CN115716884B

CN115716884B - Polygonatum cyrtonema polysaccharide and preparation method and application thereof

Info

Publication number: CN115716884B
Application number: CN202211446797.9A
Authority: CN
Inventors: 王国栋; 韩军; 邵太丽; 刘伟; 柳春燕; 袁平川; 陈静
Original assignee: Wannan Medical College
Current assignee: Wannan Medical College
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-09-29
Anticipated expiration: 2042-11-18
Also published as: CN115716884A

Abstract

The invention provides a polygonatum cyrtonema polysaccharide, a preparation method and application thereof, wherein the polygonatum cyrtonema polysaccharide comprises four monosaccharides in a molar ratio of: fructose: glucose: mannose: galactose=88.1: 9.7:1.6:0.3; compared with the prior art, the uniform polysaccharide component obtained from polygonatum cyrtonema can obviously improve oxidative stress and intestinal flora structural imbalance of NAFLD, and can be used as a natural ingredient in medicines for treating NAFLD.

Description

Polygonatum cyrtonema polysaccharide and preparation method and application thereof

Technical Field

The invention belongs to the field of plant polysaccharide, and particularly relates to a polygonatum cyrtonema polysaccharide, a preparation method and application thereof.

Background

Polysaccharides are one of the four basic substances constituting life, and are usually linked by more than 10 monosaccharides through different types of glycosidic bonds. The natural polysaccharide has various biological effects such as blood lipid reduction, blood sugar reduction, antioxidation, anti-tumor, immunoregulation, anti-inflammatory and the like, can directly influence organism substances and energy metabolism, regulate intestinal flora composition and maintain physical health, and has important medicinal value.

Non-alcoholic fatty liver disease (Non-alcoholic fatty liver disease, NAFLD) generally refers to a clinically pathological syndrome caused by excluding alcohol and other factors with defined liver damage. Histological is mainly characterized by diffuse hepatocyte bullous fat. The prevalence of NAFLD has increased year by year in recent years and has been considered one of the major public health problems worldwide. At present, no effective medicine for treating NAFLD is clinically available, and antioxidants, insulin sensitizers, lipid regulators and the like are generally adopted for clinical treatment of NAFLD.

The rhizoma polygonati contains rich polysaccharide components, has complex chemical structure, different sources and different varieties, has great difference in polysaccharide content, structure and activity, and the polygonatum cyrtonema (Polygonatum cyrtonema Hua, PCH) is a perennial herb in the genus polygonatum of the family Liliaceae, has the effects of tonifying kidney, replenishing essence and nourishing yin to moisten dryness, is one of 3 types of rhizoma polygonati collected in the Chinese pharmacopoeia of 2020 edition, and is widely distributed in southern areas of China. Polygonatum cyrtonema polysaccharide is the main active component in Polygonatum cyrtonema, and has been proved to have pharmacological activities of antioxidation, blood sugar reduction, blood lipid reduction, atherosclerosis resistance, tumor resistance, fatigue resistance and the like.

Disclosure of Invention

The invention aims to provide polygonatum cyrtonema polysaccharide and a preparation method thereof, wherein a uniform component named PCP1 of polygonatum cyrtonema polysaccharide is extracted and separated from rhizomes of polygonatum cyrtonema, and the uniform component mainly consists of fructose, glucose and mannose.

The invention also aims to provide an application of the polygonatum cyrtonema polysaccharide in preparing medicines for treating non-alcoholic fatty liver.

The specific technical scheme of the invention is as follows:

polygonatum cyrtonema polysaccharide with weight average molecular weight of 4650Da and number average molecular weight of 4420Da; purity 98.68%; the polygonatum cyrtonema polysaccharide comprises four monosaccharides in the molar ratio of: fructose: glucose: mannose: galactose=88.1: 9.7:1.6:0.3; the main chain of the polygonatum cyrtonema polysaccharide mainly comprises → 1) -beta-D-Fruf- (2 → 1, 6) -beta-D-Fruf- (2 → and a small amount of → 6) -alpha-D-Glcp- (1 → 4) -beta-D-Manp- (1 → beta-D-Glcp- (1 → and the side chain mainly comprises beta-D-Fruf- (2 → C-6 position connected at → 1, 6) -beta-D-Fruf- (2-), wherein Fruf represents fructofuranose, glcp represents glucopyranose, and Manp represents mannopyranose.

The invention provides a preparation method of polygonatum cyrtonema polysaccharide, which comprises the following steps:

1) Drying and crushing fresh rhizome of Polygonatum cyrtonema to obtain rhizome powder of Polygonatum cyrtonema;

2) Extracting rhizome powder of Polygonatum cyrtonema Fabricius with water under heating, centrifuging, and retaining supernatant; concentrating the supernatant, adding absolute ethyl alcohol, precipitating with alcohol at low temperature, centrifuging, and retaining the precipitate to obtain rhizoma polygonati polysaccharide;

3) Adding water into the polygonatum cyrtonema polysaccharide, heating for re-dissolving, centrifuging, decoloring supernatant, concentrating decolored liquid, filtering by a filter membrane, removing protein by using a Sevag reagent, dialyzing at low temperature, and finally, freeze-drying the obtained polysaccharide solution to obtain primarily purified polygonatum cyrtonema polysaccharide powder;

4) Dissolving the primarily purified polygonatum cyrtonema polysaccharide powder in water, filtering, eluting with deionized water, concentrating the eluent, dialyzing at low temperature, and freeze-drying to obtain polygonatum cyrtonema polysaccharide water-washing component powder;

5) Dissolving the polygonatum cyrtonema polysaccharide water-washed component powder in water, eluting, concentrating, dialyzing, and freeze-drying to obtain the polygonatum cyrtonema polysaccharide uniform component PCP1.

The step 1) is specifically as follows: the fresh rhizome of Polygonatum cyrtonema Fabricius is cut into slices with the diameter of 6-8 mm after being cleaned and beard removed, and then is dried for 72-96 hours at the temperature of 60 ℃, and then is crushed by an electric crusher and is sieved by a 60-mesh sieve, so that rhizome powder of Polygonatum cyrtonema Fabricius with uniform particle size is obtained.

Preferably, the fresh rhizome of Polygonatum cyrtonema is selected from the rhizome of Polygonatum cyrtonema.

Step 2), heating and extracting at 75 ℃ for 1.5-2 hours;

preferably, in the step 2), heating and extracting for 2 times, centrifuging after the first extraction, reserving supernatant, continuously adding deionized water into the sediment, and extracting for 1.5-2 hours according to the same method;

the alcohol precipitation under the low-temperature condition in the step 2) is specifically as follows: alcohol precipitation in a refrigerator at 4 ℃ for 12h; during alcohol precipitation, absolute ethyl alcohol is added to ensure that the volume fraction of the ethyl alcohol in the mixed solution is 75 percent;

preferably, step 2) specifically comprises: adding deionized water into rhizome powder of Polygonatum cyrtonema Fabricius, wherein the ratio of the solution to the solid is 1:20-24; heating to 75 ℃ and extracting for 1.5-2 h; after cooling, centrifuging for 15-20 min at 5000-8000 r/min to retain supernatant, continuously adding 1000-1200 mL of deionized water into the precipitate to repeatedly extract for 1.5-2 h, centrifuging for 15-20 min at 5000-8000 r/min, and combining the supernatants; concentrating to 200-300 mL, slowly adding absolute ethanol until the volume fraction of the ethanol is 75%, precipitating with ethanol in a refrigerator at 4 ℃ for 12h, and centrifuging at 8000r/min for 20min to obtain Polygonatum cyrtonema Sieb crude polysaccharide;

the step 3) is specifically as follows: adding 200-300 mL of deionized water into the polygonatum cyrtonema rhizome crude polysaccharide, heating to 60 ℃ and slowly stirring to re-dissolve the polygonatum cyrtonema rhizome crude polysaccharide until the solution is clear, centrifuging for 15min at 12000-15000 r/min after the precipitate is not dissolved continuously, taking supernatant, decoloring with D101 macroporous resin, concentrating decolored liquid to 1/10-1/5 of the volume of the original solution, filtering with a 0.22 mu m microporous filter membrane, removing protein with a Sevag reagent, determining with a Bradford method, proving that the protein is removed completely, removing the Sevag reagent by rotary evaporation at 60 ℃, filtering and sterilizing with a 0.22 mu m microporous filter membrane, and dialyzing for 72h at 0-4 ℃ with a dialysis bag with a cut-off molecular weight of 1000Da to remove small molecular substances; finally, the polysaccharide solution is freeze-dried for 72 hours at the temperature of minus 80 ℃ to obtain the primarily purified polygonatum cyrtonema polysaccharide powder.

The step 4) is specifically as follows: weighing 200mg of primarily purified polygonatum cyrtonema polysaccharide powder, adding 20-40 mL of deionized water for dissolution, filtering by a 0.22 mu m microporous membrane, loading the mixture on a DEAE-52 cellulose anion exchange chromatographic column, eluting by using deionized water at a flow rate of 0.4mL/min, collecting one tube every 10min, detecting whether the polysaccharide is completely eluted by using an anthrone-sulfuric acid reagent, concentrating eluent to 1/10-1/5 of the original volume after the polysaccharide is completely eluted, dialyzing for 72 hours by using a dialysis bag with a molecular weight cutoff of 1000Da at a temperature of 0-4 ℃, and freeze-drying for 72 hours at a temperature of-80 ℃ to obtain polygonatum cyrtonema polysaccharide water-washed component powder;

the step 5) is specifically as follows: weighing 30mg of polygonatum cyrtonema polysaccharide water-washing component powder, adding 4-6 mL of deionized water for dissolving at normal temperature, filtering by a 0.22 mu m microporous filter membrane, loading to a Sephadex G-75 gel chromatographic column, eluting by using the deionized water at the flow rate of 0.38mL/min, collecting one tube every 10min, concentrating eluent to 1/10-1/5 of the original volume, dialyzing for 72h by using a dialysis bag with the molecular weight cutoff of 1000Da at the temperature of 0-4 ℃, and freeze-drying for 72h at the temperature of-80 ℃ to obtain the polygonatum cyrtonema polysaccharide uniform component PCP1.

The invention also aims to provide an application of the polygonatum cyrtonema polysaccharide in preparing medicines for treating non-alcoholic fatty liver. Polysaccharides can repair the intestinal barrier by alleviating oxidative stress damage, balancing intestinal flora structure, and promoting the production of short chain fatty acids. Compared with the prior art, the uniform polysaccharide component PCP1 obtained from Polygonatum cyrtonema Fabricius can obviously improve oxidative stress and intestinal flora structural imbalance of NAFLD, and can be used as a natural component in medicines for treating NAFLD.

Drawings

FIG. 1 shows the purity, molecular weight, and infrared detection results of PCP 1; FIG. A shows the elution profile of a DEAE-52 cellulose column; b is an elution curve of the Sephadex G-75 gel chromatographic column; c is the absolute molecular weight distribution map of PCP 1; d is an infrared spectrogram of PCP 1; e is a High Performance Gel Permeation Chromatography (HPGPC) analysis chart of PCP 1;

FIG. 2 is a diagram showing the results of monosaccharide composition of PCP 1; in the figure, A is a graph of the results of standard monosaccharides; b is a graph showing the result of hydrolysis of PCP1 with 2M TFA at 121℃for 2h; c is a graph showing the result of hydrolyzing PCP1 with 2.5M TFA at 60℃for 1 h; wherein, standard monosaccharide: fucose (Fuc), arabinose (Ara), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA);

FIG. 3 shows the methylation results of PCP 1; wherein A is a gas chromatography-mass spectrometry (GC-MS) total ion flow diagram of PCP 1; b is a secondary fragment ion flow diagram;

FIG. 4A shows PCP1 ¹ H spectrogram;

FIG. 4B is a schematic view of ¹³ C, spectrogram;

FIG. 4C is a DEPT-135 spectrum;

FIG. 4D is a COSY spectrum;

FIG. 4E is a spectrum of HSQC;

FIG. 4F is a HMBC spectra;

FIG. 5 is a main sugar chain structure diagram of PCP 1;

FIG. 6 is a graph showing the effect of PCP1 on liver injury and lipid metabolism; wherein A is H & E and oil red O staining chart; b is a graph of changes in liver alanine Aminotransferase (ALT) levels; c is a graph of changes in liver aspartate Aminotransferase (AST) levels; d is liver index (the percentage of wet liver weight of mice at the end of the experiment to body weight); e is a serum Triglyceride (TG) level change map; f is a serum Total Cholesterol (TC) level profile; g is a graph of serum low density lipoprotein cholesterol (LDL-C) level change; h is a graph of serum high density lipoprotein cholesterol (HDL-C) level change; data are expressed as mean ± s.d. (n=6); p <0.05, < P < 0.01 compared to ND group; compared with the HFD group, the # P is less than 0.05, and the # P is less than 0.01;

FIG. 7 is the effect of PCP1 on oxidative stress and oral glucose tolerance in mice; wherein A is a liver superoxide dismutase (SOD) level change chart; b is a liver Glutathione (GSH) level change map; c is a graph of liver Malondialdehyde (MDA) level change; d is a graph of the oral glucose tolerance test result of the mice; e is the area under the curve of the oral glucose tolerance test time-blood glucose curve; data are expressed as mean ± s.d. (n=6); p <0.05, < P < 0.01 compared to ND group; compared with the HFD group, the # P is less than 0.05, and the # P is less than 0.01;

FIG. 8 shows the effect of PCP1 on the overall structure of the intestinal flora; wherein a is a diversity analysis: chao1, observed features, simpson and Shannon index plots; b is an unweighted principal coordinate analysis (PCoA) score map based on UniFrac; c is a Venn diagram; data are shown as mean ± s.d. (n=5); p <0.05, < P < 0.01 compared to ND group; compared with HFD group, #P < 0.05;

FIG. 9 is the effect of PCP1 on intestinal flora and short chain fatty acid levels at the phylum and genus levels; wherein A is a graph of the relative abundance of the flora at the portal level; b is the ratio of the relative abundance of Firmics/bacterioides; c is a relative abundance change of several representative genera; d is the LEfSe analysis result; e is the effect of PCP1 on fecal short chain fatty acids; data are shown as mean ± s.d. (n=5); p <0.05, < P < 0.01 compared to ND group; compared with the HFD group, #P <0.05, #P < 0.01.

Detailed Description

Materials and reagents used in the invention:

fresh Jiuhua rhizoma Polygonati dug from Jiuhua mountain of Anhui province was identified by a Liu Chunyan researcher (national academy of medical science in Anhui, china) as Polygonatum cyrtonema (Polygonatum cyrtonema Hua, PCH). DEAE-52 anion exchange cellulose and Sephadex G-75 Sephadex gel (Beijing Soy Co., ltd.). Triglyceride (TG), total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), superoxide dismutase (SOD), glutathione (GSH), and Malondialdehyde (MDA) kits (institute of biotechnology, made by tokyo). Unless otherwise indicated, all other reagents were analytical grade.

Example 1

A preparation method of polygonatum cyrtonema polysaccharide PCP1 comprises the following steps:

1) The fresh rhizome of the Polygonatum cyrtonema Fabricius is cut into slices with the diameter of 6-8 mm after being cleaned and beard removed, is dried for 96 hours at the temperature of 60 ℃, is crushed by an electric crusher, and is sieved by a 60-mesh sieve to obtain rhizome powder of the Polygonatum cyrtonema Fabricius with uniform particle size;

2) Weighing 50g of rhizome powder of Polygonatum cyrtonema Fabricius, adding 1200mL of deionized water, heating to 75 ℃, and extracting for 2h; centrifuging at 8000r/min for 15min after cooling, reserving supernatant, continuously adding 1200mL of deionized water into precipitate for repeated extraction for 2h, centrifuging at 8000r/min for 15min, combining the supernatant of the two times, concentrating to 200-300 mL under reduced pressure at 60 ℃, adding absolute ethyl alcohol until the final volume fraction of the ethanol is 75%, centrifuging at 8000r/min for 20min after 12h of alcohol precipitation in a refrigerator at 4 ℃ to obtain polygonatum cyrtonema rhizome crude polysaccharide;

3) Adding 300mL of 60 ℃ deionized water into the polygonatum cyrtonema rhizome crude polysaccharide prepared in the step 2), heating and stirring (re-dissolving) until the solution is clear, centrifuging for 15min at 15000r/min after the precipitate is not dissolved continuously, taking supernatant, decoloring with D101 macroporous resin, concentrating decolored liquid to 1/10-1/5 of the volume of the original solution, filtering with a 0.22 mu m microporous membrane, removing protein with a Sevag reagent, determining by a Bradford method, proving that the protein is removed completely, removing the Sevag reagent by rotary evaporation at 60 ℃, sterilizing by using a 0.22 mu m microporous membrane, and dialyzing for 72h in a refrigerator at 4 ℃ by using a dialysis bag with a cutoff molecular weight of 1000Da to remove micromolecule substances; finally, the polysaccharide solution is freeze-dried for 72 hours at the temperature of minus 80 ℃ to obtain the primarily purified polygonatum cyrtonema polysaccharide powder.

4) Purifying: weighing 200mg of primarily purified polygonatum cyrtonema polysaccharide powder, adding 20-40 mL of deionized water for dissolution, filtering by a 0.22 mu m microporous membrane, loading into a DEAE-52 cellulose anion exchange chromatography column, eluting by using deionized water at a flow rate of 0.4mL/min, collecting a tube every 10min, detecting whether the polysaccharide is completely eluted by using an anthrone-sulfuric acid reagent, concentrating eluent under reduced pressure to 1/10-1/5 of the original volume at 60 ℃ after the polysaccharide is completely eluted, dialyzing for 72h by using a dialysis bag with a molecular weight cutoff of 1000Da at a refrigerator at 4 ℃, and freeze-drying at-80 ℃ to obtain polygonatum cyrtonema polysaccharide water-washed component powder;

5) Weighing 30mg of polygonatum cyrtonema polysaccharide water-washing component powder, adding 4-6 mL of deionized water for dissolution, filtering by a 0.22 mu m microporous filter membrane, loading to a Sephadex G-75 gel chromatographic column, eluting by using the deionized water at the flow rate of 0.38mL/min, collecting a tube every 10min, concentrating eluent to 1/10-1/5 of the original volume, dialyzing for 72h in a refrigerator by using a dialysis bag with the molecular weight cutoff of 1000Da, and freeze-drying at the temperature of minus 80 ℃ for 72h to obtain the polygonatum cyrtonema polysaccharide uniform component PCP1.

The purity of the prepared polygonatum cyrtonema polysaccharide uniform component PCP1 is detected, and the purity is specifically as follows:

the purity of PCP1 was checked using an LC-10Avp High Performance Gel Permeation Chromatography (HPGPC) system (Shimadzu corporation). Chromatographic column: TSKgel G3000PWXL (7.8 mmID. Times.300 mm, tosoh Corp, japan); a detector: RID-10A differential refractive detector; mobile phase: deionized water; flow rate: 0.7mL/min; column temperature: 40 ℃; sample injection amount: 20. Mu.L.

The detection experiment flow comprises the following steps: accurately weighing PCP1 sample, dissolving in deionized water to prepare 5mg/mL solution, filtering with 0.22 μm microporous membrane, sampling 20 μl, and detecting PCP1 purity.

Results: extracting rhizoma polygonati multiflori rhizome powder with water, precipitating with ethanol, decolorizing, and removing protein to obtain rhizoma polygonati multiflori crude polysaccharide with yield of 8.37% and protein content of 0.87% (< 1%); the crude polysaccharide is separated and purified by DEAE-52 and Sephadex G-75 columns to obtain the polysaccharide homogeneous component PCP1 of Polygonatum cyrtonema, the yield is 5.6%. Elution curves are shown in fig. 1 a and B, indicating that PCP1 is the main and readily available component of polygonatum cyrtonema polysaccharide. HPGPC analysis (E in FIG. 1) showed that PCP1 exhibited a single symmetrical narrow peak with a retention time of 12.17min and purity of 98.68%, indicating that PCP1 was a homogeneous polysaccharide of high purity and concentrated molecular weight.

The absolute molecular weight of PCP1 was further determined:

the absolute molecular weight of PCP1 was determined using a gel chromatography-differential-multi-angle laser light scattering (SEC-MALLS-RI) system. Gel exclusion chromatographic column: ohpak SB-805HQ (300X 8 mm), ohpak SB-804HQ (300X 8 mm), ohpak SB-803HQ (300X 8 mm) are connected in series; differential detector: optilab T-rEX (Wyatt technology, U.S.); excitation light scattering detector: DAWN HELEOS ii (Wyatt technology, usa); mobile phase: 0.1mol/L sodium nitrate solution; flow rate: 0.4mL/min; column temperature: 45 ℃; sample injection amount: 100. Mu.L.

The experimental procedure is as follows: accurate weighing of PCP1 samples dissolved in a solution containing 0.02% NaN ₃ In the aqueous solution of sodium nitrate of 0.1mol/L, 1mg/mL of sample solution is prepared, and 100 mu L of sample solution is taken for machine detection after the sample solution is filtered by a microporous filter membrane of 0.22 mu m.

Results: as shown in FIG. 1C, PCP1 was a polysaccharide having a concentrated molecular weight distribution, as determined by the SEC-MALLS-RI system, having a weight average molecular weight (Mw) and a number average molecular weight (Mn) of 4650Da and 4420Da, respectively, and a polydispersity index (Mw/Mn) of about 1.05, again.

The infrared spectrum analysis of PCP1 is specifically as follows:

the experimental procedure is as follows: accurately weighing PCP1 powderPowder 1 (+ -0.05) mg was mixed with analytically pure potassium bromide powder, ground and pressed into a tablet form, and 4000-400cm was measured on an infrared spectrometer (Nexus IS10FT-IR, thermo Nicolet, USA) ^-1 Spectrum in the wavelength range.

Results: the infrared analysis (D in FIG. 1) showed that 3390cm ^-1 There is characteristic O-H stretching vibration peak, 2940cm ^-1 C-H stretching vibration peak exists nearby, 1650cm ^-1 And 1420cm ^-1 The absorption peaks at 1130 and 1020cm are due to the presence of bound water in the polysaccharide ^-1 The absorption peak at this point was due to the stretching vibration of the C-O-C glycosidic bond, which suggests the presence of furanose glycosidic bond in PCP1. Furthermore, 930cm ^-1 The presence of the absorption peak indicates the presence of a pyran type glycoside bond in PCP1. Thus, the infrared analysis results suggest that both furan-type and pyran-type glycosidic linkages may be present in PCP1.

Further analysis of PCP1 monosaccharide composition was performed as follows:

two methods were used to detect the monosaccharide composition of PCP1.

The method comprises the following steps: PCP1 sample 5 (+ -0.05) mg was accurately weighed, 1mL of 2M trifluoroacetic acid (TFA) solution was added and hydrolyzed at 121℃for 2h, nitrogen was purged, and the mixture was blown dry. Adding methanol for cleaning, drying, and repeating methanol cleaning for 2-3 times. After dissolution with deionized water, 5. Mu.L (51.8. Mu.g/mL) was taken and checked on a machine.

The second method is as follows: PCP1 sample 5 (+ -0.05) mg was accurately weighed, 1mL of 2.5M TFA solution was added and hydrolyzed at 60℃for 1h, and then nitrogen was introduced and dried. Adding methanol for cleaning, drying, and repeating methanol cleaning for 2-3 times. After dissolution with deionized water, 5. Mu.L (51.8. Mu.g/mL) was taken and checked on a machine. Wherein standard monosaccharides were accurately weighed and formulated into a 50 μg/mL control solution, and the standard monosaccharides used were fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid, guluronic acid (Shanghai Ala Biotechnology Co., ltd.).

Ion chromatography parameter setting: chromatograph: thermo ICS5000 ion chromatograph (Thermo Fisher Scientific, usa); chromatographic column: dionex ^TM CarboPac ^TM PA20 (150 mm. Times.3 mm,10 μm); mobile phase a:0.1mol/L sodium hydroxide solution; mobile phase B:0.1mol/L sodium hydroxide solution, 0.2mol/L sodium acetate solution; flow rate: 0.5mL/min; column temperature: 30 ℃; elution gradient: 0min A/B phase (95:5, V/V), 30min A/B phase (80:20, V/V), 30.1min A/B phase (60:40, V/V), 45min A/B phase (60:40, V/V), 45.1min A/B phase (95:5, V/V), 60min A/B phase (95:5, V/V); sample injection amount: 5. Mu.L.

Results: comparing the ion chromatograms of standard monosaccharides (a in fig. 2), conventional hydrolysis (B in fig. 2), and low-temperature hydrolysis (C in fig. 2), PCP1 is mainly composed of four monosaccharides in the molar ratio: fructose: glucose: mannose: galactose=88.1: 9.7:1.6:0.3.

methylation analysis of PCP1 is specifically as follows:

analysis was performed using gas chromatography-mass spectrometry (GC-MS, 7890A-5977B, agilent corporation, USA). Model of automatic sampler: G4567A; a gas chromatograph (Agilent 7890A, agilent Co., U.S.A.), the sample injection amount is 1. Mu.L, the split ratio is 10:1, and the carrier gas is high-purity helium gas; temperature: the initial temperature of the column oven is kept at 140 ℃ for 2min, and the temperature is programmed to be 230 ℃ at 3 ℃/min and kept for 3min. Quadrupole mass spectrometer (Agilent 5977B, agilent company, usa), electron bombardment ion source (EI) and MassHunter workstation, analyte detection in full scan mode, mass scan range (m/z): 30-600.

The experimental procedure is as follows: the monosaccharide composition analysis results suggest that PCP1 is a neutral polysaccharide and fructose is the main component, so the methylation experiment was designed as follows: accurately weighing 2 (+ -0.05) mg PCP1 sample, dissolving in 500 mu L anhydrous DMSO, adding 1mg solid sodium hydroxide, and reacting for 0.5h. 50. Mu.L of methyl iodide solution was added and reacted for 1 hour, and after extraction with 500. Mu.L of methylene chloride, the sample was evaporated to dryness. Adding 100 mu L of 2mol/L TFA, hydrolyzing at 60 ℃ for 0.5h, dissolving the dried hydrolysate in 50 mu L of 2mol/L ammonia water, adding 50 mu L of 1mol/L sodium borohydride, and reacting at 25 ℃ for 2.5h; then, 20. Mu.L of acetic anhydride was added thereto and reacted at 100℃for 2.5 hours to terminate the reaction. Finally, after adding 500 mu L of dichloromethane and mixing uniformly, centrifuging, discarding the water phase, repeating the water washing for 3 times, and taking the dichloromethane phase for machine detection.

Results: based on the GC-MS data signal of PCP1 in FIG. 3, the methylation analysis results shown in Table 1 were obtained by comparing the partial methylated sugar alcohol acetate (PMAAs) public databases.

TABLE 1 methylation results of PCP1

Nuclear magnetic resonance analysis of PCP 1:

the experimental procedure is as follows: 20mg of PCP1 sample was weighed and dissolved in 0.5mL of D ₂ O (99.9%). Acquisition at 25℃using a JNM-ECZ600R/S1 spectrometer (600 MHz, JEOL, japan) ¹ H NMR、 ¹³ C NMR、 ¹ H- ¹ H correlation Spectrum [ ] ¹ H- ¹ H COSY), hydrocarbon correlation spectroscopy (HSQC), and hydrocarbon remote correlation spectroscopy (HMBC).

Results: ¹ the H NMR spectrum (FIG. 4A) shows that the proton signal is concentrated in the range of δ3.4-5.4 and that there are multiple anomeric hydrogen signals in the region of δ4.5-5.4, indicating the presence of multiple sugar residues. ¹³ The signal of the C NMR spectrum (FIG. 4B) was concentrated in the delta 55-110 region, and three strong signals were found in the anomeric carbon domains, delta 103.52, 103.77, 104.08, but disappeared in the DEPT-135 spectrum (FIG. 4C), indicating that the three anomeric carbons were quaternary carbons, and there was no hydrogen related signal in the HSQC spectrum (FIG. 4E). Furthermore, from monosaccharide composition and literature reports, it can be inferred that the anomeric carbon signals δ 103.52, 104.08, 103.77 are respectively assigned to the C-2 signals of → 1) - β -D-Fruf- (2 → 1, 6) - β -D-Fruf- (2 → β -D-Fruf- (2- & gt residues) and are labeled as residues A, B, C. Furthermore, ¹ h (delta 3.4-4.2) and ¹³ the strong sugar ring signal in the C (delta 55-90) NMR spectrum is mainly represented by the chemical shift characteristics of H and C on the fructose sugar ring, and can be combined ¹ H- ¹ The H COSY spectrum ascribed it (FIG. 4D). In combination with the relevant literature ¹ H、 ¹³ C、DEPT-135、 ¹ H- ¹ H COSY, HSQC and HMBC NMR spectra, fructose residue A, B, C ¹ H and ¹³ c chemical shifts are shown in table 2.

There are significant correlation signals in the HSQC spectrum, including delta 5.34/92.21, 4.72/100.27, 4.68/104.36 (FIG. 4E). Combining literature and HSQC spectra, it can be deduced that the simultaneous presence of δ5.34/92.21, 4.72/100.27, 4.68/104.36 is → 6) - α -D-Glcp- (1 → 4) - β -D-Manp- (1 → and β -D-Glcp- (1 → H-1/C-1 related signals of residues, and labeled sequentially as residues D, E, F. → 1) - β -D-Fruf- (2 → (residues A) and → 1, 6) - β -D-Fruf- (2 → (residues B) indicates that the structure of PCP1 is likely to have a side chain, the order of attachment of residues can be determined from the HMBC spectra (FIG. 4F), the related signals δ3.63/104.08 (AH-1/BC-2) indicate that H-1 of residue A is linked to C-2 of residue B; the correlation signal delta 3.80/103.52 (A H-1/AC-2) indicates that H1 of residue A is linked to C2 of residue A, the correlation signal delta 3.65/103.77 (A H-1/C-2) indicates that H-6 of residue B is linked to C2 of residue C, the correlation signal (D H-1/AC-2) at delta 5.34/103.52 indicates that H-1 of residue D is linked to C2 of residue A, the correlation signal at delta 4.68/78.13 (E H-1/F C-4) indicates that H-1 of residue E is linked to C4 of residue F, the correlation signal at delta 4.11/100.27 (D H) The relevant signal at 6/F C-1) indicates that H-6 of residue D is linked to C-1 of residue F. Thus, it can be inferred that the skeleton of PCP1 is mainly composed of → 1) - β -D-Fruf- (2 → 1, 6) - β -D-Fruf- (2 → 6) - α -D-Glcp- (1 → 4) - β -D-Manp- (1 → and β -D-Glcp- (1 → residues, and that the side chain is β -D-Fruf- (2 → residues linked to → 1, 6) - β -D-Fruf- (2 → residues, the linking position being at C-6.

Because of the readily decomposable nature of fructose, monosaccharide composition and methylation analysis cannot very accurately reflect the proportion of monosaccharides, and we therefore combine NMR spectra to determine the proportion of individual sugar residues in PCP1. At the position of ¹³ In the C spectrum, the integrated ratio of the C-2 signal peak at residue A, B, C was about 3:1:1, indicating that residue A, B, C was present in PCP1 in a ratio of about 3:1:1; at the position of ¹ In the H spectrum, the ratio of the H1 signal peak integral of residue D, E, F to the H3 signal peak integral of fructose was about 1:1:1:20, indicating that the ratio of the proportion of residue D, E, F to the total fructose in PCP1 was about 1:1:1:20, and thus, it was inferred that the proportion of residue A, B, C, D, E, F in PCP1 was about 11:4:4:1:1:1. A possible sugar chain structure of PCP1 is shown in FIG. 5.

TABLE 2 chemical shift assignment (ppm) of C and H for PCP1

Example 2

The application of the polygonatum cyrtonema polysaccharide PCP1 in the preparation of medicaments for treating non-alcoholic fatty liver diseases is specifically studied as follows:

in vivo study of PCP1 on the improvement effect of non-alcoholic fatty liver mice:

1) Animal and experimental design:

animal experiments were approved by the ethical committee for animal experiments (LLSC-2021-041) of the southern Anhui medical college.

Male C57BL/6J mice of 6 weeks old were purchased from Qinglong mountain laboratory animal center (Jiangsu Nanjing, china) (license number: SCXK (Yu) 2020-0005). The mice were kept in a constant temperature and humidity environment with a humidity of 50-55% and a temperature of 22-25 ℃ for 12h of light/dark cycles with free diet drinking water. Adaptively feeding for 1 week, 6 mice were fed normal feed, i.e. normal group diet group (ND); the remaining 18 mice were fed 8 weeks high fat diet (1% cholesterol, 8% lard, 5% egg yolk powder, 10% sucrose and 76% basal diet) to induce the mouse NAFLD model. After successful modeling, the mice were randomly divided into 3 groups of 6 animals each, i.e., a high fat diet model group (HFD), a high fat diet +200mg/kg PCP1 group (PCP 1-L), a high fat diet +400mg/kg PCP1 group (PCP 1-H). ND and HFD groups were perfused once daily with normal saline, and PCP1-L and PCP1-H groups mice were perfused once daily with PCP1 for 6 weeks. At the end of the experiment, all mice were euthanized after 12h of fasting. Mouse blood was collected, centrifuged at 3000r/min at 4℃for 10min to obtain serum, and the liver was weighed and stored in a-80℃refrigerator.

2) Improvement effect of PCP1 on liver injury and blood lipid disorders caused by HFD:

histopathological examination: the liver of the mice at the same part is resected and then soaked in 4% paraformaldehyde solution for more than 24 hours, embedded by paraffin or OCT to prepare 8 mu m sections, which are respectively used for hematoxylin-eosin (H & E) and oil red O staining and observed under an optical microscope.

Measurement of blood lipid levels: taking 0.3-0.6 mL of mouse serum, directly measuring by using a commercial kit, adding physiological saline into 3-5 g of mouse liver tissue at the temperature of 4 ℃ to prepare homogenate, and measuring the biochemical index of blood fat by using the commercial kit.

Results: as shown in fig. 6 a, the livers of HFD mice exhibited extensive balloon-like lesions, with a disturbed hepatic cable distribution around the central vein, and fat was severely accumulated, as compared to the ND group. In addition, liver index, ALT, AST levels were significantly elevated in HFD group mice, indicating long-term HFD damage to the mice liver (B-D in fig. 6). However, 400mg/kg PCP1 significantly reduced the increase in liver weight, cavitation and fat accumulation, while also alleviating liver damage, compared to the HFD group. The biochemical index test results showed that long-term HFD resulted in significant increases in TG, TC and LDL-C levels and significant decreases in HDL-C levels in the serum of mice (E-H in FIG. 6) compared to the ND group. However, this trend was significantly reversed after 6 weeks of PCP1 treatment, especially with high doses of PCP1, suggesting that NAFLD mice may be ameliorated by PCP1.

3) Effect of PCP1 on improvement of oral glucose tolerance (OGTT) and oxidative stress in mice:

OGTT experiment procedure: after all mice were fasted and not watered for 10 hours, a drop of tail vein blood was collected to measure blood glucose levels (0 min), and then a 50% glucose solution (2 g/kg) was filled to measure blood glucose levels of the mice at 30, 60, 90, 120min, respectively.

Measurement of oxidative stress biochemical index: 3-5 g of mouse liver tissue was taken and added with physiological saline at 4℃to prepare homogenate, and GSH, MDA, SOD levels in the liver tissue were measured using a commercial kit.

Results: HFD at 14 weeks significantly reduced SOD and GSH activity in the mouse liver and increased MDA activity (a-C in fig. 7) compared to ND group, indicating severe oxidative stress in the mouse liver. However, PCP1-H significantly increased SOD and GSH levels, and decreased MDA levels (P < 0.05) compared to HFD mice. Furthermore, 400mg/kg PCP1 also significantly enhanced the glucose regulating ability of HFD mice (D in FIG. 7), as evidenced by the area under the time-blood glucose curve calculation (E in FIG. 7). The above data indicate that PCP1 can improve oxidative stress and oral glucose tolerance in NAFLD mice.

4) Intestinal flora 16S rRNA analysis:

the experimental procedure is as follows: on the last three days of the experiment, faeces were collected and flash frozen in liquid nitrogen. Total DNA of feces was extracted and PCR amplification of 16S rRNA gene was performed. The PCR products were purified using Qiagen gel extraction kit (Germanown, U.S.A.), and were subjected to library quality inspection and sequencing. Through quality control, 50,000 high quality sequences were obtained for each sample, with sequences with a similarity higher than 97% being assigned to the same operational classification unit (OTUs). Alpha and beta diversity was analyzed using the QIIME platform (1.7.0 version).

Results: the results of the alpha diversity (Chao 1, observed species, simpson, shannon index) analysis shown in FIG. 8A show that PCP1-H improves the species richness and diversity of HFD mouse bacterial communities. PCoA and Venn analyses shown in fig. 8B and C indicate that three groups of mice intestinal flora were separated from each other and shared 9.52% OTUs due to the effects of diet and PCP1. The diversity analysis result shows that PCP1 can influence the intestinal flora structure of NAFLD mice and improve the microbial diversity and richness of NAFLD mice intestinal bacteria on the whole.

Second, PCP1 improves imbalance in the intestinal flora structure of NAFLD mice from different levels. The results of the portal level study showed that the Firmicutes, bacteroides (bacterioides), proteus (Proteobacteria) and actinomycetes (actionbacteria) are the main portal in the intestinal flora of mice (fig. 9 a). In the HFD group, the ratio of the relative abundance of the firmicutes to the bacteroides was significantly increased (B in FIG. 9), suggesting that severe obesity or adiposity process occurred in the mice. However, PCP1 significantly reduced the ratio of the relative abundance of firmicutes to bacteroides, indicating that PCP1 has a good anti-obesity effect. The results of the genus level analysis showed that PCP1-H significantly increased the relative abundance of the genera Bifidobacterium, allobaculum, ruminococcus and Adlercreutzia, etc., and decreased the relative abundance of the genera Lactobacillus, bacteroides, helicobacter, etc., compared to the HFD group (C in fig. 9), wherein the relative abundance of allobaculom was inversely correlated with the diabetic pathological process caused by high-fat diet; the relative abundance of bifidobacteria was significantly reduced in the gut of high cholesterol diet mice. This suggests that PCP1 may exert a promoting effect on certain beneficial bacteria, helping to regulate intestinal flora structure. The results of the LEfSe analysis showed 5 and 12 significantly different species in the HFD group and PCP1-H group, respectively (D in fig. 9). Taken together, the above data indicate that PCP1 can significantly improve intestinal bacterial composition in NAFLD mice at both the phylum and genus levels.

5) Short chain fatty acid analysis:

the experimental procedure is as follows: short chain fatty acids were quantified in mouse faeces by GC-MS method with the aid of Agilent 7890A/5975C (Agilent company, USA). Briefly, 100mg of fecal sample was mixed with 1mL of 0.005mol/L sodium hydroxide solution and 50. Mu.L of 2-methylbutyric acid and reacted at 4℃for 2 hours. The reaction mixture was centrifuged and was mixed with distilled water and isopropanol/pyridine solution (3:2, V/V) for derivatization. Then, the sample was extracted with n-hexane, and GC-MS analysis was performed on the machine with a sample injection amount of 1. Mu.L.

Results: as shown in fig. 9E, the isobutyrate and isovalerate content of the HFD group was significantly reduced compared to the ND group, while PCP1 significantly improved this trend. At the same time, PCP1 also promotes the production of acetate, propionate and valerate. These results indicate that PCP1 can significantly promote the production of short chain fatty acids, in particular isobutyrate and isovalerate.

In conclusion, the invention obtains a homogeneous neutral polysaccharide PCP1 from PCH, and the weight average molecular weight is 4650Da. The polygonatum cyrtonema polysaccharide comprises four monosaccharides in the molar ratio of: fructose: glucose: mannose: galactose=88.1: 9.7:1.6:0.3.PCP1 backbone mainly consists of → 1) - β -D-Fruf- (2 → 1, 6) - β -D-Fruf- (2 → and a small amount of → 6) - α -D-Glcp- (1 → 4) - β -D-Manp- (1 → β -D-Glcp- (1 → and side chains mainly consist of β -D-Fruf- (2 → linked to the C-6 position of → 1, 6) - β -D-Fruf- (2 → in vivo studies show that PCP1 exhibits significant protective effects on HFD-induced NAFLD mice, mainly in reducing liver injury in mice, alleviating lipid metabolism disorder, alleviating oxidative stress, promoting short chain fatty acid production and improving the structure of intestinal flora.

Claims

1. The polygonatum cyrtonema polysaccharide is characterized by comprising four monosaccharides in a molar ratio of: fructose: glucose: mannose: galactose=88.1: 9.7:1.6:0.3;

the main chain of the polygonatum cyrtonema polysaccharide mainly comprises → 1) -beta-D-Fruf- (2 → 1, 6) -beta-D-Fruf- (2 → and a small amount of → 6) -alpha-D-Glcp- (1 → 4) -beta-D-Manp- (1 → beta-D-Glcp- (1 → and the side chain mainly comprises beta-D-Fruf- (2 → C-6 position connected at → 1, 6) -beta-D-Fruf- (2-;

the weight average molecular weight of the polygonatum cyrtonema polysaccharide is 4650Da, and the number average molecular weight is 4420Da.

2. A method for preparing the polysaccharide of polygonatum cyrtonema of claim 1, which comprises the following steps:

3. The method according to claim 2, wherein the alcohol precipitation under the low temperature condition in step 2) is specifically: alcohol precipitation in a refrigerator at 4 ℃ for 12h; and during alcohol precipitation, absolute ethyl alcohol is added to enable the volume ratio of the ethyl alcohol in the mixed solution to be 75%.

4. The preparation method according to claim 2, wherein the step 3) specifically comprises: adding 200-300 mL of deionized water into the polygonatum cyrtonema crude polysaccharide, heating to 60 ℃, slowly stirring to re-dissolve the polygonatum cyrtonema crude polysaccharide until the solution is clear, centrifuging for 15min at 12000-15000 r/min after the precipitate is not dissolved continuously, taking supernatant, decoloring with D101 macroporous resin, concentrating decolored liquid to 1/10-1/5 of the volume of the original solution, filtering with a 0.22 mu m microporous membrane, removing protein with a Sevag reagent, determining by a Bradford method, proving that the protein is removed completely, then removing the Sevag reagent by rotary evaporation at 60 ℃, filtering and sterilizing by using a 0.22 mu m microporous membrane, and dialyzing for 72h at 4 ℃ by using a dialysis bag with a cut-off molecular weight of 1000Da to remove micromolecule substances; finally, the polysaccharide solution is freeze-dried for 72 hours at the temperature of minus 80 ℃ to obtain the primarily purified polygonatum cyrtonema polysaccharide powder.

5. The preparation method according to claim 2, wherein the step 4) is specifically: weighing 200mg of primarily purified polygonatum cyrtonema polysaccharide powder, adding 20-40 mL of deionized water for dissolution, filtering by a 0.22 mu m microporous membrane, loading the mixture on a DEAE-52 cellulose anion exchange chromatographic column, eluting by using deionized water at a flow rate of 0.4mL/min, collecting one tube every 10min, detecting whether the polysaccharide is completely eluted by using an anthrone-sulfuric acid reagent, concentrating eluent to 1/10-1/5 of the original volume after the polysaccharide is completely eluted, dialyzing for 72h by using a dialysis bag with a molecular weight cutoff of 1000Da at a temperature of 0-4 ℃, and freeze-drying for 72h at a temperature of-80 ℃ to obtain the polygonatum cyrtonema polysaccharide water-washed component powder.

6. The preparation method according to claim 2, wherein the step 5) comprises the following steps: weighing 30mg of polygonatum cyrtonema polysaccharide water-washing component powder, adding 4-6 mL of deionized water for dissolving at normal temperature, filtering by a 0.22 mu m microporous filter membrane, loading to a Sephadex G-75 gel chromatographic column, eluting by using the deionized water at the flow rate of 0.38mL/min, collecting one tube every 10min, concentrating eluent to 1/10-1/5 of the original volume, dialyzing for 72h by using a dialysis bag with the molecular weight cutoff of 1000Da at the temperature of 0-4 ℃, and freeze-drying for 72h at the temperature of-80 ℃ to obtain the polygonatum cyrtonema polysaccharide uniform component PCP1.

7. Use of a polysaccharide from polygonatum cyrtonema of claim 1, for the preparation of a medicament for the treatment of non-alcoholic fatty liver disease.