JP7715365B2 - Viral infection treatment containing exosomes derived from Eucommia leaves - Google Patents

Description

The present invention relates to a product made from processed Eucommia leaves. More specifically, the present invention relates to a viral infection ameliorating agent containing a Eucommia leaf processed product, and an ameliorating agent based on small RNAs (miRNA, siRNA, piRNA) among the RNAs encapsulated in Eucommia leaf exosomes. The present invention also relates to an oral composition, pharmaceutical composition, food composition, etc., containing the viral infection ameliorating agent and an ameliorating agent based on small RNAs (miRNA, siRNA, piRNA) among the RNAs encapsulated in Eucommia leaf exosomes.

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 30-150 nm are called exosomes, and their function in vivo has been rapidly elucidated in recent years. Tetraspanins, four-spanning membrane proteins, are abundantly expressed on the membrane surface of exosomes, and molecules such as CD9 and CD63 are commonly known as exosome markers. Exosomes contain proteins, lipids, nucleic acids, and other components. 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 messenger RNA (mRNA) and small ncRNAs (miRNA, siRNA, piRNA) that play important functional roles, and be passed on to other cells. As soon as this was suggested, related research has rapidly accelerated in recent years, and continues to this day.

Exosomes contain RNA, which can be broadly divided into two types depending on whether it is translated or not. Proteins are synthesized from translated RNA. This corresponds to messenger RNA (mRNA), which copies protein-coding sequences from DNA and carries the DNA's genetic information to the ribosomes responsible for protein synthesis. Small RNAs (miRNA, siRNA, piRNA), on the other hand, are functional nucleic acids that are encoded on the genome and undergo a multi-step production process to ultimately become microRNAs 20 to 25 bases in length. These short small RNAs (miRNA, siRNA, piRNA) are classified as functional ncRNAs (a general term for RNAs that are not translated into proteins) and have been shown to play an important role in biological phenomena by regulating the expression of other genes. Small RNAs (miRNA, siRNA, piRNA) bind to their target mRNAs with imperfect homology, generally recognizing the 3'UTR of target genes, destabilizing the target mRNA and suppressing translation, thereby inhibiting protein production. Transcriptional repression mediated by small RNAs (miRNA, siRNA, piRNA) has been shown to play important roles in a wide range of biological processes, including development, cell proliferation and differentiation, apoptosis, and metabolism.

Exosomes and small RNAs (miRNA, siRNA, piRNA) are involved in regulating epithelial-mesenchymal transition (the process by which epithelial cells lose their cell polarity and adhesive function with surrounding cells and dedifferentiate into undifferentiated mesenchymal-like cells, thereby acquiring the ability to migrate and invade) and cancer stem cells, and were expected to have beneficial effects on breast cancer, lung cancer, colon cancer, and diabetes. In June 2019, Tokyo Medical University and others identified two miRs (miR-509-5p and miR-1243) involved in epithelial-mesenchymal transition (EMT). This revealed their beneficial effects on breast cancer, lung cancer, colon cancer, and diabetes.

Du Zhong (Eucommia ulmoides) is a deciduous woody plant native to central China, classified as a single species in the family Eucommia, genus Eucommia, and is a standing tree that can reach a height of 20m. Compared to plants in the Theaceae family that are commonly known as tea, du Zhong does not contain any caffeine and its ingredients are also different. Du Zhong leaves have been widely used as a beverage since the 1980s. Du Zhong bark is used as a medicine, and in China it is used as an herbal medicine effective for "high blood pressure, lower back pain, joint pain, kidney disease, liver disease, stress, decreased energy, difficulty in diuresis, and forgetfulness." The inventors have developed a food product with high functionality and nutritional value by preventing fermentation of these Eucommia leaves immediately after harvesting and then powdering them. This high functionality has been demonstrated by significant improvements in lung cancer, colon cancer, breast cancer, blood sugar levels, allergies, viruses, the ability to inhibit melanin production in B16 cells, and atopy. To this end, the inventors conducted cancer research with Ibaraki University and Yokohama City University, and discovered a new compound, eucomisin A, that eliminates cancer stem cells, for which a patent application has been filed (see Patent Document 1).

Japanese Patent Application Laid-Open No. 2017-002031

Exosomes (30-150 nm) are just the tip of the iceberg of extracellular vesicles, and much remains unknown. Future research will likely unfold using a variety of approaches, from biology to clinical research. However, plant-derived exosomes are found in extremely low concentrations in plants, and various plants around the world are being tested. In the field of biology, products containing large amounts of exosomes are necessary and sought after for advancing research. In June 2019, the inventors, working with Tokyo Medical University, discovered that Eucommia ulmoides leaf powder contained 5.72 x ¹⁰⁸ exosome particles per mg (solvent: PBS) and 2.37 x ¹⁰⁸ exosome particles (solvent: MilliQ Water).

Furthermore, they found that using a basic alkaline dissolving solution to extract exosomes resulted in twice the amount of exosomes obtained. They also found that the tips of Eucommia leaves contain large amounts of exosomes, and succeeded in obtaining an extract with a high content of Eucommia leaves. They also found that the Eucommia-derived exosomes obtained in this way have an extremely high inhibitory effect on melanin production in B16 cells, making them an effective ingredient in cosmetics.

The present invention aims to provide an agent that uses natural ingredients, has few side effects, is safe even when taken over a long period of time, and is effective in treating viral infections, breast cancer, lung cancer, colon cancer, diabetes, allergies, dementia, and hyperglycemia through the control of epithelial-mesenchymal transition by small RNAs (miRNA, siRNA, piRNA) contained in Eucommia leaf exosomes, and in preventing arteriosclerosis, as well as a medicine or food containing the same.

The inventors have conducted extensive research to solve the above problems and have discovered that an exosome composition containing small RNAs (miRNA, siRNA, piRNA) from a processed Eucommia leaf product regulates epithelial-mesenchymal transition, resulting in viral infections, and that a health-improving agent containing small RNAs (miRNA, siRNA, piRNA) contained in Eucommia leaf exosomes is effective in treating breast cancer, lung cancer, colon cancer, diabetic allergies, dementia, hyperglycemia, and arteriosclerosis. Furthermore, the present invention relates to the viral infections and health-improving agents containing small RNAs (miRNA, siRNA, piRNA) contained in Eucommia leaf exosomes that regulate epithelial-mesenchymal transition, leading to the completion of the present invention. That is, according to one aspect of the present invention, there is provided a health-improving agent containing a processed Eucommia leaf product for treating viral infections and regulating epithelial-mesenchymal transition, including small RNAs (miRNA, siRNA, piRNA) among the RNAs encapsulated in Eucommia leaf exosomes, and for improving breast cancer, lung cancer, colon cancer, diabetic allergies, dementia, hyperglycemia, and arteriosclerosis. That is, the present invention includes the following embodiments.

(1) A viral infection treatment agent containing exosomes derived from Eucommia leaves.
(2) The viral infection ameliorating agent according to (1), wherein the exosomes derived from Eucommia ulmoides leaf contain small RNAs (miRNA, siRNA, piRNA).
(3) The viral infection ameliorating agent according to (1) or (2), which is in the form of a tablet, capsule, or drink.
(4) A pharmaceutical product comprising the improving agent according to any one of (1) to (3).
(5) A food or drink containing the improving agent according to any one of (1) to (3).
(6) The food or drink according to claim 5, which is a functional food, health food, health supplement, nutritional supplement, health food, specific health food, or nutritional functional food, containing the improving agent according to any one of (1) to (3).
(7) A method for producing a processed product of mulberry leaves, comprising the steps of immediately placing the mulberry leaves in a refrigerator at -10 to 0°C for 1 to 3 hours after harvesting them to cool them down, and then removing the mulberry leaves from the refrigerator, maintaining the humidity in a dry environment of 1% to 50% by dehumidifying or blowing air, and drying the leaves at a room temperature of 0 to 100°C for 24 hours.
(8) The method for producing a processed product of mulberry leaves described in (7), wherein the drying process includes steps such as cutting and roughly crushing the mulberry leaves during drying, a step of applying heat such as far infrared rays at 50°C to 400°C to the mulberry leaves for 5 minutes, and a step of crushing the mulberry leaves thus obtained at 100°C or less, crushing them to 5 to 25 μm, and extracting mulberry exosomes.

The improving agent of the present invention achieves functional improvement effects through the expression of small RNAs (miRNA, siRNA, piRNA) in Eucommia exosomes, which regulate epithelial-mesenchymal transition (EMT). In addition to improving viral infections, the regulation of EMT by small RNAs (miRNA, siRNA, piRNA) also improves breast cancer, lung cancer, colon cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis. Furthermore, these small RNAs (miRNA, siRNA, piRNA) not only act singly but also exert a combined functional effect, resulting in a comprehensive and synergistic improvement in health recovery. Furthermore, Eucommia has been used as a food for 2,000 years, and is considered one of the three major herbal medicines due to its lack of side effects and long-term use. Based on dietary experience, it is an extremely safe drug, supplement, and food.

FIG. 1 shows a method for extracting exosomes and RNA from powdered tea. Figure 2 shows the results of exosome recovery and particle number measurement for Du Zhong tea (Hekishan) and Du Zhong tea + mulberry tea (Zuicha). Figure 3 shows a graph measuring the size of exosome particles in Du Zhong tea (Hekishan) and Du Zhong tea + mulberry tea (Zuicha). Figure 4 shows the amount of RNA per exosome particle in Duzhong tea (Hekishan) and Duzhong tea + mulberry tea (Zuicha). Figure 5(A) shows a graph of RNA extraction from exosomes of Eucommia tea (Hekishan), and Figure 5(B) shows a graph of RNA extraction from exosomes of Eucommia tea + mulberry tea (Zuicha). FIG. 6 shows the non-coding RNA category Small (miRNA). FIG. 7 shows an outline of an infection experiment using respiratory virus PIV3 (parainfluenza type 3). FIG. 8 shows the results of examining the ability to suppress PIV3 infection (SEAP). FIG. 9 shows the results of examining the ability to suppress PIV3 infection (RT-PCR).

In order to solve the above problem, the inventors conducted extensive research and found that fresh Eucommia leaves were dried immediately after harvest in a specific environment below negative temperatures for 3 hours, preferably for about 2 hours, and then returned to room temperature from below negative temperatures.The ambient temperature was then kept between 0°C and 100°C, more preferably 0-50°C or less, even more preferably 0°C-10°C, and even more preferably 0°C-5°C, and dehumidified for 24 hours, with the humidity kept at 60% or less, preferably 50% or less, and preferably 30% or less, and the drying temperature kept at 100°C or less, preferably 60°C or less, preferably 30°C or less, and even more preferably 5°C or less, and the Eucommia leaves were then left in the process for 24 hours, yielding dried leaves with a moisture content of 3%. During this drying process, the material is dried using heat such as far infrared rays at 50 to 400°C for 5 minutes, or more preferably, a drying process incorporating far infrared rays for 1 minute. After drying, the material is roughly crushed and then further crushed to particles of 10 to 25 microns, preferably 5 to 15 microns, to obtain Eucommia powder.

To obtain an exosome sample using the thus obtained green powder of Eucommia leaf, Eucommia tea was added to PBS and vortexed, then stirred and dissolved at 2,000 x g for 20 minutes at room temperature. The supernatant was then removed and further stirred and dissolved at 12,000 x g for 20 minutes. The supernatant was then ultracentrifuged and filtered through a 0.22 μm filter. 1 mL of the supernatant was stirred in an ultracentrifuge at 200,000 x g for 70 minutes, then transferred to a pellet and suspended in PBS to obtain the sample (see Figure 1).

This "du zhong powder" was dissolved by adding du zhong tea to PBS and stirring at 2,000 x g for 20 minutes at room temperature. The supernatant was then removed and further dissolved by stirring at 12,000 x g for 20 minutes. The supernatant was then ultracentrifuged and suspended in PBS, and 1.60 x ¹⁰⁸ exosomes were detected. The amount of RNA encapsulated in the exosomes was also examined. Furthermore, it was confirmed that one exosome contained 6 total RNA (x ¹⁰⁴ pg/particle). These tests were compared between two samples using du zhong powder alone and one using du zhong tea and mulberry tea powder.

Next, the size and quantity of exosome particles were measured using qNano for two samples: 100% Eucommia (Hekisan) and Eucommia tea + mulberry tea (Zuicha). As shown in Figure 3 and Table 1, the particle number for 100% Eucommia (Hekisan) was 1.60 x 10 ⁸ . For Eucommia tea + mulberry tea (Zuicha), it was 1.11 x 10 ⁹ . The average particle size for Eucommia tea was 158 nm at its highest. For Eucommia tea + mulberry tea, it was 145 nm.

Next, we examined the amount of RNA per grain for two samples: 100% Eucommia (Hekisan) and Eucommia tea + mulberry tea (Zuicha). Eucommia had 6.5 total RNA (x ¹⁰⁴ pg/particle). Eucommia tea + mulberry tea (Zuicha) had 1.5 total RNA (x ¹⁰⁴ pg/particle) (see Figure 4). These figures show that mulberry contains almost no RNA.

Next, we measured the size of RNA particles in two samples: A, 100% Eucommia (Hekisan), and B, Eucommia tea + mulberry tea (Zuicha). Graphs A and B clearly show that miRNA is contained (Figure 5). For reference, Figure 6, which shows the non-coding RNA category Small (miRNA), shows that 20-30 nt is small RNA (miRNA), and their known functions are translation control, mRNA stability control, and genome stability control.

Through the action of these small RNAs (miRNA, siRNA, piRNA), we provide ameliorative and preventative agents, as well as pharmaceuticals or foods containing them, that have an ameliorative effect in preventing viral infections, breast cancer, lung cancer, colon cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis. As a large-scale purification and processing method for providing preventative agents and pharmaceuticals or foods containing them, we developed a production method using a tangential flow system that combines ultrafiltration and filtration to purify 1g/batch of exosomes from a liter-sized exosome solution extracted from Eucommia powder in a form that is easy to formulate.

Furthermore, the inventors tested the effectiveness of Eucommia powder against respiratory virus (parainfluenza virus type 3) infection to verify its antiviral effect. PIV3 was added to a sample containing 1 mg of Eucommia powder in 1 ml of PBS at an MOI (multiple of infection) of 1. Upon viral infection, placenta-derived secretory alkaline phosphatase (SEAP) was secreted into the supernatant. After adding PIV3, the cells were cultured for 48 hours and assayed using the RT-PCR method.
(See Figure 7)

To investigate the inhibitory effect of Eucommia on PIV3 infection, the SEAP value after incubation was examined. The results are shown in Figures 8 and 9. The SEAP value after incubation was 658110.5 when Eucommia powder was used, and 1045350.667 when Eucommia powder was not used. This indicates that Eucommia powder inhibited infection with parainfluenza virus type 3 at the indicated concentrations.

Claims (1)

A method for producing an agent for ameliorating a viral infection comprising exosomes derived from Eucommia ulmoides leaves, the exosomes containing at least 6.5 x ¹⁰ pg of RNA per particle, comprising:
a step of immediately placing the harvested Eucommia leaves in a refrigerator at -10 to 0°C for 1 to 3 hours to cool them down; and a step of subsequently removing the leaves from the refrigerator and drying them for 24 hours at a room temperature of 0 to 10°C while maintaining the humidity in a dry environment of 1% to 50% by dehumidification or ventilation.
A step of finely pulverizing the dried Eucommia leaves to 5 to 25 microns to obtain Eucommia powder;
A method for producing an agent for improving viral infections, comprising: