Cell-sized liposome, estimating method of relative procyanidin concentration using cell-sized liposome, and procyanidin concentration measurement method
Patent Information
- Application Number
- JP2023129777
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-08-09
- Publication Date
- 2025-02-21
Smart Images
Figure 2025025204000001_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to a method for estimating relative procyanidin concentration using cell-sized liposomes and a method for measuring procyanidin concentration, and in particular to a technique that can estimate the relative concentration of procyanidins contained in apple juice, etc., 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 of apple cultivation in Aomori Prefecture is 20,400 hectares, accounting for more than half of the total area in Japan. However, the cultivation area has been decreasing over the years, and maintaining and securing its position as a major producing area of the apple industry, which plays an important role not only in the regional industrial economy but also in culture and tourism, is an important issue for Aomori Prefecture. In this region, research has been accumulating that the functional ingredients contained in apples are effective in maintaining health and preventing disease. For example, "Prime Apple!" (variety: Fuji, registered trademark) has been registered as a functional food as a fruit, and this is due to the function of procyanidin in reducing visceral fat.
[0003] Procyanidin contained in apples is the main component of the polyphenols contained in apples (collectively known as apple polyphenols). Apple polyphenols are composed of various components, but procyanidin accounts for approximately 60% of them (from the Aomori Prefecture Apple Council website). Procyanidin is known to have particularly high antioxidant properties and also has fat-reducing effects, and apples, which contain a lot of procyanidin, are sold as functional foods.
[0004] HPLC (High Performance Liquid Chromatography) is used in the procyanidin concentration measurement test. This equipment is not only very expensive to purchase, but also has the disadvantage of high running costs because a high-purity organic solvent is used for each measurement. In addition, it takes several hours to start up the equipment, and a calibration curve is created by running multiple standard samples, and then the concentration measurement is finally performed, which takes a considerable amount of time.
[0005] Conventionally, patent applications and the like have been filed regarding techniques for measuring procyanidins. For example, Patent Document 1, which is listed below, discloses a method for quantifying procyanidins (such as n-polymers of catechin: n≧1, n is an integer) in food and drink 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 also discloses a method for quantifying procyanidins separated by size exclusion chromatography by a fluorescence detection method. Furthermore, Non-Patent Document 1 by the present inventor discloses a technique for estimating the concentration by adding a procyanidin extract at the stage of preparing a lipid film when preparing cell-sized liposomes, and observing the state of the phase-separated domain. [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, procyanidin, known as an apple polyphenol and a functional health component, has been attracting attention, and it is expected that the procyanidin content of processed apple foods will be labeled to add high value to the foods. However, HPLC (high performance liquid chromatography) used to measure procyanidin has problems in that it is expensive in both initial and running costs, and requires considerable time and effort. It would be convenient to have a simple analytical method in the form of a preliminary analysis, that is, a so-called screening method, for selecting samples for precision analysis by HPLC, which does not need to be as accurate as HPLC but can be performed at the lowest possible cost.
[0009] Liposomes, which are used in many experiments and research as the most simplified biomembrane model, are formed when phospholipids, which have both hydrophilic and hydrophobic fatty chains, self-assemble with the hydrophilic part facing outward to form a lipid bilayer membrane, which then closes into a bag shape. Liposomes with diameters of several μm to several tens of μm are roughly the same size as living cells and are called cell-sized liposomes (giant liposomes). Cell-sized liposomes are useful in research into the elucidation of the control mechanisms of biomembranes, due to the advantages that they can be directly observed with an optical microscope and can be used to create experimental models that mimic cells.
[0010] It has also been revealed that there are phase-separated domains (raft domains) on cell membranes that are rich in saturated lipids and cholesterol and that play a role in signal transmission, etc., and that these raft domains move by sticking together and separating in association with signal transmission between cells. It has been found that similar domain structures are observed in liposomes artificially made from lipids, and research in this field is also being actively conducted. 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) accumulation dynamics of membranes are related to signal transmission using intracellular calcium ions as an indicator, and that changing the structure of lipids and sterols changes 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 using the observed videos and images, and has published the results. 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 also clarified that domain structures are generated when membranes containing antioxidants are subjected to oxidative stress. In other words, when a solution of polyphenols such as resveratrol and theaflavin, which are known as antioxidant functional components, is mixed with cell-sized liposomes, membrane dynamics such as membrane contraction and oscillation are induced, and phase-separated domains appear during observation (HHT. Phan, T. Yoda et al., Biochim. Biophys. Acta, Biomembr. 2014). In addition, it has been revealed by fluorescence microscopy that concanavalin A, a functional component contained in sword beans that activates the immune system, causes the accumulation of domain structures on the cell membrane and activates intracellular signaling (S. Yabuuchi et al., 2017 J. Biosci. Bioeng.).
[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 undergo phase separation 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 above-mentioned Non-Patent Document 1, the inventor reported a technique for estimating the concentration of cell-sized liposomes by adding apple juice at the stage of preparing a lipid film and observing the state of the phase-separated domain. However, these methods involve a procedure of vacuum drying the organic solvent after film preparation, which may result in volatilization and evaporation of procyanidin. In addition, a procedure of extracting procyanidin is required.
[0015] The problem that the present invention aims to solve is to provide a simple analytical method for procyanidin concentration in the form of a preliminary analysis for selecting samples for precise analysis by HPLC, that is, a so-called screening method, based on the progress and results of research using liposomes and the state of the prior art related to the measurement of functional component procyanidin, which does not need to be as accurate as HPLC, but can be performed at the lowest possible cost and in a short time.In other words, to provide a method for easily distinguishing a simple measurement method that utilizes the obvious difference characterized by the interaction between procyanidin and membrane by observation, using cell-sized liposomes.Furthermore, in particular, the present application aims to provide a method that can suppress the volatilization and volatilization of procyanidin by adding a procyanidin-containing concentration measurement target such as apple juice at a time other than when film is made, and can obtain information on relative concentration without performing an extraction operation from the sample, thereby saving labor. [Means for solving the problem]
[0016] The inventors have carried out research with the above problem in mind. When preparing cell-sized liposomes, we tried adding apple juice at the hydration stage. First, we found that when apple juice was added during hydration to prepare cell-sized liposomes containing only DOPC, micrometer-scale liposomes were not observed. We then tried again by diluting apple juice with pure water, and found that cell-sized liposomes were formed at a 100-fold dilution. We also found that when diluted apple juice was added at the hydration stage to prepare cell-sized liposomes containing only DOPC, the size was significantly smaller than when hydrated with pure water, and further found that the size increased when a solution containing procyanidin was added to the diluted apple juice at the hydration stage. This was consistent with a report (T. Yoda, Chemistry Select 2022) that when procyanidin was added to the film in advance, the size increased depending on the concentration.
[0017] Therefore, it was speculated that even if diluted apple juice was added during hydration instead of during film preparation, procyanidins in apple juice were taken up during the cell-sized liposome formation stage. It was also discovered that the ratio of phase-separated domain structures differed when diluted apple juice was added during hydration compared to when a solution in which diluted apple juice had previously been partially replaced with procyanidins was added. It was found that this difference in ratio, specifically the procyanidin content in the sample, could be determined by the ratio of solid ordered / liquid disordered, that is, the ratio of phase separation.
[0018] In addition to the above findings obtained by the experiment using diluted apple juice, the cost reduction effect was also confirmed by trial calculation, and it was confirmed that the cell-sized liposomes can be used as a procyanidin concentration analysis tool. That is, in order to analyze the procyanidins contained in diluted apple juice, etc., the relative concentration of procyanidins is estimated in a short time by observing the ratio of domain structures of cell-sized liposomes containing unsaturated lipids, more preferably cell-sized liposomes containing unsaturated lipids and saturated lipids, which are prepared by adding diluted apple juice at the time of hydration (Note that the reason why "unsaturated lipids" and "saturated lipids" are used here, and not "unsaturated lipids such as DOPC" or "saturated lipids such as DPPC", is that the cell-sized liposomes according to the present invention can also be prepared by using lipids other than the phospholipids that are the main subject of the description of this application, such as glycolipids. In other words, these are also within the scope of the present invention). The present invention thus completed, that is, the invention claimed in this application as a means for solving the above problems, or at least the invention disclosed therein, is as follows.
[0019] [1] A cell-sized liposome comprising a lipid component system using one or more types of unsaturated phospholipids, and a sample or procyanidin for which a procyanidin concentration is to be estimated, the cell-sized liposome producing a different phase-separation domain generation pattern depending on the procyanidin concentration, and thus being capable of being used to estimate the procyanidin concentration from the phase-separation domain generation pattern. [2] The cell-sized liposome according to [1], characterized in that the lipid component system contains one or more types of saturated phospholipids. [3] The cell-sized liposome according to [2], characterized in that the lipid component system contains one or more types of sterol 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 phospholipid:saturated phospholipid=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], further comprising a fluorescent reagent added thereto for detecting phase-separated domains. [9] The cell-sized liposome according to claim 8, wherein the fluorescent reagent is one or more of a solid ordered phase (So phase) staining reagent, a liquid disordered phase (Ld phase) staining reagent, and a sterol lipid staining reagent.
[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 a relative concentration of procyanidin in an object to be estimated using a cell-sized liposome for obtaining phase-separation domain information according to any one of [1], [2], [3], [4], and [5], which is constituted using a procyanidin standard sample having a known concentration, comprising: observing the cell-sized liposome to be estimated using a concentration estimation object, which is the cell-sized liposome for obtaining phase-separation domain information according to any one of [1], [2], [3], [4], and [5], which is constituted using a sample to be estimated using a procyanidin concentration; reading the state of phase-separation domain formation, i.e., whether or not a phase-separation domain is formed or the state in which a phase-separation domain is formed, or the state of cell-sized liposome formation; and estimating the relative concentration of procyanidin contained in the object to be estimated using information on the procyanidin concentration dependency of the state of phase-separation domain formation in the liposome for obtaining phase-separation domain information or the state of cell-sized liposome formation.
[13] The method for estimating a relative procyanidin concentration according to
[12] , wherein the subject of concentration estimation is apple juice or a processed apple juice product.
[14] A method for measuring procyanidin concentration, comprising first estimating the relative procyanidin concentrations of a plurality of samples by the method for estimating relative procyanidin concentrations described in
[12] , and then performing precise measurement of the procyanidin concentrations of some of the samples that have been subjected to the procyanidin concentration estimation process by high performance liquid chromatography.
[15] The method for measuring procyanidin concentration according to
[14] , wherein the subject of concentration measurement is apple juice or a processed apple juice product. Effect of the Invention
[0022] Cell-sized liposomes of the present invention, a method for estimating relative procyanidin concentration using cell-sized liposomes, and a method for measuring procyanidin concentration teeth As configured as described above, it is possible to provide a simple measurement method utilizing clear differences characterized by observation due to the interaction between procyanidins and membranes, and a simple discrimination method using cell-sized liposomes. Specifically, it is possible to analyze procyanidin concentrations at significantly lower cost and in a shorter time than in the past.
[0023] Conventional measurement methods requiring expensive HPLC have the problem of requiring a long time for equipment startup, etc., but 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 the cell-sized liposomes are observed under a fluorescent microscope. As long as the suitable conditions for the combination of lipids constituting the cell-sized liposomes are determined, the concentration of the contained procyanidins can be estimated by only observing for a short period of time. In addition, according to the present invention, the volatilization and evaporation of procyanidins can be suppressed, and the relative concentration information can be easily obtained without performing an extraction operation from the sample, thereby saving labor.
[0025] As an effective example of use, the present invention can be used for preliminary analysis to select samples for precision analysis by HPLC, i.e., for simple procyanidin concentration determination in a screening-like manner, and is highly useful. Therefore, by combining the method of the present invention with HPLC, a rapid, low-cost, and precise analysis system can be realized. [Brief description 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 phase-separated domain generation pattern in the cell-sized liposome of the present invention. [Figure 1-3] FIG. 2 is a conceptual diagram showing another basic structure of the cell-sized liposome of the present invention, which also contains saturated phospholipids. [Diagram 2] FIG. 2 is a conceptual diagram showing another configuration of the cell-sized liposome of the present invention, which also contains a sterol lipid. [Diagram 3] 1 shows the chemical structural formulae of lipids, which are examples of components of the cell-sized liposome of the present invention, and procyanin B2. [Figure 4] FIG. 1 is an explanatory diagram showing an example of a method for producing the cell-sized liposome of the present invention. [Diagram 5] FIG. 1 is an explanatory diagram conceptually showing the configuration of a procyanidin concentration estimation method of the present invention. [Figure 6]1 is a flow chart showing the configuration of the method for measuring procyanidin concentration of the present invention (the following figures relate to the Examples). [Figure 7] 1 is a graph showing the phase-separated domain structural composition in reference liposomes according to Examples (Cases 1, 2, and 3). [Figure 8] 1 is a graph showing the phase transition temperature, i.e., domain disappearance temperature, measurement results for each cell-sized liposome according to the examples (Cases 1, 2, and 3). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The present invention will now be described in detail 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 of the present invention is a cell-sized liposome composed of a lipid component system using one or more kinds of unsaturated phospholipids 2 (the figure shows an example in which only one kind of unsaturated phospholipid 2 is used, but even one kind can sufficiently achieve the intended effect of the present invention), and a sample or procyanidin 8 for which the procyanidin concentration is to be estimated, and the main structure of the cell-sized liposome 10 is that it produces a different phase-separation domain generation pattern depending on the procyanidin concentration, and is thus used to estimate the procyanidin concentration from the phase-separation domain generation pattern. Note that (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 liposome 10 produces and exhibits 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 procyanidin contained therein. 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 formation pattern.
[0029] The cell-sized liposome of the present invention having the structure shown in FIG. 1 will be described below. The fact that phase-separated domains can be observed under fluorescence observation with a single type of unsaturated lipid, such as DOPC, is believed to be due to active oxygen generated during fluorescence observation. That is, in fluorescence observation, excitation light is irradiated and the fluorescence emitted from the fluorescent dye is observed, and active oxygen is generated at that time, which oxidizes and breaks the unsaturated bond of the unsaturated lipid, i.e., part of the double bond. As a result, two types (or several types) of lipids exist in the system: unoxidized unsaturated lipids and oxidized saturated lipids, which are observed as phase separation in the lipid membrane.
[0030] In fact, unsaturated lipids such as DOPC have a low phase transition temperature, and liposomes can 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 cannot be affected by oxidation by active oxygen during fluorescence observation, so it is thought that the desired formation of phase-separated domains cannot be obtained with liposomes whose lipid component system is composed only of saturated phospholipids.
[0031] FIG. 1-2 is a photograph showing an example of a phase-separated domain generation pattern in the cell-sized liposome of the present invention. These are micrographs showing typical patterns, among 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. As a result of observation using a fluorescent microscope, in the present invention, the So / Ld domain structure pattern shown in C can be particularly preferably used for concentration estimation. Observation using a fluorescent microscope will be described further below.
[0032] 1-3 is a conceptual diagram 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 using one or more kinds of unsaturated phospholipids 12 (the figure shows an example of only one kind of unsaturated phospholipid 12), as well as one or more kinds of saturated phospholipids 13 (the figure shows an example of only one kind of saturated phospholipid 13. The desired effect of the present invention can be sufficiently obtained even with one kind), and a sample or procyanidin 18 for which the procyanidin concentration is to be estimated. The main structure of the cell-sized liposome is that it generates different phase-separated domain generation patterns depending on the concentration of ethyl caproate 18, and is used to estimate procyanidins from the phase-separated domain generation patterns.
[0033] The cell-sized liposome 110 of this configuration also produces and exhibits different phase-separation domain generation patterns depending on the concentration of procyanidin 18 contained therein, and the procyanidin concentration contained therein can be estimated using the phase-separation 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-separation domain generation pattern. In order to more fully achieve the intended object of the present invention, the cell-sized liposome 110 of this invention, which is composed of unsaturated lipids and saturated lipids, is more desirable than the cell-sized liposome 10 shown in FIG. 1.
[0034] 2 is a conceptual diagram showing another structure of the cell-sized liposome of the present invention, which also contains a sterol lipid. As shown in (a) of the figure, the cell-sized liposome 210 is a lipid component system containing one or more kinds of unsaturated phospholipids 22 (the figure shows an example of only one kind of unsaturated phospholipid 22), one or more kinds of saturated phospholipids 23 (the figure shows an example of only one kind of saturated phospholipid 23), and one or more kinds of sterol lipids 25 (the figure shows an example of only one kind of sterol lipid 25. The desired effect of the present invention can be sufficiently obtained even with one kind), and a sample or procyanidin 28 to be estimated as a procyanidin concentration, and the main structure is that different phase-separated domain generation patterns are generated depending on the concentration of procyanidin 28, and thus the cell-sized liposome 210 is used to estimate the procyanidin concentration from the phase-separated domain generation patterns.
[0035] The cell-sized liposome 210 having such a configuration also produces and exhibits different phase-separation domain generation patterns depending on the concentration of procyanidin 28 contained therein, and therefore the phase-separation domain generation pattern can be used to estimate the concentration of procyanidin contained therein. 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-separation domain generation pattern.
[0036] Sterol lipids are not essential elements for forming 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, so the hydration temperature can be lowered. In the present invention, heating is performed during hydration to prepare cell-sized liposomes, but by performing hydration at a lower temperature, the thermal decomposition of procyanidins can be suppressed.
[0037] In addition, sterol lipids such as cholesterol have the effect of increasing the fluidity of phospholipids, which allows for better mixing of lipids. This reduces the bias in the composition of each liposome produced, and increases the uniformity of the liposomes produced. As a result, the reproducibility of the implementation of the ethyl caproate relative concentration estimation method is improved. There are also other advantages when using a specific fluorescent reagent, which will be described later (see below).
[0038] On the other hand, there is also an advantage in not intentionally including cholesterol in the lipid component system. This is the case when observing mainly the change in the So / Ld phase separation structure, as in the ethyl caproate relative concentration estimation method of the present invention described later with reference to FIG. 5 and the like. By not including cholesterol in the lipid component system, almost all of the obtained cell-sized liposomes are So / Ld, so when observing mainly the change in the So / Ld phase separation structure, the phase separation domain generation pattern can be easily 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 read the discrimination pattern.
[0039] In particular, the present invention can provide 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. In addition to the above configuration, cell-sized liposomes can also be provided having a lipid component system containing cholesterol (Chol), a sterol lipid. In the following description, cell-sized liposomes having either a lipid component system of DOPC+DPPC or a lipid component system of DOPC+DPPC+Chol will be mainly described.
[0040] Fig. 3 shows the chemical structure 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 is one of the procyanidin dimers and is used as a standard substance for concentration measurement.
[0041] When the lipid component system of the cell-sized liposome of the present invention is used at room temperature, the molar composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system can be unsaturated phospholipid:saturated phospholipid = 75 to 10: 25 to 90. Alternatively, when the lipid component system is used at room temperature, the molar composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system can be configured to be equal. Here, room temperature may be about 20°C, which is generally understood, or may be 22±2.0°C, which is more preferable.
[0042] As an example of a specific lipid species, the lipid component system of the cell-sized liposome of the present invention has a molar composition when used at room temperature 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 the 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 in 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), DOPC:DPPC:Chol=42.5~50 : 42.5~50 : remaining amount For example, when Chol is 10, the So / Ld domain structure can be obtained in a molar composition range of DOPC 0 to 65 and DPPC 25 to 90. As described above, DOPC and DPPC may be used in equal amounts. In the examples described later, DOPC and DPPC are used in equal amounts (50:50).
[0045] The phase-separated domains themselves are observed even when cholesterol is 15 or more. For example, when cholesterol is 20 to 40 and phospholipid is 60 to 80, Lo / Ld (liquid-ordered phase / liquid-disordered phase) domains are observed. However, no correlation was found between the domain formation ratio and the procyanidin concentration in this domain pattern.
[0046] The cell-sized liposome of the present invention may be configured to have a fluorescent reagent 9 or the like added thereto for detecting phase-separated domains, such as the cell-sized liposome 10' in Fig. 1, Fig. 1-3, and Fig. 2(b). The presence or absence of a phase-separated domain structure is determined by fluorescent observation of the cell-sized liposome, and the fluorescent reagent 9 is added for that purpose and does not affect the formation of phase separation. The fluorescent reagent 9 may be one or more of the following for staining a liquid ordered phase (Lo phase), a liquid disordered phase (Ld phase), or a sterol lipid stain to constitute the cell-sized liposome 10'.
[0047] In the examples described below, Rhodamine B 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine Triethylammonium Salt (Rhodamine DHPE) is added as a fluorescent reagent at 1% molar concentration of lipids. This is a relatively commonly used 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 a fluorescent reagent for staining the DPPC-rich So or Lo phases. These have different fluorescent wavelengths, so they can be used separately or two types can be used at the same time.
[0048] In addition, the cholesterol portion can be fluorescently stained, for example, with NBD-Cholesterol. In the present invention, which uses the So / Ld domain structure, the So domain, which is rich in DPPC, is stained. When using NBD-Cholesterol, it is believed that the degree of fluorescent staining will be better if the lipid component system contains cholesterol. Of course, in the present invention, fluorescent reagents other than those described above can also be used.
[0049] FIG. 4 is an explanatory diagram showing an example of a method for producing the cell-sized liposomes of the present invention. As shown in the figure, the cell-sized liposomes can be produced by the static hydration method. Alternatively, after producing a lipid film, a sample whose concentration is to be estimated can be added when hydrating the cell-sized liposomes. That is, the lipids constituting the liposomes are dissolved in a solvent (chloroform CH(Cl)3 in the example shown in the figure) in a container such as a test tube, the solvent is evaporated to form a lipid membrane on the inner wall of the container, and the lipid membrane is hydrated with a diluted solution of apple juice, which is the target sample, to produce the liposomes.
[0050] In the examples described below, the lipid membrane and the sample to be measured were heated to about 50°C during hydration, and cell-sized liposomes were prepared using the static hydration method. This is a process for making the lipids and procyanidins uniform in the lipid membrane by heating the temperature to above 41°C, which is the melting point of DPPC. Note that about 50°C is merely an example, and it goes without saying that the present invention is not limited to this.
[0051] The phase separation domain structure is determined by the components in the membrane, such as the constituent lipids, and not by the production method. Therefore, the specific production method of the cell-sized liposome of the present invention is not limited, and a conventionally known method may be used as appropriate. For example, there are electroformation method, droplet method, and its new method (Patent No. 6031711), but in the present invention, the static hydration method can be particularly preferably used. 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 there is a test tube, gas, and a specified solution, and is optimal for the present invention.
[0052] 5 is an explanatory diagram conceptually showing the configuration of the procyanidin concentration estimation method of the present invention. The cell-sized liposome of the present invention having any of the configurations described above can be used as a liposome used in estimating the concentration of an estimation target having an unknown procyanidin concentration, i.e., as a "liposome for obtaining phase-separation domain information." In this figure, the "liposome for obtaining phase-separation domain information" is abbreviated to "reference liposome," and this abbreviation will also be used in the following explanation.
[0053] 5, the center column in the table on the left conceptually shows reference liposomes 10a, 10b, ..., 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 of the liposomes 10a, 10x, etc. The procyanidin concentrations n=a, etc. of the reference liposomes 10a, etc. are known, while the procyanidin concentrations n=x, etc. of the 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 a relative concentration n=x, etc. of a procyanidin in a concentration estimation subject using the above-mentioned phase-separation domain information acquisition liposome (reference liposome) 10a, etc. The reference liposome 10a, etc. is constructed by adding a procyanidin standard with a known concentration, such as n=a, to the concentration estimation subject. The 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 Fig. 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. Putative target liposome 10x containing procyanidin 8x at a concentration n=x (unknown) has domain generation pattern Dx, and putative target liposome 10y containing procyanidin 8y at a concentration n=y (unknown) has domain generation pattern Dy.
[0056] The procyanidin concentration estimation method of the present invention is a method of observing the reference liposomes 10a, 10b, ...under a fluorescent microscope to read the phase separation domain generation patterns Da, Db, ..., i.e., the phase separation domain generation status, such as the presence or absence of phase separation domain generation or the pattern when a phase separation domain is generated, or alternatively, the cell-sized liposome generation status; observing the concentration estimation subject cell-sized liposomes 10x, 10y, ...under a fluorescent microscope to read the phase separation domain generation patterns Dx, Dy, ..., i.e., the phase separation domain generation status, such as the presence or absence of phase separation domain generation or the pattern when a phase separation domain is generated, or alternatively, the cell-sized liposome generation status; and estimating the relative concentration of procyanidin contained in the concentration estimation subject by referring to the procyanidin concentration-dependence information of the phase separation domain generation status in the reference liposome 10a etc. or the cell-sized liposome generation status.
[0057] That is, in this procyanidin concentration estimation method, the phase-separated domain generation patterns Da, Db, ... in the reference liposome 10a, etc., have procyanidin concentration dependency, and this pattern information is compared to estimate the relative concentrations in the estimation subject liposome 10x, etc., that is, the relative ranking of the concentrations of the procyanidins contained in the concentration estimation subject, and the concentration range is estimated. In Fig. 5, an estimation result in which the concentrations of a, x, y, and b are highest in this order is shown on the right side of the figure as an example. At the same time, an 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 procyanidin concentrations are estimated for all samples N to be subjected to concentration estimation using the procyanidin concentration estimation method described above for a plurality of samples, and then a precision measurement process P20 in which the procyanidin concentrations of some of the samples n that have already been subjected to procyanidin concentration estimation processing are precisely measured by HPLC.
[0059] This method for measuring procyanidin concentration is a method in which a simple procyanidin concentration determination is performed in a concentration estimation process P10 for preliminary analysis to select samples for precision analysis by HPLC in the precision measurement process P20, in the form of a screening, and after the concentration estimation process P10 has determined whether the procyanidin content in the samples is relatively high or low, the precision measurement process P20 is performed on a portion of the samples n. Therefore, a rapid, low-cost, and precise analysis system can be realized.
[0060] The procyanidin concentration estimation method and procyanidin concentration measurement method of the present invention described above can be used for all estimation / measurement objects expected to contain procyanidins, and it goes without saying that the methods of the present invention can be applied to apple juice and processed products containing procyanidins. EXAMPLES
[0061] The following describes examples of the present invention, but the present invention is not limited thereto. Note that the examples are provided by outlining some of the experimental results that led to the completion of the present invention. [Research topic] Development of a simple method for detecting procyanidins 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.
[0062] [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) As a result, a liposome having an So / Ld domain structure as shown in A in FIG. 1-2 above is obtained.
[0063] 2. Fluorescence microscopy 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) In addition, it can also be observed with WIY (excitation wavelength 545-580 nm, fluorescence wavelength 610 nm).
[0064] [3. Experiment using apple juice] It has been reported that the PB2 concentration in apple juice is 5-15mg / 100g (Research on functional components of apples and green onions produced in Nagano Prefecture). The inventors also obtained a value of 8.3mg / 100g in a previous analysis. In this study, we prepared cell-sized liposomes using apple juice diluted 100 times in the hydration step, observed the phase-separated domain structure, and attempted to estimate the procyanidin concentration.
[0065] The samples used in the experiment are as follows: 1 apple juice Manufacturer: Aomori Prefectural 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
[0066] The lipid film formation for the production of cell-sized liposomes was carried out as follows. The lipid was adjusted to a concentration of 2 mM as a chloroform solution, and a total of 40 μL of solution was added. Hydration was carried out after evaporating and drying the organic solvent. Below, the cases used in the experiment are listed and the conditions are described. Case 1 (Apple Juice 1) Case 2 (Apple juice 1 + PB2) Case 3 (pure water)
[0067] Case 1 Apple juice 1 It has been reported that the PB2 concentration in apple juice is 5-15mg / 100g (study on functional components of apples and green onions produced in Nagano Prefecture). The inventors have also obtained a value of 8.3mg / 100g in a previous analysis. In this study, based on the above literature value, the PB2 concentration in apple juice was assumed to be 10mg / 100g, and the juice was diluted 100 times before use. According to this assumption, the concentration of the diluted solution should be 0.1mg / 100g, and 400μL of the diluted solution was added to prepare cell-sized liposomes. The weight of procyanidin in the cell-sized liposome solution based on this assumption is as follows: 0.1 / 100×0.4×1000=0.4μg (After converting to 400μL, adjust the units)
[0068] Case 2 Apple juice 1+PB 10% of the apple juice 1 was replaced with PB 20μM solution, and 400μL was added during hydration. The amount of solution added was 10% of the total amount of 400μL, i.e. 40μL. Therefore, the weight of procyanidin in the cell-sized liposome solution based on the above assumptions is as follows. 4×0.9+20 / 1000 / 1000×40×578.52 ≒ 0.9μg (The first term is obtained by reducing apple juice 1 by 10%, and the second term is multiplied by the volume added after adjusting the units, and then multiplied by the molecular weight of procyanidin B2, 578.52. The sum of the first and second terms is rounded off to obtain an approximate weight.)
[0069] Case 3 Pure water Pure water was used. Therefore, the molar weight of PB in the cell-sized liposome solution was 0 μg.
[0070] 4. Experimental Results 7 is a graph showing the phase-separated domain structure composition in the reference liposomes according to the examples (cases 1, 2, and 3). As a result of the fluorescence microscopic observation, as shown in the figure, in the reference liposomes using pure water in case 3, the So / Ld domain was about 89%, the Lo / Ld domain was about 3%, and homogenous was about 8%. Since the PB2 concentration in pure water was naturally 0 mol%, it was confirmed that the obtained estimation result was appropriate in light of Non-Patent Document 1.
[0071] In Case 1 Apple Juice 1, the So / Ld domain was approximately 40%, the Lo / Ld domain was approximately 25%, and the homogenous domain was approximately 35%.
[0072] In case 2, apple juice 1 + PB, the So / Ld domain was approximately 36%, the Lo / Ld domain was approximately 8%, and the homogenous domain was approximately 56%.
[0073] 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.
[0074] As described above, the results this time showed that the order of highest procyanidin concentration was Case 2, Case 1, and Case 3. Also, the order of lowest ratio of So / Ld domain observed in this example was Case 2, Case 1, and Case 3, which were in order of relative concentration. Also, since the procyanidin concentration was lower in Case 1 Apple juice 1 than in Case 2 Apple juice 1 + PB, 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.
[0075] From the above, it was confirmed that the method of the present invention can be used to simply rank the relative concentrations of procyanidins. Furthermore, it was confirmed that the method of the present invention can easily estimate the concentration range of procyanidins.
[0076] 5. Effectiveness of Fluorescence Microscopy It should be noted that the implementation of the fluorescence microscopy according to the present invention is guaranteed without any problems. 8 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 25° C. or higher, and it has been confirmed that there is no problem with fluorescence microscopy observation at room temperature.
[0077] [6. Estimation of cost reduction effect] 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)
[0078] 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) 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 1H + vacuum drying 3H + hydration 4H)) Measurement: 1 hour (time required to observe 60 samples) Total: 9 hours
[0079] 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 about 13,000,000 yen and a running cost of about 40,000 yen. In addition, the time required for measurement is estimated to be shortened to about half a day (note that glassware, pipettes, plastic tubes, filters, and syringes are inexpensive and do not make a difference, so they are not included in this calculation).
[0080] [7. Supplementary Note: Estimation based on liposome diameter] In this experiment, the diameter of the cell-sized liposomes produced by apple juice (case 1) and apple juice + PB (case 2) was found to be larger in the latter case. The tendency for the size to increase was also observed in a previous report by the inventor (T. Yoda, Chemistry Select 2022). From this, we would like to add that the relative concentration of procyanidins can be estimated not only by the So / Ld domain generation status, but also by comparing the generation status of cell-sized liposomes, especially the size (increase or decrease). [Industrial Applicability]
[0081] According to the cell-sized liposomes, the method for estimating relative procyanidin concentration, and the method for measuring procyanidin concentration of the present invention, procyanidin concentration can be analyzed at a significantly lower cost and in a shorter time than in the past, and therefore the invention is highly industrially applicable, particularly in the fields of food component analysis, food quality control, and all related fields. [Explanation of symbols]
[0082] 2, 12, 22...Unsaturated phospholipids 8, 8a, 8b, 8x, 8y, 18, 28... Procyanidin 9, 19, 29…Fluorescent reagents for detecting phase-separated domains 10, 10', 110, 110', 210, 210'...cell-sized liposomes 10a, 10b...cell-sized liposomes (reference liposomes) 10x, 10y...cell-sized liposomes (estimated target liposomes) 13, 23...Saturated phospholipids 25...Sterol lipids Da, Db, Dx, Dy...Phase separation domain generation pattern N: All samples for which concentration is estimated n: A portion of the sample that has been subjected to concentration estimation processing P10…Relative concentration estimation process P20…Precision measurement process
Claims
1. A lipid component system comprising one or more unsaturated phospholipids; and The sample or procyanidin for which the procyanidin concentration is to be estimated A cell-sized liposome comprising: Different procyanidin concentrations resulted in different phase-separated domain formation patterns. This results in cell-sized liposomes that can be used to estimate procyanidin concentration from phase-separated domain formation patterns.
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, characterized in that the lipid component system contains one or more sterol lipids.
4. The cell-sized liposome according to claim 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, dipalmitoylphosphotidylcholine (DPPC).
5. The cell-sized liposome according to claim 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, dipalmitoylphosphotidylcholine (DPPC), and a sterol lipid, cholesterol.
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, 3, 4 or 5, wherein the molar concentration composition of the unsaturated phospholipid and the saturated phospholipid in the lipid component system is equal when used at room temperature.
8. 6. The cell-sized liposome according to claim 1, further comprising a fluorescent reagent added thereto for detecting phase-separated domains.
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 a sterol lipid.
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, characterized in that 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 a relative concentration of procyanidin in a subject for concentration estimation using a cell-sized liposome for obtaining phase-separation domain information according to any one of claims 1 to 5, which is constituted using a procyanidin standard having a known concentration, and using the liposome for obtaining phase-separation domain information, comprising: A cell-sized liposome for which a procyanidin concentration is to be estimated is observed under a fluorescent microscope, the cell-sized liposome being the subject of concentration estimation, the cell-sized liposome being the subject of concentration estimation according to any one of claims 1, 2, 3, 4, and 5, The state of phase-separated domain formation, i.e., whether or not a phase-separated domain is formed, or the state in which such a domain is formed, or the state of cell-sized liposome formation, is read; With reference to procyanidin concentration-dependent information on the phase-separation domain formation status or the cell-sized liposome formation status in the phase-separation domain information-obtaining liposome, Estimating the relative concentration of procyanidins contained in a concentration estimation target A method for estimating a relative procyanidin concentration, comprising:
13. The method for estimating a relative procyanidin concentration according to claim 12, wherein the subject of concentration estimation is apple juice or a processed apple juice product.
14. A method for measuring procyanidin concentration, comprising: first estimating the relative procyanidin concentrations of a plurality of samples by the method for estimating the relative procyanidin concentration of claim 12; and then performing precise measurement of the procyanidin concentrations of some of the samples that have been subjected to the procyanidin concentration estimation process by high performance liquid chromatography.
15. The method for measuring procyanidin concentration according to claim 14, wherein the subject of concentration measurement is apple juice or a processed apple juice product.
Citation Information
Patent Citations
RU2009-156813