WO2015056982A1 - 만능 줄기세포의 유도 방법 및 상기 방법에 의해 제조된 만능줄기세포 - Google Patents

만능 줄기세포의 유도 방법 및 상기 방법에 의해 제조된 만능줄기세포 Download PDF

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Publication number
WO2015056982A1
WO2015056982A1 PCT/KR2014/009702 KR2014009702W WO2015056982A1 WO 2015056982 A1 WO2015056982 A1 WO 2015056982A1 KR 2014009702 W KR2014009702 W KR 2014009702W WO 2015056982 A1 WO2015056982 A1 WO 2015056982A1
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Prior art keywords
cells
extract
stem cells
composition
plant
Prior art date
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Ceased
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PCT/KR2014/009702
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English (en)
French (fr)
Korean (ko)
Inventor
김아름
김수나
박원석
권유욱
박영배
김효수
백재승
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Amorepacific Corp
SNU R&DB Foundation
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Amorepacific Corp
SNU R&DB Foundation
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Publication date
Application filed by Amorepacific Corp, SNU R&DB Foundation filed Critical Amorepacific Corp
Priority to EP14853540.4A priority Critical patent/EP2977446B1/en
Priority to JP2016548991A priority patent/JP6606085B2/ja
Priority to HK16108463.3A priority patent/HK1220487B/zh
Priority to CN201480019489.5A priority patent/CN105722974B/zh
Priority to EP17164428.9A priority patent/EP3275999B1/en
Priority to US14/779,136 priority patent/US20160215269A1/en
Publication of WO2015056982A1 publication Critical patent/WO2015056982A1/ko
Anticipated expiration legal-status Critical
Priority to US15/377,464 priority patent/US10251824B2/en
Ceased legal-status Critical Current

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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present specification relates to a method for inducing pluripotent stem cells by reprogramming and / or dedifferentiating adult differentiated cells, to pluripotent stem cells prepared by the method and to a composition comprising the pluripotent stem cells.
  • the present disclosure also relates to a pharmaceutical composition or cosmetic composition comprising pluripotent stem cells.
  • the present invention also relates to a composition for stem cell activation, skin cell proliferation, skin regeneration or anti-aging.
  • Stem cells are cells capable of differentiating into the various cells constituting biological tissues, which collectively refer to the undifferentiated cells obtained before the differentiation obtained in each tissue of the embryo, fetus and adult.
  • pluripotent stem cells refer to stem cells having versatility capable of differentiating into all three germ layers of endoderm, mesoderm, and ectoderm.
  • stem cells In classifying stem cells, depending on the anatomical location, the function of the cell, the type of antigen expressed on the cell surface, the transcription factors, the proteins produced by the cell, and the stem cells They can be sorted according to the specific cell types they can produce.
  • the most commonly used and relatively clear classification of these various classifications depends on the individual from which the stem cells were isolated. When separated from the embryo (embryo) it can be divided into embryonic stem cells (ESry cells) and adult stem cells (adult stem cells) when separated from the adult.
  • embryonic stem cells embryonic stem cells
  • adult stem cells adult stem cells
  • ES cells embryonic stem Embryonic stem cells
  • adult stem cells adult stem cells
  • Embryonic stem cells formed from this inner cell mass are one individual. It can be said to be a pluripotent stem cell that has the potential to differentiate into the cells of all the tissues that make up. In other words, embryonic stem cells are undifferentiated cells capable of unlimited proliferation, can differentiate into all cells, and unlike adult stem cells, can make germ cells and can be inherited for the next generation.
  • Embryonic stem cells such pluripotent stem cells
  • Embryonic stem cells have serious religious and ethical problems of embryo destruction in the cell manufacturing process, and because of their lack of immunocompatibility between individuals due to limited embryos, transplant rejection reactions when developed as cell therapeutics can not avoid.
  • various methods have been attempted to artificially prepare cells with pluripotent cells such as embryonic stem cells or embryonic stem cells using adult-derived cells.
  • Representative methods include somatic cell nuclear transfer (SCNT), cell fusion with ES cell, and reprogramming by defined factor.
  • SCNT somatic cell nuclear transfer
  • the somatic cell nuclear transfer method has a big limitation in that its efficiency is very low and a large number of eggs are needed which can contain ethical problems.
  • the cell fusion method has a big problem in terms of cell stability because the induced cells have two more pairs of genes.
  • the most recently introduced technology, the specific factor injection method uses a virus containing a carcinogen and has a serious problem of cancer cell formation.
  • the present inventors have obtained dedifferentiated stem cells as extracts of induced pluripotent stem cells (iPS) derived from animals, but there are some limitations.
  • iPS induced pluripotent stem cells
  • One aspect of the present invention is to solve the problems of the prior art as described above, to propose a method for producing a pluripotent stem cells with no ethical problems and secured stability, safety.
  • the present invention proposes a method of inducing human-derived dedifferentiated stem cells that were difficult to realize in the prior art.
  • the present inventors have developed a method that can induce pluripotent stem cells having the same genetic background as the adult from which the cells are separated using adult-derived cells, and thus from adult-derived cells having various genetic backgrounds. By deriving the same result, it is suggested that the method of the present invention is a suitable invention capable of producing pluripotent stem cells.
  • an aspect of the present invention provides a method for inducing stem cells by treatment of adult extracts, plant extracts, plant stem cell extracts or compositions containing them, such as shikimic acid, shikimic acid do.
  • one aspect of the present invention comprises the steps of extracting an extract comprising the active ingredient in plant stem cells or all kinds of induced pluripotent plant stem cells induced by various methods; Injecting the extract into adult-derived cells; And it provides a method for producing a custom pluripotent stem cells comprising the step of producing a pluripotent cells, such as embryonic stem cells through the culture of the cells injected with the extract.
  • an aspect of the present invention comprises the steps of injecting Shikimic acid, plant extracts containing Shikimic acid, plant stem cell extract or composition comprising them into adult-derived cells; And it provides a method for producing a stem cell further comprising the step of culturing cells injected with shikimic acid, extract or composition.
  • the extract may be a callus extract.
  • the method may further comprise the step of treating the cell membrane permeation promoting compound for the adult cells before the injection of the extract.
  • the cell membrane permeation promoting compound may include streptolysin O and digitonin, but is not limited thereto, as long as it facilitates the injection of the shikimic acid or extract according to the present invention into the cell membrane.
  • the method may further comprise the step of culturing the adult-derived cells injected with the extract to the support cell layer to further culture.
  • the support cell may include, but is not limited to, STO cells.
  • an aspect of the present invention comprises the steps of extracting an extract containing the active ingredient in plant stem cells, callus or all kinds of induced pluripotent plant stem cells induced by various methods; Injecting the extract into adult-derived cells; The extract-injected cells are cultured in a general cell culture solution and transferred to a culture condition with a feeder cell layer, and further comprising culturing in embryonic stem cell culture, to provide a customized pluripotent stem cell production method.
  • one aspect of the present invention provides a stem cell produced by the method.
  • one aspect of the present invention provides a composition comprising a stem cell.
  • the present invention by using Shikimic acid, plant extracts or plant stem cell extracts containing the same, reprogramming and / or dedifferentiating the differentiated cells of the adult inducible Pluripotent stem cell (inducible Pluripotent stem cell) : iPSC), a method for inducing a pluripotent stem cell prepared by the method and a cell therapy comprising the pluripotent stem cells.
  • the plant extract or plant stem cell extract containing shikimic acid used in the present invention is Sequoiadendron giganteum , yellow iris ( Iris pseudoacorus ), fat pork (pork potato) ( Helianthus tuberosus ), silver blue spruce (blue spruce) ) ( Picea pitchs ), Glauka spruce (white spruce) ( Picea glauca ), Eucalyptus ( Eucalyptus sieberiana ), Eucalyptus regnans , Western cypress ( Thuja plicata ), Date palm (Red Ceda, Phoenix dactylifera ), dahlia Dahlia variabilis , luminous tree ( Malus baccata ), Pear ( Pyrus communis ), wheat ( Triticum ), pine ( Pinus densifloraa ), gom ( Pinus thunbergii ), Isolum ( Ilicium anisatum ), Magnolia
  • the present invention also relates to a composition for stem cell activation, skin regeneration, or anti-aging or a pharmaceutical or cosmetic composition comprising the stem cells prepared by the above method.
  • the best method of producing stem cells, plant stem cells or plants including extracts of all kinds of induced Pluripotent Plant Stem cells derived from various methods, shikimic acid, shikimic acid Extracts or compositions comprising them can be used, and this method will also be a wide variety of fabrication methods that can be applied to all cells of all species, including humans of various genetic backgrounds.
  • the method of the present invention by using a plant-derived stem cell extract, it is possible to manufacture a custom pluripotent stem cells with no ethical problems and secured safety.
  • FIG. 1 is a schematic diagram showing the entire experimental process of inducing pluripotent stem cells according to the method of the present invention.
  • Figure 2 is a diagram showing induced pluripotent stem cells observed on day 5 after treatment with plant stem cell extract.
  • FIG. 3 is a diagram showing induced pluripotent stem cells observed on day 8 of treatment with plant stem cell extracts and on day 2 of transfer to feeder cells.
  • FIG. 4A is a diagram showing induced pluripotent stem cells observed after 4 passages after transfer to feeder cells at 32 days of treatment with plant stem cell extracts. 4B shows a typical embryonic stem cell.
  • FIG. 5 is a diagram showing the alkaline phosphatase staining results of induced pluripotent stem cells observed after four passages after transfer to feeder cells after treatment with plant stem cell extracts.
  • FIG. 6A is a diagram showing induced pluripotent stem cells observed after 7 passages after transfer to feeder cells at 50 days of treatment with plant stem cell extracts and alkaline phosphatase staining of the cells. The figure shows the result.
  • FIG. 6B is a diagram showing the results of staining of human pluripotent stem cells and alkaline phosphatase induced by 4 factor virus of Yamanaka.
  • Figure 7 is a diagram showing the gene expression of pluripotent stem cells induced by the method of the present invention.
  • Shikimic acid represents Shikimic acid having a structure of the present formula [Formula 1].
  • Figure 9 is a schematic diagram showing the entire experimental process of inducing pluripotent stem cells using the plant stem cell extract containing Shikimic acid or the same according to the method of the present invention.
  • FIG. 10 is a diagram showing the expression of Oct3 / 4 gene in HDF after treatment with shikimic acid or sequoia callus extract.
  • Figure 11 shows the expression of Oct3 / 4 gene in HDF after treatment with sikimic acid at different concentrations.
  • FIG. 12 is a diagram showing the increase in the expression of ALP after treatment with shikimic acid or sequoia callus extract.
  • Figure 13 is a diagram showing the increase in the expression of ALP after treatment with sikimic acid by concentration.
  • FIG. 14 is a diagram showing the colony generating ability of the dermal cells after treatment with shikimic acid or sequoia callus extract.
  • 15 is a graph showing colony production of dermal cells after treatment with shikimic acid or sequoia callus extract.
  • 16 is a diagram showing the colony generating ability of the dermal cells after treatment with shikimic acid by concentration.
  • Figure 17 is a graph showing the colony generating capacity of dermal cells after treatment with shikimic acid by concentration.
  • 18 is a graph showing the increase in the proliferative capacity of dermal cells after treatment with shikimic acid or sequoia callus extract.
  • Korean Patent Application No. 10-2013-0123860 filed October 17, 2013, is incorporated herein by reference in its entirety for all purposes. This application also claims the benefit of Korean Patent Application No. 10-2013-0123860, which is hereby incorporated by reference in its entirety.
  • c) providing a method of inducing a customized pluripotent stem cell comprising the step of culturing the cells injected with the extract, and producing a pluripotent cell such as an embryonic stem cell.
  • one aspect of the present invention is Shikimic acid; Plant extracts comprising shikimic acid; Plant stem cell extract comprising shikimic acid; Or it provides a method for producing a stem cell comprising treating the composition containing them to adult derived cells.
  • an aspect of the present invention comprises the steps of injecting Shikimic acid, plant extracts containing Shikimic acid, plant stem cell extracts containing Shikimic acid or a composition containing them to adult-derived cells; And it provides a method for producing a stem cell further comprising the step of culturing cells injected with shikimic acid, extract or composition.
  • the extract herein may be a callus extract.
  • the plant extract or plant stem cell extract may comprise shikimic acid, caffeic acid or ferulic acid.
  • “Simkimic acid” as used herein may have the following general formula (1), and means the broadest concept including precursors, derivatives, and the like thereof.
  • the molecular weight of the shikimic acid may also be 174.15 g / mol.
  • caffeic acid may have the following Chemical Formula 2, and means the broadest concept including a precursor derivative thereof, and the like, and may have a molecular weight of 180.16 g / mol. Caffeic acid also means phenolic compounds contained in fruits such as pears, including coffee beans, and medicinal plants such as basil, thyme, bananas, tarragon, oregano, and dandelion.
  • ferulic acid may have the following Chemical Formula 3, and means the broadest concept including a precursor derivative thereof, and the like, and may have a molecular weight of 191.18 g / mol. Ferulic acid is also a precursor to lignin, which forms the cell walls of plants, and is abundantly contained in plant cell walls and is found in plant seeds such as wheat, oats, coffee, apples, oranges, and peanuts.
  • 'stem cells' refers to master cells that can be regenerated without limitation to form specialized cells of tissues and organs.
  • Stem cells are developable pluripotent or pluripotent cells. Stem cells can divide into two daughter cells, or one daughter cell and one derived ('transit') cell, and then proliferate into mature, fully formed cells of the tissue.
  • the term 'embryonic stem cell' refers to a cell having pluripotency as a cell cultured after separation from an inner cell mass (blast cell) of blastocyt, which is an early development after fertilization. Refers to.
  • iPS induced pluripotent stem cells or induced pluripotent stem cells (iPS) extract' refers to pluripotent stem cells or induced pluripotent stem cells induced or cultured in various ways in vitro
  • Induced Pluripotent Stem cells (iPS) refers to a material that is separated by a method such as centrifugation after crushing finely by a physical-chemical method.
  • plant stem cell is a cambium-derived plant stem cell.
  • it includes pure plant stem cells (CMC Cambial Meristematic Cell) in the physically intact state in the formation layer sandwiched between the water and phloem of the plant.
  • plant stem cells is the broadest concept as including all kinds of induced pluripotent plant stem cells derived by various methods.
  • callus (callus, or callus, wound tissue, or oily tissue) is an undifferentiated irregular cell mass that is representative of tumor tissue formed by meristems formed around the wound when the plant is injured. to be. Plants are composed of meristems that divide into cells and permanent tissues that do not. Cells are formed when the cells of the meristem are first placed in the nutrient medium and grown. Thereafter, an embryonic embryo is formed and differentiated into plants. This callus is also commonly called “plant stem cells.” There is no restriction
  • pluripotent stem cell' is a multifunctional (pluripotent) that can be differentiated into all three germ layers constituting the living body (endoderm), mesoderm (mesoderm), ectoderm (ectoderm) Refers to stem cells). Embryonic stem cells are traditionally called stem cells in this category.
  • custom pluripotent stem cells' used in the present invention refers to the genetically identical donor cells and induced pluripotent stem cells used to make pluripotent stem cells, which are derived from a custom pluripotent stem cell originated from a donor cell. Indicate that it is a cell
  • 'custom pluripotent stem cells', 'dedifferentiated stem cells', 'induced pluripotent stem cells' may be used interchangeably.
  • the term 'adult-derived cell' refers to a cell derived from an adult that is born and alive as opposed to an embryonic cell.
  • the term "differentiation” refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. It means to change.
  • a relatively simple system is divided into two or more qualitatively different sub systems. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons.
  • Phosphorus difference or as a result, is a state divided into subclasses or subclasses that can be distinguished qualitatively.
  • the plant may be a sequoia.
  • the plant stem cells may be callus (callus).
  • the plant stem cells may be sequoia stem cells.
  • the plant stem cell extract may be a callus extract.
  • the extract may be Sequoia callus extract.
  • the sequoia may be a sequoiadendron giganteum .
  • the extract or composition may comprise shikimic acid.
  • the composition may comprise a concentration of 10-MuM to 30 mM simkimic acid based on the total volume of the composition.
  • the composition is based on the total volume of the composition of the shikimic acid, plant extract or plant stem cell extract 10uM or more, 20uM or more, 30uM or more, 50uM or more, 100uM or more, 1mM or more, 5mM or more It may include 10mM or more, 20mM, or 30mM or more, and may include 1M or less or 100M or less.
  • the shikimic acid included in the composition may be included at a concentration of at least 0.1 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM or at least 0.9 mM based on the total volume of the composition or at most 5 mM, at most 4 mM, at most 3 mM, 2 mM Or less, 1.5 mM or less, 1.4 mM or less, 1.3 mM or less, 1.2 mM or less, or 1.1 mM or less.
  • Shikimic acid may be included in a concentration of 0.8mM to 1.2mM based on the total volume of the composition. Most preferably, the shikimic acid may be included at a concentration of 1 mM based on the total volume of the composition.
  • the plant extract or plant stem cell extract may comprise from 0.0001% (w / v) to 45% (w / v) of the sikimic acid based on the total volume of the extract.
  • shikimic acid When included in this range of shikimic acid may have an excellent Oct3 / 4 gene expression effect, ALP expression effect, fibroblast proliferation effect.
  • the plant extract or plant stem cell extract herein is based on the total volume of the extract Shikimic acid, 0.001% (w / v) or more, 0.01% (w / v) or more, 0.1% ( w / v) or more, 1% (w / v) or more, 5% (w / v) or more, 10% (w / v) or more, 15% (w / v) or more, 20% (w / v) or more , 25% (w / v) or more, or 30% (w / v) or more, 45% (w / v) or less, 40% (w / v) or less, 38% (w / v) Up to 36% (w / v), up to 34% (w / v), up to 33% (w / v), or up to 32% (w / v).
  • the plant extract or plant stem cell extract herein may comprise from 32% (w / v) to 34% (w / v) of the shikimic acid based on the total volume of the extract, more preferably Preferably 32.75% (w / v).
  • the composition is a plant extract or plant stem cell extract based on the total volume of the composition in consideration of the concentration of the shikimic acid and the extract of the concentration of shikimic acid contained in the extracts described above And may be at a concentration of contained shikimic acid based on the total volume of the composition.
  • the plant extract or plant stem cell extract included in the composition based on the total volume of the composition is at least 0.001 ug / ml, at least 0.01 ug / ml, at least 0.1 ug / ml, at least 1 ug / ml, at least 10 ug / ml , Over 50ug / ml, over 100ug / ml, over 0.3mg / ml, over 0.4mg / ml, over 0.5mg / ml, over 0.6mg / ml, over 0.7mg / ml, over 1mg / ml, over 1.5mg / ml Or more, 2 mg / ml, 5 mg / ml or more, 10 mg / ml or more, 20 mg / ml or more, or 50 mg / ml or more and 1 g / ml or less, or 10 g / ml,
  • Measurement of shikimic acid included in the plant extract or plant stem cell extract herein is 250 * 4.60 mm, 5 micron as a Gemini 5u C18 110 A (5 ⁇ m, 4.6 X 250nm, Phenomenex) column using Waters 1525 ⁇ Binary HPLC Pump It may have a standard of, and may be measured by using the mobile solvent A as water (containing 0.1% TFA) and the solvent B as acetonitrile (containing 0.1% TFA).
  • “Sequoia” herein includes redwood ( Sequoia sempervirens ), Big Tree ( Sequoiadendron giganteum ) or Metasequoia glyptostroboides .
  • Redwood also known as the American Cedar, is a pine tree, the cypress family, and grows only on the northwest coast of California, the southwest of Oregon, and New Zealand. It is 2500 to 3000 years old and the largest tree in the world with a maximum height of 112m. 2.5 to 4.5 m in diameter, 50 to 100 m in height, and bark 20 to 30 cm thick. Leaves are similar to yeast, 1-3cm in length, with sharp tips and distinct veins. The leaf surface is dark green and the back side is white.
  • the Big Tree ( Sequoiadendron giganteum ) is the only species in the Sequoia dendron, growing at 1500 to 2500 meters above sea level in the western Sierra Nevada Mountains of California, USA, 3.5 to 6 meters in diameter, 60 to 90 meters in height, and around 10 meters in diameter. .
  • the leaves are similar to cedar and 1cm long, arranged in a spiral shape, but the leaves on the mature branches look like scales.
  • Metasequoia glyptostroboides is a tree of the cypressaceae family and is the only surviving species in the genus Metasequoia. In the country of origin, it grows up to 35m of toil and the bark is grayish brown and stripped vertically. The branches spread to the side, and the leaves are opposite each other in a line, and are 10 ⁇ 23mm long and 1.5 ⁇ 2mm wide. It has a sharp tip and leaves of brown and red color. Flowers bloom in February-March, male and female, and male flowers are gunshot inflorescences hanging from the ends of axillary branches. There are 20 stamens. Female flowers hang on the ends of branches and bloom in March. Fruits are coniferous, 18 ⁇ 25mm long, round, ripe and brown, with oval seeds with wings. It is a deciduous coniferous tree and its origin is Sichuan, Hubei, China, and is distributed in Korea and China.
  • Callus is an undifferentiated irregular cell mass, which is typical of tumor tissue formed by the meristem that forms around the wound when the plant is injured. Plants are composed of meristems that divide into cells and permanent tissues that do not. Cells are first formed by culturing cells in nutrient media and growing. Thereafter, an embryonic embryo is formed and differentiated into plants. Callus is also called 'plant stem cells'.
  • extract includes all materials obtained by extracting the components of natural products, regardless of the extraction method, extraction solvent, extracted components or the form of the extract, and extracts the substances obtained by extracting the components of natural products. It is a broad concept that includes all of the materials that can be obtained by processing or processing in a method.
  • Sequoia as used herein may be included in the form of an extract, or may be included as a ground powder of the herbal medicine, or dry ground powder of the herbal medicine, but is not limited thereto.
  • the sequoia used in the present specification is not limited to the method of obtaining, and may be grown and used or purchased and commercially available, and may mean all of them including the ground and the underground of the sequoia.
  • the ground portion may include sequoia fruit, leaves and stems, and the underground portion may include roots, but is not limited thereto.
  • plant extract includes all substances obtained by extracting a plant component, such as sequoia, regardless of the extraction method, the extraction solvent, the extracted component or the form of the extract, and the heat in the process of extracting the component. , Including a material obtained by an extraction method including a process of treating with an acid, a base, an enzyme, and the like, and extracting a plant component such as a sequoia It is a broad concept that includes all of the materials that can be obtained by treatment.
  • plant stem cell extract includes all substances obtained by culturing stem cells of plants such as sequoias and extracting the active ingredient, regardless of the extraction method, extraction solvent, extracted component or extract form. It is an active ingredient of a stem cell of a plant such as sequoia, which includes a substance obtained through an extraction method including a process of treating with heat, acid, base, enzyme, etc. in extracting the component. It is a broad concept that includes all materials that can be obtained by extracting the material obtained by extracting and then processing or processing by other methods.
  • the method of the present invention includes culturing plant stem cells or all kinds of induced pluripotent plant stem cells derived by various methods and preparing extracts therefrom, which can be carried out according to general methods known in the art. .
  • culturing plant stem cells or all kinds of induced pluripotent plant stem cells induced by various methods and treating them with proteolytic enzymes collecting suspended solids or reacting and filtering plant stem cells at 65 ° C. for 2 hours.
  • the protein or shikimic acid derived from each cell can be extracted.
  • callus powder may be used.
  • Sequoia callus extract in the present specification is i) inducing callus (callus) from Sequoia; ii) solid cell culture of the callus to establish a stem cell line; iii) producing a large amount of the active ingredient through suspension cell culture; And it can be obtained through the step of extracting the produced active ingredient.
  • a method of extracting an active ingredient from Sequoia callus may be extracted by a plant culture method of a cell line derived from a tissue explant of the plant, such as an extraction method known from Korean Patent Publication No. 2007-0113193. It can be extracted by adding a mixture of alcohols of 5-carbon atoms or less to a stable plant cell line derived from Sequoia, but is not limited to the above extraction method.
  • sequoia callus extract or sequoia callus powder may be used commercially available herein.
  • the sequoia callus extract may be prepared by dissolving sequoia callus powder in a solvent, wherein the solvent includes at least one selected from the group consisting of water, an organic solvent, and a mixture of water and an organic solvent.
  • the water may include distilled water or purified water
  • the organic solvent may be selected from the group consisting of alcohols, acetone, ether, ethyl acetate, diethyl ether, ethyl methyl ketone, and chloroform, including C 1 to C 5 lower alcohols.
  • BG butylene glycol
  • EtOH ethanol
  • DMSO dimethyl sulfoxide
  • the concentration of the shikimic acid or protein extract derived from the plant is preferably 10 ug / ml to 1 mg / ml based on the total volume of the composition including the extract, more preferably about 500 ug / ml. If the concentration of the protein extract is out of the above range because the induction efficiency of the customized pluripotent stem cells or the cells treated with the extract may be killed.
  • the method of the present invention comprises the step of injecting the protein extract isolated from plant stem cells or all kinds of induced pluripotent plant stem cells induced by various methods into adult derived cells.
  • the adult-derived cells may include human dermal fibroblasts or neonatal human dermal fibroblasts.
  • cell membrane permeation promoting compounds e.g., streptolysin O or digitonin
  • permeabilization reversible small holes
  • the method for producing pluripotent stem cells comprising
  • the cell pellet is resuspended in embryonic stem cell culture medium and seeded in a dish;
  • the present invention includes the steps of culturing cells injected with shikimic acid, extracts or compositions, to produce cells with pluripotency, such as embryonic stem cells.
  • the adult-derived cells injected with them are cultured using a general cell culture medium, and then transferred to a culture condition with a feeder cell layer to perform further culture. Can be.
  • DMEM normal cell cultures
  • FBS normal cell cultures
  • 10-100U / ml penicillin, 20-80mg / ml streptomycin are used until colonies are formed after the injection of the above-mentioned sikimic acid, extract or composition.
  • the colonies were formed, and then transferred to the culture conditions with the feeder cell layer and subcultured every 5-8 days in the embryonic stem cell culture medium, and the medium was exchanged daily.
  • Supporting cells of the present invention may include, but are not limited to, STO cells.
  • Pluripotent stem cells induced by the extract were 15-25% KSR (Knockout serum replacement), 1-4 mM L-glutamine, 0.05-0.2 mM non-essential amino acids, 0.05-0.2 in DMEM (Dulbecco's Modified Eagle Medium) / F12 Cultures can be incubated with addition of mM ⁇ -mercaptoethanol, 30-70 U / ml penicillin, 30-70 mg / ml streptomycin, and 1-30 ug / ml bFGF. It will be readily appreciated by those skilled in the art that the concentration of the compound added to the DMEM may vary within a range capable of achieving the effects of the present invention.
  • the present invention comprises the steps of a) extracting the protein from the plant stem cells or all kinds of induced pluripotent plant stem cells induced by various methods;
  • the method of the present invention is indistinguishable from embryonic stem cells from adult-derived cells using shikimic acid, plant extracts, plant stem cells or all kinds of induced pluripotent plant stem cell extracts derived from various methods or compositions containing them. It is characterized by the ability to make cells with such pluripotency.
  • the inventors confirmed that pluripotent stem cells are induced through the method of the present invention.
  • the pluripotent stem cells induced by the present invention in the shape of the cells are almost identical to the embryonic stem cells (see FIG. 4).
  • the characteristic gene Nanog, Oct4
  • one aspect of the present invention provides a pluripotent stem cell induced by the method which is an aspect of the present invention.
  • the present inventors have confirmed that the self-renewal, which is a characteristic of stem cells, by successive passages of 8-12 times through the above method (see FIG. 6).
  • one aspect of the present invention provides a composition comprising pluripotent stem cells prepared according to the method of an aspect of the present invention.
  • One aspect of the present invention relates to a composition comprising one or more of shikimic acid, a plant extract comprising shikimic acid, and a plant stem cell extract comprising shikimic acid as an active ingredient.
  • the composition may be a pharmaceutical composition, a food composition or a cosmetic composition.
  • the composition may be a composition for stem cell activation, skin regeneration or anti-aging.
  • a method of administering a composition comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more thereof to an individual in need of stem cell activation It may be related to a stem cell activation method comprising.
  • the administration may be in accordance with the method or dosage of administration described herein.
  • the present invention comprises administering to a subject in need of skin regeneration, shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising one or more thereof. It may be related to the skin regeneration method.
  • the present invention comprises administering to a subject in need of anti-aging, shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract comprising shikimic acid, or a composition comprising one or more thereof. It may be related to an anti-aging method.
  • the present invention provides a stem cell activation use, skin regeneration use, or anti-aging of shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract containing shikimic acid, or a composition comprising at least one of them. It may be related to the use.
  • the present invention includes a stem extract, a plant extract comprising a shikimic acid, a plant stem cell extract containing a shikimic acid, or one or more thereof for use in stem cell activation, skin regeneration, or anti-aging. It may be related to the composition.
  • the composition may be a cell therapeutic agent.
  • the cell therapeutic agent may be used for the formation of hepatocytes, adipocytes, bone cells, chondrocytes, muscle cells, neurons, cardiomyocytes, vascular endothelial cells and the like.
  • the present invention comprises administering shikimic acid, a plant extract comprising shikimic acid, a plant stem cell extract comprising shikimic acid, or a composition comprising one or more thereof to an individual in need of cell therapy. It may be related to a method of cell treatment.
  • the present invention may be directed to a cell therapeutic use of shikimic acid, a plant extract comprising shikimic acid, a plant stem cell extract comprising shikimic acid, or a composition comprising one or more thereof.
  • the present invention may be directed to shikimic acid, a plant extract comprising shikimic acid, a plant stem cell extract comprising shikimic acid, or a composition comprising one or more thereof for use in cell therapy.
  • the term 'cell therapeutic agent' refers to a medicinal product (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention of cells and tissues prepared by isolation, culture, and special manipulation from humans.
  • a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as proliferating, selecting, or otherwise altering the biological properties of a living autologous, allogeneic, or heterologous cell in order to restore it.
  • Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.
  • One aspect of the present invention provides a food composition comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract or pluripotent stem cells according to the present specification.
  • the composition may contain other components and the like that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention.
  • additives such as perfumes, pigments, fungicides, antioxidants, preservatives, humectants, thickeners, inorganic salts, emulsifiers and synthetic polymer materials may be further included to improve physical properties.
  • the food composition includes, but is not limited to, health food compositions, functional food compositions, supplements, processed food compositions, food additives and the like.
  • One aspect of the present invention provides a cosmetic composition
  • a cosmetic composition comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract or pluripotent stem cells according to the present specification.
  • the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base.
  • compositions obtained by dispersing the oil phase in solutions, gels, solids, pasty anhydrous products, aqueous phases, emulsions obtained by dispersing the aqueous phase in oil phases, multi emulsions, suspensions, micro emulsions, micro Capsules, microgranulocytes, ionic (liposomal) and nonionic vesicle dispersants, foams, aerosols or patches containing further compressed propellant may be used.
  • These compositions can be prepared according to conventional methods in the art.
  • the cosmetic composition may contain other ingredients in addition to the above substances in a range that does not impair the main effect, and preferably may have a synergistic effect on the main effect, and other cosmetics in addition to the active ingredient of the present invention Depending on the formulation of the composition or the purpose of use, those skilled in the art can appropriately select and blend without difficulty.
  • the cosmetic composition of the present invention may include other ingredients which are usually formulated into the cosmetic composition as necessary in addition to the active ingredient, and examples thereof include oil-fat ingredients, humectants, emollients, surfactants, organic and inorganic pigments, organic Powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, stabilizers, thickeners, glycerin, pH regulators, alcohols, pigments, flavorings, blood circulation accelerators, cooling agents, limiting agents, purified water and the like.
  • Other compounding components that may be included in the cosmetic composition is not limited thereto, and the compounding amount of the above components is possible within a range that does not impair the object and effect of the present invention.
  • the formulation of the cosmetic composition is not particularly limited and may be appropriately selected as desired.
  • supple lotion, nourishing lotion, essence, nourishing cream, massage cream, pack, gel, makeup base, foundation, powder, lipstick, patch, spray, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water One selected from the group consisting of cleanser, hair shampoo, hair conditioning, hair treatment, hair essence, hair lotion, scalp hair tonic, scalp essence, hair gel, hair spray, hair pack, body lotion, body cream, body oil and body essence It may be prepared in the above formulation, but is not limited thereto.
  • One aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising shikimic acid, a plant extract containing shikimic acid, a plant stem cell extract or pluripotent stem cells according to the present specification.
  • Such pharmaceutical compositions may contain, in addition to the active ingredient, preservatives, stabilizers, hydrates, emulsifiers, salts and / or buffers for osmotic pressure control, diluents (e.g.
  • lactose dextrose, sucrose, mannitol, sorbitol, cellulose or glycine
  • Glidants such as silica, talc, stearic acid and magnesium or calcium salts or polyethylene glycols thereof or binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose or polyvinylpi
  • binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose or polyvinylpi
  • Pharmaceutical adjuvant or other therapeutically useful substances such as rolidine).
  • other pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, or sweeteners such as starch, agar, alginic acid or sodium salts thereof may be included.
  • compositions can be formulated in various oral or parenteral dosage forms according to conventional methods.
  • the oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, and the like, and these formulations include surfactants in addition to the active ingredients.
  • Diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine
  • glidants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols.
  • Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt
  • Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners.
  • the tablets can be prepared by conventional mixing, granulating or coating methods.
  • parenteral administration agent may be, for example, formulations such as injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, and patches, but are not limited thereto. no.
  • the pharmaceutical composition according to an aspect of the present invention may be administered parenterally, rectally, topically, transdermally, subcutaneously, and the like.
  • the composition is based on the formulation and criteria suitable for each administration. In particular, in the case of intravenous injection, all additives not suitable for this are excluded, and the purity of the composition itself is also very high.
  • the applied amount of the active ingredient will vary depending on the age, sex, weight, disease, pathological condition, route of administration and prescriber's judgment of the subject to be treated. Application amount determination based on these factors is within the level of skill in the art. Typical dosages are preferably 30 ug / ml to 1 mg / ml, but are not limited thereto.
  • pluripotent stem cells prepared by the method according to the present invention it is preferable to be used in the formulation of the injection in the pharmaceutical composition.
  • Pluripotent stem cells prepared by the method according to the present invention are injected into the skin similarly to botox that is commonly used for the purpose of removing wrinkles by injection into the skin to activate skin stem cells, and to promote the proliferation of skin cells, etc. Through the effects of skin regeneration, anti-aging, improve elasticity, it can exhibit such effects as wrinkles.
  • One aspect of the present invention also provides a composition including, but not limited to, shikimic acid, a plant extract comprising shikimic acid, a plant stem cell extract, or an experimental reagent or medium composition comprising pluripotent stem cells.
  • callus powder as a plant-derived stem cell extract.
  • Callus powder that can be used in the present invention is not limited and may be purchased commercially.
  • Sequoia callus extract induces callus from the leaves of Sequoia and solid cultures to establish stem cell lines and suspension cell culture to produce active ingredients in large quantities. After production it is extracted and obtained. Extracting the active ingredient from the sequoia callus can be extracted by the plant culture method of cell lines derived from the tissue explant of the plant, such as the extraction method known in Korea Patent Publication No. 2007-0113193, although it can be eluted by adding a mixture of alcohols of 5-carbon atoms or less to a stable plant cell line derived from Sequoia, it is not limited to the above extraction method.
  • sequoia callus power (commercially available from BiofdNC) was used. Specifically, human dermal fibroblasts (human dermal fibroblasts) were made into individual cells using trypsin-EDTA, followed by washing with cold PBS (phosphate buffered saline). The cell pellet was resuspended to 100 ⁇ l / 100,000 cells of cold Ca2- and Mg2-free Han's balanced salt solution (HBSS) and transferred to a 1.5 ml tube.
  • HBSS Han's balanced salt solution
  • digitonin (20ug / ml) and transport solution 110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate, 1 mM EGTA instead of SLO
  • 200ul of 2 mM DTT, proteaseinhibitor cocktail, 20 mM HEPES pH7.3 200ul of 2 mM DTT, proteaseinhibitor cocktail, 20 mM HEPES pH7.3
  • the sample was reacted for 50 minutes in a 37 °C water bath, mixed up and down once every 10 minutes.
  • the reaction sample is placed on ice and 200 ⁇ l of cold HBSS is added, followed by centrifugation at 120 g for 5 minutes at a swing-out rotor at 4 ° C.
  • the cell pellet was resuspended with 200 ⁇ l of plant stem cell extract to 1 ⁇ l / 1000 cells.
  • Callus powder was used as a plant stem cell extract (500 ug / ml).
  • ATP-regeneration system (10 mM creatine phosphate, and 25 g / ml creatine kinase), 1 mM nucleotide triphosphate, respectively, was added and reacted for 1 hour in a 37 ° C. water bath, mixed up and down once every 10 minutes.
  • 1 ml of ES cell medium containing 2 mM CaCl 2 was added to reseal the plasma membrane and reacted in a 37 ° C. incubator for 2 hours.
  • the cell pellet was resuspended in embryonic stem cell culture medium and seeded in a dish coated with 0.1% gelatin.
  • Example 2 A step of producing cells with pluripotency, such as embryonic stem cells, by culturing the cells injected with the extract
  • DMEM Dulbecco's Modified Eagle Medium
  • FIG 1 is a schematic diagram showing the entire experimental process of inducing pluripotent stem cells according to the method of the present invention.
  • 2 to 4 show that pluripotent stem cells were induced at 5, 10 and 32 days of culture, respectively, and FIGS. 5 and 6 show alkaline phosphatase at 32 and 50 days of culture, respectively.
  • the pluripotent stem cells induced according to the method of the present invention show alkaline phosphatase staining positive (purple) characteristic of embryonic stem cells.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • pluripotent stem cells hiPS
  • hES embryonic stem cells
  • Sequoia callus extract dissolved in a solvent mixed with BG and EtOH in the sequoia extract obtained according to the method of Example 4 was analyzed by using an HPLC machine.
  • LC spectrum of the sequoia extract is as shown in Figure 8 and the results are shown in Table 1 below.
  • % Area represents% (w / v) of the substance contained in the sequoia extract.
  • Neonatal human dermal fibroblasts (NHDF-Neonatal) (CC-2509, Lonza, USA) were treated with 10 ⁇ M and 10 mM of shikimic acid, respectively. NHDF-Neonatal without any treatment was set as negative control.
  • Shikimic acid the main component of Sequoia callus extract
  • the shikimic acid or the extract including the same according to the present invention is injected into the somatic cells, the Oct3 / 4 gene can be expressed to induce customized pluripotent stem cells.
  • Test Example 3-1 Test for Confirming Increased Expression of Alkaline Phosphatase (ALP), a Stem Cell Activity Marker
  • ALP Alkaline Phosphatase
  • the HDF treated with Sequoia callus extract has a lot of stained portion compared to normal (HDF), indicating that the expression of ALP is significantly increased.
  • the treatment of sequoia callus extract can confirm that the ALP is expressed more than the treatment with shikimic acid.
  • ALP is expressed in cells three days after the expression of genes such as Oct4, a marker known to play an important role in the early stage of dedifferentiated stem cell formation ( marker). Therefore, when the sequoia callus extract according to the present invention is injected into the somatic cells, the expression of ALP is significantly increased, and since the Oct4 gene is expressed, the customized pluripotent stem cells can be induced.
  • Sequoia callus extract according to the present invention has a remarkable effect of activating stem cells in the light of promoting the expression of the stem cell activity marker ALP.
  • Test Example 3-2 Experiment of Confirming Increased Expression of Alkaline Phosphatase (ALP), a Stem Cell Activity Marker
  • NHDF- neo four exit (Neonatal) (CC-2509, Lonza, USA) acid 10uM, 10mM Sikkim gae to 1x10 6 were each treated.
  • NHDF-Neonatal without any treatment was set as negative control.
  • 100,000 cells were attached to 6-well plates, and on day 12, 7 days later, fixed with 3.8% formaldehyde in PBS for 15 minutes at room temperature, followed by diluting 200ul of NBT / BCIP® ALP substrate solution in 10 ml of ALP buffer.
  • 0.5ml of the solution was treated 20 hours later and observed 40 times with an Olympus CKX41 optical microscope. The results obtained through observation are shown in FIG. 13.
  • ALP is a marker known to play an important role in the early stage of dedifferentiated stem cell formation as it begins to be expressed three days after the gene such as Oct4. . Therefore, when the shikimic acid and the plant extract containing the same according to the present invention are injected into the somatic cells, the expression of ALP is significantly increased, and also the Oct4 gene is expressed, thereby inducing customized pluripotent stem cells.
  • the Shikimic acid and the plant extract containing the same according to the present invention shows a remarkable effect of activating stem cells in the light of promoting the expression of the stem cell activity marker ALP.
  • NHDF- neo leaving four was filtered with an embodiment (CC-2509, Lonza, USA ) 5x10 5 gae buffer Sami Billy hybridization buffer (Permeablization buffer) and digi tonin (digitonin) process and 0.4um filter to 10ug / ml in The sequoia callus extract according to 4 (BG and EtOH mixed solvent) 100ppm, 200ppm and 20ppm DMSO solution was treated. NHDF-Neonatal without any treatment was set as negative control. On day 10 of the passage, 200 cells were adhered to a 60 mm plate, and on day 23, ice-cold methanol was washed on ice with ice-cold PBS, and stored at -20 ° C.
  • sequoia callus extract according to the present invention can be confirmed that significantly promote the proliferation of fibroblasts.
  • the sequoia extract according to the present invention has an effect of promoting skin regeneration.
  • 200 cells were attached to a 60 mm plate, and on day 17, ice-cold methanol was washed on ice with ice-cold PBS and stored at -20 degrees. The cells were fixed for 10 minutes. 1% crystal violet in Ethanol stock solution was diluted 1/10 in PBS to make an experimental solution. After treatment for 5 to 10 minutes, the cells were dyed and washed 4 times with PBS.
  • shikimic acid according to the present invention significantly promotes the proliferation of fibroblasts.
  • sekimic acid and sequoia extract containing the same according to the present invention have an effect of promoting skin regeneration.
  • sequoia callus extract according to the present invention can be confirmed that significantly promote the division of fibroblasts.
  • the sequoia extract according to the present invention in view of significantly promoting fibroblast division of dermal cells, the sequoia extract according to the present invention can be seen that exhibits the effect of promoting skin regeneration.
  • Sikkim acid or sequoia callus extract according to Example 4 40ug, vitamin E 9mg, vitamin C 9mg, palm oil 2mg, vegetable hardened oil 8mg, beeswax 4mg and lecithin 9mg are mixed and mixed according to a conventional method to prepare a soft capsule filler Manufacture. 400 mg per capsule is filled to prepare a soft capsule.
  • a soft capsule sheet is prepared at a ratio of 66 parts by weight of gelatin, 24 parts by weight of glycerine, and 10 parts by weight of sorbitol solution and filled with the filler to prepare a soft capsule containing 400 mg of the composition according to the present invention.
  • Sikkim acid or sequoia callus extract according to Example 4 40 ⁇ g, vitamin E 9mg, vitamin C 9mg, galactooligosaccharide 200mg, lactose 60mg and maltose 140mg were mixed and granulated using a fluidized bed dryer and then sugar ester (sugar ester) 6 mg is added. Tablets are prepared by tableting 500 mg of these compositions in a conventional manner.
  • Sikkim acid or 40 ug of sequoia callus extract according to Example 4 vitamin E 9mg, vitamin C 9mg, anhydrous glucose of 250mg and starch 550mg were mixed, molded into granules using a fluidized bed granulator, and then packed into a fabric To prepare.
  • Injectables were prepared by conventional methods according to the compositions set forth in Table 4 below.
  • Table 4 Compounding ingredient content Sikkim acid or sequoia callus extract according to example 4 40 ⁇ g Sterile Distilled Water for Injection Quantity pH regulator Quantity
  • Injections are prepared in the above amounts per ampoule (2 ml) according to the conventional method for preparing injections.
  • composition shown in Table 5 was prepared in the conventional method for the flexible cosmetic.
  • Table 5 Compounding ingredient Content (% by weight) Sikkim acid or sequoia callus extract according to example 4 0.2 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxy Vinyl Polymer 0.1 Fiji-12 nonylphenyl ether 0.2 Polysorbate 80 0.4 ethanol 10.0 Triethanolamine 0.1 Preservative, coloring, flavoring Quantity Purified water Remaining amount
  • Nutritional longevity was prepared according to the composition described in Table 6 below in a conventional manner.
  • Table 6 Compounding ingredient Content (% by weight) Sikkim acid or sequoia callus extract according to example 4 1.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxy Vinyl Polymer 0.1 Beeswax 4.0 Polysorbate 60 1.5 Caprylic / Capric Triglycerides 5.0 Squalane 5.0 Sorbitassquioleate 1.5 Liquid paraffin 0.5 Cetearyl Alcohol 1.0 Triethanolamine 0.2 Preservative, coloring, flavoring Quantity Purified water Remaining amount
  • Nutritional cream was prepared in a conventional manner according to the composition shown in Table 7.
  • Table 8 Compounding ingredient Content (% by weight) Sikkim acid or sequoia callus extract according to example 4 2.0 glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 45.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / Capric Triglycerides 3.0 Beeswax 4.0 Cetearyl Glucoside 1.5 Sesqui oleic acid sorbitan 0.9 Vaseline 3.0 paraffin 1.5 Preservative, coloring, flavoring Quantity Purified water Remaining amount
  • Table 9 Compounding ingredient Content (% by weight) Sikkim acid or sequoia callus extract according to example 4 0.2 glycerin 4.0 Polyvinyl alcohol 15.0 Hyaluronic acid extract 5.0 Beta Glucan 7.0 Allantoin 0.1 Nonyl Phenyl Ether 0.4 Polysorbate 60 1.2 Ethanol preservative 6.0 Preservative, coloring, flavoring Quantity Purified water Remaining amount
  • Table 10 Compounding ingredient content Sikkim acid or sequoia callus extract according to example 4 20 ⁇ g Vitamin A Acetate 70 ⁇ g Vitamin E 1.0mg Vitamin B1 0.13mg Vitamin B2 0.15mg Vitamin B6 0.5mg Vitamin B12 0.2 ⁇ g Vitamin c 10mg Biotin 10 ⁇ g Nicotinic acid amide 1.7mg Folic acid 50 ⁇ g Calcium Pantothenate 0.5mg Ferrous sulfate 1.75mg Zinc oxide 0.82 mg Magnesium carbonate 25.3 mg Potassium phosphate monobasic 15 mg Dicalcium Phosphate 55 mg Potassium citrate 90 mg Calcium carbonate 100mg Magnesium chloride 24.8 mg
  • composition ratio of the said vitamin and mineral mixture was mixed and consisted with the component suitable for a healthy food in a preferable Example, the compounding ratio may be arbitrarily modified.
  • Table 11 Compounding ingredient content Sikkim acid or sequoia callus extract according to example 4 20 ⁇ g Citric acid 1000 mg oligosaccharide 100 g Taurine 1 g Purified water Remaining amount
  • the above ingredients are mixed according to a conventional method for preparing a health beverage, then stirred and heated at 85 ° C. for about 1 hour, and then the resulting solution is filtered and sterilized.

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EP3808411A1 (en) * 2019-10-16 2021-04-21 Mesoestetic Pharma Group, S.L. Cosmetic compositions and use thereof
KR102299417B1 (ko) * 2019-12-12 2021-09-07 (주)아모레퍼시픽 노화 피부세포 주변 환경변화 유도 촉진용 조성물
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KR102783281B1 (ko) * 2021-09-01 2025-03-21 주식회사 뉴셀팜 푸른여로 추출물을 이용한 피부 주름 개선 및 피부 미백용 조성물
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