Seaweed-derived extract, organism-applied product using the same, and method for extracting seaweed-derived exosome

Abstract

To scientifically prove a pharmacological effect and to promote effective use for a fucoidan reported effective for increasing an immune strength and anticancer activity.SOLUTION: A raw material, Mozuku, is hydrolyzed (boiled) to provide a low molecular fucoidan. Then, a nano-level particle group is extracted from the low molecular fucoidan through super-centrifugal separation or the like using a supercentrifuge 1. The major part of the particle group is within the size of 50-200 nm, and composed of exosome carrying miRNA or meRNA. Exosome derived from the fucoidan acts both for apoptosis-induction for an abnormal organ suffering from ulcer or cancer and reproduction for promoting growth of a normal organ, and safe and resistant / non-dependent. Thus, the exosome derived from the fucoidan can be adopted for various products such as a medicine.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to bioactive products such as medicines and dietary supplements, and seaweed-derived extracts for use therein, as well as a method for extracting seaweed-derived exosomes. [Background technology]

[0002] Fucoidan, extracted from seaweed, has been reported to have pharmacological effects such as anti-inflammatory, anti-ulcer, and immune-boosting properties, and various studies have been conducted on its application in medicines, supplements, and cosmetics (e.g., Patent Documents 1 and 2). In particular, the anti-inflammatory, anti-ulcer, and anti-cancer properties of fucoidan have attracted medical attention, and some clinics have been administering it to cancer patients, earning high praise. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2016-42804 A [Patent Document 2] Patent No. 6859017 Summary of the Invention [Problem to be solved by the invention]

[0004] Fucoidan is a type of sulfated polysaccharide found mainly in seaweed, and as mentioned above, it has been reported to have pharmacological and bioactive effects such as anti-inflammatory and anti-ulcer effects, and to improve immunity. However, the mechanism by which it exerts its pharmacological effects, such as anti-inflammatory effects, has not been elucidated. Fucoidan is often sold commercially as a supplement in the form of a drink, but the mechanism of its pharmacological action has not been elucidated, so its application to medicine has not progressed. It is also unclear whether all commercially available supplements have high bioactive effects, and this chaotic situation has led to skepticism about its effectiveness.

[0005] Commercially available fucoidan products are generally made from mozuku seaweed, and L-fucose, the main component of fucoidan, is a high molecular weight compound (polysaccharide fiber) with a molecular weight of about 200,000. There are many conflicting theories regarding the relationship between molecular weight and effectiveness, including the theory that it is effective because it is a high molecular weight, the theory that it is effective when it is reduced to a molecular weight of less than 1,000, and the theory that it is effective because of a molecular weight between the two.

[0006] In considering this point, since commercially available fucoidan products are basically beverages, it is necessary to observe the effects after drinking, but since fucoidan comes into direct contact with ulcers in the digestive system, even if it has a healing effect on ulcers, the causal relationship between the healing effect and the molecular weight cannot be clearly established. On the other hand, if a person without ulcers feels that their immunity has improved after drinking fucoidan, it can be said that fucoidan is absorbed through the intestinal wall, but since it needs to be broken down into smaller molecules in order to be absorbed through the intestinal wall, it is understood that breaking down into smaller molecules is a condition for obtaining the pharmacological effects of fucoidan.

[0007] Daiichi Sangyo Co., Ltd., a co-applicant of this application, has developed an external cream containing low molecular weight fucoidan and disclosed it in Patent Document 2. From this example, it can be understood that low molecular weight fucoidan exerts an anti-inflammatory effect, and from this, it can be considered that the effect of improving immunity after absorption into the body and the effect of healing digestive ulcers are due to the low molecular weight. Therefore, the theory that low molecular weight contributes to the pharmacological effect of fucoidan is reasonable.

[0008] The next question is whether the depolymerization of fucoidan is the end point or the starting point of its pharmacological effect. In other words, does the depolymerized fucoidan exert a pharmacological effect by itself, or is there another active ingredient that exerts a pharmacological effect, and is the depolymerization of the active ingredient easier to work? In this regard, considering that it is difficult to imagine the mechanism by which L-fucose, the main component of fucoidan, exerts a pharmacological effect when depolymerized, it is speculated that there may be an active substance or ingredient other than L-fucose.

[0009] In other words, although fucoidan (or seaweed) contains large amounts of active substances and ingredients that have anti-inflammatory and immune-boosting effects, under normal conditions these active substances and ingredients are protected by cells etc., so even if a large amount is ingested as food, for example, they do not exert their pharmacological effects. It can be hypothesized that by breaking down into smaller molecules, the active substances and ingredients emerge from within and exert their pharmacological effects, or the amount of active substances and ingredients that appear increases dramatically, allowing the pharmacological effects to be clearly expressed.

[0010] If the active substances and ingredients that exert pharmacological effects could be identified, their application in medicines would become easier, and humans and animals would receive immeasurable benefits. Therefore, it can be said that there is a demand to elucidate the active substances and ingredients that exert pharmacological effects such as anti-inflammatory effects, and to promote their use. The present invention is intended to meet this demand. [Means for solving the problem]

[0011] One of the noteworthy effects of fucoidan is its apoptosis-inducing effect, which destroys abnormal cells such as cancer cells (causing them to self-destruct). The apoptosis-inducing effect is built into the cells of all living organisms as a genetic information program, and when this function is functioning properly, it prevents cells from becoming cancerous.

[0012] Regarding the apoptosis-inducing effect of fucoidan, it is speculated that the reason why fucoidan exerts its apoptosis-inducing effect on human and animal cells is because the effective substances and ingredients contained in fucoidan have the function of repairing abnormalities in the genetic programs of human and animal cells, and therefore it can be speculated that the effective substances and ingredients of fucoidan are also highly likely to have genetic programs (genetic information transmission function). The inventors of this application continued their research based on this speculation, and have completed the present invention.

[0013] That is, the inventors of the present application focused on exosomes (extracellular vesicles: EVs) contained in fucoidan and identified exosomes as the main active substance / component of pharmacological action, thereby completing the present invention.

[0014] The present invention includes many configurations, which are specified in each claim. The invention of claim 1 is as follows: "A seaweed-derived extract for use in a bioproduct for humans or animals, containing exosomes of 50-200 nm in size as the main component." The structure is as follows.

[0015] The invention of claim 2 is an embodiment of claim 1 in terms of concentration. "The exosomes or exosome-like particles contain 500 million or more per ml." The structure is as follows.

[0016] The invention of claim 3 is as follows: "Bio-effecting products for use in humans or animals, Its main component is exosomes extracted from seaweed, measuring 50 to 200 nm in size. The structure is as follows.

[0017] The invention of claim 4 is an example of the development of claim 3. "The bioactive product is at least one of the following: medicines, quasi-drugs, dietary supplements, cosmetics, soft drinks, and confectioneries, and is intended for use by humans or animals." The structure is as follows.

[0018] Claim 4 exemplifies a bioactive product, but the appearance varies depending on the product. For example, the product may have a liquid, granular, powdery, creamy, or other appearance. Fucoidan-derived exosomes may be added to confectionery such as candy or candy, and in this case, the product will have the appearance of the confectionery. In addition, each product may freely add ingredients other than fucoidan-derived exosomes.

[0019] The invention of claim 5 specifies the raw material in claim 2 or 3, "Mozuku seaweed is used as the seaweed." It has the following characteristics:

[0020] The invention of claim 6 relates to a method for extracting exosomes derived from seaweed, "One or more types of seaweed are used as the raw material, and low molecular weight fucoidan is produced by hydrolyzing the seaweed under heating with or without the addition of enzymes, and then exosomes are separated from the low molecular weight fucoidan by ultracentrifugation or ultrafiltration." The structure is as follows.

[0021] The invention of claim 7 is a preferred embodiment of claim 6. "The seaweed used is one or more types of brown algae, including mozuku, hibamata, kombu, hijiki, wakame, einkorn, arame, and kurome." It has the following characteristics: Effect of the Invention

[0022] Exosomes (extracellular vesicles) are vesicles derived from endosomal membranes released from cells and contain lipids, proteins, and nucleic acids as their constituent components. Of particular interest are the miRNA (microRNA), mRNA (messenger RNA), and DNA contained in the nucleic acid cargo.

[0023] Recent research has revealed that exosomes have the function of transmitting information between cells (or to cells). In this case, the information can have positive or negative effects. For example, exosomes derived from breast milk play a positive role in promoting the immune system of infants, while exosomes derived from cancer cells are bad exosomes that play a negative role in promoting the proliferation of cancer cells through the action of inflammatory cytokines and miRNA.

[0024] There are research reports showing that exosomes derived from ginger have an inhibitory effect on alcohol-induced liver damage in humans, which coincides with the phenomenon that turmeric is effective against hangovers (however, excessive intake seems to have the potential to actually cause liver dysfunction (out-of-control)).

[0025] Regarding exosomes derived from cytotoxic T cells, which are well-known as killer cells, it has been reported that they are selectively taken up by cancer-derived mesenchymal stem cells, where they induce apoptosis and suppress the spread of cancer. It is also believed that exosomes derived from mesenchymal stem cells have an apoptotic effect. Since mesenchymal stem cells are multipotent cells that have the ability to differentiate into any type of cell, such as bone or fat, if exosomes can be used to proliferate mesenchymal stem cells, it is expected that this will not only promote the healing of affected areas, but will also contribute to regenerative medicine.

[0026] In light of the actions and functions of exosomes, the doubts are cleared if the actions and effects (pharmacological effects) of fucoidan, such as anti-inflammatory, anti-ulcer, and immune-boosting effects, which have traditionally been said to be the effects and efficacy of fucoidan, are attributed to exosomes. Therefore, it can be said that the actions and effects of anti-inflammatory and anti-ulcer effects can be ensured and enhanced by extracting and concentrating fucoidan-derived (seaweed-derived) exosomes and administering them to humans or animals, and this is realized by the present invention.

[0027] Although it is not yet fully understood how fucoidan-derived exosomes act on cells and tissues in the affected area, it is speculated that, after exosomes are received by receptors in the affected tissues, the signaling components such as miRNA and mRNA (or DNA) loaded on the exosomes are taken up by the affected cells and tissues, and act on the genes in the affected area to restore the program (base sequence or base composition) to a normal state or be translated into functional proteins. In other words, it is speculated that fucoidan-derived exosomes have a messenger function and signaling function that act on a wide variety of genes in human and animal tissues to rewrite their programs.

[0028] Exosomes derived from the affected area (bad exosomes that spread the disease) should also be released from the affected area, but when the fucoidan-derived exosomes enter the tissue, it is speculated that the bad exosomes will have nowhere to go and will either die (not be accepted by the tissue) or be attacked and destroyed by the fucoidan-derived exosomes (losing their program transmission function).

[0029] All cells have their own exosomes, and if the intrinsic exosomes function normally, cancer would not occur. However, when intrinsic exosomes stop functioning and cells become cancerous, fucoidan-derived exosomes act on the cells and perform the same function as intrinsic exosomes. This means that fucoidan-derived exosomes have the receptivity to be taken up by various tissues of humans and animals, and the ability to substitute for intrinsic exosomes and perform the same functions.

[0030] This suggests that fucoidan-derived exosomes are versatile, and it is possible to speculate that the mechanism by which fucoidan-derived exosomes act on affected areas to repair tissue may involve a connection with mesenchymal stem cells, which are versatile cells, although this remains merely a hypothesis.

[0031] In other words, when fucoidan-derived exosomes are adsorbed to receptors in tissue, components such as miRNA and mRNA are released, which induces apoptosis and kills the tissue itself. In parallel with this, a mechanism occurs in which mesenchymal stem cells proliferate due to the programming function of mRNA, and these cells undergo self-transformation (differentiation) into normal cells, and it is presumed that eventually the entire affected area will be replaced by new normal cells.

[0032] In other words, it is speculated that the repair of affected areas by fucoidan-derived exosomes is carried out through the proliferation of mesenchymal stem cells, which are pluripotent cells. Since tissue repair through mesenchymal stem cells is also related to regenerative medicine, we would like to continue this research further.

[0033] It is unclear whether the apoptosis-inducing effect of fucoidan-derived exosomes is a genetic program effect or a destructive effect of chemicals such as enzymes, but considering that normal tissue is not destroyed, it is highly likely to be a program effect. In any case, apoptosis induction and repair of the target tissue occur in parallel, and the amount of bad exosomes released decreases in inverse proportion to the progress of both, creating a virtuous cycle of healing.

[0034] In Japan, colon cancer is one of the leading causes of death for both men and women, and a simple and highly accurate test method would be expected to significantly reduce the mortality rate. The fucoidan-derived exosomes of the present invention can also be used as a marker for colon cancer screening. For example, assuming that fucoidan-derived exosomes bind to bad exosomes derived from colon cancer, if a person drinks fucoidan-derived exosomes as a test drug, separates stool with an ultracentrifuge, and finds that a certain percentage of particles with a mass larger than the original mass of the fucoidan-derived exosomes are present, it can be assumed that the person has colon cancer. Therefore, it is suitable to use this method in combination with a fecal occult blood test.

[0035] Claims 1 and 2 and claims 3 and 4 are related to materials and products, but the extract of claim 1 has a high concentration of exosomes, so a small amount can be highly effective when applied to the products described in claims 3 and 4. The extract of claim 1 can be applied to products after dilution or concentration, but it can also be used as it is as a dietary supplement or soft drink. In this case, a concentration of about 500 million exosomes per ml, as specified as the lower limit in claim 2, is likely to be effective in boosting immunity, etc.

[0036] In the present invention, since the fucoidan-derived exosomes can be purified to remove impurities to a negligible level, when it is made into medicine, it can be embodied in various forms such as injection solutions, drip solutions, catheter infusion solutions, tablets, ointments (creams), microcapsules, etc. Therefore, when applied to medicines, it can expand the options for treatment methods and greatly expand the range of diseases to be treated.

[0037] Specifically, the concentration of exosomes, which are active substances and ingredients, can be significantly increased, so when used for anti-inflammatory and anti-ulcer purposes as in the past, the therapeutic effect can be significantly improved. In addition, it is easy to prepare solid formulations such as tablets, powders, and granules, which expands the options for treatment and reduces the burden on patients. The ability to formulate in an easily portable form is good news for patients and improves the therapeutic effect. Insurance coverage through drug certification reduces the burden on patients, so the practical contribution is significant.

[0038] As mentioned above, it is speculated that fucoidan-derived exosomes support the differentiation of mesenchymal stem cells into normal tissues, and by administering them into the bloodstream as an intravenous drug, it is expected that they will exert the disease-healing functions of cells that make up the blood and lymph.

[0039] For example, the universal program function may contribute to the cure of blood cancers such as leukemia by promoting the proliferation of hematopoietic stem cells and their differentiation into normal cells. In addition, HIV proliferates in D4 cells, which constitute white blood cells and control immune functions, and it is expected that the administration of high-purity exosomes derived from fucoidan in an intravenous liquid form may promote the normalization of D4 cells and lead to the elimination of HIV. It can also be said that the universal program function of fucoidan-derived exosomes may be able to destroy HIV itself or the bad exosomes released from it.

[0040] Regarding cancer, there are currently a variety of treatment methods available, including radiation therapy, administration of anticancer drugs, immunotherapy, and surgical removal of the affected area. However, there is the difficult problem of drug resistance, and all of these methods aim to destroy or remove cancer cells, so they cannot be said to be constructive.

[0041] In contrast, treatment using fucoidan-derived exosomes actively supports the tissue regeneration mechanism of human and animal tissues from the outside, destroying cancer cells and promoting the proliferation of normal cells at the same time, and there is no problem with resistance even with continued use, and there is no functional impairment after treatment, making it an excellent treatment method. Another advantage is that it can be used to treat even when the disease has progressed to the point where surgery is impossible (metastasis).

[0042] The exosomes of the present invention can be used in quasi-drugs, dietary supplements, and cosmetics, and the effects of these products can be enhanced by increasing the concentration of exosomes as active substances and ingredients. Various seaweeds can be used as raw materials, but mozuku is the best in terms of the fucoidan content. In this case, domestic seaweed from various regions such as Okinawa and Aomori can be used. Of course, seaweed from overseas can also be used, for example, South Pacific seaweed from Tonga can be used.

[0043] Although it is not clear how exosomes exist inside fucoidan, there is no doubt that they are protected by tissues in a state where they cannot be easily separated (isolated). However, as in claim 6, if exosomes are first depolymerized and then centrifuged or filtered, a large amount of exosomes can be extracted. Therefore, claim 6 makes it possible to effectively utilize exosomes derived from fucoidan.

[0044] Among seaweeds, brown algae have a high content of fucoidan, so exosomes can be extracted efficiently by using brown algae as a raw material as in claim 7. As mentioned in claim 5, it is particularly suitable to use mozuku seaweed. [Brief description of the drawings]

[0045] [Figure 1] 1 is an experimental graph showing SOD3 activity in undiluted fucoidan solution and blood. [Diagram 2] FIG. [Diagram 3] FIG. 1 is a diagram showing the particle size and number of particles separated by ultracentrifugation. [Figure 4] (A) and (B) are graphs showing the effect of fucoidan-derived exosomes on liver dysfunction, and (C) to (J) are micrographs showing the effect of stained tissue on liver dysfunction. [Diagram 5] 1 is a graph showing the NK cell activation effect of fucoidan-derived exosomes. [Figure 6] This is a graph showing the inhibitory effect of fucoidan-derived exosomes on COVID-19 adsorption to the ACE2 receptor on human cells. [Figure 7] 1 is a graph showing the content of 5-aminolevulinic acid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Next, the present invention will be described in detail with reference to the drawings.

[0047] (1) Physiological effects of fucoidan and exosomes derived from it Now, one of the causes of lifestyle-related diseases in humans is oxidative stress, and the removal of active oxygen generated by oxidative stress is achieved by a type of enzyme called superoxide dismutase (SOD). However, if the amount of SOD decreases, the removal of active oxygen becomes insufficient, and through cell damage, the body becomes more susceptible to lifestyle-related diseases such as cancer. To put it simply, a decrease in SOD can be said to be a decrease in immunity.

[0048] Therefore, the amount of SOD is a barometer of health, and by knowing the relationship between the amount of SOD and substances taken into the body from the outside, the effect of the substances taken in can be understood. Therefore, we had 10 subjects drink low molecular weight fucoidan and measured the change in the amount of SOD3 in their blood. The results are shown in the graph in Figure 1.

[0049] The low molecular weight fucoidan used was Power Fucoidan CG (made in Tonga) sold by Daiichi Sangyo Co., Ltd., a co-applicant of this application. As shown in the legend in the lower right, blood was taken at five time points (before drinking, 30 minutes, 1 hour, 2 hours, and 4 hours after drinking) and the amount of SOD3 was measured.

[0050] In other words, the SOD3 activity (Colorimetric Activity: SOD3·A) due to low molecular weight fucoidan was measured. The dosage of Power Fucoidan CG was 100g, blood was drawn twice at each time point, and the average value was used as the measurement value. M written to the right of the subject's number indicates male, and F indicates female. The numbers indicate age.

[0051] The sample, Power Fucoidan CG, is made from mozuku seaweed from the Kingdom of Tonga, which has been hydrolyzed with enzymes to reduce its molecular weight. The molecular weight of fucoidan is approximately 500, and the sulfate group is approximately 19% by weight of the fucose ratio.

[0052] The amount of SOD3 in the blood of a healthy adult is 1.95-4.60U / ml, but the blood SOD3 levels rose in all subjects after taking Power Fucoidan CG, which shows that low molecular weight fucoidan has the ability to remove active oxygen (strong immune system enhancement).

[0053] It is said that the amount of SOD drops sharply around the age of 40, and the basic SOD3 levels of each subject match this general view (although there are individual differences). Furthermore, the D1-D3, D5, D6, and D7 groups had lower normal SOD3 levels than the other groups, but the SOD3 activity levels were visibly higher after taking Power Fucoidan CG. This shows that low molecular weight fucoidan exerts a high SOD3 generation function.

[0054] The subjects on D10 were young and had originally high SOD3 values ​​(activity), so even if they ingested low molecular weight fucoidan, there was almost no effect. This means that even if low molecular weight fucoidan is ingested, SOD3 is not overproduced, and from this it can be understood that there is a function in the human body (blood) that balances the amount of SOD3 produced with the amount of active oxygen, and that even if too much low molecular weight fucoidan is ingested, no physiological adverse effects will appear.

[0055] From the results in Figure 1, it can be seen that low molecular weight fucoidan contributes to the production of SOD3 in the blood, but what needs to be considered is the mechanism by which fucoidan taken as a drink and absorbed from the large intestine supports the production of SOD3 in the blood.

[0056] Therefore, the first thing to consider is its relationship with hematopoietic stem cells. It is assumed that SOD3 in the blood is secreted from hematopoietic stem cells, and that SOD3 also contributes to the normal differentiation of hematopoietic stem cells. Therefore, it is assumed that low molecular weight fucoidan acts on hematopoietic stem cells to promote the secretion of SOD3, or that low molecular weight fucoidan promotes the generation of hematopoietic stem cells. If so, it is understood that low molecular weight fucoidan has a programming function that acts on hematopoietic stem cells.

[0057] And since it cannot be assumed that L-fucose itself, which is a polysaccharide, has a programming function (information transmission function) that acts on hematopoietic stem cells, it is highly likely that it is the miRNA and mRNA stored in the fucoidan-derived exosomes that act on hematopoietic stem cells. Although SOD3 and fucoidan are completely different substances, if we take into account the programming function of fucoidan-derived exosomes, the mechanism by which SOD3 activity increases after fucoidan intake can be clearly demonstrated.

[0058] (2) Production of fucoidan-derived exosomes Figure 2 shows a simple method for producing fucoidan-derived exosomes. In this method, mozuku seaweed is used as the raw material. Mozuku seaweed is collected from the sea, washed, and foreign matter is removed. The seaweed is then put into a pot, either in its original size or cut into appropriate pieces, and hydrolyzed by boiling. Next, impurities such as surface fibrous matter are filtered and purified to obtain a low molecular weight fucoidan stock solution. During hydrolysis, enzymes or alcohol can be added to aid in decomposition.

[0059] Next, the purified fucoidan stock solution is enclosed in a kit and set in an ultracentrifuge, and exosomes are separated by applying an acceleration of 100,000 G or more. In this embodiment, an ultracentrifuge for analysis is used, but in the mass production process, a separation ultracentrifuge or a TFF system that combines ultrafiltration and anion exchange resin is used.

[0060] Figure 3 shows an example of the relationship between particle size and amount of particles separated by ultracentrifugation. Each example has a particle size of approximately 50 to 200 nm, and it is presumed that most particles of this size are exosomes. Since it is impossible to clarify the composition and structure of all the separated particles, the separated particle group should more accurately be called fucoidan-derived exosome-like particles, but in this application, they are treated as fucoidan-derived exosomes.

[0061] Since the curve in Figure 3 is a trace of the outline of a figure consisting of many plots, the total number of particles is calculated as the integrated value of the number of plots, but in all measurements, there were more than 5 billion exosomes per ml. Theoretically, it can be estimated that there are 80 billion exosomes per ml at the above-mentioned L-fucose content, but this actually accounts for less than 10% of the total amount of exosomes that can be separated (isolated). Therefore, improving the separation rate remains a future challenge.

[0062] In this embodiment, an ultracentrifuge is used as a method for separating exosomes (or exosome-like particles), but separation methods using membrane filters (e.g., ultrafiltration) and separation methods using molecular sieves are also worth considering.

[0063] The state of exosomes in fucoidan, whether they are molecularly bound to tissues such as L-fucose or whether they exist entangled within the tissue, has not been clarified, but since exosomes can be extracted without fucose fractionation, it is assumed that exosomes exist independently and evenly throughout the fucoidan extract. Therefore, ultrafiltration seems to be a promising option for mass production.

[0064] It is also worth considering a multiple filtration method that combines microfiltration and ultrafiltration, or a combination of centrifugation and filtration. We would also like to research separation methods using chemicals such as enzymes and alcohols. In other words, we would like to research a cheap and efficient extraction method.

[0065] (3) Evaluation of fucoidan-derived exosomes (1) Regarding liver function in humans and animals, when liver damage occurs, the secretion of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) increases, and their blood concentrations rise. Therefore, as one of the means to confirm the pharmacological effects of fucoidan-derived exosomes, the amount of AST and ALT (alanine aminotransferase) was measured.

[0066] In the experiment, 8-week-old female mice were used as test subjects, and the hepatitis-inducing agent concanavalin A (ConA) was administered (injected) at a rate of 20 mg per kg of body weight. Then, fucoidan-derived exosomes were injected, and blood was taken 24 hours later to measure AST and ALT in the serum, and the state of the HE chromosome in the liver tissue was observed under a microscope.

[0067] Fucoidan-derived exosomes were extracted from Tongan mozuku and from domestic mozuku. As comparative examples, subjects without ConA and subjects administered phosphate-buffered saline (PBS) were prepared. Each subject had four individuals, and the average values ​​were used for (A) and (B).

[0068] From Figures 4(A) and (B), it can be seen that both AST and ALT values ​​were lower than when ConA was not administered and when PBS was administered. This shows that hepatocytes destroyed by ConA were repaired by fucoidan-derived exosomes.

[0069] In Figure 4, (C) and (D) are paired photographs of normal liver tissue (not administered ConA). (C) is an HE stained photograph, and (D) is a positive immunological photograph. (E) and (H) are photographs of abnormal tissue with ConA administered, (E) is an HE stained photograph, and (H) is a positive immunological photograph. (F) and (I) are paired photographs of tissue administered with exosomes (EV) extracted from Tongan mozuku. (F) is an HE stained photograph, and (I) is a positive immunological photograph. (G) and (J) are paired photographs of tissue administered with exosomes (EV) extracted from domestic mozuku. (G) is an HE stained photograph, and (J) is a positive immunological photograph.

[0070] In the positive immunostaining photographs (D), (H), and (J), the dots are helper T cells, and it can be seen that they have accumulated on the surface of CD4, which is one of the cell surface antigens. Helper T cells are differentiated from hematopoietic stem cells, and from the photograph (H), it can be seen that they have accumulated in large numbers on the surface of CD4 as an immune response to ConA.

[0071] Looking at the HE stained photographs, it can be seen that the tissues (F) and (G) administered with fucoidan-derived exosomes are very close to the normal tissue (C) when compared with the abnormal tissue (E). In other words, it can be seen that the tissues have become dense and normalized.

[0072] The same is true for the positive immunostaining images, and it can be seen that the tissues (I) and (J) administered with fucoidan-derived exosomes are very close to the normal tissue (D) when compared with the abnormal tissue shown in (H). In other words, it can be seen that the tissue has become dense and normalized, resulting in a decrease in T cells.

[0073] Thus, from the results shown in Figure 4, it can be seen that fucoidan-derived exosomes contribute significantly to the repair of liver cells.

[0074] (4) Evaluation of fucoidan-derived exosomes (2) One of the main components of the immune system of humans and animals is the NK cell (natural killer cell), a type of granular lymphocyte. NK cells have the ability to directly destroy foreign substances that invade the body, and the greater their quantity and activity, the stronger the immune system of humans and animals.

[0075] NK cells are a kind of mother ship rich in cytoplasm, and are equipped with a protein called perforin and an enzyme called granzyme as weapons against invaders such as viruses. When an invader that they consider to be an enemy approaches, they release perforin to make a hole in the invader, and then granzyme penetrates the invader's tissue and induces apoptosis. Therefore, the invader is not only destroyed but also cannot proliferate.

[0076] The immune power of fucoidan has long been claimed, but we conducted an experiment to see how fucoidan-derived exosomes act on NK cells, the male of the immune system. The results are shown in the graph in Figure 5.

[0077] In the experiment, the amount of granzyme secreted by untreated NK cells and that secreted by NK cells treated with a sample were measured, and the amount of granzyme secreted was made relative to the amount of granzyme in the untreated case, which was set at 1.0. This is shown in a graph in Figure 5 (A). In the test, a kit for a FRET substrate (Förster resonance energy transfer substrate) was used to detect the activity of granzyme B as fluorescence.

[0078] As mentioned above, granzymes are a powerful weapon of NK cells, and the greater their number, the stronger the immune system becomes. As shown in Figure 5(A), when fucoidan-derived exosomes were administered, the relative amount of granzymes in NK cells was about 8 times that of the untreated case, and about 4 times that of the case where mesenchymal stem cell-derived NK cells (MSC-EV) were administered. This shows that fucoidan-derived exosomes improve the granzyme release function of NK cells.

[0079] Furthermore, an increase in the amount of granzymes means that the NK cells have a stronger ability to attack foreign invaders. Figure 5(A) shows that when fucoidan-derived exosomes are taken up by NK cells, the NK cells' ability to attack foreign invaders increases by about four times compared to when mesenchymal stem cell-derived exosomes are added.

[0080] FasL (Fas ligand), a type II transmembrane protein, is an inducer of apoptosis in tissues. The apoptosis-inducing effect through FasL is parallel to the apoptosis-inducing effect through perforin, and it invades the target tissue and induces apoptosis in the target tissue through signal transduction. Therefore, the expression of FasL in NK cells is favorable for immune function.

[0081] Therefore, we also measured the amount of FasL expressed. For the measurement, a human FasL / CD measurement ELISA kit (Cosmo Bio Co., Ltd.) was used to measure the expression level by colorimetry. In other words, the expression value of FasL in the case of untreated exosomes to which no exosomes were added was set at 1.0, and the expression level in the case of adding fucoidan-derived exosomes was displayed as an index. This is shown in Figure 5(B), and as shown in Figure 5(B), administration of fucoidan-derived exosomes resulted in approximately 2.7 times the amount of FasL expression compared to the case of no administration.

[0082] In this way, it is clear that fucoidan-derived exosomes promote the function of NK cells, which scientifically supports the general assessment that fucoidan enhances immunity. What is important is that fucoidan-derived exosomes are accepted by NK cells without being recognized as a foreign enemy, and this acceptability allows for a wide range of applications in medicines.

[0083] Although it has been confirmed that fucoidan-derived exosomes act on NK cells to increase the release of granzymes and FasL, the mechanism has not yet been elucidated. Considering that NK cells are extremely large compared to fucoidan-derived exosomes, it is speculated that fucoidan-derived exosomes are more likely to promote the release of granzymes and FasL by acting on the numerous cytoplasms that make up NK cells, rather than acting on the center of NK cells.

[0084] In addition to the positive effect of inducing apoptosis in invaders and cancer cells, FasL has also been reported to have negative effects such as destroying self-tissues through excessive proliferation and showing resistance to apoptosis in invaded tissues, and some reports suggest that it may be the cause of fulminant hepatitis. As mentioned above, liver dysfunction due to excessive intake of turmeric-derived exosomes has been reported, and the cause may be due to the runaway FasL.

[0085] In contrast, comparing Figure 5 (A) and (B), it appears that fucoidan-derived exosomes properly control the proliferation of FasL. In other words, fucoidan-derived exosomes appear to contribute to proliferation within the range of the program originally built into NK cells. Therefore, even if taken in excess, there are no side effects and it can be said to be extremely safe.

[0086] (5) Evaluation of fucoidan-derived exosomes (3) The surface of human tissues contains receptors that receive signals from the outside. Receptors are composed of specific proteins and there are multiple types. Coronaviruses, which cause respiratory infections, bind to human tissues using an enzyme called ACE2 (angiotensin-converting enzyme 2) as a receptor, and the virus that is attached to the receptor uses human cells to grow (eat away at tissues). A typical example of this is COVID-19, which has spread around the world since 2019, and the inventors of this application also measured the COVID-19 binding inhibitory function of fucoidan-derived exosomes and other substances against ACE2.

[0087] The results are shown in Figure 6. The samples used in the measurements were PNT2 (human prostate-derived cells), the protein solution MPER, a Power Fucoidan stock solution made from Tongan mozuku, mesenchymal stem cell-derived NK cells (MSC·EV), and Power Fucoidan exosomes extracted from domestically produced mozuku.

[0088] The kit used was the "COVID-19 Spike-ACE2 Binding Assay Kit" manufactured by Funakoshi Co., Ltd., and the amount of sample bound to the company's "SARS-Cov-2 Spike" was measured using a colorimetric method, which was taken as the amount of ACE2.

[0089] Furthermore, while no significant binding inhibitory effect was observed for PNT2 and MPER, MSC·EVs, undiluted fucoidan, and fucoidan-derived exosomes exhibited significant binding inhibitory function. In particular, fucoidan-derived exosomes exhibited a binding inhibition rate of nearly 50%. From this, it can be inferred that fucoidan-derived exosomes, when atomized by spray or the like and inhaled into the respiratory system, may be effective in preventing infection caused by COVID-19 (or other coronaviruses).

[0090] Although the apoptosis-inducing effect of fucoidan-derived exosomes on coronaviruses such as COVID-19 has not been tested, in light of the messenger function of fucoidan-derived exosomes, it is possible that they may have a high killing effect. If a killing effect is observed, for example, it will be possible to carry fucoidan-derived exosomes in a mask and eliminate the virus before it enters the respiratory system or after it is expelled.

[0091] 5-aminolevulinic acid (5-ALA) is a type of natural amino acid (base) known as a fundamental substance of life, and is used in supplements and medicines due to its physiological effects such as fatigue recovery. It is also used as a cancer diagnostic marker, taking advantage of its unique optical properties during the process of tetramerization to heme.

[0092] As shown in Figure 7, it was found that exosomes extracted from Power Fucoidan (PF) made from mozuku also contain a certain amount of 5-ALA. Although the content is lower than that of red wine, it is highly likely that the 5-ALA contained in the fucoidan-derived exosomes contributes to immune regulation functions. In other words, it can be assumed that although the content is low, the physiological and pharmacological effects are very high.

[0093] To be more precise, the samples shown in Figure 7 are the brand of sake Dassai (Asahi Shuzo Co., Ltd.), the natto is sold at convenience stores, the red wine is Bourgoone Pinot Noir 2018 (produced in France), and the Japanese tea is Tsukiji Uogashi Tea (produced by Uogashi Meicha Co., Ltd.).

[0094] (6). Conclusion and Others As explained above, fucoidan-derived exosomes act on human and animal tissues and cells to induce apoptosis and promote normal cell proliferation. For this reason, they are expected to be used in a wide range of applications, including drugs for treating various diseases and injuries such as inflammation, ulcers, and cancer, preventive drugs for lifestyle-related diseases, supplements for maintaining and promoting health, and skin care products that restore the skin to its original state.

[0095] Furthermore, notable advantages of fucoidan-derived exosomes include their acceptability to human and animal tissues and cells without rejection, their safety with no adverse effects (e.g. side effects) even when used in excess, their durability with no resistance even when used continuously, their independence as they support the inherent functions of the target tissue, and their versatility to act on a wide variety of tissues. These characteristics mean that they can contribute to society by supplying products in a variety of ways for a variety of uses.

[0096] Of particular note is its versatility to act on a wide variety of tissues and its ability to support self-repair by correcting the genetic information of the target tissue. By applying this to stem cells, which are pluripotent cells, it is expected that it will contribute to the treatment of various diseases and regenerative medicine.

[0097] In the manufacturing method of the embodiment, the raw material is hydrolyzed to produce low molecular weight fucoidan and then centrifuged, but it is also possible to wash the raw material such as mozuku seaweed and then grind it into fine particles and then place it in a centrifuge or filter without going through the hydrolysis step.

[0098] In addition, the purified exosomes are obtained in the form of a solution, but when commercializing the product, they can be used as is or after concentration. When used as an injection or infusion solution, they can be concentrated and mixed with a solvent such as physiological saline or distilled water according to standard methods. When used as a tablet or powder, they can be mixed with an amorphous excipient and then dried and compressed into tablets or adjusted to a powder shape according to standard methods. [Industrial Applicability]

[0099] The present invention can be embodied in a technology related to fucoidan-derived exosomes, and therefore has industrial applicability. [Explanation of symbols]

[0100] 1 Ultracentrifuge 2 Kits

Claims

1. A seaweed-derived extract for use in a bioactive product for humans or animals, the extract containing exosomes or exosome-like particles having a size of 50-200 nm as a major component. Seaweed extract.

2. The exosome or exosome-like particle contains 500 million or more per ml.

2. The seaweed-derived extract according to claim 1.

3. A bio-effecting product for use in humans or animals, comprising: Its main component is exosomes extracted from seaweed, measuring 50-200 nm in size. Bioactive products.

4. The bioactive product is at least one of a medicine, a quasi-drug, a dietary supplement, a cosmetic, a soft drink, and a confectionery, and is intended for use by humans or animals. A bioactive product as claimed in claim 3.

5. Mozuku seaweed is used as the seaweed.

5. A bioactive substance according to claim 3 or 4.

6. One or more types of seaweed are used as a raw material, and the seaweed is hydrolyzed under heating with or without the addition of an enzyme to produce low molecular weight fucoidan, and then exosomes are separated from the low molecular weight fucoidan by ultracentrifugation or ultrafiltration. Method for extracting exosomes from seaweed.

7. As the seaweed, one or more types of brown algae including mozuku, hibamata, kombu, hijiki, wakame, einkorn, arame, and kurome are used. The method for extracting exosomes derived from seaweed according to claim 6.

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