Perinatal appendage extract and method for producing perinatal appendage extract

Abstract

To provide a perinatal appendage extract in which functional protein-based macromolecules, functional protein-based micromolecules, and bioactive substances contained in perinatal appendages are maintained without damage, and the effectiveness of functional protein-based macromolecules, functional protein-based micromolecules, and bioactive substances contained in perinatal appendages can be fully exerted.SOLUTION: A perinatal appendage extract is extracted from a perinatal appendage, which is at least one selected from the group consisting of umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, and decidua), and contains undamaged functional protein-based macromolecules and undamaged functional protein-based micromolecules, as well as undamaged bioactive substances (extracellular matrix proteins and cytokines).SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a perinatal appendage extract produced from perinatal appendages obtained from a mother at the time of childbirth, and to a method for producing a perinatal appendage extract from perinatal appendages obtained from a mother at the time of childbirth. [Background technology]

[0002] A method for producing a placenta extract is disclosed that has an absorbance of 0.15 or less at a wavelength of 390 nm and a nitrogen content of 0.10% by mass or more (see Patent Document 1). This method for producing a placenta extract includes an extraction step of performing subcritical treatment of mammalian (pig, cow, horse, sheep, wild boar) placenta at a treatment temperature of 160 to 200°C, a pressure equal to or greater than the saturated vapor pressure, and a treatment time of 5 to 30 minutes to obtain a subcritically treated product, a decolorization step of decolorizing the subcritically treated product obtained by the extraction step, and a solid-liquid separation step of separating the subcritically treated product decolorized by the decolorization step into an extract and a raw material residue. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-100998 A Summary of the Invention [Problem to be solved by the invention]

[0004] The method for producing a placenta extract disclosed in Patent Document 1 involves subcritical treatment of the placenta of mammals (pig, cow, horse, sheep, wild boar) at a treatment temperature of 160 to 200°C and at a pressure equal to or higher than the saturated vapor pressure. As a result, the active ingredients contained in the placenta are exposed to a subcritical atmosphere and decomposed or damaged, resulting in a decrease in the effectiveness of the active ingredients and a decrease in the amount of the active ingredients, making it impossible to utilize the active ingredients effectively.

[0005] An object of the present invention is to provide an extract of perinatal appendages in which functional proteinaceous macromolecules, functional proteinaceous low-molecules, and physiologically active substances contained in perinatal appendages are kept in a state with less damage, and the effectiveness of the functional proteinaceous macromolecules, functional proteinaceous low-molecules, and physiologically active substances contained in the perinatal appendages can be maximally utilized. Another object of the present invention is to effectively utilize perinatal appendages discarded after childbirth, keep functional proteinaceous macromolecules, functional proteinaceous low-molecules, and physiologically active substances contained in the perinatal appendages in a state with less damage, and provide a method for producing an extract of perinatal appendages that can maximally utilize the effectiveness of the functional proteinaceous macromolecules, functional proteinaceous low-molecules, and physiologically active substances.

Means for Solving the Problems

[0006] A first premise of the present invention for solving the above problems is an extract of perinatal appendages produced from perinatal appendages obtained from a mother at the time of childbirth.

[0007] The characteristics of the extract of perinatal appendages of the present invention in the first premise are that the extract of perinatal appendages is extracted from perinatal appendages and contains functional proteinaceous macromolecules with less damage and functional proteinaceous low-molecules with less damage, and also contains physiologically active substances with less damage.

[0008] As an example of the extract of perinatal appendages according to the present invention, the physiologically active substances contained in the extract of perinatal appendages are extracellular matrix proteins and cytokines.

[0009] As another example of the extract of perinatal appendages according to the present invention, the perinatal appendages are at least one of umbilical cord, umbilical cord blood, placenta, and fetal membranes (amnion, chorion, decidua).

[0010] As another example of the extract of perinatal appendages according to the present invention, the perinatal appendages are perinatal appendages obtained from a mother at the time of childbirth by cesarean section.

[0011] In another example of the perinatal appendage extract according to the present invention, the perinatal appendage extract is extracted from the perinatal appendage by enzymatically hydrolyzing the perinatal appendage with a specific connective tissue degrading enzyme.

[0012] In another embodiment of the perinatal appendage extract according to the present invention, the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase.

[0013] The second premise of the present invention for solving the above problem is a method for producing a perinatal adjunct extract, which produces a perinatal adjunct extract from perinatal adjuncts obtained from a mother's body at the time of birth.

[0014] The perinatal appendage extract manufacturing method of the present invention in the second premise is characterized in that it comprises a perinatal appendage obtaining step of obtaining perinatal appendages separated from the mother at birth when the mother gives birth to a newborn baby, an enzymatic hydrolysis extraction step of decomposing the perinatal appendages obtained in the perinatal appendage obtaining step with a connective tissue decomposing enzyme and extracting a perinatal appendage extract from the perinatal appendages, and an extract sterilization step of sterilizing the perinatal appendage extract extracted in the enzymatic hydrolysis extraction step.

[0015] In one example of the method for producing a perinatal accessory extract according to the present invention, the perinatal accessory extract contains functional proteinaceous polymers and functional proteinaceous small molecules that are less damaged, as well as physiologically active substances that are less damaged.

[0016] In another example of the method for producing a perinatal appendage extract according to the present invention, the physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines.

[0017] As another example of the perinatal appendage extract manufacturing method according to the present invention, the perinatal appendage extract manufacturing method includes, after the extract sterilization step, testing the content of functional proteinaceous polymers and functional proteinaceous small molecules in the perinatal appendage extract sterilized by the extract sterilization step, and testing the content of physiologically active substances in the perinatal appendage extract sterilized by the extract sterilization step.

[0018] In another embodiment of the method for producing a perinatal appendage extract according to the present invention, the extract sterilization step sterilizes the perinatal appendage extract using a filter.

[0019] In another example of the method for producing a perinatal appendage extract according to the present invention, the extract sterilization step sterilizes the perinatal appendage extract by irradiating the perinatal appendage extract with gamma rays.

[0020] In another embodiment of the method for producing a perinatal appendage extract according to the present invention, the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase.

[0021] In another example of the method for producing a perinatal accessory extract according to the present invention, the perinatal accessory is at least one of umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, decidua).

[0022] In another example of the method for producing a perinatal adjunct extract according to the present invention, the perinatal adjunct is a perinatal adjunct obtained from a mother at the time of birth by Caesarean section. Effect of the Invention

[0023] According to the perinatal appendage extract of the present invention, it is extracted from the perinatal appendage obtained from the mother at the time of birth, contains a large amount of functional proteinaceous polymers and functional proteinaceous small molecules with little damage, contains a large amount of physiologically active substances with little damage, and can make the most of the effectiveness of the disease-healing power, disease-preventing power, antiviral properties, antibacterial properties, etc., possessed by the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage. The perinatal appendage extract allows the use of the active ingredients of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage, and is highly effective in treating various diseases and in preventing various diseases. By using the perinatal appendage extract, various diseases can be treated, and by using the perinatal appendage extract, various diseases can be prevented.

[0024] The physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines, and it is possible to effectively utilize these physiologically active substances. By utilizing these physiologically active substances, it is possible to exert excellent effects in the treatment of various diseases as well as in the prevention of various diseases, and by using the perinatal appendage extract, it is possible to treat various diseases and by using the perinatal appendage extract, it is possible to prevent various diseases.

[0025] A perinatal appendage extract, in which the perinatal appendage is at least one of the umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, decidua), can make maximum use of the effectiveness of the disease-curing, disease-preventing, antiviral, antibacterial, etc., properties of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, decidua), and therefore exhibits excellent effects in treating various diseases due to the multiple active ingredients contained in the perinatal appendages, as well as in preventing various diseases. Various diseases can be treated by using the perinatal appendage extract, and various diseases can be prevented by using the perinatal appendage extract.

[0026] A perinatal appendage extract is a perinatal appendage obtained from the mother during birth by Caesarean section. During normal childbirth, the perinatal appendage may become contaminated with certain bacteria as it passes through the birth canal, but clean perinatal appendages are obtained by Caesarean section, and a clean perinatal appendage extract can be obtained using the clean perinatal appendages. The use of the clean perinatal appendage extract can reliably treat various diseases, and the use of the clean perinatal appendage extract can reliably prevent various diseases.

[0027] The perinatal appendage extract is extracted from the perinatal appendages by enzymatically hydrolyzing the perinatal appendages with a specific connective tissue degrading enzyme.The perinatal appendage extract uses a connective tissue degrading enzyme used in cell purification, and the perinatal appendages are enzymatically hydrolyzed with that connective tissue degrading enzyme, so that the enzymatic hydrolysis of the perinatal appendages proceeds slowly, preventing damage to the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages and preventing the death of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances. The perinatal appendage extract extracted using connective tissue degrading enzymes contains a large amount of functional proteinaceous polymers and functional proteinaceous small molecules that are little damaged, as well as a large amount of physiologically active substances that are little damaged. Therefore, the effectiveness of the disease-healing and disease-preventing abilities, antiviral properties, antibacterial properties, etc., of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages can be maximized, and various diseases can be treated by using the perinatal appendage extract, as well as preventing various diseases by using the perinatal appendage extract.

[0028] The perinatal appendage extract in which the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase, by using collagenase, liberase, and dispase as the connective tissue degrading enzyme, the enzymatic degradation of the perinatal appendage proceeds gently, and it is possible to reliably prevent damage to the functional proteinaceous macromolecules, functional proteinaceous low-molecular substances, and physiologically active substances contained in the perinatal appendage, and it is possible to reliably prevent the death of the functional proteinaceous macromolecules, functional proteinaceous low-molecular substances, and physiologically active substances. The perinatal appendage extract extracted using collagenase, liberase, and dispase contains a large amount of functional proteinaceous macromolecules with little damage and a large amount of functional proteinaceous low-molecular substances with little damage, and also contains a large amount of physiologically active substances with little damage. Therefore, the effectiveness such as the disease-curing power, disease-preventing power, antiviral property, and antibacterial property of the functional proteinaceous macromolecules, functional proteinaceous low-molecular substances, and physiologically active substances contained in the perinatal appendage can be maximally utilized, and various diseases can be treated by using the perinatal appendage extract, and various diseases can be prevented by using the perinatal appendage extract.

[0029] According to the method for producing a perinatal appendage extract according to the present invention, a perinatal appendage separated from the mother at the time of the mother's delivery of a newborn is obtained, the obtained perinatal appendage is decomposed by a connective tissue degrading enzyme to extract a perinatal appendage extract from the perinatal appendage, and the extracted perinatal appendage extract is sterilized to produce a perinatal appendage extract. Therefore, the produced perinatal appendage extract contains a large amount of active ingredients contained in the perinatal appendage, and it is possible to produce a perinatal appendage extract that can maximally utilize the effectiveness of the perinatal appendage. The method for producing a perinatal appendage extract exhibits excellent effects in the treatment of various diseases and excellent effects in the prevention of various diseases, and it is possible to produce a perinatal appendage extract that can treat various diseases and prevent various diseases. The method for producing a perinatal appendage extract produces a perinatal appendage extract from the perinatal appendage that is discarded after delivery, so that the perinatal appendage can be effectively utilized.

[0030] The method for producing a perinatal accessory extract containing less damaged functional proteinaceous polymers and less damaged functional proteinaceous small molecules as well as less damaged physiologically active substances can produce a perinatal accessory extract that contains a large amount of less damaged functional proteinaceous polymers and less damaged functional proteinaceous small molecules as well as a large amount of less damaged physiologically active substances in the produced perinatal accessory extract, and can make it possible to make the most of the disease-curing and disease-preventing abilities, antiviral and antibacterial properties, etc., of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal accessory. The method for producing a perinatal accessory extract can make a perinatal accessory extract that is highly effective in treating and preventing various diseases, and is capable of treating and preventing various diseases.

[0031] The method for producing a perinatal appendage extract in which the physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines can effectively utilize these physiologically active substances and produce a perinatal appendage extract that is highly effective in treating and preventing various diseases, and is capable of treating and preventing various diseases.

[0032] A perinatal appendage extract production method which tests the content of functional proteinaceous polymers and functional proteinaceous small molecules contained in a sterilized perinatal appendage extract, and tests the content of physiologically active substances contained in a sterilized perinatal appendage extract, tests the content of functional proteinaceous polymers and functional proteinaceous small molecules contained in the perinatal appendage extract and the content of physiologically active substances contained in the perinatal appendage extract, and thereby proves that the perinatal appendage extract contains a large number of functional proteinaceous polymers and functional proteinaceous small molecules, that the perinatal appendage extract contains a large number of physiologically active substances, and that the perinatal appendage extract produced by the perinatal appendage extract production method contains a large number of active ingredients possessed by physiologically active substances.

[0033] The method for producing a perinatal appendage extract using a filter sterilizes the perinatal appendage extract by passing the perinatal appendage extract through a filter, thereby making it possible to sterilize the perinatal appendage extract without damaging the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage extract, and thus making it possible to produce a clean perinatal appendage extract containing less damaged functional proteinaceous polymers, less damaged functional proteinaceous small molecules, and less damaged physiologically active substances.

[0034] The method for producing a perinatal appendage extract in which a perinatal appendage extract is sterilized by irradiating the perinatal appendage extract with gamma rays, can sterilize the perinatal appendage extract without damaging the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage extract, and can produce a clean perinatal appendage extract containing less damaged functional proteinaceous polymers, less damaged functional proteinaceous small molecules, and less damaged physiologically active substances.

[0035] In the method for producing a perinatal appendage extract, the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase, by using collagenase, liberase, or dispase as the connective tissue degrading enzyme, the enzymatic decomposition of the perinatal appendages proceeds slowly, thereby reliably preventing damage to the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages, and reliably preventing the death of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances, and a perinatal appendage extract can be produced that can maximize the effectiveness of the disease-healing and disease-preventing abilities, antiviral properties, antibacterial properties, etc., of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages.

[0036] The method for producing a perinatal accessory extract, in which the perinatal accessory is at least one of the umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, and decidua), can produce a perinatal accessory extract that can fully utilize the effectiveness of the disease-curing ability, disease-preventing ability, antiviral activity, antibacterial activity, and the like, of functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, and decidua). The method for producing a perinatal accessory extract can produce a perinatal accessory extract that is capable of treating and preventing various diseases, and that exhibits excellent effects in treating and preventing various diseases due to the various multiple active ingredients contained in the perinatal accessory.

[0037] In the method for producing a perinatal appendage extract, the perinatal appendages are perinatal appendages obtained from the mother at the time of birth by Caesarean section. During normal childbirth, the perinatal appendages may become contaminated with certain bacteria as they pass through the birth canal. However, clean perinatal appendages are obtained by Caesarean section, and a clean perinatal appendage extract can be produced using the clean perinatal appendages, making it possible to produce a clean perinatal appendage extract that can reliably treat and prevent various diseases. [Brief description of the drawings]

[0038] [Figure 1] Diagram illustrating perinatal appendages. [Diagram 2] FIG. 2 is a diagram showing an example of each step carried out in a method for producing a perinatal appendage extract. [Diagram 3] FIG. 2 is a diagram showing another example of each step performed in the method for producing a perinatal appendage extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The perinatal accessory substance extract and the method for producing the perinatal accessory substance extract according to the present invention will be described in detail below with reference to the accompanying drawings. Fig. 1 is a diagram illustrating the perinatal accessory substance.

[0040] The perinatal appendage extract according to the present invention is produced by extraction from perinatal appendages 12 obtained from a mother 11 (human mother) at the time of the birth of a newborn 10. As the perinatal appendages 12, umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c) shown in Fig. 1 are used.

[0041] In addition to using perinatal appendages 12 obtained from mother 11 by normal childbirth, perinatal appendages 12 obtained from mother 11 during birth by Caesarean section are also used. It is preferable to use perinatal appendages 12 obtained by Caesarean section. During normal childbirth, perinatal appendages 12 may become contaminated with certain bacteria as they pass through birth canal 16, but when perinatal appendages 12 are obtained by Caesarean section, the perinatal appendages 12 are clean, and a clean perinatal appendage extract can be obtained using the clean perinatal appendages 12.

[0042] The umbilical cord 13 is approximately 50 cm long and 2 cm thick, with two arteries and one vein running through it. The umbilical cord 13 is a pipe that transports substances between the placenta 14 and the fetus 11, and floats in the amniotic fluid. The two umbilical arteries of the umbilical cord 13 anastomose once immediately after entering the placenta 14, and then branch out in a tree-like shape within the placenta 14.

[0043] Umbilical cord blood is blood collected from the umbilical cord 13. Umbilical cord blood is rich in hematopoietic stem cells and mesenchymal stem cells. Umbilical cord blood is collected immediately after birth, after the newborn 11 and the umbilical cord 13 are separated. The placenta 14 comes out of the mother's body 11 shortly after the birth of the newborn 10, but in many cases, it is collected before the afterbirth. In one example of a specific collection method, the umbilical cord 13 is cut close to the newborn 10 after birth. The surface of the umbilical cord 13 separated from the newborn 10 is disinfected, a collection needle connected to a collection bag by a tube is inserted into the umbilical blood vessels, and the umbilical cord blood that flows out naturally due to gravity is collected in the collection bag. The umbilical cord 13 is cut as close to the newborn 11 as possible, and finally, the tube is squeezed to collect all of it.

[0044] The placenta 14 is a disk-shaped tissue with a diameter of about 20 cm and a certain thickness, weighs about 400 to 500 g, and is attached to the uterine wall. The disk-shaped shape is formed from the hairy part of the chorion and the decidua basalis. The placenta 14 is connected to the umbilical cord 13 of the fetus 10, and connects the fetus 10 and the mother 11 to exchange various nutrients and oxygen. The placenta 14 comes out of the mother 11 after the birth of the newborn 10. The intervillous space (space) formed between the hairy part of the chorion and the decidua basalis of the placenta 14 is filled with the blood of the mother, which causes the placenta 14 to become thick. In the intervillous space (space), a protrusion called a villous shaft extends from the chorion toward the decidua basalis. Capillaries extending from the fetus 10 run inside the villous shaft, and absorb nutrients from the mother 11 through the villous epithelium.

[0045] The amniotic membrane 15 (amniotic membrane 15a, chorion 15b, decidua 15c) is a membrane that contains the fetus 10 and amniotic fluid, and forms a boundary with the inside of the uterus, covering the fetus 10 side of the placenta 14. It has a three-layer structure consisting of the amniotic membrane 15a, chorion 15b, and decidua 15c, and protects the fetus 10 from viruses, bacteria, etc. The amniotic membrane 15a that constitutes the amniotic membrane 15 is the innermost of the amniotic membranes, is filled with amniotic fluid, and protects the fetus 10 from external shocks and from viruses, bacteria, etc. that invade from the outside. The components that constitute the amniotic membrane 15a are proteins such as collagen and laminin, and it is a thin, translucent membrane about 1 mm thick that is elastic.

[0046] The chorion 15b constituting the amniotic membrane 15 is tissue derived from the fetus 10, and envelops the amniotic membrane 15a from the outside. In the early stages of pregnancy, the chorion 15b is entirely covered with villi, but as pregnancy progresses, the rest degenerates, leaving only a portion of the villi that will form the placenta 14. The degenerated portion of the villi is the chorionic non-hairy portion, which becomes an element that constitutes the amniotic membrane 15. On the other hand, the portion with the remaining villi is the chorionic hairy portion, which comes into contact with the decidua on the mother's side 11 and becomes part of the placenta 14.

[0047] The decidua 15c that constitutes the amniotic membrane 15 is separated from the uterus at the time of birth. It was originally the endometrium, which thickened and proliferated due to the implantation of the fertilized egg, and changed into the decidua. The decidua 15c is classified into the decidua basalis, which is the basal part where the fertilized egg implants, the decidua capsularis, which is formed when the fertilized egg penetrates the endometrium and covers the surface again, and the decidua parietalis, which covers the inside of the uterine cavity.

[0048] The perinatal appendage extract is extracted from the perinatal appendage 12 by enzymatically decomposing the perinatal appendage 12 with a connective tissue decomposing enzyme. At least one of collagenase, liberase, and dispase is used as the connective tissue decomposing enzyme. Collagenase is an enzyme that decomposes collagen, a type of protein. Collagenase is classified into animal collagenase and bacterial collagenase. Animal collagenase is one of a group of enzymes called matrix metalloproteinase. Bacterial collagenase is a crude collagenase secreted from Clostridium bacteria, an anaerobic bacterium that does not require oxygen. Since highly active collagenase may cause excessive cell death, it is necessary to reduce the amount of collagenase or add BSA or serum to stabilize the cells.

[0049] Liberase is made by blending highly purified collagenase with neutral proteases, dispase or thermolysin, Liberase can efficiently dissociate various tissues and cells, and has low endotoxin content, which improves the viability of the isolated cells.

[0050] Dispase is a neutral amino endoprotease that cleaves the N-terminal peptide bonds of non-polar amino acid residues. Dispase is a neutral amino endoprotease that cleaves the N-terminal peptide bonds of non-polar amino acid residues. It has mild proteolytic activity and is useful for the isolation and passaging of primary cells while preserving the integrity of the cell membrane. Dispase has a Zn-ion in the active center. 2+ Its activity is CaCa 2+The enzyme is stabilized by the addition of 1,000 mg ...

[0051] The perinatal appendage extract extracted from the perinatal appendage 12 by enzymatic degradation with connective tissue degrading enzymes contains many functional proteinaceous polymers and many functional proteinaceous small molecules with little damage, as well as many physiologically active substances with little damage, since the perinatal appendages are degraded by the mild proteolytic activity of the connective tissue degrading enzymes. "Low damage" includes an undamaged state and damage that does not impair the effects of functional proteinaceous polymers, functional proteinaceous small molecules, or physiologically active substances. The physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines.

[0052] Extracellular matrix proteins are non-cellular components present in all tissues and organs. They not only provide a physical scaffold for cells, but also provide important biochemical and biomechanical cues required for tissue morphogenesis, differentiation, and homeostasis. Extracellular matrix proteins are composed of proteins and polysaccharides, and each tissue has its own unique composition and morphology of extracellular matrix proteins. The uniqueness of extracellular matrix proteins is formed during the developmental stage of tissues and is influenced by dynamic and reciprocal biochemical and biophysical interactions between cells and with the microenvironment, such as proteins.

[0053] Cell adhesion to extracellular matrix proteins is mediated by extracellular matrix protein receptors such as integrins. Cell adhesion mediates cytoskeletal binding to extracellular matrix proteins and is also involved in cell migration through extracellular matrix proteins. In addition, they direct essential morphological and physiological functions such as induction of signal transduction and control of gene translation by binding to growth factors and interacting with cell surface receptors. Extracellular matrix proteins are composed of two types of polymers: fibrous proteins and proteoglycans. The main fibrous proteins of extracellular matrix proteins are collagen, elastin, fibronectin, and laminin. Proteoglycans form a hydrated gel in tissues and fill the gaps in the extracellular matrix.

[0054] Collagen is the major protein component of connective tissues and basement membranes and exists in many types with different tensile strengths and tissue distributions. Stiffness, flexibility, and structural changes in body tissues are due to changes in collagen composition, as well as cellular restriction and compartmentalization. The basement membrane surrounding the vascular endothelium is a specialized thin network of extracellular matrix proteins with many functions. This membrane is composed of proteins and glycoproteins (collagen, laminin, entactin, fibronectin, heparin sulfate, perlecan, etc.) and acts as a physical barrier between the epithelium and the underlying tissue. It provides a scaffold for cell surfaces through integrins, receptor kinases, and cell surface proteoglycans, and guides cell differentiation, supports architecture, and restricts the migration of normal cells. Collagen is a major inhibitor of tumor cell migration, as it is the main structural element of basement membranes and tissue scaffolding proteins.

[0055] Fibronectin is an important high molecular weight glycoprotein that binds to integrins and the extracellular matrix proteins collagen, fibrin, and heparin. Fibronectin is present in soluble form in plasma and is composed of two 250 kDa subunits linked by disulfide bonds. Insoluble fibronectin is a large complex of cross-linked subunits. There are several isoforms of fibronectin, all synthesized from a single gene. The structure of these isoforms is made up of three repeat internal regions, which are distinguished by differences in length and the presence or absence of disulfide bonds. Alternative splicing of pre-mRNA leads to combinations of the three regions, which also create variable regions. Fibronectin is involved in wound healing processes and can be used as a therapeutic agent. Fibronectin is one of the few proteins whose production increases with age, independent of the associated pathology. In addition to this, polymerized fibronectin inhibits tumor growth, angiogenesis, and metastasis.

[0056] Laminins are large molecular weight, non-collagenous, basement membrane glycoproteins with diverse biological functions including differentiation, migration, and adhesion of normal and tumor cells. The proteins consist of three distinct polypeptide chains that are linked to each other by disulfide bonds to form a cruciform molecule with one long arm and three short arms. The α-2 chain is a subunit of laminin-2 (merosin) and laminin-4 (S-merosin). Due to their cell-binding ability (via membrane-associated integrins), laminins are effective substrates for cell migration and neurite outgrowth. In placental laminins, the A chain is replaced by merosin, and in laminins near neuromuscular junctions, the B chain is replaced by S-laminin (synaptic laminin). Due to their diverse binding properties, numerous laminin subtypes have been identified. For example, laminin-5 is a basement membrane extracellular matrix that provides an attachment substrate for both adhesion and migration for a wide variety of cell types, including epithelial cells, fibroblasts, neurons, and leukocytes. Compared to fibronectin, collagen, or vitronectin, cells of epithelial origin adhere faster and spread more extensively on laminin-5. Furthermore, laminin can be used for most applications at low coating concentrations of less than 1 μg / mL, approximately 10-fold lower than other extracellular matrix proteins.

[0057] Cytokines are proteins secreted mainly from immune system cells, and exert physiological effects in extremely small amounts through specific receptors present on the surface of target cells, and act as intercellular signaling, often acting locally. Cytokines are produced by various cells, and each molecule exerts a variety of physiological effects on each target cell, but the effects of different cytokines often overlap. In addition, cytokines form complex networks and control the entire immune system by interacting with each other in cooperation and antagonism. In particular, the phenomenon in which the production of one cytokine induces other cytokines in response to it is called the cytokine cascade, and is involved in inflammatory responses, etc.

[0058] There are many types of cytokines, many of which are related to biological defense, such as immune and inflammatory responses, but some are also related to cell proliferation and differentiation, cell death, and healing. Representative cytokines include chemokines (CCLs), interleukins (ILs), interferons (IFNs), lymphokines, colony-stimulating factors (granulocyte colony-stimulating factor: G-CSF, erythropoietin, etc.), tumor necrosis factors (TNFs), and growth factors (EGFs, FGFs, TGF-β, etc.). Cytokines that cause various inflammatory symptoms in the body, such as TNF-α and IL-6, are called inflammatory cytokines, while cytokines that suppress inflammatory symptoms, such as IL-10 and TGF-β, are called anti-inflammatory cytokines.

[0059] Cytokines are small protein molecules that are stimulated, synthesized, and secreted by immune cells (monocytes, macrophages, T cells, B cells, NK cells, etc.) and some non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.). Many cytokines promote or restrict each other in the body, forming a complex immunoregulatory network. Certain cytokines exert their biological effects in autocrine, paracrine, endocrine, and other ways, and have various properties such as pleiotropy, overlap, antagonism, and synergy.

[0060] Chemokines, a type of cytokine, are basic, heparin-binding proteins with molecular weights of 8,000 to 12,000. Chemokines contain four well-conserved cysteine ​​residues, and disulfide bonds are formed between the first and third and between the second and fourth residues. They are broadly classified into two major subfamilies, CXC and CC, based on the motif formed by the two N-terminal residues. In CXC, one other amino acid is interposed between the two cysteines, but in CC, they are consecutive. Furthermore, the existence of molecular species called C chemokines (only the second and fourth cysteine ​​residues are present) and CX3C chemokines (three amino acids are interposed) has been revealed, and they are classified into the C subfamily and the CX3C subfamily, respectively.

[0061] The three-dimensional structures of chemokines are similar to each other. The N-terminus shows an unstable structure and is important for signal transduction after binding to the receptor. The central region, stabilized by two disulfide bonds, forms three antiparallel β-sheets and is involved in high-affinity binding to the receptor. The C-terminal region forms an α-helix and is the region that binds to heparan sulfate present in the extracellular matrix and cell membranes.

[0062] CXC chemokines are broadly divided into a group that has an ELR motif immediately before CXC and a group that does not. CXC chemokines with an ELR motif, such as IL-8, Gro, NAP-2, and ENA-78, are mapped to chromosome 4 and are potent chemotactic factors for neutrophils. These chemokines act via CXCR1 and CXCR2. On the other hand, CXC chemokines without an ELR motif, such as IP-10, MIG, I-TAC, SDF-1, and BLG, chemotact lymphocytes. SDF-1 also chemotacts monocytes. PF-4 also chemotacts fibroblasts.

[0063] IP10, MIG, and I-TAC act on CXCR3, while SDF-1 and BLC act on CXCR4 and CXCR5, respectively. CXC chemokines with ELR motifs exhibit angiogenic activity, while CXC chemokines with some non-ELR motifs, such as PF-4, IP-10, and MIG, exhibit antiangiogenic activity. The CC subfamily, which has the largest number of members, includes the MCP group and MIP group, which are mapped to chromosome 17. These mainly chemotactic monocytes, but also chemotactic subsets of lymphocytes. The MCP group acts mainly through CCR2 and CCR3, while the MIP group acts mainly through CCR1 and CCR5. Some, such as RANTES, act across CCR1, CCR3, and CCR5.

[0064] CCR3 is highly selectively expressed on eosinophils and basophils, and has specific ligands such as eotaxin. Furthermore, chemokines such as RANTES and the MCP group also act on CCR3, making it the receptor with the most ligands. This supports the idea that eosinophils, which are important effector cells against parasitic infections, are the most important target cells for chemokines in the host defense mechanism. In addition, a series of CC chemokines such as TARC, SLC, and LARC act specifically on lymphocytes and dendritic cells, respectively, but the relationship between the ligand and receptor is relatively specific for these chemokines. This is because each chemokine specifically controls the migration of a specific lymphocyte subset. The C chemokine SCM-1 / Lymphotactin and the CX3C chemokine Fractalkine migrate NK cells and CD8+ T cells. These also act via the specific receptors XCR1 and CXCR1.

[0065] Chemokines play various biological roles in addition to the migration of leukocytes and lymphocytes. Immune system chemokines play an essential role in the formation and maintenance of lymphatic tissues. CXC chemokines with ELR motifs exhibit angiogenic activity, which is thought to be involved in wound healing and angiogenesis in cancer tissues. In addition, many chemokines are also involved in regulating hematopoiesis in the bone marrow, as they suppress colony formation of hematopoietic precursor cells in vitro. Some chemokines are thought to play an important role in organ formation during fetal period, and in particular, SDF-1 and its receptor CXCR4 play an essential role in the formation of B cells in the fetal liver, the formation of myeloid cells in the bone marrow, the formation of the ventricular membranous septum, the formation of the large vascular system in the gastrointestinal tract, and the formation of layered structures in the cerebellum. Chemokines also play an important role in the uterine cyclical changes and changes during parturition, which are considered to be non-infectious inflammatory responses. Some chemokine receptors, such as CXCR4 and CCR5, are known to act as essential coreceptors during infection of CD4+ T cells and macrophages by immunodeficiency virus (HIV).

[0066] Interleukins are cytokines secreted by cells involved in immunity, and are very important molecules for immune function. While immune function is not possible without interleukins, they are also related to diseases such as autoimmune diseases and immunodeficiencies. Some molecules are classified as monokines or lymphokines. Interleukin 1 is secreted by macrophages, is involved in inflammatory reactions, and belongs to a group called inflammatory cytokines. Interleukin 2 is secreted by T cells, has the effect of promoting the proliferation and differentiation of T cells themselves, and is an interleukin used in cancer immunotherapy. Interleukin 3 is an interleukin secreted by T cells, is involved in bone marrow stem cells, and activates differentiation signals.

[0067] Interleukin 4 is an interleukin that plays an important role in allergic reactions, and is involved in the proliferation of B cells and the differentiation of T cells and mast cells. Interleukin 5 acts on B cells to secrete Iga (immunoglobulin A). Interleukin 6 is an interleukin that is involved in the development of inflammation and immune diseases, and is produced by T cells and macrophages. Interleukin 7 is involved in the survival, differentiation, and maintenance of homeostasis of immune cells. B cells, T cells, and NK cells are greatly affected by interleukin 7.

[0068] Interleukin 8 is involved in the properties and behavior of neutrophils. Interleukin 9 has the effect of stimulating mast cells. Interleukin 10 has the effect of inhibiting the production of Th1 cytokines. Interleukin 11 induces the production of acute phase proteins in the immune system. Interleukin 12 stimulates NK cells and induces their differentiation. Interleukin 13 induces the proliferation and differentiation of B cells, and inhibits the production of inflammatory cytokines by macrophages by suppressing Th1 cells. Interleukin 14 induces the proliferation of activated B cells. It also suppresses antibody production by B cells. Interleukin 15 is secreted from peripheral blood monocytes and epithelial cells, and is involved in the activation of killer T cells and the induction of proliferation and differentiation of B cells. Interleukin 17 induces the production of inflammatory cytokines. Interleukin 18 induces the production of interferon gamma.

[0069] Among other immune-related cells, the differentiation of T cells is induced by interleukin 2, 4, and 7, that of B cells by interleukin 7, 13, and 15, that of NK cells by interleukin 7 and 12, and that of mast cells by interleukin 4. Interleukins bind to interleukin receptors present in cells and activate the signal transduction pathway. The receptors protrude from the cell membrane, and when interleukins bind to the binding site on the outside of the cell membrane, the receptor site within the cell membrane becomes activated and transmits an activation signal into the cytoplasm.

[0070] Interferons are a family of cytokines secreted by host cells to regulate immune responses. Signaling proteins are usually released by host cells in the presence of pathogens and function to eradicate the pathogen by alerting neighboring uninfected cells to activate appropriate cellular defense mechanisms. Interferons are classified into three types (type I, type II, and type III) depending on the various receptors they bind to. Each type of interferon induces a specific immune response. Furthermore, interferon-mediated signaling promotes increased expression (upregulation) of major histocompatibility class I and II molecules (MHCI, MHCII), activating many downstream signaling cascades resulting in antiviral defense mechanisms. Since then, interferons have been used as a treatment for viral infections such as hepatitis C and hepatitis B viruses.

[0071] Type I interferons bind to specific cell surface receptors known as IFN-α / β (IFNAR1, IFNAR2) and act as a warning system to uninfected cells. In humans, type I interferons are the largest IFN family and include IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω. They are produced by many cell types, including plasmacytoid dendritic cells and fibroblasts. One of the major functions of type I interferons is the inactivation of eukaryotic translation initiation factor 2a (eIF-2a), thereby inhibiting the synthesis of viral proteins. Type I interferons activate RNaseL, cleaving ssRNA in the cytoplasm and inhibiting viral replication. IFN-α is used to treat hairy cell leukemia, and IFN-β is used as a therapeutic agent to slow the progression of multiple sclerosis.

[0072] Type II interferons (IFN-γ in humans) bind to the IFN-γ receptor complex (IFNGR1, IFNGR2) and are involved in immune and inflammatory responses. They are produced by activated T cells and natural killer (NK) cells. When type II interferons are released by type 1 helper T cells (Th1 cells), they recruit leukocytes to the site of infection, leading to increased inflammation.

[0073] Type III interferons include IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4, and, like type I interferons, are involved in the inhibition of viral infection. Type III interferons bind to receptors IFRL1 and IL-10R2, which are distinct from type I receptors. Type III interferons, although less well understood than type I and type II interferons, are associated with the JAK-STAT pathway and, like type I interferons, are synthesized in response to host detection of pathogen-associated molecular patterns (PAMPs).

[0074] Human Toll-like receptor (TLR) homologs are a class of pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs). These transmembrane receptors are found on the cell surface and in endosomes. Upon binding to PAMPs, TLRs initiate a signaling cascade that leads to the activation of interferon genes and subsequent protein secretion from the cell. RIG-I-like receptors (RLRs) are another class of PRRs that function as cytosolic sensors for the detection of viral RNA. Activation of RLRs leads to increased expression (upregulation) of interferon regulatory factor-3 (IRF-3), IRF-7, and NF-κB, which are transcription factors that lead to the induction of type I interferons and proinflammatory cytokines.

[0075] Lymphokines are soluble proteins produced by T lymphocytes in response to stimulation by specific antigens or mitogens and released outside the cell. They do not themselves act as effector molecules to induce cellular immunity, but are messenger molecules that act on various cells such as macrophages and neutrophil lymphocytes, and induce responses through these cells. As a mediator of cellular immunity in host defense, macrophage-activating factor (MAF) is one of the lymphokines that has been studied. Macrophage-activating factor is a lymphokine that activates macrophages to kill foreign pathogenic microorganisms and malignant tumor cells that develop in the body, thereby defending the body.

[0076] Macrophage-activating factor (MAF) is a lymphokine that is produced and released by lymphocytes specifically in response to specific antigenic stimulation of cellular immunity, and non-specifically in response to mitogen stimulation such as ConA (concanavalin A) and PHA (phytohaemagglutinin). For macrophage-activating factor to participate in host defense, it first binds to a receptor on macrophages to activate them, and then causes various functional and morphological changes in the macrophages to produce activated macrophages. These macrophages have characteristics not seen in normal macrophages. It is not possible to express the activation of macrophages by macrophage-activating factor in a unified manner. Various cell functions and morphological changes are used as indicators of activation. In some cases, the activity of killing microorganisms within cells and the activity of killing tumor cells in vitro are used as indicators of host defense, but increased glucose consumption and the expression of unique ruffles can also be used as indicators of macrophage activation.

[0077] Macrophage activating factor, as a mediator of cellular immunity, acts in host defense through activated macrophages, and its effect is to kill or inhibit the growth of foreign intracellular parasitic pathogenic microorganisms in the body and to eliminate tumor cells that occur in the body. Many macrophage activation reactions are related to host defense reactions, but the reactions that are most directly related are in vitro antimicrobial action and antitumor cell activity. As far as cellular immunity is concerned, the former is mainly intracellular killing, while the latter is extracellular killing, but the effector molecules derived from activated macrophages may be the same or different.

[0078] The characteristic ruffles of macrophages activated by macrophage-activating factor may be related to phagocytosis, which is accompanied by a series of oxygen-related biochemical reactions, and there is a fairly strong relationship between enhanced oxidative metabolism and the killing of intracellular microorganisms. The intracellular bactericidal effect appears as early as 2 hours after treatment of human macrophages with lymphokines, and macrophage activation proceeds quite rapidly.

[0079] Regarding the oxidative metabolism accompanying the phagocytosis of activated macrophages, for example, when neutrophils come into contact with microorganisms, oxygen consumption increases, and most of the absorbed oxygen is converted to superoxide anion (O2-) by the action of oxidase, H2O2 is produced, and further, OH, a powerful oxidant, is formed. The involvement of OH in the bactericidal action of activated phagocytes is also evident from the fact that the phagocytic bactericidal action is blocked by the addition of inhibitors such as superoxide dismutase. Furthermore, as a result of the formation of NADP+ during phagocytosis, glucose oxidation occurs via the hexose monophosphate (HMP) cycle. On the other hand, in peritoneal and alveolar macrophages of several animals, increased oxygen consumption and activation of the HMP shunt during phagocytosis have been observed, and furthermore, the release of H2O2 and the production of O2- have also been observed. Thus, H2O2 is considered to be a potent effector molecule for the bactericidal action of activated macrophages. In vitro tumor cell lytic and killing activity or cytotoxicity are used as indicators of macrophage activation by MAF / MIF. In this case, activated macrophages with antitumor properties acquire the ability to contact, recognize and destroy tumor cells not only in vitro but also in vivo, without harming nontumor cells. Furthermore, tumor-killing macrophages activated by MAF / MIF can lyse many types of target tumor cells in many different animal species by a non-immunological mechanism that requires contact between the macrophage and the target tumor cells.

[0080] Antitumor activity As an index of macrophage activation by MAF / MIF, tumoricidal activity or cytotoxicity in vitro is used. In this case, activated macrophages with antitumor activity acquire the ability to contact, recognize and destroy tumor cells not only in vitro but also in vivo, without harming nontumor cells. In addition, tumor-killing macrophages activated by MAF / MIF can lyse many types of target tumor cells in many different animal species by a non-immunological mechanism that requires contact between the macrophage and the target tumor cells.

[0081] Activation of macrophages to have antitumor activity requires the completion of a series of reactions, not a single reaction between the signal and the target cell. When macrophages were treated in vitro with lymphokines, cytotoxicity appeared within 4 hours, reached a maximum at 8-12 hours, and gradually decreased thereafter. Thus, although macrophage cytotoxicity was short-lived, the addition of LPS as a trigger signal resulted in a stronger antitumor effect. When the signal sequence was reversed, first LPS and then lymphokine, macrophages were not activated. Furthermore, a synergistic effect of lymphokines and LPS was observed in the synthesis and release of prostaglandin E2 (PGE2) by activated macrophages, but treatment of macrophages with lymphokines for 6 hours and then with LPS for 1 hour was more effective for activation than treatment of both simultaneously. Macrophages that are fully activated through a series of reactions starting from the initial stimulus ultimately produce and release effector molecules that exert antitumor properties. These effector molecules include proteases, complement component C3a, arginase, thymidine, H2O2, interferon, and prostaglandins.

[0082] Colony-stimulating factors, which are cytokines, stimulate the differentiation and colony formation of immature cells in vitro. Colony-stimulating factors stimulate various hematopoietic cell lines to form various cell colonies at the differentiation stage in semi-solid medium. Based on the differences in the cell types they stimulate, they are classified as granulocyte CSF (G-CSF), macrophage CSF (M-CSF), granulocyte-macrophage CSF (GM-CSF), multi-CSF (IL-3), stem cell factor (SCF), and erythropoietin (EPO). Granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF) promote the differentiation and proliferation of hematopoietic stem cells into granulocytes, macrophages, eosinophils, and basophils. Macrophage CSF (M-CSF) promotes the differentiation and proliferation of monocyte and macrophage precursor cells.

[0083] Tumor necrosis factor (TNF), like IL-1 and IL-6, is a powerful inflammatory cytokine derived from activated macrophages that has tumor necrosis activity, and was later found to be the same substance as cachectin, a cachexia-inducing factor. Lymphotoxin is a substance derived from activated lymphocytes that shows structural homology to tumor necrosis factor. Both bind to the same receptor on target cells and share a high degree of commonality in terms of physiological activity. Currently, the former is generally referred to as TNF-α and the latter as TNF-β, and when simply referred to as TNF, it means TNF-α. Its physiological activities are extremely diverse, and it has been shown to be the same substance as cachectin, a cachexia-inducing factor. 2 TNF is considered to be one of the mediators of inflammatory reactions, as it induces fever and various inflammatory reactions through the production of collagenase and other enzymes. Excessive production of TNF is closely related to the pathology of collagen diseases and related diseases, Kawasaki disease, meningitis, malaria, cachexia, AIDS, multiple organ failure, etc. Tumor necrosis factor plays an increasingly important role in the pathogenesis of these diseases, as anti-TNF therapy is highly effective in chronic inflammatory diseases such as Crohn's disease and rheumatoid arthritis.

[0084] Growth factors, a type of cytokine, are a general term for endogenous proteins that promote the proliferation and differentiation of specific cells in the body. They regulate various cellular and physiological processes and act as intercellular signaling substances by specifically binding to receptor proteins on the surface of the target cells. Growth factors differ from hormones (growth hormone, insulin, thyroid hormone, etc.) in that hormones are produced in specific organs (pituitary gland, pancreas, thyroid gland, etc.) and act systemically via the bloodstream, whereas growth factors are secreted by various cells and act near or locally to the secreting cells.

[0085] Growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-1, IGF-2), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). Platelet-derived growth factor (PDGF) is a liquid factor stored in granules in platelets, and is a growth factor for vascular smooth muscle cells and fibroblasts. It is involved in arteriosclerosis, wound healing, and other conditions, and is deeply involved in cancer. Epidermal growth factor (EGF) binds to the epidermal growth factor receptor, activating the receptor and causing cells to differentiate and grow. The epidermal growth factor receptor is involved in carcinogenesis, cancer growth, invasion, metastasis, and other conditions.

[0086] Insulin-like growth factors (IGF-1, IGF-2) are single-chain polypeptides with homology to insulin. IGF-1 is synthesized in the liver in a growth hormone (GH)-dependent manner and secreted into the blood. They promote cell proliferation and bone growth. In contrast to insulin-like growth factor I (IGF-1), which is the main growth factor in adults, IGF-2 is the main growth factor in fetuses. Fibroblast growth factor (FGF) is the most powerful angiogenic factor that acts directly on vascular endothelial cells. Since angiogenesis is closely related to the cause and progression of diseases themselves, research is being conducted into its production mechanism and mechanism of action. Fibroblast growth factor (FGF) is present in considerable amounts in tissues with active angiogenesis, but it only acts when angiogenesis is required.

[0087] Vascular endothelial growth factor (VEGF) is produced around blood vessels and acts specifically on vascular endothelium through paracrine secretion to promote proliferation (angiogenesis induction factor) and suppress the formation of tumor blood vessels. Nerve growth factor (NGF) is known as a neurotrophic factor (neurotrophin) and is taken up by nerve terminals through paracrine secretion in nerve cells and muscle cells, where it is involved in the formation of neuronal synapses. Hepatocyte growth factor (HGF) acts on many epithelia, promoting proliferation and cell movement. It inhibits apoptosis. It is essential for organ formation and regeneration and repair of tissue damage. Blood concentrations are abnormally high in fulminant hepatitis and myocardial infarction. It shows high levels in liver disease, renal failure, and cancer, and shows a negative correlation with patient prognosis.

[0088] The perinatal appendage extract is extracted from perinatal appendages 12 (at least one of umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) obtained from the mother at the time of birth, and contains a large amount of functional proteinaceous polymers and functional proteinaceous small molecules that are little damaged, as well as a large amount of physiologically active substances (extracellular matrix proteins (collagen, elastin, fibronectin, laminin) and cytokines (chemokines, interleukins, interferons, lymphokines, colony-stimulating factors, tumor necrosis factors, growth factors)) that are little damaged, and can make maximum use of the effectiveness of the disease-curing and disease-preventing abilities, antiviral properties, antibacterial properties, etc., of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages 12.

[0089] The perinatal appendage extract can utilize active ingredients such as functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances (extracellular matrix proteins (collagen, elastin, fibronectin, laminin) and cytokines (chemokines, interleukins, interferons, lymphokines, colony-stimulating factors, tumor necrosis factors, growth factors)) contained in the perinatal appendages 12 (at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)), and is highly effective in treating and preventing various diseases. By using the perinatal appendage extract, various diseases can be treated, and by using the perinatal appendage extract, various diseases can be prevented.

[0090] During normal childbirth, the perinatal appendages 12 (at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) may become contaminated with certain bacteria, but when the perinatal appendages 12 are obtained from the mother during birth by cesarean section, a clean perinatal appendage extract can be obtained using the clean perinatal appendages 12.

[0091] The perinatal appendage extract uses a connective tissue degrading enzyme (at least one of collagenase, liberase, and dispase) used in cell purification, and the connective tissue degrading enzyme is used to enzymatically degrade the perinatal appendages 12 (at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)). This allows the enzymatic decomposition of the perinatal appendages 12 to proceed slowly, preventing damage to the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages 12 and preventing the death of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances.

[0092] Fig. 2 is a diagram showing an example of each step P-1 to P-4 performed in the perinatal accessory substance extract production method. The perinatal accessory substance extract production method shown in Fig. 2 produces a perinatal accessory substance extract through a perinatal accessory substance acquisition step P-1, an enzymatic hydrolysis extraction step P-2, an extract sterilization step (filter sterilization) P-3, and a component inspection step P-4.

[0093] First, a perinatal appendage acquisition step P-1 is performed. In the perinatal appendage acquisition step P-1, perinatal appendages 12 separated from a mother (human mother) at the time of birth when the mother gives birth to a newborn baby are acquired. In the perinatal appendage acquisition step P-1, as described above, the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, decidua 15c) are acquired as the perinatal appendages 12. In addition, in the perinatal appendage acquisition step P-1, it is preferable to acquire perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, decidua 15c) acquired from the mother during birth by Caesarean section. During normal birth, the perinatal appendages 12 may become contaminated with certain bacteria as they pass through the mother's birth canal, but clean perinatal appendages 12 are acquired by Caesarean section. However, perinatal appendages 12 separated from the mother during normal birth may also be obtained.

[0094] In the perinatal appendage acquisition step P-1, the acquired perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c) are placed in a container (not shown) such as a tray (stainless steel tray or plastic tray) of a predetermined volume, and then a cell cryopreservation liquid is poured into the container. The cell cryopreservation liquid is poured in an amount sufficient to completely immerse the perinatal appendages 12 placed in the container. After the entire perinatal appendages 12 are immersed in the cell cryopreservation liquid, the container is left as is for a predetermined time (3 to 4 hours). The cell cryopreservation liquid may be dimethyl sulfoxide (DMSO) or glycerol, which are effective substances for protecting cells and cell organelles, or may be polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, or trehalose. The cell cryopreservation liquid prevents ice crystals from forming inside the cells when the perinatal appendages 12 (cells) are frozen.

[0095] Dimethyl sulfoxide (DMSO) is cell membrane permeable and promotes cell dehydration, slowing the growth rate of ice crystals and inhibiting ice crystal formation. Use research grade DMSO and glycerol, and sterilize before use. Dimethyl sulfoxide (DMSO) is sterilized by filtration using a 0.2 μm nylon syringe filter or Teflon PTEF syringe filter that has been washed with alcohol and rinsed with dimethyl sulfoxide. Glycerol is sterilized in an autoclave at 121°C for 15 minutes. To avoid the risk of contamination, the cryoprotectant after sterilization is stored in small aliquots for single use.

[0096] After leaving the container containing the perinatal appendage 12 and the cell cryopreservation solution for a predetermined time, the container is placed in a freezer or refrigerator and frozen to -20°C to freeze the perinatal appendage 12. The perinatal appendage 12 is frozen slowly over a predetermined time (3 to 12 hours). The perinatal appendage 12 may also be frozen in a short period of time. By slowly freezing the perinatal appendage 12 over a predetermined time, damage to the perinatal appendage 12 is minimized. The perinatal appendage 12 is preserved in a frozen state. The perinatal appendage 12 contained in the container may also be frozen without using a cell cryopreservation solution.

[0097] After the perinatal appendage acquisition step P-1, an enzymatic decomposition and extraction step P-2 is carried out. In the enzymatic decomposition and extraction step P-2, the perinatal appendage 12 acquired in the perinatal appendage acquisition step P-1 is decomposed by a connective tissue decomposition enzyme, and a perinatal appendage extract is extracted from the perinatal appendage 12. As the connective tissue decomposition enzyme, a connective tissue decomposition enzyme obtained by blending one or more of collagenase, liberase, and dispase is used.

[0098] In the enzymatic decomposition and extraction process P-2, a container is removed from a freezer or refrigerator, and the frozen perinatal appendages 12 are removed from the container. The frozen perinatal appendages are moved to a processing space while being frozen with dry ice. Next, the perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) are finely ground while still frozen by hitting with a meat tenderizer or meat hammer. Alternatively, the frozen perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) are put into a meat grinder (mincing machine), and the perinatal appendages 12 are finely ground while still frozen.

[0099] After the perinatal appendages 12 are ground, the perinatal appendages 12 are placed in a new container such as a tray, and a connective tissue decomposition enzyme (at least one of collagenase, liberase, and dispase) is injected into the inside of the tray. The container containing the ground perinatal appendages 12 and into which the connective tissue decomposition enzyme has been injected is left at room temperature for a predetermined time. In the container, the ground perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) are enzymatically decomposed by the connective tissue decomposition enzyme. By using at least one of collagenase, liberase, and dispase as the connective tissue decomposition enzyme, the enzymatic decomposition of the perinatal appendages 12 proceeds slowly and gently. The enzymatic decomposition time of the perinatal appendages 12 is 3 to 24 hours.

[0100] If the ground perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) are rapidly decomposed by enzymes, the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages 12 will be damaged, and some of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances will be destroyed. However, by using collagenase, liberase, and dispase, the enzymatic decomposition of the perinatal appendages 12 proceeds slowly and gently, so that the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages 12 are not damaged, and some of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances are not destroyed.

[0101] In the enzymatic hydrolysis and extraction process P-2, the perinatal appendages 12 (umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, decidua 15c)) are enzymatically hydrolyzed, and then the enzymatically hydrolyzed perinatal appendages 12 are centrifuged in a centrifuge. When the enzymatically hydrolyzed perinatal appendages 12 are centrifuged, a perinatal appendage extract is separated from the perinatal appendages 12, and the perinatal appendage extract (supernatant) accumulates at the top of the perinatal appendages 12. The perinatal appendage extract that has accumulated at the top of the perinatal appendages 12 is collected using a syringe or pipette.

[0102] After the enzymatic decomposition and extraction step P-2, the extract sterilization step P-3 is carried out. In the extract sterilization step P-3, the collected perinatal appendage extract (perinatal appendage extract extracted by the enzymatic decomposition and extraction step P-2) is filter sterilized (filter sterilization). In the extract sterilization step P-3, a nylon syringe filter with a pore size of 0.22 μm or 0.1 μm or a Teflon PTEF syringe filter with a pore size of 0.22 μm or 0.1 μm is used. In the filter sterilization (filter sterilization) using a syringe filter, the perinatal appendage extract to be filtered is filled into a syringe, and then the package is aseptically peeled off. Next, the syringe is attached to the filter unit and the device is removed from the package. Hold the syringe with the filter facing up and push in a few drops to remove the air so that the syringe membrane is filled with the perinatal appendage extract. At this time, be careful not to contaminate the underside of the filter unit with your fingers. The plunger is then pressed to deliver the filtered perinatal appendage extract.

[0103] The perinatal appendage extract sterilized by the extract sterilization process P-3 contains many functional proteinaceous macromolecules with little damage, many functional proteinaceous small molecules with little damage, and many physiologically active substances with little damage. The physiologically active substances are extracellular matrix proteins and cytokines. The cytokines include chemokines (CCL), interleukins (IL), interferons (IFN), lymphokines, colony stimulating factors (granulocyte colony stimulating factor: G-CSF, erythropoietin, etc.), tumor necrosis factors (TNF), growth factors (EGF, FGF, TGF-β, etc.), etc.

[0104] After the extract sterilization step P-3, a component inspection step P-4 is carried out. The component inspection step P-4 inspects the content of functional proteinaceous polymers and functional proteinaceous small molecules contained in the perinatal accessory extract sterilized by the extract sterilization step P-3, and inspects the content of physiologically active substances contained in the perinatal accessory extract sterilized by the extract sterilization step P-3.

[0105] The content of functional proteinaceous macromolecules and functional proteinaceous small molecules in the extract of perinatal appendages, and the content of physiologically active extracellular matrix proteins in the extract of perinatal appendages are measured by ultraviolet spectrophotometry, Bradford method, WST method, Biuret method, Lowry method, and BCA method. Alternatively, they are measured by a fluorescence method that quantifies proteins by using reactions with primary amines or reactions with surfactants that coat proteins. Alternatively, they are measured by protein quantification using polyacrylamide gel electrophoresis with protein staining by CBB or fluorescent dye.

[0106] Among the amino acids that make up proteins, tyrosine, tryptophan, and phenylalanine have the property of absorbing ultraviolet light at around 280 nm because they have aromatic groups such as benzene rings. In ultraviolet absorptiometry, protein concentration is quantified by measuring the absorbance of the protein at 280 nm using this property. The Bradford method is a method for quantifying proteins using Coomassie Brilliant Blue G250 (CBB250), a triphenylmethane dye. When CBB250 is added to a protein solution under acidic conditions, CBB250 binds to the protein through non-covalent bonds due to electrostatic interactions between basic amino acid residues (arginine, lysine, histidine) and N-terminal amino acids in the protein and CBB250, and hydrophobic interactions between CBB250 and aromatic amino acids. At this time, the maximum absorption wavelength of CBB250 shifts to 465 nm to 595 nm, and the color tone changes from reddish purple to blue, so the protein is quantified by measuring the change in absorbance at 595 nm.

[0107] The WST method is a protein quantification method using a water-soluble tetrazolium salt (WST8). WST8 generates formazan when reduced by amino acids in protein (cysteine, leucine, tryptophan). The generated formazan turns blue in an aqueous alkali solution, so proteins are quantified by measuring the absorbance at 650 nm, which is the maximum absorption wavelength of the formazan. When an oligopeptide or a protein consisting of three or more amino acids is mixed with a Cu(II) solution under alkaline conditions, the nitrogen atom in the protein or peptide chain coordinates with Cu(II), reducing Cu(II) to Cu(I), causing the solution to turn reddish purple. The degree of coloring due to the reaction increases with the number of peptide bonds in the protein. The Biuret method utilizes this phenomenon to measure the absorbance at 540 nm and calculates the protein concentration using a calibration curve created in advance.

[0108] The Lowry method is an improvement of the Biuret method, developed to improve detection sensitivity. It is a method for quantifying proteins that combines the Biure reagent with the Folin-Ciocalten reagent (phosphomolybdic acid and phosphotungstic acid dissolved in an acidic solution), which was developed for the purpose of detecting phenols. In principle, when the Biure reagent is added to a protein solution under alkaline conditions, Cu(II) in the Biure reagent forms a complex with the peptides that make up the protein. Next, when the Folin-Ciocalten reagent is added, phosphotungstic acid and phosphomolybdic acid are reduced by tryptophan, tyrosine, and cysteine ​​in the protein, causing the sample solution to turn blue and absorbing light at around 650 to 750 nm. The absorbance at this time is measured and compared with a calibration curve created with standard proteins to quantify the protein. The BCA method is an improved version of the Lowry method, and can perform quantitative analysis of proteins even in the presence of surfactants such as SDS and TritonX, which are used to solubilize proteins. The principle is the same as in the Biuret and Lowry methods: proteins form complexes with Cu(II) under alkaline conditions, and Cu(II) is reduced to Cu(I) by cysteine, tyrosine, and tryptophan in the protein. The amount of Cu(I) produced by reduction is proportional to the amount of protein. Next, bicinchoninic acid (BCA), a colorimetric reagent with high selectivity for Cu(I), is added. Two BCA molecules coordinate with Cu(I) to form a blue-purple complex that exhibits strong absorption at 562 nm. The absorbance at this time is measured, and colorimetric analysis of proteins is performed using a calibration curve created with quasi-proteins.

[0109] Fluorescamine, one of the proteins that reacts with primary amines, does not emit fluorescence in the first place, but when it reacts with primary amines in proteins, it forms a derivative that emits blue-green fluorescence (maximum fluorescence wavelength 495 nm). Proteins are quantitatively analyzed by measuring the fluorescence intensity at this time and comparing it with a calibration curve prepared with standard proteins. o-Phthalaldahyde (OPA), one of the proteins that reacts with primary amines, reacts with primary amines in proteins at room temperature in the presence of a reducing agent such as 2-mercaptoethanol and quickly forms a blue fluorescent substance. Its maximum fluorescence wavelength is 455 nm, and the fluorescence intensity at this wavelength is measured and compared with a calibration curve prepared with standard proteins to perform quantitative analysis. 3(4-carboxybenzoyl)quinoline 2-carboldefyde (CBQCA), one of the proteins that reacts with primary amines, is non-fluorescent when unreacted, but when it reacts with primary amines in proteins at room temperature for 1 hour in the presence of cyanide ions, it forms a green fluorescent substance. The maximum fluorescence wavelength is 550 nm, and the fluorescence intensity at this wavelength is measured and compared with a calibration curve prepared using standard proteins to perform quantitative analysis of the protein.

[0110] Protein quantification utilizes the reaction with a surfactant that coats the protein. NanOrange solution containing the surfactant is non-fluorescent when used alone, but when incubated with the protein for 10 minutes at 90-95°C, the surfactant denatures the protein and the surfactant is adsorbed to the protein surface. When the surfactant that coated the protein reacts with a fluorescent reagent, fluorescence with a maximum wavelength at 590 nm is generated. Proteins are quantified by comparing the intensity of the generated fluorescence with a standard curve.

[0111] Staining proteins with CBB is a method of staining proteins using Coomassie Brilliant Blue R250 (CBB R250), a triphenylmethane dye. After washing the gel after electrophoresis, the gel is immersed in a CBB solution and shaken for several tens of minutes, causing the CBB to bind to the protein, resulting in the appearance of a blue band derived from the protein. If necessary, destaining with deionized water or the like can be performed to reduce the background and obtain a clearer electrophoretic pattern. Quantitative analysis is possible because there is a linear relationship between protein concentration and signal intensity of the protein band.

[0112] The protein staining method using fluorescent dyes has a higher detection sensitivity than the protein staining method using CBB, and is therefore effective in quantifying extremely small amounts of protein. There are two types of protein staining methods using fluorescent dyes: SYPRORuby and OrioleTM fluorescent gel stain. The detection sensitivity of SYPRORuby protein gel staining reagent is 0.25ng, which is extremely high compared to the CBB method. In addition, the staining process after electrophoresis requires only three steps: gel fixation → staining → decolorization, and the time required for staining is completed in 90 minutes by combining it with the microwave method. Furthermore, the protein quantification range spans three orders of magnitude, so protein quantification can be performed over a wide concentration range. The detection sensitivity of OrioleTM fluorescent gel stain is 0.5 to 1ng, making it an effective method for detecting extremely small amounts of protein. In addition, the staining protocol requires only a 90-minute staining step, and does not require protein fixation, gel washing, or decolorization, making it easy to stain proteins. Furthermore, since the protein quantification range spans three orders of magnitude, proteins can be quantified over a wide dynamic range.

[0113] 3 is a diagram showing another example of each step P-1 to P-4 performed in the perinatal appendage extract production method. The perinatal appendage extract production method shown in FIG. 3 produces a perinatal appendage extract through a perinatal appendage acquisition step P-1, an enzymatic decomposition and extraction step P-2, an extract sterilization step (gamma ray sterilization) P-3, and a component inspection step P-4. The perinatal appendage extract production method shown in FIG. 3 differs from that in FIG. 2 in that gamma ray sterilization is performed in the extract sterilization step P-3, and the other steps are the same as those in the perinatal appendage extract production method in FIG. 2. Therefore, the explanation of the perinatal appendage acquisition step P-1, the enzymatic decomposition and extraction step P-2, and the component inspection step P-4 will be omitted.

[0114] In the extract sterilization step P-3 of the perinatal appendage extract manufacturing method in Figure 3, the collected perinatal appendage extract (the perinatal appendage extract extracted by the enzymatic decomposition extraction step) is irradiated with gamma rays to sterilize the perinatal appendage extract (gamma ray sterilization). 137Sterilization utilizes the effect on living organisms of the gamma rays emitted during gamma decay of Cs and other elements. Unless the energy of the gamma rays irradiated is extremely strong, the amount of new radioactivity that the irradiated object gains is negligible. Gamma ray sterilization does not significantly damage the material of the irradiated object, and does not leave behind any harmful substances that are associated with chemical sterilization.

[0115] The content of physiologically active cytokines in the perinatal appendage extract is determined by measuring the amount of cytokines in the perinatal appendage extract using an existing measurement method for cytokines (secreted extracellularly) contained in the perinatal appendage extract (cell culture supernatant).

[0116] The method for producing a perinatal appendage extract involves obtaining perinatal appendages 12 (at least one of umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c)) that are separated from the mother at the time of birth when the mother gives birth to a newborn baby, decomposing the obtained perinatal appendages 12 with a connective tissue decomposing enzyme to extract a perinatal appendage extract from the perinatal appendages 12, and sterilizing the extracted perinatal appendage extract to produce a perinatal appendage extract. Therefore, the produced perinatal appendage extract contains active ingredients (functional proteinaceous substances with little damage) contained in the perinatal appendages 12. It is possible to produce a perinatal appendage extract that contains a large amount of polymers, functional proteinaceous small molecules with little damage, and physiologically active substances with little damage (extracellular matrix proteins (collagen, elastin, fibronectin, laminin) and cytokines (chemokines, interleukins, interferons, lymphokines, colony-stimulating factors, tumor necrosis factors, growth factors)) and that can make the most of the disease-healing and disease-preventing abilities, antiviral properties, antibacterial properties, etc., of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage 12.

[0117] The method for producing a perinatal appendage extract is highly effective in treating and preventing various diseases, and can produce a perinatal appendage extract capable of treating and preventing various diseases. The method for producing a perinatal appendage extract produces a perinatal appendage extract from at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, and decidua 15c) that are discarded after birth, and can therefore effectively utilize the perinatal appendages 12 that would otherwise be discharged after birth.

[0118] The method for producing a perinatal appendage extract uses collagenase, liberase, and dispase as connective tissue degrading enzymes, which allows for gradual enzymatic decomposition of (at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, decidua 15c)), thereby reliably preventing damage to the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendages 12, reliably preventing the death of the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances, and producing a perinatal appendage extract that contains a large amount of less damaged functional proteinaceous polymers, less damaged functional proteinaceous small molecules, and less damaged physiologically active substances.

[0119] In the method for producing a perinatal appendage extract, when the perinatal appendage 12 (at least one of the umbilical cord 13, umbilical cord blood, placenta 14, and fetal membrane 15 (amnion 15a, chorion 15b, decidua 15c)) is obtained from the mother during birth by Caesarean section, the perinatal appendage 12 may become contaminated with certain bacteria as it passes through the birth canal during normal childbirth. However, by obtaining clean perinatal appendage 12 by Caesarean section, a clean perinatal appendage extract can be produced using the clean perinatal appendage 12, and a clean perinatal appendage extract that can reliably treat and prevent various diseases can be produced.

[0120] The perinatal appendage extract manufacturing method tests the content of functional proteinaceous polymers and functional proteinaceous small molecules contained in the perinatal appendage extract, as well as the content of physiologically active substances contained in the perinatal appendage extract, and thereby shows that the perinatal appendage extract contains many functional proteinaceous polymers and functional proteinaceous small molecules, that the perinatal appendage extract contains many physiologically active substances, and that the perinatal appendage extract produced by the perinatal appendage extract manufacturing method contains many active ingredients possessed by physiologically active substances.

[0121] The perinatal appendage extract production method shown in Figure 2 sterilizes (filter sterilizes) the perinatal appendage extract by passing the perinatal appendage extract through a filter, so that the perinatal appendage extract can be sterilized without damaging the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage extract, and a clean perinatal appendage extract can be produced that contains less damaged functional proteinaceous polymers, less damaged functional proteinaceous small molecules, and less damaged physiologically active substances.

[0122] The perinatal appendage extract production method shown in Figure 3 involves irradiating the perinatal appendage extract with gamma rays to sterilize the perinatal appendage extract (gamma ray sterilization), so that the perinatal appendage extract can be sterilized without damaging the functional proteinaceous polymers, functional proteinaceous small molecules, and physiologically active substances contained in the perinatal appendage extract, and a clean perinatal appendage extract can be produced that contains less damaged functional proteinaceous polymers, less damaged functional proteinaceous small molecules, and less damaged physiologically active substances. [Explanation of symbols]

[0123] 10 Newborns 11 Mother (human mother) 12 Perinatal Appendages 13 Umbilical Cord 14 Placenta 15 Egg membrane 15a amnion 15b chorion 15c decidua 16 Birth canal

Claims

1. In a perinatal accessory extract produced from perinatal accessories obtained from a mother at the time of childbirth, The perinatal appendage extract is characterized in that it is extracted from the perinatal appendage and contains functional proteinaceous polymers and functional proteinaceous small molecules that are less damaged, as well as physiologically active substances that are less damaged.

2. The perinatal appendage extract according to claim 1, wherein the physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines.

3. The perinatal appendage extract according to claim 2, wherein the perinatal appendage is at least one of the umbilical cord, umbilical cord blood, placenta, and fetal membrane (amnion, chorion, and decidua).

4. The perinatal appendage extract according to claim 3, wherein the perinatal appendage is obtained from a mother at the time of birth by Caesarean section.

5. The perinatal appendage extract according to claim 4, wherein the perinatal appendage extract is extracted from the perinatal appendage by enzymatically hydrolyzing the perinatal appendage with a specific connective tissue degrading enzyme.

6. The perinatal appendage extract according to claim 5, wherein the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase.

7. A method for producing a perinatal appendage extract from perinatal appendages obtained from a mother at the time of birth, comprising: The perinatal appendage extract production method is characterized by comprising a perinatal appendage obtaining step of obtaining perinatal appendages separated from the mother at the time of birth when the mother gives birth to a newborn baby, an enzymatic hydrolysis extraction step of decomposing the perinatal appendages obtained in the perinatal appendage obtaining step with a connective tissue decomposing enzyme and extracting the perinatal appendage extract from the perinatal appendages, and an extract sterilization step of sterilizing the perinatal appendage extract extracted in the enzymatic hydrolysis extraction step.

8. A method for producing a perinatal appendage extract as described in claim 7, wherein the perinatal appendage extract contains functional proteinaceous polymers and functional proteinaceous small molecules that are less damaged, as well as physiologically active substances that are less damaged.

9. The method for producing a perinatal appendage extract according to claim 8, wherein the physiologically active substances contained in the perinatal appendage extract are extracellular matrix proteins and cytokines.

10. The method for producing a perinatal appendage extract as described in claim 9, further comprising, after the extract sterilization step, inspecting the content of functional proteinaceous macromolecules and functional proteinaceous small molecules contained in the perinatal appendage extract sterilized by the extract sterilization step, and inspecting the content of physiologically active substances contained in the perinatal appendage extract sterilized by the extract sterilization step.

11. The method for producing a perinatal appendage extract according to claim 10, wherein the extract sterilization step sterilizes the perinatal appendage extract using a filter.

12. The method for producing a perinatal appendage extract according to claim 10, wherein the extract sterilization step sterilizes the perinatal appendage extract by irradiating the perinatal appendage extract with gamma rays.

13. The method for producing a perinatal appendage extract according to claim 11 or 12, wherein the connective tissue degrading enzyme is at least one of collagenase, liberase, and dispase.

14. The method for producing a perinatal appendage extract according to claim 13, wherein the perinatal appendage is at least one of an umbilical cord, umbilical cord blood, a placenta, and a fetal membrane (amnion, chorion, decidua).

15. The method for producing a perinatal adjunct extract according to claim 14, wherein the perinatal adjunct is obtained from a mother at the time of birth by Caesarean section.

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