Preparation method and application of Guangdong hand polysaccharide for promoting growth of muscle fibers

Abstract

The invention discloses a preparation method and application of Guangdong's hand polysaccharide for promoting growth of muscle fibers, fresh Guangdong's hand is washed, sliced and dried, Guangdong's hand is ground into powder by a crusher for later use, and the Guangdong's hand powder and ultrapure water are fully and uniformly mixed; centrifuging the reaction liquid after standing overnight and precipitating, and then retaining the precipitate to obtain crude polysaccharide of the fructus citri sarcodactylis; and freeze-drying the obtained crude polysaccharide in a vacuum freeze-drying machine, and finally storing in a drying tank. Removing protein by a Sevage method to obtain a pure polysaccharide solution; and carrying out rotary evaporation concentration on the dialyzed polysaccharide solution, and freeze-drying to obtain deproteinized crude polysaccharide. According to the preparation method and application of the Guangdong hand polysaccharide capable of promoting the growth of the muscle fibers, the diameter of myotubes can be remarkably increased through the Guangdong hand polysaccharide, the ATP concentration of muscle cells can be improved, the application range of the Guangdong hand polysaccharide is widened, and the Guangdong hand polysaccharide can play roles in improving the immune level and resisting cancers clinically; and the application of the traditional Chinese medicine composition in crowds with sarcopenia, athletes and the like can be obviously expanded.

Description

Technical Field

[0001] The invention relates to the technical field of natural polysaccharide extraction, and in particular to a preparation method and application of a citronella polysaccharide for promoting muscle fiber growth. Background Art

[0002] As one of the common traditional Chinese medicinal materials in my country, Guangdong Foshu has the effects of relieving cough and reducing phlegm, promoting salivation and stimulating appetite. With the continuous deepening of its pharmacological research, the anti-inflammatory, antioxidant, anti-aging and blood sugar-lowering effects of Guangdong Foshu have gradually become known to the public.

[0003] However, the above-mentioned preparation method and application of existing Guangdong bergamot polysaccharides still have the following problems in actual use: At present, the research on Guangdong bergamot is mostly about the extraction, identification and application of bergamot essential oil, and polysaccharide is one of the main active ingredients of Guangdong bergamot polysaccharides, with an average polysaccharide content of 5.56%. There are few studies on its extraction and physiological effects, especially the lack of structural analysis and physiological effects of purified Guangdong bergamot polysaccharides. At present, the extraction methods of plant polysaccharides are mostly hot water extraction, ultrasound-assisted extraction, enzyme-assisted extraction, etc. Studies have shown that the extraction rate of Guangdong bergamot polysaccharides by experimental hot water extraction is about 9.35%, and the extraction rates of Guangdong bergamot polysaccharides by composite enzyme method and ultrasonic extraction method are 4.13% and 3.18%, respectively. The work efficiency and product quality of industrial extraction of Guangdong bergamot polysaccharides cannot be guaranteed.

[0004] Therefore, we proposed a preparation method and application of Foeniculum vulgare polysaccharide for promoting muscle fiber growth in order to solve the above-mentioned problems. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of a bergamot polysaccharide for promoting muscle fiber growth, in order to solve the problem that the current research on bergamot is mostly about the extraction, identification and application of bergamot essential oil, and polysaccharide is one of the main active ingredients of bergamot polysaccharide, the average polysaccharide content is 5.56%, and there are few studies on its extraction and physiological effects, especially the lack of structural analysis and physiological effects of purified bergamot polysaccharide. At present, the extraction methods of plant polysaccharides are mostly hot water extraction, ultrasound-assisted extraction, enzyme-assisted extraction, etc. Studies have shown that the extraction rate of bergamot polysaccharide by experimental hot water extraction is about 9.35%, and the extraction rates of bergamot polysaccharide by composite enzyme method and ultrasonic extraction method are 4.13% and 3.18%, respectively, which cannot guarantee the working efficiency and product quality of industrial extraction of bergamot polysaccharide.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing a bergamot polysaccharide for promoting muscle fiber growth, comprising the following steps: S1: crude extraction of polysaccharides from bergamot; Preparation of materials required for preparation: washed, sliced ​​and dried bergamot, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of bergamot polysaccharide; S21: Wash the fresh bergamot fruit, slice it and dry it, grind it into powder with a crusher for later use, and mix the bergamot powder and ultrapure water in a ratio of 1:40; S22: extracting the mixed solution of Guang Fo Shou powder under an ultrasonic environment of 400W for 20min, and centrifuging the extract at a speed of 4000r / min for 20min; S23: After the supernatant is concentrated, anhydrous ethanol is added to make the final volume fraction of ethanol in the solution 80%, and the solution is placed at 4°C for 24 hours; S24: centrifuging the reaction solution after overnight precipitation at 4000 r / min for 20 min, retaining the precipitate to obtain crude polysaccharide of Foeniculum villosum, and removing the residual organic solvent in the crude polysaccharide by a rotary evaporator; S25: freeze-drying the obtained crude polysaccharide in a vacuum freeze dryer for 24 hours, and finally storing it in a drying tank; S3: Purification of polysaccharide from Cibotium barometz; S31: First, the protein in the crude polysaccharide of Citrus grandis was removed by the Sevage method. A 1 / 4 volume of Sevage solution (chloroform: n-butanol = 4:1) was added to the polysaccharide solution of Citrus grandis and mixed thoroughly for 30 minutes. The mixture was shaken and allowed to stand for 30 minutes. The mixture was then centrifuged at 4000 r / min for 5 minutes to remove the protein in the crude polysaccharide, thereby obtaining a relatively pure polysaccharide solution of Citrus grandis. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer. The polysaccharide solution of Foeniculum vulgare after removing the protein is poured into an eggplant-shaped bottle of a rotary evaporator, and after rotary evaporation at 40°C for 10 hours, dialyzed for 48 hours; S33: The dialyzed polysaccharide solution is concentrated by rotary evaporation and freeze-dried for 24 hours to obtain deproteinized crude polysaccharide; S4: Isolation of polysaccharides from Cibotium barometz; S41: Fill the activated DEAE-dextran gel A-25 into a 55 mm*30 cm column, and slowly and evenly add it into the 6 mg / ml citronella crude polysaccharide solution after equilibration; S42: Start the constant flow pump and set the flow rate to 4.8 ml / min, and collect one tube of eluate every three minutes; S43: Use different concentrations of NaCl solution (0-0.8 mg / ml) for gradient elution, and measure the polysaccharide content at 490 nm, draw an elution curve, divide the purified components according to the peak value, combine the eluates in the tubes of the same component, concentrate and dialyze for 3 days to remove salt; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

[0007] An application of bergamot polysaccharide for promoting muscle fiber growth, characterized in that C2C12 myoblasts are cultured in a specific DMEM culture medium until they differentiate into myotubes.

[0008] Preferably, after the differentiation of the C2C12 myoblasts, they were intervened with different concentrations of Psoralea corylifolia polysaccharide for 24 hours and divided into a control group and 50 and 100 μg / ml Psoralea corylifolia polysaccharide intervention groups. The myotube morphology was observed by MyHC staining and the cell growth was evaluated by ATP detection. The results showed that Psoralea corylifolia polysaccharide promoted the growth of muscle cells.

[0009] Compared with the prior art, the beneficial effects of the present invention are: the preparation method and application of the bergamot polysaccharide for promoting muscle fiber growth, the bergamot polysaccharide can significantly increase the diameter of myotubes, improve the ATP concentration of muscle cells, and broaden the application scope of the bergamot polysaccharide. It can not only improve the immune level and anti-cancer effects in clinical aspects, but also significantly expand its application in people with sarcopenia, athletes and the like. The specific contents are as follows: 1. During the purification step, high molecular weight polysaccharides are retained from the polysaccharides of Foeniculum vulgare. High molecular weight polysaccharides have better stability and fermentation persistence, and show good advantages in immune regulation, anti-oxidation, and promoting intestinal health.

[0010] 2. Compared with other extraction methods, it is simpler to operate and easier to implement on an industrial scale. The use of water as a solvent for extraction has less impact on the ecological environment. The treatment of wastewater after extraction is relatively simple. Hot water extraction has higher extraction efficiency because hot water can effectively penetrate the plant cell wall, dissolve and release polysaccharides in the cell, and ultrasonically break the plant cell wall, which helps the next step of hot water extraction to have higher efficiency.

[0011] 3. The polysaccharide of Foeniculum foetidum was obtained by ultrasound-assisted hot water extraction and ethanol precipitation, and was treated with sevage and dialysis methods for preliminary polysaccharide purification. The different components of Foeniculum foetidum polysaccharide were further separated by DEAE dextran A-25 exchange column and Sephadex G-100 dextran gel column to obtain relatively pure Foeniculum foetidum polysaccharide. Finally, by intervening in C2C12 cells, it was confirmed that Foeniculum foetidum polysaccharide has a growth-promoting effect on muscle fibers.

[0012] 4. The C2C12 cell experiment verified that Foeniculum vulgare polysaccharide has certain potential in promoting protein synthesis in the body. This shows that Foeniculum vulgare polysaccharide can not only play a role in improving immune levels and anti-cancer in clinical practice, but also significantly expand its application in people with sarcopenia, athletes and other groups.

[0013] 5. After two-step separation and purification using DEAE ion exchange column and dextran gel molecular column, a single-component pure polysaccharide of Foeniculum villosum was obtained, which provides a more comprehensive understanding of the monosaccharide composition and structure analysis of Foeniculum villosum polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the myotube morphology of the present invention; Figure 2 Schematic diagram of ATP concentration of the present invention. DETAILED DESCRIPTION

[0015] 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.

[0016] See also Figure 1-Figure 2 , the present invention provides the following technical solutions: Example 1: In order to solve the problems of low efficiency and poor product quality in the existing industrial extraction of bergamot polysaccharides, this example discloses the following technical content: a preparation of bergamot polysaccharides that promote muscle fiber growth, comprising the following steps: S1: crude extraction of polysaccharides from bergamot; Preparation of materials required for preparation: washed, sliced ​​and dried bergamot, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of bergamot polysaccharide; S21: Wash the fresh bergamot fruit, slice it and dry it, grind it into powder with a crusher for later use, and mix the bergamot powder and ultrapure water in a ratio of 1:40; S22: extracting the mixed solution of Guang Fo Shou powder under an ultrasonic environment of 400W for 20min, and centrifuging the extract at a speed of 4000r / min for 20min; S23: After the supernatant is concentrated, anhydrous ethanol is added to make the final volume fraction of ethanol in the solution 80%, and the solution is placed at 4°C for 24 hours; S24: centrifuging the reaction solution after overnight precipitation at 4000 r / min for 20 min, retaining the precipitate to obtain crude polysaccharide of Foeniculum villosum, and removing the residual organic solvent in the crude polysaccharide by a rotary evaporator; S25: freeze-drying the obtained crude polysaccharide in a vacuum freeze dryer for 24 hours, and finally storing it in a drying tank; S3: Purification of polysaccharide from Cibotium barometz; S31: First, the protein in the crude polysaccharide of Citrus grandis was removed by the Sevage method. A 1 / 4 volume of Sevage solution (chloroform: n-butanol = 4:1) was added to the polysaccharide solution of Citrus grandis and mixed thoroughly for 30 minutes. The mixture was shaken and allowed to stand for 30 minutes. The mixture was then centrifuged at 4000 r / min for 5 minutes to remove the protein in the crude polysaccharide, thereby obtaining a relatively pure polysaccharide solution of Citrus grandis. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer. The polysaccharide solution of Foeniculum vulgare after removing the protein is poured into an eggplant-shaped bottle of a rotary evaporator, and after rotary evaporation at 40°C for 10 hours, dialyzed for 48 hours; S33: The dialyzed polysaccharide solution is concentrated by rotary evaporation and freeze-dried for 24 hours to obtain deproteinized crude polysaccharide; S4: Isolation of polysaccharides from Cibotium barometz; S41: Fill the activated DEAE-dextran gel A-25 into a 55 mm*30 cm column, and slowly and evenly add it into the 6 mg / ml citronella crude polysaccharide solution after equilibration; S42: Start the constant flow pump and set the flow rate to 4.8 ml / min, and collect one tube of eluate every three minutes; S43: Use different concentrations of NaCl solution (0-0.8 mg / ml) for gradient elution, and measure the polysaccharide content at 490 nm, draw an elution curve, divide the purified components according to the peak value, combine the eluates in the tubes of the same component, concentrate and dialyze for 3 days to remove salt; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

[0017] An application of bergamot polysaccharide for promoting muscle fiber growth, characterized in that C2C12 myoblasts are cultured in a specific DMEM culture medium until they differentiate into myotubes.

[0018] After C2C12 myoblasts were differentiated, they were intervened with different concentrations of bergamot polysaccharide for 24 hours and divided into a control group and 50 and 100 μg / ml bergamot polysaccharide intervention groups. The myotube morphology was observed by MyHC staining and cell growth was evaluated by ATP detection. The results showed that bergamot polysaccharide promoted muscle cell growth.

[0019] The bergamot polysaccharide prepared in this example for promoting muscle fiber growth is different from the current research on the antioxidant activity and immune activity of bergamot polysaccharide. There are few related studies on promoting growth-related cytokines and enhancing body metabolism, especially the lack of research on the physiological characteristics of muscle cells. The bergamot polysaccharide extracted in this example promotes muscle fiber growth and is beneficial to improving muscle strength and endurance, providing a scientific basis and technical support for the entry of bergamot polysaccharide into the nutritional supplement market.

[0020] 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 bergamot polysaccharide for promoting muscle fiber growth, characterized in that: The steps include: S1: crude extraction of polysaccharides from bergamot; Preparation of materials required for preparation: washed, sliced ​​and dried bergamot, ultrapure water, crusher, anhydrous ethanol, ultrasonic extractor, rotary evaporator, vacuum freeze dryer and dry storage tank; S2: Method for crude extraction of bergamot polysaccharide; S21: Wash the fresh bergamot fruit, slice it and dry it, grind it into powder with a crusher for later use, and mix the bergamot powder and ultrapure water in a ratio of 1:40; S22: extracting the mixed solution of Guang Fo Shou powder under an ultrasonic environment of 400W for 20min, and centrifuging the extract at a speed of 4000r / min for 20min; S23: After the supernatant is concentrated, anhydrous ethanol is added to make the final volume fraction of ethanol in the solution 80%, and the solution is placed at 4°C for 24 hours; S24: centrifuging the reaction solution after overnight precipitation at 4000 r / min for 20 min, retaining the precipitate to obtain crude polysaccharide of Foeniculum villosum, and removing the residual organic solvent in the crude polysaccharide by a rotary evaporator; S25: freeze-drying the obtained crude polysaccharide in a vacuum freeze dryer for 24 hours, and finally storing it in a drying tank; S3: Purification of polysaccharide from Cibotium barometz; S31: First, the protein in the crude polysaccharide of Citronella pubescens was removed by the Sevage method. A 1 / 4 volume of Sevage solution was added to the polysaccharide solution of Citronella pubescens and the mixture was thoroughly mixed for 30 minutes. The mixture was then shaken and allowed to stand for 30 minutes. The mixture was then centrifuged at 4000 r / min for 5 minutes to remove the protein in the crude polysaccharide, thereby obtaining a relatively pure polysaccharide solution of Citronella pubescens. S32: After absorbing the supernatant, repeat the above steps until there is no middle layer. The polysaccharide solution of Foeniculum vulgare after removing the protein is poured into an eggplant-shaped bottle of a rotary evaporator, and after rotary evaporation at 40°C for 10 hours, dialyzed for 48 hours; S33: The dialyzed polysaccharide solution is concentrated by rotary evaporation and freeze-dried for 24 hours to obtain deproteinized crude polysaccharide; S4: Isolation of polysaccharides from Cibotium barometz; S41: Fill the activated DEAE-dextran gel A-25 into a 55 mm*30 cm column, and slowly and evenly add it into the 6 mg / ml citronella crude polysaccharide solution after equilibration; S42: Start the constant flow pump and set the flow rate to 4.8 ml / min, and collect one tube of eluate every three minutes; S43: Use different concentrations of NaCl solution for gradient elution, and measure the polysaccharide content at 490nm, draw an elution curve, divide the purified components according to the peak value, combine the eluates in the tubes of the same component, concentrate and dialyze for 3 days to remove salt; S44: freeze-drying to obtain fraction FCP-D1, and then purifying using Sephadex G-200 column to obtain fraction FCP-G1.

2. An application of bergamot polysaccharide for promoting muscle fiber growth, characterized in that: C2C12 myoblasts were cultured in a specific DMEM medium until they differentiated into myotubes.

3. The use of the bergamot polysaccharide for promoting muscle fiber growth according to claim 2, characterized in that: After the C2C12 myoblasts were differentiated, they were intervened with different concentrations of bergamot polysaccharide for 24 hours and divided into a control group and 50 and 100 μg / ml bergamot polysaccharide intervention groups. The myotube morphology was observed by MyHC staining and cell growth was evaluated by ATP detection. The results showed that bergamot polysaccharide promoted muscle cell growth.

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