Gastrodia elata polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicine polysaccharide, in particular to gastrodia elata polysaccharide and a preparation method and application thereof.The gastrodia elata polysaccharide is TMP-IIa, and the preparation method of the gastrodia elata polysaccharide comprises the steps that main components of gastrodia elata are subjected to water extraction and alcohol precipitation, degreasing, deproteinization, decolorization, purification and separation in combination with fiber column chromatography, gel filtration chromatography and the like, and the uniform polysaccharide TMP-IIa is obtained; the gastrodia elata polysaccharide has a molecular weight of 960kDa, and a glycosidic bond connection mode in the molecule is > 1) Glcp < 2 >. The gastrodia elata polysaccharide provided by the invention is a novel polysaccharide with a specific molecular weight and structural composition, is extracted from gastrodia elata for the first time, has an effect of delaying D-gal (D-galactose) induced PC-12 cell senescence, and can be applied to anti-senescence 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 has no leaves or roots. It is a parasitic plant that coexists with Armillaria mellea of ​​the Trichoderma family and uses the hyphae or hyphae secretions of Armillaria mellea as its nutritional source. Gastrodia elata has a history of more than 2,000 years of medicinal use, and was first recorded in the Shennong Herbal Classic during the Qin and Han dynasties. Gastrodia elata is rich in phenolic substances, gastrodin, gastrodia polysaccharides, proteins, multiple amino acids and abundant trace elements. It has analgesic, anti-depressant, anti-hypertensive, anti-epileptic, anti-inflammatory, anti-thrombotic, antioxidant, anti-aging effects, and improves learning and memory and microcirculation. It also has the effects of enhancing cellular immunity and the body's nonspecific immunity.

[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 object of Gastrodia elata polysaccharides is 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 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 a preparation method and application thereof. The polysaccharide of Gastrodia elata 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 polysaccharide of Gastrodia elata is subjected to a water extraction and alcohol precipitation method to obtain a precipitate, which is dissolved in water, the protein is removed, and the precipitate is concentrated, dialyzed and dried to obtain a crude polysaccharide of Gastrodia elata. The crude polysaccharide of Gastrodia elata is dissolved in water and subjected to column separation to obtain the uniform polysaccharide of Gastrodia elata. 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 a preparation method and application thereof. 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 with fiber column chromatography, gel filtration chromatography and the like for purification and separation to obtain a uniform polysaccharide TMP-Ⅱa, wherein the monosaccharide composition is glucose, the molecular weight is 960 kDa, 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, a glycosidic bond connection mode of →1)Glcp(2→, and a structural formula as shown below:

[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] The crude polysaccharide of Gastrodia elata is obtained by 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] Further, the water extraction, alcohol precipitation, degreasing, deproteinizing and decolorizing treatment comprises the following steps:

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

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

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

[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 times.

[0025] Furthermore, when the aqueous solution is decolorized, the mass ratio of activated carbon to solution is 1-2%. When the activated carbon is decolorized, 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 application 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 tubers of Gastrodia elata in Jinzhai for the first time.

[0033] The specific steps of the method are as follows: 500g of dried tubers of Jinzhai Gastrodia elata are decocted with hot water, filtered to obtain a filtrate, and the filtrate is extracted multiple times using petroleum ether and Sevage reagent, and the aqueous solution is collected, the aqueous solution is fully mixed with activated carbon, allowed to stand, filtered to remove the activated carbon, and 80% ethanol is used for precipitation. The precipitate is freeze-dried to obtain the crude polysaccharide TMP of Gastrodia elata. DEAE-52 anion exchange column chromatography is used, and the eluent is NaCl solution of different concentrations to obtain two components (TMP-Ⅰ and TMP-Ⅱ). TMP-Ⅱ is selected to have the best activity, and this component is dialyzed for 48 hours through a 2000Da dialysis bag, and then freeze-dried to obtain a relatively pure TMP-Ⅱ. The polysaccharide TMP-Ⅱ is then purified by G-200 dextran gel column chromatography, and the eluent is a pure aqueous solution to obtain a component (TMP-Ⅱa), and then freeze-dried to obtain a uniform polysaccharide TMP-Ⅱa, with an extraction rate 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°C, and the number of extractions 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] When the aqueous solution is decolorized, the mass ratio of activated carbon to solution is 1%. When the activated carbon is decolorized, 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, the alcohol precipitation time is 12 hours, and the 80% ethanol is ethanol with a volume fraction of 80%, ethanol accounting for 80% and water accounting for 20%.

[0040] Among them, the concentration of crude polysaccharide solution is 20 mg / mL, the specification of DEAE-52 anion exchange column chromatography is 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 by 0 mol / L NaCl solution and TMP-Ⅱ eluted by 0.1 mol / L NaCl solution, and the 0 mol / L NaCl solution is pure water.

[0041] The concentration of the polysaccharide TMP-Ⅱ solution is 10 mg / mL, the specification of the G-200 polysaccharide gel column chromatography is 1.6×70 cm, the eluent used is a pure water solution, the flow rate is 0.2 mL / min, and the components obtained 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 PC-12 cell senescence, and can be applied to 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, clear glycosidic bond linking mode, etc. The singleness of the glycosidic bond linking mode 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 preparation method of Gastrodia elata polysaccharide, and obtains Gastrodia elata crude polysaccharide by water extraction and alcohol precipitation, defatting, deproteinizing and decolorizing treatment, and purifies by using DEAE-52 anion exchange column chromatography and G-200 dextran gel column chromatography, and the obtained polysaccharide is TMP-Ⅱa. The preparation process conditions are mild, the operation is simple, and the environment is 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 descriptions 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 a 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 diagram 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 monosaccharide composition of Gastrodia elata polysaccharide TMP-Ⅱa in a specific embodiment of the present invention;

[0051] Figure 7 In the formula, 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] Fig. 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; Fig. 9A is the relationship between Gastrodia elata polysaccharide TMP-Ⅱa and PC12 cell viability without D-galactose induction; Fig. 9 B is the relationship between TMP-Ⅱa and PC12 cell viability after D-galactose induction;

[0055] Fig.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] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are 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.

[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 fully 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 alcohol precipitation temperature is 4-5°C, and the alcohol precipitation time is 12 hours. The precipitate is collected and freeze-dried to obtain the crude Gastrodia elata 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 with the change of the eluent volume, the concentration of the NaCl solution is selected, 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 eluent is detected by phenol-sulfuric acid colorimetry. The eluent containing polysaccharides 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, and the 0 mol / L NaCl solution is pure water.

[0061] (3) See Figure 2 B, by Figure 2 B shows that polysaccharide TMP-Ⅱ is a uniform component. The concentration of polysaccharide TMP-Ⅱ solution is 10 mg / mL. G-200 polysaccharide gel column chromatography (1.6×70 cm) is used. Pure water solution is used as eluent. The flow rate is 0.2 mL / min. The polysaccharide content in the eluent is detected by phenol-sulfuric acid colorimetry. The eluent containing polysaccharides is collected to obtain Gastrodia elata polysaccharide extract, and 1 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, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and 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, respectively, and place them in clean anhydrous test tubes. If it is less than 2.0 mL, add pure water to 2.0 mL, 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, use 1.0 mL of water for the same treatment as a blank control, and draw a standard curve. Weigh 5.0 mg of TMP-Ⅱa freeze-dried powder, place in a 10 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, take 0.5 mL of polysaccharide solution, dilute to different concentrations, treat by phenol-sulfuric acid method, measure the absorbance of the sample solution, and 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] It was determined that the carbohydrate content of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 was 89.2%. The instrument used was a SHIMADZUUV-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, dilute to 10 mL to prepare m-hydroxybiphenyl solution. Accurately weigh 5.0 mg of anhydrous galacturonic acid, dissolve it in pure water and dilute it with 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 with pure water to 1 mL, place the test tube in an ice bath, add 5.0 mL of the cooled borate solution dropwise, shake and mix well, place it in a boiling water bath for 20 minutes, take it out and cool it to room temperature. Add 100 μL of m-hydroxybiphenyl solution to each tube, mix well and let it stand for 30 minutes. Accurately weigh 5.0 mg of TMP-Ⅱa lyophilized powder, place it in a 10 mL volumetric flask, add water to dissolve and dilute to the mark, take 1 mL of polysaccharide solution, place it in a 10 mL test tube, and repeat the above steps. Determine the absorbance of the sample solution, and calculate the acid polysaccharide content according to the standard curve. The linear relationship diagram of the absorbance of the galacturonic acid standard solution can be found in Figure 3 B.

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

[0071] (3) using UV scanning to measure the ultraviolet absorption of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1;

[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) Analyzing the characteristic groups of the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 using an infrared spectrometer

[0075] Weigh about 2.0 mg of dried Gastrodia elata polysaccharide TMP-Ⅱa, mix with 200 mg of KBr powder, press into tablets, and then scan and analyze 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 can be found in Figure 4 B.

[0076] After testing, the gastrodia 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 the 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 (7.8×300mm) chromatographic column; mobile phase: double distilled water; injection volume: 10μL; flow rate: 1.0mL / min; column temperature: 35℃; differential refractive index detector. The HPGPC diagram of Gastrodia elata polysaccharide TMP-Ⅱa can be found in Figure 5 .

[0079] After testing, the Gastrodia elata polysaccharide TMP-Ⅱa obtained in Example 1 is 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 1.0 mg / mL Tianma polysaccharide TMP-Ⅱa aqueous solution, polysaccharide solution (1.0 mL) and Congo red solution (100 μM) at a ratio of 1:1. Then add 1.0 mL NaOH solution, establish reaction systems with different NaOH concentrations (0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5 M), scan the reaction solution at 400-600 nm UV wavelength, Congo red UV comparison chart of Tianma polysaccharide TMP-Ⅱa can be found in Figure 6 .

[0082] After testing, it was found that the spatial configuration of the obtained Gastrodia elata polysaccharide TMP-Ⅱa was not a triple helix structure. The instrument used was a SHIMADZUUV-2550 UV-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.

[0086] Weigh 20 mg of Gastrodia elata polysaccharide TMP-Ⅱa, add 1.5 mL of ultrapure water to fully dissolve, 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 spin bottle, add an appropriate amount of methanol, evaporate to dryness, add methanol again to dissolve, repeat the spin drying several times, and finally dissolve the spin-dried material with 2 mL of pure water, take 1 mL of polysaccharide hydrolysis 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 well, 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 for stirring 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 water system 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 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 method

[0089] Take 25 mg of Gastrodia elata polysaccharide and completely dissolve it in 99.9% D2O, put it into a nuclear magnetic resonance tube, and perform NMR measurement.

[0090] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; OneNMRprobe (5mm); 1H spectrum working frequency 599.81MHz; 13C spectrum working 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 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 PC-12 cell senescence

[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 Solebao 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 (Virginia, USA); BCA kit ( P0010S), anti-fluorescence quenching sealing fluid (P0126-5ml) were purchased from Shanghai Biotech Biotechnology Co., Ltd. (Shanghai, China); cell proliferation-toxicity detection kit (CellCountlngKlt-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), catalase (CAT) assay kit (visible light method) (ammonium molybdate method) (A007-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); mouse p21 ELISA kit (JL54563), mouse p53 ELISA kit (JL12305) were purchased from Jianglai Biotechnology (Shanghai, China); PALL66485 nitrocellulose membrane, PALL (New York, USA); 30% Acr -Bis, 1M Tris-HCl, 1.5M Tris-HCl, primary antibody diluent, and 5× protein loading buffer were purchased from Bio-Technology 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); anhydrous ethanol, 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 medium supplemented with 10% premium fetal bovine serum (FBS) at 37°C and 5% CO 2 . Subsequent experiments were performed when the cells grew to 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 The cells were inoculated in a 96-well plate. After the cells reached a certain density, gradient doses (2.5-640 μg / mL) of TMP-Ⅱa were added and incubated for a certain period of time. After that, the cells were incubated with CCK8 at 37°C and 5% CO2 for 1 hour, and the absorbance (OD) was read at 450nm using an ELISA reader to preliminarily obtain the safe dose of Gastrodia elata polysaccharide.

[0104] PC-12 cells were plated at 1×10 5 The amount of cells was inoculated in a 96-well plate, and D-galactose was added to intervene to induce cell senescence. After that, a safe dose (2.5-160μg / mL) of TMP-Ⅱa was added and incubated for a certain time. After that, the cells were incubated with CCK8 at 37°C and 5% CO2 for 1h, and the absorbance (OD) was read at 450nm using a microplate reader to preliminarily obtain the optimal intervention dose range of Gastrodia polysaccharide. After that, PC-12 cells were inoculated in a 6-well plate, and D-galactose solution and the optimal intervention dose of Gastrodia polysaccharide were added to the cells and incubated for a certain time, and cell proteins were extracted. Western blot was used to detect aging markers p53, p21 and inflammatory factors IL-6, NLRP3 and NFKB to determine the optimal intervention dose of Gastrodia 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 group was given normal growth medium (high-glucose DMEM medium containing 10% special grade fetal bovine serum), the model group and NAC intervention group were given growth medium containing 40mM D-galactose, and the Gastrodia polysaccharide intervention group was given growth medium containing 40mM D-galactose and 20μg / mL Gastrodia polysaccharide, and cultured together until the end of the experiment; in addition, 2h before the end of the experiment, the NAC-1μM intervention group, NAC-2μM intervention group, and NAC-4μM intervention group were given NAC (1μM, 2μM, 4μM) intervention culture for 2h. After the culture, the cells were collected for subsequent analysis.

[0107] 2.4 Detection of mtROS levels

[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 warmed 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 Green staining working solution and 1 μL Hoechst33342 mixed working solution, incubate at 37°C in the dark for 30 minutes, wash the cells 2-3 times with preheated PBS buffer, 3 minutes each time; (5) Sealing: drop 10 μL of anti-fluorescence quenching sealing solution in the center of the slide, take out the cell slide placed in the 6-well plate, and stick the reverse side (the side containing the attached cells) to the sealing solution (to prevent bubbles in the bonding area, which affects image acquisition). Subsequently, a laser scanning confocal microscope is used to observe and collect images.

[0109] 2.5 Oxidation kit assay

[0110] Cell sample preparation: The cultured cell suspension was inoculated into different 6-well plates for culture, and D-galactose and Gastrodia polysaccharide TMP-Ⅱa or NAC were given for intervention. After the intervention, 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 12000rpm for 10 minutes at 4°C, and the supernatant was taken, and the cell culture medium was collected simultaneously. Finally, the levels of CAT and MDA in cells and the level of extracellular LDH were determined using commercial kits (Nanjing Jiancheng) according to the manufacturer's instructions; the levels of aging markers p21 and p53 were determined using ELISA kits (Jianglai).

[0111] 2.6 Western Blot detection of protein expression level

[0112] First, the cultured cell samples were dissolved in the prepared RIPA solution (with protease inhibitors and phosphatase inhibitors added in proportion) to extract the proteins. Equal amounts of protein samples were separated by 15% SDS polyacrylamide gel and then transferred to the NC membrane by electrophoresis. After blocking with 5% BSA solution for 2 hours, the membrane was washed three times in Tris-buffered saline (TBS-T) containing 0.1% Tween-20 for 5 minutes each time. Then, the membrane was incubated with different primary antibodies in a 4°C refrigerator overnight. The next day, the NC membrane was washed three times with TBS-T and incubated with horseradish peroxidase (HRP)-coupled secondary antibodies at room temperature for 1 hour. After that, the protein-antibody complex was detected by chemical reaction with the HRP coupled to the secondary antibody through the emitter coupled logic (ECL) substrate (Tanon5200Multi chemiluminescence imaging analysis system). Finally, the band signal intensity was analyzed using Image-Proplus6.0 software.

[0113] 2.7 Statistical analysis

[0114] All data were expressed as 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 PC-12 cell senescence

[0117] like Fig. 9 As shown in A, in the CCK8 test, when the dose 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 dose of Gastrodia elata polysaccharide TMP-Ⅱa. Fig. 9 As shown in 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, with the effects at doses of 10 μg / mL and 20 μg / mL being significant (p<0.05).

[0118] Ineffective ROS control on mitochondrial supercomplexes leads to altered ROS signaling, thereby mediating cellular stress responses to age-dependent damage. Therefore, sustained increases in mitochondrial ROS levels are potentially critical for inducing and maintaining the cellular aging process. Fluorescence microscopy of the effect of Gastrodia elata polysaccharide TMP-ⅡC on mitochondrial ROS levels in PC-12 cells is shown in Figure 2. Fig.10 As shown in the figure, 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 shown by the significantly weakened green fluorescence intensity around the cell nucleus; among them, the 20μg / mL dose was the best. This result shows that the anti-aging effect of Gastrodia elata polysaccharide is related to the inhibition of mitochondrial ROS levels.

Claims

1. A gastrodia elata polysaccharide, characterized in that: The molecular weight of the gastrodia elata polysaccharide is 900 - 1000kDa, the glycosidic bond connection mode is →1)Glcp(2→, the structural formula is as follows: n is a positive integer.

2. The Gastrodia elata polysaccharide according to claim 1, characterized in that The molecular weight is 960 kDa.

3. A method for preparing Gastrodia elata polysaccharide according to claim 1 or 2, characterized in that: The preparation method comprises: The crude polysaccharide of Gastrodia elata is obtained by water extraction, alcohol precipitation, defatting, deproteinization and decolorization. The crude Gastrodia elata polysaccharide is purified by anion exchange column chromatography and dextran gel column chromatography to obtain Gastrodia elata polysaccharide.

4. The preparation method according to claim 3, characterized in that: The water extraction, alcohol precipitation, degreasing, deproteinizing and decolorizing treatment includes the following steps: Take Gastrodia elata, decoct with water, filter to obtain filtrate, use petroleum ether and Sevage reagent to extract the filtrate for multiple times, collect the aqueous solution, mix the aqueous solution with activated carbon, let it stand, filter to remove the activated carbon, use ethanol to precipitate, and obtain Gastrodia elata crude polysaccharide.

5. The preparation method according to claim 3, characterized in that: Purification by anion exchange column chromatography and dextran gel column chromatography includes the following steps: The crude Gastrodia elata polysaccharide is chromatographed using a DEAE-52 anion exchange column, the eluent is a 0.1-0.5 mol / L NaCl solution, the chromatographic polysaccharide is dialyzed through a dialysis bag, freeze-dried after dialysis, and then chromatographed using a dextran gel column, the eluent is pure water, and then freeze-dried to obtain Gastrodia elata polysaccharide.

6. The preparation method according to claim 4, characterized in that: Gastrodia elata uses dried tubers, and the volume ratio of Gastrodia elata to water is 1:(3-6). After decocting with water, the tubers are filtered 1-5 times.

7. The preparation method according to claim 4, characterized in that: The Sevage reagent is obtained by mixing chloroform and n-butanol, wherein 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.

8. The preparation method according to claim 4, 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.

9. The preparation method according to claim 4, characterized in that: The ethanol is 80% ethanol, the volume ratio of filtrate to 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%, ethanol accounting for 80% and water accounting for 20%.

10. Use of the Gastrodia elata polysaccharide according to claim 1 or 2 in anti-aging drugs.

Images