Corn silk glucomannan, preparation method and application thereof

Abstract

The application discloses corn silk glucan, a structure formula of which is [→4)-β-D-Glcp-(1→]n, wherein n is 6-60, and a molecular weight range is 1-9.8 kDa. By extracting effective components in corn silk, an extract is obtained, the extract is precipitated by 30-90% ethanol, corn silk crude polysaccharide is obtained, and after the corn silk crude polysaccharide is dissolved, the corn silk crude polysaccharide is sequentially subjected to biological membrane dialysis, ion exchange resin column and HPLC-ELSD separation and purification, and corn silk glucan is obtained. The corn silk glucan and zeaxanthin are compounded to form a glucan compound, the glucan compound can stabilize insulin and improve the activity of the insulin in mouse islet cells, and the glucan compound has 2-5 times stronger hypoglycemic activity than that of zeaxanthin alone, and therefore, the glucan compound has important significance for researching new hypoglycemic drugs or health-care food for assisting hypoglycemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a corn silk glucan, a preparation method and application thereof. BACKGROUND

[0002] At present, in addition to insulin injection, the main treatment for diabetes in clinic is oral hypoglycemic drugs, some of which are chemically synthesized hypoglycemic drugs, but have the disadvantages of great development difficulty and easy to produce adverse reactions, and the others are hypoglycemic active ingredients extracted from natural products. In recent years, natural polysaccharides derived from plants have gradually become a hot spot in the field of biological macromolecules at home and abroad due to their high safety and numerous pharmacological activities.

[0003] Corn silk is the style and stigma of Zea mays L. (corn) in the family Poaceae. It is recorded in various versions of pharmacopoeia and is recorded in pharmacopoeia that it has the effects of diuresis, swelling, liver and gallbladder. Corn silk polysaccharide is one of the important components of corn silk and is usually considered to have high galacturonic acid (HG) and rhamnose lactose uronic acid (RGI and RGII).

[0004] The currently reported corn silk polysaccharide skeleton type is (1→6)-α-D-glucose-(1→3)-α-L-arabinose, (1→4)-β-D-galactose and (1→3,5)-β-D-mannose, with β-D-xylose and α-L-rhamnose as terminal units. How to further extract more targeted polysaccharide components from corn silk and how to improve the performance of new polysaccharide components have important significance for the development and research of hypoglycemic drugs and health care foods for assisting hypoglycemic. SUMMARY

[0005] The present application aims to at least solve one of the problems in the prior art, and for this purpose, the present application provides a corn silk glucan, a preparation method and application thereof.

[0006] According to one aspect of the present application, a corn silk glucan is provided, and the structural formula is as follows: wherein n is 6-60, and the molecular weight range is 1-9.8 kDa.

[0007] According to another aspect of the present application, a method for preparing the above corn silk glucan is provided, comprising: extracting effective components in corn silk to obtain an extract; precipitating the extract with ethanol with a concentration of 30-90% to obtain corn silk crude polysaccharide; and sequentially subjecting the dissolved corn silk crude polysaccharide to biological membrane dialysis, ion exchange resin column and HPLC-ELSD separation and purification to obtain the corn silk glucan.

[0008] Preferably, the effective components in corn stigma are extracted by the following steps: boiling water extraction, filtration, 60℃ reduced pressure concentration, addition of chloroform and n-butanol mixture solution, centrifugation, and 50℃ reduced pressure concentration.

[0009] According to still another aspect of the present application, a method for preparing a glucan complex is provided, comprising: mixing solution A and solution B in a specific ratio; the solution A contains corn stigma glucan as described above; the solution B contains zeaxanthin.

[0010] Preferably, the corn stigma glucan is prepared by the following steps: extracting the effective components in corn stigma to obtain an extract; precipitating the extract with 30% or 50% ethanol to obtain corn stigma crude polysaccharide; and sequentially subjecting the dissolved corn stigma crude polysaccharide to biomembrane dialysis, ion exchange resin column and HPLC-ELSD separation and purification to obtain the corn stigma glucan.

[0011] Preferably, the specific ratio is 1:2-10, the concentration of the corn stigma glucan in the solution A is 2.5-5 mg / ml, and the concentration of the zeaxanthin in the solution B is 2 mg / ml.

[0012] Preferably, after mixing the solution A and the solution B in the specific ratio, the final concentration of the glucan complex is 100 μg / ml or 200 μg / ml.

[0013] Preferably, the solvent of the solution A and the solution B is DMSO pure water solution.

[0014] Preferably, the concentration of the DMSO pure water solution is 5%.

[0015] According to still another aspect of the present application, the glucan complex prepared by the above method is used in the preparation of a blood sugar lowering drug or a health food for assisting blood sugar lowering.

[0016] Specifically, the method for preparing corn silk glucan comprises: taking fresh or dried corn silk medicinal materials, crushing, reflux extraction with deionized water, filtering, concentrating the water extract, and removing proteins by Sevage method (chloroform: n-butanol 4:1) for three times. The obtained concentrated solution is centrifuged to remove the filter residue, and corn silk crude polysaccharide (CSGC) aqueous solution is obtained. The obtained aqueous solution is adjusted to 30%, 50%, 70%, and 90% ethanol concentrations in turn, and is centrifuged to obtain corn silk fractionated polysaccharide precipitates (CSGC30, CSGC50, CSGC70, and CSGC90). The precipitates are redissolved with an appropriate amount of deionized water, and are freeze-dried, respectively. After total sugar (the sugar content is determined according to the 2020 edition of the pharmacopoeia by the phenol-sulfuric acid colorimetric method) and total protein (the protein content in the sample is determined by the BCA method according to the 2020 edition of the pharmacopoeia) content determination, it is determined that the polysaccharide crude extract does not contain protein and nucleic acid components, and is pure crude polysaccharide. The crude polysaccharide is separated and purified by ion exchange resin column (DEAE52, water), biological membrane dialysis (MWCO: 100000-1000 Da), and gel column (Sephadex G-75, Sephacryl S-200) chromatography combined with HPLC-ELSD detection to obtain the required series of homogeneous glucan.

[0017] The present application has the following beneficial effects compared with the prior art.

[0018] The present application separates corn silk glucan from corn silk , wherein n is 6-60, and the molecular weight range is 1-9.8 kDa. The glucan complex formed by the glucan and zeaxanthin has 2-5 times stronger hypoglycemic activity than zeaxanthin alone in mouse islet cells by stabilizing insulin and improving its activity, which has important significance for studying new hypoglycemic drugs or health care foods for assisting hypoglycemic. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings described herein are used to provide further understanding of the present application, and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application, and do not constitute an improper limitation on the present application. In the drawings:

[0020] Figure 1 HPLC spectrum of CSGs pure product is shown;

[0021] Figure 2 PMP-HPLC spectrum of CSGs pure product is shown;

[0022] 3 IR spectrum of CSG-1 pure product is shown;

[0023] Figure 4 UV spectrum of CSG-1 pure product is shown;

[0024] Figure 5 NMR spectra of CSG-1 pure are shown;

[0025] Figure 6 ITC spectra of CSGs pure self-assembled with zeaxanthin are shown; and

[0026] Figure 7 Hypoglycemic activity results of complexes formed by CSGs with zeaxanthin are shown. DETAILED DESCRIPTION

[0027] The following examples are set forth in order to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application. It is understood that the examples below are intended to be exemplary only and are not intended to limit the scope of the application. Unless otherwise indicated, the materials used in the following examples are commercially available or are prepared by known methods.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular numerical ranges are recited herein, it is contemplated as specifically disclosing every value and sub-range within the open-ended range. Every weight percent value or range disclosed herein is also disclosed as a weight / weight percent value or range. These smaller ranges are: (1) from the same first endpoint to the same second endpoint as the larger range; (2) from either endpoint to any point in between; and (3) from any point in between to the contrary endpoint. These smaller ranges are also disclosed in the following embodiments and are individually and specifically disclosed as disclosed in this paragraph.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the patents, patent applications, publications, and descriptions are cited.

[0030] Many modifications and variations of this application of the application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean inclusion, but not limited to, the listed materials and method steps. The percent by weight of ethanol, unless otherwise specified, is the volume fraction. The technical solutions of the present application are further illustrated by the following examples.

[0032] Example 1

[0033] Corn silk polysaccharide extraction, separation, purification and structure identification: take corn silk dry powder 440 kg, add 4.4 L pure water, boiling water extraction 2 h, extract three times. Filter, combine the filtrate, 60 ℃ reduced pressure concentration to 5 L, the concentrated solution is used (chloroform: n-butanol 4:1) to remove protein, a total of three times, centrifugation (3500 rpm, 20 min), remove protein and other impurities precipitate, the supernatant is concentrated under reduced pressure at 50 ℃, to get the concentrated solution, add 95% ethanol to the concentrated solution, slow to fast stirring, the alcohol final concentration reaches 30, 50, 70, 90%, overnight, centrifugation (3500 rpm, 20 min), the obtained precipitate is added with 1 L hot water, -80 ℃ pre-freezing 2 h, freeze-drying, get corn silk crude polysaccharide CSGC30 5.5 g, CSGC50 7.3 g, CSGC70 15.3 g, CSGC90 26.0 g. Take CSGC400 mg, add 5 mL water, heat to dissolve, cool to room temperature, use biological membrane dialysis (MWCO:100000-1000 Da) in turn, collect the dialysate and the outside liquid, concentrate under reduced pressure, HPLC-ELSD detection, get the desired concentrated solution, the concentrated solution is used DEAE-52 chromatographic column (80×4 cm) in turn, distilled water elution, automatic sample collector collects each component, HPLC-ELSD detection is used to combine the same components, each component is concentrated under reduced pressure, and then vacuum freeze-dried to obtain four corn silk polysaccharide components CSG30, CSG50, CSG70 and CSG90 with different molecular weights. Further, the CSG50 is passed through DEAE-SepharoseFast Flow, Sephadex G-75 and Sephacryl S-200 column chromatography repeatedly, eluted with water, and the automatic sample collector is used to detect and combine the separated and purified CSG-1 every other tube by HPLC-ELSD. The polysaccharide linkage is determined by methylation analysis; infrared spectrum analysis; ultraviolet and optical analysis; and NMR spectrum determination. The structure of the uniform polysaccharide is determined as follows: the molecular weight is about 3.2 KDa, the monosaccharide composition is glucose, and the polysaccharide repeating sequence is 1,4-β-D-glucose (A) and 1,4-β-D-glucose (B) as the main chain, and the structure is as follows.

[0034]

[0035] Structure characterization: the relative molecular weight and purity of the product obtained in Example 1 are determined by high performance liquid chromatography (HPLC), and the purity identification result is shown in Figure 1 It can be seen from Figure 1 that the obtained product is a structure-uniform sugar.

[0036] Component analysis: monosaccharide composition analysis is carried out by PMP pre-column derivatization high performance liquid chromatography, and the result is shown in Figure 2As shown (the order of the legend is consistent with the order of the corresponding experimental data).

[0037] Characteristic absorption peak analysis: such as Figure 3 As shown, the product exhibits the characteristic absorption peak of carbohydrates, at 3422 cm⁻¹. -1 The appearance of a peak indicates the stretching vibration of the OH group within the carbohydrate molecule or in the -OH group of the molecule. The peak is at 2926 cm⁻¹. -1 The absorption peak is due to the CH stretching vibration. (1649 cm⁻¹) -1 The absorption peak at 1400-1200 cm⁻¹ is due to the C=O stretching vibration of -CHO. -1 The absorption peaks between 1200-1000 cm⁻¹ belong to the CH angle vibration. -1 The absorption peaks between them are caused by two CO stretching vibrations, one of which is COH and the other is COC (including intracyclic ether condensation and aliphatic ether condensation).

[0038] The product was dissolved in water and scanned at ultraviolet wavelengths of 210-400 nm. The resulting spectrum is shown below. Figure 4 As shown, no characteristic absorption of nucleic acids and proteins was observed at wavelengths of 260 nm and 280 nm.

[0039] In summary Figure 5 The nuclear magnetic resonance results shown indicate that the product structure is as follows: , where n is 6-60 and the molecular weight ranges from 1-9.8 kDa.

[0040] Example 2: Preparation of corn silk dextran complex

[0041] Dissolve 5 mg of homogeneous corn silk dextran (CSG30, 50, 70, 90) in 1-2 ml of 5% DMSO to prepare a dextran aqueous solution with a concentration of 2.5-5 mg / ml. Filter through a 0.25 μm filter and set aside. Dissolve an appropriate amount of zeaxanthin (ZEA) in 5% DMSO to prepare a 2 mg / ml solution. Filter through a 0.25 μm filter and set aside. In a 5% DMSO buffer solution, mix the prepared dextran solution with the zeaxanthin (ZEA) solution at a ratio of 1:2-10 to form a dextran complex. Prepare and use immediately.

[0042] Example 3: ITC analysis of corn silk dextran complex

[0043] The dextran solution and zeaxanthin (ZEA) solution prepared in Example 2 above were added to the titration needle and titration cell respectively in proportion. The formation parameters of the corn silk complex were analyzed using an isothermal microcalorimeter (ITC). The results are as follows: Figure 6 As shown.

[0044] Example 4: Hypoglycemic activity of corn silk glucomannan complex and zeaxanthin compound

[0045] Specific method of hypoglycemic activity: the complex prepared in the above Example 2 and zeaxanthin (ZEA) alone were respectively mixed with min6 cells in a 96-well plate (the final concentration of the complex was 100, 200, 400 μg / ml, and the final concentration of zeaxanthin was 12.5, 25, 50 μg / ml) for 12 h, 3 repeated holes were set for each group, and blank control holes without drugs and acarbose positive control group were set. Take the cell supernatant, follow the kit operation instruction to carry out the experiment, 15 min after the reaction is terminated, the OD value is measured by the enzyme label instrument, the detection wavelength is 450 nm, and the insulin protein activity is calculated. The results are shown in Figure 7 As shown, when the following steps are taken: extracting effective components in corn silk to obtain an extract; precipitating the extract with ethanol with a concentration of 30% or 50% to obtain corn silk crude polysaccharide; sequentially subjecting the dissolved corn silk crude polysaccharide to biological membrane dialysis, ion exchange resin column and HPLC-ELSD separation and purification to obtain corn silk glucomannan; and when the final concentration of the glucomannan complex is 200 μg / ml (30% ethanol precipitation) or 100 μg / ml (50% ethanol precipitation), the insulin is stabilized and its activity is improved in mouse islet cells, producing a hypoglycemic activity 2-5 times stronger than that of zeaxanthin alone.

[0046] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a dextran complex, characterized in that, include: Mix solution A and solution B in a ratio of 1:2-10; Solution A contains corn silk dextran, the structural formula of which is: , where n is 6-60, and the molecular weight ranges from 1-9.8 kDa; Solution B contains zeaxanthin; The concentration of corn silk dextran in solution A is 2.5-5 mg / ml, and the concentration of zeaxanthin in solution B is 2 mg / ml.

2. The method according to claim 1, characterized in that, The corn silk dextran is prepared by the following steps: Extract the active ingredients from corn silk to obtain an extract; The extract was precipitated with ethanol at a concentration of 30%-90% to obtain crude corn silk polysaccharide; The dissolved corn silk crude polysaccharide was sequentially purified by biomembrane dialysis, ion exchange resin column and HPLC-ELSD to obtain the corn silk dextran.

3. The method according to claim 2, characterized in that, After mixing solution A and solution B in a ratio of 1:2-10, the final concentration of the dextran complex is 100 μg / ml or 200 μg / ml.

4. The method according to claim 2, characterized in that, The solvent for both solution A and solution B is pure aqueous solution of DMSO.

5. The method according to claim 4, characterized in that, The concentration of the DMSO pure aqueous solution is 5%.

6. The use of the dextran complex prepared by the method according to any one of claims 1 to 5 in the preparation of hypoglycemic drugs or health foods that assist in lowering blood sugar.

Images