CN103040754A - Resveratrol nano-liposome and preparation method thereof - Google Patents

Abstract

The invention discloses a resveratrol nano liposome and a preparation method thereof. The method comprises the steps of: mixing and dissolving resveratrol, film materials, antioxidants and organic solvents, removing the organic solvents while forming a film, adding a buffer for hydration, and obtaining crude resveratrol liposomes Suspension, the resulting coarse suspension is subjected to high-pressure micro-fluidization treatment to obtain the resveratrol nano-liposome. The invention adopts a high-pressure micro-jet method to prepare the resveratrol nano-liposome through the steps of dissolution, lipid formation, sufficient hydration, and micro-jet dispersion. The resveratrol nano liposome is uniform, small in particle size, high in encapsulation efficiency and good in stability.

Description

Resveratrol nano liposome and preparation method thereof

technical field

The invention belongs to the technical field of functional health food, and relates to a resveratrol nano liposome and a preparation method thereof.

Background technique

Resveratrol, also known as stilbenol, is a phytoalexin produced by plants to resist external stimuli such as ultraviolet rays, fungi, virus infection or mechanical damage. Hormone, nerve and liver protection, anti-radiation, cough and asthma, lower blood pressure, improve microcirculation, treat AIDS and shock and other physiological functions, it is listed as "100 most popular and effective anti-aging drugs" by the "Anti-aging Bible" of the United States. aging substances". However, due to the unstable chemical properties of resveratrol, it is easy to decompose when exposed to light and heat, and its water solubility is poor. Therefore, it has disadvantages such as poor oral absorption, low bioavailability, and difficulty in lasting effect, which limits its application to a certain extent.

As a new type of drug carrier, nanoliposome is a kind of bilayer vesicle with a structure similar to a biological membrane, which is made by encapsulating the target substance with lecithin, cholesterol, etc., and has good stability, The characteristics of strong targeting, delayed release, and no immunotoxicity have become the focus of modern medicine and food functional chemistry in recent years. Therefore, the use of nano-liposomes for the encapsulation and delivery of resveratrol is conducive to improving its bioavailability and sustained release, and promoting its permeation and absorption, thereby further expanding its application in food, medicine, cosmetics and other industries. scope of application.

At present, the preparation methods of nanoliposomes mainly include film dispersion method, reverse evaporation method, ethanol injection method, ultrasonic method, high-pressure homogenization method, etc., but there are organic reagent residues, easy oxidation of phospholipid components, high production cost and long cycle , the preparation process is difficult to control and other shortcomings, and it is difficult to scale up to industrial production.

Contents of the invention

The object of the present invention is to provide a kind of resveratrol nano liposome and preparation method thereof.

The method for preparing resveratrol nanoliposomes provided by the invention comprises the following steps: mixing and dissolving resveratrol, membrane materials, antioxidants and organic solvents, removing the organic solvents while forming a film, and then adding a buffer The hydration is carried out to obtain a thick suspension of the resveratrol liposome, and the obtained thick suspension is subjected to high-pressure micro-jet treatment to obtain the resveratrol nano-liposome.

In the above method, the method of mixing and dissolving is ultrasonic; the method of removing the organic solvent while forming a film is rotary evaporation under reduced pressure.

The material constituting the film material is selected from at least one of soybean lecithin, cephalin, sphingomyelin, phosphatidylethanolamine, cholesterol, sodium taurocholate, β-sitosterol and cholesterol acetyl ester, preferably soybean lecithin and at least one type of cholesterol;

The antioxidant is selected from at least one of vitamin E, butylated hydroxyanisole, dibutyl hydroxytoluene and dibutyl hydroxytoluene, preferably vitamin E;

The organic solvent is at least one selected from absolute ethanol, methanol, chloroform, diethyl ether and petroleum ether, preferably ethanol.

The dosage ratio of the resveratrol, film material, antioxidant, organic solvent and buffer is 0.05~1g: 1~2g: 0.04~0.5g: 200~500mL: 150~350mL, preferably 0.1g: 1.1g: 0.1g: 500mL: 250mL, specifically 0.05-0.1g: 1.1-1.5g: 0.04-0.1g: 200-500mL: 150-250mL or 0.1-1g: 1.0-1.1g: 0.1-0.5g: 500mL: 250- 350mL or 0.05-1.0g: 1.0-1.5g: 0.04-0.5g: 200-500mL: 150-350mL or 0.05g: 1.5g: 0.04g: 200mL: 150mL or 1g: 1.0g: 0.5g: 500mL: 350mL;

The buffer is a phosphate buffer with a pH value of 7.4, and the concentration is 0.002-0.02mol/L, preferably 0.01mol/L, specifically 0.002-0.01mol/L.

In the ultrasonic step, the ultrasonic frequency is 28-100Hz, preferably 45Hz, specifically 45-100HZ or 28-45HZ, and the power is 120-300W, preferably 240W, specifically 120-240W or 240-300W; the time is 3-5min , specifically 3-4min or 4-5min, the temperature is 40-60°C, preferably 45°C, specifically 40-45°C or 45-60°C;

In the decompression rotary evaporation step, the temperature is 40~60°C, preferably 45°C, specifically 40-45°C or 45-60°C, and the vacuum degree is -0.09~-0.1MPa, specifically -0.09 or -0.01 or -0.08 or -0.08 to -0.01 or -0.09 to -0.01 or -0.09 to -0.08MPa;

In the hydration step, the temperature is 40-60°C, preferably 45°C, specifically 40-45°C or 45-60°C, and the time is 20-45min, preferably 30min, specifically 20-30min or 30-45min;

In the high-pressure micro-jet treatment step, the treatment pressure is 10000-30000PSI, preferably 18000PSI, specifically 10000-18000PSI or 18000-30000PSI, and the number of cycles is 1-6 times, preferably 3 times, specifically 1-3 times or 3- 6 times;

In order to wash the lipid film obtained by vacuum rotary evaporation into the hydration medium, so as to facilitate the complete washing of the film, and avoid part of the lipid film from adhering to the eggplant-shaped bottle wall, which is difficult to wash off, the above-mentioned preparation of resveratrol nano-lipid The method for the body may further comprise: adding glass beads to the buffer solution after the dissolving step and before the hydration step.

The resveratrol nanoliposome prepared by the above method also belongs to the scope of protection provided by the present invention. The particle diameter of the liposome is 24.53~219.2nm, the Zeta potential is -24.6~-65mV, and the encapsulation efficiency is 34.40%~89.30%, specifically 75.27%, 88.27%, 88.68%, 75.27%-88.68%, 75.27%-88.27% or 88.27%-88.68%;

The pH value is 7.33~7.47, and the inhibition rates to human liver cancer cells HEPG-2 and human gastric cancer cells AGS are 31.58%~76.85% and 11.49%~58.37%, respectively.

The characteristics of the resveratrol nanoliposome provided under the preferred conditions of the present invention are: the appearance is a uniform milky white suspension, and the electron microscope is round or oval microspheres, the particles are dispersed and independent, and the particle size is (76.09±1.38) nm, Zeta potential (-53.90±2.26) mV, encapsulation efficiency 88.27%, pH value (7.35±0.035), and the inhibition rates of human liver cancer cells HEPG-2 and human gastric cancer cells AGS were 76.85% and 58.37%, respectively .

In addition, the application of the above-mentioned resveratrol nanoliposomes provided by the present invention in the preparation of products for inhibiting tumor cells also belongs to the protection scope of the present invention. Wherein, the tumor cells are specifically liver cancer cells or gastric cancer cells; the liver cancer cells are specifically human liver cancer cells HEPG-2; and the gastric cancer cells are specifically human gastric cancer cells AGS.

The present invention adopts the high-pressure micro-jet method to prepare the resveratrol nano-liposome through steps such as dissolving, lipid formation, sufficient hydration, and micro-jet dispersion, and has the following beneficial effects:

1. The preparation method is simple and convenient to operate, easy to control, clean and safe, has no toxic organic solvent residue, and can be produced continuously.

2. The prepared nano-liposome has uniform shape, small particle size, high encapsulation efficiency and good stability, which realizes the carrying and sustained release of resveratrol, changes its solubility, and improves its bioavailability.

3. The liposome provided by the present invention contains antioxidants, which avoids the toxic and side effects caused by the oxidation of drugs in vivo and in vitro, and reduces adverse reactions.

Description of drawings

Fig. 1 is the transmission electron micrograph of resveratrol nano liposome.

Figure 2 is a particle size distribution diagram of resveratrol nanoliposomes.

Fig. 3 is the Zeta potential diagram of resveratrol nano liposome.

Fig. 4 is the spectrogram of resveratrol determined by high performance liquid chromatography (HPLC);

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The raw materials can be obtained from open commercial channels unless otherwise specified.

Embodiment 1, preparation of resveratrol nano liposome

Accurately weigh 0.1g of resveratrol, 1g of soybean lecithin, 0.1g of cholesterol and 0.1g of antioxidant VE, add 500mL of absolute ethanol, and ultrasonicate for 3 minutes in a 45°C water bath under the conditions of 45HZ and 240W until fully dissolved. Ethanol was removed by rotary evaporation under reduced pressure (vacuum degree -0.09MPa) until a uniform lipid film was formed on the wall of the eggplant-shaped bottle. Add 250 mL of 0.01 mol/L phosphate buffer (pH=7.4) and an appropriate amount of glass beads, and rotate and hydrate for 30 min in a water bath at 45°C to obtain a resveratrol liposome suspension. The resveratrol crude suspension is dispersed in a high-pressure microfluidic instrument, and circulated three times under the condition of 18000 PSI to obtain the resveratrol nanoliposome.

Embodiment 2, preparation of resveratrol nano liposome

Accurately weigh 0.05g resveratrol, 1g egg yolk lecithin, 0.5g sodium taurocholate and 0.04g antioxidant butylated hydroxyanisole, add 200mL of anhydrous methanol, and ultrasonicate for 5min in a water bath at 40°C under the conditions of 28HZ and 120W. Fully dissolve, remove ethanol by rotary evaporation under reduced pressure (vacuum degree -0.01MPa) in a water bath at 40°C until a uniform lipid film is formed on the wall of the eggplant-shaped bottle. Add 150 mL of 0.002 mol/L phosphate buffer (pH=7.4) and an appropriate amount of glass beads, and rotate and hydrate for 45 min in a water bath at 40°C to obtain a resveratrol liposome suspension. The resveratrol crude suspension is dispersed in a high-pressure micro-fluidizer, and circulated 6 times under the condition of 10,000 PSI to obtain the resveratrol nanoliposome.

Embodiment 3, preparation of resveratrol nano liposome

Accurately weigh 1g of resveratrol, 1.8g of cephalin, 0.2g of β-sitosterol and 0.5g of antioxidant dibutyl hydroxytoluene, add 500mL of petroleum ether, and ultrasonicate for 4 minutes in a water bath at 60°C under the conditions of 100HZ and 300W until fully dissolved Ethanol was removed by rotary evaporation under reduced pressure (vacuum degree -0.08MPa) in a water bath at 60°C until a uniform lipid film was formed on the wall of the eggplant-shaped bottle. Add 350 mL of 0.002 mol/L phosphate buffer (pH=7.4) and appropriate amount of glass beads, rotate and hydrate in a water bath at 60°C for 20 min to obtain a resveratrol liposome suspension. The resveratrol crude suspension is dispersed in a high-pressure microfluidic instrument, and circulated once under the condition of 30,000 PSI to obtain the resveratrol nanoliposome.

Embodiment 4, the quality evaluation of resveratrol nano liposome

Morphological observation: Dilute the resveratrol nanoliposomes prepared in Example 1 to a lower concentration with 0.01mol/L phosphate buffer (pH=7.4), drop them on a copper grid, and use filter paper to absorb excess liquid. After staining with uranyl acetate for 3 minutes, the morphology of the product was observed under a transmission electron microscope after staining three times. As shown in Figure 1, it was round or oval microspheres with dispersed and independent particles, and the particle size was <100nm.

Particle size and Zeta potential measurement: Take an appropriate amount of resveratrol nanoliposomes, filter through a 0.45um filter membrane, and use a Malvern Zeta potential meter to measure the particle size and Zeta potential. The measured particle size is (76.09±1.38) nm, Zeta potential (-53.90±2.26) mV, the particle size distribution diagram is shown in Figure 2, and the Zeta potential diagram is shown in Figure 3.

The pH value measurement result was 7.35±0.035.

Determination of Encapsulation Efficiency EE (%):

(1) HPLC method to establish the standard curve of resveratrol:

The chromatographic conditions are measuring instruments: Waters1525 type high performance liquid chromatography (equipped with ultraviolet and differential detectors); chromatographic column: C ₁₈ column, 150mm×4.6mm, 5um; mobile phase: acetonitrile/water/glacial acetic acid=25:75 : 0.09 (V/V), flow rate 0.7mL/min, detection wavelength 306nm, injection volume 10uL, column temperature 30°C.

Drawing of standard curve: Accurately weigh 12.5 mg (accurate to 0.0001 g) of resveratrol standard substance, dissolve it in methanol and dilute to 250 mL to obtain 50 mg/L resveratrol standard stock solution. Accurately pipette 1, 2, 4, 6, 8, 10mL of resveratrol standard stock solution, dilute with methanol and set the volume to 50mL to obtain a series of standard working solutions, with the peak area as the ordinate and the concentration as the abscissa , draw the standard curve, and get the regression equation:

y=1.02×10 ⁵ x+0.904×10 ⁵ (r ² =0.9998)

In the formula: y is the peak area, x is the concentration of resveratrol (ug/mL).

(2) Determination of encapsulation efficiency EE (%): Take an appropriate amount of resveratrol nanoliposomes, and measure its mass concentration C ₁ by HPLC. Take 5 mL of nanoliposomes in a dialysis bag with a molecular cut-off of 8000-14000 D, dialyze in 200 mL of 0.01 mol/L phosphate buffer (pH=7.4) for 24 hours, and change the dialysate every 2 hours. The dialyzed nanoliposomes were taken out, and their mass concentration C ₂ was determined by HPLC. Calculate its encapsulation rate EE (%)=C ₂ /C ₁ ×100%.

As shown in Figure 4, the peak area of resveratrol measured by HPLC before dialysis was 460868AU·min, and the peak area of resveratrol after dialysis was 407868AU·min, and the concentrations C ₁ and C ₂ were calculated by substituting into the standard curve , according to EE (%)=C ₂ /C ₁ ×100%, the encapsulation efficiency is 88.27%.

According to the same method as above, the encapsulation efficiencies of the resveratrol liposomes obtained in Examples 2 and 3 were measured to be 88.68% and 75.27%, respectively.

In vitro antitumor activity assay:

Well-growing tumor cells, human liver cancer cells HEPG-2 (purchased from Shanghai Fusheng Reagent Co., Ltd. AGS cells), and human gastric cancer cells AGS (purchased from Shanghai Ruiqi Biotechnology Co., Ltd.) were collected, and RPMI-2 containing 10% fetal bovine serum was collected. 1640 or DMEM medium to make 6×10 ⁴ /ml cell suspension, inoculated in 96-well plate, 100 μl per well, cultured in 37°C, 5% CO ₂ incubator for 24 hours, then added the drug solution to be tested (final drug concentration 6.25, 12.5, 25, 50, 100 μg/ml), set 3 parallel wells for each concentration, and set a blank control at the same time. After culturing for 48 hours, discard the supernatant, add 10 μl of MTT solution (prepared in 5 mg/ml RPMI-1640 medium) to each well and continue culturing for 4 hours, add 200 μl DMSO to each well, shake well, and use a Bio-Tek MQX200 microplate reader at a detection wavelength of 570 nm, The absorbance (A) value was measured at a reference wavelength of 450nm, and the inhibition rate was calculated: (A _{blank control} -A _sample )/A _{blank control} ×100.

The half-inhibitory concentration (IC ₅₀ ) of resveratrol liposomes on human liver cancer cells HEPG-2 was determined to be 52.183ug/mL, and the inhibition rate reached 76.85% under the condition of 100ug/mL concentration; The half-inhibitory concentration (IC ₅₀ ) was 46.809ug/mL, and the inhibition rate reached 58.37% under the condition of 50ug/mL concentration.

Claims (10)

1. A method for preparing resveratrol nano-liposomes, comprising the steps of: resveratrol, film material, antioxidant and organic solvent are mixed and dissolved, film forming removes the organic solvent while adding buffer The hydration is carried out to obtain a thick suspension of the resveratrol liposome, and the obtained thick suspension is subjected to high-pressure micro-jet treatment to obtain the resveratrol nano-liposome. 2. The method according to claim 1, characterized in that: the method of mixing and dissolving is ultrasonic; the method of removing the organic solvent while forming a film is rotary evaporation under reduced pressure. 3. according to the described method of claim 1 and 2, it is characterized in that: the material that constitutes described membrane material is selected from soybean lecithin, cephalin, sphingomyelin, phosphatidylethanolamine, cholesterol, sodium taurocholate, β- At least one of sitosterol and cholesterol acetyl ester, preferably at least one of soybean lecithin and cholesterol; The antioxidant is selected from at least one of vitamin E, butylated hydroxyanisole, dibutyl hydroxytoluene and dibutyl hydroxytoluene, preferably vitamin E; The organic solvent is at least one selected from absolute ethanol, methanol, chloroform, diethyl ether and petroleum ether, preferably ethanol. 4. The method according to any one of claims 1-3, characterized in that: the dosage ratio of the resveratrol, film material, antioxidant organic solvent and buffer solution is 0.05~1g: 1~2g: 0.04~ 0.5g: 200~500mL: 150~350mL, preferably 0.1g: 1.1g: 0.1g: 500mL: 250mL; The buffer is a phosphate buffer with a pH value of 7.4, and the concentration is 0.002-0.02mol/L, preferably 0.01mol/L. 5. The method according to any one of claims 1-4, characterized in that: in the ultrasonic step, the ultrasonic frequency is 28~100Hz, preferably 45Hz, the power is 120~300W, preferably 240W; the time is 3~5min, The temperature is 40~60°C, preferably 45°C; In the decompression rotary evaporation step, the temperature is 40~60°C, preferably 45°C, and the vacuum degree is -0.09~-0.1MPa, preferably -0.1MPa; In the hydration step, the temperature is 40-60°C, preferably 45°C, and the time is 20-45min, preferably 30min; In the high-pressure micro-fluidic treatment step, the treatment pressure is 10000-30000 PSI, preferably 18000 PSI, and the number of cycles is 1-6 times, preferably 3 times. 6. The method according to any one of claims 1-5, characterized in that: the method further comprises: adding glass beads to the buffer solution after the dissolving step and before the hydration step. 7. the resveratrol nanoliposome prepared by the arbitrary described method of claim 1-6. 8. The liposome according to claim 7, characterized in that: the particle diameter of the liposome is 24.53 ~ 219.2nm, preferably 76.09 ± 1.38nm; Zeta potential is -24.6~-65mV, preferably -53.90±2.26mV; The encapsulation rate is 34.40%~89.30%, preferably 88.27%; The pH value is 7.33~7.47, preferably 7.35±0.035; The inhibitory rates to human liver cancer cells HEPG-2 and human gastric cancer cells AGS are 31.58%-76.85% and 11.49%-58.37%, preferably 76.85% and 58.37%, respectively. 9. The application of the resveratrol nanoliposome according to claim 7 or 8 in the preparation of products for inhibiting tumor cells. 10. The application according to claim 9, characterized in that: the tumor cells are specifically liver cancer cells or gastric cancer cells; The liver cancer cells are specifically human liver cancer cells HEPG-2; The gastric cancer cells are specifically human gastric cancer cells AGS.

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