KR102163509B1 - Cosmetic Composition Comprising Extracts of Pine cone - Google Patents

Abstract

The present invention provides the effect of improving skin anti-inflammatory, skin regeneration, skin clarity, and skin contour improvement by containing red pine extract as an active ingredient in the cosmetic composition containing red pine cone extract as an active ingredient, hot water, alcohol, and candles. It relates to a cosmetic composition that can be used as functional cosmetics such as antibacterial, anti-inflammatory, and rejuvenating by using the extract extracted through the critical extraction method as an active ingredient of cosmetics.

Description

Cosmetic Composition Comprising Extracts of Pine cone}

The present invention relates to a cosmetic composition containing red pine pine cone extract as an active ingredient, and more particularly, antibacterial, anti-inflammatory, regeneration, etc. of cosmetics using the extract extracted through hot water, alcohol, supercritical extraction method using red pine pine cone extract. It relates to a cosmetic composition that can be confirmed that it can be used as the same functional cosmetics.

In recent years, with the rapid development of biotechnology, numerous mechanisms involved in skin aging and regeneration and components that play a decisive role in the mechanism have been identified. Among the ingredients, technologies for developing differentiated ingredients by searching for ingredients that can be used as cosmetic ingredients, and evaluating their effects have also greatly developed, and applications such as new functional bio cosmetics are being actively studied.

Pine (Pinus densiflora) is an evergreen coniferous arboreous tree of the coniferous plant, and is an evergreen coniferous arboreous tree that grows in the Far East such as Korea, Japan, and China. Pine cones are pine cones and are also called pine cones and pine cones. Small scales containing clusters of fruits and seeds are gathered in a round shape. The scales of pine cones are spread or folded depending on humidity. , The lower the humidity and the higher the unfolded state, the more it becomes a pangolin. The types of pine trees are divided into leaf shape and bark color, and most of the pine trees that grow wild in Korea are divided into two-leaf-shaped red pine, haesong, and banyan. In particular, when the color of the bark of a tree is red, it is called red pine, and when the color of a sword is light, it is called haesong or gomsol.

As constituents that can be used in pine trees, a small amount of protein, lipids, and ash are known in addition to moisture, sugar, and fiber, and all parts such as pine needles, pine cones, and rosin peel are used as medicinal and health foods. With the development of separation and purification technology for active ingredients, many studies are being conducted to develop products of commercial value such as pesticides, medicines, flavors, and antibacterial agents using pine trees.

The content of various compounds extracted from most plants varies depending on the part of the plant, growth environment, and harvest time, but in the same type of plant, the kind of compound is almost the same.

In particular, the content and purity of ingredients vary depending on the extraction method, and it is a method that overcomes the problems of organic solvents that are applied to the extraction and purification of expensive natural medicines.It is a supercritical fluid extract method that facilitates solvent recovery and does not have residual solvents. ) Is commercially useful. The supercritical fluid refers to a fluid that exists in a single phase above a critical temperature and pressure that has both liquid and gas characteristics. A substance in a supercritical state can change its physical and chemical properties continuously by changing its temperature and pressure near the critical point. The supercritical fluid is mainly composed of carbon dioxide or carbon dioxide and a small amount of an auxiliary solvent, and the critical temperature and pressure are low, so that extraction can be performed under mild conditions.

In addition, since there is no toxicity, flammability, reactivity and corrosiveness with the material to be extracted, carbon dioxide is commonly used as an extraction solvent, and the extract can be used in various ways in food, cosmetics, and pharmaceuticals. According to the results of previous studies, polyphenol-lignin components in pine cone extract are effective in anti-allergic and immune system diseases, and antioxidant and antibacterial effects have been reported.

On the other hand, human skin undergoes various physical and chemical changes during the aging process. The causes are largely divided into intrinsic aging and photo-aging, and studies on this have been actively conducted. UV rays, stress, disease conditions, environmental factors, wounds, and aging can be caused by activating free radicals.If such a condition intensifies, it destroys the antioxidant defenses existing in the living body and damages cells and tissues, leading to adult diseases. And promotes aging.

More specifically, skin cells and tissues are destroyed by oxidation of lipids, proteins, polysaccharides, and nucleic acids, which are major components of the skin, resulting in skin aging. Particularly, protein oxidation is severe in the skin's connective tissues, such as collagen, hyaluronic acid, elastin, proteoglycan, fibronectin, etc. If this gets worse, mutations in DNA lead to mutations, cancer, and immune function decline.

Therefore, free radicals generated during the body's metabolic process, UV irradiation, and free radicals mediated by inflammatory reactions are eliminated to protect the cell membrane, and cells that have already been damaged must be regenerated by active metabolism to proliferate the skin. Can recover quickly and maintain healthy skin.

In addition to these free radicals, an enzyme called matrix metalloproteinase (MMP), which is a collagen-degrading enzyme, is involved in aging. In other words, the synthesis and decomposition of extracellular matrix such as collagen in vivo is properly regulated, but as aging progresses, collagen synthesis decreases, and the expression of matrix metal proteolytic enzyme (MMP), an enzyme that degrades collagen, is promoted to the skin. And wrinkles are formed. In addition, such degrading enzymes may be activated by UV irradiation.

Therefore, there is a need for development of a substance capable of regulating or inhibiting the expression of MMP in which activation is induced in cells. Most of the ingredients used so far as materials for cosmetics simply inhibit enzyme activity.

Although many studies have been conducted on the essential oil components of the pine family, research on the ingredients using supercritical extracts of pine cones from red pine, which grows most in Korea, is insufficient.

This study examines the possibility of using red pine cones as a cosmetic through component analysis of the extract obtained by the supercritical extraction method, and intends to use them as data for development as a functional material in the cosmetics industry.

1.A pine cone extract having antibacterial and antifungal activity, and cosmetic composition comprising the same for improving skin disease (Patent et al. No. 10), which has antibacterial and antifungal activity, and an active ingredient thereof. -0721421) 2. Cosmetic Composition Comprising Extracts of Pinecone of Pinus sylvestris (Patent Registration No. 10-1817129) 3. Moisturizing cosmetic composition containing the rosin, the needles of red pine and the root of the red pine (Patent Registration No. 10-1608258)

The present invention has been conceived to solve the above problems, and its purpose is to use red pine pine cone extract as a functional cosmetic such as antibacterial, anti-inflammatory, regeneration of cosmetics using extracts extracted through hot water, alcohol, and supercritical extraction methods. It is to provide a cosmetic composition containing red pine pine cone extract that can be utilized as an active ingredient.

In addition, the present invention is to provide a cosmetic composition having the effect of improving skin anti-inflammatory, skin regeneration, skin transparency, and skin contour improvement by containing red pine extract as an active ingredient.

According to a feature of the present invention for achieving the above object, the total polyphenol content is measured by analyzing the extract obtained by supercritical fluid extract (SFE) extraction by pulverizing dried red pine cones and measuring the total polyphenol content. SFE through the safety of supercritical (SFE) extract by confirming the function, confirming the cell viability and anti-inflammatory effect through cytotoxicity evaluation, and preparing an emulsion of the effective substance for recovery of the damaged area through cell proliferation and migration. Provides a cosmetic composition containing red pine pine cone extract as an active ingredient in which the extract obtained from is used as a raw material for anti-inflammatory and regenerative cosmetics.

In addition, the present invention provides a cosmetic composition having the effect of improving skin anti-inflammatory, skin regeneration, skin transparency improvement, and skin contour improvement by containing red pine extract as an active ingredient.

The cosmetic composition containing the red pine pine cone extract according to the present invention as an active ingredient has an anti-inflammatory effect due to low nitric oxide expression in the anti-inflammatory experiment, and as a result of observing the stability for 30 days by preparing an emulsion containing pine cone extract, the SFE extract was added. The emulsion provides stability at pH and high temperature (40℃), so it can be used as a raw material for anti-inflammatory and regenerative cosmetics.

In addition, the cosmetic composition using the red pine pine cone extract according to the present invention as an active ingredient can selectively extract the extract through hot water, alcohol, and supercritical extraction methods, and can be used as functional cosmetics such as antibacterial, anti-inflammatory, and regeneration. .

In addition, the cosmetic composition comprising the red pine cone extract according to the present invention as an active ingredient contains red pine extract as an active ingredient to improve skin anti-inflammatory, skin regeneration, skin transparency, and skin contour improvement.

1 is a graph of the sugar composition of the cellulose composition of the hot export of red pine pine cones according to an embodiment of the present invention.
Figure 2 is a graph of the components of Dgitoxigenin and Stigmast of red pine pine cone alcohol extract according to an embodiment of the present invention.
3 is a component graph of supercritical extraction of red pine cones according to an embodiment of the present invention.
Figure 4 is a graph comparing the antioxidant activity measurement according to the extraction method of red pine pine cones according to an embodiment of the present invention.
5 is a graph comparing cell viability according to the method of extracting red pine cones according to an embodiment of the present invention
6 is a graph comparing the measurement of nitric oxide activity according to the method of extracting red pine cones according to an embodiment of the present invention.
7 is an evaluation result of cell growth and promotion effect according to the method of extracting red pine cones according to an embodiment of the present invention.
8 is a graph of PH conversion of an emulsion containing red pine pine cone extract according to an embodiment of the present invention.
9 is a phase separation result according to the temperature change of the emulsion containing red pine pine cone extract according to an embodiment of the present invention.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The cosmetic composition containing the red pine cone extract according to the present invention as an active ingredient is extracted with hot water (WE), alcohol (EE), and supercritical method (SFE) in order to utilize the pine cone as a cosmetic material, and each extraction method The main components of the obtained extract were analyzed by GC/MS. As a result of measuring the polyphenol content, WE was 180.75±1.83 mg/g, EE was 252.69±0.34 mg/g, and SFE was 270.10±0.97 mg/g.When the antioxidant power was measured using the DPPH method, it was obtained with SFE. The extract was confirmed to have high antioxidant activity even at low concentration (0.125%). As a result of measuring the antibacterial effect of pine cone extract, 12.1 ± 1.04 mm in Staphylococcus aureus and 11.8 ± 0.45 mm in Staphylococcus epidermidis were found in SEF extract.

As a result of the cytotoxicity evaluation, the cell viability of more than 80% was confirmed in all extracts, and the expression level of nitric oxide was low in the anti-inflammatory experiment, which was confirmed through the anti-inflammatory effect. As a result of observing and testing stability for 30 days by preparing an emulsion containing pine cone extract, it was confirmed that the emulsion containing the SFE extract was stable at pH and high temperature (40°C), and the extract obtained from the pine cone as SFE was anti-inflammatory. And it was confirmed that it is applied as a raw material for recycled cosmetics.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited to these examples. The following examples are only illustrative of the present invention, and the scope of the present invention is not limited by the following examples, and based on the present invention, those skilled in the art who belong only to the relevant technical branch can appropriately modify and use them. ,

1. Experimental materials and methods

end. Experiment material

The pinecone, which is a sample of the present invention, is an immature pinecone, which was collected from red pine pines cultivated in Goesan, Chungbuk between the end of July and the beginning of August. After removing the woody part from the top of the immature pine cone, it was washed with tap water and distilled water, and then naturally dried indoors. Mixer crushed pine cones were extracted by three methods: hot water extraction, alcohol extraction, and supercritical extraction.

1) Manufacture of pine cone extract

A) Water extract (WE)

Hot water extraction was performed using a pulverized pine cone using a rotary vacuum concentration extractor. After adding 300 ml of 3rd distilled water to the concentrated extraction tank, 30 g of crushed pinecone was extracted in a constant temperature water bath for 6 hours while maintaining at 90°C and 100°C by heating. After the extraction was completed, the pine cone and the extract were separated and concentrated in vacuo. The concentrate was freeze-dried to prepare a powder form.

B) Ethanol extract (EE)

Alcohol extraction was performed using a rotary vacuum concentrated extractor using pulverized pine cones. After adding 30 ml of 70% ethanol to the concentrated extraction tank, 30 g of the pine cone crushed product was extracted in a constant temperature water bath for 2 hours while maintaining at 90°C and 100°C by heating. After the extraction was completed, the pine cone crushed product and the extract were separated and concentrated in vacuo. The concentrate was stored at room temperature.

C) Supercritical fluid extract (SFE)

Supercritical fluid extraction was performed using a supercritical fluid extraction device (ISA-SEFE-0500-0700-080, Ilshin Autoclave, Daejeon, Korea). After the dried pine cones were pulverized into 100 mesh, 500 g of pine cones were injected into the reactor, and the pressure was 400 bar, the temperature was 50°C, and the co-solvent ethanol flow rate was extracted for a total of 4 hours under the conditions of 3 ml/60 minutes. Silver was extracted under the conditions of 40 bar, temperature 40°C, and carbon dioxide (CO ₂ ) flow rate of 60 ml/1 minute.

2) reagent

(+)-α-Tocopherol, L-ascorbic acid, arbutin, EDTA, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical used for free radical scavenging activity was purchased from Sigma (USA) and used. For various solvents such as ethanol (EtOH), methanol (MeOH), ethyl acetate (EtOAc), and hexane, commercially available special reagents were used.

RAW 264.7 cells, a type of macrophage used for cytotoxicity measurement, were purchased from Korean Cell Line Bank (KCLB) and used in the experiment. Cell culture reagents such as Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), and penicillin-streptomycin were purchased from Invitrogen (Gibco™, Carlsbad, CA, USA). Other reagents were used without additional treatment of grade 1 or higher.

I. Experimental method

1) GC / MS (Gas chromatography / Mass spectrometer) analysis

GC/MS equipment (GC-2010, Shimadzu Co., Kyoto, Japan) was used to analyze the components of hot water, alcohol, and supercritical extracts. After sufficiently stirring each extract in DMSO, the suspended matter was removed using a centrifuge and filtered through a micro filter (0.45 μm) to prepare a sample. Component analysis was conducted under the following conditions. The column is BD-5 (60mm×0.25mm×0.25mm), the carrier gas is He with a flow rate of 1mL/min, the injection temperature is 250℃, the split ratio is 10:1, the oven temperature is increased from 50 to 300℃/3℃, The components were analyzed under the condition of 1 µl of injection volume, and the components were quantified under the conditions of a mass range of 28 to 550 in a mass selective detector (MSD) and a scan mode in the acquisition mode.

2) Measurement of physiological activity of pine cone extract

A) Measurement of total polyphenol compound content

0.5 g of the extracted sample was added to 10 ml of solvent, shaken for 20 minutes, centrifuged for 10 minutes, extracted three times with paper filter paper, and the final volume was determined to be 50 ml. 2 ml of 2% Na ₂ CO ₃ was added to 100 μl of the extract, vortexed, and left for 2 minutes. 100 µl of 50% Folin-Ciocalteau's phenol reagent is added, vortexed and left for 30 minutes, and the absorbance is measured at 750 nm.

Gallic acid (Sigma Chemical Co., St.Louis, MO, USA) was prepared at a concentration of 0 ~ 200㎍/mL and analyzed by the same method as the sample, and the total phenol content of the sample extract was calculated from a standard calibration curve. It is expressed as gallic acid equivalents (mg GAE/g extract).

B) Measurement of antioxidant activity

The electron donating ability to measure the antioxidant effect was measured. 1 ml of 0.2 mM DPPH was added to 2 ml of each sample solution, stirred, and left in the dark for 30 minutes, and the absorbance was measured at 517 nm. The electron donating ability was expressed as the rate of decrease in absorbance of the added and non-added sample solutions.

A: absorbance of the control group (DPPH solution)

B: absorbance of the sample

3) Evaluation of antibacterial activity

The antibacterial activity of pine cone extract was measured by disc diffusion assay. The plate medium for antibacterial test was plated with 100 µl of passaged Staphylococcus aureus (SA) and Staphylococcus epidermis (SE) and dissolved in a liquid medium with 1% of the extract (WE, EE, SFE) on a paper disc (6.5mm, diameter). After slowly absorbing, drying, and incubating for 24 hours at 37°C in close contact on a flat plate, and then measuring the clear zone around the disc to compare the antibacterial activity.

4) Cell viability measurement

A) MTT assay measurement

The extract was first dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, MO, USA), then diluted in cell culture medium and treated by concentration. The cytotoxicity evaluation of the sample was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5 -diphenyl -2H-tetrazolium bromide (MTT) with reference to Hwang et al. 12. Macrophage RAW 264.7 cells were dispensed into a 96 well plate at a concentration of 1×10 ⁴ cells/mL, stabilized in a cell incubator (37° C., 5% CO ² ), and cultured for 48 hours by treating the extracts by concentration.

After that, the MTT reagent was treated and incubated for 3 more hours to be reduced by the enzymatic action of viable cells, and then the culture medium was removed, and DMSO was added to each well to dissolve the resulting formazan crystals, and then the Bio kinetics reader (ELISA, BioTek, US) Absorbance was measured at 560nm using.

A: absorbance of the experimental group

B: absorbance of the control group

5) Anti-inflammatory effect measurement

A) Nitric oxide inhibitory activity measurement

The amount of nitric oxide produced from Macrophage RAW 264.7 cells was measured using a griess reagent for NO ₂ ^- present in the cell culture medium. 100 µl of the cell culture supernatant and 100 µl of Griess reagent were mixed and reacted for 10 minutes, and the absorbance was measured at 540 nm. The amount of NO ₂ -was quantified using the standard curve of NaNO ₂ .

6) cell migration

To evaluate whether or not to promote cell migration by the test substance, HaCaT cells were dispensed into a 48 well plate at a concentration of 5×10 ⁴ cells/ml, stabilized in a cell incubator (37℃, 5% CO ₂ ), and cultured for 24 hours. After that, the cell monolayer was scratched with a p200 pipet tip to create an empty space, and cultured for 24 hours with a medium containing no serum, and the degree of cell migration of the experimental group was compared with the control group using an inverted microscope.

7) Manufacture of emulsion containing pine cone extract

A) Manufacture of emulsion

Preparation of an emulsion containing pine cone extract was prepared according to [Table 1]. The aqueous phase and the oil phase were divided and dissolved by heating to 80℃, followed by first emulsification for 15 minutes at 5,000rpm using a homomixer, and cooling to 40℃ for secondary emulsification. WE, EE, and SFE extracts were added, emulsified at 3,000 rpm for 5 minutes, and cooled to 30°C.

Emulsion extract ingredients International Nomenclature Cosmetic Ingredient Sclerotium Gum
Glycerin
CetearylOlivate/SorbitanOlivate
Wate
HydrogenatedLecithin/C12-16Alcohols/PalmiticAci
GlycerylStearate, Lipophil
GlycerylStearateS
Dimethicon
Ethanol
Pine cone(WE,EE,SFE)extract

8) Measurement of stability of emulsion containing pine cone extract

A) pH measurement

The emulsion stored at 25°C was measured using a pH meter Metrohm 691 (Metrohm UK Co., Switzerland). (1,7,14,21,28 days)

B) Stability test according to temperature

The stability test method according to the temperature of the emulsion containing pinecone extract was stored in temperature conditions (0, 25, 40℃) and the state change according to the change over time was visually evaluated. (1,7,14,21,28) days)

3. Test results and discussion

1) Pine cone extraction yield

Pine cones were completely dried and then crushed to proceed with extraction. The moisture content of the pine cone was measured to be 9.8%. The yield according to the extract method was confirmed as hot water extract (WE) 23.32%, alcohol extract (EE) 32.12%, supercritical extract (SFE) 20%. The high yield of the general solvent extract can be said to be a result of higher extraction selectivity of SFE compared to the general solvent extraction method.

2) GC/MS component evaluation

As a result of component analysis of the sample obtained by the WE method, phenolic compounds such as quinic acid (23.54%), methyl mannose (20.12%), and methyl glucose (22.86%) were identified as major compounds. Since the pinecone is composed of woody cellulose, the sugar component was confirmed as shown in FIG. 1 through hot water extraction.

Pine cone chemical composition by hot water extract Compound name R.Time Area (%) Catechol 15.050 3.72 5-APDB 15.261 2.08 2-Methoxy-4-vinylphenol 16.637 1.87 Silane 20.593 3.25 5,5-Dimethyl-1,5-oxasilonan-9-one 20.742 1.02 Methylmannose 22.168 20.12 Quinic acid 22.8 23.54 Methylglucose 25.914 22.86 Andrographolide 35.614 1.31 1-Phenanthrenemethanol 36.499 1.72 Methyl abietate 37.162 1.19

When extracted by the EE method, dehydroabietic acid (20.41%), known as a pineapple compound, Kauran's compound (6.71%), which has anti-inflammatory effects, and the main methyl abietate (2.26%) compound of pine rosin were detected.

In addition, vitamin A retinol acetate (2.78%) and Dgitoxigenin and Stigmast components extracted from the bark were found in trace amounts as shown in FIG. 2.

Pine cone chemical composition from alcohol extract Compound name R.Time Area (%) Methane, sulfinylbis 15.407 21.96 Dehydroabietic acid 33.4,34.8,37, 20.4 Cycloheptane 34.762 4.84 Kaura-5,16-dien-18-ol 40.920 6.71 Androstatriene 36.356 2.12 Methyl abietate 37.177 2.26 Retinol acetate 37.535 2.78 Medroxyprogesterone acetate 41.678 1.51 Digitoxigenin 42.349 1.33 Stigmast-5-en-3-ol 48.034 1.16

In the case of the SFE method, most fat-soluble compounds were observed. Andrographolide (1.84%), Pimara-8(14), 15-dien-19-oicacid (5.84%), 17-(acetyloxy)-kauran-18-al (1.47%), which is a bio-active substance and known as a natural antibiotic and anti-inflammatory agent. The diterpene component of was confirmed.

Phenanthrene (7.72%) alkaloid components and vitamin A (40.06%) compounds used as drugs were detected. It was confirmed as shown in FIG. 3 that the organic acid Abietic acid (21.73%), which constitutes the main component of the pine resin identified in the general solvent extraction method, contained more content than that extracted with the general solvent.

Pine cone chemical composition by supercritical extract Compound name R.Time Area (%) 1-Phenanthrene carboxylic acid 33.621 2.92 Andrographolide 34.258 1.84 Pimara-8(14),15-dien-19-oicacid 34.531 5.84 1-Phenanthrene carboxaldehyde 35.267 7.72 Naphthalene 35.986 13.18 Vitamin A 36.956 40.06 Abietic acid 37.767 21.73 17-(acetyloxy)-kauran-18-al 41.132 1.47

3) Measurement of physiological activity of pine cone extract

A) Total polyphenol content

Polyphenol chemicals present in plants are compounds that have a phenolic hydroxyl group, and are generically referred to as flavonoids, anthocyanins, tannins, and catechins, and are generic names for compounds that are components such as pigments, astringent taste, and bitter taste.

Flavonoids belong to the category of phenolic compounds and are present in almost all parts of plant leaves, flowers, fruits, stems and roots, and are effective in physiological activities such as antiviral, anti-inflammatory, anti-cancer, as well as antioxidants that remove reactive oxygen species (ROS). It is reported that there is. It is used as an indirect indicator of antioxidant activity through the content of phenolic compounds and flavonoids, which are widely distributed secondary metabolites in the plant world.

As a result of measuring the content of polyphenolic components according to the pine cone extraction method, the hot water (WE) extract sample showed 180.75±1.83 mg GAE/g extract content, and the alcohol (EE) extract sample showed 252.69±0.34 mg GAE/g extract content. Done. Supercritical (SFE) extract sample was 270.10±0.97mg GAE/g extract, yielding the highest content.

In a study by Ryu et al., 232.99±0.18 mg GAE/g extract content was reported when immature pine cones were extracted twice with 70% ethanol, and the polyphenol content of 185.24±0.18 mg GAE/g extract was also reported in the hot water extracted sample of the present invention. Was calculated. The present invention has been shown to be somewhat different, which is considered to be due to differences in the sampling time of the sample and the altitude and temperature of the region.

Total polyphenol concentration in pine cone extracts Extacts Total phenolic compound
(mg/g) WE 180.75±1.83 EE 252.69±0.34 SFE 270.10±0.97

Each value is expressed as mean±SD (n=4).

B) Measurement of antioxidant activity

1,1-Diphenyl-2-picrylhydrazyl (DPPH), which has a dark purple color, is a relatively stable free radical that is reduced by antioxidants and aromatic amines to decolorize it. This is used to search for antioxidants from various natural materials. It is being used a lot.

As shown in FIG. 4, high scavenging activity was exhibited in the EE and SFE extraction methods except for the HW extraction method. HW extract was found to be 47.17±0.79 at 0.125%, 51.41±1.05% at 0.25%, 59.6±1.05 at 0.5%, and 75.42±1.8% at 1%.

The EE extract and SFE extract were 115±7.79% and 117±17.19% at low concentration (0.125%), indicating high scavenging ability at low concentration. It was confirmed that the antioxidant activity of the extracts obtained from SFE was contained in a large amount, and the antioxidant activity was higher than that of the commonly used WE and EE extracts.

4) Evaluation of antibacterial activity

The antimicrobial activity of pine cones was compared with the concentration of Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) by disc diffusion assay.

As shown in Table 6, the clear zone of the WE extract showed antimicrobial activity of 7.2 ± 0.24 mm in SA and 6.8 ± 0.74 mm in SE.

The EE extract showed clear zones of 9.6 ± 0.86 mm in SA and 8.2 ± 0.36 mm in SE. SFE extract showed high antibacterial activity of 12.1 ± 1.04 mm in SA and 11.8 ± 0.45 mm in SE. In SFE, extracts were found to have higher antibacterial effects than WE and EE extracts.

Disc diffusion method of antibacterial against SA and SE after 24h Sample Clear zone diameter (mm) Staphylococcus aureus Staphylococcus epidermidis WE 7.2 ± 0.24 6.8 ± 0.74 EE 9.6 ± 0.86 8.2 ± 0.36 SFE 12.1 ± 1.04 11.8 ± 0.45

5) cell viability

MTT assay is one of the widely used methods for cytotoxicity and cell proliferation because it uses a 96-well plate and can easily read many samples using ELISA for test results. The MTT assay is evaluated by measuring purple fomazan produced by the dehydrogenase of mitochondria in cells.

As shown in the accompanying Figure 5, in order to observe the stability according to the concentration of the pine cone extract is the result of the MTT assay.

In the case of the WE extract, a growth rate of 0.125% to over 100% was confirmed. Cell viability of more than 90% was confirmed in the EE extract, and 80.1% cell viability was confirmed at a concentration of 1% in the case of the sample obtained by the SFE extraction method.

6) Nitric oxide activity measurement

Lipopolysaccharide (LPS), the outer membrane component of Gram-negative bacteria, responds to early infection of macrophages and plays a pivotal role in host defense, but excessive LPS stimulation in macrophages causes tumor necrosis factor-α (TNF-α), interleukin (IL). It secretes pro-inflammatory mediators such as -1β and IL-6, and secretes inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2).

NO is produced from inducible NO synthase (iNOS) when macrophages are activated, and it is also known to kill bacteria or remove tumors, but excessive production causes inflammation, causing tissue damage, genetic mutation, and nerve damage.

As a result of performing a nitric oxide assay to confirm the amount of NO generation, as shown in FIG. 6, when only LPS was treated alone, the amount of NO generation increased by about 3 times compared to the negative control, and the concentration of WE, EE and SFE When treated separately, it was confirmed that the amount of NO production decreased in a concentration-dependent manner in both extracts.

7) cell migration

When a scratch is applied to the cell monolayer, the surrounding cells repair the damaged area through cell proliferation and migration. In other words, cell migration has great significance in the process of wound healing along with cell proliferation.

The effect of the extract on cell growth and migration was evaluated in order to experimentally verify whether pine cone extract (WE, EE, SFE) helps wound healing.

The cell migration promoting effect was evaluated with the cell scratch assay, which is recognized as a direct and economical evaluation method among in vitro wound healing experiments. As a result, as shown in FIG. 7, the experimental group treated with the extract obtained from SFE among the pine cone extract recovered faster than the control group, so that the extract obtained from SFE among the pine cone extract was a useful material for wound healing.

8) Measurement of stability of emulsion containing pine cone extract

A) pH measurement

As a result of measuring the pH of each extract-containing emulsion stored at room temperature (25° C.) for 28 days, there was no change in pH according to the observation period of the emulsion containing WE, EE, and SFE extracts as shown in FIG. 8.

B) Stability test according to temperature

In order to investigate the spontaneous deterioration and chemical and physical changes during the manufacture of cosmetic formulations, temperature stability evaluations of 0, 25, and 40°C were performed as shown in [Table 7].

Results of stability test of the emulsion containing pine cone extracts in constant temperature conditions (0, 25, 40℃) Sample Temperature 0 Day 1 Day 7 Day 14 Day 21 Day 28 Day
WE 0 O O O O O O 25 O O O O O O 40 O O O O X X
EE 0 O O O O O O 25 O O O O O O 40 O O O O O O
SPE 0 O O O O O O 25 O O O O O O 40 O O O O O O

O: Stable, X: Unstable

At high temperature, the appearance of rancidity, color change, viscosity, evaporation, suspension precipitation, turbidity (transparency), and separation were observed, and at low temperature, chemical and physical changes such as coagulation, precipitation, turbidity (transparency), crystal precipitation, and separation were observed.

As a result of checking the stability according to temperature by storing the emulsion containing pine cone extracts (WE, EE, SFE) in a constant temperature water bath of 0℃, 25℃, and 40℃, the emulsion prepared by adding SFE extract as shown in FIG. 9 Only at the temperature of 0 ℃, 25 ℃, 40 ℃ showed stability until 28 days after production without changes in color, color development, discoloration, aggregation, rancidity, etc. It was confirmed that the phase separation occurred in the emulsion to which the WE and EE extracts were added after 2 weeks.

4. Conclusion

The present invention evaluated the physiological activity of pine cones according to the extraction method and evaluated the possibility of application to cosmetics. That is, in the evaluation of the physiological activity of the pine cone, the total polyphenol content of the pine cone was 180.75±1.83 mg/g for WE, 252.69±0.34 mg/g for EE, and 270.10±0.97 mg/g for SFE.

In particular, the extract obtained with SFE showed relatively high radical scavenging ability, and showed excellent antibacterial effect against Staphylococcus aureus and Staphylococcus epidermis. As a result of the cytotoxicity evaluation of each extract, a cell viability of 80% or more was confirmed. In the anti-inflammatory experiment, the extract obtained with SFE confirmed the highest anti-inflammatory activity.

In addition, as a result of observing the wound healing ability through cells, it was confirmed that the cell migration and proliferation rate were high in the SFE-treated group. Through this, it was confirmed that the development of cosmetics using pine cone extract can be used as functional cosmetics such as antibacterial, anti-inflammatory, and regeneration.

In the above, although it has been described in detail through specific embodiments of the present invention, the terms specifically used herein are used only for the purpose of describing the present invention, and limit the meaning or limit the scope of the present invention described in the claims. It was not used for. Therefore, a person of ordinary skill in the art can make various modifications from this to various modifications and equivalent other embodiments.

Claims (3)

The extract obtained by pulverizing the dried red pine cones and analyzing the extract obtained by supercritical fluid extract (SFE) was analyzed by GC/MS to measure the total polyphenol content to confirm antioxidant activity, and cell viability and anti-inflammatory through cytotoxicity evaluation. Through the safety of supercritical (SFE) extract, the extract obtained from SFE is used as a raw material for anti-inflammatory and regenerative cosmetics by confirming the effect and making an emulsion of the effective substance for recovery of damaged areas through cell proliferation and migration. Cosmetic composition containing red pine cone extract as an active ingredient, characterized in that.
delete delete

Images