KR100549097B1 - Method for extracting vitexin and isovitexin from Vigna Radiata L. and extract thereof - Google Patents

Abstract

The present invention relates to bitexin and isobitexin extract derived from mung bean and its extraction method, and more particularly, the extraction method of the present invention is the step of separating the crude extract by reflux cooling method by immersing mung bean powder in 80% methanol ; Degreasing the crude extract with methylene chloride; Fractionating the sample by solvent fractionation of the remaining aqueous layer degreased with ethyl acetate and concentration of the supernatant in vacuo; And separating the vitexin and isobitexin from the partially purified sample at the same time by using Sephadex LH-20 open column, so that the nontexin and isobitexin components contained in the green beans can be easily and quickly extracted in large quantities. In addition, the vitexin and isobitexin extract of the present invention separated in this way can be used as a raw material of various whitening cosmetics because it is effective to prevent the accumulation of waste of the skin and inhibit aging.

Mung beans, flavonoids, bitexins, isobitexins, extraction methods, solvent fractions, open columns

Description

Method for extracting vitexin and isovitexin from Vigna Radiata (L.) and extract etc}

Figure 1 schematically shows the extraction process of bitexin and isobitexin derived from mung bean of the present invention.

FIG. 2 is a high-performance liquid chromatography (HPLC) result of mung beans used in the present invention, where (A) represents non-texin and (B) represents isobitex.

Figure 3 shows the chromatogram of the bitexin (top) and isobitaxin (bottom) separated by Sephadex LH-20 according to the extraction method of the present invention.

The present invention relates to an extraction method for extracting the non-texin and isobitexin components contained in mung beans easily and quickly in large quantities, and more particularly, to a solvent by ethyl acetate without using expensive equipment. The present invention relates to a method for simultaneously extracting bitexin and isobitexin derived from mung beans using only a fraction and an open column.

Mungbean ( Vigna Radiata (L.) Wilczek or Mungbean) has been widely used as an antipyretic or antidote in herbal medicine since ancient times, and in the literature such as Dongbobogam, it is used as a soap to clean the face, remove skin impurities, and is effective for dry skin Many prescriptions related to skin beauty have been described as it eliminates blemishes, removes sweat bands that occur in summer, and is effective for swelling, boils and mumps caused by penetration of dermatophytes in the stratum corneum of the skin.

The skin care effect of mung beans is due to vitexin and isobitexin contained in mung beans, and these bioactive substances are a kind of flavonoid-C-glycoside. Vitexin and isobitexin components have the function of removing free radicals and sebum of the skin, which are toxic components in the body, and are known to be effective in preventing the accumulation of waste products in the skin and inhibiting aging. As a study on the bitexin and isobitexin, a method of separating the active substance contained in the ethanol extract of mung beans or separating the bitexin and isobitexin contained in the water extract of mung beans has been reported. As a method of separating vitexin from lichens, isobitexin from rice ethanol extract, and isolates of vitexin and isobitexin from buckwheat ethanol extract.

On the other hand, Korean Patent Registration No. 151247, which is a patent related to the present invention, discloses a method for separating the extract containing vitexin and isobitexin from mung bean using 50-80% aqueous ethyl alcohol solution, but extracting by the above method. Since the mung bean extract is not a pure separation method of bitexin and isobitexin, a large amount of carbohydrates, ash, etc. are present in the obtained whole extract. Meanwhile, as an example of using mung bean extract in cosmetics for skin beauty, Korean Patent Publication 2003-30327 describes a whitening cosmetic composition containing mung bean skin extract, and Korean Patent Publication 2002-45300 discloses a powder face wash composition, Korean Patent Publication 2000-38844 discloses a whitening cosmetic composition containing mung bean extract, and Korean Patent Laid-Open Publication No. 1999-26091 discloses a skin cosmetic composition, respectively, all of which contain crude extracts of mung beans that are not pure bitexin and isobitexin components. It is a problem.

Therefore, there is an urgent need for the development of a method for extracting only the large amounts of skin-active substances, betaxin and isobitaxin, from mung beans. In particular, until now, research on mung bean components has been mainly conducted on primary metabolites such as proteins, fats, and carbohydrates, and is insufficient on secondary metabolites, and on crops such as breeding of varieties with high bioactive components. This is especially true given the lack of access.

Accordingly, the present inventors have made intensive studies to develop a method of extracting high purity vitexin and isobitexin from mung bean with the above problems in mind, and as a result, solvents are used to extract extracts using solvent points, which have different polarities and chemical properties. By partially purifying and using an open column, it was confirmed that the high purity non-taxin and isobitexin components can be extracted easily and quickly at the same time, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a novel extraction method for purely separating high purity vitexin and isobitexin from mung beans using a solvent fraction and an open column.

Another object of the present invention is to provide a non-texin extract derived from mung bean which is extracted by the above extraction method and contains a non-texin as an active ingredient.

On the other hand, another object of the present invention is to provide an extract of Miso beans derived from mung bean which is extracted by the above extraction method and contains isobitaxin as an active ingredient.

In order to achieve the above object, the method of extracting bitexin and isobitexin derived from mung bean according to the present invention comprises immersing mung bean powder in 80% methanol (MeOH) to separate crude extract by reflux cooling; Degreasing the crude extract with methylene chloride; Fractionating the sample by performing solvent separation on the remaining aqueous solution layer after degreasing with ethyl acetate and concentrating the supernatant in vacuo; And simultaneously separating vitexin and isobitexin from the partially purified sample using a Sephadex LH-20 open column.

On the other hand, the present invention is characterized by another extract of mung bean-derived bitexin extracted by the above method containing the non-texin as an active ingredient.

In addition, the present invention is characterized by another extract of Miso beans derived from mung bean which is extracted by the above method and contains isobitaxin as an active ingredient.

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

The chemical structures of the skin active substances Vitexin and Isobitexin contained in mung beans are represented by the following Chemical Formula 1.

Vitexin and isobitaxin belonging to flavonoid-C-glycoside are a kind of physiologically active substance that functions to remove sebum of free radical and skin sebum.

Therefore, the present invention is characterized in that it provides a novel extraction method for extracting high-purity vitexin and isobitexin from mung beans that can be easily encountered in daily life. According to the method of the present invention, by using a solvent fraction, it is possible to extract vitexin and isobitexin easily from mung beans, and to separate quickly in large quantities because vitexine and isobitexin are simultaneously separated using an open column.

The extraction method of the present invention will be described in detail with reference to each step as follows.

The first step is a crude extract separation step using methanol, finely ground green beans, and methanol (MeOH) is used to 80% methanol. Mung bean powder is immersed in 80% methanol and boiled for 12 hours by reflux cooling to inhibit the enzymatic reaction in mung beans, and then concentrated using a rotary vacuum dryer to obtain a crude extract of mung beans.

Next, in the defatting step, methylene chloride (dichloromethane, CH ₂ Cl ₂ ) is used as the degreasing solvent in the present invention. Methylene chloride has been shown to be the most efficient by several experiments in which the crude extract is returned to the separatory funnel and induce degreasing with methylene chloride. It is preferable to perform degreasing about 3 times every 12 hours.

After the degreasing is completed, the remaining aqueous solution is solvent fractionated with ethyl acetate (CH ₃ COOC ₂ H ₅ ), and the purified supernatant, ie, ethyl acetate, is concentrated in vacuo to partially purify the sample.

The final step of the extraction method of the present invention is the pure separation of bitexin and isobitexin using an open column (7 × 70 cm) filled with Sephadex. Sephadex is one of the fillers used in open columns and can be semi-permanently reused, allowing fast mass extraction by separating both bitexin and isobitexin at the same time. In the present invention, only methanol is used as the eluate for Sephadex.

Vitexine extract and isobitexine extract of the present invention extracted by the above method is almost 100% high purity material is effective in preventing the accumulation of skin waste and prevent skin aging, so it is used in various skin cosmetics including whitening cosmetics It can be used as.

Hereinafter, the configuration and operation effects of the present invention through the examples will be described in more detail. However, it is obvious to those skilled in the art that the present invention is not limited only to these examples.

<Example 1>

Investigation of the contents of vitexin and isobitexin in mung bean

end. Quantitative and Qualitative Analysis

0.5 g of dried mung bean powder (100 mesh or less) was mixed with 10 ml of 70% ethanol and extracted for about 1 hour and 30 minutes in an 80 ° C water bath, which was passed through a micro-cylinder (0.45 µm) for use as a sample for quantitative HPLC It was. HPLC was used for Shimadzu system (LC-10AD pump, SCL-10A controller, SPD-M10A diode array detector, CLASS-LC10 software) and the conditions are shown in Table 1 below. HPLC analysis results are shown in FIG. 2, and chromatograms of standards were obtained as shown in FIG. 2. For qualitative analysis of each peak, compounds were identified after mass was confirmed using LC-Mass.

HPLC Conditions for Investigation of the Contents of Vitexin and Isobitexin in Mung Bean Flavonoids Detection time Condition Bitexin 6 minutes Mobile phase: MeOH: H ₂ O: EtOAC = 34.6: 60.0: 5.4 flow rate: 1 mL / min detector: photo diode array 254 nm column: PHENOMENEX C ₁₈ column, 250 × 4.60 mm, 4 μm, with BioRad Biosil Micro-Guard (C ₁₈ guard column) Temperature: Room temperature Isobitexin 7 minutes Same as above

I. Analysis

The results of quantifying bitexin and isobitexin for 195 mung bean resources are shown in Table 2 below. The content of bitexin was 2.39 mg / g to 11.48 mg / g depending on the resource, and there was a difference of up to 4.8 times among the resources. The content of isobitaxin contained 0.97 mg / g to 4.40 mg / g depending on the resource, and there was a difference of up to 4.5 times among the resources. The total flavonoid content was 3.36 mg / g to 15.88 mg / g depending on the resource, up to 4.7 times the difference between the resources. In particular, KM99004-4B-2 (Suwon No. 28 / KM94004, '03 yield trial No. 6), which is being grown by the Jeonnam Agricultural Research and Development Institute, contained the most (15.88 mg / g). This strain can be said to be of value as a breeding breed or breed for functional breeding.

Quantitative Results of Vitexin and Isovitex on 195 Green Bean Resources Flavonoids Content (mg / g) 2.00 or less 2.01-4.00 4.01-6.00 6.01-8.00 8.01 to 10.00 10.01 or later Mean ± S.D. Distribution (%) Bitexin 0 17.4 37.5 41.5 3.1 0.5 5.63 ± 1.50 Isobitexin 26.7 72.8 0.5 0 0 0 2.33 ± 0.63

<Example 2>

Mass extraction

0.5 kg of mung bean powder was added to each of two round flasks (5 L), and 80% methanol (MeOH) was added to 2 L each. The mixture was boiled under reflux for 12 hours and concentrated in a rotary vacuum dryer to prepare a crude extract. . The crude extract was added to a separatory filter, and degreased with 0.5 liter of methylene chloride three times at 12-hour intervals. The aqueous layer was separated by solvent fractionation with ethyl acetate, and the supernatant (ethyl acetate layer) was concentrated in vacuo. A partially purified sample was obtained. Partially purified samples were fractionated using 80% MeOH in an open column (7 × 70 cm) filled with Sephadex LH-20 to obtain 320 (7 mL / tube) elution (separation). In the 70 fractions, the vitexin and the 150 to 170 fractions were purely separated. Chromatograms of the isolated vitexins and isobitexins are shown in FIG. 3.

As described above, the present invention is a simple and rapid method for extracting large quantities of vitexin and isobitexin contained in mung beans, and extracting high purity materials using only solvent fraction and open column without using expensive equipment. It is expected that green beans will be developed as a source of these materials as well as to produce new agricultural crops, and the market for high-purity, high value-added functional processed products is expanding rapidly. Will be high. On the other hand, high purity bitexin and isobitexin extracted in this manner may be used as an additive of various cosmetics including whitening cosmetics.

On the other hand, while the above has been described with reference to the preferred embodiments of the present invention, those skilled in the art to which the present invention pertains without departing from the spirit and scope of the present invention described in the claims below It will be understood that modifications and changes can be made.

Claims (3)

Immersing the mung bean powder in 80% methanol to separate the crude extract by reflux cooling; Degreasing the crude extract with methylene chloride; Fractionating the sample by performing solvent separation on the remaining aqueous solution layer after degreasing with ethyl acetate and concentrating the supernatant in vacuo; And A method for extracting bitexin and isobitexin from mung beans, comprising simultaneously separating vitexin and isobitaxin from the partially purified sample using a Sephadex LH-20 open column. delete delete

Images