KR100827072B1 - An efficent isolation process for betulin of high purity from birch bark - Google Patents

Abstract

A process for isolating betulin from birch(Betula platyphylla) bark is provided to improve economical efficiency of isolation, and purity of betulin by rapidly performing the isolation process and reducing or eliminating the consumption amount of filling materials in the column. A process for isolating betulin from birch bark comprises the steps of: treating the pulverized birch bark with ultrasonic wave to prepare the crude extract of birch bark; purifying the crude extract of birch bark with n-hexane to remove nonpolar materials; and subjecting the purified crude extract of birch bark to flash chromatography using n-hexane:ethylacetate(10:1 and 4:1(v/v)) as elution solvent.

Description

An efficent isolation process for Betulin of high purity from birch bark}

1 is a TLC chromatogram for comparison of crude extract components prepared by sonication and conventional flux treatment according to the present invention.

2 is an aliquot TLC chromatogram at the time of 1-2 extraction in the bereline separation by flash chromatography according to the present invention,

3 is an aliquot TLC chromatogram at the time of 3-7th extraction in beryllin separation by flash chromatography according to the present invention,

4 is an aliquot TLC chromatogram at the time of 9-10th extraction in beryllin separation by flash chromatography according to the present invention,

Fig. 5 is an aliquot TLC chromatogram of 11-12 th extraction in beryllin separation by flash chromatography according to the present invention.

The present invention relates to a method for efficiently extracting high-purity betulin from birch bark, and more particularly, to prepare a crude extract by sonication to economically obtain high-purity betulin from the birch bark in a short time, and a flash The present invention relates to an economical method for extracting benin, comprising berylnin separation by chromatographic treatment, and thereby berylnin.

Applicant has described the optimized bevetlin extraction method from birch bark through Korean Patent Application No. 2006-43046. Technical matters disclosed in the preceding application may be referred to herein by reference. According to the preceding application, a benzine high yield extraction process is mentioned. In summary, a method of extracting benin from birch bark, comprising: preparing a crude extract by solvent extraction of a birch bark crushed sample; Purifying with a nonpolar organic solvent to remove the nonpolar material from the crude extract; And using the purified crude extract as an eluting solvent using n-hexane: ethyl acetate as a solvent to separate benzine by column chromatography. And beechlin at least 95% high purity.

Applicant has studied the economic extraction method of betulin since the preceding application. In the case where the supply of birch husks, the source of beetlin extraction, was facilitated, attention was paid to the improved extraction method based on the extraction cost, although the yield of beetlin was lower than that obtained by the prior method. In the preceding extraction process, the supply cost of the birch shell is incomparably lower than the price of beryllin, which will have a negligible effect on the cost of beryllin production, whereas most of the production cost is the labor cost and the organic solvent and column filling used for extraction. It depends on the amount of material consumed. Although organic solvents can be reused for most of the recovery device, the filling material (mainly silica gel) is not reusable and the cost of the material itself is high, so the prior patent may not be an economical extraction method. Noted. Therefore, the most effective method for lowering the extraction cost may be to reduce labor costs by shortening the production time and to reduce the use of solvents and fillers or not to use the fillers.

The present invention is the result of research to achieve the above object.

An object of the present invention relates to a method of economically extracting high-purity beculine, a high-potential potential, from functional birch casings grown in Korea.

An object of the present invention is to provide a method for extracting benin from birch bark,

Preparing a crude extract from the birch bark crushed sample using ultrasonic waves; Purifying with a nonpolar organic solvent to remove the nonpolar material from the crude extract; And separating bevetlin by flash chromatography using n-hexane: ethyl acetate as the eluent as the purified crude extract, which can be achieved by high purity bevetlin extraction from birch bark. Can be. On the other hand, the non-polar organic solvent can be used to remove the non-polar material before flash chromatography using n-hexane, the elution solvent by betaine isolation is n-hexane: ethyl acetate = 10: 1 (v / v) primary N-hexane: ethyl acetate = 4: 1 (v / v) can be applied secondarily.

The present invention also relates to high purity betulin with a purity of 98% or more prepared by the extraction method.

Hereinafter, the present invention will be described in detail by way of examples, but is not limited thereto.

First, as a test material, domestic birch ( Betula ) born about 30 years platyphylla ) bark and samples were taken from Cheonan, Chungju, and Youngdong, respectively. The outer skin part (white part) of the collected birch bark was separated, air dried and oven-dried, and the powdered extract samples were prepared with a grinder.

First step: preparation of crude extract by ultrasonic wave

1g crushed bark is placed in a 50mL flask and 30mL of chloroform is added to seal the inlet with parafilm. Crude extracts were prepared in 20, 30 minute divisions. The extraction results are shown in Table 1.

Crude extract weight by sonication Treated water Processing time 10 minutes 20 minutes 30 minutes One 0.34 g 0.33 g 0.37 g 2 0.36 g 0.36 g 0.31 g 3 0.36 g 0.36 g 0.35 g

In Table 1, the crude extract extraction yield by ultrasound showed the same tendency as 30 minutes treatment with only 10 minutes treatment, and the extraction yield was about 35%. This yield is not significantly different from the crude extract yield (about 36.5%) obtained by the Reflux method using chloroform in the preceding application referred to by reference, but considering the treatment time (1/6 reduction), etc. Economically significant productivity extraction method. On the other hand, thin film chromatogram (TLC, TLC sample concentration: 5mg / 2mL CHCl3, spotting number of samples on TLC plate: 12 times) for the comparison of the crude extracts obtained by ultrasonic and Reflux results 1 is shown. As can be seen in Figure 1, these components were found to be similar without significant difference between the crude extract by the ultrasonic treatment capable of a short time treatment and the reflux treatment required for a long time. From the crude extract analysis result, the crude extract preparation step from the birch bark using ultrasonic waves can reduce the amount of solvent used or extraction time more than the reflux method.

Second Process: Crude Extract Purification Step

As an organic solvent for purification, hexane ( n- Hexane), which is not easily dissolved, was used, and nonpolar substances were removed.

Third Process: Flash Separation step of betrinin by chromatography

The column was manufactured and sold by Biotage, USA, with a 23.5 cm (15 cm internal silica gel fill length) and 4 cm diameter Si 40 + M 2146-1 product and its accessories. Metering's FMI LAB PUMP PM 6014 and CV200 models were used. The application flow rate was 20 mL / min and the crude purified bark crude extract used for one extraction was 3 g. Unlike column chromatography, which is referred to as the comparative example below, in flash chromatography, it could be used repeatedly for 12 extractions without replacing the column. Place the primary purified crude extract in a sample vessel set connected to the column for beryllin purification, begin to pressure-inject the solvent, and take out 150 mL of n- Hexane: Ethyl acetate mixed solvent in a 10: 1 (v / v) formulation. , the solvent 4: 1 (n -Hexane: Ethyl acetate , v / v) in wet enough to take the column out 500mL replaced with a mixed solvent removed the remaining non-polar material conditions, 4: 1 (n -Hexane: Ethyl acetate, v / v) mixed solvent was flowed and separated into 21 80 mL Erlenmeyer flasks continuously. This aliquot was 1.68 L and the aliquot time was 40 minutes, which was significantly reduced compared with the column chromatography method of the comparative example below. After extraction once, the column was washed with methanol under the same pressure, and then extracted. As a result of TLC, the first two extractions included pure betholin from preparative Erlenmeyer flasks Nos. 7 to 11 (FIG. 2), and the third to seventh extractions from flasks 3 to 9 (FIG. 3). Pure betholin was identified from flasks 1 to 7 in the eighth to tenth extractions (FIG. 4) and from flasks 1 to 5 in the eleventh to twelfth extractions (FIG. 5). Drying gave bevetlin.

Comparative Example : Column Chopping chyulrin separated using chromatography

According to the same method mentioned in the prior art described by reference, several crude crude extracts (3.23-3.99 g) from 10 g of bark were first purified with hexane and then each Silica gel (Sigma-Aldrich, 70). -230 mesh) was extracted by applying to a column (diameter 2.5cm, length 60cm). 450 g of silica gel was mixed with hexane to prepare the column for each batch, and the column was prepared by flowing about 2 L of hexane while all solvent outlets of the column were opened. After closing the solvent outlet, carefully put the primary purified crude extract powder (3.03 ~ 3.84g) in the upper layer of the prepared column, and add 50 ml flask of 10: 1 (v / v) mixture of n -Hexane: Ethyl acetate mixed solvent. That is, after receiving 300 mL, n- Hexane: Ethyl acetate = 4: 1 (v / v) mixed solvent was used for 320 50 mL flasks, that is, 16 L. Elution time was about 12 minutes per flask. The total fraction time for each crude extract was 4012 minutes (65.2 hours). The amount of elution solvent used for one extraction was n -Hexane 13.0737L and Ethyl acetate 3.2273L, respectively. Silica gel used in column chromatography is generally discarded after one time use in this comparative example because the performance is greatly reduced when used once. Each flask solution was checked by TLC, and the fractions in which only foreign substances were seen but only berylrin were combined, and the solvent was vacuum dried to obtain 1.85 to 2.53 g of beryllin. As can be seen from the results, the extraction yield was significantly different in each case, which means that the preparation state of the extraction column was manually changed according to the technical difference of the experimenter.

Bedouin  For separation column  And Flash Chromatographic  Efficiency comparison

Table 2 shows a comparison between the column chromatography as a comparative example and the extraction method using the flash chromatography implemented in the third step of the present invention. As a result, the use of n- Hexane and Ethyl acetate was 7 times higher and the extraction time was 27 times longer than that of flash chromatography. The cost of the column filler used in the present invention was also 10 times higher when using column chromatography than on the basis of the number of times available. However, Methanol, which is not used in the column chromatography method, was used in the flash chromatography method at 500 mL per time, and the column chromatography method was 1.6 ~ 2.2 times higher than the case using the column chromatography method in the amount of betulin extracted per time. It was about high. However, considering all the above, the application of the flash chromatography method according to the present invention is a significantly more efficient method than the column chromatography method.

Comparison of Solvent Extraction, Filler Cost and Number of Uses, Extraction Time, and Querlin Extract Comparison Column Chromatography (Comparative Example) Flash Chromatography Extraction solvent n -Hexane 13074 mL / time 1880 mL / time Ethyl acetate 3227 mL / time 450 mL / time Methanol not used 500 mL / time Column Filler (Silica Gel) Cost * \ 60,570 / time \ 65,000 / piece Number of column fillers used 1 time 10-12 times Fraction extraction time 65.2 hours / time 2.4 hours / time Extract amount of betulin (> 98%) per 1g crude extract 0.46-0.63 g 0.21-0.38 g

* The filler (silica gel) cost is based on the bulk capacity of column chromatography for 5kg (as of November, \ 673,000-06) and the amount used (450g / time), and for flash chromatography for commercially available packs. Price per piece.

extraction Bethelin  water

Gas chromatography (GC) was used to measure the purity of bevetlin extracted by flash chromatography and comparative column chromatography according to the present invention. Squalene was used as an internal standard. As derivatization reagent, Sylon BTZ was used. The instrument used for GC analysis was the HP 5890 (U.S.A) model, the analysis column was DB5 (60m x 320μm i.d.) and the Detector was FID. The flow rate of helium was 1 ml / min and the injection volume was 1 μL. The analysis temperature program is maintained at 250 ° C. for 2 minutes, then the temperature is increased to 5 ° C./min. Up to 303 ° C., the same temperature is maintained for 1.5 minutes, and the temperature is increased to 5 ° C./min. Up to 310 ° C. for 25 minutes. did. Total measurement time was 40 minutes. The purity of beryllin extracted by column chromatography was 95.2%, and the purity of berylline extracted by flash chromatography was 98.9%.

As can be seen from the experimental results, through the extraction method according to the present invention, the present invention is applied to the preparation method of the crude extract by applying an ultrasonic treatment as compared to the bevetlin extraction method by the prior application by the applicant of about 1 / It was possible to shorten it to 6 or less, and also, by applying the flash chromatography in the beryllin separation step, it was possible to economically produce more than 98% of high purity beryllin in a very short time.

The present invention relates to crude extract preparation by sonication, removal of non-polar substances to crude extract, and bevetlin extraction method by flash chromatography on birch bark powder. The present invention can reduce the preparation time of crude extract to about 1/6 or less, and can also economically produce high purity betulin of 98% or more in a very short time by applying flash chromatography in the benzine separation step. to be.

Claims (4)

In the method of extracting beech from birch bark, Preparing a crude extract from the birch bark crushed sample using ultrasonic waves; Purifying with n-hexane to remove nonpolar material from the crude extract; And The purified crude extract using n-hexane: ethyl acetate as the elution solvent, characterized in that the step of separating the beryllin by flash chromatography, characterized in that the high purity beryl extract from birch bark. delete The elution solvent is n-hexane: ethyl acetate = 10: 1 (v / v) is applied first, and n-hexane: ethyl acetate = 4: 1 (v / v) in the secondary A high-purity bevelin extraction method from birch bark, characterized by the application. delete

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