Application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs

Abstract

This invention discloses the application of *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs, belonging to the field of bio-fermentation and immunomodulation technology. The *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder is prepared by fermenting ginseng with *Lactobacillus rhamnosus*. The rare ginsenosides include at least one of Ck, Rk1, Rh4, and Rg5. The immunomodulatory drug is used to improve cyclophosphamide-induced immunodeficiency. The amount of *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder added to the immunomodulatory drug is 10%-50% (mass fraction). The preparation method of the *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder is as follows: S1, ginseng pretreatment; S2, strain activation; S3, fermentation culture; S4, freeze-dried powder preparation. This invention has the technical effects of high bioavailability of ginseng active ingredients, significant immunomodulatory effect, good safety, and effective improvement of cyclophosphamide-induced immunodeficiency.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation and immunomodulation technology, and particularly relates to the application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs. Background Technology

[0002] Ginseng, a traditional and precious Chinese medicinal herb, contains a variety of active ingredients and possesses various pharmacological effects such as enhancing immunity, combating fatigue, and anti-oxidation, making it widely used in the pharmaceutical and health product fields. However, many of the active ingredients in ginseng, such as ginsenosides, have large molecular structures, resulting in low bioavailability. Furthermore, some components are not easily absorbed by the body when consumed directly, limiting the full realization of its immunomodulatory effects.

[0003] Probiotic fermentation is an effective means to improve the bioavailability of active ingredients in natural products. Lactobacillus rhamnosus, as a safe probiotic, has been widely used in the food and pharmaceutical fields, and it has effects such as regulating intestinal flora balance and enhancing the body's immune function. While there have been attempts to combine probiotics with ginseng in existing technologies, most have used simple mixing methods and have not achieved the conversion and enhancement of ginseng's active ingredients through fermentation. Furthermore, there are no reports on the preparation of freeze-dried powder from Lactobacillus rhamnosus-fermented ginseng, and on its regulatory effects and related mechanisms on immunocompromised individuals.

[0004] Cyclophosphamide is a commonly used immunosuppressant in clinical practice, widely applied in cancer treatment, autoimmune disease treatment, and organ transplant rejection. However, long-term or excessive use can lead to weakened immune function, resulting in adverse reactions such as weight loss, atrophy of immune organs, and abnormal secretion of immune factors, severely impacting patients' quality of life and treatment outcomes. Currently, there are no safe and effective intervention products for cyclophosphamide-induced immunosuppression. Therefore, developing a natural product formulation that can effectively regulate the body's immune function and improve immunosuppression is of significant clinical importance and application value. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an application of *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs. This method possesses advantages such as high bioavailability of ginseng active ingredients, significant immunomodulatory effects, and good safety. It can effectively improve cyclophosphamide-induced immunodeficiency, solving the problems of low bioavailability of ginseng active ingredients and lack of safe and effective intervention for cyclophosphamide-induced immunodeficiency in the prior art.

[0006] This invention is achieved by using a *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs. The *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder is prepared by fermenting ginseng with *Lactobacillus rhamnosus*. The freeze-dried powder contains rare ginsenosides, total phenols, and flavonoid active ingredients generated by fermentation and transformation of *Lactobacillus rhamnosus*. The rare ginsenosides include at least one of Ck, Rk1, Rh4, and Rg5.

[0007] As a preferred embodiment of the present invention, the immunomodulatory drug is used to improve cyclophosphamide-induced immunodeficiency.

[0008] As a preferred embodiment of the present invention, the amount of Lactobacillus rhamnosus fermented ginseng freeze-dried powder added to the immunomodulatory drug is 10%-50% (mass fraction).

[0009] As a preferred embodiment of the present invention, the dosage form of the immunomodulatory drug is powder, capsule, tablet, oral liquid or granule.

[0010] As a preferred embodiment of the present invention, the method for preparing the *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder is as follows: S1. Ginseng pretreatment: Take ginseng raw materials, wash and crush them, add deionized water to make ginseng suspension, sterilize and cool to room temperature for later use. S2. Activation of bacterial strain: Lactobacillus rhamnosus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 24 hours to obtain activated Lactobacillus rhamnosus bacterial culture. S3, Fermentation culture: The bacterial solution in S2 was inoculated into the ginseng suspension in S1 at an inoculation rate of 3%, and fermented at 40℃ for 72 hours, with stirring once every 8 hours during the fermentation process to obtain the fermentation broth. S4. Preparation of freeze-dried powder: The fermentation broth is centrifuged, the supernatant is collected, freeze-dried, pulverized and sieved to obtain freeze-dried ginseng fermented with Lactobacillus rhamnosus.

[0011] As a preferred embodiment of the present invention, in step S1, the concentration of the ginseng suspension is 100-200 g / L, and the sterilization conditions are 121°C for 15-20 min.

[0012] As a preferred embodiment of the present invention, in step S4, the centrifugation conditions are 4℃, 8000-10000r / min, 10-15min, the freeze-drying conditions are 50℃ to -40℃, vacuum degree 10-20Pa, drying time 24-36h, and the sieving conditions are using an 80-100 mesh sieve.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses Lactobacillus rhamnosus to ferment ginseng, transforming the macromolecular active ingredients in ginseng into small molecule components that are easily absorbed by the human body through microbial metabolism. This significantly improves the bioavailability and immunomodulatory effects of ginseng's active ingredients, and its immunomodulatory effect is more significant compared to unfermented ginseng. The ginseng freeze-dried powder fermented with Lactobacillus rhamnosus of the present invention has high safety and no obvious toxic side effects. It can effectively repair the weight loss caused by cyclophosphamide, improve the immune organ index, increase the content of immune-related factors such as CD8+, IgG, and IgM in serum, activate the MyD88, TLR-4, and NF-κB signaling pathways, regulate the expression of immune-related genes (LR-2, TLR-4, IL-2, IL-3, and IL-6), improve the body's immune function from multiple levels, and has a significant regulatory effect on immunodeficiency. The preparation method described in this invention is simple, convenient to operate, and has low production cost, making it suitable for large-scale industrial production. Furthermore, the freeze-dried powder formulation has good stability, is easy to store and transport, and has broad application prospects. This invention combines the probiotic efficacy of Lactobacillus rhamnosus with the immunomodulatory effects of ginseng, achieving a synergistic effect of "probiotics + natural products," providing a new direction for the development of drugs that regulate immune function, and in particular, providing a safe and effective drug intervention for the immunosuppressive adverse reactions caused by cyclophosphamide treatment. Attached Figure Description

[0014] Figure 1 This is a schematic diagram illustrating the effect of fermented ginseng freeze-dried powder provided in this embodiment of the invention on the body weight of mice.

[0015] Figure 2 This is a schematic diagram illustrating the effect of the fermented ginseng freeze-dried powder provided in this embodiment of the invention on blood biochemical indicators in mice: A represents immunoglobulin G (IgG); B represents the CD8 molecule (CD8+); C represents immunoglobulin M (IgM).

[0016] Figure 3 This is a schematic diagram of the expression of MyD88, TLR-4, and proteins in liver tissue provided in the embodiments of the present invention.

[0017] Figure 4 This is a schematic diagram illustrating the effect of the fermented ginseng freeze-dried powder provided in this embodiment of the invention on the expression of proteins related to the Toll-like receptor signaling pathway in mice: A represents the grayscale value of NF-κB / β-actin protein expression; B represents the grayscale value of TLR-4 / β-actin protein expression; C represents the grayscale value of MyD88 / β-actin protein expression.

[0018] Figure 5 This is a schematic diagram illustrating the effect of the fermented ginseng freeze-dried powder provided in this embodiment of the invention on related genes in mouse liver tissue. Detailed Implementation

[0019] To further understand the invention's content, features, and effects, the following embodiments are provided, and detailed descriptions are given in conjunction with the accompanying drawings.

[0020] The structure of the present invention will now be described in detail with reference to the accompanying drawings. Example

[0021] Preparation of ginseng freeze-dried powder fermented with Lactobacillus rhamnosus Ginseng pretreatment: Take ginseng raw materials, clean them, crush them and pass them through an 80-mesh sieve, add deionized water to make a ginseng suspension with a concentration of 150g / L, place it in a high-pressure steam sterilizer, sterilize it at 121℃ for 18min, and then cool it to room temperature for later use. Strain activation: Lactobacillus rhamnosus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 24 hours to obtain activated Lactobacillus rhamnosus bacterial culture. Fermentation culture: The activated Lactobacillus rhamnosus bacterial solution was inoculated into the above ginseng suspension at an inoculation rate of 3%, and fermented at 40℃ for 72 hours, with stirring once every 8 hours to obtain the fermentation broth; Preparation of freeze-dried powder: The fermentation broth was placed in a benchtop high-speed refrigerated centrifuge and centrifuged at 4℃ and 9000r / min for 12min. The supernatant was collected and poured into a freeze dryer and dried at -45℃ and 15Pa vacuum for 30h. After drying, the broth was crushed and passed through a 100-mesh sieve to obtain freeze-dried ginseng fermented with Lactobacillus rhamnosus. The ginseng was sealed and stored for later use. Example

[0022] Experiment on the regulatory effect of Lactobacillus rhamnosus fermented ginseng freeze-dried powder on immunocompromised mice 2.1 Test Materials Test sample: Lyophilized ginseng powder fermented with Lactobacillus rhamnosus prepared in Example 1; Experimental animals: 32 male ICR rats, 2-3 weeks old, with a body weight of (20.0±1.0) g.

[0023] 2.2 Test Methods Animal grouping and administration: After 1 week of acclimatization feeding, 32 mice were randomly divided into 4 groups of 8 mice each: blank group, model group, ginseng group, and fermented ginseng group. Except for the blank group, the other groups were injected with cyclophosphamide (100 mg / kg) for 7 consecutive days to establish a mouse model of immunodeficiency. After successful modeling, ginseng freeze-dried powder fermented with Lactobacillus rhamnosus was continuously administered by gavage for 24 days. The mice were fasted for 24 hours before slaughter, and 1.0 mL of blood was collected from the eyeballs. The mice were euthanized by cervical dislocation, and the blood, spleen, thymus, and liver were collected for later use.

[0024] Mouse body weight and immune organ index determination: 24 hours after the last administration, the mice were weighed, 1.0 mL of blood was collected from the eyeballs, the experimental animals were euthanized by cervical dislocation, and the spleen, thymus and liver were removed. The connective tissue was removed and the mice were washed with physiological saline. The residual liquid was absorbed with filter paper, and the weight of the spleen, thymus and liver was weighed. The organ index was calculated (organ index = organ weight / mouse body weight).

[0025] Blood biochemical index determination: The blood sample was centrifuged in a low-temperature high-speed centrifuge (4℃, 10000r / min) for 10 min, and the supernatant serum was separated. The contents of immunoglobulin G (IgG), immunoglobulin M (IgM) and CD8 molecules (CD8+) in the serum were detected by an enzyme-linked immunosorbent assay (ELISA) reader according to the instructions of the CD8+ kit, IgG kit and IgM kit.

[0026] Western blot analysis of proteins: 100 mg of liver tissue was taken, minced, and mixed with lysis buffer (RIPA lysis buffer + PMSF solation + protein inhibitor cocktail). After homogenization, the supernatant was collected by centrifugation. The protein concentration was determined using a BCA protein quantification kit. After denaturation, SDS-PAGE electrophoresis, membrane transfer, and blocking were performed. Primary antibodies (MyD88, TLR-4, NF-κB, β-actin) were added and incubated overnight at 4°C. After rinsing, secondary antibodies were added and incubated in the dark. The membrane was then developed and exposed using ECL chemiluminescence. β-actin was used as an internal control. ImageJ software was used to analyze the grayscale of protein bands and calculate the relative expression level of proteins.

[0027] Real-time polymerase chain reaction (RT-qPCR) detection: RNA extraction: 100 mg of mouse liver was minced in a 1.5 mL EP tube, and 1 mL of TRIzol homogenate was added. 200 μL of chloroform was added to each EP tube, followed by vortexing and incubation at room temperature for 20 min, and centrifugation at 4℃ / 12000 rpm for 15 min. The supernatant was collected, and isopropanol was added. The mixture was shaken and incubated at room temperature for 10 min, followed by centrifugation at 10000 rpm / min for 10 min. The supernatant was discarded, and 1 mL of pre-cooled 75% ethanol was added to the EP tube. The white precipitate was washed off by inverting the tube, and the waste liquid was discarded. The EP tube was placed in a clean bench and allowed to dry at room temperature for 20 min. 20 μL of RNase-free water was added to completely dissolve the RNA. After mixing, the OD values ​​of the RNA concentration at 260 nm and 280 nm were measured using NanoDrop. The samples were stored at -20℃. cDNA synthesis and reverse transcription were performed according to the FastKing cDNA first-strand synthesis kit instructions. The reverse transcription volume was 20 μL. The reaction mixture was prepared according to the gene DNA removal system in Table 1 and thoroughly mixed. After brief centrifugation, the mixture was incubated at 42°C for 3 min and placed on ice. The reverse transcription mixture was prepared according to the reverse transcription system in Table 2. After preparation, the mixture was added to the reaction solution of the gDNA removal step, thoroughly mixed, incubated at 42°C for 15 min, incubated at 95°C for 3 min, and then placed on ice to obtain cDNA for subsequent RT-qPCR detection.

[0028] Real-Time Quantitative PCR (Real-time-qPCR) – The experiment was conducted following the procedures outlined in the Talent qPCR PreMix (SYBR Green) kit. The RT-qPCR reaction system was prepared on ice (see Table 3). The experimental procedure was as follows: pre-denaturation 95℃, 3 min; denaturation 95℃, 5 s; annealing 60℃, 15 s, for a total of 45 cycles. β-actin was used as the internal control gene, with three replicates per gene. A 2 -ΔΔCt The expression levels of the relevant target genes in this experiment were relatively quantified using the method described in Table 4. Primer information is detailed in Table 4.

[0029]

[0030]

[0031] Statistical analysis: All data are expressed as mean ± standard deviation. SPSS was used for difference analysis to test for statistical differences, and Duncan's multiple range test was employed. P <0.05) or ( P<0.01) assess substantial differences between groups and use different letters (a, b, c, d, e) to represent differences between groups, with a representing the largest and e representing the smallest.

[0032] 2.3 Test Results Mouse body weight change: by Figure 1 It was found that the cyclophosphamide-induced immunosuppression model significantly inhibited the normal growth of mice. The weight gain of mice in the model group was slow and even stagnated or slightly decreased in the later stage, which was in stark contrast to the continuous and stable weight gain trend of the blank group. After gavage administration of ginseng, the weight of mice in the ginseng group gradually improved in the later stage of the experiment and slightly exceeded that of the model group, showing a certain recovery effect on the weight loss caused by immunosuppression, but the effect was relatively slow. Although the fermented ginseng group had the lowest initial weight, it had the fastest weight gain rate throughout the experiment, and the cumulative increase was significantly higher than that of the model group and the ginseng group. This indicates that fermentation treatment may more effectively promote the weight recovery under immunosuppression by improving the bioavailability of ginseng active ingredients, and shows better intervention potential in improving metabolism and combating immune damage.

[0033] Immune organ indices: The thymus, as the core site for the development, differentiation, and maturation of T lymphocytes, is responsible for transporting mature T cells to peripheral immune organs and tissues, thereby participating in cellular immune responses. The spleen, the largest peripheral immune organ in the body, is rich in immune cells such as lymphocytes and macrophages, playing a key role in humoral immunity. When the thymus and spleen atrophy, their weight decreases, the number of immune cells decreases accordingly, and immune activity also declines. Therefore, the thymus index and spleen index are often used as important indicators for assessing immune function. Their levels can directly reflect the functional status of the two organs and indirectly reflect the strength of the body's immunity. The experimental results showed that the thymus of the control group mice was normal in morphology, pale red with a slight yellow tinge, soft in texture, and generally butterfly-shaped; the spleen was of moderate size, soft in texture, and elastic. The immune organ indices of the mice in each treatment group are shown in Table 5. After cyclophosphamide induction, the thymus index and spleen index of the model group mice decreased to 0.758 and 1.556, respectively, with a significant reduction in organ volume and weight, indicating significant impairment of immune function. In contrast, after intervention with fermented ginseng freeze-dried powder, the weight and index of the thymus and spleen of the mice recovered. Among them, the fermented ginseng group showed the most significant effect, with the thymus index and spleen index increasing to 1.874 and 3.371, respectively, which were close to the level of the control group. This indicates that fermented ginseng freeze-dried powder can effectively improve the immune organ indices of immunocompromised mice, promote the recovery of damaged immune cells, and thus enhance the overall immune function of the body.

[0034]

[0035] Blood biochemical indicators: When the body experiences weakened immune function, various indicators will decrease, such as CD8+, IgG, and IgM. CD8+, IgG, and IgM are all important components of the immune system. CD8 molecules are key markers on the surface of T cells, related to T cell antigen recognition and activation. CD8 molecules can enhance T cell receptor (TCR)-mediated antigen reactivity, thereby regulating the type and intensity of the immune response. IgG is the most abundant immunoglobulin in serum, participating in various humoral immune processes: antigen neutralization, complement activation and complement-dependent cytotoxicity (CDC), target action of phagocytosis, and antibody-dependent immunodeficiency. Sex cells mediate cytotoxicity (ADCC) and hypersensitivity reactions; their fragment antigen-binding domain (Fab) binds to specific antigens, while their fragment crystallizable domain (Fc) binds to different receptors on the surface of different immune cells, thus determining the type of immune response triggered by antigen binding; immunoglobulin (Ig) M is the first antibody isotype to appear during evolution, ontogeny, and immune responses; IgM is not only the host's first line of defense against infection, but also plays an important role in immune regulation and immune tolerance; to investigate the immunomodulatory effect of lyophilized fermented ginseng powder on immunocompromised mice, the levels of CD8+, IgG, and IgM in the serum of mice in each group were detected; Depend on Figure 2 (A, B, C) It can be seen that, compared with the blank group, the injection of cyclophosphamide suppressed the immune function of mice, resulting in the inhibition of T cell activation, proliferation and differentiation, and a decrease in CD8+ content, leading to a decrease in antibody synthesis and secretion, and a corresponding decrease in the secretion of IgG and IgM. After intervention with fermented ginseng freeze-dried powder by gavage, the levels of CD8+, IgG and IgM in the serum of mice increased significantly. Compared with the model group, the level of CD8+ in the fermented ginseng group increased by 50.96%; the level of IgG increased by 99.34%; and the level of IgM increased by 233.33%. It can be seen that the fermented ginseng group had the best effect. This indicates that fermented ginseng freeze-dried powder can improve the content of immune function-related factors, thereby gradually restoring the body's immune level.

[0036] Expression of Toll-like receptor signaling pathway-related proteins: Effects of fermented ginseng freeze-dried powder on the expression levels of Toll-like receptor signaling pathway-related proteins in immunocompromised mice; Figure 3 , Figure 4As shown in (A, B, C), compared with the blank group, the expression levels of MyD88, TLR-4, and NF-κB proteins in the model group mice were significantly reduced; however, after intervention with ginseng supplementation and gavage administration of fermented ginseng freeze-dried powder, the expression levels of MyD88, TLR-4, and NF-κB proteins increased; among them, the fermented ginseng group showed the most significant effect, with the expression levels of MyD88, TLR-4, and NF-κB proteins increasing by 35.82%, 177.08%, and 29.46% respectively compared with the model group; MyD88, TLR-4, and NF-κB... Signaling pathways are the core signaling axes for maintaining immune homeostasis. Normal transmission of these pathways is crucial for the body to achieve an effective immune response, resist pathogen invasion, and maintain immune homeostasis. MyD88 is located in the cytoplasm and contains an N-terminal death domain (DD) and a C-terminal TIR domain. The TIR domain can bind to the TIR domain of TLR-4, while the death domain is responsible for recruiting downstream signaling molecules to facilitate signal transduction. After TLR-4 recognizes its ligand, it must first use TIRAP / MAL as a bridging molecule to recruit MyD88 for initiation. The TLR-4 signaling pathway activates the key transcription factor NF-κB, initiating the expression of downstream pro-inflammatory and immune-related genes. NF-κB (nuclear factor-κB), as a key transcription factor downstream of the TLR-4-MyD88 signaling pathway, plays a core regulatory role in maintaining immune homeostasis. It mainly regulates the transcription of immune-related genes, participates in the regulation of immune cell proliferation, differentiation, and apoptosis, ensuring that the body generates an appropriate immune response and avoiding excessive or insufficient immune function. TLR-4 (Toll-like receptor 4), as an important pattern recognition receptor in the innate immune system, plays a crucial role in maintaining immune homeostasis. It can recognize pathogen-related molecular patterns, initiate downstream signal transduction, regulate the proliferation, differentiation, and survival of immune cells, and thus generate a rapid and effective immune response, providing important protection for the body to resist the invasion of external pathogens. After intervention with fermented ginseng freeze-dried powder by gavage, the expression levels of the three proteins gradually increased, the immune function of mice improved, and the body's immune function was enhanced. This indicates that fermented ginseng freeze-dried powder can activate the MyD88, TLR-4, and NF-κB signaling pathways and maintain the stability of the immune system.

[0037] Immune-related gene expression: RT-qPCR expression results, such as Figure 5As shown, compared with the blank group, the expression levels of TLR-2, TLR-4, IL-2, and IL-3 mRNA in the liver of mice in the model group were significantly decreased and the expression level of IL-6 mRNA was significantly increased after injection of cyclophosphamide. After intervention with different fermented ginseng freeze-dried powders by gavage, the expression levels of TLR-2, TLR-4, IL-2, and IL-3 mRNA in mice in each group increased and the expression level of IL-6 mRNA decreased. Among them, the fermented ginseng and taurine groups had the most significant effects, increasing by 171.53%, 136.70%, 106.05%, and 106.62% respectively compared with the control group, and decreasing by 58.03%. In the body's immune system, cytokines mediate immune responses and play an important role in protecting and maintaining immune homeostasis. TLR-2 and TLR-4 can enhance antigen presentation ability, recognize various pathogen-related molecules, and initiate the activation of immune cells and immune responses. Interleukin-2 (IL-2) is an important immunomodulatory cytokine, mainly promoting the growth of CD4+ T helper cell subsets and CD4+ T cells. T cells regulate activation, proliferation, and differentiation; interleukin-3 (IL-3) is a cytokine with multi-lineage potential and a broad range of target cells, playing a crucial role in hematopoiesis; interleukin-6 (IL-6) is a major pro-inflammatory cytokine in the IL-6 family; it signals through glycoprotein 130 (gp130) and membrane-bound or soluble IL-6 receptors (IL-6R); cyclophosphamide-induced immunodeficiency in mice leads to a decrease in the number of immune cells or abnormal immune function, resulting in a reduced ability to recognize pathogens, thus potentially decreasing the expression levels of TLR-2 and TLR-4; while IL-2 is mainly composed of active... The production of T cells by cyclophosphamide leads to a decrease in IL-2 secretion, affecting T cell proliferation and differentiation, and resulting in a decline in cellular immune function. Simultaneously, cyclophosphamide damage to immune cells leads to a decrease in IL-3 secretion, affecting the proliferation and differentiation of hematopoietic stem cells and the development and maturation of various immune cells. Conversely, when the body's immune function is impaired, inflammatory factors such as IL-6 respond to immune damage by increasing their secretion. This indicates that fermented ginseng freeze-dried powder can enhance the ability of immune cells to recognize pathogens, promote the recovery of immune cell function, improve the activity and proliferation of immune cells, restore the body's immune function, and enhance the body's immune defense capabilities.

[0038] 2.4 Experimental Conclusions The experimental results of this embodiment show that *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder can effectively repair the weight loss in mice caused by cyclophosphamide, improve the immune organ index, increase the content of immune-related factors such as CD8+, IgG, and IgM in serum, activate the TLR-4 / MyD88 / NF-κB signaling pathway, regulate the expression of immune-related genes, and have a significant regulatory effect on cyclophosphamide-induced immunodeficiency. Moreover, the effect is better than that of unfermented ginseng, and it can be used as an effective ingredient to prepare drugs that regulate immune function. Example

[0039] Preparation of immunomodulatory drugs from ginseng freeze-dried powder fermented with Lactobacillus rhamnosus 3.1 Preparation of Immune-Regulating Capsules Formula: 200g of freeze-dried ginseng fermented with Lactobacillus rhamnosus, 80g of lactose, and 20g of starch; Preparation method: Mix the above raw materials evenly, pass them through an 80-mesh sieve, and fill them into empty capsules using a capsule filling machine. Each capsule contains 0.2g of freeze-dried fermented ginseng powder. Seal and package to obtain immune-regulating capsules.

[0040] 3.2 Preparation of oral solutions for regulating immune function Formula: 100g of freeze-dried ginseng fermented with Lactobacillus rhamnosus, 50g of honey, and 10,000mL of deionized water; Preparation method: Add Lactobacillus rhamnosus fermented ginseng freeze-dried powder to deionized water, stir to dissolve, add honey, continue stirring until uniform, filter, fill, sterilize at 121℃ for 15 minutes, cool and package to obtain immune-regulating oral liquid.

[0041] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0042] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of *Lactobacillus rhamnosus*-fermented ginseng freeze-dried powder in the preparation of immunomodulatory drugs, characterized in that... The Lactobacillus rhamnosus fermented ginseng freeze-dried powder is prepared by fermenting ginseng with Lactobacillus rhamnosus. The freeze-dried powder contains rare ginsenosides, total phenols and flavonoid active ingredients generated by fermentation and transformation of Lactobacillus rhamnosus. The rare ginsenosides include at least one of Ck, Rk1, Rh4 and Rg5.

2. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 1 in the preparation of immunomodulatory drugs, characterized in that, The immunomodulatory drugs are used to improve cyclophosphamide-induced immunodeficiency.

3. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 1 in the preparation of immunomodulatory drugs, characterized in that, The amount of the Lactobacillus rhamnosus fermented ginseng freeze-dried powder added to the immunomodulatory drug is 10%-50% (mass fraction).

4. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 1 in the preparation of immunomodulatory drugs, characterized in that, The dosage form of the immunomodulatory drug is powder, capsule, tablet, oral liquid or granule.

5. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 1 in the preparation of immunomodulatory drugs, characterized in that, The preparation method of the Lactobacillus rhamnosus fermented ginseng freeze-dried powder is as follows: S1. Ginseng pretreatment: Take ginseng raw materials, wash and crush them, add deionized water to make ginseng suspension, sterilize and cool to room temperature for later use. S2. Activation of bacterial strain: Lactobacillus rhamnosus was inoculated into MRS medium and cultured at 37°C under anaerobic conditions for 24 hours to obtain activated Lactobacillus rhamnosus bacterial culture. S3, Fermentation culture: The bacterial solution in S2 was inoculated into the ginseng suspension in S1 at an inoculation rate of 3%, and fermented at 40℃ for 72 hours, with stirring once every 8 hours during the fermentation process to obtain the fermentation broth. S4. Preparation of freeze-dried powder: The fermentation broth is centrifuged, the supernatant is collected, freeze-dried, pulverized and sieved to obtain freeze-dried ginseng fermented with Lactobacillus rhamnosus.

6. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 5 in the preparation of immunomodulatory drugs, characterized in that, In step S1, the concentration of the ginseng suspension is 100-200 g / L, and the sterilization conditions are 121℃ for 15-20 min.

7. The application of Lactobacillus rhamnosus fermented ginseng freeze-dried powder as described in claim 5 in the preparation of immunomodulatory drugs, characterized in that, In step S4, the centrifugation conditions are 4℃, 8000-10000r / min, 10-15min, the freeze-drying conditions are 50℃ to -40℃, vacuum degree 10-20Pa, drying time 24-36h, and the sieving conditions are using an 80-100 mesh sieve.

Images