Preparation method and application of fructus amomi purified polysaccharide for enhancing muscle function

Abstract

The invention discloses a preparation method and application of fructus amomi purified polysaccharide capable of enhancing a muscle function, fructus amomi roots are washed, sliced and dried, the dried fructus amomi roots are ground into powder by a crusher for later use, the fructus amomi powder and ultrapure water are uniformly mixed, and the mixture is extracted and centrifuged in an ultrasonic environment; centrifuging the reaction liquid after overnight precipitation, retaining the precipitate to obtain crude amomum villosum polysaccharide, removing the residual organic solvent in the crude polysaccharide by using a rotary evaporator, finally freeze-drying the crude polysaccharide in a vacuum freeze-drying machine, and storing the freeze-dried crude polysaccharide in a drying tank. According to the preparation method and application of the fructus amomi purified polysaccharide capable of enhancing the muscle function, research reports of fructus amomi on a muscle system are not found at present, research of intervention of fructus amomi polysaccharide in muscle cells C2C12 and amyotrophy resistance is not found, the fructus amomi polysaccharide can be applied to improvement of the muscle function, and the fructus amomi polysaccharide with a clear structure after purification is used, so that the fructus amomi polysaccharide can be applied to improvement of the muscle function. The application of the fructus amomi polysaccharide in amyotrophy is clearly confirmed.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of purified Amomum villosum polysaccharide, in particular to a preparation method and application of purified Amomum villosum polysaccharide for enhancing muscle function. Background Art

[0002] Amomum villosum is a specialty of Yangchun City, Guangdong Province, and a national geographical indication product of China. Its flowers, fruits, roots, stems and leaves can all be used as medicine, and the fruit is the most precious. The fruit of Amomum villosum is oval or ovate, brown on the surface, densely covered with thorn-like protrusions, thin and soft peel, and the seeds are rich in volatile oils. It has the unique quality of "plump and firm, fragrant and with all five flavors". According to the theory of traditional Chinese medicine, Amomum villosum is warm in nature and pungent in taste, and belongs to the spleen, stomach and kidney meridians. It has significant effects, mainly including promoting qi, regulating the middle, harmonizing the stomach, invigorating the spleen, removing dampness and stagnation, relieving pain and stabilizing the fetus. It can effectively relieve symptoms such as spleen and stomach deficiency and cold, and stomach qi reflux in clinical practice, and has significant therapeutic effects on the treatment of abdominal distension, stomach fruit and food stagnation, choking and vomiting. As the active ingredient of Amomum villosum, Amomum villosum polysaccharide has the effects of improving lipid metabolism, resisting oxidative stress, regulating gastrointestinal function and potentially stabilizing the fetus. It can significantly lower blood lipids, enhance the activity of antioxidant enzymes, and promote gastrointestinal health, and has good development prospects.

[0003] At present, people use ultrasonic extraction and enzymatic hydrolysis to extract Amomum villosum polysaccharides. Ultrasonic extraction is a method that increases the penetration of solvents through the cavitation effect generated by ultrasound to promote the extraction of target components. It has the advantages of low extraction temperature, high efficiency and short time, and can effectively protect the functional components of Amomum villosum polysaccharides.

[0004] However, the above-mentioned extraction and application of existing Amomum villosum polysaccharides still have the following problems in actual use: the extraction rate of Amomum villosum polysaccharides is still low, and it is either time-consuming, low in yield, or high in cost; the structural research of Amomum villosum polysaccharides 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 Amomum villosum polysaccharides in the field of nutrition.

[0005] Therefore, we proposed a method for preparing and applying a purified polysaccharide of Amomum villosum for enhancing muscle function, so as to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing purified polysaccharides of Amomum villosum for enhancing muscle function and application thereof, in order to solve the problems that the extraction rate of Amomum villosum polysaccharides is still low, and the extraction process is either time-consuming, low in yield, or high in cost; the structural research of Amomum villosum polysaccharides focuses on monosaccharide composition, side chain position, etc., and there is less research 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 Amomum villosum polysaccharides in the field of nutrition.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for preparing and applying a purified polysaccharide of Amomum villosum for enhancing muscle function, comprising the following steps: S1: crude extraction of polysaccharides from Amomum villosum; Preparation of materials required: washed, sliced ​​and dried Amomum villosum root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of polysaccharides from Amomum villosum; S21: Wash the root of Amomum villosum, slice it, dry it, grind it into powder with a crusher for later use, and mix the Amomum villosum 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 to retain the precipitate to obtain crude polysaccharide of Amomum villosum, and the residual organic solvent in the crude polysaccharide is removed by a rotary evaporator; S24: freeze-drying the crude polysaccharide in a vacuum freeze dryer for 24 h and storing it in a drying tank; S3: Purification of Amomum villosum polysaccharide; S31: First, the protein in the crude polysaccharide of Amomum villosum 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 Amomum villosum, 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 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 Amomum villosum 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 Amomum villosum polysaccharide; S4: Isolation of polysaccharides from Amomum villosum; S41: The pre-activated DEAE-dextran gel A-25 is loaded into the column. After a period of equilibration, a specific concentration of Amomum villosum crude polysaccharide solution is slowly added to 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: With the tube number as the horizontal coordinate and the absorbance as the vertical coordinate, the elution curve of the polysaccharide is obtained. After analyzing the elution curve data, the purified components are divided according to the peak value and the eluent concentration. The eluents corresponding to the tubes of the same purified component are combined, concentrated to a certain volume, transferred to a dialysis bag, and dialyzed with distilled water for 3 days to remove salt impurities in each component. After the dialysis is completed, each component is collected and freeze-dried; S44: The same method was used for column loading, balancing, sample loading, elution, determination, dialysis and freeze-drying of Sephadex G-200 to obtain purified Amomum villosum polysaccharide.

[0008] The invention discloses an application of purified polysaccharide from Amomum villosum for enhancing muscle function. 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 changed every day and the induction was continued for 5 days until myotubes were formed.

[0009] Preferably, the Amomum villosum polysaccharide intervention: using purified Amomum villosum polysaccharide at different concentration gradients to intervene in cells for 24 hours.

[0010] Preferably, the morphology of C2C12 myotubes is observed; and the ATP level in myotube cells is detected.

[0011] Compared with the prior art, the beneficial effects of the present invention are: the preparation method and application of the purified Amomum villosum polysaccharide for enhancing muscle function can not only apply the Amomum villosum polysaccharide to improve muscle function, but also use the Amomum villosum polysaccharide with a clear structure after purification, which clearly confirms the application of the Amomum villosum polysaccharide in muscular atrophy. The specific contents are as follows: 1. Its effect in alleviating muscular atrophy has been verified, providing new ideas for drug treatment to alleviate muscular atrophy and providing technical support for the application of Amomum villosum polysaccharide in the nutritional supplement market.

[0012] 2. The data were processed using SPSS24.0 software, and the measured 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a structural formula diagram of the purified polysaccharide of Amomum villosum of the present invention; Figure 2 This is a schematic diagram of the MOP comparison of the present invention; Figure 3 It is a schematic diagram of ATP content of the present invention. DETAILED DESCRIPTION

[0014] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the implementation rules 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. See also Figure 1-Figure 3 , the present invention provides the following technical solutions: In order to solve the problem that the existing spring Amomum villosum purified 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 spring Amomum villosum purified polysaccharide for enhancing muscle function and its application, comprising the following steps: S1: crude extraction of polysaccharides from Amomum villosum; Preparation of materials required: washed, sliced ​​and dried Amomum villosum root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of polysaccharides from Amomum villosum; S21: Wash the root of Amomum villosum, slice it, dry it, grind it into powder with a crusher for later use, and mix the Amomum villosum 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 to retain the precipitate to obtain crude polysaccharide of Amomum villosum, and the residual organic solvent in the crude polysaccharide is removed by a rotary evaporator; S24: freeze-drying the crude polysaccharide in a vacuum freeze dryer for 24 h and storing it in a drying tank; S3: Purification of Amomum villosum polysaccharide; S31: First, the protein in the crude polysaccharide of Amomum villosum 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 Amomum villosum, 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 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 Amomum villosum 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 Amomum villosum polysaccharide; S4: Isolation of polysaccharides from Amomum villosum; S41: The pre-activated DEAE-dextran gel A-25 is loaded into the column. After a period of equilibration, a specific concentration of Amomum villosum crude polysaccharide solution is slowly added to 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: With the tube number as the horizontal coordinate and the absorbance as the vertical coordinate, the elution curve of the polysaccharide is obtained. After analyzing the elution curve data, the purified components are divided according to the peak value and the eluent concentration. The eluents corresponding to the tubes of the same purified component are combined, concentrated to a certain volume, transferred to a dialysis bag, and dialyzed with distilled water for 3 days to remove salt impurities in each component. After the dialysis is completed, each component is collected and freeze-dried; S44: The same method was used for column loading, balancing, sample loading, elution, determination, dialysis and freeze-drying of Sephadex G-200 to obtain purified Amomum villosum polysaccharide.

[0015] 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.

[0016] Intervention with Amomum villosum polysaccharide: Purified Amomum villosum polysaccharide was used to intervene in cells at different concentration gradients for 24 hours.

[0017] Index detection: observe the morphology of C2C12 myotubes; detect the ATP level in myotube cells.

[0018] 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.

[0019] Cell differentiation: cells were divided into 2.5*10 6 The cells were seeded at a density of 1000 cells / mL 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.

[0020] Intervention with Amomum villosum polysaccharide: Purified Amomum villosum polysaccharide was used to intervene in cells at different concentration gradients (50ug / ml, 100ug / ml) for 24 hours.

[0021] 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.

[0022] Statistical analysis: SPSS 24.0 software was used for processing, and 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.

[0023] 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.

[0024] Cell differentiation: cells were divided into 2.5*10 6 The cells were seeded at a density of 100 cells / mL 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] Intervention with Amomum villosum polysaccharide: Purified Amomum villosum polysaccharide was used to intervene in cells at different concentration gradients (50ug / ml, 100ug / ml) for 24 hours.

[0026] Cellular ATP content was measured using an ATP assay kit following the manufacturer's instructions (Beyotime Biotechnology).

[0027] Statistical analysis: SPSS 24.0 software was used for processing, and 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.

[0028] 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 purified polysaccharide of Amomum villosum for enhancing muscle function, characterized in that: The steps include: S1: crude extraction of polysaccharides from Amomum villosum; Preparation of materials required: washed, sliced ​​and dried Amomum villosum root, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of polysaccharides from Amomum villosum; S21: Wash the root of Amomum villosum, slice it, dry it, grind it into powder with a crusher for later use, and mix the Amomum villosum 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 to retain the precipitate to obtain crude polysaccharide of Amomum villosum, and the residual organic solvent in the crude polysaccharide is removed by a rotary evaporator; S24: freeze-drying the crude polysaccharide in a vacuum freeze dryer for 24 h and storing it in a drying tank; S3: Purification of Amomum villosum polysaccharide; S31: First, the protein in the crude polysaccharide of Amomum villosum 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 Amomum villosum, 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 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 Amomum villosum 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 Amomum villosum polysaccharide; S4: Isolation of polysaccharides from Amomum villosum; S41: The pre-activated DEAE-dextran gel A-25 is loaded into the column. After a period of equilibration, a specific concentration of Amomum villosum crude polysaccharide solution is slowly added to 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: With the tube number as the horizontal coordinate and the absorbance as the vertical coordinate, the elution curve of the polysaccharide is obtained. After analyzing the elution curve data, the purified components are divided according to the peak value and the eluent concentration. The eluents corresponding to the tubes of the same purified component are combined, concentrated to a certain volume, transferred to a dialysis bag, and dialyzed with distilled water for 3 days to remove salt impurities in each component. After the dialysis is completed, each component is collected and freeze-dried; S44: The same method was used for column loading, balancing, sample loading, elution, determination, dialysis and freeze-drying of Sephadex G-200 to obtain purified Amomum villosum polysaccharide.

2. An application of purified polysaccharide of Amomum villosum for enhancing muscle function, 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 use of the purified polysaccharide of Amomum villosum for enhancing muscle function according to claim 2, characterized in that: Intervention with Amomum villosum polysaccharide: Purified Amomum villosum polysaccharide was used to intervene in cells at different concentration gradients for 24 hours.

4. The use of the purified polysaccharide of Amomum villosum for enhancing muscle function according to claim 2, characterized in that: Index detection: observe the morphology of C2C12 myotubes; detect the ATP level in myotube cells.

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