Ultrahigh pressure assisted preparation method and application of black bean seed coat polysaccharide with anti-oxidation and anti-fatigue effects

Abstract

Black bean seed coats as byproducts are mostly abandoned or used as feed, and huge waste of resources is caused. Research finds that black bean seed coats contain rich active ingredients such as anthocyanin, haematochrome, polyphenol and dietary fiber, but extraction of polysaccharide in the black bean seed coats and oxidation resistance of the polysaccharide are rarely reported. The black bean seed coat polysaccharide is extracted, the chemical structure, biological activity and related molecular mechanism of the black bean seed coat polysaccharide are explored, industry and standard systems related to a black bean processing supply chain are perfected, the problem of insufficient research and development design capability is solved, and the core competitiveness of products is improved. A certain theoretical basis and a reliable scientific basis are provided for future development and application of the black bean seed coat polysaccharide, and development and application of related technologies are promoted. Along with deep research, black bean polysaccharide is expected to become an important functional food raw material and health care product component.

Description

Technical Field

[0001] The present invention relates to a plant polysaccharide, and more particularly to a black soybean seed coat polysaccharide with antioxidant and anti-fatigue effects and a preparation method thereof. Background Art

[0002] In modern society, the dual pressures of work and life, combined with the impact of a complex and ever-changing social environment, have led to approximately 70% of the population being in a sub-healthy state. Sub-health is a state between disease and health, specifically manifested as a dual reduction in psychological and physiological functions. Common characteristics include weakened vitality, slow reaction, and reduced adaptability. In daily life, "fatigue" has become the most common manifestation of the sub-healthy state. Fatigue can either be a symptom accompanying various diseases such as iron-deficiency anemia, malignant tumors, and sclerosis syndrome, or it can exist as a disease alone. With the rapid development of the times, fatigue is quietly becoming an "invisible killer" threatening human health. The World Health Organization has listed fatigue as one of the important factors endangering human health in the 21st century, which has also attracted extensive attention from all walks of life regarding the research on fatigue. Relevant surveys show that more than half of the people often feel fatigued, and more than one-third of them clearly state that fatigue has led to a decline in their quality of life and a significant reduction in work efficiency.

[0003] More and more evidence shows that polysaccharides extracted from plants have the advantages of low toxicity and high efficiency in anti-fatigue. For example, epimedium polysaccharide has anti-fatigue effects; polygonatum polysaccharide delays exercise-induced fatigue by regulating the content of DA and 5-HT in the brains of mice, and schisandra polysaccharide significantly improves the anti-fatigue and hypoxia tolerance of mice; litchi polysaccharide can increase glycogen reserves in mice, reduce the accumulation of urea nitrogen and lactic acid in mice after exercise, and has good anti-fatigue effects.

[0004] Black soybean (Glycine max (L.) merr) belongs to the legume family, is of the Papilionaceae family, and is also called black bean, winter bean, and black soybean. Black soybean contains rich nutrients such as protein, vitamins, crude fiber, and short peptides. Its nutritional value is both balanced and comprehensive. As people pay more and more attention to black soybean, different deep processing methods have been carried out on black soybean, and various types of black soybean products have been developed and produced. During the large-scale processing and oil extraction of black soybean, the black soybean seed coat is mostly discarded or used as feed as a by-product, resulting in a huge waste of resources. Research has found that the black soybean seed coat contains rich active ingredients, such as anthocyanins, red pigments, polyphenols, dietary fiber, etc. However, there are few reports on the extraction and antioxidant properties of polysaccharides in the black soybean seed coat. This study aims to isolate and purify the crude polysaccharide from the black soybean seed coat, find a homogeneous polysaccharide component, clarify the in vitro antioxidant effect of the crude polysaccharide, and further study the anti-fatigue mechanism of the crude polysaccharide, providing a scientific basis for the application of black soybean seed coat polysaccharide in anti-fatigue. Summary of the Invention

[0005] Objective of the invention: The objective of the present invention is to invent a preparation method of black bean seed coat polysaccharide, clarify its anti-fatigue effect, deeply study the homogeneous polysaccharide components, and develop the application of black bean seed coat polysaccharide in the preparation of anti-fatigue health products or drugs.

[0006] Technical solution:

[0007] 1. Preparation of crude polysaccharide from black bean seed coat

[0008] Take the dried black bean seed coat, pulverize it, add distilled water according to a certain solid-liquid ratio and mix evenly. Add 3% cellulase to the mixture, carry out water bath at 50 °C for 2 h, extract at 300 MPa, concentrate, and freeze-dry to obtain the crude polysaccharide of black bean seed coat.

[0009] 2. Purification of black bean seed coat polysaccharide

[0010] The purity of polysaccharide directly affects the quality and effect of polysaccharide. Therefore, the black bean seed coat polysaccharide is further purified. The specific process is as follows: The purity of polysaccharide directly affects the quality and effect of polysaccharide. Therefore, the black bean seed coat polysaccharide is further purified. The specific process is as follows:

[0011] (1) Decoloration

[0012] Use macroporous adsorption resin (D101 type) to decolorize the above crude polysaccharide. Soak the macroporous adsorption resin in 95% ethanol for 24 h, pour it into a chromatography column (10 cm × 90 cm), and elute and balance with distilled water for 12 h. Dissolve the crude polysaccharide in water, load the sample and adsorb overnight, elute with distilled water, concentrate the eluate under reduced pressure, and freeze-dry to obtain the decolorized crude polysaccharide.

[0013] (2) Deproteinization

[0014] Take the decolorized crude polysaccharide, dissolve it in distilled water, place it in a separatory funnel, add 1 / 2 volume of Sevag reagent (reagent ratio: chloroform: n-butanol = 5:1) of the sugar solution, then shake vigorously for 5 min, let it stand and separate layers, centrifuge (3000 r / min), collect the supernatant, repeat the operation until no denatured protein layer appears, concentrate under reduced pressure, add 10 times the amount of 95% ethanol, let it stand at 4 °C overnight, centrifuge, and freeze-dry to obtain the deproteinized black bean seed coat polysaccharide, named BSPS (Black bean seed coat polysaccharide, BSPS).

[0015] (3) Calculation of protein removal rate

[0016] Protein removal rate = (protein content before protein removal - protein content after protein removal) / protein content before protein removal * 100%.

[0017] 3. Determination of black bean seed coat polysaccharide content

[0018] The phenol-sulfuric acid method was used to detect the total sugar content in the prepared black bean seed coat polysaccharide, with glucose as the standard, and the 3,5-dinitrosalicylic acid method was used to determine the reducing sugar content in the prepared black bean seed coat polysaccharide, with glucuronic acid as the standard. Description of the Drawings Figure 1 Schematic diagram of the glucose standard curve provided by the present invention; Figure 2 Schematic diagram of the glucuronic acid standard curve provided by the present invention; Figure 3 Schematic diagram of the gradient elution curve of the crude polysaccharide from black bean seed coat through a DEAE-cellulose column provided by the present invention; Figure 4 Schematic diagram of the elution curve of polysaccharide fraction F-1 through a Sepharose CL-4B chromatographic column provided by the present invention; Figure 5 Schematic diagram of the elution curve of polysaccharide fraction F-2 through a Sepharose CL-4B chromatographic column provided by the present invention; Figure 6 Schematic diagram of the elution curve of polysaccharide fraction F-3 through a Sepharose CL-4B chromatographic column provided by the present invention; Figure 7 Schematic diagram of the molecular weight of the crude polysaccharide from black bean seed coat provided by the present invention; Figure 8 Schematic diagram of the determination of the DPPH scavenging ability of the black bean seed coat polysaccharide provided by the present invention; Figure 9 Schematic diagram of the determination of the hydroxyl radical scavenging ability of the black bean seed coat polysaccharide provided by the present invention; Figure 10 Schematic diagram of the determination of the reducing ability of the black bean seed coat polysaccharide provided by the present invention; Figure 11 Schematic diagram of the determination of the superoxide anion scavenging ability of the black bean seed coat polysaccharide provided by the present invention. Specific Embodiments

[0019] (1) Determination of the total sugar content in the prepared black bean seed coat polysaccharide by the phenol-sulfuric acid method

[0020] (1) Reagent preparation

[0021] ① Preparation of phenol solution: Take refined phenol and add distilled water to prepare a 6% phenol solution for standby.

[0022] ② Preparation of standard solution: Accurately weigh 0.5000 g of glucose, make up the volume to 100 mL in a volumetric flask, take 1 mL of the solution from it and make up the volume to 50 mL with water to obtain a 100 μg / mL standard solution.

[0023] (2) Preparation of Standard Curve

[0024] Using a micropipette, respectively take 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of the 100 μg / mL standard solution and make up the volume to 50 mL in a volumetric flask. Then take 1 mL from each and put them into test tubes. Add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid to each test tube, shake the test tubes, mix well, let stand, and measure the absorbance value at 490 nm using a 722-type ultraviolet spectrophotometer. Calculate the regression equation, as Figure 1 is a schematic structural diagram of the glucose standard curve provided by the present invention.

[0025] (3) Determination of the Total Sugar Content in Black Bean Seed Coat Polysaccharide

[0026] Prepare the test sample into a 200 μg / mL solution, then take 1 mL of the sample, and the other steps are the same as those for the preparation of the standard curve. After the detection, calculate the total sugar content in the black bean seed coat polysaccharide.

[0027] (II) Determination of the Reducing Sugar Content in the Prepared Black Bean Seed Coat Polysaccharide by the 3,5-Dinitrosalicylic Acid Method

[0028] (1) Preparation of Reagents

[0029] ① Preparation of m-hydroxybiphenyl: Weigh m-hydroxybiphenyl and dissolve it in a 5 mg / mL NaOH solution to make a mass concentration of 1.5 mg / mL, and store it in the dark at 4 °C.

[0030] ② Preparation of sodium tetraborate sulfuric acid solution: Take sodium tetraborate and make it into a 0.125 mol / L solution with concentrated sulfuric acid, and store it at room temperature.

[0031] ③ Preparation of glucuronic acid standard solution: Take 1 mg of galacturonic acid and mix it evenly with 10 mL of distilled water in an EP tube to obtain a DGal-A solution with a concentration of 0.1 mg / mL.

[0032] (2) Preparation of Glucuronic Acid Standard Curve

[0033] Using a micropipette, respectively take 0 μL, 5 μL, 10 μL, 20 μL, 30 μL, and 40 μL of the 0.1 mg / mL standard solution for dilution to obtain a concentration gradient of the solution. Drop 4 μL of sulfamic acid and 250 μL of concentrated sulfuric acid into the galacturonic acid solution, heat it in a water bath for 20 min, quickly cool it, and add 4 μL of m-hydroxybiphenyl. React for 15 min, then drop 200 μL of the solution into a 96-well plate and measure the OD value at a wavelength of 525 nm. Draw a standard curve with the abscissa being the D-GalA concentration (μg / mL) and the ordinate being the absorbance value, as Figure 2 is a schematic structural diagram of the glucuronic acid standard curve provided by the present invention.

[0034] (3) Determination of the content of polysaccharide uronic acid

[0035] Take 40 μL of the sample and make it into a 0.1 mg / mL sample solution by adding 60 μL of distilled water. The remaining steps are operated according to the standard curve steps. Do two parallel experiments and measure the absorbance at 525 nm. Substitute into the standard curve to obtain the average value to get the content of uronic acid.

[0036] (4) Calculation formula for the polysaccharide yield

[0037] The polysaccharide yield Y (%) of HEIDOUZHONGPI = (WE × (CT - CR)) / WP × 100 (1.2)

[0038] WE is the weight of the purified polysaccharide, WP is the weight of the black bean seed coat pretreatment sample used in each experiment, and CT and CR are the contents of total sugar and reducing sugar in the purified polysaccharide respectively.

[0039] 4. Separation and purification of polysaccharides

[0040] (1) Use an anion exchange resin DEAE - cellulose column to separate and purify BSPS. After soaking the DEAE - cellulose in water, soak it in 0.5 M NaOH for 1 h, then wash it with dH2O until neutral, then soak it in 0.5 M HCl for 1 h, wash it with dH2O until neutral, and then load the column (remove air bubbles by vacuum before loading the column). After loading the column, balance the cellulose column with 2 volumes of 2 M NaCl and dH2O at a flow rate of 2 mL / min. Weigh 500 μg of BSPS and dissolve it in 25 mL of dH2O. After the sample is dissolved, centrifuge and take the supernatant and slowly add it to the cellulose column. Use dH2O to elute to obtain uncharged neutral sugar (BSPS - N). After the elution is completed, use a gradient mixer to pump in 0 - 1 M NaCl for gradient elution to obtain charged acidic sugar (BSPS - A) at a flow rate of 1 mL / min, and collect it with an automatic collector for 5 tubes for 5 min. Use the phenol - sulfuric acid method to measure the absorbance of the collected solution and draw a gradient elution curve to determine the appropriate elution salt concentration for acidic sugar. Weigh 2 g of BSPS, add distilled water to prepare a 20 mg / mL polysaccharide solution, centrifuge at 4500 rpm for 20 min at room temperature, and discard the precipitate. After pretreatment with 0.5 M NaOH and 0.5 M HCl, wash the DEAE - cellulose with water until neutral, filter to remove air bubbles and then load the column. After installing the DEAE - cellulose column device, balance it, load the sample, elute the neutral sugar of the black bean seed coat with distilled water, and then elute the acidic sugar of the black bean seed coat with the salt concentration obtained from the linear elution curve. After vacuum rotary evaporation, dialysis, and freeze - drying of the collected polysaccharide eluate, where F - 1 is the neutral polysaccharide component and F - 2 and F - 3 are acidic polysaccharides, as Figure 3 It is a schematic diagram of the gradient elution curve structure of the crude polysaccharide from black bean seed coat provided by the present invention through the DEAE - cellulose column.

[0041] (2) The polysaccharide components F-1, F-2, and F-3 were eluted through a Sepharose CL-4B chromatographic column to obtain single components. The black bean seed coat polysaccharide was prepared into a 20 mg / mL solution, loaded onto the column, and sampled automatically. It was eluted with deionized water and 0.1, 0.2, and 0.3 mol / L sodium chloride. The absorbance value of the polysaccharide was detected at 490 nm by the phenol-sulfuric acid method, and an elution distribution curve was plotted. The eluate was collected in segments, combined, dialyzed, concentrated, and freeze-dried for 48 h to obtain 4 kinds of white and shiny homogeneous black bean seed coat polysaccharides, which were named according to the Latin name of the black bean seed coat polysaccharide as BSPS-1, BSPS-2, and BSPA-3, as Figure 4 This is a schematic diagram of the elution curve structure of the polysaccharide component F-1 provided by the present invention through a Sepharose CL-4B chromatographic column. Figure 5 This is a schematic diagram of the elution curve structure of the polysaccharide component F-2 provided by the present invention through a Sepharose CL-4B chromatographic column. Figure 6 This is a schematic diagram of the elution curve structure of the polysaccharide component F-3 provided by the present invention through a Sepharose CL-4B chromatographic column.

[0042] The homogeneity and molecular weight of the polysaccharide were analyzed by high performance liquid gel permeation chromatography (HPGPC). The high performance liquid chromatography system was equipped with a refractive index detector (RID) and a Superdex 200 10 / 300 GL gel filtration chromatographic column. 1 mg of the polysaccharide sample was weighed and placed in a 1.5 mL microcentrifuge EP tube, and 200 μL of filtered ultrapure water (ddH2O) was added to completely dissolve the polysaccharide sample. It was filtered through a 0.22 μm inorganic phase filter membrane and then subjected to HPGPC detection.

[0043] HPGPC analysis: A Shimadzu HPLC system (equipped with a CTO-20A pump and a RID-10A refractive index detector), a TSK-gel G-3000PWXL chromatographic column (7.8 mm × 300 mm), with the mobile phase being NaCl (0.15 mol / L)-ddH2O, a flow rate of 0.6 mL / min, an injection volume of 20 μL, and a detection wavelength of 245 nm. The molecular weight of the crude black bean seed coat polysaccharide is as Figure 7 This is a schematic diagram of the molecular weight structure of the crude black bean seed coat polysaccharide provided by the present invention.

[0042] Study on the antioxidant activity of black bean seed coat polysaccharide

[0043] Determination of DPPH free radical scavenging ability

[0044] Prepare sample solutions at 5 gradients of 0.5, 1, 2, 5, and 10 mg / mL. Add 0.6 mL of the polysaccharide sample solution to 2.4 mL of 0.1 mM DPPH solution, and carry out the reaction for 30 min in the dark. Select Vc as the positive control group, measure the absorbance at a wavelength of 517 nm, and take the average value of 3 parallel experiments. The formula is as follows:

[0045] In the formula: A0 is the absorbance of dH2O + DPPH; A1 is the absorbance of the sample solution + DPPH; A2 is the absorbance of the sample solution + absolute ethanol.

[0046] Experimental results

[0047] By measuring the scavenging ability of BSPS on DPPH radicals, the antioxidant effect of BSPS was analyzed. As can be seen from Figure 8 When the concentration of BSPS increased from 0.5 mg / mL to 10 mg / mL, the scavenging ability of DPPH gradually increased, and overall it had good reducing ability. When its concentration reached the highest, the scavenging ability could reach 98.68%, which was infinitely close to Vc. It indicated that BSPS had good scavenging ability on DPPH radicals. By measuring the scavenging ability of BSPS on hydroxyl radicals, the antioxidant effect of BSPS was analyzed. The results are as Figure 8 This is the schematic diagram of the measurement structure of the DPPH scavenging ability of the black soybean seed coat polysaccharide provided by the present invention. As the concentration of BSPS increased from 0.5 mg / mL to 10 mg / mL, the hydroxyl radical scavenging rate gradually increased, but even when the concentration reached the highest, the scavenging rate was only 82.6%, which did not reach the lowest scavenging rate of Vc.

[0048] Measurement of hydroxyl radical scavenging ability

[0049] Prepare sample solutions at 5 gradients of 0.5, 1, 2, 5, and 10 mg / mL. Take 500 μL of sample solutions with different concentrations and mix them with 1 mL of FeSO4, H2O2, and salicylic acid, and carry out the reaction for 30 min under dark conditions at room temperature of 25°C. Select Vc as the positive control group, measure the absorbance at a wavelength of 530 nm, and take the average value of 3 parallel experiments. The formula is as follows:

[0050] In the formula: A0 is the absorbance value of 500 μL dH2O + 1 mL salicylic acid + 1 mL H2O2; A1 is the absorbance value of 500 μL sample solution + 1 mL salicylic acid + 1 mL H2O2; A2 is the absorbance value of 500 μL sample solution + 1 mL salicylic acid + 1 mL dH2O

[0051] Experimental results

[0052] The antioxidant effect of BSPS was analyzed by measuring its scavenging ability against hydroxyl radicals. The results are as follows Figure 9 This is a schematic diagram of the measurement structure of the hydroxyl radical scavenging ability of the black bean seed coat polysaccharide provided by the present invention. As the concentration of BSPS increased from 0.5 mg / mL to 10 mg / mL, the hydroxyl radical scavenging rate gradually increased. However, even when the concentration reached the highest level, the scavenging rate was only 82.6%, which did not reach the lowest scavenging rate of Vc.

[0053] Determination of reducing power

[0054] Polysaccharide solutions with 5 gradient concentrations of 0.5, 1, 2, 5, and 10 mg / mL were prepared respectively. 1 mL of the sample was added to 2.5 mL of PBS and potassium ferricyanide solution and soaked at 50 °C for 20 min. 1 mL of 10% TCA mixture was stirred evenly and centrifuged at 8000 rpm / min for 5 min. The supernatant was taken and dH2O and FeCl3 solution were added in sequence, left for 10 min, and the absorbance was measured at a wavelength of 700 nm. Vc was selected as the positive control group. Each sample was subjected to 2 parallel experiments and the average value was taken finally. The formula is as follows:

[0055] Experimental results

[0056] The antioxidant effect of BSPS was studied by measuring its reducing power against ferric ions. The results Figure 10 This is a schematic diagram of the measurement structure of the reducing power of the black bean seed coat polysaccharide provided by the present invention. As the concentration of BSPS increased, the ferric ion scavenging rate gradually increased. When the concentration rose to 10 mg / mL, compared with the reducing power of Vc against Fe3+, the reducing power of BSPS was relatively poor

[0057] In the formula: A0 is the absorbance value of the water-added control; A1 is the absorbance value of the polysaccharide sample solution.

[0058] Determination of superoxide anion radical scavenging ability

[0059] Sample solutions with 5 gradient concentrations of 0.5, 1, 2, 5, and 10 mg / mL were prepared. Different concentrations of the sample solution were added to 50 μL of NBT, NADH, and PMS, and the reaction was carried out for 30 min under light shielding. The absorbance was measured at a wavelength of 570 nm. Vc was selected as the positive control group. Each analyte was subjected to 3 parallel experiments and the average value was taken finally. The formula is as follows:

[0060] Wherein: A0 is the absorbance value of 50 μL dH2O + 50 μL NBT + 50 μL NADH + 50 μL PMS; A1 is the absorbance value of 50 μL sample solution + 50 μL NBT + 50 μL NADH + 50 μL PMS; A2 is the absorbance value of 50 μL sample solution + 50 μL NBT + 50 μL NADH + 50 μL dH2O.

[0061] Experimental results

[0062] The scavenging effect of BSPS on superoxide anion free radicals was studied, and its antioxidant activity was analyzed. The results Figure 11 This is a schematic diagram of the determination structure of the superoxide anion scavenging ability of black bean seed coat polysaccharide provided by the present invention. As the concentration of BSPS increases, its scavenging ability for superoxide anion also increases. When the concentration is 10 mg / mL, the scavenging rate can reach 95.4%. It shows that BSPS has good ability to scavenge superoxide anion

[0063] Experimental study on the anti-fatigue effect of black bean seed coat polysaccharide

[0064] The anti-fatigue effect of BSPS was studied using an exercise-induced fatigue model.

[0065] SPF-grade Kunming mice (20 ± 2 g), 6 - 8 weeks old, half male and half female, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., and its license number is SCXK(Xiang)2016 - 0002. Coenzyme Q10 (batch number: 140611): Eisai Pharmaceutical Co., Ltd.; BSPS (self-made).

[0066] Preparation of drugs and reagents:

[0067] (1) Preparation of BSPS

[0068] A certain amount of BSPSP was taken, and the high-dose (200 mg / kg), medium-dose (100 mg / kg), and low-dose (50 mg / kg) groups of each component were prepared into drug solutions with concentrations of 1%, 0.5%, and 0.25% respectively according to the dosing volume (0.2 mL / 10 g body weight), and used immediately after preparation.

[0069] Establishment of an exercise-induced fatigue model:

[0070] The exercise-induced fatigue model uses weight-bearing swimming as the exercise method. Swimming is an instinctive exercise method for mice, and the exercise ability of mice can be fully exerted through appropriate temperature and a certain space. Inject water into the swimming tank (50 cm × 40 cm × 40 cm), and the water depth should be such that the mice cannot rest with their tails on the bottom of the swimming tank. Place a mercury thermometer with a measuring range of 0-100 °C in the swimming tank to control the water temperature at 22 ± 2 °C. Place each group of mice in the swimming tank for weight-bearing swimming experiments. Fatigue is likely to occur after continuous exercise. The judgment criterion for exercise-induced fatigue is that the mice swim until exhausted, that is, the mouse's head sinks into the water for 8 seconds and no longer surfaces. The mice are trained in weight-bearing swimming every day. After the modeling is completed, they are returned to the cage to rest. Clean the swimming tank and replenish the feed in the cage. Continue the modeling for 14 days.

[0071] Grouping and drug administration:

[0072] Select 150 Kunming mice, with a temperature of 22 ± 2 °C and a humidity of 50-70%. After raising for 3 days to adapt to the environment, the mice are randomly divided into 15 groups, with 10 mice in each group, and the treatments are as follows:

[0073] Table 1 Grouping and drug administration of exercise-induced fatigue mice

[0074]

[0075] Changes in body weight and general conditions

[0076] Compared with the normal group, the body weight of the mice in the model group was significantly reduced (P<0.01). Compared with the model group, there were no significant changes in the body weight of each drug administration group, and there was no significant difference. In addition, compared with the normal group, the mice in the model group showed listlessness, dry and dull hair, less activity, lighter colors of the auricles and tails, reduced food intake, and loose stools. And each dose group of black soybean seed coat polysaccharide could effectively improve the above conditions.

[0077]

[0078] Note: Compared with the normal group, #P<0.05, ##P<0.01;

[0079] 4. Experimental methods

[0080] All mice were given drugs by gavage for 12 consecutive days. Thirty minutes after the last drug administration, the mice swam without load. In four large stainless-steel barrels with a diameter of 80 cm, the water depth was 45 cm. Hot water was continuously added to keep the water temperature at 25 ± 1 °C. At the same time, the air conditioner was turned on to keep the room temperature at 25 °C. After swimming for 38 minutes, the mice were taken out, immediately wiped with blotting paper, and dried with a hair dryer. Then they were put into the barrel to rest for 20 minutes, and blood was collected from the orbital cavity. The obtained blood samples were used for the determination of serum urea nitrogen and lactate dehydrogenase. After blood collection, the animals were sacrificed by cervical dislocation. Immediately, the hind limb muscles and livers were taken, rinsed with physiological saline, dried with filter paper, weighed, and then placed in pre-cooled physiological saline to remove their blood, and dried with blotting paper again for weighing, waiting to be used.

[0081] Experimental results

[0082] Compared with the normal group, the body weight of the mice in the model group was significantly reduced (P < 0.01). Each drug administration group was compared with the model group, and there was no obvious change in body weight, and there was no significant difference. In addition, compared with the normal group, the mice in the model group showed listlessness, dry and dull hair, less movement, lighter color of the auricles and tails, reduced food intake, and loose stools. And each dose group of black bean seed coat polysaccharide could effectively improve the above situation. For 14 days of weight-bearing swimming modeling, a 5% lead block of the mouse body weight was tied to the tail root of the mouse (to make the mouse swim with load and quickly reach the state of exhaustion), and the loaded mice were put into a swimming box with a water temperature of 22 ± 2 °C and a water depth of 30 cm for weight-bearing swimming. Drugs were given during the modeling. The normal group and the model group were given physiological saline by gavage according to body weight for 14 days, once a day. After 14 days, the exhaustion time of the mice's weight-bearing swimming was recorded.

[0083] The experimental data were imported into SPSS statistical software, and the one-way ANOVA test method was used for analysis. The measurement data were expressed as mean ± standard deviation (x ± s), and P < 0.05 was considered to be statistically significant.

[0084] The weight-bearing swimming time indirectly reflects the muscle tolerance. Compared with the normal group, the exhaustion time of the mice's weight-bearing swimming in the model group was significantly shortened (P < 0.01). Compared with the model group, VTP1-H and VTP1-M could prolong the exhaustion time of the mice's weight-bearing swimming (P < 0.01). Among the same dose groups, VTP1 (high-dose group, medium-dose group, low-dose group) significantly increased the exhaustion time of the mice's weight-bearing swimming (P < 0.01), indicating that VTP1 has strong muscle tolerance and anti-fatigue activities.

[0085] Table 2 Effects of VTP1 on the exhaustion time of weight-bearing swimming in fatigued mice (x ± s, n = 10)

[0086]

[0087] Note: Compared with the blank group, * represents p < 0.05, ** represents p < 0.01.

[0088] Effect of black soybean seed coat polysaccharide on blood urea nitrogen (BUN) content

[0089]

[0090] Compared with the blank control group, #P < 0.05, ##P < 0.01

[0091] Effect of black soybean seed coat polysaccharide on liver glycogen (LG) and muscle glycogen (MG)

[0093]

[0094] Compared with the blank control group, #P < 0.05, ##P < 0.01.

Claims

1. A method for ultra-high pressure-assisted preparation of black bean seed coat polysaccharide with antioxidant and anti-fatigue effects, characterized in that, It includes the following steps: (1) Preparation of black bean seed coat polysaccharide: Take the dried black bean seed coat, crush it, add distilled water according to a certain solid-liquid ratio and mix evenly. Add 3% cellulase to the mixture and carry out water bath at 50 °C for 2 h. Then carry out extraction under 300 MPa, concentrate, and freeze-dry to obtain the crude polysaccharide of black bean seed coat polysaccharide; (2) Purification of black bean seed coat polysaccharide: The purity of polysaccharide directly affects the quality and effect of polysaccharide. Therefore, the black bean seed coat polysaccharide is further purified. The specific process is as follows: a. Use macroporous adsorption resin (D101 type) to decolorize the above crude polysaccharide. Immerse the macroporous adsorption resin in 95% ethanol for 24 h, then pour it into a chromatography column (10 cm × 90 cm), wash and balance it with distilled water for 12 h. Dissolve the crude polysaccharide in water, load the sample and adsorb it overnight, wash it with distilled water, concentrate the eluate under reduced pressure, and freeze-dry to obtain the decolorized crude polysaccharide; b. Deprotein: Take the decolorized crude polysaccharide, dissolve it in distilled water, place it in a separatory funnel, add 1 / 2 volume of Sevag reagent (the reagent ratio is chloroform: n-butanol = 5:1) of the sugar solution, then shake it violently for 5 min, let it stand for layering, centrifuge (3000 r / min), collect the supernatant, repeat the operation until there is no denatured protein layer, concentrate under reduced pressure, add 10 times the amount of 95% ethanol, let it stand overnight at 4 °C, centrifuge, and freeze-dry to obtain the deproteinized black bean seed coat polysaccharide, named BSPS(,BSPS); c. Calculation of protein removal rate: Protein removal rate = (protein content before protein removal - protein content after protein removal) / protein content before protein removal * 100%. The purity of polysaccharide directly affects the quality and effect of polysaccharide. Therefore, the black bean seed coat polysaccharide is further purified. The specific process is as follows: a. Use anion exchange resin DEAE-cellulose column to separate and purify BSPS. After soaking the DEAE-cellulose in water, soak it in 0.5 M NaOH for 1 h, then wash it with dH2O to neutrality, then soak it in 0.5 M HCl for 1 h, wash it with dH2O to neutrality, and then carry out column packing (remove air bubbles under vacuum before column packing). After the column packing is completed, balance the cellulose column with 2 times the volume of 2 M NaCl and dH2O, and the flow rate is 2 mL / min. Weigh 500 μg of BSPS and dissolve it in 25 mL of dH2O. After the sample is dissolved, centrifuge and take the supernatant and slowly add it to the cellulose column. Use dH2O to elute to obtain uncharged neutral sugar (BSPS-N). After the elution is completed, use a gradient mixer to pump in 0-1 M NaCl for gradient elution to obtain charged acidic sugar (BSPS-A), and the flow rate is 1 mL / min. Use an automatic collector to collect 5 tubes for 5 min. Use the phenol-sulfuric acid method to measure the absorbance of the collected solution, draw a gradient elution curve, and determine the appropriate elution salt concentration of acidic sugar. b. Weigh 2 g of BSPS, add distilled water to prepare a polysaccharide solution with a concentration of 20 mg / mL, centrifuge at 4500 rpm for 20 min at room temperature, and discard the precipitate. After DEAE-cellulose is pretreated with 0.5 M NaOH and 0.5 M HCl, wash it with water until neutral, filter to remove air bubbles, and then load it into a column. After the DEAE-cellulose column is set up and balanced, load the sample, elute the neutral sugar of black bean seed coat with distilled water, and then elute the acidic sugar of black bean seed coat with the salt concentration obtained from the linear elution curve. After the collected polysaccharide eluate is subjected to vacuum rotary evaporation, dialysis, and freeze-drying, among them, F-1 is the neutral polysaccharide component while F-2 and F-3 are the acidic polysaccharides.

2. The preparation method of black bean seed coat polysaccharide with antioxidant and anti-fatigue effects according to claim 1, characterized in that, The determination of the polysaccharide content in black bean seed coat includes the following steps: (1) Reagent preparation a. Preparation of phenol solution: Take refined phenol and add distilled water to prepare a 6% phenol solution for standby; b. Preparation of standard solution: Accurately weigh 0.5000 g of glucose, make up the volume to 100 mL in a volumetric flask, take 1 mL of the solution from it and make up the volume to 50 mL with water to obtain a standard solution of 100 μg / mL: (2) Preparation of standard curve Use a micropipette to take 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, and 3 mL of the 100 μg / mL standard solution respectively and make up the volume to 50 mL in a volumetric flask. Take 1 mL of each and put it into a test tube, add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid to each test tube, shake the test tube, mix well, let it stand, measure the absorbance value at 490 nm with a 722-type ultraviolet spectrophotometer, and calculate the regression equation; (3) Determination of the total sugar content in black bean seed coat polysaccharide Prepare the test sample into a 200 μg / mL solution, then take 1 mL of the sample, and the other steps are the same as those for the preparation of the standard curve. After the detection, calculate the total sugar content of the black bean seed coat polysaccharide; (4) Determination of the reducing sugar content in the prepared black bean seed coat polysaccharide by the 3,5-dinitrosalicylic acid method: Configuration of reagents: a. Preparation of m-hydroxybiphenyl: Weigh m-hydroxybiphenyl and use a 5 mg / mL NaOH solution to prepare a solution with a mass concentration of 1.5 mg / mL, and store it in the dark at 4 °C; b. Preparation of sodium tetraborate sulfuric acid solution: Take sodium tetraborate and use concentrated sulfuric acid to prepare a 0.125 mol / L solution, and store it at room temperature; c. Preparation of glucuronic acid standard solution: Accurately weigh 0.5000 g of glucuronic acid and make up the volume to 100 μg / mL with distilled water Preparation of glucuronic acid standard curve: Use a micropipette to take 0 μL, 5 μL, 10 μL, 20 μL, 30 μL, and 40 μL of the 0.1 mg / mL standard solution respectively for dilution to obtain a concentration gradient of the solution. Drop 4 μL of sulfamic acid and 250 μL of concentrated sulfuric acid into the galacturonic acid solution, heat it in a water bath for 20 min, quickly cool it, and add 4 μL of m-hydroxybiphenyl. React for 15 min, drop 200 μL of the solution into a 96-well plate, and measure the OD value at a wavelength of 525 nm. Draw a standard curve with the concentration of D-GalA (μg / mL) on the abscissa and the absorbance value on the ordinate. (5) Determination of polysaccharide uronic acid content: Take 40 μL of the sample and add 60 μL of distilled water to prepare a 0.1 mg / mL sample solution. The remaining steps are carried out according to the standard curve procedure. Do two sets of parallel experiments and measure the absorbance at 525 nm. Substitute into the standard curve to obtain the average value and get the uronic acid content. (6) Calculation formula for polysaccharide yield: The yield of black bean seed coat polysaccharide Y(%) = (WE × (CT - CR)) / WP × 100 WE is the weight of the purified polysaccharide, WP is the weight of the black bean seed coat pretreatment sample used in each experiment, and CT and CR are the contents of total sugar and reducing sugar in the purified polysaccharide, respectively.

3. The preparation method of black bean seed coat polysaccharide with antioxidant and anti-fatigue effects according to claim 1, characterized in that, The steps for further separation and purification of black bean seed coat polysaccharide include: (1) Use an anion exchange resin DEAE-cellulose column to separate and purify BSPS. After soaking the DEAE-cellulose in water, soak it in 0.5 M NaOH for 1 h, then wash it with dH2O to neutrality, then soak it in 0.5 M HCl for 1 h, wash it with dH2O to neutrality, and then load the column (remove air bubbles by vacuum before loading the column). After the column loading is completed, balance the cellulose column with 2 volumes of 2 M NaCl and dH2O at a flow rate of 2 mL / min. Weigh 500 μg of BSPS and dissolve it in 25 mL of dH2O. After the sample is dissolved, centrifuge and take the supernatant and slowly add it to the cellulose column. Use dH2O to elute to obtain uncharged neutral sugar (BSPS-N). After the elution is completed, use a gradient mixer to pump in 0 - 1 M NaCl for gradient elution to obtain charged acidic sugar (BSPS-A) at a flow rate of 1 mL / min, and collect it in 5 tubes by an automatic collector for 5 min. Use the phenol-sulfuric acid method to measure the absorbance of the collected solution and draw a gradient elution curve to determine the appropriate elution salt concentration for acidic sugar. Weigh 2 g of BSPS, add distilled water to prepare a 20 mg / mL polysaccharide solution, centrifuge at 4500 rpm for 20 min at room temperature, and discard the precipitate. After pretreatment with 0.5 M NaOH and 0.5 M HCl, wash the DEAE-cellulose to neutrality, filter to remove air bubbles, and then load the column. After installing the DEAE-cellulose column device, balance it, load the sample, wash the neutral sugar of black bean seed coat with distilled water, and then elute the acidic sugar of black bean seed coat with the salt concentration obtained from the linear elution curve. After vacuum rotary evaporation, dialysis, and freeze-drying of the collected polysaccharide eluate, among them, F-1 is the neutral polysaccharide component, while F-2 and F-3 are acidic polysaccharides. Use polysaccharide components F-1, F-2, and F-3 to elute single components through a Sepharose CL-4B chromatographic column. Prepare a 20 mg / mL solution of black bean seed coat polysaccharide, load the sample, connect the sample with an automatic sampler, elute with deionized water and 0.1, 0.2, 0.3 mol / L sodium chloride, use the phenol-sulfuric acid method to track and detect the absorbance value of polysaccharide at 490 nm, draw an elution distribution curve, collect in segments, combine the elution solution, dialyze, concentrate, and freeze-dry for 48 h to obtain 4 kinds of homogeneous polysaccharides of black bean seed coat that are white and shiny, and name them according to the Latin name of black bean seed coat polysaccharide: named BSPS-1, BSPS-2, BSPA-3 respectively; (2) The polysaccharide components F-1, F-2, and F-3 were eluted through a Sepharose CL-4B chromatographic column to obtain single components. The black bean seed coat polysaccharide was prepared into a 20 mg / mL solution, loaded onto the column, and sampled automatically. It was eluted with deionized water and 0.1, 0.2, and 0.3 mol / L sodium chloride. The absorbance value of the polysaccharide was detected at 490 nm by the phenol-sulfuric acid method, and an elution distribution curve was plotted. The eluate was collected in segments, combined, dialyzed, concentrated, and freeze-dried for 48 h to obtain 4 kinds of homogeneous black bean seed coat polysaccharides that were white and shiny. They were named according to the Latin name of the black bean seed coat polysaccharide as BSPS-1, BSPS-2, and BSPA-3 respectively.

4. For the black bean seed coat polysaccharide with antioxidant and anti-fatigue effects described in claim 3, the homogeneity and molecular weight of the polysaccharide were analyzed by high performance liquid gel permeation chromatography (HPGPC). The high performance liquid chromatography system was equipped with a refractive index detector (RID) and a Superdex 200 Increase 10 / 300 GL column. Weighed 1 mg of the polysaccharide sample and placed it in a 1.5 mL microcentrifuge EP tube, added 200 μL of filtered ultrapure water (ddH2O) to completely dissolve the polysaccharide sample. Filtered through a 0.22 μm inorganic phase filter membrane, and then subjected to HPGPC detection.

5. The crude polysaccharide from black bean seed coat with antioxidant and anti-fatigue effects according to claim 1, characterized in that, The crude black bean seed coat polysaccharide was applied to the in vitro antioxidant effect.

6. Use of the crude black bean seed coat polysaccharide with antioxidant and anti-fatigue effects prepared according to claims 1 and 2 in the preparation of anti-fatigue health products or drugs.

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