TWI399209B - Use of porcine lung extract as metrix metalloproteinase inhibitor - Google Patents

Description

Use of pig lung extract as matrix metalloproteinase inhibitor

The present invention relates to the novel use of porcine lung extract as a matrix metalloproteinase (MMP) inhibitor, particularly for inhibiting MMP-1, MMP-2 or MMP-9.

Metalloproteinases are supergroups of proteases (enzymes), the number of which has increased dramatically in recent years. Based on structural and functional considerations, these enzymes have been classified into several ethnic groups and sub-populations. Examples of metalloproteinases include matrix metalloproteinases (MMPs) such as collagenase (MMP-1, MMP-8, MMP-13), gelatinase (MMP-2, MMP-9), and matrix lysin (MMP-3, MMP10, MMP-11), interstitial lysin (MMP-7), metallo-elastase (MMP-12), enamel lysin (MMP-19), MT-MMP (MMP-14, MMP-15, MMP-16) , MMP-17); a lysin or enamel lysin or MDC group, including secretases and efflux enzymes, such as TNF-converting enzymes (ADAM10 and TACE): a group of astaxanthin, including some enzymes, such as procollagen treatment Protease (PCP); and other metalloproteinases, such as aggregating proenzymes, endothelin converting enzyme populations and angiotensin converting enzyme populations. Among them, MMP is known to be associated with various diseases. For example, when MMP is involved in angiogenesis or cancer metastasis in cancer tissues, its expression level will increase or enzymes will be activated. In addition, for ulcer formation, chronic articular rheumatism, osteoporosis, Decomposition of the extracellular matrix in various conditions such as periodontal disease also plays a role. Furthermore, MMP, which is activated by external stimuli such as ultraviolet rays, will maintain the decomposition of important components of the skin structure. Recently, it has been particularly valued because it is an aging promoting factor activated by ultraviolet rays, especially MMP-1. MMP-2 and MMP-9 are particularly valued for two reasons. One is that the substrate of these MMPs is an important structural component of the skin, and the other is that the long-term exposure of the skin to factors that stimulate the pathological state of these MMPs. For example, inflammatory reactions, oxidative stress, and ultraviolet light. Therefore, it is currently believed that selective inhibition of MMP-1, MMP-2 and MMP-9 is more beneficial and effective than inhibiting all metalloproteinases in slowing skin aging.

MMP-1 is a commonly known collagen degrading enzyme. In order to regulate important factors of photoaging, the ultraviolet exposure of fibroblasts to sunlight causes a large amount of matrix metalloproteinases to be expressed, thereby degrading the extracellular matrix (ECM), that is, MMP- 1 is responsible for collagen degradation of dermal tissue.

The physiological importance of the action of MMP-2 (de-enzyme 72-kDa) has also been confirmed. Although whether MMP-2 plays a direct role in the decomposition of type I collagen has not been finalized, type I collagen has been confirmed as one of its glycolysis, and type IV collagen (basement membrane), type VII Collagen (dermis-skinned anchor microfibers), gelatin (deteriorated type I collagen), elastin and fibrin are also the yeasts of MMP-2. As the age of the body increases, the amount of enzyme activity in the skin of MMP-2 increases. Therefore, MMP-2 also plays an important role in the shrinkage of the extracellular matrix.

MMP-9 (gelatinase B; 92 kDa type IV collagenase; 92 kDa gelatinase) is a secreted protein which was first purified in 1989 and then vegetatively propagated and sequenced. The performance of MMP-9 is normally limited to a few cell types, including germ layer, osteoclasts, neutrophils, and macrophages. However, its performance can be induced by several mediators in such identical cells, and in other cell types, including exposure of such cells to growth factors or cytokines. It is the same mediator that is often associated with triggering an inflammatory response. Like other secreted MMPs, MMP-9 is released as an inactive zymogen, which then divides to form an enzyme-active enzyme. The protease required for this activation in vivo is unknown. The balance of active MMP9 to inactive enzymes is further regulated in vivo by interaction with a naturally occurring protein TIMP-1 (a tissue inhibitor of metalloproteinase-1). The TIMP-1 line binds to the C-terminal region of MMP-9, resulting in inhibition of the catalytic site of MMP-9. The balance of ProMMP-9 induced behavior, the division of ProMMP-9 into active MMP9, and the presence of TIMP-1 are combined to determine the amount of catalytically active MMP-9 present at a localized location. MMP-9 with proteolytic activity attacks the host, including gelatin, elastin, and native type IV and type V collagen; it is not active against native type I collagen, proteoglycan or laminbumin. There is a growing body of data that is associated with the role of MMP-9 in different physiology and pathology processes. Physiological roles include the invasion of the embryonic germ layer through the uterine epithelium in the early stages of embryo transfer; a role in bone growth and development; and the inflammatory cells that migrate from the blood vessels to the tissue. MMP-9 release measured using enzyme immunoassay was significantly improved in fluids, and in AM supernatants from untreated asthma patients, as compared to those from other individual populations. Increased MMP-9 performance has also been found in certain other pathological conditions, thus linking MMP-9 to disease processes such as COPD, arthritis, tumor metastasis, Alzheimer's disease, multiple sclerosis And plaque rupture in atherosclerosis, leading to acute coronary symptoms, such as myocardial infarction.

Due to the safety of natural products, there is still a need to develop natural products that inhibit MMP.

Prior to the present invention, there was no report or prior art indicating or speculating that pig lung extract has the effect of inhibiting matrix metalloproteinases, whereas in the present invention, it has been unexpectedly found that pig lung extract is effective in inhibiting matrix metalloproteinase activity. By inhibiting the activity, particularly the activity of MMP-1, MMP-2 or/and MMP-9, the decomposition of elastin is reduced, thereby improving/slowing/preventing skin aging.

It is an object of the present invention to provide a use of a pig lung extract for the preparation of a composition for inhibiting matrix metalloproteinases.

Unless otherwise defined herein, the scientific terms used in connection with the present invention shall have the meaning as commonly understood by one of ordinary skill in the art. The meaning and scope of the term should be clear; however, if there is any potential ambiguity, the definitions provided herein take precedence over the definition of any dictionary or external source. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

The term "porcine lung extract" means an extract obtained by extracting pig lungs, wherein the pig lungs are not limited to fresh or frozen pig lungs. In a preferred embodiment, the method for extracting pig lung extract of the present invention is (a) adding the buffer to the pH of about 6.0-9.0 after the pig lung is minced; (b) adding the enzyme to the enzyme in step (a) Solution, after enzymatic hydrolysis, the enzyme is inactivated; (c) adding acid solution to acid precipitation in step (b), collecting the filtrate by filtration, and (d) adding an organic solvent to the filtrate, collecting the precipitate to obtain pig lung Extracts.

In a preferred embodiment, the pig lung system is removed by removing the tracheal portion. In a preferred embodiment, the pH in step (a) of the present invention is about 6.0-8.0, 6.5-8.0, 6.0-7.5, or about 7.0. In a preferred embodiment, the buffer used in step (a) of the present invention includes, but is not limited to, sodium citrate. In a preferred embodiment, the enzyme used in step (b) of the present invention includes, but is not limited to, pineapple enzyme or protease M or a combination thereof. In a preferred embodiment, the acid used in step (c) of the present invention is a strong acid including, but not limited to, hydrochloric acid, perchloric acid, and trifluoroacetic acid. In a preferred embodiment, the organic solution used in step (d) of the present invention includes, but is not limited to, methanol, ethanol or isopropanol, more preferably ethanol, preferably food grade ethanol.

In a preferred embodiment of the present invention, the pig lung extract of the present invention inhibits MMP activity, and the MMP is preferably MMP-1, MMP-2 or MMP-9. In another preferred embodiment, the pig lung extract of the present invention inhibits the activity of MMP-1, MMP-2 and MMP-9 simultaneously.

In another aspect, the porcine lung extract of the present invention can improve, slow or prevent skin aging, particularly skin aging, by inhibiting the activity of MMP.

The term "preventing" means preventing or preventing the onset of symptoms, or improving or reverting to the symptoms that have already occurred.

The term "aging of the skin" includes such signs of aging including, but not limited to, skin fragility; loss of collagen or elastin; loss of estrogen balance in the skin; skin atrophy; appearance or depth of lines or wrinkles, including fine lines; Discoloration, including dark circles; sagging skin; skin fatigue or stress, such as skin protrusion due to environmental stress such as pollution or temperature changes; dry skin; exfoliation of the skin; cell aging; loss of skin tension, elasticity or gloss; Loss of skin fastness; coarse cortical; loss of skin elasticity or rebound resilience.

Benefits and improvements in skin aesthetics can be demonstrated by any reduction in pore size; improvement in skin tone, radiance, transparency or tension; promotion of antioxidant activity; skin fastness, swelling, flexibility or Improvement of softness; improvement of procollagen or collagen production; promotion of skin texture or re-deformation; improvement of skin barrier repair or function: improvement of skin contour appearance; repair of skin gloss or brightness; Supplementation of essential nutrients or components that are reduced by aging or menstruation; improvement in connectivity in skin cells; increase in cell proliferation or reproduction; increase in metabolism of skin cells due to aging or menopause; Improvement in wetting; promotion or acceleration of cell turnover; enhancement of skin thickness; increase in skin elasticity or resilience; and enhancement of shedding.

In another preferred embodiment, the pig lung extract of the present invention, wherein the composition further comprises a carrier required to form the composition.

The term "carrier" or "carrier acceptable in cosmetics" means a diluent, excipient or the like which is well known to those skilled in the art.

The composition of the present invention may further comprise an active ingredient which is useful for preventing skin aging, such as vitamin A acid, vitamin A alcohol, arbutin, hyaluronic acid, epidermal growth factor and other natural whitening ingredients. The composition of the present invention may further comprise a conventional cosmetic active agent, such as other conventional low pigmentation agents, such as hydroquinone, ascorbic acid or licorice extract; anti-inflammatory agents; anti-acne agents, such as salicylic acid; exfoliating agents, such as alpha - hydroxy acid, beta-hydroxy acid, keto acid, oxyacid or oxyacid; ascorbyl-phosphonium-cholesterol; sunscreen, such as oxybenzophenone, octyl methoxycinnamate, octyl sulphate, eight Alkenene, titanium dioxide, zinc oxide, butyl methoxy benzhydryl methane, methylene bis-benzotriazolyl tetramethyl butyl phenol (MBBT); or an anti-aging agent; or any combination thereof.

In another preferred embodiment, the compositions of the present invention comprise a final concentration of about 50 μ g / mL to 500 μ g / mL in lung extract. Preferably, the compositions of the present invention comprises about 200 μ g / mL to 400 μ g / mL in lung extract. Best system comprising about 300 μ g / mL in lung extract.

The pig lung extract of the present invention can be used as it is, or can be concentrated, diluted, filtered, etc. as needed, and decolorized and deodorized with activated carbon or the like. Further, the extracted solution is subjected to a treatment such as concentration drying, spray drying, freeze drying, or the like, and may be used in the form of a dried product.

The pig lung extract of the present invention can be applied to medicines, quasi-drugs, cosmetics or foods for preventing, inhibiting or improving symptoms of skin aging, and can use the above-mentioned extracts as they are, or without damaging the extracts. , ingredients used in common pharmaceuticals, quasi-drugs, cosmetics or foods: excipients, stabilizers, preservatives, binders, disintegrating agents, hydrocarbons, fatty acids, alcohols, esters, surfactants, Metal soap, pH adjuster, preservative, perfume, moisturizer, powder, UV absorber, tackifier, pigment, antioxidant, whitening agent, chelating agent, grease or wax.

The pig lung extract of the present invention may be in the form of a powder, a pill, a tablet, an injection, a suppository, an emulsion, a capsule, a granule, a liquid (including an expectorant, a fluid elixirs, an alcohol, a suspension, a lemonade). Etc.), lotion, cream, lotion, gel, spray, body cream, detergent, bath, base, powder, lipstick, ointment, moisturizing cloth, paste, plaster, essential oil, candy Ingots or drinks, etc.

The following examples are not to be construed as limiting the invention in any way. Modifications and variations of the embodiments discussed herein may be made without departing from the spirit and scope of the invention, and still fall within the scope of the invention.

Example 1: Extraction method of pig lung extract

The frozen pig lungs were thawed, washed with water and then removed. The trachea was removed and cut into pieces of about 10 cm in size. The meat grinder was ground to a non-bulk structure and added to the pig lung 2 times the weight of the extract (0.02 M citric acid). Sodium pH 7.0±0.2), mix well with a blender, and weigh 5% (W/W) pineapple enzyme (2000 GDU/g, ST BIO) and 0.1% (w/w) protease M from the lungs of minced pigs. Enzyme (5500 untis / g, Amano), stir well with a mixer, react in a water bath at 50 ° C for 3 hours, after the reaction is completed, the enzyme is inactivated in a water bath at 90 ° C for 10 minutes, and then placed in ice water to cool to 25 ° C, added After stirring 1.5% (W/W) 6N hydrochloric acid, the mixture was allowed to stand at room temperature for 1 hour to carry out an acid precipitation reaction. The above samples were directly subjected to plate filtration using a 1 μm aperture paperboard to collect the filtrate. It was precipitated in 3 volumes of 95% ethanol (food grade) and placed in a cold room at 4 ° C for 12 hours. The precipitate was collected by centrifugation at 8000 (xg) for 30 minutes, and the precipitate was again dissolved in 0.02 M sodium citrate pH 7.0.

Example 2: Toxicity of Porcine Lung Extract to Fibroblasts and Effect of Inhibiting MMP-1

MMP-1 isozyme analysis

1. Cell culture and toxicity analysis

(1) Cell culture: A total volume of 0.1 mL of 1×10 ⁴ WS1 (BCRC 60300) cells was plated in a 96-well plate in a-MEM medium containing 10% FBS, and the supernatant was removed overnight to obtain a sample of known concentration. The serum-containing medium was formulated into 5, 0.125, 0.25, 0.5, 1 mg/mL 5 analytical doses, each of 3 replicates. Then, it was filtered through a 0.2 μm filter membrane, and 0.1 mL was added to the cells. After 24 hrs, the supernatant was removed, and then a serum-free a-MEM medium containing 10 ng/mL TNF- was prepared to be 0, 0.125, 0.25. , 0.5, 1 mg / mL 5 analytical doses each 3 replicates, filtered through a 0.2 μm filter membrane, 0.1 mL was added to the cells, and after 24 hours of treatment, the supernatant was collected and stored at -80 ° C as a follow-up MMP- 1 The same enzyme enzyme map analysis sample.

(2) Toxicity analysis: The cells remaining in the 96-well plate were added to a mixture of 100 mL of serum-containing medium and 20 mL of MTS, and reacted at 37 ° C for 3 hours to analyze cell viability at OD.490/630 nm. The MMP-1 isozyme map was analyzed for the highest concentration with a survival rate higher than 80% (defined as non-toxic).

2. Analysis of the same enzyme map

(1) Preparation of enzyme electrophoresis gel and electrophoresis analysis

The solution was prepared as a 10% SDS-PAGE electrophoresis gel containing 0.2% casein. Take 4 μL of sample dye (200 mM Tris-HCl, pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol) and mix 20 μL of sample (sample dye: sample=1: 5), placed at room temperature for 10 minutes, loaded into the above prepared electrophoresis gel for electrophoresis analysis, to fix 80 volts to the end of electrophoresis.

(2) Synergic enzyme map reaction

The finished film was immersed in Wash buffer I and shaken at room temperature for 60 minutes. Then, it was washed with a washing buffer II (Wash buffer II) and shaken at room temperature for 40 minutes. Finally, it was allowed to react at 37 ° C for 20 hours in a reaction buffer.

(3) Dyeing, defection and strip analysis

The reaction-completed film was placed in a staining solution (0.15% Coomassie Blue R250) for 60 minutes. It was then placed in a de-staining solution (40% methanol, 10% acetic acid) for about 30 minutes and shaken until a clear band appeared. Finally, immerse in RO water for 10 minutes. The optical density values of the transparent strips were quantified using a Bio-Rad image processing system.

From the toxicity test chart of the pig lung extract of Fig. 1A on fibroblasts, even if the concentration of the pig lung extract reaches 400 μg/mL, the cells are not toxic and the cells are killed. As can be seen from Fig. 1B, for the control group, the higher the concentration of the pig lung extract, the more the MMP-1 was inhibited. Figure 1C is a graph showing the inhibition rates of MMP-1 in different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMP-1, while the control group does not have the same Inhibition.

Example 3: Toxicity of Porcine Lung Extract to Fibroblasts and Effect of Inhibiting MMP-2/-9

MMP-2/-9 isozyme analysis

1. Cell culture and toxicity analysis

(1) Cell culture

A total volume of 0.1 mL of 1×10 ⁴ NIH/3T3 (BCRC 60008) cells was seeded in a 96-well plate in DMEM medium containing 10% FBS, and the supernatant was removed overnight to obtain a serum-containing medium. Prepared into 5, 0.125, 0.25, 0.5, 1 mg / mL 5 analytical doses, each 3 replicates, then filtered through a 0.2μm filter membrane, 0.1 mL was added to the cells, after 24 hrs, the supernatant was removed, and then the supernatant was removed. The DMEM medium containing no serum but containing TNF- was prepared into 0, 0.125, 0.25, 0.5, 1 mg/mL, 5 analytical doses, 3 replicates, filtered through a 0.2 μm filter membrane, and 0.1 mL was added to the cells. After 24 hours of treatment, the supernatant was collected and stored at -80 ° C as a sample for subsequent MMP-2/-9 isozyme mapping.

(2) Cytotoxicity assay: The cytotoxicity assay test procedure of the same example.

2. Analysis of the same enzyme map

(1) Preparation of enzyme electrophoresis gel and electrophoresis analysis

A 10% SDS-PAGE electrophoresis gel was prepared by containing a animal glue/casein solution. Take 4 μL of sample dye (200 mM Tris-HCl, pH 6.8, 8% SDS, 0.4% bromophenol blue, 40% glycerol) and mix 20 μL of sample (sample dye: sample = 1:5), set After 10 minutes at room temperature, the electrophoresis gel prepared above was injected for electrophoresis analysis to fix 80 volts to the end of electrophoresis.

(2) Synergic enzyme map reaction

The step is the same as the enzyme reaction of the second embodiment.

(3) Dyeing, defection and strip analysis

The procedure is the same as in the dyeing, de-staining and strip analysis of Example 2.

Figure 2A shows the toxicity test of pig lung extract on fibroblasts. The concentration of pig lung extract is up to 300 μg/mL, which does not cause toxicity to cells and kills cells. It is confirmed that pig lung extract is for cells. safe. As can be seen from Fig. 2B, for the control group, the higher the concentration of the pig lung extract, the more the MMP-2 and MMP-9 were inhibited. Figure 2C shows the inhibition rates of MMP-2 in different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMP-2, while the control group does not have the same Inhibition. Figure 2D is a graph showing the inhibition rates of MMP-9 in different concentrations of pig lung extract and control group, showing that the higher concentration of pig lung extract has the effect of inhibiting MMP-9, while the control group does not have the same Inhibition.

Fig. 1A is a graph showing the toxicity test of pig lung extract on fibroblasts; Fig. 1B and C are colloidal electrophoresis patterns and graphs of various concentrations of pig lung extracts for inhibiting MMP-1 activity.

Figure 2A is a graph showing the toxicity test of pig lung extract on fibroblasts; Figures 2B to 2D are colloidal electropherograms and graphs showing inhibition of MMP-2 and/or MMP-9 activity by various concentrations of pig lung extract.

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Claims (16)

A use of a pig lung extract for preparing a composition for inhibiting matrix metalloproteinase (MMP) activity, wherein the pig lung extract is obtained by the following steps: (a) adding the buffer to the pig lung after crushing Adjusting the pH value of about 6.0-9.0; (b) adding enzyme to the step (a) to keep the enzyme hydrolyzed, and then enzymatically heat up to inactivate the enzyme; (c) adding the acid solution to acid precipitation in step (b), filtering The filtrate was collected, and (d) an organic solvent was added to the filtrate, and the precipitate was collected to obtain a pig lung extract. The use of claim 1, wherein the pig lung of the step (a) is fresh or frozen pig lung. The use according to claim 1, wherein the pH of the step (a) is about 6.0 to 8.0. The use according to claim 1, wherein the buffer in the step (a) is sodium citrate. The use according to claim 1, wherein the enzyme in the step (b) is a pineapple enzyme or a protease M or a combination thereof. The use according to claim 1, wherein the temperature of the heat retention in the step (b) is about 50 degrees C. The use of claim 1, wherein the temperature rise temperature in the step (b) is about 90 degrees C. The use according to claim 1, wherein the acid in the step (c) is hydrochloric acid, perchloric acid or trifluoroacetic acid. The use according to claim 1, wherein the organic solvent in the step (d) is methanol, ethanol or isopropanol. The use of claim 1, wherein the MMP is MMP-1, MMP-2 or MMP-9. The use according to claim 1, wherein the pig lung extract system simultaneously inhibits MMP-1, MMP-2 and MMP-9. The use of claim 1, wherein the composition that inhibits matrix metalloproteinase (MMP) activity improves, slows or prevents skin aging. The use of claim 1 wherein the composition further comprises a carrier which is required to form the composition. The use of claim 1, wherein the extract is present in the composition in an amount from about 50 μg/mL to 500 μg/mL. The use according to claim 1, wherein the composition is a pharmaceutical, a cosmetic, or a food. A pig lung extract obtained by extracting the following steps: (a) adding the buffer to the pig lung to adjust the pH to about 6.0-9.0; (b) adding the enzyme to the step (a) to keep the enzyme hydrolyzed, the enzyme After the solution, the temperature is increased to inactivate the enzyme; (c) after the acid solution is added to the acid precipitation in the step (b), the filtrate is collected by filtration, and (d) An organic solvent is added to the filtrate, and the precipitate is collected to obtain a pig lung extract.