Preparation method and application of anti-fatigue polygonatum oligosaccharide

Abstract

The present invention belongs to the field of plant oligosaccharides, and specifically relates to a preparation method and application of anti-fatigue polygonatum oligosaccharide, wherein a water extraction and alcohol precipitation process is used to obtain a small molecule clear paste of polygonatum sibiricum, and then a macroporous resin is used to enrich the oligosaccharide components in the clear paste, and the type of the macroporous adsorption resin and its process parameters are optimized to obtain polygonatum oligosaccharide. The polygonatum oligosaccharide prepared by the present invention can improve fatigue from various aspects, can be applied to anti-fatigue preparations, and can be used in combination with other nutrients to produce a synergistic effect.

Description

Technical Field

[0001] The invention belongs to the field of plant oligosaccharides, and particularly relates to a preparation method and application of anti-fatigue polygonatum oligosaccharides. Background Art

[0002] With the accelerated pace of life, fatigue has become an important factor affecting people's quality of life. Fatigue is the main manifestation of sub-health, a pre-disease state and pathogenic signal, which can lead to a series of physiological changes in the body, such as sleep disorders, endocrine disorders, metabolic disorders and immune dysfunction. If long-term fatigue is not relieved in time, it will lead to aging, anxiety, depression, cancer, Parkinson's disease and other diseases, seriously affecting the patient's life and work. Therefore, it has attracted widespread attention from scholars at home and abroad, and the development of anti-fatigue drugs has also become a research focus.

[0003] Traditional Chinese medicine carbohydrate compounds are an important anti-fatigue active substance, including polysaccharides and oligosaccharides. Carbohydrate compounds are commonly found in traditional Chinese medicines and are of high content and complex types. However, due to the limitations of the clinical use of traditional Chinese medicines, carbohydrate components are easily overlooked and lost during the preparation of traditional Chinese medicines. However, with the development of science and technology and the promotion and application of new technologies and new ideas in the research of traditional Chinese medicine, it has been found that traditional Chinese medicine carbohydrates and their derivatives have a variety of biological activities, such as anti-fatigue, immune enhancement, anti-oxidation, protection of organs and nerves, and improvement of intestinal function.

[0004] Oligosaccharides, also known as oligosaccharides, are short-chain sugars composed of monosaccharides and have a variety of biological activities. In recent years, studies have found that oligosaccharides are widely present in medicinal plants. They are not only used as energy storage components, but also play a key role in the treatment of various diseases. Studies have found that the carbohydrate components in Polygonatum sibiricum are the main active ingredients of Polygonatum sibiricum, and Polygonatum sibiricum polysaccharides and oligosaccharides generated by hydrolysis also have strong biological activities. Polygonatum sibiricum oligosaccharides have antiviral effects and enhance the immune function of mice. It has also been reported that a single fructose, glucose and sucrose mixture in Polygonatum sibiricum has blood-tonifying activity. Oligosaccharides are divided into naturally formed oligosaccharides and intermediate products of polysaccharide hydrolysis. Among them, disaccharides are the most common. Common oligosaccharides include maltooligosaccharides, fructooligosaccharides, mannooligosaccharides, xylooligosaccharides, chitosan oligosaccharides, and cello-oligosaccharides. Studies have shown that oligosaccharides can be quickly absorbed by the human body and quickly restore human liver glycogen after exercise. They have the characteristics of easy digestion, low sweetness, and low osmotic pressure, and have the functions of extending energy supply time, enhancing body endurance, and resisting fatigue. After excessive physical exertion or long-term intense exercise, the human body is prone to dehydration, low blood sugar, high body temperature, and a series of physiological changes and symptoms such as affected muscle nerve conduction and brain dysfunction. Eating oligosaccharides can not only maintain blood sugar levels and reduce the production of blood lactic acid, but also ensure insulin balance. Summary of the invention

[0005] The present invention mainly provides an anti-fatigue polygonatum oligosaccharide, an index method of the oligosaccharide, and the application of the oligosaccharide in anti-fatigue drugs, health products, food or beverages. The specific scheme is as follows:

[0006] An anti-fatigue polygonatum oligosaccharide, composed of 4.5±0.05% glucose, 73.1±

[0007] It is composed of 0.69% fructose, 17.8±0.19% sucrose and 1.8±0.03% melibiose.

[0008] A method for preparing the above-mentioned anti-fatigue polygonatum oligosaccharide comprises extracting polygonatum sibiricum slices with water and precipitating them with alcohol to obtain a small molecule clear paste, freeze-drying the small molecule clear paste to obtain a freeze-dried powder; and enriching the oligosaccharides in the freeze-dried powder with a macroporous resin to obtain polygonatum oligosaccharide.

[0009] Furthermore, the macroporous resin is one of D-101, HPD-100, HPD-400, NKA-9 or S-8 resin.

[0010] Furthermore, the preparation steps of the small molecule clear paste include: taking 1 kg of Polygonatum multiflorum slices, crushing them, passing through a 4-mesh sieve, drying at 50°C for 5 hours, adding water with a solid-liquid ratio of 1:15 to soak for 3 hours, boiling and extracting at 100°C for 2 times, and concentrating the liquid under reduced pressure. Take the concentrated liquid, add 95% ethanol to an alcohol content of 70%, place it at 4°C overnight, filter, take the filtrate, and concentrate the filtrate to obtain the small molecule clear paste.

[0011] Further, the oligosaccharide enrichment in the lyophilized powder using a macroporous resin comprises the following steps:

[0012] (1) pretreating the macroporous resin to obtain a pretreated resin;

[0013] (2) The freeze-dried powder is dissolved in water to obtain a solution of Polygonatum odoratum extract. The pretreated resin is added with a solution of Polygonatum odoratum extract having a mass concentration of 5 to 20 mg / mL, and static adsorption is performed for 12 to 48 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum odoratum oligosaccharide.

[0014] Furthermore, the mass ratio of the freeze-dried powder to the pretreated resin is 1:

[0015] (15~25).

[0016] Furthermore, the pretreatment of the macroporous resin includes the following steps: fully swelling the macroporous resin; then, washing the resin with ethanol for multiple times until the washing liquid is added to a test tube containing three times the amount of water and no longer becomes turbid; finally, thoroughly washing the resin with water until all ethanol taste is removed.

[0017] Furthermore, the sufficient swelling is to immerse the macroporous resin in 95% ethanol for 24 hours.

[0018] A use of the above-mentioned anti-fatigue polygonatum oligosaccharide in the preparation of anti-fatigue medicines, health products, foods or beverages.

[0019] Further, it is used in combination with nutrients.

[0020] By adopting the above scheme, the method of the present invention has the following advantages:

[0021] 1. The appearance of the polygonatum oligosaccharide obtained by the preparation method of the present invention is a white flocculent powder, which is significantly different from that before enrichment and does not contain the linear straight-chain inulin-type oligosaccharide mainly present in the polygonatum medicinal material.

[0022] 2. The Polygonatum oligosaccharide of the present invention can improve chronic fatigue of mice in multiple aspects. It can significantly enhance the climbing time, running endurance and weighted swimming time of mice, which directly reflects the effect of Polygonatum oligosaccharide on improving chronic fatigue.

[0023] 3. The single fructose, glucose and sucrose components in the Polygonatum oligosaccharide of the present invention can play a blood tonic role when mixed and used together. In addition, they can also be used as nutritional elements to enhance the glycogen content of the liver and muscles, thereby improving sports endurance and reducing the occurrence of sports fatigue in the body.

[0024] 4. The anti-fatigue effect of the combined use of Polygonatum oligosaccharide and nutrients of the present invention is better than that of the single use of nutrients or Polygonatum oligosaccharide, and the anti-fatigue effect when used in combination is better than that of compound nutrients, indicating that the combined use of Polygonatum oligosaccharide and nutrients can play a synergistic role.

[0025] 5. The combination of the polygonatum oligosaccharide of the present invention and nutrients can increase the immune capacity of immune organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the HPLC spectrum of the product of Example 1 and the standard substance (a. Example 1, b. fructose, c. glucose, d. sucrose, e. melibiose).

[0027] Figure 2 The figure shows the effect of the Polygonatum sibiricum oligosaccharide of Example 1 on the climbing time (a), the number of electric shocks (b) and the weighted swimming time (c) of chronic fatigue mice (n=8, data are expressed as mean±standard deviation).

[0028] Figure 3 The effects of the Polygonatum sibiricum oligosaccharide of Example 1 on blood urea nitrogen (a), blood lactic acid (b), liver glycogen (c) and muscle glycogen (d) in chronic fatigue mice (n=8, data are expressed as mean±standard deviation).

[0029] Figure 4The effect of the Polygonatum sibiricum oligosaccharide combined with nutrients in Example 1 on the climbing time (a), the number of electric shocks (b) and the weighted swimming time (c) of mice with exercise fatigue (n=8, data are expressed as mean±standard deviation).

[0030] Figure 5 The effects of the Polygonatum sibiricum oligosaccharide combined with nutrients in Example 1 on blood urea nitrogen (a), blood lactate (b), lactate dehydrogenase (c), liver glycogen (d) and muscle glycogen (e) in mice with exercise fatigue (n=8, data are expressed as mean±standard deviation). DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0032] Embodiment 1:

[0033] (1) Take 1 kg of Polygonatum multiflorum slices, crush them, pass through a 4-mesh sieve, dry at 50°C for 5 hours, add water at a solid-liquid ratio of 1:15 and soak for 3 hours, boil and extract twice at 100°C, and concentrate the liquid under reduced pressure. Take the concentrated liquid, add 95% ethanol to an alcohol content of 70%, place it at 4°C overnight, filter, take the filtrate, and concentrate the filtrate to obtain a small molecule clear paste; freeze-dry the small molecule clear paste to obtain a freeze-dried powder;

[0034] (2) Soak the NKA-9 macroporous resin in 95% ethanol for 24 hours to fully swell; then, wash the resin with ethanol several times until the washing solution is no longer turbid when added to a test tube containing three times the amount of water; finally, wash the resin thoroughly with water until all ethanol smell is removed;

[0035] (3) The lyophilized powder is dissolved in water to obtain a solution of Polygonatum odoratum paste. The pretreated resin with a mass 20 times that of the lyophilized powder is added with a solution of Polygonatum odoratum paste with a mass concentration of 20 mg / mL. The resin is statically adsorbed for 12 h. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum odoratum oligosaccharide.

[0036] Embodiment 2:

[0037] The difference from Example 1 is that step (2) uses D-101 macroporous resin.

[0038] Embodiment 3:

[0039] The difference from Example 1 is that HPD-100 macroporous resin is used in step (2).

[0040] Embodiment 4:

[0041] The difference from Example 1 is that HPD-400 macroporous resin is used in step (2).

[0042] Embodiment 5:

[0043] The difference from Example 1 is that S-8 macroporous resin is used in step (2).

[0044] Embodiment 6:

[0045] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 15 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 5 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is performed for 12 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0046] Embodiment 7:

[0047] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 15 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 10 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is carried out for 24 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0048] Embodiment 8:

[0049] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 15 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 20 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is performed for 48 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0050] Embodiment 9:

[0051] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 20 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 5 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is carried out for 24 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0052] Embodiment 10:

[0053] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum odoratum paste, 20 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 10 mg / mL of Polygonatum odoratum paste solution, and static adsorption is performed for 48 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum odoratum oligosaccharide.

[0054] Embodiment 11:

[0055] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 25 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 5 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is performed for 48 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0056] Embodiment 12:

[0057] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 25 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 10 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is performed for 12 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0058] Embodiment 13:

[0059] The difference from Example 1 is that: (3) the lyophilized powder is dissolved in water to obtain a solution of Polygonatum sibiricum paste, 25 times the mass of the lyophilized powder is taken, and the pretreated resin is added with a mass concentration of 20 mg / mL of Polygonatum sibiricum paste solution, and static adsorption is carried out for 24 hours. The fully saturated resin is filtered to dryness, the sugar-enriched filtrate is collected, and the filtrate is concentrated and dried to obtain Polygonatum sibiricum oligosaccharide.

[0060] Fructose was used as a standard to determine the total sugar content in the oligosaccharides obtained in each example, and the yield was calculated. The results are shown in Table 1 below.

[0061] Table 1:

[0062] Total sugar (Fru) content / % Yield / % Example 1 90.48 66.47 Example 2 51.64 61.52 Example 3 63.57 68.59 Example 4 59.64 62.40 Example 5 64.07 43.85 Example 6 90.58 53.71 Example 7 87.13 57.97 Example 8 82.15 54.32 Example 9 88.65 65.33 Example 10 97.59 43.52 Embodiment 11 92.01 50.03 Example 12 86.52 40.95 Embodiment 13 99.49 54.81

[0063] It can be seen from Table 1 above that by adopting the preparation method of the present invention, the total sugar content of the product is high and the yield is also high.

[0064] Example sample test:

[0065] HPLC-ELSD qualitative analysis: Take 10 mg of Polygonatum oligosaccharide of Example 1 in a 15 mL centrifuge tube, add 10 mL of water to completely dissolve it, and filter it through a 0.22 μm water membrane for later use. Take glucose, fructose, sucrose and melibiose standards to prepare 1 mg / mL reference solution for later use. Chromatographic conditions: XBridge BEHAmide (4.6×250 mm), mobile phase is acetonitrile (A)-water (B), isocratic elution (A: B=70:30), column temperature 30°C, flow rate 1.0 mL·min -1 , injection volume 20 μL, evaporative light scattering (ELSD) detector, evaporation temperature 90 °C, gas flow rate 1.6 L·min -1 The relative content of each component is shown in Table 2 below.

[0066] Table 2:

[0067] Small molecule clear ointment freeze-dried powder Example 1 Product Element Relative content% Relative content% glucose 1.8±0.01 4.5±0.05 fructose 69.6±0.50 73.1±0.69 sucrose 13.8±0.10 17.8±0.19 Melibiose 1.6±0.08 1.8±0.03

[0068] From the above table 2 and Figure 1 It can be seen that the sample of Example 1 contains a large amount of fructose and sucrose, a small amount of glucose and melibiose, and no oligosaccharides with relatively large molecular weights have been identified, nor have inulin-type oligosaccharides been found. It may be that the selection of conditions such as temperature and time during the extraction process causes some oligosaccharides with relatively large molecular weights to be hydrolyzed into monosaccharides.

[0069] Single Polygonatum oligosaccharide mouse experiment:

[0070] ICR male healthy mice (weighing 18-22 g) were randomly divided into 6 groups after one week of adaptive feeding, with 8 mice in each group, including blank control group (CG), model group (MG), positive drug group (ginseng extract, 500 mg / kg, PG), low-dose group of Polygonatum sibiricum oligosaccharide of Example 1 (500 mg / kg, HJOF-L), medium-dose group (1000 mg / kg, HJOF-M) and high-dose group (1500 mg / kg, HJOF-H).

[0071] Each group was given drugs by gavage at a volume of 10 mL / kg every day for 21 consecutive days. The CG and MG groups were gavaged with distilled water. Except for the CG group, the other five groups were intraperitoneally injected with LPS (4 mg / kg) 1 hour before administration on the 14th, 17th, and 20th days to create an LPS-induced chronic fatigue mouse model. The CG group was injected with an equal dose of normal saline. On the 20th day, the mice were fasted for 12 hours, weighed before gavage every day, and the mental state of the mice was recorded.

[0072] 30 minutes after administration on the 15th day, the mice were placed on a vertically fixed plexiglass rod with a diameter of 8 mm and a length of 25 cm, which put the muscles of the mice in a static tension state. Subsequently, the time it took for the mice to fall from the glass rod due to muscle fatigue was recorded.

[0073] On day 18, 30 minutes after drug administration, the mice were placed on a treadmill. First, the mice were properly trained to ensure that they understood how to escape from the electrified area. Subsequently, the speed of the treadmill was set to 20r / min and the current was adjusted to the highest level. Next, the number of times the mice were shocked within 5 minutes was recorded to evaluate their reaction and endurance performance.

[0074] 30 minutes after the last administration, the tails of mice in each group were tied with a lead sheet equivalent to 7% of their body weight. Then, the mice were placed in a swimming box with a water depth of 30 cm and a water temperature maintained at 25±1°C in order to perform a weighted swimming test.

[0075] After swimming, blood was collected and mice were killed immediately. After blood collection, it was placed in an EP tube containing 0.5% sodium heparin and immediately centrifuged at 12000rpm / min for 15min at 4℃. The supernatant was collected and transferred to a new 1.5mL EP tube and stored in a -80℃ refrigerator for subsequent detection of relevant biochemical indicators, including blood urea nitrogen and blood lactate. The liver and muscle of mice were obtained by dissection, and the liver and muscle tissues were used for glycogen content determination. GraphPad Prism 8 software was used for statistical analysis, and the experimental data were expressed as mean ± standard deviation. ) indicated that the groups were compared using one-way analysis of variance, and pairwise comparisons were verified using LSD tests, with p < 0.05 indicating statistical significance.

[0076] The results are as follows:

[0077] After LPS modeling, mice showed reduced appetite, lethargy, reduced activity, weight loss, and rough hair. Compared with the model group, the mice in the group receiving Polygonatum sibiricum oligosaccharide were in relatively better condition. Figure 2 As shown in Figure 2-a, compared with the blank group, the time for mice in the model group to climb the pole was significantly reduced; compared with the model group, the time for mice in the medium and high dose groups of Polygonatum sibiricum oligosaccharide to climb the pole was significantly prolonged. Figure 2 As shown in Figure 2-b, compared with the blank group, the number of electric shocks in the model group mice while running increased significantly, indicating that the mice were in a state of obvious fatigue; compared with the model group, the number of electric shocks in the medium and high dose groups of Polygonatum sibiricum oligosaccharide mice while running decreased significantly. Figure 2 -c, compared with the blank group, the weighted swimming time of mice in the model group was significantly reduced; compared with the model group, the weighted swimming time of mice in the medium and high dose groups of Polygonatum sibiricum oligosaccharide was significantly reduced.

[0078] During exercise, the body produces metabolites such as blood urea nitrogen (BUN) and blood lactic acid (BLD), which reduce the body's exercise endurance. The more obvious the increase in BUN and BLA, the worse the body's adaptation to exercise endurance. Figure 3 -a and 3-b show that compared with the blank group, the BUN and BLD contents of the mice in the model group were significantly increased, and compared with the model group, the BUN and BLD contents of the mice in the medium and high dose groups of Polygonatum sibiricum oligosaccharide were significantly decreased, indicating that a certain dose of Polygonatum sibiricum oligosaccharide can reduce the body load and play an anti-fatigue role by clearing related metabolites during exercise.

[0079] Hepatic glycogen (HG) and muscle glycogen (MG) are the main forms of glycogen that provide energy to the body. Glycogen reserves are positively correlated with the body's anti-fatigue ability. The more glycogen reserves, the stronger the body's anti-fatigue ability. Figure 3 -c and 3-d show that compared with the blank group, the HG and MG contents in the model group were significantly reduced, indicating that in the chronic fatigue state, mice consumed more glycogen after exercise. The HG and MG contents in the medium and high dose groups of Polygonatum sibiricum oligosaccharide were significantly higher than those in the model group, indicating that in the chronic fatigue state, the mice in the drug-treated group consumed less glycogen after exercise.

[0080] Nutrient combination mouse experiment:

[0081] ICR male healthy mice (weight 18-22g) were randomly divided into 6 groups after one week of adaptive feeding, with 8 mice in each group, including blank control group (Control, CG), model group (Model, MG), positive drug group (compound nutrient, RB), single nutrient group (TA), polygonatum oligosaccharide group (HJOF) and polygonatum oligosaccharide combined with nutrient group (HJOF-TA). Among them, the single nutrient group used taurine; the formula of the compound nutrient group was: 4.4g / L taurine, 2.4g / L glucuronolactone, 0.32g / L caffeine, 0.08g / L vitamin B3, 0.02g / L vitamin B6, 0.02 vitamin B5, 0.02mg / L vitamin B12, 86g / L fructose and 21g / L glucose.

[0082] The CG and MG groups were gavaged with distilled water, the RB group was gavaged with a compound nutrient drink (50 mL / kg), and the TA group (1000 mg / kg), HJOF group (1000 mg / kg) and HJOF-TA group (1000 mg / kg, HJOF:TA=1:1) were gavaged with a volume of 10 mL / kg. On the 13th day, the mice were fasted for 12 h, weighed before gavage every day, and the mental state of the mice was recorded.

[0083] Except for the CG group, the other five groups of mice were subjected to a pole climbing test 30 minutes after drug administration on the 12th day; a running test was performed 30 minutes after drug administration on the 13th day; and a weighted swimming test was performed 30 minutes after the last drug administration. The specific operating steps were carried out by referring to the above-mentioned method of the single Polygonatum oligosaccharide mouse experiment for behavioral experiments and biochemical index determination.

[0084] The results are as follows:

[0085] After a period of administration, the mice in the compound nutrient group and each medication group had smoother hair and better mental state. Figure 4 As shown, the climbing time of mice in the compound nutrient group and the group treated with Polygonatum oligosaccharide combined with nutrients was significantly increased compared with the group not treated; compared with the single nutrient group and the Polygonatum oligosaccharide group, the climbing time of the group treated with Polygonatum oligosaccharide combined with nutrients was significantly increased, indicating that the combination of the two played a synergistic role, and the effect was better than that of the compound nutrient group. Compared with the model group, the number of running electric shocks of mice in each drug-treated group decreased, and the Polygonatum oligosaccharide combined with nutrients group had the most significant effect. Compared with the model group, the weighted swimming time of mice in each drug-treated group increased; compared with the compound nutrient group, the weighted swimming time of mice in the group treated with Polygonatum oligosaccharide combined with nutrients increased more significantly. It can be seen from behavioral experiments that the Polygonatum oligosaccharide combined with nutrients group has a significant anti-fatigue effect, and the effect is better than the use of either alone.

[0086] like Figure 5 As shown in the results, during exercise, proteins and other substances are degraded to generate BUN, and excessive accumulation will aggravate fatigue. Compared with the blank group, the plasma BUN content of mice in the model group increased significantly; compared with the model group, the plasma BUN content of mice in each drug-treated group was significantly reduced, and the effect of the group of Polygonatum oligosaccharide combined with nutrients was better than that of single use. When the body is hypoxic, glycogen provides energy to the body through the glycolysis pathway and produces metabolites LD, which accumulate in the body and change the microenvironment in the body, thereby aggravating fatigue. Compared with the blank group, the BLD content of mice in the model group increased, while the compound nutrient group and the group of Polygonatum oligosaccharide combined with nutrients could reduce the BLD content in the body. In addition, the lactate dehydrogenase activity in mice in the compound nutrient group and the group of Polygonatum oligosaccharide combined with nutrients increased significantly. During the exercise of the body, when oxygen is sufficient, glycogen is broken down into glucose to provide energy for the body. Compared with the model group, Polygonatum oligosaccharide combined with nutrients can significantly increase the liver glycogen content of mice, thereby enhancing the exercise endurance of mice. It can be seen that Polygonatum oligosaccharide combined with nutrients can relieve fatigue of the body by improving fatigue-related biochemical indicators.

[0087] It can be seen from the above experiments that the polygonatum oligosaccharide combined with nutrients of the present invention can improve the sports fatigue of mice in many aspects. It can significantly enhance the time of climbing poles, running endurance and weighted swimming time of mice, which directly reflects the effect of polygonatum oligosaccharide combined with nutrients in improving sports fatigue, and the effect is better than that of single use, indicating that the combination of the two can play a synergistic effect. Secondly, polygonatum oligosaccharide combined with nutrients can activate immune organs and enhance the body's immune ability, thereby producing a certain anti-fatigue effect. In addition, polygonatum oligosaccharide combined with nutrients can also enhance the glycogen content of liver and muscles, increase the activity of lactate dehydrogenase, and reduce the accumulation of lactic acid and urea nitrogen metabolites produced during fatigue.

[0088] For those skilled in the art, various other corresponding changes and deformations can be made according to the technical solutions and concepts described above, and all of these changes and deformations should fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing anti-fatigue Polygonatum oligosaccharide, characterized in that: The method comprises 4.5±0.05% glucose, 73.1±0.69% fructose, 17.8±0.19% sucrose and 1.8±0.03% melibiose in mass percentage; extracting the polygonatum cyrtonema slices with water and subjecting them to alcohol precipitation to obtain a small molecule clear paste, freeze-drying the small molecule clear paste to obtain a freeze-dried powder; and enriching the oligosaccharides in the freeze-dried powder with a macroporous resin to obtain polygonatum cyrtonema oligosaccharide; The macroporous resin is NKA-9; The preparation steps of the small molecule clear paste include: taking 1 kg of Polygonatum cyrtonema decoction pieces, crushing them, passing them through a 4-mesh sieve, drying them at 50°C for 5 hours, adding water with a solid-liquid ratio of 1:15 to soak them for 3 hours, boiling and extracting them twice at 100°C, and concentrating the medicinal liquid under reduced pressure; taking the concentrated medicinal liquid, adding 95% ethanol to an alcohol content of 70%, placing it at 4°C overnight, filtering, taking the filtrate, and concentrating the filtrate to obtain the small molecule clear paste.

2. The method for preparing the anti-fatigue polygonatum oligosaccharide according to claim 1, characterized in that: The oligosaccharide enrichment from the lyophilized powder using a macroporous resin includes the following steps: (1) pretreating the macroporous resin to obtain a pretreated resin; (2) Dissolve the freeze-dried powder in water to obtain a solution of Polygonatum odoratum extract. Take the pretreated resin and add a solution of Polygonatum odoratum extract with a mass concentration of 5-20 mg / mL. Perform static adsorption for 12-48 h. Filter the fully absorbed resin to dryness, collect the sugar-enriched filtrate, and concentrate the filtrate to dryness to obtain Polygonatum odoratum oligosaccharide.

3. The method for preparing the anti-fatigue polygonatum oligosaccharide according to claim 2, characterized in that: The mass ratio of the freeze-dried powder to the pretreated resin is 1:(15-25).

4. The method for preparing the anti-fatigue polygonatum oligosaccharide according to claim 2, characterized in that: The pretreatment of the macroporous resin comprises the following steps: fully swelling the macroporous resin; then, washing the resin with ethanol for multiple times until the washing liquid is added to a test tube containing three times the amount of water and no longer becomes turbid; finally, washing the resin thoroughly with water until all ethanol taste is removed.

5. The method for preparing the anti-fatigue polygonatum oligosaccharide according to claim 4, characterized in that: The sufficient swelling is achieved by immersing the macroporous resin in 95% ethanol for 24 hours.

6. Use of the anti-fatigue polygonatum oligosaccharide prepared by the method according to claim 1 in the preparation of anti-fatigue drugs, health products, foods or beverages.

7. Use of the anti-fatigue polygonatum oligosaccharide prepared according to the method of claim 6 in the preparation of anti-fatigue drugs, health products, foods or beverages, characterized in that: Use with nutrients.

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