Duzhong leaf powder and its manufacturing method

Abstract

To provide a powdery composition that contains a high proportion of plant-derived exosome and a method for producing the same.SOLUTION: Eucommia ulmoides leaf powder comprises exosome derived from Eucommia ulmoides leaves, wherein the number of particles of extractable exosome is at least 2.37×108 / mg.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a powdered processed product of Eucommia ulmoides leaves and a method for producing the same. [Background technology]

[0002] Recent research has revealed that extracellular vesicles (EVs) are used for intercellular communication. They are classified into exosomes, microvesicles (MVs), and other types based on their size and biological function. Among these, vesicles with diameters of 50–150 nm are called exosomes, and their in vivo functions have been rapidly elucidated in recent years. Among exosome research, the relationship with cancer has received particular attention. Because cancer cells are known to release 20% more exosomes than normal, exosomes are considered an important tool in cancer tissue. Furthermore, because exosomes are released in the early stages of cancer, even before cancer symptoms appear, research is underway to utilize them as markers for early cancer detection. Conversely, exosomes produced from mesenchymal stem cells and plant-derived exosomes have been shown to have tumor-suppressing effects, drawing attention as research subjects.

[0003] The discovery of "exosomes" dates back about 30 years ago, but at the time they were only recognized as waste products of cells, and their function and significance remained unknown for a long time. However, as soon as it was shown in 2008 that exosomes could contain nucleic acid substances including mRNA and miRNA and be passed on to other cells, related research has accelerated in recent years and continues to this day.

[0004] In recent years, researchers have focused on the presence of miRNAs released extracellularly via exosomes. Aiming to establish non-invasive and highly sensitive biomarkers, they have been exploring diagnostic and prognostic markers using miRNAs in the serum of patients with hematopoietic tumors, such as leukemia and malignant lymphoma. miRNAs are small 18-22 nt RNAs known to bind to mRNAs intracellularly and regulate their transcription. When this research began around 2008, the mechanisms and significance of the extracellular presence of miRNAs in body fluids, including serum, were not fully understood. However, recent collaborative research between the Departments of Hematology and Molecular Pathology at Tokyo Medical University has revealed significant decreases in miR-92a in the serum of patients with acute leukemia, malignant lymphoma, and myeloma, and a number of other studies have been published (see, for example, Non-Patent Document 1).

[0005] Recently, it has become clear that exosomes are involved in various biological phenomena, such as cell differentiation and aging, and immune system regulation, and there is no doubt that this research will continue to develop in the future. Exosomes are just the tip of the iceberg among extracellular vesicles, and many aspects remain unknown. Research will likely be conducted using a variety of approaches, from biology to clinical research, and in the field of biology, compositions containing large amounts of exosomes are necessary and sought after for advancing research.

[0006] Eucommia leaves are one of the three major traditional Chinese medicines, and are rich in geniposidic acid, eucomisin A, asperuloside, and polyphenols, making them an extremely useful food with many nutritional components. Therefore, it is preferable to eat the entire eucommia leaf; however, because eucommia leaves contain a hard rubber component called gutta verka, they are difficult to grind, making it difficult to obtain fine, uniform particles. Furthermore, to efficiently extract the beneficial components contained in eucommia leaves, a technique is needed to powder them without raising the temperature as much as possible. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kadota Osamu et al., The forefront of extracellular vesicle and exosome research: Aiming for clinical application, Japanese Pharmacological Journal, 2017, Vol. 149, No. 3, pp. 119-122 Summary of the Invention [Problem to be solved by the invention]

[0008] Plant-derived exosomes are found in extremely small amounts in plants, and various plants have been tested around the world. An objective of the present invention is to discover a powdery composition containing a high content of plant-derived exosomes and a method for producing the same. [Means for solving the problem]

[0009] In order to solve the above problems, the present inventors conducted extensive research and, as a result of investigating a method for producing a eucommia leaf powder that would exhibit a vivid green color when dried and pulverized, discovered a method for producing eucommia leaf powder that contains a large amount of eucommia leaf-derived exosomes. That is, the present invention includes the following embodiments.

[0010] (1) Eucommia leaf powder containing exosomes derived from Eucommia leaves, the number of extractable exosome particles being at least 2.37 × 10 8 Eucommia leaf powder characterized by having a concentration of 100mg / mg. (2) Eucommia leaf powder described in (1) exhibits a green color with a hue of 5GY to 10G, a brightness of 5 to 8, and a saturation of 6 to 10 in the Munsell color system. (3) A heating process in which the raw Eucommia leaves are heated to 50-70°C to prevent fermentation, a cutting process in which the heated Eucommia leaves are cut into rectangles with sides measuring 1mm-3cm, and a hot air drying process in which the cut Eucommia leaves are dried with hot air at 30-60°C to reduce their moisture content to 20% or less. A method for producing mulberry leaf powder, comprising a freeze-drying process in which the mulberry leaves after hot air drying are pre-freezed at -15 to -25°C and then freeze-dried at a temperature below -70°C, and a grinding process in which the freeze-dried mulberry leaves are ground using a grinder to a particle size of 1 to 40 μm. (4) A manufacturing method described in (3), in which the fermentation heating step maintains the fresh Eucommia leaves at 65°C for 2 minutes. (5) A manufacturing method according to (3) or (4), wherein the moisture content of the eucommia leaves after hot air drying is 5 to 15%. (6) An extraction step of extracting the Eucommia leaf powder obtained by the method according to any one of (3) to (5) with purified water, phosphate buffered saline, or alkaline ionized water to obtain an extract, and a step of recovering exosomes from the precipitate obtained by ultracentrifugation of the extract, wherein at least 2.37 × 10 exosomes are recovered from 1 mg of Eucommia leaf powder. 8 A method for producing exosomes derived from Eucommia leaves, characterized by the ability to recover individual exosomes. [Effects of the Invention]

[0011] The Eucommia ulmoides leaf powder of the present invention exhibits a vivid green color because heat denaturation during the manufacturing process is minimized. Furthermore, the freeze-drying process destroys the structure of hard rubber components such as geniposidic acid, resulting in a fine, water-soluble, and smooth powder. Therefore, it is possible to provide Eucommia ulmoides leaf powder that is easy to extract exosomes from, has a high exosome content, and does not lose other nutritional components. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a process diagram illustrating an outline of the method for producing Eucommia leaf powder of this embodiment. [Figure 2] Figure 2 is a process diagram outlining the method for producing exosomes from Eucommia leaf powder in this embodiment. [Figure 3] Figure 3 is a chart showing the results of measuring the particle number and particle size distribution of exosomes extracted from the Eucommia leaf powder of this embodiment with Milli-Q water. [Figure 4] Figure 4 is a chart showing the results of measuring the particle number and particle size distribution of exosomes extracted with PBS from the Eucommia leaf powder of this embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0013] Next, various embodiments of the present invention will be described with reference to the drawings. Note that the various embodiments described below do not limit the scope of the invention as claimed, and not all of the elements and combinations thereof described in the various embodiments are necessarily essential to the solution of the present invention.

[0014] (Morium leaf powder) According to one embodiment of the present invention, the Eucommia leaf powder contains exosomes derived from Eucommia leaves, and the number of extractable exosome particles is at least 2.37 × 10 8 This value is the number of exosomes contained in the Eucommia leaf powder of this embodiment when extracted with water. On the other hand, when extracted with phosphate-buffered saline (PBS), the number of exosomes per 1 mg of Eucommia leaf powder is 5.72 × 10 8 When extracted with alkaline ionized water, 2.85 x 10 exosomes were extracted per 1 mg of Eucommia leaf powder. 9 Therefore, the number of exosome particles that can be extracted from the Eucommia leaf powder of this embodiment is preferably about 5 × 10 8 More preferably, about 1.0 × 10 9 The upper limit of the number of exosome particles contained in the Eucommia ulmoides leaf powder of this embodiment is not particularly limited, but it will usually be about 100 million to 1 billion particles / mg.

[0015] The number of exosomes and their average particle size can be measured by any method known in the art, including wet methods that observe exosomes in solution using electrical resistive nanopulse (qNANO) and nanoparticle tracking, and dry methods that observe exosomes while maintaining their morphology using a transmission electron microscope.

[0016] In another aspect, the Eucommia leaf powder of this embodiment exhibits a vivid green color when in powder form or when dissolved in water, and can be consumed as a sweet Eucommia tea. In a typical embodiment, the green color of this Eucommia tea beverage is within the range of hue 5GY to 10G, value 5 to 8, and saturation 6 to 10 on the Munsell color system.

[0017] The Munsell color system is a type of numerical representation of color based on three attributes: a color cube. Color charts are arranged using cylindrical coordinates, with lightness (V) on the vertical axis, hue (H) on the circumferential axis, and saturation (C) on the radial axis. A spatial arrangement of these color charts is called a color cube. Hue (H) is represented by the first two digits, with the first digit ranging from 0 to 10 and representing the deviation from the hue represented by the second digit, such as Y (yellow), YR (yellow-red), or R (red). Next, lightness (V) is represented by a number ranging from 0 (complete black) to 10 (complete white). Finally, saturation (C) is represented by a number starting from 0 (achromatic) and increasing as the color becomes more vivid. Note that a slash ( / ) is placed between the lightness and saturation numbers for distinction. The Munsell color system is measured based on JIS Z 8721 (1944).

[0018] (Method for producing Duzhong leaf powder) In another embodiment of the present invention, the method for producing Eucommia leaf powder can be, for example, based on the steps shown in Figure 1. Specifically, the method includes a heating step S101 for heating fresh Eucommia leaves, a cutting step S102 for cutting the Eucommia leaves whose fermentation has been inhibited by heating, a hot air drying step S103 for reducing the moisture content of the cut Eucommia leaves to about 20%, a freeze-drying step S104, and a crushing step S105 for crushing the freeze-dried Eucommia leaves. Each step is described in detail below.

[0019] The raw material, Eucommia ulmoides (du zhong) leaves, refers to those harvested and not yet subjected to heat-drying. They may be cultivated or naturally grown. For example, fresh current-year leaves before they fall off (e.g., fresh leaves harvested in Kanagawa Prefecture between April and October, preferably between May and August, and more preferably between July and August), can be used. Eucommia ulmoides (du zhong) leaves are prone to self-destruction and deteriorate rapidly. Furthermore, cut Eucommia ulmoides (du zhong) leaves deteriorate rapidly upon cutting. Therefore, fermentation must be prevented immediately by heating the harvested leaves. Specifically, fresh Eucommia ulmoides (du zhong) leaves are harvested early in the morning without damaging them. To prevent fermentation immediately, in the heating step S101, they are heated at 50°C to 70°C, preferably at approximately 65°C, for 1 to 10 minutes, preferably approximately 5 minutes. The heating method is not particularly limited, but can be, for example, by blowing hot air from a heater heated to 350°C. After heating, it is preferable to rapidly cool the product with cold air at 0 to 5°C to remove residual heat, in order to maintain the high exosome content in the resulting powder and its vivid green color.

[0020] In the cutting step S102, the heat-treated Eucommia leaves are cut into rectangles with sides measuring 1 mm to 3 cm using a cutter. To facilitate drying in the subsequent process, the size of the cut Eucommia leaves is preferably 1 cm square or less, and more preferably 5 mm square or less.

[0021] In the hot air drying step S103, the cut Eucommia leaves are dried with hot air at 30 to 60°C, and the moisture content is preferably reduced to 20% or less. In order to more efficiently perform the subsequent freeze-drying step, it is more preferable that the moisture content after hot air drying be approximately 5 to 15%.

[0022] In the freeze-drying process S104, the Eucommia leaves, whose moisture content has been reduced by hot air drying, are stored in a freezer at -15 to 25°C for 10 days to thoroughly pre-freeze them over time. By freezing them over a long period of time, the ice balls formed by the moisture in the Eucommia leaves can be frozen more finely. This produces finely granular ice, resulting in finer cell destruction. The leaves are then placed in a vacuum freeze dryer and frozen at -70°C, preferably -80°C, for 6 hours. The frozen Eucommia leaves are then slowly sublimated and dried (sublimated) at -10°C under high vacuum for 6 hours. Once dried, the Eucommia leaves are preferably heated to 30°C under high vacuum for 8 hours to further dry them. This reduces the moisture content to 1%.

[0023] In the pulverization step S105, an airflow pulverizer is used in an environment where the temperature is kept between 0° C. and 50° C. or less to obtain powder with an average particle size of 1 to 40 microns. As for pulverization conditions, it is preferable to perform the pulverization at 35° C. or less to obtain powder with a particle size of approximately 10 μm or less.

[0024] (Exosome manufacturing method) In another embodiment, the method for producing exosomes includes a solvent extraction step S201 in which the Eucommia leaf powder obtained by the above-mentioned method is extracted with purified water, phosphate-buffered saline, or alkaline ionized water to obtain an extract, and an ultracentrifugation / recovery step S202 in which exosomes are recovered from the precipitate obtained by ultracentrifuging this extract.

[0025] The extraction solvent used in the solvent extraction step S201 is not particularly limited as long as it is water or an aqueous solution, and examples thereof include purified water (Milli-Q water), phosphate buffered saline (PBS), and alkaline ionized water.

[0026] As used herein, the term "alkaline ionized water" refers to water containing alkaline ions obtained by electrolysis, and is produced separately from acidic water using an ionized water purifier or the like. The pH of this alkaline ionized water should be approximately pH 8 to 11. This allows for more efficient extraction and recovery of exosomes from Eucommia leaves. The pH value of the alkaline ionized water can be adjusted appropriately by changing the electrolysis current value of the ionized water purifier.

[0027] In an exemplary embodiment, the ultracentrifugation / recovery step S202 may be carried out through a series of ultracentrifugation steps including ultracentrifugation using a sucrose cushion density gradient at 100,000 to 200,000×g for 1 to 6 hours and ultracentrifugation using an iodixanol density gradient. TM (Optiprep) Multi-Purpose Density Gradient Centrifugation Media and other media are commercially available.

[0028] (Action and effect) As shown in the examples below, compared to grapefruit-derived exosomes, which are known to have a high content of plant-derived exosomes, Eucommia leaves contain approximately 200 times more exosomes. Therefore, the Eucommia leaf powder of this embodiment may more effectively exert the pharmacological effects of bioactive substances such as miRNA and proteins contained in Eucommia leaf-derived exosomes. Furthermore, Eucommia leaf-derived exosomes can be prepared in large quantities, which is advantageous for use in research on drug delivery systems, etc. [Example]

[0029] (Example 1) Production of Eucommia Leaf Powder Three kilograms of fresh eucommia leaves were exposed to hot air from a heater heated to 350°C for two minutes, instantly raising the surface temperature to 65°C to prevent fermentation. The cooled eucommia leaves were cut into 3mm strips parallel to the thick central vein using a cutter, and then the strips were cut perpendicularly to 3mm strips to obtain 3mm square leaves. The finely cut eucommia leaves were then dried with hot air at 45°C for 20 minutes. The moisture content of the dried eucommia leaves was measured using an infrared moisture meter FD-610 (Kett Electric Laboratory Co., Ltd.) and found to be 10%. These were then stored in a -20°C freezer for 10 days in preparation for vacuum freeze-drying, allowing the eucommia leaves to be thoroughly pre-frozen over time.

[0030] Finely chopped and pre-frozen Eucommia leaves were placed in a freeze dryer (Tokyo Rikaki Co., Ltd., Model FD0780) and kept at -80°C for 6 hours. The water was then evaporated under high vacuum at -10°C for 6 hours, followed by slow freeze-drying. The dried Eucommia leaves were then heated to 30°C under the same high vacuum for 8 hours to further dry them. This resulted in dried Eucommia leaves with a moisture content of 1%.

[0031] The freeze-dried Eucommia leaves were pulverized in an airflow pulverizer at a temperature of 35°C or less to obtain a fine powder with a particle size of 9 μm. When the obtained powder was expressed in the Munsell color system using a standard color chart conforming to JIS Z 8721 "Color representation method - representation by three attributes," it exhibited a green hue of 5G, a brightness of 5-8, and a chroma of 6-10.

[0032] (Example 2) Extraction of exosomes from Eucommia ulmoides leaf powder 250 mg of the Eucommia leaf powder produced in Example 1 was added to 100 mL of purified water (Milli-Q water), PBS, and alkaline ionized water, respectively, and stirred at room temperature for 2 hours to completely dissolve. After standing at room temperature for another hour, the supernatant of each solution was collected and centrifuged at high speed at 2000 g for 10 minutes. The supernatant was recovered and filtered through a 0.22 μm filter, and 55 mL of the resulting filtrate was ultracentrifuged at 100,000 × g for 1 hour. The precipitate obtained by ultracentrifugation was suspended in 120 μL of PBS, and the particle count was measured using a nanoparticle tracking system.

[0033] The precipitate suspended in PBS was further diluted with PBS and analyzed using the NanoSight nanoparticle analysis system (LM10, software version 2.03) manufactured by Japan Quantum Design Co., Ltd. As shown in Table 1, the measured particle count was multiplied by the dilution factor to calculate the particle concentration and total particle count in the 120 μL solution recovered by ultracentrifugation. Furthermore, the number of particles per 1 mg of Eucommia leaf was calculated from the amount of Eucommia leaf powder used in the 55 mL of filtrate used in ultracentrifugation.

[0034] Figure 3 shows the results of measuring the particle number and particle size distribution of exosomes extracted with Milli-Q water. The average particle size was 129.3 nm, and the concentration of the measurement sample (150-fold diluted) was 1.81 × 10 9 The average particle size was 131.9 nm, and the concentration of the sample (500-fold diluted) was 1.31 × 10 9 From these results, the number of particles contained in 1 mg of Eucommia leaf powder was 2.37 × 10 8 (solvent: MilliQ Water), 5.72 x 10 8 (solvent PBS) and 2.85 x 10 9 (alkaline ionized water). Also, one teaspoon (approximately 1.5 g) contains 8.57 x 10 11 (solvent: PBS), 3.55 x 10 11 (solvent: MilliQ Water) and 4.275 x 10 12It was estimated that alkaline ionized water contained exosomes in the amount of particles equivalent to 10 ...

[0035] [Table 1]

[0036] (Comparative Example 1) The dried grapefruit powder was extracted with purified water in the same manner as in Example 2, and then exosomes were collected from the precipitate obtained by ultracentrifugation. The obtained exosome solution contained 2 × 10 9 In contrast, the exosome solution extracted and recovered from Eucommia leaf powder at the same time had a concentration of 5 × 10 11 The amount was approximately 250 times that of grapefruit.

Claims

[Claim 1] A method of producing a fermented food product, comprising: a heating step of heating fresh Eucommia leaves at 50-70°C to prevent fermentation; a cutting step of cutting the heat-treated Eucommia leaves into rectangular shapes with each side having a length of 1 mm to 3 cm; a hot air drying step of drying the cut Eucommia leaves with hot air at 30 to 60°C to reduce the moisture content to 20% or less; a freeze-drying step of pre-freezing the hot-air-dried Eucommia leaves at −15 to −25° C. and then freeze-drying them at a temperature of −70° C. or lower; a grinding step of grinding the freeze-dried Eucommia leaves using a grinder to a particle size of 1 to 40 μm; an extraction step of extracting the Eucommia leaf powder produced by the method comprising the steps of: extracting the Eucommia leaf powder with purified water, phosphate buffered saline, or alkaline ionized water to obtain an extract; A step of recovering exosomes from the precipitate obtained by ultracentrifugation of the extract; and from 1 mg of the Eucommia leaf powder, at least 2.37 x 10 ８ A method for producing exosomes derived from Eucommia ulmoides leaves, characterized in that 2.85 x 10 9 exosomes can be recovered from each sample.

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