Preparation method and application of rhizoma bletillae leaf polysaccharide

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of bletilla striata leaf polysaccharide, and the preparation method comprises the following steps: (1) drying and crushing bletilla striata leaves, and degreasing with ethanol; (2) carrying out ultrasonic-assisted water extraction, concentrating, and adding 95% ethanol with the volume being 4 times that of the concentrated solution for precipitation; and (3) carrying out freeze thawing impurity removal, AB-8 macroporous resin decoloration, DEAE-52 column chromatography and Sephadex G-150 purification so as to obtain the polysaccharide BsP-L2-1 with a uniform new structure. According to the invention, high-valued component discovery of non-medicinal part resources of rhizoma bletillae is realized, and the obtained polysaccharide has remarkable antioxidant activity and myocardial protection effect, can effectively relieve doxorubicin-induced cardiotoxicity, is high in safety, and has important application value in development of anti-myocardial injury drugs or health care products.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a method for preparing polysaccharides from Bletilla striata leaves and their applications. Background Technology

[0002] Bletilla striata, a traditional Chinese medicinal herb, has been extensively studied for the hemostatic and anti-inflammatory effects of its tuber polysaccharides. However, the leaves, which account for more than 25% of the plant's biomass, are usually discarded as waste, resulting in resource waste. Recent studies have shown that Bletilla striata leaves contain abundant polysaccharide components, second only to the tubers, and possess potential development value.

[0003] Doxorubicin is a commonly used antitumor drug in clinical practice, but its severe cardiotoxicity greatly limits its application. Oxidative stress in cardiomyocytes and mitochondrial damage are the main mechanisms of its toxicity. Currently, the only FDA-approved drug, dextromethorphan (dextromethorphan), can alleviate some of the toxicity, but it suffers from problems such as high dosage and other toxic side effects. Natural polysaccharides, due to their multi-target and low-toxicity characteristics, have become a research hotspot for the prevention and treatment of drug-induced myocardial injury. However, the different components of Bletilla striata tuber polysaccharides show significant differences in activity, and systematic research on leaf polysaccharides is still lacking, resulting in the ineffective utilization of this resource. Preliminary experiments have found that crude leaf polysaccharides have good cardioprotective activity. Therefore, there is an urgent need to develop a method for efficiently extracting cardioprotective polysaccharides from Bletilla striata leaves to fill this technological gap. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing polysaccharides from Bletilla striata leaves and their application, in order to solve the problems mentioned in the background art, such as serious waste of Bletilla striata leaf resources, lack of highly effective and low-toxic cardioprotective active ingredients, and the lack of ideal prevention and treatment methods for doxorubicin cardiotoxicity.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing polysaccharide from Bletilla striata leaves, comprising the following steps: (1) drying and pulverizing Bletilla striata leaves, and defatting them with 95% ethanol; (2) using distilled water for hot extraction under ultrasonic assistance, combined with centrifugal filtration, combining the filtrates and concentrating them to 20% of the volume of the supernatant, and then adding 4 times the volume of 95% ethanol for precipitation; (3) after redissolving the obtained precipitate, it is purified by freeze-thaw method, decolorization and deproteinization with AB-8 macroporous resin, DEAE-52 cellulose column chromatography and Sephadex G-150 gel column purification to obtain high-purity homogeneous new structural polysaccharide BsP-L2-1.

[0006] Preferably, in step (1), the ethanol degreasing treatment is repeated at least 3 times, the material-to-liquid ratio is 1:3, the ultrasonic treatment time is 4 hours each time, and the residue after degreasing is dried at 50°C to evaporate the ethanol.

[0007] Preferably, in step (2), the hot water extraction temperature is 80°C, the material-to-liquid ratio is 1:30, the ultrasonic-assisted extraction time is 4 hours, the extraction is repeated 3 times, the extract is combined and concentrated under reduced pressure to less than 20% of the original volume, and then 4 times the volume of 95% ethanol is added and left to stand overnight at 4°C to precipitate.

[0008] Preferably, in step (3), the freeze-thaw method for removing impurities involves preparing a 10 mg / mL aqueous solution of crude polysaccharide, repeatedly freezing and thawing it, and then centrifuging it at 4000 r / min for 3 minutes to remove insoluble impurities and residual proteins.

[0009] Preferably, the AB-8 macroporous adsorption resin is soaked in 5% HCl, 5% NaOH and 95% ethanol for 6 hours each before use, and then washed with water until neutral and free of alcohol odor; the sample loading flow rate is 5 mL / min, used to remove impurities such as pigments and proteins.

[0010] Preferably, the DEAE-52 cellulose column chromatography employs gradient elution with NaCl aqueous solutions of 0.00, 0.10, 0.20, 0.30, 0.40, and 1.00 mol / L as the eluent. The flow rate is 1.00 mL / min, and 10 mL is collected from each tube. The polysaccharide content is determined by the phenol-sulfuric acid method, and 24 to 41 tubes of eluent are collected to obtain the component BsP-L2.

[0011] Preferably, during the Sephadex G-150 gel column purification, the BsP-L2 group is prepared into a 5 mg / mL solution, with distilled water as the mobile phase, a flow rate of 0.30 mL / min, and 5 mL is collected from each tube. The symmetrical elution peaks of tubes 19 to 29 are collected, and after lyophilization, homogeneous polysaccharide BsP-L2-1 is obtained.

[0012] Preferably, the prepared polysaccharide BsP-L2-1 is an acidic heteropolysaccharide, free of binding proteins, with a total sugar content of (3.38±2.81)%, exhibiting a single symmetrical peak in the HPGPC-ELSD spectrum, and a molecular weight greater than 5×10⁻⁶. 5 Da.

[0013] Preferably, the polysaccharide BsP-L2-1 is composed of mannose, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 6.02 : 1.74 : 2.70 : 10.00 : 4.32, and contains both α- and β-glycosidic bonds in its structure, and has a highly branched and triple helix conformation.

[0014] Preferably, the polysaccharide BsP-L2-1 exhibits a 72.37% ABTS free radical scavenging rate at a concentration of 6 mg / mL, significantly reduces the content of reactive oxygen species (ROS) and malondialdehyde (MDA) levels in H9c2(2-1) cardiomyocytes induced by doxorubicin, and exerts a cardioprotective effect by maintaining mitochondrial membrane potential stability. Furthermore, it shows no developmental toxicity to zebrafish embryos at a concentration of 900 μg / mL, making it suitable for preparing drugs, functional foods, or health products that alleviate doxorubicin cardiotoxicity.

[0015] Compared with existing technologies, the beneficial effects of this invention are as follows: The method for extracting and purifying polysaccharide BsP-L2-1 from Bletilla striata leaves provided by this invention addresses the problems of long-term waste of Bletilla striata leaves, low resource utilization, and the lack of safe and effective protective measures against doxorubicin cardiotoxicity. It has the advantages of simple operation, mild conditions, and good reproducibility. The obtained polysaccharide has high purity and uniform structure, exhibiting unique structural characteristics compared to other Bletilla striata polysaccharides. It possesses both significant antioxidant activity and cardioprotective effects, effectively inhibiting doxorubicin-induced ROS accumulation, MDA elevation, and mitochondrial damage. Furthermore, it shows no embryotoxicity at a concentration of 900 μg / mL, demonstrating good safety. This provides a new raw material and feasible pathway for the development of anti-myocardial injury drugs or functional health products. Attached Figure Description

[0016] Figure 1 This is a process flow diagram of a method for preparing Bletilla striata leaf polysaccharide according to the present invention; Figure 2 This invention relates to a method for preparing polysaccharides from Bletilla striata leaves, including separation, purification, and purity analysis. Chromatography and spectra; Figure 3 The image shows the structural characterization spectrum of the product obtained by the preparation method of Bletilla striata leaf polysaccharide of the present invention. Figure 4 This is a high-order structure and morphology analysis image of the product obtained by the preparation method of Bletilla striata leaf polysaccharide of the present invention. Figure 5 The image shows the results of antioxidant activity determination of the polysaccharide obtained by the preparation method of Bletilla striata leaf polysaccharide of the present invention; Figure 6 The figure shows the evaluation results of the cardioprotective effect of the polysaccharide obtained by the preparation method of Bletilla striata leaf polysaccharide of the present invention. Figure 7 This image shows the safety evaluation test results of zebrafish embryos using the polysaccharide obtained from the preparation method of Bletilla striata leaf polysaccharide according to the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Extraction and purification of a homogeneous polysaccharide derived from Bletilla striata leaves The steps of sampling, defatting, extraction, impurity removal and separation of Bletilla striata polysaccharides are as follows: Figure 1 As shown.

[0019] 1. Polysaccharide extraction Bletilla striata leaves were collected, washed, dried at 50℃, pulverized, and passed through a 100-mesh sieve. They were then soaked in 95% ethanol at a ratio of 1:3 and ultrasonically extracted for 4 hours. The residue was collected by filtration, and this process was repeated three times. The defatted Bletilla striata leaf powder was placed in an oven to evaporate the ethanol. Distilled water was added at a ratio of 1:30, and the mixture was ultrasonically extracted at 80℃ for 4 hours. The residue was filtered, and the extraction was repeated three times. The extracts were collected and combined, concentrated under reduced pressure to 20% of the original volume, and then 4 times the volume of 95% ethanol was added. After mixing, the mixture was allowed to precipitate overnight. The precipitate was collected, dried, and the crude polysaccharide from Bletilla striata leaves was obtained and stored at -20℃ for later use. The extraction rate was calculated to be 5.05% based on the weight of the extracted material.

[0020] 2. Freeze-thaw method for impurity removal After reconstitution of the crude polysaccharide, a solution of approximately 10 mg / mL was prepared. The solution was centrifuged at 4000 r / min for 3 min, and the supernatant was collected. This supernatant was then repeatedly frozen and thawed before centrifugation to remove residual protein and other insoluble impurities. Finally, the supernatant was collected.

[0021] 3. Impurity removal using macroporous adsorption resin The macroporous adsorption resin AB-8 was soaked in 5% HCl for 6 hours to allow it to fully swell, then rinsed with pure water until neutral. Next, it was soaked in 5% NaOH for 6 hours, then rinsed with pure water until neutral. Finally, the resin was soaked in 95% ethanol for 6 hours and washed with pure water until no alcohol odor remained. An appropriate amount of resin was packed into a column (6 cm diameter × 40 cm height) using the wet packing method. 200 mL of the supernatant was then passed through the column at a flow rate of 5 mL / min. The adsorption process was then repeated with fresh AB-8 macroporous resin following the same procedure. Samples that had undergone protein and pigment removal via AB-8 were collected, concentrated appropriately, and then lyophilized for later use.

[0022] 4. Isolation of polysaccharides The purified polysaccharides were dissolved in distilled water to prepare a 5 mg / mL solution. Separation was performed using a DEAE-52 cellulose column (6.5 cm × 10 cm) with gradient elution using 0.00, 0.10, 0.20, 0.30, 0.40, and 1.00 mol / L NaCl solutions (flow rate 1.00 mL / min), collecting 10 mL of eluent per tube. The absorbance at 490 nm was measured using the phenol-sulfuric acid method, and elution curves were plotted. (See figure). Figure 2 a. Collect the eluent from tubes 18-23, concentrate, dialyze, and lyophilize to obtain the polysaccharide fraction BsP-L1. Repeat the same process for tubes 24-41 to obtain BsP-L2. Reconstitute the main polysaccharide BsP-L2 fraction into a 5 mg / mL solution, further purify it using a Sephadex G-150 gel column (25 mm × 25 cm), and elute with distilled water at a flow rate of 0.30 mL / min (5 mL / tube). Measure the absorbance at 490 nm again and plot the elution curve. The elution curve is shown in [Figure 1]. Figure 2 b. Collect the symmetrical elution peaks from tubes 19-29, concentrate and freeze-dry to obtain purified polysaccharide BsP-L2-1, concentrate and store at 4℃ for later use.

[0023] Implementation Case 2: Extraction, purification, and structural characterization of a homogeneous polysaccharide derived from Bletilla striata leaves. 1. In this implementation case, the extraction and purification of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Implementation Case 1, and will not be repeated here. Through this step, the purified polysaccharide BSP-L2-1 was finally obtained.

[0024] 2. Purity analysis and structural characterization of purified polysaccharides 2.1 Determination of total polysaccharide content Weigh 40.10 mg of standard glucose and prepare a 4.01 mg / mL glucose solution. Dilute this solution sequentially to obtain a series of standard solutions with concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 mg / mL. Prepare a 2.5 mg / mL solution of Bletilla striata leaf polysaccharide as a test solution. Take 30 µL of each standard or sample solution, add 30 µL of phenol and 200 µL of sulfuric acid solution, and mix. Heat at 80℃ for 3 min. After the reaction, transfer 200 µL of the mixture to a 96-well plate and measure the absorbance at 490 nm using a Spectra Max i3x multi-mode microplate reader. Plot a standard curve and obtain the linear regression equation y = 0.6897x + 0.0995, R² = 0.9967. Similarly, measure the absorbance of the polysaccharide BsP-L2-1 sample and substitute it into the formula to calculate the total polysaccharide content, which is approximately (93.38 ± 2.81)%. This result indicates that BsP-L2-1 is a high-purity polysaccharide.

[0025] 2.2 UV spectral analysis Weigh 5 mg of Bletilla striata leaf polysaccharide, dissolve it in pure water to prepare a 5 mg / mL solution, add 20 μL to a 96-well plate, and perform a full-wavelength UV-Vis scan in the wavelength range of 200–400 nm. The UV-Vis spectral results are as follows. Figure 2 c. By analyzing the absorbance at 260 nm (characteristic absorption peak of nucleic acids) and 280 nm (characteristic absorption peak of proteins / peptides), the presence of residual impurities such as nucleic acids, proteins, or peptides in the polysaccharide sample was assessed. The absorption peaks of the polysaccharide component BsP-L2-1 between 260 nm and 280 nm were almost negligible, indicating that the purity of polysaccharide BsP-L2-1 was very high, and the content of impurities such as nucleic acids and proteins was very low.

[0026] 2.3 Homogeneity and Molecular Weight Determination The polysaccharide from Bletilla striata leaves was prepared into a 1 mg / mL solution, filtered through a 0.45 μm aqueous filter membrane, and then packaged into a liquid chromatography vial. Purity and molecular weight were analyzed using high-performance liquid chromatography-gel permeation chromatography-evaporative light scattering detector (HPGPC-ELSD). The specific steps are as follows: A Shimadzu LC-20A HPLC system was used, with a TSK-gel GMPWxl column (7.8 mm × 30 cm), an ELSD detector (Alltech, Deerfield, USA), and deionized water as the mobile phase (flow rate: 0.5 mL / min). The ELSD detection parameters were set as follows: nitrogen carrier gas (pressure 350–400 kPa), drift tube temperature 115℃, and injection volume 5 µL. The purity of the polysaccharide was evaluated based on the number, shape, and proportion of the main peak. Simultaneously, T-series dextran (T-5, T-10, T-40, T-70, T-500) were selected as standards and analyzed using HPGPC-ELSD under the same conditions. The molecular weight of the polysaccharide was analyzed by comparing its retention time with that of a standard polysaccharide. The retention time of BsP-L2-1 in the chromatogram was 8.311 min (…). Figure 2 d), the corresponding molecular weight calculation result is greater than that of T-500, the standard dextran with the largest molecular weight (molecular weight is 5×10). 5 This indicates that the molecular weight of BsP-L2-1 is greater than 5 × 10⁻⁶. 5 This value indicates that BsP-L2-1 is a high molecular weight polysaccharide component.

[0027] In addition, such as Figure 2As shown in Figure d, the HPGPC-ELSD analysis results indicate that the polysaccharide BsP-L2-1 component exhibits only a single, symmetrical peak in the chromatogram, with a good peak shape and no tailing, indicating excellent sample homogeneity. Furthermore, combined with the total sugar content determination results and UV spectral analysis data, these findings confirm that BsP-L2-1 is a polysaccharide component with high purity and uniform molecular weight distribution.

[0028] 2.5 Monosaccharide component analysis The monosaccharide composition of Bletilla striata root polysaccharide was determined by pre-column derivatization high-performance liquid chromatography (PMP-HPLC) using 1-phenyl-3-methyl-5-pyrazolone. First, 2 mg of the polysaccharide sample was dissolved in 1 mL of 3 mol / L trifluoroacetic acid, sealed under nitrogen, and heated at 90 °C for 6 h. After cooling, methanol was added for co-distillation to remove the trifluoroacetic acid. The acid-hydrolyzed polysaccharide was dissolved in 0.6 mol / L NaOH along with standard monosaccharides such as mannose, glucose, galactose, and arabinose. PMP solution was added for derivatization at 70 °C, followed by neutralization with an equal volume of 0.6 mol / L HCl. Extraction was then performed with chloroform, repeated three times. The aqueous layer was collected and filtered through a 0.22 µm filter membrane for later use. The hydrolyzed polysaccharide sample and the standard polysaccharide sample were analyzed separately using an Agilent 1100 HPLC system. The chromatographic conditions were as follows: the column was a Supersil AQ-C18 column (250 mm × 4.60 mm, 5 μm, Dalian, China); the mobile phase was 17% acetonitrile and 83% PBS (0.05 mol / L, pH 7.2, v / v) with isocratic elution; the flow rate was 1 mL / min; the detection wavelength was 245 nm; the column temperature was 30℃; and the injection volume was 10 µL.

[0029] The results are as follows Figure 3 As shown in a and Table 1, this polysaccharide is mainly composed of mannose (Man), galacturonic acid (GalA), glucose (Glc), galactose (Gal), and arabinose (Ara), with a molar ratio of 6.02:1.74:2.70:10.00:4.32. In terms of composition, galactose (Gal) has the highest proportion, accounting for approximately 40% of the total monosaccharide, indicating that the core structure of this polysaccharide is likely dominated by galactose. This is followed by Man (approximately 24%) and Ara (approximately 18%), while Glc (approximately 11%) and GalA (approximately 7%) are present in relatively low amounts. This monosaccharide composition is also the first time it has been found in Bletilla striata polysaccharides. Table 1. Molar ratio of BsP-L2-1 monosaccharide components Mannose Galacturonic acid glucose Galactose Arabic sugar BSP-L2-1 6.02 1.74 2.70 10.00 4.32 2.6 FT-IR Spectroscopic Analysis Weigh 2–3 mg of the dried polysaccharide sample, compress it into a pellet using the KBr method, and scan it in the infrared region of 4,000–450 cm⁻¹ using a Varian 1000 Fourier transform infrared spectrometer (Scimitar series, Varian Inc., USA).

[0030] The results are as follows Figure 3 As shown in b, the infrared spectrum of BsP-L2-1 exhibits typical polysaccharide characteristic absorption peaks: the broad peak at 3385.25 cm⁻¹ corresponds to the stretching vibration of the hydroxyl group (OH) on the sugar chain; the peak near 2927.18 cm⁻¹ originates from the stretching vibration of the CH bond; the peak at 1728.71 cm⁻¹ originates from the stretching vibration of C=O; and the absorption peaks at 1632.68 cm⁻¹ and 1423.47 cm⁻¹ are generally related to the stretching vibration of the carboxyl group (-COOH), indicating that the polysaccharide may contain a carboxyl group (-COOH). Combined with the monosaccharide composition analysis results, the carboxyl group at this location may originate from the carboxyl group of galacturonic acid. In the fingerprint region, the strong absorption peak at 1073.44 cm⁻¹ is attributed to the stretching vibration of the pyran ring of glucose residues; the absorption peak at 895.28 cm⁻¹ is generally related to the skeletal bending of the galactose ring and may also indicate the presence of an α-configuration glycosidic bond. Based on the monosaccharide composition analysis, galactose is the most abundant polysaccharide, so it is speculated that the absorption peak at this location is related to the bending of the galactose ring skeleton.

[0031] 2.7 1H NMR Spectroscopy Approximately 20 mg of the purified Bletilla striata leaf polysaccharide was dissolved in 0.5 mL of D2O. Spectra were acquired at room temperature using an Agilent 400 M nuclear magnetic resonance spectrometer (Agilent, USA), and processed and analyzed using MestReNova software (Mestrelab Research Inc.). The proton NMR spectrum is shown below. Figure 3 As shown in c. Eight anomeric hydrogen signals were detected in the anomeric hydrogen fingerprint region of this polysaccharide, with chemical shifts of δ 5.64, 5.22, 5.05, 5.01, 4.95, 4.87, 4.82, and 4.80 ppm. According to literature reports, signals with chemical shifts in the range of 4.4–5.0 ppm are attributed to β-glycosidic anomeric hydrogens, signals in the range of 5.0–5.4 ppm correspond to α-glycosidic anomeric hydrogens, and signals in the range of 3.4–4.2 ppm are hydrogens at the C2–C6 positions of the sugar ring. The above analytical results indicate that Bletilla striata leaf polysaccharide BsP-L2-1 is a heteropolysaccharide containing both α- and β-glycosidic bond configurations.

[0032] 3. High-level structural and morphological features 3.1 I2-KI Analysis The presence or absence of an absorption peak at 565 nm after a polysaccharide sample reacts with iodine reagent and is determined by ultraviolet scanning within the 300-800 nm range. An absorption peak indicates fewer branches and shorter side chains, while the absence of a peak indicates more branches and longer side chains. The I2-KI method is as follows: Polysaccharide from Bletilla striata leaves and standard starch were prepared into 2.5 mg / mL solutions. 100 μL of each solution was placed in a 1.5 mL centrifuge tube. An equal volume of I2-KI solution (0.02 g I2 + 0.20 g KI dissolved in 100 mL water) was added to each group. After mixing, the solution was transferred to a cuvette, and the color difference between the polysaccharide mixture and the starch positive control was observed. The ultraviolet-visible absorption spectrum (UV-Vis) of the polysaccharide-I2-KI mixture in the 300-800 nm wavelength range was measured. The presence or absence of an absorption peak at 565 nm after a polysaccharide sample reacts with iodine reagent and is determined by ultraviolet scanning within the range of 300 to 800 nm. An absorption peak indicates fewer branches and shorter side chains, while the absence of an absorption peak indicates more branches and longer side chains.

[0033] The results are as follows Figure 4 As shown in Figure a, BsP-L2-1 shows no absorption peak at 565 nm, indicating that the polysaccharide BsP-L2-1 contains many branches and long side chains. The reference standard (starch) shows an absorption peak at 565 nm. Figure 4 b) indicates that it contains fewer branches and shorter side chains. Furthermore, no blue reaction occurred between I2-IK and BsP-L2-1 solution, while the starch reference standard showed a color reaction with I2-IK, indicating that BsP-L2-1 does not contain starch.

[0034] 3.2 Congo Red Analysis Congo red, an acidic dye, can form polysaccharide complexes with polysaccharides having a triple helix conformation. This results in a redshift of the maximum wavelength when scanned under 475–515 nm ultraviolet light. Alkaline solutions of a certain concentration can disrupt the hydrogen bonds maintaining this structure, thus reducing the maximum wavelength. The analysis of Congo red is as follows: Prepare a 1.5 mol / L NaOH solution and dilute it to concentration gradients of 0.00, 0.30, 0.60, 0.90, 1.20, and 1.50 mol / L; separately prepare an 80 µmol / L Congo red solution and polysaccharide sample solutions of 2.5 mg / mL and 5 mg / mL; take six 0.5 mL centrifuge tubes, add 50 µL of polysaccharide solution (2.5 or 5 mg / mL) and 50 µL of Congo red solution to each tube, then add 50 µL of NaOH solution of different concentrations (0.00–1.50 mol / L), mix well, and let stand at room temperature for 10 min. Meanwhile, ultrapure water was used as a blank control instead of polysaccharide solution (two independent batches of experiments); each mixture was scanned in the range of 475–515 nm using ultraviolet light, and all absorption peak wavelengths were recorded. A curve was plotted with the final concentration of NaOH solution as the x-axis and the absorption peak wavelength as the y-axis.

[0035] The results are as follows Figure 4 As shown in c, in this experiment, the wavelength of the BsP-L2-1 sample solution red-shifted when the NaOH concentration was 0–0.1 mol / L, indicating that BsP-L2-1 has a triple helix structure and forms a polysaccharide complex with Congo red. The maximum wavelength decreased when the NaOH concentration was between 0.1 and 0.2 mol / L, indicating that the hydrogen bonds maintaining the triple helix conformation were broken.

[0036] 3.3 Electron Microscope Scanning A small amount of polysaccharide sample powder was taken and a thin layer of gold atoms (Au) was sputtered on under reduced pressure. The sample was then imaged and photographed using a Hitachi SU8010 scanning electron microscope (SEM) at an accelerating voltage of 3 kV at magnification of 1000x and 10000x.

[0037] Scanning electron microscope morphology as follows Figure 4 As shown in d and 4e. At a magnification of 1000× ( Figure 4 d) The microstructure of polysaccharide BsP-L2-1 mainly exhibits an irregular, elongated, sheet-like morphology, with slightly curled edges and a relatively smooth overall surface. Further magnification to 10000× ( Figure 4 e) Fine features of its microstructure can be observed, namely, the polysaccharide sheets are composed of multiple small, irregular sheet-like structures tightly nested together, with some areas showing minor bulges, possibly due to local aggregation of molecular chains or differences in crystallinity. This nested structure of small sheets may be related to intermolecular hydrogen bonding or hydrophobic interactions, and the smoothness of the surface can reflect the aggregated state of the polysaccharide.

[0038] Implementation Case 3: Application of a homogeneous polysaccharide derived from Bletilla striata leaves in antioxidant activity 1. In this implementation case, the extraction and purification of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Implementation Case 1, and will not be repeated here. Through this step, high-purity homogeneous polysaccharide BSP-2L-1 was finally obtained.

[0039] 2. In this embodiment, the structural characterization of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Embodiment 2, and will not be repeated here. Through this step, a novel high-purity homogeneous polysaccharide BSP-2L-1 from Bletilla striata leaves was finally obtained.

[0040] 3. ABTS free radical scavenging activity 60 μL of polysaccharide solutions of different concentrations were respectively added to 96-well microplates, 200 μL of ABTS reagent was added, and the mixture was incubated at 37℃ for 5 min. The absorbance was measured at 734 nm using a Spectra Max i3x multi-functional microplate reader. Water was used as a negative control instead of the sample, and ethanol was used as a blank control instead of the ABTS solution. All experiments were repeated three times. ABTS free radical scavenging activity was calculated using the following formula: ABTS free radical scavenging activity (%) = (1 - (Asample - Ablank)) / (Acontrol) × 100%, where Acontrol is the negative control, Ablank is the blank control, and Asample is the sample measurement value. The results are as follows: Figure 5 As shown in Figure a, in the ABTS free radical scavenging experiment, the scavenging rate significantly increased with increasing polysaccharide concentration (0–6 mg / mL). When the concentration reached 6 mg / mL, the scavenging activity reached (72.37 ± 1.94)%, significantly higher than the other two free radical scavenging systems, indicating that BsP-L2-1 had the strongest inhibitory effect on ABTS⁺.

[0041] 4. DPPH free radical scavenging activity The isolated polysaccharide samples were dissolved in water to prepare a 6.0 mg / mL stock solution, which was then serially diluted to obtain six different concentrations. 60 µL of each concentration of polysaccharide solution was added to a 96-well microplate, followed by 200 µL of 0.004% (v / v) DPPH ethanol solution. After mixing, the solution was incubated at 37°C for 5 min. The absorbance was measured at 520 nm using a Spectra Max i3x multi-plate reader. Water was used as a negative control instead of the sample, and ethanol was used as a blank control instead of the DPPH solution. BHT was used as a standard antioxidant reference. All experiments were independently repeated three times. DPPH free radical scavenging activity was calculated using the following formula: DPPH free radical scavenging activity (%) = (Acontrol - (Asample - Ablank)) / (Acontrol) × 100%, where Acontrol is the negative control, Ablank is the blank control, and Asample is the sample measurement. Results are as follows: Figure 5 As shown in b, in the DPPH free radical scavenging experiment, its scavenging rate at 6 mg / mL was (37.94±3.25)%, which is about 50% of the ABTS scavenging activity, indicating that its inhibitory ability on DPPH is moderate.

[0042] 5. Hydroxyl radical scavenging activity The Fenton-salicylic acid method was used to determine the hydroxyl radical scavenging activity: 7.5 mM FeSO4 (water-soluble, with a small iron nail added for oxidation prevention), 7.5 mM salicylic acid (anhydrous ethanol-soluble), and 1% H2O2 were used as the reaction system; 150 µL of FeSO4 and 150 µL of 1% H2O2 were added to 150 µL of polysaccharide samples of different concentrations and 150 µL of salicylic acid, and the mixture was incubated at 40℃ for 15 min before the absorbance (Ax) was measured at 510 nm; Ax0 (blank control) was measured using water instead of H2O2, and A0 (negative control) was measured using water instead of the sample, with BHT as a positive control; the hydroxyl radical (•OH) scavenging rate was calculated using the formula: Hydroxyl radical scavenging rate (%) = (A0 - (AX - AX0)) / (A0) × 100%, where A0 is the negative control, AX0 is the blank control, and AX is the sample measurement value. The results are as follows: Figure 5 As shown in c, in the hydroxyl radical scavenging activity experiment, the scavenging effect on hydroxyl radicals was relatively weak, with a scavenging rate of (23.46±3.31)% at 6 mg / mL.

[0043] Example 4: Application of a homogeneous polysaccharide derived from Bletilla striata leaves in resisting doxorubicin (DOX)-induced cardiocytotoxicity. 1. In this implementation case, the extraction and purification of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Implementation Case 1, and will not be repeated here. Through this step, the homogeneous polysaccharide BSP-2L-1 was finally obtained.

[0044] 2. In this embodiment, the structural characterization of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Embodiment 2, and will not be repeated here. Through this step, a novel homogeneous polysaccharide, BSP-2L-1, was finally obtained.

[0045] 3. In this implementation case, the in vitro antioxidant activity determination of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in implementation case 3, and will not be repeated here. Through this step, a high-purity homogeneous polysaccharide BSP-2L-1 from Bletilla striata leaves with significant antioxidant application potential was finally obtained.

[0046] 4. Assay of BSP-2L-1's anti-doxorubicin-induced cardiomyocyte cytotoxicity activity 4.1 Cell culture, processing, and cell viability testing H9c2(2-1) cardiomyocytes were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator, with the medium being changed every two days. When the cell confluence reached 80%–90%, the cells were passaged using 0.25% trypsin. After the cells adhered to the plate again and grew to 70% confluence, they were divided into three groups: a control group, a Dox group, and a Bletilla striata polysaccharide treatment group (Dox + polysaccharide group). After being seeded and cultured for another 24 h, the cells were induced with the drugs for another 24 h.

[0047] The effect of BsP-L2-1 on cardiomyocyte proliferation was examined using SRB staining. Three groups of cells were stained, and the results showed that compared with the control group ( Figure 6 (a) and (6b) The viability and number of H9c2(2-1) cells in the Dox group were significantly reduced, indicating that Dox has a serious impact on cardiomyocytes and greatly disrupts their growth. However, after treating the cells with BsP-L2-1 at a concentration of 2.00 mg / mL for 24 hours, this reduction in the number of Dox-induced cells was alleviated.

[0048] 4.2 Detection of malondialdehyde (MDA) and reactive oxygen species (ROS) levels Due to the imbalance between the antioxidant defense system and ROS activation, Dox induces oxidative stress, which is one of the root causes of Dox-dependent cardiotoxicity. To verify whether BsP-L2-1 alleviates DOX-induced oxidative stress in H9c2(2-1), ROS and MDA content were measured.

[0049] H9c2(2-1) cardiomyocytes were fed with 0.8 × 10⁻⁶ cells. 4Cells were seeded at a density of 1 / 2 well in 96-well plates and cultured until 70% confluence. Then, the cells were treated as follows: the Dox group received 40 µg / mL Dox; the Dox + polysaccharide group received 40 µg / mL Dox and 2 mg / mL Bletilla striata leaf polysaccharide; and the Control group received an equal volume of culture medium. After 24 h, the culture medium was discarded, and the cells were fixed with 10% TCA for 12 h. After washing with deionized water and drying, SRB staining solution was added and incubated at room temperature in the dark for ≥1 h. The cells were then washed with 1% acetic acid and dried. 100 µL of 10 mM Tris-HCl buffer was added to each well, and the dye was dissolved by shaking. The absorbance was measured using a microplate reader. Simultaneously, cells were collected, and the MDA levels in each group were measured according to the kit instructions.

[0050] The ROS assay procedure was as follows: DCFH-DA was diluted to 10 μmol / L with serum-free medium and added to the cells of each group after treatment. After mixing, the cells were incubated at 37°C for 20 min. The medium was discarded and the cells were washed with PBS. Fresh medium was then added and the fluorescence signal was observed under a fluorescence microscope.

[0051] The results are as follows Figure 6 As shown in c and 6d, DOX significantly induced oxidative stress, as evidenced by the elevated ROS levels confirmed by the green fluorescence accumulation reaction, while BsP-L2-1-treated cells showed significantly reduced ROS levels. MDA is a hallmark product of lipid peroxidation, primarily produced by the breakdown of polyunsaturated fatty acids in the cell membrane under the attack of ROS and free radicals. Since MDA generation is directly related to lipid peroxidation, MDA detection is often used as a marker of the degree of oxidative stress, as shown in the results... Figure 6 e shows that Dox promotes MDA generation, while BsP-L2-1 significantly alleviates this phenomenon.

[0052] 4.3 Mitochondrial membrane potential (MMP) measurement MMPs serve as a marker for assessing mitochondrial damage. Normal control cells show red fluorescence, while cells with decreased mitochondrial membrane potential show green fluorescence; the ratio of these two fluorescence values ​​reflects the degree of mitochondrial membrane potential reduction. Working solution was prepared according to the JC-1 kit instructions. After treatment, appropriate amounts of JC-1 working solution were added to each group of cells, mixed well, and incubated at 37°C for 30 min. After incubation, cells were washed with JC-1 staining buffer, added to culture medium, and observed under a microscope. Results are as follows: Figure 6 f and 6g showed that DOX reduced cellular MMP levels. In contrast, the green fluorescence in the BsP-L2-1 group gradually weakened, indicating increased MMP levels. DOX induces mitochondrial dynamic imbalance, thus affecting cell function and state. BsP-L2-1 alleviated the degree of DOX-induced decrease in mitochondrial membrane potential and can prevent apoptosis by protecting mitochondrial function. Implementation Case 5: Safety Evaluation of a Homogeneous Polysaccharide Derived from Bletilla striata Leaves 1. In this implementation case, the extraction and purification of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Implementation Case 1, and will not be repeated here. Through this step, the homogeneous polysaccharide BSP-2L-1 was finally obtained.

[0053] 2. In this embodiment, the structural characterization of a homogeneous polysaccharide derived from Bletilla striata leaves is the same as in Embodiment 2, and will not be repeated here. Through this step, a novel homogeneous polysaccharide, BSP-2L-1, was finally obtained.

[0054] 3. In this implementation case, the in vitro antioxidant activity and cardioprotective activity of a homogeneous polysaccharide derived from Bletilla striata leaves were determined in the same manner as in Implementation Cases 3 and 4, and will not be repeated here. Through this step, a high-purity homogeneous polysaccharide BSP-2L-1 from Bletilla striata leaves with significant potential for antioxidant and cardioprotective applications was finally obtained.

[0055] 4. Safety evaluation of BSP-2L-1 To assess the safety of BsP-L2-1, this invention evaluated its effects on zebrafish embryonic development. The specific procedure was as follows: Healthy 4 hpf zebrafish embryos of uniform condition were selected and cultured. Embryos were transferred to six-well plates (20 embryos / well) and exposed to culture media containing 0 (control), 100, 300, 600, and 900 μg / mL BsP-L2-1 at a constant temperature of 25.0 ± 0.5℃. The exposure solution was changed every 24 hours. During the experiment, the number of embryos that died and hatched was recorded every 8 hours, and morphological developmental changes (including somites, eye sacs, etc.) were observed every 24 hours. After 24 hpf, pericardial area and heart rate (N≥3) were measured daily using microscopic video (30 seconds / embryo). The pH was maintained between 7.0 and 7.5 throughout the process, and the absence of heartbeat and yolk sac turbidity were used as the criteria for embryonic death.

[0056] The results showed that at 24 hbf, high concentrations of polysaccharide (900 μg / mL) promoted zebrafish embryonic development, but there was no significant difference in hatching progress between the concentration groups and the control group over time (p>0.05) (Fig. 7a), and the survival rate remained at 100% (Fig. 7b). Heart rate measurements at 20 s (control group 153±18 bpm vs treatment group 129–162 bpm) (Fig. 7c) and pericardial area measurements (maintained at 0.05±0.01 mm² in each group) showed no statistically significant differences (p>0.05) (Fig. 7e). Morphological observation showed that all embryos developed normally to Kimmel stage 5, with no abnormalities observed in yolk sac absorption, eye development, somitosis formation, or pigment deposition, and a developmental malformation rate of <5% (Fig. 7d). These data consistently indicate that within the concentration range set in this experiment, BsP-L2-1 did not exhibit significant embryotoxicity, developmental delay, or cardiac function / morphological abnormalities, suggesting that this polysaccharide has good developmental safety.

[0057] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing polysaccharides from Bletilla striata leaves, characterized in that: The following steps are included: (1) After drying and pulverizing the leaves of Bletilla striata, defatting them with 95% ethanol; (2) Under ultrasonic assistance, hot extraction is performed using distilled water, combined with centrifugal filtration, the filtrates are combined and concentrated to 20% of the volume of the supernatant, and then 4 times the volume of 95% ethanol is added for precipitation; (3) After redissolving the obtained precipitate, it is purified by freeze-thaw method, decolorization and deproteinization with AB-8 macroporous resin, DEAE-52 cellulose column chromatography and Sephadex G-150 gel column purification to obtain high purity and uniform new structural polysaccharide BsP-L2-1.

2. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: In step (1), the ethanol degreasing treatment is repeated at least 3 times, the material-to-liquid ratio is 1:3, the ultrasonic treatment time is 4 hours each time, and the residue after degreasing is dried with ethanol at 50°C.

3. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: In step (2), the hot water extraction temperature is 80℃, the material-to-liquid ratio is 1:30, the ultrasonic-assisted extraction time is 4 hours, the extraction is repeated 3 times, the extract is combined and concentrated under reduced pressure to less than 20% of the original volume, and then 4 times the volume of 95% ethanol is added and left to stand overnight at 4℃ to precipitate.

4. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: In step (3), the freeze-thaw method for removing impurities involves preparing a 10 mg / mL aqueous solution of crude polysaccharide, repeatedly freezing and thawing it, and then centrifuging it at 4000 r / min for 3 minutes to remove insoluble impurities and residual proteins.

5. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: Before use, the AB-8 macroporous adsorption resin is soaked in 5% HCl, 5% NaOH and 95% ethanol for 6 hours each, and then washed with water until neutral and free of alcohol odor. The sample loading flow rate is 5 mL / min, which is used to remove impurities such as pigments and proteins.

6. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: The DEAE-52 cellulose column chromatography employed gradient elution with NaCl aqueous solutions of 0.00, 0.10, 0.20, 0.30, 0.40, and 1.00 mol / L at a flow rate of 1.00 mL / min. 10 mL was collected from each tube, and the polysaccharide content was determined using the phenol-sulfuric acid method. The fraction BsP-L2 was obtained by collecting 24–41 tubes of eluent.

7. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: During the Sephadex G-150 gel column purification, the BsP-L2 group was prepared into a 5 mg / mL solution. Distilled water was used as the mobile phase, the flow rate was 0.30 mL / min, and 5 mL was collected from each tube. The symmetrical elution peaks of tubes 19 to 29 were collected and then lyophilized to obtain homogeneous polysaccharide BsP-L2-1.

8. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: The obtained polysaccharide BsP-L2-1 is an acidic heteropolysaccharide, containing no binding proteins, with a total sugar content of (3.38±2.81)%. Its HPGPC-ELSD spectrum shows a single symmetrical peak, and its molecular weight is greater than 5×10⁻⁶. 5 Da.

9. The method for preparing Bletilla striata leaf polysaccharide according to claim 1, characterized in that: The polysaccharide BsP-L2-1 is composed of mannose, galacturonic acid, glucose, galactose and arabinose in a molar ratio of 6.02 : 1.74 : 2.70 : 10.00 : 4.

32. It contains both α- and β-glycosidic bonds and has a highly branched and triple-helical conformation.

10. The application of a Bletilla striata leaf polysaccharide according to any one of claims 1-9, characterized in that: The polysaccharide BsP-L2-1 exhibits a 72.37% ABTS free radical scavenging rate at a concentration of 6 mg / mL. It can significantly reduce the content of reactive oxygen species (ROS) and malondialdehyde (MDA) levels in H9c2(2-1) cardiomyocytes induced by doxorubicin, and exerts a cardioprotective effect by maintaining the stability of mitochondrial membrane potential. Furthermore, it shows no developmental toxicity to zebrafish embryos at a concentration of 900 μg / mL. It is suitable for preparing drugs, functional foods, or health products that alleviate the cardiotoxicity of doxorubicin.

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