Preparation method and application of centella asiatica carbon nanodots capable of inducing thyroid cancer cell B-CPAP apoptosis and resisting oxidation

Abstract

The invention discloses a preparation method and application of centella carbon nanodots capable of inducing thyroid cancer cell B-CPAP apoptosis and resisting oxidation and relates to the field of carbon dots. The preparation method of the centella carbon nanodots comprises the steps that a centella extract is taken, ultrapure water is added to be mixed, then the mixture is transferred into a high-pressure reaction kettle with a polytetrafluoroethylene lining, a reaction is conducted for 6-8 h at the temperature of 160-180 DEG C, and the centella carbon nanodots are obtained. And cooling and filtering to obtain the centella carbon nanodots capable of inducing thyroid cancer cell B-CPAP apoptosis and resisting oxidation. The compound is used for preparing anti-thyroid cancer drugs or anti-oxidation drugs. The centella asiatica carbon nanodot prepared by the invention has a growth inhibition effect on B-CPAP cells and has an anti-thyroid cancer effect. The centella asiatica carbon nanodots also have an anti-oxidation function and a skin allergy function.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care products, and particularly relates to a preparation method and application of centella asiatica carbon nanodots with the ability to induce apoptosis of thyroid cancer cells B-CPAP and antioxidant activity. Background Art

[0002] Skin aging is a complex process involving the combined action of multiple internal and external factors. As people age, the cells and tissues of the human body gradually undergo degenerative changes, and the skin is no exception. Over time, the elasticity and moisture retention of the skin gradually decline, and wrinkles and sagging gradually appear. In addition, environmental factors such as ultraviolet rays, pollutants, and poor eating habits also accelerate the process of skin aging. To slow down skin aging, people have adopted various methods, and using skin care products is a common way. Currently, many anti-aging skin care products have emerged on the market, and many of them contain centella asiatica extracts. Due to the multiple biological activities and antioxidant effects of centella asiatica, it has become a favored natural anti-aging ingredient. Using skin care products containing centella asiatica extracts can help people improve skin problems and delay the process of skin aging. In addition to using skin care products, maintaining a healthy lifestyle, such as having a good diet, getting enough sleep, and exercising appropriately, also helps slow down skin aging.

[0003] Thyroid cancer is the most common malignant tumor of the thyroid gland, accounting for about 1% of all malignant tumors in the body. Thyroid cancer includes four pathological types: papillary carcinoma, follicular carcinoma, anaplastic carcinoma, and medullary carcinoma. Among them, papillary carcinoma, which has a relatively low malignancy and a better prognosis, is the most common. The incidence of thyroid cancer is related to region, race, and gender to a certain extent. Females have a higher incidence, and the male-to-female ratio is 1:2 - 4. It can occur at any age, but is more common in young and middle-aged people. The vast majority of thyroid cancers occur in one lobe of the thyroid gland and are often single tumors. Medullary carcinoma is a malignant tumor originating from thyroid C cells and is a moderately malignant tumor, accounting for about 3% - 8% of thyroid malignancies. The cancer cells of medullary carcinoma have a consistent morphology and no papillary or follicular structure. Its classification mainly comes from the European Organization for Research and Treatment of Cancer (EORTC), the National Thyroid Cancer Treatment Cooperative Study Group (NTCTCS), and thyroid cancer surveillance. The familial type accounts for about 20%, with an average age of about 50 years. The cancer mass is often solitary, mostly confined to one lobe of the thyroid gland, with a relatively hard texture, clear edges, and a variable course (from several months to more than 10 years). It metastasizes through lymph nodes, often to the cervical lymph nodes, and can cause compressive symptoms and metastatic masses. When there is recurrence and metastasis, it can reappear, and CT measurement can be used to screen family members. Ret gene mutation analysis has been used to diagnose this disease and screen high-risk subjects among family members. However, 30% of patients only have elevated blood CT (up to 15 years in some cases) without recurrence of the lesion. Anaplastic carcinoma is a tumor with a relatively high malignancy, accounting for 10% - 15% of thyroid cancers. Its characteristics are low degree of differentiation, fast growth rate, short course, rapid development, and easy metastasis. The prognosis of anaplastic carcinoma is usually poor, and the survival time is short.

[0004] Carbon dots have excellent water solubility and can easily be compatible with the surrounding environment. At the same time, carbon dots also have excellent photostability and can remain stable even under strong light. More commendably, carbon dots have good biocompatibility, are non-toxic and harmless to organisms, making them have broad application prospects in the biomedical field. The method for preparing carbon dots is simple and easy to implement, and the raw materials are rich in sources, making the production cost of carbon dots low, which is conducive to large-scale application.

[0005] Centella asiatica is a traditional Chinese medicine with extremely high medicinal value and relatively extensive pharmacological activities. Research shows that in the field of natural medicines, Centella asiatica is widely used in the treatment of diseases such as hepatitis, liver cirrhosis, and liver cancer, and is also applied in health care. This is mainly due to the rich components contained in Centella asiatica, such as flavonoids, polysaccharides, alkaloids, etc. These components have various pharmacological effects such as antioxidant, anti-inflammatory, anti-tumor, and immunomodulatory effects. Active components in Centella asiatica such as asiaticoside can stimulate the proliferation of fibroblasts and activate the SMAD signaling pathway, increase the production of type I collagen, have the activity of wound healing, and at the same time can reduce the formation of stretch marks.

[0006] In the field of cosmetics, Centella asiatica is also widely used. Due to its good moisturizing, antioxidant, anti-inflammatory and other effects, it can effectively improve the quality of the skin, so it is used by many cosmetics manufacturers in the production of skin care products. Centella asiatica extract has an obvious promoting effect on the deposition of extracellular matrix proteins, can promote the proliferation of fibroblasts, increase the synthesis of collagen, reduce the activity of metalloproteinases, thereby increasing the deposition of collagen and the level of intracellular free proline. At the same time, Centella asiatica extract also has the skin care effects of anti-inflammatory and anti-allergic, can inhibit the inflammatory response induced by LPS, inactivate macrophages in the skin, thereby reducing skin inflammation and atopic reactions.

[0007] Generally speaking, Centella asiatica has a wide range of applications in the fields of natural medicine and cosmetics. Its unique biological activity functions and pharmacological effects make it have unique value in disease treatment and skin care. Summary of the Invention

[0008] The present invention provides a preparation method and application of Centella asiatica carbon dots with the ability to induce apoptosis of thyroid cancer cells B-CPAP and antioxidant activity.

[0009] An application of Centella asiatica carbon dots of the present invention, wherein the Centella asiatica carbon dots are used to prepare anti-thyroid cancer drugs or antioxidant drugs.

[0010] Further, the mass concentration of the Centella asiatica carbon dots is 50-200 μg / mL.

[0011] Further, the preparation method of the Centella asiatica carbon dots is as follows:

[0012] Take Centella asiatica extract, add ultrapure water and mix, then transfer it to a polytetrafluoroethylene-lined autoclave, and react at a temperature of 160-180 °C for 6-8 h. After cooling, filter to obtain Centella asiatica carbon dots with the ability to induce apoptosis of thyroid cancer cells B-CPAP and antioxidant activity.

[0013] Further, the preparation method of the Centella asiatica extract is as follows:

[0014] Take fresh Centella asiatica, wash, remove impurities, crush, and sieve to obtain Centella asiatica powder; dissolve it in water, and extract it with ethanol with a volume percentage of 70% according to the material-liquid ratio of 1:10. After extraction, concentrate and dry to obtain Centella asiatica extract; among them, the extraction conditions are to extract 3 times at room temperature, 4 hours each time.

[0015] Further, the filtration is carried out with a 0.22 nm filter membrane.

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

[0017] The centella asiatica carbon dots prepared by the present invention have a growth inhibitory effect on B-CPAP cells and have an anti-thyroid cancer effect. The centella asiatica carbon dots also have antioxidant and skin allergy functions. Description of the Drawings

[0018] Figure 1 It is a structural characterization diagram of centella asiatica carbon dots; among them, Figure 1 a is a photograph of centella asiatica carbon dots under 365 nm ultraviolet light irradiation; Figure 1 b is an electron micrograph of centella asiatica carbon dots; Figure 1 c is the ultraviolet absorption diagram of centella asiatica carbon dots and centella asiatica; Figure 1 d is the excitation wavelength diagram of centella asiatica carbon dots; Figure 1 e is the vibration diagram of the O-H bond and C═C double bond of centella asiatica carbon dots; Figure 1 f is the XPS spectrum of centella asiatica carbon dots. Detailed Embodiments

[0019] 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 relevant technical field understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified, which does not deviate from the spirit and scope of the content of the present invention.

[0020] Example 1

[0021] Take 1 mL of centella asiatica extract, add it to 150 mL of ultrapure water, mix it by ultrasonic for 1 minute, then divide it into 20 ml inner liners. After tightening the lid of the inner liner, put the inner liner into the corresponding reaction kettle, and tighten the lid of the reaction kettle to avoid problems during the heating process. First, adjust the oven temperature to 170°, and when the oven temperature rises to the set temperature, put the reaction kettle into the oven, start timing, take it out after reacting for 7 hours, place it in a non-closed space to cool down, make good marks to avoid other people being scalded, and remember to take it out carefully with several layers of dry towels wrapped around it to avoid scalding. Do not open the lid immediately after taking it out, as it will cause the overheated liquid to splash out and scald yourself. You need to wait until it cools down before taking it out. Suck the prepared carbon dot solution into a centrifuge tube with a syringe. Then filter it with a 0.22 nm filter membrane, and put the filtered medicine into the refrigerator, and set the storage temperature to -4°C.

[0022] Structural Characterization of Centella Asiatica Carbon Dots Prepared in this Example

[0023] The centella asiatica carbon dot solution is yellow, and it emits blue-green fluorescence under 365 nm ultraviolet light irradiation. From Figure 1 c, it can be found that the centella asiatica carbon dot solution has an ultraviolet absorption peak at about 290 nm more than the centella asiatica solution. From Figure 1It can be seen that the optimal excitation wavelength of the asiaticoside carbon dots is ~370 nm, and the optimal emission wavelength is ~450 nm. From Figure 1 e, the vibration peaks of the O-H bond and C=C double bond of the asiaticoside carbon dots can be seen. Figure 1 XPS spectrum of the asiaticoside carbon dots in f: High-resolution XPS spectra of the high-resolution asiaticoside carbon dots, C1s spectrum (h), O 1s spectrum (i). Analysis of the moisturizing, anti-thyroid cancer, and anti-aging activities of the asiaticoside carbon dots.

[0024] Example 2

[0025] Anti-thyroid cancer activity test of the asiaticoside carbon dots in Example 1:

[0026] The in vitro cytotoxicity of the asiaticoside carbon dots was detected by the Cell Counting Kit-8 assay, and the CCK-8 method was used to detect the toxicity of the asiaticoside carbon dots to cells. Taking B-CPAP cells as an example, the B-CPAP cells were cultured to the logarithmic growth phase, and the appropriate cell density was 3000 - 5000 cells / well. Then, the B-CPAP cells were transferred to a 96-well cell culture plate for culture. Then, 180 μL of the cell suspension per well was placed in an incubator containing 5% CO2 at 37 °C. When the cells were completely attached to the cell bottle wall, 20 μL of asiaticoside carbon dots with different concentrations was added. Next, the asiaticoside carbon dot concentrations of each well of cells were 0, 50, 100, 150, 200 μg / mL (6 replicates for each concentration). After incubating the asiaticoside carbon dots and B-CPAP cells for 48 h, 20 μL of CCK-8 was added to each well. The 96-well cell culture plate was placed in the incubator for 30 min, and the optical density (OD) was measured at a wavelength of 450 nm using a microplate reader (Infinite M200PRO, Tecan, Switzerland). The viability of B-CPAP cells was calculated based on the OD value. These results indicate that the asiaticoside carbon dots have a growth inhibitory effect on B-CPAP cells and have a certain anti-thyroid cancer effect. In subsequent experiments, the mechanism of action, the optimal concentration of action, and the possible clinical application value of the asiaticoside carbon dots can be further explored.

[0027] Verification of the repair effect of asiaticoside carbon dots on damaged mouse cancer: To verify the repair effect of asiaticoside carbon dots on damaged mouse cancer, the research team carefully designed an experimental plan. They first prepared the asiaticoside carbon dots and confirmed their structure and properties through a series of characterization methods. Subsequently, the asiaticoside carbon dots were applied to the skin of damaged mice to observe their repair effect on cancer.

[0028] Example 3

[0029] Antioxidant test of the asiaticoside carbon dots in Example 1:

[0030] (1) DPPH radical scavenging rate:

[0031] 1. Take 3 mL of the test solution and mix it with 3 mL of 2×10 -4 mol / L DPPH solution (Tube A1): This step is to test whether the antioxidants in the test solution can scavenge DPPH radicals. The concentration and volume of the DPPH solution are preset to ensure the consistency of experimental conditions.

[0032] 2. Take 3 mL of absolute ethanol and mix it with 3 mL of 2×10 -4 mol / L DPPH solution (Tube A2): This step is to obtain a control group, that is, a sample containing only DPPH solution and solvent (absolute ethanol). This control group is used to correct the absorbance of the DPPH solution itself and the absorbance changes that may be caused by the solvent in the experiment.

[0033] 3. Take 3 mL of absolute ethanol and mix it with 3 mL of the test solution (Tube A3): This step is to test the absorbance of the test solution itself, so as to exclude the influence of the test solution solvent on the experimental results. Since the absorbance may change due to the reaction when the test solution and DPPH solution are mixed in Tube A1, it is necessary to measure the absorbance of the test solution alone for correction.

[0034] 4. After reacting for 30 minutes at 30°C in the dark: Place the mixed samples in an environment at 30°C and react in the dark for 30 minutes to ensure that there is enough time for the antioxidants and DPPH radicals to react.

[0035] 5. Using distilled water as the blank group, measure the absorbance values of Tubes A1, A2, and A3 at 517 nm, and record them as OD A1, ODA2, and OD A3 respectively:

[0036] Distilled water is used as the blank group because it does not contain any substances that may affect the absorbance. Measure the absorbance at a wavelength of 517 nm because the absorbance change of DPPH radicals is most obvious at this wavelength. Record the absorbance values of Tubes A1, A2, and A3 respectively, and these values will be used for subsequent calculations.

[0037] By comparing the values of OD A1, OD A2, and OD A3, the scavenging rate of antioxidants in the test solution for DPPH radicals can be calculated, thereby evaluating the antioxidant performance of the test solution.

[0038] The DPPH radical scavenging rate is calculated according to the following formula:

[0039] DPPH scavenging rate (%) = (OD A2 + OD A3 - OD A1) / OD A2 × 100%

[0040] (2) Determination of superoxide anion scavenging:

[0041] 1. Pipette 1 mL of the test solution, add 9 mL of 0.05 mol / L Tris-HCl-EDTA buffer with pH = 8.2, and mix well: This step is to dilute the test solution and adjust the pH value to an appropriate range for subsequent reactions.

[0042] 2. React in a water bath at 25 °C for 15 min:

[0043] Place the mixed sample in a water bath at 25 °C and react for 15 minutes to ensure that there is sufficient time for the antioxidants and superoxide ions in the sample to react.

[0044] 3. After the reaction, take 3 mL of this reaction solution, add 0.1 mL of 45 mmol / L pyrogallol solution (prepared with 0.01 mol / L hydrochloric acid), mix well, and let it stand for 3 min: Pyrogallol will autoxidize to produce superoxide ions under acidic conditions, and this autoxidation process can be inhibited by adding antioxidants. In the experiment, the concentration and volume of pyrogallol are preset to ensure the consistency of experimental conditions.

[0045] 4. Measure the absorbance A1 at 340 nm, and at the same time, measure the absorbance value A2 of the mixture without adding pyrogallol. Use distilled water instead of the sample solution to obtain the absorbance value A3, with distilled water as the blank: Measure the absorbance at 340 nm because the absorbance change of superoxide ions is most obvious at this wavelength. Record the absorbance A1 of the sample with added pyrogallol, the absorbance A2 of the sample without added pyrogallol, and the absorbance A3 of distilled water respectively. Calculate the superoxide anion scavenging rate according to the following formula:

[0046] Scavenging rate of superoxide anion (%) = [A3 - (A1 - A2)] / A3 × 100%

[0047]

[0048]

[0049] As can be seen from the above table, the centella asiatica carbon dots prepared by the present invention have good antioxidant effects.

[0050] Example 4

[0051] The skin allergy test process of the centella asiatica carbon dots in Example 1 is as follows:

[0052] 1. First, conduct skin tests on 7 volunteers to ensure that the volunteers have no abnormal conditions such as skin allergies.

[0053] 2. After the experiment starts, the volunteers apply the test sample on their faces every morning and evening, and the application area should be uniform. During the experiment, the subjects shall not apply other cosmetics on the experimental area to ensure the accuracy of the experimental results.

[0054] 3. The volunteers are tested respectively after continuously using the test sample for 0 days, 14 days, and 30 days. The test contents include:

[0055] a. Observation of skin condition: Observe the changes in skin color, luster, fineness, etc.

[0056] b. Measurement of skin moisture content: Use professional instruments to measure the skin moisture content to evaluate the influence of the test sample on the skin moisturizing effect.

[0057] c. Measurement of skin sebum secretion: Use professional instruments to measure the skin sebum secretion amount to evaluate the control effect of the test sample on skin sebum secretion.

[0058] d. Questionnaire survey: Distribute questionnaires to collect the usage feelings of the volunteers during the experiment, such as comfort, ease of use, smell, etc.

[0059] 4. Data collection and analysis: Statistically analyze the experimental data to evaluate the efficacy and safety of the test sample.

[0060] 5. Compile an experimental report: Organize the experimental data and results, write an experimental report, and summarize the advantages and disadvantages of the test sample.

[0061] Through the above steps, we can comprehensively evaluate the effect of the test sample on the volunteers' skin, provide a scientific basis for product improvement and promotion. At the same time, it can also provide useful skin care product recommendations for consumers.

[0062] Test results:

[0063]

[0064]

[0065] According to the usage feelings of the subjects, it shows that the carbon nanodots with moisturizing efficacy of the present invention can reduce wrinkles and improve the roughness and lubricity of the skin.

Claims

1. An application of Centella asiatica carbon nanodots, characterized in that The Centella asiatica carbon nanodots are used for preparing anti-thyroid cancer drugs or antioxidant drugs.

2. The application of Centella asiatica carbon nanodots according to claim 1, characterized in that The mass concentration of the Centella asiatica carbon nanodots is 50-200 μg / mL.

3. The use of Centella asiatica carbon nanodots according to claim 1 or 2, characterized in that The preparation method of the Centella asiatica carbon nanodots is as follows: Take Centella asiatica extract, add ultrapure water to mix, transfer to a polytetrafluoroethylene-lined high-pressure reactor, react for 6 to 8 hours at a temperature of 160 to 180°C, cool and filter to obtain Centella asiatica carbon nanodots that can induce apoptosis of thyroid cancer cells B-CPAP and have anti-oxidation.

4. The application of Centella asiatica carbon nanodots according to claim 3, characterized in that The preparation method of the Centella asiatica extract is as follows: Take fresh Centella asiatica, wash, remove impurities, crush and sieve to obtain Centella asiatica powder; add water to dissolve, use 70% ethanol by volume in a ratio of 1:10 between solid and liquid, concentrate and dry after extraction to obtain Centella asiatica extract; wherein, the extraction condition is to extract 3 times at room temperature, each time for 4 hours.

5. The application of Centella asiatica carbon nanodots according to claim 3, characterized in that The filtration is performed using a 0.22 nm filter membrane.

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