Nostoc sphaeroids kutz alcohol-soluble neutral polysaccharide and preparation method thereof
By extracting and purifying the alcohol-soluble neutral polysaccharide GXM-1 from *Ge Xian Mi*, the problem of insufficient research on it in the existing technology was solved, and the anti-fatigue effect of the polysaccharide in the wine was realized.
Patent Information
- Application Number
- CN202411067242.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2025-01-10
AI Technical Summary
There is limited research on alcohol-soluble neutral polysaccharides from *Gexinmi* in the current technology, and there is a lack of understanding of their structure and biological activity, especially their application in wine, which has not been reported.
Alcohol-soluble neutral polysaccharides were extracted and purified from *Gnaphalium affine* using methods such as ultrasonic treatment, enzymatic hydrolysis, multi-step chromatographic purification, and freeze-drying. Specific steps included enzymatic hydrolysis, adjustment of the alcohol content of the polysaccharide solution, centrifugation, dialysis, and chromatography, to obtain homogeneous alcohol-soluble neutral polysaccharide GXM-1 from *Gnaphalium affine*.
The purification and structural identification of alcohol-soluble neutral polysaccharides from *Gynostemma pentaphyllum* were achieved, providing a reference for their stable application in wine and demonstrating significant anti-fatigue activity.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of natural extracts, and in particular to an alcohol-soluble neutral polysaccharide of scutellaria baicalensis and a preparation method thereof. Background Art
[0002] Gexianmi, scientifically known as Nostoc sphericalensis, is a special lower algae plant that can be used as both medicine and food. There are many rosary-shaped algae filaments embedded in a gelatinous polysaccharide matrix, forming a spherical or ellipsoidal original plant that is dark green and can reach a diameter of 2 cm. According to the "Supplement to Compendium of Materia Medica" and "Yao Xing Kao", Gexianmi can be eaten and used as medicine. Gexianmi is rich in polysaccharides. Some research reports have pointed out that Gexianmi polysaccharides have good antioxidant, anti-tumor and immunity-enhancing effects.
[0003] In recent years, people have gradually paid attention to the research on the active ingredients of Pueraria lobata. The results show that the polysaccharides in Pueraria lobata have extremely high biological activity, and the research is focused on the water-soluble polysaccharides of Pueraria lobata, and the research on alcohol-soluble polysaccharides is less. Mo Kaiju optimized the extraction of Pueraria lobata by alkaline enzyme composite pretreatment to obtain water-soluble Pueraria lobata polysaccharides, and its monosaccharide composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, arabinose and galacturonic acid methyl ester. From the current research situation, although some researchers have identified the structures of some Pueraria lobata polysaccharides, there is no public research on the alcohol-soluble neutral polysaccharides in Pueraria lobata. Summary of the invention
[0004] In view of this, the present invention proposes an alcohol-soluble neutral polysaccharide of Miscanthus puerariae and a preparation method and application thereof, aiming to fill the research gap in this field.
[0005] The technical solution of the present invention is achieved as follows: The present invention provides a method for preparing alcohol-soluble neutral polysaccharides from Miscanthus puerariae, which comprises the following steps:
[0006] Step 1, adding dried kudzu rice powder into ultrapure water, ultrasonic treatment, then adding complex enzyme, and adjusting the pH of the reaction system to 5.0 with hydrochloric acid, after enzymatic treatment, heating to inactivate the complex enzyme, filtering, and obtaining a crude kudzu rice polysaccharide extract;
[0007] Step 2, concentrating the crude polysaccharide extract of Pueraria lobata, adjusting the alcohol content to 40%, standing, centrifuging to obtain a supernatant, adjusting the alcohol content to 80%, standing to precipitate polysaccharides, and freeze-drying;
[0008] Step 3, dissolving the polysaccharide obtained in step 2 into a solution, then passing the solution through a polyamide resin column, and collecting the permeate;
[0009] Step 4: Concentrate the permeate to 1 / 10-1 / 20 of the original volume, and use Sevag reagent to remove protein from the concentrated permeate;
[0010] Step 5: The permeate from which the protein has been removed is loaded onto a DEAESeplifeA25 dextran gel chromatography column, and then graded and eluted using 0 mol / L, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, and 0.7 mol / L sodium chloride solutions, respectively, in order. The eluate is collected using a fully automatic fraction collector, and 10 ml is collected in each sample tube. Each fraction is measured using the phenol-sulfuric acid method throughout the process. The elution curve is plotted with absorbance as the ordinate and the number of collection tubes as the abscissa, and the sample of the sample tube corresponding to the first elution curve peak is collected;
[0011] Step 6: Combine the samples collected in step 5, desalt them through a dialysis bag, and then freeze-dry them in a vacuum to obtain alcohol-soluble neutral polysaccharides from Miscanthus puerariae.
[0012] In some embodiments, in step 1, 95% ethanol is used to adjust the alcohol content.
[0013] In some embodiments, in step 1, the method of inactivating the complex enzyme includes heating to 100° C. and keeping the temperature for 10 minutes.
[0014] In some embodiments, before ultrasonic treatment, the mass ratio of iris lobata to ultrapure water is 1:(10-20), and after ultrasonic treatment, ultrapure water is added to the solution system. After the ultrapure water is added, the mass ratio of iris lobata to ultrapure water is 1:(100-200).
[0015] In some embodiments, in step 1, the complex enzyme includes pectinase and cellulase, and the mass ratio of pectinase:cellulase is 2:1.
[0016] In some embodiments, in step 1, the added amount of the complex enzyme is 1.5%-2.5% of the mass of the Gexian rice flour, the enzymolysis temperature is 30-65° C., and the enzymolysis time is 1-4 h.
[0017] In some embodiments, in step 1, the ultrasonic power is 120-160 W, and the ultrasonic time is 30-60 min.
[0018] In some embodiments, in step 2, the concentration treatment is to concentrate the crude polysaccharide extract of Pueraria lobata to 1 / 30-1 / 50 of the original volume, the rotation speed of the centrifugal treatment is 3000-5000 rpm, and the centrifugation time is 5-15 min.
[0019] In some embodiments, in step three, the polysaccharide obtained in step two is dissolved to prepare a solution with a concentration of 5-10 mg / ml.
[0020] In the above embodiment, the solvent used in preparing the solution is ultrapure water.
[0021] In some embodiments, in step 4, the volume ratio of Sevag solvent to the concentrated permeate is (1-2):1.
[0022] In the above embodiment, the Sevag solvent used is prepared by mixing chloroform and n-butanol in a volume ratio of 5:1.
[0023] In some embodiments, in step six, the molecular weight of the dialysis bag is 8000-12000 Da.
[0024] In a second aspect, the present invention further provides an alcohol-soluble neutral polysaccharide of G. puerariae prepared by the above preparation method, and the polysaccharide is named GXM-1.
[0025] In some embodiments, the egg soup composition of the prepared alcohol-soluble neutral polysaccharide GXM-1 from Pueraria lobata includes fucose, rhamnose, arabinose, galactose, and glucose, and the molar ratio of the above monosaccharides is 1.31:3.40:0.57:2.87:91.85, and the weight average molecular weight is 2.690×10 6 Da, number average molecular weight is 1.454×10 6 Da, peak molecular weight is 8.694×10 5 Da, molecular weight distribution range is 7×10 5 Da-6×10 6 Da, its bonding structure connection modes include t-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p), 4,6-Gal(p), 3,4,6-Glc(p), and the relative molar ratio is 21.54:3.49:50.21:14.59:8.38:1.78.
[0026] In a third aspect, the present invention further provides a use of the alcohol-soluble neutral polysaccharide of Miscanthus puerariae in wine. Adding the alcohol-soluble neutral polysaccharide of Miscanthus puerariae into wine can make the wine have anti-fatigue activity.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] The present invention extracts and purifies uniform alcohol-soluble polysaccharide GXM-1 from G. raphani, and obtains its monosaccharide composition and bonding structure through structural identification, thus providing a reference for the stable application of G. raphani polysaccharide in wine. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0030] Figure 1 This is the elution curve of GXM-1 polysaccharide in Example 1;
[0031] Figure 2 is the standard ion chromatogram of GXM-1 in Example 1 (A);
[0032] Figure 3 Ion chromatogram of GXM-1 in Example 1 (B)
[0033] Figure 4 is the infrared spectrum of GXM-1 in Example 1;
[0034] Figure 5 The total ion chromatogram reconstructed by GC-MS of the methylated fragment of GXM-1 in Example 1;
[0035] Figure 6 is the high performance gel permeation chromatogram of GXM-1 in Example 1;
[0036] Figure 7 is the molecular weight distribution diagram of GXM-1 in Example 1;
[0037] Figure 8 This is a graph showing the effect of GXM-1 on blood ureaamine in mice in Example 1;
[0038] Fig. 9 This is the image of the effect of GXM-1 on mouse blood lactate in Example 1. DETAILED DESCRIPTION
[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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 creative work are within the scope of protection of the present invention.
[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.
[0041] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.
[0042] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.
[0043] Example 1
[0044] This example describes a preparation method and application of an alcohol-soluble neutral polysaccharide GXM-1 from Miscanthus puerariae, and the specific steps of the method are as follows:
[0045] (1) First, accurately weigh 30 g of dry kudzu rice flour (passed through an 80-mesh sieve) and add it to 300 mL of ultrapure water. Place it in an ultrasonic machine for ultrasonication (120 W) for 30 min, then add ultrapure water to 3 L, add 0.45 g of complex enzyme (pectinase: cellulase = 2:1) to the reaction system, adjust the pH of the reaction system to 5.0 with 0.5 mol / L hydrochloric acid, perform enzymolysis in a water bath at 30°C for 1 h, heat the solution to 100°C, maintain it for 10 min to inactivate the complex enzyme, and filter it to obtain 2.6 L of kudzu rice crude polysaccharide extract sample.
[0046] (2) After 2.6L of the crude polysaccharide extract of Puerariae chinensis was concentrated to 86mL, 95% ethanol was added to adjust the alcohol content of the Puerariae chinensis concentrate to 40%, and the mixture was allowed to stand overnight. The supernatant was obtained by centrifugation at 3000r / min for 5min, and 95% ethanol was added to adjust the alcohol content of the supernatant to 80%, and the mixture was allowed to stand overnight. The precipitated polysaccharide was collected and freeze-dried to obtain 5g of the sample. Then, the freeze-dried Puerariae chinensis polysaccharide was diluted with ultrapure water to a concentration of 5mg / mL, applied to a polyamide resin column, the permeate was collected, and the permeate was concentrated to 100mL. 100mL of Sevag reagent was used to mix and shake the permeate, and the protein was removed repeatedly once. Then, the mixture was applied to a DEAESeplifeA25 polysaccharide gel chromatography column at a loading flow rate of 0.5BV / h, and then graded elution was performed using 0, 0.1, 0.3, 0.5, and 0.7mol / L sodium chloride solutions in sequence. The eluate was collected using a fully automatic fraction collector, with 10 mL collected in each tube. Each fraction was measured using the phenol-sulfuric acid method throughout the process, and an elution curve was drawn with absorbance as the ordinate and the number of collected tubes as the abscissa. The samples in the sample tubes corresponding to the peaks of the elution curve were collected, and the samples were combined and desalted using a dialysis bag with a molecular weight of 8000 Da, and vacuum freeze-dried to obtain 178 mg of a sample of alcohol-soluble neutral polysaccharide GXM-1 from Pueraria lobata.
[0047] Example 2
[0048] This example describes a preparation method and application of an alcohol-soluble neutral polysaccharide GXM-1 from Miscanthus puerariae, and the specific steps of the method are as follows:
[0049] (1) First, accurately weigh 30 g of dry kudzu rice flour (passed through an 80-mesh sieve) and add it to 600 mL of ultrapure water. Place it in an ultrasonic machine for ultrasonication (160 W) for 60 min, then add ultrapure water to 6 L, add 0.75 g of complex enzyme (pectinase: cellulase = 2:1) to the reaction system, adjust the pH of the reaction system to 5.0 with 0.5 mol / L hydrochloric acid, perform enzymolysis in a water bath at 65 ° C for 4 h, heat the solution to 100 ° C, keep it for 10 min to inactivate the complex enzyme, and filter to obtain 5.6 L of kudzu rice crude polysaccharide extract sample.
[0050] (2) After 5.6L of the crude polysaccharide extract of Pueraria lobata was concentrated to 112mL, 95% ethanol was added to adjust the alcohol content of the Pueraria lobata concentrate to 40%, and the mixture was allowed to stand overnight. The supernatant was obtained after centrifugation at 5000r / min for 15min, and 95% ethanol was added to adjust the alcohol content of the supernatant to 80%, and the mixture was allowed to stand overnight. The precipitated polysaccharide was collected and freeze-dried to obtain 5.1g of the sample. Then, the freeze-dried Pueraria lobata polysaccharide was diluted with ultrapure water to a concentration of 5mg / mL, applied to a polyamide resin column, the permeate was collected, and the permeate was concentrated to 51mL. 102mL of Sevag reagent was used to mix and shake the permeate, and the protein was removed repeatedly for 3 times. Then, the mixture was applied to a DEAESeplifeA25 polysaccharide gel chromatography column at a loading flow rate of 0.5BV / h, and then graded elution was performed using 0, 0.1, 0.3, 0.5, and 0.7mol / L sodium chloride solutions in sequence. The eluate was collected using a fully automatic fraction collector, with 10 mL collected in each tube. Each fraction was measured using the phenol-sulfuric acid method throughout the process, and an elution curve was drawn with absorbance as the ordinate and the number of collected tubes as the abscissa. The samples in the sample tubes corresponding to the peak of the elution curve were collected, and the samples were combined and desalted using a 12000Da dialysis bag, and vacuum freeze-dried to obtain 181 mg of the alcohol-soluble neutral polysaccharide GXM-1 sample of Pueraria lobata.
[0051] Example 3
[0052] This example describes a preparation method and application of an alcohol-soluble neutral polysaccharide GXM-1 from Miscanthus puerariae, and the specific steps of the method are as follows:
[0053] (1) First, accurately weigh 30 g of dry kudzu rice flour (passed through an 80-mesh sieve) and add it to 450 mL of ultrapure water. Place it in an ultrasonic machine for ultrasonication (140 W) for 45 min, then add ultrapure water to 4.5 L, add 0.6 g of complex enzyme (pectinase: cellulase = 2:1) to the reaction system, adjust the pH of the reaction system to 5.0 with 0.5 mol / L hydrochloric acid, perform enzymolysis in a water bath at 50°C for 2.5 h, heat the solution to 100°C, maintain it for 10 min to inactivate the complex enzyme, and filter to obtain 4.2 L of kudzu rice crude polysaccharide extract sample.
[0054] (2) After 4.2L of the crude polysaccharide extract of Puerariae lobata was concentrated to 105mL, 95% ethanol was added to adjust the alcohol content of the Puerariae lobata concentrate to 40%, and the mixture was allowed to stand overnight. The supernatant was obtained after centrifugation at 4000r / min for 10min, and 95% ethanol was added to adjust the alcohol content of the supernatant to 80%, and the mixture was allowed to stand overnight. The precipitated polysaccharide was collected and freeze-dried to obtain 4.98g of the sample. Then, the freeze-dried Puerariae lobata polysaccharide was diluted with ultrapure water to a concentration of 7.5mg / mL, applied to a polyamide resin column, the permeate was collected, and the permeate was concentrated to 45mL. 68mL of Sevag reagent was used to mix and shake the permeate, and the protein was removed twice. Then, the mixture was applied to a DEAESeplifeA25 polysaccharide gel chromatography column at a loading flow rate of 0.5BV / h, and then graded elution was performed using 0, 0.1, 0.3, 0.5, and 0.7mol / L sodium chloride solutions in sequence. The eluate was collected using a fully automatic fraction collector, with 10 mL collected in each tube. Each fraction was measured using the phenol-sulfuric acid method throughout the process, and an elution curve was drawn with absorbance as the ordinate and the number of collection tubes as the abscissa. The samples in the sample tubes corresponding to the peaks of the elution curve were collected, and the samples were combined and desalted using a 10,000 Da dialysis bag, and vacuum freeze-dried to obtain 180 mg of a GXM-1 sample of alcohol-soluble neutral polysaccharide from Mie kudzuvine.
[0055] Structural analysis of the polysaccharide samples prepared in the above examples:
[0056] (1) Monosaccharide composition
[0057] ThermoICS5000+ ion chromatography system (ICS5000+, ThermoFisherScientific, USA) was used to analyze and detect the monosaccharide components of the alcohol-soluble neutral polysaccharide sample GXM-1 of Example 1 using an electrochemical detector. The ion chromatogram is shown in FIG. Figure 2 The results showed that the monosaccharide composition of the alcohol-soluble neutral polysaccharide sample GXM-1 from Pueraria lobata included fucose, rhamnose, arabinose, galactose, and glucose, and the molar ratio was 1.31:3.40:0.57:2.87:91.85.
[0058] (2) Infrared spectroscopy
[0059] Infrared spectrum results of GXM-1 sample in Example 1 are shown Figure 3 and Figure 4As shown, the absorption band at 3600-3200cm-1 is the stretching vibration absorption peak of -OH. The absorption peak in this area is the characteristic peak of sugars. Specifically: 3272.48cm-1 is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. The absorption peak at 2930.75cm-1 is attributed to the stretching vibration of CH. There is an absorption peak at 1016.97cm-1, which is attributed to the stretching vibration of CO.
[0060] (3) Methylation analysis
[0061] In Example 1, GXM-1 was subjected to methylation, acid hydrolysis, NaBD4 reduction and acetylation and then analyzed by gas chromatography-tandem mass spectrometry. The reconstructed total ion current of the methylated fragments is shown in Figure 5 According to the monosaccharide composition of GXM-1, the chromatographic peaks with higher molar content in the methylated fragments of GXM-1 were compared with the standard spectrum. The results of the methylation experiment are shown in Table 1.
[0062] Table 1 Results of bonding structure analysis of polysaccharide samples
[0063]
[0064] The results of methylation analysis showed that the bonding structure connection modes of GXM-1 included t-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p), 4,6-Gal(p), and 3,4,6-Glc(p), with the relative molar ratios of 21.54:3.49:50.21:14.59:8.38:1.78.
[0065] (4) High performance gel permeation chromatography analysis
[0066] The high performance gel permeation chromatogram of GXM-1 in Example 1 is as follows Figure 6 and Figure 7 As shown in the figure, GXM-1 has a sample peak at 16-28min, and the polydispersity coefficient of the peak is 1.850, indicating that GXM-1 is a uniform polysaccharide. The weight average molecular weight (Mw) of GXM-1 is 2.690×10 6 Da, number average molecular weight (Mn) is 1.454×10 6 Da, peak molecular weight (Mp) is 8.694×10 5 Da, molecular weight distribution range is (7×10 5 -6×10 6 Da).
[0067] In summary, GXM-1 has the following characteristics: The alcohol-soluble neutral polysaccharide GXM-1 from Pueraria lobata contains fucose, rhamnose, arabinose, galactose, and glucose in a molar ratio of 1.31:3.40:0.57:2.87:91.85, and the weight average molecular weight (Mw) is 2.690×10 6 Da, number average molecular weight (Mn) is 1.454×10 6 Da, peak molecular weight (Mp) is 8.694×10 5 Da, molecular weight distribution range is (7×10 5 -6×10 6 Da), and its bonding structure connection mode includes t-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p), 4,6-Gal(p), 3,4,6-Glc(p), and the relative molar ratio is 21.54:3.49:50.21:14.59:8.38:1.78.
[0068] (5) Anti-fatigue activity measurement
[0069] Animal grouping and dosing: 180 mice were pre-fed with Co60 radiation sterilized experimental mouse growth and reproduction pellets for 4 days. After 4 days, the mice were randomly divided into 9 groups, with 20 mice in each group, 10 of which were used for the mouse weight-bearing swimming experiment and 10 for the biochemical index detection. The 9 groups were blank control group (KB), negative control group (YX), positive control group (YZ), low-dose group of wine 1 (GD-1-JT), medium-dose group of wine 1 (GZ-1-JT), and high-dose group of wine 1 (GG-1-JT). The blank control group was sterile water, the negative control group was 12.5 mL / kg·bw of premium light-fragrance liquor base wine (aging period of 3 years, alcohol content 50.0% vol), the positive control group was 0.5 g / kg·bw icariin extract, and the low, medium and high dose groups of liquor 1 were premium light-fragrance liquor base wine added with 1.2, 2.4 and 4.8 mg / kg·bw of kudzu vine alcohol-soluble polysaccharide component 1, respectively. The mice were gavaged once a day, the weight of the mice was measured every 7 days, and the gavage was continued for 28 days.
[0070] Weighted swimming test: One hour after the last administration of the drug, mice were loaded with 5% of their body weight of lead at the base of their tails in batches and placed in a Morris water maze (water depth 40 cm, diameter 120 cm, water temperature 25±1°C). The time from the start of swimming to the time when the mice did not float to the surface for 10 seconds was recorded.
[0071] Determination of biochemical indices: 1 hour after the last administration, the mice were placed in a Morris water maze (water depth 40 cm, diameter 120 cm, water temperature 25±1°C) to swim for 40 min. After resting for 1 hour, they were anesthetized with ether. Blood was collected from the eyeballs and centrifuged in a high-speed refrigerated centrifuge (4°C, 3000 r / min) for 15 min to obtain serum. The biochemical indices were determined according to the methods of the lactate kit and the urea nitrogen kit.
[0072] Experimental results:
[0073] Effects of GXM-1 on body weight of mice
[0074] As can be seen from Table 2, during the 28-day experiment, the body weight of mice in each group increased steadily. Compared with the blank control group, negative control group and positive control group, the total increase in body weight of mice in each dose group of liquor 1 was within the normal range, with no statistically significant difference (P < 0.05). This indicates that during the experiment, the premium light-fragrance liquor base liquor and GXM-1 had no significant effect on the body weight of mice.
[0075] Table 2 Effect of GXM-1 on body weight of mice
[0076]
[0077] Effects of GXM-1 on weight-bearing swimming time in mice
[0078] The anti-fatigue effect can be directly characterized by exercise endurance, which is usually evaluated by the length of time mice can swim under load. As shown in Table 3, 28 days after administration, compared with the blank control group, the weighted swimming time of mice in the negative control group, low-dose group and high-dose group of Liquor 1 was significantly reduced (P < 0.05), which indicates that alcohol intake in mice may cause reduced exercise endurance. Compared with the negative control group, the weighted swimming time of mice in the positive control group and medium-dose group of Liquor 1 was significantly increased (P < 0.05), which indicates that at medium-dose concentrations, GXM-1 in the liquor can significantly improve the exercise endurance of mice and has a certain anti-fatigue effect.
[0079] Table 3 Effect of GXM-1 on weight-bearing swimming time of mice
[0080] Group Weighted swimming time / min KB 11.14±2.63** YX 6.01±1.01## YZ 8.23±2.34* GD-1-JT 7.10±2.18# GZ-1-JT 8.08±1.91* GG-1-JT 7.12±1.12#
[0081] Note: # indicates that there is a statistically significant difference compared with the blank control group (P < 0.05), ## indicates that there is a very significant difference compared with the blank control group (P < 0.01), * indicates that there is a statistically significant difference compared with the negative control group (P < 0.05), ** indicates that there is a very significant difference compared with the negative control group (P < 0.01), the same below.
[0082] Effects of GXM-1 on biochemical indicators of mice During high-intensity exercise, proteins and other substances in the mouse body will metabolize to produce blood urea nitrogen, and the concentration of blood urea nitrogen will increase due to exercise fatigue. Therefore, the concentration of blood urea nitrogen is one of the key indicators for assessing the degree of fatigue. Figure 8 and Fig. 9 It can be seen that compared with the blank control group, the blood urea nitrogen levels of mice in the negative control group, low-dose group and high-dose group of wine body 1 increased significantly (P < 0.05), and the blood urea nitrogen levels of mice in the positive control group and medium-dose group of wine body 1 did not change significantly. Compared with the negative control group, the blood urea nitrogen levels of mice in the positive control group and medium-dose group of wine body 1 decreased significantly (P < 0.05), while the blood urea nitrogen levels of mice in the low-dose group and high-dose group of wine body 1 did not change significantly. When mice continue to exercise, they will gradually become hypoxic, and anaerobic metabolism will occur at this time, leading to an increase in the blood lactate concentration in the body. If GXM-1 can inhibit the accumulation of lactic acid and accelerate the conversion of lactic acid, it means that it has an anti-fatigue effect. Compared with the negative control group, the blood lactate levels of mice in the positive group and the medium-dose group of wine 1 were significantly reduced by 19.56% and 19.43% respectively (P < 0.05). Compared with the blank control group, the blood lactate levels of mice in the negative control group and the low- and high-dose groups of wine 1 were significantly increased (P < 0.05), while there was no significant change in the positive group and the medium-dose group of wine 1. In summary, alcohol intake in mice will accelerate physical fatigue, and adding a medium dose of GXM-1 to wine can significantly increase exercise endurance and show a good anti-fatigue effect.
[0083] Based on the above data analysis results, the alcohol-soluble neutral polysaccharide GXM-1 prepared in the embodiment of the present invention includes fucose, rhamnose, arabinose, galactose, and glucose, and the molar ratio is 1.31:3.40:0.57:2.87:91.85, and the weight average molecular weight (Mw) is 2.690×10 6 Da, number average molecular weight (Mn) is 1.454×10 6 Da, peak molecular weight (Mp) is 8.694×10 5 Da, molecular weight distribution range is (7×10 5 -6×10 6 Da), and its bonding structure connection mode includes t-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p), 4,6-Gal(p), 3,4,6-Glc(p), and the relative molar ratio is 21.54:3.49:50.21:14.59:8.38:1.78, and the neutral polysaccharide has obvious anti-fatigue activity in wine.
[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 alcohol-soluble neutral polysaccharide from Miscanthus puerariae, characterized in that: The steps include: Step 1, adding dried kudzu rice powder into ultrapure water, ultrasonic treatment, then adding complex enzyme, and adjusting the pH of the reaction system to 5.0 with hydrochloric acid, after enzymatic treatment, heating to inactivate the complex enzyme, filtering, and obtaining a crude kudzu rice polysaccharide extract; Step 2, concentrating the crude polysaccharide extract of Pueraria lobata, adjusting the alcohol content to 40%, standing, centrifuging to obtain a supernatant, adjusting the alcohol content to 80%, standing to precipitate polysaccharides, and freeze-drying; Step 3, dissolving the polysaccharide obtained in step 2 into a solution, then passing the solution through a polyamide resin column, and collecting the permeate; Step 4: Concentrate the permeate to 1 / 10-1 / 20 of the original volume, and use Sevag reagent to remove protein from the concentrated permeate; Step 5: The permeate from which the protein has been removed is loaded onto a DEAESeplifeA25 dextran gel chromatography column, and then graded and eluted using 0 mol / L, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L, and 0.7 mol / L sodium chloride solutions, respectively, in order. The eluate is collected using a fully automatic fraction collector, and 10 ml is collected in each sample tube. Each fraction is measured using the phenol-sulfuric acid method throughout the process. The elution curve is plotted with absorbance as the ordinate and the number of collection tubes as the abscissa, and the sample of the sample tube corresponding to the first elution curve peak is collected; Step 6: Combine the samples collected in step 5, desalt them through a dialysis bag, and then freeze-dry them in a vacuum to obtain alcohol-soluble neutral polysaccharides from Miscanthus puerariae.
2. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step 1, the complex enzyme includes pectinase and cellulase, and the mass ratio of pectinase:cellulase is 2:
1.
3. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step 1, the added amount of the complex enzyme is 1.5%-2.5% of the mass of the kudzu rice flour, the enzymolysis temperature is 30-65° C., and the enzymolysis time is 1-4 h.
4. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step 1, the ultrasonic power is 120-160W, and the ultrasonic time is 30-60min.
5. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step three, the polysaccharide obtained in step two is dissolved to prepare a solution with a concentration of 5-10 mg / ml.
6. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step 4, the volume ratio of Sevag solvent to the concentrated permeate is (1-2):
1.
7. The method for preparing the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 1, characterized in that: In step six, the molecular weight of the dialysis bag is 8000-12000Da.
8. An alcohol-soluble neutral polysaccharide from Artemisia argyi, characterized in that: The preparation method is adopted according to any one of claims 1 to 7.
9. The alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 8, characterized in that: Its monosaccharide composition includes fucose, rhamnose, arabinose, galactose, and glucose, and the molar ratio is 1.31:3.40:0.57:2.87:91.85, and the weight average molecular weight is 2.690×10 6 Da, number average molecular weight is 1.454×10 6 Da, peak molecular weight is 8.694×10 5 Da, molecular weight distribution range is 7×10 5 Da-6×10 6 Da, its bonding structure connection modes include t-Glc(p), 6-Glc(p), 4-Glc(p), 3,4-Glc(p), 4,6-Gal(p), 3,4,6-Glc(p), and the relative molar ratio is 21.54:3.49:50.21:14.59:8.38:1.
78.
10. The use of the alcohol-soluble neutral polysaccharide of Miscanthus puerariae as claimed in claim 8 or 9, characterized in that: Application as an additive in wine.