Application of rhodomyrtus tomentosa polysaccharide P6 in preparation of antibacterial and antioxidant drugs

Abstract

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

Description

Technical Field

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

[0002] Polysaccharides belong to the third largest category of biopolymers (carbohydrates). They are polymerized sugar high-molecular carbohydrates 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, biological activity, 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, immunomodulatory function, blood sugar lowering, tumor inhibition, anti-aging and anti-fatigue.

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

[0004] Previously, the inventors have isolated four myrtle polysaccharides with lipid-lowering effects, namely P1, P2, P3 and P4, which are all composed of monosaccharides such as ribose, rhamnose, arabinose, xylose, mannose, glucose and galactose, but the content of each component is different. The relative binding rate of the polysaccharides to sodium taurocholate, sodium glycocholate and sodium cholate is between 30% and 50%, showing a good lipid-lowering effect.

[0005] In short, 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 and health foods;

[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 and health foods.

[0011] The myrtle polysaccharide P6 contains the following molar percentages of monosaccharides: 4.97% rhamnose, 11.32% arabinose, 4.64% galactose, 4.43% glucose, and 74.63% galacturonic acid; the composition of the monosaccharides is calculated by the liquid chromatography external standard method, and after the standard samples of different concentrations are injected, a standard curve is drawn with the peak area as the value; under completely the same conditions, the sample solution of the same volume as the reference solution is accurately injected, and according to the signal of the component to be tested, its concentration is found from the standard curve, or calculated by 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 to remove proteins and dialysis freeze-drying;

[0016] (3) separation of myrtle fruit polysaccharides by ion exchange column chromatography;

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

[0018] The step (1) is as follows: crushing the myrtle fruit, adding ultrapure water, and adding papain at 50-55°C, enzymolysis for more than 3 hours, concentrating the extract, adding several times 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, applying the sample to 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 0.3M sodium chloride eluate 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) is pretreated as follows before use: the filler is filtered and rinsed with distilled water. Since bubbles will be generated during the rinsing process, it is then degassed 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] The 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 stand, remove the organic layer and the middle flocculent white precipitate, add Sevag reagent again to the upper aqueous phase to remove protein until there is no obvious protein precipitation, add several times 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) is 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) is chromatographed on a dextran G-100 gel column, eluted with ultrapure water, and the eluted solution is collected by a branch pipe, and polysaccharides with high content and relatively concentrated distribution are collected, dialyzed, concentrated, and freeze-dried. The obtained polysaccharide is 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 h.

[0036] The medicine and health food also contain acceptable excipients and other effective ingredients that play a synergistic role;

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

[0038] The experimental results of the present invention show that myrtle polysaccharide P6 has an antibacterial effect, and the polysaccharide exhibits different inhibitory activities on different bacteria, mainly inhibiting Shigella and Salmonella. Myrtle polysaccharide P6 also has a certain ability to scavenge free radicals in vitro. Taking 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, and the antioxidant capacity increases with the increase of concentration.

[0039] The present invention obtains myrtle polysaccharide P6 by water extraction and alcohol precipitation, removes protein by Sevag method, and separates and purifies by DEAE-Sepharose fast flow ion exchange chromatography column and dextran gel chromatography column. Compared with the prior art, 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, thus expanding the potential application scope 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 This is the full-wavelength UV scan of myrtle polysaccharide P6.

[0047] Figure 2 This is the infrared spectrum of myrtle polysaccharide P6.

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

[0049] Figure 4 The inhibitory activity of myrtle polysaccharide P6 against different bacteria.

[0050] Figure 5 In vitro antioxidant activity of Myrtle polysaccharide P6. DETAILED DESCRIPTION

[0051] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0052] Example 1

[0053] A method for extracting, separating, purifying and refining polysaccharides from myrtle fruits comprises the following steps:

[0054] (1) Water extraction and enzyme-assisted extraction: Weigh 100.00 g of myrtle fruit powder, add ultrapure water, the solid-liquid ratio is 1:20, add 1% papain at 55°C, enzymolysis for 3 hours, and extract twice in total; the extracted solution obtained by filtration is concentrated under reduced pressure at 60°C using a rotary evaporator, and after the concentrated solution is cooled, 4 times the volume of 95% ethanol is added, the mixture is mixed and placed at 4°C overnight to precipitate crude polysaccharides;

[0055] (2) Protein removal: Protein is removed by the Sevag method. The crude polysaccharide precipitate is dissolved in ultrapure water, and 1 / 4 volume of the sugar solution is added with Sevag reagent (V chloroform: V n-butanol = 5:1). The mixture is shaken vigorously for 5 minutes and then allowed to stand. The organic layer and the flocculent white precipitate in the middle are removed, and the upper aqueous phase is again deproteinized by adding the organic phase reagent by the aforementioned method until no obvious protein precipitate is present. The Sevag method is repeated ten to fifteen times until the denatured protein no longer appears, and 4 times the volume of 95% ethanol is added again for alcohol precipitation.

[0056] (3) Dialysis and freeze-drying: The crude polysaccharide precipitated in the above step is re-dissolved in ultrapure water and dialyzed (3000 Da) 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) Separation of Myrtle Fruit Polysaccharides by DEAE-Sepharose Fast Flow Ion Exchange Column Chromatography: The freeze-dried crude polysaccharide obtained in the previous step was dissolved to 5 mg / mL with ultrapure water and loaded onto a DEAE-Sepharose Fast Flow column; ultrapure water, 0.1 M, 0.2 M, 0.3 M, and 0.4 M sodium chloride were used for gradient elution in sequence; 10 ml of the elution was collected in each tube at an elution rate of 2 mL / min, and the total sugar content was determined in each tube; when the chromatography column was regenerated, it was first salt-washed with 1 M sodium chloride and then washed with water; then it was washed with 1 M sodium hydroxide and finally washed with ultrapure water until neutral; the total sugar content of the components eluted with each gradient solution in the DEAE column chromatography was determined, and the 0.3 M sodium chloride eluate was concentrated for further purification;

[0058] (5) Separating the purified myrtle polysaccharide by dextran gel column chromatography: The polysaccharide concentrate obtained in the above step was chromatographed on a dextran G-100 gel column and eluted with ultrapure water; the polysaccharide was collected in separate tubes at an elution rate of 0.2 mL / min, with 5 mL in each tube; polysaccharides with high content and relatively concentrated distribution were collected, dialyzed using a 3000D dialysis bag at 4°C for 72 hours, and finally concentrated and freeze-dried to obtain myrtle polysaccharide P6.

[0059] Example 2

[0060] A myrtle polysaccharide P6 solution with a concentration of 1 mg / mL was prepared, and a full ultraviolet spectrum scan was performed in the range of 200 to 800 nm using an ultraviolet-visible spectrophotometer.

[0061] according to Figure 1 The results show that there is no absorption peak near 520nm, indicating that the refined polysaccharide does not contain pigment impurities. There are no characteristic absorption peaks at 260nm and 280nm, 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 was used to determine the monosaccharide composition and content of Myrtle polysaccharide P6, and a standard curve was drawn by preparing monosaccharide standards of different concentrations.

[0064] (1) Preparation of standard substances: Accurately weigh a certain mass of 13 standard substances, namely fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, mannuronic acid and guluronic acid, and dissolve them in ultrapure water to prepare a 10 mg / mL standard solution stock solution. Then, mix the 13 standard solution stock solutions to prepare a standard mixed standard with a maximum concentration of 40 μg / mL, and then prepare a suitable concentration gradient.

[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 neutral with 0.5 M sodium hydroxide, dilute to an appropriate multiple, 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 PA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. The mobile phases are: A (H2O), B (0.1M NaOH) and C (0.1M NaOH, 0.2M NaAc), the flow rate is 0.5ml / min; the column temperature is 30℃. 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 was performed on the myrtle polysaccharide P6 obtained in Example 1. 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 used in series, with a column temperature of 45°C, an injection volume of 100 μL, a mobile phase of A (0.02% NaN3, 0.1 M NaNO3), a flow rate of 0.6 mL / min, and an elution gradient of isocratic 75 min.

[0073] The light scattering information of macromolecules was detected by gel chromatography-differential refractive index-multi-angle laser light scattering system, 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 polysaccharide

[0076]

[0077] Example 5

[0078] The myrtle polysaccharide P6 obtained in Example 1 was measured by Fourier transform 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 at 700-4000 cm -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 -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 a non-esterified polysaccharide; 1593 cm -1 The peak at 1410 cm is the asymmetric stretching vibration of C=O. -1 The symmetric stretching vibration of C=O is at 1236cm. These two peaks are characteristic peaks of protonated carboxyl groups in uronic acid. -1Nearby is the S=O absorption peak of sulfate, and it is speculated that polysaccharide P6 contains sulfate groups. In the "fingerprint spectrum" area of ​​polysaccharides 1200-1000cm -1 Range, 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 on 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. -1 There is an obvious 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 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 of 4.70ppm is attributed to D2O. 13In the C NMR spectrum, the chemical shift of the α-configuration isomeric carbon usually appears in 98–103ppm, while the spectrum of the β-configuration is usually displayed in 103-110ppm. It can be seen that P6 has a strong signal at 98ppm, indicating that the α-configuration is dominant, which is consistent with the results of the hydrogen spectrum. In the carbon spectrum, obvious signals appear at 68ppm to 78ppm, which are attributed to the chemical shift of C2-C6. When pectin is characterized by nuclear magnetic resonance, the isomeric proton signal range of →4)-α-GalpA-(1→ is 4.89-5.25ppm, and the corresponding carbon resonance is 98.4-103.0ppm. The C6 characteristic signal of the →4)-α-GalpA-(1→ residue in pectin is assigned to 175.9-178.5ppm. The signal of P6 at 176ppm is attributed to the carboxyl carbon of the unesterified carbonyl and methyl-esterified carbonyl 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 streaking twice, a single colony was taken and cultured in a liquid culture medium in a shake flask for 5-8 hours. The bacterial solution was diluted to a concentration of 1×10 7 CFU / mL nearby, for future use.

[0086] (2) Determination of inhibition zone: The antibacterial effect was determined by the Oxford cup method. Take 200 μL of the prepared bacterial suspension and spread it on the plate. Then put 4 Oxford cups in the inoculated plate, add 200 μL of different mass concentrations of myrtle polysaccharide P6 solution respectively, use sterile water as a blank control, and culture in an incubator at 37°C for 10 hours. Measure the size of the inhibition zone and quantify the antibacterial effect.

[0087] according to Figure 4 The results showed that polysaccharides exhibited different inhibitory activities against different bacteria, and myrtle polysaccharide P6 had a better inhibitory effect 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 with 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 in parallel 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 i Determine 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, and 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 . Ultrapure water was used instead of hydrogen peroxide to measure the background absorbance of the sample (A j ); ultrapure water was used to measure the blank absorbance value (A0); vitamin C at the same concentration was used as a positive control. Three parallel tests 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 ); ultrapure water was used to measure the blank absorbance value (A0); vitamin C at the same concentration was used as a positive control. Each concentration was repeated 3 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 (ABTS) (7 mM) and potassium persulfate (2.45 mM) solution and store in the dark for 12-16 hours to obtain the ABTS stock solution. Before use, dilute the ABTS solution with phosphate buffer (10 mM, pH = 7.4) and keep the absorbance of the solution at 734 nm at 0.700 ± 0.02 to obtain the ABTS working solution. Take 0.5 mL of sample solution of different concentrations and add 1.5 mL of ABTS working solution. After reacting at room temperature in the dark for 15 minutes, 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 ); ultrapure water was used to measure the blank absorbance value (A0); vitamin C at the same concentration 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 in 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 shows that myrtle polysaccharide P6 has a certain ability to scavenge DPPH free radicals, hydroxyl free radicals, superoxide anion free radicals and ABTS free radicals, and its antioxidant capacity increases 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The application of myrtle polysaccharide P6 in the preparation of antibacterial drugs and health foods, characterized in that: 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.

2. The use according to claim 1, characterized in that: The antibacterial activity refers to inhibiting the proliferation of Shigella and Salmonella.

3. The application of myrtle polysaccharide P6 in the preparation of antioxidant drugs and health foods, characterized in that: 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.

4. The use according to any one of claims 1 to 3, characterized in that: 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.

5. The use according to any one of claims 1 to 3, characterized in that: The separation and purification method of Myrtle polysaccharide P6 comprises the following steps: (1) Extraction of crude polysaccharides by water extraction and enzyme-assisted method; (2) Sevag method to remove proteins and dialysis freeze-drying; (3) separation of myrtle fruit polysaccharides by ion exchange column chromatography; (4) Purification of myrtle polysaccharide by dextran gel column chromatography; The step (1) is as follows: crushing the myrtle fruit, adding ultrapure water, and adding papain at 50-55°C, enzymolysis for more than 3 hours, concentrating the extract, adding several times 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.

6. The use according to claim 5, 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 organic layer and the middle flocculent white precipitate were removed. Sevag reagent was added to the upper aqueous phase again to remove protein until there was no obvious protein precipitation, and several times volume of ethanol was added for alcohol precipitation; then dialyzed and freeze-dried.

7. The use according to claim 5, characterized in that: The step (4) is: The sodium chloride elution concentrate obtained in step (3) is chromatographed on a dextran G-100 gel column and eluted with ultrapure water. The eluted solution is collected in separate tubes. Polysaccharides with high content and relatively concentrated distribution are collected, dialyzed, concentrated and freeze-dried. The obtained polysaccharide is named P6.

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

9. The use according to claim 6 or 7, characterized in that: The dialysis was performed in a 3000Da dialysis bag at 4°C for 72 hours.

10. The use according to any one of claims 1 to 3, characterized in that: The medicine and health food also contain acceptable auxiliary materials and other effective ingredients with synergistic effects.

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