KR20150115096A - Gardenia extract manufacturing method and Cosmetic compositions containing the extract of gardenia - Google Patents

Abstract

The present invention relates to a method for producing a gardenia extract and a cosmetic composition containing the extract and, more specifically, to a method for producing a gardenia extract, including: a first step of hydrolyzing at 40-60 deg. C by adding polysaccharide hydrolase into a mixture of gardenia fruits and distilled water; a second step of inactivating the polysaccharide hydrolase by treating at high temperature above 90 deg. C; and a third step of filtering out the extract obtained from the second step and then processing the same into powder after concentration and drying, and also relates to a cosmetic composition containing the gardenia extract. Therefore, the present invention ensures production of various cosmetic compositions which show excellent skin friendliness and protect skin from ultraviolet rays.

Description

TECHNICAL FIELD The present invention relates to a method for producing a gardenia extract, and a cosmetic composition containing the gardenia extract,

The present invention relates to a method for producing a gardenia extract and a cosmetic composition containing the gardenia extract. More particularly, the present invention relates to a method for producing a gardenia extract which can effectively protect skin cells from ultraviolet rays, and a cosmetic composition containing the gardenia extract.

Ultraviolet light is a major cause of skin photoaging and photoprotection. Ultraviolet rays are composed of ultraviolet ray A (320 to 400 nm), ultraviolet ray B (280 to 320 nm) and ultraviolet ray C (200 to 280 nm) , Photoaging, immunosuppression, skin cancer, and the like.

In addition, ultraviolet light has been identified as a major cause of photoaging involving inflammatory cytokines and matrix metalloproteinases (MMPs). The constituents of cytokines and other water-soluble elements are low in the keratinocytes of the human body Various stimuli such as endotoxins and ultraviolet light can induce the expression of inflammatory cytokines and induce apoptosis of keratinocytes by internal pathways including direct DNA damage, activated cell death signals, and external pathways including the development of ROS .

In addition, ultraviolet rays have been identified as a major cause of skin aging, which is caused by wrinkles, sagging, blistering, roughness, loss of skin tone, and so much research has been conducted to develop more effective and safer raw materials for mitigating skin damage caused by ultraviolet rays It is true.

On the other hand, photoaging is related to changes in the composition of skin cells and collagen. matrix metalloproteinase (MMP) is a zinc endopeptidase that plays an important role in altering protein molecules including collagen type 1 in the extracellular matrix. Furthermore, the expression of MMPs such as MMP-1, -2, -3, and -9 is known to be increased in skin fibroblasts exposed to ultraviolet light.

These MMPs can regulate the process of remodeling connective tissue associated with symptoms due to wrinkles and other photoaging. Therefore, the goal of pharmacological treatment of MMPs is known to be a useful strategy to reduce the photoaging process.

That is, MMPs increased by ultraviolet B are mainly signals of activation of cytokine receptors by ultraviolet light, activation of mitogen-activated protein kinases such as ERK, JNK, p38 kinase, and thus activation of transcription factors such as AP-1 complex Mediated by the delivery path.

The promoters of the MMP-1 and MMP-3 genes have an AP-1 binding site that is activated by the activated AP-1 complex. Although the initial process of signal transduction is not fully described, several evidence suggests that DNA damaged by ultraviolet light acts as the first signal.

Previous studies have shown that the medium obtained from keratinocytes exposed to ultraviolet light effectively increases the expression of MMP-1 in fibroblasts through the effect of secretion, so that expression of MMP-1 in skin fibroblasts inhibits activation of epidermal keratinocytes by ultraviolet light Which is known to be regulated indirectly.

Japanese Patent Application Laid-Open No. 10-2013-0055330 Japanese Patent Application Laid-Open No. 10-2013-0048885

Conventionally, a sunscreen agent is mainly used to prevent skin damage due to ultraviolet rays.

Conventional ultraviolet screening agents can be classified into two types of chemical blocking agents and physical blocking agents prior to their functional groups. The chemical blocking agents absorb ultraviolet rays by capturing the sunlight energy in the molecule, Blocking agents are substances with physical properties that reflect and disperse ultraviolet light, such as zinc oxide, titanium oxide, iron oxide, magnesium oxide, and the like.

The physical barrier agent of the ultraviolet screening agent is a substance having physical properties of reflecting and dispersing ultraviolet ray, such as zinc oxide, titanium oxide, iron oxide, magnesium oxide, etc. These substances are excellent in ultraviolet ray shielding effect, Most commercialized sunscreens are chemical blockers made up of chemical blocking agents as the main constituents.

Since the chemical blocking agent as described above is composed of an artificial chemical component, when the ultraviolet ray blocking agent is frequently used, the chemical blocking agent causes skin irritation and causes skin irritation. Therefore, In the case of weak infants and young children, skin irritation is a concern and the use of sunscreen agents is avoided.

Particles such as zinc oxide, titanium dioxide, and benzophenone-3 (oxiphenone), which are the main components of ultraviolet screening agents, form a protective film on the skin to prevent ultraviolet rays from penetrating the skin. However, Since such chemical components accumulate in the human body through the skin, there is a possibility of serious damage to the main body of human body. Therefore, it is inevitable to develop a raw material for a cosmetic composition that can safely protect the skin from ultraviolet rays.

Disclosure of the Invention The present invention was made in order to overcome the above-mentioned problems of the prior art. It is a first step of adding a polysaccharide hydrolyzing enzyme to a mixture of a gardenia fruit and distilled water to proceed hydrolysis at 40 to 60 DEG C, A third step of treating the extract at a temperature of 90 ° C or higher to inactivate the extract, filtering the extract obtained in the second step, and concentrating and drying the extract to powder.

In addition, by constructing a cosmetic composition including the gardenia extract, it is possible to achieve the object of the present invention, which is capable of effectively protecting skin from ultraviolet rays while having excellent skin-affinity using a natural component.

The present invention reduces the oxidative stress, apoptosis and inflammation reaction induced by ultraviolet ray B, which is a natural component of the gardenia extract, so that it is possible to manufacture various cosmetic compositions which are excellent in skin affinity and can protect the skin from ultraviolet rays have.

1 is a graph showing the absorption spectrum of a plant extract according to the present invention
FIG. 2 is a graph showing the effect of the extract of the gardenia and the comparative group on the cell viability of HaCaT keratinocytes according to the present invention
FIG. 3 is a graph showing the effect of the extract of the gardenia and the comparative group on the cytotoxicity induced by ultraviolet ray B in keratinocytes according to the present invention
FIG. 4 is a graph showing the effect of the extract of the gardenia and the comparative group on the expression of mRNA of inflammatory cytokines induced by ultraviolet B in HaCaT cells according to the present invention
FIG. 5 is a graph showing changes in expression of MMP-1 protein in fibroblasts by conditioned medium of HaCaT cells according to presence or absence of plant extracts upon exposure to ultraviolet ray B according to the present invention
FIG. 6 is a graph showing the effect of the extract of the gardenia extract on caspase-9 and caspase-3 in HaCaT cells exposed to ultraviolet ray B according to the present invention
FIG. 7 is a graph showing the effect analysis of a gardenia extract against lipid peroxidation induced by ultraviolet B in HaCaT cells according to the present invention
Figure 8 is a HPLC specific intrinsic graph of the groomer and comparative group extracts according to the present invention.

The gardenia extract of the present invention can be obtained by various known extraction methods commonly used in the art to which the present invention belongs. Preferably, the gardenia fruit is extracted with distilled water.

Extraction of distilled water is performed by adding 1 liter of distilled water to 100 g of gardenia and then extracting at 80 to 100 ° C. More preferably, the gardenia fruit is extracted simultaneously with the polysaccharide hydrolyzing enzyme treatment.

That is, in the enzyme extraction, 0.1 to 10 parts by weight of the cellulase and the amylase enzyme selected in advance are added to 100 parts by weight of the gardenia, and extracted at 40 to 60 ° C.

The extract of the present invention may be produced in a powder state by an additional process such as vacuum distillation and lyophilization or spray drying.

In addition, the extract of the present invention includes not only the extract obtained by the above-mentioned distilled water, but also the extract obtained by extraction solvent such as ethanol, and the extract obtained by a conventional purification process.

For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation according to size, charge, hydrophobicity or affinity) The obtained fraction is also included in the extract of the present invention.

The extract of the present invention is also included in the extract of the present invention as an extract obtained through subcritical and supercritical extraction methods which are widely used as methods for extracting useful components from natural products.

In the present invention, the thus-prepared gardenia extract may be used as a raw material for a cosmetic composition by various known methods commonly used in the art to which the present invention belongs. Particularly, as a raw material for a cosmetic composition for protecting skin from ultraviolet rays Can be used.

The concrete action of the gardenia extract according to the present invention on the skin protection function from ultraviolet rays will be described below with reference to experimental examples.

Prior to the experiment, the extracts of Ehrwald (B) and Rhubarb (C) selected as comparative groups for comparison with the gardenia extract and the gardenia extract were prepared as in the following examples and comparative examples, respectively.

≪ Example 1 >

One liter of distilled water was added to 100 g of gardenia fruit, and the mixture was extracted at 80 ° C for 2 hours. The extract was filtered with a filter paper, and then concentrated under reduced pressure and lyophilized to obtain 12 g of gardenia extract. (Yield: 12%)

≪ Comparative Example 1 &

1 liter of distilled water was added to 100 g of yellow radish roots, and the mixture was extracted at 80 ° C for 2 hours. The extract was filtered with a filter paper, and then concentrated under reduced pressure and lyophilized to obtain 10 g of an extract. (Yield: 10%)

≪ Comparative Example 2 &

One liter of distilled water was added to 100 g of rhubarb root, and the mixture was extracted at 80 ° C. for 2 hours. The extract was filtered with a filter paper, and then concentrated under reduced pressure and lyophilized to obtain 9.2 g of a rhubarb extract. (Yield: 9.2%)

< Experimental Example  1>

Accompanying drawings, Figure 1 is gardenia (A) and the comparative group extract the hwangbyeok (B), rhubarb (C) gardenia As shown in the figure as showing an absorption spectrum graph of an extract (A), hwangbyeok (B), rhubarb (C) extract Was dissolved in PBS (Phosphate Buffered Saline) at a concentration of 10, 30, or 100 microgram / mL and the absorption spectrum was measured at 10-100 g / mL. Using a Shimadzu UV- 1650PC ultraviolet spectrophotometer, (B ) , and rhubarb (C) extracts, which are comparative group extracts, were measured.

As a result of the measurement, these extracts exhibited similar absorption rates in the region of ultraviolet ray B (280 to 320 nm), and the gardenia extract showed the highest absorption in ultraviolet ray A (320 to 400 nm) and blue ray region.

< Experimental Example  2>

FIG. 2 is a graph showing an effect analysis effect on the cell survival rate of the gardenia (A) in the HaCaT keratinocyte and the comparative group extracts of the yellow wall and the rhubarb extract. The plant extracts were treated for 24 hours at the concentrations assigned to the cells, Cell viability was determined by experimental method and cell viability was expressed as a percentage of control. (means SEs; n = 3). #p <0.05 vs. Control (Vehicle).

HaCaT cells (human immortal keratinocyte line) were cultured in DMEM / F-12 containing 10% fetal bovine serum and 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 g / mL amphotericin B, 10 g / mL hydrocortisone And cultured in medium (GIBCO-BRL, Grand Island, NY, USA). Cells were incubated at 37 ° C in a humidified atmosphere containing 5% CO ₂ and 95% air.

Cell viability was analyzed by MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) method. This assay is based on the activity of mitochondrial dehydrogenase in living cells which reduces light yellow MTT by accumulating dark blue formazan crystals in the cells.

Briefly, cells were washed with PBS and incubated for 3 hours in 1 mL of culture medium containing 1 mg / mL MTT. The medium was then discarded and isopropanol treated with formazan dissolved in the cells. The solution was transferred to a microplate and formazan was quantified by measuring the absorbance at 595 nm.

The cytotoxicity of the extract was compared with that of human corneal HaCaT cells at a concentration of 10 to 100 microgram / mL for 24 hours.

As shown in FIG. 2, the extracts of the gardenia extract showed no cytotoxicity up to 100 microgram / mL, while the extracts of the wild wall extract showed a slight cytotoxicity at this concentration and the extracts of rhodamine showed cytotoxicity at a concentration of 30 microgram / mL.

< Experimental Example  3>

FIG. 3 is a graph showing an effect analysis of cytotoxicity against ultraviolet B-induced cytotoxicity in keratinocytes according to the present invention and a comparative group extract of Hwangwol and Rhubarb extracts, 15 mJ / cm2 (A). (10, 30, or 100 microgram / mL) dissolved in PBS upon exposure to ultraviolet B 10 mJ / cm 2.

Cells were cultured for 24 h and cell viability was determined by MTT assay. Cell survival was expressed as a percentage of control (mean SEs, n = 3). # p < UV non-irradiated control group. * p < 0.05 UV irradiation control (vehicle).

HaCaT cells were inoculated in a 6-well plate at 200,000 cells per well and cultured for 48 hours until the cells were attached to the bottom of the wells at least 80%.

The cells were then washed twice with PBS and exposed to ultraviolet B under PBS containing the test material.

Ultraviolet B treatment was performed in a cell culture hood using an ultraviolet B lamp (Model UV B-18, ULTRA * LUM. Inc., Claremont, CA, USA). The intensity of the radiation was measured using an ultraviolet light illuminometer (Model UV 340A, Lutron Electronic Enterprise Co., Taipei, Taipei, Taiwan) and ultraviolet light was irradiated to the culture plate cells at an intensity of 80 W / cm 2.

However, the treatment duration varied for a specific amount of ultraviolet radiation of ultraviolet B (5, 10, 15 mJ / cm 2). After the treatment, the PBS was discarded and replaced with the culture medium, and the cells were cultured for 1 day. Cells were subjected to various experiments as described below. The conditioned medium obtained after cell culture was harvested and used to treat human skin fibroblasts.

As shown in A of FIG. 3, HaCaT cell viability decreased with exposure to ultraviolet light.

In addition, as shown in FIG. 3B of the accompanying drawings, the gardenia extract increased the survival rate of cells exposed to ultraviolet rays in a concentration-dependent manner. The extracts of W. wall showed similar cytoprotective effects, but no effect was observed in the case of R.

< Experimental Example  4>

FIG. 4 is a graph showing the effect of the extracts of the gardenia (A) on the expression of inflammatory cytokines induced by ultraviolet B in HaCaT cells according to the present invention and the comparative group extracts of Ehrlom (B ) and rhubarb (C) A and B irradiate HaCaT with ultraviolet ray B at 5 to 15 mJ / cm 2.

C and D were irradiated with ultraviolet B 10mJ / ㎠ in the presence of plant extract (10, 30, or 100 microgram / mL) and cultured for 24 hours, and mRNA expression was measured by qRT-PCR. GAPDH was used for comparison purposes. Data are presented as control reference multiples. (means SEs; n = 3). UV non-irradiated control group. * p < 0.05 UV irradiation control (vehicle).

Expression of inflammatory cytokines by plant extracts was investigated in HaCaT cells cultured for 24 hours after exposure to ultraviolet B of 5 ~ 15 mJ / ㎠ light intensity. MRNA of whole cells was extracted and mRNA level of cytokine was measured by qRT-PCR.

Total cellular RNA was extracted using an RNeasy kit (Qiagen, Valencia, Calif., USA). To prepare cDNA, 1 microgram of cellular RNA was reverse transcribed using High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA). This kit used a random hexamer primer and MultiScribe reverse transcriptase.

The polymerase chain reaction was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems) with SYBR Green PCR Master Mix (Applied Biosystems), 60 ng of cDNA, and reaction mixture (20 microliter) containing 2picomole gene- specific primer sets (Macrogen, .

The primers used for the PCR analysis were as follows. 5-CCT GTC CTG CGT GTT GAA AGA-3 (forward) and 5-GGG AAC TGG GCA GAC TCA AA-3 (reverse), Tumor Necrosis Factor (IL-1) (GeneBank accession number, NM_000576.2) 5-ATG GGG GGG CCG ATG GA-3 (reverse) and GAPDH (NM_002046.3) 5-TGC TCCA TCC-3 (forward) AAG GTG AAG GTC G-3 (forward) and 5-GGG GTC ATT GAT GGC AAC AA-3 (reverse).

The reaction was carried out under the following conditions. 50 to 2 min, 95 to 10 min, followed by 40 amplification cycles (95 to 15 sec, 60 to 1 min) at the dissociation step. One peak was confirmed by a melting curve analysis showing the homogeneity of the amplification product. Compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which is an internal control, IL-1 and TNF- mRNA expression levels were calculated using the relative critical cycle (CT) method.

As shown in FIG. 4, interleukin-1 (IL-1) and TNF- mRNA expression was significantly increased.

In addition, the 30 ~ 100 microgram / mL of gardenia extracts attenuated the expression of inflammatory cytokines in HaCaT cells exposed to ultraviolet B, while the extract of Wanpari showed less effect.

< Experimental Example  5>

FIG. 5 is a graph showing an analysis of expression changes of MMP-1 protein in fibroblasts by a conditioned medium of HaCaT cells according to the presence or absence of a plant extract upon exposure to ultraviolet ray B according to the present invention. , HaCaT cells were irradiated with ultraviolet B at 5 to 15 mJ / cm 2 and then cultured for 24 hours. The conditioned medium of HaCaT cells irradiated with ultraviolet B is treated with human dermal fibroblasts for 24 hours.

FIG. 5B shows the results of treatment with HaCaT cells treated with various plant extracts dissolved in PBS at 10 mJ / cm 2 exposure to ultraviolet B and cultured for 24 hours. The conditioned medium irradiated with the ultraviolet ray B is cultured in human dermal fibroblasts for 24 hours. The expression of MMP-1 protein in fibroblasts was confirmed by Western blot method using b-actin as a reference. (means SEs; n = 3). # p < UV non-irradiated control group. * p < 0.05 UV irradiation control (vehicle).

Human dermal fibroblasts isolated from adult skin were obtained from Cascade Biologics (Portland, OR, USA). Cells were cultured in Iscoves Modified Dulbeccos medium (GIBCO-BRL, Grand Island, NY, USA) containing 10% fetal bovine serum, 100 U / mL penicillin, 0.1 mg / mL streptomycin, 0.25 g / mL amphotericin B, Gt; 37 C &lt; / RTI &gt; in a humidified atmosphere containing CO ₂ and 95% air.

In the experiment on the paracrine effect of the factors secreted from the keratinocytes, the conditioned medium obtained from the HaCaT cells treated with ultraviolet B was completely replaced and the fibroblasts were cultured for 24 hours before the analysis.

All cell lysates from Western blotting were prepared using lysis buffer (10 mM Tris-Cl, pH 7.4, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 1 mM EDTA, 0.5% Triton X-100, protease inhibitor cocktail). The lysates were denatured with protein and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated by size were transferred to polyvinylidene fluoride membranes, incubated with appropriate primary antibodies overnight at 4 ° C, and secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature.

Immunoreactive bands were detected using a picoEPD Western Reagent kit (ELPIS-Biotech, Daejeon, Korea) and analyzed for densitometry. The primary antibody of MMP-1 was purchased from Calbiochem (San Diego, CA, USA). Rabbit polyclonal caspase-3 and rabbit polyclonal caspase-9 antibodies were purchased from Cell Signaling (Danvers, MA, USA). The mouse monoclonal-actin antibody was used in Sigma-Aldrich (St. Louis, Mo., USA).

As shown in FIG. 5A, the conditioned media of HaCaT cells exposed to UV light B of different light intensities were cultured in human dermal fibroblasts for 24 hours, and the expression of MMP-1 in fibroblasts was determined by the intensity dependence of ultraviolet B Respectively.

5B, when the conditioned medium of the HaCaT keratinocytes treated with the gardenia extract was exposed to the ultraviolet B and the treated medium of the HaCaT keratinocytes exposed to the ultraviolet B without the gardenia extract, And decreased the expression of MMP-1 in fibroblasts.

< Experimental Example  6>

FIG. 6 is a graph showing an analysis of the effect of the gardenia extract on caspase-9 and caspase-3 in HaCaT cells exposed to ultraviolet ray B according to the present invention. 15 mJ / cm &lt; 2 &gt;.

Fig. 6B shows the results of exposure to 10, 30, or 100 microgram / mL of the extracts of the gardenia extract when exposed to 10 mJ / cm2 of ultraviolet ray B and cultured for 24 hours. The active forms of cleavage, procasepase-9 and procaspase-3, were analyzed by Western blot. Data are presented as control reference multiples. (means SEs; n = 3). # p < UV non-irradiated control group. * p < 0.05 UV irradiation control (vehicle).

All cell lysates from Western blotting were prepared using lysis buffer (10 mM Tris-Cl, pH 7.4, 120 mM NaCl, 25 mM KCl, 2 mM EGTA, 1 mM EDTA, 0.5% Triton X-100, protease inhibitor cocktail). The lysates were denatured with protein and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins separated by size were transferred to polyvinylidene fluoride membranes, incubated with appropriate primary antibodies overnight at 4 ° C, and secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature.

 Immunoreactive bands were detected using a picoEPD Western Reagent kit (ELPIS-Biotech, Daejeon, Korea) and analyzed for densitometry. The primary antibody of MMP-1 was purchased from Calbiochem (San Diego, CA, USA). Rabbit polyclonal caspase-3 and rabbit polyclonal caspase-9 antibodies were purchased from Cell Signaling (Danvers, MA, USA). The mouse monoclonal-actin antibody was used in Sigma-Aldrich (St. Louis, Mo., USA).

As shown in FIG. 6A, ultraviolet B increased the cleaved active form of caspase-9 and caspase-3 according to the intensity of light.

In addition, as shown in FIG. 6B of the accompanying drawings, the cleaved caspase-9 and caspase-3 cleaved by ultraviolet B during ultraviolet B exposure were significantly decreased in the medium containing the gardenia extract.

< Experimental Example  7>

FIG. 7 is a graph showing an analysis of the effect of the gardenia extract on lipid peroxidation induced by ultraviolet B in HaCaT cells according to the present invention. FIG. 7A is a graph showing the effect of HaCaT cells on ultraviolet B at 5 to 15 mJ / Respectively.

FIG. 7B shows the results of exposure to ultraviolet B (10, 30, or 100 microgram / mL) of the extracts of the gardenia extract when exposed to 10 mJ / cm 2 of ultraviolet ray B and cultured for 24 hours. Lipid peroxidation was determined by quantitative TBARS. Data were expressed as a control reference multiple (means SEs; n = 3). Control value was 0.33 nmol TBARS per mg protein. # p < UV non-irradiated control group. * p < 0.05 UV irradiation control (vehicle).

The lipid peroxidation method was quantified as TBARS (2-thiobarbituric acid-reactive substances), a lipid peroxidation indicator substance. Cells were treated with lysis buffer (20 mM Tris-Cl, 2.5 mM EDTA, 1.0% SDS, pH 7.5). Cell lysates (200 mg protein in 0.1 mL) mixed with 0.9 mL of 1.0% phosphoric acid and 1.0 mL of 0.9% 2-thiobarbituric acid (Sigma-Aldrich) were heated in a boiling water bath for 45 minutes.

Cell lysates were treated in the same manner with the standard solution of 1,1,3,3-tetramethoxypropane (Sigma-Aldrich), a precursor of malondialdehyde. After cooling, 1.5 mL of 1-butanol was added and the mixture was centrifuged at 13000 rpm for 15 minutes to separate into two layers. The fluorescence intensity of the 1-butanol layer was measured using a Gemini EM fluorescence microplate reader (Molecular devices, Sunnyvale, Calif., USA) at 590 nm after stimulation at 540 nm.

As shown in FIG. 7A of the accompanying drawings, lipid peroxidation was increased in HaCaT cells exposed to ultraviolet ray B, and the increase was decreased according to the concentration of the gardenia extract as shown in FIG. 7B.

< Experimental Example  8>

8 is a graph showing the HPLC specific intrinsic graph of the extract of the gardenia and the comparative group according to the present invention, Typical HPLC chromatograms at 280 nm (A, B, C) and at 440 nm (D, E, F). The crocin peak was confirmed by comparison with the standard product crocin.

HPLC analysis was performed on a HPLC analysis of the extract using a 321 pump and a Gilson HPLC system (Gilson, Inc. Middleton, Wis., USA) equipped with a UV / VIS 151 detector.

10 mL of an aqueous solution of the test material (1.0 mg / mL) was injected. Separation was performed on a 5 mm Hector-M C18 column (4.6 mm x 250 mm) (RS tech co., Daejeon, Korea).

The mobile phase consisted of 0.5% formic acid (A) and acetonitrile (B). The concentration gradient was programmed as follows. 0-20 min, linear gradient 20-80% B; 2035 min, 80% B, and the flow rate was maintained at 0.5 mL / min.

The ultraviolet detectors were set at 280 nm and 400 nm. Purified crocin was purchased from Sigma-Aldrich (St. Louis, Mo., USA) and its purity was determined by spectrophotometry using an extinction coefficient of 443 = 89,000 M [18].

A typical HPLC pattern of extracts of gardenia, hwangwol, and rhubarb is shown in Fig. 8 of attached drawings, and the purchased chromic acid is used as a reference material because it is known as a main dye component of gardenia. The chromic content of the gardenia extract was measured as 1.7%. Crocine was not detected in the extracts of yellow moss and rhubarb.

In the above experimental data, the data are expressed as mean ± SE of three or more independent experiments. Similarity between group differences was measured using Student t-test. Duncan's multiple-range test is applied when the difference is significant with Student t-test (p <0.05). Statistical significance is judged at p values <0.05.

According to the above experimental example, the gardenia extract had a broad absorption range of ultraviolet light and had minimal effect on the cell viability of cultured HaCaT cells.

In addition, the gardenia extract decreased the death of HaCaT cells by UV exposure.

In particular, the expression of MMP-1 in human dermal fibroblasts increases when HaCaT cells exposed to ultraviolet B are treated with a conditioned medium and cytokines such as interleukin-1 (IL-1) and TNF- It is known that the gardenia extract inhibits the expression of cytokines in human dermal fibroblasts by decreasing the secretion and secretion effects of HaCaT cell cytokines in a dose-dependent manner. .

In addition, the gardenia extract inhibited lipid peroxidation as an index of oxidative stress induced in UVB. Therefore, the gardenia extract is considered to be useful for protecting skin cells from oxidative stress, inflammation, apoptosis and the like caused by ultraviolet rays.

Gardenia extract also showed antioxidant and anti - apoptotic effects in HaCaT cells exposed to UVB.

These results suggest that the extract may inhibit damage and inflammatory responses induced by UVB in skin cells. Therefore, cosmetic compositions containing the extract of the gardenia can effectively protect the skin from ultraviolet rays.

On the other hand, the outer surface of the gardenia fruit has many components such as cellulose, hemicelluloses and the like, and contains a lot of starch ingredients therein, and these polysaccharide ingredients hinder effective extraction of the active ingredient.

In general, an acid or an alkali solution is used at high temperature as a conventional method for hydrolyzing an insoluble polysaccharide component, but this method has many problems such as a large amount of environmental wastewater and corrosion of a container.

Insoluble polysaccharide components can be hydrolyzed very efficiently by using polysaccharide hydrolytic enzymes such as cellulase and amylase.

The cellulase or amylase used in the present invention can be any commercially available.

In the present invention, the cellulase (Celluclast) of Novo Nordisk and the amylase of Cellbio were used.

Through the present invention, it has been found that when extracting using these polysaccharide hydrolyzing enzymes, the yield of the gardenia extract is high and the content of the effective ingredient is high, and the present invention has been devised.

In the case of the conventional distilled water extraction method (Example 1), the yield of the extract was 12%, and the content of crocin as an active ingredient of the extract was 1.7%.

However, according to the enzyme extraction method of the present invention, the yield of extract increased up to 20.3% and the content of active ingredient increased to 2.6%.

Therefore, the total yield of the active ingredient increased more than twice from 0.20% to 0.53%.

The method of extracting the sponge enzyme of the present invention will now be described with reference to the following examples.

&Lt; Example 2 >

1 liter of distilled water was added to 100 g of gardenia fruit, and 0.2 g of cellulase was added thereto. The mixture was extracted at 50 ° C for 16 hours and heat-treated at 95 ° C for 5 minutes to inactivate the enzyme. After filtration through a filter paper, To thereby obtain 19.8 g of gardenia extract. (Yield: 16.5%)

&Lt; Example 3 >

1 g of distilled water was added to 100 g of gardenia fruit, 1 g of cellulase was added thereto, and the mixture was extracted at 50 ° C. for 24 hours. The mixture was heat-treated at 95 ° C. for 5 minutes to inactivate the enzyme. The mixture was filtered through a filter paper, 19.2 g of gardenia extract was prepared. (Yield: 17.2%)

<Example 4>

1 liter of distilled water was added to 100 g of gardenia fruit, and 5 g of cellulase was added thereto. The mixture was extracted at 50 ° C. for 12 hours and heat-treated at 95 ° C. for 5 minutes to inactivate the enzyme. The mixture was filtered through a filter paper and concentrated under reduced pressure, 18.0g of gardenia extract was prepared. (Yield: 18.0%)

&Lt; Example 5 >

One liter of distilled water was added to 100 g of gardenia fruit, 0.4 g of amylase was added, and the mixture was extracted at 40 ° C for 12 hours. The mixture was heat-treated at 95 ° C for 5 minutes to inactivate the enzyme. After filtration through a filter paper, 18.0g of gardenia extract was prepared. (Yield: 16.0%)

&Lt; Example 6 >

1 g of distilled water was added to 100 g of gardenia fruit, 2 g of amylase was added thereto, and the mixture was extracted at 40 ° C. for 24 hours. The mixture was heat-treated at 95 ° C. for 5 minutes to inactivate the enzyme. The mixture was filtered through a filter paper and concentrated under reduced pressure. 17.5 g of the extract was prepared. (Yield: 17.5%)

&Lt; Example 7 >

1 g of distilled water was added to 100 g of gardenia fruit, 10 g of amylase was added thereto, and the mixture was extracted at 40 ° C. for 16 hours. The mixture was heat-treated at 95 ° C. for 5 minutes to inactivate the enzyme. The mixture was filtered through a filter paper and concentrated under reduced pressure. 18.3 g of the extract was prepared. (Yield: 18.3%)

&Lt; Example 8 >

1 g of distilled water was added to 100 g of gardenia fruit, followed by addition of 1 g of cellulase and 2 g of amylase, extraction at 40 ° C. for 16 hours, heat treatment at 95 ° C. for 5 minutes to inactivate the enzyme, filtration through a filter paper, And lyophilized to obtain 18.9 g of gardenia extract. (Yield: 18.9%)

< Experimental Example  9>

The yield and the croccine content of the extracts prepared in Examples 1 to 8 are shown in Table 1. The content of crocin was analyzed by HPLC using the method described in Experimental Example 8.

Comparison of production efficiency of extract Example Enzyme treatment concentration Yield of extract (%) Active ingredient content (%) Remarks Example 1 - Example 2 Cellular 0.2% 12.0 1.7 Example 3 Cellular 1% 16.5 2.5 Example 4 Cellular 5% 17.2 2.4 Example 5 Amylase 0.4% 16.0 1.8 Example 6 Amylase 2% 17.5 2.5 Example 7 Amylase 10% 18.3 2.5 Example 8 Cellular 1% + Amylase 2% 20.3 2.6

Therefore, it was confirmed that the extract yield and the content of crocin pigment increased when the enzyme was extracted by using cellulase and amylase alone or in combination. It was also confirmed that the cellulase concentration was about 1% of the weight of the gardenia fruit juice, the amylase concentration was about 2% of the weight of the gardenia fruit juice, the extraction yield was lower than that, and the extraction efficiency was not significantly improved , And it was confirmed that the extraction efficiency was higher when they were mixed.

The method for producing the gardenia extract of the present invention is preferably used either alone or in combination with a cellulase (cellulase lysis enzyme) or an amylase (starch lyase) when preparing the extract by immersing the gardenia fruit in water .

For the preparation of the composition of the cosmetic composition containing the gardenia extract, a gardenia extract prepared by the method of [Example 8] was typically used.

When using the gardenia extract in cosmetics, its concentration is very important, and the amount of cosmetics per unit area is 100 times smaller than the amount of the cell culture per unit area. In view of this, the use concentration of the gardenia extract in cosmetics is about 100 times higher than that in the cell treatment Is required.

Therefore, when used in cosmetics, it is preferable to use the aqueous ginger extract of the present invention (croccine content 1.7 to 2.6%) at a concentration of about 1% or less. When the concentration is lower than the above range, the ultraviolet toxicity defense effect is weak. Or the product formulation, the present invention has been made to contain the ginger extract at a concentration of about 0.5 to 2% according to the cosmetic formulation.

A preferred embodiment for producing cosmetics using the gardenia extract prepared according to the present invention will be described. The following examples illustrate the weight percentage of each component relative to the weight of the manufactured cosmetic product, and are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples .

&Lt; Example 9 - Preparation of soft skin lotion (skin) 1 >

0.5% by weight of the gardenia extract (Example 8), 7.0% by weight of glycerin, 4.0% by weight of 1,3-butylene glycol, 0.3% by weight of hyaruronic acid, 0.1% by weight of allantoin, 0.1% by weight of DL-panthenol, 0.02% by weight of ethanol, 8.0% by weight of ethanol, 0.4% by weight of Polysorbate 20, octyldodeces-160 softener lotion.

&Lt; Example 10 - Flexible lotion (skin) production 2 >

1.0 wt% of gardenia extract, 7.0 wt% of glycerin, 4.0 wt% of 1,3-butylene glycol, 0.3 wt% of hyaruronic acid, 0.1 wt% of allantoin, 0.1 wt% of DL-panthenol, EDTA-2Na 0.02% by weight of ethanol, 8.0% by weight of ethanol, 0.4% by weight of Polysorbate 20, octyldodeces-160 softener lotion.

&Lt; Example 11 - Preparation of nutritional cream >

(Example 8) 1.0% by weight of wax extract, 0.5% by weight of cetostearyl alcohol, 1.1% by weight of glyceryl monostearate, 0.3% by weight of paraxybenzoyl ester, 1.1% by weight of cyclomethicone, 0.3% by weight of dimethicone, 1.0% by weight of jojoba oil, 2.0% by weight of squalane, 2.0% by weight of octyldodecanol, 3.0% by weight of liquid paraffin, 3.0% by weight of glycerin, 4.0% by weight of glycerin, 3.0% by weight of 1,3-butylene glycol, 0.08% by weight of Carbamo 940, and purified water having a small amount of pigment, fragrance and balance.

&Lt; Example 12 - Preparation of nutritional cream >

, 2.0 wt% of gardenia extract (Example 8), 0.7 wt% of beeswax, 0.5 wt% of cetostearyl alcohol, 1.1 wt% of glyceryl monostearate, 0.3 wt% of paroxybenzoyl ester and polyoxyethylene sorbitan triisostearate 1.1% by weight of cyclomethicone, 0.3% by weight of dimethicone, 1.0% by weight of jojoba oil, 2.0% by weight of squalane, 2.0% by weight of octyldodecanol, 3.0% by weight of liquid paraffin, 3.0% by weight of glycerin 4.0% by weight of glycerin, 3.0% by weight of 1,3-butylene glycol, 0.08% by weight of Carbamo 940, and purified water having a small amount of pigment, fragrance and balance.

Meanwhile, the cosmetic composition containing the gardenia extract may be developed into various products such as a cream, a lotion, a sun cream, a serum, a face toner, a cleansing milk, a body milk, a serum and the like, I will.

Claims (6)

A first step of adding a polysaccharide hydrolase to a mixture of gardenia fruit and distilled water to proceed hydrolysis at 40 to 60 ° C, a second step of inactivating the polysaccharide hydrolase by high temperature treatment at 90 ° C or higher, And a third step of filtering the extract obtained in step (a), and concentrating and drying the powder to obtain a powdery extract.
The method according to claim 1,
Wherein the polysaccharide hydrolase is a cellulase, amylase or a mixture thereof.
The method according to claim 1,
Wherein the polysaccharide hydrolase is used in an amount of 0.1 to 10% by weight of a gardenia fruit, respectively, or a mixture thereof.
A first step of adding a polysaccharide hydrolase to a mixture of gardenia fruit and distilled water to proceed hydrolysis at 40 to 60 ° C, a second step of inactivating the polysaccharide hydrolase by high temperature treatment at 90 ° C or higher, And a third step of filtering the extract obtained in step (1) and concentrating and drying the powder to obtain a powder.
5. The method of claim 4,
Wherein the gardenia extract comprises 0.5 to 2% by weight of the cosmetic composition.
The cosmetic composition according to claim 4, wherein the cosmetic composition is intended to protect skin cells against ultraviolet rays.

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