) with increasing concentrations of plant extract.Lesion-like symptoms were observed in the epithelial layer of the cells.Te architecture of the primary gill lamellae and the secondary gill lamellae was clearly distinct in the control sample (Figure 18).However, with increasing concentration of extracts, the lamellae were found to curl, causing congestion in the gill structures.Tis could be another reason for the hemorrhagic conditions observed in the groups of zebrafsh treated with diferent concentrations of the extracts.Changes in the gills of fsh exposed to diferent concentrations of M. charantia leaf extracts of M. charantia difered signifcantly from those of nonexposed control fsh.Fusion of the tip of the primary lamellae is observed at sample concentrations of 50 mg and 100 mg/L (Figures 14  and 15), whereas detachment of the lamellae is observed at 200 mg/L and 400 mg/L of M. charantia Green leaf extract (Figures 16 and 17).6 Scientifca

Hemorrhage.
As zebrafsh is considered an efective model for vascular development, hemorrhagic conditions for M. charantia extracts were examined.Te fsh sufered from hemorrhage with all 4 higher concentrations.Te incidence of hemorrhage might be due to rupture of the blood vessels.Tis was subjected twice, for two diferent healthy Danio rerio, but resulted in the presence of blood spots near the ventral region, especially near the fns and jaw.Signs of hemorrhage with restlessness were also confrmed in zebrafsh treated with the extracts.Leakage or damage to blood vessels might be due to ruptured blood vessels.It was also observed that these fsh were unable to regain their health immediately after the withdrawal of the extracts; however, a slight discoloration was observed in a few samples.Te integrity of the vessel was disturbed and a defect in the vascular wall was observed on both the ventral sides of the fsh and beneath the gill regions (Figures 19 and  20).Te marked changes in tissue discoloration may be due to changes in the architecture of endothelial cells that form the barrier between blood and surrounding tissue (

Acetylcholinesterase (AChE) Enzyme
Assay.Acetylcholinesterase (AChE) is one of the most efective enzymes in the nervous system.It is intensely concentrated at the cholinergic synapses.Acetylcholinesterase has been associated with certain neuromuscular disorders such as glaucoma, myasthenia gravis, and most recently Alzheimer's disease.Clinically, moderate inhibition of acetylcholinesterase is efective in the treatment of Alzheimer's disease or reduced expression of fewer receptors, as in the case of myasthenia gravis, it is a possible way to tackle this disease      Scientifca 7 [6].Te increased amount of acetylcholinesterase may cause diferent neurological disorders due to overexpression and thus reduce the amount of acetylcholine in the cholinergic synapses.Tis may lead to lesions in the nervous systems.Acetylcholinesterase is involved in various metabolic activities of the organism that include cell cycle regulation and processes related to Alzheimer's disease.However, more studies need to be conducted to prove the direct relationship between the increase in AChE and nervous disorders.Te increased concentration of acetylcholine esterase in tissues indirectly refects a reduced concentration of acetylcholine that causes local and systemic infammation.
When the increase in acetylcholinesterase concentrations was observed, acetylcholine levels decreased, and this can lead to a reduction in the anti-infammatory properties exercised by acetylcholine.Tus, an increase in the concentration of acetylcholine esterase in plasma CSF, leukocytes, RBC, platelets, and other tissues indirectly afects the reduced concentration of acetylcholine and the increase in local and systemic infammation.Tis infammation can be attributed to the toxic contribution of the M. charantia extracts to zebrafsh.According to Marroquín-Segura et al., increased acetylcholinesterase activity in plasma, RBC, CSF, leukocytes, platelets, and other tissues can be considered a reliable source for identifying acute, chronic, and lowgrade systemic infammation [10].

Succinate Dehydrogenase Enzyme.
Studies by Rustin et al. have revealed that inherited defects of mitochondrial succinate dehydrogenase in humans are associated with striking variable clinical presentations ranging from the early onset of encephalomyopathy to susceptibility to tumors in adulthood [7].Inactivation of the enzyme succinate dehydrogenase can lead to accumulation of succinate in the mitochondria causing leakage to the cytosol.Tis can result in the inhibition of a group of prolyl hydroxylase enzymes (PHDs).Depending on the inhibition of PHD, two newly recognized pathways that support tumourigenic efects can become functional, that is, afected cells become resistant to certain apoptotic signals and/or activate a pseudohypoxic response that enhances glycolysis and is conveyed by hypoxia-inducible factor [11].
Previous studies by others also support the view that inhibition of SDH will result in tumorigenesis in animals and humans [12].Te same view was supported by the present study conducted in Danio rerio with extracts of the M. charantia plant.In this study, it was clearly proved that higher concentrations of BGG and BGW plant extracts have decreased the activity of SDH enzymes, thus causing malfunctions in the development of Danio rerio.
Te plant extracts of Momordica charantia, although considered to have medicinal properties, were found to have toxic efects when used in higher concentrations.An attempt was made to analyze the toxic levels of plant extracts in the present study.It was clear from the experiments that M. charantia plant extracts were found to be safe up to the dose of 40 mg/kg body weight.Te inactivation of SDH activity was found to be crucial at 60 mg/kg, which could lead to tumor development at later stages.Terefore, it is safe to control the use of M. charantia leaves.Controlling the use of the leaves of the plant will help prevent diferent medical complications caused by the use of M. charantia.4.3. Histopathological Variations.Danio rerio exposed to a sublethal concentration of M. charantia extract showed signifcant adverse changes in the liver, intestinal tract, brain, and gills.Te liver is the main storage organ of lipids and the site of metabolic activities in fsh that helps in detoxifcation.Te organ is also associated with the biotransformation process.However, when the liver was exposed to M. charantia plant extracts, it showed hepatocyte degeneration characterized by cytoplasmic vacuolization.Exposure to the extract resulted in the appearance of a large number of hepatocytes with pyknotic nuclei and hemorrhage surrounding the central vein and severe necrosis in the    8 Scientifca liver.Te results of our study could be correlated with previous studies conducted in Danio rerio exposed to sublethal concentrations of pesticides [13].
M. charantia is used as an alternative medicine that has been used primarily to lower blood glucose levels in patients with diabetes mellitus.Studies have revealed that the components of the M. charantia extract appear to have structural similarities to animal insulin.Clinical trials found that M. charantia extracts have a moderate hypoglycemic efect.Although the extracts of M. charantia fruits were found to have moderate hypoglycemic efects in many of the studies conducted, the early efects of plant fruit extracts have not been thoroughly studied.Some studies have reported the advanced efects of the hypoglycemic compound convulsion of M. charantia in children, reduced fertility in mice, a favism-like syndrome, increased gamma-glutamyl transferase and alkaline phosphatase levels in animals, and headaches [14].Since there were no studies conducted on the toxicology of the plant extracts of M. charantia that are widely used by the common people, the present study was an attempt to understand the toxic levels of the plant extracts from leaves and fruits of M. charantia.
Fish have the ability to adapt to changes in their environment by increasing the distance of difusion to their blood.Tey do it by forming a barrier through proliferating epithelial cells and fusion of primary and secondary lamellar cells [15].However, when the gills of catfsh (Clarias gariepinus) were exposed to extracts of Parkia biglobosa, fruits showed the appearance of lesions [16] and, in some cases, epithelia proliferation in response to epithelial damage [17].Functional loss or morphological damage was observed in afected gill structures of fsh exposed to various toxic elements [18][19][20].

Hemorrhage.
In the present study, hemorrhage was observed on the ventral side of zebrafsh treated with a 60 mg/mL plant extract of M. charantia.Tis hemorrhage was caused by damage to the pillar cells and the subsequent loss of their supportive properties.Similar fndings were reported in a previous study by Camargo and Martinez [21].As a result of this damage, blood cells leaked into the epithelial layer, which could have afected the free fow of blood and subsequently caused an impairment of gaseous and ionic exchange in fsh.Furthermore, recovery from gill damage was found to be slow and difcult.An increase in the cellular layers of the lamellar epithelium could be attributed to an increase in the mitotic divisions of the lamellar epithelium.According to Kantham and Richards [22], this increase in epithelial thickness is a natural response to retard the bloodstream.In addition, we observed lamellar fusion as a result of the thickening of the gill flament epithelium.Te fusion and hyperplasia of gill lamellae could have been caused by toxins present in the M. charantia extract, which modify the glycoproteins present in the mucus that cover the cells and alter the negative charge of the epithelium, thus favoring adhesion to adjacent lamellae.Tis conclusion is consistent with earlier reports by Ferguson [23].

Conclusions
Te present study aimed to evaluate the toxicity of two varieties of Momordica charantia (bitter gourd) in zebrafsh larvae and investigate their histopathological changes.Te study found that the extracts of both the green and whitefruited varieties of Momordica charantia exhibited signifcant efects on the enzyme activity of acetylcholinesterase (AChE) and succinate dehydrogenase (SDH) in zebrafsh.Te AChE activity increased with higher concentrations of the extracts, while the SDH activity decreased.Furthermore, histological analysis revealed structural variations in the liver, intestine, and gills of zebrafsh after treatment with the extracts.Tese fndings contribute to understanding the safety profle of Momordica charantia and highlight its potential toxicity in animal models.Further research is warranted to explore the underlying mechanisms and potential therapeutic applications of this medicinal plant.
were altered in samples treated with an extract that may infuence the function of the intestine.Te intestinal villi looked diferent in the fsh exposed to leaf extracts.Columnar epithelial cells were found to be in maximum proportion in all treatment groups and in control samples.Tere could be damage in mucous-secreting cells that afects the functionality of intestinal cells(Figures 9,10,11,12,13).

3. 3 . 4 .
Histopathology of Gills.Te kind and occurrence of histopathological lesions in fsh exposed to diferent concentrations of M. charantia leaf extracts are shown in the

Figure 2 :
Figure 2: Neurodegeneration with the treatment of 60 mg/mL of leaf extracts of the green-fruited variety (BGG) of M. charantia.

Figure 3 :
Figure 3: Neurodegeneration with the treatment of 60 mg/mL of leaf extracts of white-fruited variety (BGW) of M. charantia.
Figures 21 and 22).Te results of the study indicate that the architecture is disturbed due to the presence of extracts from M. charantia.

Figure 19 :
Figure 19: Hemorrhage was observed on the ventral side of the fsh.

Figure 20 :
Figure 20: Hemorrhage was observed under the gills of the fsh.

Figure 21 :
Figure 21: Discoloration on the skin of Danio rerio treated with the green leaf extract.

Figure 22 :
Figure 22: Discoloration on the skin of Danio rerio treated with the white leaf extract.

Table 1 :
Acetylcholinesterase (AChE) activity with diferent concentrations of the extract.L1, leaf extract of green variety; L2, leaf extract of white variety; F1, fruit extract of green variety; F2, fruit extract of white variety.Results are means of triplicates (mean ± SD).Te various superscript letters indicated signifcant diferences between the samples at p ≤ 0.05.

Table 2 :
Succinate dehydrogenase (SDH) activity with diferent concentrations of the extract.
Results are means of triplicates (mean ± SD).Te various superscript letters indicate that there was a signifcant diference between the samples at 0.05.