TWI825324B - Anti-aging uses of citrus extract ingredients - Google Patents

Abstract

The present invention reveals that hesperetin has the effect of prolonging lifespan and slowing down the symptoms of aging. It acts as a Cisd2 activator and achieves the effect of fighting aging-related symptoms by increasing the expression of the Cisd2 gene through continuous application. Its efficacy is supported by a number of experimental results. .

Description

Anti-aging uses of citrus extract ingredients

The invention relates to the anti-aging use of citrus extract components.

Aging is a common issue faced by all people. Currently, the cellular and molecular characteristics of aging have been widely recognized, including: mitochondrial dysfunction, DNA damage, telomere loss, epigenetic changes, and protein homeostasis. loss, nutrient sensing disorders, cell failure, stem cell failure and changes in intercellular communication. However, at present, there are few drugs that can fight aging symptoms and extend healthy lifespan.

Although the molecular pathways responsible for aging are not yet understood, previous studies have shown that mitochondrial dysfunction plays a critical role in driving age-related pathophysiology, altering energy metabolism in organisms. , thereby affecting the regulation of insulin sensitivity and glucose homeostasis, and even accelerating the aging of mammals. In addition, mitochondrial dysfunction and metabolic disorders including reduced oxygen consumption and ATP production have also been observed in elderly subjects with low physical fitness, which are risk factors for death.

Previous studies have used genetic mouse models to explore genes with the effect of extending lifespan and discovered related pathways that regulate the lifespan of mammals. Among them, Cisd2 (CDGSH iron sulfur domain 2) is currently one of the most representative and important genes that extend the lifespan of mammals. , previous studies have confirmed that the Cisd2 gene has the effect of delaying human aging through the Gompertz mortality rate model, and confirmed that Cisd2 deficiency shortens lifespan and leads to premature aging in mice. In addition, it is worth noting that during the normal aging process, it has been detected that the expression of Cisd2 in mice gradually decreases with age. Some studies have also shown that the continuous and stable expression of the Cisd2 gene in mice through gene transfer technology can help Dramatically extending the median and maximum lifespan of mice without any apparent harmful side effects. Cisd2 also protects mitochondria from aging damage, maintains mitochondrial function, and slows down aging-related metabolic changes. Although evidence from multiple studies shows that Cisd2 is an important lifespan-extending gene, whether Cisd2 can be targeted by drugs and activated by compounds to achieve the effect of slowing aging is an important question that remains to be clarified in this field.

The expression level of Cisd2 will gradually decrease during the natural aging process. The inventor of this case hopes to find a small compound that can be used to activate the Cisd2 gene clinically to achieve the effects of extending lifespan and anti-aging.

Hesperetin is one of the components extracted from citrus and is a citrus flavonoid. It has antioxidant effects. There are currently no reports on the use of hesperetin to extend lifespan, and there are no studies on hesperetin. The relationship between it and Cisd2.

In view of the shortcomings of the prior art, the present invention reveals through experiments that there is an interactive relationship between hesperetin and Cisd2, and further evaluates the feasibility of hesperetin as a Cisd2 activator clinically, so as to achieve the effect of extending healthy lifespan and slowing down aging. Efficacy is a subject that those skilled in the art are eager to explore.

One object of the present invention is to provide a use of a citrus extract component for preparing an anti-aging pharmaceutical composition. The citrus extract component is hesperetin, which achieves anti-aging effects by increasing the expression of the Cisd2 gene. effect.

Among them, the anti-aging includes extending life span and slowing down the symptoms of aging.

Among them, the symptoms of aging include liver function damage, metabolic function decline, fat accumulation, muscle loss or heart aging symptoms.

Among them, the decrease in metabolic function includes a decrease in systemic oxygen consumption, a decrease in carbon dioxide production rate, a decrease in heat production efficiency, a decrease in insulin sensitivity, or a decrease in the constant regulation function of glucose.

Among them, the fat accumulation includes total body fat accumulation or visceral fat accumulation.

Among them, the muscle loss includes decrease in skeletal muscle mass, decrease in skeletal muscle strength, decrease in net body weight, mitochondrial degeneration, muscle fiber degeneration or muscle ultrastructural damage.

Wherein, the muscle from which the muscle is lost is skeletal muscle.

Wherein, the skeletal muscle includes femoral muscle or gastrocnemius muscle.

Among them, the symptoms of cardiac aging include reduced ejection fraction, increased cardiac performance index, increased perivascular fibrosis, arrhythmia or ultrastructural damage.

Among them, the ultrastructural damage includes deterioration of myocardial discs, mitochondria or sarcomeres.

In order to achieve the above purpose, the dosage of the pharmaceutical composition is 200-800mg/60kg/day, preferably 400-600mg/60kg/day, and the best is 491mg/60kg/day.

In order to achieve the above purpose, the pharmaceutical composition can be formulated into dosage forms including liquid nutritional supplements, oral liquids, beverages, powders, capsules, lozenges, pills, granules, jelly, chewing gum, injections or other food and pharmaceutical products. Acceptable dosage forms.

To achieve the above purpose, the pharmaceutical composition may further include pharmaceutically acceptable carriers, excipients, and diluents.

The purpose of the present invention is to evaluate the feasibility of hesperetin as a Cisd2 activator to achieve an anti-aging solution. The inventor of this case has demonstrated through relevant experiments that hesperetin does have the clinical effect of prolonging lifespan and improving The effect of aging-related symptoms, and further confirmed that the mechanism of action of hesperetin is related to Cisd2, it is regarded as a pharmaceutical activator of Cisd2 to achieve the effect of extending healthy lifespan, while alleviating age-related functional decline and structural damage.

Some embodiments of the present invention further explore its efficacy in detail, and confirm that hesperetin can effectively improve the changes in metabolic function indicators caused by aging, including: improving symptoms such as decline in metabolic function, reducing fat accumulation, reducing muscle loss, and slowing down heart aging. It helps slow down the aging process in naturally aging mammals and rejuvenates their aging tissues, thereby extending healthy lifespan.

All technical and scientific terms used in this specification, unless otherwise defined, have meanings commonly understood by those of ordinary skill in the art; as used herein, the abbreviation "ALT" refers to alanine aminotransferase (alanine aminotransferase); the abbreviation "AST" refers to aspartate aminotransferase (aspartate aminotransferase); the abbreviation "BUN" refers to blood urea nitrogen (blood urea nitrogen); the abbreviation "CKMB" refers to creatine kinase-myocardial band); the abbreviation "CRE" refers to creatinine; the abbreviation "TCHO" refers to total cholesterol; the abbreviation "TriG" refers to triglycerides; the abbreviation "WBC" refers to white blood cells (White blood cells); the abbreviation "RBC" refers to red blood cells; the abbreviation "HGB" refers to hemoglobin; the abbreviation "HCT" refers to hematocrit; the abbreviation "MCH" refers to Mean corpuscular hemoglobin; the abbreviation "MCHC" refers to the mean corpuscular hemoglobin concentration; the abbreviation "MCV" refers to the mean corpuscular volume; the abbreviation "RDW" refers to the red blood cell distribution width ( red cell distribution width); the abbreviation "RET" refers to reticulocytes count.

In various embodiments of the present invention, unless otherwise specified, the hesperetin dose provided in the food of the hesperetin treatment group (Hes) is 100 mg/kg/day, and the excipient (Veh) group is administered In addition, the excipient system was 10% propylene glycol, all mice used were male, the old mice were 19 to 21 months old (19-21-mo), and the young mice were 3 Months old (3-mo), the treatment time lasted from 3 to 6 months, the quantitative data were expressed as mean ± standard deviation, and the statistical values were analyzed by Student's T test (* p <0.05 ; ** p <0.005), and the materials used in the present invention are all commercially available and easily available materials.

The embodiments described below are only for illustrating the technical ideas and characteristics of the present invention. Their purpose is to enable those with ordinary knowledge in the field to understand the content of the present invention and implement it accordingly. They should not be used to limit the patent scope of the present invention. That is to say, all equivalent changes or modifications made in accordance with the spirit disclosed in the present invention should still be covered by the patent scope of the present invention.

Example 1. In vivo toxicity test of hesperetin

The present invention first applies hesperetin to mice and tests its toxicity. In this example, elderly mice are used for testing, and hesperetin (100 mg/kg/day) is continuously provided in their food. After the mouse life span is 26 At one month old, serum biochemical analyzes and complete blood count analyzes (CBC analyses) were performed to measure the changes in various values in the mice.

The results of serum biochemical analysis are shown in Figure 1. In this experiment, 20-month-old mice were treated for 6 months, and serum samples were collected for analysis at 26 months of age. The relevant in vivo serum parameters include: (A) Damage indicators related to liver function (ALT and AST); (B) Kidney function (BUN) and kidney injury (CRE) indicators; (C) Damage indicators related to cardiac function (CKMB); (D) Fasting 2 and Blood glucose level after 6 hours; (E) Serum insulin level after 6 hours of fasting; (F) Fat metabolism indicators (TCHO and TriG levels in serum); (G) Related electrolyte indicators in serum, including: Ca ^{2 +} , Levels of Mg ^{2 +} , Na ⁺ , K ⁺ and Cl ^- . Among them, no detection was observed after 6 months of hesperetin treatment in aged mice (from 20 to 26 months of age) compared with gender- and age-matched controls treated with vehicle (Veh). In addition, as shown in Figure 1A, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the hesperetin-administered group showed a downward trend, which revealed that the hesperetin-administered group had Improves liver function damage indicators.

The results of complete blood cell count analysis are shown in Figure 2. In this experiment, 19-month-old mice were treated for 7 months, and whole blood samples were collected for analysis at the age of 26 months. The related whole blood cell parameters Including: (A) White blood cell (WBC) parameters; (B) Red blood cell (RBC) and hemoglobin (HGB) parameters; (C) Platelet (Platelet) parameters. The results showed that similar analysis results to those of young mice (3 months old) could be observed in old mice after hesperetin administration. Among them, as shown in Figure 2A, in the white blood cell analysis, the results were similar to those of Veh-treated old mice. Compared with mice, hesperetin increased the percentage of lymphocytes (Lymphocytes) and decreased neutrophils (Neutrophils); in addition, as shown in Figure 2B, in red blood cell analysis, administration of hesperetin increased the number of RBC count and HGB concentration in old mice, i.e., hesperetin has the effect of improving age-related hematological changes without being toxic.

In summary, mice treated with hesperetin can improve liver function damage caused by aging, slow down the abnormalities of neutrophils and lymphocytes caused by aging, and improve the age-related decline of RBC and HGB. In addition, , serum biochemistry analysis and complete blood count analysis collectively showed that hesperetin had no detectable toxicity during long-term treatment of mice.

Example 2: Hesperetin as Cisd2 Activator and extends lifespan

In this example, aged mice (21 months old) were divided into three groups, namely: untreated aged mice (No treatment, Ctrl), aged mice treated with excipients (Veh) and Hesperetin was administered to aged mice (Hesperetin) and the survival rate of each group was monitored. The results are shown in Figure 3. The median median of mice treated with hesperetin was compared to mice treated with vehicle. Lifespan increased by 2.25 months, extending it by about 8.7% (from 25.95 months to 28.2 months on average), while maximum lifespan increased by 4.1 months, extending by about 13.9% (from 29.5 months to 33.6 months on average) months), that is, hesperetin significantly extended the lifespan of aged mice (p = 0.04).

In addition, as shown in Figure 4, in naturally aging old mice (26 months old), the Cisd2 protein in the femoral muscle (Femoris), gastrocnemius (Gastrocnemius) and cardiac muscle (Cardiac muscle) was lower than that in young mice. levels were significantly reduced, however, hesperetin significantly increased Cisd2 protein levels in all these tissues.

In summary, hesperetin treatment has the effect of extending the lifespan of naturally aging mice from the late stage, and is related to the enhancement of Cisd2 expression in aged mice.

Example 3: Hesperetin can improve the decline in systemic energy metabolism caused by aging

In this example, 23.5-month-old and 28-month-old old mice were treated with hesperetin (Hes) for 1.5 and 6 months respectively, and compared with those treated with vehicle (Veh) and young mice ( 3 months old) were compared to explore the changes in their whole-body energy metabolism. As shown in Figures 5A-5C, compared with young mice, the whole-body oxygen consumption of old mice (28 months old) treated with vehicle ( VO ₂ ), CO ₂ production (VCO ₂ ) and heat production (Heat) were all significantly reduced; however, 6 months of hesperetin treatment could significantly slow down systemic oxygen consumption and CO ₂ in aging-related old mice. Reduction in production volume and heat generation. In addition, regarding the symptoms of aging, as shown in Figures 5D-5F, in the Veh treatment group, the metabolic rate of 28-month-old mice during the dark period was lower than that of 23.5-month-old mice, which was in line with expectations. This is consistent with the results, showing that energy metabolism does decrease with aging; however, as shown in Figure 5G-5I, in the Hes treatment group, older (28-month-old) mice (hesperetin treated for 6 months ) was significantly higher than that of 23.5-month-old mice (hesperetin treatment for 1.5 months), which shows that hesperetin can rejuvenate aging mice in terms of energy metabolism, including increasing VO ₂ and VCO ₂ and the average level of heat generated.

In summary, hesperetin treatment can effectively slow down the decline in systemic energy metabolism caused by aging, including the decrease in systemic oxygen consumption, carbon dioxide production rate, and thermogenesis efficiency.

Example 4: Hesperetin can slow down fat accumulation and decline in metabolic function caused by aging

In this example, mice in each group were subjected to micro-computed tomography (Micro-CT) analysis and quantification, and the results are shown in Figure 6; where, as shown in Figure 6A, the yellow circle represents the visceral fat area to be quantified, and Compared with young mice (3 months old), the total body fat and visceral fat of old mice (28 months old) treated with vehicle increased significantly. However, the elderly mice treated with hesperetin for 6 months had significantly higher body fat and visceral fat. mice can significantly slow down the accumulation of total body fat and visceral fat associated with aging; in addition, as shown in Figure 6B, compared with the vehicle-treated control group, the net body weight (body weight) of aged mice treated with hesperetin lean mass) was significantly increased and returned to results similar to those of young mice, which means that hesperetin treatment can also slow down muscle loss and fat accumulation related to aging.

In addition, this example further conducted glucose tolerance test (GTT) and insulin tolerance test (ITT) for each group, and the results are shown in Figure 6; among them, after three months of administration, the results are as shown in Figure 7A, fasting for 6 hours There was no significant difference in basal blood glucose levels. However, the ITT study showed that the insulin sensitivity of the hesperetin-treated (from 23.5 months to 26.5 months) group was significantly improved, indicating that hesperetin can improve age-related changes in aged mice. Decreased insulin sensitivity. In addition, in the group treated with hesperetin for 6 months (from 22 months to 28 months), the results of which are shown in Figure 7B, a significant decrease in basal blood glucose and a significant improvement in GTT study results were detected, indicating that hesperetin Glucose can improve the glucose homeostasis regulation function in aged mice. The above results indicate that hesperetin can improve glucose homeostasis and increase insulin sensitivity in aged mice.

In summary, hesperetin treatment can effectively slow down fat accumulation, net weight loss, insulin sensitivity and glucose constant regulation function caused by aging.

Example 5: Hesperetin can slow down the symptoms of muscle loss caused by aging

In this example, mice in each group were first subjected to a roller test and a grip strength test. The results are shown in Figure 8. Among them, the roller test results are shown in Figure 8A. Compared with the Veh group, the group administered hesperetin was significantly improved. The residence time on the roller was determined; in addition, the grip strength test results are shown in Figure 8B. Compared with the 23-month-old mice (treated for three months), the 26-month-old mice (treated for six months) The grip strength performance was better, showing that continuous application of hesperetin can gradually restore skeletal muscle vitality. The above experiments show that continuous administration of hesperetin can significantly improve the decline in muscle strength caused by aging.

In this example, Masson's trichrome staining was performed on the femoral muscles and gastrocnemius muscles of mice in each group. The results are shown in Figure 9, and the quantitative results of intact and degenerated muscle fibers are shown in Figure 10; wherein, Figure 9A is Representative photomicrographs of intact (Intact) and degenerating (Degenerating) muscle fibers in the femoral and gastrocnemius muscles; the results of this example are shown in Figure 9B. There are obvious skeletal muscles of 26-month-old mice. Degeneration and fibrosis, however, hesperetin treatment can slow down these age-related histopathological deleterious changes in skeletal muscle. The quantification results are shown in Figure 10. In hesperetin-treated aged mice, the The percentage of degenerated muscle fibers in the femoral and gastrocnemius muscles was significantly reduced, indicating that hesperetin can effectively improve muscle fiber degeneration caused by aging.

This embodiment further uses transmission electron microscopy (TEM) to analyze the ultra-structural damages of skeletal muscle. The results are shown in Figure 11, where "MD" marked in the figure means mitochondrial degeneration ( Mitochondrial degeneration), "*" in the figure indicates fibrosis, and "ZLb" in the figure indicates Z-line breakdown; among them, 24-month-old elderly mice treated with Veh (Figure 11B ), compared with young mice (Figure 11A), it has obvious mitochondrial degeneration, and the structure of its triad is also disturbed. The triad system is surrounded by terminal cisterns (cisternae) on both sides. ). In addition, myofibril degeneration (myofibril degeneration), Z-line decomposition and obvious fibrosis can also be detected in the aged control mice (Veh); however, as shown in the figure As shown in 11C, after 3 months of hesperetin treatment, it effectively weakened and partially restored the effects of age-related ultrastructural damage, that is, the intact triplet structure and sarcomeres were retained, and the mitochondria were reduced. degeneration, thereby developing toward a more youthful ultrastructural pattern of skeletal muscle.

In summary, hesperetin treatment can effectively slow down the decline in muscle strength, muscle quality, muscle fiber degeneration, mitochondrial degeneration and muscle ultrastructural damage caused by aging.

Example 6: Hesperetin can slow down the symptoms of cardiac aging caused by aging

This example demonstrates that hesperetin can slow down the symptoms of cardiac aging caused by aging by analyzing cardiac ultrasound images, electrocardiograms, transmission electron microscopy images and histopathological analyzes of mice in each group. , the characteristics of cardiac aging in the hearts of aged mice include: reduced ejection fraction, increased cardiac performance index, increased arrhythmias, and increased perivascular fibrosis.

Figure 12 shows the cardiac ultrasound images of mice in each group. The ejection fraction and myocardial function index results obtained from the analysis are shown in Figure 13. After three months of hesperetin treatment, at the age of 24 months, old In aged mice, the ejection fraction increased significantly, while the myocardial performance index decreased significantly. The results showed that hesperetin can effectively improve the ejection fraction and myocardial performance index of aging hearts.

This example further analyzed the 5-minute continuous electrocardiogram tracings of each group to explore the effect of hesperetin on aging-related arrhythmias and electrical dysregulation. As shown in Figure 14, the patients treated with hesperetin Elderly mice reduce the occurrence of age-related arrhythmias; this example analyzes the QT interval and Tpeak-Tend interval of each group based on their electrocardiograms. The results are shown in Figure 15. Hesperetin treatment can significantly improve the elderly mice. The anomaly of QT interval and Tpeak-Tend interval.

This embodiment further detects the degree of perivascular fibrosis of the heart by detecting collagen through Sirius Red/Fast Green staining. The results are shown in Figure 16. In naturally aging mice and 24 Significant pericardial fibrosis was detected in the aged mice of the Veh group. However, after 3 months of hesperetin treatment, pericardial fibrosis was significantly reduced, confirming that hesperetin can also improve the health of elderly mice. Decreased pericardial fibrosis.

In addition, this example further analyzes changes at the ultrastructural level through transmission electron microscopy (TEM) to observe the integrity of three types of cell connections in the intercalated disc (ICD), including: gap junctions and desmosomes. and fascial adhesion, the results are shown in Figure 17, in which “FA” in the figure refers to fascia adherens, “GJ” in the figure refers to gap junction; “ DS" refers to desmosome; "MF" in the figure refers to myelin figure; "MD" in the figure refers to mitochondrial degeneration, and "*" in the figure refers to Refers to the degeneration and disorder of myofibrils; among them, as shown in Figure 17A, in 3-month-old mice, three types of cell connections were easily identified; on the other hand, in aged mice (24 months In the model of age), as shown in Figures 17B and 17C, severe ultrastructural changes were detected, including disruption of fascial adhesion, extension and rupture of gap junctions, and partial degeneration of desmosomes. Degeneration and degeneration were also detected. Expanded mitochondria, and myofibrils were disorganized; however, as shown in Figure 17D, after three months of treatment with hesperetin in the food of elderly mice, the ultrastructural damage to the heart caused by these aging The damage has basically disappeared, and its cardiac ultrastructure is similar to the characteristics observed in the hearts of 3-month-old mice, that is, hesperetin improves the age-related deterioration of myocardial discs, mitochondria, and sarcomeres; Taken together, hesperetin delays age-related symptoms of cardiac aging.

Example 7: Hesperetin acts in a Cisd2-dependent manner

This example knocks out the Cisd2 gene in the skeletal muscle and cardiac muscle of mice to generate Cisd2 mcKO mice (MCK-Cre; Cisd2 f/f) mice to confirm that hesperetin operates in a Cisd2 gene-dependent manner. Its anti-aging effects. In this example, 3-month-old normal (WT) and Cisd2 mcKO mice were treated with hesperetin for 4 months and sacrificed at 7 months of age. The electrocardiogram was monitored after 3 months of hesperetin treatment. As shown in Figure 18, Cisd2 mcKO mice showed obvious symptoms of premature aging at 3 months of age, and in skeletal muscle, hesperetin lost its beneficial effect on improving muscle function and pathological damage, and Figure 19 As shown, Cisd2 mcKO mice showed obvious degenerative loss and abnormal round and contractile fibers in skeletal muscles (femoral and gastrocnemius) with or without hesperetin treatment. . On the other hand, in the heart research analysis, as shown in Figure 20, the group treated with hesperetin still found typical arrhythmia (missing beat) and prolongation and irregularity of the PR interval, and Figure 21 As shown, the hesperetin-treated group still developed histopathological damage and fibrosis.

In summary, in the absence of the Cisd2 gene, hesperetin loses its beneficial effect on improving electrical dysfunctions and pathological damage (i.e., myocardial damage and fibrosis). This result reveals that hesperetin is Cisd2-dependent. Sexual methods exert their anti-aging effects.

The present invention first reveals through serum biochemical analysis and complete blood cell count analysis that the administration of hesperetin is not toxic to organisms, can improve liver function damage caused by aging, and slow down the abnormalities of neutrophils and lymphocytes caused by aging. Improves age-related decline in RBC and HGB.

The present invention further confirms through experiments that hesperetin has the effect of significantly extending lifespan, and this effect is related to increasing the level of Cisd2 protein in tissues, and can effectively slow down the systemic energy changes caused by aging. The decrease in mass metabolism includes a decrease in systemic oxygen consumption, a decrease in carbon dioxide production rate, and a decrease in heat production efficiency. At the same time, through quantitative micro-computed tomography analysis, glucose tolerance test, and insulin tolerance test, it was confirmed that hesperetin can slow down muscle loss related to aging. Fat accumulation, net weight loss, insulin sensitivity, and glucose homeostasis are reduced.

Furthermore, through the roller test and the grip strength test, the present invention reveals that hesperetin has the effect of improving the decline in muscle strength caused by aging, and through the Masson's trichrome staining results of the femoral muscle and gastrocnemius muscle, it reveals its effect in improving the muscle fiber degeneration caused by aging. , while analyzing the ultrastructure of skeletal muscle through transmission electron microscopy (TEM), it was confirmed that treatment with hesperetin can effectively slow down muscle fiber degeneration, mitochondrial degeneration and muscle ultrastructural damage caused by aging.

The present invention also uses cardiac ultrasound images, electrocardiograms, transmission electron microscopy images and histopathological analysis to confirm that hesperetin can slow down the symptoms of cardiac aging caused by aging, including reduced ejection fraction, increased cardiac performance index, vascular Increased peripheral fibrosis, arrhythmias, and deterioration of myocardial discs, mitochondria, and sarcomeres caused by aging.

The present invention further explores the mechanism of action of hesperetin, and uses roller test, grip strength test, electrocardiogram and histopathological analysis to confirm that hesperetin exerts its anti-aging effect in a Cisd2-dependent manner.

Among them, the dose used in the present invention is a dose acceptable to humans, and the equation is developed according to the interspecies dose conversion from animal to human studies (Nair AB, Jacob S.A simple practice guide for dose conversion between animals and human.J Basic Clin Pharm.7 , 27-31 2016.) Calculation, the equivalent dose of the present invention for human body is 200-800mg/60kg/day, preferably 400-600mg/60kg/day, and the best is 491mg/60kg/day.

In summary, the present invention reveals that hesperetin has the effect of prolonging lifespan and slowing down symptoms of aging. As a Cisd2 activator, it is applied to living bodies to increase the expression of Cisd2 genes to achieve the effect of fighting aging-related symptoms, and through Multiple clinical trial results support its efficacy.

The above detailed description is a specific description of the embodiments of the present invention. However, the embodiments are not used to limit the patent scope of the present invention. Any equivalent implementation or modification that does not depart from the technical spirit of the present invention shall be included in this case. within patent scope.

In addition, this case is not only innovative in terms of technical ideas, but also has many novel, useful and valuable functions. It fully meets the statutory requirements for invention patents of novelty and advancement. I submit an application in accordance with the law, and I sincerely request your office to approve this invention patent. Applications are made to encourage inventions and to be grateful.

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Figure 1 is a graph showing the results of serum biochemical analyzes of mice in each group; Figure 2 is a graph showing the results of complete blood count analyzes (CBC analyses) of mice in each group; Figure 3 Figure 4 is a graph showing changes in the lifespan of mice in each group; Figure 4 is a graph showing changes in Cisd2 expression levels in femoral muscle, gastrocnemius and myocardium of mice in each group; Figure 5 is a graph showing the changes in the expression of Cisd2 in mice in each group during the light and dark periods. Changes in oxygen consumption (VO ₂ ), CO ₂ production (VCO ₂ ), and heat production (Heat); Figure 6 shows micro computed tomography (Micro-CT) of mice in each group Analysis and quantitative results graph; Figure 7 is a graph showing the comparative experimental results of glucose tolerance test (GTT), insulin tolerance test (ITT) and 6-hour fasting basal blood glucose levels of mice in each group ; Figure 8 is a comparison result of the rotarod test and grip strength test of mice in each group; Figure 9 is Masson's trichrome staining of the femoral muscle and gastrocnemius muscle of mice in each group. (Masson's trichrome stain) results; Figure 10 is a quantitative result of the intact and degenerated muscle fibers of the femoral muscle and gastrocnemius muscle of mice in each group; Figure 11 is a transmission electron microscope of the gastrocnemius muscle of mice in each group ( Transmission electron microscopy (TEM) analysis results; Figure 12 is the echocardiography images of mice in each group; Figure 13 is the ejection fraction and ejection fraction obtained from the cardiac ultrasound images. Chart of myocardial performance index analysis results; Figure 14 shows the electrocardiography (ECG) of mice in each group and its continuous 5-minute waterfall plots; Figure 15 shows the mice in each group QT interval and Tpeak-Tend interval measurement results; Figure 16 is the Sirius Red/Fast Green staining results of mice in each group; Figure 17 is the myocardium of mice in each group The transmission electron microscope (TEM) analysis results of Three-color staining results; Figure 20 is the electrocardiogram of mice in each group and its continuous 5-minute waterfall chart; Figure 21 is the QRS interval, QT interval and Tpeak-Tend interval measurement results of mice in each group; Figure 22 is a diagram showing Masson's trichrome staining results of myocardial sections of mice in each group.

Claims (8)

A citrus extract component is used to prepare a pharmaceutical composition for increasing Cisd2 gene expression. The citrus extract component is hesperetin, and the human dose of hesperetin is 400-600mg/60kg/day. The use described in item 1 of the patent application, wherein the pharmaceutical composition can resist aging. The anti-aging includes extending life span and slowing down the symptoms of aging. The symptoms of aging include decreased metabolic function and fat accumulation in the elderly. , net weight loss, mitochondrial degeneration or cardiac aging symptoms; this extension of life is an increase in life span. For the use described in item 2 of the patent application, the symptoms caused by aging include a decrease in systemic oxygen consumption, a decrease in carbon dioxide production rate, a decrease in heat production efficiency, a decrease in insulin sensitivity, or a decrease in the constant regulation function of glucose in the elderly. The use described in item 2 of the patent application, wherein the fat accumulation is visceral fat accumulation. For the use described in item 2 of the patent application, the symptoms of cardiac aging include reduced cardiac ejection fraction, increased cardiac performance index, increased perivascular fibrosis, arrhythmia or cardiac ultrastructural damage in the elderly. The use described in item 2 of the patent application, wherein the cardiac aging symptoms are Cisd2-dependent cardiac aging symptoms. For the use described in Item 5 of the patent application, the ultrastructural damage to the heart includes deterioration of myocardial discs, mitochondria or sarcomeres. For the uses described in Item 1 or 2 of the patent application, the human dose of hesperetin is 491mg/60kg/day.