KR101036266B1 - extraction method of Asiasari radix containing whitening active components - Google Patents

Abstract

Sessin extract comprising the whitening active ingredient of the present invention comprises a drying step of drying the sacin sample; Pulverizing the dried sacin sample and filtering the sieve; Re-tangling the distilled water into the sieve sample filtered through the sieve and boiling repeatedly; Centrifuging the re-melt, filtered and concentrated to extract the hot water extract hot water extract; A first extraction step of distilling the hot water extract into an ethyl acetate layer and a distilled water layer and dividing the distilled water layer into a butanol layer and a distilled water layer to obtain an extract corresponding to each solvent; Crude extract formation step of distilling the ethyl acetate layer, butanol layer, distilled water layer under reduced pressure to obtain a semi-solid crude extract of the ethyl acetate layer, butanol layer, distilled water layer; A second extraction step of obtaining the extracts by dissolving the semi-solid crude extracts in distilled water and then fractionating by using ODS (octadesylsilane) open column chromatography; It includes.

Sessin extract, herbal medicine, whitening effect

Description

Extraction method of sessin extract containing whitening active ingredient {extraction method of Asiasari radix containing whitening active components}

The present invention relates to a sessin extract, and more particularly, to a method for extracting a sessin extract comprising a whitening active ingredient that effectively inhibits tyrosinase and inhibits melanin biosynthesis.

In addition to skin damage such as direct burns, skin damage caused by ultraviolet rays may cause more problems due to secondary phenomena caused by oxides, ie, active oxygen, generated by ultraviolet rays penetrated into the skin.

The harmful oxygen substance formed in the skin damages the DNA of the cell itself and affects the DNA replication process, thereby causing a risk of mutating genetic information, thereby causing various skin diseases and skin cancers.

Therefore, in the body, as a natural defense mechanism, abnormal inflammatory substances caused by harmful oxygen generated by ultraviolet rays are expressed in keratinocytes. Promotes the synthesis of melanin in the melanosome.

However, the melanin that occurs at this time does not disappear in the future, it remains in the skin cells causing pigmentation, and if this phenomenon is intensified, it may cause social problems such as extreme popularity in women.

Therefore, many efforts are being made to find effective and safe whitening materials.

However, the current whitening materials such as arbutin, hydroquinone, kojic acid, etc., it is difficult to maintain the efficacy in use, and ascorbic acid has a problem that the use is limited because it greatly affects the stability of the product. there was.

In addition, according to the Republic of Korea Patent Registration 10-0825929, the herbal medicine extract having a whitening effect and a method of preparing the extract and the cosmetic composition comprising the same as the herbal medicine extract extracted by mixing the white paper, Baekchul, Baekyeong, Baekyeong and Sessin as well as sessin The production process is complicated and takes a long time by including the other extracts, and accordingly there is a problem that the cost increases.

An object of the present invention is to provide a method for extracting sessin extract having a whitening activity of the extract extracted only from sessin plants, not to prepare an extract having a whitening effect, including other extracts other than sessin to be devised to solve the above problems. It is about.

 Another object of the present invention relates to a method for extracting sessin extract containing a whitening active ingredient having excellent stability of the product and less worry of side effects by being extracted from plants as a natural substance having an effect on skin whitening.

In order to achieve the above object, the extraction method of sessin extract containing the whitening active ingredient of the present invention, a drying step of drying the sessin sample; Pulverizing the dried sacin sample and filtering the sieve; Re-tangling the distilled water into the sieve sample filtered through the sieve and boiling repeatedly; Centrifuging the re-melt, filtered and concentrated to extract the hot water extract hot water extract; A first extraction step of distilling the hot water extract into an ethyl acetate layer and a distilled water layer and dividing the distilled water layer into a butanol layer and a distilled water layer to obtain an extract corresponding to each solvent; Crude extract formation step of distilling the ethyl acetate layer, butanol layer, distilled water layer under reduced pressure to obtain a semi-solid crude extract of the ethyl acetate layer, butanol layer, distilled water layer; And a second extraction step of obtaining each extract by dissolving the semi-solid crude extracts in distilled water and then fractionating the same by using ODS (octadesylsilane) open column chromatography. It includes.

According to a preferred embodiment, the re-tangling step is characterized in that the distilled water in the medicine bath and re-tangled twice for 2 hours 30 minutes.

According to a preferred embodiment, the first extraction step is characterized in that the hot water extract is put into a separatory funnel with a Teflon stopper is attached and fractional extraction from a separatory steam shaker.

According to a preferred embodiment, the fraction extraction is characterized by increasing the recovery rate of the active ingredient by repeatedly extracting five times in the order of ethyl acetate layer, butanol layer, distilled water layer.

According to a preferred embodiment, the sacin extract extracted in the second extraction step is characterized in that the crude extract relating to the ethyl acetate layer is fractionated.

According to a preferred embodiment, the crude extract of the ethyl acetate layer is characterized in that it is fractionated using any one of 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH as eluent.

According to a preferred embodiment, the crude extract of the ethyl acetate layer is fractionated using 60% methanol.

The method of extracting sessin extract comprising the whitening active ingredient of the present invention by the above-mentioned solution means inhibits tyrosinase, an enzyme that forms melanin pigment, and inhibits melanin biosynthesis in B16 melanoma cells to inhibit cells in the skin. Protect and skin whitening function is excellent.

Hereinafter, with reference to the accompanying drawings, it will be described in more detail with respect to the extraction method of the body extracts containing the whitening active ingredient of the present invention.

1 is a flowchart showing a process of extracting sessin extract having a whitening effect.

Drying step (S100)

The drying step is a step of drying the sacin sample in a vacuum oven for 5 to 7 hours.

The herbaceous perennial herb is called perennial herb and grows in the fertile and humid shade under the valley broad leaf tree of 100 ~ 1,700m above sea level, especially in the central and northern parts of Korea.

Dried roots in early summer are called sesins and contain essential oils containing 2 to 3% of asarinin tube sesamin, as well as kakuol, lignin compounds, flavonoids, coumarins, trace amounts of saponins and alkaloids. It is mainly used for acne or blemishes.

The sacin sample can be purchased from the herbal medicine, the sacin sample purchased is dried for 5-7 hours in a 40 ~ 60 ℃ vacuum oven.

Filter step (S200)

The filtering step is a step of crushing the dried sessin sample into a grinder and then sieving.

The dry weight of the sacin sample is put into a grinder and pulverized, and then filtered using a 45-125 μm mesh net.

The sissin sample filtered through the sieve is used as an extract material for hot extracts.

 Ash step (S300)

 The re-melting step is to put distilled water in the sissin sample filtered through the sieve and to boil repeatedly.

According to a preferred embodiment, the re-tangling step is preferably put into 8L distilled water in the bath containing the sessin sample, and re-ashing twice for 2 hours 30 minutes, but is not necessarily limited thereto.

Hot water extraction step (S400)

The hot water extraction step is a step of extracting the hot water extract by centrifugation, filtration and concentration of the re-melting solution.

Hot-water extraction method is a method of separating the residue and the supernatant using gauze when the volume of the water to be cut off after adding water to the sample, and then repeating the above process by adding water to the residue again. to be.

In this step, the sacin sample is placed in a shaker with distilled water and the boiled re-tangled liquid is centrifuged for 20-30 minutes at 4000 rpm.

And filtered using a 0.1 ~ 0.4 ㎛ filter and concentrated under reduced pressure at 30 ~ 50 ℃ to obtain hot-extracts (hot-extracts).

First extraction step (S500)

The first extraction step is a step of obtaining the extract corresponding to each solvent by fractionating the hot water extract into an ethyl acetate (EtoAc) layer and a distilled water layer, and further distilled the distilled water layer into a butanol layer and a distilled water layer.

According to a preferred embodiment, the extract is put into the separatory funnel with Teflon stuffer and the fraction is extracted in a separatory filter shaker.

Separation funnel is a glass mechanism that uses the difference in specific gravity of two liquids that do not mix with each other to open and separate the liquid below.

In other words, if the specific gravity of each liquid is different, the light is separated upwards and the heavy ones are separated.

This fractionation process is preferably repeated extraction five times in the order of ethyl acetate (EtoAc), butanol (BuOH), distilled water.

The reason for the repeated extraction five times is that the recovery rate of the active ingredient desired can be increased.

According to a preferred embodiment, the tyrosinase activity can be measured to determine the presence of a whitening active ingredient in the ethyl acetate layer, butanol layer, distilled water layer.

In general, tyrosinase acts as a tyrosine hydroxylase that oxidizes L-tyrosine to form L-DOPA, and is also known as an important enzyme that synthesizes melanin by acting as a DOPA oxidant that oxidizes DOPA to form DOPA quinones. have.

Therefore, since the melanin synthesis is made through the action of tyrosinase (tyrosinase) present in the melanosomes, it can be known whether or not the whitening activity according to the activity of tyrosinase.

In other words, increased activity of tyrosinase leads to increased melanin synthesis, resulting in a dark brown skin due to the brown pigment and black pigment produced in melanocytes, and decreased activity of tyrosinase results in melanin pigment production. The whitening effect can be expected by suppressing.

In the present invention, the fractionated ethyl acetate layer, butanol layer, and distilled water layer are each filtered with a 0.1 to 0.5 μm filter and concentrated under reduced pressure at 30 to 50 ° C.

Freeze-drying was performed to measure the weight and then subjected to tyrosinase activity.

As a result, all of the ethyl acetate layer, butanol layer, and distilled water layer showed whitening activity. In particular, the ethyl acetate layer showed active whitening activity, and the butanol layer and distilled water layer showed slightly weakening activity.

 Crude extract formation step (S600)

The crude extract forming step is a step of obtaining a semi-solid crude extract by distilling the ethyl acetate layer, butanol layer, distilled water layer under reduced pressure, respectively.

Generally, distillation refers to a method of recovering and condensing steam generated by heating a mixed solution by evaporating a portion thereof, and distillation under reduced pressure refers to a method of distilling under a reduced pressure.

 In other words, by separating the mixture in the liquid state using the difference in boiling point, the physical separation can be made without chemical reaction.

Therefore, the ethyl acetate layer, butanol layer and distilled water layer may be distilled under reduced pressure to obtain a crude extract in a semi-solid state separated from impurities.

Second extraction step (S700)

The second extraction step is after dissolving the crude extract in distilled water ODS (octadesylsi

lane) A step of obtaining an extract by fractionation using open column chromatography.

In more detail, 1 to 5 g of the crude extract is dissolved in distilled water and divided into several fractions using ODS (octadesylsilane) open column chromatography.

Fractionation refers to the separation of samples from one side of a column and the other side of the column when performing cromatography.

According to a preferred embodiment, the column is a glass filter column, the fixed phase is ODS (YMC gel silica, 50㎛), 100%, 20% MeOH, 40% MeOH, 60% MeOH, 80% distilled water as the eluent Fraction extraction using any one of MeOH, 100% MeOH, dichloromethane (dichloromethane).

The fraction extract thus extracted is the final body extract, which contains a whitening active ingredient.

In order to confirm this, the fractional extracts may be concentrated under reduced pressure and lyophilized to measure the dry weight, followed by an activity experiment.

Example

First, Sessin was purchased from Korean herbal medicines.

The sacin sample was dried in a vacuum oven at 50 ° C. for 6 hours, and then sieved to a 40 × 120 mesh sieve by pulverizing a dry weight of 300 g with a grinder.

The sieve sample filtered through the sieve was placed in a shaker with 8 L of distilled water and re-wound twice each for 2 hours and 30 minutes, followed by centrifugation at 4000 rpm for 20 minutes.

The centrifuge was filtered with a 0.2 μm filter and concentrated under reduced pressure at 40 ° C. to obtain hot extracts (hot water extracts).

Then, hot extracts (hot water extracts) were added to a separatory funnel to which a Teflon stopper was attached, and the separatory funnel was mounted on a separator top shaker to perform fractionation.

The fractionation was first separated into an ethyl acetate (EtoAc) layer and a distilled water layer, and then the distilled water layer was separated into a butanol (BuOH) layer and a distilled water layer.

This fraction extraction process was repeated five times to increase the recovery rate of the active ingredient.

Then, the fractionated ethyl acetate layer, butanol layer, and distilled water layer were distilled under reduced pressure, respectively, to obtain 21.6 g, 41.2 g, and 37.1 g of semi-solid extrudates.

2.0 g of each semi-solid extract was dissolved in distilled water, and then divided into several fractions using ODS (octadesylsilane) open column chromatography.

In this case, a glass filter column 66 x 200 mm is used, the stationary phase is ODS (YMC gel silica, 50 μm), and the eluent is any one of 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, and 100% MeOH. Sessine extract was extracted using.

Experimental Example 1

When fractionating the ethyl acetate layer in the above embodiment, the methanol content of the succinic extract extracted by varying the methanol content (20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH) in the eluent was determined. We examined the effects of tyrosinase inhibition (tyrosinase).

Table 1 is a table showing the results.

Referring to Table 1, starting with 100% distilled water as the eluent, the activity experiment was performed while increasing methanol by 20%.

As a result, when the methanol content in the eluent was 20, 40, 80%, the highest tyrosinase inhibition value was 20.26, 28.75, 24.20 and 31.21 when the methanol content was 60%, and the methanol content was 60%. .

Therefore, the fractionation of ethyl acetate layer with ODS-60% MeOH can best inhibit the tyrosinase activity, and as a result it can be seen that the best whitening effect by inhibiting melanin production.

Table 1

methanol% 0 20 40 60 80 100 tyrosinase
inhobition
-
20 . 26
28.76
31.21
24.20
7.01

Experimental Example 2

Sessin extract extracted from the ODS-60% MeOH was treated by B16 melanoma cells at different concentrations, and the effect of inhibiting melanin synthesis was examined.

To this end, cell extracts were treated with 0, 1ug / ml, 5ug / ml, 10ug / ml, and 20ug / ml in B16 melanoma and incubated for 5 days.

Graph 1 below shows the result.

Looking at the experimental results, it can be seen that melanin synthesis was inhibited in proportion to the concentration of sessin extract, melanin synthesis is inhibited by about 60% at a concentration of 20㎛ / ml.

Graph 1

 The present invention described above is not limited to the above-described embodiment and the accompanying drawings, and in the technical field to which the present invention pertains that substitutions, modifications, and conversions can be made without departing from the technical spirit of the present invention. It will be evident to those who have knowledge of.

1 is a flow chart showing a process for extracting a body extract having a whitening effect, according to a preferred embodiment of the present invention.

Claims (7)

In the extraction method of sessin extract, A drying step of drying the sacin sample; Pulverizing the dried sacin sample and filtering the sieve; Re-tangling the distilled water into the sieve sample filtered through the sieve and boiling repeatedly; Centrifuging the re-melt, filtered and concentrated to extract the hot water extract hot water extract; A first extraction step of distilling the hot water extract into an ethyl acetate layer and a distilled water layer and dividing the distilled water layer into a butanol layer and a distilled water layer to obtain an extract corresponding to each solvent; Crude extract formation step of distilling the ethyl acetate layer, butanol layer, and distilled water layer under reduced pressure to obtain semi-solid crude extracts for ethyl acetate layer, butanol layer, and distilled water layer; and dissolving each semi-solid crude extract in distilled water, followed by ODS. a second extraction step of obtaining an extract by fractionation using (octadesylsilane) open column chromatography; Extracting method of sessin extract comprising a whitening active ingredient, characterized in that it comprises a. The method of claim 1, The re-melting step, Distilled water is added to the medicine bath, extracting method of sessin extract containing the whitening active ingredient, characterized in that the re-tangling twice for 2 hours 30 minutes. The method of claim 1, The first extraction step, The hot water extract is placed in a separatory funnel with a Teflon stopper attached to the sessin extract containing a whitening active ingredient, characterized in that the fraction is extracted in a separatory filter shaker. The method of claim 3, The fraction extraction is Ethyl acetate layer, butanol layer, distilled water layer repeated extraction five times in order to increase the recovery rate of the active ingredient sessin extract containing the whitening active ingredient. The method of claim 1, Sessin extract extracted in the second extraction step, A method of extracting sessin extract comprising a whitening active ingredient, characterized in that the crude extract of the ethyl acetate layer is fractionated. The method of claim 5, The crude extract relating to the ethyl acetate layer is 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, 100% MeOH extraction method using a sessin extract comprising a whitening active ingredient, characterized in that fractionated using the eluent. The method of claim 6, The crude extract of the ethyl acetate layer is extracted using a whitening active ingredient, characterized in that the fraction is fractionated using 60% methanol.

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