Ophiopogon japonicus polysaccharide extraction method and application of ophiopogon japonicus polysaccharide in preparation of antioxidation and hypoglycemic drugs

Abstract

The invention discloses an extraction method of ophiopogon japonicus polysaccharide, which comprises the following steps: (1) drying ophiopogon japonicus, crushing and sieving to obtain ophiopogon japonicus powder; (2) mixing the radix ophiopogonis powder and water in a weight ratio of 1: (15-20), sterilizing, inoculating pediococcus pentosaceus, lactobacillus plantarum and lactobacillus fermentum mixed bacteria, fermenting and culturing at the temperature of 30-40 DEG C, adjusting the pH value to 5-6, and culturing for 10-15 hours; (3) performing centrifugal solid-liquid separation on the fermentation liquor obtained in the step (2), discarding solids, and collecting supernate; (4) removing protein through an isoelectric point method, adjusting the pH value to 2.5, centrifuging when floccules appear, and collecting supernate; (5) adjusting the pH value of the liquid to be neutral, and adding macroporous resin to dynamically adsorb pigment substances; (6) adding absolute ethyl alcohol, and carrying out alcohol precipitation overnight; and (7) collecting the precipitate, redissolving, dialyzing, collecting the dialyzed solution, and freeze-drying to obtain the ophiopogon japonicus polysaccharide. The ophiopogon japonicus polysaccharide is extracted by using a combined fermentation method of pediococcus pentosaceus, lactobacillus plantarum and lactobacillus fermentum, and the extraction efficiency is high.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction of Chinese herbal medicines, and particularly relates to a method for extracting ophiopogon japonicus polysaccharide. More specifically, it is a method for extracting ophiopogon japonicus polysaccharide by co-fermentation of multiple strains of bacteria. Background Art

[0002] Ophiopogon japonicus (L.f.) Ker Gawl is a herbaceous plant of the genus Ophiopogon in the family Liliaceae, with oval or spindle-shaped small tuberous roots. Its original name was Mai Men Dong, and now it is distributed in Guangdong, Guangxi, Fujian, Zhejiang and other places. Ophiopogon japonicus was included in the catalog of homologous medicines and foods in 2024. The Chinese nation has always had the concept of homologous medicines and foods. Eating it on an empty stomach is a food, and eating it by patients is a medicine. With the progress of modern technology and people's pursuit of a healthy life, the concept of homologous medicines and foods has received more extensive attention and application, which provides us with a new perspective to view health problems. Research shows that ophiopogon japonicus contains components such as polysaccharides, high isoflavones, steroidal saponins and amino acids. Among them, ophiopogon japonicus polysaccharide is one of the effective components for ophiopogon japonicus to exert its pharmacological effects. It belongs to the category of plant polysaccharides and has various pharmacological activities such as hypoglycemic, anti-tumor, anti-myocardial ischemia, immune enhancement, and anti-inflammatory.

[0003] At present, the main methods for extracting ophiopogon japonicus polysaccharide include solvent extraction method, ultrasonic-assisted extraction method, pulsed electric field extraction method, microwave extraction method, high-pressure and high-temperature extraction method, etc. However, due to problems such as materials and equipment, the production cost is too high, which is not conducive to the development of small, medium and micro enterprises and is not suitable for large-scale extraction and preparation of ophiopogon japonicus polysaccharide. Therefore, providing a method for extracting ophiopogon japonicus polysaccharide with high extraction efficiency and suitable for industrial production has become an urgent problem to be solved in the prior art. Summary of the Invention

[0004] An object of the present invention is to provide a method for extracting ophiopogon japonicus polysaccharide with high extraction efficiency and suitable for industrial production.

[0005] The technical solution adopted by the present invention is as follows:

[0006] A method for extracting ophiopogon japonicus polysaccharide, comprising the following steps:

[0007] (1) Dry ophiopogon japonicus, crush it and sieve it to obtain ophiopogon japonicus powder;

[0008] (2) Mix ophiopogon japonicus powder and water at a weight ratio of 1:(15 - 20), sterilize it, and then inoculate Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum. The co-fermentation culture temperature is 30°C - 40°C, adjust the pH to 5 - 6, and the culture time is 10 - 15 h;

[0009] (3) Centrifuge the fermentation broth obtained in step (2) for solid-liquid separation, discard the solid, and collect the supernatant;

[0010] (4) Remove proteins by the isoelectric point method, adjust the pH to 2.5, flocculates appear, centrifuge, and collect the supernatant;

[0011] (5) Adjust the pH of the liquid to neutral, add macroporous resin for dynamic adsorption of pigment substances;

[0012] (6) Add absolute ethanol and precipitate overnight;

[0013] (7) Collect the precipitated substance for re-dissolution, dialysis, collect the solution after dialysis and freeze-dry it, which is the purified Ophiopogon japonicus polysaccharide.

[0014] Among them, the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum are mixed by the Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid in a volume ratio of (1 - 1.5):(1 - 1.5):(1 - 1.5), and the bacterial liquid concentrations of the Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid are all 1×10 7 –2×10 7 cfu / mL.

[0015] Among them, the inoculation amount of the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum in the step (2) is 5vt% - 15vt%.

[0016] Among them, the step (1) is specifically: Ophiopogon japonicus is thoroughly dried in an oven at 50°C for 48 h, ground into powder with a Chinese herbal medicine grinder, and sieved through a 60-mesh sieve.

[0017] Among them, the reagent for adjusting the pH in the step (2) is 1wt% hydrochloric acid.

[0018] Among them, the reagent for adjusting the pH in the step (4) is 20wt% hydrochloric acid.

[0019] Among them, the reagent for adjusting the pH in the step (5) is 20wt% sodium hydroxide solution.

[0020] Among them, the macroporous resin in the step (5) comes from Ruida Henghui, and the model is macroporous resin AB-8.

[0021] Among them, the volume ratio of the liquid after removing pigments to ethanol in the step (6) is 1:4.

[0022] Among them, in the dialysis in the step (1), substances with a molecular weight cut-off of 1000 Da are retained, and Ophiopogon japonicus polysaccharide with a molecular weight above 1000 Da is collected.

[0023] The present invention also provides the application of the Ophiopogon japonicus polysaccharide prepared by the above extraction method in the preparation of antioxidant and hypoglycemic drugs.

[0024] Compared with the prior art, the extraction method of ophiopogon japonicus polysaccharide provided by the present invention is as follows: (1) Dry the ophiopogon japonicus, crush it and sieve it to obtain ophiopogon japonicus powder; (2) Mix the ophiopogon japonicus powder and water at a weight ratio of 1:(15-20), sterilize it, and then inoculate Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum for mixed fermentation. The culture temperature is 30°C - 40°C, adjust the pH to 5 - 6, and the culture time is 10 - 15 h; (3) Centrifuge the fermentation broth obtained in step (2) for solid-liquid separation, discard the solid, and collect the supernatant; (4) Remove proteins by the isoelectric point method, adjust the pH to 2.5, flocculates will appear, centrifuge, and collect the supernatant; (5) Adjust the pH of the liquid to neutral, add macroporous resin for dynamic adsorption of pigment substances; (6) Add absolute ethanol and precipitate overnight; (7) Collect the precipitate for re-dissolution, dialysis, and collect and freeze-dry the solution after dialysis, which is the purified ophiopogon japonicus polysaccharide.

[0025] The present invention uses a method of combined fermentation of three bacteria, Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum, to extract ophiopogon japonicus polysaccharide, with high extraction efficiency and suitable for industrial large-scale production. The ophiopogon japonicus polysaccharide extracted by this method has a unique molecular weight and monosaccharide composition. The molecular weight of this polysaccharide is about 3000 Da, and the monosaccharide composition is mannose and glucose, with a molar ratio of 3.1691:96.8309. The ophiopogon japonicus polysaccharide extracted by this method has a fluffy and uniform porous morphology. Compared with the existing extraction and preparation methods, the ophiopogon japonicus polysaccharide extracted by this extraction and preparation method has antioxidant ability and the effect of reducing blood sugar in vitro. Description of the Drawings

[0026] Figure 1 is the process flow chart of the extraction method of ophiopogon japonicus polysaccharide of the present invention;

[0027] Figure 2 is the graph showing the effect of the mixed bacteria fermentation time on the extraction rate of ophiopogon japonicus polysaccharide;

[0028] Figure 3 is the graph showing the effect of different extraction methods on the extraction rate of ophiopogon japonicus polysaccharide;

[0029] Figure 4 is the standard curve for the determination of the content of ophiopogon japonicus polysaccharide;

[0030] Figure 5 is the determination result of the molecular weight of ophiopogon japonicus polysaccharide;

[0031] Figure 6 is the determination result of the standard product of the monosaccharide composition of ophiopogon japonicus polysaccharide;

[0032] Figure 7 is the determination result of the monosaccharide composition of ophiopogon japonicus polysaccharide;

[0033] Figure 8 is the determination result of the infrared spectrum of ophiopogon japonicus polysaccharide;

[0034] Figure 9 SEM analysis results of wheat polysaccharide;

[0035] Figure 10 Total antioxidant capacity results of Ophiopogon japonicus polysaccharide;

[0036] Figure 11 DPPH scavenging rate results of Ophiopogon japonicus polysaccharide;

[0037] Figure 12 Hydroxyl radical scavenging rate results of Ophiopogon japonicus polysaccharide;

[0038] Figure 13 Ferric ion reducing ability results of Ophiopogon japonicus polysaccharide;

[0039] Figure 14 Glycosidase inhibition rate results of Ophiopogon japonicus polysaccharide;

[0040] Figure 15 Amylase inhibition rate results of Ophiopogon japonicus polysaccharide. Detailed implementation mode

[0041] To better demonstrate the technical solution and beneficial effects of the present invention, the technical solution of the present invention will be further described below in conjunction with embodiments, but it is not a limitation on the protection scope of the present invention.

[0042] Raw material description

[0043] Ophiopogon japonicus: The tuberous roots of Ophiopogon japonicus are produced in Sichuan and purchased from Jiangsu Yancheng Aikelisi Food Co., Ltd. (batch number: 20240705)

[0044] Aspergillus niger: ATCC16404

[0045] Saccharomyces cerevisiae: ATCC9763

[0046] Pediococcus pentosaceus: ATCC8041, JD.com

[0047] Lactobacillus plantarum: ATTCC14917

[0048] Lactobacillus fermentum: ATCC14931

[0049] Macroporous resin: Macroporous resin AB-8, Ruida Henghui

[0050] Dialysis membrane: MD44-1000, Bickerman

[0051] The sources of the remaining substances are commercially available.

[0052] Example 1

[0053] A method for extracting Ophiopogon japonicus polysaccharide, comprising the following steps, as Figure 1 shown:

[0054] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder using a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0055] 2) Mix the Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, sterilize, and then inoculate with a mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum for fermentation. The mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is formed by mixing Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid at a volume ratio of 1:1:1. The bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid are all 1×10 7 cfu / mL, the inoculation amount of the mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is 10 vt%, the culture temperature is 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0056] 3) The fermentation broth obtained in step 2) is separated by centrifugation to separate the solid and liquid, discard the solid, and collect the supernatant;

[0057] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0058] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances;

[0059] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and let it precipitate overnight;

[0060] 7) Collect the precipitated substance, redissolve it, dialyze, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0061] Example 2

[0062] A method for extracting Ophiopogon japonicus polysaccharide, comprising the following steps:

[0063] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder using a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0064] 2) Mix the Ophiopogon japonicus powder and water at a material-liquid ratio of 1:15, sterilize, and then inoculate with Pediococcus pentosaceus fermentation, Lactobacillus plantarum fermentation, and a mixed culture of Lactobacillus fermentum for fermentation. The mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is formed by mixing Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid at a volume ratio of 1.5:1:1.5. The bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid are all 2×10 7 cfu / mL, the inoculation amount of the mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is 5 vt%, the culture temperature is 30 °C, adjust the pH to 6 with 1 wt% hydrochloric acid, and the culture time is 10 h;

[0065] 3) The fermentation broth obtained in step 2) is subjected to centrifugal solid-liquid separation, the solid is discarded, and the supernatant is collected.

[0066] 4) The protein is removed by the isoelectric point method. The pH is adjusted to 2.5 with 20 wt% hydrochloric acid, flocculants appear, and then centrifugation is carried out.

[0067] 5) The pH of the liquid is adjusted to neutral with 20 wt% sodium hydroxide solution, and macroporous resin is added for dynamic adsorption of pigment substances.

[0068] 6) Absolute ethanol is added according to a volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and alcohol precipitation is carried out for 12 hours.

[0069] 7) The precipitated substance is collected, redissolved, dialyzed, and substances with a molecular weight cut-off greater than 1000 Da are retained. The solution after dialysis is collected and freeze-dried to obtain purified Ophiopogon japonicus polysaccharide.

[0070] Example 3

[0071] A method for extracting Ophiopogon japonicus polysaccharide, comprising the following steps:

[0072] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder with a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve.

[0073] 2) Ophiopogon japonicus powder and water are mixed at a material-liquid ratio of 1:20. After sterilization, it is inoculated with Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum for mixed fermentation. The mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum are composed of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid mixed at a volume ratio of 1:1.5:1. The bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid are all 2×10 7 cfu / mL, the inoculation amount of the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is 15 vt%, the culture temperature is 40 °C, the pH is adjusted to 5.5 with 1 wt% hydrochloric acid, and the culture time is 15 h.

[0074] 3) The fermentation broth obtained in step 2) is subjected to centrifugal solid-liquid separation, the solid is discarded, and the supernatant is collected.

[0075] 4) The protein is removed by the isoelectric point method. The pH is adjusted to 2.5 with 20 wt% hydrochloric acid, flocculants appear, and then centrifugation is carried out.

[0076] 5) The pH of the liquid is adjusted to neutral with 20 wt% sodium hydroxide solution, and macroporous resin is added for dynamic adsorption of pigment substances.

[0077] 6) Absolute ethanol is added according to a volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and alcohol precipitation is carried out overnight.

[0078] 7) Re-dissolve the precipitated matter, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, collect the solution after dialysis and freeze-dry it to obtain the purified Ophiopogon japonicus polysaccharide.

[0079] Comparative Example 1

[0080] This comparative example discloses an ultrasonic-assisted enzymatic hydrolysis extraction method for Ophiopogon japonicus polysaccharide. The specific steps are as follows:

[0081] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a Chinese herbal medicine grinder, and sieve it through a 60-mesh sieve.

[0082] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, and subject it to ultrasonic cell disruption for 20 - 40 minutes with a power of 40 - 60. After ultrasonic extraction, add papain, pectinase, and cellulase. The weight ratio of papain, pectinase, and cellulase is 1:1:1, and the enzyme addition amount is 2.5 wt%. Enzymatic hydrolysis is carried out at a temperature of 55 °C for 6 hours and then inactivate the enzyme.

[0083] 3) Centrifuge the extraction solution obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant.

[0084] 4) Remove proteins by the isoelectric point method. Adjust the pH to 2.5 with 20 wt% hydrochloric acid, and centrifuge when flocculates appear.

[0085] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances.

[0086] 6) Add anhydrous ethanol at a volume ratio of 1:4, that is, the volume ratio of the liquid after removing pigments to ethanol is 1:4, and carry out overnight alcohol precipitation.

[0087] 7) Re-dissolve the precipitated matter, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, collect the solution after dialysis and freeze-dry it to obtain the purified Ophiopogon japonicus polysaccharide.

[0088] Comparative Example 2

[0089] This comparative example discloses an extraction method for Ophiopogon japonicus polysaccharide using the high-pressure and high-temperature method. The specific steps are as follows:

[0090] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a Chinese herbal medicine grinder, and sieve it through a 60-mesh sieve.

[0091] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, and react at 120 - 130 °C and 0.1 MPa for 30 minutes.

[0092] 3) Centrifuge the extraction solution obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant.

[0093] 4) Remove proteins by isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, floccules appear, and then centrifuge.

[0094] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances.

[0095] 6) Add absolute ethanol according to the volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and carry out overnight alcohol precipitation.

[0096] 7) Collect the precipitated substances for redissolution, dialysis, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0097] Comparative Example 3

[0098] This comparative example discloses an extraction method of Ophiopogon japonicus polysaccharide by high-pressure high-temperature and acid addition method. The specific steps are as follows:

[0099] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a traditional Chinese medicine grinder, and sieve it through a 60-mesh sieve.

[0100] 2) Mix Ophiopogon japonicus powder and water with a material-liquid ratio of 1:20, adjust the pH value to 3 with 10% hydrochloric acid solution, and act at 120 - 130 °C and 0.1 MPa for 15 - 30 minutes.

[0101] 3) Centrifuge the fermentation broth obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant.

[0102] 4) Remove proteins by isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, floccules appear, and then centrifuge.

[0103] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances.

[0104] 6) Add absolute ethanol according to the volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and carry out overnight alcohol precipitation.

[0105] 7) Collect the precipitated substances for redissolution, dialysis, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0106] Comparative Example 4

[0107] This comparative example discloses an extraction method of Ophiopogon japonicus polysaccharide by yeast fermentation method. The specific steps are as follows:

[0108] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a traditional Chinese medicine grinder, and sieve it through a 60-mesh sieve.

[0109] 2) Mix the Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20. After sterilization, inoculate the yeast solution with a concentration of 1×10 7 cfu / mL, with an inoculation amount of 10 vt%, a culture temperature of 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and a culture time of 14 h;

[0110] 3) Centrifuge the fermentation broth obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant;

[0111] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculants appear, and centrifuge;

[0112] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances;

[0113] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and perform overnight alcohol precipitation;

[0114] 7) Collect the precipitated substance for re-dissolution, dialysis, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0115] Comparative Example 5

[0116] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by Aspergillus niger fermentation method, and the specific steps are as follows:

[0117] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, grind it into powder with a traditional Chinese medicine grinder, and sieve it through a 60-mesh sieve;

[0118] 2) Mix the Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20. After sterilization, inoculate the Aspergillus niger solution with a concentration of 1×10 7 cfu / mL, with an inoculation amount of 10 vt%, a culture temperature of 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and a culture time of 14 h;

[0119] 3) Centrifuge the extract obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant;

[0120] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculants appear, and centrifuge;

[0121] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances;

[0122] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after pigment removal to ethanol is 1:4, and perform overnight alcohol precipitation;

[0123] 7) Resuspend the precipitated material, dialyze it, retaining substances with a molecular weight cut-off greater than 1000 Da, collect the solution after dialysis and freeze-dry it to obtain the purified Ophiopogon japonicus polysaccharide.

[0124] Comparative Example 6

[0125] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using the fermentation method of Pediococcus pentosaceus. The specific steps are as follows:

[0126] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a Chinese herbal medicine grinder, and sieve it through a 60-mesh sieve.

[0127] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, inoculate with a Pediococcus pentosaceus liquid with a bacterial liquid concentration of 1×10 7 cfu / mL after sterilization, with an inoculation amount of 10 vt%, a culture temperature of 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and a culture time of 14 h.

[0128] 3) Centrifuge the fermentation broth obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant.

[0129] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates will appear, and centrifuge.

[0130] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances.

[0131] 6) Add anhydrous ethanol in a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and perform alcohol precipitation overnight.

[0132] 7) Resuspend the precipitated material, dialyze it, retaining substances with a molecular weight cut-off greater than 1000 Da, collect the solution after dialysis and freeze-dry it to obtain the purified Ophiopogon japonicus polysaccharide.

[0133] Comparative Example 7

[0134] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using the fermentation method of Lactobacillus plantarum. The specific steps are as follows:

[0135] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a Chinese herbal medicine grinder, and sieve it through a 60-mesh sieve.

[0136] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, inoculate with a Lactobacillus plantarum liquid with a bacterial liquid concentration of 1×10 7 cfu / mL after sterilization, with an inoculation amount of 10 vt%, a culture temperature of 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and a culture time of 14 h.

[0137] 3) The extract obtained in step 2) is subjected to centrifugal solid-liquid separation, the solid is discarded, and the supernatant is collected;

[0138] 4) Remove the protein by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0139] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances;

[0140] 6) Add anhydrous ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and perform overnight alcohol precipitation;

[0141] 7) Collect the precipitated substance, redissolve it, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0142] Comparative Example 8

[0143] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using Lactobacillus fermentum fermentation method. The specific steps are as follows:

[0144] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, pulverized with a traditional Chinese medicine pulverizer, and sieved through a 60-mesh sieve;

[0145] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, sterilize it, and then inoculate Lactobacillus fermentum liquid with a cell concentration of 1×10 7 cfu / mL, the inoculation amount is 10%, the culture temperature is 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0146] 3) The extract obtained in step 2) is subjected to centrifugal solid-liquid separation, the solid is discarded, and the supernatant is collected;

[0147] 4) Remove the protein by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0148] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances;

[0149] 6) Add anhydrous ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and perform overnight alcohol precipitation;

[0150] 7) Collect the precipitated substance, redissolve it, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0151] Comparative Example 9

[0152] This comparative example discloses a method for extracting ophiopogon japonicus polysaccharide by using a mixed bacteria fermentation method with different ratios. The specific steps are as follows:

[0153] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder with a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0154] 2) The ophiopogon japonicus powder and water with a material-liquid ratio of 1:20 are mixed. After sterilization, a mixed bacteria fermentation of pentococcus pentosaceus, lactobacillus plantarum, and lactobacillus fermentum is inoculated. The mixed bacteria of pentococcus pentosaceus, lactobacillus plantarum, and lactobacillus fermentum are formed by mixing pentococcus pentosaceus liquid, lactobacillus plantarum liquid, and lactobacillus fermentum liquid according to a volume ratio of 2:1:1.5. The bacterial liquid concentrations of pentococcus pentosaceus liquid, lactobacillus plantarum liquid, and lactobacillus fermentum liquid are all 1×10 7 cfu / mL. The inoculation amount of the mixed bacteria of pentococcus pentosaceus, lactobacillus plantarum, and lactobacillus fermentum is 10 vt%, the culture temperature is 35 °C, the pH is adjusted to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0155] 3) The extract obtained in step 2) is separated by centrifugation to separate solid and liquid, the solid is discarded, and the supernatant is collected;

[0156] 4) Proteins are removed by the isoelectric point method. The pH is adjusted to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and then centrifugation is carried out;

[0157] 5) The pH of the liquid is adjusted to neutral with 20 wt% sodium hydroxide solution, and macroporous resin is added for dynamic adsorption of pigment substances;

[0158] 6) Absolute ethanol is added according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and alcohol precipitation is carried out overnight;

[0159] 7) The precipitated substance is collected, redissolved, dialyzed, and substances with a molecular weight cut-off greater than 1000 Da are retained. The solution after dialysis is collected and freeze-dried to obtain the purified ophiopogon japonicus polysaccharide.

[0160] Comparative Example 10

[0161] This comparative example discloses a method for extracting ophiopogon japonicus polysaccharide by using a mixed bacteria fermentation method with different ratios. The specific steps are as follows:

[0162] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder with a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0163] 2) The ophiopogon japonicus powder and water with a material-liquid ratio of 1:20 are mixed. After sterilization, a mixed bacteria fermentation of pentococcus pentosaceus, lactobacillus plantarum, and lactobacillus fermentum is inoculated. The mixed bacteria of pentococcus pentosaceus, lactobacillus plantarum, and lactobacillus fermentum are formed by mixing pentococcus pentosaceus liquid, lactobacillus plantarum liquid, and lactobacillus fermentum liquid according to a volume ratio of 1:0.5:1. The bacterial liquid concentrations of pentococcus pentosaceus liquid, lactobacillus plantarum liquid, and lactobacillus fermentum liquid are all 1×107 cfu / mL. The inoculation amount of the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum was 10 vt%, the culture temperature was 35 °C, the pH was adjusted to 5 with 1 wt% hydrochloric acid, and the culture time was 14 h;

[0164] 4) The extract obtained in step 2) was separated by centrifugation to separate the solid and liquid, the solid was discarded, and the supernatant was collected;

[0165] 7) The protein was removed by the isoelectric point method. The pH was adjusted to 2.5 with 20 wt% hydrochloric acid, flocculates appeared, and centrifugation was performed;

[0166] 10) The pH of the liquid was adjusted to neutral with 20 wt% sodium hydroxide solution, and macroporous resin was added for dynamic adsorption of pigment substances;

[0167] 13) Absolute ethanol was added in a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol was 1:4, and alcohol precipitation was carried out overnight;

[0168] 16) The precipitated substance was collected, redissolved, dialyzed, and substances with a molecular weight cut-off greater than 1000 Da were retained. The solution after dialysis was collected and freeze-dried to obtain purified Ophiopogon japonicus polysaccharide.

[0169] Comparative Example 11

[0170] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using a mixed bacteria fermentation method with different ratios. The specific steps are as follows:

[0171] 25) Ophiopogon japonicus was thoroughly dried in an oven at 50 °C for 48 h, ground into powder with a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0172] 28) The Ophiopogon japonicus powder and water were mixed at a material-liquid ratio of 1:20, sterilized, and then inoculated with a mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum for fermentation. The mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum were composed of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid mixed in a volume ratio of 1.5:2:1. The bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid were all 1×10 7 cfu / mL. The inoculation amount of the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum was 10 vt%, the culture temperature was 35 °C, the pH was adjusted to 5 with 1 wt% hydrochloric acid, and the culture time was 14 h;

[0173] 33) The extract obtained in step 2) was separated by centrifugation to separate the solid and liquid, the solid was discarded, and the supernatant was collected;

[0174] 36) The protein was removed by the isoelectric point method. The pH was adjusted to 2.5 with 20 wt% hydrochloric acid, flocculates appeared, and centrifugation was performed;

[0175] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances;

[0176] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and carry out overnight alcohol precipitation;

[0177] 7) Collect the precipitated substance, redissolve it, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0178] Comparative Example 12

[0179] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using a mixed bacteria fermentation method with different ratios. The specific steps are as follows:

[0180] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, grind it into powder with a traditional Chinese medicine grinder, and sieve it through a 60-mesh sieve;

[0181] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, sterilize it, and then inoculate it with a mixed bacteria fermentation of Pediococcus pentosaceus and Lactobacillus plantarum. The mixed bacteria of Pediococcus pentosaceus and Lactobacillus plantarum are formed by mixing Pediococcus pentosaceus liquid and Lactobacillus plantarum liquid at a volume ratio of 1.5:1. The bacterial liquid concentrations of Pediococcus pentosaceus liquid and Lactobacillus plantarum liquid are both 1×10 7 cfu / mL, the inoculation amount of the mixed bacteria of Pediococcus pentosaceus and Lactobacillus plantarum is 10 vt%, the culture temperature is 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0182] 3) Centrifuge the extraction solution obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant;

[0183] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0184] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances;

[0185] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and carry out overnight alcohol precipitation;

[0186] 7) Collect the precipitated substance, redissolve it, dialyze it, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0187] Comparative Example 13

[0188] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide by using a mixed bacteria fermentation method with different ratios. The specific steps are as follows:

[0189] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder using a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0190] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, sterilize, and then inoculate with a mixed culture of Pediococcus pentosaceus and Lactobacillus fermentum for fermentation. The mixed culture of Pediococcus pentosaceus and Lactobacillus fermentum is composed of Pediococcus pentosaceus liquid and Lactobacillus fermentum liquid mixed at a volume ratio of 1.5:1.5. The bacterial liquid concentrations of both Pediococcus pentosaceus liquid and Lactobacillus fermentum liquid are 1×10 7 cfu / mL, the inoculation amount of the mixed culture of Pediococcus pentosaceus and Lactobacillus fermentum is 10 vt%, the culture temperature is 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0191] 3) The extract obtained in step 2) is separated by centrifugation to separate solid and liquid, discard the solid, and collect the supernatant;

[0192] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0193] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances;

[0194] 6) Add anhydrous ethanol at a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and carry out overnight alcohol precipitation;

[0195] 7) Collect the precipitated substance for re-dissolution, dialysis, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0196] Comparative Example 14

[0197] This comparative example discloses a method for extracting Ophiopogon japonicus polysaccharide using a mixed culture fermentation method with different ratios. The specific steps are as follows:

[0198] 1) Ophiopogon japonicus is thoroughly dried in an oven at 50 °C for 48 h, ground into powder using a traditional Chinese medicine grinder, and sieved through a 60-mesh sieve;

[0199] 2) Mix Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20, sterilize, and then inoculate with a mixed culture of Lactobacillus plantarum and Lactobacillus fermentum for fermentation. The mixed culture of Lactobacillus plantarum and Lactobacillus fermentum is composed of Lactobacillus plantarum liquid and Lactobacillus fermentum liquid mixed at a volume ratio of 1:1.5. The bacterial liquid concentrations of both Lactobacillus plantarum liquid and Lactobacillus fermentum liquid are 1×10 7 cfu / mL, the inoculation amount of the mixed culture of Lactobacillus plantarum and Lactobacillus fermentum is 10 vt%, the culture temperature is 35 °C, adjust the pH to 5 with 1 wt% hydrochloric acid, and the culture time is 14 h;

[0200] 3) The extract obtained in step 2) is subjected to centrifugal solid-liquid separation, the solid is discarded, and the supernatant is collected;

[0201] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0202] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin to dynamically adsorb pigment substances;

[0203] 6) Add absolute ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and precipitate overnight with alcohol;

[0204] 7) Collect the precipitated substance for redissolution, dialysis, retain substances with a molecular weight cut-off greater than 1000 Da, and collect and lyophilize the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharide.

[0205] Comparison of the extraction rate of Ophiopogon japonicus polysaccharide

[0206] Calculate the extraction rate of Ophiopogon japonicus polysaccharide. The polysaccharide extraction rate = (V × C × N × 10 -3 ) / M × 100% where V: the volume of constant volume (mL), C: the concentration (μg / mL), N: the dilution factor, M: the mass of the sample (mg). Measure and calculate the extraction rate of Ophiopogon japonicus polysaccharide in the examples and comparative examples respectively, and prepare a chart as Figure 2 shown, where different letters represent significant differences.

[0207] It can be seen that the extraction rate of the example is significantly higher than that of the comparative example.

[0208]

[0209]

[0210] Verification experiment 1

[0211] Study the effect of fermentation time on the extraction rate of Ophiopogon japonicus polysaccharide

[0212] A method for extracting Ophiopogon japonicus polysaccharide, including the following steps, as Figure 1 shown:

[0213] 1) Thoroughly dry Ophiopogon japonicus in an oven at 50 °C for 48 h, powder it with a Chinese herbal medicine grinder, and sieve it through a 60-mesh sieve;

[0214] 2) Mix the Ophiopogon japonicus powder and water at a material-liquid ratio of 1:20. After sterilization, inoculate with a mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum for fermentation. The mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is formed by mixing Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid at a volume ratio of 1:1:1. The bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid, and Lactobacillus fermentum liquid are all 1×10 7 cfu / mL. The inoculation amount of the mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum is 10 vt%, the culture temperature is 35 °C, adjust the pH to 6 with 1 wt% hydrochloric acid, and the culture times are 0 h, 14 h, 16 h, 18 h, and 20 h respectively to judge the influence of time on the polysaccharide yield;

[0215] 3) Centrifuge the fermentation broth obtained in step 2) for solid-liquid separation, discard the solid, and collect the supernatant;

[0216] 4) Remove proteins by the isoelectric point method, adjust the pH to 2.5 with 20 wt% hydrochloric acid, flocculates appear, and centrifuge;

[0217] 5) Adjust the pH of the liquid to neutral with 20 wt% sodium hydroxide solution, and add macroporous resin for dynamic adsorption of pigment substances;

[0218] 6) Add anhydrous ethanol according to a volume ratio of 1:4, that is, the volume ratio of the liquid after decolorization to ethanol is 1:4, and carry out overnight alcohol precipitation;

[0219] 7) Collect the precipitated substance for re-dissolution, dialysis, retain substances with a molecular weight cut-off of 1000 Da, collect Ophiopogon japonicus polysaccharides above 1000 Da, and collect and freeze-dry the solution after dialysis to obtain purified Ophiopogon japonicus polysaccharides.

[0220] Calculate the extraction rate of Ophiopogon japonicus polysaccharides. Polysaccharide extraction rate = (V×C×N×10 -3 ) / M×100% where V: fixed volume (mL), C: concentration (μg / mL), N: dilution factor, M: sample mass (mg), prepare a graph as Figure 3 shown. It can be seen that when the inoculation and culture time is 14 h, the extraction rate of Ophiopogon japonicus polysaccharides is the highest.

[0221] Verification experiment 2

[0222] 1. The detection method of Ophiopogon japonicus polysaccharides is as follows:

[0223] (1) Preparation of glucose standard curve

[0224] Absorb 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of 100 mg / mL glucose standard solution into colorimetric tubes, and make up the volume to 1 mL with water respectively. Then add 1.0 mL of 5% phenol solution to each colorimetric tube, and then add 5.0 mL of concentrated sulfuric acid. Shake well, let stand for 10 min, then place in a water bath at 30 °C for 20 min. Using the blank as a control, measure the absorbance at a wavelength of 490 nm. Plot the standard curve with absorbance as the ordinate and glucose content as the abscissa to obtain the regression equation. The results are shown in Figure 4 。

[0225] (2) Determination of polysaccharide content in samples

[0226] Take 1 mL of the prepared sample aqueous solution and place it in a 10 mL dry colorimetric tube respectively. The remaining operations are the same as (1), and measure the absorbance at a wavelength of 490 nm.

[0227] (3) Calculation of Ophiopogon japonicus polysaccharide content

[0228] Polysaccharide content (%) = m / W × dilution factor × 100%

[0229] m: Glucose content (mg) calculated according to the regression equation

[0230] w: Dry weight of the sample (mg)

[0231] 2. Molecular weight determination

[0232] First, dissolve the Ophiopogon japonicus polysaccharide (5 mg) in Example 2 in NaNO3 (0.1 M) by heating (100 °C). Then, 100 μL of the polysaccharide supernatant is obtained after centrifugation (14000 rpm, 10 min). The solution is detected by gel permeation chromatography connected with an RI detector and a multi-angle laser light scattering detector. Three chromatographic columns OhpakSB-805 / SB-804 / SB-803HQ are used in combination. The detection conditions are that the mobile phase is 0.1 M NaNO3 and the flow rate is 0.4 mL / min. Finally, it is analyzed with ASTRA 6.1 software. The results are shown in Figure 5 。

[0233] 3. Monosaccharide composition analysis

[0234] First, dissolve the polysaccharide sample (5 mg) in TFA solution (1 mL) at 121 °C for 2 h. Then, dry the acid-hydrolyzed polysaccharide solution using a nitrogen evaporator. Next, add methanol to the dried polysaccharide sample and then blow it dry. This process is repeated 3 times. Finally, dissolve the polysaccharide sample in sterile water and transfer it to a chromatographic vial for detection. Take 100 mg each of fucose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, and glucuronic acid and add them to sterile water in turn. Quantitatively dissolve them in a 10 mL volumetric flask to prepare a mixed standard solution. Gradient dilute the mixed standard solution to 1 / 100, 5 / 100, 10 / 1000, 20 / 1000, 30 / 100, 40 / 100, 50 / 100, 60 / 100 to obtain the standard working solution. Finally, transfer the diluted solution to a chromatographic vial for detection. The results of the monosaccharide standards are shown in Figure 6 , and the monosaccharide composition of Ophiopogon japonicus polysaccharide is shown in Figure 7 .

[0235] 4. Infrared spectroscopy analysis

[0236] Vacuum dry the polysaccharide for 48 h, with a ratio of 1:150 to potassium bromide. Grind it and press it into a tablet, and scan its absorption spectrum from 400 to 4000 -1 using a Fourier transform infrared spectrometer. The results are shown in Figure 8 . It shows that Ophiopogon japonicus polysaccharide has obvious polysaccharide characteristics, with a strong stretching absorption peak near 3400 cm-1, which is caused by the stretching vibration of O-H. The signal near 2900 cm-1 shows C-H stretching vibration. The strong peak near 1630 cm-1 is caused by the stretching vibration of O-H. The strong peak near 1400 cm-1 represents the vibration of C-H.

[0237] 5. Scanning electron microscopy analysis

[0238] Spray gold on the freeze-dried Ophiopogon japonicus polysaccharide powder and observe it at an appropriate magnification. The observation results are shown in Figure 9 , and the microscopic morphology of Ophiopogon japonicus polysaccharide presents an irregular flaky structure, with nanoscale pores distributed on its surface and accompanied by irregular folds. This porous characteristic is related to the formation of the hydrogen bond network of the polysaccharide molecular chain.

[0239] 6. Determination of antioxidant capacity

[0240] (1) Determination of antioxidant capacity

[0241] Refer to the kit (BL859A) to determine the total antioxidant capacity of the sample. The results are shown in Figure 10 .

[0242] (2) Determination of DPPH free radical scavenging ability

[0243] Take 500 μL of the sample solution (2, 4, 6, 8, 10 mg / ml) and mix it with 500 μL of absolute ethanol and 125 μL of 0.02% DPPH mixture. React in the dark for 25 min and measure the absorbance at 510 nm. Use V C as the positive control. The results are shown in Figure 11 .

[0244] DPPH radical scavenging rate = (1 - (A1 - A2) / A0) × 100

[0245] In the formula:

[0246] A0 is the absorbance of 500 μL of the sample solution mixed with 500 μL of absolute ethanol and 125 μL of 0.02% DPPH mixture.

[0247] A1 is the absorbance of 500 μL of the sample solution mixed with 625 μL of absolute ethanol.

[0248] A2 is the absorbance of 500 μL of H2O mixed with 500 μL of absolute ethanol and 125 μL of 0.02% DPPH mixture.

[0249] (3) Hydroxyl radical scavenging ability

[0250] Take 500 μL of the sample solution (2, 4, 6, 8, 10 mg / ml) and mix it with 500 μL of 9 mmol / L FeSO4 and 500 μL of salicylic acid - 50% ethanol (9 mmol / L) mixture. Add 500 μL of 0.03% H2O2 and mix well. Incubate in a water bath at 37 °C for 1 h. Let it stand at room temperature for 10 min and measure the absorbance at 510 nm. Use V C as the positive control. The results are shown in Figure 12 .

[0251] Hydroxyl radical scavenging rate = (1 - (A1 - A2) / A0) × 100

[0252] In the formula:

[0253] A0 is the absorbance of 500 μL of the sample solution mixed with 500 μL of 9 mmol / L FeSO4 and 500 μL of salicylic acid - 50% ethanol (9 mmol / L) mixture.

[0254] A1 is the absorbance of 500 μL of H2O mixed with 500 μL of 9 mmol / L FeSO4 and 500 μL of salicylic acid - 50% ethanol (9 mmol / L) mixture.

[0255] A2 is the absorbance of 500 μL of the sample solution mixed with 500 μL of 9 mmol / L FeSO4 and 50% ethanol solution.

[0256] (4) Determination of reducing ability

[0257] Take 500 μL of sample solution (2, 4, 6, 8, 10 mg / ml), 500 μL of PBS buffer solution (pH = 6.6), and 500 μL of 1% K3[Fe(CN)6] solution, and mix them evenly. After incubating in a water bath at 50 °C for 20 min, add 500 μL of 10% trichloroacetic acid and mix well. Take 500 μL of the mixed solution, add 500 μL of H2O and 100 μL of 0.1% FeCl3, let it stand at room temperature for 10 min, and measure the absorbance at 700 nm. Using VC as a positive control, judge the reducing ability of the polysaccharide by comparing the absorbance of the sample and VC. The results are shown in Figure 13 .

[0258] 7. In vitro hypoglycemic activity

[0259] (1) Determination of α-amylase activity inhibition rate

[0260] Prepare DNS solution in a brown bottle and use it after one week. First, prepare PBS with pH 6.8 and 0.1 mol / L, and then use PBS to prepare 5% starch solution, 20 U / mL amylase solution, and polysaccharide purification component solutions with concentrations of 2, 4, 6, 8, 10 mg / ml and the positive drug acarbose solution. First, add 300 μL of the polysaccharide solution and 400 μL of the enzyme solution to the sample group A1 test tube, add the same volume of water and enzyme solution to the blank group A0, and add the same volume of the corresponding concentration of polysaccharide solution and buffer to the control group A2. Immediately place them in a biochemical incubator and incubate at 37 °C for 10 min. Then add 300 μL of soluble starch solution to all test tubes, shake well, and place them in a biochemical incubator and incubate at 37 °C for 15 min. Then add 2 mL of DNS solution to the test tubes for color development, and inactivate the enzyme at high temperature on an induction cooker for 10 min. Finally, make up to 10 mL with PBS and measure the absorbance at 540 nm. Calculate the α-amylase activity inhibition rate according to the following formula. The results are shown in Figure 14 .

[0261] α-amylase inhibition rate = (1 - (A1 - A2) / A0) × 100

[0262] (2) Determination of α-glucosidase activity inhibition rate

[0263] Prepare 2.5 mmol / L PNPG solution, 0.25 U / mL α-glucosidase solution, and polysaccharide purification component solutions at 2, 4, 6, 8, and 10 mg / mL, as well as the positive drug acarbose solution with PBS (pH 6.8, 0.1 mol / L), and then perform spotting reactions in a 96-well plate. Add 50 μL of the polysaccharide solution and 50 μL of the enzyme solution to sample group A1, add the same volume of buffer and enzyme solution to blank group A0, add the same volume of the polysaccharide solution at the corresponding concentration and buffer to control group A2. Immediately place them in a biochemical incubator at 37 °C for 10 min, then add 50 μL of the PNPG solution to all of them. After mixing the well plate by oscillation, place it in the biochemical incubator at 37 °C for 30 min, measure the absorbance at 405 nm, and conduct 3 parallel experiments. Calculate the inhibition rate of α-glucosidase activity according to the following formula. The results are shown in Figure 15 .

[0264] α-glucosidase inhibition rate = (1 - (A1 - A2) / A0) × 100.

Claims

1. A method for extracting Ophiopogon japonicus polysaccharide, characterized in that The following steps are involved: (1) drying the ophiopogon japonicus, crushing and sieving to obtain ophiopogon japonicus powder; (2) mixing ophiopogon powder and water in a weight ratio of 1: (15-20), sterilizing, inoculating Pediococcus pentosaceus, Lactobacillus plantarum, and Lactobacillus fermentum, fermenting at a temperature of 30° C. to 40° C., adjusting the pH to 5-6, and culturing for 10-15 h; (3) The fermentation liquid obtained in step 2) is centrifuged to separate solid and liquid, the solid is discarded, and the supernatant is collected; (4) removing proteins by isoelectric point method, adjusting pH to 2.5, the floccules appeared, centrifuging, and collecting the supernatant; (5) adjusting the pH of the liquid to neutral and adding a macroporous resin to dynamically adsorb the pigment; (6) Add anhydrous ethanol and precipitate overnight; (7) collecting the precipitated material, re-dissolving it, dialyzing it, and collecting the dialyzed solution and freeze-drying it to obtain the purified Ophiopogon japonicus polysaccharide.

2. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The mixed culture of Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus fermentum is prepared by mixing Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid and Lactobacillus fermentum liquid in a volume ratio of (1-1.5): (1-1.5): (1-1.5), and the bacterial liquid concentrations of Pediococcus pentosaceus liquid, Lactobacillus plantarum liquid and Lactobacillus fermentum liquid are all 1×10 7 – 2×10 7 cfu / mL.

3. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: In the step (2), the inoculation amount of the mixed bacteria of Pediococcus pentosaceus, Lactobacillus plantarum and Lactobacillus fermentum is 5vt%-15vt%.

4. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The step (1) specifically comprises: thoroughly drying the Ophiopogon japonicus in an oven at 50° C. for 48 hours, grinding the powder with a Chinese medicinal material grinder, and sifting through a 60-mesh sieve.

5. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The reagent for adjusting pH in step (2) is 1 wt % hydrochloric acid.

6. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The reagent for adjusting pH in step (4) is 20 wt % hydrochloric acid.

7. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The reagent for adjusting pH in step (5) is 20wt% sodium hydroxide solution.

8. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: The macroporous resin in step (5) comes from Reda Henghui, and its model is macroporous resin AB-8.

9. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: In the step (6), the volume ratio of the depigmented liquid to ethanol is 1:

4.

10. The method for extracting Ophiopogon japonicus polysaccharide according to claim 1, characterized in that: In the step (1), the dialysis is performed to cut off substances with a molecular weight of 1000 Da, and Ophiopogon japonicus polysaccharides above 1000 Da are collected.

11. Use of the Ophiopogon japonicus polysaccharide prepared by the extraction method according to any one of claims 1 to 10 in the preparation of antioxidant and hypoglycemic drugs.

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