KR20180124733A - Process for Producing Deoxypodophilotoxin in Quantity - Google Patents

Abstract

The present invention relates to a method for extracting high-purity deoxypodophyllotoxin in quantity by using a column and an alcohol solvent from a substance to be extracted containing the deoxypodophyllotoxin. Conventional purification methods require complicated steps and have a disadvantage in that the yield of deoxypodophyllotoxin finally obtained is low. However, the present invention does not have a process of concentrating after alcohol extraction of the prior art, and the separation of the solvent is not required. Therefore, the present invention is suitable for simple and large-scale extraction of the deoxypodophyllotoxin contained in anticancer drugs and the like.

Description

Technical Field [0001] The present invention relates to a process for producing Deoxypodophyllotoxin in Quantity

The present invention relates to a method for massive extraction and production of deoxyglyptyltoxin from an object to be extracted.

Deoxypodophyllotoxin (deoxypodophyllotoxin) is a compound of the genus Pulsatilla koreana ], Juniperus chinensis L.), Hanbaek ( Thujopsis dolabrata ), soil smuggling ( Bridelia tulasneana ) or the like. Deoxyplastin toxin inhibits apoptosis by stopping the cell cycle or controlling the expression of proliferating proteins in various cancer cells such as cervical cancer HeLa cells, lung cancer A549 cells, colon cancer HCT116 cells and skin cancer B16F10 cells. Or inhibits angiogenesis of cancer cells, resulting in an anticancer effect. In addition to the insecticidal effects of oriental armyworms, they have been reported to have analgesic, antiinflammatory effect, whitening effect to suppress skin pigmentation caused by UV, and antiviral effect against herpes simplex virus. In particular, deoxyglycolipotoxin has excellent antitumor activity and is reported to be the main ingredient of SB injection of natural anti - cancer drug in Korea. This is advantageous in that it can be widely used for medicines, cosmetics, insecticides, livestock feed, etc. due to the anti-cancer effect, insecticidal effect, whitening effect and anti-inflammatory effect of deoxyglycolipotoxin.

Deoxyglyptyltoxin is purified from methanol extract or ethanol extract from Baekguyong, Jeonho, and Hwangbok after complicated separation such as silica gel column chromatography. However, there has been no reports of separation and purification in large quantity in high purity.

Accordingly, an object of the present invention is to provide a method for extracting deoxyglycolipotoxin, which is a useful substance, in a large amount and at a high purity, while extracting a simple deoxyglycolipotoxin.

In order to solve the above problems, the inventor of the present invention has invented an extraction method which can extract a simple but highly deoxyglycolipotoxin in a large amount and a high purity.

1. extracting the object to be extracted with 100% alcohol and adding distilled water to prepare an extract of 80% alcohol;

Passing the extract through a column;

Passing 80% alcohol through the column to remove unadsorbed material;

100% alcohol is passed through a column to elute deoxyglycolipotoxin which has been adsorbed, thereby extracting deoxyglyptyltoxin.

2. The method according to claim 1, wherein the alcohol is methanol or ethanol.

The method of massextraction of deoxyglyptyltoxin of the present invention is simple in purification step compared with the conventional purification method through a complicated separation such as methanol or ethanol extraction and silica gel column chromatography, This is possible. In addition, since the extract is not adsorbed in the aqueous solution state, the amount of resin required is small.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a simplified view of a method for extracting deoxypluginous phytotoxin in large quantities from Duanion HP-20 resin and methanol solution according to Example 1-2 of the present invention.
2 is a graph comparing high performance liquid chromatography (HPLC) graphs of the methanol extract, Diaion HP-20 resin passage liquid and 100% methanol eluent and deoxyglycolipotoxin standard material extracted according to Example 1-2 of the present invention Fig.
FIG. 3 is a graph showing the results of ¹ H-NMR analysis of a substance appearing at 9 minutes retention in 100% methanol eluate of Diaion HP-20 resin according to Example 1-2 of the present invention.
FIG. 4 is a graph showing the anticancer effect of deoxyplastin toxin isolated and purified according to Example 1-2 of the present invention through MTT assay. FIG.
FIG. 5 is a simplified view of a method of extracting deoxypluginous phytotoxin in a large amount from the Korean Pharmacopoeia using a Diaion HP-20 resin and an ethanol solution according to Example 2 of the present invention.
FIG. 6 shows a comparison of high performance liquid chromatography (HPLC) graphs of the ethanol extract, Diaion HP-20 resin passage liquid and 100% ethanol eluent and deoxyglycolipotoxin standard material extracted according to Example 2 of the present invention .
FIG. 7 is a graph showing the results of ¹ H-NMR analysis of a substance appearing at a retention time of 9 minutes in a 100% ethanol eluate of Diaion HP-20 resin according to Example 2 of the present invention. FIG.

The present invention relates to a method for preparing an extract, comprising the steps of: passing an 80% alcohol extract of an object to be extracted through a chromatography column to adsorb deoxyplugin toxin to the column;

Passing 80% alcohol through the column to elute the non-adsorbed material;

100% alcohol is passed through the column to elute deoxyglycolipotoxin which has been adsorbed, thereby providing a method for extracting deoxyglycolipotoxin.

In the step of passing the 80% alcohol extract of the object to be extracted through the chromatography column, the deoxyglycolyticoxin is adsorbed on the column, and the other ingredients are separated through the column. Thereafter, 80% alcohol is passed through the column to remove any remaining unadsorbed material. Finally, 100% alcohol is passed through the column to elute deoxyglycolipotoxin adsorbed. As a result, an alcohol solution in which deoxyglycolipotoxin is dissolved in high purity is obtained.

The alcohol can be evaporated or separated in the obtained alcohol solution to purify deoxyglycolipotoxin.

In the present invention, the alcohol refers to all compounds having at least one -OH group, and preferably the alcohol is methanol or ethanol.

In the present invention, the subject to be extracted may be any one containing deoxyglycolipotoxin. For example, the subject to be extracted of the present invention may be selected from the group consisting of Korean pepper, white pepper, juniper, Japanese white pine, Japanese white pine, and Korean soil, but is not limited thereto.

In the present invention, the columns should have a smaller grafting with deoxyplastin rather than 100% alcohol with greater gravity than deoxyglycolipotoxin, rather than 80% alcohol. Examples thereof include activated carbon, Diaion HP-20 resin, and Amberlite XAD-20 resin. However, if it satisfies the above conditions, it is possible to use other than activated carbon, Diaion HP-20 resin and Amberlite XAD-20 resin.

Example

Hereinafter, preferred embodiments, comparative examples, and experimental examples are provided to facilitate understanding of the present invention. However, the following examples, comparative examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, comparative examples and experimental examples.

EXAMPLES Example 1. Extraction of deoxyglycolipotoxin with methanol

Example 1-1. Selection of extraction solvent

To select the solvent used for extraction, hot water extraction, 50% methanol extraction and 100% methanol extraction were performed and the results were compared. Specifically, 10 g of the extract was subjected to hot water extraction at 100 ° C. for 10 minutes or methanol extraction at room temperature for 24 hours using 50% methanol and 100% methanol, and then the solid content of the extract was measured using a reduced pressure centrifugal evaporator (SpeedVac) And the content of deoxyglycolipotoxin contained in the solvent extracted with butanol was measured by high performance liquid chromatography (HPLC). The content of deoxyglycolipotoxin contained in the extracts was different from that of the extracts obtained by the method of hot water extraction and methanol extraction according to the content of methanol. The results are summarized in the following Table 1 Respectively.

Extraction method Extract
Solids content Of butanol extract
Solids content Of butanol extract
Deoxyplastin toxin content Yield (%)
(DPT / solid content) Hot water extraction 2.95 g 0.12 g 15.95 mg 0.54 50% methanol extract 2.67 g 0.14 g 67.20 mg 2.52 100% methanol extract 1.57 g 0.15 g 66.79 mg 4.25

That is, the content of the solid extracted by the hot water extraction method was the highest, but the content of deoxyglycolipotoxin was the lowest. In the 50% methanol extraction method, not only the content of the extracted solid was high but also the content of deoxyglycolipotoxin was high. In the 100% methanol extraction method, the content of the extracted solid matter was small, but the content of deoxyglycolyticoxine contained in the extract was similar to that of the 50% methanol extraction method. Thus, the yield of 4.25% (Content of deoxyplugin toxin / content of solid content). Based on these results, 100% methanol was selected as the most effective solvent for extracting deoxyplastin toxin.

Examples 1-2. Large-scale extraction of deoxyglyptyltoxin from the former

10 kg of the dry bulb was crushed finely with a large-capacity mixer, and 45 liters of 100% methanol was added to the milled mixture and extracted at room temperature for 2 days. After the first extract was recovered, 45 liters of 100% methanol was added again, and the second and third extraction were carried out under the same conditions. After completion of the third extraction, the precipitate of the 100% methanol extract was removed with a filter paper, and distilled water was added thereto to adjust the concentration of methanol to 80%. Lipophilic substances including deoxyglycolyticoxin were adsorbed on a Diaion HP-20 column, which had been equilibrated with 80% methanol in advance, through a methanol extract of the former, which had been diluted with 80% methanol to a final volume of about 4.5 liters. After all of the extract was passed through, 20 l of 80% methanol was used to rinse the unadsorbed material on the Diaion HP-20. Then, 100% methanol was used to elute deoxyglycolyticoxin adsorbed on Diaion HP-20. 100% methanol eluate containing deoxyplastin toxin was removed by methanol with a vacuum rotary evaporator and analyzed by high performance liquid chromatography to obtain 29.7 g of deoxyglycolyticoxin fraction having a purity of 80%. The extraction process of this embodiment is shown in FIG. 1 in a simplified manner.

COMPARATIVE EXAMPLE 1 After the conventional extraction method, methanol extraction, purification was carried out through complicated separation such as silica gel column chromatography

First, 500 g of the extract was extracted with methanol, and the methanol was evaporated with a rotary evaporator to leave only the solid component. The remaining solid was added with water and ethyl acetate (ethylacetate) in an equal volume and only the ethyl acetate layer was removed. A silica gel was added to the ethyl acetate layer, and ethyl acetate was removed by a rotary evaporator to adsorb the solid component on silica gel. The solid component was added to a silica gel column previously packed, and hexane and ethyl acetate were mixed in a ratio of 3: 1, To obtain a fraction containing deoxyplugin toxin. The fraction containing eluted deoxyplateletoxin was adsorbed onto C ₁₈ -reversed phase silica gel in the same manner as described above, and deoxyglycolipotoxin was eluted with 60% methanol as a developing solvent using a packed column. The fractions containing deoxyplastin toxin were concentrated on a rotary evaporator, added to a Sepadex LH-20 column and eluted with 100% methanol as eluent. The fractions containing eluted deoxyplastin toxin were separated and fractionated on a C ₁₈ column and 60% methanol using high performance liquid chromatography (HPLC) under developing solvent conditions to obtain pure deoxyglycolipotoxin 50 mg.

The results are shown in Table 2 below.

Refining method Amount of initial volume Extract
Solids content Of the final extract
Solids content Of the final extract
DPT content Purity (%)
(DPT / solid content) Comparative Example 1 500 g 79 g 52 mg 50 mg > 95 pure separation) Examples 1-2 10 kg 1,065g 37 g 29.7 g 80

In the case of the purity of deoxypluginous phytotoxin after the final purification, the purification method of the comparative example showed a pure water separation of not less than 95%, and the purification method of the example according to the present invention showed a high purity of 80% Respectively. In particular, considering the yield from the extract (total deoxypluginous phytotoxin content of the final extract / solid content of the extract), the purification method of the comparative example was 0.06%, but according to the purification method of the example according to the present invention, the efficiency of 2.8% It was possible to purify oxytocin phytotoxin.

Experimental Example 1-1. HPLC analysis of deoxyglycolipotoxin extracted and purified by the method of Example 1-2

The fraction passed through the Diaion HP-20 column and the fraction eluted in 100% methanol adsorbed on Diaion HP-20 were compared with the standard deoxyglycolyticoxin and subjected to high performance liquid chromatography analysis. The analytical conditions of the deoxysuggestinoctoxin standard material, the methanol extract of the former, the Diaion HP-20 resin passage liquid, and the 100% methanol eluate in the Diaion HP-20 resin are as follows. YL9100 HPLC column from Yeungin Scientific was using Waters Spherisorb ODS2 (Ø 4.6x150 mm, 5 μm) and 60% methanol was eluted with a flow rate of 0.8 mL / min and detected at UV 240 nm. The results of the analysis are shown in Fig. In the methanol extract of prodrug, a peak of deoxyglycolyticoxine was observed at a retention time of 9 minutes, but deoxyglycolyticoxine was not observed in the Diaion HP-20 resin solution. On the other hand, in the 100% methanol eluate of Diaion HP-20 resin, a peak of deoxyglycolyticoxine was observed at a retention time of 9 minutes, and it was found that deoxyglycolyticoxine was purely separated by Diaion HP-20 resin. The high-performance liquid chromatography analysis was carried out by proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) in order to identify the substance appearing at 9 minutes retention, and the results are shown in FIG. The spectrum of this material was the same as that of the standard deoxyplateletoxin, so that it was confirmed that the main component of the 100% methanol eluate of Diaion HP-20 resin is deoxyglycolyticoxin.

Experimental Example 1-2. Anticancer effect test of deoxyglycolipotoxin extracted and purified by the method of Example 1-2

Deoxyglobulatoxin is known to have anticancer effects in various cancer cells including prostate cancer cells, breast cancer cells and lung cancer cells. In the purification method according to the present invention, the effect of deoxyglycolipotoxin isolated from the prior art was confirmed by MTT assay. PC-3 cells, a human prostate cancer cell line, were seeded in a 48-well plate for cell culture at a concentration of 1 × 10 ⁴ cells / well and stabilized for 24 hours. Then, deoxyribonucleotides at concentrations of 2.75, 7.5, 15 and 30 ng / The grape pylotoxin was treated. After 24 hours of reaction, 250 μl of 0.5 μg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) reagent was added and reacted at 37 ° C for 2 hours. After the reaction was over, and after removing the supernatant was dispensed to each of a dimethylsulfoxide (DMSO) to 250 μl The resulting formazan moved by 100 μl in 96-well plate, VERSA _MAX Cell viability was measured by measuring the absorbance at 570 nm using a microplate reader. The results are shown in FIG. 4. As is known in the art, it was confirmed that deoxyplastin toxin isolated from Korean Prolactin using the purification method using Diaion HP-20 resin according to the present invention had an anticancer effect in a human prostate cancer cell line Respectively.

Example 2 Mass Extraction of Deoxyglypylotoxin from Ethanol-Use

The extraction procedure was carried out as follows using ethanol as an extraction solvent instead of methanol. 30 ㎏ of dry bulb was finely pulverized with a large-capacity blender, and 80 ℓ of 100% ethanol was added to the pulverized precursor and extracted at room temperature for 2 days. After the first extract was recovered, 80 liters of 100% ethanol was added again, and the second and third extraction were carried out under the same conditions. After completion of the third extraction, the precipitate of the 100% ethanol extract was removed with a filter paper, and distilled water was added thereto to adjust the concentration of ethanol to 80%. A lipophilic substance including deoxyglycolyticoxin was adsorbed on the Diaion HP-20 column, which had been pre-equilibrated with 80% ethanol in advance, through a Diaion HP-20 column, which had been diluted with 80% ethanol. After all of the extract was passed through, 30 L of 80% ethanol was used to rinse the unadsorbed material on Diaion HP-20. Then, 100% ethanol was used to elute deoxyglycolyticoxin adsorbed on Diaion HP-20. The 100% ethanol eluate containing deoxyplastin toxin was removed by ethanol with a vacuum rotary evaporator and analyzed by high performance liquid chromatography to obtain 100.6 g of a deoxyplastin toxin fraction having a purity of 80%. The extraction process of this embodiment is shown in FIG. 5 in a simplified manner.

COMPARATIVE EXAMPLE 2 After the ethanol extraction, which is a conventional extraction method, purification through a complicated separation such as silica gel column chromatography

First, 500 g of the extract was extracted with ethanol, and the ethanol was evaporated with a rotary evaporator to leave only the solid component. The remaining solid was added with water and ethyl acetate (ethylacetate) in an equal volume and only the ethyl acetate layer was removed. A silica gel was added to the ethyl acetate layer, and ethyl acetate was removed by a rotary evaporator to adsorb the solid component on silica gel. The solid component was added to a silica gel column previously packed, and hexane and ethyl acetate were mixed in a ratio of 3: 1, To obtain a fraction containing deoxyplugin toxin. The fraction containing eluted deoxyplateletoxin was adsorbed onto C ₁₈ -reversed phase silica gel in the same manner as described above, and deoxyglycolipotoxin was eluted with 60% ethanol as a developing solvent using a packed column. The fraction containing deoxyplastin toxin was concentrated on a rotary evaporator, added to a Sepadex LH-20 column and eluted with 100% ethanol as developing solvent. The fractions containing eluted deoxyplastin toxin were separated and fractionated on a C ₁₈ column and 60% ethanol using developing solvent (HPLC) and high performance liquid chromatography (HPLC) to obtain pure deoxyplastin 50 mg.

The results are shown in Table 3 below in comparison with Examples 1-2 of the present invention.

Refining method Amount of initial volume Extract
Solids content Of the final extract
Solids content Of the final extract
DPT content Purity (%)
(DPT / solid content) Comparative Example 2 500 g 79 g 52 mg 50 mg > 95 pure separation) Example 2 30 kg 3,197 g 125.2 g 100.6 g 80

In the case of the purity of deoxypluginous phytotoxin after the final purification, the purification method of the comparative example showed a pure water separation of not less than 95%, and the purification method of the example according to the present invention showed a high purity of 80% Respectively. In particular, considering the yield from the extract (total deoxypluginous phytotoxin content of the final extract / solid content of the extract), the purification method of the comparative example was 0.06%, but according to the purification method of the example according to the present invention, the efficiency was 3.1% It was possible to purify oxytocin phytotoxin.

Experimental Example 2. HPLC analysis of deoxyglycolipotoxin extracted and purified by the method of Example 2

The fraction passed through the Diaion HP-20 column and the fraction eluted with 100% ethanol adsorbed on Diaion HP-20 were compared with the standard deoxyglycolyticoxin and subjected to high performance liquid chromatography analysis. The standard conditions for deoxyplastin toxin, ethanol extract of Jeongho, Diaion HP-20 resin, and Diaion HP-20 resin are as follows. YL9100 HPLC column from Yeungin Scientific was using Waters Spherisorb ODS2 (Ø 4.6x150 mm, 5 μm) and 60% ethanol was eluted with a flow rate of 0.8 mL / min and detected at UV 240 nm. The results of the analysis are shown in Fig. Peak of deoxyglycolipotoxin was observed at 9 minutes retention time in the total ethanol extract but deoxyglycolyticoxine was not observed in Diaion HP-20 resin solution. On the other hand, in the 100% ethanol eluate of Diaion HP-20 resin, a peak of deoxyglycolyticoxine was observed at a retention time of 9 minutes, and it was found that deoxyglycolyticoxine was purely separated by Diaion HP-20 resin. The high-performance liquid chromatography analysis was carried out by proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) in order to identify the substance appearing at a retention time of 9 minutes, and the results are shown in FIG. The spectrum of this material is the same as that of the standard deoxyplateletoxin, indicating that the main component of the 100% ethanol eluate of Diaion HP-20 resin is deoxyglycolyticoxin.

Claims (4)

Passing an 80% alcohol extract of the object to be extracted through a chromatography column to adsorb deoxyglycolipotoxin to the column;
Passing 80% alcohol through the column to elute the non-adsorbed material;
100% alcohol is passed through the column to elute deoxyglycolipotoxin that has been adsorbed. The method of claim 1, wherein the alcohol is methanol or ethanol. 2. The method according to claim 1, wherein the object to be extracted is at least one selected from the group consisting of Korean pepper, white radish, jade tree, Japanese white cabbage, Japanese white bean, and Korean soil. The method of claim 1, wherein the column is filled with a filler selected from the group consisting of activated carbon, Diaion HP-20 resin and Amberlite XAD-20 resin.

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