Gastrodia elata polysaccharide and its preparation method and application

Abstract

The present invention relates to the field of traditional Chinese medicine polysaccharides, and in particular to a gastrodia elata polysaccharide and a preparation method and application thereof. The polysaccharide is TMP-IIa. The preparation method comprises the following steps: subjecting main components of the gastrodia elata to water extraction and alcohol precipitation, defatting, deproteinizing and decolorizing, and combining purification and separation with fiber column chromatography, gel filtration chromatography and the like to obtain a uniform polysaccharide TMP-IIa. The monosaccharide composition of the polysaccharide is glucose, the molecular weight is 960 kDa, and the glycosidic bond connection mode in the molecule is →1)Glcp(2→. The gastrodia elata polysaccharide provided by the present invention is a polysaccharide with a specific molecular weight and structural composition, which is extracted from the gastrodia elata for the first time, has the effect of delaying D-gal (D-galactose)-induced PC-12 cell senescence, and can be used in anti-aging drugs.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine polysaccharides, and in particular to a gastrodia elata polysaccharide and a preparation method and application thereof. Background Art

[0002] Gastrodia elata is a leafless and rootless parasitic plant that lives in symbiosis with Armillaria mellea (Armillaria officinalis), a member of the Trichodermaaceae family. It utilizes the fungus's hyphae or secretions as a nutrient source. Gastrodia elata has a medicinal history spanning over 2,000 years, with the earliest record being in the Shennong Herbal Classic, a book from the Qin and Han dynasties. Rich in phenolic compounds, gastrodin, polysaccharides, proteins, various amino acids, and abundant trace elements, Gastrodia elata has analgesic, antidepressant, antihypertensive, anti-epileptic, anti-inflammatory, anti-thrombotic, antioxidant, and anti-aging properties, as well as improving learning and memory and microcirculation. It also enhances cellular immunity and the body's nonspecific immune system.

[0003] Gastrodia elata polysaccharides generally have poor water solubility, and their metabolic processes and mechanisms of action in the body are mostly unknown. At the same time, the main research objects of Gastrodia elata polysaccharides are crude polysaccharides, and there is a lack of mature purification methods. The chemical structure of the Gastrodia elata polysaccharides contained is unclear, and the quality is difficult to control.

[0004] Chinese patent CN118684790A discloses an active component of Gastrodia elata polysaccharide and its preparation method and application. This Gastrodia elata polysaccharide is a homogeneous polysaccharide with a molecular weight of 42.58×10 4 Da~45.28×10 4 Da, the structure includes 1-Glcp, 1,6-Glcp, 1,4-Glcp, 1,3,4-Glcp and 1,4,6-Glcp, obtained by water extraction and alcohol precipitation, deproteinization and decolorization, and dialysis. This Gastrodia elata polysaccharide has good activity and can be used in the preparation of Parkinson's disease drugs, foods or health products. Although the above polysaccharide is a homogeneous polysaccharide, its molecular weight is 42.58×10 4 Da~45.28×10 4 Da contains a variety of glycosidic bond connection methods, and the polysaccharide obtained by the above preparation method is similar to the crude polysaccharide of Gastrodia elata. How to accurately analyze the structure of the above polysaccharide, how to effectively extract and purify the above polysaccharide, and how to evaluate its biological activity all face great difficulties.

[0005] Chinese patent CN114805629A discloses a uniform polysaccharide of Gastrodia elata, and its preparation method and application. The Gastrodia elata polysaccharide is a uniform polysaccharide with a molecular weight of 24kDa-26kDa, and is composed of terminal-linked glucose, 1,4-linked glucose, 1,6-linked glucose, 1,3,4-linked glucose and 1,4,6-linked glucose in a molar ratio of 5.14:6.91:4.10:1.00:3.84. The Gastrodia elata polysaccharide is subjected to a water extraction and alcohol precipitation method to obtain a precipitate, which is dissolved in water, protein is removed, and the product is concentrated, dialyzed and dried to obtain a crude Gastrodia elata polysaccharide. The crude Gastrodia elata polysaccharide is dissolved in water and subjected to column separation to obtain the uniform Gastrodia elata polysaccharide. The polysaccharide is mainly used in drugs for treating or preventing obesity, diabetes, inflammation, metabolic disorders, cancer, progeria, ALS, IBD, hypertension, or autism.

[0006] In summary, it is particularly important to develop a new Gastrodia elata polysaccharide with a clear structure and determined pharmacological activity. Summary of the Invention

[0007] The object of the present invention is to provide a Gastrodia elata polysaccharide and its preparation method and application. The polysaccharide is TMP-Ⅱa. The preparation method comprises the following steps: subjecting the main components of Gastrodia elata to water extraction and alcohol precipitation, defatting, deproteinizing and decolorizing, and combining purification and separation with fiber column chromatography, gel filtration chromatography, etc. to obtain a uniform polysaccharide TMP-Ⅱa, wherein the monosaccharide composition is glucose with a molecular weight of 960kDa, and the glycosidic bond connection mode in the molecule is →1)Glcp(2→.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A Gastrodia elata polysaccharide is a homogeneous polysaccharide with a molecular weight of 900-1000 kDa. The glycosidic bond connection mode is →1)Glcp(2→, and the structural formula is as follows:

[0010] In the structural formula, Glcp represents a glucose residue, -(1→ and →2)- represents a glycosidic bond, and n is a positive integer between 5550 and 6165.

[0011] Furthermore, the molecular weight is 960kDa.

[0012] A method for preparing Gastrodia elata polysaccharide, characterized in that the preparation method comprises:

[0013] Gastrodia crude polysaccharide is obtained through water extraction, alcohol precipitation, defatting, deproteinization and decolorization.

[0014] The crude Gastrodia elata polysaccharide is purified by anion exchange column chromatography and dextran gel column chromatography to obtain Gastrodia elata polysaccharide.

[0015] Furthermore, the water extraction, alcohol precipitation, degreasing, deproteinizing and decolorizing treatment includes the following steps:

[0016] Take Gastrodia elata, decoct it with water, filter to obtain a filtrate, use petroleum ether and Sevage reagent to extract the filtrate multiple times, collect the aqueous solution, mix the aqueous solution with activated carbon, let it stand, filter to remove the activated carbon, and use ethanol to precipitate to obtain Gastrodia elata crude polysaccharide.

[0017] Furthermore, the anion exchange column chromatography and the dextran gel column chromatography purification comprise the following steps:

[0018] The crude Gastrodia elata polysaccharide was chromatographed using a DEAE-52 anion exchange column, with the eluent being a 0.1-0.5 mol / L NaCl solution. The chromatographed polysaccharide was dialyzed through a dialysis bag, freeze-dried after dialysis, and then chromatographed using a G-200 dextran gel column, with the eluent being pure water. The polysaccharide was then freeze-dried to obtain Gastrodia elata polysaccharide.

[0019] Preferably, during chromatography using a DEAE-52 anion exchange column, the concentration of the NaCl solution is 0.1-0.2 mol / L, and further the concentration of the NaCl solution is 0.1 mol / L.

[0020] Furthermore, the dried tubers of Gastrodia elata are used, the volume ratio of Gastrodia elata to water is 1:(3-6), the decoction temperature is 100° C., and the number of extractions is 1-5 times.

[0021] Preferably, during the water extraction process, the volume ratio of Gastrodia elata to water is 1:(4-5), and the number of filtrations after decoction is 2-4 times; further, the volume ratio of Gastrodia elata to water is 1:4; the number of filtrations after decoction is 3 times.

[0022] Furthermore, the Sevage reagent is obtained by mixing chloroform and n-butanol, the volume ratio of chloroform to n-butanol is (4-6):1; the extraction volume ratio of the filtrate to petroleum ether is (1-2):1, and the number of extractions is 1-3 times; the extraction volume ratio of the filtrate to the Sevage reagent is (4-5):1, and the number of extractions is 1-3 times.

[0023] Preferably, during the deproteinization process, the Sevage reagent is a mixture of chloroform and n-butanol in a ratio of 5:1, the extraction volume ratio of the filtrate to petroleum ether is 1:1, the extraction volume ratio of the filtrate to the Sevage reagent is 5:1, and the number of extractions is 3.

[0024] Preferably, during the petroleum ether extraction process, the extraction volume ratio of the filtrate to the petroleum ether is 1:1, and the number of extractions is 3.

[0025] Furthermore, when decolorizing the aqueous solution, the mass ratio of activated carbon to solution is 1-2%. When decolorizing with activated carbon, the aqueous solution is heated to 40-50° C. and then fully stirred.

[0026] Preferably, during the decolorization process, the mass ratio of activated carbon to solution is 1%, and the decolorization temperature is 50°C.

[0027] Furthermore, the ethanol is 80% ethanol, the volume ratio of the filtrate to 80% ethanol is 1:(3-4), the alcohol precipitation temperature is 4-5°C, and the alcohol precipitation time is 10-12 hours.

[0028] Preferably, during the alcohol precipitation process, the volume ratio of the filtrate to 80% ethanol is 1:4, the alcohol precipitation temperature is 4°C, and the alcohol precipitation time is 12 hours.

[0029] The present invention also protects the use of the above-mentioned Gastrodia elata polysaccharide in anti-aging drugs.

[0030] The present invention also provides a method for preparing Gastrodia elata polysaccharide TMP-Ⅱa;

[0031] The Gastrodia elata polysaccharide TMP-Ⅱa is a polysaccharide with uniform composition, and its monosaccharide composition is glucose.

[0032] The molecular weight of Gastrodia elata polysaccharide TMP-Ⅱa is 960kDa, and it is isolated from the tuber of Gastrodia elata in Jinzhai for the first time.

[0033] The method comprises the following steps: decocting 500 g of dried tubers of Jinzhai Gastrodia elata with hot water, filtering to obtain a filtrate, extracting the filtrate multiple times with petroleum ether and Sevage reagent, collecting the aqueous solution, thoroughly mixing the aqueous solution with activated carbon, allowing it to stand, filtering to remove the activated carbon, and precipitating it with 80% ethanol. The precipitate is freeze-dried to obtain the crude Gastrodia elata polysaccharide TMP. Chromatography is performed on a DEAE-52 anion exchange column using different concentrations of NaCl as the eluent to obtain two fractions (TMP-I and TMP-II). The TMP-II fraction with the highest activity is selected and dialyzed for 48 hours using a 2000 Da dialysis bag. The fraction is then freeze-dried to obtain relatively pure TMP-II. The polysaccharide TMP-II is then purified using a G-200 dextran gel column with a pure aqueous solution as the eluent to obtain a single fraction (TMP-IIa). This fraction is then freeze-dried to obtain a homogeneous polysaccharide TMP-IIa with an extraction yield of 0.26%.

[0034] Among them, the Gastrodia elata samples are the dried tubers of Jinzhai Gastrodia elata.

[0035] Among them, the volume ratio of Gastrodia elata sample to hot water is 1:4, the hot water temperature is 100℃, and the extraction times is 3 times.

[0036] The Sevage reagent is a mixture of chloroform and n-butanol in a ratio of 5:1.

[0037] The extraction volume ratio of the filtrate to petroleum ether was 1:1, and the number of extractions was 3 times; the extraction volume ratio of the filtrate to Sevage reagent was 5:1, and the number of extractions was 3 times.

[0038] The mass ratio of activated carbon to solution during decolorization of the aqueous solution is 1%. During decolorization of the activated carbon, the aqueous solution is heated to 50° C. and then fully stirred.

[0039] The volume ratio of the filtrate to 80% ethanol is 1:4, the alcohol precipitation temperature is 4-5° C., and the alcohol precipitation time is 12 hours. The 80% ethanol is ethanol with a volume fraction of 80%, with ethanol accounting for 80% and water accounting for 20%.

[0040] Among them, the concentration of the crude polysaccharide solution is 20 mg / mL, the specifications of the DEAE-52 anion exchange column chromatography are 3.5×30 cm, the eluent used is 0, 0.1, 0.2, 0.3, and 0.5 mol / L NaCl solution, the flow rate is 2.0 mL / min, and the two components obtained are TMP-Ⅰ eluted with 0 mol / L NaCl solution and TMP-Ⅱ eluted with 0.1 mol / L NaCl solution, and the 0 mol / L NaCl solution is pure water.

[0041] Among them, the concentration of polysaccharide TMP-Ⅱ solution is 10 mg / mL, the specification of G-200 dextran gel column chromatography is 1.6×70 cm, the eluent used is pure water solution, the flow rate is 0.2 mL / min, and the obtained components are TMP-Ⅱa eluted with pure water solution.

[0042] The present invention has the following beneficial effects: the Gastrodia elata polysaccharide provided by the present invention is a novel polysaccharide with a specific molecular weight and structural composition, extracted from Gastrodia elata for the first time, has the effect of delaying D-gal (D-galactose)-induced senescence of PC-12 cells, and can be used in anti-aging drugs; the Gastrodia elata polysaccharide provided by the present invention has the characteristics of clear monosaccharide composition, determined molecular weight, uniform polysaccharide components, and clear glycosidic bond linkage. The single glycosidic bond linkage reduces the structural variation of the polysaccharide chain during biosynthesis or processing, thereby maintaining the consistency and stability of the polysaccharide chain; the present invention provides a method for preparing Gastrodia elata polysaccharide, which is obtained by water extraction and alcohol precipitation, defatting, deproteinizing and decolorization treatment, and is purified by DEAE-52 anion exchange column chromatography and G-200 dextran gel column chromatography. The obtained polysaccharide is TMP-Ⅱa. The preparation process conditions are mild, the operation is simple, and it is environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present method or the technical solutions in the prior art, a brief introduction will be given below to the drawings required for use in the implementation cases or the description of the prior art. Obviously, the drawings described below are some implementation cases of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is a flow chart of the preparation process of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention;

[0045] Figure 2 This is the chromatographic elution diagram of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention; Figure 2 A is the elution diagram of DEAE-52 anion exchange column chromatography, Figure 2 B is the elution diagram of G-200 dextran gel column chromatography;

[0046] Figure 3 is a linear relationship diagram of the standard solution in a specific embodiment of the present invention; Figure 3 A is the linear relationship diagram of glucose standard solution, Figure 3 B is the linear relationship diagram of galacturonic acid standard solution.

[0047] Figure 4 This is a spectrum of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention; Figure 4 A is the ultraviolet spectrum, Figure 4 B is the infrared spectrum;

[0048] Figure 5 This is the HPGPC diagram of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention;

[0049] Figure 6 This is a Congo red ultraviolet comparison diagram of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention;

[0050] Figure 7 HPLC chart of the monosaccharide composition of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention;

[0051] Figure 7 In the chart, 1: mannose; 2: glucuronic acid; 3: galacturonic acid; 4: rhamnose; 5: PMP; 6: glucose; 7: galactose; 8: xylose; 9: fucose

[0052] Figure 8 This is the NMR diagram of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention,

[0053] Figure 8 A. Hydrogen spectrum; Figure 8 B. Carbon spectrum; Figure 8 C. HSQC spectrum; Figure 8 D.1H-1H COSY spectrum; Figure 8 E.HMBC spectrum;

[0054] Figure 9 This is a graph showing the relationship between Gastrodia elata polysaccharide TMP-Ⅱa and PC12 cell viability in a specific embodiment of the present invention; Figure 9A is the relationship between Gastrodia elata polysaccharide TMP-Ⅱa and PC12 cell viability without D-galactose induction; Figure 9 B is the relationship between TMP-Ⅱa and PC12 cell viability after D-galactose induction;

[0055] Figure 10 This is a fluorescence microscopy image of the effect of Gastrodia elata polysaccharide TMP-Ⅱa on mitochondrial ROS levels in PC-12 cells in a specific embodiment of the present invention. DETAILED DESCRIPTION

[0056] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0057] Example 1

[0058] See also Figure 1 The preparation steps of Gastrodia elata polysaccharide are as follows:

[0059] (1) The tuber of Gastrodia elata is decocted with hot water, the volume ratio of Gastrodia elata to hot water is 1:4, the temperature of the hot water is 100°C, filtered to obtain a filtrate, the filtrate is extracted with an equal volume of petroleum ether, the two are fully mixed, the layers are allowed to separate, the water layer is collected, the operation is repeated three times, and the water layer is finally collected. The water layer is mixed with Sevage reagent in a ratio of 5:1, the two are fully mixed, the layers are allowed to separate, the water layer is collected, the operation is repeated three times, and the water layer is finally collected. An appropriate amount of activated carbon is added, the mixture is stirred at 50°C for 15 minutes, and then the activated carbon is filtered using a sand core funnel, and the aqueous solution is retained. 80% ethanol is used for precipitation, the volume ratio of the aqueous solution to 80% ethanol is 1:4, the precipitation temperature is 4-5°C, and the precipitation time is 12 hours. The precipitate is collected and freeze-dried to obtain Gastrodia elata crude polysaccharide TMP.

[0060] (2) See Figure 2 A, in Figure 2In A, the red curve indicates that the absorbance changes with the change of the eluent volume; the blue dotted line indicates that the concentration of the NaCl solution is selected as the volume of the eluent, the concentration of the crude polysaccharide TMP solution is 20 mg / mL, DEAE-52 anion exchange column chromatography (3.5×30 cm) is used, and the eluent is 0, 0.1, 0.2, 0.3, and 0.5 mol / L NaCl solution, the flow rate is 2.0 mL / min, and the polysaccharide content in the eluate is detected by phenol-sulfuric acid colorimetry. The eluate containing polysaccharide is collected to obtain the Gastrodia elata polysaccharide extract, and two components are obtained, namely TMP-Ⅰ eluted with 0 mol / L NaCl solution and TMP-Ⅱ eluted with 0.1 mol / L NaCl solution. The 0 mol / L NaCl solution is pure water.

[0061] (3) See Figure 2 B, by Figure 2 B shows that the polysaccharide TMP-Ⅱ is a homogeneous component. The concentration of the polysaccharide TMP-Ⅱ solution is 10 mg / mL. G-200 dextran gel column chromatography (1.6×70 cm) is used, and the eluent is a pure water solution with a flow rate of 0.2 mL / min. The polysaccharide content in the eluate is detected by phenol-sulfuric acid colorimetry. The eluate containing polysaccharides is collected to obtain the Gastrodia elata polysaccharide extract, and one component is obtained, which is TMP-Ⅱa eluted with pure water solution.

[0062] After the above steps, the extraction rate of Gastrodia elata polysaccharide TMP-Ⅱa is 0.26%.

[0063] Example 2

[0064] The physicochemical properties of Gastrodia elata polysaccharide are analyzed as follows:

[0065] (1) The carbohydrate content of the Gastrodia elata polysaccharide TMP-IIa obtained in Example 1 was determined by the phenol-sulfuric acid method:

[0066] Take 20.0 mg of anhydrous glucose dried to constant weight and place it in a 100 mL volumetric flask. Dissolve it in water and dilute to the mark. Shake well to prepare a 0.2 mg / mL glucose reference solution. Accurately measure 0.1 mL, 0.15 mL, 0.2 mL, 0.25 mL, 0.3 mL, 0.35 mL, 0.4 mL, 0.5 mL, 0.6 mL, and 0.7 mL of the reference solution and place them in clean, anhydrous test tubes. If the amount is less than 2.0 mL, add pure water to 2.0 mL and shake well. Take 0.25 mL of each solution, add 0.25 mL of 6% phenol aqueous solution, shake well, then add 1.25 mL of concentrated sulfuric acid and shake well. Let it stand for 30 minutes. Measure the sample absorbance at 490 nm. Perform the same treatment with 1.0 mL of water as a blank control and draw a standard curve. Weigh 5.0 mg of TMP-Ⅱa freeze-dried powder and place it in a 10 mL volumetric flask. Dissolve it in water and dilute to the mark. Shake well. Take 0.5 mL of polysaccharide solution and dilute it to different concentrations. Treat it with phenol-sulfuric acid method and measure the absorbance of the sample solution. Calculate the total sugar content according to the standard curve. The linear relationship diagram of the absorbance of glucose standard solution can be found in Figure 3 A.

[0067] The carbohydrate content of the Gastrodia elata polysaccharide TMP-IIa obtained in Example 1 was determined to be 89.2%. The instrument used was a SHIMADZU UV-2550 UV-visible spectrophotometer.

[0068] (2) The m-hydroxybiphenyl method was used to determine the uronic acid content of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1.

[0069] Accurately weigh 477.0 mg of sodium tetraborate and dissolve it in 50 mL of concentrated sulfuric acid to prepare a borate solution. Accurately weigh 15.0 mg of m-hydroxybiphenyl and dissolve it in 0.5% NaOH solution. The volume is adjusted to 10 mL to prepare a m-hydroxybiphenyl solution. Accurately weigh 5.0 mg of anhydrous galacturonic acid, dissolve it in pure water, and dilute to volume in a 10 mL volumetric flask to obtain a 0.5 mg / mL galacturonic acid standard solution. Accurately pipette 0, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.8, and 1.0 mL of the galacturonic acid standard solution into 10 mL test tubes. Fill each tube to 1 mL with pure water. Place the test tubes in an ice bath and add 5.0 mL of the cooled borate solution dropwise. After vortexing to mix, place the tubes in a boiling water bath for 20 minutes. Remove and cool to room temperature. Add 100 μL of the m-hydroxybiphenyl solution to each tube, mix thoroughly, and let stand for 30 minutes. Accurately weigh 5.0 mg of TMP-Ⅱa lyophilized powder and place it in a 10 mL volumetric flask. Dissolve it in water and dilute to the mark. Take 1 mL of the polysaccharide solution and place it in a 10 mL test tube. Repeat the above steps. Measure the absorbance of the sample solution and calculate the acid polysaccharide content according to the standard curve. For the linear relationship diagram of the absorbance of the galacturonic acid standard solution, see Figure 3 B.

[0070] The uronic acid content of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was determined to be 6.2%. The instrument used was a SHIMADZU UV-2550 UV-visible spectrophotometer.

[0071] (3) UV absorption of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was measured by UV scanning;

[0072] The UV spectrum of Gastrodia elata polysaccharide TMP-Ⅱa can be found in Figure 4 A.

[0073] After testing, it was found that the UV scanning spectrum of the obtained Gastrodia elata polysaccharide TMP-Ⅱa had no absorption peaks of pigments, proteins and nucleic acids. The instrument used was a SHIMADZUUV-2550 UV-visible spectrophotometer with a scanning range of 200-800nm.

[0074] (4) Analyze the characteristic groups of Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 using infrared spectrometer

[0075] Weigh about 2.0 mg of dried Gastrodia elata polysaccharide TMP-Ⅱa, mix it with 200 mg of KBr powder, press it into tablets, and then scan and analyze it on a L1600400 Spectrum TWO FT-IR Fourier transform infrared spectrometer with a scanning range of 4000 cm -1 ~400cm -1 The infrared spectrum of Gastrodia elata polysaccharide TMP-Ⅱa is shown in Figure 4 B.

[0076] After testing, the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 has a characteristic peak of polysaccharide at 3427cm -1 and 1350cm -1 The absorption peak at 1386 cm is caused by the stretching vibration of -OH. -1 The absorption peak at 1600 cm -1 The absorption peak at 1106 cm is caused by the water in the polysaccharide. -1 The absorption peak is caused by the vibration of the glycosidic bond in the polysaccharide molecule, corresponding to the COC stretching vibration, which is a characteristic peak of polysaccharides and is at 990cm -1 The absorption peak at 840 cm is caused by C-OH bending vibration. -1 The absorption peak is the characteristic peak of α-glycosidic bond.

[0077] (5) The HPGPC method was used to determine the uniformity and relative molecular weight of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1.

[0078] Test conditions: Agilent 1260 Infinity system; SRTSEC-150 (7.8 × 300 mm) column; mobile phase: double-distilled water; injection volume: 10 μL; flow rate: 1.0 mL / min; column temperature: 35°C; differential refractive index detector. The HPGPC diagram of Gastrodia elata polysaccharide TMP-Ⅱa is shown in Figure 5 .

[0079] After testing, the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was a uniform polysaccharide with a relative molecular mass of kDa.

[0080] (6) Congo red test analysis of the spatial configuration of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1

[0081] Prepare a 1.0 mg / mL aqueous solution of Gastrodia elata polysaccharide TMP-Ⅱa. Mix the polysaccharide solution (1.0 mL) with Congo red solution (100 μM) at a ratio of 1:1. Then add 1.0 mL of NaOH solution to establish reaction systems with different NaOH concentrations (0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 M). Scan the reaction solution at a UV wavelength of 400-600 nm. For a Congo red UV comparison chart of Gastrodia elata polysaccharide TMP-Ⅱa, see Figure 6 .

[0082] After testing, it was found that the spatial configuration of the obtained Gastrodia elata polysaccharide TMP-Ⅱa was not a triple helical structure. The instrument used was a SHIMADZUUV-2550 ultraviolet-visible spectrophotometer with a scanning range of 400-600nm.

[0083] Example 3

[0084] The chemical structure of Gastrodia elata polysaccharide is identified as follows:

[0085] (1) The monosaccharide composition of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was determined by PMP-HPLC method.

[0086] Weigh 20 mg of Gastrodia elata polysaccharide TMP-Ⅱa and add 1.5 mL of ultrapure water to fully dissolve it. Slowly add 1.5 mL of 4 mol / L trifluoroacetic acid and stir at 110°C for 5 hours. Transfer the reaction solution to a spinner flask, add an appropriate amount of methanol, and evaporate it to dryness. Add methanol again to dissolve it, and repeat the spin drying process several times. Finally, dissolve the spin-dried material in 2 mL of pure water. Take 1 mL of the polysaccharide hydrolyzed solution for derivatization reaction, add 1 mL of 0.5 mol / L PMP methanol solution and 1 mL of 0.3 mol / L NaOH solution in sequence, shake thoroughly, and stir at 70°C for 1 hour. After cooling, 1 mL of 0.3 mol / L HCl was added for neutralization, and then 2 mL of dichloromethane was added and stirred at room temperature for 5 minutes. The mixture was allowed to stand for stratification and extracted 3 times. The aqueous layer was collected and passed through a 0.45 μm aqueous membrane. The volume was adjusted to 5 mL with ultrapure water. 100 μL was transferred to a clean injection bottle, and 800 μL of ultrapure water and 100 μL of methanol were added. After mixing, the mixture was tested by HPLC. The monosaccharide composition HPLC chart of Gastrodia elata polysaccharide TMP-Ⅱa can be found in the following figure: Figure 7 .

[0087] After testing, it was found that the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was composed of glucose.

[0088] (2) Determination of the chemical structure characteristics of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 by NMR

[0089] 25 mg of Gastrodia elata polysaccharide was completely dissolved in 99.9% D2O, placed in a nuclear magnetic resonance tube, and subjected to NMR determination.

[0090] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; OneNMRprobe (5mm); 1H spectrum operating frequency 599.81MHz; 13C spectrum operating frequency 150.84MHz; measured at 25℃ 1 H. 13 C, COSY, HSQC, HMBC spectra, NMR spectra of Gastrodia elata polysaccharide TMP-Ⅱa see Figure 8 The 5.31ppm value in the H-NMR spectrum indicates that the configuration of the terminal carbon is α. Combined with the relevant information in the COSY, HSQC, and HMBC spectra, it is further confirmed that the monosaccharide in Gastrodia elata polysaccharide is glucose.

[0091] After testing, the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 is composed of →1)Glcp(2→. The specific NMR spectrum structure analysis is shown in Table 1 below.

[0092] Table 1 NMR analysis of Gastrodia elata polysaccharide TMP-Ⅱa

[0093]

[0094] Experimental Example 1: Verification of the efficacy of Gastrodia elata polysaccharide in delaying D-gal-induced senescence of PC-12 cells

[0095] 1 Experimental materials and instruments

[0096] 1.1 Experimental Materials

[0097] Gastrodia elata polysaccharide (TMP-Ⅱa) was produced in the laboratory.

[0098] 1.2 Experimental Reagents

[0099] D-galactose (CAS No. 59-23-4, product number D8310, purity: ≥99.0%) was purchased from Beijing Solaibao Technology Co., Ltd. (Beijing, China); N-Acetyl-L-cysteine ​​(NAC, HY-B0215) was purchased from MedChemExpress (MCE) Biotechnology Co., Ltd. (New Jersey, USA); MitoSOX TMGreen (M36006) was purchased from Thermo Fisher Scientific Inc. (Massachusetts, USA); Hoechst 33342 (H288601) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); high-glucose DMEM liquid culture medium, penicillin / streptomycin (double antibody) solution, and trypsin 0.25% Trypsin (25200-056) were purchased from HyClone, a subsidiary of Thermo Fisher Scientific Inc. (Logan, Utah, USA); special fetal bovine serum (FB25011) was purchased from Clark Bioscience Inc. (Virginia, USA); BCA kit ( P0010S), anti-fluorescence quenching mounting solution (P0126-5 ml) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. (Shanghai, China); cell proliferation-toxicity detection kit (CellCount 1ngKlt-8, BA350B), 1× PBS buffer (cell culture) (BL302A) were purchased from Biosharp Biotechnology Co., Ltd. (Hefei, Anhui); antibodies p53 (CAT No. 60283-2-Ig) and p21 (CAT No. 28248-1-AP) were purchased from Proteintech Biotechnology (Chicago, USA), NLRP3 (WL02635) was purchased from Wanlei Biotechnology (Shenyang, China); IL -6 (RB20104UC), NFκB (222013) were purchased from Zhengneng Biotechnology (Chengdu, China); lactate dehydrogenase (LDH, A020-2-2), malondialdehyde (MDA) assay kit (TBA method) (A003-1-2), and catalase (CAT) assay kit (visible light method) (ammonium molybdate method) (A007-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); mouse p21 enzyme-linked immunosorbent assay kit (JL54563) and mouse p53 enzyme-linked immunosorbent assay kit (JL12305) were purchased from Jianglai Biotechnology (Shanghai, China); PALL66485 nitrocellulose membrane was purchased from PALL (New York, USA); 30% Acr -Bis, 1 M Tris-HCl, 1.5 M Tris-HCl, primary antibody diluent, and 5× protein loading buffer were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); SDS, TRIS, PMSF, 10% PAGE gel coagulant, and high-efficiency RIPA lysis buffer were purchased from Solebao Technology Co., Ltd. (Beijing, China); PBS buffer powder was purchased from Aorui Dongyuan Biotechnology Co., Ltd. (Wuxi, China); DMSO was purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China); horseradish peroxidase-labeled anti-rabbit and anti-mouse IgG were purchased from Zhongshan Jinqiao Biotechnology Co., Ltd. (Beijing, China); and anhydrous ethanol was purchased from Qiangsheng Functional Chemistry Co., Ltd. (Jiangsu, China). 2 Experimental Methods

[0100] 2.1 Cell culture

[0101] PC-12 cells were cultured in high-glucose DMEM supplemented with 10% premium fetal bovine serum (FBS) at 37°C under 5% CO 2 . Subsequent experiments were performed when the cells reached the logarithmic growth phase.

[0102] 2.2 Cytotoxicity testing and dose screening of Gastrodia elata polysaccharide TMP-Ⅱa

[0103] PC-12 cells were plated at 1×10 5 Cells were seeded in 96-well plates at a specific density. After reaching a certain cell density, a gradient of TMP-IIa (2.5-640 μg / mL) was added and incubated for a specified period of time. The cells were then incubated with CCK8 at 37°C, 5% CO₂ for 1 hour. The absorbance (OD) was read at 450 nm using a microplate reader to determine a safe dose of Gastrodia elata polysaccharide.

[0104] PC-12 cells were plated at 1×10 5 Cells were seeded in 96-well plates and treated with D-galactose to induce cellular senescence. Subsequently, a safe dose of TMP-Ⅱa (2.5-160 μg / mL) was added and incubated for a specified period of time. The cells were then incubated with CCK8 for 1 hour at 37°C and 5% CO2. The absorbance (OD) was read at 450 nm using a microplate reader to preliminarily determine the optimal intervention dose range for Gastrodia elata polysaccharide. PC-12 cells were then seeded in 6-well plates and incubated with D-galactose solution and the optimal intervention dose of Gastrodia elata polysaccharide for a specified period of time. Cell proteins were extracted, and western blot analysis was performed to detect senescence markers p53 and p21, as well as inflammatory factors IL-6, NLRP3, and NFKB, to determine the optimal intervention dose of Gastrodia elata polysaccharide.

[0105] 2.3 Inhibitor intervention experiments

[0106] In order to verify the key role of reactive oxygen levels, the above-mentioned cell model was intervened with Gastrodia elata polysaccharide and combined with ROS inhibitor N-Acetyl-L-cysteine ​​(NAC). The PC-12 cells were divided into 6 groups, namely normal group (Control group), model group (Model group), Gastrodia elata polysaccharide intervention group (TMP-Ⅱa-20μg / mL group), NAC-1μM intervention group (NAC-1μM group), NAC-2μM intervention group (NAC-2μM group), and NAC-4μM intervention group (NAC-4μM group). The normal control group was given a normal growth medium (high-glucose DMEM medium supplemented with 10% premium fetal bovine serum). The model and NAC intervention groups were given a growth medium supplemented with 40 mM D-galactose, while the Gastrodia elata polysaccharide intervention group was given a growth medium supplemented with 40 mM D-galactose and 20 μg / mL Gastrodia elata polysaccharide. Cells were cultured together until the end of the experiment. Additionally, 2 hours before the end of the experiment, the NAC-1 μM, NAC-2 μM, and NAC-4 μM intervention groups were treated with NAC (1 μM, 2 μM, and 4 μM), respectively, for 2 hours. After the incubation period, cells were harvested for subsequent analysis.

[0107] 2.4 mtROS level detection

[0108] (1) Cell treatment: The cultured cell suspension was inoculated into different 6-well plates and treated with D-galactose, Gastrodia elata polysaccharide TMP-Ⅱa or NAC; (2) Stock solution preparation: MitoSOX TM Green is in powder form and needs to be returned to room temperature before use. TM Add 10 μL of DMSO solution to Green and mix well to obtain 1 mM MitoSOX. TM Green stock solution; (3) Working solution preparation: aspirate a small amount of liquid from the stock solution into a 5 mL centrifuge tube, add an appropriate amount of preheated PBS buffer to make the final concentration 1 μM; (4) Add probe working solution: drop 500 μL of 1 μM MitoSOX into a 6-well plate TM A mixture of Green staining working solution and 1 μL Hoechst 33342 working solution was incubated at 37°C in the dark for 30 minutes. The cells were washed 2-3 times with preheated PBS buffer for 3 minutes each time. (5) Sealing: 10 μL of anti-fluorescence quenching sealing solution was dripped into the center of the slide. The cell slide placed in the 6-well plate was taken out and the reverse side (the side containing adherent cells) was attached to the sealing solution (to prevent bubbles in the attachment area that affect image acquisition). Subsequently, a laser scanning confocal microscope was used to observe and collect images.

[0109] 2.5 Oxidation kit determination

[0110] Cell sample preparation: The cultured cell suspension was inoculated into different 6-well plates and treated with D-galactose, Gastrodia elata polysaccharide TMP-Ⅱa, or NAC. After the treatment, cell lysis buffer (RIPA:PMSF = 100:1) was added to each well to lyse the cells. After standing for half an hour, the cells were centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected and the cell culture medium was collected simultaneously. Finally, the levels of CAT and MDA in the cells and the level of extracellular LDH were measured using commercially available kits (Nanjing Jiancheng) according to the manufacturer's instructions; the levels of senescence markers p21 and p53 were measured using ELISA kits (Jianglai).

[0111] 2.6 Western Blot detection of protein expression levels

[0112] First, cultured cell samples were dissolved in a prepared RIPA solution (with protease and phosphatase inhibitors added in appropriate proportions) to extract proteins. Equal amounts of protein samples were separated on 15% SDS-polyacrylamide gels and then electrophoretically transferred to NC membranes. After blocking with 5% BSA for 2 hours, the membranes were washed three times for 5 minutes each in Tris-buffered saline (TBS-T) containing 0.1% Tween-20. The membranes were then incubated with different primary antibodies overnight at 4°C. The following day, the NC membranes were washed three times with TBS-T and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein-antibody complexes were then detected using an emitter-coupled logic (ECL) substrate that reacted with the HRP-conjugated secondary antibody (Tanon 5200 Multi Chemiluminescence Imaging Analysis System). Finally, band signal intensity was analyzed using Image-Proplus 6.0 software.

[0113] 2.7 Statistical analysis

[0114] All data are expressed as mean ± standard deviation (mean ± SD) established in different experiments and analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test in IBM SPSS Statistics 25. Graphs were drawn using GraphPad Prism 8.0.1 software, and p < 0.05, p < 0.01, or p < 0.001 were considered statistically significant.

[0115] 3 Experimental results

[0116] 3.1 Verification of the effect of Gastrodia elata polysaccharide TMP-Ⅱa on delaying D-galactose-induced senescence in PC-12 cells

[0117] like Figure 9 As shown in A, in the CCK8 test, when the dosage of Gastrodia elata polysaccharide TMP-Ⅱa was in the range of 2.5-160μg / mL, the cell survival rate was similar to that of the blank group; while in the range of 360-640μg / mL, the cell survival rate was higher than that of the blank group, showing a proliferation-promoting phenomenon. Therefore, we selected 2.5-160μg / mL as the safe dosage of Gastrodia elata polysaccharide TMP-Ⅱa. Figure 9 As shown in Figure B, D-galactose can induce a weakening of the proliferation ability of PC-12 cells (p<0.01), while Gastrodia elata polysaccharide can partially restore the proliferation ability of PC-12 cells, among which the effects at doses of 10μg / mL and 20μg / mL are significant (p<0.05).

[0118] Ineffective ROS control at the mitochondrial supercomplex leads to altered ROS signaling, mediating cellular stress responses to age-related damage. Therefore, sustained increases in mitochondrial ROS levels are potentially crucial for inducing and maintaining cellular aging. Fluorescence microscopy images of the effects of Gastrodia elata polysaccharide TMP-ⅡC on mitochondrial ROS levels in PC-12 cells are shown in Figure 2. Figure 10 As shown in the results, compared with the D-galactose group, the mtROS levels in the Gastrodia elata polysaccharide TMP-Ⅱa-10μg / mL group and the TMP-Ⅱa-20μg / mL group were significantly reduced, as evidenced by a significant decrease in the green fluorescence intensity around the cell nucleus; the 20μg / mL dose was the most effective. This result suggests that the anti-aging effect of Gastrodia elata polysaccharide is related to the inhibition of mitochondrial ROS levels.

Claims

1. A method for preparing Gastrodia elata polysaccharide, characterized in that: The preparation method comprises: The crude polysaccharide of Gastrodia elata is obtained by water extraction and alcohol precipitation, defatting, deproteinizing and decolorizing. The method comprises the following steps: taking Gastrodia elata, decocting with water, filtering to obtain a filtrate, extracting the filtrate with petroleum ether and Sevage reagent multiple times, collecting the aqueous solution, thoroughly mixing the aqueous solution with activated carbon, standing, filtering to remove the activated carbon, and precipitating with ethanol to obtain the crude polysaccharide of Gastrodia elata; the dried tubers of Gastrodia elata are used, the volume ratio of Gastrodia elata to water is 1:4, the volume ratio of Gastrodia elata to water is 1:4, the number of times of filtration is 3, the Sevage reagent is a mixture of chloroform and n-butanol in a ratio of 5:1, the volume ratio of extraction of the filtrate to petroleum ether is 1:1, the volume ratio of extraction of the filtrate to Sevage reagent is 5:1, and the number of times of extraction is 3; The method comprises the following steps: performing anion exchange column chromatography and dextran gel column chromatography on the crude Gastrodia elata polysaccharide to purify the crude Gastrodia elata polysaccharide, wherein the crude Gastrodia elata polysaccharide is chromatographed on a DEAE-52 anion exchange column, wherein the eluent is a 0.1-0.5 mol / L NaCl solution, dialyzing the chromatographed polysaccharide through a dialysis bag, freeze-drying the polysaccharide after dialysis, performing further chromatography on a dextran gel column, wherein the eluent is pure water, and freeze-drying the polysaccharide to obtain the Gastrodia elata polysaccharide; The molecular weight of the Gastrodia elata polysaccharide is 900-1000 kDa, and the glycosidic bond connection mode is →1)Glcp(2→, and the structural formula is as follows: , n is a positive integer.

2. The preparation method according to claim 1, characterized in that When decolorizing the aqueous solution, the mass ratio of activated carbon to solution is 1-2%. When decolorizing with activated carbon, the aqueous solution is heated to 40-50°C and then fully stirred.

3. The preparation method according to claim 1, characterized in that The ethanol is 80% ethanol, the volume ratio of the filtrate to the 80% ethanol is 1:(3-4), the alcohol precipitation temperature is 4-5°C, the alcohol precipitation time is 10-12 hours, and the 80% ethanol is ethanol with a volume fraction of 80%, with ethanol accounting for 80% and water accounting for 20%.

4. Use of the Gastrodia elata polysaccharide obtained by the preparation method of Gastrodia elata polysaccharide according to any one of claims 1 to 3 in the preparation of anti-aging drugs.

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