CN114732839A

CN114732839A - Application of total ginsenoside carbon nanodots in preparation of medicine preparation for resisting neuroblastoma and inhibiting neuroblastoma cells

Info

Publication number: CN114732839A
Application number: CN202210085829.0A
Authority: CN
Inventors: 姜英男; 田腾辉; 张哲�; 肖利智; 朱迪夫; 匡玉兰
Original assignee: Changchun University of Chinese Medicine
Current assignee: Changchun University of Chinese Medicine
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-07-12

Abstract

The invention provides application of total ginsenoside carbon nanodots in preparing a medicinal preparation for resisting neuroblastoma and/or inhibiting neuroblastoma cells. The carbon points of the obtained total saponins of ginseng have specific inhibition effect on neuroblastoma and neuroblastoma cells (SH-SY5Y cells), and have no obvious proliferation or inhibition effect on other cells. The carbon dots of the total ginsenoside have abundant structures such as a large number of hydroxyl groups, carbonyl groups and the like on the surface, and abundant surface groups, thereby being beneficial to subsequent modification and compounding.

Description

Application of total ginsenoside carbon nanodots in preparation of medicine preparation for resisting neuroblastoma and inhibiting neuroblastoma cells

Technical Field

The invention relates to the technical field of total ginsenoside carbon nanodots, in particular to application of total ginsenoside carbon nanodots in the field of preparation of a medicinal preparation for resisting neuroblastoma and/or inhibiting neuroblastoma cells.

Background

Ginseng, as a plant-derived Chinese herbal medicine, has enjoyed the reputation of "the king of Baicao" since ancient times. The modern clinical application proves that ginseng has various therapeutic effects, and is mainly attributed to that ginseng contains a large amount of various bioactive substances, such as ginsenoside, volatile oil, polysaccharide and the like. The ginsenoside is the most main bioactive substance in ginseng, and many researches prove that the ginsenoside has good effects on resisting tumors, oxidation and inflammation, resisting aging, resisting arrhythmia, inhibiting apoptosis, improving immunologic function and the like. Researchers have now isolated over 60 ginsenosides from various ginseng products, which can be classified into 3 types according to their chemical structures: protopanaxadiol type, protopanaxatriol type and oleanolic acid type. Wherein, the ginseng total saponin is the total saponin prepared by extracting and processing ginseng root, the content of the ginsenoside Rb1, Rb2, Rb3, Rc, Rd, Re, Rf and Rg1 is higher, and is about 70 percent of the ginseng total saponin.

The processing of ginseng has a long history, mainly adopts a high-temperature steaming method, and different processing methods are different. In the Zhongdao recorded in February and in the first ten days of August, the southern and northern Liu Chong, the root of the herb was removed by scraping with a bamboo knife and drying suddenly, so that there was no chance of catching wind. The method of removing four edges and reed heads and blackening is adopted in Lei Gong Pao Zhi Lun, the ' cutting and baking method ' is adopted in Tang Dynasty ' Waitai secret essences, the ' charcoal making, baking and micro-frying ' is claimed in Song Dynasty ' Ben Cao Tu Jing ', the ' Mi Zhi De Xiao Fang ' is adopted in Yuan Dynasty, the ' Zi Tu Shen ' is obtained by steaming for the first time recorded in Ming Dynasty ' Ben Meng Ri ', and the ' Wu Ling Zhi and Chuan ' and other special preparation methods are adopted in Qing Dynasty. Modern ginseng processing mainly comprises sun drying and steaming, and main processed products comprise sun-dried ginseng, red ginseng, sugared ginseng and white dried ginseng.

As a carbon nanomaterial that is emerging, carbon nanodots (C-dots) have optical characteristics that depend on the excitation wavelength with which the emission peak is red-shifted with the excitation light. Due to the characteristics of low preparation cost, small size, good water solubility, high medicinal activity, high biocompatibility and the like, C-dots are always a research hotspot in the field of nano materials in recent years, and show unique advantages and application prospects in the field of biological medicine. The preparation methods of C-dots are various, and most researches mainly take hydrothermal reaction, and also take microwave reaction, ultrasonic reaction and the like. The C-dots have a carbon core structure as the center and abundant functional groups on the surface, and can interact with functional diagnosis and treatment reagents such as targeting ligands, medical imaging contrast agents, chemical drugs, photo-thermal sensitive conversion reagents and the like to form a compound, so that the effects of the C-dots and the compound thereof in fluorescence imaging, biosensing, drug loading, gene transfer and tumor treatment are being widely developed and reported.

However, the biological functional systems are formed by compounding C-dots with unique fluorescence properties and functional substances through various ways, so that the complex structural composition not only increases the difficulty and cost of design and research, but also reduces the advantages of the ultra-small size C-dots in water solubility and biocompatibility to a certain extent, and increases the difficulty of the maintenance and subsequent action of the stability of the subsequent system.

Therefore, how to further study the functions of ginseng in the above aspects and solve the technical problems in the prior art has become one of the focuses of great concern of many prospective researchers.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide an application of ginsenoside carbon nanodots in the field of pharmaceutical preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells. The carbon nanodots of the total saponins of panax ginseng provided by the invention have activity on SH-SY5Y cells, have the advantages of high biocompatibility, small size, good water solubility, medicinal activity and the like, have good time, pH and salt fluorescence stability, and can be used as an effective cell fluorescence probe and a medicinal reagent.

The invention provides application of total ginsenoside carbon nanodots in the field of preparation of pharmaceutical preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells.

Preferably, the diameter of the carbon nanodots of the total saponins of panax ginseng is 1-50 nm;

the surfaces of the ginsenoside carbon nanodots contain hydroxyl and/or carbonyl;

the carbon nanodots containing total ginsenoside have stability when the pH value is 5.8-8.0.

Preferably, the carbon nanodots containing ginsenoside has stability in NaCl and/or KCl solution;

the neuroblastoma cell is an SH-SY5Y cell;

the carbon points of the total ginsenoside have specific inhibition effect on neuroblastoma and/or SH-SY5Y cells.

Preferably, the concentration of the NaCl and/or KCl solution is 0.2-2 mol/L;

the stability includes fluorescence stability;

the diameter of the ginsenoside carbon nanodots is 1-10 nm.

Preferably, the ginsenoside carbon nano-dots have no proliferation or inhibition effect on one or more of HeLa cells, HepG2 cells and BV2 cells.

Preferably, the preparation method of the carbon nanodots containing total saponins of panax ginseng comprises the following steps:

1) mixing the total ginsenoside with water to obtain a mixed solution;

2) and carrying out hydrothermal reaction on the mixed solution obtained in the step to obtain the carbon point aqueous solution of the total ginsenoside.

Preferably, the concentration of the total ginsenoside in the mixed solution is 0.1-10 mg/mL;

the temperature of the hydrothermal reaction is 90-220 ℃;

the time of the hydrothermal reaction is 0.5-12 h;

the hydrothermal reaction also comprises a filtering step;

the carbon point water solution of the total ginsenoside is a transparent liquid with traditional Chinese medicine flavor.

Preferably, the ginsenoside carbon nanodots further comprise ginsenoside carbon nanodot fluorescent probes;

the fluorescent probe comprises a cellular fluorescent probe;

the fluorescent probe is one or more of a cell tracking, cell labeling and cell imaging fluorescent probe.

Preferably, the pharmaceutical preparation comprises carbon dots of the total saponins of panax ginseng and pharmaceutically acceptable auxiliary materials;

the pharmaceutical preparation has specific inhibition effect on neuroblastoma and/or SH-SY5Y cells.

Preferably, the pharmaceutical formulation does not have a proliferative or inhibitory effect on one or more of HeLa cells, HepG2 cells and BV2 cells;

the dosage form of the pharmaceutical preparation comprises an oral preparation, an injection, a suppository, an inhalant or a dosage form which can be directly applied to tumors;

in the medicinal preparation, the mass content of the total ginsenoside carbon nanodots is 1-100%.

The invention provides application of total ginsenoside carbon nanodots in the field of preparation of pharmaceutical preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells. Compared with the prior art, neuroblastoma is the most common extracranial tumor in children and the most common tumor in infants. Nearly half of neuroblastoma occurs in infants and young children under the age of 2. Neuroblastoma accounts for approximately 6-10% of childhood tumors, with a mortality rate of 15% of childhood tumors. Mortality per million population for children under 4 years of age is 10; for children aged 4-9 years, the mortality rate per million population is 4 cases. Neuroblastoma is a neuroendocrine tumor that can originate at any of the nerve ridge sites of the sympathetic nervous system. The most common site of occurrence is the adrenal gland, but can also occur in the nervous tissue of the neck, chest, abdomen and pelvic cavity. A few human tumors are known that spontaneously regress from undifferentiated malignant tumors to completely benign tumors. One of them is neuroblastoma.

The application of the carbon nanodots containing the total saponins of panax ginseng in the field of preparing the medicinal preparation for resisting neuroblastoma and/or inhibiting neuroblastoma cells is creatively obtained, the carbon dots containing the total saponins of panax ginseng are rich in a large number of structures such as hydroxyl, carbonyl and the like on the surface, rich in surface groups, beneficial to subsequent modification and compounding, and good in time, pH value and salt stability; and cell activity experiments prove that the carbon points of the ginsenoside have specific inhibition effect on specific cells.

The invention combines the advantages of the biological and medicinal activity of the total ginsenoside and the high biocompatibility, small size, good water solubility, medicinal activity and the like of the carbon nanodots (C-dots), adopts the most effective medicinal component of the total ginsenoside of the ginseng, establishes the preparation process of the carbon dots of the total ginsenoside by a hydrothermal method, and has the advantages of rich groups on the surface of the carbon dots of the total ginsenoside, proper particle size, uniform dispersion and obvious fluorescence excitation dependence; meanwhile, the carbon dots of the total ginsenoside have good time, pH value and fluorescence stability of salt. Cell experiment results show that the carbon points of the total saponins of panax ginseng have obvious inhibition effect on neuroblastoma cells (SH-SY5Y cells).

The preparation method is simple, mild in condition and strong in controllability, is suitable for industrial development and application, can obtain the ultra-small fluorescent ginsenoside carbon dots, and has good size distribution, water solubility, stable optical performance and fluorescence excitation dependence. The carbon points of the total ginsenoside are subjected to various cell fluorescence imaging and cell activity analysis, so that the foundation is laid for the subsequent research and application of the carbon points of the total ginsenoside in the aspect of biomedicine, and the development of the traditional Chinese medicine ginseng serving as a reaction carbon source for the preparation of C-dots and the application and research of the biological medicine property is facilitated.

Experimental results show that a large number of hydrophilic groups and abundant functional groups exist on the surface of the carbon dots of the obtained ginsenoside, and the ginsenoside is proved to have good water solubility; the carbon points of the total ginsenoside have no obvious proliferation or inhibition effect on neuroblastoma and neuroblastoma cells (SH-SY5Y cells) and other cells (HeLa cells, HepG2 cells and BV2 cells); the results lay the foundation for researching the specific inhibition effect of the carbon points of the total saponins of panax ginseng on SH-SY5Y and developing the application of the carbon points of the total saponins of panax ginseng in the aspect of biological fluorescent labeling probes.

Drawings

FIG. 1 is a TEM image of carbon points GS-CDs @3h, GS-CDs @ s5h, GS-CDs @6h and GS-CDs @10h of ginsenoside prepared in example 1 of the present invention;

FIG. 2 is a graph of the UV-VIS absorption spectrum of the GS solution before hydrothermal reaction;

FIG. 3 is a chart of the UV-VIS absorption spectra of GS-CDs @3h, GS-CDs @ s5h, GS-CDs @6h and GS-CDs @10 h;

FIG. 4 is a three-dimensional fluorescence spectrum of GS-CDs @3h, GS-CDs @ s5h, GS-CDs @6h and GS-CDs @10 h;

FIG. 5 is a graph of the Fourier infrared spectra of GS (orange), GS-CDs @3h (purple), GS-CDs @5h (blue), GS-CDs @6h (green), and GS-CDs @10h (red);

FIG. 6 is a graph of the fluorescence attenuation curves and curve fit equations for GS-CDs @3h, GS-CDs @5h, GS-CDs @6h and GS-CDs @10 h;

FIG. 7 is a schematic representation of the X photoelectron spectra of GS-CDs @3h, GS-CDs @5h, GS-CDs @6h and GS-CDs @10h with the C1s peak separation and O1s peak separation;

FIG. 8 shows the change of GS-CDs in HeLa cell viability;

FIG. 9 shows the change of GS-CDs in cell viability upon HepG 2;

FIG. 10 shows the change of the viability of GS-CDs on BV2 cells;

FIG. 11 shows the change of the cell viability of GS-CDs acting on SH-SY 5Y;

FIG. 12 is a fluorescence microscope image of SH-SY5Y cells and GS-CDs (100. mu.g/mL) incubated at different times (30min, 2, 4, 6,12,24,48 h);

FIG. 13 shows the cell viability of SH-SY5Y cells incubated with GS-CDs for various periods of time (6,12,24,48 h);

FIG. 14 is a graph of SH-SY5Y cells undergoing apoptosis after 48h incubation with GS-CDs (10, 20, 30, 50. mu.g/mL) at different concentrations;

FIG. 15 is a correlation of the detection map for tumor growth experiments;

fig. 16 is a graph showing H & E staining patterns (n ═ 4) of major organs (heart, lung, spleen, liver, and kidney) of mice in the control group, control group + GS-CDs group, model group, cisplatin group, and GS-CDs group;

FIG. 17 is a graph of statistical weights for control (grey dashed line), control + group GS-CD (black dashed line), model (red dashed line), cisplatin (blue dashed line), and GS-CD group (purple dashed line) mice. Data are mean ± s.d. (n ═ 4).

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art. In the present invention, all the raw materials are in vitro raw materials.

All the raw materials of the invention are not particularly limited in purity, and the invention preferably adopts analytically pure or meets the medical purity standard or the standard in the field of preparing the ginseng total saponin carbon nanodots.

All the raw materials, the marks and the acronyms thereof belong to the conventional marks and acronyms in the field, each mark and acronym is clear and definite in the field of related application, and the raw materials can be purchased from the market or prepared by a conventional method by the technical staff in the field according to the marks, the acronyms and the corresponding application.

The size of the carbon nanodots containing total ginsenoside is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, the uniformity of the carbon nanodots containing total ginsenoside is better improved, functional groups on the surface of the carbon nanodots are guaranteed, biocompatibility, stability and water solubility are improved, and further specific cell activity and fluorescence excitation dependence are guaranteed, the diameter of the carbon nanodots containing total ginsenoside is preferably 1-50 nm, more preferably 1-40 nm, more preferably 2-30 nm, specifically 2-10 nm, or 2-8 nm, or 3-7 nm, or 4-6 nm, such as less than or equal to 8nm, or less than or equal to 5 nm.

The invention has no special limitation on the components on the surface of the carbon nanodots of the total ginsenoside in principle, and the skilled person can select and adjust the carbon nanodots according to the actual application condition, the raw material condition and the product requirement.

The invention has no particular limitation on the pH value for stabilizing the carbon nanodots of the total ginsenosides in principle, and technicians in the field can select and adjust the carbon nanodots according to actual application conditions, raw material conditions and product requirements, in order to better improve the uniformity of the carbon nanodots of the total ginsenosides, ensure functional groups on the surface of the carbon nanodots, improve biocompatibility, stability and water solubility and further ensure specific cell activity and fluorescence excitation dependence characteristics, the carbon nanodots of the total ginsenosides preferably have stability when the pH is 5.8-8.0, more preferably have stability when the pH is 6.2-7.6, and more preferably have pH 6.4-7.2.

The type of the solution for stabilizing the ginsenoside carbon nanodots is not particularly limited in principle, and a person skilled in the art can select and adjust the solution according to the actual application condition, the raw material condition and the product requirement.

The concentration of the NaCl solution and/or the KCl solution is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, the uniformity of the ginsenoside carbon nanodots is better improved, functional groups on the surfaces of the carbon nanodots are guaranteed, biocompatibility, stability and water solubility are improved, and further specific cell activity and fluorescence excitation dependence are guaranteed, and the concentration of the NaCl solution is preferably 0.2-2 mol/L, more preferably 0.5-1.7 mol/L, and more preferably 0.8-1.4 mol/L. The concentration of the KCl solution is preferably 0.2-2 mol/L, more preferably 0.5-1.7 mol/L, and more preferably 0.8-1.4 mol/L. Or a mixed salt solution of the two satisfies the above conditions.

The invention is a complete and refined integral preparation process, and aims to better improve the uniformity of the carbon nanodots of the ginsenoside, ensure functional groups on the surface of the carbon nanodots, improve biocompatibility, stability and water solubility, and further ensure the specific cell activity and fluorescence excitation dependence characteristics, wherein the stability preferably comprises fluorescence stability.

The invention has no special limitation on the property of the carbon nanodots capable of taking the ginsenoside, and the technicians in the field can select and adjust the carbon nanodots according to the actual application condition, the raw material condition and the product requirement, in order to better improve the uniformity of the carbon nanodots of the ginsenoside, ensure functional groups on the surface of the carbon nanodots, improve the biocompatibility, the stability and the water solubility, and further ensure the specific cell activity and the fluorescence excitation dependence property, the carbon dots of the ginsenoside preferably have the specific inhibition effect on neuroblastoma and SH-SY5Y cells.

The carbon dots of the total ginsenoside carbon are preferably free from proliferation or inhibition on one or more of HeLa cells, HepG2 cells and BV2 cells, and more preferably free from proliferation or inhibition on HeLa cells, HepG2 cells and BV2 cells.

The invention also provides a preparation method of the carbon nanodots containing the ginsenoside carbon, which comprises the following steps:

1) mixing the total ginsenoside with water to obtain a mixed solution;

The selection and composition of the raw materials in the preparation method of the carbon nanodots containing total ginsenoside carbon and the corresponding optimization principle can correspond to the selection and composition of the raw materials corresponding to the carbon nanodots containing total ginsenoside carbon and the corresponding optimization principle, and are not described in detail herein.

The invention firstly mixes the ginseng total saponin with water to obtain mixed liquor.

The concentration of the total ginsenoside in the mixed solution is not particularly limited in principle, and a person skilled in the art can select and adjust the concentration according to the actual application condition, the raw material condition and the product requirement, in order to better improve the uniformity of the carbon nanodots of the total ginsenoside, ensure functional groups on the surface of the carbon nanodots, improve the biocompatibility, the stability and the water solubility and further ensure the specific cell activity and the fluorescence excitation dependence characteristic, the concentration of the total ginsenoside in the mixed solution is preferably 0.1-10 mg/mL, more preferably 0.5-6 mg/mL, and more preferably 1-4 mg/mL.

Finally, carrying out hydrothermal reaction on the mixed solution obtained in the step to obtain the carbon point aqueous solution of the total ginsenoside.

The temperature of the hydrothermal reaction is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, the uniformity of the carbon nanodots of the ginsenoside is better improved, functional groups on the surface of the carbon nanodots are guaranteed, biocompatibility, stability and water solubility are improved, and specific cell activity and fluorescence excitation dependence characteristics are further guaranteed, and the temperature of the hydrothermal reaction is preferably 90-220 ℃, more preferably 100-200 ℃, more preferably 110-200 ℃, more preferably 140-180 ℃, and more preferably 150-175 ℃.

The hydrothermal reaction time is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, the uniformity of the carbon nanodots of the ginsenoside is better improved, functional groups on the surface of the carbon nanodots are guaranteed, biocompatibility, stability and water solubility are improved, and specific cell activity and fluorescence excitation dependence are further guaranteed, and the hydrothermal reaction time is preferably 0.5-12 hours, more preferably 2-10 hours, and more preferably 4-8 hours.

The invention is a complete and refined integral preparation process, and preferably comprises a filtering step after the hydrothermal reaction in order to better improve the uniformity of the carbon nanodots of the total saponins of panax ginseng, ensure functional groups on the surface of the carbon nanodots, improve biocompatibility, stability and water solubility and further ensure specific cell activity and fluorescence excitation dependence characteristics.

The invention is a complete and refined integral preparation process, and in order to better improve the uniformity of the carbon nanodots of the total ginsenoside, ensure functional groups on the surface of the carbon nanodots, improve biocompatibility, stability and water solubility and further ensure the specific cell activity and fluorescence excitation dependence characteristics, the water solution of the carbon nanodots of the total ginsenoside is preferably transparent liquid with traditional Chinese medicine taste.

In the invention, the carbon nanodots of total saponins of panax ginseng also comprise a fluorescent probe of the carbon nanodots of total saponins of panax ginseng.

The invention is not particularly limited to the specific category of the fluorescent probe in principle, and the skilled person can select and adjust the fluorescent probe according to the actual application situation, the raw material situation and the product requirement, in order to better improve the uniformity of the carbon nanodots of ginsenoside, ensure the functional groups on the surface of the carbon nanodots, improve the biocompatibility, the stability and the water solubility, and further ensure the specific cell activity and the fluorescence excitation dependence characteristic, the fluorescent probe preferably comprises a bio-based fluorescent probe, more specifically, the fluorescent probe is preferably one or more of a tracking cell, a labeling cell and a cell imaging, and more preferably, the fluorescent probe is more than one of a tracking cell, a labeling cell and a cell imaging.

In the invention, the pharmaceutical preparation preferably comprises carbon dots of the total saponins of panax ginseng and pharmaceutically acceptable auxiliary materials.

The specific efficacy of the pharmaceutical preparation is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, so that the uniformity of the carbon nanodots of the total ginsenoside is better improved, the functional groups on the surface of the carbon nanodots are ensured, the biocompatibility, the stability and the water solubility are improved, the specific cell activity and the fluorescence excitation dependence characteristic are further ensured, and the pharmaceutical preparation preferably has a specific inhibition effect on neuroblastoma and SH-SY5Y cells.

The invention has no particular limitation on the cells without proliferation or inhibition effects of the pharmaceutical preparation in principle, and the skilled person can select and adjust the pharmaceutical preparation according to the actual application situation, the raw material situation and the product requirement, in order to better improve the uniformity of the carbon nanodots of the total ginsenoside, ensure the functional groups on the surface of the carbon nanodots, improve the biocompatibility, the stability and the water solubility, and further ensure the specific cell activity and the fluorescence excitation dependence characteristic, the pharmaceutical preparation preferably has no proliferation or inhibition effects on one or more of HeLa cells, HepG2 cells and BV2 cells, and more preferably has no proliferation or inhibition effects on HeLa cells, HepG2 cells and BV2 cells.

The invention has no particular limitation on the dosage form of the medicine in principle, and the skilled person can select and adjust the dosage form according to the application condition, the product structure and the product performance requirements, the invention aims to better improve the uniformity of the carbon nanodots of the ginsenoside, ensure the functional groups on the surface of the carbon nanodots, improve the biocompatibility, the stability and the water solubility, and further ensure the specific cell activity and the fluorescence excitation dependence characteristic, the dosage form of the medicine preferably comprises an oral preparation, an injection, a suppository, an inhalant or a dosage form which can be directly applied to tumor treatment, and specifically can be capsules, microcapsules, tablets, granules, pills, dispersed powder, liquid preparations, decocted extract, suspending agents, syrups, gels, aerosols, patches, liposomes, oral liquid, intravenous injection or intramuscular injection.

In the pharmaceutical preparation, the mass content of the carbon nanodots of ginsenoside is not particularly limited in principle, and can be selected and adjusted by a person skilled in the art according to actual application conditions, raw material conditions and product requirements, so that the uniformity of the carbon nanodots of ginsenoside is improved, functional groups on the surface of the carbon nanodots are ensured, biocompatibility, stability and water solubility are improved, and specific cell activity and fluorescence excitation dependence are ensured, and in the pharmaceutical preparation, the mass content of the carbon nanodots of ginsenoside is preferably 1-100%, more preferably 20-80%, more preferably 40-60%, and also 5-100%, or 10-100%, or 15-100%.

The steps of the invention provide the application of the carbon nanodots containing the total saponins of panax ginseng in the field of preparing the pharmaceutical preparation for resisting neuroblastoma and/or inhibiting neuroblastoma cells. The obtained carbon nanodots containing total ginsenoside carbon have the advantages that the surface is rich in a large number of structures such as hydroxyl, carbonyl and the like, surface groups are rich, subsequent modification and compounding are facilitated, the carbon nanodots have good time, pH value and salt stability, the particle size distribution is uniform, and the particles are good in dispersibility in water and are not easy to aggregate; and cell activity experiments prove that the carbon points of the ginsenoside have specific inhibition effect on SH-SY5Y cells.

The application of the carbon nanodots containing the total saponins of panax ginseng in the field of preparing medicinal preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells is obtained, the surfaces of the carbon nanodots containing the total saponins of panax ginseng are rich in a large number of structures such as hydroxyl, carbonyl and the like, the surface groups are rich, the subsequent modification and compounding of the carbon nanodots are facilitated, and the carbon nanodots also have good time, pH value and salt stability; and cell activity experiments prove that the carbon points of the ginsenoside have specific inhibition effect on specific cells.

The invention combines the advantages of the biological and medicinal activity of the total ginsenoside and the high biocompatibility, small size, good water solubility, medicinal activity and the like of the carbon nanodots (C-dots), adopts the most effective medicinal component of the total ginsenoside of the ginseng, establishes the preparation process of the carbon dots of the total ginsenoside by a hydrothermal method, and has the advantages of rich groups on the surface of the carbon dots of the total ginsenoside, proper particle size, uniform dispersion and obvious fluorescence excitation dependence; meanwhile, the carbon dots of the total ginsenoside have good time, pH value and fluorescence stability of salt. Cell experiment results show that the carbon points of the total saponins of panax ginseng have obvious inhibition effect on neuroblastoma and neuroblastoma cells (SH-SY5Y cells).

The preparation method is simple, mild in condition and strong in controllability, is suitable for industrial development and application, can obtain the ultra-small fluorescent ginsenoside carbon dots, and has good size distribution, water solubility, stable optical performance and fluorescence excitation dependence. The carbon points of the total ginsenoside are subjected to various cell fluorescence imaging and cell activity analysis, so that a foundation is laid for the subsequent research and application of the carbon points of the total ginsenoside in the aspect of biomedicine, and the carbon points of the total ginsenoside are beneficial to developing the traditional Chinese medicine ginseng as a reaction carbon source for the preparation of C-dots and the application and research of the properties of biomedicine.

The carbon nanodots (GS-CDs) of the total saponins of panax ginseng prepared by the invention are in a quasi-spherical shape, have good dispersibility and uniform size, have the particle size distribution of 2.0-4.0 nm and have strong blue-green fluorescence. The UV-vis and FL peaks of GS-CDs gradually increased with increasing reaction time.

FTIR spectral characterization and analysis prove that the surface of GS-CDs is rich in certain hydroxyl and carbonyl structures, so that the GS-CDs are beneficial to effective subsequent interaction with organisms through functional groups. MS analysis shows that the prepared GS-CDs still contain ginsenoside structures, which indicates that the main body of the GS-CDs is formed by combining ginsenoside and contains the bioactivity of ginseng.

In vitro biological activity research proves that GS-CDs have specific inhibition effect on SH-SY5Y cells and can be taken up by SH-SY5Y cells. In addition, the total apoptosis rate of the GS-CDs solution on SH-SY5Y cells is obviously increased along with the increase of the administration concentration. The synthesized GS-CDs solution is administrated by intravenous injection on the tail of a mouse, and the GS-CDs can obviously inhibit the growth of neuroblastoma compared with a model group and a cis-platinum group.

The carbon ginseng total saponin nanodots prepared by the hydrothermal synthesis method under certain conditions have small size, uniform distribution, rich surface groups and good fluorescence property, and can be used as an effective cell fluorescence probe; and the main body of the carbon ginseng total saponin nanodots is formed by combining ginsenosides, shows better biological activity, has obvious inhibition effect on SH-SY5Y cells and neuroblastoma, and provides good research basis and application prospect for the subsequent preparation and development of traditional Chinese medicine nano-drugs.

Experimental results show that a large number of hydrophilic groups and abundant functional groups exist on the surfaces of the carbon points of the obtained ginsenoside, and the ginsenoside is proved to have good water solubility; the carbon points of the total saponins of ginseng have specific inhibition effect on neuroblastoma and SH-SY5Y cells, and have no obvious proliferation or inhibition effect on other cells (HeLa cells, HepG2 cells and BV2 cells); the results lay the foundation for researching the specific inhibition effect of the carbon points of the total saponins of panax ginseng on SH-SY5Y and developing the application of the carbon points of the total saponins of panax ginseng in the aspect of biological fluorescent labeling probes.

To further illustrate the present invention, the following examples are provided to describe the application of the carbon nanodots of ginsenoside provided in the present invention in the field of preparing pharmaceutical preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells in detail, but it should be understood that these examples are carried out on the premise of the technical solution of the present invention, and the detailed embodiments and specific procedures are given for further illustration of the features and advantages of the present invention, but not for limiting the claims of the present invention, and the scope of protection of the present invention is not limited to the following examples.

Ginseng radix total saponin (extracted from stem and leaf, UV > 80%), Shanghai leaf Biotech limited.

The method takes the total ginsenoside powder as a carbon source, the reaction concentration is 1.5mg/mL, the reaction time is controlled (1-10h) at the reaction temperature of 170 ℃, and different GS-CDs solutions are obtained by adopting a hydrothermal synthesis method. And (3) performing coarse filtration treatment on the solution by using a 0.22-micron filter membrane, and then performing centrifugal purification on the solution by using an ultrafiltration tube to obtain a light yellow GS-CDs solution.

In vitro biological activity research of the carbon nanodots containing the total saponins of panax ginseng: adopting a Cell Counting Kit-8 method to examine the biological activity of GS-CDs aiming at different types of tumor cells, including human cervical cancer cells (HeLa), human liver cancer cells (HepG2), mouse microglia cells (BV2) and human neuroblastoma cells (SH-SY5Y cells); adopting a fluorescence imaging technology to shoot cells which are incubated by GS-CDs and cells for different times to take fluorescence imaging images; and detecting the apoptosis condition of the GS-CDs on the cells by using an apoptosis kit and a flow cytometer.

The invention obtains through the experimental result that GS-CDs have obvious inhibition effect on SH-SY5Y cells, so that a corresponding neuroblastoma tumor-bearing mouse model is established. After screening, the materials are randomly divided into three groups: control group, cis-platinum group and administration group. Observing the weight change condition of the mouse; growth curve and tumor inhibition rate of tumors; HE staining to detect pathological changes of the tumor; detecting the expression of related protein by an immunohistochemical method; TMT quantitative proteomics studies. The inhibitory effect of GS-CDs on neuroblastoma was examined.

Example 1

Preparation of carbon nanodots of total saponins of panax ginseng

GS-CDs are synthesized by a hydrothermal synthesis method. Precisely weighing 1.5mg of total ginsenoside powder, placing in a beaker containing 10mL of distilled water, and stirring to dissolve completely. Then transferring the solution into a polytetrafluoroethylene-lined high-pressure reaction kettle (15mL), and heating in an oven at 170 ℃ for 1-10h (1h is an interval). After the reaction time reached, the reaction vessel was placed in cold water and rapidly cooled (15 min). Firstly, the obtained solution is roughly filtered by using a 0.22 mu m polyether sulfone membrane to remove larger particles, then the filtered solution is added into an activated ultrafiltration tube (3000MWCO) to carry out centrifugal purification (5000rpm,15min), and unreacted micromolecular organic matters are removed, so that a corresponding light yellow GS-CDs solution is obtained. The GS-CDs solution was stored at-20 ℃.

At the reaction temperature of 170 ℃, a series of GS-CDs with different reaction time are prepared by taking total ginsenoside powder as a raw material and adopting a hydrothermal synthesis method. The sizes and surface appearances of GS-CDs @3h, GS-CDs @5h, GS-CDs @6h and GS-CDs @10h obtained under different hydrothermal reaction times (3h, 5h, 6h and 10h) are characterized by using a transmission electron microscope.

As shown in fig. 1. Wherein the HRTEM image inserted in the upper right corner shows the lattice and lattice spacing of the four GS-CDs, respectively.

The TEM image of FIG. 1 shows that GS-CDs are all round, well dispersed and relatively uniform in size. The particle size of GS-CDs @3h is concentrated at 2.95 +/-0.72 nm, the particle size of GS-CDs @5h is concentrated at 2.75 +/-0.66 nm, the particle size of GS-CDs @6h is concentrated at 3.00 +/-0.64 nm, and the particle size of GS-CDs @10h is concentrated at 3.24 +/-0.62 nm. The upper right-hand insert HRTEM image shows a lattice of four GS-CDs. GS-CDs @3h has substantially no crystal lattice, and probably has short reaction time, and the formed carbon nano-dot structure is incomplete. GS-CDs @5h has a certain lattice structure, and the lattice spacing is 0.219 nm. GS-CDs @6h and GS-CDs @10h both have distinct lattices with lattice spacings of 0.218 and 0.207 nm, respectively. This indicates that the self-assembly of ginsenoside molecules tends to be orderly arranged and the structure of CDs is completely formed with longer reaction time.

See fig. 2 and 3. The UV absorption of GS solution is weak, while the absorption of GS-CDs solution is strong at the wavelength of 280 nm. And the longer the reaction time, the higher the absorption peak. The upper right inset of FIG. 3 is a photograph of four GS-CDs under sunlight (left) and 365nm ultraviolet light (right), respectively. GS-CDs with different reaction times all have stronger blue-green fluorescence, and the brightness is slightly increased along with the increase of the reaction time.

As shown in fig. 4. Wherein, in fig. 4, a. is a contour diagram; B. and (4) equidistant projection.

FIG. 4A is a three-dimensional contour plot of fluorescence spectra of GS-CDs at different reaction times, showing that there is only one fluorescence emitting material in each system, indicating a single product. As shown in the isometric drawing of the three-dimensional fluorescence spectrum of FIG. 4B, all four GS-CDs showed significant excitation dependence, with an optimal emission wavelength of 440nm at an optimal excitation wavelength of 360 nm. The longer the reaction time, the higher the fluorescence intensity of the obtained GS-CDs.

As shown in fig. 5. As shown in the infrared spectrum of FIG. 5, GS-CDs were found at 3370, 2925, 2853, 1780-1550, 1458, 1386, 1075, and 1040cm^-1There are absorption peaks, which are attributed to the vibration and rotation of O-H, C-H, C ═ O and C-O. Compared with GS with almost no v (C ═ O) absorption, the absorption peak intensity at v (C ═ O) of 4 GS-CDs is obviously enhanced, because oxidation reaction of-OH on GS generates C ═ O. Almost all absorption peaks of GS-CDs @3h are stronger than other GS-CDs, except for the absorption at ν (C ═ O). Probably because the heating time is short, the reaction is incomplete, and the reaction sites on the tetracyclic triterpene are in an active state. The intensities of the absorption peaks at v (O-H) and v (C-O) were slightly reduced for GS-CDs @5H and GS-CDs @6H as compared to GS-CDs @ 3H. It is possible that the degree of crosslinking of the ring formed by the self-assembly of the molecules increases due to the increase in reaction time. In the infrared spectrogram of GS-CDs @10h, except a v (C ═ O) peak, the intensities of other absorption peaks are lower than those of other three GS-CDs. Probably because the reaction time is increased to 10h, the glycosyl group on the side chain of the ginsenoside molecule is dissociated to the maximum extent, the dissociation of the glycosyl group side chain can enable the formed GS-CDs structure to be more ordered and compact, and the GS-CDs @10h is the most rigid. It is inferred that the longer the reaction time is, the higher the crosslinking degree is, the more compact and ordered the GS-CDs structure is, and the ultraviolet absorption and the fluorescence emission are enhanced therewith.

The steady state/transient state fluorescence spectrometer measures the fluorescence lifetime and quantum yield of the GS-CDs. As shown in fig. 6. FIG. 6 is a fluorescence attenuation curve of these four GS-CDs. Fitting with a third-order decay exponential function gave three fluorescence lifetimes (τ), indicating that GS-CDs has three fluorescence centers. The average fluorescence lifetime of GS-CDs @3h is 5.67ns, the average fluorescence lifetime of GS-CDs @5h is 5.81ns, the average fluorescence lifetime of GS-CDs @6h is 5.53ns, and the average fluorescence lifetime of GS-CDs @10h is 5.56 ns. At the same time, we also obtained quantum yields of GS-CDs @3h, GS-CDs @5h, GS-CDs @6h and GS-CDs @10h of 0.19%, 0.14%, 0.19% and 0.22%, respectively.

The elemental composition and functional groups of GS-CDs were further characterized and analyzed by X-ray photoelectron spectroscopy (XPS). As shown in fig. 7. As shown in FIG. 7, the XPS spectra of all four GS-CDs show two major peaks at 285.1 and 531.6eV, corresponding to the elements C1s and O1s, respectively. The C1s band has three peaks near 288.5, 285.6, and 284.5eV, which are assigned to the C-O, C-O, C-C related carbon peaks, respectively. The O1s band has two peaks near 534.3 and 532.6 eV, which belong to the C ═ O and C — O related oxygen peaks, respectively.

The ginsenoside molecules typically have a low content of pi electrons, either carbon-carbon double bonds, carbon-carbon triple bonds or carbon groups. The major sources of ultraviolet absorption and fluorescence emission of the prepared GS-CDs systems are not these classical conjugated structures. The occurrence and increase of uv absorption and fluorescence emission should be related to the cross-linking enhanced emission effect. Under hydrothermal conditions (170 ℃), free GS molecules crosslink to form carbon cores via a ring-buckle reaction with rapid collisions between particles. During the reaction process, the main structure of the tetracyclic triterpene of the GS molecule is not greatly changed, but the active site on the main structure is combined with H₂O reacts, so that C ═ O groups increase in the system (fig. 5). As the reaction time increases, the intermolecular buckle crosslinking reaction and carbonization degree also increase, and the structure of GS-CDs becomes more and more dense (FIG. 1). The more and more C ═ O and C — O groups in the system are fixed, their oscillation and rotation are limited, leading to an increase in radiative transitions. Thus, this non-classical conjugation leads to enhanced UV absorption and also enhanced fluorescence emission of GS-CDs.

The result shows that the GS-CDs are circular, have good dispersibility and uniform size, have particle size distribution of 2.0-4.0 nm and have stronger blue-green fluorescence. GS-CDs @3h has no obvious crystal lattice, and GS-CDs @5h, GS-CDs @6h and GS-CDs @10h have crystal lattices, and the lattice spacing is about 0.21nm, which shows that the longer the reaction time is, the more complete the structure of the carbon nano-dots is formed. In the hydrothermal reaction, the main structure of the tetracyclic triterpene of the GS molecule is not changed, and the GS-CDs are formed by crosslinking the molecules through a buckle ring reaction along with rapid collision among particles. As the reaction time increases, the degree of rigidity of GS-CDs increases, and the ultraviolet absorption and fluorescence emission of GS-CDs increase, which is related to the crosslinking-enhanced emission effect.

Example 2

Cell type and culture conditions

All cells were from ATCC. Human neuroblastoma cells (SH-SY5Y) were cultured in DMEM/F-12 medium, human cervical cancer cells (HeLa) and mouse microglia cells (BV2) were cultured in DMEM medium, and human liver cancer cells (HepG2) were cultured in MEM-EBSS medium. These media were supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin. All cells were cultured at 37 ℃ in 5% CO₂In a humidified cell incubator.

Cell culture

Resuscitation

The ultra-clean bench needs to start an ultraviolet lamp for irradiating for 30min before the experiment begins; when the centrifugal pipe rack is used, the ultraviolet lamp is turned off, the fluorescent lamp and the fan are turned on, the alcohol lamp is ignited, the inner surfaces of the pipettor and the super clean bench are wiped by alcohol cotton balls, and the centrifugal pipe rack is arranged. Cell culture flasks, complete medium (basal medium: FBS: penicillin-streptomycin mixture: 9:1:0.1), EP tubes and different standard tips sterilized in autoclave (121 ℃, 1h), and sterile water at 37 ℃ were prepared. Corresponding cells were taken out from the liquid nitrogen tank, the cap of the cell cryopreservation tube was held by forceps, the lower half portion of the cryopreservation tube was placed in sterile water at 37 ℃ and rotated in the same direction to rapidly melt the cell cryopreservation solution, the cryopreservation solution was taken up by a 1mL pipette, placed in an EP tube, and after the centrifuge was set to a flat condition, centrifugation (1200rmp/min, 5min) was carried out. Writing cell names, algebra, names and dates on a cell culture bottle, adding complete culture medium with corresponding volume into the cell culture bottle, centrifuging, removing supernatant, sucking 1mL of complete culture medium and an EP tube, resuspending cell precipitates, adding the cell precipitates into the cell culture bottle, and shaking up; standing at 37 deg.C for 5% CO₂Culturing in a cell culture box. After living cells adhere to the wall and the cell morphology can be observed, liquid is changed, and dead cells floating in the culture medium are removed, so that the cells which are just recovered can have sufficient nutrition and a better living environment.

Passage of the culture

And (4) observing the growth condition of the cells in advance, and carrying out passage when the cell fusion degree reaches 80-90%. Complete medium, PBS and 2mL of pancreatin were placed at room temperature while ultra-clean bench irradiation uv. And taking out the cells from the incubator, putting the cells into a prepared super clean bench, discarding the culture medium in the bottle, adding 2mL of PBS (phosphate buffer solution) to wash the cells, taking out the dead cells and the culture medium remained in the bottle, and washing for 1-3 times. Discarding PBS, adding 2mL pancreatin, covering a bottle cap, stably placing a cell bottle, observing under a microscope, adding an equal volume of complete culture medium to neutralize the pancreatin when cells become round and bright, and stopping digestion. The bottom cells were blown out with a 1mL pipette, and the cell suspension was transferred to 2 EP tubes prepared in advance and centrifuged (1200rmp/min, 5 min). At this point, the date and generation number on the old cell flask were changed, the same information was written on the new cell flask, and the corresponding volume of complete medium was added. After centrifugation, the supernatant was discarded, 1mL of complete medium was added to each of 2 EP tubes, the cells were resuspended, added to two cell flasks, shaken well, placed at 37 ℃ and 5% CO₂Culturing in a cell culture box.

Freezing and storing

When the cell fusion degree reached 80 to 90%, the cells were frozen, and a freezing tube and a freezing solution (FBS: DMSO: 9:1) were prepared. The cell processing mode is the same as that of passage, the centrifuged cells are resuspended by 1mL of the cryopreservation solution and transferred into a cryopreservation tube, and the name, name and date of the cells are written on the cryopreservation tube. Placing the tube in a liquid nitrogen tank at-20 deg.C for 30min and-20 deg.C for 1 hr and-80 deg.C overnight.

CCK-8 method for detecting cell viability

Culturing the cells to a logarithmic growth phase, culturing the cells to a 96-well cell culture plate according to a proper cell density, wherein the volume of cell suspension in each well is 180 mu L, culturing the cells in a cell culture box for about 6h, adding 20 mu L of GS-CDs with different concentrations when the cells are completely attached to the wall, wherein the final concentrations of the GS-CDs acting on the cells in each well are respectively 0, 10, 20, 30 and 50 mu g/mL (each concentration is provided with 6 multiple wells), and continuously placing the cells in the culture box for culturing. After GS-CDs were incubated with cells for 48h, 20. mu.L of CCK-8 was added to each well, reacted in an incubator for about 30min, and the Optical Density (OD) at 450nm was measured using a microplate reader. Cell viability was calculated from the OD values of the experimental and control groups. Meanwhile, the CCK-8 method can be used for detecting the change of the cell viability of GS-CDs after the GS-CDs act on the cells for different time (6,12,24 and 48 hours), and the experimental operation steps are the same as the above.

Cellular fluorescence imaging

And (4) taking a cell fluorescence imaging image of the intracellular uptake of the GS-CDs by using a fluorescence inverted microscope. Place the circular microscope coverslip in a 6-well cell culture plate, add 2mL of cell suspension, 1 x 10⁶Cells/well, cultured in incubator for 24 h. The supernatant medium was aspirated and replaced with medium containing a solution of GS-CDs at a final concentration of 100. mu.g/mL. After incubating GS-CDs with the cells for 30min, 2h, 4h, 6h, 12h, 24h, 48h, respectively, the supernatants were aspirated and the cells were washed 3 times with PBS. Next, the cell morphology was fixed with 4% paraformaldehyde solution for 15min, and the cells were washed 3 times with PBS. A small amount of DAPI staining solution was added, the reaction was carried out at room temperature for 5min, the cells were washed 3 times with PBS, and the PBS solution was finally blotted dry. Preparing a glass slide, dripping 10 mu L of an anti-fluorescence attenuation sealing tablet on the glass slide, taking out the glass slide in a 6-hole cell culture plate, carefully sucking the residual PBS solution on the glass slide, buckling one surface with cells on the glass slide to prepare a cell climbing sheet, storing the cell climbing sheet in a dark place, and shooting a cell fluorescence imaging image.

Detection of apoptosis by flow cytometry

And detecting the apoptosis condition of GS-CDs with different concentrations after the GS-CDs act on the cells for 48 hours by using an FITC coupled Annexin-V apoptosis detection kit I. Cells in logarithmic growth phase, 1 x 10⁶Cell density per cell/well was cultured in 6-well cell culture plates and in an incubator for 24 h. The supernatant medium was then aspirated and replaced with medium containing different concentrations of GS-CDs in GS-CDs at final concentrations of 0, 10, 20, 30 and 50. mu.g/mL per well of cells, respectively, and incubated for 48 h. Thereafter, the cells were carefully collected and the procedure was performed according to the instructions of the apoptosis detection kit. The stained cells were analyzed by flow cytometry. Total count of 1 x 10 per sample⁴And (4) cells.

Cytotoxicity of carbon nanodots of total saponins of panax ginseng

Cell Counting Kit-8 method is adopted to detect the Cell activity change condition of GS-CDs obtained under different reaction time (1-10h) after respectively acting on human cervical cancer cells (HeLa), human liver cancer cells (HepG2), mouse microglia cells (BV2) and human neuroblastoma cells (SH-SY5Y) for 48 h. As shown in figures 8, 9, 10 and 11.

The situation that SH-SY5Y cells take the carbon nanodots of the ginsenoside

In view of the fact that the reaction time is 6h in the range of 1-10h, GS-CDs have obvious inhibition effect on SHSY5Y cells, and therefore GS-CDs @6h are adopted later and are designated as GS-CDs later. Experimental studies were performed as representative. The GS-CDs system does not need to introduce other fluorescent substances for tracking and labeling while having effective medicinal effect, combines the fluorescence characteristics of the GS-CDs self structure, and further discusses the inhibition effect of the GS-CDs on SHSY5Y cells. The entry of GS @ CDs into the cells was monitored by fluorescence inverted microscopy at different incubation times. See fig. 12. As shown in fig. 12, the cell morphology at 12h had begun to change in bright field; at 24h, the cells tend to be round; at 48h, the number of viable cells in the field of microscope was significantly reduced. With the increase of the incubation time, the cells gradually shrink and become round, which indicates that GS-CDs play an inhibiting role on the cells, and the cells gradually die. From the fluorescence images, it can be seen that GS-CDs are able to enter the cell and are mainly concentrated in the cytoplasm. From 30min to 6h, the fluorescence intensity of the system is gradually enhanced, which shows that the cell internalization quantity of the CDs is continuously increased along with the time; from 6h to 12h, the fluorescence intensity of the system is reduced. This is probably due to the intracellular reaction of GS-CDs, whose rigid structure is destroyed; after 24h incubation the fluorescence of the system was very weak. By 48h, the system had little detectable fluorescence. This suggests that GS-CDs may be metabolized by the cells themselves and excreted outside the cells by exocytosis, while exerting their pharmacological effects inside the cells. The change of the fluorescence intensity of the GS-CDs after incubation with the cells indicates that the GS-CDs have time dependence on the inhibition effect of the cells.

Therefore, the change of the SH-SY5Y cell viability after incubation with GS-CDs for different times is simultaneously examined. See fig. 13. After 6h and 12h incubation, GS-CDs have little inhibition effect on SH-SY5Y cells; at 24h of incubation, significant inhibition occurred at higher GS-CDs concentrations (50. mu.g/mL), and at 48h of incubation, GS-CDs showed strong inhibition of SH-SY5Y cells. This is substantially consistent with the results of fig. 12.

Carbon nanodots of total saponins of panax ginseng induce SH-SY5Y apoptosis

And detecting the apoptosis condition of SH-SY5Y cells after 48h incubation with GS-CDs with different concentrations by using flow cytometry. The apoptosis rates of GS-CDs at the administration concentrations of 20, 30 and 50 μ g/mL are respectively 18.59%, 25.06% and 59.66%, which are higher than that of Control 8.4%. The higher the concentration administered, the more significant the rate of apoptosis. See fig. 14. The results in FIG. 14 show that strong SH-SY5Y cell inhibition can be achieved only when the intracellular GS-CDs concentration reaches a certain level (50. mu.g/mL) and the action time is sufficient (48 h).

Example 3

In vivo bioactivity of ginsenoside carbon nanodots

Animal culture

All animal experimental procedures were in compliance with all relevant ethical regulations and were approved by the animal care and welfare committee of the university of medicine, vinpocetine. Male BALB/c nude mice at 4 weeks of age were purchased from Beijing Huafukang Biotech, Inc. and housed in a specific pathogen-free laboratory.

Subcutaneous seed tumor

To assess the ability of GS-CDs to inhibit human neuroblastoma in vivo, SH-SY5Y cells were inoculated into the right axilla (3X 10 cells per mouse) of male BALB/c nude mice (5 weeks old)⁶Individual cells), a subcutaneous neuroblastoma mouse model was established. The volume of the tumor reaches 60mm³On the left and right sides, experiments can be performed.

Experimental groups and procedures

Tumor-bearing mice were randomly divided into 3 groups (n ═ 5): model group (tail vein injection of normal saline), cisplatin group (intraperitoneal injection, 5mg/kg cisplatin injection once every four days) and GS-CDs group (tail vein injection, 8mg/kg GS-CDs once every 36 h), wherein the model group and the GS-CDs group are synchronously administrated. Recording the weight of the mice from the first administration, and taking a picture of the tumor growth condition of the mice, wherein the recording is performed every two days; and scanning the tumor-bearing mice by using a small animal Micro-CT imaging system once every four days, monitoring the growth change condition of the tumor, and accurately measuring and calculating the tumor volume. After day 13 of treatment, all nude mice were sacrificed for anatomical and histopathological analysis.

To investigate the in vivo biosafety of GS-CDs, we randomly divided healthy male BALB/c nude mice (5 weeks old) into two groups (n-5): control group (tail vein injection of physiological saline) and control + GS-CDs group (tail vein injection, 8mg/kg of GS-CDs). The dosing period and number of doses were the same as above and the body weight of the mice was recorded. After day 13 of treatment, all nude mice were sacrificed for anatomical and histopathological analysis.

Results and analysis of the experiments

To assess the ability of GS-CDs to inhibit human neuroblastoma in vivo, BALB/c nude mice were used to carry human neuroblastoma tumors and animal models.

As shown in fig. 15. In fig. 15a, tumor growth in mice on

days

1, 5, 9, and 13 of ct scan (n ═ 4), with a scale bar of 1 cm. Fig. 15b is a statistical plot of relative tumor volumes of model (pink bars), cisplatin (blue bars), and GS-CDs (purple bars) group mice calculated by CT scan of the largest tumor sections. Data are photographs of tumor dissected mice on day 13 (n-4) in the mean ± s.d. (n-4) fig. 15c. model (top), cisplatin (middle), gs-cd group (bottom) at scale bar 1 cm. FIG. 15D, shows a statistical plot of tumor weights in model (pink bars), cisplatin (blue bars), and GS-CDs (purple bars) groups of mice. Data are mean ± s.d. (n ═ 4). Figure 15E H & E (left), antigen CD31 (middle) and antigen NSE (right) stained mouse neuroblastoma tissues at day 13 in the model (upper), cisplatin (middle) and GS-CDs (lower) groups with a 100 μm bar. FIG. 15F is a graph showing statistical comparison of CD31 immunohistochemically stained neuroblastoma tissues from mice in the model (pink bars), cisplatin (blue bars), GS-CDs (purple bars) groups. Figure 15g is a graph of a statistical comparison of neuroblastoma tissues immunohistochemically stained for the mouse antigen NSE in the model (pink bars), cisplatin (blue bars), GS-CDS (purple bars) groups.

FIG. 15A is a CT scan of the growth changes of mouse tumors; the growth change of the mouse tumor was observed by CT scanning with a small animal CT imaging system at 1d, 5d, 9d, and 13d of the tumor growth experiment, respectively (fig. 15A). FIG. 15B is a comparison of tumor volumes calculated from the maximum tumor section of CT scan for the model group, cisplatin group and GS-CDs group. Over time, the tumor growth trend was evident in the model group, and the tumor growth was slowed in the cisplatin group compared to the model group. The growth rate of the tumor of the GS-CDs group mouse is the slowest, and the tumor volume is the smallest. Figure 15C is a graph of the end of the 13d experimental period, dissecting each group of mice and comparing tumors. FIG. 15D is a graph comparing the model, cisplatin and GS-CDs groups, and it is apparent that the GS-CDs group had significantly less tumor volume and weight than the other two groups (FIG. 15D). The data all indicate that GS-CDs can effectively inhibit the growth of human neuroblastoma and generate substantial in vivo curative effect.

To evaluate the pathological lesion of the GS-CDs to the tumor, dissected human neuroblastoma was stained and analyzed (fig. 15E, F, G). Human neuroblastoma is a malignant tumor and grows rapidly. Therefore, after the test period is finished, the lump is large in volume, and the mouse body cannot provide sufficient nutrients, so that blood supply inside the tumor is insufficient, and cells inside the tumor are necrotized. Meanwhile, the blood supply outside the tumor is rich, and the tumor can keep growing rapidly. Thus, it was observed in HE images that tumor cells of each group were destroyed to some extent, including the model group. Neovascularization is a necessary condition for tumor growth, and by labeling vascular endothelial cells in tumors with antisense CD31 (endothelial cell adhesion molecule), it can be seen that the expression level of CD31 in the GS-CDs group is significantly lower than that in the other groups. The antigen NSE (neuron-specific enolase) is a marker of neuroblastoma, and the expression level of GS-CDs is lower than that of other groups.

See fig. 16 and 17. H & E images and statistical plots of body weight changes for the major organs of mice in the control, control + GS-CDs, model, cisplatin, and GS-CDs groups, as shown in FIGS. 16 and 17, respectively. H & E staining results showed that there was less damage to the major organs in each group of mice. The control + GS-CDs mice had a slightly lower body weight at the initial stage compared to the body weight of the control mice. The body weight of the control + GS-CDs mice exceeded the control over time. This indicates that the prepared GS-CDs have little toxic effect on the body. The weight gain of the GS-CDs group mice was very significant compared to the weight of the model group mice. This also indicates that GS-CDs do not produce toxic effects during neuroblastoma therapy. Because the growth of mouse tumor can be effectively inhibited, the health condition of the mouse is improved, and the body weight of the GS-CDs group mouse at day 13 is close to that of the control group. There was no doubt that the magnitude of weight loss was most significant in the cisplatin group mice. However, no significant organ abnormalities were observed in the pathological images of mice in the cisplatin group. Experimental observations show that cisplatin-injected mice are depressed and reluctant to feed for a period of time compared to cisplatin-injected mice. This is probably due to severe irritation of the mouse gut and stomach by cisplatin, resulting in significant weight loss.

The results show that gs-cd has high biocompatibility in the treatment process, and can be safely and effectively used for treating neuroblastoma. It is emphasized that human neuroblastoma is mainly harmful to less immune infants. For children who need careful protection during treatment, the side effects caused by the gs-cd, a nano-drug constructed based on natural herbal extracts, will be greatly reduced. Thus, the acceptance rate of infants will be much higher, which will be more beneficial for the treatment of the disease.

The invention takes GS as an important point, takes the GS as a single reactant, and adopts a one-step hydrothermal method to synthesize GS-CDs. The size of GS-CDs is concentrated in the range of 2-4 nm, the GS-CDs have good water solubility, and the optimal excitation wavelength and the optimal emission wavelength are respectively 360nm and 440 nm. At a low concentration of 50. mu.g/mL, the inhibition rate of GS-CDs on SH-SY5Y cells was-45%. In the in vivo experiment, the GS-CDs group mice showed the slowest tumor growth rate and the smallest tumor volume compared to the model group and the cisplatin group. In addition, GS-CDs showed benign response characteristics of normal body weight and active activity after frequent interventions. Therefore, the low-cost GS-CDs have simple preparation process and good curative effect, and have good application prospect in clinical treatment of fragile children human neuroblastoma.

The present invention provides carbon nanodots containing ginsenoside carbon, which are described in detail above, and the application of the carbon nanodots containing ginsenoside carbon in the field of preparation of pharmaceutical preparations for resisting neuroblastoma and/or inhibiting neuroblastoma cells, and the principle and embodiments of the present invention are described herein by using specific examples, which are provided only for helping to understand the method and the core concept thereof, including the best mode, and also for enabling any person skilled in the art to practice the present invention, including making and using any device or system, and performing any combination of the methods. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other embodiments are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Claims

1. Application of ginsenoside carbon nanodots in preparing a medicinal preparation for resisting neuroblastoma and/or inhibiting neuroblastoma cells.

2. The use of claim 1, wherein the total ginsenoside carbon nanodots have a diameter of 1 to 50 nm;

3. The use of claim 1, wherein the ginsenoside carbon nanodots are stable in NaCl and/or KCl solution;

the neuroblastoma cell is an SH-SY5Y cell;

4. The use according to claim 1, wherein the concentration of the NaCl and/or KCl solution is 0.2-2 mol/L;

the stability includes fluorescence stability;

the diameter of the ginsenoside carbon nanodots is 1-10 nm.

5. The use of claim 1, wherein the ginsenoside carbon nanodots do not have a proliferative or inhibitory effect on one or more of HeLa cells, HepG2 cells, and BV2 cells.

6. The use of claim 1, wherein the preparation method of the carbon nanodots containing ginsenoside, comprises the following steps:

1) mixing the total ginsenoside with water to obtain a mixed solution;

7. The application of claim 4, wherein the concentration of the total saponins of panax ginseng in the mixed solution is 0.1-10 mg/mL;

the temperature of the hydrothermal reaction is 90-220 ℃;

the time of the hydrothermal reaction is 0.5-12 h;

the hydrothermal reaction also comprises a filtering step;

8. The use of any one of claims 1 to 7, wherein the ginsenoside carbon nanodots further comprise ginsenoside carbon nanodot fluorescent probes;

the fluorescent probe comprises a cellular fluorescent probe;

9. The use of any one of claims 1 to 7, wherein the pharmaceutical preparation comprises carbon dots of total saponins of panax ginseng, and pharmaceutically acceptable excipients;

10. The use of claim 9, wherein the pharmaceutical formulation has no proliferative or inhibitory effect on one or more of HeLa cells, HepG2 cells, and BV2 cells;

the dosage form of the pharmaceutical preparation comprises oral preparation, injection, suppository, inhalant or dosage form which can be directly applied to tumors;