Coix seed oligosaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to coix seed oligosaccharide and a preparation method and application thereof, oligosaccharide CSO is separated and purified from coix seeds, the molecular weight of the oligosaccharide CSO is 1847 Da, the oligosaccharide CSO is composed of glucose and trace galactose, and the molar ratio of the glucose to the trace galactose is 96.5: 3.5. The preparation method comprises the following steps: performing water reflux extraction, alcohol precipitation, degreasing and deproteinization on coix seeds to obtain coix seed polysaccharide, and treating the coix seed polysaccharide with alpha-amylase and glycosidase in sequence; carrying out chromatographic separation by adopting a DEAE-52 ion exchange column; and eluting by adopting a Sephadex G-25 column to obtain the CSO. The CSO is subjected to structural analysis by adopting infrared and gel chromatography, and the CSO is determined to be composed of glucose and trace galactose in a molar ratio of 96.5: 3.5. The oligosaccharide has good uric acid lowering activity, blood sugar lowering activity and anti-inflammatory activity, is low in toxic and side effects and has a good development prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to coix seed oligosaccharide and its preparation method and application Background Art

[0002] Coix seeds are the dried mature seeds of Coix lacryma-jobi L. var. mayuen (Roman.) Stapf, a plant of the Liliaceae family. They have the effects of promoting water and moisture, strengthening the spleen and stopping diarrhea, removing numbness, draining pus, and detoxifying and dispersing knots. At the same time, coix seeds are also considered as a functional food and are widely used. Coix seeds contain many chemical components, mainly including polysaccharides, fatty acids, esters, phenols, amino acids, proteins, etc. Among them, starch-based polysaccharides are the main components of coix seeds, which play a variety of pharmacological effects in coix seeds, including lowering uric acid, lowering blood sugar, anti-tumor, regulating intestinal microbiota, anti-inflammatory, etc. However, the mechanism of action of coix seeds on lowering uric acid is still unclear, and the water solubility of coix seed polysaccharides is poor. Therefore, it is of great significance to find new, safe and efficient uric acid-lowering ingredients.

[0003] Coix seed oligosaccharides, also known as oligosaccharides, are low-polymer sugars composed of 3-10 monosaccharide molecules connected by α-1,4 glycosidic bonds. At present, the common extraction processes of coix seed oligosaccharides include enzymatic hydrolysis, biological method, solvent extraction, ultrasonic assisted method and steam explosion technology. However, these methods have disadvantages such as uncontrollable degradation process and generation of harmful by-products, which become a huge obstacle to the industrial preparation of coix seed oligosaccharides; at the same time, there has been no report that coix seed oligosaccharides have the effect of lowering uric acid. Summary of the invention

[0004] The purpose of the present invention is to provide a coix seed oligosaccharide and a preparation method and application thereof, so as to overcome the shortcomings of the prior art, fully explore the polysaccharide medicinal substances of coix seed, study the polysaccharide components of coix seed, and separate a new oligosaccharide component. The obtained oligosaccharide component has strong uric acid-lowering activity and low toxic and side effects, and has good development prospects.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a method for preparing coix seed oligosaccharide, comprising the following steps:

[0007] (1) mixing coix seed powder with water for extraction to obtain coix seed extract, concentrating the extract and adding ethanol for precipitation to obtain coix seed polysaccharide;

[0008] (2) mixing coix seed polysaccharide with water and performing enzymolysis, and terminating the enzyme reaction by heating to obtain a coix seed polysaccharide solution;

[0009] (3) filtering the coix seed polysaccharide solution and extracting it to obtain a coix seed polysaccharide extract, adding ethanol to precipitate it after concentration, adding water to re-dissolve it, and freeze-drying it to obtain coix seed crude oligosaccharide;

[0010] (4) mixing coix seed crude oligosaccharides with water, performing ion exchange chromatography separation, and eluting with water / NaCl to obtain water-eluted oligosaccharides;

[0011] (5) The water-eluted oligosaccharides are separated by gel filtration chromatography and eluted with water to obtain coix seed oligosaccharides.

[0012] In some other embodiments, in step (1), the mixing ratio of coix seed powder and water is 1:(10-15) g / mL, the extraction temperature is 55-65°C, and the extraction time is 2-5h.

[0013] In some other embodiments, in step (2), the mixing ratio of coix seed polysaccharide to water is 1: (30-60) mg / mL; the enzymatic hydrolysis is performed by sequentially using α-amylase and α-1,4-glucose hydrolase;

[0014] The conditions for heating to terminate the enzyme reaction are: heating temperature is 90-100° C. and time is 5-10 min.

[0015] In some other embodiments, in step (2), the conditions of the α-amylase treatment are: the addition amount of α-amylase is 1.5-3.0wt.%, the enzyme activity is 3000-5000U / g, the temperature is 50-60°C, and the time is 2-3h;

[0016] The conditions for the glycosidase treatment are: the addition amount of α-1,4-glucose hydrolase is 1.0-2.0wt.%, the enzyme activity is 50,000-100,000 U / g, the temperature is 50-60°C, and the time is 30-60min.

[0017] In some other embodiments, in step (3), the extraction is performed by sequentially adding petroleum ether and Sevage reagent;

[0018] In step (1) or step (3), the volume concentration of ethanol is 75-85%, the precipitation temperature is 1-5°C, and the precipitation time is 10-20h.

[0019] In some other embodiments, in step (4), the mixing ratio of coix seed crude oligosaccharide and water is 1: (30-60) mg / mL;

[0020] The conditions for the ion exchange chromatography separation are: using a DEAE-52 column, and eluting with water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution, 0.3 mol / L NaCl aqueous solution, and 0.4 mol / L NaCl, respectively.

[0021] In some other embodiments, in step (5), the conditions for gel filtration chromatography separation are: using a Sephadex G-25 column and a water flow rate of 0.1-0.2 mL / min.

[0022] In a second aspect, the present invention provides coix seed oligosaccharide prepared by the method for preparing coix seed oligosaccharide according to the first aspect, wherein the molecular weight of the coix seed oligosaccharide is 1847 Da, and the coix seed oligosaccharide consists of glucose and galactose.

[0023] In some other embodiments, the molar ratio of glucose to galactose is 96.5:3.5.

[0024] In a third aspect, the present invention provides use of the coix seed oligosaccharide described in the second aspect in the preparation of drugs with uric acid lowering activity, blood sugar lowering activity or anti-inflammatory activity.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention separates and purifies an oligosaccharide CSO from coix seed, and the obtained oligosaccharide has good uric acid-lowering activity, blood sugar-lowering activity and anti-inflammatory activity, and has low toxic and side effects, and has good development prospects.

[0027] (2) The present invention adopts a combination of a hyperuricemia animal model and drug administration, and through the determination of serum uric acid content and H&E staining of kidney sections, clarifies the mechanism of the uric acid-lowering activity of coix seed oligosaccharides, providing a scientific approach. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0029] Figure 1 The elution curve of crude oligosaccharides from coix seeds monitored by the phenol-sulfuric acid method in the embodiment of the present invention is shown;

[0030] Figure 2 This is the elution curve of oligosaccharides in the water elution part monitored by the phenol-sulfuric acid method in the embodiment of the present invention;

[0031] Figure 3 is the infrared spectrum of coix seed oligosaccharide in the embodiment of the present invention;

[0032] Figure 4The results of the uric acid lowering activity of coix seed oligosaccharide in the examples of the present invention are shown;

[0033] Figure 5 The results of coix seed oligosaccharide in reducing renal damage in the examples of the present invention;

[0034] Figure 6 This is a kidney section image of H&E staining of coix seed oligosaccharide in an embodiment of the present invention;

[0035] Figure 7 Graph showing the hypoglycemic activity of coix seed oligosaccharide in an example of the present invention, wherein (a) is the viability of HepG2 cells, and (b) is the glucose consumption. DETAILED DESCRIPTION

[0036] Those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Specific conditions are not specified in the examples, and the conventional conditions or conditions recommended by the manufacturer are used. The components used without indicating the manufacturer are all conventional products available on the market.

[0037] Example 1

[0038] 1. The preparation method of coix seed oligosaccharide, the steps are as follows:

[0039] (1) Coix seed (Baiweitang Chinese Medicine Piece Co., Ltd.) was crushed to obtain coix seed powder (mesh size 20-40). Coix seed powder (500 g) was extracted with distilled water (1:10, w / v, g / mL) at 60°C for 3 h. The extract was then filtered, concentrated, and precipitated with 80% ethanol (v / v) at 4°C overnight to obtain coix seed polysaccharide (51 g).

[0040] (2) Coix seed polysaccharide was dissolved in deionized water (1:50, w / v), treated with 2% α-amylase (enzyme activity 4000 U / g) at 60°C for 2 h, then treated with 1% α-1,4-glucose hydrolase (enzyme activity 100,000 U / g) for 40 min, and then heated at 100°C for 5 min to terminate the enzyme reaction.

[0041] (3) After filtration, petroleum ether was used for extraction 5 times to remove the fat-soluble components in the water extract. Subsequently, the polysaccharide aqueous solution after the removal of fat was extracted 5 times with Sevage reagent (chloroform: n-butanol = 4:1, v / v) to remove the protein mixed in the polysaccharide. The polysaccharide aqueous solution after the removal of protein was concentrated under reduced pressure to remove the residual organic solvent. Then, 80% ethanol (v / v) was used for precipitation overnight, and the precipitated oligosaccharides were filtered to obtain, and then re-dissolved in water and freeze-dried (-20°C) to obtain coix seed crude oligosaccharides (36.9 g, relative to 500 g coix seed powder, the yield of coix seed crude oligosaccharides was 36.9 / 500 = 7.38%).

[0042] (4) The coix seed crude oligosaccharide was mixed with deionized water to prepare a coix seed crude oligosaccharide solution with a concentration of 50 mg / mL, and ion exchange chromatography was performed on a DEAE-52 column (6.3×45 cm), and eluted with water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution, 0.3 mol / L NaCl aqueous solution, and 0.4 mol / L NaCl aqueous solution, respectively. Each gradient elution volume was 1.5 L, and one tube was collected every 50 mL. The elution curve was monitored by the phenol-sulfuric acid method. Figure 1 As shown, 30 tubes were collected at a wavelength of 490 nm to obtain oligosaccharides (27.5 g) at the water elution site with relatively high absorbance.

[0043] (5) The oligosaccharides in the water elution part were further separated by gel filtration chromatography using a Sephadex G-25 column (1.6×100 cm), and distilled water was used for elution. The flow rate of distilled water was 0.2 mL / min, the elution volume was 1.5 L, and one tube was collected for every 50 mL. A total of 30 tubes were collected at a wavelength of 490 nm. The elution curve was monitored using the phenol-sulfuric acid method. Figure 2 As shown, the oligosaccharides collected in tubes 17 to 24 with higher absorbance were freeze-dried to obtain coix seed oligosaccharides (CSO, 19.4 g).

[0044] During the research, the inventors also used other extraction methods for coix seed oligosaccharides. The specific extraction process is as follows:

[0045] The coix seed medicinal material was crushed to obtain coix seed powder, and the coix seed powder (500 g) was extracted with distilled water (1:5, w / v, g / mL) at 50°C for 3 h. The extract was then filtered, concentrated, and precipitated with 80% ethanol (v / v) at 4°C overnight to obtain coix seed polysaccharide (34 g).

[0046] Coix seed polysaccharide was dissolved in deionized water (1:50, w / v), treated with 1% α-amylase (enzyme activity 4000 U / g) at 60°C for 2 h, then treated with 0.5% α-1,4-glucose hydrolase (enzyme activity 100,000 U / g) for 40 min, and then heated at 100°C for 5 min to terminate the enzyme reaction.

[0047] After filtration, petroleum ether was used for extraction 5 times to remove the fat-soluble components in the water extract. Subsequently, the polysaccharide aqueous solution after the removal of fat was extracted 5 times with Sevage reagent (chloroform: n-butanol = 4:1, v / v) to remove the protein mixed in the polysaccharide. The polysaccharide aqueous solution after the removal of protein was concentrated under reduced pressure to remove the residual organic solvent. Then 80% ethanol (v / v) was used for precipitation overnight, and the precipitated oligosaccharides were filtered to obtain the precipitated oligosaccharides, which were re-dissolved in water and freeze-dried (-20°C) to obtain coix seed crude oligosaccharides (21.3g, relative to 500g coix seed powder, the yield of coix seed crude oligosaccharides was 21.3 / 500 = 4.26%). It can be seen that the yield of coix seed crude oligosaccharides in this extraction method is low, resulting in poor quality and effect of the final product coix seed oligosaccharides.

[0048] 2. Structural Identification of Coix Seed Oligosaccharides

[0049] The structural characteristics of coix seed oligosaccharide (CSO) were preliminarily analyzed by Fourier transform infrared spectroscopy. Figure 3 As shown. Figure 3 It can be seen that coix seed oligosaccharide is at 3235cm -1 A large stretching peak appears at 2924cm -1 There is an absorption peak at 1631cm, corresponding to the stretching vibration of OH and CH, which is a typical feature of sugar absorption spectrum. -1 The peak at 1433 cm is attributed to the presence of bound water in the sugar. -1 and 1350cm -1 The absorption peaks at 1200-1000cm -1 Peaks in the range (1124cm -1 、1085cm -1 and 1045cm- 1 ) is attributed to COH, CC and COC stretching vibrations, which is consistent with the pyranose ring structure. 907cm -1 and 840cm -1 The peaks at indicate the presence of β- and α-glucopyranose bonds.

[0050] High performance gel permeation chromatography (HPGPC) was used to separate and determine the molecular weight and distribution of coix seed oligosaccharides (CSO). The HPGPC chromatogram of coix seed oligosaccharides (CSO) showed a single symmetrical peak, and the molecular weight of coix seed oligosaccharides was determined to be 1847Da. Monosaccharide composition analysis showed that coix seed oligosaccharides were composed of glucose and trace galactose, with a molar ratio of 96.5:3.5.

[0051] 3. Pharmacological Experiments

[0052] 1. Study on the uric acid-lowering activity of coix seed oligosaccharide (CSO)

[0053] (1) Establishment of hyperuricemia animal model

[0054] Specific pathogen-free (SPF) male Kunming mice (from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into normal group (KB), model group (250 mg kg -1 ·d -1 Potassium oxonate + 250mg kg -1 ·d -1 Hypoxanthine), positive drug group (4 mg kg -1 Allopurinol) and drug-treated groups (low dose 50 mg / kg CSO-L; medium dose 100 mg / kg CSO-M; high dose 200 mg / kg CSO-H).

[0055] The modeling and drug administration were combined. The mice in the model group, positive drug group, and drug administration group were intraperitoneally injected with hypoxanthine and given potassium oxonate by gavage. One hour later, they were given drugs (different doses of coix seed oligosaccharide) and allopurinol (positive control drug) by gavage according to the dose. The mice in the normal group and model group were given an equal amount of normal saline by gavage for 3 weeks. After 20 days of drug administration, the mice were fasted but not watered. On the 21st day, the blood of the mouse eyeballs was collected 1 hour after the modeling agent was administered, and the serum to be tested was obtained by centrifugation and stored at -20℃ for later use.

[0056] (2) Determination of uric acid-lowering activity

[0057] HPLC chromatography was used to analyze and determine the uric acid content in mouse serum, and the uric acid content in mouse serum was calculated. The HPLC liquid phase conditions were: the chromatographic column was Waters Summetry C18 (4.6μm×250mm, 5μm); the mobile phase A was 0.52mmol / L sodium pentane sulfonate and 0.20mol / L potassium phosphate aqueous solution, and the pH was adjusted to 4.0 by adding HPLC grade phosphoric acid, and the mobile phase B was acetonitrile; the separation conditions were isocratic 5% acetonitrile; the detection wavelength was 254nm, the flow rate was 1mL / min, and the column temperature was 25°C. With different concentrations of uric acid standards (Shanghai Aladdin Biochemical Technology Co., Ltd. U105582) as the abscissa and the peak area as the ordinate, a standard curve of peak area (y) and uric acid concentration (x) was established, and the regression equation of uric acid content established was y=3390.8x-5.57. By bringing the measured peak area (y) into the standard curve, the uric acid content generated by the system can be calculated.

[0058] (3) Experimental results

[0059] The results of the test on the activity of coix seed oligosaccharide in lowering uric acid are as follows Figure 4 As shown, from Figure 4It can be seen that the serum uric acid (serum UA) level in the model group (Model) reached 240.54 μmol / L, which was about 3.76 times that of the normal group (Normal), confirming that the hyperuricemia (HUA) model was successfully established. CSO treatment in the drug-treated group can significantly reduce the serum UA level, and the high-dose group (CSO-H) has the most obvious effect, with serum UA reduced to 53.27 μmol / L. Compared with the normal group (Normal), the liver xanthine oxidase (XOD) activity in the model group (Model) was significantly increased to 13.27 U / gprot (p<0.01), while the coix seed oligosaccharide administration group reduced the XOD activity and dose-dependently, among which the high-dose group (CSO-H) had the most obvious effect, which was reduced to 9.56 U / gprot, indicating that coix seed oligosaccharide has the effect of reducing uric acid.

[0060] The test results of coix seed oligosaccharide on alleviating kidney damage are as follows Figure 5 As shown, from Figure 5 It can be seen that the kidney coefficient of the model group (Model) was significantly increased (p<0.01), indicating kidney damage caused by HUA. In contrast, treatment with coix seed oligosaccharide in the drug-treated group significantly reduced the kidney coefficient (p<0.01), indicating that it has a protective effect on the kidney health of HUA mice. Serum creatinine (serum CR) and BUN (markers of kidney damage and glomerular filtration efficiency) were also significantly increased in the model group (Model) (p<0.01). However, compared with the model group (Model), the administration of coix seed oligosaccharide in the drug-treated group reduced serum CR and BUN levels (p<0.01). This shows that coix seed oligosaccharide has the effect of alleviating kidney damage.

[0061] (4) Histopathological analysis

[0062] H&E stained kidney sections of HUA mice Figure 6 As shown in Figure 3, the protective effect of coix seed oligosaccharide on HUA mice was further demonstrated by histopathological analysis. Figure 6 It can be seen that the H&E stained kidney sections of the normal group (Normal) showed normal glomeruli, clear tubular boundaries, and intact epithelial cells. In contrast, the model group (Model) showed tubular dilatation, epithelial cell shedding, tubular structural destruction, and inflammatory infiltration. Treatment with coix seed oligosaccharide in the drug-treated group effectively alleviated these changes and restored the integrity of the tubules and epithelial cells, thus demonstrating the protective effect of coix seed oligosaccharide on HUA mice.

[0063] 2. Study on the hypoglycemic activity of coix seed oligosaccharide (CSO)

[0064] (1) Cell culture

[0065] HepG2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin and incubated at 37°C with 5% CO2. When the HepG2 cells reached about 80% density, the medium was removed and the cells were washed twice with 1×PBS. Subsequently, trypsin was added to promote cell detachment, and the cells were subcultured at a ratio of 1:2.

[0066] (2) Effects on HepG2 cell viability and glucose consumption

[0067] First, HepG2 cells were seeded in 96-well plates (2×10 4 Cells / well) were added and incubated in a CO2 incubator at 37°C for 24 h. Afterwards, the medium was replaced with DMEM supplemented with 0.6 mM palmitic acid (PA), and the cells were incubated for an additional 24 h. Finally, DMEM containing 0.6 mM PA containing CSO (0.40, 0.80, and 1.20 mg / mL) was re-added and incubated for 24 h. Cell viability was assessed using the MTT assay. Glucose consumption was determined using the glucose oxidase method according to the kit manufacturer's instructions.

[0068] (3) Experimental results

[0069] The results of the effects of coix seed oligosaccharide on HepG2 cell viability and glucose consumption are as follows Figure 7 As shown, (a) is the viability of HepG2 cells, and (b) is the glucose consumption. HepG2 cells (IR-HepG2) were treated with 0.6 mM PA for 24 h to induce insulin resistance in HepG2 cells. Cell viability analysis showed that different concentrations of CSO had little effect on cytotoxicity ( Figure 7 a). Figure 7 As shown in Figure b, the glucose uptake in the model group was significantly reduced (p<0.05), indicating that the IR-HepG2 cell model was successfully established. After treatment with different concentrations of CSO (0.40, 0.80, and 1.20 mg / mL), the glucose consumption in IR-HepG2 cells increased in a dose-dependent manner. Specifically, at CSO concentrations of 0.80 and 1.20 mg / mL, glucose consumption increased to 8.43 mM and 8.95 mM, respectively, which were approximately 1.43 and 1.51 times that of the model group. These findings indicate that CSO can enhance glucose consumption in IR-HepG2 cells.

[0070] 3. Study on the anti-inflammatory activity of coix seed oligosaccharide (CSO)

[0071] (1) Toxicity of CSO to RAW264.7 mouse macrophages

[0072] The MTT method was used for evaluation. RAW264.7 mouse macrophages were collected, counted, and inoculated into 96-well cell culture plates (2×10 5 / well), cultured for 24 hours. The experiment was divided into a negative control group, an LPS group ((lipopolysaccharide)), a drug-treated group, and a positive control group (dexamethasone). Only culture medium was added to the negative control group, only 1 μg / mL of LPS was added to the LPS group, 1 μg / mL of LPS and 20 μM of dexamethasone were added to the positive control group, and 1 μg / mL of LPS and 20 μM of coix seed oligosaccharide (CSO) were added to the drug-treated group. Freshly prepared serum-free culture medium containing 5.0 mg / mL MTT was added to each well, and after continued culture at 37°C for 4 hours, the supernatant was removed overnight. 150 μL of DMSO was added to each well to dissolve the formazan precipitate. The optical density at 570 nm was measured on a Perkin Elmer EnSpire microplate reader.

[0073] (2) Determination of anti-inflammatory activity

[0074] RAW264.7 mouse macrophages were collected, counted, and inoculated into 96-well cell culture plates (2×10 5 / well). Using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture, the cells were attached to the wall for 24 hours in an incubator at 37°C and 5% CO2 saturated humidity. The experiment was divided into a negative control group, an LPS group, a drug-treated group, and a positive control group (dexamethasone). Among them, only the culture medium was added to the negative control group, only 1μg / mL of LPS was added to the LPS group, 1μg / mL of LPS and dexamethasone (2μM) were added to the positive control group, and 1μg / mL of LPS (lipopolysaccharide) and CSO (concentration of 0.8mg / mL) were added to the drug-treated group. 60μL of the supernatant was taken out, and after adding Griess reagent for 10 minutes, the OD value of each compound was measured at a wavelength of 570nm using a Perkin Elmer EnSpire microplate reader. The measurement was repeated 3 times to calculate the corresponding NO generation inhibition rate.

[0075] (3) Experimental results

[0076] The results of the toxicity and anti-inflammatory activity of the negative control group, coix seed oligosaccharide (CSO) and the positive control group (dexamethasone) on RAW264.7 cells are shown in Table 1.

[0077] Table 1 Results of toxicity and anti-inflammatory activity assays on RAW264.7 cells

[0078] Cell survival rate (%) NO generation inhibition rate (%) Negative control 100 - CSO 97.5 55.2 Dexamethasone 98.7 86.3

[0079] As shown in Table 1, coix seed oligosaccharide (CSO) has no cytotoxicity to RAW264.7 mouse macrophages, and exhibits an inhibitory effect on the NO release of RAW264.7 mouse macrophages induced by LPS (lipopolysaccharide), with an inhibition rate of 55.2%, indicating that coix seed oligosaccharide (CSO) has anti-inflammatory activity.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing coix seed oligosaccharide, characterized in that: The following steps are involved: (1) mixing coix seed powder with water for extraction to obtain coix seed extract, concentrating the extract and adding ethanol for precipitation to obtain coix seed polysaccharide; (2) mixing coix seed polysaccharide with water and performing enzymolysis, and terminating the enzyme reaction by heating to obtain a coix seed polysaccharide solution; (3) filtering the coix seed polysaccharide solution and extracting it to obtain a coix seed polysaccharide extract, adding ethanol to precipitate it after concentration, adding water to re-dissolve it, and freeze-drying it to obtain coix seed crude oligosaccharide; (4) mixing coix seed crude oligosaccharides with water, performing ion exchange chromatography separation, and eluting with water / NaCl to obtain water-eluted oligosaccharides; (5) The water-eluted oligosaccharides are separated by gel filtration chromatography and eluted with water to obtain coix seed oligosaccharides.

2. The method for preparing coix seed oligosaccharide according to claim 1, characterized in that: In step (1), the mixing ratio of coix seed powder and water is 1:(10-15) g / mL, the extraction temperature is 55-65° C., and the extraction time is 2-5 h.

3. The method for preparing coix seed oligosaccharide according to claim 1, characterized in that: In step (2), the mixing ratio of coix seed polysaccharide to water is 1: (30-60) mg / mL; the enzymatic hydrolysis is performed by sequentially using α-amylase and α-1,4-glucose hydrolase; The conditions for heating to terminate the enzyme reaction are: heating temperature is 90-100° C. and time is 5-10 min.

4. The method for preparing coix seed oligosaccharide according to claim 3, characterized in that: In step (2), the conditions of the α-amylase treatment are: the addition amount of α-amylase is 1.5-3.0wt.%, the enzyme activity is 3000-5000U / g, the temperature is 50-60°C, and the time is 2-3h; The conditions for the glycosidase treatment are: the addition amount of α-1,4-glucose hydrolase is 1.0-2.0wt.%, the enzyme activity is 50,000-100,000 U / g, the temperature is 50-60°C, and the time is 30-60min.

5. The method for preparing coix seed oligosaccharide according to claim 1, characterized in that: In step (3), the extraction is performed by sequentially adding petroleum ether and Sevage reagent; In step (1) or step (3), the volume concentration of ethanol is 75-85%, the precipitation temperature is 1-5°C, and the precipitation time is 10-20h.

6. The method for preparing coix seed oligosaccharide according to claim 1, characterized in that: In step (4), the mixing ratio of coix seed crude oligosaccharide and water is 1: (30-60) mg / mL; The conditions for the ion exchange chromatography separation are: using a DEAE-52 column, and eluting with water, 0.1 mol / L NaCl aqueous solution, 0.2 mol / L NaCl aqueous solution, 0.3 mol / L NaCl aqueous solution, and 0.4 mol / L NaCl, respectively.

7. The method for preparing coix seed oligosaccharide according to claim 1, characterized in that: In step (5), the gel filtration chromatography separation conditions are: using a Sephadex G-25 column and a water flow rate of 0.1-0.2 mL / min.

8. Coix seed oligosaccharide prepared by the method for preparing coix seed oligosaccharide according to any one of claims 1 to 7, characterized in that: The molecular weight of the coix seed oligosaccharide is 1847Da, and the coix seed oligosaccharide consists of glucose and galactose.

9. The coix seed oligosaccharide according to claim 8, characterized in that: The molar ratio of glucose to galactose is 96.5:3.

5.

10. Use of the coix seed oligosaccharide according to claim 8 or 9 in the preparation of drugs with uric acid lowering activity, blood sugar lowering activity or anti-inflammatory activity.

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