LT6699B - Method for production of proliposomes by using up to 5% ethanol and the use thereof for encapsulation of lipophilic substances - Google Patents

Abstract

The present invention relates to a process for the production of proliposomes using up to 5% ethanol and their use for encapsulation of lipophilic substances. The materials used in the present invention comprise, but are not limited to, vitamins, minerals and plant extracts. Lipid encapsulated nutrients are made from lecithin, phosphatidylcholine and the like. The resulting product is a microcapsules in the form of liposomes, consisting of a single layer or multiple layers of phospholipid bilayer. The resulting product has little or no alcohol. The product according to the present invention is used for oral administration and has been shown to increase absorption and thus the bioavailability of nutrients.

Description

TECHNICAL FIELD

The invention relates to the field of food supplements, and more particularly to oral vitamins, minerals and herbal / plant extracts encapsulated in lipid blisters. Such microencapsulated preparations increase the solubility, absorption of nutrients in the gastrointestinal tract, their bioavailability and circulation in the blood.

BACKGROUND OF THE INVENTION

Food supplements are usually oral hard or semi-hard tablets, capsules or soft gel capsules. Such solid formulations are relatively inexpensive, easy to manufacture, convenient to use, and stable. Despite growing evidence that conventional food supplements do not provide the required nutrient uptake, they still occupy the majority of the pharmaceutical industry (Human Nutrition, Thirteenth Edition, Oxford University Press 2017; Blood, Volume 92, No. 4, August 15, 1998, psi 1191 -1198; ACG Case Reports Journal: Iron Pill-Induced Gastritis, 2013-10-01).

In addition to not providing an adequate supply of nutrients to the body, these preparations are not resistant to stomach acids and enzymes, so only a small part of the nutrients remain undegraded, reaching the small intestine, where they are absorbed.

Because conventional preparations do not provide adequate protection, nutrients can come in contact with and irritate the gastric mucosa. For example, iron supplements are known to cause gastrointestinal irritation, which is often manifested by nausea (ACG Case Reports Journal: Iron Pill-Induced Gastritis, 2013-10-01).

Dry plant extract preparations in standard capsules or tablets usually have a solubility problem - the bioactive substances in plants are poorly soluble in water and therefore difficult for intestinal enterocytes to absorb. The body's defense mechanisms quickly neutralize even a small amount of absorbed phytochemicals.

Not only the recommended amount of food supplements or nutrients consumed is important, but also their bioavailability (bioavailability). Bioavailability is a technical term meaning that less than 100% of the nutrients consumed are absorbed. Bioavailability is highly dependent on several factors such as digestion, dissolution, solubilization, absorption, organ uptake and release, enzymatic transformation, secretion and excretion, nutrient utilization (Bioavailability of Nutrients, https://www.researchqate.net/publication/ 3Q1702721 Bioavailability of Nutrients). Macronutrients, i. The bioavailability of carbohydrates, fats and proteins is usually high - more than 90% of the consumed macronutrients are absorbed and used by the body. On the other hand, the absorption and utilization of consumed trace elements such as vitamins and minerals, bioactive phytochemicals vary widely.

The increasing focus on uptake, stability, and protection of nutrients from degradation has led to the search for new nutrient transport systems. One of these methods is microencapsulation in liposomes.

Liposomes are spherical, closed vesicles composed of amphipathic phospholipids. Liposomes have been extensively studied and used to transport a variety of compounds. The liposome has at least one closed lipid bilayer membrane with a cavity for the liquid medium in the center. Liposomes are biocompatible carriers produced by a variety of methods. Liposomes can be monolayer, that is, composed of a single bilayer membrane, or multilayer, consisting of two or more concentrically arranged bilayer membranes.

Liposomes are mostly made from naturally occurring ingredients, so their application as a new preparation to food systems is easy to implement. Liposomes are usually composed of lecithin from natural sources such as soybeans, sunflower, eggs. These lecithins are close in structure to the plasma membrane of animal cells, so the liposome is easily recognized by small intestinal cells - the liposomes are easily absorbed by intestinal enterocytes. Three simultaneous uptake processes are thought to be possible: fusion to the cell membrane, active liposome endocytosis, and facilitated diffusion.

Improved nutrient uptake by several times is one of the most important advantages of microencapsulated products. Because phospholipids are amphipathic, liposomes can encapsulate both water- and lipid-soluble nutrients. They stabilize encapsulated materials and are suitable for encapsulation, transport and release of poorly water-soluble compounds such as lipid-soluble vitamins such as vitamin D3, vitamin K, or phytochemicals such as curcumin, resveratrol at the target site.

Due to the poor solubility in water in which lipids, and more specifically lecithin, are dispersed, lipids are usually dissolved in aqueous alcoholic solutions. In cases where the solvent is alcohol, it is recommended that the final alcoholic strength be not more than 5% by weight of the final product.

The product of the invention is prepared on the basis of a process for the production of proliposomes, in which phospholipids are dissolved in ethanol and heated to a mixture of proliposomes. When water is added to such a mixture, the proliposome mixture is converted into a liposome mixture (Journal of Pharmacy and Pharmacology: A Simple Method for the Preparation of Liposomes for Pharmaceutical Applications: Characterization of the Liposomes, 1991-03).

The objectives are to produce non-alcoholic proliposomes or proliposomes with the lowest possible ethanol content and the highest possible amount of encapsulated hydrophilic and lipophilic compounds, and to obtain proliposomes with the highest possible release of encapsulated compounds for use in food and cosmetics.

SUMMARY OF THE INVENTION

The present invention provides an encapsulated food supplement formulation. The materials used in the invention include, but are not limited to, vitamins, minerals, and plant extracts. Lipid encapsulation materials are made of lecithin, phosphatidylcholine, and the like. The resulting product is a liposome-shaped microcapsule composed of a single-layer or multilayer phospholipid bilayer. The resulting product has little or no alcohol. The product of the present invention is administered orally and has been shown to increase nutrient uptake and bioavailability.

DESCRIPTION OF THE FIGURES Fig. shows the total concentration of curcumin in the microcapsule suspension at 25 ° C and 40 ° C on days 1 and 90;

fig. depicts the release of vitamin D3 from a microcapsule at 25 ° C and 40 ° C;

fig. shows the total concentration of vitamin D3 in the microcapsule suspension at 25 ° C and 40 ° C on days 1 and 90;

fig. shows the concentration of vitamin D3 in the blood serum of rats during the 24 days of the experiment.

DETAILED DESCRIPTION OF THE INVENTION

Preparation of liposomes

Most known pharmaceutical liposome preparation processes are not suitable for dietary supplement liposomes, which are standard for oral administration, largely due to the use of non-nutritional ingredients in the pharmaceutical liposome composition.

In addition, many known preparation processes use heating, such as solubilizing lipids by heating, which raises the temperature of the lipid solution. Such processes may affect the performance or efficacy of the substance, compound or composition, especially those that are sensitive to heat and / or oxidation. When preparing liposomes, it is necessary to dissolve the lipids in organic solvents such as methanol, chloroform or ethanol. There are currently market requirements to remove residual organic solvents from liposomes or not to use ethanol in the production of liposomes. Therefore, additional processes are required in the production of liposomes for food supplements and pharmaceuticals. Various methods for preparing liposomes and encapsulating therapeutic materials are described, for example, in U.S. Pat. 3932657, 4311712, 5891465 and 5013556. Known methods include the reverse evaporation method described in U.S. Pat. 4235871.

Lipids used to form liposome compositions include vesicle-forming lipids having hydrocarbon chains, typically fatty acid tails, and a polar head group. These are phospholipids such as phosphatidylcholine, sphingolipids such as ceramide. Fatty acid chains may be saturated or unsaturated having one, two or more double bonds in the hydrocarbon chain. Phosphatidylcholine is a phospholipid with choline in the head group. More than one type of lipid can be used to make liposomes. The compositions can be varied to achieve the desired level of flow or stiffness.

The present invention describes improved proliposomal methods for the production of large and small monolayer and / or multilayer liposomes consisting of a mixture of phospholipids with encapsulated functional food elements such as curcumin, vitamins D3 and 1.1, and provides efficiency indicators for encapsulation by these methods. Improved nutrient uptake from microencapsulated products has been confirmed by preclinical in vivo studies.

The preparation of a microcapsule suspension using the proliposome method is given below.

The components of the formulation of the present invention include purified soy, sunflower or egg lecithin in a concentration of 0.10 to 20.0% by weight; α-tocopherol and / or other antioxidant in a concentration of 0.01 to 0.25% by weight; optionally up to 5% by weight of ethanol is used; vitamins, food supplements, plant extracts and other biologically active ingredients in concentrations ranging from 0.007% to 20.0% by weight; purified water from 50.0 to 99.9% by weight.

In addition to having encapsulated materials in the phospholipid bilayer, the compositions of the present invention may also include one or more food additives, including, but not limited to, flavoring agents, such as sweeteners, aromatic food additives; a stabilizer such as xylitol; preservatives such as potassium sorbate, sodium benzoate; buffers and acidity regulators.

The components - lecithin, ethanol, tocopherol acetate - are mixed in a high-capacity flask using a magnetic stirrer. After lipid dissolution, lipid- or water-soluble nutrients are added and mixed. An aqueous solution of citric acid is added with stirring. The mixture is heated to not more than 70 ° C, stirred and allowed to cool with constant stirring. The components - preservatives, sweeteners and citric acid - are added to the mixture and mixed. The solution is homogenized to obtain smaller and more uniform microcapsules.

Within the above limits, the procedure for preparing a liposomal curcumin is given below as one embodiment of the present invention:

1) Curcumin feedstock and lipids with a phosphatidylcholine content of> 50% and α-tocopherol are dissolved in 96% ethanol to a maximum of 5% ethanol in the final product. mixing is activated, maximum speed is set.

2) Add 0,01% citric acid solution in a gentle stream.

3) The temperature of the liposome mixture is heated to not more than 70 ° C; when this temperature is reached (measured by placing the thermometer in the mixture), the mixture is heated and stirred for up to 30 minutes;

4) The mixture is cooled to room temperature (~ 25 ° C) without stopping stirring;

5) Potassium sorbate (up to 0.2% by weight) and xylitol (up to 10% by weight) are dissolved in 0.01% citric acid solution. The resulting solution is instilled into the liposome mixture in 3 portions. After each stage, 10 min. maximum vortex mixing. This ensures the homogeneity of the liposomes;

6) Add 0.01% citric acid solution dropwise until the final volume of the preparation is reached. Stir at maximum speed for 10 min .;

7) Homogenize the solution to obtain homogeneous liposomal microcapsules;

8) The resulting solution is mixed with a mixture of glycerol (up to 3% by weight) and xanthan gum (up to 0.5% by weight).

Liposome size was measured by the dynamic light scattering method, metal contamination of the suspensions was analyzed by full-reflection X-ray fluorescence (TXRF) spectrometry, and phosphatidylcholine content was analyzed by thin-layer chromatography (TLC).

The following are examples with reference to Figure 1-4. Fig. 1 shows the stability of microencapsulated curcumin and vitamin D3 at different storage temperatures over 90 days. These examples are intended to illustrate the invention without limiting it, and may be applied to other phytochemicals, vitamins and minerals.

Liposomal curcumin and vitamin D3 samples were stored at 25 ° C and 40 ° C for 90 days; release of encapsulated compounds from microcapsules was observed. Total bioactive compounds were determined on days 1 and 90.

example. Release of microencapsulated curcumin

As shown in Figure 1, no release of curcumin was observed during 90 days of storage at 25 ° C and 40 ° C.

example. Release of microencapsulated vitamin D3 Fig. shows the release of vitamin D3 at 25 ° C and 40 ° C. Up to 22% of vitamin D3 was released from the microcapsules. Vitamin D3 excretion began to decrease from day 22 and degradation of released vitamin D3 was observed.

fig. shows the total concentration of vitamin D3 in the microcapsule suspension at 25 ° C and 40 ° C on days 1 and 90 of storage.

and a summary of 2 examples

The microencapsulated curcumin sample was the most stable because no release from the microcapsule was detected during 90 days of storage at 25 ° C and 40 ° C. The case of microencapsulated vitamin D3 was different, as on day 11 of storage at 25 ° C and 40 ° C, the release of 15% and 22% of vitamin D3 was detected, respectively. The concentration of vitamin D3 released began to decrease after 22 days, and after 90 days it was no longer detectable, probably due to the breakdown of released vitamin D3. The microencapsulated vitamin D3 remained stable, which means that the present invention protects the encapsulated nutrients from degradation.

example. The bioavailability of vitamin D3 example shown in Figure 4 shows the bioavailability of microencapsulated vitamin D3 in rats. This example is intended only to illustrate the invention and its advantages and can be applied to other phytochemicals, vitamins and minerals.

Serum levels of vitamin D3 in rats over the 24 days of the study: rats were divided into 3 groups; from day 1 to day 7, rats were fed 2000 IU / kg of vitamin D3 in different forms: microencapsulated, oil-based, and micellar.

With microencapsulated vitamin D3, steady-state serum vitamin D3 levels were maintained for the longest time even after discontinuation of vitamin D3. Microencapsulated vitamin D3 was more bioavailable than oil-based vitamin D3 and almost twice as bioavailable as micellar vitamin D3.

This study demonstrates better bioavailability of vitamins, minerals, and phytochemicals using microencapsulation technology. The invention is characterized by a higher uptake of the active substances, as well as a prolonged effect due to the uptake of the substances through the lymphatic system, in contrast to the normal absorption of nutrients directly into the blood. The method of the present invention is superior to the use of standard oral preparations, micellarized oral preparations and oil-based preparations, as the latter are much less absorbed and transfer nutrients directly to the liver for presystemic metabolism. The liver has innate mechanisms of neutralization of certain nutrients, especially phytochemicals, due to exposure to cytochromes P450. Microencapsulated materials avoid such presystemic metabolism.

Claims (4)

DEFINITION OF THE INVENTION A process for the production of proliposomes, comprising dissolving a liposome-forming amphipathic lipid and a lipid or water-soluble bioactive compound to obtain liposome-shaped microcapsules, comprising the steps of: (a) components: lecithin, tocopherol acetate and, optionally, ethanol are mixed in a high-capacity flask using a magnetic stirrer; (b) after reconstitution of the lipids, lipid-soluble or water-soluble nutrients such as vitamin D3, vitamin K, curcumin, resveratrol and others are added and mixed; (c) an aqueous solution of citric acid is added with stirring; (d) the resulting mixture is heated to not more than 70 ° C, stirred and allowed to cool; (e) the components preservatives, sweeteners and citric acid are added to the cooled mixture and mixed; f) Homogenize the solution to obtain small liposomal microcapsules of uniform size. 2. The method according to claim 1, characterized in that from 0.10 to 20.0% by weight of purified soy, sunflower or egg lecithin is added; from 0.01 to 0.25% by weight of α-tocopherol and / or other antioxidant; optionally ethanol up to 5% by weight; from 0.007 to 20.0% by weight of vitamins, food supplements, plant extracts and other biologically active ingredients; from 50.0 to 99.9% by weight of purified water. A method according to any preceding claim for the preparation of liposomal curcumin, comprising the steps of: (a) curcumin raw material and lipids, phosphatidylcholine> 50%, and α-tocopherol are dissolved in 96% ethanol to a maximum of 5% ethanol in the final product; (b) stirring is started, the maximum speed is set to avoid foaming of the solution; (c) a 0,01% citric acid solution is added in a gentle stream; (d) the mixture is heated to not more than 70 ° C; (e) when this temperature is reached, the mixture is heated and stirred for up to 30 minutes; (f) cooling the mixture to room temperature without interruption; (g) potassium sorbate (up to 0,2% by weight) and xylitol (up to 10% by weight) dissolved in 0,01% citric acid solution; h) The resulting solution is instilled into the liposome mixture in 3 portions over 10 minutes after each step. maximum vortex mixing, thus ensuring liposome homogeneity; (i) a 0.01% citric acid solution is added dropwise until the final volume of the preparation is reached and the mixture is stirred for 10 minutes; j) homogenizing the resulting solution to obtain homogeneous liposomal microcapsules; (k) the solution is mixed with a mixture of glycerol (up to 3% by weight) and xanthan gum (up to 0,5% by weight). Use of a method according to any preceding claim for the encapsulation of lipophilic substances.