A novel polysaccharide with antibacterial effect and its preparation method and application

Abstract

The present invention discloses a novel Polygonatum odoratum polysaccharide with antibacterial efficacy, a preparation method thereof, and an application thereof, and belongs to the field of biomedical engineering materials. The present invention provides a novel Polygonatum odoratum polysaccharide, which is mainly composed of fructose, glucose, and mannose, and has an average molecular weight of 3.0 kDa. The preparation method of the above-mentioned Polygonatum odoratum polysaccharide comprises the steps of ethanol impurity removal, distilled water extraction, concentration, and ethanol precipitation. The preparation method is simple and efficient, and the purity of the obtained Polygonatum odoratum polysaccharide is as high as 99.4%. The results of antibacterial experiments show that the novel Polygonatum odoratum polysaccharide provided by the present invention has significant antibacterial activity against Staphylococcus aureus, and can be used for the treatment of Staphylococcus aureus infection. Moreover, the Polygonatum odoratum polysaccharide provided by the present invention is of natural origin, has lower side effects and higher safety than synthetic drugs, is suitable for long-term use, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering materials, and in particular to a novel polygonatum sibiricum polysaccharide with antibacterial efficacy, a preparation method thereof and an application thereof. Background Art

[0002] Bacterial infections are a pressing medical issue, compounded by the overuse of antibiotics and the emergence of multidrug-resistant bacteria. While more advanced antibiotics are being developed, long-term use of these drugs can lead to adverse reactions such as hepatotoxicity, limiting their application.

[0003] Polysaccharides are a class of linear or branched carbohydrates linked by glycosidic bonds, formed by the condensation reaction of multiple monosaccharides. Traditional Chinese medicine (TCM) polysaccharides are known for their diverse biological activities, including anti-inflammatory, immunomodulatory, and antioxidant effects. Structural elucidation of TCM polysaccharides is challenging due to their unique structural characteristics, such as the presence of multiple monosaccharides, complex linkages, and heterogeneous molecular sizes. These diverse structures are often closely related to the polysaccharide's biological function.

[0004] Polygonatum sibiricum is an important plant with both medicinal and edible properties. Rich in polysaccharides, it is considered an effective adjunct to improving hepatic dyslipidemia and human microbial imbalance caused by an unhealthy diet. In recent years, Polygonatum sibiricum polysaccharides, the main active ingredient in Polygonatum sibiricum, have attracted considerable attention due to their remarkable pharmacological activities. Studies have shown that polysaccharides from Polygonatum sibiricum exhibit a variety of biological activities, including anti-inflammatory, immunomodulatory, anti-osteoporosis, and anti-diabetic activities. These effects may be closely related to their unique structural properties. However, research on the antimicrobial potential of Polygonatum sibiricum polysaccharides remains limited, and further exploration of their potential applications in this field is warranted. Summary of the Invention

[0005] The present invention aims to provide a novel Yunnan Polygonatum sibiricum polysaccharide with antibacterial efficacy, its preparation method, and application, to address the problems of the prior art. The polysaccharide provided by the present invention is prepared by repeatedly extracting the root of Polygonatum sibiricum with ethanol and distilled water, followed by precipitation, centrifugation, dissolution, and freeze-drying, with a purity of up to 99.4%. The preparation method is simple, and the obtained Yunnan Polygonatum sibiricum polysaccharide has a significant inhibitory effect on Staphylococcus aureus, and can be used to treat infections caused by Staphylococcus aureus, showing good application potential.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a polygonatum yunnanensis polysaccharide with antibacterial efficacy. The polygonatum yunnanensis polysaccharide has an average molecular weight of 3.0 kDa and is composed of 91.3% fructose, 6.9% glucose, 0.9% mannose, and a total amount of 0.9% galacturonic acid, galactose, xylose, and arabinose.

[0008] The present invention provides a method for preparing the polygonatum dahliae polysaccharide, comprising the following steps:

[0009] The root material of Polygonatum dahliae was mixed with 60% ethanol by volume, and heated at 60°C for 0.5 h, repeated three times, and filtered to collect the residue.

[0010] The filter residue was mixed with distilled water, and the mixture was heated at 100°C for 2 h, and the extraction was repeated 3 times. The extract was collected, centrifuged, the supernatant was collected, and the concentrate was collected;

[0011] The concentrated solution is mixed with anhydrous ethanol, and the mixture is centrifuged overnight to collect the precipitate. The precipitate is redissolved in water and freeze-dried to obtain the Polygonatum yunnanensis polysaccharide.

[0012] Preferably, the mass volume ratio of the Polygonatum odoratum root material to the 60% volume fraction of ethanol is 1g:10mL.

[0013] Preferably, the mass volume ratio of the Polygonatum odoratum root material to the distilled water is 1g:10mL.

[0014] Preferably, the concentration is to concentrate the extract until the volume ratio of the concentrate to the distilled water is 3:10.

[0015] Preferably, the volume ratio of the concentrate to the anhydrous ethanol is 1:4.

[0016] The present invention also provides an application of the polygonatum dahliae polysaccharide in preparing an antibacterial agent for inhibiting Staphylococcus aureus and / or Escherichia coli.

[0017] The present invention also provides an antibacterial agent for inhibiting Staphylococcus aureus and / or Escherichia coli, wherein the antibacterial agent contains the polygonatum sibiricum polysaccharide as the only effective ingredient; and the concentration of the polygonatum sibiricum polysaccharide is not less than 200 mg / mL.

[0018] The present invention also provides a use of the polygonatum yunnanensis polysaccharide in preparing a medicine for treating Staphylococcus aureus and / or Escherichia coli infection.

[0019] The present invention also provides a drug for treating Staphylococcus aureus and / or Escherichia coli infection, wherein the drug contains the polygonatum sibiricum polysaccharide as the only active ingredient; the concentration of the polygonatum sibiricum polysaccharide is not less than 200 mg / mL.

[0020] The present invention discloses the following technical effects:

[0021] (1) Highly effective antibacterial effect: The Polygonatum dahliae polysaccharide provided by the present invention has significant antibacterial activity against Staphylococcus aureus, and can effectively inhibit the growth and reproduction of the bacterium, providing a new natural candidate for antibacterial treatment.

[0022] (2) High-purity preparation: Through a simple extraction and purification process, a polysaccharide with a purity of up to 99.4% was obtained, ensuring the stability and reliability of its biological activity.

[0023] (3) Natural origin and high safety: As a traditional medicinal and edible plant, the polysaccharide components of Polygonatum sibiricum are derived from natural sources. Compared with synthetic drugs, it has lower side effects and higher safety, and is suitable for long-term use.

[0024] (4) Simple preparation process: The preparation method of the present invention is simple and easy to operate, which not only reduces production costs but also has good industrial production potential.

[0025] (5) Broad application prospects: In addition to the treatment of Staphylococcus aureus infection, Polygonatum dahliae polysaccharide may also have other potential antibacterial effects and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 is the high performance gel permeation chromatogram of PKP in Example 2;

[0028] Figure 2 is a regression equation diagram of the molecular weight of PKP and the retention time of dextran in Example 2;

[0029] Figure 3 This is a chromatogram of the monosaccharide composition of the PMP derivatization analysis of the mixed polysaccharide in Example 3;

[0030] Figure 4 This is a chromatogram of the monosaccharide composition of the PMP derivatization analysis of the Polygonatum odoratum polysaccharide in Example 3;

[0031] Figure 5 This is a chromatogram of the monosaccharide composition of the PMP non-derivatized analysis of the mixed polysaccharide in Example 3;

[0032] Figure 6 This is a chromatogram of the monosaccharide composition of the PMP-derivatized analysis of the Polygonatum odoratum polysaccharide in Example 3;

[0033] Figure 7 is the PKP in Example 4 1 H- 13 Overlay of C HSQC and HMBC spectra;

[0034] Figure 8 The figure shows the in vitro antibacterial effect of PKP on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in Example 5;

[0035] Figure 9 The figure shows the in vitro antibacterial effect of PKP on Staphylococcus aureus and Escherichia coli in Example 5; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] This study purified and obtained a novel Polygonatum yunnanensis polysaccharide from Polygonatum yunnanensis. The detailed structure of the polysaccharide was elucidated through monosaccharide composition analysis, high-performance gel permeation chromatography (HPGPC), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Its antibacterial activity was further confirmed using in vitro bacterial counts against Staphylococcus aureus. These experimental results provide new insights into the development and application of Polygonatum yunnanensis polysaccharide and demonstrate its potential as a potential antibacterial agent.

[0042] The formula of the TSB liquid culture medium / TSB solution used in the present invention is: 17.0 g of trypticase, 5.0 g of sodium chloride, 3.0 g of soybean papain digest, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose monohydrate, pH 7.3±0.2, and the volume is adjusted to 1 L with deionized water.

[0043] Example 1

[0044] 200 g of Polygonatum kingianum root material was obtained and extracted three times with 2.0 L of 60% ethanol at 60°C for 0.5 h each time to remove terpenes and flavonoids. The solid residue was extracted three times with 2.0 L of distilled water at 100°C for 2 h each time. The combined aqueous extracts were centrifuged to remove insoluble matter, concentrated to 600 mL, and precipitated overnight with 2400 mL of anhydrous ethanol. After centrifugation, the precipitate was dissolved in water and freeze-dried to obtain 35.92 g of the precipitate, which was successfully prepared as Polygonatum kingianum polysaccharide (PKP).

[0045] Example 2

[0046] The purity and average molecular weight of PKP were determined by high performance gel permeation chromatography (HPGPC) using a TSK PWxl G3000 column (7.8×300 mm) with a mobile phase of 0.1 mol / L NaCl at a flow rate of 0.6 mL / min. The molecular weight was estimated using a calibration curve of standard dextran (molecular weights of 1, 3, 6, 10, 40, and 100 kDa) and NaCl. The calibration curve was obtained by linear regression with the logarithm of the relative molecular mass Mp of the standard (logMp) as the ordinate and the retention time of the corresponding chromatographic peak as the abscissa. The molecular weight distribution of PKP showed a main peak at a retention time of 13.4 min, indicating that it is a high-purity homogeneous polysaccharide (e.g. Figure 1 shown).

[0047] The regression equation is Kav = -0.34×lgMw + 1.758.

[0048] Wherein, Mw is weighted average molecular weight; Kav is effective partition coefficient.

[0049] Kav=(V e -V0) / (V t -V0); where V e =Flow×retention time, V t =Flow×internal water volume RT, V0=Flow×external water volume RT, where Flow is 0.6 mL / min, internal water volume RT is 16.03 min, and external water volume RT is 9.83 min.

[0050] It can be obtained that Kav=(retention time-9.83) / 6.2.

[0051] The regression equation has high correlation, R 2 = 0.997 (e.g. Figure 2 Based on a standard dextran reference, the average molecular weight of the major polysaccharide was 3.0 kDa.

[0052] Example 3

[0053] The monosaccharide composition of PKP was determined using two high-performance liquid chromatography (HPLC) methods. Fructose (Fru) was quantified using hydrophilic interaction chromatography coupled with a charged aerosol detector (HILIC-CAD). Other monosaccharides (including glucose (Glc), galactose (Gal), arabinose (Ara), mannose (Man), rhamnose (Rha), glucuronic acid (GlcA), and galacturonic acid (GalA)) were determined using 1-phenyl-3-methyl-5-pyrazolone derivatization HPLC (PMP-HPLC).

[0054] PMP derivatization analysis chromatographic Figure 3-Figure 4 As shown in Figure 4, one prominent peak and three smaller peaks were identified by PMP pre-column derivatization, which were glucose (Glc), mannose (Man), galacturonic acid (GalA), and galactose (Gal).

[0055] 1 mg of PKP was hydrolyzed with 1 mL of 0.1 mol / L trifluoroacetic acid at 80°C for 30 min. The hydrolyzate was analyzed by HPLC using a Dionex UltiMate™ 3000 system equipped with an ACQUITY UPLC BEH Amide column (2.1 × 150 mm) and a Corona CAD detector. The flow rate was set to 0.1 mL / min, and the mobile phase consisted of acetonitrile and 100 mM ammonium acetate (86:14 by volume). PMP derivatization analysis chromatogram was as follows: Figure 5-Figure 6As shown in Figure 3, according to the integrated area of ​​the chromatographic peaks by HILIC-CAD method, fructose (Fru) accounts for the largest proportion, followed by glucose (Glc).

[0056] Overall, PKP is composed of fructose (91.3%), glucose (6.9%), and mannose (0.9%), with small amounts of galacturonic acid, galactose, xylose, and arabinose.

[0057] Example 4

[0058] 10 mg of polysaccharide sample was dissolved in 0.5 mL of D2O (99.8%) and stirred overnight.

[0059] The NMR spectra were acquired using a Bruker AVANCE NEO Ascend 600 NMR spectrometer (Bruker, Karlsruhe, Germany) at 25 °C equipped with a 5 mm broadband observation probe. 1 H NMR (600 MHz) and 13 C NMR (150 MHz) recording 1 H- 13 C HMBC and HMQC spectra. All data were processed and analyzed using Bruker standard software, and chemical shifts were referenced to acetone. The structural characteristics of PKP were further elucidated by NMR spectroscopy. 1 H and 13 The C NMR results are shown in Table 1.

[0060] according to 1 H- 13 C HSQC and HMBC spectra, chemical shifts were assigned to individual residues. The spectrum of PKP showed typical fructan peaks. For the Glcp residues, the HSQC spectra of HC1, HC3, HC4, HC5, and HC6 were 1 H and 13The C chemical shift is clearly visible, while the HC2 signal is weak but detectable at δ69.1 / 3.38 ppm. The chemical shift of HC1 is δ92.0 / 5.33 ppm, indicating the presence of α-Glcp. For fructose, two key splitting signals in the HSQC spectrum (δ81.0 / 3.78 ppm and δ79.9 / 3.85 ppm) are assigned to HC5 of β-2,1-Fruf and β-2,1-Fruf, respectively. Splitting signals (δ74.1 / 4.02 ppm and δ75.2 / 4.01 ppm) are assigned to HC4 of β-2,1-Fruf and β-2,1-Fruf, respectively. However, the signals of HC1,6 and HC2 (δ102.2-104.3 ppm) overlap significantly in the HMPC spectrum, making accurate distinction impossible. Based on the integration of independent HC5 signals, the ratio of β-2,1-Fruf to β-2,6-Fruf is approximately 2.5:1. Combined with the analysis of monosaccharide composition, the structure of PKP is speculated to be Figure 7 shown.

[0061] Table 1 PKP 1 H and 13 C NMR results

[0062]

[0063] Example 5

[0064] Single colonies were picked from the culture dishes of Escherichia coli and Staphylococcus aureus, inoculated into 10 mL of TSB liquid medium, and cultured overnight at 37°C. According to the experimental requirements, the overnight culture solution was diluted 1000 times to a bacterial concentration of 1×10 6 CFU / mL.

[0065] Solution preparation: Dissolve the Polygonatum yunnanensis polysaccharide prepared in Example 1 in TSB solution to prepare Polygonatum yunnanensis polysaccharide solutions with final concentrations of 100, 200, 300, 400, 500, and 600 mg / mL. Mixed culture: Take 200 μL of sample solution and place it in a 24-well plate, and inoculate 200 μL of bacterial suspension in the same logarithmic growth phase. For the positive control, add 200 μL of TSB solution to 200 μL of bacterial suspension in the same logarithmic growth phase and mix well by pipetting. For the negative control group, add 400 μL of TSB solution. Co-culture at 37°C for 24 h, and dilute the co-cultured suspension by 10 4 100 μL of each sample was plated on solid culture medium and incubated in a 37°C incubator for 12 h.

[0066] Finally, the number of single colonies on each plate was counted and the antibacterial rate was calculated. Figure 8-Figure 9As shown in Tables 2 and 3, the bactericidal rates of 200, 300, and 400 mg / mL of Polygonatum sibiricum polysaccharide against Staphylococcus aureus were 73.2%, 82.6%, and 89.8%, respectively; and the bactericidal rates of 200, 300, 400, 500, and 600 mg / mL of Polygonatum sibiricum polysaccharide against Escherichia coli were 27.6%, 47.7%, 54.7%, 62.5%, and 72.1%, respectively. The results indicate that Polygonatum sibiricum polysaccharide has a certain antibacterial effect against both Staphylococcus aureus and Escherichia coli, and that the antibacterial effect of Polygonatum sibiricum polysaccharide against Staphylococcus aureus at equal concentrations is superior to that against Escherichia coli.

[0067] Table 2 Experimental statistics of Escherichia coli inhibition rate

[0068]

[0069] Table 3 Experimental statistics of Staphylococcus aureus inhibition rate

[0070]

[0071] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A polysaccharide from Polygonatum yunnanensis with antibacterial efficacy, characterized in that: The average molecular weight of the polygonatum yunnanensis polysaccharide is 3.0 kDa, and it is composed of 91.3% fructose, 6.9% glucose, 0.9% mannose, and a total of 0.9% galacturonic acid, galactose, xylose, and arabinose; The preparation method of the polygonatum dahliae polysaccharide comprises the following steps: The root material of Polygonatum dahliae was mixed with 60% ethanol by volume, and heated at 60°C for 0.5 h, repeated three times, and filtered to collect the residue. The filter residue was mixed with distilled water, and the mixture was heated at 100°C for 2 h, and the extraction was repeated 3 times. The extract was collected, centrifuged, the supernatant was collected, and the concentrate was collected; The concentrated solution is mixed with anhydrous ethanol, and the mixture is centrifuged overnight to collect the precipitate, and the precipitate is redissolved in water and freeze-dried to obtain the polygonatum odoratum polysaccharide; The mass volume ratio of the polygonatum root material to the 60% volume fraction ethanol is 1g:10mL; The mass volume ratio of the polygonatum root material to the distilled water is 1g:10mL; The concentration is to concentrate the extract until the volume ratio of the concentrate to the distilled water is 3:10; The volume ratio of the concentrated solution to the anhydrous ethanol is 1:

4.

2. A use of the polygonatum dahliae polysaccharide as claimed in claim 1 in the preparation of an antibacterial agent for inhibiting Staphylococcus aureus and / or Escherichia coli.

3. An antibacterial agent for inhibiting Staphylococcus aureus and / or Escherichia coli, characterized in that: The antibacterial agent contains the polygonatum sibiricum polysaccharide according to claim 1 as the only active ingredient; the concentration of the polygonatum sibiricum polysaccharide is not less than 200 mg / mL.

4. Use of the polygonatum yunnanensis polysaccharide according to claim 1 in the preparation of a medicament for treating Staphylococcus aureus and / or Escherichia coli infection.

5. A drug for treating Staphylococcus aureus and / or Escherichia coli infection, characterized in that: The drug contains the polygonatum sibiricum polysaccharide described in claim 1 as the only active ingredient; the concentration of the polygonatum sibiricum polysaccharide is not less than 200 mg / mL.

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