KR101065020B1 - Composition for treating burn comprising kaempferol - Google Patents

Abstract

The present invention relates to a composition for burn treatment comprising kaempferol as an active ingredient.

Burns, camphor roll

Description

Composition for treating burn containing camphor roll as active ingredient {Composition for treating burn comprising kaempferol}

The present invention relates to a composition for treating burns, comprising camphor roll as an active ingredient.

The present invention is derived from a study conducted as part of the Health and Medical Technology R & D Project Hospital Specialization Research Center Project of the Ministry of Health, Welfare and Family Affairs.

In general, flavonoids are the most common polyphenolic compounds present in nature. The basic structure of polyphenol-based pravonoids has a diphenylpropane backbone including single prabonol, prabonol, flavonone, and flavone (Chen, CC., Et al., Mol. Pharmacol . 66: 683- 693, 2004; Lotito, SB. And Frei, B., J. Biol. Chem. 281: 37102-37110, 2006). Various flavonoids have various biological functions such as antioxidant function, anti-inflammatory function and inhibitory effect of cellular signaling protein (Comalada, M., et al., Biochem. Pharmacol. 72: 1010-1021, 2006; Fang, SH et al., Bioorg. Med. Chem. 13: 2381-2388, 2005). The various biological functions of these flavonoids have attracted attention because they can be used as therapeutic agents for cancer, cardiovascular diseases, autoimmune diseases, infectious diseases (Kempuraj, D., et al., Br. J. Pharmacol. 145: 934-944, 2005; Pang, JL, et al., Biochem.Pharmacol . 71: 818-826, 2006). However, the exact mechanism of action that represents the biological function of flavonoids has not been elucidated.

TNF-α is a cytokine associated with inflammatory responses produced in activated macrophages, leaflets, endothelial cells, mast cells, etc. (Bradley, JR. J. Pathol. 214: 149-160, 2008; Rahman, MM and McFadden, G , PLoS Pathog . 2: 66-77, 2006). Overproduction of TNF-α is closely related to the development of allergic diseases such as rheumatoid arthritis, psoriasis, Crohn's disease, and asthma (Kim, HJ., Et al., Exp. Mol. Med . 40: 167-175, 2008 Muppidi, JR, et al., 4: 461-465, 2004; Palladino, MA et al., Nat. Rev. Drug Discov . 9: 736-746, 2003). TNF-α was found to activate the transcription factor NF-κB through signaling through the TNF-α receptor (Zhang, G., Curr. Opin. Struct. Biol. 2: 154-160, 2004; Karin, M and Greten FG., Nat. Rev. Immunol. 10: 749-759, 2005). The use of antioxidants flavonoids to inhibit the activation of NF-κB by TNF-α (Park, JS., Et al., J. Agric. Food Chem. 54: 2951-2956, 2006) Samhan-Arias, AK et al., Free Radic. Biol. Med. 37: 48-61, 2004). However, the mechanism of flavonoids that inhibit the activation of NF-κB has not yet been accurately identified.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a flavonoid for burn treatment.

In order to achieve the above object, the present invention provides a composition for treating or preventing burns comprising camphor roll as an active ingredient.

In one embodiment of the invention, the effective amount of the camphor roll is a cell non-toxic burn treatment composition, characterized in that 1 to 200μg / kg body weight. As can be seen from Example 4 of the present invention and the a) of FIG. 4, the effective amount is a value obtained by converting the range without cytotoxicity (about 10 to 200 μM) through the experiment into μg per kg body weight.

The compositions of the present invention can be administered by methods known to those skilled in the art. Dosage forms include, but are not limited to, enteral (oral) administration, extra enteral (intravenous, subcutaneous and intramuscular) administration and topical use. The method of treatment according to the invention comprises a method of internally or locally administering to a patient in need thereof in an amount effective to treat a camphorol obtained from a single or complex feedstock such as a composite, natural product or combination thereof. .

The present invention includes an improved method for separating and purifying camphorol obtained from plants containing the compounds according to the invention. The method of the present invention includes a method of extracting an extract from a plant containing camphorol; b) neutralizing and concentrating the extract extracted by the above method; And c) a method for purifying the neutralized and concentrated extract. In a preferred embodiment of the invention the extract is purified using a method selected from the group consisting of recrystallization, precipitation, solvent partition separation and / or chromatography separation. The present invention provides a commercially viable method for the separation and purification of camphorol with desirable physiological activity.

As used herein, the term "treatment" includes treatment and prevention. When used in animals as well as humans means "treatment".

"Pharmaceutically or therapeutically effective dose or amount means a dosage sufficient to induce a desired biological result. Such a result may be a reduction in the cause of signs, symptoms or disease or other desired changes in the biological system. Can be.

The manufacture of a product for administration in the form of a pharmaceutical may be carried out by a variety of methods well known to those skilled in the art. Camperol is a herb powder in the form of natural materials; Solvent and / or supercritical fluid extracts of various concentrations; Compounds concentrated and purified through recrystallization, column separation, solvent phase separation, precipitation and other methods; It can be formulated into a monolith and / or mixture containing camphor rolls that have been sufficiently synthesized and prepared by synthesis.

Various delivery systems known in the art can be used to administer the pharmaceutical compositions of the present invention. Examples include aqueous solutions, capsules with liposomes, particulates and microcapsules.

Pharmaceutical administration methods may include effective administration methods such as arterial injection, inhalable spray, orally active formulation, transdermal ionization or suppository, but the pharmaceutical composition of the present invention may be applied to the skin, inhaled spray or It can be administered by parenteral methods by injection. Preferred carriers include physiological saline, but other pharmaceutically acceptable carriers may also be used. As a preferred embodiment,

And carriers and camphorol to form a formulation which is physiologically compatible and slowly released. The main solvent in the carrier may be water soluble or water insoluble in nature. In addition, the carrier may include pharmaceutically acceptable excipients to control or maintain the pH, osmotic pressure, viscosity, transparency, color, sterility, stability, dissolution rate or flavor of the agent. Similarly, the carrier may include pharmaceutically acceptable excipients to control or maintain stability, dissolution rate, release or adsorption of the ligand. Such excipients are substances commonly used to formulate pharmaceuticals for parenteral administration in the form of single or multiple drugs.

Once the therapeutic composition is formulated, it is stored in sterile vials in the form of solutions, suspensions, gels, emulsifiers, solid or dehydrated powders or lyophilized powders; Or in direct encapsulation and / or tablet form with other inert carriers for oral administration. Such formulations may be stored in ready-to-use form or in powder form for use in water before administration. Methods of administering a formulation containing a systemic delivery composition are administered via the intestine, subcutaneous, intramuscular, intravenous, parenteral, vaginal or rectal.

The amount of the composition effective for treating burns of the present invention depends on the extent of the burn or the nature of the abnormal condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used for optimal dosage identification. The exact amount administered in the formulation will depend on the route of administration, the severity or progress of the disease or adverse condition, and will depend upon the circumstances of the physician and patient. Effective dosages are determined by response curves according to dosages derived from ex vivo or animal model test systems.

Can be extrapolated from For example, the effective dosage of the present invention is readily determined by administering the present compositions in graded amounts to observe the desired effect.

The method of treatment according to the present invention comprises administering camphorol alone and / or multiple mixtures obtained from a single raw material or multiple raw materials in or locally to a patient in need of a therapeutically effective amount. The purity of the plurality of camphorol alone and / or mixtures is within the range of 0.01 to 100%, but not limited to, depending on the method used to obtain the compound.

In a preferred embodiment, the dosage of the pharmaceutical composition containing camphorol is effective and nontoxic to administer in an amount selected in the range of 0.001 to 200 mg / kg body weight, preferably 1 to 200 μg / kg body weight. Those skilled in the art using routine clinical tests can determine the optimal dosage depending on the individual disease to be treated.

The composition comprising the camphorol of the invention can also be administered by any suitable method of administration known in the art. Camperol of the present invention may be administered to the upper airway as steam or spray. In addition, the camphorol of the present invention may be administered by aerosolization, nebulization or atomization. example

When the camphorol of the invention is administered by aerosolization, atomization or atomization, it is administered in the form of droplets having a diameter in the range from about 1 micrometer to about 10 micrometers. Methods and devices useful for aerosolization, atomization and atomization are known in the art. For example, medical nebulizers have been used to deliver metered doses of physiologically active liquid into an inhalation stream for inhalation by the administerer (see, eg, US Pat. No. 6,598,602). The medical nebulizer may be operable to produce liquid droplets that form an aerosol with the inhaler. In other circumstances, a medical nebulizer may be used to inject water droplets to supply a gas having a suitable wet content to the administrator, in which the intake stream is a mechanical breathing aid, for example a respirator, a ventilator or an anesthetic delivery system. It is particularly useful when provided by.

Exemplary nebulizers are described in WO 95/01137, which describes a handheld device that is operated to inject droplets of medical liquid into, for example, a passing air stream (inhalation stream) generated by inhalation of the injector through a mouthpiece. have. Other examples provide respiratory control and augmentation for patients with respiratory failure and medicinal liquid particles in the airway and

US Pat. No. 5,388,571 describing a positive pressure ventilator system comprising a nebulizer for delivery into the alveoli of the lung. U.S. Patent 5,312,281 describes an ultrasonic nebulizer capable of atomizing water or liquid at low temperatures and reportedly controlling the size of the mist. In addition, US Pat. No. 5,287,847 describes an aerodynamic spraying facility for delivering medicinal aerosols to newborns, children, and adults, with measurable flow rates and output volumes. In addition, US Pat. No. 5,063,922 describes an ultrasonic atomizer.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

In the present invention, the present inventors studied that the flavonoid camphorol effectively inhibits the expression of IL-8 expressed by TNF-α stimulation in HEK 293 cells. The inventors confirmed that camphorol is not cytotoxic, unlike other flavonoids. Camphorol inhibited the production of free radicals produced by stimulation of TNF-α, and inhibited the degradation of IκBα and the activation of NF-κB. In the present invention, the present inventors confirmed that kampeferol is effective in treating burns by inhibiting the inflammatory response in the burned skin tissue in which the inflammatory response is induced.

The present inventors confirmed that camphorol is not cytotoxic and inhibits the inflammatory response caused by TNF-α among various types of flavonoids, and is treated in burn tissue when camphorol is treated to burn-induced skin tissue. It has been shown to be effective.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples.

However, the following examples are described for the purpose of illustrating the present invention, and the scope of the present invention should not be construed as being limited by the following examples.

Example 1 Analysis of Efficacy of Flavonoids Inhibiting IL-8 Expression by TNF-α

1) Construction of IL-8 Promoter- Luc Reporter Plasmid

To amplify the IL-8 promoter region (-135 bp to +46 bp), human genomic DNA was used as a template and PCR was performed using the following primer sets.

 5 'primer (SEQ ID NO: 1)  5'- GTGAGATCTGAAGTGTGATGACTCAGG-3 ' 3 'primer (SEQ ID NO: 2) 5'- GTGAAGCTTGAAGCTTGTGTGCTCTGC-3 '

Table 1 shows primers for the IL-8 promoter region.

The amplified IL-8 promoter region fragment was inserted into pGL3-Basic plasmid (Promega) digested with Bgl II and Hin dIII. This produced the IL-8 promoter- Lu reporter plasmid (Wu GD et al., J. Biol. Chem ., 272: 2396-2403, 1997).

2) Promoter Activation Assay: Luciferase Activity Assay

HEK 293 cells (ATCC, Rockviller, MID) were dispensed into 12-well plates at a concentration of ⁵ × 10 ⁴ cells / well and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator. The cells were co-transfected with the IL-8 promoter- Lu reporter plasmid prepared in 1) and the pRL-null plasmid (Promega). Then, incubated for 24 hours at 37 ℃, 5% CO ₂ incubator. Flavonoids (10, 20, 40, 80 μM / well) were treated in each well, incubated for 1 hour in a 37 ° C., 5% CO ₂ incubator, treated with TNF-α (5 ng / ml) for 8 hours Incubated for At this time, the control group was treated with PBS. Subsequently, PLB (passive lysis buffer) of the Dual-luciferase reporter assay system (Promega) was added to each well at a concentration of 100 μl / well to disrupt the cells. Luciferase assay was performed using the supernatant (15 μl) obtained by centrifuging the cell lysate. Luciferase activity was measured using a TD-20 / 20 (Turner designs) luminometer. The activity of each promoter following treatment with TNF-α is shown as relative to the control. That is, the activity of the control group is expressed as a fold activation.

3) RT-PCR

After pretreatment of camphorol to HEK 293 cells and TNF-α treatment, expression of IL-8 was confirmed using RT-PCR. Total RNA was isolated from cells using TRIzol (Invitrogen). The total RNA (5 μg) was then treated with M-MLV reverse transcriptase (invitrogen) to prepare cDNA. Thus prepared cDNA as a template and RT-PCR was performed using the following specific primer set of IL-8.

 Forward primer (SEQ ID NO: 3)  5'-ATG ACT TCC AAG CTG GCC GTG GCT-3 ' Reverse primer (SEQ ID NO: 4) 5'-TCT CAG CCC TCT TCA AAA ACT TCT-3 '

Table 2 shows the primer sequences for IL-8.

PCR amplification was carried out for a total of 25 repetitions with a cycle of DNA denaturation at 95 ° C for 30 seconds, primer annealing at 57 ° C for 40 seconds, and extension for 1 minute at 72 ° C. After the PCR reaction was completed, the PCR product amplified on a 1% agarose gel was confirmed.

Example 2: Analysis of Effect of Flavonoids on IκBα Degradation and NF-κB Activation Inhibition by TNF-α

1) Western blotting

It was confirmed by Western blotting that IκBα was degraded by stimulation of TNF-α and IκBα was inhibited by pretreatment of flavonoids. Equal amounts of proteins were separated by 12.5% SDS-polyacrylamide gel electrophoresis and electrotransferred with a polyvinyridine fluoride membrane (Millipore, Bedford, Mass.). The membranes were blocked with tris-buffer saline containing 0.05% Tween 20 and 2% bovine serum albumin at room temperature for 1 hour and incubated with anti-IκBα antibody or anti-phospho-IκBα antibody for 2 hours. Immunoreactive proteins include horseradish peroxidase-coupled secondary antibody (Jackson ImmunoResearch Laborities, Inc. , West Grove, PA) and enhanced chemumiminisense reagent (Amersham Pharmacia Biotech, Piscataway, NJ) Detected by.

2) confocal microscope

It was confirmed through confocal microscopy that NF-κB was activated by stimulation of TNF-α and that activation of NF-κB was inhibited by pretreatment of flavonoids. We previously reported Kwon, HJ, et al. (2003). Biochem. Biophys. Res. Commun. NF-κB p65 was detected using confocal microscopy as described in 311, 129-138. HEK 293 cells (5 × 10 ⁴ ) were cultured directly in glass coverslips in 24-well plates. After 24 hours, flavonoids were pretreated for 1 hour and treated with TNF-α for 30 minutes, and the cells were then fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. To identify cell nuclei, we used DNA staining (0.5 mg / ml Hoechst No. 33258; Sigma, St. Louis, Mo.). Coverslips were mounted in Fluoromount-G (Southern Biotechnology Associates, Inc., Birmingham, AL). Samples were scanned with a Zeiss LSM 510 laser scanning confocal device attached to an Axiovert 100 microscope using Plan-Apochromat 100X / Oil DIC Objective (Carl Zeiss, Germany).

Example 3 Low Efficacy Analysis of Free Radical Production by Flavonoids by TNF-α

It was confirmed through confocal microscopy that free radicals are produced by stimulation of TNF-α and free radicals are inhibited by pretreatment of flavonoids. Intracellular free radicals were measured using an oxidation-sensitive fluorescent probe, dihydrorhodamine 123.

Example 4: MTT assay

HEK 293 cells were inoculated at 1 × 10 ⁴ / well, flavonoids treated with 0-160 μM and incubated at regular intervals for 48 hours with MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium The cell viability was observed by bromide assay. MTT solution (0.2 mg / ml in DMSO) was added to each well and incubated in 37 ° C., 5% CO ₂ incubator for 4 hours. The culture solution was discarded and the formazan crystal blue formed on the cells by MTT reaction was dissolved in DMSO. The amount of formazan was measured at 595 nm using a microplate reader.

Example 5 Analysis of Burn Treatment Efficacy of Camperol

1) Causes burns in mouse

The back of the 6-week-old BALB / c mouse was epilated and left for 24 hours to heal fine wounds on the skin. After anesthesia, a plastic plate was placed on the back of the mouse and the skin was soaked in 100 ° C. water for 8 seconds.

2) skin tissue analysis

Camperol of 100 μg / kg body weight mice was applied per mouse at 12 hour intervals after 1 hour of burn induction. After anesthesia, the mice were anesthetized 5, 10, and 30 days after the induction of burns, and the skin of the burned site was biopsied using a 2 mm punch. After fixing in 4% formalin solution, it was filled with paraffin and cut to a thickness of 4 μm. The tissue was observed by staining with hematoxylin-eosin.

3) RT-PCR

Expression of TNF-α in mouse skin tissues was confirmed using RT-PCR. Total RNA was isolated from cells using TRIzol (Invitrogen). The total RNA (5 μg) was then treated with M-MLV reverse transcriptase (invitrogen) to prepare cDNA. Thus prepared cDNA as a template and RT-PCR was performed using the following specific primer set of TNF-α.

 Forward primer (SEQ ID NO: 5)  5'-TCT CAT CAG TTC TAT GGC CC-3 ' Reverse primer (SEQ ID NO: 6) 5'-GGG AGT AGA CAA GGT ACA AC-3 '

Table 3 shows the primer sequences for TNF-α.

PCR amplification was carried out for a total of 25 repetitions with a cycle of DNA denaturation at 95 ° C for 30 seconds, primer annealing at 57 ° C for 40 seconds, and extension for 1 minute at 72 ° C. After the PCR reaction was completed, the PCR product amplified on a 1% agarose gel was confirmed.

The result of the above example is as follows.

Flavonoids (kaempferol, quercetin, fisetin, chrysin) reduced the production of IL-8 expressed by stimulation of TNF-α. Flavonoids were confirmed through luciferase assay that the concentration-dependent inhibition of promoter activation of IL-8 by TNF-α (Fig. 1a). Inhibition of IL-8 promoter activation by fisetin and chrysin was the highest. In addition, flavonoids were confirmed by RT-PCR that inhibits the mRNA expression of IL-8 by TNF-α (Fig. 1c).

TNF-α stimulates the TNF-α receptor to phosphorylate IκBα to degrade IκBα, and NF-κB is activated and transported to the nucleus. After pretreatment with flavonoids, treatment with TNF-α inhibited the phosphorylation of IκBα and degradation of IκBα (FIG. 2A). In addition, it was observed by confocal microscopy that the activation of NF-κB by TNF-α was inhibited by pretreatment of flavonoids (FIG. 2B). It has been reported that TNF-α activates NF-κB by increasing the production of free radicals. The production of such active oxygen could be confirmed by the use of dihydrorhodamine 123, an oxidation-sensitive fluorescence probe, to inhibit flavonoids (FIG. 3).

As a result of confirming through MTT assay whether the flavonoids used in the present invention cause cytotoxicity, it was observed that quercetin, fisetin, and chrysin inhibit cell survival of HEK 293 in a concentration-dependent manner. Camperol, on the other hand, did not affect the survival of HEK 293 cells (FIG. 4).

When water at 100 ° C. is exposed to the skin of a mouse, a burn occurs in the skin tissue and abnormality of the skin tissue occurs. After burn induction, the inflammatory cells in the dermal layer of skin tissues were found to gather after 10 days. In addition, it was confirmed that after 30 days it is formed thick in the keratinocyte layer. On the other hand, the application of camphorol every 12 μg / kg body weight mice after 12 hours of burn-induced burn inhibited the migration of inflammatory cells to the skin tissue, and the keratinocyte layer was similar to normal mouse skin tissue. (FIG. 5B).

When water at 100 ° C. was exposed to the skin of the mouse, it was confirmed through RT-PCR that TNF-α expression increased after 10 days while the skin tissue was burned. After inducing burns, camphorol was applied to mice at 100 μg / kg body weight every 12 hours. After 10 days, it was confirmed that expression of TNF-α was suppressed in the skin tissue (FIG. 6).

Figure 1a shows the effect of flavonoids on the process of activation of the IL-8 promoter on TNF-α in HEK 293 cells through the luciferase assay. It was confirmed that kaempferol, quercetin, fisetin, and chrysin inhibit the activation of IL-8 promoter by TNF-α in a concentration dependent manner. Figure 1b was confirmed by RT-PCR when the expression of IL-8 increased when TNF-α treatment HEK 293 cells. Figure 1c shows that the expression of IL-8 is affected by flavonoids when HEK 293 cells are pretreated with kaempferol, quercetin, fisetin, and chrysin at 40 μM for 1 hour and treated with TNF-α for the indicated time. RT-PCR It was confirmed through.

2a shows that IκBα is phosphorylated and degraded when TNF-α is treated in HEK 293 cells, and pretreatment of flavonoids inhibits phosphorylation and degradation of IκBα by Western blotting. In addition, in FIG. 2B, NF-κB is activated and moved to the nucleus when TNF-α is treated in HEK 293 cells. Pretreatment of flavonoids inhibits NF-κB activation by confocal microscopy.

3 is confirmed by confocal microscopy using dihydrorhodamine 123, an oxidation-sensitive fluorescent probe, in which TNF-α is treated in HEK 293 cells, and pre-treatment of flavonoids inhibits the production of free radicals. The microscope confirmed it.

4 shows 10-160 μM of kaempferol (A), quercetin (B), fisetin (C), and chrysin (D) in HEK 293 cells, respectively, and MTT (3- (4,5-dimethylthiazol-2) for 48 hours. -yl) -2,5-diphenyltetrazolium bromide) assay showed that cell viability was the only one of the various flavonoids that was not toxic to camphorol.

FIG. 5A shows that the BALB / c mouse's back area is depilated cleanly, left for 24 hours to heal fine wounds of the skin, and after anesthesia, a plastic plate is placed on the back area of the mouse and the skin is soaked in 100 ° C. water for 8 seconds. . 1 hour after the induction of burns, camphor rolls were applied at 12 hour intervals to visually observe skin tissues for 30 days. FIG. 5B shows that when water at 100 ° C. is exposed to the skin of a mouse, an inflammatory reaction occurs in the skin tissue while the skin tissue is burned. On the other hand, the application of camphorol every 12 μg / kg body weight mice after 12 hours of burn-induced burn inhibited the migration of inflammatory cells to the skin tissue, and the keratinocyte layer was similar to normal mouse skin tissue. It was.

6 is a skin tissue was taken after 5 days, 10 days, 30 days by applying a camphor roll at 12 hour intervals 1 hour after the burn induction on the back of the BALB / c mice. RNA was isolated from skin tissues and expression of TNF-α was confirmed by RT-PCR.

<110> Hallym University Industrial Academic Cooperation Foundation <120> Composition for treating burn comprising kaempferol <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gtgagatctg aagtgtgatg actcagg 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtgaagcttg aagcttgtgt gctctgc 27 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atgacttcca agctggccgt ggct 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tctcagccct cttcaaaaac ttct 24 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tctcatcagt tctatggccc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gggagtagac aaggtacaac 20  

Claims (2)

Cell non-toxic burn treatment or prevention composition containing 1 to 200 μg / kg body weight of camphorol as an active ingredient. delete

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