Agent for improving viral infectious diseases containing exosomes derived from Eucommia ulmoides leaves

Abstract

To provide a viral infection improving agent that comprises plant-derived exosome useful in medicines or foods, which are extremely low in plants and are being investigated in various plants around the world.SOLUTION: There is provided a viral infection improving agent in which exosome derived from Eucommia leaf is small RNA (miRNA, siRNA, or piRNA).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a product processed from Eucommia ulmoides leaves. More specifically, it relates to an agent for improving viral infections containing a processed product of Eucommia ulmoides leaves, and an agent based on small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in Eucommia ulmoides leaf exosomes. Further, the present invention relates to an oral intake composition, a pharmaceutical composition, a food composition, etc. containing the agent for improving viral infections and the agent based on small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in Eucommia ulmoides leaf exosomes.

Background Art

[0002] Recent research has shown that extracellular vesicles (EVs) are used for cell - to - cell communication. Depending on their size and biological functions, they are classified into Exosomes, Microvesicles (MVs), etc. Among them, vesicles with a diameter of 30 - 150 nm are called exosomes, and in recent years, the understanding of their functions in vivo has advanced rapidly. On the membrane surface of exosomes, many four - transmembrane proteins called tetraspanins are expressed, and molecules such as CD9 and CD63 are known as common exosome markers. Inside, they contain proteins, lipids, nucleic acids, etc. The discovery of "exosomes" dates back about 30 years. At first, they were only recognized as unwanted substances in cells, and their functions and significance remained unclear for a long time. However, in 2008, as soon as it was shown that messenger RNA (mRNA) and nucleic acid substances containing small ncRNAs (miRNAs, siRNAs, piRNAs) miRNA, which play important functional roles, are encapsulated in exosomes and may be transferred to other cells, related research has accelerated rapidly in the past few years and has reached the present stage.

[0003] Exosomes contain RNA, which can be broadly classified into two types depending on whether it is translated or not. Proteins are synthesized from the RNA that undergoes translation. Messenger RNA (mRNA), which copies the DNA sequence encoding the protein and transports the genetic information of DNA to the ribosomes responsible for protein synthesis, corresponds to this. On the other hand, small RNAs (miRNA, siRNA, piRNA) are functional nucleic acids encoded on the genome and finally become microRNAs 20 to 25 bases in length through a multi-step generation process. These small RNAs (miRNA, siRNA, piRNA) with a short chain length are classified as functional non-coding RNAs (a general term for RNAs that are not translated into proteins), and it has been revealed that they play important roles in biological phenomena by regulating the expression of other genes. Small RNAs (miRNA, siRNA, piRNA) bind to their target mRNAs with incomplete homology, generally recognize the 3'UTR of the target gene, destabilize the target mRNA, and suppress protein production by inhibiting translation. It has been clarified that the transcriptional repression mediated by small RNAs (miRNA, siRNA, piRNA) plays an important role in a wide range of biological processes such as development, cell proliferation and differentiation, apoptosis, or metabolism.

[0004] Exosomes and small RNAs (miRNA, siRNA, piRNA) are also involved in the regulation of epithelial-mesenchymal transition (a process that controls the process by which epithelial cells lose their cell polarity and adhesion function with surrounding cells and dedifferentiate into undifferentiated mesenchymal-like cells to acquire migratory and invasive abilities) and the control of cancer stem cells, and were expected to have improvement effects on breast cancer, lung cancer, colorectal cancer, and diabetes. However, in June 2019, two types of miRs (miR-509-5p and miR-1243) related to epithelial-mesenchymal transition (EMT) were identified by the University of Tokyo Medical Science and others. As a result, the improvement effects on breast cancer, lung cancer, colorectal cancer, and diabetes were clarified.

[0005] Eucommia ulmoides is a deciduous woody plant classified into a single species of a single genus in the Eucommiaceae family, originating from central China, and is an upright tree reaching a height of 20 m. Compared with the plant of the Theaceae family generally called tea, Eucommia ulmoides contains no caffeine at all and has different components. The leaves of Eucommia ulmoides have been popular as a beverage since the 1980s. The bark of Eucommia ulmoides is treated as a medicine and is used in China as a Chinese herbal medicine effective for "hypertension, low back pain, joint pain, kidney disease, liver disease, stress, loss of energy, difficulty in urination, forgetfulness". The inventors have developed a food with high functional components and nutritional components by immediately preventing fermentation and further pulverizing the leaves of Eucommia ulmoides after harvesting. Its high functionality has significantly obtained improvement effects such as lung cancer, colorectal cancer, breast cancer, blood glucose level, allergy, virus, inhibitory effect on B16 cell melanin production, and atopy. Therefore, the inventors have conducted cancer research with the University of Tsukuba, Yokohama City University, etc., discovered a novel compound, eucommicin A, which eliminates cancer stem cells, and filed a patent application (see Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] Exosomes (30 - 150 nm) are just the tip of the iceberg among "extracellular vesicles", and many parts remain unexplained. In the future, research will be carried out by various approaches from biology to clinical research. However, plant-derived exosomes are contained in plants at an extremely low rate, and various plants around the world are being tested. Products containing a large amount of "exosomes" in the field of biology are necessary and required for further research. The inventors found that in June 2019, in the green powder of Eucommia ulmoides leaves and Tokyo Medical University, the number of exosome particles per 1 mg was 5.72×10 8individuals (solvent PBS), 2.37×10 8 individuals (solvent MilliQ Water) are found to be contained.

[0008] Furthermore, it was found that when a basic alkaline solution is used as a lysate to extract exosomes, the amount of exosomes obtained is doubled. Also, it was found that the tip part of Eucommia ulmoides leaves contains a large amount of exosomes, and an extract with a high content rate of exosomes derived from Eucommia ulmoides leaves was successfully obtained. In addition, the exosomes derived from Eucommia ulmoides thus obtained have an extremely high inhibitory effect on B16 cell melanin production and were found to be an effective raw material for cosmetics.

[0009] The present invention uses natural raw materials, has few side effects and is safe even when ingested over a long period. It provides an agent for improving viral infectious diseases, breast cancer, lung cancer, colorectal cancer, diabetes, allergy, dementia, hyperglycemia, an agent for preventing arteriosclerosis, a pharmaceutical or food containing the same, which are brought about by the regulation of epithelial-mesenchymal transition of small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in exosomes of Eucommia ulmoides leaves.

Means for Solving the Problems

[0010] In order to solve the above problems, the present inventors conducted intensive research. As a result, it was found that an exosome composition containing small RNAs (miRNAs, siRNAs, piRNAs) of Eucommia ulmoides leaf extract can control epithelial-mesenchymal transition, and is related to viral infections, breast cancer, lung cancer, colorectal cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis by a health improvement agent containing small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in Eucommia ulmoides leaf exosomes. Furthermore, the present invention found a health improvement agent for controlling epithelial-mesenchymal transition containing small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in the above viral infections and Eucommia ulmoides leaf exosomes, and completed the present invention. That is, according to one aspect of the present invention, there is provided an agent for improving breast cancer, lung cancer, colorectal cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis as a health improvement agent for controlling epithelial-mesenchymal transition containing small RNAs (miRNAs, siRNAs, piRNAs) among the RNAs encapsulated in Eucommia ulmoides leaf exosomes, which contains Eucommia ulmoides leaf extract. That is, the present invention includes the following embodiments.

[0011] (1) An agent for improving viral infections containing exosomes derived from Eucommia ulmoides leaves. (2) The agent for improving viral infections according to (1), wherein the exosomes derived from Eucommia ulmoides leaves contain small RNAs (miRNAs, siRNAs, piRNAs). (3) The agent for improving viral infections according to (1) or (2), which is in the form of tablets, capsules, or drinks. (4) A pharmaceutical product containing the agent according to any one of (1) to (3). (5) A food or drink containing the 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, insurance function food, specific insurance food, or nutritional function food containing the agent according to any one of (1) to (3). (7) After harvesting Eucommia ulmoides leaves, immediately place the fresh leaves in a refrigerator at -10 to 0 °C for 1 to 3 hours to remove the rough heat, and then take out the fresh leaves from the refrigerator, and keep the humidity in the drying environment at 1% or more and 50% or less by dehumidification or blowing, and dry at room temperature of 0 °C or more and 100 °C or less for 24 hours, including the manufacturing method of the Eucommia ulmoides leaf processed product. (8) The drying process includes processes such as cutting and rough pulverization of Eucommia ulmoides leaves during drying, further applying heat from far-infrared rays or the like at 50 °C to 400 °C to the Eucommia ulmoides in the process for 5 minutes, and further pulverizing the obtained Eucommia ulmoides leaves at 100 °C or less to 5 to 25 μm to extract Eucommia exosomes, including the manufacturing method of the Eucommia ulmoides leaf processed product according to (7).

Effect of the Invention

[0012] The improving agent according to the present invention is due to the improving effect of the functional epithelial-mesenchymal transition control expression action by the small RNAs (miRNAs, siRNAs, piRNAs) of the RNA of Eucommia exosomes. It has not only the improving effect on viral infections, but also the improving effects on breast cancer, lung cancer, colorectal cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis by the epithelial-mesenchymal transition control of small RNAs (miRNAs, siRNAs, piRNAs). In addition, these small RNAs (miRNAs, siRNAs, piRNAs) not only act singly but also have a combined functional effect, and comprehensively synergistically improve health for health recovery. Also, it has been used as a food since 2000 years ago, has no side effects, and has no harm even after long-term use, so it is counted as one of the three major traditional Chinese medicines, and it is a very reassuring drug, supplement, and food from the food experience.

Brief Explanation of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] In order to solve the above problems, as a result of intensive studies, the inventors immediately dried the fresh Eucommia ulmoides Oliv. leaves for 3 hours in a specific environment below minus after harvesting, preferably for about 2 hours of drying, returned from below minus to room temperature, and adjusted the environmental temperature from 0°C to 100°C, more preferably to 0 to 50°C or lower, more preferably to 0°C to 10°C, and even more preferably to 0°C to 5°C at room temperature, carried out dehumidification for 24 hours, maintained the humidity at 60% or lower, preferably 50% or lower, preferably 30% or lower, maintained the drying temperature at 100 degrees or lower, preferably 60°C or lower, preferably 30°C or lower, and even more preferably 5°C or lower, left the Eucommia ulmoides Oliv. in the process for 24 hours, and obtained dry leaves with a moisture content of 3%. During such drying, drying was carried out by heat from far-infrared rays, etc. from 50°C to 400°C for 5 minutes, more preferably for 1 minute of drying including a drying process by far-infrared rays, etc., and after drying, coarse pulverization was carried out, and further fine pulverization was carried out to 10 to 25 microns, preferably to 5 to 15 microns to obtain Eucommia ulmoides Oliv. powder.

[0015] Using the Eucommia ulmoides leaf powder obtained in this way, in order to obtain an exosome specimen, Eucommia ulmoides tea was added to PBS and vortexed, then stirred and dissolved at 2,000×g for 20 minutes at room temperature. The supernatant was taken and further stirred and dissolved at 12,000×g for 20 minutes. The supernatant was ultracentrifuged, and the supernatant was further filtered through a 0.22μm filter. 1 mL of the filtered supernatant was stirred at 200,000×g for 70 minutes in an ultracentrifuge, and the pellet was taken and suspended in PBS to obtain a specimen (see Figure 1).

[0016] This "Eucommia ulmoides powder" was added to PBS with Eucommia ulmoides tea and stirred and dissolved at 2,000×g for 20 minutes at room temperature. Then the supernatant was taken and further stirred and dissolved at 12,000×g for 20 minutes. The supernatant was taken again and ultracentrifuged, and then suspended in PBS to obtain 1.60×10 8 exosomes. Furthermore, the quantity of RNA encapsulated in the exosomes was examined. Furthermore, it was examined that 6 Total RNA (×10 4 pg / particle) of RNA was contained in one particle of the exosomes. These examinations were carried out by comparing two cases, one using only Eucommia ulmoides powder and the other using Eucommia ulmoides tea + mulberry tea powder.

[0017] Next, the size and quantity of exosome particles were measured using qNano for two specimens, Eucommia ulmoides 100% (Bishan) and Eucommia ulmoides tea + mulberry tea (Ruicha). As shown in Figure 3 and Table 1, for Eucommia ulmoides 100% (Bishan), the number of particles was 1.60×10 8 . For Eucommia ulmoides tea + mulberry tea (Ruicha), it was 1.11×10 9 . For Eucommia ulmoides tea, the average particle size was 158 nm at the maximum value. For Eucommia ulmoides tea + mulberry tea, it was 145 nm.

[0018]

Table 1

[0019] Next, the amount of RNA per particle was examined for two specimens, Eucommia ulmoides 100% (Bishan) and Eucommia ulmoides tea + mulberry tea (Ruicha). For Eucommia ulmoides, it was 6.5 Total RNA (×10 4pg / particle). Eucommia ulmoides Oliv. tea + Morus alba L. tea (Rui tea) was 1.5Total RNA (×10 4 pg / particle) (see Figure 4). Judging from this number, it can be seen that almost no RNA is contained in Morus alba L..

[0020] Next, the sizes of RNA particles were measured for two specimens, A, 100% Eucommia ulmoides Oliv. (Bishan), and B, Eucommia ulmoides Oliv. tea + Morus alba L. tea (Rui tea). It is clear from the two graphs of A and B that miRNA is contained (Figure 5). Also, referring to Figure 6 of the non-coding RNA category Small (miRNA), it can be seen that 20 - 30 nt are small RNAs (miRNAs), and their functions are as described for known functions, namely translational control, mRNA stability control, and genomic stability control.

[0021] The functions of this small RNA (miRNA, siRNA, piRNA) bring about an improvement effect on the prevention of viral infections, breast cancer, lung cancer, colorectal cancer, diabetes, allergies, dementia, hyperglycemia, and arteriosclerosis, and provide an improving agent, a preventive agent, a medicine or food containing the same. As a method for mass purification and processing for providing a preventive agent, a medicine or food containing the same, a production method by a tangential flow system combining ultrafiltration and filtration for mass purification of exosomes of 1 g / batch in a form easy to formulate was carried out from an exosome solution of litter size extracted from Eucommia ulmoides Oliv. powder.

[0022] In addition, the inventors conducted a test on the effect of the antiviral action of Eucommia ulmoides Oliv. powder to verify its effect on respiratory virus (parainfluenza virus type 3) infection. PIV3 was introduced into a specimen with a concentration of 1 mg of Eucommia ulmoides Oliv. powder in 1 ml of PBS at an MOI (infection efficiency) = 1. When the virus infects, placenta-derived secreted alkaline phosphatase (SEAP) is secreted into the supernatant. After introducing PIV3, the cells were cultured for 48 hours and assayed by RT-PCR method. (see Figure 7)

[0023] To examine the ability to inhibit PIV3 infection, the SEAP after culture was examined. The results are shown in Figs. 8 and 9. The SEAP value after culture was 658110.5 when eucommia powder was used, whereas it was 1045350.667 when eucommia powder was not used. From this, it was found that eucommia powder inhibited the infection of parainfluenza virus type 3 at the indicated concentration.

Claims

【Claim 1】 An exosome derived from Eucommia ulmoides leaves, the method for producing an agent for improving viral infectious diseases, comprising the exosome containing at least 6.5 × 10 ４ pg of RNA per particle After harvesting Eucommia ulmoides leaves, immediately place the fresh leaves in a refrigerator at -10 to 0°C for 1 to 3 hours to remove the wild heat, and then take out the fresh leaves from the refrigerator and keep the humidity in the drying environment at 1% or more and 50% or less by dehumidification or blowing, and dry at room temperature of 0°C or more and 10°C or less for 24 hours; A step of finely pulverizing the dried Eucommia ulmoides leaves to 5 to 25 microns to obtain Eucommia powder; A method for producing an agent for improving viral infectious diseases, comprising the above steps.

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