A kind of tangerine peel polysaccharide and its preparation method, identification method and application

Abstract

The present invention provides a polysaccharide derived from Citrus aurantium dulcis (Ligurianthus chinensis) and its preparation method, identification method, and application, belonging to the technical fields of medicine and health foods. The polysaccharide derived from Citrus aurantium dulcis (Ligurianthus chinensis) has a molecular weight of 11917 Da. The preparation method is simple, the reaction conditions are mild, and large-scale production is possible. Furthermore, the chemical structure of the highly purified polysaccharide obtained is identified and clarified, providing a structural basis for exploring its pharmacological activity mechanism. Furthermore, the pure polysaccharide obtained from the present invention lays a foundation for the development of polysaccharide-derived Citrus aurantium dulcis (Ligurianthus chinensis) as a pharmaceutical, health food, or functional food, as well as for quality control and in-depth research into its structure-activity relationship and mechanism of action.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and health food, and particularly relates to a tangerine peel polysaccharide and a preparation method, an identification method and an 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, ultimately 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 also aggravates tissue and organ dysfunction and structural damage. This myocardial injury caused by reperfusion 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 found widely in animals, plants, and microorganisms. Due to their outstanding antioxidant and immunomodulatory activities, natural polysaccharides play a vital role in a variety of diseases, including cancer, neurological disorders, and cardiovascular diseases. Increasing evidence suggests that polysaccharides can effectively mitigate myocardial injury induced by ischemia / reperfusion. Therefore, identifying potential therapeutic agents from natural polysaccharides is expected to be an effective strategy for preventing and treating myocardial injury induced by ischemia / reperfusion.

[0004] Citrus grandis polysaccharide is a component of the dried outer peel of the Rutaceae plant, Citrus grandis Tomentosa. It has a pungent and bitter taste and a warm nature. It enters the lung and spleen meridians. It regulates qi, relieves fullness, and eliminates dampness and resolves phlegm. Clinically, it is used to treat cough with excessive sputum, alcohol-induced food indigestion, nausea, and abdominal distension. The chemical components of Citrus grandis polysaccharide include polysaccharides, flavonoids, volatile oils, and coumarins. Modern pharmacological studies have shown that polysaccharides, as one of the main active ingredients in Citrus grandis, have a wide range of pharmacological effects, including antioxidant, anti-inflammatory, hypoglycemic, and cardioprotective activities. Current research on Citrus grandis polysaccharide has primarily focused on optimizing its extraction process and anti-inflammatory activity. Few reports exist on its homogeneous polysaccharide structure and cardioprotective effects. Therefore, it is necessary to provide a method for extraction, purification, and structural identification to lay the foundation for quality control of Citrus grandis polysaccharide and further research on its protective activity against ischemia-reperfusion injury. 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 tangerine peel polysaccharide having the following structural formula:

[0008]

[0009] in It is L-furanose arabinose, 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 tangerine peel into small segments to obtain tangerine peel segments;

[0012] S2 water extraction: add the dried 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 concentrate, ethanol is added to the concentrate, and the precipitate is collected to obtain crude polysaccharide;

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

[0015] Preferably, the length of the cut tangerine peel segments 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 the following steps:

[0018] S31: concentrating the extract under reduced pressure to obtain a first concentrated solution, adding ethanol to a volume concentration of a% and allowing the solution to stand, collecting the precipitate and 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% and allowing the solution to stand, 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 volume concentration of ethanol is c%, 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 10-28 hours, 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, performing gradient elution with 0-2M NaCl, using the phenol-sulfuric acid method to draw an elution curve, collecting the sugar part 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 using the phenol-sulfuric acid method, collecting the sugar part according to the elution curve, concentrating, freeze-drying, and obtaining the finished tangerine peel polysaccharide.

[0024] In a third aspect, the present invention provides a method for identifying tangerine peel polysaccharide, comprising the following steps:

[0025] (1) Take a sample of tangerine peel polysaccharide, completely hydrolyze it with acid, and then derivatize the hydrolyzate with PMP and detect 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) Take the tangerine peel polysaccharide sample, methylate, hydrolyze, reduce, acetylate, and perform GC-MS detection;

[0028] (4) Take a sample of tangerine peel polysaccharide and dissolve it in D2O for nuclear magnetic resonance analysis;

[0029] (5) The polysaccharide samples of tangerine peel 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 manufacturing 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 carry out preliminary separation of tangerine peel polysaccharide, which has significant effect. In addition, the preparation method is simple, the reaction conditions are mild, and it can be produced on a large scale;

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

[0034] 3. The present invention identifies the structure of the purified polysaccharide, 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 tangerine peel polysaccharide obtained by the present invention has well-preserved components, a 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 tangerine peel polysaccharide in the fields of medicine, health care products, etc.

[0036] 5. At the same time, the present invention lays the foundation for the development 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 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 This 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 HSQC spectrum of the tangerine peel polysaccharide of the present invention;

[0042] Figure 6 This 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 polysaccharide of tangerine peel on the viability of AC16 cells induced by OGD / R;

[0045] Figure 9 This is the effect of the polysaccharide of tangerine peel of the present invention on the oxidative stress damage of AC16 cells induced by OGD / R;

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

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

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

[0049] To address the problems of the prior art, the present invention provides a polysaccharide derived from Citrus aurantium dulcis (Ligurianthus officinalis) and its preparation method, identification method, and application. The preparation method is simple, requires mild reaction conditions, and can be produced on a large scale. Furthermore, the chemical structure of the highly purified polysaccharide obtained is identified, its components clarified, and a structural basis for exploring its pharmacological activity mechanism is provided. Furthermore, the pure polysaccharide obtained by the present invention lays the foundation for the development of Citrus aurantium dulcis (Ligurianthus officinalis) polysaccharide drugs, quality control, and in-depth research into its structure-activity relationship and mechanism of action.

[0050] The present invention provides a tangerine peel polysaccharide. The inventor obtains the tangerine peel polysaccharide by purifying and separating a crude tangerine peel polysaccharide, and obtains the molecular weight of the tangerine peel polysaccharide to be 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-arabinofuranosyl, wherein n+6m=34. In other embodiments, 6≤n+6m≤68, wherein n and m are positive integers.

[0054] At the same time, the present invention also provides a preparation method of tangerine peel polysaccharide.

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

[0056] S1 Cutting: Cut the dried peel of the dried tangerine peel into small segments, wash with water, and dry them to obtain the dried tangerine peel segments;

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

[0058] S3 graded alcohol precipitation:

[0059] The extract obtained in step S2 was concentrated under reduced pressure to obtain a concentrated solution 1; ethanol was added to the concentrated solution 1 until the ethanol volume concentration was a%, and the solution was allowed to stand, and the precipitate and supernatant were collected to obtain crude polysaccharide CG1 and 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 solution was allowed to stand, and the precipitate and the supernatant were collected to obtain crude polysaccharide CG2 and supernatant 2;

[0061] The supernatant 2 was concentrated under reduced pressure to obtain a concentrated solution 3; ethanol was added to the concentrated solution 3 until the ethanol volume concentration was c%, and the solution was allowed to stand, and the precipitate was 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 deproteinization, dialyzed, and freeze-dried to obtain Citrus aurantium polysaccharide;

[0065] Secondary purification:

[0066] The polysaccharide CG3 after 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 fractions were collected according to the elution curve, concentrated, and freeze-dried; then they were dissolved in water, centrifuged, and the supernatant was collected;

[0067] The supernatant is subjected to molecular sieve gel column chromatography and 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] Through research, the inventors discovered that the optimal length of Citrus aurantium dulcis segments is 1 to 8 cm. Furthermore, the present invention combines water extraction with graded alcohol precipitation, using ethanol concentrations from low to high to perform preliminary separation of Citrus aurantium dulcis polysaccharides. High-concentration ethanol can separate highly polar, water-soluble polysaccharides from less polar, water-soluble polysaccharides, resolving the issue of traditional water-boiling extraction methods that make subsequent separation complex and difficult.

[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 a method for identifying tangerine peel polysaccharide, comprising the following steps:

[0072] (1) Take a sample of tangerine peel polysaccharide, completely hydrolyze it with acid, and then derivatize the hydrolyzate with PMP and detect 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) Take the tangerine peel polysaccharide sample, methylate, hydrolyze, reduce, acetylate, and perform GC-MS detection;

[0075] (4) Take a sample of tangerine peel polysaccharide and dissolve it in D2O for 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 dulcis and explore new sources of medicine, the inventors have obtained the present invention through extensive experimental research: using dried Citrus aurantium dulcis peel as raw material, a water extraction and alcohol precipitation method is used to separate and obtain crude polysaccharides, the extracted crude polysaccharides are deproteinized, and then the crude Citrus aurantium dulcis polysaccharides are purified by ion exchange chromatography and gel molecular sieve column chromatography to prepare a pure Citrus aurantium dulcis polysaccharide for the first time. The physicochemical properties, molecular weight, and monosaccharide composition of the pure polysaccharide are systematically analyzed and identified, and the structural information of the Citrus aurantium dulcis polysaccharide is successfully obtained. The Citrus aurantium dulcis polysaccharide of the present invention is an arabinan, the main chain of which 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 chains are composed of α-L-Araf-(1→ and →5)-α-L-Araf-(1→).

[0078] The present invention also relates to the use of the obtained tangerine peel polysaccharide in the preparation of ischemia-reperfusion injury protective drugs or health products, and evaluates the ischemia-reperfusion injury protective activity 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 embodiments described 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 making creative efforts 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, quickly rinse with cold water, and air 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, and dry the residue to 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 supernatant 1 was concentrated under reduced pressure at 60°C, ethanol was added to a volume concentration of 40% and allowed to stand at room temperature for 24 hours. The mixture was then centrifuged and the precipitate and supernatant were collected to obtain crude polysaccharide CG2 and supernatant 2.

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

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

[0089] Example 2: Tangerine peel 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 tangerine peel 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 eluent conditions with different salt concentrations, of which the elution peak was the 0.1 M NaCl elution portion (the elution curve was tracked using the phenol-sulfuric acid method during the elution process, and the sugar fractions were collected according to the elution curve). The resulting eluates were concentrated and freeze-dried to obtain polysaccharide LC2;

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

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

[0094] (2) 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), and place in an oil bath at 120°C for hydrolysis for 6 hours. Cool to room temperature, repeatedly add methanol and spin dry to remove TFA, dissolve in deionized water to 1 mL, centrifuge, and aspirate 100 μL of each sample solution. Add 100 μL of 0.3M NaOH solution, then add 100 μL of 0.5M PMP methanol solution, mix well, and 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, and repeat this extraction twice. The aqueous phase is filtered through a 0.45 μm filter membrane and then used for HPLC 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 83:17); detection wavelength: 250 nm; flow rate: 0.8 mL / min; injection volume: 20 μL.

[0100] 2. Infrared spectroscopy detection

[0101] 2.0 mg of the dried test material was ground with KBr and pressed into pellets. The pellets were 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 dried test material into a reaction flask, add 8 mL of anhydrous DMSO, then add 800 mg of dry sodium hydroxide, and sonicate for 30 min. Under ice bath conditions, add 3.0 mL of iodomethane in the dark, and add it three times, each time in an ice bath for 30 min. After the reaction is completed, add 2 mL of distilled water to decompose the residual iodomethane, and add 1 mL of chloroform for extraction, and centrifuge to obtain the chloroform layer.

[0104] After complete methylation, the sample was placed in a stoppered test tube and hydrolyzed with 2 mol / L TFA in a constant-temperature oil bath at 120°C for 6 hours. Evaporate to dryness under reduced pressure, repeatedly add methanol and spin dry until the pH is neutral. The hydrolyzed product was then reduced with 20 mg of NaBH₄ at 40°C for 30 minutes. The reaction was terminated with 100 μL of glacial acetic acid, and the sample was spin dried under reduced pressure. Acetylation was then performed by adding 2 mL of acetic anhydride and pyridine. The reaction was maintained at 95°C with magnetic stirring for 2 hours. Methanol was then added three times, spin dried, and dissolved in 1 mL of chloroform. The sample was then washed three times with an equal volume of distilled water. The aqueous layer was removed, and the chloroform layer was evaporated in a fume hood for GC-MS analysis.

[0105] 4. Nuclear Magnetic Resonance Analysis

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

[0107] 5. Molecular Conformation Analysis

[0108] The polysaccharide of the present invention was dissolved into a 5 mg / mL solution, and 20 μL of the solution was injected into a high performance liquid chromatography (HPLC) system equipped with a G-3000PWXL gel column (Tosoh Biosep, Tokyo, Japan). Detection was performed using a multi-angle laser light scattering (MALLS) detector (λ = 663 nm) (DAWN HELEOS II, Wyatt, Santa Barbara, CA, USA) and a refractive index (RID) detector. Elution was performed with a 0.1 M NaCl solution at a flow rate of 0.5 mL / min at room temperature. Data were analyzed using WyattASTRA 6.1.5.22 software.

[0109] The solution of tangerine peel polysaccharide (0.5 mg / mL) and Congo red solution (50 μmol / L) were mixed thoroughly with NaOH solution (0.00-0.60 M) in a concentration gradient, 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 in the HPLC spectrum, it can be seen that the tangerine peel polysaccharide prepared by the present invention contains only arabinose. (Chromatographic peak order: 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. After hydrolysis and reductive acetylation, GC-MS detection was performed. 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 passed 1 H NMR, 13 The chemical shifts of the carbon atoms and hydrogen atoms of the sugar residues of the tangerine peel polysaccharide of the present invention were assigned by C NMR and HSQC, and then the connection sequence was confirmed by HMBC. 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 below.

[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 human myocardial cell ischemia-reperfusion injury

[0125] (1) Test materials: the polysaccharide of tangerine peel of the present invention.

[0126] (2) Experimental subjects: AC16 cells (provided by Shanghai Cell Bank, Chinese Academy of Sciences).

[0127] (3) Test methods:

[0128] 1. Experimental modeling:

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

[0130] 2. Medication

[0131] During 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 assay

[0133] The CCK-8 assay was used to investigate the effects of the present invention's tangerine peel polysaccharide on AC16 cell viability in an OGD / R model. Following treatment, 10 μL of CCK-8 solution was added to each well and incubated at 37°C for 2 hours. Absorbance was measured using a microplate reader 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] Specific detection kits were used to detect the effects of the present invention's tangerine peel polysaccharide 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. First, cells from the control group, OGD / R group, and OGD / R + present invention's tangerine peel polysaccharide group were collected and washed with cold PBS solution. Subsequently, the cells were sonicated in lysis buffer and centrifuged at 12,000 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 manufacturer's instructions.

[0136] After 24 hours of pretreatment with the tangerine peel polysaccharide of the present invention, each cell group was rinsed once with heated DMEM / F12 medium and then incubated with 500 μL of DCFH-DA (10 μM) at 37°C in the dark for 30 minutes. Following incubation, the staining solution was replaced with PBS buffer, and the cells were harvested 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 Rapid RNA Extraction Kit according to the manufacturer's instructions. RNA was then reverse-transcribed into cDNA using a reverse transcription kit and amplified by PCR to detect ANP and BNP mRNA expression levels.

[0139] 6. Western Blot Analysis

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

[0141] (IV) Experimental results

[0142] Please see the attached experimental results Figures 8 to 12 After treatment with different concentrations of the tangerine peel 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, and the expression levels of ANP and BNP mRNA were reduced. The polysaccharide also exerted a protective effect against ischemia-reperfusion injury by regulating the Nrf2 / Keap1, IRE1 / GRPC78 and Bax / Bcl-2 signaling pathways.

[0143] In summary, the pure tangerine peel polysaccharide prepared by the present invention can restore 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] Anything not described in detail in the present invention is well known 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 solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified and replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An application of Citrus aurantium polysaccharide in the manufacture of a drug for protecting against ischemia-reperfusion injury, characterized in that: The tangerine peel polysaccharide is obtained by a preparation method comprising the following steps: S1 shearing: cutting the peel of the tangerine peel into small segments to obtain tangerine peel segments; S2 water extraction: add the dried tangerine peel segments into water, heat and extract, and filter to obtain the extract; S3 alcohol precipitation: the extract is concentrated to obtain a concentrate, ethanol is added to the concentrate, and the precipitate is collected to obtain crude polysaccharide; S4 purification: Purify the crude polysaccharide by chromatography to obtain the finished product of tangerine peel polysaccharide. The alcohol precipitation step is a graded alcohol precipitation step, comprising the following steps: S31: concentrating the extract under reduced pressure to obtain a first concentrated solution, adding ethanol to a volume concentration of 20%, allowing the solution to stand, and collecting the precipitate and 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 40%, allowing the solution to stand, and collecting the precipitate and 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 80%, the solution is allowed to stand, and the precipitate and supernatant are collected to obtain a third crude polysaccharide. The temperature during reduced pressure concentration is 40~70℃ and the standing time is 10~28h; The chromatography process includes: firstly taking the third crude polysaccharide after the first purification for ion exchange column chromatography, performing gradient elution with 0-2M NaCl, collecting the elution part of 0.1M sodium chloride, concentrating and freeze-drying, 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 by phenol-sulfuric acid method, collecting the sugar part according to the elution curve, concentrating and freeze-drying to obtain the finished tangerine peel polysaccharide.

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

3. The use according to claim 1, wherein The parameters of water extraction are: the amount of water added is 5 to 15 times the weight of the tangerine peel segment, the heating temperature is 60 to 100°C, and the extraction time is 1 to 10 hours.

4. The use according to claim 1, wherein The purification steps are as follows: taking the third crude polysaccharide for a first purification to remove protein, and then taking the purified third crude polysaccharide for chromatography to obtain the finished tangerine peel polysaccharide.

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