Cell-sized liposome, method for estimating concentration of procyanidin using cell-sized liposome, method for estimating concentration of procyanidin using observation result of cell-sized liposome and calibration curve, and method for measuring concentration of procyanidin

Abstract

To provide a simple procyanidin analyzing method capable of being performed at a low cost and having a form such as preliminary analysis or screening for selecting a sample to be subjected to precise analysis by HPLC (high performance liquid chromatography).SOLUTION: The cell-sized liposome 110 is a cell-sized liposome composed of a lipid component system using one or more kinds of saturated phospholipids 13 in addition to one or more kinds of unsaturated phospholipids 12, and a sample or procyanidin 18 of a procyanidin concentration estimation object, and is used for procyanidin concentration estimation from a phase separation domain generation pattern by generating a different phase separation domain generation pattern by the concentration of the procyanidin 18. The lipid component system of the cell-sized liposome 110 may include a sterol-based lipid such as cholesterol. Further, a calibration curve can be used for concentration estimation.SELECTED DRAWING: Figure 1-3

Description

[Technical Field]

[0001] The present invention relates to cell-sized liposomes, a method for estimating procyanidin concentration using cell-sized liposomes, a method for estimating procyanidin concentration using observation results of cell-sized liposomes and a calibration curve, and a method for measuring procyanidin concentration, and in particular to a technology that can estimate the concentration of procyanidin contained in apple juice or the like at low cost and in a relatively short time. [Background technology]

[0002] Apple cultivation is a major industry in Aomori Prefecture. According to the 2021 Crop Statistics Survey (Ministry of Agriculture, Forestry and Fisheries), the area under apple cultivation in Aomori Prefecture is 20,400 hectares, accounting for more than half of the total area in Japan. However, this area has been declining over the years, and maintaining and securing the prefecture's position as a major apple-producing region, which plays an important role not only in the local industrial economy but also in culture and tourism, is a key issue for Aomori Prefecture. Research in the region has been accumulating that the functional components contained in apples are effective in maintaining health and preventing disease. For example, "Prime Apple!" (Fuji variety, registered trademark) has been registered as a functional food, specifically for the fruit's ability to reduce visceral fat, thanks to the properties of procyanidins.

[0003] Procyanidins contained in apples are the main component of the polyphenols contained in apples (collectively known as apple polyphenols). Apple polyphenols are made up of various components, but procyanidins account for approximately 60% of them (from the Aomori Prefecture Apple Council website). Procyanidins are known to have particularly high antioxidant properties and also have fat-reducing effects, and apples, which contain a lot of procyanidins, are sold as functional foods.

[0004] Procyanidin concentration measurement tests use HPLC (high-performance liquid chromatography). This equipment is not only very expensive to purchase, but also has the disadvantage of high running costs because it requires the use of high-purity organic solvents for analysis for each measurement. Furthermore, it takes several hours to set up the equipment, and multiple standard samples must be run to create a calibration curve before concentration measurements can be performed, which takes a considerable amount of time.

[0005] Patent applications and the like have been filed for techniques for measuring procyanidins. For example, Patent Document 1, cited below, discloses a method for quantifying procyanidins (such as n-polymers of catechin: n≧1, where n is an integer) in foods and beverages as a quantitative method capable of specifically and accurately measuring procyanidins, characterized in that procyanidins separated by size exclusion chromatography are quantified by a fluorescence detection method. This document also discloses a method for quantifying procyanidins separated by size exclusion chromatography by a fluorescence detection method. Furthermore, Non-Patent Document 1, written by the present inventor, discloses a technique for estimating the concentration of procyanidins by adding a procyanidin extract during the lipid film preparation stage when preparing cell-sized liposomes and observing the state of the phase-separated domains. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2009-156813 A "Method for quantifying procyanidins" [Non-patent literature]

[0007] [Non-Patent Document 1] Membranes (2022) Vol. 12, p. 943 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, procyanidins, known as apple polyphenols and functional health components, have been attracting increasing attention, and labeling the procyanidin content of processed apple foods and other products is expected to add value to these foods. However, HPLC (high-performance liquid chromatography) used to measure procyanidins has problems such as high initial and running costs, as well as the considerable time and effort required. It would be convenient to have a simple analytical method for preliminary analysis, or so-called screening, to select samples for precise HPLC analysis, which does not need to be as accurate as HPLC but can be performed as inexpensively as possible.

[0009] Liposomes, the most simplified model of biological membranes used in many experiments and research, are formed by the self-assembly of phospholipids, which have both hydrophilic and hydrophobic fatty chains, with the hydrophilic portion facing outward to form a lipid bilayer membrane, which then closes into a sac-like shape. Liposomes with diameters ranging from a few micrometers to several tens of micrometers are roughly the same size as living cells and are called cell-sized liposomes (giant liposomes). Cell-sized liposomes are useful for research into the elucidation of biological membrane control mechanisms, due to their advantages, such as their ability to be directly observed with an optical microscope and their use in creating experimental models that mimic cells.

[0010] Furthermore, it has been revealed that the cell membrane contains phase-separated domains (raft domains) rich in saturated lipids and cholesterol, which are responsible for signal transduction and other functions. Raft domains are thought to move, adhering and separating in response to intercellular signaling. Similar domain structures have also been observed in artificially constructed liposomes from lipids, and research in this area is also in full swing. These studies using biological model membranes (artificial cell membranes) and human-derived cells have revealed that the three-dimensional dynamics and two-dimensional (domain structure) assembly dynamics of membranes are related to signal transduction using intracellular calcium ions as an indicator, and that altering the structure of lipids and sterols alters the temperature response of the domain structure.

[0011] The inventor has previously conducted research to clarify the function of cell membranes by observing the movement and phase separation structure of biological model membranes and cell-sized liposomes under a microscope, and has published the results using the observed videos and images. For example, he discovered that the concentration of procyanidin affects the membrane fluidity and formation size of cell-sized liposomes (T. Yoda, Chemistry Select 2022).

[0012] The inventors have also demonstrated that domain structures form in membranes containing antioxidants when subjected to oxidative stress. Specifically, mixing cell-sized liposomes with solutions of polyphenols known for their antioxidant properties, such as resveratrol and theaflavin, induces membrane dynamics, such as membrane contraction and fluctuation, resulting in the emergence of phase-separated domains during observation (HHT. Phan, T. Yoda et al., Biochim. Biophys. Acta, Biomembr. 2014). Furthermore, fluorescence microscopy has revealed that concanavalin A, a functional component found in sword beans that activates the immune system, induces the accumulation of domain structures in the cell membrane and activates intracellular signaling (S. Yabuuchi et al., J. Biosci. Bioeng. 2017).

[0013] When cell-sized liposomes are prepared by mixing equal amounts of the unsaturated phospholipid 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the saturated phospholipid dipalmitoylphosphatidylcholine (DPPC), they undergo phase separation into a DOPC-rich liquid disordered phase domain (Ld domain) and a DPPC-rich solid ordered phase domain (So domain). Furthermore, when cholesterol is added to this mixture, many cell-sized liposomes are observed that phase separate into a DOPC-rich liquid disordered phase domain (Ld domain) and a DPPC- and cholesterol-rich liquid ordered phase domain (Lo domain).

[0014] In the aforementioned Non-Patent Document 1, the inventors reported a technique for estimating the concentration of cell-sized liposomes by adding apple juice during the lipid film preparation stage and observing the state of the phase-separated domains. However, these methods involve a step of vacuum drying the organic solvent after film preparation, which can result in the volatilization and evaporation of procyanidins. Furthermore, a procyanidin extraction step is required.

[0015] The problem to be solved by the present invention is to provide a simple analytical method for procyanidin concentration, in the form of a preliminary analysis (i.e., a so-called screening) for selecting samples for precise analysis by HPLC, which does not necessarily require the same level of accuracy as HPLC, but can be performed at the lowest possible cost and in a short time, taking into account the progress and results of research using liposomes and the state of the art in measuring the functional component procyanidin. In other words, the present invention provides a simple method for easily distinguishing between procyanidin concentration using cell-sized liposomes, utilizing clear differences characterized by observation due to the interaction between procyanidin and membranes. Furthermore, the present invention particularly aims to provide a labor-saving method in which the volatilization and evaporation of procyanidin is suppressed by adding a procyanidin-containing substance, such as apple juice, to the sample at a stage other than film production, thereby obtaining relative concentration information without extracting the sample. [Means for solving the problem]

[0016] The inventors have carried out research with the above problem in mind. To prepare cell-sized liposomes, we attempted to add apple juice during the hydration step. First, we found that when apple juice was added during the hydration step to prepare cell-sized liposomes containing only DOPC, micrometer-scale liposomes were not observed. Therefore, we attempted again by diluting apple juice with pure water, and found that cell-sized liposomes were formed at a 100-fold dilution. Furthermore, when preparing cell-sized liposomes containing only DOPC, adding diluted apple juice during the hydration step resulted in significantly smaller liposome sizes than when hydrated with pure water. Furthermore, adding a solution containing pre-prepared procyanidins to diluted apple juice during the hydration step increased the liposome size. This is consistent with a previous report (T. Yoda, Chemistry Select 2022) that showed that liposome size increased depending on the concentration of procyanidins added to films.

[0017] Therefore, it was inferred that even if diluted apple juice was added during hydration instead of during film preparation, procyanidins in the apple juice were incorporated during the cell-sized liposome formation stage. We also discovered that the proportion of phase-separated domain structures differed between the case where diluted apple juice was added during hydration and the case where a solution in which part of the diluted apple juice had been replaced with procyanidins was added first. We found that this difference in proportion, specifically the procyanidin content in the sample, could be determined by the ratio of solid-ordered to liquid-disordered phases, i.e., the proportion of phase separation.

[0018] In addition to the above findings obtained through experiments using diluted apple juice, we also confirmed the cost reduction effect through trial calculations, demonstrating that cell-sized liposomes can be used as a tool for analyzing procyanidin concentrations. Specifically, to analyze procyanidins contained in diluted apple juice, etc., this method involves observing the proportion of domain structures in cell-sized liposomes containing unsaturated lipids, more preferably cell-sized liposomes containing unsaturated and saturated lipids, prepared by adding diluted apple juice during hydration, and quickly estimating the relative concentration of procyanidins. (Note: The use of "unsaturated lipids" and "saturated lipids" here, rather than "unsaturated lipids such as DOPC" or "saturated lipids such as DPPC," indicates that the cell-sized liposomes of the present invention can also be prepared using lipids other than the phospholipids described herein, such as glycolipids. These lipids are also within the scope of the present invention.) The present invention, thus completed, i.e., the invention claimed in this application as a means for solving the above-mentioned problems, or at least the invention disclosed therein, is as follows:

[0019] [1] A lipid component system comprising one or more unsaturated phospholipids; and The sample to be estimated for procyanidin concentration or procyanidin A cell-sized liposome comprising: Different procyanidin concentrations resulted in different phase-separated domain formation patterns. This allows cell-sized liposomes to be used to estimate procyanidin concentration from the phase-separated domain formation pattern. [2] The cell-sized liposome according to [1], characterized in that the lipid component system contains one or more saturated phospholipids. [3] The cell-sized liposome according to claim 2, wherein the lipid component system contains one or more sterol-based lipids. [4] The cell-sized liposome according to [1], characterized in that the lipid component system is composed of an unsaturated phospholipid, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), and a saturated phospholipid, Dipalmitoylphosphatidylcholine (DPPC). [5] The cell-sized liposome according to [1], characterized in that the lipid component system is composed of an unsaturated phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a saturated phospholipid dipalmitoylphosphatidylcholine (DPPC), and a sterol lipid cholesterol.

[0020] [6] The cell-sized liposome according to any one of [2], [3], [4], and [5], characterized in that, when used at room temperature, the molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is unsaturated phospholipids:saturated phospholipids=75 to 10:25 to 90. [7] The cell-sized liposome according to any one of [2], [3], [4], and [5], characterized in that, when used at room temperature, the molar concentration composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is equal. [8] The cell-sized liposome according to any one of [1], [2], [3], [4], and [5], characterized in that a fluorescent reagent for detecting phase-separated domains is added. [9] The cell-sized liposome according to [8], characterized in that the fluorescent reagent used is one or more of a reagent for staining a solid-ordered phase (So phase), a reagent for staining a liquid-disordered phase (Ld phase), or a reagent for staining a sterol-based lipid.

[10] The cell-sized liposome according to any one of [1], [2], [3], [4], and [5], characterized in that it is produced by a static hydration method.

[11] The cell-sized liposome according to

[10] , wherein after the lipid film is prepared, a sample whose concentration is to be estimated is added during hydration of the cell-sized liposome.

[0021]

[12] A method for estimating the relative concentration of procyanidin in a subject for concentration estimation, comprising using a cell-sized liposome according to any one of [1], [2], [3], [4], and [5], which is configured using a procyanidin standard having a known concentration, as a liposome for obtaining information on phase-separation domains, and using the liposome for obtaining information on phase-separation domains, The cell-sized liposomes to be used for estimating the concentration of procyanidins are prepared using a sample to be used for estimating the concentration of procyanidins, and the cell-sized liposomes are subjected to fluorescence microscopic observation. The state of phase-separated domain formation, i.e., whether or not a phase-separated domain is formed, or the state in which it is formed, or the state of cell-sized liposome formation, is read, referring to procyanidin concentration-dependent information on the phase-separation domain formation status or cell-sized liposome formation status in the liposome for obtaining phase-separation domain information, Estimate the relative concentration of procyanidins contained in the target substance A method for estimating procyanidin concentration,

[13] The method for estimating a procyanidin concentration according to

[12] , wherein a calibration curve based on the observation results of the liposome for obtaining phase-separated domain information is used to estimate the procyanidin concentration.

[14] When estimating the procyanidin concentration, the following <lr>The method for estimating a procyanidin concentration according to

[12] , characterized in that a calibration curve based on the observation results of the liposome for obtaining phase-separated domain information is used. <lr>Liposomes for obtaining information on phase-separated domains containing sugars and / or acids as components that are components to be included in the concentration estimation target

[15] The above <lr>The liposome for obtaining information on the phase separation domain is as follows: <s>or / and The method for estimating a procyanidin concentration according to

[14] , characterized in that it is at least one of the following: <s>The sugar may be one of fructose, sucrose, glucose, or sorbitol, or any two or more thereof. < / s> <s> The acid contains one or more of the following: malic acid, citric acid, acetic acid, and caproic acid.

[16] The method for estimating a procyanidin concentration according to

[12] ,

[13] ,

[14] , or

[15] , wherein the subject of concentration estimation is apple juice or a processed apple juice product.

[17] A method for measuring procyanidin concentration, comprising first estimating the procyanidin concentrations of a plurality of samples by the procyanidin concentration estimation method according to any one of

[12] ,

[13] ,

[14] , and

[15] , and then precisely measuring the procyanidin concentrations of some of the samples that have been subjected to the procyanidin concentration estimation process by high performance liquid chromatography.

[18] The method for measuring procyanidin concentration according to

[17] , wherein the subject of concentration measurement is apple juice or a processed apple juice product. [Effects of the Invention]

[0022] The cell-sized liposomes, procyanidin concentration estimation method using cell-sized liposomes, procyanidin concentration estimation method using observation results of cell-sized liposomes and a calibration curve, and procyanidin concentration measurement method of the present invention are configured as described above, and therefore provide a simple measurement method that utilizes clear differences characterized by observation due to the interaction between procyanidins and membranes, and a simple discrimination method using cell-sized liposomes. Specifically, procyanidin concentration can be analyzed at significantly lower cost and in a shorter time than conventional methods. In particular, the procyanidin concentration estimation method using a calibration curve can further improve the accuracy and precision of concentration estimation and measurement.

[0023] Conventional measurement methods, which require expensive HPLC, have the problem of requiring a long time for equipment setup, etc. However, the concentration estimation method of the present invention makes it possible to use a relatively inexpensive fluorescence observation microscope as the measurement device, reducing not only the initial cost but also the running cost, making it easy to analyze the procyanidin concentration of apple juice, etc., and enabling measurements to be performed quickly.

[0024] In the present invention, cell-sized liposomes are prepared by incorporating the procyanidin to be measured and then observed under a fluorescence microscope. Once the optimal conditions for the combination of lipids constituting the cell-sized liposomes are determined, the procyanidin concentration can be estimated simply by short-term observation. Furthermore, the present invention can suppress the volatilization and evaporation of procyanidins, and can easily obtain information on relative concentrations without extracting them from samples, thereby saving labor.

[0025] As an effective example of its use, the present invention can be used for preliminary analysis to select samples for precision analysis by HPLC, or for simple procyanidin concentration determination in a screening-like manner, which is highly useful. Therefore, by combining the method of the present invention with HPLC, a rapid, low-cost, and precise analytical system can be realized. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. [Figure 1-2] FIG. 1 is a photograph showing an example of a pattern of phase-separated domains formed in the cell-sized liposomes of the present invention. [Figure 1-3] FIG. 1 is a conceptual diagram showing another basic structure of the cell-sized liposome of the present invention, which also contains saturated phospholipids. [Figure 2] FIG. 1 is a conceptual diagram showing another configuration of the cell-sized liposome of the present invention, which also contains a sterol lipid. [Figure 3] 1 shows the chemical structural formulas of lipids and procyanin B2, which are examples of constituent elements of the cell-sized liposome of the present invention. [Figure 4] FIG. 1 is an explanatory diagram showing an example of a method for producing the cell-sized liposome of the present invention. [Figure 5] FIG. 1 is an explanatory diagram conceptually showing the configuration of the procyanidin concentration estimation method of the present invention. [Figure 6] FIG. 1 is a flow chart showing the configuration of the procyanidin concentration measurement method of the present invention. [Figure 6-2] 1 is a flow chart showing the configuration of the procyanidin concentration estimation method of the present invention (the following figures relate to the Examples). [Figure 7] 1 is a graph showing the structural composition of phase-separated domains in reference liposomes according to Examples (Cases 1, 2, and 3). [Figure 8] 1 is a graph showing a calibration curve prepared based on the phase-separated domain structure composition when the lipid composition is 1 in cell-sized liposomes for estimating procyanidin concentrations in Examples (Cases 1, 2, and 3). [Figure 9] 1 is a graph showing a calibration curve prepared based on the phase-separated domain structure composition when the lipid composition is 2 in cell-sized liposomes for estimating procyanidin concentrations in Examples (Cases 1, 2, and 3). [Figure 10] 1 is a graph showing a calibration curve prepared based on the phase-separated domain structure composition when the lipid composition is 3 in cell-sized liposomes for estimating procyanidin concentrations in Examples (Cases 1, 2, and 3). [Figure 11] 1 is a graph showing a calibration curve prepared based on the phase-separated domain structure composition when lipid composition 1 is used and sugar is contained in cell-sized liposomes for estimating procyanidin concentrations in Examples (Cases 1, 2, and 3). [Figure 12] 1 is a graph showing the results of measuring the phase transition temperature, i.e., the domain disappearance temperature, of each cell-sized liposome according to Examples (Cases 1, 2, and 3). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will be described in detail below with reference to the drawings. FIG. 1 is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. As shown in (a) of the figure, the cell-sized liposome 10 is a cell-sized liposome composed of a lipid component system using one or more types of unsaturated phospholipids 2 (the figure shows an example in which only one type of unsaturated phospholipid 2 is used; the desired effects of the present invention can be sufficiently achieved with just one type) and a sample or procyanidin 8 for which procyanidin concentration is to be estimated. Its main structure is that it generates different phase-separated domain formation patterns depending on the procyanidin concentration, and can be used to estimate procyanidin concentration from the phase-separated domain formation patterns. (b) of the figure shows a cell-sized liposome 10' of the present invention containing a fluorescent reagent 9 as a component, which will be described later.

[0028] With this configuration, the cell-sized liposomes 10 exhibit different phase-separated domain formation patterns depending on the concentration of procyanidin 8 contained therein, and the phase-separated domain formation patterns can be used to estimate the concentration of the contained procyanidin. At the very least, by observing the phase-separated domain formation patterns, it is possible to determine whether the procyanidin content of a sample is relatively high or low.

[0029] The cell-sized liposome of the present invention having the structure shown in FIG. 1 will now be described in more detail. The ability to observe phase-separated domains under fluorescence observation with a single unsaturated lipid, such as DOPC (described below), is believed to be due to reactive oxygen species generated during fluorescence observation. Specifically, during fluorescence observation, excitation light is applied to observe the fluorescence emitted from the fluorescent dye, which generates reactive oxygen species that oxidize and cleave the unsaturated bond, i.e., part of the double bond, of the unsaturated lipid. As a result, two types (or several types) of lipids exist in the system: unoxidized unsaturated lipids and oxidized saturated lipids. This is observed as phase separation in the lipid membrane.

[0030] In fact, unsaturated lipids such as DOPC have a low phase transition temperature, allowing liposomes to be prepared without heating, and the formation of phase-separated domains can be easily confirmed. However, saturated lipids such as DPPC do not have unsaturated bonds and are therefore not susceptible to oxidation by reactive oxygen species during fluorescence observation. Therefore, liposomes composed solely of saturated phospholipids are unlikely to produce the desired phase-separated domains.

[0031] Figures 1-2 are photographs showing examples of phase-separated domain formation patterns in cell-sized liposomes of the present invention. These are micrographs showing typical patterns, of which A is an So (solid-ordered phase) / Ld (liquid-disordered phase) domain structure, B is an Lo (liquid-ordered phase) / Ld (liquid-disordered phase) domain structure, and C is a uniform membrane vesicle. Observations using a fluorescence microscope have shown that in the present invention, the So / Ld domain structure pattern shown in C can be particularly suitably used for concentration estimation. Observations using a fluorescence microscope will be discussed further below.

[0032] 1-3 are conceptual diagrams showing another basic structure of the cell-sized liposome of the present invention. As shown in (a) of the figure, the cell-sized liposome 110 is a cell-sized liposome composed of a lipid component system that uses one or more types of unsaturated phospholipids 12 (the figure shows an example in which only one type of unsaturated phospholipid 12 is shown) and one or more types of saturated phospholipids 13 (the figure shows an example in which only one type of saturated phospholipid 13 is shown; the desired effect of the present invention can be sufficiently achieved with just one type), and a sample whose procyanidin concentration is to be estimated or procyanidin 18. The cell-sized liposome generates different phase-separated domain formation patterns depending on the concentration of procyanidin 18, and is therefore primarily configured to be used for estimating procyanidins from the phase-separated domain formation patterns.

[0033] The present cell-sized liposome 110 having such a configuration also produces and exhibits different phase-separated domain generation patterns depending on the concentration of procyanidin 18 contained therein, and the procyanidin concentration contained therein can be estimated using this phase-separated domain generation pattern. At the very least, it is possible to determine whether the procyanidin content of a sample is relatively high or low by observing the phase-separated domain generation pattern. Furthermore, in order to more fully achieve the intended object of the present invention, the present cell-sized liposome 110, which is composed of unsaturated lipids and saturated lipids as components, is a more desirable configuration than the cell-sized liposome 10 shown in FIG. 1.

[0034] 2 is a conceptual diagram showing another configuration of the cell-sized liposome of the present invention, which also contains a sterol-based lipid. As shown in (a) of the figure, the cell-sized liposome 210 is a cell-sized liposome composed of a lipid component system containing one or more types of unsaturated phospholipids 22 (the figure shows an example in which only one type of unsaturated phospholipid 22 is shown), one or more types of saturated phospholipids 23 (the figure shows an example in which only one type of saturated phospholipid 23 is shown), and one or more types of sterol-based lipids 25 (the figure shows an example in which only one type of sterol-based lipid 25 is shown; the desired effect of the present invention can be sufficiently achieved with just one type), and a sample or procyanidin 28 for which procyanidin concentration is to be estimated. The cell-sized liposome generates different phase-separated domain formation patterns depending on the concentration of procyanidin 28, and is therefore primarily configured to be used for estimating procyanidin concentration from the phase-separated domain formation patterns.

[0035] The cell-sized liposome 210 having such a configuration also produces and exhibits different phase-separated domain generation patterns depending on the concentration of procyanidin 28 contained therein. Therefore, the concentration of procyanidin contained therein can be estimated using this phase-separated domain generation pattern, and at least, it is possible to determine whether the procyanidin content of a sample is relatively high or low by observing the phase-separated domain generation pattern.

[0036] Sterol lipids are not essential elements for constructing the cell-sized liposomes of the present invention. However, there are advantages to using sterol lipids such as cholesterol. First, the phase transition temperature is lowered, allowing for a lower hydration temperature. In the present invention, heating is performed during hydration to prepare cell-sized liposomes, but by performing hydration at a lower temperature, thermal decomposition of procyanidins can be suppressed.

[0037] Furthermore, sterol lipids such as cholesterol have the effect of increasing the fluidity of phospholipids, resulting in better mixing of lipids. This reduces the bias in the composition of each liposome produced, increasing the uniformity of the liposomes produced. As a result, the reproducibility of the implementation of the procyanidin relative concentration estimation method is improved. There are also other advantages when using specific fluorescent reagents (described below).

[0038] On the other hand, there are advantages to intentionally not including cholesterol in the lipid component system. This is the case when primarily observing changes in the So / Ld phase-separated structure, as in the method for estimating the relative procyanidin concentration of the present invention, which will be explained later with reference to Figure 5 and other figures. By not including cholesterol in the lipid component system, almost all of the resulting cell-sized liposomes are So / Ld. Therefore, when primarily observing changes in the So / Ld phase-separated structure, the phase-separated domain formation pattern can be clearly distinguished. When the concentration of added cholesterol is low, the So / Ld structure is dominant, but some Lo / Ld phase separation is mixed in, making it more complicated to interpret the distinguishing pattern.

[0039] In particular, the present invention can produce cell-sized liposomes having a lipid component system using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as the unsaturated phospholipid and dipalmitoylphosphatidylcholine (DPPC) as the saturated phospholipid. Furthermore, in addition to this configuration, cell-sized liposomes can also be produced with a lipid component system that includes cholesterol (Chol), a sterol lipid. The following explanation will mainly focus on cell-sized liposomes having a lipid component system of either DOPC + DPPC or DOPC + DPPC + Chol.

[0040] Figure 3 shows the chemical structures of lipids, which are examples of components of the cell-sized liposome of the present invention, and procyanidin B2 (PB2). In the figure, A is DOPC, B is DPPC, C is cholesterol, and D is PB2. Procyanidin B2 (PB2) is a procyanidin dimer and is used as a standard substance for concentration measurement.

[0041] When used at room temperature, the lipid component system of the cell-sized liposomes of the present invention can have a molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system of unsaturated phospholipids:saturated phospholipids = 75-10:25-90. Alternatively, when used at room temperature, the lipid component system may have an equal molar composition of unsaturated phospholipids and saturated phospholipids. Here, room temperature may be the generally understood temperature of about 20°C, or, more preferably, 22±2.0°C.

[0042] As an example of a specific lipid species, the lipid component system of the cell-sized liposome of the present invention, when used at room temperature, has a molar concentration composition in which both DOPC and DPPC are 45 or more, and if the lipid component system also contains cholesterol, the cholesterol can be the remaining amount.

[0043] In the present invention, the most desirable phase-separated domain composition for observation to estimate procyanidin concentration is the So / Ld domain, as described above. The molar concentration composition at which this domain is observed has been reported in the following examples. DOPC:DPPC:Chol = 37.5~50 : 37.5~50 : 0~15 DOPC:DPPC:Chol = 75~10 : 25~90 : 0~10

[0044] The reason why the composition range differs depending on the reported examples is that the domain formation is affected by temperature. According to the results of the inventor's experiments at room temperature (around 20°C), The ratio is DOPC:DPPC:Chol = 42.5-50: 42.5-50: balance. For example, if Chol is 10, the So / Ld domain structure can be obtained in a molar composition range of DOPC 0-65 and DPPC 25-90. As mentioned above, DOPC and DPPC can also be used in equal amounts. In the examples described below, DOPC and DPPC are used in equal amounts (50:50).

[0045] Phase-separated domains are also observed even when cholesterol is 15 or higher. For example, when cholesterol is 20 to 40 and phospholipids are 60 to 80, Lo / Ld (liquid-ordered / liquid-disordered) domains are observed. However, no correlation was confirmed between the domain formation rate and procyanidin concentration in this domain pattern.

[0046] As shown in Figures 1, 1-3, and 2(b), the cell-sized liposomes of the present invention may be configured to contain a fluorescent reagent 9 for detecting phase-separated domains. The presence or absence of a phase-separated domain structure is determined by fluorescent observation of the cell-sized liposomes, and the fluorescent reagent 9 is added for this purpose and does not affect the formation of phase separation. The fluorescent reagent 9 may be one or more of the following: a liquid-ordered phase (Lo phase) staining reagent, a liquid-disordered phase (Ld phase) staining reagent, or a sterol-based lipid staining reagent.

[0047] In the examples described below, the fluorescent reagent used is rhodamine B (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt) (rhodamine DHPE), which is added at 1% molar concentration of lipid. This is a relatively common fluorescent reagent for staining the DOPC-rich Ld phase. On the other hand, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4yl)(ammonium salt) (NBD-PE) is used to stain the DPPC-rich So or Lo phases. Because these have different fluorescent wavelengths, they can be used separately or simultaneously.

[0048] In addition, NBD-Cholesterol, for example, is used to fluorescently stain the cholesterol portion. In the present invention, which uses the So / Ld domain structure, the DPPC-rich So domain is stained. When using NBD-Cholesterol, it is thought that the degree of fluorescent staining will be better if the lipid component system contains cholesterol. Of course, fluorescent reagents other than those described above can also be used in the present invention.

[0049] FIG. 4 is an explanatory diagram showing an example of a method for preparing cell-sized liposomes of the present invention. As shown in the figure, the cell-sized liposomes can be prepared by the static hydration method. Alternatively, after preparing a lipid film, a sample whose concentration is to be estimated can be added during hydration of the cell-sized liposomes. Specifically, liposomes can be prepared using the following basic method: dissolving the lipids constituting the liposomes in a solvent (chloroform CH(Cl)3 in the example shown in the figure) in a container such as a test tube, evaporating the solvent to form a lipid film on the inner wall of the container, and then hydrating the lipid film with a diluted apple juice sample, which is the target sample.

[0050] In the examples described below, the lipid membrane and the sample to be measured were heated to approximately 50°C during hydration, and cell-sized liposomes were prepared using the static hydration method. This is a process to ensure that the lipids and procyanidins are uniform in the lipid membrane by heating the temperature to above 41°C, the melting point of DPPC. Note that approximately 50°C is merely an example, and it goes without saying that the present invention is not limited to this.

[0051] The phase-separated domain structure is determined not by the production method but by the components of the membrane, such as the constituent lipids. Therefore, the specific method for producing the cell-sized liposomes of the present invention is not limited, and any conventionally known method may be used as appropriate. Examples include the electroformation method, the droplet method, and a new method thereof (Patent No. 6031711). In the present invention, the static hydration method is particularly suitable. The static hydration method is a generally well-known method, and has the advantage that it can be easily produced without the need for special equipment, as long as a test tube, gas, and the specified solution are available, making it ideal for the present invention.

[0052] 5 is an explanatory diagram conceptually illustrating the configuration of the procyanidin concentration estimation method of the present invention. The cell-sized liposomes of the present invention having any of the configurations described above can be used as liposomes used in estimating the concentration of an estimation target whose procyanidin concentration is unknown, i.e., as "liposomes for obtaining phase-separation domain information." In this diagram, the "liposomes for obtaining phase-separation domain information" are abbreviated as "reference liposomes," and this abbreviation will also be used in the following description.

[0053] 5, the center column of the table on the left conceptually shows reference liposomes 10a, 10b, ..., and the right column conceptually shows "estimated target liposomes" 10x, 10y, ..., which are liposomes of cell size for which relative concentrations are to be estimated, and the left column shows the concentration n of procyanidin contained in each liposome 10a, 10x, etc. The procyanidin concentrations n=a, etc. of reference liposomes 10a, etc. are known, while the procyanidin concentrations n=x, etc. of estimated target liposomes 10x, etc. are unknown.

[0054] As shown in the figure, the procyanidin relative concentration estimation method of the present invention is a method for estimating the relative concentration n=x, etc. of procyanidin in a concentration estimation subject using the above-mentioned liposome for obtaining phase-separation domain information (reference liposome) 10a, etc. Reference liposome 10a, etc. is constructed by adding a procyanidin standard with a known concentration, such as n=a, to the concentration estimation subject. Furthermore, estimation subject liposome 10x, etc. is a cell-sized liposome constructed using the concentration estimation subject whose relative concentration is to be estimated, such as apple juice or a processed product thereof.

[0055] In the central table of Figure 5, the domain structure generation patterns (domain generation patterns) of each liposome in the table on the left are shown as columns, and the differences in concentration are shown as rows. That is, reference liposome 10a containing procyanidin 8a at a concentration n = a has domain generation pattern Da, and reference liposome 10b containing procyanidin 8b at a concentration n = b has domain generation pattern Db. Furthermore, putative target liposome 10x containing procyanidin 8x at an unknown concentration n = x has domain generation pattern Dx, and putative target liposome 10y containing procyanidin 8y at an unknown concentration n = y has domain generation pattern Dy.

[0056] The procyanidin concentration estimation method of the present invention involves observing the reference liposomes 10a, 10b, etc. under a fluorescent microscope to determine the phase-separation domain generation patterns Da, Db, etc., i.e., the state of phase-separation domain generation, such as whether or not a phase-separation domain has been generated or the pattern that would occur if a phase-separation domain has been generated, or alternatively, determining the state of cell-sized liposome generation; observing the cell-sized liposomes 10x, 10y, etc., whose concentration is to be estimated under a fluorescent microscope to determine the phase-separation domain generation patterns Dx, Dy, etc., the state of phase-separation domain generation, such as whether or not a phase-separation domain has been generated or the pattern that would occur if a phase-separation domain has been generated, or alternatively, determining the state of cell-sized liposome generation; and estimating the relative concentration of procyanidin contained in the object whose concentration is to be estimated by referring to the procyanidin concentration-dependence information of the phase-separation domain generation status or cell-sized liposome generation status in the reference liposome 10a, etc.

[0057] That is, in this procyanidin concentration estimation method with such a configuration, since the phase-separated domain generation patterns Da, Db, ... in the reference liposome 10a, etc., are procyanidin concentration dependent, this pattern information is compared, and the relative concentrations in the estimation subject liposome 10x, etc., i.e., the relative ranking of the procyanidin concentrations contained in the concentration estimation subject, and the concentration range is estimated. In Figure 5, as an example, one estimation result is shown on the right side of the figure, in which the concentrations are highest in the order of a, x, y, and b. At the same time, the estimation result is also obtained that the unknown concentrations x and y are both lower than the known concentration a and higher than b.

[0058] 6 is a flow diagram showing the configuration of the procyanidin concentration measurement method of the present invention. As shown in the figure, this procyanidin concentration measurement method is composed of a concentration estimation process P10 in which the procyanidin concentrations of all samples N to be subjected to concentration estimation are estimated using the procyanidin concentration estimation method described above, and then a precise measurement process P20 in which the procyanidin concentrations of some samples n that have already undergone procyanidin concentration estimation processing are precisely measured by HPLC.

[0059] This procyanidin concentration measurement method involves a concentration estimation process P10 in which a simple procyanidin concentration determination is performed in the form of a preliminary analysis, a sort of screening, to select samples for precision analysis by HPLC in the precision measurement process P20. After determining whether the procyanidin content in the samples is relatively high or low in the concentration estimation process P10, precision measurement process P20 is performed on a portion of the samples n. This allows for the realization of a rapid, low-cost, and precise analytical system.

[0060] The procyanidin concentration estimation method and procyanidin concentration measurement method of the present invention described above can be used for all estimation and measurement targets that are expected to contain procyanidins, and it goes without saying that these methods of the present invention can also be applied to apple juice containing procyanidins, or processed products thereof.

[0061] Here, the procyanidin concentration estimation method of the present invention, which has already been explained, will be explained again. 6-2 is a flow diagram showing the configuration of the procyanidin concentration estimation method of the present invention. As shown in (i) in the figure, the procyanidin concentration estimation method comprises a pattern reading process Q510 that performs fluorescence microscopic observation of cell-sized liposome 510x (liposome to be estimated) to determine whether or not a phase-separated domain has been formed, or reads a phase-separated domain generation pattern 5Dx that represents the pattern that will be observed if a phase-separated domain has been formed, and a concentration estimation process Q520 that estimates the concentration of procyanidin contained in the object to be estimated by referencing procyanidin concentration-dependent information Ref of the phase-separated domain generation pattern in liposome 510a (reference liposome) for obtaining phase-separated domain information.

[0062] In this procyanidin concentration estimation method having such a configuration, in the pattern reading process Q510, the cell-sized liposome 510x to be subjected to concentration estimation is observed under a fluorescent microscope to read the phase-separated domain generation pattern 5Dx, and then in the concentration estimation process Q520, the procyanidin concentration-dependent information Ref of the phase-separated domain generation pattern in the liposome 510a for obtaining phase-separated domain information is referenced to estimate the concentration of procyanidin contained in the object to be subjected to concentration estimation, and an estimated concentration Est is obtained.

[0063] As shown in (ii) in the figure, in concentration estimation step Q52C of the procyanidin concentration estimation method, when estimating the procyanidin concentration, a calibration curve Ccv based on the observation results of liposome 510a for obtaining phase-separation domain information can be used as procyanidin concentration-dependent information Ref of the phase-separation domain production pattern.

[0064] As a result, in the concentration estimation process Q52C, procyanidin concentration is estimated using the calibration curve Ccv to obtain the estimated concentration EsC, which further improves the accuracy and precision of concentration estimation and measurement, enabling concentration measurement with a consistent level of precision. The calibration curve Ccv can be created by focusing on the proportion of cell-sized liposomes that have not phase-separated, and can also be created using multiple lipid species of cell-sized liposomes. The actual use of the calibration curve as information on the procyanidin concentration dependence of the phase-separated domain generation pattern will be described later in the Examples section [5. Concentration Estimation Experiments and Results Using the Calibration Curve]. [Example]

[0065] Examples of the present invention will be described below, but the present invention is not limited to these examples. Note that the examples will be explained by outlining some of the experimental results that led to the completion of the present invention. [Research topic] Development of a simple procyanidin detection method using observation of the phase-separated domain structure of cell-sized liposomes [Research purpose] The goal is to create a phase-separated domain structure that depends on the concentration of procyanidin, the main component of apples and apple juice, and to use this to establish a procyanidin concentration estimation technique, i.e., a screening technique for precise measurement.

[0066] [Experimental details] [1. Basics of cell-sized liposome production] The method for preparing cell-sized liposomes was as shown in Figure 4 above. The lipid component system was composed as follows: The fluorescent reagent added was one that stains the DOPC-rich Ld phase. DOPC / DPPC / Chol=45:45:10 +Lissamine Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt(Rhodamine DHPE) 1% (stained liquid disordered domain) This results in a liposome having an So / Ld domain structure as shown in A in Figure 1-2 above.

[0067] 2. Fluorescence Microscopic Observation of Phase-Separated Domain Structure The conditions for fluorescence microscopic observation of the phase-separated domain structure were as follows. Microscope used: Olympus BX51 Observation conditions: WIG (excitation wavelength 530-550 nm, fluorescence wavelength 575 nm) It can also be observed with WIY (excitation wavelength 545-580 nm, fluorescence wavelength 610 nm).

[0068] [3. Experiment using apple juice] The PB2 concentration in apple juice has been reported to be 5-15 mg / 100 g (research into functional components of apples and green onions produced in Nagano Prefecture). The inventors also obtained a value of 8.3 mg / 100 g in a previous analysis. In this study, we prepared cell-sized liposomes using a 100-fold diluted apple juice in the hydration step, observed their phase-separated domain structure, and attempted to estimate the procyanidin concentration.

[0069] The samples used in the experiment are as follows: 1 apple juice Manufacturer: Aomori Prefecture Rural Industrial Agricultural Cooperative Association (JA Aoren) Brand name: Kibo no Shizuku (registered trademark) PB2 pure product (standard product) Manufacturer: Fujifilm Wako Pure Chemical Corporation Product name: Procyanidin B2

[0070] The lipid film formation for cell-sized liposomes was carried out as follows. The lipid was prepared as a chloroform solution with a concentration of 2 mM, and a total of 40 μL of solution was added. The organic solvent was evaporated to dryness, after which hydration was carried out. Below, we list the cases used in the experiment and describe the conditions. Case 1 (Apple Juice 1) Case 2 (Apple juice 1 + PB 2) Case 3 (pure water)

[0071] Case 1 Apple Juice 1 The PB2 concentration in apple juice has been reported to be 5-15 mg / 100 g (research on functional components of apples and green onions from Nagano Prefecture). The inventors also obtained a value of 8.3 mg / 100 g in a previous analysis. In this study, based on the literature value, the PB2 concentration in apple juice was assumed to be 10 mg / 100 g, and the juice was diluted 100 times before use. Based on this assumption, the concentration should be 0.1 mg / 100 g. 400 μL of this diluted solution was added to prepare cell-sized liposomes. Based on this assumption, the procyanidin weight in the cell-sized liposome solution is as follows: 0.1 / 100×0.4×1000=0.4μg (After converting to 400 μL, adjust the units accordingly)

[0072] Case 2: 1 apple juice + 2 PBs 10% of the apple juice 1 was replaced with a 20 μM solution of PB2, and 400 μL of this solution was added during hydration. The amount of solution added was 10% of the total 400 μL, or 40 μL. Therefore, based on the above assumptions, the weight of procyanidins in the cell-sized liposome solution is as follows: 4×0.9+20 / 1000 / 1000×40×578.52 ≒ 0.9μg (The first term was obtained by reducing apple juice 1 (case 1) by 10%, and the second term was calculated by adjusting the units, multiplying it by the added volume, and then multiplying it by the molecular weight of procyanidin B2 (PB2), 578.52. The sum of the first and second terms was rounded off to obtain an approximate weight of 0.9 μg.)

[0073] Case 3 Pure water Pure water was used, and therefore the molar weight of PB2 in the cell-sized liposome solution was 0 μg.

[0074] 4. Experimental Results 7 is a graph showing the phase-separated domain structural composition of the reference liposomes according to the examples (Cases 1, 2, and 3). As a result of fluorescence microscopic observation, as shown in the figure, the reference liposomes using pure water in Case 3 contained approximately 89% So / Ld domains, approximately 3% Lo / Ld domains, and approximately 8% homogenous. Since the PB2 concentration in pure water was naturally 0 mol%, the obtained estimation results were confirmed to be valid in light of Non-Patent Document 1.

[0075] In Case 1 Apple Juice 1, the So / Ld domain accounted for approximately 40%, the Lo / Ld domain for approximately 25%, and the homogenous domain for approximately 35%.

[0076] In case 2, apple juice 1 + PB2, the So / Ld domain was approximately 36%, the Lo / Ld domain was approximately 8%, and the homogenous domain was approximately 56%.

[0077] In Non-Patent Document 1, the inventors reported a technique for estimating the concentration of cell-sized liposomes by adding apple juice extract during the lipid film preparation stage and observing the state of the phase-separated domains.

[0078] As mentioned above, the results of this experiment showed that the order of highest procyanidin concentration was Case 2, Case 1, and Case 3. Furthermore, the order of lowest proportion of So / Ld domains observed in this experiment was Case 2, Case 1, and Case 3, showing the relative concentration order. Furthermore, because the procyanidin concentration was lower in Case 1 Apple Juice 1 than in Case 2 Apple Juice 1 + PB2, it was found that the procyanidin concentration in Case 1 Apple Juice 1 was lower than the concentration of pure procyanidin added in Case 2.

[0079] From the above, it was confirmed that the method of the present invention can be used to simply rank the relative concentrations of procyanidins, and further confirmed that the method of the present invention can simply estimate the concentration range of procyanidins.

[0080] 5. Concentration estimation experiment using calibration curve and results The calibration curve is then used to estimate the procyanidin concentration in the sample to be measured. As explained above in [1. Basics of Cell-Sized Liposome Preparation], the method for preparing cell-sized liposomes is shown in Figure 4. The lipid component systems were composed of the following three types. Similarly, the fluorescent reagent added was rhodamine DHPE, which stains the DOPC-rich Ld phase. Lipid composition1. DOPC / DPPC=50:50 +Rhodamine DHPE 1% Lipid composition 2. DOPC / DPPC / Chol=45:45:10 +Rhodamine DHPE 1% Lipid composition 3. DOPC / DPPC / Chol=40:40:20 +Rhodamine DHPE 1%

[0081] Hydration of the target sample for estimating procyanidin concentrations using the three lipid compositions described above and reference liposomes using sample solutions with known concentrations were prepared. When preparing these reference liposomes, four sample solutions were used for hydration: pure water alone, and sample solutions containing procyanidin concentrations of 5 mg / 100 mL, 10 mg / 100 mL, and 15 mg / 100 mL. A calibration curve was prepared by observing the reference liposomes encapsulating sample solutions with these four different procyanidin concentrations. For hydration of the reference liposomes prepared for calibration, the sample solutions with known concentrations were the pure PB2 described above in [3. Experiments using apple juice] and pure water.

[0082] In lipid compositions 2 and 3, Lo / Ld domains were observed in addition to So / Ld domains, but both were considered to be phase-separated cell-sized liposomes. On the other hand, homogenous cell-sized liposomes, which were uniformly stained with fluorescent dye and had not undergone phase separation, were also observed. In this experiment, phase-separated cell-sized liposomes and homogenous cell-sized liposomes without phase separation were counted separately.

[0083] As mentioned above, in Non-Patent Document 1, the inventors reported that when preparing cell-sized liposomes, apple juice extract was added during the lipid film preparation stage and the state of phase-separated domains was observed, and that the higher the procyanidin concentration in the lipid membrane, the more difficult it was to observe a phase-separated domain structure. This was confirmed by the results of this experiment. In other words, it was shown that the higher the procyanidin concentration in the sample solution used for hydration, the more difficult it was to observe So / Ld domains or Lo / Ld domains.

[0084] Furthermore, they discovered that the proportion of homogenous cell-sized liposomes in the observed cell-sized liposomes increased linearly in proportion to the procyanidin concentration, meaning that it was possible to create a calibration curve in which the proportion of homogenous cell-sized liposomes was plotted on the vertical axis and the procyanidin concentration in the sample solution on the horizontal axis. 8, 9, and 10 show graphs of calibration curves prepared under conditions of lipid composition 1, lipid composition 2, and lipid composition 3, respectively.

[0085] To create the calibration curves shown in Figures 8, 9, and 10, cell-sized liposomes were prepared and observed at least three times, with at least 60 samples observed per observation. The average values ​​and standard errors are shown as error bars when creating the calibration curves.

[0086] At the same time, cell-sized liposomes were prepared by hydration using samples for estimating procyanidin concentrations. The samples for estimating procyanidin concentrations were Case 1 (juice), Case 2 (juice + PB2), and Case 3 (pure water), as described in [3. Experiments using apple juice]. The procyanidin concentrations in each case were 10 mg / 100 mL, 22.5 mg / 100 mL, and 0 mg / 100 mL, as previously described.

[0087] The estimated concentrations of procyanidins using the calibration curve showed different values ​​depending on the lipid composition, namely: Lipid composition 1 Case 1 (juice) 19.31mg / 100mL Case 2 (juice + PB2) 29.99mg / 100mL Case 3 (pure water) 3.3 mg / 100 mL Lipid composition 2 Case 1: 8.08 mg / 100 mL Case 2: 32.68 mg / 100 mL Case 3 -0.99 mg / 100 mL Lipid composition 3 Case 1: 25.02 mg / 100 mL Case 2: 39.03 mg / 100 mL Case 3: 0.15 mg / 100 mL

[0088] In the case of pure water in Case 3, the error was within ±4 mg / 100 mL for all lipid compositions, and in Case 1 (juice), the estimated concentration was within ±4 mg / L for lipid composition 2. In the other cases, although the concentrations were different from the actual concentrations, the relative rankings were accurate for all lipid compositions.

[0089] [5.-2 Consideration of sugar components when creating a calibration curve] It has been reported that the formation of cell-sized liposomes is affected by the sugar used for hydration (K. Tsumoto et al., Colloids and Surfaces B 2009), and that sugar and hydrogen ion concentration affect the formation of phase-separated domain structures (Guo et al., Langmuir 2021).

[0090] Therefore, to create the calibration curve, five samples were used for hydration in the production of cell-sized liposomes: the four main sugars found in apples, namely fructose, sucrose, glucose, and sorbitol, as well as a sugar mixture of these. The sugar mixture was mixed in the following proportions, based on the actual components of apples. Fructose / sucrose / glucose / sorbitol = 40:20:15:5

[0091] Each sample solution was adjusted to a sugar content of 13 degrees Brix, and the stock solution containing procyanidins was diluted 100-fold with pure water as described above in [3. Experiments using apple juice] and used for hydration to prepare cell-sized liposomes. The lipid composition used here was lipid composition 1 described above in [1. Basics of preparing cell-sized liposomes]. Lipid composition 1 had a small standard error and high linearity, so the estimated procyanidin concentration was close to the actual concentration.

[0092] Figure 11 shows the calibration curves prepared under each sugar addition condition. Specifically, this graph shows the calibration curves prepared based on the phase-separated domain structure composition when lipid composition 1 is used and sugar is included in the cell-sized liposomes used to estimate the procyanidin concentration in the examples (Cases 1, 2, and 3). There were six test groups in total, including a sample that did not contain any sugar. These groups were: A. pure water; B. fructose; C. sucrose; D. glucose; E. sorbitol; and F. sugar mixture.

[0093] Reference liposomes were prepared using a sample solution containing one of the four sugars or a sugar mixture, and the procyanidin concentrations estimated using the calibration curves created from the observations were found to have a fairly small error from the actual concentrations. That is, in all three cases, regardless of which sugar or sugar mixture was used, the error from the actual concentrations was approximately ±5 mg / 100 mL, which was a fairly small error.

[0094] [5.-3 Consideration of acid components when creating a calibration curve] Considering the sugar components mentioned above, it was suggested that the error could be reduced by using a sample solution used for hydration during the preparation of cell-sized liposomes with a composition closer to that of the target for concentration estimation. Therefore, in addition to using any of the five sugars mentioned above in [5.-2 Consideration of sugar components in creating a calibration curve] (the four main sugars contained in apples and a sugar mixture), tests were conducted by adding acid to bring the hydrogen ion concentration to pH 3.0, which is similar to that of apple juice.

[0095] Specifically, five samples were used for hydration in the production of cell-sized liposomes: four main acids found in apples (malic acid, citric acid, acetic acid, and caproic acid), as well as an acid mixture made by combining these. The mixing ratio of the acid mixture was set as follows, based on the actual components of apples. Malic acid / citric acid / acetic acid / caproic acid = 80:4:2:0.2

[0096] Each sample solution was adjusted to a sugar content of 13 degrees Brix. The procyanidin-containing stock solution was then diluted 100-fold with pure water as described in [3. Experiments using apple juice] to prepare the sample solution, which was then used for hydration to prepare cell-sized liposomes. The lipid composition used was lipid composition 1 described above in [1. Basics of preparing cell-sized liposomes]. Lipid composition 1 had a small standard error and high linearity, so the estimated procyanidin concentration was close to the actual concentration. In this study, there were 36 test plots, including five sugars, five acids, and a blank (pure water) for each sugar and acid.

[0097] Reference liposomes were prepared using a sample solution containing one of four sugars or sugar mixtures and one of four acids or sugar mixtures, and the procyanidin concentrations estimated using the calibration curves created from the observations were found to have a fairly small error from the actual concentrations. That is, in Cases 1, 2, and 3, regardless of which sugar or sugar mixture, and which acid or acid mixture was used, the error from the actual concentrations was approximately ±5 mg / 100 mL, a fairly small error. Furthermore, the closer the sugar and acid component composition (mixing ratio) was to apple juice, the smaller the error from the actual concentrations tended to be.

[0098] Furthermore, even when using only one of the four acids or acid mixtures without using sugar or sugar mixtures for hydration to prepare cell-sized liposomes, the error between the estimated procyanidin concentration and the actual concentration was small when using a calibration curve created from the observation results of reference liposomes. That is, in Cases 1, 2, and 3, the error between the estimated procyanidin concentration and the actual concentration was small regardless of which acid or acid mixture was used. Furthermore, the closer the acid component composition (mixing ratio) was to apple juice, the smaller the error between the estimated procyanidin concentration and the actual concentration tended to be (not shown for [5.-3] above).

[0099] [5.-4 Summary of the calibration curve method] As a result of the above experiments, the following was found. To optimize the calibration curve, more accurate estimation results can be obtained by mixing more types of sugar and acid components contained in apple juice, the concentration of which is to be estimated, when adjusting the sample solution used for hydration in the production of reference liposomes. However, the more types of sugar and acid added, the greater the cost and the burden of addition procedures.

[0100] Therefore, if one wishes to know the relative ranking of concentrations in apple juice, which is the subject of concentration estimation, one can simply prepare reference liposomes using pure water without adding components such as sugar or acid contained in apple juice.

[0101] Furthermore, when it is desired to obtain an estimated concentration with a certain degree of accuracy, at least one of the following eight components may be used to prepare the reference liposome: sugars such as fructose, sucrose, glucose, and sorbitol; or acids such as malic acid, citric acid, acetic acid, and caproic acid. When it is desired to obtain a more accurate estimated concentration, it is also possible to use one or more of these sugars and acids.

[0102] If you want to further improve the accuracy and precision of the estimated concentration, you can use a mixture of sugars and acids to approximate as closely as possible the component composition of the apple juice that is the target of concentration estimation. In other words, if you mix the above four types of sugars in the same way as the composition of apple juice, Fructose / sucrose / glucose / sorbitol = 40:20:15:5 The sugar mixture is mixed in the above proportions and adjusted to a sugar content of 13 degrees, and the above four types of acids are mixed in the same manner as the composition of apple juice. Malic acid / citric acid / acetic acid / caproic acid = 80:4:2:0.2 An acid mixture is prepared by mixing the above ratios and the sample stock solution is adjusted to pH 3.0. By diluting this sample stock solution 100 times and adding it during hydration in the production of reference liposomes, a value closer to the actual concentration and closer to the accurate procyanidin concentration can be determined using the calibration curve method.

[0103] 6. Effectiveness of Fluorescence Microscopy It should be noted that the implementation of the fluorescence microscopic observation according to the present invention is guaranteed without any problems. 12 is a graph showing the phase transition temperature, i.e., the domain disappearance temperature, of each cell-sized liposome according to the examples (Cases 1, 2, and 3). As shown in the figure, in each case, the temperature range at which the generated domains disappear is generally above 25°C, confirming that there is no problem with fluorescence microscopy observation at room temperature.

[0104] [7. Estimation of cost reduction effects] The costs (expenses and time) were estimated for the conventional measurement method using HPLC and the method of the present invention. 1) Existing method (HPLC) Initial cost HPLC equipment: 15,730,000 yen (Agilent) Running costs Acetone gallon bottle 6,000 yen Acetonitrile Gallon Bottle 17,000 yen PB2 92,200 yen Total: 115,200 yen Measurement time 1-2 days (including equipment setup and calibration sample measurement)

[0105] 2) The method of the present invention (cell-sized liposomes) Initial cost Microscope 1,146,260 yen Fluorescent unit: 1,400,000 yen (both manufactured by Olympus) Total: 2,546,260 yen Running costs Lipid DOPC 8,800 yen DPPC 9,000 yen Chol 5,000 yen Rhodamine DHPE 50,900 yen Solvent: Chloroform 2,850 yen Total: 76,550 yen Measurement time Preparation: 8 hours (liposome preparation (film preparation 1 hour + vacuum drying 3 hours + hydration 4 hours)) Measurement: 1 hour (time required to observe 60 samples) Total: 9 hours

[0106] As described above, the method of the present invention is estimated to be cheaper than the conventional method using HPLC, with an initial cost of approximately 13,000,000 yen and a running cost of approximately 40,000 yen. Furthermore, the measurement time is estimated to be shortened to approximately half a day. (Note that glassware, pipettes, plastic tubes, filters, syringes, sugar, and acid are inexpensive and do not affect the cost, so they have not been included in this calculation.)

[0107] [8. Supplementary Information - Estimation by Liposome Diameter] In this experiment, the diameter of cell-sized liposomes produced by apple juice (Case 1) and apple juice + PB (Case 2) was found to be larger in the latter case. This tendency in size was also observed in a previous report by the inventor (T. Yoda, Chemistry Select 2022). Therefore, we note that the relative procyanidin concentration can be estimated not only by the So / Ld domain production status, but also by comparing the production status of cell-sized liposomes, particularly the size (increase or decrease). [Industrial Applicability]

[0108] The cell-sized liposomes, the procyanidin concentration estimation method using the cell-sized liposomes, the procyanidin concentration estimation method using the observation results of the cell-sized liposomes and a calibration curve, and the procyanidin concentration measurement method of the present invention enable procyanidin concentration analysis to be performed at significantly lower cost and in a shorter time than conventional methods. In particular, the procyanidin concentration estimation method using a calibration curve can further improve the accuracy and precision of concentration estimation and measurement. Therefore, this invention has high industrial applicability, particularly in the fields of food component analysis, food quality control, and all related fields. [Explanation of symbols]

[0109] 2, 12, 22...unsaturated phospholipids 8, 8a, 8b, 8x, 8y, 18, 28... Procyanidins 9, 19, 29…Fluorescent reagents for detecting phase-separated domains 10, 10', 110, 110', 210, 210'...cell-sized liposomes 10a, 10b, 510a...cell-sized liposomes (reference liposomes) 10x, 10y, 510x...cell-sized liposomes (estimated target liposomes) 13, 23...Saturated phospholipids 25...Sterol lipids Ccv...calibration curve Da, Db, Dx, Dy, 5Dx...phase separation domain generation pattern EsC, Est...Estimated concentration N: All samples for which concentration estimation is required n: A portion of the sample that has been subjected to concentration estimation processing P10: Relative concentration estimation process P20…Precision measurement process Q510...Pattern reading process Q520, Q52C…Concentration estimation process Ref…Procyanidin concentration dependence of phase-separated domain formation pattern< / s> < / s> < / lr> < / lr> < / lr>

Claims

1. A lipid component system comprising one or more unsaturated phospholipids; and The sample to be estimated for procyanidin concentration or procyanidin A cell-sized liposome comprising: Different procyanidin concentrations resulted in different phase-separated domain formation patterns. This allows cell-sized liposomes to be used to estimate procyanidin concentration from the phase-separated domain formation pattern.

2. 2. The cell-sized liposome of claim 1, wherein the lipid component system contains one or more saturated phospholipids.

3. The cell-sized liposome according to claim 2, wherein the lipid component system contains one or more sterol-based lipids.

4. The cell-sized liposome according to claim 1, characterized in that the lipid component system is composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), which is an unsaturated phospholipid, and Dipalmitoylphosphotidylcholine (DPPC), which is a saturated phospholipid.

5. The cell-sized liposome according to claim 1, characterized in that the lipid component system is composed of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), which is an unsaturated phospholipid, dipalmitoylphosphotidylcholine (DPPC), which is a saturated phospholipid, and cholesterol, which is a sterol lipid.

6. The cell-sized liposome according to any one of claims 2, 3, 4, and 5, characterized in that, when used at room temperature, the molar composition of unsaturated phospholipids and saturated phospholipids in the lipid component system is unsaturated phospholipid:saturated phospholipid=75-10:25-90.

7. 6. The cell-sized liposome according to claim 2, wherein the unsaturated phospholipid and the saturated phospholipid in the lipid component system have an equal molar concentration when used at room temperature.

8. 6. The cell-sized liposome according to claim 1, wherein a fluorescent reagent for detecting phase-separated domains is added.

9. The cell-sized liposome described in claim 8, characterized in that the fluorescent reagent used is one or more of a reagent for staining a solid ordered phase (So phase), a reagent for staining a liquid disordered phase (Ld phase), or a reagent for staining sterol-based lipids.

10. 6. The cell-sized liposome according to claim 1, 2, 3, 4, or 5, which is prepared by a static hydration method.

11. The cell-sized liposome according to claim 10, wherein after the lipid film is prepared, a sample whose concentration is to be estimated is added during hydration of the cell-sized liposome.

12. A method for estimating the relative concentration of procyanidin in a subject for concentration estimation using the cell-sized liposome according to any one of claims 1 to 5, which is constructed using a procyanidin standard having a known concentration, as a liposome for obtaining phase-separation domain information, comprising: Fluorescence microscopic observation of the cell-sized liposomes to be used for estimating the concentration of procyanidins, which are the cell-sized liposomes according to any one of claims 1 to 5 and are formed using a sample to be used for estimating the concentration of procyanidins; The state of phase-separated domain formation, i.e., whether or not a phase-separated domain is formed, or the state in which it is formed, or the state of cell-sized liposome formation, is read, referring to procyanidin concentration-dependent information on the phase-separation domain formation status or cell-sized liposome formation status in the liposome for obtaining phase-separation domain information, Estimate the relative concentration of procyanidins contained in the target substance A method for estimating procyanidin concentration,

13. 13. The method for estimating a procyanidin concentration according to claim 12, wherein a calibration curve based on the observation results of the liposome for obtaining phase-separated domain information is used in estimating the procyanidin concentration.

14. The method for estimating a procyanidin concentration according to claim 12, characterized in that, in estimating the procyanidin concentration, a calibration curve based on the observation results of the liposome for obtaining phase-separated domain information described in <Lr> below is used. <Lr> Liposome for obtaining information on phase-separated domains containing sugar and / or acid as components, which are components included in the target for concentration estimation

15. The method for estimating a procyanidin concentration according to claim 14, wherein the liposome for obtaining information on phase-separated domains described in <Lr> is at least one of the following <S> and / or : <S> A sugar containing one or more of fructose, sucrose, glucose, and sorbitol. A composition containing one or more of malic acid, citric acid, acetic acid, and caproic acid as the acid.

16. The method for estimating a procyanidin concentration according to claims 12, 13, 14, and 15, wherein the subject of concentration estimation is apple juice or a processed apple juice product.

17. A method for measuring procyanidin concentration, comprising: first estimating the procyanidin concentrations of a plurality of samples by the procyanidin concentration estimation method according to any one of claims 12, 13, 14, and 15; and then precisely measuring the procyanidin concentrations of some of the samples that have been subjected to the procyanidin concentration estimation process by high performance liquid chromatography.

18. 18. The method for measuring procyanidin concentration according to claim 17, wherein the subject of concentration measurement is apple juice or a processed apple juice product.

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