KR20210050750A - Method of selective extracting natrual material with simplified extract process - Google Patents

Abstract

A method for extracting a non-polar natural material is disclosed. The method for extracting a non-polar natural material according to one embodiment of the present invention includes: a primary extraction step of precipitating a natural raw material in ethanol for a time period of within 72 hours to perform cold extraction; a secondary extraction step of mixing and stirring a phase separation composition including a lipophilic solubilizer and water in the primary extract to perform phase-separation; and a step of separating the upper layer of the phase-separated solution to obtain a non-polar natural material. Accordingly, the non-polar natural material can be extracted relatively easily with high efficiency using a dissolution-emulsion extract (DEE) method.

Description

Selective extraction method of natural substances with simplified extraction process{METHOD OF SELECTIVE EXTRACTING NATRUAL MATERIAL WITH SIMPLIFIED EXTRACT PROCESS}

The present invention relates to a method for extracting a selective natural substance with a simplified extraction process, and more particularly, inducing a physical emulsification of an optional ingredient (non-polar/polar, poorly soluble/water-soluble, HLB) or a substance among natural substances It relates to a technology for extracting and concentrating/separating by dissolving and solubilizing, and to a method for extracting a selective natural substance with a simplified extraction process using a dissolution-emulsion extraction (DEE).

Extraction is a method of dissolving and separating components contained in solid or liquid raw materials with a solvent.It is a general extraction method. If the raw material is solid, the solid-liquid extraction method is used, and if the raw material is liquid, the liquid-liquid Use the extraction method.

Liquid-liquid extraction (LLE) is the separation of certain components in a mixture from other components by applying a solvent to a liquid mixture containing a solute.

In order to extract non-polar substances in natural substances, a liquid-liquid extraction method is generally used. In liquid-liquid extraction, non-polar solvents such as nucleic acid (hexane), chloroform (methane dichloride), ethyl acetate, etc. are toxic. It is very strong, and there is a problem in that the extraction process is very inefficient and complex due to several repetitive extraction processes and a drying process for removing the non-polar solvent, and the process is long, the extraction efficiency is low, and the process is long, resulting in a high production cost.

For example, in the process of extracting Brevilin A from Centipedea minima, the raw material is extracted multiple times with methanol, and hexane is mixed in the extract for a plurality of fractions, and chloroform is mixed for a plurality of fractions, Ethyl acetate is mixed and extracted through a plurality of fractions and butyl alcohol is mixed and extracted through a plurality of fractions, and when extraction is performed, about 25 mg of Brevilin A is extracted from 300 g of dry weight of medium-sized scallop, so the extraction amount is very low and the efficiency is very low. Patent Document 1)

As a result of the experiment with another extraction method, it was found that the extraction efficiency was lower in the case of resin extraction, and the extraction was not possible or the extraction efficiency was very low in the case of saponification/unsaponification extraction.

Patent Literature 1 discloses the extract of S. snailfish, and in this case, natural substances were extracted by the supercritical carbon dioxide extraction method, but the conditions for use of this extraction method are complicated and the extraction time takes a considerable amount of time, and the extraction efficiency is also very high. There is a low problem.

Accordingly, there is a need to develop a novel extraction method capable of easily extracting natural substances having nonpolar properties from natural substances and the like with high efficiency and utilizing the extracts easily.

Patent Document 1: Chinese Patent Publication 104840457 B

Non-Patent Document 1: Arch Pharm Res Vol 29, No 1, 64-66, 2006.

Embodiments of the present invention are intended to provide a method of extracting natural substances using a dissolution-emulsion extraction (DEE) method.

According to the method of selectively extracting natural substances according to an embodiment of the present invention, the first extraction step of cold-spit extraction by precipitating natural raw materials in ethanol for less than 72 hours, lipophilic solubilizing agent and water in the first extract. Mixing and stirring the phase separation composition comprising a second extraction step of phase separation and separating the upper layer of the phase-separated solution to obtain a non-polar natural material.

In addition, according to an embodiment of the present invention, before the second extraction step, there is provided a method for extracting a non-polar natural substance, characterized in that water is added to the first extract.

In addition, according to an embodiment of the present invention, a non-polar natural material is obtained by separating the upper layer of the phase-separated solution and re-phase separation by adding ethanol, and then separating the ethanol layer of the re-phase-separated solution. Provides a method for extracting substances.

In addition, according to an embodiment of the present invention, prior to the second extraction step, there is provided a method for extracting a non-polar natural substance, characterized in that the alcohol in the first extract is removed.

In addition, according to an embodiment of the present invention, after the second extraction step, there is provided a method for extracting a non-polar natural substance, characterized in that the alcohol in the second extract is removed.

In addition, according to an embodiment of the present invention, there is provided a method for extracting a non-polar natural material, characterized in that the temperature is raised when the phase separation composition is mixed and stirred in the second extraction step to concentrate the non-polar natural material.

In addition, according to an embodiment of the present invention, the upper layer of the phase-separated solution provides a method for extracting a non-polar natural material, characterized in that it contains a lipophilic solubilizing agent and a non-polar natural material.

In addition, according to an embodiment of the present invention, the non-polar natural material provides a method for extracting a non-polar natural material, characterized in that it comprises Brevilin A or a derivative thereof.

In addition, according to an embodiment of the present invention, the non-polar natural substance may inhibit or inhibit the JAK-STAT signaling process, or activate the Wnt signaling process to increase stem cells in the hemocytosis system and hair follicles. It provides a method for extracting non-polar natural substances.

In addition, according to an embodiment of the present invention, the natural material raw material includes any one or more selected from the group consisting of plants of the genus C. Centipeda minima , Litsea glutinous , plants of the genus Arnica and plants of the genus Helenium. It provides a method for extracting non-polar natural substances.

In addition, according to an embodiment of the present invention, the phase separation composition, 1 to 90% by weight of a lipophilic solubilizing agent; And it provides a method for extracting a non-polar natural material containing 10 to 99% by weight of water.

In addition, according to an embodiment of the present invention, the phase separation composition provides a method for extracting a non-polar natural substance, characterized in that it further comprises 60% by weight or less of a nonionic surfactant.

Embodiments of the present invention relate to a method of extracting natural substances from raw materials such as wild grass, and in particular, non-polar natural substances can be extracted relatively easily with high efficiency using a dissolution-emulsion extraction (DEE) method. .

In addition, since the liquid-liquid extraction method using toxic solvents is not used, steps such as drying can be omitted.Since the solvent is a material harmless to the human body, the extract containing natural substances is separated and immediately produced as a product without any other steps. It can be used, and the active substance concentration can be increased due to the limiting micelle concentration, and the absorption rate of the product can be increased by microemulsion.

1A is a view for explaining a comparison between a liquid-liquid extraction method, which is an existing natural substance extraction method, and a dissolution-emulsification extraction method (DEE) according to an embodiment of the present invention.
1B is a table comparing the characteristics of various extraction methods.
Figure 2 is a three-phase state diagram for explaining the composition ratio of the phase separation composition according to an embodiment of the present invention.
3 is a view for explaining the composition of the phase separation composition according to embodiments of the present invention.
4 is a view for explaining the behavior of the phase separation composition according to an embodiment of the present invention.
5 is a photograph showing the results of a human body application test of a hair tonic product made of a natural substance extracted according to an embodiment of the present invention.
6 and 7A to 7C are views showing changes in the amount of hair loss and the amount of hair growth as a man uses the hair tonic prepared according to an embodiment of the present invention for 4 weeks.
8 and 9A to 9C are views showing changes in the amount of hair loss and the amount of hair growth as a woman uses the hair tonic prepared according to an embodiment of the present invention for 24 weeks.

Hereinafter, a method for selectively extracting a natural substance in which an extraction process is simplified according to embodiments of the present invention, a natural substance extracted according to the method, and a use thereof will be described.

Prior to that, the terminology used herein is for reference only to specific embodiments and is not intended to limit the invention. In addition, the singular forms used herein also include plural forms unless the phrases clearly represent the opposite meaning. In addition, the meaning of'comprising' or'containing' as used herein specifies a specific characteristic, area, integer, step, action, element or component, and other specific characteristic, area, integer, step, action, element, or It does not exclude the addition of ingredients.

In the present invention, terms such as first and second are used to describe various components, and the terms are used only for the purpose of distinguishing one component from other components.

In addition, terms used in the present specification are only used to describe exemplary embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise", "include", or "have" are intended to designate the existence of a feature, number, step, element, or combination of the implemented features, but one or more other features or It is to be understood that the possibility of the presence or addition of numbers, steps, elements, or combinations thereof is not preliminarily excluded.

The present invention will be described in detail below and exemplify specific embodiments, as various modifications can be made and various forms can be obtained. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

Hereinafter, the present invention will be described in more detail.

In the present invention, a method of extracting a natural substance refers to a non-polar natural substance as a dissolution-emulsion extraction (DEE).

The extraction method of a non-polar natural substance according to an embodiment of the present invention includes a first extraction step of precipitating a natural material raw material in ethanol for within 72 hours to extract cold needles, and a phase separation comprising a lipophilic solubilizing agent and water in the first extract. And a second extraction step of phase-separating the composition by mixing and stirring, and a step of separating the upper layer of the phase-separated solution to obtain a non-polar natural material.

According to the dissolution-emulsification extraction method, a non-polar natural substance is extracted from a natural material, a phase separation composition is mixed, dissolved and emulsified, and then a non-polar natural substance, which is an active substance, is extracted through phase separation.

1A is a view for explaining a comparison between a liquid-liquid extraction method, which is an existing natural substance extraction method, and a dissolution-emulsification extraction method (DEE) according to an embodiment of the present invention. 1B is a table comparing the characteristics of various extraction methods.

As described above, in order to extract non-polar substances in natural substances, a liquid-liquid extraction method is generally used. In liquid-liquid extraction, solvents are nucleic acid (hexane), chloroform (methane dichloride), and ethyl acetate. As such, as a non-polar solvent, the toxicity is very strong, the extraction process is complicated, and the extraction efficiency is low, resulting in a high production cost and a long process.

First, the natural product raw material is washed with water, and the natural product raw material is precipitated in ethanol for less than 72 hours and subjected to a first extraction step of cold needle extraction.

In one embodiment, before the second extraction step, water may be added to the first extract, and the upper layer of the solution phase-separated in the second extraction step is separated and then phase-separated again by adding ethanol, and then re-phase separation. By separating the ethanol layer of the resulting solution, a non-polar natural substance can be obtained. The ethanol layer may be separated to obtain an active fraction from which the solubilizer was separated. Here, the second ethanol extraction is performed by dissolving the active substance in the ethanol layer as much as possible in the ethanol composition of 90% or less using the solubilizing agent is not soluble in alcohol 90% or less, and separating the solubilizing agent (Labrafac) to separate only the separated natural active fraction. This is a method of obtaining an oral or highly pure active fraction.

In another embodiment, before the second extraction step, alcohol in the primary extract may be removed.

In one embodiment of the present invention, the natural material raw material may be used as a plant of the genus Middle Saffron (Centipeda minima ), a plant of the Samputuri (Litsea glutinous ), a plant of the genus Arnica and a plant of the genus Helenium.

The natural material used in the examples of the present invention was Jungdae Garipul, and Goesan Herbal Organic Agricultural Products Cooperative (https://natural-herb.co.kr) purchased and used domestic Abul vinegar (Chongdae Garipul, Yeongcheon). .

A number of literatures have been found that abulin vinegar ( Centipeda minima ) contains Brevilin A, and some literature reports that the shampoo tree ( Litsea glutinous ) also contains Brevilin A. In addition, plants of the genus Arnica ( Arnica longifolia , Arctium lappa , Arnica chamissonis subsp . Foliosa , Arnica chamissonis Less. ) And the genus Helenium (Helenium autumnale L, Helenium alternifolium, Helenium pinnatifidum , Helenium vernale , Helenium brevifolium , and Helenium flexuosum ) has also been found to contain some Brevilin A.

For example, the natural raw materials, in particular, scallop, contain a non-polar natural substance, Brevilin A or a derivative thereof. The non-polar natural substance extracted from S. scallop contains Brevilin A and various derivatives thereof.

The non-polar natural substance, which is an active ingredient extracted from S. sphenoid, can inhibit or inhibit the JAK-STAT signaling process, or activate the Wnt signaling process to increase stem cells in the hemocytosis system and hair follicles.

Brevilin A is a natural substance, and it is widely known to inhibit JAK (janus kinase activity) and STAT3 signaling in cancer cells ( PLoS One 8 (5) (2013) e63697 ).

That is, Brevilin A inhibits and inhibits JAK-STAT signaling, and is effective in autoimmune diseases such as rheumatoid arthritis.

Overall, Brevilin A is a major component of S. scallop, and studies on anticancer activity using it have been mainly conducted, but there are no studies on hair loss and hair growth.

For example, Brevilin A inhibits or inhibits the JAK-STAT signaling process, such as colds, scabies, diabetes, nominal, eczema, coloriasis, rhinitis, swelling, asthma, sinusitis, dermatitis, hazelnutrition, seawater, fever and pertussis (pediatric diseases). ) In addition, autoimmune diseases including rheumatoid arthritis and cancer or tumors, and adult alopecia, alopecia areata, alopecia areata, neurogenic alopecia, alopecia areata, tricotillomania, malignant alopecia, female type alopecia, male pattern alopecia, androgenic It can be effective in the treatment of autoimmune diseases such as hair loss, telogen hair loss, tinea head, total head alopecia, hypochondriasis, hereditary alopecia or general alopecia.

For example, the non-polar natural substance includes any one or more of the following compounds, which shows the chemical structure of Brevilin A and its derivatives ( P. Wu et al. / Phytochemistry 76 (2012) 133-140 ). .

In the method for extracting a non-polar natural substance according to an embodiment of the present invention, first, a natural substance is extracted from a natural substance to prepare a first extract.

As the extraction solvent, several solvents such as water and alcohol may be selected and used, and alcohol was used in the present invention.

For example, the extraction can be used by selecting any one of a method such as hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, and elution, and additionally, a conventional fractionation process can be performed. .

In the extraction according to the embodiments of the present invention, cold needle extraction may be performed by immersing a natural material raw material in cold water. For example, as a raw material for natural products, the scallop splinter itself may be used, or a powder obtained by pulverizing it may be used.

Thereafter, the step of removing the alcohol component may be selectively performed by concentrating the first extract according to the first extraction under reduced pressure. Although there is no difference in achieving the present invention even if it goes to the next step using the first extract that has not undergone the step of concentrating, it may be desirable to remove unnecessary alcohol components in terms of more clear phase separation and extraction efficiency.

Thereafter, a phase separation composition is added to the first extract and mixed.

Figure 2 is a three-phase state diagram for explaining the composition ratio of the phase separation composition according to an embodiment of the present invention. 3 is a view for explaining the composition of the phase separation composition according to embodiments of the present invention. 4 is a view for explaining the behavior of the phase separation composition according to an embodiment of the present invention.

The phase separation composition may include a lipophilic solubilizer and water. For example, the phase separation composition, 1 to 90% by weight of a lipophilic solubilizing agent; And 10 to 99% by weight of water. The lipophilic solubilizing agent may be included in an amount of 50 times or less, preferably 30 times or less compared to the content of the non-polar natural material contained in the primary extract.

For example, the phase separation composition may further include a nonionic surfactant. The phase separation composition may contain 40% by weight or less of a nonionic surfactant.

Referring to Figures 2 and 3, Figure 2 shows a state appearing in the mixture according to the components of the phase separation composition of the present invention, phase separation according to the composition ratio of the lipophilic solubilizer, nonionic surfactant, and water, It indicates that the state of the solution changes due to microemulsion and emulsion. In Fig. 2, Labrafac is one of the lipophilic solubilizers, and Labrasol is one of the nonionic surfactants.

The composition ratio of the phase separation composition of the present invention was set according to the composition ratio of the phase-separated region based on the three-phase diagram of FIG. 2. The phase separation compositions according to the embodiments of the present invention are physically accelerated and mixed with a specific substance to emulsify in a short period of time, and at that time, a specific substance suitable for the composition is solubilized, and when separated over time, the fraction containing the solubilized specific substance is separated. Therefore, the non-polar natural substance, which is the active substance contained in the raw material of the natural substance, is first extracted and then mixed with the phase-separating composition to form a single layer of a lipophilic solubilizer and a non-polar natural substance. In addition, the remaining water and other components are contained in the other layer, causing phase separation.

Examples of the present invention relate to a technology for extracting and concentrating/separating an optional component (non-polar/polar, poorly soluble/water soluble, HLB) or material among natural products by inducing physical emulsification and rapidly dissolving and solubilizing them. It relates to a method of extracting selective natural substances using the extraction method (Dissolution-Emulsion Extract, DEE), and using this, in a mixture of non-polar/polar substances, poorly soluble/water-soluble substances, or substances with high HLB/low HLB, Optionally, a substance having any one property can be extracted or vice versa.

In the embodiments of the present invention, when the phase separation composition contains a nonionic surfactant, in the step of mixing the phase separation composition, the temperature may be increased and mixed for clear phase separation. For example, the mixture obtained by mixing the phase separation composition with the first extract may be stirred while maintaining a temperature of 30 to 50°C, preferably while maintaining a temperature of 35 to 45°C.

5 is a view for explaining the behavior of the phase separation composition according to an embodiment of the present invention. Referring to FIG. 5, when mixed with the phase separation composition in the first extract, Brevilin A and its derivatives, that is, non-polar natural substances, are extracted and dissolved in a nonionic surfactant or a lipophilic solubilizing agent. At this time, if additional high-speed mixing is performed, the surface area of the surfactant and solubilizing agent in the mixture increases, and some non-polar natural substances that are not extracted or dissolved are mostly dissolved, resulting in an instantaneous critical micelle concentration (CMC). , When the cloud point is reached as the temperature of the mixed solution increases, unlike the ionic surfactant, the nonionic surfactant exhibits a distinct compositional polarity according to the ideal gas law as the temperature increases, and the aqueous layer in the mixed solution The phase separation between the fruit and oil layers clearly occurs, and concentration occurs as much as the volume of the lipid surfactant.

In particular, the method of phase separation by raising the temperature can be advantageous when the addition of a lipophilic solubilizing agent having a relatively high HLB (hydrophile-lipophile balance) value, for example, Labrafac having an HLB value of 3 or less, preferably HLB is close to 1. For example, the nonionic surfactant is Labrasol, Crodex A-PA-(RB), Geleol, Gelot 64, Suppocire AS2X, Suppocire BML, Synperonic PE/F 127, Synperonic PE/L 44 and Tefose 63. It may include any one or more selected from the group.

For example, the lipophilic solubilizer is BRIJ 010-SS-(RB), Capryol 90, Capryol PGMC, Gantrez ES-435, Gantrez S-97 BF, Gelucire 43/01, Gelucire 44/14, Gelucire 50/13 , Gelucire 48/16, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Labrafil M 1944 CS, Labrafil M 2125 CS, Labrafil M 2130 CS, Lauroglycol 90, Lauroglycol FCC, Monosteol, Peceol, Pharmasolve, Plurol Oleique CC497, Span 20- LQ-(SG), Span 60-PA-(SG), upper Refined Oleic Acid-LQ-(JP), Super Refined Tween 80A-LQ-(MH), Surfadone LP-300, Transcutol HP, Transcutol P, TWEEN 60 -SS-(SG), TWEEN 80 HP-LQ-(MH), TWEEN 80-LQ-(SG), and Vitamin E TPGS.

The upper layer of the phase-separated solution is separated to obtain a fraction containing a target active substance, a non-polar natural substance.

An emulsified product may be prepared by additionally mixing an emulsifying composition in the microemulsion range, which is outside the range of the phase separation composition, to the phase separation layer, which is a high concentration active fraction extracted according to the above methods.

In particular, when a nonionic surfactant with a relatively high HLB value, for example, Labrasol having an HLB value of 12 or higher, is added to the active fraction at a high concentration, it may act advantageously in promoting absorption and increasing skin permeability through a microemulsion. I can.

Natural substances extracted according to the embodiments of the present invention and products containing the same are not used in a liquid-liquid extraction method using a toxic solvent, so steps such as drying can be omitted, and the solvent is not affected by the human body. Since it is a harmless substance, the extract containing natural substances can be separated and used as a product immediately without any other steps, and the concentration of the active substance can be increased due to the limiting micelle concentration, and the absorption rate of the product can be increased with microemulsion.

In general, the use of surfactant increases the absorption rate by increasing the solubility of the compound by using the CMC of the surfactant, and increases the absorption rate at the absorption site by reducing the size of the material to be absorbed as well as increasing the solubility of the microemulsion. have.

Products manufactured in this way are hair tonic, hair conditioner, hair lotion, hair nutrition lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream, hair wax, hair aerosol , Hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservation agent, hair dye, hair wave agent, hair bleaching agent, hair gel, hair glaze, hairdressing agent, hair lacquer, hair moisturizer, Products such as hair mousse, hair spray, etc., can be prepared in formulations such as cream, gel, patch, spray, ointment, warning agent, lotion, liner, pasta and cataplasma as external skin preparation, liquid, solid , It can be suitably used in gaseous form

Such a product is effective in improving various diseases to which the effects of Brevilin A and its derivatives can be applied. , Seawater, fever and pertussis (pediatric diseases), as well as rheumatoid arthritis, lupus and autoimmune diseases including cancer or tumors, and adult alopecia, alopecia areata, alopecia areata, neurogenic alopecia, alopecia areata, tricotillomania, It can be effective in the treatment of autoimmune diseases such as malignant alopecia, female type alopecia, male type alopecia, androgenic alopecia, telogen alopecia, tinea head, alopecia totalis, hypotrichosis, hereditary alopecia or general alopecia.

Hereinafter, it will be described in more detail through examples of the method for extracting a non-polar natural substance of the present invention.

In the extraction method of the present invention, cold needle extraction and alcohol extraction of natural raw materials, phase separation by mixing a phase separation composition in the extract, and separation of some layers to obtain a non-polar natural material.

Example

The raw material for natural products is Jungdae Garipul, and Goesan Herbal Organic Agricultural Products Cooperative (https://natural-herb.co.kr) purchased domestically produced Abul vinegar (Chongdae Garipul, Yeongcheon), washed and dried.

1,000 g of heavy scallop hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by cold extraction with alcohol to obtain a first extract. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol.

After mixing the primary extract and the phase separation composition concentrated under reduced pressure according to the composition ratio shown in Table 1 below, high-speed mixing (using a homogenizer) for 60 minutes, transferred to a fractionation funnel (separator), stirred for 30 minutes with a stirrer, and heated up Then, the aqueous layer was removed and separated.

Primary extract content Water content Lipophilic solubilizer content Non-polar natural substance content Parts by weight Parts by weight Content compared to non-polar natural substances Parts by weight Example 1 10mg/g (1%) 25 60 60 15 Example 2 10 84 6 Example 3 25 65 40 10 Example 4 10 86 4 Example 5 25 70 20 5 Example 6 10 88 2 Example 7 25 66 35 9 Example 8 10mg/g (0.2%) 90 0 60 10 Example 9 50 44 6 Example 10 93 0 40 7 Example 11 50 46 4 Example 12 96 0 20 4 Example 13 50 48 2 Example 14 50 46 35 4 Example 15 2mg/g (1%) 90 0 60 10 Example 16 93 0 40 7 Example 17 96 0 20 4 Example 18 94 0 35 6

Thereafter, the temperature was raised to 60 degrees, left for 6 hours, and then the separated aqueous layer was removed and separated. After allowing to stand for 6 hours, the phase separated supernatant was separated.

The content of Brevilin A in the supernatant was analyzed by HPLC (Water 2960 HPLC). HPLC analysis conditions are shown in Table 2 below.

Item Condition Column SUPERSIL COLUMN ODS-I, 4.6 X 250mm, 5㎛ Mobilie phase Solvent A
(45%) Solvent B
(55%) 0.1% formic acid in water Methanol Oven temp. 40℃ Flow rate 1ml/min. Injection volume 20µl Run time 30 min. Wave length 224nm

Accordingly, the Brevilin A content of each example is shown in Table 3.

1,000 g of heavy scallop hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by cold extraction with alcohol to obtain a first extract. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol, and the extract was analyzed by HPLC, and it was confirmed that the extract contained Brevilin A at a concentration of 2 mg/mL (0.20% content compared to hay).

Brevilin A content in the extracted layer (mg) Brevilin-A Concentration Concentration Concentration Rate in Extraction Layer (ppm) Yield (%) of indicator components compared to the primary extract Example 1 185.25 12,350 74.1 Example 2 73.2 12,200 73.2 Example 3 195.5 19,550 78.2 Example 4 84.1 21,025 84.1 Example 5 133.5 26,700 53.4 Example 6 56.1 28,050 56.1 Example 7 218.75 25,000 87.5 Example 8 89.6184 8,298 49.8 Example 9 45.2 7,533.33 45.2 Example 10 109.126 14,667.4 58.7 Example 11 57.1 14,275 57.1 Example 12 41.2877 10,752 21.5 Example 13 23.1 11,550 23.1 Example 14 63 18,000 63.0 Example 15 128.498 11,898 71.4 Example 16 154.397 20,752.3 83.0 Example 17 107.274 27,936 55.9 Example 18 173.392 26,351.3 92.2

Comparative Example 1-LLE

The raw material for natural products is Jungdae Garipul, and Goesan Herbal Organic Agricultural Products Cooperative (https://natural-herb.co.kr) purchased domestically produced Abul vinegar (Chongdae Garipul, Yeongcheon), washed and dried.

1,000 g of heavy scallop hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by cold extraction with alcohol to obtain a first extract. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol to obtain a second extract. The second extract was filtered and evaporated to dryness to obtain 0.6 g of an extract.

500 mL of water (DW) and 1000 mL of nucleic acid (Cyclohexane) were mixed, stirred with a vortex mixer for 1 minute, and placed in a separating funnel to separate the nucleic acid layer. And the nucleic acid layer obtained by repeating 3 times was concentrated and analyzed by HPLC.

In the above, the nucleic acid fraction layer was removed, and 500 mL of chloroform (MC) was mixed with the remaining aqueous layer extract, and the chloroform (MC) layer was separated by placing it in a separating funnel. And the chloroform (MC) layer obtained by repeating three times was concentrated and analyzed by HPLC.

In the above, the chloroform (MC) fraction layer was removed, and 500 mL of ethyl acetate (EtAC) was mixed with the remaining aqueous layer extract, and the ethyl acetate (EtAC) layer was separated by putting it in a separating funnel. And the ethyl acetate (EtAC) layer obtained by repeating three times was concentrated and analyzed by HPLC.

The ethyl acetate (EtAC) fraction layer was removed from the above, and the remaining aqueous layer extract was concentrated and analyzed by HPLC.

The obtained chloroform (MC) layer was fractionated under mobile phase (MC:M, 100:1 ~ 1:1) conditions through Silica gel column chromatography, further separated, and analyzed by HPLC, which is shown in Table 4.

Comparative Example 2-RESIN

The raw material for natural products is Jungdae Garipul, and Goesan Herbal Organic Agricultural Products Cooperative (https://natural-herb.co.kr) purchased domestically produced Abul vinegar (Chongdae Garipul, Yeongcheon), washed and dried.

1,000 g of heavy scallop hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by cold extraction with alcohol to obtain a first extract. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol to obtain a second extract. The second extract was evaporated to dryness after a filter, and 1000 ml of water and 100 g of resin (HP-20) were mixed, stirred for 1 minute with a stirrer, and incubated at 100 rpm for 1 hour. Thereafter, the first supernatant was separated by centrifugation (including water), and 300 ml of ethanol was mixed with the remaining resin. Centrifugation was performed again to separate the second supernatant (including ethanol). Each of the separated supernatant was concentrated, resuspended in ethanol in the same amount, filtered using a syringe filter, and analyzed by HPLC.

Brevilin A content (mg) Brevilin A yield compared to primary extract (%) Comparative Example 1 650.1 (Cyclohexane layer) 32.55 862.4 (MC layer) 42.12 310.6 (EtOAC layer) 15.53 167.2 (water floor) 8.35 Comparative Example 2 Brevilin A content (mg) Brevilin A yield compared to primary extract (%) 82.7 (1st supernatant, aqueous layer) 4.13 16.28 (second supernatant, ethanol layer) 49.35 Comparative Example 1 Brevilin A content (mg) Brevilin A yield compared to MC layer (%) 0 (Chromatography, 1fr.) 0 7.32 (Chromatography, 2fr.) 0.85 10.83 (Chromatography, 3fr.) 1.26 20.21 (Chromatography, 4fr.) 2.34 68.9 (Chromatography, 5fr.) 7.99 98.7 (Chromatography, 6fr.) 11.44 140.7 (Chromatography, 7fr.) 16.31 210.6 (Chromatography, 8fr.) 24.42 164.7 (Chromatography, 9fr.) 19.10 65.3 (Chromatography, 10fr.) 7.57 22.1 (Chromatography, 11fr.) 2.56 10.8 (Chromatography, 12fr.) 1.25 6.2 (Chromatography, 13fr.) 0.72

Manufacturing Example 1

The supernatant (second extract) separated according to Example 7 was concentrated (naturally concentrated at elevated temperature) to not exceed 120% of the volume of the added lipophilic solubilizing agent, and Labrasol 5g, Labrafac, as an emulsifying composition in the concentrated supernatant. A microemulsion was prepared by mixing 4.4g, 0.5g of Lecithin, and 0.1g of D-pantenol. Accordingly, a microemulsion of 12mg/mL, 10mL, and a tonic formulation having a yield of 87.5% were prepared. These emulsions contain safe excipients, not organic solvents, and can be used as various types of products such as ointments and tonics on their own without any additional process (Preparation Example 1).

Secondary extract, weight (%) (DEE, Example 7) Labrasol, weight (%) Lecithin, weight (%) D-pantenol, weight ((%) 50 44 5 One

Manufacturing Example 2

The supernatant (second extract) separated according to Example 7 was concentrated (naturally concentrated at elevated temperature) so as not to exceed 120% of the volume of the added lipophilic solubilizing agent, and mixed with 90% ethanol in the concentrated supernatant to provide lipophilicity. A fraction containing Brevilin A and derivatives from which the solubilizing agent was removed was prepared by layer-separating the solubilizing agent.

3.2 g of the tertiary extract from which the lipophilic solubilizing agent has been removed was mixed at high speed in the following composition to prepare a tonic formulation for sensitive skin of 3.4 mg/mL and 100 mL.

Tertiary extract (solubilizer removed) weight (%) Ethanol (95%) Oleic acid, weight (%) Lecithin, weight (%) D-pantenol, weight ((%) 34 50 10 5 One

When performing the secondary extraction (Dissolution Emulsion Extract or Lipid-based drug(fr.) extract systems.) from the primary extract of various concentrations, braviline-A depends on the lipophilic solubilizer (Lipds sulubilizer) regardless of the concentration amount. This is a photograph to explain the state that it can be separated and extracted by selectively concentrating and its derivatives, and has a more suitable performance for a trace extraction method.

The phase separation composition of the present invention can extract all natural substances contained in the extract, substantially regardless of the concentration of the non-polar natural substance to be extracted contained in the primary extract. That is, it can be used to efficiently extract or remove a trace amount of material. That is, although the concentration of the non-polar natural substance to be extracted is lowered, the phase-separated region is reduced in the same or similar to this, but it was found that the desired substance can be separated by sufficiently performing phase separation. Most of the pesticides remaining in fruits and vegetables are non-polar (poorly soluble/lipophilic). They are not washed well and are deposited on the fibers of plants. In order to remove pesticides remaining in these plants, it is possible to use the phase separation composition.

Experimental example

The hair tonic prepared according to Preparation Example 1 was measured for the amount of hair loss and the change in the amount of hair growth according to the use of the hair tonic for a certain period of time in general men and women. Hair Tonic was used in such a way that the hair was cleanly applied to the scalp after shampooing according to the user's level. Here, since it is somewhat difficult to accurately measure the amount of hair loss, it was determined by the number of strands of hair filtered after a shower, and the amount of hair growth was determined by the hair density in the same area measured by a hair analyzer.

6 and 7A to 7C are views showing changes in the amount of hair loss and the amount of hair growth as a man uses the hair tonic prepared according to an embodiment of the present invention for 4 weeks.

8 and 9A to 9C are views showing changes in the amount of hair loss and the amount of hair growth as a woman uses the hair tonic prepared according to an embodiment of the present invention for 24 weeks. 6 to 9C, it can be seen that the amount of hair loss gradually decreases as the hair tonic was used in a certain amount, and it was found that the amount of hair loss increased somewhat as the use was stopped during the experiment period. In particular, in the case of a female experimenter, most of the hair tonics of a certain amount or more were periodically used as shown in FIG. 8, and as a result, it was found that the amount of hair loss was continuously decreased. In FIGS. 7A to 7C and 9A to 9C, the amount of hair growth in both experimenters increased, and the hair density increased by about 19% as in FIG. 9B in the case of male, and the amount of hair loss was about 36 as in FIG. 7C. % Decreased, and in the case of the female case, the hair density increased by about 15% as in FIG. 9B, and the amount of hair loss decreased by about 29% as in FIG. 9C. In addition, after 4 weeks, when looking at the harmonic average for this amount of hair loss, the experimenters with the initial amount of hair loss of 10 to 150 strands were found to be stabilizing to 50 to 60 strands when looking at the harmonic average of the amount of hair loss after 4 weeks.

As described above, although exemplary embodiments of the present invention have been described, the present invention is not limited thereto, and those of ordinary skill in the art are within the scope not departing from the concept and scope of the following claims. It will be appreciated that various changes and modifications are possible in

Claims (12)

A first extraction step of precipitating the natural material raw material in ethanol for within 72 hours to extract cold needles;
A second extraction step of phase separation by mixing and stirring a phase separation composition comprising a lipophilic solubilizer and water in the first extract solution; And
A method for extracting a non-polar natural material comprising the step of separating the upper layer of the phase-separated solution to obtain a non-polar natural material. The method of claim 1,
Before the second extraction step, the method of extracting a non-polar natural substance, characterized in that water is added to the first extract. The method of claim 2,
A method of extracting a non-polar natural substance, characterized in that the upper layer of the phase-separated solution is separated, ethanol is added to phase-separate again, and then the ethanol layer of the re-phase-separated solution is separated to obtain a non-polar natural substance. The method of claim 1,
Prior to the second extraction step, the method of extracting a non-polar natural substance, characterized in that the alcohol in the first extract is removed. The method of claim 1,
After the second extraction step, the method of extracting a non-polar natural substance, characterized in that the alcohol in the second extract is removed. The method of claim 1,
A method of extracting a non-polar natural material, characterized in that the temperature is raised when the phase separation composition is mixed and stirred in the second extraction step to concentrate the non-polar natural material. The method of claim 1,
The upper layer of the phase-separated solution comprises a lipophilic solubilizer and a non-polar natural substance. The method of claim 1,
The method of extracting a non-polar natural material, characterized in that the non-polar natural material comprises Brevilin A or a derivative thereof. The method of claim 8,
The method of extracting a non-polar natural substance, characterized in that the non-polar natural substance inhibits or inhibits the JAK-STAT signaling process or activates the Wnt signaling process to increase stem cells in the hemocytosis system and hair follicles. The method of claim 1,
The natural material raw material is a method for extracting a non-polar natural substance, characterized in that it contains at least one selected from the group consisting of plants of the genus Centipeda minima , Litsea glutinous , plants of the genus Arnica and plants of the genus Helenium. The method of claim 1,
The phase separation composition, 1 to 90% by weight of a lipophilic solubilizing agent; And 10 to 99% by weight of water. The method of claim 11,
The phase separation composition further comprises 60% by weight or less of a nonionic surfactant.

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