CN111214458A - Curcuma rhizome-derived exosome-like nanoparticle and preparation method thereof - Google Patents
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Abstract
According to the turmeric-derived exosome-like nano-particles and the preparation method thereof, turmeric tissue protoplasm is centrifuged at the temperature of 4 ℃, so that the obtained tissue supernate keeps the activity of substances; carrying out resuspension and dispersion on the obtained precipitate by using a phosphate buffer solution, carrying out discontinuous sucrose gradient centrifugation on a resuspension solution to form discontinuous density gradient, separating substances with different molecular weights by using the action of centrifugal force, then collecting liquid placed between liquid layers with 30% sucrose concentration and 45% sucrose concentration, wherein the liquid layer contains high curcumin content, then washing the liquid until the liquid is in a clear state, and then carrying out resuspension and dispersion on the precipitate to obtain turmeric-derived exosome-like nanoparticles; the method has simple process flow and is easy to realize.
Description
Technical Field
The invention belongs to the technical field of turmeric extraction, and particularly relates to preparation and application of turmeric-derived exosome-like nanoparticles.
Background
Turmeric, a plant of the family Zingiberaceae of the order Musaceae, is often consumed by humans because of its various pharmacological actions, such as anti-inflammatory, antibacterial, and antioxidant properties, and its low toxicity. Research shows that the turmeric is helpful for protecting heart, protecting memory and reducing the incidence of Alzheimer's disease, and the turmeric is a powerful anti-inflammatory perfume and can promote the recovery of inflammatory diseases, such as arthritis and inflammatory intestinal diseases. In the traditional Chinese medicine theory of China, the theory of homology of medicine and food is widely accepted, and the turmeric is added into the diet to promote the healthy liver to detoxify, prevent the oxidation of cholesterol and further help to reduce the risk of atherosclerosis and apoplexy.
Curcumin is a relatively low molecular weight polyphenol compound which is separated from turmeric for the first time in 1870, is a main active ingredient of the turmeric, has a strong anti-inflammatory effect, is a very strong antioxidant, and is listed as a 3 rd generation anti-cancer chemopreventive by the national cancer institute. But because curcumin is hardly dissolved in water, the solubility in water is only 11 ng/mL; the oral administration bioavailability is low, 40% of the oral natural curcumin is excreted with feces; the curcumin in the intravenous injection mouse (10mg/kg) needs 28.1 plus or minus 5.6 hours to degrade, and the curcumin in the oral administration mouse (500mg/kg) needs 44 plus or minus 7.5 hours to degrade; and the activity is unstable, and the clinical significance of the medicine is greatly limited.
In recent years, researches show that turmeric has a natural nano drug-carrying system exosome structure, and active substances such as curcumin, membrane protein, intracellular protein and nucleic acid are contained in the turmeric, so that the turmeric has good biocompatibility. The turmeric-derived exosome-like nanoparticles can fully utilize the biological activity of curcumin, improve the physicochemical property of curcumin, improve the solubility and dissolution rate of insoluble drugs, improve gastrointestinal absorption, achieve the target purpose in vivo, avoid the use of organic solvents in the preparation process of nano drugs, and overcome the problem that the nano drugs cannot be produced in large batch. The turmeric-derived exosome-like nano-particles are used as a natural material with good edibility and antioxidant activity, and have huge clinical medicinal potential.
Disclosure of Invention
The invention aims to provide a turmeric-derived exosome-like nanoparticle and a preparation method thereof, the nanoparticle is loaded with a plurality of active substances such as curcumin, membrane protein, intracellular protein and nucleic acid naturally, can be used for treating ulcerative colitis, and is in an oral preparation form, so that the patient compliance is high.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of turmeric-derived exosome-like nanoparticles, which comprises the following steps:
step 4, carrying out heavy suspension on the precipitate obtained in the step 3 by using a phosphate buffer solution to obtain a heavy suspension;
and 5, carrying out sucrose gradient centrifugation on the heavy suspension obtained in the step 4, wherein the mass concentrations of sucrose are respectively 8%, 30% and 45%, and obtaining liquid stratification.
And 6, collecting a liquid layer between liquid layers corresponding to the liquid layers with the mass concentration of the sucrose of 30% and 45%, washing the collected liquid until the liquid is clear, and then re-suspending and dispersing the precipitate to obtain the turmeric-derived exosome-like nanoparticles.
Preferably, in step 2, the process conditions of the centrifugal treatment are: centrifuging at a speed of 6000-15000 rpm for 1-5 h.
Preferably, in step 3, the process conditions of the centrifugal treatment are: centrifuging for 1-5 h at a centrifugation speed of 50000-80000 rpm.
Preferably, in step 4, the phosphate buffer has a molarity of 0.01M and a PH of 7.4.
Preferably, in step 5, the process conditions of the centrifugal treatment are: centrifuging for 1-5 h at a centrifugation speed of 50000-80000 rpm.
A turmeric-derived exosome-like nanoparticle is prepared based on the preparation method of the turmeric-derived exosome-like nanoparticle.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the turmeric-derived exosome-like nano-particles provided by the invention comprises the following steps of centrifuging turmeric tissue protoplasm at the temperature of 4 ℃, so that the obtained tissue supernate keeps the activity of substances; carrying out resuspension and dispersion on the obtained precipitate by using a phosphate buffer solution, carrying out discontinuous sucrose gradient centrifugation on a resuspension solution to form discontinuous density gradient, separating substances with different molecular weights by using the action of centrifugal force, then collecting liquid placed between liquid layers with 30% sucrose concentration and 45% sucrose concentration, wherein the liquid layer contains high curcumin content, then washing the liquid until the liquid is in a clear state, and then carrying out resuspension and dispersion on the precipitate to obtain turmeric-derived exosome-like nanoparticles; the method has simple process flow and is easy to realize.
The turmeric-derived exosome-like nanoparticle provided by the invention is naturally loaded with various active substances such as curcumin, membrane protein, intracellular protein and nucleic acid, can be used for treating ulcerative colitis, and is in an oral preparation form, so that the patient compliance is high.
Drawings
Fig. 1 is a schematic diagram of the preparation process and mechanism of action of turmeric-derived exosome-like nanoparticles.
Figure 2 is a transmission electron microscope image of turmeric-derived exosome-like nanoparticles.
Figure 3 is a graph of the results of the particle size distribution of turmeric-derived exosome-like nanoparticles.
Figure 4 is a Zeta potential diagram of turmeric-derived exosome-like nanoparticles.
Figure 5 is a graph of the compositional analysis of the content of turmeric-derived exosome-like nanoparticle.
Figure 6 is an in vivo fluorescence imaging of turmeric-derived exosome-like nanoparticles.
Figure 7 is a cytographic image of turmeric-derived exosome-like nanoparticles.
Figure 8 is a graph of the anti-inflammatory effect of turmeric-derived exosome-like nanoparticles.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1
Specifically, the preparation method of the turmeric-derived exosome-like nanoparticle provided by the invention comprises the following steps:
wherein the centrifugation speed is 6000-15000 rpm, and the centrifugation time is 1-5 h; preferably, the centrifugation rate is 20000rpm and the centrifugation time is 3 h;
wherein the centrifugation speed is 50000-80000 rpm, and the centrifugation time is 1-5 h; preferably, the centrifugation rate is 65000rpm and the centrifugation time is 3 h;
step 4, carrying out heavy suspension on the precipitate obtained in the step 3 by using a phosphate buffer solution to obtain a heavy suspension; the sediment adheres to the wall after centrifugation and needs to be subjected to heavy suspension dispersion;
wherein the phosphate buffer solution has a molar concentration of 0.01M and a pH of 7.4, and is prepared from 3.473g of Na2HPO4·12H2O, 0.226g of NaH2PO4·12H2O and 0.9g of NaCl tri-distilled water are added to reach the volume of 1000 ml.
And 5, performing sucrose gradient centrifugation on the heavy suspension obtained in the step 4, wherein the concentration of sucrose is 8%, 30% and 45%. Discontinuous sucrose gradient centrifugation is a relatively common centrifugation method, an 8% concentration layer cannot be removed, substances with different molecular weights need to be separated, and three sucrose solutions with different concentrations of 8%, 30% and 45% are very obvious in boundary and easy to distinguish during centrifugation; when the sucrose solution is centrifuged, a discontinuous density gradient is formed in the centrifugal tube, and substances with different molecular weights can be separated by the action of centrifugal force
The sucrose is prepared in a solvent of Tris-HCl, the pH value of the Tris-HCl is 7.2, and the volume concentration of the solvent is 20 mM/L.
Wherein the centrifugation speed is 50000-80000 rpm, and the centrifugation time is 1-5 h; preferably, the centrifugation rate is 65000rpm and the centrifugation time is 3 h;
and 6, collecting liquid between 30% and 45%, washing for three times until the liquid is clear, then re-suspending and dispersing the precipitate to obtain turmeric-derived exosome-like nanoparticles, placing the turmeric-derived exosome-like nanoparticles into PBS, and storing at-80 ℃ for a long time.
Turmeric-derived exosome-like nanoparticles (TDNPs 2) were prepared using any of the methods described above.
As a result:
test 1: and (3) carrying out appearance characterization on the turmeric-derived exosome-like nanoparticles by using a transmission electron microscope.
Test 2: 50uL of turmeric-derived exosome-like nanoparticle stock solution was diluted to 1mL, and the average particle size, particle size distribution and charge state were analyzed using a dynamic light scattering particle sizer and a laser particle sizer.
Test 3: to explore the composition of the contents of turmeric-derived exosome-like nanoparticle, we performed high performance liquid chromatography analysis on it.
The transmission electron microscope results show that the nanoparticles are in a saucer-like structure similar to exosomes, as shown in fig. 2; the results of the dynamic light scattering particle size analyzer show that the nanoparticles have uniform particle size, the average particle size is 177.9nm, and the polymer dispersibility index is 0.203, as shown in FIG. 3; the detection result of the laser particle size analyzer shows that the turmeric-derived exosome-like nanoparticles have negative charges, and the Zeta potential is-21.7 mV, as shown in figure 4; the content of curcumin in the turmeric-derived exosome-like nanoparticles was determined by using a high performance liquid chromatograph, and the result showed that the content of curcumin active ingredient in turmeric-derived exosome-like nanoparticles TDNPs2 was high, as shown in fig. 5.
Example 2
The preparation method of this example is the same as that of example 1
Targeted research of curcuma derived exosome-like nanoparticles
Test 4: oral administration of TDNPs2 targets the colon, and the colon cell population specifically takes up TDNPs2
In vivo fluorescence imaging is carried out on mice at 6h and 24h after the oral administration of the nano-particle TDNPs2, and the result shows that the fluorescence intensity is strongest at the colon part at 6h after the oral administration of the TDNPs2, which indicates that the oral administration of the TDNPs2 can target the colon, and is shown in figure 6.
Test 5: cell imaging experiments
We applied the nanoparticles of the present invention to intestinal epithelial cells Colon-26 and RAW264.7 for fluorescence imaging, and the results are shown in FIG. 7.
The specific operation steps are as follows: 2mg/ml of nanoparticles are added into a culture solution in which Colon-26 or RAW264.7 cells are cultured for 4 hours in a carbon dioxide incubator at 37 ℃, fixed by 4% paraformaldehyde, stained by FITC and DAPI, and imaged by a confocal microscope. Fluorescence imaging was performed by blue channel (λ ex 405 nm; λ em 425-475nm), green channel (λ em 590 nm; λ ex 460-550 nm) and red channel (λ ex 561 nm; λ em 570-620nm) on Colon-26 or RAW264.7 cells. As a result, it was found that the turmeric-derived exosome-like nanoparticles were specifically taken up by Colon-26 or RAW264.7 into cells after incubation with intestinal epithelial cells Colon-26 or RAW264.7, as compared to the control group without incubation of nanoparticles, as shown in fig. 7.
Example 3
The preparation method of this example is the same as that of example 1
Anti-inflammatory effects of turmeric-derived exosome-like nanoparticles TDNPs2
Finally, the above examples illustrate the nanoparticles by way of general and specific embodiments, but without limitation, and although the invention has been described by reference to preferred embodiments thereof, it will be understood by those of ordinary skill in the art that the invention is not limited thereto. Therefore, such modifications and improvements are intended to be included within the scope of this patent without departing from the spirit of the invention.
Claims (6)
1. A method for preparing turmeric-derived exosome-like nanoparticles, comprising the steps of:
step 1, preparing turmeric tissue primary pulp;
step 2, centrifuging the tissue protoplasm obtained in the step 1 at 4 ℃ to obtain tissue supernatant;
step 3, centrifuging the tissue supernatant collected in the step 2 at 4 ℃ to obtain a precipitate;
step 4, carrying out heavy suspension on the precipitate obtained in the step 3 by using a phosphate buffer solution to obtain a heavy suspension;
step 5, carrying out sucrose gradient centrifugation on the heavy suspension obtained in the step 4, wherein the mass concentrations of sucrose are respectively 8%, 30% and 45%, and obtaining liquid stratification;
and 6, collecting a liquid layer between liquid layers corresponding to the liquid layers with the mass concentration of the sucrose of 30% and 45%, washing the collected liquid until the liquid is clear, and then re-suspending and dispersing the precipitate to obtain the turmeric-derived exosome-like nanoparticles.
2. The method for preparing turmeric-derived exosome-like nanoparticles according to claim 1, wherein in the step 2, the centrifugal treatment process conditions are as follows: centrifuging at a speed of 6000-15000 rpm for 1-5 h.
3. The method for preparing turmeric-derived exosome-like nanoparticles according to claim 1, wherein in step 3, the centrifugal treatment process conditions are as follows: centrifuging for 1-5 h at a centrifugation speed of 50000-80000 rpm.
4. The method for preparing turmeric-derived exosome-like nanoparticles according to claim 1, wherein in step 4, the phosphate buffer has a molarity of 0.01M and a PH of 7.4.
5. The method for preparing turmeric-derived exosome-like nanoparticles according to claim 1, wherein in step 5, the centrifugal treatment process conditions are as follows: centrifuging for 1-5 h at a centrifugation speed of 50000-80000 rpm.
6. A turmeric-derived exosome-like nanoparticle prepared based on a method of preparing a turmeric-derived exosome-like nanoparticle according to any one of claims 1 to 5.
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Cited By (2)
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CN111569082A (en) * | 2020-06-11 | 2020-08-25 | 四川大学 | Oral delivery system for protein-loaded polypeptide drug exosomes |
CN113689456A (en) * | 2021-08-18 | 2021-11-23 | 山东大学 | Exosome particle size analysis device and method based on deep learning |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN111569082A (en) * | 2020-06-11 | 2020-08-25 | 四川大学 | Oral delivery system for protein-loaded polypeptide drug exosomes |
CN113689456A (en) * | 2021-08-18 | 2021-11-23 | 山东大学 | Exosome particle size analysis device and method based on deep learning |
CN113689456B (en) * | 2021-08-18 | 2023-07-25 | 山东大学 | Exosome particle size analysis device and exosome particle size analysis method based on deep learning |
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