Novel polygonatum kingianum polysaccharide with antibacterial effect as well as preparation method and application of novel polygonatum kingianum polysaccharide

Abstract

The invention discloses novel polygonatum kingianum polysaccharide with an antibacterial effect and a preparation method and application thereof, and belongs to the field of biomedical engineering materials. The invention provides a novel polygonatum kingianum 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 polygonatum kingianum polysaccharide comprises the steps of ethanol impurity removal, distilled water extraction, concentration, ethanol precipitation and the like, the preparation method is simple, convenient and efficient, and the purity of the obtained polygonatum kingianum polysaccharide is as high as 99.4%. Bacteriostatic experiment results show that the novel polygonatum kingianum polysaccharide provided by the invention has remarkable antibacterial activity on staphylococcus aureus and can be used for treating staphylococcus aureus infection, and the polygonatum kingianum polysaccharide provided by the invention is a natural source, has lower side effects and higher safety compared with synthetic drugs, and is suitable for industrial production. The method is suitable for long-term use and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering materials, and particularly to a novel polygonatum kingianum polysaccharide with antibacterial efficacy, its preparation method and application. Background Art

[0002] Bacterial infection is an urgent problem in medical treatment. Especially, the abuse of antibiotics and the emergence of multi-drug resistant bacteria have made this problem more complex and severe. Although more and more advanced antibiotics are under research and development, the long-term use of these drugs may cause adverse reactions such as hepatotoxicity, which limits their scope of application.

[0003] Polysaccharides are a class of linear or branched carbohydrates linked by glycosidic bonds, formed by the condensation reaction of various monosaccharides. Chinese herbal polysaccharides are well-known for their diverse biological activities, including anti-inflammatory, immunomodulatory and antioxidant effects. Due to their unique structural features, such as the presence of multiple monosaccharides, complex linkage patterns and the heterogeneity of molecular sizes, the structural analysis of Chinese herbal polysaccharides is extremely challenging. These different structures are often closely related to the biological functions of polysaccharides.

[0004] Polygonatum is an important plant with both medicinal and edible uses, rich in polysaccharides, and is considered an effective adjuvant for improving hepatic dyslipidemia and human microbiota dysbiosis caused by unhealthy diets. In recent years, polygonatum polysaccharide, as the main active ingredient of polygonatum, has attracted much attention due to its significant pharmacological activities. Research shows that polysaccharides from polygonatum plants have various biological activities such as anti-inflammatory, immunomodulatory, anti-osteoporotic and anti-diabetic effects, and these effects may be closely related to their unique structural characteristics. However, the research on the antibacterial potential of polygonatum polysaccharides is still very limited, and it is worthy of further exploring its application prospects in this field. Summary of the Invention

[0005] The object of the present invention is to provide a novel polygonatum kingianum polysaccharide with antibacterial efficacy, its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The polysaccharide provided by the present invention is prepared by repeatedly extracting the roots of polygonatum with ethanol and distilled water, followed by precipitation, centrifugation, dissolution and freeze-drying treatment, and its purity is as high as 99.4%. The preparation method has a simple process, and the obtained polygonatum kingianum 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 kingianum polysaccharide with antibacterial efficacy. The average molecular weight of the Polygonatum kingianum 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.

[0008] The present invention provides a preparation method of the above-mentioned Polygonatum kingianum polysaccharide, comprising the following steps:

[0009] Mix the Polygonatum kingianum root material with ethanol at a volume fraction of 60%, heat and extract at 60 °C for 0.5 h, repeat 3 times, filter, and collect the filter residue;

[0010] Mix the filter residue with distilled water, heat and extract at 100 °C for 2 h, repeat 3 times, collect the extract, centrifuge, take the supernatant, concentrate, and collect the concentrated solution;

[0011] Mix the concentrated solution with absolute ethanol, leave overnight, centrifuge to collect the precipitate, redissolve the precipitate in water, and freeze-dry to obtain the Polygonatum kingianum polysaccharide.

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

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

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

[0015] Preferably, the volume ratio of the concentrated solution to the absolute ethanol is 1:4.

[0016] The present invention also provides an application of the above-mentioned Polygonatum kingianum polysaccharide in the preparation of 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, and the antibacterial agent uses the above-mentioned Polygonatum kingianum polysaccharide as the only active ingredient; the concentration of the Polygonatum kingianum polysaccharide is not less than 200 mg / mL.

[0018] The present invention also provides an application of the above-mentioned Polygonatum kingianum polysaccharide in the preparation of a drug 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, and the drug uses the above-mentioned Polygonatum kingianum polysaccharide as the only active ingredient; the concentration of the Polygonatum kingianum polysaccharide is not less than 200 mg / mL.

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

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

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

[0023] (3) Natural source and high safety: As a traditional plant with both medicinal and edible uses, polygonatum kingianum has a natural source for its polysaccharide component. 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 has a simple process and is easy to operate. It not only reduces production costs but also has good potential for industrial production.

[0025] (5) Wide application prospects: In addition to the treatment of Staphylococcus aureus infections, polygonatum kingianum polysaccharide may also have other potential antibacterial effects and has wide application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

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

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

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

[0030] Figure 4 It is the monosaccharide composition chromatogram of the PMP derivatization analysis of polygonatum kingianum polysaccharide in Example 3;

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

[0032] Figure 6 It is the monosaccharide composition chromatogram of the PMP non-derivatization analysis of polygonatum kingianum polysaccharide in Example 3;

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

[0034] Figure 8 In vitro antibacterial effect diagrams of PKP against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in Example 5;

[0035] Figure 9 Statistical chart of the in vitro bacteriostatic rate effect of PKP against Staphylococcus aureus and Escherichia coli in Example 5; where *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively. Detailed implementation manners

[0036] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0038] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0039] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0040] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0041] The present invention purified and obtained a novel Polygonatum kingianum polysaccharide from Polygonatum kingianum. Through techniques such as monosaccharide composition analysis, high performance gel permeation chromatography (HPGPC), high performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy, the detailed structure of this polysaccharide was analyzed. The antibacterial activity was further confirmed by in vitro bacterial counting experiments using Staphylococcus aureus. These experimental results provide a new perspective for the development and application of Polygonatum kingianum polysaccharide and demonstrate its application prospect as a potential antibacterial drug.

[0042] The formula of the TSB liquid medium / TSB solution used in the present invention is: 17.0 g of tryptone, 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 made up to 1 L with deionized water.

[0043] Example 1

[0044] 200 g of root raw materials of Polygonatum kingianum were obtained and extracted 3 times with 2.0 L of ethanol with a volume fraction of 60% at 60 °C for 0.5 h each time to remove terpenoids and flavonoids. The solid residue was extracted 3 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 substances, concentrated to 600 mL, and 2400 mL of absolute ethanol was added to precipitate overnight. After centrifugation, the precipitate was dissolved in water and freeze-dried to obtain 35.92 g of the precipitate, and thus Polygonatum kingianum polysaccharide (PKP) was successfully prepared.

[0045] Example 2

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

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

[0048] Among them, Mw: weight-average molecular weight; Kav: effective distribution coefficient.

[0049] Kav=(V e -V 0 ) / (V t -V 0 ); where V e =Flow×retention time, V t =Flow×inner water volume RT, V 0 =Flow×outer water volume RT, where Flow is 0.6 mL / min, inner water volume RT is 16.03 min, and outer 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 a high correlation, R 2 = 0.997 (as Figure 2 shown). According to the standard dextran reference, the average molecular weight of the main polysaccharide is 3.0 kDa.

[0052] Example 3

[0053] The monosaccharide composition of PKP was determined by two high-performance liquid chromatography (HPLC) methods. Hydrophilic interaction chromatography-charged aerosol detector (HILIC-CAD) method was used to quantify fructose (Fru). 1-Phenyl-3-methyl-5-pyrazolone derivatization HPLC (PMP-HPLC) method was used to determine other monosaccharides (including glucose [Glc], galactose [Gal], arabinose [Ara], mannose [Man], rhamnose [Rha], glucuronic acid [GlcA] and galacturonic acid [GalA]).

[0054] The PMP derivatization analysis chromatogram is as Figures 3 - 4 shown. By PMP pre-column derivatization method, a significant peak and three smaller peaks were identified as glucose (Glc), mannose (Man), galacturonic acid (GalA) and galactose (Gal), respectively.

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

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

[0057] Example 4

[0058] Take 10 mg of the polysaccharide sample and dissolve it in 0.5 mL of D 2 O (99.8%), and stir overnight.

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

[0060] According to 1 1H- 13 13C HSQC and HMBC spectra, chemical shifts were assigned to single residues. The spectra of PKP showed typical fructan peaks. For Glcp residues, in the HSQC spectrum, the 1 1H and 13The C chemical shift is clearly visible, while the HC2 signal is weak but can be detected at δ 69.1 / 3.38 ppm. The chemical shift of HC1 is δ 92.0 / 5.33 ppm, indicating the presence of α-Glcp. For fructose, the two key split signals (δ 81.0 / 3.78 ppm and δ 79.9 / 3.85 ppm) in the HSQC spectrum are attributed to HC5 of β-2,1-Fruf and β-2,1-Fruf respectively. The split signals (δ 74.1 / 4.02 ppm and δ 75.2 / 4.01 ppm) are attributed to HC4 of β-2,1-Fruf and β-2,1-Fruf. However, the signals of HC1,6 and HC2 (δ 102.2 - 104.3 ppm) overlap severely in the HMPC spectrum and cannot be accurately distinguished. According to the integration of the independent HC5 signal, the ratio of β-2,1-Fruf to β-2,6-Fruf is approximately 2.5:1. Combining with the monosaccharide composition analysis, the structure of PKP is speculated as Figure 7 as shown.

[0061] Table 1 1 H and 13 C nuclear magnetic resonance results

[0062]

[0063] Example 5

[0064] Pick single colonies from the culture dishes of Escherichia coli and Staphylococcus aureus and inoculate them into 10 mL of TSB liquid medium, and culture overnight at 37 °C. According to the experimental requirements, dilute the overnight culture broth 1000 times to a bacterial concentration of 1×10 6 CFU / mL.

[0065] Prepare the solution: Dissolve the polygonatum kingianum polysaccharide prepared in Example 1 in TSB solution to prepare polygonatum kingianum polysaccharide solutions with final concentrations of 100, 200, 300, 400, 500, and 600 mg / mL. Mixed culture: Take 200 μL of the sample solution and place it in a 24-well plate, and inoculate 200 μL of the bacterial suspension in the same logarithmic growth phase. The positive control is supplemented with 200 μL of TSB solution in 200 μL of the bacterial suspension in the same logarithmic growth phase and pipetted and mixed evenly. The negative control group drops 400 μL of TSB solution. Co-culture at 37 °C for 24 h, dilute the co-cultured suspension 4 times, and take 100 μL respectively for plating on solid medium. Incubate the plates in a constant temperature incubator at 37 °C for 12 h.

[0066] Finally, count the number of single colonies on each plate and calculate the antibacterial rate. The antibacterial results are as Figures 8 - 9As shown in Table 2 - Table 3: The bactericidal rates of Polygonatum kingianum polysaccharide at 200, 300, and 400 mg / mL against Staphylococcus aureus were 73.2%, 82.6%, and 89.8% respectively; the bactericidal rates of Polygonatum kingianum polysaccharide at 200, 300, 400, 500, and 600 mg / mL against Escherichia coli were 27.6%, 47.7%, 54.7%, 62.5%, and 72.1% respectively. According to the result data, Polygonatum kingianum polysaccharide has certain antibacterial effects on both Staphylococcus aureus and Escherichia coli, and the antibacterial effect of Polygonatum kingianum polysaccharide at the same concentration against Staphylococcus aureus is better than that against Escherichia coli.

[0067] Table 2 Experimental statistics of the antibacterial rate of Escherichia coli

[0068]

[0069] Table 3 Experimental statistics of the antibacterial rate of Staphylococcus aureus

[0070]

[0071] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Polygonatum odoratum polysaccharide with antibacterial efficacy, characterized in that: The average molecular weight of the polygonatum cyrtonema polysaccharide is 3.0 kDa, and it 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.

2. A method for preparing Polygonatum cyrtonema polysaccharide according to claim 1, characterized in that: The following steps are involved: The root material of Polygonatum cyrtonema was mixed with 60% ethanol by volume, and heated to 60℃ for extraction for 0.5 h, repeated 3 times, filtered, and the residue was collected; 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 taken, and the concentrate was collected. The concentrated solution is mixed with anhydrous ethanol, left overnight, and the precipitate is collected by centrifugation. The precipitate is redissolved in water, and freeze-dried to obtain the Polygonatum cyrtonema polysaccharide.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the polygonatum cyrtonema root material to the 60% volume fraction of ethanol is 1g:10mL.

4. The preparation method according to claim 2, characterized in that: The mass volume ratio of the polygonatum cyrtonema root material to the distilled water is 1g:10mL.

5. The preparation method according to claim 2, characterized in that: The concentration is to concentrate the extract until the volume ratio of the concentrate to the distilled water is 3:

10.

6. The preparation method according to claim 2, characterized in that: The volume ratio of the concentrated solution to the anhydrous ethanol is 1:

4.

7. Use of the Polygonatum cyrtonema polysaccharide as claimed in claim 1 in the preparation of an antibacterial agent for inhibiting Staphylococcus aureus and / or Escherichia coli.

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

9. Use of the polygonatum cyrtonema polysaccharide as claimed in claim 1 in the preparation of a medicament for treating Staphylococcus aureus and / or Escherichia coli infection.

10. 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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