JP7822566B2 - Duzhong leaf powder and its manufacturing method - Google Patents

Description

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

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 in recent years, their function in the body has been rapidly elucidated. 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, before cancer symptoms appear, research is underway to utilize them as a marker for early cancer detection. Conversely, exosomes produced from mesenchymal stem cells and plant-derived exosomes have been shown to have cancer-suppressing effects, drawing attention as research subjects.

The discovery of "exosomes" dates back approximately 30 years ago, but initially they were simply recognized as waste products of cells, and their function and significance remained unknown for a long time. However, in 2008, it was suggested that exosomes may contain nucleic acid substances, including mRNA and miRNA, and be passed on to other cells. As soon as this was demonstrated, related research has accelerated rapidly in the last few years, continuing to this day.

In recent years, attention has been focused on the presence of miRNA released extracellularly via exosomes, and efforts have been made to establish non-invasive, highly sensitive biomarkers by searching for diagnostic and prognostic markers using miRNA in the serum of patients with hematopoietic tumors, such as leukemia and malignant lymphoma. miRNAs are a type of small RNA consisting of 18-22 bases, and are known to bind to mRNA intracellularly and regulate its transcription. When this research began around 2008, the mechanism and significance of the presence of miRNA in body fluids (extracellular), including serum, was not well understood. However, recent collaborative research between the Departments of Hematology and Molecular Pathology at Tokyo Medical University has revealed a significant decrease in miR-92a in the serum of patients with acute leukemia, malignant lymphoma, and myeloma, and a number of other reports have been published (see, for example, Non-Patent Document 1).

Recently, it has become clear that exosomes are involved in a variety of biological phenomena, including 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, but in the field of biology, compositions containing large amounts of exosomes are necessary and sought after for advancing research.

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. For this reason, 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, and it is difficult to obtain fine, uniform particles. Furthermore, in order to efficiently extract the useful components contained in eucommia leaves, a technique is needed to powder them without raising the temperature as much as possible.

Kadota Osamu et al., The forefront of extracellular vesicle and exosome research: Aiming for clinical application, Japanese Pharmacology Journal, 2017, Vol. 149, No. 3, pp. 119-122

Plant-derived exosomes are found in extremely small amounts in plants, and various plants around the world have been tested. The objective of the present invention is to discover a powdered composition containing a high content of plant-derived exosomes and a method for producing the same.

In order to solve the above-mentioned problems, the inventors conducted extensive research and, while 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. Specifically, the present invention includes the following embodiments.

(1) A Duzhong leaf powder containing exosomes derived from Duzhong leaves, characterized in that the number of extractable exosome particles is at least 2.37 × 10 ⁸ / 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 step in which the raw 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-3 cm; and a hot air drying step in which the cut Eucommia leaves are dried with hot air at 30-60°C to reduce their moisture content to 20% or less.
The method for producing eucommia leaf powder includes a freeze-drying step in which the eucommia leaves after hot air drying are pre-freezed at -15 to -25°C and then freeze-dried at a temperature of -70°C or lower, and a crushing step in which the eucommia leaves after freeze-drying are crushed using a crusher to have a particle size of 1 to 40 μm.
(4) A manufacturing method described in (3), in which the fresh Eucommia leaves are maintained at 65°C for 2 minutes during the fermentation heating step.
(5) A manufacturing method described in (3) or (4), in which the moisture content of the eucommia leaves after hot air drying is 5 to 15%.
(6) A method for producing exosomes derived from Eucommia ulmoides leaves, comprising: an extraction step of extracting Eucommia ulmoides 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 x ¹⁰ exosomes can be recovered from 1 mg of Eucommia ulmoides leaf powder.

The Eucommia ulmoides leaf powder of the present invention exhibits a vibrant green color because denaturation due to heating is minimized during the manufacturing process. Furthermore, the freeze-drying process destroys the structure of hard rubber components such as geniposidic acid, resulting in a finely granulated, smooth powder that dissolves easily in water. Therefore, it is possible to provide Eucommia ulmoides leaf powder that is easy to extract the exosomes contained in this Eucommia ulmoides leaf powder, contains a high amount of exosomes, and does not lose other nutritional components.

FIG. 1 is a process diagram illustrating an outline of the method for producing Eucommia leaf powder of this embodiment. Figure 2 is a process diagram outlining the method for producing exosomes from Eucommia leaf powder of this embodiment. FIG. 3 is a chart showing the results of measuring the particle number and particle size distribution of exosomes extracted from the Eucommia ulmoides leaf powder of this embodiment with Milli-Q water. 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.

Next, each embodiment of the present invention will be described with reference to the drawings. Note that the embodiments described below do not limit the invention as defined in the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

(Morium leaf powder)
According to one embodiment of the present invention, the Eucommia leaf powder contains exosomes derived from Eucommia leaves and is characterized in that the number of extractable exosome particles is at least 2.37 x 10 ⁸ particles/mg. 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), 5.72 x 10 ⁸ exosomes are extracted per 1 mg of Eucommia leaf powder, and when extracted with alkaline ionized water, 2.85 x 10 ⁹ exosomes can be obtained per 1 mg of Eucommia leaf powder. Therefore, the number of extractable exosome particles contained in the Eucommia leaf powder of this embodiment is preferably at least about 5 x 10 ⁸ particles/mg, and more preferably at least about 1.0 x 10 ⁹ particles/mg. The upper limit of the number of exosome particles contained in the Eucommia leaf powder of this embodiment is not particularly limited, but is typically around 10 to 1 billion particles/mg.

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

In another aspect, the Eucommia leaf powder of this embodiment exhibits a vivid green color when in powder form or dissolved in water, and can be consumed as 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.

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. The first digit ranges from 0 to 10 and represents the deviation from the second digit's 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). Finally, chroma (C) is represented by a number starting from 0 (achromatic) and increasing in number as the color becomes more vivid. Note that a "/" (slash) is inserted between the lightness and saturation numbers for distinction. The Munsell color system is measured in accordance with JIS Z 8721 (1944).

(Method for producing Eucommia 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.

The raw material, Eucommia ulmoides leaves, refer to those harvested and not yet subjected to heat-drying. They may be cultivated or naturally grown. For example, current-year leaves before they fall off, harvested in Kanagawa Prefecture between April and October, preferably between May and August, and more preferably between July and August, can be used. Eucommia leaves are prone to self-destruction and deteriorate quickly. Furthermore, cut Eucommia leaves deteriorate rapidly upon cutting. Therefore, fermentation must be prevented immediately by heating the harvested leaves. Specifically, Eucommia ulmoides 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 quickly cool the product with cold air at 0-5°C to remove residual heat, in order to maintain the high exosome content in the resulting powder and its vivid green color.

In the cutting process S102, a cutter is used to cut the heat-treated du zhong leaves into rectangular pieces with sides measuring 1 mm to 3 cm. To facilitate drying in the subsequent process, the size of the cut du zhong leaves is preferably 1 cm square or less, and more preferably 5 mm square or less.

In the hot air drying step S103, the cut Eucommia leaves are dried with hot air at 30-60°C, preferably reducing the moisture content to 20% or less. To more efficiently perform the subsequent freeze-drying step, it is more preferable to reduce the moisture content after hot air drying to approximately 5-15%.

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, allowing them to be thoroughly pre-frozen over time. By freezing slowly, the ice balls formed by the moisture within 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) under high vacuum at -10°C 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 allows the moisture content to reach 1%.

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, resulting in powder with an average particle size of 1 to 40 microns. The pulverization conditions are preferably 35°C or less, to obtain powder with a particle size of approximately 10 μm or less.

(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-described 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.

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), alkaline ionized water, etc.

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 as needed by changing the electrolysis current value of the ionized water purifier.

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. Commercially available density gradient solutions include, for example, OptiPrep ^™ multi-purpose density gradient centrifugation media.

(Action and effect)
As will be 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 potently 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 1: Production of Eucommia Leaf Powder. 3 kg of fresh Eucommia leaves were exposed to hot air from a heater heated to 350°C for 2 minutes, instantly raising the leaf surface to 65°C to prevent fermentation. The cooled Eucommia leaves were cut into 3 mm strips parallel to the thick vein in the center of the leaf using a cutter, and then the strips were cut perpendicularly to 3 mm strips to obtain 3 mm 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 (manufactured by 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.

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 Eucommia leaves dried in this way 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%.

The freeze-dried Eucommia leaves were pulverized in an airflow pulverizer at a temperature of 35°C or below, yielding a fine powder with a particle size of 9 μm. When expressed in the Munsell color system using a standard color chart in accordance with JIS Z 8721 "Color representation - representation by three attributes," the resulting powder exhibited a green hue of 5G, a brightness of 5-8, and a saturation of 6-10.

Example 2: Extraction of exosomes from Eucommia ulmoides leaf powder. 250 mg of the Eucommia ulmoides leaf powder prepared 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 2,000 g for 10 minutes. The supernatant was collected and filtered through a 0.22 μm filter. 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.

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 leaves was calculated from the amount of Eucommia leaf powder used in the 55 mL filtrate used in ultracentrifugation.

Figure 3 shows the results of measuring the particle count 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 dilution) was 1.81 x ¹⁰ particles/mL. Figure 4 shows the results of measuring the particle count and 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 x ¹⁰ particles/mL. From these results, the number of particles contained per mg of Eucommia leaf powder was 2.37 x ¹⁰ particles (solvent Milli-Q Water), 5.72 x ¹⁰ particles (solvent PBS), and 2.85 x ¹⁰ particles (solvent alkaline ionized water). Furthermore, it was calculated that one teaspoon (approximately 1.5 g) contains 8.57 × 10 ¹¹ particles (solvent: PBS), 3.55 × 10 ¹¹ particles (solvent: MilliQ Water), and 4.275 × 10 ¹² particles (alkaline ionized water).

(Comparative Example 1)
Dried grapefruit powder was extracted with purified water using the same method as in Example 2, and exosomes were recovered from the precipitate obtained by ultracentrifugation. The resulting exosome solution had a concentration of 2 x ¹⁰ cells/mL. In contrast, the exosome solution extracted and recovered from Eucommia ulmoides leaf powder at the same time had a concentration of 5 x ¹⁰ cells/mL, approximately 250 times the amount of grapefruit.

Claims (1)

A heating process in which the Eucommia leaves are heated to 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 capable of recovering at least 2.37 x ¹⁰ to 2.85 x ¹⁰ exosomes from 1 mg of the Eucommia leaf powder.