CN121445819A - A method for preparing an active food-medicine homology compound nutrient and its application - Google Patents

Abstract

This application discloses a method for preparing an active medicinal and edible homologous compound nutrient. The method includes: initial culture of a Ganoderma lucidum monoclonal isolate on potato dextrose agar plates, followed by transfer to a specific liquid culture medium for shaking culture; centrifugation to detect β-glucosidase activity reaching 100 mU/mL, collection of the supernatant, addition of Polygonatum sibiricum, Ganoderma lucidum, and mulberry leaf powder for biotransformation; inoculation with Lactobacillus plantarum, followed by freeze-drying with a freeze-drying protectant and sieving to obtain the active medicinal and edible homologous compound nutrient. This method breaks down the macromolecular active components of Ganoderma lucidum, Polygonatum sibiricum, and mulberry leaf into smaller molecules through biotransformation, solving the problem of poor intestinal absorption of traditional materials. Furthermore, the prepared active medicinal and edible homologous compound nutrient can restore the number of neutrophils, macrophages, and T cells, while upregulating the expression of pro-inflammatory genes such as TNF-α, IL-12A, and IFN-γ, simultaneously restoring innate and adaptive immune function. Moreover, its natural source ensures high safety and its applicability in immune function regulation scenarios.

Description

Preparation method and application of active medicinal and edible composite nutrient

Technical Field

The invention belongs to the field of preparation of medicine-food homologous composite nutrients, and particularly relates to a preparation method and application of active medicine-food homologous composite nutrients.

Background

The immune system is critical for host defense, immune surveillance and tissue homeostasis. The immune function of cancer patients is often impaired by tumor microenvironment or cytotoxic treatments such as chemotherapy, radiation therapy, etc. Immunosuppression of such patients may lead to increased risk of infection, slow healing process and poor therapeutic effect. Therefore, there is a need for safe and effective drugs to support or restore immune function, which should ideally be natural components, which can exert therapeutic benefit and have good safety.

In the theory of traditional Chinese medicine, certain natural substances are classified according to the principle of homology of medicine and food, and can be used as a therapeutic drug and also be integrated into daily diet to promote health and prevent diseases. Among them, ganoderma lucidum, rhizoma polygonati and mulberry leaf have a long history of application in eastern asia medicine because they have the property of enhancing immunity. These natural substances are rich in bioactive components such as polysaccharides, flavonoids, glycoproteins, etc., which have been shown to regulate immune responses, reduce inflammation and provide antioxidant protection.

However, these natural materials often contain bulky, structurally complex molecules that are difficult for the human body to efficiently absorb. In the case of Ganoderma lucidum, the original form of Ganoderma lucidum is wrapped with a hard spore coat, and the Ganoderma lucidum must be broken to release the bioactive components. Rhizoma Polygonati and folium Mori are used as traditional medicinal plants, and are rich in polysaccharide, flavonoid, etc. However, due to the presence of high molecular weight polysaccharides, glycosylated flavonoids and poor lipophilicity, their oral bioavailability is limited, which can hinder intestinal absorption.

Disclosure of Invention

In order to solve the problems that the traditional medicinal and edible materials such as ganoderma lucidum, rhizoma polygonati, mulberry leaf and the like have complex macromolecular structures, low oral bioavailability and limited immunoregulation effect, the application designs a preparation method and application of the active medicinal and edible composite nutrient, so as to enhance the absorption of ganoderma lucidum, rhizoma polygonati and mulberry leaf when the active medicinal and edible composite nutrient is used for enhancing immunity.

A preparation method of active medicinal and edible composite nutrient comprises the following steps:

Step S1, culturing a ganoderma lucidum monoclonal isolate by adopting a potato dextrose agar plate, and transferring an agar block containing active growth mycelium into a liquid culture medium for culturing;

S2, placing the liquid culture prepared in the step S1 in a conical flask, and performing shake culture in a dark environment at 30 ℃;

S3, quantitatively measuring the activity of the beta-glucosidase in the culture in the step S2 by adopting a beta-glucosidase detection kit, centrifuging the culture when the activity of the enzyme exceeds 100 mU/mL, and collecting supernatant;

s4, supplementing 1.5% w/v of rhizoma polygonati powder, 1.5% w/v of ganoderma lucidum powder and 1.5% w/v of mulberry leaf powder into the supernatant collected in the step S3, and carrying out shaking culture for 24 hours to carry out bioconversion;

S5, centrifuging the culture solution after bioconversion in the step S4, and collecting supernatant;

step S6, inoculating lactobacillus plantarum into the supernatant collected in the step S5, and enabling the final concentration of the lactobacillus plantarum to reach 1 multiplied by 10 ⁷ CFU/mL to obtain an active medicine-food homologous composite nutrient precursor;

Step S7, adding a freeze-drying protective agent, namely adding 10% w/v maltodextrin, 10% w/v mannitol and 2% w/v- (+) trehalose dihydrate into the active medicinal and edible composite nutrient precursor in the step S6, and fully stirring until the active medicinal and edible composite nutrient precursor is uniformly dispersed;

And S8, freeze-drying the mixed solution obtained in the step S7 by adopting a laboratory freeze dryer until the mixed solution reaches constant weight, ensuring that the freeze-dried powder is uniform and free of polysaccharide precipitation, and grinding and sieving the mixed solution to 100-mesh particle size to obtain the active medicine-food homologous compound nutrient.

Preferably, in step S1, the liquid medium comprises 40g/L D-glucose, 5g/L peptone, 5g/L yeast extract, 0.46g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate.

Preferably, in step S2, the dark environment of the shake culture needs to be kept free from illumination interference throughout, and the rotation speed of the shake culture is 100 rpm.

Preferably, in step S3, the quantitative determination of the β -glucosidase activity is performed within 1 hour after the culture is sampled.

Preferably, in step S4, the rhizoma Polygonati powder, the Ganoderma powder, and the folium Mori powder are all sieved with 80 mesh sieve and added into the supernatant.

Preferably, in step S6, the lactobacillus plantarum is actively cultured in MRS medium to the logarithmic growth phase before being inoculated.

Preferably, in step S8, the pre-freezing temperature of the freeze drying is-40 ℃ and the pre-freezing time is 4 hours.

An active medicinal and edible composite nutrient product is 100-mesh granular freeze-dried powder, and comprises biotransformed rhizoma polygonati saponin, ganoderan, mulberry leaf flavone active ingredients and Vege-start60 strain lactobacillus plantarum with a final concentration of 1 multiplied by 10 ⁷ CFU/mL.

An active medicinal and edible composite nutrient product can be used for preparing medicines for increasing the number of neutrophils and medicines for increasing the number of macrophages.

An active medicine-food homologous composite nutrient product can be used for preparing medicines for increasing the number of T cells and preparing medicines for pro-inflammatory genes.

The application has the following advantages and effects:

The application relates to a preparation method of active medicinal and edible composite nutrient, which comprises the steps of carrying out initial culture on a ganoderma lucidum monoclonal isolate by a potato dextrose agar plate, transferring to a specific liquid culture medium for shake culture, detecting that the activity of beta-glucosidase reaches 100 mU/mL, centrifuging to obtain supernatant, adding rhizoma polygonati, ganoderma lucidum and mulberry leaf powder for bioconversion, inoculating lactobacillus plantarum, adding a freeze-drying protective agent, freeze-drying and sieving to obtain the active medicinal and edible composite nutrient. According to the method, macromolecular active ingredients of ganoderma lucidum, rhizoma polygonati and mulberry leaf are decomposed into small molecules through biotransformation, the problem of poor intestinal absorption of the traditional material is solved, the prepared active medicinal and edible composite nutrient can restore the numbers of neutrophils, macrophages and T cells, simultaneously up-regulate the expression of tnf-alpha, il-12a and ifn-gamma pro-inflammatory genes, synchronously restore the innate and adaptive immune functions, has high safety of natural sources, and can be used for regulating the immune functions.

The foregoing description is only an overview of the present application, and is intended to provide a better understanding of the technical means of the present application, so that the present application may be practiced according to the teachings of the present specification, and so that the above-mentioned and other objects, features and advantages of the present application may be better understood, and the following detailed description of the preferred embodiments of the present application will be presented in conjunction with the accompanying drawings.

The above and other objects, advantages and features of the present application will become more apparent to those skilled in the art from the following detailed description of the specific embodiments of the present application when taken in conjunction with the accompanying drawings.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale.

FIG. 1 is a graph showing the effect of AMCN provided by the application on neutrophils in zebra fish larvae;

FIG. 2 is a graph showing the effect of AMCN provided by the application on zebra fish larvae macrophages;

FIG. 3 is a graph showing the effect of AMCN provided by the application on T cells of zebra fish larvae;

FIG. 4 is a graph showing the effect of the qRT-PCR assay AMCN on the relative expression of the tnf- α, il-12a and ifn- γ genes in zebra fish larvae.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments of the present application. In the following description, specific details such as specific configurations and components are provided merely to facilitate a thorough understanding of embodiments of the application. It will therefore be apparent to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the application. In addition, descriptions of well-known functions and constructions are omitted in the embodiments for clarity and conciseness.

It should be appreciated that reference throughout this specification to "one embodiment" or "this embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Thus, the appearances of the "one embodiment" or "this embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Furthermore, the present application may repeat reference numerals and/or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed.

The term "and/or" herein is merely one kind of association relation describing the association object, and indicates that three kinds of relations may exist, for example, a and/or B may indicate that a alone exists, B alone exists, and a and B exist simultaneously, and the term "/and" herein is another kind of association object relation describing that two kinds of relations may exist, for example, a/and B may indicate that a alone exists, and a and B exist separately, and in addition, a character "/" herein generally indicates that the association object is an "or" relation.

The term "at least one" is used herein to describe only one association relationship of associated objects, and means that three relationships may exist, for example, at least one of A and B may mean that A exists alone, while A and B exist together, and B exists alone.

It is further noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprise," "include," or any other variation thereof, are intended to cover a non-exclusive inclusion.

Example 1 this example mainly describes a specific design of a method for preparing an active pharmaceutical and dietetic homologous composite nutrient, comprising:

Production of active pharmaceutical and edible composite nutrient AMCN:

the initial culture of the ganoderma lucidum monoclonal isolate adopts a potato dextrose agar plate. The agar block containing actively growing mycelium was transferred to a medium containing 40 g/l D-glucose, 5 g/l peptone, 5 g/l yeast extract, 0.46 g/l potassium dihydrogen phosphate and 0.5 g/l magnesium sulfate heptahydrate. 25 ml of the liquid culture was placed in a 250 ml Erlenmeyer flask and incubated in a dark environment at 30℃with shaking at a speed of 100 revolutions per minute.

The beta-glucosidase activity detection adopts a beta-glucosidase detection kit to carry out quantitative determination. When the enzyme activity exceeded 100 mU/mL, the culture broth was centrifuged at 5000 rpm for 10 minutes to collect the supernatant. Subsequently, 1.5% (w/v) of Polygonatum sibiricum powder was added to the supernatant, and 1.5% (w/v) of Ganoderma lucidum powder and 1.5% (w/v) of mulberry leaf powder were added. The mixture was further incubated under the same conditions for 24 hours to promote bioconversion. After the bioconversion was completed, the broth was centrifuged again at 5000 rpm for 10 minutes. The supernatant was inoculated with Lactobacillus plantarum at a final concentration of 1X 10 ⁷ CFU/mL to prepare AMCN. The lactobacillus plantarum strain Vege-start 60 used is a probiotic starter specifically optimized for fermentation.

Subsequently, 10% (mass/volume) maltodextrin, 10% (mass/volume) mannitol, and 2% (mass/volume) D- (+) trehalose dihydrate were added to the final supernatant as lyoprotectants. The mixture was thoroughly stirred for at least one hour to ensure uniform dispersion. After the mixing was completed, the solution was subjected to freeze-drying treatment using a laboratory freeze-dryer until a constant weight was reached. Special care was taken during the procedure to ensure that the lyophilized powder was homogeneous and no polysaccharide precipitated. All lyophilized powders were weighed and weight data recorded, and the powders were subsequently ground and sieved to obtain a particle size of about 100 mesh. The final product was designated AMCN. Freeze-dried AMCN powder is packaged by a1 kg aluminum foil bag and then is stored in a room temperature environment, so that direct sunlight is avoided.

Example 2. This example mainly describes the effect verification of an active pharmaceutical and food homologous compound nutrient designed by the application:

To evaluate the immunomodulatory potential of active pharmaceutical and dietary homologous complex nutrients, we studied using the zebra fish immunosuppressive model. The model mimics the general hematopoietic dysfunction and immune cell depletion characteristics of cancer patients. Zebra fish, which is an important model organism for vertebrate immune studies, has increasingly been shown to have not only highly conserved hematopoietic pathways with optically transparent properties at early developmental stages, but also transgenic lines capable of expressing fluorescent markers in critical immune cell populations. The immune system of zebra fish includes innate components such as macrophages and neutrophils, and adaptive components such as T lymphocytes, allowing for a comprehensive analysis of immune responses in vivo.

In this study, we first evaluated AMCN for in vivo toxicity to ensure their safety. The changes in macrophages, neutrophils and T cells after AMCN treatment were then quantified by a transgenic zebra fish model. Finally, we analyzed immune-related genes using real-time quantitative PCR (qRT-PCR) techniques, and explored the molecular mechanisms behind these immune-enhancing effects.

1. Zebra fish rearing and embryo collection

The breeding and propagation method of zebra fish is described with reference to our previous study. Briefly, adult zebra fish were raised in a 28 ℃ circulating water system with a 14 hour light/10 hour dark photoperiod and fed three times daily. During embryo collection, male zebra fish and female zebra fish are matched in a ratio of 1:1 for evening period and separated by a baffle plate. The next morning the partition was removed to complete spawning within the first hour after the beginning of the light cycle. Fertilized larvae were collected and incubated in 1×e3 medium supplemented with methylene blue (0.1%). Samples were incubated at a concentration of 3ppm in a 28.5 ℃ incubator until use.

2. Acute toxicity assessment

Wild type AB strain zebra fish were raised under standard conditions in a 28 ℃ constant temperature aquarium grade broth. Embryos are randomly inoculated into six well plates, 30 tails per well, with AMCN of treatment fluid added at concentrations of 12.5, 25, 50, 4000, 100, 200 and 400 μg/mL, respectively.

All groups were exposed to 20 μg/mL cyclophosphamide for 48 hours to induce immunosuppression, except for the normal control (untreated). All groups were incubated at 28℃for 72 hours. Death was recorded daily and the dead embryos were immediately removed to prevent effects due to decomposition.

3. Immunomodulatory Activity assessment

1. Neutrophil count analysis of zebra fish model

Myeloperoxidase (mpo) is a well known marker of neutrophils in zebra fish. Transgenic zebra fish (mpo: EGFP) is a transgenic strain which can realize specific marking and in vivo visualization of neutrophils by regulating and controlling expression of Enhanced Green Fluorescent Protein (EGFP) through mpo promoter. Transgenic zebra fish larvae of 2 days of age were randomly aliquoted into 6 well plates (30 per well) and given AMCN treatments at 12.5, 25 and 50 μg/mL concentrations, respectively. The experiments were divided into three groups, normal control, model and treatment. Each of the remaining groups, except the normal control group, received a treatment with cyclophosphamide at 20 μg/mL to induce immunosuppression. All treatments were continued for 48 hours at 28 ℃ constant temperature. After treatment, 10 larvae were randomly selected for each group and imaged under a fluorescence microscope. Neutrophil count in tail vein was quantitatively analyzed using NIS-Elements software.

2. Macrophage level analysis of zebra fish model

Macrophage specific gene 1 (mpeg 1) is a gene unique to zebra fish. Transgenic strain (mpeg 1: EGFP) zebra fish drives EGFP expression through mpeg1 promoter, and can realize in vivo macrophage specificity visualization. The experiments were performed by selecting 30 transgenic (mpeg 1: EGFP) zebra fish larvae of 2 days old, randomly grouped under the same conditions as described above. Following the established treatment protocol, the larvae were exposed to AMCN solutions at concentrations of 12.5, 25 and 50 μg/mL, respectively, with or without cyclophosphamide added, and subsequently incubated in a 28 ℃ environment for 48 hours. Each group of 10 tail larvae was subjected to microscopic imaging, and after observation with MZX81 fluorescence microscope, fluorescence intensity of tail vein macrophages was quantitatively analyzed by NIS-Elements software.

3. Analysis of T cell levels in zebra fish models

Recombinant activating gene 2 (rag 2) is a well-known T lymphocyte specific marker in thymus and is highly conserved in vertebrates such as mice, humans and zebra fish. Tg (rag: dsRed) zebra fish is a transgenic line whose rag promoter drives expression of discoid red fluorescent protein (dsRed), thereby allowing specific detection of lymphoid progenitor cells, particularly thymic T cells. In the experiment, 30 Tg (rag 2:2 dsRed) zebra fish (2 day old) were randomly grouped in 6-well plates, treated with different concentrations of AMCN (12.5, 25 and 50. Mu.g/mL) under the same conditions, respectively, with or without cyclophosphamide added as described above. After incubation for 48 hours at 28 ℃, 10 juvenile fish were selected for subsequent observation in each group.

The fluorescence intensity levels of T cells in the tail vein were quantified using NIS-Elements software, with random selection for imaging.

4. Gene expression analysis

The study uses green fluorescent neutrophil transgenic zebra fish larvae (2 days old) for immune gene expression detection. Experimental treatment method referring to the 3.1 th node, after larvae were cultured in a 28℃incubator for 48 hours, total RNA of each set of larvae was extracted according to the protocol using RNeasy universal RNA extraction kit. RNA concentration and purity were measured by UV-Vis spectrophotometry and 2. Mu.g RNA samples were taken for reverse transcription using the first strand cDNA synthesis kit, with a final reaction volume of 20.0. Mu.L. The relative expression levels of tumor necrosis factor-alpha (tnf-alpha), interleukin-12 alpha (il-12 a) and interferon-gamma (ifn-gamma) of the pro-inflammatory genes were detected by using a real-time quantitative PCR (qRT-PCR) technique, and beta-actin was used as an internal control. Three replicates were set up for all experiments and specific primer information is detailed in table 1.

TABLE 1 primer sequences used in qPCR

Gene primer sequences (5 'to 3')

Beta-actin pre-thymine-guanine-glutamine-guanine-glutamine-bird purine-glutamine-guanine

Reverse CTCGTGGATACCGCAAGATTC

Tnf-alpha front GCGCTTTTCTGAATCCTACG

Reverse TGCCCAGTCTGTCTCCTTCT

Il-12 alpha front AACTCCTACAAGCCCAGCAC

Reverse ACTACCGTGTGTGTCAAACGAA

Ifn-gamma front CTTTCCAGGCAAGTGCAGA

Reverse thymine-guanine-cytosine-adenine-guanine-cytosine-thymine-cytosine

5. Statistical analysis

The study used a one-way anova in combination with a dunnity post-hoc test to statistically compare the normal control, AMCN treatment and model groups. All quantitative data are expressed as mean ± Standard Error (SE). Data analysis was performed blindly using GraphPadPrism software, with a statistical significance criterion of p-value <0.05.

6. Results

(1) AMCN increase the number of neutrophils in zebra fish models

To determine whether AMCN regulates the immune system in our immunodeficient zebra fish model, we first examined its effect on neutrophils, a key effector of innate immunity

Immunization-Tg (mpo: EGFP) larvae were used. Referring to FIG. 1, FIG. 1 shows the effect of AMCN on neutrophil count in zebra fish larvae. FIG. 1 (A) representative images of Tg (mpo: EGFP) of zebra fish larvae (after 4 days) after AMCN treatment for 48 hours. The white dotted line area indicates the area for quantitative analysis. Scale bar = 100 μm. FIG. 1 (B) quantitative analysis of the number of neutrophils in the tail vein. Data are expressed as mean ± standard deviation (n=10 per group), compared to model group,p<0.01,P <0.001 (single factor analysis of variance combined with Dunnett post hoc test). Compared with the normal control group, the number of neutrophils in the model group is reduced by 30% (figures 1A & B), and successful establishment of the immunodeficiency model is confirmed. Notably, AMCN treatments increased neutrophil count by-14% relative to the model group.

(2) AMCN increase the number of macrophages in zebra fish models

Next, we assessed AMCN the effect on macrophages using Tg (mpeg 1: EGFP) transgenic zebra fish larvae. Macrophages are another key cell type responsible for phagocytic debris in zebra fish. Referring to FIG. 2, FIG. 2 is a graph showing the effect AMCN on zebra fish larval macrophage levels. FIG. 2 (A) representative images of Tg (mpeg 1: EGFP) zebra fish larvae (after 4 days) after AMCN hours of treatment. The white dotted line area indicates the area for quantitative analysis. Scale bar = 100 μm. FIG. 2 (B) is a quantitative analysis of macrophage level by tail vein fluorescence intensity. Data are expressed as mean ± standard deviation (n=10 per group) compared to model groupp<0.001,P <0.0001 (single factor analysis of variance combined with Dunnett post hoc test). In the normal control group, the number of macrophages was highest, reflecting the basal level of zebra fish larvae (fig. 2a & b). Compared with the normal control group, the macrophage quantity in the model group is reduced by 54 percent. Notably, AMCN treatment significantly restored macrophage numbers, increasing them by 57% relative to the model group.

(3) AMCN increase the level of T cells in zebra fish models

Next, we studied AMCN effect on T cells using Tg (rag 2:dsred) zebrafish larvae. Because T cells in thymus are tightly aggregated, the number of single cells cannot be counted accurately, and therefore fluorescence intensity is used as a substitute index of the abundance of the T cells. Referring to FIG. 3, FIG. 3 is a graph showing the effect AMCN on the T cell level of zebra fish larvae. FIG. 3 (A) is a representative image of a zebra fish larvae (4 days old) at Tg (rag 2: dsR-ed) after AMCN hours of treatment. The white dotted line area represents the thymus quantification area. Scale bar = 100 μm. FIG. 3 (B) fluorescent intensity quantification of thymic T cell levels. Data are expressed as mean ± standard deviation (n=10 per group), compared to model group,p<0.01,P <0.0001 (single factor analysis of variance combined with Dunnett post hoc test).

Compared with a normal control group, the thymus fluorescence intensity of the model group is reduced by 45%, which indicates that the number of T cells is obviously reduced. Notably, AMCN treatment significantly increased the fluorescence intensity of the model group by 61%, indicating that the T cell population was effectively restored.

(4) AMCN increase of proinflammatory genes in zebra fish model

Given that AMCN treatment can raise the levels of key immune cell types in the zebra fish model, we further investigated AMCN mechanisms affecting the immune system by evaluating several pro-inflammatory cytokine genes known to be involved in regulating innate immunity, namely tnf- α, il-12a and ifn- γ.

Referring to FIG. 4, FIG. 4 shows the effect of AMCN on the relative expression of the tnf- α, il-12a and ifn- γ genes in zebra fish larvae by qRT-PCR. Data are expressed as mean ± standard deviation (n=3 per group),p<0.05,p<0.01,p<0.001,P <0.0001 compared to model group (single factor analysis of variance combined with Dunnett post hoc test). Our qRT-PCR showed that the expression of tnf- α was significantly reduced in the model group compared to the normal control group, whereas AMCN treatment significantly increased the expression of tnf- α (fig. 4, left panel) similar to that of tnf- α, and the expression levels of il-12a and ifn- γ were also significantly lower in the model group than in the normal control group. Whereas AMCN treatment resulted in statistically significant increases in the expression levels of these genes (figure 4, right panel). These findings indicate that AMCN can effectively improve the immune function of zebra fish models by enhancing the expression of key pro-inflammatory cytokine genes.

In the immunodeficient zebra fish model, the present example investigated the immunoregulatory effect of the bioconversion complex nutrients AMCN of ganoderma lucidum, sealwort and mulberry leaf. The results of the studies of the present application provide strong evidence that AMCN promotes the restoration of innate and adaptive immune functions by restoring the function of neutrophil, macrophage and T cell populations while enhancing key pro-inflammatory cytokine gene expression including tumor necrosis factor-alpha (tnf-alpha), interleukin-12 a (il-12 a) and interferon-gamma (ifn-gamma). These findings highlight the great potential of AMCN as a natural drug that is effective against immunosuppression, a common and clinically significant complication that is prevalent in cancer and other chronic diseases.

The innate immune cells such as neutrophils and macrophages form the first line of defense against pathogens, and can remove cell debris and regulate and control adaptive immune response through signal transduction. In the immunodeficient zebra fish model we constructed, the number of these two cells was significantly reduced, which is highly consistent with the immunosuppressive phenomenon. After AMCN treatment, the number of the two types of cells is obviously increased, which indicates that the medicine can effectively recover the innate immunity. Among these, neutrophils are critical for acute antimicrobial defense, and their number decreases dramatically associated with increased risk of infection during chemotherapy in cancer patients. By partially restoring neutrophil levels AMCN may help alleviate complications associated with neutropenia. Also, macrophage involvement in tissue repair and antigen presentation demonstrated AMCN's ability to restore macrophages.

The study in this example shows that AMCN plays a broader role in maintaining immune homeostasis and promoting immune recovery following cytotoxic stress.

This explanation is further supported by the simultaneous elevation of the expression levels of tnf-alpha and il-12 a. The tnf-alpha produced by activated macrophages and like cells can promote inflammatory responses, enhance phagocytic function, and recruit more immune effectors. il-12a, a key cytokine linking innate immunity and adaptive immunity, can drive the differentiation of naive T cells into Th1 effector cells. AMCN up-regulate the expression of these cytokines suggests that their immunostimulatory effect is not limited to restoration of cell numbers, but extends to functional activation of the innate immune pathway.

The adaptive immune system (especially T cells) plays a central role in long-term immunoprotection and anti-tumor surveillance. In our zebra fish model, the number of thymic T cells was significantly reduced under immunodeficiency conditions, consistent with impaired adaptive immune function. Notably, AMCN treatment successfully restored T cell levels-this phenomenon strongly justifies the therapeutic effect by observing a significant increase in thymus fluorescence intensity in transgenic (rag 2: dsRed) juvenile fish. This finding is remarkable because adaptive immune recovery is generally slower and less complete than innate immune recovery in immunocompromised states such as after chemotherapy or after transplantation.

The elevation of interferon-gamma (IFN-gamma) expression levels provides additional support for this mechanism. Ifn-gamma, which is an immune factor secreted mainly by T cells and natural killer cells, can not only enhance antigen presenting functions, activate macrophages, but also participate in antiviral and antitumor defense mechanisms. Elevated ifn-gamma levels were detected in AMCN treated zebra fish, indicating that the treatment was able to restore not only T cell numbers, but also functional activity. Taken together, these results demonstrate that AMCN exerts an immunomodulatory effect by modulating the synergy of the innate and adaptive immune systems, a property of great value to help patients with immunodeficiency recover.

AMCN is a remarkable feature of using bioconversion to process ganoderma lucidum, sealwort and mulberry leaves. These natural products

The material is rich in bioactive components, but has the problem of low oral bioavailability due to high molecular weight and complex glycosylation structure. By microbial or enzymatic bioconversion processes, macromolecules can be broken down into more readily absorbable metabolites, and sometimes new compounds with enhanced pharmacological activity can be produced. Studies have shown that bioconverted ganoderan exhibits stronger immune stimulating and anti-tumor effects than raw materials. Similarly, flavonoid metabolites produced by fermentation of mulberry leaves and sealwort also show superior antioxidant and immune supporting activities. Our studies have found that AMCN, by virtue of these advantages, exhibits an expected effect on immune repair over raw materials.

Immunosuppression remains a major obstacle facing cancer treatment. Chemotherapy-induced neutropenia, lymphopenia, and immune failure can increase the risk of infection, delay the progression of recovery, and reduce the response to immunotherapy. At present, clinical intervention means aiming at immunosuppression, such as granulocyte colony stimulating factor (G-CSF) or cytokine therapy, has remarkable curative effect, but is accompanied with high cost and side effects such as bone pain, fatigue, excessive inflammatory response and the like. Nutritional immunomodulators derived from medicinal and edible substances (e.g., AMCN) provide safer, more accessible adjuvant therapy regimens for susceptible patient populations. The preparation with the dual properties of food and medicine makes the preparation an ideal choice for people who need long-term administration, such as cancer survivors, elderly patients and the like, and in the groups, the safety and the tolerance are always the primary consideration.

The above description is only of the preferred embodiments of the present invention and it is not intended to limit the scope of the present invention, but various modifications and variations can be made by those skilled in the art. Variations, modifications, substitutions, integration and parameter changes may be made to these embodiments by conventional means or may be made to achieve the same functionality within the spirit and principles of the present invention without departing from such principles and spirit of the invention.

Claims (10)

1. The preparation method of the active medicinal and edible composite nutrient is characterized by comprising the following steps of:

Step S1, culturing a ganoderma lucidum monoclonal isolate by adopting a potato dextrose agar plate, and transferring an agar block containing active growth mycelium into a liquid culture medium for culturing;

S2, placing the liquid culture prepared in the step S1 in a conical flask, and performing shake culture in a dark environment at 30 ℃;

S3, quantitatively measuring the activity of the beta-glucosidase in the culture in the step S2 by adopting a beta-glucosidase detection kit, centrifuging the culture when the activity of the enzyme exceeds 100 mU/mL, and collecting supernatant;

s4, supplementing 1.5% w/v of rhizoma polygonati powder, 1.5% w/v of ganoderma lucidum powder and 1.5% w/v of mulberry leaf powder into the supernatant collected in the step S3, and carrying out shaking culture for 24 hours to carry out bioconversion;

S5, centrifuging the culture solution after bioconversion in the step S4, and collecting supernatant;

step S6, inoculating lactobacillus plantarum into the supernatant collected in the step S5, and enabling the final concentration of the lactobacillus plantarum to reach 1 multiplied by 10 ⁷ CFU/mL to obtain an active medicine-food homologous composite nutrient precursor;

Step S7, adding a freeze-drying protective agent, namely adding 10% w/v maltodextrin, 10% w/v mannitol and 2% w/v- (+) trehalose dihydrate into the active medicinal and edible composite nutrient precursor in the step S6, and fully stirring until the active medicinal and edible composite nutrient precursor is uniformly dispersed;

And S8, freeze-drying the mixed solution obtained in the step S7 by adopting a laboratory freeze dryer until the mixed solution reaches constant weight, ensuring that the freeze-dried powder is uniform and free of polysaccharide precipitation, and grinding and sieving the mixed solution to 100-mesh particle size to obtain the active medicine-food homologous compound nutrient.

2. The method for preparing an active pharmaceutical/food homologous compound nutrient according to claim 1, wherein in step S1, the liquid medium comprises 40g/L D-glucose, 5g/L peptone, 5g/L yeast extract, 0.46g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate.

3. The method for preparing active pharmaceutical/food homologous compound nutrients according to claim 1 or 2, wherein in step S2, the dark environment of the shake culture is kept clear of the light interference throughout, and the rotational speed of the shake culture is 100 rpm.

4. The method for preparing an active pharmaceutical/dietetic complex nutrient according to claim 1, wherein in step S3, the quantitative determination of the β -glucosidase activity is performed within 1 hour after sampling the culture.

5. The method for preparing active pharmaceutical and edible composite nutrients according to claim 1, wherein in step S4, the rhizoma polygonati powder, the ganoderma lucidum powder and the mulberry leaf powder are added to the supernatant after sieving with a 80-mesh sieve.

6. The method for preparing active pharmaceutical/dietetic composite nutrients according to claim 1, wherein in step S6, the lactobacillus plantarum is activated in MRS medium to logarithmic phase before inoculation.

7. The method for preparing an active pharmaceutical/edible complex nutrient according to claim 1, wherein in step S8, the pre-freezing temperature of the freeze drying is-40 ℃ and the pre-freezing time is 4 hours.

8. The active pharmaceutical and edible composite nutrient product prepared by the method according to any one of claims 1 to 7, wherein the active pharmaceutical and edible composite nutrient product is 100-mesh granular freeze-dried powder, and comprises bioconverted rhizoma polygonati saponin, ganoderma lucidum polysaccharide and mulberry leaf flavone active ingredients, and Vege-start60 strain lactobacillus plantarum with a final concentration of 1×10 ⁷ CFU/mL.

9. An active pharmaceutical and dietetic homologous compound nutrient product prepared by the method according to any of claims 1-7, wherein the product is used for preparing a medicament for increasing the number of neutrophils and a medicament for increasing the number of macrophages.

10. The active pharmaceutical and edible composite nutrient product prepared by the method according to any one of claims 1 to 7, which is used for preparing medicines for increasing the number of T cells and medicines for preparing pro-inflammatory genes.