HG type pectin as well as preparation method and application thereof

Abstract

The invention discloses HG type pectin as well as a preparation method and application thereof. A crude extract is extracted from a plant raw material and is subjected to multistage separation and purification, and the single-component HG type pectin with the activity of improving dyslipidemia is obtained through screening. In addition, by constructing an animal model, it is verified that the HG type pectin has the effect of improving dyslipidemia. The HG type pectin has a wide application prospect in the fields of foods, medicines and the like for preventing, improving or treating dyslipidemia.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to HG type pectin and a preparation method and application thereof. Background Art

[0002] Pectin, as an active ingredient of some functional edible and medicinal plants, has a variety of pharmacological activities such as liver protection, anti-inflammatory, immunomodulatory, antioxidant, and anti-tumor. The relationship between its biological activity and structural characteristics is reflected in molecular weight, monosaccharide composition, glycosidic bonds and their connection methods, functional groups, and spatial configuration. The structural characteristics of pectin not only affect its physical and chemical properties such as solubility, stability, and bioavailability, but also affect its biological activity. At the same time, the composition and fine structure of pectin vary greatly due to differences in extraction conditions.

[0003] However, there are still some problems with the current pectin extraction and application technology. For example, after pectin is extracted, it needs to be purified to remove impurities such as pigments and inorganic salts. Appropriate purification agents and methods need to be selected during the purification process, otherwise it will lead to pectin loss and structural changes. Therefore, it is still necessary to study new pectin extraction and purification processes to obtain pectin components with specific structures and higher biological activity, thereby promoting the sustainable development of the pectin industry. Summary of the invention

[0004] In order to solve at least part of the technical problems in the prior art, the present invention extracts crude extracts from plant raw materials and performs multi-stage separation and purification to screen out a single component HG pectin having the activity of improving dyslipidemia. Specifically, the present invention includes the following contents.

[0005] In a first aspect of the present invention, there is provided an isolated HG pectin, the main chain of which has a glycosidic bond as shown in →4)-α-D-GalAp-(1→, the terminal α-D-Galp-(1→ is connected to the main chain via →3,4)-α-D-GalAp-(1→, and the HG pectin has a structure as shown in Formula I: Formula I; Here, n is a natural number selected from 1 to 30.

[0006] In certain embodiments, the HG pectin according to the present invention has a molecular weight of 12931 Da.

[0007] The second aspect of the present invention provides a pharmaceutical composition comprising the HG pectin of the present invention.

[0008] In certain embodiments, the pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier.

[0009] In certain embodiments, according to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, a preservative and an antioxidant.

[0010] The third aspect of the present invention provides a method for preparing the HG pectin according to the present invention, which comprises the following steps: (1) extracting the plant material with an aqueous solvent at 50-90° C. for 100-200 min to obtain an extract, adding a precipitant to precipitate, and obtaining a crude extract; (2) The crude extract is subjected to multi-stage separation and purification to obtain the HG pectin.

[0011] In certain embodiments, according to the preparation method of the present invention, the plant raw material includes at least one of sea buckthorn, citrus, lemon, grapefruit, apple, pear, cherry, carrot and ginseng.

[0012] In certain embodiments, the preparation method according to the present invention further comprises a step of crushing the plant material before the extraction step.

[0013] In certain embodiments, according to the preparation method of the present invention, the precipitant comprises at least one of ethanol, methanol, propanol, isopropanol, acetone and methyl ethyl ketone.

[0014] In certain embodiments, according to the preparation method of the present invention, the multi-stage separation and purification includes protein removal, resin adsorption, anion exchange chromatography and non-ionic gel chromatography separation.

[0015] In certain embodiments, according to the preparation method of the present invention, the multi-stage separation and purification comprises: Preliminarily removing proteins and pigments from the crude extract to obtain a first purified product; Separating the first purified product on an anion chromatography column, collecting the eluted fractions, and taking the fraction capable of lowering blood lipid levels as the second purified product; Using water as a mobile phase, the second purified product is passed through a non-ionic gel filtration column to obtain a third purified product, and the fraction capable of lowering blood lipid levels is used as a fourth purified product; The fourth purified product is passed through a non-ionic gel filtration column again using water as the mobile phase to obtain the HG pectin.

[0016] In a fourth aspect of the present invention, there is provided a use of HG pectin in the preparation of a drug for preventing, treating or improving dyslipidemia, wherein the main chain thereof has a glycosidic bond as shown in →4)-α-D-GalAp-(1→, the terminal α-D-Galp-(1→ is connected to the main chain via →3,4)-α-D-GalAp-(1→, and the HG pectin has a structure as shown in Formula I: Formula I; Here, n is a natural number selected from 1 to 30.

[0017] The present invention extracts crude extracts from plant raw materials and performs multi-stage separation and purification to screen out a single component HG pectin with the activity of improving dyslipidemia. In addition, the present invention verifies that the HG pectin has the effect of improving dyslipidemia by constructing an animal model. The HG pectin of the present invention has broad application prospects in the fields of food, medicine, etc. for preventing, improving or treating dyslipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1-2 The isolation and screening of HG-type pectin (designated "SPC2A") is shown.

[0019] Figure 3-4 The results of structural and compositional analysis of SPC2A are shown.

[0020] Figure 5-7 The results of nuclear magnetic resonance analysis of SPC2A are shown.

[0021] Figure 8-10 The morphology, three-dimensional structure and thermogravimetric analysis results of SPC2A are shown.

[0022] Figure 11-13 Shown are the effects of SPC2A on body weight gain and dyslipidemia in zebrafish.

[0023] Figure 14-17 Shown is the effect of SPC2A on lipid accumulation in zebrafish liver. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] HG Pectin In one aspect of the present invention, there is provided an HG pectin, the main chain of which has a glycosidic bond as shown in →4)-α-D-GalAp-(1→, the terminal α-D-Galp-(1→ is connected to the main chain via →3,4)-α-D-GalAp-(1→, and the HG pectin has a structure as shown in Formula I: Formula I; in, n is a natural number selected from 1 to 30.

[0028] In a preferred embodiment, n is selected from a natural number of 1 to 30, preferably 1 to 25, also preferably 1 to 20, further preferably 1 to 15, more preferably 1 to 13, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. In a specific embodiment, n is 13.

[0029] The present invention carries out structural analysis and characterization of the obtained HG pectin. In a specific embodiment, high performance gel permeation chromatography (HP-GPC) is used to measure the molecular weight, and the HG pectin of the present invention has a number average molecular weight of 12931 Da.

[0030] In a specific embodiment, HG pectin is subjected to infrared spectroscopy analysis, and the IR mode is used for detection, with a scanning wave number range of 4000 cm -1 -400 cm -1 , scanning number 32 times. The HG pectin of the present invention is at 3412 cm -1There is the OH stretching vibration peak of sugar molecules at 1740 cm -1 There is a stretching vibration peak of the amide carbonyl C=O produced by uronic acid at 1608 cm -1 The stretching vibration peak of the free carboxylic acid carbonyl group is at 1417 cm -1 There is a stretching vibration peak of CO at 1048 cm -1 and 1018 cm -1 There is a pyran ring absorption peak at 890-970 cm -1 There is an α-glycosidic bond absorption peak between them.

[0031] In a specific embodiment, the specific chemical structure of HG pectin is determined by nuclear magnetic resonance analysis, and the nuclear magnetic resonance spectrometer is used to measure 1 H NMR, 13 C NMR, COSY, NOESY, HSQC, HMBC and TOCSY. The results show that the HG pectin of the present invention has a hydrogen spectrum signal between 3.0-5.5 ppm, a sugar ring proton signal of δ3.2-4.0 ppm, and an end proton peak distributed in the region of 4.3-5.8 ppm, including signal peaks of δ5.71, 5.23, 5.02, 4.85 and 4.52.

[0032] In a specific embodiment, the HG pectin of the present invention has an anomeric carbon region between δ93-180, wherein the HG pectin of the present invention has anomeric carbon signal peaks including δ108.18, 101.34, 100.38, 97.48, and 93.62, and signal peaks distributed in the 60-85 ppm region, including signal peaks in δ72.1, 71.31, 70.70, 71.69, 72.7, 82.55, 80.28, 71.83, 69.4, 70.05, 79.15, 72.65, 72.82, 73.28, 79.19, 72.75, 69.6, 75.3, 79.12, and 72.7.

[0033] In a specific embodiment, the HG pectin of the present invention has an anomeric carbon signal peak of δ100.38 and an anomeric hydrogen signal peak of δ5.02 in the HSQC spectrum, the signal of H1-2 is 5.02 / 3.67; the signal of H2-3 is 3.67 / 3.93; the signal of H3-4 is 3.93 / 4.32; the signal of H4-5 is 4.32 / 4.99, wherein H1, H2, H3, H4, H5 are δ5.02, 3.67, 3.93, 4.32, 4.99, respectively, and the corresponding C1-5 are 100.38, 69.40, 70.05, 79.15, 72.65.

[0034] In a specific embodiment, the HG pectin of the present invention has an anomeric hydrogen integral of →4)-α-D-GalAp-(1→) and →3,4)-α-D-GalAp-(1→ in a ratio of 3:1 in the H spectrum.

[0035] In a specific embodiment, the HG type pectin of the present invention has a signal peak related to the C4 of the anomeric hydrogen of the glycosidic bond →4)-α-D-GalAp-(1→) according to the one-dimensional and two-dimensional nuclear magnetic resonance spectra, indicating the existence of a linking mode of →4)-α-D-GalAp-(1→4)-α-D-GalAp-(1→), and the anomeric hydrogen of →4)-α-D-GalAp-(1→ has correlation peaks with the C4 of the glycosidic bond →3,4)-α-D-GalAp-(1→, respectively, indicating the existence of a glycosidic bond →4)-α-D-GalAp-(1→3,4)-α-D-GalAp-(1→.

[0036] Pharmaceutical composition One aspect of the present invention provides a pharmaceutical composition comprising the HG pectin of the present invention and a pharmaceutically acceptable carrier.

[0037] In the present invention, the pharmaceutically acceptable carrier includes at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, a preservative and an antioxidant. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion vehicles, etc.; fillers include, but are not limited to starch, lactose, mannitol, microcrystalline cellulose, etc.; absorbents include, but are not limited to, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents include, but are not limited to, water, ethanol, etc.; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, microcrystalline cellulose, etc.; disintegrants include, but are not limited to, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium lauryl sulfate, micropowdered silica gel, talc, etc.; sweeteners include, but are not limited to, sucralose, acesulfame, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, aspartame, etc.; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, etc.; antioxidants include, but are not limited to, ascorbic acid, methionine, etc.

[0038] In the present invention, a pharmaceutically acceptable carrier is involved in carrying or delivering a drug from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable" if it is compatible with the other ingredients of the formulation (e.g., HG pectin) and does not harm the patient.

[0039] Preparation method One aspect of the present invention provides a method for preparing the HG pectin of the present invention, which comprises steps (1) and (2), which are described in detail below.

[0040] The step (1) of the present invention is to extract the plant raw material with an aqueous solvent at 50-90° C. for 100-200 minutes to obtain an extract, and then add a precipitant to precipitate to obtain a crude extract.

[0041] In a preferred embodiment, the plant material is mixed with 5-20 times (preferably 6-19 times, further preferably 7-18 times, more preferably 8-17 times, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 times) of an aqueous solvent, subjected to a crushing treatment (for example, wall crushing at a speed of 5000-15000 r / min for 20-60 s), and extracted at 50-90° C., preferably 52-88° C., further preferably 54-86° C., further preferably 56-84° C., more preferably 58-82° C., more preferably 60-80° C., for example 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80° C. for 100-200 min, preferably 110-190 min, and further preferably 120-180 min, for example, 120, 130, 140, 150, 160, 170, 180 min, to obtain an extract, filter (for example, filter through a 100-300 mesh sieve), centrifuge the filtrate, retain the supernatant and concentrate it to a volume of 5-10 times (for example, 5, 6, 7, 8, 9, 10 times) the original fresh medicinal material, add a precipitant to precipitate, stand at room temperature in the dark for more than 8 hours, centrifuge again to discard the supernatant, add water to the precipitate until it is completely dissolved, thoroughly evaporate to remove the alcohol taste and concentrate it to a volume of 3-8 times (for example, 3, 4, 5, 6, 7, 8 times) the original fresh medicinal material to obtain a crude extract. It is understandable that in order to better extract the active ingredient, the extraction process can be repeated several times.

[0042] In the present invention, aqueous solvents include but are not limited to distilled water, deionized water, reverse osmosis water, ultrapure water, etc.; plant raw materials include but are not limited to sea buckthorn, citrus, lemon, grapefruit, apple, pear, cherry, carrot, ginseng, etc.; precipitants include but are not limited to ethanol, methanol, propanol, isopropanol, acetone, methyl ethyl ketone, etc.

[0043] Step (2) of the present invention is a step of subjecting the crude extract to multi-stage separation and purification to obtain the HG pectin. In a preferred embodiment, the multi-stage separation and purification comprises protein removal, resin adsorption, anion exchange chromatography and non-ionic gel chromatography.

[0044] In a preferred embodiment, the protein removal process is carried out by the Sevag method. In a specific embodiment, the crude extract obtained in step (1) is mixed with chloroform-n-butanol and shaken for 20-40 minutes to obtain an aqueous phase. It is understood that in order to better remove the protein, the above mixing and shaking process can be repeated.

[0045] In a preferred embodiment, resin adsorption includes adding the above-mentioned aqueous phase to a macroporous adsorption resin, adjusting the pH to acidic (for example, pH 3, 4, 5, 6), decolorizing at 50-70°C (preferably 52-68°C, also preferably 54-66°C, for example 54, 56, 58, 59, 60, 62, 64, 66°C) for 70-150 min (preferably 80-140 min, also preferably 90-130 min, for example 90, 100, 110, 120, 130 min), filtering to remove the macroporous resin, centrifuging to remove impurities, and lyophilizing to obtain a first purified product.

[0046] In a preferred embodiment, an aqueous solvent is used to dissolve the first purified product, and a 0.45 μM aqueous filter membrane is used to remove impurities and then loaded onto an anion chromatography column (examples of which include but are not limited to DEAE-cellulose DE-52 column, TSKgelBioAssist Q column, TSKgel DEAE-5PW column, TSKgel DEAE-2SW column, TSKgel DEAE-3SW column, TSKgel SuperQ-5PW column, TSKgel QAE-2SW column, Hamilton RCX-30 column, Hypersil SAX column, etc.), and eluted with an aqueous solvent, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 0.7 M NaCl, respectively. The peak position and the content of the purified product are detected tube by tube by the phenol-sulfuric acid method, an elution curve is drawn, the main fractions of the elution are collected, the supernatant is dialyzed (3500 Da), and lyophilized to obtain different purified products.

[0047] In order to obtain a purified product with pharmacological activity, the fraction that can lower blood lipid levels is screened, and the fraction that can lower blood lipid levels is used as the second purified product for subsequent purification steps. The fraction that can lower blood lipid levels refers in particular to a purified product that can significantly reduce the amount of triglycerides and / or total cholesterol.

[0048] In a preferred embodiment, water is used as the mobile phase, and a non-ionic gel filtration column (examples of which include but are not limited to Sephacryl S-300 HR column, polystyrene gel chromatography column, porous silica ball gel chromatography column, Bio-Beads series gel chromatography column, Toyopearl series gel chromatography column, etc.) is used to further separate and purify the second purified product aqueous solution, and the peak position and the content of the purified product are detected tube by tube using the m-hydroxyphenyl method, and the purified fractions are combined and collected to obtain a third purified product, and the fraction capable of lowering blood lipid levels is used as the fourth purified product.

[0049] It is understandable that the fourth purified product can be directly used as the HG pectin of the present invention, or as a preferred technical solution, water is used as the mobile phase to make the fourth purified product pass through a non-ionic gel filtration column again to obtain HG pectin with higher content and purity.

[0050] application One aspect of the present invention provides use of the HG pectin of the present invention in the preparation of a medicament for preventing, treating or improving dyslipidemia.

[0051] In the present invention, the term "prevention, treatment or improvement" refers to therapeutic treatment and preventive or prophylactic measures, the purpose of which is to prevent or slow down (reduce) undesirable physiological changes or disorders, such as elevated blood lipids. Beneficial or desired clinical results include, but are not limited to, the following results, whether detectable or undetectable, including relief of symptoms, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete). Those in need of treatment include those who already suffer from hyperlipidemia or blood lipid-related diseases or those who need to prevent or improve hyperlipidemia or blood lipid-related diseases.

[0052] In the present invention, dyslipidemia includes at least one of abnormal total cholesterol, abnormal total triglycerides, abnormal low-density lipoprotein cholesterol, abnormal high-density lipoprotein cholesterol, abnormal apolipoprotein A1 and fatty liver.

[0053] Example 1 The following is an exemplary illustration of the extraction and screening process of SPC2A.

[0054] 1. Extraction of SPC2A Fresh sea buckthorn berries were added with 12 times the amount of distilled water, broken in a wall-breaking machine at a speed of 9000 r / min for 40s, and heated at 70°C for 150 minutes, and the heating extraction was repeated twice. The extract was filtered with a 200-mesh silk cloth, the filtrate was centrifuged, and the supernatant was concentrated to eight times the volume of the original fresh medicinal material. Then, 4 times the volume of the supernatant was added to ethanol (95%, v / v) for precipitation, and the precipitate was placed at room temperature and away from light for more than 8 hours. The supernatant was discarded by centrifugation again, and the precipitate was added with water until it was completely dissolved, and the alcohol taste was completely evaporated and concentrated to five times the volume of the original fresh medicinal material to obtain a crude extract. The extraction rate under this process was 1.2% and the purity was 54%.

[0055] 2. Isolation and Purification of SPC2A The protein mixture in the crude extract was removed according to the Sevag method. Take the crude extract solution, add 1 / 4 volume of chloroform-n-butanol (volume ratio of 4:1), shake thoroughly for 30 minutes, and separate the aqueous phase from the chloroform phase. Add chloroform-n-butanol solution equivalent to 1 / 4 of its volume to the aqueous phase, repeat twice, and leave the water layer in the separatory funnel. Use macroporous adsorption resin to remove the pigment in the crude extract solution, add 1 / 5 mass of macroporous adsorption resin, adjust the pH to 5, decolorize at 60℃ for 110 min, filter to remove the macroporous resin, centrifuge the crude extract solution to remove impurities, and freeze-dry to obtain the seabuckthorn crude extract, at this time the purity is 67%.

[0056] The crude extract of seabuckthorn was re-dissolved in distilled water and separated on a DEAE-cellulose DE-52 column. Weigh 10 g of crude extract of seabuckthorn, dissolve it in 50 mL of distilled water, remove impurities with a 0.45 μM water filter membrane, and load it. Elute with distilled water, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 0.7 M NaCl, respectively. The peak position and sugar content of 700 tubes of eluted products were detected tube by tube by phenol-sulfuric acid method, and the elution curve was drawn. The main fractions of the elution were collected, and the supernatant was dialyzed (3500 Da), and freeze-dried to obtain SPA, SPB, SPC, SPD purified components ( Figure 1 A). Using high-fat diet-induced hyperlipidemia zebrafish larvae as a model, triglycerides and total cholesterol were used as indicators to screen out the SPC component with the best effect in regulating blood lipids ( Figure 2 ).

[0057] SPC was further purified on a Sephacryl S-300HR column using a tertiary aqueous solution as the mobile phase (flow rate 1.0 mL / min). 40 mg of SPC was weighed and dissolved with 2 mL of tertiary water. The sample was loaded after impurities were removed through a 0.22 μM water filter. The peak position and sugar content of the 40 tubes of eluted products were detected tube by tube using the m-hydroxybenzene method. The elution curve of SPC on the ion exchange column was drawn, and the products were combined and collected according to the elution curve. After multiple purifications, the same components were collected and freeze-dried to obtain purified fractions (SPC1, SPC2) ( Figure 1 SPC2 was separated and purified again on a Sephacryl S-300 HR column using distilled water as the mobile phase to obtain the final product SPC2A ( Figure 1 C). Using the hyperlipidemia zebrafish larvae model, the same method was used to screen out SPC1 and SPC2, which had similar effects on regulating total cholesterol (p<0.05), but the SPC2 component had a more significant effect on regulating total triglycerides (p<0.01) ( Figure 2 C, D).

[0058] Example 2 The structure and composition analysis of SPC2A are shown below.

[0059] 1. Protein content detection Accurately weigh 50 mg of bovine serum albumin and dissolve it in an appropriate amount of distilled water, dilute to 100 mL and shake well. Add 0 μL, 0.4 μL, 0.8 μL, 1.2 μL, 1.6 μL, 2.0 μL, and 2.4 μL of protein stock solution to 6 clean test tubes, and make up the total volume to 15 μL with deionized water. Then add 5 mL of Coomassie brilliant blue solution, shake well, place at room temperature for 5 min, and measure the absorbance at a wavelength of 595 nm. With protein concentration as the abscissa and absorbance as the ordinate, the standard curve regression line equation is y=14.178x+0.0117, and the fitting degree R²=0.9944. According to the standard curve, the protein content in the purified sample is calculated to be 0.001 mg / mL, and the protein removal of seabuckthorn crude extract can effectively remove most of the protein in SPC2.

[0060] 2. UV spectroscopy analysis Weigh 1.00 mg of SPC2A accurately and dissolve it in distilled water to a concentration of 1 mg / mL. Use distilled water as a control to detect the sample's absorption light curve in the range of 190-400 nm and observe the absorbance of SPC2A at 280 nm and 260 nm. Perform a full wavelength scan of SPC2A, which shows no characteristic absorption peaks at 230 nm and 260 nm ( Figure 3A), indicating that SPC2A contains almost no protein, which is consistent with the result of Coomassie Brilliant Blue assay.

[0061] 3. Molecular weight analysis High performance gel permeation chromatography was used for molecular weight determination. Chromatographic conditions (chromatographic column: BRT105-103-101 (8×300 mm); detector: differential detector RID-20A; mobile phase: 0.05 M NaCl solution; flow rate: 0.7 mL / min; column temperature: 40°C; injection volume: 25 μL). Samples were injected in order from small to large relative molecular weight, retention times were recorded, standard curves of lgMp-RT (Mp peak molecular weight), lgMw-RT (Mw weight average molecular weight), and lgMn-RT (Mn number average molecular weight) were drawn, and the molecular weight calculation formula was obtained. Samples were tested under the same conditions as above.

[0062] The chromatogram of SPC2A shows a single symmetrical peak ( Figure 3 B), indicating that its composition is single and the purity is good. According to the molecular weight determination method, a linear regression equation was established with a series of molecular weight dextran as the standard to obtain the calibration curves of lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), and lgMn-RT (number average molecular weight). The equation of the lgMp-RT calibration curve is: y = -0.1345x + 9.4624, R² = 0.9967; the equation of the lgMw-RT calibration curve is: y = -0.1361x + 9.514, R² = 0.997; the equation of the lgMn-RT calibration curve is: y = -0.1354x + 9.4855, R² = 0.9965. According to the standard curve, the calculation formula is obtained to calculate the molecular weight of each sample. The retention time of 39.694 min in the SPC2A chromatogram is substituted into the regression equation, and the number average molecular weight of SPC2A is calculated to be approximately 12931 Da. The dispersion coefficient (Mw / Mn) is used to describe the width of the molecular weight distribution. According to HP-GPC analysis, the dispersion of SPC2A is 1.001, which is close to 1, indicating that its molecular weight distribution is narrow and the molecular weight is relatively uniform.

[0063] 4. Infrared spectroscopy analysis The purified sample was mixed with potassium bromide at a mass ratio of 1:100 and ground into powder. The sample was pressed into tablets by a tablet press and detected in IR mode with a scanning wave number range of 4000 cm -1 -400 cm -1 , scan times 32 times.

[0064] Infrared spectrum analysis of SPC2A revealed that at about 3412 cm -1The broad peak at 1740 cm -1 The stretching vibration of the amide carbonyl C=O is the absorption peak produced by uronic acid; at 1608 cm -1 The absorption peak at 1417 cm -1 The absorption peak at is the stretching vibration of CO ( Figure 4 In addition, at 1048 cm -1 and 1018 cm -1 The significant absorption peak is the pyran ring in the SPC2A structure, at 890-970 cm -1 The weaker absorption peak between them is the α-glycosidic bond.

[0065] 5. Monosaccharide composition analysis After SPC2A was hydrolyzed by adding trifluoroacetic acid, the monosaccharide composition in the hydrolyzate was determined by ion chromatography. Chromatographic conditions (chromatographic column: Thermo Scientific™ Dionex™ CarboPac™M PA100; mobile phase A: 20 mM NaOH; mobile phase B: 100 mM NaOH + 500 mM NaAc; flow rate: 1 mL / min; column temperature: 30°C). Ion chromatography analysis of the crude extract sample after acid hydrolysis showed that the main monosaccharide was GalA (galacturonic acid), with only a small amount of Rha (rhamnose) and Ara (arabinose) ( Figure 4 B).

[0066] Example 3 The structural analysis of SPC2A is shown below.

[0067] 1. Methylation Analysis Weigh 20 mg of SPC2A and dilute to 4 mL with 3x water. Add 800 μL of 0.2 M morpholineethanesulfonic acid (MES) and 1.6 mL of 500 mg / mL EDC solution and react at 25°C for 3 h. Add 4 mL of 4 M imidazole-HCl and weigh 168 mg of sodium borotritiate directly into the reaction solution and react overnight at room temperature. The next day, add 2 mL of glacial acetic acid in an ice bath to terminate the reaction. The reaction solution was dialyzed for desalination and freeze-dried.

[0068] Take 2 mg of freeze-dried SPC2A in 0.5 mL DMSO (after drying with molecular sieves), ultrasonically dissolve it in a stoppered glass tube, add 20 mg of dry NaOH under nitrogen protection, ultrasonically dissolve it completely, continue to add 0.3 mL of iodomethane under nitrogen protection, stir and react for 2.5 h at room temperature in the dark, add 0.3 mL of ultrapure water and stir for 0.5 h to terminate the reaction. Continue to add 1 mL of dichloromethane to wash the reaction tube and transfer all to a clean glass test tube, add 3 mL of ultrapure water to the glass tube for extraction, take the lower organic phase, extract it repeatedly with ultrapure water 3 times, add excess NaSO 4 After absorbing excess water, transfer to a clean glass tube and blow dry with nitrogen. Repeat the above reaction three times until the hydroxyl groups are completely replaced.

[0069] Add 1 mL of 2 M TFA to the methylated sample and react at 110°C for 2 h. After the hydrolysis is completed, place it in an evaporating dish and heat it in a 70°C water bath to evaporate it. Add a small amount of methanol to evaporate it. Repeat 5 times until the pH of the aqueous solution is neutral. Then add 0.3 mL of ultrapure water and 1 drop of 1% ammonia water to adjust the pH to alkaline. Add 5 mg of NaBD to the hydrolyzate. 4 Mix well, stir at room temperature, and reduce for 12 h. After the reaction is completed, adjust the pH to neutral with acetic acid. After evaporating the reaction solution, add methanol to wash and evaporate repeatedly, repeat 3 times. Then add 1 mL of acetic anhydride: pyridine (1: 1 = v / v) to react at 90 ° C for 2 h, add 1 mL, ultrapure water to terminate the reaction. Then extract twice with 1 mL of dichloromethane, shake thoroughly, collect the dichloromethane phase and evaporate to dryness. Finally, add 0.6 mL of dichloromethane to dissolve, filter with a 0.22 μm organic filter membrane, and perform GC-MS analysis. The gas chromatograph quadrupole time-of-flight high-resolution mass spectrometer 8890-7250 was used for detection, and the capillary column was HP-5MS (0.25 μm, 30 m×0.25mm).

[0070] 2. NMR Analysis Weigh 50 mg of SPC2A sample, dissolve it in 0.5 mL of heavy water and freeze-dry it. Then dissolve the freeze-dried powder in 0.5 mL of heavy water and continue freeze-drying. Repeat the above process to fully exchange active hydrogen. Then dissolve the sample in 0.5 mL of heavy water and place it in a 600 MHz nuclear magnetic resonance instrument at room temperature 25°C. 1 H NMR, 13 C NMR, COSY, NOESY, HSQC, HMBC and TOCSY.

[0071] The hydrogen spectrum signals are mainly concentrated between 3.0-5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks δ5.71, 5.23, 5.02, 4.85, and 4.52 are concentrated in the 4.3-5.8 ppm region ( Figure 5 A).

[0072] Carbon spectrum analysis 13 C NMR (201 MHz, D 2 O): The NMR carbon spectrum signals are mainly concentrated between 60-120 ppm. By observing the carbon spectrum, it can be seen that the main anomeric carbon signal peaks δ108.18, 101.34, 100.38, 97.48, 93.62 anomeric carbon regions are mainly between δ93-180. And δ72.1, 71.31, 70.70, 71.69, 72.7, 82.55, 80.28, 71.83, 69.4, 70.05, 79.15, 72.65, 72.82, 73.28, 79.19, 72.75, 69.6, 75.3, 79.12, 72.7 main signal peaks are distributed in the 60-85 ppm region ( Figure 5 B). According to the monosaccharide composition results, the purified product is mainly composed of galacturonic acid, indicating that it is mainly galacturonic acid polysaccharide, namely HG type pectin.

[0073] By HSQC spectrum ( Figure 6 B), the anomeric carbon signal can be observed to be δ100.38, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ5.02. By HH-COSY( Figure 6 A), the signal of H1-2 is 5.02 / 3.67; the signal of H2-3 is 3.67 / 3.93; the signal of H3-4 is 3.93 / 4.32; the signal of H4-5 is 4.32 / 4.99; indicating that H1, H2, H3, H4, H5 are δ5.02, 3.67, 3.93, 4.32, 4.99, respectively, and the corresponding C1-5 is 100.38, 69.40, 70.05, 79.15, 72.65. Therefore, this signal belongs to the glycosidic bond →4)-α-GalpA-(1→. According to similar rules and combined with HMBC( Figure 7 A) and TOCSY( Figure 7 B), all glycosidic bond signals were assigned as shown in Table 1.

[0074] The integration of anomeric hydrogen in the H spectrum revealed that the ratio of →4)-α-D-GalAp-(1→ and →3,4)-α-D-GalAp-(1→ was about 3:1. Therefore, in the HMBC spectrum, the glycosidic bond signals of SPC2A were assigned according to the one-dimensional and two-dimensional NMR spectra; the anomeric hydrogen of the glycosidic bond →4)-α-D-GalAp-(1→ had a related signal peak with its own C4; indicating the existence of a linking mode of →4)-α-D-GalAp-(1→4)-α-D-GalAp-(1→. The anomeric hydrogen of →4)-α-D-GalAp-(1→ had related peaks with the C4 of the glycosidic bond →3,4)-α-D-GalAp-(1→, indicating the existence of a glycosidic bond →4)-α-D-GalAp-(1→3,4)-α-D-GalAp-(1→.

[0075] In summary, the main glycosidic bond structure of HG pectin is as follows: the main chain connection is the glycosidic bond of →4)-α-D-GalAp-(1→, and the terminal α-D-Galp-(1→) is connected to the main chain through the O-3 bond of →3,4)-α-D-GalAp-(1→, the structural formula is as follows Figure 7 C, where n is 13.

[0076] Table 1 Example 4 The morphology, three-dimensional structure, and thermogravimetric analysis of SPC2A are shown below.

[0077] 1. Scanning Electron Microscope Analysis About 5 mg of dried SPC2A was taken and adhered to a conductive carbon film containing a double-sided adhesive, placed in the sample chamber of an ion sputtering instrument, and sprayed with gold for about 40 s. After the sample was taken out, it was placed in the observation chamber of an ultra-high resolution scanning electron microscope with an accelerating voltage of 5 kV.

[0078] Under the microscope of 500x and 1000x, the samples are mainly in the form of flakes with relatively smooth surfaces. When further magnified to 2000x, it can be seen that some flake samples have a small amount of attachments on their surfaces. After magnification to 5000x, the smooth surface is more obvious ( Figure 8 ).

[0079] 2. Atomic Force Microscopy Analysis The ultrastructure of the sample was measured using a Bruker ICON atomic force microscope. SPC2A was dissolved in ethanol water (20 μg / mL), placed in a 60°C water bath, and shaken for 120 min. The treated solution (10 μL) was placed on a mica sheet and dried at 25°C for 12 h. AFM scanning was performed at room temperature in the tapping mode with a scanning range of 5*5 μm.

[0080] Planar and three-dimensional images of SPC2A Fig. 9 As shown. Among them, SPC2A is mainly linear and a small amount of irregular round particles, randomly distributed and of different sizes. The height of SPC2A is -4.8-3.8 nm, which is inconsistent with the average height of single-chain polysaccharides, indicating that SPC2A is easier to aggregate and entangle in the solvent, and its higher aggregates may be due to the existence of van der Waals forces and other forces between its chains. It can be seen that the uneven surface structure of SPC2A has sample aggregation and stacking.

[0081] 3. Congo Red Analysis The Congo red experiment was used to determine whether SPC2A was a triple helix chain structure. Weigh 5 mg of SPC2A, add 10 mL of distilled water and an equal volume of 50 μmol / L Congo red reagent. After mixing, prepare different concentrations of NaOH solutions. Specifically, gradually add 2 mol / L NaOH solution to gradually increase the final concentration of NaOH in the solution from 0.0 mol / L to 0.5 mol / L, and scan with a UV-visible recording spectrometer to measure the maximum absorption wavelength under each NaOH concentration condition, and plot with NaOH concentration as the horizontal axis and the maximum absorption wavelength as the vertical axis. Among them, deionized water was used instead of HG pectin solution as a blank control.

[0082] The results are as follows Fig.10 As shown in Figure A, compared with the blank Congo red solution, the maximum absorption wavelength of the sample in Congo red has basically not changed, that is, in the range of sodium hydroxide concentration of 0.1-0.5 mol / L, in the same sodium hydroxide solution, the maximum absorption wavelength of Congo red has not changed significantly after adding HG pectin, that is, the absorption has not undergone obvious red shift, indicating that HG pectin has a disordered structure.

[0083] 4. TG / DSC Thermogravimetric Analysis The thermal stability of the sample was analyzed by thermogravimetric analyzer. 3 mg of SPC2A was accurately weighed and placed in a crucible. After compaction, the temperature was raised from 25°C to 800°C at a rate of 10°C / min under nitrogen protection using a thermogravimetric analyzer.

[0084] From the thermogravimetric curve Fig.10 Figure B shows that the overall thermogravimetric curve is divided into three stages: the first weight loss stage is 25-206.4℃, SPC2A mainly loses free water, and the weight loss rate is 36.3%; the second stage is about 206.5-301.9℃, there is an obvious weight loss step in this stage, and the weight loss rate reaches 45.4%; the third stage is about 302.0-787.0℃, there is no obvious weight loss step in this stage, and the weight loss rate reaches 69.4%; the total weight loss rate of the sample is 69.4%. This shows that in this temperature range, HG pectin decomposes and the chemical bonds are destroyed.

[0085] From the differential scanning calorimetry curve, it can be seen that SPC2A shows a downward trend from room temperature to 259.7℃, indicating that SPC2A exhibits heat release at this temperature, and shows an upward trend from 259.8-275.5℃, indicating that SPC2A exhibits heat absorption at this temperature. Then SPC2A shows a straight downward trend from 275.6-602.4℃, indicating that SPC2A exhibits heat release at this temperature, and shows an upward trend from 602.5-679.9℃, indicating that SPC2A exhibits heat absorption at this temperature. Finally, the sample shows a downward trend from 680.0-707.0℃, indicating that SPC2A exhibits heat release at this temperature, and shows a straight upward trend from 707.1-787.0℃, indicating that SPC2A exhibits heat absorption at this temperature.

[0086] Example 5 The efficacy evaluation of SPC2A is shown below.

[0087] 1. Experimental Methods Three-month-old male zebrafish AB strain were purchased from the Chinese Zebrafish Resource Center (Wuhan, China) and maintained in a zebrafish culture system under the conditions of 28°C, 0.2% edible sea salt, pH 6.9-7.2, and conductivity 480-510 mS·cm -1 , hardness 53.7-71.6 mg / L CaCO 3 180 adult zebrafish (male) were divided into 6 groups, namely, blank control group, high-fat diet group (HFD), SPC2A low-dose group (SPC2A-10) (HG pectin aqueous solution), SPC2A medium-dose group (SPC2A-50), SPC2A high-dose group (SPC2A-100), positive group (benzafibrate), with 30 in each group. Throughout the experimental period, zebrafish in the blank control group were fed with a normal diet (ND) consisting of brine shrimp larvae bait, and zebrafish in the HFD group were fed with a high-fat diet prepared by adding 7% (w / w) egg yolk powder and 7% (w / w) cholesterol (Shanghai Feixi Biological Co., Ltd.) to the normal diet. The other four groups were fed a high-fat diet for the first 10 days. On the 11th day, the zebrafish in the bezafibrate group began to be fed with a positive drug group (70 mg / kg per fish) prepared by adding bezafibrate to a high-fat diet, and the SPC2A group began to be fed with a SPC2A treatment group (10, 50, and 100 mg / kg per fish) prepared by adding SPC2A to a high-fat diet. Feeding was continued twice a day for 50 days. The administration method was as follows Fig.11 A.

[0088] At the end of the 60-day experimental period, the zebrafish were fasted overnight, anesthetized in an ice-water mixture, weighed, and the tail was quickly cut off between the anal fin and the caudal fin with a blade. The dorsal artery blood was drawn with a micropipette and transferred into a centrifuge tube. The centrifuge tube was rinsed with sodium heparin in advance, left to stand at room temperature for 15 min, and centrifuged at 1500 g for 5 min to collect plasma. The zebrafish was dissected under a stereomicroscope, and the liver was quickly separated and placed in a microcentrifuge tube, frozen in liquid nitrogen, and stored at -80°C for kit detection. Another part of the liver was fixed in 4% paraformaldehyde for pathological observation.

[0089] 2. Effects on weight gain and dyslipidemia in zebrafish The zebrafish weight and blood lipid abnormalities showed that the high-fat diet group zebrafish had an enlarged abdomen and a wider body shape compared with the normal diet group. Drug treatment can alleviate this obesity trend, especially the high-dose SPC2A group and bezafibrate ( Fig.11 At the same time, compared with the blank control group, the body weight of zebrafish in the HFD group increased significantly, and SPC2A intervention significantly reduced the weight gain caused by the high-fat diet ( Fig.12 Of A). It is noteworthy that there was no significant difference in the feeding time among the groups ( Fig.12 B). This suggests that SPC2A can reduce dyslipidemia caused by a high-fat diet without affecting appetite and energy intake. By observing the plasma lipid profile, it was found that compared with the blank control group, the plasma total cholesterol (TC), total triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels in the HFD group were significantly increased, while the high-density lipoprotein cholesterol (HDL-C) level was significantly decreased ( Fig.13 ). Compared with the HFD group, SPC2A intervention significantly reduced TC and LDL-C levels and increased HDL-C levels without obvious dose-dependent relationship (p<0.05). However, SPC2A intervention significantly reduced TG levels in a dose-dependent manner (p<0.05).

[0090] 3. Effects on lipid accumulation in zebrafish liver Long-term feeding of high-fat diets can induce lipid metabolism disorders in zebrafish, and the liver is prone to hepatic steatosis, which increases the susceptibility to liver damage. In this example, the liver weight of zebrafish fed a high-fat diet was increased compared with that of zebrafish fed a normal diet, and SPC2A treatment further reduced the liver weight of zebrafish fed a high-fat diet ( Fig.14 A). Compared with the N blank control group, the high-fat diet induced a significant increase in the liver weight index (liver weight / body weight) of zebrafish, and bezafibrate and SPC2A treatment inhibited the increase in the liver weight index ( Fig.14B). There were statistical differences in liver TC and TG levels between the blank control group and the HFD group. SPC2A and bezafibrate could reduce liver TC and TG levels. In zebrafish with dyslipidemia induced by a high-fat diet, the TG-lowering effect of SPC2A was more significant than the TC-lowering effect ( Fig.14 C, D). Compared with the normal group, the plasma AST and ALT activities of zebrafish in the HFD group were increased, while the ALT and AST activities in the plasma of zebrafish after SPC2A intervention were significantly lower than those in the HFD group ( Fig.14 E, F). These results suggest that SPC2A can regulate dyslipidemia in zebrafish and protect against liver damage to some extent.

[0091] To further confirm the effect of SPC2A on lipid accumulation in the liver of zebrafish with dyslipidemia, the appearance and microstructure of zebrafish liver were examined. Fig.15 As shown in the figure, from the perspective of liver morphology under the microscope, the liver of zebrafish in the HFD group was swollen, the color became lighter, and fatty degeneration appeared, while the liver color and morphology of zebrafish in the SPC2A intervention group showed a trend of recovery, and the liver was in a relatively healthy state. HE staining showed that the liver cells of zebrafish in the HFD group were significantly damaged and a large number of lipid droplets were produced. SPC2A can significantly reduce the fatty degeneration and balloon-like lesions of the liver caused by a high-fat diet, and reduce the accumulation of lipid droplets in the liver ( Fig.16 ). Nile red staining further revealed lipid accumulation in zebrafish livers in the HFD group, and this feature was significantly reduced in liver sections of the SPC2A group (p<0.0001)( Fig.17 ).

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A HG pectin, characterized in that Its main chain has a glycosidic bond as shown in →4)-α-D-GalAp-(1→, and the terminal α-D-Galp-(1→ is connected to the main chain through →3,4)-α-D-GalAp-(1→. The HG type pectin has a structure as shown in Formula I: Formula I; Wherein, n is a natural number selected from 1 to 30.

2. The HG pectin according to claim 1, characterized in that Its molecular weight is 12931 Da.

3. A pharmaceutical composition, characterized in that It comprises the HG pectin according to claim 1 or 2.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, which comprises at least one of a diluent, a filler, an absorbent, a wetting agent, a binder, a disintegrant, a lubricant, a sweetener, a preservative and an antioxidant.

5. The method for preparing HG pectin according to claim 1 or 2, characterized in that: The following steps are involved: (1) extracting the plant material with an aqueous solvent at 50-90° C. for 100-200 min to obtain an extract, adding a precipitant to precipitate, and obtaining a crude extract; (2) The crude extract is subjected to multi-stage separation and purification to obtain the HG pectin.

6. The preparation method according to claim 5, characterized in that: The plant raw material comprises at least one of sea buckthorn, citrus, lemon, grapefruit, apple, pear, cherry, carrot and ginseng.

7. The preparation method according to claim 5, characterized in that: The multi-stage separation and purification includes protein removal, resin adsorption, anion exchange chromatography and non-ionic gel chromatography separation.

8. The preparation method according to claim 5, characterized in that: The multi-stage separation and purification comprises: Preliminarily removing proteins and pigments from the crude extract to obtain a first purified product; Separating the first purified product on an anion chromatography column, collecting the eluted fractions, and taking the fraction capable of lowering blood lipid levels as the second purified product; The second purified product is passed through a nonionic gel filtration column using water as a mobile phase to obtain a third purified product, and the fraction capable of lowering blood lipid levels is used as a fourth purified product.

9. The preparation method according to claim 5, characterized in that: The precipitant includes at least one of ethanol, methanol, propanol, isopropanol, acetone and methyl ethyl ketone.

10. Use of HG pectin in the preparation of a drug for preventing, treating or improving dyslipidemia, characterized in that: The main chain of the HG pectin has a glycosidic bond as shown in →4)-α-D-GalAp-(1→, and the terminal α-D-Galp-(1→ is connected to the main chain through →3,4)-α-D-GalAp-(1→. The HG pectin has a structure as shown in Formula I: Formula I; Wherein, n is a natural number selected from 1 to 30.

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