CN113632978A

CN113632978A - PPAR action substance and method for producing the same

Info

Publication number: CN113632978A
Application number: CN202110786799.1A
Authority: CN
Inventors: 宮本哲也; 供田洋; 安原義; 杜艳萍
Original assignee: Gong Benzheye
Current assignee: Gong Benzheye
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2021-11-12

Abstract

The present invention provides a PPAR active substance and a method for producing the same. The manufacturing method comprises the following steps: providing a powdered dried plant or food product comprising a lipoxygenase enzyme, said lipoxygenase enzyme comprising at least one of cyclooxygenase, lipoxygenase and cytochrome P450; placing the dried powder in water or buffer solution, activating lipid metabolism enzyme under the action of polyunsaturated fatty acid and/or polyunsaturated fatty acid ester as substrate to perform enzymatic reaction to obtain substance containing PPAR action, and obtaining composition containing PPAR action substance. The method does not purify or separate enzyme from plant body, but directly reacts concentrated high-concentration enzyme of dried plant body with endogenesis or exogenously added substrate to generate HODE and oxo-ODE, and then the HODE and oxo-ODE are heated and dried to obtain dried food with good safety and preservation property, and no raw material waste or waste is generated, thus being an environment-friendly method.

Description

PPAR action substance and method for producing the same

Technical Field

The invention relates to the field of PPAR, in particular to a PPAR acting substance and a preparation method thereof.

Background

PPARs (peroxisome proliferator-activated receptors) are ligand-activated receptors in the nuclear hormone receptor family, three subtypes of which have been found in different species: α, β and γ. It controls many intracellular metabolic processes, for example, and is involved in lipid and lipoprotein metabolism, in vitro carbohydrate balance, and in Ligand-induced nuclear receptors (Ligand-induced nuclear receptors). It is essentially a class of ligand-dependent transcriptional regulators.

The ligands for PPARs can be classified into natural ligands and synthetic ligands according to their origin. All PPARs are activated to varying degrees by fatty acids and their derivatives, and participate in the regulation of HDL/Chol levels. PPAR α is selectively activated by polyunsaturated fatty acids (PUFA) and their derivatives, eicosanoids. The products of arachidonic acid after the action of cyclooxygenase and lipoxygenase (the catalytic substrates of the arachidonic acid are linoleic acid, linolenic acid, arachidonic acid and the like) are natural ligands of PPAR gamma. Linolenic acid oxidative metabolites derived from the LDL metabolic pathway 9-hydroxyoctadecadienol (9-HODE) and 13-hydroxyoctadecadienol (13-HODE) were confirmed to be PPAR γ agonists.

Oxylipins oxidized lipids are bioactive lipids produced by oxidation of polyunsaturated fatty acids (PUFAs). Oxidized lipid synthesis is tightly regulated and functions in a paracrine or autocrine fashion. Free PUFAs are produced by three enzyme families: cyclooxygenase (COX), Lipoxygenase (LOX) and cytochrome P450(CYP) are broken down into oxidized lipids. COX enzymes can produce some hydroxyl metabolites, such as Arachidonic Acid (AA) to 11-HETE and linoleic acid LA to 9-HODE. LOX enzyme forms the hydroxyl metabolite of LA, HODEs.

Linoleic Acid (LA) is also metabolized by CYP epoxidases via hydroxylation to form a metabolite called hydroxyoctadecadienoic acid (HODEs). 13-HODE has anti-inflammatory effects in inflammatory diseases through its action as PPAR γ agonist. 13-HODE increases prostacyclin biosynthesis.

The ligands of PPAR (also called PPAR acting substances, or PPAR agonists) reported include the aforementioned hydroxyoctadecadienoic acid (HODE), ketooctadecadienoic acid (oxo-ODE), and the like. These ligands, when bound to PPAR, activate PPAR to exhibit a wide range of pharmacological effects, and thus many health foods or supplements contain such components. The prior art related to PPAR active substances and methods for producing the same include the following patent documents and non-patent documents:

1) patent documents:

patent document 1: japanese patent laid-open No. 2020 and 92697;

patent document 2: japanese patent No. 6661121;

patent document 3: japanese patent No. 6008983;

patent document 4: japanese patent laid-open No. 2019-17370;

patent document 5: japanese patent laid-open No. 2017-209053;

patent document 6: japanese patent No. 4700601.

2) Non-patent documents:

non-patent document 1: kim, y, et al, 2012, "patent PPAR α Activator Derived from Tomato Juice,13-oxo-9, 11-octadienoic Acid, deletions Plasma and hepatitis trigyceride in oxide diabetes Mice" PLOS ONE 7(2), e 31317;

Non-patent document 2: bio L.pharm.Bull. Vol.forty-second, 937-943, (2019).

Jp 2020-a 92697 describes only a method for producing an oxygenated fatty acid or a composition of an oxygenated fatty acid comprising allowing a lipoxygenase to act on a fatty acid in the presence of a reducing agent and in the presence of a reduced amount of dissolved oxygen, and does not describe the use of dried plants at all.

Japanese patent No. 6661121 describes a method of producing a fermentation liquid by fermenting a fermentation medium produced from dried plants such as dried soybeans, medlar, jujube, and turmeric, and honey in a fermentation tank. This process requires a large amount of fermentation broth and the waste stream also requires post-treatment.

Japanese patent No. 6008983 describes a method for producing ketooctadecadienoic acid by fermenting round soybeans or processed round soybeans. Large amounts of fermentation broth and waste liquid work-up are required.

Japanese patent application laid-open No. 2019-17370 describes a method of producing a composition containing a hydroxy derivative of an unsaturated fatty acid by subjecting beer lees to an alkali treatment to obtain an alkaline extract, acidifying the alkaline extract, and then preparing the composition. A large amount of waste is generated.

Japanese patent application laid-open No. 2017-209053 describes a method for producing an oxidized derivative of an unsaturated fatty acid comprising a peroxide of an unsaturated fatty acid or the like by bringing an unsaturated fatty acid having a specific structure into contact with at least 1 plant water extract. A large amount of waste is generated.

In Japanese patent No. 4700601, a method of extracting beta-glucan from crushed grains and a method of purifying the same are described. Beta-glucan is the target.

In non-patent document 1, it is clarified that oxo-ODE is a PPAR agonist and its role in fat burning.

In non-patent document 2, the effect of 9-HODE on various cancer cells was investigated to elucidate the carcinostatic activity.

Disclosure of Invention

The main object of the present invention is to provide a PPAR active substance and a method for producing the same, which solve the problem that the conventional method for producing a PPAR active substance requires complicated steps such as purification and extraction.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method for producing a PPAR action substance, the method comprising: providing a dried substance containing a lipoxygenase enzyme, wherein the lipoxygenase enzyme comprises any one or more of cyclooxygenase, lipoxygenase and cytochrome P450, and the dried substance is a powdered dried plant or a powdered dried food; placing the dried powder in water or buffer solution, activating lipid metabolism enzyme under the action of polyunsaturated fatty acid and/or polyunsaturated fatty acid ester as substrate to perform enzymatic reaction to obtain substance containing PPAR action, and obtaining composition containing PPAR action substance.

Further, providing a dried substance containing a lipid-metabolizing enzyme includes: crushing a plant containing lipid metabolism enzyme after non-heating drying to obtain a powdery dried plant; or crushing the plant containing lipid metabolism enzyme, and then performing non-heating drying to obtain powdery dried plant; or crushing the dried food to obtain powdered dried food.

Further, the polyunsaturated fatty acids and/or polyunsaturated fatty acid esters are endogenously or exogenously added to the dried material; preferably, the polyunsaturated fatty acid is at least one of the group consisting of linoleic acid, linolenic acid and arachidonic acid; preferably, the polyunsaturated fatty acid ester is a triglyceride.

Further, the dried substance is placed in water or a buffer containing a reduced substance.

Further, the reducing substance is at least one or more selected from the group consisting of reducing sugar, ascorbic acid, tocopherol and astaxanthin; preferably, the reducing sugar is glucose; further, the concentration of the reducing substance in the water or the buffer solution is 1.5-2.5 mg/mL.

Further, the mass-to-volume ratio of the powdery dry substance to water or buffer solution is 1: 10-1: 3 in terms of mg: μ L; preferably, the time of the enzymatic reaction is 30min to 300min, more preferably 30min to 90 min; preferably, the pH value of the enzymatic reaction is 6-10.

Further, the production method further comprises, after the production of the composition containing a PPAR action substance: the activity of the lipid-metabolizing enzyme and the enzyme-inhibiting substance in the composition is lost.

Further, the lipid-metabolizing enzyme and the enzyme-inhibiting substance in the composition are inactivated by heating.

In order to achieve the above object, according to one aspect of the present invention, there is provided a PPAR active substance which is a composition containing the PPAR active substance produced by the above production method; or a mixture containing the composition.

By applying the technical scheme of the invention, the improved manufacturing method of the PPAR acting substance breaks through the existing improvement thought, enzymes are not purified or separated from the plant body, but the enzymes in the plant body directly react with polyunsaturated fatty acids/esters contained in the plant or externally added to generate HODE and oxo-ODE, thereby obtaining the composition containing the PPAR acting substance. Furthermore, by using dried plants and dissolving them in a powdered state with water or a buffer solution, it is possible to increase the water permeability and thus to activate the enzymes in the plant body in a highly concentrated state, to increase the reaction rate, and to suppress side reactions and to effectively promote the reaction between the enzymes and the polyunsaturated fatty acids/esters, thereby increasing the production of HODE and oxo-ODE.

The method for producing HODE and oxo-ODE by using plant as reaction medium, in the preferred embodiment, the method for producing the same adjusts the supplementary material components in the health care product under the condition of not involving purification and separation of HODE and oxo-ODE, but fully utilizes the original edibility of edible plant, and can be changed into edible products by directly drying after obtaining the composition of PPAR action substance containing HODE and oxo-ODE, thereby not generating waste and wasting raw materials, and being an environment-friendly method for producing the same.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 is a graph showing HPLC detection results under different processing conditions in example 2 of the present invention;

FIG. 2 shows the effect of the addition or absence of linoleic acid to the production of HODE in different samples according to example 10 of the present invention;

FIG. 3 shows the effect of the addition or absence of linoleic acid on the production of oxo-ODE for different samples in example 10 of the present invention;

FIG. 4 shows the effect of pH difference on HODE generation in example 10 of the present invention;

FIG. 5 shows the effect of pH difference on oxo-ODE production in example 10 of the present invention.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Interpretation of terms:

PPAR-acting substances: this application refers to compositions containing hydroxyoctadecadienoic acid (HODE), ketooctadecadienoic acid (oxo-ODE). Among them, HODE and oxo-ODE are known ligands of PPAR, and exhibit a wide range of pharmacological effects by activating PPAR after binding to PPAR, and thus are added to many health foods or supplements.

As mentioned in the background, the prior art processes for the production of PPAR-acting substances such as HODE generally require treatment of linoleic acid with crude or purified lipoxygenase or peroxidase from plant extracts to form hydroxyHODE and preparation of the oxo derivative oxo-ODE by the action of a dehydrogenase, followed by purification and isolation of HODE. This method is complicated, and the production cost of such PPAR active substances is relatively high.

In order to improve the above-described situation, in one exemplary embodiment of the present application, there is provided a method for producing an improved PPAR action substance, the method comprising: providing a dried substance containing a lipid-metabolizing enzyme, wherein the lipid-metabolizing enzyme comprises any one or more of cyclooxygenase, lipoxygenase and cytochrome P450, and the dried substance is a powdered dried plant or a powdered dried food; the dried substance is placed in water or buffer solution, and lipid metabolism enzyme is activated under the action of polyunsaturated fatty acid and/or polyunsaturated fatty acid ester to perform enzymatic reaction to generate substance containing PPAR action, thereby obtaining composition containing substance containing PPAR action.

The improved method for producing the PPAR acting substance breaks through the existing improvement idea, does not purify or separate enzyme from the plant body, but directly reacts the enzyme in the plant body with polyunsaturated fatty acid (such as linoleic acid contained in the plant or added linoleic acid) and/or polyunsaturated fatty acid ester (such as triglyceride endogenous to the plant or added triglyceride) contained in the plant or added externally to generate HODE and oxo-ODE, thereby obtaining the composition containing the PPAR acting substance. Furthermore, by using dried plants and dissolving them in a powdered state with water or a buffer solution, it is possible to increase the water permeability and thus to activate the enzymes in the plant body in a highly concentrated state, to increase the reaction rate, and to suppress side reactions and to effectively promote the reaction between the enzymes and the polyunsaturated fatty acids/esters, thereby increasing the production of HODE and oxo-ODE.

The method for producing HODE and oxo-ODE by using plant as reaction medium, in the preferred embodiment, the method does not relate to the regulation of supplementary food components in health care products under the condition of purifying and separating HODE and oxo-ODE, but fully utilizes the original edibility of edible plants, and can be changed into edible products by directly drying after obtaining the composition of PPAR action substances containing HODE and oxo-ODE, thereby generating no waste and no waste of raw materials, and being an environment-friendly method for producing.

The plants and foods used in the production method of the present invention may be any plants or fungi containing lipoxygenase, such as beans, barley, rice, wheat, solanaceae, cucurbitaceae, cruciferae, umbelliferae, and mushrooms. Preferably edible plants or food products such as dried vegetables (dried from fresh vegetables or prepared into food products by drying), such as soybean, shiitake mushroom, radish, bell pepper, medlar, tomato, barley, etc. The dry powder is at least one selected from dry vegetable group or mixture thereof.

In a preferred embodiment, the composition containing the PPAR active substance of HODE and oxo-ODE produced by the production method of the present application may be used as a health food directly after drying, or as an additive component of a health food (for example, by mixing the composition containing the PPAR active substance of the present application with other components).

In the above manufacturing method, the reaction can be effectively promoted by using the powder of the dried plant or food, and the obtaining method includes but is not limited to the following methods: crushing the plant containing the lipid metabolism enzyme after non-heating drying to obtain a powdery dried plant; or crushing the plant containing lipid metabolism enzyme, and then performing non-heating drying to obtain powdery dry plant; or crushing the dried food to obtain powdered dried food.

In addition, the dried sample may be pulverized while adding physiological saline (or a buffer solution) under stirring. Further, homogenization and freeze-drying may be used, but it is most preferable to use the powder as it is obtained by various pulverization methods after freeze-drying.

The non-heat drying is performed in consideration of the fact that most plants are rich in lipid-metabolizing enzymes such as lipoxygenase, and the optimum temperature for the oxidation reaction is 20 ℃ to 30 ℃ and is inactivated at 90 ℃. Thus, enzymes tend to be reduced in activity or even inactivated when dried by heating. Drying without heating, such as freeze drying or natural drying, helps to retain the enzyme activity. The enzyme is stable when dried in the tissue. When the powder is crushed, the operations such as crushing and rolling destroy the texture and improve the water permeability. Further, from the viewpoint of being useful as an additive component in a health product, it is more preferable to perform drying by freeze-drying, which is advantageous in retaining color tone, flavor, and the like.

As described above, the production method of the present application utilizes the enzyme of the plant itself to react with the polyunsaturated fatty acid and/or the polyunsaturated fatty acid ester added by itself or from an external source. Compared with the method of only using own polyunsaturated fatty acid and/or polyunsaturated fatty acid ester, the production amount of HODE and oxo-ODE can be remarkably increased by adding exogenous polyunsaturated fatty acid and/or polyunsaturated fatty acid ester such as linoleic acid or triglyceride. Polyunsaturated fatty acids include, but are not limited to, linoleic acid, linolenic acid, and arachidonic acid, and polyunsaturated fatty acids include, but are not limited to, monoglycerides, triglycerides, and the like.

In the case of the dried plant or dried food, in view of the fact that it has been dried, it contains a lower amount of antioxidant substances than the plant of fresh origin, and in order to further avoid oxidation during the course of the reaction, during the drying of the composition of PPAR action substances at a later stage, or during long-term storage, in a preferred embodiment of the present application, the dried substance is placed in water or a buffer containing a reducing substance during the enzymatic reaction by the above-mentioned lipoxygenase, lipoxygenase and cytochrome P450 or other lipid metabolizing enzymes. Dissolving the dried material in water or buffer solution containing reducing substance can remove oil-soluble and/or water-soluble oxidizing substance, and further improve enzyme activity.

Here, any substance that is reduced and edible may be used in the present application. From the viewpoints of known and experimentally verified effects of the present application, the preferable reducing substance is at least one or more selected from the group consisting of reducing sugar, ascorbic acid or tocopherol, astaxanthin; more preferably, the reducing sugar is glucose.

The specific concentration of the reducing substance in water or buffer varies depending on the kind and physical properties (e.g., fat solubility or water solubility) of the reducing substance. In a preferred embodiment of the present application, the concentration of the reducing substance in water or in the buffer is 1.5-2.5 mg/mL.

In the production method of the present application, the temperature and pH of the enzymatic reaction may be the temperature and pH at which the hydroxy fatty acid and the oxygen-containing fatty acid are produced. For example, in the case of soybean, the reaction temperature is 0 ℃ to 60 ℃, preferably 20 ℃ to 35 ℃, and the reaction pH is 4 to 11, which can be determined according to the yield of the product and the subsequent use. The reaction time may be appropriately selected depending on the type and form of the dried plant material.

The concentration of relevant enzyme in dry plant or food is high, and when the enzyme is dissolved in water or buffer solution in the form of powder, the tissue structure is destroyed, so that the permeability of the water or buffer solution is improved, thereby facilitating the dissolution of endogenous or exogenous polyunsaturated fatty acid/ester in the water or buffer solution, and activating enzyme such as lipoxygenase in the plant to perform enzymatic reaction. Optimizing the amount of added water or buffer solution is helpful to further improve the efficiency of enzymatic reaction and improve the yield angle of HODE and oxo-ODE. Specifically, the mass-to-volume ratio of the dry substance to water or buffer solution in mg: μ L may be any one of 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, and 1:1, and in a preferred embodiment of the present invention, the mass-to-volume ratio of the dry substance to water or buffer solution in mg: μ L is controlled to be 1:10 to 1:3, more preferably 1:4 to 1:3, and most preferably 1:3, and dissolution is performed in a volume 3 times the mass of the dry substance powder, so that the obtained amounts of HODE and oxo-ODE are also the highest, and the amount of water is further increased, and the amount of production is reduced compared with the amount of 3 times the water.

Further, by comparing the amounts of HODE and oxo-ODE produced over a long period of time without passing through the reaction, it was found that the amounts were relatively high in the case where the time for the enzymatic reaction was 30min to 300min, and the reaction time was further prolonged, for example, up to 20 hours, and the yield was not increased accordingly. More preferably 30min to 90min, most preferably 60 min.

In addition, the sequence of mixing the exogenously added polyunsaturated fatty acids and water or buffer with the dried plant powder was also investigated in this application and it was found that different addition sequences had less influence on the amount of product produced. The pH value condition of the enzymatic reaction is preferably 6-10. The pH value can be adjusted reasonably in the reaction to obtain the optimal pH value according to different plant varieties or sources.

As mentioned above, the PPAR active substance obtained by the production method of the present application is in the form of a composition which can be directly consumed without isolation and purification, and therefore, from the viewpoint of edible use and long-term storage, it is preferable that the production method further comprises, after producing a composition containing the PPAR active substance: the activities of the lipid-metabolizing enzyme and the enzyme-inhibiting substance in the composition are lost, and the purpose is to maintain the long-term stability of the PPAR-acting substance in the composition and to prevent degradation or other reactions leading to deterioration. In a preferred embodiment, the inactivation of the lipid-metabolizing enzyme and the reducing substance in the composition by heating is a simple and convenient method without destroying the desired active ingredient, and the safety and preservability of the product as an edible product are improved.

In a second exemplary embodiment of the present application, there is provided a PPAR active substance, which is a composition containing a PPAR active substance produced by the above production method; or a mixture containing the composition. The PPAR active substance is a composition containing HODE and oxo-ODE, and the dried food produced by the method of the present application is excellent in storage stability and safety, and therefore can be added to supplements, foods and drinks, cosmetics, quasi-drugs, pharmaceuticals, feeds, and the like to exert its effects.

The advantageous effects of the present application will be described in detail below with reference to specific embodiments. The plant materials used in the following examples are all commercially available products (mushrooms, radishes, paprika, wolfberries, tomatoes, etc. are all purchased from towns, and the specific quality and storage conditions of the varieties are not clear), and the variety, the place of production, or the source may be different, but the scheme of the present application is not limited thereto. In the following examples, the sources of the respective raw materials are as follows:

example 1

Preparation of HODE based on dried vegetables or crops

The dried vegetables include: lentinus Edodes, radix Raphani, color pepper, fructus Lycii, and fructus Lycopersici Esculenti. Drying the crops: barley glutinous rice seed and soybean.

The dried vegetable or dried crop containing the lipoxygenase was pulverized, 200mg of each powder was taken, 200. mu.L of linoleic acid and 600. mu.L of purified water were added to the powder and stirred to suspend, and then reacted at room temperature for 1 night. A portion was diluted 10-fold with EtOH and the amount of HODE produced was measured by HPLC. The results of the measurements are shown in the following table:

as a result, the generation of HODEs is found, and specific products are as follows: fr 1: 13- (Z, E) -HODE, Fr 2: 9- (E, Z) -HODE, Fr 3: 13- (Z, Z) -HODE, Fr 4: 9- (Z, Z) -HODE (where Z, E structure refers to Z on the same side of the double bond as the two larger groups to which the two carbon atoms of the double bond are attached, and E on both sides of the double bond). Also P1, P2 and P3.

Here, the oxo-ODE is an oxo derivative of linoleic acid, and HODE is a compound in which-OH is changed to O by the action of dehydrogenase. The PPAR acting substance has anti-obesity effect. The literature states that 4 oxo-ODEs exist, 3 peaks are detected in the application, and the average molecular weights of the peaks in the mass spectrum are all 294, which is consistent with the molecular weight of the oxo-ODE, and the UV absorption is also considered as an absorption peak of 280nm instead of 235nm, which is consistent with the literature report. After the peaks are separated and purified, the structural distinction of 3 peaks is not obvious due to isomerization, so that the application is indicated by P-1, P-2 and P-3. The isomerization is described in the literature as follows: 13-oxo-ODE,11-oxo-ODE, 9-oxo-ODE (see www.chemspider.com/chemical-structure.8014803. html).

Meanwhile, it was found that HODE was produced when the dry powder was mixed with linoleic acid and water and allowed to react at room temperature for 1 night, but the amount of HODE produced was relatively small if linoleic acid was not added (see, specifically, example 10).

Table 1:

example 2

Study of HODE in original soybeans

Since HODE was detected when raw soybeans were pulverized and mixed with water, in order to investigate whether HODE was contained in the raw soybeans, 250mg of soybean powder obtained by pulverizing raw soybeans with a coffee bean mill was mixed with lmL of pure water in this example, and different treatments were investigated.

1: the mixture was mixed with 1mL of purified water, and 1mL of EtOH was added thereto, followed by immediate extraction to prepare an HPLC sample.

2: the mixture was mixed with 1mL of purified water, and EtOH 1mL of 4M NaOH 150. mu.L was added thereto to hydrolyze the mixture at 90 ℃ for 60 minutes to prepare an HPLC sample.

3: after mixing with 1mL of purified water and reacting for 1 night, 1mL of EtOH was added and extracted immediately to prepare an HPLC sample.

4: after mixing with 1mL of purified water and reacting for 1 night, 1mL of EtOH was added, and 150. mu.L of 4M NaOH was added to hydrolyze at 90 ℃ for 60 minutes to prepare an HPLC sample.

5: after mixing with 2mL of 50% EtOH and reacting for 1 night, 150. mu.L of 4M NaOH was added and the mixture was hydrolyzed at 90 ℃ for 60 minutes to prepare an HPLC sample.

And (3) taking the HPLC samples subjected to different treatments for HPLC detection, wherein the conditions of the HPLC detection are as follows:

A chromatographic column: TSK ODS 80TM 4.6x150mm, column temperature 30 ℃;

solvent: m/100 HCl: CH (CH)₃CN：CH₃OH (volume ratio 46: 41: 13) (referring to the volume ratio of the three components per 100 volumes of mobile phase) at a flow rate of 1 mL/min;

and (3) detection: 235nm 0.04AUFS (AUFS is an abbreviation for Absorbance Units Full Scale, indicating detection sensitivity of HPLC)

The results are shown in FIG. 1. As can be seen from the results in fig. 1, when pure water was added to the dried soybean powder and reacted for 1 night, HODEs were formed. Extraction with EtOH was able to extract free HODE but not glyceride bound HODE. Thus, by hydrolysis with NaOH to produce free HODE, it can be obtained by EtOH extraction. This also confirms the fact that lipoxygenase in plants described in the textbook acts not only on free linoleic acid but also on triglycerides.

From the detection results of this embodiment, the following conclusions are drawn: 1) the starting raw soybeans do not contain HODEs; 2) the bound HODEs are formed by reacting lipoxygenase and peroxidase directly with triglycerides, not free HODEs.

Example 3

Detection of different varieties of soybeans

HODE (. mu.g/g) in soybean powder prepared with soybean oil was compared using 100g of soybean powder of round soybeans and long soybeans.

Since it was not found in the EtOH extraction, it was not free HODE but produced in the form of glyceride. Depending on the kind of beans, HODE is produced differently. From the following results, the yield of HODE was higher in the long soybeans than in the round soybeans.

Table 2: the amount of HODE produced (μ g/g) from soybean oil (soybean oil contains various fatty acids such as linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, etc., wherein oleic acid is present in the form of glyceride at the most) in round soybeans and long soybeans

	Fr1	Fr2	Fr3	Fr4
					Round soybean	Without treatment	18	6	9	10
Hydrolysis	460	174	141	106
					Long soybean	Without treatment	28	12	9	10
Hydrolysis	993	372	216	164

Example 4

Study on mixing sequence of Soybean powder, Soybean oil (ingredients as above) and Water

HODE was prepared by changing the mixing order of soybean powder 100g (A), water (pH)300g (B), soybean oil 100g (C). The method comprises the following specific steps:

table 3:

from the results shown in the above table, 1) the order of addition of soybean oil and buffer had little effect on the amount of reaction product produced. 2) As for the pH value of the reaction, the pH value varies from 6 to 10 depending on the species and the state of the starting enzyme (e.g., origin, freshness), and the optimum pH value can be adjusted during the reaction.

Example 5

Investigation of reaction time

Soybean powder 200mg, linoleic acid 100 μ L and physiological saline solution 600 μ L were used for reaction.

Table 4:

HODE was not detected in 200mg of untreated soy flour, but was only detected by the above-described combination treatment (i.e., free HODE was detected without alkali treatment, but both free and bound HODE were detected by extraction when alkali treatment was performed. When the reaction time was increased to 60 minutes, the amount of the formed product was the highest and substantially decreased thereafter. The reaction time reached 300 minutes and 20 hours, and the amounts of the formed products were about the same. The oxo-ODE, similar to HODE, also increases more when the reaction time is 60 minutes, and then gradually decreases; after 20 hours, the amount produced was less than 300 minutes.

Example 6

Reactivity studies in soy flour and soy extract were performed to find the optimum amount of water.

The reactivity in crude enzyme extracted from whole soybean meal and soybean meal was compared against dried tomato powder as a control.

1) Placing the soybean powder in 10 times volume of water (namely, the volume of the water is 10 times of the mass of the soybean powder) to react for 60 minutes;

2) dissolving soybean powder with 10 times volume of water, centrifuging to obtain soybean crude enzyme, and reacting the soybean crude enzyme in 10 times volume of water for 60 minutes;

3) Placing soybean powder in water with 3 times volume for reaction for 60 minutes as a control;

4) using the dry tomato powder as a control, the tomato powder was placed in 10 volumes of water for 60 minutes.

The results of the measurements are shown in the following table:

table 5:

in HODE, soybean flour, soybean crude enzyme and tomato reacted in 10 times of water for 60 minutes, and the amount of reaction product decreased, but the amount of product formed between 3 samples hardly changed.

Example 7

Ascorbic acid is added into the reaction system

The production of HODE when ascorbic acid was added to physiological saline was compared in order to prevent oxidation during the reaction, drying and storage. The reaction was carried out for 60 minutes using soybean powder 200mg, linoleic acid 100. mu.L, and physiological saline (containing ascorbic acid) 600. mu.L, and then ascorbic acid was added to the physiological saline to a concentration of 0.5mg/mL to 2.5mg/mL, and the HODE formation was examined, and the results are shown in the following Table.

Table 6:

as can be seen from the above table, the amount of ascorbic acid produced is relatively large when the concentration of ascorbic acid in physiological saline is 1.5mg/mL or more.

Example 8

The effect of ascorbic acid addition on reaction time was investigated

The results of comparing the reaction time and the amount of HODE produced (peak height) in the case of 200mg of soybean powder, 600. mu.L of physiological saline containing 2.5mg/m L of ascorbic acid, and 100. mu.L of linoleic acid are shown in the following table.

Table 7:

as can be seen from the above results, even if ascorbic acid is added, the reaction time is preferably about 60 minutes for both HODE and oxo-ODE to be formed, i.e., the addition of ascorbic acid does not result in an increase in the reaction time.

Example 9

Addition of tocopherol

Ascorbic acid is a water-soluble antioxidant substance, and tocopherol is a substance having an antioxidant effect of a fat-soluble lipid, and the effect of adding tocopherol on the production of HODE is examined in this example.

When 200mg of soybean powder was reacted with 50. mu.L of linoleic acid and physiological saline containing 2.5mg/mL of ascorbic acid for 1 hour, HODE and oxo-ODE produced by adding 1.0mg, 2.5mg and 5.0mg of tocopherol, respectively, were compared.

Table 8:

fr4 overlapped with the inclusions and could not be measured. P-3 generation is a trace.

As can be seen from the above table, when 5mg of tocopherol was added to the reaction of 200mg of soybean powder and 50. mu.L of linoleic acid, the production of HODE was enhanced.

Examples 8 and 9 show that reaction inhibition by oxidation can be prevented by adding water-soluble ascorbic acid and fat-soluble tocopherol.

In the above examples, soybeans were pulverized in each experiment, and whether the reaction was proceeding or stopped was judged from the overall tendency. However, there may be some differences in specific conditions, such as specific sources of soybeans (commercially available products, all stored at room temperature, but the market storage state is unclear), drying methods and specific drying degrees, particle sizes of the pulverized samples, and stirring rates and times. Thus, the amounts of HODE produced detected under the same conditions in different examples were different.

Example 10

The production of HODE and oxo-ODE was examined in the presence or absence of polyunsaturated fatty acids in plants other than soybean.

1) Drying a sample:

soybean and barley: naturally dried soybean and barley were used directly.

The kiwi fruit, the eggplant and the tomato are mature and can not form slices, so the freeze-dried fruit juice is prepared.

Other samples were cut into sections and freeze-dried.

2) Crushing a dried sample:

the dried sample was pulverized in a mortar. Although some fibers are difficult to crush, they are crushed as much as possible to allow them to be stored uniformly in a sample bottle until use.

3) Preparation of PPAR agonist in Dry samples

After drying 20 samples to powder: mu.L of linoleic acid and 30. mu.L of tocopherol (100mg/mL of a solution in LETOH) were added to 50mg of the sample powder, mixed and stirred, and then 125. mu.L of ascorbic acid (10mg/mL of physiological saline) was added thereto, mixed well, reacted at room temperature for 20 hours, and then lyophilized. To this end, 30. mu.L of LEtOH was added to 50mg of the sample powder and mixed with stirring, 125. mu.L of physiological saline was added and kneaded, and the mixture was reacted at room temperature for 20 hours and then freeze-dried. To each dried sample was added 1ml of LEtOH, sonicated for 30 minutes, and then centrifuged at 10000rpm for 10 minutes to prepare a supernatant as an HPLC sample.

4) HPLC detection conditions:

a chromatographic column: TSK-ODS 80TM 4.6x150mm Room temperature;

solvent: m/100HCl: CH3CN: CH3 OH: 47:43:10, flow rate: 0.8 mL/min;

and (3) detection: HODE: 235 nm; oxo-ODE: 280 nm.

5) The results are shown in the following table and in FIGS. 2 and 3:

HODE comparison: fr1:13- (Z, E) -HODE, Fr2:9- (Z, E) -HODE. 13- (E, E) -HODE and 9- (E, E) -HODE were omitted because of low yield and poor separability. In addition, the oxo-ODE was compared at 3 peak heights.

Table 9: amount of HODE produced by the reaction with and without linoleic acid (. mu.g/g)

In addition, soybeans and eggplants were treated in the same manner at pH7.4 and pH11 and compared by reacting for 1 hour, and the results are shown in the following table and fig. 4 and 5.

Table 10:

HODE was formed according to the difference in pH (reaction time 1 hour. mu.g/g)

	Fr1	Fr2	P-1	P-2	P-3
						Soybean pH11	7974	4407	30	16	3
Soybean pH7.4	2230	2177	15	14	3
						Eggplant pH11	2098	3725	7	15	2
Eggplant pH7.4	2413	3620	11	22	5

As shown in tables 9 and 10 above and FIGS. 2 to 3, the amount of HODE produced varied from sample to sample, and soybean and eggplant produced a large amount of HODE. Without the addition of linoleic acid, most of the yields were less than 10% of the added amount. It can be seen that the large amount of HODE production is due to polyunsaturated fatty acids added by the enzyme in the sample.

In addition, this example clarifies the effect of pH differences on Fr1 and Fr 2. In soybean, Fr1 is the main product at pH11, and the production rates of Fr1 and Fr2 are almost the same at pH 7.4. In eggplant, more Fr2 is produced regardless of pH.

For oxo-ODE, a similar pattern of formation to HODE is shown, with oxo-ODE-1 (i.e., P-1) being presumed to be 13- (Z, E) -oxo-ODE and oxo-ODE-2 (i.e., P-2) being 9- (Z, E) -oxo-ODE.

From the results of this example, since the content of Fr1 and Fr2 can be adjusted by using each sample alone or in combination, flavoring agents having different tastes of consumers such as flavor and taste are added, and thus, a wide variety of utilization methods of nutriments, health foods, food improvers, and the like can be realized.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: a plant/food containing lipoxygenase is freeze-dried or naturally dried to concentrate enzymes in the plant, the plant is crushed or rolled to destroy tissues (or is crushed and dried), the powder is mixed with water or buffer solution containing tocopherol and/or ascorbic acid and other reducing substances to remove or inhibit water-soluble or fat-soluble oxidizing substances, lipoxygenase and other lipoxygenase are activated under the action of endogenous or exogenous addition of linoleic acid, linolenic acid, triglyceride and other polyunsaturated fatty acids/esters, and the lipoxygenase and other lipoxygenase are subjected to enzymatic reaction to generate combined HODE and oxo-ODE, so that a composition product containing HODE and oxo-ODE is obtained. Because the adopted plants are edible, the plant can be directly used as an edible product without separation and purification.

In order to improve safety and long-term preservability as an edible product, enzymes such as lipoxygenase and enzyme inhibitors may be inactivated by heating after completion of the reaction, thereby producing a dry food.

Although the products of the present application may be consumed directly, other special requirements for the isolation of HODE and oxo-ODE from the products made herein are not excluded. If it is desired to extract a large amount of HODE and oxo-ODE obtained by the present application using the plant itself as a reaction medium and adjusting the operation for subsequent use (e.g., extraction to prepare other foods or health products with other active ingredients), the extraction and separation can be performed in the manner as described in the above examples, e.g., hydrolysis with alkali followed by extraction with ethanol.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for producing a PPAR-acting substance, the method comprising:

Providing a dried substance containing a lipid-metabolizing enzyme including any one or more of cyclooxygenase, lipoxygenase and cytochrome P450, the dried substance being a powdered dried plant or a powdered dried food;

and (3) putting the powder of the dried product into water or a buffer solution, and activating the lipid metabolism enzyme under the action of a substrate polyunsaturated fatty acid and/or polyunsaturated fatty acid ester to perform an enzymatic reaction to generate a substance containing the PPAR action, thereby obtaining the composition containing the PPAR action substance.

2. The method according to claim 1, wherein the step of providing the dried substance containing the lipid-metabolizing enzyme comprises:

crushing the plant containing the lipid metabolism enzyme after non-heating drying to obtain the powdery dried plant; or

Crushing the plant containing the lipid metabolism enzyme, and then carrying out non-heating drying to obtain the powdery dried plant; or

And crushing the dried food to obtain the powdery dried food.

3. The production method according to claim 1, wherein a polyunsaturated fatty acid and/or a polyunsaturated fatty acid ester is added endogenously or exogenously to the dry substance;

Preferably, the polyunsaturated fatty acid is at least one of the group consisting of linoleic acid, linolenic acid, arachidonic acid, EPA and DHA;

preferably, the polyunsaturated fatty acid ester is an alcohol ester, more preferably a triglyceride.

4. The production method according to any one of claims 1 to 3, wherein the dried substance is placed in water or a buffer containing a reduced substance.

5. The method according to claim 4, wherein the reducing substance is at least one or more selected from the group consisting of reducing sugars, ascorbic acid, tocopherol, and astaxanthin;

preferably, the reducing sugar is glucose.

6. The method according to claim 5, wherein the concentration of the reduced matter in the water or the buffer solution is 1.5 to 2.5 mg/mL.

7. The method according to claim 4, wherein the mass-to-volume ratio of the powdery dry substance to the water or the buffer solution is 1:10 to 1:3 in terms of mg: μ L;

preferably, the time of the enzymatic reaction is 30min to 300min, more preferably 30min to 90 min;

preferably, the pH value of the enzymatic reaction is 6-10.

8. The method according to claim 4, wherein the method further comprises, after the step of producing the composition containing the PPAR-acting substance: the activity of the lipid-metabolizing enzyme and the enzyme-inhibiting substance in the composition is lost.

9. The method according to claim 6, wherein the lipid-metabolizing enzyme and the enzyme-inhibiting substance in the composition are inactivated by heating.

10. A PPAR active substance, which is a composition containing a PPAR active substance produced by the production method according to any one of claims 1 to 9; or mixtures containing said compositions.