Oral capsule-like body product for resisting organ failure and aging

Abstract

The invention provides an oral thylakoid product for resisting organ failure and aging, which is prepared by the following steps: extracting thylakoid from plants including spinach, arabidopsis thaliana, lettuce, cabbage, green algae or wheat seedlings, redissolving the thylakoid into a buffer salt solution A, and carrying out ultrasonic treatment for 10-20 minutes under the ultrasonic power of 200-500W to prepare a suspension; adjusting the concentration of the suspension to 0.5-2 mg / ml, and obtaining a nano capsule-like body with the particle size of 200-500 nm by using a microfluidic technology; the buffer salt solution A is prepared from 10 to 100 mmol / L of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), 1 to 2 mmol / L of ethylenediamine tetraacetic acid (EDTA) disodium, 0.5 to 4 mmol / L of magnesium chloride (MgCl2), 1 to 10 mmol / L of ascorbic acid and 100 to 500 mmol / L of sapoeda sodium (SNAC), and the pH value of the buffer salt solution A is adjusted to 7.5 to 8.0; and the concentration of the capsule in the buffer salt solution A is 100-400 mg / mL. According to the product, self QPRT enzyme of the thylakoid is used for efficiently catalyzing de novo synthesis of NAD + of whole body tissues and organs, so that multiple physiological processes such as energy synthesis enhancement, oxidative stress reduction and inflammatory reaction relief are finally realized, organ failure is efficiently reversed, and organ aging is relieved.

Description

TECHNICAL FIELD

[0001] The present application provides a kind of cystoid product, especially relates to an oral cystoid product against organ failure and aging. BACKGROUND

[0002] With the aggravation of population aging, organ failure and aging-related diseases have become a global health problem. Organ failure or aging itself is a process accompanied by oxidative stress injury and acute and chronic inflammation caused by energy metabolism disorder. Therefore, "anti-aging or anti-organ failure" is largely a physiological level to resist these two forces by effective means. Current, whether it is the pathological aging and failure of clinical tissue and organs, or the physiological aging and functional degradation of the whole body system with age in daily life, the drug approach is only from one aspect of reducing oxidative stress or resisting inflammation, it is difficult to comprehensively restore both aspects as a whole, and for severe organ failure, such as acute liver failure or renal failure, there is no effective specific drug, and only symptomatic treatment, and organ replacement therapy is used for severe cases. Therefore, it is urgent to develop a specific drug that can comprehensively restore tissue cell energy metabolism and inflammation homeostasis, thereby fundamentally restoring tissue and organ function and vitality, and promoting the development of anti-aging and failure drugs.

[0003] Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme and signaling molecule involved in various biological processes, including energy metabolism, DNA repair, and gene expression regulation. Its levels are closely related to aging and various diseases. Studies have shown that NAD+ levels decline significantly with age, which is closely associated with the development of various age-related diseases. As a core regulator of energy metabolism, NAD+ can promote various biological functions, including fatty acid oxidation, mitochondrial repair, vascular normalization, and immune phenotype remodeling. Increasing evidence suggests that NAD+ deficiency is a fundamental cause of organ failure and premature aging. Current clinical trials primarily focus on precursor supplementation, demonstrating that targeting NAD+ is an important research direction for delaying aging or treating age-related diseases. However, these clinical trials are all in early stages, with long treatment cycles, high doses, and unknown efficacy. Currently, methods to increase NAD+ levels mainly include supplementing with NAD+ precursors such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). However, these methods have significant limitations: first, the absorption rate is low, requiring high-dose supplementation; second, with age and organ function decline, the activities of key enzymes for NAD+ synthesis, such as NAMPT and NMNAT, decrease, while the activities of enzymes that break down NAD+ increase, leading to poor prodrug supplementation effects; and third, long-term exogenous supplementation may inhibit the activity of NAD+ synthases in the body through negative feedback mechanisms, leading to dependence and withdrawal symptoms upon discontinuation. Therefore, developing more effective NAD+ enhancement strategies is of great significance.

[0004] Studies have found that most tissues and organs primarily rely on the de novo synthesis pathway mediated by QPRT to synthesize NAD+, rather than the precursor-replenishment retrieval pathway. More importantly, during functional decline, QPRT expression decreases while NAD+-consuming enzyme expression increases. Therefore, supplementing QPRT in cells during organ failure or aging, thereby fundamentally increasing NAD+ levels in tissues and cells, may be an important pathway to reverse organ failure and alleviate aging.

[0005] We know that NAD+, as an important carrier of energy conversion, is widely present in plant cells and plays a crucial role in regulating electron transport and antioxidant defense. In recent years, plant-derived thylakoids have attracted widespread attention due to their unique biological characteristics. Thylakoids are the main site of photosynthesis in plants, containing abundant photosynthetic pigments, proteins, and enzyme systems. Plant cells may have evolved a large number of QPRTs within the thylakoid membrane to promote de novo NAD+ synthesis, while simultaneously evolving numerous stress-resistant and antioxidant enzyme systems to work synergistically with NAD+ to resist the significant oxidative stress encountered during natural cell growth. More importantly, plant thylakoids possess a natural biomembrane structure that effectively protects the degradation sites of functional protein systems, allowing them to maintain high activity and function even in environments with various complex degradative enzymes. This inspires us to potentially develop orally administered biological agents through formulation optimization and other methods, effectively circumventing degradation by intestinal and pepsin enzymes. Current research on thylakoids indicates that, firstly, the lack of specific preparation processes for oral administration leads to poor stability and low bioavailability of thylakoids in the gastrointestinal environment; secondly, the failure to effectively control the particle size and dispersibility of thylakoids affects their distribution and effects in vivo; and thirdly, the lack of in-depth research on their animal mechanisms of action makes it difficult to use them rationally.

[0006] Currently, oral NAD+ precursor supplements such as NMN have low absorption rates, require high doses, and the levels of enzymes related to the synthesis of NAD+, such as MAMPT and NMNAT, decline during aging and decline, while the activity of hydrolytic enzymes remains high, resulting in limited NAD+ enhancement and poor responsiveness. Therefore, developing a new product that can effectively increase NAD+ levels, improve gut-liver axis function, and avoid the drawbacks of oral NAD+ precursor supplements such as NMN has significant scientific and practical value. Summary of the Invention

[0007] To overcome the shortcomings of existing technologies, this invention provides an orally administered thylakoid product. Through an efficient preparation process, an orally available plant-based thylakoid formulation is obtained. This product utilizes the thylakoid's own QPRT enzymes to efficiently catalyze the de novo synthesis of NAD+ in tissues and organs throughout the body, reshaping the energy metabolism processes of tissue parenchymal cells and immune cells. Furthermore, through its antioxidant enzyme system and metabolic influence on the gut microbiota, it enhances the antioxidant capacity of cells, ultimately achieving multiple physiological processes such as enhanced energy synthesis, reduced oxidative stress, and alleviated inflammatory responses. This effectively reverses organ failure and slows down organ aging.

[0008] This invention provides an oral thylakoid product for combating organ failure and aging. Thylakoids are extracted from plants including spinach, Arabidopsis thaliana, lettuce, cabbage, green algae, or wheat seedlings, and reconstituted in buffer salt solution A. The solution is then sonicated at 200–500 W for 10–20 min to prepare a suspension. The concentration of this suspension is adjusted to 0.5–2 mg / mL, and microfluidic technology is used to obtain nano-thylakoids with a particle size of 200–500 nm. Buffer salt solution A comprises 10–100 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1–2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 0.5–4 mmol / L magnesium chloride (MgCl2), 1–10 mmol / L ascorbic acid, and 100–500 mmol / L sodium sapolixalate (SNAC), with the pH adjusted to 7.5–8.0. The concentration of the thylakoids in buffer salt solution A is 100–400 mg / mL. It is worth noting that, compared with nano-thylakoids obtained by traditional biomembrane extruders, the technical solution provided by this invention can improve the QPRT enzyme gating bioavailability of the oral thylakoid product by 2 times, and improve the particle stability in the gastrointestinal microenvironment by 50%, and increase the QPRT enzyme activity by 1.5 times.

[0009] Preferably, in the oral thylakoid product provided by the present invention, the microfluidic technology uses a hydrodynamic flow focusing chip, the width and / or depth of the chip channel is between 100 and 500 μm, and the flow rate of the suspension in the chip is controlled at 10 to 30 μL / min to control the thylakoid particle size; nano-thylakoids are obtained by ultracentrifugation at 50,000 to 80,000 rpm.

[0010] Preferably, in the oral thylakoid product provided by the present invention, the buffer salt solution A comprises 50 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 1 mmol / L magnesium chloride (MgCl2), 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), and the pH is adjusted to 8.0.

[0011] Preferably, in the oral thylakoid product provided by the present invention, the concentration of thylakoids in buffered saline solution A is 200 mg / mL, the ultrasonic power is 300 W, the channel width and / or depth of the microfluidic chip is 300 μm, the flow rate of the suspension is controlled at 30 μL / min, the ultracentrifugation speed is 60000 rpm, and the average particle size of the nanothylakoids is 300 nm.

[0012] The inventors discovered that thylakoids extracted from plants, including spinach, Arabidopsis thaliana, lettuce, cabbage, green algae, or wheat seedlings, can be dissolved in a specific buffer salt solution and then subjected to ultrasonication and microfluidic particle formation technology at appropriate power. The resulting nano-thylakoids with specific particle sizes can retain QPRT enzyme activity to the maximum extent, making them suitable for preparing oral thylakoid products. This ensures their stability and bioavailability in the gastrointestinal environment, while effectively controlling the particle size and dispersibility of the thylakoids further enhances their specific biological activity.

[0013] Preferably, the oral thylakoid product provided by the present invention is characterized in that the nano-thylakoid is lyophilized to obtain a solid powder nano-thylakoid, and the solid powder nano-thylakoid is reconstituted in a buffer salt solution B for oral administration; the buffer salt solution B comprises 200-500 mmol / L sucrose, 0.5-2 mmol / L MgCl2, 1-5 mmol / L ascorbic acid, and 100-500 mmol / L sodium saporixate (SNAC), and the pH is adjusted to 7.5-8.0; the concentration of the thylakoid in the buffer salt solution B is 20-80 mg / mL.

[0014] Preferably, in the oral thylakoid product provided by the present invention, the buffer salt solution B comprises 330 mmol / L sucrose, 1 mmol / L MgCl2, 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), and the pH is adjusted to 8.0.

[0015] Through creative work, the inventors discovered that adding a specific concentration of sodium sapordine (SNAC) to buffer salt solutions A and B increases the bioavailability of the prepared oral thylakoid QPRT enzyme by 3 times, significantly higher than that of ordinary oral buffer solutions. This also confirms that a specific concentration of SNAC can enhance the bioavailability of oral QPRT enzyme nanoformulations, achieving unexpected technical results.

[0016] Preferably, in the oral thylakoid product provided by the present invention, the concentration of thylakoids in buffered saline solution B is 20–60 mg / mL.

[0017] The inventors discovered that by using ultrasonic and microfluidic particle-forming techniques to form suitable particle sizes from thylakoids extracted from plants including spinach, Arabidopsis thaliana, lettuce, cabbage, green algae, or wheat seedlings, and then freeze-drying them at low temperatures to obtain freeze-dried solid powder-like nano-thylakoids, and then reconstituted them in a specific solution, an oral thylakoid product can be prepared that achieves unexpected oral effects containing QPRT enzymes, maximizing the preservation of the thylakoids' own QPRT enzyme activity and exhibiting unique biological activity.

[0018] Preferably, the oral thylakoid product provided by the present invention accumulates in the intestine and liver, and is used to prepare products that regulate the gut-liver axis.

[0019] Preferably, the oral thylakoid product provided by this invention increases the level of probiotics in the gut while reducing intestinal inflammation and oxidative stress. This oral thylakoid product also enhances liver nicotinamide adenine dinucleotide (NAD+) levels, reduces inflammation, and restores oxidative stress and liver function indicators, making it suitable for preparing anti-organ failure formulations. Through the inventors' inventive efforts, the prepared oral thylakoids remain intact in the gut after oral administration, retaining their own QPRT enzyme activity and efficiently reaching the liver to reverse acute liver failure. Further research by the inventors has revealed that this oral thylakoid product can effectively alleviate drug-induced acute liver failure in mice, restore liver function, and reduce mortality caused by acute liver failure, thus providing a valuable window of opportunity for other therapies for acute organ failure.

[0020] Preferably, the oral thylakoid product provided by this invention reduces peripheral blood inflammation and oxidative stress indicators, improves metabolic capacity, and can be used to prepare anti-aging products. Through the inventors' inventive work, it was discovered that this oral thylakoid product can significantly reduce various aging indicators in physiologically aging mice, including oxidative stress and inflammation, and significantly alleviate the aging process. This oral thylakoid product can promote NAD+ synthesis, increase probiotic content, and reduce peripheral blood pro-inflammatory and oxidative stress levels, and can be used to prepare anti-aging products. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 Transmission electron microscopy analysis and particle size analysis of oral thylakoid products;

[0023] Figure 2 To compare the protein integrity of extracted thylakoids with that of orally administered thylakoid products and the particle size integrity in a simulated oral environment;

[0024] Figure 3 The accumulation and distribution of orally administered granule products in the heart, liver, spleen, lungs, and intestines;

[0025] Figure 4Comparison of NAD+ in the liver, pro-inflammatory cytokine TNF-α, anti-inflammatory cytokine IL-10, and GSH in the small intestine of healthy mice, liver failure mice, and mice with oral thylakoid products plus liver failure mice;

[0026] Figure 5 Comparison of serum ALT and AST concentrations in healthy mice, liver failure mice, and mice treated with thylakoid products and liver failure mice;

[0027] Figure 6 To analyze the oxygen consumption and energy expenditure capacity of aging model mice and mice using oral thylakoid products combined with aging model mice during the day and night. Detailed Implementation

[0028] To further illustrate the present invention, embodiments are given below. It should be noted that these embodiments are entirely illustrative. The purpose of providing these embodiments is to fully demonstrate the meaning and content of the present invention, but they do not limit the present invention to the scope of these embodiments.

[0029] Example 1

[0030] This invention provides an oral thylakoid product, wherein thylakoids are extracted from plants including spinach, Arabidopsis thaliana, lettuce, cabbage, green algae, or wheat seedlings, and reconstituted in buffer salt solution A. The mixture is then sonicated at 200–500 W for 10–20 min to prepare a suspension. The concentration of the suspension is adjusted to 0.5–2 mg / mL, and microfluidic technology is used to obtain nano-thylakoids with a particle size of 200–500 nm. Buffer salt solution A comprises 10–100 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1–2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 0.5–4 mmol / L magnesium chloride (MgCl2), 1–10 mmol / L ascorbic acid, and 100–500 mmol / L sodium sapolixalate (SNAC), and the pH is adjusted to 7.5–8.0. The concentration of the thylakoids in buffer salt solution A is 100–400 mg / mL. In one embodiment, the microfluidic technology employs a hydrodynamic flow focusing chip with channels of 100–500 μm width and / or depth. The flow rate of the suspension within the chip is controlled at 10–30 μL / min to control the thylakoid particle size, and nano-thylakoids are obtained by ultracentrifugation at 50,000–80,000 rpm. In a more preferred embodiment, buffer solution A comprises 50 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 1 mmol / L magnesium chloride (MgCl2), 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), adjusted to pH 8.0. The thylakoids were prepared in buffer salt solution A at a concentration of 200 mg / mL, with an ultrasonic power of 300 W, a channel width and / or depth of 300 μm in the microfluidic chip, a suspension flow rate of 30 μL / min, and an ultracentrifugation speed of 60,000 rpm. The average particle size of the nanothylakoids was 300 nm.

[0031] In one embodiment, the obtained nano-thylakoids are lyophilized to obtain solid powder nano-thylakoids. The solid powder nano-thylakoids are then reconstituted in a buffer salt solution B for oral administration. The buffer salt solution B comprises 200–500 mmol / L sucrose, 0.5–2 mmol / L MgCl2, 1–5 mmol / L ascorbic acid, and 100–500 mmol / L sodium saporixate (SNAC), adjusted to a pH of 7.5–8.0. The concentration of the thylakoids in the buffer salt solution B is 20–80 mg / mL. In a more preferred embodiment, the buffer salt solution B comprises 330 mmol / L sucrose, 1 mmol / L MgCl2, 5 mmol / L ascorbic acid, and 300 mmol / L sodium saporixate (SNAC), adjusted to a pH of 8.0. The concentration of the thylakoids in the buffer salt solution B is 20–60 mg / mL.

[0032] Through creative work, the inventors discovered that adding a specific concentration of sodium saporthoxam (SNAC) to buffer salt solutions A and B increases the bioavailability of the prepared gnaphaloids by 3 times, significantly higher than that of ordinary gnaphaloid buffer solutions. This also confirms that a specific concentration of SNAC can enhance the bioavailability of oral QPRT enzyme nanoparticles, achieving unexpected technical results.

[0033] The oral thylakoid product provided by this invention accumulates in the intestine and liver, and can be used to prepare products that regulate the gut-liver axis. This oral thylakoid product increases the level of beneficial bacteria in the gut while reducing intestinal inflammation and oxidative stress levels. It also enhances hepatic nicotinamide adenine dinucleotide (NAD+) levels, reduces inflammation, and restores oxidative stress and liver function indicators, making it suitable for preparing anti-organ failure formulations. Through the inventors' inventive efforts, the prepared oral thylakoids remain intact in the intestine after oral administration, retaining their own QPRT enzyme activity and efficiently reaching the liver to reverse acute liver failure. Further research by the inventors revealed that this oral thylakoid product can effectively alleviate drug-induced acute liver failure in mice, restore liver function, and reduce mortality caused by acute liver failure, thus providing a valuable window of opportunity for other therapies for acute organ failure. Simultaneously, the oral thylakoid product provided by this invention can reduce peripheral blood inflammation and oxidative stress indicators and improve metabolic capacity, making it suitable for preparing anti-aging products.

[0034] Example 2

[0035] This embodiment provides an oral thylakoid product, which extracts thylakoids from lettuce and reconstitutes them in a buffer salt solution A. Buffer salt solution A comprises 10 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 0.5 mmol / L magnesium chloride (MgCl2), 1 mmol / L ascorbic acid, and 100 mmol / L sodium sapolixalate (SNAC), with the pH adjusted to 7.5.

[0036] In the preparation process, the extracted thylakoids were prepared into a suspension with a concentration of 100 mg / mL in buffer salt solution A. Subsequently, this suspension was sonicated at a power of 200 W for 10 minutes to prepare a mixed suspension. The concentration of this suspension was adjusted to 0.5 mg / mL. A fluid dynamics focusing chip was selected, employing microfluidic technology. The chip's channels had a width and depth of 100 μm, and the flow rate of the suspension within the chip was controlled at 10 μL / min to regulate the thylakoid particle size. Finally, ultracentrifugation at 50,000 rpm yielded nanothylakoids with an average particle size of 200 nm.

[0037] The obtained nano-thylakoids were lyophilized to obtain solid powder nano-thylakoids. For use, the solid powder nano-thylakoids were reconstituted in buffer salt solution B for oral administration. Buffer salt solution B consisted of 200 mmol / L sucrose, 0.5 mmol / L MgCl2, 1 mmol / L ascorbic acid, and 100 mmol / L sodium saporthase (SNAC), with the pH adjusted to 7.5. The concentration of the thylakoids in buffer salt solution B was 20 mg / mL.

[0038] Example 2

[0039] This embodiment provides an oral thylakoid product, which extracts thylakoids from spinach and reconstitutes them in a buffer salt solution A. Buffer salt solution A comprises 100 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 4 mmol / L magnesium chloride (MgCl2), 10 mmol / L ascorbic acid, and 500 mmol / L sodium sapolixalate (SNAC), with the pH adjusted to 8.0.

[0040] In the preparation process, the extracted thylakoids were prepared into a suspension with a concentration of 400 mg / mL in buffer salt solution A. Subsequently, this suspension was sonicated at a power of 500 W for 20 minutes to prepare a coarse suspension. The concentration of this coarse suspension was adjusted to 2 mg / mL. A fluid dynamics focusing chip was used, employing microfluidic technology. The chip's channels had a width and depth of 500 μm, and the flow rate of the suspension within the chip was controlled at 30 μL / min to regulate the thylakoid particle size. Finally, ultracentrifugation at 80,000 rpm yielded nanothylakoids with an average particle size of 500 nm.

[0041] The obtained nano-thylakoids were lyophilized to obtain solid powder nano-thylakoids. For use, the solid powder nano-thylakoids were reconstituted in buffer salt solution B for oral administration. Buffer salt solution B comprised 500 mmol / L sucrose, 2 mmol / L MgCl2, 5 mmol / L ascorbic acid, and 500 mmol / L sodium saporixate (SNAC), with the pH adjusted to 8.0. The concentration of thylakoids in buffer salt solution B was 80 mg / mL. In a preferred embodiment, the concentration of thylakoids in buffer salt solution B was 60 mg / mL.

[0042] This oral thylakoid product accumulates in the gut and liver, and can be used to regulate the gut-liver axis. The product increases the level of beneficial gut bacteria while reducing intestinal inflammation and oxidative stress. Furthermore, it enhances hepatic nicotinamide adenine dinucleotide (NAD+) levels, reduces inflammation, and restores oxidative stress and liver function indicators, making it suitable for developing anti-organ failure formulations.

[0043] This oral thylakoid product can also reduce peripheral blood inflammation and oxidative stress markers, and improve metabolic capacity, making it suitable for the development of anti-aging products. Functional testing results showed that after using the product, the gut microbiota diversity of the experimental subjects significantly increased, with the proportion of beneficial bacteria such as Bifidobacteria and Lactobacillus increasing by more than 30%; Figure 4 , 5 Comparative data of mouse indicators in healthy models, liver failure models, and mice using oral thylakoid products combined with liver failure models show that, by preserving QPRT enzyme activity, this oral thylakoid product increased NAD+ levels in the livers of mice in the liver failure model by nearly 100%, and reduced liver function indicators ALT and AST by two-thirds and one-half, respectively, within 6 hours; the level of the inflammatory factor TNF-α decreased by 2.5 times, the level of the anti-inflammatory factor IL-10 increased by about 100 times, and the level of the antioxidant stress indicator GSH increased by 50%.

[0044] Example 4

[0045] This embodiment provides an oral thylakoid product, which extracts thylakoids from cabbage and reconstitutes them in buffer salt solution A.

[0046] First, thylakoids were extracted from cabbage. Specifically, cabbage leaves were selected, washed, chopped, and homogenized with buffer solution. The thylakoid precipitate was then obtained by centrifugation.

[0047] Next, the extracted thylakoids were reconstituted in buffer solution A. Buffer solution A consisted of 50 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid, 2 mmol / L disodium ethylenediaminetetraacetate, 1 mmol / L magnesium chloride, 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), with the pH adjusted to 8.0. The concentration of thylakoids in buffer solution A was then adjusted to 200 mg / mL.

[0048] Then, the reconstituted thylakoid solution was sonicated at 300W for 15 minutes. During sonication, the sample temperature was kept below 4°C to prevent denaturation of thylakoid proteins, thus preparing a suspension.

[0049] The concentration of the suspension was adjusted to approximately 1 mg / ml. A fluid dynamics focusing chip was selected, employing microfluidic technology. The chip's channels had a width and depth of 300 μm, and the flow rate of the suspension within the chip was controlled at 30 μL / min to control the thylakoid particle size and ensure uniformity. Ultracentrifugation at 60,000 rpm ultimately yielded nanothylakoids with an average particle size of 300 nm. Figure 1 As can be seen from the transmission electron microscopy analysis and particle size analysis, the particle size distribution range of the product is 260-300 nm, and the polydispersity index is less than 0.2, indicating that the product has good particle size uniformity.

[0050] The prepared nano-thylakoids exhibit good stability, remaining stable for over 30 days at 4°C. In vitro tests simulating gastrointestinal fluid showed good stability in gastric juice, effectively protecting internal active ingredients such as the QPRT enzyme from gastric acid degradation. In the intestinal fluid environment, the product slowly releases its internal active substances, facilitating absorption. The enzyme activity of QPRT in the simulated intestinal microenvironment remained similar to that before oral administration, consistently at 45 U / L.

[0051] Example 5

[0052] In this embodiment, a further preparation method for an oral thylakoid product is provided, including the following steps:

[0053] S1: Freeze-drying process:

[0054] The nano-thylakoids prepared in Example 4 were freeze-dried to obtain solid powder nano-thylakoids. Specifically, the prepared nano-thylakoid solution was placed in a freeze-drying bottle and first pre-frozen at -40°C for 4 hours. Then, it was dried under a vacuum of 10 Pa for 24 hours, followed by a secondary drying under a vacuum of 5 Pa for 12 hours, finally yielding white, fluffy solid powder nano-thylakoids.

[0055] S2: Reconstitution treatment:

[0056] The solid powder nano-thylakoids were reconstituted in buffer salt solution B for oral administration. Specifically, the solid powder nano-thylakoids obtained in the above steps were added to the pre-prepared buffer salt solution B and gently stirred at room temperature for 15 minutes to ensure complete dissolution.

[0057] In this embodiment, buffer salt solution B comprises 330 mmol / L sucrose and 1 mmol / L MgCl₂. z The solution contains 5 mmol / L ascorbic acid and 300 mmol / L sodium saporide (SNAC), and the pH is adjusted to 8.0. Buffer solution B is prepared as follows: First, weigh 113.1 g sucrose, 0.095 g MgCl2, and 0.881 g ascorbic acid, dissolve them in 900 mL deionized water, then add 90.4 g sodium saporide (SNAC), mix thoroughly, adjust the pH to 8.0 using 1 M NaOH solution, and finally bring the volume to 1000 mL with deionized water.

[0058] In this embodiment, the concentration of nano-thylakoids in buffer salt solution B is 42 mg / mL. Specifically, 4.2 g of solid powder nano-thylakoids was weighed and added to 100 mL of the above-prepared buffer salt solution B, and gently stirred until completely dissolved to obtain an oral thylakoid product with a concentration of 42 mg / mL.

[0059] Efficacy verification:

[0060] The prepared oral thylakoid products were stored at 4°C for 30 days, and their stability was tested periodically. The results showed that the thylakoids reconstituted in buffer salt solution B maintained good stability during storage, with particle size variation not exceeding 5% and encapsulation efficiency remaining above 95%. Figure 2 A comparison of the integrity of proteins such as QPRT in extracted thylakoids and oral thylakoid products, as well as particle size integrity in simulated oral environments, showed that lyophilized and reconstituted thylakoids exhibited 10-fold improved stability at room temperature compared to untreated thylakoids, with no significant change in activity within 24 hours, resulting in a longer shelf life. Animal experiments demonstrated that the bioavailability of lyophilized and reconstituted thylakoids reached 55% after oral administration, significantly higher than that of directly administered traditional formulations—extracted thylakoids (15% bioavailability). Furthermore, the solid powder-like nano-thylakoids obtained through lyophilization facilitate long-term storage and transportation, greatly improving the product's practicality.

[0061] Example 6

[0062] For example, the oral thylakoid product provided in Example 5 of this invention, such as Figure 3 As shown, this product can accumulate in the intestines and liver after oral administration, effectively regulating the function of the gut-liver axis and having an anti-organ failure effect.

[0063] Animal experiments have verified that, 24 hours after oral administration of this type of cyst product, the four major phyla of bacteria in the intestines of experimental animals—Bacteroides, Firmicutes, Actinomycetes, and Proteus—returned to normal compared to the disordered state in the non-oral group. Figure 4 A comparison of NAD+ levels in the liver, pro-inflammatory cytokine TNF-α, and anti-inflammatory cytokine IL-10, and GSH levels in the small intestine of healthy mice, liver failure mice, and mice treated with oral thylakoid products combined with liver failure showed that oral thylakoids, by maintaining QPRT enzyme activity, promoted NAD+ elevation, downregulated pro-inflammatory cytokine TNF-α, upregulated anti-inflammatory cytokine IL-10, and increased antioxidant stress levels. The level of the antioxidant stress marker GSH was elevated to a level similar to that of the healthy group (3 μM). Figure 5 Comparison of serum ALT and AST concentrations in healthy mice, liver failure mice, and mice with oral thylakoid products combined with liver failure mice showed that oral thylakoids effectively alleviated acute liver failure in mice, allowing liver function to gradually return to normal within 6 hours. The liver function indicators ALT and AST in mice decreased by 2 / 3 and 1 / 2, respectively, and liver inflammation and oxidative damage were significantly reduced.

[0064] This oral thylakoid product, as a regulator of the gut-liver axis, effectively prevents and treats organ failure by increasing gut probiotic levels, reducing intestinal inflammation and oxidative stress, enhancing liver NAD+ levels, and improving liver function indicators. It can be used to develop anti-organ failure formulations. Clinical trials have shown that this product demonstrates good therapeutic effects in patients with liver dysfunction, inflammatory bowel disease, and multiple organ dysfunction syndrome, with good safety and no significant adverse reactions.

[0065] Example 7

[0066] The thallus-like product provided in Example 5 of this invention can reduce peripheral blood inflammation and oxidative stress indicators, improve metabolic capacity, and be used to prepare anti-aging products.

[0067] The efficacy verification experiment results of this oral thylakoid product showed that:

[0068] 1. Improvement of inflammatory markers: In aged mice, after 28 days of continuous oral administration of this type of capsule product, the level of the inflammatory factor TNF-α in the peripheral blood of the subjects decreased by 45.5%, and the level of the anti-inflammatory factor IL-10 increased by 60.4%, indicating that the product has a significant anti-inflammatory effect.

[0069] 2. Improvement of oxidative stress indicators: After 28 days of continuous oral administration of this type of capsule product, the content of 8-hydroxy-2′-deoxyguanosine (8-OHdG, a marker of oxidative stress) in the peripheral blood of aged mice decreased by 40.6%, and GSH increased by 4 times, indicating that the product has a significant antioxidant effect.

[0070] 3. Improved metabolic capacity: After 28 days of continuous oral administration of this type of capsule product, if... Figure 6 Analysis of oxygen consumption and energy expenditure in aging models, mice treated with oral thylakoid products, and aging model mice during the day and night showed that the animals' oxygen consumption increased by an average of 20.7%, and the mice's energy expenditure increased by 18.7%, indicating that the product can effectively improve metabolic capacity.

[0071] 4. Improvement of aging markers: After 28 days of continuous oral administration of this type of capsule product, the level of growth differentiation factor-15 (GDF-15, an aging stress response cytokine) in the peripheral blood of aging mice decreased by 15.5%, indicating that the product has the effect of delaying cell aging.

[0072] This oral thylakoid product has the following advantages: Firstly, the oral thylakoid product prepared using a specific process can protect its internal active ingredients, such as QPRT enzymes, from destruction by gastric acid and digestive enzymes, thus improving bioavailability. Secondly, this thylakoid product can promote the cellular uptake of the active ingredient QPRT enzymes, enhancing their distribution and function in the body. Furthermore, by retaining its own QPRT enzyme activity, this oral thylakoid product efficiently catalyzes the de novo synthesis of NAD+ in tissues and organs throughout the body, reshaping the energy metabolism processes of tissue parenchymal cells and immune cells. Through its antioxidant enzyme system and its influence on gut microbiota metabolism, it enhances the antioxidant capacity of cells, ultimately achieving multiple physiological processes such as enhanced energy synthesis, reduced oxidative stress, and alleviated inflammatory responses, effectively reversing organ failure and alleviating organ aging.

[0073] In summary, this oral thylakoid product effectively slows down the aging process by reducing peripheral blood inflammation and oxidative stress indicators and improving metabolic capacity, providing a novel and highly effective product for the anti-aging field.

[0074] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An oral thylakoid product for combating organ failure and aging, characterized in that, Thylakoids were extracted from plants including spinach, Arabidopsis thaliana, lettuce, cabbage, green algae, or wheat seedlings, and reconstituted in buffer salt solution A. The solution was then sonicated at 200–500 W for 10–20 min to prepare a suspension. The concentration of the suspension was adjusted to 0.5–2 mg / mL, and microfluidic technology was used to obtain nano-thylakoids with a particle size of 200–500 nm. Buffer salt solution A comprised 10–100 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1–2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 0.5–4 mmol / L magnesium chloride (MgCl2), 1–10 mmol / L ascorbic acid, and 100–500 mmol / L sodium sapogenide (SNAC), with the pH adjusted to 7.5–8.

0. The concentration of the thylakoids in buffer salt solution A was 100–400 mg / mL.

2. An oral thylakoid product as described in claim 1, characterized in that, The microfluidic technology uses a hydrodynamic flow focusing chip, the width and / or depth of the channels in the chip are between 100 and 500 μm, and the flow rate of the suspension in the chip is controlled at 10 to 30 μL / min to control the thylakoid particle size; the nano-thylakoids are obtained by ultracentrifugation at 50,000 to 80,000 rpm.

3. An oral thylakoid product as described in claim 1, characterized in that, The buffer salt solution A comprises 50 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 2 mmol / L disodium ethylenediaminetetraacetic acid (EDTA), 1 mmol / L magnesium chloride (MgCl2), 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), adjusted to pH 8.

0.

4. An oral thylakoid product as described in claim 2, characterized in that, The concentration of the thylakoids in buffer salt solution A is 200 mg / mL, the ultrasonic power is 300 W, the channel width and / or depth of the microfluidic chip is 300 μm, the flow rate of the suspension is controlled at 30 μL / min, the ultracentrifugation speed is 60,000 rpm, and the average particle size of the nanothylakoids is 300 nm.

5. An oral thylakoid product as described in claim 1, characterized in that, The nano-thylakoids were lyophilized to obtain solid powder nano-thylakoids, which were then reconstituted in buffer salt solution B for oral administration. Buffer salt solution B comprised 200–500 mmol / L sucrose, 0.5–2 mmol / L MgCl2, 1–5 mmol / L ascorbic acid, and 100–500 mmol / L sodium saporixate (SNAC), with the pH adjusted to 7.5–8.

0. The concentration of the thylakoids in buffer salt solution B was 20–80 mg / mL.

6. An oral thylakoid product as described in claim 5, characterized in that, The buffer salt solution B comprises 330 mmol / L sucrose, 1 mmol / L MgCl2, 5 mmol / L ascorbic acid, and 300 mmol / L sodium sapolixalate (SNAC), with the pH adjusted to 8.

0.

7. An oral thylakoid product as described in claim 5, characterized in that, The concentration of the thylakoids in buffer salt solution B is 20–60 mg / mL.

8. An oral thylakoid product as described in claim 1, characterized in that, The oral thylakoid product accumulates in the intestine and liver and is used to prepare products that regulate the gut-liver axis.

9. An oral thylakoid product as described in claim 8, characterized in that, The oral thylakoid product increases the level of probiotics in the gut while reducing the level of inflammation and oxidative stress in the gut; the oral thylakoid product increases the level of nicotinamide adenine dinucleotide (NAD+) in the liver, reduces the level of inflammation, and restores oxidative stress and liver function indicators, and is used to prepare formulations for treating organ failure.

10. An oral thylakoid product as described in claim 1, characterized in that, The oral thylakoid product reduces peripheral blood inflammation and oxidative stress indicators and improves metabolic capacity, and is used to prepare anti-aging products.

Images