Cell-sized liposomes, method for estimating procyanidin concentration, and method for measuring procyanidin concentration

Abstract

To provide a simple procyanidin analysis method that can be performed at a low cost, and in the form of preliminary analysis for selecting a sample for precision analysis in HPLC, that is, screening.SOLUTION: Cell size liposomes 10, 10' are composed of a lipid component system in which one or more phospholipids 2, 3 and one or more sterol-based lipids 5 are used in a constant composition, and procyanidin 8, where different phase separation domain generation patterns are generated depending on the concentration of procyanidin 8, which can be used for estimating the procyanidin concentration from the phase separation domain generation pattern. The phospholipids 2, 3 are unsaturated phospholipid or saturated phospholipid.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to cell-size liposomes, a method for estimating procyanidin concentration, and a method for measuring procyanidin concentration, and particularly relates to a technology capable of estimating the content concentration of procyanidin contained in apple juice or the like at low cost and in a relatively short time.

Background Art

[0002] Apple cultivation is a major industry in Aomori Prefecture. According to the 2018 statistics, the cultivated area of apples in Aomori Prefecture is 20,800 hectares, accounting for half of the country. However, the cultivated area has been decreasing over the years, and maintaining and securing the position as the main production area of the apple industry, which plays an important role not only in the regional industrial economy but also in the cultural and tourism aspects, is an important issue for Aomori Prefecture. In this region, research has been accumulating on the effectiveness of functional components contained in apples in maintaining health and preventing diseases. For example, "Prime Apple!" (variety = Fuji, registered trademark) has obtained registration as a functional display food as a fruit, and its target is the function of procyanidin to reduce visceral fat.

[0003] Procyanidin contained in apples is the main component of polyphenols (general name: apple polyphenols) contained in apples. Apple polyphenols are composed of various components, and about 60% of them are "procyanidins" (from the website of the Aomori Apple Countermeasure Council). Procyanidin is particularly known for its high antioxidant function and also has a fat-reducing effect, and apples themselves containing a large amount of procyanidin are sold as functional display foods. Under such circumstances, in order to enhance the commercial value of products such as apples and apple juice, the Hirosaki Industrial Research Institute related to the applicant organization has conventionally measured procyanidin concentration as one of the commissioned test business items.

[0004] HPLC (High-Performance Liquid Chromatography) is used in procyanidin concentration measurement tests. This equipment is not only very expensive to purchase, but also has the disadvantage of high running costs because it uses high-purity organic solvents for analysis with each measurement. Furthermore, it takes several hours to start up the equipment, and a calibration curve must be created by running multiple standard samples before the concentration measurement can finally be performed, so it is a considerably time-consuming process.

[0005] Regarding the measurement techniques for procyanidins, patent applications and other documents have been filed in the past. For example, Patent Document 1, cited below, discloses a quantitative method for specifically and accurately measuring procyanidins in food and beverages (such as n polymers of catechin: n≧1, where n is an integer), characterized by quantifying procyanidins separated by size exclusion chromatography using fluorescence detection. This document also discloses a method for quantifying procyanidins separated by size exclusion chromatography using fluorescence detection. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2009-156813, "Method for the Quantitative Determination of Procyanidins" [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] As mentioned above, procyanidins, known as apple polyphenols and possessing health-promoting functional properties, are attracting increasing attention, and it is expected that the value of apple processed foods and other products can be enhanced by indicating their procyanidin content. However, HPLC, used for measuring procyanidins, has the problem of being expensive both in terms of initial and running costs, as well as requiring considerable effort and time. It would be convenient to have a simpler analytical method, such as a screening method, that can be performed at the lowest possible cost, even if it does not require the same level of precision as HPLC, to select samples for precise HPLC analysis.

[0008] Liposomes, the most simplified biological membrane model used in many experiments and studies, are formed when phospholipids, which possess both hydrophilic and hydrophobic fatty acid chains, self-assemble with their hydrophilic parts facing outwards to form a lipid bilayer, which then closes into a sac-like structure. Liposomes with a diameter of several micrometers to tens of micrometers are almost the same size as living cells and are called cell-size liposomes (giant liposomes). Cell-size liposomes are useful for research such as elucidating the regulatory mechanisms of biological membranes due to their advantages, such as being observable directly with an optical microscope and being usable in the creation of experimental models that mimic cells.

[0009] Furthermore, it has been revealed that the cell membrane contains phase-separated domains (raft domains) rich in saturated lipids and cholesterol, which play a role in signal transduction and other functions. These raft domains are thought to move by attaching and detaching in conjunction with intercellular signal transduction. Similar domain structures have been observed in liposomes artificially created from lipids, and research in this area is also actively being conducted. These studies using biological model membranes (artificial cell membranes) and human-derived cells have revealed that the three-dimensional dynamics of the membrane, the two-dimensional (domain structure) accumulation dynamics, and signal transduction using intracellular calcium ions as indicators are involved, and that changing the structure of lipids and sterols alters the temperature response of the domain structure.

[0010] The inventor has previously conducted research to elucidate 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 findings from these observation videos and images. For example, he discovered that cholesterol plays a more important role than unsaturated lipids in the dynamics of biological model membranes in response to oxidative stress (T. Yoda et al., Chem. Lett. 2010), detected oxidized cholesterol produced by oxidative stress, and discovered that biological model membranes containing oxidized cholesterol exhibit remarkable temperature responsiveness through microscopic observation of biological model membrane vesicles.

[0011] Furthermore, we revealed that domain structures are generated when membranes containing antioxidants are subjected to oxidative stress. Specifically, we discovered an example where cell-sized liposomes were mixed with solutions of polyphenols such as resveratrol and theaflavins, which are known as antioxidant functional components, causing membrane dynamics such as membrane contraction and oscillation, and resulting in the appearance of phase-separated domains during observation (HHT.Phan,T.Yoda et al.,Biochim.Biophys.Acta,Biomembr.2014). In addition, fluorescence microscopy has revealed that concanavalin A, a functional component found in sword beans that activates immunity, causes the accumulation of domain structures on cell membranes and activates intracellular signal transduction (S.Yabuuchi et al.,2017 J.Biosci.Bioeng.).

[0012] When domains on cell-sized liposomes are prepared by mixing equal amounts of the unsaturated lipid 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the saturated lipid Dipalmitoylphosphatidylcholine (DPPC), they undergo phase separation into a liquid disordered phase domain (Ld domain) rich in DOPC and a solid ordered phase domain (So domain) rich in DPPC. Furthermore, it has been shown that when cholesterol is added, many cell-sized liposomes exhibit phase separation into a liquid disordered phase domain (Ld domain) rich in DOPC and a liquid ordered phase domain (Lo domain) rich in both DPPC and cholesterol.

[0013] The problem that this invention aims to solve is, based on the progress and results of previous research using liposomes and the status of conventional technologies for measuring the functional component procyanidin, to provide a simple procyanidin analysis method that can be performed as low-cost and in a short time as possible, even if it does not have the same precision as HPLC, for preliminary analysis to select samples for precise analysis by HPLC, in other words, a simple procyanidin analysis method in the form of a so-called screening. In other words, it aims to provide a simple measurement method that utilizes the clear differences characterized by observation due to the interaction between procyanidin and membrane, and to easily distinguish them using cell-sized liposomes. [Means for solving the problem]

[0014] The inventors conducted research to address the above-mentioned problem. When observing the phase separation domain structure of the membrane when a procyanidin solution was added during the preparation of cell-sized liposomes, they first discovered that mixing procyanidin with DOPC alone significantly increased the size of the cell-sized liposomes, and that the proportion of the domain structure differed depending on the procyanidin concentration. Based on this proportion, they found that the procyanidin content in samples such as apple juice could be determined by the ratio of liquid-ordered phase separation to liquid-disordered phase separation.

[0015] Furthermore, experiments using apple juice confirmed the above, and cost reduction effects were also confirmed through calculations, confirming that cell-sized liposomes can be used as a tool for analyzing procyanidin concentration. In other words, in order to analyze procyanidins contained in apple juice, etc., the procyanidin content can be estimated in a short time by observing the ratio of the domain structure of cell-sized liposomes containing extracted procyanidins, unsaturated lipids, saturated lipids, and cholesterol. The present invention thus completed, that is, the invention claimed in this application, or at least the invention disclosed, as a means of solving the above problems, is as follows.

[0016] [1] A cell-sized liposome composed of a lipid component system in which one or more phospholipids and one or more sterol lipids are used in a fixed composition, and a procyanidin, The phospholipids are unsaturated phospholipids and saturated phospholipids. The lipid component system consists of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), and cholesterol (Chol). The ratio of these is DOPC / DPPC / Chol = 4:4:2. A cell-sized liposome characterized by generating different phase separation domain formation patterns depending on the procyanidin concentration, which can then be used to estimate the procyanidin concentration from the phase separation domain formation pattern.

[0017] 〔2〕A cell-sized liposome according to 1 〕, characterized in that a fluorescent reagent for detecting a phase separation domain is added. 〔 3 〕 The cell-sized liposome according to 2 〕, characterized in that one or more of them are used as the fluorescent reagent for staining the liquid ordered phase (Lo phase), the liquid disordered phase (Ld phase), or the sterol-based lipid. 〔 4 〕 The cell-sized liposome according to any one of 〔1〕、〔2〕、〔3〕 〕, characterized in that it is produced by the static hydration method. 〔 5 〕 The cell-sized liposome according to any one of 〔1〕、〔2〕、〔3〕、〔4〕 〕, characterized in that the procyanidin has a known concentration and is used in the concentration estimation of an object to be estimated with an unknown procyanidin concentration as a liposome for obtaining phase separation domain information.

[0018] 〔 6 〕 〔1〕、〔2〕、〔3〕、〔4〕、〔5〕 A method for estimating the procyanidin concentration, which uses the cell-sized liposome according to any one of 〔1〕、〔2〕、〔3〕、〔4〕、〔5〕 〕 as a liposome for obtaining phase separation domain information and estimates the procyanidin concentration in the object to be estimated. One or more of the phase separation domain information acquisition liposomes configured using a procyanidin standard with a known concentration are used. In the cell-sized liposome according to any one of to 〕, an extract of the object to be estimated is added instead of procyanidin to form a cell-sized liposome for estimating the concentration of the object to be estimated, and fluorescence microscopy is performed to observe the phase separation domain generation pattern, that is, the presence or absence of phase separation domain generation or the pattern when it is generated, and the procyanidin concentration-dependent information of the phase separation domain generation pattern in the above-mentioned phase separation domain information acquisition liposome obtained in advance is referred to, and the procyanidin concentration contained in the object to be estimated is estimated. 〔 7 〕 The object to be estimated is an apple or a processed product of an apple. 6The procyanidin concentration estimation method described in 〔 〕. 〔 8 〕 For a plurality of samples, first, the procyanidin concentration is estimated by the procyanidin concentration estimation method described in any of 〔 〕, 〔 〕, and then, for some of the samples for which the procyanidin concentration estimation process has been completed, precise measurement of the procyanidin concentration by high performance liquid chromatography is performed. A procyanidin concentration measurement method characterized by this. Furthermore, the following inventions are also disclosed. [1'] A cell-sized liposome composed of a lipid component system comprising one or more phospholipids and one or more sterol lipids in a fixed composition, and a procyanidin, characterized in that it produces different phase separation domain generation patterns depending on the procyanidin concentration, and is used to estimate the procyanidin concentration from the phase separation domain generation pattern. [1'-2] The cell-sized liposome according to [1], characterized in that the phospholipids are unsaturated phospholipids and saturated phospholipids. [1'-3] The cell-sized liposome according to [2], characterized in that the lipid component system consists of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), and cholesterol. [1'-4] The cell-sized liposome according to [3], characterized in that, when used at room temperature, the molar concentration composition of the lipid component system is such that DOPC and DPPC are both 30 to 45, and cholesterol is the remainder. [1'-5] The cell-sized liposome according to [4], characterized in that DOPC and DPPC are in equal amounts.

Advantages of the Invention

[0019] Since the cell-sized liposome, procyanidin concentration estimation method, and procyanidin concentration measurement method of the present invention are configured as described above, according to these, it is possible to provide a simple measurement method using the clear difference characterized by observation of the interaction between procyanidin and the membrane, and a simple discrimination method using cell-sized liposomes. Specifically, it is possible to analyze the procyanidin concentration at a significantly lower cost and in a shorter time than in the past.

[0020] ​​​​Conventional measurement methods requiring expensive HPLC have the problem of requiring long periods of time for equipment startup, etc. However, the concentration estimation method according to the present invention allows the use of a relatively inexpensive fluorescence observation microscope as the measuring instrument, reducing not only initial costs but also running costs, making it easier to analyze the procyanidin concentration of apple juice and other substances, and enabling rapid measurement. The present invention involves creating cell-sized liposomes incorporating the procyanidin to be measured and observing them with 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 performing observation for a short period of time.

[0021] Furthermore, as an effective application, the present invention can be used for preliminary analysis to select samples for precise analysis using HPLC, essentially as a screening tool for determining procyanidin concentration, demonstrating its high utility. According to the present invention, it is possible to determine whether the procyanidin content in a sample is approximately 20% or less relative to the lipids. Therefore, a rapid, low-cost, and precise analytical system can be realized by combining the present invention with HPLC. [Brief explanation of the drawing]

[0022] [Figure 1] This is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. [Figure 2] This is a photographic diagram showing an example of the phase separation domain generation pattern in cell-sized liposomes according to the present invention. [Figure 3] These are examples of lipids, which are components of the cell-sized liposomes of the present invention, and the chemical structural formula of procyanin ginseng B2. [Figure 4] This is an explanatory diagram showing an example of a method for producing cell-sized liposomes according to the present invention. [Figure 5] This is a conceptual diagram illustrating the configuration of the procyanidin concentration estimation method of the present invention. [Figure 6]This is a flowchart illustrating the configuration of the procyanidin concentration measurement method of the present invention. (The following figures relate to examples.) [Figure 7] This graph shows the phase separation domain structure and composition in reference liposomes at various concentrations. [Figure 8] This graph shows the structural composition of the phase separation domain in the estimated target liposomes related to the examples (Cases 1, 2, and 3). [Modes for carrying out the invention]

[0023] The present invention will be described in detail below, with the help of drawings. Figure 1 is a conceptual diagram showing the basic structure of the cell-sized liposome of the present invention. As shown in (a) in the figure, the cell-sized liposome 10 is a cell-sized liposome composed of a lipid component system in which one or more phospholipids 2 and 3 and one or more sterol lipids 5 are used in a fixed composition, and procyanidin 8. The main feature is that different phase separation domain generation patterns are produced depending on the concentration of procyanidin 8, and this is used to estimate the procyanidin concentration from the phase separation domain generation pattern. With this configuration, the cell-sized liposome 10 exhibits different phase separation domain generation patterns depending on the concentration of procyanidin 8 contained therein. Therefore, the concentration of procyanidin contained can be estimated using this phase separation domain generation pattern. At a minimum, it is possible to determine whether or not the procyanidin content of the sample is approximately 20% of the lipids by observing the phase separation domain generation pattern.

[0024] The cell-sized liposome 10 of the present invention includes liposomes for obtaining phase separation domain information, which are prepared by adding procyanidin at a known concentration, as well as cell-sized liposomes for concentration estimation, which are formed by adding extracts from samples with an unknown procyanidin concentration, i.e., samples that are certain to contain procyanidin but whose concentration is unknown. Further details will be described later.

[0025] Figure 2 is a photographic diagram showing examples of phase separation domain generation patterns in cell-sized liposomes according to the present invention. These are micrographs showing typical patterns, of which A is a homogeneous membrane vesicle, B is a Lo (liquid ordered phase) / Ld (liquid disordered phase) domain structure, and C is a So (solid ordered phase) / Ld (liquid disordered phase) domain structure. Observation using a fluorescence microscope shows that, in particular, the Lo / Ld domain structure pattern shown in B can be suitably used for concentration estimation in the present invention. Observation using a fluorescence microscope will be described further later.

[0026] The cell-sized liposomes of the present invention can be configured to use both unsaturated and saturated phospholipids as phospholipids. In particular, the present invention can use 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) as the unsaturated phospholipid and Dipalmitoylphosphatidylcholine (DPPC) as the saturated phospholipid, and can also use cholesterol (Chol) as the sterol lipid in a lipid component system. The following description will mainly focus on this lipid component system.

[0027] Figure 3 shows the chemical structures of lipids and procyanidin B2 (PB2), which are examples of components of the cell-sized liposomes of the present invention. In the figure, A is DOPC, B is DPPC, C is cholesterol, and D is PB2. Procyanidin B2 is one of the dimers of procyanidins and is used as a standard substance for concentration measurement.

[0028] The lipid component system of the cell-sized liposomes of the present invention can have a molar concentration composition of 30 to 45 for both DOPC and DPPC when used at room temperature, with cholesterol being the remainder. In the present invention, the most desirable phase separation domain composition for observation to determine the procyanidin concentration is the Lo / Ld domain, as described above. Conventional reports have shown that this domain can be observed at the following molar concentration compositions. Note that the unit is mole percent (the same applies to the molar concentration composition ratios below). DOPC:DPPC:Chol=30~45:30~45:10~40 DOPC:DPPC:Chol=32.5~42.5:32.5~42.5:15~35

[0029] The reason why the composition range differs among the reported examples is that domain formation is affected by temperature. According to the inventor's results at room temperature (around 20°C), DOPC:DPPC:Chol=30~45:30~45:Remaining amount For example, if Chol is set to 20, a Lo / Ld domain structure can be obtained in a molar concentration composition range of DOPC 60-30 and DPPC 20-50. Of course, DOPC and DPPC can also be used in equal amounts. In the examples described later, DOPC and DPPC are used in equal amounts (40).

[0030] Furthermore, the phase separation domain itself can be observed even in the absence of cholesterol. For example, when cholesterol is 10 or less and phospholipid is 45 or more or 42.5 or more, the So / Ld (solid ordered phase / liquid disordered phase) domain is observed. However, in this domain pattern, no correspondence was confirmed between the domain formation rate and the procyanidin concentration.

[0031] As shown in Figure 1(b) above, the cell-sized liposome 10' of the present invention can be configured with a fluorescent reagent 9 added for detecting the phase separation domain. The presence or absence of the phase separation domain structure is determined by fluorescence observation of the cell-sized liposome, but the fluorescent reagent 9 is added for this purpose and does not act on phase separation formation. The cell-sized liposome 10' can be constructed using one or more of the fluorescent reagents 9 selected from those for liquid ordered phase (Lo phase) staining, liquid disordered phase (Ld phase) staining, or sterol lipid staining.

[0032] In the examples described later, Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt (Rhodamine DHPE) is added as a fluorescent reagent at a concentration of 1 mole percent of the lipid. This is a relatively common fluorescent reagent used to stain 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 as a fluorescent reagent to stain the DPPC-rich Lo phase. Since these have different fluorescence wavelengths, they can be used interchangeably or both can be used simultaneously.

[0033] Furthermore, for the cholesterol portion, for example, NBD-Cholesterol can be used as a fluorescent stain. In this invention, which uses the Lo / Ld domain structure, the DPPC-rich Lo domain is stained. Of course, in this invention, it is also possible to use fluorescent reagents other than those described above.

[0034] Figure 4 is an explanatory diagram illustrating an example of the method for producing cell-sized liposomes according to the present invention. As illustrated in the figure, these cell-sized liposomes can be produced by dissolving lipids for liposome composition in a solvent (chloroform CH(Cl)3 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 with distilled water. In the examples described later, cell-sized liposomes were produced using this static hydration method.

[0035] The phase separation domain structure is determined by components in the membrane, such as constituent lipids, rather than by the manufacturing method. Therefore, the specific method for producing the cell-sized liposomes of the present invention is not limited, and conventionally known methods may be used as appropriate. For example, there are electroformation, droplet methods, and newer methods thereof (Patent No. 6031711), but 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 of being easy to use, requiring no special equipment other than test tubes, gas, and a predetermined solution, making it ideal for the present invention.

[0036] Figure 5 is a conceptual diagram illustrating the configuration of the procyanidin concentration estimation method of the present invention. Any cell-sized liposome of the present invention configured as described above can be used as a "phase separation domain information acquisition liposome" for estimating the concentration of a target substance whose procyanidin concentration is unknown, provided that the contained procyanidin is of known concentration. As the procyanidin contained in this "phase separation domain information acquisition liposome," a procyanidin standard (pure product) can be used. In this figure, the "phase separation domain information acquisition liposome" is abbreviated as "reference liposome," and this abbreviation will also be used in the following explanation.

[0037] In Figure 5, the reference liposome groups 10a, 10b, ... are conceptually shown in tabular form on the left, and the cell-sized liposome 10x of the present invention, in a form with an unknown procyanidin concentration, is shown on the right. As shown in the figure, the procyanidin concentration estimation method of the present invention is a method for estimating the procyanidin concentration in a target substance using the above-mentioned phase separation domain information acquisition liposome (reference liposome) 10a, etc. One or more reference liposomes 10a, etc., composed of procyanidin standards with known concentrations are used.

[0038] The table in the figure shows, with each column representing the concentration n of the procyanidin contained in the reference liposome 10a, the reference liposome containing a predetermined concentration n of procyanidin, and the domain structure generation pattern (domain generation pattern) of the reference liposome, and each row representing the difference in concentration. Specifically, reference liposome 10a containing procyanidin 8a with concentration n=a has domain generation pattern Da, and reference liposome 10b containing procyanidin 8b with concentration n=b has domain generation pattern Db.

[0039] On the other hand, the cell-sized liposome 10x of the present invention, which is formed by adding an extract of the substance to be concentrated instead of procyanidin in any of the cell-sized liposome configurations described with reference to Figures 1-4 shown on the right, contains procyanidin or a comparable element 8x, and has a phase separation domain generation pattern Dx as observed by fluorescence microscopy.

[0040] The present invention provides a method for estimating the concentration of procyanidins in a target cell-sized liposome 10x, which is used for concentration estimation. This method involves microscopic observation of the target cell-sized liposome 10x, reading the phase separation domain generation pattern Dx, i.e., whether or not a phase separation domain is generated or the pattern if it is generated, and then referencing the procyanidin concentration-dependent information of the phase separation domain generation patterns Da, Db, ... in a previously obtained reference liposome 10a, etc., to estimate the concentration of procyanidins contained in the target substance. Hereafter, "target cell-sized liposome for concentration estimation" will also be simply referred to as "target liposome for estimation."

[0041] With this configuration, in this procyanidin concentration estimation method, the target liposome 10x is observed under a fluorescence microscope to read the phase separation domain generation pattern Dx, that is, whether or not a phase separation domain is generated, or the pattern if it is generated. Then, the phase separation domain generation pattern Dc is referenced and compared with the procyanidin concentration-dependent information of phase separation domain generation patterns Da, Db, ... in reference liposomes 10a, etc. obtained in advance, and the concentration in the reference liposome having a matching phase separation domain generation pattern is estimated to be the concentration of procyanidin contained in the substance whose concentration is to be estimated.

[0042] Figure 6 is a flowchart showing the configuration of the procyanidin concentration measurement method of the present invention. As shown in the figure, the procyanidin concentration measurement method consists of a concentration estimation process P10 in which the procyanidin concentration is first estimated for all samples N to be measured using the procyanidin concentration estimation method described above, and then a precision measurement process P20 in which the procyanidin concentration is precisely measured by HPLC for some of the samples n that have undergone the procyanidin concentration estimation process.

[0043] This procyanidin concentration measurement method involves performing a simplified procyanidin concentration determination in the concentration estimation process P10, which serves as a preliminary screening process to select samples for precise analysis using HPLC in the precise measurement process P20. In the concentration estimation process P10, it is determined whether the procyanidin content in the sample is approximately 20% or less of the lipids, and then the precise measurement process P20 is performed on a selection of samples n. Therefore, a rapid, low-cost, and precise analytical system can be realized.

[0044] The procyanidin concentration estimation method and procyanidin concentration measurement method of the present invention described above can be used for all objects that are expected to contain procyanidins, and it goes without saying that apples, which are fruits containing procyanidins, or processed products thereof, are also subject to the application of these methods of the present invention. [Examples]

[0045] The following describes examples of the present invention, but the present invention is not limited thereto. The examples will consist of a brief overview of some of the experimental results leading to the completion of the present invention. Furthermore, the experiments were based on the method described in K. Sugahara et al., 2015 Chem. Lett., with Lidcaine and Tetracaine replaced by PB2 in the same report. [Research topic] Development of a simple detection method for procyanidins contained in apples, etc., using observation of the phase separation domain structure of artificial biomechanism membranes. [Research purpose] To create a phase separation domain structure that depends on the concentration of procyanidins, the main component of apples and apple juice, and to establish a procyanidin concentration estimation technique, i.e., a screening technique for precise measurement, using this structure.

[0046] [Details of the experiment] [1. Basics of Cell-Sized Liposome Production] The method for preparing cell-sized liposomes is shown in Figure 4 above. The composition of the lipid component system was as follows. In addition, a fluorescent reagent that stains the DOPC-rich Ld phase was used as the added reagent. DOPC / DPPC / Chol=4:4:2 +Lissamine Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt(Rhodamine DHPE) 1% (stained liquid disordered domain) This yields liposomes having an Lo / Ld domain structure, as shown in B in Figure 2 above.

[0047] [2. Procyanidin Mixing Conditions] The mixing conditions for pure procyanidin B2 were as follows: 0%(Control), 10%, 20%, 30%, 40%, 50% The final concentration of the lipid component system and PB2 was 0.2 mM, in a volume of 400 μL. 2 μM of Rhodamine DHPE was added to this to create reference liposomes for each concentration. Specifically, the volumes used for mixing are as follows: • Control: DOPC / DPPC / Chol / PB2=16 / 16 / 8 / 0 ·PB2_10%: DOPC / DPPC / Chol / PB2=16 / 16 / 8 / 4.44 ·PB2_50%: DOPC / DPPC / Chol / PB2 = 16 / 16 / 8 / 40 DOPC only: DOPC / DPPC / Chol / PB2=40 / 0 / 0 / 0

[0048] [3. Fluorescence microscopy observation of phase-separated domain structure] The conditions for fluorescence microscopy observation of the phase-separated domain structure are as follows: Microscope used: Olympus BX51 Observation conditions: WIG (excitation wavelength 530-550 nm, fluorescence wavelength 575 nm) Furthermore, observation is also possible using WIY (excitation wavelength 545-580 nm, fluorescence wavelength 610 nm).

[0049] [4. Reference liposomes] Figure 7 is a graph showing the phase separation domain structure composition in reference liposomes at each procyanidin concentration. The observed proportions of domain structures are shown as homogeneous membrane vesicles (white), Lo / Ld domain structure (light gray), and So / Ld domain structure (dark gray). As shown in the figure, the correspondence between each concentration and the phase separation domain structure composition is as follows.

[0050] Concentration 0%: Lo / Ld domain accounts for approximately 90%. 10% concentration: Lo / Ld domain approximately 50%, So / Ld domain less than 2%. Concentration 20%: Lo / Ld domain approximately 15% Concentration 30%: Lo / Ld domain approximately 15% Concentration 40%: Lo / Ld domain approximately 5% Concentration 50%: Lo / Ld domain approximately 20%

[0051] [5. Experiment using PB2 extracted from apple juice] It has been reported that the PB2 concentration in apple juice is 5-15 mg / 100g (research on functional components of apples and leeks from Nagano Prefecture). The inventor also obtained a value of 8.3 mg / 100g in a previous analysis. In this study, we decided to create cell-sized liposomes using PB2 extracted from apple juice, observe their phase separation domain structure, and attempt to estimate the procyanidin concentration. The apple juice used is as follows: Manufactured by Aomori Prefectural Agricultural Cooperative Federation (JA Aoren) Brand name: Kibou no Shizuku (Drops of Hope)

[0052] The extraction method for PB2 using acetone was as described in the above-mentioned literature (Research on Functional Components of Apples and Leeks from Nagano Prefecture). Specifically, 17.5 mL of acetone was added to a predetermined amount of sample apple juice, the solution was made up to 25 mL with Milli-Q (ultrapure water), and the mixture was extracted using a filter to obtain each sample (Case 1, 2, and 3). Case 1 (Apple Juice) 5mL of juice Case 2 (2x diluted apple juice): 2.5mL of juice Case 3 (Pure Water) Juice 0mL

[0053] The molecular weights of each component of cell-sized liposomes are as follows: DOPC:786 DPPC:734 Chol:387 PB2: 578 The weights of each component of cell-sized liposomes were calculated when the lipid content was 0.2 mM / 400 μL. Since the lipid weight in 0.2 mM / 400 μL is 0.08 mM, the following results are obtained. DOPC:786×0.08×400 / 1000 / 1000×1000 =25.15μg DPPC:687×0.08×400 / 1000 / 1000×1000 =21.98μg Chol:387×0.04×400 / 1000 / 1000×1000 = 6.192μg Furthermore, PB2 with a mol concentration of 10% is PB2=578×0.02×400 / 1000 / 1000=4.624μg

[0054] Case 1: Apple Juice Based on the above literature values, the concentration of PB2 extracted from apple juice, whose concentration was unknown, was assumed to be 10 mg / 100 g and used after diluting it five times. 20 mL of this diluted solution, which resulted in a concentration of 2 mg / 100 g under this assumption, was added to prepare cell-sized liposomes. The weight of procyanidin based on this assumption was: 2×20 / 1000 / 1000×1000=0.04mg=40μg Therefore, the molar concentration is, 40 / 1000×1000×1000 / 400 / 578=0.17mM That is the case.

[0055] Since the total lipid concentration in 400 μL is 0.2 mM and the procyanidin concentration is 0.17 mM, the procyanidin concentration in the cell-sized liposomes produced will be: 0.17 / (0.2+0.17)×100 = 45.9 mol% ≈ approximately 50 mol% This is how it is calculated. Based on this, the phase separation domain structure is expected to consist of approximately 20% Lo / Ld domains, as can be seen in Figure 7.

[0056] For reference, the calculated weights of each cell-sized liposome component mentioned above, converted to 100g for Case 1, are as follows. Breakdown: DOPC (40% 0.08mM) 6.29 mg per 100g DPPC (40% 0.08mM) 6.87 mg per 100g Chol (20% 0.04mM) 1.55 mg per 100g PB2 (10% 0.17mM) 14.71 mg per 100g

[0057] Case 2: 2x diluted apple juice The apple juice was diluted beforehand to a PB2 concentration of 5 mg / 100 g. Based on the above literature values, the unknown PB2 concentration extracted from the apple juice was assumed to be 10 mg / 100 g. This was diluted fivefold to 1 mg / 100 g, and 20 mL was added to prepare cell-sized liposomes. The procyanidin weight based on this assumption was: 1×20 / 1000 / 1000×1000=0.02mg=20μg Therefore, the molar concentration is, 20 / 1000×1000×1000 / 400 / 578=0.085mM That is the case. If we consider the concentration to be roughly half that of Case 1, the procyanidin concentration in the resulting cell-sized liposomes is estimated to be around 33 mol%. In that case, the phase separation domain structure is expected to have less than 10% Lo / Ld domains, as shown in Figure 7.

[0058] Case 3: Pure water The apple juice used is additive-free, and pure water is used. Therefore, the procyanidin concentration in the resulting cell-sized liposomes is 0 mol%. In that case, the phase separation domain structure is expected to consist of approximately 90% Lo / Ld domains, as can be seen in Figure 7.

[0059] [6. Experimental Results] Figure 8 is a graph showing the phase separation domain structure composition in the estimated target liposomes for the examples (Cases 1, 2, and 3). As shown in the figure, in the estimated target liposome using pure water in Case 3, fluorescence microscopy revealed that the So / Ld domain was approximately 5% and the Lo / Ld domain was approximately 90%. Comparing this with the graph of the reference liposome in Figure 7, it matched the phase separation domain structure composition of PB2 concentrations of 0% or 10%. In other words, the estimated result was that the procyanidin concentration (content) in pure water in Case 3 was 10 mol% or less relative to the lipids. Since the procyanidin concentration in pure water is naturally 0 mol%, the obtained estimation result was confirmed to be valid.

[0060] In Case 1 apple juice (Pure juice), the domains were difficult to observe, but approximately 8% of the Lo / Ld domains could still be confirmed by fluorescence microscopy. Comparing this with the reference liposome graph in Figure 7, it was found that the phase separation domain structure composition did not match that of PB2 concentrations of 0% or 10%, and that it only matched concentrations of 20% or higher. In other words, the estimated procyanidin concentration (content) in Case 1 apple juice was estimated to be 20 mol% or more relative to the lipids. As already mentioned, calculations based on the amount of PB2 contained in a standard juice, derived from dilution and addition, result in a concentration of approximately 50 mol%. Therefore, the obtained estimation result was confirmed to be valid.

[0061] In Case 2, the two-times diluted apple juice was even more difficult to observe, with less than 2% of Lo / Ld domains being confirmed by fluorescence microscopy. Comparing this with the reference liposome graph in Figure 7, it was found that the phase separation domain structure composition did not match that of PB2 concentrations of 0% or 10%, but only matched concentrations of 20% or higher. In other words, the estimated procyanidin concentration (content) in the two-times diluted apple juice of Case 2 was estimated to be 20 mol% or more relative to the lipids. As already mentioned, calculations based on the amount of PB2 contained in a standard juice, derived from the dilution and addition amount, result in a concentration of approximately 33 mol%. Therefore, the obtained estimation result was confirmed to be valid.

[0062] [7. Estimation of cost reduction effects] We estimated the costs (expenses and time) for the conventional measurement method using HPLC and the method of the present invention. 1) Existing methods (HPLC) Initial cost HPLC system: 15,730,000 yen (manufactured by 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 instrument setup and calibration curve sample measurement)

[0063] 2) 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 Corporation) Total A: 2,546,260 yen Furthermore, if lipids and PB2 necessary for database creation are included, Lipids and PB2: 159,800 yen Total B: 2,706,060 yen Running costs Lipid-rich DOPC: 8,400 yen DPPC 9,000 yen Chol 9,400 yen Rhodamin 40,800 yen Acetone, gallon bottle, 6,000 yen Total: 73,600 yen Measurement time Preparation: 8 hours (liposome preparation (film preparation 1H + vacuum drying 3H + hydration 4H)) Measurement: 1 hour (time required to observe 60 samples) Total: 9 hours

[0064] As described above, the present invention is estimated to have an initial cost of approximately 13,000 yen and a running cost of approximately 40,000 yen lower compared to the conventional HPLC method. Furthermore, the measurement time is estimated to be reduced to about half a day. (Note that glassware, pipettes, plastic tubes, filters, and syringes are inexpensive and do not make a difference, so they were omitted from this calculation.) [Industrial applicability]

[0065] The cell-sized liposome, procyanidin concentration estimation method, and procyanidin concentration measurement method of the present invention enable the analysis of procyanidin concentration at a significantly lower cost and in a shorter time than conventional methods. Therefore, this invention has high industrial applicability, particularly in the fields of food nutrition analysis, food quality control, and all related fields. [Explanation of Symbols]

[0066] 2, 3...phospholipids 5…Sterol lipids 8, 8a, 8b, 8c, 8d, 8n... Procyanidins 8x...Procyanidin, or a comparable element. 9…Fluorescent reagent for detecting phase separation domains 10, 10'... Cell-sized liposomes 10a, 10b, 10c, 10d... Cell-sized liposomes (reference liposomes) 10x…Cell-sized liposomes (estimated target liposomes) Da, Db, Dc, Dd, Dx... Phase-separated domain generation patterns N...All samples subject to concentration estimation n...Some samples that have undergone concentration estimation processing. P10…Concentration estimation process P20…Precision measurement process

Claims

1. A cell-sized liposome composed of a lipid component system in which one or more phospholipids and one or more sterol lipids are used in a fixed composition, and a procyanidin, The phospholipids are unsaturated phospholipids and saturated phospholipids. The lipid component system consists of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), Dipalmitoylphosphodidylcholine (DPPC), and cholesterol (Chol). The ratio of these is DOPC / DPPC / Chol = 4:4:

2. A cell-sized liposome characterized by generating different phase separation domain formation patterns depending on the procyanidin concentration, which can then be used to estimate the procyanidin concentration from the phase separation domain formation pattern.

2. The cell-sized liposome according to claim 1, characterized in that a fluorescent reagent for detecting phase separation domains is added.

3. The cell-sized liposome according to claim 2, characterized in that one or more of the fluorescent reagents selected from those for liquid ordered phase (Lo phase) staining, liquid disordered phase (Ld phase) staining, or sterol-based lipid staining are used.

4. A cell-sized liposome according to any one of claims 1, 2, or 3, characterized in that it is produced by a static hydration method.

5. The cell-sized liposome according to any one of claims 1, 2, 3, or 4, characterized in that the procyanidin is of known concentration and is used as a liposome for obtaining phase separation domain information in estimation of the concentration of a target substance for which the procyanidin concentration is unknown.

6. A method for estimating the procyanidin concentration in a substance to be concentrated, wherein one or more of the cell-sized liposomes described in any of Claims 1, 2, 3, 4, or 5 are used as liposomes for acquiring phase separation domain information, the cell-sized liposomes for acquiring phase separation domain information being composed of procyanidin standards of known concentration, the cell-sized liposomes for concentration estimation formed by adding an extract of the substance to be concentrated in place of procyanidin to the cell-sized liposomes described in any of Claims 1, 2, 3, 4, or 5 are observed under a fluorescence microscope, the phase separation domain generation pattern, i.e., whether or not a phase separation domain is generated or the pattern if it is generated, is read, and the concentration of procyanidin contained in the substance to be concentrated is estimated by referring to procyanidin concentration-dependent information of the phase separation domain generation pattern in the above-mentioned liposomes for acquiring phase separation domain information obtained in advance.

7. The method for estimating the procyanidin concentration according to claim 6, characterized in that the target of concentration estimation is an apple or a processed apple product.

8. A method for measuring procyanidin concentration, characterized by first estimating the procyanidin concentration of multiple samples using the procyanidin concentration estimation method described in either claim 6 or 7, and then performing a precise measurement of the procyanidin concentration of some of the samples that have undergone the procyanidin concentration estimation process using high-performance liquid chromatography.

Images