Lycium barbarum extract with enhanced immunological effect and method for preparing the same

Abstract

This invention provides a stable and effective immune-enhancing extract of Lycium barbarum. The preparation method of the Lycium barbarum extract includes the following steps: (1) Fresh Lycium barbarum fruit is mixed with an organic acid aqueous solution of 1%~3% g / mL at a ratio of 2:1 g / mL and pulped. The organic acid is selected from citric acid, lactic acid, or malic acid. (2) The pulp is enzymatically inactivated and sterilized. (3) The pulp is filtered through a 200-mesh plate and frame filter. (4) The filtrate is enriched by macroporous resin and eluted sequentially with water and 60% v / v ethanol. The macroporous resin is selected from D101 and / or AB-8. The 60% v / v ethanol eluent is collected, concentrated at low temperature to remove alcohol, and the Lycium barbarum extract is obtained. This invention also provides a relatively unique preparation process for the extract. Studies have found that the amount of citric acid added in this invention has a significant effect on the immune-enhancing effect. When the concentration of the citric acid aqueous solution is 1%~3% g / mL, the immune-enhancing effect is significantly improved compared to not using citric acid or a higher concentration (4%).

Description

Technical Field

[0001] This invention relates to the field of the efficacy of natural plants. Background Technology

[0002] Goji berries (Lycium chinense Miller) are perennial woody plants belonging to the genus Lycium in the Solanaceae family. They are a nutritious and health-promoting vegetable and a valuable traditional Chinese medicine. The "Shennong's Classic of Materia Medica" records: "Goji berries treat evil qi in the five internal organs, thirst, and rheumatism. Long-term consumption strengthens tendons and bones, promotes longevity, and improves resistance to cold and heat."

[0003] Currently, reports from both domestic and international sources indicate that some components of wolfberry possess immune-enhancing activities. Examples include "Zhang Xueyan et al., Research progress on chemical components and pharmacological effects of wolfberry and prediction analysis of quality markers, Chinese Journal of Traditional Chinese Medicine, 2024" and "You Jiaqi. Bioactivity study of wolfberry glycopeptides and their polyphenol complexes, East China University of Science and Technology, 2025".

[0004] Existing technologies mainly report the immune-enhancing activities of Lycium barbarum polysaccharides and glycopeptides, but do not provide a feasible preparation scheme for immune-enhancing products. Summary of the Invention

[0005] This invention aims to provide a wolfberry extract that can effectively remove impurities and can be produced stably, for use in enhancing immunity.

[0006] Specifically, this invention provides the use of wolfberry extract in the preparation of immune-enhancing products, wherein the preparation method of the wolfberry extract includes the following steps:

[0007] (1) Fresh wolfberry fruit, add 1%~3% g / mL aqueous solution of organic acid at a ratio of 2:1 g / mL, and pulp. The organic acid is selected from citric acid, lactic acid or malic acid;

[0008] (2) The fruit pulp is enzymatically inactivated and sterilized;

[0009] (3) 200-mesh plate and frame filter;

[0010] (4) The filtrate was enriched by macroporous resin and eluted sequentially with water and 60% v / v ethanol. The macroporous resin was selected from D101 or /

[0011] AB-8; collect the 60% v / v ethanol eluent, concentrate at low temperature to remove alcohol, and then obtain the wolfberry extract.

[0012] Among these methods, pulping fresh goji berries with a low-concentration organic acid aqueous solution offers the following advantages:

[0013] (1) Synergistic color protection and stabilization: Organic acids at low concentrations can regulate pH, inhibit polyphenol oxidase activity, prevent the oxidation and browning of polyphenols and other components during the pulping of fresh wolfberry, and help maintain the stability of some acid-sensitive active ingredients.

[0014] (2) Assisted extraction and protection: A mild acidic environment may be more conducive to the dissolution of certain glycosides or acidic polysaccharides, while avoiding the damage of active ingredients by strong acids or high temperatures, providing a more complete material basis for subsequent steps.

[0015] The advantages of enzymatic inactivation and sterilization of fruit pulp are as follows:

[0016] (1) Termination of degradation reaction: Rapidly inactivate endogenous enzymes (such as pectinase, glycosidase, etc.) in the fruit pulp to prevent them from degrading the target components (such as polysaccharides and glycosides) during subsequent processing or storage, thus ensuring the originality and consistency of the extract components.

[0017] (2) Control microorganisms in advance: Eliminating microbial contamination before purification not only ensures the hygiene and safety of the product, but also avoids the consumption or transformation of active ingredients by microbial activities, thereby improving the purity and quality stability of the final product.

[0018] The use of 200-mesh plate and frame filters as a preliminary solid-liquid separation method reflects a consideration of balancing process efficiency and cost.

[0019] (1) High-efficiency coarse separation: While maintaining a large throughput, it effectively removes large particulate impurities such as fruit pulp residue and seed fragments, reducing the burden on subsequent fine purification steps, preventing resin blockage, and improving the overall process smoothness and efficiency.

[0020] (2) Retaining target components: This mesh size selection avoids the loss of small and medium molecular weight active ingredients (such as some polysaccharides and flavonoids) that may be caused by excessive filtration, and achieves an optimized balance between efficiency and yield in the early stage of purification.

[0021] Among these steps, the enrichment and elution using macroporous resin is a key purification process, and the selection of its parameters is highly targeted.

[0022] (1) Resin screening: D101 or / and AB-8 type macroporous resins were specifically selected. These two resins have specific adsorption capacity for common hydrophilic and weakly polar active ingredients in wolfberry (such as wolfberry polysaccharides, betaine, and glycosides), which can effectively enrich substances and remove a large amount of water-soluble impurities such as sugars and inorganic salts.

[0023] (2) Gradient elution strategy: A simple gradient of "water washing + 60% ethanol elution" is adopted. Water washing can remove highly polar impurities; precise elution with a specific concentration of 60% ethanol (v / v) can selectively desorb the target components, resulting in an extract with a higher proportion of active ingredients and fewer impurities.

[0024] The advantages of using low-temperature alcohol removal are as follows: the eluent is concentrated at low temperature to remove ethanol, which minimizes the decomposition or inactivation of heat-sensitive components (such as certain volatile active substances or heat-labile glycosides) during the concentration process, thus ensuring the activity of the final product.

[0025] Furthermore, comparative analysis revealed that the amount of citric acid added in this invention significantly affects the immune-enhancing effect. When the concentration of the citric acid aqueous solution is between 1% and 3% g / mL, the immune-enhancing effect is significantly improved compared to not using citric acid or at a higher concentration (4%). In particular, when the concentration of the citric acid aqueous solution is 1%, it is significantly superior to other citric acid extracts at the same or even higher concentrations in improving the CD4+ / CD8+ ratio and increasing erythrocytes, / and hemoglobin, / and NK cells. Therefore, this invention ultimately preferably uses a citric acid aqueous solution of 1% to 3% g / mL, and more preferably 1%.

[0026] The elevated levels of CD4+ and NK cells indicate that lymphocyte subsets are selectively activated and proliferating.

[0027] Optimizing the CD4+ / CD8+ ratio is the core of immune balance.

[0028] CD4+ T cells (elevated): As the "commanders of the immune system," they are responsible for activating other immune cells (such as B cells producing antibodies and macrophages phagocytizing) and secreting cytokines to coordinate the entire immune response. An increased number of CD4+ T cells usually indicates an enhanced immune response.

[0029] CD8+ T cells (decreased or relatively reduced in proportion): As "killer cells," they are responsible for eliminating virus-infected or cancerous cells. In certain states of enhanced immunity (rather than acute infection), their numbers may remain stable or increase less than that of CD4+ cells, resulting in a relatively "decreased" proportion. This increase in the ratio (CD4+ / CD8+) is, in most cases, interpreted as the immune system being in a better responsive and regulated state, a positive signal of improved immune function. Detailed Implementation

[0030] Example 1

[0031] Wash fresh Qaidam wolfberries, add 1% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0032] UHT was used for enzyme inactivation and sterilization at a temperature of 125°C for 12 seconds.

[0033] Plate and frame filter, filtration accuracy 200 mesh;

[0034] Purification was performed using a chromatography column, with the column material being a mixture of macroporous resins: D101 and AB-8 in a 1:1 ratio.

[0035] The elution solvents were water and 60% ethanol. The water was used to elute for 3-5 column volumes, and the 60% ethanol was used to elute for 3-5 column volumes. The 60% ethanol eluent was then collected.

[0036] Low-temperature concentration to remove alcohol, temperature 50~60℃ (solid content measured ≥8%).

[0037] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 121°C for 6 seconds.

[0038] Example 2

[0039] Wash fresh Qaidam wolfberries, add 3% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0040] UHT was used for enzyme inactivation and sterilization at a temperature of 118°C for 20 seconds.

[0041] Plate and frame filter, filtration accuracy 200 mesh;

[0042] Purification was performed using a chromatography column, with the column material being a mixture of macroporous resins: D101 and AB-8 in a 1:1 ratio.

[0043] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0044] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0045] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 115℃ for 10 seconds.

[0046] Example 3

[0047] Wash fresh Qaidam wolfberries, add 2% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0048] UHT was used for enzyme inactivation and sterilization at a temperature of 121°C for 15 seconds.

[0049] Plate and frame filter, filtration accuracy 200 mesh;

[0050] Purification was performed using a chromatography column, with the column material being a mixture of macroporous resins: D101 and AB-8 in a 1:1 ratio.

[0051] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0052] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0053] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 118°C for 8 seconds.

[0054] Comparative Example 1

[0055] Wash fresh goji berries from the Qaidam Basin, add water at a 2:1 ratio, and blend into a pulp.

[0056] UHT was used for enzyme inactivation and sterilization at a temperature of 125°C for 12 seconds.

[0057] Plate and frame filter, filtration accuracy 200 mesh;

[0058] Purification was performed using a chromatography column, with the column material being a mixture of macroporous resins: D101 and AB-8 in a 1:1 ratio.

[0059] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0060] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0061] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 121°C for 6 seconds.

[0062] Comparative Example 2

[0063] Wash fresh Qaidam wolfberries, add 4% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0064] UHT was used for enzyme inactivation and sterilization at a temperature of 118°C for 20 seconds.

[0065] Plate and frame filter, filtration accuracy 200 mesh;

[0066] Purification was performed using a chromatography column, with the column material being a mixture of macroporous resins: D101 and AB-8 in a 1:1 ratio.

[0067] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0068] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0069] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 115℃ for 10 seconds.

[0070] Experiment Example 1: Immunity Enhancement Test

[0071] 1 Materials and Methods

[0072] 1.1 Samples: The samples are in liquid form, with a specification of 100 mL / bag, and are prepared by Example 1, Comparative Example 1, and Comparative Example 2 respectively.

[0073] 1.2 Experimental animals: 56 male SD rats of SPF grade were provided by Shandong Mingyue Experimental Animal Technology Co., Ltd. (Quality certificate number: 370726250100609087); 10 months old, production license number: SCXK(Lu)2022 0006.

[0074] 1.3 Experimental conditions:

[0075] 1.3.1 Experimental environment: The experimental animal room is a barrier system, with a use license number: SYXK(Shaan)2023 - 011, temperature 20°C - 26°C, relative humidity 30% - 70%.

[0076] 1.3.2 Animal feed: The animal maintenance feed is provided by Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., production license number: Su Feed License (2024)01008, feed certificate numbers: 20 - SM250328093, 20 - SM250509209.

[0077] 1.3.3 Animal bedding: The corn cob bedding for experimental animals is provided by Dezhou Gumei Agricultural Technology Co., Ltd., bedding certificate numbers: GM20250304003, GM20250506005.

[0078] 1.4 Equipment and reagents: JA3003 electronic balance, STX6201ZH electronic balance, JA21001 electronic balance, T500 electronic balance, SP - 56P ultraviolet - visible spectrophotometer, DL - 5B low - speed refrigerated centrifuge, Shenzhen Mindray BC - 5000Vet automatic blood cell analyzer, TGL - 16M table - top high - speed refrigerated centrifuge, DZKW - S - 6 electrothermal constant temperature water bath, DHP - 9402 constant temperature incubator, MX - S Czech mixer, JXFSTPRP - 24 automatic sample rapid grinder, DEM - 3 plate washer, DZKW - D - 2 electrothermal constant temperature water bath, RT - 6100 microplate reader, SC - 3610 low - speed centrifuge, DHP - 9162A electrothermal constant temperature incubator, pipette, pipette tips, test tubes, dissection instruments, etc.; blood cell analysis reagents (produced by Shenzhen Mindray Bio - medical Electronics Co., Ltd.); absolute ethanol, ethyl acetate, glacial acetic acid.

[0079] 1.5 Experimental Grouping and Dosage Design: Male aged SD rats were divided into four groups based on MDA levels: a high-dose group of Lycium barbarum extract, a low-dose group, a control group (Group 1 and Group 2), and a model control group, with 12 rats in each group. Dosage: 15 mL / kg BW for the high- and low-dose groups, 5 mL / kg BW for the control group, 15 mL / kg BW for the control group (Group 1 and Group 2), with a dosage of 5 mL twice daily. All groups were supplemented with purified water, while the model control group received the same volume of purified water.

[0080] 1.6 Experimental methods: Different concentrations of the test sample were administered to the dosage group, while the same volume of solvent (pure water) was administered to the model control group. The samples were administered by gavage for 30 consecutive days. After the last gavage, the samples were weighed, and blood was collected from the abdominal aorta to measure hematological parameters (hemoglobin, red blood cell count, white blood cell count and differential, hematocrit, platelet count, etc.) and immune cell typing (CD8+, CD4+, NK cells).

[0081] 1.7 Statistical Analysis of Experimental Data: SPSS 24 software was used for analysis of variance. However, the homogeneity of variance test must be performed first according to the procedure for analysis of variance. If the variances are homogeneous, the F-value is calculated. <F 0.05 Conclusion: There was no significant difference between the means of each group; F-value ≥ F 0.05 If P ≤ 0.05, perform statistical analysis by pairwise comparison of means among multiple dose groups and a control group; perform appropriate variable transformation on non-normal or unequal variance data until they meet the requirements of normality or homogeneity of variance, and then use the transformed data for statistical analysis; if the transformation still does not achieve the goal of normality or homogeneity of variance, use the rank-sum test for statistical analysis.

[0082] 2. Results:

[0083] Rats were administered different doses of "Lycium barbarum extract" by gavage for 30 days. The results of various indicators were compared with those of the model control group. The results are as follows:

[0084] 2.1 Effects of Lycium barbarum extract on rat body weight

[0085] As shown in Table 1, there were no significant differences in body weight and total weight gain between the dosage groups and the model control group at each stage (P>0.05).

[0086] Table 1. Effects of Lycium barbarum extract on rat body weight

[0087]

[0088] 2.2 Effects of Lycium barbarum extract on rat hematology

[0089] As shown in Tables 2-3, compared with the model control group, the hematological test results of rats in each dose group showed significant differences in the three indicators of red blood cells (RBC), hemoglobin (HGB), and hematocrit (HCT) in the high-dose group (P<0.05); significant differences in the two indicators of lymphocyte percentage (Lymph) and neutrophil percentage (Neut) between the high- and low-dose groups (P<0.01); and significant differences in the two indicators of monocyte percentage (Mono) in the high-dose group (P<0.01).

[0090] Table 2 Effects of Lycium barbarum extract on hematological parameters in rats

[0091]

[0092] Note: WBC stands for white blood cells, RBC for red blood cells, HGB for hemoglobin, PLT for platelets, and HCT for hematocrit. * indicates P < 0.05 compared to the model control group.

[0093] Table 3. Effects of Lycium barbarum extract on hematological parameters in rats.

[0094]

[0095] Note: Lymph refers to lymphocytes, Neutral cells to neutrophils, Mono to monocytes, Eos to eosinophils, and Baso to basophils. ** indicates P < 0.01 compared to the model control group.

[0096] 2.3 Effects of Lycium barbarum extract on rat T cell subsets / NK cells

[0097] As shown in Table 4, compared with the model control group, the differences in TCD4+ and CD8+ in the medium-dose group and the differences in NK cell indicators in the high- and low-dose groups were significant (P<0.05).

[0098] Table 4. Effects of Lycium barbarum extract on rat T cell subsets / NK cells

[0099]

[0100] Note: * indicates P<0.05 compared with the model control group.

[0101] 3. Summary

[0102] The "goji berry extract" was administered at doses of 15 mL / kg BW and 5 mL / kg BW, with a volume of 5 mL per dose, twice daily for 30 consecutive days. The experimental results are as follows:

[0103] Compared with the model control group, the hematological test results of rats in each dose group showed significant differences in red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT) between the high and low dose groups (P<0.05); significant differences in lymphocyte percentage and neutrophil percentage between the medium and low dose groups (P<0.01); and significant differences in monocyte percentage between the medium dose group (P<0.01).

[0104] Compared with the model control group, the differences in CD4+ and CD8+ in the high-dose group and the differences in NK cell indices between the high-dose and low-dose groups were statistically significant (P<0.01).

[0105] In conclusion, wolfberry extract has a good effect on enhancing immunity.

Claims

1. Use of a wolfberry extract in the manufacture of a product for enhancing immunity, wherein, The preparation method of the wolfberry extract includes the following steps: (1) Fresh wolfberry fruit, add 1%~3% g / mL aqueous solution of organic acid at a ratio of 2:1 g / mL, and pulp. The organic acid is selected from citric acid, lactic acid or malic acid; (2) The fruit pulp is enzymatically inactivated and sterilized; (3) 200-mesh plate and frame filter; (4) The filtrate was enriched by macroporous resin and eluted sequentially with water and 60% v / v ethanol. The macroporous resin was selected from D101 and / or AB-8. The 60% v / v ethanol eluent was collected and concentrated at low temperature to remove alcohol, thus obtaining the wolfberry extract.

2. The use according to claim 1, characterized in that, The enhanced immunity includes improving the CD4+ / CD8+ ratio, or increasing red blood cells, or / and hemoglobin, or / and NK cells.

3. The use according to claim 1, characterized in that, The organic acid is selected from citric acid.

4. The use according to claim 1, characterized in that, UHT was used for enzyme inactivation and sterilization.

5. The use according to claim 1, characterized in that, The macroporous resin is selected from a mixture of D101 and AB-8, where D101:AB-8 = 1:1 w / w.

6. The use according to claim 1, characterized in that, Elute with water for 3-5 column volumes, then elute with 60% v / v ethanol for 3-5 column volumes.

7. The use according to claim 1, characterized in that, Low-temperature concentration is carried out at a temperature of 50~60℃ until the solid content is ≥8%.

8. A method for preparing wolfberry extract with immune-enhancing effects, comprising the following steps: (1) Fresh wolfberry fruit, add 1%~3% g / mL aqueous solution of organic acid at a ratio of 2:1 g / mL, and pulp. The organic acid is selected from citric acid, lactic acid or malic acid; (2) The fruit pulp is enzymatically inactivated and sterilized; (3) 200-mesh plate and frame filter; (4) The filtrate was enriched by macroporous resin and eluted sequentially with water and 60% v / v ethanol. The macroporous resin was selected from D101 and / or AB-8. The 60% v / v ethanol eluent was collected and concentrated at low temperature to remove alcohol, thus obtaining the wolfberry extract.

9. The wolfberry extract prepared by the method of claim 8.

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