Comparative Study of Herbal Plants on the Phenolic and Flavonoid Content, Antioxidant Activities and Toxicity on Cells and Zebrafish Embryo

Figure 01 - Life Cycle of Zebra Fish Embryo Development

1. Fertilization (0 hours post-fertilization, hr)

• Occurs externally in water following the release of eggs by the female and sperm by the male.
• A translucent chorion (protective membrane) envelops the fertilized egg as it develops into a single-cell zygote.

2. Cleavage Stage (0.75 – 2.25hr)

• Rapid cell division, or cleavage, occurs in the zygote without any expansion.
• On top of the yolk, it creates a blastodisc of cells.
• The developmental stages of the embryo are one cell, two cells, four cells, eight cells, sixteen cells, and thirty-two cells.

3. Blastula Stage (2.25 – 5.25 hr)

• The cells proliferate and disperse throughout the yolk.
• A sheet of cells called blastoderm is formed.
• The yolk syncytial layer (YSL) develops where the yolk and blastoderm meet.

4. Gastrula Stage (5.25 – 10 hr)

• Three major cell movements take place: convergence-extension, involution, and epiboly.
• There are three layers of germ:

• Ectoderm → skin, nervous system
• Mesoderm → bones, muscles, and circulatory system
• Endoderm → pancreas, liver, and gut

• The head-to-tail orientation, or body axis, is determined.

5. Segmentation Stage (10 – 24 hr)

• Somites, or blocks of mesoderm, form along the body as body segmentation starts.
• The neural tube and notochord develop.
• The eyes, brain, and tail bud emerge.

6. Pharyngula Stage (24 – 48 hr)

• The embryo assumes the distinctive shape of a fish.
• The heart begins to beat (~24–26 hpf).
• Circulation, pigmentation, and organogenesis (organ creation) start.

7. Hatching Stage (48 – 72 hr)

• The embryo develops into a larva that can swim freely.
• It uses hatching enzymes to emerge from the chorion.
• The development of the swim bladder, fins, and sensory systems begins.

8. Larval to Adult Stage (3 days – several months post-fertilization)

• Fins, scales, and reproductive organs develop gradually.
• It takes three to four months to acquire sexual maturity.⁵

Model Organisms: The Zebrafish

The zebrafish (Danio rerio) has grown in significance in scientific studies since the 1960s. It is a useful model for researching human genetics and illness because of its various features.

• Model creatures are non-human organisms that researchers examine in order to reveal biology's hidden patterns.
• In the 1960s, zebrafish (Danio rerio) were first used as model organisms.

Figure 02 - Model Organisms of Zebrafish

The Zebrafish (Danio rerio)

• In the 1960s, zebrafish (Danio rerio) were first used as model organisms.
• Males are typically torpedo-shaped and thin, with a hint of pink or yellow.
• Because of the eggs they carry, females are larger and less pink than males. ⁶

Limitation of using the zebrafish

• Because of their simpler architecture and fewer organ systems, zebrafish are not ideal for investigating every facet of mammalian physiology.
• Because zebrafish frequently contain many copies of genes (paralogs), certain genes could not be functional.

Derivatives For Zebrafish Models

1. Quercetin derivatives in zebrafish model

Quercetin derivatives have protective qualities in the zebrafish model of cisplatin-induced toxicity. To better understand the pharmacological and toxicological consequences of quercetin derivatives, more research has been done utilizing zebrafish models. When compared to quercetin itself, these compounds show better solubility, stability, and bioavailability, which boosts their biological activity. Quercetin and its modified forms have demonstrated promise in lowering oxidative stress, preventing drug-induced organ damage, and modifying developmental pathways in zebrafish. This deformity is probably associated with mitochondrial malfunction, which could lead to anomalies.

Figure 03 - Quercetin Derivatives for Zebra Fish Models

Figure (a) illustrates the neuroprotective and antioxidant effects of quercetin and its derivatives in zebra fish.

Figure (b) illustrates how to preserve ATP levels and safeguard mitochondria, two essential components of brain health.

2. NARINGENIN derivatives for zebrafish models

One kind of polyphenolic chemical that falls under the category of flavonoids is naringenin. In zebrafish embryos and larvae, these derivatives show antioxidant, anti-inflammatory, and cytoprotective effects. They also allow researchers to evaluate dose-dependent toxicological responses and developmental safety. These compounds share a distinctive chemical structure consisting of two aromatic rings connected by a three-carbon bridge (C6–C3–C6) that forms an oxygen-containing heterocyclic ring.¹⁰

Figure 04 - Naringenin Derivatives of Zebra Fish Model

Using a zebrafish (Danio rerio) model,

figure (a) illustrates the anti-seizure action of naringenin and its methylated counterparts. It shows how drugs are discovered and how to screen for possible neuroprotective or anti-epileptic properties.

Figure (b) Zebrafish-Based Screening and Naringenin Structure

Chemical Structure:

Naringenin's and its glycoside form's molecular structures.

Using Zebrafish as a Model,

Drug effects are tested on zebrafish eggs and larvae.

Spontaneous tail-induced vibration, or STIV production, is a sign of seizure-like behavior.

Cell growth: to track possible neurotoxicity or neuroprotection.

Molecular alterations can be seen using optical examination of differential marker expression.

KAEMPFEROL derivatives for zebrafish modals

The naturally occurring flavonoid kaempferol (Kaem) [3, 5, 7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is found in a wide range of therapeutic plants and herbs. This bioactive substance is important in therapeutic research and the development of natural medicine since it demonstrates a wide range of pharmacological actions, such as strong anti-inflammatory, antioxidant, anticancer, and antiulcer properties.⁷ Kaempferol derivatives are useful for researching developmental, neurotoxic, and cardiotoxic reactions in zebrafish models because they are potent regulators of oxidative stress, cell death, angiogenesis, and metabolic processes.¹¹

Applications of Kaempferol Derivatives in Zebrafish Models

Osteogenesis (Bone Formation):

It has been demonstrated that kaempferol (Kaem) and its Kaem-Zn complex increase caudal fin growth and encourage bone mineralization in adult zebrafish vertebrae.

Neuroprotection

Kaempferol improves motor function in zebrafish by lowering oxidative stress and neuroinflammation and mitigating striatal damage brought on by 3-nitropropionic acid (NPA). ⁷

Hesperidin derivatives for zebrafish modal

Hesperidin from citrus fruits has a broad variety of pharmacological effects and is known to interact with several cellular pathways.  Zebrafish larvae exposed to 1% alcohol exhibit a variety of morphological defects as well as behavioral alterations. The behavioral abnormalities and morphological flaws were both lessened by pretreatment with the flavonoid hesperidin.  This illustration demonstrates how hesperidin, a naturally occurring substance found in citrus fruits, aids in preventing alcohol-induced liver damage. It accomplishes this by lowering endoplasmic reticulum stress, lipid imbalance, and metabolic malfunction and DNA damage.

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Figure 05- Hesperidin Derivatives for Zebra Fish Model

General toxicological investigation linking human and zebrafish model

1. Developmental toxicity

Three distinct testing protocols are outlined in the FDA guidelines:

• Segment I assesses fertility and reproductive function.
• Segment II evaluates teratogenic effects and developmental toxicity.
• Perinatal and postnatal development is the emphasis of Segment III.

In order to address the use of zebrafish and other in vitro models for researching developmental toxicity, the Developmental and Reproductive Toxicology Technical Committee of the Health and Environmental Sciences Institute (HESI) held a workshop in 2008.The study of embryonic and fetal development, with a primary focus on abnormalities or embryo-fetal lethality (MEFL), is known as developmental toxicity.⁸

2. Reproductive toxicity

Mice have historically been the most popular mammalian model for testing the reproductive toxicity of different medications and chemicals. Studying the hazardous potential of organisms was based on histopathological examination of the testes and ovaries, as well as assessment of sperm quality, measurement of egg production, and other associated data.⁸

Benefits of reproductive toxicity

• The fertility should be high.

• It should develop quickly and resemble humans genetically.
• It needs to be economical.

Zebrafish Genome Compared to Human

The zebrafish (Danio rerio) has become a popular model in recent years for researching genetic diseases in vertebrates. Zebrafish are a creative and practical choice for study since, in comparison to other laboratory animals, they have a number of advantages. They are an effective and dependable instrument for scientific research because of their quick development, transparent embryos, and striking genetic resemblance to humans.⁹

Table 01: Zebrafish genome compared to human

Figure 06 - Quercetin Derivatives for Zebra Fish Models

The type of exposure by using the zebrafish model

1. Acute exposure

A zebrafish embryo goes through a number of crucial stages during development, including zygote, cleavage, blastula, gastrula, segmentation, pharyngeal, and hatching.

Following fertilization, the critical phases of zebrafish development advance quickly.

• The zygote stage (0 h) is when fertilization occurs, creating a freshly formed egg.
• The blastula stage, which lasts for about two hours, is when epiboly begins. Rapid, coordinated cell divisions gradually slow down and become asynchronous during the mid-blastula transition.
• The gastrula stage, which lasts for about five hours, is characterized by significant morphogenetic processes that form the epiblast, hypoblast, and embryonic axis, such as involution, convergence, and extension.
• The segmentation stage, which lasts for about ten hours, is when somite creation, pharyngeal arch primordia, neuromas, primary organ formation, first body movements, and tail development all start.
• Pharyngeal stage (around 24 hours), this is the phonotypic stage, when the embryo's body axis straightens from its previous curve around the yolk sac.
• The hatching stage, which lasts for around 48 hours, is when rapid organ morphogenesis is finished and cartilage forms in the head and pectoral fins. Individuals hatch asynchronously.
• Early larval stage (about 72 hours): the larvae exhibit active avoidance behaviors, have inflated swim bladders, and are able to perceive and locate food. This time frame is appropriate for evaluating hatching¹⁰.
• Early larval stage (about 72 hours): the larvae exhibit active avoidance behaviors, have inflated swim bladders, and are able to perceive and locate food. This time frame is appropriate for evaluating hatching¹⁰.    

2. Chronic Exposure

Zebrafish have an average lifespan of two to three years and grow quickly, usually reaching sexual maturity in three to four months. Zebrafish can therefore be used in long-term exposure studies to evaluate chronic toxicity.According to the study, hepatic tissue damage resulted from a 45-day exposure to Microcystis aeruginosa and total ammonia nitrogen, which altered lipid synthesis, breakdown, and transport. Long-term exposure also had an impact on muscle metabolism, altering levels of fatty acids, glycolipids, and glycerophospholipids, which resulted in lipid accumulation, nutrition loss, and damage to muscle structure.

Restricting long-term exposure

But the absence of the placenta makes it impossible to research the impacts of the placenta.

3. Combined exposure

Assessing the impact of cumulative exposure in zebrafish becomes crucial in several situations. Assessing the toxicity of co-occurring substances is one crucial scenario. For example, Lee and associates investigated the combined toxicity of homosalate and avobenzone, two popular UV-filtering components in sunscreens, on male zebrafish. According to the study, homosalate inhibited androgen activity through a steroid hormone pathway and produced estrogen-like effects. These anti-androgenic effects were amplified when avobenzone was also present. Ten

Second, evaluating combined exposure is crucial for researching combination toxicity. Chinese herbal remedies, which are usually taken as multi-component mixes rather than single compounds in practical application, are a pertinent example.¹⁰

Table 02: Difference between zebrafish Embryo and Human Embryo

Zebrafish model for human disease

Because of their genetic resemblance to humans, transparent embryos, outward development, and affordability, zebrafish are an ideal model for human disorders. Because they allow researchers to monitor disease progression and test possible therapeutic therapies in an entire animal system, they are used to examine a wide range of ailments, including malignancies, neurodegenerative diseases, metabolic diseases, and skeletal abnormalities.¹¹

The zebrafish has emerged as one of the most useful and versatile models for studying human diseases. Because of their similar genetic makeup to humans and the ease with which their genes can be altered, zebrafish models are particularly useful.¹¹

Zebrafish models are now a valuable resource for researching several cancers, including pancreatic cancer, leukaemia, and melanoma. Researchers have created models that closely resemble real tumours by introducing human cancer-causing genes into the zebrafish genome or modifying genes linked to tumour growth.

Some of the diseases studied using these models include:

• Parkinson’s disease
• Alzheimer’s disease
• Epilepsy
• Dravet syndrome (DS)

Characteristics of Zebra fish

Figure No. 08 - Characteristics of Zebra Fish

Lifespan of zebra fish

In captivity, zebrafish usually live for two to three years, but under the right circumstances, they can live for more than five years. They are frequently an annual species in the wild.

• In captivity, the average lifespan is two to three years, but under the right circumstances, it might be up to five years.
• In the wild, they are frequently annual species, which means they only have a single year to survive.
• Average lab lifespan: Research indicates that the average lifespan is approximately 42 months, or 3.5 years.
• Maximum lifetime in the lab: Some people can survive for 58 to 66 months (4.8 to 5.5 years).

Automated Microinjection of Zebrafish Embryos

A fine-tipped needle is used in the process of microinjection [1,2] to introduce foreign materials into cells, including proteins, RNA, DNA, sperm, and medicinal compounds. Numerous facets of live cells, including as signal transduction, cellular genetic structure, and gene expression, have been extensively studied with it.¹⁴

Microinjection is less harmful and helps maintain the biological activity of cells as compared to other conventional physical delivery techniques like electroporation, viral vectors, gene guns, ultrasound-mediated delivery, sperm-mediated delivery, hydrodynamic delivery, and electrophoresis (DEP). Microinjection produces less toxicity and better maintains the biological activity of cells than other conventional physical delivery methods like electroporation, viral vectors, gene guns, ultrasound-mediated delivery, sperm-mediated delivery, hydrodynamic delivery, and electrophoresis (DEP)..¹⁴

Figure 02 - Automated Microinjection of Zebrafish Embryo