CN118546270A - Method for directly extracting lycium barbarum polysaccharide from fresh lycium barbarum - Google Patents

Abstract

The present invention belongs to the technical field of preparation of natural drug extracts, and relates to a method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum. The present invention directly takes fresh Lycium barbarum, processes it to obtain Lycium barbarum homogenate; then passes it through a disc centrifuge to remove solid insoluble matter and obtain a supernatant; adds ethanol to the supernatant, precipitates it with alcohol, separates the precipitate Lycium barbarum alcohol precipitation polysaccharide; then adds water to dissolve it, ultrafilters it, removes the filtrate, and obtains refined Lycium barbarum polysaccharide. The present invention does not need to dry fresh Lycium barbarum, which can save energy and reduce production costs; and can realize the processing of large quantities of fresh Lycium barbarum in a short time, improve production efficiency and also improve the utilization rate of Lycium barbarum, and avoid fresh Lycium barbarum from moldy deterioration and environmental pollution due to untimely treatment; the present invention does not involve a heating process, does not need to boil and extract Lycium barbarum, and is conducive to the complete preservation of the structure and spatial conformation of Lycium barbarum polysaccharide while saving energy and reducing consumption, so as to better retain its biological activity.

Description

A method for directly extracting wolfberry polysaccharides from fresh wolfberries

Technical Field

The invention belongs to the technical field of preparation of natural medicine extracts, and particularly relates to a method for directly extracting wolfberry polysaccharides from fresh wolfberries.

Background Art

Lycium barbarum polysaccharide is a water-soluble polysaccharide extracted and separated from the ripe fruit of Lycium barbarum L., a plant of the Solanaceae family. It is the most important biologically active substance in wolfberry. Modern research has shown that Lycium barbarum polysaccharide has multiple biological activities such as antioxidant, anti-aging, immunomodulatory, anti-cancer, neuroprotective, anti-diabetic and liver function improvement.

The preparation of Lycium barbarum polysaccharides is generally achieved by using dried Lycium barbarum as raw material through water extraction, alcohol precipitation, and membrane separation. However, drying fresh Lycium barbarum takes a long time and is easily polluted by the environment; Lycium barbarum has a high sugar content and is prone to mold and deterioration if not handled in time; drying Lycium barbarum requires special drying equipment and consumes a lot of energy; at the same time, when Lycium barbarum polysaccharides are extracted from dried Lycium barbarum as raw material, heating reflux method is often used to improve mass transfer efficiency. Therefore, from the perspective of technical economics, the production cycle of extracting Lycium barbarum polysaccharides from dried Lycium barbarum is long and the investment cost is high. Modern research has found that Lycium barbarum polysaccharides are also attached to proteins. Long-term heating may affect the structure and stereo conformation of the protein, thereby affecting the biological activity.

Lycium barbarum polysaccharide is a primary metabolite in wolfberry, which exists naturally in wolfberry. Some researchers have tried to extract Lycium barbarum polysaccharide from fresh wolfberry. Chinese patent CN1263108A "Lycium barbarum polysaccharide extraction and purification process" discloses a Lycium barbarum polysaccharide extraction and purification process. Fresh wolfberry juice is first separated by membrane to remove substances with a molecular weight greater than 500,000 Daltons (Da), and then substances with a molecular weight of 10,000 to 100,000 Da are retained. Lycium barbarum polysaccharide is then obtained by column chromatography. This method uses fresh wolfberry as raw material and uses ultrafiltration membrane to prepare Lycium barbarum polysaccharide. However, due to the viscous nature of wolfberry juice, it is impossible to directly process wolfberry juice using membrane separation technology during actual operation, so it is not feasible. Chinese patent CN101502316A "A method for extracting Lycium barbarum polysaccharides" discloses a method for extracting Lycium barbarum polysaccharides. After fresh Lycium barbarum is homogenized, microwave boiling is performed for 10 to 60 minutes, purified water is added to cool to 50 to 80°C, ultrasonic treatment is performed for 20 to 90 minutes, centrifugation is performed, the supernatant is taken, reduced pressure concentration is performed, and ethanol is added until the alcohol content reaches 80 to 90% for precipitation to obtain Lycium barbarum polysaccharides. This method uses microwave heating in the process of extracting Lycium barbarum polysaccharides, which may still affect the protein on Lycium barbarum polysaccharides. Moreover, as the molecular weight of Lycium barbarum polysaccharides increases, the water solubility decreases accordingly. This method may cause the loss of high molecular weight Lycium barbarum polysaccharides after adding purified water and centrifugation.

The above existing methods for extracting wolfberry polysaccharides have the following problems: (1) the polysaccharides are obtained by using dried wolfberries as raw materials through water extraction, alcohol precipitation, and membrane separation. However, drying fresh wolfberries is time-consuming and easily polluted; drying wolfberries requires special drying equipment and consumes a lot of energy; (2) When extracting wolfberry polysaccharides from dried wolfberries, in order to improve mass transfer efficiency, a heating reflux method is often used, which has a long production cycle and high investment costs; and long-term heating during the extraction process may affect the structure and stereo conformation of the protein attached to the wolfberry polysaccharide, thereby affecting its biological activity; (3) The prior art CN1263108A "Wolfberry Polysaccharide Extraction and Purification Process" uses fresh wolfberries as raw materials and adopts ultrafiltration membrane to prepare wolfberry polysaccharides. Due to the viscous nature of wolfberry juice, it is not feasible to process; (4) The prior art CN101502316A "A Method for Extracting Wolfberry Polysaccharides" uses microwave heating during the extraction of wolfberry polysaccharides, which may still affect the biological activity of the protein on the wolfberry polysaccharide. In addition, this method may cause the loss of high molecular weight wolfberry polysaccharides after adding purified water and centrifugation.

Therefore, it is necessary to study a method for directly preparing Lycium barbarum polysaccharides using fresh Lycium barbarum as raw material to overcome the defects in the prior art.

Summary of the invention

In view of the defects existing in the prior art, the inventor team of the present invention has developed an extraction method based on the long-term research results on wolfberry polysaccharides and their extraction methods, combined with the development status of the wolfberry industry and the feasibility of the process, which can directly extract wolfberry polysaccharides from fresh wolfberries without heating during the entire extraction process.

The object of the present invention is to provide a method for directly extracting wolfberry polysaccharides from fresh wolfberries.

In order to achieve the purpose of the present invention, the inventor adopts the following technical solution:

A method for directly extracting wolfberry polysaccharides from fresh wolfberries: taking fully mature fresh wolfberries and processing them to obtain wolfberry homogenate; passing the wolfberry homogenate through a centrifuge to remove solid insoluble matter to obtain a supernatant; adding ethanol to the supernatant to perform alcohol precipitation, separating the obtained precipitate to obtain wolfberry alcohol precipitation polysaccharide; further dissolving the obtained wolfberry alcohol precipitation polysaccharide in water, ultrafiltering, removing the filtrate to obtain refined wolfberry polysaccharide.

The relative density of the wolfberry homogenate is 1.01-1.05 at room temperature.

The processing method comprises adding water to fresh wolfberries and passing them through a colloid mill for homogenization, or squeezing and crushing the fresh wolfberries with a juicer, filtering to remove the skin and seeds, adding water to dilute, and passing them through a colloid mill for homogenization.

Lycium barbarum polysaccharides are macromolecules existing in organelles. When fresh wolfberries are passed through a colloid mill, the wolfberries are broken and their cell structures are destroyed, which is conducive to the outflow of the macromolecules of the wolfberry polysaccharide and improves the mass transfer efficiency.

The centrifuge is a disc centrifuge or a tubular centrifuge.

Further preferably, the adding of water is adding deionized water or purified water.

The ethanol is 95% by volume.

The alcohol precipitation is to add ethanol to make the alcohol content of the supernatant reach 80-90%, and then stand at 10-25° C. for 12-48 hours; the alcohol content is a volume percentage.

Further preferably, the alcohol precipitation is performed by adding ethanol to make the alcohol content of the supernatant of the wolfberry homogenate reach 80%.

The ultrafiltration is performed by filtering with an ultrafiltration membrane, and the molecular cutoff value of the ultrafiltration membrane is 3000-100000 Da.

The technical principles of the present invention are as follows:

Lycium barbarum homogenate processed from fresh wolfberries is a mixed multiphase dispersion system with water as the main solvent. As a water-soluble polysaccharide containing protein, Lycium barbarum polysaccharide naturally exists in Lycium barbarum homogenate. If ethanol is added to the Lycium barbarum homogenate to reduce the polarity of the mixed solvent, Lycium barbarum polysaccharide can be precipitated and separated from other components. However, Lycium barbarum homogenate has a high viscosity and also contains substances such as fiber and pectin. These macromolecular substances will agglomerate into lumps. If ethanol is added suddenly, it may lead to excessive local ethanol concentration, too fast precipitation, impurities wrapped, and affect product quality. Therefore, in the process of preparing wolfberry homogenate, it is necessary to add water to dilute it to adjust the density of the obtained wolfberry homogenate, and it is necessary to use a colloid mill to disperse the clumps formed by macromolecular substances so that the active substances in the wolfberries are evenly distributed in the wolfberry homogenate. Before alcohol precipitation, the obtained wolfberry homogenate needs to be passed through a disc centrifuge to separate and remove solid insoluble matter, and the supernatant of the wolfberry homogenate is taken. At this time, ethanol is added to the supernatant for alcohol precipitation, which can promote sufficient and complete precipitation, which is beneficial to ensure the quality consistency of the obtained wolfberry polysaccharide.

Compared with the prior art, the present invention has the following advantages.

1. The present invention directly uses fresh wolfberry as raw material to extract wolfberry polysaccharide, does not need to dry the wolfberry, and can save energy; and can realize the processing of a large number of fresh wolfberries in a short time, thereby improving production efficiency, while improving the utilization rate of wolfberries, and also avoiding the mold and deterioration of fresh wolfberries due to untimely processing and the resulting environmental pollution.

2. The process route adopted by the present invention does not involve a heating process and does not require the wolfberry to be boiled and extracted, which saves energy and reduces consumption while being conducive to the complete preservation of the structure and spatial conformation of wolfberry polysaccharides, thereby better retaining its biological activity.

3. The present invention can obtain Lycium barbarum polysaccharides through extrusion crushing, homogenization, alcohol precipitation, and solid-liquid separation. The process route is simple, the cost is low, the process has obvious applicability and economy, and has broad application prospects.

4. Lycium barbarum polysaccharides are macromolecules existing in cell organelles. According to the process provided by the present invention, when the polysaccharides are crushed by a colloid mill, the cell structure is destroyed, which is conducive to the outflow of macromolecules and improves the mass transfer efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an infrared spectrum of the wolfberry alcohol-precipitated polysaccharide prepared from fresh wolfberry as raw material, the refined wolfberry polysaccharide, and the wolfberry alcohol-precipitated polysaccharide prepared from dried wolfberry as raw material obtained in Example 1.

FIG. 2 is a gel permeation chromatogram of the wolfberry alcohol-precipitated polysaccharide prepared from fresh wolfberry as raw material, the refined wolfberry polysaccharide and the wolfberry alcohol-precipitated polysaccharide prepared from dried wolfberry as raw material obtained in Example 1.

3 is a graph showing the maximum ultraviolet absorption peak changes of the wolfberry alcohol-precipitated polysaccharide prepared with fresh wolfberry as raw material, the refined wolfberry polysaccharide and the wolfberry alcohol-precipitated polysaccharide prepared with dried wolfberry as raw material after combining with Congo red obtained in Example 1.

DETAILED DESCRIPTION

The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings, but the content of the present invention is not limited thereto.

Example

Example 1

Since polysaccharides in traditional Chinese medicine or natural medicine usually exist in the form of mixtures, it is difficult to keep the chemical composition of polysaccharides obtained by different extraction and separation processes consistent. The inventor team of the present invention has shown through research that the biological activities of Lycium barbarum polysaccharides, such as anti-aging, immunomodulation, anti-cancer, neuroprotection, anti-diabetes and improvement of liver function, are all related to their antioxidant activity. Therefore, in the process research of extracting Lycium barbarum polysaccharides using fresh Lycium barbarum as raw material, the inventors used simple and easy to operate, reproducible in vitro antioxidant activity as an indicator for process screening.

The inventor team screened the relative density of wolfberry homogenate and the alcohol precipitation process, and compared the differences between wolfberry polysaccharides obtained from fresh wolfberries and those prepared from dried wolfberries.

1. Screening of relative density of wolfberry homogenate.

During the screening process, the relative density, total solids content, polysaccharide content, iron ion reducing ability (FRAP) of the test solution, and the free radical scavenging ability of 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) of the test solution were determined. The specific methods and test results are described as follows.

1.1 Instruments and Materials

Instruments: Multiskan FC microplate reader, manual pipette (100~1000μL, 0.5~5mL), specific gravity bottle, colloid mill.

Materials: Fresh wolfberry, deionized water, ethanol, L-ascorbic acid, ferrous sulfate (FeSO ₄ ), 2,4,6-tri(2-pyridyl)triazine (TPTZ), ferric chloride (FeCl ₃ ), acetate buffer (pH 3.5), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate (K ₂ S ₂ O ₈ ).

1.2 Test methods

Preparation of test solution: Take fully mature fresh wolfberry, squeeze and crush, remove the skin and seeds, and obtain wolfberry pulp. Weigh 100.00g of wolfberry pulp, add different amounts of deionized water, and slurry it with a colloid mill. Determine the relative density of the wolfberry homogenate. Pass the wolfberry homogenate through a disc centrifuge to remove the solid insoluble matter and obtain the supernatant; add ethanol to the supernatant to make the alcohol content of the wolfberry homogenate supernatant reach 85%, let it stand overnight, and take the precipitate. Dissolve the precipitate with water and make up to volume in a 100mL volumetric flask as the test solution.

Preparation of reference solution of wolfberry puree (without homogenization): Take fully mature fresh wolfberries, squeeze and crush them, remove the skin and seeds, and obtain wolfberry puree. Weigh 100.00g of wolfberry puree and measure the relative density of wolfberry puree. Pass the wolfberry puree through a disc centrifuge to remove solid insolubles and obtain a supernatant; add ethanol to the supernatant to make the alcohol content of the wolfberry homogenate supernatant reach 85%, let it stand overnight, take the precipitate, dissolve the precipitate with water and make up to 100mL volumetric flask as the reference solution of wolfberry puree. Determine the relative density, total solids, polysaccharide content, FRAP value, and ABTS value of wolfberry homogenate according to the following determination methods.

Determination of relative density of wolfberry homogenate: According to the relative density determination method (General Rule 0601) of the "Chinese Pharmacopoeia" (Volume 4 of the 2020 edition), at room temperature, fill the density bottle with wolfberry homogenate, use filter paper to remove the liquid overflowing from the side tube, immediately cover it, accurately weigh it, subtract the weight of the density bottle, and obtain the weight of the wolfberry homogenate. Then pour out the wolfberry homogenate, wash the density bottle, fill it with freshly boiled deionized water, and then measure the weight of water at the same temperature according to the above method, and calculate the relative density of the wolfberry homogenate according to the following formula.

The relative density of wolfberry homogenate = weight of wolfberry homogenate/weight of water.

Determination of total solids: refer to the first method of the extract determination method (General Rule 2201) of the Chinese Pharmacopoeia (Volume 4 of the 2020 edition), accurately measure 5.0 mL of wolfberry homogenate, place it in a dry constant weight evaporating dish, evaporate to dryness in a water bath, dry at 105°C for 3 hours, cool in a desiccator for 30 minutes, and quickly and accurately weigh the weight. Then recover ethanol from the supernatant after alcohol precipitation, dissolve the residue with water and make up to volume in a 100 mL volumetric flask, accurately measure 5.0 mL of the solution, and weigh it according to the above method, and calculate the total solid content according to the following formula.

The content of total solids = [(the weight of total solids in wolfberry homogenate - the weight of total solids in supernatant) / the weight of total solids in wolfberry homogenate] × 100%.

Determination of polysaccharide content: Refer to the determination method of wolfberry polysaccharide content under "wolfberry" in the "Chinese Pharmacopoeia" (Volume 1 of the 2020 edition), and use anhydrous glucose as the reference substance to determine the content of polysaccharides in the precipitate. Accurately measure 1.0 mL of the test solution, place it in a stoppered test tube, add 1.0 mL of water, accurately add 1.0 mL of 5% phenol solution, shake well, quickly and accurately add 5.0 mL of sulfuric acid, shake well, let it stand for 10 minutes, quickly cool to room temperature, use the corresponding reagent as a blank, and measure the absorbance at a wavelength of 490 nm according to the UV-visible spectrophotometry (General Rule 0401), read the weight (mg) of glucose in the test solution from the standard curve, and calculate according to the following formula to obtain the content of wolfberry polysaccharide in terms of glucose (C ₆ H ₁₂ O ₆ ).

The content of polysaccharides in the test solution = [(weight of glucose in the test solution × 100)/total solids] × 100%.

Determination of the ferric ion reducing ability (FRAP) of the test solution: With FeSO ₄ as the reference substance, prepare different concentrations of FeSO ₄ solution (0.050mmol/L~2.000mmol/L), accurately pipette 0.20mL into a test tube, add 3.90mL of freshly prepared FRAP working solution, mix thoroughly, incubate at 37°C for 10min, use deionized water as the blank, and measure the absorbance at 593nm. Draw a standard curve with FeSO ₄ concentration as the horizontal axis and absorbance as the vertical axis. Then accurately pipette 0.20mL of the test solution into the test tube, and follow the preparation method of the standard curve, starting from "add 3.90mL of freshly prepared FRAP working solution", use ascorbic acid solution as the positive control, measure the absorbance according to the law, and read the FeSO ₄ value corresponding to the test solution from the standard curve.

Determination of the free radical scavenging ability of the test solution 2,2'-azobis(3-ethylbenzothiazole-6-sulfonic acid) diammonium salt (ABTS): Accurately pipette 0.20 mL of the test solution of different concentrations into a test tube, add 3.90 mL of freshly prepared ABTS working solution, mix thoroughly, incubate at room temperature for 5 minutes, measure the absorbance at 734 nm using deionized water as a blank and ascorbic acid solution as a positive control, calculate the ABTS free radical scavenging rate (%) according to the following formula, and calculate the _IC50 value.

ABTS free radical scavenging rate (%) = [1-(absorbance of experimental group-absorbance of background group)/absorbance of blank group] × 100%.

1.3 Test results

The viscosity of wolfberry homogenate is relatively large, and direct alcohol precipitation may cause the local ethanol concentration to be too high, and the precipitation may form too quickly, encapsulating small molecules. In addition, wolfberry homogenate also contains substances such as fiber and pectin. These macromolecular substances will agglomerate into blocks, affecting the mass transfer of ethanol in the dispersed system. Therefore, in the process of preparing wolfberry homogenate, it is necessary to add water to dilute to adjust the density of the obtained wolfberry homogenate, and it is necessary to use a colloid mill to disperse the agglomerates formed by macromolecular substances so that the active substances in wolfberry are evenly distributed in the wolfberry homogenate. Before alcohol precipitation, the obtained wolfberry homogenate needs to be passed through a disc centrifuge to separate and remove solid insolubles, and the supernatant of the wolfberry homogenate is taken. At this time, ethanol is added to the supernatant for alcohol precipitation, which can promote the precipitation to be sufficient and complete, thereby ensuring the quality of wolfberry polysaccharides. The results of the determination of total solids, polysaccharide content, FRAP value and ABTS free radical inhibition ability of wolfberry homogenates with different relative densities are shown in Table 1.

Table 1 lists the total solids, polysaccharide content and in vitro antioxidant capacity of the wolfberry homogenate with relative density adjusted by adding deionized water and the original wolfberry pulp without homogenization. From the data in the table, the wolfberry homogenate with relative density adjusted has significant differences in total solids and ABTS free radical scavenging ability compared with the original wolfberry pulp ( p <0.05), but there is no difference in polysaccharide content and reducing ability, indicating that the original wolfberry pulp is beneficial to improve the solid yield and polysaccharide content after colloid mill homogenization, and the scavenging effect on free radicals is enhanced. When preparing wolfberry polysaccharides with fresh wolfberries as raw materials, it is necessary to adjust the relative density and perform homogenization. After adjusting the relative density, there is no significant difference in total solids, polysaccharide content and in vitro antioxidant capacity of the wolfberry homogenate. When preparing wolfberry polysaccharides, the relative density can be adjusted to 1.01~1.05 according to actual conditions. If more deionized water is added to make the wolfberry homogenate thinner, the amount of water used will be too large, which will lead to an increase in the amount of ethanol used, and the process economy is poor.

2. Screening of alcohol precipitation process.

The alcohol precipitation process is an effective means to obtain macromolecular substances such as polysaccharides. Ethanol is added to the supernatant of wolfberry homogenate to change the ethanol concentration (volume percentage of ethanol) in the mixed solvent system, reduce the solubility of wolfberry polysaccharides therein, and precipitate from the dispersed system in the form of a precipitate, thereby separating from other substances. After adding ethanol to the supernatant of wolfberry homogenate, the ethanol concentration in the homogenate is different, and the precipitated wolfberry polysaccharides are also different. In order to screen the process of obtaining wolfberry polysaccharides from fresh wolfberries, the inventors still used in vitro antioxidant activity as an indicator to compare the iron ion reducing ability (FRAP), ABTS free radical scavenging ability and oxygen free radical adsorption capacity (ORAC) of the precipitates obtained with different ethanol concentrations (alcohol precipitation concentrations), and determined the ethanol concentration (volume percentage of ethanol) during alcohol precipitation.

2.1 Instruments and Materials

Instruments: Multiskan FC microplate reader, manual pipette (100~1000μL, 0.5~5mL), colloid mill.

Materials: fresh wolfberry, deionized water, ethanol, L-ascorbic acid, phosphate buffer (pH 7.4), sodium fluorescein, 2,2-azobisisobutylamidine dihydrochloride (AAPH), and other reagents are the same as those described under "Screening of relative density of wolfberry homogenate".

2.2 Test methods

Preparation of test solution: Take fully mature fresh wolfberry, squeeze and crush, remove the skin and seeds, and obtain wolfberry pulp. Weigh 100.00g of wolfberry pulp, add deionized water, colloid mill homogenize, adjust the relative density to 1.05 (room temperature), then pass the wolfberry homogenate through a disc centrifuge, remove the solid insoluble matter, and obtain the supernatant; add ethanol to the supernatant to make the alcohol content of the wolfberry homogenate supernatant reach 50%, 60%, 70%, 80%, 85%, and 90%, respectively, let it stand overnight, separate the precipitate, dissolve the precipitate with water and make up to volume in a 100mL volumetric flask as the test solution.

Determination of relative density of wolfberry homogenate: Same as described under "Screening of relative density of wolfberry homogenate".

Determination of total solids: Same as described under “Screening of relative density of wolfberry homogenate”.

Determination of polysaccharide content: Same as described in “Screening of relative density of wolfberry homogenate”.

Determination of the iron ion reducing ability (FRAP) of the test solution: Same as described under "Screening of relative density of wolfberry homogenate".

Determination of the 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging ability of the test solution: as described under "Screening of relative density of wolfberry homogenate".

Determination of Oxygen Radical Adsorption Capacity (ORAC) of Test Solution: Test solution was diluted to different concentrations with phosphate buffer (pH 7.4) and measured in black 96-well plates. Each well contained 20 µL of sample or L-ascorbic acid standard of different concentrations and 200 µL of sodium fluorescein solution (final concentration 0.96 µM). The blank solution was 75 mM phosphate buffer. The 96-well plate was placed on a multi-function microplate reader. After incubation at 37 °C for 20 min, 20 µL of 119.4 mM AAPH was added to each well, and the fluorescence decay curve was measured under fluorescence. The fluorescence conditions were: excitation wavelength 485 nm, emission wavelength 535 nm, and measurement every 2 min for a total of 66 cycles. The ORAC value results are reported as micromolar equivalents of L-ascorbic acid per gram of sample dry weight (DW), and the calculation formula is shown below.

Wherein: AUC _sample is the area under the sample curve, AUC _standard is the area under the standard curve, AUC _blank is the area under the blank control curve, CONC _sample is the sample concentration, and CONC _standard is the standard concentration.

2.3 Test results

The test results of total solid content, polysaccharide content, FRAP value of iron ion reducing ability, ABTS free radical scavenging ability and oxygen free radical adsorption capacity (ORAC) of the alcohol precipitated materials obtained by ethanol precipitation with different concentrations are shown in Table 2.

As shown in Table 2, the total solid and polysaccharide contents of the alcohol precipitates obtained by ethanol precipitation with different concentrations are quite different. With the increase of alcohol precipitation concentration, the total solid and polysaccharide contents increase accordingly, while the total solid and polysaccharide contents of 80%, 85% and 90% ethanol precipitations are not much different. Due to the different alcohol precipitation concentrations, the molecular structures of the obtained polysaccharides are also different, resulting in different antioxidant activities. As can be seen from Table 2, in the in vitro antioxidant activity, with the increase of ethanol concentration, the reducing ability (FRAP value) of the alcohol precipitate also increases accordingly, and the FRAP value of the 90% alcohol precipitation product is the largest; the oxygen free radical absorption capacity and reducing ability of Lycium barbarum polysaccharides show the same trend, and the ORAC value of the 90% alcohol precipitation product is also the largest, while the 85% ethanol precipitation shows the strongest ABTS free radical scavenging ability. However, there is no significant difference between the three in vitro antioxidant activities of the 80%, 85% and 90% alcohol precipitation products. There is no significant difference in the ABTS free radical scavenging ability of the products obtained by precipitation at all ethanol concentrations. Therefore, considering the total solids, polysaccharide content and antioxidant activity, when ethanol precipitation is performed, adjusting the ethanol concentration to 80~90% can obtain alcohol-precipitated polysaccharides with outstanding antioxidant capacity. From the perspective of technical and economic efficiency, choosing 80% ethanol precipitation is more advantageous.

3. Comparison of the differences between Lycium barbarum polysaccharides obtained from fresh Lycium barbarum and Lycium barbarum polysaccharides prepared from dried Lycium barbarum.

In order to verify the difference between wolfberry polysaccharides obtained from fresh wolfberry and wolfberry polysaccharides prepared from dried wolfberry, the inventors prepared wolfberry alcohol-precipitated polysaccharides from fresh wolfberry and further refined them to obtain refined wolfberry polysaccharides. At the same time, the same batch of fresh wolfberry was dried and wolfberry alcohol-precipitated polysaccharides were prepared by water extraction and alcohol precipitation, with an alcohol precipitation concentration of 80%.

The inventors conducted Fourier infrared spectroscopy, gel permeation chromatography, determination of monosaccharide composition and Congo red experiment, and compared the chemical structures and stereo conformations of wolfberry alcohol-precipitated polysaccharides prepared from fresh wolfberry and dried wolfberry as raw materials, as well as refined wolfberry polysaccharides prepared from fresh wolfberry as raw materials. The results are shown in Figures 1, 2 and 3. The monosaccharide composition of wolfberry alcohol-precipitated polysaccharides prepared from fresh wolfberry and wolfberry alcohol-precipitated polysaccharides prepared from dried wolfberry were compared. The results are shown in Table 3.

FIG. 1 is an infrared spectrum of the wolfberry alcohol-precipitated polysaccharide prepared from fresh wolfberry as raw material, the refined wolfberry polysaccharide and the wolfberry alcohol-precipitated polysaccharide prepared from dried wolfberry as raw material obtained in Example 1. As can be seen from the infrared spectrum in Figure 1, the wolfberry alcohol precipitated polysaccharide and refined wolfberry polysaccharide prepared from fresh wolfberry as raw material and the wolfberry alcohol precipitated polysaccharide prepared from dried wolfberry as raw material all present the typical chemical structure of polysaccharides, such as the absorption peak 3369cm ^-1 from the stretching vibration of hydroxyl group, the absorption peak 2936cm ^-1 caused by methylene, the absorption peak of CO in the carboxyl group 1612cm ^-1 , the absorption peak of CN 1421cm ^-1 , the absorption peaks 1055cm ^-1 and 920cm ^-1 caused by the pyran ring, and the absorption peaks of 816cm ^-1 and 777cm ^-1 of glycosidic bonds, indicating that the wolfberry polysaccharide prepared from fresh wolfberry as raw material and the wolfberry polysaccharide produced from dried wolfberry as raw material have little difference in chemical structure.

Fig. 2 is a gel permeation chromatogram of the alcohol-precipitated Lycium barbarum polysaccharide prepared from fresh Lycium barbarum as raw material, the refined Lycium barbarum polysaccharide and the alcohol-precipitated Lycium barbarum polysaccharide prepared from dried Lycium barbarum as raw material obtained in Example 1. As shown in Fig. 2, the main chromatographic peaks of the alcohol-precipitated Lycium barbarum polysaccharide prepared from fresh Lycium barbarum and the alcohol-precipitated Lycium barbarum polysaccharide prepared from dried Lycium barbarum are consistent, and the alcohol-precipitated Lycium barbarum polysaccharide prepared from fresh Lycium barbarum contains more polysaccharides with high molecular weight than the alcohol-precipitated Lycium barbarum polysaccharide prepared from dried Lycium barbarum.

Figure 3 is a graph showing the maximum ultraviolet absorption peak changes of the wolfberry alcohol precipitated polysaccharide prepared from fresh wolfberry as raw material, the refined wolfberry polysaccharide and the wolfberry alcohol precipitated polysaccharide prepared from dried wolfberry as raw material after being combined with Congo red obtained in Example 1. As shown in Figure 3, all three exhibited maximum ultraviolet absorption in 0.05 mol/L sodium hydroxide solution, and as the concentration of the sodium hydroxide solution increased, the maximum ultraviolet absorption peak decreased, indicating that all three had a triple helical structure.

Table 3 is a comparison of the monosaccharide compositions of Lycium barbarum alcohol precipitated polysaccharides prepared from fresh Lycium barbarum and Lycium barbarum alcohol precipitated polysaccharides prepared from dried Lycium barbarum. From the data in Table 3, it can be seen that the monosaccharide compositions of the two are basically the same.

Similarly, the inventors also compared the antioxidant activities of alcohol-precipitated polysaccharides prepared from fresh wolfberry, refined wolfberry polysaccharides and alcohol-precipitated polysaccharides prepared from dried wolfberry using FRAP, ABTS and ORAC. The results are shown in Table 4.

As shown in Table 4, there is no significant difference in the in vitro antioxidant activity of alcohol-precipitated polysaccharides prepared from fresh wolfberry and dried wolfberry, while the refined wolfberry polysaccharide prepared from fresh wolfberry showed better iron ion reducing ability (FRAP) and oxygen free radical adsorption capacity (ORAC). It can be seen that the preparation of wolfberry polysaccharide from fresh wolfberry is simple in process, saves energy, and can be used to process fresh wolfberry in large quantities and in a short time, with good process applicability and process economy.

Example 2

Take 1000g of fully mature fresh wolfberry, add 1000g of purified water, use a colloid mill to homogenize, and measure the relative density to be 1.01 (room temperature). Pass the obtained wolfberry homogenate through a disc centrifuge to remove the solid insoluble matter to obtain a supernatant; add ethanol to the supernatant to make the alcohol content reach 85%, place it at 10℃ for 24h, filter it, and dry it to obtain 215g of brown yellow powder wolfberry alcohol precipitation polysaccharide. The polysaccharide content is 8.50±0.32% measured by phenol-sulfuric acid method. Accurately weigh 1.000g of wolfberry alcohol precipitation polysaccharide powder, dissolve it in water and make it constant in a 5mL volumetric flask. The FRAR value is 0.71±0.02mmol/L, the IC ₅₀ for inhibiting ABTS free radicals is 4.70±0.15mg/mL, and the ORAC value is 8.28±0.07μmol/g.

Example 3

Take 1000g of fully mature fresh wolfberry, add 500g of purified water, use a colloid mill to homogenize, and measure the relative density to be 1.03 (room temperature). Pass the obtained wolfberry homogenate through a disc centrifuge to remove the solid insoluble matter to obtain a supernatant; add ethanol to the supernatant to make the alcohol content reach 90%, place it at room temperature for 12h, filter it, and dry it to obtain 205g of brown-yellow powder wolfberry alcohol precipitation polysaccharide. The polysaccharide content is 8.47±0.29% measured by the phenol-sulfuric acid method. Accurately weigh 1.000g of wolfberry alcohol precipitation polysaccharide powder, dissolve it in water and make it constant in a 5mL volumetric flask. The FRAR value is 0.66±0.02mmol/L, the IC ₅₀ for inhibiting ABTS free radicals is 5.30±0.10mg/mL, and the ORAC value is 7.74±0.11μmol/g.

Example 4

Take 10kg of fully mature fresh wolfberry, add 1000g of purified water, use a colloid mill to homogenize, and measure the relative density to be 1.05 (room temperature). Pass the obtained wolfberry homogenate through a disc centrifuge to remove the solid insoluble matter to obtain a supernatant; add ethanol to the supernatant to make the alcohol content reach 80%, place it at 10℃ for 48h, filter it, and dry it to obtain 226g of brown-yellow powder wolfberry alcohol precipitation polysaccharide. The polysaccharide content measured by phenol-sulfuric acid method is 9.07±0.30%. The wolfberry alcohol precipitation polysaccharide product is re-dissolved with an appropriate amount of purified water, and the ultrafiltration membrane is used to cut off the material with a molecular weight of 3000~100000Da, and dried to obtain 25.7g of light brown-yellow powder refined wolfberry polysaccharide. The polysaccharide content in the refined wolfberry polysaccharide is 43.76±1.48% measured by phenol-sulfuric acid method. 1.000 g of refined wolfberry polysaccharide powder was accurately weighed, dissolved in water and fixed to volume in a 5 mL volumetric flask. The FRAR value was measured to be 1.04±0.05 mmol/L, the _IC50 for inhibiting ABTS free radicals was 3.49±0.08 mg/mL, and the ORAC value was 9.92±0.18 μmol/g.

Example 5

The alcohol-precipitated Lycium barbarum polysaccharide and the refined Lycium barbarum polysaccharide obtained in Example 4 were tested for in vitro inhibition of α-glucosidase activity.

Sample preparation: Lycium barbarum alcohol-precipitated polysaccharide and refined Lycium barbarum polysaccharide were prepared into a 1 mg/mL stock solution with phosphate buffer (pH=7.0). α-Glucosidase (Shanghai Ruiyong Biotechnology Co., Ltd., 100U, stored at -20℃) was prepared into a 1 U/mL stock solution with phosphate buffer (pH=7.0), stored at -20℃, and diluted into a 0.2 U/mL solution with phosphate buffer (pH=7.0) before use. Positive control acarbose (Shanghai Aladdin Biochemical Technology Co., Ltd., stored dry at 4℃) was prepared into a 1 mg/mL stock solution with phosphate buffer (pH=7.0). 4-Nitrophenyl-α-D-pyranoglucopyranoside (Alfa Aesar (China) Chemical Co., Ltd., stored dry at 4℃) was prepared and diluted into a 2.5 mmol/L solution with phosphate buffer (pH=7.0). Na ₂ CO ₃ was prepared into a 0.2 mol/L solution with reverse osmosis water.

Determination of α-glucosidase inhibitory activity: 80 μL of 0.2 U/mL α-glucosidase solution was precisely added to the test tube, and then 50 μL of 1, 0.5, 0.25, 0.125, and 0.0625 mg/mL solutions of Lycium barbarum polysaccharide were added respectively, with acarbose as the positive control. After pre-incubation at 37°C for 10 min, 20 μL of p-nitrophenyl-α-D-pyranoglucoside solution was added, and then incubated for another 20 min. The terminator Na ₂ CO ₃

The reaction was terminated with 50 μL of the solution, and the absorbance of the mixed solution was read at 405 nm to determine the inhibition rate and calculate IC ₅₀ .

The results showed that both the alcohol-precipitated and purified Lycium barbarum polysaccharides exhibited inhibitory activity against α-glucosidase, with IC ₅₀ values of 0.22±0.02mg/mL and 0.18±0.03mg/mL, respectively.

Example 6

Take 10kg of fully mature fresh wolfberry, squeeze and crush it with a juicer, filter and remove the skin and seeds, add 900g of purified water to dilute, use a colloid mill to homogenize, and obtain a wolfberry homogenate, and the relative density is 1.05 (room temperature); pass the wolfberry homogenate through a disc centrifuge, separate and remove solid insolubles, and obtain a supernatant; add ethanol to the supernatant to make the alcohol content reach 80%, place it at 10℃ for 48h, filter, and dry to obtain 226g of brown-yellow powder wolfberry alcohol precipitation polysaccharide, and the polysaccharide content measured by phenol sulfuric acid method is 9.07±0.30%. The wolfberry alcohol precipitation polysaccharide product is re-dissolved with an appropriate amount of purified water, and the ultrafiltration membrane is used to cut off the material with a molecular weight of 3000~100000Da, and dried to obtain 25.2g of light brown-yellow powder refined wolfberry polysaccharide, and the polysaccharide content in the refined wolfberry polysaccharide is 43.15±1.52% measured by phenol sulfuric acid method. 1.000 g of refined wolfberry polysaccharide powder was accurately weighed, dissolved in water and fixed to volume in a 5 mL volumetric flask. The FRAR value was measured to be 1.02±0.05 mmol/L, the _IC50 for inhibiting ABTS free radicals was 3.42±0.06 mg/mL, and the ORAC value was 9.91±0.15 μmol/g.

The above is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any technical solution implemented within the scope of the present invention, or any possible changes and modifications made by any person skilled in the art using the above disclosed method content, all belong to the protection scope of the present invention.

Claims (10)

1. A method for directly extracting wolfberry polysaccharides from fresh wolfberries, characterized by: taking fully mature fresh wolfberries and processing them to obtain wolfberry homogenate; passing the wolfberry homogenate through a centrifuge to remove solid insoluble matter to obtain a supernatant; adding ethanol to the supernatant for alcohol precipitation, separating the obtained precipitate to obtain wolfberry alcohol precipitation polysaccharide; dissolving the obtained wolfberry alcohol precipitation polysaccharide in water, ultrafiltering, removing the filtrate, and obtaining refined wolfberry polysaccharide. 2. A method for directly extracting wolfberry polysaccharides from fresh wolfberries as described in claim 1, characterized in that the relative density of the wolfberry homogenate is 1.01~1.05 at room temperature. 3. A method for directly extracting wolfberry polysaccharides from fresh wolfberries as described in claim 1, characterized in that: the processing method is to add water to the fresh wolfberries and pass them through a colloid mill for homogenization, or to squeeze and crush the fresh wolfberries with a juicer, filter out the skin and seeds, dilute with water, and pass them through a colloid mill for homogenization. 4. A method for directly extracting wolfberry polysaccharides from fresh wolfberries as described in claim 1, characterized in that the fresh wolfberries are squeezed and crushed with a juicer, filtered to remove the skin and seeds, diluted with water, and homogenized with a colloid mill. 5. The method for directly extracting wolfberry polysaccharides from fresh wolfberries as claimed in claim 1, characterized in that the centrifuge is a disc centrifuge or a tubular centrifuge. 6. A method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum as claimed in claim 1, characterized in that the ethanol is 95% by volume ethanol. 7. A method for directly extracting wolfberry polysaccharides from fresh wolfberries as claimed in claim 1, characterized in that: the alcohol precipitation is to add ethanol to make the alcohol content of the wolfberry homogenate supernatant reach 80-90%; then stand at 10-25°C for 12-48 hours; the alcohol content is the volume percentage. 8. A method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum as claimed in claim 6, characterized in that: the alcohol precipitation is performed by adding ethanol to make the alcohol content of the supernatant reach 80%. 9. The method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum as claimed in claim 1, characterized in that the adding of water is adding deionized water or purified water. 10. The method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum according to claim 1, wherein the ultrafiltration is performed using an ultrafiltration membrane, and the molecular cutoff value of the ultrafiltration membrane is 3000-100000 Da.