KR101046166B1 - Cosmetic composition having melanin synthesis inhibitory activity, containing raspberry root extract prepared using supercritical carbon dioxide - Google Patents

Abstract

The present invention relates to a cosmetic composition having a melanin synthesis inhibitory activity containing the extract of the root of the herb oil extract prepared using supercritical carbon dioxide. More specifically, the present invention exhibits higher activity and color tone improvement when extracting a useful substance having melanin synthesis inhibitory activity from the roots of Matsutake Sprout, compared to conventional solvent extraction methods (ethanol, methanol, water). Cytotoxicity relates to a cosmetic composition containing a botanical herb extract extracted by a method using alleviating supercritical carbon dioxide.

Oil herb, supercritical extract, melanin, color tone, cytotoxicity, cosmetics

Description

Cosmetic composition with melanin synthesis inhibitory activity containing the extract of the root oil extract from supercritical carbon dioxide {Cosmectic composition for improving an anti-melanogenesis activity comprising Peucedanum japonicum Thunb. root extract manufactured by Supercritical Carbon dioxide extraction}

The present invention relates to a cosmetic composition having a melanin synthesis inhibitory activity, containing the extract of the root of the herb oil herb prepared using supercritical carbon dioxide. More specifically, the present invention is extracted from the roots of the oil from the roots of the oil extract produced by the method using supercritical carbon dioxide to increase the melanin synthesis inhibitory activity, improve color tone, and alleviate cytotoxicity It relates to a cosmetic composition containing the extract.

Boiled oil herb ( Peucedanum japonicum Thunb.) Is a perennial plant belonging to the mountain family, and it grows on the beaches of East Asia such as Japan, Taiwan, China, and the Philippines in the world. The young sprouts of rape oil herb are used for edible foods such as herbs, and the root is one of the medicinal plants used for the windbreak of herbal medicine together with Glehnia littoralis Fr. Schm . Pharmacological activities such as anticonvulsant, antiallergic effect ( Aida Y et al., Pharmacol , 20 , 343-351, 1998) and antiplatelet activity ( Chen IS et al., Phytochemistry , 41 , 525-530, 1996) It is reported. In addition, Rutin, Neochlorogenic Acid, Cryptochlorogenic Acid, Chlorogenic Acid, etc. have been identified from the leaves of these leaves, and these are the components showing antioxidant activity ( Masashi H et al., J. Agric. Food Chem., 51 , 5255-5261, 2003).

Melanin is a pigment that is widely distributed in animals and plants. In humans, it determines the skin color and plays an important role in protecting the human body from harmful ultraviolet rays and free radicals (Jong Eon Chin et.al., 2006). The melanin pigment is synthesized from L-tyrosine through several stages of oxidation, and its biosynthetic mechanism is tyrosinase DHICA oxidase (Trosinase related protein-1, TRP-1) and DOPAchrome tautomerase. (tyrosinase related protein-2, TRP-2) and Catechol-O-methyltransferase (COMT) are involved (Arcoa P. et.al., 1993, Paval S. et. al., 1993, Jimenez Cervants et.al., 1994) Among these, tyrosinase oxidizes L-tyrosine and dopa (3,4-dihydroxyphenylalanine (DOPA)), thereby guiding dopa (DOPA) and dopaquinone (DOPA-). quinone). Since most of the skin color and skin pigmentation are determined by the amount and distribution of melanin in keratinocytes, it is important to inhibit melanin formation in order to test the whitening effect of the whitening material. Arbutin (Maeda K. et al., 1996, Chakraborty AK. Et al., 1998), ascorbic acid, and derivatives thereof (Ando S. et., Et al.) Inhibit the activity of tyrosinase related to the biosynthesis mechanism of melanin pigment. al., 1993, Kameyama K. et al., 1996), kojic acid (Cabanes J et al., 1991) and many other natural products have been reported, but these materials have the disadvantage of being unstable or of low efficacy.

On the other hand, Japanese patent (application number: 2002184099) has already been reported that the inhibitory activity of melanin synthesis of the ethanol extract of radish herb root. However, the present inventors have obtained the ethanol extract of radish herb root and concentrated to a concentration level of 0.5 ~ 1.0% (weight / volume), which is suitable for use as a cosmetic raw material, and then confirm whether it can be used as a main efficacy ingredient of the whitening cosmetics. As a result, I found some problems. First, in order to achieve sufficient efficacy in the whitening cosmetics there was a limit that can not be prescribed the raw material at a high concentration. In other words, the development of whitening cosmetics containing ethanol extracts from the roots of white oil with whitening activity is generally predominantly 10 times higher than the effective concentration in cell experiments. to be. However, the ethanol extracts of the root oil ethanol extract prepared for use as a cosmetic raw material had a dark red hue, and this was prescribed at a level of about 5 to 10% for the development of a whitening cosmetic. As a result, the formulation of the developed whitening cosmetic was dark. It was tinted. Therefore, in order to solve this problem, the present inventors investigated purified fractions showing the highest melanin synthesis inhibitory activity from the root ethanol extract of radish sprouts. As a result, the Hexane fraction, in which the nonpolar components were concentrated and purified, showed the highest melanin synthesis inhibitory activity compared to the fractions obtained using ethyl acetate, butanol, water, and the like. It was found that the activity was increased. When the hexane fraction was prepared at a concentration of 0.5-1.0% (weight / volume), although the color tone did not improve significantly, it could be prescribed at a lower concentration in the whitening cosmetic composition due to the increased activity. However, in this case, the problem that the cytotoxicity is increased by about 10 times in the cultured skin cell conditions. That is, there was a possibility of causing a skin safety problem by the high concentration of extract formulations. Due to these current problems, even if the extract of the root oil extract is excellent in whitening activity has not been used smoothly as the main ingredient of the whitening cosmetics. Therefore, it is necessary to develop cosmetic raw materials with high melanin synthesis inhibitory activity from botanical herb sprouts while improving color tone, while reducing cytotoxicity.

Accordingly, the present inventors have made high-purity purified cosmetic raw materials with high melanin synthesis inhibitory activity while improving color tone, while reducing cytotoxicity. In this case, it was confirmed that the dark red extract color tone of ethanol-based organic solvent extraction was alleviated, and that the increase in cytotoxicity in the case of organic solvent purification using hexane was reduced while the melanin synthesis inhibitory activity was improved. The present invention was completed.

The present inventors prepared the ethanol extract and hexane purified fraction thereof to investigate the use of the oil herb root as a whitening cosmetic raw material, and then investigated the melanin synthesis inhibitory efficacy and cytotoxicity, respectively, and also titrated for using them as cosmetic raw materials. The color tone in the state concentrated to concentration was investigated. In addition, the present inventors have continued to overcome the dark red color tone and cytotoxicity increase, which is a problem of the ethanol extract and the hexane fraction of the ethanol extract roots from these results, and the present inventors have a high content of the bran extract produced using supercritical carbon dioxide. It was found that the color tone was improved while having melanin synthesis inhibitory activity, while the cytotoxicity was alleviated, and thus it was suitable for use as a cosmetic raw material.

In other words, the supercritical carbon dioxide was added to the root biomass of radish herb root at conditions of 300 bar and 60 ° C to inhibit the melanin synthesis activity, cytotoxicity, and color tone of the supercritical extract fractions. Hexane) extract compared with the case of the extract to determine the optimum extraction conditions to achieve the object of the present invention.

The supercritical carbon dioxide extraction condition of the present invention was indirectly confirmed in the fractional experiments by the organic solvent type, which is a condition for extracting the non-polar components of the root oil of the oil. In other words, it showed the best melanin synthesis inhibitory activity in the hexane fraction of the root of the oil herb, in general, it is known that the plant biofraction obtained using hexane contains most of the non-polar components insoluble in water, including the lipid component of the plant. Therefore, the supercritical carbon dioxide extraction condition applied in the present invention is to apply the conditions of 300bar, 60 ℃ that can efficiently extract the lipid components from the plant material. To this end, as disclosed in European Patent No. 925,724, an extraction apparatus consisting of an extraction tank, a heat exchanger, a pump, a carbon dioxide storage tank, a chiller (cooling condenser), a pressure reducing separator, and a pressure reducing valve is used. In the extracting device, the extraction tank serves to extract the root of the boiled oil herb, the pump serves to pressurize the supercritical carbon dioxide, the heat exchanger serves to heat the supercritical carbon dioxide, The pressure reducing valve serves to decompress the supercritical carbon dioxide emitted from the extractor, and the pressure reducing separator completely decompresses the supercritical carbon dioxide, thereby separating the gaseous carbon dioxide, and the chiller converts the decompressed carbon dioxide into the supercritical carbon dioxide. It serves to convert, and the carbon dioxide reservoir serves to store supercritical carbon dioxide.

In order to extract a specific component from the root of the oil herb using the extracting device, the raw material is first introduced into the extraction tank, the supercritical carbon dioxide passed through the pump and the heat exchanger is introduced into the bottom of the extraction tank, and then The mixture of carbon dioxide is discharged to the top of the extraction tank, and the discharged mixture is decompressed through a pressure reducing valve, and the carbon dioxide is separated and collected in a gas phase in a pressure reducer, and the extracted components are collected. At this time, the separated carbon dioxide is converted to the liquid carbon dioxide through the chiller, stored in the carbon dioxide storage tank, and is then supplied to the extractor using a pump and a heat exchanger to extract a specific component in a circulating manner.

Root extract produced by the present invention has a high melanin synthesis inhibitory activity while improving the color tone, while the cytotoxicity is reduced, high purity purified extract compared to the general extraction method using an organic solvent to the human body By using a supercritical extraction method that is harmless and environmentally friendly, it can be used as a food ingredient as well as a cosmetic ingredient.

The present invention relates to a cosmetic composition having a melanin synthesis inhibitory activity, containing the extract of the root of the herb oil herb prepared using supercritical carbon dioxide. More specifically, the present invention exhibits higher activity when extracting useful substances having melanin synthesis inhibitory activity from the roots of seaweed oil using supercritical carbon dioxide than conventional general solvent extraction methods (ethanol, methanol, water). At the same time it relates to a cosmetic composition containing the extract of root oil extract produced by the method using supercritical carbon dioxide to improve the color tone and alleviate cytotoxicity. Supercritical carbon dioxide extraction method of the root oil of the present invention is more harmless to the human body compared to the general extraction method using an organic solvent, the extract developed as being friendly to the natural environment is not only functional cosmetic raw materials but also as food raw materials It can be widely used.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of Root Ethanol Extract of Peucedanum japonicum Thunb.

Root oil roots were harvested from the coast of Jeju Island and used for the experiment. The sample was washed with purified water, dried and ground. After extracting 150 g of dried crushed boiled herb root with 80 ml of ethanol for 3 hours at room temperature using ultrasonic waves, Advantech NO. 4 (Toyo Roshi Kaisha, Ltd.) was filtered through a filtrate. The filtrate was dried under a reduced pressure concentrator to obtain a dry weight of 34.7 g.

Example 2: Preparation of Solvent Fraction of Root Ethanol Extract of Peucedanum japonicum Thunb.

Solvent fractionation was carried out for the ethanol extracts of the root oil herb obtained in Example 1.

Solvent fractionation of 10 g of ethanol extracts from the root of radish oil was carried out in a conventional manner using hexane, chloroform, ethyl acetate, butanol, and water, followed by 3.45 g of hexane fraction, 0.19 g of chloroform fraction, 0.047 g of ethyl acetate fraction, and 0.30 of butanol fraction. g, 4.19 g of water fractions were obtained.

Example 3: Inhibitory Effect of Extra-cellular Melanin (ECM) Synthesis in B16 melanoma Cells on the Root Ethanol Extract of Peucedanum japonicum Thunb.

The inhibitory effect of extra-cellular melanin on the extract of Root Sprout Root prepared in Example 1 was measured as follows. In this experiment, the experiment was carried out with arbutin, a whitening functional raw material, currently announced by KFDA as a positive control group. Mouse-derived B16 melanoma cells were inoculated to 5 × 10 ³ cells in 96-well plates, and cultured in a 37 ° C. CO ₂ incubator for 24 hours, followed by replacement with medium supplemented with botanical herb extract. After further incubation for 72 hours, the melanin released into the medium was measured for absorbance at 405 nm using a microplate reader.

  Inhibitory Effect of Extra-cellular Melanin (ECM) Synthesis in B16 melanoma Cells on Root Ethanol Extracts
sample Melanin synthesis inhibitory effect (%) IC ₅₀
(Μg / ml) Treatment concentration (㎍ / ㎖) 10 30 50 80 100 Miso herb root ethanol extract 6.4 11.3 26.8 73.6 88.4 58.5 Arbutin 18.4 - 39.3 - 55.7 78.7

As shown in Table 1, the ethanol extracts of the root oil ethanol extract showed better melanin synthesis inhibitory activity than the arbutin, a whitening functional ingredient, which was notified to the KFDA.

Example 4 Inhibitory Effect of Pecandanum japonicum Thunb. Root Ethanol Extract on Intracellular Cellular Melanin (ICM) Synthesis in B16 melanoma Cells

The inhibitory effect of intra-cellular melanin synthesis on the extract of the Root Sprout Root prepared in Example 1 was measured as follows. In this experiment, the experiment was carried out with arbutin, a whitening functional raw material, currently announced by KFDA as a positive control group. Mouse-derived B16 melanoma cells were seeded in 6-well plates to be 1 × 10 ⁵ cells and cultured in a 37 ° C. CO ₂ incubator for 24 hours, followed by replacement with medium supplemented with botanical oil herb extract. After further incubation for 48 hours, cells were harvested from each well and collected by centrifugation at 1200 rpm for 5 minutes. The supernatant was removed, 1N NaOH / 10% DMSO solution was added to dissolve melanin in an 80 ° C. water bath, and the absorbance was measured at 405 nm using a microplate reader.

 Inhibitory Effects of Root Ethanol Extracts on Intracellular Cellular Melanin (ICM) Synthesis in B16 melanoma Cells
sample Melanin synthesis inhibitory effect (%) IC ₅₀
(Μg / ml) Treatment concentration (㎍ / ㎖) 10 50 100 Miso herb root ethanol extract 4.94 31.28 56.80 86.45 Arbutin 1.30 8.86 23.56 > 100

As shown in Table 2, even in B16 melanoma cells, ethanol extracts from the root oil ethanol extract showed better melanin inhibitory activity than Arbutin, a whitening functional ingredient, which was notified by the KFDA.

Example 5: Determination of Cytotoxicity of Root Ethanol Extract of Peucedanum japonicum Thunb.

Mouse-derived B16 melanoma cells were inoculated to 5 × 10 ³ cells in 96-well plates and incubated in a 37 ° C. CO ₂ incubator for 24 hours, followed by replacement with medium supplemented with botanical herb extract. After 48 hours of further incubation, the medium was removed and washed with DBS (D-phosphate buffered saline), followed by MTT (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) 500 Μg / ml was added at 50 μl per well. After reacting for 4 hours at 37 ° C. under 10% CO ₂ , MTT solution was removed, and 200 μl of DMSO (dimethyl sulfoxide) was added per well to dissolve formazan precipitate formed by reacting with living cells, followed by microplate reader at 540 nm. Absorbance was measured.

MTT experiment was performed on B16 melanoma cells to determine whether the inhibitory effect of melanin synthesis of the ethanol extracts from the root oil extracts was an error caused by cytotoxicity. As shown in FIG. It was confirmed that there was no cytotoxicity in the concentration of ml.

Example 6 Inhibitory Effect of Extra-cellular Melanin (ECM) and Synthesis in B16 melanoma Cells on Hexane Fraction of Root Ethanol Extract of Peucedanum japonicum Thunb.

As a result of measuring the inhibitory effect of extra-cellular melanin (ECM) on solvent fractions of the root ethanol extract of Peucedanum japonicum Thunb. Prepared in Example 2, the inhibition of melanin synthesis was lower than that of ethanol extract in other fractions except Hexane fraction. Although effective or cytotoxic, the hexane fraction showed much higher melanin synthesis inhibition than ethanol extract in the concentration range without cytotoxicity.

Inhibitory Effect of Extra-cellular Melanin (ECM) Synthesis in B16 melanoma Cells on Hexane Fraction of Root Ethanol Extract of Peucedanum japonicum Thunb. Sprout Root Hexan Fraction Melanin synthesis inhibitory effect (%) IC ₅₀
(Μg / ml) Treatment concentration (㎍ / ㎖) One 2 4 8 10 ECM 5.25 25.66 55.88 88.86 92.40 3.6

As shown in Table 3, it was confirmed that the inhibitory effect of melanin synthesis in the extra-cellular to the Hexane fraction of the ethanol extracts of the root oil ethanol extract was about 17 times higher than that of the ethanol extract of the co-agent.

Example 7 Cytotoxicity Measurement of Hexane Fraction of Root Ethanol Extract of Peucedanum japonicum Thunb.

MTT experiment was performed on B16 melanoma cells to determine whether the inhibitory effect of melanin synthesis on the Hexane fraction of the ethanol extracts of the Sprout Sprout Root was an error caused by cytotoxicity. Fractions were confirmed to be cytotoxic at a concentration of 10 μg / ml.

Example 8 Supercritical Carbon Dioxide Extraction of Root Sprouts

Using supercritical carbon dioxide, an effective fraction having melanin synthesis inhibitory activity was separated and extracted from the roots of the oil herb. Using dry crushed samples of the same root oil sprouts used in Example 1, fractions were obtained at constant extraction time under supercritical carbon dioxide extraction conditions of 300 bar and 60 ° C., and the melanin synthesis inhibitory activity and cytotoxicity of these fractions The color tone and the like of the supercritical extract fractions were compared with those of the previous organic solvent (ethanol, hexane) extracts. As shown in the figure, in the case of supercritical carbon dioxide extraction of the root of the oil herb, the yield according to the extraction time was about 10.4%. These results were higher than 7.0 ~ 8.8% for hexane organic solvent extraction.

Example 9 Inhibitory Effect of Radical Sprout Root Supercritical Carbon Dioxide Extract on Extra-cellular Melanin (ECM) Synthesis in B16 melanoma Cells

Embodiments Conger oil herbs (Peucedanum japonicum Thunb.) Roots supercritical carbon dioxide extracts prepared from 8 was prepared in the fractions in accordance with the extracted time. Among these fractions, the highest extra-cellular melanin (ECM) inhibitory effect was obtained in the fraction obtained by the extraction time of 17.4 minutes (Fraction 1) and the fraction obtained after 34.8 minutes (Fraction 2). As shown in Table 4, the highest melanin synthesis inhibition rate was shown in fraction 1, and this result was about 16 times higher than the ethanol extract, although somewhat lower than the hexane fraction of the co-agent.

Inhibitory Effect of Extra-cellular Melanin (ECM) Synthesis in B16 melanoma Cells on the Root Supercritical Carbon Dioxide Extract of Peucedanum japonicum Thunb. Oil Sprout Root Supercritical Carbon Dioxide Extract Fraction Melanin synthesis inhibitory effect (%) IC ₅₀
(Μg / ml) Treatment concentration (㎍ / ㎖) 5 10 20 40 80 100 Fraction 1 48.34 66.07 89.97 96.88 100.29 97.37 5.4 Fraction 2 1.25 21.30 60.93 90.64 97.18 91.61 17.2

Example 10 Measurement of Cytotoxicity of B16 melanoma Cells of Radix Sprout Root Supercritical Carbon Dioxide Extract

MTT experiment was performed on B16 melanoma cells to determine whether the inhibitory effect of melanin synthesis on the supercritical carbon dioxide extracts of Boiled Sprout Roots was an error caused by cytotoxicity. All of the fractions (Fraction 1, Fraction 2) with excellent melanin synthesis inhibitory activity selected during the extraction process were found to have no cytotoxicity at a concentration of 40 µg / ml. These results showed that the hexane fraction, which had the highest inhibitory effect on melanin synthesis, alleviated the increase of cytotoxicity, which was pointed out as a problem, about 4 times compared to the extract concentration. In other words, the root oil supercritical carbon dioxide extract (Fraction 1), which showed a melanin synthesis inhibitory effect similar to that of the hexane fraction, was able to secure skin safety even when prescribed at a concentration of about 4 times higher when prescribed as a whitening cosmetic composition. . In addition, as can be seen in Fig. 5, the herb root supercritical carbon dioxide extract has a lighter tint at the same concentration (concentration range that can be used as a cosmetic raw material, 0.5 ~ 1%) than the ethanol extract and the hexane fraction. These results imply that saemul root supercritical carbon dioxide extract satisfies the conditions to be used as the main active ingredient in whitening cosmetics.

Example 11 Inhibitory Effects of Tyrosinase / TRP-1 / TRP-2 on B16 melanoma Cells from Radix Root Supercritical Carbon Dioxide Extracts

Total RNA extraction was homogenized using TRIzol-reagent (invitrogen, USA), and then centrifuged (12000 rpm, 15 min) by adding chloroform. The same amount of isopropanol was added to the supernatant and centrifuged (12000 rpm, 5 min) to precipitate RNA, washed with 75% ethanol treated with 0.1% DEPC (diethylpyrocarbonat), dried and dissolved in 0.1% DEPC distilled water. RNA was quantified by measuring absorbance at 260 nm, and RNA having a value in the range of absorbance 260 / absorbance 280 nm in the range of 1.6 to 1.9 was selected and used. cDNA synthesis ^{Improm-Ⅱ TM cDNA kit (promega} , USA) for 25, total RNA of 1㎍ by oligo (dT) primer, dNTP ( 0.5μM), 1unit RNase inhibitor and ^{Improm-Ⅱ TM reverse transcriptase (2U} ) using The reaction was stopped by heating at 5 ° C. for 5 minutes, at 42 ° C. for 60 minutes, and at 75 ° C. for 10 minutes.

Polymerase chain reaction (PCR) was performed to amplify tyrosinase, TRP-1, TRP-2, β-actin from cDNA, 1 μl cDNA, 4 μM of 5 'and 3' primers, 10X buffer (10 mM Tris-HCl, pH 8.3, PCR was performed using 50 mM KCl, 0.1% Triton X-100), 250 μM dNTP, 25 mM MgCl ₂ , 1 unit Taq poylmerase (promega, USA). PCR amplification was carried out in 25 cycles of 94 ℃ 30 seconds, 57 ~ 62 ℃ 45 seconds, 72 ℃ 45 seconds. The product produced by PCR was subjected to electrophoresis on 1.2% agarose gel, the density of each band was used Quantity One 1-D Image Analysis Software (Bio-rad. USA). The primer sequences of tyrosinase, TRP-1, TRP-2 and β-actin are as follows.

Tyrosinase:

Forward primer; 5'-GGC CAG CTT TCA GGC AGA GGT-3 '

Reverse primer; 5'-TGG TGC TTC ATG GGC AAA ATC-3 '

TRP-1:

Forward primer; 5'-GCT GCA GGA GCC TTC TTT CTC-3 '

Reverse primer; 5'-AAG ACG CTG CAC TGC TGG TCT-3 '

TRP-2:

Forward primer; 5'-GGA TGA CCG TGA GCA ATG GCC-3 '

Reverse primer; 5'-CGG TTG TGA CCA ATG GGT GCC-3 '

β-actin:

Forward primer; 5'-GAG ACC TTC AAC ACC CCA GCC-3 '

Reverse primer; 5'-GGC CAT CTC TTG CTC GAA GTC-3 '

As shown in FIG. 6, the supercritical carbon dioxide extract from the root of the Sprouts was inhibited by inhibiting the gene expression of Tyrosinase and TRP-1 involved in melanogenesis in melanogenesis cells.

1 is a graph showing the cytotoxicity of the ethanol extract of radish herb root.

Figure 2 is a graph showing the cytotoxicity of the Hexane fraction of the ethanol extract of radish herb root.

3 is a graph showing a change in yield of the root oil supercritical carbon dioxide extract over the course of extraction.

Figure 4 is a graph showing the cytotoxicity measurement results of the root oil supercritical carbon dioxide extract of radish sprouts.

Figure 5 is a comparison of the color tone at the same concentration conditions of ethanol extract and hexane fraction of coarse oil herb root supercritical carbon dioxide extract and co-agent.

Figure 6 is a graph showing the inhibitory effect of Tyrosinase / TRP-1 / TRP-2 mRNA expression of the herb root supercritical carbon dioxide extract.

Claims (2)

A cosmetic composition containing a botanical herb extract, which has excellent color tone and reduced cytotoxicity, prepared by a manufacturing process comprising the following steps. ① extracting the active ingredient by applying supercritical carbon dioxide to the root of seaweed oil at the conditions of 35 to 80 ℃ and 200 to 700 bar; ② collecting and separating carbon dioxide into a gaseous phase from the mixture of the botanical herb extract and supercritical carbon dioxide. The cosmetic composition according to claim 1, wherein the temperature in the extraction step is 60 ° C to 80 ° C and the pressure is 300 to 600 bar.

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