Use of a traditional Chinese medicine composition in the preparation of a product for improving immune function

Abstract

This invention, entitled "Application of a Traditional Chinese Medicine Composition in the Preparation of Products that Improve Immune Function," belongs to the technical field of traditional Chinese medicine and health food. The technical problem to be solved is to provide a traditional Chinese medicine composition with immunomodulatory effects. This composition is made from the following raw materials in the indicated weight ratios: 2 parts jujube, 2 parts polygonatum, and 1 part atractylodes. Experiments show that the traditional Chinese medicine composition of this invention can significantly promote the proliferation of T and B lymphocytes and increase the CD3+ of the spleen. + CD4 + CD8 + T cells and CD19 + The formula increased the proportion of B cells and dose-dependently elevated serum hemolysin levels. The overall formula was superior to individual herbs or herbal pairs (jujube-Polygonatum, jujube-Atractylodes, Polygonatum-Atractylodes) in promoting lymphocyte proliferation and regulating T cell subsets. This provides a foundation for developing drugs or functional foods that enhance immunity.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine and health food technology, specifically to the application of a traditional Chinese medicine composition in the preparation of products that enhance immunity. Background Technology

[0002] For understanding the technical content of this invention: The immune system is the core system responsible for executing immune responses, immune defense, and immune surveillance, maintaining homeostasis. Under physiological conditions, the immune system effectively eliminates foreign pathogens and mutated cells through the synergistic effect of innate immunity's non-specific recognition and adaptive immunity's specific response. When the body's immune function declines due to factors such as aging, malnutrition, mental stress, chronic diseases, or medications, the number or activity of T lymphocytes, B lymphocytes, and natural killer cells becomes abnormal, and the secretion of immunoglobulins and cytokines becomes unbalanced. This leads to a significant decrease in the body's immune defense function, resulting in a significant increase in susceptibility to pathogenic microorganisms. In severe cases, it can induce a series of pathological changes such as infectious diseases, tumors, and autoimmune diseases. Traditional Chinese medicine believes that immune dysregulation is often due to "spleen deficiency and impaired transport and transportation, resulting in insufficient defensive qi." "The spleen governs defensive qi," meaning that spleen deficiency leads to insufficient production of qi and blood, weakened defensive qi, and ultimately, damage to the body's vital energy and decreased immune function.

[0003] Currently, commonly used immunostimulants in clinical practice—including chemically synthesized agents (such as levamisole, cimetidine, and pidotimod), biological agents and cytokines (such as thymosin, transfer factor, interferon, and interleukin), and microbial-derived agents (such as BCG and bacterial lysates)—have certain limitations in terms of efficacy, safety, and stability. For example, recombinant cytokines (such as IL-15) have a half-life of only about one hour in vivo and low bioavailability; allogeneic interferon, transfer factor, or microbial-derived mannan peptides have all been reported to induce severe allergic reactions; recombinant human interferon β can induce drug-resistant antibodies, accelerate drug clearance, and affect efficacy. Traditional Chinese medicine and its active ingredients can synergistically regulate immune cell activity and cytokine secretion through multiple targets, possessing the advantages of definite efficacy and few adverse reactions, and their immunomodulatory value is increasingly valued. Existing immune-enhancing products derived from traditional Chinese medicine often employ coarse powder or decoction processes, resulting in insufficient dissolution of active ingredients and limited absorption by the human body. Some products also contain hormones or chemical enhancers, posing safety risks with long-term use, thus limiting their application as natural, safe, and highly effective immune-enhancing products. Therefore, developing a medicine or health food specifically designed to enhance immunity has significant market value and broad application prospects.

[0004] This traditional Chinese medicine composition consists of three herbs: jujube, polygonatum, and atractylodes. Modern pharmacological studies have shown that jujube, polygonatum, and atractylodes all possess varying degrees of immunomodulatory effects. However, existing research focuses on the immunomodulatory effects of single herbs or the jujube-polygonatum pair. The differences in efficacy between the combined formula and individual herbs, the contribution of each single herb to the overall immune-enhancing effect of the formula, and the synergistic effects of the combination remain unclear.

[0005] Relevant patent documents retrieved: This document, published in China (publication number: China), on May 15, 2013, discloses a preparation of a Polygonatum sibiricum-jujube herb pair, with a weight ratio of Polygonatum sibiricum to jujube of 1:1. The preparation is obtained by concentrating the aqueous extracts of Polygonatum sibiricum and jujube after decocting them three times. This preparation is said to have a synergistic effect in enhancing non-specific immune activity.

[0006] Relevant non-patent literature retrieved: The journal is titled "Chinese Journal of Traditional Chinese Medicine", and the article titled "Mechanism of the Polygonatum-Atractylodes Rhizome Drug Pair Regulating Lung Cancer Progression via the Apex1 / STAT3 Axis", Volume 39, Issue 10, published in October 2024. This article discloses that the Polygonatum-Atractylodes Rhizome drug pair can inhibit the proliferation of non-small cell lung cancer cells and slow down the progression of primary lung cancer in mice by inhibiting the Apex1 / STAT3 signaling pathway, thereby arresting the cell cycle of lung cancer cells. However, it does not disclose the related content on improving immunity.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: There are no reports that a compound of jujube, polygonatum, and atractylodes can improve immunity. Summary of the Invention

[0008] The purpose of this invention is to provide: The application of a traditional Chinese medicine composition in the preparation of products that enhance immunity is disclosed. This composition is made from jujube, polygonatum, and atractylodes. The three herbs work synergistically to enhance cellular immunity, humoral immunity, and non-specific immunity. Jujube tonifies the middle energizer and replenishes qi, polygonatum strengthens the spleen and moistens the lungs, and atractylodes dries dampness and invigorates the spleen. The combined effect of these three herbs is nourishing without causing stagnation, thus achieving the functions of strengthening the spleen and lungs, and harmonizing qi and blood. When the spleen and stomach function well, the defensive qi is abundant; when the lung qi is firm, the pores are secure; and when the body's vital energy is stored internally, pathogens cannot invade, thereby enhancing the body's immune function.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Definitions of standard chemical terms can be found in the Chinese Pharmacopoeia.

[0012] Unless otherwise stated, conventional methods within the scope of the art shall be used.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] The term "jujube" as used in this article refers to the dried, ripe fruit of the jujube plant (Ziziphus jujuba Mill.), belonging to the Rhamnaceae family. The fruit is harvested in autumn when ripe and then dried.

[0016] The term "Huangjing" as used in this article refers to the dried rhizome of *Polygonatum kingianum* Coll. et Hemsl., *Polygonatum sibiricum* Red., or *Polygonatum cyrtonema* Hua, all belonging to the Liliaceae family. Depending on its shape, it is commonly known as "large Huangjing," "chicken-head Huangjing," or "ginger-shaped Huangjing." It is harvested in spring and autumn, the fibrous roots are removed, it is washed, briefly blanched in boiling water or steamed until thoroughly cooked, and then dried.

[0017] The term "Atractylodes" as used in this article refers to the dried rhizome of *Atractylodes lancea* (Thunb.) DC. or *Atractylodes chinensis* (DC.) Koidz., both belonging to the genus *Atractylodes* of the Asteraceae family. It has the effects of drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. It is mainly used to treat dampness obstructing the middle jiao, abdominal distension, diarrhea, edema, beriberi, rheumatic pain, common cold due to wind-cold, night blindness, and blurred vision.

[0018] The term "ConA" used in this article refers to Concanavalin A, a T lymphocyte mitogen used to induce T lymphocyte proliferation.

[0019] The term "LPS" used in this article refers to a B lymphocyte mitogen used to induce a B lymphocyte proliferative response.

[0020] The term "serum hemolysin" as used in this article refers to specific antibodies (mainly IgM) produced by B cells after the body is stimulated by antigens, and humoral immune function is evaluated by the half-hemolysin value (HC50).

[0021] The term "NK cell" used in this article refers to natural killer cells, an important type of lymphocyte in the innate immune system that has the ability to non-specifically kill virus-infected cells and tumor cells.

[0022] In a first aspect, the present invention provides: the application of a traditional Chinese medicine composition in the preparation of products that enhance immunity.

[0023] The traditional Chinese medicine composition consists of jujube, polygonatum, and atractylodes. Preferably, the weight ratio of jujube, polygonatum, and atractylodes is 2:2:1; Preferably, the preparation method of the traditional Chinese medicine composition is as follows: weigh out jujube, polygonatum and atractylodes according to the formula, soak them in water, then heat and reflux to extract, obtain extract, add water to the residue and heat and reflux to extract, obtain extract, combine the extracts, filter, concentrate to obtain clear extract, which is the traditional Chinese medicine composition; More preferably, the liquid-to-solid ratio of water to the total mass of jujube, polygonatum, and atractylodes is 8-12 ml:1 g, the reflux extraction time is 90-120 min, the extraction is performed 1-3 times, the extraction yield of the extract is >50%, the total polysaccharide content of the extract is >42%, and the extraction yield is the dry weight of the extract / total mass of jujube, polygonatum, and atractylodes. Most preferably, the ratio of water added to the total mass of jujube, polygonatum, and atractylodes is 11.3 ml: 1 g, the heating and reflux extraction time is 79 min, the extraction is performed twice, the extraction yield of the extract is 68-72%, and the total polysaccharide content of the extract is 63-64%.

[0024] Preferably, the concentration of the extract is 1g crude drug / mL.

[0025] Preferably, the product can promote the proliferation of T lymphocytes and B lymphocytes; Preferably, the product can improve CD3 + CD4+ CD8 + T cells and CD19 + B cell ratio.

[0026] Preferably, the product can enhance the killing activity of NK cells; Preferably, the product can increase serum hemolysin levels.

[0027] Preferably, the product is a pharmaceutical or health product.

[0028] Preferably, the product further includes pharmaceutically or food-grade excipients; More preferably, the pharmaceutically or food-grade excipients include one or more of fillers, disintegrants, surfactants, binders, and lubricants.

[0029] For example, the filler includes one or more of starch, sorbitol, dextrin, lactose, microcrystalline cellulose, mannitol, and pregelatinized starch.

[0030] For example, the disintegrant includes one or more of sodium carboxymethyl starch, croscarmellose sodium, croscarmellose, and hydroxypropyl cellulose.

[0031] For example, the surfactants include, but are not limited to, sodium dodecyl sulfate, oleic acid, stearic acid, sodium hexadecyl sulfate, lauric acid, etc.

[0032] For example, the adhesive includes, but is not limited to: starch paste, hydroxypropyl methylcellulose, sodium starch paste, povidone, etc.

[0033] For example, the lubricant includes, but is not limited to, talc, magnesium stearate, polyethylene glycol, etc.

[0034] Preferably, the pharmaceutically or food-grade excipients may also include any one or more of suspending agents, solubilizers, flavoring agents, preservatives, antioxidants, and stabilizers.

[0035] For example, the suspending agent includes, but is not limited to: gum arabic, hydroxypropyl cellulose, methyl cellulose, tragacanth gum, bentonite, sodium carboxymethyl cellulose, bentonite, etc.

[0036] For example, the co-solvents include, but are not limited to: lactobionic acid, malic acid, hydrochloric acid, acetic acid, lactic acid, phosphoric acid, glucoheponic acid, propionic acid, boric acid, glucuronic acid, gluconic acid, citric acid, tartaric acid, threononic acid, 2,5-dihydroxybenzoic acid, acidic amino acids, etc.

[0037] For example, the flavoring agents include, but are not limited to: cyclamate, sorbitol, mannitol, sodium saccharin, acetylsupan-k, aspartame, glycerin, sucrose, simple syrup, aromatic syrup, etc.

[0038] For example, the preservatives include, but are not limited to: sorbic acid and its salts, phenol, chlorocresol, cresol, thymol, benzoic acid and its salts, propionic acid, formaldehyde, boric acid and its salts, phenethyl alcohol, chlorobutanol, glutaraldehyde, benzyl alcohol, chloroform, chlorhexidine, etc.

[0039] For example, the antioxidants and stabilizers include, but are not limited to: thiosulfates, sulfurous acid, glutathione, dimercaprol, bisulfites, sulfites, metabisulfites, dithionites, gallic acid and its salts, thiourea, mercaptoacetic acid and its salts, thiolactic acid and its salts, thiodipropionic acid and its salts, caffeine or its pharmaceutical salts, ferulic acid or its pharmaceutical salts, ascorbic acid and its salts, di-tert-butyl-p-phenol, nicotinamide, tartaric acid, phosphates, 2,5-dihydroxybenzoic acid or its salts, salicylic acid or its salts, isoascorbic acid and isoascorbic acid salts, pharmaceutical salts of acetate, citrates, EDTA and its salts, etc.

[0040] More preferably, the dosage form of the product is tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, or patches; Example 1 of this invention supports the scope of protection for the application of at least one traditional Chinese medicine composition in the preparation of products that enhance immunity.

[0041] "The application of a traditional Chinese medicine composition in the preparation of products that enhance immunity" is summarized from the foregoing explanation and / or the traditional Chinese medicine composition and its preparation method in Example 1. Therefore, those skilled in the art can reasonably presume that the above-mentioned technical features, their subordinate concepts, substantially equivalent technical means, and technical means that can be replaced by conventional technical means and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0042] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. Compared with the prior art, the present invention has better technical effects in promoting the proliferation of T and B lymphocytes.

[0043] 2. Compared with existing technologies, the present invention retains the three medicinal ingredients of jujube, polygonatum, and atractylodes in the existing traditional Chinese medicine compound (Zhengyuan Dan), but optimizes the ratio of medicinal materials, changing the mass ratio of the medicines from 1:1:1 to 2:2:1, thus increasing the proportion of jujube and polygonatum. Animal model results show that after continuous administration of the traditional Chinese medicine composition of the present invention for 20 days, it can significantly promote the proliferation of T and B lymphocytes and increase the CD3+ of the spleen. + CD4 + CD8 + T cells and CD19 + The formula enhances B cell ratio, increases NK cell killing activity, and dose-dependently increases serum hemolysin levels. Its efficacy in improving immune function is superior to existing traditional Chinese medicine compound formulas, and is close to or slightly better than the positive control drug. The entire formula is superior to any single herb or herb pair in promoting lymphocyte proliferation and regulating T cell subsets; the three herbs work synergistically to enhance cellular and humoral immunity.

[0044] 3. This invention uses the total polysaccharide content and extraction yield of the traditional Chinese medicine composition as evaluation indicators. A combination of single-factor experiments, response surface methodology, and an entropy weight-TOPSIS comprehensive evaluation model was employed to optimize the water extraction process. Single-factor experiments showed that the liquid-to-solid ratio, extraction time, and number of extractions significantly affected the extraction effect. Based on this, the response surface methodology factors were determined to be a liquid-to-solid ratio of 8:1 to 12:1, an extraction time of 60 to 120 min, and 1 to 3 extractions. Entropy weight calculations indicated that the extraction yield and total polysaccharide content together constituted the core evaluation indicators. The quadratic polynomial model constructed based on Box-Behnken design was significant and showed good fit. The order of influence of each factor was: extraction time > liquid-to-solid ratio > number of extractions. The optimal extraction process for the traditional Chinese medicine composition of this invention, with a water volume of 424 mL, an extraction time of 79 min, and 2 extractions, predicted a total polysaccharide content of 63.21% and an extraction yield of 70.26%. The relative error between the measured and predicted values ​​in three batches of verification experiments was less than 2%, indicating a stable and reliable process.

[0045] Furthermore, based on the present invention: Based on Example 1, this invention optimizes and improves the proportions and processes of jujube, polygonatum, and atractylodes to produce a traditional Chinese medicine composition. It was found that the various ingredients in this composition work synergistically to enhance cellular and humoral immunity, and can significantly enhance NK cell killing activity, thus preparing a product that enhances immunity. Attached Figure Description

[0046] Figure 1 This is the standard curve for glucose.

[0047] Figure 2 These are the results of a single-factor experiment.

[0048] Figure 3 For response surface 3D surface plot and contour plot.

[0049] Figure 4 The results show the enhancing effect of the traditional Chinese medicine composition on the immune function of normal mice (x±SD, n=10), where A represents the T lymphocyte proliferation result and B represents the T cell (CD3+) proliferation result. + The result of the proportional change is that C is CD4. + Results of T cell proportion changes, D represents CD8 + Results of T cell proportion changes, E represents B cells (CD19) + The results show the proportional changes, with F representing the half-hemolysin value and G representing the NK cell activity value. Compared with the normal group: p<0.05, p<0.01, p<0.001, p<0.0001. Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0051] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0052] Specific information on raw materials and reagents is shown in Table 1: Table 1

[0053] Example 1: Optimization of preparation process and preparation of traditional Chinese medicine composition using response surface methodology 1. Optimization of the preparation process of traditional Chinese medicine compositions using response surface methodology 1.1 Method for determining total sugar content 1.1.1 Construction of the glucose standard curve Following the preparation method for the glucose standard curve in the National Pharmacopoeia, accurately weigh 33 mg of anhydrous glucose reference standard and place it in a 100 mL volumetric flask. Dissolve in water, dilute to the mark, and shake well (each 1 mL contains 0.33 mg of anhydrous glucose). Pipette 0.00, 0.10, 0.20, 0.30, 0.40, 0.50, and 0.60 mL of glucose stock solution into colorimetric tubes, and add water to bring the volume to 1 mL. Add 0.5 mL of 5% phenol solution to each tube sequentially, shake well, and then quickly add 2.5 mL of concentrated sulfuric acid. Shake well, let stand for 10 min, heat at 99℃ for 30 min, cool to room temperature, and then add 200 μL to a 96-well plate. Measure the absorbance using a microplate reader at the maximum absorption wavelength of 490 nm and plot the standard curve.

[0054] 1.1.2 Determination of total sugar content in the test sample Take the concentrated water extract and bring the volume to 100 mL in a volumetric flask. Take 1 mL of the diluted extract and dilute it 5000 times. Take 200 μL of the diluted solution and add 800 μL of distilled water to bring the volume to 1 mL. Add 0.5 mL of 5% phenol solution sequentially, shake well, and quickly add 2.5 mL of concentrated sulfuric acid. Shake well, let stand for 10 min, heat at 99℃ for 30 min, cool to room temperature, and then add 200 μL to a 96-well plate. Measure the absorbance at the maximum absorption wavelength of 490 nm using a microplate reader.

[0055] 1.2 Calculation of total polysaccharide content The total polysaccharide content is calculated according to formulas (1) and (2).

[0056] Total polysaccharide mass (mg) = c v p(1) Total polysaccharide content (%) = (total polysaccharide mass / total mass of medicinal materials in the Chinese herbal composition) x 100% (2) c represents the concentration of polysaccharides in the extract (mg / mL); v represents the volume of the extract (mL); and p represents the dilution factor.

[0057] 1.3 Single-factor investigation of the early stage of extraction process of traditional Chinese medicine composition Liquid-to-solid ratio: Weigh 37.5g of jujube, polygonatum, and atractylodes slices (medicinal material mass ratio of 2:2:1) into 3 portions, and place them in 500mL round-bottom flasks respectively. Add purified water according to the liquid-to-solid ratio of 4ml:1g, 8ml:1g, and 12ml:1g. After soaking for 30 min, heat and reflux to extract twice, 60 min each time. Combine the filtrates, concentrate, freeze dry, calculate the extraction yield, and determine the total sugar content. Set up 3 parallel experiments for each level.

[0058] Extraction time: Weigh 37.5g of jujube, polygonatum, and atractylodes slices (medicinal material mass ratio of 2:2:1) into 3 portions, place them in 500 mL round bottom flasks, add ultrapure water at a liquid-to-material ratio of 8ml:1g, soak for 30 min, then heat and reflux to extract twice, with extraction times of 30, 60, and 90 min each time. Combine the filtrates, concentrate, freeze dry, calculate the extraction yield, and determine the total sugar content. Set up 3 parallel experiments for each level.

[0059] Extraction times: 37.5g of jujube, polygonatum, and atractylodes slices (medicinal material mass ratio of 2:2:1) were weighed into 3 portions and placed in 500 mL round-bottom flasks. Ultrapure water was added at a liquid-to-material ratio of 8ml:1g. After soaking for 30 min, the mixture was heated and refluxed for extraction 1, 2, and 3 times, with each extraction time being 60 min. The filtrates were combined, concentrated, freeze-dried, and the extraction yield was calculated. The total sugar content was also determined. Three parallel experiments were set up for each level.

[0060] 1.4 Entropy Weight-TOPSIS Joint Response Surface Design Optimization Method Entropy weighting is an objective weighting method based on information entropy. Its core principle is to use information entropy to measure data uncertainty. The smaller the entropy value, the greater the degree of variation of the indicator, the more information it provides, and the greater its weight should be. First, range standardization is used to standardize the original data xij, transforming the standardized data yij into a weight pij. Then, the entropy value ej of each indicator is calculated, and the difference coefficient gj = 1 - ej is calculated. The weight wj is then determined based on the difference coefficient.

[0061] First, the weights of each indicator are determined using the entropy weight method, and then the TOPSIS method is used to calculate the distance between each indicator and the positive ideal solution. Distance from the negative ideal solution Finally, the relative proximity is calculated according to formula (3).

[0062] (3) Based on the results of the single-factor experiments, the levels of response surface methodology (RSM) factors were determined, and a response surface factor level table was prepared (see Table 2). According to the set levels of influencing factors, Design Expert 11 software was used, and Box-Behnken was selected for the RSM design. Factor A was the liquid-to-solid ratio, Factor B was the extraction time, and Factor C was the number of extractions. The evaluation indicators were total sugar content and yield. The yield and total polysaccharide content were comprehensively scored using entropy-weighted TOPSIS, and the optimal extraction process was calculated based on the results.

[0063] Table 2 Response Surface Factor Level Table

[0064] 1.5 Determination and Verification Experiment of Optimal Process Parameters Based on the response surface methodology analysis, the optimal extraction parameters for the traditional Chinese medicine composition were predicted. Three parallel validation experiments were conducted using these optimal parameters. The extraction yield, total polysaccharide content, and relative similarity were calculated and compared with the model predictions. The relative error was calculated to verify the reliability of the model and the stability of the optimal process.

[0065] 1.6 Statistical Methods Data are expressed as mean ± standard deviation. Statistical analysis was performed using Graph Pad Prism 9 software. One-way ANOVA Dunnet's test was used for comparisons among multiple groups, and Kruskal-Wallis test was used for unequal variances. P < 0.05 was considered statistically significant.

[0066] 1.7 Results 1.7.1 Results of the glucose standard curve The results of the glucose standard curve are shown below. Figure 1 A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis. The equation of the standard curve is y = 0.0131x - 0.0739, R² = 0.9979. The curve shows a good linear relationship between glucose concentration and absorbance in the range of 0–100 mg / mL.

[0067] 1.7.2 Results of Single-Factor Experiments The results of the single-factor experiment are shown in Figure 2 Within the liquid-to-solid ratio range of 4:1 to 12:1, the dry extract weight showed a significant upward trend with increasing liquid-to-solid ratio, reaching its maximum at 12:1, indicating that increasing the solvent volume can promote the dissolution of active ingredients. Since the dry extract weight continued to increase during this period without a clear plateau, 8:1, 10:1, and 12:1 were selected as response surface methodology (RSM) levels. Within the extraction time range of 30-90 min, the dry extract weight increased with prolonged extraction time, indicating that appropriately extending the extraction time is beneficial for component dissolution. Since there is still room for improvement in the extraction efficiency at 90 min, considering the needs of subsequent industrial production, 60, 90, and 120 min were selected as RSM levels. Within the extraction number range of 1-3 times, the dry extract weight increased with increasing extraction number, reaching its maximum at 3 extractions and essentially reaching a plateau; therefore, 1, 2, and 3 extractions were selected as RSM levels.

[0068] 1.7.3 Significance Test of Response Surface Experiment Results and Model Fit (1) Indicator weights and their determination In this study, total polysaccharide content and extraction yield were selected as two factors, and the entropy weight method was used to determine the weights. The entropy weight calculation results are shown in Table 3.

[0069] Table 3 Entropy weight calculation results

[0070] (2) Results of response surface methodology Based on the results of the single-factor experiments, a three-factor, three-level response surface methodology was conducted using a Box-Behnken design, resulting in 17 sets of experimental data, as shown in Table 4. Based on the raw data in Table 4 and the entropy weights in Table 3, the comprehensive score for each experimental point was calculated using the TOPSIS method, as shown in Table 5. Experiment 16 (A=1, B=1, C=0) had the highest comprehensive score, reaching 100.00, indicating that the extraction effect was optimal under the conditions of a liquid-to-solid ratio of 12:1, an extraction time of 120 min, and two extractions. Experiment 10 (A=0, B=-1, C=-1) had the lowest comprehensive score, indicating that the extraction effect was worst under the conditions of an extraction time of 30 min and one extraction.

[0071] Table 4 Response Surface Design and Measured Index Values

[0072] Table 5 Entropy weight TOPSIS score values ​​for response surface methodology experiments

[0073] (3) Response surface model fitting and significance test The TOPSIS scoring results with entropy weight are shown in Table 5. Using the comprehensive score as the response value, a binomial regression was performed to fit the liquid-to-solid ratio, extraction time, and number of extractions. The results of the analysis of variance are shown in Table 6. The results show that the model is significant; the lack-of-fit term is not significant, and the model has a high degree of fit with the experimental results, making it suitable for further prediction and optimization.

[0074] As shown in Table 6, the order of influence of each factor on the extraction effect is: extraction time > liquid-to-solid ratio > number of extractions, with extraction time being the most critical factor.

[0075] Table 6. Analysis of Variance for Experimental Models

[0076] 1.7.4 Response Surface Analysis and Validation of Optimal Process Parameters Design-Expert 11.0 software was used to generate 3D surface plots and contour plots of the response surface. The results are shown in [Figure 1]. Figure 3 The binomial form of the model is shown below: Y = 51.086 + 7.787A + 33.851B + 4.473C - 1.809AB - 4.175AC - 0.465BC + 12.497A² - 5.098B² - 6.945C². Based on the binomial formula, a response surface plot and contour plot are generated to show the impact of liquid-to-solid ratio, extraction time, and number of extractions on the overall extraction score.

[0077] The response surface methodology shows that the overall score initially increases and then levels off with increasing extraction time and water volume, while steadily improving with increasing extraction cycles. The order of influence of each factor on the extraction effect is: extraction time > liquid-to-solid ratio > extraction cycles, consistent with the analysis of variance conclusions. Based on the regression model, the extreme points were solved at a 95% confidence level, determining the optimal extraction process parameters as: water volume 424 mL, extraction time 79 min, and extraction cycles of 2. Under these conditions, the model predicts a total polysaccharide content of 63.21% and an extraction yield of 70.26%.

[0078] To verify the predictive ability of the model and the stability of the optimal process, three batches of parallel validation experiments were conducted according to the above-mentioned optimal process parameters. The results showed that the average measured total polysaccharide content was 63.58%, the average extraction yield was 69.87%, and the relative error between the comprehensive score calculated by the entropy weight-TOPSIS method and the model prediction value was less than 2%, indicating that the model has high prediction accuracy and the determined optimal extraction process is stable and reliable, and can be used as a basis for optimizing the extraction process.

[0079] In summary, the extraction process of the traditional Chinese medicine composition of the present invention, with a liquid-to-solid ratio of 8:1 to 12:1, an extraction time of 90 to 120 minutes, and 1 to 3 extractions, can achieve a total polysaccharide content of >42% and an extraction yield of >50%.

[0080] 2. Preparation method of traditional Chinese medicine composition Weigh 15g of jujube, 15g of polygonatum, and 7.5g of atractylodes lancea slices (medicinal material mass ratio of 2:2:1), add 424mL of purified water, soak the medicinal materials for 30min, and extract twice by reflux, 79min each time. Combine the two extracts, filter, and concentrate to a concentration of 1g crude drug / mL to obtain the Chinese herbal composition (hereinafter referred to as JP), and store it frozen at -20°C.

[0081] Comparative Example 1 Weigh out 12.5g of jujube, 12.5g of polygonatum, and 12.5g of atractylodes lancea slices (medicinal material mass ratio of 1:1:1), and use the same preparation method as in Example 1 to obtain the clear paste.

[0082] Comparative Example 2 The difference from Example 1 is that 37.5g of jujube was taken as the single herb and prepared using the same method as in Example 1 to obtain the clear paste.

[0083] Comparative Example 3 The difference from Example 1 is that 37.5g of Polygonatum sibiricum was taken and prepared using the same method as in Example 1 to obtain a clear extract.

[0084] Comparative Example 4 The difference from Example 1 is that 37.5g of Atractylodes lancea alone was taken and prepared using the same method as in Example 1 to obtain a clear extract.

[0085] Comparative Example 5 The difference from Example 1 is that jujubes and polygonatum were weighed in a mass ratio of 1:1, with a total mass of 37.5g. The same preparation method as in Example 1 was used to obtain the extract.

[0086] Comparative Example 6 The difference from Example 1 is that jujubes and Atractylodes lancea were weighed at a mass ratio of 2:1, with a total mass of 37.5g. The same preparation method as in Example 1 was used to obtain the extract.

[0087] Comparative Example 7 The difference from Example 1 is that Polygonatum sibiricum and Atractylodes lancea were weighed at a mass ratio of 2:1, with a total mass of 37.5g. The same preparation method as in Example 1 was used to obtain the extract.

[0088] Example 1: Enhancing effect of traditional Chinese medicine composition on immune function in normal mice Male C57BL / 6J mice, weighing 18g±2g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in an SPF-grade animal barrier system at an indoor temperature of 22±2°C and a relative humidity of 50±5%. All animals had free access to food and water and underwent acclimatization for 3 days. The animal experiments were approved by the Animal Management and Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.

[0089] Mice were randomly divided into 6 groups using random number sequences generated by Microsoft Excel software: normal group, positive drug group, low-dose group of Example 1, medium-dose group of Example 1, high-dose group of Example 1, and high-dose group of Comparative Example 1, with 10 mice in each group. The normal group was administered 20 mL / kg / day of distilled water by gavage, and the positive drug group was administered 20 mL / kg / day of pidotimod oral solution by gavage. The low-dose, medium-dose, and high-dose groups of Example 1 were administered 1.5 g / kg / day, 3 g / kg / day, and 6 g / kg / day of crude drug by gavage, respectively, with an administration volume of 20 mL / kg / day. The Comparative Example 1 group was administered the corresponding extract by gavage at a dose of 6 g / kg / day, with an administration volume of 20 mL / kg / day. All groups were administered the drugs continuously for 21 days.

[0090] Statistical analysis was performed using GraphPad Prism 9.0 software. The data were first tested for normality using the Shapiro-Wilk method, and for homogeneity of variance using the Brown-Forsythe method. Normally distributed data were expressed as mean ± standard deviation. Mean squared (±SD) is used, and non-normal data are represented by the median (25th and 75th percentiles) [M (P25, P75)]. Comparisons among multiple groups followed this strategy: If the data were normally distributed and homogeneous in variance, one-way ANOVA and Tukey's post-hoc test were used; if the data were normally distributed but heterogeneous in variance, Brown-Forsythe and Welch ANOVA and Dunnett's T3 test were used; if the data did not conform to a normal distribution, the Kruskal-Wallis test and Dunn's multiple comparisons were used. P <0.05 indicates a statistically significant difference.

[0091] The detection of immune function indicators followed the methods for enhancing immunity in the "Technical Specifications for Inspection and Evaluation of Health Foods (2023 Edition)," comprehensively evaluating the immune function from three dimensions: cellular immunity, humoral immunity, and non-specific immunity (NK cell activity). For cellular immunity, the ConA and LPS-induced mouse spleen lymphocyte transformation assay was used to detect T lymphocyte and B lymphocyte proliferation; for humoral immunity, the serum hemolysin assay was used; and for non-specific immunity, the lactate dehydrogenase (LDH) release method was used to measure NK cell activity.

[0092] Mouse spleen lymphocyte population detection: 100 μL of spleen cell suspension was stained for live and dead cells according to the Zombie NIR™ FixableViability Kit instructions. After washing with 0.1% BSA PBS, the cells were incubated with the corresponding fluorescently labeled antibody in the dark for 30 min. After washing with 0.1% BSA PBS, the cells were collected and resuspended in 300 μL FACS buffer for flow cytometry analysis.

[0093] The results are as follows Figure 4 As shown, compared with the normal group, the ConA-induced T lymphocyte proliferation levels in each dose group and the positive drug group in Example 1 were significantly increased ( P <0.01), and the effect of Example 1 in promoting T lymphocyte proliferation showed a dose-dependent relationship.

[0094] The traditional Chinese medicine composition prepared in Example 1 can significantly upregulate T cells (CD3). + ), B cells (CD19) + CD4 + T cells and CD8+ T cell ratio ( P <0.05), suggesting that it has a certain regulatory effect on the distribution of lymphocyte subsets under physiological conditions.

[0095] Example 1: The NK cell activity in each dose group and the positive drug group was significantly higher than that in the normal group ( P <0.05, indicating that it can effectively improve the activity of mouse NK cells.

[0096] Compared with the normal group, all dose groups in Example 1 significantly increased serum hemolysin levels ( P The concentration of the herbal medicine composition prepared in Example 1 is <0.05%, and the effect is dose-dependent. This suggests that the herbal composition prepared in Example 1 has a certain positive regulatory effect on the humoral immune function of normal mice. Compared with Comparative Example 1, Example 1 can significantly increase the proliferation capacity of lymphocytes and upregulate the proportion of T or B lymphocytes.

[0097] Detection Example 2 1. Experiments on the proliferation of T lymphocytes and B lymphocytes induced by ConA and LPS in mouse spleen cells. Specific experimental methods: C57BL / 6J mice were euthanized by cervical dislocation and immersed in 75% alcohol for 5 minutes. Spleens were aseptically harvested under a laminar flow hood. A sterile 70µm nylon mesh cell filter was placed on a 50mL centrifuge tube. The spleen was placed on the nylon mesh and ground. The filter was washed with 1% BSA PBS, and the spleen cell suspension was collected. The cells were centrifuged at 300g for 10 minutes, and the supernatant was discarded. Red blood cell lysis buffer was added, and the cells were resuspended. The cells were incubated at room temperature for 5-10 minutes. 1% BSA PBS was added, and the cells were washed twice by centrifugation. The white precipitate after centrifugation was collected as lymphocytes. The cells were resuspended in 10% FBS RPMI 1640 medium. Viable cells (above 95%) were counted using trypan blue. The cell concentration was adjusted to 5×10⁶ cells / mL. 5Cells / well were seeded in 96-well plates. In the experiment on the effect of traditional Chinese medicine on the proliferative activity of mouse ConA-induced spleen lymphocytes, the following groups were set up: a blank control group, control groups for each test drug (each containing either Example 1 or Comparative Examples 1-7, with 4-5 concentrations of the same drug), a ConA control group, ConA + Example 1 (with 4-5 concentrations selected for Example 1), ConA + Comparative Example 1 (with 4-5 concentrations selected for Comparative Example 1), ConA + Comparative Example 2 (with 4-5 concentrations selected for Comparative Example 2), ConA + Comparative Example 3 (with 4-5 concentrations selected for Comparative Example 3), ConA + Comparative Example 4 (with 4-5 concentrations selected for Comparative Example 4), ConA + Comparative Example 5 (with 4-5 concentrations selected for Comparative Example 5), ConA + Comparative Example 6 (with 4-5 concentrations selected for Comparative Example 6), and ConA + Comparative Example 7 (with 4-5 concentrations selected for Comparative Example 7). In the experiment on the effect of traditional Chinese medicine on the proliferative activity of mouse LPS-induced spleen lymphocytes, the experimental groups were the same as in the ConA induction experiment. The mixture was continuously incubated in an incubator for 48-72 hours, and the OD value was determined using the MTT assay. The combined drug effect was evaluated using the Chou-Talalay method. Jujube, Polygonatum, and Atractylodes were mixed in the original formula ratio (2:2:1) to form the complete formula. The pharmacodynamic effects of each of the three herbs individually, the complete formula, and formulas without any of the three herbs (jujube, Polygonatum, and Atractylodes) were measured at different concentration gradients. The dose-response curves for each group were fitted using the Chou-Talalay method, and the combination index (CI) of the complete formula at different effect levels (Fa=0.5, 0.75, 0.9) was calculated. CI<1, =1, and >1 represent synergistic, additive, and antagonistic effects, respectively.

[0098] 2. Effects of drugs on the phagocytic function of RAW264.7 macrophages Cells were seeded in 96-well plates (1×10⁻⁶ cells / well). 5 Cells were cultured for 12 h at a time (1 cell / well). Different doses of the drug used in Experiment 1 and Comparative Examples 1-7 were added, with LPS (1 μg / mL) used as a positive control. Cells were cultured for 24-48 h, and 100 μL of 0.1% neutral red staining solution was added to each well. The cells were incubated at 37 °C for 2 h, washed three times with PBS, and 100 μL of ethanol-acetic acid lysis buffer (ethanol: glacial acetic acid = 1:1) was added to each well. After shaking, the OD value was measured at 540 nm, and the phagocytic rate was calculated using the formula. The combination index was calculated using the aforementioned method.

[0099] Verification of technical effectiveness and / or analysis of solutions to technical problems: The traditional Chinese medicine composition prepared in this invention, through the synergistic combination of Polygonatum sibiricum, Atractylodes lancea, and Ziziphus jujuba, exhibits superior enhancing effects on cellular immunity, humoral immunity, and non-specific immune function compared to any single herb or the Ziziphus jujuba-Polygonatum sibiricum pair, Ziziphus jujuba-Atractylodes lancea pair, and Atractylodes lancea-Polygonatum sibiricum pair. The three herbs demonstrate a synergistic effect. The entire formula significantly promotes the proliferation of T and B lymphocytes and increases spleen CD3 levels. + CD4 + CD8 + T cells and CD19 + It increases the proportion of B cells and enhances NK cell activity and serum hemolysin levels.

[0100] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of products that improve immune function, characterized in that, The traditional Chinese medicine composition consists of jujube, polygonatum, and atractylodes, with the weight ratio of jujube, polygonatum, and atractylodes being 2:2:

1.

2. The application according to claim 1, characterized in that, The preparation method of the traditional Chinese medicine composition is as follows: weigh out jujube, polygonatum and atractylodes according to the formula, soak them in water, then heat and reflux to extract, and obtain an extract. Add water to the filter residue and heat and reflux to extract again, and obtain an extract. Combine the extracts, filter, and concentrate to obtain a clear paste, which is the traditional Chinese medicine composition.

3. The application according to claim 2, characterized in that, The soaking time is 0.5-1 hour, the liquid-to-solid ratio of water to the total mass of Atractylodes lancea, Polygonatum sibiricum and jujube is 8-12 ml:1 g, the heating and reflux extraction time is 90-120 min, the extraction is performed 1-3 times, the extraction yield of the extract is >50%, and the total polysaccharide content of the extract is >42%.

4. The application according to claim 3, characterized in that, The soaking time is 0.5 hours, the liquid-to-solid ratio of water to the total mass of Atractylodes lancea, Polygonatum sibiricum and jujube is 11.3 ml: 1 g, the heating and reflux extraction time is 79 min, the extraction is performed twice, the extraction yield of the extract is 68-72%, and the total polysaccharide content of the extract is 63-64%.

5. The application according to claim 1, characterized in that, The product can promote the proliferation of T lymphocytes and B lymphocytes.

6. The application according to claim 1, characterized in that, The product described can improve CD3 + CD4 + CD8 + T cells and CD19 + B cell ratio.

7. The application according to claim 1, characterized in that, The product described can enhance the killing activity of NK cells.

8. The application according to claim 1, characterized in that, The product described can increase serum hemolysin levels.

9. The application according to claim 1, characterized in that, The product in question is a medicine or health product.

10. The application according to claim 1, characterized in that, The product also includes pharmaceutically or food-grade excipients, and the dosage form of the product is tablet, sugar-coated tablet, film-coated tablet, enteric-coated tablet, capsule, hard capsule, soft capsule, oral liquid, lozenge, granule, powder, pill, powder, ointment, pill, suspension, solution, injection, suppository, ointment, plaster, cream, spray, drop, or patch.

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