KR100765436B1 - Methods for increasing aurantio-obtusin content in Cassiae Semen, and the method for extraction of aurantio-obtusin having increased contents - Google Patents

Abstract

The present invention is a method of increasing the content of aurantio-optucin in the ethanol extract of the defect by treating the ethanol extract of the Aspergillus kawachii- derived coenzyme solution, using the solubility difference and various chromatographies for the ethanol extract Provided are a method for purifying thio-optucin, and a dietary supplement or pharmaceutical composition prepared by mixing a carrier, an excipient, a diluent, and the like with the purified aurathio-optucin.

According to the present invention, it can be extracted by increasing the content of the aurantio-optucin active ingredient, such as anti-mutation, phytopathogenic inhibition, as a result can provide a health supplement food composition and a pharmaceutical composition with improved therapeutic effect have.

Defender, Aurantio Optusin

Description

Methods for increasing aurantio-obtusin content in Cassiae Semen, and the method for extraction of aurantio-obtusin having increased contents }

FIG. 1 is a flowchart illustrating a method of increasing the content of Aurantio-Ottocin as an active ingredient in the ethanol extract of the Clarifier as an embodiment of the present invention, and then purifying Aurantio-Ottocin from the extract.

Figure 2a is an HPLC chromatogram showing the content of the aurathio-optucin in the ethanol extract of the defect before treatment with Aspergillus kawachii- derived coenzyme solution.

FIG. 2B is an HPLC chromatogram showing the content of aurantio-optucin in ethanol extract of Cassiae after inert enzyme treatment.

Figure 2c is an HPLC chromatogram showing the content of aurathio-optucin in the ethanol extract of the defect after treatment with Aspergillus kawachii- derived coenzyme solution of the present invention.

Figure 3 is a hydrogen nuclear magnetic resonance spectrum of the aurantio-optucin purified by one embodiment of the present invention.

4 is a carbon nuclear magnetic resonance spectrum of the aurantio-optucin purified by one embodiment of the present invention.

Figure 5 is a graph showing the measurement of the aurantio-optucin content change over time when the Aspergillus kawachii- derived coenzyme solution treatment.

The present invention relates to a method for increasing the content of aurathio-optucin in the ethanol extract of the defect and to a method for purifying the aurathio-optucin increased by the method, more specifically Aspergillus kawachii Increase the content of aurantio-optucin, an active ingredient having antimutagenicity, phytopathogenic inhibition, etc., from the extract of L. vulgaris treated with derived coenzyme solution, and effectively purify the active ingredient, aurathio-optucin, from the extract. It is about how to.

In general, the Cassiae Semen is a year-old herbaceous plant of legumes (leguminosae) that grows about 1m in length, with yellow flowers in June-August, and its fruit harvested in August-September. Fruits of the name tuber are pointed on one side, yellowish brown, greenish brown, 4-7mm long, 2-3mm wide, and have a hexagonal shape, known as aurantio-obtusin. Has been reported to exhibit various physiological activities such as antimutation and phytopathogenic inhibition.

Aurantio-optucin is represented by the following formula (1).

Literature on the antimutagenic action of aurantio-optucin is described in J. S., Choi; H. J., Lee; K. Y., Park; J. O., Ha; "In vitro antimutagenic effects of anthraquinone aglycones and naphthopyrone glycosides from Cassia tora" by S. S., Kang. Planta medica (1997), 63 (1), 11-4. The literature on phytopathogenic inhibitory activity is Yu, Dazhao; Yang, Xiaojun; Ni, Hanwen; Yang, Lijun; Wang, Shaonan; Zhao, Yongyu; "Use of anthraquinone derivatives as pesticides for controlling plant diseases." By Zhang, Hanyi. PCT Int. Appl. (2005), 16.

However, to date any literature on techniques for increasing the content of aurantio-optucin in the extracts of the Cassiae extract and further on extracting aurathio-optucins from the extracts of the Cassiae with this increased aurathio-optucin content Has not been found.

Accordingly, an object of the present invention is to provide a method for increasing the content of aurathio-optucin, the active ingredient in the ethanol extract of the defect.

Another object of the present invention is to provide a method for purifying aurantio-optocin from ethanol extracts of the cultivars treated to increase the aurantio-optucin content.

Still another object of the present invention is to provide an auxiothio-optucin extract of such increased content, thereby providing a health supplement composition and pharmaceutical composition effective in antimutation, phytopathogenic inhibition, and the like.

According to the first aspect of the present invention,

Provided is a method for increasing the content of aurantio-optucin in the ethanol extract of the Clarifier, characterized in that the ethanol extract of the Clarifier is treated with Aspergillus kawachii- derived coenzyme solution for at least 50 minutes at 37 ° C.

According to the second aspect of the present invention,

The ethanol extract of the Cultivator with the increased content of aurantio-optucin by the method of the first aspect was dispersed in water, extracted by distillation with one solvent selected from the group consisting of dichloromethane and ethyl acetate, and concentrated to give an organic solvent solubility. Obtaining a fraction; And

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Concentrate the organic solvent soluble fraction, and then use a solvent selected from the group consisting of methanol, hexane, ethyl acetate, acetic acid, formic acid, chloroform, benzene, and a solvent mixed with two or more selected from them. Purifying aurantio-optucin using chromatography using a carrier selected from the group consisting of liquid and Sephadex (A). Is provided.

According to the third aspect of the present invention,

Provided are a health supplement composition comprising an aurathio-optucin, a carrier, an excipient, and a diluent purified by the method of the second aspect.

According to the fourth aspect of the present invention,

There is provided a pharmaceutical composition comprising aurantio-optucin, a carrier, an excipient and a diluent purified by the method of the second aspect.

Hereinafter, with reference to the accompanying drawings, the present invention will be described in detail.

In order to increase the content of the active ingredient aurantio-optucin in the deficiency, as shown in FIG. 1, the deficiency is first extracted with ethanol, wherein the ethanol concentration is 80-95%, preferably 95 Use%. This extract is then treated with Aspergillus kawachii- derived coenzyme solution. On the other hand, Aspergillus kawachii used in the present invention mainly produces β-glucosidase, and the optimum temperature of this enzyme is known to be 37 ° C. (Β-glucosidase produced by Aspergillus usamii D5 isolated from fermented foods. Purification and Characterization of Master's Thesis at Kyungpook National University. Therefore, when the ethanol extract of the cultivars is treated with Aspergillus kawachii- derived coenzyme solution, the treatment temperature is about 37 ° C, and preferably 37 ± 2 ° C. In addition, when the ethanol extract of the cultivars was treated with Aspergillus kawachii- derived coenzyme solution at 37 ° C., when the change of the content of the aurathio-optucin over time was measured, the aurathio -op was treated after at least 50 minutes. It was confirmed that the content of the transmission was increased the most. This result is described in FIG. 5.

An appropriate amount, preferably about three times as much water as the obtained dry matter, is added to the dried product, followed by dispersion. Then, an appropriate amount of the dried product obtained by weight, preferably the same amount as the dichloromethane, chloroform, ethyl acetate, ether, Partition extraction with ethanol, preferably dichloromethane.

The obtained extract was dried by distillation, distillation under reduced pressure, or spray dryer, and then separated by silica gel, reverse phase silica gel, Sephadex, preferably column chromatography using silica gel, and the like. Pure fractions are obtained.

Thus obtained aurantio-optucin can be formulated according to a known method by adding a pharmaceutical, cosmetic or pharmaceutically acceptable excipient, carrier, diluent and the like.

Carriers, excipients and diluents are conventionally used as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrroly Money, cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. It is not.

In addition to the above components, it may further include commercially available lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

On the other hand, as an example of the formulation, the active ingredient may be mixed or diluted with the carrier, or may be contained in capsules, sachets, paper or other types of carriers, where the carrier is a solid, semisolid that can serve as a diluent. Or a liquid substance and can act as a vehicle, excipient or medium for the active ingredient. The resulting formulation may be in the form of tablets, pills, dispersants, granules, elixirs, suspensions, emulsifiers, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders and ointments, oral or Parenteral administration.

The aurantio-optucin preparations obtained in this manner can be released rapidly, continuously or, as needed, with the active ingredient after administration to a patient as a dietary supplement or as a pharmaceutical composition.

For example, the daily dosage of aurathio-optucin as the active ingredient is usually within the range of about 0.1-20 mg per kg of body weight, and when used as a dietary supplement, water or ethanol extract It is sufficient in the range of 100 mg to 10 g in terms of equivalent.

Example

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail with reference to drawings. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto.

Example 1 Content of Aurantio-Ottocin in Ethanol Extracts of Enzymatically Treated Cassia

As shown in the flow chart of Figure 1, to collect the crushed crushed, 13 kg of crushed crushed to extract the reflux with 95% ethanol at 80 ℃ and filtered, the filtrate was concentrated under reduced pressure to obtain 120 g of the extract.

The extract thus obtained was dispersed in 1 L of distilled water, and then treated with the same amount of Aspergillus kawachii- derived crude enzyme solution at 37 ° C. for 50 minutes, and lyophilized.

The dried product thus obtained was dispersed in 1 L of distilled water, and then extracted three times with the same amount of dichloromethane, followed by concentrating by mixing only the soluble portion in dichloromethane.

Then, silica gel column chromatography (Merck, Art No. 7734, column size: 5.5 × 40.0 cm, eluent: nucleic acid-acetone = 50-1 was given to the slope of 0-100 for the dichloromethane soluble fraction. Elution) to obtain 10 fractions, of which the 10th fraction contained auranthio-optucin. The 10th fraction was again subjected to silica gel column chromatography (Merck, Art No. 7734, eluent: nucleic acid-acetone = eluted at 8-1 starting from 20-1) to obtain 5 fractions. Of which the fourth fraction contained aurantio-optucin. This fourth fraction was subjected to silica gel column chromatography (Merck, Art No. 7734, eluent: nucleic acid-acetone = eluted with 5-1 starting from 15-1) to obtain 5 fractions. Fourth of these fractions contained aurantio-optucin. This fourth fraction was subjected to silica gel column chromatography (Merck, Art No. 7734, eluent: nucleic acid-acetone = eluted at 10-1 starting from 15-1) to obtain four fractions. The third fraction contained pure only aurathio-optucin, and 0.38 g of aurathio-optucin with 95% purity on HPLC was obtained.

The content of aurantio-optucin in the ethanol extract of the clarifier ethanol extract obtained by repeating the above procedure was 72.31 ± 1.58 mg per gram of ethanol extract.

HPLC chromatograms for the solubilizer ethanol extracts thus obtained were measured and shown in FIG. 2C.

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of the obtained ethanol extracts were measured, and the results are shown in FIGS. 3 and 4, respectively. The results of the respective nuclear magnetic resonance spectra are represented by aurathio-optucin. Since the properties were exactly the same, it was confirmed that the material extracted by the above method was Aurathio-Optucin.

Comparative Example 1-Content of Aurantio-Ottocin in Insoluble Enzyme-treated Ethanol Extract

The same procedure as in Example 1 was repeated, except that an enzyme which was deactivated by heating at 121 ° C. for 15 minutes instead of Aspergillus kawachii- derived coenzyme solution was used for 15 minutes. Toxin was extracted, and the extracted content of aurathio-optucin was 24.55 ± .06 mg / g.

In addition, HPLC chromatograms for the solubilizer ethanol extracts thus obtained were measured and shown in FIG. 2A.

Comparative Example 2 Content of Aurantio-Ottocin in Unethanolated Extract of Ethanol Extract

Except for the enzymatic treatment, the same procedure as in Example 1 was repeated to extract the aurathio-optucin, and the extracted aurathio-optucin content was about 135 mg.

In addition, HPLC chromatograms for the solubilizer ethanol extracts thus obtained were measured and shown in FIG. 2B.

Through the results of Example 1 and Comparative Example 1, by the enzymatic treatment of the ethanol extract of the crystallizer as in the present invention, the aurantio-optocin content in the ethanol extract of the crystallizer is about 2.8 times as compared to the case of inactive enzyme treatment. It was confirmed that the increase to about 2.8 times compared to the case without the enzyme treatment.

 In addition, as can be seen in Figures 2a to 2c, HPLC chromatograms of aurathio-optocin (Fig. 2a and 2b) for the clarifier ethanol extract without any treatment and the ethanol extract treated with inactive enzymes is very small In contrast to the aurathio-optocin peak, the HPLC chromatogram (Fig. 2c) of the ethanol extract of the clarifier obtained in Example 1 showed that the peak was very large.

It is apparent to those skilled in the art that the present invention is not limited to the above-described embodiments, and that various modifications and changes can be made without departing from the spirit of the present invention.

In other words, the present invention illustrates the increase in the amount of aurantio-optucin by using the deficiency as an example, but the same or similar effects may be obtained through the same Aspergillus kawachii- derived coenzyme solution treatment on similar plants containing the aurathio-optucin . You can get it. Therefore, such modifications or variations are intended to fall within the claims of the present invention.

According to the present invention, it is possible to extract by increasing the content of the active ingredient aurantio-optucin, such as anti-mutation, phytopathogenic inhibition, resulting in a food supplement and pharmaceutical composition for health supplements improved economical function or therapeutic effect Can be provided.

Claims (11)

A method of increasing the content of aurantio-optucin in an ethanol extract of the nickname , comprising treating the ethanol extract of the nickname with an Aspergillus kawachii derived coenzyme solution at about 37 ° C. for at least 50 minutes. delete The ethanol extract of the cultivars with increased content of aurantio-optucin, prepared according to the defined method of claim 1, was dispersed in water, partitioned and extracted with one solvent selected from the group consisting of dichloromethane and ethyl acetate. To obtain an organic solvent soluble fraction; And The organic solvent soluble fraction is concentrated, and then silica gel, reverse phase silica gel, high-speed liquid using a solvent selected from the group consisting of methanol, hexane, ethyl acetate, acetic acid, formic acid, chloroform, benzene, and a solvent mixed with two or more selected from them. And purifying auraantio-optucin using chromatography using a carrier selected from the group consisting of Sephadex; Purification method of aurantio-optucin in the ethanol extract of the bladder comprising a. A health supplement food composition comprising a carrier, an excipient and a diluent in 100 mg to 10 g of the aurantio-optucin purified by the method of claim 3. The method of claim 4, wherein the carrier, excipient and diluent are lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpi Characterized in that it is selected from the group consisting of lollidon, cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil Health supplement food composition. The method of claim 4 or 5, wherein the tablets, pills, dispersants, granules, elixirs, suspensions, emulsifiers, solutions, syrups, aerosols, soft and hard gelatin capsules, sterile injectable solutions, sterile packaged powders or ointments Health supplement food composition having a form. The health supplement food composition according to claim 4 or 5, further comprising one or more selected from the group consisting of a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. delete delete delete delete

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