Lycium ruthenicum polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses lycium ruthenicum polysaccharide as well as a preparation method and application thereof, and relates to the technical field of deep processing and application of natural products. According to the method disclosed by the invention, plant cell walls are destroyed under mild conditions by utilizing an enzyme method through an enzyme method-assisted aqueous two-phase extraction technology so as to release polysaccharide, and the polysaccharide is efficiently separated and purified by combining an aqueous two-phase system, so that the yield and the activity retention rate are improved. The high selectivity of an enzyme method is combined with the mild distribution characteristic of an aqueous two-phase system, so that the use of an organic solvent can be reduced, the environmental pollution can be reduced, and the biological activity of the lycium ruthenicum polysaccharide in the prevention and treatment of diseases related to retinal endothelial cell injury can be reserved. The lycium ruthenicum polysaccharide prepared by the method can be used for preparing auxiliary treatment preparations for ophthalmic diseases such as diabetic retinopathy and macular degeneration.

Description

Technical Field

[0001] This invention relates to the field of deep processing and application technology of natural products, and more specifically, to a black goji berry polysaccharide, its preparation method, and its application. Background Technology

[0002] Black goji berries ( Lycium ruthenicum *Lycium chinense* (Murr.) is a rare wild plant belonging to the genus *Lycium* in the Solanaceae family. It is widely distributed in the arid and cold regions of the Qinghai-Tibet Plateau and Northwest China. Its fruit is believed to nourish the liver and kidneys, improve eyesight, and combat fatigue. Modern pharmacological studies have shown that black goji berries are rich in various bioactive components, such as polysaccharides, anthocyanins, flavonoids, amino acids, and various trace elements. Among these, polysaccharides have attracted widespread attention due to their excellent bioactivity in antioxidation, anti-inflammation, immunomodulation, hypoglycemia, hypolipidemia, and neuroprotection. In recent years, in particular, with the increasing aging population and environmental pollution, the incidence of ophthalmic diseases related to retinal endothelial cell damage, such as diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal vein occlusion, has continued to rise. The main pathological mechanisms of these diseases involve increased oxidative stress and retinal microvascular endothelial dysfunction, leading to blood-retinal barrier disruption, vascular leakage, and decreased visual function. Existing studies have confirmed that black goji berry polysaccharide (LPR) can effectively alleviate oxidative damage to retinal endothelial cells through mechanisms such as scavenging free radicals, inhibiting the release of inflammatory factors, and improving mitochondrial function, demonstrating its potential value in adjunctive intervention for ophthalmic diseases.

[0003] In the fields of food processing and pharmaceutical development, black goji berry polysaccharides can be added as functional ingredients to beverages, dairy products, formula powders, and health foods, and can also be developed into pharmaceutical compositions with health-promoting and adjuvant therapeutic effects. However, current extraction methods for black goji berry polysaccharides mainly rely on traditional hot water extraction, acid-base extraction, and alcohol precipitation, which suffer from low extraction efficiency, high energy consumption, complex operating procedures, and easy destruction of active ingredients, severely restricting their large-scale application in food processing and pharmaceutical development. Therefore, there is an urgent need for an efficient, mild, and environmentally friendly extraction and purification technology to achieve the industrial production of black goji berry polysaccharides while maintaining their biological activity, thereby providing a stable and high-quality source of raw materials for the adjuvant treatment of ophthalmic diseases such as diabetic retinopathy and macular degeneration.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a black goji berry polysaccharide, its preparation method, and its application. This method can not only reduce the use of organic solvents and reduce environmental pollution, but also retain the bioactivity of black goji berry polysaccharide in preventing and treating diseases related to retinal endothelial cell damage, providing a high-quality raw material for adjuvant treatment of ophthalmic diseases such as diabetic retinopathy and macular degeneration.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a method for preparing black goji berry polysaccharide, comprising the following steps: preparing dried black goji berries into black goji berry powder; adding water, cellulase, and pectinase to the black goji berry powder, and carrying out an enzymatic hydrolysis reaction under water bath shaking conditions, and inactivating the reaction in a boiling water bath after the reaction is completed; adding PEG and (NH4)2SO4 to the enzymatic hydrolysate, mixing evenly, and performing a first centrifugation, collecting the supernatant as black goji berry extract; concentrating the black goji berry extract and preliminarily purifying it, performing a second centrifugation, collecting the precipitate, dissolving it in water, adding chloroform-n-butanol solution, shaking, performing a third centrifugation, collecting the supernatant for a second purification by dialysis, performing a fourth centrifugation, collecting the precipitate, and freeze-drying it to obtain black goji berry polysaccharide powder.

[0007] In some preferred embodiments, the dried black goji berries are dried in a hot air dryer at a temperature below 60°C until the moisture content of the dried black goji berries is less than 8%, and then pulverized using a high-speed pulverizer and passed through a 60-mesh sieve to obtain black goji berry powder.

[0008] In some preferred embodiments, the amount of cellulase added is 1.0-2.5% of the mass of the black goji berry powder; the amount of pectinase added is 1.0-2.5% of the mass of the black goji berry powder.

[0009] In some preferred embodiments, the enzymatic hydrolysis reaction is carried out at a temperature of 40-60°C for 2-3 hours and at an oscillation frequency of 100-250 rpm.

[0010] In some preferred embodiments, the molecular weight of the PEG is 6000-8000, and its addition amount is 15%-30% of the enzymatic hydrolysate; the addition amount of the (NH4)2SO4 is 15%-24% of the enzymatic hydrolysate.

[0011] In some preferred embodiments, the preliminary purification includes the following steps: concentrating the black goji berry extract to 1 / 3-1 / 4 of its original volume, adding anhydrous ethanol to a final concentration of 80%-85%, and allowing it to stand for 24-48 hours.

[0012] In some preferred embodiments, the first centrifugation is performed at a speed of 5000-6000 r / min for 10-15 min; the second, third, and fourth centrifugations are all performed at a speed of 4000-5000 r / min for 20-25 min.

[0013] In some preferred embodiments, the secondary purification via dialysis includes the following steps: dialysis of the supernatant, concentration of the dialysate followed by the addition of anhydrous ethanol to a final concentration of 80%-85%, and standing for 24-48 hours; the molecular weight cutoff for dialysis is 3.5-4.5 kDa, the temperature is 2-8°C, and the time is 48-60 hours.

[0014] Secondly, the present invention provides a black goji berry polysaccharide prepared by the above preparation method.

[0015] Thirdly, the present invention provides the application of black goji berry polysaccharide in the preparation of drugs for the prevention or treatment of ophthalmic diseases.

[0016] The present invention has the following beneficial effects: This invention proposes an enzymatic-assisted aqueous two-phase extraction technology. Enzymes are used to break down plant cell walls under mild conditions to release polysaccharides, which are then efficiently separated and purified using an aqueous two-phase system, thereby improving yield and activity retention. The combination of the high selectivity of the enzymatic method and the mild partitioning characteristics of the aqueous two-phase system not only reduces the use of organic solvents and environmental pollution but also preserves the bioactivity of black goji berry polysaccharides in preventing and treating diseases related to retinal endothelial cell damage. This provides a high-quality raw material for adjuvant treatments of diabetic retinopathy, macular degeneration, and other ophthalmic diseases. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram showing the results of the antioxidant activity determination of black goji berry polysaccharides in this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] The following is a detailed description of a black goji berry polysaccharide provided by the present invention, its preparation method, and its application.

[0021] Existing extraction methods for black goji berry polysaccharides (LRP) (such as hot water extraction, acid-base extraction, and alcohol precipitation) have several shortcomings. Firstly, due to the dense cell wall structure of black goji berry fruits, traditional methods struggle to fully break down the cell walls during extraction, resulting in incomplete polysaccharide release and low yield. Secondly, commonly used high-temperature or strong acid-base conditions easily damage the spatial configuration and glycan structure of black goji berry polysaccharides, significantly reducing their antioxidant and anti-inflammatory bioactivities. Furthermore, existing methods yield crude polysaccharides with high impurity content; proteins, pigments, and low-molecular-weight sugars are difficult to remove, affecting functional verification and subsequent processing. Simultaneously, the use of organic solvents is substantial, leading to high energy consumption, environmental pollution, and excessively high production costs. Especially in applications related to the prevention and treatment of diabetic retinopathy, age-related macular degeneration, and other retinal endothelial cell damage, the effectiveness of black goji berry polysaccharides depends on their structural integrity and high purity, which traditional processes struggle to simultaneously meet the demands for high yield, high activity, and low environmental impact. Therefore, this invention proposes a method for preparing black goji berry polysaccharides using enzymatic-assisted aqueous two-phase extraction technology. The method utilizes enzymes to disrupt plant cell walls under mild conditions to release polysaccharides, combined with an aqueous two-phase system for efficient separation and purification, thereby improving yield and activity retention. The combination of the high selectivity of the enzyme method and the mild partitioning characteristics of the aqueous two-phase system not only reduces the use of organic solvents and environmental pollution but also preserves the bioactivity of black goji berry polysaccharides in preventing and treating diseases related to retinal endothelial cell damage, providing a high-quality raw material for adjuvant treatment of diabetic retinopathy, macular degeneration, and other ophthalmic diseases.

[0022] In a first aspect, this application provides a method for preparing black goji berry polysaccharide, which includes the following steps: S1. Prepare dried black goji berries into black goji berry powder.

[0023] In some preferred embodiments, mature, plump, and mold-free dried black goji berries are selected as raw materials. Impurities (such as fruit stems, leaves, and foreign matter) are removed, and the berries are rinsed with tap water and then thoroughly washed with deionized water, and drained of surface moisture. The clean berries are pre-dried in a 60 ℃ hot air drying oven to constant weight to reduce the moisture content of the raw material, keeping it below 8% to prevent microbial growth and polysaccharide degradation during subsequent extraction. The dried sample is then pulverized using a high-speed pulverizer and passed through a 60-mesh sieve to obtain a uniform fine powder. This powder is then placed in a sealed, light-proof container and stored at 4 ℃ for later extraction.

[0024] S2. Add water, cellulase and pectinase to black goji berry powder, carry out enzymatic hydrolysis under water bath shaking conditions, and inactivate in boiling water bath after the reaction is completed.

[0025] In some preferred embodiments, the amount of cellulase added is 1.0-2.5% of the mass of the black goji berry powder; the amount of pectinase added is 1.0-2.5% of the mass of the black goji berry powder.

[0026] Preferably, the amount of cellulase added is 2.08% of the mass of the black goji berry powder; and the amount of pectinase added is 2.09% of the mass of the black goji berry powder.

[0027] An appropriate amount of cellulase can effectively hydrolyze the cellulose components of the cell wall of black goji berries, promoting the release of intracellular polysaccharides. However, when the enzyme concentration is too high, the hydrolysis in the system may be too vigorous, and some high-molecular-weight polysaccharides may be further degraded into oligosaccharides or monosaccharides and lost. At the same time, high enzyme concentration may lead to changes in the viscosity of the reaction system and competition between enzyme molecules, thereby affecting the release and stability of polysaccharides. An appropriate amount of pectinase can degrade the cell wall protopectin and middle lamella structure, promoting polysaccharide release. However, when the enzyme concentration is too high, some polysaccharides may be degraded by the enzyme system, or the intracellular components (such as proteins and monosaccharides) produced after cell wall disruption may increase, affecting the distribution and recovery of polysaccharides in the aqueous two-phase system, resulting in a decrease in yield.

[0028] In some preferred embodiments, the enzymatic hydrolysis reaction is carried out at a temperature of 40-60°C for 2-3 hours and at an oscillation frequency of 100-250 rpm.

[0029] The extinguishing process in a boiling water bath involves placing the enzyme in water at 95-100℃ and heating it for 5-15 minutes. This temperature is much higher than the denaturation temperature of enzymes, which can rapidly and thoroughly destroy the tertiary structure of the enzyme protein, causing it to be permanently inactivated, thereby accurately terminating the reaction and preventing deviations in subsequent analytical results.

[0030] S3. Add PEG and (NH4)2SO4 to the enzymatic hydrolysate, mix well, and then centrifuge for the first time. Collect the supernatant as the black goji berry extract.

[0031] In some preferred embodiments, the molecular weight of the PEG is 6000-8000, and its addition amount is 15%-30% of the enzymatic hydrolysate; the addition amount of the (NH4)2SO4 is 15%-24% of the enzymatic hydrolysate.

[0032] Preferably, the PEG has a molecular weight of 6000 and is added at 18.13% of the enzymatic hydrolysate; the (NH4)2SO4 is added at 15.29% of the enzymatic hydrolysate.

[0033] Appropriate salting-out can alter the solubility of polysaccharides in the solvent system, causing them to preferentially allocate to a specific aqueous phase, thereby improving recovery efficiency. However, when the salt concentration is too high, the ionic strength of the system is too great, which may lead to the co-precipitation of some impurities, increasing sedimentation losses, or inhibiting the phase separation efficiency of the aqueous two-phase system, thus reducing the extraction rate. A suitable PEG concentration can enhance the enrichment of the target product in the lower phase; however, excessive PEG content will increase the system viscosity, reduce the mass transfer rate, and may also cause some polysaccharides to be retained or adsorbed at the phase interface, thereby reducing the recovery rate.

[0034] In some embodiments of the present invention, the first centrifugation is performed at a speed of 5000-6000 r / min for a time of 10-15 min.

[0035] S4. After concentrating and preliminarily purifying the black goji berry extract, perform a second centrifugation, collect the precipitate, dissolve it in water, add chloroform and n-butanol solution, shake, and perform a third centrifugation. Collect the supernatant solution for a second purification by dialysis, perform a fourth centrifugation, collect the precipitate, and freeze-dry it to obtain black goji berry polysaccharide powder (LRP).

[0036] Specifically, in some embodiments of this application, the rotation speed of the second, third and fourth centrifugations is 4000-5000 r / min, and the time is 20-25 min.

[0037] In some preferred embodiments, the preliminary purification includes the following steps: concentrating the black goji berry extract to 1 / 3-1 / 4 of its original volume, adding anhydrous ethanol to a final concentration of 80%-85%, and allowing it to stand for 24-48 hours.

[0038] First, reduced pressure evaporation removes most of the water from the extract, reducing its volume and increasing the concentration of polysaccharides and other solutes, facilitating subsequent alcohol precipitation. Second, the solubility of polysaccharides in high-concentration ethanol is significantly reduced, causing them to precipitate from the solution through hydrogen bond disruption and hydration film reduction. Some small-molecule soluble impurities (such as monosaccharides, oligosaccharides, and phenols) remain in the ethanol supernatant, achieving preliminary purification. Finally, sufficient settling time ensures that the polysaccharides fully aggregate to form larger precipitate particles, facilitating centrifugal separation.

[0039] In some preferred embodiments, the secondary purification via dialysis includes the following steps: dialysis of the supernatant, concentration of the dialysate followed by the addition of anhydrous ethanol to a final concentration of 80%-85%, and standing for 24-48 hours; the molecular weight cutoff for dialysis is 3.5-4.5 kDa, the temperature is 2-8°C, and the time is 48-60 hours.

[0040] The process involves using a dialysis bag to trap large polysaccharides while allowing smaller molecules such as salts and residual organic solvents to diffuse out for further purification. Next, high-concentration anhydrous ethanol is used to remove soluble small-molecule impurities after dialysis, ensuring complete polysaccharide precipitation and minimizing losses.

[0041] Secondly, the present invention provides a black goji berry polysaccharide, which is prepared by the above-mentioned preparation method. The black goji berry polysaccharide comprises the following components by weight percentage: 81.96%-92.3% neutral sugar, 8.89%-15.33% uronic acid and 0.14%-0.38% protein, with high purity and good structural integrity.

[0042] Thirdly, the present invention provides the application of black goji berry polysaccharide in the preparation of drugs for the prevention or treatment of ophthalmic diseases.

[0043] The black goji berry polysaccharide prepared by this invention has significant in vitro and cellular antioxidant activity, making it suitable for the development of high-quality natural functional factors in the fields of functional foods, health products, and prevention and treatment of ophthalmological diseases. At the same time, it takes into account environmental friendliness and controllable production costs, and has significant value for promotion and application.

[0044] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0045] Example 1 This embodiment provides a method for preparing black goji berry polysaccharide, including the following steps: S1. Place the clean, dried black goji berries in a 60 ℃ hot air drying oven to pre-dry to constant weight, ensuring the moisture content is below 8%. After drying, the sample is pulverized using a high-speed pulverizer and passed through a 60-mesh sieve to obtain uniform fine powder. Store the powder in a sealed, light-proof container at 4 ℃.

[0046] S2. Add water, 2.08% (by weight of the black goji berry powder) of cellulase and 2.09% (by weight of the black goji berry powder) of pectinase to the black goji berry powder, shake in a 50°C water bath for 160 minutes, terminate the reaction, and then inactivate in a boiling water bath for 10 minutes.

[0047] S3. Add 18.13% PEG and 15.29% (NH4)2SO4 (by weight of the enzyme hydrolysate) to the enzyme hydrolysate, mix well, centrifuge at 5000 r / min for 10 minutes, and collect the lower layer extract for subsequent experiments.

[0048] S4. Concentrate the black goji berry extract to 1 / 3 of its original volume using a rotary evaporator, then add anhydrous ethanol to a final concentration of 80%. After standing for 24 h, centrifuge at 4000 r / min for 20 min and collect the precipitate. Dissolve the precipitate in distilled water, add a chloroform-n-butanol mixture (chloroform:n-butanol = 4:1, volume ratio) at a 1:1 ratio, shake for 20 min, and centrifuge at 4000 r / min for 20 min. Collect the uppermost layer of solution and dialyze at 4 ℃ for 48 h. Concentrate the dialysate, add anhydrous ethanol again to a final concentration of 80%, let stand for 24 h, and centrifuge at 4000 r / min for 20 min to collect the precipitate. Freeze-dry to obtain crude black goji berry polysaccharide powder.

[0049] S5. The content of polysaccharides in black goji berries was determined by the phenol-sulfuric acid method. The absorbance at 490 nm was measured and compared with the standard curve to obtain the quantification.

[0050] Black goji berry polysaccharide yield W (%) = (C × V × N) / M × 100 In the formula: W represents the yield of black goji berry polysaccharide, %; C represents the mass concentration of black goji berry polysaccharide solution calculated based on absorbance value, mg / mL; N represents the dilution factor of the solution; V represents the volume of polysaccharide solution, mL; and M represents the sample amount of black goji berries, mg.

[0051] The final yield of black goji berry polysaccharide was 35.33%.

[0052] Example 2 This embodiment provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of cellulase added is 1.8% of the mass of the black goji berry powder.

[0053] The final yield of black goji berry polysaccharide was 27.61%.

[0054] Example 3 This embodiment provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of pectinase added is 1.8% of the mass of the black goji berry powder.

[0055] The final yield of black goji berry polysaccharide was 27.32%.

[0056] Example 4 This embodiment provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of PEG added is 15% of the mass of the enzymatic hydrolysate.

[0057] The final yield of black goji berry polysaccharide was 30.51%.

[0058] Example 5 This embodiment provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of (NH4)2SO4 added is 17% of the mass of the enzymatic hydrolysate.

[0059] The final yield of black goji berry polysaccharide was 28.83%.

[0060] Comparative Example 1 This comparative example provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of cellulase added is set to 0.5% of the mass of the black goji berry powder.

[0061] The final yield of black goji berry polysaccharide was 22.53%.

[0062] Comparative Example 2 This comparative example provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of pectinase added is set to 0.5% of the mass of the black goji berry powder.

[0063] The final yield of black goji berry polysaccharide was 21.01%.

[0064] Comparative Example 3 This comparative example provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of PEG added is set to 35% of the mass of the enzymatic hydrolysate.

[0065] The final yield of black goji berry polysaccharide was 23.91%.

[0066] Comparative Example 4 This comparative example provides a method for preparing black goji berry polysaccharide. The remaining steps are the same as in Example 1, except that the amount of (NH4)2SO4 added is set to 26.4% of the mass of the enzymatic hydrolysate.

[0067] The final yield of black goji berry polysaccharide was 24.31%.

[0068] Experimental Example 1 The freeze-dried powder of black goji berry polysaccharide prepared in Example 1 was used as a sample to determine its antioxidant capacity.

[0069] (1) DPPH free radical scavenging rate Three groups of reaction solutions were set up to determine the DPPH free radical scavenging rate: blank control group (containing 1 mL of 0.2 mmol / L DPPH solution and 1 mL of anhydrous ethanol), sample group (containing 1 mL of DPPH solution and 1 mL of sample solution), and calibration group (containing 1 mL of sample solution and 1 mL of anhydrous ethanol). The corresponding reaction reagents were added according to the group, and the mixture was reacted at room temperature in the dark for 30 min. The absorbance of HRLP was measured at 517 nm and recorded as A0, A1, and A2, respectively. The DPPH free radical scavenging rate (S) of the sample was calculated according to formula (1).

[0070] S=[1-(A1-A2) / A0]×100%(1) DPPH free radical scavenging results are as follows Figure 1 As shown in Figure -A, the half-maximal scavenging concentration (IC50) of LRP for lipophilic DPPH radicals is... 50 The concentration was 0.26 mg / mL, which is about 3.7 times that of vitamin C.

[0071] (2) Determination of hydroxyl radical scavenging rate Three reaction systems were set up to determine the scavenging rate of hydroxyl radicals: blank control group (containing 1 mL 8 mmol / L FeSO4 solution, 1 mL 8 mmol / L salicylic acid solution, 1 mL 8 mmol / L H2O2 and 1 mL distilled water), sample group (replacing the distilled water in the blank control with an equal amount of sample solution) and correction group (replacing H2O2 with an equal amount of distilled water and adding sample solution). The corresponding reaction reagents were added according to the group, and the reaction was carried out in a water bath at 37 ℃ for 1 h. The absorbance of the sample was measured at 510 nm and recorded as A0, A1 and A2 respectively. The scavenging rate of hydroxyl radicals was calculated according to formula (1).

[0072] The results of hydroxyl radical scavenging rate are as follows: Figure 1 As shown in -B, for highly reactive hydroxyl radicals, IC 50 The concentration was 0.52 mg / mL.

[0073] (3) ABTS + Determination of free radical scavenging rate Three reaction systems were set up: a blank control group (2 mL ABTS⁺ working solution and 0.2 mL distilled water), a sample group (2 mL ABTS⁺ working solution and 0.2 mL sample solution), and a calibration group (equal amounts of distilled water were used instead of H₂O₂ and equal amounts of sample solution were added). The corresponding reaction reagents were added according to the group, and the mixture was allowed to stand in the dark for 30 min. After centrifugation at 10,000 r / min for 5 min, the supernatant was collected and the absorbance was measured at 734 nm, and recorded as A0, A1, and A2, respectively. ABTS⁺ was calculated according to formula (1). + Free radical scavenging rate.

[0074] ABTS + Free radical scavenging results are as follows Figure 1 As shown in -C, for ABTS with strong hydrophilicity... + Free radicals, IC 50 The concentration was reduced to 0.15 mg / mL, indicating that LRP has better reaction potential in hydrophilic systems.

[0075] (4) Determination of iron ion reducing power 30 μL of sample and 265 µL of ferric ion reducing antioxidant power (FRAP) working solution were reacted at 37 °C for 30 min. The OD value was measured at 593 nm and recorded as A1. The OD value was measured using the solvent instead of the sample and recorded as A0. The ferric ion reducing power (T) was calculated according to formula (2).

[0076] T = A1 - A0 (2) The results of the iron ion reducing power are as follows Figure 1 As shown in Figure -D, when the LRP concentration is 0.60 mg / mL, its reducing power is equivalent to that of 0.10 mg / mL VC, which is about 1 / 6 of VC. Although the reducing power is not as high as VC, the trend is consistent with the free radical scavenging experiment, indicating that LRP has certain electron donor and iron ion reducing effects.

[0077] The results above indicate that LRP possesses scavenging capabilities against various types of free radicals, with a particularly strong effect on hydrophilic ABTS free radicals, and also exhibits some potential for metal ion reduction. These results suggest that LRP's in vitro antioxidant capacity stems from a multi-pathway synergy, including direct free radical scavenging, electron / hydrogen atom transfer, and metal ion chelation. Although its activity is lower than that of small-molecule antioxidants such as vitamin C, as a naturally derived macromolecular polysaccharide, it offers advantages such as high safety and multiple target mechanisms of action, demonstrating promising application prospects in functional foods and natural antioxidants.

[0078] Experimental Example 2 The freeze-dried powder of black goji berry polysaccharide prepared in Example 1 was used as a sample to determine its protective effect on retinal endothelial cells.

[0079] Human retinal microvascular endothelial cells (hRMECs, passage number ≤10) were cultured in DMEM / F12 (1:1) medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (100 U / mL penicillin + 100 μg / mL streptomycin) at 37 ℃, 5% CO2, and saturated humidity. The medium was changed every 2–3 days. When the cell confluence reached 80%, the cells were digested and passaged using 0.25% trypsin-EDTA solution, with the passage ratio controlled at 1:2–1:3 to ensure cell stability.

[0080] hRMECs in logarithmic growth phase and in good condition were seeded into 96-well plates, 6-well plates, or culture dishes according to experimental requirements. After the cells were cultured until they adhered stably and reached an appropriate density, they were divided into a control group (no treatment), a model group (H2O2 stimulation), a low-concentration group (50 μg / mL), a medium-concentration group (100 μg / mL), a high-concentration group (200 μg / mL) of black goji berry polysaccharide, and a positive control group (100 μmol / L vitamin C treatment). The polysaccharide treatment group and the positive control group were pretreated with the corresponding concentration of black goji berry polysaccharide solution or vitamin C solution for 24 h before model establishment. Subsequently, except for the control group, all other groups were replaced with fresh culture medium containing 200 μmol / L hydrogen peroxide (H2O2) and incubated at 37℃ and 5% CO2 for 2 h to induce the formation of the oxidative stress injury model. After model induction, samples were immediately collected and analyzed according to different detection items.

[0081] (1) Cell viability assay (CCK-8 assay) and lactate dehydrogenase (LDH) release assay First, discard the cell culture medium, wash twice with PBS, add 90 μL of fresh culture medium and 10 μL of CCK-8 working solution, incubate at 37°C for 1–2 h, and measure the OD value at 450 nm. Cell viability calculation formula: Cell viability (%) = (A1 - A0) / (A2 - A0) × 100% Where A0 is the OD value of the control group; A1 is the OD value of the sample group; and A2 is the OD value of the calibration group.

[0082] Next, cell supernatants from each group were collected, and OD values ​​were measured at a wavelength of 490 nm according to the kit instructions to reflect changes in cell membrane integrity.

[0083] The results are shown in Table 1. Compared with the control group, the cell viability of the H2O2 model group decreased significantly, from 100% to 54.2% ± 3.1%, and the LDH release increased significantly, from 98.3 ± 5.6 U / L to 215.6 ± 12.4 U / L. Treatment with black goji berry polysaccharides significantly improved cell viability and reduced LDH release in a dose-dependent manner; among them, the high concentration group (200 μg / mL) restored cell viability to 85.4% ± 2.5% and reduced LDH release to 126.5 ± 8.9 U / L, which was close to that of the positive control (VC, 87.6% ± 2.1% viability, 119.3 ± 7.8 U / L LDH).

[0084] (2) Detection of intracellular ROS levels (DCFH-DA probe method) Cells were washed twice with PBS, and then incubated with 10 μmol / L DCFH-DA working solution at 37 °C for 30 min, gently shaking every 10 min. After incubation, cells were washed three times with PBS, and the fluorescence intensity was detected using a fluorescence microplate reader (Ex 488 nm / Em 525 nm).

[0085] The results are shown in Table 1. H2O2 induction significantly increased intracellular ROS levels, while black goji berry polysaccharide effectively inhibited excessive ROS production. The ROS level in the high concentration group was 44.2% lower than that in the model group, and the ROS level in the medium concentration group was 30.1% lower, which was similar to the effect of the VC group.

[0086] (3) Determination of antioxidant enzyme activity and lipid peroxidation level Cells were lysed and centrifuged (12000 rpm, 10 min) to collect the supernatant. The activities of SOD, CAT, GSH-Px and the content of MDA were detected according to the kit instructions. The results were standardized according to protein concentration.

[0087] The results are shown in Table 1. In the H2O2 model group, the activities of SOD, CAT, and GSH-Px decreased significantly (by 38.4%, 42.6%, and 36.7%, respectively), while the MDA level increased significantly. BLP treatment significantly restored antioxidant enzyme activity and reduced MDA accumulation, with the high-concentration group showing the best recovery effect, which was not significantly different from the VC group.

[0088] Table 1. Protective effect of LRP against H2O2-induced oxidative damage to retinal endothelial cells

[0089] Note: Different letters in the superscript of data in the same column indicate significant differences (P<0.05), while the same letter or no letter indicates no significant differences (P>0.05).

[0090] In summary, this invention achieves efficient extraction and purification of polysaccharides from black goji berries under mild conditions by employing an enzymatically assisted aqueous two-phase system. This solves the problems of easy structural damage, low yield, poor purity, and heavy environmental burden associated with traditional high-temperature or strong acid / alkali conditions for polysaccharides. The obtained crude polysaccharide contains 87.13%±5.17% neutral sugars and 12.11%±3.22% uronic acid, with protein accounting for only 0.26%±0.12%, exhibiting high purity and good structural integrity.

[0091] The results from Examples 1-5 and Comparative Examples 1-4 show that the highest yield of 35.33% was achieved in Example 1 when the amount of dual enzymes added was 2.08%. Cellulase and pectinase significantly broke down the cell structure barrier and promoted polysaccharide dissolution by synergistically degrading the cellulose skeleton and pectin of plant cell walls. However, in Comparative Examples 1 and 2, the amount of dual enzymes added was reduced to 0.5%, which resulted in insufficient enzyme addition and incomplete cell wall degradation, leading to a significant decrease in yield to about 22%. PEG and ammonium sulfate, as components of the aqueous two-phase extraction system, directly affect the phase separation effect and polysaccharide partitioning behavior. When the PEG addition increases from 15% to the optimal 18.13%, the yield increases from 30.51% to 35.33%. However, excessive addition disrupts the phase equilibrium, causing more polysaccharides to remain in the aqueous phase rather than the precipitated phase, resulting in a decrease in recovery rate to 23.91%. Similarly, the optimal salt concentration of ammonium sulfate is 15.29%, promoting polysaccharide partitioning to the PEG-rich phase. However, excessively high concentrations may lead to protein denaturation or excessive salting out, causing polysaccharide co-precipitation or loss, reducing the yield to 24.31%. Therefore, the addition amounts of cellulase, pectinase, PEG, and ammonium sulfate show a clear dose-response relationship on the yield of black goji berry polysaccharides, and their mechanism of action is based on the principles of enzymatic hydrolysis and aqueous two-phase extraction.

[0092] In vitro activity evaluation results showed that this LRP exhibited significant concentration-dependent antioxidant activity in scavenging DPPH, hydroxyl radicals, ABTS radicals, and in FRAP iron reduction assays. Specifically, the IC50 values ​​for DPPH, hydroxyl radicals, and ABTS radicals were significantly higher. 50The concentrations of LRP were 0.26, 0.52, and 0.15 mg / mL, respectively. Although these concentrations were generally lower than those of the small-molecule antioxidant vitamin C, LRP exhibited superior scavenging efficiency in hydrophilic free radical systems. FRAP results showed that its reducing power at 0.60 mg / mL was approximately 1 / 6 that of vitamin C, suggesting that the antioxidant mechanism of this polysaccharide may involve a synergistic effect of multiple pathways, including direct free radical scavenging, hydrogen / electron transfer, and metal ion chelation. Furthermore, LRP significantly enhanced the activity of H2O2-induced oxidative stress-induced retinal endothelial cells, reduced lactate dehydrogenase release, decreased reactive oxygen species accumulation, and restored the activity of antioxidant enzymes (SOD, CAT, GSH-Px) and reduced the level of lipid peroxidation product MDA. Its high-dose effects were comparable to those of vitamin C. Therefore, the black goji berry polysaccharide prepared in this invention possesses significant in vitro and cellular antioxidant activity, making it suitable for the development of high-quality natural functional factors in functional foods, health products, and the prevention and treatment of ophthalmic diseases. It also considers environmental friendliness and controllable production costs, demonstrating significant potential for widespread application.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing polysaccharides from black wolfberry, characterized in that, Includes the following steps: Dried black goji berries were prepared into black goji berry powder; water, cellulase and pectinase were added to the black goji berry powder, and enzymatic hydrolysis was carried out under water bath shaking conditions. After the reaction was completed, the enzyme was inactivated in a boiling water bath; PEG and (NH4)2SO4 were added to the enzymatic hydrolysate, mixed evenly, and centrifuged for the first time. The supernatant was collected as black goji berry extract. After concentrating and initially purifying the black goji berry extract, a second centrifugation was performed, the precipitate was collected, dissolved in water, and then chloroform-n-butanol solution was added. After shaking, a third centrifugation was performed, the supernatant was collected, and a second purification was performed by dialysis. A fourth centrifugation was performed, the precipitate was collected, and freeze-dried to obtain black goji berry polysaccharide powder.

2. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The dried black goji berries are dried in a hot air dryer at a temperature below 60°C until the moisture content of the dried black goji berries is less than 8%. They are then pulverized using a high-speed pulverizer and passed through a 60-mesh sieve to obtain black goji berry powder.

3. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The amount of cellulase added is 1.0-2.5% of the mass of the black goji berry powder; the amount of pectinase added is 1.0-2.5% of the mass of the black goji berry powder.

4. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The enzymatic hydrolysis reaction is carried out at a temperature of 40-60℃ for 2-3 hours, with an oscillation frequency of 100-250 rpm.

5. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The PEG has a molecular weight of 6000-8000 and is added at 15%-30% of the enzymatic hydrolysate; the (NH4)2SO4 is added at 15%-24% of the enzymatic hydrolysate.

6. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The preliminary purification includes the following steps: concentrating the black goji berry extract to 1 / 3-1 / 4 of its original volume, adding anhydrous ethanol to a final concentration of 80%-85%, and letting it stand for 24-48 hours.

7. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The first centrifugation is performed at a speed of 5000-6000 r / min for 10-15 min; the second, third, and fourth centrifugations are all performed at a speed of 4000-5000 r / min for 20-25 min.

8. The method for preparing black wolfberry polysaccharide according to claim 1, characterized in that, The secondary purification via dialysis includes the following steps: dialysis of the supernatant, concentration of the dialysate followed by the addition of anhydrous ethanol to a final concentration of 80%-85%, and standing for 24-48 hours; the molecular weight cutoff for dialysis is 3.5-4.5 kDa, the temperature is 2-8℃, and the time is 48-60 hours.

9. A black goji berry polysaccharide prepared by the method described in any one of claims 1-8, characterized in that, The black goji berry polysaccharide comprises the following components by weight percentage: 81.96%-92.3% neutral sugar, 8.89%-15.33% uronic acid, and 0.14%-0.38% protein.

10. The use of the black goji berry polysaccharide as described in claim 9 in the preparation of a medicament for the prevention or treatment of ophthalmic diseases.

Images