Method for producing a proanthocyanidin-containing composition

Abstract

The present invention aims to provide a method for producing a proanthocyanidin-containing composition having high antiviral activity. [Solution] A method for producing a proanthocyanidin-containing composition by purifying proanthocyanidins from ginger lily juice (A), comprising: (1) a solution (AB) containing ginger lily juice (A) and a first preservative (B), and porous particles (C) coexisting with the porous particles (C) to adsorb proanthocyanidins onto the porous particles (C) to obtain adsorbed porous particles (D) on which the proanthocyanidins have been adsorbed; and (2) a solution (EF) containing the adsorbed porous particles (D), a second preservative (E), and ethanol (F). A method for producing a proanthocyanidin-containing composition, comprising the step (2) of desorbing the proanthocyanidin from adsorbed porous particles (D) by allowing the coexistence of the two, wherein the content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight based on the weight of the solution (AB), and the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight based on the weight of the solution (EF).

Description

Technical Field

[0001] The present invention relates to a method for producing a proanthocyanidin-containing composition.

Background Art

[0002] Proanthocyanidins contained in Alpinia zerumbet squeezed juice, etc. are known to have an excellent control effect against various plant viruses and plant pathogens (Patent Document 1). Proanthocyanidins are expected to be widely used because they can safely control plant diseases without causing phytotoxicity such as leaf atrophy and spot formation, but it is difficult to recover and produce them in large quantities, and the development of a production method has been desired.

[0003] Generally, chromatography is used when recovering a specific component from plant squeezed juice or the like. Patent Document 2 describes a method in which at least two adsorbents are allowed to act on an extract or squeezed juice of a plant body to adsorb a specific component and then separated with an ethanol solution. However, general chromatography does not have sufficient recovery efficiency, and proanthocyanidins obtained by the method described in Patent Document 2 have a problem of inferior antiviral activity.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] An object of the present invention is to provide a method for producing a proanthocyanidin-containing composition having high antiviral activity.

Means for Solving the Problems

[0006] The inventors of this invention arrived at the present invention as a result of diligent research to solve the above problems. In other words, the present invention relates to a method for producing a proanthocyanidin-containing composition by purifying proanthocyanidins from ginger lily juice (A), comprising: (1) a step of coexisting a solution (AB) containing ginger lily juice (A) and a first preservative (B) with porous particles (C) to adsorb proanthocyanidins onto the porous particles (C) to obtain adsorbed porous particles (D) on which the proanthocyanidins have been adsorbed; and (2) a step of coexisting a solution (EF) containing the adsorbed porous particles (D) with a second preservative (E) and ethanol (F). The method for producing a proanthocyanidin-containing composition comprises a step (2) of desorbing the proanthocyanidin from the adsorbed porous particles (D) by allowing the coexistence of the first preservative (B) and the second preservative (E) in the solution (EF) to obtain a proanthocyanidin-concentrated solution (G), wherein the content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight based on the weight of the solution (AB), and the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight based on the weight of the solution (EF). [Effects of the Invention]

[0007] According to the manufacturing method of the present invention, a proanthocyanidin-containing composition having high antiviral activity can be obtained. [Modes for carrying out the invention]

[0008] The present invention provides a method for producing a proanthocyanidin-containing composition by purifying proanthocyanidins from ginger lily juice (A), comprising: (1) a step of coexisting a solution (AB) containing ginger lily juice (A) and a first preservative (B) with porous particles (C), allowing proanthocyanidins to be adsorbed onto the porous particles (C) to obtain adsorbed porous particles (D) on which the proanthocyanidins have been adsorbed; and (2) a step of combining the adsorbed porous particles (D) with a second preservative (E) and ethanol (F The method for producing a proanthocyanidin-containing composition comprises a step (2) of desorbing the proanthocyanidin from the adsorbed porous particles (D) by coexisting with a solution (EF) containing the first preservative (B) in the solution (AB), wherein the content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight based on the weight of the solution (AB), and the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight based on the weight of the solution (EF).

[0009] In the present invention, "proanthocyanidin" refers to a compound in which multiple flavan-3-ols, such as catechin and epicatechin, are bonded together.

[0010] In this invention, the term "ginger lily" includes ginger lilies of the genus Alpinia (Alpinia) of the family Zingiberaceae, such as the island ginger lily of Okinawa Prefecture and the island ginger lily of Amami Oshima (Alpinia zerumbet (Pers.) BLBurtt&R.M.Sm.), the large-flowered ginger lily (Alpinia zerumbet (Pers.) BLBurtt&R.M.Sm.) of Kita Daito Island, Minami Daito Island and Miyako Island, the Tachibana ginger lily, the large-flowered ginger lily of Taiwan, the Urai ginger lily, and the Tunlu ginger lily, as well as hybrids thereof.

[0011] In the present invention, the ginger lily juice (A) can be obtained, for example, by physically crushing the ginger lily plant using a crusher or juicer. The resulting crude juice may be used after removing impurities by, if necessary, processing it with a centrifuge or filtering it with filter paper or a filter. The concentration of proanthocyanidins contained in the ginger lily juice (A) is preferably 0.1 mg / mL or higher, more preferably 0.5 mg / mL or higher, even more preferably 1 mg / mL or higher, then even more preferably 5 mg / mL or higher, and particularly preferably 10 mg / mL or higher, from the viewpoint of purification efficiency, concentration efficiency before and after purification, and ease of application after purification. It is also preferably 50 mg / mL or lower.

[0012] In the present invention, concentration treatment may be performed to adjust the concentration of proanthocyanidins contained in the ginger lily juice (A). Concentration methods include heating concentration, vacuum concentration, ultrafiltration concentration, and dialysis membrane concentration. Of these, vacuum concentration is preferred from the viewpoint of concentration efficiency and the heat resistance of proanthocyanidins.

[0013] In the present invention, the first preservative (B) and the second preservative (E) include those that have antioxidant and microbial growth inhibitory effects on proanthocyanidins. Examples include sulfite compounds (e.g., sodium pyrosulfite, potassium pyrosulfite, sodium sulfite, potassium sulfite, sodium bisulfite, potassium bisulfite, etc.), gallic acid, sodium thiosulfate, ascorbic acid, ascorbyl palmitate, erythorbic acid, tocopherol, butylhydroxyanisole, dibutylhydroxytoluene, disodium edetate gallic acid, eugenol, cinnamic acid, coumarin, carvacrol, etc. Of these, sulfite compounds are preferred from the viewpoint of having bactericidal effects in addition to antioxidant effects, more preferably sodium pyrosulfite, potassium pyrosulfite and gallic acid, and particularly preferably potassium pyrosulfite. The first preservative (B) and the second preservative (E) may each be used individually, or two or more may be used in combination. Furthermore, the first preservative (B) and the second preservative (E) may be the same, or they may be different.

[0014] In the present invention, porous particles (C) do not have functional groups such as ion exchange groups, are porous, and have fine continuous pores (micropores), and can be adsorbed by van der Waals forces, for example. The average value of the most frequent pore diameter of the porous particles (C) is preferably 10 to 120 nm, and more preferably 20 to 50 nm, from the viewpoint of purifying proanthocyanidins with high antiviral activity and removing low molecular weight sugars and high molecular weight proteins through purification. In this invention, the average value of the most frequent radius of the pore diameter can be measured by the carrier gas method (JIS Z 8831-2).

[0015] Porous particles (C) exhibit different degrees of hydrophilicity and hydrophobicity depending on the type of resin material, for example, aromatic resin particles, substituted aromatic resin particles, (meth)acrylic resin particles, etc. Aromatic resin particles include, for example, cross-linked styrene resin particles. Commercially available examples include, for example, Diaion® HP-10, HP-20, HP-21, HP-30, HP-40, and HP-50 (manufactured by Mitsubishi Chemical Corporation); Amberlight® XAD-4, XAD-16, XAD-1180, and XAD-2000 (manufactured by Organo Corporation); and Sepabeads® SP-825, SP-800, SP-850, and SP-875 (manufactured by Mitsubishi Chemical Corporation). Substituted aromatic resin particles include those made from highly hydrophobic resins in which bromine atoms or the like are bonded to the aromatic nuclei of an aromatic polymer. Examples of commercially available products include Sepabeads® SP-205, SP-206, and SP-207 (manufactured by Mitsubishi Chemical Corporation). (Meth)acrylic resin particles include those using highly hydrophilic resins with a (meth)acrylic acid ester polymer as their backbone. Examples of commercially available products include Diaion® HP1MG and HP2MG (manufactured by Mitsubishi Chemical Corporation) and Amberlight® XAD-7 (manufactured by Organo Corporation). Among these, from the viewpoint of the adsorption efficiency of proanthocyanidins, aromatic resin particles are preferred, more preferably styrene-divinylbenzene resin, and particularly preferably Diaion HP-20.

[0016] The production method of the present invention includes a step (1) of coexisting a solution (AB) containing Alpinia zerumbet squeezed juice (A) and a first preservative (B) with porous particles (C) to adsorb proanthocyanidins onto the porous particles (C) to obtain adsorbent porous particles (D) adsorbed with proanthocyanidins. In the present invention, by coexisting Alpinia zerumbet squeezed juice (A) and porous particles (C) in the presence of the first preservative (B) to adsorb proanthocyanidins onto the porous particles (C), it is possible to remove impurities such as sugars and proteins that hinder formulation without reducing the antiviral activity of proanthocyanidins. The content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight, preferably 0.05 to 0.3% by weight, more preferably 0.075 to 0.2% by weight, and particularly preferably 0.1 to 0.15% by weight based on the weight of the solution (AB). When the content of the first preservative (B) in the solution (AB) is 0.02% by weight or more, it is possible to prevent loss and decomposition of proanthocyanidins due to spoilage, and when it is 0.5% by weight or less, it is possible to prevent decomposition of proanthocyanidins due to a decrease in pH caused by the first preservative.

[0017] In step (1), from the viewpoint of the storage stability of proanthocyanidins, the content of the first preservative (B) is preferably 0.05 to 0.3% by weight, more preferably 0.06 to 0.1% by weight based on the weight of the solution (AB).

[0018] In step (1), from the viewpoint of purification efficiency, the weight ratio of the porous particles (C) to the weight of proanthocyanidins contained in the solution (AB) is preferably 4.2 to 42 mg / g, more preferably 12 to 20 mg / g.

[0019] In step (1), from the viewpoint of the stability of proanthocyanidins, the temperature of the solution (AB) is preferably 4 to 30°C.

[0020] In the present invention, examples of step (1) include: (1i) a method of preparing a solution (ABC) containing a solution (AB) and porous particles (C), and allowing the solution (AB) and the porous particles (C) to coexist in the solution (ABC) to obtain adsorbed porous particles (D); (1ii) a method of filling the porous particles (C) into a column or the like and passing the solution (AB) through the column to obtain adsorbed porous particles (D). Among these, from the viewpoint of purification efficiency, the method of (1ii) is preferred.

[0021] In the method of (1i), from the viewpoint of the stability of proanthocyanidin monomer, the coexistence time of the solution (AB) and the porous particles (C) is preferably 2 to 12 hours, more preferably 2 to 5 hours. In the method of (1i), from the viewpoint of the thermal stability of proanthocyanidins, the temperature of the solution (ABC) is preferably 4 to 30°C, more preferably 15 to 30°C. In the method of (1i), as a method for obtaining the adsorbed porous particles (D) from the solution (ABC), general methods for separating a solid and a liquid, such as filtration, can be used.

[0022] In the method of (1ii), from the viewpoint of the stability of proanthocyanidins, it is preferable to fill the porous particles (C) into the column so that the solution (AB) is passed through the column from the start of liquid passage for 2 to 12 hours until the liquid passage is completed at a space velocity SV of 0.5 to 2 ( / hr).

[0023] The production method of the present invention has a step (2) of allowing the adsorbed porous particles (D) and a solution (EF) containing a second preservative (E) and ethanol (F) to coexist to desorb proanthocyanidins from the adsorbed porous particles (D) to obtain a proanthocyanidin concentrated solution (G). In the present invention, by allowing the solution (EF) to act on the adsorbed porous particles (D) in the presence of the second preservative (E), it is possible to maintain the stability of proanthocyanidins and prevent a decrease in activity. In the present invention, the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight, preferably 0.005 to 0.1% by weight, more preferably 0.0075 to 0.05% by weight, and particularly preferably 0.01 to 0.03% by weight, based on the weight of the solution (EF). A content of 0.0025% by weight or more of the second preservative (E) in the solution (EF) prevents oxidation of proanthocyanidins during liquid passage, and a content of 0.2% by weight or less prevents oxidative decomposition by the second preservative (E) when concentrating the purified proanthocyanidins.

[0024] The solution (EF) is not particularly limited as long as it contains the second preservative (E) and ethanol (F), but it is preferable that it also contains water from the viewpoint of selectively eluting high molecular weight proanthocyanidins with high antiviral activity. When the solution (EF) is an aqueous solution, the ethanol content in the solution (EF) is preferably 20 to 50% by volume, and more preferably 30 to 40% by volume, based on the volume of the solution (EF), from the viewpoint of selectively eluting high-molecular-weight proanthocyanidins with high antiviral activity.

[0025] In step (2), the temperature of the solution (EF) is preferably 4 to 30°C from the viewpoint of the temperature stability of proanthocyanidins.

[0026] The weight ratio of the solution (EF) to the weight of the adsorbent porous particles (D) is preferably 50 to 500 g / L, and more preferably 100 to 200 g / L, from the viewpoint of preventing physical loss of proanthocyanidins due to shear.

[0027] In the present invention, step (2) includes (2i) a method of desorbing proanthocyanidins from adsorbent porous particles (D) by having the adsorbent porous particles (D) and the solution (EF) coexist in a solution (DEF) containing a second preservative (E) and ethanol (F), and (2ii) a method of packing the adsorbent porous particles (D) into a column or the like and passing the solution (EF) through the column to desorb the proanthocyanidins. Of these, method (2ii) is preferred from the viewpoint of purification efficiency.

[0028] In method (2i), the temperature of the solution (DEF) is preferably 4 to 30°C from the viewpoint of the temperature stability of proanthocyanidins.

[0029] In method (2ii), from the viewpoint of the stability of proanthocyanidins, it is preferable to pack the column with adsorbed porous particles (D) so that the solution (EF) is passed through the column 2 to 12 hours after the start of flow, or at a space velocity of 0.5 to 5 ( / hr).

[0030] In the present invention, there may be a step of concentrating the proanthocyanidins in the proanthocyanidin concentrate solution (G), for example, by vacuum concentration or freeze-drying. From the viewpoint of ease of application after purification, it is preferable to have a step (3) of distilling off the ethanol in the proanthocyanidin concentrate solution (G).

[0031] In step (3), the temperature of the proanthocyanidin concentrate (G) is preferably 25 to 80°C, and more preferably 50 to 70°C, from the viewpoint of concentration efficiency and the temperature stability of proanthocyanidins. In step (3), the absolute pressure used when processing the proanthocyanidin concentrate (G) is preferably 80 to 140 kPa, and more preferably 80 to 110 kPa, from the viewpoint of concentration efficiency and the stability of the proanthocyanidins.

[0032] The proanthocyanidin content in the proanthocyanidin-containing composition obtained by the production method of the present invention is preferably 5 to 60% by weight, and more preferably 10 to 30% by weight, based on the weight of the composition, from the viewpoint of storage stability and ease of application after purification.

[0033] The proanthocyanidin-containing composition obtained by the production method of the present invention has high antiviral activity and is therefore useful, for example, as a pest control agent.

[0034] When using the proanthocyanidin-containing composition obtained by the production method of the present invention as a control agent, applicable plant viral diseases include, for example, diseases caused by infection with viruses of the genus Tobamovirus, Potexvirus, Carlavirus, Cucumovirus, Calmovirus, Potivirus, Tospovirus, or Begomovirus.

[0035] Examples of viruses belonging to the Tobamovirus genus include tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), cucumber green spotted mosaic virus (KGMMV), pepper microspotted virus (PMMoV), watermelon green spotted mosaic virus (CGMMV), tomato mottle mosaic virus (ToMMV), and tomato brown rugose fruit virus. Examples include ToBRFV virus, and examples of Potexvirus viruses include plantain mosaic virus (PlAMV) and potato X virus (PVX). Examples of Carlavirus viruses include potato M virus (PVM). Examples of Cucumovirus viruses include cucumber mosaic virus (CMV). Examples of Calmovirus viruses include melon necrotic spot virus (MNSV). Examples of Potivirus viruses include potato Y virus (PVY) and plum ring spot virus (PPV). Examples of Tospovirus viruses include watermelon gray spot virus (WSMoV). Examples of Begomovirus viruses include tomato yellow leaf curl virus (TYLCV), but are not limited to these.

[0036] The plants to which the proanthocyanidin-containing composition obtained by the production method of the present invention is applied as a control agent are not particularly limited as long as they are infected with the above-mentioned plant viruses, but for example, Solanaceae plants (tobacco, tomato, eggplant, potato, bell pepper, chili pepper, petunia, etc.), Cucurbitaceae plants (cucumber, melon, pumpkin, melon, watermelon, etc.), Poaceae plants (rice, barley, wheat, corn, sugarcane, sorghum, sorghum, turfgrass, etc.), Brassicaceae plants (Chinese cabbage, cabbage, radish, bok choy, komatsuna, broccoli) Examples include rapeseed, Arabidopsis thaliana, legumes (soybeans, peanuts, peas, green beans, broad beans, etc.), roses (strawberries, apples, pears, peaches, plums, roses, cherries, etc.), morning glory (sweet potatoes, etc.), lilies (leeks, onions, lilies, tulips, etc.), daisies (lettuce, chrysanthemums, gerberas, etc.), grapes (grapes, etc.), carnations (carnations, etc.), orchids (cattleyas, cymbidiums, etc.), gentians (lisianthus, etc.), and plumbagins (statice, etc.).

[0037] When the proanthocyanidin-containing composition obtained by the production method of the present invention is used as a control agent, it can be applied to a wide range of plants, including Solanaceae, Cucurbitaceae, Poaceae, Brassicaceae, Fabaceae, Rosaceae, Convolvulaceae, Liliaceae, Asteraceae, Vitaceae, Caryophyllaceae, Orchidaceae, Gentianaceae, and Plumbaginaceae. For information on the relationship between plant viruses and host plants, please refer to the Japanese Plant Disease Database (Agricultural Bioresource Gene Bank).

[0038] This specification contains the following information:

[0039] This disclosure (1) is a method for producing a proanthocyanidin-containing composition by purifying proanthocyanidins from ginger lily juice (A), Step (1) involves coexisting a solution (AB) containing ginger lily juice (A) and a first preservative (B) with porous particles (C), allowing proanthocyanidins to be adsorbed onto the porous particles (C), thereby obtaining adsorbed porous particles (D) on which the proanthocyanidins have been adsorbed. The process includes step (2) of obtaining a proanthocyanidin concentrated solution (G) by coexisting the adsorbent porous particles (D) with a solution (EF) containing a second preservative (E) and ethanol (F), thereby desorbing the proanthocyanidin from the adsorbent porous particles (D). The content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight based on the weight of the solution (AB). This is a method for producing a proanthocyanidin-containing composition, wherein the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight based on the weight of the solution (EF).

[0040] Disclosure (2) is a method for producing a proanthocyanidin-containing composition according to Disclosure (1), wherein in step (1), the weight ratio of the porous particles (C) to the weight of the proanthocyanidin contained in the solution (AB) is 4.2 to 42 mg / g.

[0041] The present disclosure (3) is a method for producing a proanthocyanidin-containing composition according to the present disclosure (1) or (2), wherein in step (1), the coexistence of the solution (AB) and the porous particles (C) is made by making the solution (AB) and the porous particles (C) coexist in a solution (ABC) containing the solution (AB) and the porous particles (C), and the time for which the solution (AB) and the porous particles (C) coexist is 2 to 12 hours.

[0042] This disclosure (4) is a method for producing the proanthocyanidin-containing composition according to this disclosure (3), wherein in step (1), the temperature of the solution (ABC) is 4 to 30°C.

[0043] The present disclosure (5) is a method for producing a proanthocyanidin-containing composition according to any one of the present disclosures (1) to (4), wherein in step (2), the coexistence of the adsorbent porous particles (D) and the solution (EF) containing the second preservative (E) and the ethanol (F) is made by coexisting the adsorbent porous particles (D) and the solution (EF) in a solution (DEF) containing the adsorbent porous particles (D) and the second preservative (E) and the ethanol (F), and the temperature of the solution (DEF) is 4 to 30°C.

[0044] Disclosure (6) is a method for producing a proanthocyanidin-containing composition according to any one of Disclosures (1) to (5), wherein in step (2), the solution (EF) is an aqueous solution containing 20 to 50% by volume of ethanol based on the volume of the solution (EF).

[0045] Disclosure (7) is a method for producing a proanthocyanidin-containing composition according to any one of Disclosures (1) to (6), comprising a step (3) of distilling off the ethanol in the proanthocyanidin concentrate (G).

[0046] Disclosure (8) is a method for producing a proanthocyanidin-containing composition according to any one of Disclosures (1) to (7), wherein the first preservative (B) is at least one selected from the group consisting of sodium pyrosulfite, potassium pyrosulfite, and gallic acid.

[0047] Disclosure (9) is a method for producing a proanthocyanidin-containing composition according to any one of Disclosures (1) to (8), wherein the second preservative (E) is at least one selected from the group consisting of sodium pyrosulfite, potassium pyrosulfite, and gallic acid.

[0048] Disclosure (10) is a method for producing a proanthocyanidin-containing composition according to any one of Disclosures (1) to (9), wherein the average value of the most frequent pore diameter of the porous particles (C) is 10 to 120 nm. [Examples]

[0049] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the following, "part" refers to "weight part".

[0050] [Example: Preparation of Alpinia zerumbet juice] The juice of Okinawa ginger lily (Alpinia zerumbet) from Kunigami District, Okinawa Prefecture (hereinafter referred to simply as "Okinawa-grown Okinawa ginger lily" unless otherwise specified) was centrifuged in a centrifuge (8000 × g, 30 minutes), and the supernatant was filtered using filter paper. Potassium pyrosulfite (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) or gallic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the obtained filtrate as the first preservative in the weight proportions shown in Tables 1 and 2 to obtain ginger lily juice.

[0051] [Examples 1-18: Purification of proanthocyanidin solution (batch method)] To the ginger lily juice (0.04 L) obtained by the method described in [Example: Preparation of Ginger Lily Juice] above, 5 g of porous synthetic adsorbent (DIAION H2O, manufactured by Mitsubishi Chemical Corporation: pore size 29 nm) was added (the weight ratio of porous synthetic adsorbent to ginger lily juice was 8 g / g), and the mixture was immersed at 25°C for 2 hours. After 2 hours, the supernatant was discarded, and the porous synthetic adsorbent was redispersed in 40 mL of 10% EtOH (Hayashi Pure Chemical Industries, Ltd.). After 10 minutes, the supernatant was discarded, and the mixture was redispersed in 20 mL of 10% EtOH. After 10 minutes, the supernatant was discarded to wash the mixture. After washing, the washed porous adsorbent was redispersed in 40 mL of 40 vol% EtOH, to which potassium pyrosulfite or gallic acid was added as a second preservative in the weight ratios shown in Tables 1 and 2, thereby eluting the proanthocyanidins adsorbed on the adsorbent. After standing at 25°C for 2 hours, the supernatant was collected. The collected supernatant was decontaminated by distillation of ethanol using an evaporator (80°C (liquid temperature 60°C), reduced pressure of 90 kPa or less) to obtain the proanthocyanidin-containing compositions according to Examples 1 to 18 derived from Alpinia zerumbet.

[0052] [Example 19: Purification of proanthocyanidin solution (column method)] A porous synthetic adsorbent (DIAION H20, manufactured by Mitsubishi Chemical Corporation) (250 g) was packed into a column, and 2 L of the ginger lily juice obtained in the above [Example: Preparation of Ginger Lily Juice] was passed through the column. Next, 0.5 L of 100 ppm potassium pyrosulfite (secondary preservative) aqueous solution was passed through the column relative to the volume of the synthetic adsorbent. Subsequently, the proanthocyanidins adsorbed on the adsorbent were eluted using an aqueous solution containing 40 vol% ethanol. At this time, the porous synthetic adsorbent, the 100 ppm potassium pyrosulfite aqueous solution, and the aqueous solution containing 40 vol% ethanol coexist in the column. The ethanol was removed from the recovered solution by distillation using an evaporator (80°C (liquid temperature 60°C), reduced pressure 90 kPa or less) to obtain the proanthocyanidin-containing composition according to Example 19 derived from ginger lily.

[0053] [Table 1]

[0054] [Table 2]

[0055] [Comparative example: Concentrated ginger lily juice] Alpinia zerumbet juice was obtained in the same manner as described in "Examples: Preparation of Alpinia zerumbet Juice" above, except that potassium pyrosulfite was added as a primary preservative in the weight proportions shown in Tables 1 and 2. Furthermore, a ginger lily juice without the first preservative was used as the proanthocyanidin-containing composition according to Comparative Example 1.

[0056] [Comparative Examples 2-7: Purification of Proanthocyanidin Solutions] The proanthocyanidin-containing compositions of Comparative Examples 2 to 7 were obtained by the same method as in the above [Examples 1 to 18: Purification of proanthocyanidin solution (batch method)], except that potassium pyrosulfite was added to EtOH as a second preservative in the weight ratios shown in Tables 1 and 2.

[0057] • Measurement of proanthocyanidin concentration (+) Catechin hydrate (manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in distilled water to prepare the standard solution. For the quantitative determination of proanthocyanidins, the method using 4-dimethylaminocinnamaldehyde (DMAC) as described in the report by Oki et al. (Tomoyuki Oki et al., Nippon Shokuhin Kagaku Kogaku Kaishi Vol.60, No6, 301-309 (2013)) was followed in general. Specifically, a calibration curve proanthocyanidin aqueous solution (10 mg / mL) was diluted 20-fold with assay solution A (ethanol:methanol:2-propanol = 90:5:5), and then a 2-fold dilution series was prepared with assay solution B (assay solution A:water = 95:5). The proanthocyanidin concentrations in the proanthocyanidin-containing compositions obtained in the examples and comparative examples, as well as in the ginger lily juice, were measured as calibration curve samples, and the recovery rate of proanthocyanidins was obtained. For the proanthocyanidin-containing compositions in the examples and comparative examples, the proanthocyanidin-containing compositions at the time of purification and after standing at 25°C for 2 weeks were diluted 20-fold with assay solution A (ethanol:methanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.):2-propanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) = 90:5:5) and used as measurement samples. The remaining percentage of proanthocyanidins after standing for 2 weeks was evaluated. The results are shown in Tables 1 and 2. Furthermore, the proanthocyanidin-containing compositions of Comparative Examples 5 and 6 spoiled and turned brown after being left standing for two weeks, making it impossible to measure the remaining percentage of proanthocyanidins.

[0058] • Molecular weight measurement of proanthocyanidins The proanthocyanidin-containing compositions obtained in the examples and comparative examples were dissolved at a concentration of 1 mg / mL in dimethylformamide dissolved in 50 mM lithium chloride for samples taken during purification and after standing at 25°C for 2 weeks. These samples were analyzed using a high-performance liquid chromatograph (e2695, Waters Japan Co., Ltd.) equipped with a UV detector (2489, Waters Japan Co., Ltd., detection wavelengths 265 nm and 283 nm) and a size exclusion chromatography column (α-3000, Tosoh Corporation) in dimethylformamide dissolved in 50 mM lithium chloride as the solvent (flow rate 0.6 mL / mL, column temperature 40°C, injection volume 25 μL). Along with the samples, standard polystyrene polymers for organic solvent-based size exclusion chromatography (PStQuick E and PStQuick F, Tosoh Corporation) were also analyzed. A calibration curve was calculated with the logarithm of the molecular weight of the standard polystyrene polymers on the vertical axis and the peak top retention time detected at 265 nm on the horizontal axis. Based on this calibration curve and the peak top retention times of the three proanthocyanidin purified products, the weight-average molecular weight of the proanthocyanidin-containing composition derived from Alpinia zerumbet was estimated. Furthermore, the purity of the proanthocyanidins was measured from the sum of the peaks of the obtained proanthocyanidins and the peaks of impurities. The results are shown in Tables 1 and 2. Furthermore, the proanthocyanidin-containing compositions of Comparative Examples 5 and 6 were spoiled and browned, and therefore this evaluation was not performed.

[0059] • Measurement of sugar content 0.5 mL of the proanthocyanidin-containing compositions of the examples and comparative examples, placed in test tubes, were mixed with 0.5 mL of Somogyi copper reagent (Sigma-Aldrich) and boiled at 100°C for 20 minutes. Next, Nelson color reagent (Sigma-Aldrich) was added, and after standing for 30 minutes, the volume was increased to 12.5 mL with pure water and thoroughly mixed. The supernatant of the resulting reaction solution was collected, and the absorbance at 500 nm was confirmed using a UV-1800 ultraviolet-visible spectrophotometer (Shimadzu Corporation). Sucrose (Fujifilm Wako Pure Chemical Industries, Ltd.) was used as the standard solution. The sugar content was then measured. The results are shown in Tables 1 and 2.

[0060] • Measurement of oxalic acid concentration Each proanthocyanidin-containing composition was diluted 6-fold with pure water to create a 50 mL beaker (Corning Corporation). The solution was then stirred using a ceramic hot stirrer (AS ONE Corporation) to maintain a temperature of 25°C. Next, a waterproof conductivity meter (Custom Corporation) was immersed in the solution, and 0.1 M calcium acetate (Fujifilm Wako Pure Chemical Industries, Ltd.) was titrated while stirring. The volume (mL) of 0.1 M calcium acetate titrated until the inflection point was reached was recorded. Subsequently, the oxalic acid concentration was calculated using the following formula (1). Oxalic acid content (mg) = 0.1M calcium acetate titration volume (mL) × 9 × dilution ratio ... (1) The results are shown in Tables 1 and 2. Oxalic acid is a plant-derived component that combines with salts (especially calcium) to form an insoluble precipitate. A low amount of oxalic acid is preferable, and since oxalic acid has a low molecular weight, it is removed along with other impurities during this purification process.

[0061] • Analysis of antiviral activity The effectiveness of proanthocyanidin-containing compositions in controlling plant viral diseases was evaluated using the tomato mosaic virus (ToMV)-bensamiana tobacco evaluation system. First, as plant material, we sowed one seed per pot of tobacco benthamiana in a mixture of peat moss (Super Mix A, Sakata Seed Co., Ltd.): white vermiculite (A-2, Asahi Kogyo Co., Ltd.): obsidian perlite (Obsidian Series No. 3, Taiheiyo Perlite Co., Ltd.) in a 2:1:1 ratio, and cultivated it at 24°C in a 24-hour light-dark cycle (16 hours of light and 8 hours of darkness). Water was added to each proanthocyanidin-containing composition to prepare a 500 ppm (500 ppm = 500 μg / mL) proanthocyanidin aqueous solution as the test drug. Water was also used as a control. Then, each of the prepared pesticides was sprayed onto the stems and leaves of the entire tobacco plant (Tabana benthamiana), 21 days after sowing, when the third leaf was still small but three true leaves had developed. The plants were then left to stand at 24°C in a 24-hour light-dark cycle (16 hours of light and 8 hours of darkness). Three days later, the mature third leaf was inoculated with ToMV and left to stand at 24°C in a 24-hour light-dark cycle (16 hours of light and 8 hours of darkness). ToMV was prepared by using 2 μg of a plasmid encoding ToMV-GFP (virus with added green fluorescent protein GFP) (pTLBN.G3; see Kubota K. et al., J Virol. 2003 Oct;77(20):11016-26) as a template, and transcribing it using an in vitro RNA transcription synthesis kit (product name: AmpliCap-Max T7 High Yield Message Maker Kits, CellScript). The RNA transcript was then diluted 30-40 times, and 10 μL of the resulting RNA transcript was applied to two locations on the third leaf of Tobacco benthamiana using carborundum (600 mesh, Nacalai Tesque Co., Ltd.) to inoculate the virus. Three days after virus inoculation, the GFP fluorescence spots corresponding to the infection and replication sites of ToMV were counted and compared with the counts in the control (water treatment group) to test the viral infection suppression rate of each drug. The infection control rate (control value) was calculated using the following formula (2). Infection suppression rate (control value) % = 100 - {(average number of fluorescent spots on treated plants) / (average number of fluorescent spots on control treated plants)} × 100 ... (2) The results are shown in Tables 1 and 2. Furthermore, the proanthocyanidin-containing compositions of Comparative Examples 5 and 6 were spoiled and browned, and therefore were not evaluated. [Industrial applicability]

[0062] The proanthocyanidin-containing composition obtained by the production method of the present invention has high antiviral activity and is therefore useful as a control agent against various plant viruses and plant pathogens.

Claims

1. A method for producing a proanthocyanidin-containing composition by purifying proanthocyanidins from Alpinia zerumbet juice (A), Step (1) involves coexisting a solution (AB) containing ginger lily juice (A) and a first preservative (B) with porous particles (C), allowing proanthocyanidins to be adsorbed onto the porous particles (C), thereby obtaining adsorbed porous particles (D) on which the proanthocyanidins have been adsorbed. The process includes step (2) of obtaining a proanthocyanidin concentrated solution (G) by coexisting the adsorbent porous particles (D) with a solution (EF) containing a second preservative (E) and ethanol (F), thereby desorbing the proanthocyanidin from the adsorbent porous particles (D). The content of the first preservative (B) in the solution (AB) is 0.02 to 0.5% by weight based on the weight of the solution (AB). A method for producing a proanthocyanidin-containing composition, wherein the content of the second preservative (E) in the solution (EF) is 0.0025 to 0.2% by weight based on the weight of the solution (EF).

2. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein in step (1), the weight ratio of the porous particles (C) to the weight of the proanthocyanidin contained in the solution (AB) is 4.2 to 42 mg / g.

3. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein in step (1), the coexistence of the solution (AB) and the porous particles (C) is such that the solution (AB) and the porous particles (C) coexist in a solution (ABC) containing the solution (AB) and the porous particles (C), and the time for which the solution (AB) and the porous particles (C) coexist is 2 to 12 hours.

4. A method for producing a proanthocyanidin-containing composition according to claim 3, wherein in step (1), the temperature of the solution (ABC) is 4 to 30°C.

5. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein in step (2), the coexistence of the adsorbent porous particles (D) and the solution (EF) containing the second preservative (E) and the ethanol (F) is such that the adsorbent porous particles (D) and the solution (EF) coexist in a solution (DEF) containing the adsorbent porous particles (D) and the solution (EF) containing the second preservative (E) and the ethanol (F), and the temperature of the solution (DEF) is 4 to 30°C.

6. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein in step (2), the solution (EF) is an aqueous solution containing 20 to 50% by volume of ethanol based on the volume of the solution (EF).

7. A method for producing a proanthocyanidin-containing composition according to claim 1, comprising the step (3) of distilling off the ethanol from the proanthocyanidin concentrate (G).

8. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein the first preservative (B) is at least one selected from the group consisting of sodium pyrosulfite, potassium pyrosulfite, and gallic acid.

9. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein the second preservative (E) is at least one selected from the group consisting of sodium pyrosulfite, potassium pyrosulfite, and gallic acid.

10. A method for producing a proanthocyanidin-containing composition according to claim 1, wherein the average value of the most frequent radius of the pore size of the porous particles (C) is 10 to 120 nm.

Images