Application of oryzanol in preparation of product for relieving exercise-induced fatigue

Abstract

The invention is applicable to the technical field of biological medicine, and relates to an application of oryzanol in preparation of a product for relieving exercise-induced fatigue, which comprises the following steps: through integration of network pharmacology, molecular docking and animal experiments, it is systematically proved for the first time that oryzanol can significantly improve exercise tolerance, enhance muscle strength, reduce oxidative damage and promote muscle repair; the anti-fatigue effect of the oryzanol is closely related to regulation and control of PI3K / AKT / FOXO signal channels, and molecular docking shows that the oryzanol can be stably combined with channel key proteins AKT1, FOXO1 and FOXO3. The invention provides a clear theoretical basis and an application scheme for developing natural, safe and efficient anti-fatigue food, health-care products or medicines taking oryzanol as an active ingredient, and also provides a set of effective method for researching the anti-fatigue effect of natural products.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of oryzanol in the preparation of products for relieving exercise fatigue. Background Technology

[0002] Exercise-induced fatigue refers to a series of physiological and psychological phenomena that occur after exercise, including decreased physical strength, reduced endurance, and impaired muscle function. Its mechanism is complex, with oxidative stress and inflammatory response currently recognized as the two core driving factors. During prolonged or high-intensity exercise, the body's energy metabolism increases dramatically, leading to the production of large amounts of reactive oxygen species (ROS) in the mitochondrial electron transport chain. When ROS production exceeds the body's endogenous antioxidant defense system's ability to eliminate it, oxidative stress is triggered. High concentrations of reactive oxygen species (ROS) not only directly attack skeletal muscle cell membranes, triggering lipid peroxidation and resulting in increased levels of its end product, malondialdehyde (MDA), but also disrupt mitochondrial membrane integrity, impairing its oxidative phosphorylation function and hindering adenosine triphosphate (ATP) synthesis. This energy supply crisis ultimately manifests as decreased muscle contractility and fatigue. Simultaneously, excessive ROS can act as signaling molecules, activating key inflammatory pathways such as nuclear factor kappa-B (NF-κB), leading to the massive release of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). These cytokines not only exacerbate local muscle tissue inflammation but may also affect the central nervous system through blood circulation, collectively contributing to a decline in athletic performance.

[0003] Currently, products on the market for relieving exercise-induced fatigue mainly include chemically synthesized supplements and naturally derived supplements. Chemically synthesized supplements have side effects (such as the potential stress creatine can put on the kidneys), while many natural supplements have problems such as unclear mechanisms of action, high costs, or limited effects, making it difficult to simultaneously achieve the dual goals of "improving exercise endurance" and "promoting muscle repair".

[0004] Oryzanol is a natural mixture extracted from rice bran oil. Its main components are esters composed of ferulic acid and phytosterols, and it is known to have antioxidant and anti-inflammatory activities. Existing studies have shown that oryzanol can exert anti-inflammatory effects by inhibiting the NF-κB pathway in models of colitis and hyperlipidemia. However, whether oryzanol can be used to relieve exercise-induced fatigue, as well as its specific targets and molecular mechanisms, require further investigation by researchers in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide an application of oryzanol in the preparation of products for relieving exercise-induced fatigue, thereby solving problems such as insufficient safety, unclear mechanisms of action, or limited effects in existing products for relieving exercise-induced fatigue. In addition, this invention also reveals the molecular mechanism by which oryzanol relieves exercise-induced fatigue by regulating the PI3K / AKT / FOXO signaling pathway, providing a solid theoretical basis and quantitative scheme for the development of related functional products.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of oryzanol in the preparation of products for relieving exercise-induced fatigue, wherein oryzanol improves exercise-induced fatigue by regulating the PI3K / AKT / FOXO signaling pathway, inhibiting oxidative stress and inflammatory response.

[0007] Furthermore, the product is an oral formulation, including capsules, tablets, or sports drink additives.

[0008] Furthermore, the effective daily dose of the oryzanol is 200 mg / kg.

[0009] Furthermore, the oryzanol regulates the PI3K / AKT / FOXO signaling pathway by binding to AKT1, FOXO1, and / or FOXO3 proteins.

[0010] Furthermore, the binding energies are all less than -7 kcal / mol.

[0011] Furthermore, the oryzanol is used in combination with vitamin E and tea polyphenols to synergistically enhance the anti-fatigue effect.

[0012] Furthermore, the improvement in exercise-induced fatigue manifests as increased exercise endurance, enhanced muscle strength, reduced oxidative stress levels, and / or promoted muscle tissue repair.

[0013] Secondly, this invention also reveals the molecular mechanism by which oryzanol alleviates exercise-induced fatigue, through the integration of network pharmacology, molecular docking, and in vitro experimental verification, including: Network pharmacology validation: PharmMapper, Swiss Target Prediction, NCBI, GeneCards®, TargetNet, and CTD databases were used to identify potential targets for oryzanol in preventing / treating fatigue. To improve prediction accuracy, data from these databases were merged and duplicate data were removed. The therapeutic target of oryzanol for fatigue prevention was submitted to the STRING database with a confidence score of 0.7. Subsequently, the topological parameters of each protein node in the interaction network were calculated using Cytoscape 3.10.3 software. GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on the candidate targets using the online tool Metascape database, and the results were visualized.

[0014] Molecular docking verification: Molecular docking was performed on the key active ingredients and key targets obtained. The 3D structure of oryzanol was downloaded from the PubChem database, and the 3D structures of AKT1, FOXO1, and FOXO3 proteins were downloaded from the PDB database. PyMOL was used to dehydrate and separate the protoligands. Then, AutoDock Tools 1.5.6 was used to convert the files to pdbqt format and locate the active pockets. Finally, molecular docking and visualization were performed using AutoDock Vina software.

[0015] In vitro experimental validation: Mouse experiments were conducted, including a control group, an exercise group, and a oryzanol group. Behavioral tests were performed on mice, including forelimb grip strength testing (measuring muscle strength), swimming testing (measuring exercise endurance), and spontaneous activity testing (measuring body activity). After euthanizing the mice, brain, heart, kidney, spleen, lung, liver, and gastrocnemius muscle tissues were collected and preserved for later use. Oxidative stress indicators such as SOD (superoxide dismutase) activity and MDA (malondialdehyde) content, as well as the levels of inflammation-related factors, were detected in the tissues. The expression levels of proteins related to the PI3K / AKT / NF-κB signaling pathway (such as AKT and NF-κB) were detected by Western blot. The interaction between oryzanol and AKT protein was verified through molecular docking and kinetic analysis.

[0016] Compared with existing technologies, the application of oryzanol provided by this invention in the preparation of products for relieving exercise fatigue has at least the following beneficial effects: 1. This invention is the first to explicitly apply oryzanol to the field of relieving exercise-induced fatigue, providing a brand-new material option for the development of novel anti-fatigue natural products.

[0017] 2. For the first time, through a systematic study involving "network pharmacology verification - molecular docking verification - experimental verification", the molecular mechanism by which oryzanol exerts its anti-fatigue effect by regulating the PI3K / AKT / FOXO signaling pathway was elucidated, achieving a breakthrough from phenomenon to essence, and providing a scientific basis for precise application and product development.

[0018] 3. Oryzanol is derived from natural grains, has high safety, and low risk with long-term use. Animal experiments have shown that it has clear effects in improving endurance, enhancing strength, anti-oxidation, and promoting repair, achieving a combination of multiple anti-fatigue benefits.

[0019] 4. Oryzanol has a wide range of sources (byproduct of rice bran oil), mature extraction technology, relatively low production cost, and is easy to achieve large-scale production and market promotion.

[0020] 5. The integrated research strategy (computational biology + experimental verification) adopted in this invention is not only applicable to oryzanol, but can also provide a reference methodological framework for the discovery of anti-fatigue activities and the study of mechanisms of action of other natural products. Attached Figure Description

[0021] To more clearly illustrate the solution of the present invention, a brief introduction will be given to the drawings used in the description of the embodiments below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a comparative diagram showing the effects of oryzanol intervention on the exercise capacity and endurance of fatigued mice in the embodiments of the present invention; Figure 2 This is a comparative diagram showing the effects of oryzanol intervention on the body weight and food intake of fatigued mice in the embodiments of the present invention; Figure 3 This is a comparative diagram showing the effects of oryzanol intervention on the morphology of muscle tissue in fatigued mice in this embodiment of the invention. Figure 4 This is a comparative diagram showing the effects of oryzanol intervention on antioxidant-related enzymes in fatigued mice in the embodiments of the present invention; Figure 5 This is a schematic diagram of the mapping results between oryzanol target and fatigue target in network pharmacology according to an embodiment of the present invention; Figure 6 This is a schematic diagram of the interaction results between oryzanol and PPI, potential anti-fatigue targets, in network pharmacology according to an embodiment of the present invention. Figure 7 This is a diagram illustrating the biological process function of oryzanol as a potential anti-fatigue target in the embodiments of the present invention. Figure 8This is a cellular component functional annotation diagram of the potential anti-fatigue target of oryzanol in the embodiments of the present invention; Figure 9 This is a molecular functional annotation diagram of the potential anti-fatigue target of oryzanol in the embodiments of the present invention; Figure 10 This is a KEGG pathway annotation diagram of the potential anti-fatigue target of oryzanol in the embodiments of the present invention; Figure 11 This is a schematic diagram illustrating the docking of oryzanol with AKT1 protein molecules in an embodiment of the present invention; Figure 12 This is a schematic diagram of the docking between oryzanol and FOXO1 protein molecules in an embodiment of the present invention; Figure 13 This is a schematic diagram of the docking between oryzanol and FOXO3 protein molecules in an embodiment of the present invention. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Example 1

[0025] Intervention experiment of oryzanol on exercise-induced fatigue mice Experimental subjects: 7-week-old male C57BL / 6J mice, weighing 23±2g, were divided into 3 groups (several mice in each group): Control group (Con): 0.5% CMC-Na (sodium carboxymethyl cellulose) was administered by gavage, without forced exercise; Exercise group (Ex): 0.5% CMC-Na was administered by gavage, while continuous forced exercise (such as swimming or treadmill exercise) was performed; Oryzanol group (Ex-Ory): 200mg / kg / d of a mixture of oryzanol and 0.5% CMC-Na was administered by gavage, while the same forced exercise as the Ex group was performed.

[0026] Experiment duration: 4 weeks in total.

[0027] Experimental steps: 1. Adaptation period: Before the start of the experiment, all mice were raised in the same environment (consistent temperature and humidity) to adapt to the diet and feeding conditions without any intervention or exercise; 2. Intervention period (weeks 1-4): Gavage procedure: Mice in the three groups were gavaged at fixed times every day, and the gavage dose was adjusted according to body weight to ensure that the intake of oryzanol in the Ex-Ory group was 200 mg / kg / day; Exercise intervention: The Ex group and the Ex-Ory group were subjected to forced exercise (such as swimming, 30 minutes each time, gradually increasing the intensity) once a day, while the Con group did not exercise; 3. Indicator Detection (Days 28-29, after intervention): Behavioral Detection: Forelimb grip strength test (to detect muscle strength), swimming test (to detect exercise endurance), and spontaneous activity test (to detect body activity); Sample Collection: After euthanizing mice, brain, heart, kidney, spleen, lung, liver, and gastrocnemius muscle tissues were collected and preserved for later use; Biochemical Indicator Detection: Oxidative stress indicators such as SOD (superoxide dismutase) activity and MDA (malondialdehyde) content, as well as the levels of inflammation-related factors, were detected in the tissues; Molecular Mechanism Detection: The expression levels of proteins related to the PI3K / AKT / NF-κB signaling pathway (such as AKT and NF-κB) were detected by Western blot; The interaction between oryzanol and AKT protein was verified by molecular docking and kinetic analysis.

[0028] In this embodiment, the effects of oryzanol on the exercise capacity and endurance of fatigued mice are as follows: Figure 1 As shown, the behavior of mice was observed using an animal behavior analysis system. Compared with the Con group, the Ex group showed a significant decrease in total movement distance, movement speed, number of activities, activity time, and linearity, and an increase in rest time. However, after supplementation with oryzanol, the total movement distance, movement speed, number of activities, activity time, and linearity of the mice significantly increased, while the rest time significantly decreased, indicating that oryzanol can significantly improve the voluntary activity ability of fatigued mice. The grip strength of the mice's forelimbs was measured using a grip strength meter. After fatigue training, the grip strength of the mice's forelimbs decreased significantly. After treatment with oryzanol, the grip strength of the mice's limbs significantly increased. Swimming time is an important indicator of exercise endurance. Compared with the Con group, fatigue significantly reduced the swimming time of the mice, resulting in a significant increase in the rest time. After oryzanol gavage, the swimming time of the mice significantly increased, and the corresponding rest time significantly decreased. This indicates that oryzanol gavage can effectively alleviate fatigue in mice.

[0029] In this embodiment, the effects of oryzanol on the body weight and food intake of fatigued mice are as follows: Figure 2 As shown, mouse body weight and food intake were measured weekly. The results showed no significant differences in body weight and food intake among the groups, indicating that the fatigue model and drug intervention did not affect the growth and development of the mice.

[0030] In this embodiment, the effect of oryzanol on the morphology of muscle tissue in fatigued mice is as follows: Figure 3 As shown, Figure 3Images of gastrocnemius muscle tissue from fatigued mice, stained with H&E (50x magnification). Histological analysis of the gastrocnemius muscle using H&E staining showed that, compared to the Con group, the Ex group exhibited typical striated muscle degeneration characteristics, such as blurred or disappearing boundaries, loose local muscle fiber structure (marked by black arrows), and disordered cell and cellular distribution. Vitamin B1 intervention significantly improved muscle fiber damage. Pathological analysis was more similar to that of the Con group.

[0031] In this embodiment, the effect of oryzanol on the glycogen content of fatigued mice is as follows: Figure 4 As shown, glycogen is the body's main energy storage substance. Based on its storage location, glycogen is mainly divided into liver glycogen and muscle glycogen. Glycogen levels in the liver and muscle tissues were measured separately. Compared to the Con group, the Ex group mice showed a significant decrease in both liver and muscle glycogen levels, likely due to fatigue and gradual energy depletion. Supplementation with oryzanol significantly increased both liver and muscle glycogen levels in the mice.

[0032] In this embodiment, the effect of oryzanol on serum lactate levels in fatigued mice is as follows: Figure 4 As shown, when exercise intensity exceeds the body's aerobic metabolic capacity, anaerobic respiration in muscles increases, leading to increased lactic acid production and its entry into the bloodstream, thus promoting fatigue. Compared to the Con group, the blood lactate levels in the Ex group mice were significantly higher, while those in the Ex-Ory group were significantly lower. Compared to the Con group, the significantly higher levels of lactate dehydrogenase in the Ex group mice indicated that the rate of lactate production exceeded the rate of metabolism, promoting fatigue. In the Ex-Ory group, LDH levels were significantly lower, alleviating the fatigue in the mice.

[0033] In this embodiment, the effect of oryzanol on oxidative damage markers in fatigued mice is as follows: Figure 4 As shown, malondialdehyde (MDA) is a lipid peroxidation product, and its level directly reflects the degree of oxidative damage in the body. Compared with the Con group, the serum MDA level in the Ex group was significantly higher, reflecting a higher degree of oxidative damage in the body under fatigue. After gavage administration of oryzanol, the MDA level in the Ex-Ory group was significantly lower than that in the Ex group, indicating that oryzanol alleviated oxidative damage caused by fatigue.

[0034] In this embodiment, the effect of oryzanol on antioxidant-related enzymes in fatigued mice is as follows: Figure 4 As shown, SOD exerts its antioxidant effect by scavenging free radicals in the body. GSH-Px protects cells from oxidative damage by scavenging peroxides in the body. Compared with the Con group, the levels of SOD and GSH-Px in the Ex group were significantly decreased, while the levels of SOD and GSH-Px in the Ex-Ory group were significantly increased. Example 2

[0035] Network pharmacology predicts potential targets and pathways for oryzanol's anti-fatigue effects. Identification of potential targets for the anti-fatigue effects of oryzanol: such as Figure 5 The diagram shows the mapping results between oryzanol targets and fatigue targets. In the PharmMapper, SEA, Swiss target prediction, NCBI, CTD, and Targetnet databases, 299, 31, 14, 614, 618, and 9 targets for oryzanol were identified, respectively. After removing redundant targets, 748 drug targets remained. In the CTD, OMIM, Digsee, and Genecard databases, 28704, 1873, 298, and 10051 fatigue-related targets were identified, respectively. After removing redundant targets, a total of 29880 fatigue-related targets were identified. The online software Venny 2.1 identified 514 oryzanol-related targets.

[0036] Construction of key target PPI network: such as Figure 6 The image shows the PPI interaction results of oryzanol as a potential anti-fatigue target. Potential therapeutic targets for oryzanol in anti-fatigue were submitted to the STRING database with a confidence level of 0.700. A main network was formed by 503 genes with at least one interaction. The degree of each protein node in the PPI network was calculated using Cytoscape 3.10.3 software. Nodes with a degree greater than twice the median (degree ≥ 210) were extracted for visualization analysis. The PPI network consists of 78 nodes and 2959 edges. Akt1 had the highest degree value of 435. Furthermore, several oxidation, inflammation, and apoptosis-related targets (Tnf, Il6, Trp53, Mapk14, Casp3, Bcl2) played important roles in the virtual network for oryzanol's anti-fatigue effect.

[0037] GO functional enrichment analysis of potential targets: GO functional enrichment analysis was performed on 514 intersecting targets using the Metasccape database to determine gene functional information, such as... Figures 7 to 9The annotation includes biological processes (BP), cellular components (CC), and molecular functions (MF). The top 20 terms for BP, CC, and MF were selected based on the number of counts. Biological process annotation revealed that oryzanol is mainly involved in biological processes such as "cellular response to nitrogenous compounds," "positive regulation of movement," "regulation of MAPK cascades," "cellular response to lipids," "regulation of transsynaptic signaling," "positive regulation of apoptosis," and "regulation of protein phosphorylation." Oryzanol may be involved in the generation of cellular components such as "receptor complexes," "pre-cell membranes," "interneuronal synapses," "extracellular plasma membranes," and "postsynaptic membranes." In terms of molecular functions, oryzanol is mainly involved in "G protein-coupled receptor activity," "protein kinase activity," "phosphotransferase activity," "kinase activity," "protein tyrosine kinase activity," "peptide receptor activity," "protein serine kinase activity," "protein serine / threonine kinase activity," "protein binding," "peptidase activity," "transcription factor binding," "enzyme activator activity," "DNA-binding transcription factor binding," and "oxidoreductase activity."

[0038] KEGG pathway enrichment analysis of potential targets: such as Figure 10 As shown, further KEGG enrichment analysis of these anti-fatigue targets of oryzanol revealed that oryzanol may be a key signal in regulating a series of biological processes induced by fatigue, including the PI3K-Akt signaling pathway, cAMP signaling pathway, calcium signaling pathway, MAPK signaling pathway, chemokine signaling pathway, TNF signaling pathway, Ras signaling pathway, FoxO signaling pathway, NF-κB signaling pathway, and PPAR signaling pathway.

[0039] Validate key targets: such as Figures 11 to 13 Based on KEGG signaling pathway annotation and existing research, the PI3K-Akt and TNF signaling pathways are significantly correlated with the occurrence and development of fatigue. Pathway visualization of 78 key targets using KEGGmapper revealed the PI3K-Akt and TNF signaling pathways. Molecular docking of oryzanol to key target proteins showed binding energies all less than -7 kcal / mol. This indicates that oryzanol may exert its anti-fatigue effect by binding to AKT1, FOXO1, and FOXO3 proteins and regulating the PI3K / AKT / FoxO pathway.

[0040] The application of oryzanol in the preparation of products for relieving exercise-induced fatigue, as described in the above embodiments, is the first time that oryzanol has been explicitly applied to the field of relieving exercise-induced fatigue, providing a novel material option for the development of new anti-fatigue natural products. This first systematic study, through "network pharmacology verification - molecular docking verification - experimental verification," elucidates the molecular mechanism by which oryzanol exerts its anti-fatigue effect by regulating the PI3K / AKT / FOXO signaling pathway, achieving a breakthrough from phenomenon to essence, and providing a scientific basis for precise application and product development. Oryzanol is derived from natural grains, has high safety, and low long-term use risk. Animal experiments have shown that it has clear effects in improving endurance, enhancing strength, anti-oxidation, and promoting repair, achieving a combination of multiple anti-fatigue benefits. Oryzanol has a wide range of sources (rice bran oil byproduct), mature extraction technology, and relatively low production costs, making it easy to achieve large-scale production and market promotion. The integrated research strategy (computational biology + experimental verification) adopted in this invention is not only applicable to oryzanol but can also provide a methodological framework for the discovery of anti-fatigue activities and the study of mechanisms of action of other natural products.

[0041] Obviously, the embodiments described above are merely preferred embodiments of the present invention, and not all embodiments. The accompanying drawings illustrate preferred embodiments of the present invention, but do not limit the scope of the patent. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the scope of patent protection of this invention.

Claims

1. The application of oryzanol in the preparation of products for relieving exercise fatigue, characterized in that, The oryzanol improves exercise-induced fatigue by regulating the PI3K / AKT / FOXO signaling pathway, inhibiting oxidative stress and inflammatory responses.

2. The application of oryzanol according to claim 1 in the preparation of products for relieving exercise fatigue, characterized in that, The product is an oral preparation, including capsules, tablets, or sports drink additives.

3. The application of oryzanol according to claim 1 in the preparation of a product for relieving exercise fatigue, characterized in that, The effective daily dose of oryzanol in C57BL / 6J mice is 200 mg / kg.

4. The application of oryzanol according to claim 1 in the preparation of a product for relieving exercise fatigue, characterized in that, The oryzanol regulates the PI3K / AKT / FOXO signaling pathway by binding to AKT1, FOXO1 and / or FOXO3 proteins.

5. The application of oryzanol according to claim 4 in the preparation of a product for relieving exercise fatigue, characterized in that, The binding energies are all less than -7 kcal / mol.

6. The application of oryzanol according to claim 1 in the preparation of a product for relieving exercise fatigue, characterized in that, The oryzanol is used in combination with vitamin E and tea polyphenols to synergistically enhance the anti-fatigue effect.

7. The application of oryzanol according to claim 1 in the preparation of a product for relieving exercise fatigue, characterized in that, The improvement in exercise-induced fatigue is manifested in increased exercise endurance, enhanced muscle strength, reduced oxidative stress levels, and / or promoted muscle tissue repair.

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