Lycium barbarum extract with anti-inflammatory effect and preparation method thereof

Abstract

The invention provides a wolfberry extract which is stable and has good anti-inflammatory activity. The invention also provides a relatively special preparation process of the extract.

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, both domestic and international reports indicate that some components of wolfberry possess anti-inflammatory activity. For example, "Song Jiantao et al., Research on the Mechanism of Effect of Wolfberry Polysaccharide on Chronic Ulcerative Colitis Based on Tryptophan Metabolism, Drug Evaluation Research, 2025", "Han Lina et al., Progress in Anti-inflammatory Effect and Mechanism of Wolfberry Polysaccharide, Modern Immunology, 2018", and "Liu Yanxia et al., Exploring the Potential Mechanism of Wolfberry in Treating Knee Osteoarthritis Based on Network Pharmacology and Molecular Docking, Hainan Medical Journal, 2023".

[0004] Existing technologies mainly report the anti-inflammatory activities of Lycium barbarum polysaccharides and quercetin, but do not provide a feasible preparation method for anti-inflammatory 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 effective anti-inflammatory purposes.

[0006] Specifically, this invention provides the use of wolfberry extract in the preparation of anti-inflammatory 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 oxidative browning of anti-inflammatory components such as polyphenols 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 pulp to prevent them from degrading the target anti-inflammatory 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 anti-inflammatory 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 anti-inflammatory 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 anti-inflammatory components (such as certain volatile active substances or heat-unstable 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 anti-inflammatory effect. When the concentration of the citric acid aqueous solution is between 1% and 3% g / mL, its anti-inflammatory effect is significantly enhanced compared to the absence of citric acid or 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 reducing CRP, and / or IL-6, and / or IL-1β, and / or TNF-α levels, and / or increasing IL-1Ra, and / or ALB levels. Therefore, this invention ultimately preferably uses a citric acid aqueous solution of 1% to 3% g / mL, and more preferably 1%.

[0026] CRP (C-reactive protein) is an acute-phase reactive protein produced by the liver. It does not directly cause inflammation, but acts as a "barometer" or "amplifier" of inflammation intensity. Its levels rise rapidly and significantly during infection, trauma, or chronic inflammatory diseases. Decreased CRP levels are a clear indication that inflammatory activity is under control. This means that the primary cause triggering inflammation (such as infection or autoimmune attack) has lessened, or that treatment (such as antibiotics or immunosuppressants) is effective.

[0027] IL-1β (interleukin-1β), TNF-α (tumor necrosis factor-α), and IL-6 (interleukin-6) are the three core pro-inflammatory cytokines, serving as the "engine" and "command signal" of the inflammatory response. Specifically: TNF-α and IL-1β are the initial initiators of the inflammatory cascade, directly causing fever, vasodilation, activation of immune cells, and inducing the production of other factors (such as IL-6); IL-6 acts as a crucial amplifier, induced by TNF-α / IL-1β, further activating more immune cells and directing the liver to synthesize acute-phase proteins such as CRP. Lowering the levels of these core pro-inflammatory cytokines is the direct goal and fundamental effect of anti-inflammatory therapy, breaking the vicious cycle of inflammatory signals and alleviating tissue damage and clinical symptoms (such as fever and pain).

[0028] IL-1Ra (interleukin-1 receptor antagonist) is a naturally occurring anti-inflammatory cytokine. Its structure is similar to IL-1β, and it competitively binds to the IL-1 receptor without activating signaling, thus blocking the pro-inflammatory effects of IL-1β. An elevated level of IL-1Ra indicates:

[0029] Activation of endogenous anti-inflammatory response: This is a negative feedback regulatory mechanism initiated by the body after sensing excessive inflammation (especially excessive IL-1β signaling). The body attempts to "neutralize" the excess IL-1β by increasing IL-1Ra to prevent uncontrolled inflammation. Shift of inflammatory balance towards resolution: In successful anti-inflammatory treatment or during natural disease recovery, a sustained or relatively elevated level of IL-1Ra indicates a favorable shift in the balance of pro-inflammatory (IL-1β) and anti-inflammatory (IL-1Ra) forces, which helps limit tissue damage and promote inflammation resolution. As a marker of disease protection or good prognosis: In some infectious or inflammatory diseases, individuals who can produce sufficient IL-1Ra often have milder symptoms and better prognoses.

[0030] Albumin (ALB) is the main plasma protein synthesized by the liver. During acute inflammation, the liver preferentially synthesizes proteins such as CRP, leading to a decrease in albumin synthesis. Simultaneously, albumin breakdown may increase, resulting in a drop in blood levels. Albumin levels decrease during the acute inflammatory phase (negative acute response), so a rebound or maintenance of normal high levels is significant. A rebound in ALB levels indicates that the liver has switched from "inflammatory emergency mode" back to "normal mode," no longer preferentially synthesizing large amounts of inflammatory proteins like CRP, but resuming the synthesis of albumin, which is crucial for maintaining normal bodily functions. Albumin synthesis requires sufficient amino acid precursors and normal liver function. Elevated levels reflect adequate nutrient intake, good absorption, and robust liver synthesis. This is directly related to an improvement in the inflammatory state. In patients with chronic diseases (such as cirrhosis, chronic kidney disease, and chronic heart failure) or severe illness, albumin levels are a strong predictor of disease severity and mortality risk. Elevated or stable levels generally indicate better clinical outcomes and survival rates. Albumin is responsible for maintaining plasma colloid osmotic pressure (preventing edema), transporting hormones, fatty acids, and drugs. Normalization of these levels means that these important physiological functions are protected, which helps in overall recovery. Detailed Implementation

[0031] Example 1

[0032] Wash fresh Qaidam wolfberries, add 1% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0033] UHT was used for enzyme inactivation and sterilization at a temperature of 125°C for 12 seconds.

[0034] Plate and frame filter, filtration accuracy 200 mesh;

[0035] 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.

[0036] 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.

[0037] Low-temperature concentration to remove alcohol, temperature 50~60℃ (solid content measured ≥8%).

[0038] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 121°C for 6 seconds.

[0039] Example 2

[0040] Wash fresh Qaidam wolfberries, add 3% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0041] UHT was used for enzyme inactivation and sterilization at a temperature of 118°C for 20 seconds.

[0042] Plate and frame filter, filtration accuracy 200 mesh;

[0043] 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.

[0044] The elution solvents were water and 60% ethanol. Five column volumes of water were used for elution, and three column volumes of 60% ethanol were used for elution.

[0045] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0046] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 115℃ for 10 seconds.

[0047] Example 3

[0048] Wash fresh Qaidam wolfberries, add 2% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0049] UHT was used for enzyme inactivation and sterilization at a temperature of 121°C for 15 seconds.

[0050] Plate and frame filter, filtration accuracy 200 mesh;

[0051] 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.

[0052] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0053] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0054] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 118°C for 8 seconds.

[0055] Comparative Example 1

[0056] Wash fresh goji berries from the Qaidam Basin, add water at a 2:1 ratio, and blend into a pulp.

[0057] UHT was used for enzyme inactivation and sterilization at a temperature of 125°C for 12 seconds.

[0058] Plate and frame filter, filtration accuracy 200 mesh;

[0059] 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.

[0060] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0061] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0062] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 121°C for 6 seconds.

[0063] Comparative Example 2

[0064] Wash fresh Qaidam wolfberries, add 4% citric acid aqueous solution at a ratio of 2:1, and blend into a pulp;

[0065] UHT was used for enzyme inactivation and sterilization at a temperature of 118°C for 20 seconds.

[0066] Plate and frame filter, filtration accuracy 200 mesh;

[0067] 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.

[0068] The elution solvents were water and 60% ethanol. Five column volumes were eluted with water, and three column volumes were eluted with 60% ethanol.

[0069] Low-temperature concentration to remove alcohol, temperature 50~60℃;

[0070] The wolfberry extract was obtained by aseptic filling after UHT sterilization at 115℃ for 10 seconds.

[0071] Test Example 1: Anti-inflammatory Activity Test

[0072] 1. Materials and Methods

[0073] 1.1 Sample: The sample is in liquid form, with a specification of 100 mL / bag, and is prepared by Example 1 (also known as "wolfberry extract" in this invention), Comparative Example 1, and Comparative Example 2 respectively.

[0074] 1.2 Experimental animals: SPF-grade male SD rats provided by Shandong Mingyue Experimental Animal Technology Co., Ltd. (Quality certificate number: 370726250100609087); 10 months old, production license number: SCXK (Lu) 2022 0006.

[0075] 1.3 Experimental conditions:

[0076] 1.3.1 Experimental environment: The experimental animal room is a barrier system, with the use license number: SYXK (Shan) 2023-011, temperature 20°C - 26°C, relative humidity 30% - 70%.

[0077] 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.

[0078] 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.

[0079] 1.4 Equipment and Reagents: JA3003 electronic balance, STX6201ZH electronic balance, JA21001 electronic balance, T500 electronic balance, SP-56P UV-Vis spectrophotometer, DL-5B low-speed refrigerated centrifuge, Shenzhen Mindray BC-5000Vet fully automated blood cell analyzer, TGL-16M benchtop high-speed refrigerated centrifuge, DZKW-S-6 electric thermostatic water bath, DHP-9402 thermostatic incubator, MX-S Cello Czech mixer. JXFSTPRP-24 fully automated rapid sample grinder, DEM-3 plate washer, DZKW-D-2 electric thermostatic water bath, RT-6100 enzyme-linked immunosorbent assay (ELISA) analyzer, SC-3610 low-speed centrifuge, DHP-9162A electric thermostatic incubator, pipettes, pipette tips, test tubes, dissecting instruments, etc.; Protein quantification kit (batch number: 20250607) (Nanjing Jiancheng Bioengineering Institute); Rat interleukin 1β (IL-1β) quantification kit, rat interleukin 6 (IL-6) quantification kit, rat tumor necrosis factor α (TNF-α) quantification kit, rat interleukin 1 receptor antagonist (IL-1Ra) quantification kit, rat albumin (ALB) quantification kit, rat C-reactive protein (CRP) quantification kit (Quanzhou Ruixin Biotechnology Co., Ltd.); Anhydrous ethanol, ethyl acetate, glacial acetic acid.

[0080] 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.

[0081] 1.6 Experimental methods: Different concentrations of test samples were administered to the dosage group, while the model control group was given the same volume of solvent (pure water). The samples were continuously administered by gavage for 30 days. After the last gavage, the samples were weighed, and blood was collected from the abdominal aorta to measure CRP, IL-6, IL-1Ra, IL-1β, TNF-α, and ALB.

[0082] 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 0.05If 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.

[0083] 2. Results:

[0084] 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:

[0085] 2.1 Effects of Lycium barbarum extract on rat body weight

[0086] 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).

[0087] Table 1. Effects of Lycium barbarum extract on rat body weight

[0088]

[0089] 2.2 Effects of Lycium barbarum extract on inflammatory factors and albumin in rats

[0090] As shown in Tables 2 and 3, compared with the model control group, the high-dose group showed highly significant differences in C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and albumin (ALB) (P<0.01, P<0.05). The low-dose group showed significant differences in interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) (P<0.05), which manifested as alleviating the inflammatory response and increasing albumin levels, such as decreasing the levels of CRP, IL-6, IL-1β, and TNF-α, and increasing the levels of IL-1Ra and ALB. Furthermore, compared with the model control group, the comparative group 1 showed significantly increased levels of CRP, IL-6, IL-1β, and TNF-α, while decreasing ALB levels (P<0.01, P<0.05).

[0091] Table 2. Effects of Lycium barbarum extract on inflammatory factors and albumin in rats.

[0092]

[0093] Note: CRP is C-reactive protein, IL-6 is interleukin-6, and IL-1Ra is an interleukin-1 receptor antagonist. ** indicates P < 0.01 compared to the model control group.

[0094] Table 3. Effects of Lycium barbarum extract on inflammatory factors and albumin in rats.

[0095]

[0096] Note: IL-1β is interleukin-1β, TNF-α is tumor necrosis factor-α, and ALB is albumin. Compared with the model control group, ** indicates P<0.01, and * indicates P<0.05.

[0097] 3. Summary

[0098] 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:

[0099] Compared with the model control group, the high-dose group showed highly significant differences in C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and albumin (ALB) (P<0.01, P<0.05). The low-dose group showed significant differences in interleukin-1 receptor antagonist (IL-1Ra), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) (P<0.05), which manifested as alleviating the inflammatory response and increasing albumin levels, such as decreasing CRP, IL-6, IL-1β, and TNF-α levels, and increasing IL-1Ra and ALB levels.

[0100] In summary, the "goji berry extract" provided by this patent has anti-inflammatory effects.

Claims

1. The use of wolfberry extract in the preparation of anti-inflammatory products, among which, 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 anti-inflammatory effects include reducing CRP, and / or IL-6, and / or IL-1β, and / or TNF-α levels, and / or increasing IL-1Ra, and / or ALB levels.

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 a wolfberry extract with anti-inflammatory 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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