Eucommia ulmoides leaf powder and method for producing the same

Abstract

The present invention provides a powdered composition containing a high concentration of plant-derived exosomes and a method for producing the same. [Solution] The method includes a heating step of heating fresh Eucommia leaves to 50-70°C to prevent fermentation, a cutting step of cutting the heated Eucommia leaves into rectangular shapes with sides of 1 mm to 3 cm in length, a hot air drying step of drying the cut Eucommia leaves with hot air at 30-60°C to reduce their moisture content to 20% or less, a freeze-drying step of pre-freezing the hot air-dried Eucommia leaves at -15-25°C and then freeze-drying them at a temperature of -70°C or lower, and a grinding step of grinding the freeze-dried Eucommia leaves using a grinder to obtain particle sizes of 1-40 μm.

Description

[Technical Field]

[0001] This 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. Based on their size and biological function, EVs are classified into exosomes, microvesicles (MVs), and other types. Among these, vesicles with a diameter of 50-150 nm are called exosomes, and their function in vivo is being rapidly elucidated in recent years. Among exosome research, the link to cancer is of particular interest. Cancer cells are known to release 20% more cancer-derived exosomes, so exosomes are considered an important tool in cancer tissue. Furthermore, because exosomes are released from the early stages of cancer, even before it shows its signs, research is progressing toward their practical application as an early detection marker for cancer. Conversely, exosomes produced from mesenchymal stem cells and plant-derived exosomes have shown cancer-suppressive effects and are attracting attention as research subjects.

[0003] The discovery of "exosomes" itself dates back about 30 years, but at the time of their discovery, 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 suggested in 2008 that exosomes may contain nucleic acid substances including mRNA and miRNA and be transferred to other cells, related research has accelerated rapidly in recent years, leading to the current state of affairs.

[0004] In recent years, attention has been focused on the presence of miRNAs released extracellularly via exosomes, and research has been conducted to search for diagnostic and prognostic markers using serum miRNAs from hematopoietic malignancies such as leukemia and malignant lymphoma, with the aim of establishing non-invasive and highly sensitive biomarkers. miRNAs are a type of small RNA molecule consisting of 18-22 nucleotides, and are known to bind to mRNA within cells and regulate its transcription. When this research began around 2008, the mechanisms and significance of the presence of miRNAs in body fluids, including serum (extracellularly), were not well understood. However, recently, collaborative research by the Department of Hematology and the Department of Molecular Pathology at Tokyo Medical University has revealed a significant reduction in miR-92a in the serum of patients with acute leukemia, malignant lymphoma, and myeloma, and this has been reported one after another (see, for example, Non-Patent Literature 1).

[0005] Recently, it has become clear that exosomes are involved in various life phenomena such as cell differentiation and aging, and the regulation of the immune system, and this research will undoubtedly continue to develop. Exosomes are just the tip of the iceberg among extracellular vesicles, and many aspects remain unexplained. Research will be conducted through various approaches, from biology to clinical research, but in the field of biology, compositions containing a large amount of exosomes are necessary and in demand for advancing research.

[0006] Eucomycin leaves are one of the three major herbal medicines and are an extremely useful food, rich in nutrients such as geniposidic acid, eucomycin A, asperuloside, and polyphenols. Therefore, it is preferable to consume the entire Eucomycin leaf, but because Eucomycin leaves contain a hard, rubbery component called guttaperca, they are not easy to grind, and it is difficult to obtain fine, uniform particles. Furthermore, in order to efficiently extract the useful components contained in Eucomycin leaves, a technology is needed to powder them while keeping the temperature as low as possible. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Osamu Kadota et al., The Cutting Edge of Extracellular Vesicle and Exosome Research: Aiming for Clinical Application, Japanese Journal of Pharmacology, 2017, Vol. 149, No. 3, pp. 119-122. [Overview of the project] [Problems that the invention aims to solve]

[0008] Plant-derived exosomes are present in very small amounts in plants, and various plants are being tested worldwide. The objective of this invention is to find a powdered composition containing plant-derived exosomes at a high concentration and a method for producing the same. [Means for solving the problem]

[0009] In order to solve the aforementioned problems, the present inventors conducted diligent research and, while investigating a method for producing Eucommia ulmoides leaf powder that exhibits a vivid green color when the leaves are dried and pulverized, discovered a method for producing Eucommia ulmoides leaf powder that contains a large amount of exosomes derived from the leaves. That is, the present invention includes the following embodiments.

[0010] (1) Eucommia leaf powder containing exosomes derived from Eucommia leaves, wherein the number of extractable exosome particles is at least 2.37 × 10 8 Eucommia ulmoides leaf powder characterized by having a concentration of granules / mg. (2) The Eucommia ulmoides leaf powder described in (1), which exhibits a green color in the Munsell color system with a hue of 5GY to 10G, a lightness of 5 to 8, and a saturation of 6 to 10. (3) A heating process in which the fresh Eucommia leaves are heated to 50-70°C to prevent fermentation; a cutting process in which the heated Eucommia leaves are cut into rectangular shapes with sides of 1 mm to 3 cm in length; 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 Eucommia ulmoides leaf powder, comprising: a freeze-drying step of pre-freezing Eucommia ulmoides leaves after hot-air drying at -15 to -25°C, and then freeze-drying them at a temperature of -70°C or lower; and a grinding step of grinding the freeze-dried Eucommia ulmoides leaves using a grinder to obtain a particle size of 1 to 40 μm. (4) The manufacturing method according to (3), wherein in the fermentation heating step, the fresh Eucommia leaves are maintained at 65°C for 2 minutes. (5) The manufacturing method according to (3) or (4), wherein the moisture content of the Eucommia leaves after hot air drying is 5 to 15%. (6) The process includes an extraction step of extracting Eucommia ulmoides leaf powder obtained by any of the methods in (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 this extract, wherein at least 2.37 × 10¹⁶ exosomes are obtained from 1 mg of Eucommia ulmoides leaf powder. 8 A method for producing exosomes derived from Eucommia ulmoides 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 its manufacturing process minimizes denaturation due to heat. Furthermore, the freeze-drying process breaks down the structure of hard, rubbery components such as geniposidic acid, resulting in a fine, water-soluble, and smooth powder. Therefore, it is possible to provide an Eucommia ulmoides leaf powder in which exosomes can be easily extracted, have a high exosome content, and do not lose other nutritional components. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a process diagram illustrating the outline of the method for producing Eucommia ulmoides leaf powder according to this embodiment. [Figure 2] Figure 2 is a process diagram illustrating the outline of the method for producing exosomes from Eucommia ulmoides leaf powder according to 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 Eucommia ulmoides leaf powder of this embodiment using MilliQ water. [Figure 4] Figure 4 is a chart showing the results of measuring the particle number and particle size distribution of exosomes extracted from Eucommia ulmoides leaf powder using PBS according to this embodiment. [Modes for carrying out the invention]

[0013] Next, each embodiment of the present invention will be described with reference to the drawings. Note that each embodiment described below does not limit the invention according to the claims, and not all of the elements and combinations thereof described in each embodiment are essential for the solution means of the present invention.

[0014] (Eucommia ulmoides leaf powder) The Eucommia ulmoides leaf powder according to one embodiment of the present invention contains exosomes derived from Eucommia ulmoides leaves, and the number of extractable exosome particles is at least 2.37×10 8 per mg. This value is the number when the exosomes contained in the Eucommia ulmoides leaf powder of this embodiment are extracted with water. On the other hand, when extracted with phosphate buffered saline (PBS), 5.72×10 8 exosomes per mg of Eucommia ulmoides leaf powder are extracted, and further, when extracted with alkaline ion water, 2.85×10 9 exosomes per mg of Eucommia ulmoides leaf powder can be obtained. Therefore, the number of extractable exosome particles contained in the Eucommia ulmoides leaf powder of this embodiment is preferably about5×10 8 per mg or more, more preferably about 1.0×10 9 per mg or more. The upper limit of the number of exosome particles contained in the Eucommia ulmoides leaf powder of this embodiment is not particularly limited, but is usually about 100 million to 1 billion per mg.

[0015] The number of exosomes and their average particle size can be measured by any measurement method in the relevant technical field. Examples of measurement methods include wet measurement methods for observing exosomes in a solution state, such as the electrical resistance nanopulse method (qNANO) and the nanoparticle tracking method, and dry measurement methods for observing the morphology of exosomes with a transmission electron microscope while maintaining their form.

[0016] In other respects, the Eucommia leaf powder of this embodiment exhibits a vivid green color 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, lightness 5 to 8, and chroma 6 to 10 in the Munsell color system.

[0017] Here, the Munsell color system is a type of numerical representation of color based on three attributes and a color cube. The color samples are arranged using cylindrical coordinates, with lightness V on the vertical axis, hue H in the circumferential direction, and chroma C in the radial direction. The spatial arrangement of these color samples is called a color cube. Hue H is indicated by the first two digits, the first digit ranging from 0 to 10 and the second digit representing the distance from the hue, such as Y (yellow), YR (yellow-red), or R (red). Next, lightness V is represented by a number ranging from 0 (completely dark) to 10 (completely pure white), and finally, chroma C is represented starting from 0 (achromatic) and increasing as the color becomes more vivid. A " / " (slash) is inserted between the lightness and chroma numbers for distinction. The Munsell color system is measured according to JIS Z 8721 (1944).

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

[0019] The Eucommia ulmoides leaves used as raw material refer to leaves harvested and before being subjected to heat drying treatment, and may be cultivated or naturally produced. For example, fresh leaves from the current year before leaf fall can be used. In Kanagawa Prefecture, for example, fresh leaves collected from April to October, preferably from May to August, and more preferably from July to August can be used. Eucommia ulmoides leaves are prone to self-destruction and deteriorate quickly. Furthermore, cut Eucommia ulmoides leaves deteriorate rapidly from the moment they are cut. Therefore, fermentation must be quickly prevented by heating the harvested leaves. Specifically, in the early morning, fresh Eucommia ulmoides leaves are harvested without damaging them, and in the heating process S101, they are heated in an environment of 50°C to 70°C, preferably at a temperature of about 65°C for about 1 to 10 minutes, preferably about 5 minutes, in order to immediately prevent fermentation. The heating method is not particularly limited, but for example, it can be carried out by blowing hot air from a heater heated to a high temperature of 350°C. After heating, it is preferable to rapidly cool the product with cold air at 0-5°C to remove residual heat, as this maintains a high exosome content in the resulting powder and its vibrant green color.

[0020] In cutting step S102, a cutting machine is used to cut the heat-treated Eucommia leaves into rectangular shapes with sides measuring 1 mm to 3 cm in length. The size of the cut Eucommia leaves is preferably 1 cm square or less, and more preferably 5 mm square or less, in order to facilitate drying in subsequent processes.

[0021] In the hot air drying process S103, it is preferable to dry the cut Eucommia leaves with hot air at 30-60°C to reduce their moisture content to 20% or less. To make the subsequent freeze-drying process more efficient, it is even more preferable that the moisture content after hot air drying be approximately 5-15%.

[0022] In the freeze-drying process S104, the Eucommia ulmoides 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 allow for thorough pre-freezing over time. Freezing over time allows for finer freezing of the ice spheres formed by the moisture within the Eucommia ulmoides leaves. This finer ice formation results in less cell damage. Furthermore, these leaves are placed in a vacuum freeze-dryer and frozen at -70°C, preferably -80°C, for 6 hours. The frozen Eucommia ulmoides leaves are then slowly sublimated and dried (sublimated) at -10°C under high vacuum for 6 hours. Preferably, the dried Eucommia ulmoides leaves are then further dried by applying heat at 30°C under high vacuum for 8 hours to enhance the degree of dryness. This allows the moisture content to be reduced to 1%.

[0023] In the grinding step S105, a powder with an average particle size of 1 to 40 microns can be obtained using an air-jet grinder in an environment where the temperature is kept between 0°C and 50°C or lower. Preferably, the grinding is performed at a temperature of 35°C or lower to obtain powder with a particle size of approximately 10 μm or less.

[0024] (Method for producing exosomes) In other embodiments, the method for producing exosomes includes a solvent extraction step S201 in which the Eucommia ulmoides leaf powder obtained by the method described above is extracted with purified water, phosphate-buffered saline, or alkaline ionized water to obtain an extract, and an ultracentrifugation / recovery step S202 in which the extract is ultracentrifuged to recover exosomes from the precipitate obtained.

[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, but examples include purified water (Milli-Q water), phosphate-buffered saline (PBS), and alkaline ionized water.

[0026] In this specification, the term "alkaline ionized water" refers to water containing alkaline ions obtained by electrolysis, and is produced separately along with acidic water using an ion water purifier or similar device. 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 ulmoides leaves. The pH value of the alkaline ionized water can be adjusted as needed by changing the electrolysis current of the ion water purifier.

[0027] The ultracentrifugation / recovery step S202 may, in exemplary embodiments, 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. The density gradient liquid may be, for example, OptiPrep TM (OptiPrep) Multi-purpose density gradient centrifugation media and other such media are commercially available.

[0028] (Effects and Benefits) As shown in the examples described later, Eucommia ulmoides leaves contain approximately 200 times more exosomes than grapefruit-derived exosomes, which are known to have a high exosome content among plant-derived exosomes. Therefore, the Eucommia ulmoides leaf powder of this embodiment may exhibit stronger pharmacological effects from physiologically active substances such as miRNAs and proteins contained in the exosomes derived from Eucommia ulmoides leaves. Furthermore, it is advantageous for preparing large quantities of Eucommia ulmoides leaf-derived exosomes for use in research on drug delivery systems and the like. [Examples]

[0029] (Example 1) Production of Eucommia ulmoides leaf powder 3 kg of fresh Eucommia ulmoides leaves were heated to 350°C and exposed to hot air for 2 minutes to instantly raise the leaf surface temperature to 65°C, thereby preventing fermentation. The cooled Eucommia ulmoides leaves were then cut into 3 mm strips parallel to the thick central vein using a cutting machine. These strips were then cut perpendicularly to the 3 mm strips to obtain 3 mm square leaves. The finely chopped Eucommia ulmoides leaves were then dried with 45°C hot air for 20 minutes. The moisture content of the dried Eucommia ulmoides leaves was measured using an infrared moisture meter FD-610 (manufactured by Kett Scientific Research Institute Co., Ltd.), and the result showed a moisture content of 10%. These were then stored in a -20°C freezer for 10 days to allow for vacuum freeze-drying, ensuring thorough pre-freezing over time.

[0030] Finely chopped and pre-frozen Eucommia leaves were placed in a freeze-dryer (Tokyo Rikaki Co., Ltd., FD0780 model) and maintained at -80°C for 6 hours. Subsequently, the moisture was evaporated slowly at -10°C under high vacuum for 6 hours, performing slow freeze-drying. The dried Eucommia leaves were then treated with heat at 30°C under the same high vacuum for 8 hours to further enhance the dryness. This resulted in dried Eucommia leaves with a moisture content of 1%.

[0031] Freeze-dried Eucommia ulmoides leaves were ground using an air-jet pulverizer at a temperature of 35°C or lower 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 compliant with JIS Z 8721 "Method of indicating color - indication by three attributes", it exhibited a green color in the range of hue 5G, lightness 5-8, and chroma 6-10.

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

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

[0034] Figure 3 shows the results of measuring the number of particles and the 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 dilution) was 1.81×10 9 particles / mL. Figure 4 shows the results of measuring the number of particles and the particle size distribution of exosomes extracted with PBS. The average particle size was 131.9 nm, and the concentration of the measurement sample (500-fold dilution) was 1.31×10 9 particles / mL. From these results, the number of particles contained per 1 mg of Eucommia ulmoides leaf powder was 2.37×10 8 particles (solvent: MilliQ Water), 5.72×10 8 particles (solvent: PBS), and 2.85×10 9 particles (solvent: alkaline ion water). Also, in calculation, one cup of teaspoon (about 1.5 g) contained 8.57×10 11 particles (solvent: PBS), 3.55×10 11 particles (solvent: MilliQ Water), and 4.275×10 12It was hypothesized that it contained exosomes with a particle count of 1 (alkaline ionized water).

[0035] [Table 1]

[0036] (Comparative Example 1) Exosomes were recovered from the precipitate obtained by ultracentrifugation after extraction of dried grapefruit powder with purified water using the same method as in Example 2. The resulting exosome solution was 2 × 10⁻⁶ 9 The concentration was 10 cells / mL. In contrast, the exosome solution extracted and recovered simultaneously from Eucommia ulmoides leaf powder was 5 × 10⁶ cells / mL. 11 This amounted to 1 unit / mL, which is about 250 times the amount found in grapefruit.

Claims

1. A heating process in which fresh Eucommia leaves are heated to 50-70°C to prevent fermentation, A cutting step in which the heat-treated Eucommia leaves are cut into rectangular shapes with sides of 1 mm to 3 cm in length, The aforementioned cut Eucommia leaves are dried with hot air at 30-60°C to reduce their moisture content to 20% or less in a hot air drying process, The aforementioned hot-air-dried Eucommia leaves are pre-frozen at -15 to -25°C, and then freeze-dried at a temperature of -70°C or lower in a freeze-drying process. The freeze-dried Eucommia leaves are ground using a pulverizer to obtain a particle size of 1 to 40 μm in a grinding step, A method for producing Eucommia ulmoides leaf powder containing [the specified ingredient].

2. The manufacturing method according to claim 1, wherein the heating step to inhibit fermentation is maintained at 65°C for 2 minutes.

3. The manufacturing method according to claim 1 or 2, wherein the moisture content of the Eucommia leaves after the hot air drying step is 5 to 15%.

4. The aforementioned Eucommia leaf powder is extractable with purified water, phosphate-buffered saline, or alkaline ionized water, and contains at least 2.37 × 10 ８ The method for producing an exosome according to claim 1 or 2, comprising exosomes / mg.

5. The manufacturing method according to claim 1 or 2, wherein the Eucommia ulmoides leaf powder exhibits a green color in the Munsell color system with a hue of 5 GY to 10 G, a lightness of 5 to 8, and a chroma of 6 to 10.

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