Calcium pulse type zingiberaceae traditional Chinese medicine exosome as well as preparation method and application thereof

Abstract

The invention discloses a calcium pulse type zingiberaceae traditional Chinese medicine exosome as well as a preparation method and application thereof. The calcium pulse type zingiberaceae traditional Chinese medicine exosome is loaded on the zingiberaceae traditional Chinese medicine exosome by taking a polyphenol compound as a calcium chelating agent, the zingiberaceae traditional Chinese medicine exosome is prepared by taking zingiberaceae traditional Chinese medicine as a raw material and sequentially carrying out drying pretreatment and ultra-high-speed centrifugation, and the obtained precipitate is the zingiberaceae traditional Chinese medicine exosome. The prepared calcium pulse type gingeaceae traditional Chinese medicine exosome avoids the problems that equipment is expensive, biological barriers are difficult to overcome, single cells are difficult to focus and the like due to the fact that a conventional physical mode is used for stimulating cell pulse type calcium adjustment, and pulse type calcium adjustment of the single cells is accurately acted on in a chemical mode for the first time; and a new nano preparation choice is provided for treatment of diseases related to calcium ions.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to calcium pulse type ginger family traditional Chinese medicine exosomes and a preparation method and application thereof. Background Art

[0002] Calcium ions (Ca 2+ )-mediated nano-modulators have multiple biological therapeutic functions and are expected to achieve breakthrough progress in disease treatment. This concept has long been proposed. However, currently, there are only a few that can truly meet the physiological conditions of cells. 2+ There are a limited number of nanoscale calcium modulators that can regulate the influx characteristics (transient and pulsatile). Most of the chemical stimuli reported so far introduce Ca 2+ The influx method is due to Ca 2+ The continuous uncontrolled influx of Ca 2+ Physically mediated nano-calcium regulators (such as heat, electricity or ultrasound) have been gradually proven to control the switch to achieve pulsed Ca overload in vitro. 2+ However, due to the skull barrier and the complex multi-cellular microenvironment of the brain, single-cell manipulation is practically impeded. 2+ Perform non-physical pulse regulation to actually control Ca2+ in single cells in vivo 2+ The physiological patterns are very limited.

[0003] Zingiberaceae Chinese medicine refers to the rhizomes of ginger plants. Its main active ingredients include 6-shogaol, 8-shogaol and 10-shogaol, which are effective in improving gastrointestinal digestion, increasing lipid oxidation metabolism and promoting hair growth. In addition, the vanillyl part of gingerol compounds can form hydrogen bonds with the T551 and E571 residues on TRPV1, thereby effectively opening the Ca2+ on the cell surface. 2+ channels (TRPV1, TRPC5, and TRPA1), are widely used as calcium agonists to increase intracellular Ca 2+ and start Ca 2+ At present, the content of gingerol compounds in the exosomes of traditional Chinese medicine extracted from ginger family is very low, and it is difficult to use it as an effective calcium agonist to increase intracellular Ca 2+ In addition, there are currently two main types of materials for treating diseases based on calcium ions: one is chemical drugs loaded with calcium membrane agonists (chemical stimulation). The advantage of this method is that it can theoretically achieve manipulation on in vivo target cells, but the disadvantage is that the release of chemical drugs is continuous and uncontrollable, which can easily cause cellular calcium overload; the other is to use physical means, which can use switches to avoid calcium overload while regulating calcium in a pulsed manner, but physical means cannot achieve good targeted regulation of single cells in vivo. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the physical stimulation of cell pulse calcium regulation in the prior art, the present invention provides a calcium pulse type ginger family Chinese medicine exosome, which is formed by loading a polyphenol compound that is more hydrophobic than gingerol compounds as a calcium chelator on the ginger family Chinese medicine exosome, and the exosome can first release a calcium agonist to preferentially increase Ca 2+ levels, the released calcium chelator subsequently reduces excess Ca 2+ The present invention has prepared a new material for chemical regulation of calcium pulses, which effectively overcomes the problems of existing chemical and physical calcium regulation.

[0005] The exosome material prepared by the present invention first releases gingerol compounds outside the cell and then releases polyphenolic compound quercetin inside the cell, which can realize intracellular Ca 2+ The goal of increasing autophagy first and then decreasing it to inhibit inflammation: gingerol compounds released first act as cell membrane Ca 2+ Activators promote the influx of extracellular calcium ions into cells and stimulate lysosomal autophagy in a calcium-dependent manner; secondly, the polyphenolic compounds released later can act as calcium chelators and bind to Ca 2+ Chelation, forming a metal polyphenol network, can achieve anti-inflammatory effects. This pulsed Ca 2+ Programmed regulatory strategies to achieve synergistic treatment of autophagy and anti-inflammation are expected to bring new treatment ideas for diseases with multifactorial pathogenic factors.

[0006] The present invention also provides a preparation method and application of the calcium pulse type Zingiberaceae traditional Chinese medicine exosomes.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the present invention discloses a calcium pulse-type Zingiberaceae Chinese medicine exosomes, wherein the calcium pulse-type Zingiberaceae Chinese medicine exosomes are loaded with polyphenol compounds as calcium chelators on the Zingiberaceae Chinese medicine exosomes, and the Zingiberaceae Chinese medicine exosomes are prepared by sequentially performing drying pretreatment and ultra-high-speed centrifugation on Zingiberaceae Chinese medicine as raw materials, and the obtained precipitate is the Zingiberaceae Chinese medicine exosomes.

[0008] The ginger family Chinese medicine exosomes are ginger family Chinese medicine exosomes rich in shogaols, and the polyphenol compounds are polyphenol compounds that are more hydrophobic than the shogaol compounds contained in the ginger family Chinese medicine exosomes.

[0009] Wherein, the ginger family Chinese medicine is any one of turmeric, alkyl oxyphylla, galangal, amomum villosum, fresh ginger, dried ginger and galangal.

[0010] Wherein, the polyphenol compound is any one of quercetin, rutin, kaempferol, luteolin and naringin.

[0011] The method for preparing calcium pulse-type ginger Chinese medicine exosomes of the present invention comprises the following steps:

[0012] (1) Drying of ginger herbal medicines: Weigh the cleaned fresh ginger, cut it into thin slices, and record the mass and sample state after drying until the sample weight is constant;

[0013] (2) adding the dried ginger Chinese medicine to a buffer solution and collecting the homogenate after stirring; collecting the supernatant after the first centrifugation; collecting the supernatant after the second centrifugation; collecting the precipitate after the third centrifugation; resuspending the precipitate in different sucrose gradient solutions after the third centrifugation, collecting the substances between the specific sucrose gradient solutions after the third centrifugation, which are the exosomes of ginger Chinese medicine;

[0014] (3) The polyphenol compounds are loaded into the exosomes of the Zingiberaceae Chinese medicine, namely, the calcium pulse-type Zingiberaceae Chinese medicine exosomes.

[0015] The drying treatment is one or more of air drying, infrared heating, freeze drying, natural shade drying, natural sun drying, microwave heating, and water bath heating.

[0016] The drying time of the blast drying is 1-50 days, and the drying temperature is 20-150°C; the wavelength of the irradiation light of the infrared heating is 700-1200nm, the irradiation light power is 2-100W, the irradiation distance is 20-100cm, and the irradiation time is 1-60min / day; the freezing temperature of the freeze drying is -15°C-80°C, and the drying time is 1-30 days; the natural shade drying is placed in a dark and ventilated place, the ambient temperature is 10-40°C, and the drying time is 1-30 days; the natural sun drying is laid in a direct sunlight environment, the ambient temperature is 10-40°C, the irradiation time is 1-8h / day, and the drying time is 1-30 days; the microwave power of the microwave heating is 500-900W, the frequency is 200-300MHz, and the drying time is 1-60min / day; the heating temperature of the water bath heating is 30-70°C, the stirring speed is 100-1000rpm, and the stirring time is 1-30 days.

[0017] Preferably, in the air drying, the cleaned ginger Chinese medicine is weighed to a mass of 1-100 g, cut into thin slices, and laid flat in an air drying oven for 1-50 days at a drying temperature of 20-150° C. The mass and sample state are recorded until the sample reaches a constant weight.

[0018] Preferably, for the infrared heating, the cleaned ginger family Chinese medicine is weighed to a mass of 1-100 g, cut into thin slices, laid flat under infrared light, the irradiation light wavelength is 700-1200 nm, the irradiation light power is 2-100 W, the irradiation distance is 20-100 cm, the irradiation time is 1-60 min / day, and the mass and sample state are recorded until the sample has a constant weight.

[0019] Preferably, for the freeze drying, the cleaned ginger Chinese medicine is weighed to a mass of 10-100 g, cut into thin slices, and laid flat in a freeze dryer at a freezing temperature of -15°C-80°C for 1-30 days. The mass and sample state are recorded until the sample reaches a constant weight.

[0020] Preferably, the natural drying is performed by weighing 1-100 g of the washed ginger Chinese medicine, cutting it into thin slices, laying it flat in a dark and ventilated place at an ambient temperature of 10-40° C. for 1-30 days, and recording the mass and sample state until the sample reaches a constant weight.

[0021] Preferably, the natural sun drying is performed by weighing 1-100 g of the washed ginger Chinese medicine, cutting it into thin slices, laying it flat in a direct sunlight environment, with an ambient temperature of 10-40° C., an irradiation time of 1-8 h / day, a drying time of 1-30 days, and recording the mass and sample state until the sample reaches a constant weight.

[0022] Preferably, in the microwave heating, 1-100 g of the cleaned ginger family Chinese medicine is weighed, cut into thin slices, and laid flat in a microwave oven. The microwave power is 500-900 W, the frequency is 200-300 MHz, the drying time is 1-60 min / day, and the mass and sample state are recorded until the sample has a constant weight.

[0023] Preferably, the water bath heating is performed, 1-100 g of the washed ginger Chinese medicine is weighed, cut into thin slices, dispersed in 10-500 mL of deionized water, the heating temperature is 30-70° C., the stirring speed is 100-1000 rpm, the stirring time is 1-30 days, and the mass and sample state are recorded until the sample has a constant weight.

[0024] The first centrifugal stratification has a centrifugal force of 2000-5000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 10-100min; the second centrifugal stratification has a centrifugal force of 10000-20000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 50-120min; the third centrifugal stratification has a centrifugal force of 150000-300000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 50-120min; the precipitate is resuspended and added to 8%, 30%, 45%, and 60% sucrose gradient solutions, 150000-300000g, a centrifugal temperature of 4-25°C, a centrifugal time of 50-120min, stratified, and the substances between the specific (8%-30%) sucrose gradient solutions are collected, which are the exosomes of traditional Chinese medicine of the ginger family.

[0025] Preferably, the ginger family Chinese medicine exosomes containing shogaols are extracted according to the following steps: adding dried ginger to a PBS buffer solution, homogenizing, and collecting the precipitate by ultracentrifugation as ginger family Chinese medicine exosomes.

[0026] Furthermore, the ginger Chinese medicine treated with drying conditions was added to PBS buffer solution, stirred in a blender for 5 minutes, and the homogenate was collected; after the first centrifugation (centrifugal force 2000g, centrifugal temperature 4°C, 10min), the supernatant was collected; after the second centrifugation (centrifugal force 10000g, centrifugal temperature 4°C, centrifugal time 120min), the supernatant was collected; after the third centrifugation (centrifugal force 150000g, centrifugal temperature 4°C, centrifugal time 120min), the precipitate was collected; the precipitate was resuspended in 8%, 30%, 45%, and 60% sucrose gradient solutions, after centrifugation (centrifugal force 150000g, centrifugal temperature 4°C, centrifugal time 120min), the material between the specific (8%-30%) sucrose gradient solutions was collected, which was the ginger Chinese medicine exosomes.

[0027] Wherein, the method for loading the polyphenol compounds into the exosomes of ginger Chinese medicine is any one of electroporation, co-incubation method, and ultrasound method.

[0028] The method for preparing calcium pulse-type Zingiberaceae traditional Chinese medicine exosomes of the present invention is used in preparing a pulse calcium regulating reagent or drug for single cells.

[0029] Wherein, the method for detecting the content of gingerol compounds in the ginger family traditional Chinese medicine exosomes rich in gingerols is mass spectrometry detection, ultra-high performance liquid chromatography detection or one or more thereof.

[0030] Furthermore, the drug release method is that most of the hydrophilic gingerol compounds are preferentially released outside the cells, thereby promoting the influx of extracellular calcium ions into the cells. Subsequently, the ginger family Chinese medicine exosomes taken up by the cells release a large amount of polyphenolic compounds inside the cells.

[0031] The present invention uses polyphenol compounds that are more hydrophobic than gingerol compounds as calcium chelators and loads them into ginger family traditional Chinese medicine exosomes to achieve different release of the drugs.

[0032] The ginger family Chinese medicine exosomes described in the present invention are made of ginger family Chinese medicine as raw materials, and are sequentially subjected to drying pretreatment and ultra-high speed centrifugation, and the resulting precipitate is ginger family Chinese medicine exosomes rich in gingerols; the drying pretreatment is one or more of forced air drying, infrared heating, freeze drying, natural shade drying, natural sun drying, microwave heating, and water bath heating, so as to significantly increase one or more gingerol compounds as calcium agonists. Further, the calcium pulse type ginger family Chinese medicine exosomes described in the present invention are polyphenol compounds that are more hydrophobic than gingerol compounds as calcium chelators, which are loaded on ginger family Chinese medicine exosomes. Due to the difference in hydrophilic and hydrophobic properties between calcium agonists and calcium chelators in calcium pulse type ginger family Chinese medicine exosomes, most of the hydrophilic gingerol compounds (calcium agonists) are preferentially released outside the cells, thereby promoting the influx of extracellular calcium ions into the cells. Subsequently, the ginger family Chinese medicine exosomes taken up by the cells release a large amount of polyphenol compounds (calcium chelators) inside the cells, thereby chelating excess intracellular calcium ions. The calcium pulse-type ginger family Chinese medicine exosomes prepared by the present invention avoid the conventional physical stimulation of cell pulse calcium regulation, which has the problems of expensive equipment, difficulty in overcoming biological barriers, and difficulty in focusing on single cells. For the first time, pulse calcium regulation that acts precisely on single cells by chemical means is realized, providing a new nano-preparation option for the treatment of calcium ion-related diseases.

[0033] The present invention uses non-physical means to develop chemical preparations for calcium pulses. Different from the existing calcium regulation technology, the present invention proposes for the first time a calcium pulse regulation based on chemical stimulation technology to achieve non-physical stimulation, which satisfies the advantages of targeted single-cell regulation, avoids calcium overload and satisfies the characteristics of physiological calcium pulse fluctuations. The present invention uses gingerol compounds in the exosomes of traditional Chinese medicine of the ginger family as calcium agonists and traditional Chinese medicine polyphenol compounds as calcium chelators. In order to achieve calcium pulse regulation, that is, the intracellular calcium ions are first increased and then decreased, the difference in hydrophilicity and hydrophobicity between gingerol compounds and traditional Chinese medicine polyphenol compounds is used, that is, the more hydrophilic gingerol compounds are first released outside the cell, preferentially activating calcium channels to first increase intracellular calcium ions, while the release of the more hydrophobic traditional Chinese medicine polyphenol compounds depends on the fusion of exosomes with cell membranes, and is almost completely released inside the cell to chelate excess calcium ions.

[0034] For ordinary ginger family Chinese medicine exosomes, the gingerol compound content is low and cannot meet the dosage requirements of calcium agonists, so it cannot be used directly to stimulate calcium membrane channels. Therefore, in the present invention, the biosynthesis process of gingerol compounds is used for the first time, and different pretreatment methods are screened to increase the content of exosome target compounds. In addition, if gingerol compounds are directly used to load directly into exosomes, the risk of calcium overload caused by the continuous release and stimulation of gingerol compounds cannot be avoided. The gingerol compounds exist in the ginger family exosomes used in the present invention, which is equivalent to exosomes loaded with gingerol compounds. Subsequent experiments also proved that ginger family exosomes cause calcium overload.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0036] The present invention uses the biosynthesis process of gingerol compounds for the first time, screens different pretreatment methods to increase the content of exosome target compounds, uses gingerol compounds in ginger family Chinese medicine exosomes as calcium agonists, and uses Chinese medicine polyphenol compounds as calcium chelators to prepare a new material for calcium pulse chemical regulation, which can achieve pulse calcium regulation of chemically stimulated cells, and provide new ideas and materials for achieving pulse calcium regulation of single cells with carriers accurately. The content of 6-gingerol compounds in the exosome material prepared by the present invention is increased, and the ginger family Chinese medicine exosomes loaded with quercetin can restore the intracellular calcium ion level to normal compared with the unloaded ginger family Chinese medicine exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The diagram is a diagram of the transformation mechanism of 6-shogaol in different pathways;

[0038] Figure 2 The extraction diagram of exosomes from ginger herbal medicine under different drying conditions;

[0039] Figure 3 The curve of the number of days required for ginger to reach constant weight under different drying treatments;

[0040] Figure 4 This is the TEM image corresponding to ginger;

[0041] Figure 5 This is the TEM image of ginger treated with air blast drying;

[0042] Figure 6 This is the TEM image of ginger treated with infrared heating;

[0043] Figure 7 This is the TEM image of freeze-dried ginger.

[0044] Figure 8 This is the TEM image of ginger that was naturally dried;

[0045] Fig. 9 This is the TEM image of ginger that was naturally dried;

[0046] Fig.10 This is the TEM image of ginger treated with microwave heating;

[0047] Fig.11 This is the TEM image of ginger treated with water bath heating;

[0048] Fig.12 BCA protein quantitative graph of exosomes of ginger herbal medicines with different drying treatments;

[0049] Fig.13 The corresponding structural diagrams of different polyphenol compounds;

[0050] Fig.14 Different polyphenols and Ca 2+ Snapshots of the chelation process at different times;

[0051] Fig.15 Different polyphenols and Ca 2+ Chelation ratio and binding energy diagram;

[0052] Fig.16 The heat map of the contents of six gingerols corresponding to the exosomes of ginger-related traditional Chinese medicines with different drying treatments;

[0053] Fig.17 The quantitative graphs of 6-shogaol and 6-shogaol corresponding to the exosomes of ginger-derived traditional Chinese medicines with different drying treatments and the conversion rate of 6-shogaol;

[0054] Fig.18 This is the zingerone content corresponding to the exosomes of ginger-derived traditional Chinese medicines with different drying treatments;

[0055] Fig.19 Schematic diagram of drug release from quercetin-loaded ginger herbal exosomes;

[0056] Fig. 20 The extracellular and intracellular release curves of 6-shogaol corresponding to the exosomes of traditional Chinese medicine of the ginger family loaded with quercetin;

[0057] Fig.21 The extracellular and intracellular release curves of quercetin corresponding to the exosomes of traditional Chinese medicine of the ginger family loaded with quercetin;

[0058] Fig. 22 In vitro pulse calcium imaging corresponding to Zingiberaceae Chinese herbal exosomes and Zingiberaceae Chinese herbal exosomes loaded with quercetin. DETAILED DESCRIPTION

[0059] The present invention is further described below with reference to the embodiments.

[0060] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0061] Example 1

[0062] 1. Drying of ginger

[0063] Take the cleaned fresh ginger and cut it into thin slices.

[0064] Air drying: weigh 50 g of cleaned fresh ginger, cut into thin slices, and place them flat in an air drying oven at a drying temperature of 60°C for 3 days. Record the mass and sample state until the sample reaches a constant weight.

[0065] Infrared heating: weigh 50 g of cleaned fresh ginger, cut into thin slices, lay flat under infrared light, with an irradiation wavelength of 850 nm, an irradiation power of 150 W, an irradiation distance of 30 cm, and an irradiation time of 30 min / day. Dry for 9 days, record the mass and sample status until the sample reaches a constant weight.

[0066] Freeze drying: Weigh 50 g of cleaned fresh ginger, cut into thin slices, and place flat in a freeze dryer. The freezing temperature is -47°C and the drying time is 5 days. Record the mass and sample state until the sample reaches a constant weight.

[0067] Natural drying in the shade: Weigh 50g of the cleaned sample, cut into thin slices, lay them flat in a dark and ventilated place at an ambient temperature of 26°C, dry them for 14 days, record the mass and sample status, until the sample weighs a constant weight.

[0068] Natural sun drying: Weigh 50g of cleaned fresh ginger, cut into thin slices, lay flat in direct sunlight at 30°C, irradiation time of 8h / day, drying time of 8 days, record the mass and sample status until the sample reaches constant weight.

[0069] Microwave heating: weigh 50 g of cleaned fresh ginger, cut into thin slices, and place flat in a microwave oven. The microwave power is 900 W, the frequency is 300 MHz, the microwave heating time is 30 min / day, the drying time is 1 day, and the mass and sample state are recorded until the sample reaches a constant weight.

[0070] Water bath heating: weigh 50 g of cleaned fresh ginger, cut into thin slices, disperse in 250 mL of deionized water, heat to 56 ° C, stir at 500 rpm, stir for 7 days, record the mass and sample state until the sample reaches a constant weight.

[0071] See also Figure 1It is shown that 6-shogaol is structurally unstable and easily undergoes aldol reverse condensation at high temperatures to generate ginger ketone and hexanal; see Figure 2 Figure 2 shows the extraction of exosomes from ginger herbal medicines under different drying conditions. Figure 3 This is the constant weight curve corresponding to different drying treatments of ginger. The results show that air drying, freeze drying and microwave drying require the shortest time, while water bath heating requires the longest drying time.

[0072] 2. Extraction of exosomes from ginger-derived Chinese medicinal herbs

[0073] 3 g of ginger dried under different drying conditions in step (1) was added to 60 mL of PBS buffer solution (pH = 7.4), stirred in a blender for 5 min, and the homogenate was collected; after the first centrifugation (centrifugal force 2000 g, centrifugal temperature 4 ° C, 10 min), the layers were separated, and the supernatant was collected; after the second centrifugation (centrifugal force 10000 g, centrifugal temperature 4 ° C, centrifugal time 120 min), the layers were separated, and the supernatant was collected; after the third centrifugation (centrifugal force 150000 g, centrifugal temperature 4 ° C, centrifugal time 120 min), the layers were separated, and the precipitate was collected; all the precipitates were resuspended in 8 mL of PBS buffer solution The top layer of the sucrose gradient solution (pH = 7.4) containing 8 mL of 8%, 30%, 45%, and 60% (mass concentration) was added to the top layer, and the layers were separated by centrifugation (centrifugal force 150000g, centrifugal temperature 4°C, centrifugal time 120min). The yellow liquid substance between the specific (8%-30%) sucrose gradient solutions was collected, which was the exosomes of ginger Chinese medicine. The number of exosomes per unit volume was determined by NTA (nanoparticle tracking analysis technology). The exosome concentration after dilution with PBS buffer solution was 1*10 9 Pieces / mL.

[0074] See also Figure 3-Figure 11 The TEM images of ginger with different drying treatments show that compared with other groups, the ginger exosomes in the air-dried group can maintain their complete vesicle structure. Fig.12 The BCA protein quantification diagram of exosomes of ginger herbal medicines with different drying treatments. Specific steps: 200 μL protein lysate and 200 μL exosomes of ginger herbal medicines with different drying treatments (1*10 9 After mixing, ultrasonic crusher was used for 30s (5 times), incubated on ice for 30min, and then centrifuged at 4°C, 12000rpm for 10min, and the supernatant after centrifugation was taken; 200μL BCA working solution and 20μL sample were added to each well, placed at 37°C, incubated for 30min, and the OD value was measured by microplate reader at a wavelength of 562nm to calculate the protein concentration. The results showed that compared with other groups, the ginger exosomes in the air-dried group retained the original protein well.

[0075] 3. Screening of polyphenol compounds

[0076] Polyphenol compounds (quercetin, luteolin, kaempferol, rutin and naringin) were optimized by gaussian software: the deprotonated structures of the five polyphenols were optimized at the 6-31g* basis set level using the B3LYP functional of the DFT method, and the RESP charges were obtained using the Multiwfn software. Dynamics simulation: All dynamics simulations were completed using gromacs-2023.2 software, and the GAFF force field was used for the Chinese medicine molecules, Ca 2+ Use the amber99 force field. 2+ and 40 polyphenols were placed in a box with a side length of 40 (theoretical calculation model construction, Fig.14 ), a total of 5 boxes were constructed, each containing a polyphenol compound. The remaining space in the box was filled with water molecules, using the TIP3P water model, and CL- was added to neutralize the system charge to keep the system neutral. The steepest gradient method was used for energy minimization (100 k mol -1 nm -1 ). Then, 0.5ns NVT and 0.5ns NPT pre-equilibrium simulations were performed respectively. Simulation parameter settings: the temperature was set to 298.15K, a V-rescale thermostat was used, and the pressure was maintained at 1 bar using a Berendsen barostat. The long-range van der Waals interaction was cut off at 10, EnerPres was used for dispersion correction, and particle-mesh Ewald was used to process electrostatic interactions. A 50ns production simulation was performed with a step size of 2fs and a trajectory saved once every 1ps. The gromacs analysis tool was used to obtain the SASA of polyphenol compounds to determine the equilibrium of the system. VMD was used to obtain snapshots of the overall trajectory and Ca at 0, 10, 20, 30, 40, and 50ns. 2+ The local coordination information of Ca and polyphenol compounds was obtained from the dynamics. The local highest coordination of Ca and each polyphenol compound was optimized and the energy was calculated in Gaussian software. The complex structure of Ca and each polyphenol compound was optimized at the 6-31g* basis set level using the B3LYP functional of the DFT method, and the binding energy was calculated at the 6-311+g** basis set, and the dispersion correction was performed using gd3bj.

[0077] Formula: E = ΔE cmple x-ΔE ca -Ex

[0078] x: represents different polyphenol compounds

[0079] See also Figure 13-Figure 15 The results showed that in the optimal chelating structure of quercetin-Ca complex, Ca 2+ The coordination ratio with quercetin is 2:3, and quercetin and Ca 2+ The highest binding energy is -85.12 kcal mol -1 In this example, the coordination ratios and binding energies of five polyphenol compounds with calcium ions were obtained through theoretical calculations. By comparing the binding energies, it was found that quercetin had the best binding performance with calcium ions, which screened out the optimal polyphenol compound for subsequent experiments.

[0080] 4. Preparation of quercetin-loaded exosomes from ginger-derived Chinese medicine

[0081] Quercetin was loaded into ginger herbal exosomes, and quercetin-loaded ginger herbal exosomes were prepared by electroporation. 0.05 mg of quercetin and 200 ul of ginger herbal exosomes with different drying treatments were mixed and loaded into a frozen electroporation cuvette, and electroporated at 1000 kV for 5 ms. After electroporation, the mixture was incubated at 37 ° C for 30 minutes to restore the exosome vesicle membrane and obtain liquid-loaded quercetin ginger herbal exosomes.

[0082] 4. Determination of gingerol content in ginger exosomes

[0083] Preparation of standard solution: Methanol is used as the solvent to prepare the standard stock solution. First, accurately weigh the 6 different gingerol standards (accurate to 0.01 mg, see Table 1 for details) on the analytical balance, transfer the weighed standards to a 10mL volumetric flask, use methanol solution to dissolve the standards and dilute to the volumetric flask, and store the prepared standard stock solution in a -20℃ refrigerator. Based on the standard stock solution, dilute it with methanol to a 1000μg / L mixed standard working solution and store it in a -20℃ refrigerator for later use; each standard solution is prepared before use. Among them, Table 1 refers to the experimental conditions of the 6 gingerols obtained by the LC-MS instrument; Table 2 is the standard curve of the 6 standards of gingerol.

[0084] Sample pretreatment: Take 1 mL of ginger exosomes with different drying treatments and dilute to 5 mL (exosomes without quercetin), vortex with a parallel vortexer for 5 minutes, sonicate for 5 minutes at room temperature, and filter through a 0.22 μm filter into a sample vial. Because the content of compounds in the samples is different, based on the need for instrument protection, compounds with high levels need to be diluted, so the extracts are diluted 5 times, 10 times, 50 times, and 1000 times respectively; the extracts after different dilutions are stored in a refrigerator at 4°C for testing.

[0085] Instrument conditions: chromatographic column C18 column, column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm; column oven temperature: 35°C; mobile phase used: (phase A) 0.1% formic acid and 2 mM ammonium formate aqueous solution, (phase B) 0.1% formic acid and 2 mM ammonium formate methanol solution; method flow rate: 0.35 mL / min; mobile phase gradient elution: 0-1 min, 85% A (15% B); 1-5 min, 50% A (50% B); 5-8 min, 20% A (80% B); 8-10 min, 0% A (100% B); 10-15 min, 0% A (100% B), sample injection volume: 5 μL.

[0086] The TSQ triple quadrupole mass spectrometry conditions are as follows: ion source type: electrospray ion source (H-ESI); scanning mode: multiple reaction monitoring (MRM); spray voltage (Ion Spray Voltage): positive ion mode 5500 V, negative ion mode 4500 V; mass spectrometry ion source temperature (Temperature): 325°C; sheath gas (Sheath Gas): 30Arb; auxiliary gas (Aux Gas): 10Arb; tail gas (Sweep Gas): 0Arb.

[0087] Table 16 gingerol compound names, retention times, MRM ion pairs and corresponding collision energies (CE),

[0088] Declustering Potential (DP) Plasma Characteristic Mass Spectrometry Conditions

[0089]

[0090] Table 2 Standard curves of 6 gingerols

[0091] name Standard curve <![CDATA[R 2 ]]> Zingerone Y=812440.17X-108511.55 0.99743 6-Gingerol Y=428152.06X-44062.04 0.99869 6-Shogaol Y＝3244525.26-714468.64 0.9958 8-Gingerol Y＝1046615.62-234155.05 0.99571 8-Shogaol Y=4260902.62X-977071.12 0.9954 10-Shogaol Y=9378868.82X-1597296.71 0.99736

[0092] The gingerol concentration in the sample was calculated as C by the standard curve of gingerol and the liquid phase test results. The gingerol content W per gram of ginger was calculated by the gingerol concentration. (A: sample dilution multiple; V: total sample volume)

[0093] W=(C*A*V) / 50

[0094] See also Figure 16-18 Compared with other groups, the air-dried group increased the content of 6-shogaol in ginger exosomes, and the content of zingerone, a byproduct produced during the drying process, was lower. The above experiments show that compared with other drying treatments, air-dried drying has three advantages: short drying time, good preservation of the original structure of exosomes, and effective increase of the content of 6-shogaol in ginger exosomes; and the polyphenol compound quercetin is loaded into ginger Chinese medicine exosomes.

[0095] 5. Determination of intracellular and extracellular release of 6-shogaol in exosomes of quercetin-loaded ginger herbal medicines

[0096] Preparation of standard solution: Methanol is used as solvent to prepare standard stock solution. First, accurately weigh the 6-gingerol and quercetin standards (accurate to 0.01 mg) on ​​the analytical balance, transfer the weighed standards to a 10 mL volumetric flask, dissolve the standards with methanol solution and adjust the volume to the volumetric flask, and store the prepared standard stock solution in a -20 ° C refrigerator. Dilute the standard stock solution with methanol to a 1000 μg / L mixed standard working solution, and store it in a -20 ° C refrigerator for use; standard solutions are prepared before use.

[0097] 5*10 SH-SY5Y cells were added 5 100 / mL were inoculated in 24-well plates and cultured for 24 h until the cells adhered to the wall. Then, MPP with a final concentration of 1 mM was added. + After 24 h of stimulation, the culture medium was removed and 1 mL of quercetin-loaded ginger herbal exosomes (at a concentration of 1*10 9 The cells were incubated with 1 mL of PBS and 1 mL of cell culture medium. The solution in each well was aspirated at 5 min, 10 min, 20 min, 30 min, 1 h, and 2 h, and the cell precipitates at each time point were collected by centrifugation. The collected culture medium containing materials at different time points was centrifuged at 14000 rpm for 30 min to separate the materials from the free drugs. 100 μL of the supernatant was added to 400 μL of methanol and filtered through a 0.22 μm organic filter membrane. The collected cell precipitates were added with 1 mL of PBS and crushed with a cell ultrasonic disruptor for 5 min, and then centrifuged at 14000 rpm for 30 min to separate the drugs in the cells. 100 μL of the supernatant was added to 400 μL of methanol and filtered through a 0.22 μm organic filter membrane. The content of 6-gingerol was detected by HPLC.

[0098] Instrument conditions: chromatographic column C18 column, column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; column oven temperature: 35°C; mobile phase used: (Phase A) acetonitrile, (Phase B) 0.1% formic acid aqueous solution; method flow rate: 1.0 mL / min; mobile phase gradient elution: 0 min, 60% A (40% B); 0-20 min, 90% A (10% B); 20-30 min, 90% A (10% B); 30-40 min, 60% A (40% B); sample injection volume: 10 μL; UV detector wavelength: 280 nm.

[0099] The concentration of 6-shogaol inside and outside the cell was calculated as C1 and the total concentration of 6-shogaol in the material was calculated as C by the standard curve of 6-shogaol and the liquid phase detection results. The percentage of 6-shogaol released inside and outside the cell was calculated by the concentration of 6-shogaol. (A: sample dilution multiple)

[0100] W=(C1*A) / C

[0101] See also Fig.19 and Fig. 20 The extracellular release of 6-shogaol from the exosomes of ginger-type traditional Chinese medicine loaded with quercetin increased with time, reaching the maximum release at about 30 minutes, and then slightly decreased within 120 minutes. The possible reason is that 6-shogaol, which is slightly soluble in water, diffuses out of the cell and is then taken up by the cell, and its content gradually decreases. The intracellular release gradually increases with time and reaches equilibrium at 30 minutes. The intracellular and extracellular release data show that 6-shogaol is mainly released extracellularly.

[0102] 6. Determination of the intracellular and extracellular release of quercetin in exosomes of zingiberaceous Chinese medicine loaded with quercetin

[0103] 5*10 SH-SY5Y cells were added 5 100 μg / mL was inoculated in a 24-well plate and cultured for 24 h until the cells adhered to the wall. Then, 1 mM MPP was added to the plate. + After 24 h of stimulation, the culture medium was removed and 1 mL of cell culture medium containing quercetin-loaded ginger herbal exosomes (concentration of 1*10 9 / mL), the culture medium of the solution in each well was aspirated at 5min, 10min, 20min, 30min, 1h, and 2h time points, and the cell precipitate at each time point was collected by centrifugation. The collected culture medium containing materials at different time points was centrifuged at 14000rpm for 30min to separate the materials and free drugs, 100μL of the supernatant was added to 400μL of methanol, and passed through a 0.22μm organic filter membrane; the cell precipitates of the collected cells were added with 1mL PBS and crushed with a cell ultrasonic disruptor for 5min, and then centrifuged at 14000rpm for 30min to separate the drugs in the cells, 100μL of the supernatant was added to 400μL of methanol, and passed through a 0.22μm organic filter membrane. The quercetin content was detected by HPLC.

[0104] Instrument conditions: chromatographic column C18 column, column length 250mm, inner diameter 4.6mm, particle size 5μm; column oven temperature: 40°C; mobile phase used: (Phase A) acetonitrile, (Phase B) 0.2% phosphoric acid aqueous solution; method flow rate: 1.0mL / min; mobile phase gradient elution: 0-15min, 31%A (69%B); sample injection volume: 10μL; UV detector wavelength: 360nm.

[0105] The concentration of quercetin inside and outside the cells was calculated as C1 and the total concentration of quercetin in the material was calculated as C by the standard curve of 6-shogaol and the liquid phase detection results. The percentage of quercetin released inside and outside the cells was calculated by the concentration of 6-shogaol. (A: sample dilution multiple)

[0106] W=(C1*A) / C

[0107] See also Fig.19 and Fig.21 The extracellular release of quercetin in the exosomes of ginger-type traditional Chinese medicine loaded with quercetin increased with time, reaching the maximum release at about 30 minutes, and then slightly decreased within 60 minutes. The extracellular release of quercetin is small, which may be due to the difficulty of quercetin, which is poorly soluble in water, to diffuse out of the cell, resulting in a small release amount. The intracellular release gradually increased with time and reached equilibrium at 60 minutes. It is significantly higher than the intracellular release of 6 gingerol. The intracellular and extracellular release data show that quercetin is mainly released intracellularly.

[0108] The above experiments prove that the hydrophilic 6-shogaol compounds (calcium agonists) in the calcium-pulse-type ginger-type Chinese medicine exosomes prepared by the present invention can be preferentially released extracellularly; subsequently, the quercetin-loaded ginger-type Chinese medicine exosomes, after being taken up by cells, release a large amount of polyphenol compounds (calcium chelators) intracellularly.

[0109] 7. In vitro calcium imaging

[0110] SH-SY5Y cells 1*10 4 100 μL / mL was inoculated in a confocal dish and cultured for 24 h until the cells attached to the wall. Then, a final concentration of 1 mM MPP+ was added for stimulation for 24 h. Then, the culture medium was removed and a 2 μM Flou-4AM calcium ion probe was added for incubation for 30 min. After 30 min, the probe solution was removed and washed three times with PBS. Finally, 200 μL PBS (containing 1.8 mM CaCl2) solution was added and monitored using a calcium imaging system. After the baseline was stable, 100 μL (final concentration) was added. 9 / mL) of quercetin-loaded ginger herbal exosomes and non-quercetin-loaded ginger herbal exosomes to monitor the changes in calcium ion signals.

[0111] See also Fig. 22 The results showed that after stimulation by exosomes from ginger herbal medicines, Ca 2+ The signal showed a pulsed fluctuation of rising and falling. After stabilization, the intracellular calcium ion content was higher than the normal value. After stimulation by the ginger herbal exosomes loaded with quercetin, Ca 2+ The signal showed a pulsed fluctuation of rising and falling, and stabilized after 400s. 2+ The intensity basically returned to the calcium ion level before stimulation. The results showed that the quercetin loading could chelate the excess calcium ions in the body and achieve calcium ion balance, further proving that the exosomes prepared by the present invention could achieve pulsed fluctuations of intracellular calcium ions, and finally the intracellular calcium ions could return to normal levels.

Claims

1. A calcium pulse type ginger Chinese medicine exosome, characterized in that: The calcium pulse type Zingiberaceae Chinese medicine exosomes are loaded with polyphenol compounds as calcium chelators on Zingiberaceae Chinese medicine exosomes. The Zingiberaceae Chinese medicine exosomes are prepared by sequentially performing drying pretreatment and ultra-high-speed centrifugation on Zingiberaceae Chinese medicine as raw materials, and the resulting precipitate is Zingiberaceae Chinese medicine exosomes.

2. The calcium pulse type Zingiberaceae Chinese medicine exosomes according to claim 1, characterized in that: The Zingiberaceae Chinese medicine exosomes are Zingiberaceae Chinese medicine exosomes rich in shogaols, and the polyphenol compounds are polyphenol compounds that are more hydrophobic than the shogaol compounds contained in the Zingiberaceae Chinese medicine exosomes.

3. The calcium pulse type Zingiberaceae Chinese medicine exosomes according to claim 1, characterized in that: The ginger family Chinese medicine is preferably any one of turmeric, alkyl oxyphylla, galangal, amomum villosum, fresh ginger, dried ginger and galangal.

4. The calcium pulse type Zingiberaceae Chinese medicine exosomes according to claim 1, characterized in that: The polyphenol compound is any one of quercetin, rutin, kaempferol, luteolin and naringin.

5. A method for preparing calcium pulse-type ginger Chinese medicine exosomes according to claim 1, characterized in that: The steps include: (1) Drying of ginger herbal medicines: Weigh the cleaned fresh ginger, cut it into thin slices, and record the mass and sample state after drying until the sample weight is constant; (2) adding the dried ginger Chinese medicine to a buffer solution and collecting the homogenate after stirring; After the first centrifugation, the supernatant was collected; after the second centrifugation, the supernatant was collected; After a third centrifugation, the precipitate was collected; After the third centrifugation, the precipitate is resuspended in different sucrose gradient solutions, and after centrifugation and stratification, the substances between specific sucrose gradient solutions are collected, which are the exosomes of traditional Chinese medicine of the ginger family; (3) The polyphenol compounds are loaded into the exosomes of the Zingiberaceae Chinese medicine, namely, the calcium pulse-type Zingiberaceae Chinese medicine exosomes.

6. The preparation method according to claim 5, characterized in that: The drying treatment is one or more of forced air drying, infrared heating, freeze drying, natural shade drying, natural sun drying, microwave heating, and water bath heating.

7. The preparation method according to claim 5, characterized in that: The drying time of the blast drying is 1-50 days, and the drying temperature is 20-150°C; the wavelength of the irradiation light of the infrared heating is 700-1200nm, the irradiation light power is 2-100W, the irradiation distance is 20-100cm, and the irradiation time is 1-60min / day; the freezing temperature of the freeze drying is -15°C-80°C, and the drying time is 1-30 days; the natural shade drying is placed in a dark and ventilated place, the ambient temperature is 10-40°C, and the drying time is 1-30 days; the natural sun drying is laid in a direct sunlight environment, the ambient temperature is 10-40°C, the irradiation time is 1-8h / day, and the drying time is 1-30 days; the microwave power of the microwave heating is 500-900W, the frequency is 200-300MHz, and the drying time is 1-60min / day; the heating temperature of the water bath heating is 30-70°C, the stirring speed is 100-1000rpm, and the stirring time is 1-30 days.

8. The preparation method according to claim 5, characterized in that: The first centrifugal stratification has a centrifugal force of 2000-5000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 10-100min; the second centrifugal stratification has a centrifugal force of 10000-20000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 50-120min; the third centrifugal stratification has a centrifugal force of 150000-300000g, a centrifugal temperature of 4-25°C, and a centrifugal time of 50-120min; the precipitate is resuspended and added to 8%, 30%, 45%, and 60% sucrose gradient solutions, 150000-300000g, a centrifugal temperature of 4-25°C, a centrifugal time of 50-120min, stratified, and the material between the 8%-30% sucrose gradient solution is collected, which is the ginger family Chinese medicine exosomes.

9. The preparation method according to claim 4, characterized in that: The method for loading the polyphenolic compounds into the exosomes of the ginger family traditional Chinese medicine is any one of electroporation, co-incubation and ultrasound.

10. Use of the method for preparing calcium pulse-type Zingiberaceae Chinese medicine exosomes according to claim 1 in preparing a pulse calcium regulating agent or drug for single cells.

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