Polysaccharides having wound healing effects and methods of making and using the same

Abstract

The present application discloses a kind of polysaccharides with wound healing effect and preparation method and use;The polysaccharide is extracted from the fresh tuber of Bletilla striata (Thunb.) Reichb. f. Separation and purification are obtained pure polysaccharide-white and total polysaccharide (BSPt) containing the mixture of polysaccharide-white and total polysaccharide (BSPt) have significant wound healing effect. These polysaccharides can significantly enhance the proliferation, differentiation and migration of C2C12 cells;Promote the epithelialization of mouse wound and the formation of new granulation tissue, accelerate wound healing. The pharmaceutical composition with the polysaccharide as active ingredient can be applied to the treatment of trauma.

Description

Technical Field

[0001] This invention relates to the extraction, isolation, and purification of a homogeneous polysaccharide with wound-healing properties—Bletilla striata polysaccharide BSPS—and a mixture containing this homogeneous polysaccharide—Bletilla striata total polysaccharide BSPt—from the fresh tubers of Bletilla striata (Thunb.) Reichb.f., the preparation method thereof, and pharmaceutical compositions using the polysaccharides as active ingredients, as well as their applications in the treatment of wounds. Background Technology

[0002] The traditional Chinese medicine Bletilla striata is the dried tuber of the orchid Bletilla striata (Thunb.) Reichb.f. It has astringent and hemostatic properties, reduces swelling and promotes tissue regeneration. It is used for hemoptysis, hematemesis, traumatic bleeding, carbuncles and boils, and chapped skin. Modern pharmacological studies have confirmed that it has hemostatic, wound healing, anticancer, antiviral, anti-inflammatory, antifibrotic and immunomodulatory effects [XRHe,XXWang,JCFang,ZFZhao,LHHuang,H.Guo,XHZheng,Bletilla striata:medicinal uses,phytochemistry and pharmacological activities,J.Ethnopharmacol.195(2017)20–38;S.Jiang,MYWang,L.Jiang,Q.Xie,HWYuan,YPYang,S.Zafar,Y.Liu,YQJian,B.Li,W.Wang,2021.The medicinal uses of the genus Bletilla in traditional Chinese medicine:A phytochemical and pharmacological review.J.Ethnopharmacol.280,114263]. Bletilla striata mainly contains polysaccharides, phenanthrene, anthraquinones, and saponins [C.Yang,T.Xia,CQWang,HYSun,YJLi,ZPGong,YTLi,L.Zheng,Y.Huang,2019.Using theUPLC-ESI-Q-TOF-MS]. Emethod and intestinal bacteria for metaboliteidentification in the nonpolysaccharide fraction from Bletilla striata.Biomed.Chromatogr.33,e4637; Res.Int.2020,5391379]. Among them, polysaccharides are its main active ingredients, which have hemostatic, wound healing, gastric protection, antitumor and immunomodulatory effects [C.Liu,KYDai,HYJi,XYJia,AJLiu,Structural characterization of a low molecular weight Bletillastriata polysaccharide and antitumor activity on H22 tumor-bearingmice.Int.J.Biol.Macromol.205(2022)553–56], and are widely used in the pharmaceutical, food and cosmetic industries [DLXu,YCPan,JSChen,2019.Chemical constituents,pharmacologic properties,and clinical applications of Bletilla striata.Front.Pharmacol.10,1168].In recent years, Bletilla striata polysaccharide has been widely studied as a carrier material for drug delivery systems due to its non-toxicity, high safety, biocompatibility, and biodegradability [WC Zhao, Q. Zhang, Y. Wang, JH Hou, Preparation, characterization and in vitro antitumor effect of cholesterol succinyl Bletilla striata polysaccharide-loaded paclitaxel nanoparticles, Biomed. Res. 28(2017) 9638–9646; LL Hu, ZCLiao, QQ Hu, KGMaffucci, Y. Qu, Novel Bletilla striata polysaccharide microneedles: fabrication, characterization, and in vitro transcutaneous drug]. delivery.Int.J.Biol.Macromol.117(2018)928–936; Q.Zhang,C.Qi,H.Wang,XFXiao,Y.Zhuang,SJGu,YHZhou,L.Wang,HJYang,WLXu,2019.Biocompatible and degradable Bletilla striata polysaccharide hemostasissponges constructed from natural medicinal herb Bletillastriata.Carbohydr.Polym.226,115304].

[0003] To date, all reported Bletilla striata polysaccharides have been isolated from the dried tubers of Bletilla striata. Compared with the dried medicinal material, the active ingredients of the fresh material are preserved more completely. In addition, all Bletilla striata polysaccharides used to evaluate wound healing are crude polysaccharides.

[0004] This invention uses modern column chromatography to separate and purify a homogeneous polysaccharide—Bletilla striata polysaccharide BSPS—from the fresh tubers of Bletilla striata. Various spectroscopic and waveletography techniques, combined with chemical analysis methods, are used to characterize its structure. In vitro mouse myoblast C2C12 cell model and in vivo mouse skin injury experiments confirm that Bletilla striata polysaccharide BSPS has wound-healing effects. Summary of the Invention

[0005] The primary objective of this invention is to provide a class of polysaccharides with wound-healing properties.

[0006] A second objective of this invention is to provide a method for extracting and isolating polysaccharides from fresh tubers of Bletilla striata (Thunb.) Reichb.f.

[0007] A third objective of this invention is to provide a method for analyzing the structure of Bletilla striata polysaccharide BSPS.

[0008] A further object of the present invention is to provide a pharmaceutical composition for treating trauma.

[0009] Another object of the present invention is to provide the use of the above-mentioned polysaccharides and compositions in the treatment of wounds.

[0010] The polysaccharide of this invention with wound-healing properties comprises a pure monopolysaccharide—Bletilla striata polysaccharide BSPS—extracted, isolated, and purified from the fresh tubers of Bletilla striata (Thunb.) Reichb.f., and a mixture containing this homogeneous polysaccharide—total Bletilla striata polysaccharide BSPt. The relative molecular weight of Bletilla striata polysaccharide BSPS is 722.90 kDa, and it is composed of glucose and mannose in a molar ratio of 1:2.5.

[0011] The Bletilla striata polysaccharide BSPS and the mixture containing this homogeneous polysaccharide—Bletilla striata total polysaccharide BSPt—provided by this invention are extracted from the fresh tubers of the orchid Bletilla striata (Thunb.) Reichb.f., and the preparation method includes the following steps:

[0012] a. Homogenize fresh tubers of Bletilla striata and extract with water.

[0013] b. Concentrate the aqueous extract, and precipitate twice with 95% ethanol. Collect the precipitate, dissolve it in distilled water, remove proteins with Sevage's reagent (chloroform: n-butanol = 4:1), dialyze, concentrate the osmotic residue under reduced pressure, and freeze-dry to obtain Bletilla striata total polysaccharide BSPt.

[0014] c. The total polysaccharide BSPt of Bletilla striata was separated by gel chromatography, eluted with sodium chloride solution, and the different eluents were collected, concentrated, desalted, and freeze-dried to obtain two fractions.

[0015] d. The first fraction was then purified by gel chromatography, eluted with sodium chloride solution, concentrated, desalted, and freeze-dried to obtain Bletilla striata polysaccharide BSPS.

[0016] The column chromatography gel mentioned in step c above is DEAE-Sephadex A-25, DEAE-Sephadex A-50, DEAE-Sepharose CL-4B, DEAE-Sepharose CL-6B, DEAE-Cellulose 32, or DEAE-Cellulose 52. The column chromatography gel mentioned in step d is Sephadex G-100, Sephadex G-150, Sephadex G-200, Sephacryl S-300, Sephacryl S-400, Sepharose CL-4B, or Sepharose CL-6B. The concentration of the sodium chloride solution mentioned in steps c and d is 0–2.0 mol / L.

[0017] The pharmaceutical composition of the present invention contains a therapeutically effective amount of Bletilla striata polysaccharide BSPS or total Bletilla striata polysaccharide BSPt as the active ingredient, and contains one or more pharmaceutically acceptable carriers.

[0018] The effective amount of Bletilla striata polysaccharide BSPS or total Bletilla striata polysaccharide BSPt of the present invention is 0.01 μg / kg to 100 mg / kg body weight.

[0019] The polysaccharide and pharmaceutical composition of the present invention can be used to treat wounds.

[0020] The pharmaceutically acceptable carriers mentioned above refer to drug carriers in the pharmaceutical field. Examples include: diluents, excipients such as water, physiological saline, glucose, mannitol, glycerol, ethanol, or mixtures thereof; fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginate, gelatin, polyvinylpyrrolidone, or mixtures thereof; humectants such as glycerol; disintegrants such as calcium carbonate and / or sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as Tween-80; and lubricants such as talc, calcium stearate, magnesium stearate, polyethylene glycol, or mixtures thereof.

[0021] The compounds of this invention can be administered in combination form to patients requiring such treatment or to individuals requiring vaccination via oral, nasal, rectal, parenteral, or transdermal routes. For oral administration, they can be formulated into conventional solid dosage forms such as tablets, powders, granules, capsules, pills, sustained-release microspheres, solid dispersions, inclusion complexes, etc., and into liquid dosage forms such as suspensions, emulsions, sols, syrups, mixtures, solutions, etc. For parenteral administration, they can be formulated into injectable solutions, aqueous or oily suspensions, emulsions, lyophilized powders, liposomes, microcapsules, microspheres, nanocapsules, nanospheres, etc. Preferred forms are tablets, coated tablets, capsules, microspheres, suppositories, and injections, with particular preference for formulations that target release at specific sites.

[0022] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical manufacturing methods. For example, the active ingredient can be mixed with one or more carriers and then formulated into the desired dosage form.

[0023] The pharmaceutical composition of the present invention preferably contains an active ingredient in a total weight ratio of 0.01% to 99.9%. Most preferably, it contains an active ingredient in a total weight ratio of 0.5% to 95%.

[0024] The polysaccharides of this invention exhibit wound healing effects, significantly enhancing the proliferation, differentiation, and migration of C2C12 cells; promoting wound epithelialization and the formation of new granulation tissue, accelerating wound healing in mice, and can be used as drugs for treating trauma. Attached Figure Description

[0025] Figure 1 This document describes the isolation, purification, molecular weight, monosaccharide composition, and physicochemical properties of BSPS from fresh Bletilla striata tubers. A is the elution curve of total Bletilla striata polysaccharide (BSPt) on a Sephadex G200 column. B is the elution curve of fraction 1 (F1) on a DEAE Sephadex A50 column. C is the HPLC chromatogram of Bletilla striata polysaccharide BSPS; the peak at 47.606 min corresponds to the NaCl mobile phase. D is the HPLC chromatogram of the monosaccharide standard PMP derivative; E is the HPLC chromatogram of the BSPS monosaccharide PMP derivative. 1: D-Mannose; 2: D-Glucosamine Hydrochloride (GlcN); 3: L-Rhamnose (Rha); 4: D-Glucuronic Acid (Glc-UA); 5: D-Galacturonic Acid (Gal-UA); 6: D-Glycerol Hydrochloride (GalN); 7: D-Glucose (Glc); 8: D-Galactose (Gal); 9: D-Xylose (Xyl); 10: L-Arabinose (Ara); 11: L-Fucose (Fuc). F is the GC-MS spectrum of the methylated product of Bletilla striata polysaccharide BSPS. G is the infrared spectrum of Bletilla striata polysaccharide BSPS.

[0026] Figure 2 These are the NMR spectra of Bletilla striata polysaccharide BSPS. A–E represent the NMR spectra of Bletilla striata polysaccharide BSPS. 1 H NMR(A), 13 CNMR(B) 1 H- 1 HCOSY(C), HSQC(D), HMBC(E), and NOESY(F); G is the repeating structural unit of Bletilla striata polysaccharide BSPS (n≈600).

[0027] Figure 3These are the conformations and ultrastructures of Bletilla striata polysaccharide BSPS. A is the result of the Congo red experiment; B is a scanning electron microscope image of Bletilla striata polysaccharide; C and D are the AFM planar image (C) and three-dimensional image (D) of Bletilla striata polysaccharide.

[0028] Figure 4 This study examines the effects of BSPS on the proliferation and differentiation of C2C12 cells. A represents the MTT assay used to detect the viability of C2C12 cells; B represents the RT-qPCR assay used to detect the expression level of the MyHC gene. b P<0.01 and c P < 0.001 vs 0 μg / mL. C represents the expression level of MYH(B-5) protein as detected by IFA.

[0029] Figure 5 This refers to the effect of BSPS on C2C12 cell migration. c P < 0.001 vs 0 μg / mL.

[0030] Figure 6 This is due to the effect of BSPS C2C12 cell invasion. a P<0.05 and b P < 0.01 vs 0 μg / mL.

[0031] Figure 7 This shows the effect of BSPS on wound healing in mice. A represents the wound healing rate in mice. B is a photograph of wound contraction in mice. C shows the histopathological changes in mouse skin tissue. Detailed Implementation

[0032] The invention is further illustrated by the following examples, but not by limiting its scope.

[0033] Example 1: Preparation of Bletilla striata polysaccharide

[0034] 1. Preparation of total polysaccharides from Bletilla striata

[0035] 500g of fresh Bletilla striata tubers were homogenized, extracted by reflux for 2 hours with 12 times the amount of water, filtered, and the residue was extracted again by reflux with water. The filtrates were combined, collected under reduced pressure to 500mL, and 8 times the volume of 95% ethanol was added. The mixture was stirred, allowed to stand at 4°C for 24 hours, and then filtered under reduced pressure using rapid filter paper. The precipitate was dissolved in distilled water, and 8 times the volume of 95% ethanol was added. This process was repeated once. The precipitate was dissolved in distilled water, and Sevage's reagent (chloroform:n-butanol = 4:1) was added at a ratio of 4:1. The mixture was shaken vigorously and centrifuged at 3500rpm for 10 minutes. The protein and the lower organic solvent layer were discarded. The supernatant was repeated until no protein was observed under UV light. The concentration was reduced under reduced pressure, and the mixture was freeze-dried to obtain total polysaccharide BSPt, with a yield of 9.24%. The total polysaccharide of Bletilla striata consists of glucose and mannose.

[0036] 2. Preparation of Bletilla striata polysaccharide BSPS

[0037] Total polysaccharides from Bletilla striata were separated using a Sephadex G-200 column, eluted with 0.1 mol / L sodium chloride, and the eluent was collected, concentrated, dialyzed (7000 Da), and lyophilized to obtain fractions F1 (81.87%) and F2 (small amount) (see appendix). Figure 1 Fraction F1 was further purified by a DEAE Sephadex A50 column, eluted with 0–2.0 mol / L sodium chloride, concentrated, desalted, and freeze-dried to obtain Bletilla striata polysaccharide BSPS (49.23%) (see attached). Figure 1 (B) The total sugar and acetyl content of Bletilla striata polysaccharide BSPS is (98.09±0.29)% and (4.71±0.02)%, respectively, and it does not contain sulfonic acid groups or uronic acid.

[0038] Example 2: Molecular weight of Bletilla striata polysaccharide BSPS

[0039] Weigh 5 mg of Bletilla striata polysaccharide BSPS, dissolve it in ultrapure water to prepare a solution with a concentration of 1 mg / mL, and filter it through a 0.45 μm filter membrane to obtain the sample solution. Separately, prepare a series of standard solutions using Dextran with different molecular weights (1000, 5000, 12000, 25000, 50000, 150000, 270000, 410000, and 670000). A Waters 1515 liquid chromatography system, a Shodex KS805 column (8.0 × 300 mm), Shodex tandem columns (OHpak SB-803HQ, Ohpak SB-804HQ, and Ohpak SB-805HQ), a Waters 2410 differential detector, 0.5 mol / L sodium chloride as the mobile phase, a flow rate of 0.65 mL / min, a column temperature of 40 °C, and an injection volume of 30 μL for analysis. The molecular weight of Bletilla striata polysaccharide BSPS is 722.90 kDa (see attached image). Figure 1 (C)

[0040] Example 3: Monosaccharide composition and molar ratio of Bletilla striata polysaccharide BSPS

[0041] Sample preparation: Weigh 5 mg of Bletilla striata polysaccharide sample, add 1 mL of 2 mol / L TFA acid solution, and heat at 121℃ for 2 hours. Purge with nitrogen and dry. Wash with 3 mL of methanol, then dry again, repeating the methanol washing three times. Dissolve in 5 mL of sterile water, transfer to a chromatographic vial, and prepare for analysis. Take 0.2 mL of monosaccharide standard solution or polysaccharide hydrolysate into a stoppered conical centrifuge tube, add 0.2 mL of 0.5 mol / L sodium hydroxide solution and 0.5 mL of 0.5 mol / L PMP methanol solution, vortex to mix, and react in a 70℃ water bath for 1 hour. After completion, add 0.2 mL of 0.5 mol / L hydrochloric acid to neutralize the remaining sodium hydroxide, add 1 mL of chloroform, and vortex extract three times to remove excess PMP. After discarding the chloroform layer, take 0.3 mL and dilute with water to 1 mL.

[0042] Detection: Thermo U3000 liquid chromatography system, ZORBAX EclipseXDB-C18 column, mobile phase acetonitrile: phosphate buffer (potassium dihydrogen phosphate 12 g / L, pH adjusted to 6.8 with 2 mol / L NaOH) isocratic elution, acetonitrile: phosphate buffer volume ratio 17:83, flow rate 0.8 mL / min, column temperature 30℃, detection wavelength 250 nm, injection volume 10 μL.

[0043] The results showed that Bletilla striata polysaccharide BSPS was composed of glucose and mannose in a molar ratio of 30.25:69.75 (ca.1:2.5). (See attached image) Figure 1 Middle D and Appendix Figure 1 (E).

[0044] Example 4: Methylation analysis of Bletilla striata polysaccharide BSPS

[0045] Sample derivatization: Weigh 10 mg of Bletilla striata polysaccharide (BSPS) sample, dissolve in 1 mL of primary water, and add 1 mL of...

[0046] 100 mg / mL carbodiimide was added, and the reaction was allowed to proceed for 2 h. Then, 1 mL of 2 mol / L imidazole and 1 mL of 30 mg / mL NaBD4 were added, and the reaction was allowed to proceed for 3 h. Finally, 100 μL of glacial acetic acid was added to terminate the reaction. The sample was dialyzed for 48 h, freeze-dried, and then subjected to methylation.

[0047] Dissolve the lyophilized sample in 500 μL of DMSO; accurately weigh 1 mg of the sample to be tested and dissolve it in 500 μL of DMSO; add 1 mg of NaOH and incubate for 30 min; add 50 μL of iodomethane solution and react for 1 h; add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water 3 times; collect the lower dichloromethane phase and evaporate to dryness, add 100 μL of 2 mol / L TFA, and react at 121 °C for 90 min. Evaporate to dryness at 30 °C, add 50 μL of 2 mol / L ammonia and 50 μL of 1 mol / L NaBD4, mix well, and react at room temperature for 2.5 h; add 20 μL of acetic acid to terminate the reaction, and blow dry with nitrogen.

[0048] Wash twice with 250 μL of methanol and dry with nitrogen; add 250 μL of acetic anhydride, vortex to mix, and react at 100 °C for 2.5 h; add 1 mL of water and let stand for 10 min; add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and repeat the washing with water 3 times.

[0049] The lower layer of dichloromethane phase was removed and analyzed using the instrument.

[0050] Detection: An Agilent 7890A-5977B gas chromatography-mass spectrometry (GC-MS) system was used. The chromatographic system employed was an Agilent 7890A GC-MS system (Agilent Technologies, USA), with an HP-5MS capillary column (30m × 0.25mm × 0.25μm, Agilent J&W Scientific, Folsom, CA, USA). High-purity helium (purity not less than 99.999%) was used as the carrier gas. The flow rate was 1.0 mL / min, and the injection port temperature was 260℃. The injection volume was 1 μL, injected as a split at a split ratio of 10:1, with a solvent delay of 2.2 min. The temperature program was as follows: 50℃ held for 1.0 min, increased to 130℃ at 50℃ / min, increased to 230℃ at 3℃ / min, and held for 2 min. The mass spectrometry system employed an Agilent 5977B quadrupole mass spectrometer (Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source had an injection port temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scanning mode was full scan (SCAN), with a mass scan range (m / z) of 30–600.

[0051] The results of BSPS methylation analysis of Bletilla striata polysaccharide are attached. Figure 1 Bletilla striata polysaccharide BSPS contains two sugar residues: →4)-D-Man p -(1→and→4)-D-Glc p -(1→. The molar ratio of mannose to glucose is 2.5:1 (Table 1).

[0052] Table 1. Results of BSPS methylation analysis of Bletilla striata polysaccharide.

[0053]

[0054] Example 5: Infrared Spectroscopic (IR) Analysis of Bletilla striata Polysaccharide BSPS

[0055] A Thermo Nicolet 5700FT-IR Fourier transform infrared spectrometer was used, KBr pellets were pressed, and the spectrometer was used at room temperature in the frequency range of 4000–400 cm⁻¹. -1 Scan within the range, with a resolution of 4cm. -1 At 3405cm -1 A broad absorption peak is observed at 2889 cm⁻¹, indicating the presence of free hydroxyl groups in BSPS (Bletilla striata polysaccharide). -1 The weak absorption band is due to the C–H stretching vibration of the sugar ring; 1735 cm⁻¹ -1 and 1614cm -1 The absorption peak indicates the presence of an ester carbonyl group; 1379 cm⁻¹ -1 This is the absorption peak for the methyl symmetry bending vibration; 1251 cm⁻¹ -1 and 1062cm -1 Absorption peaks of stretching vibrations of C–O–C and C–O–H, respectively (see attached diagram). Figure 1 (G).

[0056] Example 6: Nuclear Magnetic Resonance (NMR) Analysis of Bletilla striata Polysaccharide BSPS

[0057] 10 mg of Bletilla striata polysaccharide (BSPS) sample was weighed and dissolved in 0.5 mL of D2O. TMS was added as an internal standard. One-dimensional NMR was measured using a Bruker AVANCE HD III 600 MHz NMR spectrometer. 1 H-NMR, 13 C-NMR, DEPT-135 and two-dimensional NMR 1 H- 1 H COSY, HSQC, HMBC, NOESY.

[0058] like Figure 2 As shown, Bletilla striata polysaccharide BSPS 1 H and 13 In the C-NMR spectrum, most of the signal is concentrated in a relatively narrow delta range. H 3.0–5.1 ppm and δ C 50–110 ppm is typical of polysaccharides. Antecedent hydrogen chemical shift δ H 4.41, 4.65 and 5.08 ppm and anomeric carbon δ C104.88 and 102.51 ppm indicate that BSPS (Blanc-based polysaccharide bisaccharide) contains three different types of monosaccharide residues. BSPS... 1 H and 13 C chemical shifts are detailed in Table 1. The repeating structural unit of Bletilla striata polysaccharide BSPS is →4)-β-D-Glc p -(1→4)-β-D-Man p -(1→4)-β-D-Man p -(1→4)-β-D-Glc p -(1→4)-β-D-Man p -(1→4)-β-D-Man p -(1→4)-β-D-Man p -(1→).

[0059] Table 2. Bacillus polysaccharide BSPS 1 H and 13 C chemical shift (δ, ppm)

[0060]

[0061] Example 6: Congo Red Experiment of Bletilla striata Polysaccharide BSPS

[0062] Weigh out the Bletilla striata polysaccharide (BSPS), dissolve it in double-distilled water to prepare a 2 mg / mL solution; take 1 mL of the polysaccharide solution and 3 mL of NaOH solutions of different concentrations, mix well, then add 1.5 mL of 0.2 mmol / L Congo red solution and 0.5 mL of double-distilled water respectively, mix well, and let stand for 1 h. Use a mixture of Congo red solution and corresponding concentrations of sodium hydroxide solution as a control, and detect the maximum absorption wavelength using a Tu-1901 UV spectrophotometer. (See attached image) Figure 3 As shown in Figure A, the maximum absorption wavelength of the Bletilla striata polysaccharide BSPS–Congo red complex is significantly greater than that of Congo red alone at the same sodium hydroxide solution concentration, suggesting that the Bletilla striata polysaccharide BSPS solution has a triple helix structure.

[0063] Example 7: Microstructural observation of Bletilla striata polysaccharide BSPS

[0064] Bletilla striata polysaccharide powder was placed on a sample holder fixed with double-sided tape and surface-sprayed with gold. The microstructure of the sample was observed using a field emission scanning electron microscope (SEM) at an accelerating potential of 3.0 kV under high vacuum conditions. A 10 μg / mL Bletilla striata polysaccharide BSPS solution was prepared using double-distilled water, and the polysaccharide solution was uniformly coated onto a mica sheet (1.0 × 1.0 cm). 2 The BSPS were allowed to air dry naturally, and their structure was observed using atomic force microscopy (AFM). Under SEM and AFM, chain-like and scaly structures were observed (see attached image). Figure 3(B) Due to the interaction between free OH groups, several chains aggregate and coil to form a spherical structure with a length of approximately 1.8 μm and a diameter of approximately 20 nm (see appendix). Figure 3 C and Appendix Figure 3 (D). These observations further support the existence of a triple helix structure in BSPS.

[0065] Example 8: Effect of Bletilla striata polysaccharide BSPS on the proliferation of C2C12 cells

[0066] Methods: C2C12 cells were used at a concentration of 1×10⁻⁶. 5 100 μL of the culture medium was seeded into 96-well plates at a concentration of 1 / mL and placed in a 37℃, 5% CO2 cell culture incubator. After 24 h of adherent culture, the culture medium was discarded, and DMEM culture medium containing different concentrations of Bletilla striata polysaccharide BSPS or 2% horse serum complete culture medium was added (final concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL), and cultured for another 48 h. The absorbance was measured at 492 nm using an ELISA reader via the MTT assay.

[0067] Results: See attached. Figure 4 As shown in Figure A, BSPS promoted the proliferation of C2C12 cells in the concentration range of 3.125–12.5 μg / mL, had no significant effect on C2C12 cell proliferation in the range of 25–100 μg / mL, while 200 and 400 μg / mL significantly inhibited C2C12 cell proliferation (P<0.001). BSPS concentrations of 3.125–25 μg / mL significantly promoted the proliferation of C2C12 cells treated with 2% horse serum (P<0.01 or P<0.001), while 50–400 μg / mL had no significant effect (P>0.05). These results indicate that Bletilla striata polysaccharide BSPS can promote the proliferation of C2C12 cells and their differentiated cells.

[0068] Example 9: Effects of Bletilla striata polysaccharide BSPS on C2C12 cell differentiation

[0069] The effect of BSPS on the expression of differentiation genes in C2C12 cells was detected by RT-qPCR. C2C12 cells (4 × 10⁻⁶) 4 Cells were seeded at 1 mL / well in 24-well plates and incubated at 37°C in a 5% CO2 incubator. After 24 h of adherent culture, the culture medium was discarded, and DMEM medium or 2% horse serum complete medium containing different concentrations of Bletilla striata polysaccharide BSPS (final concentrations of 0, 25, 50, and 100 μg / mL) was added, and the cells were cultured for another 24 h. Cell samples were collected, total RNA was extracted using TRIzol reagent, and the expression level of the myosin heavy chain MyHC gene was detected by RT-qPCR.

[0070] IFA was used to analyze the expression level of MYH(B-5) protein in C2C12 cells. C2C12 cells (4 × 10⁻⁶) 4 The sample was seeded at 1 mL / well in a 24-well plate and cultured at 37°C in a 5% CO2 cell culture incubator. After 24 h of cell adhesion, the cells were treated with DMEM medium containing different concentrations of BSPS (final concentrations of 0, 25, 50, and 100 μg / mL) for 72 h. The samples were collected, the culture medium was discarded, and the cells were washed three times with PBS. 4% general-purpose tissue fixative was added to each well for fixation for 15 min, followed by three washes with PBS. 0.5% Triton X-100 (diluted with PBS buffer) was added to each well for permeation for 20 min, followed by three washes with PBS. Blocking buffer (PBS buffer containing 5% BSA) was added to each well, and the cells were blocked at room temperature for 30 min, followed by three washes with PBS. 300 μL of MYH(B-5) primary antibody (1:500) was added to each well, and the cells were incubated overnight in a humidified chamber at 4°C, then warmed to 37°C for 1 h, followed by three washes with PBS. Add 300 μL of goat anti-mouse IgG secondary antibody (1:1000, diluted with blocking buffer) to each well, incubate at room temperature in the dark for 2 h, and wash three times with PBS. Add 200 μL of DAPI (1:1000) to each well, incubate at room temperature in the dark for 5 min, wash three times with PBS, observe under a fluorescence microscope, and take pictures.

[0071] As attached Figure 4 As shown in Figure B, Bletilla striata polysaccharide BSPS upregulated the expression level of the MyHC gene in C2C12 cells and myotube cells in a concentration-dependent manner (P<0.05, P<0.01, or P<0.001). (See attached figure.) Figure 4 As shown in Figure C, the number of green fluorescent cells increased with increasing concentration of Bletilla striata polysaccharide BSPS, indicating that Bletilla striata polysaccharide BSPS can upregulate the expression level of MYH(B-5) protein in C2C12 cells. These results suggest that BSPS can induce and promote C2C12 cell differentiation.

[0072] Example 10: Effect of Bletilla striata polysaccharide BSPS on the migration ability of C2C12 cells

[0073] C2C12 cells (4×10) 4Cells were seeded at 1 mL / well in 24-well plates and cultured at 37°C in a 5% CO2 incubator. After 48 h, a 1 mL pipette tip was used to slowly and evenly scratch the cells along the vertical lines. After scratching, the culture medium was aspirated, and DMEM culture medium containing different concentrations of Bletilla striata polysaccharide (BSPS) was added (final concentrations of 0, 25, 50, and 100 μg / mL). At 0 h, 24 h, 48 h, and 72 h, the 24-well plates were observed and photographed under an inverted microscope. The width of the cell scratches was measured using Oplenic software, and cell migration rate was calculated. Cell migration rate = (initial scratch width – scratch width at a specific time) / initial scratch width × 100%.

[0074] As attached Figure 5 The results showed that Bletilla striata polysaccharide BSPS could increase the migration rate of C2C12 cells in a concentration- and time-dependent manner (P < 0.05, P < 0.01, or P < 0.001), indicating that BSPS can promote the migration ability of C2C12 cells.

[0075] Example 11: Effect of Bletilla striata polysaccharide BSPS on the invasive ability of C2C12 cells

[0076] Cell invasion assays were performed using Corning 24-well Transwell chambers. Matrix gel (1:6) was diluted with DMEM medium and added to the upper chamber at 100 μL / well, then incubated overnight at 37°C to allow the matrix gel to solidify. C2C12 cells were mixed with different concentrations of Bletilla striata polysaccharide BSPS (final concentrations of 0, 25, 50, and 100 μg / mL) at a concentration of 5 × 10⁻⁶ mcg / mL. 4 Cells were seeded per well in the upper chamber of the microplate chamber, and 0.7 mL of DMEM medium containing 10% FBS was added to the lower chamber. The chambers were incubated at 37°C with 5% CO2 for 24 h. Cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet at room temperature for 10 min. Non-invasive cells were removed with cotton swabs, and the cells were observed and photographed using an Olympus CKX31 inverted microscope. Five random fields of view were recorded for each sample. Images were analyzed using ImageJ software. Invasion rate was expressed as the fold change in the number of migrating cells compared to the control group.

[0077] As attached Figure 6 As shown, the average migration rates of C2C12 cells treated with BSPS concentrations of 25, 50, and 100 mg / mL were 1.84, 2.40, and 4.65 times that of the control cells, respectively. This indicates that BSPS concentrations of Bletilla striata enhance the invasiveness of C2C12 cells in a concentration-dependent manner.

[0078] Example 12: The effect of Bletilla striata polysaccharide BSPS on wound healing in mice

[0079] Preparation of Bletilla striata polysaccharide BSPS / Vaseline paste: Weigh 500 mg of the polysaccharide sample and dissolve it in 10 mL of double-distilled water to prepare a 50 mg / mL Bletilla striata polysaccharide BSPS solution; separately, take 10 mL of double-distilled water and sterilize it by steam for 30 min. Weigh two portions of Vaseline (50 g each) and sterilize them by dry heat at 180℃ for 2 h. Mix the Bletilla striata polysaccharide BSPS solution and double-distilled water separately with the Vaseline and stir for 30 min until completely mixed. After cooling, solidify to form a thin paste. This yields the negative control drug and the test drug, which are stored at 4℃ for later use.

[0080] Model establishment: Seventy-five mice were randomly divided into five groups: a negative control group, a positive control group, and low, medium, and high dose groups of Bletilla striata polysaccharide (BSPS), with 15 mice in each group and three cages in each group. Mice were anesthetized by intraperitoneal injection of 0.20 mL of 0.375% sodium pentobarbital solution per 10 g body weight. After anesthesia, the mice's back fur was moistened with water, and then depilatory cream was applied to the backs. The cream was spread evenly with a scraper. After 10 minutes, water was added, and the back fur was gently scraped off with a scraper. The back skin was then cleaned with a cotton ball soaked in water to prevent prolonged residue from damaging the skin. The back skin was dried with a paper towel. Then, the back skin was pinched with one hand, and two symmetrical, full-thickness circular wounds with a diameter of 10 mm were created on the back of the mice using a punch.

[0081] Administration: Weigh out 0.1 g of double-distilled water / Vaseline paste (negative control), 0.05 g, 0.1 g, and 0.2 g of BSPS / Vaseline paste, and 0.1 g of recombinant bovine basic fibroblast growth factor gel (positive control), and divide each into two portions. Apply the two portions evenly to the surface and edges of two wounds on the back of mice using a small spatula. Change the dressing once a day at a fixed time for 12 consecutive days.

[0082] Observation of wound healing in mice: Two days after drug administration, the diameter of the mouse wounds was photographed and measured daily using a ruler-type distance measuring instrument, and the wound healing rate was calculated.

[137] Wound healing rate (%) = (Initial wound size – Wound size on the day of measurement) / Initial wound size × 100%.

[0083] Histopathological observation of wound tissue: Five mice in each group were sacrificed on the 4th and 8th days after administration and 24 hours after the last administration. Wound tissue was collected, fixed in 4% neutral buffered formalin solution, embedded in paraffin, cut into 4μm thick sections, stained with hematoxylin and eosin (H&E), and observed and photographed using an Olympus CKX31 inverted microscope.

[0084] Bletilla striata polysaccharide BSPS significantly reduced wound area in mice in a dose- and time-dependent manner (see attached image). Figure 7 (A). BSPS can also accelerate hair growth, especially at high doses, which is comparable to the healing efficiency of rb-bFGF (Appendix). Figure 7 (B) Histopathological examination showed that BSPS not only reduced inflammatory cell infiltration and epidermal and subcutaneous necrosis in a dose- and time-dependent manner, but also accelerated the formation of granulation tissue in traumatic skin (see appendix). Figure 7 (C). These results indicate that BSPS can reduce inflammatory cell infiltration, promote epidermal regeneration, and promote wound healing.

Claims

1. A polysaccharide with wound-healing properties, characterized in that... It comes from Bletilla striata. Bletilla striata A pure monopolysaccharide, Bletilla striata polysaccharide BSPS, was extracted, isolated, and purified from the fresh tubers of (Thunb.) Reichb. f. The relative molecular weight of Bletilla striata polysaccharide BSPS is 722.90 kDa, and it is composed of glucose and mannose in a molar ratio of 1:2.

5. The polysaccharide BSPS can be used for skin wound treatment, promoting myoblast proliferation, differentiation, and migration, alleviating inflammatory infiltration and epidermal and subcutaneous tissue necrosis, and accelerating the formation of granulation tissue, epidermal regeneration, and hair growth in wounded skin.

2. The preparation method of the polysaccharide with wound-healing effect according to claim 1, the specific steps of which are as follows: a. Homogenize the fresh tubers of Bletilla striata, add water, and extract; b. The concentrated aqueous extract was precipitated twice with 95% ethanol, the precipitate was collected, dissolved in distilled water, deproteinized with Sevage reagent, dialyzed, the osmotic residue was concentrated under reduced pressure, and freeze-dried to obtain total polysaccharide BSPt from Bletilla striata. c. The total polysaccharide BSPt of Bletilla striata was separated by gel chromatography, eluted with sodium chloride, and the different eluents were collected, concentrated, desalted, and freeze-dried to obtain two fractions. d. The first fraction was then purified by gel chromatography, eluted with sodium chloride solution, concentrated, desalted, and freeze-dried to obtain Bletilla striata polysaccharide BSPS.

3. The preparation method according to claim 2, characterized in that the gel chromatography column in step c is DEAE-Sephadex A-25, DEAE-Sephadex A-50, DEAE-Sepharose CL-4B, DEAE-Sepharose CL-6B, DEAE-Cellulose 32 or DEAE-Cellulose 52.

4. The preparation method according to claim 2, characterized in that the gel chromatography column in step d is Sephadex G-100, Sephadex G-150, Sephadex G-200, Sephacryl S-300, Sephacryl S-400, Sepharose CL-4B or Sepharose CL-6B.

5. A pharmaceutical composition for treating trauma, comprising a therapeutically effective amount of the Bletilla striata polysaccharide BSPS of claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that... The effective dose of Bletilla striata polysaccharide BSPS is 0.01 mg / kg to 100 mg / kg body weight.

7. The use of the polysaccharide according to claim 1 in the preparation of a wound treatment drug.

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