KR101420267B1 - Pharmaceutical composition for preventing and treating inflammation comprising polyamine as effective component - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating inflammation containing polyamine as an active ingredient.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating inflammation comprising polyamine as an active ingredient,

The present invention relates to a pharmaceutical composition for preventing and treating inflammation containing polyamine as an active ingredient.

Inflammation is a plasma-induced dynamic process and cell event that responds to an infection by pathogens, harmful substances such as chemicals and tissue damage. The inflammatory process is necessary for immune surveillance, optimal repair and post-injury regeneration. However, persistent, excessive or inappropriate inflammation is the cause of a number of diseases including rheumatoid arthritis, psoriasis and inflammatory bowel disease. It is also the primary causative agent for diseases such as cancer, diabetes and cardiovascular disease (Lucas et al., 2006 British Journal of Pharmacology 147, 232-240). However, chronic inflammation is an important contributing factor in the prevalence and mortality rate.

Locally, inflammation occurs in the classic form of swelling, redness, fever, and often pain. Common causes of inflammation Providers are on one side of a wide range of adverse conditions that lead to inflammation, which triggers white blood cell recruitment and induction or activation of inflammatory mediators such as quinine, cyclooxygenase products and cytokines. Many of these molecules are made locally and have been demonstrated to be associated with tissue inflammation and are thus a major target for therapeutic intervention in a variety of diseases (Medzhitov, R., 2008, Origin and Physiological Roles of Inflammation. Nature 454 , 428-435).

High levels of inflammatory cytokines, reactive oxygen species, and nitric oxide have been suggested to contribute to pathophysiological mechanisms associated with a variety of inflammatory skin disorders. It has been widely recognized that skin inflammation is produced and maintained by the interaction of various inflammatory cell populations that migrate to the site of inflammation in response to the release of soluble pro-inflammatory mediators such as cytokines, prostaglandins and leukotrienes (Lee et al., 2003 Toxicol, Appl. Pharmacol., 192, 294-306).

Clinical treatment of inflammatory diseases depends on drugs belonging to the non-steroidal or steroidal chemical family (Yonathan et al., 2006 Journal of Ethnopharmacology 108, 462-470). However, steroids can disrupt numerous cytokine networks that are accompanied by lymphocyte function, resulting in immunosuppression, and long-term topical use can reduce collagen synthesis, leading to skin atrophy . Because of these risks, new therapeutic approaches have been intensively studied.

The ongoing search for potentially effective and safe anti-inflammatory drugs can not be overemphasized. The use of herbal extracts and nutritional supplements as alternative or adjunctive medicines for conventional chemotherapy for the treatment of inflammatory diseases has been well documented in Ayurveda, an alternative medicine that has been practiced predominantly in the Indian continent for 5,000 years System (Rao et al., 2006 Indian Journal of Pharmacology 38, 346-349).

Polyamines (PA) are biogenic polyvalent cation amines present in all eukaryotic cells with multifunctional characteristics during growth and play a substantial role in the regulation of cell proliferation and differentiation. They electrostatically interact with multivalent anionic macromolecules such as DNA and RNA, and proteins regulate the action of different components of the cell membrane and regulate intracellular messenger systems such as protein-kinase (C. Loeser, 2000 Br J. Nutr 84 (Suppl 1) (2000) S55-S58). At present, the daily uptake of polyamines necessary for human growth and development is well known. PA is involved in various processes of cell growth and cell differentiation, but the precise function of PA in mammalian cells has not been elucidated yet. PA is also an important regulator of immune function and has some anti-inflammatory and immunosuppressive activity. SPM (spermine) has evolved to limit excessive production of macrophage-induced inflammatory mediators. SPM inhibits LPS-induced nitric oxide release in the murine macrophage cell line J774.2 and inhibits TNF-a synthesis in LPS-activated human peripheral blood mononuclear cells (PBMC) and murine macrophage-like cell line RAW 264.7. SPM regulates the production of IL-12 and IL-10 in mouse splenocytes and inhibits the release of IFN-y. It has also been reported that SPD (spermidine) regulates the production of TNF-α and MCP-1 in NR8383 macrophages (Francisco et al., 2003 Cellular Immunology 86-94). However, the effectiveness of polyamines for antiinflammation and macrophage activation has not yet been thoroughly studied.

Korean Patent Laid-Open Publication No. 2003-0070085 discloses a drug comprising polyamine as an active substance, and Korean Patent Publication No. 2002-0080691 discloses a sugar-specific peptide having strong binding force and a method for producing the same.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and the present inventors have found that the treatment of three polyamine (Put, Spd, Spm) concentrates reduces the ear and foot edema of the mouse and also prevents the development of LPS (lipopolysaccharide) stimulating mouse macrophage- Confirming that it inhibits the production of inflammatory cytokines (TNF-α and IL-1β).

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing and treating inflammation containing polyamine as an active ingredient.

According to the present invention, since the pharmaceutical composition of the present invention has an anti-inflammatory activity effect, it can be very usefully used in the functional food, cosmetics and pharmaceutical industry related to the development of a preventive and therapeutic agent for inflammation.

Figure 1 shows the efficacy of polyamines on carrageenan (Carr) induced hind paw edema. 50 μl of 1% Carr was injected 30 minutes after the injection (ip) of putrescine, sppermin, spermine (1, 2.5 and 5 mg / kg) and indomethacin (5 mg / kg) Intravenous injection. Data were expressed as mean ± SD (n = 3). Values with different shoulders (a and b) are significant ( p <0.05).
Figure 2 shows the efficacy of polyamines and indomethacin on tissue MDA (A) and NO (B) concentrations of the foot fluids in mice. Data were expressed as means ± SD (n = 3). Values with different shoulders (a and b) are significant ( p <0.05).
Figure 3 shows the efficacy of polyamines on TPA-induced ear thickness and water content in BLAB / c mice. Mice were pretreated with the indicated concentrations of polyamine and 1% hydrocortisone (HC) for 30 min, and TPA (4 mM) was applied to induce skin inflammation. After 4 h, the increase in ear thickness (A) and skin water content (B) was measured. Each column represents the mean ± SD (n = 3). Values with different shoulders (a and b) are significant ( p <0.05).
Figure 4 shows the epithelial, dermal and cartilaginous layers of the histological flakes of the mouse ear skin biopsy. All pictures were taken at 100x magnification, except for 4B and D, which were shot at 40x magnification. (A) untreated; (B) TPA; (C) HC; (DF) phentresin, 50, 100, 250 [mu] g / ml; (GI) spumidine, 50, 100, 250 占 퐂 / ml; (JL) spermine, 50, 100, 250 占 퐂 / ml.
Figure 5 shows the area measurements for evaluating the thickness of the epithelium and dermis. Data are expressed as mean SD (n = 10). Values with different shoulders (a and b) are significant ( p <0.05).
Figure 6 shows the efficacy of polyamines on IL-1? (A) and TNF-? (B) concentrations of the cell supernatants of Raw 264.7 macrophages induced by bacterial LPS. Data are expressed as mean SD (n = 10). Values with different shoulders (a and b) are significant ( p <0.05).

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing and treating inflammation containing polyamine as an active ingredient.

In the pharmaceutical composition for preventing and treating inflammation according to an embodiment of the present invention, the polyamine may be putrescine, spermidine or spermine, but is not limited thereto.

The pharmaceutical compositions containing the polyamines of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The pharmaceutical dosage forms of the pharmaceutical compositions of the present invention may 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.

The pharmaceutical compositions containing polyamines according to the present invention can be prepared in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions And can be used as formulations. Examples of carriers, excipients and diluents that can be included in the polyamine-containing composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. 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, sucrose), 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 administration 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. The base of suppositories may be witepsol, macrogol, tween, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In addition, the present invention provides a health functional food comprising a food-acceptable food-aid additive exhibiting an effect of preventing or improving inflammation. Examples of foods to which the pharmaceutical composition can be added include various foods, beverages, gums, tea, vitamin complexes, and health functional foods.

It can also be added to foods or beverages for the purpose of preventing or improving inflammation. The amount of the pharmaceutical composition in the food or drink may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, .

The health functional beverage composition of the present invention is not particularly limited to the other components other than those containing the pharmaceutical composition as an essential ingredient at the indicated ratio and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tautatin, stevia extract, for example, 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, the pharmaceutical composition of the present invention may also be used as a cosmetic preparation, a preservative, a preservative, a preservative, a preservative, a preservative, a preservative, a preservative, A salt thereof, an organic acid, a protective colloid thickener, a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like. In addition, the pharmaceutical compositions of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These ingredients may be used independently or in combination, and 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 pharmaceutical composition of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

One. Chemicals

12-O-tetradecanoylphorbol-13-acetate (TPA), hydrocortisone, 1,1-dihydroxypyrrolidine, 3,3-tetramethoxypropane, thiobarbituric acid, trichloroacetic acid, sulfanilamide, naphthylethylenediamine dihydrochloride and phosphoric acid were purchased from Sigma-Aldrich (St. Louis, Mo., USA).

2. Experimental animals

Five-week-old female BALB / c mice (18-20 g) were purchased from Dong Yang Bio Co., Ltd. (Seoul, Korea). Animals were placed in a polypropylene cage (3 mice per mouse) and maintained in a freely accessible standard laboratory diet and water. They stayed at least 12 hours before the experiment a week in a place where they were air-conditioned with light and dark cycles. Room temperature (about 23 ° C) and humidity (about 60%) were automatically adjusted. All experimental procedures were carried out in accordance with the Daegu University Guide for Care and Use of Laboratory Animals.

3. λ- Carrageenan  ( Carr ) -Induced foot edema

Carr-induced hind paw edema model was used to measure anti-inflammatory activity (Chang et al., 2009 Evidence-Based Complementary and Alternative Medicine, doi: 10.1093 / ecam / nep027). Animals were randomly assigned to 12 groups (n = 3). Controls were given normal saline (intraperitoneal; ip .). The other seven groups were Carr-treated, Positive Control (Carr + Indomethacin), Putrescine (Carr + PUT: 1, 2 and 5 mg / kg), Spurmidine (Carr + SPM: 1, 2 and 5 mg / kg), and animals were injected 30 minutes before injection of 1% Carr (50 [mu] L) (1, 2.5 and 5 mg / kg), ip with indomethacin or normal saline. The paw thickness was measured with a pocket thickness gauge (Mitotoyo, Japan) at intervals of 1, 2, 3 and 4 h immediately after the Carr injection and after the administration of the edematogenic agent in the range of 0 to 9 mm, Graded at intervals and measured. The induced swelling was evaluated as ab (where a is the volume of the right hind paw after Carr treatment and b is the contraceptive of the right hind paw prior to Carr treatment). Indomethacin (Indo) was used as a positive control. After 4 h, the animals were sacrificed, the Carr-induced edema was cut and the right hind paw tissue was taken at the calcane bone level at 4 hours. The right hind paw tissue was rinsed with ice-cold normal saline, immediately placed in cold normal saline, which was four times its volume, and homogenized at 4 ° C. The homogenate was then centrifuged at 12,000 x g for 5 minutes. The supernatant was obtained and stored in a -20 &lt; 0 &gt; C refrigerator for MDA assay and NO measurement.

4. Whole protein assay

The protein concentration of the samples was measured by a Bradford dye-binding assay (Bio-Rad, Hercules, Calif.).

5. MDA  black

MDA from Carr-inducible edema foot has some variations on the thiovarbitalic acid reactant (TRARS) method according to Chang et al., 2009 Evidence-Based Complementary and Alternative Medicine, doi: 10.1093 / ecam / nep027 Was measured. Briefly, the tissue supernatant was mixed with two volumes of cold 10% TCA to precipitate the protein. The remaining supernatant was reacted with 0.67% thiobarbituric acid in a heat bath for 10 minutes. MDA reacted with thiobarbituric acid at high temperature of acid and formed red-complex TBARS. The absorbance of TBARS was measured at 532 nm. 1,1,3,3-tetramethoxypropane was used for standard curve production.

6. Measurement of nitric oxide / nitrite

The nitrite levels in the baths, reflecting intracellular nitric oxide synthase activity, were measured by the Griess reaction. The stock solution was mixed with an equal volume of Griess reagent (1% sulfapanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) and incubated at room temperature for 10 minutes. A standard curve was made using sodium nitrite and the concentration of nitrite was measured from the absorbance at 540 nm.

7. In the mouse TPA - Test for inducible inflammation

12-O-Tetradecanoylphorbol-13-acetate (TPA) causes skin irritation and consequently increases ear thickness and skin water content in BALB / c mice. TPA was dissolved in acetone: olive oil (4: 1) and used as skin inflammation inducer. A volume of 10 [mu] l was provided on both the inner and outer surfaces of the ear to induce skin inflammation. 10 μl of sample solution and normal saline as a control was applied topically 30 minutes before TPA treatment. Hydrocortisone (HC), which is currently used in the treatment of various inflammatory skin diseases, was used as a positive control. Ear thickness was measured with a pocket thickness gauge (Mitotoyo, Japan) ranging from 0 to 9 mm, graded at 0.01 mm intervals, and measurements were made at the center of the ear. Ear thickness was measured before treatment (a) and 4 h after TPA treatment (b = TPA alone; b '= TPA and samples). The following values were then calculated:

TPA alone induced edema A (b - a);

TPA and sample - induced edema B (b '- a).

After ear thickness measurements, the animals were anesthetized, 6 mm ² diameter ear punch biopsies were collected, individually weighed in a Mettler-Toledo (AE-163) electronic balance, and the weight was determined as the Wet weight. The ears were dried in a drying oven for 24 h, weighed again, and the weight was determined as dry weight. The skin water content was calculated by subtracting the dry weight from the wet weight and dividing it by the dry weight, expressed as H ₂ O / mg dry weight.

8. Morphological analysis of mouse ear tissue

For morphological evaluation of skin inflammation, the treated ear and control derived biopsies of the mice in each treatment group were collected, fixed in 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4), and calcined . The fixed tissue was cut into a series of thin layers with a thickness of 5.0 쨉 m using a microtome (LEICA RM 2135, Nussloch, Germany). The flakes were stained with Harry's hematoxylin-eosin and their lengths were measured using an optical microscope and representative areas were selected for quantitative optical microscopic analysis of cellular mediated inflammatory response.

9. Cell culture

The mouse macrophage cell line RAW 264.7 (ATCC, TIB-71) was purchased from the American Type Culture Collection (ATCC) (USA). Cells were 10% fetal bovine serum (FBS, Sigma, USA) supplemented with a Dulbecco's Modified Eagle Medium CO ₂ at 37 ℃ in plastic containers containing (Gibco-BRL, Gaithersburg, MD , USA) Cultured in an incubator (5% CO _{2 in} air) and diluted 1: 5 with 0.05% trypsin-0.02% EDTA in phosphate-buffered saline (DPBS) free of Ca ^{2 +} -, Mg ^{2 +} Subcultured.

For the experiments, cells were cultured in 12-well plates at 1 x ¹⁰ cells / well. Treated for 4 h with freshly prepared PUT (5, 10, 25 μM), SPM (5, 10, 25 μM) and SPD (5, 10, 25 μM) diluted in sterile saline 10 ng / ml LPS (Sigma, St. Louis, USA) for 3 h. The cells were collected using a scraper and centrifuged (600 g x 5 min). The supernatants for TNF [alpha] and IL-1 [beta] measurements were frozen at -80 [deg.] C until ELISA assay.

10. For cytokine measurement ELISA

Levels of TNF [alpha] and IL-1 [beta] in cell supernatants were measured by sandwich ELISA using polystyrene micro ELISA plates and TNF [alpha] and IL-1 [beta] ELISA kits (Invitrogen, Frederick, MD, USA). Monoclonal anti-mouse TNF [alpha] / IL-1 [beta] antibodies were used as capture and specific biotin conjugates were added as secondary antibodies. Recombinant mouse TNF? / IL-1? Was used as a protein standard. These ELISA techniques allowed a minimum detection value of 16 pg / mL for both cytokine kits. The assay was performed according to the manufacturer's instructions. Absorbance was measured on an ELISA plate detector (Bio-Tek instrument co., WA, USA). TNF-α and IL-1β results were expressed as percentage of secretion provided by LPS-stimulated untreated macrophages.

11. Statistical analysis

Data were presented as mean ± SD (n = 3) and analyzed using the Statistical Analysis System (SPSS, Version 11) ANOVA procedure. Significant differences between the treatment averages were determined using Duncan's multiple-interval test. The significance of the difference was set at p <0.05 level.

Example  1. γ- Carrageenan  ( Carr ) -Induced edema

The anti-inflammatory activity of polyamines was tested using? -Carrageenan (Carr) -induced edema. Indomethacin significantly inhibited the development of Carr-induced foot edema 3 h after treatment, but spermine (5 mg / kg, 5 mg / kg) and spermidine (2.5 mg / mg / kg) and spumidine (5 mg / kg) were higher than those of the positive control group. Putrescine (5 mg / kg) also suppressed swelling after 4 h of treatment (Fig. 1).

Example  2. MDA  ( malondialdehyde For level measurement Polyamine  effectiveness

MDA levels in the edema feet induced by Carr were significantly higher. However, MDA levels were significantly lowered at 5 mg / kg indomethacin treatment as well as polyamine (Fig. 2). Polyamine treatment (1, 2.5 and 5 mg / kg) significantly inhibited Carr-induced MDA levels compared to the Carr group. Spermidine (2.5 and 5 mg / kg), spermine (2.5 mg / kg) reduced MDA levels as well as indomethacin, while spermine (5 mg / kg) decreased more than the positive control. Putrescine (5 mg / kg) moderately inhibited MDA production (Figure 2A).

Example  3. NO  Suppression of production

The antiinflammatory activity of polyamines was studied in vivo by analyzing their inhibitory effectiveness against chemical mediators released into the body fluids. Poutresin, spermidine, and spermine (2.5, 5 mg / kg) resulted in a significant reduction in liquid NO levels and were similar to the control group. Sulfamine (5 mg / kg) showed the highest activity among the tested compounds, even better than the control. The levels of NO in carrageenan, indomethacin, putrescine, spermidine and spermine (2.5, 5 mg / kg) were 22.36 ± 1.0, 2.74 ± 1.0, 5.63 ± 1.0, 4.08 ± 0.078, 4.17 ± 0.078 and 3.47 0.078, 3.81 ± 0.078, and 1.44 ± 0.078 μM (FIG. 2B).

Example  4. TPA  Inductive edema

To investigate whether polyamines could reduce inflammation in the skin, a TPA-induced skin inflammation model was attempted to assess the potential anti-inflammatory efficacy of different concentrations of polyamines topically applied in vivo. Increased skin thickness is often the first characteristic of skin irritation and local inflammation. The parameters are markers of a number of processes that occur during skin inflammation, including increased vascular permeability, intradermal edema and swelling, and proliferation of epithelial keratinocytes. Exposure of TPA to the mouse ear resulted in a noticeable increase in both skin thickness (Figure 3A) and skin water content (Figure 3B) in BALB / c mice.

Figure 3A shows that TPA (4 mM) results in a substantial increase in ear thickness, and topical application of polyamines significantly inhibits such increase in ear thickness. In the ear thickness measurement, a nearly 10-fold increase was observed for the TPA-inducible ear (0.63 ± 0.05 mm) compared to the untreated mouse (0.06 ± 0.01 mm). Treatment with puromycin (250 μg / ml), spupride (250 μg / ml) and spermine (100 μg / ml, 250 μg / ml) resulted in 0.33 ± 0.02, 0.31 ± 0.01, 0.33 ± 0.01 and 0.21 ± Lt; / RTI &gt; and 0.03 mm, respectively. The efficacy of spumidine (250 [mu] g / ml) and spermine (250 [mu] g / ml) on the TPA-induced ear was also comparable to that of 1% HC (0.31 0.1 mm) used as a positive control. The increase in TPA-induced skin water content was also inhibited by polyamine treatment in a concentration dependent manner (Figure 3B). Treatment with spumidine (250 [mu] g / ml) and spermine (250 [mu] g / ml) resulted in a reduction in water content of about 50 and 53%, respectively, in the TPA-induced skin inflammation model. However, treatment with hydrocortisone resulted in a 42% reduction in water content in the TPA-induced ear. Putrecine (250 [mu] g / ml) also resulted in a 37% reduction in skin water content. As a result shown in Figures 3A and 3B, the inhibitory effectiveness of polyamines on TPA-induced skin inflammation was better than that of 1% HC.

Example  5. Your organization  Shape examination

H & E-stained ear flakes from TPA-inducible animals were investigated. TPA application resulted in a noticeable increase in ear thickness with clear evidence of swelling, epidermal hyperplasia, and substantial inflammatory cell invasion in the dermis, accompanied by connective tissue disruption (Fig. 4A, B). By histological comparison, polyamine treatment reduced ear thickness and associated pathologic markers (D-L in FIG. 4C) to an extent comparable to the positive control hydrocortisone (HC).

To further demonstrate the effectiveness of polyamines for TPA-induced skin inflammation, the thickness of the epithelial and dermal regions of mouse ear tissues was measured. As shown in Fig. 5, the thickness of the epithelial region of the sputumine treated (100 / / ml) ear tissue (16.2 賊 2.1 탆 ² ) was the same as that of the control group, and the concentrations of spumidine (250 / / ml) (15.8 ± 2.6 ㎛ ² , 15.7 ± 1.9 ㎛ ² , and 13.1 ± 2.3 ㎛ ² , respectively) of the treated ear tissues were much better than those of the control group. Putrecine (250 ug / ml) also reduced epithelial thickness to a certain level (20.26 ± 2.0 ㎛ ² ). In addition, the thickness of the ear dermal area of the essential oil treated ear provides evidence that spermine exhibits the highest anti-inflammatory activity among all treated polyamine samples (FIG. 5). The thicknesses of the dermal regions for HC and puromycin (250 ㎍ / ml), spupmidin (100 ㎍ / ml, 250 ㎍ / ml) and spermine (100 ㎍ / ml, 250 ㎍ / ml) were 155.6 ± 20.4, 178.7 ± 24.1, 157.2 ± 28.6, 111.53 ± 21.4, 108.86 ± 26.2 and 83.13 ± 22.1.

Example  6. Raw  264.7 In the macrophage IL -1β and TNF for the secretion of -α pole Effectiveness of the riamine

To confirm anti-inflammatory activity of polyamines, IL-1 [beta] and TNF-alpha levels in cells were evaluated using ELISA experiments because IL-1 [beta] and TNF- [alpha] act as the main marker molecules following stimulation with LPS. As expected, pre-treatment with sppermin and suffermin (10, 25 [mu] M) significantly inhibited IL-1 [beta] and TNF- [alpha] expression in Raw 264.7 macrophages in rats and inhibition efficacy was not significant at low concentrations 6A, 6B). Furthermore, ELISA experiments revealed that both IL-1 [beta] and TNF-a production were inhibited in a dose-dependent manner by all polyamines. However, putrescine was not as active as spermidine and spermine in inhibiting cytokine production. In contrast, lower concentrations of spermine (5 [mu] M) also significantly inhibited TNF-a production (Fig. 6B). These results indicate that the production of cytokines may depend on the amino group present in the backbone of the particular polyamine.

Claims (2)

A pharmaceutical composition for preventing and treating 12-O-tetradecanoylphorbol-13-acetate (TPA) -induced skin inflammation containing 50 to 250 占 퐂 / ml of spermine as an active ingredient. delete

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