Method for producing a composition for enhancing the infectivity of arbuscular mycorrhizal fungi

Abstract

The present invention provides a method for producing a composition that acts on arbuscular mycorrhizal fungi to enhance their ability to infect plants and promote symbiosis between arbuscular mycorrhizal fungi and plants, and provides a method for producing the composition that offers improved production efficiency compared to conventional techniques. [Solution] An extract is obtained by extracting a plant of the Gentianaceae family with 40-60% by weight of ethanol-containing alcohol (ethanol content of the ethanol-containing alcohol is 80% by weight or more), and the obtained extract is used as a composition for enhancing the infectivity of arbuscular mycorrhizal fungi.

Description

[Technical Field]

[0001] This invention relates to a method for producing a composition that enhances the infectivity of arbuscular mycorrhizal fungi to plants and promotes symbiosis. [Background technology]

[0002] Approximately 90% of land plants live in symbiosis with fungi in their roots. This symbiosis helps plants efficiently utilize essential elements and other nutrients in the soil. When such fungi invade plant roots and establish a symbiotic relationship, the symbiotic structure formed between the fungi and the plant is called a "mycorrhizae," and the symbiotic phenomenon is called "mycorrhizal symbiosis."

[0003] Arbuscular mycorrhizal fungi (AM), also known as veesicular arbuscular mycorrhizal fungi (VA), are a type of endomycorrhizal fungi in which hyphae penetrate the cell walls of plant roots and interact with the cytoplasm. Most agricultural plants are thought to benefit from symbiosis with arbuscular mycorrhizal fungi, improving the efficiency of their utilization of phosphorus (more specifically, possibly in the form of phosphate) and other nutrients in the soil. In this symbiosis, regardless of differences in plant and mycorrhizal fungal species, a common structure called an arbuscule is formed. The arbuscule is a structure formed by hyphae that penetrate the contour of the cell wall of the plant root, invaginate (not "penetrate") the plant cell membrane, and become significantly branched and tree-like. This tree-like structure of the arbuscule significantly increases the interaction area between the mycorrhizal fungi and the plant cytoplasm. On the other hand, mycorrhizal fungal hyphae that extend and branch outside the plant cell wall enable the acquisition of nutrients from a wider soil area and increase the opportunity for infection of new plant root regions.

[0004] Phosphorus is an essential element for living organisms, and mycorrhizal symbiosis allows plants to grow even in soils with relatively low phosphorus levels. There is a global problem of phosphate rock depletion, which could affect the production of phosphorus-based fertilizers, and Japanese soil has a high phosphorus adsorption capacity, making it difficult for plants to absorb phosphorus. Therefore, there is a growing expectation for the benefits of more actively utilizing mycorrhizal fungi in agriculture.

[0005] However, although mycorrhizal fungal soil conditioners (VA mycorrhizal fungal materials) that use arbuscular mycorrhizal fungi as active ingredients are already commercially available, they are expensive and not always satisfactory in terms of the stability of their effects. Consequently, despite being the only microbial resource specified in the Soil Fertility Improvement Law, VA mycorrhizal fungal materials have not become widespread.

[0006] Patent Document 1 describes a method for promoting symbiosis between plants and mycorrhizal fungi by applying a gibberellin synthesis inhibitor in an amount used as a growth inhibitor or less. Patent Document 2 describes an agent for promoting infection by arbuscular mycorrhizal fungi in plants, comprising chitin oligosaccharides and / or chitin nanofibers. Patent Document 3 describes a symbiosis promoter for arbuscular mycorrhizal fungi, comprising oxidized glutathione and / or cystathionine as active ingredients.

[0007] Non-patent document 1 describes how the plant hormone gibberellin promoted the formation of Paris-type mycorrhizae by arbuscular mycorrhizal fungi in lisianthus, while suppressing the formation of Arum-type mycorrhizae in chives and other plants. Non-patent document 2 describes how the plant component strigolactone promotes hyphal branching of arbuscular mycorrhizal fungi.

[0008] Strigolactone is a particularly promising arbuscular mycorrhizal fungal infection promoter in terms of its stability of effect, but it also has undesirable effects, such as suppressing the branching of the plant itself and stimulating seed germination of parasitic plants like Striga and Orobanchus, which infest agriculturally important crops. It also has drawbacks such as being unstable in water and being very expensive.

[0009] The environments in which plants, such as agricultural or horticultural plants, are grown are diverse, and therefore it is beneficial to provide new options for compositions and methods that can be used to promote symbiosis between plants and mycorrhizal fungi. In particular, (1) there is a need to enhance the usefulness of VA mycorrhizal fungal materials, and (2) since the response of plants to substances that stimulate mycorrhizal fungi to send signals, such as gibberellins, can vary greatly depending on the plant species, it may be preferable to provide compositions and methods that can act directly on the mycorrhizal fungi rather than compositions and methods that are thought to act mainly on the plant side, as described in Patent Documents 1 and 2.

[0010] Conventionally, research and development has been conducted on compositions that enhance the ability of arbuscular mycorrhizal fungi to infect plants and promote symbiosis between arbuscular mycorrhizal fungi and plants. For example, Patent Document 1 proposes a technique for using a composition containing secoiridoid glycosides, iridoid glycosides, or combinations thereof as active ingredients to enhance the infectivity of arbuscular mycorrhizal fungi. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2023 / 167242

[0012] On the other hand, there is room for improvement in such compositions and their manufacturing methods from the standpoint of manufacturing efficiency and the long-term shelf life of the resulting compositions. [Overview of the project] [Problems that the invention aims to solve]

[0013] Under these circumstances, a continuing technical challenge is to provide a method for producing a composition that acts on arbuscular mycorrhizal fungi to enhance their ability to infect plants and promote symbiosis between arbuscular mycorrhizal fungi and plants, and which offers improved production efficiency compared to conventional techniques.

[0014] Therefore, the object of the present invention is to provide a method for producing a composition that acts on arbuscular mycorrhizal fungi to enhance their ability to infect plants and promote symbiosis between arbuscular mycorrhizal fungi and plants, and which offers improved production efficiency of the composition compared to the prior art. [Means for solving the problem]

[0015] As a result of diligent research, the inventors have found that the above-mentioned problems can be solved by extracting plants of the Gentianaceae family with 40-60% by weight of ethanol-containing alcohol, and using the obtained extract as a composition for enhancing the infectivity of arbuscular mycorrhizal fungi. In other words, the gist of the present invention is as follows.

[0016] [1] A method for producing a composition for enhancing the infectivity of arbuscular mycorrhizal fungi, comprising a glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton, comprising an extraction step of obtaining an extract by extracting a plant of the Gentianaceae family with 40-60% by weight of ethanol-containing alcohol, The ethanol-containing alcohol contains 80% by weight or more of ethanol. The aforementioned manufacturing method. [2] The manufacturing method according to [1], further comprising an immersion step of immersing the gentian plant in ethanol-containing alcohol at a concentration of 50 to 100% by weight, prior to the extraction step. [3] The manufacturing method according to [1] or [2], further comprising a dilution step after the extraction step to obtain a dilution by diluting the extract. [4] In the dilution step, the extract is diluted so that the ethanol-containing alcohol concentration in the diluted product is 1 to 20% by weight, according to the production method described in [3]. [5] The composition for enhancing the ability of arbuscular mycorrhizal fungi infection further contains secoiridoid glycosides, iridoid glycosides or a combination thereof, according to the production method described in any one of [1] to [4]. [6] The glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton is any one selected from the group consisting of gentiopicroside, swertiamarin, loganin, geniposide and oleuropein, according to the production method described in any one of [1] to [5]. [7] A composition for enhancing the ability of arbuscular mycorrhizal fungi infection obtained by the production method described in any one of [1] to [6]. [Advantages of the Invention]

[0017] According to the present invention, there is provided a method for producing a composition that acts on arbuscular mycorrhizal fungi, enhances the ability of arbuscular mycorrhizal fungi to infect plants, and promotes the symbiosis between arbuscular mycorrhizal fungi and plants, and a production method with improved production efficiency compared to the prior art can be provided.

[0018] [Method for Producing Composition for Enhancing Ability of Arbuscular Mycorrhizal Fungi Infection] According to one aspect of the present invention, there is provided a method for producing a composition (hereinafter, also simply referred to as "the composition of the present invention") that acts on arbuscular mycorrhizal fungi, enhances the ability of arbuscular mycorrhizal fungi to infect plants, and promotes the symbiosis between arbuscular mycorrhizal fungi and plants (hereinafter, also simply referred to as "AM fungal infection ability"). (Hereinafter, also simply referred to as "the production method of the present invention").

[0019] [Composition for Enhancing AM Infection Ability] The composition of the present invention is an AM-infectious composition and contains as an active ingredient a glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton. The description of this glycoside also applies to the secoiridoid glycoside and iridoid glycoside described above. In this specification, the term "3,4-dihydropyran ring skeleton" means the ring skeleton of compound 3,4-dihydropyran, that is, a heterosix-membered ring formed from one oxygen atom and five carbon atoms, having one unsaturated (i.e., double bond) carbon atom adjacent to the oxygen atom. The structure of compound 3,4-dihydropyran is shown below. [ka]

[0020] In the present invention, the positions of atoms in the 3,4-dihydropyran ring skeleton are indicated by the numbering shown in the structural formula above. The compound name "3,4-dihydropyran" originally indicates that the double bond between the 3rd and 4th positions of the parent compound pyran is saturated, and additional hydrogen atoms are bonded to the 3rd and 4th positions, respectively. However, the term "3,4-dihydropyran ring skeleton" used herein does not require that hydrogen atoms be bonded to the 3rd and 4th positions, respectively. For example, embodiments in which no hydrogen atoms are bonded to the 3rd or 4th position are possible.

[0021] In fact, the 3,4-dihydropyran ring skeleton contained in the glycoside in this embodiment can be modified with a wide variety of substituents, and it has been found that even when modified with such a variety of substituents, the activity that enhances the infectivity of AM bacteria is maintained. Therefore, it is thought that the common 3,4-dihydropyran ring skeleton itself contributes to the activity together with the glucopyranose ring described later. Accordingly, the term "glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton" in this specification includes those that have various substituent modifications in addition to this basic structure.

[0022] For example, in some embodiments, the substituent (R 1 ) at the 3-position of the 3,4-dihydropyran ring skeleton can be an alkyl group having 1 to 6 carbon atoms or an unsaturated chain hydrocarbon group having 2 to 6 carbon atoms, and can be, for example, a vinyl group or an ethylidene group. In another embodiment, the substituent (R 1 ) at the 3-position and the substituent (R 2 ) at the 4-position of the 3,4-dihydropyran ring skeleton can be linked together to form a saturated or unsaturated five-membered ring. That is, this five-membered ring will be condensed with the 3,4-dihydropyran ring skeleton. In yet another embodiment, the 5-position (R 3 ) of the 3,4-dihydropyran ring skeleton can have an ester group (-COO-R 11 ), where R 11 can be an alkyl group having 1 to 6 carbon atoms such as methyl or an alkenyl group having 2 to 6 carbon atoms. Alternatively, R 11 can be linked to the substituent (R 2 ) at the 4-position of the 3,4-dihydropyran ring skeleton to form a saturated or unsaturated hetero six-membered ring. That is, this hetero six-membered ring will be condensed with the 3,4-dihydropyran ring skeleton. In this particular embodiment, the 4-position of the 3,4-dihydropyran ring skeleton may further have a hydroxyl group. In a particular embodiment, the above alkyl group or unsaturated hydrocarbon group at the 3-position (R 1 ) and the above hetero six-membered ring fused at the 4- and 5-positions can coexist. In another particular embodiment, the above ester group (R 3 ) that does not form a ring at the 5-position and the above five-membered ring fused at the 3- and 4-positions can coexist. In another particular embodiment, the above alkyl group or unsaturated hydrocarbon group at the 3-position (R 1 ) and the above ester group (R 3 ) that does not form a ring at the 5-position can coexist. In this particular case, the substituent (R 2 ) at the 4-position is typically a carboxymethyl group or its ester (-CH2-COOR 12 ). R 12This can be, for example, an alkyl group having 1 to 6 carbon atoms, or an arylalkyl group, and the divalent alkyl portion can be, for example, a C1 to C6 group (e.g., ethyl). In some embodiments, the hydrogen atom at position 6 of the 3,4-dihydropyran ring skeleton is not substituted (i.e., R 4 (=H)

[0023] The general formula (I) below represents the glycoside in which a glucopyranose ring is O-glycosidically linked to the 3,4-dihydropyran ring skeleton described above. [ka]

[0024] In the glycosides of each embodiment described above, an oxygen atom is bonded to the 2 position of the 3,4-dihydropyran ring skeleton, and glucopyranose (β-D-glucopyranose) (R) is bonded via this oxygen atom. 5 ) is linked by a glycosidic bond. That is, glucopyranose (or glucose) is linked to the 2nd position of the 3,4-dihydropyran ring skeleton by an O-glycosidic bond at the anomeric carbon (i.e., the 1st carbon of pyranose).

[0025] Examples of suitable glycosides as active ingredients that can be used in the embodiments described above include gentiopicroside, swertiamarin, loganin, geniposide, and oleuropein, and any combination of these may be included as the active ingredient. These glycosides are encompassed by general formula (I). Gentiopicroside is particularly preferred due to its high activity and high content in gentian plants, which allows for efficient extraction. These compounds appear to be stable in water. Therefore, in one preferred embodiment, the composition of the present invention includes gentiopicroside as a glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton.

[0026] The structural formula of gentiopicroside is shown below. [ka]

[0027] The structural formula of swertiamarin is shown below. [ka]

[0028] The structural formula of geniposide is shown below. [ka]

[0029] The structural formula of loganin is shown below. [ka]

[0030] The structural formula of oleuropein is shown below. [ka]

[0031] In one embodiment, the composition of the present invention may include, in addition to the glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton described above, a secoiridoid glycoside, an iridoid glycoside, or a combination thereof. The groups of secoiridoid glycosides and iridoid glycosides themselves are well known and are mainly found in plants. Iridoid glycosides are characterized in that a five-membered ring is condensed to the 3,4-dihydropyran ring skeleton described above at the [c] condensation position of pyrane in the skeleton, and glycosides are formed at the 2-position of the 3,4-dihydropyran ring skeleton. Furthermore, a secoiridoid glycoside is an iridoid glycoside in which the five-membered ring is cleaved and replaced by another group.

[0032] (extraction process) The present invention's manufacturing method includes a step of extracting an extract from a plant of the Gentianaceae family with 40-60% by weight of ethanol-containing alcohol (i.e., an extraction step).

[0033] The "ethanol-containing alcohol" used in the extraction process is not particularly limited as long as it contains 80% or more ethanol by weight. It may be ethanol itself (i.e., 100% ethanol by weight), or a mixture of ethanol and other liquid components. Examples of other liquid components include water and alcohols other than ethanol. Examples of ethanol-containing alcohols include industrial alcohol and brewing alcohol.

[0034] Examples of plants belonging to the Gentianaceae family include, but are not limited to, Gentiana scabra, Gentiana manshurica, Gentiana triflora, Gentiana lutea, Swertia japonica, and any combination thereof. Gentianaceae plants may be used whole or in part. Parts of Gentianaceae plants may include, for example, roots, stems, rhizomes, leaves, and flowers, or combinations thereof (e.g., underground parts, above-ground parts, or the whole plant). Of these, roots are particularly preferred. For example, the main component of Ryutan (gentian root), a crude drug made from dried gentian roots and rhizomes, is known to be a glycoside such as gentiopicroside, in which glucopyranose is O-glycosidically linked to the 2-position of a 3,4-dihydropyran ring skeleton. Furthermore, all parts of the Swertia japonica plant—flowers, leaves, stems, and roots—possess a strong bitter taste, and a decoction of the dried entire plant is consumed as an extremely bitter "Swertia japonica tea." The bitter components are mainly glycosides such as swertiamarin, in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton. In fact, the inventors have found that aqueous or organic solvent extracts from dried plants of the Gentianaceae family can provide mycorrhizal fungal infection-enhancing activity, similar to isolated compounds. This is thought to be mainly due to the glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton. In other words, the glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton, which are the active ingredients of compositions for enhancing arbuscular mycorrhizal fungal infection activity, can be provided in the form of extracts from plants of the Gentianaceae family.

[0035] Furthermore, gentian plants may be used in a dried state. A dried material is defined as having a moisture content of 15% by mass or less. Drying and / or fragmenting gentian plants is preferable because it destroys the plant tissue and promotes the release or extraction of active compounds.

[0036] In the extraction process, plants of the Gentianaceae family are extracted with ethanol-containing alcohol at a concentration of 40-60% by weight, preferably 45-60% by weight, and more preferably 50-60% by weight. By extracting plants of the Gentianaceae family with the above-mentioned ethanol-containing alcohol concentration, an extract containing a large amount of glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton can be obtained compared to extraction under other conditions. Furthermore, by extracting plants of the Gentianaceae family with the above-mentioned ethanol-containing alcohol concentration, it is possible to suppress the decrease in the amount of glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton contained in the extract when the resulting extract is stored for a long period of time. Therefore, by extracting plants of the Gentianaceae family with the above-mentioned ethanol-containing alcohol concentration, the storage stability of the composition of the present invention can be improved.

[0037] In the extraction process, the amount of ethanol-containing alcohol mixed with the gentian plant is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type and part of the gentian plant. For example, the amount of ethanol-containing alcohol per 10g of gentian plant can be 150-300g, 40-70g, etc.

[0038] In one embodiment, the extraction step uses ethanol-containing alcohol with an ethanol content of 80% by weight. In another embodiment, the extraction step uses ethanol-containing alcohol with an ethanol content of 85% by weight. In yet another embodiment, the extraction step uses ethanol-containing alcohol with an ethanol content of 85.5% by weight.

[0039] In the extraction process, the temperature of the ethanol-containing alcohol mixed with the gentian plant is not particularly limited as long as the effects of the present invention are achieved, and can be set appropriately depending on the type and part of the gentian plant, but is preferably 5 to 35°C, more preferably 15 to 25°C. By setting the temperature of the ethanol-containing alcohol in the extraction process within the above range, glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton can be sufficiently extracted, while suppressing the risks associated with the volatilization of the ethanol-containing alcohol.

[0040] The extraction time is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type and part of the Gentianaceae plant, the temperature of the ethanol-containing alcohol, the extraction temperature, etc. For example, it can be 5 minutes to 24 hours, 10 minutes to 1 hour, 10 to 40 minutes, 10 to 30 minutes, 15 to 25 minutes, etc., preferably 15 to 25 minutes, and particularly preferably 20 minutes.

[0041] The extraction process may be carried out under light-shielding conditions as necessary. Furthermore, during the extraction process, operations to promote the extraction of glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton (e.g., stirring) may or may not be performed, but such operations are preferred. Specifically, the extraction process is carried out in a mixer equipped with stirring blades while stirring the gentian plant and ethanol-containing alcohol.

[0042] After extraction under the conditions described above, the mixture is separated by filtration or centrifugation to obtain the filtrate or supernatant as the extract. Methods for filtering the mixture include, for example, filtering the mixture using filter paper and plastic filters, or filtering (rough filtering) the mixture using nonwoven fabric or a metal strainer, and then further filtering it using a plastic filter.

[0043] The obtained extract can be used as is in the composition of the present invention, or it can be fractionated by a method known to those skilled in the art (for example, by utilizing differences in molecular weight, affinity column binding, solvent solubility, etc.) to purify and isolate the glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton, and use it. Alternatively, the obtained extract or product can be mixed with other components acceptable in the field of fertilizers to form the composition of the present invention. Examples of other components acceptable in the field of fertilizers include preservatives and fertilizer components. The timing of mixing the obtained extract or product with other components acceptable in the field of fertilizers is not particularly limited and can be appropriately set depending on the type and amount of other components acceptable in the field of fertilizers, but if the manufacturing method of the present invention includes a dilution step described later, it can be mixed in the dilution step. The obtained extract or product can also be provided as a solution (for example, an aqueous solution) or in a dry state (for example, attached to or mixed with a granular solid substrate as described later) and can be sprayed or mixed into the soil. Alternatively, the composition can be applied directly to cultured AM bacteria.

[0044] (Soaking process) The manufacturing method of the present invention may further include, in addition to the extraction step described above, an immersion step in which a plant of the Gentianaceae family is immersed in ethanol-containing alcohol. The ethanol-containing alcohol used in the immersion step may be the same as that described in the "extraction step" section above. The immersion step is performed before the extraction step. In the immersion step, operations to promote the extraction of glycosides in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton (e.g., stirring) may or may not be performed, but it is preferable not to perform such operations.

[0045] In the immersion step, the concentration of the ethanol-containing alcohol into which the gentian plants are immersed is 50 to 100% by weight, preferably 55 to 100% by weight, more preferably 60 to 100% by weight, even more preferably more than 60% by weight and 100% by weight or less, and particularly preferably 100% by weight. By immersing the gentian plants in ethanol-containing alcohol of the above concentration before the extraction step, the storage stability of the composition of the present invention can be improved.

[0046] In the immersion process, the amount of ethanol-containing alcohol mixed with the gentian plant is not particularly limited as long as the effects of the present invention are achieved, and can be appropriately set depending on the type and part of the gentian plant. For example, the amount of ethanol-containing alcohol per 10g of gentian plant can be 150-300g, 40-70g, etc.

[0047] In one embodiment, an ethanol-containing alcohol with an ethanol content of 80% by weight is used in the immersion step. In another embodiment, an ethanol-containing alcohol with an ethanol content of 85% by weight is used in the immersion step. In yet another embodiment, an ethanol-containing alcohol with an ethanol content of 85.5% by weight is used in the immersion step.

[0048] In the immersion process, the temperature of the ethanol-containing alcohol mixed with the gentian plant is not particularly limited as long as the effects of the present invention are achieved, and can be set appropriately depending on the type and part of the gentian plant, but is preferably 5 to 35°C, more preferably 15 to 25°C. By setting the temperature of the ethanol-containing alcohol in the immersion process within the above range, the storage stability of the composition of the present invention can be improved, while the risk caused by the volatilization of the ethanol-containing alcohol can be suppressed.

[0049] The immersion time is not particularly limited as long as the effects of the present invention are achieved, and can be set appropriately depending on the type and part of the Gentianaceae plant, the temperature of the ethanol-containing alcohol, the immersion temperature, etc., but is preferably 10 to 60 minutes, more preferably 15 to 50 minutes, and even more preferably 20 to 40 minutes.

[0050] (Dilution process) The manufacturing method of the present invention may further include a step of diluting the extract obtained in the extraction step described above to obtain a dilution.

[0051] In the dilution step, the solution used to dilute the extract is not particularly limited as long as the effects of the present invention are achieved, but for example, water can be used. Furthermore, when mixing the extract with other components acceptable in the fertilizer field as described above in the dilution step, the other components acceptable in the fertilizer field can be dissolved in the solution used to dilute the extract and then mixed with the extract.

[0052] In the dilution step, the extract is diluted so that the concentration of ethanol-containing alcohol in the dilution is preferably 1 to 20% by weight, more preferably 2 to 15% by weight, and even more preferably 2 to 10% by weight. For example, if the ethanol content in the ethanol-containing alcohol in the extraction step is 80% by weight, in the dilution step the extract is diluted so that the concentration of ethanol in the dilution is preferably 0.8 to 16% by weight, more preferably 1.6 to 12% by weight, and even more preferably 1.6 to 8% by weight. Also, if the ethanol content in the ethanol-containing alcohol in the extraction step is 85% by weight, in the dilution step the extract is diluted so that the concentration of ethanol in the dilution is preferably 0.85 to 17% by weight, more preferably 1.7 to 12.75% by weight, and even more preferably 1.7 to 8.5% by weight. Furthermore, if the ethanol content in the ethanol-containing alcohol during the extraction process is 85.5% by weight, in the dilution process, the extract is diluted so that the ethanol concentration in the dilution is preferably 0.855 to 17.1% by weight, more preferably 1.71 to 12.825% by weight, and even more preferably 1.71 to 8.55% by weight. By diluting the extract so that the ethanol-containing alcohol in the dilution falls within the above range, it is possible to suppress the decrease in the abundance of glycosides in the diluted extract (i.e., the dilution) in which glucopyranose is O-glycosidically bonded to the 2-position of the 3,4-dihydropyran ring skeleton, thereby improving the storage stability of the composition of the present invention.

[0053] In one preferred embodiment, the manufacturing method of the present invention includes an immersion step of immersing a plant of the Gentianaceae family in ethanol-containing alcohol at a concentration of 50 to 100% by weight, an extraction step of extracting an extract from the immersed Gentianaceae plant with ethanol-containing alcohol at a concentration of 40 to 60% by weight, and a dilution step of diluting the obtained extract so that the ethanol-containing alcohol concentration in the resulting dilution is 1 to 20% by weight. [Examples]

[0054] The embodiments of this disclosure will be described in more detail below with reference to examples, but these examples are merely illustrative, and the present invention is not limited to these examples.

[0055] Example 1: Investigation of ethanol-containing alcohol concentration in the extraction process The ethanol-containing alcohol concentration in the extraction process was investigated according to the following procedure. First, industrial alcohols with an ethanol content of 85.5% by weight (100% by weight, 80% by weight, 60% by weight, 40% by weight, and 20% by weight) were prepared as ethanol-containing alcohols. Furthermore, dried chips of gentian (Nakaya Hikojuro Pharmacy), a crude drug made from the dried roots and rhizomes of gentian (Gentiana spp.), were prepared. Next, 20 g of dried gentian chips were pulverized for 1 minute using a benchtop grinder (Y-308B, manufactured by Yamamoto Electric Co., Ltd.) to obtain dried gentian powder. 180 g of each concentration of industrial alcohol was added to the obtained dried gentian powder, and the mixture was stirred for 1 hour to extract the liquid. Next, the liquid was filtered through filter paper and a 0.45 μm filter to obtain gentian extract. Finally, gentiopicroside in the obtained gentian extract was quantified by HPLC. The results are shown in Table 1. In the table, "GPS" refers to gentiopicroside.

[0056] [Table 1]

[0057] The results shown in Table 1 indicate that extracts obtained by adding 40-60% by weight of industrial alcohol to dried gentian powder achieved particularly high gentiopicroside recovery. On the other hand, extracts obtained by extraction with 80% by weight of industrial alcohol showed a slightly lower gentiopicroside recovery compared to extracts obtained with 40% or 60% by weight of industrial alcohol. Furthermore, extracts obtained by extraction with 100% by weight of industrial alcohol showed a significantly lower gentiopicroside recovery compared to extracts obtained with 40% or 60% by weight of industrial alcohol. In addition, extracts obtained by extraction with 20% by weight of industrial alcohol produced excessive foam, making filtration impossible.

[0058] Example 2: Investigation of the effect of the immersion process on storage stability The effect of the immersion process on the storage stability of the extract was investigated according to the following procedure. First, 2 g of dried chips of gentian (Nakaya Hikojuro Pharmacy), a crude drug made from the dried roots and rhizomes of gentian (Gentiana spp.), were processed according to the same procedure as in Example 1 to obtain dried gentian powder. Next, the obtained dried gentian powder was immersed in 44.8 g of 100% by weight industrial alcohol (ethanol content 85.5% by weight) for 30 or 60 minutes. Next, water was added to the immersed material so that the industrial alcohol concentration became 40% by weight, and extraction was carried out with stirring for 20 minutes to obtain the extract. Next, the obtained extract was filtered through filter paper and a 0.45 μm filter to obtain gentian extract. On the other hand, gentian extract produced by conventional methods was obtained according to the same procedure as above, except that the immersion process was omitted. Next, gentiopicroside in the obtained gentian extract was quantified by HPLC. Next, the obtained gentian extract was placed in a glass bottle and stored at a constant temperature of 40°C for accelerated testing, and gentiopicroside was quantified by HPLC at three different time points. The results are shown in Table 2. In Table 2, "April 22" refers to the time before storage at a constant temperature of 40°C, while "May 13," "May 30," and "June 19" refer to the time after storage at a constant temperature of 40°C.

[0059] [Table 2]

[0060] The results shown in Table 2 indicate that by performing an immersion step using industrial alcohol before the extraction step, the storage stability of the resulting gentian extract is improved, and the decrease in the abundance of gentiopicroside contained in the gentian extract can be suppressed.

[0061] Example 3: Investigation of the effect of the dilution process on storage stability The effect of dilution steps on the storage stability of extracts was investigated according to the following procedure. First, dried chips of gentian (Gentiana spp.), a crude drug made from dried roots and rhizomes, were processed according to the same procedure as in Example 1 to obtain dried gentian powder. Next, 550 g of 40% by weight industrial alcohol was added to 10 g of the obtained dried gentian powder, and the mixture was stirred for 1 hour to extract the extract. Next, the obtained extract was diluted with water to obtain a gentian extract at an industrial alcohol concentration (i.e., ethanol-containing alcohol concentration) of 10% by weight. On the other hand, a gentian extract was obtained according to the same procedure as above, except that the dilution step was omitted. Next, gentiopicroside in the obtained gentian dilution was quantified by HPLC. Next, the obtained gentian dilution was placed in a glass bottle and stored at a constant temperature of 40°C for accelerated stability testing, and gentiopicroside was quantified by HPLC at three different time points. The results are shown in Table 3. In Table 3, "immediately after preparation" refers to the time before the product is left to stand and stored at a constant temperature of 40°C, while "4 days later," "15 days later," and "21 days later" refer to the time after the product has been left to stand and stored at a constant temperature of 40°C (number of days after standing storage).

[0062] [Table 3]

[0063] The results shown in Table 3 indicate that the storage stability of the gentian extract is improved by the dilution step after the extraction step, and the decrease in the abundance of gentiopicroside contained in the gentian extract can be suppressed.

Claims

1. A method for producing a composition for enhancing the infectivity of arbuscular mycorrhizal fungi, comprising a glycoside in which glucopyranose is O-glycosidically bonded to the 2-position of a 3,4-dihydropyran ring skeleton, comprising an extraction step of obtaining an extract by extracting a plant of the Gentianaceae family with 40 to 60% by weight of ethanol-containing alcohol, The ethanol-containing alcohol contains 80% by weight or more of ethanol. The aforementioned manufacturing method.

2. The manufacturing method according to claim 1, further comprising an immersion step of immersing the gentian plant in ethanol-containing alcohol at a concentration of 50 to 100% by weight, prior to the extraction step.

3. The manufacturing method according to claim 1, further comprising a dilution step after the extraction step to obtain a dilution by diluting the extract.

4. The manufacturing method according to claim 3, wherein in the dilution step, the extract is diluted so that the ethanol-containing alcohol concentration in the dilution is 1 to 20% by weight.

5. The manufacturing method according to claim 1, wherein the composition for enhancing the infectivity of arbuscular mycorrhizal fungi further comprises a secoiridoid glycoside, an iridoid glycoside, or a combination thereof.

6. The method for producing the product according to claim 1, wherein the glycoside having a glucopyranose O-glycosidically linked to the 2-position of the 3,4-dihydropyran ring skeleton is one selected from the group consisting of gentiopicroside, swertiamarin, loganin, geniposide, and oleuropein.

7. A composition for enhancing the infectivity of arbuscular mycorrhizal fungi, obtained by a manufacturing method described in any one of claims 1 to 6.

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