KR20190060677A - Method for preparing nanoparticles - Google Patents

Abstract

The present invention relates to: a method for manufacturing nanoparticles; nanoparticles manufactured by the same; and a composition comprising nanoparticles. More specifically, the present invention relates to the method for manufacturing nanoparticles containing one or more plant extracts in a simplified process without adding any toxic substances and surfactants. In addition, the present invention relates to nanoparticles manufactured by the same and the composition comprising nanoparticles.

Description

[0001] METHOD FOR PREPARING NANOPARTICLES [0002]

The present invention relates to a process for preparing nanoparticles and a composition comprising the nanoparticles and nanoparticles thus prepared. More specifically, the present invention relates to a method for preparing nanoparticles containing one or more plant extracts in a simplified process without adding any toxicants and surfactants. The present invention also relates to a pharmaceutical composition for treating or preventing cancer, which comprises the nanoparticles thus prepared, and nanoparticles, or a composition for improving or preventing cancer.

In order to avoid the side effects brought about by conventional chemotherapy, there have been reported many anticancer drugs using one or two or more plant extracts. However, even though plant extracts are harmless to human body, they are very low in solubility even though they are in the spotlight as alternative medicine. Oral administration is limited, or it is difficult to target a desired target site because of low drug transferring ability, or to use a high dose Or storage stability is not ensured.

Accordingly, various efforts have been made in the art to solve these problems, one of which is the application of nanotechnology such as nanoparticle formation. The size and surface charge of the nanoparticles affect the adhesion of the particles and their interaction with the cell, which ultimately leads to changes in the nanoparticle's cellular uptake path and efficiency. In addition, the shape of the nanoparticles depends on the interaction with the nanoparticle preparation method, the inducing agent and the stabilizer used in the production of the nanoparticles, and affects the binding ability to the target receptor and the ability to capture by the macrophage.

An example of the prior art on a method for producing such nanoparticles is disclosed in Japanese Patent Application Laid-Open No. 10-2009-0112444 (2009.10.28), which is an invention for providing an edible composition containing broccoli nanoparticles. However, this prior art includes a step of dissolving edible lecithin oil in a small amount in CH ₂ Cl ₂ or chloroform, drying the broth, dispersing the broccoli water-soluble extract in the oil phase with an ultrasonic disperser, The use of a solvent other than water causes particle agglomeration phenomenon, which means that nanoparticles are not produced or nanoparticles are not homogeneously produced.

Therefore, in the method for producing nanoparticles, it is an important concern in the art to obtain nanoparticles having excellent properties by optimizing the process conditions.

Japanese Patent Application Laid-Open No. 10-2009-0112444 (Oct. 28, 2009)

The present inventors have made intensive efforts to develop a novel method for producing nanoparticles capable of solving the problems of the prior art, and have completed the present invention. The method of the present invention can be applied not only to various plant extracts but also to two or more complex plant extracts in addition to a single plant extract. Therefore, the present invention is not limited to solving the problems of the prior arts, It is expected to be possible.

In one aspect of the present invention, the method for preparing nanoparticles of the present invention comprises the following steps:

a) Preparing a stock solution by dissolving one or more plant extracts in water,

b) Adding the stock solution to water at a temperature of 70 DEG C to 100 DEG C by a dropping or spraying method, and

c) Applying ultrasonic pulverization.

Preferably, the plant extract of step a) is prepared by a spray drying method.

Preferably, the dropping or spraying method of step b) includes adding the stock solution at a predetermined rate. The predetermined speed means adding at a constant speed or at an already determined speed. For example, 0.1 to 1.0 ml / min, but is not limited thereto.

Preferably, the temperature of the water to which the storage liquid is added in step b) is 100 ° C.

Preferably, the solution produced by applying ultrasonic pulverization in the step c) is lyophilized. Such freeze-dried nanoparticles are excellent in storage stability and are easy to store until they are actually used. Optionally, reduced pressure concentration can be performed prior to the lyophilization.

In one aspect of the present invention, the nanoparticles prepared according to the production method of the present invention can be contained as an active ingredient in a pharmaceutical composition.

The pharmaceutical composition according to the present invention can prevent or treat cancer. It will be readily appreciated by those skilled in the art that the disease to be prevented or treated may vary depending on which extract is contained in the nanoparticles contained in the pharmaceutical composition according to the present invention, It is to be understood that the invention is not limited thereto.

The cancer may be a solid tumor and is preferably selected from the group consisting of brain tumor, stomach cancer, liver cancer, lung cancer, thyroid cancer, breast cancer, cervical cancer, colon cancer, pancreatic cancer, rectal cancer, colorectal cancer, prostate cancer, kidney cancer, melanoma, Cancer. ≪ / RTI >

As used herein, the term " treatment " refers to any action that improves or alleviates the symptoms of cancer upon administration of the composition of the present invention. The term " prophylactic ", as used herein, refers to all actions that inhibit or delay the onset of cancer by administration of the composition of the present invention.

The content of the plant extract nanoparticles in the pharmaceutical composition of the present invention can be appropriately adjusted according to the symptoms of the disease, the progress of symptoms, the condition of the patient, and the like. For example, from 0.0001 to 99.9% by weight, from 0.1 to 90% by weight, from 1 to 80% by weight, from 1 to 70% by weight, from 1 to 60% by weight, or from 1 to 50% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further contain additional components in addition to the plant extract nanoparticles for enhancing the efficacy. Alternatively, the pharmaceutical compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Examples of carriers, excipients and diluents that may be contained in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral.

The pharmaceutical composition of the present invention may preferably be an oral dosage form. In addition, the pharmaceutical compositions according to the present invention may be formulated into suitable dosage forms for topical administration using techniques well known to those skilled in the art, and in the case of topical dosage forms, the formulations may include external preparations, foams, suppositories, have. In one embodiment, the pharmaceutical composition of the present invention may be formulated as an external preparation by mixing the extract with a base, which is well known in the art and commonly used. The external preparation may contain emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions and tinctures.

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as pills, powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method have. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, Can be mixed and prepared. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

The pharmaceutical composition of the present invention is administered to a subject in a pharmaceutically effective amount. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment for medical treatment, and the effective dose level will depend on the species and severity, Sex, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and body weight. Specifically, the composition of the present invention may be administered at a dose of 0.1 to 100 mg / kg once or several times a day depending on the severity of the cancer patient. In addition, the dose may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and particularly, the severity of the patient. Accordingly, the dosage is not limited in any way to the scope of the present invention.

In another aspect of the present invention, nanoparticles prepared according to the method of the present invention may be included in a food composition. The food composition of the present invention can be used to improve or prevent cancer. It will be apparent to those skilled in the art that the food composition of the present invention may be subject to different conditions depending on which extract is contained in the nanoparticles contained in the food composition of the present invention will be. Thus, it should be understood that the subject condition in which the food composition is intended to be improved or prevented is not limited to cancer.

In the food composition according to the present invention, the plant extract nanoparticles and the cancer are as mentioned in the above pharmaceutical composition, respectively, unless otherwise specified.

The term " improvement " means any action that decreases the degree of symptom associated with a condition in which the cancer is being treated, for example, delaying the onset or deterioration of symptoms or ameliorating symptoms.

The food may be a health functional food. The term " health functional food " means food prepared and processed in the form of tablets, capsules, powders, granules, liquids, and circles using raw materials and components having useful functions in the human body. The term "functional" as used herein means that the structure and function of the human body have a beneficial effect on health uses such as controlling nutrients or physiological actions.

The food composition according to the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there are no side effects that may occur when a food is used as a raw material for a long time, and the portability is excellent. Therefore, the food composition of the present invention is useful for improving or improving the bone disease treatment effect It can be taken as an adjuvant.

The amount of the plant extract nanoparticles contained as an active ingredient in the food composition according to the present invention can be suitably determined according to the intended use (prevention, improvement, or therapeutic treatment). Generally, the plant extract nanoparticles of the present invention may be contained in an amount of 0.001 to 20% by weight, 0.001 to 15% by weight or 0.001 to 10% by weight in the raw material composition when the food is prepared. In the case of health drinks, 0.01 to 2 g, specifically 0.02 to 2 g, more specifically 0.3 to 1 g, may be added based on 100 mL. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the above amount can also be used below the above-mentioned range.

The content of the plant extract nanoparticles according to the present invention added to the food composition in the course of preparing the food composition can be appropriately adjusted.

The food composition of the present invention may further comprise an additional component in addition to the plant extract nanoparticles for enhancing efficacy.

The food composition may be any one selected from the group consisting of pills, tablets, granules, powders, capsules, and liquid solutions.

The kind of the food is not particularly limited. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, and includes foods in a conventional sense.

The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary food. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.

When the food composition of the present invention is a beverage composition, there are no particular limitations on the liquid component other than the above-mentioned ingredients in the indicated proportions as an essential ingredient, and various flavors or natural carbohydrates, .

Since the manufacturing method according to the present invention does not add any toxic chemicals and surfactants, the nanoparticles produced by the present invention are harmless to the human body. In addition, even if the nanoparticles produced by the present invention are contained in a pharmaceutical composition for treating or preventing cancer or contained in a food composition for improving or preventing cancer, it is possible to suppress or prevent side effects or adverse effects due to impurities contained in the manufacturing process It is possible to block.

When an organic solvent such as methanol, acetone, dichloromethane or the like is used, particle agglomeration phenomenon occurs, so that nanoparticles of a certain size are not homogeneously produced, do not show spherical shape, and can not be separated as nanoparticles. On the other hand, according to the production method of the present invention, the nanoparticles produced without using an organic solvent are very advantageous in that they do not show any aggregation of particles and can obtain nanoparticles with high yield.

Furthermore, the manufacturing method according to the present invention has a simple and cost-effective process as a whole process, and has a short total process time, which is very advantageous for industrial application.

The nanoparticles prepared according to the preparation method of the present invention are superior in the transporting ability of the target site, the homogeneous distribution in the cell, and the bioavailability, compared with the particles of the general size, and thus they are advantageously used in terms of pharmacological, . In addition, since the nanoparticles of the present invention are stable and no harmful substances are used in the manufacturing process, human toxicity can be excluded in advance.

In addition, the nanoparticles produced by the present invention are uniform in size in the nanometer range and are provided in a circular shape. In addition, the nanoparticles prepared according to the method of the present invention and having a specific size and shape are highly capable of being homogeneously distributed in the cells. Therefore, the nanoparticles produced by the present invention can be homogeneously provided to a subject in terms of pharmacological effect or food effect. Since ordinary particles have a size in the micrometer range, intracellular internalization and homogeneous distribution are difficult, the endocytosis process observed at the time of using the nanoparticles of the present invention does not proceed, and the shape is irregular. Therefore, It is difficult. For this reason, regular particles should be used at higher concentrations than nanoparticles in order to achieve the desired effect, leading to cytotoxicity and side effects.

Particularly, since the method for producing nanoparticles can be applied not only to a single plant extract but also to a combination of two or more complex plant extracts, it is possible to universally utilize the manufacturing method according to the present invention for a conventionally known or commercially available product .

Fig. 1 shows the UV-visible spectroscopic absorption spectrum of the nanoparticles obtained in Example 1. Fig.
Figure 2 shows an HR-TEM image. FIG. 2A shows an untreated control, FIG. 2B shows several nanoparticles dispersed in the cytoplasm, FIGS. 2C and 2D show nanoparticles distributed in the nucleus, and FIGS. 2E and 2F show nano- 2G and 2H show nanoparticles present inside the cell membrane.
FIG. 3A is a graph showing cell survival rates according to concentrations as a result of MTT assay on HepG2 liver cancer cells and HBM normal cells. 'BRM270' refers to the general particles obtained in Comparative Example 1, and 'BRM270 nanoparticle' refers to the nanoparticles obtained in Example 1.
FIG. 3B is a graph showing cell survival rate as a result of MTT assay for HeLa cervical cancer cells.
FIG. 4 is a microphotograph for the morphological analysis of HepG2 liver cancer cells, A549 lung cancer cells, HeLa cervical cancer cells, and HBM normal cells.
FIG. 5 shows DAPI assay results for confirming apoptosis on HepG2 liver cancer cells. 'BRM270 normal' refers to the general particles obtained in Comparative Example 1, and 'BRM270 NP' refers to the nanoparticles obtained in Example 1.
Fig. 6 shows the UV-visible spectroscopic absorption spectrum of the nanoparticles obtained in Example 2. Fig.
7 shows an FE-SEM micrograph of the general particles of Comparative Example 2 and the nanoparticles of Example 2. Fig.
8 is a graph showing cell survival rate as a result of MTT assay for HeLa cervical cancer cells.
9 is a photomicrograph of the morphological analysis of Cal72 osteosarcoma cells and HeLa cervical cancer cells.
10 shows FE-SEM micrographs of the general particles of Comparative Example 3 and the nanoparticles of Example 3. Fig.
11 is an FE-SEM micrograph of the general particles of Comparative Example 4 and the nanoparticles of Example 4. Fig.
12 shows FE-SEM micrographs of the general particles of Comparative Example 5 and the nanoparticles of Example 5. Fig.
13 shows FE-SEM micrographs of the general particles of Comparative Example 6 and the nanoparticles of Example 6. Fig.
14 is a graph showing cell survival rate as a result of MTT assay for HeLa cervical cancer cells.
Fig. 15 is a microphotograph for morphological analysis of HeLa cervical cancer cells.
16 is an FE-SEM micrograph of the general particles of Comparative Example 7 and the nanoparticles of Example 7. Fig.
17 shows an FE-SEM micrograph of the general particle of Comparative Example 8, and the right side of FIG. 17 shows an FE-SEM micrograph of the nanoparticle of Example 8. [
18 is a graph showing cell survival rate as a result of MTT assay on HepG2 liver cancer cells.
FIG. 19 shows a micrograph and DAPI assay results for morphological analysis of HepG2 liver cancer cells.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Comparative Example 1

21.13 wt.%, 21.13 wt.%, 18.3 wt.%, 18.3 wt.%, 14.09 wt.% Of Saururus chinensis , Prunella vulgaris , Rhizopus, Portulaca oleracea , , And 7.05% by weight. Here, gold refers to the roots of Scutellaria baicalensis , root refers to the roots of Lithospermum officinale , and cheongpy refers to the peel of citrus ( Citrus nippokoreana ).

The mixed material was placed in a broad bean sprout, placed in an extractor, and 8 times of extracted water was added thereto, followed by extraction at 95 to 100 ° C for 10 to 14 hours. The resulting extract was filtered with a filter, transferred to a stirring tank, and concentrated to a sugar content of 10 brix using a concentrator.

Example 1

Saururus chinensis, golden, persimmon, obtusa, cheongpije, and kwangwol were boiled, respectively, and spray-dried to produce liquid extracts.

Each of the liquid extracts thus prepared was compounded in the same ratio to produce a total of 10 mg of a solution, which was then dissolved in 20 ml of water to obtain a stock solution.

1 ml of the obtained stock solution was added to 50 ml of boiling water by a dropwise method or a spray method at a flow rate of 0.2 ml / min for 5 minutes. Considering both the formation of the nanoparticles and the homogeneity of the size, it is preferred that the stock solution is added at a constant flow rate, i. E. At a predetermined rate. In contrast, when the stock solution was added at one time, particle agglomeration occurred, which made it impossible to effectively separate the nanoparticles. In addition, if the flow velocity fluctuates greatly, the size of the nanoparticles formed is not homogeneous, which hinders the consistency of the effect exhibited by the nanoparticles.

After continuously stirring at 200 - 800 rpm for 30 minutes at room temperature, an ultrasonicator was applied at 100.0 W and 30.0 kHz for 2 hours. After sonication, a clear yellow orange solution was obtained, which was concentrated to 5 ml under reduced pressure at 40 < 0 > C using a rotary evaporator.

The concentrated sample was lyophilized to obtain a brown nanoparticle powder.

Experimental Example 1

In this experimental example, the particle size, shape and nano particle size of the product obtained according to Example 1 were confirmed by various experiments.

(1) UV-visible spectroscopy

1 mg of the lyophilized powder obtained in Example 1 was dissolved in 1 ml of distilled water to make an aqueous solution, and spectroscopic analysis was performed at a wavelength of 200 to 800 nm using a Perkin Elmer LAMBDA 25 spectrophotometer.

The resulting UV-visible spectra are shown in FIG. The spectrum of FIG. 1 shows a sharp peak, especially at a wavelength of 283.3 nm. This means that the lyophilized powder obtained in Example 1 is very stable and very well dispersed in distilled water.

(2) Field-Emission Scanning Electron Microscopy (FE-SEM)

In order to confirm the surface morphology, particle size and shape of the lyophilized powder obtained in Example 1, FE-SEM was carried out using a JSM-6700F instrument.

To this end, 1 mg of the product of Example 1 and 1 mg of the product of Comparative Example 1 were each dissolved in 10 ml of distilled water, then spread on a carbon tape and dried under a stream of nitrogen. Then, a 200 Å thick platinum layer was coated on an osmium plasma sputter coater OPC 80 T under vacuum conditions.

As a result of the analysis of the obtained micrograph, it was confirmed that the lyophilized powder obtained in Example 1 was perfectly spherical and uniformly showed a diameter of 23.0 to 70.0 nm.

(3) Difference according to the type of solvent to which the storage liquid is added

When the stock solution was added to an organic solvent such as methanol, acetone, or dichloromethane without adding water to the water in the process of Example 1, spherical homogeneous nanoparticles could not be obtained due to particle agglomeration. In addition, the manufacturing method according to the present invention does not add any toxic chemicals, surfactants, impurities, etc. in the manufacturing process in order to produce nano-particles harmless to the human body. Therefore, it is not within the scope of the present invention to add a stock solution made from a plant extract to an organic solvent in the method for producing nanoparticles.

(4) Difference according to the temperature of the water to which the storage liquid is added

When the stock solution was added to water at a different temperature without adding it to the boiling water in the process of Example 1, the following results were observed.

The temperature of the water to which the stock solution is added The size of the generated particles The shape of the generated particles One 40 ℃ 1.71-2.24 nm Irregular shape 2 50 ℃ 160-285 nm polygon 3 60 ° C 91-116 nm hexagon 4 70 ℃ 77-92 nm rectangle 5 80 ℃ 64-81 nm rectangle 6 100 ℃ 23-70 nm rectangle

In Table 1, the shape of particles generated from the case where the temperature of the water to which the storage liquid is added is 70 ° C or higher, is spherical. In addition, when the temperature of the water to which the storage liquid is added is below 60 ° C, the particle size is smaller or larger than the nano range.

When the shape of the particle is not spherical or deviates from the size of the nanometer range, the nanoparticle to be obtained in the present invention can not exhibit excellent biocompatibility and target transfer ability. For example, if the size of the nanoparticles is too small, the effect of the chemotherapeutic agent is not effective because the tumor is easily leaked from the tumor tissue into the capillary vessel during the chemotherapy, and if the size of the nanoparticles is too large, the reticuloendothelial system The capture by the macrophage can not be avoided.

As demonstrated in the experimental examples to be described later, the nanoparticles obtained according to the production method of the present invention have no phenomenon of leaking from the tumor tissue to the capillary blood vessels, and are free from macrophage capturing phenomenon, .

Experimental Example 2

In this experimental example, the size and shape of the nanoparticles obtained according to the manufacturing method of the present invention using HR-TEM (High Resolution-Transmission Electron Microscope), and the intracellular distribution and internalization were analyzed.

(1) Size and shape of nanoparticles

In order to confirm the size and shape of the nanoparticles, the nanoparticles of Example 1 and the general particles of Comparative Example 1 were ultrasonically dissolved in double-distilled water and then deposited on a carbon-coated copper grid, I let it evaporate.

As a result of the HR-TEM analysis, the general particles of Comparative Example 1 were polygons having an irregular contour, whereas the nanoparticles of Example 1 were perfectly spherical and the average diameter was measured to be about 40 to 70 nm. When the nanoparticles are spherical, they can not only enhance the interaction with the cell membrane during long-term circulation in the blood vessels, but also minimize the absorption by the macrophages, increase the binding capacity to the target receptor, It is possible to induce the biological distribution. Therefore, the nanoparticles obtained according to the production method of the present invention are significantly more advantageous in terms of bioavailability than non-spherical nanoparticles.

(2) Intracellular distribution and internalization of nanoparticles

Next, in order to analyze the intracellular distribution and internalization of nanoparticles, experiments were conducted as follows.

6.0x10 ⁵ HepG2 liver cancer cells were seeded in each culture dish and adhered and adapted for 1 day. The nanoparticles of Example 1 were exposed to each culture dish for 12 hours. The cells were then harvested by trypsinization, fixed in 2.5% glutaraldehyde, and stored at 4 < 0 > C until use.

As a result of HR-TEM image analysis, it was observed that the nanoparticles reached the nuclear peripheral region of the cells one hour after treating HepG2 liver cancer cells with the nanoparticles of Example 1, and the cells continuously absorbed the nanoparticles. This demonstrates that the nanoparticles of the invention are very quickly internalized in the cells after treatment.

Figure 2A shows an untreated control. Figure 2B shows several nanoparticles dispersed in the cytoplasm. In particular, Figures 2C and 2D show nanoparticles distributed in the nucleus, which is the first to identify the location of nanoparticles at the subcellular level.

2E and 2F show nanoparticles present on the cell membrane during entry of the nanoparticles into the cell. In particular, in FIG. 2F, an endosome-like structure containing nanoparticles was found near the cell membrane, and this endosomal-like structure migrated into the Seno interior. Figures 2G and 2H show nanoparticles present inside the cell membrane.

The small size of the nanoparticles according to the present invention facilitates endocytosis, and endocytosis is a very favorable process for cell internalization. Accordingly, it has been observed that nanoparticles are accumulated in cell membranes and cytoplasm in larger amounts, which means that the components constituting the nanoparticles can effectively function on the desired cells.

Experimental Example 3

In this Example, the effect of the nanoparticles obtained according to the production method of the present invention was confirmed.

(1) MTT assay

In order to comparatively analyze the cytotoxic effect of the nanoparticles of Example 1, the general particles of BRM270 and doxorubicin, HepG2 liver cancer cells, HeLa cervical cancer cells and HBM (human bone marrow) Cell viability and proliferation rate were confirmed by assay.

For this, each cell was inoculated into 96-well plates at a concentration of 1 x 10 ⁴ cells / well. The cells were incubated overnight, and the nanoparticles of Example 1, the common particles of Comparative Example 1, and doxorubicin were each treated at a concentration ranging from 2.0 to 12.0 μg / mL. Here, doxorubicin is a strain of Streptomyces peucetius var. It is a kind of antracycline antibiotic substance produced by caesius. It is also called adriamycin and has been reported to exhibit excellent anticancer effect on malignant lymphoma, digestive cancer, lung cancer, and breast cancer. In this example, doxorubicin was used as a positive control.

After 24 hours, the cells were washed twice with PBS and treated with 5.0 mg / mL of a solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) Incubated for 4 hours together. The cells were then lysed with 100.0 μL of DMSO. The absorbance at 570.0 nm of the cell lysate was measured using a bio-assay reader (Bio-Rad, Berkeley, Calif., USA).

The results are shown in Fig.

FIG. 3A shows the results of treating the hepatocellular carcinoma cells and HBM normal cells with the nanoparticles of Example 1, the common particles of Comparative Example 1, and doxorubicin, respectively. As can be seen from FIG. 3A, the nanoparticles of Example 1 were significantly superior to the general particles and doxorubicin of Comparative Example 1 in cancer cell proliferation inhibitory effect. In addition, while doxorubicin exhibits side effects that inhibit the growth and proliferation of normal cell HBM, the nanoparticles of Example 1 have very little effect on growth and proliferation of normal cell HBM, and thus have a high selectivity to the target.

FIG. 3B shows the results of treating the HeLa cervical cancer cells with the nanoparticles of Example 1, the common particles of Comparative Example 1, and doxorubicin, respectively. As can be seen from FIG. 3B, the nanoparticles of Example 1 are significantly superior to the general particles of Comparative Example 1 and doxorubicin in inhibiting cancer cell proliferation.

Therefore, the nanoparticles of Example 1 exhibit minimal cytotoxicity and can exhibit the maximum therapeutic effect. This makes it possible to lower the effective amount of the nanoparticles of Example 1 and thus the effective amount of the nanoparticles to be ingested or administered to the subject is lowered, so that the nanoparticles according to the present invention are very advantageous in terms of convenience and cost.

(2) cytomorphological analysis

The anticancer effect of the nanoparticles of Example 1 and the general particles of Comparative Example 1 was analyzed in terms of cytomorphology.

HepG2 liver cancer cells, A549 lung cancer cells, HeLa cervical cancer cells, and HBM normal cells were cultured in 24-well plates, respectively. The cells were treated with the nanoparticles of Example 1 and the common particles of Comparative Example 1, respectively, and after 24 hours had elapsed, the cells were observed using a microscope.

As can be seen in FIG. 4, in the untreated control, a clear cytoskeleton was identified regardless of the cell type, indicating that the cells maintained a healthy morphology. Similarly, for the HBM normal cells, both the nanoparticles of Example 1 and the normal particles of Comparative Example 1 were found to have clear cytoskeletons.

On the other hand, when HepG2 liver cancer cells, A549 lung cancer cells and HeLa cervical cancer cells were treated with the nanoparticles of Example 1 and the common particles of Comparative Example 1, cell shrinkage was observed, and apoptotic cells and cell debris were increased. Particularly, in the group treated with the nanoparticles of Example 1, more apoptotic cells and cell debris were observed than the group treated with the normal particles of Comparative Example 1. [

(3) DAPI assay

In order to compare the anticancer effects of the nanoparticles prepared in Example 1 with those of the common particles of Comparative Example 1, DAPI assays were performed to confirm the apoptosis of HepG2 liver cancer cells.

For this, 1x10 ⁶ HepG2 hepatoma cells were seeded in 6-well microtiter plates. After overnight incubation, cells were treated with the nanoparticles of Example 1 and the normal particles of Comparative Example 1, respectively. Cells were then washed with PBS, fixed with 4.0% paraformaldehyde for 10 minutes, and incubated for 5 minutes with DAPI (4,6-diamidino-2-phenylindole). After washing three times with PBS, the cells were observed under a fluorescence microscope.

As a result, apoptosis was induced in 44.4% of HepG2 liver cancer cells treated with the nanoparticles of Example 1 at a minimum effective concentration of 12.0 μg / mL. On the other hand, in the group treated with the common particles of Comparative Example 1 at the same concentration, apoptosis was induced only in 12.5% of HepG2 liver cancer cells (FIG. 5).

In addition, the nanoparticles of Example 1 caused chromatin condensation in apoptotic cells and nuclear DNA fragmentation in non-apoptotic cells.

Example 2 and Comparative Example 2

Saururus chinensis was boiled and then spray-dried to produce a liquid extract. 10 mg of the liquid extract thus prepared was dissolved in 20 ml of water to obtain a stock solution. 1 ml of the stock solution was added to 50 ml of boiling water at a constant flow rate for 5 minutes by dripping or spraying. The resulting solution was continuously stirred at 200-800 rpm for 30 minutes at room temperature and then ultrasonically pulverized for 2 hours at 100.0 W and 30.0 kHz. After the ultrasonic treatment, the mixture was concentrated to 5 ml under reduced pressure at 40 ° C using a rotary evaporator, and lyophilized. The lyophilized powder thus obtained was used as the sample of Example 2. [

On the other hand, the lyophilized powder obtained in the same manner as in Example 2 was used as a sample of Comparative Example 2, except that the Saururus chinensis extract was dripped into methanol.

Experimental Example 4

As to the product obtained according to Example 2, it was confirmed whether it corresponds to nanoparticles or nanoparticles.

(1) UV-visible spectroscopy

UV-visible spectroscopic analysis was performed in the same manner as in Experimental Example 1, and the obtained spectrum was shown in FIG.

The absorption spectrum of FIG. 6 shows a sharp peak at 660 nm. This means that the lyophilized powder obtained in Example 2 is well dispersed in the distilled water and is very stable.

(2) FE-SEM

FE-SEM was performed in the same manner as in Experimental Example 1, and the obtained microphotograph was shown in FIG.

7, the diameter of the lyophilized powder obtained in Comparative Example 2 was measured to be 1.153 占 퐉, which does not correspond to nanoparticles. On the other hand, in the photograph of the bottom of FIG. 7, the diameters of 67.6 nm, 70.1 nm, 66.3 nm and 72.4 nm of the lyophilized powders obtained in Example 2 were homogeneously measured and confirmed to be spherical nanoparticles. Such a nanoparticle form is advantageous for exhibiting the anticancer effect described later.

(3) MTT assay

To compare the effects of the nanoparticles of Example 2, the common particles of Comparative Example 2, and doxorubicin on HeLa cervical cancer cells, MTT assays were performed in the same manner as in Experimental Example 3 to confirm cell viability and proliferation rate . The results are shown in Fig.

As can be seen from Fig. 8, it can be seen that the nanoparticles of Saururus chinensis extracts are much more effective in inhibiting the proliferation of cervical cancer cells than both the normal particles (microsize) and doxorubicin of Saururus chinensis extracts.

(4) cytomorphological analysis

To compare the effects of the nanoparticles of Example 2 and the general particles of Comparative Example 2 on Cal72 osteosarcoma cells and HeLa cervical cancer cells, cytomorphological analysis was performed in the same manner as Experimental Example 3 above. The results are shown in Fig.

As can be seen in FIG. 9, in the untreated control group, cells that maintained a clean cytoskeleton and healthy morphology were identified in both osteosarcoma cells and cervical cancer cells. On the other hand, when treated with the nanoparticles of Example 2 and Comparative Example 2, apoptotic cells and cell debris were observed, especially in the nanoparticle-treated group of Example 2.

Example 3 and Comparative Example 3

A lyophilized powder was obtained by applying the same method as in Example 2 and Comparative Example 2, except that roots of Scutellaria baicalensis were used as a single material. These were used as the samples of Example 3 and Comparative Example 3, respectively.

Experimental Example 5

FE-SEM was performed in the same manner as in Experimental Example 1 to confirm whether the samples of Example 3 and Comparative Example 3 were nanoparticles, and the obtained microphotographs are shown in FIG.

In the top photograph of FIG. 10, the sample of Comparative Example 3 had a particle diameter of 4.476 μm, which is not a nanoparticle. On the other hand, in the photograph of the bottom of FIG. 10, the diameters of 79.7 nm, 75.3 nm, 80.0 nm, 84.4 nm and 80.3 nm of the sample of Example 3 were homogeneously measured and confirmed to be spherical nanoparticles. Such a nanoparticle form is advantageous for exhibiting an anticancer effect.

Example 4 and Comparative Example 4

Freeze-dried powder was obtained by applying the same method as in Example 2 and Comparative Example 2, except that Prunella vulgaris was used as a single material. These were used as samples of Example 4 and Comparative Example 4, respectively.

Experimental Example 6

FE-SEM was performed in the same manner as in Experimental Example 1 to confirm whether the samples of Example 4 and Comparative Example 4 were nanoparticles. The obtained micrographs are shown in FIG.

In the top photograph of FIG. 11, the sample of Comparative Example 4 was measured within a range of particle diameters of 5.76 μm to 8.75 μm, which does not correspond to nanoparticles. On the other hand, in the photograph of the bottom of FIG. 11, the diameter of the sample of Example 4 was measured homogeneously within the range of 66.3 nm to 72.4 nm, and it was confirmed that it was a spherical nanoparticle. Such a nanoparticle form is advantageous for exhibiting an anticancer effect.

Example 5 and Comparative Example 5

A lyophilized powder was obtained in the same manner as in Example 2 and Comparative Example 2, except that Arnebia euchroma was used as a single material. These were used as samples of Example 5 and Comparative Example 5, respectively.

Experimental Example 7

FE-SEM was performed in the same manner as in Experimental Example 1 to confirm whether the samples of Example 5 and Comparative Example 5 were nanoparticles. The obtained micrographs are shown in FIG.

In the top photograph of Fig. 12, the sample of Comparative Example 5 had a particle diameter of 0.794 mu m, which is not a nanoparticle. On the other hand, in the photograph of the bottom of FIG. 12, the diameter of the sample of Example 5 was measured uniformly within the range of 75.1 nm to 77.0 nm, and it was confirmed that it was a spherical nanoparticle. Such a nanoparticle form is advantageous for exhibiting an anticancer effect.

Example 6 and Comparative Example 6

Freeze-dried powder was obtained by applying the same method as in Example 2 and Comparative Example 2, except that the dermis of Citrus unshiu markovich was used as a single material. These were used as samples of Example 6 and Comparative Example 6, respectively.

Experimental Example 8

For the product obtained according to Example 6, whether or not it corresponds to nanoparticles and the effect as nanoparticles were confirmed.

(1) FE-SEM

FE-SEM was performed in the same manner as in Experimental Example 1, and the obtained micrograph is shown in Fig.

In the top photograph of FIG. 13, the sample of Comparative Example 6 had a particle diameter ranging from 0.105 μm to 0.479 μm, which is not a nanoparticle. On the other hand, in the photograph of the bottom of Fig. 11, the diameter of the sample of Example 4 was measured uniformly within the range of 45.5 nm to 64.5 nm, and it was confirmed that it was a spherical nanoparticle. Such a nanoparticle form is advantageous for exhibiting the anticancer effect described later.

(2) MTT assay

In order to compare the effects of the nanoparticles of Example 6, common particles of Comparative Example 6, and doxorubicin against HeLa cervical cancer cells, MTT assays were performed in the same manner as in Experimental Example 3 to confirm cell viability and proliferation rate . The results are shown in Fig.

As can be seen from Fig. 14, it can be seen that the nanoparticles of mandarin dandruff single extract are much more effective in inhibiting the proliferation of cervical cancer cells than both normal particles (microsize) and doxorubicin of mandarin dandruff single extract. The nanoparticles of the dandelion dandelion extract showed a specific proliferation inhibitory effect at a concentration of 10 to 100 μg / ml and an IC ₅₀ value of 30 μg / ml only in cancer cells without affecting normal cells.

(3) cytomorphological analysis

To compare the effects of the nanoparticles of Example 6, the common particles of Comparative Example 6, and doxorubicin on HeLa cervical cancer cells, cytomorphological analysis was performed in the same manner as in Experimental Example 3 above. The results are shown in Fig.

The largest number of apoptotic cells and cell debris were observed in the nanoparticle-treated group of Example 6.

Example 7 and Comparative Example 7

Freeze-dried powder was obtained by applying the same method as in Example 2 and Comparative Example 2, except that Portulaca oleracea was used as a single material. These were used as samples of Example 7 and Comparative Example 7, respectively.

Experimental Example 9

In order to confirm whether the samples of Example 7 and Comparative Example 7 were nanoparticles, FE-SEM was performed in the same manner as in Experimental Example 1, and the obtained micrographs are shown in FIG.

In the top photograph of FIG. 16, the sample of Comparative Example 7 had a particle diameter ranging from 0.137 μm to 0.331 μm, which is not a nanoparticle. On the other hand, in the photograph of the bottom of FIG. 16, it was confirmed that the sample of Example 7 exhibited a perfect sphere having a diameter of 25.1 nm to 28.8 nm uniformly. Such a nanoparticle form is advantageous for exhibiting an anticancer effect.

Example 8

The freeze-dried powder was obtained by applying the same method as in Example 2 to the roots of red ginseng. This powder was brown and used as the sample of Example 8.

Experimental Example 10

With respect to the sample of Example 8, whether or not the sample corresponds to nanoparticles and the effect as nanoparticles were confirmed.

(1) FE-SEM

FE-SEM was performed in the same manner as in Experimental Example 1, and the obtained micrograph is shown in Fig.

17 shows the microsized red ginseng root particles (Comparative Example 8), and the right side of FIG. 17 shows the red ginseng root nanoparticles of Example 8 having a complete spherical shape with a diameter of 50-65 nm.

(2) MTT assay

Planting the HepG2 liver cancer cells in a 96-well 1x10 ⁴ concentration of cells / well to the plate, and was kept for 48 hours in a culture medium. MTT, i.e., (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium-bromide) was then added to each well at a concentration of 0.5 mg / mL.

HepG2 liver cancer cells were incubated at 37 DEG C for 4 hours, and then the supernatant was removed. Then 700 μL of DMSO was added per well to dissolve the formazan product. The absorbance was measured at 540 nm using a microplate reader (BioRad, Hercules, Calif.). The results are shown in FIG.

As can be seen from FIG. 18, the survival rate of HepG2 hepatocellular carcinoma cells was also decreased in a concentration-dependent manner by the red ginseng particles, but it can be confirmed that the red ginseng nanoparticles have a much lower cell survival rate than the red ginseng common particles.

(3) DAPI assay

To compare the anticancer effect of the red ginseng nanoparticles and the red ginseng common particles of Example 8, DAPI assays were performed to confirm the apoptosis of HepG2 liver cancer cells.

For this, HepG2 hepatoma cells were seeded on sterile cover glasses located in a four-chamber slit. After confirming that HepG2 hepatoma cells were grown to about 70% confluence, red ginseng common particles, red ginseng nanoparticles and doxorubicin were each treated at a concentration of 10 μg / ml and incubated for 24 hours.

After the incubation was completed, the cells were washed with PBS and HepG2 liver cancer cells were fixed with cold methanol for 5 minutes at room temperature. Subsequently, the cells were washed with PBS, treated with a solution of DAPI (4,6-diamidino-2-phenylindole) at a concentration of 2 mg / ml in PBS, and incubated at room temperature for 10 minutes. FIG. 19 shows a microscope photograph of a nucleus taken using a fluorescence microscope (Olympus).

As a result, as can be seen from FIG. 19, the photograph of the case treated with red ginseng nanoparticles showed more pronounced chromatin condensation phenomenon, which means that cell apoptosis is active. In particular, it was confirmed that red ginseng nanoparticles showed a more improved cell apoptosis effect than doxorubicin used as a positive control.

Claims (9)

A method for producing nanoparticles comprising the steps of:
a) dissolving one or more plant extracts in water to produce a stock solution;
b) adding the stock solution to water at a temperature of 70 ° C to 100 ° C in a dropping or spraying manner; And
c) applying ultrasonic pulverization. The method according to claim 1,
Wherein the dripping or spraying method of step b) comprises adding the stock solution at a predetermined rate. The method according to claim 1,
Wherein the temperature of water in step b) is 100 < 0 > C. The method according to claim 1,
And lyophilizing the product obtained in step c). 5. The method of claim 4,
Characterized in that the product is concentrated under reduced pressure before being lyophilized. The method according to claim 1,
A surfactant and a toxic substance are not added. A nanoparticle produced by the method of any one of claims 1 to 6. A pharmaceutical composition for treating or preventing cancer, comprising nanoparticles produced by the method of any one of claims 1 to 6. A food composition for improving or preventing cancer comprising nanoparticles prepared by the method of any one of claims 1 to 6.

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