KR102551398B1 - Composition for promoting the generation of exosomes derived from stem cells containing Citrus junos extracts - Google Patents

Abstract

The present invention relates to a composition for promoting the production of stem cell-derived exosomes containing a natural product extract. Since this increases, it is possible to more efficiently produce high-quality stem cells and stem cell-derived exosomes, which can be usefully used for related research and development and commercialization.

Description

Composition for promoting the generation of exosomes derived from stem cells containing Citrus junos extracts}

The present invention relates to a stem cell-derived exosome production-promoting composition comprising a natural product extract, and more particularly, to a natural product extract that increases the production of stem cell-derived exosomes by enhancing the exosome-producing ability of stem cells during stem cell culture. It relates to a composition for promoting stem cell-derived exo-some production comprising a.

Microvesicles (Extracellular Vesicles) are vesicles composed of a spherical lipid-bilayer with a size of 30 to 1000 nm including microvesicles and exosomes.

The lipid bilayer of exosomes has a phospholipid bilayer structure similar to that of the cell of origin (donor cell). It is a component of substances secreted by cells to the outside of the cell and performs functional roles such as cell-cell communication and cellular immune mediation. It is known.

Exosomes contain cell-specific constituents that reflect the unique biological functions of the cell of origin (donor cell), and include various soluble proteins, extrinsic proteins, and transmembrane protein components in addition to phospholipids, mRNAs, and miRNAs.

These exosomes are released from all animal cells, including mast cells, lymphocytes, astrocytes, platelets, nerve cells, endothelial cells, and epithelial cells, and are found in various body fluids such as blood, urine, mucus, saliva, bile, ascites, and cerebrospinal fluid. do. Exosomes can pass through the Blood-Brain Barrier (BBB) and have high selective permeability to the extent that they can pass through the cell membrane of epidermal cells and endothelial cells, making them a drug delivery system (DDS), a nanocarrier for specific drugs. It is also used for development.

It is known that exosomes and microvesicles secreted from mesenchymal stem cells are involved in cell-to-cell communication and show regenerative medicine therapeutic efficacy of stem cells.

It is known to have a trophic effect on paracrine factors secreted from cells without long-term survival after stem cells are transplanted into the body, and these factors include growth factors, chemokine ), cytokines, and the like are secreted by extracellular vesicles such as exosomes, and these exosomes are derived from stem cells. Therefore, exosomes are used to characterize stem cells and evaluate their therapeutic efficacy.

Recently, studies on the treatment effects of various diseases are being actively conducted using exosomes secreted by mesenchymal stem cells without using mesenchymal stem cells themselves, and academia and industry are using this to overcome the limitations of existing stem cell therapies. is expected to be a new alternative that can overcome the

Accordingly, the inventors of the present invention found that the natural product extract according to the present invention promotes the production of stem cell-derived exosomes, and when fermented turmeric, citron, and blueberry extracts are pretreated with stem cells, the ability of stem cells to produce exosomes and the level of proteins in exosomes are improved. It was confirmed that the effect of increasing the content and producing uniform and high-purity exosomes was excellent.

Accordingly, an object of the present invention is to provide a composition for promoting stem cell-derived exosome production comprising a natural product extract.

Another object of the present invention is to provide stem cell-derived exosomes pretreated with natural product extracts.

Another object of the present invention is to provide a method for producing stem cell-derived exosomes comprising a pretreatment step of culturing stem cells in a cell culture medium containing a natural product extract.

The present invention relates to a composition for promoting the production of stem cell-derived exosomes containing a natural product extract. The uniformity and purity of is improved.

Accordingly, the present inventors confirmed that the composition for promoting the production of stem cell-derived exosomes, including a natural product extract, greatly increased the number of exosomes produced from stem cells and the content of proteins in the exosomes when the stem cell culture was pretreated.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a composition for promoting stem cell-derived exosome production comprising at least one selected from the group consisting of tomato extract, raspberry extract, fermented turmeric extract, citron extract and blueberry extract.

In one embodiment of the present invention, the extract may include one or more selected from the group consisting of fermented turmeric extract, citron extract, and blueberry extract.

In the present invention, the extract includes a crude solvent extract, a specific solvent-soluble extract (solvent fraction), and a solvent fraction of the crude solvent extract, and the extract may be in the form of a solution, concentrate or powder.

In the present invention, fermented turmeric is obtained by fermenting turmeric tuberous roots of the ginger family as they are or by removing the perianth and steaming and drying turmeric. Turmeric is native to and cultivated in Southeast Asia including southern China, India and Okinawa, and is also cultivated in southern Korea do. The main component of turmeric is curcumin and its analogs, which show strong antioxidant activity and are used as a medicine to treat inflammation. It is known to treat, clear the mind, get rid of chest and abdominal fullness, promote bile secretion and treat gallbladder stones, promote bile secretion and excretion, and reduce plaque formation in the coronary arteries.

In one embodiment of the present invention, fermented turmeric may be fermented through known lactic acid bacteria.

Lactic acid bacteria refers to a group of bacteria that produce lactic acid as a major product of carbohydrate metabolism.

In the present invention, fermented turmeric is Aspergillus oryzae, Monascus ruber, Monascus pilosus, Monascus purpureus, Lactococcus, lactose from the group consisting of Lactobacillus, Leuconostoc, Propionibacterium, Enterococcus, Bifidobacterium, Streptococcus and Pediococcus It may be fermented through the selected lactic acid bacteria, but is not limited thereto.

In one embodiment of the present invention, fermented turmeric is inoculated with a strain in a dried and sterilized turmeric sample at 0.1 to 40%, 1 to 20%, for example, 1 to 15% (v/v%), compared to powder. It may be manufactured.

In one embodiment of the present invention, the fermented turmeric is 1 to 60 ℃ to 50 ℃, for example, 20 to 40 ℃ at a relative humidity of 20 to 90%, 40 to 80%, for example, 50 to 70% It may be fermented.

In one embodiment of the present invention, the fermented turmeric extract may be obtained by treating fermented turmeric powder with a solvent.

A polar solvent or a non-polar solvent may be used as a solvent used in fermented turmeric extraction. Polar solvents include water, methanol, methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol, acetic acid, dimethylformamide (DMF) and DMSO ( dimethyl sulfoxide). Non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkanes, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1 -chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1 ,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride and THF.

When a mixture of water and alcohol is used as a solvent used in the preparation of fermented turmeric extract, 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), or It may be an aqueous solution of 70% or more and less than 100% (v/v) of straight-chain or branched alcohol having 1 to 4 carbon atoms.

In the present invention, the citron extract may be a crude extract obtained by extracting citron with water and at least one solvent selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

When a mixture of water and alcohol is used as a solvent used in the preparation of the crude extract of citron, it is 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), or It may be an aqueous solution of 70% or more and less than 100% (v/v) of straight-chain or branched alcohol having 1 to 4 carbon atoms.

The alcohol aqueous solution may be at least one selected from the group consisting of an aqueous methanol solution, an ethanol aqueous solution, a propanol aqueous solution, and a butanol aqueous solution.

Citron extract according to the present invention may be a solvent fraction obtained by fractionating the crude solvent extract with an additional solvent, for example, a solvent using at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol in the crude solvent extract It may be a fraction.

For example, citron extract is a solvent crude extract obtained by extracting citron with one or more solvents selected from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms, and 1 selected from the group consisting of ethyl ether, ethyl acetate, and butanol. It may be a solvent fraction using more than one solvent.

In the present invention, the blueberry extract may be a crude extract obtained by extracting blueberries with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

When a mixture of water and alcohol is used as a solvent used in the preparation of crude extract of blueberry, 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), 30% More to less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), Or it may be an aqueous solution of 70% or more to less than 100% (v/v) of straight-chain or branched alcohol having 1 to 4 carbon atoms.

The blueberry extract according to the present invention may be a solvent fraction obtained by fractionating the crude solvent extract with an additional solvent, for example, using one or more solvents selected from the group consisting of ethyl ether, ethyl acetate, and butanol in the crude solvent extract. It may be a solvent fraction.

For example, the blueberry extract is a solvent crude extract obtained by extracting blueberries with one or more solvents selected from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms in the group consisting of ethyl ether, ethyl acetate, and butanol. It may be a solvent fraction using one or more selected solvents.

In the present invention, the tomato extract may be a crude extract obtained by extracting tomatoes with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

When a mixture of water and alcohol is used as a solvent used in the preparation of a crude extract of tomato, 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), or It may be an aqueous solution of 70% or more and less than 100% (v/v) of straight-chain or branched alcohol having 1 to 4 carbon atoms.

The alcohol aqueous solution may be at least one selected from the group consisting of an aqueous methanol solution, an ethanol aqueous solution, a propanol aqueous solution, and a butanol aqueous solution.

The tomato extract according to the present invention may be a solvent fraction obtained by fractionating the crude solvent extract with an additional solvent, for example, a solvent using at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol in the crude solvent extract It may be a fraction.

For example, tomato extract is a solvent crude extract obtained by extracting tomatoes with at least one solvent selected from the group consisting of water and a straight-chain or branched alcohol having 1 to 4 carbon atoms, ethyl ether, ethyl acetate, and 1 selected from the group consisting of butanol. It may be a solvent fraction using more than one solvent.

In the present invention, raspberry extract may be a crude extract obtained by extracting raspberry with water and one or more solvents selected from the group consisting of straight-chain or branched alcohols having 1 to 4 carbon atoms.

In the case of using a mixture of water and alcohol as a solvent used in the preparation of crude extract of Bokbunja, 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), or 30% or more to less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), or It may be an aqueous solution of 70% or more and less than 100% (v/v) of straight-chain or branched alcohol having 1 to 4 carbon atoms.

The alcohol aqueous solution may be at least one selected from the group consisting of an aqueous methanol solution, an ethanol aqueous solution, a propanol aqueous solution, and a butanol aqueous solution.

The black raspberry extract according to the present invention may be a solvent fraction obtained by fractionating the crude solvent extract with an additional solvent, for example, using one or more solvents selected from the group consisting of ethyl ether, ethyl acetate, and butanol in the crude solvent extract. It may be a solvent fraction.

For example, bokbunja extract is a solvent crude extract obtained by extracting bokbunja with at least one solvent selected from the group consisting of water and a straight-chain or branched alcohol having 1 to 4 carbon atoms, ethyl ether, ethyl acetate, and 1 selected from the group consisting of butanol. It may be a solvent fraction using more than one solvent.

The extraction method used in the present invention may be any method commonly used, and may be, for example, cold brewing, hot water extraction, ultrasonic extraction, or reflux cooling extraction, but is not limited thereto.

In one embodiment of the present invention, the composition for promoting the production of exosomes derived from stem cells including fermented turmeric extract, citron extract, and blueberry extract may further include a known exosome production promoting material other than the extract.

The term "Exosome" in the present specification is a cell-derived endoplasmic reticulum, which is present in the body fluids of almost all eukaryotic organisms, and refers to an endoplasmic reticulum having a diameter of about 30-100 nm, which is larger than LDL protein but much smaller than red blood cells. do. It is well known that exosomes can be released from cells when multivesicular bodies fuse with cell membranes or can be released directly from cell membranes, and perform important and specialized functions such as coagulation and intercellular signaling.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into two or more different types of cells while having self-renewal ability as an undifferentiated cell.

In one embodiment of the present invention, the stem cells may be autologous or allogeneic stem cells, may be derived from any type of animal, including humans and non-human mammals, may be adult-derived stem cells, and embryo-derived stem cells. may be stem cells. For example, the stem cells may be selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells (iPSC), but are not limited thereto.

In one embodiment of the present invention, adult stem cells are derived from human or animal tissue-derived adult stem cells, human or animal tissue-derived mesenchymal stromal cells, and human or animal tissue-derived induced pluripotent stem cells. It may be selected from the group consisting of mesenchymal stem cells, but is not limited thereto.

The term "embryonic stem cell" in the present specification refers to cells extracted during the development of an embryo, and cultured in vitro by extracting inner cell mass from a blastocyst embryo, just before a fertilized egg is implanted in the mother's uterus. it means one

Embryonic stem cells refer to cells that are pluripotent or totipotent capable of differentiating into cells of all tissues of an individual and have self-renewal ability, in a broad sense. means that embryoid bodies derived from embryonic stem cells are also included.

In one embodiment of the present invention, stem cells may include embryonic stem cells of any origin, such as human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, etc., but are not limited thereto.

As used herein, the term "adult stem cell" refers to cells extracted from umbilical cord blood, bone marrow, blood, etc. of a grown adult, and refers to cells immediately before differentiation into cells of a specific organ, and develops into tissues within the body when necessary. It refers to cells in an undifferentiated state that have the ability to

In one embodiment of the present invention, adult stem cells include adult stem cells derived from various tissues of humans or animals, mesenchymal stromal cells derived from human or animal tissues, and induced pluripotent stem cells derived from various tissues of humans or animals. It may include mesenchymal stem cells and pluripotent stem cells derived from cells, but is not limited thereto.

In one embodiment of the present invention, human or animal or animal tissue may be selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion, and placenta, but is not limited thereto.

In one embodiment of the present invention, stem cells derived from various tissues of humans or animals are a group consisting of hematopoietic stem cells, mammary stem cells, intestinal stem cells, vascular endothelial stem cells, neural stem cells, olfactory nerve stem cells and testicular stem cells. It may be selected from, but is not limited thereto.

In one embodiment of the present invention, the stem cells may be adipose mesenchymal stem cells.

The term "induced pluripotent stem cell (iPSC)" as used herein refers to cells induced to have pluripotent differentiation potential through an artificial dedifferentiation process from differentiated cells, and includes "dedifferentiated stem cells" and the like. can be used with the same meaning.

The artificial dedifferentiation process is performed by using a virus-mediated or non-viral vector using retroviruses, lentiviruses, and Sendai viruses, by introducing non-virus-mediated dedifferentiation factors using proteins and cell extracts, or by using stem cell extracts, It may include a reverse differentiation process by a compound or the like.

induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have a similar cell shape, similar gene and protein expression, have pluripotency in vitro and in vivo, and form teratomas. When inserted into the blastocyst of a mouse, a chimera mouse is formed, and germline transmission of the gene is possible.

In one embodiment of the present invention, induced pluripotent stem cells may include induced pluripotent stem cells of any origin, such as human, monkey, pig, horse, cow, sheep, dog, cat, mouse, rabbit, etc., but are not limited thereto. It is not.

As used herein, the term "production promotion" means that the production, production, release, etc. of a specific substance is increased within the same time compared to the control group. Specifically, the number of exosomes produced from stem cells and the exosomes It means that the content of protein, RNA, etc. is increased.

In one embodiment of the present invention, the composition of the present invention can be used for stem cell culture as a medium composition, and is administered in vivo with stem cells as a pharmaceutical composition for promoting in vivo exosome production, or It can enhance the in vivo efficacy of stem cells by administering precedent or follow-up.

The term "enhancement of the in vivo efficacy of stem cells" in the present specification is based on the premise that the efficacy of stem cells for the purpose of treatment (in vivo efficacy) for specific diseases such as arthritis or neuropathy is mediated by exosomes released by stem cells. As a result, when the composition of the present invention is administered together with stem cell administration, the production of exosomes from stem cells is promoted, and as a result, the disease treatment effect (in vivo efficacy) of stem cells is enhanced.

In one embodiment of the present invention, the medium composition refers to a composition containing essential components necessary for the growth, survival and proliferation of cells in vitro, and is a medium for stem cell culture commonly used in the art. including DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10) , DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), KnockOut DMEM , E8 (Essential 8 Medium), commercially prepared media or artificially synthesized media may be used, but is not limited thereto.

In one embodiment of the present invention, the medium composition generally includes a carbon source, a nitrogen source, and trace element components, and may further include amino acids, antibiotics, and the like.

In one embodiment of the present invention, the medium composition may be prepared by adding ranifibranor, a component of the present invention, to a conventional stem cell culture medium.

In one embodiment of the present invention, the fermented turmeric extract, citron extract or blueberry extract is 1 to 1000ug/ml, 1 to 900ug/ml, 1 to 800ug/ml, 1 to 700ug/ml, 1 to 600ug/ml in the medium. , 1 to 500ug/ml, 1 to 400ug/ml, 1 to 300ug/ml, 1 to 200ug/ml, 50 to 300ug/ml, 50 to 250ug/ml, 50 to 200ug/ml, 50 to 150ug/ml, 1 to 100ug/ml concentration, for example, but may be added at a concentration of 100ug/ml, but is not limited thereto.

Another aspect of the present invention is a stem cell-derived exosome pretreated with at least one selected from the group consisting of fermented turmeric extract, citron extract and blueberry extract.

Descriptions of common contents between the composition according to the present invention and the stem cell-derived exosomes are omitted to avoid excessive complexity in the present specification.

Another aspect of the present invention is a method for producing stem cell-derived exosomes comprising the following steps:

A culture step of culturing stem cells in a cell culture medium containing at least one selected from the group consisting of fermented turmeric extract, citron extract and blueberry extract.

In one embodiment of the present invention, the method for producing stem cell-derived exosomes may further include the following steps:

An additional culture step of further culturing in a cell culture medium after washing the cultured stem cells; and

Separation step to isolate exosomes.

The culturing step of the method for producing stem cell-derived exosomes according to the present invention is a process of applying a stimulus to the stem cells by pre-treating the stem cells with a natural product. By this process, the stem cells produce more exosomes than the stem cells that have not been pretreated with the material, and the protein content in the exosomes is also increased.

In one embodiment of the present invention, the additional culture of the additional culture step is further cultured for 12 hours to 120 hours, 24 hours to 96 hours, 48 hours to 96 hours, or 60 hours to 84 hours, for example, 72 hours. It may be cultured, but is not limited thereto.

In the separation step of the method for producing stem cell-derived exosomes according to the present invention, the culture medium of the stem cells additionally cultured in the additional culture step is centrifuged at 3000xg for 5 to 25 minutes to remove remaining cells and cell residues, After taking the supernatant and high-speed centrifugation at 1,000-2,000xg for 10-70 minutes, the supernatant is removed to obtain exosomes remaining in the lower layer.

The present invention relates to a composition for promoting the production of stem cell-derived exosomes containing a natural product extract. Since this increases, it is possible to more efficiently produce high-quality stem cells and stem cell-derived exosomes, which can be usefully used for related research and development and commercialization.

1 is a view briefly showing a manufacturing process of a natural product extract according to an embodiment of the present invention.
Figure 2 is a graph showing the toxicity evaluation results when the natural product extract according to an embodiment of the present invention is treated with stem cells.
3 is a graph showing the protein concentration in exosomes of adipose mesenchymal stem cell-derived exosomes pretreated with a natural product extract according to an embodiment of the present invention.
4 is a graph showing the size distribution of exosomes derived from adipose mesenchymal stem cells pretreated with a natural product extract according to an embodiment of the present invention.
5 is a photograph visually showing the distribution of exosome particles derived from adipose mesenchymal stem cells pretreated with a natural product extract according to an embodiment of the present invention.
6 is a photograph confirming CD63, an exosome-specific marker, by Western blot in exosomes derived from adipose mesenchymal stem cells pretreated with a natural product extract according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Preparation of natural product extract

1-1. Preparation of Blueberry Extract

Preparation of Blueberry Water Extract (VW)

After obtaining blueberries from Damyang, Jeollanam-do, and drying and pulverizing blueberries, 100g was put into 2L of 100% water (20 times the dry weight), extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge. Impurities were filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

Preparation of Blueberry Ethanol Extract (VE)

After obtaining blueberries from Damyang, Jeollanam-do, and drying and pulverizing blueberries, 100g was put in 2L of 70% ethanol solvent (20 times the dry matter weight), extracted under reflux for 3 hours at 250 ° C, cooled, and the precipitate was removed using a centrifuge. The following impurities were filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

1-2. Preparation of raspberry extract

Preparation of raspberry water extract (RW)

After obtaining bokbunja from Gwangyang, Jeonnam, drying and pulverizing bokbunja, 100 g of bokbunja was put in 2L of 100% water (20 times the dry weight), extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge. It was filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

Preparation of raspberry ethanol extract (RE)

After obtaining bokbunja from Gwangyang, Jeollanam-do, drying and pulverizing bokbunja, 100g was put in 2L of 70% ethanol solvent (20 times the dry matter weight), extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge, followed by impurities. was filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

1-3. Preparation of Tomato Extract

Preparation of tomato water extract (LW)

Tomatoes from Hwasun, Jeollanam-do were obtained, and 100 g of dried and pulverized tomatoes were added to 2 L of 100% water (20 times the dry weight), extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifugal separator. It was filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

Preparation of tomato ethanol extract (LE)

Tomatoes obtained from Hwasun, Jeollanam-do were obtained, dried, and 100 g of pulverized tomatoes were put in 2L of 70% ethanol solvent (20 times the dry weight), extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge, followed by impurities. was filtered to obtain a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50 °C and 750 mmHg, and dried at -80 °C using a lyophilizer. After drying, it was stored in a -20 ° C freezer until used in experiments.

1-4. Preparation of fermented turmeric extract

Production of fermented turmeric

Turmeric purchased from the Jindo Turmeric Farming Association located in Jisan-myeon, Jindo-gun, Jeollanam-do was cut, dried, and pulverized, and 100 kg of dried turmeric powder was used as a raw material. As the strain used for fermentation, Hwanggukgyun (Aspergillus oryzae) purchased from Chungmu Fermentation was used. After washing turmeric three times with running water, the surface was dried and cut (thickness less than 5 mm). Then, the cut turmeric is dried for 24 hours (moisture content less than 5%) with an electrothermal dryer (Daeshin Gong), and then sieved through a 100 mesh sieve through the processes of 1st grinding (controlling), 2nd grinding (crude powder), 3rd grinding (fine powder) The turmeric powder that passed through was hermetically packed and stored at room temperature to prepare turmeric powder. The prepared turmeric powder was sterilized using a solid fermenter (3ton, manufactured by Innobio) owned by the Gyeongbuk Bio Industry Research Institute located in Andong, Gyeongbuk. (sterilization condition 121 ℃, 1.0 atm, 20min). After the sterilization of the turmeric powder was completed, the fermented turmeric was prepared by inoculating the Hwangguk strain at 2% (v / v%) of the powder in the fermentation tank and performing fermentation at a temperature of 25 ° C, moisture 60%, and fermentation time of 36 hours.

Preparation of fermented turmeric water extract (FW)

1 L of purified water was added to 50 g of prepared fermented turmeric, followed by heating and extraction in hot water at 250 ° C for 3 hours, concentrating the filtrate with a vacuum concentrator (Hanshin Co., Ltd., HS-2000), and then drying the fermented turmeric extract by heating. obtained and used in the following experiments.

Preparation of fermented turmeric ethanol extract (FE)

1 L of ethanol was added to 50 g of prepared fermented turmeric, followed by heating and extraction in hot water at 250 ° C for 3 hours, concentrating the filtrate with a vacuum concentrator (Hanshin Co., Ltd., HS-2000), and then drying the fermented turmeric extract by heating. obtained and used in the following experiments.

1-5. Preparation of Citron Extract

Preparation of Citron Water Extract (CW)

After purchasing citron from Wando, Jeollanam-do, drying and pulverizing the pulp and skin, 50 g of citron was added to 1 L of 100% water, extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge, and impurities were filtered and obtained a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50°C and 750 mmHg, and dried at -80°C using a lyophilizer to obtain a dried extract.

Preparation of Citron Ethanol Extract (CE)

After purchasing citron from Wando, Jeollanam-do, drying and pulverizing the pulp and skin, 50 g of citron was put into 1 L of 100% ethanol, extracted under reflux for 3 hours at 250 ° C, cooled, and then the precipitate was removed using a centrifuge, and impurities were filtered and obtained a filtrate. Thereafter, the filtrate was concentrated under reduced pressure at 50°C and 750 mmHg, and dried at -80°C using a lyophilizer to obtain a dried extract.

Example 2: Separation of stem cell-derived exosomes according to extract treatment

Extract pre-treatment

Adipose-derived stem cells were cultured in DMEM high glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 5% CO2 and 37°C, and the plates were 90% full. when subcultured. After culturing for 2 days, washing with PBS and replacing with exosome-depleted media, blueberry water extract, blueberry ethanol extract, raspberry water extract, raspberry ethanol extract, tomato water extract, tomato ethanol extract, fermentation Turmeric water extract, fermented turmeric ethanol extract, citron water extract and citron ethanol extract were respectively treated. At this time, the natural product extract was treated to be 50 μg/ml, 100 μg/ml or 200 μg/ml, respectively. The culture medium was recovered after two days.

Exosome isolation

Exosomes were isolated according to the same process as in FIG. 1 . Exosomes were isolated using the ExoQuick kit. The collected culture medium is centrifuged at 3000xg for 15 minutes to remove cell residues, and only the supernatant is transferred to a new tube. Add 2 ml of exoquick reagent per 10 ml of culture medium, mix well, and store in the refrigerator. The next day, centrifugation was performed at 1500xg for 30 minutes, the supernatant was discarded, and an appropriate amount of PBS was added to the settled exosomes to loosen them well and stored at -20 ° C.

Example 3: Evaluation of toxicity of extracts to stem cells

To evaluate the toxicity of the extract prepared in Example 1 on adipose mesenchymal stem cells, an MTT assay was performed.

Stem cells treated with blueberry water extract, blueberry ethanol extract, raspberry water extract, raspberry ethanol extract, tomato water extract, tomato ethanol extract, fermented turmeric water extract, fermented turmeric ethanol extract, citron water extract and citron ethanol extract The cytotoxicity was evaluated by measuring the absorbance at 550 nm using the MTT kit.

MTT assay results As can be seen in Figure 2, blueberry water extract, blueberry ethanol extract, tomato water extract, tomato ethanol extract, fermented turmeric water extract, fermented turmeric ethanol extract, citron water extract, citron ethanol extract are all 100ug/ml It was confirmed that it did not exhibit toxicity to adipose mesenchymal stem cells up to the concentration.

Example 4: Increase in the amount of exosome-derived protein according to extract treatment

BSA standards with concentrations of 0, 25, 125, 250, 500, 750, 1000, 1500, and 2000ug/ml were prepared. BCA reagents A and B were prepared according to the number of samples at a ratio of 50:1. In a microplate, put 15ul each of standard, blueberry water extract, blueberry ethanol extract, fermented turmeric water extract, fermented turmeric ethanol extract, citron water extract, and citron ethanol extract into wells, and add 200ul of BCA reagent A+B mixture. and absorbance was measured at 550 nm.

CON CW CE FW FE VW VE Concentration of exosome-derived protein (ug/mL) 1189.815 926.11 997.963 1006.481 1078.7 906.85 1221.3

As can be seen from the experimental results, Table 1 and FIG. 3, it was confirmed that the concentration of exosome-derived proteins in the adipose mesenchymal stem cell-derived exosomes pretreated with blueberry ethanol extract was significantly higher than that of the control group.

Example 5: Confirmation of improved uniformity of exosomes according to extract treatment

The size and uniformity of exosomes were measured using a ZetaSizer ZS90 (Malvern Instruments, UK). Each sample was diluted with 1:100 PBS (Phosphate buffered saline) and measured. (Refractive Index = 1.330, Viscosity = 0.8882, Temperature = 25°C).

processing material Average particle size (r.nm) Degree of Dispersion (PDI) Control 49.87 0.696 CW 42.85 0.598 CE 37.26 0.539 FW 54.24 0.656 FE 87.37 0.589 VW 24.18 0.505 VE 50.27 0.520

As a result of the experiment, as can be seen in Table 2, the average size of the extract-treated adipose mesenchymal stem cell-derived exosomes was all measured to be 200 nm or less. Considering that the size of exosomes is generally determined to be 200 nm or less, it was confirmed that all exosomes derived from adipose mesenchymal stem cells pretreated with the extract according to the present invention exhibit the characteristics of exosomes. In addition, as shown in Table 2, the degree of dispersion of exosomes in adipose mesenchymal stem cells pretreated with citron water extract, citron ethanol extract, fermented turmeric water extract, fermented turmeric ethanol extract, blueberry water extract and blueberry ethanol extract compared to the control group ( PDI) was confirmed to be close to a single substance. Furthermore, the dispersity of the blueberry water extract and the blueberry ethanol extract was close to 0.5, confirming that the uniformity of exosomes was the best.

Example 6: Confirmation of increase in the number of exosomes according to extract treatment

The size and distribution of the particles were measured using a NS300 (Malvern) 488 nm laser. All samples were diluted with PBS within the range of 1×10 ⁸ to 1×10 ⁹ particles/ml, and the final volume was unified to 1 ml to measure the distribution of particles.

processing material Particle size (nm) Concentration (NO/ml) Control 95.9 ± 94.5 1.75×10 ¹¹ ± 7.95×10 ¹⁰ CW 123.5 ± 3.4 2.23×10 ¹¹ ± 1.4×10 ¹⁰ CE 116.4 ± 18.0 2.58×10 ¹¹ ± 1.10×10 ¹⁰ FW 113.4 ± 8.3 2.16×10 ¹¹ ± 9.95×10 ⁹ FE 107.6 ± 6.4 1.95×10 ¹¹ ± 4.99×10 ⁹ VW 95.5 ± 7.5 1.34×10 ¹¹ ± 1.11×10 ¹⁰ VE 102.5 ± 9.5 3.22×10 ¹¹ ± 6.85×10 ¹⁰

As a result of the analysis, as shown in FIGS. 4 to 5 and Table 3, it was confirmed that the exosomes derived from adipose mesenchymal stem cells pretreated with each extract had a diameter of around 100 nm. Accordingly, the exosomes derived from adipose mesenchymal stem cells pretreated with the extract It was confirmed that all exhibit the characteristics of exosomes. In addition, it was confirmed that the production of stem cell-derived exosomes increased when the citron water extract, the citron ethanol extract, the blueberry water extract, and the blueberry ethanol extract were pretreated, compared to the control group. In particular, in the case of blueberry ethanol extract, it was confirmed that the exosome generating ability of stem cells was most significantly improved.

Example 7: Confirmation of expression of exosome markers according to extract treatment

After adjusting the concentration to the same concentration through exosome protein quantification by the BCA method, electrophoresis was performed at 300 mA using a 5% stacking and 10% seperating gel. The protein was transferred from the gel to the membrane through the transfer process and blocked for one hour. The primary antibody, CD63 antibody, was attached and incubated overnight at 4°C. After washing, the secondary antibody was attached, and the unbound secondary antibody was washed away with a washing buffer, and then the band was measured.

Experimental results, as shown in FIG. 6, exosome-specific markers in stem cell-derived exosomes pretreated with citron water extract, citron ethanol extract, fermented turmeric water effluent, fermented turmeric ethanol extract, blueberry water extract, and blueberry ethanol extract It was confirmed that phosphorus CD63 was expressed.

Claims (9)

In the composition for promoting the production of stem cell-derived exosomes containing citron extract,
Wherein the stem cells are selected from the group consisting of embryonic stem cells, adult stem cells and induced pluripotent stem cells (iPSC), stem cell-derived composition for promoting exosome production. According to claim 1, wherein the composition further comprises at least one selected from the group consisting of tomato extract, raspberry extract, fermented turmeric extract and blueberry extract, the composition. delete The composition according to claim 1, wherein the adult stem cells are adipose mesenchymal stem cells. The composition according to claim 2, wherein the citron extract, tomato extract, raspberry extract, fermented turmeric extract and blueberry extract are water extracts or ethanol extracts. delete delete In the method for producing stem cell-derived exosomes comprising the following steps,
Method for producing stem cell-derived exosomes, wherein the stem cells are selected from the group consisting of embryonic stem cells, adult stem cells, and induced pluripotent stem cells (iPSC):
A culture step of culturing stem cells in a cell culture medium containing citron extract. The method of claim 8, wherein the cell culture medium further comprises fermented turmeric extract or blueberry extract.

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