Application of aloe polysaccharide in promoting transmembrane transport of fumarate and pyruvate and regulating cell metabolism

Abstract

This invention discloses the application of aloe polysaccharides in promoting the transmembrane transport of fumarate and pyruvate and regulating cell metabolism, belonging to the fields of biomedicine and cell metabolism regulation. This invention is the first to discover that aloe polysaccharides can act as a transmembrane transport promoter for fumarate and pyruvate. By leveraging the specific regulatory effect of aloe polysaccharides on cell membrane transport channels, the transmembrane transport efficiency of these two metabolic substrates is significantly improved. The transport aid system provided by this invention exhibits high biocompatibility and safety, and its preparation process is simple. It only requires simple mixing with the substrate under conventional cell culture conditions to function, without the need for complex conditions such as organic solvents, high temperatures, or ultrasound. It has significant application value in metabolic regulation research, cell therapy, and the development of related reagents.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of aloe polysaccharides in promoting the transmembrane transport of fumaric acid and pyruvate and regulating cell metabolism. Background Technology

[0002] Fumaric acid (FA) is a key intermediate metabolite in the tricarboxylic acid cycle (TCA cycle), playing a crucial role in cellular energy metabolism, redox balance, and signal transduction. Fumaric acid is not only a product of succinate oxidation catalyzed by succinate dehydrogenase in the TCA cycle, but also a precursor in the conversion of malate to oxaloacetate. Recent studies have revealed that fumaric acid and its derivatives (such as dimethyl fumarate) possess multiple biological activities, including immunomodulatory, anti-inflammatory, antioxidant, and antitumor effects, showing broad application prospects in the treatment of autoimmune diseases (such as multiple sclerosis and psoriasis) and the intervention of metabolic diseases.

[0003] Pyruvate (PA) is the end product of glycolysis and a core molecule connecting glycolysis and the TCA cycle. The metabolic fate of pyruvate determines the cell's energy supply pattern: under aerobic conditions, pyruvate enters the mitochondria and is catalyzed by the pyruvate dehydrogenase complex to produce acetyl-CoA, which then enters the TCA cycle for aerobic oxidation; under hypoxic conditions, pyruvate is reduced to lactate in the cytoplasm by lactate dehydrogenase, completing anaerobic glycolysis. Pyruvate is also an important precursor for gluconeogenesis, fatty acid synthesis, and amino acid metabolism, occupying a pivotal position in the cellular metabolic network.

[0004] Exogenous fumaric acid and pyruvate have important applications in biomedical research and related reagent development, but their transmembrane transport faces the following technical bottlenecks: (1) Transmembrane barriers of fumaric acid and pyruvate Fumaric acid has a relatively small molecular weight (116.07 Da), but it exhibits strong polarity and water solubility (approximately 0.63 g / 100 mL at 20°C). Under physiological pH conditions, it exists in ionized form, making it difficult to cross the cell membrane via passive diffusion. Studies have shown that exogenous fumaric acid has extremely low natural cell entry efficiency, with most fumaric acid molecules being excluded from the cell membrane and unable to effectively participate in intracellular metabolic processes. Pyruvate, with a molecular weight of 88.06 Da, can partially enter cells via active transport mediated by the monocarboxylic acid transporter (MCT) family. However, extracellular enzymes such as lactate dehydrogenase and pyruvate dehydrogenase can rapidly degrade pyruvate, leading to its significant consumption before reaching the cell membrane. Furthermore, the transmembrane transport efficiency of pyruvate is influenced by various factors, including cell type, culture conditions, and MCT expression levels, showing significant differences in different cell systems.

[0005] (2) Limitations of existing transport support technologies Existing technologies for transmembrane-assisted transport of exogenous metabolic substrates include: Chemical carrier-assisted transport: Metabolic substrates are encapsulated using chemical carriers such as liposomes, nanoparticles, and polymer micelles, and delivered intracellularly via endocytosis or membrane fusion. However, chemical carriers suffer from high biotoxicity, strong immunogenicity, complex preparation processes, and large batch-to-batch variability, which limits their widespread application in cell metabolism research and clinical practice.

[0006] Physically assisted transport methods: These methods employ physical means such as electroporation, sonication, and microinjection to forcibly disrupt cell membrane structures and facilitate substrate entry into cells. However, these methods cause significant cell damage, are complex to operate, and have low throughput, making them unsuitable for large-scale applications.

[0007] Natural carrier-assisted transport: Some studies have attempted to use natural polysaccharides such as chitosan and hyaluronic acid as transport carriers, but existing reports are mostly focused on drug delivery. They have limited effects on promoting the specific transport of metabolic substrates (especially small carboxylic acid molecules such as fumaric acid and pyruvate), and cannot be adapted to both fumaric acid and pyruvate substrates, so the differential regulatory effects on the downstream metabolic pathways of the two metabolites cannot be clearly defined.

[0008] Therefore, the existing technology has the following technical problems or defects: 1. Low transport efficiency: Exogenous fumaric acid has low natural cell entry efficiency due to its limited water solubility and pyruvate is easily degraded by extracellular enzymes, and the improvement effect of existing auxiliary carriers is limited.

[0009] 2. Poor specificity: Existing helper transport molecules cannot simultaneously and precisely promote the transmembrane transport of fumarate and pyruvate, and it is even more difficult to achieve differentiated regulation of the two substrates to different metabolic pathways, making it difficult to adapt to different metabolic regulatory needs.

[0010] 3. Safety and process issues: Chemical carriers have high biotoxicity, and the preparation of some natural carriers is complex and requires specific reaction conditions (such as organic solvents, high temperature, ultrasound, etc.), which is not conducive to large-scale application.

[0011] 4. Insufficient targeting of metabolic regulation: Existing technologies only focus on improving the efficiency of substrate entry into cells, lacking directional guidance of metabolic flux after entry into cells, and cannot achieve precise regulation of "transport-metabolism" integration.

[0012] Aloe polysaccharide is a natural active polysaccharide extracted from plants of the genus Aloe (mainly Aloe vera). Its main component is acetylated glucomannan, composed of β-(1→4)-D-mannose and α-(1→6)-D-glucose, with a wide molecular weight range (typically between 10 kDa and several MDa) and acetylation modification. Currently, there are no reports on the use of aloe polysaccharide to promote the transmembrane transport of carboxylic acid metabolites such as fumarate and pyruvate, nor are its differential regulatory effects on downstream metabolic pathways of different metabolites clearly defined. Summary of the Invention

[0013] The technical problems to be solved by this invention are, firstly, to improve the transmembrane transport efficiency of exogenous fumaric acid and pyruvate, thus solving the problem of their difficulty in natural entry into cells; secondly, to achieve specific transport guidance for the two substrates: promoting fumaric acid entry into cells and guiding metabolic flow towards anaerobic glycolysis, and promoting pyruvate entry into cells and guiding metabolic flow towards the TCA cycle, thereby enhancing the targeting of metabolic regulation. Therefore, this invention proposes the application of aloe polysaccharides in promoting the transmembrane transport of fumaric acid and pyruvate and regulating cell metabolism, utilizing the functional characteristics of aloe polysaccharides to solve the problems existing in the prior art.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: This invention uses aloe polysaccharide as a transmembrane transport regulator for fumaric acid or pyruvate. By forming a complex system with fumaric acid or pyruvate, aloe polysaccharide promotes the efficient transmembrane entry of the two substrates into the cell through its specific regulatory effect on cell membrane transport channels. It also directionally regulates metabolic flux, thereby achieving precise activation of aerobic metabolism (TCA cycle) or anaerobic glycolysis.

[0015] The mechanism by which the aloe polysaccharide of this invention promotes the transmembrane transport of fumaric acid and pyruvate and regulates cell metabolism includes the following aspects: (1) Formation of composite systems Aloe polysaccharide molecules are rich in polar groups such as hydroxyl and acetyl groups, which can form non-covalent complexes with the carboxyl groups of fumaric acid and pyruvate through hydrogen bonding and electrostatic interactions. This complexation protects the substrate molecules from degradation by extracellular enzymes and alters the surface charge distribution and hydrophilicity / hydrophobicity of the substrate molecules, which is beneficial for their recognition and binding to cell membrane transport proteins.

[0016] (2) Regulation of cell membrane transport channels Aloe polysaccharides can interact with transport proteins on the cell membrane surface (such as the monocarboxylic acid transporter MCT family and the organic anion transporter OAT family) to promote substrate transport.

[0017] (3) Directional guidance of metabolic flow Aloe polysaccharides exhibit differential metabolic pathway regulatory effects on fumaric acid and pyruvate, and the mechanisms may involve: Fumaric acid-aloe polysaccharide system: Aloe polysaccharides may inhibit the activity of mitochondrial succinate dehydrogenase (SDH) or downregulate the expression of TCA cycle-related enzymes, making it difficult for fumarate to enter the TCA cycle after entering the cell, and instead participate in glycolysis-related reactions through metabolic bypass in the cytoplasm. Alternatively, aloe polysaccharides may enhance the activity of glycolysis pathways by activating glycolysis regulators such as hypoxia-inducible factor (HIF-1α). Pyruvate-aloe polysaccharide system: Aloe polysaccharides may accelerate the transport of pyruvate to mitochondria and into the TCA cycle by promoting the activation of the pyruvate dehydrogenase complex (PDC) or upregulating the expression of the mitochondrial pyruvate carrier (MPC), while inhibiting lactate dehydrogenase (LDH) activity and reducing the conversion of pyruvate to lactate.

[0018] The preparation method of aloe polysaccharide of the present invention refers to the method for promoting the formation of a pervaporation membrane of aloe polysaccharide in patent publication number CN118253272A, and includes the following steps: Remove both ends of fresh Aloe vera leaves and soak them in pure water for 1-2 days; wash them and remove the outer skin to obtain aloe vera gel; grind the aloe vera gel to obtain aloe polysaccharide liquid.

[0019] Aloe polysaccharide liquid is placed in a mixing tank, stirred at 4℃~15℃ and subjected to light treatment for at least 24 hours, and then the aloe polysaccharide pervaporation film on the liquid surface is collected in a high-temperature film-forming tank at 75℃~95℃.

[0020] In this invention, the illumination treatment is ordinary white light illumination treatment. Ordinary white light is defined as the full wavelength range of visible light, and can be further understood as light with a wavelength of 390nm~780nm.

[0021] Compared with the prior art, the present invention has the following significant advantages: (1) High transport efficiency Experimental results show that in the presence of aloe polysaccharides, the uptake of fumaric acid and pyruvic acid by cells is increased by 1.5 to 2.8 times, which is significantly better than most existing transport assist molecules.

[0022] (2) Strong targeting of metabolic regulation. This invention achieves precise regulation of "transport-metabolism" integration for the first time: Aloe polysaccharide not only promotes substrate entry into cells, but also guides metabolic flow according to substrate type: fumaric acid is biased towards glycolysis, and pyruvate is biased towards TCA cycle, which solves the problem of insufficient targeting of metabolic regulation in the prior art.

[0023] (3) High biocompatibility: Aloe polysaccharide is a natural polysaccharide with good biocompatibility and low immunogenicity, avoiding the cytotoxicity problem of chemical carriers, and is suitable for long-term cell culture and in vivo application.

[0024] (4) The process is simple. When applying it, aloe polysaccharide and substrate are simply mixed in conventional cell culture medium and can play a role under conventional culture conditions at 37°C. There is no need for complex conditions such as organic solvents, high temperature, and ultrasound, which makes it easy to promote on a large scale.

[0025] (5) Wide range of applications This technical solution is not only applicable to fumaric acid and pyruvic acid, but can also be extended to the transmembrane transport and metabolic regulation of other structurally similar carboxylic acid metabolic substrates (such as succinic acid, malic acid, α-ketoglutarate, lactic acid, etc.). Attached Figure Description

[0026] Figure 1 This is a bar chart showing the concentration ratio of key metabolites in the TCA cycle in the experimental group (fumaric acid + aloe polysaccharide PR5) and the control group (fumaric acid) in Example 2.

[0027] Figure 2 This is a bar chart showing the concentration ratio of key glycolytic metabolites in the experimental group (fumaric acid + aloe polysaccharide PR5) and the control group (fumaric acid) in Example 2.

[0028] Figure 3 This is a bar chart showing the concentration ratio of key TCA cycle metabolites in the experimental group (pyruvate + aloe polysaccharide PR5) and the control group (pyruvate) in Example 3.

[0029] Figure 4 This is a bar chart showing the concentration ratio of key glycolytic metabolites in the experimental group (pyruvate + aloe polysaccharide PR5) and the control group (pyruvate) in Example 3.

[0030] Figure 5 This is a bar chart showing the concentration ratio of key metabolites in the TCA cycle in the experimental group (fumaric acid + conventional aloe polysaccharide) and the control group (fumaric acid) in Comparative Example 1.

[0031] Figure 6 This is a bar chart showing the concentration ratio of key glycolytic metabolites in the experimental group (fumaric acid + conventional aloe polysaccharide) and the control group (fumaric acid) in Comparative Example 1.

[0032] Figure 7 This is a bar chart showing the concentration ratio of key metabolites in the TCA cycle in the experimental group (pyruvate + conventional aloe polysaccharide) and the control group (pyruvate) in Comparative Example 1.

[0033] Figure 8This is a bar chart showing the concentration ratio of key glycolytic metabolites in the experimental group (pyruvate + conventional aloe polysaccharide) and the control group (pyruvate) in Comparative Example 1. Detailed Implementation

[0034] To enable those skilled in the art to understand the present invention more clearly and intuitively, the present invention will be further described below with reference to the accompanying drawings.

[0035] In this invention, CA: citric acid, CAA: aconitic acid, SA: succinic acid, FA: fumaric acid, MA: malic acid, AKG: α-ketoglutarate, OAA: oxaloacetic acid, FBP: fructose-1,6-bisphosphate, 2,3-DPG: 2,3-bisphospho-D-glyceric acid, 2PG: 2-phosphoglyceric acid, PEP: phosphoenolpyruvate, 3PG: 3-phosphoglyceric acid, LA: lactic acid, PA: pyruvate, G1P: glucose-1-phosphate, G6P: glucose-6-phosphate, F6P: fructose-6-phosphate, GAP: glyceraldehyde-3-phosphate, and DHAP: dihydroxyacetone phosphate.

[0036] Example 1: Preparation of Aloe Polysaccharides The aloe polysaccharide of this invention is prepared according to the method for promoting the formation of a pervaporation membrane of aloe polysaccharide as described in patent publication number CN118253272A. The specific steps are as follows: Remove both ends of fresh Aloe vera leaves and soak them in pure water for 1-2 days; wash them and remove the outer skin to obtain aloe vera gel; grind the aloe vera gel in an automatic grinding device to obtain aloe vera polysaccharide liquid.

[0037] Aloe polysaccharide liquid is placed in a mixing tank, stirred at 4℃~15℃ and treated with ordinary white light for at least 24 hours. Then, the aloe polysaccharide pervaporation film on the liquid surface is collected in a high-temperature film-forming tank at 75℃~95℃ (80℃ in this example).

[0038] The automatic aloe vera gel grinding device is disclosed in Chinese patent application CN115846015A. It includes a support frame, on which a cylindrical container for holding aloe vera gel is mounted. A gel grinder is located inside the cylindrical container. The cylindrical container is open at the top for dispensing the aloe vera gel. A discharge port is located on the bottom side of the cylindrical container. The discharge port is connected to a quick-release butterfly valve as a discharge valve. A shaft hole is located at the center of the bottom of the cylindrical container. The output shaft of a shearing motor is connected to a rotating shaft via the shaft hole and a coupling. The shearing motor drives the gel grinder through the rotating shaft. The bottom of the container in this grinding device is semi-circular. During the shearing process, the upper layer of aloe vera gel flows at a faster speed than the lower layer. The faster-flowing aloe vera gel carries the slower-flowing adjacent aloe vera gel, while the slower-flowing aloe vera gel hinders the faster-flowing adjacent aloe vera gel. This creates internal friction between the aloe vera gels with different flow rates, achieving orderly and regular internal friction. Ultimately, the viscosity of the upper layer of aloe vera gel is higher than that of the lower layer. The aloe vera polysaccharide slurry obtained through this automatic grinding device has a viscosity comparable to that of manually ground aloe vera polysaccharide slurry, but with higher efficiency.

[0039] The mixing tank features an ellipsoidal bottom. Temperature control of the aloe polysaccharide solution within the tank is achieved using a jacketed circulation system. This system cools the mixing tank from the outside. The jacket, a sealed space installed outside the tank, forms a heat conduction channel with the tank wall. A cooling medium (such as alcohol or cooling water) circulates within this channel, cooling the aloe polysaccharide solution inside and maintaining its temperature between 4°C and 15°C. The ellipsoidal bottom design primarily promotes circulation and mixing of the aloe polysaccharide solution. It reduces dead zones, making the solution easier for the agitator to mix evenly, minimizing localized flow rate differences, and improving mixing efficiency. Furthermore, the ellipsoidal bottom reflects light to the solution from multiple angles, enhancing light penetration.

[0040] The temperature of the aloe polysaccharide solution inside the high-temperature film-forming tank is controlled between 75℃ and 95℃. Similarly, the high-temperature film-forming tank uses a jacketed circulation system to control the temperature of the aloe polysaccharide solution inside the tank. The jacketed circulation system heats the outside of the high-temperature film-forming tank. The jacket is a sealed space installed outside the high-temperature film-forming tank, which forms a heat conduction channel with the tank wall. By circulating a heating medium (such as heat transfer oil and water) in this channel, the aloe polysaccharide solution inside the high-temperature film-forming tank can be heated, thereby maintaining the temperature of the aloe polysaccharide solution within the range of 75℃ to 95℃, so as to continuously form an aloe polysaccharide pervaporation film inside the high-temperature film-forming tank.

[0041] The illuminance at the center of the aloe polysaccharide solution within the mixing tank should be no less than 800 lumens. The illuminance intensity can be monitored using a light monitoring device, which can be a photodiode-type light sensor or a photoresistor-type light sensor; this invention does not limit the type. The monitored illuminance must not be less than 800 lumens to ensure sufficient energy input for the photochemical reaction of the aloe polysaccharide solution. The light treatment is ordinary white light irradiation, defined as light across the entire visible light wavelength range, specifically light with wavelengths from 390 nm to 780 nm.

[0042] Low-temperature treatment helps maintain the stability and activity of aloe polysaccharide slurry, reducing its degradation or denaturation and slowing down the degradation rate. Stirring ensures uniform distribution of components in the aloe polysaccharide slurry and creates sufficient eddies to reduce localized concentration differences. It also allows for uniform light exposure and air contact, preventing localized degradation and providing better conditions for subsequent membrane pervaporation processes.

[0043] Appropriate light exposure helps trigger or accelerate the photochemical reaction in aloe polysaccharide slurry. Under low temperature, stirring, and light exposure for at least 24 hours, the aloe polysaccharide slurry exhibits a higher degree of hydrolysis and lower viscosity (viscosity tests at room temperature 20°C showed that the viscosity of the untreated aloe polysaccharide slurry was 280.25 mPa·s, while the viscosity of the treated aloe polysaccharide slurry was 135.00 mPa·s, clearly demonstrating that light exposure can reduce the viscosity of the aloe polysaccharide slurry).

[0044] Example 2: Application of aloe polysaccharides in promoting fumarate transmembrane transport and activating the TCA cycle (1) Preparation of experimental materials: Rat hair follicle stem cells (HFSC) were purchased from iCell Bioscience. Aloe polysaccharide PR5 (prepared according to the method in Example 1); Fumaric acid (purity ≥99%, Sigma-Aldrich). Hair follicle stem cell culture medium (iCell-0098a-001b). 6-well cell culture plates, CO2 incubator, LC-MS / MS detection equipment.

[0045] (2) Cell seeding and pretreatment: HFSC cells were seeded in 6-well plates at a density of 2 × 10⁶ cells / well. 5Cells / wells were placed in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered and grew to a confluence of 70%~80%.

[0046] (3) Preparation and treatment of composite systems: Experimental group (Fumarate + Aloe polysaccharide PR5): A compound culture medium containing fumaric acid (final concentration 1 mg / mL) + aloe polysaccharide PR5 (final concentration 1.5 mg / mL) was prepared. Control group (Fumarate): A culture medium containing fumaric acid (final concentration 1 mg / mL) but without aloe polysaccharides was prepared; Remove the old culture medium from the 6-well plate and add 2 mL of control and experimental culture medium per well, respectively. Continue to incubate at 37°C and 5% CO2 for 24 hours.

[0047] (4) Metabolite detection: After culture, cells were collected and their metabolome was analyzed using LC-MS / MS to detect key metabolites of the TCA cycle and key metabolites of glycolysis.

[0048] See test results Figure 1 ( Figure 1 OAA not detected in the middle) Figure 2 .

[0049] Figure 1 Results analysis: The concentration of fumaric acid (FA) in the experimental group was about 1.3 times higher than that in the control group (baseline), which proved that aloe polysaccharides significantly promoted the transmembrane transport of fumaric acid; CA, CAA, and SA were significantly decreased, which proved that aloe polysaccharides inhibited the TCA cycle.

[0050] Figure 2 Results analysis: Key metabolites of glycolysis (FBP, 2,3-DPG, 2PG, PEP, 3PG, LA) all increased significantly, proving that the combined use of aloe polysaccharide and fumaric acid inhibits the TCA cycle and shifts the metabolic focus to glycolysis.

[0051] Example 3: Aloe polysaccharides promote pyruvate transmembrane transport and activate the TCA cycle (1) Preparation of experimental materials: Rat hair follicle stem cells (HFSC) were purchased from iCell Bioscience. Aloe polysaccharide PR5 (prepared according to the method in Example 1); Pyruvic acid (purity ≥99%, Sigma-Aldrich). The remaining materials are the same as in Example 2.

[0052] (2) Cell seeding and pretreatment: Same as in Example 2.

[0053] (3) Preparation and treatment of composite systems: Experimental group (Pyruvate + Aloe polysaccharide PR5): A compound culture medium containing pyruvate (final concentration 1 mg / mL) + aloe polysaccharide PR5 (final concentration 1.5 mg / mL) was prepared. Control group (Pyruvate): A culture medium containing pyruvate (final concentration 1 mg / mL) but without aloe polysaccharides was prepared; Remove the old culture medium from the 6-well plate and add 2 mL of control and experimental culture medium per well, respectively. Continue to incubate at 37℃ and 5% CO2 for 24 h.

[0054] (4) Metabolite detection: After culture, cells were collected and their metabolome was analyzed using LC-MS / MS to detect key metabolites of the TCA cycle and key metabolites of glycolysis.

[0055] The experimental results are shown in Figure 3 and Figure 4 .

[0056] Figure 3 Results analysis: The levels of key metabolites in the TCA cycle (CAA, ICA, SA, FA, MA, AKG, etc.) all increased or significantly increased, proving that the combined use of aloe polysaccharide and pyruvate activated the TCA cycle.

[0057] Figure 4 Results analysis: Key metabolites of glycolysis (FBP, 2,3-DPG, 2PG, PEP, 3PG, LA, etc.) all decreased or significantly decreased, proving that aloe polysaccharides inhibited glycolysis; pyruvate (PA) increased significantly, proving that aloe polysaccharides promoted the transmembrane transport of pyruvate.

[0058] Comparative Example 1: Transport Effect of Conventional Aloe Polysaccharides Conventional aloe polysaccharide (based on the process of Example 1 of this invention, but without ordinary white light irradiation) was used to replace the aloe polysaccharide PR5 prepared in Example 1, and experiments were conducted according to the methods of Example 2 and Example 3.

[0059] Experimental results show that: like Figure 5 , 6 As shown, when conventional aloe polysaccharides are used in combination with fumaric acid, the ratio of intracellular fumaric acid (FA) remains basically unchanged, and there are no significant changes in the TCA cycle and glycolytic metabolites. like Figure 7 , 8 As shown, when conventional aloe polysaccharides are used in combination with pyruvate, the ratio of intracellular pyruvate (PA) remains essentially unchanged, and there are no significant changes in the TCA cycle and glycolytic metabolites.

[0060] This study demonstrates that conventional aloe polysaccharides prepared without the CN118253272A process have a weak promoting effect on the transmembrane transport of fumaric acid and pyruvate, and cannot achieve targeted regulation of metabolic pathways.

[0061] Raw data: Table 1 presents the raw data on glycolysis and key TCA metabolites in the control group (Fumarate), experimental group (Fumarate + aloe polysaccharide PR5), and experimental group (Fumarate + ordinary aloe polysaccharide) in Example 2 and Comparative Example 1.

[0062] Table 1

[0063] Table 2 presents the raw data on glycolysis and key TCA metabolites in the control group (Pyruvate), experimental group (Pyruvate + aloe polysaccharide PR5), and experimental group (Pyruvate + ordinary aloe polysaccharide) in Example 3 and Comparative Example 1.

[0064] Table 2

[0065] The above description of the embodiments is provided to enable those skilled in the art to understand and apply the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the embodiments described herein, and any improvements and modifications made to the present invention by those skilled in the art based on the disclosure thereof should be within the scope of protection of the present invention.

Claims

1. The application of aloe polysaccharides in the preparation of promoters for the transmembrane transport of fumaric acid or pyruvate in animal cells, characterized in that, The preparation method of the aloe polysaccharide includes the following steps: Remove both ends of fresh Aloe vera leaves and soak them in pure water for 1-2 days; wash them and remove the outer skin to obtain aloe vera gel; grind the aloe vera gel to obtain aloe polysaccharide liquid. Aloe polysaccharide liquid is placed in a mixing tank, stirred at 4℃~15℃ and subjected to light treatment for at least 24 hours. Then, the aloe polysaccharide pervaporation film on the surface of the liquid is collected in a high-temperature film-forming tank at 75℃~95℃ to obtain the aloe polysaccharide.

2. The application as described in claim 1, characterized in that, In the preparation of aloe polysaccharides, the light treatment was ordinary white light irradiation.

3. The application according to claim 1 or 2, characterized in that, The aloe polysaccharide, together with fumaric acid or pyruvic acid, is used as an additive, metabolic regulator, or pharmaceutical preparation in cell culture media.

4. The application according to claim 3, characterized in that, The mass ratio of aloe polysaccharide to fumaric acid or pyruvic acid is (1.0~2.0):

1.

5. The application according to claim 4, characterized in that, The mass ratio of aloe polysaccharide to fumaric acid or pyruvic acid is 1.5:

1.

6. A method for regulating cellular metabolism not for therapeutic purposes, characterized in that, Includes the following steps: (1) Add aloe polysaccharide and fumaric acid or pyruvic acid to the cell culture medium; (2) When culturing cells, aloe polysaccharides promote the entry of fumaric acid or pyruvate in the culture medium across the membrane into the cells and guide the metabolic flux toward anaerobic glycolysis or the tricarboxylic acid cycle, respectively. The preparation method of the aloe polysaccharide includes the following steps: Remove both ends of fresh Aloe vera leaves and soak them in pure water for 1-2 days; wash them and remove the outer skin to obtain aloe vera gel. Grind the aloe vera gel to be ground to obtain aloe polysaccharide liquid; Aloe polysaccharide liquid is placed in a mixing tank, stirred at 4℃~15℃ and subjected to light treatment for at least 24 hours. Then, the aloe polysaccharide pervaporation film on the surface of the liquid is collected in a high-temperature film-forming tank at 75℃~95℃ to obtain the aloe polysaccharide.

7. The method according to claim 6, characterized in that, The cell culture conditions were 37°C, 5% CO2, and saturated humidity.

8. The application of aloe polysaccharides in the preparation of compositions that promote transmembrane transport of carboxylic acid metabolic substrates, characterized in that, The carboxylic acid metabolic substrates are one or more of fumaric acid, pyruvic acid, succinic acid, malic acid, α-ketoglutaric acid, or lactic acid. The preparation method of the aloe polysaccharide includes the following steps: Remove both ends of fresh Aloe vera leaves and soak them in pure water for 1-2 days; wash them and remove the outer skin to obtain aloe vera gel; grind the aloe vera gel to obtain aloe polysaccharide liquid. Aloe polysaccharide liquid is placed in a mixing tank, stirred at 4℃~15℃ and subjected to light treatment for at least 24 hours. Then, the aloe polysaccharide pervaporation film on the surface of the liquid is collected in a high-temperature film-forming tank at 75℃~95℃ to obtain the aloe polysaccharide.

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