Use of myrica rubra polysaccharide P6 in preparation of antibacterial and antioxidant medicine

Abstract

The application discloses application of myrtle polysaccharide P6 in preparation of antibacterial and antioxidant medicines, wherein the myrtle polysaccharide P6 contains monosaccharides in the following molar percentage: rhamnose 4.97%, arabinose 11.32%, galactose 4.64%, glucose 4.43% and galacturonic acid 74.63%; the number average molecular weight (Mn) is 36.96 kDa, and the weight average molecular weight (Mw) is 59.15 kDa. The application firstly finds that the myrtle polysaccharide has antibacterial and antioxidant effects, and expands the potential application range of the myrtle polysaccharide.

Description

Technical Field

[0001] The invention belongs to the field of natural products (drugs), and particularly relates to the application of myrtle polysaccharide P6 in the preparation of antibacterial and antioxidant drugs. Background Art

[0002] Polysaccharides belong to the third category of biopolymers (carbohydrates). They are polymeric sugars composed of sugar chains linked by glycosidic bonds, with at least more than 10 monosaccharides. Compared with other synthetic polymers, natural polysaccharides have many advantages, such as safety, stability, hydrophilicity, biocompatibility, bioactivity, easy functionalization, bioadhesion properties and biodegradability, and are used in many fields. In the field of food and beverage, polysaccharides are widely used. For example, plant polysaccharides can be used as natural sweeteners, thickeners, stabilizers and emulsifiers to make various foods and beverages. Polysaccharides are also used in the industrial field. In cosmetics, polysaccharides can be used as moisturizers, thickeners and stabilizers. In the papermaking and textile fields, polysaccharides can be used as adhesives, sizing agents and printing agents. In the petroleum field, polysaccharides can be used as drilling fluid stabilizers, oilfield chemicals, etc. to improve the efficiency and safety of oil extraction. In the field of medicine and health, polysaccharides have multiple functions, such as antioxidant, antibacterial, anti-diabetic, anti-parasitic, anticoagulant, anti-inflammatory, anti-cancer, lipid-lowering, immune regulation, blood sugar lowering, tumor inhibition, anti-aging and anti-fatigue.

[0003] Myrtle (Rhodomyrtus tomentosa (Aiton) Hassk.), a shrub of the Myrtaceae family, has important edible and medicinal uses. Myrtle fruit is a nutritionally comprehensive source of dietary fiber, vitamin E, phenolic compounds, and important elements such as manganese, copper, chromium, and iron.

[0004] Previously, the inventors had isolated four myrtle polysaccharides, P1, P2, P3, and P4, with lipid-lowering effects. These polysaccharides are composed of monosaccharides such as ribose, rhamnose, arabinose, xylose, mannose, glucose, and galactose, but the content of each component varies. Their relative binding rates for sodium taurocholate, sodium glycocholate, and sodium cholate range from 30% to 50%, demonstrating a significant lipid-lowering effect.

[0005] In conclusion, the current research on the pharmacology of myrtle is still in its preliminary stage, and myrtle still has broad development prospects in the pharmaceutical field. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of myrtle polysaccharide P6 in the preparation of antibacterial and antioxidant drugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] Application of myrtle polysaccharide P6 in the preparation of antibacterial drugs;

[0009] The antibacterial effect particularly refers to inhibiting the proliferation of Shigella and Salmonella.

[0010] Application of myrtle polysaccharide P6 in the preparation of antioxidant drugs.

[0011] The myrtle polysaccharide P6 contains the following monosaccharides in molar percentages: 4.97% rhamnose, 11.32% arabinose, 4.64% galactose, 4.43% glucose, and 74.63% galacturonic acid. The monosaccharide composition is calculated using the liquid chromatography external standard method. After injecting standards of different concentrations, a standard curve is plotted using the peak area as the value. Under completely identical conditions, a sample solution of the same volume as the reference solution is accurately injected, and the concentration of the component to be tested is determined from the standard curve based on its signal, or calculated using a regression equation.

[0012] The number average molecular weight (Mn) of the myrtle polysaccharide P6 is 36.96 kDa, and the weight average molecular weight (Mw) is 59.15 kDa.

[0013] The above-mentioned method for separating and purifying myrtle polysaccharide P6 comprises the following steps:

[0014] (1) Extraction of crude polysaccharides by water extraction and enzyme-assisted method;

[0015] (2) Sevag method for protein removal and dialysis freeze-drying;

[0016] (3) Ion exchange column chromatography to separate myrtle fruit polysaccharides;

[0017] (4) Purification of myrtle polysaccharide by dextran gel column chromatography.

[0018] The step (1) comprises: crushing the myrtle fruit, adding ultrapure water, adding papain at 50-55° C., enzymolyzing for more than 3 hours, concentrating the extract, adding several times the volume of ethanol, mixing, and placing at 4° C. for precipitation for more than 12 hours to obtain crude polysaccharide;

[0019] In step (1), the ultrapure water is added for extraction, and the material-liquid ratio of each extraction is preferably 1:20;

[0020] The concentration of papain in step (1) is preferably 1%.

[0021] The step (3) is as follows: dissolving the lyophilized crude polysaccharide in step (2) with ultrapure water, loading the sample onto a DEAE-Sepharose Fast Flow column (agarose gel column); gradient eluting with ultrapure water, 0.1M, 0.2M, and 0.3M sodium chloride solutions in sequence, collecting the solution in separate tubes, and concentrating and purifying the 0.3M sodium chloride eluate;

[0022] The DEAE-Sepharose Fast Flow column (agarose gel column) described in step (3) was pretreated as follows before use: the filler was filtered and rinsed with distilled water. Since bubbles were generated during the rinsing process, the column was then deaerated with ultrasound and allowed to stand.

[0023] The crude polysaccharide described in step (3) is dissolved in ultrapure water, and the dissolved concentration is preferably 5 mg / mL;

[0024] The elution rate in step (3) is preferably 2 mL / min.

[0025] Said steps (2) and (4) can be carried out according to the methods of the prior art;

[0026] Preferably, steps (2) and (4) can be carried out according to the methods described in Chinese invention patents ZL201810986675.6, ZL201810987061.X, ZL201810987144.9 and ZL201810987874.9.

[0027] Particularly preferably, the step (2) is:

[0028] Dissolve the crude polysaccharide precipitate in ultrapure water, add Sevag reagent, shake vigorously and let it stand, remove the organic layer and the flocculent white precipitate in the middle, add Sevag reagent again to the upper aqueous phase to remove protein until no obvious protein precipitate is found, add several times the volume of ethanol to precipitate; then dialyze and freeze-dry;

[0029] The Sevag reagent in step (2) is composed of chloroform and n-butanol in a volume ratio of 5:1;

[0030] The amount of Sevag reagent added in step (2) is preferably 1 / 4 of the volume of the sugar solution;

[0031] The dialysis in step (2) was performed at 4°C (3000 Da) for 72 hours.

[0032] Particularly preferably, the step (4) is:

[0033] The sodium chloride elution concentrate obtained in step (3) was passed through a dextran G-100 gel column for chromatography, eluted with ultrapure water, and the eluted fraction was collected in a separate tube. The polysaccharide with high content and relatively concentrated distribution was collected, dialyzed, concentrated, and freeze-dried. The obtained polysaccharide was named P6;

[0034] The elution flow rate in step (4) is preferably 0.2 mL / min;

[0035] The dialysis conditions described in step (4) are to transfer the concentrated fraction into a dialysis bag (3000 Da) and dialyze at 4°C for 72 hours.

[0036] The medicine also contains acceptable excipients and other effective ingredients that act synergistically;

[0037] The medicine can be in various dosage forms, such as tablets, granules, capsules, oral liquids, etc.

[0038] Experimental results of the present invention demonstrate that myrtle polysaccharide P6 has antibacterial effects, exhibiting varying inhibitory activity against different bacteria, primarily inhibiting Shigella and Salmonella. Myrtle polysaccharide P6 also has a certain ability to scavenge free radicals in vitro. Using vitamin C as a positive control, myrtle polysaccharide P6 has a certain ability to scavenge DPPH free radicals, hydroxyl free radicals, and ABTS free radicals. Its antioxidant capacity increases with increasing concentration.

[0039] The present invention obtains myrtle polysaccharide P6 by water extraction and alcohol precipitation, deproteinizes by the Sevag method, and separates and purifies it using a DEAE-Sepharose fast flow ion exchange chromatography column and a dextran gel chromatography column. Compared with the existing technology, the present invention has the following advantages and effects:

[0040] (1) The present invention is the first to discover that myrtle polysaccharide has antibacterial and antioxidant effects, which expands the potential application range of myrtle polysaccharide.

[0041] (2) The present invention establishes a complete and feasible technical route for the extraction, separation and purification, structural characteristics and biological activity research of myrtle polysaccharides, and provides technical guidance for the extraction, separation and purification of myrtle fruit polysaccharides from wild plant resources.

[0042] (3) The water extraction and alcohol precipitation method used in the present invention can complete a large number of polysaccharide extraction operations with low cost, good repeatability, high yield, and is suitable for industrial large-scale production.

[0043] (4) The DEAE-Sepharose fast flow column chromatography method used in the present invention has stable physical and chemical properties, good mechanical properties, large exchange capacity, can be cleaned in place, has high flow rate and loading capacity, and is suitable for purification of large amounts of crude products.

[0044] (5) The dextran gel column chromatography method used in the present invention has high separation efficiency and can efficiently purify biomacromolecules and complex mixtures. It is easy to operate and does not require special high-pressure equipment and professional skills.

[0045] (6) The present invention creatively combines the water extraction and alcohol precipitation method of polysaccharides with the chromatography column separation and purification method, and obtains better process parameters, providing technical guidance and new ideas for the extraction, separation and purification of myrtle polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is the UV full wavelength scanning diagram of Myrtus communis L. polysaccharide P6.

[0047] Figure 2 is the infrared spectrum diagram of Myrtus communis L. polysaccharide P6.

[0048] Figure 3 is the nuclear magnetic resonance spectrum diagram of Myrtus communis L. polysaccharide P6 (upper: 1H spectrum; lower: 13C spectrum).

[0049] Figure 4 is the inhibitory activity of Myrtus communis L. polysaccharide P6 on different bacteria.

[0050] Figure 5 is the in-vitro antioxidant activity of Myrtus communis L. polysaccharide P6. DETAILED DESCRIPTION

[0051] The application will be further described in detail below with reference to the examples and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0052] Example 1

[0053] A method for extracting and separating and purifying polysaccharide from Myrtus communis L. fruit, comprising the following steps:

[0054] (1) Water extraction and enzyme-assisted extraction: 100.00 g of Myrtus communis L. fruit powder is weighed, and ultrapure water is added, with a material-liquid ratio of 1:20. 1% papain is added at 55°C, and enzyme hydrolysis is carried out for 3 hours. The extraction is carried out twice. The extracted solution is concentrated under reduced pressure at 60°C using a rotary evaporator. After the concentrated solution is cooled, 4 times the volume of 95% ethanol is added, mixed, and placed at 4°C overnight to precipitate the crude polysaccharide.

[0055] (2) Protein removal: The Sevag method is used to remove the protein. The crude polysaccharide precipitate is dissolved in ultrapure water, and 1 / 4 volume of Sevag reagent (V chloroform:V n-butanol=5:1) is added. After vigorous shaking for 5 minutes, it is left to stand. The organic layer and the intermediate white flocculent precipitate are removed, and the upper aqueous phase is again subjected to the aforementioned method of adding organic phase reagent to remove protein until no obvious protein precipitate is present. The Sevag method is repeated 10 to 15 times until denatured protein no longer appears, and 4 times the volume of 95% ethanol is again added for alcohol precipitation.

[0056] (3) Dialysis and freeze-drying: The crude polysaccharide precipitate obtained in the above step is dissolved in ultrapure water, and dialysis (3000 Da) is carried out at 4°C for 72 hours to remove residual organic reagents and small molecular impurities in the solution. After dialysis, the solution is concentrated and freeze-dried to obtain freeze-dried crude polysaccharide.

[0057] (4) DEAE-Sepharose Fast Flow ion exchange column chromatography to separate the Myrica rubra polysaccharide: the freeze-dried crude polysaccharide obtained in the foregoing step is dissolved in ultrapure water to 5 mg / mL, and then is loaded into a DEAE-Sepharose Fast Flow column; ultrapure water, 0.1 M, 0.2 M, 0.3 M, 0.4 M sodium chloride gradient elution is used in sequence; the elution speed is 2 mL / min, and each tube is collected for 10 mL; the total sugar content is determined; when the column is regenerated, first, 1 M sodium chloride is used for salt washing, then water washing, then 1 M sodium hydroxide is used for cleaning, and finally, ultrapure water is used for cleaning until neutral; the total sugar content of each gradient solution eluted in the DEAE column chromatography is determined, and the 0.3 M sodium chloride eluent is concentrated for further purification;

[0058] (5) Sephadex gel column chromatography to separate the Myrica rubra refined polysaccharide: the polysaccharide concentrated solution obtained in the foregoing step is subjected to Sephadex G-100 gel column chromatography, and ultrapure water is used for elution; the elution speed is 0.2 mL / min, and each tube is collected for 5 mL; the polysaccharide with high content and concentrated distribution is collected, and is dialyzed in a 3000D dialysis bag at 4°C for 72 hours; finally, it is concentrated and freeze-dried to obtain the Myrica rubra polysaccharide P6.

[0059] Example 2

[0060] A Myrica rubra polysaccharide P6 solution with a concentration of 1 mg / mL is prepared, and a full scan of ultraviolet spectrum is performed in the range of 200-800 nm by using a UV-visible spectrophotometer.

[0061] According to Figure 1 The result analysis shows that there is no absorption peak near 520 nm, indicating that the refined polysaccharide does not contain pigment impurities. There is no characteristic absorption peak at 260 nm and 280 nm, indicating that the protein and nucleic acid in the polysaccharide are completely removed, and the polysaccharide is a pure polysaccharide.

[0062] Example 3

[0063] The external standard method is selected to determine the monosaccharide composition and content of the Myrica rubra polysaccharide P6, and a standard curve is drawn by preparing monosaccharide standard solutions with different concentrations.

[0064] (1) Preparation of standard: 13 kinds of standard substances of fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid are accurately weighed, dissolved in ultrapure water to prepare 10 mg / mL standard solution mother liquor, and then mixed to prepare a standard mixture with a highest concentration of 40 μg / mL, and then an appropriate concentration gradient is prepared.

[0065] (2) Sample pretreatment: Weigh an appropriate amount of the myrtle polysaccharide P6 obtained in Example 1, add 125 μL of sulfuric acid solution (72%) for acid hydrolysis, incubate at 30°C for 1 h, then add 1.35 ml of water, vortex mix, heat at 121°C for 2 hours, adjust to neutrality with 0.5 M sodium hydroxide, dilute appropriately, and transfer to a chromatographic bottle for testing.

[0066] (3) Sample detection: Thermo ICS 5000+ ion chromatography system was used for sample injection, and the monosaccharide components were analyzed and detected using an electrochemical detector. The measurement conditions were as follows: Dionex TM CarboPac TM A PA20 (150 x 3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μl. The mobile phases were A (H2O), B (0.1 M NaOH), and C (0.1 M NaOH, 0.2 M NaAc), with a flow rate of 0.5 ml / min. The column temperature was 30°C. Elution gradient: 0 min phase A / phase B / phase C (95:5:0, V / V), 26 min phase A / phase B / phase C (85:5:10, V / V), 42 min phase A / phase B / phase C (85:5:10, V / V), 42.1 min phase A / phase B / phase C (60:0:40 V / V), 52 min phase A / phase B / phase C (60:40:0, V / V), 52.1 min phase A / phase B / phase C (95:5:0, V / V), 60 min phase A / phase B / phase C (95:5:0, V / V).

[0067] The measured monosaccharide composition of the refined myrtle fruit polysaccharide is shown in Table 1 below:

[0068] Table 1 Monosaccharide composition of Myrtle polysaccharide P6

[0069]

[0070] Example 4

[0071] The polysaccharide molecular weight analysis of the myrtle polysaccharide P6 obtained in Example 1 was performed. The myrtle polysaccharide P6 was dissolved in a 0.1M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) at a concentration of 1 mg / mL and filtered through a filter with a pore size of 0.45 μm before sample detection.

[0072] Gel exclusion chromatography columns Ohpak SB-805HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) were connected in series. The column temperature was 45°C, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1 M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0073] A gel chromatography-differential refractive index-multi-angle laser light scattering system was used to detect the light scattering information of macromolecules, and the absolute molecular weight of each component was calculated according to the Mark-Houwink equation.

[0074] The molecular weight of the purified component of Myrtle polysaccharide P6 was measured and is shown in Table 2 below:

[0075] Table 2 Molecular weight of refined myrtle fruit polysaccharides

[0076]

[0077] Example 5

[0078] The myrtle polysaccharide P6 obtained in Example 1 was subjected to Fourier infrared spectroscopy. 1 mg of myrtle polysaccharide P6 sample was weighed and mixed with KBr powder, fully ground and pressed into 1 mm particles, and infrared spectrometer was used to measure the concentration of the polysaccharide. -1 The infrared spectra of the polysaccharide samples were obtained in the frequency range of .

[0079] according to Figure 2 The results show that the infrared spectrum of myrtle polysaccharide P6 is at 3301cm -1 A broad absorption peak appears near 2937 cm, which is due to the stretching vibration of the hydroxyl group in the sugar residue; -1 A small peak appears near the C–H asymmetric stretching vibration. These two peaks are typical polysaccharide absorption peaks; 1740 cm -1 No obvious absorption of carboxylic acid ester was observed, indicating that the polysaccharide was non-esterified polysaccharide; 1593 cm -1 The peak at 1410 cm is the asymmetric stretching vibration of C=O. -1 The two peaks are the characteristic peaks of the protonated carboxyl group in uronic acid. -1 Nearby is the S=O absorption peak of sulfate group, and it is speculated that polysaccharide P6 contains sulfate group. In the “fingerprint spectrum” area of ​​polysaccharide 1200-1000cm -1 Within the range of 1141cm -1 The absorption peak at 1094 cm comes from the asymmetric C—O—C stretching vibration. -1 The absorption peak at 1010 cm comes from the C—O—C stretching vibration of the sugar ring. -1 The absorption peak at 835cm comes from the stretching vibration of the side chain C—C—O. The appearance of three absorption peaks in this region indicates the presence of pyranose. -1 and 889cm -1 The characteristic absorption at 768 cm is attributed to the presence of α and β configurations in P6. -1There is a clear absorption peak at , which is the characteristic absorption of the symmetrical stretching vibration of the D-pyranose ring. In summary, it can be inferred that P6 is an acidic polysaccharide, mainly containing α-pyranose bonds.

[0080] Example 6

[0081] Fourier transform nuclear magnetic resonance analysis was performed on the myrtle polysaccharide P6. 20 mg of the myrtle polysaccharide P6 sample in Example 1 was taken at room temperature and completely dissolved in 0.8 mL of D2O. 1 H and 13 C NMR spectra were collected by Fourier transform nuclear magnetic resonance spectrometer at frequencies of 600 MHz and 151 MHz, respectively.

[0082] according to Figure 3 Analysis shows that 1 In the H NMR spectrum, protons mainly exist in the range of 3.5-5.5ppm, but the overlap and interference between proton signals affect the analysis. Therefore, the characteristic signals of the first carbon protons appearing in the 4.8-5.5ppm region in the hydrogen spectrum are mainly analyzed to determine the glycosidic bond configuration. It is generally believed that the proton displacement on the first carbon greater than 4.90ppm is an α-type glycoside, and less than 4.90ppm is a β-type glycoside. It is speculated that polysaccharide P6 mainly has an α-type glycoside configuration. Combined with the monosaccharide composition results, 4.98ppm corresponds to the first carbon proton on galacturonic acid. The strong absorption peak at 4.70ppm is attributed to D2O. In 13 In the C NMR spectrum, chemical shifts for the α-configured isomeric carbon atoms typically appear in the range of 98–103 ppm, while those for the β-configured are typically found in the range of 103–110 ppm. A strong signal for P6 at 98 ppm indicates a predominant α-configuration, consistent with the H-spectroscopy results. Significant signals appear between 68 and 78 ppm in the C-spectrum, attributed to chemical shifts at C2–C6. When characterizing the structure of pectin by NMR, the isomeric proton signals for (→4)-α-GalpA-(1→) range from 4.89 to 5.25 ppm, with corresponding carbon atoms at 98.4–103.0 ppm. The characteristic C6 signal for the (→4)-α-GalpA-(1→) residue in pectin is assigned to 175.9–178.5 ppm. The P6 signal at 176 ppm is attributed to the carboxyl carbon of both the unesterified carbonyl group and the methyl-esterified carbonyl group in α-GalAp.

[0083] Example 7

[0084] Antibacterial test of myrtle polysaccharide P6

[0085] (1) Bacterial culture: Six pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Candida albicans, Pseudomonas aeruginosa, Salmonella, and Shigella flexneri, were inoculated from the slant culture medium and streaked onto nutrient agar medium. After two consecutive streakings, a single colony was taken and cultured in a liquid culture medium with a shake flask for 5-8 hours. The bacterial solution was diluted to a concentration of 1×10 7 CFU / mL or so, set aside.

[0086] (2) Determination of inhibition zone: The antibacterial effect was determined using the Oxford cup method. 200 μL of the prepared bacterial suspension was spread onto a plate. Four Oxford cups were then placed in the inoculated plate, and 200 μL of different concentrations of myrtle polysaccharide P6 solution was added to each of the four Oxford cups. Sterile water was used as a blank control. The plates were incubated at 37°C for 10 h, and the size of the inhibition zone was measured to quantify the antibacterial effect.

[0087] according to Figure 4 Analysis showed that polysaccharides exhibited different inhibitory activities against different bacteria, and myrtle polysaccharide P6 had better inhibitory effects on Shigella and Salmonella.

[0088] Example 8

[0089] Antioxidant Test of Myrtle Polysaccharide P6

[0090] (1) Determination of DPPH free radical scavenging rate: 1 mL of myrtle polysaccharide P6 solution with different concentration gradients (50, 100, 150, 200, 250 μg / mL) was added to 1 mL of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, 0.2 mmol / L) ethanol solution and reacted at room temperature in the dark for 30 min. The absorbance value A of the polysaccharide solution at each concentration was measured at 517 nm. i The background absorbance of the sample (A j ), the blank absorbance value (A0) was measured with ultrapure water, and the same concentration of vitamin C was used as a positive control. Each concentration was repeated 3 times and the average value was taken. The DPPH free radical scavenging rate was calculated according to formula (1). The 50% inhibition concentration (IC 50 ) is expressed as the amount of sample that reacted with half of the DPPH radicals.

[0091]

[0092] Where: A0 is the blank absorbance; A iMeasure the absorbance of the sample; A j is the sample background absorbance

[0093] (2) Determination of hydroxyl radical scavenging rate: 0.5 mL of Myrtle polysaccharide P6 solution of each concentration was taken, and 0.5 mL of 9.0 mmol / L ferrous sulfate and 9.0 mmol / L salicylic acid ethanol solution were added respectively. Finally, 0.25 mL of 8.8 mmol / L hydrogen peroxide was added to start the reaction. The reaction was carried out at 37°C for 30 min, and the absorbance value A was measured at a wavelength of 510 nm. i The background absorbance of the sample (A j ); the blank absorbance (A0) was measured with ultrapure water; the same concentration of vitamin C was used as a positive control. Three replicates were performed for each concentration, and the average value was taken. The hydroxyl radical scavenging rate was calculated according to formula (1).

[0094] (3) Determination of superoxide anion radical scavenging rate: 2 mL of each concentration gradient of myrtle polysaccharide P6 sample was mixed with 4.5 mL of Tris-HCl buffer (1 M, pH 8.2), and then placed in a 37°C water bath for 30 minutes. Finally, 1 mL of 2 mM pyrogallol was added and allowed to react for 10 minutes, and then the absorbance value A at 320 nm was measured. i Ultrapure water was used instead of pyrogallol to measure the background absorbance of the sample (A j ); the blank absorbance (A0) was measured with ultrapure water; the same concentration of vitamin C was used as a positive control. Each concentration was repeated three times, and the average value was taken. The superoxide anion radical scavenging rate was calculated according to formula (1).

[0095] (4) Determination of ABTS free radical scavenging rate: Mix 2.2-azido-bis(3-ethylbenzothiazole-6-sulfonic acid) (7 mM) and potassium persulfate (2.45 mM) solution and store in the dark for 12-16 hours to obtain ABTS stock solution. Before use, dilute the ABTS solution with phosphate buffer (10 mM, pH = 7.4) and maintain the absorbance of the solution at 734 nm at 0.700 ± 0.02 to obtain ABTS working solution. Take 0.5 mL of sample solution with different concentrations, add 1.5 mL of ABTS working solution, and react at room temperature in the dark for 15 minutes. Then measure the absorbance value of the sample at 734 nm. i ;Use ultrapure water to replace ABTS and measure the background absorbance of the sample (A j ); the blank absorbance (A0) was measured with ultrapure water; the same concentration of vitamin C was used as a positive control. Three parallel experiments were performed for each concentration, and the average value was taken. The ABTS free radical scavenging rate was calculated according to formula (1).

[0096] (5) Determination of total reducing power: Take 0.5 mL of different concentrations of myrtle polysaccharide P6 sample solution, add 1.0 mL of phosphate buffer (0.2 mmol / L, pH = 6.6) and 1.0 mL of 1.0% potassium ferricyanide solution, mix well, and place in a 50°C water bath for 30 minutes. After cooling to room temperature, add 1.0 mL of 10% trichloroacetic acid solution, mix well, centrifuge at 4000 r / min for 15 minutes, take 2 mL of supernatant into another test tube, add an equal volume of ultrapure water and 0.4 mL of 0.1% ferric chloride solution, mix well, and react in the dark for 30 minutes. Measure the absorbance at 700 nm. The greater the absorbance, the stronger the reducing power. Use the same concentration of vitamin C as a positive control. The total reducing power is calculated according to formula (2).

[0097] Total reducing power = A-A0 formula (2)

[0098] Where: A is the absorbance value of the sample solution after reaction; A0 is the absorbance value of the blank control.

[0099] according to Figure 5 Analysis showed that myrtle polysaccharide P6 had a certain scavenging ability against DPPH free radicals, hydroxyl free radicals, superoxide anion free radicals and ABTS free radicals, and its antioxidant capacity increased with increasing concentration.

[0100] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. The application of myrtle polysaccharide P6 in the preparation of antibacterial drugs is characterized by: The myrtle polysaccharide P6 contains the following monosaccharides in the following molar percentages: rhamnose 4.97%, arabinose 11.32%, galactose 4.64%, glucose 4.43%, and galacturonic acid 74.63%; The antibacterial effect refers to the inhibition of the proliferation of Shigella and Salmonella; The number average molecular weight (Mn) of the myrtle polysaccharide P6 is 36.96 kDa, and the weight average molecular weight (Mw) is 59.15 kDa.

2. The use according to claim 1, characterized in that: The method for separating and purifying myrtle polysaccharide P6 comprises the following steps: (1) Extraction of crude polysaccharides by water extraction and enzyme-assisted method; (2) Sevag method for protein removal and dialysis freeze-drying; (3) Ion exchange column chromatography to separate myrtle fruit polysaccharides; (4) Purification of myrtle polysaccharide by dextran gel column chromatography; The step (1) comprises: crushing the myrtle fruit, adding ultrapure water, adding papain at 50-55° C., enzymolyzing for more than 3 hours, concentrating the extract, adding several times the volume of ethanol, mixing, and placing at 4° C. for precipitation for more than 12 hours to obtain crude polysaccharide; The step (3) is as follows: the crude polysaccharide after freeze-drying in step (2) is dissolved in ultrapure water, and the sample is loaded onto a DEAE-Sepharose Fast Flow column; ultrapure water, 0.1M, 0.2M, and 0.3M sodium chloride solutions are used for gradient elution in sequence, and the 0.3M sodium chloride eluate is collected in separate tubes, and the 0.3M sodium chloride eluate is concentrated and purified.

3. The use according to claim 2, characterized in that: The step (2) is: The crude polysaccharide precipitate was dissolved in ultrapure water, and Sevag reagent was added. After vigorous shaking, the mixture was allowed to stand and the organic layer and the flocculent white precipitate in the middle were removed. Sevag reagent was added again to the upper aqueous phase to remove protein until no obvious protein precipitate was found. Several times the volume of ethanol was added for alcohol precipitation. The mixture was then dialyzed and freeze-dried.

4. The use according to claim 2, characterized in that: The step (4) is: The sodium chloride elution concentrate obtained in step (3) was passed through a dextran G-100 gel column for chromatography, eluted with ultrapure water, and the eluted solution was collected in separate tubes. The polysaccharides with high content and relatively concentrated distribution were collected, dialyzed, concentrated, and freeze-dried. The obtained polysaccharide was named P6.

5. The use according to claim 2, characterized in that: The concentration of papain in step (1) is 1%.

6. The use according to claim 3 or 4, characterized in that: The dialysis was performed in a 3000Da dialysis bag at 4°C for 72 hours.

7. The use according to claim 1, characterized in that: The medicine also contains acceptable excipients and other effective ingredients that have synergistic effects.

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