KR100998534B1 - Nano­particles containing water­soluble extracts from berberis koreana entrapped into gelatin, a manufacturing method thereof and a decreasing method of cytotoxicity using the same - Google Patents

Abstract

The present invention is a water-soluble barberry ( Berberis ) collected in gelatin (gelatin) The present invention relates to nanoparticles including extracts, a method for preparing the same, and a method for reducing cytotoxicity by adjusting the elution rate of water-soluble barberry extract using the same. The nanoparticles of the present invention, a method for producing the same, and a method for reducing cytotoxicity using the same, the cytotoxicity unique to the barberry when utilizing the water-soluble barberry extract as a functional material such as neuronal protection and inhibitors of obesity and diabetes In order to reduce the release rate and concentration in the body in a constant control and at the same time there is an excellent effect capable of expressing high physiological activity.

Nanoparticles, barberry, obesity, diabetes, gelatin, cytotoxicity

Description

Nanoparticles containing water-soluble barberry extract collected in gelatin, a method of manufacturing the same, and a method for reducing cytotoxicity using the same

The present invention is a water-soluble barberry ( Berberis ) collected in gelatin (gelatin) The present invention relates to nanoparticles including extracts, a method for preparing the same, and a method for reducing cytotoxicity by adjusting the elution rate of water-soluble barberry extract using the same.

Barberry is a deciduous shrub of barberry and is known to protect nerve cells and regulate body temperature. Berberine, the main component of barberry extract, activates various enzymes in the muscles of the human body, It is known to have an effect of inhibiting obesity and diabetes by improving insulin sensitivity and lowering blood sugar.

However, to date, systematic studies for applying the effect as a functional material have been insufficient, and the main reason is due to the cytotoxicity that occurs in a certain concentration of barberry extract.

Nanotechnology, on the other hand, is a technology that controls the nanometer level, and has recently been applied in various fields such as electronics and materials engineering, as well as chemical engineering, medicine, and pharmacy. The application of technology to the manufacture of pharmaceuticals is emerging as a new means of treating diseases.

The application of nanotechnology to medicines increases the duration of active ingredients in vivo, and the use of these materials is gradually increasing because nanomaterialized ingredients can be adjusted to operate at desired times and places.

However, the conventional nanoparticles are not only synthetic polymers such as polycaprolactone (PCL) and polyglycolide-co-lactide (PGLA), but also organic solvents, etc. Since there was a problem that can not be applied directly to the human body.

 The present invention has been made to solve the above problems, the object of the present invention is nanoparticles of low-utility water-soluble barberry extract due to cytotoxicity, despite high expected activity, the water-soluble barberry extract is a biological The present invention provides a nanoparticle comprising a water-soluble barberry extract capable of controlling the cytotoxicity caused by excess by being released at a constant rate and a method of preparing the same, and a method of adjusting the cytotoxicity using the same.

In order to achieve the object of the present invention as described above,

The present invention provides a nanoparticle comprising a water-soluble barberry extract collected in gelatin.

In addition, the present invention provides a method for producing the nanoparticles comprising the following steps:

 (1) extracting the crushed barberry with an aqueous solvent, followed by filtration, concentration and drying to obtain a powder, and dissolving the powder in an aqueous solvent to obtain a water-soluble barberry extract;

(2) dissolving gelatin in distilled water, followed by evaporation and drying to form liposomes; And

(3) adding the water-soluble barberry extract of step (1) to the liposome form gelatin obtained in step (2), and then dispersing and homogenizing.

In addition, the present invention provides a method for reducing cytotoxicity by controlling the elution rate of the water-soluble barberry extract using the nanoparticles.

The nanoparticles of the present invention, a method for producing the same, and a method for reducing cytotoxicity using the same provide cytotoxicity unique to barberry when the water-soluble barberry extract is used as a functional material for protecting nerve cells and inhibiting obesity and diabetes. In order to reduce the release rate and concentration in the body in a constant control and at the same time there is an excellent effect capable of expressing high physiological activity.

The present invention is characterized by providing a nanoparticle comprising a water-soluble barberry extract collected in gelatin.

In the preparation of the nanoparticles of the present invention, gelatin used to collect the water-soluble barberry extract is obtained by inducing hot water of collagen, a natural protein constituting animal skin, tendons, cartilage, etc. In one kind, it expands only in cold water, but melts in warm water to become a sol, and at a concentration of 2-3% or more, it becomes an elastic gel at room temperature. This state is called jelly, and it is widely used to form and harden by mixing it with food using its coagulation property. The gel returns to the sol when heated, and unlike collagen, it acts like trypsin or pepsin. Although gelatin is a protein, it has little or no nutritional important amino acids such as tryptophan, and its nutritional value is low. It is mainly used for photosensitive membranes, adhesives, hemostatic agents, processed foods, medicinal capsules, and microbial incubators. When the gelatin is used as a material for trapping the nanoparticles of the present invention, it is thicker than the oil phase of the nanoparticles having different film thicknesses and changes in surface charges so that the amount of elution is reduced and it is possible to adjust it constantly.

Nanoparticles of the present invention is composed of liposomes to form the nanoparticles by collecting the oil phase of the water-soluble barberry extract expected to be physiological activity, the prepared nanoparticles are formed in the range of 50 ~ 300nm, preferably 200nm or less It can be used by screening through filtration or the like. The size of the nanoparticles can be controlled in the range of 50 to 300nm through the use of gelatin and the formation of the operating conditions.

In addition, the present invention is characterized by providing a method for producing the nanoparticles comprising the following steps:

 (1) extracting the crushed barberry with an aqueous solvent, followed by filtration, concentration and drying to obtain a powder, and dissolving the powder in an aqueous solvent to obtain a water-soluble barberry extract;

(2) dissolving gelatin in distilled water, followed by evaporation and drying to form liposomes; And

(3) adding the water-soluble barberry extract of step (1) to the liposome form gelatin obtained in step (2), and then dispersing and homogenizing.

Referring to the method of nanoparticles by gelatin of the water-soluble barberry extract of the present invention in detail by process.

[Step 1: Extraction Process]

The barberry is pulverized to an appropriate size and extracted by conducting a conventional extraction including hot water extraction using water and an aqueous extracting solvent such as ethanol to elute the active ingredient, and the obtained water-soluble barberry extract is known in the art. It is powdered and quantified through conventional concentrated and lyophilized processes.

[Step 2: Measuring Cytotoxicity]

To evaluate the biotoxicity of the water-soluble barberry extract, cytotoxicity is measured by the usual cytotoxicity measurement method using the extract, and based on the result, the highest concentration at which the cytotoxicity is not prominent is determined.

[Step 3: Water Soluble Barberry  Manufacturing process of extract]

The samples obtained through the first and second steps are dissolved in an aqueous solvent at a concentration below the maximum concentration determined through the second step to prepare a water-soluble extract. Preferred water-soluble solvents are distilled water. At this time, the final concentration of the water-soluble extract is preferably to be 0.6mg / ml or less.

[Step 4: Using Gelatin Liposomes  Manufacture process]

Edible gelatin corresponding to 0.05 ~ 0.2% by weight of water-soluble barberry extract to be nanoparticles is dissolved in distilled water of 40 ℃ or more. The solution in which gelatin is dissolved is dried by evaporation of the whole distilled water under reduced pressure by a conventional method using a rotary evaporator. Preferable evaporation temperature is 20-60 degreeC. Through the above process, gelatin forms a liposome-like membrane capable of collecting substances.

[Step 5: Water Soluble Barberry  Extract Captured Liposomes  Manufacture process]

The water-soluble barberry extract is poured into the dried gelatin, hydrated by hand shaking, and in order to prevent denaturation of the water-soluble barberry extract, it is preferably a homogenizer for 30 to 40 ° C. for 50 to 60 minutes. Homogenizer and microfludizer are used to homogenize and disperse.

[Step 6: Nanoparticle Separation Process]

When the nanoparticles obtained through the process are passed through a micro filter, particles of several tens to 300 nm can be obtained. Sepadex gel G-50 or G-75 can be used to separate the material and nanoparticles.

In addition, the present invention is characterized by using the nanoparticles, by providing a method for reducing the cytotoxicity by controlling the rate of intracellular dissolution of water-soluble barberry extract.

Hereinafter, the present invention will be described in more detail with reference to Examples.

[ Example ]

1. Water Soluble Barberry  Preparation of Extract

50 g of the crushed barberry sample was taken from the dried weight, and 10 times (w / v) of distilled water was added, followed by extraction at 100 ° C. for 12 hours. Extract was obtained by filtration under reduced pressure, concentration, lyophilization.

2. Determine Optimal Concentration

In order to confirm the cytotoxicity and the optimum use concentration of barberry samples, cytotoxicity was measured in human normal cells using the powder sample of 1 above. Cells were used for experiments by adapting and culturing in RPMI 1640 medium to which 10% bovine serum medium was added using HEK293, a human kidney cell, and 3- (4,5-dimethylthiazol-2-yl)-for measuring cytotoxicity. 2,5-diphenyl tetrazoilum bromide (MTT) assay was performed according to a conventional method.

The cytotoxicity was measured by adjusting the concentration of 0.2, 0.4, 0.6, 0.8 and 1.0mg / ml, respectively, and the cytotoxicity increased with the administered concentration. It was confirmed that the cytotoxicity to the normal cells is rapidly increased (Fig. 1). Through this, the barberry extract was found to be preferably used at a concentration of 0.6mg / ml or less.

3. Preparation of Nanoparticles

When applying the barberry water-soluble extract identified in step 2 directly to the proposed use concentration can not expect a large functionality was performed nanoparticles process to enable usability and functionality in vivo.

25 mg of edible gelatin was dissolved in 10 ml of hot water at 50 ° C., and then the whole amount of hot water was evaporated and dried using a reduced pressure rotary evaporator. Through the above process, gelatin was formed into a membrane having a liposome structure. 50 ml of barberry extract of 0.5 mg / ml concentration was poured into dried gelatin and hand shaked to hydrate the lipids. The liposome solution thus prepared was placed in a water bath at 40 ° C. and dispersed by adding ultrasonic waves for 1 hour using an ultrasonic disperser.

4. Identification of Nanoparticles

In order to determine the size-specific distribution of the barberry water-soluble nanoparticles prepared by the process of step 3 using an image analyzer (analyzer), it was confirmed that more than 70% of the nanoparticles are formed to a size of 200 or less (Fig. 2).

5. Comparative measurement of emission amount of nanoparticles

In order to confirm the release rate control ability of the nanoparticles collected by the gelatin prepared by the process of step 3 was compared with the nanoparticles prepared using other capture oil phase and the amount of release over time. As a result, it was confirmed that the gelatin nanoparticles showed the smallest emission amount and showed a constant value (FIG. 3).

6. Application of Nanoparticles to Cells

In order to confirm the action mechanism in vivo of the barberry water-soluble nanoparticles prepared in 3 above, the cultured cells were treated with fluorescent particles loaded nanoparticles and observed by confocal microscopy (FIGS. 4A to 4C).

7. Cytotoxicity Measurement

To determine the product applicability of the barberry water-soluble nanoparticles prepared in step 3 was measured for toxicity at the cellular level. Cytotoxicity was performed by the same method as the cytotoxicity of the extract, and compared with other nanoparticles and compared with the nanoparticles prepared by the nanoparticles using the PCL and PGLA nanoparticles process. As a result, it was confirmed that the water-soluble barberry extract nanoparticles using the gelatin of the present invention exhibited lower cytotoxicity than other nanoparticles using conventional materials (FIG. 5).

As described above, the nanoparticles of the present invention, a method for producing the same, and a method for reducing cytotoxicity using the same may be directly applied to food and medicine without a separate process, and thus may be applied to a functional product manufacturing process using barberry.

1 is a graph showing the cytotoxicity of normal water-soluble barberry extract of the present invention.

Figure 2 is a size heat distribution of the nanoliposomes prepared through nanoparticles of the water-soluble barberry extract of the present invention.

3 is a graph showing the amount of release over time of nanoparticles prepared using gelatin, lecithin, and chitosan, respectively.

Figures 4a to 4c is a photograph of the observation mechanism of the intracellular release of the fluorescent material-loaded nanoparticles by confocal microscopy. Figure 4a before the nanoparticles injection, Figure 4b at the nanoparticle administration, Figure 4c shows 36 hours after the nanoparticle administration.

5 is a graph showing cytotoxicity in normal cells of nanoparticles prepared through a process using gelatin, PCL, and PGLA, respectively.

Claims (5)

delete delete Method for preparing nanoparticles comprising a water-soluble barberry extract collected with gelatin comprising the following steps: (1) extracting the crushed barberry with an aqueous solvent, followed by filtration, concentration and drying to obtain a powder, and dissolving the powder in an aqueous solvent to obtain an aqueous barberry extract having a concentration of 0.6 mg / ml or less; (2) dissolving the gelatin of 0.05 ~ 0.2% by weight of the water-soluble barberry extract obtained in step (1) in distilled water of 40 ℃ or more, and then evaporated and dried at 20 ~ 60 ℃ using a rotary evaporator to form liposomes ; And (3) adding the water-soluble barberry extract of step (1) to the liposome form gelatin obtained in step (2), and then homogenizing by dispersing for 50 to 60 minutes at 30 ~ 40 ℃ using an ultrasonic disperser . The method of claim 3, wherein the water-soluble solvent of step (1) is distilled water or ethanol. delete

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