JP6683450B2 - Extraction method of condensed tannin with high degree of polymerization - Google Patents

Description

  The present invention relates to a method for extracting a condensed tannin having a high degree of polymerization, a method for producing a condensed tannin having a high degree of polymerization using the extraction method, and a method for producing a composite agent containing the condensed tannin having a high degree of polymerization. .

Tannin is a natural phenolic compound that is abundantly contained in specific parts of various limited plants, and has astringent action, which is the property of aggregating biopolymers such as proteins, so it has been used for a long time in skin tanning. It has been used as a tanning agent and an antidiarrheal agent.
It is also used as a mordant, a clarifying agent for sake and the like, an antiseptic, a component of ink, etc. Also, it is contained in various edible plants (treated products) such as tea-treated products and herbs. It has been used as a food, a luxury item, etc.

Tannin includes a hydrolyzable tannin in which polyhydric phenolic acid such as gallic acid is ester-bonded to a polyhydric alcohol such as sugar having a plurality of hydroxyl groups, and a condensed tannin in which a plurality of flavonoids are bonded by a carbon-carbon single bond. Exists.
Of these, condensed tannin is sometimes called proanthocyanidin and is a substance represented by the formula (1) described later.
Condensed tannin has, in addition to the above-mentioned actions and properties, actions and properties such as anti-oxidant properties that suppress aging and cancer, mental stability, adhesion (improvement), and adsorptivity to adsorb substances, and It is useful because it has various excellent actions and properties that are not present in phenolic resin, which is an artificial product (synthetic product) having similar chemical structure and physical properties.

In the condensed tannin, the various actions and properties described above depend on the degree of polymerization of the condensed tannin, and there are many actions and properties which are said to be superior to the condensed tannin having a high degree of polymerization.
For example, Non-Patent Document 1 describes that “condensed tannin with a high degree of polymerization” is superior in protein adsorption ability and protein insolubilization ability.

  However, the extraction of condensed tannins with a high degree of polymerization may not be performed in good yield or may be decomposed during the extraction, as compared with the extraction of tannin which is a monomer or condensed tannins with a low degree of polymerization. , Was extremely difficult to extract.

In general, the extraction method includes, for example, a steam distillation method using steam at 100 ° C .; a hydrothermal extraction method using pressurized hot water; a direct immersion in water or an organic solvent under heating and / or pressure for extraction. Direct extraction method: Extraction method by adding and compressing oily component; Supercritical extraction method using supercritical fluid such as carbon dioxide; Direct extraction by depressurizing at low temperature without using extraction medium Low temperature decompression extraction method; subcritical water extraction method in which temperature and pressure are adjusted to specific ranges to extract with water; and the like are known.
For example, Patent Document 1 describes a method for extracting condensed tannin from a natural resource using a lower alcohol or a “mixed solvent of lower alcohol and water”.
Further, Patent Document 2 describes a method of extracting tannin from a wood-based molding material using hot water at 110 ° C. pressurized in an autoclave.

However, in the above-mentioned method, condensed tannins cannot be extracted in good yield, and they cannot be completely removed when the remaining extraction medium such as an organic solvent used for extraction is an obstacle. However, there are various problems such as decomposition or denaturation, loss of the feature of being all natural products by using an extraction medium, easy corrosion of the extraction cell (container), and excessive cost.
Further, even if the whole condensed tannin can be extracted in good yield, the extraction efficiency of the condensed tannin having a large degree of polymerization is low, that is, the proportion of the condensed tannin having a large degree of polymerization in the whole extracted condensed tannin is large. It was low.

On the other hand, “superheated steam”, which is steam having a temperature higher than 100 ° C., is used for treating wastes, decomposing / extracting unnecessary inclusions, and improving physical properties of useful substances.
For example, Patent Document 3 and Non-Patent Document 2 describe a steam generator having a temperature higher than 100 ° C., and Patent Document 4 produces a high specific surface area activated carbon by treating a porous carbon material with superheated steam. How to do is described. Further, Patent Document 5 describes a method of treating a lignocellulosic material with superheated steam to decompose and remove hemicellulose.

  A superheated steam extraction method of extracting with steam higher than 100 ° C., that is, using steam at a temperature higher than 100 ° C. (referred to as “T ° C.”) at normal pressure, and hence “saturated vapor pressure of water at T ° C.” It is not known that the method of extracting with (a mixed gas of) steam at a low pressure is effective for the extraction of tannin, and it is particularly effective in the circumstances and the point that "condensation tannin with a high degree of polymerization" can be efficiently extracted. Not known to be.

  In recent years, high value-added natural products such as tannin have attracted more and more attention, and their application fields are expanding. In other words, not only is there a demand for physical properties superior to artificial products (synthetic products), but there is an increasing demand for unprecedented actions and properties, including natural products and synthetic products, but this is not possible with known technology. Was enough.

  In particular, regarding condensed tannins with a high degree of polymerization, even though they are expected to have unprecedented actions and properties, they cannot be extracted and confirmed (identified), so their content (presence) in the plant is clear. However, there is nothing known about it, and nothing was known about its efficient extraction method.

JP 2002-302497 A JP, 2009-154437, A JP, 2014-152953, A JP, 2009-292670, A JP, 2013-176922, A

Mookiah Saminathan, et al, Molecules 2014, 19, 7990-8010. Dai-ichi Kogyo Kogyo Co., Ltd., website as of September 10, 2015, http://www.dhf.co.jp/

  The present invention has been made in view of the above background art, and an object thereof is to provide a method for extracting a condensed tannin having a large degree of polymerization, with good yield while suppressing decomposition during extraction. It is intended to provide a method for producing a “condensed tannin having a high degree of polymerization” and a method for producing a “composite agent containing a condensed tannin having a high degree of polymerization”.

  The present inventor has conducted extensive studies to solve the above-mentioned problems, and by extracting with superheated steam, "extraction method using an organic solvent that requires a post-process", "pressurized extraction" The present invention is completed by finding that condensed tannins having a large degree of polymerization can be extracted in a high yield while suppressing decomposition, modification, polymerization, etc., as compared with other extraction methods described above such as Came to.

  That is, the present invention provides a method for extracting a condensed tannin having a high degree of polymerization, which comprises extracting using superheated steam from a part of a plant containing the condensed tannin. is there.

  The present invention also provides a method for producing a condensed tannin having a high degree of polymerization, which is characterized by using the above extraction method.

  Further, the present invention is a method for producing a composite agent containing a condensed tannin having a high degree of polymerization, which uses the above-mentioned “method for producing a condensed tannin having a high degree of polymerization”, wherein the composite agent is an antioxidant. A method for producing a composite agent, which is a composite agent selected from the group consisting of agents, tranquilizers, adhesives and adsorbents.

According to the present invention, the above-mentioned problems and the above-mentioned problems can be solved, and condensed tannins having a high degree of polymerization can be efficiently extracted from a plant. Condensed tannin with a degree of 7 or more) can be extracted extremely efficiently.
The extraction method of the present invention has a high extraction efficiency of condensed tannins having a high degree of polymerization as compared with other extraction methods, in other words, the condensed tannins having a high degree of polymerization occupy the entire extracted condensed tannins. The percentage is large. For example, condensed tannins having a high degree of polymerization (particularly condensed tannins of 7-mer or more) can be satisfactorily extracted as compared with a relatively good subcritical water extraction method.

In addition, a 9-mer condensed tannin could be confirmed (identified) for the first time. The "confirmed 9-mer condensed tannin" (n = 9 compound in the formula (1) described below) is new as the confirmed / identified "product", but was extracted for the first time by the present invention. , The structure (degree of polymerization) was confirmed.
The one immediately after extraction is a mixture of condensed tannins having different degrees of polymerization, but the condensed tannin having a number average molecular weight of more than 1250, specifically, a number average molecular weight of 1680 was obtained for the first time by the extraction method of the present invention. Such condensed tannin can exhibit various excellent actions and properties described above and below.

Further, according to the present invention, in addition to being able to extract a condensed tannin with a high degree of polymerization in good yield, since superheated steam is used for extraction, a post-treatment after extraction is performed as compared with the extraction method using an organic solvent. Is easy, and the remaining extraction medium such as an organic solvent is less likely to cause an obstacle.
For example, when an organic solvent is used for extraction, it may not be possible to completely remove the organic solvent, or the obtained extract may lose the characteristic that "all are natural products and water". However, according to the present invention, such a problem does not occur. That is, according to the present invention, "no organic solvent" or "no use other than natural products" can be achieved.
However, in the present invention, it is essential to use superheated steam, but, for example, when purification after extraction is necessary, when a post-process is performed, etc., the use of an organic solvent in the process after extraction is excluded. Not something to do.

According to the extraction method of the present invention, it is possible not to use an organic solvent, so that it is possible to suppress environmental load.
Further, it is considered that the condensed tannin having a high degree of polymerization is easily extracted, and thus the condensed tannin having a high degree of polymerization is likely to be dissolved and extracted. It is conceivable that "unnecessary polymerization of" condensed tannin having a high degree of polymerization (for example, a degree of polymerization of 7 or more) "is difficult to decompose.
Also, the extraction does not cost too much.
Furthermore, if the “part of the plant containing condensed tannins” to be extracted is sought in the waste, a useful product called condensed tannin with a high degree of polymerization can be obtained and the waste can be treated. .

  The complexing agent containing the “condensation tannin with a high degree of polymerization” produced by using the extraction method of the present invention has a low degree of polymerization with a condensation tannin or a monomeric tannin, and compared with a synthesized phenol resin. The tannins can be used in a wider range of applications because of their superior action and properties. For example, skin tanning agents, protein astringents, antidiarrheals, mordants, fining agents, preservatives, ink ingredients, seasonings, health foods, luxury items, antioxidants, tranquilizers, adhesive (improvement) agents. , It can be preferably used for applications such as adsorbents.

It is a "conceptual diagram including an apparatus" showing an example of the extraction method of the present invention. 2 is a MALDI mass spectrometric spectrum of condensed tannins obtained in Example 1 of the present invention and Comparative Example 1. (A) The extraction method of the present invention using the superheated steam of Example 1. (B) Subcritical water extraction method using the subcritical water of Comparative Example 1. 4 is a MALDI mass spectrometric spectrum of condensed tannins obtained in Comparative Example 2 and Comparative Example 3. (A) Organic solvent extraction method using the organic solvent of Comparative Example 2. (B) Hydrothermal extraction method using pressurized hot water of Comparative Example 3. 3 is a MALDI mass spectrometric spectrum of condensed tannin extracted by changing the temperature of superheated steam in Example 2. (A) 100 ° C (b) 120 ° C (c) 150 ° C (d) 180 ° C

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific forms and can be arbitrarily modified within the scope of the technical idea.

The present invention is a method for extracting condensed tannin having a high degree of polymerization, which is characterized in that extraction is performed using superheated steam from a plant part containing the condensed tannin.
Here, the meaning of "extraction method of condensation type tannin having a high degree of polymerization" is, as compared with other extraction methods, the average or maximum degree of polymerization of condensed tannin in the extract is a method of extracting a higher tannin. Is the meaning.

<Chemical structure of condensed tannin>
As the condensed tannin contained in plants, a compound represented by the following formula (1) has been confirmed. Therefore, specific examples of the "condensed tannin" in the present invention include those represented by the formula (1).

[In the formula (1), R ¹ and R ² each represent a hydrogen atom (-H) or a hydroxyl group (-OH) which may be different from each other, and R ³ represents a hydrogen atom (-H) or a hydroxyl group (-H). OH) or shows a gallic acid residue _{(-OC (= O) C 6} H 2 (OH) 3), R 1, R 2 and ^{R 3} may be the same or different every n repeating units , N is an integer of 2 or more. ]

The “gallic acid residue” in the formula (1) is a group represented by the following formula (2).

<< Condensed tannin with high degree of polymerization >>
The “condensed tannin having a high degree of polymerization” is preferably a condensed tannin having a degree of polymerization of 4 or more (4 ≦ n in the formula (1)), and a condensed tannin having a degree of polymerization of 6 or more. Tannin is more preferable, and condensed tannin having a polymerization degree of 8 or more (8 ≦ n in the formula (1)) is particularly preferable. The specificity of the extraction method of the present invention is exhibited when the degree of polymerization is not less than the above. Condensed tannins having different degrees of polymerization may be mixed in the extract.

  In other words, the present invention is preferably a “condensed tannin extraction method” containing a condensed tannin with a degree of polymerization of 4 or more, and more preferably an extracted method containing a condensed tannin with a degree of polymerization of 6 or more. It is particularly preferable that the extraction method includes a condensed tannin having a degree of polymerization of 8 or more.

Further, according to the extraction method of the present invention, it is possible to extract the condensed tannin of 9-mer. The “9-mer condensed tannin” (the compound of n = 9 in the formula (1)) is novel as an isolated and confirmed (identified) product. As the extract by the extraction method of the present invention, a mixture of condensed tannins of all n (degree of polymerization) of 3 ≦ n ≦ 9 has been confirmed.
Therefore, an embodiment of the present invention is also the above-mentioned extraction method, wherein the condensed tannin to be extracted contains at least all condensed tannins having a degree of polymerization of 4 (more preferably 7) to 9.

The extract may be a mixture of condensed tannins having different degrees of polymerization. The present invention is preferably a method of extracting condensed tannins having a number average molecular weight of 1200 or more, more preferably a method of extracting condensed tannins of 1300 or more, and a method of extracting condensed tannins of 1500 or more. Particularly preferred is a method of extracting condensed tannin of 1600 or more, and further preferred.
By the extraction method of the present invention, a condensed tannin having a number average molecular weight of 1600 or more, specifically, a number average molecular weight of 1640 to 1680 was obtained as a "product" for the first time.

  The three-dimensional structure of the condensed tannin represented by formula (1) (each of the monomers forming the tannin) is not particularly limited as long as it is contained in the plant part.

In addition, since condensed tannins having a high degree of polymerization were extracted by the present invention, it was confirmed from structural analysis that all were extracted as R ¹ , R ² and R ³ (combination) in formula (1). Even if it does not exist, it is considered that “ease of extraction and difficulty” of “condensed tannin with high degree of polymerization” does not change regardless of R ¹ , R ² and R ³ (combination). Generally, condensed tannins with a high degree of polymerization are extracted. "
That, ^{R 1} is OH, ^{R 2} is H, so ^{R 3} is condensed tannins with a high degree of polymerization condensed tannin of OH is extracted, ^{R 1} is OH, ^{R 2} is H, ^{R 3} is not OH It is clear that condensed tannins having a high degree of polymerization can be extracted from the entire condensed tannins.
Moreover, since condensed tannins having a high degree of polymerization were actually extracted from acacia, even condensed condensed tannins having different structures (combination of R ¹ , R ² and R ³ ) extracted from plants other than acacia could be polymerized. It is clear that highly condensed tannins are extracted.
Therefore, from the examples described below, the present invention can be said to be a method for extracting condensed tannins having a high degree of polymerization among the condensed tannins represented by the formula (1).

<Superheated steam>
In the extraction method of the present invention, condensed tannins are extracted from a plant part containing condensed tannins using superheated steam.
Here, "superheated steam" means steam (mixed gas) having a temperature higher than 100 ° C (hereinafter, the temperature is referred to as "T ° C") and a pressure lower than "saturated vapor pressure of water at T ° C". That is.
It is preferable that the superheated steam at the time of extraction be at normal pressure (extract at normal pressure) because of the simplicity of the extraction device. Note that the atmospheric pressure (1 atm) is lower than the “saturated vapor pressure of water at T ° C., which is a temperature higher than 100 ° C.”.

  Superheated steam has features such as a large heat capacity, a low dielectric constant, a low enthalpy, a high constant pressure specific heat, and a high heat efficiency because it has radiative heat transfer and condensation heat transfer in addition to convective heat transfer. Although the action and principle of the extraction in the present invention are not limited, it is considered that the extraction method of the present invention can extract the condensed tannin having a high degree of polymerization because it utilizes the above characteristics.

  Superheated steam can be obtained by a conventional method, and in the present invention, the superheated steam obtained by a conventional method is also used. Superheated steam can be obtained by further applying heat to steam at 100 ° C. For the method and apparatus for generating superheated steam, for example, those described in Patent Document 3 and Non-Patent Document 2 are used. Further, for example, commercially available products such as "DHF Super-HI" manufactured by Dai-ichi Kogyo Kogyo Co., Ltd. are also suitably used.

  FIG. 1 shows an example of the extraction method of the present invention including an outline of a superheated steam generator. Although not limited, specifically, water is heated to 100 ° C. under normal pressure by a usual method, and the obtained saturated steam is further heated by a heating device. As a heating method, a method of using heat generated by burning liquid fuel, a method of using heat generated by burning gas fuel, a method of using heat generated by an electric heater, a method of induction heating, etc. Is mentioned.

Among them, the method of induction heating is preferable. In FIG. 1, a high-frequency current is passed from a high-frequency heating power source to a high-frequency heating device by a high-frequency cable, and steam of 100 ° C. is heated by the high-frequency heating device to become superheated steam.
As a method of induction heating, a conductive heating element composed of a metal tube is superheated by induction heating, and saturated steam (100 ° C.) is passed through the heating element without applying pressure. A method of generating superheated steam is preferably used.
As shown in FIG. 1, it is preferable to constantly monitor the temperatures of the heating element and the superheated steam, send a signal to the temperature monitoring box, and feed back to the high frequency heating power source.

<Plants to be extracted>
In the present invention, the "condensed tannin with a high degree of polymerization" is extracted by using superheated steam from "the part of the plant containing condensed tannin", but the plant used as the extraction target is not particularly limited. Is a plant belonging to a genus selected from the group consisting of Acacia, Scutellaria, Chestnut, Pine, Rheum, Grape and Nikkei, condensed tannin (condensed tannin with high degree of polymerization) It is preferable because it has a large amount. Also, when the plant is to be disposed of as waste (medium), it is preferable because it is easily obtained as waste.

  For example, plants belonging to the genus Acacia include various acacia, mimosa and the like, plants belonging to the genus Pine include various pine trees, radiata pine, etc., and plants belonging to the genus Chestnut include Shivaguri, Yamaguri And the like, as plants belonging to the genus Scutellaria, persimmons of various varieties, and the like, as plants belonging to the genus Rheum, such as rhubarb, as plants belonging to the genus Grape, various grapes and the like. Examples of plants belonging to the genus Nikkei include cinnamon (keihi) and the like.

  Although it is not limited, most preferably, it is a plant belonging to the genus Acacia, and specific examples thereof include Ginkgo acacia (hana acacia), Fusa acacia, Morisima acacia, Acacia mangium, Golden mimosa, and the like.

<Plant parts to be extracted>
In the present invention, it is extracted from a specific part of the plant, but the part of the plant used as the extraction target is not particularly limited, and is selected from the group consisting of bark, wood part, pericarp, fruit, seed, leaf and pod. The parts that have been removed are listed. Condensed tannins are often contained in these sites. The bark is particularly preferable because it contains a large amount of condensed tannin.
In addition, when obtaining the waste (medium) for the part of the plant to be extracted, after using the woody part inside the tree as wood, a large amount of bark is obtained as waste. Bark is particularly preferred.

<Condensed tannins with high degree of polymerization obtained from acacia plants>
A preferred embodiment of the present invention is a method for extracting a condensed tannin having a high degree of polymerization, which is characterized by extracting from a site (preferably bark) of a plant of the genus Acacia (preferably bark) using superheated steam. And the condensed tannin (or a mixture having a different polymerization degree) represented by the following formula (1) obtained by using the extraction method. Speaking of condensed tannin contained in a site (preferably bark) of a plant of the genus Acacia (preferably acacia), the chemical structure of its repeating unit is specified.

  That is, the present invention also provides an isolated condensed tannin, which is represented by the following formula (1) extracted from acacia bark with superheated steam.

[In the formula (1), R ¹ and R ² each represent a hydrogen atom (-H) or a hydroxyl group (-OH) which may be different from each other, and R ³ represents a hydrogen atom (-H) or a hydroxyl group (-H). OH) or shows a gallic acid residue _{(-OC (= O) C 6} H 2 (OH) 3), R 1, R 2 and ^{R 3} may be the same or different every n repeating units , N is an integer of 2 or more. ]
In particular, in the above formula (1), a condensed tannin in which n is a mixture of 7, 8 and 9 (in particular, a condensed tannin in which n = 9) has been newly found.

The isolated condensed tannin represented by the above formula (1) extracted from acacia bark with superheated steam has the following formula (1), wherein R ¹ is a hydroxyl group (—OH) and R ² is A condensed tannin that is a hydrogen atom (-H), R ³ is a hydroxyl group (-OH) as a main component, and n is a mixture of 7, 8 and 9 (particularly a condensed tannin of n = 9). Was newly found.

<Process of extraction method / extraction conditions>
The extraction method of the present invention is to extract condensed tannin with a high degree of polymerization by bringing superheated steam into contact with the extraction target, and specifically, for example, the following steps may be performed for extraction. Particularly preferred.
(1) A step of crushing the plant (preferably only the part of the plant containing condensed tannin) (2) A step of filling the extraction cell (extraction container) with the one obtained in the step (1) (3) Step (4) of passing superheated steam through the extraction cell (container), step (4) of collecting the mixture of the superheated steam obtained in step (3) and the extract, and (5) mixing the mixture recovered in step (4) into a liquid. Dissolving to obtain the whole solution (6) Purifying condensed tannin from the whole solution (7) Separating the condensed tannin into condensed tannins having different degrees of polymerization

  Among the above steps (1) to (7), steps (2), (3) and (4) are essential in the extraction method of the present invention.

  Although the step (1) is not essential, it is preferably performed when the plant is larger than the extraction cell or in order to enhance the extraction efficiency. The crushing can be performed by a conventional method. When using plants as waste, remove unnecessary substances and crush appropriately.

  The step (2) is a step of filling the plant (the part thereof) into the extraction cell (extraction container). The filling amount is determined depending on (the part of) the plant to be treated, the amount of condensed tannin to be obtained, and the like. The extraction cell (extraction container) is preferably suitable for extraction under normal pressure, and the size (volume) is determined by the filling amount of the plant, the amount of condensed tannin to be obtained, and the like.

Step (3) is a step of passing the superheated steam through the extraction cell (container), but it is essential that the temperature of the superheated steam is higher than 100 ° C. The temperature is preferably 105 ° C or higher and 500 ° C or lower, more preferably 110 ° C or higher and 300 ° C or lower, particularly preferably 115 ° C or higher and 250 ° C or lower, and further preferably 120 ° C or higher and 200 ° C or lower.
If the temperature of the superheated steam is too low, the extraction rate of the condensed tannin fraction may decrease, the extraction speed may decrease, or the condensed tannin with a high degree of polymerization may not be extracted, and if the temperature of the superheated steam is too high, The condensed tannin may be thermally decomposed or polymerized.

The flow rate of the superheated steam is not particularly limited, but is preferably 1 to 300 L / (min · g), more preferably 10 to 200 L / (min · g), and 50 to 160 L / (per 1 g of the extraction target (sample). Min.g) is particularly preferred.
When the flow rate is too low, the extraction speed becomes slow, and the extraction time must be lengthened to secure the yield, and the sample is at a high temperature during that time, so that condensation type tannin decomposes and polymerizes at the same time. There is.
On the other hand, if the flow rate is too high, the superheated steam may be wasted, the extract may be diluted, and it may take time to concentrate the extract.

  The extraction time depends on the mass of the extraction target (sample), the flow rate of superheated steam, and the like, and is not particularly limited, but is preferably 1 minute to 60 minutes, more preferably 2 minutes to 30 minutes, and preferably 5 minutes to 20 minutes. Particularly preferred. If the extraction time is too short, the condensed tannin may not be completely extracted and may remain.If the extraction time is too long, the condensed tannin will be completely extracted, which is a waste of time, and the extract will be diluted and extracted. It may take time to concentrate the product.

It is also preferable to arrange extraction regions having different temperature ranges of superheated steam in multiple stages and to extract the superheated steam in order from the lowest temperature.
Extraction of (low molecular weight) impurities and low molecular weight condensed tannins with relatively low temperature superheated steam, followed by extraction of high molecular weight (high degree of polymerization) condensed tannins with relatively high temperature superheated steam By doing so, it is possible to shorten the time of keeping the high temperature state and suppress the thermal decomposition of the condensation type tannin having a high degree of polymerization, and the condensation type tannin having a high degree of polymerization can be obtained by first extracting and removing impurities and low molecular weight components. There is an effect that it can be easily extracted.

  The temperature of the former stage in the multi-stage arrangement is not particularly limited, but is preferably higher than 100 ° C and 200 ° C or lower, and particularly preferably 105 ° C to 120 ° C. The temperature of the latter stage is preferably 110 ° C to 500 ° C, particularly preferably 120 ° C to 300 ° C.

Step (4) is a step of collecting the mixture of superheated steam and the extract. Since the superheated steam becomes liquid water at the time of recovery, the step (4) is a step of recovering the mixture of water and the extract.
The recovery method is not particularly limited, but a liquid such as water is added to the extraction cell containing the extract obtained in step (3), and the mixture is stirred or washed to obtain a (water) solution or dispersion. A method of collecting the liquid is preferable.
Since the extract may adhere to the cell wall surface of the extraction cell or the surface of the plant used for the extraction, the above method is preferable in that case. The water is preferably distilled water.

  Further, as shown in FIG. 1, it is preferable to add a mechanism for introducing a gas into a liquid to cool and trap the mixture. The liquid used for the trap is preferably water, a water / ethanol mixed liquid or the like, and water is particularly preferable.

The step (5) is a step of once dissolving the mixture recovered in the step (4) in a liquid to obtain the whole solution. The step (5) is not an essential step in the present invention, and the one to be obtained contains a compound such as hydrolyzable tannin, low polymerization degree or monomeric tannin, gallic acid (derivative), and other impurities. It can be omitted if it is acceptable. It can be omitted if the desired product is sufficiently filtered by insoluble matter.
The liquid which is once dissolved is a solvent in which condensed tannin is dissolved, and examples thereof include a water / ethanol mixed solvent and water. Those that do not dissolve in the liquid are preferably filtered off.

Step (6) is a step of purifying condensed tannin from the above-mentioned whole solution. Step (6) is not an essential step in the present invention, and can be omitted in the case described in step (5), when purification is not necessary, or the like.
Examples of the purification include a method using a purification column. For example, it is preferred that the condensed tannin fraction is allowed to flow out with a water / acetone mixed solvent or the like, and the low molecular weight fraction is allowed to flow out with ethanol, an ethanol / water mixed solvent or the like. Specifically, for example, the method described in the examples can be mentioned.

The step (7) is a step of separating the condensed tannin into condensed tannins having different degrees of polymerization. Step (7) can be omitted if there is no need for separation. Depending on the application, it may be preferable to use only the high molecular weight component, and in such a case, the step (7) is performed.
GPC (Gel Permeation Chromatography) or the like is used for the separation.

<Method for producing condensed tannin with high degree of polymerization>
The present invention is also a method for producing condensed tannin having a high degree of polymerization, which is characterized by using the above-mentioned extraction method.
The "condensed tannin having a high degree of polymerization" produced may be purified to a single degree of polymerization or is a mixture of condensed tannins having a large (number average) molecular weight. Good.

<Method for producing composite agent containing condensed tannin with high degree of polymerization>
Further, the present invention is a method for producing a composite agent containing a condensed tannin having a high degree of polymerization, using the method for producing a condensed tannin having a high degree of polymerization, wherein the composite agent is an antioxidant, It is also a method for producing a composite agent, which is a composite agent selected from the group consisting of tranquilizers, adhesives and adsorbents.

  It is clear that condensed tannin is used as the above-mentioned agent, and it is clear that condensed tannin having a high degree of polymerization may be useful as the above-mentioned agent.

  Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples as long as the gist thereof is not exceeded. Hereinafter, unless otherwise specified, numerical values such as “ratio” and “concentration” are based on “mass”.

Example 1
<Extraction method of the present invention (extraction with superheated steam)>
Extraction was performed using the apparatus and system whose conceptual diagram is shown in FIG.
The waste bark of Acacia mangium of the genus Acacia was crushed to a maximum particle size of about 0.1 mm, and 200 mg of the crushed bark was used as a sample and filled in a 15 mL extraction cell.
Next, superheated steam having a temperature of 120 ° C. was generated using a high frequency heating device (“DHF Super-HI” manufactured by Daiichi High Frequency Industry Co., Ltd.), and the flow rate was set to 18 L / min, and the superheated steam was introduced into the extraction cell. . That is, superheated steam was flowed through the extraction cell at a flow rate of 90 L / (min · g) per sample of unit mass. The extraction was carried out for 10 minutes.

As described above, the superheated steam was caused to flow at a flow rate of 90 L / (min · g) for 10 minutes, and then the extraction cell was cooled to 25 ° C.
Then, distilled water was added to the extraction cell containing the extract and stirred, and the extract adhering to the wall surface of the extraction cell and the surface of acacia was washed out and recovered as a (water) solution.

<< Analysis of extract >>
The obtained solution was swelled by adding 50 mL of ethanol / water (4: 1) to 15 g of Sephadex LH20 (manufactured by GE Healthcare Japan) and swelling it into a column (length 300 mm, inner diameter 20 mm, Shibata Scientific Co., Ltd.). (Manufactured by the company) to prepare a slurry.
Then, the extract solution obtained above was injected and supported on the upper part of the column. Next, a mixed solvent of ethanol / water (4: 1) was poured in to elute low molecular weight components such as hydrolyzable tannin other than condensed tannin and gallic acid. This process was continued until the color of the eluent became clear.

  A mixed solvent of acetone / water (7: 3) was poured into the column to elute the condensed tannin component. This process was continued until the color of the eluent became clear.

The ratio of the dry mass of the extracted component to the mass of the entire bark sample filled in the extraction cell was defined as the "extraction rate", and the ratio of the dry mass of the insoluble material to the mass of the entire bark sample was defined as the "insoluble matter ratio" (hereinafter , With the same definition).
The condensed tannin fraction had an extraction rate of 7.14% by mass, and the low molecular weight fraction had a total extraction rate of insoluble matter of 5.06% by mass.

  The main components of the low molecular weight fraction are considered to be hydrolyzable tannin and gallic acid. The insoluble matter is considered to be a decomposed product of condensed tannin, such as methyl 1,3,5-trimethoxybenzoate and 1- (2-methoxyether) -3,4,5-trimethoxybenzene.

  The condensed tannin fraction was obtained using Bruker's Autoflex to obtain a MALDI mass spectrometric spectrum. DHBA (2,5-dihydroxybenzoic acid) was used as a matrix, and cesium iodide (CsI) was used as an ionization aid.

FIG. 2A shows the MALDI mass spectrum of the obtained condensed tannin fraction.
Compared with the subcritical water extraction method described below in Comparative Example 1, although the peak areas of the 3-5 mer are almost the same for both extraction methods, the 7 mer and the 8 mer are compared, The extraction method of the present invention was larger. Further, although the 9-mer was not confirmed by the subcritical water extraction method, the 9-mer was confirmed by the extraction method of the present invention.
That is, the 7-mer, 8-mer and 9-mer had higher extraction efficiency and a higher content ratio in the entire condensed tannin fraction according to the present invention.

The number average molecular weight calculated from the peak area of the mass spectrum was 1680.
In addition, when compared with the subcritical water extraction method between "mixtures of hexameric or higher condensed tannins", the content ratio of "mixture of hexameric or higher condensed tannins" is higher in the extraction method of the present invention. Was significantly higher.

In Example 1, the condensed tannin extracted from the bark of the genus Acacia mangium has the formula (1), wherein R ¹ is a hydroxyl group (—OH) and R ² is a hydrogen atom (—H). , R ³ has a hydroxyl group (—OH) as a main component. The same applies to every n repeating units regardless of the degree of polymerization.

Comparative Example 1
<Subcritical water extraction method (extraction with subcritical water)>
Using a sample similar to that used in Example 1, subcritical water was extracted according to a conventional method at 150 ° C. for 10 minutes.

The condensed tannin fraction had an extraction rate of 12.0% by mass, and the low molecular weight fraction had a total extraction rate of insoluble matter of 4.70% by mass.
The condensed tannin fraction was treated in the same manner as in Example 1 to obtain a MALDI mass spectrum. The results are shown in Fig. 2 (b).

The extraction rate of the condensed tannin fraction was higher than that of the extraction method using superheated steam (7.14% by mass) of Example 1, but as described above in Example 1, as an extraction method of condensed tannin having a high degree of polymerization, Was superior to the extraction method of the present invention (extraction method using superheated steam).
The maximum degree of polymerization was up to the octamer, and the 9-mer confirmed in Example 1 was not confirmed.
The number average molecular weight measured and calculated in the same manner as in Example 1 was 1250, which was smaller than the number average molecular weight 1680 in Example 1.

Comparative example 2
<Organic solvent extraction method (extraction with water-acetone mixed solvent)>
Using a sample similar to that used in Example 1, a water-acetone mixed solvent (1: 1) was used as an organic solvent in an amount of 10 times (mass) of the sample, and at room temperature (25 ° C.) according to a conventional method. The mixture was stirred for 3 days and extracted.

The condensed tannin fraction has an extraction rate of 16.6% by mass, the low molecular weight fraction has an extraction rate of 1.6% by mass, and the insoluble matter ratio is 0% by mass based on the principle of the organic solvent extraction method.
The condensed tannin fraction was obtained in the same manner as in Example 1 to obtain a MALDI mass spectrum. The results are shown in Fig. 3 (a).

Although the extraction rate of the condensed tannin fraction was higher than that of the extraction method using superheated steam (7.14% by mass) of Example 1, the same as “Comparison of Example 1 and Comparative Example 1” described in Example 1 above. As the method for extracting condensed tannin having a high degree of polymerization, the extraction method of the present invention (extraction method using superheated steam) was superior.
The maximum degree of polymerization was up to the octamer, and the 9-mer confirmed in Example 1 was not confirmed.
The number average molecular weight measured and calculated in the same manner as in Example 1 was 1240, which was smaller than the number average molecular weight 1680 in Example 1.
Further, a post-process for removing the organic solvent from the extract is required, and even if the post-process is performed, the remaining amount of the organic solvent cannot be completely eliminated.

Comparative Example 3
<Hydrothermal extraction method (extraction with pressurized water at 100 ° C or higher)>
200 mL of the sample similar to that used in Example 1 was filled inside a stainless tube having a length of 12 cm and an internal volume of 10 mL, and extracted with 6 mL of distilled water according to a conventional method.
The extraction temperature was changed in the range of 100 ° C. to 180 ° C., and the extraction time was changed between 10 minutes and 30 minutes for extraction.
It was then filtered and freeze dried. 500 mL of the obtained dried solid matter was dissolved in 10 mL of a mixed solvent of water / ethanol = 1/4 (mass ratio), and the insoluble matter was removed by filtration.
The extract was purified from the obtained solution using the same purification column as in Example 1.
A low molecular weight fraction was obtained in the same manner as in Example 1, and a condensed tannin fraction was obtained in the same manner as in Example 1.

The extraction rate of the condensed tannin fraction is 9.4 to 12.0 mass% in the extraction temperature of 100 ° C. to 180 ° C. and the extraction time of 10 minutes to 30 minutes. And the insoluble matter ratio were all 3.1 to 6.7 mass%.
The condensed tannin fraction was obtained in the same manner as in Example 1 to obtain a MALDI mass spectrum. The results are shown in Fig. 3 (b).

Although the extraction rate of the condensed tannin fraction was higher than that of the extraction method using superheated steam (7.14% by mass) of Example 1, the same as “Comparison of Example 1 and Comparative Example 1” described in Example 1 above. As the method for extracting condensed tannin having a high degree of polymerization, the extraction method of the present invention (extraction method using superheated steam) was superior.
The maximum degree of polymerization was up to the octamer, and the 9-mer confirmed in Example 1 was not confirmed.
Further, the number average molecular weight measured and calculated similarly was 1250, which was smaller than the number average molecular weight 1680 in Example 1.

Example 2
<Change in extraction temperature (temperature of superheated steam)>
In Example 1, the temperature of the superheated steam was changed to 100 ° C., 120 ° C., 150 ° C. and 180 ° C. instead of 120 ° C., and in Example 1, the flow rate was changed from 18 L / min to 13 L / min. Was extracted in the same manner as in Example 1 for 10 minutes.
Then, the evaluation was performed in the same manner as in Example 1.

The extraction rate of the condensed tannin fraction is in the range of 5.5 to 7.1% by mass at 100 ° C., 120 ° C., 150 ° C. and 180 ° C., and the extraction rate of the low molecular weight fraction and the insoluble matter rate. And the total was 2.4 to 2.9% by mass.
The extraction ratio of the condensed tannin fraction and the "total of the extraction ratio of the low molecular weight fraction and the insoluble matter ratio" were almost the same in the range of 100 ° C to 180 ° C.

MALDI mass spectrometry spectra were obtained for each condensed tannin fraction in the same manner as in Example 1.
The results are shown in Fig. 4. The extraction temperature (temperature of superheated steam) is 100 ° C. in FIG. 4 (a), 120 ° C. in FIG. 4 (b), 150 ° C. in FIG. 4 (c), and 180 ° C. in FIG. 4 (d). The MALDI mass spectrum showed a difference depending on the extraction temperature.

In each of the MALDI mass spectrometry spectra, a series of peak groups was clearly observed in the region up to the mass-to-charge ratio (m / z) 2800. In each peak group, the strongest peak was detected at every m / z 288, and this interval corresponded to the mass of the flavan-3-ol unit having 5 hydroxyl groups in the molecule.
Further, as shown in the enlarged view of FIG. 4 (a), the ion peaks constituting each group are observed with an interval of m / z16, and this interval is the increment when the hydroxyl group is substituted. Match.

  Combining these results and the value of m / z of each ion, a series of peak groups is a cesium ion addition of condensed tannin consisting of a combination of a plurality of flavan-3-ol units having different numbers of hydroxyl groups contained. It was assigned as a molecule.

  In addition, in "Fig. 4 (a) 100 ° C extraction", the maximum degree of polymerization was the octamer, but the extraction method of the present invention is shown in Fig. 4 (b) 120 ° C, (c) 150 ° C and (d). 9) Condensed tannin of 9-mer was observed in common at 180 ° C. In each case, it was found that the condensed tannins having a high degree of polymerization were extracted while maintaining the polymer structure.

The maximum polymerization degree and number average molecular weight of the extracted condensed tannins are summarized in Table 1 below.

As shown in Table 1, in each of the extraction methods using superheated steam, the number average molecular weight was about 1600, which was a substantially constant value.
On the other hand, when water vapor at 100 ° C. was used, the number average molecular weight was 1500, which was significantly low. It is considered that this is because sufficient heat energy could not be obtained at 100 ° C., and condensed tannin with a high degree of polymerization could not be extracted.
Steam having a temperature higher than 100 ° C has higher heat energy than steam having a temperature of 100 ° C, and since these steams provide an extraction field having a relatively low dielectric constant, condensed tannins having a high degree of polymerization are efficient. It is thought that it was able to be extracted.

Furthermore, the number average molecular weight of the condensed tannin obtained using superheated steam is 340 to 390 as compared with the number average molecular weight 1250 of the condensed tannin obtained by the subcritical water extraction method of Comparative Example 1. Was getting higher.
It is considered that the reason for this is that in the subcritical water extraction method, since the extraction is carried out at high temperature and high pressure, side reactions such as decomposition and polymerization of condensed tannin proceed.
From the above, it was found that when superheated steam is used, it is possible to perform extraction under milder conditions than the subcritical extraction method, and it is possible to extract condensed tannin with a higher degree of polymerization.

Example 3
Extraction was performed in the same manner as in Example 2 except that the temperature of superheated steam in Example 2 was changed as follows.
When extracted at 280 ° C, the condensed tannin contained in the bark of acacia decomposed or polymerized more than when extracted at 120 to 180 ° C, but the number average molecular weight was about 1600 and up to a 9-mer. It could be confirmed.
Further, when extracted at 320 ° C., the number average molecular weight was high, but only octamers were confirmed.
In addition, when the extraction was carried out at 500 ° C, the condensation type tannin contained in the bark of acacia was decomposed and polymerized, the number average molecular weight was lowered, and it was of course that the 9-mer could not be confirmed. The tannin yield was reduced.

  The method for extracting condensed tannins using superheated steam of the present invention is capable of extracting condensed tannins having a high degree of polymerization, and thus "condensed tannins having a high degree of polymerization" obtained using the extraction method is skin tanned. , Protein astringents, mordants, fining agents, preservatives, inks, seasonings, (health) foods, antioxidants, tranquilizers, adhesive (improving) agents, adsorbents, etc. Is.

Claims (8)

A method for extracting a condensed tannin extract containing a 9-mer condensed tannin, which comprises extracting the condensed tannin from a plant part using superheated steam at an extraction temperature of 120 C. or more and 280.degree. C. or less, and an extraction time of 1 minute or more and 60 minutes or less . The extraction method according to claim 1, wherein the condensed tannin extract to be extracted contains at least all condensed tannins having a degree of polymerization of 4 to 9. The extraction method according to claim 1 or 2, wherein the condensed tannin contained in the condensed tannin extract to be extracted has a number average molecular weight of 1200 or more. The extraction method according to any one of claims 1 to 3 , wherein extraction regions having different temperature ranges of superheated steam are arranged in multiple stages and extraction is performed in order from the lowest temperature of the superheated steam. The extraction method according to any one of claims 1 to 4 , wherein the plant part is a part selected from the group consisting of bark, wood part, pericarp, fruit, seed, leaf and pod. The plant according to any one of claims 1 to 5 , wherein the plant is a plant belonging to a genus selected from the group consisting of Acacia, Scutellaria, Chestnut, Pinus, Rheum, Grape and Nikkei. The extraction method described in. A method for producing a condensed tannin extract containing a 9-mer condensed tannin, which comprises using the extraction method according to any one of claims 1 to 6 . A method for producing a complexing agent containing a 9-mer condensed tannin, which comprises using the method for producing a condensed tannin extract containing a 9-mer condensed tannin according to claim 7 .
A method for producing a composite agent, wherein the composite agent is a composite agent selected from the group consisting of an antioxidant, a tranquilizer, an adhesive and an adsorbent.