Polygonatum sibiricum polysaccharide, preparation method and application of polygonatum sibiricum polysaccharide in treatment of hyperlipemia

Abstract

Polysaccharide is a main bioactive component in rhizoma polygonati. According to the research, the rhizoma polygonati polysaccharide PSPF is obtained by adopting DEAE-sepharose FF and Sephadex LH-20 column chromatography, and the rhizoma polygonati polysaccharide PSPF is characterized through gel permeation chromatography, monosaccharide analysis, FT-IR (Fourier transform infrared spectroscopy), NMR (nuclear magnetic resonance) and GC-MS (gas chromatography-mass spectrometry) methylation analysis. And the weight-average molecular weight of the PSPF is 3592 Da. The result shows that the PSPF is mainly composed of fructose, and the fructose has a main chain of-> 1)-beta-D-fructofuranose-(2) and a side chain of-> 6)-beta-D-fructofuranose-(2). The PSPF can reduce the blood fat of an acute hyperlipidemia model, the serum HDL-C is obviously increased, and the oxidation and decomposition of fatty acid can be promoted, which is related to the up-regulation of AMPK and CPT-1 and the low expression of SREBP-1c, ACC and FAS; therefore, the PSPF is an apparent food polysaccharide for controlling lipid.

Description

Technical Field

[0001] The invention relates to the technical field of natural polysaccharides, and in particular to a polygonatum polysaccharide, a preparation method and application thereof in treating hyperlipidemia. Background Art

[0002] Polygonatum Rhizoma is a monocotyledonous plant of the genus Polygonatum Mill, family Liliaceae, order Asparagales. The Chinese Pharmacopoeia (2020 edition) systematically divides Polygonatum into three types according to the connection mode of rhizome nodules, appearance morphology, and thin layer identification method, namely Polygonatum sibiricum Red. (P. sibiricum), Polygonatum kingianum Coll. et Hemsl. (P. kingianum), and Polygonatum cyrtonema Hua. (P. cyrtonema). According to different shapes, it is commonly known as "chicken head Polygonatum", "big Polygonatum", and "ginger-shaped Polygonatum". The dried rhizome of Polygonatum is rich in carbohydrates, has a sweet taste and fragrance, and is considered to be a new source of nutrients. As an important part of the medicine and food, the rhizome of Polygonatum has high medicinal and edible value. The "Famous Doctors' Records" first recorded that Polygonatum has the effect of medicine and food. Polygonatum sibiricum is sweet and easy to eat, can satisfy hunger, and also has the effect of nourishing yin. Polygonatum sibiricum polysaccharide is the main active ingredient of Polygonatum sibiricum extract with antioxidant, immunomodulatory, hypoglycemic, and anti-tumor effects. In addition, Polygonatum sibiricum polysaccharide is widely used as a drug supplement due to its low toxicity and few side effects (Luo et al., 2022; Hu et al., 2023; Wang et al., 2020). However, the different activities of Polygonatum sibiricum polysaccharide are closely related to its components (Ji, Yan, Hou, Shi, & Liu, 2020). Polygonatum sibiricum polysaccharide is composed of monomeric sugar units connected by glycosidic bonds. Different glycosidic bond structures and molecular weights may change the pharmacological effects and mechanisms of Polygonatum sibiricum polysaccharide (Liu et al., 2015; Wang et al., 2022). At present, the study of the structure and composition of Polygonatum sibiricum polysaccharide is still in its early stages (Hu et al., 2023).

[0003] Hyperlipidemia is a disease with high concentrations of low-density lipoprotein cholesterol (LDL-C) or triglycerides (TC), which plays a key role in the progression of cardiovascular diseases (CVD), including atherosclerosis, non-alcoholic fatty liver disease, etc. (Zhao et al., 2016). At present, hyperlipidemia has become a target of increasing attention in the early stages of cardiovascular disease and an important component of the prevention and treatment strategies of cardiovascular disease (Shi, Wang, Wang, & Feng, 2016). Therefore, the establishment of a suitable animal model of hyperlipidemia is crucial for its treatment. At present, the main models of hyperlipidemia include hyperlipidemia induced by high-fat diet, hyperlipidemia induced by fat emulsion, hypolipidemia induced by egg yolk emulsion, and hypercholesterolemia induced by Triton WR1339 (Levine, & Saltzman, 2007). In recent years, the hyperlipidemia model induced by egg yolk emulsion has been used in the development of hyperlipidemia-related drugs due to its advantages of short time and high success rate (Ademuywa, Fasogbon, Bamidele, & Ukpo, 2023; Kong, Liu, Li, & Chen, 2018). In this study, the hyperlipidemia model was induced by egg yolk emulsion and serum-related indicators were detected. The results showed that the acute hyperlipidemia model was successfully constructed.

[0004] Classic clinical treatments for hyperlipidemia include statins and fibrates. However, these drugs may cause considerable liver dysfunction and musculoskeletal side effects (Collins et al., 2016; Karr, 2017; Luo, Yang, & Song, 2020). In recent years, safe and effective natural drug active ingredients have been widely developed and applied in the treatment of acute hyperlipidemia. Polysaccharides are natural macromolecules widely found in different plants, showing good prospects in immunomodulation, anti-inflammatory and hypoglycemic effects (Fernandes, & Coimbra, 2023). In addition, recent studies have also shown that polysaccharides even have antioxidant functions, thereby better treating hepatocyte steatosis (Ti et al., 2022). Studies have shown that polysaccharides may be effective natural foods or drugs for lowering blood lipids. Researchers have found that Polygonatum sibiricum polysaccharides have multiple pharmacological effects such as antioxidant, hypoglycemic, immunomodulatory, and anti-tumor. However, there are few reports on the structural analysis and bioactivity evaluation of Polygonatum sibiricum polysaccharides. More importantly, there are few studies on the relationship between the structure of Polygonatum sibiricum polysaccharide PSP (PSPF for short) and its lipid-lowering effects and mechanisms. Summary of the invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a polygonatum polysaccharide PSPF, the structural formula of which is as follows:

[0006]

[0007] Where n = 3 to 4,

[0008] The molecular weight of the polygonatum polysaccharide PSPF is 3592Da.

[0009] Furthermore, the monosaccharide composition of the polygonatum polysaccharide PSPF is glucose (Glc) and fructose (Fru), and the molar ratio is 2.707:97.292.

[0010] Furthermore, the glycosidic bonds of the polygonatum polysaccharide PSPF are composed of Fruf-(2→, Glcp-(1→, →6)-Fruf-(2→, →1)-Fruf-(2→, →6)-Glcp-(1→, →1,6)-Fruf-(2→, and the molar ratio is: 25.318:0.252:18.109:36.050:2.455:17.815.

[0011] The polygonatum polysaccharide PSPF in the present invention is obtained by water extraction, alcohol precipitation, drying, impurity removal, DEAE-sepharoseFF and Sephadex LH-20 column chromatography from the dried rhizomes of Polygonatum Mill and Polygonatum sibiricum Red. of the Liliaceae family, and separation and purification to obtain the uniform polygonatum polysaccharide PSPF. It is characterized by monosaccharide analysis, FT-IR, NMR and GC-MS methylation analysis. The weight average molecular weight of PSPF is 3592Da. The results show that PSPF is mainly composed of fructose, which is a kind of polysaccharide with a →1)-β-D-fructofuranose-(2→ main chain and →6)-β-D-fructofuranose-(2→ side chain.

[0012] The second aspect of the present invention provides a polygonatum polysaccharide extract, which contains the above-mentioned polygonatum polysaccharide PSPF. It is understandable that the polysaccharide extracts obtained by various methods contain many other impurities, such as proteins, pigments, vitamins, salts, etc., and it is necessary to gradually separate and purify the polysaccharide through processes such as alcohol precipitation → impurity removal → column chromatography to obtain polysaccharides with relatively high purity. In addition, if high-purity uniform polysaccharides are to be obtained, different separation methods and technologies must be combined to achieve this. Therefore, the extracts containing polygonatum polysaccharide PSPF in each of the above steps are polygonatum polysaccharide extracts. Previous studies have shown that polygonatum polysaccharide extracts contain saponins, flavonoids and alkaloid compounds. Therefore, as long as the above-mentioned polygonatum polysaccharide extracts contain polygonatum polysaccharide PSPF, they may also contain a small amount of other active ingredients in the polygonatum extraction process, which are all covered within the scope of the polygonatum extract.

[0013] The third aspect of the present invention provides a method for extracting polygonatum polysaccharide, comprising the following steps:

[0014] Step (1) water extraction: take the raw material of Polygonatum sibiricum, extract it with boiling water for 1-1.5h, and add 8-15ml of water per gram of raw material of Polygonatum sibiricum to the liquid-to-solid ratio, filter, repeat the extraction 2-3 times, and combine the filtrate to obtain an extract;

[0015] Step (2) concentration: the extract of step (1) is concentrated at a temperature of 70-90°C until the original medicinal material content is 1 g / ml to obtain a concentrated solution. In step (2), the concentration instrument is a rotary evaporator at a temperature of 80°C and 30 rpm.

[0016] Step (3) alcohol precipitation: adding anhydrous ethanol to the concentrated solution of step (2) for alcohol precipitation, the volume ratio of the concentrated solution to anhydrous ethanol is (1-1.5): (4-6), centrifuging, taking the precipitate, freeze-drying, and obtaining the crude polygonatum polysaccharide PSPF; the centrifugation condition is 12000r / min, and the centrifugation is 10min.

[0017] Step (4) impurity removal: dissolve the crude polygonatum polysaccharide PSPF in step (3) in water, with a liquid-to-solid ratio of 10-20 ml of water per mg of crude polygonatum polysaccharide, centrifuge, take the supernatant, filter with a microporous membrane, and obtain the filtrate; the centrifugation conditions are 8000 r / min, centrifugation for 10 min, and the microporous membrane is 0.45 μm.

[0018] Step (5) DEAE-sepharose FF ion exchange column separation: The filtrate of step (4) is subjected to DEAE-sepharose FF ion exchange column separation, the sample loading amount is 0.5%-1% of the column volume, 0, 0.2, 0.5 and 1.0M NaCl gradient elution, and the ultrapure water eluate is collected, 3 ml is collected in each tube, and 1 column volume is eluted to obtain the eluate of HJ-E1 crude polysaccharide of polygonatum sibiricum; The pretreatment of DEAE-Sepharose FF is specifically carried out according to the following steps: The DEAE-Sepharose FF gel suspension in the Buchner funnel is connected to a vacuum pump and drained, and repeatedly washed with pure water until the filler has no alcohol taste, and the obtained precipitated gel is poured into a beaker, and 1 / 2 to 1 times the volume of the precipitated gel is added with distilled water, stirred, and then poured into a φ3.0×50cm XK chromatography column, wet packed, and the peristaltic pump is started at a flow rate of 250cm / h, and ultrapure water is rinsed to balance the gel surface.

[0019] Step (6) Sephadex LH-20 polyacrylamide gel column chromatography purification: The crude polygonatum polysaccharide HJ-E1 eluate of step (5) is subjected to Sephadex LH-20 polyacrylamide gel column chromatography purification, with the sample loading amount being 0.5% to 1% of the column volume, eluted with ultrapure water, the eluate is collected, and freeze-dried to obtain uniform polygonatum polysaccharide PSPF.

[0020] Further, the raw material of Polygonatum sibiricum is taken from the dried rhizome of Polygonatum Mill Polygonatum sibiricum Red. of Liliaceae. The raw material of Polygonatum sibiricum can be purchased from a commercial store or obtained by planting it yourself. The raw material of Polygonatum sibiricum of the present invention is purchased from Hebei Shunquanlong Pharmaceutical Co., Ltd., batch number 20210301, which is the dried rhizome of Polygonatum sibiricum Red. of Liliaceae.

[0021] It is understood that the polygonatum polysaccharide extract of the present invention refers to the substance obtained by extracting the dried rhizome of polygonatum, and the water extract refers to the component obtained by extracting with water, which can be solid after drying or not dried as liquid, and it should be understood that both are within the scope of protection of the present invention. The polygonatum polysaccharide extract of the present invention can be prepared by existing methods, for example: boiling water decoction, enzymolysis, low temperature and high pressure water extraction, microwave water extraction, ultrasonic water extraction, etc., which should not be understood as limiting the present invention. In the embodiment of the present invention, the polygonatum polysaccharide extract is obtained by boiling water decoction.

[0022] Furthermore, the preparation process of the polygonatum polysaccharide PSPF also includes a step (0) before water extraction, pretreatment: taking the dried rhizome of polygonatum and cutting it into blocks less than 3×3×3 cm.

[0023] The above step (0) is a conventional pretreatment, and its purpose is to cut the rhizome of the raw material of polygonatum into pieces for easy extraction. If the raw material of polygonatum is purchased in small pieces, no pretreatment step is required. If the raw material of polygonatum is freshly picked, it needs to be cleaned, for example: the raw material of polygonatum is cleaned once or multiple times with water and / or deionized water to completely remove impurities such as dirt and dust on the surface, and then dried and cut into pieces.

[0024] Another aspect of the present invention provides the use of the above-mentioned Polygonatum sibiricum polysaccharide PSPF or an acceptable salt thereof as an active ingredient in the preparation of medicines, health products or foods for treating symptoms, conditions or diseases related to hyperlipidemia.

[0025] Furthermore, the hyperlipidemia is acute hyperlipidemia, such as reducing blood lipids of acute hyperlipidemia mice induced by egg yolk emulsion, reducing serum TC, TG and LDL-C levels, and increasing serum HDL-C concentration.

[0026] Furthermore, the polygonatum polysaccharide PSPF or its acceptable salt as an active ingredient regulates hyperlipidemia by activating the AMPK target. Further studies have shown that PSPF can promote the oxidation and decomposition of fatty acids, which is associated with the upregulation of AMPK and CPT-1 and the low expression of SREBP-1c, ACC and FAS. These findings indicate that PSPF is a drug, health product or food polysaccharide that controls symptoms, conditions or diseases associated with hyperlipidemia.

[0027] The present invention has the following advantages over the prior art:

[0028] 1. In this study, crude polysaccharides from polygonatum were first extracted, and then the extracted crude polysaccharides were purified to obtain uniform polygonatum polysaccharide PSPF with a weight-average molecular weight of 3592 Daltons. The purified PSPF was studied by methylation, infrared spectroscopy, monosaccharide structure and nuclear magnetic resonance. The PSPF is mainly composed of fructose, which is a polysaccharide with a →1)-β-D-Fruf-(2→ main chain and →6)-β-D-Fruf-(2→ side chain. It is a completely new polysaccharide structure. However, previous studies have shown that polygonatum polysaccharide consists of a glucosidic bond connecting mannose (Manp) and glucose (Glcp) (1→4) (Li et al., 2020), which can be attributed to the deviation of the source of polygonatum polysaccharide and extraction technology.

[0029] 2. PSPF can reduce the content of TC, TG and LDL-C, and partially increase the content of HDL-C in serum and liver. Therefore, PSPF has a certain lipid-lowering effect, which provides a new breakthrough for the treatment of PSPF as a natural lipid-lowering drug.

[0030] 3. The progression of hyperlipidemia is partly attributed to lipid metabolism disorders regulated by signaling pathways, and AMPK is an important regulator. Long-term hyperlipidemia can inhibit AMPK activity and affect the expression of its downstream related targets ACC, FAS, SREBP-1c and CPT-1. SREBP-1c is a subtype of sterol regulatory element binding protein (SREBPs) and is the main transcriptional regulator of genes involved in fat synthesis, mainly affecting fatty acid synthesis and triglyceride accumulation. Activation of AMPK can downregulate the expression of SREBP-1c, thereby inhibiting the synthesis of TG and fatty acids. It can also reduce the expression of ACC and FAS, thereby accelerating the oxidative decomposition of fatty acids and preventing lipid synthesis. CPT-1 is regulated by AMPK, which can limit fatty acid oxidation and directly inhibit the oxidative decomposition of fatty acids. After PSPF treatment, the mRNA levels of AMPK and lipid decomposition-related factor CPT-1 were significantly increased, and the lipid synthesis-related factors SREBP-1, FAS and ACC were significantly decreased, indicating that PSPF may improve hepatic lipid deposition in acute hyperlipidemia mice by inhibiting lipid synthesis and promoting lipid decomposition. Therefore, PSPF can alleviate hyperlipidemia by reducing lipid deposition in egg yolk emulsion-induced acute hyperlipidemic mice. AMPK-related cell signaling mechanisms contribute to the lipid metabolism effects of PSPF in hyperlipidemic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the anti-hyperlipidemia effect of Polygonatum sibiricum polysaccharide PSPF.

[0032] Figure 2 This is the preparation of Polygonatum sibiricum polysaccharide PSPF. (A) Ion exchange column chromatography elution curve of Polygonatum sibiricum polysaccharide PSPF. (B) Chromatographic elution curve of Polygonatum sibiricum polysaccharide PSPF gel column. (C) HPGPC chromatogram of Polygonatum sibiricum polysaccharide PSPF.

[0033] Figure 3 These are the ion chromatograms of monosaccharide composition in Polygonatum sibiricum Polysaccharide PSPF. (A) is the mixed standard ion chromatogram, (B) is the polysaccharide ion chromatogram of Polygonatum sibiricum Polysaccharide PSPF, and (C) is the FT-IR spectrum of Polygonatum sibiricum Polysaccharide PSPF.

[0034] Figure 4 This is the NMR spectrum of Polygonatum sibiricum polysaccharide PSPF. (A) 1H NMR spectrum; (B) 13C NMR spectrum; (C) Dept135 spectrum; (D) HSQC analysis; (E) COSY analysis; (F) HMBC analysis; (G) TCOSY analysis.

[0035] Figure 5 This is the structural link diagram of Polygonatum sibiricum Polysaccharide PSPF. (A) Structural diagram of Polygonatum sibiricum Polysaccharide PSPF. (B) Structural formula of Polygonatum sibiricum Polysaccharide PSPF.

[0036] Figure 6 It is that Polygonatum sibiricum polysaccharide PSPF can alleviate acute hyperlipidemia in mice. Effects of Polygonatum sibiricum polysaccharide PSPF on serum TC (A), serum TG (B), serum LDL-C (C), serum HDL-C (D), liver TC (E), liver TG (F), liver LDL-C (G), liver HDL-C (H) and liver index (I) in hyperlipidemia mice; results of liver tissue H&E (J) and Oil Red O staining (K) (200x). Note: Data are expressed as mean ± SD (n = 6), ### compared with NC group P < 0.001, compared with Model group * P < 0.05, ** P < 0.01 and *** P < 0.001.

[0037] Figure 7 are the relative mRNA expression levels of AMPK (A), SREPB1-c (B), ACC (c), FAS (D), and CPT-1 (E) in the liver of hyperlipidemic mice (n=3), ###P<0.001 compared with the NC group *P<0.05 and ***P<0.001 compared with the Model group. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0039] Embodiment 1:

[0040] Step (0): Cut the purchased dried rhizomes of Polygonatum sibiricum into small pieces smaller than 3×3×3 cm, add 9 ml of water per gram of Polygonatum sibiricum raw material to mix, and soak overnight.

[0041] Step (1): boil it and keep it at a slight boiling state for 1 hour, then filter it with gauze. Repeat the above steps (0) and (1) twice, then combine the obtained filtrates, take the combined upper extract, and discard the precipitate.

[0042] Step (2) Concentrate the extract at 80° C. to 1 / 6 to 1 / 4 of the original volume to obtain a concentrated solution.

[0043] Step (3) The concentrate was precipitated with anhydrous ethanol at a ratio of 1:4 (v / v) overnight, and then centrifuged at 12000 r / min for 10 min, and the supernatant was discarded to obtain a precipitate. Finally, the precipitate was dried in a freeze-vacuum drying oven for 48 hours, and then ground into powder to obtain crude polygonatum sibiricum polysaccharide PSPF.

[0044] Step (4) Considering that the water elution component of the Polygonatum sibiricum polysaccharide PSPF is the main component, the Polygonatum sibiricum polysaccharide PSPF sample is dissolved in an appropriate amount of ultrapure water, centrifuged at 8000 rpm for 10 minutes, the precipitate is removed, and filtered with a 0.45 μm microporous membrane to obtain a filtrate.

[0045] Step (5) Take the filtrate and load it onto a balanced DEAE-sepharose FF ion exchange column for separation, with the sample loading amount being 1% of the column volume. After all sample solutions enter the ion exchange column, gradient elution is performed using different NaCl concentrations of 0, 0.2, 0.5 and 1.0 M NaCl gradient. The elution separation curve obtained is as follows: Figure 2 As shown in A. HJ-E1, HJ-E2, HJ-E3 and HJ-E4 were eluted with four fractions, and the 2.0M NaCl eluate was measured with anthrone-sulfuric acid. No obvious color reaction was observed in the 2.0M NaCl eluate, indicating that the crude polysaccharide was completely eluted. The fraction eluted with ultrapure water had the highest polysaccharide content. Therefore, HJ-E1 was selected for gel filtration column chromatography purification.

[0046] Step (6) Sephadex LH-20 polysaccharide gel column chromatography purification, the sample loading amount is 1% of the column volume. After all sample solutions enter the gel column, they are eluted and separated with ultrapure water, and the eluent is collected by an automatic partial collector, 3 mL is collected in each tube, and 1 column volume is eluted. Combined with the differential detector for online detection and collection, the eluent is collected in the same elution peak area, with high peak and good symmetry. Within the retention time of 55-65 minutes, the peak is very good and the content is very high ( Figure 2 B). The purified polysaccharide was repeatedly enriched by gel filtration column chromatography. The collected eluate fractions were concentrated under vacuum and freeze-dried to obtain purified Polygonatum sibiricum polysaccharide PSPF. The PSPF concentration was determined by anthrone-sulfuric acid method.

[0047] High performance gel permeation chromatography (HPGPC) was used to determine the purity and molecular weight of the extracted polygonatum polysaccharide. The specific detection conditions were as follows: Waters 2707 autosampler, 2424 evaporative light scattering detector (ELS), TSKgel GMPWXL column (7.8mm×300mm), column temperature 35℃, injection volume 20μL, ultrapure water as mobile phase, flow rate 0.6mL / min.

[0048] A short peak appeared at retention time 48.254 min ( Figure 2 C), which is the mobile phase salt peak; the symmetrical single peak at 42.526min is caused by Polygonatum cyrtonema polysaccharide, which indicates that the polysaccharide sample has good homogeneity. According to the standard calibration curve, the molecular weight of Polygonatum cyrtonema polysaccharide is calculated to be 3592Da.

[0049] Embodiment 2:

[0050] Structural identification of Polygonatum sibiricum polysaccharide PSPF in Example 1:

[0051] (1) Monosaccharide composition of PSPF:

[0052] The purified Polygonatum sibiricum polysaccharide PSPF of Example 1 was decomposed into monosaccharides by acid hydrolysis, and then the composition and content of monosaccharides in the polysaccharide were determined by ion chromatography. Briefly, a 10 mg / mL mixed standard mother solution was first prepared. After dissolving with sterile water, 100 mg of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, mannose, mannuronic acid and galacturonic acid were added to a volumetric flask (10 mL). The above mixed standard mother solution was diluted to obtain a working solution (100 μg / mL), which was loaded into a 1.5 mL EP tube and a standard curve was prepared by the gradient method. Next, 5 mg of PSPF was weighed in an asymmetric flask, TFA solution (2 M) was added, heated (60° C.), and dried with nitrogen. Finally, the sample was dissolved in sterile water and transferred to a chromatographic bottle for analysis.

[0053] Further chemical composition studies ( Figure 3 AB) Ion exchange chromatography showed that PSPF was mainly composed of Fru, Glc, Gal-UA and Gal. Fru was the enriched component in PSPF, accounting for 94.30%, and the Glc content was 5.43%. Then, the molecular weight of PSPF was determined based on HPGPC, and calculated according to the formula 1g-Mp=-0.1688T+10.6871 (R2=0.9926), LogMw=-0.1772T+11.0909 (R2=0.9936), Log Mn=-0.1685T+10.5937 (Rμ=0.9945) (Mp: peak position, Mw: weight average, Mn: number average). The Mp, Mw and Mn of PSPF were 3226, 3592 and 2680Da respectively.

[0054] (2) FT-IR analysis of PSPF:

[0055] The mixture of dried PSPF powder and anhydrous KBr powder was used for the tablets’ Fourier transform infrared spectrophotometer (Thermo Scientific, USA) measurement (4000-400 cm -1 ) and at 4000-400cm -1 Infrared spectrum scans were recorded within the range.

[0056] From the FT-IR spectrum of PSPF ( Figure 3C), characterized by infrared spectroscopy, which is a typical feature of polysaccharides (Coimbra, António, Mário, Rutledge, & Delgadillo, 1998; Wiercigroch et al., 2017). 3389.08 cm -1 The broad and strong absorption peak at 2935.33 cm is the stretching vibration of carbohydrate OH, and the smaller absorption peak of methyl or methylene CH appears at 2935.33 cm -1 The peak should be carbohydrate CH at 1400-1200 cm -1 The sample is a polysaccharide. -1 Polysaccharide bound water at 1131.27cm -1 and 1027.42cm -1 The peaks between 1100 and 1010 cm correspond to the stretching vibration peaks of the polysaccharide backbone COC and COH. -1 There are two strong absorption peaks at 932.04, 875.45 and 819.37 cm -1 ) indicates the presence of β-fructosyl stretching vibration (Liu et al., 2014). Therefore, PSPF is a neutral polysaccharide with a mainly β-type structure and contains a large number of furanose rings.

[0057] (4) PSPF methylation study

[0058] Weigh 2-3 mg PSPF sample to dissolve in DMSO (500 μL), add NaOH (1 mg) and incubate for 30 min, add 50 μL iodomethane solution to react for 60 min, and add CH2Cl2 to centrifuge. After washing the organic phase with ddH2O three times, remove the lower CH2Cl2 phase and evaporate to dryness, add 100 μL 0.5M TFA to react at 90°C for 60 min, and evaporate to dryness at 30°C. Continue to add 50 μL 2MNH3.H2O and 50 μL NaBD4 (30 mg / mL) to mix evenly and react for 150 minutes, and add 20 μL acetic acid to terminate the reaction. After adding acetic anhydride (250 μL) to the residue, vortex mix, react at 100°C for 2.5 hours, add 1 mL ddH2O and wait for about 10 minutes, add 500 μL CH2Cl2, vortex mix, centrifuge and discard the aqueous phase. It was washed three times with ddH2O and the lower layer of CH2Cl2 phase was collected and analyzed by GC / MS.

[0059] Methylation analysis was used to preliminarily determine the monosaccharide linkage order of PSPF. Based on relative retention time (RT) and mass spectrometry, the literature data (Wang et al., 2022) were compared with the database of the Center for Complex Carbohydrate Research at the University of Georgia (https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html), the type of sugar residues corresponding to each chromatographic peak was analyzed, and the corresponding content was calculated using the chromatographic peak area of ​​the corresponding sugar residue type (Table 2).

[0060] Table 2 PSPF methylation analysis

[0061]

[0062] (5) Nuclear Magnetic Resonance Analysis

[0063] PSPF was dissolved in deionized water and TMSP was added as an internal standard. NMR (1H- and 13C), DEPT-135 and two-dimensional 1H-1H COSY, HSQC, HMBC, NOESY were evaluated using a 600MHz Bruker NMR spectrometer. Calibration: HDO hydrogen δH = δ4.70ppm, TMSP methyl carbon δC = δ-1.80ppm

[0064] The structural characteristics of PSPF were explored in depth. The hydrogen spectrum signals of polysaccharides are mostly between δ3.0ppm and δ5.5ppm, and the resonance region of heterocyclic protons (H-1) is in the range of δ4.0 to 5.5. In addition, the chemical shift of 1H of α-type sugars is usually greater than 4.95ppm, while the chemical shift less than 4.95ppm indicates that it is a β-type sugar (He et al., 2022).

[0065] In the 1H-NMR spectrum ( Figure 4 A), there is no obvious strong anomeric hydrogen (H-1) signal around δ4.2ppm to δ5.5ppm in the anomeric H region, only a weak signal at 5.27ppm. Other hydrogen signals are concentrated in the region of δ4.5ppm to δ3.0ppm, and the signals overlap severely, making them difficult to distinguish.

[0066] PSPF 13 C-NMR spectrum ( Figure 4 B) shows fewer spectral lines than the 1H-NMR spectrum, and several anomeric carbons appear in the anomeric carbon region of δ90-110 ppm, among which there is a strong and obvious signal at δ103-105 ppm. However, the peak letters in the δ103-105 ppm abnormal carbon region disappear in the DEPT-135 diagram ( Figure 4 C), there are no cross peaks and connected hydrogens in the HSQC spectrum ( Figure 4D). Therefore, it is speculated that this part belongs to the seasonal carbon signal, indicating that PSPF contains a large amount of ketolase. Combining the monosaccharide composition and methylation analysis, fructose has the highest content. This type of heterocarp carbon is consistent with the NMR signal characteristics of fructose residues. The signal peak in this area is the heterocarp carbon (C-2) of the furanose residue, and the main signal peaks are δ104.25ppm, δ104.74ppm, δ104.91ppm and δ105.08ppm, indicating that the fructose residue is in β configuration and there are four different types of fructose residues in the sample. Based on numerous reports in the literature (Chen & Tian, ​​2003; Li, Xiao, Chen, & Xu, 2023; Yan, Zhang, Wang, & Zhang, 2019; Zhao et al., 2019; Zhang et al., 2021; Zhang et al., 2013) and methylation analysis, the C-2 signals of the anomeric carbon of fruf-(2→→1)-β-D-Fruf-(2→, β-D-Fruf-(2→, →1, 6)-β-D-Fruf-(2→ and →6)-β-D-Fruf-(2→) are marked as fructose residues A, B, C and D, respectively. According to the DEPT-135 spectrum, there is an obvious inverted peak signal in the δ60-65ppm region, which is consistent with the C-1 and C-6 signal characteristics of fructose residues. It can be inferred that the C-1 and C-6 signal regions belong to fructose residues. 13In C-NMR, DEPT-135 and HSQC spectra, there are five relatively independent signal peaks at δ80-83ppm, δ76-78ppm, δ75-76ppm, δ62-65ppm and δ60-62ppm. Combined with literature reports (Abid et al., 2019; Cai, Liu, Li, Lu, 2019; Li et al., 2017; Sun et al., 2022), they are respectively attributed to the C-5, C-3, C-4, C-6 and C-1 signal peaks of fructose residues. In the HSQC spectrum, there are two cross peaks (δ3.71 / 82.20ppm and δ3.79 / 81.32ppm) in the typical fructose characteristic signal H-5 / C-5, indicating that the fructose residues in PSPF have multiple types of linkage patterns. Combining the methylation analysis results with the reference report (Lopez, Mancilla-Margalli, & Mendoza-Diaz, 2003), the cross peak of H-5 / C-5 δ 3.72 / 82.20 ppm can be attributed to →1)-β-D-Fruf-(2→) and β-D-Fruf-(2→, and the cross peak of H-5 / C-5 3.79 / 81.32 ppm belongs to →6)-β-D-Fruf-(2→) and →1,6)-β-D-Fruf-(2→. Taking →1)-β-D-Fruf-(2→) as an example, it is marked as A. Since there is no hetero-head hydrogen signal in the hydrogen spectrum of fructan, the chemical shift of fructan H-5 can be determined to be δ 3.72 ppm by HSQC spectrum.

[0067] Then, using the cross peaks of COSY spectrum A H-4 / A H-5 (δ 3.96 / 3.71 ppm), H-3 / H-4 (δ 4.09 / 3.96 ppm), H-5 / H-6a and H-5 / H-6b, the signals of H-4, H-3 and H-6a / 6b were obtained to be δ 3.96 ppm, δ 4.09 ppm and δ 3.61 / 3.68 ppm ( Figure 4 E). Since there is no heterogeneous hydrogen signal on C-2 (δ104.25ppm), the chemical shift of H-1a / 1b is inferred to be δ3.54 / 3.75ppm using the strong absorption peaks (δ3.54 / 104.25ppm and δ3.75 / 104.25ppm) in the HMBC spectrum. The chemical shifts of C-1, C-2, C-3, C-4, C-5 and C-6 are δ61.85ppm, δ104.25ppm, δ78.25ppm, Δ75.42ppm, δ82.20ppm and δ63.38ppm, respectively (Table 3).

[0068] Table 3 Various sugar residues in PSPF 1 H and 13 Chemical shift assignment of C

[0069]

[0070] In the HSQC spectrum, there is a cross peak between the abnormal signal δ5.27ppm (H-1) and δ93.13ppm (C-1), which belongs to the terminal group signal of α-Glcp. Then, the H-2, H-3, H-4, H-5 and H-6a / 6b signals are obtained through the COSY spectrum cross peak. According to the above results, it can be inferred that the chemical shifts of H-2, H-3 and H-4 are δ3.41ppm, δ3.62ppm, δ3.40ppm, δ3.80ppm and δ3.67 / 3.93ppm respectively. In addition, the chemical shifts of C-1 to C-6 on the sugar ring can be attributed to the HSQC correlation spectrum: δ93.13ppm, δ72.11ppm, δ73.52ppm, δ70.21ppm, δ7.089ppm, δ61.11ppm. Then, it was inferred that the sugar moiety was →6)-α-D-Glcp-(1→, labeled as G according to methylation analysis and previous studies (Lopez, Mancilla-Margalli, & Mendoza-Diaz, 2003; Zhao et al., 2019; Zhang et al., 2021), and its chemical changes are shown in Table 3. According to 1 Based on the integral values ​​of the signals of the terminal hydrogen of Glcp and 3 or 4 hydrogen protons on fructan in the H NMR spectrum, it was calculated that the integral ratio of Glcp sugar group to fructan was 1:26, indicating that the polysaccharide sample might be fructan with an average degree of polymerization of 27.

[0071] 1 H and 13 C chemical shift based on 1 H. 13 C, DEPT-135, HSQC, HMBC, and NOESY chromatographic analysis, and refer to relevant fructan literature reports (Lopez, Mancilla-Margalli, & Mendoza-Diaz, 2003; Zhao et al., 2019; Zhang et al., 2021), representing fructose residues A, B, C, and D and glucose residue G, respectively. HMBC ( Figure 4 F) and NOESY spectra ( Figure 4G) As shown in the figure: (1) In the HMBC spectrum, there is a peak (GH-1 / a C-2) between the sugar residue G (δ5.27 ppm) and the sugar residue a (δ104.25 ppm). In the NOESY spectrum, there is a cross peak (GH-1 / AH-1a) between the sugar residue GH-1 (δ5.27 ppm) and the sugar residue A H-1a (δ3.54 ppm). Since there is no heterogeneous hydrogen signal in the hydrogen spectrum of the furan ring →1)-β-D-Fruf-(2→ shows HMBC correlation, this indicates that →1,1)-β-D-Fruf-(2→1)-α-D-Glcp-(6→ crossroads (2) There are cross signal peaks in the HMBC spectrum of C H-1a / A C-2 (δ3.63 / 104.25ppm) and C H-1b / A C-2 →1)-β-D-Fruf-(2→ and →1,6)-β-D-Fruf-(2→) There is a β(2→1) connection; (3) There are A H-1a / C C-2 (δ3.54 / 104.91ppm) and A H-1b / C C-2 in the HMBC spectrum C-2 (δ3.75 / 104.91ppm→1,6)-β-D-Fruf-(2)→ and →1)-β-D-Fruf-(2)→ have β(2→1) connection; (4) HMBC spectrum exists in the cross signal peak CH-6b / D C-2 (δ 3.69 / 15.08 PPM), indicating that →66)-β-D-Fruf-(2→ and →6)-β-D-Fruf-(2→) have β(2→6) connection; (5) →6)-β-D-Fruf-(2→ and →6)-β-D-Fruf-(2-β(2→6) cross signal peaks in the HMBC spectrum could not be found. The molar ratios of sugar residues A, B, C, D and G are (22.260+13.790):(15.632+9.686):17.81:(11.194+6.915):2.455=36.050:25.318: 17.815:18.109:2.455=14.684:10.313:7.257:7.376:1.00, where the molar ratio of fructose residues A, C and D is approximately 2:1:1. Based on the integral value of the proton signal of the hydrogen of the Glcp terminal group and the proton signal of the Fruf sugar group at 3 or 4 sites, it can be calculated that the integral ratio of the Glcp sugar group to the Fruf sugar group is 1:38, which is consistent with the data of the methylation results. Based on the above findings, the initial structure of PSPF may consist of a →1)-β-D-Fruf-(2→ main chain and →6)-β-D-Fruf-(2→ side chain. Possible chemical structures are as follows Figure 5 shown.

[0072] Embodiment 3:

[0073] Effects of Polygonatum sibiricum Polysaccharide PSPF in Example 1 on Acute Hyperlipidemia Mice and Its Related Mechanisms

[0074] (1) Acute hyperlipidemia mice and PSPF treatment

[0075] Thirty-six healthy KM male mice were randomly divided into 6 groups (40±2g, n=6). After 1 week of adaptive feeding, they were divided into NC mice group (blank), acute hyperlipidemia mice group (Model), low-dose PSPF mice group (1000mg / kg / d) (L-PSPF), medium-dose PSPF mice group (1500mg / kg / d) (M-PSPF), high-dose PSPF mice group (2000mg / kg / d) (H-PSPF), and SIM mice group (positive drug control group, simvastatin). Mice in NC, Model, and other groups were gavaged with PSPF or simvastatin or saline every day for 6 consecutive days. Two hours after the last administration, except for the NC group, all other groups were intraperitoneally injected with a 75% egg yolk emulsion solution (20 mL / kg body weight) (Kong, Liu, Li, & Chen, 2018) that caused experimental hyperlipidemia. The 75% egg yolk emulsion was: 45 mL of fresh egg yolk + 15 mL of normal saline, mixed into an emulsion, and stored at 4°C for use. The blank group was intraperitoneally injected with an equal volume of normal saline. Fasted overnight and drank freely. After 21 hours of the intervention experiment, each group was gavaged again to give the same treatment as the previous 6 days. Two hours after the last administration, blood was collected to detect related indicators of hyperlipidemia. Finally, the weight of the liver tissue was recorded, and the liver, intestines, and other organs of the mice were collected for future analysis. These animal studies were provided by the Laboratory Animal Care and Use Committee of Chongqing Medical University and followed the Chinese Guidelines for Laboratory Animal Care.

[0076] (2) Detection of blood and liver lipid levels in mice

[0077] The eyeballs of mice were removed, blood was collected, and serum was extracted. According to the instructions of the kit, the concentration of lipoprotein in the liver and serum of mice was detected.

[0078] (3) Mouse liver tissue morphology

[0079] The dissected fresh mouse liver was immersed in 4% paraformaldehyde solution, embedded in paraffin and sliced, and then H&E stained. The lesions were recorded under appropriate magnification using a fluorescent microscope. Another part of the fixed liver was made into frozen sections for Oil Red O staining. The pathological changes were observed under a microscope.

[0080] (4) Detection of relative messenger RNA expression in mouse liver

[0081] Total RNA was extracted using the TRIzol conventional one-step method and reverse transcribed into cDNA for qRT-PCR analysis. mRNA content was defined in the 2-ΔΔCt formula using the SYBR Green q-PCR assay. Specific primers are listed in Table 1.

[0082] Table 1 Description of main primers

[0083]

[0084] (5) Statistical analysis

[0085] Data analysis was performed using SPSS Professional Edition (SPSS Inc., USA). The data were expressed as mean ± SD, and P value < 0.05 was considered significant.

[0086] (6) Results

[0087] a. Effects of PSPF on biochemical and liver indicators:

[0088] Hyperlipidemia is caused by abnormal lipid metabolism in serum and liver, which is mainly manifested by increased serum lipid concentrations (LDL-C, TC and TG) and decreased serum HDL-C concentrations. The effects of PSPF intervention on serum indicators of hyperlipidemia mice are as follows: Figure 6 AD). The serum lipid concentration in the Model group was significantly higher than that in normal mice (P < 0.001), indicating that the hyperlipidemia mouse model was successfully established. In addition, PSPF can reduce serum lipid levels, especially in the H-PSPF group. At the same time, the serum HDL-C concentrations of L-PSPF, M-PSPF and H-PSPF were significantly higher than those in the Model group (P < 0.001), with the increase rates being 44.04%, 46.36% and 62.66%, respectively. In addition, PSPF also has a decreasing effect on liver TC, TG, LDL-C and liver index, and an increasing effect on liver HDL-C ( Figure 6 EI). After oral administration for 6 days, PSPF showed a significant therapeutic effect on hyperlipidemia.

[0089] b. Effects of PSPF on liver morphological changes in mice with acute hyperlipidemia

[0090] In order to further clarify the therapeutic effect of PSPF on acute hyperlipidemia, morphological staining was used to perform pathological staining on mouse liver. Figure 6As shown in JK, a small amount of lipid droplets were occasionally seen in the Model group, the arrangement of hepatic cords was disordered, the volume of some hepatocytes increased, the cytoplasm was loose, the nuclei of some hepatocytes were significantly enlarged, and diffuse red lipid droplets appeared in the field of view. The lipid droplets fused in the adjacent hepatocytes, and most of the hepatocyte nuclei were accumulated to one side by orange-red lipid droplets. After PSPF intervention, the fatty degeneration of mouse hepatocytes was improved, the number of red lipid droplets and lipid vesicles in the cytoplasm decreased, the hepatic cords were arranged relatively neatly, and the volume of hepatocytes decreased, especially in high-dose PSPF.

[0091] c. Mechanism of action of PSPF on hyperlipidemia mice

[0092] The progression of hyperlipidemia is often associated with the neutralization of lipid metabolism in the liver. AMPK is a key target for in vivo. Activation of AMPK can downregulate the expression of SREBP-1c (steroid regulatory element binding protein 1c), thereby inhibiting the synthesis of fatty acids (Ha, Jang, Chung, & Yoon, 2016; Li et al., 2014; Li et al., 2011; Ruderman, Carling, Prentki, & Caccedo, 2013). In addition, the signaling pathways of lipid metabolism were also detected. Compared with the Model group, the mRNA level of AMPK was significantly enhanced in M-PSPF (P < 0.05) and H-PSPF (P < 0.01) ( Figure 7 AE), indicating that PSPF can regulate hyperlipidemia by activating the AMPK signaling pathway. At the same time, the mRNA levels of SREBP-1c, ACC, and FAS were significantly reduced, indicating that PSPF can improve liver fat deposition in mice by reducing the expression of lipid synthesis-related factors (SREBP-1c, ACC, and FAS), and the CPT-1 mRNA level in the liver of mice was significantly increased, indicating that PSPF can improve liver fat deposition in mice by increasing the expression of lipid metabolism-related factor CPT-1 in the acute hyperlipidemia model. In short, PSPF can improve hyperlipidemia in mice by promoting the classical AMPK signaling pathway, increasing the mRNA level of lipid decomposition-related factor CPT-1, reducing the mRNA levels of lipid synthesis-related factors SREBP-1c, FAS, and ACC, inhibiting lipid synthesis, and promoting lipid decomposition. See Figure 1 .

[0093] The above is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited to the above content. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A Polygonatum sibiricum polysaccharide PSPF, characterized in that: The structural formula of Polygonatum sibiricum polysaccharide PSPF is as shown in Formula Ⅰ: Where n = 3 to 4, The molecular weight of the polygonatum polysaccharide PSPF is 3592Da.

2. The polygonatum polysaccharide PSPF according to claim 1, characterized in that: The monosaccharide composition of the polygonatum polysaccharide PSPF is glucose and fructose, and the molar ratio is 2.707:97.

292.

3. The polygonatum polysaccharide PSPF according to claim 1, characterized in that: The glycosidic bonds of the polygonatum polysaccharide PSPF are composed of Fruf-(2→, Glcp-(1→, →6)-Fruf-(2→, →1)-Fruf-(2→, →6)-Glcp-(1→, →1,6)-Fruf-(2→), and the molar ratio is: 25.318:0.252:18.109:36.050:2.455:17.

815.

4. A Polygonatum sibiricum polysaccharide PSPF extract, characterized in that: The polygonatum polysaccharide PSPF extract contains the polygonatum polysaccharide PSPF described in claim 1.

5. A method for extracting polygonatum polysaccharide PSPF, characterized in that: The following steps are included: Step (1) water extraction: take the raw material of Polygonatum sibiricum, extract it with boiling water for 1-1.5h, and add 8-15ml of water per gram of raw material of Polygonatum sibiricum to the liquid-to-solid ratio, filter, repeat the extraction 2-3 times, and combine the filtrate to obtain an extract; Step (2) concentration: concentrating the extract of step (1) at a temperature of 70-90° C. until the original medicinal material content is 1 g / ml to obtain a concentrated solution; Step (3) alcohol precipitation: adding anhydrous ethanol to the concentrated solution of step (2) for alcohol precipitation, wherein the volume ratio of the concentrated solution to the anhydrous ethanol is (1-1.5):(4-6), centrifuging, taking the precipitate, and freeze-drying to obtain the crude polygonatum polysaccharide PSPF; Step (4) impurity removal: redissolve the crude polygonatum polysaccharide PSPF in step (3) in water, with a liquid-to-solid ratio of 10-20 ml of water per mg of crude polygonatum polysaccharide, centrifuge, take the supernatant, filter with a microporous membrane, and obtain a filtrate; Step (5) DEAE-sepharose FF ion exchange column separation: the filtrate of step (4) is subjected to DEAE-sepharose FF ion exchange column separation, the sample loading amount is 0.5% to 1% of the column volume, 0, 0.2, 0.5 and 1.0 M NaCl gradient elution, and the ultrapure water eluate is collected to obtain the eluate of the crude polysaccharide HJ-E1 of polygonatum sibiricum; Step (6) Sephadex LH-20 polyacrylamide gel column chromatography purification: The crude polygonatum polysaccharide HJ-E1 eluate of step (5) is subjected to Sephadex LH-20 polyacrylamide gel column chromatography purification, with the sample loading amount being 0.5% to 1% of the column volume, eluted with ultrapure water, the eluate is collected, and freeze-dried to obtain uniform polygonatum polysaccharide PSPF.

6. The method for extracting polygonatum polysaccharide PSPF according to claim 5, characterized in that: The polygonatum raw material is taken from the dried rhizome of Polygonatum Mill and Polygonatum sibiricum Red of Liliaceae.

7. The method for extracting polygonatum polysaccharide PSPF according to claim 5, characterized in that: The preparation process of the polygonatum polysaccharide PSPF also includes a step (0) before water extraction, pretreatment: taking the dried rhizome of polygonatum and cutting it into blocks less than 3×3×3 cm.

8. Use of Polygonatum sibiricum polysaccharide PSPF or an acceptable salt thereof as an active ingredient in the preparation of medicines, health products or foods for treating symptoms, conditions or diseases associated with hyperlipidemia.

9. The use according to claim 8, characterized in that: The hyperlipidemia is acute hyperlipidemia.

10. The use according to claim 8, characterized in that: The polygonatum polysaccharide PSPF or an acceptable salt thereof is used as an active ingredient to regulate hyperlipidemia by activating the AMPK target.

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