Green preparation method of attenuated monkshood carbon dots and application of attenuated monkshood carbon dots in sepsis treatment

Abstract

The invention belongs to the technical field of biological medicine, and relates to a green preparation method of attenuated monkshood carbon dots and application of the attenuated monkshood carbon dots in sepsis treatment, the green preparation method comprises the following steps: S1, using monkshood as a raw material for pretreatment, the pretreatment comprises water washing, slicing, drying in the sun and crushing; s2, taking the raw radix aconiti lateralis praeparata pretreated in the S1 as a precursor and water as a solvent, performing microwave heating to 150 DEG C, and keeping the temperature for 30 minutes; s3, centrifuging the mixture obtained in the step S2 to remove large-particle impurities, performing suction filtration by using a filter membrane to obtain supernatant, and further removing impurities; s4, dialyzing the mixture obtained in S3 in pure water in a dialysis bag for not less than 3 days, performing suction filtration through a filter membrane for further purification, and freeze-drying the purified liquid in a freeze dryer to obtain the target monkshood carbon dots; the purpose of attenuating the toxicity of the raw radix aconiti lateralis praeparata is achieved through the carbon dot technology, the preparation method is green and environmentally friendly, no toxic organic solvent exists, preparation is simple, and large-scale production can be achieved; meanwhile, active ingredients of radix aconiti carmichaeli can be effectively reserved, and toxicity is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a green preparation method of attenuated aconite carbon dots and application thereof in the treatment of sepsis. BACKGROUND

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which can lead to organ dysfunction and high mortality. Currently, antibiotics and hormone drugs are commonly used in clinical treatment, but there are problems such as drug resistance, limited efficacy and side effects.

[0003] Aconitum carmichaelii Debx is a traditional Chinese medicine with the effects of dispelling cold and relieving pain, restoring yang and rescuing reversal. The various alkaloids contained in aconite have anti-inflammatory and immunoregulatory effects. However, aconite contains double-ester diterpene alkaloids, which have significant cardiotoxicity and neurotoxicity risks. The traditional administration methods such as long decoction and oral administration are difficult to achieve the purpose of first aid in sepsis-related capillary leakage syndrome, and aconite still has hepatotoxicity and other toxicity risks after long decoction. These have seriously restricted the application of aconite in sepsis and other critical conditions.

[0004] Carbon dots (CDs) as a new type of nanomaterial have broad prospects in drug delivery, imaging and antioxidant due to their good biocompatibility, fluorescence characteristics and enzyme simulation activity. During the generation process under high temperature and pressure, the biomass can be restructured, and part of the functional groups of the source drug is still retained, which endows it with the unique biological efficacy of the source drug.

[0005] Therefore, there is a need for a method to reduce the toxicity of aconite and expand its application in sepsis and other critical conditions to solve the above technical problems. SUMMARY

[0006] The application provides the following technical scheme: a green preparation method of attenuated aconite carbon dots, comprising the following steps:

[0007] Step S1, using aconite as raw material for pretreatment, the pretreatment includes: water washing, slicing, drying, and crushing;

[0008] Step S2, using the aconite pretreated in step S1 as a precursor, water as a solvent, and microwave heating to 150 DEG C for 30 min;

[0009] Step S3, centrifuging the mixture obtained in step S2 to remove large particle impurities, using a filter membrane to perform suction filtration to obtain supernatant, and further removing impurities;

[0010] Step S4, the mixture obtained in step S3 is dialyzed in pure water in a dialysis bag for not less than 3 days, and then further purified by filtration through a filter membrane, and the purified liquid is freeze-dried in a freeze dryer to obtain the target aconite carbon dots.

[0011] Preferably, in the step S3, the centrifugal speed is 4000 rpm, and the filter membrane is a 0.22 mu m aperture PES water system filter membrane.

[0012] Preferably, in the step S4, the dialysis bag is a 500D dialysis bag, and the filter membrane is a 0.22 mu m aperture PES water system filter membrane.

[0013] More preferably, in the step S4, the obtained aconite carbon dots are stored at 4 DEG C.

[0014] Preferably, in the step S1, the aconite is raw aconite.

[0015] The application also discloses application of the attenuated aconite carbon dots in treatment of sepsis.

[0016] The application has the following beneficial effects:

[0017] The application realizes the attenuated purpose of raw aconite through carbon dot technology, and has the advantages of green and environmental protection, no toxic organic solvent, simple preparation, and realization of large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a green preparation method flowchart of the attenuated aconite carbon dots of the application;

[0019] Figure 2 is a schematic diagram of retaining effective functional groups of aconite of the application;

[0020] Figure 3 is a schematic diagram of reducing toxicity of aconite of the application;

[0021] Figure 4 is a schematic diagram of treatment effect of aconite carbon dots of the application in sepsis;

[0022] Figure 5 is a schematic diagram of prevention and treatment mechanism of aconite carbon dots of the application in sepsis.

[0023] Among them, Figure 1 Among them, a: aconite carbon dot preparation flowchart; b: morphology and particle size of aconite carbon dots under a transmission microscope, and lattice morphology and interval under a high-power lens; c: blue-green fluorescence emitted by aconite carbon dots under ultraviolet light irradiation.

[0024] Figure 2 a: Fourier transform infrared spectroscopy; b: 1 H nuclear magnetic resonance; c: X-ray photoelectron spectroscopy analysis of full spectrum; d: X-ray photoelectron spectroscopy analysis of C1s, O1s, N1s; e: structural pattern; f: antioxidant capacity detection of carbon dots of aconite - DPPH scavenging capacity; g: antioxidant capacity detection of carbon dots of aconite - superoxide anion electron paramagnetic resonance spectrum.

[0025] Figure 3 a: survival rate (A-De is the control of aconite water decoction for 30 min, 0.22 μm filtration and freeze-drying, A-CDs is carbon dots of aconite); b: content of toxic ingredients aconitine, mesaconitine and hypaconitine (Aconitum is raw aconite, A-De is aconite water decoction control, A-CDs is carbon dots of aconite).

[0026] Figure 4 a: in the LPS-induced sepsis model, carbon dots of aconite A-CDs can significantly improve the survival rate of mice; b: carbon dots of aconite can significantly reduce the secretion level of TNF-α, IL-6 and IL-1β in the serum of sepsis mice; c: carbon dots of aconite can significantly improve the vascular leakage of lung tissue of sepsis mice and reduce the content of Evans Blue in lung tissue; d: carbon dots of aconite can help restore the expression continuity of vascular endothelial tight junction protein in lung tissue of sepsis mice. DETAILED DESCRIPTION

[0027] The related technologies in the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0028] Reference Figures 1 to 5 , the specific embodiments are as follows:

[0029] ① Select high-quality raw aconite as raw material, and go through pretreatment steps such as water washing, slicing, drying, and crushing;

[0030] ② Take raw aconite as precursor and water as solvent, and place them in a high-pressure reaction kettle, and heat them in a microwave reactor (150℃ for 30 min);

[0031] ③ Remove large particle impurities by centrifugation at 4000 rpm for 10 min, and take the supernatant to be filtered by a 0.22 μm pore size PES water system filter membrane to further remove impurities;

[0032] ④The 500D dialysis bag was dialyzed in pure water for 3 days, and then further purified by 0.22 μm pore size PES water system filter membrane by suction filtration. The purified liquid was freeze-dried in a freeze dryer to obtain the target aconite carbon dots, which were stored at 4°C.

[0033] ⑤The obtained aconite carbon dots were used in a mouse LPS sepsis model, which showed obvious therapeutic effects of improving survival rate, reducing inflammatory response, and improving capillary leakage.

[0034] In this embodiment, raw aconite is used as the main carbon source raw material. Different origins and different processing degrees of aconite (such as light aconite tablets and black aconite tablets) or other traditional Chinese medicines rich in similar diterpenoid alkaloids (such as Sichuan aconite and grass aconite) can also be used as carbon sources. Through similar green preparation processes, carbon dots with similar functions can also be obtained. Through carbonization treatment, the original toxic components (double ester diterpenoid alkaloids, etc.) in aconite are effectively degraded or removed, while the active ingredients such as anti-inflammatory and antioxidant are maximally retained. This is the core of the process of the present application. Aconite carbon dots not only retain the efficacy of aconite, but also exhibit nanoscale enzyme activity and free radical scavenging function, which can effectively reduce the oxidative stress level in the sepsis model.

[0035] In this embodiment, hydrothermal synthesis and microwave-assisted carbonization are preferred, but other mild green carbonization processes such as low-temperature calcination, enzyme-catalyzed carbonization, or biomass anaerobic hydrolysis can also be used to prepare carbon dots to achieve the same purpose of attenuation and function retention. In the process of preparing carbon dots, water hydration and microwave assistance are used to avoid the use of organic solvents, high-temperature calcination, or heavy metal catalysts, ensuring the low toxicity and high biocompatibility of the carbon dots. This green preparation is the technical basis for achieving attenuation, safety, and environmental protection, and is a key process link.

[0036] In addition to dialysis, other physical or chemical purification methods such as ultrafiltration, molecular sieving, gel chromatography, membrane separation, or resin adsorption can also be used to remove residual toxic components to achieve the same purification effect. Through physical purification methods such as centrifugation, suction filtration, and dialysis, residual small molecule toxic impurities are removed to improve the safety and consistency of the carbon dots. The purification process is a key guarantee for ensuring the repeatability and controllability of the preparation results.

[0037] In this embodiment, surface functional group modification (such as carboxylation, amination, and polyethylene glycolation) can be used to improve the water solubility and biocompatibility of the carbon dots. If other biocompatible modification methods such as grafting chitosan, polylactic acid, or peptide coupling are used, similar technical effects can also be achieved.

[0038] The attenuated aconite carbon dots of this embodiment are used for the auxiliary treatment of sepsis-related capillary leakage syndrome, and can also be applied to the treatment of diseases related to systemic inflammatory response syndrome (SIRS) and other diseases that require anti-inflammatory and antioxidant treatment.

[0039] Examples

[0040] This embodiment takes a mouse sepsis model induced by bacterial lipopolysaccharide (LPS) as an example to specifically illustrate the application process and effect of the attenuated aconite carbon dots prepared by the present application in treating sepsis and its concurrent capillary leakage syndrome.

[0041] Step 1, establishment of a sepsis model:

[0042] Select 8-10-week-old male mice (SPF level, 21-25 g) and inject LPS (20 mg / kg) intraperitoneally to establish a sepsis model. This model has a high mortality rate and obvious inflammatory response, and is suitable for evaluating acute drug efficacy.

[0043] Step 2, survival rate analysis:

[0044] Randomly divide the successfully modeled mice into groups: ① model control group: intraperitoneally inject the same volume of PBS; ② aconite carbon dot treatment group: intraperitoneally inject aconite carbon dots (10, 40, 80 mg / kg) once, respectively; ③ positive control group: intraperitoneally inject dexamethasone (DXM, 1 mg / kg) once. Continuously observe the clinical manifestations and survival of the mice within 7 days. The results show that the mortality rate of the model group mice within 7 days is as high as 90%; the mortality rates of the aconite carbon dot 10, 40, and 80 mg / kg groups are 70%, 30%, and 0%, respectively; and the mortality rate of the DXM group is 10%. This indicates that aconite carbon dots have a dose-dependent effect of improving survival rate, and the high-dose group is superior to the dexamethasone control group.

[0045] Step 3, detection of inflammatory factor levels:

[0046] Randomly divide the successfully modeled mice into groups: ① model control group: intraperitoneally inject the same volume of PBS; ② aconite carbon dot treatment group: intraperitoneally inject aconite carbon dots (40, 80 mg / kg) once, respectively; ③ positive control group: intraperitoneally inject dexamethasone (DXM, 1 mg / kg) once. Sacrifice the mice in each group at 24 h after LPS injection, collect the serum, and use the ELISA method to detect the levels of inflammatory factors IL-1β, IL-6, and TNF-α in the serum. The results show that the levels of each inflammatory factor in the serum of the model group mice are significantly increased, the aconite carbon dot treatment groups significantly inhibit the release of the above inflammatory factors, especially the 80 mg / kg group, which has a stronger inhibitory effect and is superior to the DXM group, with a clear dose-effect relationship.

[0047] Step 4, analysis of vascular leakage:

[0048] The vascular permeability was evaluated by Evans Blue dye leakage experiment: 22h after the LPS-induced model was constructed, Evans Blue dye (2%, 4ul / g) was injected into the tail vein, and 2h later, 0.3% pentobarbital was injected intraperitoneally for anesthesia, and PBS (20ml, slowly) was perfused through the heart to remove the circulating dye, and the lung tissue was taken out and photographed; if the tissue was blue, it indicated that the blood vessels leaked, and the bluer the color, the more obvious the blood vessel leakage. The lung tissue of the LPS sepsis model group of mice was obviously blue, and the color became lighter after the treatment of the carbon dots of aconite, especially the 80mg / kg group. The lung tissue of each group of mice was dried (60℃, 48h), formamide (10ul / mg) was added, homogenized, 60℃ water bath for 24h, centrifuged at 14000rpm for 10min; the absorbance of the supernatant at 620nm was measured; the dye content in the tissue was calculated by comparing with the standard curve of Evans Blue; the higher the content, the more obvious the vascular leakage of the tissue. The experimental results showed that the Evans Blue content in the lung tissue of the sepsis model group of mice was significantly increased, and the carbon dots of aconite treatment could significantly reduce the dye accumulation in the lung tissue, and the 80mg / kg group had the best effect. The above results all showed that it had a good effect on relieving the vascular leakage related to sepsis.

[0049] Step 5, expression analysis of vascular endothelial tight junction protein:

[0050] In order to further verify the repair effect of the carbon dots of aconite on the vascular endothelial barrier function: the lung tissue of each group of mice was taken out 24h after modeling, fixed with 4% paraformaldehyde, embedded with paraffin, sectioned, and the expression of tight junction protein ZO-1 was detected by immunofluorescence method, and the cell nucleus was stained with DAPI; the expression and distribution of ZO-1 in the lung vascular endothelium were observed by fluorescence microscope. The results showed that the expression of ZO-1 in the lung tissue of the model group was weakened and discontinuous, while the expression of ZO-1 was obviously enhanced and distributed continuously after the treatment of the carbon dots of aconite, which indicated that it could reduce the leakage by maintaining the structure of the vascular endothelium.

[0051] This example fully verified the application effect of the attenuated carbon dots of aconite prepared by the application in the sepsis model. The carbon dots of aconite have the advantages of injectability, good survival rate improvement, strong anti-inflammatory effect and repair function of vascular endothelium, and show superiority in the treatment of sepsis related capillary leakage syndrome. The effect has clear dose dependence, and is better than the traditional glucocorticoid treatment method, which provides a new strategy for the nanization of traditional Chinese medicine and its application in acute and critical conditions.

[0052] In summary, the carbon dot technology is used to achieve the attenuation purpose of raw aconite in the application, the preparation method is green and environmentally friendly, there is no toxic organic solvent, the preparation is simple and can realize large-scale production; at the same time, the application can effectively retain the active ingredients of aconite and reduce the toxicity; the application shows good anti-inflammatory and repair effect on vascular endothelial damage in the sepsis model.

[0053] It should be emphasized that the above are only the preferred embodiments of the present application, and do not limit the present application in any form. Any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application are still within the scope of the technical solutions of the present application.

Claims

1. A green preparation method of attenuated aconite carbon dots, characterized in that: The following steps are involved: Step S1, performing pre-treatment on aconite root as a raw material, wherein the pre-treatment comprises: washing, slicing, drying, and crushing; Step S2: using the aconite root pretreated in step S1 as a precursor and water as a solvent, heating the mixture to 150° C. in a microwave oven and maintaining the temperature for 30 min; Step S3: centrifuging the mixture obtained in step S2 to remove large particles of impurities, and filtering the mixture using a filter membrane to obtain the supernatant to further remove impurities; Step S4: dialyze the mixture obtained in step S3 in pure water using a dialysis bag for no less than 3 days, and then further purify it by filtration through a filter membrane. The purified solution is freeze-dried in a freeze dryer to obtain the target aconite carbon dots.

2. The green preparation method of attenuated aconite carbon dots according to claim 1, characterized in that: In step S3, the centrifugal speed is 4000 rpm, and the filter membrane is a PES water filter membrane with a pore size of 0.22 μm.

3. The green preparation method of attenuated aconite carbon dots according to claim 1, characterized in that: In step S4, the dialysis bag is a 500D dialysis bag, and the filter membrane is a PES water filter membrane with a pore size of 0.22 μm.

4. The green preparation method of attenuated aconite carbon dots according to claim 3, characterized in that: In step S4, the obtained aconite carbon dots are stored at 4°C.

5. The green preparation method of attenuated aconite carbon dots according to claim 1, characterized in that: In step S1, the aconite root is raw aconite root.

6. Application of attenuated aconite carbon dots in the treatment of sepsis, characterized in that: The attenuated aconite carbon dots are prepared using the green preparation method of the attenuated aconite carbon dots according to any one of claims 1 to 5.

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