Andrographis paniculata carbon dot nano-enzyme and application thereof

Abstract

The invention belongs to the technical field of carbon nano-enzymes, and particularly relates to an andrographis paniculata carbon dot nano-enzyme and application thereof. According to the andrographis paniculata carbon dot nano-enzyme prepared by a hydrothermal method, the particle size is 1-5nm, and the andrographis paniculata carbon dot nano-enzyme is relatively small in size and uniform in particle size, so that the andrographis paniculata carbon dot nano-enzyme has good biocompatibility, not only has good oxidation resistance, but also can be used for treating inflammatory diseases in vivo. The andrographis paniculata carbon dot nano-enzyme provided by the invention has a scavenging effect on various free radicals such as ABTS free radicals, DPPH free radicals, hydroxyl free radicals, superoxide anions and the like, and can be used for treating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon nanozymes, and particularly relates to andrographis carbon dot nanozymes and their applications. Background Art

[0002] Due to their ability to mimic the catalytic activity of natural enzymes, low cost, high stability and durability, nanozymes are gradually becoming substitutes for natural enzymes and have been widely used in industries, medicine, biology and other fields. Currently, the developed nanozymes exhibit high catalytic activity for the scavenging of reactive oxygen and reactive nitrogen species. Among them, carbon dot nanozymes are widely used. Carbon dot nanozymes are carbon nanomaterials with a diameter less than 10 nm.

[0003] Currently, carbon dot nanozymes (GH-CDzymes) prepared from glucose and histidine show effective scavenging ability for reactive oxygen, reactive nitrogen and stable free radicals by surface modification groups and N element doping to improve catalytic activity. This ability can be attributed to multiple antioxidant mechanisms conferred by their unique structures. Peas and Eucommia ulmoides grown under salt stress conditions can be significantly (p<0.001) relieved of oxidative damage to biological components including chlorophyll, proline, carbohydrates and proteins caused by salt stress after treatment with carbon dot nanozymes, and the functions of redox enzymes in vivo can be repaired. In order to further provide more types of carbon dot nanozymes, further research on other types of carbon dot nanozymes is needed. Summary of the Invention

[0004] To solve the above problems, the present invention provides andrographis carbon dot nanozymes and their applications.

[0005] The first aspect of the present invention provides andrographis carbon dot nanozymes for antioxidant use, and the andrographis carbon dot nanozymes are prepared by hydrothermal carbonization of andrographis. The particle size of the andrographis carbon dot nanozymes is 1 nm to 5 nm.

[0006] In the present invention, andrographis in traditional Chinese herbal medicines is used as a carbon precursor, and carbon dot nanozymes are synthesized by a hydrothermal method. The synthesis method is simple and fast, the raw material source is inexpensive, and the synthesis process is green and pollution-free. Among them, the particle size of the andrographis carbon dot nanozymes is 1 nm to 5 nm. Due to their small size and particle size uniformity, they have good biocompatibility, can not only have good antioxidant properties, but also can be used for the treatment of inflammatory diseases in vivo.

[0007] In another preferred embodiment, the specific process of the hydrothermal method is as follows: Disperse andrographis powder in water to obtain a dispersion. After subjecting the dispersion to hydrothermal reaction at 210°C to 240°C for 8 h to 12 h, the supernatant was obtained by centrifugation and, after purification, the andrographis carbon dot nanozyme was obtained.

[0008] In another preferred embodiment, the purification refers to filtering the supernatant and then dialyzing it using a dialysis bag with a molecular weight cut-off of 1000 Da.

[0009] The second aspect of the present invention provides the application of the described andrographis carbon dot nanozyme in the preparation of antioxidant products for scavenging free radicals.

[0010] In another preferred embodiment, the free radicals are at least one of ABTS free radical, DPPH free radical, hydroxyl free radical and superoxide anion.

[0011] The third aspect of the present invention provides the application of the described andrographis carbon dot nanozyme in the preparation of anti-inflammatory drugs.

[0012] In another preferred embodiment, the drug uses the andrographis carbon dot nanozyme as the sole active ingredient.

[0013] In another preferred embodiment, the anti-inflammatory drug is an anti-ulcerative colitis drug.

[0014] In the present invention, andrographis in traditional Chinese herbal medicines is used as a carbon precursor to synthesize carbon dot nanozymes by a hydrothermal method. The synthesis method is simple and fast, the raw material source is inexpensive, the synthesis process is green and pollution-free, and the requirements for instruments and equipment are low, having good commercial potential. The andrographis carbon dot nanozymes prepared in the present invention have a scavenging effect on various free radicals such as ABTS free radical, DPPH free radical, hydroxyl free radical and superoxide anion. The andrographis carbon dot nanozymes prepared in the present invention are small and uniform in size, have good biocompatibility, can achieve the treatment of various inflammatory diseases, especially have a better treatment effect on ulcerative colitis, and can effectively relieve ulcerative colitis by regulating the intestinal microbiota and scavenging ROS. Description of the Drawings

[0015] Figure 1 is the electron micrograph of andrographis carbon dot nanozyme, where a in the figure is the lattice diagram of andrographis carbon dot nanozyme.

[0016] Figure 2 is the fluorescence spectrum diagram of andrographis carbon dot nanozyme.

[0017] Figure 3 is the scavenging result diagram of andrographis carbon dot nanozyme on ABTS free radical; among them, A is the scavenging result diagram of ABTS free radical at different wavelengths, and B is the scavenging result diagram of ABTS free radical by andrographis carbon dot nanozyme at different concentrations.

[0018] Figure 4 Figure showing the scavenging effect of andrographolide carbon dot nanozyme on DPPH radicals; among them, A is the figure showing the scavenging effect on DPPH radicals at different wavelengths, and B is the figure showing the scavenging effect on DPPH radicals by andrographolide carbon dot nanozymes at different concentrations.

[0019] Figure 5 Figure showing the scavenging effect of andrographolide carbon dot nanozyme on superoxide anions; among them, A is the figure showing the scavenging effect on superoxide anion radicals at different wavelengths, and B is the figure showing the scavenging effect on superoxide anion radicals by andrographolide carbon dot nanozymes at different concentrations.

[0020] Figure 6 Figure showing the scavenging effect of andrographolide carbon dot nanozyme on hydroxyl radicals; among them, A is the figure showing the scavenging effect on hydroxyl radicals at different wavelengths, and B is the figure showing the scavenging effect on hydroxyl radicals by andrographolide carbon dot nanozymes at different concentrations.

[0021] Figure 7 Figure showing the therapeutic effect of andrographolide carbon dot nanozyme on ulcerative colitis.

[0022] Figure 8 Figure showing the effect of andrographolide carbon dot nanozyme on pro-inflammatory cytokines; among them, A is the figure showing the effect on TNF-α, B is the figure showing the effect on IL-6, and C is the figure showing the effect on IL-1β. Detailed implementation manners

[0023] Next, the technical solutions in the present invention will be clearly and completely described in combination with the specific implementation of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] In each embodiment of the present invention, the methods are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial channels unless otherwise specified.

[0025] Example 1 Preparation of andrographolide carbon dot nanozyme: Weigh 150 mg of andrographolide powder and disperse it in 10 mL of ultrapure water. Stir it with a magnetic stirrer for 30 min. Then add the mixed solution into the inner liner of a polytetrafluoroethylene reaction kettle, seal it completely, and carry out a hydrothermal reaction at 210 °C for 8 h. After the reaction is completed, wait for the temperature of the reaction kettle to drop to room temperature. Take out the mixture and centrifuge it at a speed of 10000 rpm for 30 min to remove impurities, and repeat centrifugation 3 times. Then filter the solution with a mixed cellulose ester membrane with a pore size of 220 nm to further remove impurities, and then dialyze it with a dialysis bag with a cut-off molecular weight of 1000 Da for 24 h to obtain andrographolide carbon dot nanozyme.

[0026] The prepared andrographis carbon dot nanozyme was scanned, and the results are as Figure 1 shown. It can be seen from the figure that the andrographis carbon dot nanozyme has a small and uniform size, and the particle size is concentrated around 1.8 nm. As can be seen from a in the figure, the andrographis carbon dot nanozyme has lattice fringes and good crystallinity.

[0027] The fluorescence spectrum analysis of the above-prepared andrographis carbon dot nanozyme was carried out, and the results are as Figure 2 shown. From Figure 2 it can be seen that the optimal excitation wavelength of the andrographis carbon dot nanozyme is 330 nm - 340 nm, and the optimal emission wavelength is 394 nm - 408 nm, showing fluorescence characteristics.

[0028] Example 2 Antioxidant research on andrographis carbon dot nanozyme The antioxidant ability of andrographis carbon dot nanozyme was tested by using a variety of free radical scavenging experiments. ABTS, as one of the most widely used reagents for measuring free radical scavenging ability, can be oxidized to green ABTS•+ under the action of an oxidant and has a characteristic absorption peak at 734 nm as Figure 3 shown. As the concentration of andrographis carbon dot nanozyme increases, the peak value of ABTS•+ at 734 nm decreases more significantly, and the scavenging rate is higher. Similarly, DPPH• has a characteristic absorption peak at 517 nm, as Figure 4 shown. As the concentration of andrographis carbon dot nanozyme added increases, the peak value decreases more significantly, and the scavenging rate is higher. Nitroblue tetrazolium (NBT) can be converted into a blue formazan compound with a prominent absorption peak at 560 nm in the presence of superoxide anions. As Figure 5 shown, superoxide anions were generated in aqueous solution by the reaction of riboflavin and methionine, and then andrographis carbon dot nanozyme at 0 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL was added respectively. It was found that it could scavenge superoxide anions and make the characteristic peak decrease significantly, and the scavenging rate was higher as the concentration increased, showing SOD enzyme activity. Similarly, 3,3',5,5'-tetramethylbenzidine (TMB) can be oxidized to oxTMB in the presence of •OH free radicals, thus having a characteristic absorption peak at 650 nm. As Figure 6As shown, •OH was generated in an aqueous solution using the classical Fenton reaction. Subsequently, 0 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL of andrographis carbon dot nanozymes were added respectively. It was found that the peak value decreased more significantly with the increase in concentration, and the scavenging rate was also higher. A variety of free radical scavenging experiments showed that andrographis carbon dot nanozymes had excellent scavenging ability for various free radicals such as DPPH radicals, ABTS radicals, hydroxyl radicals, and superoxide anions.

[0029] Example 3 Study on the Anti-ulcerative Colitis of Andrographis Carbon Dot Nanozymes Male C57BL / 6 mice were provided by the Medical Experimental Animal Center of Xi'an Jiaotong University. The mice were randomly divided into a control group denoted as CK, a DSS group, an andrographis extract group denoted as the Extract group, and an andrographis carbon dot nanozyme group denoted as the CDs group. An ulcerative colitis model was induced by continuously allowing the mice to freely drink water containing 2.5 wt% dextran sulfate sodium (DSS) for 7 days. Then, for another 4 days, the andrographis extract group was orally administered andrographis extract once a day, with a dosing dose of 20 mg / kg; the andrographis carbon dot nanozyme group was orally administered andrographis carbon dot nanozymes once a day, with a dosing dose of 20 mg / kg. After 4 days of gavage, with the help of colonoscopy, the treatment progress of ulcerative colitis in the mouse model was closely observed. The andrographis extract was prepared by mixing 10 g of andrographis powder with 200 mL of water, extracting at 100 °C for 1 h, filtering, and taking the filtrate.

[0030] As Figure 7 shown, after the administration of andrographis carbon dot nanozymes, the intestinal inflammatory state of the mice was significantly alleviated. It shows that andrographis carbon dot nanozymes can effectively reduce the ulcer symptoms of the intestine, strongly promote the repair of the intestinal mucosa, and significantly improve the intestinal inflammatory state. As found in the detection of intestinal inflammatory factor levels Figure 8 shown, andrographis carbon dot nanozymes can significantly reduce the levels of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Andrographis carbon dot nanozymes can significantly reduce the expression of pro-inflammatory cytokines and improve the intestinal inflammatory level. Thus, it can be shown that andrographis carbon dot nanozymes can effectively alleviate the damage caused by the oxidative environment of in vivo inflammatory diseases and show great potential in realizing the treatment of in vivo inflammation.

[0031] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. Andrographis paniculata carbon dot nanozyme for anti-oxidation, characterized in that: The Andrographis paniculata carbon dot nanozyme is prepared by carbonizing Andrographis paniculata through a hydrothermal method; The particle size of the Andrographis paniculata carbon dot nanozyme is 1 nm to 5 nm.

2. The Andrographis paniculata carbon dot nanozyme according to claim 1, characterized in that: The specific process of the hydrothermal method is as follows: dispersing andrographolide powder in water to obtain a dispersion; The dispersion was subjected to hydrothermal reaction at 210° C. to 240° C. for 8 h to 12 h, and then the supernatant was collected by centrifugation and purified to obtain the Andrographis paniculata carbon dot nanozyme.

3. The Andrographis paniculata carbon dot nanozyme according to claim 2, characterized in that: The purification refers to filtering the supernatant and then dialyzing it.

4. The use of the Andrographis paniculata carbon dot nanozyme according to claim 1 in the preparation of antioxidant products, characterized in that: The antioxidant product is used to scavenge free radicals.

5. The use of the Andrographis paniculata carbon dot nanozyme in the preparation of antioxidant products according to claim 4, characterized in that: The free radical is at least one of an ABTS free radical, a DPPH free radical, a hydroxyl free radical and a superoxide anion.

6. Use of the Andrographis paniculata carbon dot nanozyme according to claim 1 in the preparation of anti-inflammatory drugs.

7. The use according to claim 6, characterized in that: The drug contains Andrographis paniculata carbon dot nanozyme as the only effective ingredient.

8. The use according to claim 6, characterized in that: The anti-inflammatory drug is an anti-ulcerative colitis drug.

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