Chinese chive vesicles for preventing and treating sarcopenia

Abstract

The invention relates to the field of biological medicine and tissue regeneration, and discloses Chinese chive vesicles capable of being used for preventing and treating sarcopenia, a preparation method of the Chinese chive vesicles comprises the following steps: selecting fresh Chinese chives, and performing pretreatment to obtain Chinese chive leaves; juicing the pretreated Chinese chive leaves to obtain Chinese chive juice; performing multi-stage centrifugal separation on the Chinese chive juice, and extracting to obtain Chinese chive vesicles; re-suspending and filtering the separated Chinese chive vesicles to prepare a Chinese chive vesicle solution; and finally, storing the leek vesicle solution in a low-temperature environment or freeze-drying and storing the leek vesicle solution. The allium tuberosum vesicles prepared by the method are rich in various bioactive factors such as miRNA, protein and lipid, have the effects of resisting inflammation, resisting oxidation, regulating mitochondrial functions, promoting satellite cell proliferation and repairing muscle fibers, can be widely applied to prevention and treatment of age-increasing related sarcopenia, diabetes related muscular atrophy and postoperative muscle rehabilitation, and can be used for preparing the allium tuberosum vesicles. High biocompatibility and wide clinical application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and tissue regeneration technology, and specifically relates to a leek vesicle that can be used to prevent and treat sarcopenia. Background Art

[0002] Sarcopenia is an aging-related disease characterized by loss of skeletal muscle mass and function. Muscle strength depends on the contractile ability of muscle fibers, which are highly dependent on mitochondria to provide them with the energy required for contraction. Mitochondrial dysfunction in muscle fibers will lead to a decline in their biological function. To maintain muscle abundance and repair damaged and atrophied muscle fibers, muscle stem cells, which are satellite cells, are required. Similarly, aging of muscle stem cells and depletion of stem cell pools will make it difficult to maintain skeletal muscle mass and repair damaged muscle fibers. Chronic, low-grade inflammation throughout the body will change the tissue microenvironment, promote and maintain muscle atrophy, and impair muscle function. How to reverse or reduce the above-mentioned factors that are not conducive to maintaining muscle fiber homeostasis and muscle tissue homeostasis has become the key to preventing and treating sarcopenia.

[0003] Leek is a common natural plant of the genus Allium in the Liliaceae family. It has a short growth cycle and strong regeneration ability. Previous studies have shown that leeks have anti-inflammatory, antioxidant, antibacterial, and anticancer activities. Although leeks have good biological effects, there is currently no precedent for their application in the treatment of sarcopenia, and how to apply this advantage at a low dose and high effect remains a challenge.

[0004] Dietary vesicle-like nanoparticles are membrane-enclosed nanoparticles (50-300 nanometers in diameter) containing proteins, lipids, and RNA. The membrane of dietary vesicles encapsulates biomolecules within the nanoparticles, thereby conferring stability and resistance to gastric and intestinal solutions and protecting biomolecules from degradation. Therefore, dietary vesicles are able to reach target tissues and exert their biological activities. For example, studies have found that oral ginger vesicles accumulate in the liver, induce the expression of detoxification / antioxidant genes in the liver, including Hmox1, Nqo1, Gclm, and Gclc, and improve alcohol-induced liver damage in mice. Grape-derived vln is taken up by intestinal stem cells and promotes their proliferation by regulating the Wnt / β-catenin signaling pathway. The bioavailability, stability, and bioactivity of these dietary vesicles give them high therapeutic potential. Leek vesicles are a plant-derived natural exosome structure rich in a variety of bioactive factors (such as miRNA, lipids, and proteins), with multiple effects of anti-inflammatory, antioxidant, and cell proliferation and migration promotion. However, the extraction technology of leek vesicle-like nanoparticles is still blank, and its biological effects are still unclear. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a leek vesicle that can be used to prevent and treat sarcopenia, and the leek vesicle is used to prevent and treat sarcopenia.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: In a first aspect, the present invention provides a method for preparing leek vesicles, comprising the following steps: S1, select leek, pre-treat it, and obtain leek leaves; S2, juicing the pretreated leek leaves to obtain leek juice; S3, treating the leek juice by multi-stage centrifugation to separate and obtain leek vesicles; S4, resuspending and filtering the separated leek vesicles to prepare a leek vesicle solution; S5. Store the leek vesicle solution in a low temperature environment or freeze-dry it for storage.

[0007] Preferably, the step of pre-treating the leek comprises: Remove the roots and dead leaves of the leeks, leaving the green leaves; Rinse the leeks at least three times in deionized water to remove surface dust and stains; Cut the chive leaves into 2-3 cm pieces.

[0008] Preferably, the step of juicing the pretreated leek leaves comprises: Mix leek leaves with phosphate buffer at a ratio of 5 mL to 20 mL per 100 g leek; Stir and crush at 20℃~30℃ for 2~5 minutes.

[0009] Preferably, the step of processing the leek juice by multi-stage centrifugation comprises: Perform the following centrifugation steps at 4°C: Centrifuge at 1000 × g for 10 min, remove the precipitate, and retain the supernatant; Centrifuge at 2000 × g for 20 min, remove the precipitate, and retain the supernatant; Centrifuge at 5000 × g for 30 min, remove the precipitate, and retain the supernatant; Centrifuge at 10,000 × g for 1 h, remove the precipitate, and retain the supernatant; The mixture was ultracentrifuged at 150,000 × g for 1.5 hours and the precipitate was collected.

[0010] Preferably, the steps of resuspending and filtering the separated leek vesicles include: The pellet obtained after ultracentrifugation was resuspended in phosphate buffer; The leek vesicle solution was obtained by filtering through 0.45 μm and 0.22 μm filter heads in turn.

[0011] Preferably, the storage of the leek vesicle solution comprises: -80℃ low temperature freezing; or After freeze-drying, store at 4°C.

[0012] In a second aspect, the present invention provides a leek vesicle, which is prepared by the preparation method described in the first aspect of the present invention.

[0013] In a third aspect, the present invention provides a composition comprising the leek vesicles described in the second aspect of the present invention and a pharmaceutically acceptable carrier or excipient.

[0014] Preferably, the leek vesicles include leek vesicle-derived products in the following forms: Vesicle solution; Freeze-dried powder; Injections; Enteric-coated sustained-release tablets; Enteric-coated capsule preparation.

[0015] In a fourth aspect, the present invention provides a use of the leek vesicles described in the second aspect of the present invention in the preparation of a drug for preventing and treating sarcopenia.

[0016] The present invention provides a leek vesicle that can be used to prevent and treat sarcopenia. It has the following beneficial effects: 1. The present invention provides a systematic method for extracting leek vesicles, which can effectively separate and purify leek vesicles through multi-stage centrifugation and fine filtration operations to ensure the integrity and functionality of its bioactive molecules. Compared with traditional plant extraction technology, the present invention can minimize impurity interference and improve extraction efficiency and product purity.

[0017] 2. The present invention designs leek vesicles into a variety of dosage forms, including vesicle solutions, freeze-dried powders, injections, enteric-coated sustained-release tablets and enteric-coated capsules, etc., which can meet the needs of different populations and clinical treatments. Especially in the treatment of sarcopenia, both oral preparations and injection preparations show good patient acceptance and efficacy adaptability, significantly improving the product's wide application and market potential.

[0018] 3. The leek vesicles in the present invention can effectively improve the muscle function and quality of patients with sarcopenia through multiple mechanisms such as regulating inflammatory factors, enhancing mitochondrial function, and promoting satellite cell proliferation. Animal experiments have shown that leek vesicles have significant therapeutic effects in preventing and reversing muscle atrophy and enhancing muscle fiber repair, and can provide a new natural treatment for patients with sarcopenia.

[0019] 4. The leek vesicles of the present invention are derived from plant-based diets, have good biocompatibility and safety, and avoid the toxic side effects that may be caused by chemical drugs. As a daily dietary ingredient, leek vesicle preparations are suitable for long-term use, especially for the prevention of chronic diseases in the elderly population, which is safer and more reliable.

[0020] 5. The leek vesicles of the present invention are active factors of natural origin, and their repair effect is significantly better than that of traditional chemical drugs. At the same time, oral administration of vesicles avoids the pain and limitations of traditional injections. On the one hand, the vesicles can pass through the barrier of the gastrointestinal tract and accumulate in the muscles along with the blood circulation. On the other hand, they can also act on the gastrointestinal flora and improve the intestinal ecology.

[0021] 6. The leek vesicles of the present invention are extracts from daily diet, have good biological activity and gastrointestinal adaptability, and improve the safety of use and patient compliance.

[0022] 7. In addition to the treatment of sarcopenia, the leek vesicles of the present invention also show good therapeutic potential in postoperative rehabilitation, muscle atrophy caused by diabetes, and other muscle degeneration-related diseases. The flexible combination of multiple dosage forms can achieve precise treatment for different disease characteristics, expanding the scope of application of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the leek vesicle extraction process of the present invention; Figure 2 This is a schematic diagram of the production process of leek vesicles according to an embodiment of the present invention; Figure 3 This is a diagram showing the behavioral results of treating sarcopenia mice with leek vesicles according to an embodiment of the present invention; Figure 4 This is a diagram showing the tissue section results of treating sarcopenia mice with leek vesicles according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Please refer to the attached Figure 1 The present invention provides a method for preparing leek vesicles, which comprises the steps of pre-treating leek, squeezing juice, centrifuging, resuspending and filtering, and storing at low temperature or freeze-drying. The method can efficiently separate and purify leek vesicles to obtain leek vesicle products with high biological activity and stability.

[0026] like Figure 1 As shown, the preparation method of the leek vesicle may include the following steps: S1, select leek, pre-treat it, and obtain leek leaves; S2, juicing the pretreated leek leaves to obtain leek juice; S3, treating the leek juice by multi-stage centrifugation to separate and obtain leek vesicles; S4, resuspending and filtering the separated leek vesicles to prepare a leek vesicle solution; S5. Store the leek vesicle solution in a low temperature environment or freeze-dry it for storage.

[0027] For step S1, in this embodiment, fresh leeks are selected as raw materials. Fresh leeks generally refer to leeks with complete leaves, bright green color, and no obvious disease spots or mechanical damage, so as to ensure that the leek vesicles extracted in the subsequent preparation process have high biological activity and purity.

[0028] It should be noted that during the selection of leeks, rotten or dehydrated leeks should be avoided as much as possible to prevent the impurities or degradation products carried therein from affecting the efficiency of subsequent extraction steps and the quality of vesicles. As an option, leeks in the tender leaf stage, i.e. leeks harvested during the growth cycle when the leaves are relatively tender and the fiber content is low, can be selected. At this time, the vesicle extraction efficiency and active factor content of the leeks are relatively high.

[0029] Specifically, the pretreatment of leek in this embodiment includes the following steps: First, remove the roots and yellow leaves of leeks. The roots and yellow leaves of leeks are often rich in sand, bacteria or other insoluble impurities, which will affect the subsequent purification efficiency, so they need to be completely removed.

[0030] After removing the roots and dead leaves, the green and complete leek leaves can be retained as the main part of subsequent processing.

[0031] In this embodiment, the leeks after removing the roots and leaves need to be thoroughly washed to remove dust, pesticides or other impurities remaining on the surface. The washing process uses flowing deionized water to reduce the external pollution that may be introduced by the residual impurities. In the specific operation, the leeks can be placed in a washing pool and gently washed with water. Exemplarily, the number of washings is usually three times, and the washing time each time is 1-3 minutes, which can be adjusted according to the initial cleanliness of the leeks.

[0032] It should be noted that after cleaning, excessive exposure of the leek to the air should be avoided as much as possible to reduce the risk of degradation of the bioactive factors due to oxidation. In some embodiments, after cleaning, the leek leaves can be evenly spread out and placed in a sterile environment for moderate drying. The drying time is generally 10 to 30 minutes to ensure that there is no excess moisture on the surface of the leek and does not affect subsequent operations.

[0033] In this embodiment, in order to facilitate the subsequent crushing and extraction operations, the leek leaves need to be cut into a suitable size. In some embodiments, the leek leaves can be cut into small segments of 2 to 3 cm, and the specific cutting size can be adjusted according to the processing capacity and extraction efficiency of the equipment.

[0034] After cutting, the leek leaves can be immediately transferred into a sterile container and temporarily stored at 4°C to ensure that the biological activity factors of the leek are not reduced due to high temperature or prolonged exposure.

[0035] It should be noted that the refrigerated storage time is generally not more than 2 hours to minimize the degradation or denaturation of the active ingredients in the leek leaves. In addition, the refrigerated container should be sealed to avoid contact with the outside air, thereby further ensuring the cleanliness and stability of the sample.

[0036] It is understandable that the purpose of the pretreatment operation in this step is to remove impurities to the maximum extent and retain the active ingredients in the leek leaves, so as to provide a guarantee for the efficient extraction and purification of the vesicles in the subsequent steps. As an implementation scheme, the specific operating parameters of this step can be appropriately adjusted according to the actual situation, such as the number of washing times, cutting size and refrigeration time, but all of them need to ensure that the treated leek leaves have high cleanliness and stable biological activity.

[0037] For step S2, in this embodiment, the leek leaves after pretreatment need to be prepared into leek juice by juicing to provide a basis for the subsequent extraction of leek vesicles. It should be noted that the juicing operation not only affects the extraction efficiency of leek vesicles, but may also have an important impact on the structural integrity and biological activity of the vesicles, so the specific operating conditions of this step need to be strictly controlled.

[0038] In one possible implementation, the juicer needs to be sterilized by high-pressure steam before use to prevent contamination by exogenous microorganisms. Specifically, the juicer can be placed in an autoclave, the sterilization temperature is set to 121°C, and maintained for 15 to 20 minutes. As an option, the sterilized juicer can be cleaned internally with anhydrous ethanol to remove possible residual organic contaminants, and the ethanol residue can be rinsed with deionized water, and then cleaned again with phosphate buffered saline (PBS) for subsequent use.

[0039] It should be noted that after the juicer is sterilized, it needs to be operated in a sterile environment to avoid secondary contamination. In some embodiments, in order to ensure the high purity of the subsequent leek vesicles, the entire juicing operation can be performed in a clean bench or a sterile room.

[0040] In this embodiment, the pretreated leek leaves are added to a phosphate buffered saline (PBS) in a weight ratio to form a mixture. Specifically, the amount of PBS added is 5 to 20 ml of PBS solution per 100 grams of leek to maintain a suitable liquid viscosity and centrifugal separation efficiency. As an option, the pH value of the PBS solution is 7.4 ± 0.2 to ensure the protection of the leek vesicle structure while avoiding the effect of the acid-base environment on the biological activity of the vesicle.

[0041] In one possible implementation, after adding PBS, a sterile glass rod is used to stir the mixture of leek leaves and PBS for 1 to 2 minutes to ensure that the leek leaves are fully soaked in the PBS solution, so that the vesicles can be efficiently released in the subsequent crushing process.

[0042] It should be noted that the use of PBS can not only effectively protect the structural integrity of leek vesicles, but also reduce the possible destructive effects of endogenous enzymes or other active ingredients in leek on the vesicles.

[0043] In some embodiments, the crushing time of the juicing operation is 2 to 5 minutes, and the specific time can be appropriately adjusted according to the cutting size of the leek leaves and the machine power. Exemplarily, the crushing speed of the juicer is 1500 to 2500 revolutions per minute (rpm) to ensure that the leek tissue can be fully crushed without destroying the structure of the vesicles due to excessive mechanical force.

[0044] As an option, the operating temperature of the juicer needs to be controlled within the range of 20℃ to 30℃ to avoid degradation of the thermosensitive bioactive factors of the leek vesicles due to excessive temperature. It is understandable that mechanical friction may generate a certain amount of heat during operation, so it is recommended to cool down appropriately during operation, such as using an ice bath or cooling device outside the juicer to maintain a suitable temperature.

[0045] In a possible implementation, after the juicing is completed, the obtained leek juice is preliminarily filtered through a filter screen or sterile gauze to remove larger fiber particles and unbroken leek leaves. It should be noted that this filtering step helps to improve the efficiency of subsequent centrifugal separation and reduce the impact of impurity particles on the purity of the vesicles.

[0046] It should be noted that the filtered leek juice should be avoided from being exposed to the air as much as possible to reduce the effect of oxidation on the activity of the leek vesicles. In some embodiments, the filtered leek juice can be immediately transferred to a sterile centrifuge tube and temporarily stored at 4°C for no more than 1 hour to ensure the quality of subsequent processing.

[0047] It can be understood that the key to step S2 is to effectively release the vesicles in the leek cells into the solution through appropriate juicing and filtration operations, while minimizing the mechanical damage or chemical degradation of the vesicles.

[0048] For step S3, in this embodiment, the leek juice after the treatment in step S2 needs to be separated by multi-stage centrifugation to effectively remove impurities and enrich the leek vesicles. It should be noted that the setting of the centrifugation step not only affects the purity and activity of the vesicles, but also directly determines the yield of the final product, so the centrifugation conditions need to be scientifically and reasonably designed.

[0049] In one possible implementation, the multi-stage centrifugation operation includes gradually increasing centrifugal force and time settings, which are specifically divided into three stages: initial separation, intermediate separation, and final enrichment. Exemplarily, each stage is performed at low temperature (4°C) to avoid degradation of vesicle active molecules due to high temperature.

[0050] Specifically, the preliminary separation stage is mainly used to remove larger particles and cell fragments in the leek juice. In some embodiments, a centrifugal force of 1000×g can be used for 10 minutes, the supernatant can be collected, and larger insoluble particles can be removed. As an option, the supernatant after the preliminary centrifugation may also contain a small amount of medium-sized particles, so the supernatant can be separated and collected again at a centrifugal force of 2000×g for 20 minutes. It should be noted that the operation at this stage is intended to fully remove larger impurities to reduce the burden of subsequent high-speed centrifugation and improve the purity of the final vesicle separation.

[0051] The intermediate separation stage in this embodiment is mainly used to remove medium particles and some soluble impurities. In some embodiments, a centrifugal force of 5000×g can be used for 30 minutes to absorb the supernatant and retain the precipitate. It should be noted that the precipitate at this stage is mainly soluble fiber and some protein aggregates in leek, while the supernatant retains most of the leek vesicles.

[0052] The final enrichment stage is a key step in the vesicle separation in this embodiment. Exemplarily, the leek vesicles can be completely precipitated from the supernatant by high-speed and ultracentrifugation operations. In some embodiments, the centrifugal force of 10,000×g is first applied for 1 hour to remove the trace large particle impurities that may remain, and then the obtained supernatant is transferred to an ultracentrifuge and centrifuged for 1.5 hours at 150,000×g to finally collect the vesicle precipitate.

[0053] It should be noted that during the ultracentrifugation process, in order to avoid the rupture or loss of vesicle precipitates, centrifuge tubes that can withstand high centrifugal forces should be used, and the filling volume of the solution in the tube should be 70% to 90% of the total volume of the centrifuge tube to avoid sample loss due to bubbles or vibration.

[0054] In another possible implementation, the precipitate obtained after ultracentrifugation can be resuspended in a sterile PBS solution and centrifuged at a low speed for a short time (eg, 2000×g for 10 minutes) to remove residual impurities or dissolved substances.

[0055] It is understandable that the specific conditions of the centrifugal operation (such as centrifugal force, time and temperature) can be adjusted according to the specific equipment and sample amount, but the general principle is to ensure that the vesicles are not damaged by mechanical force and the purity meets the experimental requirements. The setting of multi-stage centrifugal operation is intended to remove impurities of different particle sizes and densities step by step, and finally obtain leek vesicles with higher purity.

[0056] For step S4, in this embodiment, the leek vesicle precipitate obtained in step S3 needs to be resuspended and filtered to prepare a uniform and high-purity leek vesicle solution, which provides a basis for subsequent storage and application. It should be noted that this step is intended to further remove residual impurities and improve the uniformity and stability of the vesicle solution, so the operating conditions and process parameters need to be strictly controlled.

[0057] In one possible implementation, the leek vesicle precipitate obtained after ultracentrifugation is resuspended in sterile phosphate buffered saline (PBS). Specifically, the volume of PBS added is usually 10 to 50 times the volume of the precipitate. For example, 10 mL of PBS can be added to the centrifuge tube for resuspension. As an option, the precipitate can be mixed by gentle manual shaking or using a vortex oscillator until a uniform suspension is formed. It should be noted that vigorous stirring or excessive shaking should be avoided during the resuspension process to avoid destroying the vesicle structure.

[0058] In another possible implementation, in order to further improve the resuspension efficiency, a micropipette can be used to fully disperse the precipitate in PBS by up and down pipetting. This method is particularly suitable for cases where the amount of precipitate is small or a high uniformity requirement is required.

[0059] As an option, the leek vesicle suspension obtained after resuspension needs to be filtered to remove possible impurity particles that are not completely separated. Specifically, the suspension can be filtered step by step using sterile filter heads with pore sizes of 0.45 μm and 0.22 μm, respectively. Exemplarily, the filtration operation can be performed in a sterile operating table to avoid the influence of external contamination on the vesicle solution.

[0060] The main purpose of 0.45 μm filtration is to initially remove larger particle impurities, such as incompletely dissolved fiber fragments or cell debris, while 0.22 μm filtration further removes smaller particles to ensure the purity and uniformity of the vesicle solution.

[0061] It can be understood that the core of step S4 is to disperse the leek vesicles into a uniform suspension through a resuspension operation, and remove possible particulate impurities through step-by-step filtration, so as to finally prepare a vesicle solution with high purity and uniform distribution.

[0062] For step S5, in this embodiment, the leek vesicle solution prepared in step S4 needs to be stored at low temperature or freeze-dried to ensure the biological activity and stability of the vesicles and provide high-quality vesicle products for subsequent use. It should be noted that the storage method selected in this step can be flexibly adjusted according to the requirements of specific use and storage time.

[0063] In a possible implementation, the leek vesicle solution can be directly dispensed into sterile cryopreservation tubes and stored at an ultra-low temperature of -80°C. Specifically, the prepared vesicle solution is equally divided into 1 to 2 ml per tube, and the cryopreservation tubes are ensured to be well sealed to prevent the sample from contacting the air.

[0064] It should be noted that the leek vesicle solution stored at -80°C is suitable for short-term storage or laboratory research. The storage time can generally reach 6 months to 1 year. During this period, repeated freezing and thawing should be avoided to maintain the integrity and biological activity of the vesicles.

[0065] In some embodiments, in order to meet the needs of long-term storage or transportation, the leek vesicle solution can be stored by freeze-drying. The core of the freeze-drying process is to remove the water in the solution under a low-temperature vacuum environment to form a stable powdered vesicle product.

[0066] In one possible implementation, the leek vesicle solution must first be quickly frozen at -80°C to ensure that the vesicle structure remains stable at low temperatures. Subsequently, the frozen solution is placed in a vacuum freeze dryer and sublimated and dried at a temperature range of -40°C to -50°C. For example, the sublimation drying time is generally 12 to 24 hours, which can be adjusted appropriately according to the sample quantity and equipment parameters.

[0067] It can be understood that the core of step S5 is to maintain the activity and stability of the leek vesicles through a reasonable preservation method, while minimizing the physical and chemical degradation of the vesicles during storage.

[0068] In this embodiment, the leek vesicles can be mixed with pharmaceutically acceptable carriers or excipients to be prepared into various pharmaceutical composition forms to facilitate different administration methods and clinical needs.

[0069] Specifically, leek vesicles can be used as the core active ingredient of vesicle solutions, lyophilized powders, injections, enteric-coated sustained-release tablets or enteric-coated capsule preparations, and used in combination with appropriate pharmaceutical excipients (such as stabilizers, sustained-release agents or protective agents).

[0070] In one possible implementation, the leek vesicle solution can be directly prepared by diluting it with PBS or saline for oral administration or laboratory research; the lyophilized powder is obtained through freeze-drying technology, which is convenient for long-term storage and transportation and suitable for subsequent dissolution and use.

[0071] In some embodiments, the injection can be prepared by dissolving the leek vesicles in sterile saline or glucose solution and sterile filtering for intramuscular or subcutaneous injection. Exemplarily, enteric-coated sustained-release tablets or enteric-coated capsules are processed by mixing lyophilized powder with appropriate excipients (such as microcrystalline cellulose or hypromellose), which can gradually release the active ingredients of the vesicles in the gastrointestinal tract to enhance the therapeutic effect.

[0072] It should be noted that the design of these compositions is optimized according to different dosing requirements, which can effectively maintain the stability and biological activity of leek vesicles while improving patient compliance in clinical applications.

[0073] In this embodiment, the derivative products of leek vesicles include the following: Vesicle solution: directly dissolve the prepared leek vesicles in PBS or other buffer solutions to prepare a liquid preparation suitable for oral administration or injection.

[0074] Freeze-dried powder: The leek vesicles are dehydrated through the freeze-drying process and made into a powdered product, which is convenient for long-term storage and transportation.

[0075] Injectable injection: Dissolve the leek vesicles in sterile saline or buffer, filter and sterilize to prepare an injectable preparation, which is suitable for the precise treatment of muscular atrophy related diseases.

[0076] Enteric-coated sustained-release tablets: Leek vesicles are made into tablets by coating enteric-coated materials, which remain stable in the gastric acid environment and gradually release the active ingredients in the small intestine.

[0077] Enteric-coated capsule preparation: The leek vesicles are used as the main active ingredient, which are mixed with appropriate excipients and filled into capsules to prepare an oral preparation suitable for intestinal absorption and long-term release.

[0078] It should be noted that these different forms of derivative products can be selected according to the needs of patients and the specific characteristics of the disease. They can provide short-term and efficient therapeutic effects and are also suitable for long-term preventive treatment.

[0079] In this embodiment, leek vesicles are mainly used to prepare drugs for preventing and treating sarcopenia, and their biological effects and mechanisms of action provide support for improving the function and quality of skeletal muscle.

[0080] In some embodiments, leek vesicles can improve the pathological state of sarcopenia through the following mechanisms: Regulating chronic inflammation: The miRNA and anti-inflammatory factors rich in leek vesicles can reduce the chronic inflammatory state around skeletal muscle and restore the homeostasis of the muscle microenvironment.

[0081] Enhance mitochondrial function: The active molecules in leek vesicles can promote the energy metabolism of muscle fiber mitochondria, thereby improving the muscle's contraction ability.

[0082] Repairing muscle fibers: Leek vesicles promote the repair of muscle fibers and reverse muscle atrophy by regulating the proliferation and differentiation of satellite cells.

[0083] In one possible application, leek vesicle solution or injection can be used to restore muscle function after surgery, improving the patient's muscle strength and mobility; freeze-dried powder or oral enteric-coated preparation is suitable for daily prevention of sarcopenia in the elderly.

[0084] It is understandable that these mechanisms of action and various preparation forms of leek vesicles give them broad application prospects in the treatment of sarcopenia and related diseases.

[0085] Example 1: Preparation of leek vesicles Step 1: Item preparation 1. Juicer preparation High pressure steam sterilized juicer; At 25°C, add 50 ml of anhydrous ethanol and run it idle to remove the ethanol; At 25°C, rinse once with deionized water, add 50 ml of deionized water, run idle to remove residual ethanol, and drain water; At 25°C, rinse once with deionized water, add 50 ml of PBS, spin, remove PBS and set aside.

[0086] 2. Leek preparation Select fresh chives, remove the roots and dead leaves, and keep the green chive leaves; Rinse with running deionized water three times to ensure that dust and stains on the surface of the leeks are washed away; Cut the leek leaves into 2-3 cm pieces with sterile scissors and store at 4°C until use.

[0087] Step 2: Extract leek vesicles 1. Crush the chives and squeeze out the juice At 25°C, fresh leek leaves were placed in a juicer, 10 mL of phosphate buffered saline (PBS) was added per 100 g of leek, and the leaves were stirred and crushed for 3 minutes, and the leek juice was collected.

[0088] 2. Centrifugation Centrifuge at 1000 × g for 10 min at 4°C and remove the supernatant; Centrifuge at 2000 × g for 20 min at 4°C and remove the supernatant; Centrifuge at 5000 × g for 30 min at 4°C and remove the supernatant; Centrifuge at 10,000 × g for 1 h at 4°C and remove the supernatant; Ultracentrifuge at 150,000×g at 4°C for 1.5 hours, discard the supernatant, and collect the precipitate.

[0089] 3. Preparation of Leek Vesicle Solution The precipitate was resuspended in 10 mL PBS and filtered through 0.45 μm and 0.22 μm filters in sequence. The filtrate obtained was the leek vesicle solution.

[0090] 4.Save The leek vesicle solution was divided into cryovials and stored at -80°C for further experiments.

[0091] The leek vesicles were freeze-dried and stored at 4°C for further experiments.

[0092] like Figure 2 Shown is a diagram of the process of making leek vesicles.

[0093] Example 2: Muscle fiber repair experiment Step 1: Prepare concentration gradient vesicle solution Tube a: 0.75 mL vesicle solution + 0.25 mL PBS; b test tube: 0.5 mL vesicle solution + 0.5 mL PBS; Tube c: 0.25 mL vesicle solution + 0.75 mL PBS; d Test tube: 1 mL PBS.

[0094] Steps 2-5: Experimental grouping, model construction and result evaluation Animal experiment groups: Prepare 25 C57BL / 6 mice; randomly grab 5 mice and mark them as the control Con group, and the remaining 20 mice as the model group; the mice in the model group are randomly divided into 4 groups, namely High group, Mid group, Low group, and Dex group, and mark them; Construction of muscle atrophy model: The mice in the model group were taken, weighed, and intraperitoneally injected with 25 mg / kg / d (dexamethasone injection concentration is 2.5 mg / ml, and the injection volume after conversion is 10 ml / kg / d) of dexamethasone for 9 days to complete the construction of the muscle atrophy model, and then intraperitoneally injected with 5 mg / kg / d (the injection volume after conversion is 2 ml / kg / d) of dexamethasone to maintain the muscle atrophy state; the mice in the control group were taken, weighed, and intraperitoneally injected with 10 ml / kg / d of PBS solution for 9 days, and then intraperitoneally injected with 2 ml / kg / d of PBS solution.

[0095] Implementation of leek vesicles: Starting from the 10th day, all mice were administered drugs by oral gavage; the High group was given test tube solution a with normal vesicle concentration, the Mid group was given test tube solution b with 1 / 2 vesicle concentration, the Low group was given test tube solution c with 1 / 4 vesicle concentration, and the Dex group was given test tube solution d without drug; similarly, the con group was given test tube solution d without drug.

[0096] Observation and evaluation of results: Weighing: days 10, 17, 24, 31, and 38; limb grip strength: day 38; gait: day 38; total open field activity distance: day 38; bone density: day 38; total body fat mass / body weight: day 38; hind limb fat mass / body weight: day 38; total body lean mass / body weight: day 38; hind limb lean mass / body weight: day 38; tibialis anterior, gastrocnemius, and quadriceps femoris sections: day 38.

[0097] like Figure 3 As shown in A, compared with the placebo group, the gait of mice administered with leek vesicles had a higher stride length, less swing, and a longer walking distance, which increased in a dose-dependent manner.

[0098] like Figure 3 As shown in B, compared with the placebo group, the mice administered with leek vesicles had higher grip strength, which increased with the dosage.

[0099] like Figure 3 As shown in C, compared with the placebo group, the mice in the leek vesicle group had more intensive and numerous activity tracks in the mine and were more active, and the degree of activity increased with the dosage.

[0100] like Figure 4As shown in the results of tissue section staining, the muscle fibers of the placebo group were thin and loose, while the mice in the group administered with leek vesicles had thicker and denser muscle fibers, and the state of these muscle fibers improved more with increasing dosage.

[0101] Conclusion: Leek vesicles have the ability to promote the regeneration and repair of atrophic muscle fibers, and this effect is dependent on the concentration of leek vesicles. In terms of disease, the vesicles have the ability to reverse the already occurred sarcopenia and promote the recovery of muscle fiber quality and strength.

[0102] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing leek vesicles, characterized in that: The following steps are involved: S1, select leek, pre-treat it, and obtain leek leaves; S2, juicing the pretreated leek leaves to obtain leek juice; S3, treating the leek juice by multi-stage centrifugation to separate and obtain leek vesicles; S4, resuspending and filtering the separated leek vesicles to prepare a leek vesicle solution; S5. Store the leek vesicle solution in a low temperature environment or freeze-dry it for storage.

2. The method for preparing leek vesicles according to claim 1, characterized in that: The step of pre-treating leeks comprises: Remove the roots and dead leaves of the leeks, leaving the green leaves; Rinse the leeks at least three times in deionized water to remove surface dust and stains; Cut the chive leaves into 2-3 cm pieces.

3. The method for preparing leek vesicles according to claim 1, characterized in that: The step of squeezing juice from the pretreated leek leaves comprises: Mix leek leaves with phosphate buffer at a ratio of 5 mL to 20 mL per 100 g leek; Stir and crush at 20℃~30℃ for 2~5 minutes.

4. The method for preparing leek vesicles according to claim 1, characterized in that: The step of processing the leek juice by multi-stage centrifugation comprises: Perform the following centrifugation steps at 4°C: Centrifuge at 1000 × g for 10 min, remove the precipitate, and retain the supernatant; Centrifuge at 2000 × g for 20 min, remove the precipitate, and retain the supernatant; Centrifuge at 5000 × g for 30 min, remove the precipitate, and retain the supernatant; Centrifuge at 10,000 × g for 1 h, remove the precipitate, and retain the supernatant; The mixture was ultracentrifuged at 150,000 × g for 1.5 hours and the precipitate was collected.

5. The method for preparing leek vesicles according to claim 1, characterized in that: The steps of resuspending and filtering the separated leek vesicles include: The pellet obtained after ultracentrifugation was resuspended in phosphate buffer; The leek vesicle solution was obtained by filtering through 0.45 μm and 0.22 μm filter heads in turn.

6. The method for preparing leek vesicles according to claim 1, characterized in that: The storage of the leek vesicle solution comprises: -80℃ low temperature freezing; or After freeze-drying, store at 4°C.

7. A leek vesicle, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 6.

8. A composition, characterized in that It comprises the leek vesicles according to claim 7 and a pharmaceutically acceptable carrier or excipient.

9. The composition according to claim 8, characterized in that The leek vesicles include leek vesicle derivatives in the following forms: Vesicle solution; Freeze-dried powder; Injections; Enteric-coated sustained-release tablets; Enteric-coated capsule preparation.

10. Use of the leek vesicles according to claim 7 in preparing a drug for preventing and treating sarcopenia.

Images