Method for preparation of liposomal glutathione derived from sunflower lecithin extract

Abstract

The present invention provides a method for preparing glutathione-encapsulated liposomes, the method comprising the steps of: (a) mixing 10 wt% of L-glutathione powder, 5 wt% of anhydrous citric acid, and 10 wt% of an ethyl alcohol solvent extract of sunflower lecithin in purified water; (b) homogenizing the mixture at 65°C and 6,500 rpm for 20 minutes to form an emulsion; (c) stirring the emulsion at 65°C and 60 rpm for 90 minutes to perform liposome rolling; and (d) cooling and freeze-drying the stirred emulsion that has undergone liposome rolling. The liposomes can be effectively used as a glutathione-containing health functional food.

Description

Method for producing liposomal glutathione derived from sunflower lecithin extract

[0001] The present invention relates to a method for producing liposomal glutathione derived from sunflower lecithin extract, and can be utilized in the field of health functional foods, etc.

[0002] The present invention relates to research conducted by Korea Liposome Co., Ltd. with the support of the Korea Bio-Specialized Center Council (Project No.: B0080207002074, Project No.: KBC-001, Research Project Name: Bioactive Agent Global Competitiveness Enhancement Project, Research Period: 2024.03.08 ~ 10.31) and with the Korea Industrial Technology Industry Promotion Agency (KIAT) of the Ministry of Trade, Industry and Energy as the project management specialized organization.

[0003] Glutathione (GSH), a type of amino acid polymer, is a representative antioxidant. It removes and safely excretes reactive oxygen species generated during metabolic processes. While all cells synthesize glutathione, the liver is the primary source. It can directly bind to reactive oxygen species or act as a catalyst, converting them into a safe form.

[0004] Animal studies have shown that oral glutathione is bioavailable and effective in increasing blood and tissue glutathione levels, potentially protecting against aging and immune dysfunction. Recent clinical trials have demonstrated that daily oral glutathione supplementation effectively enhances glutathione levels in oral buccal cells and various intracellular and extracellular blood compartments.

[0005] Liposomes have been used as effective drug delivery vehicles, enabling more efficient absorption and delivery of both hydrophilic and lipophilic substances, while also enhancing protection against oxidation and degradation. Because glutathione is destroyed in the acidic environment of the stomach, oral liposomal glutathione can be an effective means of delivering glutathione in vivo. Liposome-based drug delivery systems are widely utilized in the health functional food industry due to their high utility. They are expected to significantly contribute to reducing social and economic management costs, particularly by improving the quality of life of the elderly.

[0006] The purpose of the present invention is to provide a method for producing liposomal glutathione derived from sunflower lecithin extract.

[0007] To solve the above problem, the present invention provides a method for preparing liposomes encapsulating L-glutathione, comprising the steps of: (a) mixing 10 wt% of L-glutathione powder, 5 wt% of anhydrous citric acid, and 10 wt% of an ethyl alcohol solvent extract of sunflower lecithin in purified water; (b) homogenizing the mixture at 65°C for 20 minutes and 6,500 rpm to form an emulsion; (c) stirring the emulsion at 65°C for 90 minutes and 60 rpm to perform liposome rolling; and (d) cooling and freeze-drying the stirred mass of liposome rolling.

[0008] In the above manufacturing method, the ethanol solvent extract of sunflower lecithin may be a primary, secondary or tertiary lecithin extract.

[0009] In one embodiment, the ethanol solvent extract of sunflower lecithin is a primary lecithin extract and is prepared through the steps of (a-1) mixing sunflower lecithin and ethyl alcohol in a weight ratio of 3:10; soaking the mixture for 72 hours; performing reduced pressure filtration and concentration on the soaked material; and drying the concentrate at 60° C. for 48 to 72 hours to obtain a primary sunflower lecithin extract.

[0010] In one embodiment, the ethanol solvent extract of sunflower lecithin is a secondary lecithin extract, and is prepared through the steps of (a-2) mixing and steeping a sunflower lecithin primary extract and ethyl alcohol in a weight ratio of 3:10; filtering and concentrating the steeped substance under reduced pressure; and drying the concentrate to obtain a sunflower lecithin secondary extract.

[0011] In one embodiment, the ethanol solvent extract of sunflower lecithin is a tertiary lecithin extract, and is prepared by the steps of (a-3) mixing and soaking a sunflower lecithin secondary extract and ethyl alcohol in a weight ratio of 3:10; filtering and concentrating the soaked product under reduced pressure; and drying the concentrate to obtain a sunflower lecithin tertiary extract.

[0012] In the method for preparing glutathione-entrapping liposomes of the present invention, a tertiary lecithin extract may preferably be used to increase the phosphatidyl content of the liposome components and improve stable and efficient entrapment of glutathione.

[0013] Additionally, in the manufacturing method of the present invention, homogenization may be performed using a homogenizer (Homo Emulsifier Disperser; HED) to form a liposome emulsion suitable for liposome rolling.

[0014] Additionally, in the manufacturing method of the present invention, the stirring may be performed in a stirrer composed of impellers having an internal gap of 5 to 100 mm for liposome rolling, and may be performed in a stirrer composed of impellers having an internal gap of about 20 mm, for example.

[0015] The present invention also provides L-glutathione-entrapped liposomes manufactured according to the above-described manufacturing method and health functional foods containing the same as a functional raw material.

[0016] The manufacturing method of the present invention utilizes a lecithin extract with improved phospholipid content to efficiently and stably encapsulate L-glutathione into liposomes, thereby improving the functional effects of glutathione, including its antioxidant properties. Therefore, the present invention can be usefully applied in the field of health functional foods utilizing liposomal glutathione.

[0017] Figure 1 is a flow chart for the process of manufacturing the lecithin extract used in the present invention.

[0018] Figure 2a is a photograph of the shape of a lecithin extract that was not concentrated in ethyl alcohol (Figure 2a), Figure 2b is a photograph of a lecithin extract that was first concentrated (Figure 2b), Figure 2c is a photograph of a lecithin extract that was second concentrated, and Figure 2d is a photograph of a lecithin extract that was third concentrated.

[0019] Figure 3a is an electron microscope photograph of glutathione-entrapping liposomes prepared using a primary lecithin extract, Figure 3b is a secondary lecithin extract, and Figure 3c is a tertiary lecithin extract.

[0020] Figure 4a is an electron microscope photograph of the morphology of glutathione-entrapping liposomes prepared using a primary lecithin extract, Figure 4b is a secondary lecithin extract, and Figure 4c is a tertiary lecithin extract after sterilization.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0022] In the present invention, glutathione (GSH), the active ingredient to be captured by liposomes, is a compound represented by the following chemical formula 1 and is the most abundant non-protein thiol in cells. More specifically, the glutathione of the present invention is L-glutathione.

[0023] [Chemical Formula 1]

[0024]

[0025] Glutathione has a variety of important functions, including drug detoxification, protection of macromolecules from oxidative damage, and maintenance of immune function. It is synthesized in the body from cysteine ​​(Cys), glutamic acid (Glu), and glycine (Gly), with cysteine ​​(Cys) being the rate-limiting substrate. Consequently, glutathione levels can be depleted when cysteine ​​(Cys) levels are limited, such as during periods of fasting. Glutathione depletion has numerous detrimental effects, including impaired immune function and increased susceptibility to xenobiotics and oxidants. Maintaining optimal tissue glutathione levels is considered crucial for maintaining health, and low glutathione levels are associated with an increased risk of diseases including cancer, cardiovascular disease, arthritis, and diabetes. Glutathione fortification represents a potentially important approach for the treatment and prevention of disorders associated with glutathione depletion.

[0026] In the present invention, liposome refers to a single-layer or multi-layer lipid-bilayer structure that is most similar to a cell membrane, which has a matrix form of a phospholipid bilayer. Liposomes can be molecular groups that form a bilayer closed membrane composed of phospholipids such as phosphatidylcholine (PC), ethanolamine (PE), serine, sphingomyelin, cardiolipin, plasmogen, phosphatidylic acid, and cerebroside, and are in equilibrium with water. Phospholipids, the basic unit constituting liposomes, are composed of an anionic or polar head portion and two non-polar hydrocarbon chains. The hydrocarbon chains vary in length, and in the case of natural phospholipids, the hydrocarbon chains are 16 or more in length and have an unsaturation degree of about one pair. Liposomes are natural or synthetic phospholipids that participate in metabolism, are interchangeable with biological membrane molecules, are biodegradable, have little toxicity to the body, and can entrap active substances without chemical binding.

[0027] In the present invention, sunflower lecithin, which is rich in phosphatidylcholine (PC), a key component of liposome formation, was used as a liposome component, lecithin. Sunflower lecithin is a natural lecithin and is emerging as a substitute for soy lecithin, which has been used in food and beverages. Sunflower lecithin has a higher phosphatidylcholine content than soy lecithin, making it suitable for manufacturing liposomes. In addition, since soy lecithin also raises the issue of GMO (Genetically Modified Organism), using sunflower lecithin eliminates concerns about GMO. In addition, sunflower lecithin has other advantages, such as a simpler extraction process than soy lecithin, easier use in food manufacturing because it melts at a lower temperature than soy lecithin, and natural freedom from gluten, soy, and dairy products.

[0028] In the liposome manufacturing method of the present invention, a lecithin raw material optimized for improving the stable entrapment rate of L-glutathione was provided by subjecting sunflower lecithin to an ethyl alcohol extraction process. For example, while the content of phosphatidylcholine in sunflower lecithin, which is generally used as a food raw material, is 18 to 22%, the lecithin extract used in the liposome manufacturing of the present invention is characterized by an increased content of phosphatidylcholine among the phospholipids used as liposome components, thereby increasing the entrapment rate of glutathione within the liposome, and various effects of L-glutathione, such as antioxidant effects, can be expected.

[0029] The anhydrous citric acid used as a surfactant in the liposome manufacturing process of the present invention is a weak organic acid also called anhydrous citric acid, which acts as a surfactant and pH regulator in the liposome manufacturing process of the present invention to aid liposome formation and contribute to the stability of the liposome. Liposome stability is closely related to the stability of the active ingredient, and liposomes are known to be most stable when the pH is 5.50 to 7.0. Citric acid maintains the pH of the solution to be slightly acidic during the liposome manufacturing process, and additionally has the effects of energy metabolism, nutrient absorption, and kidney stone prevention (literature [Andre O. (2001) Handbook of Cosmetic Science and Technology. Marcel Dekker, Inc., pp205], etc.).

[0030] In the mixing process of the present invention, anhydrous citric acid also plays a role in helping to form liposomes well by the affinity between the hydrophilic head and hydrophobic tail of the phospholipid after the lecithin is dispersed in the aqueous solution in the manufacturing process of the present invention. In the mixing process, other components besides anhydrous citric acid may be used as surfactants. However, for the purpose of the mass-production liposome manufacturing method of the present invention, no surfactant components other than anhydrous citric acid may be used.

[0031] The method for manufacturing L-glutathione-entrapped liposomes according to the present invention forms an emulsion by sufficiently homogenizing or dispersing liposomes using anhydrous citric acid as a surfactant without using an organic solvent or ultrasonic treatment from raw materials (L-glutathione, anhydrous citric acid, sunflower lecithin ethyl alcohol solvent extract).

[0032] In the present invention, homogenization is a process of mixing or emulsifying two or more substances by applying mechanical force, and can be performed using an agitator, a homogenizer, a homomixer, a homogenizer (Homo Emulsifier Disperser; HED), or a stirrer. By the homogenization, a liposome emulsion suitable for liposome rolling, which is a key process of subsequent stirring, is formed.

[0033] The liposome rolling process that occurs during the stirring process is a key process of the present invention for stably entrapping the entrapping substance, L-glutathione, into liposomes. However, unpredictable variables arise in liposome stability depending on the rotational force of the stirring process, the temperature of the rolling process, and the rolling time. To control the above variables, the homogenization and stirring process of the liposome stock solution can be performed using a reactor for mass production of liposomes suggested in a previous study of the present applicant (see Patent Registration No. 10-2259975). The reactor is prepared for mass production of liposomes on an industrial scale and can be used for the stirring reaction of the glutathione-entrapping liposomes according to the present invention.

[0034] In one embodiment, the reactor is provided with an impeller stirrer for mass production of liposomes, and the impeller stirrer comprises (1) an impeller having a cylindrical outer impeller and a plurality of inner impellers arranged concentrically therein, (2) a central shaft penetrating the center of the impeller, and (3) a connecting portion physically connecting the outer impeller, the inner impellers, and the central shaft, openings are positioned at the bottom and side of the outer impeller and the inner impeller, and a power transmission portion capable of receiving rotational power is positioned at the end of the central shaft, the spacing between the impellers is 5 to 100 mm, the number of impellers is 3 to 20, and rolling of liposomes is performed in the inner space of the impeller partitioned by the outer impeller and the plurality of inner impellers. In one embodiment, after injecting an emulsion feedstock for producing entrapped liposomes into the reactor through an inlet, liposome rolling can be performed in the internal space of an impeller at 35 to 70°C for 0.5 to 2 hours. In the present invention, the impeller refers to a blade that mixes a mixed solution used to stir the emulsified liposome stock solution, and can be used in a shape such as a paddle shape, a propeller shape, a screw shape, a turbine shape, etc., and the spacing between the impellers refers to the average spacing of each blade sign.

[0035] When the glutathione-entrapping liposome manufactured according to the present invention is manufactured in the form of a food such as a powder stick or jelly stick, it can improve the taste and flavor and solve difficulties in food manufacturing compared to liposomes using conventional sunflower lecithin.

[0036] The health functional food according to the present invention refers to a food manufactured and processed using raw materials or ingredients with useful functions for the human body so that, in addition to providing nutrition, it efficiently exhibits a bioregulatory function. The health functional food may include one or more additional additives in addition to the liposomes encapsulating glutathione according to the present invention. The additives include excipients, stabilizers, preservatives, buffers, etc., which are intended to improve the stability and bioavailability of the composition, maintain the quality of the preparation during storage or use, and improve economic efficiency by controlling the physical properties of the health functional food. However, since the technical characteristics of the present invention are not determined by the addition of the additives, it goes without saying that the liposome composition according to the present invention may not include the additives. In this case, by not including a specific additive, it may have the additional advantage of not having side effects from chemical substances that may be caused by the use of the additive.

[0037] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0038] Manufacturing Example 1. Manufacturing of sunflower lecithin extract

[0039] Sunflower lecithin was purchased from SHANKAR. Ethyl alcohol, the extraction solvent, was purchased from Deoksan General Science (Ethyl Alcohol, 94.0%). 300 g of sunflower lecithin was mixed with 1,000 ml of ethyl alcohol, which is approximately 3.3 times the volume, and the container was covered with aluminum foil to seal the container. The mixture was soaked at room temperature for 72 hours, and then filtered under reduced pressure using filter paper. The filtrate was concentrated using a rotary vacuum evaporator. The concentrated liquid extract was concentrated by evaporating the remaining solvent in a 60°C drying oven for 48 to 72 hours to obtain a primary lecithin extract.

[0040] The above first concentrated lecithin ethanol extract (EtLC) was completely dissolved again in 1000 ml of ethyl alcohol, filtered using a vacuum filtration device as before, and then first and second concentrations were performed using a rotary vacuum evaporator and a drying oven to obtain a second lecithin extract. Finally, the extraction process was performed once more with ethyl alcohol to obtain a third lecithin extract (Fig. 1).

[0041] As a result, it was confirmed that the sunflower extract that went through the first concentration (Fig. 2b), second concentration (Fig. 2c), and third concentration (Fig. 2d) showed a raw material shape with a more yellow color compared to the sunflower lecithin that did not go through concentration (Fig. 2a).

[0042] Manufacturing Example 2. Preparation of glutathione-entrapped liposome stock solution

[0043] L-glutathione powder having a glutathione content of 99.8% was used as glutathione for the production of glutathione-entrapped liposomes. 10 wt% of the glutathione powder, 5 wt% of anhydrous citric acid, 10 wt% of a lecithin extract, and 75 wt% of purified water were mixed to form a glutathione-entrapped liposome stock solution. The lecithin extract used was the primary, secondary, or tertiary lecithin extract obtained in Manufacturing Example 1, and the liposome stock solutions produced accordingly were named primary, secondary, and tertiary liposome stock solutions, respectively.

[0044] Manufacturing Example 3. Manufacturing of glutathione-entrapped liposomes by homogenization and stirring processes.

[0045] Glutathione-entrapped liposomes were prepared by homogenizing and stirring the liposome stock solution obtained in Manufacturing Example 2. For the stable production of glutathione-entrapped liposomes, a reactor manufactured through the applicant's prior research was used (see Patent No. 10-2259975).

[0046] In one embodiment, the mixture was homogenized at 65°C, 6,500 rpm, and 20 minutes using a homogenizer (Homo Emulsifier Disperser; HED) installed within the device to disperse the solute. Thereafter, the mixture was stirred at 65°C, 60 rpm, and 90 minutes using a stirrer installed within the device to induce liposome formation. To ensure stable formation of glutathione-entrapped liposomes, effective liposome rolling is necessary, and for this purpose, the gap between the impellers in the internal space of the stirrer was set to approximately 20 mm.

[0047] Once liposome production was complete, the liposome solution was cooled in ice water for 30 minutes to stabilize the liposomes. The liposome L-glutathione solution was then placed on a freeze-dryer shelf, freeze-dried at -70°C for 72 hours, and then powdered.

[0048] The L-glutathione-entrapped liposome products prepared according to the primary, secondary, or tertiary lecithin stock solutions obtained in Manufacturing Example 2 were named primary, secondary, and tertiary liposomes, respectively.

[0049] Experimental Example 1. Analysis of component content in sunflower lecithin extract

[0050] Nuclear magnetic resonance (NMR) analysis was performed to analyze the phosphatidylcholine content in the sunflower lecithin extract obtained in Manufacturing Example 1. Cholic acid sodium salt and deuterium oxide (99.9%) were purchased from Deoksan General Science. Disodium dihydrogen ethylenediaminetetraacetic acid dihydrate (EDTA 2Na) and phosphoserine (PSER) were obtained from Merk, and Tris-HCl buffer (1 M, pH 7.0) was purchased from LabPharmService. EDTA solution (100 mg / g) was prepared by dissolving 2.5 g of EDTA 2Na in 25 ml of Tris-HCl buffer solution (1 M, pH 7.0). Internal standard solution (1 mg / mL) was prepared by dissolving 10 mg of phosphoserine and 2 ml of EDTA solution, then adding Tris-HCl (1 M, pH 7.0) buffer solution to make up to 20 ml. Surfactant solution (20%) for sample pretreatment was prepared by dissolving 20 g of sodium cholate in 100 ml of deuterium oxide.

[0051] For NMR sample preparation, the sample was added with a surfactant solution to adjust the concentration to 0.5% to 2%. 1 ml of the internal standard solution was added and shaken at 50°C for 1 hour. The mixture was then centrifuged at 15,000 rpm for 10 minutes. The pH was adjusted to 6.9±0.4 with 1 M hydrogen chloride (HCl) or 1 M sodium hydroxide (NaOH), and 600 μl of the supernatant was transferred to a p-tube.31 PNMR was measured. Phosphoserine is available as a standard substance (SI) and in this study 31 Phospholipids were used as a reference standard for PNMR quantification. To accurately quantify phospholipids, it is important to obtain an accurate area ratio from the signals generated by phospholipids and phosphoserine (internal standard). Pulse interval time is the most important parameter for accurately determining the area of ​​the NMR signal. This was used to calculate the appropriate pulse interval time after measuring the T1 of the focusing signal. Verification was performed using a secondary lecithin extract (PC 70 sample). Each sample was dissolved in a surfactant solution, and the T1 parameter was measured using the reverse recovery method. The peak of the P compound was detected using a 90° RF pulse with a 2-s relaxation time. The T1 value for the peak was measured using an inversion recovery pulse sequence, a two-pulse sequence in which the spin population was inverted by a 180° pulse. Recovery was monitored using a 90° observation pulse followed by a variable delay. Relaxation times ranged from 5 to 40 seconds, and the same number of scans were performed for each of the 10 τ values ​​used until a sufficient signal-to-noise ratio was achieved. The T1 value is 31 The peak intensity integrated in P inversion recovery was determined.

[0052] Sample of sunflower lecithin extract 31 PNMR spectrum was measured at 242.81 MHz using a 5 mm BBO probehead. 31The spectra were collected using a Varian 600 MHz solution NMR spectrometer for P. The following parameters were used: probe frequency, 242.81 MHz; probe temperature, 30°C; spectral width, 19531.2 ppm; FID data points, 65,536; spinning, 15 Hz; dummy scans, 4; 1 H decoupling, Waltz16; pulse angle, 90°; pulse width, 12.2 μs. The pulse sequence used was s2pul, which is used for general NMR measurements, quantitative measurements, homonuclear and heteronuclear decoupling, NOE, and solvent suppression experiments. All spectra were acquired using VnmrJ software, and the following parameters were used: scans, 128; acquisition time, 5.4520 s; relaxation delay, 20.0 s; The total acquisition time was measured at 56 min.

[0053] All obtained spectra were peak-selected and integrated using Mnova software, and the substances were analyzed using the phosphorus spectrum table. The spectral peaks of the sunflower lecithin extract sample were classified as PSER (3.79 ppm), PE (-0.29 ppm), PS (-0.38 ppm), PI (-0.56 ppm), and PC (-0.85 ppm). The relative abundance of each sample was estimated as the relative percentage of the integrated area in the corresponding region to the total spectral area in the corresponding region.

[0054] The formula for calculating phosphatidyl content is as follows:

[0055]

[0056] Value A = Phospholipid content (%)

[0057] I IS = Area ratio of phosphoserine

[0058] I A = Area ratio of phospholipids

[0059] N IS = Number of phosphorus atoms in phosphoserine

[0060] N A = Number of phosphorus atoms in phospholipids

[0061] M IS = molecular weight of phosphoserine

[0062] M A = molecular weight of phospholipid

[0063] W IS = mass of phosphoserine

[0064] W A = mass of phospholipids

[0065] P IS = Purity of phosphoserine (%)

[0066] As a result of the measurement, the peak areas and molar concentrations of the 1st, 2nd, and 3rd sunflower lecithin extracts are as shown in the table below (PC: Phosphatidylcholine, PE: Phosphatidylethanolamine, PS: Phosphatidylserine, PI: Phosphatidylinositol).

[0067] [Table 1]

[0068]

[0069] To accurately determine the phospholipid class, a surfactant solution of cholic acid was used. 31 A PNMR quantification method was optimized and its performance verified. Pulse interval time and scan number were optimized to obtain NMR signals suitable for quantitative calculations. The optimized pulse interval time can be successfully applied to quantify phospholipid-rich samples such as lecithin produced from soybeans, egg yolk, and dietary supplements. Using phosphoserine as an internal standard, this content test method achieved traceability to reference materials (SI). 31P NMR also allows simultaneous analysis of many phospholipid classes, which is difficult to achieve using 1H NMR. Optimized 31 P NMR was successfully applied to quantify phospholipid levels in three types of polar lipid samples. As a result, the phospholipid content of the primary sunflower lecithin extract was found to be PC_54.52%, PE_1.57%, and PS_4.72%, and the contents of the secondary sunflower lecithin extract 70% were confirmed to be PC_67.55%, PS_5.15%, and PI_0.95%. In addition, the contents of the tertiary sunflower lecithin extract were confirmed to be PC_90.98%, PS_2.14%, and PI_0.38%.

[0070] Experimental Example 2. Analysis of liposome particle size, polydispersity index, and zeta potential

[0071] The particle size, polydispersity index (PDI), and zeta potential of solid-phase (powder) glutathione-entrapped liposomes were analyzed using a nanoparticle analyzer (NanoBrook 90Plus PALS, Brookhaven Instruments Corp., NY). Particle size was analyzed using the principle of dynamic light scattering. The analysis was repeated five times in total, and the results are shown in the table below.

[0072] [Table 2]

[0073]

[0074] The average particle size of the manufactured solid (powder) liposomal L-glutathione was 204.0 ~ 233.4 nm and the average polydispersity index was 0.236 ~ 0.284, which means that liposomes of relatively similar particle sizes were distributed. It is known that as the absolute value of zeta potential increases by more than 30 mV, the repulsion between colloidal particles increases, indicating a more stable colloidal state. The zeta potential of the solid (powder) liposomal L-glutathione was measured to be approximately -30.26 ~ -37.22 mV on average, which is higher than the general stable range of zeta potential, ±30 mV, indicating that it is electrostatically stable.

[0075] Experimental Example 3. Analysis of Glutathione Entrapment Rate in Liposomes

[0076] The glutathione-entrapped liposome powder obtained through the above manufacturing example was diluted 10-fold with PBS (Phosphate buffer saline), stirred, and then the supernatant and precipitate were separated three times at 17,000 rpm for about 20 minutes using a high-speed centrifuge (LZ-1730R, LABOGENE, Seoul, South Korea). Then, each solvent was evaporated using a rotary vacuum concentrator (Rotavapor™ R-300, BUCHI, Switzerland). A small amount of 10% Triton X-100 was added to the container containing the separated and dried liposomes and the container from which the supernatant was evaporated, and stirred. 10% Triton X-100 acts to destroy the liposomes, causing the L-glutathione inside to leak out. After liposomes were disrupted, they were filtered through a 0.2 μm syringe filter and quantitatively analyzed using high-performance liquid chromatography (HPLC, Agilent 1100, USA). The HPLC analysis conditions for L-glutathione were as follows.

[0077] [Table 3]

[0078]

[0079] Encapsulation Efficiency (EE %) was calculated using the following equation.

[0080]

[0081] As a result of the experiment, the effective substance entrapment rate of the glutathione-entrapping liposome obtained in the manufacturing example was measured as follows.

[0082] [Table 4]

[0083]

[0084] Experimental Example 4. Observation of liposome shape

[0085] To observe the shape of liposomes of liposomal L-glutathione, an electron microscope (Cryo-TEM, 120kV Talos L120C Cryo-Electron Microscope) was used. The solid (powder) sample was dissolved in purified water and the liposome size was observed. As a result, electron microscope images of Figs. 3a, 3b, and 3c were obtained for the primary, secondary, and tertiary glutathione-entrapping liposomes according to the manufacturing example, respectively, and the particle size was similar to that analyzed by a particle size analyzer, which was approximately 200 to 300 nm.

[0086] Experimental Example 5. Stability of liposomal L-glutathione under sterile conditions.

[0087] Liposomal L-glutathione can be used as a food ingredient. When used as a food ingredient, liposomal L-glutathione lyophilized powder can be dissolved in purified water (aqueous solution) and processed into a jelly stick-type food. In this case, the presence or absence of liposomes was analyzed using an electron microscope under food sterilization conditions (80-95℃, 30-40 minutes). The sterilization conditions were 95℃, 40 minutes, and the shape was observed after double-boiling the diluted liposomal L-glutathione powder solution.

[0088] As a result, electron microscope photographs of Figs. 4a, 4b, and 4c were obtained for the primary, secondary, and tertiary glutathione-entrapping liposomes according to the manufacturing example, respectively, and it was observed that the size of the liposomes was approximately 200 to 250 nm even after sterilization, and that the shape of the liposomes was also maintained.

Claims

1. (a) A step of mixing 10 wt% of L-glutathione powder, 5 wt% of anhydrous citric acid, and 10 wt% of ethyl alcohol solvent extract of sunflower lecithin in purified water; (b) a step of homogenizing the mixture at 65°C for 20 minutes at 6,500 rpm to form an emulsion; (c) performing liposome rolling by stirring the emulsion at 60 rpm for 90 minutes at 65°C; and (d) a step of cooling and freeze-drying the above liposome-rolled stirred material; A method for producing a liposome containing L-glutathione.

2. A method for producing an ethanol solvent extract of sunflower lecithin in accordance with claim 1, wherein the method comprises the steps of: (a-1) mixing sunflower lecithin and ethyl alcohol in a weight ratio of 3:10; soaking the mixture for 72 hours; performing reduced pressure filtration and concentration on the soaked material; and drying the concentrate at 60°C for 48 to 72 hours to obtain a sunflower lecithin primary extract.

3. In the second paragraph, the ethanol solvent extract of sunflower lecithin is prepared by a method for preparing the sunflower lecithin, comprising the steps of: (a-2) mixing and soaking a sunflower lecithin primary extract and ethyl alcohol in a weight ratio of 3:10; filtering and concentrating the soaked product under reduced pressure; and drying the concentrate to obtain a sunflower lecithin secondary extract.

4. In the third paragraph, the ethanol solvent extract of sunflower lecithin is prepared by the steps of: (a-3) mixing and soaking a sunflower lecithin secondary extract and ethyl alcohol in a weight ratio of 3:10; filtering and concentrating the soaked product under reduced pressure; and drying the concentrate to obtain a sunflower lecithin tertiary extract.

5. A manufacturing method in which, in the first paragraph, homogenization is performed using a homogenizer (Homo Emulsifier Disperser) to form a liposome emulsion suitable for liposome rolling.

6. A manufacturing method in which, in paragraph 5, stirring is performed in a stirrer composed of impellers having an internal gap of 5 to 100 mm for liposome rolling.

7. L-glutathione entrapping liposome prepared according to any one of claims 1 to 6.

8. Food containing the liposome of Article 7 as a raw material.

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