JP6747931B2 - Anti-inflammatory agent caused by endotoxin derived from enterobacteria - Google Patents

Description

The present invention relates to a smoldering inflammation inhibitor that suppresses an increase in fragility due to aging, prevents the health of skin and hair, and prevents deterioration of body function.

In today's aging society, cancers, neurodegenerative diseases such as Alzheimer's disease, arteriosclerotic diseases, autoimmune diseases, etc. are clearly increasing, which is a cause of hindering the healthy aging of the people. There is. The onset, progression, and aggravation of these diseases have been considered to be caused by smoldering inflammation.

Smoldering inflammation is chronic inflammation that continues to smolder in the body, and various diseases such as cancer, Alzheimer's disease, arteriosclerosis, obesity, etc., which increase with age, and aging itself, Evidence is emerging that the symptoms may be advanced by inflammatory changes.

In addition to chronic diseases, smoldering inflammation that increases with aging causes various physiological reserves to deteriorate, vulnerability to environmental factors increases, tiredness becomes easy, skin loses elasticity and wrinkles are engraved. ,, more gray hair, thinner hair, and decreased physical function. External factors include ultraviolet rays, climate change, dryness, pollutants, foods and additives, and intrinsic factors include mental stress, sleep and fatigue.

Factors that increase vulnerability to environmental factors with aging include hyperglycemia, insulin resistance, chronic inflammation, low muscle mass, high levels of interleukins 1, 2, 6, interferon γ, and TNF-α. Has been pointed out.

The body is equipped with the ability to maintain homeostasis. However, it cannot completely return to its original state with aging, and changes in morphology are also likely to cause cell damage. Although functional deterioration occurs due to cell damage, in response to cell damage, reactions such as regeneration, hypertrophy, and hyperplasia occur to supplement the function of the damaged part.

Regeneration is the replacement of cells/tissues lost in the living body with the original cells/tissues. In addition, hypertrophy causes the cells to increase in volume and become larger than normal, resulting in large tissue and organ shapes. In the case of myocardium or striated muscle, hypertrophy occurs because it is a non-dividing cell that does not undergo cell division in adults. On the other hand, hyperplasia is an increase in the number of cells that make up a tissue/organ.

Cells that divide as long as an individual continues to live (dividing cells) include epithelial cells of the epidermis and mucous membranes (skin, oral cavity, cervix, digestive tract, trachea/bronchi, bladder, etc.) in contact with the outside world, and bone marrow Hematopoietic cells and the like. The parenchymal cells of the liver, pancreas, kidney, fibroblasts, smooth muscle cells, and vascular endothelial cells usually remain in the G0 phase, but they may start to divide depending on the stimulus. Particularly, the liver has a high regenerative capacity.

It is well known that internal organ disorders appear on the skin. The liver has the function of detoxifying by metabolizing and decomposing unnecessary substances such as harmful substances and additives that have entered the body through food and respiration. However, as the liver is said to be a silent organ, the disorder does not appear as a symptom until it becomes severe. The total number of human dogs in 2014 has increased dramatically so that one in three people can find abnormal values in liver function.

Even if the number of liver functions is in the normal range, if there is a year-to-year change, there is a possibility that the cell self-renewal ability and repair action in the liver are inhibited. When self-renewal ability and repair action are impaired and the areas of reduced liver function increase, if the liver does not function well and metabolism is not possible until the disease does not occur, harmful substances will gradually decline in various functions around the body. By causing, the physiological reserve capacity becomes weakened. As a result, abnormalities appear on the skin, causing skin troubles and worsening of the complexion, and age-related changes appear as a decline in appearance.

Therefore, it is considered that the development of a method for suppressing the increase in fragility due to aging, that is, a method for suppressing chronic inflammation or smoldering inflammation will make a great contribution to the prevention of physical changes and functional deterioration associated with aging.

Japanese Patent Fair 7-68122 Republished WO95/30412 Republished WO99/45900 Patent No. 5688163 JP, 2015-27968, A

Kazutomo Imahori, Tamio Yamakawa (1998): Biochemistry Dictionary, 3rd Edition, Tokyo Kagaku Dojin Tsutomu Goto, Masayuki Ikeda, Ichiro Hara (1994): Handbook of Industrial Poisoning (enlarged edition), Ito Dental Publishing Takaya Murakami, Toxicology Letters 94(1998) 137-144 Ellison et a1. , Brain Res (1987) pages 389-392. Marshall et a1. , Neurosci Res Commun 18(1996) pp. 87-96

Increased fragility with aging affects the maintenance of homeostasis, causing aging changes of skin and hair and deterioration of physical function. The problem to be solved by the present invention is to provide a method for suppressing the increase in fragility due to aging, thereby making the skin healthy and restoring healthy skin, and preventing deterioration of physical function.

The present invention provides an age-related smoldering inflammation inhibitor, which comprises ethanolamine and an amino acid as active ingredients.

The present invention also provides an age-related smoldering anti-inflammatory agent containing ethanolamine and amino acid contained in a mammalian placenta extract, mammalian breast milk or mammalian liver extract as active ingredients.

The present invention also provides the age-related smoldering inflammation inhibitor, wherein the amino acid contains one or more of glycine, serine, and threonine.

In addition, the present invention provides a healthy skin by ingesting ethanolamine and one or more of glycine, serine, or threonine to a subject who needs to suppress smoldering inflammation with aging. A method for regaining healthy skin and/or a method for preventing deterioration of physical function are provided.

In addition, the present invention, as an active ingredient of the smoldering inflammation inhibitor with aging, a placenta extract of mammals, milk of mammals or glycine, serine or threonine contained in the extract of liver of mammals. One or more uses are provided.

In order to suppress the increase in vulnerability due to aging, a means for preventing the deterioration of liver function can be considered. For that purpose, it is important to maintain the homeostasis of liver function, and it is known that ethanolamine is involved in the regeneration of hepatocytes. Ethanolamine is a constituent component of phosphatidylethanolamine (PE), which is the most common phospholipid in the body next to choline-containing phospholipids, and serine, which is one of the amino acids. It is produced by hydrolysis of PE with phospholipase D and decarboxylation of serine. , Constantly exist in the living body (see Non-Patent Documents 1 and 2).

Ethanolamine is used in the synthesis of phosphatidylethanolamine (PE), which is a phospholipid, and is a factor necessary for keeping the phospholipid composition of the cell membrane normal, and functions to keep the biological reaction through the cell membrane normal. Are doing

Ethanolamine is known to have a hepatoprotective action and a hepatocyte proliferation promoting action from a test using a carbon tetrachloride-induced liver injury model (see Non-Patent Document 3). Further, it was reported that PE in the brain of Alzheimer's disease patients, particularly the temporal cortex, the frontal cortex and the hippocampus, decreased to 40-60% of the normal level (see Non-Patent Document 4), and ethanolamine in Alzheimer's disease A role has been suggested (see Non-Patent Document 5).

Also, for the treatment of diseases associated with abnormal accumulation of collagen by an ethanolamine derivative (see Patent Document 1), the treatment of diseases in which the function of cytokines is reduced (see Patent Document 2), and the health of skin (see Patent Document 3). Proposed to be useful.

It is generally known that ethanolamine and its metabolite, ethanolamine phosphate, can be added to a medium as a cell growth factor enhancer. Ethanol phosphate is an active ingredient that enhances acetaldehyde dehydrogenase activity, and is known to be useful for liver system diseases such as hangover and intestinal mucosal disorders. Furthermore, ethanolamine phosphate may be a biomarker for depression (see Patent Document 4), and ethanolamine phosphate has an anti-inflammatory action and an antioxidant action, and thus prevents inflammatory diseases. Alternatively, it has been proposed to be useful for treatment (see Patent Document 5).

On the other hand, ethanolamine is also a raw material for pH adjusters used in the production of synthetic detergents and cosmetics, rust preventives contained in cleaning agents for machinery, solvents, gas absorbents, fiber softeners, and chemical substances. In these applications, it is corrosive to the skin and eyes and is corrosive even after ingestion, causing abdominal pain, burning sensation, and collapse, so it is not used in food.

Therefore, as a result of diligent study, it was possible to reduce the required amount of ethanolamine not only by the protective action by amino acids but also by a synergistic effect by combining amino acids with ethanolamine which cannot be taken orally by itself due to strong irritation. It was found that it can be ingested.

In addition, as a result of diligent studies of resources containing ethanolamine that exist in nature, by adding glycine, serine or threonine as amino acids in addition to ethanolamine, the increase in vulnerability due to aging is suppressed, and smoldering inflammation with aging The suppression effect was found.

Furthermore, by ingesting one or more of ethanolamine and glycine, serine, or threonine, the function of internal organs such as digestive organs and liver caused by smoldering inflammation associated with aging is reduced, and the elasticity of the skin is reduced accordingly. It was also found that it has an improving effect on skin conditions such as wrinkles increase.

Furthermore, they have found that the placenta extract of mammals, breast milk of mammals, or the extract of liver of mammals contain ethanolamine and the above amino acids, and completed the present invention.

The present invention relates to a cytokine (eg, interleukin) in which a smoldering anti-inflammatory agent containing ethanolamine and one or more amino acids consisting of glycine, serine or threonine shows a high value with an increase in aging vulnerability. 1β and 6 and TNF-α) and iNOS (inducible nitric oxide synthase) induced by inflammation and stress can be suppressed, and by suppressing liver fibrosis, increase in vulnerability due to aging can be suppressed. As a result, the skin can be made healthy and healthy skin can be regained, and deterioration of physical function can be prevented.

Effect of porcine placenta extract on the induction of inflammatory cytokines by LPS stimulation of dTHP-1 cells Effect of porcine placenta extract on production of soluble collagen by TGFβ stimulation of LX-2 cells (calculated per living cells) Effects of pig placenta extract on drug-induced liver injury model Effect of porcine placenta extract on acute alcoholism model Effect of pig placenta extract on chronic alcoholic liver injury model

The smoldering anti-inflammatory agent of the present invention is a naturally occurring resource containing ethanolamine and one or more amino acids consisting of glycine, serine and threonine as active ingredients, or an extract extracted from the material thereof. is there.

For example, a placenta extract can be mentioned, and as the placenta extract derived from pig or horse, a commercially available product from Snowden Co., Ltd. can be used. Approximately 20 to 2000 μg of ethanolamine, 20 to 200 mg of glycine, 10 to 100 mg of serine, and 10 to 100 mg of threonine are contained in 1 g of the placenta extract derived from pig. In addition, mammalian breast milk, liver extract and the like can also be used.

The smoldering inflammation inhibitor of the present invention includes ethanolamine and an extract of a material containing one or more amino acids consisting of glycine, serine or threonine, as a functional component, for example, "vitamins". (Retinols, liver oils, β-carotene, vitamin Ds, vitamin Es, thiamine salts, vitamin B1 derivatives, riboflavin, riboflavin butyrate, vitamin B6s, vitamin B12s, vitamin Cs, nicotinic acids, pantothenic acids, Biotin, etc.), “nutrients” (eg potassium/magnesium aspartate, inositol, inositol hexanicotinate, ursodesoxycholic acid, cysteines, orotic acid, γ-oryzanol, calcium salts, glucuronic acid, etc.), “herbal extract” Kind" (blue skull cap, valerian, heaven gate winter, yellow radish, Centella asiatica, ginseng, carrot, oats, eleuthero, Hypericum perforatum, hops, lemon balm, mugwort, roses, passionflower, litchi, basil, ginseng, Ashwagandha, guguru, Longan Meat, Ginkgo, Sadako, Cuckoo Chologi, Damiana, Chamomile, Lime, Lavender, etc.), “Amino Acids and Proteins” (aspartic acid, cystine, phenylalanine, taurine, tryptophan, casein hydrolyzate, lactoferrin, etc.), “Minerals” "(Calcium, sulfur, magnesium, zinc, selenium, iron, etc.), "Mushrooms" (Shiitake extract, Yamatake mushrooms, etc.), "Seeds, germs, fruits" (pomegranate, acerola, prunes, blueberries, sesame, etc.), " It is possible to combine "health tea extract" (hub tea, beetroot tea, dokudami tea, etc.), and honey, glucosamine, rice-derived ceramide and hyaluronic acid, N-acetylglucosamine, bilberry, soy isoflavone, hesperidin, and the like.

In addition, since it can be expected to be effective transdermally for external application, for the purpose of skin regeneration, "anti-inflammatory component" (dipotassium glycyrrhizinate, glycyrrhetinic acid, etc.), "blood circulation promoting component" (benzyl nicotinate, tocopherol, etc.), "Tissue repair component" (heparin analogue, allantoin, tocopherol acetate, etc.), "whitening component" (ascorbic acid, ascorbate, magnesium ascorbate phosphate, ascorbyl glucoside, cysteine, glutathione, glutathione salt, N- Acylated glutathione, glutathione ester, hydroquinone, hydroquinone salt, hydroquinone glycoside, ferulic acid, ferulic acid salt, isoferulic acid, isoferric acid salt, caffeic acid, caffeic acid salt, resorcinols, caffeine, Tannin, verapamil, tranexamic acid, licorice extract, glabridin, tocopherol acetate, glycyrrhizic acid, arbutin, kojic acid, ellagic acid, chamomile extract, linoleic acid, oleic acid, linolenic acid, etc.), "hyaluronic acid production promoting component" (N-acetylglucosamine, glucosamine, retinol, retinol palmitate, retinoic acid, marjoram extract, mint extract, green mint extract, apple mint extract, citrus extract, bugle extract, swordfish extract, xenothera extract, perilla extract, aojiso extract, Chili Menjiso extract, Perilla extract, Lemon perilla extract, Mebuki extract, Hikiokoshi extract, Magwa extract, Mokkogo extract, Wax extract, Panoki extract, Kozo extract, Casinoki extract, Fig extract, Oitabi extract, Anahusa extract, Oohaosa extract, Pterus chinensis extract, Ogonorii extract. , Macsa extract, ginseng extract, arame extract, wakame extract, hijiki extract, knotdrak extract, derbyria extract, etc.), “component having a collagenase activity inhibitory action” (retinol, retinol palmitate, retinoic acid, ascorbic acid or a salt thereof, Ascorbyl palmitate, Ascorbyl dipalmitate, Ascorbyl stearate, Ascorbyl stearate, Sodium ascorbyl phosphate, Ascorbyl phosphate magnesium, Soybean extract, Birch extract, Fuji tea extract, Turmeric extract, Button pie extract, Silk peptide etc.) Etc. can be combined.

The smoldering inflammation inhibitor of the present invention may contain ethanolamine in an amount of 1 μg to 100 mg per adult per day. The smoldering inflammation inhibitor of the present invention is not particularly limited, but may be administered 1 to 7 days a week.

The formulation of the present invention may be in the form of foods for the sick, health foods, functional foods, foods for specified health uses, dietary supplements and supplements.

As the inhibitor of the present invention, the smoldering inflammation inhibitor of the present invention can be used as it is.

The inhibitor of the present invention may further contain any component. For example, the smoldering inflammation inhibitor of the present invention can be provided in a form containing a base acceptable for pharmaceuticals and foods, a carrier, an excipient, a binder, a disintegrating agent, a lubricant, a coloring agent and the like. ..

Examples of carriers and excipients used in medicines and foods include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate and crystalline cellulose.

Examples of the binder include starch, gelatin, syrup, gum tragacanth, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose and carboxymethyl cellulose.

Examples of the disintegrant include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose and the like.

In addition, examples of the lubricant include magnesium stearate, hydrogenated vegetable oil, talc, macrogol and the like. Further, as the colorant, any colorant which is allowed to be added to the food can be used.

In addition, the smoldering inflammation inhibitor of the present invention, if necessary, sucrose, gelatin, purified shellac, gelatin, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, phthalic acid cellulose acetate, hydroxypropyl methylcellulose. One or more layers may be coated with phthalate, methyl methacrylate, methacrylic acid polymer or the like.

Moreover, a pH adjuster, a buffer, a stabilizer, a solubilizer and the like may be added as necessary.

Further, it can be provided as a preparation in any form. For example, as a preparation for oral administration, tablets such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets, troches, pills, capsules including powders and soft capsules, granules, suspensions, emulsions, syrups including dry syrup, And a liquid such as an elixir.

When an extract from a mammal or the like is used, since the extract has a characteristic acridness, it may be used as a masking agent for the extract or as a film coating agent coated with a coating agent. it can.

Further, the smoldering anti-inflammatory agent of the present invention can be used as it is as it is dissolved in a solvent such as water, alcohol and 1,3-butylene glycol alone or appropriately mixed, but it is usually used in the production of pharmaceuticals or cosmetics. Various ointments, lotions, W/O type creams, O/W type creams, liquids, etc. can be easily added by an ordinary method by adding an emulsifier, an emulsion stabilizer, a gelling agent, a wetting agent, a preservative, or other additives. Skin external preparation. In addition, as the shape of cosmetics, various viscosity such as liquid and paste can be mentioned, such as lotion, lotion, cream, milk, gel, pack, impregnation sheet, impregnation mask, bath salt, mist and spray. You can

When used externally, the blending ratio of the smoldering anti-inflammatory agent of the present invention is 0.001 to 95 wt% (abbreviation of% by weight) based on the total amount of the external preparation, but considering the expression of effects and economical efficiency. It is preferably 0.005 to 50 wt%.

The present invention will be described in detail below with reference to test examples.
[Test Example 1]
Using a normal placental placenta of pigs, after sterilization using the material homogenized after washing, 1.0% by weight of proteolytic enzyme was added to the mixture while stirring with water, and the mixture was kept warm overnight for digestion and centrifuged. The enzyme digestion solution was collected. The enzyme digestive juice was concentrated and spray-dried to obtain a pig placenta extract. It was confirmed that the obtained pig placenta extract contained 400 μg of ethanolamine, 75 mg of glycine, 25 mg of serine, and 25 mg of threonine in 1 g.

[Test Example 2]
The liver damage caused by endotoxin is caused by a component derived from intestinal bacteria that penetrates into the blood in a trace amount. Usually, except for exposure to large amounts of endotoxin due to serious infectious diseases such as sepsis, endotoxin slightly contaminated in blood rarely transits to chronic inflammation. On the other hand, diabetes, long-term alcohol intake, high levels of fatigue and exposure to chemicals accelerate the intestinal permeability to transfer endotoxins derived from intestinal bacteria into the blood, and these habits and diseases. It is known that, since hypersensitivity to endotoxin stimulation is induced in liver Kupffer cells (resident macrophage lineage) and the like, strong inflammation is caused in the liver and liver damage is induced.

Therefore, LPS stimulation was applied to human macrophage-like cells (dTHP-1) in the presence of a placenta extract, and the expression levels of inflammatory cytokines and inflammatory response factors were evaluated by Realtime PCR.

Human monocyte-like cell line (THP-1) was suspended in RPMI (invitrogen) + 10% FBS (invitrogen), final concentration of 10 nM PMA (sigma) was added, and the conditions were 37°C and 5% CO ₂ After incubation for 36 hours, the cells were differentiated into human macrophage-like cells (dTHP-1).
The pig placenta extract of Test Example 1 was added to a medium containing RPMI1640 (Sigma) + 5% FBS (Invitrogen) at a final concentration of 0.5-30 mg/ml, and pretreated for 2 hours, and then LPS-05 (Sigma). Was added to the cells at a final concentration of 30 ng/ml and incubated for 5 hours.
After washing the cells with PBS(-), total RNA was extracted according to the protocol attached to the product of the RNeasy Mini kit (Qiagen). Total RNA was subjected to cDNA synthesis using ReverTra Ace qPCR RT Master Mix with gDNA Remover kit (TOYOBO) according to the protocol attached to the product.
For cDNA, the gene was quantified by Realtime PCR analysis using Fast SYBR Green Master Mix (Life technologies) and StepOne Plus (Life technologies) according to the conditions described in the product protocol.
The following primers were used.
IL-1beta (Primer Set ID: HA237369, Takara Bio), IL-6 (HA209655), TNFalpha (HA198263), iNOS (HA032546).

The results are shown in Figure 1. When dTHP-1 was stimulated with LPS, the expression levels of inflammatory cytokines were 78×10 ⁴ times higher with IL-6, 64×10 ² times higher with IL-1beta, and 106 times higher with TNFalpHa than untreated, respectively. A large increase was observed.

On the other hand, when the pig placenta extract of 10, 20, and 30 mg/ml was treated with LPS, IL-6 was 67×10 ⁴ times, 4×10 ⁴ times, and 1.4×10 ⁴ times, respectively, as compared with the untreated case. , IL-1beta was 36×10 ² times, 35×10 ² times, 4×10 ² times, and TNFalpHa was 78 times, 43 times, and 21 times, respectively, and almost a dose-dependent decrease was observed.

When cells were treated only with 10, 20 and 30 mg/ml of porcine placenta extract, IL-6 was 2.2×10 ⁴ times, 1.2×10 ⁴ times and 1. 4×10 ⁴ times, IL-1beta 3.7×10 ² times, 6.4×10 ² times, 6.5×10 ² times, TNFalpHa 14 times, 22 times, 12 times respectively Was given.

These results suggest that the porcine placenta extract suppresses the inflammatory response caused by endotoxin (LPS) mainly derived from membrane components such as Escherichia coli. These results suggest that the porcine placenta extract has a palliative effect on such inflammatory organ damage.

[Test Example 3]
Liver fibrosis is a disease caused by excessive accumulation of extracellular matrix mainly composed of collagen in an organ, and is a reaction caused to hepatocyte damage due to almost all causes. In particular, when hepatocytes are stimulated by hepatitis virus, endotoxin, alcohol, fatty liver, etc., inflammatory cells accumulate from the peripheral blood and bone marrow in the damaged area, releasing TGFb, which promotes fibrosis, and hepatic stellate cells are released. It is known that when activated, it causes excessive accumulation of extracellular matrix such as collagen, and the fibrosis of organs progresses.

Human liver stellate cell line (LX-2) was stimulated with TGFβ as a model of inflammatory response in the presence of porcine placenta extract, and the total amount of collagen produced was measured.

LX-2 cells seeded at 50% cell density were cultivated in DMEM medium (Sigma) + 2% FBS (invitrogen) medium for 24 hours, and pre-cultured for 3 hours after changing to Opti-MEM Serum Free Medium (invitrogen). After that, the porcine placenta extract of Example 1 was added at a final concentration of 1-100 mg/ml, pretreated for 2 hours, and 2 ng/ml recombinant TGFβ (Militenyi) was added to the cells at a final concentration of 2 ng/ml. After further culturing for 12 hours, the culture supernatant was collected. The remaining cells were subjected to cell viability measurement using Cell Counting Kit-8 (Dojindo) according to the conditions described in the product protocol. From the recovered culture supernatant, using the QuickZyme Soluble Collagen Assay (QuickZyme bioscience), according to the conditions described in the product protocol, the fluctuation of the total soluble collagen in the supernatant was treated with untreated (0%) and 2 ng/ml TGFβ ( The increase rate (%) was expressed from the value of only 100%.

The results are shown in Figure 2. Stimulation of LX-2 with 2 ng/ml TGFb increased the production of soluble collagen by about 90 times compared to unstimulated, but by adding TGFb stimulation with pig placenta extract at 10, 50, 100 mg/ml, the concentration was increased. The production was suppressed 82-fold, 62-fold, and 54-fold, respectively, in a dependent manner. When only the pig placenta extract was added to the cells, 16-fold, 15-fold, and 8-fold fluctuations were observed, respectively.

From these results, the pig placenta extract was found to have an inhibitory effect on TGFb-stimulated collagen overproduction from stellate cells, which is a major cause of fibrosis.

[Test Example 4]
The liver is an organ that occupies the most volume in the body, and its functions are diverse. In particular, the detoxification and excretion function of foods, therapeutic drugs, and waste products produced in the body is important, and it is known that once the liver function deteriorates, various organs and skin in the body are affected. .. In the following test examples, various drug models were used to evaluate the effects of placental extract and ethanolamine on liver injury.

The effect of placenta extract and ethanolamine on liver dysfunction was evaluated using a chronic drug-induced liver injury model with long-term high-dose acetaminophen, which is known as a component of cold medicines.

C57BL/6 (female, 6 weeks old) mice were given an intraperitoneal administration of 0.2 g/kg acetaminophen (Sigma) four times a week after 16 hours of fasting. At the same time, oral administration of the pig placenta extract of Example 1 at 350 mg/kg 6 times a week or the ethanolamine at 0.35 mg/kg 6 times a week was performed for 12 weeks. After completion of the treatment, plasma was collected from the mouse, and the liver deviating enzymes ALT and AST in the blood, which are indicators of liver injury, were measured using transaminase CII-Test Wako (Wako Pure Chemical Industries) according to the conditions described in the product protocol. Carried out. In addition, the liver was also collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The tissue was stained with hematoxylin-eosin and immunohistochemically stained with F4/80 antibody (Biorad), From the image acquired after the speculum, M
Using ethamolph (Molecular device), the accumulation rate of degenerated hepatocytes with loss of cytoplasm and macrophage/Kupffer cell marker (F4/80), which is one of the indicators of liver inflammation, was evaluated.

Results are shown in FIG. FIG. 3 shows representative photographic data of liver HE staining at the end of treatment in the first row, and representative photographic data of immunostaining of liver macrophages/Kupffer cells in the second row. In addition, the digitized data of the above images are shown in the third row, and the measurement data of the blood deviating enzyme (AST, ALT) in the liver at the end of the treatment are shown in the fourth row. AAP is acetaminophine and Etn is ethanolamine.

Long-term high-dose acetaminophen increased mean degenerate liver cells by 55.6 fold, F4/80 strongly positive cells by 7.4 fold, AST by 3.6 fold, ALT by ALT It increased by 3.2 times. On the other hand, in mice treated with placenta extract or ethanolamine together, degenerated liver cells increased by an average of 17.1 times and 16.2 times, respectively, and F4/80 strongly positive cells were 3 times higher than that of the non-treated mice. The increase was 0.8 times, 7.2 times, AST was 2.5 times, 2.3 times, and ALT was 1.9 times and 1.8 times.

From these results, it was confirmed that the administration of porcine placenta extract and ethanolamine, which is an index excluding the inflammatory index, has an inhibitory effect on liver damage to acetaminophen-induced liver damage.

[Test Example 5]
As a drinking model, an acute alcohol poisoning model was created by forced ethanol administration, and the effects of placental extract and ethanolamine on the alcohol metabolism rate in the liver and the behavioral inhibition caused by the feeling of drunk and vomiting were evaluated.

C57BL/6 (female, 6 weeks old) mice were fasted for 12 hours and then orally administered with 100 mg/kg of the porcine placenta extract of Example 1 or 0.35 mg/kg of ethanolamine, or sterile water. .. After 0.5 hours, ethanol suspended in a physiological saline solution of 3.5 g/kg was forcibly orally administered, and after the lights were turned off, the locomotor activity was measured by Supermex (Muromachi Kikai). Similarly, C57BL/6 (female, 6 weeks old) mice were fasted for 12 hours and then orally administered with 100 mg/kg of the pig placenta extract of Example 1 or 0.35 mg/kg of ethanolamine or sterile water. After 0.5 hours, ethanol suspended in 3.5 g/kg of physiological saline was orally administered by force, plasma was collected 2 hours later, and ethanol and metabolites of ethanol remaining in the blood were collected. The amount of acetaldehyde, which is a cause of vomiting, headache and various modulations, was measured by F-kit ethanol/acetaldehyde (JK International).

The results are shown in Fig. 4. EtOH is 3.5 g/kg ethanol, Etn is 100 μg/kg ethanolamine, and 100 mg/kg placenta extract.

Immediately after the light was turned off, the amount of exercise was actively increased in the group that did not receive ethanol, but in the ethanol-administered group, almost no exercise was observed until 7 hours after administration, and slight exercise was observed until 9 hours thereafter. It was only. On the other hand, in the group that had been pre-administered with the placenta extract, active movement was observed from the 6th hour, and recovery was observed at 7 hours and a half until it became equivalent to the ethanol-unadministered group. In the ethanolamine pre-administration group, an increase in the exercise amount was recognized 6 hours after the administration as in the placenta extract, but the exercise amount was about 1/3 of that in the non-administration group from beginning to end.

Further, when the blood concentration of ethanol was measured 2 hours after the administration of ethanol, the administration of the placenta extract and ethanolamine showed almost no difference in alcohol concentration as compared with the sterile water administration group. On the other hand, when the concentration of acetaldehyde, which is the cause of sickness in metabolites, was measured, a significant decrease of 32% was observed only in the placenta extract-administered group.

From these results, it was confirmed that the pretreatment with the extract of the placenta pig improved the behavioral inhibition associated with acute alcohol poisoning and the detoxification of blood acetaldehyde in the liver, which is the cause of the behavioral inhibition. On the other hand, in the corresponding ethanolamine administration group, a weak improvement tendency was shown, but no significance was observed.

[Test Example 6]
As a chronic alcoholic liver injury model, a long-term alcohol administration model was prepared, and the effects of placenta extract and ethanolamine on liver dysfunction were evaluated.

C57BL/6 (female, 6-week-old) mice were continuously fed a sample in which 5% ethanol was mixed with Lieber-DeCarli liquid diet (EPS Yasushin) for 3 months, and then 31.5% ethanol was added. After the oral administration of 20 ml/kg by gavage, the mixture was incubated at 37°C for 9 hours, and then blood and liver were collected. At the same time, in addition to ethanol administration, a group was also prepared in which 10-1000 mg/kg of the porcine placenta extract of Example 1 or 10-1000 μg/kg of ethanolamine or sterilized water was orally administered daily in addition to ethanol administration. As a control group, a group in which ethanol was not administered was given a sample in which maltodextrin (Sigma) corresponding to the calorie of ethanol was mixed with Lieber-DeCarli liquid diet.

After completion of the treatment, plasma was collected from the mice, and blood hepatic deviation enzymes ALT and AST, which are indicators of liver injury, were measured using transaminase CII-Test Wako according to the conditions described in the product protocol. The liver was also collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The tissue was stained with hematoxylin-eosin and immunohistochemically stained with F4/80 antibody according to a standard method. From the acquired images, the accumulation rate of the macrophage/Kupffer cell marker (F4/80), which is one of the indicators of degenerated hepatocytes with loss of cytoplasm and liver inflammation, and the progression of alcoholic fatty liver progression in Methamolph. The number of lipid droplets in the liver having a diameter of 10 μm or more, which is an index, was evaluated.

Results are shown in FIG. Pair is an ethanol non-administration group (maltodextrin is administered as a control), Etn is 100 μg/kg ethanolamine, 100 mg/kg placenta extract, and F4/80 is a macrophage/Kupffer cell marker.

The number of liver cells degenerated by long-term administration of ethanol was 54 times on average, 4.1 times for F4/80 strongly positive cells, 3.8 times for AST, 5.5 times for ALT, compared to the group not administered with ethanol. The number of fat droplets increased 51 times. In mice treated with placenta extract in combination with ethanol, the number of denatured liver cells, F4/80 strongly positive cells, AST, ALT, and 100 mg/kg or more placental extract were administered in mice treated with sterile water. There was a dose-dependent decrease in the number of lipid droplets in the group. On the other hand, when the corresponding ethanolamine was used in combination, the number of degenerated liver cells, AST and ALT tended to decrease, whereas the number of lipid droplets tended to increase.

From these results, it was confirmed that the pig placenta extract has an inhibitory effect on chronic alcoholic liver damage caused by long-term administration of alcohol. On the other hand, regarding ethanolamine, since the number of ALT and degenerated hepatocytes is remarkably reduced, it is considered that hepatocyte protective action is observed, but there is no action on fatty liver and inflammation, or The possibility of exacerbation is suggested.

From Test Examples 4, 5, and 6, administration of ethanolamine alone shows a certain protective action against drug-induced liver damage including alcohol, but it is difficult to expect a sufficient effect, and skin quality is improved. Is considered the same. Therefore, in order to expect a therapeutic effect, it is considered desirable to provide ethanolamine in the form of containing some auxiliary components such as placenta extract.

The present invention will be described in more detail by the following examples, but the present invention is not limited thereto.

Example 1 (solution)
(component)
(1) 90 g of porcine placenta extract of Test Example 1
(2) Trisodium citrate 10 g
(3) Sucralose 0.6 g
(4) Citric acid 10 g
(5) Fragrance 6 g
(6) Water residue (total 3000 g)

(Manufacturing method)
The above (1) to (5) are sequentially added to 2000 g of water to dissolve, and water is added to make 3000 g. Next, after heating the mixture to 95° C., 10 g of the mixture was filled in each container to obtain a drink-type liquid preparation of Example 1.

Example 2 (capsule)
(component)
(1) Placenta extract of Test Example 1 300 mg
(2) Glycine 10 mg
(3) Nicotinic acid amide 5 mg
(4) L-lysine hydrochloride 5 mg
(5) L-arginine 5 mg
(6) L-histidine 5 mg
(7) Calcium stearate 4 mg
(8) Vitamin B1 2 mg
(9) Vitamin B6 2mg
(10) Vitamin B2 2mg
(11) Folic acid 0.2mg
(12) Vitamin D3 1mg
(13) Vitamin B12 0.5mg
(14) Crystalline cellulose 38.3 mg
(Total 380mg)

(Manufacturing method)
A 10,000 times amount of each of the components (1) to (14) described above is weighed and mixed, and 380 mg each is filled in pullulan capsule No. 1 to obtain a capsule of Example 3.

Example 3 (tablets)
(Prescription)
(1) Pig placenta extract of Example 1 25 parts by weight (2) Lactose 69 parts by weight (3) Synthetic aluminum silicate 5 parts by weight (4) Magnesium stearate 1 part by weight

(Production method)
The above components are mixed, and the mixture is tableted with a tableting machine to give 300 mg of each tablet to give a tablet containing 75 mg of the pig placenta extract.

Claims (1)

An anti-inflammatory agent caused by endotoxin derived from intestinal bacteria, which comprises a mammalian placenta extract as an active ingredient.