CN115678550B - 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 fields of nano engineering and animal virology, and particularly relates to matrine carbon quantum dots, 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 provided by the invention has the characteristics of smaller particle size, better biocompatibility, lower biotoxicity and the like, has a better inhibition effect on proliferation of arteriviridae viruses including porcine reproductive and respiratory syndrome viruses, can be used for preventing and treating porcine reproductive and respiratory syndrome, and promotes healthy development of pig industry.
Description
Technical Field
The invention belongs to the technical fields of nano engineering and animal virology, and particularly relates to matrine carbon quantum dots, a preparation method and application thereof.
Background
Carbon quantum dots (CDs), also known as carbon dots or carbon nanodots, are zero-dimensional carbonaceous nanomaterial with a size below 10nm, and 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, biological marking, photodynamic therapy, drug delivery and the like at present.
Matrine (matrine) is alkaloid extracted from plants such as Sophora, cassia and Sophora of Leguminosae, and has various biological activities including antiparasitic, antibacterial, antifungal and antiviral effects. Porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV) belongs to the genus arterivirus of the family arterividae, and mainly causes reproductive disorders such as abortion, stillbirth, mummy, weak baby, and the like in pregnant sows, respiratory diseases and death of piglets. PRRSV has been seriously detrimental to the health development of the world pig industry since discovery due to the lack of safe and effective vaccines and specific therapeutic drugs, resulting in a significant economic loss to the world pig industry. Matrine has been reported to have a certain inhibitory effect on PRRSV proliferation 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 arterividae viruses compared with matrine.
The invention provides matrine carbon quantum dots, the diameter of the matrine carbon quantum dots is 1.3-3.7 nm, the lattice spacing is 0.24nm, the optimal excitation wavelength is preferably 380-390 nm, and the optimal emission wavelength is preferably 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 pressurized condition, dissolving the heated product, and purifying to obtain solution containing matrine carbon quantum dots.
Preferably, the heating temperature is 140-220 ℃ and the heating time is 1-6 h.
Preferably, the heating is performed in a reaction kettle.
Preferably, the purification comprises: cooling and dissolving the product, centrifuging the dissolved solution to remove precipitate and obtain a product solution;
and filtering and dialyzing the product solution by using a filter membrane to obtain a solution containing the matrine carbon quantum dots.
Preferably, the pore size of the filter membrane for filtration is 0.22. Mu.m.
Preferably, the dialysis uses dialysis bags with a molecular weight cut-off of 500 Da.
The invention also provides application of the matrine carbon quantum dot or the matrine carbon quantum dot prepared by the preparation method in inhibiting proliferation of arterividae virus.
The invention also provides application of the matrine carbon quantum dot or the matrine carbon quantum dot prepared by the preparation method in preparation of medicines for preventing and treating porcine reproductive and respiratory syndrome.
The invention also provides a medicine for preventing and treating porcine reproductive and respiratory syndrome, and the active ingredients of the medicine comprise matrine carbon quantum dots or matrine carbon quantum dots prepared by the preparation method.
The beneficial effects are that:
the invention provides matrine carbon quantum dots, the diameter of the matrine carbon quantum dots is 1.3-3.7 nm, the lattice spacing is 0.24nm, the optimal excitation wavelength is preferably 380-390 nm, and the optimal emission wavelength is preferably 460-465 nm. The matrine carbon quantum dot has small particle size, high water solubility and good biocompatibility, retains main functional groups of matrine, has better inhibition effect on proliferation of arteriviridae viruses including porcine reproductive and respiratory syndrome viruses, can be used for preventing and treating porcine reproductive and respiratory syndrome, and promotes healthy development of 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 diagram of a process for synthesizing matrine carbon quantum dots (matrine-CDs) in example 1 of the present invention;
FIG. 2 shows the results of the characterization 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 spectrum diagram of matrine-CDs;
FIG. 3 shows fluorescence excitation and emission spectra of matrine-CDs of examples 1-5 according to test example 1 of the present invention;
FIG. 4 shows the results of the characterization of matrine-CDs in example 1 of test example 1 according to the present invention, wherein A is ultraviolet absorption spectrum, fluorescence excitation and emission spectrum, B is Fourier transform infrared spectrum (FT-IR), C is XPS full scan spectrum, D-F is C1s spectrum, N1s spectrum, O1s spectrum of matrine-CDs, respectively;
FIG. 5 shows cytotoxicity test results of the matrine-CDs (B-F) and matrine (A) of examples 1 to 5 according to the present invention, wherein the abscissa indicates the concentration of matrine or matrine-CDs and the ordinate indicates the relative viability of cells;
FIG. 6 shows the TCID of the matrine-CDs and matrine inhibiting PRRSV proliferation in examples 1-5 of test example 3 of the present invention 50 And (5) detecting a result.
FIG. 7 shows the TCID of matrine-CDs and matrine inhibiting PRRSV proliferation in example 1 of test example 3 of the present invention 50 Detecting a result;
FIG. 8 shows the results of an indirect Immunofluorescence (IFA) assay of the effect of matrine-CDs of example 1 on PRRSV proliferation, on the scale of 50 μm, tested in accordance with the present invention, when PRRSV WUH3 strain is inoculated for 12 h;
FIG. 9 shows the results of an indirect Immunofluorescence (IFA) assay of the effect of matrine-CDs of example 1 on PRRSV proliferation, on the scale of 50 μm, tested in accordance with the present invention, at 24h of inoculation of PRRSV WUH3 strain;
FIG. 10 shows the results of an indirect Immunofluorescence (IFA) assay of the effect of matrine-CDs of example 1 on PRRSV proliferation, on the scale of 50 μm, tested in accordance with the present invention, when PRRSV WUH3 strain is inoculated for 36 h;
FIG. 11 is a result of a Westernblot analysis of matrine-CDs inhibition of PRRSV proliferation in example 1 at a concentration of 125 μg/mL in test example 3 of the present invention;
FIG. 12 is a result of a Westernblot analysis of matrine-CDs inhibition of PRRSV proliferation in example 1 at a concentration of 250 μg/mL in test example 3 of the present invention;
FIG. 13 is a result of a Westernblot analysis of matrine-CDs inhibitory PRRSV proliferation in example 1 at a concentration of 500 μg/mL in test example 3 of the present invention.
Detailed Description
The invention provides matrine carbon quantum dots, the diameter of which 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.
The average diameter of 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 465nm preferably. The matrine carbon quantum dot surface comprises hydroxyl and carbonyl, and the hydroxyl is a functional group which is not contained in matrine, so that the solubility of the matrine carbon quantum dot can be enhanced. The matrine carbon quantum dot preferably comprises three elements of C, N and O; the contents of the three elements are preferably 78.11%, 10.67% and 11.22% in this order in percentage. The matrine carbon quantum dot preferably comprises functional groups C-H, O =c-NH, C-N and-OH, wherein-OH is a functional group which is 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 pressurized condition, and purifying the heated product to obtain solution containing matrine carbon quantum dots.
The matrine is preferably ground to obtain matrine powder. The method of the present invention is not particularly limited, and a conventional method in the art may be used. The particle size of the matrine powder is not particularly limited, and the matrine powder is in a powder state conventionally recognized in the art. The purity of matrine according to the present invention is preferably 98%. The source of matrine is not particularly limited, and the matrine can be obtained by adopting a conventional commercial product 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 even 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 according to the invention is preferably carried out in a reaction kettle; in an embodiment of the invention, the heating is performed in a reactor lined with polytetrafluoroethylene. The matrine is partially carbonized under the heating condition.
After the heating, the product obtained by heating is purified to obtain the solution containing the matrine carbon quantum dots.
The purification according to the invention preferably comprises: cooling the product, dissolving the product with water, centrifuging the dissolved solution to remove precipitate and obtain a product solution; and filtering and dialyzing the product solution by using a filter membrane to obtain a solution containing the matrine carbon quantum dots.
The product obtained by heating is preferably dissolved after cooling. The temperature after cooling according to the invention is preferably room temperature, more preferably 25 ℃. The solvent for dissolution according to the present invention is preferably water, more preferably deionized water. The mass volume ratio of the product 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 ultrasonic wave is preferably 0.5 to 2 hours, more preferably 1 hour.
After the dissolution, the invention preferably removes the precipitate by centrifugation of the dissolved solution to obtain a product solution. The rotational speed of the centrifugation according to the invention is preferably 4000-13000 rpm, more preferably 12000rpm; the time of 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 a microporous filter membrane, and the pore size of the filter membrane is preferably 0.22 mu m. The filter according to the invention is preferably an aqueous filter.
After the filtration, the solution obtained by filtering the microporous filter membrane is preferably dialyzed to obtain the solution containing the matrine carbon quantum dots. The dialysis according to the invention preferably comprises dialysis of the solution obtained by filtration for 2 to 10 hours, with water being exchanged every 2 hours, using dialysis bags with a molecular weight cut-off of 100 to 1000 Da. The molecular weight cut-off of the dialysis bag is preferably 500Da; the dialysis time is preferably 8 hours.
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 application of the matrine carbon quantum dot or the matrine carbon quantum dot prepared by the preparation method in inhibiting proliferation of arterividae virus. The arteriviridae virus is preferably porcine reproductive and respiratory syndrome virus.
Based on the application of the matrine carbon quantum dot in inhibiting the proliferation of the arterividae virus, 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 the medicine for preventing and treating the porcine reproductive and respiratory syndrome. The matrine carbon quantum dot is prepared from matrine serving as a raw material, has higher biocompatibility and lower biotoxicity than the conventional matrine, has smaller particle size, has better inhibition effect on proliferation of arterividae viruses including porcine reproductive and respiratory syndrome viruses, can be used for preparing medicines for preventing and treating porcine reproductive and respiratory syndrome, and has better application prospect.
The invention also provides a medicine for preventing and treating porcine reproductive and respiratory syndrome, and the active ingredients of the medicine comprise matrine carbon quantum dots or matrine carbon quantum dots prepared by the preparation method.
For further explanation of the present invention, the technical solutions provided by the present invention are described in detail below with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used, unless otherwise specified, are commercially available.
In the examples of the present invention, the partial reagents used are 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 the components are as follows:
DMEM cell basal medium: after 13.3g of DMEM powder was dissolved in 950mL of ultrapure water and stirred until completely dissolved, 3.7g of NaHCO was added 3 And 5.9g HEPES, adjusting pH to 6.8 with 1mol/L HCl, fixing volume to 1L, filtering with microporous membrane (0.22 μm), sterilizing, and storing at 4deg.C;
DMEM cell growth fluid: adding new born calf serum, penicillin with the final concentration of 100U/mL, 100 mug/mL and streptomycin into DMEM cell basal culture solution according to the volume ratio of 10%, and preserving at 4 ℃ for later use;
DMEM cell maintenance fluid: adding new born calf serum, penicillin with final concentration of 100U/mL and streptomycin with final concentration of 100 mug/mL into DMEM cell basal culture solution according to the proportion of 2%, and preserving at 4 ℃ for standby.
Example 1
The schematic diagram of the synthesis flow of matrine carbon quantum dots is shown in figure 1.
The preparation method of matrine carbon quantum dots (matrine-CDs) comprises the following steps:
1) Matrine (purity 98%, M109803, purchased from Shanghai aladine biochemical technologies, inc.) was ground in an agate mortar and mixed well;
2) Transferring 0.2g matrine into a reaction kettle (the lining of which is polytetrafluoroethylene) with the volume of 25mL, and reacting for 2h at 200 ℃ in an oven;
3) After the product is cooled to room temperature (25 ℃), opening the reaction kettle, adding 10mL of deionized water, and carrying out dissolution assistance for 1h under the ultrasonic condition of 40KHz and 100W;
4) Centrifuging at 12000rpm for 1h, and collecting supernatant;
5) Filtering the supernatant collected in the step 4) with a water-phase filter membrane of 0.22 μm to further remove the precipitate;
6) Putting the solution obtained in the step 5) into a dialysis bag with the molecular weight cut-off of 500Da, dialyzing with ultrapure water, replacing the ultrapure water every 2 hours, and dialyzing for 8 hours;
7) Collecting the solution in the dialysis bag, and vacuum freeze-drying to obtain purified matrine-CDs.
Example 2
Matrine carbon quantum dots were prepared by the method of example 1, except that the temperature in step 2) was 140 ℃.
Example 3
Matrine carbon quantum dots were prepared by the method of example 1, except that the temperature in step 2) was 160 ℃.
Example 4
Matrine carbon quantum dots were prepared by the method of example 1, except that the temperature in step 2) was 180 ℃.
Example 5
Matrine carbon quantum dots were prepared by the method of example 1, except that the temperature in step 2) was 220 ℃.
Test example 1
Characterization of matrine-CDs was performed as follows:
the size and morphology of the matrine-CDs synthesized in example 1 were observed by high resolution transmission electron microscopy, and the synthesized matrine-CDs were found to be spherical, well dispersed, uniformly distributed in particle size, 2.4nm in average diameter, and 0.24nm in lattice spacing. XRD diffraction showed good matrine crystallinity, while matrine-CDs crystallinity decreased, as shown in FIG. 2.
As a result of measuring the fluorescence spectra of the matrine-CDs synthesized in examples 1 to 5 by using a fluorescence spectrophotometer, as shown in FIG. 3, it was found that the optimal excitation wavelengths of the matrine-CDs synthesized at different temperatures were substantially uniform, and the optimal emission wavelengths were also substantially uniform at 380 to 390nm and at 460 to 465nm.
Further detection of the ultraviolet absorption spectrum and fluorescence spectrum of matrine-CDs prepared in example 1 using an ultraviolet-visible spectrophotometer and a fluorescence spectrophotometer revealed a small peak at 297nm, a distinct blue fluorescence at 465nm, and an optimal excitation wavelength of 390nm, as shown in fig. 4 a.
Fourier transform infrared spectra (FT-IR) of matrine-CDs synthesized in example 1 were examined and compared to matrine as shown in fig. 4B: as a result, matrine-CDs were found to be 3425cm -1 There appears a broad peak (-OH) that is absent from matrine, other major peaks such as C-H (2935 cm -1 )、O＝C-NH(1631cm -1 ) And C-N (1417 cm) -1 ) Both matrine and matrine-CDs are consistent, indicating that the main functional groups of matrine are preserved during the preparation of matrine-CDs.
For further analysis of the elemental composition and surface functional groups of matrine-CDs, the matrine-CDs synthesized in example 1 were characterized by X-ray photoelectron spectroscopy (XPS) and the results are shown in FIGS. 4C-F:
XPS full scan spectra show matrine-CDs mainly contains C, N, O elements with peaks 284eV, 399eV and 531eV respectively, and elemental compositions 78.11%, 10.67% and 11.22% respectively (C in FIG. 4); high resolution C1s can be deconvolved into 3 peaks c=o (286.8 eV), C-O (285.2 eV), C-C/C-N (284.2 eV) (D in fig. 4); whereas high resolution N1s can be resolved into 2 peaks, which can be designated pyrroic-N (398.8 eV), amino-N (400.6 eV) (E in FIG. 4); 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) (F in fig. 4), respectively.
XPS is matched with the characterization result of FT-IR, and the fact that hydroxyl and carbonyl exist on the surface of matrine-CDs synthesized by the invention is confirmed, and a new hydrophilic group-hydroxyl (-OH) appears.
Test example 2
Cytotoxicity detection of matrine-CDs is performed as follows:
the effect of matrine, matrine-CDs prepared in examples 1-5, on MARC-145 cell (from China Center for Type Culture Collection (CCTCC) at university of Wuhan, wuhan) was examined by the CCK-8 (from Shanghai Biyun biotechnology Co., ltd., C0042) method. MARC-145 was thin in good growth statusCells are inoculated in 96-well plates, 100 mu L of cells are used in each well, and the cell quantity is 0.5 to 2 multiplied by 10 5 The culture solution is sucked and discarded after the cells grow into a single layer;
setting test groups: drug-treated wells, cell control wells, and blank control wells.
Drug treatment wells: comprises a matrine group and a matrine-CDs group (including matrine-CDs prepared in examples 1-5);
matrine group is: matrine containing different concentrations (2000. Mu.g/mL, 1750. Mu.g/mL, 1500. Mu.g/mL, 1250. Mu.g/mL, 1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL and 125. Mu.g/mL) was added to a 96-well plate from which the culture solution was pipetted, 100. Mu.L of matrine was used per well, and 6 wells were made per drug concentration;
matrine-CDs group: matrine-CDs containing different concentrations (2000. Mu.g/mL, 1750. Mu.g/mL, 1500. Mu.g/mL, 1250. Mu.g/mL, 1000. Mu.g/mL, 500. Mu.g/mL, 250. Mu.g/mL and 125. Mu.g/mL) were added to 96-well plates from which the culture broth was pipetted, 100. Mu.L each, and 6 multiplex wells were made for each drug concentration;
cell control wells: 6 compound holes are arranged without drug treatment;
blank control wells: no cells and no drug treatment are added, and 6 compound holes are arranged;
96-well plates containing drug-treated wells, cell control wells and blank control wells were incubated at 37℃with 5% CO 2 The cell culture was continued for 48 hours in the incubator. Adding 10 μl of CCK-8 reagent into each well, incubating the 96-well plate in 37 ℃ incubator for 1h, and measuring the absorbance OD of each well at 450nm with a multifunctional enzyme-labeled instrument 450nm . Calculate the average OD for each group 450nm The viability of untreated control cells was set at 100% based on the average OD of each cell group 450nm The values calculate cell viability.
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 indicates matrine or matrine-CDs concentration and the ordinate indicates relative viability of cells.
From the fitting calculation of fig. 5 in combination with GraphPad Prism 7, it can be derived that: matrine CC 50 (half cytotoxicity concentration) 1437. Mu.g/mL, CC for matrine-CDs in examples 1-5 50 1575. Mu.g/mL, 1503. Mu.g/mL, 1513. Mu.g/mL, 1496. Mu.g/mL and 1478. Mu.g/mL in this order, indicate that matrine-CDs and matrine have little toxicity to MARC-145 cells and matrine-CDs have less cytotoxicity.
Test example 3
The effect of matrine-CDs on PRRSV proliferation 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 with matrine effect
1)TCID 50 The effect of matrine-CDs on PRRSV proliferation was examined in examples 1-5
(1) MARC-145 cells were inoculated into 24-well cell culture plates, 1mL per well, with cell numbers of 0.5-2X 10 5 individual/mL;
(2) when the cell grows to 80% -90% and is fused, absorbing the cell culture solution, adding cell maintenance solution containing matrine of 500 mug/mL or matrine-CDs prepared at different temperatures, 1mL of each hole, setting 3 compound holes at each concentration, and setting 5% CO at 37 DEG C 2 Incubating in an incubator for 2 hours;
(3) adding matrine or matrine-CDs corresponding to the step (2) into PRRSVWUH3 strain (GenBank accession number: HM853673; separated and stored in a separate room for the virus of the key laboratory of agricultural microbiology national center of China university of agriculture) virus solution, wherein the final concentration is 500 mug/mL respectively, uniformly mixing, and then reacting for 1h at 4 ℃;
(4) inoculating the treated cells with PRRSV in an amount of 0.5MOI, incubating at 37℃for 1h, pipetting away the inoculum, rinsing the cells 3 times with 1 XPBS;
(5) adding 1mL of a cell maintenance solution containing matrine or matrine-CDs corresponding to step (2) per well at 37℃with 5% CO 2 Continuously culturing in an incubator;
(6) respectively collecting samples after culturing for 36h, repeatedly freezing and thawing for three times, and performing TCID 50 And (5) detecting. At the same time set without addingControl group (Control) of drug treatment.
⑦TCID 50 The specific operation of the detection is as follows: MARC-145 cells were seeded into 96-well cell culture plates and placed in 5% CO at 37 ℃C 2 Culturing in an incubator. After the cells grew to 90% confluence, the samples to be tested were serially diluted 10-fold in 1.5mLEP tubes with DMEM cell maintenance solution from 10 -1 To 10 -8 Different dilutions of virus were inoculated into columns 1-8 of a 96 well cell microplate, 8 wells were inoculated per dilution, 100 μl per well, and equal amounts of DMEM cell maintenance solution were added to columns 9-10 as controls. 96-well cell culture plates were placed at 37℃in 5% CO 2 Culturing in incubator is continued, and the number of cells producing CPE is recorded, and observed day by day, until cytopathy is stable. Calculation of TCID of virus according to Reed-Muench method 50 The results are shown in FIG. 6.
As can be seen from fig. 6: when the concentrations of matrine and matrine-CDs are 500 mug/mL, the proliferation inhibition effect of matrine-CDs prepared in examples 1-5 on PRRSV is stronger than that of matrine, and especially the matrine-CDs prepared in example 1 by adopting a heating temperature of 200 ℃ has the strongest inhibition effect on PRRSV proliferation.
2) By TCID using the procedure in step 1) 50 Further examining the effect of different concentrations of matrine-CDs synthesized in example 1 on PRRSV proliferation at different infection times
The difference is that:
the cell maintenance solution added in the step (2) contains matrine or matrine-CDs of 500 μg/mL, 250 μg/mL and 125 μg/mL respectively;
in the step (6), samples are collected after culturing for 12h, 24h and 36h respectively, and TCID is carried out after repeated freeze thawing for three times 50 And (5) detecting.
The results are shown in FIG. 7.
As can be seen from FIG. 7, both matrine and matrine-CDs can effectively inhibit PRRSV proliferation in MARC-145 cells, and the inhibition has time and dose dependence, and the inhibition effect is most obvious with 12h, the inhibition effect of matrine-CDs is significantly stronger than matrine, and high concentration of matrine-CDs (500 mug/mL) can completely inhibit PRRSV proliferation at 3 time points (12 h, 24h, 36h after infection) detected.
3) IFA detection of the Effect of matrine-CDs synthesized in example 1 on PRRSV proliferation
(1) MARC-145 cells were inoculated into 24-well cell culture plates in which cell climbing sheets were previously placed, 1mL per well, and the cell amount was 0.5 to 2X 10 5 individual/mL;
(2) when the cell growth reaches 80% -90% fusion, the cell culture solution is sucked, and cell maintenance solution containing matrine or matrine-CDs with different concentrations (500 mug/mL, 250 mug/mL, 125 mug/mL) is added, 1mL is added in each hole, and 5% CO is added at 37 DEG C 2 Incubating in an incubator for 2 hours;
(3) adding matrine or matrine-CDs into PRRSV WUH3 strain virus liquid to final concentration of 500 μg/mL, 250 μg/mL and 125 μg/mL respectively, mixing well, and reacting at 4deg.C for 1h;
(4) inoculating the PRRSV treated in the step (3) to the treated cells in an amount of 0.5MOI, incubating for 1h at 37 ℃, sucking off the inoculum, and rinsing the cells 3 times with 1 XPBS;
(5) adding 1mL of a cell maintenance solution containing matrine or matrine-CDs at a concentration corresponding to step (2) per well, 5% CO at 37 DEG C 2 Continuously culturing in an incubator;
(6) and (5) respectively collecting samples after culturing for 12 hours, 24 hours and 36 hours, and performing IFA detection. Control wells without drug treatment (Control) and cell Control wells without drug treatment or toxin (Mock) were also set.
(7) The specific operation of IFA detection is: sucking and discarding cell culture solution, rinsing cells 3 times with PBS for 5 min/time, fixing cells with 4% paraformaldehyde for 15min, permeabilizing with pre-cooled methanol at-20deg.C for 10min, rinsing cells 3 times with pre-cooled PBS for 5 min/time after permeabilization; blocking with PBS containing 5% BSA at room temperature for 60min, adding a murine monoclonal antibody against PRRSVN protein (the murine monoclonal antibody against PRRSVN protein was prepared and stored in a separate room for the virus of the national key laboratory of agricultural microbiology, university of agricultural, china, see Jiang Y, fang L, luo R, xiao S, chen H.2010.N-acetylpenicillamine inhibits the replication of porcine reproductive and respiratory syndrome virus in vitro. Veterinary Research Communications 34:607-617.) and incubating the cells 3 times with PBS; fluorescein Isothiocyanate (FITC) (AlexaFluor 488) labeled rabbit anti-mouse IgG (purchased from Jackson ImmunoResearch, inc, cat# 200-002-037) was added, incubated at 37℃for 1h in the absence of light, and the cells were rinsed 3 times with PBS; DAPI (4', 6-diamidino-2-phenylindole) was added, the reaction was carried out at room temperature in the absence of light for 10min, and the cells were rinsed 3 times with PBS; the slide was taken out, fixed on a slide glass with a slide sealing liquid, observed under an Olympus Fluoview ver.3.1 laser confocal microscope, and photographed, and the results are shown in fig. 8 to 10.
From FIGS. 8 to 10, it was found that both matrine and matrine-CDs were effective in inhibiting PRRSV proliferation at 3 different time points (12 h, 24h, 36 h) after inoculation of PRRSV WUH3 strain, and that the effect of matrine-CDs was significantly stronger than matrine. Only the low dose group (125 mug/mL) of matrine-CDs treated group can observe that a small number of cells fluoresce 36 hours after virus inoculation, and the rest of cells in the holes have no fluorescent reaction; the inhibition effect of matrine on PRRSV is obvious in time and dose dependence, the inhibition effect on early viral replication is most obvious, and only high-dose (500 mug/mL) matrine can completely inhibit the proliferation of viruses 12h after virus infection.
4) Western blot detection of the Effect of matrine-CDs synthesized in example 1 on PRRSV proliferation
(1) MARC-145 cells were inoculated into 6-well cell culture plates, 3mL per well, with cell numbers of 0.5-2X 10 5 individual/mL;
(2) when the cell growth reaches 80% -90% fusion, the cell culture solution is sucked, and cell maintenance solution containing matrine or matrine-CDs with different concentrations (500 mug/mL, 250 mug/mL, 125 mug/mL) is added, 3mL is added in each hole, and 5% CO is added at 37 DEG C 2 Incubating in an incubator for 2 hours;
steps (3) - (6) are synchronized with step 3);
(7) the specific operation of Western Blot detection is: the cell culture solution was aspirated, the cells were rinsed 3 times with PBS, 300. Mu.L of cell lysate was added to each well, and the cells were scraped with the cells. The lysed cell suspension was aspirated with a disposable 1mL syringe, added to a 1.5mL EP tube, and blotted approximately 20 times with the syringe. Adding a corresponding volume of 5 XSDS-PAGE loading buffer, boiling for 10min, centrifuging at 12000rpm for 10min, taking supernatant, performing SDS-PAGE electrophoresis, transferring PVDF membrane (polyvinylidene fluoride membrane) after electrophoresis, sealing by TBST containing 10% skimmed milk, taking a murine monoclonal antibody against PRRSVN/nsp2 protein (the murine monoclonal antibody against PRRSV N/nsp2 protein is prepared and stored in a national key laboratory virus laboratory of agricultural microbiology of China university) as a primary antibody, performing Western Blot detection by taking HRP-labeled rabbit anti-mouse IgG as a secondary antibody, and performing image acquisition by a chemiluminescence imaging system, wherein the results are shown in figures 11-13.
From FIGS. 11 to 13, it was found that 3 concentrations (500. Mu.g/mL, 250. Mu.g/mL, 125. Mu.g/mL) of matrine-CDs completely inhibited the expression of N and nsp2 proteins of PRRSV at different time points (12 h, 24h and 36h after infection), while matrine was effective in inhibiting the expression of N and nsp2 proteins of PRRSV at different time points, but only high concentrations (500. Mu.g/mL) of matrine completely inhibited the expression of N and nsp2 proteins of PRRSV at 12h after infection, and the results were consistent with the IFA detection results, indicating that matrine-CDs significantly inhibited PRRSV proliferation more than matrine.
From the above results, it can be seen that both matrine and matrine-CDs inhibit PRRSV proliferation in MARC-145 cells, but matrine-CDs are significantly more potent than matrine.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
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;
the preparation method of the matrine carbon quantum dot comprises the following steps: heating matrine powder under pressurized condition to obtain heated product;
cooling the heated product, dissolving, centrifuging the dissolved solution to remove precipitate, and obtaining a product solution;
filtering and dialyzing the product solution by using a filter membrane to obtain a solution containing the matrine carbon quantum dots;
the heating temperature is 140-220 ℃ and the heating time is 1-6 h.
2. The matrine carbon quantum dot according to claim 1, wherein the heating is performed in a reaction kettle.
3. The matrine carbon quantum dot according to claim 1, wherein the pore size of the filter membrane is 0.22 μm.
4. The matrine carbon quantum dot according to claim 1 or 3, wherein the dialysis uses a dialysis bag having a molecular weight cut-off of 500 Da.
5. The use of matrine carbon quantum dots as claimed in any one of claims 1 to 4 in the preparation of a medicament for inhibiting proliferation of a virus of the arterividae family.
6. The use of matrine carbon quantum dots according to any one of claims 1-4 in the preparation of a medicament for preventing and treating porcine reproductive and respiratory syndrome.
7. A medicament for preventing and treating porcine reproductive and respiratory syndrome, which is characterized in that the active ingredient of the medicament comprises matrine carbon quantum dots as claimed in any one of claims 1 to 4.
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