JP2019199453A - Composite containing caffeoylquinic acid compound - Google Patents

Abstract

To provide a technology that enables simple isolation and purification of dicaffeoylquinic acid or tricaffeoylquinic acid.SOLUTION: A method for producing a composite containing a caffeoylquinic acid compound includes a step of mixing at least one kind of caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid with animal protein in an aqueous medium to form a composite insoluble in the aqueous medium from the caffeoylquinic acid compound and the animal protein. In the production method, the caffeoylquinic acid compound is preferably derived from sweet potato foliage.SELECTED DRAWING: None

Description

  The present invention relates to a caffeoylquinic acid compound, and more particularly to formation of a complex of a caffeoylquinic acid compound and a protein.

  Caffeoylquinic acid compounds are polyphenolic substances having physiological functionality, which are contained in sweet potatoes, propolis (bees), coffee beans, and the like. In particular, dicaffeoylquinic acid and tricaffeoylquinic acid, which are abundant in the stems and leaves of sweet potato, have an Alzheimer-type dementia prevention effect (Patent Document 1), an ATP production promoting effect (Patent Document 2), and a neurogenesis promoting effect ( Patent Document 3), blood pressure lowering action (Non-Patent Document 1), human cancer cell growth inhibitory effect (Non-Patent Document 2), anti-diabetic action (Non-Patent Document 2), etc. have been found and attracted attention.

  On the other hand, regarding formation of a complex of a polyphenolic substance and a protein, for example, Patent Document 4 discloses that a tea polyphenol can be used in a stable state by making a tea polyphenol a complex with a protein. It is described that it is expected to be used in the fields of pharmaceuticals, foods, etc., because the taste is also reduced.

  Further, for example, in Patent Document 5, catechins are extracted from a tea leaf extract without using an expensive column gel by mixing an aqueous tea leaf extract and a soy protein extract and adjusting the acidity. It is possible to prepare a polyphenol-protein complex by separation, and the produced complex is made from tea catechin and soybean protein, and is described as a material excellent in safety and functionality as food. Yes.

  In addition, for example, Patent Document 6 describes a method for producing a polyphenol-containing protein comprising a water-insoluble complex of polyphenol and protein. According to the production method, a heterogeneous complex due to ramping (dama formation) or the like is described. It is described that formation is prevented, a uniform complex of polyphenol and protein can be obtained, and the bitterness and astringency of the polyphenol-containing protein obtained by the production method is greatly reduced.

International Publication No. 2007/091613 Japanese Patent No. 5403582 JP 2017-145215 A Japanese Patent Laid-Open No. 02-202900 JP 2002-68991 A JP 2012-51840 A

Koji Ishiguro, Makoto Yoshimoto, Hitoto Hamada, Shinya Takagaki, “Blood pressure-lowering effect of sweet potato stems and leaves”, Journal of Japan Society for Food Science and Technology, Vol. 54 (2007) No. 1, p. 45-49 Makoto Yoshimoto, “Pharmacological Action of Polyphenols in Sweet Potato Stalks and Leaves”, Food Industry, Vol. 48 (2005) 6 p. 69-75

  However, regarding the technology for isolating and purifying dicaffeoylquinic acid and tricaffeoylquinic acid from bases derived from natural products, for example, column purification using a synthetic adsorbent after extraction with hot water or hydrous ethanol There is a known method of performing such a method, but such a method has a problem in that it is expensive to manufacture and industrially mass-produces. Moreover, the techniques described in Patent Documents 4 to 6 have not been focused on caffeoylquinic acid compounds.

  Therefore, an object of the present invention is to provide a technique that makes it possible to more easily isolate and purify dicaffeoylquinic acid and tricaffeoylquinic acid.

  As a result of intensive studies to solve the above problems, the present inventors have found that dicaffeoylquinic acid and tricaffeoylquinic acid form a complex with respect to a specific protein. It came to be completed.

  That is, the present invention provides, in a first aspect, at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid and an animal protein in an aqueous medium. And a method for producing a complex containing a caffeoylquinic acid compound, comprising the step of forming an insoluble complex in the aqueous medium with the caffeoylquinic acid compound and the animal protein.

  According to the present invention having the above structure, dicaffeoylquinic acid and tricaffeoylquinic acid form a complex with a specific protein and precipitate as an insoluble matter in an aqueous medium. For example, caffeoylquinic acid compounds such as dicaffeoylquinic acid and tricaffeoylquinic acid can be easily isolated and purified even from primary extracts of plants.

  In the said manufacturing method, it is preferable that the said caffeoylquinic acid compounds are derived from a sweet potato.

  In the above production method, the animal protein is preferably a water-soluble protein.

  In the above production method, the animal protein is preferably one or more selected from the group consisting of gelatin, water-soluble collagen, water-soluble elastin, water-soluble keratin, and collagen peptide.

  Moreover, in the said manufacturing method, it is preferable that the said animal protein is 1 type (s) or 2 or more types selected from the group which consists of a pig origin gelatin and a fish origin gelatin, and especially a pig origin gelatin is preferable.

  On the other hand, the present invention provides, in a second aspect, a method for producing a caffeoylquinic acid compound, comprising a step of treating the complex containing a caffeoylquinic acid compound obtained by the above production method with a water-containing organic solvent. To do.

  According to the present invention having the above-described configuration, the caffeoylquinic acid compound-containing complex obtained by the above-described production method is treated with a water-containing organic solvent, so that dicaffeoylquinic acid or triflic acid is contained in the solvent. Caffeoylquinic acid compounds such as caffeoylquinic acid are eluted, while the protein that has formed the complex hardly elutes or only part of the protein is eluted. Can be increased.

  Moreover, this invention provides the manufacturing method of a caffeoylquinic acid compound which includes the process of processing the caffeoylquinic acid compound containing complex obtained by said manufacturing method with a proteolytic enzyme in the 3rd viewpoint. To do.

  According to the present invention having the above-described structure, the caffeoylquinic acid compound-containing complex obtained by the production method described above is treated with a proteolytic enzyme, so that the protein that has formed the complex is treated after the treatment. Almost decomposed or at least partially decomposed, so the above complex decomposes, so that caffeoylquinic acid compounds such as dicaffeoylquinic acid and tricaffeoylquinic acid are eluted again into the solution. Can be made. And if desired, the degree of purification can be further increased.

  Furthermore, the present invention provides, in a fourth aspect, caffeoylquinic acids formed from gelatin and at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid. A compound-containing complex is provided.

  According to the present invention having the above-described configuration, it is possible to provide useful materials that are safe even if taken orally by humans, and that can be applied to various fields such as foods, pharmaceuticals, cosmetics, and animal feeds. it can.

  According to the present invention, there is provided a technique that makes it possible to very easily isolate and purify dicaffeoylquinic acid and tricaffeoylquinic acid.

FIG. 1 shows a measurement chart when the solution A is subjected to HPLC and a measurement chart when the liquid part solution obtained by solid-liquid separation after complex formation in the sample 15 is subjected to HPLC.

  In general, a compound in which a carboxyl group of caffeic acid is ester-bonded with a hydroxy group of quinic acid is known as a caffeoylquinic acid compound. The present invention includes dicaffeoylquinic acid in which two molecules of caffeic acid are bonded to one molecule of quinic acid, and tricylate in which three molecules of caffeic acid are bonded to one molecule of quinic acid. This is particularly applicable to caffeoylquinic acid.

  Specifically, as a dicaffeoylquinic acid derived from a natural product and based on this, for example, 3,4-di-O-caffeoylquinic acid represented by the following chemical formula (1), There are 3,5-di-O-caffeoylquinic acid represented by chemical formula (2) and 4,5-di-O-caffeoylquinic acid represented by chemical formula (3) below.

  Similarly, tricaffeoylquinic acid derived from a natural product and based on it is 3,4,5-tri-O-caffeoylquinic acid represented by the following chemical formula (4).

  However, the caffeoylquinic acid compounds to which the present invention is applied are not limited to the compounds specifically described above, and as described above, two molecules of caffeic acid are bonded to one molecule of quinic acid. Or a compound in which three molecules of caffeic acid are bonded to one molecule of quinic acid. Moreover, as said caffeoylquinic acid compound, it may be applied with respect to a single type of compound, and may be applied with respect to the mixture of a multiple types of compound. Further, the caffeoylquinic acid compound to which the present invention is applied may be a chemically synthesized product or may be derived from a natural product. Examples of the natural product containing the caffeoylquinic acid compound include sweet potato, mugwort, burdock, coffee, and black walnut.

  In a preferred embodiment of the present invention, the caffeoylquinic acid compound is derived from sweet potato. Particularly preferred is a sweet potato stem and leaf. In general, a primary extract extracted from sweet potato stems and leaves with water, a water-containing organic solvent (preferred organic solvents are ethanol, methanol, isopropanol) and the like contains caffeoylquinic acid compounds (chlorogenic acid (5-cafe) as polyphenolic substances. Monocaffeoylquinic acid, dicaffeoylquinic acid, and tricaffeoylquinic acid)), and above-mentioned 3,4-di-O-caffeoylquina. The content of acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid is abundant. In addition, the above-mentioned 3,4,5-tri-O-caffeoylquinic acid is also contained, although the content is lower than that of dicaffeoylquinic acid.

  The sweet potato cultivar used in the preferred embodiment of the present invention is not particularly limited. It ’s so good.

  Even when the present invention is applied to a primary extract or the like of such a plant, the above caffeoylquinic acid compounds can be simply and easily extracted from an extract containing various and various contaminants. It can be separated and purified. Since plant extracts often contain a lot of polysaccharide components, in such cases, the polysaccharide is first precipitated by alcohol treatment with ethanol, methanol, isopropanol or the like. In addition, it is more preferable to remove it by solid-liquid separation (more preferably, a polysaccharide removal treatment is included). According to this, the purification efficiency of the said caffeoylquinic acid compounds from the primary extract etc. of the plant in which various and various impurities are contained can be improved more.

  On the other hand, as the protein used in the present invention, when mixed with at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid in an aqueous medium, the aqueous medium Insoluble in water, any compound capable of forming a complex with the caffeoylquinic acid compound may be used, but considering that it is well dispersed at the molecular level in an aqueous medium where the complex is formed, at least It is preferable to have water solubility to such an extent that aggregation does not occur in water at room temperature. In addition, when the complex is formed, in consideration of making it insoluble in the aqueous medium, it is obtained from a natural product such as animals and plants once in a form insoluble in water from its base. It is more preferable that the protein has improved water solubility by various treatments such as heating / pressurizing treatment, acid / alkali treatment, enzyme treatment, and any combination of these treatments. More specifically, according to the examples described later, water-soluble proteins derived from animal proteins such as gelatin, water-soluble collagen, water-soluble elastin, water-soluble keratin, and collagen peptide are exemplified. As the protein, a single type of protein may be used, or a plurality of types of proteins may be used in combination.

  In the present invention, the protein is preferably gelatin. Generally, gelatin is derived from animal collagen and extracted into a water-soluble form by chemical and / or physical means such as acid treatment, heat treatment, alkali treatment, and pressure treatment. Depending on the origin, the properties are greatly different. For example, gelatin derived from pigs, cows, fish and the like is widely used, but in the present invention, pigs and fish-derived gelatin are preferred, and among these, pig-derived gelatin is particularly preferred. As shown in the examples described later, pork-derived gelatin has a high ability to form a complex with the caffeoylquinic acid compound and to precipitate it as an insoluble matter in an aqueous medium. Jelly strength is known as one of the quality standards of gelatin. When the protein is gelatin in the present invention, the jelly strength is preferably 80 to 330 (g), and 230 to 330 (g). It is particularly preferred that If gelatin having such a quality within the range of jelly strength is used, a complex with the caffeoylquinic acid compound can be effectively formed. In addition, as shown in the below-mentioned Examples, the higher the jelly strength, the higher the ability to form a complex with the caffeoylquinic acid compound and precipitate as an insoluble matter in an aqueous medium.

  The above jelly strength is the Japanese Industrial Standard JIS K 6503: 2001 “Niwa and Gelatin” as a standard measurement method. According to the standard measurement method, gelatin is dissolved in water and adjusted to a concentration of 6.67 w / v%. The jelly surface prepared by cooling at 10 ° C. for 17 hours is assumed to mean the load required to push it down by a half inch (12.7 mm) diameter plunger by 4 mm. In the specification, it has the same meaning and can be measured in the same manner.

  In the present invention, the caffeoylquinic acid compound and the protein are mixed in an aqueous medium to form an insoluble complex in the aqueous medium. As a result, the caffeoylquinic acid compound is incorporated into the insoluble matter precipitated in the aqueous medium, while other impurities remain in the solution. Thus, the caffeoylquinic acid compound is easily isolated. Can be purified. And after formation of the said composite_body | complex, the composite_body | complex can be collect | recovered by a suitable solid-liquid separation means suitably. Examples of the solid-liquid separation means include a centrifugal separator and filter filtration. In addition to or instead of this, moisture may be removed by appropriate drying means, or the aqueous medium and impurities may be removed. Examples of the drying means include spray drying, vacuum drying, freeze drying and the like.

  The aqueous medium is not particularly limited as long as it can provide a place for forming a complex, but is preferably composed mainly of water, and more specifically, the complex is formed. It is preferable that 50 mass% or more of water is contained in the whole system in that case, and it is more preferable that 80 mass% or more is contained. However, other components may be optionally included as long as they are components that do not substantially adversely affect the action and effect of the present invention, such as simply isolating and purifying the caffeoylquinic acid compounds.

  The mode of mixing in the aqueous medium is not particularly limited as long as it facilitates contact between the caffeoylquinic acid compound and the protein. Typically, for example, The caffeoylquinic acid compound (in the case of plural kinds, in terms of the total mass) is preferably contained in an amount of 0.1 to 30% by mass, and more preferably 1 to 20% by mass. Moreover, it is preferable that 0.1-30 mass% is contained, and it is more preferable that 1-20 mass% is contained for the said protein (in the case of multiple types, it converted into the total mass). Further, the mass ratio of the caffeoylquinic acid compound (in the case of plural types converted to the total mass) and the protein (in the case of plural types converted to the total mass) is 10: 1 to 1. : 10 is preferable, and 5: 1 to 1: 5 is more preferable. In particular, when gelatin, more preferably pork-derived gelatin, is used as the protein, it is preferable that 1 to 20% by mass of the gelatin is contained in the entire system during mixing, and 2 to 10% by mass is preferably contained. More preferably. Moreover, it is preferable that mass ratio of the said caffeoylquinic acid compounds (in the case of a plurality of types converted into the total mass) and the gelatin is 7: 1 to 1: 7, and 4: 1 to 1: 4 is more preferable. If the abundance of the caffeoylquinic acid compound or the protein is too low, the complex success rate and the insoluble precipitation efficiency may decrease. If the abundance of the caffeoylquinic acid compound or the protein is too high, contact with the partner compound may not be facilitated due to aggregation of the high abundance compound itself in the aqueous medium. . And it is preferable to fully form a composite by holding at 10 to 30 ° C. for 0.5 to 72 hours. At the time of mixing, the solution forming the system may be appropriately stirred by an appropriate stirring means as desired.

  In addition, when mixing in the said aqueous medium, control of pH is not especially required, but if it is advantageous to form a complex, it may be controlled.

  The complex obtained as described above is the above-mentioned caffeoyl oil formed of at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid and a protein. It is a complex containing a quinic acid compound. In this case, as described above, the composite may be appropriately recovered from the aqueous medium by an appropriate solid-liquid separation means or may be appropriately dried by an appropriate drying means as described above. Although there is no restriction | limiting in particular in a form, Typically, the said caffeoylquinic acid compounds (in the case of multiple types, it is the total mass conversion) that 1-90 mass% is contained in solid content, for example. Preferably, 10 to 50% by mass is contained. Moreover, it is preferable that 10-99 mass% is contained in solid content, and it is more preferable that 50-90 mass% is contained in the said protein (in the case of multiple types converted into the total mass). Further, the mass ratio of the caffeoylquinic acid compound (in the case of plural types converted to the total mass) and the protein (in the case of plural types converted to the total mass) is 10: 1 to 1. : 10 is preferable, and 7: 1 to 1: 7 is more preferable. In particular, when gelatin, more preferably pork-derived gelatin, is used as the protein, the gelatin content is preferably 5 to 70% by mass, more preferably 10 to 60% by mass. . Moreover, it is preferable that mass ratio of the said caffeoylquinic acid compound (in the case of a plurality of types converted into the total mass) and the gelatin is 10: 1 to 1:10, and 6: 1 to 6: 1 is more preferable.

  In a preferred embodiment of the present invention, the composite obtained as described above may be treated with a water-containing organic solvent. As a result, the caffeoylquinic acid compound elutes in the solvent, while the protein forming the complex hardly elutes or is partly eluted. The degree of purification of can be increased. As the water-containing organic solvent to be used, a solvent comprising ethanol, methanol, isopropanol, acetone or the like may be appropriately selected and used. However, in view of elution efficiency, it is particularly preferable to use water-containing ethanol or water-containing acetone. Moreover, as the moisture content, it is preferable that it is 5-70 v / v%, and it is more preferable that it is 20-50 v / v%. A water-containing organic solvent may be used in the form of a water-containing solvent by using a plurality of types of organic solvents together in the elution process, and performing a step-by-step elution process with a plurality of types of water-containing organic solvents, The obtained eluate may be combined later.

  The mode of elution with the water-containing organic solvent is not particularly limited as long as the contact between the complex and the water-containing organic solvent is promoted. Typically, for example, 1 part by mass of the complex is used. On the other hand, it is preferable to elute the said water-containing organic solvent using 5-70 mass parts, and it is more preferable to elute using 10-50 mass parts. Moreover, it is preferable that the elution from the complex is sufficiently carried out, for example, by holding at 20 to 50 ° C. for 0.5 to 24 hours. If desired, the solution constituting the elution system may be stirred by an appropriate stirring means. In addition, the elution process may be performed while performing ultrasonic treatment. According to this, elution efficiency can be improved more.

  The liquid part of the eluate obtained from the water-containing organic solvent obtained as described above can be recovered by appropriate solid-liquid separation means. Thereby, the protein which does not elute to a liquid part can be removed. Examples of the solid-liquid separation means include centrifugation, centrifugal filtration, and filter filtration. In addition to or instead of this, moisture may be removed by appropriate drying means, or the aqueous medium and impurities may be removed. Examples of the drying means include spray drying, vacuum drying, freeze drying and the like.

In a preferred embodiment of the present invention, the complex obtained as described above may be treated with a proteolytic enzyme. Thus, after the treatment, the protein that has formed the complex is almost decomposed or at least partly decomposed, and thus the complex is decomposed. Therefore, the caffeoylquinic acid compound is again added to the solution. Can be eluted. The enzyme to be used may be appropriately selected from proteinase or peptidase. However, in view of the degradation efficiency of the protein used in the present invention, aspartic protease (for example, maitake protease) or cysteine protease (for example, papain). Etc. are preferably used.
The conditions for the treatment with the proteolytic enzyme are appropriately determined according to the conditions optimally known for the enzyme used.

  The enzyme-treated product obtained as described above can be appropriately removed by an appropriate separation means to remove the enzyme degradation product, thereby recovering the caffeoylquinic acid compound. For example, since the enzymatic degradation products are amino acids, a preferable method for separating them is a method using an ion (cation) exchange resin. By passing through a column packed with ion (cation) exchange resin, amino acids in the treated product are adsorbed and removed. If the eluate is concentrated under reduced pressure, the above-mentioned caffeoylquinic acid compounds with a higher degree of purification can be obtained. It is done.

  The caffeoylquinic acid compound-containing complex and the caffeoylquinic acid compound provided by the present invention are useful as useful materials applicable to various fields such as foods, pharmaceuticals, cosmetics, animal feeds and the like. is there. In particular, for example, as described above, caffeoylquinic acid compounds are known to have various functionalities, and thus are useful as supplements having such functionalities. Furthermore, it is useful as a food additive or a hair-growth cosmetic product (see Japanese Patent Application Laid-Open No. 2015-036370).

  As described above, according to the present invention, at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid contained in the starting material is used as an aqueous medium. In this technology, a complex with a specific protein is formed to make it insoluble in the aqueous medium, so that it can be isolated and purified very easily from other contaminant components contained in the starting material. It is possible to provide a technique for producing useful materials at a lower cost on an industrial scale. In the present specification, “isolated and purified” means that at least a part of other contaminant components other than the caffeoylquinic acid compounds contained in the starting material is removed. . More specifically, among other contaminating components other than the caffeoylquinic acid compounds contained in the starting material, the content concentration (% by mass) per solid content contained in the starting material is calculated, It means that the content concentration is reduced by an amount of 50% by mass or more, more typically it means that it is reduced by an amount of 50-98% by mass, and even more typical. Means that the amount is reduced by 80 to 98% by mass.

  Hereinafter, the present invention will be specifically described with reference to test examples, but the present invention is not limited to the forms of these test examples.

  First, Solution A to Solution C shown below were prepared.

(Solution A) Aqueous solution containing dicaffeoylquinic acid A commercially available sweet potato stem-and-leaf extract (trade name “Polyphemin”, produced by Gosei Kosan Co., Ltd.) was used. This sweet potato stalk-and-leaves extract is a liquid composition containing about 2% by weight of a caffeoylquinic acid compound, and dicaffeoylquinic acid is the main component of the caffeoylquinic acid compound contained, Contains chlorogenic acid (5-caffeoylquinic acid) and tricaffeoylquinic acid (hereinafter simply referred to as “sweet potato stem-leaf extract”).
Column purification was performed in order to increase the purity of dicaffeoylquinic acid, which is the main component of the sweet potato stem-and-leaf extract. Specifically, the extract derived from the sweet potato foliage was adsorbed on an adsorption column packed with a synthetic adsorbent (MCI gel CHP20P (manufactured by Mitsubishi Chemical Corporation)) as an adsorption resin. Next, the non-adsorbed contaminants that were not adsorbed on the adsorbent resin were removed by washing the adsorbent column with about 3 times the capacity of the adsorbent resin used. Furthermore, impurities containing chlorogenic acid as a main component were removed by elution with water-containing ethanol (containing 20 v / v% ethanol) twice the volume of the adsorption resin used. Finally, the elution fraction of dicaffeoylquinic acid was collected by elution with water-containing ethanol (containing 40 v / v% ethanol) twice the volume of the adsorption resin used.

  The obtained elution fraction of dicaffeoylquinic acid was concentrated with an evaporator and dried to obtain a solid containing dicaffeoylquinic acid. As a yield, when 1 kg of the extract derived from sweet potato foliage was used, the obtained solid was 14 g. As a result of HPLC analysis of the content in the solid using the sample, this solid was found to have three types of dicaffeoylquinic acid isomers, namely 3,4-di-O-caffeoylquinic acid, 3,5. It was a mixture of -di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid, and the total of the three types was 90% by mass or more. Chlorogenic acid and tricaffeoylquinic acid were hardly detected. This solid was dissolved in degassed distilled water to obtain a 5% by mass aqueous solution of dicaffeoylquinic acid.

(Solution B) Aqueous solution containing tricaffeoylquinic acid The amount of tricaffeoylquinic acid contained in the sweet potato stem-and-leaf extract is extremely small. Therefore, tricaffeoylquinic acid was synthesized by the method described in Table 2014/168139 to prepare a 5% by mass aqueous solution thereof.

(Solution C) Polysaccharide-Removed Extract Solution The sweet potato stem-and-leaf extract is a liquid composition containing about 2% by mass of a caffeoylquinic acid compound as described above. The composition contains various impurities, and particularly contains a lot of water-soluble polysaccharides. Then, the process for reducing content of a polysaccharide was performed. Specifically, 200 mL of ethanol was added to 100 g of the extract derived from the sweet potato foliage and stirred for about 1 hour. Thereafter, the mixture was allowed to stand overnight at room temperature, and the precipitated water-insoluble polysaccharide was suction filtered. When filtering, commercially available cellulose powder was used as a filter aid on the filter paper. The filtrate was concentrated under reduced pressure to obtain about 9 g of a solid. By dissolving it in water to obtain a 25 g solution, the concentration of dicaffeoylquinic acid could be adjusted to about 5% by mass.

(Test Example 1) Formation test of complex with dicaffeoylquinic acid by various water-soluble polymer compounds Precipitate by forming complex in water with polyphenol such as tannin (mainly polyphenol having pyrogallol group in the molecule) Fourteen water-soluble polymer compounds known to be selected were selected and dissolved in distilled water to prepare a 5% by mass aqueous solution.

  Table 1 shows the origin of the 14 types of water-soluble polymer compounds used. The polymer compounds used in Samples 3, 4, and 7 were prepared based on the referenced literature and then freeze-dried and used as a powder. All the polymer compounds used for the samples other than those were obtained as powders. Depending on the polymer compound used, some turbidity was sometimes generated in the 5 mass% aqueous solution. In this case, the turbid component was precipitated using a centrifuge, and the supernatant was used.

  10 mL of these 5 mass% aqueous solutions and 2 mL of solution A (aqueous solution containing 5 mass% dicaffeoylquinic acid) were mixed, and the presence or absence of precipitation was observed. In either case, the pH of the mixed solution was not adjusted.

  As a result, as shown in Table 1, the solution A containing dicaffeoylquinic acid was less likely to form a complex than tannin, and the change beyond turbidity was caused by the aqueous solution derived from animal protein. Samples 1 to 6 using gelatin, water-soluble collagen, water-soluble elastin, collagen peptide (all derived from pigs), water-soluble keratin (derived from feathers) and hydrolyzed keratin (derived from wool). Among them, only Sample 1 using gelatin formed a complex and clearly caused precipitation.

(Test Example 2) Formation test of complex with dicaffeoylquinic acid by gelatin derived from various raw materials In Test Example 1, it was found that gelatin easily forms a complex with dicaffeoylquinic acid. We tested how complex formation differs with different gelatin types. The gelatin used is shown in Table 2. In the same manner as in Test Example 1, 10 mL of gelatin aqueous solution prepared to 5% by mass and 2 mL of solution A (aqueous solution containing 5% by mass of dicaffeoylquinic acid) were mixed and observed.

  As a result, as shown in Table 2, the raw material is also important for gelatin. In sample 15 using pork (pig skin) raw material, precipitation is generated, and in sample 16 using fish raw material gelatin, two layers are separated (the upper layer is The complex) showed clear complex formation, but the sample 17 of bovine (cow bone) raw material gelatin was not separated into two layers, and complex formation could not be confirmed.

(Test Example 3) Quantitative determination of residual dicaffeoylquinic acid and effect of gelatin jelly strength Samples 15 and 16 prepared in Test Example 2 were allowed to stand for 12 hours after the complex formation test. Liquid separation was performed, and HPLC analysis was performed on the obtained liquid part solution. And the residual rate (percentage with respect to the original mass) with respect to the mass of the dicaffeoylquinic acid contained in 2 mL of solution A before complex formation was calculated | required. Further, in the case of using gelatin having different jelly strength, the residual rate was similarly determined after the complex was formed under the same conditions as in Test Example 2.

  As a result, as shown in Table 3, the dicaffeoylquinic acid remaining in the liquid part solution obtained by solid-liquid separation after the formation of the complex was 4.6% in the case of Sample 15. In the case of Sample 16, the residual rate was 36%. This was well correlated with the fact that pork raw material gelatin tends to form a complex with dicaffeoylquinic acid as compared with fish raw material gelatin as shown in Test Example 2.

  Further, from the results shown in Table 3, it has been clarified that, in gelatin derived from the same base, a higher jelly strength is more advantageous for complex formation. The effect of jelly strength was greater with fish gelatin. Furthermore, even when the jelly strength was comparable, it was found that pork raw material gelatin is more advantageous for complex formation with dicaffeoylquinic acid than fish raw material gelatin (comparison of sample 16 to sample 18). And comparison of sample 20 to sample 19).

  FIG. 1 shows a measurement chart when the solution A is subjected to HPLC, and a measurement chart when the liquid part solution obtained by solid-liquid separation after complex formation in the sample 15 is subjected to HPLC.

(Test Example 4) Formation test of complex with tricaffeoylquinic acid by gelatin Pork raw material gelatin used in Sample 19 of Test Example 3 (jelly strength: 235-265 g, trade name “APH-250” Nitta Gelatin Stock 10 mL of a 5% by mass aqueous solution thereof and 2 mL of Solution B (an aqueous solution containing 5% by mass of tricaffeoylquinic acid) were mixed and the presence or absence of precipitation was observed.

  As a result, in the same manner as observed for dicaffeoylquinic acid in Sample 1 in Test Example 1 and in Sample 15 in Test Example 2, tricaffeoylquinic acid was also mixed with pork raw material gelatin, A clear precipitate was produced. This was subjected to solid-liquid separation by centrifugation, and the obtained liquid part solution was quantified by HPLC analysis. As a result, the residual ratio (original mass) with respect to the mass of tricaffeoylquinic acid contained in 2 mL of the solution B before complex formation. %) Was 2.0%. Therefore, it was clarified that tricaffeoquinic acid easily forms a complex by mixing with gelatin, like dicaffeoylquinic acid.

(Test Example 5) Formation test of complex using extract derived from sweet potato foliage Pork raw material gelatin used in Sample 19 of Test Example 3 (jelly strength: 235-265 g, trade name “APH-250” manufactured by Nitta Gelatin Co., Ltd.) ), 10 mL of a 5% by mass aqueous solution thereof, and Solution C (an aqueous solution in which the concentration of dicaffeoylquinic acid is adjusted to about 5% by mass after removing the polysaccharide from the sweet potato stem-and-leaf extract) When 2 mL was mixed, a precipitate was formed. This was subjected to solid-liquid separation by centrifugation, and the obtained liquid part solution was quantified by HPLC analysis. As a result, the residual ratio (original mass) with respect to the mass of dicaffeoylquinic acid contained in 2 mL of the solution C before complex formation. %) Was 38%. Therefore, it was considered that the composition in which the polysaccharide was simply removed from the sweet potato stem and leaf extract, that is, such an ancestor extract, may contain a component that inhibits complex formation.

  Therefore, a test in which a 5% by mass aqueous solution of the above pork raw material gelatin (jelly strength: 235 to 265 g, trade name “APH-250” manufactured by Nitta Gelatin Co., Ltd.) is increased to 20 mL and added in 10 mL portions (1 hour) As a result, the residual ratio of dicaffeoylquinic acid was reduced to 5%. Therefore, it was revealed that 95% of dicaffeoylquinic acid can be incorporated as a gelatin complex from a composition in which polysaccharides have been removed from an extract derived from sweet potato foliage, that is, from such an ancestral extract. On the other hand, the solution C contains a small amount of chlorogenic acid (5-caffeoylquinic acid). According to the result of subjecting the liquid part solution after the solid-liquid separation to HPLC analysis, it was not taken into the complex at all. It remained in the liquid part solution.

  From the above results, it was shown that such a complex formation method can be a simple isolation and purification method for dicaffeoylquinic acid and tricaffeoylquinic acid.

  In the case of an extract derived from sweet potato stalks and leaves that does not remove polysaccharides (no alcohol treatment), a complex formation test was performed under the same conditions. As a result, the residual ratio of dicaffeoylquinic acid was 20%. Therefore, although the amount of incorporation into the gelatin complex has been slightly reduced, dicaffeoylquinic acid and tricaffeoylquinic acid can be easily and simply used even in crude extracts containing more diverse components such as polysaccharides. It was shown that this could be a method of separation and purification.

Test Example 6 Formation Test of Complex with Chlorogenic Acid Using Gelatin Chlorogenic acid (5-caffeoylquinic acid) was obtained as a reagent to prepare a 5% by mass aqueous solution of chlorogenic acid. 2 mL of the mixture and 10 mL of a 5% by mass aqueous solution of the above-described pork raw material gelatin prepared in Test Example 4 and Test Example 5 (jelly strength: 235 to 265 g, trade name “APH-250” manufactured by Nitta Gelatin Co., Ltd.) After mixing and allowing to stand for 24 hours, no precipitation occurred. Even after being left for 1 week, even turbidity did not occur. This suggests that chlorogenic acid does not form a complex with gelatin.

(Test Example 7) Preparation of dicaffeoylquinic acid / gelatin complex The pork raw material gelatin prepared in Test Examples 4 to 6 (jelly strength: 235 to 265 g, trade name “APH-250” manufactured by Nitta Gelatin Co., Ltd.) A precipitate obtained by mixing 400 g of a 5% by weight aqueous solution (gelatin content 20 g) and 80 g of a solution A (aqueous solution containing 5% by weight dicaffeoylquinic acid) (4 g dicaffeoylquinic acid content) Centrifuged. The obtained precipitate was washed with water and filtered and then freeze-dried to obtain 23.2 g of a solid. The amount of dicaffeoylquinic acid contained in the solid was estimated to be 3.6 g from HPLC analysis of the liquid part solution separated into the solid and liquid.

(Test Example 8) Recovery of dicaffeoylquinic acid using a water-containing organic solvent from the composite 5 g of the composite obtained in Test Example 7 was added to 25 mL of water-containing ethanol (containing 50 v / v% ethanol) or water-containing acetone. Each was added to 25 mL (containing 50 v / v% acetone), irradiated with ultrasonic waves for about 30 minutes with an ultrasonic cleaner, and then allowed to stand at room temperature for 24 hours. Then, the solution was poured into 50 mL of ethanol cooled to 5 ° C., stirred for about 30 minutes, and centrifuged to remove the precipitate. The solid obtained by concentrating and drying the solid was subjected to HPLC analysis to quantify the amount of dicaffeoylquinic acid. As a result, 0.65 g and 0.70 g of dicaffeoylquinic acid having a purity of 90% by mass or more were obtained. The recoveries were 84% and 90%, respectively, as a percentage of the amount of dicaffeoylquinic acid contained in 5 g of the complex used, and the use of water-containing acetone was slightly more than the use of water-containing ethanol. The recovery rate was excellent.

  From the above results, it has been clarified that high-purity dicaffeoylquinic acid can be prepared through a step of treating the complex with gelatin with a water-containing organic solvent.

(Test Example 9) Regeneration of dicaffeoylquinic acid by enzymatic decomposition of the complex 50 mL of water was added to 5 g of the complex obtained in Test Example 7, and 2 mg of proteinase K (manufactured by Wako Pure Chemical Industries) was added thereto. It was left in a thermostatic container at 0 ° C. for about 2 days. The obtained solution was subjected to HPLC analysis to quantify the amount of dicaffeoylquinic acid. As a percentage of the amount of dicaffeoylquinic acid contained in 5 g of the complex used, 98% dicaffeoylquinic acid was obtained. Was eluted in the solution.

  From the above results, it has been clarified that dicaffeoylquinic acid with high purity can be prepared through a step of treating the complex with gelatin with a proteolytic enzyme.

Claims (8)

  At least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid and an animal protein are mixed in an aqueous medium, and the caffeoylquinic acid compound and the above compound are mixed. A method for producing a complex containing a caffeoylquinic acid compound, comprising a step of forming an insoluble complex in an aqueous medium with an animal protein.   The production method according to claim 1, wherein the caffeoylquinic acid compound is derived from sweet potato.   The production method according to claim 1 or 2, wherein the animal protein is a water-soluble protein.   4. The animal protein according to claim 1, wherein the animal protein is one or more selected from the group consisting of gelatin, water-soluble collagen, water-soluble elastin, water-soluble keratin, and collagen peptide. Manufacturing method.   The manufacturing method according to any one of claims 1 to 4, wherein the animal protein is one or more selected from the group consisting of porcine-derived gelatin and fish-derived gelatin.   A method for producing a caffeoylquinic acid compound, comprising a step of treating the complex containing a caffeoylquinic acid compound obtained by the production method according to claim 1 with a water-containing organic solvent.   A method for producing a caffeoylquinic acid compound, comprising a step of treating the complex containing a caffeoylquinic acid compound obtained by the production method according to claim 1 with a proteolytic enzyme.   A caffeoylquinic acid compound-containing complex formed of at least one caffeoylquinic acid compound selected from the group consisting of dicaffeoylquinic acid and tricaffeoylquinic acid and gelatin.