Chuanxingsu polysaccharide having a therapeutic mycoplasmal pneumonia effect, and a preparation method and application thereof

Abstract

The application discloses a kind of prunus mira polysaccharide with the effect of treating mycoplasma pneumonia and its preparation method and application, the prunus mira polysaccharide is homogeneous polysaccharide, monosaccharide composition is rhamnose, arabinose, glucose, galactose, xylose, molar ratio is 12.5:11.15:17.89:6.57:1.5;The prunus mira polysaccharide structure is as shown below.Experiments prove that in the mycoplasma pneumoniae infection mouse model, prunus mira polysaccharide can effectively alleviate the body weight loss caused by mycoplasma pneumonia infection, and the effect of high-dose group is more significant.Prunus mira polysaccharide can significantly reduce lung tissue inflammatory cell infiltration and alveolar structure destruction, reduce lung tissue pathological score, and can effectively reduce lung tissue mycoplasma load, and the bacteriostatic effect of high-dose group is most significant.

Description

Technical Field

[0001] This invention relates to polysaccharides, their preparation and uses, and specifically to a Sichuan apricot polysaccharide, its preparation method, and its application in the prevention and treatment of mycoplasma pneumonia. Background Technology

[0002] Mycoplasma pneumoniae pneumonia (MPP) is caused by Mycoplasma pneumoniae (… Mycoplasma pneumoniae Mycoplasma pneumoniae (MPP) is a common community-acquired pneumonia, predominantly affecting children and adolescents. In recent years, its prevalence has rebounded in many areas, with a high incidence during autumn and winter. Currently, macrolides, fluoroquinolones, and tetracyclines are the main drugs used clinically to treat mycoplasma infection. However, my country has the highest macrolide resistance rate among Mycoplasma pneumoniae globally. Reports indicate that the resistance rates of Mycoplasma pneumoniae to erythromycin and roxithromycin are as high as 71.2% and 73.1%, respectively, while the resistance rate to azithromycin is approximately 25.0%. The persistently high proportion of Mycoplasma pneumoniae resistant to macrolide antibiotics poses a serious challenge to traditional antibiotic treatment.

[0003] Chuanxing Qingfei Syrup is a traditional Chinese medicine compound preparation composed of nine ingredients: loquat leaf, ophiopogon japonicus, rehmannia glutinosa, licorice root, bitter almond, platycodon grandiflorus, peppermint, mulberry leaf, and fritillaria cirrhosa. It has been used clinically for nearly 30 years. However, the effective material basis for the actual effect of this compound preparation is still unclear. Summary of the Invention

[0004] Objective of the Invention: The objective of this invention is to provide a structurally defined apricot polysaccharide and its preparation method. Another objective of this invention is to provide the application of the apricot polysaccharide in the treatment of mycoplasma pneumonia.

[0005] Technical solution: The apricot polysaccharide of this invention is a homogeneous polysaccharide, with a monosaccharide composition of rhamnose, arabinose, glucose, galactose, and xylose in a molar ratio of 12.5:11.15:17.89:6.57:1.5; the structure of the apricot polysaccharide is shown below: .

[0006] The aforementioned apricot polysaccharide has a weight-average molecular weight of 16,500 to 16,900 Da. Preferably, the weight-average molecular weight of the apricot polysaccharide is 16,700 Da.

[0007] The preparation method of the aforementioned apricot polysaccharide includes the following steps: (1) Weigh out loquat leaves, bitter almonds, fritillaria cirrhosa, ophiopogon japonicus, rehmannia glutinosa, licorice, platycodon grandiflorus, mint, and mulberry leaves; (2) Soak bitter almonds in water, mix with mint, heat and distill, and collect the distillate; (3) Combine the remaining seven medicinal materials with the dregs from step (2), add water to decoct and extract, filter the decoction, let it stand, and take the supernatant to concentrate; (4) Combine the distillate obtained in step (2) with the concentrate obtained in step (3), remove the protein by repeated freeze-thaw cycles, and add hydrogen peroxide for decolorization; (5) The decolorized solution was precipitated with ethanol, the precipitate was collected by centrifugation, dialyzed with a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and freeze-dried to obtain crude polysaccharide; (6) The crude polysaccharide was purified by cellulose column, eluted with water, and the main neutral components were collected. The polysaccharide was then dialyzed and freeze-dried to obtain apricot polysaccharide.

[0008] Preferably, dialysis uses a 3500Da dialysis bag.

[0009] The preparation method of the apricot polysaccharide is as follows: Step (1) Weigh 200-300 parts of loquat leaves, 30-50 parts of bitter apricot kernels, 3-5 parts of fritillaria cirrhosa, 30-50 parts of ophiopogon japonicus, 30-50 parts of rehmannia glutinosa, 30-50 parts of licorice, 15-30 parts of platycodon grandiflorus, 5-15 parts of peppermint, and 5-15 parts of mulberry leaves according to the weight.

[0010] The preparation method of the apricot polysaccharide is as follows: step (2) soak bitter apricot kernels in warm water for 10-30 h.

[0011] The preparation method of the apricot polysaccharide, the decolorization conditions in step (4) are as follows: the polysaccharide is added to hydrogen peroxide and decolorized in a water bath at 40~60℃ for 3~12 h.

[0012] The preparation method of the apricot polysaccharide, the conditions for ethanol precipitation in step (5) are: add ethanol to a final ethanol concentration of 60-90%, and let stand overnight at 1-10℃.

[0013] The preparation method of the apricot polysaccharide, in step (6), the crude polysaccharide is purified by DEAE-52 cellulose column; the conditions for purification by DEAE-52 cellulose column are: sample concentration 5~15 mg / mL, elution flow rate 0.5~1.5 mL / min, and phenol-sulfuric acid method is used for tracking and detection.

[0014] The application of the apricot polysaccharide in the preparation of drugs for treating mycoplasma pneumonia.

[0015] The application described herein includes oral formulations.

[0016] The glycosidic bonds of the apricot polysaccharide include 1,4-linked β-galactose, 1,3,6-linked β-glucose, and 1,2,4-linked α-rhamnose, and have one or more of β-1,3,6-glucose side chains and α-1,2,4-rhamnose and α-1,5-arabinose side chains.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The apricot polysaccharide provided by this invention has a well-defined structure and high purity (≥95%), with a weight-average molecular weight of 16700 Da. It is composed of monosaccharides rhamnose, arabinose, glucose, galactose, and xylose in a molar ratio of 12.5:11.15:17.89:6.57:1.5, exhibiting diverse glycosidic bond linkages, making it a novel and homogeneous polysaccharide. Experiments have shown that in a mouse model of Mycoplasma pneumoniae infection, apricot polysaccharide effectively alleviates weight loss caused by Mycoplasma pneumoniae infection (P<0.05), with a more significant effect in the high-dose group (P<0.01); it significantly reduces inflammatory cell infiltration and alveolar structural damage in lung tissue, lowering lung pathological scores; and it effectively reduces the mycoplasma load in lung tissue, with the high-dose group showing the most significant antibacterial effect. Therefore, apricot polysaccharide can serve as an effective candidate ingredient for developing novel anti-mycoplasma pneumonia drugs, especially providing a new treatment option for patients with mycoplasma pneumoniae resistant to macrolide antibiotics. Attached Figure Description

[0018] Figure 1 Diagram showing the separation and collection of Prunus armeniaca polysaccharides;

[0019] Figure 2 The chromatogram of HPGPC for Prunus armeniaca polysaccharide is shown.

[0020] Figure 3 The diagram shows the results of monosaccharide composition analysis of Prunus armeniaca polysaccharide; the upper diagram is the pre-column derivatization high-performance liquid chromatogram of the Prunus armeniaca polysaccharide; the lower diagram is the pre-column derivatization high-performance liquid chromatogram of the monosaccharide standard.

[0021] Figure 4 This is a schematic diagram of the proposed structure of apricot polysaccharide. Detailed Implementation

[0022] Example 1: Identification of the Anti-inflammatory Effective Components of Chuanxing Qingfei Syrup (1) Separation of polysaccharide and volatile fractions of Sichuan apricot kernel for clearing lung heat Weigh out 250 g of loquat leaves, 50 g of bitter almonds, 5 g of fritillaria cirrhosa, 50 g of ophiopogon japonicus, 50 g of rehmannia glutinosa, 50 g of licorice root, 30 g of platycodon grandiflorus, 15 g of peppermint, and 15 g of mulberry leaves. Soak bitter almonds in warm water for 24 hours, mix with peppermint, heat and distill, collect 100 mL of distillate and store separately; combine the remaining seven ingredients (loquat leaves, etc.) with the above two dregs, add water and decoct twice, 2 hours each time, combine the decoctions, filter, let the filtrate stand, and concentrate the supernatant to an appropriate volume. Combine the above distillate and concentrate, mix well, and obtain the concentrated extract of Sichuan apricot kernel for clearing lung heat.

[0023] The aqueous extract concentrate was added to ethanol to a final concentration of 75%–85%, allowed to stand overnight at low temperature, and then centrifuged. The resulting precipitate was reconstituted, dialyzed, and freeze-dried to obtain the polysaccharide-enriched fraction. The ethanol in the resulting alcohol precipitate supernatant was recovered and concentrated, then dissolved again in anhydrous ethanol to obtain the depolysaccharide-free fraction. The aforementioned distillate was then appropriately concentrated to obtain the volatile fraction. To ensure subsequent pharmaceutical validation, the final dissolved volumes of the polysaccharide fraction, the depolysaccharide-free fraction, and the volatile fraction were kept consistent with the volume of the Sichuan Apricot Kernel Lung-Clearing Aqueous Extract Concentrate.

[0024] (2) Identification of the anti-inflammatory activity of polysaccharide and volatile fractions of Chuanxing Qingfei Syrup SPF-grade BALB / c female mice (6-8 weeks old, weighing 18-22 g) were used. Mice were randomly divided into 6 groups (n=5 per group): blank control group, infection group, and groups receiving concentrated aqueous extract of *Apricot Kernel and Apricot Blossom Lung-Clearing Dehydrated Extract* (5 ml / kg / d), polysaccharide fraction of *Apricot Kernel and Apricot Blossom Lung-Clearing Dehydrated Extract* (5 ml / kg / d), and volatile fraction of *Apricot Kernel and Apricot Blossom Lung-Clearing Dehydrated Extract* (5 ml / kg / d). *Mycoplasma pneumoniae* strain MP129 (10 μL / kg) was inoculated intranasally. 8 A mycoplasma pneumonia infection model was established (CCU / animal). Each treatment group was administered medication by gavage 2 days before modeling, once daily for 7 consecutive days, after which lung tissue was collected. Lung tissue was embedded in paraffin, sectioned, stained with H&E, and observed under a light microscope. Pathological scoring was performed based on indicators such as inflammatory cell infiltration and edema in the lung tissue. The results (Table 1) showed that the pneumonia model group had significantly higher scores. The concentrated extract of Chuanxing Qingfei syrup significantly reduced inflammatory cell infiltration and edema in the lung tissue. The polysaccharide-rich portion group also significantly reduced inflammatory cell infiltration and alveolar structure destruction in the lung tissue. The depolysaccharide-free portion group also inhibited inflammatory cell infiltration in the lung tissue to some extent. However, the volatile portion group had no significant effect. This indicates that the polysaccharide-rich portion is one of the important active components in Chuanxing Qingfei syrup for anti-pneumonia.

[0025]

[0026] Example 2: Extraction, separation and purification of apricot polysaccharides Weigh out 250 g of loquat leaves, 50 g of bitter almonds, 5 g of fritillaria cirrhosa, 50 g of ophiopogon japonicus, 50 g of rehmannia glutinosa, 50 g of licorice root, 30 g of platycodon grandiflorus, 15 g of peppermint, and 15 g of mulberry leaves. Soak bitter almonds in warm water for 24 hours, mix with peppermint, heat and distill, collect 100 mL of distillate, and store separately. Combine the remaining seven ingredients (loquat leaves, etc.) with the above two dregs, add water and decoct twice, 2 hours each time, combine the decoctions, filter, let the filtrate stand, and concentrate the supernatant to an appropriate volume. Combine the above distillate and concentrate, mix well, and add purified water to 1000 mL.

[0027] The concentrated aqueous extract was added to ethanol to a final concentration of 75%–85%, allowed to stand overnight at low temperature, and then centrifuged. The resulting precipitate was reconstituted, dialyzed, and freeze-dried to obtain the polysaccharide-rich fraction. For further decolorization and deproteinization, the crude polysaccharide was dissolved in water to adjust the final polysaccharide concentration to 25 mg / mL. After repeated freeze-thaw centrifugation to remove the precipitate, 10% (v / v) H2O2 was added, and the solution was decolorized for 8 h in a 50°C water bath. The decolorized solution was then precipitated again with ethanol (anhydrous ethanol was added to a final concentration of 80%, and the solution was allowed to stand overnight at 4°C). The precipitate was collected by centrifugation, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, dialyzed in running water for 48 h, and then freeze-dried to obtain decolorized and deproteinized apricot polysaccharide.

[0028] The obtained polysaccharides were dissolved in ultrapure water (10 mg / mL) and loaded onto a DEAE-52 cellulose column. Elution was performed with distilled water at a flow rate of 1.0 mL / min, collecting 5 mL from each tube. The sugar content in each tube was determined using the phenol-sulfuric acid method: 1.0 mL of eluent was taken, 1.0 mL of 6% phenol was added, followed by the rapid addition of 5.0 mL of concentrated sulfuric acid. The mixture was mixed, allowed to stand for 30 min, and the absorbance was measured at 490 nm. The absorbance of each collected tube was used to determine the sugar content. Figure 1 The separation and collection diagram is shown. The main neutral components were collected, the eluents were combined, concentrated under reduced pressure, and placed in a dialysis bag with a molecular weight cutoff of 3500 Da. The mixture was dialyzed in running water for 48 h and then freeze-dried to obtain purified Prunus armeniaca polysaccharide (purity ≥95%).

[0029] Example 3 Structural characterization of Prunus armeniaca polysaccharides (1) Determination of purity and molecular weight of apricot polysaccharide A 5 mg / mL solution of Prunus armeniaca polysaccharide was accurately prepared. High-performance gel permeation chromatography (HPGPC) was used as a standard to determine the purity and molecular weight of the polysaccharides, using different molecular weight dextran (Mw: 2700 Da, 5250 Da, 9750 Da, 13050 Da, 36800 Da, 64650 Da). Chromatographic conditions: TSKgel G3000PWxl column (7.8 mm × 300 mm), mobile phase: pure water, flow rate: 0.5 mL / min, column temperature: 40℃, injection volume: 10 μL, detection: refractive index detector (RID). Results showed that Prunus armeniaca polysaccharide exhibited a single symmetrical peak on the HPGPC chromatogram. Figure 2 The result indicates that it has high purity; according to the standard curve, the weight-average molecular weight of Prunus armeniaca polysaccharide is 16700 Da.

[0030] (2) Monosaccharide composition analysis of Prunus armeniaca polysaccharide Accurately weigh 2 mg of apricot polysaccharide and analyze it using equimolarly prepared monosaccharide standards (mannose, rhamnose, glucosamine, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose) on a CarboPac PA-20 anion exchange column (3 mm × 150 mm, Dionex) with a pulsed amperometric detector (PAD; Dionex ICS 5000). + The system was used for detection. The flow rate was 0.5 mL / min; the injection volume was 5 μL; solvent system A: (ddH2O), solvent system B: (0.1 mol / L NaOH), solvent system C: (0.1 mol / L NaOH, 0.2 M NaAc); the gradient program was as follows: the volume ratio of solutions A, B, and C was 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. The results showed that the polysaccharide of Prunus armeniaca mainly contained rhamnose, arabinose, glucose, galactose, and xylose. Figure 3 The molar ratio is 12.5:11.15:17.89:6.57:1.5.

[0031] (3) Glycosidic bond linkage analysis of Prunus armeniaca polysaccharide Methylation analysis of Prunus armeniaca polysaccharides was performed using a modified Hakomori method. 5 mg of dried polysaccharide sample was dissolved in 0.5 mL of anhydrous DMSO, and 5 mg of NaOH powder was added. The mixture was stirred overnight at room temperature. 0.1 mL of iodomethane was added, and the reaction was allowed to proceed for 2 h. The reaction was terminated by adding 1 mL of water, followed by extraction with dichloromethane, washing three times with water, and then evaporating to dryness. The methylated product was subjected to acid hydrolysis (2 mol / L TFA, 110 °C, 4 h), reduction with NaBD4 (room temperature, 2.5 h), and acetylation with acetic anhydride (100 °C, 2.5 h) before analysis by GC-MS. GC conditions: HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), temperature program: initial 140 °C, increased to 230 °C at 3 °C / min, held for 3 min. Results are as follows: Figure 4 The results showed that the polysaccharide from Sichuan apricot contains the following glycosidic bond linkages, including 1,4-linked β-galactose, 1,3,6-linked β-glucose, and 1,2,4-linked α-rhamnose, and has multiple linkages such as β-1,3,6-glucose side chains, α-1,2,4-rhamnose side chains, and α-1,5-arabinose side chains (Table 2). The proposed structural diagram is shown below. Figure 4 As shown.

[0032]

[0033] Example 4: Therapeutic effect of apricot polysaccharide on a mouse model of mycoplasma pneumonia. SPF-grade BALB / c female mice (6-8 weeks old, weighing 18-22 g) were used. Mice were randomly divided into 5 groups (n=10 per group): blank control group, infection group, positive control group (azithromycin, 20 mg / (kg·d)), high-dose apricot kernel polysaccharide group (400 mg / (kg·d)), and low-dose apricot kernel polysaccharide group (200 mg / (kg·d)). Mycoplasma pneumoniae MP129 strain (10 μg / kg·d) was inoculated intranasally. 8 A mycoplasma pneumonia infection model was established using mice from CCU (Central Cubic Mechanism / mouse). Prunus armeniaca polysaccharide was dissolved in physiological saline. Each treatment group was administered the drug via gavage starting 2 days before infection, once daily for 7 consecutive days, with daily mouse weight recorded.

[0034] (1) Effect of Prunus armeniaca polysaccharide on body weight of infected mice The results (Table 3) showed that mice with Mycoplasma pneumoniae infection experienced a significant decrease in body weight, while the treatment group with apricot kernel polysaccharide effectively alleviated the weight loss caused by Mycoplasma pneumoniae infection (P<0.05), with the high-dose group of apricot kernel polysaccharide showing a more significant effect (P<0.01).

[0035]

[0036] (2) Effects of apricot polysaccharide on pathological damage in lung tissue of infected mice Lung tissue was embedded in paraffin, sectioned, and stained with H&E for observation under a light microscope. In the control group, the alveolar structure was clear and intact, with no obvious inflammatory cell infiltration. In the infected group, the alveolar structure was severely damaged, with diffuse infiltration of numerous neutrophils, lymphocytes, and other inflammatory cells, and consolidation in some areas. The degree of inflammatory cell infiltration was significantly reduced in the groups treated with apricot kernel polysaccharide, and the alveolar structure damage was significantly improved, especially in the high-dose apricot kernel polysaccharide group. Pathological scoring was performed based on the degree of inflammatory cell infiltration and alveolar structure damage in the lung tissue. The results (Table 4) showed that the score in the infected group was significantly higher, while the scores in both the high- and low-dose apricot kernel polysaccharide groups were significantly lower than those in the infected group.

[0037]

[0038] (3) Effect of Prunus armeniaca polysaccharide on mycoplasma load in lung tissue of infected mice Approximately 500 mg of lung tissue was placed in a sterile homogenization tube, and 1 mL of mycoplasma culture medium was added. The homogenate was thoroughly homogenized on ice to prepare a tissue homogenate. The homogenate was serially diluted with mycoplasma culture medium. 20 μL of each dilution was evenly spread on the surface of mycoplasma-specific SP-4 solid culture medium. The plates were incubated upside down at 37°C and 5% CO2 for 7–14 days. After incubation, the Mycoplasma pneumoniae colonies on the plates were counted. Plates with colony counts between 30 and 300 were selected for calculation, and the final result was expressed as colony forming units (CFU / mL) per milliliter of lung homogenate.

[0039] The results (Table 5) showed that the mycoplasma load in the lung tissue of infected mice was significantly higher than that in the control group. Compared with the infected group, the mycoplasma load in the lung tissue of the azithromycin positive control group and all groups treated with apricot kernel polysaccharide showed a significant decrease, with the high-dose apricot kernel polysaccharide group showing the most significant antibacterial effect.

[0040]

[0041] The above experimental results indicate that oral administration of apricot kernel polysaccharide can effectively improve the clinical symptoms of mice infected with mycoplasma pneumonia, significantly reduce lung tissue pathological damage, and decrease the pathogen load in the lungs. This demonstrates that apricot kernel polysaccharide has a clear therapeutic effect on mycoplasma pneumonia.

Claims

1. A type of apricot polysaccharide, characterized in that, The apricot polysaccharide is a homogeneous polysaccharide with a monosaccharide composition of rhamnose, arabinose, glucose, galactose, and xylose in a molar ratio of 12.5:11.15:17.89:6.57:1.

5. The structure of the apricot polysaccharide is shown below: 。 2. The apricot polysaccharide according to claim 1, characterized in that, The weight-average molecular weight of the apricot polysaccharide is 16,500 to 16,900 Da.

3. A method for preparing the apricot polysaccharide according to claim 1, characterized in that, Includes the following steps: (1) Weigh out loquat leaves, bitter almonds, fritillaria cirrhosa, ophiopogon japonicus, rehmannia glutinosa, licorice, platycodon grandiflorus, mint, and mulberry leaves; (2) Soak bitter almonds in water, mix with mint, heat and distill, and collect the distillate; (3) Combine the remaining seven medicinal materials with the dregs obtained in step (2), add water to decoct and extract, filter the decoction, let it stand, and take the supernatant to concentrate; (4) Combine the distillate obtained in step (2) and the concentrate obtained in step (3), remove the protein by repeated freeze-thaw cycles, and add hydrogen peroxide to the combined liquid or crude polysaccharide solution for decolorization; (5) The decolorized solution was precipitated with ethanol, the precipitate was collected by centrifugation, dialyzed with a dialysis bag with a molecular weight cutoff of 3000~4000 Da, and freeze-dried to obtain crude polysaccharide; (6) The crude polysaccharide was purified by cellulose column, eluted with water, and the main neutral components were collected. The polysaccharide was then dialyzed and freeze-dried to obtain apricot polysaccharide.

4. The method for preparing apricot polysaccharide according to claim 3, characterized in that, Step (1) Weigh out 200-300 parts of loquat leaves, 30-50 parts of bitter almonds, 3-5 parts of fritillaria cirrhosa, 30-50 parts of ophiopogon japonicus, 30-50 parts of rehmannia glutinosa, 30-50 parts of licorice, 15-30 parts of platycodon grandiflorus, 5-15 parts of mint, and 5-15 parts of mulberry leaves according to the weight.

5. The method for preparing apricot polysaccharide according to claim 3, characterized in that, Step (2) Soak bitter almonds in warm water for 10-30 hours.

6. The method for preparing apricot polysaccharide according to claim 3, characterized in that, The decolorization conditions described in step (4) are as follows: the polysaccharide is added to hydrogen peroxide and decolorized in a water bath at 40~60℃ for 3~12 h.

7. The method for preparing apricot polysaccharide according to claim 3, characterized in that, The conditions for ethanol precipitation in step (5) are: add ethanol to a final concentration of 60-90%, and let stand overnight at 1-10°C.

8. The method for preparing apricot polysaccharide according to claim 3, characterized in that, Step (6) The crude polysaccharide was purified by DEAE-52 cellulose column; the conditions for purification by DEAE-52 cellulose column were: sample concentration of 5~15 mg / mL, elution flow rate of 0.5~1.5 mL / min, and phenol-sulfuric acid method for monitoring and detection.

9. The use of the apricot polysaccharide according to claim 1 in the preparation of a medicament for treating mycoplasma pneumonia.

10. The application according to claim 9, characterized in that, The drug includes oral formulations.

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