Compositions and their use in the manufacture of products for protecting mitochondria

Abstract

The present application relates to the technical field of health food, and particularly relates to a composition and application thereof in preparing a product for protecting mitochondria. The present application provides a composition, raw materials of which include Haematococcus pluvialis, Lycium ruthenicum, Cordyceps militaris and nicotinamide. The composition has the effects of improving the oxidative stress level under mitochondrial dysfunction and delaying the aging related to mitochondria. Compared with single components, the components in the composition cooperate with each other, produce a synergistic effect, and significantly improve the effect of protecting mitochondrial function.

Description

Technical Field

[0001] This invention relates to the field of health food technology, and in particular to compositions and their application in the preparation of products that protect mitochondria. Background Technology

[0002] Mitochondria are organelles found in most cells, enclosed by two membranes, and are the cells' energy-producing structures. In eukaryotes, mitochondria are the site of oxidative metabolism, where carbohydrates, fats, and amino acids are ultimately oxidized to release energy, playing a crucial role in energy conversion. Mitochondria are also the site of the tricarboxylic acid cycle and oxidative phosphorylation. Furthermore, mitochondria store calcium ions, regulate membrane potential and control programmed cell death, regulate cell proliferation and metabolism, and synthesize cholesterol and certain heme pigments. Studies have demonstrated that mitochondrial dysfunction is associated with various physiological and pathological processes, such as aging, Parkinson's disease, diabetes, and neuropathic pain.

[0003] Currently, mitochondrial function is mainly evaluated by measuring mitochondrial oxygen consumption, membrane potential, ATP levels, and reactive oxygen species levels. Slc25a46- / - mice are mice with the Slc25a46 nuclear gene encoding mitochondrial outer membrane proteins knocked out. Their main characteristics include a shortened lifespan of no more than two months, significantly impaired motor function, gastrocnemius muscle atrophy, and an imbalance in redox levels in the brain and liver. The mechanism of multiple organ dysfunction in Slc25a46 knockout mice may be related to mitochondrial dysfunction, mainly manifested as mitochondrial structural damage and reduced respiratory chain enzyme activity. This invention uses Slc25a46 gene knockout mitochondrial-damaged aging mice as a model organism for mitochondrial dysfunction to verify the function of the composition or extract described in this invention. This model has significant phenotypic advantages (including short lifespan, small body size, slow movement, mitochondrial dysfunction, oxidative stress damage, phenotypic stability, clear mechanism, high reproducibility, and short modeling time). This mouse model has significant advantages in practical applications, helping to study the mechanisms of aging in energy metabolism, mitochondrial structure, mitochondrial function, oxidative stress, and evaluating the efficacy of health foods that delay aging. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a composition and its application in the preparation of products that protect mitochondria, the composition being able to improve the level of oxidative stress under mitochondrial dysfunction and delay mitochondrial-related aging, and being able to be used to prepare products that protect mitochondrial function.

[0005] The composition provided by this invention comprises the following raw materials: Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide.

[0006] In this invention, the mass ratio of Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide is (100-450):(200-800):(50-150):(0.1-10).

[0007] In this embodiment of the invention, the mass ratio of Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide is (120-400):(300-700):(60-120):(0.1-5).

[0008] In some embodiments, the mass ratio of Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide is (150-300):(400-650):(80-110):(0.1-0.5).

[0009] In some specific embodiments, the mass ratio of Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide is 180:435:100:0.2.

[0010] The composition of this invention contains Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide. The components work together to produce a synergistic effect, significantly improving the protection of mitochondrial function.

[0011] The present invention also provides an extract prepared from the said composition.

[0012] The method for preparing the extract of the present invention includes:

[0013] Haematococcus pluvialis extract was prepared by extracting Haematococcus pluvialis with ethanol;

[0014] Preparation of black goji berry extract by water extraction;

[0015] Cordyceps militaris extract was prepared by water extraction.

[0016] The extract was prepared by mixing the Haematococcus pluvialis extract, black goji berry extract, Cordyceps militaris extract and nicotinamide.

[0017] In this invention, the ethanol extraction of Haematococcus pluvialis includes: refluxing Haematococcus pluvialis with 80 times its weight of 70wt% ethanol for 4 hours;

[0018] The water extraction method for preparing black goji berry extract includes: extracting black goji berries by reflux with 10 parts by weight of water for 5 hours;

[0019] The water extraction of Cordyceps militaris includes: extracting Cordyceps militaris fruiting bodies by boiling and refluxing with 8 times their weight of water 3 times, each time for 1.5 hours, combining the extracts and concentrating and drying them;

[0020] The application of the composition, extract, or extract prepared by the extraction method of the present invention in the preparation of products that improve oxidative stress levels and delay aging.

[0021] In this invention, the improvement of oxidative stress level refers to the improvement of oxidative stress level under mitochondrial dysfunction, mainly by increasing SOD enzyme activity.

[0022] In this invention, the delay in aging refers to increasing the activity of NAMPT enzyme produced under mitochondrial dysfunction.

[0023] In this invention, the delay in aging refers to increasing the expression level of respiratory chain complex proteins under mitochondrial dysfunction.

[0024] In this invention, the mitochondrial dysfunction is mitochondrial dysfunction caused by the deletion or low expression of the Slc25a46 gene.

[0025] The Slc25a46 gene encodes a protein of the mitochondrial outer membrane and plays a crucial role in mitochondrial dynamics. This invention uses Slc25a46 gene knockout mice with mitochondrial damage to validate the function of the compositions or extracts described herein. This model exhibits significant phenotypic advantages (including short lifespan, small body size, slow movement, mitochondrial dysfunction, oxidative stress damage, phenotypic stability, clear mechanism, high reproducibility, and short model establishment time). In practical applications, this mouse model is helpful for studying the mechanisms of aging in energy metabolism, mitochondrial structure, mitochondrial function, and oxidative stress, as well as evaluating the efficacy of anti-aging health foods, demonstrating significant advantages.

[0026] The use of the composition, extract, or extract obtained by the extraction method of the present invention in the preparation of products that protect mitochondria.

[0027] The present invention also provides a product for protecting mitochondria, comprising the composition described in the present invention, the extract described in the present invention, or the extract obtained by the extraction method described in the present invention.

[0028] In this invention, the product is food, medicine, or health product.

[0029] The food that protects mitochondria includes the composition or extract of the present invention or the extract obtained by the extraction method, and acceptable ingredients in the food.

[0030] The health supplement that protects mitochondria includes the composition or extract described in this invention, or the extract prepared by the extraction method described in this invention, and excipients acceptable in the health supplement.

[0031] The mitochondrial-protecting drug includes the composition of the present invention, the extract of the present invention, or the extract prepared by the extraction method thereof, and pharmaceutically acceptable excipients.

[0032] The pharmaceutically acceptable excipients are one or a mixture of two or more of the following: fruit powder, flavoring, sweetener, acidulant, filler, lubricant, preservative, suspending agent, food coloring, diluent, emulsifier, disintegrant, or plasticizer.

[0033] The dosage form of the drug or health product is tablets, pills, oral liquids, capsules, syrups, drop pills, or granules. Preferably, the capsules are hard capsules or soft capsules. Preferably, the tablets are oral tablets or oral tablets. Oral tablets refer to tablets intended for oral administration; most of these tablets contain drugs absorbed through the gastrointestinal tract to exert their effects, while some tablets contain drugs that exert their effects locally in the gastrointestinal tract. In some embodiments provided by this invention, the oral tablets are ordinary compressed tablets, dispersible tablets, effervescent tablets, chewable tablets, coated tablets, or sustained-release tablets.

[0034] This invention provides a composition comprising Haematococcus pluvialis, black goji berries, Cordyceps militaris, and nicotinamide. This composition improves oxidative stress levels in cases of mitochondrial dysfunction and delays mitochondrial-related aging. Compared to individual components, the components in this composition work synergistically to significantly enhance the protective effect against mitochondrial function. Attached Figure Description

[0035] Figure 1.a Effects of the examples on the morphology and viability of fibroblasts in Slc25a46- / - and WT primary embryos;

[0036] Figure 1.b Effects of Comparative Example 1 on the morphology and viability of fibroblasts in Slc25a46- / - and WT primary embryos;

[0037] Figure 1.c Effects of Comparative Example 2 on the morphology and viability of fibroblasts in Slc25a46- / - and WT primary embryos;

[0038] Figure 1.d Effects of Comparative Example 3 on the morphology and viability of fibroblasts in Slc25a46- / - and WT primary embryos;

[0039] Figure 1.e Effects of Comparative Example 4 on the morphology and viability of fibroblasts in Slc25a46- / - and WT primary embryos;

[0040] Figure 2 The results show the detection of SOD enzyme activity in the brain tissue of Slc25a46- / - mice in the blank group, the intervention group of the example, and the intervention groups of comparative examples 1-4;

[0041] Figure 3The results show the detection of SOD enzyme activity in the liver tissue of Slc25a46- / - mice in the blank group, the intervention group of the example, and the intervention groups of comparative examples 1-4;

[0042] Figure 4 The results show the expression levels of mitochondrial respiratory chain complex proteins in the liver tissue of Slc25a46- / - mice in the blank group, the intervention group of the example, and the intervention groups of comparative examples 1-4.

[0043] Figure 5 The results show the detection of NAMPT enzyme activity in the liver tissue of Slc25a46- / - mice in the blank group, the intervention group of the example, and the intervention groups of comparative examples 1-4. Detailed Implementation

[0044] This invention provides compositions and their application in the preparation of products protecting mitochondria. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art will clearly be able to modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0045] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0046] In a specific embodiment, the natural product composition and individual raw materials of the present invention, which have mitochondrial protective function, were used in vivo experiments in a Slc25a46 gene knockout mitochondrial injury mouse model to explore the mitochondrial function protection of the natural product composition and individual raw materials. Mitochondrial dysfunction can lead to decreased mitochondrial membrane potential, reduced ATP synthesis, and increased oxidative stress, which can directly or indirectly affect the normal function of the entire cell, and thus have a certain impact on organs such as the liver (the central station of metabolism in the body) and the brain (the organ with the highest metabolic rate in the body, and which obtains most of its energy through aerobic metabolism).

[0047] Specifically, in addition to conducting cytotoxicity (MTT) experiments on the formulation and individual ingredients, the efficacy of the natural product composition and individual ingredients was evaluated using oxidative stress and anti-aging enzyme activities in the aforementioned animal models. To evaluate oxidative stress levels and anti-aging capabilities, the activity of SOD (superoxide dismutase) in the brain and liver tissues of Slc25a46- / - mice after intervention with the formulation and individual ingredients, the expression level of mitochondrial respiratory chain complex protein in the liver tissue, and the activity of NAMPT (nicotinamide phosphoribosyltransferase) were measured.

[0048] The present invention will be further illustrated below with reference to the embodiments:

[0049] Example

[0050] The composition was prepared according to the following formula: Haematococcus pluvialis, black goji berries, Cordyceps militaris and nicotinamide in a mass ratio of 180:435:100:0.2.

[0051] Preparation methods include:

[0052] 1. Preparation method of Haematococcus pluvialis extract: 1 part by weight of Haematococcus pluvialis is added to 80 parts by weight of 70wt% ethanol, and the mixture is refluxed in a water bath at 40℃ for 4 hours. After filtration, concentration and vacuum drying, the extract is obtained.

[0053] 2. Preparation method of black goji berry extract: Wash black goji berries clean, add 1 part by weight of black goji berries to 10 parts by weight of water, reflux extract in a 50℃ water bath for 5 hours, filter, and spray dry the resulting precipitate to obtain the extract.

[0054] 3. Preparation method of Cordyceps militaris powder: The fruiting bodies of Cordyceps militaris are boiled and refluxed three times with 8 times their weight of water for 1.5 hours each time. The extract is collected and concentrated into an extract under vacuum at 69±3℃ (specific gravity 1.05-1.07). Then, it is spray dried (inlet air temperature 140-190℃, outlet air temperature 70-90℃) and passed through an 80-mesh sieve.

[0055] The Haematococcus pluvialis extract, black goji berry extract, Cordyceps militaris powder, and nicotinamide were mixed according to the formula to prepare the composition.

[0056] Comparative Example 1

[0057] Preparation method of Haematococcus pluvialis extract: 1 part by weight of Haematococcus pluvialis is added to 80 parts by weight of 70 wt% ethanol, and the mixture is refluxed in a water bath at 40°C for 4 hours. After filtration, concentration and vacuum drying, the extract is obtained.

[0058] Comparative Example 2

[0059] Preparation method of black goji berry extract: Wash black goji berries clean, add 1 part by weight of black goji berries to 10 parts by weight of water, reflux extract in a 50℃ water bath for 5 hours, filter, and spray dry the resulting precipitate to obtain the extract.

[0060] Comparative Example 3

[0061] Preparation method of Cordyceps militaris powder: The fruiting bodies of Cordyceps militaris are boiled and refluxed three times with 8 times their weight of water for 1.5 hours each time. The extract is collected and concentrated into an extract under vacuum at 69±3℃ (specific gravity 1.05-1.07). Then, it is spray dried (inlet air temperature 140-190℃, outlet air temperature 70-90℃) and passed through an 80-mesh sieve.

[0062] Comparative Example 4

[0063] Purchase commercial food-grade nicotinamide.

[0064] Efficacy verification

[0065] I. Obtaining and culturing primary embryonic fibroblasts (MEF)

[0066] MEF cells were derived from wild-type fetal mice (WT) and Slc25a46 homozygous fetal mice (Slc25a46- / -). Pregnant mice were anesthetized and euthanized, and mouse embryos were obtained through dissection. After obtaining the fetal mouse torso, the tissue was washed three times with PBS. The cleaned mouse embryonic tissue was transferred to a culture dish, minced, and 2 mL of trypsin was added to the tissue fragments. Mincing continued for a few minutes, and then 5 mL of trypsin was added to the culture dish. The mixture was vigorously pipetted up and down and placed in an incubator for 20-30 minutes. The minced tissue was removed from the incubator, and approximately 20 mL of LMEF medium was added. The tissue mixture was transferred to each culture flask and placed in an incubator for overnight incubation. Fibroblast growth was then observed periodically, and the medium was changed as needed. When the cells covered 90% of the culture flask, the mouse embryonic fibroblasts were collected for subsequent experiments.

[0067] II. In vitro cytotoxicity test of the natural product composition formulation described in this invention

[0068] MTT experiment:

[0069] 100 μL of cells were prepared at a concentration of 3.5 × 10⁻⁶. 5 WT mouse cells and Slc25a46- / - mouse cells were seeded in 96-well plates and incubated at 37°C for 24 h in a CO2 incubator. Cells were then treated with a series of test solutions containing different concentrations of 0, 62.5, 125, 250, 500, 1000, and 2000 μg / mL of natural product compositions (examples) and single-ingredient solutions (comparative examples 1-4) for 24 h. 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated for another 4 h. The supernatant was discarded, and 150 μL of DMSO was added to each well to fully dissolve the formazan crystals. The absorbance was measured at 570 nm using a multi-mode microplate scanner. The effect of each concentration of test solution on cell viability was calculated with the blank control as 100%.

[0070] like Figure 1.a As shown, the formulations in the examples had no effect on cell morphology; at concentrations of 125-2000 μg / mL, they were non-toxic to cells; and at concentrations of 1000 and 2000 μg / mL, they promoted the growth of WT cells, with a concentration-dependent effect.

[0071] like Figure 1.b As shown, Comparative Example 1, i.e., Haematococcus pluvialis, had no effect on cell morphology; it promoted the growth of Slc25a46- / - cells and WT cells at concentrations ranging from 125 to 2000 μg / mL, and there was a concentration effect.

[0072] like Figure 1.c As shown, Comparative Example 2, i.e., black goji berries, had no effect on cell morphology; at 1000 μg / mL, they promoted the growth of Slc25a46- / - cells, with no concentration effect; and at concentrations of 125-2000 μg / mL, they all promoted the growth of WT cells, showing a concentration effect.

[0073] like Figure 1.d As shown in Comparative Example 3, Cordyceps militaris had no effect on cell morphology; at low concentrations (125-500 μg / mL), it had no significant effect on cell growth; and at high concentrations (1000-2000 μg / mL), it had an inhibitory effect.

[0074] like Figure 1.e As shown in Comparative Example 4, nicotinamide had no effect on cell morphology; however, it promoted the growth of Slc25a46- / - and WT cells at concentrations of 1000 and 2000 μg / mL, with no concentration effect.

[0075] Regarding cell morphology: Within the same concentration range, the formulations and comparative examples 1-4 (single formulations) had no significant effect on the morphology of Slc25a46- / - and WT cells. Regarding cell viability, the formulations had no effect on Slc25a46- / - cell viability within the range of 125-2000 μg / mL, but higher concentrations promoted WT cell proliferation. Comparative examples 1, 2, and 4, within the single formulation range of 125-2000 μg / mL, had no effect on the viability of both Slc25a46- / - and WT cells, and higher concentrations also promoted WT cell proliferation. However, although low concentrations of comparative example 3 (Cordyceps militaris) had no effect on the viability of either cell type, high concentrations of this ingredient had an inhibitory effect on both cell types, demonstrating toxicity; therefore, dosage should be carefully monitored. The formulations were composed of comparative examples 1-4 in specific proportions. Therefore, at the same total concentration, the contents of Haematococcus pluvialis, Lycium barbarum, Cordyceps militaris, and nicotinamide in the formulations were lower than the contents of the corresponding raw materials in the comparative examples. In summary, the results of the content of the formulation (examples) and the single ingredients (comparative examples 1-4), as well as their effects on cell morphology and viability, show that the examples exhibit better safety and stability than the comparative examples, demonstrating the superiority of the scientific formulation ratio and exploring the safety of the formulation.

[0076] III. Experiments on the in vivo protection of mitochondrial function by the natural product composition of the present invention.

[0077] 1. Laboratory animals

[0078] Animal subjects used in the experiment were 1-month-old Slc25a46 gene knockout (Slc25a46- / -) mice, divided into a blank solvent control group (n=3) and experimental groups (n=4 in each group). Mice were fed a complete nutritional diet formulated with AIN93G from Jiangsu Xietong Biotechnology Co., Ltd. under the same SPF-grade conditions. They were administered the diet by gavage daily for 10 days. The experimental groups received the test substance formulation at a dose of 400 mg / kg / day, as did Comparative Group 1 (100 mg / kg / day), Comparative Group 2 (244 mg / kg / day), Comparative Group 3 (56 mg / kg / day), and Comparative Group 4 (500 mg / kg / day). The blank solvent control group received water as the corresponding solvent. After the gavage experiment, brain and liver tissues were collected from the mice for physiological and biochemical analysis to assess oxidative stress levels and anti-aging capabilities.

[0079] 2. Biochemical detection of mouse brain and liver tissues

[0080] Add 9 volumes of physiological saline (ice) to the tissue at a ratio of 1:9 (tissue weight (g):volume (mL)). Mince the tissue with surgical scissors and homogenize it into a 10% tissue homogenate using a homogenizer (homogenization time 10 s / time, 5 s interval, 3-5 times consecutively, performed on ice). Centrifuge at 2500-3000 rpm for 10 min, and collect the supernatant for subsequent assays. SOD activity in brain and liver plasma was detected strictly according to the instructions of the superoxide dismutase (SOD) assay kit (catalog number: A001-3, WST-1 method, Nanjing Jiancheng Bioengineering Institute). Statistical analysis was performed using Prism software.

[0081] The higher the SOD enzyme activity, the better its antioxidant effect. (See Table 1 and...) Figure 2-3 As shown, the SOD activity in the brain and liver tissues of the formulation (example) intervention group was significantly higher than that in the blank group (p<0.001), indicating that the formulation intervention can effectively improve the antioxidant level in the brain and liver tissues of Slc25a46- / - homozygous mice. Simultaneously, the SOD enzyme activity in the brain and liver tissues of the example intervention group was significantly higher than that in comparative groups 1-4 (p<0.01), while there were no significant differences between any pairwise comparative groups. In summary, the above analysis indicates that the formulation has good antioxidant capacity, and its antioxidant effect is significantly higher than that of any single ingredient.

[0082] 3. Detection of protein expression levels of mitochondrial respiratory chain complexes in mouse liver tissue

[0083] 100 mg of mouse liver tissue was weighed and lysed with lysis buffer containing protease and phosphorylated protease inhibitors according to the specified ratio. Proteins were collected and extracted using standard methods. The protein concentration was detected using a BCA kit. SDS-PAGE electrophoresis gel was prepared, followed by protein electrophoresis, wet transfer, and blocking. Antibody incubation: Based on the protein size indicated by the protein marker, the NC membrane was cut to the required molecular weight. The cut NC membrane was placed in the antibody incubation chamber, and the primary antibody (OXPHOS, 1:1000) solution was added to completely submerge the NC membrane. The incubation chamber was then placed in a shaker at 4°C and incubated overnight. The next morning, the primary antibody solution was recovered and stored at -20°C. The NC membrane was washed three times with TBST buffer, approximately 10 min each time. After washing, secondary antibody was added, and the membrane was incubated at room temperature on a shaker for approximately 1.5 h. The NC membrane was then washed three more times with TBST buffer. The protein developing instrument was pre-cooled, and the protein developing solution A:B was prepared fresh and used immediately. First, use filter paper to absorb excess buffer from the membrane. Then, immerse the NC membrane in a container containing developing solution in the dark. Next, place the membrane into the instrument for exposure imaging. Select the exposure function with the optimal exposure time for a single image. Keep the exposed protein band image and analyze the data using ImageJ software. Calculate the protein expression level based on the grayscale value.

[0084] The respiratory chain (complex IV) represents the fundamental function of mitochondria. It is a continuous reaction system formed by arranging a series of hydrogen and electron transfer reactions in a specific sequence. The main function of the respiratory chain is to generate energy through its own hydrogen and electron carriers and a series of redox processes. For example... Figure 4 As shown in the figure (values ​​marked in the figure = complex gray level / β-actin gray level in the same column, are relative values), compared with the control group, the levels of mitochondrial respiratory chain complexes I, III, IV, and V proteins in the intervention group of the examples were significantly increased, especially I, IV, and V, indicating that the examples can improve the electron transport and ATP synthesis functions of the damaged mitochondrial respiratory chain. The expression level of respiratory chain complex IV protein in each single-component intervention group (comparative examples 1-4) was slightly higher than that in the control group, especially comparative example 2 (black goji berry group), but all were lower than those in the example group. In summary, compared with the comparative examples, the examples (formulas) can improve the function of damaged mitochondria.

[0085] 4. Detection experiment of NAMPT enzyme activity in mouse liver tissue

[0086] A certain amount of mouse liver tissue was weighed, homogenized, and the supernatant was collected. The procedure was strictly followed according to the instructions of the Mouse Nicotinamide Phosphoribosyltransferase (NAMPT) ELISA Kit (catalog number: JL24460, ELISA, Jianglai Biotechnology). Standard wells, blank wells, and sample wells were set up. 50 μL of different concentrations of standard were added to each standard well, 50 μL of the test sample was added to each sample well, and 50 μL of sample diluent was added to each blank well. 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each of the blank, standard, and sample wells. The reaction wells were sealed with sealing film and incubated in a 37°C water bath for 60 min. The waste liquid was discarded, and the plate was patted dry on absorbent paper. Each well was filled with washing buffer (350 μL), allowed to stand for 1 min, the washing buffer was discarded, and the plate was patted dry on absorbent paper. This washing process was repeated 5 times. 50 μL each of substrate A and B were added to each well. The OD value of each well was measured at 450 nm within 15 min. Record the data and perform statistical analysis using Prism software.

[0087] Nicotinamide phosphoribosyltransferase (NAMPT) is divided into intracellular and extracellular nicotinamide phosphoribosyltransferases, which are essential for the biosynthesis of NAD+. + As the rate-limiting enzyme, NAMPT plays a very important role. Studies have found that increasing NAMPT levels can promote NAD+ uptake. + Synthesized to exert anti-aging and life-extending effects. (See Table 1 and...) Figure 4 As shown, compared with the control group, the NAMPT enzyme activity in the liver tissue of mice in the example group was significantly higher than that in the control group (p < 0.05). Meanwhile, the NAMPT enzyme activity in the example group was significantly higher than that in comparative groups 1, 2, and 4 (p < 0.05), but there was no significant difference compared with comparative group 3. These results indicate that the example (formulation) has the potential to exert anti-aging and life-extending effects, and its effect is superior to that of a single ingredient.

[0088] Table 1

[0089]

[0090] Note: Blank group vs. Implementation Example, # ", p<0.05; " ## ", p<0.01; " ### ", p<0.001;

[0091] Example vs. Comparative Example * ", p<0.05; " ** ", p<0.01; " *** ", p<0.001.

[0092] The combined results of MTT toxicity assay, SOD enzyme activity assay, mitochondrial complex protein expression assay, and NAMPT enzyme activity assay indicate that the formulation, within a certain concentration range, has no effect on the morphology of mitochondrial damaged (Slc25a46- / -) and normal (WT) cells, but can enhance the proliferation capacity of both damaged and normal cells; and increases the activity of the antioxidant enzyme SOD, the expression level of mitochondrial respiratory chain complex protein, and promotes NAD synthesis in Slc25a46- / - mouse tissues after intervention. + The increased NAMPT enzyme activity can improve oxidative stress levels under mitochondrial impairment and mitochondrial damage-related aging, making it suitable for use in products with mitochondrial protective effects. Furthermore, comparing the performance of the formulation and single ingredients in the aforementioned test indicators revealed that the formulation is safer, more stable, and more effective than the single ingredient, demonstrating the importance and scientific basis of ingredient selection and formulation.

[0093] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for improving oxidative stress levels under mitochondrial dysfunction and delaying mitochondrial-related aging, characterized in that, The mixture is prepared from the following raw material components in the following mass ratio: Haematococcus pluvialis: black goji berries: Cordyceps militaris: nicotinamide = 180:435:100:0.

2. According to the raw material ratio, Haematococcus pluvialis is extracted by refluxing with 80 parts by weight of 70 wt% ethanol for 4 hours to obtain Haematococcus pluvialis extract; black goji berries are extracted by refluxing with 10 parts by weight of water for 5 hours to obtain black goji berry extract; Cordyceps militaris fruiting bodies are extracted by boiling and refluxing with 8 parts by weight of water three times, 1.5 hours each time, and the extracts are combined, concentrated, and dried to obtain Cordyceps militaris powder; the Haematococcus pluvialis extract, black goji berry extract, Cordyceps militaris powder, and nicotinamide are mixed to obtain the composition.

2. A product for improving oxidative stress levels under mitochondrial dysfunction and delaying mitochondrial-related aging, characterized in that, Includes the composition of claim 1.

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