Anoectochilus roxburghii polysaccharide and application thereof in preparation of cartilage injury repair medicine

Abstract

The invention discloses a Zhejiang anoectochilus roxburghii polysaccharide and application thereof in preparation of cartilage injury repair medicine.The Zhejiang anoectochilus roxburghii polysaccharide is characterized in that a crude polysaccharide AZJP is extracted from the Zhejiang anoectochilus roxburghii polysaccharide through a hot water extraction method, an anion exchange column DEAE-celluse-52 and a gel permeation chromatographic column Sephadex G-200 are used for separating the AZJP, and the Zhejiang anoectochilus roxburghii homogeneous polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c are obtained; measuring UV (ultraviolet), IR (infrared), molecular weight, monosaccharide composition, methylated sugar residues, NMR (nuclear magnetic resonance) signals, triple helix structures, SEM (scanning electron microscope) and AFM (atomic force microscopy) surface microstructures and cartilage repair activity; the four homogeneous polysaccharides have remarkable cartilage repair activity on transgenic zebrafish, are superior to a positive drug alendronate sodium, and have application prospects in development of drugs for treating osteoarticular cartilage injuries.

Description

Technical Field

[0001] The present invention relates to the field of extraction, separation, purification, structural characterization and activity evaluation of natural plant polysaccharides, and in particular to a Zhejiang-produced roxburghii polysaccharide with cartilage repairing effect, and its separation and characterization, as well as use in preparing a cartilage damage repairing drug. Background Art

[0002] Osteoarthritis is a cartilage degenerative disease that mainly affects the elderly and is characterized by cartilage damage. It is currently believed that the pathogenesis of osteoarthritis is usually accompanied by changes in cartilage composition and increased sensitivity of cartilage to physical destructive forces, and the cartilage is gradually eroded and loses its integrity. Clinical studies have shown that long-term use of the glucocorticoid dexamethasone (Dex) increases the likelihood of patients developing osteoarthritis and may even accelerate the disease progression in patients with confirmed osteoarthritis. Therefore, further research is needed on safe and effective articular cartilage repair drugs that can replace dexamethasone.

[0003] Polysaccharides from natural sources are a class of non-toxic and safe substances, which may contain some polysaccharide components with bone and joint injury repair. The separation and purification of polysaccharides is relatively complicated, especially it is difficult to separate uniform polysaccharides, which generally requires repeated process optimization. In addition, the activity of polysaccharides is closely related to their structural characteristics, including molecular weight, sugar residues, degree of branching and branch chain conformation. Anoectochilus roxburghii is distributed in Jinhua, Zhejiang Province. According to folk records, it is often used to treat digestive system diseases such as stomachache and gastroenteritis. In addition, Anoectochilus roxburghii is also believed to have the effect of clearing heat and detoxifying, and is often used to treat symptoms such as sore throat, skin eczema and boils. Due to its potential effects on regulating immunity and anti-fatigue, Anoectochilus roxburghii is gradually considered to be a tonic medicinal material. In recent years, modern pharmacological studies have found that Anoectochilus roxburghii has liver protection, hypoglycemic and anti-inflammatory activities. However, the separation and structural identification of Anoectochilus roxburghii polysaccharides have not been reported so far.

[0004] The zebrafish genome is about 70% similar to the human genome, and about 84% of human disease-causing genes have homologous genes in zebrafish. Therefore, the pathogenic mechanisms of many genetic diseases can be effectively reproduced in zebrafish. Through gene editing technology (such as CRISPR / Cas9), specific genes can be knocked out or mutated in zebrafish to simulate human diseases, such as cartilage damage.

[0005] The present invention optimizes the extraction and separation process conditions and parameters, purifies four novel uniform polysaccharides from the Anoectochilus roxburghii produced in Zhejiang, adopts NMR, IR, GC-MS and other methods to characterize the structures, and evaluates the cartilage damage repair effects of the four Anoectochilus roxburghii uniform polysaccharides on a transgenic zebrafish model. The results show that the effects are significantly better than those of the positive drug alendronate sodium, and the polysaccharides can be used for the preparation of cartilage damage repair drugs. Summary of the invention

[0006] In view of the problems that the fine structure of Anoectochilus roxburghii polysaccharide produced in Zhejiang is less studied and the biological activity is not extensive enough, the present invention provides a Anoectochilus roxburghii polysaccharide produced in Zhejiang and its use in preparing a cartilage damage repair drug.

[0007] The present invention uses hot water extraction to extract the polysaccharide of Anoectochilus roxburghii from Zhejiang, and separates and purifies it for the first time by combining DEAE-cellulose-52 filler with Sephadex G-200 gel permeation chromatography filler, thereby obtaining four new monomer polysaccharides of Anoectochilus roxburghii from Zhejiang. Then, UV, IR, molecular weight, monosaccharide composition, methylated sugar residues, NMR signal, triple helix structure, SEM and AFM surface microstructure and cartilage repair activity are determined, laying a foundation for further realizing the development and application of Anoectochilus roxburghii from Zhejiang in medicines and functional foods.

[0008] The technical solution of the present invention is as follows:

[0009] A polysaccharide of Anoectochilus roxburghii produced in Zhejiang Province is obtained by separation as follows:

[0010] S1. Take the original medicinal material of Anoectochilus roxburghii produced in Zhejiang, grind it, extract it with water, precipitate it with alcohol, collect the polysaccharide precipitate, and redissolve it in water to obtain the original polysaccharide solution;

[0011] The specific operation is as follows: take the original medicinal material of Anoectochilus roxburghii produced in Zhejiang, crush it and pass it through a 50-mesh sieve, mix the medicinal material powder with water at a solid-liquid ratio of 1:10-30 (g / mL), heat it to 90-110℃ and extract it for 1.5-2.5h, filter it, repeat the extraction of the filter residue 1-2 times, combine the extracts, concentrate it under reduced pressure, centrifuge it, take the supernatant, add ethanol, precipitate it at 4℃ for 8-12h, collect the polysaccharide precipitate by centrifugation, and redissolve it in water to obtain the original polysaccharide solution;

[0012] S2, sequentially subjecting the original polysaccharide solution to deproteinization by the CaCl2-NaOH method, decolorization by the activated carbon method, dialysis, and freeze-drying to obtain crude polysaccharide AZJP;

[0013] The specific operation is as follows: take the original polysaccharide solution, adjust the pH to 8-9 with NaOH solution, heat to 80-90℃, add CaCl2 solid to a final concentration of 40-60g / L, boil for 25-35min, cool to room temperature, filter, adjust the filtrate pH to 6-8 with hydrochloric acid, add anhydrous ethanol, leave for 8-12h, centrifuge, collect the precipitate, add water to dissolve and repeat the above operation until the protein content is less than 5%; re-dissolve the deproteinized precipitate with water, add activated carbon, stir and decolorize at 45-55℃ for 35-45min, then centrifuge to remove the activated carbon; transfer the decolorized solution to a dialysis bag with a molecular weight cutoff of 3500Da, dialyze at 3-5℃ for 24-72h, and then freeze-dry to obtain crude polysaccharide AZJP;

[0014] In this step, the CaCl2-NaOH method can not only remove the protein in the original polysaccharide solution, but also remove most of the pigments in the solution. After further decolorization and purification by activated carbon method, the crude polysaccharide AZJP is obtained by dialysis and freeze-drying. The phenol-sulfuric acid method can be used to determine the content of crude polysaccharide of Zhejiang-produced golden thread lotus, and the extraction rate can be calculated.

[0015] S3, using DEAE-cellulose-52 anion exchange column filler to preliminarily separate the crude polysaccharide AZJP, using 0.0-0.1 mol / L NaCl aqueous solution as eluent for gradient elution, monitoring by phenol-sulfuric acid method and drawing the elution curve, combining the eluates of each component according to the elution curve, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain polysaccharides AZJP-1 and AZJP-2;

[0016] AZJP-3 and AZJP-4 can be further eluted using 0.2-0.5 mol / L NaCl aqueous solution. Figure 1 , Attachment Figure 2 It is shown in the figure that AZJP-3 and AZJP-4 were not studied in this application and therefore will not be discussed;

[0017] The specific operation is as follows: the crude polysaccharide AZJP is dissolved in distilled water to prepare a 5-15 mg / mL AZJP solution, filtered through a 0.45 μm water-based microporous filter membrane, and then added to a chromatographic column (26×300 mm) filled with DEAE-cellulose-52 filler for separation, with a sample volume of 5-15 mL, and gradient elution is performed using 0.0 and 0.1 mol / L NaCl aqueous solutions as eluents, respectively, at a flow rate of 1-2 mL / min, and monitored using the phenol-sulfuric acid method and an elution curve is drawn, and the eluates of each component are combined according to the elution curve, and the polysaccharides AZJP-1 and AZJP-2 are obtained by reduced pressure concentration, dialyzing at 3-5°C for 24-72 h, and freeze-drying;

[0018] The phenol-sulfuric acid method is as follows: take 1.0 mL of the eluate sample, add 1.0 mL of distilled water, then add 1.0 mL of 6% phenol, quickly add 5.0 mL of concentrated sulfuric acid, mix thoroughly, let stand for 30 minutes, and measure the absorbance at 490 nm;

[0019] DEAE-cellulose-52 filler was treated as follows before use: distilled water was added to the new DEAE-cellulose-52 filler, stirred evenly, soaked for 2.5-3.5 hours to remove surface impurities, filtered under reduced pressure, then soaked in 0.4-0.6 mol / L HCl solution for 1.5-2.5 hours, filtered under reduced pressure, washed with distilled water until neutral, soaked in 0.4-0.6 mol / L NaOH solution for 1.5-2.5 hours, filtered under reduced pressure, washed with distilled water until neutral; the treated filler was soaked in 15-25% EtOH and stored in an environment of 3-5°C for a long time;

[0020] Used DEAE-cellulose-52 filler can be regenerated and reused. The regeneration method is as follows: the used DEAE-cellulose-52 filler is first rinsed with a saturated NaCl solution to remove impurities adsorbed in the filler, then rinsed with distilled water, and then soaked in 0.4-0.6mol / L HCl solution and 0.4-0.6mol / L NaOH solution for 1.5-2.5h, respectively, filtered under reduced pressure, washed with distilled water until neutral, and stored in a 15-25% ethanol solution at 3-5°C;

[0021] S4. Separate the polysaccharide AZJP-1 using gel permeation chromatography filler Sephadex G-200, elute with 0.15-0.25 mol / L NaCl aqueous solution as eluent, monitor and draw the elution curve using phenol-sulfuric acid method, combine the eluates of each component according to the elution curve, concentrate under reduced pressure, dialyze, and freeze-dry to obtain the Zhejiang-produced Anoectochilus roxburghii polysaccharide AZJP-1a; separate the polysaccharide AZJP-2 using the same method to obtain the Zhejiang-produced Anoectochilus roxburghii polysaccharides AZJP-2a, AZJP-2b, and AZJP-2c;

[0022] The specific operation is as follows: Sephadex G-200 column (26×600mm) is used to separate polysaccharides AZJP-1 and AZJP-2 respectively, with a sample concentration of 5-15 mg / mL and a sample volume of 5-20 mL, eluted with 0.15-0.25 mol / L NaCl aqueous solution as the eluent, and a flow rate of 0.2-0.8 mL / min, monitored by phenol-sulfuric acid method and an elution curve is drawn, and the eluates of each component are combined according to the elution curve, concentrated under reduced pressure, dialyzed at 3-5°C for 24-72h, and freeze-dried to obtain Zhejiang-produced Anoectochilus roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b, and AZJP-2c;

[0023] The gel permeation chromatography filler Sephadex G-200 is pretreated as follows before use: at room temperature, the Sephadex G-200 gel powder is soaked in distilled water for 24-72 hours, and stirred continuously to ensure that the gel is swollen, and the gel fragment particles floating on the upper layer are removed.

[0024] The relative molecular weights of the polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c produced in Zhejiang Province are 387.083 kDa, 947.069 kDa, 3.989 kDa and 3.045 kDa, respectively. The structures of the four polysaccharides are characterized by using UV, FT-IR, HPLC, HPGPC, GC-MS, 1D-NMR, 2D-NMR, FESEM and AFM techniques.

[0025] AZJP-1a is composed of Manp-(1→, Galp-(1→, →4)-Galp-(1→, →4)-Manp-(1→, →3,4)-Manp-(1→ and →4,6)-Manp-(1→; the galactose content is 2.83% and the mannose content is 96.10%.

[0026] AZJP-2a is composed of Araf-(1→, Arap-(1→, →3)-Araf-(1→, Glcp-(1→, Galp-(1→, →4)-Galp-(1→, →4)- Glcp-(1→, →3)-Galp-(1→, →3,4)-Glcp-(1→, →2,4)-Glcp-(1→, →4,6)-Galp-(1→ and →3,6)-Galp-(1→; glucose content is 19.47%, galactose content is 53.22%, and arabinose content is 23.46%.

[0027] AZJP-2b is composed of Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Galp-(1→ and →4,6)-Glcp-(1→; the glucose content is 84.17%, the galactose content is 11.32%, and the arabinose content is 3.52%.

[0028] AZJP-2c is composed of Glcp-(1→,→4)-Galp-(1→,→4)-Glcp-(1→,→3,4)-Glcp-(1→ and →4,6)-Glcp-(1→; glucose content is 97.52%.

[0029] The surface microstructure revealed that polysaccharides AZJP-1a and AZJP-2a were branched network structures, while AZJP-2b and AZJP-2c were porous strip-like structures.

[0030] The roxburghii roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c produced in Zhejiang Province of the present invention can be used for preparing drugs for repairing cartilage damage.

[0031] The present invention uses transgenic fluorescent zebrafish (strain Tg (Col2a1a:eGFP)) as an in vitro model of cartilage damage, and uses a digital high-throughput microfluidic zebrafish chip system to quantify the cartilage fluorescence intensity of zebrafish, and evaluates the cartilage repair activity of four types of Anoectochilus roxburghii monomer polysaccharides produced in Zhejiang. The Anoectochilus roxburghii polysaccharides isolated and obtained by the present invention have a novel and unique basic structure and cartilage repair activity, and can be used to prepare new drugs for treating cartilage damage caused by osteoarthritis, or as intermediates for drugs for treating osteoarthritis or plant raw materials for functional foods.

[0032] Compared with the prior art, the present invention has the following characteristics:

[0033] 1) The present invention optimizes the separation and purification process of the polysaccharide produced in Zhejiang Province for the first time, and provides a suitable method for separating the uniform polysaccharide produced in Zhejiang Province.

[0034] 2) The present invention is the first to use a DEAE-cellulose-52 anion exchange column combined with a Sephadex G-200 gel permeation chromatography column to perform multiple separation and purification of the Zhejiang-produced Anoectochilus roxburghii polysaccharides to obtain Zhejiang-produced Anoectochilus roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c with uniform structures.

[0035] 3) The present invention is the first to conduct cold field scanning electron microscopy (FESEM) experiments on the polysaccharides of A. roxburghii produced in Zhejiang, and found that AZJP-1a and AZJP-2a have network branched structures to varying degrees, and AZJP-2b and AZJP-2c have porous strip-like and filamentous structures.

[0036] 4) The present invention is the first to conduct atomic force microscopy (AFM) experiments on the polysaccharides of Aromatic Thread Lotus produced in Zhejiang Province, and found that the four polysaccharides have different degrees of branching and aggregation, which is consistent with the detection results of GC-MS, NMR and FESEM.

[0037] 5) The present invention is the first to conduct a Congo red test on the polysaccharides of A. roxburghii produced in Zhejiang, and found that these polysaccharides do not have a triple helix structure.

[0038] 6) The present invention reports for the first time that the polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c produced in Zhejiang can effectively enhance the fluorescence intensity of zebrafish head cartilage, have good cartilage repair promoting ability, and are more effective than the positive drug alendronate sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 :Flow chart of extraction, separation and purification of polysaccharides from Anoectochilus roxburghii produced in Zhejiang.

[0040] Figure 2 :Eluent curves of polysaccharides from Anoectochilus roxburghii produced in Zhejiang. (A) Elution curve of crude polysaccharide from Anoectochilus roxburghii produced in Zhejiang on DEAE-cellulose-52 column; (B) Elution curve of AZJP-1 on Sephadex G-200 column; (C) Elution curve of AZJP-2 on Sephadex G-200 column; (D) Physical pictures of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0041] Figure 3 : Structural analysis diagrams of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c. (A) HPGPC spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c; (B) IR spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c; (C) Monosaccharide composition spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0042] Figure 4 : Surface microstructure of polysaccharides from Anoectochilus roxburghii produced in Zhejiang. (A) Congo red test results of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c; (B) FESEM images of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c; (C) AFM images of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0043] Figure 5 : Methylation GC-MS spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0044] Figure 6 : 1D-NMR and 2D-NMR spectra of AZJP-1a. (A) 1H-NMR spectrum; (B) 13C-NMR spectrum; (C) DEPT-135 spectrum; (D) HSQC spectrum; (E) HMBC spectrum; (F) COSY spectrum.

[0045] Figure 7 : 1D-NMR and 2D-NMR spectra of AZJP-2a. (A) 1H-NMR spectrum; (B) 13C-NMR spectrum; (C) DEPT-135 spectrum; (D) HSQC spectrum; (E) HMBC spectrum; (F) COSY spectrum.

[0046] Figure 8: 1D-NMR and 2D-NMR spectra of AZJP-2b. (A) 1H-NMR spectrum; (B) 13C-NMR spectrum; (C) DEPT-135 spectrum; (D) HSQC spectrum; (E) HMBC spectrum; (F) COSY spectrum.

[0047] Fig. 9 : 1D-NMR and 2D-NMR spectra of AZJP-2c. (A) 1H-NMR spectrum; (B) 13C-NMR spectrum; (C) DEPT-135 spectrum; (D) HSQC spectrum; (E) HMBC spectrum; (F) COSY spectrum.

[0048] Fig.10 : Structures of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0049] Fig.11 : Abdominal view of the zebrafish cartilage injury model of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c.

[0050] Fig.12 : Effects of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c on zebrafish cartilage injury model. DETAILED DESCRIPTION

[0051] The present invention is fully and clearly described below through specific embodiments and in conjunction with the accompanying drawings. Obviously, the protection scope of the present invention is not limited to this.

[0052] The present invention uses a new extraction, separation and purification method to obtain four monomer polysaccharides with different basic structures from the Anoectochilus roxburghii, and conducts a complete and systematic structural characterization of them. Since the Anoectochilus roxburghii has the effect of treating bone-related diseases, a transgenic fluorescent zebrafish cartilage injury model is constructed, and a high-throughput digital zebrafish chip system is used to evaluate the cartilage repair effects of the four monomer polysaccharides of the Anoectochilus roxburghii, laying a foundation for the development of osteoarthritis treatment drugs and functional foods.

[0053] In the following embodiments,

[0054] The Zhejiang-produced golden thread lotus comes from Zhejiang Jinhua Academy of Agricultural Sciences;

[0055] DEAE-cellulose-52 chromatographic column was purchased from Whatman, UK;

[0056] Sephadex G-200 chromatographic column was purchased from Beijing Ruida Henghui Technology Development Co., Ltd.

[0057] Example 1 Extraction, separation and purification of polysaccharide monomers from Anoectochilus roxburghii

[0058] The whole herb of dried Anoectochilus roxburghii was used as the raw material, crushed by a powder grinder and sieved (50 mesh). The sieved powder was mixed with distilled water at a ratio of 1:20 and stirred evenly, transferred to a round-bottom flask and heated to reflux at 100℃ for 2h, extracted twice, cooled in the middle, filtered with gauze, mixed the two extracts, concentrated to one twentieth of the original volume, centrifuged (1500 rpm, 10 min), took the supernatant, added 4 times the amount of ethanol (to make the ethanol volume fraction reach 80%), and placed at 4℃ overnight. The solution obtained by alcohol precipitation at 4℃ overnight was centrifuged (1500 rpm, 10 min), the polysaccharide precipitate was collected, and the precipitate was redissolved in water at a ratio of 1:20. Take 200ml of the original solution of Anoectochilus roxburghii polysaccharide from Zhejiang, adjust it to pH 8-9 with 2% NaOH solution, and heat it to 85℃. The CaCl2 solid was adjusted to a concentration of 5% (50 g / L), boiled for 30 min, cooled to room temperature and filtered, the filtrate was adjusted to pH 7 with dilute hydrochloric acid (1 mol / L), 4 times the volume of anhydrous ethanol was added to reach an ethanol concentration of 80%, left overnight, and centrifuged at 1500 rpm / min for 10 min to precipitate polysaccharides. The above operation was repeated 3 times after dissolving in water, and the protein content must be less than 5%. 2.5% activated carbon was added to the above deproteinized polysaccharide solution, stirred and decolorized at 50°C for 40 min, and then centrifuged (10000 rpm, 10 min) and repeated 2-3 times until the activated carbon was completely removed. The deproteinized and depigmented polysaccharide solution was transferred to a dialysis bag (molecular weight cutoff: 3500Da), dialyzed at 4°C for 48 h, and the dialysate was replaced every 8 h to ensure that the small molecules in the solution were completely removed. The dialyzed polysaccharide solution was freeze-dried to obtain the Zhejiang-produced Jinxianlian crude polysaccharide AZJP.

[0059] AZJP was dissolved in distilled water to obtain a 10 mg / mL crude polysaccharide solution, which was filtered with a 0.45 μm water-based microporous filter membrane. 10 mL of the crude polysaccharide solution was added to a chromatographic column equipped with DEAE-cellulose-52 (26×300 mm) for separation. 0.0 and 0.1 mol / L NaCl were used as mobile phases for elution, respectively, and the flow rate was maintained at 1.5 mL / min. The effluent components were collected in test tubes, 5 mL per tube, and monitored by the phenol-sulfuric acid method (1.0 mL of polysaccharide sample was taken, 1.0 mL of distilled water was added, and then 1.0 ml of 6% phenol was added, and 5.0 ml of concentrated sulfuric acid was quickly added, mixed thoroughly, allowed to stand for 30 minutes, and the absorbance was measured at 490 nm). The effluent curve was drawn. The components were combined according to the results of the effluent curve, concentrated under reduced pressure, dialyzed at 4°C for 48 hours, and freeze-dried. Polysaccharides AZJP-1 and AZJP-2 were obtained.

[0060] AZJP-1 and AZJP-2 have high contents, so they will be further separated and purified. A Sephadex G-200 column (26×600mm) was used, 0.2mol / L NaCl was used as the eluent, the flow rate was maintained at 0.5mL / min, the sample concentration was adjusted to 10mg / mL, and the sample loading volume was 10mL. The effluent components were collected in test tubes, 5mL per tube, and monitored by the phenol-sulfuric acid method to draw the effluent curve. The components were combined according to the effluent curve, concentrated under reduced pressure, dialyzed at 4℃ for 48h, and freeze-dried. Polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c were obtained.

[0061] Figure 1 The overall process of extracting, separating and purifying monomeric polysaccharides from the raw medicinal materials of Anoectochilus roxburghii was systematically described. The yield of crude polysaccharides from Anoectochilus roxburghii relative to dry medicinal materials was 8.19%. Four components (AZJP-1, AZJP-2, AZJP-3, and AZJP-4) were separated from the crude polysaccharides from Anoectochilus roxburghii by DEAE-cellulose-52 chromatography column, and then the two components with higher contents, AZJP-1 and AZJP-2, were separated by Sephadex G-200 column to obtain four monomeric components (AZJP-1a, AZJP-2a, AZJP-2b, and AZJP-2c). After HPGPC detection, it was found that the four polysaccharides all showed a single symmetrical peak ( Figure 3 The results are shown in Figure 5A), and it is shown that they have different molecular weights for subsequent monosaccharide composition, FT-IR, GC-MS methylation analysis, NMR analysis and surface microstructure analysis.

[0062] Example 2 Specific structural characterization of the monomer polysaccharide of Anoectochilus roxburghii from Zhejiang

[0063] (1) HPGPC molecular weight determination

[0064] The purity and relative molecular weight of AZJP-1a, 2a, 2b and 2c polysaccharides from Anoectochilus roxburghii were determined by high performance gel permeation chromatography (HPGPC). Waters e2695 high performance liquid chromatograph (Waters Company), chromatographic column TSKgelG3000PWxl (7.8 mm×300mm, 7μm, Sigma-Aldrich Company), mobile phase was pure water, flow rate was 0.5ml / min, injection volume was 10μL, detector was differential refractive index detector (ELSD), column temperature was 40℃, and running time was 30min.

[0065] The gel chromatograms of the polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c produced in Zhejiang Province are shown in Figure 2. Figure 3As shown in A, there are four single and symmetrical peaks. According to the retention time of each of the four polysaccharides and the standard curve of dextran (lgMw=-0.4399Rt+10.8886, R 2 = 0.9917), the relative molecular weights of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c were calculated to be 387.083 kDa, 947.069 kDa, 3.989 kDa and 3.045 kDa, respectively.

[0066] (2) Fourier transform infrared (FT-IR) analysis

[0067] Take 2 mg of sample and mix it with 200 mg of KBr. Compress the mixture into tablets and analyze the mixture with a Fourier transform infrared spectrophotometer at 400-4000 cm -1 Infrared measurements were performed in the range (Thermo SIENTIFIC Nicolet iS50 FT-IR, USA).

[0068] Figure 3 B is the infrared spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c. 3425.27cm -1 The strong broad absorption band at 2920.14cm belongs to the stretching vibration peak of -OH. -1 The CH stretching vibration peak belonging to -CH3 or -CH2 is 1732.52 cm -1 There is no absorption peak or the absorption peak is weak, indicating that there is no uronic acid or the content of uronic acid is small, 1636.74cm -1 The absorption peak may come from the absorbed water, which is caused by the binding of water, 1409.99cm -1 and 1380.80cm -1 The absorption peak at 1244.41 cm -1 Due to the presence of pyranose, 1200-1000 cm -1 The three characteristic absorption peaks between (1154.06cm -1 ,1079.22cm -1 and 1047.04cm -1 ) may be caused by the COC and COH stretching vibrations in pyranose and furanose, 934.04 cm -1 、869.69cm -1 and 812.06cm -1 The absorption peak at 829.28 cm is a typical signal of furan ring. -1 Indicates the presence of α-configuration glycosidic bond, 617.50 -1 and 573.34cm-1 Indicates the presence of pyranose.

[0069] (3) HPLC monosaccharide composition analysis

[0070] The monosaccharide composition of Anoectochilus roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c was determined by PMP pre-column derivatization combined with high performance liquid chromatography. Weigh 4 mg of dried polysaccharide sample accurately, place it in a hydrolysis tube, fill it with nitrogen, add 2 mL of 3 mol / L trifluoroacetic acid, hydrolyze it at 110 ° C for 6 h, cool it to room temperature, add 5 mL of methanol to dissolve it, dry it under reduced pressure, repeat three times, then add 1 mL of pure water to dissolve it, centrifuge it (10000 rpm, 10 min), take 100 μL of supernatant, add 100 μL of 0.3 mol / L NaOH aqueous solution and 100 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP), react at 70 ° C for 0.5 h, then add 105 μL of 0.3 mol / L HCl for neutralization, finally add an equal amount of chloroform, vortex, centrifuge it (3000 rpm, 10 min), take the supernatant, discard the chloroform layer, repeat three times. Obtain the PMP-derivative labeled sample to be tested, filter it with a 0.22 μm microporous filter membrane, and enter HPLC for analysis.

[0071] Weigh 1 mg of each monosaccharide standard (rhamnose, xylose, arabinose, glucose, galactose, galacturonic acid, mannose, fructose and glucuronic acid) accurately into an EP tube, add 1 mL of pure water to dissolve, centrifuge (10000 rpm, 10 min), take 100 μL of supernatant, add 100 μL of 0.3 mol / L NaOH aqueous solution and 100 μL of 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP), react at 70 ° C for 0.5 h, then add 105 μL of 0.3 mol / L HCl for neutralization, and finally add an equal amount of chloroform, vortex, centrifuge (3000 rpm, 10 min), take the supernatant, discard the chloroform layer, and repeat three times. Obtain the PMP-derivatized monosaccharide reference, filter with a 0.22 μm microporous filter membrane, and enter HPLC for analysis.

[0072] The chromatograph was Agilent 1260 high performance liquid chromatograph; the chromatographic column was Kromasil 100-5-C 18 Chromatographic column (4.6mm id×250mm, 5μm); mobile phase is acetonitrile:0.05M phosphate buffer = 17.5:82.5 (pH=6.8); isocratic elution, flow rate is 1.0mL / min; UV detection wavelength is 250nm; column temperature is 30℃; injection volume is 10μL; running time is 90min.

[0073] The monosaccharide compositions of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c of Anoectochilus roxburghii polysaccharides were separated and analyzed by PMP pre-column derivatization and reversed-phase high performance liquid chromatography. Figure 3 As shown in C. The monosaccharide composition results of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c are shown in Table 1. It can be seen from the table that mannose accounts for the majority of AZJP-1a, and galactose accounts for only about 3%, indicating that AZJP-1a may be a mannan. The monosaccharide composition of AZJP-2a, AZJP-2b and AZJP-2c is the same, and the content of each monosaccharide component is very different. As shown in Table 1, AZJP-2a is a heteropolysaccharide, mainly composed of glucose, galactose and arabinose, accounting for 19.47:53.22:23.46 respectively. As the molecular weight decreases, the proportion of glucose content in the latter two components gradually increases, and the percentage of galactose and arabinose gradually decreases. Among them, the monosaccharide composition of AZJP-2b is glucose: galactose: arabinose = 84.17:11.32:3.56. The monosaccharide composition of AZJP-2c has become glucose accounting for 97.59%. We also found that the uronic acid content of AZJP-1a was lower than that of the fraction separated from AZJP-2, which is consistent with the fact that AZJP-1 was eluted by pure water, while AZJP-2 needed to be eluted by a higher concentration of salt solution.

[0074] Table 1 Monosaccharide composition of Anoectochilus roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c

[0075]

[0076] (4) Congo red triple helix structure analysis

[0077] 2.0 mg of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c were accurately weighed and dissolved in 2 mL of deionized water, and 2 mL of 100 μL of Congo red aqueous solution were added to obtain a polysaccharide-Congo red mixed solution. Different amounts of sodium hydroxide were used to prepare sodium hydroxide solutions with a concentration of 0-0.5 M. The mixture was allowed to stand at room temperature for 10 min, and then the UV-visible spectrum in the wavelength range of 200-800 nm was recorded with a UV spectrophotometer, and the maximum absorption wavelength was determined based on the UV-visible spectrum. Subsequently, the sodium hydroxide concentration was used as the horizontal coordinate and the maximum absorption wavelength of each sample was used as the vertical coordinate to draw a graph. 2 mL of deionized water was mixed with Congo red and sodium hydroxide to prepare control solutions of different gradients, and the maximum absorption wavelength was recorded using a UV spectrophotometer, and a graph was drawn for comparison with the Congo red curve of the actual polysaccharide.

[0078] Congo red detection is a common method for determining whether a triple helix structure exists in polysaccharides. This phenomenon can be attributed to the fact that Congo red can form a complex with polysaccharides with a triple helix structure. Therefore, compared with Congo red, the maximum absorption wavelength of the complex will be red-shifted. Figure 4 As shown in A, after combining with any Anoectochilus roxburghii monomer polysaccharide under any NaOH concentration conditions, the maximum absorption wavelength did not show an obvious red shift, indicating that AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c do not have a triple helical structure.

[0079] (5) Cold field scanning electron microscopy (FESEM) analysis

[0080] First, prepare the sample: use antistatic tweezers to pick up a small amount of freeze-dried samples of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c, and evenly distribute them on the conductive glue attached to the sample stage. Gently vibrate the sample stage to disperse the polysaccharide, and blow it gently with an ear bulb to remove the sample that is not on the conductive glue. Then put the sample stage into an ion sputtering instrument for hot stamping to make it conductive, and finally transfer the sample stage to an S-8020 scanning electron microscope (FESEM, Hitachi) to observe the surface microstructure of the polysaccharide. Under high vacuum conditions, the sample was observed at a voltage of 5.0 kV and magnified 5000 times to obtain a scanning electron microscope image of the polysaccharide produced in Zhejiang.

[0081] FESEM was used to examine the morphological characteristics of polysaccharides. Figure 4As shown in B, the results showed that AZJP-1a had a mesh structure with a large number of surface pores, which may be due to the presence of more branched interactions in the polysaccharide. AZJP-2a had a rough surface and a tight mesh structure, with small and fewer surface pores and irregular clumps on the surface. This indicates that AZJP-2a has a higher molecular weight and branching degree, indicating that it has sugar chains that interact more tightly. AZJP-2b has a ribbon-like structure, and pores can be observed on the surface of the ribbon-like structure, which may lead to the enhanced water solubility of AZJP-2b. Similarly, AZJP-2c was observed to have many pores on the surface of the filaments at 5000×, which may be the reason for its strong water solubility. It was observed that AZJP-2c was irregularly coiled and showed filamentous cross-linking. Although this structure is related to the formation of triple helix, AZJP-2b and 2c may not be sufficient to achieve this process due to their lower molecular weight. The cartilage protective effect of these four polysaccharides is related to their branched ribbon structure and branched mesh structure to a certain extent. Studies have shown that polysaccharides with branched network structures can play an anti-osteoporosis role to a certain extent. Similarly, most agents used for cartilage repair are natural polysaccharides (such as chondroitin sulfate, hyaluronic acid, xanthan gum, etc.) that are non-toxic, non-irritating, biocompatible, and biodegradable. Although AZJP-2b and 2c appear to have the necessary conditions for forming a triple helical structure (curled filaments) according to scanning electron microscopy images, due to their low molecular weight, the destruction of hydrogen bonds ultimately prevents the formation of a triple helical structure. This finding is consistent with the results of the Congo red test.

[0082] (6) Atomic force microscopy (AFM) analysis

[0083] Lyophilized AZJP-1a, AZJP-2a, AZJP-2b, and AZJP-2c polysaccharide samples were dissolved in deionized water to a concentration of 5 μg / mL. The solution was then dropped onto a freshly cleaved mica plate and dried overnight at 25-30 °C. AFM images of the samples were observed on a Bruker Dimension Edge AFM system (Bruker, Germany) operating in tapping mode. Images were acquired using a NanoScope.

[0084] Atomic force microscopy is considered an important technique for observing polysaccharide morphology. In order to directly observe the conformational features (shape and size) of AZJP-1a, 2a, 2b, and 2c, we tested them at a concentration of 5 μg / mL. Figure 4The results shown in C indicate that the main morphology of AZJP-1a is short branched structures that interact to form a mesh-like porous morphology. In contrast, AZJP-2a exhibited interactions between long branches at the same concentration, showing a reduced number of pores and a dense mesh structure. Both AZJP-2b and AZJP-2c showed ribbon-like long-chain structures with short branches. The morphologies of all four polysaccharides showed peak-like aggregations, and their heights were significantly higher than the height of a single polysaccharide chain (0.1-1 nm). The results showed that all four polysaccharides were involved in molecular aggregation, which also indicated the presence of branched structures, with different degrees of branching and lengths of branches for each polysaccharide. It was observed that these structures were entangled with each other to form aggregates of various morphologies, presenting a mesh-like structure. These findings are consistent with the results observed by scanning electron microscopy.

[0085] (7) Methylation GC-MS analysis

[0086] The specific process of polysaccharide methylation is as follows: take an appropriate amount of polysaccharide and place it in a ball mill for crushing and grinding, then accurately weigh 3 mg of polysaccharide in an EP tube, place it in a freeze dryer for drying for 5 hours, and then place it in a dryer for drying overnight for use. Grind the sodium hydroxide into a fine powder with a mortar, vacuum dry it at 80°C for 8 hours, and then transfer it to a dryer for drying overnight for use. Put the dried polysaccharides into a dry 10mL round-bottom flask, seal it, fully replace it with argon, add 500μL of ultra-dry dimethyl sulfoxide, seal and stir until the polysaccharide is completely dissolved, then add 5mg of dry sodium hydroxide powder, seal and stir overnight to allow the hydroxyl groups on the polysaccharide chain to react completely with sodium hydroxide. Then add 100μL of iodomethane to the reaction solution, react overnight, and then add 1mL of water to terminate the reaction. After the methylation reaction, 2 mL of dichloromethane was added for extraction, vortexed and centrifuged (3000 rpm, 10 min) to collect the lower layer of extract, and then 3 mL of pure water was added, vortexed and centrifuged (3000 rpm, 10 min), and repeated three times. The dichloromethane layer was taken and dried at 30 ° C. The methylation product was detected by Fourier transform infrared spectrometer. If the peak at 3500 cm -1There is no hydroxyl peak on the left and right, indicating that the methylation is complete; if the methylation is incomplete, methylation needs to be performed again. Add 2mL of 2M trifluoroacetic acid to the methylated product, replace with argon under 110℃ oil bath conditions, condense and reflux for hydrolysis reaction for 12h, cool to room temperature after the reaction, spin dry, dissolve with 1-2mL of methanol, spin dry, repeat three times to reduce the residual trifluoroacetic acid in the product. Add 200 μL of 2M ammonia water and 200μL of 1M sodium borodeuteride to the hydrolyzate, mix well, and react under argon protection for 24h. After the reaction is completed, add 20μL of acetic acid to the reaction system to terminate the reaction, spin dry, add 1mL of methanol, wash twice, and spin dry. Add 250 μL of acetic anhydride to the reaction system, mix well, react in an oil bath at 100°C for 12 hours, cool to room temperature after the reaction, add 1 mL of water and let stand for 10 minutes, add 500 μL of dichloromethane, vortex mix well, centrifuge (3000 rpm, 10 minutes), discard the aqueous phase, and repeat the water washing three times. Take the dichloromethane layer, remove excess water with anhydrous sodium sulfate, obtain partially methylated alditol acetate (PMAAs), pass through a 0.22 μm microporous filter membrane, and perform GC-MS detection.

[0087] GC-MS experimental conditions: chromatographic column: Agilent HP5-ms (30m×0.32mm, 0.25μm, Agilent); injection port temperature: 250°C; interface temperature: 250℃; carrier gas: high-purity helium; carrier gas flow rate: 1.5 mL / min; programmed temperature: the initial temperature of the column oven is 140°C, maintained for 2 min, increased to 230°C at a rate of 3℃ / min, and maintained for 3 min; injection volume: 1μL; split ratio 10:1; MS detector; EI ionization 70eV; mass spectrometry scanning range m / z 30-600.

[0088] The methylation GC-MS spectra of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c are shown in Figure 5As shown in Table 2, the results of methylation GC-MS analysis illustrate the types of sugar residues and the corresponding relative percentages of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c. Specifically, AZJP-1a is composed of Manp-(1→, Galp-(1→, →4)-Galp-(1→, →4)-Manp-(1→, →3,4)-Manp-(1→ and →4,6)-Manp-(1→), and AZJP-2a is composed of Araf-(1→, Arap-(1→, →3)-Araf-(1→, Glcp-(1→, Galp-(1→, →4)- Galp-(1→, →4)- AZJP-2c is composed of Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Glcp-(1→, →2,4)-Glcp-(1→, →4,6)-Galp-(1→ and →3,6)-Galp-(1→), AZJP-2b is composed of Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Galp-(1→ and →4,6)-Glcp-(1→), and AZJP-2c is composed of Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Glcp-(1→ and →4,6)-Glcp-(1→.

[0089] Table 2 Methylation analysis results of four polysaccharide monomers of Anoectochilus roxburghii from Zhejiang

[0090]

[0091] (8) NMR analysis

[0092] Weigh 50 mg of AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c, add 1 mL of heavy water to fully dissolve, freeze-dry, repeat the exchange with heavy water twice, dry the freeze-dried samples in a freeze dryer overnight, and finally dissolve in 0.6 mL of heavy water, add 2 μL of internal standard substance acetone ( 1 H = 2.225 ppm, 13 C = 31.45 ppm), after being fully dissolved, transfer to the NMR tube, fill it to a height of 3-4 cm, and let it stand at room temperature before testing. The test temperature is 298K. The test items are one-dimensional 1 HNMR, DEPT135 and 13 CNMR, 2D COSY, HSQC, HMBC and NOESY.

[0093] The monosaccharide composition and methylation analysis results were combined with the one-dimensional and two-dimensional nuclear magnetic resonance results to clarify the basic structures of the polysaccharides AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c produced in Zhejiang. The anomeric hydrogen and anomeric carbon signals of AZJP-1a are mainly concentrated in δ4.4-5.5ppm ( Figure 6 A) and between 90-110ppm. 13 CNMR( Figure 6 Medium B), HSQC ( Figure 6 D) and COSY ( Figure 6 Based on the results of the spectrum of (F) in (C), and referring to the methylation results, monosaccharide composition and related references, it is speculated that sugar residue A is α-D-Manp-(1→), sugar residue B is α-D-Galp-(1→), sugar residue C is →4)-β-D-Galp-(1→, sugar residue D is →4)-α-D-Manp-(1→, sugar residue E is →3,4)-α-D-Manp-(1→, and sugar residue F is →4,6)-β-D-Manp-(1→. The measured 1H and 13C chemical shifts are shown in Table 3. According to the 13C and 1H chemical shifts of each sugar residue in the sample, the connection order between each sugar residue was characterized by HMBC spectrum ( Figure 6 Specifically, H1 of residue A has cross peaks with C3 and C4 of sugar residue E, C4 and C6 of sugar residue F at δ 5.40 / 74.51, 5.40 / 74.10, 5.40 / 77.44 and 5.43 / 69.04 ppm; H1 of sugar residue B has cross peaks with C3 and C4 of sugar residue E, C4 and C6 of sugar residue F at δ 5.00 / 74.51, 5.00 / 74.10, 5.00 / 77.44 and 5.00 / 69.04 ppm; H1 of sugar residue C has cross peaks with C3 and C4 of sugar residue E, C4 and C6 of sugar residue F at δ 4.68 / 78.92 and 4.68 / 77.44 ppm; H1 of sugar residue D has cross peaks with C3 and C4 of sugar residue E, C4 and C6 of sugar residue F at δ 5.43 / 78.92, 5.43 / 78.94 and 5.43 / 78.94 ppm. There are cross peaks with C4 of sugar residues C and D, C3 and C4 of sugar residue E, and C4 and C6 of sugar residue F at δ 5.43 / 78.08, δ 5.43 / 74.51, δ 5.43 / 74.10, δ 5.43 / 77.44 and δ 5.43 / 69.04 ppm respectively; H4 of sugar residue D has cross peaks with C1 of sugar residues E and F at δ 3.86 / 99.54 and δ 3.86 / 100.83 ppm; H1 of sugar residue E has cross peaks with C3 and C4 of sugar residue E at δ 5.27 / 74.51 and δ 5.27 / 74.10 ppm; H1 of sugar residue F has a cross peak at C4 of sugar residue F at δ 4.67 / 77.44 ppm.

[0094] Based on the one-dimensional and two-dimensional NMR information and the analysis of methylation results, it was inferred that the main chain of AZJP-1a was mainly composed of →4)-β-D-Galp-(1→, →3,4)-α-D-Manp-(1→, →4,6)-β-D-Manp-(1→, and →4)-α-D-Manp-(1→. The side chain was mainly composed of α-D-Manp-(1→, and a small amount of α-D-Galp-(1→ was connected to O-4, O-3, and O-6 of →3,4)-α-D-Manp-(1→ and →4,6)-β-D-Manp-(1→ residues. Therefore, the possible structure of the polysaccharide chain is as follows Fig.10 As shown in AZJP-1a.

[0095] Table 3 AZJP-1a, AZJP-2a, AZJP-2b and AZJP-2c 13 C / 1 H NMR chemical shift

[0096]

[0097] like Figure 7 Middle (AB) and Figure 7 As shown in E, 1 H. 13 Twelve signals corresponding to sugar residues in AZJP-2a were found in the anomeric C / H region of C NMR and HSQC spectra, including 5.29 / 110.54, 4.54 / 105.08, 5.29 / 110.79, 5.00 / 99.72, 5.00 / 100.25, 4.68 / 105.72, 5.40 / 101.29, 5.04 / 100.83, 5.43 / 101.08, 5.01 / 99.94, 4.67 / 106.25, and 4.54 / 104.90. The signals belonged to α-L-Araf-(1→(residue A), α-L-Arap-(1→(residue B), →3)-α-L-Araf-(1→(residue C), α-D-Glcp-(1→(residue D), α-D-Galp-(1→(residue E), →4)-β-D-Galp-(1→(residue F), →α-D-Galp-(1→(residue F), →4)-β-D-Galp-(1→(residue G), →3,4)-α-D-Glcp-(1→(residue I), →2,4)-α-D-Glcp-(1→(residue J), ​​→4,6)-β-D-Galp-(1→(residue K) and →3,6)-β-D-Galp-(1→(residue L) respectively. The remaining H / C signals were analyzed by COSY ( Figure 7 F) and HSQC ( Figure 7D) and are summarized in Table 3. These results are consistent with the cross-peak signal of HMBC ( Figure 7 Middle E) shows that AZJP-2a has longer branches and a more complex structure than AZJP-1a, with →4)-α-D-Glcp-(1→, →3, 6)-β-D-Galp-(1→, →3, 4)-α-D-Glcp-(1→, →4, 6)-β-D-Glcp-(1→, →2, 4)-α-D-Glcp-(1→, and →4)-β-D-Galp-(1→ as the main chain, and is substituted by branches of different lengths at different positions. The possible structure of the polysaccharide chain is shown in the figure. Fig.10 As shown in AZJP-2a.

[0098] The NMR chemical shifts of AZJP-2b and AZJP-2c are shown in Table 3 and are marked in Figure 8 and Fig. 9 of 1 D and 2 In the D NMR spectrum, the main chain of AZJP-2b is mainly composed of →4)-α-D-Galp-(1→(residue B), →4)-α-D-Glcp-(1→(residue C), →3,4)-β-D-Galp-(1→(residue D) and →4,6)-α-D-Glcp-(1→(residue E), with chemical shifts of 5.5.00 / 99.94, 5.43 / 101.09, 4.67 / 105.74, respectively. and 5.40 / 101.25. α-D-Glcp-(1→(residue A) is connected to the O-4 position of →4,6)-β-D-Galp-(1→(residue B) and the O-3 position of →3,4)-α-D-Glcp-(1→(residue C), with chemical shifts of 5.00 / 99.94, 5.43 / 101.09, 4.67 / 105.74 and 5.40 / 101.25, respectively. The possible polysaccharide chain structure of AZJP-2b is shown in Fig.10 The main chain of AZJP-2c is mainly composed of →4)-β-D-Galp-(1→(residue B), →4)-α-D-Glcp-(1→(residue C), →3,4)-α-D-Glcp-(1→(residue D) and →4,6)-α-D-Glcp-(1→(residue E), with chemical shifts of 4.68 / 105.75, 5.43 / 101.09, 5.00 / 99.99 and 5.40 / 101.18, respectively. α-D-Glcp-(1→(residue A) is connected to the O-3 position of →3,4)-α-D-Glcp-(1→(residue B) and the O-6 position of →4,6)-α-D-Glcp-(1→(residue C). Therefore, the possible structure of the polysaccharide chain is as follows Fig.10As shown in AZJP-2c in Figure 1, it is clear that the polysaccharide chain structures of AZJP-2b and AZJP-2c are simpler than those of AZJP-1a and AZJP-2a. The above results show that the polysaccharide chain structures of AZJP-2b and AZJP-2c are surprisingly similar, and the multi-branched structure of the above polysaccharides also indicates their potential therapeutic effects in the treatment of bone diseases.

[0099] Example 3 Cartilage repair activity of polysaccharide monomers of Anoectochilus roxburghii from Zhejiang Province

[0100] Transgenic fluorescent zebrafish cartilage injury model: Zebrafish larvae were placed in 6-well plates and divided into 15 groups (n=30 / group) for drug treatment from 3 dpf to 9 dpf. The groups were as follows: 0.1% DMSO (blank control), 12.5μM dexamethasone (Dex) (Dex cartilage injury model group), 12.5μM Dex + 0.308μM alendronate (ALN) (positive control), 12.5μM Dex + 24 (L), 48 (M) or 96 (H) μg / mL AZJP-1a, AZJP-2a, AZJP-2b or AZJP-2c (polysaccharide treatment group). Zebrafish were observed under a fluorescent microscope at 9dpf, and the zebrafish were washed three times with PBS 1×. Then the zebrafish were anesthetized with 0.016% MS-222. The zebrafish were oriented ventrally using a digital high-throughput zebrafish microfluidic chip platform and transported to a stereo fluorescence microscope (NIB950-FL, Nexcope) for observation, and then photographed with a digital camera. The relative fluorescence intensity (RFI) of the head cartilage of each zebrafish was quantified by morphometric analysis (ImageJ, Bethesda, USA). The cartilage fluorescence intensity of 8 zebrafish in each group was quantified, and the total signal of each zebrafish was averaged.

[0101] Zebrafish was used as a model organism to evaluate the efficacy of AZJP-1a, 2a, 2b and 2c in cartilage repair. The fluorescence intensity of the craniofacial cartilage of zebrafish larvae was observed and calculated under a fluorescence microscope. Fig.11 and Fig.12The fluorescence intensity of the craniofacial area of ​​zebrafish at 9 dpf is shown, reflecting the cartilage repair ability of the polysaccharide. From 3 dpf to 9 dpf, the fluorescence intensity of craniofacial cartilage in the Dex cartilage injury model group was significantly reduced compared with the normal group (P < 0.001). AZJP-1a, AZJP-2b, and AZJP-2c could significantly increase the fluorescence intensity of craniofacial cartilage in a concentration-dependent manner. We observed that AZJP-1a-H increased the fluorescence intensity of craniofacial cartilage (P < 0.05), but there was no statistical significance between ALN ​​(positive control) and AZJP-1a (L and M). Compared with AZJP-1a, AZJP-2a had a stronger cartilage repair ability and showed the best efficacy at a therapeutic concentration of 48 μg / ml (P < 0.01). The reason may be that AZJP-2a has more and longer side chains than AZJP-1a, which is easy to form a dense mesh structure and can be used as a promising material for preparing cartilage tissue bioengineering. At the same time, AZJP-2a has a larger molecular weight. However, when the polysaccharide concentration is too high, the mesh pore size will become smaller due to aggregation, making it difficult for cells to penetrate and degrade the material, which will eventually lead to a weakening of the cartilage repair ability, which may be the reason why the efficacy of AZJP-2a is reduced at high concentrations. It has been observed that at high concentrations, both AZJP-2b and AZJP-2c can significantly enhance the fluorescence intensity of the larval craniofacial region. This may be due to the low molecular weight of these two compounds, good cell permeability, and the ability to promote the metabolism of chondrocytes, thereby promoting cartilage repair.

Claims

1. A polysaccharide of Anoectochilus roxburghii produced in Zhejiang, characterized in that: Separate as follows: S1. Take the original medicinal material of Anoectochilus roxburghii produced in Zhejiang, grind it, extract it with water, precipitate it with alcohol, collect the polysaccharide precipitate, and redissolve it in water to obtain the original polysaccharide solution; S2, sequentially subjecting the original polysaccharide solution to deproteinization by the CaCl2-NaOH method, decolorization by the activated carbon method, dialysis, and freeze-drying to obtain crude polysaccharide AZJP; S3, using DEAE-cellulose-52 anion exchange column filler to preliminarily separate the crude polysaccharide AZJP, using 0.0-0.1 mol / L NaCl aqueous solution as eluent for gradient elution, monitoring by phenol-sulfuric acid method and drawing the elution curve, combining the eluates of each component according to the elution curve, concentrating under reduced pressure, dialyzing, and freeze-drying to obtain polysaccharides AZJP-1 and AZJP-2; S4. Separate the polysaccharide AZJP-1 using gel permeation chromatography filler Sephadex G-200, and elute with 0.15-0.25 mol / L NaCl aqueous solution as eluent, monitor with phenol-sulfuric acid method and draw the elution curve, combine the eluates of each component according to the elution curve, and obtain Zhejiang Anoectochilus roxburghii polysaccharide AZJP-1a through reduced pressure concentration, dialyzation and freeze-drying; separate the polysaccharide AZJP-2 using the same method to obtain Zhejiang Anoectochilus roxburghii polysaccharides AZJP-2a, AZJP-2b and AZJP-2c.

2. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that The operation of S1 is as follows: take the original medicinal material of Anoectochilus roxburghii produced in Zhejiang, crush it and pass it through a 50-mesh sieve, mix the medicinal material powder with water at a solid-liquid ratio of 1g:10-30mL, heat it to 90-110℃ and extract it for 1.5-2.5h, filter it, repeat the extraction of the filter residue 1-2 times, combine the extracts, concentrate under reduced pressure, centrifuge, take the supernatant, add ethanol, precipitate it at 4℃ for 8-12h, collect the polysaccharide precipitate by centrifugation, and redissolve it in water to obtain the original polysaccharide solution.

3. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that The S2 operation is as follows: take the original polysaccharide solution, adjust the pH to 8-9 with NaOH solution, heat to 80-90°C, add CaCl2 solid to a final concentration of 40-60g / L, boil for 25-35min, cool to room temperature, filter, adjust the filtrate pH to 6-8 with hydrochloric acid, add anhydrous ethanol, leave for 8-12h, centrifuge, collect the precipitate, add water to dissolve and repeat the above operation until the protein content is less than 5%; re-dissolve the deproteinized precipitate with water, add activated carbon, stir and decolorize at 45-55°C for 35-45min, then centrifuge to remove the activated carbon; transfer the decolorized solution to a dialysis bag with a molecular weight cutoff of 3500Da, dialyze at 3-5°C for 24-72h, and then freeze-dry to obtain crude polysaccharide AZJP.

4. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that The S3 operation is as follows: the crude polysaccharide AZJP is dissolved in distilled water to prepare a 5-15 mg / mL AZJP solution, which is filtered through a 0.45 μm water-based microporous filter membrane and added to a chromatographic column filled with DEAE-cellulose-52 filler for separation. The sample volume is 5-15 mL, and 0.0 and 0.1 mol / L NaCl aqueous solutions are used as eluents for gradient elution at a flow rate of 1-2 mL / min. The phenol-sulfuric acid method is used for monitoring and the elution curve is drawn. The eluates of each component are combined according to the elution curve, and the polysaccharides AZJP-1 and AZJP-2 are obtained by concentrating under reduced pressure, dialyzing at 3-5°C for 24-72 h, and freeze-drying.

5. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that: The operation of S4 is as follows: Use Sephadex G-200 chromatographic column to separate polysaccharides AZJP-1 and AZJP-2 respectively, with a sample concentration of 5-15 mg / mL and a sample volume of 5-20 mL. Use 0.15-0.25 mol / L NaCl aqueous solution as the eluent for elution, and a flow rate of 0.2-0.8 mL / min. Monitor using the phenol-sulfuric acid method and draw an elution curve. Combine the eluates of each component according to the elution curve, concentrate under reduced pressure, dialyze at 3-5°C for 24-72 h, and freeze-dry to obtain Zhejiang-produced golden thread vine polysaccharides AZJP-1a, AZJP-2a, AZJP-2b, and AZJP-2c.

6. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that: The relative molecular weights of Zhejiang-produced Anoectochilus roxburghii polysaccharides AZJP-1a, AZJP-2a, AZJP-2b, and AZJP-2c are 387.083 kDa, 947.069 kDa, 3.989 kDa, and 3.045 kDa, respectively.

7. The polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1, characterized in that: AZJP-1a is composed of Manp-(1→, Galp-(1→, →4)-Galp-(1→, →4)-Manp-(1→, →3,4)-Manp-(1→ and →4,6)-Manp-(1→; galactose content is 2.83%, mannose content is 96.10%; AZJP-2a is composed of Araf-(1→, Arap-(1→, →3)-Araf-(1→, Glcp-(1→, Galp-(1→, →4)-Galp-(1→, →4)- Glcp-(1→, →3)-Galp-(1→, →3,4)-Glcp-(1→, →2,4)-Glcp-(1→, →4,6)-Galp-(1→ and →3,6)-Galp-(1→; glucose content is 19.47%, galactose content is 53.22%, and arabinose content is 23.46%; AZJP-2b is composed of Glcp-(1→, →4)-Galp-(1→, →4)-Glcp-(1→, →3,4)-Galp-(1→ and →4,6)-Glcp-(1→; glucose content is 84.17%, galactose content is 11.32%, and arabinose content is 3.52%; AZJP-2c is composed of Glcp-(1→,→4)-Galp-(1→,→4)-Glcp-(1→,→3,4)-Glcp-(1→ and →4,6)-Glcp-(1→; glucose content is 97.52%.

8. Use of the polysaccharide of Anoectochilus roxburghii produced in Zhejiang as claimed in claim 1 in the preparation of drugs for repairing cartilage damage.

Images