Compound red date extract with immune regulation function as well as preparation method and application of compound red date extract

Abstract

The invention relates to a compound red date extract with an immune regulation function as well as a preparation method and application thereof. The composite red date extract is prepared by taking red dates, astragalus membranaceus, Chinese yam, poria cocos, sea-buckthorn and medlar as raw materials through extraction, concentration and drying, and can be prepared into health-care food with an immune regulation function. Experiments prove that the compound red date extract prepared by the invention can enhance lymphopoiesis, delayed allergy, antibody generation, macrophage phagocytosis and NK cell activity, comprehensively improve the immune function and meet the health-care function requirement of enhancing immunity. The immunomodulatory activity of the compound red date extract is researched for the first time, and the compound red date extract has the advantages of being simple in production process, prominent in health-care function, small in dosage, safe to use, capable of being produced on a large scale and the like, thereby having wide application prospects in the aspect of preparing medicines and health-care foods for improving immunity.

Description

Technical Field

[0001] This invention belongs to the field of health food technology, specifically relating to a compound jujube extract with immune-regulating function, its preparation method, and its application. Background Technology

[0002] Sub-health is a state between illness and health. Due to increasingly fierce social competition and the accelerated pace and increased stress of life and work, sub-health has become a common condition, affecting people's quality of daily life, work, and learning abilities. Therefore, effectively preventing and improving sub-health has become an urgent problem to be solved by the medical community.

[0003] Immunity is the body's ability to resist external pathogens and maintain internal homeostasis; it's essentially the body's immune system's self-protective capacity. The primary task of immunity is to resist bacterial or viral infections, maintaining normal growth, development, and health. Weakened immunity is a common symptom and sign of sub-health; therefore, improving immunity is crucial for overcoming sub-health and reducing the likelihood of illness. Immune enhancement is the most direct, rapid, and effective method for improving immunity, and its basic methods include passive and active immunity. The immune system is a complex, sensitive, and orderly network system; any deficiency or abnormality in any link will lead to impaired immune defense. Therefore, maintaining the normal physiological function of the immune system is an indispensable and important aspect of improving immune function. Currently, there are many nutritional supplements that regulate immune function, but most have a single mechanism of action. Therefore, developing products that comprehensively enhance immunity has broad market prospects.

[0004] Modern scientific research shows that jujube, astragalus, wolfberry, poria cocos, yam, and sea buckthorn all have immune-enhancing effects, but studies on the combined use of these six ingredients to enhance immunity have not been reported. This invention is the first to study a compound jujube extract and its immunomodulatory activity prepared from jujube, astragalus, yam, poria cocos, sea buckthorn, and wolfberry. It has advantages such as simple production process, prominent health benefits, low dosage, safe use, and suitability for large-scale production. Therefore, it has broad application prospects in the preparation of drugs and health foods that improve immunity. Summary of the Invention The purpose of this invention is to provide a compound jujube extract with immunomodulatory functions and its preparation method. This invention uses jujube, astragalus, yam, poria cocos, sea buckthorn, and wolfberry as raw materials, and obtains a compound jujube extract through extraction, concentration, and drying. This extract can be made into a health food with immunomodulatory functions. Animal experiments have verified that the compound jujube extract prepared by this invention has a significant immune-enhancing effect.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A compound jujube extract with immunomodulatory function and its preparation method are disclosed. The extract is prepared by extracting, concentrating and drying a combination of jujube, astragalus, yam, poria cocos, sea buckthorn and wolfberry.

[0006] Preferably, the raw material composition comprises, by weight: 6-15 parts of jujube, 9-30 parts of astragalus, 15-30 parts of yam, 10-15 parts of poria cocos, 3-10 parts of sea buckthorn, and 6-12 parts of wolfberry.

[0007] More preferably, the raw material composition comprises, by weight: 15 parts red dates, 12 parts astragalus, 15 parts yam, 10 parts poria cocos, 3 parts sea buckthorn, and 9 parts wolfberry.

[0008] This invention also provides a method for preparing a compound jujube extract with immunomodulatory function, comprising the following steps: 1) Extraction: Weigh each raw material composition according to the weight parts, add water with a total material-to-liquid ratio of 1:6-1:18, soak, decoct and extract 1-3 times, extract for 1-2 hours, filter and combine the supernatants to obtain the total extract; 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 50-60℃ to obtain a concentrate that is concentrated to 1 / 20 to 1 / 10 of the original total extract volume. 3) Drying and pulverizing: The concentrate obtained in step 2) is vacuum dried at a temperature of 50-60℃ and pulverized to obtain compound jujube extract.

[0009] After obtaining the above-mentioned compound jujube extract, the present invention can be prepared into various dosage forms, such as tablets, capsules, granules, powders or pills, as needed using conventional preparation methods. It can also be oral liquids, drops, decoctions, ointments, gels, etc. The application of the compound jujube extract with immunomodulatory function obtained by the method described in this invention in the preparation of health food products that enhance immunity.

[0010] The raw material composition used in this invention consists of six medicinal materials: jujube, wolfberry, astragalus, yam, poria cocos, and sea buckthorn. The efficacy and bioactivity of each individual medicinal material as recorded in the 2020 edition of the Chinese Pharmacopoeia are described below: Red dates: Red dates ( Ziziphus Jujuba Mill. Ziziphus jujuba is the fruit of a plant in the Rhamnaceae family, genus Ziziphus. It has the effects of tonifying the middle energizer and replenishing qi, nourishing blood and calming the mind, nourishing yin and moistening dryness, and strengthening the body. It contains sugars, proteins, alkaloids, triterpenes, saponins, flavonoids, cyclic adenosine monophosphate, cyclic guanosine monophosphate, as well as amino acids, vitamins, minerals, and other nutrients and active substances. Modern research shows that it has various biological activities such as antioxidation, immune regulation, antitumor, hypoglycemia, hypolipidemia, and regulation of vaginal flora.

[0011] Goji berries: Goji berries are the fruit of the Solanaceae family, specifically the Ningxia wolfberry plant (Lycium barbarum). Lycium barbarum L The dried, ripe fruit of the plant has the effects of nourishing the liver and kidneys, and improving eyesight. It is rich in polysaccharides, betaine, pigments, proteins and other active ingredients, and has the effects of immune regulation, anti-oxidation, anti-aging, relieving eye fatigue and protecting the liver.

[0012] Astragalus: Astragalus is a species of Fabaceae (family Leguminosae). Astragalus membranaceus Fisch The dried root of Astragalus membranaceus has the effects of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting diuresis and reducing swelling, generating fluids and nourishing blood, and promoting blood circulation and relieving pain. It mainly contains three types of substances: saponins, flavonoids, and polysaccharides, which have the effects of enhancing immunity, calming nerves, improving sleep, improving memory, anti-oxidation, anti-tumor, anti-radiation, anti-thrombosis, and protecting the liver.

[0013] Yam: Yam is a plant belonging to the Dioscoreaceae family (Dioscorea). Dioscorea opposita Thunb The dried rhizome of [the plant] has the effects of tonifying the spleen and stomach, promoting body fluid production and benefiting the lungs, and tonifying the kidneys and astringing essence. It mainly contains a variety of functional components such as polysaccharides, sterols, glycoproteins, saponins, allantoin, polyphenols, and flavonoids, which have the effects of enhancing immunity, lowering blood sugar, anti-oxidation, and improving gastrointestinal function.

[0014] Poria cocos: Poria cocos is a fungus belonging to the Polyporaceae family. Poria cocos (Schw.) Wolf The dried sclerotium of the fungus has diuretic, spleen-strengthening, and calming effects. It mainly contains polysaccharide compounds, triterpenes and their esters, which enhance immunity, lower blood sugar and blood lipids, protect the liver, improve memory, and increase tolerance to hypoxia.

[0015] Sea buckthorn: Sea buckthorn is a plant of the Elaeagnaceae family (Hippophae rhamnoides). Hippophae rhamnoides L The dried, ripe fruit of the plant has the effects of strengthening the spleen and aiding digestion, relieving cough and expectoration, and promoting blood circulation and removing blood stasis. It contains flavonoids, sterols, vitamins, proteins, volatile oils, amino acids, and sugars, and has the effects of protecting the gastric mucosa, protecting the liver, anti-oxidation, enhancing immunity, and lowering blood lipids.

[0016] As can be seen from the above description of each medicinal material in the raw material composition used in this invention, they all have the function of enhancing immunity. Experiments have shown that the compound jujube extract prepared by this invention can enhance lymphocyte proliferation, delayed-type hypersensitivity reaction, antibody production, macrophage phagocytosis and NK cell activity, comprehensively improve immune function, and meet the health care function requirements of health food to enhance immunity. Attached Figure Description Figure 1 Cellular immune function assay - Mouse spleen lymphocyte transformation experiment.

[0017] Figure 2 Cellular immune function assay - delayed-type hypersensitivity test.

[0018] Figure 3 Humoral immune function assay - Measurement of serum hemolysin.

[0019] Figure 4 Mononuclear-macrophage function assay - mouse carbon clearance assay.

[0020] Figure 5 NK cell activity assay. Detailed Implementation To further understand the present invention, the present invention will be described in detail below with reference to embodiments. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0021] Example 1 1) Extraction: Weigh 6 parts of red dates, 9 parts of astragalus, 30 parts of yam, 10 parts of poria cocos, 3 parts of sea buckthorn and 6 parts of wolfberry according to the weight ratio. Add water with a total material-to-liquid ratio of 1:6, soak for 1 hour, decoct and extract once, extract for 1 hour, filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 50°C to obtain a concentrate that is 1 / 10 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 60°C and pulverized to obtain compound jujube extract with an extraction rate of 18.5%.

[0022] Example 2 1) Extraction: Weigh 8 parts of red dates, 11 parts of astragalus, 15 parts of yam, 12 parts of poria cocos, 6 parts of sea buckthorn and 12 parts of wolfberry according to the weight ratio, add water with a total material-to-liquid ratio of 1:6, soak for 1 hour, decoct and extract twice, extract for 1.5 hours, filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 60°C to obtain a concentrate that is 1 / 20 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 60°C and pulverized to obtain compound jujube extract with an extraction rate of 35.3%.

[0023] Example 3 1) Extraction: Weigh 14 parts of red dates, 9 parts of astragalus, 15 parts of yam, 10 parts of poria cocos, 10 parts of sea buckthorn and 6 parts of wolfberry according to the weight ratio. Add water with a total material-to-liquid ratio of 1:12. Soak for 1 hour, then decoct and extract once for 2 hours. Filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 50°C to obtain a concentrate that is 1 / 15 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 60°C and pulverized to obtain compound jujube extract with an extraction rate of 31.7%.

[0024] Example 4 1) Extraction: Weigh out 8 parts of red dates, 15 parts of astragalus, 16 parts of yam, 15 parts of poria cocos, 4 parts of sea buckthorn and 8 parts of wolfberry by weight, add water with a total material-to-liquid ratio of 1:12, soak for 1 hour, decoct and extract 3 times, extract for 1.5 hours each time, filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 55°C to obtain a concentrate that is 1 / 20 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 60°C and pulverized to obtain compound jujube extract with an extraction rate of 37.8%.

[0025] Example 5 1) Extraction: Weigh 15 parts of red dates, 12 parts of astragalus, 15 parts of yam, 10 parts of poria cocos, 3 parts of sea buckthorn and 9 parts of wolfberry according to the weight ratio. Add water with a total material-to-liquid ratio of 1:18, soak for 1 hour, decoct and extract 3 times, extract for 1 hour each time, filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 60°C to obtain a concentrate that is 1 / 10 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 50°C and pulverized to obtain compound jujube extract with an extraction rate of 43.5%.

[0026] Example 6 1) Extraction: Weigh 6 parts of red dates, 30 parts of astragalus, 15 parts of yam, 10 parts of poria cocos, 3 parts of sea buckthorn and 6 parts of wolfberry according to the weight ratio. Add water with a total material-to-liquid ratio of 1:18, soak for 1 hour, decoct and extract twice for 2 hours each time, filter and combine the supernatants to obtain the total extract. 2) Concentration: The total extract obtained in step 1) is concentrated under vacuum at a temperature of 50°C to obtain a concentrate that is 1 / 10 of the original extract. 3) Drying and pulverizing: The concentrate obtained in step 2) was vacuum dried at 55°C and pulverized to obtain compound jujube extract with an extraction rate of 34.5%.

[0027] Implementation of Section 7: Evaluation Experiment on Efficacy of Enhancing Immunity According to the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)" issued by the State Administration for Market Regulation, the National Health Commission, and the National Medical Products Administration, the determination method for whether a sample helps enhance immunity is used is as follows: A positive result in any two of the four aspects—cellular immune function, humoral immune function, mononuclear macrophage function, and NK cell activity—indicates that the tested sample has an effect on enhancing immunity. The following experiments were conducted according to Part II, Functional Testing Methods, of the "Methods for Functional Testing and Evaluation of Health Foods (2023 Edition)".

[0028] 1. Cellular immune function detection: including lymphocyte transformation assay and delayed-type hypersensitivity reaction.

[0029] Lymphocyte transformation experiment: C57 female mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were randomly divided into groups (n=10) and administered the compound jujube extract (40, 200, 1000 mg / kg), positive control drug (Yupingfeng granules 250 mg / kg), and physiological saline control by gavage for 30 consecutive days. Spleens were aseptically harvested and placed in a petri dish containing an appropriate amount of sterile Hank's solution. The spleen was gently ground with forceps to prepare a single-cell suspension. The suspension was filtered through a 200-mesh sieve or ground with four layers of gauze, washed twice with Hank's solution, and centrifuged for 10 min each time (1000 r / min). The cells were then resuspended in 1 mL of complete culture medium, and the viable cell count was determined by staining with tyrosine blue (should be above 95%). The cell concentration was adjusted to 3 × 10⁻⁶ cells / kg. 6 Cells / mL. Divided into ConA stimulation wells (7.5 μg / mL) and control wells, proliferation activity was detected using the MTT assay. The difference in OD570 between the ConA wells and the control wells was calculated, and the differences between groups were compared. Results are shown below. Figure 1 As shown in the figure, the results indicated that the OD difference in the medium (200 mg / kg) and high (1000 mg / kg) dose groups of the compound jujube extract was significantly higher than that in the control group (p<0.01), at 0.274±0.02 and 0.137±0.01, respectively (compared to 0.065±0.01 in the control group). This suggests that the compound jujube extract can specifically enhance the proliferation capacity of T lymphocytes in C57 female mice.

[0030] Delayed-type hypersensitivity assay (sheep erythrocyte (SRBC)-induced DTH in mice (plantar thickening method)): Female C57 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were randomly divided into groups (n=10) and administered the compound jujube extract obtained in Example 5 (40, 200, 1000 mg / kg), positive control drug (Yupingfeng granules 250 mg / kg), and physiological saline control by gavage for 30 consecutive days. Mice were immunized with 4% SRBC (0.1 mL / mouse). Four days later, the thickness of the left hind paw was measured, followed by a subcutaneous injection of 20% SRBC (20 μL). Measurements were repeated 24 hours later. The difference in paw thickness before and after challenge was calculated to assess DTH intensity. The results are as follows: Figure 2 As shown in the figure. The experimental results showed that the difference in foot-to-foot length in each dose group of the extract (40-1000 mg / kg) was significantly increased compared with the control group (0.757±0.15 mm) (1.160-1.753 mm, p<0.05). This indicates that continuous oral administration of compound jujube extract for 30 days can enhance the cellular immune response of C57 female mice.

[0031] 2. Humoral immune function assay: Measurement of serum hemolysin C57 female mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were randomly divided into groups (n=10) and administered the compound jujube extract obtained in Example 5 (40, 200, 1000 mg / kg), positive control drug (Yupingfeng granules 250 mg / kg), and physiological saline control by gavage for 30 consecutive days.

[0032] Sheep blood was collected and washed three times with physiological saline, centrifuged for 10 min each time (2000 r / min). The compacted SRBCs were resuspended in physiological saline to a 2% (v / v) cell suspension. 0.2 mL was injected intraperitoneally into each C57 female mouse (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) for immunization. Four to five days later, the eyeballs were removed, and blood was collected in centrifuge tubes. After approximately 1 hour, the clotted blood was separated from the tube wall to allow serum to be fully separated. The collected serum was centrifuged for 10 min (2000 r / min) and collected. The serum was serially diluted with physiological saline. 100 μL of serum at different dilutions was placed in each well of a micro-hemagglutination assay plate, followed by 100 μL of 0.5% (v / v) SRBC suspension. The mixture was thoroughly mixed, placed in a moistened dish, covered, and incubated at 37°C for 3 h. The degree of hemagglutination was observed. Antibody volume was calculated based on the agglutination grade, and differences between groups were compared. The results are as follows: Figure 3 As shown in the figure, compared with the control group, the antibody counts induced by low (40 mg / kg), medium (200 mg / kg), and high (1000 mg / kg) concentrations of compound jujube extract and positive control drug were significantly higher than those in the control group. This indicates that continuous gavage administration of compound jujube extract for 30 days can enhance humoral immune function in C57 female mice.

[0033] 3. Mononuclear-macrophage mouse carbon clearance experiment.

[0034] Female C57 mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.) were randomly divided into groups (n=10) and administered the compound jujube extract obtained in Example 5 (40, 200, 1000 mg / kg), the positive control drug (Yupingfeng granules 250 mg / kg), and physiological saline as a control by gavage for 30 consecutive days. Indian ink (10 mL / kg) was injected via the tail vein, and blood samples were collected at 2 min and 10 min to measure OD600. The phagocytic index was used to represent the carbon clearance capacity of the mice. The phagocytic index α was calculated using the following formula. A significantly higher phagocytic index in the test sample group than in the control group indicates a positive result for this experiment.

[0035] ; ; Compared with the control group, low (40 mg / kg), medium (200 mg / kg), and high (1000 mg / kg) concentrations of compound jujube extract significantly enhanced the phagocytic index of mononuclear macrophages, as shown in the results. Figure 4 As shown. Figure 4 The results showed that continuous oral administration of compound jujube extract for 30 days could enhance the phagocytic function of mononuclear macrophages in mice.

[0036] 4. NK cell activity assay.

[0037] Spleen effector cells (2×10⁻⁶) were prepared according to the method described in "1. Cellular immune function detection". YAC-1 target cells were passaged and cultured, and the cell concentration was adjusted to 4 × 10⁶ cells / mL using RPMI 1640 complete culture medium. 5 / mL. Take 100 μL each of target cells and effector cells (effector-target ratio 50:1) and add them to a 96-well culture plate; add 100 μL each of target cells and culture medium to the target cell spontaneous release well, and add 100 μL each of target cells and 1% NP40 or 2.5% Triton to the target cell maximum release well; each of the above is set up with three parallel wells, and cultured in a 37℃, 5% CO2 incubator for 4 h. Then, centrifuge the 96-well culture plate at 1500 r / min for 5 min, and aspirate 100 μL of supernatant from each well into a flat-bottomed 96-well culture plate, and add 100 μL of LDH matrix solution. React for 3-10 min depending on the room temperature. Add 30 μL of 1 mol / L HCl to each well, and measure the optical density (OD) at 490 nm using a microplate reader. The results are as follows. Figure 5As shown in the results, compared with the control group, low (40 mg / kg), medium (200 mg / kg), and high (1000 mg / kg) concentrations of compound jujube extract significantly enhanced the phagocytic index of mononuclear macrophages. NK cell activity in the low (40 mg / kg), medium (200 mg / kg), and high (1000 mg / kg) concentrations of compound jujube extract and the positive control group administered by gavage for 30 consecutive days was significantly higher than that in the control group.

[0038] The above examples demonstrate that compound jujube extract (200-1000 mg / kg) comprehensively enhances immune function by synergistically boosting lymphocyte proliferation, DTH response, antibody production, macrophage phagocytosis, and NK cell activity, thus meeting the requirements for immune-enhancing health benefits. The functional tests used the compound jujube extract obtained in Example 5 as the test sample. Subsequent tests of compound jujube extracts obtained in other examples also yielded the same test results as the compound jujube extract obtained in Example 5, and will not be repeated here.

[0039] All aspects not described in detail in this invention can be covered using conventional technical knowledge in the field.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications or equivalent substitutions to the technical solutions of the present invention do not depart from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A compound red date extract having immunomodulatory function, characterized in that, The compound red date extract is prepared from red dates, astragalus, Chinese yam, poria cocos, sea buckthorn and medlar.

2. The compound red date extract according to claim 1, characterized in that, The raw materials comprise, by weight, 6-15 parts of red dates, 9-30 parts of astragalus, 15-30 parts of Chinese yam, 10-15 parts of poria cocos, 3-10 parts of sea buckthorn and 6-12 parts of medlar.

3. The compound red date extract according to claim 1, characterized in that, The raw materials comprise, by weight, 15 parts of red dates, 12 parts of astragalus, 15 parts of Chinese yam, 10 parts of poria cocos, 3 parts of sea buckthorn and 9 parts of medlar.

4. A preparation method of a compound red date extract with immunomodulatory function, the preparation method comprising the following steps: 1) Extraction: the raw materials according to any one of claims 1-3 are weighed, water is added at a total liquid ratio of 1:6-1:18, soaking and decoction extraction is performed 1-3 times, extraction is performed for 1-2 hours, the supernatant is filtered and combined to obtain a total extract; 2) Concentration: the total extract obtained in step 1) is vacuum concentrated at a temperature of 50-60℃ to obtain a concentrated solution concentrated to 1 / 20-1 / 10 of the volume of the original total extract; 3) Drying and pulverization: the concentrated solution obtained in step 2) is vacuum dried at a temperature of 50-60℃, and pulverized to obtain the compound red date extract.

5. Use of the compound red date extract according to any one of claims 1-3 or the compound red date extract obtained by the preparation method of claim 4 in the preparation of health-care food for enhancing immunity.

6. Use according to claim 5, characterized in that, The dosage form of the health-care food is tablets, capsules, granules, powder or pills.

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