KR20090076536A - Essential oil of lotus flower extract for treating dermatological diseases and method for preparing the same - Google Patents

Abstract

A Nelumbo nuficera extract having treatment effect on dermatitis is provided to promote melanogenesis, enhance tyrosinase activity and prevent and treat pigmentary disorder. A Nelumbo nuficera extract is isolated with n-hexane and have treatment effect on pigmentary disorder including vitiligo, white lentigo, depigmentation after inflammation, and photosensitive skin disorder. The Nelumbo nuficera is petals or stamens of Nelumbo nuficera. A Nelumbo nuficera extract essential oil is obtained by extracting and filtering the Nelumbo nuficera with n-hexane, evaporating at 40~60°C and removing hexane in vacuum. The essential oil comprises palmitic acid, linoleic acie, and their methyl ester. A therapeutic composition of pigmentary skin disorder comprises the essential oil of Nelumbo nuficera extract or palmitic acid methyl ester.

Description

Essential oil of lotus flower extract for treating dermatological diseases and method for preparing the same}

The present invention is a lotus ( Nelumbo) having a skin disease treatment effect nuficera ) extract and its essential oils, methods for their preparation and use as a therapeutic agent for skin diseases, more specifically lotus extract obtained by extracting lotus flower with hexane and its essential oil (lotus flower essential oil) and (1) petals or stamens of lotus flower Extracted with hexane; (2) filtered to remove hexane; (3) dissolved in dimethylsulfooxide; The present invention relates to a method for obtaining essential oil of lotus extract as a process and to use the same for the prevention or treatment of pigment-related skin diseases including vitiligo and the like.

Lotus flower ( Nelumbo nuficera ) is a perennial aquatic plant with yellow flowers. It is not only widely used as a food ingredient but also as a medicinal herb in eastern Asia, especially in China. Specifically, the seed of the lotus has been applied to various uses such as tissue inflammation, cancer and leprosy treatment and poisons. In addition, lotus embryos have been reported to alleviate acute inflammation in vivo and their roots have antioxidant effects (Jin-Tuarn L., Ying-Shu L., Chien-Jung L., Ann-Ru W., Food Chem. Tox ., 45, 486-493 (2007); Dongmei et. al ., 2007). Lotus leaf is also used as a traditional Chinese medicine with hemostatic action and is known to relieve hyperlipidemia in experimental animals (la Cour et. al ., 1995).

In addition, the stamen of lotus flower can be made into fragrant Chinese tea leaves when dried, and has been shown to have antioxidant effect in kidney cells (Jung et al ., 2003). Also, in Vietnam, the petals of lotus flowers are used to enhance the aroma of tea leaves. However, until now, there has been no report that lotus affects melanin formation.

Melanocytes play an important role in preventing skin cancer by protecting the skin from sunlight through the process of synthesizing melanin and transporting it to the skin epidermal cells (keratinocytes). Melanin is derived from tyrosine oxidized by a series of enzymes including tyrosinase and is an important factor in determining skin color. Specifically, melanin is synthesized in melanosomes containing specific enzymes necessary for melanin production, of which the tyrosinase gene family plays an important role in regulating melanin formation. do. This tyrosinase gene family includes tyrosinase, tyrosinase-related protein (TRP-1), microphthalmia-associated transcription factor M (MITF-M) and dopachrome tautomerase DCT and TRP-2. First, tyrosinase is a specific enzyme that catalyzes the rate regulation step in melanin biosynthesis, and microphthalmia-related transcription factor M also regulates the expression of tyrosinase TRP-1 and TRP-2. It is a melanocyte-specific transcription factor involved in pigmentation (Busca and Ballotti, 2000).

In humans, the basic skin color is genetically determined, but skin color can change for several reasons. Uneven skin color can be caused by hyper or underpigment, which can be very stressful for many patients. Therefore, it is very necessary to develop natural medicines for preventing or treating such under or hyperpigmentation.

Accordingly, the present inventors have continued efforts to develop new skin disease treatments, and as a result, lotus ( Nelumbo Lotus extract and essential oils obtained by hexane extraction of nuficera ) promote melanin formation, enhance enzymatic activity of tyrosinase, induce the expression of MITF-M protein and tyrosinase, and the palmitic acid methyl esters isolated from them. The present invention was successfully completed by confirming that they can be used for the prevention or treatment of pigment-related skin diseases.

The research was carried out by a fund (code: SC3270) from the Stem Cell Research Center of the 21st Century Frontier Research Program supported by the Ministry of Science and Technology.

REFERENCES

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2) Dongmei Y., Qiushuang W., Leqin K., Jianmei j., Tiejin Y., Asia Pac . J. Clin . Nutr ., 16, 158-163 (2007).

3) la Cour B, Molgaard P, Yi Z., J. Ethnopharmacol ., 46 (2), 125-129 (1995).

4) Borgi W., Bouraoui A., Chouchane N., J. Ethpharm ., 112 (2), 228-231 (2007).

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7) Oka M., Ichihashi M., Chakraborty AK., J. Invest . Dermatol ., 106 (2), 377-378 (1996).

8) Takahashi H., Parsons PG., J. Invest . Dermatol ., 98 (4), 481-487 (1992).

9) Englaro W., Rezzonico R., Durand-ClM., Lallemand D., Ortonne JP., Ballotti R., J. Biol . Chem ., 270 (41), 24315-24320 (1995).

10) Roser B., Robert B., Pigment Cell Res ., 13, 60-69 (2000).

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18 (3), 1489-1497 (1998).

12) Nishizuka Y., Science , 258 (5082), 607-614 (1992).

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18) Solano F., Martinez-Liarte JH, Jimenez-Cervantes C., Garcia-Borron JC, and Lozano JA, Biochem . Biophys . Res . Commun ., 204, 12431250 (1994).

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20) Tsukamoto K., Jackson IJ, Urabe K., Montague PM, and Hearing VJ, EMBO J. , 11, 519526 (1992).

21.Pawelek JM, and Murray M., Cancer Res ., 46, 493497 (1986).

Fang D., Kute T., and Setaluri V., Pigment Cell Res ., 14, 132139 (2001).

23) Hornyak TJ, Hayes DJ, and Ziff EB, J. Invest . Dermatol ., 115, 106112 (2000).

The present invention is a lotus ( Nelumbo) having a skin disease treatment effect nuficera ) extract and its essential oils, their preparation and their use for the treatment of skin diseases.

In order to achieve the above object, the present invention is a lotus extract, characterized in that it has a therapeutic effect on pigment-related skin diseases, including vitiligo, white spots, depigmentation after inflammation, white hair, photosensitivity skin diseases obtained by extracting the lotus flower with an organic solvent (lotus flower extract). It is preferable to use n-hexane as the organic solvent, and the lotus flower is preferably used in the dried petals (petals) or stamens (stamens).

In addition, the present invention is obtained by extracting the lotus flower with n-hexane, filtered and evaporated hexane and then completely removed hexane in vacuum, vitiligo, white spots, post-inflammatory depigmentation, white hair, pigments including photosensitive skin diseases It provides essential oil of lotus flower extract, which is characterized by having a therapeutic effect on skin diseases. The lotus is preferably used in the dried form of lotus petals (petals) or stamens (stamens). In addition, the lotus extract essential oil may include palmitic acid, linoleic acid, palmitoleic acid and linolenic acid and their methyl esters.

The lotus extract essential oil induces melanin synthesis, increases the enzymatic activity of tyrosinase, and also enhances the expression of tyrosinase enzyme, and thus is particularly useful for the prevention or treatment of vitiligo, which requires the formation of melanin. In addition to vitiligo, it can be used for the prevention or treatment of various pigment-related skin diseases that require a process of increasing the enzyme activity of tyrosinase or enhancing the expression of tyrosinase enzyme. Such pigment related skin diseases may include white spots, post-inflammatory depigmentation, white hair, and photosensitive skin diseases. The lotus extract essential oil includes, as an active ingredient, one or more selected from palmitic acid, linoleic acid, palmitoleic acid and linolenic acid and their methyl esters.

In addition, the present invention (1) the lotus is extracted with n-hexane; (2) filtration to evaporate hexane; (3) completely removing hexane again in a vacuum; It provides a process for obtaining the lotus extract essential oil as a process.

As the lotus flower, it is preferable to use dried flower petals or stamens. In addition, n-hexane in the process (1) is preferably extracted at room temperature for about 48 hours, n-hexane in the process (2) is preferably evaporated at 40-60 ℃, process (3) N-hexane is preferably removed under vacuum for about 30 minutes. In addition, after the process (3), the lotus extract essential oil is preferably dissolved in dimethyl sulfooxide (DMSO) and is preferably used by filtering.

In addition, the present invention provides a composition for treating pigment-related skin diseases, including vitiligo, white spots, post-inflammatory depigmentation, white hair, photosensitivity skin diseases containing the lotus flower extract (lotus flower extract) as an active ingredient.

In addition, the present invention provides a composition for treating pigment-related skin diseases, including vitiligo, white spots, post-inflammatory depigmentation, white hair, photosensitive skin diseases, containing the essential oil of lotus flower extract as an active ingredient. do.

The present invention provides palmitic acid and its methyl ester having a therapeutic effect on pigment-related skin diseases, including vitiligo, white spots, post-inflammatory depigmentation, white hair, photosensitive skin diseases, purely isolated from the lotus extract or the lotus extract essential oil. do.

In addition, the present invention is a pigment-related skin containing vitiligo, white spots, depigmentation after inflammation, white hair, photosensitive skin diseases containing palmitic acid and its methyl ester as an active ingredient purely isolated from the lotus extract or the lotus extract essential oil Provided are compositions for treating diseases.

The composition for treating skin diseases containing the lotus extract or the lotus extract essential oil of the present invention as an active ingredient may be used independently or as a pharmaceutical additive with a pharmaceutically acceptable carrier or excipient according to the intended use or suitable for administration to humans. It can be used in all other forms. Carriers that may be included in the pharmaceutical composition for treating dermatological diseases of the present invention may include extenders, high fiber additives, encapsulating agents, lipids, and the like, and examples of such carriers are well known in the art.

The therapeutic agent for skin diseases containing the lotus extract or the lotus extract essential oil of the present invention as an active ingredient may be administered according to the degree of disease progression, age, sex, physical condition, administration period, administration method, patient's weight, meal, discharge rate, and the like. It may be administered differently.

In addition, the composition for treating skin diseases containing palmitic acid and its methyl ester of the present invention purely isolated from the lotus extract or the lotus extract essential oil as an active ingredient may be used and administered in the same manner as described above.

The present invention (1) extract the petals or stamens of the lotus with hexane; (2) filtered to remove hexane; (3) dissolved in dimethylsulfooxide; In the process of preparing the essential oil of the lotus extract, the essential oil of the lotus extract and palmitic acid methyl ester purified from it to promote melanogenesis (melanogenesis), enhance the enzymatic activity of tyrosinase and induce the expression of related proteins Since the melanin content can be adjusted, it can be effectively used to prevent or treat various pigment-related skin diseases including vitiligo.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1>

Manufacture of Lotus Extract and Its Essential Oils

In the present invention, the lotus flower including petals and stamens ( Nelumbo nuficera ) was purchased at a traditional market in Ilsan, Gyeonggi-do. The lotus was dried and crushed in 87 g of 2 g of n-hexane at room temperature for 48 hours and filtered again. Obtained from which the filtrate was completely removed in a vacuum for a couple of hexane of room temperature, and then evaporating the hexane in 50 ℃ (in vacuo) condition for 30 minutes. As a result, 1.2 g of yellow essential oil was obtained in a clean state. The lotus essential oil thus obtained was dissolved in dimethylsulfooxide (DMSO) at a concentration of 10 mg / ml before being treated with melanocytes, and then filtered using a 0.2 μm syringe filter.

< Example  2>

Analysis of Essential Oils Containing Lotus Extracts ( GC Of MS  analysis)

In the present invention, in order to qualitatively and quantitatively analyze the essential oil components included in the lotus extract, gas chromatography and mass spectrometry (GC / MS) were performed as follows. First, a Hewlett Packard 5890 series II instrument was used in connection with an automatic mass meter (Automass 50 (JEOL)).

The operating conditions are that the column is fused with a silica capillary column and uses TC-wax (Hewlett Packard) with a size of 60 mx 0.25 mm and a film thickness of 0.25 μm, and the column temperature is increased to 5 ° C. per minute up to 150 ° C. and 15 per minute up to 300 ° C. The temperature was adjusted to 40-300 ° C. to raise to 300 ° C. for 5 minutes. In addition, the conditions of the injector 180 degreeC, the nitrogen of a carrier gas 30 cm / s flow rate, the column head pressure 180 kPa, the injection volume 0.5 ml, the ionization energy 70eV, the ion source temperature 200 degreeC, etc. were analyzed. The chemicals obtained were analyzed by comparing retention time and mass spectra with MS data library (NBS library). Relative quantification of each component was determined by calculating the peak area of the TIC chromatogram.

< Example  3>

Investigation of the effects of lotus extract essential oil on melanin formation

In the present invention, the effect of essential oil obtained from lotus extract on human melanocytes cultured by cells was investigated by measuring the melanin content and the like using MTT assay.

(1) human skin Melanocyte  culture

In the present invention, in order to obtain human melanocytes, skin samples removed during cesarean section and abortion surgery were used for cell culture. These cells were treated with bovine pituitary extract, fetal calf serum, bovine insulin, hydrocortisol, bFGF and bovine transferrin (# S-001-) in 254 medium (# M-254-500; Cascade Biologics, Portland, OR, US). 5; Cascade Biologics, Portland, OR, USA), heparin, pobol-12-myristate 13-acetate (phorbol-12-myristate 13-acetate; # S-002-5; Cascade Biologics, Portland, OR, USA) Supplemented and suspended cells therein.

(2) MTT  Method

In the present invention, in order to examine the proliferation of melanocytes, using the MTT assay kit (R & D Systems, Minneapolis, MN, USA) and the absorbance of each sample was measured at a wavelength of 570 nm. The lotus extract essential oil obtained above was treated with melanocytes, and at the same time, MTT analysis was performed by dividing each essential oil component, forskolin 10 μM treatment group, and a negative control group.

(3) melanin content measurement

The melanin content of the melanocytes cultured above was measured according to the method of Oka et al. (Oka et al., 1996). Specifically, the cell pellet was dissolved in 1 M NaOH, heated to 80 ° C. for 2 hours, and analyzed for color change at wavelength 475 nm. To determine the actual amount of melanin formed from the same number of cells, the total melanin amount in each pellet was divided by the number of melanocytes.

(4) Effect of Lotus Extract Essential Oil

Melanocytes became dark brown when treated with lotus extract essential oil (10 μg / ml) of the present invention, which was correlated with increased melanogenesis (see FIG. 1A, lane 2). This pigmentation is similar to the effect of treatment with 10 μM of phoscholine, an activator of adenyllate cyclase (Englaro et. al ., 1995), the cAMP pathway is one of the most important signaling pathways in melanin formation. However, when treated with phospholine or essential oil, there was a difference in the size of the cell pellet, it was observed that the cells treated with phospholine pellets larger than the cells treated with the lotus extract essential oil (Fig. 1A). Therefore, the lotus extract essential oil can increase the melanin content, but did not affect the number of melanocytes at all.

In addition, to investigate the effect of the lotus extract essential oil on the melanogenesis process, melanocytes were treated at four different concentrations (1.25, 2.5, 5 and 10 μg / ml). As a result, the melanin content was found to increase dose-dependently as the lotus extract essential oil was treated (FIG. 1B). In fact, the melanin content of each cell at the concentration of the essential oil 10 μg / ml reached 2.1 times compared to the negative control, it was confirmed that even higher compared to the result of treatment with forskolin (Fig. 1B). In addition, the cell number under all concentrations did not change according to the essential oil treatment, but when treated with phoscholine cell number increased compared to the control (Fig. 1B). As a result, it was confirmed that the lotus extract essential oil enhances melanin formation but does not affect cell proliferation at all.

< Example  4>

Lotus extract essential oils Melanocyte  Investigate effect on specific enzyme expression

The effect of the melanin formation of the lotus extract essential oil of the present invention was measured and evaluated the expression level of MITF, tyrosinase, TRP-1 and TRP-2 protein using Western blot and RT-PCR technique.

RT - PCR

Total RNA of melanocytes was isolated using QuickGene RNA cultured cell HC kit (Life Science, Minato-ku, Tokyo, Japan). cDNA was synthesized from total RNA using First Strand cDNA Synthesis Kit for RTPCR (AMV; Boehringer Mannheim, Germany). Human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a control gene.

  The primer sequence of MITF-M is forward primer: 5'-ATGCTGGAAATGCTAGAATATAAT-3 ', reverse primer: 5'-ATCATCCATCTGCATACAG-3',

  The primer sequence for TrP-1 is forward primer: 5'-TGGCAAAGCGCACAACTCACCC-3 ', reverse primer: 5'-AGTGCAACCAGTAACAAAGCGCC-3,

  Primer sequence for TrP-2 is forward primer: 5'-GCACACATGTAACCTCTGTG-3 ', reverse primer: 5'-TCATATAAGCAGGCTTGGCC-3',

  The primer sequence for tyrosinase is forward primer: 5'-TTGGCATAGACTCTTCTTGT TGCGG-3 ', reverse primer: 5'-CAAGGAGCCATGACCAGATCCG-3',

  The primer sequence for GAPDH is forward primer: 5'-TCCACCACCCTGTTGCTGTA-3 'and reverse primer: 5'-ACCACAGTCCATGCCATCAC-3'.

The apial amplification was 40 ul, and the composition was 10X buffer, 2.5m MgCl ₂ , 250 M dNTPs, 100 ng each PCR primer, 0.5 U Taq DNA polymerase, and 20-50 ng DNA, and Thermal Cycler 9600 (Applied Biosystems , Foster city, CA, USA) was used for 1 minute at 94 degrees, 1 minute at 55 ° C to 62 ° C, 1 minute at 72 ° C, and thus 35-40 cycles were performed. The DNA fragments amplified by PCR were separated by electrophoresis on 2% agarose gel.

PCR amplification sequences gene forward reverse size (bp) Ta ( cycle MITF-M 5'-ATGCTGGAAATGCTAGAATATAAT-3 '(SEQ ID NO: 1) 5'-ATCATCCATCTGCATACAG-3 '(SEQ ID NO: 2) 300 58 40 tyrosinase 5'-TTGGCATAGACTCTTCTTGTTGCGG-3 '(SEQ ID NO: 3) 5'-CAAGGAGCCATGACCAGATCCG-3 '(SEQ ID NO: 4) 812 62 40 TRP1 5'-TGGCAAAGCGCACAACTCACCC-3 '(SEQ ID NO: 5) 5'-AGTGCAACCAGTAACAAAGCGCC-3 '(SEQ ID NO: 6) 1027 55 35 TRP2 5'-GCACACATGTAACCTCTGTG-3 '(SEQ ID NO: 7) 5'-TCATATAAGCAGGCTTGGCC-3 '(SEQ ID NO: 8) 711 57 40 GAPDH 5'-TCCACCACCCTGTTGCTGTA-3 '(SEQ ID NO: 9) 5'-ACCACAGTCCATGCCATCAC-3 '(SEQ ID NO: 10) 400 55 35

Weston Blot  analysis

Cultured melanocytes treated with BV or forskolin, treated or untreated with inhibitors were like ice containing 50 mM Tris-base (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20 and protease inhibitors. Homogenized in cold homogenization buffer. The same amount as the extracted protein (30 g) was dissolved via 10% SDS-PAGE and transferred to nitrocellulose membrane. After incubation in a blocking solution of 5% nonfat dry milk in Tris-buffered saline (TTBS) containing 10 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20, the membrane was anti-phospho at 4 ° C. -MAPK, MAPK, CREB, phospho-CREB (rabbit polyclonal; Cell Signaling Technology, Beverly, MA), MITF (mouse monoclonal; Abcam, Cambridge, UK) or tyrosinase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight with antibodies and diluted 1: 1000 in blocking solution. The membrane is then further incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescent solution (ECL kit; Amersham Life Science, Buckinghamshire, England) Treated with. The signal was then captured on an Image Reader (LAS-3000; Fuji Photo Film, Tokyo, Japan). To monitor the amount of protein loaded in each lane, the membranes were rerobed with mouse monoclonal anti-actin antibody (Sigma, St. Louis, MO, USA) and proceeded in the manner described above. Protein bands were analyzed by densitometry.

(One) Tyrosinase  Activity analysis

Tyrosinase activity was analyzed by measuring DOPA oxidase activity as a modification of the conventional Takahashi method (Takahashi et. al ., 1992). First the cells were washed twice with PBS buffer and dissolved in phosphate buffer containing 1% Triton X-100. The cell extract was centrifuged at 10,000 xg for 10 minutes and only the clear supernatant was separated therefrom. The cell solution obtained here was quantified by the Bradford method to adjust the concentration in the digestion buffer, and then 90 μl of each fraction was placed in a 96-well plate and 10 μl of L-DOPA solution was added. The resulting reaction solution was reacted at 37 ° C. and absorbance at 475 nm was measured every 10 minutes for at least 1 hour using an ELISA reader.

(2) Investigation of the effects of lotus extract essential oil on melanin formation

First, when the lotus extract essential oil was treated at a concentration of 10 μg / ml, the expression of MITF-M and TRP-2 mRNA gradually increased until 2 hours, while the expression of tyrosinase and TRP-1 protein remained almost unchanged ( 2B). However, protein levels by MITF-M protein and tyrosinase expression increased steadily beyond 5 days of culture (FIG. 2C). Therefore, mRNA expression and protein expression correlated with the expression of MITF-M protein, and tyrosinase was not related.

In addition, by examining the signal transduction process that regulates the expression of MITF-M protein, it was confirmed that its expression is up-regulated by CREB protein (cAMP response element binding protein) simultaneously with PKA (Bertolotto et. al ., 1998a). In addition, CREB proteins have been reported to be activated by ERK (Englaro et. al ., 1995). Therefore, in order to investigate whether lotus extract activates signal transduction process in human melanocytes, phosphorylation degree of ERK and CREB proteins was measured using phosphospecific enzymes. At this time, the melanocytes were treated with essential oil isolated from lotus extract 10μg / ml.

As a result, it was found that phosphorylation of ERK and CREB proteins increased at 30 minutes and this level was maintained up to 2 hours after treatment with the essential oil (FIG. 2A). Therefore, it was confirmed that the MITF-M protein induced by lotus in melanocytes was expressed through activation of ERK and CREB proteins.

< Example  5>

Analysis of Ingredients in Lotus Extract Essential Oil

In the present invention, in order to investigate the main components affecting melanin formation, lotus extract essential oil was analyzed using the GC-MS system. Table 2 below shows nine compounds that occupy about 0.7% or more of the total essential oil content, the main components of which are methyl hexadecanoate (palmitic acid methyl ester) and methyl cis, cis, 9,12 Octadecadienoate (methyl cis, cis, 9,12-octadeca dienoate; linoleic acid methyl ester) accounted for 22.66% and 11.16%, respectively. Saturated fatty acid linoleic acid has been reported to reduce melanin content in melanoma cells, and palmitic acid as unsaturated fatty acid increases melanin content (Ando et al ., 2004). Therefore, even when the methyl group is attached to all of the fatty acids in the lotus extract essential oil, melanin formation may be increased when the palmitic acid methyl ester is large.

For reference, the chemical structural formula of methyl hexadecanoate is as follows.

Composition of lotus extract essential oil ingredient Retention time (minutes) content(%) Heptadecadiene 36.47 1.23 8-Heptadecene 36.57 1.13 Heptadecane 36.84 0.69 9-methylhexadecanoate (palmitioleic acid methyl ester) 39.05 7.55 Methylhexadecanoate (palmitic acid methyl ester) 39.17 22.66 Methyl cis, cis-9,12-octadecadienoate (Linoleic acid methyl ester) 40.85 11.16 Methyl 9,12,15-octadecatrienoate (Linolenic acid methyl ester) 40.90 5.16 heneicosane 41.05 5.55 Methyloctadecanoate 41.14 1.04

< Example  6>

Investigation of the effects of palmitic acid methyl ester on melanin formation

In the present invention, the effects of melanin formation were analyzed as palmitic acid methyl ester and linoleic acid methyl est, which are the main components affecting tyrosinase activity and expression. First, palmitic acid methyl ester and linoleic acid methyl ester were treated with melanocytes, and the results were examined and compared with each other. Indeed, it was found that linoleic acid and its methyl ester slightly reduced tyrosinase expression, tyrosinase activity and melanin content (Nishizuka Y., Science , 258 (5082), 607-614, 1992) (see FIG. 3). On the other hand, palmitic acid and its methyl esters induced a dose-dependent significant increase in tyrosinase expression, tyrosinase activity and melanin content (see FIG. 3). However, palmitic acid methyl ester was found to continuously improve melanin formation induced by palmitic acid. Also in cytotoxicity, it was found that these essential oil components have a toxicity that is not much greater than that of each free fatty acid. Thus, the functional activity of these derivative compounds is presumed to be somewhat different from that of the original compounds.

Figure 1 shows the effect of the lotus extract and essential oils of the present invention on melanin formation in human melanocytes. Figure 1A shows the effect of the essential oil lotus extract essential oil of the cell pellet, Display 1 is not treated anything, Display 2 is 10 μg / ml of the lotus extract essential oil, Display 3 is treated with 10 μM phospholine for 5 days will be. Figure 1B shows the melanin content and cell number of melanocytes cultured for 5 days with no treatment (left), forskolin (right) and lotus extract essential oils (1.25, 2.5, 5 and 10 μg / ml) without any treatment. It is shown.

Figure 2 shows the effect of the lotus extract and essential oils of the present invention on the expression of MITF protein and tyrosinase in human melanocytes. Figure 2A shows the phosphorylation degree of ERK and CREB protein when the lotus extract essential oil is treated with 10 μg / ml for 0.5, 1 or 2 hours, Figure 2B shows 10 μg of lotus extract essential oil for 0.5, 1 or 2 hours When treated with / ml, it shows the expression of MITF-M, TRP-1, TRP-2, tyrosinase and GAPDH mRNA, Figure 2C treated with lotus extract essential oil at 10 μg / ml for 1, 3 or 5 days In case of MITF and tyrosinase, β-actin protein expression is shown. However, C represents melanocytes which did not process anything.

Figure 3 shows the effects of the lotus extract of the present invention and the essential components of the essential oils on the enzymatic activity and expression of tyrosinase. However, LA represents linolenic acid, mLA represents linolenic acid methyl ester, PA represents palmitic acid, and mPA represents palmitic acid methyl ester.

<110> Dongguk University Industry-Academic Cooperation Foundation <120> Essential oil of lotus flower extract for treating dermatological          disease and method for preparing the same <130> DP-2007-0182 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> foward primer for MITF-M <400> 1 atgctggaaa tgctagaata taat 24 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for MITF-M <400> 2 atcatccatc tgcatacag 19 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer for tyrosinase <400> 3 ttggcataga ctcttcttgt tgcgg 25 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for tyrosinase <400> 4 caaggagcca tgaccagatc cg 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> foward primer for TRP1 <400> 5 tggcaaagcg cacaactcac cc 22 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRP1 <400> 6 agtgcaacca gtaacaaagc gcc 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for TRP2 <400> 7 gcacacatgt aacctctgtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for TRP2 <400> 8 tcatataagc aggcttggcc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for GAPDH <400> 9 tccaccaccc tgttgctgta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for GAPDH <400> 10 accacagtcc atgccatcac 20  

Claims (11)

Lotus flower extract (lotus flower extract) characterized by having a therapeutic effect of pigment-related skin diseases, including vitiligo, white spots, post-inflammatory depigmentation, white hair, photosensitive skin diseases, obtained by extracting the lotus with n-hexane. The method of claim 1, The lotus flower is a lotus extract, characterized in that the dried petals (petals) or stamens (stamens). It is obtained by extracting the lotus with n-hexane, filtering and evaporating the hexane, and then completely removing hexane in a vacuum state. Pigment-related skin including vitiligo, white spot, post-inflammatory depigmentation, white hair, and photosensitive skin disease Essential oil of lotus flower extract, characterized by the therapeutic effect of the disease. The method of claim 3, wherein The lotus extract essential oil is a lotus extract essential oil, characterized in that it comprises palmitic acid, linoleic acid, palmitole and linolenic acid and their methyl ester. (1) extracting the lotus with n-hexane; (2) filtration to evaporate hexane; (3) completely removing hexane again in a vacuum; Process for obtaining the essential oil lotus extract of claim 3. The method of claim 5, The lotus is a manufacturing method characterized in that the dried lotus petals or stamens. The method of claim 5, N-hexane in the process (2) is 40 ~ 60 ℃ Method of producing a characterized in that the evaporation. The method of claim 5, After the step (3), the method of producing a lotus extract essential oil, characterized in that in dimethylsulfooxide (DMSO) to dissolve. Claim 1, characterized in that containing the lotus extract as an active ingredient, vitiligo, white spots, post-inflammatory depigmentation, white hair, photosensitive skin diseases, the composition for treating skin diseases. A composition for treating pigment-related skin diseases, including vitiligo, white spots, post-inflammatory depigmentation, white hair, and photosensitive skin diseases, comprising the lotus extract essential oil of claim 3 as an active ingredient. The lotus extract of claim 1 and the lotus extract of claim 3, palmitic acid and palmitic acid methyl ester, which is purely separated from essential oils, characterized by vitiligo, white spot, post-inflammatory depigmentation, white hair, photosensitive skin Pigment related skin disease treatment composition comprising a disease.

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