A method for directly extracting wolfberry polysaccharides from fresh wolfberries

Abstract

The present invention belongs to the technical field of preparation of natural drug extracts, and relates to a method for directly extracting wolfberry polysaccharides from fresh wolfberries. The present invention directly takes fresh wolfberries, processes them to obtain wolfberry homogenate; then passes them through a disc centrifuge to remove solid insoluble matter and obtain a supernatant; adds ethanol to the supernatant, precipitates them with alcohol, and separates the precipitate wolfberry alcohol-precipitated polysaccharide; then adds water to dissolve, ultrafilters, removes the filtrate, and obtains refined wolfberry polysaccharide. The present invention does not require drying fresh wolfberries, which can save energy and reduce production costs; and can achieve the processing of large quantities of fresh wolfberries in a short time, thereby improving production efficiency while also improving the utilization rate of wolfberries, and avoiding mold and deterioration of fresh wolfberries due to untimely processing and causing environmental pollution; the present invention does not involve a heating process, and does not require boiling and extracting wolfberries, 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 their biological activity.

Description

Technical Field

[0001] 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

[0002] Lycium barbarum polysaccharide is obtained from the Solanaceae plant Ningxia Lycium barbarum Lycium barbarum The water-soluble polysaccharide extracted and isolated from the ripe fruit of Lycium barbarum L. is the most important bioactive substance in Lycium barbarum. Modern research has shown that Lycium barbarum polysaccharides have multiple biological activities, including antioxidant, anti-aging, immunomodulatory, anti-cancer, neuroprotective, anti-diabetic and liver function improvement.

[0003] Lycium barbarum polysaccharides (Lycium barbarum polysaccharides) are typically prepared from dried wolfberries through a process involving water extraction, alcohol precipitation, and membrane separation. However, sun-drying fresh wolfberries is time-consuming and susceptible to environmental pollution. Lycium barbarum polysaccharides are high in sugar and can easily become moldy and spoil if not promptly processed. Drying wolfberries requires specialized drying equipment, which consumes significant energy. Furthermore, to improve mass transfer efficiency, a heating reflux method is often used to extract Lycium barbarum polysaccharides from dried wolfberries. Therefore, from a technical and economic perspective, extracting Lycium barbarum polysaccharides from dried wolfberries requires a long production cycle and high investment costs. Modern research has revealed that Lycium barbarum polysaccharides are also bound to proteins, and prolonged heating can affect their structure and conformation, thereby impacting their biological activity.

[0004] Lycium barbarum polysaccharides (Lycium barbarum polysaccharides) are primary metabolites naturally present in wolfberries. Researchers have attempted to extract Lycium barbarum polysaccharides using fresh wolfberries as a raw material. Chinese patent CN1263108A, "Lycium barbarum polysaccharide extraction and purification process," discloses a Lycium barbarum polysaccharide extraction and purification process. Fresh wolfberry juice is first subjected to membrane separation to remove substances with a molecular weight greater than 500,000 Daltons (Da), followed by a retention of substances with a molecular weight of 10,000 to 100,000 Da. Lycium barbarum polysaccharides are then obtained through column chromatography. This method uses fresh wolfberries as a raw material and employs ultrafiltration membranes to prepare Lycium barbarum polysaccharides. However, due to the viscous nature of wolfberry juice, direct membrane separation technology cannot be used to process the juice directly in practice, making it unfeasible. Chinese patent CN101502316A, "A Method for Extracting Lycium Barbarum Polysaccharides," discloses a method for extracting Lycium Barbarum polysaccharides. Fresh wolfberries are homogenized and then microwaved for 10-60 minutes. Purified water is added to cool the mixture to 50-80°C, followed by ultrasonic treatment for 20-90 minutes. The mixture is centrifuged, the supernatant is collected, concentrated under reduced pressure, and ethanol is added until the alcohol content reaches 80-90% for precipitation, thereby obtaining Lycium Barbarum polysaccharides. This method uses microwave heating during the extraction of Lycium Barbarum polysaccharides, which may still affect the proteins on the Lycium Barbarum polysaccharides. Furthermore, as the molecular weight of Lycium Barbarum polysaccharides increases, their water solubility decreases accordingly. This method, followed by centrifugation after the addition of purified water, may result in the loss of high-molecular-weight Lycium Barbarum polysaccharides.

[0005] The problems with the above-mentioned existing methods for extracting wolfberry polysaccharides are as follows: (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 by the environment; 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, the 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 three-dimensional conformation of the protein attached to the wolfberry polysaccharide, thereby affecting its biological activity; (3) The existing technology CN1263108A "Wolfberry Polysaccharide Extraction and Purification Process" uses fresh wolfberries as raw materials and adopts ultrafiltration membrane to prepare wolfberry polysaccharide. Due to the viscous nature of wolfberry juice, it is not feasible to process it; (4) The existing technology CN101502316A "A Method for Extracting Wolfberry Polysaccharide" uses microwave heating during the extraction of wolfberry polysaccharide, which may still affect the biological activity of the protein on the wolfberry polysaccharide, and the method may cause the loss of high molecular weight wolfberry polysaccharide after adding purified water and centrifugation.

[0006] 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

[0007] In response to the defects in the existing technology, 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 current development status of the wolfberry industry and the feasibility of the process. This method can directly extract wolfberry polysaccharides from fresh wolfberries without heating during the entire extraction process.

[0008] The object of the present invention is to provide a method for directly extracting wolfberry polysaccharides from fresh wolfberries.

[0009] In order to achieve the purpose of the present invention, the inventors adopted the following technical solutions:

[0010] A method for directly extracting wolfberry polysaccharides from fresh wolfberries comprises the following steps: taking fully mature fresh wolfberries and processing them to obtain a 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-precipitated polysaccharide; further dissolving the obtained wolfberry alcohol-precipitated polysaccharide in water, ultrafiltrating, and removing the filtrate to obtain refined wolfberry polysaccharide.

[0011] The relative density of the wolfberry homogenate is 1.01-1.05 at room temperature.

[0012] The processing method comprises the following steps: 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, diluting with water, and passing them through a colloid mill for homogenization.

[0013] Lycium barbarum polysaccharides are macromolecules that exist in organelles. When fresh wolfberries are passed through a colloid mill, the wolfberries are broken and their cell structure is destroyed, which is conducive to the outflow of the macromolecules of Lycium barbarum polysaccharides and improves the mass transfer efficiency.

[0014] The centrifuge is a disc centrifuge or a tubular centrifuge.

[0015] More preferably, the adding of water is adding deionized water or purified water.

[0016] The ethanol is 95% by volume.

[0017] The alcohol precipitation is performed by adding ethanol to make the alcohol content of the supernatant reach 80-90%, and then standing at 10-25° C. for 12-48 hours; the alcohol content is expressed as a volume percentage.

[0018] Further preferably, the alcohol precipitation is performed by adding ethanol to make the alcohol content of the wolfberry homogenate supernatant reach 80%.

[0019] The ultrafiltration is performed using an ultrafiltration membrane having a molecular cutoff value of 3000 to 100000 Da.

[0020] The technical principles of the present invention are as follows:

[0021] Lycium barbarum homogenate, processed from fresh goji berries, is a mixed multiphase dispersion system with water as the primary solvent. Lycium barbarum polysaccharides, a water-soluble polysaccharide containing protein, are naturally present in the goji berry homogenate. Adding ethanol to the goji berry homogenate reduces the polarity of the mixed solvent, allowing the polysaccharides to precipitate and separate from other components. However, goji berry homogenate has a high viscosity and also contains substances such as fiber and pectin, which can cause these macromolecules to aggregate into clumps. Adding ethanol suddenly can lead to excessively high local ethanol concentrations, causing precipitates to form too quickly, trapping impurities and compromising 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 a colloid mill is needed 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.

[0022] Compared with the prior art, the present invention has the following advantages.

[0023] 1. The present invention directly uses fresh wolfberries as raw materials to extract wolfberry polysaccharides, does not require wolfberry drying, and can save energy. It can also achieve the processing of large quantities of fresh wolfberries in a short time, thereby improving production efficiency and the utilization rate of wolfberries. It also avoids the mold and deterioration of fresh wolfberries due to untimely processing and the resulting environmental pollution.

[0024] 2. The process adopted by the present invention does not involve a heating process and does not require boiling and extracting wolfberries, 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 their biological activity.

[0025] 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, and it has obvious process applicability and economy, and has broad application prospects.

[0026] 4. Lycium barbarum polysaccharides are macromolecules present in organelles. According to the process provided by the present invention, when they are crushed with a colloid mill, the cell structure is destroyed, which is conducive to the outflow of macromolecules and improves mass transfer efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The infrared spectra 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 are shown.

[0028] Figure 2The gel permeation chromatograms 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 are shown.

[0029] Figure 3 This 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 combining with Congo red. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings, but the content of the present invention is not limited thereto. Example

[0031] Example 1

[0032] Because polysaccharides in traditional Chinese medicine or natural medicines typically exist as mixtures, it's difficult to maintain consistent chemical composition when using different extraction and separation processes. Research by the inventors of the present invention has shown that the biological activities of Lycium barbarum polysaccharides, including anti-aging, immunomodulation, anti-cancer, neuroprotection, anti-diabetes, and liver function improvement, are all related to their antioxidant activity. Therefore, in their research on extracting Lycium barbarum polysaccharides from fresh wolfberries, the inventors used simple, reproducible in vitro antioxidant activity as a metric for process screening.

[0033] 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.

[0034] 1. Screening of relative density of wolfberry homogenate.

[0035] During the screening process, the relative density, total solids content, polysaccharide content, ferric 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 below.

[0036] 1.1 Instruments and Materials

[0037] Instruments: Multiskan FC microplate reader, manual pipette (100-1000 μL, 0.5-5 mL), pycnometer, colloid mill.

[0038] Materials: fresh wolfberry, deionized water, ethanol, L-ascorbic acid, ferrous sulfate (FeSO4), 2,4,6-tris(2-pyridyl)triazine (TPTZ), ferric chloride (FeCl3), acetate buffer (pH 3.5), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulfate (K2S2O8).

[0039] 1.2 Test methods

[0040] Preparation of the test solution: Take fully ripe fresh wolfberries, crush them, and remove the skin and seeds to obtain wolfberry puree. Weigh 100.00 g of the wolfberry puree and add varying amounts of deionized water. Homogenize the mixture using a colloid mill and measure the relative density of the wolfberry homogenate. Pass the wolfberry homogenate through a disc centrifuge to remove insoluble solids to obtain a supernatant. Add ethanol to the supernatant to a concentration of 85% alcohol. Let the mixture stand overnight. Collect the precipitate, dissolve it in water, and dilute to volume in a 100 mL volumetric flask to prepare the test solution.

[0041] Preparation of a reference solution of wolfberry puree (unhomogenized): Take fully ripe fresh wolfberries, crush them, and remove the skin and seeds to obtain wolfberry puree. Weigh 100.00 g of the wolfberry puree and measure its relative density. Pass the wolfberry puree through a disc-type centrifuge to remove insoluble solids, obtaining a supernatant. Add ethanol to the supernatant to bring the alcohol content of the wolfberry homogenate to 85%. Allow to stand overnight, collect the precipitate, dissolve it in water, and dilute to volume in a 100 mL volumetric flask. This serves as the reference solution of wolfberry puree. Determine the relative density, total solids content, polysaccharide content, FRAP value, and ABTS value of the wolfberry homogenate using the following methods.

[0042] Determination of relative density of wolfberry homogenate: According to the relative density determination method (General Rule 0601) of the "Chinese Pharmacopoeia" (Part IV of the 2020 edition), at room temperature, fill the pycnometer 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 pycnometer, and obtain the weight of the wolfberry homogenate. Then pour out the wolfberry homogenate, wash the pycnometer, fill it with freshly boiled deionized water, and then measure the weight of water at the same temperature according to the above method. Calculate the relative density of the wolfberry homogenate according to the following formula.

[0043] The relative density of wolfberry homogenate = weight of wolfberry homogenate / weight of water.

[0044] Determination of Total Solids: Refer to Method 1 of the Extract Determination Method (General Chapter 2201) in the Chinese Pharmacopoeia (2020 Edition, Part IV). Accurately measure 5.0 mL of wolfberry homogenate and 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. Recover ethanol from the supernatant after alcohol precipitation. Dissolve the residue in water and dilute to volume in a 100 mL volumetric flask. Accurately measure 5.0 mL of the solution and weigh again as above. Calculate the total solids content according to the following formula.

[0045] Total solid content = [(the weight of the total solids of the wolfberry homogenate - the weight of the total solids of the supernatant) / the weight of the total solids of the wolfberry homogenate] × 100%.

[0046] Determination of polysaccharide content: Refer to the method for determining the content of wolfberry polysaccharides under the item "Wolfberry" in the "Chinese Pharmacopoeia" (Part 1, 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, and 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 method (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 polysaccharides in glucose (C6H 12 O6) content.

[0047] The content of polysaccharide in the test solution = [(weight of glucose in the test solution × 100) / total solids] × 100%.

[0048] Ferric Ion Reducing Ability (FRAP) Assay for the test solution: Prepare FeSO₄ solutions of varying concentrations (0.050 mmol / L to 2.000 mmol / L) using FeSO₄ as the reference. Accurately pipette 0.20 mL of FeSO₄ solutions into a test tube. Add 3.90 mL of freshly prepared FRAP working solution and mix thoroughly. Incubate at 37°C for 10 minutes. Measure absorbance at 593 nm using deionized water as a blank. Construct a standard curve with FeSO₄ concentration as the abscissa and absorbance as the ordinate. Then, accurately pipette 0.20 mL of the test solution into a test tube. Following the standard curve preparation procedure, starting with "Add 3.90 mL of freshly prepared FRAP working solution," measure absorbance using ascorbic acid solution as the positive control. Read the FeSO₄ value corresponding to the test solution from the standard curve.

[0049] Determination of the free radical scavenging ability of the test solution 2,2'-azino-bis(3-ethyl-benzothiazole-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 with deionized water as blank and ascorbic acid solution as positive control, and calculate the ABTS free radical scavenging rate (%) according to the following formula to calculate IC 50 value.

[0050] ABTS free radical scavenging rate (%) = [1-(absorbance of experimental group-absorbance of background group) / absorbance of blank group] × 100%.

[0051] 1.3 Test results

[0052] Lycium barbarum homogenate has a high viscosity, and direct alcohol precipitation may result in high local ethanol concentrations, resulting in rapid precipitation and the encapsulation of small molecules. Furthermore, Lycium barbarum homogenate contains substances such as fiber and pectin, which can aggregate into large molecules and affect the mass transfer of ethanol within the dispersion. Therefore, during the preparation of Lycium barbarum homogenate, dilution with water is necessary to adjust the density of the resulting Lycium barbarum homogenate. A colloid mill is also used to disperse any large molecules, ensuring that the active substances in the Lycium barbarum are evenly distributed throughout the homogenate. Furthermore, before alcohol precipitation, the resulting Lycium barbarum homogenate is passed through a disc centrifuge to remove insoluble solids. Adding ethanol to the supernatant for alcohol precipitation promotes complete precipitation, thereby ensuring the quality of Lycium barbarum polysaccharides. The total solids content, polysaccharide content, FRAP values, and ABTS free radical inhibition capacity of Lycium barbarum homogenates of different relative densities are shown in Table 1.

[0053]

[0054] Table 1 lists the total solids, polysaccharide content, and in vitro antioxidant capacity of the wolfberry homogenate after adding deionized water to adjust the relative density and the original wolfberry puree without homogenization. The data in the table show that the wolfberry homogenate after adjusting the relative density has significant differences in total solids and ABTS free radical scavenging capacity compared with the original wolfberry puree ( p<0.05), while there was no difference in polysaccharide content and reducing capacity. This suggests that colloid mill homogenization of wolfberry pulp is beneficial for improving solid yield and polysaccharide content, and enhancing the scavenging effect of free radicals. When preparing wolfberry polysaccharides from fresh wolfberries, it is necessary to adjust the relative density and perform homogenization. After adjusting the relative density, there was 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.

[0055] 2. Screening of alcohol precipitation process.

[0056] Alcohol precipitation is an effective method for obtaining macromolecular substances such as polysaccharides. By adding ethanol to the supernatant of a goji berry homogenate, the ethanol concentration (volume percentage of ethanol) in the mixed solvent system is altered, reducing the solubility of the goji berry polysaccharides, allowing them to precipitate from the dispersed system, thereby separating them from other substances. Adding ethanol to the supernatant of the goji berry homogenate results in different precipitates of goji berry polysaccharides depending on the ethanol concentration in the homogenate. To screen for a process for obtaining goji berry polysaccharides from fresh goji berries, the inventors used in vitro antioxidant activity as a metric, comparing the iron reducing power (FRAP), ABTS free radical scavenging capacity, and oxygen radical adsorption capacity (ORAC) of precipitates obtained at different ethanol concentrations (ethanol precipitation concentrations), and determining the optimal ethanol concentration (volume percentage of ethanol) for alcohol precipitation.

[0057] 2.1 Instruments and Materials

[0058] Instruments: Multiskan FC microplate reader, manual pipette (100-1000 μL, 0.5-5 mL), colloid mill.

[0059] 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".

[0060] 2.2 Test methods

[0061] Preparation of the test solution: Take fully mature fresh wolfberries, crush them, and remove the skin and seeds to obtain wolfberry puree. Weigh 100.00 g of wolfberry puree and add deionized water. Homogenize using a colloid mill to adjust the relative density to 1.05 (room temperature). Then, pass the wolfberry puree through a disc-type centrifuge to remove insoluble solids to obtain a supernatant. Add ethanol to the supernatant to adjust the alcohol content of the wolfberry homogenate supernatant to 50%, 60%, 70%, 80%, 85%, and 90%, respectively. Let stand overnight, collect the precipitate, dissolve it with water, and dilute to volume in a 100 mL volumetric flask to prepare the test solution.

[0062] Determination of relative density of wolfberry homogenate: Same as described under "Screening of relative density of wolfberry homogenate".

[0063] Determination of total solids: Same as described under “Screening of relative density of wolfberry homogenate”.

[0064] Determination of polysaccharide content: Same as described in “Screening of relative density of wolfberry homogenate”.

[0065] Determination of the iron reducing ability (FRAP) of the test solution: Same as described under "Screening of relative density of wolfberry homogenate".

[0066] Determination of the 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radical scavenging ability of the test solution: Same as described under "Screening of relative density of wolfberry homogenate".

[0067] Oxygen Radical Adsorption Capacity (ORAC) of the test sample solutions was determined by diluting the sample solutions to various concentrations in phosphate buffer (pH 7.4) and measuring them in black 96-well plates. Each well contained 20 µL of sample or L-ascorbic acid standard at various concentrations and 200 µL of sodium fluorescein solution (final concentration 0.96 µM). A blank solution consisted of 75 mM phosphate buffer. The 96-well plate was incubated at 37°C for 20 minutes. Then, 20 µL of 119.4 mM AAPH was added to each well, and fluorescence decay curves were measured under a fluorometer. Fluorescence conditions were: excitation at 485 nm, emission at 535 nm, with measurements taken every 2 minutes for 66 cycles. ORAC values ​​are reported as micromolar equivalents of L-ascorbic acid per gram dry weight (DW) of sample, calculated using the following formula.

[0068]

[0069] Where: 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, CONCsample is the sample concentration, CONC standard is the standard concentration.

[0070] 2.3 Test results

[0071] The test results of total solid content, polysaccharide content, FRAP value of iron ion reducing ability, ABTS free radical scavenging ability and oxygen radical adsorption capacity (ORAC) of the alcohol-precipitated materials obtained by precipitation with different concentrations of ethanol are shown in Table 2.

[0072]

[0073] Table 2 shows that the total solids and polysaccharide contents of the precipitates obtained by ethanol precipitation at different concentrations varied significantly. With increasing ethanol precipitation concentration, both total solids and polysaccharide contents increased accordingly, while the total solids and polysaccharide contents of the precipitates obtained with 80%, 85%, and 90% ethanol precipitations showed little difference. Due to the different ethanol precipitation concentrations, the molecular structures of the polysaccharides obtained also varied, leading to differences in antioxidant activity. Table 2 also shows that the reducing capacity (FRAP value) of the precipitates increased with increasing ethanol concentration, with the 90% ethanol precipitation product having the highest FRAP value. The oxygen free radical absorbance capacity and reducing capacity of Lycium barbarum polysaccharides showed similar trends, with the 90% ethanol precipitation product also having the highest ORAC value, while the 85% ethanol precipitation product exhibited the strongest ABTS free radical scavenging activity. However, no significant differences were found among the 80%, 85%, and 90% ethanol precipitation products in these three in vitro antioxidant activities. There was no significant difference in ABTS free radical scavenging activity among the products precipitated 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.

[0074] 3. Comparison of the differences between Lycium barbarum polysaccharides obtained from fresh Lycium barbarum and those prepared from dried Lycium barbarum.

[0075] In order to verify the difference between the wolfberry polysaccharides obtained from fresh wolfberry and those 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 using the water extraction and alcohol precipitation method with an alcohol precipitation concentration of 80%.

[0076] The inventors conducted Fourier infrared spectroscopy, gel permeation chromatography, determination of monosaccharide components and Congo red experiments, and compared the chemical structure and stereo conformation of Lycium barbarum alcohol-precipitated polysaccharides prepared from fresh and dried Lycium barbarum berries, as well as the refined Lycium barbarum polysaccharide prepared from fresh Lycium barbarum berries. Figure 1 、 Figure 2 、 Figure 3 The monosaccharide composition of Lycium barbarum alcohol-precipitated polysaccharides prepared from fresh Lycium barbarum and Lycium barbarum alcohol-precipitated polysaccharides prepared from dried Lycium barbarum was compared, and the results are shown in Table 3.

[0077] Figure 1 The infrared spectra of the Lycium barbarum alcohol-precipitated polysaccharide prepared from fresh Lycium barbarum as raw material, the refined Lycium barbarum polysaccharide and the Lycium barbarum alcohol-precipitated polysaccharide prepared from dried Lycium barbarum as raw material obtained in Example 1 are shown in FIG. Figure 1 The infrared spectra in the figure show that the alcohol-precipitated Lycium barbarum polysaccharide prepared from fresh Lycium barbarum fruit, the refined Lycium barbarum polysaccharide and the alcohol-precipitated Lycium barbarum polysaccharide prepared from dried Lycium barbarum fruit all present the typical chemical structure of polysaccharides, such as the absorption peak at 3369cm-1 derived from the stretching vibration of hydroxyl groups. -1 , the absorption peak caused by methylene is 2936cm -1 The absorption peak of CO in the carboxyl group is 1612 cm -1 , the absorption peak of CN is 1421cm -1 , the absorption peak caused by the pyran ring is 1055cm -1 , 920cm -1 etc., and the absorption peak of glycosidic bond at 816 cm -1 and 777cm -1 , which shows that the chemical structure of wolfberry polysaccharide prepared from fresh wolfberry as raw material is very little different from that produced from dried wolfberry as raw material.

[0078] Figure 2 This is the gel permeation chromatogram of the Lycium barbarum alcohol-precipitated polysaccharide prepared from fresh Lycium barbarum as raw material, the refined Lycium barbarum polysaccharide and the Lycium barbarum alcohol-precipitated polysaccharide prepared from dried Lycium barbarum as raw material obtained in Example 1. Figure 2 It can be seen that the main chromatographic peaks of the wolfberry alcohol-precipitated polysaccharides prepared from fresh wolfberry fruits and the wolfberry alcohol-precipitated polysaccharides prepared from dried wolfberry fruits are consistent. The wolfberry alcohol-precipitated polysaccharides prepared from fresh wolfberry fruits contain more high-molecular-weight polysaccharides than the wolfberry alcohol-precipitated polysaccharides prepared from dried wolfberry fruits.

[0079] Figure 3 This is a graph showing the maximum UV 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 combining with Congo red. Figure 3It can be seen that all three exhibit maximum ultraviolet absorption in 0.05 mol / L sodium hydroxide solution. As the concentration of the sodium hydroxide solution increases, the maximum ultraviolet absorption peak decreases, indicating that all three have a triple helical structure.

[0080] Table 3 is a comparison of the monosaccharide compositions of Lycium barbarum ethanol-precipitated polysaccharides prepared from fresh Lycium barbarum berries and Lycium barbarum ethanol-precipitated polysaccharides prepared from dried Lycium barbarum berries. As can be seen from the data in Table 3, the monosaccharide compositions of the two are basically the same.

[0081]

[0082] 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.

[0083]

[0084] As shown in Table 4, there was no significant difference in the in vitro antioxidant activity of alcohol-precipitated polysaccharides prepared from fresh and dried wolfberries, while the refined wolfberry polysaccharide prepared from fresh wolfberries exhibited better ferric ion reducing power (FRAP) and oxygen free radical adsorption capacity (ORAC). This indicates that preparing wolfberry polysaccharides from fresh wolfberries is a simple and energy-saving process, suitable for processing fresh wolfberries in large quantities and within a short period of time, and has good process applicability and cost-effectiveness.

[0085] Example 2

[0086] Take 1000g of fully mature fresh wolfberry, add 1000g of purified water, and use a colloid mill to homogenize. The relative density is measured to be 1.01 (room temperature). The obtained wolfberry homogenate is passed through a disc centrifuge to remove solid insoluble matter to obtain a supernatant. Ethanol is added to the supernatant to make the alcohol content reach 85%. The mixture is placed at 10°C for 24 hours, filtered, and dried to obtain 215g of brown powdered wolfberry alcohol-precipitated polysaccharide. The polysaccharide content is measured by the phenol-sulfuric acid method to be 8.50±0.32%. Accurately weigh 1.000g of wolfberry alcohol-precipitated polysaccharide powder, dissolve it in water and dilute it to a 5mL volumetric flask. The FRAR value is measured to be 0.71±0.02mmol / L, and the IC value for inhibiting ABTS free radicals is 0. 50 The ORAC value was 4.70±0.15mg / mL and 8.28±0.07μmol / g.

[0087] Example 3

[0088] Take 1000g of fully mature fresh wolfberry, add 500g of purified water, and use a colloid mill to homogenize. The relative density is measured to be 1.03 (room temperature). The obtained wolfberry homogenate is passed through a disc centrifuge to remove solid insoluble matter to obtain a supernatant. Ethanol is added to the supernatant to make the alcohol content reach 90%. The mixture is placed at room temperature for 12 hours, filtered, and dried to obtain 205g of brown powder wolfberry alcohol-precipitated polysaccharide. The polysaccharide content is measured to be 8.47±0.29% by the phenol-sulfuric acid method. 1.000g of wolfberry alcohol-precipitated polysaccharide powder is accurately weighed, dissolved in water and diluted to a 5mL volumetric flask. The FRAR value is measured to be 0.66±0.02mmol / L, and the IC value for inhibiting ABTS free radicals is 0. 50 The ORAC value was 5.30±0.10 mg / mL and 7.74±0.11 μmol / g.

[0089] Example 4

[0090] 10 kg of fully mature fresh wolfberries were added to 1000 g of purified water and homogenized using a colloid mill. The relative density was measured to be 1.05 (at room temperature). The resulting wolfberry slurry was then passed through a disc centrifuge to remove insoluble solids, resulting in a supernatant. Ethanol was added to the supernatant to a concentration of 80% alcohol. The supernatant was then incubated at 10°C for 48 hours, filtered, and dried to yield 226 g of alcohol-precipitated wolfberry polysaccharide (Lycium barbarum) in a brownish-yellow powder. The polysaccharide content was determined to be 9.07 ± 0.30% using the phenol-sulfuric acid method. The alcohol-precipitated L. barbarum polysaccharide was reconstituted with an appropriate amount of purified water and filtered using an ultrafiltration membrane with a molecular weight cutoff of 3000–100,000 Da. The resulting powder was dried to yield 25.7 g of purified L. barbarum polysaccharide (Lycium barbarum) in a light brownish-yellow powder. The polysaccharide content was determined to be 43.76 ± 1.48% using the phenol-sulfuric acid method. 1.000 g of refined Lycium barbarum polysaccharide powder was accurately weighed, dissolved in water and diluted to a volume of 5 mL in a volumetric flask. The FRAR value was measured to be 1.04 ± 0.05 mmol / L, and the IC value for inhibiting ABTS free radicals was 1.04 ± 0.05 mmol / L. 50 The ORAC value was 3.49±0.08mg / mL and 9.92±0.18μmol / g.

[0091] Example 5

[0092] 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.

[0093] Sample preparation: Lycium barbarum alcohol-precipitated polysaccharides and purified Lycium barbarum polysaccharides were prepared in phosphate buffer (pH 7.0) to a 1 mg / mL stock solution. α-Glucosidase (Shanghai Ruiyong Biotechnology Co., Ltd., 100 U, stored at -20°C) was prepared in phosphate buffer (pH 7.0) to a 1 U / mL stock solution, stored at -20°C, and diluted to a 0.2 U / mL solution in phosphate buffer (pH 7.0) immediately before use. Acarbose, a positive control (Shanghai Aladdin Biochemical Technology Co., Ltd., stored dry at 4°C), was prepared in phosphate buffer (pH 7.0) to a 1 mg / mL stock solution. 4-Nitrophenyl-α-D-glucopyranoside (Alfa Aesar (China) Chemical Co., Ltd., stored dry at 4°C) was prepared in phosphate buffer (pH 7.0) and diluted to a 2.5 mmol / L solution. Na₂CO₃ was prepared in reverse osmosis water to a 0.2 mol / L solution.

[0094] 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. Acarbose was used as a positive control. After pre-incubation at 37°C for 10 minutes, 20 μL of p-nitrophenyl-α-D-pyranoglucopyranoside solution was added, and the mixture was incubated for another 20 minutes. The terminator Na2CO3 was added.

[0095] 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 the IC 50 .

[0096]

[0097] The results showed that both the alcohol-precipitated and purified Lycium barbarum polysaccharides showed inhibitory activity against α-glucosidase, IC 50 0.22±0.02 mg / mL and 0.18±0.03 mg / mL respectively.

[0098] Example 6

[0099] 10 kg of fully mature fresh wolfberries were crushed in a juicer, filtered to remove the skin and seeds, diluted with 900 g of purified water, and homogenized using a colloid mill to obtain a wolfberry homogenate with a relative density of 1.05 (room temperature). The wolfberry homogenate was then passed through a disc centrifuge to separate and remove insoluble solids, resulting in a supernatant. Ethanol was added to the supernatant to a concentration of 80% alcohol. The supernatant was then incubated at 10°C for 48 h, filtered, and dried to obtain 226 g of alcohol-precipitated wolfberry polysaccharide, a brown-yellow powder. The polysaccharide content was determined to be 9.07 ± 0.30% by the phenol-sulfuric acid method. The alcohol-precipitated wolfberry polysaccharide product was reconstituted with an appropriate amount of purified water, filtered using an ultrafiltration membrane with a molecular weight cutoff of 3,000 to 100,000 Da, and dried to obtain 25.2 g of purified wolfberry polysaccharide, a light brown-yellow powder. The polysaccharide content was determined to be 43.15 ± 1.52% by the phenol-sulfuric acid method. 1.000 g of refined Lycium barbarum 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, and the IC value of inhibiting ABTS free radicals was 1.02 ± 0.05 mmol / L. 50 It was 3.42±0.06mg / mL, and the ORAC value was 9.91±0.15μmol / g.

[0100] The foregoing description is merely 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 solution that can be modified and varied by a person skilled in the art using the above-disclosed method, falls within the scope of protection of the present invention.

Claims

1. A method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum, characterized by: Fully mature fresh wolfberries are taken, water is added, and the mixture is passed through a colloid mill for homogenization to obtain a wolfberry homogenate with a relative density of 1.01-1.05; the wolfberry homogenate is passed through a disc centrifuge or a tubular centrifuge to remove solid insoluble matter to obtain a supernatant; 95% ethanol is added to the supernatant to adjust the alcohol content to 80-90%, and the mixture is allowed to stand at 10-25°C for 12-48 hours for alcohol precipitation, and the precipitate is separated to obtain wolfberry alcohol-precipitated polysaccharide; the obtained wolfberry alcohol-precipitated polysaccharide is dissolved in deionized water or purified water, filtered through an ultrafiltration membrane with a molecular cutoff value of 3000-100000 Da, and the filtrate is removed to obtain refined wolfberry polysaccharide.

2. The method for directly extracting Lycium barbarum polysaccharides from fresh Lycium barbarum according to claim 1, wherein: The alcohol precipitation is performed by adding ethanol to make the alcohol content of the supernatant reach 80%.

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