Encyclopedia
Please note this is a comparison between Version 1 by An Zhu and Version 2 by Camila Xu.
Natural products are small molecules naturally produced by multiple sources such as plants, animals, fungi, bacteria and archaea. They exert both beneficial and detrimental effects by modulating biological targets and pathways involved in oxidative stress and antioxidant response. Natural products’ oxidative or antioxidative properties are usually investigated in preclinical experimental models, including virtual computing simulations, cell and tissue cultures, rodent and nonhuman primate animal models, and human studies.
- natural products
- Caenorhabditis elegans
- antioxidation
- oxidative stress
1. Introduction
1.1. Oxidation and Antioxidation under Physiological and Pathological Conditions
1.1. Oxidation and Antioxidation under Physiological and Pathological Conditions
Redox homeostasis is central to life, ranging from bioenergetics to metabolism and biological functions [1]. To defend against oxidative damage, organisms have evolved defenses that primarily rely on antioxidant enzymes, the supply of their substrates and repairing the damage. Antioxidant defenses can be enhanced through physiological signaling, dietary components and potential pharmaceutical interventions, thereby improving the capacity to scavenge oxidants and electrophiles. In 1954, Commoner et al. first described the occurrence of oxidative damage in a biological environment [2]. In 1985, the concept of “oxidative stress” was first applied to the biological logic system, which is defined as any oxidative damage in which excessive production of ROS or inadequate antioxidant defense occurs [3].
An imbalance between the production of oxidants and antioxidant defenses leads to damage to biological systems. This involves the chemistry of reactions of reactive species derived from oxygen, so-called ‘oxidative stress.’ Oxidative stress has been shown to participate in a variety of diseases, including cardiovascular disease, degenerative disease and cancer, and multiple mechanisms by which oxidants contribute to cellular damage have been revealed [4,5,6]. However, the degree of oxidative stress involved in the pathology of diseases is quite variable. This variability makes it difficult to improve the antioxidant effects of therapy. Most of the antioxidant defense in cells is provided not by either exogenous or endogenous small molecules acting as scavengers but by antioxidant enzymes using their specific substrates to reduce oxidants. The therapeutic use of small molecules has been disappointing, largely due to overoptimism and incorrect assumptions about how antioxidants work [7]. Furthermore, antioxidant enzymes react with oxidants thousands to millions of times faster than small molecules and provide the predominant antioxidant defense [7,8]. Therefore, the major therapeutic opportunities lie in preventing the production of oxidants that directly damage macromolecules, inhibiting the downstream signaling of oxidants that leads to inflammation or cell death, and increasing both antioxidant enzymes and their substrates. Currently, clinical trials based on this approach are underway [4]. A greater understanding of the mechanisms of action of antioxidants and where and when they are effective may provide a rational approach to addressing oxidative stress.
Redox homeostasis is central to life, ranging from bioenergetics to metabolism and biological functions [1]. To defend against oxidative damage, organisms have evolved defenses that primarily rely on antioxidant enzymes, the supply of their substrates and repairing the damage. Antioxidant defenses can be enhanced through physiological signaling, dietary components and potential pharmaceutical interventions, thereby improving the capacity to scavenge oxidants and electrophiles. In 1954, Commoner et al. first described the occurrence of oxidative damage in a biological environment [2]. In 1985, the concept of “oxidative stress” was first applied to the biological logic system, which is defined as any oxidative damage in which excessive production of ROS or inadequate antioxidant defense occurs [3].
1.2. Role of Oxidation and Antioxidation Based on the Complex Composition of Natural Products
In the pharmaceutical industry, the approved medicinal products are mostly composed of a single molecule or combinations of single molecules whose pharmacological properties and safety are characterized during the preclinical phase and then validated in human trials [9]. The plants, animals, fungi, bacteria and archaea are a source of drugs of natural origin. Each of them contains hundreds of compounds, which belong to different classifications, such as diterpenoid, flavonoid, coumarin, steroid, hydrocarbon, carboxylic acid, ester, aldehyde, alcohol, ketone, ether, epoxide, phenol and so on [10]. Although many approved drugs isolated from natural sources have been proven to be involved in oxidation or antioxidation in experimental models and human studies [11], their mechanisms of action are usually not yet fully elucidated since it is difficult to identify the effective compounds and dosages. For example, researchers have demonstrated that Glycyrrhizae radix extract is beneficial in preventing oxidative damage in Caenorhabditis elegans (C. elegans). However, this extract is a mixture of all water-soluble compounds from Glycyrrhizae radix, so it is still unknown which compound plays an antioxidant role in this protective effect [12]. The composition of natural products is complex, thus posing a challenge to clarify their role in the oxidation and antioxidation process clearly. Along with the improved sensitivity of trace detection techniques (e.g., high-resolution mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy, ultra-performance liquid chromatography), an increasing number of newly discovered compounds have been isolated from natural products [13]. By means of plane and spatial structure analysis methods (e.g., nuclear magnetic resonance, atomic spectroscopy, circular dichroism spectrum, single crystal X-ray diffraction), the structures of these novel compounds can be resolved, making it possible to reveal the oxidation and antioxidation effects and mechanisms [14,15].
An imbalance between the production of oxidants and antioxidant defenses leads to damage to biological systems. This involves the chemistry of reactions of reactive species derived from oxygen, so-called ‘oxidative stress.’ Oxidative stress has been shown to participate in a variety of diseases, including cardiovascular disease, degenerative disease and cancer, and multiple mechanisms by which oxidants contribute to cellular damage have been revealed [4][5][6]. However, the degree of oxidative stress involved in the pathology of diseases is quite variable. This variability makes it difficult to improve the antioxidant effects of therapy. Most of the antioxidant defense in cells is provided not by either exogenous or endogenous small molecules acting as scavengers but by antioxidant enzymes using their specific substrates to reduce oxidants. The therapeutic use of small molecules has been disappointing, largely due to overoptimism and incorrect assumptions about how antioxidants work [7]. Furthermore, antioxidant enzymes react with oxidants thousands to millions of times faster than small molecules and provide the predominant antioxidant defense [7][8]. Therefore, the major therapeutic opportunities lie in preventing the production of oxidants that directly damage macromolecules, inhibiting the downstream signaling of oxidants that leads to inflammation or cell death, and increasing both antioxidant enzymes and their substrates. Currently, clinical trials based on this approach are underway [4]. A greater understanding of the mechanisms of action of antioxidants and where and when they are effective may provide a rational approach to addressing oxidative stress.
1.2. Role of Oxidation and Antioxidation Based on the Complex Composition of Natural Products
In the pharmaceutical industry, the approved medicinal products are mostly composed of a single molecule or combinations of single molecules whose pharmacological properties and safety are characterized during the preclinical phase and then validated in human trials [9]. The plants, animals, fungi, bacteria and archaea are a source of drugs of natural origin. Each of them contains hundreds of compounds, which belong to different classifications, such as diterpenoid, flavonoid, coumarin, steroid, hydrocarbon, carboxylic acid, ester, aldehyde, alcohol, ketone, ether, epoxide, phenol and so on [10]. Although many approved drugs isolated from natural sources have been proven to be involved in oxidation or antioxidation in experimental models and human studies [11], their mechanisms of action are usually not yet fully elucidated since it is difficult to identify the effective compounds and dosages. For example, researchers have demonstrated that Glycyrrhizae radix extract is beneficial in preventing oxidative damage in Caenorhabditis elegans (C. elegans). However, this extract is a mixture of all water-soluble compounds from Glycyrrhizae radix, so it is still unknown which compound plays an antioxidant role in this protective effect [12]. The composition of natural products is complex, thus posing a challenge to clarify their role in the oxidation and antioxidation process clearly. Along with the improved sensitivity of trace detection techniques (e.g., high-resolution mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy, ultra-performance liquid chromatography), an increasing number of newly discovered compounds have been isolated from natural products [13]. By means of plane and spatial structure analysis methods (e.g., nuclear magnetic resonance, atomic spectroscopy, circular dichroism spectrum, single crystal X-ray diffraction), the structures of these novel compounds can be resolved, making it possible to reveal the oxidation and antioxidation effects and mechanisms [14][15].
1.3. Advantages of C. elegans as a Model of Oxidation and Antioxidation Assessment
C. elegans was introduced to science as a model organism for development and neurobiology in 1965 [16]. Nematodes share approximately 60–80% of genes and 12 signaling pathways with humans [17][18], and notes on gene function can be obtained from the WormBase online consortium. C. elegans shows many advantages, such as self-fertilization, a short life cycle, a small and transparent body, ease of culture, simple operation and low cost, without limitations in ethics, showing great potential as an alternative model for the 3R principle [19][20]. Despite their simple structure, nematodes have complete muscle, subcutaneous tissue, nervous system, gut, gonads, glands and excretory system, and many basic physiological processes and oxidative stress responses of higher organisms are also highly conserved in nematodes [21][22]. Therefore, nematodes have great potential as models for evaluating the pharmacological and toxicological effects in humans [23], and researchers systematically summarize the advantages and disadvantages of C. elegans in the pharmacotoxicology research field and compare this model with other classical models, namely Drosophila, zebrafish, yeast, cell and mammalian in
1.3. Advantages of C. elegans as a Model of Oxidation and Antioxidation Assessment
C. elegans was introduced to science as a model organism for development and neurobiology in 1965 [16]. Nematodes share approximately 60–80% of genes and 12 signaling pathways with humans [17,18], and notes on gene function can be obtained from the WormBase online consortium. C. elegans shows many advantages, such as self-fertilization, a short life cycle, a small and transparent body, ease of culture, simple operation and low cost, without limitations in ethics, showing great potential as an alternative model for the 3R principle [19,20]. Despite their simple structure, nematodes have complete muscle, subcutaneous tissue, nervous system, gut, gonads, glands and excretory system, and many basic physiological processes and oxidative stress responses of higher organisms are also highly conserved in nematodes [21,22]. Therefore, nematodes have great potential as models for evaluating the pharmacological and toxicological effects in humans [23], and we systematically summarize the advantages and disadvantages of C. elegans in the pharmacotoxicology research field and compare this model with other classical models, namely Drosophila, zebrafish, yeast, cell and mammalian in
Table 1. In recent decades, nematodes have become very popular for high-throughput drug screening. This allows the drug discovery process to be studied throughout the life cycle and the manipulation of individual genes or genomes, such as N-ethyl-N-nitrosourea or ethyl methanesulfonate mutations, RNA interference (RNAi) and CRISPR [24]. Nematodes have been applied to the mechanism of action of addictive drugs [25], the pharmacological effects of neurodegenerative diseases such as Alzheimer’s disease (AD) drugs [26], and the neurotoxicity of anticancer drugs [27]. Currently, a large number of mutants have been used to study the molecular mechanism of effective components of natural products. For example, ursolic acid affects the stress response of nematodes by disturbing genes expressions of dopamine receptors [28][29], as also applies in the toxicological mechanism of the extract of Peganum harmala L. seeds [30], and the intestinal toxicity mechanism of Euphorbia factor L1 [31], etc. In addition, there are many reports on the effectiveness of antioxidants in C. elegans, as they can be used to establish an antioxidant stress response model for the assessment of antioxidant capacities in vivo [32][33][34][35].
. In recent decades, nematodes have become very popular for high-throughput drug screening. This allows the drug discovery process to be studied throughout the life cycle and the manipulation of individual genes or genomes, such as N-ethyl-N-nitrosourea or ethyl methanesulfonate mutations, RNA interference (RNAi) and CRISPR [24]. Nematodes have been applied to the mechanism of action of addictive drugs [25], the pharmacological effects of neurodegenerative diseases such as Alzheimer’s disease (AD) drugs [26], and the neurotoxicity of anticancer drugs [27]. Currently, a large number of mutants have been used to study the molecular mechanism of effective components of natural products. For example, ursolic acid affects the stress response of nematodes by disturbing genes expressions of dopamine receptors [28,29], as also applies in the toxicological mechanism of the extract of Peganum harmala L. seeds [30], and the intestinal toxicity mechanism of Euphorbia factor L1 [31], etc. In addition, there are many reports on the effectiveness of antioxidants in C. elegans, as they can be used to establish an antioxidant stress response model for the assessment of antioxidant capacities in vivo [32,33,34,35].
Table 1.
Advantages and disadvantages in the pharmacotoxicology of C. elegans and other models.
| Life Cycle | Metabolism | High-Throughput Screening | Costing | Live Imaging | Ethics and Welfare | 3R | Phylogenetics | Cognitive Behavior | Homology with Human | Immune System | Genetic Manipulation | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| C. elegans | Very short lifespan (approximately 3 weeks), small body (1 mm), short reproductive cycle (3.5d) and large broodsize | As a multicellular organism composed of the brain, pharynx, intestine, gonads, | Available | Easy and low-cost in infrastructure and maintenance | Available | √ | √ | Different anatomical systems (no brain structure and immune system, etc.) | Extremely simple cognitive behaviors | Approximately 60–80% homologous genes to human; 12 of the 17 signal pathways in humans are conserved in nematodes | No immune system | Highly amenable to genetic manipulations |
| Drosophila | 3 months | The metabolism of the whole body exists, lack of blood circulatory system, and blood–brain barrier, might cause inconsistent and unpredictable results when applied to humans | Available | Low-cost in infrastructure and maintenance | Unavailable | √ | √ | Simple and asymmetric brain structure | Relatively simple cognitive behaviors | Approximately 70% of the genes related to disease conditions in mammals are also present in Drosophila | Lack of an adaptive immune system | Highly amenable to genetic manipulations |
| Zebrafish | Fertilizing 200–300 eggs every 5–7 days, an equivalent longevity and generation time to mice (3–5 m) | Some major differences related to anatomy and physiology associated with an aquatic species, but most organs perform the same functions as their human counterparts and exhibit well-conserved physiology | Available | Relatively expensive in infrastructure and maintenance (compared to Drosophila and C. elegans) | Unavailable | √ | √ | A vertebrate animal model, | Limited cognitive behavioral assays | Approximately 70% homologous genes to human; over 80% of known human disease genes have orthologues in zebrafish | Complete immune system | Genetic tools yet to be comprehensive (compared to Drosophila and C. elegans) |
| Yeast | 3 days | Unlikely as a suitable model | Available | Available | √ | √ | A single-celled organism | - | 70% homologous genes to human; has no physiologic relevance to humans, but with many mitochondrial proteins that are orthologous to human proteins | - | Powerful genetic model, | |
| Cell | Stable cell lines can be passed on for tens of generations | Cells alone are no longer metabolized in the whole body. | Available | Available | √ | √ | - | - | Human-derived cells as a research model | - | Amenable to genetic manipulations | |
| Mammalian | Years | The metabolic process of the body is close to that of human beings. | Large-scale studies are limited | Costly in infrastructure and | Unavailable | × | × | Phylogenetically close to human | Complex cognitive analysis | Almost 100% human homolog genes found in rodents | Complete immune system | Costly in genetic manipulations |
An oxidant is formed as a normal product at a relatively low concentration during aerobic metabolism but is produced at ascending rates under pathological conditions. A quasi-steady state is maintained via intricate patterns of oxidant and antioxidant balance, and the disruption will jeopardize normal biological functions [36][37]. Natural products represent rich sources of bioactive compounds for pharmacology and toxicology, and phytochemicals show great potential in oxidation or antioxidation, as proven by numerous experimental models and human studies. Moreover, aqueous and organic solvent extracts, as well as secondary metabolites, play vital roles in oxidative stress by means of different cellular and molecular mechanisms. As a rapidly developed model organism, C. elegans has been introduced into the oxidation and antioxidation assessment of natural products for decades. The results based on C. elegans correlate with traditional experiments on rodents and rabbits [38].
2. Establishment of Oxidative Stress Model in C. elegans
The lifespan of C. elegans is always proportional to its resistance to environmental stress, and stress resistance is dominant in the lifespan of C. elegans [39]. Under heat stress, cells show a heat shock response and induce gene expression to prevent cell degeneration and enhance heat resistance. Heat shock experiments usually observe the survival time of nematodes by placing them at a temperature higher than the suitable living environment for some time. This index has been widely used to evaluate the protective effect of substances. For example, Lin et al. exposed nematodes to traditional Chinese herbal tea for 4 days, transferred them to 35 °C and recorded death per hour, indicating this treatment extended the average lifespan of nematodes significantly [40]. The same method was also used to evaluate the antistress effects of Cyclocarya paliurus polysaccharide leaf extract and piceatannol, and the researchers came to similar conclusions [41][42]. There are also some different treatment methods in heat stress experiments; for example, Lu et al. studied the protective effect of calycosin on nematodes under heat stress. On the third day, adult nematodes were cultured at 36 °C for 4 h and then transferred to 20 °C, and their survival was recorded every day [43]. The oxidative stress model of nematodes involves observing the survival of nematodes by exposing them to strong oxidants. Paraquat is a common stimulator of oxidative stress. Nematodes were exposed to NGM plates containing 5–50 mM paraquat, and the survival rate was monitored every 12 h [40][41][42][44]. Alternatively, the paraquat concentration was adjusted to 300 mM, and the survival time was recorded every 1 h [45]. The hydrogen peroxide (H2O2) test can also be used for the oxidative stress model. Nematodes were exposed to 0.8 mM H2O2 for 7 h to calculate the survival rate or incubated in 4–12 mM H2O2 for 4 h, and the number of surviving nematodes was recorded every 30 min [40][41][46][47]. Oxidative stress can also be tested using juglone, exposing nematodes to lethal juglone at 250 μM for 3 h, 300 μM for 1 h or 150 mM for 24 h, and then the survival rates of nematodes were recorded [28][29][48][49]. The heat shock response in C. elegans has revealed three related neuroendocrine signaling pathways: the nuclear hormone receptor pathway, transforming growth factor-β pathway and IGF/insulin-like signaling pathway [50]. Among them, the IIS pathway is the most thoroughly studied and has been demonstrated to play an important role in the signaling regulation of oxidative stress in C. elegans. Lin et al. and Shen et al. proved that the improvement of stress resistance mediated by phytomedicine was positively correlated with the activation of IIS pathway [41][42]. To investigate whether forkhead box protein O (DAF-16), a key regulator of antioxidation or heat stress, plays a role in this process, the subcellular distribution of DAF-16 in the TJ356 mutant was observed. The transfer of DAF-16 from the cytoplasm to the nucleus could be inhibited under stress [40]. The mechanism of Cyclocarya paliurus (C. paliurus) polysaccharide enhancing nematode heat tolerance was related to heat shock transcription factor 1 (HSF-1) without affecting the expression of DAF-16 in TJ356 but changed the fluorescence expression of SOD-3::GFP, and altered the expression of heat stress-related genes hsp-16.1 and hsp-16.2, suggesting that the HSF-1 pathway was necessary to improve heat tolerance. The longevity promoter skinhead-1 (SKN-1) regulates oxidative stress resistance. Under H2O2-induced oxidative stress, C. paliurus polysaccharide did not shorten the longevity of SKN-1 mutants, suggesting that the C. paliurus polysaccharide-mediated oxidative stress is dependent on SKN-1 [41].An oxidant is formed as a normal product at a relatively low concentration during aerobic metabolism but is produced at ascending rates under pathological conditions. A quasi-steady state is maintained via intricate patterns of oxidant and antioxidant balance, and the disruption will jeopardize normal biological functions [36,37]. Natural products represent rich sources of bioactive compounds for pharmacology and toxicology, and phytochemicals show great potential in oxidation or antioxidation, as proven by numerous experimental models and human studies. Moreover, aqueous and organic solvent extracts, as well as secondary metabolites, play vital roles in oxidative stress by means of different cellular and molecular mechanisms. As a rapidly developed model organism, C. elegans has been introduced into the oxidation and antioxidation assessment of natural products for decades. The results based on C. elegans correlate with traditional experiments on rodents and rabbits [38].
2. Establishment of Oxidative Stress Model in C. elegans
The lifespan of C. elegans is always proportional to its resistance to environmental stress, and stress resistance is dominant in the lifespan of C. elegans [39]. Under heat stress, cells show a heat shock response and induce gene expression to prevent cell degeneration and enhance heat resistance. Heat shock experiments usually observe the survival time of nematodes by placing them at a temperature higher than the suitable living environment for some time. This index has been widely used to evaluate the protective effect of substances. For example, Lin et al. exposed nematodes to traditional Chinese herbal tea for 4 days, transferred them to 35 °C and recorded death per hour, indicating this treatment extended the average lifespan of nematodes significantly [40]. The same method was also used to evaluate the antistress effects of Cyclocarya paliurus polysaccharide leaf extract and piceatannol, and the authors came to similar conclusions [41,42]. There are also some different treatment methods in heat stress experiments; for example, Lu et al. studied the protective effect of calycosin on nematodes under heat stress. On the third day, adult nematodes were cultured at 36 °C for 4 h and then transferred to 20 °C, and their survival was recorded every day [43].
The oxidative stress model of nematodes involves observing the survival of nematodes by exposing them to strong oxidants. Paraquat is a common stimulator of oxidative stress. Nematodes were exposed to NGM plates containing 5–50 mM paraquat, and the survival rate was monitored every 12 h [40,41,42,44]. Alternatively, the paraquat concentration was adjusted to 300 mM, and the survival time was recorded every 1 h [45]. The hydrogen peroxide (H2O2) test can also be used for the oxidative stress model. Nematodes were exposed to 0.8 mM H2O2 for 7 h to calculate the survival rate or incubated in 4–12 mM H2O2 for 4 h, and the number of surviving nematodes was recorded every 30 min [40,41,46,47]. Oxidative stress can also be tested using juglone, exposing nematodes to lethal juglone at 250 μM for 3 h, 300 μM for 1 h or 150 mM for 24 h, and then the survival rates of nematodes were recorded [28,29,48,49].
The heat shock response in C. elegans has revealed three related neuroendocrine signaling pathways: the nuclear hormone receptor pathway, transforming growth factor-β pathway and IGF/insulin-like signaling pathway [50]. Among them, the IIS pathway is the most thoroughly studied and has been demonstrated to play an important role in the signaling regulation of oxidative stress in C. elegans. Lin et al. and Shen et al. proved that the improvement of stress resistance mediated by phytomedicine was positively correlated with the activation of IIS pathway [41,42]. To investigate whether forkhead box protein O (DAF-16), a key regulator of antioxidation or heat stress, plays a role in this process, the subcellular distribution of DAF-16 in the TJ356 mutant was observed. The transfer of DAF-16 from the cytoplasm to the nucleus could be inhibited under stress [40]. The mechanism of Cyclocarya paliurus (C. paliurus) polysaccharide enhancing nematode heat tolerance was related to heat shock transcription factor 1 (HSF-1) without affecting the expression of DAF-16 in TJ356 but changed the fluorescence expression of SOD-3::GFP, and altered the expression of heat stress-related genes hsp-16.1 and hsp-16.2, suggesting that the HSF-1 pathway was necessary to improve heat tolerance. The longevity promoter skinhead-1 (SKN-1) regulates oxidative stress resistance. Under H2O2-induced oxidative stress, C. paliurus polysaccharide did not shorten the longevity of SKN-1 mutants, suggesting that the C. paliurus polysaccharide-mediated oxidative stress is dependent on SKN-1 [41].
