Potential role of different animal models for the evaluation of bioactive compounds

Abstract

Animal models are crucial in biomedical research, facilitating the understanding of human diseases at the molecular and cellular levels. At the same time, animal models aid in molecular screening for drug discovery and development. In this review article, we extensively discuss two critical points: the importance of animal models and the bioactive compounds. During the discussion of the importance of animal models, we explore how they aid in understanding disease mechanisms and progression, as well as genetic diseases, drug discovery and development, and drug repurposing. To discuss the importance of bioactive compounds, we illustrate their impact on human health and disease, as well as their industrial applications. Finally, we discuss the various studies on bioactive compounds that have been conducted using different animal models. To highlight the various studies on bioactive compounds using animal models, we categorized them under two headings: mammalian animal models and non-mammalian animal models. Again, for mammalian animal models, we explained various studies on bioactive compounds in mice, rats, rabbits, guinea pigs, hamsters, ferrets, and gerbils. Similarly, for non-mammalian animal models, we illustrated the different studies on bioactive compounds using zebrafish, Drosophila melanogaster, and Caenorhabditis elegans. The work will be beneficial in various ways in the field. First, using animal models, researchers can develop effective next-generation therapies for a number of human diseases utilizing bioactive compounds. Second, during drug development using bioactive compounds, researchers can assess the efficacy, safety, and pharmacokinetics of these compounds in an animal model. Third, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of these bioactive compounds can be studied in animal models before clinical trials. Ultimately, the scientific combination between bioactive compounds and animal models will facilitate the advancement of scientific and biomedical discoveries implicating bioactive compounds.

Introduction

The remarkable resemblance between humans and animals (mammals) in terms of both anatomical as well as physiological characteristics provoked scientists to use animals to study diverse human disease pathophysiology and investigate the efficacy of novel therapies and their side effects before their human application^[1]. Animal models are extensively used in various biomedical research fields, including pharmacology, cancer biology and therapeutics, toxicology, neuroscience, endocrinology, and palliative medicine^[1–4]. Animal models have also been used as a vital tool for surgeries and implants, which are used frequently in biomedical research and development^[5,6]. Animal models are helping in human and animal research to gain a deeper understanding of the area. At the same time, the key findings of the animal models research can provide a better outcome of the research^[7]. Selecting an ideal animal model is crucial for biomedical research. The primary criteria rely on the pathophysiological similarities between human and selected animal models, the extent to which the disease conditions are mimicked in humans, the prolonged lifespan, compatibility with the tested treatment regimes, and the large availability and size of the chosen animal species^[3,8]. In this regard, fruit flies (Drosophila sp.), zebrafish (Danio rerio), frogs (Xenopus sp.), reptiles, mice, rats, guinea pigs, rabbits, hamsters, cats, birds, dogs, monkeys, pigs are used as animal model throughout the world due to their phylogenetic similarities to humans^[9–11]. Moreover, the selection of an inappropriate animal model leads to erroneous experiments, incorrect outcomes, and the misallocation of resources, as well as the misuse of lives^[12]. To overcome such problems, scientists have generated specialized transgenic animal models by directly incorporating genetic information into embryos via foreign DNA injections or by using retroviral vectors^[13]. The incorporation of human cells into recipient animal models enables scientists to investigate pathogenic actions similar to those in the human body^[14]. It is necessary to study the potential of tested drugs or other medical devices in more than one animal model to validate their efficacy in alleviating human disease^[3,15]. Using animal models, researchers can understand the effect of temperature, light, humidity, animal diet, and medications in an experiemnt and any hange of these parameters affects experimental outcomes. Additionally, the exact genetic makeup (e.g., genetic diversity, manipulability) of the used animal models is essential to ascertain the evaluation of only one factor being investigated^[5]. Nowadays, the implementation of genetically modified or transgenic animals, nanotechnology, and artificial intelligence promotes significant advancements in medical research^[16,17]. Despite numerous advantages, the use of animals in research remains a highly controversial topic and poses significant ethical challenges, including the need for strict laws, guidelines, and norms^[18]. Additionally, the use and maintenance of laboratory animals are thoroughly monitored by a strict bioethical committee, adhering to the principles of the 3Rs, i.e., reduce, replace, and refine procedures to improve the conditions of laboratory animals^[19,20]. Several biochemical components have been tested for drug development in the different animal models from time to time^[21]. In addition to bioactive compounds, biosimilars and vaccines can also be tested in animal models before clinical trials. Numerous successful studies have been conducted on these molecules using animal models^[22,23]. Along with the animal model, analytical technique helps in diagnosing and treating diseases. A combination of these two will help improve the performance of the animal model. One such technique is non-invasive radiographic techniques in diagnosing and treating malignant tumors^[24].

HIGHLIGHTS

• Animal models can help in screening for drug discovery and development using bioactive compounds.

• The efficacy, safety, and pharmacokinetics of bioactive compounds can be evaluated in animal models.

• Animal models can be used to evaluate the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of a bioactive compound.

• Overall, animal models help in the progression of bioactive compound research.

A bioactive compound refers to any substance that exhibits biological activity, particularly in living organisms. Based on the substance used, its doses, and bioavailability, the effect can be positive or negative. It has been reported that consumption of bioactive compounds up to a certain level is beneficial to human health, helping to prevent and treat diverse disease conditions^[25,26]. Fruits, vegetables, whole grains, spices, plants, and their derived foods and beverages are vital sources of diverse bioactive compounds, including polyphenols (flavonoids, phenolic acids, non-flavonoids), carotenoids, phytosterols, terpenoids, alkaloids, glucosinolates, etc^[27–29]. The phenolic compounds are generally plant secondary metabolites produced under both normal and stressed conditions^[30]. Besides plants, marine microorganisms, such as bacteria, fungi, microalgae, and myxomycetes, also serve as an enriched source of natural bioactive chemicals^[31]. The agro-wastes or byproducts produced from various food industries, in the form of peels, leaves, shells, seeds, steam, pomace, skins, husks, barks, and straws, also contain bioactive compounds^[32]. However, a considerable amount of agricultural residues are discarded as agro-waste and are associated with environmental disposal problems. Therefore, the implementation of sustainable green extraction methods to extract and recover value-added bioactive compounds from these agro-wastes helps to develop various valorized items, including functional foods and supplements^[32]. The bioactive compounds possess efficient antioxidant, anti-inflammatory, antiproliferative, antibacterial, antiviral, and immunostimulatory activity to exert their health-promoting effect^[33]. Reports have revealed that a diet enriched with polyphenols can delay the occurrence of oxidative stress-induced metabolic diseases, cardiovascular disease, cancer, and brain dysfunction^[34,35]. Resveratrol, a bioactive compound present in grapes and red wines, displays anticancer action^[36]. Naringenin (flavanone) and quercetin (flavonol) present in apples, oranges, onions, and grapefruit lowered cancer cell proliferation and cardiovascular damage^[37–39]. Quercetin also possesses anti-inflammatory and antibacterial actions^[40]. Capsaicin, a bioactive constituent of chili peppers, is used to treat non-communicable diseases, including diabetes, obesity, and dyslipidemia^[41]. Phytochemicals found in spices and herbs exert antiobesity and lipid-lowering action^[42,43]. Therefore, consumers’ preference for functional food products has increased sharply in recent years. Functional food refers to food items that offer health benefits with the help of its constituent bioactive compounds besides satisfying hunger^[44–46]. However, it has been noted that plant-derived natural products are one of the essential sources for drug discovery and development. Therefore, plant-derived bioactive compounds are being used regularly and are finally being tested in various animal models^[47]. In a mini-review, Gonzales and Valerio listed different medicinal plants from Peru that have been tested for anticancer activities. The three crucial endemic plant materials they have listed are Dragon’s Blood (Croton lechleri), Maca (Lepidium sp), and Cat’s Claw (Uncaria sp). Along with their anticancer activity, these plants have been scientifically examined for a wide range of therapeutic uses^[48]. Quercetin is phytochemicals and plant derive flavonoid. Nam et al have explained the role of bioactive quercetin in oncotherapy^[49]. On the other hand, Kim et al have explained the therapeutic role of aging and regeneration in tendons, muscles, and bone. They also attempted to explain how quercetin might stimulate redox signaling pathways associated with musculoskeletal disorders^[50]. Sharma et al have illustrated the role of flavonoids in bone loss. They also discussed the molecular mechanism by which flavonoids support bone loss. In addition to the molecular mechanism, they also discuss the targeted delivery of flavonoids in bone loss^[51]. In other work, Sharma et al have demonstrated that nanoparticles encapsulating quercetin are effective for treating breast cancer, as shown in vivo using a xenograft mouse model^[52]. Razon et al assessed the effects of fenugreek seed powder and oyster mushroom powder on type 2 diabetic rat model (T2D Animal Model) to understand the impact of bioactive compounds on type 2 diabetes. In the study using an animal model, the bioactive compounds have a strong binding affinity to α-glucosidase, indicating that these bioactive compounds from the plant may control blood glucose levels (postprandial)^[53]. However, to test the activity of the bioactive compounds, different animal models are necessary.

Artificial Intelligence (AI) has been progressing rapidly and has been explored in various sectors periodically. Its applications are being examined across various areas^[54,55]. AI can be utilized in the evaluation of bioactive compounds in animal models, thereby enhancing efficiency and prediction accuracy. However, it needs to understand how we can use AI in the field. The extensive review article aims to illustrate the importance of animal models, the importance of bioactive compounds, and different studies on bioactive compounds using different animal models. To highlight the different studies on bioactive compounds using animal models, we discussed those under two headings: mammalian animal models and non-mammalian animal models. Again, for mammalian animal models, we discussed various studies on bioactive compounds in mice, rats, rabbits, guinea pigs, hamsters, ferrets, and gerbils. Similarly, for non-mammalian animal models, we illustrated the different studies on bioactive compounds using zebrafish, Drosophila melanogaster, and C. elegans. Finally, the article illustrated recommendations on the use of AI in the field of Animal models and the use of bioactive compounds.

Materials and methods

An extensive review article aims to examine the role of various animal models in evaluating bioactive compounds. For this review article, we searched the different literature to discuss the role of various animal models in evaluating bioactive compounds. For the study, we used four databases: Google Scholar, Web of Science, PubMed, and PubMed Central. We used various keywords or keyword combinations for data mining, including “bioactive compounds,” “animal models,” “disease mechanisms,” “drug discovery and development,” “genetic diseases,” “genetic effects,” “mutations,” and others. Significant and highly cited references were used to write this extensive review article.

Importance of animal models

Animal models have several importance to study the disease and disease progression, drug discovery and development, animal models are utilized in the study of human diseases due to their resemblances to humans in various aspects, including genetics, anatomy, and physiology.

Studying disease mechanisms and disease progression

Animal models can mimic various aspects of human biology. Therefore, animal models are significant in understanding the molecular mechanism of disease (Table 1). It enables researchers to study disease processes in a living system that is similar to humans without affecting human health. The animal model also provides insight into the molecular mechanisms of disease. It also aids in the investigation and identification of key molecular pathways^[56]. Lamichhane et al demonstrated that animal models are vital for understanding the disease mechanisms^[57]. Fieber effectively describes the importance of aquatic animal models for studying human diseases^[58]. At the same time, it can also be used for researching various aspects of human diseases, from investigating disease progression to diagnosis and treatment^[59–61]. McGonigle and Ruggeri explained how an animal model is essential for understanding novel therapeutic agents. They also illustrated the role of animal models in studying pharmacokinetic/pharmacodynamic (PK/PD) relationships^[60]. However, selecting the right animal model is very much crucial. It is essential to select the right animal model for specific disease research. Swearengen discussed how the right animal model could be chosen for infectious disease research to understand host-pathogen interactions. It will help us extend our understanding at the molecular level^[62].

Table 1.

Different significant diseases and their importance in animal models

Sl. No.	Disease name/types	Importance in animal models	Remarks	Reference
1	Streptozotocin (STZ) induced diabetes	It shown clinical symptoms in animals that looks alike diabetes in human	The physiochemical characteristics and toxicities of streptozotocin source for mortality to the animals	[63]
2	Restoration of limbs	Very quick, high regenerative capacity, least ethical regulations about the zebrafish	Not ideal look like as human model	[64,65]
		The trial result can simply be shifted to human and good for preclinical trial in dog and horse model	Required more maturity period, and the rearing cost is expensive	[66]
3	Cartilage defect	Different animal models (dogs, deer, sheep and goat) research carried out	The sheep are the best suitable models for cartilage thickness, and joint biomechanics substances	[67]
4	Bacterial infection	Toxicity and efficacy of antibacterial also be considered	The animal model cannot guess human response to specific component	[68]
5	Reperfusion injury of the spinal cord, and ischemia	Diverse animal models are defensible	It need several animal models (rabbit, mouse, and pig)	[69]
6	Cancer	The preclinical trials of mAbs for cancer treatment in animal models are essential to extent the clinic	In animal studies the mAbs are very less adapted	[70]
7	Alzheimer’s disease	The best practices to discover the mechanisms of Alzheimer’s disease and study it’s treatment methods	For sporadic Alzheimer’s It not directly linked to genetic mutation, whereas multiple environmental factors are tangled	[71]
8	Type 2 diabetes (T2D)	Used for disease’s mechanisms, recognize potential therapeutic targets, and assess the effectiveness of new treatments in a controlled environment before testing in human body	The distinct animal model categories to assess the effectiveness of therapeutic interventions against T2D	[72]
9	Cardiovascular diseases	Pathophysiology of cardiovascular condition to study complex disease mechanisms and test new therapeutic strategies under controlled situations	Animal models may not perfectly replicate human conditions, and the findings from animal studies may not always translate directly to humans	[73]
10	Obesity	It understand the physiological and molecular progressions that affect energy balance and add to the development of obesity	The differences in physiology and metabolism between animal species and humans, it may limit the direct applicability of from animal models to humans	[74]

Studying inherited traits, genetic effects, mutations, and genetic diseases

Animal models aid in studying inherited traits, genetic effects, mutations, and genetic diseases^[75,76]. Many traits related to disease may be passed down through generations. The animal models assist researchers in comprehending how these traits are inherited and expressed. Therefore, it helps us to understand the effects of a single gene in genetic disorders^[76]. Mutational studies are essential for understanding the disease formation mechanism. The animal model aids in studying the impact of mutations on disease development and progression. It is also crucial to understanding mutations and genetic diseases, such as cancers, deafness, blindness, skin pigmentation diseases, glaucoma, and neurodegenerative disorders (such as Huntington’s or Alzheimer’s disease). Several methods have been developed to understand the relationship between mutations and genetic disorders. The methods involve large-scale mutation screens^[77,78], transgenesis^[79,80], gene knockouts and knock-ins^[81,82].

Studying drug discovery and development

Animal models have been utilized to study drug discovery and development, from identifying drug targets to the clinical testing of drugs^[83–85]. These are vital for pre-clinical screening of a newly developed drug molecule^[83,86]. Animal models are used to discover molecules for specific diseases, like a neural disease (Alzheimer’s disease)^[15], and to facilitate the discovery of anticancer drugs^[87]. It can evaluate the ADME (absorption, distribution, metabolism, and excretion) of newly developed drug candidates^[1,88]. It will also assist researchers in evaluating the efficacy, safety, and toxicity of newly developed drugs, as well as understanding their PKs and PDs^[89]. The model indirectly aids in the approval of clinical trials and the regulatory approval process by the FDA. Without the study results of the newly developed drugs in animal models, especially regarding toxicity results, the country’s regulatory authority will not provide permission for the next steps, such as clinical trials^[90].

Studying drug repurposing

From a known drug, drug repurposing uses an existing or investigational drug that can identify a new molecular target, allowing the drug to be used for another symptom^[91]. In this case, the ADME and pharmacological properties of the drug are well understood in the context of drug repurposing^[92]. On the other hand, information about the dose is needed to understand fully. Different animal models, such as the mouse model and zebrafish model, have been used for drug repurposing^[93–95].

Importance of bioactive compounds

Bioactive compounds encompass carotenoids, polyphenols, peptides, minerals, dietary fibers, vitamins, and various other compounds that exhibit several beneficial effects. These compounds have several advantages, especially two broad importance health benefits and industrial applications.

Human health and disease

Bioactive compounds show the vast health benefits and also exhibit the disease presentation properties^[95,96]. These compounds exhibit anti-inflammatory and antioxidant properties. In these plants, phenolic compounds are closely linked to their antioxidant activity, primarily due to their redox properties and their ability to hinder the production of reactive oxygen species (ROS)^[97–100]. One such example is the work of Anorue et al, which reported the use of cashew leaf extract (Anacardium occidentale), rich in plant-derived bioactive compounds. These compounds have significant anti-inflammatory and antioxidant properties^[99]. In addition to these properties, the compounds exhibit antimicrobial and other beneficial effects. It contributes to overall health improvement and also disease prevention^[100]. If we can summarize the benefits of the bioactive compounds, they can be listed as antioxidant activity, anti-inflammatory effects, antimicrobial properties, and therapeutic roles such as antiobesity effects, anticancer, and antidiabetic effects, among others. Therefore, the bioactive compounds have immense benefits^[96,101].

Applications in industry

Bioactive compounds are also employed in various industries, including the pharmaceutical sector, food and beverage industry, and cosmetics industry. Some bioactive compounds are utilized in drug development for the treatment of various diseases. Therefore, it has vast applications in the pharmaceutical sector^[100,102]. Bioactive compounds have numerous applications in the food and beverage industries, as they offer high nutritional value and contribute to food preservation^[103–105]. Bioactive compounds are used in cosmetic products for their skin-protecting and antiaging properties. It holds enormous value in the cosmetic industry^[106].

Different studies on bioactive compounds using different animal models

Various researches have been performed over time to understand the effects of bioactive compounds using different animal models to understand the therapeutic properties or other properties of bioactive compounds, such as antioxidants, anti-inflammatory, anticancer, cardioprotection, neuroprotection, and antimetabolic disorder (Fig. 1). On the other hand, several bioactive compound such as hesperidin, quercetin, gallic acid, lycopene, lutein, resveratrol, capsaicin, catechin, rutin, curcumin, etc. have been tested different animal models from time to time (Table 2). Similarly, several bioactive compounds and their structures with properties have been described by different researchers over time (Table 3)^[107–109]. Some critical studies of bioactive compounds using significant animal models are as follows:

Figure 1.

A schematic diagram illustrates the bioactive compounds and their effects using various animal models to understand their therapeutic properties, as well as other properties such as antioxidant, anti-inflammatory, anticancer, cardioprotective, neuroprotective, and antimetabolic disorder effects.

Table 2.

A list of bioactive compounds that have been tested in different animal models from time to time

Bioactive Compounds	Animal Models
	Mice	Rat	Zebrafish	Rabbit	Drosophila	Guinea pig	Hamster	Ferret	Gerbils	Reference
Hesperidin	✓	✓	✓	✓	✓	✓	✓	-	✓	[110–116]
Quercetin	✓	✓	✓	✓	✓	✓	✓	-	✓	[117–121, 46–214]
Gallic Acid	✓	✓	✓	✓	✓	✓	-	-	-	[125, 135, 190,215–217]
Lycopene	✓	✓	✓	✓	✓	✓	✓	✓	✓	[127, 138, 218–224]
Lutein	✓	✓	✓	✓	✓	✓	-	✓	✓	[130, 142, 225–229]
Resveratrol	✓	✓	✓	✓	✓	✓	✓	✓	✓	[134, 194, 230–234]
Capsaicin	✓	✓	✓	✓	✓	✓	-	✓	✓	[197, 235–240]
Catechin	✓	✓	✓	✓	✓	✓	✓	-	✓	[196, 241–247]
Rutin	✓	✓	✓	✓	✓	✓	✓	-	-	[162, 168, 248–251]
Curcumin	✓	✓	✓	✓	✓	✓	✓	-	✓	[169, 252–257]

Table 3.

Different important bioactive compounds with structure and their properties

Sl. No.	Name of bioactive compounds	Structure	Properties
1.	Quercetin		It inhibits or retard oxidation reactions, and counteracts the damaging effects of oxidation in animal tissues
2.	Curcumin		Anti-inflammatory agents that are non-steroidal in nature, it also have analgesic, antipyretic, and platelet-inhibitory actions
3.	Resveratrol		The resveratrol include its anti-inflammatory and antioxidant effects, platelet aggregation inhibitors and enzyme inhibitors in animal tissue
4.	Rutin		Rutin have high antioxidant, anti-inflammatory, antidiabetic, and anticancer effects. It also exhibits neuroprotective, nephroprotective, and hepatoprotective properties
5.	Catechin		It scavenge free radicals, inhibit lipid peroxidation, and modulate various cellular pathways.
6.	Capsaicin		Capsaicin shown analgesic, anti-inflammatory, antioxidant, and anticancer effects. It can also play a role in cardiovascular health, gastrointestinal issues, and dermatological conditions

Mammalian animal model

Several mammalian animal models have been used from time to understand the therapeutic and other properties of bioactive compounds. The animal models are mice, rats, rabbits, guinea pigs, hamsters, ferrets, and gerbils (Fig. 2). Reported critical studies of bioactive compounds using significant mammalian animal models are noted, which are as follows:

Figure 2.

The diagram illustrates the different mammalian animal models used to study bioactive compounds. The mammalian models are mice, rats, rabbits, guinea pigs, hamsters, ferrets, and gerbils.

Mice

Mice are considered one of the most prevalent animal models used in biomedical research due to their small size, short lifespan, rapid reproductive cycle, and extensive knowledge of their anatomical, physiological, and genetic makeup, which facilitates the manipulation of their genetics. They are used as models of human diseases to test the therapeutic potential, toxicity, and side effects of novel treatments or drugs. Literature reports suggest that mice have been used to study a wide range of conditions, including diabetes, cataracts, obesity, Alzheimer’s disease, cancer, Parkinson’s disease, muscular dystrophy, deafness, hypertension, seizures, cardiovascular disease, and acquired immunodeficiency syndrome^[7]. Hesperidin, a bioflavonoid^[122] present in citrus fruits, olive oil, and tea, displayed a neuroprotective effect in the APP/PS1 mice model. Administration of 40 mg/kg hesperidin via the intragastric route for up to 90 consecutive days in APP/PS1 mice improved cognitive impairment by reducing oxidative stress and elevating the activity of antioxidant genes, such as SOD, catalase, HO-1, and GSH-Px. Additionally, hesperidin treatment reduces inflammation, as evidenced by a decline in TNF-α levels and RAGE/NF-κB activity, as well as the activation of the Akt/Nrf2 pathway^[110]. 100 mg/kg dose of hesperidin inhibits lung cancer proliferation in male C57BL/6N mice by elevating the protein expression of pinX1, which inhibits telomerase catalytic activity to exert its antitumor action^[117]. The hypoglycemic, hypolipidemic, and antioxidant property of quercetin was investigated in C57BL/KsJ-db/db mice receiving quercetin through their diet^[123]. Quercetin demonstrated its antitumor action in mice bearing CT-26 and MCF-7 tumors by inducing apoptosis, offering an increased lifespan compared to the untreated control group^[124]. Gallic acid, with the help of its anti-inflammatory potential, reduced arthritis scores and hind paw volume in collagen-induced arthritis mouse models at 40 and 80 mg/kg doses^[125]. Oral administration of 100 mg/kg dose of gallic acid in Swiss albino mice before exposure to gamma radiation showed its radioprotective effect. It can minimize radiation-induced weight loss and DNA damage, restore antioxidant enzyme levels, prevent micronucleus formation, and more^[126]. Administration of lycopene, a carotenoid in high-fat diet (HFD)-fed mice, inhibited the expression of Srebp-1c, FAS, ACC-1, and STAT3 phosphorylation in the liver, improved the lipid profile, lowered the expression of IL-1β, TNF-α, and C-reactive proteins, which ultimately leads to prevention of lipid accumulation, insulin resistance, inflammation, metabolic disorders^[127]. Lycopene administration at a dose of 3-6 mg/kg body weight for five consecutive weeks in a BALB/c nude xenograft mouse model of colorectal tumor markedly augmented apoptosis, reduced the protein expression of p21 and proliferating cell nuclear antigen (PCNA), and increased the p21CIP1/WAF1 cell cycle inhibitor protein. It also induced E-cadherin protein and inhibited β-catenin protein, along with a reduction in MMP-9, COX-2, PGE2, and ERK1/2 phosphorylation^[128]. Oral administration of lycopene at 1-10 mg/kg body weight dose once/day for 7 days in male Balb/c nude mice reduced prostate cancer progression, improved mice survival by reducing tumor regulatory T cells (Tregs), increasing tumor inflammatory cells (Tc1, Th1, Tc17, Th17 cells), decreasing level of serum inflammatory markers (TNF-α, IL-1, IL-6, IL-8)^[129]. Lutein prevented the disruption of tight junctions in the retinal pigment epithelium. It decreased the elevated ROS level, which was increased due to light (2000 lux, 3h) induced age-related macular degeneration^[130]. Oral administration of lutein in mice for 4 weeks improved femoral bone mass, suppressed bone resorption as well as influenced bone formation^[258]. Lutein supplementation decreased the formation of atherosclerotic lesions^[259] and protect skin from UV-induced inflammation, skin damage, swelling, and hyperplasia in hairless mice models^[260]. Oral administration of myrcene at a dose of 50 mg/kg body weight in post-adrenalectomized mice resulted in a reduction in proinflammatory cytokine levels and immunomodulatory factors, such as IFN-γ and NF-κB. It boosted the antioxidant machinery by increasing catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) levels while decreasing MDA levels in treated mice compared to untreated mice, thereby lessening renal inflammation^[261]. Borneol, a bicyclic monoterpene, attenuated cerulein-induced oxidative stress as well as inflammation in an acute pancreatitis mice model via downregulation of NF-κB and upregulation of Nrf2 signaling^[262]. Geraniol, a monoterpenic alcohol, offers antioxidant, anti-inflammatory, immunomodulatory, and antibacterial action to protect against methicillin-resistant Staphylococcus aureus infections in mice^[263]. Citral, found in citratus essential oil, offers an anti-inflammatory effect in a carrageenan-induced hyperplasia and pleurisy mouse model^[264]. Carvacrol decreased the levels of inflammatory biomarkers (TNF-α, IL-4, IFN-γ, IL-5, IL-13). It increased the levels of antioxidant molecules (GSH, SOD) to confer protection against ovalbumin-induced bronchial asthma in rats^[265].

Rat

Rats, like mice, are small in size, have a short developmental time, a known genetic makeup and microbial status, resemble human diseases, and have a tractable nature, making them one of the most suitable animal models. Rats are used to study genetics, infectious diseases, metabolic diseases, nutrition, immunology, neurology, behavior, and more. They are vigorously used for drug discovery, testing their toxicity, effectiveness, etc^{[10,266,267,131]}. With the administration of hesperidin Sprague-Dawley rats with a dose 100 mg/kg/day in alleviates methotrexate-induced neurotoxic effects by lowering oxidative stress and inducing hippocampal neurogenesis^[111]. It has been reported that hesperidin lowers cardiovascular risk factors such as increased blood pressure and hypercholesterolemia. Oral administration of hesperidin resulted in a decrease in systolic blood pressure (SBP) dose-dependently in spontaneously hypertensive rats (SHR). Hesperidin, administered at a dose of 50 mg/kg when injected intraperitoneally in SHR, resulted in a significant decline in SBP^[132]. Hesperidin lowers liver weights, hepatic steatosis, serum cholesterol level, and adipose tissue in male Wistar rats fed with a high-cholesterol diet to treat fatty liver and hypercholesterolemia^[133]. Administration of quercetin at 15 mg/kg dose in streptozotocin-(STZ) induced diabetes rats for 8 weeks boosts the action of antioxidant enzymes, reduces the ROS level, lowers the TUNEL-positive apoptotic cell numbers, improves PCNA activity to protect testicular DNA damage^[118] Another study revealed that the administration of resveratrol in STZ-induced diabetic rats protects pancreatic beta cells, improves insulin sensitivity, decreases blood glucose levels, and decreases oxidative stress^[134]. Gallic acid offers neuroprotective action in rat models of traumatic brain injury by lowering lipid peroxidation and IL-1β, TNF-α, IL-6, and malondialdehyde in the brain^[135]. Oral administration of naringenin in diabetic rats at 50 mg/kg/day dose reduced GSH levels and antiapoptotic markers, suggesting its antioxidant, antiapoptotic capability as well as improves neurodegeneration by reducing tropomyosin-related-kinase B, brain-derived neurotrophic factor, in the diabetic retina^[136]. Oral supplementation of lycopene in HFD induced rats significantly decreased the accumulation of triglycerides, low-density lipoprotein (LDL), cholesterol, β-hydroxybutyrate, etc., along with elevating hepatic PPAR-γ level. Additionally, lycopene upregulated the enzymatic activities of SOD, CAT, and GR. It increased GSH levels while downregulating the levels of nitric oxide (NO), lipid peroxide (LPO), and myeloperoxidase (MPO) to alleviate hepatic oxidative stress. Lycopene also alleviated the levels of inflammatory markers, such as TNF-α and IL-1β, which were induced by HFD administration. Lycopene supplementation also exerts an antifibrotic effect, as evident by reduced expression of TGF-β and α-SMA, as well as a cardioprotective effect through decreasing the atherogenic index^[137]. Another study revealed that lycopene administration at a dose of 25 mg/kg body weight in male Wistar albino rats with a HFD could ameliorate the HFD-induced alterations that lead to different metabolic disorders and liver injury through its antioxidant and anti-inflammatory actions, suggesting its potential to improve obesity^[138]. Daily usage of lycopene at a concentration of 50-150 mg/kg body weight was also found to reduce the risk of developing cancer by inducing SOD, CAT, and GPx activities, along with boosting immunity, in a N-methyl-N′-nitro-N-nitrosoguanidine-stimulate rat gastric carcinoma model^[139]. Lycopene intervention at a dose of 25 mg/kg body weight for 4 weeks in N-nitroso methyl benzylamine-induced esophageal cancer in F344 rats facilitates apoptosis by upregulating the expression of PPAR-γ, caspase-3, and diminishing inflammation through lowering NF-κB and COX-2 protein expression, thereby exerting its anticancer action^[140]. The anticataract activity displayed by lutein was induced by eicosapentaenoic acid and docosahexaenoic acid. Lutein improved the enzymatic activities of antioxidants (SOD, CAT, GST, GPx) and lowered malondialdehyde levels in serum and lens of cataract-bearing male Wistar rat pups. It also reduced serum eicosanoids, such as leukotriene B4, leukotriene C4, and PGE2, as well as cytokines like TNF-α and IL-1β, and C-reactive proteins^[141]. Ovariectomized female Sprague-Dawley Wistar rats treated with body weight 50 mg/kg doses of lutein for 4 weeks protected rats from osteoporosis by improving antioxidant status and anti-inflammatory responses. Lutein treatment in ovariectomized rats decreased lipid peroxidation, prevented the activation of NF-κB, and downregulated the levels of inflammatory markers (IL-6, TNF-α, IL-8). Additionally, it induced the initiation of Nrf2-mediated antioxidant gene expression (NQO1, HO-1)^[142]. Ginsenoside Rg1, a bioactive terpenoid present in roots, rhizomes, and aerial parts of Panax ginseng, protects from stress-induced major depression disorders upon its intraperitoneal administration at 40 mg/kg dose in rats^[143]. Perillyl alcohol, a natural monoterpene, exhibited promising oxidative stress-neutralizing, inflammation-reducing, antiproliferative effects in monocrotaline-induced pulmonary arterial hypertension in rats^[144]. Treatment with 25, 50, and 100 mg/kg body weight doses of limonene in ethanol-induced gastric ulcers in a male Wistar rat model, exerting a gastroprotective effect via a decline in myeloperoxidase activity, elevation of antioxidant enzyme activities, and upregulation of an anti-inflammatory mechanism^[145]. α-pinene (50 and 100 mg/kg) ameliorated neuroinflammation, as evident by the reduction in TNF-α and IL-1β, and prevented apoptosis by downregulating the Bax marker (proapoptotic marker) and upregulating the expression of the Bcl-2 marker (antiapoptotic) in the ischemic rat brain^[146].

Rabbits

Rabbits have been employed as a suitable animal model in atherosclerosis, osteoporosis, teratogenicity testing of novel compounds, and immunology research^[147–149]. It has been utilized as a human pregnancy model for the development of polyclonal antibodies due to its larger blood volume^{[10, 150, 151]}. Its larger body size made it well-adapted for the surgical implantation of biomedical devices^{[10, 152, 153]}. Topical administration of hesperidin in rabbit hypertrophic scar model showed a reduction in scar formation^[112]. Oral administration of quercetin at 25 mg/kg dose displayed enhanced SOD, tissue inhibitor of metalloproteinases activity, reduced matrix metalloproteinase-13 activity in the surgical-induced osteoarthritis rabbit model to offer protection from oxidative stress and inflammation^[119]. Quercetin, owing to its antioxidant effect, maintains the semen quality in rabbits by reducing heat stress-induced damage^[154]. Sadraei and Tabesh reported the relaxant effect of quercetin in rabbit-isolated bladders^[155]. The coadministration of quercetin (25 mg/kg) with a hypercholesterolemic diet in rabbit’s downregulates the inflammatory mediators, such as cyclooxygenase, lipoxygenases, nitric oxide synthase, myeloperoxidase, and C-reactive protein levels, thereby decreasing inflammation^[156]. Guava leaves extract supplementation at 20 mg/kg diet in rabbits lowered total cholesterol, LDL, HDL, VLDL, and triglycerides levels as well as increased antioxidant genes to mitigate ROS with the help of its constituent bioactive compounds such as gallic acid, catechin, ferulic acid, caffeic acid^[157].

Guinea pig

Guinea pigs (Cavia porcellus) are small-sized rodents widely used in the investigation of infectious diseases (such as tuberculosis and syphilis), asthma, and cholesterol metabolism^[158,159]. In a carrageenan-induced paw edema model in guinea pigs the oral administration of hesperidin at a dose of 40 mg/kg body weight revealed its anti-inflammatory potential^[113]. Upon oral administration in guinea pigs, trans-resveratrol improved the activity of detoxifying enzymes, such as cardiac catalase and DT-diaphorase, providing a cardioprotective effect against ROS-induced menadione toxicity^[160]. Resveratrol also functions as a relaxant in guinea pigs in a dose-dependent manner by inducing fundic relaxation via nitric oxide and ATP-sensitive potassium channels^[161]. Quercetin decreased scleral remodeling in myopic guinea pigs by its inhibition in PERK-EIF2α signalling^[120]. Quercetin and rutin exert antiasthmatic effects in ovalbumin-sensitized conscious guinea pigs^[162].

Hamster

Hamsters belonging to the order Rodentia are used in research due to their anatomical and physiological resemblance and susceptibility to developing human infections. They are predominantly used in the treatment of infectious diseases, cancer, cardiovascular disease, metabolic disorders, and reproductive endocrinology, among others^[163–166]. Pre-treatment with α-glucosyl hesperidin reduces the extent of oral mucositis, a side effect of 5-fluorouracil-mediated cancer chemotherapy, without compromising its anticancer activity^[167]. Quercetin displayed a chemoprotective effect in 7,12-dimethylbenz[a]anthracene-induced HBP carcinoma not only by reducing tumor incidence and burden but also upregulating the expression of caspase, cytochrome C, BAX, BID, BAD, RECK, TIMP-2 and downregulating the expression of Bcl-2, BCL-xL, MMPs, HIF1α, VEGF and its receptors, HDAC-1 indication its proapoptotic, antiproliferative, antiangiogenic, antimetastatic potential^[121]. Short-term treatment with a high concentration of rutin in a hypercholesterolemic Golden Syrian hamster model reduced the level of triglycerides in plasma, along with no alteration in total cholesterol and high-density lipoprotein cholesterol levels. It also did not produce any immunotoxic effect^[168]. Curcumin supplementation with a HFD in male hamsters for up to 12 weeks resulted in a decline in cholesterol levels in serum and liver, inhibits cholesterol absorption in the intestine, and prevents bile cholesterol supersaturation^[161].

Ferret

The ferret (Mustela putorius furo) belongs to the order Carnivora and is a small, non-rodent animal applied in biomedical study since the early twentieth century. Its social behavior along with early sexual maturity as compared to that of the other large animals, hardy nature, diet, vomiting ability, requirement of low amount of test sample, susceptibility to various human teratogens, human influenza virus, similarities to human respiratory tracts, etc makes it one of the best model organisms. Ferrets are used as animal models for studying influenza, infectious respiratory diseases, gastrointestinal diseases, cystic fibrosis, lung cancer, cardiovascular research, neuroscience, and toxicology^[170]. Daily supplementation of lycopene at 30 and 90 mg/kg body weight doses for 22 weeks in NNK-exposed ferrets resulted in the inhibition of inflammation, chronic bronchitis, emphysema, the incidence of preneoplastic lesions, and lung cancer. It also prevented cholesterol accumulation by upregulating the transcription of PPARα, LXRα, ABCA1, and ABCG1 in the lung, which are associated with reverse cholesterol transport^[171]. Another report showed that lycopene could reduce the expression of p53, cyclin D1, and PCNA and increases the expression of p21, Bax-1, and cleaved caspase 3 to exert its antiproliferative and apoptosis-inducing action in gastric mucosa of ferrets^[172]. Lycopene supplementation at doses of 1.1 mg/kg and 4.3 mg/kg body weight for nine consecutive weeks in a smoke-induced ferret lung cancer model upregulated IGFBP-3 levels in plasma, reducing the risk of lung cancer and providing protection against lung squamous metaplasia^[173]. Administration of β-cryptoxanthin, a natural carotenoid in smoke-induced ferrets, downregulated NF-κB activation and reduced TNF-α level, preventing oxidative DNA damage to reduce inflammation and oxidative stress in the lung^[174].

Gerbils

Among 100 different species of gerbils documented, the Mongolian gerbil is most frequently used for research purposes. The small, long-tailed, burrowing, herbivorous rodent is considered a notable model for studying cerebral ischemia, stroke, epilepsy, and infectious diseases, among others^{[10, 175]}. Their susceptibility to viral, bacterial, and parasitic pathogens, as well as their nonaggressive nature and unique anatomical and physiological characteristics, make them suitable laboratory research animals^[176]. Z-ajoene, present in the ethanolic extract of garlic, when administered orally at a 25 mg/kg concentration in male gerbils before forebrain ischemia, decreased lipid peroxidation in the hippocampal CA1 region, protecting I/R-induced delayed neuronal death and gliosis^[177]. Oral treatment with caffeine at 0.2% in drinking water for four consecutive weeks in Mongolian gerbils markedly upregulated the binding of the adenosine A1-receptor ligand [3H] cyclohexyl-adenosine to different regions of brain encompassing the hippocampal CA1 area, displayed minimal neuronal damage such as reduced ischemic necrosis in pyramidal cells of CA1 region to mitigate ischemic hippocampal damage^[178]. Chlorogenic acid attenuated ischemia-induced hippocampal damage by boosting the neuroprotective activity of PEP-1-rpS3, as reported in the gerbil model^[179]. Additionally, pretreatment with 30 mg/kg chlorogenic acid in gerbils ameliorated ischemia-induced cognitive deficits by decreasing the levels of proinflammatory cytokines, such as TNF-α and IL-2, and increasing the levels of anti-inflammatory cytokines, including IL-4 and IL-13, while also boosting the enzymatic activity of antioxidant molecules^[180].

Non-mammalian animal model

Several non-mammalian animal models have been used from time to understand the therapeutic and other properties of bioactive compounds. The significant non-mammalian animal models are zebrafish, Drosophila melanogaster, and C. elegans (Fig. 3). Reported critical studies of bioactive compounds using different animal models are noted, which are as follows:

Figure 3.

The diagram illustrates the different non-mammalian animal models used to study bioactive compounds. The significant non-mammalian animal models are zebrafish, Drosophila melanogaster, and Caenorhabditis elegans.

Zebrafish

Zebrafish (Danio rerio) is extensively used to study human diseases, including developmental biology, cancer, neurological disorders, metabolic disorders, genetic disorders, and cardiovascular disease, due to its genomic resemblance to humans (approximately 70%)^{[94,181–185]}. Additionally, their rapid development, high reproductive rate, large number of offspring, low maintenance, regenerative potential, ease of genetic manipulation, and transparent embryos offer extra benefits^[186]. Intraperitoneal administration of quercetin in lipopolysaccharide-induced zebrafish at 50 and 100 mg/kg doses for 7 days mitigated oxidative stress and neuroinflammation, as evident from the reduction in lipid peroxidation, AChE, TNF-α, and IL-1β^[187]. Geethanjali et al explored the neuropharmacological potential of silibinin and naringenin against bisphenol A-induced neurotoxicity and oxidative damage in a zebrafish model^[188]. Silibinin and naringenin also reduce acute stress-induced anxiety in zebrafish^[189]. Pretreatment with gallic acid (10 mg/L) inhibited ethanol-induced oxidative stress and degradation of extracellular nucleotides through regulating nucleoside triphosphate phosphohydrolase (NTPDase) activity^[190]. Hesperidin also protects from acute alcoholic liver diseases, as investigated in zebrafish larvae exposed to 350 mM ethanol. Here, hesperidin markedly reduced the expression of HMGCR, CYP2Y3, CYP3A65, FASN, and FADS2, along with improved hepatic morphological damage^[114]. Zebrafish pretreated with hesperidin and berberine prevents memory impairment induced by pentylenetetrazole^[191].

Drosophila melanogaster

The short life cycle, high fecundity, ease of genetic manipulation, and low maintenance requirements make the fruit fly Drosophila melanogaster a suitable model organism for scientific research. They are commonly used for genetics, embryonic development, neurobiology, behavior, and aging studies^[192]. Dietary phytochemicals, such as gallic acid, quercetin, and limonene, demonstrated antioxidant and antimutagenic effects in the induced D. melanogaster model. In the phytochemicals-treated flies, an upregulation in the level of CAT, SOD glutathione S-transferase (GST), GSH, and downregulation in lipid peroxidation (MDA content) was observed as compared to the urethane treated group. The phytochemical treatment also resulted in a significant reduction in SLRL (sex-linked recessive lethal) mutations compared to the urethane-treated group^[193]. Supplementation with resveratrol and aloe vera extract in larval diet increased the longevity of adult flies, prevented neurodegeneration, and increased locomotor activities along with upregulation in the detoxifying antioxidant enzymes activity (SOD)^[194]. Pomegranate juice supplementation also extends the lifespan of adult flies, i.e., 18% in males and 8% in females. Additionally, the fecundity rate, resistance to free radical-induced stress, and fungal infection were increased^[195]. Catechins present in green tea increase SOD and CAT activities in D. melanogaster, promoting a prolonged lifespan^[196]. Capsaicin treatment confers protection against Parkinson’s disease in flies by causing a delay in climbing ability^[197].

Caenorhabditis elegans

Caenorhabditis elegans is a free-living nematode primarily found in nutrient-rich and bacterial environments. They are widely used as an in vivo model to investigate the diseases such as Alzheimer’s and Parkinson’s disease. However, studies have also revealed that C. elegans can be used in many food-derived bioactive compounds for better health^[198]. Over the past few years, the C. elegans aging model has been utilized for various metabolic, aging, and longevity pathways to achieve health benefits through bioactive phytochemicals. Significant genes of IIS pathways get protected and facilitated by epigallocatechin gallate (EGCG) and epicatechin from oxidative stress, thereby enhancing longevity^[199]. Among many bioactive compounds, resveratrol has the property of reducing lipid activity, thereby improving lifespan and mitigating oxidative stress. Aranaz et al also revealed that phenolic compounds and phenolic acids induced in C. elegans significantly decrease the fat content in the intestine^[200].

Recommendation on the use of AI in the field

AI is used significantly in different fields. AI has already been used to study various aspects of animals. AI is used to study the anatomy of animals^[201]. AI-powered imaging has immense potential to revolutionize animal diagnostics and surgery, especially in veterinary science. AI-powered techniques have been used in anatomical imaging, which helps in diagnostics and surgery for animals in veterinary science^[202]. On the other hand, several studies have been conducted from time to time using AI or ML (machine learning) or DL (deep learning)^[203–208]. A large language model (LLM) is a subset of DL. Researchers reported that LLM or ChatGPT has helped in different areas of medical education, including veterinary anatomy education^[209,210]. AI can be used in the area of animal models in evaluating bioactive compound which will enhance the efficiency, prediction accuracy. AI-powered tools, technologies, and datasets will predict better outcomes in animal studies, leading to more effective and targeted research.

Limitation

We have tried to cover most of the literature in this field. Literature reviews are related to the time. In the literature, we aimed to provide a snapshot of the research landscape at the time of the review. However, future studies may alter the conclusions of the study. Such an example is the entry of AI in this field. New developments in this field may alter the conclusion. Another challenge arises during organizing and synthesizing the literature from numerous studies into a meaningful and coherent narrative.

Future directions

Although animal models are valuable, they have certain limitations, such as high susceptibility to specific diseases and a relatively short lifespan (Table 4). However, it has been found that animal models are crucial to evaluating bioactive compounds. Ongoing advancements in animal models, such as transgenic or knockout models, enable researchers to conduct more sophisticated studies using these models, which will aid in various drug discovery studies involving bioactive compounds shortly. For refined studies, employing in vitro and in silico approaches, alongside next-generation research, will be immensely beneficial. Researchers should employ in vitro and in silico approaches in the future, utilizing bioactive compounds, from drug discovery to elucidating disease mechanisms.

Table 4.

Importance of different animal models and limitations in biological and medical science

Sl. No.	Name of animal models	Importance in biological and medical science	Limitations	Reference
1.	Rabbit	Used as model organism for surgically created osteoarthritis, study of drugs, cholesterol research, wound healing purpose, Alzheimers disease and cardiovascular diseases	High susceptibility to specific diseases, difficulties for genetic manipulation, with much rearing cost	[268–272]
2.	Golden hamster	Employed for reproductive research (having short gestation period), important for micro-circulation research, infection and cancer model, vaccine research	Short lifespan, having developmental block in in vitro studies	[273–275]
3.	Rats	Stress-free breeding, less animal handling and rearing care, minimum genetic variations	Not considerate as a suitable animal model for inflammation study	[276–278]
4.	Mice	Act as model for wide range of human diseases, including cancer, heart disease, infectious diseases, and genetic disorders; used for precision medicine research		[3,279]
5.	Ferret	A well-suited for studying respiratory viruses and several human diseases (cardiovascular disease, cystic fibrosis, and certain types of cancer)	It require specific care and husbandry practices and prone to certain diseases	[170,280]
6.	Zebrafish	High reproductive rate it enabling large-scale genetic studies and drug screenings. Best model for cardiovascular diseases, metabolic disorders, including obesity and diabetes	Dissimilarity of some organs like the respiratory system and the reproductive system	[3,281]
7.	Fruit fly	Fundamental genetic mechanisms, including gene regulation, development, and evolution, suitable diseases model like Parkinson’s and Alzheimer’s	It showing genetic redundancy and lack of adaptive immune system	[3,282]
8.	Caenorhabditis elegans	It used for studied to understand the nervous system and behavior as shown similarities in neural circuitry and genetic makeup with humans	It highly temperature sensitive and lacking of complex anatomy	[283]

Conclusion

Biomedical research relies on the use of animal models, which facilitate understanding of disease pathogenesis at the molecular level and aid in testing new therapies. For in vivo studies, the significance of animal models is undeniable for any biological or medical research. Similarly, the significance of animal models in studying bioactive compounds is unquestionable. In addition to studying disease mechanisms for drug discovery, animal models have also been utilized in tissue engineering and vaccine research^[211,284]. On the other hand, researchers are discussing the potential replacement of animal models. They are working towards the “3Rs” approaches: “Replacement,” “Reduction,” and “Refinement”^[212]. Animal models remain crucial for pre-clinical to clinical research. However, there is a shift toward animal-free technologies. Although several scientists have highlighted that animal models are essential for scientific discovery and biomedical progress^[5].

Here, we aim to draw on proof provided by diverse examples to demonstrate the scope of studying bioactive compounds in a wide range of animal models. With ongoing investment in bioactive compound research, animal models of disease offer experimental resilience that can serve as effective human disease models, enhancing our knowledge of disease pathogenesis and facilitating the development of innovative and effective next-generation therapies for a wide range of human diseases using bioactive compounds.

Ethical approval

This article does not require any human/animal subjects to acquire such approval.

Consent

Informed consent was not required for this review article.

Sources of funding

This study received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author contributions

A.D.: Validation, Data curation, Investigation, Writing – Original Draft, Writing – review & editing. M.B.: Validation, Investigation, figure development. C.C.: Conceptualization, Data curation, Investigation, Writing – Original Draft, Writing – review & editing. A.D.: Validation, Investigation, supervision.

All authors critically reviewed and approved the final version of the manuscript.

Conflicts of interest disclosure

All authors report no conflicts of interest relevant to this article.

Guarantor

Chiranjib Chakraborty.

Research registration unique identifying number (UIN)

Not applicable.

Provenance and peer review

Not commissioned, internally peer-reviewed.

Data availability statement

The data in this correspondence article are not sensitive in nature and are accessible in the public domain. The data are therefore available and not of a confidential nature.