Plant source dry mitochondria as well as preparation method and application thereof

Abstract

The invention relates to plant source dry-state mitochondria for cell repair and aging resistance as well as a preparation method and application of the plant source dry-state mitochondria. The plant source dry-state mitochondria can effectively improve functions of mitochondria, reduce the damage degree of cells, inhibit cell aging and effectively repair skin wounds. According to the method, the mitochondria is extracted from healthy plant tissues, so that the production cost is reduced, and a harsh quarantine step is not needed. According to the present invention, the dry-state processing enables the mitochondria storage condition to be loose, the transportation and storage difficulty and the transportation and storage cost can be further reduced, the plant source mitochondria subjected to the dry-state processing has characteristics of flexible dosage form, wide auxiliary material application range, and meeting of the design requirements of various products, and the dry-state processing can further remove the nucleic acid in the extract and eliminate the inflammation of the product; in the research process, the inventor also finds that after the plant source mitochondria is subjected to dry-state processing, the mitochondria function can be more effectively improved, the cell damage degree is reduced, cell aging is inhibited, and the skin wound surface is effectively repaired.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a plant-derived dry mitochondria for cell repair and anti-aging, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the energy production center within cells, are crucial for maintaining cellular function. They produce adenosine triphosphate (ATP) through oxidative phosphorylation, providing the necessary energy for various cellular biochemical reactions and maintaining the normal physiological functions of skin cells. Oxidative stress is a key factor in skin aging, directly affecting skin health. With age, cellular oxidative stress levels significantly increase, impairing mitochondrial function, leading to reduced ATP production, which in turn reduces the proliferation and repair capacity of skin cells, ultimately accelerating skin aging.

[0003] In recent years, fresh animal-derived mitochondria delivery has emerged as a novel cell repair strategy. Multiple studies have shown that mitochondria from healthy animals can enhance mitochondrial function, regulate cellular energy metabolism by restoring ATP levels in aging cells, promote cell proliferation, migration, and differentiation, thereby improving skin cell vitality, enhancing the structure and function of aging skin, and slowing down the skin aging process. This demonstrates the significant potential of mitochondria in repairing cell damage and combating aging.

[0004] Currently, fresh animal-derived mitochondria have been proposed as an effective ingredient for treating skin aging. They can improve skin aging characteristics in several ways: First, they can effectively repair mitochondrial function, improve ATP production, and regulate cellular energy metabolism. They can also prevent DNA and protein damage by reducing oxidative stress levels, while enhancing skin cell vitality, promoting cell proliferation and migration, and improving skin regeneration.

[0005] However, fresh animal-derived mitochondria often need to be extracted from fresh animal tissues, resulting in high production costs. The animal tissues used as raw materials may contain pathogenic microorganisms, thus requiring stringent quarantine requirements and significant expenditure on animal quarantine. Even so, it is still possible that pathogenic microorganisms cannot be completely eliminated; for example, some mitochondria from cadaveric xenografts still pose a risk of disease transmission, and the nucleic acids contained in the extracts may cause inflammatory responses. Furthermore, fresh animal-derived mitochondria need to maintain good activity during transportation and storage, requiring stringent preservation conditions, leading to high supply chain costs and stringent requirements for excipients. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a plant-derived dry mitochondria for cell repair and / or anti-aging, its preparation method and application, to solve the problems of high production cost, high quarantine requirements and potential disease transmission risks of existing fresh animal-derived mitochondria.

[0007] The technical solution to achieve the above objectives is as follows.

[0008] The first aspect of the present invention is to provide a method for preparing plant-derived dry mitochondria for cell repair and / or anti-aging, comprising the following steps: S1 Selecting healthy plant tissue, grinding, centrifuging to separate, and obtaining a precipitate;

[0009] The precipitate obtained by treating S2 with nuclease yielded plant-derived mitochondria.

[0010] S3 processes plant-derived mitochondria in a dry state to obtain plant-derived dry mitochondria.

[0011] In some embodiments, the dry processing is freeze-drying, critical point drying, drying in an inert gas environment, or natural air drying, preferably freeze-drying.

[0012] In some of these embodiments, the centrifugation separation involves centrifuging the ground healthy plant tissue at 1000 rcf-8000 rcf for 5 min-20 min, and then at 11000 rcf-21000 rcf for 5 min-20 min.

[0013] In some preferred embodiments, the centrifugation separation involves centrifuging the ground healthy plant tissue at 3000 rcf-5500 rcf for 10 min-15 min, and then centrifuging at 15000 rcf-18000 rcf for 10 min-15 min.

[0014] In some of the embodiments, the precipitate obtained by nuclease treatment includes adding water and nuclease to the precipitate, with an enzyme activity concentration of 25 U / ml-50 U / ml, reacting at 20℃-40℃ for 15 min-60 min, terminating the reaction, and centrifuging at high speed to obtain the plant-derived mitochondria.

[0015] In some of these embodiments, the reaction was terminated by adding an EDTA solution with a concentration of 1 mM to 10 mM.

[0016] In some of these embodiments, the aforementioned high-speed centrifugation is performed at a rotation speed of 11000 rcf-21000 rcf for 5 min-20 min.

[0017] The plant tissue can be petals, pulp, leaves, fleshy stems, etc.

[0018] In some of these embodiments, the healthy plant tissue is healthy tissue from plants of the genera Rosa, Solanum, Aloe, Zingiber, Centella, Coreopsis, or Calendula.

[0019] In some preferred embodiments, the healthy plant tissue is healthy tissue from plants of the genus Aloe or Zingiber.

[0020] In some preferred embodiments, the freeze-drying includes adding water to plant-derived mitochondria, mixing well, freezing into ice blocks at below 0°C, transferring the ice blocks to a freeze dryer, and completely drying them under conditions of vacuum ≤0.1 mBar and cold trap temperature ≤-40°C to obtain plant-derived dry mitochondria.

[0021] In some preferred embodiments, the mass ratio of water added to the plant-derived mitochondria is 1:1 to 1:5.

[0022] A second aspect of the present invention is to provide a plant-derived dry mitochondria prepared by the aforementioned method.

[0023] A third aspect of the present invention is to provide the application of the aforementioned plant-derived dry mitochondria in the preparation of products for repairing cellular mitochondrial damage.

[0024] A fourth aspect of the present invention is to provide the use of the aforementioned plant-derived dry mitochondria in the preparation of products for repairing cell damage and / or anti-aging of the skin.

[0025] A fifth aspect of the present invention is to provide a cell repair and / or anti-aging product, the active ingredient of which includes the above-mentioned plant-derived dry mitochondria, and pharmaceutically acceptable excipients.

[0026] The product may be a drug, which is formulated into a stable and controllable dosage form by matching different excipients as needed, for the treatment of functional cell disorders caused by skin damage due to natural aging or exogenous factors.

[0027] During the research, the inventors discovered that after removing nucleic acids from plant-derived mitochondria and combining them with dry processing, the resulting plant-derived dry mitochondria can more effectively improve mitochondrial function, reduce the degree of cell damage, inhibit cell aging, and effectively repair skin wounds.

[0028] The plant-derived dry mitochondria prepared in this invention also have the following advantages:

[0029] Mitochondria can be extracted from healthy plant tissues. Compared to animal tissues, plant tissues are easier to obtain, reducing production costs. In addition, plant tissues do not pose a risk of disease and do not require stringent quarantine procedures, making them more widely applicable.

[0030] Dry processing allows for more flexible mitochondrial preservation conditions, further reducing the difficulty and cost of transportation and storage. The plant-derived mitochondrial formulations after dry processing are flexible, have a wide range of applicable excipients, and can meet the needs of various product designs. In addition, dry processing can further remove nucleic acids from the extract and eliminate the inflammatory properties of the product. Attached Figure Description

[0031] Figure 1 It is the improvement of intracellular mitochondrial function by exogenous mitochondria.

[0032] Figure 2 It is the regulation of the degree of cell damage by exogenous mitochondria.

[0033] Figure 3 It is the regulation of intracellular aging marker expression by exogenous mitochondria.

[0034] Figure 4 It is the regulation of rat tissue aging by exogenous mitochondria.

[0035] Figure 5 It is the repair of rat wound tissue by exogenous mitochondria.

[0036] Figure 6 It describes the uptake of dry plant-derived mitochondria by senescent cells before and after nucleic acid removal.

[0037] Figure 7 It represents the particle size change of mitochondria from dry plant sources before and after the removal of nucleic acids.

[0038] Figure 8 It refers to the change in DNA concentration after removing nucleic acids using enzyme preparations of different concentrations.

[0039] Figure 9 This study investigates the effect of different water addition ratios during freeze-drying on mitochondrial quality. Detailed Implementation

[0040] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0041] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0042] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0043] In this article, the enzyme concentration of 25 U / ml refers to the activity concentration of the enzyme preparation solution, that is, each milliliter of solution can hydrolyze 25 micromoles of nucleic acid substrate per minute under standard conditions.

[0044] The present invention will be further described in detail below with reference to specific embodiments.

[0045] Example 1: Preparation of plant-derived dry mitochondria

[0046] Healthy plant tissues: petals of rose (Rosa), pulp of potato (Solanum melanoxylon), leaves of aloe (Aloe vera), fleshy stems of ginger (Zingiber officinale), leaves of centella asiatica (Centella asiatica), petals of snow chrysanthemum (Coreopsis grandiflora), and petals of calendula (Calendula officinalis).

[0047] S1. Take healthy plant tissues separately, add protease inhibitors (Merck's EDTA-free cOmplete™ Mini protease inhibitor mixture, one inhibitor tablet per 10 ml of extract) to prevent the target substance from being hydrolyzed by proteases, grind thoroughly, filter out larger tissue fragments, and obtain the extract. Centrifuge at 5000 rcf for 15 min to remove impurities. Then centrifuge at 16000 rcf for 15 min to obtain mitochondrial precipitate.

[0048] S2. Deionized water and nuclease (the main nuclease selected is the Benzonase® series nuclease, such as Benzonase® Nuclease from Merck, catalog number E1014-25KU) are mixed at a volume ratio of 9:1 to prepare the enzyme preparation. The enzyme preparation is added to the mitochondrial precipitate to the enzyme activity concentration of 25 U / ml-50 U / ml, mixed well, and reacted at 37℃ for 40 min to remove excess nucleic acid. The reaction is terminated by adding an equal volume of 5 mM EDTA solution. The mixture is centrifuged at 16000 rcf for 15 min. The resulting precipitate is the plant-derived mitochondria (Mito).

[0049] The process for screening enzyme concentrations is as follows:

[0050] Enzyme preparations of different concentrations (5 U / ml-50 U / ml) were added to 40 μg of mitochondrial precipitate and mixed well. The mixture was reacted at 37°C for 40 min to remove excess nucleic acid. The reaction was then terminated by adding an equal volume of 5 mM EDTA solution. The mixture was centrifuged at 16000 rcf for 15 min to obtain the precipitate. The DNA content in the precipitate was detected using the dsDNA HS assay kit (Yeasen, catalog number: 12640ES60). The DNA content of 40 μg of untreated mitochondrial precipitate was used as the control group.

[0051] Test results as follows Figure 8 Based on the DNA concentration, the final enzyme concentration was selected as 25 U / ml-50 U / ml.

[0052] S3. Add water to plant-derived mitochondria (Mito), mix well, freeze into ice blocks below 0℃, transfer to a freeze dryer, and completely dry them under conditions of vacuum degree ≤0.1 mBar and cold trap temperature ≤-40℃ to obtain plant-derived dry mitochondria (LyoMito).

[0053] We found that the amount of water added affects mitochondrial quality. Mitochondrial precipitate was mixed with water at a ratio of mitochondrial mass (μg):water volume (μl) of 1:1 / 1:5 / 1:10 / 1:20, and then frozen at below 0°C. The mixture was then transferred to a freeze dryer and completely dried under vacuum ≤0.1 mBar and cold trap temperature ≤-40°C to obtain plant-derived dry mitochondria (LyoMito). The mitochondrial mass (mass of the powder after dry treatment) of different proportions was then measured to observe whether any loss occurred during the drying process.

[0054] Test results as follows Figure 9 As shown, the water ratio added to plant-derived mitochondria was ultimately selected to be 1:1 to 1:5 by mass.

[0055] The plant-derived mitochondria (Mito) and plant-derived dry mitochondria (LyoMito) used in the following examples are all derived from this example.

[0056] Example 2: Improvement of intracellular mitochondrial function by exogenous mitochondria

[0057] 1. Source of raw materials

[0058] 1.1 Animal-derived mitochondria (Mito-animal-derived) are derived from rat adipose tissue, and the preparation method is as follows:

[0059] Select adipose tissue from healthy animals, grind it, and centrifuge at 1000-8000 rcf for 15 min. Add mitochondrial isolation reagent A (from a tissue mitochondrial isolation kit, manufacturer: Beyotime, product number C3606, 100 μL per 10 mg of tissue) and homogenize thoroughly. Centrifuge at 400-800 rcf for 4 min, and take the intermediate clear liquid and centrifuge at 11000-21000 rcf for 15 min to obtain mitochondrial precipitate.

[0060] 1.2 The experimental cells were derived from healthy rat fibroblasts.

[0061] 2. Experimental Methods

[0062] 2.1 Grouping

[0063] This experiment was divided into five main groups: Control group, Sen group, Mito-animal source group, Mito-plant source group, and LyoMito-plant source group. The Mito-plant source group and LyoMito-plant source group were further divided into seven subgroups: Rosa group, Solanum group, Aloe group, Ginger group, Centella asiatica group, Coreopsis group, and Calendula group.

[0064] 2.2 Operation

[0065] (1) Induction of senescent cells: D-galactose (product number G100367, manufacturer: Aladdin) was added to high-glucose DMEM medium (product number C11995500BT, manufacturer: Gibco, USA) containing 10% FBS and 1% penicillin antibiotics to prepare high-glucose DMEM medium with a D-galactose content of 40 mg / ml (hereinafter referred to as D-galactose-containing medium). Senescent rat fibroblasts were obtained by co-incubating healthy rat fibroblasts with D-galactose-containing medium for 48 h.

[0066] (2) Cell seeding: The cells were resuspended in the culture medium and seeded in 24-well plates, with 20,000 cells seeded in each well. The Control group was seeded with healthy rat fibroblasts in high-glucose DMEM medium, while the Sen group, Mito-animal source group, Mito-plant source group (a total of seven groups), and LyoMito-plant source group (a total of seven groups) were seeded with aged rat fibroblasts in D-galactose-containing medium.

[0067] (3) Mitochondria co-incubation: The Mito-animal source group, Mito-plant source group and LyoMito-plant source group were co-incubated with cells at 37°C for 48 h with mitochondria of the corresponding source. The incubation concentration was 40 μg / ml (40 μg of mitochondria were added to each ml of cell culture medium).

[0068] (4) Staining and imaging: After fixing the cells, stain them with MitoTracker Red. After 30 minutes, image them under a confocal microscope. The imaging results are as follows: Figure 1 As shown.

[0069] Under normal circumstances, mitochondrial function is maintained at a certain level, which helps maintain mitochondrial structural integrity, ATP synthesis, and cellular respiration. However, when cells are damaged or undergo physiological changes, mitochondrial function decreases. Lower levels of mitochondrial function may be associated with impaired mitochondrial function, apoptosis, and other abnormal physiological states. Under fluorescence imaging, mitochondria with higher levels of function appear as a deeper red. Figure 1 It can be seen that the Sen group had the lowest red fluorescence intensity, indicating the weakest mitochondrial function. Compared with the Sen group, the Mito-animal source group, the Mito-plant source group, and the LyoMito-plant source group showed significantly stronger red fluorescence, and the LyoMito-plant source group showed the best effect, indicating that LyoMito may restore mitochondrial function in senescent cells to some extent and has the best therapeutic effect.

[0070] Example 3: Regulation of cell damage by exogenous mitochondria

[0071] Except for the staining and imaging steps, everything else is the same as in Example 2. In this example, DCFH-DA was used to stain the cells, and imaging was performed under a confocal microscope 30 minutes later. The imaging results are as follows. Figure 2 As shown.

[0072] The degree of cell damage is closely related to cellular senescence; the higher the degree of cell damage, the deeper the green color in fluorescence imaging. Intracellularly, increased oxidative stress can cause damage to intracellular proteins, lipids, and nucleic acids, leading to mitochondrial and cellular dysfunction and structural damage, thereby affecting physiological processes such as cell growth, proliferation, and apoptosis, and accelerating the process of cellular senescence. Figure 2 As can be seen, the Sen group exhibited the highest green fluorescence intensity, indicating the highest degree of cell damage. Compared to the Sen group, the Mito-animal-derived group, the Mito-plant-derived group, and the LyoMito-plant-derived group showed significantly weaker green fluorescence, with the LyoMito-plant-derived group showing the best effect. This suggests that LyoMito may have repaired cell damage to some extent, and with the best repair effect.

[0073] Example 4: Regulation of intracellular aging marker expression by exogenous mitochondria

[0074] Except for the staining and imaging steps, everything else was the same as in Example 2. In this example, SA-β-Gal was used to stain the cells, and images were taken under an optical microscope after overnight staining. The imaging results are as follows. Figure 3 As shown.

[0075] SA-β-Gal (senescence-associated β-galactosidase) is a commonly used biomarker for detecting cellular senescence. SA-β-Gal primarily appears blue in senescent cells, while showing no significant expression in young cells; therefore, it can be used for the visual detection of senescent cells. Figure 3 It can be seen that the Control group had almost no blue cells, indicating that it was in a non-senescent state. The Sen group showed a large number of blue cells, indicating that most cells were in a senescent state. Compared with the Sen group, the Mito-animal source group, Mito-plant source group, and LyoMito-plant source group had significantly fewer blue cells, and the LyoMito-plant source group showed the best effect, indicating that LyoMito may inhibit cell senescence to a certain extent, and the inhibitory effect is the best.

[0076] Example 5: Regulation of tissue aging in rats by exogenous mitochondria

[0077] The mitochondrial source is the same as in Example 2.

[0078] Grouping: The Control group consisted of young, healthy rats, while the Sen, Mito-animal, Mito-plant, and LyoMito-plant groups consisted of aged rats. The Mito-plant and LyoMito-plant groups were further divided into seven subgroups: Rosa, Solanum, Aloe, Zingiber, Centella, Coreopsis, and Calendula.

[0079] The specific experimental procedure is as follows:

[0080] (1) Mitomitochondria of the corresponding origin were subcutaneously injected into the back of aged rats in the Mito-animal group, Mito-plant group (seven groups in total), and LyoMito-plant group (seven groups in total). The injection concentration was 40 μg / ml. The injection frequency was once every 3 days.

[0081] (2) The above rats were raised under the same conditions as the Control group and Sen group for 10 days and then sacrificed. The back wound tissue was removed with scissors.

[0082] (3) Preparation of frozen sections: The excised tissue was washed twice in PBS to remove excess blood, placed in cryovials and flash-frozen in liquid nitrogen. After being taken out of the dissection room, it was stored in a freezer at -80°C. On the day of embedding, OCT embedding medium was added to a disposable plastic embedding box to eight-tenths full. The tissue was immersed in the embedding medium with forceps, with the cut surface of the tissue in close contact with the bottom of the embedding medium. The tissue was flash-frozen in liquid nitrogen until the embedding medium turned completely white. Frozen tissue sections with a thickness of 10 μm were prepared using a cryostat. The sections could be temporarily stored in a freezer at -80°C.

[0083] (4) Warming and washing: Take the frozen sections out of the -80℃ freezer and warm them for 15 min. Wash them in PBS 3 times for 5 min each time to remove excess OCT embedding agent.

[0084] (5) Fixation, staining and observation: The tissue was fixed with fixative for 15 min at room temperature, and then stained with SA-β-Gal overnight. After staining, the tissue was mounted with anti-fluorescence quenching mounting medium and observed under an optical microscope. The imaging results are as follows. Figure 4 As shown.

[0085] Similar to cell experiments, the inventors stained frozen sections of fresh tissue with SA-β-Gal to assess the regulatory effects of Mito and LyoMito on rat tissue senescence. The areas stained blue in the image represent SA-β-Gal positive regions, which are considered to be areas of senescent tissue. Figure 4 It can be seen that the blue area is the largest in the Sen group. The proportion of blue area in the Mito-animal source group, Mito-plant source group and LyoMito-plant source group is significantly lower than that in the Sen group, and the decrease is more obvious in the LyoMito-plant source group. Its proportion of blue area is close to that of the Control group (normal tissue), indicating that LyoMito can most effectively inhibit the aging of rat tissues.

[0086] Example 6: Repair of rat wound tissue by exogenous mitochondria

[0087] The mitochondrial origin and grouping were the same as in Example 5.

[0088] The specific experimental procedure is as follows:

[0089] (1) Mito-animal-derived rats from the Mito-plant-derived group (seven subgroups in total) and the LyoMito-plant-derived group (seven subgroups in total) were subcutaneously injected into the back of their rats with mitochondria of the corresponding origin. The concentration of mitochondria injected was 40 μg / ml. The injection frequency was once every 3 days.

[0090] (2) The above rats were raised under the same conditions as the Control group and Sen group for 10 days and then sacrificed. The back wound tissue was removed with scissors.

[0091] (3) Preparation of paraffin sections: Place the tissue into a paraffin embedding cassette and mark it with a pencil. Place it in 4% paraformaldehyde and fix it in a 4℃ refrigerator for 24 h. Rinse thoroughly with tap water for 15 min to remove excess paraformaldehyde. Fix the tissue in the embedding cassette and place it in the cylinder of a dehydrator. Select the set program and perform automatic dehydration. Embed the tissue using a paraffin embedding machine. The embedded tissue paraffin blocks can be stored at room temperature. When sectioning, fix the paraffin block on the sample head. After trimming, set the section thickness to 6 mm. Use a brush and tweezers to gently spread the section on the water surface of the slide spreader. Gently insert the glass slide under the section and lift the glass slide obliquely upwards to allow the section to adhere to the glass slide. Bake the glass slide in a 65℃ oven for 3 h to allow the tissue to adhere to the glass slide.

[0092] (4) Dewaxing and rehydration: After dewaxing and rehydration, the paraffin sections are soaked in the following solutions in sequence: anhydrous ethanol for 5 min → 90% ethanol for 2 min → 70% ethanol for 2 min → 50% ethanol for 2 min → distilled water for 2 min.

[0093] (5) H&E staining: After staining the sections with hematoxylin staining solution for 5 minutes, remove them and soak them in tap water for 10 minutes to wash away excess staining solution. Wash them again with distilled water, differentiate them with hydrochloric acid ethanol differentiation solution for 2 seconds, rinse them with tap water for 10 minutes, and stain them with eosin staining solution for 30 seconds.

[0094] (6) Dehydration and clearing: The stained sections were soaked in the following solutions in sequence: 70% ethanol for 10 seconds → 80% ethanol for 10 seconds → 90% ethanol for 10 seconds → anhydrous ethanol for 10 seconds → xylene clearing for 5 minutes → replace with fresh xylene and clear for another 5 minutes.

[0095] (7) Mounting and observation: Mount the slides with neutral resin. Scan the slides with a pathological slide scanner. The results are as follows: Figure 5 As shown.

[0096] The degree of wound repair is directly related to skin thickness. Skin at the wound site is thinner due to impaired cell function, while the skin at better repair sites is thicker. H&E staining can reveal extensive cytoplasmic, nuclear, and extracellular matrix characteristics, and in this experiment, it was used to investigate the extent of mitochondrial repair in rat wound tissue. Figure 5 As can be seen from the H&E staining analysis of the wound tissue, new skin tissue was generated in the Mito-animal group, Mito-plant group and LyoMito-plant group. Moreover, the skin thickness of the LyoMito-plant group was close to that of the Control group (normal tissue), indicating that LyoMito can most effectively repair rat skin wounds.

[0097] Example 7: Uptake of dry plant-derived mitochondria by senescent cells before and after nucleic acid removal.

[0098] 7.1 Experimental Objective

[0099] Plant-derived mitochondria have a much larger molecular weight than animal-derived mitochondria, mainly because the genome in plant-derived mitochondria is much larger. Therefore, during our experiments, we hypothesized that the large amount of nucleic acids in plant-derived mitochondria might affect the ability of senescent cells to take up mitochondria, and thus conducted endocytosis experiments to verify this hypothesis.

[0100] 7.2 Experimental Procedure

[0101] (1) Plant-derived mitochondria were extracted according to the method in Example 1. The extracted fresh plant-derived mitochondria were stained with mitotracker-red (manufacturer Beyotime, product number C1035) at 37°C for 30 min. They were then divided into two groups for dry treatment. One group had nucleic acid removed, while the other group did not have the removal operation performed.

[0102] (2) Senescent rat fibroblasts were seeded on confocal dishes, with 50,000 cells seeded per cell. After the number of mitochondria in the two groups was determined by flow cytometry, the cells were fed at a ratio of 1:30 to the number of particles, and the mitochondria and senescent rat fibroblasts were co-incubated for 24 hours to allow the cells to fully take up the mitochondria.

[0103] (3) Finally, the cells were imaged using a confocal microscope, where green represents the cytoskeleton and red represents mitochondria. The group with nucleic acid not removed was named the LyoMito+mtDNA group (the preparation method does not include step S2 of Example 1), and the group with nucleic acid removed was named the LyoMito group (the preparation method includes step S2 of Example 1).

[0104] 7.3 Experimental Results

[0105] Experimental results are as follows Figure 6 The LyoMito group showed a significantly higher number of mitochondria compared to the LyoMito+mtDNA group, indicating that senescent cells are more effective at uptake of dry plant-derived mitochondria after nucleic acid removal.

[0106] Example 8: Particle size changes in dry plant-derived mitochondria before and after nucleic acid removal

[0107] 8.1 Experimental Objective

[0108] We hypothesize that the removal of nucleic acids promotes endocytosis because the molecular weight of plant-derived mitochondria decreases, resulting in a smaller particle size, which may be one reason why they are more easily taken up by cells. Therefore, we measured the particle size of plant-derived mitochondria before and after nucleic acid removal.

[0109] 8.2 Experimental Procedure

[0110] Plant-derived mitochondria were extracted according to the method in Example 1 and divided into two groups for dry treatment. One group had its nucleic acid removed (the preparation method included step S2 of Example 1) and was named the LyoMito group. The other group did not undergo the removal operation (the preparation method did not include step S2 of Example 1) and was named the LyoMito+mtDNA group. The particle size of the plant-derived mitochondria before and after nucleic acid removal was detected using a particle size analyzer.

[0111] 8.3 Experimental Results

[0112] Experimental results are as follows Figure 7 As shown, the particle size of the LyoMito group was significantly smaller than that of the LyoMito+mtDNA group.

[0113] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing plant-derived dry mitochondria for cell repair and / or anti-aging, characterized in that: Includes the following steps: S1 Select healthy plant tissue, grind it, centrifuge it to obtain a precipitate; The precipitate obtained by treating S2 with nuclease yielded plant-derived mitochondria. S3 processes plant-derived mitochondria in a dry state to obtain plant-derived dry mitochondria.

2. The preparation method according to claim 1, characterized in that: The dry processing includes freeze drying, critical point drying, drying in an inert gas environment, or natural air drying, with freeze drying being the preferred method.

3. The preparation method according to claim 1, characterized in that: The centrifugation separation described in step S1 includes centrifuging the ground healthy plant tissue at 1000 rcf-8000 rcf for 5 min-20 min, and then centrifuging at 11000 rcf-21000 rcf for 5 min-20 min.

4. The preparation method according to claim 3, characterized in that: The centrifugation separation includes centrifuging the ground healthy plant tissue at 3000 rcf-5500 rcf for 10 min-15 min, and then centrifuging at 15000 rcf-18000 rcf for 10 min-15 min.

5. The preparation method according to claim 1, characterized in that: The precipitate obtained by nuclease treatment includes adding water and nuclease to the precipitate, with an enzyme activity concentration of 25 U / ml-50 U / ml, reacting at 20℃-40℃ for 15 min-60 min, terminating the reaction, and centrifuging at high speed to obtain the plant-derived mitochondria.

6. The preparation method according to any one of claims 2-5, characterized in that: The freeze-drying process includes adding water to plant-derived mitochondria, mixing them, freezing them into ice blocks at below 0°C, transferring them to a freeze dryer, and completely drying them under conditions of vacuum degree ≤0.1 mBar and cold trap temperature ≤-40°C to obtain plant-derived dry mitochondria; preferably, the mass ratio of water added to plant-derived mitochondria is 1:1 to 1:

5.

7. The preparation method according to claim 1, characterized in that: The healthy plant tissue is healthy tissue from plants of the genera Rosa, Solanum, Aloe, Zingiber, Centella, Coreopsis, or Calendula.

8. Plant-derived dry mitochondria prepared by any one of the methods described in claims 1-7.

9. The use of plant-derived dry mitochondria as described in claim 8 in the preparation of products for repairing cellular mitochondrial damage; or the use of plant-derived dry mitochondria as described in claim 8 in the preparation of products for repairing cell damage and / or skin anti-aging.

10. A cell repair and / or anti-aging product, characterized in that, Its active ingredients include the plant-derived dry mitochondria as described in claim 8, and pharmaceutically acceptable excipients.

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