KR102110966B1 - Process for Producing Deoxypodophilotoxin in Quantity - Google Patents

Abstract

The present invention relates to a method for extracting deoxypodophyllotoxins of high purity in large quantities using an alcohol solvent and a column from an extraction target containing deoxypodophyllotoxins. Conventional purification methods require complicated steps and have a disadvantage in that the yield of deoxydophyllotoxin finally obtained is low. However, in the present invention, there is no process of concentration after extraction of the alcohol of the previous issue, and the solvent is single, so no separation process is required. Therefore, the present invention is suitable for simple and large-scale extraction of deoxypodophyllotoxins contained in anticancer agents and the like.

Description

Mass production of deoxypodophilotoxin {Process for Producing Deoxypodophilotoxin in Quantity}

The present invention relates to a method for extracting and producing deoxypodophyllotoxins in large quantities from an extraction target.

Deoxypodophyllotoxins include Baekduong (Galliflower [ Pulsatilla root of koreana ), Juniper ( Juniperus chinensis L.), Na Hanbaek ( Thujopsis dolabrata ), Bridelia tulasneana ). Deoxypophylotoxin stops the cell cycle in various cancer cells such as HeLa cells of cervical cancer, A549 cells of lung cancer, HCT116 cells of colon cancer, B16F10 cells of skin cancer, or apoptosis by regulating the expression of proliferative proteins in cells. Induces or inhibits the blood vessel production of cancer cells, causing anti-cancer action. In addition, not only the insecticidal effect on the pests such as oriental armyworm, but also analgesic, anti-inflammatory effect, whitening effect to suppress skin pigmentation by UV, and antiviral effect on herpes simplex virus were reported. In particular, deoxypodophyllotoxin has excellent anti-tumor activity and has been reported as the main component of the first natural anti-cancer drug SB injection in Korea. Due to the anti-cancer effect, insecticidal effect, whitening effect, and anti-inflammatory effect of deoxypodophyllotoxin, it has the advantage that it can be widely used in drugs, cosmetics, pesticides, and animal feed.

Deoxypodophyllotoxins are purified through complex separation such as silica gel column chromatography after extraction of methanol or ethanol from Baekduong, Jeonho, and cypress trees, but there are no reports of separation and purification in bulk with high purity.

Accordingly, an object of the present invention is to provide a simple method for extracting deoxypodophyllotoxin while being able to extract a useful substance, deoxypodophyllotoxin, in bulk and with high purity.

In order to solve the above problems, the inventors of the present invention have invented an extraction method capable of extracting deoxypodophyllotoxin in a large amount and with high purity, and the means for solving the problems of the present invention are as follows:

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

Passing the extract through a column;

Removing the unadsorbed material by passing 80% alcohol through the column;

A method for extracting deoxypodophyllotoxins comprising; eluting deoxypodophyllotoxins adsorbed by passing 100% alcohol through a column.

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

The bulk extraction method of deoxypodophyllotoxin of the present invention is simple compared to the conventional extraction method, methanol or ethanol extraction, followed by purification through complex separation such as silica gel column chromatography, and the purification step is simple, and high purity and bulk extraction This is possible. In addition, since the extract is not adsorbed in the aqueous solution, the amount of resin required is small.

1 is a simplified diagram showing a method for extracting deoxydodophyllotoxin in bulk from a previous call using Diaion HP-20 resin and methanol solution according to Example 1-2 of the present invention.
Figure 2 is a high-performance liquid chromatography (HPLC) graph comparing the methanol extract, Diaion HP-20 resin passed through and 100% methanol eluate and deoxypodophyllotoxin standard extracted according to Example 1-2 of the present invention It is a diagram showing that.
Figure 3 is a diagram showing the results of analyzing the material appearing in 9 minutes of retention in the 100% methanol eluate of Diaion HP-20 resin according to Example 1-2 of the present invention by ¹ H-NMR.
4 is a view showing the results of confirming the anti-cancer effect of deoxypodophyllotoxin separated and purified according to Example 1-2 of the present invention through an MTT test method.
5 is a simplified diagram showing a method for extracting deoxydophyllotoxin in large quantities from a previous issue using Diaion HP-20 resin and ethanol solution according to Example 2 of the present invention.
FIG. 6 shows a comparison of high performance liquid chromatography (HPLC) graphs of ethanol extract extracted according to Example 2 of the present invention, Diaion HP-20 resin pass-through solution, and 100% ethanol eluate and deoxypodophyllotoxin standard. It is.
7 is a view showing the result of analyzing the material appearing in 9 minutes of retention in the 100% ethanol eluate of Diaion HP-20 resin according to Example 2 of the present invention by ¹ H-NMR.

The present invention comprises the steps of adsorbing deoxypodophyllotoxin to the column by passing an 80% alcohol extract of the extraction target through a chromatography column;

Eluting unadsorbed material by passing 80% alcohol through the column;

It provides a method for extracting deoxypodophyllotoxin comprising; elution of deoxypodophyllotoxin adsorbed by passing 100% alcohol through the column.

In the step of passing the 80% alcohol extract of the extraction target through the chromatography column, deoxypodophyllotoxin is adsorbed to the column, and other components are removed through the column. Thereafter, 80% alcohol is passed through a column to remove the remaining unadsorbed material. Finally, 100% alcohol is passed through a column to elute deoxypodophyllotoxins adsorbed, and as a result, an alcohol solution in which deoxypodophyllotoxins are dissolved in high purity is obtained.

Deoxypodophyllotoxin can be purified by evaporating or separating alcohol from the obtained alcohol solution.

In the present invention, the alcohol refers to all compounds having one or more -OH groups, preferably the alcohol is methanol or ethanol.

In the present invention, the object to be extracted may be any one containing deoxypodophyllotoxin. For example, the extraction target of the present invention may be selected from the group consisting of Jeonho, Baekduong, juniper, side cypress, Nahanbaek and tomilsu, but is not limited thereto.

In the present invention, the column should have a higher attraction force with deoxypodophyllotoxin than 80% alcohol, and a smaller attraction force with deoxypodophyllotoxin than 100% alcohol. Examples include activated carbon, Diaion HP-20 resin, and Amberlite XAD-20 resin, but other materials other than activated carbon, Diaion HP-20 resin, and Amberlite XAD-20 resin may be used as long as the above conditions are satisfied.

Example

Hereinafter, preferred examples, comparative examples, and experimental examples are presented to help understanding of the present invention. However, the following examples, comparative examples, and experimental examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by examples, comparative examples, and experimental examples.

Example 1. Extraction of deoxypodophyllotoxin using 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 compared. Specifically, 10 g of hot water was extracted at 100 ° C for 10 minutes, or 50% methanol and 100% methanol were used to extract with methanol at room temperature for 24 hours, and then the solid content of the extract was extracted using a reduced pressure centrifugal evaporator (SpeedVac). Was measured and the content of deoxydophyllotoxin contained in the solvent-extracted extract with butanol was measured using high performance liquid chromatography (HPLC). Through the hot water extraction method and the methanol extraction method according to the content of methanol, the solid content of the extract extracted from the previous issue was different, and there was also a difference in the content of deoxypodophyllotoxin contained therein, and the results are summarized in Table 1 below. Did.

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

That is, the content of solids extracted by the hot water extraction method was the highest, but the content of deoxydophyllotoxin was the lowest. The 50% methanol extraction method had a high content of deoxypodophyllotoxin as well as a high content of extracted solids. The 100% methanol extraction method had a small amount of solids to be extracted, but the content of deoxydophyllotoxin contained in the extract was similar to the 50% methanol extraction method, yielding the highest yield of 4.25% among the three extraction methods used. (Content of deoxydophyllotoxin / content of solids). Based on these results, 100% methanol was selected as a solvent for extracting deoxydophyllotoxin most efficiently.

Example 1-2. Bulk extraction of deoxydophyllotoxin from Jeonho

After crushing 10 kg of dried pre-cooked with a large-capacity blender, 45 l of 100% methanol was added to the crushed pre-cooked and extracted at room temperature for 2 days. After recovering the primary extract, 45ℓ of 100% methanol was added again to perform secondary and tertiary extraction under the same conditions. After completion of the third extraction, the precipitate of 100% methanol extract was removed with filter paper and distilled water was added to adjust the concentration of methanol to 80%. A fat-soluble substance containing deoxypodophyllotoxin was adsorbed by passing a methanol extract of the previous dilution with 80% methanol to a Diaion HP-20 column having a volume of about 4.5 liters previously equilibrated with 80% methanol. After passing all the extracts, the material not adsorbed to Diaion HP-20 was washed with 20 l of 80% methanol. Subsequently, 100% methanol was used to elute deoxydophyllotoxin adsorbed on Diaion HP-20. Deoxypodophyllotoxin-containing 100% methanol eluate was removed with a vacuum rotary evaporator (evaporator) and analyzed by high performance liquid chromatography to obtain 29.7 g of deoxypodophyllotoxin fraction of 80% purity. The extraction process of this embodiment is simplified to FIG. 1.

Comparative Example 1. Result of purification through complex separation such as silica gel column chromatography after methanol extraction, which is an existing extraction method

First, 500 g of the whole number was extracted with methanol, and then methanol was evaporated with a rotary evaporator, and only solid components remained. Water and ethylacetate were added in the same amount to the remaining solid components, and then only the ethyl acetate layer was taken. Silica gel is added to the ethyl acetate layer, ethyl acetate is removed by rotary evaporation, the solid components are adsorbed on the silica gel, and then added to a pre-filled silica gel column to develop a solution in which hexane and ethyl acetate are mixed in a 3 to 1 solution. Eluted with to obtain a fraction containing deoxydophyllotoxin. The fraction containing the eluted deoxypodophyllotoxin was adsorbed to a C ₁₈ -reverse silica gel in the same manner as described above, and the deoxypodophyllotoxin was eluted using 60% methanol as a developing solvent using a packed column. The fraction containing deoxypodophyllotoxin was concentrated by rotary evaporation, added to a Sepadex LH-20 column, and eluted with 100% methanol as a developing solvent. The fraction containing the eluted deoxypodophyllotoxin is separated and fractionated by using a high performance liquid chromatography (HPLC) C ₁₈ column, 60% methanol in a developing solvent condition, and fractionated to pure deoxypodophyllotoxin 50 mg was obtained.

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

Purification The amount of the initial call Of extract
Solid content Of final extract
Solid content Of final extract
DPT content Purity (%)
(DPT / solid content) Comparative Example 1 500 g 79 g 52 mg 50 mg > 95 pure separation) Example 1-2 10 kg 1,065 g 37 g 29.7 g 80

In the case of the purity of deoxypodophyllotoxin after the final purification, the purification method of the comparative example showed more than 95% pure separation, and the purification method of the example according to the present invention was high purity of 80% despite purifying a large number of simple methods. It was shown. In particular, considering the yield from the whole extract (deoxypodophyllotoxin content of the final extract / solid content of the extract), the purification method of the comparative example was 0.06%, but by the purification method of the embodiment according to the present invention, the efficiency was 2.8%. Oxypodophyllotoxin could be purified.

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

The high-performance liquid chromatography analysis was performed by comparing the fraction passed through the Diaion HP-20 column and the fraction adsorbed on Diaion HP-20 and eluted in 100% methanol compared to the standard deoxypodophyllotoxin. Analysis conditions of deoxypodophyllotoxin standard, methanol extract of Jeonho, Diaion HP-20 resin pass-through, and 100% methanol eluate from Diaion HP-20 resin are as follows. In YL9100 HPLC of Youngin Science, a column was used Waters Spherisorb ODS2 (Ø 4.6x150 mm, 5 μm). As a developing solvent, 60% methanol was eluted at a flow rate of 0.8 mL / min and eluted to detect UV 240 nm. The analysis results are shown in FIG. 2. In the methanol extract of Jeonho, a peak of deoxydodophyllotoxin was observed at a retention time of 9 minutes, but deoxypodophyllotoxin was not observed in the Diaion HP-20 resin pass-through. On the other hand, in the 100% methanol eluate of Diaion HP-20 resin, the peak of deoxydodophyllotoxin was observed at 9 minutes of retention, indicating that deoxypodophyllotoxin was purely separated by Diaion HP-20 resin. In the high performance liquid chromatography analysis, the proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) was used to identify the substance appearing at 9 minutes of retention, and the results are shown in FIG. 3. The spectrum of the substance was the same as that of the standard substance deoxypodophyllotoxin, which confirmed that the main component of the 100% methanol eluate of Diaion HP-20 resin was deoxypodophyllotoxin.

Experimental Example 1-2. Experimental anticancer effect of deoxypodophyllotoxin extracted and purified by the method of Example 1-2

Deoxypophylotoxin is known to exhibit anti-cancer effects in various cancer cells, including prostate cancer cells, breast cancer cells, and lung cancer cells. The purification method according to the present invention confirmed whether deoxypodophyllotoxin isolated from the previous one has this effect through the MTT test method. PC-3 cells, a human prostate cancer cell line, were dispensed into 48-well plates for cell culture at a concentration of 1 × 10 ⁴ cells / well, stabilized for 24 hours, and then deoxy at a concentration of 2.75, 7.5, 15, 30 ng / ml. Grapephyllotoxin was treated. After the reaction for 24 hours, 250 μl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) reagent was added at a concentration of 0.5 mg / ml, and reacted at 37 ° C. for 2 hours. After the reaction is over, remove the supernatant and dispense 250 μl of dimethylsulfoxide (DMSO) into the generated formazan, transfer 100 μl to a 96-well plate, and then transfer to VERSA _MAX. The absorbance at 570 nm was measured using a microplate reader to measure cell viability. The results are shown in FIG. 4, and it was confirmed that the deoxyphodophyllotoxin isolated from the former using the purification method using Diaion HP-20 resin according to the present invention shows anti-cancer effects in human prostate cancer cell lines as previously known. Did.

Example 2. Method for bulk extraction of deoxypodophyllotoxin from electrophoresis using ethanol

Ethanol was used as an extraction solvent instead of methanol to perform an extraction procedure in the following manner. After 30 kg of dried pre-garment was finely crushed with a large-capacity mixer, 80 l of 100% ethanol was added to the crushed pre-warm and extracted at room temperature for 2 days. After recovering the primary extract, 80ℓ of 100% ethanol was added again to perform secondary and tertiary extraction under the same conditions. After completion of the third extraction, the precipitate of the 100% ethanol extract was removed with filter paper and distilled water was added to adjust the concentration of ethanol to 80%. A dialysis HP-20 column with a volume of about 6.6 liters previously equilibrated in 80% ethanol was passed through an ethanol extract of a final concentration diluted with 80% ethanol to adsorb fat-soluble substances including deoxypodophyllotoxins. After passing all the extracts, the material not adsorbed to Diaion HP-20 was washed with 30 l of 80% ethanol. Subsequently, 100% ethanol was used to elute deoxydophyllotoxin adsorbed on Diaion HP-20. Deoxypodophyllotoxin-containing 100% ethanol eluate was removed by ethanol with a vacuum rotary concentrator (evaporator) and analyzed by high performance liquid chromatography to obtain 100.6 g of deoxypodophyllotoxin fraction of 80% purity. The extraction process of this embodiment is simplified and illustrated in FIG. 5.

Comparative Example 2. Results of purification through complex separation such as silica gel column chromatography after ethanol extraction, which is an existing extraction method

First, 500 g of the whole number was extracted with ethanol, and then ethanol was evaporated using a rotary evaporator, and only solid components remained. Water and ethylacetate were added in the same amount to the remaining solid components, and then only the ethyl acetate layer was taken. Silica gel is added to the ethyl acetate layer, ethyl acetate is removed by rotary evaporation, the solid components are adsorbed on the silica gel, and then added to a pre-filled silica gel column to develop a solution in which hexane and ethyl acetate are mixed in a 3 to 1 solution. Eluted with to obtain a fraction containing deoxydophyllotoxin. The fraction containing the eluted deoxypodophyllotoxin was adsorbed onto C ₁₈ -reverse silica gel in the same manner as above, and deoxypodophyllotoxin was eluted using 60% ethanol as a developing solvent using a packed column. The fraction containing deoxypodophyllotoxin was concentrated by rotary evaporation, added to a Sepadex LH-20 column, and eluted with 100% ethanol as a developing solvent. The fraction containing the eluted deoxypodophyllotoxin is separated and fractionated by using a high performance liquid chromatography (HPLC) C ₁₈ column, 60% ethanol in a developing solvent condition, and fractionated to pure deoxypodophyllotoxin 50 mg was obtained.

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

Purification The amount of the initial call Of extract
Solid content Of final extract
Solid content Of 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 deoxypodophyllotoxin after the final purification, the purification method of the comparative example showed more than 95% pure separation, and the purification method of the example according to the present invention was high purity of 80% despite purifying a large number of simple methods. It was shown. In particular, considering the yield from the whole extract (deoxypodophyllotoxin content of the final extract / solid content of the extract), the purification method of the comparative example was 0.06%, but by the purification method of the embodiment according to the present invention, the efficiency was 3.1%. Oxypodophyllotoxin could be purified.

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

The high-performance liquid chromatography analysis was performed by comparing the fraction passed through the Diaion HP-20 column and the fraction adsorbed on Diaion HP-20 and eluted in 100% ethanol compared to the standard deoxypodophyllotoxin. Analysis conditions of deoxypodophyllotoxin standard, ethanol extract of Jeonho, Diaion HP-20 resin passage, and 100% ethanol eluate from Diaion HP-20 resin are as follows. In the YL9100 HPLC of Youngin Science, a column was used Waters Spherisorb ODS2 (Ø 4.6x150 mm, 5 μm). As a developing solvent, 60% ethanol was eluted at a flow rate of 0.8 mL / min and eluted to detect UV 240 nm. The analysis results are shown in FIG. 6. In the ethanol extract of Jeonho, a peak of deoxypodophyllotoxin was observed at a retention time of 9 minutes, but deoxypodophyllotoxin was not observed in the Diaion HP-20 resin passage. On the other hand, in the 100% ethanol eluate of Diaion HP-20 resin, the peak of deoxypodophyllotoxin was observed at 9 minutes of retention, indicating that deoxypodophyllotoxin was purely separated by Diaion HP-20 resin. In the high performance liquid chromatography analysis, the proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) was used to identify the substance appearing at 9 min of retention, and the results are shown in FIG. 7. The spectrum of the material was the same as that of the standard deoxypodophyllotoxin, and it was confirmed that the main component of the 100% ethanol eluate of Diaion HP-20 resin was deoxypodophyllotoxin.

Claims (4)

Adsorbing deoxypodophyllotoxin to the column by passing an 80% by volume alcohol extract of the extraction target through a chromatography column filled with a filler selected from the group consisting of activated carbon, Diaion HP-20 resin and Amberlite XAD-20 resin;
Eluting the unadsorbed material by passing 80% by volume alcohol through the column;
The step of elution of deoxypodophyllotoxin adsorbed by passing the 100% by volume alcohol through the column; includes,
The alcohol is methanol or ethanol,
The extraction target is one or more selected from the group consisting of Jeonho, Baekduong, juniper, cypress, Nahanbaek and tomilsu,
Deoxypodophyllotoxin extraction method. delete delete delete

Images