Preparation method and application of liposome composition

Abstract

The invention provides a preparation method and application of a liposome composition. The liposome composition disclosed by the invention comprises a water-soluble functional component, phospholipid, fatty acid ester and epigallocatechin gallate. The invention also provides a preparation method of the liquid liposome, the liposome gel and the liposome powder containing the components. The entrapment efficiency of the water-soluble liposome is improved, so that the water-soluble liposome has a slow-release effect, the intestinal retention rate is improved, and the hygroscopicity of the liposome powder can be obviously reduced when the liposome powder is prepared into powder.

Description

Technical Field

[0001] The present invention relates to the fields of common food, health food, medicine and cosmetics, and in particular to a method for preparing a liposome composition and application thereof. Background Art

[0002] Liposomes are an important drug delivery system that has the main functions of improving the bioavailability and stability of the delivered active ingredients and achieving controlled release.

[0003] However, conventional liposomes with water-soluble ingredients have poor stability, low encapsulation rate, easy to absorb moisture, produce gas and change color, and have a narrow application range. They are usually not acid-resistant and heat-resistant, and cannot be sterilized using conventional sterilization processes, which limits the development of liposomes in industrial oral liquid products and food. There are two main reasons for the low encapsulation rate of liposomes prepared with water-soluble ingredients: first, due to the good solubility of water-soluble ingredients in water, the particle size in water is also small, generally around 20-30nm, while the conventional liposome structure is generally 100-200nm. This particle size difference makes it easy for water-soluble ingredients to leak from the gaps in the phospholipid bilayer; second, because the inner cavity structure is small, the proportion of water-soluble ingredients that can enter the inner cavity is relatively small, which also causes the low encapsulation rate of liposomes prepared with water-soluble ingredients.

[0004] Common methods for preparing liposomes include organic solvent injection method, thin film dispersion method, etc. These methods all require the use of organic reagents, may have solvent residues, and have high technical requirements for the workshop, which increases production costs and is not conducive to large-scale industrial production.

[0005] Liposome technology is mostly used in medicine. The embedded membrane materials used are usually high PC content phospholipids (PC>75), modified phospholipids (hydrogenated phospholipids), etc., but they are relatively expensive. Cholesterol, Tween, etc. are usually added to the membrane material to embed the phospholipid bilayer to make the liposome structure more stable. However, cholesterol cannot be added in the food field, which limits its application. At the same time, Tween, as a synthetic surfactant, has a good emulsification effect, but it has certain safety risks to the intestines and their health, which does not conform to the development trend of the modern health food industry. Summary of the invention

[0006] In order to solve the problems existing in the prior art that conventional water-soluble component liposomes have poor storage stability, low encapsulation rate, no sustained-release effect, low bioacceptability or low retention rate after passing through the gastrointestinal tract, and the prepared liposome powder is easy to absorb moisture and has poor dosage form applicability.

[0007] The first aspect of the present invention provides a liposome composition, which comprises phospholipids, fatty acid esters and epigallocatechin gallate.

[0008] In certain embodiments, the composition further comprises water.

[0009] In certain embodiments, the composition further comprises an effective ingredient.

[0010] In certain embodiments, the active ingredient is a water-soluble ingredient.

[0011] In certain embodiments, the water-soluble ingredients include water-soluble vitamins, water-soluble minerals, water-soluble plant extracts, water-soluble protein peptides and / or water-soluble non-protein thiols.

[0012] In certain embodiments, the water-soluble vitamin comprises vitamin C.

[0013] In certain embodiments, the water-soluble minerals include magnesium threonate and / or ferric pyrophosphate.

[0014] In certain embodiments, the water-soluble plant extract includes elderberry.

[0015] In certain embodiments, the water-soluble protein peptides include collagen peptides.

[0016] In certain embodiments, the water-soluble non-protein thiol comprises glutathione.

[0017] In certain embodiments, the composition comprises a water-soluble component, a phospholipid, a fatty acid ester, and epigallocatechin gallate (EGCG).

[0018] In certain embodiments, the fatty acid ester includes one or more of mono- and di-glycerol fatty acid esters, citric acid fatty acid glycerides, lactic acid fatty acid glycerides, sucrose fatty acid esters, polyglycerol fatty acid esters and beeswax.

[0019] In certain embodiments, the water-soluble component has an amount of 0.2-45 parts by weight, such as 5-30 parts by weight, such as about 15 parts by weight.

[0020] In certain embodiments, the phospholipids are present in an amount of 0.4-6.5 parts by weight, such as 0.4-5, 2-6.5 or 2-5 parts by weight.

[0021] In certain embodiments, the fatty acid ester has an amount of 0.5-3.5 parts by weight, such as 1-3 parts by weight.

[0022] In certain embodiments, the weight portion of epigallocatechin gallate (EGCG) is 0.03-0.9, such as 0.04-0.8.

[0023] In certain embodiments, the phospholipid has a PC value of 30-70.

[0024] In certain embodiments, the phospholipid has a PC value of 40-70.

[0025] In certain embodiments, the phospholipid has a PC value of 55-70.

[0026] In certain embodiments, the water has an amount of 0.2-90 parts by weight.

[0027] In certain embodiments, the composition further comprises one or more components selected from the group consisting of a sour taste regulator, a hydrophilic adjuvant, a flavor and a sweetener.

[0028] In certain embodiments, the composition does not include one or more of the following ingredients: cholesterol, ethanol, and Tween.

[0029] In certain embodiments, the sour taste regulator is selected from one or more of citric acid, lactic acid and malic acid.

[0030] In certain embodiments, the flavor is a water-soluble flavor, such as lemon flavor.

[0031] In certain embodiments, the sweetener is a sugar sweetener and / or a non-sugar sweetener, such as sucralose.

[0032] In certain embodiments, the hydrophilic excipient is selected from one or more of dextrin, sugar alcohol and sodium starch octenylsuccinate.

[0033] In certain embodiments, the sour taste regulator is 0.01-5 parts by weight.

[0034] In certain embodiments, the hydrophilic auxiliary material is 0.5-60 parts by weight.

[0035] In certain embodiments, the flavor is 0.04-1.5 parts by weight.

[0036] In certain embodiments, the sweetener is 0.02-0.3 parts by weight.

[0037] In certain embodiments, the liposome composition is a liquid liposome; preferably, the water-soluble component is dissolved in 0.2-90 parts by weight of water.

[0038] In certain embodiments, the liposome composition is a liposome gel; preferably, the composition comprises water in an amount 1-2 times the weight of the water-soluble components.

[0039] In certain embodiments, the liposome composition is a liposome powder.

[0040] The second aspect of the present invention provides a method for preparing the liquid liposomes described in the first aspect of the present invention, comprising the following steps: 1) mixing phospholipids and fatty acid esters and heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring component A and component B to form a uniform system, adding EGCG and stirring evenly, and high-pressure homogenization to obtain liquid liposomes.

[0041] In certain embodiments, the weight portion of water in step 2) is more than 2 times the weight portion of the water-soluble component.

[0042] In certain embodiments, in step 3), component A and component B are stirred and mixed into a uniform system, EGCG and one or more of the optional sourness regulator, flavor and sweetener are added and stirred evenly, and liquid liposomes are obtained by high pressure homogenization.

[0043] The third aspect of the present invention provides a method for preparing the liposome gel described in the first aspect of the present invention, comprising the following steps: 1) mixing phospholipids and fatty acid esters and heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring and mixing component A and component B into a uniform system, adding EGCG and stirring evenly, and high-pressure homogenization to obtain liposome gel.

[0044] In certain embodiments, the weight of the water in step 2) is 1-2 times that of the water-soluble component.

[0045] In certain embodiments, in step 3), component A and component B are stirred and mixed into a uniform system, EGCG and a sourness regulator are added and stirred evenly, and liposome gel is obtained by high pressure homogenization.

[0046] In certain embodiments, flavors and sweeteners are added to the liposome gel obtained in step 3), and then the gel is canned and sterilized.

[0047] The fourth aspect of the present invention provides a method for preparing the liposome powder described in the first aspect of the invention, comprising the following steps: 1) mixing phospholipids and fatty acid esters, heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring component A and component B to form a uniform system, adding EGCG and stirring evenly, and high-pressure homogenization to obtain liquid liposomes; 4) adding hydrophilic excipients and optionally sweeteners, mixing, sterilizing, and drying to obtain liposome powder; optionally, adding flavors after drying.

[0048] In certain embodiments, in step 3), component A and component B are stirred and mixed into a uniform system, EGCG and a sourness regulator are added and stirred evenly, and liquid liposomes are obtained by high-pressure homogenization.

[0049] The fifth aspect of the present invention provides a product comprising the composition.

[0050] In certain embodiments, the product is selected from one or more of oral liquid, soft capsule, gel, soft candy, jelly, powder, tablet, and hard capsule.

[0051] The sixth aspect of the present invention provides use of the composition described in the first aspect of the present invention in the preparation of food, cosmetics or medicines.

[0052] In certain embodiments, the food is a health food.

[0053] In certain embodiments, the combination is prepared into one or more selected from oral liquid, soft capsule, gel, soft candy, jelly, powder, tablet, hard capsule.

[0054] The method for preparing the liposome composition and the prepared liposomes of the present invention have the following advantages over the prior art:

[0055] (1) The liposome composition has a high encapsulation efficiency, a sustained release effect and a high intestinal retention rate.

[0056] (2) Liposomes can form a self-gel system before adding hydrophilic excipients, which has high stability.

[0057] (3) The preparation process of liposome powder is simple and safe, suitable for encapsulating and carrying a variety of water-soluble biological active ingredients, and the material is not easy to absorb moisture during storage, which can expand the scope of use of liposome powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Schematic diagram of liposome structure.

[0059] Figure 2 This is a comparison chart of the appearance stability of liposome samples.

[0060] Figure 3 This is a transmission electron microscopy image of the vitamin C liposome liquid in Example 9.

[0061] Figure 4 This is a transmission electron micrograph of the vitamin C liposome powder after reconstitution in Example 9.

[0062] Figure 5 This is a picture of the vitamin C liquid liposomes before drying in Example 34.

[0063] Figure 6 This is a picture of the vitamin C liposome gel of Example 32.

[0064] Figure 7 This is a picture of the vitamin C liposome powder of Example 30. DETAILED DESCRIPTION

[0065] The following definitions are provided to facilitate understanding of the present invention by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Preferred materials and methods are described herein, but any methods and materials similar to or equivalent to those described herein may be used in the practice of the present invention's tests. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0066] Definition of terms

[0067] Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. In addition, unless otherwise specified, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known per se, which will be understood by those skilled in the art.

[0068] As used herein, the term "liposome" is an artificial membrane, which is composed of a phospholipid bilayer and an inner cavity. The phospholipid bilayer is a ring-shaped structure formed by a hydrophilic head and a hydrophobic tail. The structure enclosed by this ring structure is the inner cavity. The water-soluble components are mainly present in the hydrophilic inner cavity, and the fat-soluble components are mainly present in the middle part of the bilayer. The structure of a liposome can be seen in Figure 1 .

[0069] As used herein, the term "vitamin C" is a water-soluble vitamin that participates in the body's complex metabolic processes, can promote growth and enhance resistance to disease, and can be used as a nutritional supplement and antioxidant.

[0070] As used herein, the term “elderberry” is a common herbal plant that is rich in flavonoids, anthocyanins, polyphenols and other substances, and has the effects of improving immunity, anti-inflammation, anti-oxidation, improving cardiovascular health and the like.

[0071] As used herein, the term "glutathione" is widely present in animals and plants, plays an important role in organisms, has a strong antioxidant effect, can participate in detoxification, protect the activity of sulfhydryl enzymes, and maintain the stability of red blood cell membrane structure.

[0072] As used herein, the term "magnesium threonate" is a magnesium salt of threonic acid, which has a cathartic effect, bowel preparation, prevention of preeclampsia during pregnancy, prevention of magnesium deficiency, and gastric acid neutralization. Its main effect is to promote fecal excretion and relieve constipation by increasing intestinal fluid secretion and promoting intestinal peristalsis.

[0073] As used herein, the term "epigallocatechin gallate", abbreviated as "EGCG", belongs to catechins. EGCG has antibacterial, antiviral, antioxidant, anti-atherosclerotic, anti-thrombotic, anti-angiogenic, anti-inflammatory and anti-tumor effects. In medicine and health care, EGCG has the functions of preventing and treating various diseases such as cancer and enhancing immunity; in the food industry, it can be used as an antioxidant, antibacterial, fresh-keeping, and deodorant; in daily chemical products, it can be used as a special function preservative and skin care agent. In the present invention, EGCG participates in the formation of the liposome structure, which can enhance the stability of the liposome structure, make the formed liposome structure clear and not easily destroyed after drying, thereby ensuring that the hygroscopicity of the formed liposome powder is significantly reduced.

[0074] As used herein, the term "fatty acid ester" is an ester generated by the reaction of the carboxyl group of a fatty acid with the hydroxyl group of an alcohol, and is a nonionic surfactant. They are usually milky white solids with good wetting, emulsifying and foaming properties. In the present invention, fatty acid esters can fill the gaps in the liposome structure, making the liposome structure more solid, and the water-soluble functional ingredients encapsulated in the liposome are not easy to leak, which significantly improves the encapsulation rate of the liposome.

[0075] As used herein, the term "PC value" refers to the content or concentration of phosphatidylcholine (PC) in phospholipids. For example, phospholipid PC50 refers to a phospholipid with a phosphatidylcholine content of 50%. DETAILED DESCRIPTION OF THE INVENTION

[0077] Liposome composition

[0078] The present invention provides liposome compositions, including essential and non-essential components.

[0079] In certain embodiments, the essential components include phospholipids, fatty acid esters and epigallocatechin gallate (EGCG). Preferably, the essential components further include functional ingredients. Preferably, the functional ingredients are water-soluble functional ingredients, including but not limited to water-soluble vitamins, water-soluble minerals, water-soluble plant extracts, water-soluble protein peptides, and water-soluble non-protein thiols. Preferably, the water-soluble ingredients include but are not limited to vitamin C, magnesium threonate, iron pyrophosphate, elderberry, glutathione, collagen peptides, etc. Preferably, the phospholipids include but are not limited to soybean lecithin, sunflower lecithin, and egg yolk lecithin. Preferably, the fatty acid esters include but are not limited to mono- and diglycerol fatty acid esters, citric acid fatty acid glycerides, lactic acid fatty acid glycerides, sucrose fatty acid esters, polyglycerol fatty acid esters, and beeswax.

[0080] In certain embodiments, the weight portion of the water-soluble component is 0.2-45, such as 5-30, such as 10-30, such as 15-30, such as about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45 parts.

[0081] In some embodiments, the phospholipid weight portion is, for example, 0.4-6.5, for example, 0.4-5, for example, 2-6.5, for example, 2-5, for example, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5.

[0082] In certain embodiments, the fatty acid ester is present in an amount of 0.2-5 parts by weight, such as 0.5-3.5, such as 1-3, such as about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, or about 5 parts by weight.

[0083] In certain embodiments, the epigallocatechin gallate (EGCG) is in an amount of 0.01-1.2, such as 0.03-0.9, such as 0.04-0.8, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.00, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, or about 1.2 parts by weight.

[0084] In certain embodiments, the phospholipid has a PC value of 30-70, such as a PC value of 40-70, such as 55-70, such as a PC value of 50-70, such as a PC value of 30, 35, 55, 60, 70.

[0085] In certain embodiments, the composition is a liquid liposome, and the essential component also includes water. Preferably, the weight portion of water is 0.2-90 parts, such as about 0.2, about 0.5, about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90 parts.

[0086] In certain embodiments, the composition is a liposome gel, and the essential component also includes water. Preferably, the weight portion of water is 0.5-3 times, such as 1-2 times, such as about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2 times the weight portion of the water-soluble component.

[0087] In certain embodiments, the composition is a liposomal powder, and the essential components do not include water or substantially do not include water.

[0088] In certain embodiments, the liposomal powder contains less than 3% water.

[0089] In certain embodiments, the optional ingredients include acidity regulators, hydrophilic adjuvants, flavors, sweeteners and / or pigments.

[0090] In certain embodiments, the acidity regulator includes but is not limited to citric acid, lactic acid, malic acid, etc., preferably citric acid. Preferably, the weight portion of the acidity regulator is 0.01-5 parts.

[0091] In certain embodiments, the hydrophilic auxiliary material includes but is not limited to the following ingredients: one or more of dextrin, sugar alcohol, sodium starch octenyl succinate, and water. Preferably, the weight portion of the hydrophilic auxiliary material is 0.5-60 parts.

[0092] Liposome preparation method

[0093] The present invention provides a method for preparing liposome compositions of different dosage forms, wherein the liposome compositions of different dosages include liquid liposomes, liposome gels and liposome powders.

[0094] In certain embodiments, the preparation method of liquid liposomes comprises the following steps: (1) mixing phospholipids and fatty acid esters, heating and dissolving, which is recorded as component A; (2) dissolving water-soluble components in water, mixing evenly, which is recorded as component B; (3) pouring A into B and stirring and mixing in a stirring device to form a uniform system, then adding EGCG, stirring evenly, then adding an acidity regulator and stirring evenly, then high-pressure homogenizing 1-2 times at 200-600 bar to prepare liquid liposomes; the stirring device can be at least one of a stirrer, a high-pressure homogenizer, a high-pressure microfluidizer, a ball mill, and a colloid mill. Preferably, the weight of water in step (2) is greater than 2 times that of the water-soluble component; preferably, the weight of water in step (2) is more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times, or more than 10 times that of the water-soluble component.

[0095] In certain embodiments, the preparation method of the liposome gel is to reduce the weight of water in step (2) of the preparation method of the liquid liposome to 1-2 times of the water-soluble component, and then prepare according to the above method, add flavors and sweeteners to the prepared liposome gel, and then fill it, and sterilize it at 80-90°C for 30-60 minutes.

[0096] In certain embodiments, the preparation method of the liposome powder is to add a hydrophilic excipient and a sweetener to the liquid liposome, wherein the hydrophilic excipient includes one or more of dextrin, sugar alcohol, and sodium starch octenyl succinate, mix them thoroughly and evenly, sterilize them at 80-90° C. for 30-60 minutes, then remove moisture by freeze drying, spray drying, etc., add flavors and mix them evenly, and then obtain the liposome powder.

[0097] Application of liposome compositions

[0098] The invention provides application of a liposome composition in preparing food, cosmetics or medicines.

[0099] In certain embodiments, the food is a health food.

[0100] In certain embodiments, the composition is prepared into one or more selected from oral liquid, soft capsule, gel, soft candy, jelly, powder, tablet, and hard capsule.

[0101] Example

[0102] The present invention will be more readily understood with reference to the following examples, which are included only to illustrate certain aspects and embodiments of the present invention and are not intended to limit the present invention.

[0103] Unless otherwise stated, the reagents used in this example are all commercially available materials or conventional materials.

[0104] Example 1 Liposome Preparation Method

[0105] 1.1 Preparation of liquid liposomes

[0106] The preparation method of liquid liposomes may include the following steps:

[0107] (1) mixing phospholipids and fatty acid esters, heating and dissolving them, and recording them as component A;

[0108] (2) dissolving the water-soluble component in water and mixing well, which is recorded as component B;

[0109] (3) Pour A into B and stir and mix thoroughly in a stirring device to form a uniform system, then add EGCG and stir thoroughly, then add the acidity regulator and stir evenly, then high-pressure homogenize 1-2 times at 200-600 bar to prepare liquid liposomes; the stirring device is a high-pressure homogenizer.

[0110] 1.2 Preparation of liposome gel

[0111] The water in step (2) of 1.1 above is reduced to 1-2 times of the water-soluble component, and then the liposome gel is prepared according to the above method, and flavors and sweeteners are added to the prepared liposome gel, and then filled and sterilized at 80-90°C for 30-60 minutes;

[0112] 1.3 Preparation of liposome powder

[0113] Add hydrophilic excipients and sweeteners to the liquid liposomes obtained in step (3) of 1.1. The hydrophilic excipients include one or more of dextrin (such as maltodextrin), sugar alcohol (such as erythritol), and sodium starch octenylsuccinate. Mix well and sterilize at 80-90° C. for 30-60 min. Then remove moisture by freeze drying, spray drying, etc., add flavors and mix well to obtain liposome powder.

[0114] Example 2 Liposome Testing Method

[0115] 2.1 Appearance evaluation

[0116] The appearance stability of the liquid liposomes prepared in step (3) of Example 1.1 was evaluated. The severity of the precipitation or stratification of the liposome stability samples was observed. The evaluation criteria were as follows: Figure 2 The degree of instability increases from right to left. -: stable, no obvious change; +: slightly unstable holes or water precipitation; ++: obviously unstable stratification or slag structure.

[0117] 2.2 Content determination

[0118] (1) Vitamin C

[0119] a. Drawing of standard curve: Accurately weigh 20 mg of vitamin C reference substance into a 25 mL volumetric flask, add water to dissolve and dilute to scale. Accurately transfer 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL into a 10 mL volumetric flask, add water to dissolve and dilute to scale to prepare a series of solutions for the standard curve.

[0120] b. Sample detection: Weigh a certain amount of liquid vitamin C liposomes into a 10ml volumetric flask, add some anhydrous ethanol and shake to break the emulsion, then add 2% oxalic acid solution to the scale line and shake well, then use 2% oxalic acid solution to absorb moisture 100 times, and determine the vitamin C content. (Mobile phase: 0.01mol / L potassium dihydrogen phosphate solution (containing 0.1% phosphoric acid): methanol = 95:5, injection volume: 5μL, flow rate: 0.6ml / min)

[0121] Vitamin C content % = C × V / m × 100%

[0122] C——Vitamin C concentration in the sample solution calculated from the standard curve, mg / mL;

[0123] V——dilution factor of sample solution, mLM——sample weight, mg.

[0124] (2) Magnesium threonate

[0125] a. Drawing of standard curve: Accurately weigh 0.1mL, 0.4mL, 1mL, 1.6mL, and 2mL of 1mg / mL magnesium stock solution into a 20mL volumetric flask, add water to dissolve and dilute to the scale to prepare a series of solutions for the standard curve.

[0126] b. Sample detection: Accurately weigh the liquid magnesium threonate liposome sample and place it in a crucible, place it in a 500℃ muffle furnace for ash treatment for 5-8 hours, add 10ml of 5% nitric acid solution to dissolve it after cooling, and then dilute it to 25ml with purified water. Detect at a wavelength of 279.08nm and calculate the magnesium threonate content in the sample.

[0127] Magnesium threonate content % = M × V / (m × 7.5%) × 100%

[0128] M————magnesium concentration in the test sample solution obtained from the standard curve, mg / mL;

[0129] V——sample dilution multiple, ml;

[0130] m——weight of sample, mg;

[0131] (3) Ferric pyrophosphate

[0132] The ferric pyrophosphate detection method is the same as the magnesium threonate detection method. The reserve solution is an iron reserve solution. The iron content in the ferric pyrophosphate is 25.5%. The ferric pyrophosphate content in the liquid ferric pyrophosphate liposome is calculated based on this content.

[0133] (4) Elderberry (anthocyanins)

[0134] a. Drawing of standard curve: Accurately weigh about 10 mg of cyanidin-3-O-glucoside reference substance, place in a 100 mL brown volumetric flask, shake well, and use as reference substance stock solution. Accurately measure 0.1, 0.5, 1.0, 2.0, and 3.0 mL of reference substance stock solution, place in a 20 mL volumetric flask, add extractant to make solutions containing 0.5, 2.5, 5, 10, and 15 of cyanidin-3-O-glucoside per 1 mL, respectively. Take the extractant as blank, measure the absorbance at a wavelength of 535 nm, and draw a standard curve with the concentration (μg / mL) of cyanidin-3-O-glucoside and the absorbance.

[0135] b. Sample detection: Weigh an appropriate amount of liquid elderberry liposomes into a 100mL brown volumetric flask, add 60mL of extractant (0.1% hydrochloric acid solution-80% ethanol solution (15:85)), ultrasonically extract for 15 minutes, let it cool to room temperature, dilute with the extractant, filter, and obtain (appropriate dilution can be performed if necessary). Take the extractant as the blank, measure the absorbance at a wavelength of 535nm, and find the concentration of cyanidin-3-O-glucoside in the sample solution from the standard curve to calculate the anthocyanin content in the sample.

[0136] Elderberry (anthocyanin content) % = C × V / (M × 1000) × 100%

[0137] C————The concentration of cyanidin-3-O-glucoside in the test sample solution obtained from the standard curve, μg / mL;

[0138] V——sample dilution multiple, mL;

[0139] M——weight of sample, mg;

[0140] (5) Glutathione

[0141] a. Drawing of standard curve: Accurately weigh 15 mg of glutathione reference substance into a 25 mL volumetric flask, dissolve it in water and dilute it to the mark. Accurately transfer 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL into a 10 mL volumetric flask, dissolve it in water and dilute it to the mark to prepare a series of standard curve solutions. (Mobile phase: phosphate buffer: methanol = 96:4, injection volume: 10 μL, flow rate: 1.0 ml / min)

[0142] b. Sample detection: Accurately weigh an appropriate amount of mixed sample (containing about 200 mg of glutathione) and place it in a 50 mL volumetric flask. Add 5 mL of methanol, vortex to disperse the sample, and then ultrasonicate for 5 minutes. Then add about 30 mL of mobile phase, ultrasonicate for 10 minutes, cool to room temperature, and adjust the volume to the scale with mobile phase, and shake well. Accurately transfer 0.5 mL to a 10 mL volumetric flask, adjust the volume to the scale with mobile phase, shake well, filter with a 0.45 um microporous filter membrane, and inject for determination.

[0143] Glutathione content % = C × V / m × 100%

[0144] C——glutathione concentration in the sample solution calculated from the standard curve, mg / mL; V——dilution factor of the sample solution, mLM——sample weight, mg.

[0145] 2.3 Determination of encapsulation efficiency

[0146] (1) Vitamin C

[0147] Accurately weigh 2 ml of liquid vitamin C liposomes and place the dialysis bag MD34 (8000-14000D) in a 70-

[0148] Activate in 80℃ purified water for 30min, put the sample into the activated dialysis bag, clamp it front and back, add 198ml 0.4% sodium bisulfite solution into the beaker, then immerse the dialysis bag with the sample completely in the solution, and perform dialysis while stirring on the stirrer. After dialysis for 4h, take 1ml of dialysate and determine the content of vitamin C according to the content determination method, that is, the content of free vitamin C in liposomes. The total vitamin C content is the total content of vitamin C in 2mL liquid vitamin C liposomes.

[0149] Encapsulation rate % = (1-free vitamin C content * 100 / total vitamin C content) * 100%

[0150] (2) Magnesium threonate

[0151] Accurately weigh 1 ml of liquid magnesium threonate liposomes, place the sample in an activated dialysis bag, clamp it front and back, add 999 ml of purified water to a beaker, then completely immerse the dialysis bag containing the sample in the solution, and perform dialysis while stirring on a stirrer. After dialysis for 4 hours, take 1 ml of dialysate and determine the content of magnesium threonate, i.e., the free magnesium threonate content in the liposomes, according to the content determination method. The total content of magnesium threonate is the content of magnesium threonate in 1 ml of liquid magnesium threonate liposomes.

[0152] Encapsulation rate % = (1-free magnesium threonate content * 1000 / total magnesium threonate content) * 100%

[0153] (3) Ferric pyrophosphate

[0154] The method is the same as magnesium threonate.

[0155] (4) Elderberry (anthocyanins)

[0156] Accurately weigh 5 ml of liquid magnesium threonate liposomes, place the sample in an activated dialysis bag, clamp it front and back, add 195 ml of pH = 4.0 phosphate buffer to a beaker, then completely immerse the dialysis bag containing the sample in the solution, and perform dialysis while stirring on a stirrer. After dialysis for 4 hours, take 1 ml of the dialysate and determine the content of anthocyanins according to the content determination method, that is, the content of free anthocyanins in the liposomes. The total anthocyanin content is the content of anthocyanins in 5 ml of liquid elderberry liposomes.

[0157] Encapsulation rate % = (1-free anthocyanin content * 40 / total anthocyanin content) * 100%

[0158] (5) Glutathione

[0159] Accurately weigh 2 ml of liquid magnesium threonate liposomes, place the sample in an activated dialysis bag, clamp it front and back, add 198 ml of 50 mM phosphate buffer with pH = 4.8 to a beaker, then completely immerse the dialysis bag containing the sample in the solution, and perform dialysis while stirring on a stirrer. After dialysis for 4 hours, take 1 ml of dialysate and determine the content of glutathione according to the content determination method, that is, the free glutathione content in the liposomes. The total glutathione content is the glutathione content in 2 ml of liquid glutathione liposomes. Encapsulation rate % = (1-free glutathione content * 100 / total glutathione content) * 100%.

[0160] 2.4 Intestinal retention rate determination

[0161] According to the INFOGEST static simulated digestion test, the digestion and absorption of vitamin C liposomes in the body, i.e., the bioaccessibility, is evaluated by simulating the digestion and absorption in the oral cavity and gastrointestinal tract. The specific operation is as follows: a certain amount of liquid liposome sample is weighed, and purified water is added to 5g to prepare the test solution; simulated saliva (SSF), gastric juice (SGF) and intestinal juice (SIF) are prepared, and the corresponding enzymes, i.e., oral mucosal protein, pepsin and pancreatic enzyme, pancreatic lipase, and bile salt are added respectively; the pH of saliva is adjusted to 6.8, the pH of gastric juice to 3.0, and the pH of intestinal juice to 7.0, and the samples are activated at 37.2°C for one hour for use. Take the test solution and add it to 4mL simulated saliva, the pH of the simulated oral digestive environment is 6.8, and the oral digestion is completed by constant temperature oscillation at 37℃ for 10min; add 8mL simulated gastric juice to the digestive solution after simulated saliva digestion, the pH of the simulated gastric digestive environment is 3.0, the temperature is constant at 37℃, and the incubation time is 1h, which completes the simulated gastric digestion; add 16mL simulated intestinal juice to the digestive solution after simulated gastric digestion, the pH of the simulated intestinal digestive environment is 7.0, and the time is 2h. During this period, the pH value should be dynamically adjusted to maintain the pH at 7.0, which completes the simulated intestinal digestion; finally, terminate the reaction in an ice bath, centrifuge at 14000rpm for 30min to obtain the upper clear liquid, which is the micelle layer. The content before digestion is the actual content of the water-soluble components before the in vitro digestion experiment; the water-soluble components before digestion and in the micelle layer are determined by the determination method.

[0162] Intestinal retention rate % = actual measured content of micelle layer (W1) / actual measured content before digestion (W0) * 100%

[0164] 2.5 Determination of hygroscopicity

[0165] Take about 100g of the dried liposome powder in a suitable basin and store it in an environment with a humidity of less than 40% and a temperature of less than 25°C. Detect the moisture content at 0h and 8h respectively. The moisture content is expressed in %. The moisture content at 0h is recorded as M1, and the moisture content at 8h is recorded as M2.

[0166] Moisture absorption rate % = M2-M1

[0167] Structure determination

[0168] Take a certain amount of liposome powder and dissolve it in purified water. Add 10 μL of sample on the copper mesh and let it settle for 1 minute, then use filter paper to absorb the excess liquid. Add 10 μL of 3% phosphotungstate dye on the copper mesh and let it settle for 1 minute, then use filter paper to absorb the floating liquid. After drying at room temperature for several minutes, perform electron microscopy detection and imaging under 80-120 kV to obtain transmission electron microscopy imaging results.

[0169] Example 3 Effect of phospholipid content on liposomes

[0170] According to the formula of each component in Table 1, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the vitamin C liposome powder was prepared by spray drying to remove moisture. The hygroscopicity was tested. The results are shown in Table 2.

[0171] Table 1 Effect of phospholipid content on liposomes

[0172]

[0173] Table 2 Effect of phospholipid content on hygroscopicity of powders

[0174]

[0175] According to Table 1 and Table 2, when the content of phospholipids is 0.4-5 parts, the encapsulation rate, intestinal retention rate and moisture absorption rate of liposomes are all at a good level. When the phospholipid content is too low, the effective ingredients cannot be effectively encapsulated, and the effective ingredients are exposed to the environment, resulting in the hygroscopicity of the efficacy; when the phospholipid content is too high, there is an excess of phospholipids that do not participate in the formation of the liposome structure, which causes the hygroscopicity of the phospholipids to be exhibited, and the moisture absorption rate is significantly increased.

[0176] Example 4 Effect of Mono- and Di-Glycerol Fatty Acid Ester Content on Liposomes

[0177] According to the formula of each component in Table 3, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the vitamin C liposome powder was prepared by spray drying to remove moisture. The hygroscopicity was tested. The results are shown in Table 4. Figure 3 and Figure 4 The transmission electron micrographs of the vitamin C liposome liquid obtained according to the formula of Example 9 and the transmission electron micrographs of the vitamin C liposome powder after reconstitution are shown respectively. It can be seen that the fingerprint membrane structure of the prepared vitamin C liquid liposome is obvious and the liposome structure is clear; the liposome structure of the prepared vitamin C liposome powder after reconstitution is clear, and the liposome structure is not destroyed due to the drying process.

[0178] Table 3 Effect of mono- and di-glycerol fatty acid ester content on liposomes

[0179]

[0180] Table 4 Effect of mono- and di-glycerol fatty acid ester content on the hygroscopicity of powders

[0181]

[0182] As shown in Tables 3 and 4, when the content of mono- and di-glycerol fatty acid esters is 0.5-3.5 parts, the appearance, encapsulation rate and intestinal retention rate of the liposomes are good, and the moisture absorption rate is also at a relatively low level. This may be due to the interaction between the hydroxyl or carboxyl groups in the fatty acid esters and the water-soluble functional ingredients to form hydrogen bonds, which increases the particle size of the water-soluble functional ingredients and hinders the overflow of the water-soluble ingredients into the liposome structure; in addition, the long-chain fatty acid groups in the fatty acid esters are similar to the glycerol and hydroxyl structures in the phospholipids, and can form a strong interaction with them, reducing the gaps in the phospholipid structure, further inhibiting the overflow of the internal water-soluble components, and thus improving the encapsulation rate, which is reflected in the significant reduction in the moisture absorption of the powder. - When the addition amount of mono- and di-glycerol fatty acid esters is 5 parts, there is still a residue after combining with phospholipids and water-soluble functional ingredients, which is reflected in the significant increase in viscosity, which cannot meet the needs of industrial production.

[0183] Example 5 Effect of EGCG content on liposomes

[0184] According to the formula of each component in Table 5, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the moisture was removed by spray drying to prepare the vitamin C liposome powder, and its hygroscopicity was tested. The results are shown in Table 6.

[0185] Table 5 Effect of EGCG content on liposomes

[0186]

[0187] Table 6 Effect of EGCG content on hygroscopicity of powder

[0188]

[0189] As shown in Tables 5 and 6, when EGCG is 0.03-0.9 parts, the liposome encapsulation rate, intestinal retention rate and moisture absorption rate are all good. This may be because EGCG contains multiple hydroxyl groups, which can be covalently bonded with the hydroxyl groups in phospholipids and fatty acid esters to limit the free rotation inside the molecule, thereby enhancing the rigidity of the liposome structure formed by phospholipids and fatty acid esters, so that the structure and the structure can withstand extrusion more, and the stirring during the preparation process will also make the structure more orderly. When the EGCG content is too high, the remaining EGCG combined with the liposome structure will exist in the outer and inner layers of the liposome structure, which will cause the repulsion between the liposome structure and the liposome structure to decrease, causing the structure to aggregate and produce instability and precipitation.

[0190] Example 6 Effect of phospholipid PC value on liposomes

[0191] According to the formula of each component in Table 7, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the vitamin C liposome powder was prepared by spray drying to remove moisture. The hygroscopicity was tested. The results are shown in Table 8.

[0192] Table 7 Effect of phospholipid PC value on liposomes

[0193] Type / content (portions) Example 20 Example 21 Example 22 Example 23 Example 24 Phospholipid PC30 4 / / / / Phospholipid PC35 / 4 / / / Phospholipid PC55 / / 4 / / Phospholipid PC60 / / / 4 / Phospholipid PC70 / / / / 4 Mono- and di-glycerol fatty acid esters 1.5 1.5 1.5 1.5 1.5 EGCG 0.2 0.2 0.2 0.2 0.2 VC 15 15 15 15 15 Citric Acid 0.03 0.03 0.03 0.03 0.03 water 50 50 50 50 50 Appearance stability - - - - - Encapsulation rate% 43.28 45.32 54.1 52.9 51.1 Intestinal retention rate % 76.2 80.0 89.3 87.7 86.7

[0194] Table 8 Effect of phospholipid PC value on the hygroscopicity of powder

[0195] Type / content (portions) Example 20 Example 21 Example 22 Example 23 Example 24 Maltodextrin 30 30 30 30 30 Moisture absorption rate % 2.64 2.58 0.72 1.26 1.39

[0196] According to Tables 7 and 8, phospholipids with different PC values ​​can achieve good encapsulation efficiency and intestinal retention rate, among which the best is the PC value in the range of 55-70.

[0197] Example 7 Effects of different fatty acid esters on liposomes

[0198] According to the formula of each component in Table 9, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the vitamin C liposome powder was prepared by spray drying to remove moisture. The hygroscopicity was tested. The results are shown in Table 10.

[0199] Table 9 Effects of different fatty acid esters on liposomes

[0200] Type / content (portions) Example 25 Example 26 Example 27 Example 28 Example 29 Phospholipid PC50 4 4 4 4 4 Citric acid fatty acid glyceride 1.5 / / / / Lactic acid glyceride / 1.5 / / / Sucrose fatty acid esters / / 1.5 / / Polyglycerol fatty acid esters / / / 1.5 / beeswax / / / / 1.5 EGCG 0.2 0.2 0.2 0.2 0.2 VC 15 15 15 15 15 Citric Acid 0.03 0.03 0.03 0.03 0.03 water 50 50 50 50 50 Appearance stability - - - - - Encapsulation rate% 52.5 53.3 53.8 49.6 50.2 Intestinal retention rate % 87.2 89.1 90.2 85.2 86.9

[0201] Table 10 Effect of different fatty acid esters on the hygroscopicity of powders

[0202] Type / content (portions) Example 25 Example 26 Example 27 Example 28 Example 29 Maltodextrin 30 30 30 30 30 Moisture absorption rate % 0.79 0.71 0.66 0.91 0.83

[0203] It can be seen from Tables 9 and 10 that different fatty acid esters can achieve good encapsulation efficiency and intestinal retention rate. The prepared liposome powder has a low moisture absorption rate, which broadens the application of subsequent products.

[0204] Example 8 Different liposome compositions

[0205] According to the formula amount of each component in Table 11, different amounts of hydrophilic excipients were added, and the vitamin C liposome powder was prepared by spray drying to remove moisture at an inlet air temperature of 165°C and an outlet air temperature of 70°C. The hygroscopicity thereof was tested, and the results are shown in Table 12. Figure 5 The picture of the vitamin C liquid liposome before drying in Example 34 is shown, and it can be seen that the formed liquid liposome system is stable and uniform; Figure 6 The picture of the vitamin C liposome gel of Example 32 is shown, and it can be seen that the formed liposome gel is smooth and uniform, and the gel structure is stable; Figure 7Display Example 30 shows a picture of vitamin C liposome powder, and it can be seen that the formed vitamin C liposome powder has a dry, uniform and fine texture.

[0206] Table 11 Effects of different liposome compositions on liposomes

[0207]

[0208]

[0209] Table 12 Effect of different liposome compositions on the hygroscopicity of powders

[0210]

[0211] According to Tables 11 and 12, different ingredients such as acidity regulators and flavor sweeteners have little effect on liposomes, and there is no significant difference in the hygroscopicity of powders prepared by adding different hydrophilic excipients to the prepared liquid liposomes.

[0212] Example 9 Effects of different functional ingredients on liposomes

[0213] According to the formula of each component in Table 13, 30 parts of maltodextrin were added, the inlet air temperature was 165°C, the outlet air temperature was 70°C, and the water was removed by spray drying to prepare each liposome powder, and its hygroscopicity was tested. The results are shown in Table 14.

[0214] Table 13 Effects of different active ingredients on liposomes

[0215]

[0216]

[0217] Table 14 Effects of different functional ingredients on the hygroscopicity of powders

[0218] Type / content (portions) Example 35 Example 36 Example 37 Example 38 Example 39 Maltodextrin 30 30 30 30 30 Moisture absorption rate % 1.31 0.54 2.43 0.57 1.25

[0219] According to Table 13 and Table 14, when the effective ingredients are elderberry, glutathione, magnesium threonate, and ferric pyrophosphate, a higher encapsulation rate can be achieved, and the intestinal retention rate and moisture absorption are both good. Since the collagen peptide adopts the Kjeldahl method for nitrogen determination, the intestinal retention rate is 100%.

Claims

1. A liposome composition, characterized in that: The composition comprises phospholipids, fatty acid esters and epigallocatechin gallate; preferably, the composition further comprises water; preferably, the composition further comprises an effective ingredient; more preferably, the effective ingredient is a water-soluble ingredient.

2. A composition according to claim 1, wherein the water-soluble ingredients include water-soluble vitamins, water-soluble minerals, water-soluble plant extracts, water-soluble protein peptides and / or water-soluble non-protein thiols; preferably, the water-soluble vitamins include vitamin C; preferably, the water-soluble minerals include magnesium threonate and / or ferric pyrophosphate; preferably, the water-soluble plant extracts include elderberry; preferably, the water-soluble protein peptides include collagen peptides; preferably, the water-soluble non-protein thiols include glutathione.

3. The composition according to claim 1 or 2, comprising a water-soluble component, a phospholipid, a fatty acid ester and epigallocatechin gallate (EGCG); preferably, the fatty acid ester comprises one or more of mono- and di-glycerol fatty acid esters, citric acid fatty acid glycerides, lactic acid fatty acid glycerides, sucrose fatty acid esters, polyglycerol fatty acid esters and beeswax; Preferably, the water-soluble component is present in an amount of 0.2-45 parts by weight, such as 5-30 parts by weight, such as about 15 parts by weight; Preferably, the weight portion of the phospholipid is 0.4-6.5, such as 0.4-5, 2-6.5 or 2-5; Preferably, the fatty acid ester has a weight of 0.5-3.5, such as 1-3; Preferably, the epigallocatechin gallate (EGCG) is 0.03-0.9 by weight, such as 0.04-0.8; Preferably, the PC value of the phospholipid is 30-70, for example, the PC value is 40-70 or 55-70; Preferably, the weight portion of water is 0.2-90.

4. The composition according to any one of claims 1 to 3, further comprising one or more components selected from the group consisting of a sourness regulator, a hydrophilic auxiliary material, a flavor and a sweetener; preferably, the sourness regulator is selected from one or more of citric acid, lactic acid and malic acid; preferably, the hydrophilic auxiliary material is selected from one or more of dextrin, sugar alcohol and sodium starch octenylsuccinate; preferably, the sourness regulator is 0.01-5 parts by weight; preferably, the hydrophilic auxiliary material is 0.5-60 parts by weight; preferably, the flavor is 0.04-1.5 parts by weight; preferably, the sweetener is 0.02-0.3 parts by weight.

5. The composition according to any one of claims 1 to 4, which is a liquid liposome; preferably, the water-soluble component is dissolved in 0.2-90 parts by weight of water; preferably, the composition comprises water in an amount greater than 2 times the weight of the water-soluble component.

6. The composition according to any one of claims 1 to 4, which is a liposome gel; preferably, the composition comprises water in an amount 1 to 2 times the weight of the water-soluble components.

7. The composition according to any one of claims 1 to 4, which is a liposome powder.

8. The method for preparing the liquid liposome according to claim 5, characterized in that: The method comprises the following steps: 1) mixing phospholipids and fatty acid esters, heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring and mixing component A and component B into a uniform system, adding EGCG and stirring evenly, and homogenizing under high pressure to obtain liquid liposomes; preferably, the weight of water in step 2) is more than twice that of the water-soluble components; preferably, step 3) comprises stirring and mixing component A and component B into a uniform system, adding EGCG and one or more of an optional acidity regulator, flavor and sweetener and stirring evenly, and homogenizing under high pressure to obtain liquid liposomes.

9. The method for preparing the liposome gel according to claim 6, characterized in that: The method comprises the following steps: 1) mixing phospholipids and fatty acid esters, heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring and mixing component A and component B to form a uniform system, adding EGCG, stirring and homogenizing them under high pressure to obtain liposome gel; preferably, the weight of water in step 2) is 1-2 times that of the water-soluble components; preferably, step 3) stirring and mixing component A and component B to form a uniform system, adding EGCG and a sourness regulator, stirring and homogenizing them under high pressure to obtain liposome gel; preferably, flavors and sweeteners are added to the liposome gel obtained in step 3), and then the mixture is canned and sterilized.

10. The method for preparing the liposome powder according to claim 7, characterized in that: The method comprises the following steps: 1) mixing phospholipids and fatty acid esters, heating and dissolving them to obtain component A; 2) dissolving water-soluble components in water to obtain component B; 3) stirring and mixing component A and component B to form a uniform system, adding EGCG and stirring evenly, and homogenizing under high pressure to obtain liquid liposomes; 4) adding hydrophilic excipients and optionally sweeteners, mixing, sterilizing, and drying to obtain liposome powder; optionally, adding flavors after drying; preferably, in step 3), stirring and mixing component A and component B to form a uniform system, adding EGCG and a sourness regulator and stirring evenly, and homogenizing under high pressure to obtain liquid liposomes.

11. A product comprising the composition according to any one of claims 1 to 7; preferably, the product is selected from one or more of oral liquid, soft capsule, gel, soft candy, jelly, powder, tablet and hard capsule.

12. Use of the composition according to any one of claims 1 to 7 in the preparation of food, cosmetics or medicines; preferably, the food is a health food; preferably, the combination is prepared into one or more selected from oral liquid, soft capsule, gel, soft candy, jelly, powder, tablet, hard capsule.

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