Preparation process and application of magnolol-loaded plant exosome

Abstract

The invention discloses a preparation process and application of magnolol-loaded plant exosomes, and relates to the technical field of plant exosomes. Magnolol is extracted from a magnolia officinalis plant material through ultrasonic assistance, the plant exosomes are obtained from fresh ginger by using an ultracentrifugation method, and the plant exosomes are further modified through palmitic acid and polyethylene glycol, so that the magnolol-loaded plant exosomes are obtained. And finally, preparing the magnolol-loaded plant exosome by virtue of an incubation method. The magnolol can be protected from being damaged by gastric acid and digestive enzymes through membrane structure wrapping of the plant exosome, the stability of the magnolol in the digestive tract is improved, and it is further verified through mouse experiments that the magnolol-loaded plant exosome can be beneficial to regulation of intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant exosomes, and specifically relates to a preparation process and application of magnolol-loaded plant exosomes. Background Art

[0002] Magnolol (MG) is an active ingredient extracted from Magnolia officinalis, a plant of the Magnoliaceae family. It has multiple pharmacological effects, including anti-inflammatory, anti-tumor, cardiovascular protection, anti-angiogenesis, anti-diabetic, hypoglycemic, antioxidant, neuroprotective, gastrointestinal protection, and activity that helps regulate intestinal flora.

[0003] Plant exosomes, also known as plant-derived exosome-like nanoparticles (PENs), are a type of nanoparticles found in plants that have physical properties and composition similar to animal cell exosomes. Compared with exosomes derived from animal cells, PELNs have higher yields, shorter extraction cycles, and because they are usually derived from edible plants, they have lower immunogenicity. PELNs can be used as carriers to deliver various therapeutic molecules to relevant parts. When PELNs are used as delivery carriers for therapeutic molecules, they often have therapeutic effects themselves, further enhancing the effect. In addition, compared with artificially synthesized nanocarriers, PELNs have significant advantages such as non-toxicity, low immunogenicity, and targeted uptake.

[0004] At present, in terms of bioavailability, due to the poor solubility of magnolol itself in water, its application in pharmaceutical preparations and treatments is limited. Therefore, improving its water solubility and stability, as well as enhancing the effect of magnolol, is an important direction of current research. In recent years, more and more studies have shown that plant exosomes, as a natural carrier, can effectively transport bioactive substances into cells, thereby achieving more accurate targeted therapy. At the same time, plant exosomes have good biocompatibility and stability, which can reduce the adverse reactions and side effects of drugs. Therefore, there is no technical problem in the prior art that magnolol is encapsulated and loaded with drugs in combination with natural plant exosomes, which helps to regulate the beneficial flora of the human body and expand its application in the health care products industry. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation process and application of plant exosomes loaded with magnolol, so as to solve the technical problem that the prior art does not combine natural plant exosomes to encapsulate and load magnolol to help regulate the beneficial flora of the human body and expand its application in the health care products industry.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a preparation process of magnolol-loaded plant exosomes, comprising the following steps:

[0008] (1) extracting magnolol from Magnolia officinalis bark or root to obtain high-purity magnolol powder;

[0009] (2) extracting plant exosomes from fresh ginger and modifying them to obtain stability-modified plant exosomes;

[0010] (3) Incubating high-purity magnolia officinalis powder with stability-modified plant exosomes to obtain magnolia officinalis-loaded plant exosomes.

[0011] Furthermore, a process for preparing plant exosomes loaded with magnolol, step (1) comprises the following process:

[0012] S1. Material preparation: crush the dried Magnolia officinalis and sieve to obtain Magnolia officinalis powder;

[0013] S2. Ultrasonic-assisted extraction: ethanol was selected as the solvent. Magnolia bark powder and ethanol were thoroughly stirred and mixed, then ultrasonicated and soaked at room temperature, followed by centrifugation and concentration to obtain Magnolia bark extract.

[0014] S3. Freeze drying: Pour the Magnolia officinalis extract into a freeze drying bottle, then place it in the cold trap of the freeze dryer, reduce the pressure of the freeze dryer to a high vacuum state, and dry for 24 to 30 hours; after drying is completed, continue to maintain a high vacuum state; after freeze drying is completed, turn off the freeze dryer and slowly return to atmospheric pressure; open the freeze drying bottle and collect the high-purity magnolia officinalis powder.

[0015] Furthermore, in the preparation process of the plant exosomes loaded with magnolia bark phenol, S1 material preparation is dried in a drying oven at 40-45°C for 24-36 hours, and then sieved through a 40-60 mesh sieve after crushing; S2 ultrasonic-assisted extraction uses 40-90% ethanol, the material-liquid ratio is 1:5-15, ultrasound is performed at 40-60°C for 20-120 minutes, and solvent extraction is performed by soaking at room temperature for 16-24 hours, centrifugation at 3000-5000rpm for 5-10 minutes, and the operation is repeated 2-3 times; S3 freeze-drying The temperature in the cold trap is -40 to -80°C, the freezing time is 8 to 12 hours, and a high vacuum state is below 0.1mbar. The high vacuum state is dried for 24 to 30 hours, and the high vacuum state is maintained for 2 to 4 hours.

[0016] Furthermore, a process for preparing plant exosomes loaded with magnolol, step (2) comprises the following process:

[0017] Q1: Take fresh ginger root and use ddH 2O washing, crushing with a juicer, adding 1× PBS buffer, and filtering through gauze to obtain the ginger crude filtrate;

[0018] Q2: Centrifuge the ginger crude filtrate, take the supernatant, discard the precipitate, and obtain ginger supernatant A;

[0019] Q3: Centrifuge the ginger supernatant A, take the supernatant, discard the precipitate, and obtain the ginger supernatant B;

[0020] Q4: Centrifuge the ginger supernatant B, take the supernatant, discard the precipitate, and obtain the ginger supernatant C; repeat this step until there is no obvious precipitate, and obtain the ginger essence filtrate;

[0021] Q5: Filter the ginger extract filtrate through a microporous filter membrane to obtain a ginger membrane filtered supernatant;

[0022] Q6: Add the supernatant filtered through the ginger filter membrane to the exosome concentrated solution and let it stand to obtain the exosome concentrated solution;

[0023] Q7: Centrifuge the exosome concentrate, discard the supernatant, and resuspend the precipitate in 1×PBS to obtain an exosome resuspension;

[0024] Q8: Transfer the exosome resuspension to a centrifuge tube, centrifuge, and take the supernatant. If there is a lot of precipitation, centrifuge several times to take the supernatant to obtain the exosome supernatant;

[0025] Q9: Transfer the exosome supernatant to the exosome purification column, centrifuge, and filter to the bottom of the exosome purification column to obtain the plant exosome solution;

[0026] Q10: mixing palmitic acid with the plant exosome solution and PBS solution to obtain palmitic acid-modified plant exosomes;

[0027] Q11: mixing polyethylene glycol-800 with the palmitic acid-modified plant exosomes to obtain a plant exosome cross-linking system;

[0028] Q12: Add the plant exosome cross-linking system to the trehalose solution, ultracentrifuge and resuspend to obtain stability-modified plant exosomes.

[0029] Furthermore, a process for preparing plant exosomes loaded with magnolol, Q1 using ddH 2O was washed three times, and 1× PBS buffer was added with the ginger root in a volume ratio of 1:0.5-1; Q2 was centrifuged at 2500-3000 r / min for 20-30 min; Q3 was centrifuged at 8000-10000 r / min for 20-30 min; Q4 was centrifuged at 12000 r / min for 25-30 min; Q5 microporous filter membrane diameter was 0.22-0.45 μm; Q6 ginger filter membrane filtered supernatant and exosome concentrated solution in a volume ratio of 4:1, and stood at 4°C for 4-6 h; Q7 was centrifuged at 12000 r / min for 50-60 min, and 1-2 mL of 1× PBS was added to resuspend the precipitate; Q8 was centrifuged at 12000 r / min for 8-10 min; Q9 was centrifuged at 12000 r / min for 2-5 min; the entire centrifugation process of plant exosome extraction was carried out at 0-4°C, and the exosome extraction and purification kit was purchased from Shanghai Yumeibo Biotechnology Co., Ltd.

[0030] Furthermore, a preparation process of plant exosomes loaded with magnolol, wherein the concentration of palmitic acid is 0.1-10 mM, preferably 5 mM, the volume ratio of palmitic acid to plant exosome solution is 1:8-12, preferably 1:10, the amount of PBS solution added is 0.5-2 times the total volume of the plant exosome solution and the palmitic acid, more preferably 1 times, the mixing temperature is 20-25°C, preferably 25°C, the mixing time is 8-16h, preferably 12h; the original concentration of polyethylene glycol-800 is 30-50%, preferably 35%; the final concentration of polyethylene glycol-800 in the plant exosome cross-linking system is 1.3-2.5%, preferably 2%; the mixing The time is 1 to 3 hours, preferably 2 hours; the original concentration of trehalose solution is 20 to 30%, preferably 25%; the final concentration of trehalose after adding to the plant exosome cross-linking system is 10 to 15%, preferably 12%; the speed of ultracentrifugation is 12000 to 13000 rpm, preferably 12500 rpm; the temperature of ultracentrifugation is preferably 0 to 4°C, more preferably 4°C; the time of ultracentrifugation is preferably 0.5 to 2 hours, more preferably 1 hour, and the resuspending solvent is preferably a trehalose solution with a concentration of 25%; the concentration of the stability-modified plant exosomes after resuspending is preferably 1 to 30 mg / mL, more preferably 15 mg / mL.

[0031] Furthermore, a process for preparing plant exosomes loaded with magnolol, wherein step (3) comprises the following process:

[0032] M1: The prepared magnolol is prepared into a magnolol solution using PBS buffer;

[0033] M2: The magnolol solution was mixed with the stability-modified plant exosomes and incubated in a refrigerator to obtain magnolol-loaded plant exosomes.

[0034] Furthermore, a preparation process of plant exosomes loaded with magnolol, wherein the concentration of magnolol solution is 5-8 mg / mL, the volume ratio of magnolol solution to plant exosomes is 1:5-30, preferably 1:20; the incubation temperature is 0-4°C; the incubation shaker speed is 50-120 rpm, more preferably 90 rpm; the incubation time is 24-36 h, more preferably 24 h; the final magnolol content in the plant exosomes loaded with magnolol is 10-25%.

[0035] The present invention also provides an application of magnolol-loaded plant exosomes in health products.

[0036] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0037] 1. The present invention extracts magnolol from magnolia bark plant material with the assistance of ultrasound. The vibration generated by ultrasound can destroy the cell wall of plant tissue, which helps to accelerate the contact between the extraction solvent and magnolol, thereby shortening the extraction time and improving the extraction efficiency.

[0038] 2. The plant exosome extraction of the present invention is based on the cell supernatant exosome extraction and purification kit, which is improved, that is, the exosomes in ginger rhizomes are prepared by combining differential centrifugation, microporous membrane filtration and a kit, which has higher purity and stability, and helps to maintain the activity of exosomes during storage and transportation. The plant exosomes are further modified by palmitic acid and polyethylene glycol, which helps to regulate intestinal flora.

[0039] 3. The present invention can protect magnolia officinalis from being destroyed by gastric acid and digestive enzymes through the membrane structure of plant exosomes, increase its stability in the digestive tract, and improve its stability in the body. In addition, plant exosomes can not only be used as drug carriers, but also have the function of helping to regulate intestinal flora, and can synergize with magnolia officinalis to further improve the efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 A schematic diagram of the preparation of magnolol-loaded plant exosomes provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments 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.

[0043] Example 1

[0044] The preparation process of magnolol-loaded plant exosomes of this embodiment comprises the following steps:

[0045] (1) extracting magnolol from Magnolia officinalis bark or root to obtain high-purity magnolol powder;

[0046] (2) extracting plant exosomes from fresh ginger and modifying them to obtain stability-modified plant exosomes;

[0047] (3) Incubating high-purity magnolia officinalis powder with stability-modified plant exosomes to obtain magnolia officinalis-loaded plant exosomes.

[0048] Specifically, step (1) includes the following process:

[0049] S1. Material preparation: wash the bark or root of Magnolia officinalis, and then dry it in a drying oven at 40°C for 36 hours. Grind the dried Magnolia officinalis material and pass it through a 40-mesh sieve to obtain Magnolia officinalis powder;

[0050] S2. Ultrasonic-assisted extraction: 40% ethanol was selected as the solvent with a solid-liquid ratio of 1:5. The magnolia bark powder and ethanol were fully stirred and mixed, and then ultrasonicated at 40°C for 120 minutes and soaked at room temperature for 16 hours. The magnolia bark phenol extract was then collected in a centrifuge cup, balanced, and centrifuged at 3000rpm at room temperature for 10 minutes. The supernatant was taken and the precipitate was discarded. The operation was repeated twice. The concentrated volume was reduced by 5 times to obtain the magnolia bark extract.

[0051] S3. Freeze-drying: Pour the Magnolia officinalis extract into a freeze-drying bottle, and then put it into the cold trap of a freeze dryer, set the temperature to -40°C, freeze for 12 hours, set the freeze dryer to a high vacuum state below 0.1 mbar, and dry for 30 hours; after drying, continue to maintain the high vacuum state for 4 hours; after freeze-drying, turn off the freeze dryer and slowly return to atmospheric pressure; open the freeze-drying bottle and collect the high-purity magnolia officinalis powder.

[0052] Step (2) includes the following process:

[0053] Q1: Take fresh ginger root and use ddH 2O was washed three times, and the ginger root was crushed in a juicer. During the process, 1× PBS buffer was added to the ginger root in a volume ratio of 1:1, and the ginger crude filtrate was obtained by coarse filtration with gauze;

[0054] Q2: Centrifuge the ginger crude filtrate at 2500r / min for 30min, take the supernatant, discard the precipitate, and obtain ginger supernatant A;

[0055] Q3: Centrifuge ginger supernatant A at 8000r / min for 30min, take the supernatant, discard the precipitate, and obtain ginger supernatant B;

[0056] Q4: Centrifuge the ginger supernatant B at 12000r / min for 30min, take the supernatant, discard the precipitate, and obtain ginger supernatant C; repeat this step until there is no obvious precipitate, and obtain ginger essence filtrate;

[0057] Q5: Filter the ginger extract filtrate twice using a microporous filter membrane with a diameter of 0.22-0.45 μm to obtain a ginger membrane filtration supernatant;

[0058] Q6: Add the supernatant filtered through the ginger filter membrane to the exosome concentrated solution, the volume ratio of the supernatant filtered through the ginger filter membrane to the exosome concentrated solution is 4:1, and stand at 4°C for 6 hours to obtain the exosome concentrated solution;

[0059] Q7: Centrifuge the exosome concentrate at 12000r / min for 60min, discard the supernatant, add 1mL 1×PBS to resuspend the precipitate to obtain the exosome resuspension;

[0060] Q8: Transfer the exosome resuspension to a centrifuge tube, centrifuge at 12000r / min for 10min, take the supernatant, there is a lot of precipitation, centrifuge multiple times to take the supernatant, and obtain the exosome supernatant;

[0061] Q9: Transfer the exosome supernatant to the exosome purification column, centrifuge at 12000r / min for 5min, and filter to the bottom of the exosome purification column to obtain the plant exosome solution;

[0062] Q10: 0.1 mM palmitic acid was mixed with the plant exosome solution and PBS solution, the volume ratio of palmitic acid to plant exosome solution was 1:8, the amount of PBS solution added was 0.5 times the total volume of the plant exosome solution and the palmitic acid, and the mixture was mixed at 20° C. for 8 h to obtain palmitic acid-modified plant exosomes;

[0063] Q11: Mix 30% polyethylene glycol-800 with the palmitic acid-modified plant exosomes for 1 hour, and adjust the final concentration of polyethylene glycol-800 to 1.3% to obtain a plant exosome cross-linking system;

[0064] Q12: The plant exosome cross-linking system was added to the trehalose solution, ultracentrifuged at 12000 rpm for 0.5 h, and resuspended with 25% trehalose solution to obtain stability-modified plant exosomes with a concentration of 1 mg / mL.

[0065] Step (3) includes the following process:

[0066] M1: The prepared magnolol was prepared into a magnolol solution using PBS buffer, and the concentration of the magnolol solution was 5 mg / mL;

[0067] M2: The magnolol solution was mixed with the stability-modified plant exosomes and incubated in a refrigerator for 24 hours. The incubation temperature was 0°C and the rotation speed was 50 rpm. The volume ratio of the magnolol solution to the plant exosomes was 1:5 to obtain plant exosomes loaded with magnolol.

[0068] Example 2

[0069] The preparation process of magnolol-loaded plant exosomes of this embodiment comprises the following steps:

[0070] (1) extracting magnolol from Magnolia officinalis bark or root to obtain high-purity magnolol powder;

[0071] (2) extracting plant exosomes from fresh ginger and modifying them to obtain stability-modified plant exosomes;

[0072] (3) Incubating high-purity magnolia officinalis powder with stability-modified plant exosomes to obtain magnolia officinalis-loaded plant exosomes.

[0073] Specifically, step (1) includes the following process:

[0074] S1. Material preparation: wash the bark or root of Magnolia officinalis, and then dry it in a drying oven at 42°C for 30 hours. Grind the dried Magnolia officinalis material and pass it through a 60-mesh sieve to obtain Magnolia officinalis powder;

[0075] S2. Ultrasonic-assisted extraction: Select 70% ethanol as the solvent with a solid-liquid ratio of 1:10. After fully stirring the Magnolia officinalis powder and ethanol, perform ultrasound at 42°C for 60 minutes and soak at room temperature for 18 hours. Then collect the magnolia officinalis phenol extract in a centrifuge cup, balance it, and centrifuge it at 4000rpm at room temperature for 8 minutes. Take the supernatant and discard the precipitate. Repeat the operation 3 times. The concentrated volume is reduced by 5 times to obtain the Magnolia officinalis extract.

[0076] S3. Freeze-drying: Pour the magnolia phenol crystal product into a freeze-drying bottle, and then put it into the cold trap of a freeze dryer, set the temperature to -60°C, freeze for 10 hours, set the freeze dryer to a high vacuum state below 0.1 mbar, and dry for 28 hours; after drying, continue to maintain the high vacuum state for 3 hours; after freeze-drying, turn off the freeze dryer and slowly return to atmospheric pressure; open the freeze-drying bottle and collect the high-purity magnolia phenol powder.

[0077] Step (2) includes the following process:

[0078] Q1. Take fresh ginger root and use ddH 2 O was washed three times, and the ginger root was crushed in a juicer. During the process, 1× PBS buffer was added to the ginger root at a volume ratio of 1:0.8, and the ginger crude filtrate was obtained by coarse filtration with gauze;

[0079] Q2, centrifuge the ginger crude filtrate at 2800r / min for 25min, take the supernatant, discard the precipitate, and obtain ginger supernatant A;

[0080] Q3, centrifuge ginger supernatant A at 9000r / min for 25min, take the supernatant, discard the precipitate, and obtain ginger supernatant B;

[0081] Q4, centrifuge the ginger supernatant B at 12000r / min for 28min, take the supernatant, discard the precipitate, and obtain ginger supernatant C; repeat this step until there is no obvious precipitate, and obtain ginger essence filtrate;

[0082] Q5, filtering the ginger extract filtrate twice with a microporous filter membrane having a diameter of 0.22-0.45 μm to obtain a ginger filter membrane filtration supernatant;

[0083] Q6. Add the supernatant filtered through the ginger filter membrane to the exosome concentrated solution, the volume ratio of the supernatant filtered through the ginger filter membrane to the exosome concentrated solution is 4:1, and stand at 4°C for 5 hours to obtain the exosome concentrated solution;

[0084] Q7, centrifuge the exosome concentrate at 12000r / min for 55min, discard the supernatant, add 2mL 1×PBS to resuspend the precipitate to obtain the exosome resuspension;

[0085] Q8. Transfer the exosome resuspension to a centrifuge tube, centrifuge at 12000r / min for 9 minutes, take the supernatant, there is a lot of precipitation, centrifuge multiple times to take the supernatant, and obtain the exosome supernatant;

[0086] Q9, transfer the exosome supernatant to the exosome purification column, centrifuge at 12000r / min for 3min, filter to the bottom of the exosome purification column to obtain the plant exosome solution;

[0087] Q10, mixing 1 mM palmitic acid with the plant exosome solution and PBS solution, wherein the volume ratio of palmitic acid to the plant exosome solution is 1:10, and the amount of PBS solution added is 1 times the total volume of the plant exosome solution and the palmitic acid, and mixing at 22° C. for 12 h to obtain palmitic acid-modified plant exosomes;

[0088] Q11, mixing 40% polyethylene glycol-800 with the palmitic acid-modified plant exosomes for 2 hours, adjusting the final concentration of polyethylene glycol-800 to 2%, and obtaining a plant exosome cross-linking system;

[0089] Q12. Add the plant exosome cross-linking system to the trehalose solution, ultracentrifuge at 13,000 rpm for 1 h, and resuspend with 25% trehalose solution to obtain stability-modified plant exosomes with a concentration of 10 mg / mL.

[0090] Step (3) includes the following process:

[0091] M1. Prepare the prepared magnolol with PBS buffer to form a magnolol solution, wherein the concentration of the magnolol solution is 6 mg / mL;

[0092] M2. The magnolol solution was mixed with the stability-modified plant exosomes and incubated in a refrigerator for 30 hours. The incubation temperature was 4°C and the rotation speed was 90 rpm. The volume ratio of the magnolol solution to the plant exosomes was 1:10 to obtain plant exosomes loaded with magnolol.

[0093] Example 3

[0094] The preparation process of magnolol-loaded plant exosomes of this embodiment comprises the following steps:

[0095] (1) extracting magnolol from Magnolia officinalis bark or root to obtain high-purity magnolol powder;

[0096] (2) extracting plant exosomes from fresh ginger and modifying them to obtain stability-modified plant exosomes;

[0097] (3) Incubating high-purity magnolia officinalis powder with stability-modified plant exosomes to obtain magnolia officinalis-loaded plant exosomes.

[0098] Step (1) includes the following process:

[0099] S1. Material preparation: wash the bark or root of Magnolia officinalis, and then dry it in a drying oven at 45°C for 24 hours. Grind the dried Magnolia officinalis material and pass it through a 60-mesh sieve to obtain Magnolia officinalis powder;

[0100] S2. Ultrasonic-assisted extraction: Select 90% ethanol as the solvent with a solid-liquid ratio of 1:15. After fully stirring the Magnolia officinalis powder and ethanol, perform ultrasound at 60°C for 20 minutes and soak at room temperature for 24 hours. Then collect the magnolia officinalis phenol extract in a centrifuge cup, balance it, and centrifuge it at 5000rpm at room temperature for 5 minutes. Take the supernatant and discard the precipitate. Repeat the operation 3 times. The concentrated volume is reduced by 5 times to obtain the Magnolia officinalis extract.

[0101] S3. Freeze-drying: Pour the magnolia phenol crystal product into a freeze-drying bottle, and then put it into the cold trap of a freeze dryer, set the temperature to -80°C, freeze for 8 hours, set the freeze dryer to a high vacuum state below 0.1 mbar, and dry for 24 hours; after drying, continue to maintain the high vacuum state for 2 hours; after freeze-drying, turn off the freeze dryer and slowly return to atmospheric pressure; open the freeze-drying bottle and collect the high-purity magnolia phenol powder.

[0102] Step (2) includes the following process:

[0103] Q1. Take fresh ginger root and use ddH 2 O was washed three times, and the ginger root was crushed in a juicer. During the process, 1× PBS buffer was added to the ginger root at a volume ratio of 1:0.5, and the ginger crude filtrate was obtained by coarse filtration through gauze;

[0104] Q2, centrifuge the ginger crude filtrate at 3000r / min for 20min, take the supernatant, discard the precipitate, and obtain ginger supernatant A;

[0105] Q3, centrifuge ginger supernatant A at 10000r / min for 20min, take the supernatant, discard the precipitate, and obtain ginger supernatant B;

[0106] Q4, centrifuge the ginger supernatant B at 12000r / min for 25min, take the supernatant, discard the precipitate, and obtain ginger supernatant C; repeat this step until there is no obvious precipitate, and obtain ginger essence filtrate;

[0107] Q5, filtering the ginger extract filtrate twice with a microporous filter membrane having a diameter of 0.22-0.45 μm to obtain a ginger filter membrane filtration supernatant;

[0108] Q6. Add the supernatant filtered through the ginger filter membrane to the exosome concentrated solution, the volume ratio of the supernatant filtered through the ginger filter membrane to the exosome concentrated solution is 4:1, and stand at 4°C for 4 hours to obtain the exosome concentrated solution;

[0109] Q7, centrifuge the exosome concentrate at 12000r / min for 50min, discard the supernatant, add 2mL 1×PBS to resuspend the precipitate to obtain the exosome resuspension;

[0110] Q8. Transfer the exosome resuspension to a centrifuge tube, centrifuge at 12000r / min for 8min, take the supernatant. If there is a lot of precipitation, centrifuge several times to take the supernatant to obtain the exosome supernatant;

[0111] Q9, transfer the exosome supernatant to the exosome purification column, centrifuge at 12000r / min for 2min, filter to the bottom of the exosome purification column to obtain the plant exosome solution;

[0112] Q10, mixing 0.1-10 mM palmitic acid with the plant exosome solution and PBS solution, wherein the volume ratio of palmitic acid to the plant exosome solution is 1:12, and the amount of PBS solution added is twice the total volume of the plant exosome solution and the palmitic acid, and mixing at 25° C. for 16 h to obtain palmitic acid-modified plant exosomes;

[0113] Q11, mixing 50% polyethylene glycol-800 with the palmitic acid-modified plant exosomes for 3 hours, adjusting the final concentration of polyethylene glycol-800 to 2.5%, and obtaining a plant exosome cross-linking system;

[0114] Q12. Add the plant exosome cross-linking system to the trehalose solution, ultracentrifuge at 12500 rpm for 2 h, and resuspend with 25% trehalose solution to obtain stability-modified plant exosomes with a concentration of 30 mg / mL.

[0115] Step (3) includes the following process:

[0116] M1: The prepared magnolol was prepared into a magnolol solution using PBS buffer, and the concentration of the magnolol solution was 8 mg / mL;

[0117] M2: The magnolol solution was mixed with the stability-modified plant exosomes and incubated in a refrigerator for 36 hours. The incubation temperature was 4°C and the rotation speed was 120 rpm. The volume ratio of magnolol solution to plant exosomes was 1:20 to obtain plant exosomes loaded with magnolol.

[0118] Embodiment 4:

[0119] The method of using magnolol-loaded plant exosomes in an oral hydrogel health product of this embodiment comprises the following steps:

[0120] Step 1: Weigh 10g of cassava starch and add it to 160g of PBS buffer to form a starch slurry, then add 20g of Bacillus subtilis-derived α-amylase to the starch slurry, stir in a 37°C water bath for 16h, stir with a magnetic stirrer at a speed of 200rpm / min, centrifuge at 2500g for 15min, add 2 times the mass of anhydrous ethanol to wash the precipitate 3 times, and dry the precipitate in a 45°C oven for 18h to obtain porous starch.

[0121] Step 2: Weigh 100 g of plant exosomes loaded with magnolia phenol and 5 g of porous starch, stir and mix at room temperature, let stand for 4 hours, gently remove the supernatant with a pipette, put it in a refrigerator at -20°C for pre-freezing for 6 hours, and then freeze-dry for 14 hours to obtain plant exosome porous starch particles loaded with magnolia phenol.

[0122] Step 3: Weigh 4g of high amylose corn starch and dissolve it in 56g of ddH 2 O, heated at 65 ° C for 20 minutes to gelatinize into gel, cooled to room temperature, then weighed 50g of high-amylose corn starch gel and 25g of magnolol-loaded plant exosome porous starch granules, mixed them evenly, put them in a refrigerator at -20 ° C for pre-freezing for 12 hours, and continued freeze-drying for 30 hours to obtain magnolol-loaded plant exosome porous starch granules coated with corn starch gel.

[0123] Step 4: Weigh 2.4 g of the magnolol-loaded plant exosome porous starch granules coated with corn starch gel, add 12.6 g of ddH 2 Stir thoroughly to dissolve it completely, weigh 0.4g sodium alginate and add it to 9.6g ddHO. 2 O to completely dissolve, then mix the two at room temperature at 400 rpm / min for 25 min, stir thoroughly to mix them evenly, and use a peristaltic pump to drip the above mixture into 50 mL of CaCl through a needle with an inner diameter of 0.51 mm. 2 The solution (1.5% w / v) solidified to form hydrogel microbeads, and the microbeads were rinsed three times with a large amount of ultrapure water to obtain magnolol-loaded plant exosome oral hydrogel.

[0124] Comparative Example 1:

[0125] Comparative Example 1 Compared with Example 3, in the preparation process of plant exosomes loaded with magnolol in Comparative Example 1, the volume ratio of magnolol solution to plant exosomes is 1:8, and other conditions remain unchanged.

[0126] Comparative Example 2:

[0127] Comparative Example 2 Compared with Example 3, in the preparation process of plant exosomes loaded with magnolol in Comparative Example 2, the volume ratio of magnolol solution to plant exosomes is 1:15, and other conditions remain unchanged.

[0128] Comparative Example 3:

[0129] Comparative Example 3 Compared with Example 3, in the preparation process of plant exosomes loaded with magnolol in Comparative Example 3, the volume ratio of magnolol solution to plant exosomes is 1:25, and other conditions remain unchanged.

[0130] Comparative Example 4:

[0131] Comparative Example 4 Compared with Example 3, in the preparation process of plant exosomes loaded with magnolol in Comparative Example 4, the volume ratio of magnolol solution to plant exosomes is 1:30, and other conditions remain unchanged.

[0132] Experimental example

[0133] The loading efficiency of the plant exosomes loaded with magnolol prepared in Example 3 and Comparative Examples 1-4 was measured, and the results are shown in Table 1:

[0134] Table 1

[0135] Group Encapsulation rate (%) Drug loading (mg / piece) Example 3 72.2 <![CDATA[63.9 / 10 11 ]]> Comparative Example 1 53.9 <![CDATA[87.0 / 10 11 ]]> Comparative Example 2 66.7 <![CDATA[70.4 / 10 11 ]]> Comparative Example 3 71.3 <![CDATA[54.6 / 10 11 ]]> Comparative Example 4 70.4 <![CDATA[40.3 / 10 11 ]]>

[0136] According to the measurement results in Table 1, it can be seen that the drug loading efficiency of the plant exosomes loaded with magnolia officinalis prepared in Example 3 of the present invention is higher than that of Comparative Examples 1-4. The experiment shows that when the volume ratio of magnolia officinalis solution to plant exosomes is 1:20, the amount of magnolia officinalis loaded on the exosomes is the largest and the encapsulation rate is the highest.

[0137] The plant exosomes loaded with magnolol prepared in Examples 1-3 and Comparative Examples 1-4 were subjected to the following experimental tests:

[0138] (1) Preparation of mouse materials: Nine cages of 8-week-old female Balb / C mice were collected, with 6 mice in each cage. Each cage was divided into 9 groups, with a total of 54 mice.

[0139] (2) Group experiment: Group I was used as the control group, and was fed with 25 g of normal feed and 5 g of normal saline per day; Group II was used as the experimental group, and was fed with 25 g of normal feed and 5 g of magnolol aqueous solution (1.5 g / mL) per day; Group III was used as the experimental group, and was fed with 25 g of normal feed and 5 g of ginger exosomes (30 mg / mL) per day; Group IV was used as the experimental group, and was fed with 25 g of normal feed and 5 g of strawberry exosomes (30 mg / mL) per day; Group V was used as the experimental group, and was fed with 25 g of normal feed and 5 g of plant exosomes loaded with magnolol prepared in Example 1 per day ; Group VI was used as the experimental group, fed with 25 g of normal feed and 5 g of the plant exosomes loaded with magnolia officinalis prepared in Example 2 every day; Group VII was used as the experimental group, fed with 25 g of normal feed and 5 g of the plant exosomes loaded with magnolia officinalis prepared in Example 3 every day; Group VIII was used as the experimental group, fed with 25 g of normal feed and 5 g of the plant exosomes loaded with magnolia officinalis prepared in Example 3 and prepared into an oral hydrogel 5 g through Example 4 every day; Group IX was used as the control group, fed with 25 g of normal feed and 5 g of the oral hydrogel prepared by ginger exosomes without magnolia officinalis through Example 4 every day, and fed for two weeks.

[0140] (3) Observation and statistical results:

[0141] Observation and statistical result 1: The condition of the rats was observed and recorded every day, and they were weighed twice a week at the same time. It was found that the mice were agile, had smooth fur, ate and drank normally, and gained weight normally during the experiment. There was no obvious abnormal pathology or death.

[0142] Observation and statistical results 2: The feces of the mice in the control group and the experimental group were sampled on the 1st day, 7th day and 14th day of the experiment, 2g of fresh feces were weighed and placed in an oven at 105°C to dry to constant weight, and the weights before and after drying were measured respectively, and the water content was calculated: feces water content (weight %) = (feces weight before drying - feces weight after drying) / feces weight before drying × 100%, and the measurement results of each group of mice were averaged. The feces water content results of each group are shown in Table 2 below:

[0143] Table 2: Water content of feces

[0144]

[0145]

[0146] The results showed that when mice were raised, compared with the control group in group I, oral administration of magnolia officinalis, ginger or strawberry exosomes could promote fecal excretion to varying degrees, among which ginger exosomes were better than strawberry exosomes in promoting fecal excretion, and plant exosomes loaded with magnolia officinalis were more effective than single magnolia officinalis or single plant exosomes. It is worth noting that the best effect was achieved when mice took plant exosome hydrogel loaded with magnolia officinalis orally, indicating that magnolia officinalis can synergistically act with plant exosomes to regulate fecal excretion in mice, and that plant exosomes loaded with magnolia officinalis can be made into hydrogel formulations to improve their utilization.

[0147] Observation and statistical results three: The feces of mice in the control group and the experimental group were sampled on the 1st, 7th and 14th days of the experiment, 2g of fresh feces were weighed and placed in a sterile ground-mouth flask containing 200mL of 0.1% peptone diluent, and the feces sample was mixed vigorously. After mixing, it was gradiently diluted into a sterilized anaerobic tube containing 9mL of 0.1% peptone diluent, and then 200μL of the appropriately diluted feces sample was added to the five solid plate culture media (BBL agar, bile glycoside sodium azide agar, crystal violet neutral red bile salt agar, MRS lactic acid bacteria agar), spread evenly, and used to culture bifidobacteria, enterococci, Escherichia coli, lactobacilli, etc., respectively. Among them, MRS lactic acid bacteria agar was placed in an anaerobic tank at 37°C for 48h, and the other three were directly placed in an aerobic incubator at 37°C for 24h. Then, the colonies on each plate were counted according to the standard plate counting method, and the average value was taken for each group of mice. The results are shown in Table 3:

[0148] Table 3 Statistics of plate counts of four types of fecal bacteria

[0149]

[0150]

[0151]

[0152] Note: The results are expressed in log10.

[0153] The results showed that when mice were raised, compared with the control group in group I, the oral administration of magnolia officinalis, ginger or strawberry exosomes could increase the beneficial intestinal bacteria to varying degrees, among which ginger exosomes were more effective than strawberry exosomes in increasing the beneficial intestinal bacteria, and the oral administration of plant exosomes loaded with magnolia officinalis was better than single magnolia officinalis or single plant exosomes. It is worth noting that the best effect was achieved when mice took plant exosome hydrogel loaded with magnolia officinalis orally, indicating that magnolia officinalis can synergize with plant exosomes to help regulate the intestinal flora of mice, and that the hydrogel formulation of plant exosomes loaded with magnolia officinalis can improve its utilization rate.

[0154] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0155] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A process for preparing plant exosomes loaded with magnolol, characterized in that: The steps include: (1) extracting magnolol from Magnolia officinalis bark or root to obtain high-purity magnolol powder; (2) extracting plant exosomes from fresh ginger and modifying them to obtain stability-modified plant exosomes; (3) Incubating high-purity magnolia officinalis powder with stability-modified plant exosomes to obtain magnolia officinalis-loaded plant exosomes.

2. The process for preparing plant exosomes loaded with magnolol according to claim 1, characterized in that: Step (1) prepares high-purity magnolol powder by crushing, ultrasonic-assisted extraction and freeze-drying dried magnolia bark.

3. The process for preparing plant exosomes loaded with magnolol according to claim 2, characterized in that: Ultrasonic-assisted extraction uses 40-90% ethanol, a solid-liquid ratio of 1:5-15, and ultrasound at 40-60°C for 20-120 min; the temperature in the freeze-drying cold trap is -40--80°C, and the freezing time is 8-12 h.

4. The process for preparing plant exosomes loaded with magnolol according to claim 1, characterized in that: Step (2) comprises washing, crushing and coarse filtering fresh ginger roots to obtain a ginger coarse filtrate; centrifuging the ginger coarse filtrate for multiple times and taking the supernatant to obtain a ginger essence filtrate; filtering the ginger essence filtrate through a microporous filter membrane to obtain a ginger membrane filtered supernatant; adding an exosome concentrated solution to the ginger membrane filtered supernatant to obtain an exosome concentrated solution; centrifuging the exosome concentrated solution for multiple times to obtain an exosome supernatant; and treating the exosome supernatant through an exosome purification column to obtain a plant exosome solution; The plant exosome solution is mixed with palmitic acid and PBS solution to obtain palmitic acid-modified plant exosomes; the plant exosomes are mixed with polyethylene glycol-800 and trehalose solution and centrifuged to obtain stability-modified plant exosomes.

5. The process for preparing plant exosomes loaded with magnolol according to claim 4, characterized in that: The diameter of the microporous filter membrane is 0.22-0.45 μm; the temperature of the centrifugation process is 0-4°C.

6. The process for preparing plant exosomes loaded with magnolol according to claim 4, characterized in that: The concentration of the palmitic acid is 0.1-10 mM, and the volume ratio of palmitic acid to plant exosome solution is 1:8-12; the original concentration of polyethylene glycol-800 is 30-50%; and the original concentration of the trehalose solution is 20-30%.

7. The process for preparing plant exosomes loaded with magnolol according to claim 1, characterized in that: Step (3) includes the following process: M1: The prepared magnolol is prepared into a magnolol solution using PBS buffer; M2: The magnolol solution was mixed with the stability-modified plant exosomes and incubated in a refrigerator to obtain magnolol-loaded plant exosomes.

8. The process for preparing plant exosomes loaded with magnolol according to claim 7, characterized in that: The concentration of the magnolol solution is 5 to 8 mg / mL, the volume ratio of the magnolol solution to the plant exosomes is 1:5 to 30; the incubation temperature is 0 to 4°C; the incubation shaker speed is 50 to 120 rpm; the incubation time is 24 to 36 hours; and the magnolol content in the plant exosomes loaded with magnolol is finally 10 to 25%.

9. Use of the plant exosomes loaded with magnolol according to any one of claims 1 to 8 in health products.

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