Application of L-rhamnose in preparation of immunity enhancing product

Abstract

The invention relates to application of L-rhamnose in preparation of an immunity enhancing product, and belongs to the technical field of biological medicines. The invention discloses new application of L-rhamnose in enhancing cellular immunity or humoral immunity for the first time. A mouse experiment effectively proves that the L-rhamnose has the functions of improving the splenocyte proliferation rate, improving the NK cell activity, improving the phagocytic ability of macrophages and improving the antibody generation ability. Compared with an existing product for enhancing immunity, the L-rhamnose has the effects on humoral immunity, cellular immunity, mononuclear-macrophage functions and NK cell viability, is comprehensive in effect and can be used for preparing the product for enhancing cellular immunity or humoral immunity. And the L-rhamnose has the characteristic of sweetness, so that the L-rhamnose can be used as a sweetening agent for enhancing cellular immunity or humoral immunity to be applied to preparation of health care products or functional foods, and has a better application prospect.

Description

Technical Field

[0001] The invention relates to application of L-rhamnose in preparing immunity enhancing products, and belongs to the technical field of biomedicine. Background Art

[0002] In recent years, as people's awareness of health has increased, the research and development of drugs, health products and functional foods that enhance immunity has gradually become a hot topic. In the existing technology, commonly used immune-enhancing products include vitamin supplements (vitamin C and vitamin D), mineral supplements (zinc and selenium), plant extracts (echinacea, elderberry and turmeric), probiotics (Lactobacillus rhamnosus), protein supplements, bee products (propolis and royal jelly), Chinese herbal supplements (Ganoderma lucidum spore powder, ginseng and cordyceps), polysaccharides (β-glucan), etc. However, these traditional products often have problems such as limited effects or limited applicable populations, and some products may cause side effects due to improper dosage control. Therefore, it is urgent to develop an efficient, safe and diversified immune enhancer.

[0003] L-rhamnose is a naturally occurring 6-deoxyhexose that is widely distributed in plant cell walls, microbial polysaccharide chains and certain biological metabolites. L-rhamnose can be extracted from the waste liquid of quercetin preparation from Sophora japonica seeds at a low price. L-rhamnose not only has a natural sweetness (33% of the sweetness of sucrose), but also has good safety and can be used as a food additive. In addition, L-rhamnose has anti-wrinkle, anti-aging, anti-glycation, anti-sensitivity and moisturizing effects, and can be widely used in the field of skin care.

[0004] According to the "Health Food Function Test and Evaluation Method (2023 Edition)", if the results of any two of the four aspects of humoral immunity, cellular immunity, monocyte-macrophage function, and NK cell activity are positive, it can be determined that the test sample has the effect of enhancing immunity. At present, there is no direct evidence that oral L-rhamnose has the effect of enhancing immunity, nor is there any core technology and actual products that use L-rhamnose in medicines, health products, or functional foods that enhance immunity. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides the use of L-rhamnose in the preparation of an immunity enhancing product.

[0006] The technical solution of the present invention is as follows: Application of L-rhamnose in preparing a product for enhancing cellular immunity or humoral immunity, wherein L-rhamnose is a main active ingredient in the product.

[0007] According to the preferred embodiment of the present invention, the molecular formula of L-rhamnose is C6H 12 O5 or C6H 12O5·H2O (L-rhamnose monohydrate).

[0008] Preferably according to the present invention, the product for enhancing cellular immunity or humoral immunity is a medicine, health product or functional food that can enhance cellular immunity or humoral immunity.

[0009] Preferably, according to the present invention, the product for enhancing cellular immunity enhances cellular immunity by increasing the proliferation rate of spleen cells through L-rhamnose.

[0010] Preferably, according to the present invention, the product for enhancing cellular immunity enhances cellular immunity by increasing the activity of NK cells through L-rhamnose.

[0011] Preferably, according to the present invention, the product for enhancing cellular immunity enhances cellular immunity by improving the phagocytic ability of macrophages through L-rhamnose.

[0012] Preferably, according to the present invention, the product for enhancing humoral immunity enhances humoral immunity by improving antibody production capacity through L-rhamnose.

[0013] A composition for enhancing cellular immunity or humoral immunity comprises L-rhamnose and / or its hydrate, and a pharmaceutically acceptable carrier or excipient; the composition contains L-rhamnose and / or its hydrate as the main active ingredient.

[0014] Preferably according to the present invention, the dosage form of the composition for enhancing cellular immunity or humoral immunity includes powder, granules, tablets, pills, capsules, sustained-release agents, rate-controlled slow-release agents or suspensions.

[0015] Preferably according to the present invention, the composition for enhancing cellular immunity or humoral immunity is administered via the gastrointestinal tract.

[0016] Preferably, according to the present invention, the administration dosage of the composition for enhancing cellular immunity or humoral immunity is 10-100 mg / kg body weight each time, 1-3 times a day.

[0017] Beneficial effects: The present invention discloses for the first time a new use of L-rhamnose for enhancing cellular immunity or humoral immunity. The present invention effectively confirms through mouse experiments that L-rhamnose has the functions of increasing the proliferation rate of spleen cells, increasing the activity of NK cells, increasing the phagocytic ability of macrophages and increasing the ability to produce antibodies. Compared with existing products that enhance immunity, L-rhamnose has effects in four aspects: humoral immunity, cellular immunity, monocyte-macrophage function and NK cell activity, and has comprehensive effects, and can be used to prepare products that enhance cellular immunity or humoral immunity.

[0018] In addition, L-rhamnose has the property of sweetness, which makes it possible to use it as a sweetener that enhances cellular immunity or humoral immunity in the preparation of health products or functional foods. It has better application prospects, and its relatively low price also makes it more popular in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the body weight change of mice in each group.

[0020] Figure 2 This is a graph analyzing the differences in the proliferation rates of spleen cells in each group of mice.

[0021] Figure 3 This is the difference analysis of NK cell activity among mice groups.

[0022] Figure 4 These are the results of differential analysis of the phagocytic index and phagocytic rate of chicken red blood cells by mouse macrophages in each group.

[0023] Figure 5 This is the result of the analysis of the difference in the number of antibody-producing cell plaques in each group of mice. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0025] The main reagents and consumables used in the examples are as follows.

[0026] Reagents: L-rhamnose (R3875-10G, Sigma-Aldrich), Hank's solution (MA0041, Meilun Biotechnology), RPMI1640 culture medium (G4531, Sewell), fetal bovine serum (164210-50, Sewell), red blood cell lysis solution (R1010, Solebao), glutamine (MAO155, Meilun), mercaptoethanol solution (M917637, McLean), concanavalin (11028-71-0, Sigma-Aldrich), CCK-8 solution (C0043, Beyotime), pH 6.8 buffer (P397573, Yuanye Biotechnology), agar (HB8274-1, Haibo Biotechnology), 0.4% Trypan blue dye (C0040, Solebol), Giemsa staining solution (G1010, Solebol), methanol (M116115, Aladdin), sterile defibrinated sheep blood (SRBC) (20240626, TX0030, Solebol), SA buffer (0625A24, IH0065, Regen Biotechnology).

[0027] Consumables: 96-well culture plates (Corning), 24-well culture plates (Corning), 200-mesh screen (abs7232, absin), microscope slides (25×75 mm, Shitai), 1 mL syringes, gavage needles, 1 mL separation gel / coagulant serum collection tubes (Sanli), vernier calipers (precision 0.02 mm), gauze, enamel trays, and microsyringes (50 μL).

[0028] Experimental animals: 167 SPF-grade Balb / c male mice, weighing 18-22 g, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., production license number: SCXK (Lu) 2022-0006. Pre-feed for 1 week, room temperature 20-25℃, humidity 40-70%, ventilation frequency 10-20 times / hour, light 12 hours light and dark alternation. Mice were free to eat, fed with maintenance feed, drinking bottles were changed at any time, and mouse cages were changed twice a week.

[0029] Instruments: electronic scale (ACS-6 kg, Shanghai Ranhao), precision electronic balance (GB / T26497-2011, Shanghai Zhuojing), desktop low-temperature centrifuge (5417R, EPPENDORF), pipette (20~200 μL, Thermo), carbon dioxide incubator (311, Thermo), biological safety cabinet (HR30-11A2, Haier), living cell analyzer (CountessⅡ, United States / LifeTechnologies), biochemical incubator (LRH-70, Shanghai Yiheng), fluorescence inverted microscope (CKX53, Olybas), microplate reader (Enspire, United States / Perkinelmer), vertical pressure steam sterilizer (YXQ-LS-50SⅡ, Shanghai Boxun), high-speed refrigerated centrifuge (Germany / ThermoFisher), balance (Shimadzu), desktop low-speed centrifuge (TDZ5, Hunan Hexi), etc.

[0030] Example 1. Construction of mouse model 1. Construction of experimental mouse model Grouping: 167 mice were placed in an environment with a temperature of 24±1℃ and a relative humidity of 60~70% with a 12-h light-dark cycle (light from 7:00 to 19:00), and randomly divided into 10 groups, namely, blank group: C1 / C2; positive control group: P1 / P2; low-dose drug administration group: A1 / A2; medium-dose drug administration group: M1 / M2; high-dose drug administration group: B1 / B2.

[0031] The details are as follows: Group C1: blank control group, 15 mice; Group C2: blank control group, 20 mice.

[0032] Group P1: positive control group, 18 mice; Group P2: positive control group, 14 mice.

[0033] Group A1: low-dose group, 18 mice; Group A2: low-dose group, 14 mice.

[0034] Group M1: medium-dose group, 15 mice; Group M2: medium-dose group, 20 mice.

[0035] Group B1: high-dose group, 18 mice; Group B2: high-dose group, 14 mice.

[0036] Administration: L-rhamnose was administered to mice by gavage at a dose of 10 mg / kg in group A1 / A2, 30 mg / kg in group M1 / M2, 100 mg / kg in group B1 / B2, sterile water was administered to mice by gavage at the same dose in group C1 / C2, and β-glucan was administered to mice by gavage at a dose of 100 mg / kg in group P1 / P2 for 5 consecutive weeks (W). During the administration process, the state of the mice (mental state, hair state, excrement state, and oral and nasal secretion state) was carefully observed, the reagents were prepared daily, and the gavage administration time was fixed every day to obtain the experimental mouse model.

[0037] 2. Body weight changes of mice in each group The mice were weighed before the experimental mouse model was constructed as the initial weight. Then, during the construction of the experimental mouse model, the mice were weighed every week. After 5 weeks of gavage (W), the mice were fasted for one week. The mice were weighed before sampling as the sampling weight. The weight measurement results of each group were averaged. The results are shown in Figure 1 shown.

[0038] Depend on Figure 1 It can be seen that there is no significant difference in the body weight of mice among the groups, indicating that L-rhamnose has no obvious toxic side effects.

[0039] Example 2: Determination of the proliferation rate of spleen cells in each group of mice The experimental mouse models of groups C1, P2, A2, M1, and B2 were selected. The mice were fasted for one week and then killed and dissected to obtain spleen tissues of mice in each group. Then, the proliferation rate of spleen cells was measured. The specific measurement method is as follows.

[0040] 1. Preparation of complete culture medium RPMI1640 culture medium was sterilized by filtration and then added with 10% fetal bovine serum, 1% glutamine (200 mmol / L), penicillin (100 U / mL), streptomycin (100 μg / L) and 5×10 -5 1 mol / L of mercaptoethanol, adjust the pH to 7.0-7.2 with sterile 1 mol / L HCl or 1 mol / L NaOH to obtain a complete culture solution.

[0041] 2. Preparation of ConA solution ConA was prepared into a 100 μg / mL solution with double distilled water, sterilized by filtration, and stored at -20°C.

[0042] 3. Preparation of spleen cell suspension The spleen tissue of each group of mice was placed in a small dish containing an appropriate amount of sterile Hank's solution, and the spleen was gently ground with tweezers to prepare a single-cell suspension; filtered through a 200-mesh sieve, centrifuged at 300 g for 5 min, the supernatant was discarded, the cell slurry was flicked up, 3 mL of red blood cell lysis buffer was added for 3 min, and 3 mL of Hank's solution was added after the red blood cells were lysed, centrifuged at 300 g for 5 min, resuspended with 1 mL of complete culture medium, and the number of live cells was counted with trypan blue staining, and the cell concentration was adjusted to 3×10 with complete culture medium. 6 / mL, and spleen cell suspension of each group of mice was obtained.

[0043] 4. Spleen Cell Culture The spleen cell suspension of each group of mice was added into two wells of a 24-well culture plate, 1 mL per well. 75 μL of ConA solution (equivalent to 7.5 μg / mL) was added to one well, and the other well was used as a control. The plates were cultured in a carbon dioxide incubator with 5% CO2 and 37°C for 72 h.

[0044] 5. Cell transformation assay One hour before the end of the spleen cell culture of each group of mice, 0.7 mL of supernatant was gently aspirated from each well, and 0.7 mL of RPMI1640 culture medium without fetal bovine serum was added. The cells were mixed by pipetting, and 200 μL / well was transferred to a 96-well plate. Three parallel wells were added with 20 μL of CCK-8 solution, and the culture was continued for 1 hour. After the culture was completed, the optical density value was measured at a wavelength of 450 nm using an ELISA reader, and the proliferation rate of spleen cells of each group of mice was calculated based on the optical density value. The results are shown in the figure. Figure 2 shown.

[0045] Depend on Figure 2 It can be seen that the proliferation rate of spleen cells in the medium and high dose groups of L-rhamnose was significantly higher than that in the blank control group, and the proliferation rate of spleen cells in the high dose group of L-rhamnose was equivalent to or slightly higher than that in the positive control group, which indicates that L-rhamnose can enhance cellular immunity by increasing the proliferation rate of immune cells.

[0046] Example 3: Determination of NK cell activity in each group of mice The experimental mouse models of groups C1, P2, A2, M1, and B2 were selected. The mice were fasted for one week and then killed and dissected to obtain spleen tissues of mice in each group, and then NK cell activity was detected. The specific method is as follows.

[0047] 1. Prepare complete culture medium and spleen cell suspension according to Example 2. Wash YAC-1 cells with Hank's solution three times, and then subculture YAC-1 cells with complete culture medium. After subculture for 24 hours, adjust the cell concentration to 4×10 5 Pieces / mL.

[0048] 2. With YAC-1 cells as target cells and spleen cells of each group of mice as effector cells, 100 μL of YAC-1 cells and spleen cells (target-effect ratio 50:1) were taken and added to a U-shaped 96-well culture plate and divided into the following groups: culture wells without cells (blank control wells), 100 μL of YAC-1 cells and complete culture medium (natural release wells), cell wells for subsequent lysis of YAC-1 cells (maximum release wells), and 100 μL of YAC-1 cells and spleen cells (reaction wells). Three parallel wells were set for each of the above items and cultured in a 37°C, 5% CO2 incubator for 4 h. 1 h before the end of the culture, the cell culture plate was removed from the cell culture incubator, and the LDH release reagent was added to the "maximum release well" in an amount of 10% of the original culture medium volume. After adding the LDH release reagent, the mixture was mixed by repeated pipetting several times, and then continued to be incubated in the cell culture incubator. After the predetermined time was reached, the cell culture plate was centrifuged at 400 g for 5 min using a multi-well plate centrifuge. Take 120 μL of the supernatant from each well and add it to the corresponding wells of a new 96-well plate. Add 60 μL of LDH detection working solution to each well, mix well, incubate at room temperature in the dark for 30 min, and then measure the absorbance at 490 nm. The absorbance of each group should be subtracted from the absorbance of the blank control well. Calculate the NK cell activity based on the results. The calculation formula is as follows. The NK cell activity results are as follows: Figure 3 shown.

[0049] .

[0050] Depend on Figure 3 It can be seen that the NK cell activity in the low, medium and high dose groups of L-rhamnose was significantly higher than that in the blank control group, and the NK cell activity in the medium and high dose groups of L-rhamnose was significantly better than that in the positive control group, which indicates that L-rhamnose can enhance cellular immunity by increasing NK cell activity.

[0051] Example 4: Determination of the phagocytic index and phagocytic rate of chicken erythrocytes by macrophages of each group of mice The specific method of the experiment on phagocytosis of chicken red blood cells by mouse peritoneal macrophages is as follows: 1. Take chicken jugular vein or arterial blood, place it in a conical flask containing glass beads (about 20), shake it in one direction for 5-10 minutes to remove fibrin, and store it in a refrigerator at 4°C. Wash it with physiological saline three times before the experiment, centrifuge it at 1500 r / min for 10 minutes, discard the supernatant, and prepare a 1% red blood cell suspension with Hank's solution according to the hematocrit.

[0052] 2. Add 1 part of Giemsa staining solution to 8 parts of PBS buffer (pH = 6.8) to obtain diluted Giemsa staining solution.

[0053] 3. Activation of mouse peritoneal macrophages The experimental mouse models of groups C1, P2, A2, M1, and B2 were selected, and 0.2 mL of 2% compressed SRBC was injected into the mice intraperitoneally 4 days before the experiment. The mice were killed by cervical dislocation, and 4 mL of complete culture medium was injected into the peritoneum. The abdomen was gently massaged 20 times to fully wash out the peritoneal macrophages, and then a small incision was made in the abdominal wall. 2 mL of peritoneal lavage fluid was drawn into a test tube with a rubber-tipped pipette (or a syringe). 0.5 mL of peritoneal lavage fluid was drawn into a test tube containing 0.5 mL of 1% chicken red blood cell suspension using a 1 mL pipette and mixed.

[0054] Use a pipette to draw 0.5 mL of the mixture and add it to the agar circle on the slide. Place it in an incubator at 37°C for 15-20 minutes.

[0055] After the incubation, the non-adherent cells were quickly washed away with saline, fixed in methanol solution for 1 minute, and stained with dilute Giemsa stain for 15 minutes. Rinse with distilled water, dry, and count the phagocytic rate and phagocytic index under a 20× microscope. The results are as follows: Figure 4 shown.

[0056] The phagocytic rate is the percentage of macrophages that phagocytize chicken red blood cells among every 100 macrophages; the phagocytic index is the average number of chicken red blood cells phagocytized by each macrophage.

[0057] Depend on Figure 4 It can be seen that the phagocytic index of chicken red blood cells by macrophages in the medium and high doses of L-rhamnose groups was significantly higher than that in the blank control group, and the phagocytic index of chicken red blood cells by macrophages in the high dose of L-rhamnose group was equivalent to or slightly higher than that in the positive control group; the phagocytic rate of chicken red blood cells by macrophages in the high dose of L-rhamnose group was significantly higher than that in the blank control group, and the phagocytic rate of chicken red blood cells by macrophages in the high dose of L-rhamnose group was equivalent to or slightly higher than that in the positive control group, which indicates that L-rhamnose can enhance cellular immunity by improving the phagocytic ability of macrophages.

[0058] Example 5: Determination of the number of antibody-producing cell plaques in each group of mice Experimental mouse models of groups C2, P1, A1, M2, and B1 were selected, and the blood of the mice was collected. After static centrifugation, the serum of each group of mice was obtained; the mice were then fasted for one week and then killed and dissected to obtain the spleen tissue of each group of mice.

[0059] Antibody-producing cell detection (Jerne modified slide method), the specific method is as follows: 1. Take 450 μL of saline in an EP tube, add 50 μL of washed SRBC, and mix to make 500 μL of 10% packed SRBC. Add 1 mL packed SRBC to 5 mL guinea pig serum, place in a 4°C refrigerator for 30 min, shake frequently, centrifuge to obtain the supernatant, aliquot, and store at -80°C. Dilute with SA buffer at 1:8 to obtain complement.

[0060] 2. Take the defibrinated sheep blood, wash it with physiological saline for 3 times, centrifuge it (2000 r / min) for 10 min each time, count the cells, and inject 5×10 SRBC into the M2 group (medium dose group) and C2 (blank group) in the first experimental mouse model and the A1 (low dose group), B1 (high dose group) and C1 (positive control group) in the second experimental mouse model. 7 ~2×10 8 To obtain the immunized mice in each group.

[0061] 3. After fasting for one week, the mice in each group were killed and dissected to obtain spleen tissues of each group of mice; then spleen cell suspensions of each group of mice were prepared according to the method described in Example 2.

[0062] 4. Add 0.1 mL of spleen cell suspension and 0.2 mL of 10% SRBC to 1 mL of complete culture medium, and then add 0.2 mL of complement. Place a fat-free slide on the table, use double-sided tape to stick a strip on both ends and the middle of the slide, and then cover it with the same slide to form two small chambers. First seal one side of the slide with melted paraffin, use a sharp pipette to take the mixed liquid and pour it into the two small chambers respectively, and then seal with wax. Place the slide in a 37°C incubator and incubate for 30 minutes before taking it out. Note that the slide must be flat and not tilted during incubation. After incubation for 30 minutes, count the number of hemolytic plaques in the control group and the experimental group under a 20× microscope. The results are as follows. Figure 5 shown.

[0063] Depend on Figure 5 It can be seen that the number of antibody-producing cell plaques in the low- and high-dose L-rhamnose groups was significantly higher than that in the blank control group, and the number of antibody-producing cell plaques in the high-dose L-rhamnose group was equivalent to or slightly lower than that in the positive control group, which indicates that L-rhamnose can enhance humoral immunity by improving antibody production ability.

[0064] In summary, the present invention has effectively confirmed through mouse experiments that L-rhamnose has the functions of increasing the proliferation rate of spleen cells, increasing the activity of NK cells, increasing the phagocytic ability of macrophages and increasing the ability to produce antibodies. Compared with existing products that enhance immunity, L-rhamnose has effects in four aspects: humoral immunity, cellular immunity, monocyte-macrophage function, and NK cell activity. It has a comprehensive effect and can be used to prepare products that enhance cellular immunity or humoral immunity.

[0065] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of L-rhamnose in the preparation of a product for enhancing cellular immunity or humoral immunity, wherein L-rhamnose is the main active ingredient in the product.

2. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The molecular formula of L-rhamnose is C6H 12 O5 or C6H 12 O5·H2O.

3. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The product for enhancing cellular immunity or humoral immunity is a medicine, health product or functional food that can enhance cellular immunity or humoral immunity.

4. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The product for enhancing cellular immunity enhances cellular immunity by increasing the proliferation rate of spleen cells through L-rhamnose.

5. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The product for enhancing cellular immunity enhances cellular immunity by increasing the activity of NK cells through L-rhamnose.

6. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The product for enhancing cellular immunity enhances cellular immunity by improving the phagocytic ability of macrophages through L-rhamnose.

7. The use of L-rhamnose according to claim 1 in preparing a product for enhancing cellular immunity or humoral immunity, characterized in that: The product for enhancing humoral immunity enhances humoral immunity by improving antibody production capacity through L-rhamnose.

8. A composition for enhancing cellular immunity or humoral immunity, characterized in that: The composition comprises L-rhamnose and / or its hydrate, and a pharmaceutically acceptable carrier or auxiliary material; the composition takes L-rhamnose and / or its hydrate as the main active ingredient.

9. The composition for enhancing cellular immunity or humoral immunity according to claim 8, characterized in that: The dosage form of the composition for enhancing cellular immunity or humoral immunity includes powder, granules, tablets, pills, capsules, sustained-release agents, rate-controlled slow-release agents or suspensions.

10. The composition for enhancing cellular immunity or humoral immunity according to claim 8, characterized in that: The composition for enhancing cellular immunity or humoral immunity is administered via the gastrointestinal tract, with an administration dose of 10 to 100 mg / kg body weight each time, 1 to 3 times a day.

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