Carbon quantum dots derived from American cockroaches, preparation method and application thereof

Abstract

The present invention provides carbon quantum dots derived from American cockroaches, and a preparation method and application thereof, and belongs to the technical field of biomedical materials. The present invention crushes dried American cockroaches, adds a first solvent to dissolve, stirs, and filters to obtain a first solution; adds a second solvent to the first solution, ultrasonicates, and heats to obtain a second solution; the second solution is cooled, centrifuged, and separated to obtain a middle layer solution, and the middle layer solution is filtered and dialyzed to obtain a third solution; the third solution is concentrated and vacuum freeze-dried to obtain carbon quantum dots derived from American cockroaches. The carbon quantum dots derived from American cockroaches prepared by the present invention have good in vitro and in vivo fluorescence imaging properties, high biocompatibility, and can resist cell inflammatory reactions.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to carbon quantum dots derived from American cockroaches, and a preparation method and application thereof. Background Art

[0002] American cockroach (Periplaneta americana) is an important Chinese medicinal material with the effects of promoting blood circulation and dispersing blood stasis, removing toxins and eliminating malnutrition, promoting diuresis and reducing swelling, and treating stomach diseases. Its main chemical components include various unsaturated fatty acids, polyols, antimicrobial peptides, polysaccharides, flavonoids, trace elements, etc. Studies have found that the above substances have antibacterial, anti-inflammatory, anti-tumor, and tissue regeneration effects. In addition to the original drug, the current market for medicinal materials market includes Kangfuxinye, Xinmailong, Ganlong Capsules, etc. The large-scale breeding of American cockroaches in China has matured, and the development of more pharmaceutical products on this basis is a current trend.

[0003] Charcoal medicine is a kind of special Chinese medicine preparation, which has a wide range of pharmacological effects such as hemostasis, anti-inflammatory, antibacterial, antiviral, lipid-lowering, liver protection, and kidney protection. Charcoal medicine is made by carbonizing Chinese medicine from different sources at high temperature. According to reports, there are more than 70 kinds of charcoal medicine, among which plant-derived Chinese medicine is the main type. Therefore, it is urgent to expand the types of charcoal medicine and broaden the sources of charcoal medicine. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide carbon quantum dots derived from American cockroaches, and a preparation method and application thereof. The carbon quantum dots derived from American cockroaches provided by the present invention use the Chinese medicinal material American cockroaches as raw materials, have good in vivo and in vitro fluorescence imaging properties, high biocompatibility, and can resist cell inflammatory reactions.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0007] S1, crushing the dried American cockroaches, adding the first solvent to dissolve, stirring, and filtering to obtain a first solution;

[0008] S2, adding the second solvent to the first solution, ultrasonicating and heating, to obtain a second solution;

[0009] S3, the second solution is cooled and centrifuged and separated to obtain a middle solution, and the middle solution is filtered and dialyzed to obtain a third solution;

[0010] S4. Concentrating the third solution and performing vacuum freeze-drying to obtain carbon quantum dots derived from the American cockroach.

[0011] Preferably, the first solvent in S1 is ethanol with a volume fraction of 75.0% to 99.5%, and the dosage ratio of the powder of the pulverized American cockroach to the first solvent is (0.8 g to 1.3 g): (0.5 mL to 1.5 mL).

[0012] Preferably, the stirring in S1 is magnetic stirring for 3 h to 5 h, and the filtration in S1 is filtration using a 0.45 μm microporous filter.

[0013] Preferably, the second solvent in S2 is water or ethanol with a volume fraction of 20%, and the dosage ratio of the powder of the crushed American cockroach to the second solvent is (0.8 g to 1.3 g): (3.5 mL to 4.5 mL).

[0014] Preferably, the ultrasonic power of the ultrasound in S2 is 300W, and the ultrasonic time is 10min to 20min; the heating in S2 is firstly pouring the ultrasonicated solution into a hydrothermal reactor, and then heating the hydrothermal reactor in an oven at 180°C to 230°C for 4h to 6h.

[0015] Preferably, the centrifugation in S3 is performed at 12000 rpm to 15000 rpm for 15 min to 25 min; the filtration in S3 is performed using a 0.22 μm microporous filter; and the dialysis in S3 is performed using a 3000 Da dialysis bag in ultrapure water for 4 h to 6 h.

[0016] Preferably, the concentration in S4 is rotary evaporation concentration, and the volume of the third solution is concentrated to 1 / 4 to 1 / 3; the vacuum freeze-drying in S4 is drying for 48h to 72h under the conditions of vacuum degree of 10Par to 30Par and temperature of -35°C to -50°C.

[0017] The present invention also provides carbon quantum dots derived from American cockroaches prepared by the method for preparing carbon quantum dots derived from American cockroaches described in the above scheme.

[0018] Preferably, the carbon quantum dots derived from American cockroaches have a particle size of 4.2nm to 7.5nm, an average particle size of 5.6nm, are mainly composed of carbon, hydrogen, oxygen, and nitrogen elements, and can exhibit blue, green, and red fluorescence in the excitation wavelength range of 420nm to 480nm, 495nm to 580nm, and 600nm to 650nm, respectively.

[0019] The present invention also provides an application of the carbon quantum dots derived from American cockroaches described in the above scheme in anti-oxidation and / or anti-cellular inflammatory drugs.

[0020] Beneficial technical effect: The present invention provides carbon quantum dots derived from American cockroaches, and a preparation method and application thereof. The present invention comprises the following steps: crushing the prepared American cockroaches, adding a first solvent to dissolve, stirring, filtering, and obtaining a first solution; adding a second solvent to the first solution, ultrasonicating, heating, and obtaining a second solution; cooling the second solution, centrifuging, and separating, obtaining a middle-layer solution, filtering, and dialyzing the middle-layer solution, and obtaining a third solution; concentrating the third solution, and vacuum freeze-drying, to obtain carbon quantum dots derived from American cockroaches. The carbon quantum dots derived from American cockroaches prepared by the present invention have good in vitro and in vivo fluorescence imaging properties, high biocompatibility, and can resist cell inflammatory reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 a is a transmission electron microscopy image of carbon quantum dots derived from American cockroaches, where the scale is 5 nm and the built-in image is the crystal morphology of a single carbon dot at high resolution; Figure 1 b is the particle size distribution diagram of carbon quantum dots derived from American cockroach;

[0022] Figure 2 a is the blue fluorescence image of carbon quantum dots derived from American cockroaches at an excitation wavelength range of 420nm to 480nm. Figure 2 b is the green fluorescence image of carbon quantum dots derived from American cockroaches at an excitation wavelength range of 495nm to 580nm; Figure 2 c is the red fluorescence image of carbon quantum dots derived from American cockroach at an excitation wavelength range of 600nm to 650nm;

[0023] Figure 3 This is the characteristic peak distribution diagram of carbon quantum dots derived from American cockroach;

[0024] Figure 4 a is a cell image under natural light; Figure 4 b is the blue fluorescence image of cells under the excitation wavelength of 450 nm; Figure 4 c is the green fluorescence image of cells under the excitation wavelength of 560nm; Figure 4 d is the red fluorescence image of cells under the excitation wavelength of 610 nm;

[0025] Figure 5 The CCK-8 assay shows the changes in cell survival rate after cells were treated with different concentrations of carbon quantum dots from American cockroaches for 48 hours, where "*" indicates p < 0.05, with significant differences;

[0026] Figure 6 DAPI staining images of different experimental groups. Figure 6 a~6f are DAPI staining images of experimental groups a~f, respectively;

[0027] Figure 7This is a flow cytometer test of the rescue effect of different concentrations of carbon quantum dots from American cockroaches on LPS-treated cells. Figure 7 a~7f are the rescue effect diagrams of experimental groups a~f respectively;

[0028] Figure 8 is the level of reactive oxygen species (ROS) in cells of different experimental groups; a~f correspond to the reactive oxygen species levels of experimental groups a~f, respectively, and “**” indicates p<0.01, which means the difference is extremely significant. DETAILED DESCRIPTION

[0029] The present invention provides a method for preparing carbon quantum dots derived from American cockroaches, comprising the following steps:

[0030] S1, crushing the prepared American cockroach, adding the first solvent to dissolve, stirring, filtering, and obtaining a first solution;

[0031] S2, adding the second solvent to the first solution, ultrasonicating and heating, to obtain a second solution;

[0032] S3, the second solution is cooled and centrifuged and separated to obtain a middle solution, and the middle solution is filtered and dialyzed to obtain a third solution;

[0033] S4. Concentrating the third solution and performing vacuum freeze-drying to obtain carbon quantum dots derived from the American cockroach.

[0034] The method comprises the following steps: firstly crushing the prepared American cockroaches, adding the first solvent to dissolve the cockroaches, stirring the cockroaches, filtering the cockroaches, and obtaining the first solution.

[0035] In the present invention, the first solvent is preferably ethanol with a volume fraction of 75.0% to 99.5%, more preferably ethanol with a volume fraction of 75.0%; the crushing is preferably crushed to 60 mesh to 80 mesh, more preferably crushed to 70 mesh; the amount ratio of the powder after the American cockroach is crushed to the first solvent is preferably (0.8g to 1.3g): (0.5mL to 1.5mL), more preferably 1.0g: 1.0mL; the stirring is preferably magnetic stirring for 3h to 5h, more preferably magnetic stirring for 4h; the filtration is preferably filtered with a 0.45μm microporous filter, and before filtering with a 0.45μm microporous filter, it is necessary to filter with a funnel. The present invention can fully dissolve the effective ingredients in the American cockroach powder into the first solution by crushing the dried American cockroach, selecting a suitable first solvent, and controlling the amount of the first solvent added, thereby increasing the yield of carbon quantum dots from the final American cockroach source.

[0036] The present invention further adds a second solvent into the first solution, performs ultrasonic treatment and heating to obtain a second solution.

[0037] In the present invention, the second solvent is preferably water or ethanol with a volume fraction of 20%, more preferably ethanol with a volume fraction of 20%; the amount ratio of the powder of the American cockroach to the second solvent is preferably (0.8g-1.3g): (3.5mL-4.5mL), more preferably 1.0g: 4.0mL. By adding the second solvent, the first solution can be diluted to facilitate the subsequent ultrasonic and hydrothermal reactions.

[0038] In the present invention, the ultrasonic power is preferably 300 W, and the ultrasonic time is preferably 10 to 20 minutes, more preferably 15 minutes. The present invention can accelerate the release, diffusion and dissolution of the effective substances of the American cockroach through ultrasound, and increase the yield of carbon quantum dots derived from the American cockroach.

[0039] In the present invention, the heating is preferably to first pour the ultrasonicated solution into a hydrothermal reactor, and then heat the hydrothermal reactor in an oven at 180°C to 230°C for 4h to 6h, more preferably in an oven at 200°C for 5h. The present invention can fully carbonize the American cockroach extract through the hydrothermal reaction to achieve the transformation of the effective ingredients therein.

[0040] The present invention further cools the second solution, centrifuges and separates it to obtain a middle-layer solution, and the middle-layer solution is filtered and dialyzed to obtain a third solution.

[0041] In the present invention, the centrifugation is preferably performed at 12000rpm to 15000rpm for 15min to 25min, more preferably at 15000rpm for 20min; after centrifugation, the solution is transferred into a separatory funnel, and three layers are obtained after standing, wherein the upper layer is oil, the middle layer is solution, and the lower layer is sediment; the filtration is preferably performed using a 0.22μm microporous filter; the dialysis is preferably performed using a 3000Da dialysis bag in ultrapure water for 4h to 6h, and the present invention removes impurities such as lipopolysaccharide and oil in the middle layer solution through dialysis.

[0042] Finally, the third solution is concentrated and vacuum-freezed to obtain the carbon quantum dots derived from the American cockroach.

[0043] In the present invention, the concentration is preferably rotary evaporation concentration, concentrated to 1 / 4 to 1 / 3 of the volume of the third solution; the instrument for rotary evaporation concentration is preferably a rotary evaporator, and the rotary evaporation is preferably rotary evaporation under the conditions of vacuum degree of 80mBar, rotation speed of 70rpm to 90rpm, and temperature of 45°C to 55°C; the vacuum freeze-drying is preferably carried out under the conditions of vacuum degree of 10Par to 30Par and temperature of -35°C to -50°C, drying for 48h to 72h, more preferably under the conditions of vacuum degree of 20Par and temperature of -40°C, drying for 60h.

[0044] The present invention also provides carbon quantum dots derived from American cockroaches prepared by the method for preparing carbon quantum dots derived from American cockroaches described in the above scheme.

[0045] In the present invention, the particle size of the carbon quantum dots derived from the American cockroach is 4.2nm~7.5nm, the average particle size is 5.6nm, and they are mainly composed of carbon, hydrogen, oxygen, and nitrogen elements. They can exhibit blue, green, and red fluorescence in the excitation wavelength range of 420nm~480nm, 495nm~580nm, and 600nm~650nm, respectively.

[0046] The present invention also provides an application of the carbon quantum dots derived from American cockroaches described in the above scheme in anti-oxidation and / or anti-cellular inflammatory drugs.

[0047] The present invention has no special limitation on the dosage form of the drug, and the carbon quantum dots derived from American cockroaches can be used in a medically acceptable dosage form. The preparation method of the drug has no special limitation, and the preparation method of the corresponding dosage form can be used. The present invention has no special limitation on the content of the carbon quantum dots derived from American cockroaches in the drug, and the content of conventional active substances in the drug can be used.

[0048] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0049] Example 1

[0050] The method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0051] 10 g of dried American cockroaches were cut into small pieces of about 1 cm to 2 cm in size, crushed in an ultra-high-speed grinder, filtered with a 70-mesh stainless steel screen, added with 10 mL of 75% ethanol, stirred at room temperature for 3 hours on a magnetic stirrer, filtered with a funnel and then filtered with a 0.45 μm microporous filter; 40 mL of 20% ethanol solution was added to the obtained filtrate, placed in an ultrasonic instrument, ultrasonicated at 300 W for 10 minutes, poured into a polyvinyl fluoride hydrothermal reactor, and then the reactor was placed in a 200° C. oven for reaction for 5 hours.

[0052] After the reaction solution is cooled, it is poured into a 50mL centrifuge tube (the volume of the solution after the reaction is transferred in whole or in batches, the same below), placed in an ultracentrifuge, and centrifuged at 15000rpm for 20min. The centrifuged solution is transferred to a separatory funnel, allowed to stand and separated to obtain a middle layer solution, and the obtained middle layer solution is filtered with a 0.22μm microporous filter, and after filtration, it is dialyzed in ultrapure water with a 3000Da dialysis bag for 4h; the solution in the dialysis bag after dialysis is first placed in a rotary evaporator, and rotary evaporated and concentrated under the conditions of a vacuum degree of 80mBar, a rotation speed of 80rpm, and a temperature of 50°C, and concentrated to 1 / 3 of the volume of the solution obtained after dialysis, and the concentrated solution is further dried for 60h under the conditions of a vacuum degree of 20Par and a temperature of -40°C to obtain purified carbon quantum dots from American cockroaches.

[0053] Example 2

[0054] The method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0055] 8 g of dried American cockroaches were cut into small pieces of about 1 cm to 2 cm in size, crushed in an ultra-high-speed grinder, filtered with a 70-mesh stainless steel screen, added with 4 mL of anhydrous ethanol, stirred at room temperature for 4 h on a magnetic stirrer, filtered with a funnel and then filtered with a 0.45 μm microporous filter; 35 mL of pure water was added to the obtained filtrate, placed in an ultrasonic instrument, ultrasonicated at 300 W for 15 min, poured into a polyvinyl fluoride hydrothermal reactor, and then the reactor was placed in an oven at 180°C for reaction for 6 h.

[0056] After the reaction solution is cooled, it is poured into a 50mL centrifuge tube, placed in an ultracentrifuge, and centrifuged at 12000rpm for 25min. The centrifuged solution is placed in a separatory funnel, allowed to stand, and separated to obtain a middle layer solution. The obtained middle layer solution is filtered with a 0.22μm microporous filter, and after filtration, it is dialyzed in ultrapure water with a 3000Da dialysis bag for 6h; the solution in the dialysis bag after dialysis is first placed in a rotary evaporator, and rotary evaporated and concentrated under the conditions of a vacuum degree of 80mBar, a rotation speed of 90rpm, and a temperature of 45°C to 1 / 4 of the volume of the solution obtained after dialysis. The concentrated solution is further dried for 48h under the conditions of a vacuum degree of 10Par and a temperature of -50°C to obtain purified carbon quantum dots from American cockroaches.

[0057] Example 3

[0058] The method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0059] 13 g of dried American cockroaches were cut into small pieces of about 1 cm to 2 cm in size, crushed in an ultra-high-speed grinder, filtered with a 70-mesh stainless steel screen, added with 15 mL of anhydrous ethanol, placed on a magnetic stirrer and stirred at room temperature for 5 hours, filtered with a funnel and then filtered with a 0.45 μm microporous filter; 45 mL of 20% ethanol solution was added to the obtained filtrate, placed in an ultrasonic instrument, ultrasonicated at 300 W for 12 minutes, poured into a polyvinyl fluoride hydrothermal reactor, and then the reactor was placed in a 230° C. oven for reaction for 4 hours.

[0060] After the reaction solution is cooled, it is poured into a 50mL centrifuge tube, placed in an ultracentrifuge, and centrifuged at 15000rpm for 15min. The centrifuged solution is placed in a separatory funnel, allowed to stand, and separated to obtain a middle layer solution. The obtained middle layer solution is filtered with a 0.22μm microporous filter, and after filtration, it is dialyzed in ultrapure water with a 3000Da dialysis bag for 5h; the solution in the dialysis bag after dialysis is first placed in a rotary evaporator, and rotary evaporated and concentrated under the conditions of a vacuum degree of 80mBar, a rotation speed of 80rpm, and a temperature of 50°C to 1 / 4 of the volume of the solution obtained after dialysis. The concentrated solution is further dried for 62h under the conditions of a vacuum degree of 30Par and a temperature of -45°C to obtain purified carbon quantum dots from American cockroaches.

[0061] Example 4

[0062] The method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0063] 9 g of dried American cockroaches were cut into small pieces of about 1 cm to 2 cm in size, crushed in an ultra-high-speed grinder, filtered with a 70-mesh stainless steel screen, added with 10 mL of 75% ethanol, stirred at room temperature for 3 h on a magnetic stirrer, filtered with a funnel and then filtered with a 0.45 μm microporous filter; 36 mL of pure water was added to the obtained filtrate, placed in an ultrasonic instrument, ultrasonicated at 300 W for 18 min, poured into a polyvinyl fluoride hydrothermal reactor, and then the reactor was placed in a 200° C. oven for reaction for 6 h.

[0064] After the reaction solution is cooled, it is poured into a 50mL centrifuge tube, placed in an ultracentrifuge, and centrifuged at 15000rpm for 20min. The centrifuged solution is placed in a separatory funnel, allowed to stand, and separated to obtain a middle layer solution. The obtained middle layer solution is filtered with a 0.22μm microporous filter, and after filtration, it is dialyzed in ultrapure water with a 3000Da dialysis bag for 4h; the solution in the dialysis bag after dialysis is first placed in a rotary evaporator, and rotary evaporated and concentrated under the conditions of a vacuum degree of 80mBar, a rotation speed of 78rpm, and a temperature of 48°C, and concentrated to 1 / 3 of the volume of the solution obtained after dialysis. The concentrated solution is further dried for 72h under the conditions of a vacuum degree of 15Par and a temperature of -35°C to obtain purified carbon quantum dots from American cockroaches.

[0065] Example 5

[0066] The method for preparing carbon quantum dots from American cockroaches comprises the following steps:

[0067] 10 g of dried American cockroaches were cut into small pieces of about 1 cm to 2 cm in size, crushed in an ultra-high-speed grinder, filtered with a 70-mesh stainless steel screen, added with 12 mL of 75% ethanol, placed on a magnetic stirrer and stirred at room temperature for 3.5 hours, filtered with a funnel and then filtered with a 0.45 μm microporous filter; 38 mL of 20% ethanol solution was added to the obtained filtrate, placed in an ultrasonic instrument, ultrasonicated at 300 W for 16 minutes, poured into a polyvinyl fluoride hydrothermal reactor, and then the reactor was placed in a 200° C. oven for reaction for 5 hours.

[0068] After the reaction solution is cooled, it is poured into a 50mL centrifuge tube, placed in an ultracentrifuge, and centrifuged at 15000rpm for 20min. The centrifuged solution is placed in a separatory funnel, allowed to stand, and separated to obtain a middle layer solution. The obtained middle layer solution is filtered with a 0.22μm microporous filter, and after filtration, it is dialyzed in ultrapure water with a 3000Da dialysis bag for 6h; the solution in the dialysis bag after dialysis is first placed in a rotary evaporator, and rotary evaporated and concentrated under the conditions of a vacuum degree of 80mBar, a rotation speed of 82rpm, and a temperature of 52°C to 1 / 4 of the volume of the solution obtained after dialysis. The concentrated solution is further dried for 65h under the conditions of a vacuum degree of 25Par and a temperature of -42°C to obtain purified carbon quantum dots from American cockroaches.

[0069] Test Example 1

[0070] 1. The carbon quantum dots derived from American cockroaches obtained in Examples 1 to 5 were observed by transmission electron microscopy (TEM), and then the particle size was measured using Nano Measurer 1.2 software.

[0071] Transmission electron microscopy analysis results of carbon quantum dots from American cockroaches are as follows Figure 1 The particle size distribution diagram is shown in a. Figure 1 As shown in b, it can be seen from the particle size distribution that the particle size of the carbon quantum dots derived from the American cockroach is 4.2nm~7.5nm, and the average particle size is 5.6nm.

[0072] 2. The carbon quantum dots derived from American cockroaches obtained in Examples 1 to 5 were taken and their photoluminescence spectra were detected using a fluorescence spectrometer (Hitachi, Japan). The detection results are as follows: Figure 2 As shown. Among them, Figure 2 a is the blue fluorescence emitted by the carbon quantum dots of American cockroaches in the excitation wavelength range of 420nm to 480nm. Figure 2b shows that the carbon quantum dots of American cockroaches exhibit green fluorescence in the range of 495nm to 580nm. Figure 2 c: The American cockroach carbon quantum dots exhibit red fluorescence within the excitation wavelength range of 600nm to 650nm.

[0073] 3. The carbon quantum dots derived from American cockroaches obtained in Examples 1 to 5 were analyzed by Fourier transform infrared spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA). The test results are as follows: Figure 3 As shown. Figure 3 It can be seen that the American cockroach carbon quantum dots mainly contain four elements: carbon, hydrogen, oxygen and nitrogen.

[0074] 4. Cell test:

[0075] The carbon quantum dots derived from American cockroaches obtained in Examples 1 to 5 were prepared into a mother solution with a concentration of 1 mg / mL using ultrapure water;

[0076] Lipopolysaccharide (LPS) was prepared into a stock solution with a concentration of 0.1 mg / mL using ultrapure water;

[0077] Cells: RAW264.7, mouse mononuclear macrophages.

[0078] (1) Cell fluorescence test:

[0079] The above-mentioned carbon quantum dot solution with a concentration of 1 mg / mL was dissolved in the culture medium at a certain dilution ratio (50 μg / mL) and then treated with cells. The cell fluorescence changes under different excitation wavelengths were detected by fluorescence microscopy (Nikon SMZ18, Japan). The detection results are as follows: Figure 4 As shown. Figure 4 It can be seen that the carbon quantum dots obtained in the present invention have good cell fluorescence imaging performance.

[0080] (2) Cytotoxicity test:

[0081] The above-mentioned carbon quantum dot solution with a concentration of 1 mg / mL was diluted to 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 600 μg / mL with culture medium, and the cells were treated for 48 h. The changes in cell survival rate under different carbon dot concentrations were detected by CCK-8. The test results are as follows: Figure 5 As shown. Figure 5 It can be seen that the carbon quantum dots derived from American cockroach obtained in the present invention do not cause obvious cytotoxicity within a concentration of 400 μg / mL.

[0082] (3) Cell apoptosis rescue assay:

[0083] This experiment set up 6 treatments in total, using a 6-well cell plate, and each treatment was repeated 3 times. The reagents of the specific experimental groups are shown in Table 1; among them, experimental group a served as the control group and did not receive any treatment; experimental groups b and c treated the cells with the corresponding reagents in Table 1 for 24 hours; experimental groups d, e, and f first treated the cells with the corresponding carbon quantum dot solutions in Table 1 for 2 hours, removed the carbon quantum dot solution, and then treated the cells with 100 ng / mL lipopolysaccharide (LPS) for 24 hours.

[0084] Table 1 Reagent usage and processing time of different experimental groups

[0085]

[0086] ① Use DAPI to stain the cells after treatment with different experimental groups. The staining results are as follows Figure 6 As shown. Figure 6 It can be seen that carbon quantum dots derived from American cockroaches can significantly inhibit LPS-induced cell apoptosis.

[0087] ② Flow cytometry was used to detect cell apoptosis in different experimental groups. The test results were as follows: Figure 7 As shown. Figure 7 It can be seen that different concentrations of carbon quantum dots derived from American cockroaches can inhibit the increase in the number of apoptotic cells.

[0088] ③ DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) was used to detect the level of reactive oxygen species (ROS) in cells of different experimental groups; the results are as follows Figure 8 As shown. Figure 8 It can be seen that the carbon quantum dots derived from American cockroaches can significantly reduce the level of reactive oxygen species in LPS-induced cells.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing carbon quantum dots from American cockroaches, characterized in that: The following steps are involved: S1, crushing the dried American cockroaches, adding the first solvent to dissolve, stirring, and filtering to obtain a first solution; S2, adding the second solvent to the first solution, ultrasonicating and heating, to obtain a second solution; S3, the second solution is cooled and centrifuged and separated to obtain a middle solution, and the middle solution is filtered and dialyzed to obtain a third solution; S4. Concentrating the third solution and performing vacuum freeze-drying to obtain carbon quantum dots derived from the American cockroach.

2. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The first solvent in S1 is ethanol with a volume fraction of 75.0% to 99.5%, and the dosage ratio of the powder of the pulverized American cockroach to the first solvent is (0.8 g to 1.3 g): (0.5 mL to 1.5 mL).

3. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The stirring in S1 is magnetic stirring for 3 h to 5 h, and the filtration in S1 is filtering with a 0.45 μm microporous filter.

4. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The second solvent in S2 is water or ethanol with a volume fraction of 20%, and the dosage ratio of the powder of the crushed American cockroach to the second solvent is (0.8g-1.3g): (3.5mL-4.5mL).

5. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The ultrasonic power of the ultrasound in S2 is 300W, and the ultrasonic time is 10min to 20min; the heating in S2 is firstly pouring the ultrasonicated solution into a hydrothermal reactor, and then heating the hydrothermal reactor in an oven at 180°C to 230°C for 4h to 6h.

6. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The centrifugation in S3 is performed at 12000 rpm to 15000 rpm for 15 min to 25 min; the filtration in S3 is performed using a 0.22 μm microporous filter; the dialysis in S3 is performed using a 3000 Da dialysis bag in ultrapure water for 4 h to 6 h.

7. The method for preparing carbon quantum dots from American cockroach according to claim 1, characterized in that: The concentration in S4 is concentrated by rotary evaporation to 1 / 4 to 1 / 3 of the volume of the third solution; the vacuum freeze-drying in S4 is dried for 48 hours to 72 hours under the conditions of a vacuum degree of 10Par to 30Par and a temperature of -35°C to 50°C. 8 . Carbon quantum dots derived from American cockroaches prepared by the method for preparing carbon quantum dots derived from American cockroaches according to any one of claims 1 to 7 .

9. The carbon quantum dots derived from American cockroach according to claim 8, characterized in that: The carbon quantum dots derived from the American cockroach have a particle size of 4.2nm to 7.5nm and an average particle size of 5.6nm. They are mainly composed of carbon, hydrogen, oxygen and nitrogen elements and exhibit blue, green and red fluorescence in the excitation wavelength range of 420nm to 480nm, 495nm to 580nm and 600nm to 650nm, respectively.

10. Use of the carbon quantum dots derived from American cockroach according to claim 8 or 9 in the preparation of antioxidant and / or anti-cellular inflammatory drugs.

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