KR102433111B1 - Method of selective extracting natrual material with simplified extract process - Google Patents

Abstract

A method for extracting non-polar natural substances is disclosed. The extraction method of a non-polar natural material according to an embodiment of the present invention includes a primary extraction step of precipitating a natural product raw material in ethanol for a period of within 72 hours and extracting it with cold needles, a lipophilic solubilizing agent and water in the primary extract A second extraction step of phase-separating the phase-separation 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. Therefore, a non-polar natural substance can be extracted relatively easily with high efficiency using a dissolution-emulsion extraction (DEE) method.

Description

Method of selective extraction of natural substances with simplified extraction process

The present invention relates to a method for extracting a selective natural material in which the extraction process is simplified, and more particularly, by inducing a physical emulsification of a selective component (non-polar/polar, poorly soluble/water-soluble, HLB) or material among natural products to quickly It relates to a technique for extraction and concentration/separation by dissolution and solubilization, and to a selective extraction method of natural substances in which the extraction process using a dissolution-emulsion extract (DEE) is simplified.

Extraction is the separation of components contained in solid or liquid raw materials by dissolving them with a solvent. extraction method is used.

Liquid-liquid extraction (LLE) is a process in which a solvent is applied to a liquid mixture containing a solute to separate a specific component in the mixture from other components.

In order to extract non-polar substances from natural substances, a liquid-liquid extraction method is generally used, and non-polar solvents such as hexane, methane dichloride, and ethyl acetate are toxic during liquid-liquid extraction. The extraction process is very inefficient and complicated due to the very strong, repeated several extraction processes and the drying process to remove the non-polar solvent, and the process is long, the extraction efficiency is low, and the production cost is high as the process is long.

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

As a result of experiments with other extraction methods, the extraction efficiency was lower in the case of resin extraction, and in the case of saponification/unsaponification extraction, it was also found that extraction was not possible or the extraction efficiency was very low.

It is disclosed in Patent Document 1 with respect to the extract of S. serrata, and natural substances are extracted here by a supercritical carbon dioxide extraction method, but this extraction method has complicated conditions for use and takes a considerable amount of time for extraction, and the extraction efficiency is also very high. There is a low problem.

Accordingly, it is necessary to develop a novel extraction method capable of easily and efficiently extracting a non-polar natural substance from natural products and the like and using the extract easily.

Patent Document 1: Chinese Patent Publication No. 104840457 B

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

Embodiments of the present invention are to provide a method of extracting a natural substance using a dissolution-emulsion extraction method (Dissolution-Emulsion Extract, DEE).

According to the method for selectively extracting natural substances according to an embodiment of the present invention, the primary extraction step of precipitating the S. serrata in ethanol for less than 72 hours and extracting cold chills, the HLB value of the primary extract is 3 or less A second extraction step of phase-separating by mixing and stirring a phase-separation composition comprising a lipophilic solubilizer and water, and a step of separating the upper layer of the phase-separated solution to obtain a non-polar natural material, in the second extraction step, 10 to 96% by weight of the primary extract, 2 to 15% by weight of a lipophilic solubilizer and the remainder of water are mixed, and the lipophilic solubilizer is Gelucire 43/01, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Lauroglycol 90 , Peceol, Plurol Oleique CC497, Surfadone LP-300 and any one or more selected from the group consisting of Transcutol HP, the non-polar natural material includes any one or more of the compounds represented by the following formula.

In addition, according to an embodiment of the present invention, before the secondary extraction step, there is provided a method of extracting a non-polar natural material, characterized in that the addition of water to the primary extract.

In addition, according to an embodiment of the present invention, the phase-separated dragon is separated, phase-separated again by adding ethanol, and then the ethanol layer of the re-phase-separated solution is separated to obtain a non-polar natural material. An extraction method is provided.

In addition, according to an embodiment of the present invention, before the secondary extraction step, there is provided a method of extracting a non-polar natural material, characterized in that removing the alcohol of the primary extract.

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

In addition, according to an embodiment of the present invention, there is provided a method of extracting a non-polar natural material, characterized in that the non-polar natural material is concentrated by increasing the temperature when mixing and stirring the phase-separation composition in the secondary extraction step.

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

delete

In addition, according to an embodiment of the present invention, 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 hematopoietic lineage and hair follicles. It provides a method for extracting non-polar natural substances.

delete

delete

In addition, according to an embodiment of the present invention, the phase separation composition provides a method of extracting a non-polar natural material, 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 a natural substance from a raw material such as wild grass, and in particular, a non-polar natural substance can be extracted relatively easily with high efficiency using Dissolution-Emulsion Extract (DEE). .

In addition, since the liquid-liquid extraction method using a toxic solvent is not used, steps such as drying can be omitted. It can be used, and the concentration of the active substance can be increased due to the limiting micellar concentration, and the absorption rate of the product can be increased by micro-emulsification.

1A is a diagram for explaining a comparison between a liquid-liquid extraction method, which is an existing extraction method of natural substances, 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.
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 the 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 human body application test result of a hair tonic product prepared with a natural material 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 hair growth as a man uses a 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 hair growth as a woman uses a hair tonic prepared according to an embodiment of the present invention for 24 weeks.

Hereinafter, a selective extraction method of a natural material in which the extraction process is simplified according to embodiments of the present invention, a natural material extracted according to this, and uses thereof, etc. will be described.

Prior to that, the terminology used herein is for the purpose of referring to specific embodiments only, and is not intended to limit the present invention. And, as used herein, the singular forms also include the plural forms unless the phrases clearly indicate the opposite. Also, as used herein, the meaning of 'comprising' or 'containing' specifies a particular characteristic, region, integer, step, operation, element or component, and other specific characteristic, region, integer, step, operation, element, or This 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, the terminology used herein is used only to describe exemplary embodiments, and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present specification, terms such as "comprise", "comprising" or "having" are intended to designate the presence of an embodied feature, number, step, element, or a combination thereof, but one or more other features or It should be understood that the existence or addition of numbers, steps, elements, or combinations thereof is not precluded in advance.

Since the present invention may have various changes and may have various forms, specific embodiments will be illustrated and described in detail below. However, this is not intended to limit the present invention to the specific disclosed form, it should be understood to include all modifications, 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, the extraction method of natural substances is called dissolution-emulsion extraction (DEE) of non-polar natural substances.

The extraction method of a non-polar natural material according to an embodiment of the present invention includes a primary extraction step of precipitating a natural product raw material in ethanol for a period of within 72 hours and extracting it with cold needles, a phase separation comprising a lipophilic solubilizer and water in the primary extract 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 material is extracted from a raw material of a natural product, a phase-separation composition is mixed to dissolve and emulsify, and a non-polar natural material as an active material is extracted through phase separation.

1A is a diagram for explaining a comparison between a liquid-liquid extraction method, which is an existing extraction method of natural substances, 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, and during liquid-liquid extraction, the solvents are nucleic acid (hexane), chloroform (methane dichloride), ethyl acetate (ethyl acetate) As a non-polar solvent, it is very toxic, the extraction process is complicated, and the extraction efficiency is low, so there are problems in that the production cost is high and the process is long.

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

In one embodiment, before the second extraction step, water may be added to the first extract, and the upper layer of the phase-separated solution in the second extraction step is separated and phase-separated again by adding ethanol, followed by re-phase separation A non-polar natural substance can be obtained by separating the ethanol layer of the obtained solution. By separating the ethanol layer, it is possible to obtain an active fraction from which the solubilizer is separated. Here, the second ethanol extraction uses the characteristic that the solubilizer does not dissolve in alcohol 90% or less, so that the active material is dissolved as much as possible in the ethanol layer in the ethanol composition of 90% or less, and only the natural active fraction separated by separating the solubilizer (Labrafac) It is a method to obtain an active fraction orally or with high purity.

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

In one embodiment of the present invention, the natural product raw material can be used in the middle head grass ( Centipeda minima ).

The natural raw material used in the examples of the present invention was medium head herb, Goesan Herb Organic Agricultural Products Cooperative (https://natural-herb.co.kr) domestic Abul vinegar (Middle head herb, Yeongcheon) was purchased and used. .

A number of documents have been found that Abul vinegar ( Centipeda minima ) contains Brevilin A, and some documents report 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 plants of the genus Helenium ( Helenium autumnale L, Helenium alternifolium, Helenium pinnatifidum , Helenium vernal , Helenium brevifolium , and Helenium flexuosum ) has also been found to contain some Brevilin A.

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

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

Brevilin A is a natural substance, and it is widely known that it inhibits 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, thereby having an effect on autoimmune diseases such as rheumatoid arthritis.

Throughout the literature, Brevilin A is a major component of mid-head grass, and studies on anticancer activity using it have been mainly conducted, but studies on hair loss and hair growth are absent.

For example, by inhibiting or inhibiting the JAK-STAT signaling process, Brevilin A inhibits or inhibits the JAK-STAT signaling process, thereby causing colds, flatulence, diabetes, epilepsy, ichthyosis, rhinitis, edema, asthma, sinusitis, dermatitis, dysentery, seawater, antipyretic and whooping cough (pediatric disease). ) as well as rheumatoid arthritis and autoimmune diseases including cancer or tumors, and senile alopecia, alopecia collectively, alopecia areata, alopecia nervosa, alopecia pityroides, tricotillomania, malignant alopecia, female pattern hair loss, male pattern hair loss, androgenetic It can be effective in the treatment of autoimmune diseases such as hair loss, telogen alopecia, ringworm of the head, frontal alopecia, hypotrichosis, hereditary alopecia or generalized hair loss.

For example, the non-polar natural material 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 of extracting a non-polar natural material according to an embodiment of the present invention, a primary extract is prepared by first extracting a natural material from a natural material raw material.

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

For example, extraction may be performed by selecting any one of methods such as hot water extraction, cold extraction, reflux extraction, solvent extraction, steam distillation, ultrasonic extraction, and elution, and additionally a conventional fractionation process may be performed. .

Extraction according to embodiments of the present invention can be extracted by immersing a natural raw material in cold water. For example, the natural product raw material may use the medium head grass itself, or a powder obtained by pulverizing it.

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

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

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.

delete

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

2 and 3, Figure 2 shows the state that appears in the mixed solution according to the components of the phase separation composition of the present invention, phase separation according to the composition ratio of the lipophilic solubilizer, the nonionic surfactant and water (Phase separation), It indicates that the state of the solution changes to microemulsion and emulsion. In FIG. 2, Labrafac is one of the lipophilic solubilizers, and Labrasol is one of the nonionic surfactants.

Based on the three-phase phase diagram of FIG. 2, the composition ratio of the phase-separation composition of the present invention was set according to the composition ratio of the region to be phase-separated. Phase separation compositions according to embodiments of the present invention are emulsified in a short-term by physically accelerated mixing with a specific material, and then solubilize a specific material suitable for the composition, and when separated over time, the fraction containing the solubilized specific material is separated It is concentrated and the layers are separated from each other. Therefore, the non-polar natural material, which is the active material contained in the natural product raw material, is first extracted and then mixed with the phase-separation composition to combine the lipophilic solubilizer and the non-polar natural material into one layer. , and the remaining water and other components are contained in another layer, resulting in phase separation.

Embodiments of the present invention relate to a technology for extracting and concentrating/separating a selective component (non-polar/polar, poorly soluble/water-soluble, HLB) or material from natural products by inducing physical emulsification to quickly dissolve and solubilize it. Dissolution-emulsification It relates to a selective extraction method of natural substances using an extraction method (Dissolution-Emulsion Extract, DEE), and by using this, a mixture of non-polar / polar substances, poorly soluble / water soluble substances or substances with high HLB / low HLB is mixed, Optionally, a material having any one property may be extracted or vice versa.

In embodiments of the present invention, when the phase-separation composition includes a nonionic surfactant, in the step of mixing the phase-separation composition, the mixture may be mixed while raising the temperature for clearer phase separation. For example, the mixture in which the phase separation composition is mixed with the primary extract may be stirred while maintaining a temperature of 30 to 50° C., and preferably may be stirred 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 primary extract, Brevilin A and its derivatives, that is, a non-polar natural material, are extracted and dissolved in a nonionic surfactant or a lipophilic solubilizer. At this time, when additional high-speed mixing is performed, the surface area of the surfactant and solubilizer in the mixture increases, and most of the non-polar natural substances that are not extracted or dissolved are dissolved, resulting in an instantaneous critical micelle concentration (CMC) state. , when the cloud point is reached as the temperature of the mixed solution increases, the nonionic surfactant, unlike the ionic surfactant, exhibits a distinct compositional polarity according to the ideal gas law as the temperature increases, and the aqueous layer in the mixed solution Phase separation occurs clearly between the oil and oil layers, and concentration occurs as much as the volume of the lipid surfactant.

In particular, the method of phase separation by raising the temperature may advantageously act upon the addition of a lipophilic solubilizer having a relatively high hydrophile-lipophile balance (HLB) value, for example, Labrafac having an HLB value of 3 or less, preferably close to 1 For example, the nonionic surfactant is composed of 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 solubilizing agent 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 may include any one or more selected from the group consisting of.

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

An emulsifying composition outside the range of the phase separation composition, which is outside the range of the phase separation composition, may be further mixed with the phase separation layer, which is a high concentration active fraction extracted according to the above methods, to prepare an emulsified product.

In particular, when a nonionic surfactant having a relatively high HLB value, for example, Labrasol having an HLB value of 12 or more, is added to the active fraction at a high concentration, it may act advantageously to promote absorption and increase skin permeability through micro emulsion. can

Products such as natural substances and compositions containing them extracted according to embodiments of the present invention can omit drying steps because they do not use a liquid-liquid extraction method using a toxic solvent, and the solvent is not harmful to the human body. Since it is a harmless substance, it is possible to separate the extract containing natural substances and immediately use it as a product without any other steps.

In general, the use of surfactants increases the absorption rate by increasing the solubility of the compound by using the CMC of the surfactant. In addition to increasing the solubility of microemulsions, the absorption rate at the absorption site can be increased by reducing the size of the material to be absorbed. have.

The products manufactured in this way are hair tonic, hair conditioner, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, 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 preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair lacquer, hair moisturizer, Products such as hair mousse and hairspray, as external preparations for skin, can be prepared in the form of creams, gels, patches, sprays, ointments, warnings, lotions, liniments, pastas and cataplasmas, etc. , can be appropriately utilized in the gas phase.

Such a product is effective in improving various diseases to which the effects of Brevilin A and its derivatives can be applied, for example, colds, flatulence, diabetes, epilepsy, ichthyosis, rhinitis, edema, asthma, sinusitis, dermatitis, and dysentery. , seawater, antipyretic and pertussis (pediatric disease), as well as rheumatoid arthritis, lupus and autoimmune diseases including cancer or tumors, and alopecia in adults, alopecia onset, alopecia areata, alopecia nervosa, alopecia pityroides, tricotylomania, It can be effective in the treatment of autoimmune diseases such as malignant alopecia, female pattern hair loss, male pattern hair loss, androgenetic alopecia, telogen hair loss, ringworm of the head, frontal alopecia, hypotrichosis, hereditary alopecia or generalized hair loss.

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

In the extraction method of the present invention, cold extraction and alcohol extraction of a natural raw material are performed, a phase separation composition is mixed with the extract to phase-separate, and some layers are separated to obtain a non-polar natural material.

Example

The raw material for natural products is medium head herb, Goesan Herb Organic Agricultural Products Cooperative (https://natural-herb.co.kr), domestic Abul vinegar (Middle head herb, Yeongcheon) was purchased, washed and dried.

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

After mixing the reduced pressure-concentrated primary extract and the phase-separation composition according to the composition ratio in Table 1 below, high-speed mixing (using a homogenizer) for 60 minutes, transfer to a fractionation funnel (separator), stirring with a stirrer for 30 minutes, and then raising the temperature The aqueous layer was removed and separated.

Primary extract content water content Lipophilic solubilizer content Content of non-polar natural substances parts by weight parts by weight Content compared to non-polar natural substances parts by weight Example 1 10 mg/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 10 mg/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 2 mg/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, and the separated aqueous layer was removed and separated after being left for 6 hours. After standing 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㎛ mobile phase Solvent A
(45%) Solvent B
(55%) 0.1% formic acid in water Methanol Oven temp. 40℃ flow rate 1 ml/min. Injection volume 20 μl Run time 30 min. Wave length 224nm

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

1,000 g of mid-head grass hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by alcohol cold extraction to obtain a primary extract. Thereafter, the extract from which ethanol was removed by concentrating the primary extract under reduced pressure was analyzed by HPLC, and it was confirmed that the extract contained Brevilin A at a concentration of 2 mg/mL (content of 0.20% compared to hay).

Brevilin A content in the extract layer (mg) Brevilin-A Concentration Concentration Concentration Rate of Extraction Layer (ppm) Yield (%) of the index component 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 medium head herb, Goesan Herb Organic Agricultural Products Cooperative (https://natural-herb.co.kr), domestic Abul vinegar (Middle head herb, Yeongcheon) was purchased, washed and dried.

1,000 g of mid-head grass hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by alcohol cold extraction to obtain a primary extract. Thereafter, the first extract was concentrated under reduced pressure to remove ethanol. Thereafter, the primary extract was concentrated under reduced pressure to remove ethanol to obtain a secondary extract. The second extract was filtered and evaporated to dryness to obtain 0.6 g of the 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 three times was concentrated and analyzed by HPLC.

After removing the nucleic acid fraction layer, 500 mL of chloroform (MC) was mixed with the remaining aqueous layer extract and placed in a separating funnel to separate the chloroform (MC) layer. And the chloroform (MC) layer obtained by repeating three times was concentrated and analyzed by HPLC.

After removing the chloroform (MC) fraction layer, 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 3 times was concentrated and analyzed by HPLC.

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

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

Comparative Example 2-RESIN

The raw material for natural products is medium head herb, Goesan Herb Organic Agricultural Products Cooperative (https://natural-herb.co.kr), domestic Abul vinegar (Middle head herb, Yeongcheon) was purchased, washed and dried.

1,000 g of mid-head grass hay was precipitated in 10 L of 80% alcohol for 48 hours, followed by alcohol cold extraction to obtain a primary extract. Thereafter, the primary extract was concentrated under reduced pressure to remove ethanol to obtain a secondary extract. The second extract was filtered and evaporated to dryness, mixed with 1000 ml of water and 100 g of resin (HP-20), stirred with a stirrer for 1 minute, 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. The second supernatant was separated (including ethanol) by centrifugation again. Each of the separated supernatant was concentrated, resuspended in the same amount of ethanol, filtered using a syringe filter, and analyzed by HPLC.

Brevilin A content (mg) Brevilin A yield (%) compared to the 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 the primary extract 82.7 (1st supernatant, aqueous phase) 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

Preparation Example 1

The supernatant (second extract) separated according to Example 7 is concentrated not to exceed 120% of the volume of the added lipophilic solubilizer (natural concentration at elevated temperature), and Labrasol 5g, Labrafac as an emulsifying composition in the concentrated supernatant A microemulsion was prepared by mixing 4.4 g, 0.5 g of Lecithin, and 0.1 g of D-pantenol. Accordingly, tonic formulations of micro emulsion 12mg/mL, 10mL, and yield of 87.5% were prepared. These emulsions contain safe excipients, not organic solvents, and can be used as products in various forms such as ointments and tonics by themselves without additional processing (Preparation Example 1).

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

Preparation 2

The supernatant (second extract) separated according to Example 7 is concentrated (natural concentration at elevated temperature) not to exceed 120% of the volume of the added lipophilic solubilizer, and mixed with 90% ethanol in the concentrated supernatant to lipophilic The solubilizing agent was separated and removed to prepare a fraction containing Brevilin A and derivatives from which the solubilizing agent was removed.

A tonic formulation for sensitive skin of 3.4 mg/mL and 100 mL was prepared by mixing 3.2 g of the tertiary extract with the lipophilic solubilizer removed at high speed with the following composition.

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

When performing secondary extraction (Dissolution Emulsion Extract or Lipid-based drug(fr.) extract systems.) from the primary extract of various concentrations, braviline-A and its derivatives can be selectively concentrated and extracted, and moreover, it is a photograph to explain the state that it has more suitable performance for a trace amount of extraction method.

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

Experimental example

Changes in the amount of hair loss and hair growth according to the use of the hair tonic prepared according to Preparation Example 1 for a certain period of time for general men and women were measured. The hair tonic was used by applying the hair directly to the scalp after shampooing the hair according to the user's level. Here, since it is rather difficult to accurately measure the amount of hair loss, it was determined by the number of strands of hair filtered after showering, and the amount of hair growth was determined by the density of hair in the same area as measured by a hair diagnostic device.

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

In the foregoing, exemplary embodiments of the present invention have been described, but the present invention is not limited thereto, and those of ordinary skill in the art will fall within the scope and concept of the following claims. It will be understood that various changes and modifications are possible.

Claims (12)

A primary extraction step of precipitating S. head grass in ethanol for less than 72 hours and extracting cold chills;
A secondary extraction step of phase separation by mixing and stirring a phase separation composition comprising a lipophilic solubilizer and water having an HLB value of 3 or less in the primary extract; and
Separating the upper layer of the phase-separated solution to obtain a non-polar natural material,
In the secondary extraction step, 10 to 96% by weight of the primary extract, 2 to 15% by weight of the lipophilic solubilizer and the remainder of water are mixed,
The lipophilic solubilizer includes at least one selected from the group consisting of Gelucire 43/01, Labrafac CC, Labrafac Lipophile WL1349, Labrafac PG, Lauroglycol 90, Peceol, Plurol Oleique CC497, Surfadone LP-300 and Transcutol HP,
The non-polar natural material is a method of extracting a non-polar natural material comprising any one or more of the compounds represented by the following formula.

According to claim 1,
Before the secondary extraction step, the extraction method of a non-polar natural material, characterized in that by adding water to the primary extract. 3. The method of claim 2,
A method of extracting a non-polar natural material, characterized in that the phase-separated solution is separated, phase-separated again by adding ethanol, and the ethanol layer of the re-phase-separated solution is separated to obtain a non-polar natural material. According to claim 1,
Before the secondary extraction step, the extraction method of a non-polar natural material, characterized in that to remove the alcohol in the primary extract. According to claim 1,
After the secondary extraction step, the extraction method of a non-polar natural material, characterized in that to remove the alcohol in the secondary extract. According to claim 1,
A method of extracting a non-polar natural material, characterized in that the non-polar natural material is concentrated by increasing the temperature when mixing and stirring the phase-separation composition in the secondary extraction step. According to claim 1,
The phase-separated solution is a non-polar natural material extraction method, characterized in that it contains a lipophilic solubilizer and a non-polar natural material. delete According to claim 1,
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 hematopoietic system and hair follicles. Extraction method of a non-polar natural substance. delete delete According to claim 1,
The phase separation composition is a non-polar natural material extraction method, characterized in that it further comprises 60% by weight or less of a nonionic surfactant.

Images