阅读说明：本技术 一种荷叶多糖的提取方法 (Method for extracting lotus leaf polysaccharide ) 是由 俞保彬 张治国 曾静 于 2020-06-01 设计创作，主要内容包括：本发明涉及植物提取技术领域,具体公开了一种荷叶多糖的提取方法。本发明所述提取方法先对荷叶加水提取,然后将水提取液用纤维素酶、菠萝蛋白酶和果胶酶组成的复合酶进行酶提取,获得多糖提取液；多糖提取液醇沉、除蛋白、脱色后,获得荷叶多糖。本发明采用由纤维素酶、菠萝蛋白酶和果胶酶组成的复合酶辅助水提取法提取荷叶多糖,在保证荷叶多糖高收率的同时,提高荷叶多糖的含量,同时保证荷叶多糖还含有较高的多糖、总黄酮等活性物质,同时还表现出较高的免疫活性,研究出一条简洁、高效、合理的提取高活性荷叶多糖的工艺路线,可为其规模化生产、高值化利用奠定基础。(The invention relates to the technical field of plant extraction, and particularly discloses a method for extracting lotus leaf polysaccharide. The extraction method comprises the steps of firstly adding water into lotus leaves for extraction, and then carrying out enzyme extraction on a water extracting solution by using a complex enzyme consisting of cellulase, bromelain and pectinase to obtain a polysaccharide extracting solution; precipitating the polysaccharide extract with ethanol, removing protein, and decolorizing to obtain folium Nelumbinis polysaccharide. The invention adopts a complex enzyme-assisted water extraction method consisting of cellulase, bromelain and pectinase to extract the lotus leaf polysaccharide, improves the content of the lotus leaf polysaccharide while ensuring the high yield of the lotus leaf polysaccharide, ensures that the lotus leaf polysaccharide also contains higher active substances such as polysaccharide, total flavone and the like, and simultaneously shows higher immunocompetence, researches a simple, efficient and reasonable process route for extracting the high-activity lotus leaf polysaccharide, and can lay a foundation for large-scale production and high-valued utilization of the lotus leaf polysaccharide.)

1. The method for extracting the lotus leaf polysaccharide is characterized by comprising the following steps:

step 1, adding water into lotus leaves for extraction to obtain a water extract;

step 2, performing enzyme extraction on the water extract by using a complex enzyme consisting of cellulase, bromelain and pectinase to obtain a polysaccharide extract;

and 3, precipitating the polysaccharide extracting solution with ethanol, removing protein and decoloring to obtain the lotus leaf polysaccharide.

2. The extraction method according to claim 1, wherein the liquid-solid ratio of water to lotus leaves in step 1 is 20: 1-40: 1.

3. The extraction method according to claim 1, wherein the mass ratio of the cellulase, the bromelain and the pectinase is (1-5): 1-4.

4. The extraction method as claimed in claim 1, wherein the total addition amount of the complex enzyme is 0.2-0.6% of the water extract.

5. The extraction method according to claim 1, wherein the enzyme extraction is an enzyme extraction at 30 to 60 ℃.

6. The extraction method according to claim 1, wherein the alcohol precipitation is ethanol precipitation.

7. The extraction method as claimed in claim 1, wherein the deproteinization adopts sevag method to remove the protein.

8. The extraction method according to claim 1, wherein the decolorization is performed by activated carbon.

9. The extraction method according to any one of claims 1 to 8, further comprising lyophilizing the obtained lotus leaf polysaccharide.

Technical Field

The invention relates to the technical field of plant extraction, in particular to a method for extracting lotus leaf polysaccharide.

Background

The lotus leaf is the leaf of the lotus of the Nymphaeaceae, belongs to perennial herb and is one of the traditional Chinese medicinal materials in China. The lotus leaf contains various bioactive components such as polysaccharide, flavone, alkaloid and the like. The traditional medicine considers that the lotus leaves are bitter and cold in nature and have the activities of clearing summer heat, promoting diuresis, growing hair, clearing yang, clearing away heart-fire, removing heat, stopping bleeding and promoting diuresis. According to the record of the compendium of materia Medica: the lotus leaf is taken to make people slim, grow primordial qi, help spleen and stomach, astringe turbid urine, dissipate blood stasis, eliminate tumor and acne. Modern pharmacological research shows that the lotus leaves have the effects of resisting oxidation, resisting aging, reducing fat and losing weight, inhibiting fatty liver, inhibiting bacteria, inhibiting HIV proliferation, resisting virus, resisting inflammation, resisting allergy and the like.

The lotus leaf polysaccharide is an important bioactive component in lotus leaves, but the development of the lotus leaf polysaccharide in China is relatively laggard, most products can only meet the requirements of health care products, and the preparation and application of the high-purity lotus leaf polysaccharide are still in the research stage. The hot water extraction method is a traditional lotus leaf polysaccharide extraction method, and the method has low yield and high energy consumption. Therefore, the development of the high-activity lotus leaf polysaccharide extraction process has important practical significance.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for extracting lotus leaf polysaccharide, which can obtain lotus leaf polysaccharide with high yield;

another object of the present invention is to provide a method for extracting lotus leaf polysaccharide, which can obtain lotus leaf polysaccharide with high content;

another object of the present invention is to provide a method for extracting lotus leaf polysaccharide, which can obtain lotus leaf polysaccharide with high immunocompetence;

another object of the present invention is to provide a method for extracting lotus leaf polysaccharide, which can obtain lotus leaf polysaccharide with high polypeptide content;

another object of the present invention is to provide a method for extracting lotus leaf polysaccharide, which can obtain lotus leaf polysaccharide with high total flavone content;

another object of the present invention is to provide a method for extracting lotus leaf polysaccharide, which can simultaneously obtain lotus leaf polysaccharide with the above beneficial effects;

in order to achieve the above purpose, the invention provides the following technical scheme:

a method for extracting lotus leaf polysaccharide comprises the following steps:

step 1, adding water into lotus leaves for extraction to obtain a water extract;

step 2, performing enzyme extraction on the water extract by using a complex enzyme consisting of cellulase, bromelain and pectinase to obtain a polysaccharide extract;

and 3, precipitating the polysaccharide extracting solution with ethanol, removing protein and decoloring to obtain the lotus leaf polysaccharide.

The invention adopts an enzyme-assisted extraction method to extract the lotus leaf polysaccharide, reduces energy consumption and improves yield while ensuring the activity of the lotus leaf polysaccharide, provides a new development idea aiming at the current situation of high economic value and low utilization rate of lotus leaves in China, researches a simple, efficient and reasonable process route for extracting the lotus leaf polysaccharide with high activity, and lays a foundation for large-scale production and high-valued utilization of the lotus leaf polysaccharide.

The water extraction step in the step 1 can refer to the current water extraction method of the lotus leaf polysaccharide, specific parameters can be different according to actual situations, preferably, the liquid-solid ratio of water to lotus leaves in the step 1 is 20: 1-40: 1(m L: g), specifically, the liquid-solid ratio can be selected to be 20:1, 30:1 or 40:1, the water extraction temperature is preferably 60-100 ℃, the water extraction temperature is 75 ℃ in the specific implementation mode of the invention, and the water extraction time can be adjusted according to actual requirements, and is generally 1.5-3 hours.

Preferably, the mass ratio of the cellulase to the bromelain to the pectinase is (1-5) to (1-4); in the range, the mass ratio of the cellulase, the bromelain and the pectinase is (1-3) to (1-4) or (3-5) to (1-4); in a specific embodiment of the invention, the mass ratio of the cellulase, the bromelain and the pectinase is 3:4:4, 1:1:1: or 5:1: 1.

Preferably, the total addition amount of the complex enzyme is 0.2-0.6% of the water extract, and in the specific embodiment of the invention, the total addition amount is 0.2%, 0.4% or 0.6%.

In the step 2 of the invention, the optimum extraction temperature of the complex enzyme is usually selected, and is generally 30-60 ℃, and more preferably 45-55 ℃; the enzyme extraction time is generally 1-4 hours or longer, preferably 3-4 hours, and in the embodiments of the present invention, 3 hours, 3.5 hours, or 4 hours may be extracted. After enzyme extraction, high-temperature enzyme deactivation is generally carried out, and concentration can be carried out according to requirements.

The alcohol precipitation in step 3 of the present invention is generally an alcohol precipitation understood in the field of plant polysaccharide extraction, and generally ethanol is used to perform alcohol precipitation on a polysaccharide extract to obtain a precipitate, and an exemplary alcohol precipitation operation is given in the present invention: adding ethanol with the concentration of 92-96% and the volume of 3-5 times of that of the polysaccharide extracting solution, standing at 4 ℃ for 12 hours, and centrifuging to collect precipitates.

In the step 3, the protein is preferably removed by adopting a sevag method, and the specific operation is as follows:

precipitating with ethanol to obtain precipitate, adding a certain amount of purified water, and redissolving with polysaccharide water solution. Adding chloroform-n-butanol at a ratio of 4:1 and appropriate amount of proteolytic enzyme according to the volume of the polysaccharide aqueous solution 1/5. The mixture is violently shaken for 20min to 30min, and denatured protein at the junction of the fat-soluble substance and the solvent layer is separated. The steps are repeated for a plurality of times until no denatured protein is formed at the two intersection points to finish deproteinizing the sample.

The decolorization in the step 3 is preferably decolorized by activated carbon, and an alcohol precipitation operation with the following exemplary properties is given in the invention: and (3) pouring activated carbon with the volume of 20-30% of the solution into the extracting solution, uniformly stirring, placing in a 80-82 ℃ constant-temperature water bath kettle, preserving the heat for 30-45 minutes, and cooling the filtrate to room temperature after hot suction filtration.

The lotus leaf polysaccharide after alcohol precipitation, deproteinization and decoloration is in a solution state and can be dried into a solid by adopting a freeze-drying method.

On the premise of ensuring the consistency of other extraction steps, the extraction process of the invention compares the extraction process only adopting cellulase and the extraction process adopting the complex enzyme consisting of cellulase, papain and pectinase, and the result shows that the lotus leaf polysaccharide obtained by the extraction method of the invention is superior to the two comparison processes in yield, total polysaccharide content, polypeptide content, total flavone content and immunocompetence.

In addition, the lotus leaf polysaccharide obtained by the extraction method of the invention is proved to have better cleaning capability on hydroxyl free radicals and DPPH and stronger oxidation resistance through the measurement of oxidation resistance.

According to the technical scheme, the lotus leaf polysaccharide is extracted by adopting a complex enzyme-assisted water extraction method consisting of cellulase, bromelain and pectinase, the content of the lotus leaf polysaccharide is improved while the high yield of the lotus leaf polysaccharide is ensured, the lotus leaf polysaccharide also contains higher active substances such as polysaccharide, total flavone and the like, and higher immunocompetence is also shown, a simple, efficient and reasonable process route for extracting the high-activity lotus leaf polysaccharide is researched, and a foundation can be laid for large-scale production and high-valued utilization of the lotus leaf polysaccharide.

Detailed Description

The invention discloses a method for extracting lotus leaf polysaccharide, which can be realized by appropriately improving process parameters by taking the contents of the lotus leaf polysaccharide as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the extraction methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the extraction methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.

In the present embodiment, the comparative tests provided were identical for each treatment group and the test conditions were consistent for each group except for the differences.

The starting materials, reagents and enzymes to which the present invention relates are commercially available unless otherwise specified.

The analysis method of the yield, the total polysaccharide content, the polypeptide content, the total flavone content and the immunological activity of the invention refers to the following steps:

1. t cell Activity assay-De-E receptor assay for immune Activity

Reagent (1) Hank's solution: mixing 0.3% potassium dihydrogen phosphate solution, 0.76% disodium hydrogen phosphate solution, 2% potassium chloride solution and 20% sodium chloride solution in sequence according to a ratio of 20:20:20:40, adding 1g of glucose, dissolving, uniformly mixing, diluting with water to 1000ml, and adjusting the pH value to 7.2-7.3 with 4% sodium bicarbonate solution (prepared at the time of clinical application).

(2) Liquid A: taking 0.420g of sodium chloride, 0.055g of citric acid and 0.766g of sodium citrate, 2.05g of glucose, adding water to dissolve and dilute to 100ml, and sterilizing.

(3) Separating liquid: is lymphocyte separating liquid.

(4) Sheep blood: 5ml of sheep venous blood is taken and added into 5ml of the donkey-hide gelatin solution, and the mixture is stored in a refrigerator.

(5) Fixing liquid: mixing 25% glutaraldehyde solution, 3.5% sodium bicarbonate solution and) Hank's solution in a ratio of 1:1:38 in sequence. (6) Stock solution of giemsa staining solution: 0.5g of giemsa dye was added to 33ml of glycerin. Heating to 55-60 ℃ until the giemsa dye is dissolved, cooling to room temperature, adding 33ml of methanol, standing at room temperature for 24 hours, and filtering with filter paper to obtain a filtrate, namely the stock solution. Sealing and storing at room temperature.

(7) Dyeing liquid: taking 2ml of stock solution of the dyeing liquor of the giemsa, adding 6ml of Hank's liquor, shaking up, centrifuging at 1500 rpm for 10 minutes, and taking supernatant for later use.

An operation method (1) of preparing a suspension of the thymus T cells of the de-E receptor comprises the steps of taking fresh pig thymus, removing fat, cutting into pieces, adding a proper amount of Hank's solution to form a cell suspension, filtering through a 100-mesh sieve, centrifuging for 3-5 minutes at 1500 rpm, discarding supernatant, adding a small amount of Hank's solution to a centrifugal tube with 1/3 filter solution, centrifuging for 20 minutes at 2000 rpm, carefully sucking out thymocytes in the middle layer, putting the thymocytes into another centrifugal tube, adding a proper amount of Hank's washing solution, shaking uniformly, centrifuging for 3-5 minutes at 1500 rpm, discarding supernatant, adding Hank's solution, washing once, adding a proper amount of Hank's solution to a precipitate, mixing uniformly, keeping the temperature in a constant-temperature water bath at 45 ℃ for 30 minutes (shaking once every 5 minutes), centrifuging for 3-5 minutes at 1500 rpm, discarding supernatant, adding a proper amount of Hank's solution, mixing uniformly, keeping the temperature in a water bath at 45 ℃ for 30 minutes, taking out, centrifuging for 3-5 minutes at 1500 rpm, discarding supernatant, using Hank's solution, counting for 3-5 minutes, finally, counting for 3 × 10ml, and diluting the concentration of the Hank's solution before each operation (finally, counting for 10 ml)⁶)～(5×10⁵) And (4) cells.

(2) Preparation of sheep erythrocyte suspension: appropriate amount of sheep blood was collected and washed 3 times with appropriate amount of Hank's solution (same as above). And (4) discarding the supernatant, adding a proper amount of Hank's solution to dilute and count so that the final concentration is 8-10 times of the concentration of the E-removed receptor thymic T cell suspension.

(3) Preparation of a test solution: the test sample was taken and prepared into 1mg solution per 1ml with Hank's solution.

(4) The determination method comprises the following steps: taking 6 small test tubes, wherein, 0.1ml Hank's solution is added in each of 3 test tubes as a control tube, 0.1ml test sample solution is added in each of the other 3 test tubes as a test tube, and 0.2ml E-receptor-free thymus T cell suspension is added in each test tube. Keeping the temperature at 37 ℃ for 1 hour, adding 0.2ml of sheep erythrocyte suspension, shaking up, centrifuging for 3 minutes at 500 revolutions per minute, putting the mixture into a refrigerator at 4 ℃ for overnight, taking out the mixture the next day, removing supernatant, adding one drop of fixing solution into each tube, shaking up lightly, standing for 10 minutes, adding 2 drops of staining solution, shaking up, standing for 15 minutes, counting, taking lymphocytes as light blue larger cells in a microscope visual field, counting the number of all lymphocytes (not less than 200) on 16 large squares of a counting plate in total, and counting the number of cells formed by E rosettes (combining with the lymphocytes of more than 3 sheep erythrocytes). The percentage of flower formation was determined and averaged. The average value of the test tube or the control tube is obtained.

Sample viability assay tube E rosette percentage-control tube E rosette percentage

2. Phenol-sulfuric acid method for measuring total polysaccharide

Phenol solution: dissolving phenol solvent in water bath at 45 ℃, accurately weighing 6g of phenol solvent, placing the phenol solvent in a 100ml volumetric flask, dissolving the phenol solvent in water and fixing the volume to the scale.

Preparing a reference substance solution: adding appropriate amount of glucose control substance into water to obtain 1.0mg/ml solution, and placing 10ml of the above solution in 100ml volumetric flask to obtain 0.1mg/ml glucose standard solution.

Preparation of a standard curve: precisely measuring 0ml, 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml of standard solution, adding the standard solution into a 20ml glass colorimetric tube, supplementing the glass colorimetric tube with distilled water to 1.0ml, then adding 1ml of phenol solution, quickly adding 5ml of sulfuric acid, standing for 10min, and then placing the colorimetric tube in a water bath at 30 ℃ for reaction for 20 min.

Preparing a test solution: diluting the sample to an appropriate amount, controlling the absorbance to be between 0.3 and 0.7, placing the sample in an appropriate volumetric flask, adding water, ultrasonically dissolving, fixing the volume to a scale, uniformly mixing, filtering for later use, and treating the sample by using a standard solution method.

And (3) determination: taking water as a accompanying blank, respectively measuring absorbance at 490nm by ultraviolet-visible spectrophotometry (general rule 0401), drawing a standard curve by taking the absorbance as a vertical coordinate and the concentration as a horizontal coordinate, and calculating the content of polysaccharide in the lotus leaf extract according to the standard curve.

3. Determination of polypeptide content by Fulin phenol method

Preparing reference solution by precisely weighing 20mg of bovine serum albumin reference, placing in a 100ml volumetric flask, adding water to dissolve, and fixing volume to obtain 0.2mg of reference solution in each 1m L.

Preparing a standard curve, precisely measuring 0ml, 0.1ml, 0.3ml, 0.5ml, 0.7ml and 0.9ml of standard solution, respectively placing the standard solution in test tubes with plugs, supplementing the standard solution to 1.0ml with distilled water, respectively adding 1ml of alkaline copper test solution, uniformly mixing, respectively adding 4m L of forrin phenol test solution, immediately mixing the mixture uniformly, placing the mixture in a water bath at 55 ℃ for accurate reaction for 5 minutes, placing the mixture in an ice-water bath for reaction for 10 minutes, and standing the mixture at room temperature for 10 minutes.

Preparing a test solution: and (3) taking a sample, diluting the sample in a proper amount, controlling the absorbance to be between 0.3 and 0.7, and treating the sample by using a standard solution method for later use.

And (3) determination: taking water as blank, irradiating with ultraviolet-visible spectrophotometry (general rule 0401), measuring absorbance at 650nm, respectively, drawing standard curve with absorbance as ordinate and concentration as abscissa, and calculating polypeptide content in folium Nelumbinis extract according to the standard curve.

4. Functional study on hydroxyl radical clearance

(1) Mechanism of reaction

The ability to scavenge. OH was tested using a Fenton reaction model. The Fenton reaction is characterized by high OH reaction activity and short survival time, and takes H as a raw material₂O₂With Fe²⁺Mixing produces OH, salicylic acid is added to the system, which can effectively trap OH and produce a coloured substance which has a maximum absorption at 510 hm. When a substance having OH scavenging effect is present in the system, it competes with salicylic acid for OH, resulting in a decrease in the amount of colored substance produced and a decrease in the color of the solution, so that the OH scavenging ability of the substance can be calculated by measuring the absorbance of the sample.

(2) Research method

1m L FeSO was added to the test tube in sequence₄The solution 1m L salicylic acid-ethanol solution and sample solution 3m L with different concentrations are added with lm L H₂O₂The reaction was started, and the reaction was carried out at 37 ℃ for 30min, and the absorbance at 510nm was measured. The lotus leaf polysaccharide pair-OH clearance rate is calculated according to the following formula:

wherein A is₀Absorbance of blank control, A_iAbsorbance after sample addition, A_jIs the background absorbance of the sample solution.

5. Functional study on DPPH free radical clearance

(1) Mechanism of reaction

DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) is a stable free radical with nitrogen as the center, and the stability mainly comes from the steric barrier of three benzene rings with resonance stabilization, so that an unpaired electron on the middle nitrogen atom cannot exert the corresponding electron pairing effect. As the space barrier of the benzene ring leads the central nitrogen atom to carry a single electron, the single electron has strong absorption at about 517nm, and the solution can be deep purple. When DPPH meets substances with antioxidant activity, single electrons of DPPH are paired, the absorption of the paired single electrons at 517nm is weakened or even disappears, and the color of the solution is lightened, and the lightening degree is in quantitative relation with the number of electrons paired with DPPH free radicals. Therefore, the clearance rate of DPPH free radicals removed by a substance can be quantitatively analyzed by spectrophotometry.

(2) Research method

Respectively transferring sample solutions with different concentrations of 2m L, adding 2m L DPPH solution, mixing uniformly, standing in the dark for 30min, and measuring the absorbance of the solution at 517nm, wherein the clearance rate of the sample on DPPH free radicals is calculated according to the following formula:

wherein A is₀Absorbance of distilled water plus DPPH solution; a. the_sAdding the absorbance of the DPPH solution into the sample solution; a. the_bThe absorbance of the sample solution added with ethanol solvent.

6. Yield (%)

The invention is further illustrated by the following examples.