White lentil flower polysaccharide, preparation method, production line and application

Abstract

The present invention relates to the field of biomedicine development, and specifically to a white lentil flower polysaccharide with antioxidant and blood sugar and lipid lowering effects, a preparation method, a production line and applications. The present invention uses ethanol to extract white lentil flower crude polysaccharide through an alcohol precipitation method, and further purifies the white lentil flower polysaccharide by hot water extraction, trichloroacetic acid deproteinization and alcohol precipitation, and characterizes the structure of the white lentil flower polysaccharide. Through DPPH free radical scavenging experiments, hydroxyl free radical scavenging experiments, ABTS + Scavenging experiments and iron ion reducing capacity assays showed that white lentil flower polysaccharides have good antioxidant activity. In vitro hypoglycemic and lipid-lowering experiments also showed that white lentil flower polysaccharides have hypoglycemic and lipid-lowering activities. The polysaccharide content in white lentil flowers is 7.05±0.74%. The prepared white lentil flower polysaccharide is a potential polysaccharide with antioxidant and hypoglycemic and lipid-lowering effects, and has good market development and application prospects in related health products or pharmaceuticals.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a white lentil flower polysaccharide, a preparation method, a production line and applications. Background Art

[0002] Hypertension, hyperlipidemia, and hyperglycemia, commonly known as the "three highs," have become the deadly "number one killer" of humans. Hyperglycemia can cause diabetes, and hyperlipidemia can cause cardiovascular and cerebrovascular diseases such as vascular embolism. Once suffering from hyperglycemia and hyperlipidemia, patients can only rely on long-term use of lipid-lowering and blood sugar-lowering drugs for treatment, which not only causes great harm to the body, but also leads to a continuous decline in body functions. The World Health Organization has clearly stated that the first line of defense against cardiovascular disease is to reduce and control the "three highs." Currently, existing technologies for treating the "three highs" mainly rely on drugs that reduce the "three highs" and dietary control. Although the effect of drugs in reducing the "three highs" is obvious, they will produce side effects, especially when taken for a long time. For example, long-term use of glucose-lowering drugs, including metformin and sulfonylurea insulin secretagogues, can cause damage, dysfunction, or failure of various tissues and organs (such as the eyes, kidneys, heart, blood vessels, and nerves). Overdose can lead to hypoglycemia, forcing patients to stop taking the drug. Another common side effect of lipid-lowering drugs, including fibrates and statins, is impacting liver function, often leading to elevated liver enzymes. Therefore, when taking Western medications, it's important to monitor liver function for damage in addition to checking blood lipids. Lipid-lowering drugs can also cause gastrointestinal side effects, including constipation, indigestion, nausea, diarrhea, anorexia, and vomiting, which can seriously impact patients' physical and mental health and quality of life.

[0003] In view of this, it is necessary to find a drug that has the effect of lowering blood sugar and lipids and has low side effects. Summary of the Invention

[0004] The present invention aims to provide a polysaccharide from white lentil flower with the functions of anti-oxidation, lowering blood sugar and blood lipid, and a preparation method thereof.

[0005] Another object of the present invention is to provide a production line for white lentil flower polysaccharide.

[0006] Another object of the present invention is to provide an application of white lentil flower polysaccharide.

[0007] To achieve the above objectives, in the first aspect, the present invention provides a white lentil flower polysaccharide, which is composed of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the molar ratio of the monosaccharides is 1:0.27:1.36:0.32:15.97:62.92:13.05:0.28:1.33.

[0008] Furthermore, the weight average molecular weight of the white lentil flower polysaccharide is 106.387 kDa.

[0009] In a second aspect, the present invention provides a method for preparing white lentil flower polysaccharide, comprising the following steps:

[0010] S1. White lentil flower crude polysaccharide: White lentil flower is precipitated in ethanol and dried to obtain white lentil flower crude polysaccharide;

[0011] S2 hot water extraction: The crude polysaccharide of white lentil flower was first extracted with hot water, and the supernatant was taken I;

[0012] S3. Protein removal: The supernatant I was deproteinized by trichloroacetic acid, and the supernatant II was obtained after centrifugation;

[0013] S4. White lentil flower polysaccharide: The supernatant II is precipitated with alcohol and then dried to obtain white lentil flower polysaccharide.

[0014] Furthermore, before the white lentil flowers are precipitated in ethanol, the method further comprises the step of pre-treating the white lentil flowers. The pre-treatment comprises: washing the white lentil flowers, drying and crushing them, and sieving them.

[0015] Preferably, the sieving is through a 50-80 mesh sieve.

[0016] Furthermore, the solid-liquid ratio (g / mL) of the white lentil flower and ethanol is 1:8-12.

[0017] Furthermore, the specific steps of alcohol precipitation of white lentil flowers are: adding 10 times the volume of ethanol to the pretreated white lentil flowers, shaking on a shaker at room temperature, centrifuging, collecting the precipitate, and drying to obtain white lentil flower crude polysaccharide.

[0018] Preferably, the volume fraction of the ethanol is 95%.

[0019] Preferably, the drying is performed at 70°C.

[0020] Furthermore, the specific steps of hot water extraction of the crude polysaccharide of white lentil flower are: adding distilled water to the crude polysaccharide of white lentil flower, heating for extraction, centrifuging after cooling, and collecting the supernatant I.

[0021] Furthermore, the solid-liquid ratio (g / mL) of the white lentil flower crude polysaccharide and distilled water is 1:15-25.

[0022] Preferably, the hot water extraction temperature is 90-100° C. and the time is 6-8 hours.

[0023] Preferably, the centrifugation is performed at 5000-7000 rpm for 5-15 min.

[0024] Furthermore, in step S3, the specific steps of removing protein with trichloroacetic acid are: adding trichloroacetic acid to the above-mentioned supernatant I, mixing, standing overnight, centrifuging, and collecting supernatant II.

[0025] Preferably, the mass fraction of trichloroacetic acid is 3%, the overnight standing is at 4° C., and the centrifugation is at 7000-9000 rpm for 5-15 min.

[0026] Preferably, the centrifugation is performed at 8000 rpm for 10 min.

[0027] Furthermore, in step S4, the specific steps of the alcohol precipitation are: adding anhydrous ethanol to the above-mentioned supernatant II, standing overnight, centrifuging, collecting the precipitate, and drying to obtain white lentil flower polysaccharide.

[0028] Preferably, the overnight standing is standing at 4° C. overnight, the centrifugation is centrifugation at 7000-9000 rpm for 5-15 min, and the drying is freeze-drying.

[0029] Preferably, the centrifugation is performed at 8000 rpm for 10 min.

[0030] Furthermore, the content of the white lentil flower polysaccharide of the present invention is 7.05±0.74%.

[0031] Furthermore, the content of uronic acid in the white lentil flower polysaccharide of the present invention is 3.09±0.80%.

[0032] Furthermore, the white lentil flower polysaccharide of the present invention has a DPPH scavenging rate of 103.213±2.508% when the polysaccharide concentration is 2.0 mg / mL.

[0033] Furthermore, the white lentil flower polysaccharide of the present invention has a significant effect on hydroxyl radicals, ABTS, and + The scavenging ability and iron ion reduction ability were 53.447±1.872%, 52.033±1.224% and 104.379±1.183μmol / L, respectively.

[0034] Furthermore, the white lentil flower polysaccharide of the present invention has an inhibition rate of 80.789±1.651% on α-amylase when the polysaccharide concentration is 4.0 mg / mL; and the binding capacity to sodium glycocholate and sodium taurocholate is 22.750±2.334% and 63.730±0.886%.

[0035] In a third aspect, the present invention provides a production line for preparing white lentil flower polysaccharide, which can achieve large-scale mass production of white lentil flower polysaccharide, overcoming the limitations of low output and low efficiency of laboratory equipment. A production line for preparing white lentil flower polysaccharide, used in the above-mentioned preparation method of white lentil flower polysaccharide, comprises a shaking kettle, an extraction kettle, a first settling tank, and a second settling tank, which are sequentially connected by pipelines and valves. The shaking kettle, the extraction kettle, the first settling tank, and the second settling tank are all connected to a centrifuge via a feed pipe.

[0036] Furthermore, in the shaking kettle, the shaking kettle body is arranged on a slide, the slide is connected to the eccentric disk through a connecting rod, and the eccentric disk is connected to the variable frequency motor to drive the slide to reciprocate horizontally;

[0037] The bottom of the shaking kettle body is circular, and a corresponding circular groove is provided on the slide seat. The bottom of the shaking kettle body is located in the circular groove. On one side of the shaking kettle body, the shaking cylinder is hinged to the shaking kettle body to drive the shaking kettle body to shake, which is combined with the horizontal reciprocating motion driven by the eccentric disk to form a shaking action.

[0038] Furthermore, shaking cylinders connected to the shaking kettle body are provided on multiple sides of the shaking kettle body, with two shaking cylinders at each location, and the two shaking cylinders are arranged in an "eight" shape. The positions where the two shaking cylinders are connected to the shaking kettle body are relatively close, while the positions away from the shaking kettle body are relatively far apart.

[0039] Furthermore, a radial slide groove is provided in the eccentric disk, and a slider pin is slidably installed in the slide groove. The slider pin is connected to the connecting rod pin. An adjusting push rod is also provided in the slide groove, and the adjusting push rod is connected to the slider pin to adjust the radial position of the slider pin in the eccentric disk.

[0040] Furthermore, in the extraction kettle, a stirring shaft is provided in the extraction kettle body, and the stirring shaft is connected to the stirring motor of the extraction kettle;

[0041] An extraction frame is fixed on the stirring shaft of the extraction kettle, a filter screen is provided on the outer wall of the extraction frame, at least one extraction baffle is provided in the extraction frame, and an extraction beater is connected to the stirring shaft of the extraction kettle through a bearing. When the stirring shaft of the extraction kettle rotates back and forth, the extraction beater repeatedly beats the material in the space isolated by the extraction baffle.

[0042] Furthermore, an ultrasonic vibrator of the extraction kettle is also provided on the extraction clapboard.

[0043] Furthermore, the structure of the sedimentation tank is that a sedimentation tank ultrasonic vibrator is provided in the sedimentation tank body, and a sedimentation tank circulation pipe is provided to connect different positions of the sedimentation tank body, and a sedimentation tank circulation pump is provided on the sedimentation tank circulation pipe.

[0044] In a fourth aspect, the present invention provides the use of the above-mentioned white lentil flower polysaccharide in the preparation of related health products or medicines with antioxidant efficacy or hypoglycemic and hypolipidemic effects.

[0045] The present invention provides a polysaccharide of white lentil flower, a preparation method, a production line and an application. The polysaccharide of white lentil flower has a special structure and has good antioxidant activity and hypoglycemic and lipid-lowering activity. During the preparation, the white lentil flower is firstly purified to remove the fatty substances therein, and then the protein is further removed to purify and obtain a new type of white lentil flower polysaccharide. The polysaccharide is purified by DPPH free radical scavenging test, hydroxyl free radical scavenging test, ABTS + The results of the scavenging and iron ion reducing power assays indicate that the polysaccharide from the white lentil flower of the present invention has good antioxidant activity. Furthermore, the results of the in vitro hypoglycemic and lipid-lowering experiments indicate that the polysaccharide from the white lentil flower of the present invention has hypoglycemic and lipid-lowering activity. The present invention provides a new resource for the development and research of antioxidant and hypoglycemic and lipid-lowering drugs.

[0046] The beneficial effects of the present invention are:

[0047] 1. The preparation method of the white lentil flower polysaccharide provided by the present invention is simple, the product is easy to separate, and the operability is strong, and it is suitable for industrial application.

[0048] 2. The present invention uses white hyacinth bean flower as raw material, which has a wide range of materials and is cheap. Moreover, as a Chinese medicinal material that can be used as both medicine and food, white hyacinth bean flower has high safety and strong acceptance, providing a material basis for the research on new uses of white hyacinth bean flower polysaccharide.

[0049] 3. The white lentil flower polysaccharide prepared by the present invention is a novel structural polysaccharide compound, and exhibits good antioxidant activity and hypoglycemic and hypolipidemic effects, providing a basis for its further application in the field of health products or medicines.

[0050] 4. The production line provided by the present invention greatly improves the extraction efficiency of white lentil flower polysaccharide and can achieve mass production of white lentil flower polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The present invention will be further described below with reference to the accompanying drawings and examples:

[0052] Figure 1 This is a process flow chart for preparing white lentil flower polysaccharide of the present invention;

[0053] Figure 2 This is a picture of white lentil flower polysaccharide obtained by the preparation process of the present invention;

[0054] Figure 3 This is the HPGPC diagram of the white lentil flower polysaccharide of the present invention;

[0055] Figure 4 This is the monosaccharide composition diagram of the white lentil flower polysaccharide of the present invention.

[0056] In the figure, Man: mannose, GlcN: glucosamine hydrochloride, Rha: rhamnose, GlcA: glucuronic acid, GalA: galacturonic acid, Glc: glucose, Gal: galactose, Xyl: xylose, Ara: arabinose;

[0057] Figure 5 This is the infrared spectrum of the white lentil flower polysaccharide of the present invention;

[0058] Figure 6 This is a scanning electron microscope image of the white lentil flower polysaccharide of the present invention.

[0059] In the figure, a is a SEM image at a magnification of 500×, b is a SEM image at a magnification of 2000×, and c is a SEM image at a magnification of 5000×;

[0060] Figure 7 3. It is a relationship diagram between the concentration of white lentil flower polysaccharide of the present invention and the DPPH free radical scavenging rate;

[0061] Figure 8 is a graph showing the relationship between the concentration of white lentil flower polysaccharide and the hydroxyl radical scavenging rate;

[0062] Figure 9 The concentration of the white lentil flower polysaccharide of the present invention is + Free radical scavenging rate relationship diagram;

[0063] Figure 10 is a graph showing the relationship between the concentration of white lentil flower polysaccharide and the iron ion reducing capacity of the present invention;

[0064] Figure 11 is a graph showing the relationship between the concentration of white lentil flower polysaccharide and α-amylase inhibitory activity;

[0065] Figure 12 It is a relationship diagram of the binding ability of white lentil flower polysaccharide of the present invention to sodium glycocholate and sodium taurocholate.

[0066] Figure 13 The present invention is a production flow chart of white lentil flower polysaccharide.

[0067] Figure 14 It is a schematic diagram of the production line of the present invention.

[0068] Figure 15 It is a structural schematic diagram of a shaking kettle in the present invention.

[0069] Figure 16 It is a structural schematic diagram of the extraction kettle in the present invention.

[0070] Figure 17 It is a top view of the extraction frame in the present invention.

[0071] Figure 18It is a top view of the sedimentation tank in the present invention.

[0072] In the figure, shaking kettle 1, shaking kettle feed port 101, shaking kettle body 102, shaking cylinder 103, discharge port 104, slide 105, connecting rod 106, adjusting push rod 107, frequency conversion motor 108, slider pin 109, eccentric disk 110, leaching kettle 2, leaching kettle stirring motor 201, leaching kettle feed port 202, leaching kettle body 203, leaching kettle stirring shaft 204, leaching frame 205, leaching kettle ultrasonic vibrator 206, bearing 207, leaching clapper 208, jacket 209, leaching baffle 210, sedimentation tank 3, sedimentation tank body 31, sedimentation tank ultrasonic vibrator 32, sedimentation tank circulation pipe 33, sedimentation tank circulation pump 34, centrifuge 4, feed pipe 5. DETAILED DESCRIPTION

[0073] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Based on the described 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.

[0074] The various commonly used chemical reagents used in the following examples of the present invention are all commercially available products.

[0075] The method for determining the content of white lentil flower polysaccharide in the following embodiment is as follows:

[0076] Polysaccharide content determination method

[0077] (1)

[0078] Where:

[0079] m1-mass of white lentil flower polysaccharide after freeze-drying (g);

[0080] m2-Weigh the mass of white lentil flowers (g).

[0081] Determination of uronic acid content

[0082]

[0083] Where, C is the sample mass concentration, mg / mL;

[0084] V is the sample volume, mL;

[0085] m is the sample mass, mg.

[0086] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0087] Example 1:

[0088] like Figure 13 As shown in, this embodiment provides a method for preparing white lentil flower polysaccharide, comprising the following steps (see the process flow chart Figure 1 ):

[0089] S1. White lentil flower pretreatment: Take 10g white lentil flower, wash it, dry it and grind it, and pass the powder through a 60-mesh sieve;

[0090] White lentil flower crude polysaccharide: add 10 times the volume of 95% ethanol at room temperature, shake at 200 rpm for 6 hours, then centrifuge the sample at 6000 rpm for 10 minutes, collect the precipitate, and dry it at 70°C to obtain white lentil flower crude polysaccharide;

[0091] S2. Hot water extraction: Add 20 volumes of distilled water to the crude polysaccharide of white lentil flower, extract at 95°C for 6 hours, cool, centrifuge at 6000 rpm for 10 minutes, and collect the supernatant I;

[0092] S3. Protein removal: Add 3% trichloroacetic acid to an equal volume of supernatant I, mix thoroughly, and incubate at 4°C overnight. Centrifuge at 8000 rpm for 10 min, and collect supernatant II.

[0093] S4. White lentil flower polysaccharide: add anhydrous ethanol to a medium volume of supernatant II, mix well, let stand at 4°C overnight, centrifuge at 8000 rpm for 10 min, collect the precipitate, and freeze-dry to obtain white lentil flower polysaccharide.

[0094] The polysaccharide content and uronic acid content of the white lentil flower in this example are shown in Table 1.

[0095]

[0096] As shown in Table 1, the polysaccharide of the white lentil flower of the present invention is an acidic polysaccharide.

[0097] Test Example 1

[0098] The structure of the white lentil flower polysaccharide prepared in Example 1 was characterized.

[0099] 1. Molecular Weight Determination

[0100] The molecular weight distribution of polysaccharides from white lentil flowers was determined by high performance gel permeation chromatography (HPGPC) using high performance gel permeation chromatography tandem columns. Figure 2 .

[0101] Chromatographic conditions: A Waters high-performance liquid chromatograph was used; the chromatographic column was three polymer-based water-soluble SEC (GFC) columns (8×300 mm) connected in series; the mobile phase was 0.05 M NaCl solution; the flow rate was 0.65 ml / min; the column temperature was 40°C; the injection volume was 30 μl; and the detector was a Waters differential detector.

[0102] Sample configuration:

[0103] Standard preparation: Accurately weigh dextran standards of different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analytical standards), add 0.05M NaCl solution to prepare 5 mg / ml dextran standard solution, filter with 0.22μm microporous filter membrane for later use, adopt HPGPC method, use high performance gel permeation chromatography tandem columns for detection, and perform linear regression with the logarithm of the relative molecular mass of the standard as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa to obtain the calibration curve.

[0104] Sample preparation: Accurately weigh 5 mg of sample, add 1 ml of 0.05 M NaCl solution to the sample to prepare a 5 mg / ml test sample solution, centrifuge at 8000 rpm for 10 min, take the supernatant and filter it with a 0.22 μm microporous membrane, then transfer the sample to a 2 ml injection vial for later use.

[0105] High performance gel permeation chromatography (HPGPC) analysis results are as follows Figure 2 As shown, according to the experimental results, the calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) were obtained:

[0106] The equation of the lgMp-RT calibration curve is: lgMp-RT=-0.1701T+10.6276, R 2 =0.9903;

[0107] The equation of the lgMw-RT calibration curve is: lgMw-RT=-0.1786T+11.0312, R 2 =0.9922;

[0108] The equation of the lgMn-RT calibration curve is: lgMn-RT=-0.1697T+10.5354, R 2 =0.9926.

[0109] The molecular weight of polysaccharide from white lentil flower was calculated according to the standard curve. w) is 106.387 kDa.

[0110] 2. Monosaccharide composition

[0111] The monosaccharide composition of white lentil flower polysaccharide was determined by pre-column derivatization method. Figure 3 .

[0112] Chromatographic conditions: ThermoU3000 liquid chromatography system, ZORBAX EclipseXDB-C18 column; mobile phase: acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, 2 M NaOH adjusted to pH 6.8), volume ratio of 17:83, isocratic elution; flow rate: 0.8 ml / min; column temperature: 30°C; detection wavelength: 250 nm; injection volume: 10 μl.

[0113] Sample configuration:

[0114] Take a clean chromatographic bottle, accurately weigh 5 mg of polysaccharide sample, add 1 ml of 2M TFA acid solution, and heat at 121°C for 2 hours; pass nitrogen and blow dry; add 3 mL of methanol to wash, blow dry again, and repeat the methanol washing 2-3 times; add 5 mL of sterile water to dissolve, and transfer to a chromatographic bottle for later use.

[0115] Take an appropriate amount of supernatant, rotary concentrate or blow dry with nitrogen; add 1 ml of 2M TFA solution, heat at 121°C for 2 hours; pass nitrogen and blow dry; add methanol to wash, blow dry again, and repeat the methanol wash 2-3 times; add sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0116] Depend on Figure 3 It can be seen that white lentil flower polysaccharide is a glucose-rich heteropolysaccharide composed of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the molar ratio of each monosaccharide is 1:0.27:1.36:0.32:15.97:62.92:13.05:0.28:1.33.

[0117] 3. Infrared analysis

[0118] Weigh 1-2 mg of the dried polysaccharide sample in a mortar, add 200 mg of KBr powder and grind evenly, press into tablets, and use a Fourier transform infrared spectrometer to scan the sample with a wavelength range of 4000-400 cm -1 , the results are shown in Figure 4 .

[0119] Depend on Figure 4 It can be seen that the polysaccharide of the present invention has a typical characteristic absorption curve of polysaccharides. -1 The stretching vibration absorption peak of OH is at 2934.34cm, which is the characteristic peak of sugars.-1 There is an absorption peak at 1667cm, which may be CH stretching vibration; -1 There is an absorption peak at 1441.78cm, which may be the absorption peak of crystal water; -1 There is an absorption peak at 1144.42 cm, which may be CH angle vibration; -1 There is an absorption peak at 1335.48cm, which may be CO stretching vibration; -1 There is an absorption peak at 1258.14 cm, which may be the C=O symmetric stretching vibration; -1 and 1015.60cm -1 There is an absorption peak at 913.05cm, which may be the OH angle vibration; -1 There is an absorption peak at 832.46 cm, which may be the CH angle vibration of the differential isomerization of the β end of the pyran ring; -1 There is an absorption peak at 740.44 cm, which may be the CH anti-angle vibration at the α end of the pyran ring; -1 The absorption peak at 679.48 cm may be due to the symmetrical ring stretching vibration of the pyran ring; -1 The absorption peak near the absorption peak is the characteristic absorption peak of α-glycosidic bond, which shows that white lentil flower polysaccharide is an acidic polysaccharide with both α-configuration and β-configuration.

[0120] 4. Scanning electron microscopy images of white lentil flower polysaccharide

[0121] The surface microstructure of polysaccharide from white lentil flower was observed by scanning electron microscopy. Figure 5 .

[0122] Experimental method: Take an appropriate amount of polysaccharide sample on a conductive carbon tape, spray gold, and place it in a scanning electron microscope for observation and photography. Set the working voltage to 10.0kV, observe the solid morphology of the sample at different magnifications, and perform corresponding clarity adjustments until the ideal observation field of view is obtained, and take photos and record.

[0123] Depend on Figure 5 It can be seen that SEM images can clearly show the changes in the surface microstructure of white lentil flower polysaccharide, and the surface microstructure is also different under different magnifications. The SEM image of Figure a shows that the surface of white lentil flower polysaccharide is relatively regular, but there are some cracks around it. The polysaccharide molecules are closely cross-linked, the interaction between them is strong, and the overall structure is relatively compact; the SEM image of Figure b shows that the surface of white lentil flower polysaccharide is relatively compact, but there are cracks in the middle part, and the overall smoothness is low; the SEM image of Figure c shows that the surface of white lentil flower polysaccharide has small protrusions and is not smooth.

[0124] Test Example 2 Evaluation of Antioxidant Ability

[0125] 1. Antioxidant activity

[0126] DPPH is very stable. When dissolved in anhydrous ethanol, the DPPH ethanol solution has a maximum absorbance at 517nm, and the absorbance is linearly related to concentration. When a scavenging agent is added, it can react with DPPH to reduce its number, lighten the sample color, and thus reduce the absorbance, which can be used to evaluate the antioxidant capacity of the sample.

[0127] Experimental method: Prepare 0.1mmol / L DPPH solution, weigh 0.2mL polysaccharide sample solution and 0.2mL 0.1mol / L DPPH solution and mix them evenly. After reacting in the dark for 30 minutes, measure the absorbance at a wavelength of 517nm.

[0128] Substitute the following formula for calculation:

[0129] ;

[0130] Where: A0 is the absorbance of 0.2 mL H2O plus 0.2 mL DPPH; A1 is the absorbance of 0.2 mL polysaccharide sample solution and 0.2 mL 50% ethanol mixture; A2 is the absorbance of 0.2 mL polysaccharide sample solution and 0.2 mL DPPH mixture.

[0131] The results are as follows Figure 6 As shown in the results, the DPPH free radical scavenging ability of white hyacinth bean flower polysaccharides increased to varying degrees with increasing concentration. When the polysaccharide concentration was 1.0 mg / mL, the scavenging rate of white hyacinth bean flower crude polysaccharide reached 98.795%, which was consistent with the scavenging rate of vitamin C. When the polysaccharide concentration was 2.0 mg / mL, the scavenging rate of white hyacinth bean flower crude polysaccharide was 103.213±2.508%, which was higher than the scavenging rate of vitamin C of 98.795%.

[0132] 2. Hydroxyl radical scavenging ability

[0133] Hydroxyl free radicals are often formed in the metabolism of the body's life activities and have extremely strong oxidative capacity. 2+ When reacting with hydrogen peroxide, OH groups will be generated, simulating the hydroxyl free radicals in the body. When reacting with salicylic acid, a purple product will be generated, and a characteristic absorption peak will appear at 510nm. The scavenging ability of the sample for hydroxyl free radicals can be detected by color development reaction.

[0134] Experimental Method: Prepare 9 mmol / L salicylic acid solution, 9 mmol / L FeSO4 solution, and 8.8 mmol / L H2O2 solution. Take 0.2 mL of the polysaccharide sample solution, 0.2 mL of the 9 mmol / L salicylic acid solution, 0.2 mL of the 9 mmol / L FeSO4 solution, and 0.2 mL of the 8.8 mmol / L H2O2 solution, respectively, mix thoroughly, and incubate in a 37°C water bath for 30 minutes. Measure the absorbance at 510 nm.

[0135] Substitute the following formula for calculation:

[0136] ;

[0137] Where: A0 is the absorbance of a mixture of 0.2 mL ethanol, 0.2 mL FeSO4, 0.2 mL salicylic acid and 0.2 mL H2O2; A1 is the absorbance of a mixture of 0.2 mL polysaccharide sample, 0.2 mL FeSO4, 0.2 mL salicylic acid and 0.2 mL H2O; A2 is the absorbance of a mixture of 0.2 mL polysaccharide sample, 0.2 mL FeSO4, 0.2 mL salicylic acid and 0.2 mL H2O2.

[0138] The results are as follows Figure 7 As shown in the results, the scavenging ability of white lentil flower polysaccharide on hydroxyl radicals showed a concentration-dependent relationship. Although white lentil flower polysaccharide had a certain scavenging rate on hydroxyl radicals at polysaccharide concentrations of 0.5, 1.0, and 2.0 mg / mL, the scavenging rate of white lentil flower polysaccharide on hydroxyl radicals was the highest at a polysaccharide concentration of 4 mg / mL, reaching 53.447±1.872%.

[0139] 3.ABTS + Free radical scavenging ability

[0140] ABTS reacts with potassium persulfate to generate ABTS free radicals, which appear green. Similarly, ABTS free radicals have a maximum absorption at 734 nm, so measuring the absorbance at 734 nm can determine their concentration. If the absorbance at 734 nm decreases after adding a polysaccharide to an ABTS free radical solution, it indicates free radical scavenging activity.

[0141] Experimental Method: Mix equal volumes of 7.4 mol / L ABTS solution and 3.8 mmol / L potassium sulfate solution. Incubate in the dark for 12 hours to obtain a stock solution. Adjust the absorbance of the stock solution to 0.70 ± 0.02 at 734 nm using PBS (pH 7.4) buffer to obtain a reaction solution. Mix 25 μL of the polysaccharide sample solution with 250 μL of the reaction solution, let it stand for 15 minutes, and measure the absorbance at 734 nm using a microplate reader.

[0142] Substitute the following formula for calculation:

[0143] ;

[0144] Where: A0 is the absorbance of 25 μL H2O and 250 μL reaction solution; A1 is the absorbance of 25 μL polysaccharide sample solution and 250 μL PBS buffer; A2 is the absorbance of 25 μL polysaccharide sample solution and 250 μL reaction solution.

[0145] The test results are as follows Figure 8 As shown, the polysaccharide of white lentil flower has an effect on ABTS + The free radical scavenging ability showed a concentration-dependent relationship. Although the polysaccharide had a certain scavenging ability against ABTS+ free radicals at polysaccharide concentrations of 0.5, 1.0, and 2.0 mg / mL, the scavenging rate of polysaccharide from white lentil flower was the highest at a polysaccharide concentration of 4 mg / mL, reaching 52.033±1.224%.

[0146] 4. Total antioxidant capacity test (FRAP method)

[0147] The FRAP method was used as an indicator of the total antioxidant capacity in the samples.

[0148] Experimental Methods: FRAP working solution was prepared by mixing 300 mmol / L acetate buffer (pH 3.5), 10 mmol / L TPTZ solution, and 20 mmol / L ferric chloride solution at a volume ratio of 10:1:1. 900 μL of FRAP working solution was mixed with 100 μL of polysaccharide sample solution and incubated at 37°C for 5 minutes. The absorbance was measured at 595 nm using a microplate reader. Under the same conditions, a concentration curve was constructed using FeSO₄ as a reference standard. The iron ion reducing capacity (μmol / L) of the polysaccharide sample was calculated based on the concentration curve.

[0149] The results are as follows Figure 9 As shown in the results, the iron ion reducing ability of white lentil flower polysaccharide was concentration-dependent. Although white lentil flower polysaccharide had a certain iron ion reducing ability at polysaccharide concentrations of 0.5, 1.0, and 2.0 mg / mL, the iron ion reducing ability of white lentil flower crude polysaccharide reached as high as 104.379±1.183 μmol / L when the polysaccharide concentration was 4.0 mg / mL.

[0150] Test Example 3 In vitro hypoglycemic and lipid-lowering effect determination

[0151] 1. Determination of blood sugar lowering ability

[0152] Experimental methods:

[0153] Sample solution preparation: Take 0.4 mL of polysaccharide solution of different concentrations, add it to 0.2 mL of 1% starch, mix well, incubate in a 40°C water bath for 5 min, add 0.2 mL of α-amylase, incubate in a 40°C water bath for 10 min, add 0.4 mL of DNS reagent, incubate in a boiling water bath for 3 min 30 s, and measure the absorbance at a wavelength of 540 nm.

[0154] Preparation of reference solution: Acarbose was used as a positive control, and the reference solution was prepared in the same manner as the sample solution.

[0155] Calculate as follows:

[0156] ;

[0157] Where: A0 is the absorbance of a mixture of 0.4 mL PBS solution, 0.2 mL α-amylase solution, 0.2 mL starch solution and 0.4 mL DNS solution; A1 is the absorbance of a mixture of 0.4 mL polysaccharide sample solution, 0.2 mL PBS solution, 0.2 mL starch solution and 0.4 mL DNS solution; A2 is the absorbance of a mixture of 0.4 mL polysaccharide sample solution, 0.2 mL α-amylase solution, 0.2 mL starch solution and 0.4 mL DNS solution.

[0158] The results are as follows Figure 10 As shown, white lentil flower polysaccharide can inhibit α-amylase in a concentration-dependent manner. Compared with the positive control acarbose, although white lentil flower polysaccharide at concentrations of 0.5 mg / mL, 1 mg / mL, and 2 mg / mL had inhibition rates on α-amylase, the inhibition rate of white lentil flower crude polysaccharide was the highest at a polysaccharide concentration of 4 mg / mL, reaching 80.789±1.651%. The results indicate that white lentil flower polysaccharide has a strong ability to lower blood sugar.

[0159] 2. Determination of lipid-lowering ability

[0160] Experimental methods:

[0161] Take 2 mL of bile salt standard solution (the concentrations of sodium glycocholate solution are 0.03, 0.06, 0.12, 0.18, 0.24, and 0.3 mmol / L, respectively; the concentrations of sodium taurocholate solution are 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 mmol / L, respectively), add 3 mL of 60% H2SO4, bathe in a constant temperature water bath at 70°C for 1 h, then bathe in ice water for 5 min, measure the absorbance at 387 nm, and prepare a standard curve.

[0162] Take 0.2 mL of polysaccharide sample solution (0.5, 1.0, 2.0, and 4.0 mg / mL), add 0.2 mL of pepsin (10 mg / mL), 0.6 mL of HCl solution (0.01 mol / L), and incubate at 37°C with shaking to simulate the gastric environment for 1 hour. Adjust the pH to 6.3 with 0.1 mol / L NaOH solution, add 0.8 mL of trypsin (10 mg / mL), and incubate for 1 hour in a simulated intestinal environment. Add 0.8 mL of bile salt (0.4 mmol / L sodium glycocholate, 0.5 mmol / L sodium taurocholate) and digest for 1 hour. Centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and measure the bile salt content.

[0163] ;

[0164] Where:

[0165] C1 is the amount of sodium glycocholate added, μmol;

[0166] C2 is the remaining amount of sodium glycocholate, μmol;

[0167] C3 is the amount of sodium taurocholate added, μmol;

[0168] C4 is the remaining amount of sodium taurocholate, μmol.

[0169] The results are as follows Figure 11 At a concentration of 4.0 mg / mL, the binding capacity of D. dorsiflora polysaccharide for sodium glycocholate and sodium taurocholate was 22.750 ± 2.334% and 63.730 ± 0.886%, respectively. These results suggest that D. dorsiflora polysaccharide has a strong lipid-lowering effect.

[0170] In summary, the present invention obtains a complex polysaccharide from white lentil flower by extracting and purifying white lentil flower, and the polysaccharide is tested by DPPH free radical scavenging test, hydroxyl free radical scavenging test, iron ion reducing ability test, ABTS + Free radical scavenging experiments showed that white lentil flower polysaccharide has good antioxidant activity; in vitro hypoglycemic and lipid-lowering experiments showed that white lentil flower polysaccharide has hypoglycemic and lipid-lowering activity, and has development potential in related health products or drugs.

[0171] Example 2:

[0172] During the research process, the inventors found that the efficiency of existing laboratory methods for producing white dwarf bean flower polysaccharide was too low to meet market demand. Therefore, a production line for preparing white dwarf bean flower polysaccharide was provided, which could achieve large-scale mass production of white dwarf bean flower polysaccharide and overcome the limitations of low output and low efficiency of laboratory equipment.

[0173] like Figure 14As shown in the figure, a production line for preparing white lentil flower polysaccharide, used for the above-mentioned preparation method of white lentil flower polysaccharide, includes a shaking kettle 1, an extraction kettle 2, a first sedimentation tank, and a second sedimentation tank connected in sequence by pipelines and valves. The shaking kettle 1, the extraction kettle 2, the first sedimentation tank, and the second sedimentation tank are all connected to a centrifuge 4 through a feed pipe 5. The connected production line is adjusted through pipelines and valves to meet the processing flow of the preparation method in Example 1. The centrifuge 4 is an intermittent production machine and needs to be cleaned before production.

[0174] To improve the efficiency of mass production, it is necessary to improve the efficiency of shaking, hot water extraction, protein removal and ethanol extraction. Figure 15 In the shaking kettle 1, the shaking kettle body 102 is arranged on a slide 105, and the bottom of the slide 105 is provided with a polytetrafluoroethylene material to reduce friction. The slide 105 is connected to the eccentric disk 110 through a connecting rod 106, and the eccentric disk 110 is connected to the frequency conversion motor 108 to drive the slide 105 to reciprocate horizontally; the frequency of the horizontal reciprocating motion of the shaking kettle body 102 is controlled by the frequency conversion motor 108.

[0175] The bottom of the shaking kettle body 102 is circular, with a corresponding circular groove formed on the slide 105. The bottom of the shaking kettle body 102 is located within the circular groove. On one side of the shaking kettle body 102, a rocking cylinder 103 is hingedly connected to the shaking kettle body 102. The rocking cylinder 103 reciprocates and telescopes to drive the shaking kettle body 102 to rock. This, combined with the horizontal reciprocating motion driven by the eccentric disk 110, forms a rocking motion. In other words, the eccentric disk 110 and the rocking cylinder 103 work together to form the rocking motion of the shaking kettle body 102. Furthermore, the rocking cylinders 103 connected to the shaking kettle body 102 are provided on multiple sides of the shaking kettle body 102. There are two rocking cylinders 103 at each location, arranged in an "eight" shape. The locations where the two rocking cylinders 103 are connected to the shaking kettle body 102 are relatively close, while the locations away from the shaking kettle body 102 are relatively far apart.

[0176] Furthermore, a radially extending slot is provided within the eccentric disk 110, into which a slider pin 109 is slidably mounted. The slider pin 109 is pin-connected to the connecting rod 106. An adjustment push rod 107 is also provided within the slot and connected to the slider pin 109 to adjust the radial position of the slider pin 109 within the eccentric disk 110. This allows the rotational speed and amplitude of the eccentric disk 110 to be adjusted, and the telescopic stroke of the rocking cylinder 103 to be adjusted. This allows the rocking motion of the rocking kettle body 102 to be adjusted according to the characteristics of the material.

[0177] Further as Figure 16 In the leaching kettle 2, a stirring shaft 204 is provided in the leaching kettle body 203, and the stirring shaft 204 is connected to the stirring motor 201 of the leaching kettle;

[0178] A leaching frame 205 is fixed to the stirring shaft 204 of the leaching kettle. The outer wall of the leaching frame 205 is equipped with a filter screen. At least one leaching baffle 210 is located within the leaching frame 205. A leaching slapper 208 is connected to the stirring shaft 204 via a bearing 207. As the stirring shaft 204 rotates back and forth, the leaching slapper 208 repeatedly slaps the material within the space isolated by the leaching baffle 210. Unlike shaking, which only retains the sediment, the hot water extraction step requires the retention of the supernatant. Therefore, the mechanical stirring and the slapping and squeezing of the material between the leaching baffle 210 and the leaching slapper 208 effectively improve the extraction efficiency. Testing has shown that the extraction time can be shortened to approximately 4 hours.

[0179] Furthermore, an ultrasonic vibrator 206 for the extraction kettle is also provided on the extraction clapper 208. With this structure, the extraction effect is further improved.

[0180] Furthermore, the first and second settling tanks, i.e., the settling tanks 3, are configured such that an ultrasonic vibrator 32 is installed within the settling tank body 31, and a settling tank circulation pipe 33 is provided to connect different locations of the settling tank body 31. A settling tank circulation pump 34 is installed on the settling tank circulation pipe 33. This structure significantly improves the efficiency of protein removal and ethanol extraction through the combined effects of circulation and the ultrasonic vibrator 32.

[0181] The above embodiments are merely preferred technical solutions of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention shall be the technical solutions set forth in the claims, including equivalent alternatives to the technical features of the technical solutions set forth in the claims. In other words, equivalent alternatives and improvements within this scope are also within the scope of protection of the present invention.

Claims

1. The application of white lentil flower polysaccharide in the preparation of related health products or medicines with hypoglycemic and hypolipidemic effects, which is characterized by: The white lentil flower polysaccharide is composed of mannose, glucosamine hydrochloride, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the molar ratio of the monosaccharides is 1:0.27:1.36:0.32:15.97:62.92:13.05:0.28:1.33; The weight average molecular weight of the white lentil flower polysaccharide is 106.387 kDa; The preparation method of white lentil flower polysaccharide comprises the following steps: S1. White Bean Flower Crude Polysaccharide: White Bean Flower was added to 10 volumes of 95% ethanol, shaken on a shaker at room temperature, centrifuged, and the precipitate was collected and dried at 70°C to obtain White Bean Flower Crude Polysaccharide; S2. Hot water extraction: Add 20 volumes of distilled water to the crude polysaccharide of white lentil flower, extract at 95°C for 6 h, cool and centrifuge, and collect the supernatant I; S3. Protein removal: The supernatant I was deproteinized by trichloroacetic acid, and the supernatant II was obtained after centrifugation; S4. White lentil flower polysaccharide: The supernatant II was precipitated with alcohol and then dried to obtain white lentil flower polysaccharide.

2. The use according to claim 1, characterized in that: In step S1, before the white lentil flowers are precipitated in ethanol, the process further includes pre-treating the white lentil flowers. The pre-treatment comprises washing the white lentil flowers, drying and crushing the flowers, and sieving the flowers.

3. The use according to claim 1, characterized in that: In step S3, the specific steps of removing protein with trichloroacetic acid are: adding trichloroacetic acid to the above-mentioned supernatant I, mixing, standing overnight, centrifuging, and collecting supernatant II.

4. The use according to claim 3, characterized in that: The mass fraction of the trichloroacetic acid is 3%, the overnight standing is standing at 4° C., and the centrifugation is centrifugation at 7000-9000 rpm for 5-15 min.

5. The use according to claim 1, characterized in that: In step S4, the specific steps of the alcohol precipitation are: adding anhydrous ethanol to the above supernatant II, standing overnight, centrifuging, collecting the precipitate, and drying to obtain white lentil flower polysaccharide.

6. The use according to claim 5, characterized in that: The overnight standing is standing at 4° C. overnight, the centrifugation is centrifugation at 7000-9000 rpm for 5-15 min, and the drying is freeze-drying.

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