CN115678550A - Matrine carbon quantum dot and preparation method and application thereof - Google Patents
Matrine carbon quantum dot and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano-engineering and animal virology, and particularly relates to a matrine carbon quantum dot and a preparation method and application thereof. The diameter of the matrine carbon quantum dot is 1.3-3.7 nm, the lattice spacing is 0.24nm, the optimal excitation wavelength is 380-390 nm, and the optimal emission wavelength is 460-465 nm. Compared with the conventional matrine, the matrine carbon quantum dot has the characteristics of smaller particle size, better biocompatibility, lower biotoxicity and the like, has better inhibition effect on the proliferation of arterivirridae viruses including porcine reproductive and respiratory syndrome viruses, can be used for preventing and treating the porcine reproductive and respiratory syndrome and promoting the healthy development of the pig industry.
Description
Technical Field
The invention belongs to the technical field of nano-engineering and animal virology, and particularly relates to a matrine carbon quantum dot and a preparation method and application thereof.
Background
Carbon quantum dots (CDs), also called carbon dots or carbon nanodots, are a class of zero-dimensional carbonaceous nanomaterials with the size of less than 10nm, have the characteristics of good photoluminescence, good biocompatibility, low biotoxicity, photobleaching resistance, low synthesis cost and the like, and are widely applied to the fields of biological imaging, biological sensing, biomarkers, photodynamic therapy, drug delivery and the like at present.
Matrine (matrine) is an alkaloid extracted from plants of Sophora, vespertilio and Sophora of Leguminosae, and has various biological activities including antiparasitic, antibacterial, antifungal and antiviral effects. Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) belongs to the genus of arterivirus of the family of arteriviruses and mainly causes reproductive disorders such as abortion, stillbirth, mummy, and weak piglets in pregnant sows, and respiratory diseases and death of piglets. Due to the lack of safe and effective vaccines and specific therapeutic drugs, PRRSV has seriously jeopardized the healthy development of the world pig industry since the discovery, and causes huge economic loss to the world pig industry. The matrine has a certain inhibition effect on the proliferation of PRRSV on MARC-145 cells, but the effect is not ideal.
Disclosure of Invention
The invention aims to provide matrine carbon quantum dots which have stronger inhibition effect on viruses of arterivirus family compared with matrine.
The invention provides a matrine carbon quantum dot, which has the diameter of 1.3-3.7 nm, the lattice spacing of 0.24nm, the optimal excitation wavelength of 380-390 nm and the optimal emission wavelength of 460-465 nm.
The invention also provides a preparation method of the matrine carbon quantum dot, which comprises the following steps: heating matrine powder under pressure, dissolving the heated product, and purifying to obtain solution containing matrine carbon quantum dots.
Preferably, the heating temperature is 140-220 ℃ and the time is 1-6 h.
Preferably, the heating is carried out in a reaction kettle.
Preferably, the purification comprises: cooling and dissolving the product, and centrifuging the dissolved solution to remove precipitates to obtain a product solution;
filtering the product solution with a filter membrane and dialyzing to obtain a solution containing the matrine carbon quantum dots.
Preferably, the pore size of the filtration membrane is 0.22 μm.
Preferably, the dialysis uses a dialysis bag with a molecular weight cut-off of 500 Da.
The invention also provides the matrine carbon quantum dot in the technical scheme or the application of the matrine carbon quantum dot prepared by the preparation method in inhibiting the proliferation of viruses of arterivirus family.
The invention also provides the application of the matrine carbon quantum dot or the matrine carbon quantum dot prepared by the preparation method in the preparation of the medicine for preventing and treating porcine reproductive and respiratory syndrome.
The invention also provides a medicament for preventing and treating porcine reproductive and respiratory syndrome, and the effective components of the medicament comprise the matrine carbon quantum dots or the matrine carbon quantum dots prepared by the preparation method.
Has the advantages that:
the invention provides a matrine carbon quantum dot, which has the diameter of 1.3-3.7 nm, the lattice spacing of 0.24nm, the optimal excitation wavelength of 380-390 nm and the optimal emission wavelength of 460-465 nm. The matrine carbon quantum dot has small particle size, high water solubility and good biocompatibility, retains the main functional group of matrine, has better inhibition effect on the proliferation of arterivirus including porcine reproductive and respiratory syndrome virus, can be used for preventing and treating porcine reproductive and respiratory syndrome and promoting the healthy development of the pig industry.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is a schematic flow chart of the synthesis of matrine carbon quantum dots (matrine-CDs) in example 1 of the present invention;
FIG. 2 is a characterization result of matrine-CDs in test example 1 of the present invention, wherein A and B are images of matrine-CDs under a transmission electron microscope, C is a particle size distribution diagram of matrine-CDs, and D is an X-ray diffraction spectrogram of matrine-CDs;
FIG. 3 is fluorescence excitation and emission spectra of matrine-CDs in examples 1-5 of the present invention in test example 1;
FIG. 4 shows the characterization results of matrine-CDs in example 1 in testing example 1 of the present invention, wherein A is the ultraviolet absorption spectrum, fluorescence excitation and emission spectrum, B is Fourier transform infrared (FT-IR) spectrum, C is XPS full scan spectrum, and D-F are the C1s spectrum, N1s spectrum and O1s spectrum of matrine-CDs, respectively;
FIG. 5 shows the results of cytotoxicity assays of matrine-CDs (B-F) and matrine (A) in examples 1-5 in test example 2 of the present invention, with the abscissa representing the concentration of matrine or matrine-CDs and the ordinate representing the relative viability of cells;
FIG. 6 shows matrine-CDs and TCID of matrine inhibiting PRRSV proliferation in examples 1-5 of test example 3 of the present invention 50 And (6) detecting the result.
FIG. 7 shows matrine-CDs and TCID of matrine inhibiting PRRSV proliferation in example 1 of test example 3 of the present invention 50 Detecting the result;
FIG. 8 shows the results of indirect immunofluorescence assay (IFA) detection of the effect of matrine-CDs on PRRSV proliferation in example 1, with a scale of 50 μm, when PRRSV WIH 3 strain in test example 3 of the present invention was inoculated for 12 h;
FIG. 9 shows the results of indirect immunofluorescence assay (IFA) assay of the effect of matrine-CDs on PRRSV proliferation in example 1, with a 50 μm scale, when the PRRSV WIH 3 strain of the present invention was inoculated for 24h in test example 3;
FIG. 10 shows the results of indirect immunofluorescence assay (IFA) assay of the effect of matrine-CDs on PRRSV proliferation in example 1, with a 50 μm scale, when the PRRSV WIH 3 strain of test example 3 of the present invention was inoculated for 36 h;
FIG. 11 shows the results of Westernblot assay for matrine-CDs in example 1 for inhibiting PRRSV proliferation at a concentration of 125. Mu.g/mL in test example 3 of the present invention;
FIG. 12 shows the results of Westernblot assay for matrine-CDs in example 1 for inhibiting PRRSV proliferation at a concentration of 250. Mu.g/mL in test example 3 according to the present invention;
FIG. 13 shows the results of Westernblot assay for matrine-CDs in example 1 to inhibit PRRSV proliferation at a concentration of 500. Mu.g/mL in test example 3 of the present invention.
Detailed Description
The invention provides a matrine carbon quantum dot, which has the diameter of 1.3-3.7 nm, the lattice spacing of 0.24nm, the optimal excitation wavelength of 380-390 nm and the optimal emission wavelength of 460-465 nm.
The average diameter of the matrine carbon quantum dots (matrine-CDs) is preferably 2.4nm. The optimal excitation wavelength of the matrine quantum dot is preferably 390nm. The optimal emission wavelength of the matrine quantum dot is preferably 465nm. The matrine carbon quantum dot surface comprises hydroxyl and carbonyl, wherein the hydroxyl is a functional group which is not contained in the matrine, and can enhance the solubility of the matrine carbon quantum dot. The matrine carbon quantum dot preferably comprises C, N and O; the content of the three elements is preferably 78.11%, 10.67% and 11.22% in this order in percentage. The matrine carbon quantum dot of the invention preferably comprises functional groups C-H, O = C-NH, C-N and-OH, wherein-OH is a functional group not contained in matrine.
The invention also provides a preparation method of the matrine carbon quantum dot, which comprises the following steps: heating matrine powder under pressure, and purifying the heated product to obtain solution containing matrine carbon quantum dots.
In the invention, the matrine is preferably ground to obtain matrine powder. The grinding mode is not particularly limited in the invention, and a conventional grinding mode in the field can be adopted. The particle size of the matrine powder is not particularly limited in the invention, and the matrine powder can be powder state which is generally recognized in the field. The purity of the matrine is preferably 98%. The source of the matrine is not particularly limited, and the matrine can be obtained by adopting conventional commercial products in the field.
After the matrine powder is obtained, the matrine powder is heated under the condition of pressurization. The heating temperature of the invention is preferably 140-220 ℃, more preferably 180-210 ℃, and more preferably 200 ℃; the heating time is preferably 1 to 6 hours, more preferably 1.5 to 4.5 hours, and still more preferably 2 hours. The heating is preferably carried out in a reaction kettle; in an embodiment of the invention, the heating is performed in a reaction vessel lined with polytetrafluoroethylene. The matrine is partially carbonized under the heating condition.
After the heating, the invention purifies the product obtained by heating to obtain the solution containing the matrine carbon quantum dots.
The purification according to the invention preferably comprises: cooling the product, dissolving the product in water, and centrifuging the dissolved solution to remove precipitates to obtain a product solution; filtering the product solution with a filter membrane and dialyzing to obtain a solution containing the matrine carbon quantum dots.
The invention preferably dissolves the product obtained by heating after cooling. The temperature after cooling according to the present invention is preferably room temperature, more preferably 25 ℃. The dissolved solvent of the present invention is preferably water, more preferably deionized water. The mass volume ratio of the product of the invention to water is preferably 0.1-1 g:10mL, more preferably 0.2g:10mL. The invention preferably assists the dissolution with ultrasound, the frequency of which is preferably 40KHz; the power of the ultrasound is preferably 100W; the time of the ultrasound is preferably 0.5 to 2 hours, more preferably 1 hour.
After the dissolution, the present invention preferably centrifuges the dissolved solution to remove the precipitate, and obtains a product solution. The rotating speed of the centrifugation is preferably 4000-13000 rpm, and more preferably 12000rpm; the time for the centrifugation is preferably 0.5 to 1.5 hours, more preferably 1 hour.
After the product solution is obtained, the product solution is preferably filtered by using a microporous filter membrane, and the pore diameter of the filter membrane is preferably 0.22 μm. The filtration membrane of the present invention is preferably an aqueous phase filtration membrane.
After the filtration, the solution obtained by the filtration of the microporous membrane is preferably dialyzed to obtain the solution containing the matrine carbon quantum dots. The dialysis of the invention preferably comprises dialyzing the filtered solution for 2-10 h by using a dialysis bag with molecular weight cutoff of 100-1000 Da, and changing water every 2h. The molecular weight cut-off of the dialysis bag is preferably 500Da; the dialysis time is preferably 8h.
After the solution containing the matrine carbon quantum dots is obtained, the invention preferably carries out vacuum freeze drying on the solution to obtain the matrine carbon quantum dots.
The invention also provides the matrine carbon quantum dot in the technical scheme or the application of the matrine carbon quantum dot prepared by the preparation method in inhibiting the proliferation of viruses of arterivirus family. The virus of the arterivirus family is preferably porcine reproductive and respiratory syndrome virus.
Based on the application of the matrine carbon quantum dot in inhibiting the proliferation of viruses of arterivirus, the invention also provides the application of the matrine carbon quantum dot or the matrine carbon quantum dot prepared by the preparation method in preparing a medicament for preventing and treating porcine reproductive and respiratory syndrome. The matrine carbon quantum dot is prepared by taking matrine as a raw material, has higher biocompatibility and lower biotoxicity compared with the conventional matrine, has smaller particle size, has better inhibition effect on the proliferation of viruses of arteriviruses including porcine reproductive and respiratory syndrome viruses, can be used for preparing medicaments for preventing and treating the porcine reproductive and respiratory syndrome, and has better application prospect.
The invention also provides a medicament for preventing and treating porcine reproductive and respiratory syndrome, and the effective components of the medicament comprise the matrine carbon quantum dots or the matrine carbon quantum dots prepared by the preparation method.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
The experimental procedures used in the following examples are conventional ones unless otherwise specified, and the materials, reagents and the like used therein are commercially available.
In the examples of the present invention, some of the reagents used were as follows:
the cell culture solution is Dulbecco's Modified Eagle Medium (DMEM) cell growth solution, and the cell maintenance solution is DMEM cell maintenance solution;
the specific preparation method and components are as follows:
DMEM cell basal medium: 13.3g of DMEM powder is dissolved in 950mL of ultrapure water, stirred until the solution is completely dissolved, and then 3.7g of NaHCO is added 3 And 5.9g HEPES, adjusting pH to 6.8 with 1mol/L HCl, diluting to 1L, filtering with microporous membrane (0.22 μm) for sterilization, and storing at 4 deg.C;
DMEM cell growth solution: adding newborn bovine serum, penicillin with final concentration of 100U/mL, 100 mu g/mL and streptomycin into DMEM cell basic culture solution according to the volume ratio of 10%, and storing at 4 ℃ for later use;
DMEM cell maintenance solution: newborn bovine serum, penicillin at a final concentration of 100U/mL and streptomycin at a final concentration of 100. Mu.g/mL were added to DMEM cell basic medium at a ratio of 2%, and stored at 4 ℃ for further use.
Example 1
The synthetic flow diagram of the matrine carbon quantum dot is shown in figure 1.
The preparation method of matrine carbon quantum dots (matrine-CDs) comprises the following steps:
1) Grinding matrine (purchased from Shanghai Allan Biotechnology, ltd., purity 98%, M109803) in agate mortar and mixing uniformly;
2) Transferring the ground 0.2g matrine into a reaction kettle (the inner lining of which is made of polytetrafluoroethylene) with the volume of 25mL, and reacting for 2 hours in an oven at the temperature of 200 ℃;
3) When the product is cooled to room temperature (25 ℃), opening the reaction kettle, adding 10mL of deionized water, and performing dissolution assistance for 1h under the ultrasonic condition of 40KHz 100W;
4) Centrifuging at 12000rpm for 1h, and collecting supernatant;
5) Filtering the supernatant collected in the step 4) by using a 0.22-micron water-phase filter membrane, and further removing precipitates;
6) Putting the solution obtained in the step 5) into a dialysis bag with the molecular weight cutoff of 500Da, dialyzing with ultrapure water, replacing the ultrapure water every 2 hours, and dialyzing for 8 hours;
7) And collecting the solution in the dialysis bag, and carrying out vacuum freeze drying to obtain the purified matrine-CDs.
Example 2
The matrine carbon quantum dot was prepared by the method in example 1 except that the temperature in step 2) was 140 ℃.
Example 3
The matrine carbon quantum dot was prepared by the method in example 1 except that the temperature in step 2) was 160 ℃.
Example 4
The matrine carbon quantum dot was prepared by the method in example 1 except that the temperature in step 2) was 180 ℃.
Example 5
The matrine carbon quantum dot was prepared by the method in example 1 except that the temperature in step 2) was 220 ℃.
Test example 1
Characterization of matrine-CDs, comprising the following steps:
the size and morphology of the matrine-CDs synthesized in example 1 were observed by a high-resolution transmission electron microscope, and it was found that the synthesized matrine-CDs were spherical, well dispersed, uniformly distributed in particle size, with an average diameter of 2.4nm and a lattice spacing of 0.24nm. XRD diffraction showed good matrine crystallinity, while matrine-CDs crystallinity decreased, as shown in FIG. 2.
The fluorescence spectra of matrine-CDs synthesized in examples 1-5 were measured by a fluorescence spectrophotometer, and as shown in FIG. 3, it was found that the optimal excitation wavelengths of matrine-CDs synthesized at different temperatures were substantially the same, all were 380-390 nm, and the optimal emission wavelengths were also substantially the same, were 460-465 nm.
Further detection of the ultraviolet absorption spectrum and the fluorescence spectrum of matrine-CDs prepared in example 1 was performed by using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer, and it was found that a small peak appeared at 297nm, blue fluorescence was clearly observed at 465nm, and the optimal excitation wavelength was 390nm, as shown in A in FIG. 4.
Fourier transform infrared spectroscopy (FT-IR) of matrine-CDs synthesized in example 1 was examined and compared with matrine as shown in B in FIG. 4: as a result, matrine-CDs were found to be 3425cm -1 There appears a broad peak (-OH) which matrine does not have, and other major peaks such as C-H (2935 cm) -1 )、O＝C-NH(1631cm -1 ) And C-N (1417 cm) -1 ) The matrine is consistent with the matrine-CDs, which shows that the main functional group of the matrine is reserved in the process of preparing the matrine-CDs.
In order to further analyze the elemental composition and surface functional groups of matrine-CDs, the matrine-CDs synthesized in example 1 was characterized by X-ray photoelectron spectroscopy (XPS), and the results are shown in fig. 4C to F:
the XPS full scan spectrum shows matrine-CDs contain mainly C, N, O, with corresponding peaks of 284eV, 399eV and 531eV, respectively, and with elemental compositions of 78.11%, 10.67% and 11.22% (FIG. 4C), respectively; high resolution C1s can be deconvoluted into 3 peaks C = O (286.8 eV), C-O (285.2 eV), C-C/C-N (284.2 eV) (D in fig. 4); while high resolution N1s can be decomposed into 2 peaks, which can be designated as pyrrolic-N (398.8 eV), amino-N (400.6 eV) (FIG. 4E); the high resolution O1s can be decomposed into 3 fitted peaks, assigned to C = O (530.3 eV), C-O (531.3 eV), O = C-O (532.1 eV), respectively (F in fig. 4).
The characterization results of XPS and FT-IR are matched, and the existence of hydroxyl and carbonyl on the surface of matrine-CDs synthesized by the invention is confirmed, and a new hydrophilic group-hydroxyl (-OH) appears.
Test example 2
The cytotoxicity detection of matrine-CDs comprises the following steps:
the effect of matrine, matrine-CDs prepared in examples 1-5 on the activity of MARC-145 cells (purchased from China Center for Type Culture Collection (CCTCC) of Wuhan university, wuhan, china) was examined by the CCK-8 (purchased from Shanghai Bintian Biotech Co., ltd., C0042). Marc-145 cells in good growth state were seeded in a 96-well plate at 100. Mu.L/well in a cell amount of 0.5 to 2X 10 5 Per mL, absorbing and discarding culture solution after the cells grow into a monolayer;
setting a test group: drug-treated wells, cell control wells, and blank control wells.
Drug treatment wells: comprises matrine group and matrine-CDs group (comprising the matrine-CDs prepared in the embodiments 1-5);
the matrine group is: adding matrine with different concentrations (2000 mug/mL, 1750 mug/mL, 1500 mug/mL, 1250 mug/mL, 1000 mug/mL, 500 mug/mL, 250 mug/mL and 125 mug/mL) into a 96-well plate with culture solution sucked away, wherein each well is 100 mug L, and each drug concentration is 6 multiple wells;
matrine-CDs group: adding matrine-CDs with different concentrations (2000 mug/mL, 1750 mug/mL, 1500 mug/mL, 1250 mug/mL, 1000 mug/mL, 500 mug/mL, 250 mug/mL and 125 mug/mL) into a 96-well plate with culture solution being discarded, wherein each well is 100 mug/mL, and each drug concentration is 6 multiple wells;
cell control wells: no medicine treatment is carried out, and 6 compound holes are arranged;
blank control wells: no cell is added, no drug is added for treatment, and 6 multiple holes are arranged;
the 96-well plate containing the drug-treated wells, the cell control wells and the blank control wells was subjected to 5% CO at 37 ℃ 2 The cell culture box is continued to culture for 48h. Add 10. Mu.L of CCK-8 reagent per well,placing the 96-well plate in a 37 ℃ incubator for incubation for 1h, and measuring the light absorption value OD of each well at the wavelength of 450nm by using a multifunctional microplate reader 450nm . Calculate the average OD of each group 450nm The survival rate of untreated control cells was set to 100%, based on the average OD of each group of cells 450nm The cell viability was calculated.
Cell viability = ((As-Ab)/(Ac-Ab)). 100%;
as: drug treatment well OD 450nm A value; ac: cell control well OD 450nm A value; ab: blank control well OD 450nm A value;
the cytotoxicity results of matrine and matrine-CDs are shown in FIG. 5, wherein the abscissa is the concentration of matrine or matrine-CDs and the ordinate is the relative viability of the cells.
From fig. 5 in combination with GraphPad Prism 7, fitting calculations can be made: CC of matrine 50 (half cytotoxic concentration) 1437. Mu.g/mL, CC of matrine-CDs in examples 1 to 5 50 The sequence is 1575 mu g/mL, 1503 mu g/mL, 1513 mu g/mL, 1496 mu g/mL and 1478 mu g/mL, which shows that both matrine-CDs and matrine have little toxicity to MARC-145 cells, and the matrine-CDs have smaller cytotoxicity.
Test example 3
Effect of matrine-CDs on PRRSV proliferation, the procedure was as follows:
by TCID 50 Indirect Immunofluorescence (IFA) and Western Blot experiments to detect the effect of matrine-CDs on PRRSV proliferation, and compare the effect with matrine
1)TCID 50 Examination of the Effect of matrine-CDs on PRRSV proliferation in examples 1-5
(1) MARC-145 cells were seeded in 24-well cell culture plates at 1 mL/well in cell volumes of 0.5-2X 10 5 Per mL;
(2) when the cells grew to 80% -90% confluency, the cell culture solution was discarded, and a cell maintenance solution containing 500. Mu.g/mL of matrine or matrine-CDs prepared at different temperatures was added at 1mL per well, 3 duplicate wells per concentration, and 5% CO at 37 ℃% 2 Incubating for 2h in an incubator;
(3) adding matrine or matrine-CDs corresponding to the step (2) into PRRSV WUH3 strain (GenBank accession number: HM853673; separated and stored in different rooms for key laboratory viruses in agricultural microbiology of Huazhong agricultural university) virus liquid until the final concentration is 500 mug/mL respectively, and reacting for 1h at 4 ℃ after uniformly mixing;
(4) inoculating the treated cells with the acted PRRSV at an MOI of 0.5, incubating at 37 ℃ for 1h, discarding the inoculum, rinsing the cells with 1 XPBS 3 times;
(5) adding 1mL of cell maintenance solution containing matrine or matrine-CDs corresponding to step (2) at 37 ℃ by 5% CO 2 Continuously culturing in an incubator;
(6) collecting samples after culturing for 36h, repeatedly freezing and thawing for three times, and performing TCID 50 And (6) detecting. A Control group (Control) was also set without drug treatment.
⑦TCID 50 The specific operation of detection is: seeding MARC-145 cells into 96 well cell culture plates, placing at 37 ℃ 5% CO 2 Culturing in an incubator. When the cells grow to 90% confluence, continuously diluting the sample to be tested in a 1.5mLEP tube by 10 times by using DMEM cell maintenance solution -1 To 10 -8 The virus solutions of different dilutions were inoculated into the 1 st to 8 th columns of the 96-well cell microplate, 8 wells were inoculated at each dilution, 100. Mu.L of each well was inoculated, and the same amount of DMEM cell maintenance solution was added to the 9 th to 10 th columns as a control. Placing a 96 well cell culture plate at 37 ℃ 5% 2 The incubation was continued in the incubator, and the number of CPE-producing wells of the cells was recorded day by day until the cytopathic effect was stable. Calculation of viral TCID by Reed-Muench method 50 The results are shown in FIG. 6.
As can be seen from fig. 6: when the concentration of matrine and matrine-CDs is 500 mu g/mL, the proliferation inhibition effect of the matrine-CDs prepared in the embodiments 1-5 on PRRSV is stronger than that of the matrine, and particularly, the proliferation inhibition effect of the matrine-CDs prepared at the heating temperature of 200 ℃ in the embodiment 1 on PRRSV is strongest.
2) Passing the TCID by the steps in step 1) 50 Further examine the effect of different concentrations of matrine-CDs synthesized in example 1 on the proliferation of PRRSV at different infection times
The difference lies in that:
the cell maintenance solution added in the step (2) respectively contains matrine or matrine-CDs of 500 mug/mL, 250 mug/mL and 125 mug/mL;
in the step (6), samples are respectively collected after 12h, 24h and 36h of culture, and TCID is carried out after repeated freeze thawing for three times 50 And (6) detecting.
The results are shown in FIG. 7.
As can be shown in FIG. 7, both matrine and matrine-CDs can effectively inhibit the proliferation of PRRSV in MARC-145 cells, and the inhibition effect is time-dependent and dose-dependent, and is most obvious in 12h inhibition effect, the inhibition effect of matrine-CDs is remarkably stronger than that of matrine, and the proliferation of PRRSV can be completely inhibited by high-concentration matrine-CDs (500 mug/mL) at 3 detected time points (12 h, 24h and 36h after infection).
3) IFA detection of the Effect of matrine-CDs synthesized in example 1 on PRRSV proliferation
(1) Marc-145 cells were seeded in a 24-well cell culture plate containing a cell slide in advance, 1mL per well, in an amount of 0.5 to 2X 10 5 Per mL;
(2) when the cells grow to 80-90% confluence, the cell culture solution is aspirated away, and cell maintenance solutions containing matrine or matrine-CDs at different concentrations (500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL) are added, 1mL per well, at 37 ℃ 5% CO 2 Incubating for 2h in an incubator;
(3) adding matrine or matrine-CDs into PRRSV WIH 3 strain virus solution to final concentrations of 500 μ g/mL, 250 μ g/mL and 125 μ g/mL respectively, mixing, and acting at 4 deg.C for 1h;
(4) inoculating the PRRSV acted in the step (3) with the treated cells in an amount of 0.5MOI, incubating for 1h at 37 ℃, removing the inoculum, and rinsing the cells with 1 × PBS for 3 times;
(5) adding 1mL of cell maintenance solution containing matrine or matrine-CDs at a concentration corresponding to step (2) at 37 ℃ by 5% CO 2 Continuously culturing in an incubator;
(6) samples were collected after 12h, 24h, and 36h of culture for IFA detection. Control wells (Control) without drug treatment and cell Control wells (Mock) without drug treatment and virus inoculation were also set.
(7) The specific operation of IFA detection is as follows: removing cell culture solution, rinsing cells with PBS for 3 times and 5 min/time, fixing cells with 4% paraformaldehyde for 15min, permeabilizing with methanol pre-cooled at-20 deg.C for 10min, and rinsing cells with PBS for 3 times and 5 min/time; blocking with 5% BSA in PBS at room temperature for 60min, adding a murine monoclonal antibody against PRRSV protein (the murine monoclonal antibody against PRRSV protein was prepared and stored in the laboratory for the central laboratory virus in the national agricultural microbiology of university of China, see Jiang Y, fan L, luo R, xiao S, chen H.2010.N-acetyl penicilla inhibitors the reproduction of pore reproduction and reproduction virus in vitro. Virus Research Communications 34) and incubating the cells at room temperature for 1h, rinsing the cells with PBS 3 times; adding Fluorescein Isothiocyanate (FITC) (AlexaFluor 488) -labeled rabbit anti-mouse IgG (purchased from Jackson ImmunoResearch, inc, cat # 200-002-037), incubating at 37 ℃ in the dark for 1h, rinsing the cells with PBS 3 times; adding DAPI (4', 6-diamidino-2-phenylindole), incubating at room temperature in the dark for 10min, rinsing the cells with PBS 3 times; the slide was taken out, fixed on a slide glass with mounting solution, observed under an Olympus fluoroview ver.3.1 laser confocal microscope, and photographed, and the results are shown in FIGS. 8 to 10.
From fig. 8 to 10, it is found that matrine and matrine-CDs can effectively inhibit the proliferation of PRRSV at 3 different time points (12 h, 24h and 36 h) after the PRRSV vhuh 3 strain is inoculated, and the effect of the matrine-CDs is significantly stronger than that of the matrine. Only the low-dose group (125 mu g/mL) in the matrix-CDs treatment group can observe a small amount of cells to fluoresce 36h after virus inoculation, and the cells in the other wells have no fluorescence reaction; the inhibition effect of matrine on PRRSV is obvious time and dosage dependence, the inhibition effect on the early stage of virus replication is most obvious, and only high dosage (500 mug/mL) of matrine can completely inhibit the virus proliferation 12h after virus infection.
4) Western blot detection of the influence of matrine-CDs synthesized in example 1 on PRRSV proliferation
(1) MARC-145 cells were seeded in 6-well cell culture plates at 3 mL/well in cell volumes of 0.5-2X 10 5 Per mL;
(2) waiting for the cells to growWhen 80% -90% of the cells were fused, the cell culture solution was aspirated off, and cell maintenance solutions containing matrine or matrine-CDs at different concentrations (500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL) were added at 3 mL/well, and CO was 5% at 37 ℃% 2 Incubating for 2h in an incubator;
step (3) to step (6) and step 3);
(7) the specific operation of Western Blot detection is as follows: the cell culture was aspirated, cells were rinsed 3 times with PBS, 300. Mu.L of cell lysate was added to each well, and cells were scraped off with a cell scraper. The lysed cell suspension was aspirated with a disposable 1mL syringe, added to a 1.5mL EP tube, and sufficiently aspirated about 20 times with a syringe. Adding a corresponding volume of 5 xSDS-PAGE sample buffer, boiling for 10min in boiling water, centrifuging for 10min at 12000rpm, taking the supernatant, carrying out SDS-PAGE electrophoresis, transferring a PVDF membrane (polyvinylidene fluoride membrane) after electrophoresis, sealing by TBST containing 10% skim milk, taking a mouse monoclonal antibody (a mouse monoclonal antibody for resisting PRRSV N/nsp2 protein is prepared and stored in a laboratory for a key laboratory virus of agricultural microbiology national university in China) of PRRSV N/nsp2 protein as a primary antibody, taking HRP-labeled rabbit anti-mouse IgG as a secondary antibody, carrying out Western Blot detection, and carrying out image acquisition by using a chemiluminescence imaging system, wherein the results are shown in figures 11-13.
From FIGS. 11 to 13, it is found that 3 concentrations (500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL) of matrine-CDs completely inhibit the expression of the N and nsp2 proteins of PRRSV at different time points (12 h, 24h and 36h after infection), while matrine can effectively inhibit the expression of the N and nsp2 proteins of PRRSV at different time points, but only high concentration (500. Mu.g/mL) of matrine can completely inhibit the expression of the N and nsp2 proteins of PRRSV at 12h after infection, and the result is consistent with the IFA detection result, which indicates that the inhibition effect of matrine-CDs on the proliferation of PRRSV is significantly stronger than that of matrine.
The results show that both matrine and matrine-CDs can inhibit the proliferation of PRRSV in MARC-145 cells, but the inhibition effect of the matrine-CDs is obviously stronger than that of the matrine.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. The matrine carbon quantum dot is characterized in that the diameter of the matrine carbon quantum dot is 1.3-3.7 nm, the lattice spacing is 0.24nm, the optimal excitation wavelength is 380-390 nm, and the optimal emission wavelength is 460-465 nm.
2. The method for preparing matrine carbon quantum dots according to claim 1, which comprises the following steps: heating matrine powder under pressure, and purifying the heated product to obtain solution containing matrine carbon quantum dots.
3. The method according to claim 2, wherein the heating is carried out at a temperature of 140 to 220 ℃ for 1 to 6 hours.
4. The production method according to claim 2 or 3, wherein the heating is performed in a reaction tank.
5. The method of claim 2, wherein the purifying comprises: cooling and dissolving the product, and centrifuging the dissolved solution to remove precipitates to obtain a product solution;
filtering the product solution with a filter membrane and dialyzing to obtain a solution containing the matrine carbon quantum dots.
6. The production method according to claim 5, wherein the pore size of the filtration membrane is 0.22. Mu.m.
7. The method of claim 5 or 6, wherein the dialysis uses a dialysis bag with a molecular weight cut-off of 500 Da.
8. Use of the matrine carbon quantum dot according to claim 1 or the matrine carbon quantum dot prepared by the preparation method according to any one of claims 2 to 7 for inhibiting the proliferation of viruses of the arterivirus family.
9. The use of the matrine carbon quantum dot of claim 1 or the matrine carbon quantum dot prepared by the preparation method of any one of claims 2 to 7 in the preparation of medicaments for preventing and treating porcine reproductive and respiratory syndrome.
10. A medicament for preventing and treating porcine reproductive and respiratory syndrome, which is characterized in that the effective component of the medicament comprises the matrine carbon quantum dot in claim 1 or the matrine carbon quantum dot prepared by the preparation method in any one of claims 2 to 7.
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