Preparation method and application of lithospermum carbon nanodot with anti-adrenal cortex cancer

Abstract

The invention discloses a preparation method and application of lithospermum carbon nanodots with an anti-adrenal cortical carcinoma function, and relates to the field of carbon dots, the lithospermum carbon nanodots are prepared by the following steps: mixing a lithospermum extract and deionized water, and carrying out a hydrothermal reaction to obtain a lithospermum carbon dot aqueous solution; the lithospermum carbon nanodots are used for preparing medicines for resisting adrenal cortex cancer. The lithospermum carbon nanodot provided by the invention has an inhibition effect on proliferation of adrenal cortex cancer cells, especially on the adrenal cortex cancer cells ACHN and 769-P. The lithospermum carbon nanodot disclosed by the invention has a remarkable proliferation inhibition effect on ACC (adrenal cortical carcinoma) cells. The adrenal cortex cancer is a rare and malignant tumor, and traditional treatment methods such as surgery and chemotherapy have certain limitations. Therefore, the development of a new targeted therapy strategy is very important. The lithospermum carbon nanodots can effectively inhibit proliferation of adrenal cortex cancer cells such as ACHN and 769-P through a certain molecular mechanism.

Description

Technical Field

[0001] The present invention relates to the field of carbon dots, and particularly to a preparation method of lithospermum carbon nanodots with anti-adrenocortical carcinoma activity. Background Art

[0002] Adrenocortical carcinoma (ACC) is a rare malignant tumor with strong invasiveness. Most patients are diagnosed with local invasion or metastasis. Its estimated incidence is 0.7×10 6 ~2.0×10 6 cases / year, with a higher incidence in the age groups of 1 - 4 years old and 40 - 50 years old, and a higher prevalence in females. ACC may be functional (referring to having hormone secretion function), causing Cushing's syndrome or virilization; or it may be non-functional, manifested as an upper abdominal mass or only accidentally discovered. The prognosis of this disease is poor, and the 5-year overall survival rate is only 15 - 44%. Tumor staging is a key prognostic factor for ACC. The expected 5-year survival rate for stage I patients is 80%, while that for stage IV patients is 13%. Approximately 2 / 3 of local disease patients relapse and require comprehensive treatment mainly based on systemic chemotherapy.

[0003] Carbon nanodots (C-dots) are quasi-spherical zero-dimensional carbon-based nanomaterials with a particle size of 1 - 20 nm, and their surfaces often contain abundant functional groups such as hydroxyl, amino, or carboxyl groups. Due to characteristics such as low preparation cost, small size, good water solubility, medicinal activity, and high biocompatibility, C-dots have been a research hotspot in the field of nanomaterials in recent years and have shown unique advantages and application prospects in multiple fields. The preparation method of carbon dots is relatively simple, the raw materials are extensive and inexpensive, and they have good water solubility, optical properties, and biocompatibility. Their roles in biosensing, drug delivery, tumor treatment, and the cosmetics field are being widely developed and reported.

[0004] As a commonly used traditional Chinese medicine, lithospermum has a long medicinal history, and its use has been recorded in medical classics of past dynasties. Lithospermum carbon nanodots have broad application prospects in the field of cancer treatment, but current research in this area is relatively scarce and needs to be continuously explored in depth. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a preparation method of lithospermum carbon nanodots with anti-adrenocortical carcinoma activity.

[0006] An application of the lithospermum carbon nanodots of the present invention, wherein the lithospermum carbon nanodots are used for preparing drugs against adrenocortical carcinoma.

[0007] Furthermore, the diameter of the lithospermum carbon nanodots is 1 - 50 nm; the surface of the lithospermum carbon nanodots contains one or more of hydroxyl groups, carbonyl groups, and carbon-carbon double bonds.

[0008] Furthermore, the preparation method of the lithospermum carbon nanodots is as follows:

[0009] 1) Mix the lithospermum extract and deionized water according to a mass ratio of 1:125 - 500 to obtain a mixed solution;

[0010] 2) Perform a hydrothermal reaction on the obtained mixed solution to obtain an aqueous solution of lithospermum carbon dots;

[0011] The temperature of the hydrothermal reaction is 170°C - 180°C; the time of the hydrothermal reaction is 8 - 9 h.

[0012] Furthermore, the preparation method of the lithospermum extract is as follows:

[0013] Take lithospermum, wash, dry, crush, and sieve it to obtain lithospermum powder; add an aqueous solution to the lithospermum powder, and use an ethanol aqueous solution with a volume percentage of 70% as a solvent for extraction according to a solid-liquid ratio of 1:10. After extraction, concentrate and dry to obtain the lithospermum extract; among them, the extraction conditions are extraction at room temperature for 3 times, 4 hours each time.

[0014] Furthermore, perform filtration after the hydrothermal reaction.

[0015] Furthermore, the mass ratio of the lithospermum to deionized water is 1:200 - 400.

[0016] The present invention has the following beneficial effects:

[0017] The lithospermum carbon nanodots of the present invention have an inhibitory effect on the proliferation of adrenocortical cancer cells, especially on adrenocortical cancer cells ACHN and 769-P. The lithospermum carbon nanodots of the present invention have a significant inhibitory effect on the proliferation of adrenocortical carcinoma (ACC) cells. Adrenocortical carcinoma is a rare and malignant tumor, and traditional treatment methods such as surgery and chemotherapy have certain limitations. Therefore, it is very important to develop new targeted treatment strategies. The lithospermum carbon nanodots can effectively inhibit the proliferation of adrenocortical cancer cells such as ACHN and 769-P through a certain molecular mechanism. Description of the Drawings

[0018] Figure 1 It is a diagram of the lithospermum carbon nanodots in Example 1 under bright field and 365 nm ultraviolet irradiation; under bright field and 365 nm ultraviolet irradiation; under bright field, the lithospermum carbon nanodots are a light yellow transparent solution; under 365 nm irradiation, the lithospermum carbon nanodots show blue fluorescence;

[0019] Figure 2It is the UV spectrum of Lithospermum erythrorhizon and the carbon nanodots of Lithospermum erythrorhizon in Example 1; Lithospermum erythrorhizon does not produce UV absorption; the carbon nanodots of Lithospermum erythrorhizon all produce UV absorption at around 280 nm;

[0020] Figure 3 It is the infrared spectrum of the carbon nanodots of Lithospermum erythrorhizon in Example 1; characteristic absorption peaks appear at around 3500 cm-1, 1700 cm-1, and 1400 cm-1 for the carbon nanodots of Lithospermum erythrorhizon. It is speculated that the structure of the carbon nanodots of Lithospermum erythrorhizon contains chemical bonds such as O-H, C=O, and C=C;

[0021] Figure 4 It is the XPS diagram of the carbon nanodots of Lithospermum erythrorhizon in Example 1; the structure of the carbon nanodots of Lithospermum erythrorhizon contains O and C atoms;

[0022] Figure 5 It is the XPS peak-fitting diagram of the carbon nanodots of Lithospermum erythrorhizon in Example 1; it can be seen from the XPS peak-fitting C spectrum that the absorption peak generated at around 284 eV may be C-C / C=O; the absorption peak generated at around 285 eV may be C-O; the absorption peak generated at around 288 eV may be C=O;

[0023] Figure 6 It is the XPS peak-fitting O spectrum of the carbon nanodots of Lithospermum erythrorhizon in Example 1; it can be seen that the absorption peak generated at around 45 eV may be C=O;

[0024] Figure 7 It is the excitation dependence diagram of the carbon nanodots of Lithospermum erythrorhizon in Example 1; the optimal excitation wavelength of the carbon nanodots of Lithospermum erythrorhizon is 370 nm; the wavelength at the maximum absorption is 430 nm;

[0025] Figure 8 It is the transmission electron microscopy image of the carbon nanodots of Lithospermum erythrorhizon in Example 1; at a scale of 20 nm, it is observed that the particle size distribution of the carbon nanodots of Lithospermum erythrorhizon is uniform Figure 9 It is the graph for determining the proliferation rate of adrenocortical cancer cells by the CCK-8 method; Figure 10 It is the graph of the inhibitory result of the carbon nanodots of Lithospermum erythrorhizon on mouse adrenocortical tumors. Detailed implementation manners

[0026] 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 by the present invention will be described in detail below. After any person skilled in the art in the technical field to which the present invention pertains understands the embodiments of the content of the present invention, they can make changes and modifications based on the technology taught by the content of the present invention, which do not depart from the spirit and scope of the content of the present invention.

[0027] The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but do not limit the present invention.

[0028] Example 1

[0029] The preparation method of a kind of lithospermum carbon nanodots with anti - adrenocortical carcinoma in this example is as follows:

[0030] 1) Mix lithospermum and deionized water to obtain a lithospermum mixture;

[0031] 2) After carrying out hydrothermal reaction on the mixture obtained in the above step, a lithospermum carbon dot aqueous solution is obtained; the temperature of the hydrothermal reaction is 170°C - 180°C; the time of the hydrothermal reaction is 8 - 9h.

[0032] Activity detection of Example 2

[0033] Experiment 1. Whitening and moisturizing experiment

[0034] After shaving the back hair of pigmented guinea pigs, irradiate with ultraviolet light 2 times every other day, repeat this operation, and detect with a skin detector until ultraviolet pigmentation is formed on the guinea pig skin. Then continuously apply lithospermum carbon nanodots for 21 days, and measure the skin water content and melanin situation on the 21st day. The control group does not apply any drug.

[0035] The experimental results are shown in Tables 1 and 2.

[0036] Table 1

[0037]

[0038] Note * represents p < 0.01 compared with the control group.

[0039] Table 2

[0040]

[0041]

[0042] Note * represents p < 0.05 compared with the control group.

[0043] It can be seen from Table 1 that the melanin content of CDs - 1 - 3 is significantly lower than that of the control group, proving that lithospermum carbon nanodots have the effect of whitening. It can be seen from Table 2 that the skin water content of the tested guinea pigs of CDs - 1 - 3 is significantly higher than that of the control group, proving that lithospermum carbon nanodots have the effect of moisturizing.

[0044] It can be seen from the experimental results that lithospermum carbon nanodots can brighten the skin color and have the effect of whitening and moisturizing.

[0045] Experiment 2. Analysis of the anti - adrenocortical carcinoma activity of lithospermum carbon nanodots

[0046] Determination of the proliferation rate of adrenocortical cancer cells by CCK - 8 method

[0047] The H295R cells were passaged 2 - 3 times and seeded in 96 - well plates at a cell density of 3×10 3 cells / well, with a total volume of 180 μL of cell culture medium in each well. After 24 hours of seeding, the growth status of the cells was observed. After the cells adhered to the wall, they were treated with shikonin carbon nanodots solutions at different concentrations (4 μg / mL, 8 μg / mL, 12 μg / mL, 16 μg / mL). After culturing for 24 h, 48 h, 72 h, 96 h, and 120 h respectively, 10 μL of CCK - 8 solution was added, and after culturing for 2 h, the absorbance (OD450) values of each well at 450 nm were measured using an enzyme - linked immunosorbent assay (ELISA) reader.

[0048] The experimental results are as Figure 9 shown.

[0049] The inhibitory effect of shikonin on the proliferation of H295R cells was detected by CCK - 8 cell proliferation assay. It can be Figure 9 seen that it has an obvious inhibitory effect on the proliferation of H295R cells, and as the concentration of the administered drug increases, the cell survival rate gradually decreases, indicating that shikonin carbon nanodots can inhibit the proliferation of adrenocortical carcinoma H295R cells.

[0050] Experiment 3: Animal experiment

[0051] H295R cells were injected subcutaneously into the skin of BALB / c nude mice to establish a tumor - bearing model. The tumor - bearing nude mice were randomly divided into three groups. Olive oil (90 mg / Kg, 5 times a week) was intraperitoneally injected as the model group, mitotane (2 mg / Kg, 5 times a week) was intraperitoneally injected as the positive control group, and shikonin carbon nanodots (2 mg / Kg, 5 times a week) were intraperitoneally injected as the drug - administered group. The experimental period was 14 days, and they were fed under the same conditions. An animal CT imaging system was used to observe the growth of the tumors in mice on the 3rd, 8th, and 14th days during one experimental period. The results are as Figure 10 shown. As time goes by, the tumor growth trend in the model group is obvious, and the tumor growth in the mitotane group is slower than that in the model group. The tumor growth in the shikonin carbon nanodot group is the slowest, and the tumor volume is the smallest. Compared with the control group, the tumor volume of mice in the shikonin carbon nanodot group is significantly reduced. The above data indicate that shikonin carbon nanodots have the effect of inhibiting the growth of adrenocortical tumors and producing significant curative effects in vivo.

Claims

1. An application of Lithospermum officinale carbon nanodots, characterized in that The lithospermum carbon nanodots are used for preparing drugs for resisting adrenocortical carcinoma.

2. The use of lithospermum officinale carbon nanodots with anti-adrenocortical carcinoma properties according to claim 1, characterized in that The diameter of the comfrey carbon nanodots is 1-50nm; the surface of the comfrey carbon nanodots contains one or more of hydroxyl groups, carbonyl groups and carbon-carbon double bonds.

3. The use of a comfrey carbon nanodot according to claim 1 or 2, characterized in that The preparation method of the lithospermum carbon nanodots is as follows: 1) mixing the lithospermum officinale extract and deionized water in a mass ratio of 1:125-500 to obtain a mixed solution; 2) subjecting the obtained mixed solution to a hydrothermal reaction to obtain an aqueous solution of lithospermum parkii carbon dots; The temperature of the hydrothermal reaction is 170° C. to 180° C.; the time of the hydrothermal reaction is 8 to 9 hours.

4. The use of comfrey carbon nanodots according to claim 3, characterized in that The preparation method of the lithospermum officinale extract is as follows: The method comprises the steps of washing, drying, crushing and sieving lithospermum officinale powder; adding an aqueous solution to obtain lithospermum officinale powder, extracting the lithospermum officinale powder using an ethanol aqueous solution with a volume percentage of 70% as a solvent at a solid-liquid ratio of 1:10, concentrating after extraction and drying to obtain a lithospermum officinale extract; wherein the extraction condition is to extract 3 times at room temperature, each time for 4 hours.

5. The use of comfrey carbon nanodots according to claim 3, characterized in that The hydrothermal reaction is followed by filtration.

6. The use of comfrey carbon nanodots according to claim 3, characterized in that The mass ratio of the lithospermum officinale to deionized water is 1:200-400.

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