KR20190010212A - Method for preparation of resveratrol nano-capsue and the nano-capsule therefrom - Google Patents

Abstract

The present invention relates to a technology for protecting unstable active ingredients from external factors such as light, oxygen, moisture, and temperature through encapsulation of resveratrol. The technology is for preventing oxidation of the active ingredients, improving stability and preservability, and enhancing physical properties as well as preparation processes. By utilizing the encapsulation using a drug delivery system (DDS) using nano-technologies, solubility can be improved through nano-scale particles while increasing the in-vivo absorption rate and enhancing the stable delivery to target organs for enhancing the bioavailability. A resveratrol solution is dissolved in 100% ethanol before using the chitosan and poly-glutamic acid (PGA) as base coating materials of nano-capsules in order to prepare the chitosan/PGA nano-particles. The specific method for preparing the nano-particles includes the steps of: (1) dissolving the resveratrol solution in 100% ethanol; (2) sufficiently dispersing the resveratrol solution in a chitosan solution and keep stirring the mixture at 1,000 rpm to prepare the chitosan/PGA nano-capsules containing the resveratrol; and (3) using a peristaltic pump to drop the PGA solution to the mixture of the chitosan and the resveratrol solution at the same time at a predetermined rate of 1.0 mL/min for preparing the chitosan/PGA nano-capsules containing the resveratrol. The size of the chitosan/PGA nano-capsules containing the resveratrol is 100-150 nm.

Description

METHOD FOR PREPARATION OF RESVERATOR NANO-CAPSUE AND THE NANO-CAPSULE THEREFOROM < RTI ID = 0.0 >

The present invention relates to a technique for protecting an unstable active ingredient from external factors such as light, oxygen, moisture, and temperature through encapsulation of resveratrol, which is effective for preventing oxidation of the active ingredient, improving stability and preservability, (DDS: Drug Delivary System), it is possible to improve the solubility through the small nano-sized particles, to increase the absorption into the body, and to make stable delivery to the target organ. Thereby increasing the bioavailability.

Resveratrol (3,4 ', 5-trihydroxystilbene) is one of the most remarkable molyphenols in recent years. It is a powerful antimicrobial substance secreted naturally. It has excellent antioxidant activity and various physiological and pharmacological activities such as anti-cancer, anti- Studies have been carried out to utilize them as functional materials for foods and cosmetics.

However, industrial utilization is low compared to various physiological activities for the following reasons.

Resveratrol

2) Due to structural instability, trans type is cis type when it is exposed to light, heat, oxygen etc. 3) Its metabolism is fast and its half life is short. The bioavailability is very low.

Particularly, the main extraction method is a hot water extraction method, but since such a hot water extraction method is performed at a high temperature of 110 캜, there is a problem in that the content of resveratrol in the extract is lowered due to denaturation of the extract or destruction of resveratrol.

Therefore, in order to expand the development of the resveratrol industry, it is urgently required to develop a technique for increasing the availability and bioavailability of resveratrol.

Patent Registration No. 10-14604020000 Patent Registration No. 10-12131410000

In order to solve such a problem, the present invention aims to improve this through ninocapsulation of resveratrol.

Encapsulation is a technique for protecting an unstable active ingredient from external factors such as light, oxygen, moisture, and temperature, thereby improving oxidation property, stability and preservability of the active ingredient, and improving physical properties and manufacturing process.

Through the use of nanoparticle drug delivery system (DDS), it is intended to increase solubility through small particles in the nano unit, increase absorption into the body, and enable stable delivery to target organs, thereby increasing bioavailability.

For purposes of the present invention,

Chitosan / PGA nanoparticles were prepared using water-soluble chitosan and poly-γ-glutamic acid (PGA) as basic coating materials for nanocapsules.

Resveratrol solution was prepared by dissolving in 100% organic solvent, preferably ethanol. To prepare resveratrol-containing chitosan / PGA nanocapsules, resveratrol solution was thoroughly dispersed in chitosan solution, and then the solution was dropped into chitosan and resveratrol mixed solution at a constant rate while stirring continuously to prepare resveratrol-containing nanocapsules (see FIG. 1) ).

At this time, it is preferable that the agitation speed is 1,000 rpm and the PGA mixing rate is 1.0 mL / min, and it is preferable to use peristaltic pump for PGA mixing.

Accordingly, the present invention is to develop a resveratrol material having improved solubility of resveratrol, maximizing antioxidant, anti-aging activity and bioavailability by preparing capsules of resveratrol by applying such nanotechnology.

Such resveratrol nanocapsules can be advantageously used for the production of functional foods, medicines and cosmetics using the same.

1 is a schematic diagram of preparation of resveratol-containing nano-capsule.
2 is a graph showing the particle size of the nanoparticles according to the concentration of the coating material.
FIG. 3 is a graph showing the dispersion of nanoparticles (PDI) according to the concentration of the coating material.
4 is a graph of solubility of resveratrol-containing nanodispersions and glass resveratrol in 8.3% ethanol.
5 is the cell permeability of resveratrol-containing chitosan / PGA capsules.
6 is a third-order graph of particle size according to chitosan and PGA concentration.
FIG. 7 is a tertiary graph of solubility according to the concentration of chitosan and PGA.
Figure 8 is a tertiary graph of UV stability with chitosan and PGA concentration.

In the present invention, chitosan and γ-PGA were selected as coating materials used in the nanocapsulation method to overcome the solubility and instability of resveratrol. The selection of safe coating materials for application of nanocapsules to cosmetics or foods is an important factor. Chitosan and PGA are widely used in the food and medicine field, and its safety has already been proved. It is biodegradable and non-toxic in the human body after ingestion It is known. In addition, since chitosan has a positive charge and PGA has a negative charge, it can be nano-encapsulated by using only two substances without a cross-linking agent.

In the resveratol nanocapsule of the present invention,

Resveratol is extracted from grapes and enzymes can be processed to increase the rate of resveratrol extraction.

The treatment enzymes were found to be more useful than yeast extracts. Yeast extracts were treated with 0.5% of the yeast extract. Therefore, the yeast extract (Yeast Extract) is preferably used at a concentration of 0.5%. (See Table 1)

Yeast extract treatment concentration

The content of resveratrol (㎍ / g) Distilled water 0.42 0.1% 0.47 0.5% 0. 52 One% 1,79

The resveratrol nanocapsules prepared as described above are useful for the production of functional foods, medicines and cosmetics using the same.

Resveratrol (> 99%, RES) used in the present invention was purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The grape extract was used in Dankook University (Gyeonggi, Korea). Chitosan (M.W. 1,000-3,000) and poly-γ-glutamic acid (PGA, M.W. 50 kDa) used as coating materials were purchased from Kittolife Co. (Seoul, Korea) and BioLeaders Corp. (Daejeon, Korea).

Chitosan / PGA nanoparticles were prepared by using water-soluble chitosan and poly-γ-glutamic acid (PGA) as the basic coating material of nanocapsules. Resveratrol solution was prepared by dissolving resveratrol in 100% ethanol. To prepare resveratrol-containing chitosan / PGA nanocapsules, the resveratrol solution was thoroughly dispersed in chitosan solution, and the PGA solution was continuously poured into the chitosan and resveratrol mixed solution at a constant rate (1.0 mL / min) while stirring (1,000 rpm) To prepare resveratrol-containing nanocapsules.

The size of resveratrol-containing chitosan / PGA nanocapsules was measured at 100-150 nm.

Physical properties of resveratrol-containing nanocapsules

Six manufacturing conditions were established to measure the physical properties of resveratrol - containing chitosan / PGA nanoparticles with varying chitosan and PGA concentration. Particle characteristics such as particle size and particle dispersity (PDI) of nanocapsules were analyzed under these manufacturing conditions (see FIGS. 2 and 3).

Particle properties such as particle size of nanocapsules, polydispersity index (PDI), derived count rate (DCR), and zeta potential were measured using a nano particle size analyzer (Marvern Zetasizer Nano ZS).

When the concentration ratio of chitosan to PGA was about 5: 1 to 10: 1, relatively uniform nanocapsules were formed. In other ratios, precipitates were formed or the particle size was unevenly formed. In the case of chitosan 0.5 mg / mL, PGA 0.05, 0.10 mg / mL, PGA 0.10 and 0.20 mg / mL when chitosan 1.0 mg / mL and PGA 0.20 and 0.40 mg / mL when chitosan 2.0 mg / It was confirmed that nanoparticles having excellent properties with low particle dispersion were produced.

The particle size of the nanocapsules was not significantly different by the change of chitosan concentration from 0.5 to 2.0 mg / mL, but it was significantly increased with increasing chitosan / PGA ratio. Regardless of chitosan concentration, the particle size was measured at about 500 nm when the chitosan / PGA ratio was 10, and about 150 nm nanocapsules were formed when the ratio was 5. PDI was measured to be 0.1 or less under all conditions regardless of chitosan, PGA concentration and chitosan / PGA ratio, and it was found that PDI had a very uniform particle size distribution.

Therefore, nanoparticles were prepared at a chitosan / PGA ratio of 5, which forms a relatively small particle size, and then used in the experiment.

- Solubility assessment

In order to measure the solubility change by nanocapsulation, a dispersion of nanocapsules containing resveratrol and a non-nanocapsulated resveratrol solution as a control group were prepared. To measure the amount of resveratrol dissolved in the two solutions, the concentration of resveratrol was gradually increased. The ethanol content of both solutions was kept the same at 8.3% in order to eliminate interference effect on ethanol. To measure the solubility in water

The two solutions were lyophilized under the same conditions and redispersed in water to determine their solubility in water. Each dispersion was filtered through a 0.45 μm MCE syringe filter and resveratrol was quantitatively analyzed by HPLC.

- Solubility evaluation results

In order to observe the change of solubility of resveratrol following chitosan / PGA nanoencapsulation, resveratrol solubility was measured while gradually increasing resveratrol concentration in the capsules. Nano-encapsulated glass resveratrol also measured the solubility at the same concentration. In order to eliminate the interference effect of ethanol contained in the preparation of nanocapsules, the concentration of ethanol in both nanocapsules and glass resveratrol was fixed at 8.3%.

The effects of nanocapsulation on the solubility of resveratrol were compared by quantitative analysis and visual observation. (See Fig. 4)

In the case of the unencapsulated glass resveratrol dispersion, the solubility was increased with increasing amount of resveratrol up to 80 μg / mL of resveratrol concentration, but the amount of resveratrol, which could be melted further, remained unchanged. However, nanocapsules have been shown to dissolve up to 200 μg / mL resveratrol, which is 2.5 times the solubility of non-encapsulated glass resveratrol. Therefore, it was confirmed that solubility of resolubilized resveratrol increases with nanocapsule formation. However, there was no difference in solubility between the concentrations of the coating materials.

As a result of visual observation, there was no visual difference according to the presence or absence of encapsulation up to the resveratrol concentration of 80 μg / mL. However, as the concentration of resveratrol gradually increased, resveratrol capsules remained slightly cloudy translucent without any change, In the case of the resveratrol concentration, the dispersion became cloudy and showed almost opaque property at the concentration of 200 μg / mL.

- Storage stability evaluation

To evaluate the storage stability of resveratrol-containing chitosan / PGA nanocapsules, the solubility of the resveratrol-containing chitosan / PGA nanocapsules was measured at 25 ° C for 15 days.

During the storage period, the solubility of non-nanocapsulated resveratrol in glass and resveratrol in nanocapsules was observed every 5 days. Resveratrol solubility of all samples before storage was the same. After 5 days of storage, the solubility of glass resveratrol was reduced by 30%, while the solubility of nanocapsulated resveratrol was reduced by 5-15%.

This is probably due to the increased solubility of resorbable nanocapsulated resveratrol compared to glass resveratrol due to the influence of hydrophilic chitosan and PGA.

Among the samples, CS0.5 PGA0.1 nanoparticles with the smallest particle size showed the greatest solubility at pH 3.5 and pH 5.5 during the storage period. Also, the larger the particle size, the lower the solubility during the storage period, and the particle size influenced the solubility.

- Cellular permeability evaluation

Confocal fluorescence microscopy was used to confirm the intestinal cell permeability of resveratrol.

In confocal microscope photographs, the larger the fluorescence intensity, the more resveratrol was absorbed into the cells, the fluorescence intensity of the nanoscopically unresponsive glass resveratrol was smaller than that of the nanocapsulated resveratrol. Through this, it was confirmed that the cell permeability was enhanced by nano-encapsulation, but the quantitative analysis was carried out because no visible difference was observed between the samples. (See FIG. 5).

The cell permeability of nano-encapsulated glass resveratrol was 3.5%, but the nanocapsulated resveratrol showed a cell permeability of 5-7%. The lower the concentration of the coating material, the higher the cell permeability, which is similar to the solubility. Therefore, it is considered that not only chitosan, which is known to open a tight junction, but also solubility affects cell permeation.

- Experiment result

The particle size was observed to increase as the chitosan / PGA ratio increased from 3 or higher to the chitosan / PGA ratio (see FIG. 6).

The concentration of the coating material influences the particle size and increases the chitosan concentration,

It is considered that particle size increases with decreasing PGA concentration.

Solubility tended to increase with decreasing chitosan concentration and PGA concentration at chitosan concentration 1.1-2.5 mg / mL (see FIG. 7).

It was confirmed that the solubility increases with decreasing particle size. The particle size and physical and chemical properties of the coating material are thought to have affected solubility.

UV stability was observed to increase with increasing chitosan concentration (see FIG. 8). The UV stability increased as the concentration of chitosan decreased from 0.5 to 1.5 mg / mL. However, no significant trend was observed with the concentration of chitosan in the range of 1.5-2.5 mg / mL. The lowest UV stability was obtained at the lowest concentration of chitosan and the highest concentration of PGA, and UV stability and particle size showed similar tendency. The larger the particle size, the higher the UV stability and the lower the solubility.

Claims (8)

Wherein the coating material is chitosan and? -PGA.
The method for preparing resveratrol nanocapsules according to claim 1, comprising the following steps:
Resveratrol solution is prepared by dissolving in 100% organic solvent;
Sufficiently dispersing the resveratrol solution in the chitosan solution to prepare the resveratrol-containing chitosan / PGA nanocapsule, and then continuing to stir; And
And simultaneously dropping the PGA solution into the chitosan and resveratrol mixed solution at a constant rate.
3. The method according to claim 1 or 2,
Wherein the concentration ratio of chitosan to PGA is about 5: 1 to 10: 1.
3. The method of claim 1 or 2, wherein the resveratrol
A method for preparing resveratrol nanocapsules, which comprises extracting resveratrol by adding 0.5% yeast extract to grapes.
The method for preparing resveratrol nanocapsules according to any one of claims 1 to 5, wherein the resveratrol nanocapsules have a size of 100-150 nm.
3. The method of claim 2, wherein the organic solvent is 100% ethanol.
2. The method for preparing resveratrol nanocapsules according to claim 2, wherein the stirring speed is 1000 rpm, the PGA addition rate is 1.0 mL / min, and a peristaltic pump is used when added.
A food or cosmetic composition comprising resveratrol nanocapsules prepared by the method of claims 1 or 2 as an active ingredient.

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