Preparation method and application of red date polysaccharide

Abstract

The present invention relates to the field of pharmaceuticals and health food technology, and specifically to a method for preparing and applying red date polysaccharides. The specific technical solution comprises the following steps: a method for preparing red date polysaccharides comprises concentrating jujubes with distilled water, removing proteins, and then precipitating with ethanol to obtain a primary ethanol precipitate containing polysaccharides; dialyzing the primary ethanol precipitate, and then precipitating with ethanol to obtain a secondary ethanol precipitate containing polysaccharides, which is then freeze-dried. The extracted red date polysaccharides can improve glucose utilization, increase GLUT4 protein expression, and prevent palmitate-induced insulin resistance and muscle damage in muscle cells, and can be used to prevent, improve, or treat anabolic or muscular diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of medicines and health-care foods, and in particular to a preparation method and application of red date polysaccharide. Background Art

[0002] Muscles in the human body perform a variety of functions, such as attaching to bones to protect them and maintaining body shape. Muscles also facilitate calcium influx, increasing bone density. However, with aging, the body's composition changes, leading to a redistribution of fat and protein. It's well known that muscle mass decreases after age 40, and by age 80, muscle mass has decreased by 50%. Muscle loss in old age is considered the most significant factor in reducing overall physical function. As we age, we experience changes in body shape, including changes in muscle and fat content, as well as skeletal deformation. The global incidence of obesity due to muscle loss in old age continues to rise, exceeding 30%. Furthermore, sarcopenia is more common in people with diabetes than in the general population, as abnormal insulin secretion affects cellular energy supply, leading to impaired muscle development.

[0003] Many factors are known to be involved in the balance between adipocyte and muscle cell differentiation. For example, glucocorticoids, such as cortisol, a natural hormone, or various synthetic cortisol analogs (including prednisone, hydrocortisone, and dexamethasone), act on the body through the glucocorticoid receptor (GR). Glucocorticoids play an important role in regulating differentiation decisions in vivo and in vitro, promoting adipogenesis and inhibiting muscle formation. Studies have shown that androgens can increase muscle mass while reducing fat mass by affecting body composition.

[0004] During the development of obesity, the increase in adipose tissue mass may be due to an increase in the size and number of adipocytes. The increase in cell number may arise from the mobilization of preadipocytes from a multipotent stem cell population or from a subpopulation of cells residing in mature white adipose tissue (WAT). Bone marrow mesenchymal stem cells (MSCs) can differentiate into a variety of cell types, including fat, muscle, cartilage, and bone. With aging, bone marrow adipogenesis accelerates in the body, while the osteogenic capacity of mesenchymal stem cells decreases. It has been suggested that mesenchymal stem cell precursors may contribute to age-related changes in body composition by differentiating into fat rather than bone.

[0005] Fat redistribution in older adults is associated with an increased risk of metabolic syndrome, which includes diabetes, hypertension, dyslipidemia, atherosclerosis, and a relative increase in intra-abdominal fat. Furthermore, muscle aging and performance decline are associated with normal aging, often with the gradual development of sarcopenia. Although skeletal muscle has the capacity to regenerate itself, this process is less active in older adults. Age-related changes in skeletal muscle tissue and the host environment are known to influence the proliferation and fusion of myoblasts in response to injury in aged animals.

[0006] Muscle underdevelopment or weakness is one of the most devastating childhood health problems. Various congenital myopathies and muscular dystrophy disorders, such as Duchenne muscular dystrophy (DMD), affect one in every 3,000 children. These disorders are often associated with inherited or spontaneous gene mutations. Children with these conditions experience numerous complications. The lack of effective treatments leads to high mortality rates, necessitating the development of new therapeutic strategies.

[0007] Muscle tissue in adult vertebrates regenerates from stem cells called satellite cells or muscle stem cells (MuSCs). Satellite cells are distributed throughout muscle tissue and, in the absence of injury or disease, remain quiescent and localized in anatomically defined niches. In addition to satellite cells, other cell types that can contribute to muscle regeneration include (but are not limited to) mesenchymal stem cells, bone marrow-derived cells, muscle interstitial cells, and mesenchymal stem cells.

[0008] Muscle atrophy refers to a decrease in muscle mass, which is evident in patients with cancer and various immunodeficiency diseases, and is even more pronounced in patients whose physical activity is very limited due to long-term bed rest, disabled people with limited mobility, and the elderly.

[0009] The synergistic effect of increased body fat and muscle loss is thought to increase the risk of functional and metabolic diseases.

[0010] Body mass index (BMI) and waist circumference (WHR) are used as markers of obesity due to their ease of clinical use, but they also have limitations as they do not reflect the effects of changes in body composition caused by age, exercise, or weight loss. In particular, muscle mass has been reported to be associated with metabolic syndrome and is considered a predictor of all-cause mortality. Recently, attention has been focused on increased body fat percentage and decreased muscle mass, as these factors are more closely related to health factors such as insulin resistance, glucose metabolism, lipid concentrations, and blood pressure. Muscle loss is associated with increased arthritis, back pain, and chronic pain; abdominal obesity can worsen urinary and fecal incontinence; and fracture injuries can increase depression in late life and even lead to mortality. Muscle loss in late life not only impairs mental health but is also a major factor in chronic diseases and decreased quality of life in older adults. Given the close relationship with these chronic diseases, preventing, improving, or treating muscle strength, hypertrophy, differentiation, regeneration, or sarcopenia could be used to curb the decline in physical activity that occurs with aging.

[0011] Muscle mass plays a crucial role in glucose and energy metabolism, as skeletal muscle is the largest organ in the body responsible for processing glucose under insulin stimulation, accounting for approximately 40-50% of body weight. However, overeating leads to the accumulation of intramuscular fat, which increases the likelihood of muscle mass loss and insulin resistance, making skeletal muscle an important target organ for diabetes and insulin resistance research.

[0012] In response to the above problems, the present invention discloses a preparation and application of jujube polysaccharide, which can prevent palmitate-induced insulin resistance and muscle damage in muscle cells, and can be used to prevent, improve or treat anabolic diseases or muscle diseases. Summary of the Invention

[0013] In view of the deficiencies of the prior art, the present invention provides a preparation method and application of red date polysaccharide.

[0014] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0015] The invention discloses a method for preparing red date polysaccharide. The method comprises the following steps: concentrating red dates with distilled water, removing protein, and then precipitating with ethanol to obtain a primary ethanol precipitate containing polysaccharide; dialyzing the primary ethanol precipitate, and then precipitating with ethanol to obtain a secondary ethanol precipitate containing polysaccharide, which is then freeze-dried.

[0016] Correspondingly, the above preparation method prepares red date polysaccharide.

[0017] Correspondingly, a composition comprises the red date polysaccharide prepared above.

[0018] Preferably, the added amount of the red date polysaccharide is 1 to 90 wt%.

[0019] Preferably, the added amount of the red date polysaccharide is 10 to 80 wt%.

[0020] Preferably, the added amount of the red date polysaccharide is 20 to 60 wt%.

[0021] Accordingly, the red date polysaccharide prepared by the above preparation method or the composition is used in the preparation of drugs for preventing, improving or treating anabolic diseases or muscle diseases.

[0022] Preferably, the anabolism comprises increasing muscle mass or stimulating muscle generation.

[0023] Preferably, the muscle disease includes any one of amyotrophic lateral sclerosis, muscular dystrophy, myotonia, ankylosing spondylosis, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Pompe disease, Canavan disease, dystonia, muscular dystrophy, myasthenia gravis, cachexia, sarcopenia and muscle fatigue.

[0024] Accordingly, the red date polysaccharide prepared by the above preparation method or the composition is used in the preparation of a drug for treating insulin resistance and muscle damage caused by palmitate in muscle cells.

[0025] The present invention has the following beneficial effects:

[0026] Researchers have discovered that a composition composed of jujube polysaccharides can improve glucose utilization and increase the expression of GLUT4 protein (a glucose transporter expressed in muscle). Furthermore, it potentially benefits mitochondrial function by alleviating palmitate-induced reductions in mitochondrial mass, increasing expression of the master regulator of mitochondrial biogenesis, Pgc-1α, and increasing expression of anabolic genes. Therefore, the jujube polysaccharide composition of the present invention can prevent palmitate-induced insulin resistance and muscle damage in muscle cells, and can be used to prevent, ameliorate, or treat anabolic or muscle diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the structural analysis diagram of red date polysaccharides;

[0028] Figure 2 This is a three-dimensional molecular structure analysis diagram of red date polysaccharides;

[0029] Figure 3 The concentration dependence of toxicity of jujube polysaccharide and palmitate on muscle cells;

[0030] Figure 4 This is the result of measuring the glucose utilization rate of red date polysaccharides in muscle cells;

[0031] Figure 5 The results of immunofluorescence analysis of jujube polysaccharide on GLUT4 expression;

[0032] Figure 6 The results of mitochondrial biogenesis and dynamics of red date polysaccharides were measured;

[0033] Figure 7 This is the expression result of mitochondria-related mRNA of red date polysaccharide;

[0034] Figure 8 Effects of red date polysaccharides on glucose metabolism, mitochondrial function and muscle function in muscle cells induced by insulin resistance;

[0035] Figure 9 The changes of myosin (MYHC) expression, fusion index and myotube diameter induced by red date polysaccharide;

[0036] Figure 10 The results show the expression of myogenic mRNA induced by red date polysaccharides. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0039] 1. The present invention discloses a method for preparing red date polysaccharides. The red dates are concentrated with distilled water to remove protein, and then precipitated with ethanol (ethanol concentration >95%, with a volume ratio of 1:3-5) to obtain a primary ethanol precipitate containing polysaccharides. The primary ethanol precipitate is dialyzed and then precipitated with ethanol (using the same ethanol precipitation process as described above) to obtain a secondary ethanol precipitate containing polysaccharides, which is then freeze-dried. Protein is removed with trichloroacetic acid. The ethanol precipitation time is set according to the polysaccharide content, specifically 1-2 hours.

[0040] 2. The present invention discloses a composition comprising the red jujube polysaccharide prepared by the above-described preparation method, i.e., the red jujube polysaccharide as an active ingredient. It also comprises one or more pharmaceutically acceptable carriers for administration. Pharmaceutically suitable and physiologically acceptable excipients, such as excipients, disintegrants, sweeteners, binders, coating agents, leavening agents, lubricants, gliding agents, or flavoring agents, may also be used for formulation.

[0041] The added amount of the red date polysaccharide is 1 to 90 wt %, more preferably 10 to 80 wt %, and even more preferably 20 to 60 wt %.

[0042] The dosage form of the composition can be granules, tablets, mini-tablets, covered tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injections. For example, when formulated in tablet or capsule form, the active ingredient can be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. In addition, if necessary, suitable binders, lubricants, disintegrants and coloring agents can also be added to the mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, sucrose or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0043] The composition is used to formulate a sterile and biocompatible liquid solution. Acceptable pharmaceutical carriers include physiological saline, sterile water, Ringer's solution, buffered physiological saline, albumin injection, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of the above components. Other conventional additives, such as antioxidants, buffers, and antibacterial agents, may also be added as needed. The composition can also be formulated into an injectable preparation, such as an aqueous solution, suspension, emulsion, etc., or into pills, capsules, granules, or tablets, with the addition of a diluent, dispersant, surfactant, binder, and lubricant.

[0044] The composition can be administered orally or parenterally. If it is administered parenterally, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal injection, etc., preferably oral administration.

[0045] The appropriate dosage of the composition of the present invention may vary depending on factors such as the formulation, mode of administration, patient age, weight, sex, morbidity, food, time of administration, route of administration, excretion rate, and reaction sensitivity. A physician with ordinary skill can easily determine and prescribe a dosage effective for the intended treatment or prevention. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001 to 10 g / kg.

[0046] Said composition can also be made into nutritional health food, in the form of tablets, capsules, powders, granules, liquids, pills, liquids, syrups, juices, suspensions, emulsions or drops. For example, when prepared in tablet or capsule form, active ingredient can be combined with oral, nontoxic, acceptable inert carriers (such as ethanol, glycerol, water etc.). In addition, if necessary or necessary, suitable binders, lubricants, disintegrants and coloring agents can also be added in the mixture. Suitable binders include but are not limited to natural sugars, such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, sucrose or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride etc. Disintegrants include but are not limited to starch, methylcellulose, agar, bentonite, xanthan gum etc. Acceptable pharmaceutical carriers for compositions formulated into sterile and biocompatible liquid solutions include physiological saline, sterile water, Ringer's solution, buffered physiological saline, albumin injection, glucose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of the foregoing. Other conventional additives, such as antioxidants, buffers, and bacteriostatic agents, may also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to further improve the properties of the formulation.

[0047] It can also be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include tea, beverages, meat, chocolate, jelly, food, candy, pizza, instant noodles, other noodles, chewing gum, candy, ice cream, alcoholic beverages, multivitamins and dietary supplements. In addition to the above-mentioned active ingredients, the composition may also contain various nutrients, vitamins, minerals (electrolytes), flavorings (such as synthetic and natural flavorings), colorants and flavor enhancers (cheese, chocolate, etc.), pectin and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0048] 3. The present invention also discloses the use of the jujube polysaccharide or composition prepared by the above-mentioned preparation method in the preparation of drugs for preventing, improving or treating anabolic diseases or muscle diseases.

[0049] Wherein, the anabolism includes increasing muscle mass or stimulating muscle generation. The muscle disease includes any one of amyotrophic lateral sclerosis, muscular dystrophy, myotonia, ankylosing spondylosis, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Pompe disease, Canavan disease, dystonia, muscular dystrophy, myasthenia gravis, cachexia, sarcopenia and muscle fatigue.

[0050] 4. The present invention also discloses the use of the jujube polysaccharide or composition prepared by the above preparation method in the preparation of drugs for treating insulin resistance and muscle damage caused by palmitate in muscle cells.

[0051] The present invention will be further described below with reference to specific embodiments.

[0052] Example 1 Preparation of Red Date Polysaccharide

[0053] 1. Extraction of Red Date Polysaccharides

[0054] The dried jujube fruit powder was mixed with distilled water at a ratio of 1:30, boiled, and concentrated to 1 / 3 of the liquid volume. The concentrate was mixed with trichloroacetic acid at a ratio of 1:1 and reacted at room temperature. The protein was removed by centrifugation (10 minutes, 8000r / min, room temperature) to obtain an ethanol precipitate containing polysaccharides (1:4, v / v, 12h). The obtained polysaccharide was dissolved in distilled water at a ratio of 1:1.5, dialyzed with a semipermeable membrane with a molecular weight cutoff of 3500Mw, and the polysaccharide was precipitated with ethanol again (1:4, v / v, 12h). The ethanol containing the secondary polysaccharide was then freeze-dried to obtain red jujube polysaccharide (JP). The structural analysis of red jujube polysaccharides can be found in Figure 1 The results of the three-dimensional molecular structure analysis of red date polysaccharides are shown in Figure 2 As shown, A is the structural formula of red date polysaccharide, B is the composition diagram of red date polysaccharide, C is the predicted molecular structure of red date polysaccharide, which contains 281 units, and D shows that red date polysaccharide has a spiral lamellar structure, and the SEM image also shows the lamellar shape.

[0055] 2. Monomer Analysis of Red Date Polysaccharides

[0056] The monomer analysis of red date polysaccharides confirmed that it is mainly composed of galacturonic acid, galactose, rhamnose, etc., as shown in Table 1 below.

[0057] Table 1 Composition of red date polysaccharides

[0058] monomer Retention time / min Molar ratio / % Fuc 3.61 0.367±0.009 Rha 7.71 9.130±0.071 Ara 8.14 3.294±0.071 Gal 10.36 9.481±0.194 Glc 11.94 4.053±0.089 Xyl 14.2 2.610±0.077 Man 14.98 1.336±0.046 Fru 16.84 0.389±0.064 Rib 18.73 0.051±0.002 GalA 34.69 68.707±0.499 GlcA 37.59 0.485±0.004 ManA 40.34 0.098±0.014

[0059] 3. The molecular weight, polydispersity and effective value analysis of red date polysaccharides are shown in Table 2.

[0060] Table 2 Analysis results of molecular weight, polydispersity and effective value of red date polysaccharides

[0061]

[0062]

[0063] Example 2

[0064] 1. Cytotoxicity Assay

[0065] The concentration-specific cytotoxicity of jujube polysaccharides and palmitate was determined using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Mouse muscle-derived C2C12 cells were differentiated into myotubes in 96-well plates and then treated with varying concentrations of jujube polysaccharides (JP) and palmitate (PA) for 24 hours. After incubation, the supernatant was removed, and the resulting formazan was dissolved in 100 μL of DMSO. The absorbance was measured at 540 nm using a microplate reader.

[0066] Figure 3 The concentration-dependent cytotoxicity of jujube polysaccharides and palmitate is shown. As shown, no cytotoxicity was observed at concentrations up to 0.75 mM PA and 400 μg / mL JP. Therefore, 0.75 mM PA and 400 μg / mL JP were used in this experiment.

[0067] 2. Improve insulin resistance in muscle cells and measure muscle physiological activity

[0068] Mouse muscle-derived C2C12 cells were differentiated into myotubes and treated with 0.75 mM PA for 24 hours to induce insulin resistance, representing a muscle insulin resistance model caused by excessive lipid accumulation. They were then treated with red jujube polysaccharides to determine whether they could alleviate insulin resistance.

[0069] (1) Measuring glucose utilization

[0070] We determined whether red jujube polysaccharides could improve glucose utilization in muscle cells. Glucose utilization was measured using a glucose assay kit (abcam, ab272532). C2C12 cells were cultured in 96-well plates, differentiated into myotubes, and then treated with PA and JP, either alone or in combination, for 24 hours. Following incubation, the supernatant was collected and the residual glucose content in the culture medium was determined according to the kit manual.

[0071] Figure 4 This graph shows the effect of red date polysaccharides on glucose utilization in muscle cells. As shown, under normal conditions, while glucose utilization isn't significantly improved, it's still elevated. Furthermore, even in the presence of palmitate-induced insulin resistance, red date polysaccharides can improve glucose utilization. In the figure, INS stands for Insulin.

[0072] (2) Measurement of GLUT4 expression

[0073] The expression of glucose transporter GLUT4 in muscle was determined by immunofluorescence.

[0074] C2C12 muscle cells were cultured on 12 mm coverslips for differentiation and then treated with PA and JP, either alone or in combination, for 24 hours. The supernatant was removed, and the cells were fixed with 10% formalin for 15 minutes and washed three times with phosphate-buffered saline (PBS). To prevent nonspecific antibody binding, the fixed cells were incubated with 5% bovine serum albumin for 30 minutes and then incubated in blocking buffer containing an Alexa fluor 594-conjugated GLUT4 antibody at 4°C for 12 hours. The blocking buffer containing the antibody was removed, the cells were rinsed with PBS, and then mounted in mounting buffer containing DAPI (4',6-diamidino-2-phenylindole, dihydrochloride). GLUT4 expression in the cells was confirmed by confocal microscopy and quantified using an image analysis program.

[0075] Figure 5 The results of immunofluorescence analysis of GLUT4 expression on red date polysaccharides are shown. As shown in the figure, red date polysaccharides can increase GLUT4 expression under both normal and insulin resistance conditions.

[0076] These experiments confirmed that jujube polysaccharides can improve glucose utilization in insulin resistance-induced muscle cells, which is achieved by increasing the expression of GLUT4.

[0077] 3. Measuring Mitochondrial Function

[0078] Mitochondria are dynamic intracellular organelles that are regulated through the processes of fusion (elongation of mitochondria) and fission (division of mitochondria). Fusion is triggered by energy demand and stress, while fission regulates mitochondrial quality by breaking up and removing damaged parts.

[0079] Mitochondrial biogenesis and dynamics are important regulators of skeletal muscle remodeling and atrophy, and reduced mitochondrial functional properties are risk factors for insulin resistance and skeletal muscle atrophy, so we measured markers of mitochondrial mass, biogenesis, and dynamics.

[0080] (1) Determination of total mitochondrial mass

[0081] To measure total mitochondrial mass, C2C12 cells were stained with Mitotracker Deep Red FM (Invitrogen, M22426). Mitotracker Red reagent was diluted to 200 nM in culture medium and incubated in myotube-differentiated C2C12 cells at 37°C for 40 minutes. After incubation, cells were photographed under a confocal microscope. Image analysis software (IMAGE J) was used to quantify the area stained with Mitotracker Red in the cell photographs.

[0082] Figure 6 The results of measurements of mitochondrial biogenesis and dynamic changes induced by red date polysaccharides are shown. As shown, under normal conditions, red date polysaccharides increased mitochondrial tracking intensity compared to the control group; palmitate alone decreased mitochondrial tracking intensity compared to the control group; and palmitate plus red date polysaccharides increased mitochondrial tracking intensity compared to the palmitate control group.

[0083] (2) Determination of mitochondrial-related gene expression

[0084] To measure the expression of mitochondrial-related genes in muscle cells at the transcript level, total RNA was extracted from cells using Trizol reagent. The extracted RNA was synthesized into cDNA using the ABI High Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions. The synthesized cDNA was analyzed by real-time PCR using SYBR Green. Relative gene expression was calculated using the 2-ΔΔCt method, and normalization was performed using the ribosomal protein lateral stalk subunit p0 (Rplp0, 36b4) as a housekeeping gene (internal control).

[0085] Figure 7 The results of the study on the effects of red date polysaccharides on the mRNA expression of mitochondrial-related genes are shown. As shown, under the action of red date polysaccharides, the master regulator of mitochondrial biogenesis, Pgc-1α, showed an upward trend in both the normal and palmitate-treated groups, while the mRNA expression of its downstream genes, Nrf1, Nrf2, and Tfa, did not change significantly. Furthermore, genes related to mitochondrial dynamics remained unchanged under the action of red date polysaccharides.

[0086] From these experiments, it can be seen that although red date polysaccharides had no obvious effects on markers of mitochondrial biogenesis, they did alleviate the palmitate-induced decline in mitochondrial mass and increased the gene expression of Pgc-1α, a master regulator of mitochondrial biogenesis, suggesting that red date polysaccharides have potential benefits on mitochondrial function.

[0087] Figure 8 described the glucose metabolism, mitochondrial function, and muscle function induced by red date polysaccharides in insulin resistance-induced muscle cells.

[0088] 4. Observe Myogenesis

[0089] During the differentiation process into muscle fibers, some embryonic precursor cells are retained as muscle satellite cells, and most become myoblasts, which differentiate into muscle cells, and then the muscle cells merge together to form multinucleated myotubes or muscle fibers. In addition, the genes expressed at each differentiation stage are also different, so in this experiment, we mainly studied the markers that appear in the later stages of muscle fiber differentiation. Muscles contain several types of muscle fibers, and each type of muscle fiber has different characteristics, such as contraction speed, fatigue, and metabolic activity. In general, the content of type 1 muscle fibers (i.e., oxidative fibers) in the muscles of patients with type 2 diabetes is lower, and several clinical trials have shown that a lower proportion of type 1 muscle fibers is associated with insulin resistance. Therefore, this study aims to study the effects of red date polysaccharides on muscles by detecting changes in muscle fiber types.

[0090] (1) Determination of changes in myogenic factors

[0091] Immunofluorescence was used to determine the changes in myotube myosin (MYHC) expression, nuclear fusion index and myotube diameter.

[0092] Cells were fixed with 10% formalin for 15 minutes, washed three times with phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 for 15 minutes. To prevent nonspecific antibody binding, the fixed cells were incubated with 5% bovine serum albumin for 30 minutes. The fixed cells were then placed in blocking buffer containing a primary anti-myosin monoclonal antibody and incubated at 4°C for 12 hours. The blocking buffer containing the primary antibody was removed, the sections were rinsed with PBS, and then incubated in blocking buffer containing a secondary antibody, Alexa Fluor 488, for 1 hour. Finally, sections were blocked with mounting buffer containing DAPI (4',6-diamidino-2-phenylindole, dihydrochloride). Fluorescence images were captured under a confocal microscope and analyzed using ImageJ for fluorescence intensity, root canal nucleation index, and root canal orifice diameter.

[0093] Figure 9 The results of changes in myosin (MYHC) expression, fusion index, and root canal diameter induced by red date polysaccharides are shown. As shown in the figure, compared with the control group, the myosin (MYHC) expression intensity, root canal diameter, and fusion index (nuclear fusion index) of palmitate-treated cells decreased, but red date polysaccharides showed a recovery trend.

[0094] (2) Measuring gene expression related to muscle growth

[0095] We also examined the expression of myogenesis-related genes. To measure the expression of myogenesis-related genes in myoblasts at the transcript level, we extracted total RNA from cells using Trizol reagent. The extracted RNA was synthesized into cDNA using the ABI High Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions. The synthesized cDNA was analyzed by real-time PCR using SYBR Green. Relative gene expression was calculated using the 2-ΔΔCt method, and ribosomal protein lateral stalk subunit p0 (Rplp0, 36b4) was used as a housekeeping gene (internal control) for normalization.

[0096] Figure 10 The results of a study evaluating the effects of red date polysaccharides on the expression of myogenesis-related genes are shown. As shown, palmitate reduced the expression of Myod mRNA, an intermediate marker of differentiation that enables undifferentiated cells to become muscle progenitors, while red date polysaccharides did not inhibit its expression. Myogenin mRNA, which enables muscle cells to differentiate and connect into final myofibers, was also reduced by palmitate, but red date polysaccharides increased its expression. Finally, an analysis of markers associated with muscle fiber type revealed a significant increase in MyhcI gene expression in slow-twitch and oxidative fibers. Furthermore, palmitate reduced the expression of Myhc2x mRNA in fast-twitch fibers, while red date polysaccharides increased its expression.

[0097] The following examples illustrate the preparation of pharmaceutical preparations using the red date polysaccharide of the present invention.

[0098] 1. Preparation of pharmaceutical preparations

[0099] 1.1 Preparation of injection

[0100] Red jujube polysaccharide 300 mg, sodium metabisulfite 3.0 mg, methyl parahydroxybenzoate 0.8 mg, propyl parahydroxybenzoate 0.1 mg, sterile distilled water for injection.

[0101] The above ingredients were mixed according to conventional methods to prepare an injection with a final volume of 2 mL, which was then filled into an ampoule and sterilized.

[0102] 1.2 Preparation of acid preparation

[0103] Red date polysaccharide 10mg, lactose 1g.

[0104] The above raw materials are mixed and placed in a sealed bag to prepare an acid preparation.

[0105] 1.3 Tablet Preparation

[0106] Red date polysaccharide 0.1mg, corn starch 100mg, lactose 100mg, magnesium stearate 2mg.

[0107] After mixing the above ingredients, tablets are prepared according to conventional tablet preparation methods.

[0108] 1.4 Capsule Preparation

[0109] Red date polysaccharide 0.1mg, corn starch 100mg, lactose 100mg, magnesium stearate 2mg.

[0110] After the above ingredients are mixed, they are filled into gelatin capsules according to conventional capsule preparation methods.

[0111] 1.5 Preparation of pills

[0112] Red date polysaccharide 1mg, lactose 1.5g, glycerol 1g, xylitol 0.5g.

[0113] After mixing the above raw materials, 4-gram pills were prepared according to conventional methods.

[0114] 1.6 Preparation of granules

[0115] Red date polysaccharide 0.15mg, soybean extract 50mg, glucose 200mg, starch 600mg.

[0116] After mixing the above ingredients, add 100 mL of 30% ethanol, dry at 60°C into granules, and then pack into small bags.

[0117] The following is an example of using the red date polysaccharide of the present invention to prepare a health drink.

[0118] 2. Preparation of health drinks

[0119] 2.1 Preparation of health drinks

[0120] 0.5 g of the red date polysaccharide of the present invention is uniformly mixed with liquid sugar (0.5%), oligosaccharide (2%), white sugar (2%), salt (0.5%), water (75%) and other auxiliary materials, pasteurized, and packed into glass bottles, PET bottles or other small packaging containers.

[0121] 2.2 Preparation of vegetable juice

[0122] 0.5 g of the red date polysaccharide of the present invention was added to 1000 mL of tomato juice or carrot juice to prepare vegetable juice.

[0123] 2.3 Juice preparation

[0124] 0.1 g of the red date polysaccharide of the present invention was added to 1000 mL of apple juice or grape juice to prepare juice.

[0125] The above composition ratio is a preferred embodiment of the mixed ingredients suitable for relatively palatable beverages, but the composition ratio can also be arbitrarily modified according to the preferences of regions and nations such as the demand level, demand country and use.

[0126] The following is an example of using the red date polysaccharide of the present invention to prepare a health food.

[0127] 3. Preparation of health foods

[0128] The method for preparing the food containing the red date polysaccharide of the present invention as an effective ingredient is as follows.

[0129] 3.1 Preparation of flour foods

[0130] 0.5-5.0 wt% of the red date polysaccharide of the present invention is added to flour, and bread, cakes, biscuits, cookies and noodles are prepared with the mixture.

[0131] 3.2 Preparation of soups and gravies

[0132] 0.1-5.0 wt% of the red date polysaccharide of the present invention is added to soup and gravy to prepare soup and gravy for health-care meat products and noodles.

[0133] 3.3 Preparation of ground beef

[0134] 10 wt% of the red date polysaccharide of the present invention is added to ground beef to prepare health-promoting ground beef.

[0135] 3.4 Preparation of dairy products

[0136] 5 to 10 parts by weight of the red date polysaccharide of the present invention are added to milk, and various dairy products such as butter and ice cream are prepared using the milk.

[0137] 3.5 Preparing pre-meal food

[0138] Brown rice, barley, glutinous rice and corn were alkalized and dried according to the disclosed method, and then powdered with a particle size of 60 mesh using a grinder.

[0139] Black beans, black sesame seeds and perilla seeds are also steamed and dried according to the public method, and then ground into powder with a particle size of 60 mesh.

[0140] The red date polysaccharide of the present invention is concentrated under reduced pressure in a vacuum concentrator, sprayed, and dried in a hot air dryer. The obtained dry product is ground with a grinder to a particle size of 60 meshes to obtain dry powder.

[0141] The cereals, seeds, fruits and red date polysaccharides prepared above are compounded in the following proportions.

[0142] Cereals (30 parts by weight of brown rice, 15 parts by weight of glutinous rice, 20 parts by weight of barley, 15 parts by weight of corn), seeds (7 parts by weight of perilla seeds, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds), red date polysaccharide of the present invention (3wt%), Ganoderma lucidum (0.5wt%), turmeric (0.5 parts by weight).

[0143] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of jujube polysaccharide in the preparation of a drug for treating muscle damage caused by palmitate in muscle cells, characterized in that: The preparation process of the red date polysaccharide comprises the following steps: mixing dried jujube fruit powder and distilled water at a ratio of 1:30, boiling, concentrating to 1 / 3 of the liquid volume, mixing the concentrate with trichloroacetic acid at a ratio of 1:1 and reacting at room temperature, removing protein by centrifugation at room temperature and 8000 r / min for 10 minutes, and then precipitating with ethanol at a volume ratio of 1:4 for 12 hours to obtain a primary ethanol precipitate containing polysaccharide; dissolving the obtained polysaccharide in distilled water at a ratio of 1:1.5, dialyzing with a semipermeable membrane with a molecular weight cutoff of 3500Mw, and then precipitating with ethanol at a volume ratio of 1:4 for 12 hours to obtain a secondary ethanol precipitate containing polysaccharide, and freeze-drying the precipitate to obtain the red date polysaccharide.

2. A composition, characterized in that: The invention comprises the red date polysaccharide prepared in the application of claim 1.

3. The composition according to claim 2, characterized in that: The added amount of the red date polysaccharide is 1 to 90 wt %.

4. The composition according to claim 3, characterized in that: The added amount of the red date polysaccharide is 10 to 80 wt %.

5. The composition according to claim 3 or 4, characterized in that: The added amount of the red date polysaccharide is 20 to 60 wt %.

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