Plant-derived free ceramides, methods of manufacture and methods of analysis thereof

Abstract

[Problem] To provide a method for simply and efficiently producing a plant-derived free ceramide using a plant raw material. [Solution] An endogenous enzyme activation step for disrupting the cellular structure of a plant other than callus to obtain a crushed plant in which glycosyl inositol phosphoceramide is used as a substrate and phospholipase C endogenous to the plant cells is activated; and a decomposition step in which the crushed plant is individually reacted with and / or other plant-derived glycosyl inositol phosphate ceramide-containing substances at a temperature in the range of 20-30 DEG C, thereby selectively decomposing the plant-derived glycosyl inositol phosphate ceramide into free ceramides using phospholipase C. The invention also relates to a method for producing such a ceramide-containing plant-derived glycosyl inositol phosphate ceramide-containing plant-derived glycosyl inositol phosphate ceramide-containing plant-derived glycosyl inositol phosphate ceramide-containing plant.

Description

[Technical field]

[0001] Claiming priority This application claims the priority of Japanese Patent Application No. 2022-162901 filed on October 11, 2022, the entire contents of which are incorporated herein by reference. For the original specification (including claims, specification and drawings) of the prior application on which the priority is claimed, the filing date of the prior application will be deemed as the priority date (reference date) and will not be affected by the newly added specification.

[0002] The present invention relates to a plant-derived free ceramide, and a preparation method and an analysis method thereof. [Prior Art]

[0003] Sphingolipids are a biological membrane component and are a general term for lipids composed of long-chain bases (hereinafter referred to as "LCB") and long-chain amino alcohols. The fatty acid bound to LCB is called free ceramide.

[0004] Sphingolipids present in plants can be divided into four categories: free LCB, free ceramide (hereinafter referred to as "Cer"), glucosylceramide (hereinafter referred to as "GlcCer"), and glycosyl inositol phosphoceramide (hereinafter referred to as "GIPC"). Among them, free LCB and free ceramide are trace components, while glucosylceramide and GIPC account for the majority of biological membrane lipids.

[0005] There is an extracellular lipid layer in the human stratum corneum that serves as a skin barrier. Free ceramides have very long chain (C20 and above) fatty acids and are the main components of the extracellular lipid layer and a substance that supports the skin barrier function. Supplementing free ceramides through the skin or orally can improve skin function.

[0006] However, the natural content of free ceramides is very low, so chemical synthetic products have traditionally been used in cosmetics.

[0007] In recent years, plant-derived ceramide has attracted much attention as a functional ingredient in skin care. Plant-derived ceramide is a functional ingredient that far exceeds substances such as hyaluronic acid, and its market size is expanding globally.

[0008] Currently, the plant-derived ceramide that is mainly used as an ingredient for functional foods is glucosylceramide. Glucosylceramide is composed of sugars bonded to long-chain (C16-18) ceramides, and its structure is different from human free ceramides, which have very long-chain fatty acids. Therefore, it has a weak effect on the skin and is not suitable for use in cosmetics. There are also problems such as low digestibility when using it as food. Glucosylceramide cannot completely replace the functions of free ceramide.

[0009] Free ceramides, which are the same type as those in human skin, can be applied directly to the skin as a cosmetic ingredient, with significant effects, and are also expected to improve digestion and absorption. However, free ceramides are a rare ingredient with no examples of natural accumulation outside of the stratum corneum of animals, so the current mainstream is to use chemical synthesis methods, including similar compounds. Free ceramides, as a safe and inexpensive plant-derived ingredient, are a particularly promising market demand in the skin care industry, and are eagerly sought after as ingredients in food, beverages, cosmetics and medicines.

[0010] Among them, GIPC is commonly found in all plants, and its content is several times that of glucosylceramide. GIPC is the most abundant ceramide-containing substance known on earth. GIPC is composed of sugar chains and very long chain pairs of ceramides. In other words, the structure of the ceramide part is the same or similar to that of human skin ceramide. However, its polysaccharide part is extremely stable, and enzymatic decomposition by animal digestive juices or intestinal bacteria has not been observed. Although GIPC is present in large quantities in the plant foods we eat daily, it is an underutilized ceramide resource in both biological and industrial fields.

[0011] The chemical structure of the ceramide skeleton and hydrophilic part of plants is very diverse, and the total number of molecular species present in a plant species can reach several hundred. In addition, the molecular composition varies depending on the plant species and tissue. It is not easy to obtain complete information using ordinary analytical methods.

[0012] As a method for producing plant-derived free ceramide, Patent Document 1 discloses a method for extracting and purifying ceramide by adding an alcohol solvent to fermentation lees, a byproduct produced when fermentation products are produced using Aspergillus koji.

[0013] Patent Document 2 discloses a method for extracting and purifying free ceramide by adding an alcohol solvent to a chestnut peel extract.

[0014] Patent Document 3 discloses a method for producing free ceramide by inducing autophagy by heat-treating a fragment of undifferentiated plant callus at 45° C. for 5 hours.

[0015] Patent Document 4 discloses a composition for digesting GIPC or GlcCer to produce free ceramide as a method for preparing free ceramide by mixing GIPC with GlcCer, wherein the composition contains a mushroom solution or a purified product thereof.

[0016] Non-patent document 1 discloses that an enzyme activity (hereinafter referred to as "GIPC-phospholipase D" or "GIPC-PLD") that specifically hydrolyzes GIPC into phytoceramide-1-phosphate (hereinafter referred to as "PC1P") was found in cabbage, and that this enzyme activity was activated when leaf tissue was ground, and it is speculated that PC1P in the body is converted into free ceramide by phosphatase in the digestive tract.

[0017] Non-patent document 2 describes a method for extracting plant ceramides called the Bligh & Dyer method. A mixture of chloroform, methanol, and water is used as the basic extraction solvent, and potassium chloride or hydrochloric acid is sometimes added. The two-phase partition between the chloroform phase and the aqueous phase can remove water-soluble and insoluble foreign matter, and is the most common lipid extraction method.

[0018] A method for extracting phytoceramide-related substances called the Markham method is described in Non-Patent Document 3. This method uses isopropyl alcohol as an extraction solvent and can effectively extract phytoceramide-related substances such as GIPC.

[0019] Non-patent document 4 discloses a method for analyzing the structural diversity of plant sphingolipids using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0020] However, the methods of Patent Documents 1 and 2 are only to obtain free ceramide already present in the raw material by repeatedly or multiple extraction and purification steps using an alcohol solution. Considering the content of free ceramide in the raw material, these methods are not efficient.

[0021] The method of Patent Document 3 requires the preparation of a culture medium and an apparatus for culturing undifferentiated plant callus in advance, which is not convenient and inefficient for mass production. In addition, the method of Patent Document 3 requires a heating step (45 to 70°C) to place the mixture under autophagy-inducing conditions. Furthermore, since autophagy is a physiological phenomenon that occurs in living cells, plant raw materials are limited to living cells.

[0022] In the method of Patent Document 4, mushroom self-solution decomposes GlcCer, which has a high utilization value, rather than selectively decomposing GIPC into free ceramide. In addition, mushroom self-solution does not require a separate preparation step. Moreover, this method is complex and requires pretreatment such as extracting GIPC and GlcCer from plant raw materials. In addition, mushrooms are not plants but fungi. The method using mushroom self-solution utilizes different enzymes and cannot be regarded as a manufacturing method for producing free ceramide using only plant-derived ingredients.

[0023] In order to finally obtain free ceramide from GIPC in the method of non-patent document 1, GIPC must first be decomposed by GIPC-PLD to obtain PC1P under in vitro conditions, and then PC1P must be dephosphorylated by phosphatase. The method of non-patent document 1 requires two different enzymes and is not very efficient.

[0024] The method of non-patent document 2 is suitable for extracting free ceramide and GlcCer, but it is not suitable for comprehensive analysis of phytoceramide-related substances because GIPC cannot be recovered from the organic phase. In addition, chloroform is highly toxic and has been avoided in recent years.

[0025] The method of non-patent document 3 currently requires a long extraction process and a large number of extraction containers. In addition, since a large amount of low-volatile aqueous solvent is used, it takes a long time to evaporate and remove the solvent. In addition, since two-phase partitioning is not performed, a large amount of impurities are present. In particular, the inclusion of polysaccharides will reduce the quantitative value of GIPC measured by LC-MS / MS.

[0026] The method of non-patent document 4 states that the content and composition of each plant sphingolipid molecule can be comprehensively grasped by combining high performance liquid chromatography (hereinafter referred to as "HPLC") and mass spectrometry using multiple reaction monitoring (hereinafter referred to as "MRM"), but does not disclose a method for simultaneously analyzing free ceramide, GIPC, and GlcCer. It is also pointed out that there are still many problems to be solved in order to simply and comprehensively analyze plant sphingolipid molecules with various structures. For example, the total number of molecular species present in a plant species reaches several hundred, and the molecular composition varies depending on the plant species and plant tissue part. However, the number of molecules that can be detected simultaneously in a typical MRM analysis is limited to about 50 to 100, so it is difficult to grasp the overall situation. [Prior art literature] [Patent Literature]

[0027] [Patent Document 1] Patent Publication No. 2012-41518 [Patent Document 2] WO2018 / 021476 [Patent Document 3] Patent Publication No. 2019-115318 [Patent Document 4] Patent Publication No. 2021-103950

Non-patent literature

[0028] [Non-patent document 1] Kita Takafumi et al.: Lipid Nutrition, Vol. 25(1), pp. 75-85, 2016 [Non-patent document 2] EG BLIGH, WJ DYER: Canadian Journal of Biochemistry and Physiology 1959 August; 37(8): 911-917 [Non-patent document 3] Jennifer E Markham et al: J Biol Chem. 2006Aug; 281(32): 22684-94 [Non-patent document 4] Hiroyuki Imai et al.: Biochemistry, Vol. 88 (1), pp. 94-104, 2016 [Summary of the invention] [Problems to be solved by the invention]

[0029] The present invention is proposed to solve the above-mentioned problems, and its purpose is to provide a method for selectively, simply and efficiently producing plant-derived free ceramide from GIPC contained in plant raw materials. Another purpose of the present invention is to provide a method for efficiently extracting free ceramide from plants. Another purpose of the present invention is to provide a method for simultaneously analyzing plant-derived free ceramide, GIPC and GlcCer in a very short time.

Methods to solve the problem

[0030] The inventors have conducted in-depth research on the problems of the prior art and found that: by applying the activity of phospholipase C with GIPC contained in plants other than callus as a substrate, plant-derived free ceramide can be produced simply and efficiently; by using a specific extraction solvent, plant-derived free ceramide can be efficiently extracted; and by combining HPLC with time-slot MRM (Schedule-MRM) (detection time difference setting), plant-derived free ceramide, GIPC and GlcCer can be simultaneously analyzed in a short time. Based on these findings, the inventors have made repeated improvements until the present invention was completed.

[0031] That is, the present invention provides the following inventions. [1] The step of activating endogenous enzymes, destroying the cell structure of the plant other than the callus tissue, and obtaining plant body fragments in which the endogenous phospholipase C of the plant cells is activated with glycosylinositol phosphoceramide as a substrate; In the decomposition step, the plant-derived glycosyl inositol phosphoceramide substance is reacted with the plant body fragments alone and / or with other glycosyl inositol phosphoceramide substances, thereby selectively decomposing the plant-derived glycosyl inositol phosphoceramide into free ceramide by using phospholipase C. [2] The method for producing plant-derived free ceramide according to [1], wherein the substance containing the glycosyl inositol phosphoceramide is any one or a combination of the following substances. (a) The plant containing glycosylinositol phosphoceramide. (b) Xenografts containing glycosylinositol phosphoceramide. (c) Glycosyl inositol phosphoceramide, which is an extract extracted from the plant. (d) Glycosyl inositol ceramide phosphate, which is an extract extracted from xenobiotic plants. [3] The method for producing plant-derived free ceramide according to [1], wherein the weight ratio of the plant fragments to the glycosyl inositol phosphoceramide-containing material in the decomposition step is 1:0 to 1:150. [4] A plant-derived free ceramide produced by the method described in any one of [1] to [3]. [5] A dietary product according to [4] containing plant-derived free ceramide. [6] A cosmetic according to [4] containing plant-derived free ceramide. [7] A pharmaceutical product according to [4], comprising plant-derived free ceramide. [8] A method for extracting free ceramide from a plant source obtained by the production method described in [1], comprising the following steps: adding a 1-butanol / methanol mixture to a sample for extraction treatment; and treating the sample under weak alkaline conditions to decompose ester lipids. 1-Butanol and a strong acid are added to the sample after the decomposition step, and the sample is separated into two layers under acidic conditions. The 1-butanol in the upper layer is recovered to obtain an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide. [9] A method for analyzing plant-derived free ceramide obtained by the production method described in [1], comprising the following steps: A 1-butanol / methanol mixture was added to the sample for extraction, and then the sample was treated under weak alkaline conditions to decompose the ester lipids; Adding 1-butanol and a strong acid to the sample after the decomposition step, separating the sample into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide; during the analysis process, introducing the extract into a liquid chromatography-tandem mass spectrometer, using a liquid chromatography with a reversed phase chromatography column to separate the free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide in the extract, and simultaneously using a mass spectrometer to perform mass spectrometry analysis on the three separated substances; In the analysis step, a time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the extract are quantitatively analyzed at a regular time. [1] A step of preparing the free ceramide produced by the production method as an analysis sample; The crushed plant bodies after heat treatment or freeze drying were used as control samples; A 1-butanol / methanol mixture was added to each sample for extraction treatment, and then the sample was treated under weak alkaline conditions to decompose ester lipids; adding 1-butanol and a strong acid to the sample after the decomposition step, separating the sample into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide; During the analysis, each extract is introduced into a liquid chromatography-tandem mass spectrometer, and free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide in the extract are separated by a liquid chromatography using a reverse phase chromatography column, and the three substances separated are subjected to mass spectrometry analysis by a mass spectrometer; In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the above-mentioned extract are simultaneously determined, and the amount of free ceramide generated is evaluated by comparing the amount of free ceramide with the amount of glycosylinositol phosphoceramide. [Effects of the invention]

[0032] According to the production method of the present invention, a method for selectively, simply and efficiently producing plant-derived free ceramide from GIPC contained in plant raw materials can be provided.

[0033] In the production method of the present invention, GIPC existing in large quantities in plants is directly decomposed by phospholipase C using endogenous GIPC in plants as substrates, without relying on heterologous enzymes or special chemical treatments. GIPC not only exists in large quantities in all plants, but also is considered to remain in large quantities in plant wastes due to its stability and poor solubility. According to the production method of the present invention, GIPC that has not been used before can be decomposed to directly generate free ceramide. Moreover, no special cultivated plant materials or complicated techniques are required, and free ceramide can be produced simply, quickly, in large quantities and at low cost.

[0034] The free ceramide obtained by the production method of the present invention can be used as an ingredient or raw material for cosmetics including functional foods, pharmaceuticals, and the like.

[0035] In addition, according to the extraction method of the present invention, a method for providing free ceramide, GIPC and GlcCer from plant sources can also be provided.

[0036] In addition, the analysis method of the present invention can provide a method for simultaneously analyzing plant-derived free ceramide, GIPC, and GlcCer in a short time.

[0037] Plants contain a wide variety of ceramide-related substances, and their molecular composition varies greatly depending on the plant species and tissues. The analytical method of the present invention can obtain comprehensive quantitative data covering all of these, and makes a great contribution to the establishment of a method for mass production of free ceramides using various plant species as raw materials.

Simple explanation of the diagram

[0038] [ Fig. 1 ] A diagram showing the overall MRM image at different time periods and the superposition of all chromatograms in the analysis method of the present invention. [ Fig. 2 ] A diagram showing the results of preliminary experiments to evaluate the extraction efficiency of phytoceramide-related substances. [Figure 3] Graph showing the results of GIPC degradation and free ceramide generation in Arabidopsis thaliana fragments after standing for 30 minutes [ Fig. 4 ] A graph showing the results of GIPC degradation and free ceramide generation after Arabidopsis thaliana fragments were left to stand for 10 to 60 minutes. [ Fig. 5 ] A diagram showing the results of GIPC degradation and free ceramide generation in various plants. [ Fig. 6 ] A diagram showing the results of decomposing GIPC in a xenobiotic plant (carrot) using Arabidopsis thaliana fragments and generating free ceramide. [ Fig. 7 ] A diagram showing the results of decomposition of soybean-derived GIPC by Arabidopsis thaliana fragments and generation of free ceramide. [Figure 8] A diagram showing the results of GIPC degradation and free ceramide generation in comparison between a solution of Flammulina velutipes and a crushed Arabidopsis thaliana, and a mixture with another heterologous plant (carrot). [ Fig. 9 ] A diagram showing the results of GIPC degradation and free ceramide generation when GIPC-PLD was added to Arabidopsis thaliana fragments. [Figure 10] Schematic diagram of the speculated mechanism of GIPC decomposition pathway. [Figure 11] shows a typical GIPC structure diagram. [Figure 12] Schematic diagram of the generation of free ceramide from GIPC by phospholipase (PLC). [ Fig. 13 ] A diagram showing the differences in reactants caused by different degrading enzymes (PLD or PLC). [ Fig. 14] is a graph showing the difference in the amount of free ceramide generated depending on the temperature conditions in the decomposition step. [Implementation Method]

[0039] The following is a description of the specific embodiments of the present invention. It should be noted that the configurations shown in the following embodiments are only examples, and the present invention is not limited to these configurations.

[0040] Method for producing free ceramide from plant sources The method for producing plant-derived free ceramide of the present invention comprises: an endogenous enzyme activation step, wherein the cell structure of the plant body except for callus tissue is destroyed to obtain a plant body fragment, wherein the plant body fragment is obtained by activating phospholipase C using glycosylinositol phosphoceramide endogenous to the plant body cells as a substrate; and A decomposition step, in which the crushed material reacts with a single and / or other substance containing plant-derived glycosyl inositol phosphoceramide at a temperature range of 20 to 30° C., thereby selectively decomposing the plant-derived glycosyl inositol phosphoceramide into free ceramide by phospholipase C.

[0041] (Activation step of endogenous enzyme) In the step of activating the endogenous enzyme, the cell structure of the plant body except the callus tissue is destroyed to obtain the activated plant body fragment phospholipase C endogenous to the plant cell with glycosylinositol phosphoceramide as substrate.

[0042] In the present invention, the plant body includes plant individuals and parts thereof, such as plant organs such as leaves, stems, roots, plant tissues, seeds and fruits. In addition, the plant body of the present invention also includes plant waste (plant residues), but does not include undifferentiated plant callus.

[0043] Examples of plant bodies include, but are not limited to, cruciferous plants (cabbage, radish, radish, broccoli, komatsuna, Arabidopsis, pakchoi, mizuna, etc.), Umbelliferae plants (carrot, celery, Japanese parsley, coriander, angelica, etc.), leguminous plants (soybean, mung bean sprouts, etc.), lily plants (onion, green onion, etc.), Lamiaceae plants (basil, red ginseng, blue shiso, perilla, etc.), cucurbitaceae plants (cucumber, watermelon, pumpkin, zucchini, etc.), various vegetable wastes, fruit peels and seeds, soybean meal, coffee grounds, brewery waste, etc.

[0044] The term "disruption" in this specification refers to a state in which the cell membrane is broken, the living cells are destroyed, and the enzymes contained in the plant body are activated. Specifically, it means that the tissue mass has been sufficiently micronized. For example, the state of disruption refers to a uniform suspension in which no masses of about several millimeters in size can be seen, and which will not be further micronized even if the disruption process is continued, i.e., a so-called homogenate state.

[0045] As a means of disruption, for example, any method can be used as long as it can generate a shear force sufficient to destroy living cells, such as a blender (Waring blender), a food processor, a homogenizer, and a mechanical method such as ultrasonic treatment. In one embodiment of the present invention, when a blender (Waring blender) is used, the material can be disrupted by processing for 1 to 10 minutes until the material reaches the disrupted state.

[0046] When crushing, the plant body can be in any state such as fresh, dried, or frozen, but it must contain water. For example, when using a dried product, add 5 to 20 times the weight of the dried product before crushing. In addition, since the cell membrane of frozen plants is damaged by freezing, if frozen plants are crushed before use, free ceramide can be effectively obtained.

[0047] The crushing time can be appropriately set according to the amount of the plant body and the crushing means.

[0048] As enzymes present in plants, phospholipase C, which is a lipid degrading enzyme, can be cited. Phospholipase C is an enzyme that is widely present in many plants and generally cleaves the glycerophosphate bond on the lipid side of glycerophospholipids. In this specification, phospholipase C that uses glycosylinositol phosphoceramide as a substrate to generate sugar chains and free ceramide may be referred to as "GIPC-phospholipase C" or "GIPC-PLC".

[0049] The main body of the protein that catalyzes the GIPC-PLC enzymatic reaction is still unclear. However, as shown in the examples described below, the amount of GIPC decreased and the amount of free ceramide increased was almost equal, while the amount of free ceramide did not increase when GIPC-PLD disclosed in Non-Patent Document 1 was added to the reaction system. Therefore, the increase in free ceramide observed in the present invention is directly generated by GIPC, which supports the existence of GIPC-PLC activity.

[0050] Among the plants, cruciferous plants, especially Arabidopsis thaliana, contain highly active GIPC-PLC. Therefore, the crushed cruciferous plants can be used as reaction substrates to efficiently produce free ceramide.

[0051] The effective temperature range of GIPC-PLC is usually 20-30° C. Therefore, the temperature condition in the activation step of the endogenous enzyme is preferably within the temperature range of 20-30° C. From the perspective of promoting the enzymatic reaction, the temperature range is preferably 20-28° C., and more preferably 20-25° C.

[0052] (Decomposition steps) In the decomposition step, the plant body fragments and / or other substances containing plant-derived glycosylinositol phosphoceramide are reacted at a temperature range of 20 to 30° C., thereby selectively decomposing the plant-derived glycosylinositol phosphoceramide into free ceramide by phospholipase C.

[0053] The reaction temperature of the decomposition step is preferably in the range of 20 to 30°C from the perspective of the active temperature range of GIPC-PLC, and is preferably in the range of 20 to 28°C from the perspective of promoting enzymatic reaction, and is more preferably 20 to 25°C.

[0054] To react the plant fragments alone means, for example, leaving the plant fragments still in a container to react with the GIPC-PLC and the GIPC in the plant.

[0055] The reaction of the plant fragments with the GIPC-containing substance derived from other plants refers to, for example, placing the plant fragments and the GIPC-containing substance derived from other plants in the same container and mixing them.

[0056] The mixing method is not particularly limited, but it is preferably mixed while stirring. Examples of such mixing devices include, but are not limited to, common stirrers (e.g., Waring stirrers, Nauta stirrers, Henschel stirrers, butterfly stirrers, etc.), food processors, homogenizers, blenders, etc.

[0057] In the present specification, "reacting the plant body fragment alone and / or with other plant-derived glycosylinositol phosphoceramide-containing substances" means reacting the plant body fragment alone, reacting the plant body fragment with other plant-derived glycosylinositol phosphoceramide-containing substances, and a combination of the above substances.

[0058] The reaction time of the decomposition step is, for example, 10 minutes to 48 hours, preferably 10 minutes to 24 hours, and can be appropriately set according to the amount of the plant body fragments and the amount of the plant-derived GIPC-containing substance.

[0059] The GIPC-containing substance is any one of the following or a combination thereof. (a) the plant containing GIPCs; (b) xenografts containing GIPCs; (c) the extract extracted from the plant, namely GIPC; (d) GIPCs extracted from xenobiotic plants.

[0060] The types of plants containing GIPC are shown in the activation step of the endogenous enzyme. The GIPC-containing plant used in the decomposition step may be a plant having weak GIPC decomposition enzyme activity, a plant having no GIPC decomposition enzyme activity, or a plant having inactivated GIPC decomposition enzyme activity.

[0061] The plant containing GIPC may be in any state of fresh, dried or frozen, but must contain water. When using a dried product, for example, 5 to 20 times the weight of the dried product is added with water.

[0062] The form of the plant containing GIPCs may be, for example, a whole block, a sheet, minced, sliced, coarsely ground, granulated, powdered, or a paste (homogenized). From the viewpoint of efficiently reacting with the plant fragment, it is preferred to pulverize the plant finely, and it is more preferred to fully pulverize the tissue block by pulverizing means until the cell membrane of the plant is ruptured and the living cells are destroyed during the activation of the endogenous enzyme, and it is more preferred to make the plant into a paste (homogenized) state.

[0063] In the decomposition step, the mixing weight ratio of the plant crushed material and the GIPC-containing material is 1:0 to 1:150. In the mixing weight ratio, 1:0 means that the GIPC contained in the plant crushed material is decomposed, that is, the plant crushed material is not a mixture but is used alone.

[0064] From the perspective of obtaining free ceramide in a short time and with high efficiency, the preferred weight ratio of the plant body fragments to the GIPC mixed component is in the range of 1:0 to 1:10, with 1:0 to 1:4 being the best.

[0065] The activation step of the endogenous enzyme and the decomposition step can be performed in sequence or simultaneously. For example, the cruciferous plant, especially Arabidopsis thaliana, contains highly active GIPC-PLC. The cruciferous plant and other GIPC substances (such as plants with low GIPC decomposition enzyme activity) are placed in the same container, mixed and crushed, and left to stand at a temperature range of 20 to 30°C to react, so that the activation step and the decomposition step of the endogenous enzyme are performed simultaneously.

[0066] The plant-derived free ceramide obtained by the production method is extracted by the following method.

[0067] Extraction method of free ceramide from plant sources The present invention relates to a method for extracting free ceramide from a plant source, which is a method for extracting free ceramide from a plant source obtained by the manufacturing method of the present invention, and comprises the following steps: Add 1-butanol / methanol mixture to the sample for extraction treatment, and treat the sample under weak alkaline conditions to decompose ester lipids; 1-Butanol and a strong acid are added to the sample after the decomposition step, and the sample is separated into two layers under acidic conditions. The 1-butanol in the upper layer is recovered to obtain an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide.

[0068] (Decomposition steps) In the decomposition step, the sample is extracted with a 1-butanol / methanol mixture and then treated under weak alkaline conditions to decompose the ester lipids.

[0069] The "sample" referred to herein refers to the plant-derived free ceramide obtained by the production method of the present invention, and refers to any sample including plant body fragments that have undergone an activation step and a decomposition step by endogenous enzymes. The sample may be used directly, or may be pretreated and / or prepared.

[0070] In the 1-butanol / methanol mixture, the volume ratio of 1-butanol to methanol is generally 1:6 to 6:1 (v:v) or any value within the range, preferably 1:4 to 4:1 (v:v) or any value within the range, more preferably 1:3 to 3:1 (v:v) or any value within the range, and even more preferably 2:1 (v:v).

[0071] The temperature for the extraction treatment by adding the 1-butanol / methanol mixed solution to the sample is usually 50° C. or higher, preferably 75° C. or higher. The reaction time is usually 5 minutes or higher, preferably 10 minutes or higher.

[0072] Alkaline agents under weak alkaline conditions include, for example, sodium hydroxide and potassium hydroxide. Alkaline agents can be used alone or in combination of two or more.

[0073] The volume ratio of the 1-butanol / methanol mixture to the alkaline agent is generally 1:6 to 6:1 (v:v) or any value within the range, preferably 1:4 to 4:1 (v:v) or any value within the range, more preferably 1:3 to 3:1 (v:v) or any value within the range, and even more preferably 1:0.3 to 1:0.6 (v:v) or any value within the range. Depending on the mixing ratio, the solution may separate into two layers. At this time, adding a small amount of methanol to mix the mixed solvent evenly can promote the decomposition of the ester lipids contained in the organic layer.

[0074] When treating under alkaline conditions, the lower limit of the reaction temperature is usually 20°C or more, preferably 30°C or more, and more preferably 45°C or more. The upper limit is usually 70°C or less, preferably 60°C or less, and more preferably 55°C or less. Therefore, the reaction temperature is usually 20-70°C, preferably 30-60°C, and more preferably 45-55°C. The reaction time is usually 5 minutes or more, preferably 10 minutes or more. Treatment under such weak alkaline conditions will decompose the ester lipids in the sample.

[0075] (Step of obtaining extract) In the step of obtaining the extract, 1-butanol and a strong acid are added to the sample that has undergone the decomposition step, the two layers are separated under acidic conditions, and the 1-butanol in the upper layer is recovered, thereby obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide.

[0076] The volume ratio of the sample after the decomposition step to 1-butanol and the strong acid is usually 1:1.5:1.5 to 1:3:5 (v:v:v) or any value within the range, preferably 1:2:3 (v:v:v).

[0077] Strong acids include inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, etc. Strong acids can be used alone or in combination of two or more.

[0078] Generally, a two-phase partition between a 1-butanol / methanol mixture and water is performed under acidic conditions, and free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide can be recovered in the 1-butanol phase, and impurities including interfering components (e.g., polysaccharides) can be removed to the aqueous phase. The extract recovered from the 1-butanol phase contains total lipids, including decomposition intermediates formed by the stepwise cleavage of the hydrophilic part of GIPC.

[0079] (Other steps) After the extract is evaporated to remove the solvent and other steps, the plant-derived free ceramide can be obtained.

[0080] The main fatty acid chain length of the plant-derived free ceramide obtained by the production method of the present invention varies somewhat depending on the plant species, but is centered around C24, similar to humans, and is mostly distributed between C22 and C26. In this respect, it is different from glucosylceramide, which is mainly composed of C16 to C20, and is also different from synthetic ceramide of a single molecule type.

[0081] The extraction method of the present invention can complete the steps from saponification to two-phase distribution in one container, thereby reducing the number of processes. In addition, since the amount of solvent used is less than that of the conventional technology, a small-capacity container can be used to extract multiple samples simultaneously. In addition, since the extract is obtained in a small amount of organic solvent by two-phase distribution, evaporation removal can be completed in a short time. The extraction method of the present invention has a better recovery rate for plant ceramide-related substances than the Markham method, and in particular, the removal of polysaccharides can improve the detection sensitivity of GIPC by LC-MS / MS. A comparison of the prior art and the extraction method of the present invention is summarized in Table 1.

[0082]

Table 1

[0083] Analytical Methods for Free Ceramides from Plant Sources1 The method for analyzing free ceramide derived from plants in the present invention is a method for analyzing free ceramide obtained by the production method, which comprises the following steps: A 1-butanol / methanol mixture was added to the sample for extraction, and the sample was treated under weak alkaline conditions to decompose ester lipids; Adding 1-butanol and a strong acid to the sample after the decomposition step, separating the sample into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide; introducing the extract into a liquid chromatography-tandem mass spectrometer, and using a liquid chromatography with a reverse phase chromatography column to separate the free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide in the extract, and using a mass spectrometer to perform mass analysis on the three separated substances; In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the extract are quantitatively analyzed at a timely manner.

[0084] (Decomposition steps) (Step of obtaining extract) The steps have been described in the detailed description of the extraction method of the present invention. In one embodiment of the present invention, a 1-butanol / methanol (volume ratio of 2:1) mixture is added to the sample, treated at 75°C or above for more than 10 minutes, then 0.3 to 1 times the amount of a strong base (such as 1N potassium hydroxide, etc.) is added, and treated at 45 to 70°C for more than 10 minutes to decompose the ester lipids, and then 1.5 to 3 times the amount (volume) of 1-butanol and 1.5 to 5 times the amount (volume) of a strong acid (such as 0.4N hydrochloric acid, etc.) are added to acidify the sample and separate the two layers, and the upper layer of 1-butanol is recovered to obtain an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide.

[0085] (Analysis steps) In the analysis step, the extract is introduced into a liquid chromatography-tandem mass spectrometer, free ceramide, GIPC and glucosylceramide in the extract are separated by liquid chromatography using a reverse phase chromatography column, and then the separated three substances are subjected to mass spectrometry analysis using a mass spectrometer. In the analysis step, time-divided MRM measurement (multiple reaction monitoring) is performed to selectively detect only the time period during which three substances are eluted from the liquid chromatograph, and the three substances contained in the extract are quantitatively analyzed at regular intervals.

[0086] The liquid chromatography-tandem mass spectrometer referred to here is, for example, a liquid chromatography-triple quadrupole mass spectrometer or a liquid chromatography quadrupole / time of flight (Q-TOF) mass spectrometer.

[0087] Typically, in such a liquid chromatography-tandem mass spectrometer, the analytical parameters can be arbitrarily determined to maximize the detection sensitivity, i.e., to obtain the best conditions for each component in the extract after liquid phase separation using a reversed phase chromatography column.

[0088] The structural types of phytoceramides and related metabolites are listed in Table 2 .

[0089]

Table 2

[0090] In the table, "Glc" in the hydrophilic part represents glucose, "OH" represents hydroxyl, "Hex" represents hexose, "HexN" represents hexosamine, and "HexNAc" represents N-acetylhexosamine. In addition, in the notation such as "t18:0" of the skeleton of ceramide, "t" represents that it has 3 hydroxyl groups, "18" represents the number of carbon atoms, and the last number "0" represents the number of double bonds. In addition, "d" refers to dihydroxy base.

[0091] Perform time-segmented MRM measurements to selectively detect only the time periods (e.g., dissolution time ± 1 minute) during which the three substances are dissolved from the liquid chromatograph. For example, the dissolution time of GIPC is 6 to 19 minutes, the dissolution time of GlcCer is 15 to 25 minutes, and the dissolution time of Cer is 20 to 30 minutes. This allows the number of overlapping molecules to be limited to less than 100, and 1,000 or more molecular species can be measured simultaneously. This large-scale simultaneous analysis of plant ceramides is unprecedented.

[0092] According to the analysis method of the present invention, the amount of the three substances present can be determined based on the obtained signal intensity, and the total amount of sphingolipids and the composition of each molecular species can be obtained. Figure 1 shows the overall image of the MRM in the time period and the superposition of all chromatograms in the analysis method of the present invention.

[0093] Analysis Method for Free Ceramides from Plant Sources2 The method for analyzing plant-derived free ceramide of the present invention comprises the following steps: a step of preparing free ceramide produced by the production method of the present invention as an analysis sample; The plant body is subjected to a heating treatment or a freeze-drying treatment and then crushed to prepare the plant body as a control sample; The step of adding a 1-butanol / methanol mixture to each sample for extraction treatment, and then treating the sample under weak alkaline conditions to decompose ester lipids; The step of adding 1-butanol and a strong acid to the sample after the decomposition step, separating the sample into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide; In the analysis step, each of the extracts is introduced into a liquid chromatography-tandem mass spectrometer, and free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide in the extract are separated by a liquid chromatography using a reverse phase chromatography column, and the separated three substances are subjected to mass spectrometry analysis by a mass spectrometer; In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the above-mentioned extract are simultaneously determined, and the amount of free ceramide is compared with the amount of glycosyl inositol phosphoceramide to evaluate the amount of free ceramide generated.

[0094] (Steps for preparing analytical samples) The plant-derived free ceramide produced by the production method of the present invention is prepared as a sample to be analyzed. The details of the production method of the present invention are as described above. Before measurement, it is recommended to pre-inactivate the enzyme in the plant body in the sample to be analyzed by heat treatment as described below.

[0095] (Steps for preparing control sample) In the step of preparing the control sample, the plant body is subjected to heat treatment or freeze-drying treatment and then crushed to prepare the control sample.

[0096] The purpose of heat treatment is to inactivate the enzymes in the plant. The treatment temperature is, for example, 80 to 100°C, preferably 95 to 100°C. The treatment time is, for example, 1 to 30 minutes, preferably 5 to 20 minutes. The heating method can be, for example, water bath or direct flame heating, but is not limited thereto.

[0097] The freeze drying step is to prevent the inherent enzymes in the plant from reacting by freeze drying. The freeze drying method is not particularly limited, and for example, a method of placing the plant in a container and freeze drying it using a freeze dryer can be appropriately adopted. Specifically, for example, the plant body can be frozen at a temperature below -40°C, vacuum treated, and dried at a temperature range of -20°C to 30°C.

[0098] The crushing means has been described in detail in the part of the manufacturing method of the present invention.

[0099] (Decomposition steps) (Step of obtaining extract) The steps have been described in detail in the part of the extraction method of the present invention.

[0100] (Analysis steps) The analysis step comprises: in the analysis step, each of the extracts is introduced into a liquid chromatography-tandem mass spectrometer, and the free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide in the extract are separated by a liquid chromatography with a reverse phase chromatography column, and then the mass spectrometer is used to perform mass spectrometry analysis on the three separated substances; In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the above-mentioned extract are simultaneously determined, and the amount of free ceramide generated is evaluated by comparing the amount of free ceramide with the amount of glycosylinositol phosphoceramide.

[0101] According to the analytical method of the present invention, the amount of the three substances present can be determined based on the obtained signal intensity, and the total amount of sphingolipids and the composition of each molecular species can be obtained. In addition, according to the analytical method of the present invention, the results of comparing the sample to be analyzed with the control sample can reveal that GIPC-PLC present in the raw material plant body decomposes GIPC and directly produces free ceramide. According to the analytical method of the present invention, a plant ceramide library is constructed, which covers all known and unknown molecular species that may exist in various plant raw materials, and the detection is focused on the elution time of a single molecule, so that more than 1,000 molecular species can be analyzed at a time. This makes it possible to quantitatively grasp the overall picture of ceramide metabolism steps and quickly and accurately evaluate the efficiency of free ceramide production. [Example]

[0102] The features of the present invention are described in more detail below through examples, but the scope of the present invention is not limited to these examples.

[0103] <Production of plant-derived free ceramide> 1. Decomposition of GIPC and generation of free ceramide in Arabidopsis thaliana fragments Example 1·Comparative Example 1 1.0 g of fresh Arabidopsis rosette leaves were crushed for about 10 minutes using a blender (trade name: Waring blender, model HGBSS, manufactured by WARING) until the tissue blocks were fully crushed into a uniform suspension, i.e., a so-called homogenate state. The resulting crushed material was allowed to stand at 25°C for 30 minutes and then reacted, and the glycosyl inositol phosphoceramide derived from Arabidopsis was selectively separated into free ceramide by endogenous phospholipase C, thereby generating free ceramide of Example 1. 1.0 g of fresh Arabidopsis rosette leaves were heat-treated at 95°C for 10 minutes to inactivate the endogenous enzyme, and then crushed using a blender under the same conditions as in Example 1. The resulting crushed material was used as Control Example 1.

[0104] <Preliminary Experiment to Evaluate the Extraction Efficiency of Phytoceramide-Related Substances> (A) Arabidopsis leaves, (B) Rice leaves were freeze-dried and crushed into powder. 30 mg of each sample was extracted using the extraction method of the present invention (method using a 1-butanol / methanol mixture), the method using chloroform (Bligh & Dyer method), and the method using isopropanol (Markham method) to obtain extracts, and then LC-MS / MS was used to quantify GlcCer, Cer, and GIPC. The recovery rate (%) was evaluated by the relative intensity when the detection intensity of the extraction method of the present invention was 100%. The Bligh & Dyer method was operated based on the method disclosed in the literature (EG BLIGH, WJ DYER: Canadian Journal of Biochemistry and Physiology 1959Aug; 37(8): 911-917). The Markham method was operated based on the method disclosed in the literature (Jennifer E Markham et al: J Biol Chem. 2006Aug; 281(32): 22684-94). The results showed that the recovery rates of GlcCer and Cer were comparable between the two methods, but the recovery rate of GIPC varied greatly depending on the method, with the extraction method of the present invention showing the best value. The results are shown in FIG2 .

[0105] <Extraction of free ceramide from plants> A 1-butanol / methanol (volume ratio 2:1) mixture was added to Example 1 and Control Example 1, and the mixture was heated at 80°C for 10 minutes. After cooling, 0.6 times (volume) of 1N potassium hydroxide was added, and the mixture was treated at 50°C for 20 minutes to decompose the ester lipids. Then, 2 times (volume) of 1-butanol and 3 times (volume) of 0.4N hydrochloric acid were added to each sample to acidify it and separate it into two layers. The upper butanol layer was collected, and the solvent was removed under reduced pressure to obtain an extract containing free ceramide, GIPC, and glucosylceramide.

[0106] <Analysis of Free Ceramide from Plants> The extract was detected and quantified using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring (MRM) mode under the following conditions. Equipment: Shimadzu LC-MS8030, ESI, MRM mode (ceramide: [M+H] + >[LCB+H] +, glucosylceramide: [M+H]+>[LCB+H]+, GIPC: [M+H]+>[Cer+H]+), nebulizer flow rate 1.5L / min, heating block temperature: 400℃, solution A: tetrahydrofuran / methanol / 5mM ammonium formate (150:100:250)+0.1% formic acid, solution B: tetrahydrofuran / methanol / 5mM ammonium formate (350:100:50)+0.1% formic acid, gradient conditions: 0 min, A / B=90:10>35 min, A / B=0:100, chromatographic column: Shimadzu GLC Sceptor C18 (3μm, 2.1×75mm), flow rate: 0.2ml / min

[0107] Under the above analytical conditions, the three substances were quantitatively analyzed simultaneously. In addition, the amount of free ceramide generated (nmol / g) was evaluated by comparing the measured values ​​of free ceramide and GIPC in the example and the control example (average value of 3 repeated measurements). As shown in Table 3 and Figure 3.

[0108]

Table 3

[0109] Comparing Example 1 with Control Example 1, it can be seen that most of the GIPC (about 80%) in Example 1 was decomposed, and free ceramide was selectively generated (Table 3, Figure 3). The results of Control Example 1 show that when the plant tissue is preheated to inactivate the endogenous enzyme, the decomposition of GIPC and the generation of free ceramide will not occur. In the prior art (Patent Document 3), it is speculated that when the differentiated plant is subjected to a heat treatment (45°C) after a crushing treatment, glucosylceramide will be decomposed by autophagy, but in Example 1, which was reacted under non-heating conditions (25°C), the amount of glucosylceramide was almost unchanged (see also Example 22 described later). It is speculated that the reason is that the homogenization caused by the crushing leads to the death of living cells.

[0110] Example 2·Comparative Example 2 1.0 g of fresh Arabidopsis rosette leaves were crushed with a blender under the same conditions as in Example 1. The resulting crushed material was allowed to stand at 25°C for 10, 20, 30 and 60 minutes for reaction, and the glycosyl inositol phosphoceramide from Arabidopsis was selectively decomposed into free ceramide by endogenous phospholipase C to generate free ceramide of Example 2. 1.0 g of fresh Arabidopsis rosette leaves were heated at 95°C for 10 minutes to inactivate the endogenous enzyme, and then crushed with a blender in the same manner, and the resulting crushed material was used as Control Example 2.

[0111] For Example 2 and Control Example 2, the same method as that of Example 1 and Control Example 1 was used to quantitatively analyze free ceramide, and the amount of free ceramide and glycosyl inositol phosphoceramide was compared to evaluate the amount of free ceramide generated (nmol / g). The results are shown in Table 4 and Figure 4.

[0112]

Table 4

[0113] Comparison between Example 2 and Control Example 2 shows that in Example 2, rapid decomposition of GIPC occurs about 10 minutes after crushing, and the reaction proceeds slowly thereafter (Table 4, FIG. 4 ).

[0114] 2. Decomposition of GIPC and generation of free ceramide in various plants Examples 3 to 12 Comparative Examples 3 to 12 Free ceramide from each plant body was prepared in the same manner as in Example 1, except that 1.0 g of each fresh plant body shown in Table 5 was used. Comparative Examples 3 to 12 were obtained in the same manner as in Comparative Example 1. The standing time (reaction time) of the crushed product in each example was 1 hour or 24 hours.

[0115]

Table 5

[0116] For Examples 3 to 12 and Comparative Examples 3 to 12, simultaneous quantitative analysis of free ceramide was performed in the same manner as in Example 1 and Comparative Example 1, and the lipid composition (mol %) was evaluated. The results are shown in FIG5 .

[0117] As shown in FIG5 , GIPC was degraded by GIPC-PLC present in various plants, and an increase in free ceramide was observed. Arabidopsis thaliana showed the highest GIPC degrading activity after 1 hour. As time went by, the decomposition of GIPC continued, and the content of free ceramide increased in all plant species. On the other hand, since the amount of glucosylceramide hardly changed over time, it was shown that the reaction observed in Examples 3 to 12 was a GIPC-specific degrading enzyme reaction that was completely different from autophagy shown in the prior art (Patent Document 3).

[0118] In addition, the distribution of fatty acid chain length (mol%) in the free ceramide composition after GIPC decomposition for 24 hours in Examples 3 to 5 was evaluated. The results are shown in Table 6.

[0119]

Table 6

[0120] As shown in Table 6, although the plant-derived free ceramide obtained by the production method of the present invention has some differences depending on the plant species, it is believed that the main component is C24, which is the same as that of the human type.

[0121] 3. Decomposition of GIPCs in Heterogeneous Plants by Arabidopsis Disrupts Example 13 Comparative Example 13 0.5 g of fresh Arabidopsis rosette leaves were mixed with 1.0 g of carrots that had been heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then the mixture was crushed using a blender (product name: Waring blender, model HGBSS, manufactured by WARING) until the tissue blocks were fully crushed into a uniform suspension or homogenous state, in other words, crushed for 3 minutes until a homogenous state. The resulting crushed material was allowed to stand at 25°C for 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours to react and generate free ceramide. 1.0 g of fresh Arabidopsis rosette leaves were heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then crushed in a blender, and the resulting crushed material was used as Control Example 13.

[0122] For Example 13 and Control Example 13, simultaneous quantitative analysis of free ceramide was performed in the same manner as in Example 1 and Control Example 1, and the amount of free ceramide generated (nmol / g) was evaluated. The composition of each plant sample is shown in Table 7, and the changes in free ceramide and GIPC over time are shown in Table 8 and Figure 6. The amounts of free ceramide and GIPC at the baseline in Figure 6 show the values ​​of Arabidopsis thaliana in Control Example 13.

[0123]

Table 7

[0124]

Table 8

[0125] In Example 13, as the standing time (reaction time) increased, GIPC in carrots decomposed and the amount converted into free ceramide also increased (Figure 6). The conversion efficiency of GIPC into free ceramide after 3, 6 and 24 hours was about 75%, about 90% and about 95%, respectively.

[0126] 4. Decomposition of soybean-derived GIPCs by Arabidopsis thaliana fragments Example 14 Comparative Example 14 10 nmol of GIPC extracted from soybean was dried under nitrogen flow, and a homogenized state of 0.1 g of Arabidopsis rosette leaves was added, and the mixture was suspended by ultrasonic treatment at 25°C for 30 seconds using an ultrasonic treatment device (product name US-2KS, manufactured by SND Co., Ltd.). The resulting suspension was allowed to stand at 25°C for 30 minutes, 1 hour, 3 hours, 6 hours, and 24 hours to react and generate free ceramide. 0.1 g of fresh Arabidopsis rosette leaves were heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then crushed with a blender. The resulting crushed material was used as Control Example 14.

[0127] For Example 14 and Control Example 14, simultaneous quantitative analysis of free ceramide was performed in the same manner as in Example 1 and Control Example 1, and the amount of free ceramide generated (nmol) was evaluated. The composition of each sample is shown in Table 9, and the changes in free ceramide and GIPC over time are shown in Table 10 and Figure 7. The amounts of free ceramide and GIPC at the baseline in Figure 7 are the values ​​of Arabidopsis thaliana of Control Example 14.

[0128]

Table 9

[0129]

Table 10

[0130] In Example 14, as the standing time increased, the soybean-derived GIPC also decomposed, and the amount of GIPC converted into free ceramide also increased ( FIG. 7 ). This indicates that free ceramide can also be produced from GIPC extracted from plant tissues.

[0131] 5. Production of free ceramide by mixing a sample containing GIPC-PLC with different types of plants Examples 15 to 20 Comparative Example 15 The mixture was mixed with 0.01 g (Example 15), 0.05 g (Example 16), 0.1 g (Example 17), 0.25 g (Example 18), 0.5 g (Example 19), and 1.0 g (Example 20) of fresh Arabidopsis rosette leaves after heating at 95°C for 10 minutes to inactivate endogenous enzymes, and the mixture was crushed in a blender until uniform. The resulting crushed material was placed at 25°C for 3 hours to react and generate free ceramide. 1.0 g of carrot (edible part) was heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then crushed with a blender. The resulting crushed material was used as Control Example 15.

[0132] Comparative Examples 1 and 2 After freezing overnight, Flammulina velutipes was placed in a thermostatic bath at 25°C for self-digestion. EDTA was added after 24 hours, and then the mixture was crushed in a food processor. The crushed mixture was subjected to solid-liquid separation, and the liquid portion was prepared as Flammulina velutipes self-solution. 1.0 g of carrots heated at 95°C for 10 minutes to inactivate endogenous enzymes were mixed with 0.25 g (Comparative Example 1) or 1.0 g (Comparative Example 2) of Flammulina velutipes self-solution, and then crushed in a blender. The resulting crushed mixture was left to stand at 25°C for 3 hours to produce free ceramide.

[0133] For Examples 15 to 20, Control Example 15, and Comparative Examples 1 to 2, free ceramide was analyzed using the same method as in Example 1 and Control Example 1, and the ratio (%) of GIPC to free ceramide was determined. The results are shown in FIG8 .

[0134] As shown in FIG8 , the conversion efficiency of GIPC to free ceramide is related to the amount of plant body fragments (enzyme-containing sample) added. In order to generate free ceramide in a short time, the weight ratio of plant body fragments to GIPC-containing substances is preferably 1:1 to 1:4, but even if the mixing ratio of plant body fragments is low, the conversion efficiency can be improved by extending the reaction time. In Comparative Example 2, the GIPC decomposition rate of the Flammulina velutipes self-solution equivalent to 1.0g of Flammulina velutipes was only about 7%, while in Example 16, the GIPC decomposition rate when 0.05g of Arabidopsis thaliana was used was about 15%. If the conversion rates of the samples containing the decomposing enzymes at the same weight equivalent are compared, a difference of about 6.33 times is observed between Example 18 and Comparative Example 1 with an equivalent of 0.25g. A difference of about 5.5 times is observed between Example 20 and Comparative Example 2 with an equivalent of 1.0g. From the above results, it can be seen that the Arabidopsis fragments of Examples 15 to 20 have higher GIPC decomposition activity than the Flammulina velutipes self-solutions in Comparative Examples 1 to 2. In addition, the Arabidopsis crushed materials of Examples 15 to 20 can be simply prepared by crushing under non-heating conditions (25°C), while the Flammulina velutipes autolyzed solutions of Comparative Examples 1 to 2 require the steps of freezing, thawing, and autolyzing the plants, as well as the extraction step. Considering this point, Examples 15 to 20 can produce plant-derived free ceramide more simply and efficiently than Comparative Examples 1 to 2, and their advantages are demonstrated.

[0135] 6. Verify the difference with the known GIPC degradation pathway (GIPC-PLD pathway) Example 21·Comparative Example 21 1.0 g of fresh Arabidopsis rosette leaves were crushed with a blender until uniform. The crushed material was allowed to stand at 25°C for 60 minutes to react and generate free ceramide. 1.0 g of fresh Arabidopsis rosette leaves were heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then crushed with a blender. The crushed material was used as control example 21.

[0136] Comparative Example 3 The GIPC-PLD gene from Arabidopsis thaliana was introduced into the E. coli expression vector pBAD-DEST49 (ThermoFisher Scientific) to obtain the recombinant GIPC-PLD enzyme from E. coli. 1.0 g of fresh Arabidopsis thaliana rosette leaves were mixed with 10 μg of the GIPC-PLD enzyme from E. coli and crushed with a blender until uniform. The resulting crushed material was allowed to stand at 25°C for 60 minutes to react and generate free ceramide.

[0137] For Example 21, Control Example 21, and Comparative Example 3, free ceramide analysis was performed in the same manner as in Example 1 and Control Example 1, and the amount of free ceramide generated (mol%) was evaluated. The results are shown in FIG9 , and the mechanism of the inferred GIPC decomposition pathway is shown in FIG10 .

[0138] As shown in FIG9 , when GIPC-PLD was added to Arabidopsis thaliana fragments, GIPC decreased and phytoceramide-1-phosphate (PC1P) increased, but free ceramide did not increase (Comparative Example 3). This result suggests that free ceramide is produced by plant fragments through the PLC pathway, and is not related to the PLD pathway mediated by PC1P ( FIG10 ). A previous report (Non-Patent Document 1) showed that GIPC-PLD that specifically hydrolyzes GIPC to PC1P was found in cabbage, and that PC1P is likely to be converted to ceramide by alkaline phosphatase in the small intestine. However, as shown in FIG10 , if the PLD pathway that produces free ceramide is an intrinsic pathway for decomposing GIPC as a decomposition pathway of GIPC, then the amount of free ceramide should also increase by adding PLD from the outside. However, the amount of free ceramide did not increase, but rather decreased slightly. This result shows that the PLC pathway is the main decomposition pathway of GIPC, and the contribution of the PLD pathway is very small and negligible.

[0139] GIPC has a structure of ceramide-phospho-inositol-oligosaccharide (Figure 11), and free ceramide is generated by breaking the phosphate bond between ceramide and phosphoric acid (Figure 12). The enzyme that cuts this part is called GIPC-PLC. From the above analysis results of free ceramide, it can be seen that the decrease in GIPC is roughly equal to the increase in free ceramide, and as shown in Comparative Example 3, free ceramide does not increase after adding GIPC-PLD to the reaction system, which indicates that the increased free ceramide is directly generated by GIPC, indicating the presence of PLC activity (Figure 13).

[0140] 7. The amount of free ceramide generated varies depending on the temperature conditions in the decomposition step. Example 22·Comparative Example 22 1.0 g of fresh Arabidopsis rosette leaves were crushed with a blender until uniform. The resulting crushed material was allowed to stand at 25°C for 3 hours to generate free ceramide. 1.0 g of fresh Arabidopsis rosette leaves were heated at 95°C for 10 minutes to inactivate endogenous enzymes, and then crushed with a blender. The resulting crushed material was used as Control Example 22.

[0141] Comparative Example 4 1.0 g of fresh Arabidopsis thaliana leaves were crushed with a blender until uniform, and the resulting crushed material was left to stand at 45°C for 3 hours to generate free ceramide.

[0142] For Example 22, Control Example 22, and Comparative Example 4, free ceramide was analyzed in the same manner as in Example 1 and Control Example 1, and the amount of free ceramide generated (nmol / g) was evaluated. The results are shown in Table 11 and Figure 14

[0143]

Table 11

[0144] As shown in FIG. 14 , the comparison between 25°C (Example 22) and 45°C (Comparative Example 4) shows that GIPC is actively decomposed under the high temperature condition of 45°C, but the amount of free ceramide generated does not increase compared with 25°C, but tends to decrease, while the decomposition rate of GIPC and GlcCer increases. It is speculated that various decomposition reactions including the decomposition reaction of free ceramide occur under the high temperature condition of 45°C. On the other hand, at 25°C, only the decomposition reaction of GIPC occurs, and the decomposition reaction of free ceramide and GlcCer does not occur. From the above, it can be seen that the appropriate temperature range for producing free ceramide using GIPC-PLC is 20 to 30°C.

[0145] The invention described in the appended claims of the original application on which the priority claim is based is set forth below. The claim numbers listed in the appendix are the same as the claim numbers originally attached to the original application. 1. A method for producing free ceramide from plant sources, comprising the following steps: The endogenous enzyme activation step comprises crushing the plant body to obtain a crushed plant body in which the endogenous phospholipase C of the plant is activated with glycosyl inositol phosphoceramide as a substrate; In the decomposition step, the plant body fragments are reacted with a substance containing plant-derived glycosylinositol phosphoceramide to decompose the glycosylinositol phosphoceramide into free ceramide. 2. The substance containing glycosyl inositol phosphoceramide is any one or a combination of the following. The method for producing free ceramide from plant source according to claim 1 is characterized in that: (a) Glycosyl inositol phosphoceramide contained in the plant. (b) Glycosyl inositol phosphoceramide contained in heterologous plants. (c) Glycosyl inositol phosphoceramide, which is an extract extracted from the plant. (d) Glycosyl inositol ceramide phosphate, which is an extract extracted from the xenobiotic plant. 3. The method for producing plant-derived free ceramide according to claim 1, wherein the decomposition step comprises mixing the plant body fragments with the glycosyl inositol phosphoceramide-containing material in a weight ratio of 1:0 to 1:150. 4. The method for producing plant-derived free ceramide according to claim 1, wherein the reaction temperature of the decomposition step is in the range of 20 to 30°C. 5. The free ceramide produced by the production method according to claim 1 is used as a sample to be analyzed; The crushed plant bodies after heating or freeze-drying were used as control samples; adding a solvent to each of the samples to obtain an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide; The extracts are introduced into a liquid chromatography-tandem mass spectrometer, and free ceramide and glycosyl inositol in the extracts are analyzed by a liquid chromatography with a reverse phase column, and phosphoceramide and glucosylceramide are separated, and at the same time, mass analysis of the three substances separated is performed by a mass spectrometer; The extraction step comprises adding a 1-butanol / methanol (volume ratio 2:1) mixed solution to each sample, treating at 75°C or above for more than 10 minutes, then adding 0.3 to 1 times (volume) of 1N potassium hydroxide, treating at 45 to 70°C for more than 10 minutes to decompose the ester lipids, adding 1.5 to 3 times (volume) of 1-butanol and 1.5 to 5 times (volume) of 0.4N hydrochloric acid to each sample after the decomposition step, acidifying and separating into two layers, and recovering the 1-butanol in the upper layer; The analytical method for free ceramide derived from plants comprises performing time-divided MRM measurement in the analytical step so as to selectively detect only the time when the three substances are eluted from the liquid chromatograph, thereby simultaneously measuring the three substances contained in each sample, and evaluating the generated free ceramide by comparing the amount of free ceramide with the amount of glycosyl inositol phosphoceramide. [Possibility of Industrial Application]

[0146] The production method of the present invention has prospects in the following two aspects. First, it is to improve the quality of existing free ceramide raw materials. For example, free ceramide can be generated from fresh agricultural products, processed products, residues, etc. of plant raw materials that are already used in food and cosmetics. In addition, the yield of free ceramide is equal to or greater than the yield of existing plant-derived glucosylceramide. The production method of the present invention can improve the functionality and added value of plant-derived free ceramide, contributing to quality improvement. Second, it is the resource utilization of plant waste. This technology has a wide range of uses and can utilize plant waste from farmers, agricultural product markets, and food industries as raw materials for ceramide. At present, agricultural waste is passively handled by cultivating soil, etc., but by actively utilizing it, we can contribute to sustainable development goals. This will also help farmers return profits and reduce raw material costs. The production method of the present invention is also useful for the production method of plant-derived free ceramide used as an ingredient or raw material for functional foods, cosmetics, pharmaceuticals, etc. The extraction method of the present invention can recover nearly 100% of all molecular species of ceramide-related substances, so it is very useful. In addition, the analysis method of the present invention is very useful because it can simultaneously analyze free ceramides, GIPC, and more than 1,000 molecular species of glucosylceramide derived from plants.

Claims

1. A method for producing free ceramide from plant sources, comprising: an endogenous enzyme activation step, destroying the cell structure of the plant body except the callus tissue to obtain a plant body fragment, wherein the plant body fragment activates phospholipase C that uses glycosylinositol phosphoceramide endogenous to the plant body cells as a substrate; and The decomposition step is to react the plant body fragments alone and / or with other plant-derived substances containing glycosyl inositol phosphoceramide at a temperature range of 20 to 30° C., thereby selectively decomposing the plant-derived glycosyl inositol phosphoceramide into free ceramide by phospholipase C.

2. The method for producing free ceramide from plant sources according to claim 1, characterized in that , the substance containing glycosyl inositol phosphoceramide is any one of the following or a combination thereof: (a) the plant containing glycosyl inositol phosphoceramide; (b) xenografts containing glycosylinositol phosphoceramide; (c) glycosyl inositol phosphoceramide as an extract extracted from the plant; (d) Glycosyl inositol phosphoceramide Glycosyl inositol phosphoceramide as an extract extracted from a xenobiotic plant.

3. The method for producing free ceramide from plant sources according to claim 1, characterized in that , the mixing weight ratio of the plant body fragments and the glycosyl inositol phosphoceramide-containing substance in the decomposition step is 1:0 to 1:

150.

4. A plant-derived free ceramide obtained by the production method according to any one of claims 1 to 3.

5. A food product comprising the plant-derived free ceramide according to claim 4.

6. A cosmetic comprising the plant-derived free ceramide according to claim 4.

7. A pharmaceutical product comprising the plant-derived free ceramide according to claim 4.

8. A method for extracting free ceramide, obtained by the manufacturing method according to claim 1, the extraction method comprising: The step of adding a 1-butanol / methanol mixture to the sample for extraction treatment, and then treating it under weak alkaline conditions to decompose ester lipids; A step of adding 1-butanol and a strong acid to the sample after the decomposition step, separating it into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide.

9. A method for analyzing free ceramide from plant sources, wherein the free ceramide from plant sources is obtained by the production method according to claim 1, the production method comprising: A step of adding a 1-butanol / methanol mixture to the sample for extraction treatment, and then treating the sample under weak alkaline conditions to decompose ester lipids; A step of adding 1-butanol and a strong acid to the sample after the decomposition step, separating the sample into two layers under acidic conditions, and recovering the 1-butanol in the upper layer to obtain an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosylceramide; In the analysis step, the extract is introduced into a liquid chromatography-tandem mass spectrometer, free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide in the extract are separated by a liquid chromatography using a reverse phase chromatography column, and the separated three substances are subjected to mass spectrometry analysis by a mass spectrometer. In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively dissolved from the liquid chromatograph, and the three substances contained in the extract are quantitatively analyzed at a timely manner.

10. A method for analyzing free ceramide from plant sources, the method comprising: A step of preparing free ceramide produced by the production method according to claim 1 as an analysis target sample; The plant body is subjected to a heating treatment or a freeze-drying treatment and then crushed to prepare a control sample; A step of adding a 1-butanol / methanol mixture to each sample for extraction treatment, and then treating the sample under weak alkaline conditions to decompose ester lipids; The steps of adding 1-butanol and a strong acid to the sample after the decomposition step, separating into two layers under acidic conditions, recovering the 1-butanol in the upper layer, and obtaining an extract containing free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide; in the analysis step, introducing each extract into a liquid chromatography-tandem mass spectrometer, separating free ceramide, glycosyl inositol phosphoceramide, and glucosyl ceramide in the extract by using a liquid chromatography with a reverse phase chromatography column, and performing mass spectrometry analysis on the three substances separated by using a mass spectrometer; In the analysis step, time-divided MRM measurement is performed to selectively detect only the time periods in which the three substances are respectively eluted from the liquid chromatograph, and the three substances contained in the above-mentioned extract are simultaneously determined, and the amount of free ceramide generated is evaluated by comparing the amount of free ceramide with the amount of glycosylinositol phosphoceramide.

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