A burdock polysaccharide with probiotic function, its preparation method and application

Abstract

The present invention relates to a burdock polysaccharide with probiotic functions, its preparation method and applications. The present invention obtains a brand-new burdock polysaccharide compound through a stepwise enzymatic hydrolysis method, with a purity of over 99%, a weight-average molecular weight Mw of 4256 Da, a number-average molecular weight Mn of 3437 Da, and an Mw / Mn of 1.24, showing good homogeneity of the burdock polysaccharide. The crude burdock polysaccharide (ALCP) and the burdock polysaccharide (ALP) of the present invention have significant inhibitory activities against pancreatic lipase, showing a dose correlation. The inhibition rate of ALCP is 45.44% at 10 mg / mL, while the IC 50 value of ALP is 8.18 mg / mL.

Description

Technical Field

[0001] The present invention belongs to the field of natural plant polysaccharides, and particularly relates to a burdock polysaccharide with probiotic functions, a preparation method thereof, and an application thereof. Background Art

[0002] Burdock (Arctium lappa L.), also known as Arctii Fructus and Great Seed, is a biennial herbaceous plant of the genus Arctium in the Asterales order and the Asteraceae family. It is cold in nature, bitter in taste, and non-toxic. Both the achene and the root can be used as medicine. As a traditional plant with dual functions of medicine and food, the burdock root is described in detail in works such as Compendium of Materia Medica, Supplementary Records of Famous Physicians, and Chinese Pharmacopoeia. Compendium of Materia Medica records that it can "open the twelve meridians and wash away the evil qi in the five internal organs", and "long-term consumption can make the body light and resistant to aging". Supplementary Records of Famous Physicians records that burdock can "treat sweating, stroke, facial swelling, diabetes, and expel water". Burdock has been praised by the Food and Agriculture Organization of the United Nations (FAO) as "one of the best health foods for humans in the 21st century" and has extremely high nutritional value, economic value, and development and application prospects.

[0003] Burdock contains compounds and volatile substances such as inulin, lignans, fatty acids, acetylenes, phytosterols, caffeoylquinic acid derivatives, flavonoids, and terpenoids. Among them, inulin is an important bioactive component in burdock. Burdock inulin has a variety of biological activities, such as anti-inflammatory, regulating the intestinal flora, lowering blood sugar, regulating lipid metabolism, antioxidant and other pharmacological effects. Inulin is a chain-like polyfructose formed by connecting D-fructofuranose through β-(1→2) glycosidic bonds, and usually contains a glucose group at the end. It is a water-soluble dietary fiber and is widely used in food, medicine, and health products. Inulin has a wide range of biological activities and can be used as a health food ingredient and natural medicine for preventing and treating tumors, coronary heart disease, diabetes, colon cancer, constipation, etc. The indigestibility and fermentability of inulin can promote the body to maintain a normal intestinal ecology, increase the fecal volume, and lower the fecal pH value, and have a good preventive effect on constipation, anorectal diseases, etc. Research has found that most of the causes of hyperlipidemia are closely related to diet. For example, excessive intake of carbohydrates can affect insulin secretion, accelerate the synthesis of very low-density lipoproteins in the liver, and easily cause hypertriglyceridemia; excessive intake of cholesterol and animal fats is likely to form hypercholesterolemia; long-term excessive intake of protein, fat, carbohydrates, and too little intake of dietary fiber are also closely related to hyperlipidemia. Inulin basically does not produce calories and can reduce blood lipids. Summary of the Invention

[0004] The present invention provides a burdock polysaccharide, characterized in that the HPLC purity of the burdock polysaccharide is above 99%, the weight-average molecular weight Mw is 4256 Da, and the number-average molecular weight Mn is 3437 Da. The high-performance liquid chromatography conditions are: Waters 2414 differential refractometer; SUGAR KS-805 (8.0×300 mm); Mobile phase: ultrapure water; Flow rate: 1 mL / min; Column temperature: 35 °C; Detector temperature: 30 °C; Injection volume: 30 μL. The infrared spectrum of the burdock polysaccharide is consistent with Figure 8 basically. The 1 1H NMR spectrum of the burdock polysaccharide is consistent with Figure 9 basically.

[0005] Another embodiment of the present invention provides the above-mentioned burdock polysaccharide, characterized in that the preparation method of the burdock polysaccharide comprises the following steps:

[0006] (1) Stepwise enzymatic hydrolysis extraction: Take burdock root powder, add water and thermostable α-amylase, and enzymatically hydrolyze at 80 °C for 1 h; cool down to 60 °C, add papain and enzymatically hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min, then cool down to 60 °C, add glucoamylase and enzymatically hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min; after keeping warm at 80 °C for 3 h, centrifuge to obtain the supernatant and precipitate; The precipitate can be optionally repeated this step 1-2 times, and the obtained supernatant can be optionally combined with the supernatant obtained for the first time;

[0007] (2) Protein removal: The supernatant obtained in step (1) is deproteinized by the Sevage method to obtain a supernatant;

[0008] (3) Alcohol precipitation: Concentrate the supernatant obtained by deproteinization in step (2) to an appropriate volume, add 2-4 times the volume of absolute ethanol, let it stand overnight, and dry the obtained precipitate to obtain crude burdock polysaccharide;

[0009] (4) Purification: Take the crude burdock polysaccharide obtained in step (3), and purify it by one or a combination of two of anion exchange column chromatography and Sephadex column chromatography to obtain the burdock polysaccharide. (The purity is above 99%, the weight average molecular weight Mw is 4256 Da, and the number average molecular weight Mn is 3437 Da. The infrared spectrum of the burdock polysaccharide is consistent with Figure 8 basically. The 1 1H NMR spectrum of the burdock polysaccharide is consistent with Figure 9 basically.)

[0010] In step (1), the amount of water used is preferably 8-12 times the mass of the burdock root powder, and the amounts of thermostable α-amylase, papain, and glucoamylase used are preferably 250 μL of thermostable α-amylase (700,000 U / mL), 75 mg of papain (200,000 U / g), and 2 mL of glucoamylase (100,000 U / mL) per 100 g of burdock root powder.

[0011] In step (1), the burdock root powder is preferably pretreated as follows: before segmented enzymatic hydrolysis, it is preferably soaked in an alcohol solution (one or more of n-butanol and ethanol solution) with a volume fraction of 70%-95% for 2-5 h to remove small molecule impurities, and a precipitate (i.e., the burdock root powder with small molecule impurities removed) is obtained.

[0012] In step (2), in the Sevage method for protein removal, the amount of the Sevage reagent is one-fourth of the volume of the supernatant, and the used Sevage reagent is n-butanol: chloroform = 1:4, by volume ratio; in step (2), the Sevage method for protein removal is optionally repeated 3-5 times until there is no denatured protein layer at the interface of the two phases.

[0013] In step (3), standing overnight is preferably carried out at 0-4 °C overnight; centrifugation or filtration is preferably used to obtain a precipitate.

[0014] In step (4), the stationary phase of the anion exchange column chromatography is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, and the flow rate is: 0.5 mL / min; the stationary phase of the Sephacryl TM S-400 column chromatography is water, and the flow rate is 0.2 mL / min.

[0015] In step (4), the specific purification operation is as follows: take the crude burdock polysaccharide obtained in step (3), perform anion exchange column chromatography, the stationary phase is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, the flow rate is: 0.5 mL / min, collect every 5 min / tube, collect 100 tubes; take samples every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluates with the same retention time and a purity above 99%, and freeze-dry to obtain the burdock polysaccharide; optionally, the eluate with a purity less than 99% is further subjected to Sephacryl TM S-400 Sephacryl column chromatography, the mobile phase is water, the flow rate is 0.2 mL / min, collect every 15 min / tube, collect 100 tubes; take samples every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluates with the same retention time and a purity above 99%, and freeze-dry to obtain the burdock polysaccharide. HPLC conditions: Waters 2414 differential refractometer; SUGAR KS-805 (8.0×300 mm); mobile phase: ultrapure water; flow rate: 1 mL / min; column temperature: 35 °C; detector temperature: 30 °C; injection volume: 30 μL.

[0016] Another embodiment of the present invention provides a method for preparing the above-mentioned burdock polysaccharide, which is characterized by including the following steps:

[0017] (1) Extraction by stepwise enzymatic hydrolysis: Take burdock root powder, add water and heat-resistant α-amylase, and hydrolyze at 80 °C for 1 h; cool down to 60 °C, add papain and hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min, then cool down to 60 °C, add glucoamylase and hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min; after holding at 80 °C for 3 h, centrifuge to obtain the supernatant and precipitate; the precipitate can be optionally repeated this step 1-2 times, and the obtained supernatant can be optionally combined with the supernatant obtained for the first time;

[0018] (2) Protein removal: The supernatant obtained in step (1) is deproteinized by the Sevage method to obtain the supernatant;

[0019] (3) Alcohol precipitation: Concentrate the supernatant obtained by deproteinization in step (2) to an appropriate volume, add 2-4 times the volume of absolute ethanol, let stand overnight, and dry the obtained precipitate to obtain crude burdock polysaccharide;

[0020] (4) Purification: Take the crude burdock polysaccharide obtained in step (3), and purify it by one or two combinations of anion exchange column chromatography and Sephadex column chromatography to obtain the burdock polysaccharide. (The purity is above 99%, the weight-average molecular weight Mw is 4256 Da, and the number-average molecular weight Mn is 3437 Da. The infrared spectrum of the burdock polysaccharide is Figure 8 basically the same. The 1 1H NMR spectrum of the burdock polysaccharide is Figure 9 basically the same.)

[0021] In step (1), the amount of water used is preferably 8-12 times the mass of the burdock root powder, and the amounts of heat-resistant α-amylase, papain, and glucoamylase used are preferably 250 μL of heat-resistant α-amylase (700,000 U / mL), 75 mg of papain (200,000 U / g), and 2 mL of glucoamylase (100,000 U / mL) per 100 g of burdock root powder.

[0022] In step (1), the burdock root powder is preferably pretreated as follows: Before stepwise enzymatic hydrolysis, it is preferably soaked in an alcohol solution (one or several of n-butanol and ethanol solution) with a volume fraction of 70%-95% for 2-5 h to remove small molecule impurities, and a precipitate (i.e., burdock root powder after removing small molecule impurities) is obtained.

[0023] In step (2), the amount of Sevage reagent used in the Sevage method for protein removal is one-fourth of the volume of the supernatant, and the Sevage reagent used is n-butanol:chloroform = 1:4, by volume ratio; in step (2), the Sevage method for protein removal can be optionally repeated for 3-5 times until there is no denatured protein layer at the interface of the two phases.

[0024] In step (3), it is preferably left to stand overnight at 0-4 °C; it is preferably centrifuged or filtered to obtain the precipitate.

[0025] In step (4), the stationary phase of the anion exchange column chromatography is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, and the flow rate is: 0.5 mL / min; the stationary phase of the Sephadex column chromatography is Sephacryl TM S-400, the mobile phase is water, and the flow rate is 0.2 mL / min.

[0026] The specific purification operation in step (4) is as follows: Take the crude burdock polysaccharide obtained in step (3), perform anion exchange column chromatography, the stationary phase is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, the flow rate is: 0.5 mL / min, collect every 5 min per tube, collect 100 tubes; sample every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluents with the same retention time and a purity above 99%, and freeze-dry to obtain the burdock polysaccharide; optionally, the eluent with a purity less than 99% is further subjected to Sephacryl TM S-400 Sephadex column chromatography, the mobile phase is water, the flow rate is 0.2 mL / min, collect every 15 min per tube, collect 100 tubes; sample every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluents with the same retention time and a purity above 99%, and freeze-dry to obtain the burdock polysaccharide. HPLC conditions: Waters 2414 differential refractometer detector; SUGAR KS-805 (8.0×300 mm); mobile phase: ultrapure water; flow rate: 1 mL / min; column temperature: 35 °C; detector temperature: 30 °C; injection volume: 30 μL.

[0027] Another embodiment of the present invention provides the application of the above-mentioned burdock polysaccharide in the preparation of a drug for inhibiting pancreatic lipase.

[0028] Another embodiment of the present invention provides the application of the above-mentioned burdock polysaccharide in the preparation of a lipid-lowering drug.

[0029] Another embodiment of the present invention provides a lipid-lowering drug, which is characterized in that the drug uses the above-mentioned burdock polysaccharide or a pharmaceutically acceptable salt thereof as an active ingredient. The drug may further include pharmaceutically acceptable excipients, and its dosage form is preferably a solid preparation, a liquid preparation or a semi-solid preparation.

[0030] The burdock root powder of the present invention can be purchased through commercial channels or obtained by the following method: Cut the fresh burdock root into slices and immediately put them into boiling water to inactivate enzymes, take out the burdock root slices and drain the water, use microwave frequency conversion drying combined with low-temperature treatment and ultrafine pulverization technology to pulverize the burdock root, and store it in a powder dryer for later use.

[0031] The water described in the present invention is preferably deionized water, distilled water, double-distilled water, purified water, etc.

[0032] Compared with the prior art, the advantages of the present invention are as follows: (1) A brand-new burdock polysaccharide compound is obtained by a segmented enzymatic hydrolysis method in the present invention, with a purity of over 99%, a weight-average molecular weight Mw of 4256 Da, a number-average molecular weight Mn of 3437 Da, and Mw / Mn of 1.24, indicating good homogeneity of the burdock polysaccharide; (2) By using specific enzymes (thermostable α-amylase, papain, glucoamylase) and strictly controlling the enzyme dosage, enzymatic hydrolysis temperature, and time, a burdock polysaccharide (ALP) with a brand-new structure (molar ratio of fructose: glucose: galactose: arabinose: rhamnose being 0.675: 0.265: 0.023: 0.016: 0.009) is obtained in the present invention; (3) The crude burdock polysaccharide (ALCP) and burdock polysaccharide (ALP) in the present invention have significant inhibitory activity against pancreatic lipase, showing a dose correlation. The inhibition rate of ALCP is 45.44% at 10 mg / mL, while the IC 50 value of ALP is 8.18 mg / mL. (4) The inhibitory activity of the burdock polysaccharide (ALP) in the present invention against pancreatic lipase is far superior to that of the burdock polysaccharide with Mw between 2400 - 3000 Da disclosed in the prior art. The reason may be that the important pharmacophores are retained in the burdock polysaccharide obtained by the segmented enzymatic hydrolysis method in the present invention, and the specific structure-activity relationship remains to be further confirmed. Brief Description of the Drawings

[0033] Figure 1 is the glucose standard curve graph;

[0034] Figure 2 is the HPLC graph of ALP;

[0035] Figure 3 is the ultraviolet-visible spectrum graph of ALP;

[0036] Figure 4 is the ion chromatogram of a 16-monosaccharide mixed standard;

[0037] Figure 5 is the ion chromatogram of ALP;

[0038] Figure 6 is the standard curve graph of Mp, Mw, and Mn of ALP (A: Mp; B: Mw; C: Mn);

[0039] Figure 7 is the HPGPC graph of ALP;

[0040] Figure 8 is the infrared spectrum graph of ALP;

[0041] Figure 9 is the 1 1H NMR spectrum of ALP (600 MHz, D2O);

[0042] Figure 10 It is a graph showing the inhibition rates of ALCP and ALP on pancreatic lipase. Specific implementation manners

[0043] For the convenience of further understanding of the present invention, the following provided examples illustrate it in more detail. However, these examples are only for better understanding the invention and are not used to limit the scope or implementation principles of the present invention. The implementation manners of the present invention are not limited to the following content.

[0044] The burdock root powder used in the examples is optionally purchased through commercial channels or obtained by the following method: After slicing fresh burdock roots, immediately put them into boiling water to inactivate enzymes, take out the burdock root slices and drain the water, crush the burdock roots by using microwave frequency conversion drying combined with low-temperature treatment and ultrafine pulverization technology, and store the powder in a powder dryer for standby.

[0045] Example 1 Preparation of burdock crude polysaccharide (abbreviation: ALCP) and burdock polysaccharide (abbreviation: ALP)

[0046] Pretreatment: Soak the burdock root powder with 5 - 10 times the volume of 85% n-butanol solution for 2 h, then filter or centrifuge to obtain a precipitate. Then soak the precipitate with 5 - 10 times the volume of 85% ethanol solution for 2 h, filter or centrifuge to obtain a precipitate, and dry it (i.e., the burdock root powder from which small molecule impurities are removed).

[0047] (1) Stepwise enzymatic hydrolysis extraction: Take 100 g of the pretreated burdock root powder, add 1 L of deionized water and 250 μL of thermostable α-amylase (700,000 U / mL), and carry out enzymatic hydrolysis at 80 °C for 1 h; cool down to 60 °C, add 75 mg of papain (200,000 U / g) and carry out enzymatic hydrolysis for 1 h; heat up to the reflux temperature, inactivate for 30 min, cool down to 60 °C, add 2 mL of glucoamylase (100,000 U / mL) and carry out enzymatic hydrolysis for 1 h; heat up to the reflux temperature, inactivate for 30 min, then keep warm at 80 °C for 3 h, and centrifuge to obtain a supernatant and a precipitate; The precipitate is optionally repeated this step 1 - 2 times, and the obtained supernatant is optionally combined with the supernatant obtained for the first time;

[0048] (2) Deproteinization: The supernatant obtained in step (1) is deproteinized by the Sevage method to obtain a supernatant; The dosage of the Sevage reagent is one-fourth of the volume of the supernatant, and the used Sevage reagent is n-butanol:chloroform = 1:4, volume ratio; Optionally repeat the deproteinization operation 3 - 5 times until there is no denatured protein layer at the interface of the two phases;

[0049] (3) Alcohol precipitation: Concentrate the supernatant obtained by deproteinization in step (2) to an appropriate volume, add 2 - 4 times the volume of absolute ethanol, and let it stand overnight at 4 °C. Dry the obtained precipitate to obtain burdock crude polysaccharide (abbreviation: ALCP);

[0050] (4) Purification: Take the crude burdock polysaccharide obtained in step (3), perform anion exchange column chromatography, the stationary phase is selected from Cellulose DEAE-52, the mobile phase is selected from water, the flow rate is: 0.5 mL / min, collect every 5 min per tube, collect 100 tubes; take samples every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluents with the same retention time and a purity above 99%, and freeze-dry to obtain the said burdock polysaccharide (abbreviated as ALP); optionally, the eluent with a purity less than 99% is further subjected to Sephacryl TM S-400 dextran gel column chromatography, the mobile phase is water, the flow rate is 0.2 mL / min, collect every 15 min per tube, collect 100 tubes; take samples every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluents with the same retention time and a purity above 99%, and freeze-dry to obtain the said burdock polysaccharide. HPLC conditions: Waters 2414 differential refractometer; SUGAR KS-805 (8.0×300 mm); mobile phase: ultrapure water; flow rate: 1 mL / min; column temperature: 35 °C; detector temperature: 30 °C; injection volume: 30 μL.

[0051] Example 2 Analysis of Crude Burdock Polysaccharide (Abbreviated as ALCP) and Burdock Polysaccharide (Abbreviated as ALP)

[0052] 1. Determination of the total sugar content of ALCP

[0053] (1) Preparation of the standard curve: Weigh the glucose standard product and prepare the standard stock solution (1 mg / mL). Before each determination, first dilute it with distilled water to a standard solution of 100 μg / mL. Take 5 EP tubes, add 0.2, 0.4, 0.6, 0.8 and 1.0 mL of 100 μg / mL glucose standard solution respectively, add distilled water to 1.0 mL, then add 0.5 mL of 6% phenol solution and 2.5 mL of concentrated sulfuric acid successively, shake well and cool at room temperature for 20 min, and measure the absorbance at 490 nm. Plot the standard curve with the glucose concentration (μg / mL) on the abscissa and the absorbance (Absorbance) on the ordinate ( Figure 1 ).

[0054] (2) Determination of the total sugar of the polysaccharide sample: Take the polysaccharide sample to be measured, prepare it into a 100 μg / mL sample solution, add 0.5 mL of 6% phenol solution and 2.5 mL of concentrated sulfuric acid successively, shake well and let it stand and cool for 20 min. Measure the absorbance value at 490 nm, and calculate the total sugar content according to the standard curve.

[0055] The glucose standard curve has a good linear relationship between 0 - 100 μg / mL, and the linear regression equation is: y = 0.0074x - 0.0007, R 2= 0.9967. The absorbance of the sample was 0.5986, and the total sugar content of the crude burdock polysaccharide was calculated to be (80.99 ± 0.04)%.

[0056] 2. Purity analysis of ALP

[0057] HPLC conditions: Waters 2414 differential refractometer; SUGAR KS-805 (8.0×300 mm); mobile phase: ultrapure water; flow rate: 1 mL / min; column temperature: 35 °C; detector temperature: 30 °C; injection volume: 30 μL. The HPLC purity was above 99% ( Figure 2 ).

[0058] 3. UV-visible spectral analysis of ALP

[0059] Prepare a 1 mg / mL ALP sample solution with distilled water and scan it in the range of 200 - 799 nm. As Figure 3 shown, there is no absorption peak at 260 - 280 nm, indicating that ALP does not contain nucleic acids and proteins.

[0060] 4. Monosaccharide composition analysis of ALP

[0061] (1) Preparation of the standard stock solution: Take 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, D-galactosamine hydrochloride, D-glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) and prepare the standard stock solution.

[0062] (2) Preparation of the mixed standard solution: Take the standard stock solutions of each monosaccharide to prepare the mixed standard solution. According to the absolute quantification method, calculate the masses of different monosaccharides and calculate the molar ratios based on the molar masses of the monosaccharides.

[0063] (3) Sample preparation and analysis: Weigh 5 mg of the sample precisely into an ampoule, add 2 mL of 3 M trifluoroacetic acid, and hydrolyze it at 120 °C for 3 h. Pipette the acid hydrolysis solution into a flask, dry it under nitrogen, add 5 mL of water and vortex to mix evenly. Pipette 200 μL, add 800 μL of deionized water, centrifuge at 12000 rpm for 5 min, and take the supernatant for injection into an ion chromatograph (IC) to analyze the monosaccharide composition. Chromatographic conditions: electrochemical detector; Dionex CarbopacTM PA20 chromatographic column (3×150 mm); mobile phase A: H2O, mobile phase B: 15 mM NaOH, mobile phase C: 15 mM NaOH and 100 mM NaOAC; flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature 30 °C.

[0064] As can be seen from Table 1, ALP mainly contains fructose: glucose: galactose: arabinose: rhamnose, and their molar concentration ratio is 0.675: 0.265: 0.023: 0.016: 0.009. Among them, fructose: glucose = 2.55: 1.

[0065] Table 1 Monosaccharide composition of ALP

[0066]

[0067] 5. Determination of the molecular weight of ALP

[0068] The molecular weight of the polysaccharide was determined by high performance gel permeation chromatography (HPGPC): The polysaccharide sample and the standard were accurately weighed. The polysaccharide sample was prepared into a 5 mg / mL solution, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a microporous membrane for later use. Chromatographic conditions: RI-10A differential detector; BRT105-104-102 series gel column (8×300 mm); mobile phase: 0.05 M NaCl solution; flow rate: 0.6 mL / min, column temperature: 40 °C; injection volume: 20 μL.

[0069] Dextran standards with different molecular weights (relative molecular weights are: 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800 and 667800 Da) were prepared into 5 mg / mL solutions, filtered through a microporous membrane and injected successively. A standard curve was plotted with the elution volume (mL) as the abscissa and the logarithm (lgMw) of the dextran weight-average molecular weight as the ordinate. Standard curves of the number-average molecular weight (Mn) and the peak-average molecular weight (Mp) were plotted using the same method. The retention time of the polysaccharide sample was recorded, and the molecular weight of the polysaccharide sample was calculated according to the molecular weight standard curve.

[0070] According to the retention times of dextran standards with different molecular weights, a relative standard curve of the polysaccharide was plotted ( Figure 6 ). The equation of the lgMp-RT calibration curve is: y = -0.1804x + 11.563, R 2 = 0.9947; the equation of the lgMw-RT calibration curve is: y = -0.1923x + 12.115, R 2 = 0.993; the equation of the lgMn-RT calibration curve is: y = -0.1782x + 11.4, R 2 = 0.9906.

[0071] Calculate the molecular weight of burdock polysaccharide ALP according to the standard curve regression equation. The molecular weight diagram and calculation results of burdock polysaccharide are shown in Table 2. As can be seen from Table 2, the weight-average molecular weight Mw of burdock polysaccharide ALP is 4256 Da, the peak molecular weight Mp is 4001 Da, the number-average molecular weight Mn is 3437 Da, and Mw / Mn is 1.24, indicating that the polysaccharide has good homogeneity.

[0072] Table 2 Molecular weight information of ALP

[0073]

[0074] 6. Infrared spectrum analysis of ALP

[0075] Take 10 mg of dry ALP sample (purity 99%) and scan the infrared spectrum in the range of 4000 - 400 cm -1 . As Figure 8 shown, the broad and round peak shape in the 3343 cm -1 region indicates the presence of intermolecular or intramolecular hydrogen bonds in the hydroxyl groups of the ALP structure; the weak signal peak at 2930 cm -1 is the asymmetric stretching vibration of C-H in -CH, -CH2, and -CH3 groups; the absorption peak at 1634 cm -1 is the stretching vibration of C=O in the polysaccharide molecule. There is no absorption at 1700 cm -1 , indicating that ALP contains almost no uronic acid, which is consistent with the analysis of monosaccharide composition; the absorption peaks at 1430 and 1332 cm -1 may be caused by the stretching vibration of carbonyl products; the characteristic absorption at 1221 cm -1 is the stretching vibration of C-O-C glycosidic bond; there are strong absorption peaks at 1131 and 1031 cm -1 , which may be the characteristic absorption of pyranoside in ALP; 1000 - 800 cm -1 is the fingerprint region of the infrared spectrum. The absorption peaks at 934, 867, and 861 cm -1 are the characteristic fingerprint absorptions of long-chain inulin; the weak sharp peak at 597 cm -1 is caused by the bending vibration of the β-pyranose ring in the sugar molecule.

[0076] 7. 1H-NMR spectrum analysis of ALP

[0077] Weigh 20 mg of ALP (purity 99%) and dissolve it in D2O. After freeze-drying, redissolve it in D2O. Repeat three times. Finally, dissolve it in 0.5 mL of D2O and measure the 1H-NMR spectrum.

[0078] The 1 1H-NMR spectrum of burdock polysaccharide ALP ( Figure 9)Displays typical hydrogen signals of polysaccharides, specifically: δ H-1 The signal of the anomeric proton of the α configuration of the glucose residue is at 5.51 (s) ppm, δ H-2 3.55 (m) and δ H-4 3.63 (m) ppm are the signals of the two hydrogens adjacent to the oxygenated methylene group of the glucose residue; the hydrogen signals of the β-fructose residue are at 3.70 - 4.33 ppm. Among them, δ H-3’ 4.33 (d, 8.4 Hz) and δ H-4’ 4.16 (t, 8.4 Hz) ppm are the signals of the hydrogens coupled to the adjacent position, δ H-1’ 3.78 (m) and 3.99 (m) ppm, δ H-6’ 3.82 - 3.70 (m) ppm are the signals of two pairs of oxygenated methylene hydrogens, δ H-5’ 3.93 (m) ppm is the signal of one hydrogen adjacent to the oxygenated methylene group; in addition, a singlet signal is also shown at 2.80 (s) ppm, and its assignment remains to be determined. Compared with the literature, the preliminary assignment of the 1 1H-NMR spectral signals of burdock polysaccharide ALP is listed in Table 3.

[0079] Table 3 1 1H-NMR spectral data of ALP (600 MHz, D2O)

[0080]

[0081] Example 3

[0082] Test of pancreatic lipase activity of crude burdock polysaccharide (abbreviated as ALCP) and burdock polysaccharide (abbreviated as ALP)

[0083]

[0084] A blank : Pancreatic lipase + buffer + lauroyl-4-nitrophenol

[0085] A control : Buffer + 4-Nitrophenyl laurate

[0086] A sample : Arctiin + Pancreatic lipase + Buffer + 4-Nitrophenyl laurate

[0087] A test : Arctiin + Buffer + 4-Nitrophenyl laurate

[0088] The test results showed that the in vitro pancreatic lipase inhibitory activities of ALP and ALCP were dose-dependent. The IC 50 value of ALP was 8.18 mg / mL, while the inhibition rate of ALCP was 45.44% at 10 mg / mL ( Figure 10 ).

Claims

1. A kind of burdock polysaccharide, characterized in that The HPLC purity of the burdock polysaccharide is above 99%, the weight-average molecular weight Mw is 4256 Da, the number-average molecular weight Mn is 3437 Da. In the burdock polysaccharide, the molar ratio of fructose: glucose: galactose: arabinose: rhamnose is 0.675: 0.265: 0.023: 0.016: 0.

009.

2. The preparation method of the burdock polysaccharide according to claim 1, characterized in that It includes the following steps: (1) Stepwise enzymatic extraction: Take burdock root powder, add water and thermotolerant α-amylase, and enzymatically hydrolyze at 80 °C for 1 h; cool down to 60 °C, add papain and enzymatically hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min, then cool down to 60 °C, add glucoamylase and enzymatically hydrolyze for 1 h; heat up to the reflux temperature, inactivate for 30 min, keep warm at 80 °C for 3 h, and then centrifuge to obtain the supernatant and precipitate; The precipitate can be optionally repeated this step 1-2 times, and the obtained supernatant can be optionally combined with the supernatant obtained for the first time; (2) Protein removal: The supernatant obtained in step (1) is deproteinized by the Sevage method to obtain the supernatant; (3) Alcohol precipitation: Concentrate the supernatant obtained by deproteinization in step (2) to an appropriate volume, add 2-4 times the volume of absolute ethanol, let it stand overnight, and dry the obtained precipitate to obtain crude burdock polysaccharide; (4) Purification: Take the crude burdock polysaccharide obtained in step (3), perform anion exchange column chromatography to purify the burdock polysaccharide.

3. The preparation method according to claim 2, characterized in that In step (1), the amount of water used is 8-12 times the mass of the burdock root powder, and the amounts of thermotolerant α-amylase, papain, and glucoamylase used are 250 μL of 700,000 U / mL thermotolerant α-amylase, 75 mg of 200,000 U / g papain, and 2 mL of 100,000 U / mL glucoamylase per 100 g of burdock root powder.

4. The preparation method according to claim 2, characterized in that In step (4), the stationary phase of the anion exchange column chromatography is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, and the flow rate is: 0.5 mL / min.

5. The preparation method according to claim 2, characterized in that The specific purification operation in step (4) is as follows: Take the crude burdock polysaccharide obtained in step (3), perform anion exchange column chromatography, the stationary phase is selected from Cellulose DEAE-32 or DEAE-52, the mobile phase is selected from water, the flow rate is: 0.5 mL / min, collect every 5 min / tube, collect 100 tubes; take samples every two tubes, filter through a microporous membrane, analyze the purity by HPLC, combine the eluents with the same retention time and purity above 99%, and freeze-dry to obtain the burdock polysaccharide.

6. Use of the burdock polysaccharide according to claim 1 in the preparation of a drug for inhibiting pancreatic lipase.

7. A lipid-lowering drug, characterized in that The drug uses the burdock polysaccharide according to claim 1 as an active ingredient.

8. The lipid-lowering drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients, and its dosage form is selected from solid preparations, liquid preparations or semi-solid preparations.

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