Targeted delivery type tea-iron composite microspheres as well as preparation method and application thereof

Abstract

The invention relates to the technical field of functional supplements, in particular to composite microspheres for regulating and controlling iron metabolism and a preparation method and application of the composite microspheres. The targeted delivery type tea-iron composite microsphere comprises an inner core containing a ferrous compound and theabrownin, the inner core is sequentially coated with sodium alginate and chitosan from inside to outside, so that the double-shell microsphere is formed; the surfaces of the double-shell microspheres are also coated with a theabrownin-probiotic metabolite composite membrane. The invention provides the tea-iron composite microsphere capable of realizing targeted delivery, effective release and synergistic interaction of iron element and tea active ingredients are realized through the design of a special structure, the immune function is remarkably enhanced while iron metabolism is regulated and controlled, the problems of low absorption efficiency, strong gastrointestinal tract irritation and the like of a traditional iron agent are effectively solved, and the tea-iron composite microsphere has a good application prospect. Therefore, a brand new solution is provided for improving intestinal health and enhancing body immunity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional supplements, in particular to a complex microsphere for regulating iron metabolism and a preparation method and application thereof. BACKGROUND

[0002] Iron, as an indispensable trace element for human body, plays a key role in oxygen transport, energy metabolism and the maintenance of catalytic activity of various enzymes. Normal iron metabolism is essential for maintaining the health of the body, and iron deficiency can lead to iron deficiency anemia, affecting the normal growth and physiological function of the body; while iron overload can cause oxidative stress damage and is closely related to the occurrence and development of various chronic diseases such as cardiovascular diseases and neurodegenerative diseases. The absorption, transport, storage and utilization of iron in the human body are subject to a delicate regulatory mechanism, in which the intestine plays a key role in the absorption of iron. The intestine is not only an important place for digestion and absorption, but also the largest immune organ in the human body. The intestinal immune system can recognize and resist the invasion of pathogens, while maintaining immune tolerance to symbiotic microorganisms. This immune balance is of great significance to intestinal health and overall health. Abnormalities in intestinal immune function, whether increased susceptibility to infection due to immunodeficiency or inflammatory bowel disease caused by excessive immune activation, can have a serious impact on quality of life. There is a close interaction between iron metabolism and intestinal immunity, and appropriate levels of iron are essential for the normal function of intestinal immune cells, while the intestinal immune status in turn affects iron absorption and transport.

[0003] At present, the products on the market for iron metabolism abnormalities and intestinal immune problems are relatively single. Commonly used are various iron supplements that can regulate iron metabolism to a certain extent. However, traditional iron agents have low absorption efficiency and cause severe gastrointestinal reactions, etc. The reason is that inappropriate intake of iron agents can cause intestinal adverse reactions, which in turn affects the absorption of normal iron elements, and long-term or improper use of iron agents can also cause iron overload risk. For example, a food-grade active protein iron supplement and its preparation method disclosed in patent CN111528482A, which forms active protein iron complex particles by embedding iron element compounds in protein fibers, can improve iron absorption and stability to a certain extent. On the one hand, the protein fibers have poor coating ability in gastric acid, and the burst release of iron compounds directly stimulates the intestinal mucosa, not only causing stress damage, but also affecting the absorption of the ingested iron agent. On the other hand, the protein fibers have poor antioxidant capacity, and long-term storage can easily lead to oxidation of the embedded ferrous compounds. Therefore, there is an urgent need to find a solution to regulate iron metabolism and promote immune function. SUMMARY

[0004] The present invention aims to overcome the shortcomings of traditional iron supplements in the prior art, such as low absorption efficiency, strong gastrointestinal irritation, and lack of response to intestinal immune disorders. It provides a targeted delivery type of tea-iron composite microspheres, its preparation method, and its application to overcome the above-mentioned defects.

[0005] To achieve the objectives of the invention described above, the present invention is implemented through the following technical solution: In a first aspect, the present invention discloses a targeted delivery type tea-iron composite microsphere, which includes a core containing ferrous compounds and theabrownins; the core is coated from the inside out with sodium alginate and chitosan to form a double-shelled microsphere; the surface of the double-shelled microsphere is also coated with a theabrownin-probiotic metabolite composite membrane.

[0006] Ferrous compounds are common iron supplements; however, in practice, direct contact between ferrous ions and the gastric mucosa can easily cause adverse reactions such as nausea and abdominal pain. Direct contact with the intestinal mucosa can easily induce inflammation and damage the intestinal barrier function. Therefore, this invention uses theaflavins and ferrous compounds together as the core of composite microspheres to achieve the simultaneous release of theaflavins and ferrous compounds in vivo. On the one hand, subsequent experiments revealed that when theaflavins and ferrous compounds act together on intestinal cells, they not only better regulate iron metabolism but also effectively reduce the expression of inflammatory factors, significantly improving iron absorption compared to traditional iron supplements while also regulating intestinal immunity. On the other hand, theaflavins have strong antioxidant properties, significantly inhibiting the oxidation of ferrous iron into poorly absorbed ferric iron, thereby improving the product's stability during processing, storage, and release in the gastrointestinal environment.

[0007] However, ferrous compounds and theabrownins, when directly mixed, form dispersions or precipitates, making direct application impossible and failing to achieve targeted delivery. Therefore, this invention sequentially coats the core with sodium alginate and chitosan, providing physical encapsulation and maintaining the microsphere morphology. Sodium alginate forms the basis for microsphere formation, immobilizing both ferrous compounds and theabrownins within the microspheres. Chitosan further coats the sodium alginate microspheres, compensating for surface defects while effectively preventing premature degradation of sodium alginate in the acidic gastric environment. This ensures the composite microspheres reach the intestines smoothly, and the sodium alginate gel network regulates the release rate of nutrients from the core.

[0008] However, chitosan is highly susceptible to moisture and oxygen in the air when exposed to air, severely affecting the product's storage stability. Therefore, it is necessary to consider how to ensure the biosafety of the microspheres while protecting the structure of the double-shelled microspheres and the normal function of the core nutrients as much as possible. To this end, this invention, based on the double-layered microspheres, also covers them with a layer of theaflavins-probiotic metabolites composite membrane. On the one hand, in vitro, the theaflavins on the outer layer directly form an antioxidant barrier on the surface of the microspheres, more effectively intercepting oxygen and free radicals in the environment. Probiotic metabolites (SCFAs) synergistically enhance antioxidant capacity. At the same time, the theaflavins-probiotic metabolites composite membrane acts as a physical barrier, preventing the sodium alginate and chitosan in the middle layer from directly contacting moisture and oxygen. On the other hand, after the microspheres enter the intestines, the probiotic metabolites can stimulate mucus secretion, strengthen the epithelial barrier, and create a more favorable environment for iron absorption. Meanwhile, the theaflavins on the outer layer can also synergistically regulate the intestinal flora, reduce pathogenic bacteria that competitively consume iron, and improve the final bioavailability of iron through physiological regulation.

[0009] In summary, the composite microspheres of the present invention can be divided into three layers, namely a core containing ferrous compounds and theabrownins, ... The microspheres consist of an inner layer formed by sodium alginate and chitosan, and an outer layer composed of a theabrownin-probiotic metabolite composite membrane. The inner layer serves as the core nutrient layer; subsequent experiments have verified that theabrownin effectively regulates iron metabolism and intestinal immunity, thereby promoting the absorption of ferrous compounds in the intestine. The inner layer, as the structural layer, confines the core nutrients within the microsphere structure, a prerequisite for long-term storage and targeted delivery. The outer layer, as the protective layer, protects the inner and outer layers from external humidity and oxygen, ensuring the targeted release of the core nutrients into the intestine. Therefore, this invention, through the above triple structure and using bio-friendly raw materials, yields an iron-supplementing composite microsphere that enables precise delivery, synergistic effects, and is friendly to the intestinal flora.

[0010] Furthermore, by mass, the ratio of the ferrous compound to theaflavins is 1:2~3.

[0011] Furthermore, the probiotic metabolites (SCFAs) include one or more of acetic acid, propionic acid, and butyric acid.

[0012] Furthermore, the ferrous compound includes one or more of ferrous sulfate, ferrous lactate, and ferrous gluconate.

[0013] Furthermore, the particle size of the composite microspheres is 100-500 nm.

[0014] Secondly, this invention also discloses a method for preparing the above-mentioned targeted delivery type tea-iron composite microspheres, comprising the following steps: S1. Ferrous compounds are mixed with theaflavins and subjected to ultrasonic treatment to form a dispersion; S2. Mix the dispersion with sodium alginate solution, and then add the mixture dropwise into calcium chloride solution to form microspheres; S3. Coat the microspheres with chitosan solution to obtain composite microspheres; S4. Coat the surface of the composite microspheres with a tea brown pigment-probiotic metabolite composite membrane and freeze-dry to obtain the above-mentioned targeted delivery type tea iron composite microspheres.

[0015] Furthermore, in step S4, the ratio of theabrownin to probiotic metabolites added is 1:1~2 by mass.

[0016] Thirdly, the present invention also discloses the application of a targeted delivery type tea-iron composite microsphere in the preparation of a drug that regulates intestinal iron metabolism.

[0017] Fourthly, this invention also discloses the application of a targeted delivery type tea-iron composite microsphere in the preparation of a drug that regulates intestinal immune homeostasis.

[0018] Fifthly, the present invention also discloses the application of a targeted delivery type tea-iron composite microsphere in the preparation of functional foods that regulate iron metabolism and intestinal immunity.

[0019] Therefore, the present invention has the following beneficial effects: (1) The present invention uses ferrous compounds and theabrownins as core nutrients, which can not only better regulate iron metabolism, but also effectively reduce the expression of inflammatory factors. Compared with traditional iron supplements, it can effectively improve iron absorption rate and play a role in regulating intestinal immunity.

[0020] (2) This invention provides a tea-iron composite microsphere with a triple structure that can achieve targeted delivery. Through the design of the special structure, the iron element and the active ingredients of tea are effectively released and synergistically enhanced. While regulating iron metabolism, it significantly enhances immune function and effectively solves the problems of low absorption efficiency and strong gastrointestinal irritation of traditional iron supplements. Thus, it provides a brand-new solution for improving intestinal health and enhancing the body's immunity. Attached Figure Description

[0021] Figure 1 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microsphere on FTH gene expression in Example 1 of the present invention.

[0022] Figure 2 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microspheres on the expression of cellular inflammatory factors (IL-4, TSLP, IL-33) in Example 1 of the present invention.

[0023] Figure 3 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microspheres on the expression of tight junction protein (ZO-1) in Example 1 of the present invention.

[0024] Figure 4 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microsphere on FTH gene expression in Example 2 of the present invention.

[0025] Figure 5 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microspheres on the expression of cellular inflammatory factors (IL-4, TSLP, IL-33) in Example 2 of the present invention.

[0026] Figure 6 This is a schematic diagram illustrating the effect of a targeted delivery type of tea-iron composite microspheres on the expression of tight junction protein (ZO-1) in Example 2 of the present invention. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0028] Example 1: The composite microspheres in this embodiment are prepared through the following steps: S1. Mix 100 mg of ferrous sulfate (FeSO4) powder and 200 mg of theabrownin powder and add to 5 mL of water. Sonicate for 30 minutes to obtain a dispersion. S2. Add the dispersion to 10 mL of sodium alginate solution (2% w / v), mix well to form a mixture, and add the mixture dropwise to 50 mL of calcium chloride solution (2% w / v), stirring to form microspheres; S3. Coat the microspheres with 10 mL of chitosan solution (1% w / v) and let stand for 30 minutes; S4. Theabrownin and acetic acid at a concentration of 15% are mixed at a mass ratio of 1:1, coated on the surface of microspheres, and freeze-dried to obtain the targeted delivery type tea-iron composite microspheres. The particle size of the prepared composite microspheres is between 100-500 nm, and the encapsulation rate is ≥90%.

[0029] The following experiments were conducted to verify the function of the targeted delivery type tea-iron composite microspheres prepared in Example 1.

[0030] Iron metabolism regulation experiment: The human intestinal epithelial cell line Caco-2 was selected and divided into two groups: a control group (normally cultured Caco-2 cells without any treatment), an iron control group (Caco-2 cells treated with ferrous sulfate, a traditional iron supplement, at a concentration of 25 μM), and a composite microsphere group (targeted delivery type tea-iron composite microspheres prepared in Example 1 were added to 6-well plates with a growth density of approximately 60%-70% of Caco-2 cells, at a concentration of 25 μM (calculated based on iron content).

[0031] Experimental steps: 1. Cell culture: Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics and placed in an incubator at 37°C and 5% CO2. Subsequent experiments were conducted after the cells reached the logarithmic growth phase.

[0032] 2. Cell treatment: Seed cells in 6-well plates and culture for 24 hours. Then, add the treatment solutions of the different groups mentioned above according to the experimental groups and continue to culture for another 24 hours.

[0033] 3. Detection indicators: The mRNA expression levels of genes encoding the heavy and light chains (FTH) in ferritin were detected using RT-qPCR.

[0034] Ferritin (FTH) is a major component of ferritin, and its main functions include storing iron in a soluble and non-toxic state to prevent iron overload from damaging cells; and regulating iron metabolism, playing a crucial role in iron uptake and release and influencing iron metabolism in different tissues. Experimental results are as follows... Figure 1 As shown, the targeted delivery type tea-iron composite microspheres prepared in Example 1 significantly improved the expression of FTH mRNA in Caco-2 cells, with a significantly better effect than the traditional iron supplement ferrous sulfate.

[0035] Intestinal epithelial cell immunomodulatory activity assay: The human intestinal epithelial cell line Caco-2 was selected and divided into three groups: a control group (normally cultured Caco-2 cells without any treatment), an iron control group (Caco-2 cells treated with ferrous sulfate, a traditional iron supplement, at a concentration of 25 μM), and a composite microsphere group (Caco-2 cells treated with the targeted delivery type tea-iron composite microspheres prepared in Example 1 at a concentration of 25 μM (calculated based on iron content).

[0036] Experimental steps: 1. Cell culture: Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics and placed in an incubator at 37°C and 5% CO2. Subsequent experiments were conducted after the cells reached the logarithmic growth phase.

[0037] 2. Cell treatment: Seed cells into 6-well plates and culture for 24 hours. Then, add the corresponding treatment solution according to the experimental groups and continue culturing for another 24 hours.

[0038] 3. Detection indicators: Intestinal immune-related indicators: RT-qPCR was used to detect the levels of inflammatory factors (IL-4, TSLP, IL-33) and the mRNA expression of tight junction protein (ZO-1) in cells.

[0039] Experimental results are as follows Figure 2 , 3 As shown, the targeted delivery tea-iron composite microspheres prepared in Example 1 significantly reduced the mRNA expression of inflammatory factors TSLP and IL-33 in Caco-2 cells, while increasing the expression of tight junction protein ZO-1, indicating that it can protect intestinal barrier function and reduce inflammatory response. In contrast, Caco-2 cells treated with the traditional iron supplement ferrous sulfate did not show significant changes in the above indicators.

[0040] Example 2: The composite microspheres in this embodiment are prepared through the following steps: S1. Mix 100 mg of ferrous sulfate (FeSO4) and 300 mg of theabrownin in water, and sonicate for 30 minutes to obtain a mixture. S2. Add the mixture to 10 mL of sodium alginate solution (2% w / v), then add it dropwise to 50 mL of calcium chloride solution (2% w / v), and stir to form microspheres; S3. Coat the microspheres with 10 mL of chitosan solution (1% w / v) and let stand for 30 minutes; S4. Theabrownin and acetic acid at a concentration of 15% are mixed at a mass ratio of 1:2, coated on the surface of microspheres, and freeze-dried to obtain the targeted delivery type tea-iron composite microspheres.

[0041] The following experiments were conducted to verify the function of the targeted delivery type tea-iron composite microspheres prepared in Example 2.

[0042] Iron metabolism regulation experiment: The human intestinal epithelial cell line Caco-2 was selected and divided into three groups: a control group (normally cultured Caco-2 cells without any treatment), an iron control group (Caco-2 cells treated with ferrous sulfate, a traditional iron supplement, at a concentration of 25 μM), and a composite microsphere group (Caco-2 cells treated with the targeted delivery type tea-iron composite microspheres prepared in Example 1 at a concentration of 25 μM (calculated based on iron content).

[0043] Experimental steps: 1. Cell culture: Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics and placed in an incubator at 37°C and 5% CO2. Subsequent experiments were conducted after the cells reached the logarithmic growth phase.

[0044] 2. Cell treatment: Seed cells in 6-well plates and culture for 24 hours. Then, add the treatment solutions of the different groups mentioned above according to the experimental groups and continue to culture for another 24 hours.

[0045] 3. Detection indicators: The mRNA expression levels of genes encoding the heavy and light chains (FTH) in ferritin were detected using RT-qPCR.

[0046] Experimental results are as follows Figure 4 As shown, the targeted delivery type tea-iron composite microspheres prepared in Example 2 significantly improved the expression of FTH mRNA in Caco-2 cells, with a significantly better effect than the traditional iron supplement ferrous sulfate.

[0047] Intestinal epithelial cell immunomodulatory activity assay: The human intestinal epithelial cell line Caco-2 was selected and divided into three groups: control group (normally cultured Caco-2 cells without any treatment); iron control group (Caco-2 cells treated with ferrous sulfate, a traditional iron supplement, at a concentration of 25 μM); and composite microsphere group (Caco-2 cells treated with targeted delivery tea-iron composite microspheres at a concentration of 25 μM (calculated based on iron content).

[0048] Experimental steps: 1. Cell culture: Caco-2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics and placed in an incubator at 37°C and 5% CO2. Subsequent experiments were conducted after the cells reached the logarithmic growth phase.

[0049] 2. Cell treatment: Seed cells in 6-well plates and culture for 24 hours. Then, add the corresponding treatment solution according to the experimental groups and continue culturing for another 24 hours.

[0050] 3. Detection indicators: Intestinal immune-related indicators: RT-qPCR was used to detect the levels of inflammatory factors (IL-4, TSLP, IL-33) and the mRNA expression of tight junction protein (ZO-1) in cells.

[0051] Experimental results are as follows Figure 5 , 6 As shown, the targeted delivery tea-iron composite microspheres prepared in Example 2 significantly reduced the mRNA expression of inflammatory factors TSLP and IL-33 in Caco-2 cells, while increasing the expression of tight junction protein ZO-1, indicating that they can protect intestinal barrier function and reduce inflammatory response. In contrast, Caco-2 cells treated with the traditional iron supplement ferrous sulfate did not show significant changes in the above indicators.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A targeted delivery type of tea-iron composite microsphere, characterized in that: Including a kernel containing ferrous compounds and theabrownins; The core is coated with sodium alginate and chitosan from the inside out, thereby forming a double-shelled microsphere; The surface of the bishelled microspheres is also coated with a theabrownin-probiotic metabolite composite membrane.

2. The targeted delivery type tea-iron composite microsphere according to claim 1, characterized in that: The ratio of the ferrous compound to the theabrownin is 1:2 to 3 by mass.

3. The targeted delivery type tea-iron composite microsphere according to claim 1, characterized in that: The probiotic metabolites include one or more of acetic acid, propionic acid, and butyric acid.

4. The targeted delivery type tea-iron composite microsphere according to claim 1, characterized in that: The ferrous compound includes one or more of ferrous sulfate, ferrous lactate, and ferrous gluconate.

5. The targeted delivery type tea-iron composite microsphere according to claim 1, characterized in that: The composite microspheres have a particle size of 100-500 nm.

6. A method for preparing targeted delivery tea-iron composite microspheres, characterized in that, Includes the following steps: S1. Ferrous compounds are mixed with theaflavins and subjected to ultrasonic treatment to form a dispersion; S2. Mix the dispersion with sodium alginate solution, and then add the mixture dropwise into calcium chloride solution to form microspheres; S3. Coat the microspheres with chitosan solution to obtain composite microspheres; S4. Coat the surface of the composite microspheres with a tea brown pigment-probiotic metabolite composite membrane, and freeze-dry to obtain a targeted delivery type tea iron composite microsphere as described in any one of claims 1-5.

7. The method for preparing targeted delivery tea-iron composite microspheres according to claim 6, characterized in that: In step S4, the ratio of theabrownin to probiotic metabolites added is 1:1~2 by mass.

8. The use of a targeted delivery type tea-iron composite microsphere as described in any one of claims 1-5 in the preparation of a drug for regulating intestinal iron metabolism.

9. The use of a targeted delivery type tea-iron composite microsphere as described in any one of claims 1-5 in the preparation of a drug for regulating intestinal immune homeostasis.

10. The application of a targeted delivery type tea-iron composite microsphere as described in any one of claims 1-5 in the preparation of functional foods that regulate iron metabolism and intestinal immunity.

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