Application of coptis extract carbon dots

Abstract

The invention discloses application of a coptis extract carbon dot, relates to the field of carbon dots, and particularly relates to application of the coptis extract carbon dot in preparation of a medicinal preparation for inhibiting human fibrosarcoma cells and / or repairing and brightening. According to the invention, the coptis chinensis extract is used as a carbon source, and the ultra-small-size coptis chinensis extract carbon nanodots are synthesized by adopting a hydrothermal method. The rhizoma coptidis paste extract carbon dots disclosed by the invention can inhibit the generation of melanin to play a whitening role, and have the effects of inhibiting the proliferation of human fibrosarcoma HT-1080 cells and promoting the apoptosis of the HT-1080 cells.

Description

Technical Field

[0001] The present invention relates to the field of carbon dots, and particularly to the application of carbon dots extracted from Coptis chinensis ointment in the preparation of pharmaceutical preparations for inhibiting human fibrosarcoma cells and for repair and brightening. Background Art

[0002] In recent years, the development of materials chemistry has greatly influenced the lifestyle of humans. As a new type of heavy-metal-free and environmentally friendly nanomaterial, fluorescent carbon nanomaterials have received increasing attention due to their unique quantum size effect, surface effect and other properties, including carbon nanodots, carbon quantum dots, carbon nanotubes, graphene oxide, graphene quantum dots, etc. Among them, carbon nanodots (CDs) are a new type of zero-dimensional carbon nanomaterial with a spherical or quasi-spherical structure, usually with a size within 10 nm. Due to their unique fluorescence characteristics, good water solubility, excellent biocompatibility and cell absorption rate, low toxicity, environmental friendliness and other advantages, they are considered to be a promising fluorescent material, and the research and application in the field of nanomedicine have developed rapidly.

[0003] Plants are important natural resources for developing drugs. In recent years, plant preparations have received increasing attention worldwide, and many components extracted from natural plants have shown good biological activities and medicinal effects. China is one of the countries with the richest plant resources in the world and is the origin of Chinese herbal medicines. Currently, there are approximately 12,000 species of medicinal plants that can be used for the treatment and prevention of diseases. However, the active ingredients in the molecular state cannot cross the cell membrane due to poor cell membrane permeability and are difficult to exert their original medicinal effects. Therefore, improving the pharmacological activities of these components remains the focus of many ongoing research works. Many studies have shown that the active ingredients of Chinese herbal medicines can form nanoaggregates to improve water solubility and biocompatibility and exert better pharmacological effects. Summary of the Invention

[0004] The present invention provides the application of carbon dots extracted from Coptis chinensis ointment to achieve the inhibition of human fibrosarcoma cells and repair and brightening.

[0005] The application of carbon dots extracted from Coptis chinensis ointment of the present invention is the application of the carbon dots extracted from Coptis chinensis ointment in the preparation of pharmaceutical preparations for inhibiting human fibrosarcoma cells and / or for repair and brightening.

[0006] Further, the repair and brightening is skin repair.

[0007] Further, the skin repair includes whitening, removing skin spots / acne marks.

[0008] Further, the mass concentration of the carbon dots extracted from Coptis chinensis ointment is 40-100 μg / mL.

[0009] Furthermore, the Coptis chinensis ointment extract carbon dots are prepared by a one-step hydrothermal method.

[0010] Furthermore, for the preparation by the one-step hydrothermal method, the specific method is as follows:

[0011] Weigh the Coptis chinensis ointment extract, add distilled water, stir evenly until dissolved, and prepare a Coptis chinensis ointment extract solution with a mass concentration of; then transfer the Coptis chinensis ointment extract solution to a polytetrafluoroethylene-lined autoclave, react at a temperature of 160-200 °C for 8-12 h, then cool, and filter the reaction solution to obtain the Coptis chinensis ointment extract carbon dots.

[0012] Furthermore, the preparation method of the Coptis chinensis ointment extract is as follows:

[0013] Collect fresh Coptis chinensis ointment, clean and remove impurities, crush, and sieve to obtain Coptis chinensis ointment powder; dissolve it in water, add ethanol with a volume percentage content of 70% for extraction according to a solid-liquid ratio of 1:10, concentrate the extract, and after concentration, perform drying treatment to obtain the Coptis chinensis ointment extract; among them, the extraction conditions are: room temperature, extraction for 4 hours.

[0014] Furthermore, the filtration is carried out using a 0.22 μm polyethersulfone filter membrane.

[0015] The present invention has the following beneficial effects:

[0016] The present invention uses the Coptis chinensis ointment extract as a carbon source and synthesizes ultra-small-sized Coptis chinensis ointment extract carbon dots by a hydrothermal method. The Coptis chinensis ointment extract carbon dots of the present invention can inhibit melanin production and play a whitening role, and also have the effects of inhibiting the proliferation of human fibrosarcoma HT-1080 cells and promoting the apoptosis of HT-1080 cells. Description of the Drawings

[0017] Figure 1 TEM image of the prepared Coptis chinensis ointment extract carbon dots;

[0018] Figure 2 Particle size distribution diagram of the Coptis chinensis ointment extract carbon dots;

[0019] Figure 3 Fluorescence spectra of the Coptis chinensis ointment extract carbon dots at different excitation wavelengths;

[0020] Figure 4 UV-visible absorption spectra of the Coptis chinensis ointment extract and the Coptis chinensis ointment extract carbon dots (Illustration: Photos of the Coptis chinensis ointment extract carbon dots under a fluorescent lamp (left) and a 365 nm ultraviolet lamp (right));

[0021] Figure 5 Infrared spectrum of the Coptis chinensis ointment extract carbon dots;

[0022] Figure 6 X-ray photoelectron spectroscopy of carbon dots extracted from Coptis chinensis ointment;

[0023] Figure 7 Cell viability graph of LO2 cells after incubation in carbon dots extracted from Coptis chinensis ointment for 48 h;

[0024] Figure 8 Cell viability graph of HT-1080 cells after incubation in carbon dots extracted from Coptis chinensis ointment for 48 h;

[0025] Figure 9 Cell apoptosis graph of HT-1080 cells in the control group (left figure) and the carbon dots group extracted from Coptis chinensis ointment (right figure);

[0026] Figure 10 Comparison photos of dissected mouse tumors in the model (upper) and carbon dots group extracted from Coptis chinensis ointment (lower), with the scale bar being 1 cm;

[0027] Figure 11 Graph of relative melanin content in B16-F10 cells after different grouping treatments. Detailed implementation method

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed in the present invention will be described in detail below. After any person skilled in the art in the relevant technical field understands the embodiments of the content of the present invention, they can make changes and modifications based on the techniques taught by the content of the present invention, and these changes and modifications do not depart from the spirit and scope of the content of the present invention.

[0029] The schematic embodiments of the present invention and their descriptions are used to explain the present invention, but do not limit the present invention.

[0030] Example 1

[0031] Preparation of carbon dots extracted from Coptis chinensis ointment in this example

[0032] One-step hydrothermal method was used to prepare carbon dots extracted from Coptis chinensis ointment. 50 mg of the extract of Coptis chinensis ointment was accurately weighed and placed in a beaker containing 10 mL of distilled water. A magnetic stirrer was put in to stir until the extract of Coptis chinensis ointment was fully dissolved, and the solution concentration was 5.0 mg / mL. Then the solution was transferred to a polytetrafluoroethylene-lined autoclave and heated in an oven at 180 °C for 10 h. After the reaction time reached, the autoclave was quickly cooled in cold water. The solution in the inner liner was roughly filtered with a 0.22 μm polyethersulfone membrane to remove larger aggregates and precipitates, obtaining the corresponding yellow carbon dot solution of the extract of Coptis chinensis ointment, which was stored at 4 °C.

[0033] 1. Physicochemical property characterization of carbon dots extracted from Coptis chinensis ointment

[0034] Instruments: Transmission electron microscope (TEM) images were obtained using a Tecnai F20 electron microscope; ultraviolet-visible absorption spectra were obtained using a UV-2550 ultraviolet-visible spectrophotometer; fluorescence spectra were obtained using an F-2700 fluorescence spectrophotometer; infrared spectra were obtained using a Vector33 Fourier transform infrared spectrometer; X-ray photoelectron spectra were obtained using an ESCALAB 250 X-ray photoelectron spectrometer.

[0035] Experimental results: The particle size of the carbon dots extracted from Coptis chinensis ointment was concentrated at 2.28 ± 0.49 nm, with a uniform size distribution ( Figure 1-2 ), and had obvious excitation-dependent characteristics. The optimal excitation wavelength was at 370 nm, and the optimal emission wavelength was 480 nm ( Figure 3 ). The carbon dots extracted from Coptis chinensis ointment were a yellow solution and had strong blue-green fluorescence under irradiation with a 365 nm ultraviolet lamp. As Figure 4 shown by the ultraviolet absorption spectrum, there was an ultraviolet absorption peak at 277 nm in the carbon dot solution of the Coptis chinensis ointment extract. As Figure 5 shown by the infrared spectrum, the carbon dots extracted from Coptis chinensis ointment had absorption bands at 3400, 2930, 1760 - 1600, 1515, 1100 - 1000 cm -1 , which were attributed to the vibrations and rotations of O-H, C-H, C=O, C=C, and C-O. As Figure 6 shown by the X-ray photoelectron spectroscopy (XPS), two main peaks appeared at 282.2 and 531.2 eV in the carbon dots extracted from Coptis chinensis ointment, corresponding to the C1s and O1s elements respectively.

[0036] 2. Biosafety of the carbon dots extracted from Coptis chinensis ointment

[0037] Human normal liver cells (LO2) were seeded into 96-well plates (density: 3.5×10 3 cells / well) and incubated for 12 h to allow the cells to attach. The medium in the 96-well plates was discarded, and 200 μL of complete medium containing different concentrations of the carbon dots extracted from Coptis chinensis ointment (0, 40, 60, 80, and 100 μg / mL) was added. The cells were then cultured routinely for 48 h. The above cell medium was discarded, and then 100 μL / well of complete medium containing 10% cell counting kit-8 (CCK-8 assay) was added. After continuing to incubate in the incubator for 1 h, the absorbance was read at 450 nm.

[0038] The CCK8 results showed that the carbon dots extracted from Coptis chinensis ointment had no inhibitory effect on LO2 cells, indicating that the carbon dots extracted from Coptis chinensis ointment had almost no biological toxicity and had good biocompatibility.

[0039] 3. Study on the inhibitory effect of the carbon dots extracted from Coptis chinensis ointment on human fibrosarcoma cells

[0040] Human fibrosarcoma HT-1080 cells (MEM medium) were seeded into 96-well plates (density: 4×10 3 cells / well) and incubated for 12 h to allow cell attachment. The medium in the 96-well plates was discarded, and 200 μL of complete medium containing carbon dots extracted from Coptis chinensis ointment at different concentrations (0, 40, 60, 80, and 100 μg / mL) was added. The cells were then cultured routinely for 48 h. After discarding the above cell medium, 100 μL of complete medium containing 10% CCK-8 assay was added per well, and the plates were incubated in an incubator for 1 h and then read at 450 nm.

[0041] The CCK8 results showed that carbon dots extracted from Coptis chinensis ointment had a strong inhibitory effect on HT-1080 cells, indicating that carbon dots extracted from Coptis chinensis ointment have the effect of inhibiting the proliferation of human fibrosarcoma cells.

[0042] 4. Detection of cell apoptosis by flow cytometry

[0043] The apoptosis of HT-1080 cells after treatment with carbon dots extracted from Coptis chinensis ointment for 48 h was detected using the FITC-conjugated Annexin-V apoptosis detection kit I. Cells were cultured at an appropriate density in 6-well cell culture plates and incubated in an incubator for 24 h to allow the cells to grow stably. Then, the supernatant medium was aspirated, and the cells were divided into a control group (added with complete medium) and a carbon dots extracted from Coptis chinensis ointment group (concentration: 100 μg / mL), and they were co-incubated for 48 h. After that, the cells were carefully collected, and the dead cells floating on the medium were also collected. The operation was carried out according to the instructions of the cell apoptosis detection kit. The stained cells were analyzed using a flow cytometer. A total of 1×10 4 cells were counted for each sample.

[0044] The results showed that carbon dots extracted from Coptis chinensis ointment could promote the apoptosis of HT-1080 cells.

[0045] 5. Study on the inhibitory effect of carbon dots extracted from Coptis chinensis ointment on human fibrosarcoma

[0046] HT-1080 cells in the logarithmic growth phase with good morphology were prepared into cell suspensions with corresponding densities using PBS and inoculated into the right axilla of male BALB / c nude mice (5 weeks old). Approximately 3×10 6 cells were inoculated into each mouse to establish a subcutaneous fibrosarcoma mouse model. When the tumor volume reached about 50 mm 3 , the tumor-bearing mice were randomly divided into 3 groups (n = 3): a model group (intragastric administration, normal saline) and a carbon dots extracted from Coptis chinensis ointment group (intragastric administration, 10 mg / kg, once a day), and the dosage was 0.1 mL / 10 g. After 18 days of treatment, all nude mice were sacrificed and dissected to remove the tumors.

[0047] The results showed that the carbon dots extracted from Coptis chinensis ointment could inhibit the growth of human fibrosarcoma.

[0048] 6. In vitro whitening activity study

[0049] Effect of carbon dots extracted from Coptis chinensis ointment on melanin production in B16-F10 cells

[0050] The cell experiment was divided into three groups: blank group (Control): B16-F10 cells + PBS; model group: B16-F10 cells + α-MSH + PBS; administration group: B16-F10 cells + α-MSH + carbon dots extracted from Coptis chinensis ointment (60 / 80 / 100 μg / mL). The cells were inoculated into 24-well plates at a density of 3×10 4 cells / well. After 24 h, different concentrations of carbon dots extracted from Coptis chinensis ointment were added and incubated for 1 h. α-MSH was added to all groups except the blank group. Then, incubation continued for 48 h. After 48 h, the old medium in the wells was removed, and the cells were washed with cold PBS. 500 μL of trypsin was added to each well and digested at 37 °C for two minutes. Then, the cell suspension was transferred to an EP tube and centrifuged at 2000 rpm / min for 5 min, and the supernatant was discarded. Then, 150 μL of 1N NaOH / 10% DMSO was added to the cell pellet, and the cells were lysed at 80 °C for 1 h. The total melanin content was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Taking the absorbance value of the blank group as a control, the relative content of melanin in each group of cells was calculated.

[0051] From the results of the measured intracellular melanin content, it can be seen that compared with the blank control group, the melanin content of B16-F10 cells stimulated with 1.5 μM αMSH was significantly higher than that of unstimulated cells (p < 0.001), indicating successful modeling. With the increase in the concentration of carbon dots extracted from Coptis chinensis ointment, the melanin content in α-MSH-stimulated B16-F10 cells decreased significantly in a dose-dependent manner (p < 0.001). This indicates that the carbon dots extracted from Coptis chinensis ointment can inhibit the production of melanin induced by α-MSH in B16-F10 cells. The carbon dots extracted from Coptis chinensis ointment can reduce the production of melanin in B16-F10 cells to achieve the whitening effect.

[0052] 7. Human repeated open application test

[0053] The human repeated open application test is a simple and feasible method for evaluating the safety of cosmetic raw materials and products. Referring to the operation in the "Technical Specifications for Cosmetics Safety" (2015 Edition), 12 eligible volunteers were selected. An appropriate area on the flexor side of the forearm of the subject was smeared with 0.05 mL (500 μg / mL) of the carbon dots extracted from Coptis chinensis ointment, twice a day for 7 consecutive days. The skin reactions were observed, and physiological saline was used as a blank control. The judgment criteria are as follows: 0 points - negative reaction; 1 point - slight erythema, skin dryness, wrinkles; 2 points - erythema, edema, papules, wheals, desquamation, fissures; 3 points - obvious erythema, edema, blisters; 4 points - severe erythema, edema, large blisters, erosion, pigmentation or hypopigmentation, acne-like changes.

[0054] Table 1 Results of the human repeated open application test

[0055]

[0056] The test sites on the forearms of 12 subjects were observed for 7 days, and the results are shown in Table 1. In the human repeated open application test, the score of the carbon dots extracted from Coptis chinensis ointment on human skin irritation was 0. According to the standard judgment, the carbon dots extracted from Coptis chinensis ointment have no irritation to human skin. Therefore, it can be determined that the carbon dots extracted from Coptis chinensis ointment are safe and effective as raw materials added to cosmetics.

[0057] 8. Efficacy evaluation

[0058] Thirty volunteers with different skin conditions were randomly selected. The carbon dots extracted from Coptis chinensis ointment were used twice a day, once in the morning and once in the evening. It was used continuously for 4 weeks. The experiment was controlled by the before and after of itself. The volunteers evaluated the experience effect.

[0059] The recognition of the effect was relatively good after the volunteers used the carbon dots extracted from Coptis chinensis ointment for 2 weeks. 100% of the subjects thought that the skin became moist and soft; more than 60% of the subjects thought that the skin looked shiny, brightened, less sallow, and the skin glowed after using the carbon dots extracted from Coptis chinensis ointment. The recognition of the effect was relatively high after the volunteers used the carbon dots extracted from Coptis chinensis ointment for 4 weeks. More than 50% of the subjects thought that the skin spots / scar marks were significantly faded, the skin was cleaner, and the number of skin spots / blemishes decreased. 100% of the subjects thought that the skin became moist and soft after using the carbon dots extracted from Coptis chinensis ointment; more than 80% of the subjects thought that the skin looked shiny, brightened, less sallow, and the skin glowed after using the carbon dots extracted from Coptis chinensis ointment. More than 70% of the subjects thought that the skin spots / scar marks were significantly faded, the skin was cleaner, and the number of skin spots / blemishes decreased.

Claims

1. Application of carbon dots from Coptis chinensis extract, characterized in that The application of the carbon dots of the coptis chinensis extract in the preparation of a pharmaceutical preparation for inhibiting human fibrosarcoma cells and / or repairing and brightening the cells.

2. The use according to claim 1, characterized in that The repairing and brightening described herein refers to skin repairing.

3. The use according to claim 1, characterized in that The skin repair includes whitening and removing spots / acne marks.

4. The use according to claim 1, 2 or 3, characterized in that The mass concentration of the carbon dots in the coptis chinensis extract is 40-100 μg / mL.

5. The use according to claim 1, 2 or 3, characterized in that The carbon dots of the coptis root extract are prepared by a one-step hydrothermal method.

6. The use according to claim 5, characterized in that The one-step hydrothermal preparation method is as follows: Weigh the coptis chinensis paste extract, add distilled water, stir and dissolve evenly to prepare a coptis chinensis paste extract solution with a mass concentration of; then transfer the coptis chinensis paste extract solution to a polytetrafluoroethylene-lined high-pressure reactor, react for 8 to 12 hours at a temperature of 160 to 200° C., and then cool. After filtering the reaction solution, the coptis chinensis paste extract carbon dots are obtained.

7. The use according to claim 1, 2 or 3, characterized in that The preparation method of the coptis root extract is as follows: Fresh coptis paste is collected, cleaned and impurities removed, crushed, and sieved to obtain coptis paste powder; after being dissolved in water, 70% ethanol by volume is added in a ratio of 1:10 between solid and liquid to extract, the extract is concentrated, and after concentration, it is dried to obtain coptis paste extract; wherein the extraction conditions are: room temperature, and extraction for 4 hours.

8. The use according to claim 3, characterized in that The filtration is carried out using a 0.22 μm polyethersulfone filter membrane.

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