Exocarpium citri grandis polysaccharide as well as preparation method, identification method and application thereof

Abstract

The invention provides exocarpium polysaccharide as well as a preparation method, an identification method and application thereof, and belongs to the technical field of medicines and health foods. The molecular weight of the exocarpium polysaccharide provided by the invention is 11917 Da. The preparation method is simple, reaction conditions are mild, and large-scale production can be achieved; the chemical structure of the obtained high-purity exocarpium polysaccharide is identified, the structure of the exocarpium polysaccharide is clarified, and a structural basis is provided for exploring the pharmacological activity mechanism of the exocarpium polysaccharide. Meanwhile, the pure exocarpium citri grandis polysaccharide obtained by the invention lays a foundation for exocarpium citri grandis polysaccharide medicines, health-care foods and functional foods, quality control of the exocarpium citri grandis polysaccharide medicines, health-care foods and functional foods, and deep research on the structure-effect relationship and action mechanism of the exocarpium citri

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and health food, and specifically relates to a tangerine peel polysaccharide and a preparation method, an identification method and application thereof. Background Art

[0002] Myocardial ischemia (MI) is one of the main causes of sudden death, usually due to thrombosis or atherosclerosis, which leads to a sharp decrease in blood flow to the heart, eventually leading to cardiovascular disease. In clinical practice, reperfusion is the preferred treatment strategy for myocardial ischemia. However, a large number of studies have shown that reperfusion after a period of ischemia not only fails to restore tissue and organ function, but aggravates tissue and organ dysfunction and structural damage. This reperfusion-induced myocardial injury is widely referred to as myocardial ischemia-reperfusion (MI / R) injury. At present, there is still a lack of mature and effective MI / R treatment methods or drugs, making the treatment of MI / R still a clinical treatment challenge worldwide.

[0003] Polysaccharides, also known as polysaccharides, are a class of bioinformatic macromolecules widely found in animals, plants and microorganisms. Natural polysaccharides play an important role in a variety of diseases, including tumors, nervous system diseases and cardiovascular system diseases, due to their outstanding antioxidant and immunomodulatory activities. Increasing evidence shows that polysaccharides can effectively reduce myocardial injury caused by ischemia / reperfusion. Therefore, searching for potential therapeutic drugs from natural polysaccharides is expected to become an effective strategy for preventing and treating myocardial injury caused by ischemia / reperfusion.

[0004] Huajuhong polysaccharide is one of the components of the dried outer peel of the Rutaceae plant Huazhou pomelo (Citrusgrandis Tomentosa). It tastes pungent and bitter, and is warm in nature. It enters the lung and spleen meridians. It regulates qi and relieves the middle, and dries dampness and resolves phlegm. It is clinically used for cough with sputum, food accumulation and alcohol damage, nausea and stuffiness, etc. The chemical components of Huajuhong mainly include polysaccharides, flavonoids, volatile oils and coumarins. At the same time, modern pharmacological studies have shown that polysaccharides, as one of the main active ingredients of Huajuhong, have a wide range of pharmacological effects and have a variety of beneficial effects, including antioxidant, anti-inflammatory, hypoglycemic and cardioprotective activities. At present, the research on Huajuhong polysaccharide mainly focuses on the optimization of its extraction process and anti-inflammatory activity. There are few reports on the uniform polysaccharide structure and myocardial protective effect of Huajuhong polysaccharide. Therefore, it is necessary to provide a method for extraction, purification and structural identification to lay a foundation for the quality control of Huajuhong polysaccharide and in-depth study of its ischemia-reperfusion injury protective activity. Summary of the invention

[0005] The purpose of the present invention is to provide a tangerine peel polysaccharide and its preparation method, identification method and application, so as to provide a basis for the application of tangerine peel polysaccharide in medicine.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] In the first aspect, the present invention provides a polysaccharide of tangerine peel having the following structural formula:

[0008]

[0009] in It is L-furanose, where n+6m=34.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned tangerine peel polysaccharide, comprising the following steps:

[0011] S1 shearing: cutting the peel of the orange peel into small segments to obtain orange peel segments;

[0012] S2 water extraction: add the tangerine peel segments into water, heat and extract, and filter to obtain the extract;

[0013] S3 alcohol precipitation: the extract is concentrated to obtain a concentrated solution, ethanol is added to the concentrated solution, and the precipitate is collected to obtain crude polysaccharides;

[0014] S4 purification: Purify the crude polysaccharide by chromatography to obtain finished tangerine peel polysaccharide.

[0015] Preferably, the length of the tangerine peel segments obtained by shearing is 1 to 8 cm.

[0016] Preferably, the parameters of water extraction are: the amount of water added is 5 to 15 times the weight of the Citrus aurantium segment, the heating temperature is 60 to 100° C., and the extraction time is 1 to 10 hours.

[0017] Preferably, the alcohol precipitation step is a graded alcohol precipitation step, comprising:

[0018] S31: concentrating the extract under reduced pressure to obtain a first concentrated solution, adding ethanol to a volume concentration of a% and standing the solution, collecting the precipitate and the supernatant to obtain a first crude polysaccharide and a first supernatant;

[0019] S32: concentrating the first supernatant under reduced pressure to obtain a second concentrated solution, adding ethanol to a volume concentration of b% of ethanol, standing the solution, collecting the precipitate and the supernatant to obtain a second crude polysaccharide and a second supernatant;

[0020] S33: The second supernatant is concentrated under reduced pressure to obtain a third concentrated solution, ethanol is added to the solution until the ethanol volume concentration is c%, and the solution is allowed to stand, and the precipitate and the supernatant are collected to obtain a third crude polysaccharide.

[0021] The temperature during reduced pressure concentration is 40-70°C, the standing time is 10-28h, and 10≤a<b<c<100.

[0022] Preferably, the purification step is: taking the third crude polysaccharide for a purification to remove protein, and then taking the purified third crude polysaccharide for chromatography to obtain the finished tangerine peel polysaccharide.

[0023] Furthermore, the chromatography process includes: first taking the third crude polysaccharide after the first purification for ion exchange column chromatography, gradient elution with 0-2M NaCl, using the phenol-sulfuric acid method to draw an elution curve, collecting sugar parts according to the elution curve, concentrating, freeze-drying, and then dissolving with water, centrifuging, taking the supernatant, and then subjecting the supernatant to molecular sieve gel column chromatography, eluting with water, detecting the elution curve with the phenol-sulfuric acid method, collecting sugar parts according to the elution curve, concentrating, freeze-drying, and obtaining finished tangerine peel polysaccharide.

[0024] In a third aspect, the present invention provides a method for identifying Citrus aurantium polysaccharides, comprising the following steps:

[0025] (1) taking a sample of tangerine peel polysaccharide, completely hydrolyzing it with acid, and then derivatizing the hydrolyzate with PMP and detecting it by liquid chromatography;

[0026] (2) taking a sample of tangerine peel polysaccharide, drying it, pressing it into tablets, and performing infrared spectroscopy detection;

[0027] (3) taking a sample of tangerine peel polysaccharide, methylating it, hydrolyzing it, reducing it, acetylating it, and performing GC-MS detection;

[0028] (4) Dissolving a sample of tangerine peel polysaccharide in D2O and performing nuclear magnetic resonance analysis;

[0029] (5) The tangerine peel polysaccharide samples were taken for molecular conformation analysis using a multi-angle light scattering system and the Congo red method.

[0030] In a fourth aspect, the present invention discloses the use of the above-mentioned Citrus aurantium polysaccharide for preparing ischemia-reperfusion injury protective drugs or health products.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The present invention adopts water extraction and alcohol precipitation method to perform preliminary separation of tangerine peel polysaccharide, which has significant effect, and the preparation method is simple, the reaction conditions are mild, and it can be mass-produced;

[0033] 2. The present invention uses column chromatography to perform secondary separation and purification of the crude polysaccharide of Citrus aurantium, with significant results, and prepares a pure product of Citrus aurantium polysaccharide for the first time;

[0034] 3. The present invention identifies the structure of the purified polysaccharide pure product, clarifies the physicochemical properties and structure of each polysaccharide component, and provides a structural basis for exploring its pharmacological activity mechanism;

[0035] 4. The pure product of Citrus aurantium polysaccharide obtained by the present invention has well-preserved components, clear structure, and controllable quality. It can enhance the protective effect of AC16 cardiomyocytes after OGD / R induction, reduce the level of lactate dehydrogenase (LDH), inhibit the oxidative stress level of AC16 cardiomyocytes, and exert myocardial protective effects by regulating the crosstalk of Nrf2 / Keap1, IRE1 / GRPC78 and Bax / Bcl-2 signaling pathways, providing a basis for the application of Citrus aurantium polysaccharide in the fields of medicine, health care products, etc.;

[0036] 5. At the same time, the present invention lays a foundation for the preparation of tangerine peel polysaccharide drugs, quality control and in-depth study of their structure-activity relationship and mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a high performance liquid chromatogram of the monosaccharide composition of the tangerine peel polysaccharide of the present invention, wherein CGP3-1 is the tangerine peel polysaccharide of the present invention;

[0038] Figure 2 is the infrared spectrum of the tangerine peel polysaccharide of the present invention;

[0039] Figure 3 The present invention is the tangerine peel polysaccharide 1 H NMR spectrum;

[0040] Figure 4 The present invention is the tangerine peel polysaccharide 13 C NMR spectrum;

[0041] Figure 5 is the HSQC spectrum of the tangerine peel polysaccharide of the present invention;

[0042] Figure 6 is the HMBC spectrum of the tangerine peel polysaccharide of the present invention;

[0043] Figure 7 This is a molecular conformation determination spectrum of the tangerine peel polysaccharide of the present invention;

[0044] Figure 8 The effect of the tangerine peel polysaccharide of the present invention on the viability of AC16 cells induced by OGD / R;

[0045] Fig. 9 The effect of the polysaccharide of tangerine peel of the present invention on the oxidative stress injury of AC16 cells induced by OGD / R;

[0046] Fig.10 The tangerine peel polysaccharide of the present invention inhibits the expression of ANP and BNP mRNA in AC16 cells induced by OGD / R;

[0047] Fig.11The tangerine peel polysaccharide of the present invention exerts a myocardial protective effect by regulating the Nrf2 / Keap1 and Bax / Bcl-2 signaling pathways;

[0048] Fig.12 The tangerine peel polysaccharide of the present invention exerts myocardial protective effect by regulating the IRE1 / GRPC78 signaling pathway. DETAILED DESCRIPTION

[0049] In order to solve the problems existing in the prior art, the present invention provides a polysaccharide of tangerine peel and a preparation method, an identification method and an application thereof. The preparation method of the present invention is simple, the reaction conditions are mild, and large-scale production can be achieved; and the chemical structure of the obtained high-purity polysaccharide of tangerine peel is identified, its components are clarified, and a structural basis is provided for exploring its pharmacological activity mechanism. At the same time, the pure polysaccharide of tangerine peel obtained by the present invention lays a foundation for the drug of tangerine peel polysaccharide, quality control and in-depth study of its structure-activity relationship and mechanism of action.

[0050] The invention provides a tangerine peel polysaccharide. The inventor obtains the tangerine peel polysaccharide by purifying and separating the crude tangerine peel polysaccharide, and obtains the molecular weight of the tangerine peel polysaccharide as 11917 Da through structural analysis.

[0051] Furthermore, the structure of the tangerine peel polysaccharide is shown in the following formula:

[0052]

[0053] in is L-furanose, wherein n+6m=34. In other embodiments, 6≤n+6m≤68, wherein n and m are positive integers.

[0054] At the same time, the invention also provides a method for preparing the tangerine peel polysaccharide.

[0055] In an exemplary preparation embodiment, the preparation method comprises the following steps:

[0056] S1 cutting: cutting the dried peel of the dried tangerine peel into small segments, washing with water, and drying to obtain the dried tangerine peel segments;

[0057] S2 water extraction: adding the Citrus aurantium segments obtained in step S1 into water, heating for extraction, filtering, and obtaining an extract and medicinal residues;

[0058] S3 graded alcohol precipitation:

[0059] The extract obtained in step S2 is concentrated under reduced pressure to obtain a concentrated solution 1; ethanol is added to the concentrated solution 1 until the volume concentration of ethanol is a%, and the concentrated solution is allowed to stand, and the precipitate and the supernatant are collected to obtain a crude polysaccharide CG1 and a supernatant 1;

[0060] The supernatant 1 was concentrated under reduced pressure to obtain a concentrated solution 2; ethanol was added to the concentrated solution 2 until the ethanol volume concentration was b%, and the concentrated solution 2 was allowed to stand, and the precipitate and the supernatant were collected to obtain crude polysaccharide CG2 and the supernatant 2;

[0061] The supernatant 2 is concentrated under reduced pressure to obtain a concentrated solution 3; ethanol is added to the concentrated solution 3 until the volume concentration of ethanol is c%, and the solution is allowed to stand, and the precipitate is collected to obtain a crude polysaccharide CG3; wherein 10≤a<b<c<100;

[0062] S4 purification:

[0063] Primary purification:

[0064] The crude polysaccharide CG3 obtained in step S3 is subjected to protein removal, dialyzation, and freeze-drying to obtain Citrus aurantium polysaccharide;

[0065] Secondary purification:

[0066] The polysaccharide CG3 after the primary purification was subjected to ion exchange column chromatography, and gradient elution was performed with 0-2M NaCl. The elution curve was drawn using the phenol-sulfuric acid method. The sugar parts were collected according to the elution curve, concentrated, and freeze-dried; then they were dissolved with water, centrifuged, and the supernatant was taken;

[0067] The supernatant is subjected to molecular sieve gel column chromatography, eluted with water, the elution curve is detected by phenol-sulfuric acid method, the sugar part is collected according to the elution curve, concentrated, and freeze-dried to obtain the tangerine peel polysaccharide product of the present invention.

[0068] The inventors have found through research that the preferred length of the tangerine peel segment is 1 to 8 cm. At the same time, the present invention combines water extraction with graded alcohol precipitation, and graded alcohol precipitation is performed from low to high ethanol concentration to preliminarily separate the tangerine peel polysaccharide. High-concentration ethanol can separate polysaccharides with high polarity and good water solubility from polysaccharides with low polarity and poor water solubility, thus solving the problem that the traditional water boiling method for extracting polysaccharides leads to complicated and difficult separation in the later stage.

[0069] In a preferred embodiment, the amount of water added in step S2 is 5 to 15 times the weight of the Citrus aurantium segment, the heating temperature is 60 to 100° C., and the extraction time is 1 to 10 hours.

[0070] Furthermore, in step S3, the reduced pressure concentration temperature is 40 to 70° C., and the standing time is 10 to 28 hours.

[0071] In addition, the present invention also provides an identification method for Citrus aurantium polysaccharide, comprising the following steps:

[0072] (1) taking a sample of tangerine peel polysaccharide, completely hydrolyzing it with acid, and then derivatizing the hydrolyzate with PMP and detecting it by liquid chromatography;

[0073] (2) taking a sample of tangerine peel polysaccharide, drying it, pressing it into tablets, and performing infrared spectroscopy detection;

[0074] (3) taking a sample of tangerine peel polysaccharide, methylating it, hydrolyzing it, reducing it, acetylating it, and performing GC-MS detection;

[0075] (4) Dissolving a sample of tangerine peel polysaccharide in D2O and performing nuclear magnetic resonance analysis;

[0076] (5) The tangerine peel polysaccharide samples were taken and the molecular conformation was analyzed using a multi-angle light scattering system and the Congo red method.

[0077] In order to further develop the valuable resource of Citrus aurantium, and to explore new sources of medicine, the inventors obtained the present invention through a large number of experimental studies: using dried Citrus aurantium peel as raw material, separating crude polysaccharides by water extraction and alcohol precipitation, deproteinizing the extracted crude polysaccharides, and then purifying the Citrus aurantium crude polysaccharides by ion exchange chromatography and gel molecular sieve column chromatography, and preparing a pure Citrus aurantium polysaccharide for the first time. The physicochemical properties, molecular weight, monosaccharide composition, etc. of the pure polysaccharide were systematically analyzed and identified, and the structural information of Citrus aurantium polysaccharide was successfully obtained, wherein the Citrus aurantium polysaccharide of the present invention is arabinan, the main chain is composed of →5)-α-L-Araf-(1→, →2,3,5)-α-L-Araf-(1→ and →3,5)-α-L-Araf-(1→, and the side chain is composed of α-L-Araf-(1→ and →5)-α-L-Araf-(1→.

[0078] The present invention also relates to the use of the above obtained tangerine peel polysaccharide in the preparation of ischemia reperfusion injury protective drugs or health products, and the ischemia reperfusion injury protective activity is evaluated through cell experiments.

[0079] The following will be combined with the specific embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] Example 1 Preparation method of tangerine peel polysaccharide

[0081] The preparation method of the tangerine peel polysaccharide comprises the following steps:

[0082] S1 cutting: cut 10 kg of dried tangerine peel into 1-3 cm pieces with scissors, wash quickly with cold water, and dry to obtain tangerine peel segments;

[0083] S2 water extraction: add 10 times the weight of water to the tangerine peel segment obtained in step S1, heat to 80°C for extraction, extract for 3 hours, collect the extract, dry the residue in the air, and obtain the extract and the residue;

[0084] S3 graded alcohol precipitation:

[0085] The extract obtained in step S2 was concentrated under reduced pressure at 60° C., and ethanol was added to a volume concentration of 20%. After standing at room temperature for 24 hours, the extract was centrifuged, and the precipitate and supernatant were collected to obtain crude polysaccharide CG1 and supernatant 1.

[0086] After the supernatant 1 was concentrated under reduced pressure at 60°C, ethanol was added to a volume concentration of 40%, and after standing at room temperature for 24 hours, the supernatant was centrifuged to collect the precipitate and the supernatant to obtain crude polysaccharide CG2 and supernatant 2;

[0087] After the supernatant 2 was concentrated under reduced pressure at 60°C, ethanol was added to a volume concentration of 80%, and after standing at room temperature for 24 hours, centrifuged, the precipitate and the supernatant were collected to obtain crude polysaccharide CG3;

[0088] S4 purification: The crude CG3 polysaccharide obtained in step S3 was deproteinized by the Sevag method. After deproteinization, the crude polysaccharide was dialyzed with a dialysis bag (molecular weight cutoff of 1000Da) and freeze-dried to obtain the citrus aurantium polysaccharide CG3.

[0089] Example 2: Citrus aurantium polysaccharide of the present invention

[0090] The tangerine peel polysaccharide of the present invention is obtained by secondary purification of the tangerine peel polysaccharide CG3 obtained in Example 1, and the specific method is as follows:

[0091] 1) Ion exchange column chromatography: 200 mg of the citrus polysaccharide CG3 obtained in Example 1 was dissolved in 5 mL of deionized water and loaded onto a DEAE-FF column. Two peaks appeared under the conditions of different salt concentrations of the eluent, of which the elution peak was the 0.1 M NaCl elution portion (the elution curve was tracked by the phenol-sulfuric acid method during the elution process, and the sugar portions were collected separately according to the elution curve). The obtained eluents were concentrated and freeze-dried to obtain polysaccharide LC2;

[0092] 2) Molecular sieve gel chromatography: The above-mentioned freeze-dried polysaccharide sample is dissolved in water, centrifuged, the supernatant is taken, and applied to a Sephadex G-75 column, eluted with water, and the elution curve is tracked using the phenol-sulfuric acid method. A single symmetrical peak appears, and the main peak is collected, concentrated, and freeze-dried to obtain the tangerine peel polysaccharide of the present invention.

[0093] Example 3 Structural analysis of the tangerine peel polysaccharide of the present invention (I) Experimental materials: the tangerine peel polysaccharide of the present invention prepared in Example 2.

[0094] (II) Test methods:

[0095] 1. Monosaccharide composition analysis

[0096] Sample processing:

[0097] Accurately weigh 4.0 mg of each test material of the tangerine peel polysaccharide sample into a stoppered test tube, add 2.0 mL of 2M trifluoroacetic acid (TFA), place in an oil bath and hydrolyze at 120°C for 6 hours. Cool to room temperature, repeatedly add methanol and spin dry to remove TFA, dissolve to 1 mL with deionized water, centrifuge, take 100 μL of sample solution, add 100 μL of 0.3M NaOH solution, then add 100 μL of 0.5M PMP methanol solution, mix, react in a 70°C water bath for 30 minutes, cool, add 105 μL of 0.3M HCL solution to neutralize, add deionized water to 1 mL, then add an equal volume of chloroform solution, shake vigorously, centrifuge, remove the chloroform phase, repeat extraction twice, take the water phase and filter it with a 0.45 μm filter membrane for HPLC injection analysis.

[0098] Chromatographic conditions:

[0099] Chromatographic column: Kromasil 100-5-C18, 4.6×250 mm, 5 μm; mobile phase: 0.1 M phosphate (pH=6.9) buffer-acetonitrile (v / v is 83:17); detection wavelength: 250 nm; flow rate: 0.8 mL / min; injection volume: 20 μL.

[0100] 2. Infrared spectrum detection

[0101] 2.0 mg of the dried test material was ground with KBr, pressed into pellets, and analyzed by Shimadzu IRAffinity-1 at 4000-400 cm -1 Scan within the range.

[0102] 3. Methylation / GC-MS Analysis

[0103] Weigh 8.0 mg of the dry test material into a reaction bottle, add 8 mL of anhydrous DMSO, then add 800 mg of dry sodium hydroxide, and ultrasonicate for 30 min. Under ice bath conditions, add 3.0 mL of methyl iodide in the dark, add three times, and ultrasonicate for 30 min each time in an ice bath. After the reaction is completed, add 2 mL of distilled water to decompose the residual methyl iodide, add 1 mL of chloroform for extraction, and centrifuge to obtain the chloroform layer.

[0104] After the methylation is complete, the sample is placed in a stoppered test tube and hydrolyzed with 2mol / L TFA in a constant temperature oil bath at 120℃ for 6h, evaporated to dryness under reduced pressure, and then repeatedly added with methanol and dried until the pH is neutral, and then 20mg of NaBH4 is reacted at 40℃ for 30min to reduce the hydrolysis product. Then 100μL of glacial acetic acid is used to terminate the reaction, the sample is dried under low pressure, and then 2mL of acetic anhydride and pyridine are added for acetylation. Keep the reaction at 95℃ with magnetic stirring for 2h. Then add methanol repeatedly 3 times, dry, dissolve with 1mL of chloroform, wash 3 times with an equal volume of distilled water, remove the water layer, and finally place in a fume hood to evaporate the chloroform layer for GC-MS analysis.

[0105] 4. Nuclear Magnetic Resonance Analysis

[0106] After repeated freeze-drying of each of the tangerine peel polysaccharide samples of the present invention, 60 mg was dissolved in 0.6 mL of D2O, placed in a nuclear magnetic resonance tube, and the results were recorded using a 400 MHz nuclear magnetic resonance instrument, Bruker AV-400. 1 HNMR, 13 C NMR, HSQC, HMBC and other spectra.

[0107] 5. Molecular conformation analysis

[0108] Take the polysaccharide of the present invention and dissolve it into a 5 mg / mL solution, and inject 20 μL of the solution into a high performance liquid chromatography (HPLC) system equipped with a G-3000PWXL gel column (Tosoh Biosep, Tokyo, Japan). Use a multi-angle laser scattering (MALLS) detector (λ=663nm) (DAWN HELEOS II, Wyatt, Santa Barbara, CA, USA) and a refractive index (RID) detector for detection. Elution was performed with a 0.1M NaCl solution at a flow rate of 0.5 mL / min at room temperature. Use WyattASTRA 6.1.5.22 software to analyze the data;

[0109] The tangerine peel polysaccharide solution (0.5 mg / mL) and Congo red solution (50 μmol / L) were mixed thoroughly with a concentration gradient of NaOH solution (0.00-0.60 M), and then each mixed solution was scanned in the range of 400-600 nm using a UV-visible spectrophotometer. The maximum absorption wavelength (λ max ) and the maximum absorption wavelength (λ max ) for comparison (iii) test results:

[0110] 1. Structural analysis of the polysaccharide of tangerine peel of the present invention

[0111] (1) Monosaccharide composition analysis

[0112] like Figure 1 As shown, it can be seen from the HPLC spectrum that the tangerine peel polysaccharide prepared by the present invention contains only arabinose. (Chromatographic peak sequence: 1: mannose, 2: rhamnose, 3: glucuronic acid, 4: galacturonic acid, 5: glucose, 6: galactose, 7: xylose, 8: arabinose, 9: fucose).

[0113] (2) Infrared spectroscopy analysis

[0114] like Figure 2 As shown, it can be seen from the infrared spectrum of the tangerine peel polysaccharide of the present invention that the tangerine peel polysaccharide of the present invention contains the infrared characteristic absorption peak of polysaccharide.

[0115] (3) Methylation / GC-MS analysis

[0116] The methylation analysis of the tangerine peel polysaccharide of the present invention was carried out, and after hydrolysis, reductive acetylation and GC-MS detection, the GC-MS spectrum showed that the tangerine peel polysaccharide of the present invention contained sugar residues such as →5)-α-L-Araf-(1→, →2,3,5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→ and α-L-Araf-(1→.

[0117] (4) Nuclear Magnetic Resonance Analysis

[0118] This test was conducted through 1 H NMR, 13 C NMR and HSQC were used to assign the chemical shifts of the carbon atoms and hydrogen atoms of the sugar residues of the tangerine peel polysaccharide of the present invention, and then HMBC was used to confirm the connection order. Figure 3-6 The present invention is the tangerine peel polysaccharide 1 HNMR, 13 CNMR, HSQC and HMBC spectra.

[0119] according to Figure 3-6 The NMR spectrum of the tangerine peel polysaccharide of the present invention is shown in Table 1.

[0120] Table 1 NMR analysis results of the tangerine peel polysaccharide of the present invention

[0121]

[0122] “a” means the signal cannot be distinguished from other signals, and “nd” means no signal was detected.

[0123] In summary: the tangerine peel polysaccharide of the present invention is an arabinose polysaccharide composed of arabinose. Methylation analysis shows that it contains sugar residues such as →5)-α-L-Araf-(1→, →2,3,5)-α-L-Araf-(1→, →3,5)-α-L-Araf-(1→ and α-L-Araf-(1→. The connection sequence between different sugar residues is obtained by two-dimensional nuclear magnetic resonance HMBC spectrum analysis. From the above analysis, the structure of the tangerine peel polysaccharide of the present invention is as shown in the structural formula herein, wherein n+6m=34.

[0124] Example 4 Study on the protective effect of pure tangerine peel polysaccharide on ischemia-reperfusion injury of human myocardial cells

[0125] (I) Test materials: the polysaccharide of tangerine peel of the present invention.

[0126] (ii) Experimental subjects: AC16 cells (provided by Shanghai Cell Bank, Chinese Academy of Sciences).

[0127] (III) Test methods:

[0128] 1. Experimental modeling:

[0129] The present invention adopts a human cardiomyocyte AC16 model. When the cell density reaches about 60%, the original AC16 cell culture medium is replaced with glucose-free DMEM. The cells are then cultured in a three-gas incubator containing 1% O2 and 5% CO2 for 8 hours. After incubation, the glucose-free DMEM is replaced with a fresh complete culture medium, and the cells are re-incubated in a CO2 incubator at 37°C and 5% CO2 for 2, 4, 8, 10, 12 and 16 hours, respectively, to induce an OGD / RAC16 cell model.

[0130] 2. Drug administration

[0131] During the oxygen-glucose deprivation / oxygen supply culture, the cells were pre-incubated with different concentrations of the present invention's Citrus aurantium polysaccharide (50, 100 and 200 μg / mL) for 24 hours.

[0132] 3.CCK-8 Experiment

[0133] The CCK-8 method was used to study the effect of the tangerine peel polysaccharide of the present invention on the viability of AC16 cells in the OGD / R model. After administration, 10 μL of CCK-8 solution was added to each well and incubated at 37° C. for 2 hours. The absorbance was detected using an enzyme marker at a wavelength of 450 nm.

[0134] 4. Detection of expression levels of LDH, MDA, T-AOC, SOD, CAT, GSH-Px, NPSH and ROS

[0135] The effects of the tangerine peel polysaccharide of the present invention on the expression levels of lactate dehydrogenase (LDH), MDA, total antioxidant capacity (T-AOC), SOD, catalase (CAT), glutathione peroxidase (GSH-Px), and non-protein sulfhydryl (NPSH) in AC16 cells were detected using a specific detection kit. First, cells from the control group, OGD / R group, and OGD / R+the tangerine peel polysaccharide group of the present invention were collected and washed with cold PBS solution. Subsequently, the cells were ultrasonically treated in a lysis buffer and centrifuged at 12000 rpm for 10 minutes (4°C). The supernatant was then collected and the expression levels of LDH, MDA, T-AOC, SOD, 315CAT, GSH-Px, and NPSH in AC16 cells were evaluated according to the respective instructions of the manufacturers.

[0136] After 24 hours of pretreatment with the polysaccharide of the present invention, each group of cells was rinsed once with heated DMEM / F12 medium, and then incubated with 500 μL DCFH-DA (10 μM) at 37°C in the dark for 30 minutes. After incubation, the staining solution was replaced with PBS buffer, and the cells were taken and observed under a fluorescence microscope. ImageJ software was used to quantify the ROS fluorescence intensity of AC16 cells.

[0137] 5. Detection of ANP and BNP mRNA expression levels

[0138] Total RNA was extracted from AC16 cells using the AFTSpin Cell Rapid RNA Extraction Kit according to the manufacturer's instructions. RNA was then reverse transcribed into cDNA using a reverse transcription kit, and then PCR amplified to detect the expression levels of ANP and BNP mRNA.

[0139] 6. Western Blot Analysis

[0140] The cells after administration were lysed with RIPA buffer to obtain total cell lysate. The protein concentration was determined using a BCA kit, and 15 μg of protein was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was sealed with a 5% skim milk powder solution for 2 hours and then rinsed with TBST solution. Subsequently, the membrane was incubated with primary antibodies for Nrf2, Keap1, HO-1, JNK, p-JNK, CHOP, GRP78, IRE1, p-IRE1, Bcl-2, Bax, NQO1, Histone H3, Cleaved-Caspase 3, Caspase3, β-Actin, and GAPDH at 4°C overnight. After removing the primary antibody and washing with TBST, the membrane was incubated with a secondary antibody for 2 hours at room temperature. After washing three times with TBST, the sample was fixed by electrochemiluminescence (ECL) and imaged by a gel imager. This experiment was repeated three times.

[0141] (IV) Experimental results

[0142] Please see the attached experimental results Figures 8 to 12 After being treated with different concentrations of the polysaccharide of the present invention, the cell viability of AC16 cardiomyocytes after OGD / R induction was restored, and the level of intracellular lactate dehydrogenase (LDH) was reduced, the oxidative stress level of AC16 cardiomyocytes was inhibited, the expression levels of ANP and BNP mRNA were reduced, and the protective effect against ischemia-reperfusion injury was exerted by regulating the Nrf2 / Keap1, IRE1 / GRPC78 and Bax / Bcl-2 signaling pathways.

[0143] In summary, the pure Citrus aurantium polysaccharide prepared by the present invention can restore the cell viability after OGD / R induction, while reducing the level of intracellular lactate dehydrogenase (LDH), inhibiting the oxidative stress level of AC16 cardiomyocytes, reducing the expression levels of ANP and BNP mRNA, and regulating the Nrf2 / Keap1, IRE1 / GRPC78 and Bax / Bcl-2 signaling pathways, thereby exerting the protective ability against ischemia-reperfusion injury.

[0144] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

[0145] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified and replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A polysaccharide of tangerine peel, characterized in that: Has the following structural formula: in It is L-furanose, where n+6m=34.

2. The method for preparing tangerine peel polysaccharide according to claim 1, characterized in that: The method comprises the following steps: S1 shearing: cutting the peel of the orange peel into small segments to obtain orange peel segments; S2 water extraction: add the tangerine peel segments into water, heat and extract, and filter to obtain the extract; S3 alcohol precipitation: the extract is concentrated to obtain a concentrated solution, ethanol is added to the concentrated solution, and the precipitate is collected to obtain crude polysaccharides; S4 purification: Purify the crude polysaccharide by chromatography to obtain finished tangerine peel polysaccharide.

3. The method according to claim 2, characterized in that The length of the cut tangerine peel segments is 1 to 8 cm.

4. The method according to claim 2, characterized in that The parameters of water extraction are: the amount of water added is 5 to 15 times the weight of the Citrus aurantium segment, the heating temperature is 60 to 100° C., and the extraction time is 1 to 10 hours.

5. The method according to claim 2, characterized in that The alcohol precipitation step is a graded alcohol precipitation step, and the steps are: S31: concentrating the extract under reduced pressure to obtain a first concentrated solution, adding ethanol to a volume concentration of a% and standing the solution, collecting the precipitate and the supernatant to obtain a first crude polysaccharide and a first supernatant; S32: concentrating the first supernatant under reduced pressure to obtain a second concentrated solution, adding ethanol to a volume concentration of b% of ethanol, standing the solution, collecting the precipitate and the supernatant to obtain a second crude polysaccharide and a second supernatant; S33: The second supernatant is concentrated under reduced pressure to obtain a third concentrated solution, ethanol is added to the solution until the ethanol volume concentration is c%, the solution is allowed to stand, and the precipitate and the supernatant are collected to obtain a third crude polysaccharide. The temperature during reduced pressure concentration is 40-70°C, the standing time is 10-28h, and 10≤a<b<c<100.

6. The method according to claim 5, characterized in that The purification steps are as follows: taking the third crude polysaccharide for a purification to remove the protein, and then taking the purified third crude polysaccharide for chromatography to obtain the finished tangerine peel polysaccharide.

7. The method according to claim 6, characterized in that The chromatography process includes: firstly taking the third crude polysaccharide after the first purification for ion exchange column chromatography, gradient elution with 0-2M NaCl, using phenol-sulfuric acid method to draw an elution curve, collecting sugar parts according to the elution curve, concentrating, freeze-drying, and then dissolving with water, centrifuging, taking the supernatant, and then performing molecular sieve gel column chromatography on the supernatant, eluting with water, detecting the elution curve with phenol-sulfuric acid method, collecting sugar parts according to the elution curve, concentrating, freeze-drying, and obtaining finished tangerine peel polysaccharide.

8. A method for identifying tangerine peel polysaccharide, characterized in that: The method comprises the following steps: (1) taking a sample of tangerine peel polysaccharide, completely hydrolyzing it with acid, and then derivatizing the hydrolyzate with PMP and detecting it by liquid chromatography; (2) taking a sample of tangerine peel polysaccharide, drying it, pressing it into tablets, and performing infrared spectroscopy detection; (3) taking a sample of tangerine peel polysaccharide, methylating it, hydrolyzing it, reducing it, acetylating it, and performing GC-MS detection; (4) Dissolving a sample of tangerine peel polysaccharide in D2O and performing nuclear magnetic resonance analysis; (5) The tangerine peel polysaccharide samples were taken for molecular conformation analysis using a multi-angle light scattering system and the Congo red method.

9. Use of the Citrus aurantium polysaccharide as claimed in claim 1 in preparing ischemia-reperfusion injury protective drugs or health products.

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