Preparation method of morinda officinalis polysaccharide for relieving amyotrophy

Abstract

The invention discloses a preparation method of Morinda officinalis polysaccharide for relieving amyotrophy, which comprises the following steps: cleaning Morinda officinalis roots, slicing, drying, grinding into powder by using a crusher for later use, concentrating supernate, adding a certain amount of absolute ethyl alcohol until the final volume fraction of ethyl alcohol in the solution is 80%, and standing at 4 DEG C for 24 hours; centrifuging the reaction liquid after overnight precipitation, and retaining the precipitate to obtain crude morinda officinalis polysaccharide; removing the residual organic solvent in the crude polysaccharide by using a rotary evaporator, finally putting the crude polysaccharide into a vacuum freeze-drying machine to be freeze-dried for 24 hours, and putting the freeze-dried crude polysaccharide into a drying tank to be stored; and analyzing out small molecular substances and salts in the polysaccharide by using a dialysis bag to obtain relatively pure morinda officinalis polysaccharide. According to the preparation method of the morinda officinalis polysaccharide for relieving amyotrophy, the morinda officinalis polysaccharide provides a new thought for medicine treatment for relieving amyotrophy and provides technical support for application of the morinda officinalis polysaccharide in the nutritional supplement market, and the application of the morinda officinalis polysaccharide in amyotrophy is proved by using the purified morinda officinalis polysaccharide with a clear structure.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of Morinda officinalis polysaccharide, in particular to a preparation method of Morinda officinalis polysaccharide for alleviating muscular atrophy. Background Art

[0002] The existing preparation methods and applications of Morinda officinalis polysaccharides for alleviating muscular atrophy still have the following problems in actual use: currently, people use ultrasonic extraction and enzymatic hydrolysis to extract Morinda officinalis polysaccharides. The ultrasonic extraction method increases the penetration of the solvent through the cavitation effect generated by ultrasonic waves. In addition to this extraction step, Morinda officinalis polysaccharides also need to undergo protein removal, pigment removal, dialysis, precipitation, column chromatography and other separation and purification operations. However, the extraction rate of the above-mentioned Morinda officinalis polysaccharide extraction methods is still low, and they are either time-consuming, low in yield, or high in cost. The structural research of Morinda officinalis polysaccharides focuses on monosaccharide composition, side chain position, etc., and there are fewer studies on advanced structures, structure-activity relationships, etc. The polysaccharide composition and molecular weight distribution obtained by different extraction methods are very different, which cannot meet market demand and is not conducive to the promotion and application of Morinda officinalis polysaccharides in the field of nutrition. Therefore, we proposed a method for preparing Morinda officinalis polysaccharide for alleviating muscular atrophy in order to solve the above-mentioned problems. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing Morinda officinalis polysaccharide for alleviating muscular atrophy, in order to solve the problems that the extraction rate of the current Morinda officinalis polysaccharide extraction methods is still low, and the extraction methods are either time-consuming, low in yield, or high in cost; the structural research of Morinda officinalis polysaccharide focuses on monosaccharide composition, side chain position, etc., and there are fewer studies on higher-order structure, structure-activity relationship, etc. The polysaccharide composition and molecular weight distribution obtained by different extraction methods are very different, which cannot meet market demand and is not conducive to the promotion and application of Morinda officinalis polysaccharide in the field of nutrition.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing Morinda officinalis polysaccharide for alleviating muscular atrophy, comprising the following steps: The steps include: S1: crude extract of Morinda officinalis polysaccharide; Preparation of materials required: washed, sliced ​​and dried Morinda officinalis root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of Morinda officinalis polysaccharides; S21: Wash the root of Morinda officinalis, slice it, dry it, grind it into powder with a crusher for later use, and mix the Morinda officinalis powder and ultrapure water in a ratio of 1:60; S22: Extract under ultrasonic environment, then transfer to 90℃ environment for reaction for 3 hours, centrifuge the reaction solution, take the supernatant and concentrate it, add a certain amount of anhydrous ethanol to make the final volume fraction of ethanol in the solution 80%, and place it at 4℃ for 24 hours; S23: After overnight precipitation, the reaction solution is centrifuged and the precipitate is retained to obtain crude Morinda officinalis polysaccharide; S24: using a rotary evaporator to remove the residual organic solvent in the crude polysaccharide, and finally placing the crude polysaccharide in a vacuum freeze dryer for 24 hours and storing it in a drying tank; S3: Purification of Morinda officinalis polysaccharide; S31: First, the protein in the crude polysaccharide of Morinda officinalis was removed by the Sevage method, and a certain concentration of crude polysaccharide solution of Foeniculum foetida and Sevage solution (chloroform: n-butanol = 4:1) were prepared. 1 / 4 of the volume of Sevage solution was added to the polysaccharide solution of Morinda officinalis, and the mixture was thoroughly mixed for 30-60 minutes, and then centrifuged. The supernatant solution was polysaccharide, the middle layer was protein, and the lower layer solution was organic solvent. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer, and pour the remaining organic solvent into a separatory funnel and let it stand overnight before it can be reused; S33: the Morinda officinalis polysaccharide solution after protein removal is poured into an eggplant-shaped bottle of a rotary evaporator, and the organic solvent in the sample solution is evaporated away in a vacuum at 40°C; S34: using a dialysis bag to analyze small molecules and salts in the polysaccharide to obtain relatively pure Morinda officinalis polysaccharide; S4: Isolation of Morinda officinalis polysaccharides; S41: loading the pre-activated DEAE-dextran gel A-25 into the column, and after a period of equilibration, slowly adding a specific concentration of Morinda officinalis crude polysaccharide solution onto the gel column; S42: Turn on the constant flow pump, set the flow rate, collect the eluate with a partial collector, collect one tube every three minutes, use a NaCl solution with a specific ion concentration as the mobile phase, and determine the polysaccharide content of the eluate in the separate tube by the phenol-sulfuric acid method; S43: using the tube number as the abscissa and the absorbance as the ordinate to obtain the elution curve of the polysaccharide, and after analyzing the elution curve data, dividing the purified components according to the peak value and the concentration of the eluent, the tubes corresponding to the same purified component; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

[0006] An in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy, cell culture and differentiation: in vitro experiments were carried out using C2C12 myoblasts. C2C12 cells were cultured in a 37°C, 5% CO2 incubator using DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cells grew to about 80%, myotube differentiation was induced using DMEM culture medium containing 2% horse serum and 1% penicillin-streptomycin solution. The culture medium was replaced every day and the induction was continued for 5 days until myotubes were formed.

[0007] Preferably, the muscular atrophy model is induced and grouped as follows: after successful differentiation of C2C12 cells, 10uM dexamethasone solution is used to induce the cells for 24 hours to establish a muscular atrophy model, and the cells are divided into 6 groups: a control group, a dexamethasone group, a dexamethasone + 10ug / ml Morinda officinalis polysaccharide group, a dexamethasone + 25ug / ml Morinda officinalis polysaccharide group, a dexamethasone + 50ug / ml Morinda officinalis polysaccharide group, and a dexamethasone + 100ug / ml Morinda officinalis polysaccharide group.

[0008] Preferably, the Morinda officinalis polysaccharide intervention: using purified Morinda officinalis polysaccharide at different concentration gradients to intervene in cells for 24 hours.

[0009] Preferably, the index detection includes: observing the morphology of C2C12 myotubes; detecting the ATP level in myotube cells, and detecting the changes in the mRNA and protein levels of SIRT1, MyHC, FoxO3, Murf1 and Atrogin-1 genes.

[0010] Compared with the prior art, the beneficial effects of the present invention are: the preparation method of Morinda officinalis polysaccharide for alleviating muscular atrophy, Morinda officinalis polysaccharide provides a new idea for drug treatment for alleviating muscular atrophy, provides technical support for the application of Morinda officinalis polysaccharide in the nutritional supplement market, and uses the purified Morinda officinalis polysaccharide with a clear structure to confirm the application of Morinda officinalis polysaccharide in muscular atrophy. The specific contents are as follows: 1. Morinda officinalis polysaccharide (MOP) can significantly prevent and alleviate dexamethasone-induced myoblast fiber atrophy. Compared with the dexamethasone-induced group, the diameter of C2C12 cell myotubes in the Morinda officinalis polysaccharide intervention group tended to thicken; Effects of different concentrations of Morinda officinalis polysaccharide (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) on the diameter of myotubes of C2C12 myoblasts in the presence of dexamethasone (10uM); Ctl group: blank control group, without Morinda officinalis polysaccharide and dexamethasone; Dex group: dexamethasone-induced group, 10uM; MOP1 (10ug / ml) group: MOP (10ug / ml) + DEX (10uM); MOP1 (25ug / ml) group: MOP (25ug / ml) + DEX (10uM); MOP1 (50ug / ml) group: MOP (50ug / ml) + DEX (10uM); MOP1 (100ug / ml) group: MOP (100ug / ml) + DEX (10uM), * indicates significant difference between the two groups (p<0.05).

[0011] 2. Morinda officinalis polysaccharide (MOP) can improve the decrease in mitochondrial ATP metabolism in myocytes caused by dexamethasone. Compared with the dexamethasone-induced group, the ATP content in C2C12 cells in the Morinda officinalis polysaccharide intervention group showed an upward trend; Effects of different concentrations of Morinda officinalis polysaccharide (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) on ATP production in C2C12 myoblasts with the addition of dexamethasone (10uM). Ctl: blank control group, without Morinda officinalis polysaccharide and dexamethasone; Dex: dexamethasone-induced group, 10uM; MOP (10ug / ml) group: MOP (10ug / ml) + DEX (10uM); MOP1 (25ug / ml) group: MOP (25ug / ml) + DEX (10uM); MOP1 (50ug / ml) group: MOP (50ug / ml) + DEX (10uM); MOP1 (100ug / ml) group: MOP (100ug / ml) + DEX (10uM); * indicates significant difference between the two groups (p<0.05). BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a structural formula diagram of the purified polysaccharide from Morinda officinalis of the present invention; Figure 2 This is a schematic diagram of the myotube morphology of the present invention; Figure 3 Schematic diagram of ATP metabolism level of the present invention. DETAILED DESCRIPTION

[0013] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the implementation regulations described 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 creative work are within the scope of protection of the present invention.

[0014] See also Figure 1-Figure 3 , the present invention provides the following technical solutions: In order to solve the problem that the existing Morinda officinalis polysaccharide extraction has not been purified, and has not been structurally identified, and the functional components that work are not clear, the following technical content is disclosed, a method for preparing Morinda officinalis polysaccharide for alleviating muscular atrophy, comprising the following steps: S1: crude extract of Morinda officinalis polysaccharide; Preparation of materials required: washed, sliced ​​and dried Morinda officinalis root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of Morinda officinalis polysaccharides; S21: Wash the root of Morinda officinalis, slice it, dry it, grind it into powder with a crusher for later use, and mix the Morinda officinalis powder and ultrapure water in a ratio of 1:60; S22: Extract under ultrasonic environment, then transfer to 90℃ environment for reaction for 3 hours, centrifuge the reaction solution, take the supernatant and concentrate it, add a certain amount of anhydrous ethanol to make the final volume fraction of ethanol in the solution 80%, and place it at 4℃ for 24 hours; S23: After overnight precipitation, the reaction solution is centrifuged and the precipitate is retained to obtain crude Morinda officinalis polysaccharide; S24: using a rotary evaporator to remove the residual organic solvent in the crude polysaccharide, and finally placing the crude polysaccharide in a vacuum freeze dryer for 24 hours and storing it in a drying tank; S3: Purification of Morinda officinalis polysaccharide; S31: First, the protein in the crude polysaccharide of Morinda officinalis was removed by the Sevage method, and a certain concentration of crude polysaccharide solution of Foeniculum foetida and Sevage solution (chloroform: n-butanol = 4:1) were prepared. 1 / 4 of the volume of Sevage solution was added to the polysaccharide solution of Morinda officinalis, and the mixture was thoroughly mixed for 30-60 minutes, and then centrifuged. The supernatant solution was polysaccharide, the middle layer was protein, and the lower layer solution was organic solvent. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer, and pour the remaining organic solvent into a separatory funnel and let it stand overnight before it can be reused; S33: the Morinda officinalis polysaccharide solution after protein removal is poured into an eggplant-shaped bottle of a rotary evaporator, and the organic solvent in the sample solution is evaporated away in a vacuum at 40°C; S34: using a dialysis bag to analyze small molecules and salts in the polysaccharide to obtain relatively pure Morinda officinalis polysaccharide; S4: Isolation of Morinda officinalis polysaccharides; S41: loading the pre-activated DEAE-dextran gel A-25 into the column, and after a period of equilibration, slowly adding a specific concentration of Morinda officinalis crude polysaccharide solution onto the gel column; S42: Turn on the constant flow pump, set the flow rate, collect the eluate with a partial collector, collect one tube every three minutes, use a NaCl solution with a specific ion concentration as the mobile phase, and determine the polysaccharide content of the eluate in the separate tube by the phenol-sulfuric acid method; S43: using the tube number as the abscissa and the absorbance as the ordinate to obtain the elution curve of the polysaccharide, and after analyzing the elution curve data, dividing the purified components according to the peak value and the concentration of the eluent, the tubes corresponding to the same purified component; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

[0015] An in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy is characterized by: cell culture and differentiation: in vitro experiments are carried out using C2C12 myoblasts, C2C12 cells are cultured in a DMEM culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and a 5% CO2 incubator, and when the cells grow to about 80%, myotube differentiation is induced using a DMEM culture medium containing 2% horse serum and 1% penicillin-streptomycin solution, the culture medium is replaced every day, and the induction is continued for 5 days until myotubes are formed.

[0016] An in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy, induction of muscular atrophy model and grouping: After successful differentiation of C2C12 cells, 10uM dexamethasone solution was used to induce the cells for 24 hours to establish a muscular atrophy model, and the cells were divided into 6 groups: control group, dexamethasone group, dexamethasone + 10ug / ml Morinda officinalis polysaccharide group, dexamethasone + 25ug / ml Morinda officinalis polysaccharide group, dexamethasone + 50ug / ml Morinda officinalis polysaccharide group and dexamethasone + 100ug / ml Morinda officinalis polysaccharide group.

[0017] Intervention with Morinda officinalis polysaccharide: Purified Morinda officinalis polysaccharide was used to intervene in cells at different concentration gradients for 24 hours.

[0018] Index detection: observe the morphology of C2C12 myotubes; detect the ATP level in myotube cells, and detect the changes in the mRNA and protein levels of SIRT1, MyHC, FoxO3, Murf1 and Atrogin-1 genes.

[0019] like Figure 1As shown in the results, Morinda officinalis polysaccharide (MOP) can significantly prevent and alleviate dexamethasone-induced myoblast fiber atrophy. Compared with the dexamethasone-induced group, the diameter of C2C12 cell myotubes in the Morinda officinalis polysaccharide intervention group tended to thicken.

[0020] Effects of different concentrations of Morinda officinalis polysaccharide (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) on the diameter of myotubes of C2C12 myoblasts in the presence of dexamethasone (10uM); Ctl group: blank control group, without Morinda officinalis polysaccharide and dexamethasone; Dex group: dexamethasone-induced group, 10uM; MOP1 (10ug / ml) group: MOP (10ug / ml) + DEX (10uM); MOP1 (25ug / ml) group: MOP (25ug / ml) + DEX (10uM); MOP1 (50ug / ml) group: MOP (50ug / ml) + DEX (10uM); MOP1 (100ug / ml) group: MOP (100ug / ml) + DEX (10uM), * indicates significant difference between the two groups (p<0.05).

[0021] like Figure 2 As shown in the results, Morinda officinalis polysaccharide (MOP) can improve the decreased mitochondrial ATP metabolism level of muscle cells caused by dexamethasone. Compared with the dexamethasone-induced group, the ATP content in C2C12 cells in the Morinda officinalis polysaccharide intervention group showed an increasing trend.

[0022] Effects of different concentrations of Morinda officinalis polysaccharide (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) on ATP production in C2C12 myoblasts with the addition of dexamethasone (10uM); Ctl: blank control group, without Morinda officinalis polysaccharide and dexamethasone; Dex: dexamethasone induced group, 10uM; MOP (10ug / ml) group: MOP (10ug / ml) + DEX (10uM); MOP1 (25ug / ml) group: MOP (25ug / ml) + DEX (10uM); MOP1 (50ug / ml) group: MOP (50ug / ml) + DEX (10uM); MOP1 (100ug / ml) group: MOP (100ug / ml) + DEX (10uM). * indicates significant difference between the two groups (p<0.05).

[0023] Example 1: Cell culture and differentiation: In vitro experiments were conducted using C2C12 myoblasts. C2C12 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C in a 5% CO2 incubator. When the cells grew to about 80%, the cells were collected for later use.

[0024] Cell differentiation: The cells were seeded at a density of 2.5*106 in a 6-well plate and placed in a cell culture incubator. When the cell confluence reached about 80%, myotube differentiation was induced using DMEM medium containing 2% horse serum and 1% penicillin-streptomycin solution. The medium was replaced every day and the induction was continued for 5 days until myotubes were formed.

[0025] Induction and grouping of muscular atrophy model: After successful differentiation of C2C12 cells, 10uM dexamethasone solution was used to induce the cells for 24 hours to establish a muscular atrophy model, and the cells were divided into 6 groups: control group, dexamethasone group, dexamethasone + 10ug / ml Morinda officinalis polysaccharide group, dexamethasone + 25ug / ml Morinda officinalis polysaccharide group, dexamethasone + 50ug / ml Morinda officinalis polysaccharide group and dexamethasone + 100ug / ml Morinda officinalis polysaccharide group.

[0026] Intervention with Morinda officinalis polysaccharide: Purified Morinda officinalis polysaccharide was used to intervene in cells at different concentration gradients (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) for 24 hours.

[0027] Measurement of C2C12 cell myotube diameter: C2C12 myotube cells were observed and photographed using an inverted microscope. Four fields of view were randomly selected from each group, and the middle 2 / 3 of each mature myotube was used as the diameter measurement site. Each myotube was measured along three different short axis directions, and the average of the three measurements was the myotube diameter.

[0028] Statistical analysis: SPSS 24.0 software was used for data processing. The measurement data were expressed as (x±s). One-way analysis of variance was used for comparison among multiple groups, and LSD test was used for further pairwise comparison; P<0.05 indicated that the difference was statistically significant.

[0029] Example 2: Cell culture: In vitro experiments were conducted using C2C12 myoblasts. C2C12 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37° C. in a 5% CO 2 incubator. When the cells grew to about 80% confluence, the cells were collected for later use.

[0030] Cell differentiation: The cells were seeded at a density of 2.5*106 in a 6-well plate and placed in a cell culture incubator. When the cell confluence reached about 80%, myotube differentiation was induced using DMEM medium containing 2% horse serum and 1% penicillin-streptomycin solution. The medium was replaced every day and the induction was continued for 5 days until myotubes were formed.

[0031] Induction and grouping of muscular atrophy model: After successful differentiation of C2C12 cells, 10uM dexamethasone solution was used to induce the cells for 24 hours to establish a muscular atrophy model, and the cells were divided into 6 groups: control group, dexamethasone group, dexamethasone + 10ug / ml Morinda officinalis polysaccharide group, dexamethasone + 25ug / ml Morinda officinalis polysaccharide group, dexamethasone + 50ug / ml Morinda officinalis polysaccharide group and dexamethasone + 100ug / ml Morinda officinalis polysaccharide group.

[0032] Intervention with Morinda officinalis polysaccharide: Purified Morinda officinalis polysaccharide was used to intervene in cells at different concentration gradients (10ug / ml, 25ug / ml, 50ug / ml, 100ug / ml) for 24 hours.

[0033] Cellular ATP content was measured using an ATP assay kit following the manufacturer's instructions (Beyotime Biotechnology).

[0034] Statistical analysis: SPSS 24.0 software was used for processing. The measurement data were expressed as (x±s). One-way analysis of variance was used for comparison among multiple groups. LSD test was used for further pairwise comparison. P<0.05 indicated that the difference was statistically significant.

[0035] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing Morinda officinalis polysaccharide for alleviating muscular atrophy, characterized in that: The steps include: S1: crude extract of Morinda officinalis polysaccharide; Preparation of materials required: washed, sliced ​​and dried Morinda officinalis root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of Morinda officinalis polysaccharides; S21: Wash the root of Morinda officinalis, slice it, dry it, grind it into powder with a crusher for later use, and mix the Morinda officinalis powder and ultrapure water in a ratio of 1:60; S22: Extract under ultrasonic environment, then transfer to 90℃ environment for reaction for 3 hours, centrifuge the reaction solution, take the supernatant and concentrate it, add a certain amount of anhydrous ethanol to make the final volume fraction of ethanol in the solution 80%, and place it at 4℃ for 24 hours; S23: After overnight precipitation, the reaction solution is centrifuged and the precipitate is retained to obtain crude Morinda officinalis polysaccharide; S24: using a rotary evaporator to remove the residual organic solvent in the crude polysaccharide, and finally placing the crude polysaccharide in a vacuum freeze dryer for 24 hours and storing it in a drying tank; S3: Purification of Morinda officinalis polysaccharide; S31: First, the protein in the crude polysaccharide of Morinda officinalis was removed by the Sevage method, and a certain concentration of crude polysaccharide solution of Foeniculum foetida and Sevage solution (chloroform: n-butanol = 4:1) were prepared. 1 / 4 of the volume of Sevage solution was added to the polysaccharide solution of Morinda officinalis, and the mixture was thoroughly mixed for 30-60 minutes, and then centrifuged. The supernatant solution was polysaccharide, the middle layer was protein, and the lower layer solution was organic solvent. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer, and pour the remaining organic solvent into a separatory funnel and let it stand overnight before it can be reused; S33: the Morinda officinalis polysaccharide solution after protein removal is poured into an eggplant-shaped bottle of a rotary evaporator, and the organic solvent in the sample solution is evaporated away in a vacuum at 40°C; S34: using a dialysis bag to analyze small molecules and salts in the polysaccharide to obtain relatively pure Morinda officinalis polysaccharide; S4: Isolation of Morinda officinalis polysaccharides; S41: loading the pre-activated DEAE-dextran gel A-25 into the column, and after a period of equilibration, slowly adding a specific concentration of Morinda officinalis crude polysaccharide solution onto the gel column; S42: Turn on the constant flow pump, set the flow rate, collect the eluate with a partial collector, collect one tube every three minutes, use a NaCl solution with a specific ion concentration as the mobile phase, and determine the polysaccharide content of the eluate in the separate tube by the phenol-sulfuric acid method; S43: using the tube number as the abscissa and the absorbance as the ordinate to obtain the elution curve of the polysaccharide, and after analyzing the elution curve data, dividing the purified components according to the peak value and the concentration of the eluent, the tubes corresponding to the same purified component; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

2. An in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy, characterized in that: Cell culture and differentiation: In vitro experiments were carried out using C2C12 myoblasts. C2C12 cells were cultured in a 37°C, 5% CO2 incubator using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. When the cells grew to about 80% confluence, myotube differentiation was induced using DMEM medium containing 2% horse serum and 1% penicillin-streptomycin solution. The culture medium was changed every day and the induction was continued for 5 days until myotubes were formed.

3. The in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy according to claim 2 is characterized in that: Induction and grouping of muscular atrophy model: After successful differentiation of C2C12 cells, 10uM dexamethasone solution was used to induce the cells for 24 hours to establish a muscular atrophy model, and the cells were divided into 6 groups: control group, dexamethasone group, dexamethasone + 10ug / ml Morinda officinalis polysaccharide group, dexamethasone + 25ug / ml Morinda officinalis polysaccharide group, dexamethasone + 50ug / ml Morinda officinalis polysaccharide group and dexamethasone + 100ug / ml Morinda officinalis polysaccharide group.

4. The in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy according to claim 2, characterized in that: Intervention with Morinda officinalis polysaccharide: Purified Morinda officinalis polysaccharide was used to intervene in cells at different concentration gradients for 24 hours.

5. The in vitro application experiment of Morinda officinalis polysaccharide for alleviating muscular atrophy according to claim 2, characterized in that: Index detection: observe the morphology of C2C12 myotubes; detect the ATP level in myotube cells, and detect the changes in the mRNA and protein levels of SIRT1, MyHC, FoxO3, Murf1 and Atrogin-1 genes.

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