KR101796370B1 - A composition for producing metal nanoparticle comprising Dendropanax Morbifera extracts and the use thereof - Google Patents
A composition for producing metal nanoparticle comprising Dendropanax Morbifera extracts and the use thereof Download PDFInfo
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- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
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Abstract
The present invention relates to a composition for preparing metal nanoparticles comprising an extract of Huangchil as an active ingredient, a method for producing metal nanoparticles using the composition, metal nanoparticles prepared by the method, and anticancer agents containing the metal nanoparticles ≪ / RTI >
The composition for the preparation of metal nanoparticles of the present invention and the method for preparing the metal nanoparticles using the same as the active ingredient of the present invention are characterized in that the preparation time is remarkably shortened and the metal nanoparticles are uniformly dispersed without addition of a reducing agent or stabilizer, Can be produced. In addition, the metal nanoparticles prepared by the above method can be utilized as anticancer agents.
Description
The present invention relates to a composition for the preparation of metal nanoparticles comprising a Huangchil extract and a use thereof, and more particularly to a composition for preparing metal nanoparticles comprising a Huangchil extract as an active ingredient, a method for producing metal nanoparticles using the composition, And the use of the metal nanoparticles for anti-cancer.
Nanotechnology is rapidly growing into an important field in modern science with potential effects in electrical engineering and biotechnology. Among them, metal nanoparticles are considered important due to their unique physico-chemical and biological particle size and shape (Colloids Surf B Biointerfaces, 107, 227-34), due to their unique electronic, magnetic, catalytic and optical properties Biomedical, and optical fields, and its importance is gradually increasing.
Among the various metals, silver has long been used for the treatment of medical diseases to treat many bacterial infections with its antibacterial properties, and silver nanoparticles in the form of nanoparticles are less cytotoxic than their ionic forms, It is known to be applied to many fields for antibacterial, anti-inflammatory or infectious prevention. Gold also has a unique and harmonious surface plasmon oscillation that can be used to detect the presence or absence of a drug, such as drug delivery, tissue or cancer remission, photo thermal therapies, immune chromatographic identification of pathogens in a sample, Vapor sensing, and the like. In addition, metal nanoparticles have biocompatibility, are easily deformed, and exhibit high cell permeability. Therefore, they are expected to be used as drug carriers (Narsireddy Amreddy et al., International Journal of Nanomedicine 2015: 10 6773? 6788, Jean-Pascal Piret et al., Nanoscale, 2012, 4, 7168-7184).
In general, chemical, physical, or biological synthesis methods are used to produce such metal nanoparticles. The chemical synthesis method is relatively simple in process, but the cost is high and the side effects of the reagent are problematic. In addition, the physical method is disadvantageous in that it is difficult to control the size of nanoparticles and requires expensive manufacturing facilities, which makes it difficult to produce effective metal nanoparticles.
For this reason, researches on a method for producing environmentally friendly metal nanoparticles containing a natural extract as an active ingredient have been extensively studied. However, the conventional method of synthesizing metal nanoparticles by bacteria or strains requires a long time for synthesis from days to days to several days. In order to stabilize the synthesized nanoparticles, a capping agent such as an additional reducing agent or a stabilizer there is a disadvantage that a capping agent is required. Therefore, development of metal nanoparticles overcoming the manufacturing time limit is still required as compared with a synthetic method which is still environmentally friendly, stable, chemical, or physical.
On the other hand, Hwangchil is a plant belonging to the genus Araliaceae. It is mainly used as a medicinal plant by using trees and leaves. Sesqui-terpene dendropanoxide is known as saponin and essential oil component. As functional ingredients, α-Cubebene, β-Elemene, β-Selinene, α-Muurolene, Germacrene D and β-Sitosterol are known. Aug; 64 (8): 547-9), liver function improvement, antioxidative action, hard tissue (bone and tooth) regeneration, immunity enhancement, nervous stability, antimicrobial action and anticancer action. However, metal nanoparticles containing Huangchil leaf extract have not yet been produced.
Under these circumstances, the inventors of the present invention have found that, in producing metal nanoparticles containing an extract of Huangchil as an active ingredient, the preparation time is remarkably shortened and the preparation can be performed stably without addition of a reducing agent or stabilizer The present inventors have completed the present invention by developing a method for producing metal nanoparticles and confirming that the composition comprising the metal nanoparticles prepared by the above method has an anticancer activity.
One object of the present invention is to provide a composition for the production of metal nanoparticles, which comprises a Huangchil extract as an active ingredient.
Another object of the present invention is to provide a method for producing metal nanoparticles, which comprises reacting the metal nanoparticle-forming composition with a metal precursor.
Still another object of the present invention is to provide the metal nanoparticles produced by the above-mentioned production method.
As one embodiment of the present invention, the present invention provides a composition for preparing metal nanoparticles, comprising a Huangchil extract as an active ingredient.
The composition for preparing metal nanoparticles of the present invention can be used for preparing metal nanoparticles having a uniform size without addition of a reducing agent or a stabilizer, because the composition contains a Huangchil extract as an active ingredient, .
The term " Dendropanax " morbifera "is a plant belonging to the family Araliaceae. Its distribution area is native to southern provinces and island regions such as Jeju Island, Wando, Bogil Island, Eocheong Island, Jindo Island, Hongdo Island, Geomundo Island and Boryeong Island. Is a pale yellow dark oily liquid with a refreshing aroma and bitter taste. Its scientific name is ' Dendropanax ' which means 'tree ginseng' morbifera ' . In the name of Hwangchil, it is said that the yellowish liquid comes out like a lacquer of lacquer when it is scratched by the yellowish bark, and it is also used as a paint. In addition, due to the efficacy of Huangchil, Huangchil's benzoin is effective for relaxation of mind and body, inhibition of various weeds, strengthening of kidneys for men and treatment of physiological impurity for women. Polyacetylene contained in Huangchil promotes the growth of immune cells, It is known that it strengthens the immune system and the bio-defense system and is known to be useful for prevention and treatment of various diseases.
In the present invention, the term "Huangchil extract" means a Huangchil component or material obtained by a conventional method using a solvent.
The Huangchil extract may be produced by a manufacturing method including, but not limited to, the following steps:
1) extracting yellowish crush with an extraction solvent;
2) cooling the extract of step 1) and filtering; And
3) Concentrating the filtered extract of step 2) under reduced pressure and drying.
In the above method, the step 1) can be used without limitation, including but not limited to cultivated or commercially available. In addition, the parts of Huangchil may include all the organs of a natural, hybrid or variant water lily, such as roots, branches, stems, leaves, flowers, fruits, and more specifically, But is not limited thereto.
In the above method, the extraction solvent may be water, C1 to C4 alcohols or a mixture thereof, as long as it is capable of producing a Huangchil extract. Specifically, ethanol, methanol or water may be used. More specifically, distilled water can be used.
In the above method, conventional methods such as filtration, hot water extraction, immersion extraction, reflux cooling extraction and ultrasonic extraction can be used as the extraction method. The hot water extraction method may be performed one to five times, and specifically, it may be repeated three times, but is not limited thereto. The extraction solvent may be added in an amount of 0.1 to 10 times, preferably 0.3 to 5 times, based on the dried yellowing, but is not limited thereto. In addition, the extraction temperature may be 20 ° C to 40 ° C, but is not limited thereto. The extraction time may be 12 to 48 hours, but is not limited thereto.
In the above method, the vacuum concentration of the step 3) may be carried out by using a vacuum decompression concentrator or a vacuum rotary evaporator. However, the vacuum distillation is not limited thereto. Specifically, the drying may be carried out under reduced pressure, vacuum, boiling, spray drying or freeze- But not limited to.
The term "metal nanoparticle" in the present invention means an ultra-fine particle having a size of 1 to 100 nm, which is made of metal, and exhibits a specific and diverse nature due to its small size. In the present invention, the metal nanoparticles may have different physiological activities depending on the production method. Specifically, the metal nanoparticles of the present invention, which are prepared using a Huangchil extract or Huangchil leaf extract, contain a physiologically active substance of each extract And can have a much better anticancer activity than metal nanoparticles prepared by other methods.
Specifically, the "metal" may be at least one selected from the group consisting of gold (Au), silver (Ag), copper (Cu), palladium (Pd), platinum (Pt), and rhodium May be gold (Au) or silver (Ag), but is not limited thereto.
In the present invention, gold nanoparticles and D-AuNP (s) are mixed and silver nanoparticles are mixed with D-AgNP (s).
In one embodiment of the present invention, the leaves of Dendropanax Morbifera were washed and dried, and then cut into pieces and heated in sterilized water to prepare extracts in which yellowish ingredients were leached into water.
In addition, the color change of the reaction mixture caused by the reduction reaction of the yellowish green leaf extract with the tetrachloro-gold (III) acid or silver nitrate solution was visually observed to confirm the synthesis of silver and gold nanoparticles (Example 3) .
Therefore, the composition of the present invention is for preparing metal nanoparticles by using Huangchil extract as an active ingredient. In particular, gold or silver nanoparticles can be prepared using Huangchil leaf extract.
According to another aspect of the present invention, there is provided a method for preparing metal nanoparticles comprising the step of reacting a metal precursor with a composition for preparing metal nanoparticles.
Since the method of the present invention uses the Huangchil extract, which is a natural extract, it is more environmentally friendly than the conventional chemical synthesis method and physical synthesis method, and the preparation time is remarkably shortened. At the same time, the metal nanoparticles are stabilized without any reducing agent or stabilizer Can be produced, and can be very usefully used in various industrial fields.
In the present invention, the term "metal precursor" means a compound added to prepare metal nanoparticles. Specifically, as the metal precursor, various kinds of compounds can be selected depending on the kind of metal of the nanoparticles to be produced.
For example, at least one selected from the group consisting of tetrachloro gold (III) acid (HAuCl 4 ), NaAuCl 3 , and AuCl 3 can be used for the preparation of gold nanoparticles, and more specifically, tetrachloro gold (III) acid (HAuCl 4 ) can be used, but it is not limited thereto.
The silver nanoparticles may be prepared by using one or more selected from the group consisting of AgBF 4 , AgCF 3 SO 3 , AgClO 4 , AgNO 3 , AgPF 6 and Ag (CF 3 COO). More specifically, Silver nitrate (AgNO 3 ) may be used, but is not limited thereto.
In addition, copper nanoparticles may be prepared from the group consisting of CuCl 2 , CuF 2 , CuBr 2 , CuI 2 , (CH 3 COO) 2 Cu, Cu (ClO 4 ) 2, Cu (NO 3 ) 2 and CuSO 4 , But is not limited thereto.
In the production process of the present invention, the concentration of the metal precursor may affect the production of metal nanoparticles. The concentration of the metal precursor of the present invention is not limited as long as it is capable of preparing metal nanoparticles using the composition for preparing metal nanoparticles. Specifically, the concentration of the metal precursor is 0.01 to 100 mM, more specifically 0.01 to 50 mM, May be 0.01 to 10 mM.
In the production process of the present invention, the reaction temperature may affect the production of metal nanoparticles. The reaction temperature of the present invention is not limited as long as it is capable of preparing metal nanoparticles with the composition for preparing metal nanoparticles, but the reaction temperature may be 10 to 100 ° C, more specifically 50 to 100 ° C.
In the production process of the present invention, the reaction time can affect the production of metal nanoparticles. The reaction time of the present invention is not limited as long as the metal nanoparticles can be prepared by using the composition for preparing metal nanoparticles. Specifically, when the metal is gold, the reaction time may be 1 minute to 1 hour, more specifically 1 minute to 50 minutes , And when the metal is silver, the reaction time may be from 30 minutes to 5 hours, more specifically from 30 minutes to 2 hours.
In a specific embodiment of the present invention, when gold or silver nanoparticles were prepared using leaf extract of Huangchil, the reaction mixture containing tetrachloro gold (III) acid completely changed to ruby red in 3 minutes ), And the reaction mixture containing silver nitrate changed to completely dark brown within 1 hour (FIG. 1A).
From the above results, it was confirmed that the silver and gold nanoparticles prepared using the leaves of Huangchil were synthesized in a very short time as compared with the metal nanoparticles prepared using other natural extracts. This suggests that the production method of the present invention can synthesize stable metal nanoparticles in an economical and environmentally friendly manner in a remarkably short time.
The method of the present invention may further include the step of recovering the metal nanoparticles by centrifuging the reaction product after the reaction is completed.
In one specific embodiment of the present invention, the composition for preparing metal nanoparticles containing Huangchil extract was added to 1 mM each of tetrachloro gold (III) acid and silver nitrate solutions, and the mixture was maintained at 80 ° C., centrifuged at 16,000 rpm for 20 minutes To prepare metal nanoparticles (Example 3).
According to still another aspect of the present invention, there is provided the metal nanoparticles produced by the above-described method.
The metal nanoparticles of the present invention can be used for medical, biological analysis, fuel, and electronic component materials. Specifically, since gold nanoparticles have inherent light conduction characteristics, they can be used for organic solar cells, sensor probes, Silver nanoparticles can be used as antibacterial agents, biosensors for quantitative detection, optical analysis and the like because of their optical properties, conductivity and antibacterial properties. In addition, the metal nanoparticles of the present invention can control the optical and electronic characteristics of the particles by changing the size, shape, chemical characteristics of the surface, or aggregation state.
The shape of the metal nanoparticles produced by the production method of the present invention may be generally spherical, but is not limited thereto.
The size of the metal nanoparticles produced by the manufacturing method of the present invention is not limited to this, but can be measured using a microscope or a device. The size of the nanoparticles generally referred to is the size measured by microscopic observation However, the measured size may be presented together with the device.
Specifically, the size of the silver nanoparticles produced by the method of the present invention may be 30 to 60 nm, more specifically, 20 to 50 nm when measured by a microscope, and 110 to 160 nm , More specifically 100 to 150 nm, but is not limited thereto.
In addition, the size of gold nanoparticles produced by the method of the present invention may be 15 to 20 nm, more specifically 5 to 10 nm when measured by a microscope and 20 to 30 nm, More specifically 10 to 20 nm, but is not limited thereto.
In one embodiment of the present invention, the size of the silver nanoparticles prepared using the Huangchil extract was measured using a TEM, and it was confirmed that the size of the silver nanoparticles was in the range of 20 to 50 nm. As a result, it was confirmed that it had a size ranging from 100 to 150 nm. Further, the size of the gold nanoparticles was measured by TEM and found to be in the range of 5 to 10 nm. As a result of measurement using the XDR apparatus, it was confirmed that the size of the gold nanoparticle was in the range of 10 to 20 nm Example 4). From the results, it can be seen that the size of the metal nanoparticles prepared using the Huangchil extract of the present invention is smaller than that of the conventional metal nanoparticles. This suggests that the metal nanoparticles of the present invention would be advantageous for intracellular absorption.
Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the metal nanoparticles.
The above "metal nanoparticles" are as described above.
The pharmaceutical composition for preventing or treating cancer according to the present invention includes metal nano-particles prepared by using Huangchil extract, specifically Huangchil leaf extract, and has excellent activity of inhibiting cancer cell proliferation, and is useful as a gene transfer agent, a biosensor, Nano carriers, and the like. However, the present invention is not limited thereto.
In addition, the pharmaceutical composition may further include a ginsenoside. Specifically, the ginsenoside may be Compound K, but is not limited thereto.
In one specific embodiment of the present invention, the silver nanoparticles of 100 / / mL inhibit the growth of A549 lung cancer cells by more than 70%, and when the silver or gold nanoparticles and the ginsenoside Compound K are simultaneously treated, Compound K (Fig. 8), which is more effective in inhibiting A549 lung cancer cell growth than in the case of single treatment. This suggests that the silver or gold nanoparticles of the present invention prepared using the Huangchil leaf extract can be used as an anticancer agent.
The term "prophylactic" of the present invention means any action that inhibits or delays cancer disease by the administration of the pharmaceutical composition.
The term "treatment" of the present invention means all the actions that improve or ameliorate the symptoms of cancer by the administration of the pharmaceutical composition.
Wherein the cancer is selected from the group consisting of lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, gastric cancer, Endometrioid cancer, thyroid cancer, pituitary cancer, adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, endometrial cancer, endometrial carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, (CNS), primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, pituitary adenoma, and the like, including, but not limited to, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cell carcinoma, But is not limited thereto.
The pharmaceutical composition may comprise a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable carrier" of the present invention may mean a carrier, excipient or diluent which does not disturb the biological activity and properties of the compound to be injected, without irritating the organism, (non-naturally occuring carrier). The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more.
The pharmaceutical composition comprising a pharmaceutically acceptable carrier may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
In particular, solid preparations 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, lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the non-aqueous solution and suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Examples of the suppository base include withexol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The pharmaceutical composition may be administered in a pharmaceutically effective amount.
The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. For example, the pharmaceutical composition may be administered such that the metal nanoparticles of the present invention are administered at a daily dose of 0.0001 to 1000 mg / kg, specifically 0.001 to 100 mg / kg.
Such administration means introducing the composition of the present invention to a patient in any appropriate manner, and the administration route of the composition can be administered through any conventional route as long as it can reach the target tissue. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal administration.
The pharmaceutical composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or two or three times. When the two active ingredients are each monoglyceride, the number of administrations may be the same or different. In addition, the composition of the present invention can be used alone or in combination with other drug therapy for the prevention or treatment of cancer. 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 term " individual " means any animal such as a mouse, a mouse, a domestic animal, or the like, including a human capable of developing or developing cancer. Specifically, it may be a mammal including a human.
The composition for the preparation of metal nanoparticles of the present invention and the method for preparing the metal nanoparticles using the same, as a natural ingredient extract, Huangchil extract is used as an effective ingredient, and the preparation time is remarkably shortened compared with the conventional method, Metal nanoparticles can be produced. In addition, since the composition comprising the metal nanoparticles produced by the above method has anticancer activity, it can be utilized as an agent for improving or treating cancer.
By 1 is observed for silver nitrate (AgNO 3) and tetrachloro-gold (III) acid changes in solution color of the inside (HAuCl 4 · 3H 2 O) and the reaction was post-experimental flasks to hwangchil leaf extract, the nanoparticles (A) Or gold nanoparticles (B). Also, it shows the generation of the silver nanoparticles (C) or gold nanoparticles (D) of the present invention using the Huangchil leaf extract through the UV-vis spectrum.
Fig. 2 is a diagram showing the structural characteristics of silver nanoparticles (AD) or gold nanoparticles (EG) of the present invention through TEM analysis.
3 shows the purity of the silver nanoparticles (AC) or gold nanoparticles (DF) of the present invention through EDAX analysis.
FIG. 4 is a view showing a diffraction pattern of silver nanoparticles (A) or gold nanoparticles (B) of the present invention through X-ray diffraction analysis.
5 is a drawing showing the result of size distribution profile based on the intensity, number and volume of silver nanoparticles (AC) or gold nanoparticles (DF) of the present invention through the operation light scattering method to be.
FIG. 6 is a graph showing the results of the cytotoxicity evaluation on human keratinocytes (HaCaT cells), showing the results of the cytotoxicity evaluation of silver nanoparticles alone of the present invention and B of the present invention alone .
FIG. 7 is a graph showing the cytotoxicity evaluation results of human lung cancer cell (A549), wherein A is the silver nanoparticle alone of the present invention and B is the result of cytotoxicity evaluation of the gold nanoparticle alone of the present invention .
FIG. 8 is a graph showing the cytotoxicity evaluation results for human lung cancer cell (A549), wherein A is a ginsenoside Compound K alone, B is a combination of silver nanoparticles of the present invention and CK, C is a gold nanoparticle Lt; / RTI > and CK.
FIG. 9 is an image showing the result of apoptosis evaluation for human lung cancer cell (A549), wherein A is silver nanoparticle alone of the present invention, B is silver nanoparticle and CK combination, C is gold nanoparticle alone, And the result of the cytotoxicity evaluation on the combination of particles and CK.
Hereinafter, the present invention will be described in more detail with reference to the following examples. These embodiments are only for illustrating the present invention, and the scope of the present invention is not construed as being limited by these embodiments.
Example 1: Material
Six-year-old leaves of Dendropanax Morbifera grown on Wando island, Republic of Korea, 34 ° 36'16 "N 126 ° 69'90" E were purchased from Serom company. Silver nitrate (AgNO 3 ) and tetrachloro gold (III) chloride trihydrate (HAuCl 4 .3H 2 O) were purchased from Sigma-Aldrich Chemicals (USA) Were purchased from Difco (MB cell, Seoul, Republic of Korea).
Example 2: Burrows Extract preparation
Singh P ( Artif Cells Nanomed Biotechnol . Dendropanax Morbifera leaf extract was prepared by the method shown in Table 1 (2015: 1-7). Healthy leaves of Huangchil were washed with deionized water, cut into small pieces and then dried. 5 g of dried leaves were placed in a 1 L beaker containing distilled water and heated for 30 minutes to extract the constituents with water. The extract was cooled at room temperature, centrifuged at 5,000 rpm for 10 minutes, filtered through Whatman filter paper (No. 2), the filtered material was removed, and a clean filtered filtrate was obtained. The total volume of the filtered liquid was made up to 100 mL and stored at 4 DEG C for the next experiment.
Example 3: Synthesis of silver and gold nanoparticles
3-1. Burrows Synthesis of Silver and Gold Nanoparticles Using Leaf Extract
For the synthesis of silver nanoparticles, 5 mL of the Yellowish Leaf Extract prepared in Example 2 was mixed with 45 mL of deionized water and added to the reaction mixture at a final concentration of 1 mM with an aqueous solution of silver nitrate (AgNO 3 ). The reaction mixture was incubated at 80 ° C for 1 hour, and then the color change of the reaction mixture was continuously observed at intervals of 10 minutes, 20 minutes, 30 minutes, 45 minutes, and 1 hour, respectively, in comparison with the control group.
For the synthesis of the gold nanoparticles, 5 mL of the yellowish leaf extract prepared in Example 2 was mixed with 45 mL of deionized water and an aqueous solution of tetrachloro gold (III) acid (HAuCl 4 .3H 2 O) was added to a final concentration of 1 mM to the reaction mixture. The reaction mixture was incubated at 80 ° C for 1 hour, and then the color change of the reaction mixture was continuously observed at intervals of 1 minute, 2 minutes, and 3 minutes, respectively, in comparison with the control group. The formed silver nanoparticles (D-AgNPs) and gold nanoparticles (D-AuNP) were centrifuged at 16,000 rpm for 20 minutes and recovered in the form of pellets, and washed several times with deionized water to remove impurities. The final product was dried and used for characterization.
3-2. Silver and gold nanoparticle synthesis results
The synthesis of the silver and gold nanoparticles prepared in Example 3-1 was observed.
As a result, as shown in Figs. 1A and 1B, the extract of Huangchu leaf extract containing AgNO 3 aqueous solution completely changed to dark brown within 1 hour at 80 ° C (Fig. 1A). Likewise, the yellowish leaf extract solution containing HAuCl 4 aqueous solution completely changed to dark ruby red within 3 minutes at 80 ° C (FIG. 1B). No change in color was observed in the control solution without AgNO 3 and HAuCl 4 .
This indicates that the silver nitrate Ag Ag + reduced to 0 by hwangchil leaf extract in the AgNO 3 aqueous solution, similarly suggest that the Au 3 + reduced into Au 0 by the hwangchil leaf extract in the HAuCl 4 aqueous solution.
Example 4: Burrows Characterization of Nanoparticles Prepared from Leaf Extracts
4-1. Analysis method
The nanoparticles prepared by using the extract of Huangchil foliage were analyzed by ultraviolet / visible spectroscopy (UV-vis), FE-transmission electron microscopy (FE-TEM) The size, shape, composition and characteristics of nanoparticles were analyzed using energy dispersive X-ray spectrometer (EDX) spectroscopy, elemental mapping, dynamic light scattering (DLS) and stability analysis.
Specifically, the silver nanoparticles and the gold nanoparticles prepared using the Huangchil leaf extract through a UV-vis spectrophotometer (Ultrospec 2100 prou) were scanned in the range of 350-800 nm and 400-800 nm, respectively. Also, element mapping (FE-transmission electron microscopy, TEM) and energy dispersive X-ray spectroscopy (EDX, JEM-2100F The shape, morphology, and dispersal of nanoparticles were analyzed by elemental mapping.
For analysis of FM-TEM, EDAX and elemental mapping, a small amount of purified nanoparticles was dropped on a carbon coated copper grid and dried in an oven at 60 DEG C to prepare a sample. The structural properties of the nanoparticles synthesized by TEM-EDX were analyzed and the distribution of the elements was analyzed by EDX and element mapping.
In addition, the size analysis profile of nanoparticles synthesized by dynamic light scattering (DLS) through a particle size analyzer (Photol, Otsuka Electronics, Japan) was analyzed. The purity of the synthesized silver nanoparticles and gold nanoparticles was measured by XRD (X-ray diffractometer D8 Advance, Bruker, Germany) at 40 ° C, 40 mA Cu · kα radiation at 6 ° / min, The step size was analyzed over a 2 [theta] range of 20-80 [deg.].
4-2. Burrows Analysis of Characteristic of Silver and Gold Nanoparticles Prepared by Leaf Extract
4-2-1. Ultraviolet / Visible Spectroscopy (UV- vis )
The synthesis of silver or gold nanoparticles prepared with Huangchil leaf extract was confirmed by UV-vis analysis.
As a result, as shown in FIGS. 1C and 1D, the silver nanoparticles and the gold nanoparticles produced through the Yellowish Leaf Extract exhibited characteristic peaks at 458 nm (FIG. 1C) and 540 nm (FIG. 1D) From this, it was confirmed that silver nanoparticles and gold nanoparticles were synthesized.
4-2-2. Field Emission scanning electron microscope (FE- 전송 transmission electron microscopy, TEM) analysis
The synthesis of silver nanoparticles or gold nanoparticles prepared using Huangchil leaf extract was confirmed by FE-TEM.
As a result, as shown in FIG. 2, the silver nanoparticles prepared through the Huangchil leaf extract exhibited a polygonal shape, and the hexagon shape and the triangle shape were smaller than those of the silver nanoparticle (Fig. 2A). It was also confirmed that the size of the particles from the high resolution lattice image for one particle represented by the scale bar in FIGS. 2B and 2C corresponded to 50 nm and 20 nm, and in FIG. 2D, It was confirmed that the crystalline characteristics of the nanoparticles are SAED patterns having a bright circular spot corresponding to (111), (200), (220), and (311).
Similarly, it was confirmed that the gold nanoparticles prepared through the Huangchil leaf extract exhibited a polygon shape and a hexagon shape with a small number (Fig. 2E). It was also confirmed that the particle size from a high resolution lattice image for one particle represented by the scale bar in FIGS. 2F and 2G corresponds to 5 nm and 10 nm, and in FIG. 2H It was confirmed that the crystal properties of the gold nanoparticles were a SAED pattern having a bright circular spot corresponding to (111), (200), (220), and (311).
4.2.3. Energy dispersive X-ray Dispersive X-ray Analysis, EDAX ) analysis
The synthesis of gold or silver nanoparticles prepared by extracts of Huangchil leaves was confirmed by EDAX analysis.
As a result, a strong signal was observed at 3 keV for silver nanoparticles and 2.2 keV for gold nanoparticles, as shown in Fig. As a result, the silver nanoparticles and gold nanoparticles were synthesized through the analysis of the characteristics of the metal nanoparticles.
4.2.4. Transmission electron microscope, TEM ) analysis
The synthesis of gold or silver nanoparticles prepared by Huangchil leaf extract was confirmed by element mapping. As a result, the distribution of the silver nanoparticles was confirmed as shown in FIG. 3C, and the distribution of the gold nanoparticles was confirmed as shown in FIG. 3F.
4.2.5. X-ray diffraction (X-ray diffraction, XRD ) analysis
Whether or not the synthesis of silver or gold nanoparticles prepared using Huangchil leaf extract and the size of the nanoparticles were confirmed by X-ray diffraction analysis.
As a result, as shown in FIG. 4, the silver nanoparticles were observed in the XRD spectrum at 38.19 °, 44.34 °, 64.48 ° corresponding to the (111), (200), (220) and (311) lattice planes The peak was confirmed at 2 θ v of 77.46 °.
The gold nanoparticles were also observed in the XRD spectrum at 2 ? V values of 38.09 °, 44.40 °, 64.56 ° and 77.60 ° corresponding to the (111), (200), (220) and (311) lattice planes I could confirm the peak. These results are similar to those of Bragges' reflection of silver and gold nanocrystals. From these results, it can be seen that silver and gold nanoparticles were produced.
Further, as a result of directly measuring the size of the metal nanoparticles of the present invention using XRD, it was confirmed that the size of silver nanoparticles was 100 to 150 nm and the size of gold nanoparticles was 10 to 20 nm.
4.2.6. dynamic Light scattering method (Dynamic Light Scattering, DLS) analysis
The size distribution profile of the gold or silver nanoparticles prepared by the extract of Huangchil Leaf according to the intensity, number and volume was confirmed by dynamic light scattering analysis. As a result, as shown in Figs. 5A to 5C, the size distribution pattern of the silver nanoparticles was confirmed, and the size distribution pattern of the gold nanoparticles was confirmed as shown in Figs. 5D to 5F.
Example 5: Anticancer efficacy of metal nanoparticles
5-1. Cell culture
Human lung adenocarcinoma (A549) cells purchased from Korean cell bank were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 IU / mL penicillin and 100 ug / mL streptomycin (Gibco-Brl. Gaithersburg , MD, USA) in a RPMI 1640 culture medium (Gen DEPOT.Inc) at 37 ° C and 5% CO 2 .
5-2. Toxicity assessment method
To evaluate toxicity of metal particles, cell viability was evaluated by MTT (3, 4, 5-dimethylthiazol-2-yl) -2-5-diphenyletrazolium bromide analysis.
Specifically, cells were inoculated into a 96-well plate (NEST®, USA) at a density of 1 × 10 5 , and after 24 hours, the cells were coated with silver nanoparticles (1, 10 and 100 μg / , 10 and 100 μg / mL), Compound K (1, 2.5, 5, 10, 25 and 50 μM),
5-4. Person keratin Toxicity to cells
In order to investigate the anticancer activity of silver or gold nanoparticles prepared from Huangchil leaf extract, cytotoxicity was evaluated using human keratinocyte Hacat cell (human keratinocyte).
As a result, as shown in FIG. 6, the silver nanoparticles inhibited cell growth at a maximum concentration of 100 占 퐂 / mL (FIG. 6A). On the other hand, gold nanoparticles did not show toxicity to the cells at all concentrations (FIG. 6B).
5-3. Human or lung cancer of silver or gold nanoparticles Toxicity to cells
In order to investigate the anticancer activity of silver or gold nanoparticles prepared from Huangchil leaf extract, cytotoxicity was evaluated using human lung cancer cell line A549.
As a result, as shown in Fig. 7, treatment of 100 ㎍ / mL silver nanoparticles for 48 hours inhibited the growth of A549 cells by more than 70% (Fig. 7A). In the case of gold nanoparticles, it was found that treatment with 100 μg / mL showed almost no toxicity to cancer cells (FIG. 7B).
5-4. In human lung cancer cells Korean silver Or synergistic effect of gold nanoparticles and CK
The synergistic effect of the combination of silver or gold nanoparticles prepared with Huangchil leaf extract and ginsenoside Compound K (CK) on A549 human lung cancer cells was examined.
As a result, as shown in FIG. 8, the ginsenoside Compound K inhibited cell growth to about 50% at a treatment concentration of 25 μM and inhibited cell growth to about 90% at a treatment concentration of 50 μM. In addition, when A549 cells were treated with a combination of silver nanoparticles and ginsenoside Compound K, a synergistic effect of further inhibiting A549 cell growth was observed. In addition, when the combination of 50 μg / mL of silver nanoparticles and 50 μM CK was combined, it was found that the growth of A549 cells was inhibited by 20% (FIG. 8B). From these results, it was confirmed that silver nanoparticles not only inhibited the growth of human lung cancer cell A549 but also enhanced the toxic efficiency of ginsenoside CK against cancer cells.
In the case of the gold nanoparticles, when the gold nanoparticles were combined with 50 μg / mL of gold nanoparticles and 50 μM CK, the growth of A549 cells was suppressed by 20% (FIG. 8C). From these results, it was confirmed that gold nanoparticles did not show toxicity to human lung cancer cells, but the toxic efficiency of ginsenoside CK against cancer cells was enhanced.
Taken together, these results indicate that A549 cells treated with metal nanoparticles and ginsenoside CK were more cytotoxic than those treated alone. In addition, although gold nanoparticles showed little cytotoxicity against A549 cells, the synergistic effect was confirmed by the combination treatment with ginsenoside CK. This suggests that metal nanoparticles enhance the permeability of A549 cell membrane and facilitate the entry of senoside CK into the cells, thereby enhancing the gastrointestinal effects of ginsenoside CK. Therefore, effective utilization of metal nanoparticles as a drug carrier for delivering drugs into cells is expected.
Example 6: For Human Lung Cancer Cells Apoptosis evaluation
The effect of silver or gold nanoparticles alone and silver or gold nanoparticles prepared with Huangchil leaf extract on apoptosis was examined through Hoechst 33258 staining.
As a result, as shown in FIG. 9, it was confirmed that when the silver nanoparticles of 100 / / mL were treated with A549 cells alone, the nuclear morphology of many cells was changed (FIG. 9A). However, in the combination treatment of 50 μg / mL silver nanoparticles and 25 μM CK, apoptosis was more induced than silver nanoparticle alone treatment (FIG. 9B). In addition, in the case of gold nanoparticles, almost no apoptosis was induced in the treatment of 100 / / mL (FIG. 9C). In the combination treatment of gold nanoparticles of 50 / / mL and 25 μM CK, The nucleus was shrunken or fragmented (Figure 9D).
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments and experiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
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| KR102031569B1 (en) * | 2018-12-14 | 2019-10-14 | 학교법인 한문화학원 | Pharmaceutical compositions for preventing or treating cancers comprising the nanoparticles of Dendropanax morbifera |
| KR102553572B1 (en) * | 2021-09-24 | 2023-07-07 | 강원대학교산학협력단 | Silver nanoparticles using sedge flower extract and method for manufacturing the same |
| KR20240011895A (en) | 2022-07-18 | 2024-01-29 | 농업회사법인 고려홍삼원 주식회사 | Gynostemma pentaphyllum extract-metal nanoparticle composite, method of manufacturing the same and use thereof |
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| KR100883016B1 (en) | 2007-05-22 | 2009-02-12 | 충북대학교 산학협력단 | Method for preparing metal nanoparticles |
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