Mitochondrial targeting group modified zwitterionic active compound as well as preparation method and application thereof

Abstract

The invention discloses a zwitterionic active compound modified by a mitochondrial targeting group as well as a preparation method and application of the zwitterionic active compound, and belongs to the technical field of precise nutrition and medicines. The preparation method comprises the following steps: synthesizing a negatively charged anionic active compound through an electrophilic addition reaction between an active compound and NaHSO3; and synthesizing the active compound modified by the mitochondrial targeting group through electrostatic coupling between the anionic active compound and the cationic mitochondrial targeting molecule. The preparation method is simple and efficient, meanwhile, the obtained product can retain the original functional activity of the active compound to the maximum extent, and meanwhile accurate positioning and efficient enrichment of the active compound in mitochondria are promoted; the active compound can effectively relieve mitochondrial homeostasis under oxidative stress and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of precision nutrition and medical technology, and specifically relates to a zwitterionic active compound modified with a mitochondrial targeting group, and a preparation method and application thereof. Background Art

[0002] Mitochondrial homeostasis under oxidative stress is closely related to the occurrence and progression of many diseases, which mainly involves the imbalance between reactive oxygen species (ROS) and the antioxidant system. Mitochondria are double-membrane organelles present in almost all mammalian cells, and play a vital role in various physiological processes such as energy homeostasis, metabolism, redox balance, and apoptosis signaling pathways. Once oxidative stress occurs, mitochondria, as the main producer of ROS, are the primary target of oxidative damage. Excessive ROS generation can cause multi-faceted damage to mitochondria, mainly including decreased mitochondrial membrane potential, mitochondrial dysfunction, inhibition of mitochondrial autophagy, and cell apoptosis, which are all risk factors for the occurrence and development of neurodegenerative diseases, inflammatory bowel disease, cancer, and metabolic disorders. Therefore, regulating mitochondrial homeostasis under oxidative stress is crucial for the intervention of various mitochondrial homeostasis-related diseases.

[0003] In recent years, increasing evidence has shown that active compounds can improve human health by regulating mitochondrial homeostasis. Active compounds can maintain mitochondrial homeostasis by correcting metabolic imbalances, reducing oxidative stress levels, and promoting biosynthesis. However, the low uptake rate within mitochondria limits the regulation of mitochondrial homeostasis by active compounds under oxidative stress. Therefore, enhancing the accumulation of active compounds in mitochondria is of great significance for addressing diseases related to mitochondrial homeostasis.

[0004] The mitochondrial targeting strategy provides the possibility for the large-scale accumulation of active compounds in mitochondria. The significant negative transmembrane potential (120-140mV) in mitochondria can drive positively charged molecules into the organelle. Generally, mitochondrial targeted active compounds can be obtained by covalent modification with lipophilic cationic compounds (such as triphenylphosphine cations, pyridinium salts and mitochondrial targeting peptides). Although, the covalent modification of (3-propylcarboxyl) triphenylphosphine bromide, berberine, and SS31 peptide has been applied to the synthesis of a variety of mitochondrial targeted active compounds, including curcumin, coenzyme Q10, proanthocyanidins, doxorubicin, etc. However, the covalent binding of lipophilic cationic compounds to active compounds still faces a series of problems that need to be solved, including harsh reaction conditions, low yield, difficult separation and purification, poor biocompatibility, etc. Therefore, there is an urgent need for an efficient, safe and reliable preparation method to achieve the modification of active compounds by lipophilic cationic compounds. Summary of the invention

[0005]

Technical issues

[0006] The present invention overcomes the disadvantages of harsh reaction conditions, low yield, difficult separation and purification, and poor biocompatibility faced by traditional covalent bond modification of active compounds and lipophilic cationic compounds, and provides a zwitterionic active compound modified with a mitochondrial targeting group, and a preparation method and application thereof.

[0007]

Technical solution

[0008] In a first aspect, the present invention provides a method for constructing a mitochondrial targeting product, which is to obtain a mitochondrial targeting product by utilizing the electrostatic coupling between a negatively charged anionic active compound and a positively charged cationic mitochondrial targeting molecule;

[0009] The structure of the negatively charged anionic active compound is A-X1, wherein X1 is a sulfonate, and A is derived from the following active compounds: β-carotene, lycopene, lutein, astaxanthin, capsanthin, zeaxanthin, fucoxanthin, curcumin, piperine, or any isomer of the above active compounds.

[0010] In one embodiment of the present invention, the sulfonate salt comprises sodium sulfonate and / or potassium sulfonate.

[0011] In one embodiment of the present invention, the positively charged cationic mitochondrial targeting molecule may specifically be (3-propylcarboxyl)triphenylphosphonium bromide.

[0012] In one embodiment of the present invention, the above construction method comprises:

[0013] S1, the active compound first undergoes an electrophilic addition reaction with NaHSO3 to synthesize a negatively charged anionic active compound;

[0014] S2. The prepared negatively charged anionic active compound is then mixed with the positively charged cationic mitochondrial targeting molecule to undergo electrostatic coupling, and the mixture is stirred at room temperature, dialyzed, and freeze-dried to obtain a mitochondrial targeting product.

[0015] In one embodiment of the present invention, the S1 specifically includes:

[0016] Under an inert atmosphere, the active compound and NaHSO3 are dissolved in an organic reagent and deionized water, respectively, the two are mixed, and reacted under heating and stirring conditions, and the negatively charged anionic active compound is collected by centrifugation and freeze drying.

[0017] Furthermore, the molar ratio of the active compound to NaHSO3 is 1:1 to 1:1.05.

[0018] Furthermore, the organic reagent for dissolving the active compound is one of tetrahydrofuran, ethylene glycol methyl ether, methanol, ethanol, and deionized water.

[0019] Furthermore, the heating temperature is 50-70°C.

[0020] Furthermore, the reaction time is 1 to 2 hours.

[0021] Furthermore, the centrifugal speed is 6000-8000 rpm, and the time is 10-20 min.

[0022] In one embodiment of the present invention, in S2, the molar ratio of the negatively charged anionic active compound to the positively charged cationic mitochondrial targeting molecule is 1:1 to 1:1.5.

[0023] In one embodiment of the present invention, in S2, the stirring time at room temperature is 1 to 3 hours.

[0024] In one embodiment of the present invention, in S2, the dialysis cut-off molecular weight is 500-1000 Da, and the dialysis time is 24-48 h.

[0025] In a second aspect, the present invention also provides a mitochondrial targeting product based on the above method.

[0026] In one embodiment of the present invention, the structure of the mitochondrial targeting product is as follows:

[0027]

[0028] Wherein A is derived from the following active compounds: β-carotene, lycopene, lutein, astaxanthin, capsanthin, zeaxanthin, fucoxanthin, curcumin, piperine, or any isomer of the above active compounds, It is a type of positively charged cationic mitochondrial targeting structure.

[0029] In one embodiment of the present invention, the structure of the mitochondrial targeting product can be:

[0030]

[0031] In one embodiment of the present invention, It refers to electrostatic coupling.

[0032] In the present invention, electrostatic coupling is a reversible non-covalent interaction that provides a new possibility for the combination of lipophilic cations and active compounds. The resulting mitochondrial targeting product carries equal amounts of positive and negative charges, similar to zwitterionic compounds. It is worth noting that the zwitterionic compounds formed by electrostatic coupling have excellent biocompatibility, good cell permeability and reversible disassembly characteristics, which will help to improve the mitochondrial targeting efficiency of active compounds on the basis of traditional covalent bond modification. In addition, there are currently few reports on the synthesis of zwitterionic compounds from active compounds.

[0033] The present invention provides use of the mitochondrial targeting product in preparing medicines for diseases related to mitochondrial homeostasis.

[0034] In one embodiment of the present invention, the use is to provide a nutritional intervention delivery system using the above-mentioned mitochondrial targeted product.

[0035] In one embodiment of the present invention, in the use, the above-mentioned mitochondrial targeted product can also be embedded in various delivery systems to provide nutritional intervention for various mitochondrial homeostasis-related diseases.

[0036]

Beneficial Effects

[0037] (1) The present invention provides a simple, efficient and safe method for constructing a mitochondrial targeting product (i.e., a zwitterionic active compound modified with a mitochondrial targeting group), and the yield of the product can reach 86.17%.

[0038] (2) The mitochondrial targeting product provided by the present invention retains the original functional activity of the active compound to the maximum extent, and also promotes the precise positioning and efficient enrichment of the active compound in the mitochondria.

[0039] (3) The mitochondrial targeted product provided by the present invention can effectively alleviate mitochondrial homeostasis under oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Example 1 Mitochondria targeting group modified zwitterionic active compound Mito-Cur 1 H NMR spectrum;

[0041] Figure 2 This is the Fourier transform infrared spectrum of Mito-Cur, a zwitterionic active compound modified with a mitochondrial targeting group in Example 1;

[0042] Figure 3 1 is a schematic diagram of mitochondrial fluorescence co-localization of Mito-Cur, an active compound modified with a mitochondrial targeting group in Example 1 (scale bar: 50 μm);

[0043] Figure 4 The anion AXT-SO3 in the synthesis of the zwitterionic active compound Mito-AXT modified by the mitochondrial targeting group in Example 2 is 1 H NMR spectrum;

[0044] Figure 5 Example 2 Mitochondria targeting group modified zwitterionic active compound Mito-AXT 1 H NMR spectrum;

[0045] Figure 6 This is the ESR spectrum of the anion AXT-SO3 in the synthesis of the zwitterionic active compound Mito-AXT modified with the mitochondrial targeting group in Example 2;

[0046] Figure 7 This is the EDS spectrum of the zwitterionic active compound Mito-AXT modified with the mitochondrial targeting group in Example 2;

[0047] Figure 8 This is the XPS spectrum of Mito-AXT, a zwitterionic active compound modified with a mitochondrial targeting group in Example 2;

[0048] Fig. 9 This is a graph showing the potential measurement results of Mito-AXT, a zwitterionic active compound modified with a mitochondrial targeting group in Example 2;

[0049] Fig.10 is the UV-visible spectrum of the zwitterionic active compound Mito-AXT modified with the mitochondrial targeting group in Example 2;

[0050] Fig.11 This is a test result diagram of Example 2 in which the zwitterionic active compound Mito-AXT modified with a mitochondrial targeting group regulates H2O2-induced cellular ROS generation (scale: 100 μm);

[0051] Fig.12 This is a test result diagram of the mitochondrial targeting group-modified zwitterionic active compound Mito-AXT restoring mitochondrial membrane potential under oxidative stress in Example 2 (scale: 100 μm);

[0052] Fig.13 This is a test result diagram of Example 2 that the zwitterionic active compound Mito-AXT modified with a mitochondrial targeting group regulates cell autophagy under oxidative stress (scale: 50 μm);

[0053] Fig.14 This is a test result diagram of Example 2 on the regulation of cell apoptosis under oxidative stress by the zwitterionic active compound Mito-AXT modified with a mitochondrial targeting group (scale: 100 μm). DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the following drawings and embodiments, but the present invention is not limited to the following embodiments.

[0055] In the present invention, the term "AXT" refers to astaxanthin; the term "AXT-SO3" refers to sulfonate-modified astaxanthin; the term "TPP" refers to the mitochondrial targeting molecule (3-propylcarboxyl)triphenylphosphonium bromide; the term "Mito-AXT" refers to AXT modified by TPP electrostatic coupling; the term "AXT-TPP" refers to AXT covalently modified by TPP; the term "Cur" refers to curcumin; the term "Mito-Cur" refers to Cur modified by TPP electrostatic coupling; the term "Cur-TPP" refers to Cur covalently modified by TPP.

[0056] The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions.

[0057] Example 1

[0058] The present invention discloses a mitochondrial targeting group-modified active compound Mito-Cur, which is prepared by the following synthetic route:

[0059]

[0060] Cur (7.37 mg, 0.02 mmol) and NaHSO3 (2.08 mg, 0.02 mmol) were dissolved in 2 mL of ethanol and distilled water, respectively; Cur and NaHSO3 solutions were then mixed and stirred at 50°C (under nitrogen atmosphere) for 2 h; Cur-SO3 was separated by centrifugation at 6000 rpm for 10 min; Cur-SO3 (8.99 mg, 0.02 mmol) and TPP (8.59 mg, 0.02 mmol) were dissolved in 2 mL of DMSO and stirred continuously at 25°C for 2 h; then dialyzed in distilled water for 48 h (molecular weight cutoff MW 500 Da) to obtain the reaction product Mito-Cur with a yield of 83.14%.

[0061] pass 1 H NMR spectroscopy was used to analyze the changes in proton signals during the synthesis of Mito-Cur.

[0062] Results Figure 1 The proton of C=C in Cur-SO3 is at 6.52ppm (He). 1The disappearance of sulfonate in the 1H NMR spectrum is attributed to the addition of sulfonate during the electrophilic addition reaction between Cur and NaHSO3. In addition, characteristic proton signals of anionic Cur-SO3 at 9.66ppm (Ha), 7.58-7.51ppm (Hc), 7.32ppm (Hd), 6.07ppm (Hf) and cationic TPP at 7.94-7.74ppm (Hb), 1.79-1.68ppm (Hg) were observed in the 1H NMR spectrum of Mito-Cur, confirming the formation of zwitterionic Mito-Cur.

[0063] The changes of chemical groups during the synthesis of Mito-Cur were characterized and analyzed by Fourier transform infrared spectroscopy.

[0064] Results Figure 2 , Cur-SO3 infrared spectrum in the fingerprint region 502.4cm -1 The characteristic absorption peak at is attributed to the presence of sulfonate groups, and the characteristic absorption peaks in the infrared spectrum of Mito-Cur that overlap with Cur-SO3 and TPP illustrate the occurrence of electrostatic coupling between Cur-SO3 and TPP.

[0065] The mitochondrial targeting ability of Mito-Cur was analyzed by colocalization of Mito-Cur with mitochondrial fluorescence. The culture medium containing Cur and Mito-Cur (20 μg / mL) was added to a 12-well plate and incubated with IEC-6 cells and IEC-6 cells pretreated with TPP (10 μg / mL) for 4 h, respectively. Subsequently, the mitochondria and nuclei of IEC-6 cells were labeled with Mito-Tracker Red (100 nmol / L) and Hoechst 33342 (10 μg / mL). Finally, the Pearson correlation coefficient (R) was calculated to determine the degree of colocalization of Mito-Cur with mitochondria.

[0066] Results Figure 3, Mito-Cur and Cur-TPP-treated cells showed stronger green fluorescence than Cur-treated cells. Meanwhile, the green fluorescence overlapped with the red fluorescence of mitochondria to produce bright orange fluorescence. Surprisingly, the green fluorescence intensity in Mito-Cur-treated cells was higher than that in Cur-TPP-treated cells. The R values ​​after incubation with Cur, Mito-Cur, and Cur-TPP were 0.69, 0.84, and 0.76, respectively. After Cur-TPP was incubated with IEC-6 cells pretreated with TPP, the R value decreased from 0.76 to 0.73, indicating that the preferential binding of TPP to mitochondria can reduce the accumulation of covalent Cur-TPP in mitochondria. Mito-Cur showed stronger mitochondrial targeting ability (R = 0.88), emitting bright orange fluorescence after overlapping with the red fluorescence of mitochondria. These results indicate that TPP modification can improve the mitochondrial targeting ability of Cur, and the mitochondrial targeting ability of zwitterionic Mito-Cur formed by electrostatic coupling is better than that of Cur-TPP formed by covalent bonds. This is because the electrostatic coupling between anionic Cur-SO3 and cationic TPP is a reversible non-covalent binding mode. This means that the electrically neutral zwitterionic Mito-Cur molecules can undergo decomposition behavior in different physiological environments. A large number of studies have shown that the mitochondrial membrane potential of IEC-6 cells is significantly reduced under oxidative stress conditions. When the mitochondrial membrane potential drops to a level close to the charge carried by anionic Cur-SO3, it may provide favorable conditions for the decomposition of zwitterionic Mito-Cur. Subsequently, anionic Cur-SO3 is enriched in mitochondria driven by cationic TPP and negatively charged mitochondria. This process helps to further improve the efficiency of TPP-modified Cur molecules targeting mitochondria based on the traditional covalent bond strategy.

[0067] Example 2

[0068] The present invention discloses a mitochondrial targeting group-modified active compound Mito-AXT, which is prepared by the following synthetic route:

[0069]

[0070] AXT (12 mg, 0.02 mmol) and NaHSO3 (2.08 mg, 0.02 mmol) were dissolved in 2 mL of tetrahydrofuran and distilled water, respectively; then the AXT and NaHSO3 solutions were mixed and stirred at 50°C (under nitrogen atmosphere) for 2 h; AXT-SO3 was separated by centrifugation at 6000 rpm for 10 min; AXT-SO3 (13.65 mg, 0.02 mmol) and TPP (8.59 mg, 0.02 mmol) were dissolved in 2 mL of DMSO and stirred continuously at 25°C for 2 h; then dialyzed in distilled water for 48 h (molecular weight cutoff MW 1000 Da) to obtain the reaction product Mito-AXT with a yield of 86.17%.

[0071] pass 1 H NMR spectroscopy was used to analyze the changes in proton signals during the synthesis of Mito-AXT. Figure 4 The proton signal of C=C of AXT molecule at 5.06ppm(b) is 1 The disappearance of sulfonate in the H NMR spectrum is attributed to the addition of sulfonate during the electrophilic addition reaction. Subsequently, the electron cloud density decreases and the proton signal of -CH3 moves from 3.31 ppm (a) to 3.47 ppm (a') upfield. Figure 5 , in Mito-AXT 1 The characteristic proton signals of cationic TPP at 12.34 ppm (HA) and 8.10-7.60 ppm (HB) and anionic AXT-SO3 at 6.90-6.12 ppm (HC) and 1.40-1.10 ppm (HD) were also observed in the H NMR spectrum, confirming the formation of zwitterionic Mito-AXT.

[0072] The effect of the AXT-SO3 synthesis process on the antioxidant activity of AXT was analyzed by ESR spectroscopy. Figure 6 , anionic AXT-SO3 showed no significant difference from AXT in terms of DPPH and hydroxyl radical capturing ability, which means that the synthesis of anionic AXT-SO3 by electrophilic addition does not affect the intrinsic antioxidant activity of AXT.

[0073] The changes in the surface element composition during the synthesis of Mito-AXT were analyzed by EDS spectroscopy. Figure 7 The characteristic S element was also detected in the anionic AXT-SO3, and the characteristic P element was also detected in the zwitterionic Mito-AXT, with contents of 3.88% and 1.84%, respectively, confirming the successful synthesis of AXT-SO3 and Mito-AXT.

[0074] XPS spectroscopy was used to analyze the changes in elemental composition during the synthesis of Mito-AXT. Figure 8 , the peak at 152.02eV in the AXT-SO3 spectrum reveals the presence of sulfur in anionic AXT-SO3. The characteristic elements of AXT are C and O, with contents of 85.64% and 14.36%, respectively; while the characteristic elements of AXT-SO3 include C, O and S, with contents of 83.91%, 15.34% and 0.75%, respectively. In the XPS spectrum of Mito-AXT, the peak at 132.08eV is attributed to the presence of phosphorus.

[0075] The potential changes during the synthesis of Mito-AXT were analyzed by a potentiostat. Fig. 9 After the anion AXT-SO3 (-31.9 mV) combined with the cation TPP (19.4 mV), the Zeta potential of Mito-AXT changed to 2.1 mV, indicating that electrostatic coupling occurred during the formation of the zwitterionic Mito-AXT.

[0076] The changes in the optical properties of Mito-AXT during its synthesis were analyzed by UV-visible spectroscopy. Fig.10 The UV-visible spectra of AXT and AXT-SO3 show typical maximum absorption peaks at 491nm. The absorption peak of the UV-visible spectrum of TPP at 268nm is attributed to the π→π* electronic transition of the aromatic group. The UV-visible spectrum of Mito-AXT shows characteristic absorption peaks of AXT-SO3 and TPP, further illustrating the formation of zwitterionic Mito-AXT.

[0077] The protective effect of Mito-AXT on H2O2-induced oxidative stress was analyzed by ROS staining. Fresh culture medium containing AXT, Mito-AXT, and AXT-TPP was added to IEC-6 cells and incubated for 24 h (the concentration of AXT was 40 μg / mL). Subsequently, 1 mL of culture medium containing 1200 μmol / L H2O2 was added and incubated for 3 h. After removing the culture medium, ROS staining was performed with DCFH-DA (10 μg / mL).

[0078] Results Fig.11, a large amount of reactive oxygen species was generated after incubation with H2O2, and strong green fluorescence was emitted. The green fluorescence intensities of Mito-AXT-treated cells and AXT-TPP-treated cells were lower than those of AXT-treated cells, indicating that TPP-modified AXT can effectively inhibit H2O2-induced ROS generation and prevent the occurrence of oxidative stress in IEC-6 cells. It is worth noting that the inhibitory effect of zwitterionic Mito-AXT on ROS generation was more obvious than that of covalent AXT-TPP. After incubation with Mito-AXT and AXT-TPP, the green fluorescence intensity was 1.3 times and 3.4 times that of the control group, respectively, which can be attributed to the improved mitochondrial targeting ability of zwitterionic Mito-AXT in IEC-6 cells. These results indicate that zwitterionic Mito-AXT formed by electrostatic coupling of anionic AXT-SO3 and cationic TPP can enhance the protective effect of AXT against H2O2-induced oxidative stress.

[0079] The regulatory effect of Mito-AXT on mitochondrial homeostasis in IEC-6 cells was analyzed by mitochondrial membrane potential staining. Fresh culture medium containing AXT, Mito-AXT, and AXT-TPP was added to IEC-6 cells and incubated for 24 h (the concentration of AXT was 40 μg / mL). Subsequently, 1 mL of culture medium containing 1200 μmol / L H2O2 was added and incubated for 3 h. After removing the culture medium, mitochondrial aggregates and monomers were stained with JC-1.

[0080] Results Fig.12 , after H2O2 treatment, a large number of JC-1 aggregates disappeared significantly, and the red fluorescence intensity was significantly weakened. At the same time, JC-1 monomers showed strong green fluorescence in IEC-6 cells. These results confirmed the disruption of mitochondrial homeostasis caused by oxidative stress, resulting in a decrease in MMP. Unfortunately, there was no significant difference in the fluorescence intensity ratio of JC-1 aggregates (red) / JC-1 monomers (green) in AXT-treated cells compared with H2O2-treated cells, indicating that free AXT had limited effect in restoring MMP under oxidative stress. Fortunately, after Mito-AXT and AXT-TPP treatment, the ratio of JC-1 aggregates / JC-1 monomers increased by 337.1% and 146.8%, respectively. In Mito-AXT-treated cells, the ratio of JC-1 aggregates / JC-1 monomers was 1.77 times that of AXT-TPP-treated cells. These results indicate that zwitterionic Mito-AXT can better protect mitochondrial homeostasis in IEC-6 cells under oxidative stress.

[0081] The regulatory effect of Mito-AXT on mitochondrial function of IEC-6 cells was analyzed by fluorescence colocalization of autophagosomes and mitochondria. Fresh culture medium containing AXT, Mito-AXT, and AXT-TPP was added to IEC-6 cells and incubated for 24 h (the concentration of AXT was 40 μg / mL). Subsequently, 1 mL of culture medium containing 1200 μmol / L H2O2 was added and incubated for 3 h. After removing the culture medium, the autophagosomes were stained with MDC.

[0082] Results Fig.13 , cells treated with H2O2 showed weak green fluorescence from autophagosomes, which may be due to the fact that excessive ROS inhibited the selective degradation of damaged mitochondria by IEC-6 cells. After incubation with AXT, IEC-6 cells showed weak green fluorescence, which was not significantly different from that with H2O2. In contrast, obvious green fluorescence was observed in cells treated with zwitterionic Mito-AXT and AXT-TPP, which overlapped significantly with the red fluorescence of mitochondria, resulting in strong orange fluorescence. The results showed that mitochondrial targeted regulation was conducive to the occurrence of mitophagy under oxidative stress, which was also the main reason for the reduction of ROS generation in oxidative stress cells incubated with Mito-AXT and AXT-TPP. Compared with cells treated with zwitterionic Mito-AXT, the fluorescence intensity of autophagosomes in cells treated with covalent AXT-TPP was reduced by 27.1%. These results indicate that zwitterionic Mito-AXT can effectively regulate the homeostasis of mitochondria under oxidative stress by promoting the occurrence of mitophagy.

[0083] The regulatory effect of Mito-AXT on mitochondrial homeostasis of IEC-6 cells was analyzed by cell apoptosis detection. Fresh culture medium containing AXT, Mito-AXT, and AXT-TPP was added to IEC-6 cells and incubated for 24 hours (the concentration of AXT was 40μg / mL). Subsequently, 1mL of culture medium containing 1200μmol / L H2O2 was added and incubated for 3 hours. After trypsin digestion of cells, cells were stained with Annexin V-FITC / PI, and cell apoptosis was detected by flow cytometry.

[0084] Results Fig.14, after H2O2 treatment, the proportions of live cells, early apoptotic cells, late apoptotic cells, and necrotic cells were 42.9%, 0.08%, 19.00%, and 38.10%, respectively, indicating that oxidative stress mainly induced late apoptosis and necrosis in IEC-6 cells. Compared with other treatments, Mito-AXT had the best intervention effect. After Mito-AXT treatment, the proportions of early apoptotic cells, late apoptotic cells, and necrotic cells decreased to 2.14%, 0.025%, and 18.00%, respectively. At the same time, the proportion of live cells increased to 79.80%. These results indicate that Mito-AXT can more effectively alleviate oxidative stress-induced cell apoptosis.

[0085] The above-mentioned specific implementation methods further illustrate the objectives, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for constructing a mitochondrial targeting product, characterized in that: The mitochondrial targeting product is obtained by utilizing the electrostatic coupling between negatively charged anionic active compounds and positively charged lipophilic cationic mitochondrial targeting molecules; The structure of the negatively charged anionic active compound is A-X1-B, wherein X1 is a sulfonate, and A is derived from the following active compounds: β-carotene, lycopene, lutein, astaxanthin, capsanthin, zeaxanthin, fucoxanthin, curcumin, piperine, or any isomer of the above active compounds.

2. The method according to claim 1, characterized in that The positively charged lipophilic cationic mitochondrial targeting molecule is (3-propylcarboxy)triphenylphosphonium bromide.

3. The method according to claim 1, characterized in that The method comprises: S1, the active compound first undergoes an electrophilic addition reaction with NaHSO3 to synthesize a negatively charged anionic active compound; S2. The prepared negatively charged anionic active compound is then mixed with the positively charged cationic mitochondrial targeting molecule to undergo electrostatic coupling, and the mixture is stirred at room temperature, dialyzed, and freeze-dried to obtain a mitochondrial targeting product.

4. The method according to claim 3, characterized in that The S1 specifically includes: Under an inert atmosphere, the active compound and NaHSO3 are dissolved in an organic reagent and deionized water, respectively, the two are mixed, and reacted under heating and stirring conditions, and the negatively charged anionic active compound is collected by centrifugation and freeze drying.

5. The method according to claim 3, characterized in that: The molar ratio of the active compound to NaHSO3 is 1:1 to 1:1.

05.

6. The method according to claim 4, characterized in that The organic reagent for dissolving the active compound is one of tetrahydrofuran, ethylene glycol methyl ether, methanol, ethanol and deionized water; the heating temperature is 50-70°C; the reaction time is 1-2h; the centrifugal speed is 6000-8000rpm, and the time is 10-20min.

7. The method according to claim 3, characterized in that In S2, the molar ratio of the negatively charged anionic active compound to the positively charged cationic mitochondrial targeting molecule is 1:1 to 1:1.5; the stirring time at room temperature is 1 to 3 hours; the dialysis cut-off molecular weight is 500 to 1000 Da, and the dialysis time is 24 to 48 hours.

8. A mitochondrial targeting product constructed by the method according to any one of claims 1 to 7.

9. A mitochondrial targeting product, the structure of which is shown below: Wherein A is derived from the following active compounds: β-carotene, lycopene, lutein, astaxanthin, capsanthin, zeaxanthin, fucoxanthin, curcumin, piperine, or any isomer of the above active compounds, It is a positively charged lipophilic cationic mitochondrial targeting structure.

10. Use of the mitochondrial targeted product according to claim 8 or 9 in the preparation of drugs for diseases related to mitochondrial homeostasis.

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