CN110903409A - Millettia speciosa polysaccharide, preparation method and application thereof in antibacterial aspect - Google Patents
Millettia speciosa polysaccharide, preparation method and application thereof in antibacterial aspect Download PDFInfo
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- CN110903409A CN110903409A CN201911238835.XA CN201911238835A CN110903409A CN 110903409 A CN110903409 A CN 110903409A CN 201911238835 A CN201911238835 A CN 201911238835A CN 110903409 A CN110903409 A CN 110903409A
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- polysaccharide
- beautiful millettia
- millettia root
- mscp1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses beautiful millettia root polysaccharide, a preparation method and application thereof in the aspect of antibiosis. The method comprises the following steps: (1) cleaning, slicing, drying, crushing and sieving the beautiful millettia root blocks, and then carrying out hot water reflux extraction to obtain a beautiful millettia root polysaccharide crude extract; (2) removing pigment from the crude extract of the beautiful millettia root polysaccharide by using macroporous resin, removing residual protein by using a Sevag reagent, and then carrying out alcohol precipitation to obtain the beautiful millettia root crude polysaccharide; (3) adding water into the radix millettiae speciosae crude polysaccharide to prepare a radix millettiae speciosae crude polysaccharide solution, then adding the solution into a DEAE-52 cellulose anion exchange column, sequentially eluting with distilled water and 0.2mol/L NaCl solution, and respectively collecting eluates to obtain radix millettiae speciosae polysaccharides MSCP1 and MSCP 2. The beautiful millettia root polysaccharide prepared by the method has a certain inhibiting effect on gram-negative bacteria, particularly escherichia coli and salmonella, so that the beautiful millettia root polysaccharide can be used for developing antibacterial materials or antibacterial agents.
Description
Technical Field
The invention relates to the field of natural active products, in particular to beautiful millettia root polysaccharide, a preparation method and application thereof in the aspect of antibiosis.
Background
Polysaccharides are an important class of biological macromolecules. In recent years, polysaccharides isolated from plants, animals, and microorganisms have been attracting much attention because they exhibit biological activities such as antioxidant, antitumor, anti-inflammatory, hypoglycemic, and immunoregulatory activities. Millettia speciosa is the main variety of south Chinese medicines, belongs to the family of Leguminosae, and is the plant Millettia dielsiana, which is mainly distributed in south China and some countries in southeast Asia. The beautiful millettia root is a medicinal and edible traditional Chinese medicine material, has the effects of treating arthritis and bronchitis, protecting liver, strengthening muscles and bones, tonifying deficiency, moistening lung and the like, and the medicinal effects are closely related to active ingredients such as flavone, polysaccharide and the like in the beautiful millettia root. However, the current research mainly focuses on the extraction and activity evaluation of flavonoids in the root mass of beautiful millettia root, and there are few reports on the separation, purification and activity evaluation of beautiful millettia root polysaccharides. Therefore, refined polysaccharide with uniform molecular weight is prepared by a series of separation and purification technologies, and the activity of the refined polysaccharide is evaluated, so that scientific basis and theoretical guidance can be provided for deep development and utilization of the beautiful millettia root polysaccharide.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a preparation method of beautiful millettia root polysaccharide.
The invention also aims to provide the beautiful millettia root polysaccharide prepared by the method.
Still another object of the present invention is to provide the use of the beautiful millettia root polysaccharide.
The purpose of the invention is realized by the following technical scheme: a preparation method of beautiful millettia root polysaccharide comprises the following steps:
(1) preparation of Millettia speciosa Roxb crude extract
Cleaning, slicing, drying, crushing and sieving the beautiful millettia root blocks to obtain beautiful millettia root block powder; then carrying out hot water reflux extraction on the millettia speciosa champ root block powder at the temperature of 80-100 ℃, centrifuging to obtain supernatant, and then carrying out reduced pressure concentration to obtain a millettia speciosa champ polysaccharide crude extract;
(2) preparation of Millettia speciosa Roxb crude polysaccharide
Adding the crude extract of beautiful millettia root polysaccharide into macroporous resin, standing for adsorption, eluting with distilled water, collecting the eluent, and concentrating under reduced pressure to obtain a concentrated solution (the beautiful millettia root crude polysaccharide after pigment removal); adding a Sevag reagent into the concentrated solution, oscillating (to remove residual protein), centrifuging, taking supernatant, and repeating for 1-5 times; adding absolute ethanol into the supernatant to perform alcohol precipitation, standing at 4 ℃, centrifuging, taking the precipitate, adding water to redissolve the precipitate, dialyzing, and freeze-drying to obtain the crude radix millettiae speciosae polysaccharide;
(3) preparation of beautiful millettia root extract polysaccharide
Adding water into the beautiful millettia root crude polysaccharide to prepare a beautiful millettia root crude polysaccharide solution, then adding the beautiful millettia root crude polysaccharide solution into a DEAE-52 cellulose anion exchange column, sequentially eluting with distilled water and 0.2mol/L NaCl solution, respectively collecting eluent, concentrating, dialyzing and freeze-drying to obtain the beautiful millettia root polysaccharide MSCP1 and MSCP 2.
The drying in the step (1) is carried out under the following conditions: drying at 60 + -5 deg.C for 15 hr.
Sieving in the step (1) is to pass through a sieve of 80-100 meshes; preferably through a 100 mesh screen.
The conditions for the hot water reflux leaching in step (1) are preferably: the material-liquid ratio is 1: 10-30 and the extraction time is 1-3 h.
The leaching frequency in the step (1) is preferably 1-3 times.
The temperature of the reduced pressure concentration in the step (1) is preferably 55-75 ℃.
The concentration under reduced pressure in the step (1) is preferably concentration under reduced pressure to 1/4 volumes of the supernatant.
The macroporous resin in the step (2) is preferably AB-8 macroporous resin.
The AB-8 macroporous resin is pretreated AB-8 macroporous resin; the pretreatment can be carried out by referring to an AB-8 macroporous resin specification, and specifically comprises the following steps: soaking in ethanol or methanol for 24-30 hr, washing with ethanol or methanol until the effluent is not white and turbid, and washing with water to remove ethanol or methanol; then soaking the mixture in 5% by mass of HCl solution for 2-4 hours, and washing the mixture with water until the pH of effluent is neutral; and finally, soaking the mixture in NaOH solution with the mass fraction of 2% for 2-4 hours, and washing the mixture with water until the pH of effluent is neutral.
The time for standing adsorption in step (2) is preferably 4 hours or more.
The concentration under reduced pressure in step (2) is preferably concentrated under reduced pressure to 1/3 volumes of the eluate.
The Sevag reagent in the step (2) is preferably chloroform and n-butanol in a volume ratio of 4: 1 mixing the obtained reagents.
The volume ratio of the concentrated solution to the Sevag reagent in the step (2) is preferably 5: 1.
the conditions for the centrifugation in step (2) are preferably: centrifuging at 4000-6000 r/min for 4-6 min.
The oscillation time in the step (2) is preferably 20-40 min.
The volume ratio of the supernatant to the absolute ethyl alcohol in the step (2) is 1: 4.
The time for standing at 4 ℃ in the step (2) is preferably 12 hours or more.
The dialysis in the steps (2) and (3) is carried out by adopting a dialysis bag with the molecular weight cutoff of 3500 Da; preferably, the dialysis is carried out for more than 48h by using a dialysis bag with the molecular weight cutoff of 3500 Da.
The concentration of the radix millettiae speciosae crude polysaccharide solution in the step (3) is 10-20 mg/mL; preferably 10 mg/mL.
And (4) eluting at the flow rate of 1-3 mL/min in the step (3).
The preparation method of beautiful millettia root polysaccharide further comprises the step of further purifying the obtained beautiful millettia root polysaccharide MSCP1 and MSCP2 after the step (3), and specifically comprises the following steps:
dissolving radix Millettiae Speciosae polysaccharides MSCP1 and MSCP2 in water to obtain radix Millettiae Speciosae polysaccharides MSCP1 and MSCP2 solutions; then adding the extract into Sephadex G-100 gel column, eluting with distilled water, collecting eluate, concentrating, dialyzing, and lyophilizing to obtain refined radix Millettiae Speciosae polysaccharides MSCP1 and MSCP 2.
The concentration of the beautiful millettia root polysaccharide MSCP1 solution is 3-3.5 mg/mL; preferably 3.3 mg/mL.
The concentration of the beautiful millettia root polysaccharide MSCP2 solution is 3-3.5 mg/mL; preferably 3.3 mg/mL.
The flow rate of the elution with distilled water is 0.1-0.3 mL/min.
The dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 3500 Da; preferably, the dialysis is carried out for more than 48h by using a dialysis bag with the molecular weight cutoff of 3500 Da.
Beautiful millettia root polysaccharide prepared by any one of the methods.
The beautiful millettia root polysaccharide is at least one of beautiful millettia root polysaccharide MSCP1 and MSCP2, and the weight average molecular weight is 9.36 multiplied by 10 respectively3Da，2.85×104Da; the MSCP1 contains glucose and xylose, and the MSCP2 contains glucose, galactose, xylose, arabinose and fucose.
The application of the beautiful millettia root polysaccharide in preparing immunoregulation type functional food or medicine.
The application of the beautiful millettia root polysaccharide in preparing antibacterial materials or antibacterial agents.
The antibiosis is to kill bacteria, inhibit the growth and/or reproduction of bacteria.
The bacteria are gram-negative bacteria; preferably at least one of Escherichia coli and Salmonella; wherein, the beautiful millettia root polysaccharide MSCP1 has good bacteriostatic effect on escherichia coli; and the beautiful millettia root polysaccharide MSCP2 has good bacteriostatic effect on salmonella.
Compared with the prior art, the invention has the following advantages and effects:
1. the method adopts a hot water reflux method to extract the beautiful millettia root block powder, and then adopts a DEAE-52 cellulose anion exchange chromatographic column and a Sephadex G-100 chromatographic column to separate and purify to obtain two beautiful millettia root polysaccharides, which have certain inhibition effects on escherichia coli and salmonella, so the method has wide prospects in the development of antibacterial agents; in addition, the beautiful millettia root polysaccharide also has the function of immunoregulation, and can be used for developing immunoregulation type functional foods or medicines.
2. The beautiful millettia root polysaccharide prepared by the invention can inhibit the growth and the propagation of escherichia coli and salmonella, can be used for preparing antibacterial materials, and has wide development value.
Drawings
FIG. 1 is a flow chart of the separation and purification of beautiful millettia root polysaccharide.
FIG. 2 is a DEAE-52 elution graph.
FIG. 3 is a Sephadex G-100 elution profile of Millettia speciosa MSCP1 and MSCP 2; wherein A is a Sephadex G-100 elution curve of the beautiful millettia root polysaccharide MSCP 1; b is the Sephadex G-100 elution curve of beautiful millettia root polysaccharide MSCP 2.
FIG. 4 is a graph of the results of molecular weight determinations of Millettia speciosa MSCP1 and MSCP 2; wherein A is the molecular weight measurement result of beautiful millettia root polysaccharide MSCP 1; and B is the result of molecular weight determination of beautiful millettia root polysaccharide MSCP 2.
FIG. 5 is a graph of the monosaccharide composition measurements of Millettia speciosa polysaccharide MSCP1 and MSCP 2; wherein A is a monosaccharide composition determination result of the beautiful millettia root polysaccharide MSCP 1; b is the monosaccharide composition determination result of beautiful millettia root polysaccharide MSCP 2; c is the monosaccharide composition determination result of the standard product.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.
1. The AB-8 macroporous resin (purchased from Shanghai leaf Biotechnology Co., Ltd.) used in the embodiment of the invention is pretreated firstly, and the specific steps are as follows:
(1) the column (column height 90cm, internal diameter 5cm) was cleaned before packing to prevent contamination of the resin by harmful substances and to drain the water in the column.
(2) Adding ethanol or methanol which is 0.4-0.5 times of the volume of the filled resin into the adsorption column, putting new resin into the column (AB-8 is filled into the chromatographic column and has the height of 65cm) to ensure that the liquid level is about 0.3m higher than the resin layer, and soaking for 24 hours.
(3) 2BV (BV means column volume) of ethanol or methanol is used for soaking for 4-5 hours, and the flow rate is 2 BV/h.
(4) Passing through the resin layer with ethanol or methanol at a flow rate of 2BV/h until the effluent is not cloudy, and washing with water at the same flow rate.
(5) And (3) passing a HCl solution with the mass fraction of 2BV and the mass fraction of 5% through the resin layer at the flow rate of 4-6 BV/h, soaking for 2-4 hours, and then washing with water at the same flow rate until the pH of the effluent is neutral.
(6) And (3) passing a 2BV NaOH solution with the mass fraction of 2% through the resin layer at the flow rate of 4-6 BV/h, soaking for 2-4 hours, and then washing with water at the same flow rate until the pH of the effluent is neutral.
2. The method for determining the content of beautiful millettia root polysaccharide by adopting a phenol-sulfuric acid method in the embodiment of the invention comprises the following steps:
drawing a standard curve: weighing 10mg of glucose dried to constant weight, diluting the glucose to a constant volume with distilled water in a 100mL brown volumetric flask to prepare a standard glucose solution with the concentration of 0.1mg/mL, respectively sucking 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL of the standard glucose solution in a test tube, supplementing the standard glucose solution to 2mL with distilled water, adding 1mL of phenol with the mass fraction of 5% and 5mL of concentrated sulfuric acid (the mass fraction is 95.5%), shaking the glucose solution uniformly, placing the glucose solution at room temperature for 20min, and measuring the absorbance value at 490nm to obtain a regression equation: y is 0.0119x-0.0023 (R)2＝0.9905)。
Preparing a sample to be detected into 1mg/mL, and measuring according to the same method, namely adding 1mL of phenol with the mass fraction of 5% and 5mL of concentrated sulfuric acid, shaking uniformly, placing at room temperature for 20min, measuring the absorbance value at 490nm, substituting the absorbance value into a regression equation, and calculating to obtain the content of the sample to be detected.
Example 1: preparation of beautiful millettia root polysaccharides MSCP1 and MSCP2
The separation and purification process of the beautiful millettia root polysaccharide is shown in figure 1:
(1) preparing a beautiful millettia root crude extract: cleaning root pieces of radix Millettiae Speciosae (produced in Zhaoqing city, Guangdong province, China), cutting into pieces, drying (drying at 60 deg.C for 15 hr), pulverizing, and sieving with 100 mesh sieve to obtain radix Millettiae Speciosae root piece powder; extracting radix millettiae speciosae block powder by a hot water reflux method under the conditions of the temperature of 80 ℃, the material-liquid ratio of 1: 10, extracting for 1 h; centrifuging after leaching, taking supernatant, repeatedly extracting for two times according to the conditions, combining the supernatants, and concentrating under reduced pressure at 55 ℃ to 1/4 volumes to obtain a radix millettiae speciosae polysaccharide crude extract;
(2) preparing the beautiful millettia root crude polysaccharide: in order to remove pigment and protein, 300mL of the crude extract of beautiful millettia root polysaccharide is added into an AB-8 macroporous resin column (the column height is 65cm, the inner diameter is 5cm), the mixture is kept stand and adsorbed for 4 hours, then the mixture is eluted by distilled water, the eluent is collected and concentrated to 100mL under reduced pressure at 55 ℃; removing residual protein by using a Sevag method, adding 1/5 volumes of Sevag reagent (chloroform: n-butyl alcohol is 4: 1, v/v) into the concentrated solution, oscillating for 20min in a shaking table (180rpm), centrifuging for 4min at 4000r/min, taking the supernatant, and repeating the operation for 5 times (namely adding 1/5 volumes of Sevag reagent into the obtained supernatant, oscillating, centrifuging, taking the supernatant, and repeating the operation for 5 times); adding 4 times volume of anhydrous ethanol into the supernatant, precipitating with ethanol, standing in a refrigerator at 4 deg.C for 12h, centrifuging at 4000r/min for 4min, collecting precipitate, adding water (30ml) to redissolve the precipitate, dialyzing with flowing water (3500 Da dialysis bag) for 48h to remove small molecular impurities, and lyophilizing to obtain radix Millettiae Speciosae crude polysaccharide;
(3) preparing beautiful millettia root extract polysaccharide:
① preparing 10mL10mg/mL the above crude polysaccharide solution of radix Millettiae Speciosae, adding DEAE-52 cellulose anion exchange column (Beijing Ding Guoshang biotechnology, Ltd.), eluting with 0, 0.2, 0.4, 0.6mol/L NaCl solution (prepared with distilled water) at flow rate of 1mL/min, collecting eluate with automatic part collector, collecting 5mL each tube, tracking and measuring polysaccharide content of eluate by phenol-sulfuric acid method, drawing elution curve (figure 2), collecting eluate with distilled water and 0.2mol/L NaCl solution as single and symmetrical peak, respectively collecting the two eluates (MSCP1 and MSCP2), concentrating to 20mL, dialyzing (molecular weight cut-off 3500Da), lyophilizing, and purifying.
② respectively dissolving 10mg of the collected components in 3mL of distilled water, adding into Sephadex G-100 gel column (Beijing Ding Guosheng biotechnology, Inc.), eluting with distilled water at an elution flow rate of 0.1mL/min, collecting the eluate with an automatic part collector, collecting 3mL of eluate per tube, tracking and determining polysaccharide content of the eluate per tube by adopting phenol-sulfuric acid method, wherein the elution curve (figure 3) is a single and symmetrical peak, which indicates that the eluate is a single component, concentrating, dialyzing (molecular weight cut-off 3500Da), and freeze-drying to obtain Millettia speciosa refined polysaccharides MSCP1 and MSCP2 respectively.
Example 2: preparation of beautiful millettia root polysaccharides MSCP1 and MSCP2
(1) Preparing a beautiful millettia root crude extract: cleaning root pieces of radix Millettiae Speciosae (produced in Zhaoqing city, Guangdong province, China), cutting into pieces, drying (drying at 60 deg.C for 15 hr), pulverizing, and sieving with 100 mesh sieve to obtain radix Millettiae Speciosae root piece powder; extracting radix millettiae speciosae block powder by a hot water reflux method under the conditions that the temperature is 90 ℃, the ratio of material to liquid is 1: 20, extracting for 2 h; centrifuging after leaching, taking supernatant, repeatedly extracting for two times according to the conditions, combining the supernatants, and concentrating under reduced pressure at 65 ℃ to 1/4 volumes to obtain a radix millettiae speciosae polysaccharide crude extract;
(2) preparing the beautiful millettia root crude polysaccharide: in order to remove pigment and protein, 300mL of the crude extract of beautiful millettia root polysaccharide is added into an AB-8 macroporous resin column (the column height is 65cm, the inner diameter is 5cm), the mixture is kept stand and adsorbed for 4 hours, then the mixture is eluted by distilled water, the eluent is collected, and the mixture is decompressed and concentrated to 100mL at 65 ℃; removing residual protein by Sevag method, adding 1/5 volume Sevag reagent (chloroform: n-butanol is 4: 1, v/v) into the concentrated solution, shaking in shaking table (180rpm) for 30min, centrifuging at 5000r/min for 5min, collecting supernatant, and repeating the operation for 5 times; adding 4 times volume of anhydrous ethanol into the supernatant, precipitating with ethanol, standing in a refrigerator at 4 deg.C for 12h, centrifuging at 5000r/min for 5min to obtain precipitate, adding water (30ml) to redissolve the precipitate, dialyzing with flowing water (3500 Da dialysis bag) for 48h to remove small molecular impurities, and lyophilizing to obtain radix Millettiae Speciosae crude polysaccharide;
(3) preparing beautiful millettia root extract polysaccharide:
① preparing 10mL10mg/mL the above crude polysaccharide solution of radix Millettiae Speciosae, adding DEAE-52 cellulose anion exchange column (Beijing Ding Guoshang biotechnology, Ltd.), eluting with 0, 0.2, 0.4, 0.6mol/L NaCl solution at a flow rate of 2mL/min, collecting the eluate with an automatic part collector, collecting 5mL each tube, determining the polysaccharide content of the eluate by phenol-sulfuric acid method, drawing an elution curve, collecting the eluates with distilled water and 0.2mol/L NaCl solution, respectively, concentrating, dialyzing (molecular weight cut-off of 3500Da), lyophilizing, and purifying.
② respectively dissolving 10mg of the collected components in 3mL of distilled water, adding into a Sephadex G-100 gel column (Beijing Ding Guosheng biotechnology, Inc.), eluting with distilled water at an elution flow rate of 0.2mL/min, collecting the eluate with an automatic part collector, collecting 3mL of eluate per tube, tracking and determining the polysaccharide content of the eluate per tube by a phenol-sulfuric acid method, wherein the elution curve is a single and symmetrical peak, which indicates that the eluate is a single component, collecting the eluate, concentrating, dialyzing (molecular weight cut-off 3500Da), and freeze-drying to obtain Millettia speciosa refined polysaccharides MSCP1 and MSCP2 respectively.
Example 3: preparation of beautiful millettia root polysaccharides MSCP1 and MSCP2
(1) Preparing a beautiful millettia root crude extract: cleaning root pieces of radix Millettiae Speciosae (produced in Zhaoqing city, Guangdong province, China), cutting into pieces, drying (drying at 60 deg.C for 15 hr), pulverizing, and sieving with 100 mesh sieve to obtain radix Millettiae Speciosae root piece powder; extracting radix millettiae speciosae block powder by a hot water reflux method under the conditions that the temperature is 100 ℃, the ratio of material to liquid is 1: 30, the extraction time is 3 hours; centrifuging after leaching, taking supernatant, repeatedly extracting for two times according to the conditions, combining the supernatants, and concentrating under reduced pressure at 75 ℃ to 1/4 volumes to obtain a radix millettiae speciosae polysaccharide crude extract;
(2) preparing the beautiful millettia root crude polysaccharide: in order to remove pigment and protein, 300mL of the crude extract of beautiful millettia root polysaccharide is added into an AB-8 macroporous resin column (the column height is 65cm, the inner diameter is 5cm), the mixture is kept stand and adsorbed for 4 hours, then the mixture is eluted by distilled water, the eluent is collected and concentrated to 100mL under the reduced pressure at the temperature of 75 ℃; removing residual protein by Sevag method, adding 1/5 volume Sevag reagent (chloroform: n-butanol 4: 1, v/v) into the concentrated solution, shaking in shaking table (180rpm) for 40min, centrifuging at 6000r/min for 6min, collecting supernatant, and repeating the operation for 5 times; adding 4 times volume of anhydrous ethanol into the supernatant, precipitating with ethanol, standing in a refrigerator at 4 deg.C for 12h, centrifuging at 6000r/min for 6min to obtain precipitate, adding water (30ml) to redissolve the precipitate, dialyzing with flowing water (3500 Da dialysis bag) for 48h to remove small molecular impurities, and lyophilizing to obtain radix Millettiae Speciosae crude polysaccharide;
(3) preparing beautiful millettia root extract polysaccharide:
① preparing 10mL10mg/mL the above crude polysaccharide solution of radix Millettiae Speciosae, adding DEAE-52 cellulose anion exchange column (Beijing Ding Guoshang biotechnology, Ltd.), eluting with 0, 0.2, 0.4, 0.6mol/L NaCl solution at flow rate of 3mL/min, collecting the eluate with automatic part collector, collecting 5mL each tube, determining the polysaccharide content of each tube by phenol-sulfuric acid method, drawing elution curve, collecting the eluates with distilled water and 0.2mol/L NaCl solution, concentrating, dialyzing (molecular weight cut-off is 3500Da), lyophilizing, and purifying.
② respectively dissolving 10mg of the collected components in 3mL of distilled water, adding into a Sephadex G-100 gel column (Beijing Ding Guosheng biotechnology, Inc.), eluting with distilled water at an elution flow rate of 0.3mL/min, collecting the eluate with an automatic part collector, collecting 3mL of eluate per tube, tracking and determining the polysaccharide content of the eluate per tube by a phenol-sulfuric acid method, wherein the elution curve is a single and symmetrical peak, which indicates that the eluate is a single component, collecting the eluate, concentrating, dialyzing (molecular weight cut-off 3500Da), and freeze-drying to obtain Millettia speciosa refined polysaccharides MSCP1 and MSCP2 respectively.
Example 4: purity determination of beautiful millettia root polysaccharides MSCP1 and MSCP2
The purity of beautiful millettia root polysaccharides MSCP1 and MSCP2 was determined by the phenol-sulfuric acid method.
Drawing a standard curve: weighing 10mg of glucose dried to constant weight, diluting the glucose to a constant volume with distilled water in a 100mL brown volumetric flask to prepare a standard glucose solution with the concentration of 0.1mg/mL, respectively sucking 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4 and 1.6mL of the standard glucose solution in a test tube, supplementing the standard glucose solution to 2mL with distilled water, adding 1mL of phenol with the mass fraction of 5% and 5mL of concentrated sulfuric acid (the mass fraction is 95.5%), shaking the glucose solution uniformly, placing the glucose solution at room temperature for 20min, and measuring the absorbance value at 490nm to obtain a regression equation: y is 0.0119x-0.0023 (R)2＝0.9905)。
Preparing 1mg/mL of MSCP1 and MSCP2, measuring according to the same method, namely adding 1mL of phenol with the mass fraction of 5% and 5mL of concentrated sulfuric acid, shaking uniformly, placing at room temperature for 20min, measuring the absorbance value at 490nm, substituting the absorbance value into a regression equation, and calculating to obtain the contents of MSCP1 and MSCP 2; according to the formula: the purity (polysaccharide content in the sample/sample mass) × 100, and the purity of each sample was calculated.
And (4) analyzing results: the purity of beautiful millettia root polysaccharide MSCP1 and MSCP2 measured by the method are 94.28% and 91.86%, respectively.
Example 5: molecular weight determination of beautiful millettia root polysaccharides MSCP1 and MSCP2
The molecular weights of the beautiful millettia root polysaccharides MSCP1 and MSCP2 were determined by means of a gel permeation chromatography system (GPC) equipped with an Agilent 1260 parallax detector, G3000PWXL(7.8x300 i.d.,5 μm) and G5000PWXL(7.8x300 i.d.,10 μm). The sample amount was 20. mu.L, the mobile phase was 0.02mol/L potassium dihydrogen phosphate, and the flow rate was 0.6 mL/min. Pullulan series glucan (6, 10, 21.7, 48.8, 113, 210, 366, 805, 2500kDa (all from Shanghai Boo's Biotechnology Co., Ltd.) was used as a standard curve, and the regression equation was that LogMolWt is 1270-2-2.73V3+0.0815V4-0.000968V5(R20.999); in the formula, MolWt represents a molecular weight, and V represents a volume.
And (4) analyzing results: the weight average molecular weights of Millettia speciosa polysaccharide MSCP1 and MSCP2 determined by the above method are 9.36 × 103Da，2.85×104Da (FIG. 4).
Example 6: monosaccharide composition determination of beautiful millettia root polysaccharides MSCP1 and MSCP2
The monosaccharide compositions of the beautiful millettia root polysaccharides MSCP1 and MSCP2 were determined by ion chromatography. 10mg of the sample are dissolved in 4mL of trifluoroacetic acid (2mol/L), hydrolyzed at 105 ℃ for 6h, evaporated under reduced pressure to remove the trifluoroacetic acid, the residue is dissolved in methanol and evaporated to dryness under reduced pressure, repeated 4 times to ensure complete removal of the residual trifluoroacetic acid. Finally, the hydrolysate was fully dissolved in ultrapure water, filtered through a 0.22 μm aqueous phase filter membrane and subjected to chromatography. Wherein the content of the first and second substances,
chromatographic conditions are as follows: chromatographic column Carbopac PAI (250X 4mm, i.d.,5cm), column temperature 30 deg.C, sample size 20. mu.L; elution gradient: 0-25min 1% NaOH (500mM), 25-40min NaOH (500mM), 40-50min 100% NaOH (500mM), flow rate 1 mL/min.
Mixing a series of standard substances (glucose, galactose, xylose, arabinose, fucose, mannose, galacturonic acid and glucuronic acid, all purchased from Shanghai Boao biotechnology Co., Ltd.), diluting into mixed standard solutions with different concentrations, drawing each monosaccharide standard curve according to peak areas, and calculating the content of each monosaccharide in the sample by referring to the peak emergence time of the monosaccharide standard substance and the standard curve. The standard curve obtained is as follows:
regression equation for glucose standards: 2.7679x +0.7206 (R)2＝0.9932)；
Regression equation for galactose standard: y 2.1461x +0.3743 (R)2＝0.9959)；
Regression equation for xylose standard: 2.7254x +0.8767 (R)2＝0.9912)；
Regression equation for arabinose standard: 1.7712x +0.4025 (R)2＝0.9954)；
Regression equation for fucose standards: 1.7987x +0.5235 (R)2＝0.9942)；
Regression equation for mannose standards: 2.3846x +0.3142 (R)2＝0.9965)；
Regression equation for galacturonic acid standard: 1.2739x-0.0298 (R)2＝0.9991)；
Regression equation for glucuronic acid standard: 2.2868x +0.0636 (R)2＝0.9992)。
And (4) analyzing results: MSCP1 contained glucose (99.58%) and xylose (0.42%), MSCP2 contained glucose (87.274%), galactose (4.043%), xylose (3.960%), arabinose (2.524%) and fucose (2.198%) (fig. 5).
Example 7: antibacterial activity determination of beautiful millettia root polysaccharide
The antibacterial activity of the beautiful millettia root polysaccharide is measured by an oxford cup antibacterial ring method.
Escherichia coli (Escherichia coli ATCC25922), Salmonella (S)Salmonella typhimurium ATCC 13311), Staphylococcus aureus (Staphylococcus aureus ATCC51650) (both purchased from ATCC) were inoculated in LB agar medium and activated in an incubator at 37 ℃ for 24 hours; selecting similar colonies, inoculating the colonies in an LB culture medium, and culturing for 6 hours in a 37 ℃ incubator; diluting a certain amount of bacterial suspension with sterile normal saline to 107cfu/mL, 200. mu.L of the diluted bacterial suspension was applied to LB agar medium. Placing the sterilized Oxford cup on a culture medium, injecting 200 μ L of sample solution filtered by an aseptic filter membrane (namely 20mg/mL solution prepared by the Millettia speciosa champ refined polysaccharides MSCP1 and MSCP2 prepared in example 1 respectively), continuously culturing for 24h in a 37 ℃ incubator, and judging the bacteriostatic activity of the sample by observing whether a bacteriostatic zone exists and measuring the size of the bacteriostatic zone.
TABLE 1 experimental results of zone of inhibition of beautiful millettia root polysaccharides to test bacteria
And (4) analyzing results: the beautiful millettia root polysaccharides MSCP1 and MSCP2 have inhibition effects on escherichia coli and salmonella, but have no inhibition effect on staphylococcus aureus.
Example 8 determination of Minimum Inhibitory Concentration (MIC)
MIC was determined by broth dilution. Respectively picking 3 similar colonies to be detected: escherichia coli (Escherichia coli ATCC25922) and Salmonella typhimurium (Salmonella typhimurium ATCC 13311) were inoculated into 10mL hydrolyzed casein (MH) broth (Qingdao Haibo Biotech Co., Ltd.), incubated at 37 ℃ for 2 hours, and the bacterial suspension concentration was adjusted to 10 using the medium5cfu/mL. Adding 100ul of broth into each well of a 96-well plate, adding 100ul of samples in the first row (namely 20mg/mL solutions prepared by MSCP1 and MSCP2 prepared from the beautiful millettia root refined polysaccharide prepared in example 1 respectively), mixing uniformly, sucking 100ul to the second row, repeating the steps until 100ul is sucked into the 10 th hole, discarding 10 concentration gradients, and adding no medicine in the 11 th row as a growth control; then 100ul of diluted bacterial suspension was added to each well. After sealing, the cells were incubated in an incubator at 37 ℃ for 16 h.
TABLE 2 experiment results of the minimum inhibitory concentration of beautiful millettia root polysaccharides to the tested bacteria
And (4) analyzing results: the lower the MIC value is, the better the bacteriostatic effect is, so the bacteriostatic effect of MSCP1 on the Escherichia coli is better than that of the Salmonella, and the bacteriostatic effect of MSCP2 on the Salmonella is better than that of the Escherichia coli.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The preparation method of the beautiful millettia root polysaccharide is characterized by comprising the following steps:
(1) preparation of Millettia speciosa Roxb crude extract
Cleaning, slicing, drying, crushing and sieving the beautiful millettia root blocks to obtain beautiful millettia root block powder; then carrying out hot water reflux extraction on the millettia speciosa champ root block powder at the temperature of 80-100 ℃, centrifuging to obtain supernatant, and then carrying out reduced pressure concentration to obtain a millettia speciosa champ polysaccharide crude extract;
(2) preparation of Millettia speciosa Roxb crude polysaccharide
Adding the crude extract of beautiful millettia root polysaccharide into macroporous resin, standing for adsorption, eluting with distilled water, collecting the eluent, and concentrating under reduced pressure to obtain a concentrated solution; adding a Sevag reagent into the concentrated solution, oscillating, centrifuging, taking supernatant, and repeating for 1-5 times; adding absolute ethanol into the supernatant to perform alcohol precipitation, standing at 4 ℃, centrifuging, taking the precipitate, adding water to redissolve the precipitate, dialyzing, and freeze-drying to obtain the crude radix millettiae speciosae polysaccharide;
(3) preparation of beautiful millettia root extract polysaccharide
Adding water into the beautiful millettia root crude polysaccharide to prepare a beautiful millettia root crude polysaccharide solution, then adding the beautiful millettia root crude polysaccharide solution into a DEAE-52 cellulose anion exchange column, sequentially eluting with distilled water and 0.2mol/L NaCl solution, respectively collecting eluent, concentrating, dialyzing and freeze-drying to obtain the beautiful millettia root polysaccharide MSCP1 and MSCP 2.
2. The method for preparing beautiful millettia root polysaccharide according to claim 1, characterized in that:
the macroporous resin in the step (2) is AB-8 macroporous resin;
the Sevag reagent in the step (2) is chloroform and n-butanol according to a volume ratio of 4: 1 mixing the obtained reagents.
3. The method for preparing beautiful millettia root polysaccharide according to claim 1, characterized in that:
the hot water reflux leaching conditions in the step (1) are as follows: the material-liquid ratio is 1: 10-30, and extracting for 1-3 h;
concentrating under reduced pressure to 1/4 volumes of supernatant as described in step (1);
concentrating under reduced pressure in the step (2) to 1/3 volumes of eluent;
the volume ratio of the concentrated solution to the Sevag reagent in the step (2) is 5: 1;
the volume ratio of the supernatant to the absolute ethyl alcohol in the step (2) is 1: 4;
the concentration of the radix millettiae speciosae crude polysaccharide solution in the step (3) is 10-20 mg/mL.
4. The method for preparing beautiful millettia root polysaccharide according to claim 1, characterized in that:
the drying in the step (1) is carried out under the following conditions: drying at 60 + -5 deg.C for 15 hr;
sieving in the step (1) is to pass through a sieve of 80-100 meshes;
leaching for 1-3 times;
the temperature of the reduced pressure concentration in the step (1) is 55-75 ℃;
the standing adsorption time in the step (2) is more than 4 h;
the oscillation time in the step (2) is 20-40 min;
the centrifugation conditions in the step (2) are as follows: centrifuging at 4000-6000 r/min for 4-6 min;
the standing time at the temperature of 4 ℃ in the step (2) is more than 12 h;
the dialysis in the steps (2) and (3) is carried out by adopting a dialysis bag with the molecular weight cutoff of 3500 Da;
and (4) eluting at the flow rate of 1-3 mL/min in the step (3).
5. The method for preparing beautiful millettia root polysaccharide according to claim 1, characterized in that: the step (3) is followed by a step of further purifying the obtained beautiful millettia root polysaccharides MSCP1 and MSCP2, which comprises the following steps:
dissolving radix Millettiae Speciosae polysaccharides MSCP1 and MSCP2 in water to obtain radix Millettiae Speciosae polysaccharides MSCP1 and MSCP2 solutions; then adding the obtained product into Sephadex G-100 gel column, eluting with distilled water, collecting eluate, concentrating, dialyzing, and lyophilizing to obtain refined radix Millettiae Speciosae polysaccharides MSCP1 and MSCP 2;
the concentration of the beautiful millettia root polysaccharide MSCP1 solution is 3-3.5 mg/mL;
the concentration of the beautiful millettia root polysaccharide MSCP2 solution is 3-3.5 mg/mL;
the flow rate of eluting with distilled water is 0.1-0.3 mL/min;
the dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 3500 Da.
6. A beautiful millettia root polysaccharide is characterized in that: prepared by the method of any one of claims 1 to 5.
7. The beautiful millettia root polysaccharide of claim 6, which is characterized in that: the beautiful millettia root polysaccharide is at least one of beautiful millettia root polysaccharide MSCP1 and MSCP 2;
the weight average molecular weights of the beautiful millettia root polysaccharides MSCP1 and MSCP2 are respectively 9.36 multiplied by 103Da，2.85×104Da。
8. Use of the beautiful millettia root polysaccharide of claim 6 or 7 for the preparation of an immunoregulatory functional food or medicament.
9. Use of the beautiful millettia root polysaccharide according to claim 6 or 7 for the preparation of an antibacterial material or an antibacterial agent, characterized in that:
the antibiosis is to kill bacteria, inhibit the growth and/or reproduction of bacteria;
the bacteria are gram-negative bacteria.
10. Use according to claim 9, characterized in that: the bacteria is at least one of escherichia coli and salmonella.
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