Preparation and application of Phellodendron chinense carbon nanoparticles

Abstract

The present invention relates to the field of medicine, and specifically to the preparation and application of Phellodendron chinense carbon nanoparticles (Phellodendron chinense carbon nanoparticles / Phellodendron chinense carbon nanoparticles / Phellodendron chinense carbon quantum dots / Phellodendron chinense carbon dots). The present invention also provides a method for extracting and separating carbon dots from Phellodendron chinense. Phellodendron chinense carbon is prepared by muffle furnace calcination method, and then high-purity Phellodendron chinense carbon dots are obtained by separation and purification and other technical methods. Pharmacological experiments have found that Phellodendron chinense carbon dots have a clear therapeutic effect on refractory psoriasis and have the activity of relieving inflammation.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the preparation and application of Phellodendron chinense carbon nanoparticles (nanoparticles / carbon quantum dots / carbon dots). Background Art

[0002] Huangbai charcoal has a history of thousands of years of application and definite clinical efficacy. In addition to its well-known hemostatic effect, its anti-inflammatory and anti-infective effects have also attracted the attention of medical scholars of all generations. Its typical clinical manifestations are the treatment of skin diseases such as sores, boils, and carbuncles. Unfortunately, its key material basis and pharmacodynamic mechanism have not been elucidated so far, which has affected the clinical application and development of Huangbai charcoal.

[0003] Psoriasis is a common, recurrent scaly papular skin disease that poses a serious threat to human physical and mental health. Currently, there is no drug or method that can cure the disease. In recent years, although some progress has been made in the treatment of psoriasis, many problems exposed in clinical practice, including drug side effects and high prices, have limited the application of these drugs.

[0004] Therefore, it is urgent to find a safe, efficient and reasonably priced drug. Phellodendron charcoal carbon dots are a new type of nanomaterial discovered from Phellodendron charcoal. Previous cell experiments have shown that they have anti-inflammatory activity. Since psoriasis is a chronic inflammatory disease, it is of great significance to study the therapeutic effect of these carbon dots on psoriasis. Summary of the invention

[0005] Definition of terms:

[0006] Phellodendron charcoal nanoparticles: a type of particles with a particle size of nanometers extracted from Phellodendron charcoal, which can also be called Phellodendron charcoal nanoparticles, Phellodendron charcoal carbon quantum dots, and Phellodendron charcoal carbon dots.

[0007] The present invention extracts and separates carbon dots (nanoparticles / nanoparticles) from Phellodendron charcoal, and for the first time discovers that the Phellodendron charcoal carbon dots have significant biological effects in treating psoriasis, and are worthy of further development and utilization.

[0008] Use of Phellodendron charcoal and its nanoparticles in preparing a composition for treating skin diseases. The skin disease is any one of contact dermatitis, eczema, urticaria, allergic skin vasculitis and drug-induced dermatitis.

[0009] The skin disease is psoriasis.

[0010] The composition may or may not contain pharmaceutically acceptable excipients.

[0011] The composition is any one of a medicine, a health product, and a food, and the composition can be prepared into a clinically acceptable oral preparation; further preferably, the clinically acceptable oral preparation is one or more of a pill, a capsule, a tablet, a granule or a liquid oral preparation; more preferably, the oral preparation is a granule.

[0012] Specifically, the technical solution of the present invention is as follows.

[0013] The present invention relates to a method for extracting, separating and purifying nanoparticles (nanoparticles / carbon quantum dots / carbon dots) from Phellodendron charcoal, which specifically comprises the following steps: preparing Phellodendron charcoal by muffle furnace calcination, extracting carbon dots from the Phellodendron charcoal with a solvent, and separating and purifying by dialysis, ultrafiltration, centrifugation, extraction and other technical methods to obtain high-purity carbon dots.

[0014] Further:

[0015] (1) Muffle furnace heating process: Phellodendron charcoal is prepared by muffle furnace calcination method, and carbon dots are extracted from Phellodendron charcoal with solvent for later use;

[0016] (2) Purification step of Phellodendron charcoal carbon dots: using deionized water as solvent, heating and extracting, concentrating, dialyzing with a dialysis bag with a molecular weight greater than 1000Da, or ultrafiltration, high-speed centrifugation, extraction and separation of the carbon dots in step (1) to obtain Phellodendron charcoal carbon dots.

[0017] Further:

[0018] The muffle furnace heating process is as follows: heat up to 60-80°C, the heating process lasts for 5 minutes, and maintains at 60-80°C for 15-35 minutes; then heat up to 350-450°C, the heating process lasts for 10-30 minutes and maintains for 0.5-1.5 hours. After the temperature drops to room temperature, open the furnace door and take out the crucible. The product in the crucible is phellodendron charcoal (PCCC).

[0019] The purification steps of Phellodendron charcoal carbon dots are as follows: PCC is boiled in a water bath with 10 times the amount of deionized water for 3 times (100°C), filtered and concentrated with a 0.22μm microporous filter membrane. Then, the concentrate is dialyzed with a 1000D dialysis membrane for 7 days with deionized water as the dialysis medium, and the solution in the bag is taken out, which is Phellodendron charcoal carbon dots.

[0020] The invention also provides the biological activity of the carbon dots of Phellodendron chinense charcoal and its application in the medical field. The carbon dots in the Phellodendron chinense charcoal have a clear therapeutic effect on refractory psoriasis and have the activity of relieving inflammation.

[0021] The particle size of the Phellodendron chinense carbon dots measured by transmission electron microscopy is less than 10 nm, and they are mainly composed of elements such as C, O and N. The invention also relates to the application of the nanoparticles in the medical field, mainly used for alleviating psoriasis symptoms and anti-inflammatory activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 .A Low-resolution transmission electron microscopy image of Phellodendron charcoal dots, with the particle size distribution map inside; B High-resolution transmission electron microscopy image of Phellodendron charcoal dots, with the magnified image inside; C Ultraviolet absorption spectrum of Phellodendron charcoal dots; D Fluorescence spectrum of Phellodendron charcoal dots.

[0023] Figure 2 .A Infrared spectrum of Phellodendron amurense carbon dots; B XPS full scan spectrum of Phellodendron amurense carbon dots; C C1s graph of Phellodendron amurense carbon dots; D O 1s graph of Phellodendron amurense carbon dots; E N 1s graph of Phellodendron amurense carbon dots.

[0024] Figure 3 .Study on the effect of Phellodendron charcoal carbon dots on imiquimod (IMQ)-induced psoriasis-like lesions on the back (AF is an enlarged image of the back skin) and ears (a) to f) of mice. The animals were divided into 6 groups: control group (Control), model group (IMQ), positive drug group (Methotrexate, MTX), and different doses of Phellodendron charcoal carbon dots administration groups [high (H), medium (M), low (L)].

[0025] Figure 4 .Study on the effects of Phellodendron charcoal carbon dots on the PASI score, (I) spleen index, (J) spleen volume and (K) body weight of mice induced by imiquimod (IMQ) on the back and ears of mice. The animals were divided into 6 groups: control group (Control), model group (IMQ), positive drug group (Methotrexate, MTX), and different doses of Phellodendron charcoal carbon dots [high (H), medium (M), low (L)].

[0026] Figure 5 .Study on the effects of Phellodendron chinense carbon dots on the levels of serum pro-inflammatory factors (A) TNF-α, (B) IL-6 and (C) IL-17A in mice induced by imiquimod (IMQ). DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments, but the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1: Preparation of Phellodendron amurense carbon dots

[0029] The same batch of Phellodendron chinense slices were purchased and crushed and weighed and then loaded into a crucible. The crucible mouth was covered with aluminum foil, the lid was tightly closed, and the crucible was placed in a muffle furnace for carbonization.

[0030] The heating process of the muffle furnace is as follows: from room temperature to 70°C, the heating process takes 5 minutes, and it is maintained at 70°C for 25 minutes; then from 70°C to 400°C, the heating process takes 20 minutes and it is maintained for 1 hour. After the temperature drops to room temperature, the door of the muffle furnace is opened, and the crucible is taken out. The product in the crucible is phellodendron charcoal (PCCC).

[0031] The purification steps of Phellodendron charcoal carbon dots are as follows: PCC is boiled in a water bath with 10 times the amount of deionized water for 3 times (100°C), filtered and concentrated with a 0.22μm microporous filter membrane. Then, the concentrate is dialyzed with a 1000D dialysis membrane for 7 days with deionized water as the dialysis medium, and the solution in the bag is taken out, which is Phellodendron charcoal carbon dots.

[0032] Example 2. Study on the effect of Phellodendron chinense carbon dots on relieving psoriasis

[0033] 1. Experimental Animals

[0034] In this experiment, SPF male Balb / c mice (weight 22.0-25.0 g) were used as experimental subjects. After 3 days of adaptive feeding at a temperature of (24.0±1.0)℃ and a relative humidity of 55-65%, the hair of the 2×2 cm area on the back was shaved. During this period, the animals were allowed to drink water and eat freely. The animal experiment was approved by the Animal Experiment Ethics Committee of Beijing University of Chinese Medicine (ethics number BUCM-4-2018101601-4006).

[0035] 2. Drug administration, modeling and sampling of experimental animals

[0036] The animals were weighed and divided into 6 groups according to a random number table: normal group, model group, positive group, and high, medium, and low doses of Phellodendron chinense carbon dots administration groups, with 10 animals in each group. During the experiment, except for the normal group, all other groups of animals were smeared with 62.5 mg of 5% imiquimod on the shaved area of ​​the back every day, and the model was established for 7 consecutive days to establish a psoriasis-like skin lesion mouse model. Drug intervention and modeling were carried out simultaneously. The normal group and the model group were given normal saline, and the positive group was given methotrexate. During the modeling period, the weight of the mice and the PASI scores (erythema, scales, protrusions and total scores) of the back lesions were monitored and recorded every day. On the 8th day after modeling, blood was collected from the eyeballs of the mice to detect the levels of pro-inflammatory factors (TNF-α, IL-6 and IL-17A), and the spleen of the mice was removed to record the spleen index.

[0037] 3. Characterization of the physicochemical properties of Phellodendron amurense carbon dots

[0038] 3.1 Transmission electron microscope image, UV absorption spectrum, and fluorescence spectrum of Phellodendron chinense carbon dots

[0039] Electron microscopy technology (high and low resolution electron microscopy), optical technology (ultraviolet visible light analysis technology), and X-ray electron spectroscopy technology were used to characterize and analyze the morphological characteristics, functional group changes and other physical and chemical characteristics of Phellodendron chinense carbon dots.

[0040] The low-resolution transmission image of Phellodendron charcoal carbon dots shows that the nanoparticles are nearly spherical and have good dispersibility in water. The particle size is between 0.5nm and 3.6nm, and the lattice spacing is 0.22nm. In addition, spectral studies have shown that Phellodendron charcoal carbon dots have more significant absorption at 270nm and 350nm, indicating that there are π-π* and n-π* transitions in the carbon dot structure. Its fluorescence spectrum shows that Phellodendron charcoal carbon dots have significant fluorescence, with maximum excitation and emission wavelengths at 330nm and 445nm, respectively, and a quantum yield of 5.63%.

[0041] 3.2 FTIR and XPS of Phellodendron amurense carbon dots

[0042] Infrared spectra show that Phellodendron chinense carbon dots contain multiple active groups such as hydroxyl, carboxyl, and amino groups, and this result is further confirmed by XPS spectra. Figure 2 (A) shows that the peaks are located at 284.7eV, 399.7eV and 531.8eV, corresponding to C1s, N1s and O1s, respectively, and the proportions of these three elements are 67.61%, 24.19% and 3.81%, respectively. Further scanning and obtaining high-resolution C1s, N1s and O1s spectra show the existence of chemical bonds such as CC / C=C, CO, O=N / C=O, verifying the existence of active groups such as hydroxyl, amino and carboxyl groups in the carbon dots.

[0043] 3.3 Effect of Phellodendron amurense carbon dots on the skin appearance of psoriasis-like mice

[0044] Figure 3 The typical symptoms of psoriatic lesions on the skin and ears of animals in different groups are shown. Compared with the control group animals, the skin and ears of mice treated with IMQ alone showed symptoms such as erythema, infiltration, thickening and scaling, which indicated the successful establishment of the model. After treatment with MTX or different doses of PCC-CD, the psoriasis-like appearance of the skin and ears could be significantly improved, indicating that Phellodendron chinense carbon dots have a protective effect on IMQ-induced psoriasis inflammation.

[0045] 3.4 Effect of Phellodendron chinense carbon dots on PASI scores of psoriasis-like mice

[0046] The PASI score (including erythema, infiltration, scaling and cumulative score) is one of the important indicators for the preliminary clinical evaluation of the severity of psoriasis. Figure 4 .AD It can be seen that starting from the second day after IMQ modeling, the animals gradually began to show mild erythema and scaling symptoms, and as the modeling time prolonged, the symptoms gradually worsened. The same situation can also be observed in the ears. Figure 4 .EH shows the psoriasis-like symptoms of the ears of mice on the 7th day of modeling. Typical erythema, infiltration, and scaling can be observed on the ears of mice in the model group. However, after intervention with Phellodendron chinense carbon dots, the PASI scores of the skin and ears were significantly reduced, with the best effect in the medium-dose group. In addition, after autopsy of mice treated with IMQ, it was observed that the spleen volume was enlarged ( Figure 4 .J) phenomenon, further weighing analysis can know its spleen index ( Figure 4 .I) significantly increased, body weight ( Figure 4 .K) was reduced, and the intervention of Phellodendron chinense charcoal carbon dots could significantly alleviate this pathological change.

[0047] 3.5 Effect of Phellodendron amurense carbon dots on imiquimod (IMQ)-induced pro-inflammatory factors in mouse serum

[0048] Studies have shown that proinflammatory factors play a very important role in the development and improvement of psoriasis. Combined with the serum indicators of clinical psoriasis patients, TNF-α, IL-6 and IL-17A were selected for testing. Figure 5 The results showed that after 7 consecutive days of IMQ modeling, the levels of these three pro-inflammatory factors in the mouse serum increased significantly. The intervention of Phellodendron chinense carbon dots can alleviate this phenomenon, and the effect of medium-dose CDs is the best.

Claims

1. Use of Phellodendron amurense carbon dots in the preparation of drugs for the treatment of psoriasis; The method for preparing the cork carbon dots comprises the following steps: Phellodendron charcoal was prepared by muffle furnace calcination method, and carbon dots were extracted from the Phellodendron charcoal with solvent; wherein, the muffle furnace heating process was as follows: heating to 60-80°C, the heating process lasted for 5 minutes, and maintained at 60-80°C for 15-35 minutes; then heating to 350-450°C, the heating process lasted for 10-30 minutes and maintained for 0.5-1.5 hours, and after the temperature dropped to room temperature, the furnace door was opened, and the crucible was taken out, and the product in the crucible was Phellodendron charcoal; after the Phellodendron charcoal was boiled in a water bath at 100°C for 3 times with 10 times the amount of deionized water, it was filtered and concentrated with a 0.22μm microporous filter membrane, and then deionized water was used as the dialysis medium, and the concentrate was dialyzed with a 1000D dialysis membrane for 7 days, and the solution in the bag was taken out, which was the Phellodendron charcoal carbon dots.

2. The use according to claim 1, characterized in that: The medicine may or may not contain pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that: The particle size of the cork carbon dots is less than 10 nm.

4. The use according to claim 1, characterized in that: The particle size of the cork carbon dots is 0.5nm~3.6nm.

5. The use according to claim 1, characterized in that: The medicine is a clinically acceptable oral preparation.

6. The use according to claim 1, characterized in that: The clinically acceptable oral preparation is a pill, capsule, tablet, granule or liquid oral preparation.

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