Application of garlic-derived nanoparticles in preparation of product for improving intestinal immune disorder

Abstract

The invention relates to application of garlic-derived nanoparticles in preparation of a product for improving intestinal immune disorder, and belongs to the technical field of biological medicines. The invention particularly discloses nano-particles prepared from natural liliaceae allium garlic rhizomes through an ultracentrifugation method or a high-pressure homogenization method, the particle size of the garlic nano-particles is 50-150 nm, and the garlic nano-particles have spherical morphology, are uniformly distributed and contain various specific proteins and lipids. The garlic-derived nanoparticles are delivered in an oral manner, so that the phenotype of immune cells in intestinal tracts, especially gamma delta T cells, can be effectively regulated and controlled, and the immune activity of the intestinal tracts is enhanced.

Description

[0001] This invention application is a divisional application filed on May 23, 2023, with application number 2023106341314 and titled "Separation and preparation of garlic-derived nanoparticles and their application in antitumor drugs". Technical Field

[0002] This invention relates to the field of biomedical technology, and in particular to the application of garlic-derived nanoparticles in the preparation of products that improve intestinal immune disorders. Background Technology

[0003] The immune system is an organism's own defense system against harmful pathogens such as bacteria and viruses, as well as abnormal cells (such as cancer cells). It contains a large number of immune cells, including T cells, B cells, natural killer cells (NK cells), and antigen-presenting cells (macrophages, dendritic cells), etc. T cells are the most numerous and functionally complex type of lymphocyte.

[0004] γδT cells are a unique type of T lymphocyte characterized by T cell receptors (TCRs) composed of γ and δ chains. They constitute the largest proportion of intestinal intraepithelial lymphocytes, accounting for approximately 40% of all T cells. γδT cells play a crucial role in connecting innate and adaptive immunity and are widely involved in various physiological and pathological processes. In recent years, their dual role in immunomodulation, inflammatory responses, and tissue repair within the intestinal immune microenvironment has gradually become a research focus. Therefore, in-depth research on intestinal γδT cells has revealed their key role in the pathogenesis of various diseases, and related molecules may become potential therapeutic targets. Regulating the phenotype and function of γδT cells to alleviate the symptoms of various diseases has become one of the hot research directions in this field. Interestingly, current research shows that extracting active ingredients or concentrated juices from various plants, such as allicin, can effectively regulate the number and function of γδT cells. This is due to the relative invariance of the TCRs of γδT cells, which are not restricted by the major histocompatibility complex (MHC), allowing them to be bound by a variety of plant-derived compounds (unlike the TCRs of αβT cells, which can only recognize specific antigens). Therefore, plant compounds such as allicin exhibit unique intervention potential in regulating the differentiation and functional homeostasis of intestinal γδT cell subsets, suggesting that they may become novel candidate agents for intestinal immune regulation.

[0005] Allicin is an important organosulfur compound found in garlic (Allium sativum), produced by the breakdown of alliin by alliinase. Its chemical name is diallyl thiosulfinate, and it possesses significant biological activities, such as antibacterial activity, antioxidant and anti-inflammatory effects, cardiovascular protection, and antitumor potential. However, despite these diverse biological activities, allicin has extremely low oral bioavailability (<5%). Allicin is highly unstable under natural conditions, particularly in neutral or alkaline environments (pH>7), and has low water solubility, making it difficult to fully dissolve in body fluids after oral administration, thus affecting absorption. Pure allicin is therefore unsuitable for direct use in water-based pharmaceuticals or beverages. It is also readily degraded in the gastrointestinal tract and easily oxidized into sulfides such as diallyl disulfide (DADS) and diallyl trisulfide (DATS). Therefore, effectively improving the oral bioavailability of allicin is a pressing issue that needs to be addressed.

[0006] Garlic-derived nanoparticles (GNPs) are a promising natural nanocarrier for efficiently delivering allicin and overcoming its poor water solubility, low stability, and insufficient bioavailability. These nanoparticles, extracted directly from garlic, possess a unique natural composition including lipids, proteins, nucleic acids, and other pharmacologically active molecules. This provides an ideal solubility environment for allicin and effectively protects it from degradation by harsh gastrointestinal conditions such as low pH and digestive enzymes through their lipid bilayer structure. The nanoscale size (50-300 nm) and negatively charged surface properties of GNPs facilitate their uptake by intestinal epithelial cells, significantly enhancing the regulatory effect of allicin on the gut. Compared to synthetic nanocarriers (such as liposomes or polymer nanoparticles), GNPs offer advantages such as natural origin, rich composition, high biocompatibility, simple preparation, and low cost. Furthermore, the endogenous bioactive components of GNPs (such as antioxidant polyphenols and antimicrobial proteins) can synergistically enhance allicin's anti-inflammatory, antibacterial, and antitumor pharmacological activities. Therefore, GNPs not only provide a stable and efficient delivery system for allicin, but also pave the way for the development of novel drug carriers based on natural nanoparticles. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides the application of garlic-derived nanoparticles in the preparation of products that improve intestinal immune disorders. This invention relates to garlic-derived nanoparticles prepared by ultracentrifugation or high-pressure homogenization, which significantly improve the oral bioavailability of allicin. Oral administration can regulate intestinal γδT cells and enhance intestinal immunity against various diseases.

[0008] This invention is achieved through the following technical solution:

[0009] The first objective of this invention is to provide the application of garlic-derived nanoparticles in the preparation of products that improve intestinal immune disorders and enhance intestinal immunity, wherein the garlic-derived nanoparticles include proteins and lipids.

[0010] The proteins include alliinase, lectins, or defensins; the lipids include ethyl docosahexaenoic acid, erucamide, or cholesterol acetate; and the active substances include allicin, polyphenolic compounds, etc.

[0011] In one embodiment of the present invention, the size of the garlic-derived nanoparticles is 50nm-150nm.

[0012] In one embodiment of the present invention, the garlic-derived nanoparticles are prepared by separation using ultracentrifugation or high-pressure homogenization.

[0013] In one embodiment of the present invention, the garlic-derived nanoparticles are uniformly distributed spherical.

[0014] In one embodiment of the present invention, the content of alliinase is >25 wt%; the content of lectin is >5 wt%; the content of defensin is >2 wt%; the content of ethyl docosahexaenoic acid is >80 wt%; the content of erucamide is >6 wt%; the content of cholesterol acetate is >1 wt%; the content of allicin in the active substance is >1.7 wt%; the content of polyphenolic compounds is >15 wt%; and the content of organic sulfur is >0.017 wt%.

[0015] In one embodiment of the invention, the product is delivered orally.

[0016] In one embodiment of the present invention, the product includes pharmaceuticals, health foods, or food.

[0017] In one embodiment of the present invention, the dosage form of the medicine is selected from tablets, granules, pills, emulsions or dry suspensions.

[0018] In one embodiment of the invention, the pharmaceutical product further includes a pharmaceutically acceptable carrier.

[0019] In one embodiment of the present invention, the carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, suspending agent, surfactant, and preservative.

[0020] In one embodiment of the present invention, the disintegrant is selected from one or more of corn starch, potato starch, croscarmellose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxymethyl cellulose, calcium carboxymethyl cellulose, and alginate.

[0021] The preparation and characterization of garlic-derived nanoparticles of the present invention include the following aspects:

[0022] (1) Fresh garlic cloves are juiced and the garlic juice is collected. The collected garlic juice is then subjected to ultracentrifugation or high-pressure homogenization, and the precipitate is collected, which is the garlic-derived nanoparticles;

[0023] (2) The garlic-derived nanoparticles obtained in step (1) were characterized, including morphological characterization, particle size, proteomics analysis, and lipidomics analysis.

[0024] The garlic-derived nanoparticles of the present invention are delivered orally to regulate intestinal γδT cells and enhance intestinal immunity against a variety of diseases.

[0025] This invention is the first to use the rhizome of garlic (Allium spp.), a plant in the Liliaceae family, as the source of nanoparticles, thus exhibiting high biosafety. Without any modification to the extracted nanoparticles, oral administration via gavage can effectively regulate the intestinal immune microenvironment and exert its immunomodulatory effect.

[0026] The technical solution of the present invention has the following advantages compared with the prior art:

[0027] 1. The garlic-derived nanoparticles of this invention are derived from garlic rhizomes. Garlic, as a plant, seasoning, and food, is beneficial to the body and has high biocompatibility.

[0028] 2. The preparation of garlic-derived nanoparticles of the present invention has good reproducibility, can be mass-produced, and is inexpensive;

[0029] 3. When the garlic-derived nanoparticles of the present invention are administered orally by gavage, they can effectively accumulate in the intestines compared to allicin or large food particles, and have no obvious toxic side effects on the body.

[0030] 4. The garlic-derived nanoparticles of the present invention can effectively regulate the intestinal immune microenvironment, regulate intestinal γδT cells, and improve the intestinal immunity against a variety of diseases. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0032] Figure 1 This is a transmission electron microscope image of garlic-derived nanoparticles in Example 1 of the present invention;

[0033] Figure 2 This is a particle size distribution diagram of the garlic-derived nanoparticles in Example 1 of the present invention;

[0034] Figure 3 This is a protein distribution diagram of the garlic-derived nanoparticles in Example 1 of the present invention;

[0035] Figure 4 This is a proteomic distribution map of the garlic-derived nanoparticles in Example 1 of the present invention;

[0036] Figure 5 Lipidomics of garlic-derived nanoparticles in Example 1 of this invention;

[0037] Figure 6 This is a flow cytometry analysis of garlic-derived nanoparticles activating γδT cells in vitro in Example 2 of this invention.

[0038] Figure 7 This invention presents the retention and enrichment of garlic-derived nanoparticles in the intestine in vivo in Example 3. A shows fluorescence imaging at different time points after oral administration of PBS, DiD (fluorescent dye), garlic microparticle-DiD, or garlic nanoparticle-DiD to C57BL / 6 mice; B shows quantitative analysis of bioluminescent signals in the intestine at different time points (3 samples per group) for different treatment groups; C shows fluorescence imaging of major organs in vitro: 24 hours after different treatments, fluorescence imaging of the heart, liver, spleen, lungs, kidneys, and intestines of C57BL / 6 mice. D represents in vitro fluorescence imaging (n=3 per group); E represents quantitative analysis of intestinal fluorescence signals 24 hours after oral administration of different granules (3 intestinal samples per group); E represents in vitro fluorescence imaging analysis of feces in C57BL / 6 mice at 0, 3, 6, 12, 24, 36, 48 and 60 hours after oral administration of garlic-derived granules; F represents quantitative analysis of bioluminescence signals in major organs (heart, liver, spleen, lung, kidney and intestine) 24 hours after oral administration of different granules (3 organ samples per group); data are expressed as mean ± standard deviation.

[0039] Figure 8 This is a diagram showing the in vivo activation of intestinal γδT cells to secrete IFN-γ by garlic-derived nanoparticles in Example 4 of the present invention.

[0040] Figure 9This is a diagram showing the in vivo activation of intestinal γδT cells by garlic-derived nanoparticles in Example 4 of the present invention; where F is the t-SNE distribution map of different T cells in the UNTX group and GNP group; G is the relative proportion analysis of T cells (including γδT cells, naive T cells, NKT cells, CD4 T cells and CD8 T cells); H is the quantitative analysis of γδT cells in the CD3+ T cell population in the four groups of samples (3 intestinal samples in each group); IJ is the pseudo-time series analysis of T cells (I) and γδT cells, naive T cells, NKT cells, CD4+ T cells and CD8+ T cells (J); K is the T cell differentiation trajectory map generated by Monocle software; L is the expression map of marker genes (including Tcrg-C1 and Trdc) generated by Monocle software in the pseudo-time series; M is the relative expression level of γδT cell activation markers and their secreted cytokines in the UNTX group and GNP group.

[0041] Figure 10 This is a diagram illustrating the in vivo regulation of IFN-γ secretion by intestinal γδT cells by garlic-derived nanoparticles in Example 4 of this invention. M represents the relative expression levels of γδT cell activation markers and their secreted cytokines in the UNTX and GNP groups; N represents the hallmark pathway of intestinal γδT cells (IFNγ response characteristic pathway); O represents the expression of IFNγ in T cells of the UNTX and GNP groups; P represents the relative proportion of IFNγ expression in various types of intestinal T cells; Q represents the relative level of IFNγ in the blood of mice in the UNTX and GNP groups over time (3 intestinal samples from each group); R represents the t-SNE distribution map of IFNγ-positive γδT cells in the intestines of the UNTX and GNP groups; and S represents the dot matrix diagram of IFNγ-positive γδT cell activation markers in the intestines of the UNTX and GNP groups.

[0042] Figure 11 For the toxicity study of garlic-derived nanoparticles in Example 5 of this invention, the changes in body weight were observed in C57(A) and Babl / c(B) mice, respectively. Detailed Implementation

[0043] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0044] The 6-8 week old female C57BL / 6 mice used in this invention were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. All mouse experiments were conducted according to the animal experimental protocols approved by the Experimental Animal Center of Soochow University.

[0045] Example 1: Preparation and characterization of garlic-derived nanoparticles.

[0046] Garlic was juiced, and the juice was collected. The collected garlic juice was then subjected to ultracentrifugation (100,000g–120,000g for 90 minutes) or high-pressure homogenization (1200bar–1500bar pressure), and the precipitate was collected as garlic-derived nanoparticles. Transmission electron microscopy (TEM) was used to characterize the internal structure of the garlic-derived nanoparticles, and the results are as follows: Figure 1 As shown, the garlic-derived nanoparticles exhibit a spherical morphology and are uniformly distributed. Dynamic light scattering (DLS) analysis results are as follows... Figure 2 As shown, the size distribution of garlic-derived nanoparticles ranges from 50 nm to 150 nm. SDS-PAGE gel electrophoresis was used to characterize the protein distribution within the garlic-derived nanoparticles, and the results are as follows. Figure 3 As shown. To further explore the types of proteins contained in garlic-derived nanoparticles, the proteomics analysis results are as follows. Figure 4 As shown, the garlic-derived nanoparticles contain various proteins, including alliinase (26.5%), lectins (5.4%), and defensins (2.8%). Lipidomics analysis results are as follows... Figure 5 As shown, the garlic-derived nanoparticles contain various lipids, mainly including ethyl docosahexaenoic acid (87.1%), erucamide (6.6%), and cholesterol acetate (1.8%). The results of the active substance analysis are shown in Table 1. The content of the active substance allicin is >1.7 wt%; the content of polyphenolic compounds is >15 wt%; and the content of organic sulfur is >0.01 wt%.

[0047] Table 1 Content of active substances

[0048]

[0049] 1ppm = 1mg / kg

[0050] Example 2: Activation effect of garlic-derived nanoparticles on γδT cells in vitro.

[0051] Garlic-derived nanoparticles were co-incubated with T cells for 24 hours, and the activation of γδT cells was analyzed by flow cytometry. The flow cytometry results are as follows: Figure 6 As shown, co-incubation with garlic-derived nanoparticles significantly increased the proportion of γδT cells. Simultaneously, CD69 expression on γδT cells was significantly enhanced, indicating that co-incubation effectively activated γδT cells by the garlic-derived nanoparticles and promoted the secretion of the cytokine IFN-γ.

[0052] Example 3: Accumulation and retention of garlic-derived nanoparticles in the intestine after oral administration.

[0053] Garlic-derived nanoparticles were administered orally via gavage to 6-8 week old female C57BL / 6 mice. The retention and distribution of these nanoparticles in the intestines were monitored at different time points. Results are as follows: Figure 7 As shown, small-molecule allicin and large-particle food chyme cannot be enriched and retained in the intestine. Only nano-sized garlic-derived nanoparticles can achieve long-term retention and enrichment in the intestine. This is mainly due to their structure, which can effectively penetrate the intestinal mucus layer and interact with intestinal immune cells.

[0054] Example 4: The regulatory effect of garlic-derived nanoparticles on γδT cells in the intestine.

[0055] Six- to eight-week-old female C57BL / 6 mice were orally administered garlic-derived nanoparticles via gavage. After a period of time following delivery, the intestines of the C57BL / 6 mice were collected, and immunological evaluation of γδT cells was performed. Results are as follows: Figure 8 As shown, immune cells in the intestines of mice in the garlic-derived nanoparticle oral gavage group were significantly activated. Specifically, this was manifested in the activation of T cells, especially γδT cells, which secreted large amounts of the cytokine IFN-γ. The image shows the in vivo activation of intestinal γδT cells by garlic-derived nanoparticles. Figure 9 As shown, the intestinal immune microenvironment of mice in the garlic-derived nanoparticle oral gavage group was fully regulated, affecting the activity of various immune cells and promoting the production of γδT cells. The figure shows the in vivo regulation of intestinal γδT cell secretion of cytokine IFN-γ by garlic-derived nanoparticles. Figure 10 As shown, under the influence of garlic-derived nanoparticles, γδT cells differentiated into the IFN-γ phenotype and significantly increased the secretion level of IFN-γ.

[0056] Example 5: Effects of garlic-derived nanoparticles on the toxicity of mice.

[0057] Six- to eight-week-old female C57BL / 6 mice and Babl / c mice were administered garlic-derived nanoparticles orally via gavage for three days, once daily at a dose of 50 mg / kg. Mouse body weight was assessed, and the results are as follows: Figure 11 As shown, there was no significant change in mouse weight, indicating that oral administration of garlic-derived nanoparticles has extremely high safety.

[0058] In summary, the garlic-derived nanoparticles of the present invention can be obtained efficiently in a simple manner and can serve as regulators of intestinal immune cells, especially γδT cells.

[0059] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Application of garlic-derived nanoparticles in the preparation of products that improve intestinal immune disorders, wherein the garlic-derived nanoparticles include proteins and lipids; The protein includes alliinase, lectin, or defensin; the lipid includes ethyl docosahexaenoic acid, erucamide, or cholesterol acetate.

2. The application according to claim 1, characterized in that, The garlic-derived nanoparticles have a size of 50nm-150nm.

3. The application according to claim 1, characterized in that, The garlic-derived nanoparticles were prepared by separation using ultracentrifugation or high-pressure homogenization.

4. The application according to claim 1, characterized in that, The garlic-derived nanoparticles are uniformly distributed spherical.

5. The application according to claim 1, characterized in that, The alliinase content is >25 wt%; the lectin content is >5 wt%; the defensin content is >2 wt%; the docosahexaenoic acid ethyl ester content is >80 wt%; the erucamide content is >6 wt%; and the cholesterol acetate content is >1 wt%.

6. The application according to claim 1, characterized in that, The product is delivered orally.

7. The application according to claim 1, characterized in that, The products include pharmaceuticals, health foods, or food products.

8. The application according to claim 7, characterized in that, The dosage form of the drug is selected from tablets, granules, pills, emulsions, or dry suspensions.

9. The application according to claim 7, characterized in that, The medicine also includes a pharmaceutically acceptable carrier.

10. The application according to claim 9, characterized in that, The carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, suspending agent, surfactant, and preservative.

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