CN113229496A - Compound of lotus root polysaccharide and polyphenol and preparation method thereof - Google Patents
Compound of lotus root polysaccharide and polyphenol and preparation method thereof Download PDFInfo
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- CN113229496A CN113229496A CN202110508275.6A CN202110508275A CN113229496A CN 113229496 A CN113229496 A CN 113229496A CN 202110508275 A CN202110508275 A CN 202110508275A CN 113229496 A CN113229496 A CN 113229496A
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Classifications
-
- 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
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- 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
-
- 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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- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Molecular Biology (AREA)
- Food Science & Technology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Sustainable Development (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of fruit and vegetable deep processing and functional food development, and particularly relates to a compound of lotus root polysaccharide and polyphenol and a preparation method thereof. The preparation method of the compound of the lotus root polysaccharide and the polyphenol comprises the following steps: exogenous polyphenol or lotus root polyphenol is compounded with lotus root polysaccharide to obtain the compound of lotus root polysaccharide and polyphenol. Based on the technical scheme, the compound of the lotus root polysaccharide and the polyphenol can be obtained, and the compound has obvious antioxidant and immunoregulatory activities.
Description
Technical Field
The invention belongs to the technical field of fruit and vegetable deep processing and functional food development, and particularly relates to a compound of lotus root polysaccharide and polyphenol and a preparation method thereof.
Background
The lotus roots are the big rhizome of lotus (Nelumbo nucifera Gaertn.), have more than two thousand years of cultivation history in China, and are the largest planting area and the highest yield of aquatic vegetables in China. According to statistics, the planting area of lotus roots in 2016 years reaches 40 kilohm2The yield reaches 1210 ten thousand tons, and the industrial development has good agricultural foundation. As a characteristic vegetable with homology of medicine and food, the lotus root has good flavor and nutrition, is both raw and cooked, and has the effects of strengthening spleen and tonifying qi, clearing heat and stopping bleeding, nourishing and soothing nerves, promoting digestion and stopping diarrhea, promoting granulation and the like. At present, processed products of lotus roots mainly comprise starch, beverages, pickled products, cans and leisure products, the types of the products are rich, the forms are various, and the development of functional products and the application of functional components are limited.
A large number of researches show that the lotus root has the effects of regulating immunity, resisting oxidation, reducing blood sugar, resisting obesity and the like, and has inseparable functional activity with polysaccharide and polyphenol. The lotus root contains abundant polysaccharide and polyphenol, and the processing byproducts (lotus root residue, lotus root peel and lotus root nodes) also have good development value, but the development and utilization of the lotus root polysaccharide and the polyphenol still have certain restrictions. The molecular weight of the Nelumbo nucifera polysaccharide is low (about 34kDa) and the molecular weight of the 58% fraction is below 6kDa, which may not form a highly reactive structure or conformation, resulting in a weak functional activity. At present, the structure-activity relationship and the structure modification of the lotus root polysaccharide are not reported. The polyphenol in lotus root exists in a free state, and has poor chemical stability and low bioavailability, so that the application of the polyphenol as an active ingredient in functional food is limited. The polyphenol and the polysaccharide can form a non-covalent complex, the combination can increase the physical stability, the biological activity and the bioavailability of the polyphenol, possibly increase the active sites of the polysaccharide and change the conformation of the polysaccharide, and simultaneously can form a synergistic effect, thereby increasing the application range of the polysaccharide and the polyphenol in functional food.
The problems existing in the prior related art are summarized as the following four points:
(1) after the lotus root tissue is crushed, phenolic substances are free and combined with polysaccharide through non-covalent interaction, so that the extraction rate of the polysaccharide and polyphenol is reduced.
(2) The molecular weight of the lotus root polysaccharide is small, the overall bioactivity is weak, and the content of the high-activity fraction obtained by the activity tracking screening method is low, so that the requirements of development and utilization cannot be met.
(3) Although the traditional chemical modification methods such as carboxymethylation, acetylation, sulfation and the like can improve the biological activity of the lotus root polysaccharide, the potential safety hazard of toxic and harmful reagent residues exists.
(4) The lotus root polyphenol has poor physical and chemical stability and low bioavailability, and has certain defects when being directly added and applied as a functional food ingredient.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a compound of lotus root polysaccharide and polyphenol and a preparation method thereof.
The technical scheme provided by the invention is as follows:
a preparation method of a compound of lotus root polysaccharide and polyphenol comprises the following steps: exogenous polyphenol or lotus root polyphenol is compounded with lotus root polysaccharide to obtain the compound of lotus root polysaccharide and polyphenol.
Based on the technical scheme, the compound of the lotus root polysaccharide and the polyphenol can be obtained, and the compound has obvious antioxidant and immunoregulatory activities.
Further, the preparation method of the lotus root polyphenol comprises the following steps: inhibiting non-covalent interaction of lotus root polyphenol and lotus root polysaccharide to obtain an extraction system containing lotus root polyphenol in a free state, and separating lotus root polyphenol from the extraction system.
Specifically, the preparation method of the lotus root polyphenol specifically comprises the following steps:
1) obtaining a lotus root polyphenol to-be-extracted solution, wherein a solvent is 80% -90% ethanol preheated to 55-65 ℃;
2) stirring and leaching the to-be-extracted solution of the lotus root polyphenol obtained in the step 1) for 2-4 hours at 45-55 ℃ to obtain a leaching solution;
3) centrifuging the leaching solution obtained in the step 2) at 45-55 ℃ to obtain a supernatant;
4) concentrating the supernatant obtained in the step 3) at 55-65 ℃ in vacuum until ethanol is completely volatilized, adding ethyl acetate for extraction, and separating to obtain an ethyl acetate extract phase;
5) and (3) concentrating the extract phase obtained in the step 4) at 55-65 ℃ in vacuum until ethyl acetate is completely volatilized, and freeze-drying the concentrated solution to obtain the lotus root polyphenol.
The above technical scheme improves the extraction rate of the lotus root polyphenol based on the non-covalent interaction inhibition of the polysaccharide-polyphenol: in the extraction process of lotus root polyphenol, the temperature of an extraction system is kept between 45 and 55 ℃, the precipitation loss of polyphenol and polysaccharide after the combination of the polyphenol and the polysaccharide through non-covalent interaction is prevented, and the oxidation and degradation of the polyphenol caused by high temperature are avoided as much as possible
More specifically, fresh lotus roots or processing byproducts (lotus root residues, lotus root peels, lotus root nodes and the like) of the fresh lotus roots are taken, washed and crushed, added with 80% -90% ethanol which is 5 times of the mass and preheated to 60 ℃ for homogenate, stirred and extracted for 3 hours at the temperature of 45-55 ℃, and centrifuged (4500r/min, 10min) to separate supernatant while the mixture is hot; concentrating the supernatant at 60 deg.C under vacuum until ethanol is completely volatilized, adding ethyl acetate for extraction, and separating ethyl acetate extract phase; vacuum concentrating the extractive solution at 60 deg.C until ethyl acetate is completely volatilized, freeze drying the concentrated solution to obtain rhizoma Nelumbinis polyphenol, evaporating, condensing, and recovering ethanol and ethyl acetate for reuse.
Further, the preparation method of the lotus root polysaccharide comprises the following steps: inhibiting the non-covalent interaction of lotus root polyphenol and lotus root polysaccharide to obtain an extraction system containing lotus root polysaccharide in a free state, and separating lotus root polysaccharide from the extraction system.
Specifically, the preparation method of the lotus root polysaccharide specifically comprises the following steps:
1) obtaining a lotus root polysaccharide to-be-extracted solution, wherein a solvent is water preheated to 55-65 ℃;
2) stirring and leaching the lotus root polysaccharide to-be-extracted solution obtained in the step 1) for 1-3 hours at 85-95 ℃ to obtain a leaching solution;
3) filtering and separating the leaching liquor obtained in the step 2) at 85-95 ℃, adding ethanol into the separation liquor, keeping the mixed liquor standing at 35-45 ℃ for 2-4 h, and precipitating starch and insoluble particles, wherein the volume ratio of the extracting liquor to the added ethanol is 2: 8-4: 6;
4) centrifuging the supernatant obtained in the step 3), adding ethanol into the supernatant, standing for 10-18 h at 3-5 ℃, and centrifuging and separating precipitates, wherein the volume ratio of the supernatant to the added ethanol is 7: 3-8: 2;
5) washing the precipitate obtained in the step 4) with alcohol, and performing vacuum drying at 45-55 ℃ to obtain the lotus root polysaccharide.
The technical scheme is based on the non-covalent interaction inhibition of polysaccharide-polyphenol, and the extraction rate of the lotus root polysaccharide is improved: in the extraction and separation process of lotus root polyphenol, 30% ethanol is added into the extracted supernatant and then the extracted supernatant is kept at 45 ℃, so that the macromolecular compound of polysaccharide and polyphenol which are combined in a non-covalent way is prevented from precipitating in an ethanol system at 35-45 ℃, and the starch is ensured to be fully precipitated and removed in the ethanol system.
More specifically, fresh lotus roots or processing byproducts (lotus root residues, lotus root peels, lotus root nodes and the like) thereof are taken, washed, crushed, added with 5 times of pure water preheated to 60 ℃ by mass for homogenate, heated to about 90 ℃, stirred and leached for 2 hours; filtering and separating an extracting solution while the extracting solution is hot by adopting a 100-200-mesh filter screen, adding ethanol into the extracting solution until the volume concentration is 30%, and standing for 3 hours at 35-45 ℃ to precipitate starch and insoluble particles; centrifuging at 4500r/min for 10min to separate supernatant, adding ethanol to volume concentration of 75%, standing at 4 deg.C for 12 hr, centrifuging to separate precipitate, sequentially washing the precipitate with 75% and 100% ethanol, and vacuum drying at 50 deg.C to obtain rhizoma Nelumbinis polysaccharide; mixing the ethanol solutions, vacuum heating at 50 deg.C, condensing, and recovering ethanol for reuse.
Specifically, the exogenous polyphenol is selected from catechin or chlorogenic acid.
Specifically, the complexing mode is non-covalent binding.
Specifically, the non-covalent binding method specifically comprises the following steps: mixing an aqueous solution of exogenous polyphenol or an aqueous solution of lotus root polyphenol with an aqueous solution of lotus root polysaccharide, adding NaCl to the concentration of 0-50 mmol/L, adjusting the pH to 5-7, stirring at 0-30 ℃ (ice bath can be adopted at 0 ℃), concentrating by adopting a membrane separation device with the molecular weight cutoff of 1000Da after stirring, and freeze-drying the concentrated solution to obtain the compound of lotus root polysaccharide and polyphenol.
The temperature, the pH, the mass ratio of the polysaccharide to the polyphenol and the ion concentration are important factors influencing the non-covalent interaction of the lotus root polysaccharide and the polyphenol, the technical scheme improves the combination rate of the polyphenol through specific parameters, prepares a high-activity lotus root polysaccharide-polyphenol compound, and can obtain enhanced antioxidant and immunoregulation activities.
More specifically, the lotus root polyphenol or commercial phenolic compounds (catechin, chlorogenic acid and the like) extracted by the method are prepared into 2mg/mL aqueous solution A, the lotus root polysaccharide in the step II is prepared into 2mg/mL aqueous solution B, the solution A and the solution B are mixed according to the volume ratio of 4: 1-1: 1, the pH value is adjusted to 5-7, and the mixture is stirred at room temperature or in an ice water bath at a low speed for 30 min; and after stirring, concentrating by adopting a membrane separation device with the molecular weight cutoff of 1000Da, and freeze-drying the concentrated solution to obtain the lotus root polysaccharide-polyphenol compound.
The invention also provides a compound of the lotus root polysaccharide and the polyphenol.
The compound of lotus root polysaccharide and polyphenol obtained based on the scheme shows excellent antioxidant and immunoregulatory activity, and is superior to that of corresponding lotus root polysaccharide. Especially in the aspect of macrophage function regulation, compared with the corresponding polysaccharide or polysaccharide-polyphenol mixture, the polysaccharide-polyphenol compound has the functions of immunostimulation on normal macrophages and anti-inflammation on macrophages induced by inflammation, and can better regulate the immune function of the macrophages. And the macrophage immunoregulation activity of the polysaccharide complex of catechin and chlorogenic acid is superior to that of the polysaccharide complex of caffeic acid, gallic acid, ferulic acid and endogenous polyphenol.
Drawings
Fig. 1 shows the infrared spectrum characteristics of the lotus root polysaccharide and its five polyphenol compounds and mixtures.
Fig. 2 is an atomic force microscope image of the lotus root polysaccharide and its five polyphenol compounds and mixtures according to the embodiment of the present invention.
Fig. 3 shows the antioxidant activity of the lotus root polysaccharide and the five polyphenol compounds and mixtures thereof provided by the embodiment of the invention.
FIG. 4 is a graph showing the effect of Nelumbo nucifera Gaertn and its five polyphenol compounds and mixtures on macrophage NO production in normal and lipopolysaccharide-induced inflammatory states, as provided by examples of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
It will be understood that when an element or component is referred to as being "connected," "positioned" or "coupled" to another element or component, it can be directly on the other element or component or intervening elements or components may also be present. The terms "left", "right", "upper", "lower" and the like as used herein are for illustrative purposes only.
Example 1
Step one, taking fresh lotus roots or processing byproducts (lotus root residues, lotus root peels, lotus root nodes and the like) of the fresh lotus roots, cleaning, crushing, adding 85% ethanol with 5 times of mass and preheating to 60 ℃ for homogenizing, stirring and leaching for 3 hours at 50 ℃, and centrifuging (4500r/min, 10min) to separate supernate while hot; and (3) concentrating the supernatant in vacuum at 60 ℃ until the ethanol is completely volatilized, adding ethyl acetate for extraction, separating to obtain an ethyl acetate extract phase, concentrating the extract phase in vacuum at 55-65 ℃ until the ethyl acetate is completely volatilized, freeze-drying the concentrated solution to obtain the lotus root polyphenol, and condensing and recycling the ethanol solution for reuse.
Step two, taking fresh lotus roots or processing byproducts (lotus root residues, lotus root peels, lotus root nodes and the like) of the fresh lotus roots, cleaning, crushing, adding 5 times of pure water preheated to 60 ℃ by mass, homogenizing, heating to about 90 ℃, stirring and leaching for 2 hours; filtering and separating the extracting solution while the extracting solution is hot by adopting a filter screen of 150 meshes, adding ethanol into the extracting solution until the volume concentration is 30%, and standing for 3 hours at 40 ℃ to precipitate starch and insoluble particles; centrifuging at 4500r/min for 10min to separate supernatant, adding ethanol to volume concentration of 75%, standing at 4 deg.C for 12 hr, centrifuging to separate precipitate, sequentially washing the precipitate with 75% and 100% ethanol, and vacuum drying at 50 deg.C to obtain rhizoma Nelumbinis polysaccharide; mixing the ethanol solutions, vacuum heating at 50 deg.C, condensing, and recovering ethanol for reuse.
Step three, preparing the lotus root polyphenol in the step one into a 1g/mL aqueous solution A, preparing the lotus root polysaccharide in the step two into a 2mg/mL aqueous solution B, mixing the solutions A and B according to the volume ratio of 1:1, adjusting the pH value to 6, and stirring at a low speed for 30min at room temperature or in an ice water bath; and after stirring, concentrating by adopting a membrane separation device with the molecular weight cutoff of 1000Da, and freeze-drying the concentrated solution to obtain the lotus root polysaccharide-lotus root polyphenol compound.
Examples 2 to 6
The procedure is as in example 1, and commercial polyphenols were used for the polyphenols: catechin, gallic acid, chlorogenic acid, caffeic acid and ferulic acid.
As shown in table 1, the molecular weight distributions of the lotus root polysaccharide and the five complexes thereof are determined by the combination of the high performance size exclusion chromatography and the differential refraction detection and the multi-angle laser scattering provided by the embodiment of the invention.
TABLE 1
As shown in fig. 1, it is the infrared spectrum characteristics of the lotus root polysaccharide and its five polyphenol compounds and mixtures provided by the embodiment of the present invention. As can be seen from the figure:
the phenolic substances (including catechin, gallic acid, chlorogenic acid, ferulic acid and caffeic acid) are in 1120cm-1And 617cm-1Characteristic absorption of the polysaccharide-polyphenol complex and infrared light not present in the polysaccharide-polyphenol complexIn the spectra, it is indicated that the polysaccharide and the polyphenol form non-covalent bonds.
Fig. 2 is an atomic force microscope image of the lotus root polysaccharide and its five polyphenol compounds and mixtures according to the embodiment of the present invention. The non-covalent binding mechanism of different phenols and lotus root polysaccharide is different, so that the aggregation behaviors among molecules are different. As can be seen, the molecular morphology of the polysaccharide complex of catechin and caffeic acid is similar to that of lotus root polysaccharide, the homopolymer is disc-shaped, but the diameter and height of the disc of the complex are larger; polysaccharide complexes of ferulic acid and chlorogenic acid form irregular clusters; the binding rate of gallic acid is low, and the binding causes the rigidity of the lotus root polysaccharide molecules to increase, so that the complex presents a loose aggregation state.
As shown in fig. 3, the antioxidant activities of the lotus root polysaccharide and the six polyphenol compounds thereof provided by the embodiment of the present invention are shown.
The test method for the DPPH free radical scavenging IC50 value is as follows:
taking 50 mu L of sample solution with different concentration gradients, adding 0.7mL of 100 mu mol/LDPPH solution, mixing uniformly, and reacting for 30min at room temperature in a dark place. The absorbance of the reaction solution was measured at 517min, methanol was used as a blank control, and three sets of replicates were performed for different sample concentrations and measurements. The DPPH radical scavenging ability is calculated by the following formula.
In the formula: a0 is the absorbance at 517nm of a mixture of 50. mu.L methanol and 0.7mL 100. mu. mol/L DPPH solution; ai is the light absorption value of a mixed solution of 50 mu L of sample solution and 0.7mL of 100 mu mol/L DPPH solution at 517 nm; aj is the absorbance at 517nm of 50. mu.L of the sample solution with 0.7mL of methanol.
And fitting a linear regression equation according to the DPPH free radical clearance rate corresponding to the concentration of the sample solution, and calculating the corresponding sample concentration when the DPPH free radical clearance rate is 50%, namely the IC50 value (mg/mL). The lower the IC50 value of the sample, the greater its antioxidant capacity.
The method for testing the total oxidation resistance of the FRAP comprises the following steps: the antioxidant capacity (mmol/g) of the sample is measured by adopting a total antioxidant capacity detection kit (FRAP method) according to the kit specification, and the higher the value is, the stronger the antioxidant capacity of the sample is.
As can be seen from fig. 3, the antioxidant performance of the complexes of the lotus root polysaccharides and the polyphenols is significantly higher than that of the lotus root polysaccharides.
As shown in fig. 4, the macrophage NO production regulating activity of the nelumbo nucifera gaertn and the six polyphenol compounds thereof provided in the present invention is shown.
The method for testing the NO production of normal macrophages comprises the following steps:
RAW264.7 cells were adjusted to a concentration of 5X 10 by DMEM medium (containing 10% volume fraction of fetal bovine serum)5cells/mL, 400. mu.L/well in 24-well plates, at 37 ℃ with 5% CO2After culturing for 3h, the culture solution is aspirated. Each well is added with 200 μ L of solution of Nelumbo nucifera polysaccharide or its complex prepared by dissolving in culture medium, and its concentration is 200 μ g/mL. Blank control wells were filled with 200. mu.L of medium, Lipopolysaccharide (LPS) control wells were filled with 200. mu.L of 1. mu.g/mL LPS medium solution. Each concentration was provided with 4 multiple wells. After the culture plate is incubated in an incubator for 24h, the cell supernatant is sucked into a 1.5mL centrifuge tube, and 300g/L ZnSO is added4Precipitating protein with the solution, centrifuging at 12000 r/min for 4min, taking 100 μ L of supernatant, adding 100 μ L of Grignard reagent into 96-well culture plate, slightly shaking for 10min at room temperature, measuring optical density at 492nm wavelength on enzyme labeling instrument, and repeating the measurement for 3 times. With NaNO2And establishing a standard curve to calculate the NO production of the macrophage.
The method for measuring the NO production of macrophages under lipopolysaccharide-induced inflammation comprises the following steps:
RAW264.7 cells were adjusted to a concentration of 5X 10 by DMEM medium (containing 10% volume fraction of fetal bovine serum)5cells/mL, 400. mu.L/well in 24-well plates, at 37 ℃ with 5% CO2After culturing for 3h, the culture solution is aspirated. Each well is added with 100 μ L of solution of lotus root polysaccharide or its complex prepared by dissolving culture medium, the concentration is 400 μ g/mL, and each well is supplemented with 100 μ L of LPS culture medium solution of 2 μ g/mL. Blank spaceControl wells were filled with 200. mu.L of medium and Lipopolysaccharide (LPS) control wells were filled with 200. mu.L of 1. mu.g/mL LPS medium solution. Each concentration was provided with 4 multiple wells. After the culture plate is incubated in an incubator for 24h, the cell supernatant is sucked into a 1.5mL centrifuge tube, and 300g/L ZnSO is added4Precipitating protein with the solution, centrifuging at 12000 r/min for 4min, taking 100 μ L of supernatant, adding 100 μ L of Grignard reagent into 96-well culture plate, slightly shaking for 10min at room temperature, measuring optical density at 492nm wavelength on enzyme labeling instrument, and repeating the measurement for 3 times. With NaNO2And establishing a standard curve to calculate the NO production of the macrophage.
As can be seen in FIG. 4A, both Nelumbo nucifera polysaccharide and its polyphenol complex stimulate NO production by normal macrophages. As can be seen from fig. 4B, the nelumbo nucifera polysaccharides can promote the generation of lipopolysaccharide-induced macrophage inflammation, but the catechin complexes and the chlorogenic acid complexes thereof can significantly inhibit the generation of inflammatory factor NO. The result shows that the non-covalent combination of catechin and chlorogenic acid can endow the lotus root polysaccharide with the macrophage bidirectional regulation function, and generate better immunoregulation effect.
The foregoing is merely a preferred embodiment of this invention and is not intended to limit the invention in any manner; those skilled in the art can readily practice the invention as shown and described in the drawings and detailed description herein; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (10)
1. A preparation method of a compound of lotus root polysaccharide and polyphenol is characterized by comprising the following steps: exogenous polyphenol or lotus root polyphenol is compounded with lotus root polysaccharide to obtain the compound of lotus root polysaccharide and polyphenol.
2. The method for preparing the lotus root polysaccharide-polyphenol compound as claimed in claim 1, wherein the method for preparing the lotus root polyphenol comprises the following steps: inhibiting non-covalent interaction of lotus root polyphenol and lotus root polysaccharide to obtain an extraction system containing lotus root polyphenol in a free state, and separating lotus root polyphenol from the extraction system.
3. The method for preparing the lotus root polysaccharide-polyphenol compound as claimed in claim 2, wherein the method for preparing lotus root polyphenol specifically comprises the following steps:
1) obtaining a lotus root polyphenol to-be-extracted solution, wherein a solvent is 80% -90% ethanol preheated to 55-65 ℃;
2) stirring and leaching the to-be-extracted solution of the lotus root polyphenol obtained in the step 1) for 2-4 hours at 45-55 ℃ to obtain a leaching solution;
3) centrifuging the leaching solution obtained in the step 2) at 45-55 ℃ to obtain a supernatant;
4) concentrating the supernatant obtained in the step 3) at 55-65 ℃ in vacuum until ethanol is completely volatilized, adding ethyl acetate for extraction, and separating to obtain an ethyl acetate extract phase;
5) and (3) concentrating the extract phase obtained in the step 4) at 55-65 ℃ in vacuum until ethyl acetate is completely volatilized, and freeze-drying the concentrated solution to obtain the lotus root polyphenol.
4. The method for preparing the lotus root polysaccharide and polyphenol compound as claimed in claim 1, wherein the method for preparing the lotus root polysaccharide comprises the following steps: inhibiting the non-covalent interaction of lotus root polyphenol and lotus root polysaccharide to obtain an extraction system containing lotus root polysaccharide in a free state, and separating lotus root polysaccharide from the extraction system.
5. The method for preparing the lotus root polysaccharide and polyphenol compound as claimed in claim 4, wherein the method for preparing lotus root polysaccharide specifically comprises the following steps:
1) obtaining a lotus root polysaccharide to-be-extracted solution, wherein a solvent is water preheated to 55-65 ℃;
2) stirring and leaching the lotus root polysaccharide to-be-extracted solution obtained in the step 1) for 1-3 hours at 85-95 ℃ to obtain a leaching solution;
3) filtering and separating the leaching liquor obtained in the step 2) at 85-95 ℃, adding ethanol into the separation liquor, keeping the mixed liquor standing at 35-45 ℃ for 2-4 h, and precipitating starch and insoluble particles, wherein the volume ratio of the extracting liquor to the added ethanol is 2: 8-4: 6;
4) centrifuging the supernatant obtained in the step 3), adding ethanol into the supernatant, standing for 10-18 h at 3-5 ℃, and centrifuging and separating precipitates, wherein the volume ratio of the supernatant to the added ethanol is 7: 3-8: 2;
5) washing the precipitate obtained in the step 4) with alcohol, and performing vacuum drying at 45-55 ℃ to obtain the lotus root polysaccharide.
6. The method for preparing the complex of lotus root polysaccharides and polyphenols according to any of claims 1 to 5, wherein: the exogenous polyphenol is selected from catechin or chlorogenic acid.
7. The method for preparing the lotus root polysaccharide and polyphenol compound as claimed in claim 6, wherein the method comprises the following steps: the complexing mode is non-covalent binding.
8. The method for preparing the complex of lotus root polysaccharides and polyphenols according to claim 7, wherein the non-covalent binding method specifically comprises the following steps: mixing an aqueous solution of exogenous polyphenol or an aqueous solution of lotus root polyphenol with an aqueous solution of lotus root polysaccharide, adding NaCl to the concentration of 0-50 mmol/L, adjusting the pH to 5-7, stirring at 0-30 ℃, concentrating by using a membrane separation device with the molecular weight cutoff of 1000Da after stirring, and freeze-drying the concentrated solution to obtain the compound of lotus root polysaccharide and polyphenol.
9. The method for preparing the complex of lotus root polysaccharides and polyphenols according to claim 8, wherein the method comprises the following steps: mixing the water solution of the exogenous polyphenol or the water solution of the lotus root polyphenol with the water solution of the lotus root polysaccharide, wherein the mass ratio of the polyphenol to the polysaccharide is (4:1) - (1: 1).
10. A compound of lotus root polysaccharide and polyphenol prepared by the preparation method according to any one of claims 6 to 9.
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