KR20160127188A - An anti-inflammatory Cosmetic Composition comprising the extracts of Allium sativum L. stems - Google Patents
An anti-inflammatory Cosmetic Composition comprising the extracts of Allium sativum L. stems Download PDFInfo
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Abstract
The present invention relates to a composition containing garlic for (Allium sativum L.) extract as an active ingredient, and more particularly to, an anti-inflammatory composition containing an extract of garlic for.
Description
The present invention relates to an anti-inflammatory composition containing garlic extract as an active ingredient and its use.
Oxidative stress is known to be closely related to aging and various diseases. In particular, it is known that it is closely related to the autoimmune reaction. Typical examples thereof include atopy, rhinitis, asthma, and allergy, which are inflammatory reactions caused by oxidative stress.
The inflammatory reaction is a mechanism to repair and regenerate the injured area when an invasion that brings about a physical change such as a physical action, a chemical substance, a bacterial infection or the like is applied to a living body or a tissue, and once the stimulus is applied, prostaglandins, hydroxyeicosatetraenoic acid, and leukotriene are released to increase the vascularization process, leading to inflammation. However, the persistent inflammatory reaction promotes mucosal injury and, in part, causes diseases such as cancer (Willoughby, D., Ann. Rheum. Dis., 34 (6) 471, 1975)
Occurs, the inflammatory response NO (nitric oxide), TNF- α (tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), prostasin Cloud Dean E2 (PGE 2), iNOS (COX-2, cyclooxygenase-2, etc.) and is involved in chronic diseases as well as inflammation. Therefore, the substance that inhibits the inflammatory reaction is a functional substance for preventing or suppressing chronic diseases Is likely to be used as
Nitric oxide (NO) is synthesized by nitric oxide synthase (NOS) from L-arginine in various tissues and cells and is involved in vasodilation, neurotransmission, blood coagulation, Regulation and so on. NOS can be divided into two groups, cNOS and iNOS. In other words, neuronal and endothelial NOS belong to cNOS, which is Ca 2 + -calmodulcin-dependent and constantly regulates NO by secretion of NO. On the other hand, iNOS is activated by cytokines such as IFN-γ, IL-1, and TNF-α or by lipopolysaccharide (LPS) of bacteria and produces a large amount of NO over a prolonged period of time. This NO plays a major role in defending the host from microorganisms or tumor cells because it has a large effect on the cytotoxic activity of macrophages. However, because of the NO produced and the inflammatory reaction such as rheumatoid arthritis deteriorates, it shows harmful action. These stimulated iNOS are present in macrophages and hepatocytes, and the NO production increases significantly and inflammation is associated with other pathogens, resulting in cytotoxicity. Furthermore, the large reaction of normal free radicals of NO with oxygen to produce nitrogen dioxide produces NO, a powerful pro-oxidant molcule that can cause loss of oxidative power. Thus inhibition of NO production in inflammatory stimuli responses can be used therapeutically in inflammatory diseases
Free radicals and other reactive oxygen species are produced by endogenous metabolites in the food or endogenous metabolism in the body. Radicals can oxidize biomolecules to cause oxidative damage leading to apoptosis or tissue damage. Atherosclerosis, cancer, emphysema, cirrhosis and arthritis are known to be associated with oxidative damage. Thus, oxidative damage plays a very important pathological role in human disease, and ingestion of foods containing antioxidant supplements or antioxidants is very important because it reduces oxidative damage in humans. Recently, natural antioxidants in foods such as vitamins C, E, sesamol, and carnosic acid have been provided to consumers.
So far, the most powerful anti-inflammatory drugs developed by the first-class drugs are steroids. However, long-term use is accompanied by side effects. Therefore, steroids in asthma treatment are surprisingly effective at first time, and they cause symptoms to disappear completely but only for a short time. Symptoms of asthma are reversed with the use of steroids. Goes. Side effects of steroids include side effects such as round polyhedronic face, retention of fluid, adrenal suppression, increased susceptibility to infection, and other psychoses, cataracts, glaucoma, peptic ulcer, delayed wound healing, (Check WA & Kaliner MA, Am. Rev. Res. Dis., 141: 44-51, 1990).
Therefore, it is necessary to study a substance which can be safely ingested safely without a separate purification process, which has an effect of suppressing inflammation, and which can be easily used for food, as a medicament containing a natural plant extract as an active ingredient.
Accordingly, the present inventors have made intensive efforts to develop a composition derived from a natural plant extract, which is effective in the treatment of inflammatory diseases, which does not cause side effects while utilizing a garlic stand recognized as a waste resource. As a result, generation inhibitory ability, and inflammatory cytokines (inflammatory cytokines) TNF-α ( tumor necrosis factor-α), IL-1β (interleukin-1β), IL-6 (interleukin-6), prostasin Cloud Dean E2 (PGE 2) and inflammatory And has the ability to inhibit the production of mediators iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2), thus completing the present invention.
The main object of the present invention is to provide a cosmetic composition for preventing and improving inflammatory diseases containing an extract of garlic as an active ingredient.
It is still another object of the present invention to provide a pharmaceutical composition for treating or preventing an inflammatory disease containing an extract of garlic as an active ingredient.
Another object of the present invention is to provide a health functional food for preventing and improving inflammatory diseases containing an extract of garlic as an active ingredient.
In order to solve the above problems, the present invention provides a cosmetic composition for prevention and improvement of inflammatory diseases containing an extract of garlic as an active ingredient.
At this time, the extract may be crude extract, polar solvent-soluble extract or nonpolar solvent-soluble extract of garlic stand.
Preferably, the crude extract is an extract which is soluble in water, including water, methanol, ethanol, butanol or a mixed solvent thereof, and the polar solvent-soluble extract is a solvent selected from water, ethanol, butanol, , And the non-polar solvent-soluble extract is preferably an extract soluble in hexane, chloroform, dichloromethane or ethyl acetate.
In particular, the garlic extract of the present invention is more preferably extracted using water, C1-C4 lower alcohol or a mixture thereof as a solvent, and most preferably a hot-water extract or an ethanol extract.
The garlic extract may be contained in an amount of 0.1 to 50% by weight based on the total weight of the composition of the present invention. Preferably, the garlic extract is used at a concentration of 90-110 g / mL, most preferably about 100 g / mL.
The inflammatory disease may be selected from the group consisting of dermatitis, allergy, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia ), Psoriasis arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases. In particular, it exhibits a beneficial effect on dermatitis or atopy.
In addition, the present invention provides, as another embodiment, a pharmaceutical composition for preventing and treating inflammatory diseases containing an extract of garlic as an active ingredient.
The present invention also provides a health functional food for preventing and improving an inflammatory disease containing an extract of garlic as an active ingredient.
Thus, the present invention provides an antioxidant and anti-inflammatory function of garlic extract, more specifically inhibiting the production of NO and inhibiting the production of inflammatory cytokines TNF-α, IL-1β, IL-6, PGE 2 and inflammatory mediators iNOS, COX-2 Production inhibiting ability.
The composition containing the extract of garlic extract according to the present invention has excellent anti-inflammatory and anti-asthmatic activity and has almost no cytotoxicity. Therefore, the composition for preventing and treating inflammation related diseases, allergy and asthma, cosmetics, Food, food additive, functional beverage, or beverage additive.
In addition, by utilizing the garlic stand recognized as a waste resource, it can be used as a basic data for discovering as a functional material by raising the utilization value of the agricultural by-product garlic stand.
1 is a graph showing the electron donating ability of garlic to hot water extract (ASSW) and ethanol extract (ASSE).
2 is a graph showing ABTS + radical scavenging activity of garlic to hot water extract (ASSW) and ethanol extract (ASSE).
FIG. 3 is a graph showing the effect of the garlic to hot water extract (ASSW) and ethanol extract (ASSE) on the viability of mouse macrophage RAW 264.7 cells.
4 is a graph showing inhibitory effects of garlic to hot water extract (ASSW) (A) and ethanol extract (ASSE) (B) on NO production in RAW 264.7 cells.
FIG. 5 is a graph showing the effect of garlic to hot water (ASSW) and ethanol extract (ASSE) on the production of LPS-stimulated cytokines [A: IL-6, B: IL-1 ?, C: TNF? , D: PGE 2 , ASSW (a), ASSE (b)].
Figure 6 is a graph showing the effect of garlic to hot-water extract (ASSW) on iNOS and COX-2 protein levels in RAW 264.7 cells.
7 is a graph showing the effect of garlic versus ethanol extract (ASSE) at iNOS and COX-2 protein levels in RAW 264.7 cells.
The terms used in the present invention are defined as follows.
"Inflammation" refers to the pathological condition of abscesses formed by the infiltration of external infectious agents (bacteria, fungi, viruses, various kinds of allergens).
"Extract" is a crude extract of garlic, a polar solvent-soluble extract or a non-polar solvent-soluble extract.
The crude extract is a solvent selected from water containing purified water, a lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol, and butanol, or a mixed solvent thereof, preferably a water and ethanol mixed solvent, more preferably 50 to 100% Includes extracts soluble in ethanol.
The "polar solvent-soluble extract" includes extracts which are soluble in water, methanol, butanol or a solvent mixture thereof, preferably water or butanol, more preferably butanol.
"Non-polar solvent-soluble extract" includes extracts which are soluble in hexane, chloroform, dichloromethane or ethyl acetate, preferably hexane, dichloromethane or ethyl acetate, more preferably in hexane or ethyl acetate solvents.
"Pharmaceutical composition" means a mixture of other chemical ingredients such as a garlic extract and a diluent or carrier of the present invention.
"Carrier" is defined as a compound that facilitates the addition of a compound into a cell or tissue. For example, dimethylsulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into cells or tissues of an organism.
A "diluent" is defined as a compound that not only stabilizes the biologically active form of the compound of interest, but also dilutes it in the water in which it is dissolved. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, since it mimics the salt state of the human solution. Since buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compounds.
"Subject" or "patient" means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included.
"Tissue or cell sample" refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any blood components; It may be a cell at any point in the pregnancy or development of the subject. Tissue samples can also be primary or cultured cells or cell lines.
An "effective amount" is an appropriate amount that affects a beneficial or desired clinical or biochemical outcome. An effective amount may be administered one or more times. For purposes of the present invention, an effective amount is an amount sufficient to temporarily alleviate, ameliorate, stabilize, reverse, slow down or slow the progression of a disease state. If the recipient animal is capable of enduring the administration of the composition, or the administration of the composition to the animal is suitable, the composition will be "pharmaceutically or physiologically acceptable ". If the dose administered is physiologically significant, it can be said that the formulation is administered in a "therapeutically effective amount ". The formulation is physiologically relevant if the presence of the formulation results in a physiologically detectable change in the recipient.
The term "treating ", unless otherwise indicated, refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder . As used herein, the term " treatment " refers to an act of treating when " treating " is defined as above.
"Health functional food" means a food having improved functionality of a general food by adding the garlic extract of the present invention to the general food. When the extract of the present invention is added to a general food, the physical properties and physiological functions of the general food will be improved, and the present invention can be broadly classified into 'health function Food ".
All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.
Hereinafter, the present invention will be described in detail.
The present invention relates to the use of garlic vinegar extracts and relates to the specific physiological activity and function exhibited by the garlic vinegar extract.
Garlic ( Allium sativum L.) are widely grown in the lilies (Lilliaceae) Allium (as Ann crops belonging to Allium) is said to Central Asia and the Mediterranean, including the provinces and our country, China, India, South America and Europe. Garlic extracts are known to be effective in the treatment of cardiovascular diseases such as hypercholesterolemia, diabetes, and thrombosis. It also has antibacterial, antioxidant and anticarcinogenic effects.
In order to grow garlic, it is necessary to remove the garlic stand from the beginning of May, but if it is not removed at this time, the bulb will not grow and the stem will be edible. As a byproduct of garlic farming, garlic stands are used for pickling at the early stage of harvest, but most of them are disposed of in garlic fields. As a result of the increase in the amount of garlic cultivation, the waste by-product, garlic, is increased. However, the research conducted to suggest the use of garlic was not enough for the production of bread with garlic.
That is, there have been some reports of antimicrobial and antioxidant effects on garlic itself, but it was first discovered by the present inventors that the antioxidative and anti-inflammatory functions of garlic bands, which are recognized as waste resources, .
The garlic extract of the present invention can be produced by a method known in the art, a modified method thereof, or a method according to the present invention.
As one specific example, it can be produced by the following method.
i) extracting the garlic strip by adding an extraction solvent;
ii) filtering the extract of step i); And
iii) Concentrating the filtered extract of step ii) under reduced pressure and drying.
The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, it is preferable to use a C1 to C4 lower alcohol, and it is more preferable to use ethanol as the lower alcohol.
As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of the dried garlic. The extraction temperature is preferably 30 to 100 DEG C, but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, but is not limited thereto. In addition, the extraction number is preferably 3 to 5 times, more preferably 3 times, but not limited thereto.
In this method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step iii), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.
As a specific example, the garlic extract or crude extract of the present invention may be prepared by mixing water, methanol, ethanol (ethanol) containing purified water of about 1 to 30 times volume, preferably 5 to 15 times volume (w / v% , Butanol and the like, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 50 to 100% ethanol, at a temperature of about 0 to 100 ° C, The extract is extracted with hot water extraction method such as cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, or heat extraction method at room temperature for 10 to 60 hours, preferably 30 to 50 hours, followed by filtration and concentration under reduced pressure The garlic extract of the present invention can be obtained.
In another embodiment, the polar solvent or non-polar solvent soluble extract of the present invention has a volume of about 1 to 150 times, preferably 5 to 100 times the volume of the crude extract, preferably 50 to 100% ethanol crude extract, / v%), and then hexane, ethyl acetate and butanol are added to the reaction solution in the order of 1 to 10 times, preferably 1 to 5 times, preferably 1 to 5 times, preferably 2 to 4 times And fractionated to obtain the polar solvent and nonpolar solvent soluble extract of the present invention.
In one embodiment of the present invention, a hot-water extract and an ethanol extract of a garlic stand were obtained.
Therefore, the present invention includes a method for producing the garlic extract.
For example, the non-exemplified extraction method according to the present invention can be used in a wide range of applications in the art. And can be successfully performed by a person skilled in the art.
As a person skilled in the art to which the present invention belongs, specific reaction conditions for the production of the garlic extract according to the present invention can be confirmed through examples which will be described later, and a detailed description thereof will be omitted.
The present invention relates to various antioxidant and / or anti-inflammatory functions of the above-prepared garlic extract.
The following specific functions were confirmed by observing the effects of the hot water extract (ASSW) and the 70% ethanol extract (ASSE) on Raw 264.7 cells induced by LPS with the LPS through the embodiments of the present invention.
(i) The garlic extract of the present invention contains a phenolic compound as an active ingredient.
Phenolic compounds are known to exhibit various physiological activities such as antioxidant, anti-cancer, anti-inflammatory and antiallergic functions since they have a phenolic hydroxyl and thus easily bind to proteins and other macromolecules. In addition, these phenolic compounds are known to exhibit anti-inflammatory properties by regulating the enzyme activity as well as the enzyme activity involved in the inflammatory reaction. Natural antioxidants such as β-carotenem vitamin E and vitamin C, trace minerals such as Se, and phenolic compounds such as vitamin C and phenol-based compounds are known to be actively exploring natural antioxidants in everyday ingesting edible plants. And their anticancer effects are also reported to be due to antioxidant ability.
In one embodiment of the present invention, the polyphenol content of the garlic to hot water extract (ASSW) is 37.08 ± 1.51 mg (TAE) / g and the polyphenol content of the ethanol extract (ASSE) is 44.7 ± 1.32 mg (TAE) / g .
(ii) The garlic extract of the present invention has antioxidative ability with excellent electron donating ability.
DPPH (1,1-diphenyl-2-picrylhydrazyl), widely used for antioxidant effects, has a stable free radical property by sharing extra electrons in the molecule. When DPPH solution is mixed with other substances, Is lost.
In the DPPH and ABTS + experiments of the present invention, the electron donating ability was increased in both the ASSW and ASSE of the garlic. In the case of DPPH, the antioxidant activity was confirmed to be half of that of Vit. C in the control group at 1,000 μg / mL. In the case of ABTS +, the effect of ASSW and ASSE was similar to that of vitamin C. And antioxidant ability.
(iii) The garlic extract of the present invention has excellent NO production inhibiting ability without exhibiting cytotoxicity.
In one embodiment of the present invention, it was confirmed that the MTT assay did not have cytotoxicity, and furthermore, the NO secretion level was significantly decreased from 25 μg / mL in both ASSW and ASSE. Especially, at the concentration of 100 μg / mL, the inhibitory effect was about 18% and 23%.
(iv) The garlic extract of the present invention inhibits the secretion of inflammatory cytokines.
The majority of skin-related diseases in the body are accompanied by inflammation due to oxidative stress, which promotes aging, further aggravates skin conditions, and inflammatory cytokines such as IL-1β, TNF-α and IL-6 .
PGE 2 is also an important inflammatory mediator that is mainly involved in the transmission of pain and fever in damaged areas and tissues, as well as NO. It is synthesized by COX-2 and when overproduced, it causes excessive immune response and causes multiple sclerosis, Parkinson's disease, Alzheimer's disease, Inflammatory diseases such as cancer.
IL-6 is a typical inflammatory cytokine that is synthesized and secreted by various factors and plays an important role in the development and progression of acute or chronic inflammatory diseases. TNF-α induces neutrophils to the site at the initial stage of inflammatory reaction, IL-1β activates macrophages, aggravates endothelial cell adhesion of lymphocytes and neutrophils, induces the production of chemokines and raises infiltration of inflammatory cells into inflammatory sites.
The garlic extract of the present invention inhibits the secretion amount of the inflammatory cytokines IL-6, TNF-α, IL-1β and PGE 2 of the macrophages in a concentration-dependent manner (Example 6). Particularly, in one embodiment of the present invention, about 55% and 60% reduction was observed at a concentration of 100 μg / mL of ASSW and ASSW against PGE 2 .
(v) The garlic extract of the present invention also inhibits iNOS and COX-2 production.
It is an inorganic vitreous substance produced from L-arginine by iNOS (Inducible Nitric Oxide Synthase). It plays an important role in regulating physiological functions such as neurotransmission, vasodilation, and immune response in a normal state. It is known that it increases permeability and causes edema and deepening of inflammation to cause various cell and tissue damage, resulting in chronic inflammatory diseases and autoimmune diseases. in iNOS expression genes by intracellular Ca 2 + concentrations and bacterial toxin or inflammation and immune response, ischemia, tissue damage, several types of cytokines that are free due to oxidative stress regardless (IL-1β, TNF-α, etc.) Is induced at the transcriptional stage, and thus, when NO is released in a large quantity, it may be involved as an important factor in the pathophysiology of the disease.
COX-2 is involved in blood coagulation, renal function, angiogenesis, and immune responses, as well as inflammatory responses. When inflammatory cytokines are secreted, COX-2 is activated. In addition, COX-2 induces the production of prostaglandin in a large amount compared to normal cells in inflammatory and malignant tumor tissues, promotes angiogenesis, promotes cell proliferation and suppresses immune function, , Suggesting that the expression of COX-2 is directly related to the pathogenicity of another disease.
It was confirmed that the expression level of iNOS was remarkably suppressed at a concentration of 100 μg / mL of the extract of the present invention, especially ASSE, and the concentration of COX-2 was decreased in a concentration-dependent manner, and particularly 50 μg / mL and 100 μg / mL And the inhibition of protein expression was confirmed in the sections.
Thus, the extract of garlic extract inhibits the production of NO and TNF-α which are rapidly increased by inflammation induction, inhibits the expression of iNOS and COX-2, and inhibits IL-1β, IL-6 and PGE 2 And has a function of significantly reducing the amount of cytokine.
Therefore, in one aspect, the present invention relates to a cosmetic composition for preventing and improving an inflammatory disease containing an extract of garlic as an active ingredient.
The term "inflammatory disease" as used herein refers to allergic diseases such as dermatitis, allergies, atopy, asthma, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, inflammatory bowel disease, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder inflammation, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases . Preferably it is dermatitis, allergy, atopy, and most preferably atopic.
The prophylactic and ameliorative effect of the inflammatory disease defined herein is characterized by inhibiting NO production and acting through the activity of inhibiting the production of TNF-α, IL-6, PGE 2, and IL-1β.
The cosmetic composition of the present invention may contain an acceptable carrier in cosmetic preparations. Herein, "an acceptable carrier for a cosmetic preparation" is a known or used compound or composition that can be contained in a cosmetic preparation, or a compound or composition to be developed in the future, which is toxic, instable or irritating It says nothing.
The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 90% by weight to about 99.99% by weight of the composition, based on the total weight thereof. However, since the ratio varies depending on the formulations described below in which the cosmetic composition of the present invention is prepared, and on the specific application site (face, neck, etc.) of the cosmetic composition and its preferable application amount, And should not be construed as limiting the scope of the invention.
Examples of the carrier include an alcohol, an oil, a surfactant, a fatty acid, a silicone oil, a wetting agent, a moisturizer, a viscous modifier, an emulsifier, a stabilizer, an ultraviolet scattering agent, an ultraviolet absorber, a coloring agent and a perfume. The compounds / compositions which can be used as the above-mentioned alcohol, oil, surfactant, fatty acid, silicone oil, humectant, humectant, viscous modifier, emulsifier, stabilizer, ultraviolet scattering agent, ultraviolet absorber, The person skilled in the art can select and use the appropriate substance / composition.
The cosmetic composition according to the present invention can be prepared in various forms such as lotion, essence, gel, emulsion, lotion, cream (water-in-oil type, water-in-water type, multiphase), solution, suspension (Soft capsules, hard capsules) with a coating such as anhydrous products (oil and glycol), gel, mask, pack, powder, or gelatin.
The composition for external application for skin may be prepared in the form of a general emulsified formulation and a solubilized formulation, in addition to the preparation method specifically disclosed in the present invention, using a conventionally known production method.
When the cosmetic composition is manufactured from a cosmetic composition, the cosmetic composition of the emulsified formulation includes nutritional lotion, cream, essence and the like. It can also be prepared in the form of adjuvants which can be applied topically or systemically, which are conventionally used in the field of dermatology by containing a dermatologically acceptable medium or base.
In addition, suitable cosmetic formulations include, for example, solutions, gels, solid or kneaded anhydrous products, emulsions obtained by dispersing an oil phase in water, suspensions, microemulsions, microcapsules, microgranules or ionic forms (liposomes) May be provided in the form of a follicular dispersing agent of the type, cream, skin, lotion, powder, ointment, spray or conical stick. It can also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant.
The present invention further relates to a method of preparing a composition for the prevention and / or treatment of cancer, comprising the step of administering an effective amount of at least one compound selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, foaming agents, Cosmetics or dermatology such as metal ion sequestrants and chelating agents, preservatives, vitamins, sunscreens, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredient commonly used in cosmetics May contain adjuvants commonly used in the art. And, the above ingredients can be introduced in amounts commonly used in the field of dermatology.
In addition, the present invention relates to a pharmaceutical composition for treating or preventing an inflammatory disease containing, as an active ingredient, a garlic extract as an active ingredient. The present invention also provides a method for treating and preventing an inflammatory disease comprising administering the pharmaceutical composition to an individual suffering from an inflammatory disease.
The pharmaceutical composition for prevention and treatment of inflammatory diseases containing the garlic extract of the present invention preferably contains the above extract in an amount of 0.1 to 50% by weight based on the total weight of the composition. Preferably, the garlic extract is used at a concentration of 90-110 g / mL, most preferably about 100 g / mL.
The pharmaceutical compositions containing the garlic extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.
In addition, the composition containing the garlic extract of the present invention can be used in combination with or in combination with medicines such as steroidal drugs, antihistamines, antiinflammatory agents and antibiotics already used.
The pharmaceutical composition containing the extract of garlic extract according to the present invention may be formulated into oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And the like.
Examples of carriers, excipients and diluents that may be contained in the composition containing the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil have.
In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.
Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose , Gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .
Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.
The amount of the composition of the present invention may vary depending on the age, sex and body weight of the patient, but may be 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, once or several times a day. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.
The pharmaceutical composition may be administered to mammals such as rats, mice, livestock, humans, and the like in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.
The pharmaceutical dosage forms of the compositions of the present invention may also be used in the form of their pharmaceutically acceptable salts and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.
On the other hand, the present invention relates to a health functional food for preventing and improving an inflammatory disease containing the garlic extract as an active ingredient from another viewpoint.
Functionality can be categorized into physical properties and physiological functions. When the garlic extract of the present invention is added to a general food, the physical properties and physiological functions of the general food will be improved. For example, the functional food for preventing and alleviating inflammation, allergy, atopy or asthma can be produced by using the anti-asthmatic, anti-allergic, anti-atopic or rhinitis-suppressing effect of the garlic extract of the present invention. In addition, functional foods and the like can be produced using the same.
That is, the compound containing a garlic extract of the present invention or a pharmaceutically acceptable salt thereof can be used as a main ingredient or an additive and an adjuvant of foods in the production of various functional foods and health functional foods.
Examples of foods to which this can be added include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, tea, vitamin complexes, and health functional foods.
In one embodiment, the extract of the present invention can be added to foods or beverages for the purpose of preventing asthma or allergic diseases. At this time, the amount of the extract in food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and 0.02 to 30 g of 100% Can be added at a ratio of 0.3 to 10 g.
The health beverage composition of the present invention may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient, as long as it contains the extract as an essential ingredient at the indicated ratio, and there is no particular limitation to the liquid ingredient. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.
In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
Further, since the composition containing the garlic extract of the present invention is a herbal medicine ingredient, it has little toxicity and side effects and can be safely used for prolonged use for preventive purposes.
Thus, the present invention provides an antioxidant and anti-inflammatory function of garlic extract, more specifically inhibiting the production of NO and inhibiting the production of inflammatory cytokines TNF-α, IL-1β, IL-6, PGE 2 and inflammatory mediators iNOS, COX-2 Production inhibiting ability.
[ Example ]
Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples
Experimental material
1. Materials and Sample Extraction
The garlic stand was purchased from domestic sources and used as a material. The solvent of the hot water extract (ASSW) was extracted three times at 85 ° C for four hours with 10 times the weight of the sample. A 70% ethanol extract (ASSE) was prepared by adding 10 times the weight of the sample and immersing it at room temperature for 24 hours The supernatant and precipitate were separated and extracted three times in the same manner. The extracts were filtered and concentrated, lyophilized and stored in a refrigerated room.
2. Reagents and instruments
(1) Antioxidant activity and anti-inflammatory measuring reagent
(1, 1-diphenyl-2-picrylhydrazyl), K 2 S 2 O 8 (potassium peroxodisulfate), and ABTS (diammonium 2,2'-azino-bis ethylbenzothiazoline-6-sulfonate), tannic acid (C 76 H 52 O 46 ), p-dimethylaminobenzaldehyde, sodium nitrate, griess reagent, ripa buffer, lipopolysaccharide, Protease inhibitors and the like are commercially available from Sigma Chemical Co. Ltd. (Tokyo, Japan), sodium carbonate anhydrate (Na 2 CO 3 ) was purchased from DUKSAN (Kyngkido, KOREA), St. Louis, MO, USA. The primary antibodies iNOS, COX-2, β-actin and secondary antibodies were purchased from Santa Cruz (Biotech, CA, USA), and the ELISA kit for PGE 2 and cytokine measurement was purchased from R & D Systems Inc , USA).
(2) Cells and reagents used for measuring cell viability
The cell line Raw 264.7 used for measuring cell viability was purchased from Korean Cell Line Bank (KCLB). Cell viability was measured by using Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), 0.25% trypsin-EDTA and 0.4% trypan blue stain (Gibco BRL Co, Grand Island, NY, USA) MTT (3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) (Sigma Chemical Co. Ltd., St. Louis, Mo., USA) set, R & D Systems, Minneapolis, MN 55413, USA).
(3) Equipment used in the experiment
The instrument was equipped with a rotary vacuum evaporator (Eyelace-1112, Japan), a centrifuge (Vision, Korea), a freeze drier (Ilsin, Korea), a microscope, a microscope (Olympus optical Co., Japan) , A CO 2 incubator (Hanb aek Scientific Co.), a pH meter (HANNA instruments, Korea), a BOD incubator (Vision scientific Co., Korea), an autoclave (Hanbaek Scientific Co., Korea), an ELISA reader USA) was used.
Experimental method .
1. Cell culture
Raw 264.7 cells used in this experiment were cultured in a DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin (100 U / mL) and subcultured at 37 ° C in a 5% CO 2 incubator.
2. To determine cytotoxicity by MTT assay
Cytotoxicity measurements were performed according to the method of Carmichael (Carmichael J et al., Cancer Res. 47 (4), 936-942). Raw 264.7 cells were seeded in a 96-well plate at a density of 5 × 10 4 cells / well in an amount of 0.18 mL, and 0.02 mL was added to each sample. The cells were cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours.
Garlic at 10, 25, 50, 100, 250, and 400 μg / mL for 24 hours. After adding 0.02 mL of MTT solution prepared at 2.5 mg / mL concentration and incubating for 4 hours, the culture solution was removed, 0.1 mL of DMSO was added to each well, reacted at room temperature for 30 minutes, and absorbance was measured at 540 nm using an ELISA reader .
Cytotoxicity measurement was expressed by the absorbance reduction rate of the sample solution addition group and the no addition group.
3. Measurement of inhibitory activity of NO (nitric oxide)
NO measurements were made as nitrite (nitrite) and nitrate (nitrate) in the supernatant of the cells.
A grease reagent (Sigma, USA), a safe form after being reduced to nitrate for nitrite, was used and 2 x 10 6 cells in 6 well plates were washed twice with 1X PBS at a confluence of 80% And then cultured for 12 hours or longer using serum-free medium. Then, 10 μg / mL of LPS (lipopolysacchride) was added to all wells except for the control group for stimulation. After 2 hours, the samples were treated by concentration. The amount of NO produced after 24 hours was measured by absorbance at 540 nm after collecting the supernatant and reacting with a grease reagent for 10 minutes.
LPS is present in the extracellular membrane of Gram-negative bacteria and increases the levels of various proinflammatory factors such as nitric oxide, inflammatory cytokines and prostaglandin E2 in macrophages or monocytes (Lee et al., Bio. Pharm. Bull. 27 (5), 617, 2004)
4. Measurement of pro-inflammatory cytokine secretion level
Raw 264.7 cells were adjusted to 2 × 10 5 cells / mL and inoculated on 6-well plates and preincubated for 24 hours. Cells were treated with 1 μg / mL of LPS and extracts for 24 hours. The amount of IL-6, TNF-α, IL-1β, and PGE 2 cytokines secreted in the cell culture was measured using an ELISA kit.
5. Measurement of protein expression by Western blotting
The cells were washed twice with 1X PBS, and 10 ml of RIPA buffer was added to 1 ml of complate mini to dissolve in 70 μl, and the mixture was centrifuged at 4 ° C and 12,000 rpm for 15 minutes. The supernatant obtained by centrifugation was quantified by Bradford assay and 20 μL of protein was separated by electrophoresis on 10% SDS-PAGE.
The separated proteins were transferred to a PVDF membrane using a Powerpac HC instrument (BIO RAD, USA) and incubated in blocking buffer (5% skim milk in TBST) for 30 minutes at room temperature. The primary antibody was diluted and reacted for 2 hours. The membrane was further washed with TBST three times at intervals of 10 minutes, and the membrane was diluted to 1: 1,000 with each of the HRP-polymerized secondary antibodies and allowed to react for 60 minutes. After washing three times, bands were identified and quantified using a Davinch-k western imaging system instrument.
Example 1: Determine total polyphenol content
First, the polyphenol contents of the garlic to hot water extract and the ethanol extract of the present invention were determined. Tannic acid was designated as a reference material, and a standard curve and a polyphenol content of the garlic strip were measured and shown in Table 1.
Thus, 37.08 mg / g of garlic to hot water extract (ASSW) and 44.7 mg / g of 70% ethanol extract (ASSE) were obtained.
Example 2 : Electron donating ability Confirm
The inventors measured the antioxidant function at the ratio of DPPH remaining through the ability of free radical scavenging activity at an absorbance of 517 nm.
As a result, the DPPH radical scavenging activity of the garlic extract of the present invention was as shown in Fig.
ASSW and ASSE, respectively. The concentration of ASSW and ASSE were increased to 50% and 50%, respectively. The electron donating ability was slightly higher in ASSE than in ASSW.
From these results, it was found that the antioxidative effect of the garlic extract of the present invention can be expected.
Example 3: ABTS + Radical Verify erase activity
ABTS [Diammonium 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate)] radical ion has been used as one of the experiments to measure the antioxidant activity of extracts using the wavelength band. It has been reported that ABTS + coloration of blue / green is obtained through the reaction of potassium persulfate and the maximum absorption rate at 645 nm, 734 nm and 815 nm wavelength band.
The ABTS + electron donating ability of the garlic to hot water extraction and 70% ethanol extract of the present invention was measured by the above-described method.
As a result, as shown in FIG. 2, the activity increased in a concentration-dependent manner in all the extracts, and 96.85% in ASSW and 97.79% in ASSE at 1,000 μg / mL. However, Vit. C of the control group showed ABTS + electron donating ability of 99.70% at 1,000 μg / mL.
Thus, it was confirmed that the activity of the garlic extract of the present invention was comparable to that of the control Vit.C at a maximum concentration of 1,000 μg / mL. Thus, it was confirmed that the ABTS + scavenging ability of the garlic extract of the present invention was excellent.
Example 4: Macrophages ( Raw 264.7) survival rate
The survival rate of macrocytotoxicity of garlic extract was investigated by MTT assay.
As a result, as shown in Fig. 3, the cell growth rate of garlic to hot-water extract (ASSW) was not affected to the cell viability up to 100 μg / mL, but the cell growth rate was decreased in a concentration-dependent manner from 250 μg / mL or more.
In addition, 70% ethanol extract (ASSE) also had no effect on cell viability up to 100 μg / mL, but the viability was suppressed depending on the treatment concentration.
Based on these results, the concentration was set at 100 μg / mL for comparison of the anti-inflammatory activity of the garlic extract of the present invention.
Example 5: NO Inhibition activity measurement result
LPS induced Raw 264.7 cells were treated with garlic extract (5, 10, 25, 50, 100 μg / mL) to determine the amount of NO in the supernatant.
As a result, as shown in FIG. 4, the amount of secretion was significantly decreased from 25 μg / mL in both ASSW and ASSE, and 18% and 23% of inhibition was observed at a concentration of 100 μg / mL.
Example 6: IL -6, IL -1β, TNF -α, PGE 2 Production inhibitory effect
The effect of the garlic extract of the present invention on the amount of inflammatory cytokine secretion of macrophages was confirmed.
As a result, as shown in Fig. 5, the garlic to ASSW (a) and ASSE (b) of the present invention significantly reduced the production of inflammatory cytokines IL-1β, IL-6 and TNF-α. In particular, IL-1β and IL-6 showed a significant decrease at concentrations of 100 μg / mL, 100 μg / mL and 100 μg / mL, respectively.
The expression level of PGE 2 was remarkably decreased at a concentration of 100 g / mL of ASSW, and the expression level of ASSE was decreased in a dose dependent manner, indicating an inhibition rate of 33.87% at 100 g / mL (D in FIG. 5).
The inhibitory effect of ASSW on the production of inflammatory cytokines was higher than that of ASSW. However, both extracts showed activity and differentiation as well as migration of different inflammatory cells.
Example 7: iNOS And COX -2 generation inhibitory effect
Western blotting was performed to examine the effect of the present invention on the expression of iNOS and COX-2 protein in garlic extract-ASSW, ASSE.
As a result, the expression of iNOS and COX-2 in the hot-water extract (ASSW) increased by LPS treatment was constant up to the concentration range of 100 μg / mL (FIG. 6)
In addition, the expression of iNOS in the extract of 70% EtOH was significantly inhibited at the concentration of 100 μg / mL, and the expression of COX-2 was decreased in a concentration-dependent manner. Especially at the concentration of 50 μg / mL and 100 μg / mL (Fig. 7).
These results indicate that the garlic-ethanol extract of the present invention exhibits a slightly higher anti-inflammatory effect, but the garlic extract of the present invention has antioxidative and anti-inflammatory effects. It could be used for the treatment and prevention of inflammatory diseases.
As described above, in the extracts of the perforated corals and garlic, which are anticipated to be similar to each other, the inhibition of NO production, inhibition of IL-6 production, inhibition of PGE2 generation, and inhibition of MDC formation, The extracts of the present invention are more effective in the treatment and prevention of inflammatory diseases.
Claims (12)
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| KR20210046408A (en) * | 2019-10-18 | 2021-04-28 | 선문대학교 산학협력단 | compositions containing by-product extract of Allium sativum L. |
| KR20220018310A (en) * | 2020-08-06 | 2022-02-15 | 선문대학교 산학협력단 | Compositions containing black garlic extract |
| KR102647912B1 (en) * | 2023-11-13 | 2024-03-14 | 이형도 | Oral composition useful for preventing and treating gum disease, including natural fermented extract as an active ingredient |
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| CN107929460A (en) * | 2017-12-01 | 2018-04-20 | 广西玉林市和兴农业科技有限公司 | Treat the Chinese medicine composition of rhinitis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20210046408A (en) * | 2019-10-18 | 2021-04-28 | 선문대학교 산학협력단 | compositions containing by-product extract of Allium sativum L. |
| KR20220018310A (en) * | 2020-08-06 | 2022-02-15 | 선문대학교 산학협력단 | Compositions containing black garlic extract |
| KR102647912B1 (en) * | 2023-11-13 | 2024-03-14 | 이형도 | Oral composition useful for preventing and treating gum disease, including natural fermented extract as an active ingredient |
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