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INTRODUCTION
Oxidative stress (OS) emerges from an imbalance between the generation and accumulation of oxygen-reactive species (ROS) and the biological system capacity to neutralize these reactive by products1. ROS, like anion superoxide (O2•-), hydrogen peroxide (H2O2) and hydroxyl radical (OH•), are significant pro-oxidant agents1. This imbalance can be countered by both endogenous and exogenous antioxidant mechanisms. Among the endogenous defense, some include endogenous antioxidants enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX)2. In contrast, exogenous sources encompass ascorbic acid (Vit C), α-tocopherol (Vit E) and diverse polyphenols3.
Both obesity and aging are complex physiological processes linked to multi-organ dysfunction that is intertwined with OS4. In skeletal muscle, atrophy coincides with mitochondrial dysfunction5 and the activation of pro-oxidant enzymes like Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases6. In visceral adipose tissue, an increase in M1-like macrophage infiltration induces a state of low-grade inflammation and pro-oxidative conditions7. This elevated basal ROS production contributes to the onset of OS-associated diseases, such as insulin resistance (IR), type 2 diabetes mellitus (T2DM), hypertension (HT), cardiovascular diseases, cancer and neurodegenerative process6.
Exercise serves as a preventive strategy against disorders linked to OS, frequently harnessed to reinforce the intrinsic antioxidant defense system8. Paradoxically, exercise also incites acute OS, particularly during muscle contraction, as numerous sources of ROS experience increased activity9. The raising of ROS stemming from exercise has diverse sources. These encompass mitochondrial origins, attributed to electron leakage from complex I and complex III, and enzymatic sources, characterized by elevated nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) activity in skeletal and cardiac muscle9. Additionally, exercise induces augmented endothelial xanthine oxidase (XO) activity, particularly evident in ischemia/reperfusion processes10.
Despite the generation of acute OS during exercise, it concurrently instigates adaptive responses. These adaptations involve the upregulation of antioxidant enzymes to enhance the antioxidant defense and mitochondrial biogenesis8, as well as the promotion of glucose uptake, achieved independently of insulin, during exercise11.
Exercise-derived ROS, primarily as H2O2, play a crucial role in prompting adaptations based on the hormesis principle12. According to this principle, a modest increase in ROS levels triggers an upregulation in both the protein content and activity of antioxidant enzymes in skeletal muscle8,9. This, in turn, leads to an enhancement of the overall redox environment and a reinforcement of antioxidant defense mechanisms8,12. These adaptive responses are often referred to as the pre-conditioning effect and are orchestrated through the PGC1-α/Nrf2 axis signaling pathway8,13. PGC-1α, acting as a transcriptional coactivator, plays a central role in recruiting and co-regulating various transcription factors that oversee the gene expression of numerous skeletal muscle proteins and factors, including Nrf213. Furthermore, ROS have the ability to induce structural changes in the Nrf2 complex, which is typically sequestered in the cytosol by Keap1. With increased ROS levels resulting from exercise, the thiol groups of Keap1 undergo covalent modifications, causing their detachment from the Nrf2 complex. This modification facilitates the translocation of Nrf2 into the nucleus, enabling it to interact with antioxidant response elements (ARE)8. As a consequence, this event catalyzes the upregulation of antioxidant enzymes such as SOD, CAT, and GPX. These enzymes play pivotal roles in preserving mitochondrial structure and function, inducing mitochondrial biogenesis, and improving oxidative metabolism8,13,14. However, it is important to note that in the context of metabolic diseases associated with chronic OS, exercise alone may not be sufficient to rectify the redox imbalance inherent in these pathological states15. Consequently, an intriguing alternative therapeutic approach in such conditions involves the supplementation of exogenous antioxidants15,16. Nevertheless, compelling evidence has shown that the preventive role of physical exercise against chronic OS can be compromised when performed in conjunction with the supplementation of specific types of antioxidants known as scavengers17. Apigenin (4’,5,7-trihydroxyflavone), a monomeric flavonoid prevalent in the Western diet, is found in various herbs, vegetables, and fruits. It has demonstrated protective effects against a range of cardiometabolic disorders, including obesity, T2DM, HT, Alzheimer’s disease, and cancer18. Furthermore, it has been shown to regulate metabolism and enhance antioxidant defenses in different cellular contexts, including adipocytes, hepatic cells, cardiomyocytes, and endothelial cells19, acting as an exercise mimetic. This review compiles several studies on the potential beneficial effects of apigenin in the context of oxidative stress, ARE, mitochondrial biogenesis and physical performance. The search for relevant articles was conducted using PubMed from the National Library of Medicine-National Institute of Health, Web of Science, and Scopus. The keywords utilized include apigenin, antioxidant, ARE, exercise, physical performance, skeletal muscle, and mitochondria. The review includes articles published between 2013 and 2023 that describe the impact of apigenin supplementation on skeletal muscle antioxidant responses, ARE, mitochondrial biogenesis, and endurance capacity or strength performance.
Antioxidant supplementation and endogenous antioxidant adaptation induced by exercise
Exogenous antioxidants can serve various roles in combating ROS. Some, often referred to as scavengers or direct antioxidants, directly engage with ROS by breaking down peroxyl radicals, alkyl and alkoxy radicals, and decomposing O2•- and H2O220. In contrast, other antioxidants, categorized as indirect antioxidants, operate by promoting the activation of transcription factors associated with ARE through inducing moderate increases in ROS levels21. This mechanism enhances the endogenous production of antioxidant enzymes. When it comes to treating metabolic diseases, certain antioxidants, such as Vit C and E, are considered promising options. Vit C interacts directly with ROS and effectively reduces lipid peroxidation in cell membranes, mitigating oxidative damage caused by ROS. On the other hand, Vit E, a fat-soluble vitamin, excels at minimizing lipid peroxidation and protecting cell membranes. These two vitamins work synergistically to scavenge ROS and prevent oxidative damage22. Vitamin C and Vitamin E have shown to reduce OS biomarkers and improve clinical parameters such as systolic and diastolic blood pressures in HT patients23.
Despite substantial evidence, attempts to amplify the adaptive effects on endogenous antioxidant defense in healthy individuals through antioxidant scavenger supplementation have proven ineffective. In fact, research has indicated that supplementation with antioxidant scavengers may reduce the expression and content of transcription factors associated with ARE, resulting in decreased levels and activity of antioxidant enzymes17,24,25. Healthy animal models engaged in exercise have shown that Vit C and Vit E supplementation, either separately or in combination, leads to reduced expression of PGC-1α and SOD in skeletal muscle, as well as diminished activity of SOD, CAT, and GPX17,25,26. Additionally, in exercising mice models, antioxidant scavengers have been found to lower aerobic capacity and decrease tolerance for long-term exercise17,27. These outcomes are likely due to Vit C and E reducing different ROS concentrations, particularly O2•-, which hinders the formation of H2O2 through SOD. ROS, especially H2O2, serves as a cellular signaling modulator, for example, in the activation of transcription factors like PGC-1α and Nrf28,13. Therefore, when antioxidant scavengers inhibit the PGC-1α/Nrf2 pathway and reduce the levels and activity of antioxidant enzymes, such as SOD, CAT, GPX and mitochondrial biogenesis17,24–26,28, physical exercise loses its preventive role against metabolic diseases linked to chronic OS when combined with antioxidant scavenger supplementation24. On the other hand, it has been demonstrated that indirect antioxidants, including polyunsaturated fatty acids29 and certain polyphenols3, can induce a pre-conditioning effect similar to exercising. This effect enhances the content of antioxidant enzymes via the PGC-1α/Nrf2 pathway in metabolic diseases associated with OS30. Nonetheless, there are some polyphenols for which the mechanisms and interactions with physical exercise concerning endogenous antioxidant defenses remain unexplored.
Apigenin: structure, absorption, and metabolism
Apigenin, a prevalent monomeric polyphenol in Western diets31, is abundant in various dietary sources, including fresh parsley (Petroselinum crispum), celery (Apium graveloens), artichoke (Cyanara cardunculus), green celery heart, vine spinach, and dry oregano, among others31. Apigenin primarily exists in glycosylated forms, with common glycosides including apigenin-7-O-glycoside, apiin, isovitexin, schaftoside, roifoline, and vitexin32,33. Apigenin’s limited solubility and low bioavailability lead to interactions with gut content and metabolism by commensal bacteria into smaller compounds34. Its oral bioavailability in rats and humans has been reported as very low35,36, with only about 5-10% of polyphenols, primarily monomers and dimers, being absorbed in the small intestine34. In an in vitro model using Caco-2 cells, apigenin permeabilities are significantly higher compared to apigenin-7-O-glycoside, emphasizing its potential for absorption. In a rat intestinal model, apigenin is quickly absorbed throughout the entire intestinal tract, with the duodenum as the primary absorption site37. Notably, in a rat-perfused intestinal model, rapid absorption of apigenin aglycone is observed38. Apigenin’s transport involves a saturable mechanism, combining both active and passive carriers in the initial digestive tract segments, with subsequent transport in the ileum and colon relying predominantly on passive mechanisms37. Similarly, apigenin glycosides are absorbed from the stomach to the gut, undergoing rapid absorption or deglycosylation, primarily within the cecum32. Moreover, apigenin serves as a prebiotic, influencing the composition and functionality of the gut microbiota, highlighting the pivotal role of the gastrointestinal tract in metabolizing and conjugating apigenin before it enters the systemic circulation and passes through the liver38.
According to the Biopharmaceutical Classification System, apigenin is categorized as a class II drug, characterized by high intestinal permeability but low solubility32. While apigenin exhibits limited solubility in non-polar solvents, ranging from 0.001 to 1.63 mg/mL, it displays high hydrophilicity at 1.35 μg/mL in purified water39. The highest solubility is achieved in phosphate buffers, recorded at 2.16 μg/mL at pH 7.5. Notably, apigenin demonstrates complete solubility in dimethylsulfoxide, surpassing the threshold of >100 mg/mL32.
After oral consumption, apigenin is absorbed systemically, circulating through enterohepatic and local intestinal pathways. Its bioavailability is estimated at approximately 30%40. Once absorbed, apigenin undergoes a comprehensive phase I and phase II metabolism, primarily involving hepatic phase I enzymes34. This phase II biotransformation occurs within enteric and enterohepatic cycles, with conjugation reactions such as glucuronidation and sulfation serving as fundamental metabolic pathways for apigenin32. Following absorption into the bloodstream and tissues, apigenin transforms into glucuronide, sulfate, or luteolin conjugates32. Glucuronidation produces three distinct -monoglucuronides, while sulfation results in a single product32. The maximum circulating concentration (Cmax) is reached within 0.5-2.5 hours, with an elimination half-life averaging 2.52 ± 0.56 hours41. Fecal excretion following oral intake provides valuable insights into assessing apigenin’s metabolic activity within the gut microbiota. This evidence underscores the gradual elimination phase of apigenin, suggesting the potential for accumulation within the body. In murine models, a diet supplemented with apigenin revealed that apigenin plasma concentrations reached a steady state after the initial 5 days42. During this phase, apigenin concentrations in plasma, liver, and small intestine mucosa were measured. Consistent with earlier findings38, these results emphasize the comprehensive distribution of apigenin within the organism.
A recent study indicates that apigenin is poorly absorbed, with only 0.5% of the ingested apigenin excreted as metabolites in urine within the 0-24-hour post-intake period41. The authors explain that the bioavailability of apigenin is highly dependent on the food matrix, making it impractical to achieve desired blood levels through dietary sources41. However, the use of semi-purified apigenin supplements in capsule form may offer a more practical means to reach target concentrations within acceptable dosage ranges for supplements and medications. Therefore, further research, including direct pharmacokinetic and clinical studies, is essential to comprehensively evaluate the potential clinical utility of apigenin.
Apigenin: evidence of endogenous antioxidant defense and physical performance
Apigenin has demonstrated its protective impact on diverse cardiometabolic disorders and has been shown to enhance antioxidant defenses in different cellular contexts19. In primary cultures of cardiac muscle cells, we observed differences when examining different concentrations of apigenin. Unlike certain flavonoids such as epicatechin, quercetin, and kaempferol, which have shown the ability to reduce H2O2, apigenin did not exhibit a corresponding reduction in this parameter43. Furthermore, other flavonoids such as hesperetin and luteolin exhibit reducing potential comparable to Vit C and Vit E44. However, it is important to note that apigenin possesses significantly lower reducing potential compared to these vitamins44.
In cell lines like 22Rv145, HeLa, SiHa, CasKi, and C33A46, an interesting phenomenon was documented where apigenin administration resulted in a reduction in mitochondrial membrane potential through a direct interaction with complex I. This interaction led to increased leakage of O2•-, prompting SOD to dismutate O2•- into H2O2. H2O2 emerged as a key inducer of cellular signaling, activating the PGC-1α and Nrf2 pathway. Additionally, after exposure to apigenin, there was a noticeable increase in CAT activity observed in HeLa, Caski, and C33A cell lines46. Similarly, in C2C12 cell lines, there was a significant elevation in the expression of PGC-1α47. In mice with metabolic syndrome induced by a high-fructose diet, apigenin activated the Nrf2 pathway and significantly improved the activity of SOD, CAT, and GPX in hepatic tissue while reducing malondialdehyde (MDA), an indicator of lipid peroxidation48.
While there have been limited investigations into the influence of apigenin on skeletal muscle and physical performance (Table 1), some notable findings have emerged. In a study involving healthy, untrained C57BL/6 mice that were administered an AIN-76A diet supplemented with 0.4% apigenin, several significant outcomes were observed47. These mice exhibited a notable increase in quadriceps muscle mass while their overall weight remained unchanged. Interestingly, the supplemented mice also covered a longer distance during a single race test compared to the control group, and this improvement was correlated with higher expression of PGC-1α in their muscles. Additionally, in a model involving aged mice and various apigenin doses for supplementation, the findings were particularly striking. The supplemented group showed significant improvements in both strength and endurance capacity compared to the control group. This effect was associated with an increased activity of SOD and GPX, along with a lower content of MDA and an improvement in total antioxidant capability in the gastrocnemius muscle. Apigenin also improved mitochondrial parameters in tibialis anterior tissue, including an increase in the number of mitochondria, mitochondrial volume, and mitochondrial diameter. In gastrocnemius muscle tissue, apigenin decreased cytochrome c content in cytosol, caspase 3 activity, and chromatin fragmentation49. Furthermore, a study involving an obese mice model with muscle atrophy revealed intriguing outcomes following apigenin supplementation. Obese mice that received supplementation displayed a higher protein content of PGC-1α in the quadriceps. In the gastrocnemius, a greater enzyme activity of citrate synthase was noted, along with an increased expression of electron transport chain proteins and mitochondrial area compared with non-supplemented obese mice. These effects became evident after an 8-week treatment period50.
Table 1 Summarized apigenin effects on endogenous antioxidant defense and physical performance.
| Author | Animal model | Intervention | ARE, Antioxidant response and Mitochondrial function | Physical performance |
|---|---|---|---|---|
| Jang 201748 | Male C57BL/6 mouse, 5- weeks old, n= 30 | Animals were divided into three groups: Normal diet group. Low-apigenin diet group (AIN-76 diet with 0,2% of apigenin). High-apigenin diet group (AIN-76A diet with 0.4% apigenin). Mice were fed with the apigenin diets for 7 weeks. Skeletal quadricep muscles were harvested for western blot analysis. |
High-apigenin diet vs Normal diet group ↑ PGC-1α (a.u.) |
High-apigenin diet vs Normal diet group ↑ Quadriceps weight (g) ↑ Running distance(m) Low-apigenin diet vs Normal diet group ↑ Running distance (m) |
| Wang 202049 | Male C57BL/6 mouse, 16 months old, n= 48. Young mice (n= 12), 6 to 9 months |
Young mice as a young control adults. Old animals were divided into four groups: Old control diet. Old + low-apigenin diet group (apigenin 25 mg·kg−1 day−1). Old + middle-apigenin diet group (apigenin 50 mg·kg−1 day−1). High-apigenin diet group (apigenin 100 mg·kg−1·day−1). Skeletal quadricep muscles were harvested for western blot analysis for PGC-1α and skeletal gastrocnemius muscles were used to determinate OS biomarkers. |
Old High-apigenin diet vs Old Normal diet group ↑ PGC-1α (a.u.) ↔ SOD (U/mg protein) ↑ GPX (mU/mg protein) ↓ MDA (nmol/mg protein) ↓ PC (nmol/mg protein) ↑ TAC (nmol/mg protein) ↑ Mitochondrial number (/µm2) ↑ Mitochondrial volume density (Fold change from young) ↑ Mitochondrial diameter (µm) ↓ Caspase 3 activity (x103 AFU/mg protein) ↓ Cytochrome C in cytosol (a.u) ↓ Chromatin fragmentation (Fold change from young) |
Old High-apigenin diet vs Old Normal diet group ↑ Grip strength (Newton) ↑ Running distance(m) |
|
Old Middle-apigenin diet vs Old Normal diet group ↑ Grip strength (Newton) ↑ Running distance(m) | ||||
|
Old Low-apigenin diet vs Old Normal diet group ↑ Grip strength (Newton) ↔ Running distance(m) | ||||
|
Old Middle-apigenin diet vs Old Normal diet group ↑ PGC-1α (a.u.) ↑ SOD (U/mg protein) ↑ GPX (mU/mg protein) ↓ MDA (nmol/mg protein) ↓ PC (nmol/mg protein) ↑ TAC (nmol/mg protein) ↑ Mitochondrial number (/µm2) ↑ Mitochondrial volume density (Fold change from young) ↑ Mitochondrial diameter (µm) ↓ Caspase 3 activity (x103 AFU/mg protein) ↓ Cytochrome C (a.u) ↓ Chromatin fragmentation (Fold change from young) |
||||
|
Old Low-apigenin diet vs Old Normal diet group ↔ PGC-1α (a.u.) ↔ SOD (U/mg protein) ↔ GPX (mU/mg protein) ↔ MDA (nmol/mg protein) ↔ PC (nmol/mg protein) ↔ TAC (nmol/mg protein) ↔ Mitochondrial number (/µm2) ↔ Mitochondrial volume density (Fold change from young) ↔ Mitochondrial diameter (µm) ↔ Caspase 3 activity (x103 AFU/mg protein) ↔ Cytochrome C (a.u) ↔ Chromatin fragmentation (Fold change from young) |
||||
| Choi 201750 | Male C57BL/6 mice, 4-weeks old | After 1 week of adaptation, the mice were divided in three groups: Normal diet group and two groups were induced obesity with a high fat diet (HFD) (60% calories as fat ad libitum for 9 weeks). Then, the diet- induced obese mice were assigned to two groups. |
HFD + apigenin group vs HFD group ↑ PGC-1α relative expression ↑ CS activity ↑ MTC I and II activity |
HFD + apigenin group vs HFD group ↑ Running distance(m) |
| HFD group | ||||
| HFD + apigenin group (0.1%, w/w) for an additional 8 weeks. Skeletal quadricep muscles were harvested for western blot analysis for PGC-1 α and skeletal gastrocnemius muscles were used to determinate CS activity and MTC I and II activity. |
↑Significative increases
↓Significative decreases
↔Non significative changes
a.u. Arbitrary units; CS, Citrate synthase; GPX, Glutathione peroxidase; HFD, High Fat Diet; MDA, Malondialdehyde; MTC I and II, Mitochondrial complex I and II; PC, Protein Carbonyl; PGC-1α, peroxisome proliferator activated receptor-Gamma Coactivator-1-alpha; SOD, Superoxide dismutase; TAC, Total antioxidant capability.
The evidence presented highlights the beneficial effects of apigenin supplementation in animal models. Similar to exercise, apigenin allows the activation of PGC-1α, favoring an improvement in the antioxidant defense enzymes of skeletal muscle49, protecting the mitochondrial function and structure, reducing markers of apoptosis dependent on the mitochondrial pathway, in addition to improving its oxidation capacity49,50 and preserving skeletal muscle function in obesity and aging, ultimately enhancing overall performance49,50 (Figure 1).
Figure 1 Hypothetical mechanism of apigenin's impact on the PGC-1α/Nrf2 pathway and its influence on physical performance during exercise.
However, the absence of available experimental exercise models represents a limitation. Studies in healthy humans evaluating the effects of apigenin and exercise without pharmacological co-treatment on antioxidant defense or physical performance were not found after searching the databases. Therefore, is imperative to deepen our understanding and knowledge regarding the impact of apigenin on antioxidant defense mechanisms.
CONCLUSIONS
Exercise has been acknowledged as both a preventive strategy and a potential inducer of OS. It can also trigger adaptive responses to enhance antioxidant defenses. Among the exogenous antioxidants, apigenin has demonstrated the ability to influence cellular redox status, improve mitochondrial function, and boost antioxidant enzyme activities. However, despite promising results in animal models, the limited availability of experimental exercise models has impeded a comprehensive understanding of how apigenin affects antioxidant defense mechanisms.
In conclusion, while exercise remains a fundamental approach for addressing OS-related disorders, there is a need for further exploration into the interplay between exercise, antioxidant supplementation, and specific compounds like apigenin. Gaining a deeper understanding of these interactions could open the door to innovative strategies for mitigating the impact of diseases associated with OS and enhancing overall well-being.
