Method for manufacturing acetylcholine-containing composition

Abstract

To provide a method for manufacturing an acetylcholine-containing composition from nightshade.SOLUTION: A method for manufacturing an acetylcholine-containing composition from nightshade includes obtaining acetylcholine from a placenta and an endocarp of a fruit of nightshade and / or a jelly part of a fully ripe fruit of nightshade.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing an acetylcholine-containing composition, for example from a Solanum plant. [Background technology]

[0002] It has been reported in recent years that choline esters have excellent functionality, such as suppressing blood pressure elevation when orally ingested (Non-Patent Document 1). Furthermore, it has been discovered that acetylcholine, a type of choline ester, is specifically and highly concentrated in eggplant fruit (Non-Patent Document 2), and eggplant is now attracting attention as a functional food. The present applicant has been working on developing eggplant cultivation techniques and functional breeding with a focus on food functionality, and has filed patent applications for a method for producing eggplant strains with high acetylcholine content (Patent Application No. 2023-073752) and a simple method for analyzing acetylcholine (Patent Application No. 2023-056633).

[0003] In the anticipated future notification of eggplant as a functional food and the development of processing and utilization technology for eggplant as a functional food material, it is thought that increasing the acetylcholine content of eggplant will be extremely effective in reducing manufacturing costs and improving its appeal.

[0004] On the other hand, Patent Document 1 describes methods of freeze-drying to powder, hot air drying to powder, and extraction with ethanol or aqueous ethanol as inexpensive processing forms for orally ingesting acetylcholine contained in eggplant.

[0005] However, in conventional technologies including Patent Document 1, the acetylcholine content of eggplant dried powder is less than 2.25 mg / g, and the acetylcholine content of the extract is less than 3.6 mg / g (per dry matter), which is low. Therefore, it is necessary to increase the content in order to reduce production costs and improve the appealing effect. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2018 / 070545 [Non-patent literature]

[0007] [Non-Patent Document 1] Nishimura, M., Suzuki, M., Takahashi, R., Yamaguchi, S., Tsubaki, K., Fujita, T., ...& Nakamura, K. (2019). Daily ingestion of eggplant powder improves blood pressure and psychological state in stressed individuals: A randomized placebo-controlled study. Nutrients, 11(11), 2797. [Non-patent document 2] Wang, W., Yamaguchi, S., Suzuki, A., Wagu, N., Koyama, M., Takahashi, A., ... & Nakamura, K. (2021). Investigation of the distribution and content of acetylcholine, a novel functional compound in eggplant. Foods, 10(1), 81. Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above-mentioned circumstances, an object of the present invention is to provide a method for producing an acetylcholine-containing composition from a plant of the genus Solanum. [Means for solving the problem]

[0009] As a result of intensive research to solve the above problems, the inventors have found that a specific part of the fruit of a plant of the genus Solanum contains a high content of acetylcholine, which has led to the completion of the present invention.

[0010] That is, the present invention includes the following. [1] A method for producing an acetylcholine-containing composition from a plant of the genus Solanum, the method comprising a step of obtaining acetylcholine from the placenta and endocarp of the fruit of a plant of the genus Solanum. [2] The method described in [1], wherein the fruit of the Solanum plant is more mature than saleable fruit or is fully ripe fruit. [3] The method described in [2], further comprising obtaining acetylcholine from the jelly portion of the fruit of a plant of the genus Solanum. [4] A method for producing an acetylcholine-containing composition from a plant of the genus Solanum, the method comprising a step of obtaining acetylcholine from the jelly portion of a fully ripe fruit of a plant of the genus Solanum. [5] The method according to any one of [1] to [4], wherein the solanum plant is eggplant (Solanum melongena L.). [Effects of the Invention]

[0011] According to the present invention, a composition containing a high amount of acetylcholine can be provided as a functional food material such as a food additive or a supplement. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the fruit weight and acetylcholine content (summer harvest) of unripe (dashed line) and fully ripe (solid line) Senryo No. 2 fruits. [Figure 2] This is a photograph showing the internal structure of a fully ripe fruit of Senryo No. 2. [Figure 3] This is a photograph showing the internal structure of a ripe Tamita fruit. [Figure 4] 10 is a photograph showing the internal structure of the ripe fruit of the high-content line AE-ACH04. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below.

[0014] The method for producing an acetylcholine-containing composition from a solanum plant according to the present invention (hereinafter referred to as "the method") comprises the step of obtaining acetylcholine from the placenta and endocarp of the fruit of a solanum plant. The method may further comprise the step of obtaining acetylcholine from the jelly portion of the fruit of a solanum plant. Alternatively, the method comprises the step of obtaining acetylcholine from the jelly portion of a fully ripe fruit of a solanum plant. The acetylcholine-containing composition obtained by the method contains a high content of acetylcholine.

[0015] Fruits of Solanum plants are preferably more mature than saleable fruits (e.g., fruits 2-4 weeks after flowering and weighing approximately 100 g), with fully ripe fruits being the most preferable. Fully ripe fruits allow for high concentrations of acetylcholine to be obtained from the jelly portion of the fruit of Solanum plants. Fully ripe fruits refer to fruits that have stopped growing approximately 2-3 months after flowering, with yellow to brown skin in varieties and lines that change skin color, and fruits with a dull skin in varieties and lines that do not change skin color.

[0016] Examples of Solanum plants include eggplant (Solanum melongena L.) and related species. Examples of eggplant include all strains and varieties, including "Senryo No. 2" (F1 variety), the native variety "Minden," and the high-acetylcholine-content strain "AE-ACH04" (Patent Application No. 2023-073752). "AE-ACH04" was deposited on February 24, 2023, with the National Institute of Technology and Evaluation (NITE-IPOD) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number FERM P-22469. Examples of eggplant-related species include horsenettle (S. carolinense), nightshade (S. nigrum), and staghorn sorrel (S. aethiopicum).

[0017] In this method, acetylcholine is obtained from the placenta and endocarp of the fruit of a plant of the genus Solanum, and / or the jelly portion (if the fruit is fully ripe).

[0018] Here, "fruit placenta" refers to the tissue inside each oocyte, bearing many seeds on its outer surface, and enlarging to fill the interior of the fruit after fertilization. "Fruit endocarp" refers to the wall tissue that originates from the inner epidermis of the carpel and forms the boundary with the oocyte. "Fruit jelly" refers to the jelly-like or liquid part found between the placenta and the wall tissue, as well as the water-soaked wall tissue.

[0019] Acetylcholine can be obtained, for example, by cutting off (trimming) parts of the fruit that contain little acetylcholine (e.g., the stalk side where the seeds are not distributed, or the pericarp (mesocarp + exocarp) (mesocarp = flesh, exocarp = skin)), separating the placenta, endocarp, and jelly parts of the fruit, and subjecting the separated parts to, for example, crushing, squeezing, extracting, filtering, or a combination thereof, to obtain an acetylcholine-containing composition.

[0020] For example, the raw separated portion can be heated in a microwave oven, then water is added and the mixture is crushed in a mixer or the like, and the resulting centrifuged supernatant or filtrate can be used as an acetylcholine-containing composition (microwave heating extraction method).

[0021] Alternatively, for example, the freeze-dried sample from the separated portion may be pulverized in a mixer or the like, and the resulting powder may be suspended in 50 times the volume of an extraction buffer (e.g., water), sealed, and heated to 98°C. The suspension is then subjected to ultrasonic treatment, and the centrifugal supernatant is collected. This supernatant can be used as an acetylcholine-containing composition (extract). This extraction process may be repeated two or more times (e.g., two or three times) for the precipitated portion. Furthermore, the resulting extract may be filtered, for example, through a filter with a pore size of 0.45 μm to remove insoluble residues (hot water extraction method after freeze-drying).

[0022] Alternatively, because it is difficult to separate only the seeds from the seed-rich parts (the fruit placenta, endocarp, and jelly), the seed-containing parts can be crushed to prepare a solution, and the insoluble parts containing the seeds can be easily removed by collecting the centrifugal supernatant or by filtering or squeezing the juice. The centrifugal supernatant collected from the crushed solution can be used as an acetylcholine-containing composition.

[0023] Furthermore, the jelly portion alone can be easily separated and recovered by squeezing or other methods. The disrupted liquid, squeezed juice, extract, filtrate, etc. may be subjected to, for example, freeze-drying to obtain a dried product. [Example]

[0024] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0025] [Experimental Method] 1. Test plant For multiple strains and cultivars of Solanum melongena L. cultivated using conventional methods, immature fruits harvested 2-5 weeks after flowering and fully ripe fruits harvested 7 weeks after flowering were used for the study. Similar cultivation was also conducted for closely related species of S. melongena (9 species, 12 strains), and the harvested fruits were used for the study. For some closely related species, plants grown wild in the Tokai region were used for the study.

[0026] 2.Extraction method a) Raw eggplant samples were microwaved in a microwave oven, then water was added and crushed in a mixer or the like, and the centrifuged supernatant or filtrate was frozen and stored as a sample for component analysis.

[0027] b) When freeze-drying raw eggplant samples, the dried sample was pulverized in a mixer or similar, and the resulting powder was suspended in 50 times the volume of extraction buffer (containing an internal standard, etc., if necessary), sealed, and heated to 98°C. This was then sonicated, and the centrifuged supernatant was collected. The precipitate was again suspended in extraction buffer, sonicated, and the centrifuged supernatant was collected. The precipitate was once again suspended in extraction buffer, sonicated, and the centrifuged supernatant was collected. The three centrifuged supernatants were mixed and filtered through a 0.45 μm pore size filter to remove insoluble residue, and the mixture was frozen and stored until analysis.

[0028] 3. Acetylcholine Analysis Quantitative analysis of acetylcholine in eggplant fruit extract was performed according to the acetylcholine concentration measurement method (Japanese Patent Application No. 2023-056633). Specifically, an 80-cm length of unmodified polyimide resin-coated fused silica tubing (75 μm inner diameter, GL Sciences) was used as the capillary. A CE system (Agilent 7100, Agilent) equipped with a photodiode array detector was used to detect major cations by indirect absorption. Depending on the content of the target component, the final dilution ratio of the analytical sample was 1:1000, and the pressure was injected at 50 mbar for 5 seconds. The electrophoresis buffer consisted of 10 mM imidazole, 5 mM 2-hydroxyisobutyric acid (HIBA), 2 mM 18-crown-6-ether, and 0.2% (v / v) acetic acid. A voltage of 25 kV was applied for 10–12 minutes. Before each run, the capillary tube was washed and equilibrated by running the electrophoresis buffer for 5 minutes. The capillary tube was cooled to 25°C. Detection was performed using an indirect absorbance method, with the reference wavelength set at 215 nm and the detection wavelength set at 310 nm, where no absorption occurs, to invert the negative peak detected at 215 nm. Triethylmethylammonium chloride (TEMA) (Tokyo Chemical Industry) was added to the sample as an internal standard, and acetylcholine chloride (Tokyo Chemical Industry), choline chloride (Tokyo Chemical Industry), and a mixed standard solution of major cation components (ammonium ion (NH), potassium ion (K), calcium ion (Ca), sodium ion (Na), and magnesium ion (Mg) (Agilent)) were used as standards. The analyte components were quantified by the internal standard method.

[0029] [Results and Discussion] 1. Examination of extraction method In this example, two extraction methods with different conveniences were used depending on the sample form and amount, as well as the target component content and storage space. These methods, microwave heating extraction (method A) and freeze-drying followed by hot water extraction (method B), were used, and the quantitative analysis of acetylcholine was compared. As a result, it was found that both methods had similar extraction efficiencies and yielded similar quantitative values ​​(data not shown). Therefore, in subsequent experiments, either extraction method was used depending on the situation.

[0030] 2. Relationship between fruit ripeness and acetylcholine content To clarify the changes in acetylcholine content with fruit maturation, we quantified acetylcholine content in fruits at a stage of maturity where seeds are not fully ripened (immature fruits) and fruits at a stage where seeds are fully ripened and have high germination potential (fully ripe fruits, harvested approximately 7 weeks after flowering). As a result (Figure 1), a high correlation was observed between fruit weight and acetylcholine content in immature fruits of the common eggplant cultivar "Senryo No. 2." Because immature fruits increase in size and weight with maturation, it is thought that the higher the maturity (i.e., the longer the maturation period after flowering), the higher the acetylcholine content. In contrast, no correlation was observed between fruit weight and acetylcholine content in fully ripe fruits. The average acetylcholine content in fully ripe fruits was generally twice that of immature fruits.

[0031] Typical commercially available eggplant fruits, such as "Senryo No. 2," weigh approximately 80-120 g and are immature fruits harvested approximately 2-4 weeks after flowering. Compared to the immature fruits (150-250 g) in this example, these fruits are at a more immature stage of maturity. Such commercially suitable fruits are considered to be "marketable fruits." Wang, W., Yamaguchi, S., Suzuki, A., Wagu, N., Koyama, M., Takahashi, A., ... & Nakamura, K. (2021). Investigation of the distribution and content of acetylcholine, a novel functional compound in eggplant. Foods, 10(1), 81, reports that the acetylcholine content in "Senryo No. 2" fruits is approximately 20 mg / kg fresh weight, which is approximately one-third of the content in the immature fruits observed in this example. This low content is likely due to the fact that marketable fruits were the subject of this study.

[0032] From the above, it can be said that if eggplant fruits that are more ripe than saleable fruits are used as the raw material, a composition containing a higher concentration of acetylcholine can be produced.

[0033] 3. Closely related species To date, only the fruits and bamboo shoots of S. melongena have been reported to contain high levels of acetylcholine, while other Solanum vegetables, such as tomatoes and bell peppers, contain very little (Wang, W., S. Yamaguchi, M. Koyama, S. Tian, ​​A. Ino, K. Miyatake, and K. Nakamura. 2020. LC-MS / MS analysis of choline compounds in Japanese-cultivated vegetables and fruits. Foods. 9:1029.). To confirm the presence of acetylcholine in eggplant-related species, we quantified acetylcholine in the fruits of 12 strains of nine species closely related to eggplant S. melongena. High concentrations of acetylcholine were detected in the fruits of 11 strains of six species, including horsenettle (S. carolinense), nightshade (S. nigrum), and oak (S. aethiopicum) (data not shown). This is the first time that high concentrations of acetylcholine have been confirmed in any species other than S. melongena and bamboo, making this study highly novel. Although the confirmed acetylcholine content varies significantly between species and strains, some species have a content exceeding that of S. melongena, making this a promising source of acetylcholine-containing compositions.

[0034] 4. Acetylcholine content in different parts of the fruit As eggplant fruit matures, the formation of internal fruit tissues, such as seeds, progresses, making it easier to visually and physically distinguish each tissue. Therefore, we used fully ripe fruit to separate and extract internal fruit tissues, and quantified the acetylcholine content of each. The results (Figures 2–4, Tables 1–3) revealed significant differences in content between the various parts of the fruit, including the common cultivar "Senryo No. 2," the native cultivar "Minden," and the high-acetylcholine line "AE-ACH04" (Patent Application No. 2023-073752). Since the acetylcholine content in the seeds was significantly lower than in other parts (Table 2), it was revealed that the acetylcholine content in the jelly and placenta around the seeds was significantly higher than in other parts. Wang, W., Yamaguchi, S., Suzuki, A., Wagu, N., Koyama, M., Takahashi, A., ... & Nakamura, K. (2021). Investigation of the distribution and content of acetylcholine, a novel functional compound in eggplant. Foods, 10(1), 81, compared acetylcholine content in different parts of the eggplant, but reported that the differences between parts were extremely small. This is thought to be due to the fact that marketable fruits at an immature stage of maturity were used.

[0035] Furthermore, the acetylcholine content of the stalk (Table 1, upper section) where the seeds are not distributed and the pericarp (mesocarp + exocarp) (corresponding to the mesocarp and exocarp) is lower than that of the area surrounding the seeds. Therefore, by trimming these low-content areas, it is possible to extract a high-acetylcholine composition using only the high-content areas as raw material. The high-content areas referred to here can be considered to include the jelly, placenta, and endocarp. Because it is difficult to separate the seeds from the high-content areas, the insoluble portion containing the seeds can be easily removed by preparing a crushed solution from the seed-containing area and recovering the centrifugal supernatant or by filtering and squeezing the juice. When the acetylcholine content per dry weight of the centrifugal supernatant recovered from the disruption solution was calculated, the maximum content in the area around the seeds was approximately 6,000 mg / kg for "Senryo No. 2" and "Minden," and extremely high at approximately 14,000 mg / kg for the high-content variety "AE-ACH04," as shown in the rightmost column of Tables 1 to 3.

[0036] In fully ripe eggplants, a jelly region was observed between the seeds and the pericarp (Figures 2 and 4), with its quantity and frequency varying significantly depending on the strain, cultivar, and cultivation conditions (data not shown). To date, the presence of a jelly region has not been reported in S. melongena, and this is the first reported case. The presence of a jelly region was observed in several strains and cultivars other than those shown in Figures 2 and 4, as well as in most closely related species tested. This suggests that this trait is common to all Solanum fruits. There are eggplant strains and related species in which the jelly region accounts for an extremely high proportion of the total fruit, approximately 50% on a fresh weight basis (data not shown). Therefore, by utilizing this multi-jelly trait, it is possible to easily separate and recover only the jelly region with a high acetylcholine content by methods such as squeezing.

[0037] [Table 1]

[0038] [Table 2]

[0039] [Table 3] [Accession number]

[0040] FERM P-22469

Claims

1. A method for producing an acetylcholine-containing composition from a Solanum plant, the method comprising the step of obtaining acetylcholine from the placenta and endocarp of the fruit of a Solanum plant.

2. 2. The method according to claim 1, wherein the fruit of the Solanum plant is a fruit that is more mature than saleable fruit or a fully ripe fruit.

3. 3. The method of claim 2, further comprising obtaining acetylcholine from the jelly portion of the fruit of a Solanum plant.

4. A method for producing an acetylcholine-containing composition from a plant of the genus Solanum, the method comprising a step of obtaining acetylcholine from the jelly portion of a fully ripe fruit of a plant of the genus Solanum.

5. The method according to any one of claims 1 to 4, wherein the Solanum plant is eggplant (Solanum melongena L.).

Images