KR20050092648A - Eextraction method for polyprenols - Google Patents

Abstract

The present invention relates to an economical and useful method for extracting and purifying plant growth regulators for steaming plants comprising polyprenol represented by the following formula (1) as an active ingredient.

 [Formula I]

Wherein m is an integer from 3 to 33 and X is a hydroxy or acetyloxy group. In particular, m is preferably an integer of 11 or 12.

Extracting and purifying plant growth regulators for plant growth from plants, especially cotton leaves, according to the present invention is economical and time efficient as compared to the existing methods, and is a method that can dramatically increase the content of polyprenol.

Description

Extraction method of polyprenol {Eextraction method for polyprenols}

The present invention relates to a method for extracting and purifying plant growth regulators for steam containing polyprenol, and more particularly, to a method for extracting polyprenol using a difference in solubility of polyprenol in at least two solvent mixtures. .

Polyprenol is known to be useful for conventional antiviral agents, immunomodulators, cosmetic anticancer agents, etc. (US 4,668,820 to Ibata, et al., Russian Patent 2005475; LL Danilov, et al., Archivum Immunologiae and Thrapiae Experimentalis, 1996, 44, 395-400; AV Sanin, et al. Abstracts of the meeting "Dolichols and Related Lipids", Aug. 11-13, 1993, Zakopane, Poland; European Patent Application 0 350 801; Japanese Patent Laid-Open No. 62-169724; Nature (London), 186, 470 (1960). US 4,613,593 describes that phosphates of dolichol, a derivative of polyprenol, are useful for liver tissue regeneration, diabetes, and the like.

Korean Patent Nos. 0405216 and 413394 disclose that polyprenole is useful as a growth regulator of a plant.

Regarding the method for producing such polyprenol, there are extraction or chemical synthesis methods from plants. As for the extraction method from plants, US 5,077,046; US5,012,018; US 4,886,904 and the like disclose a method for extracting polyprenol having 14 to 22 isoprenoid units from Ginkgo biloba or Cedrus deodara. The method includes extracting an organic solvent soluble material from the leaves of Ginkgo biloba; Hydrolyzing the extract with an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide, if necessary; The extract is composed of a step of separating by chromatography, thin film chromatography, and the like. US Pat. No. 4,668,820 discloses polyprenol having isoprenold units 13 to 21 from the Pinus L. of the family Pinaceae in a similar manner to the above extraction method, if necessary, hydrolysis, and esterification and / Or transesterification. Also, US 4,791,105 describes the extraction of polyprenol of isoprenoid units 15-25, and US 4,564,477 isoprenoid units 15-24.

US 5,714,645 and US 5,981,811 describe methods for chemical synthesis or modification of polyprenol.

However, these methods were not economical because the polyprenol content of the final product was low or the process was complicated or expensive. In addition, silica gel column chromatography (TLC) and thin layer chromatography (TLC) have been used to increase the content, but when using column chromatography or TLC, it cannot be used at all at the plant-level mass purification method. there was.

Therefore, while the present inventors are conducting research on an effective purification method of plant growth regulators that can overcome the above problems, the method of the present invention finds an economical method while dramatically increasing the content of polyprenol. The present invention has been completed.

It is an object of the present invention to provide an effective extraction and purification method of plant growth regulators that can enhance the germination, increase and growth effects of plants.

In order to achieve the above object of the present invention, the present invention provides a method for extracting polyprenol, which is present in a natural plant and composed of isoprenoid units, preferably represented by the following formula (1).

[Formula 1]

Wherein m is an integer from 3 to 33 and X is a hydroxy or acetyloxy group

to be. In the above formula, m is preferably 11 or 12.

Specifically, to achieve the object of the present invention, the present invention comprises the steps of separating the organic solvent solubles by extracting the polyprenol from a plant containing a polyprenol consisting of isoprenoid units; Hydrolyzing the separated organic solvent soluble material; In the method for extracting a polyprenol from the plant consisting of separating the hydrolyzed solution using an organic solvent mixture, after separating the organic solvent soluble material, using a mixture of two or more organic solvents It provides a method for extracting a polyprenol further comprising the step of precipitating the polyprenol using the difference in solubility between the organic solvents. As such, in the precipitation step, polyprenol is precipitated and other impurities are not precipitated, thereby increasing the purity of polyprenol, thereby making it unnecessary to perform chromatographic processes, which are essential in the prior art.

The method is preferably

1) extracting the organic solvent solubles from the plant leaves using an organic solvent and separating the organic solvent solubles by removing the organic solvent;

      2) precipitating polyprenol using the organic solvent solubility of the separated organic solvent soluble material using two or more organic solvent mixtures;

3) removing the organic solvent from the precipitate and drying;

4) Hydrolyzed from the dried precipitate using a suitable organic solvent, methanol, ethanol, isopropyl alcohol, etc., preferably in an isopropyl alcohol environment, using a suitable base, preferably an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, an aqueous pyrogallol solution, or the like. Doing;

5) A method of extracting polyprenol from a plant leaf consisting of separating the polyprenol by using the hydrolyzed solution using an appropriate organic solvent mixture.

In addition, the present invention comprises the steps of extracting the organic solvent soluble material from the cotton leaf using an organic solvent and separating the organic solvent soluble material by removing the organic solvent; Hydrolyzing the separated material; In the method for extracting polyprenol from the plant consisting of separating the hydrolyzed solution using an organic solvent mixture, after extracting the organic solvent soluble material, the solvent polarity index (4.76) It provides a method for extracting polyprenol further comprising the step of precipitating the polyprenol using the solubility difference between the organic solvents using two or more organic solvent mixture satisfies the condition of 5.50.

The organic solvent mixture is ethyl acetate and methanol or isopropyl alcohol and methanol in a ratio of 1: 1 to 10 (that is, a higher amount of methanol), preferably about 1.2: about 1.8 ratio, the polypre The knol is preferably undecaprenol or dodecaprenol.

The present invention is characterized by using two or more organic solvent mixtures of the organic solvent soluble material and other steps, for example, separating the organic solvent soluble material, hydrolysis step, separation step can use a known method. have. In this sense, reference is made to the contents of the patents relating to the method for extracting polyprenol from the plants described in the prior art of the present invention.

 Hereinafter, the present invention will be described in detail.

Polyprenol having a structural formula of Formula 1 is a linkage of isoprenoids that form a huge group in nature and are responsible for various biochemical functions. These polyprenols are known to act as components of quinones in electron transfer systems, components of microbial cell membranes, components of photosynthetic pigments such as carotenoids and chlorophylls, and hormones such as gibberellin and brassinosteroids (Taniguchi et al., Proc. Natl. Acad. Sci . USA 97: 131712-13177, 2000). In addition, it is well soluble in organic solvents such as ethanol, methylene chloride, chloroform, nucleic acid and acetone, is non-toxic, and has long-term storage for 6-12 months (Stone et al . Biochem. J. 102: 325 -330, 1967).

The polyprenol consisting of the isoprenoid units is present in various forms and sizes, as represented by Formula 1. Polyprenol may have four double bond carbons in its molecular structure, cis and trans. For example, polyprenol present in cotton leaves contains significant amounts of undecaprenol and dodecaprenol having 11 and 12 isoprenoid units, and their geometry is as follows.

[Undecaprenol]

[Dodecaprenol]

According to a preferred embodiment of the present invention, first, cotton (cotton plant), horse chestnut (horse chestnut), tobacco (tobacco plant), lords and ladies, birch

(silver birch, gingko, soy bean leaves, etc.), preferably organic leaves (ethanol, acetone, normal nucleic acid, benzene, chloroform, methylene chloride, etc.) from their leaves, preferably cotton leaves To separate the organic solvent solubles. As the organic solvent, from an economical point of view, it is preferable to use methylene chloride.

The polyprenol is then precipitated by adding two or more organic solvent mixtures to the organic solvent soluble material.

In the case of the preferred undecaprenol or dodecaprenol to be extracted in the present invention, the mixture of two or more organic solvents preferably satisfies the conditions of 4.76 to 5.00, based on the solvent polarity index. Examples thereof include ethyl acetate and methanol, isopropyl alcohol and methanol. As a more specific example, a mixed solution of ethyl acetate and methanol as an organic solvent in a ratio of at least 1: 1, preferably in a ratio of 1: 1 to 10, more preferably in a ratio of about 1.2: about 1.8 (solvent polarity index 4.82) Is prepared and added. After the above mixed solution is added, the mixture is left at a temperature of about −10 ° C. to about −20 ° C. for at least 2 hours to induce polyprenol to precipitate. As the ethyl acetate organic solvent group, 1-propanol, 2-propanol and the like can be used. Since the solubility in the precipitation step is greatly affected by the temperature, it is important to select the combination of the solvent and the precipitation temperature in consideration of the solubility change according to the temperature of each solvent as well as the difference in solubility between each solvent.

The reason for precipitation due to the difference in solubility of polyprenor solvent is that undecaprenol and dodecaprenol generally dissolve well in solvents such as low polar normal nucleic acid, and higher polar solvents do not dissolve well. There is this. Therefore, as the polarity of the organic solvent mixture is gradually increased by using two or more solvents, undecaprenol and dodecaprenol remain as precipitates without being dissolved. In addition, by finding the optimum organic solvent mixing ratio for inducing such precipitation, the organic solvent mixing ratio for obtaining the highest polyprenol content is selected.

In mixing organic solvents, it is not easy to determine the type of organic solvent and the mixing ratio between solvents according to the type of polyprenol to be extracted. For example, in the case of polyprenol having 10 isoprenoid monomers, precipitation of polyprenol easily occurs when the ratio of ethyl acetate and methanol is 2: 1, that is, when the ratio of ethyl acetate is higher. . But. In the case of undecaprenol or dodecaprenol which is a preferred embodiment of the present invention, precipitation of polyprenol hardly occurs when the ratio of ethyl acetate is higher, and precipitation occurs when the ratio of methanol is higher.

Even when three or more types of organic solvents are used as the organic solvent mixture for precipitation, the solvent polarity index can be applied if the above conditions are satisfied.

Subsequently, an organic solvent, preferably isopropyl alcohol or ethyl acetate, is added to the precipitate-induced material to dissolve well, followed by addition of a base, preferably, sodium hydroxide or aqueous potassium hydroxide solution. The reason why the aqueous solution of sodium hydroxide is added is that polyprenol, which is generally found in plants, exists in the form of esters combined with acetic acid, so it is necessary to make it in the form of alcohol through acid or base hydrolysis. As well as capreno and dodecaprenol, there are many other polyprenoles, so that hydrolysis of polyprenoles with fewer isoprenoid groups via acids or bases is possible. This is because it is necessary to hydrolyze various fatty acid impurities.

Subsequently, an active charcoal may be added to the hydrolyzed solution according to a known method to adsorb the hydrolyzed impurities, and may be filtered out using celite, diatomaceous earth, perlite, calcium silicate, magnesium silicate and the like.

Subsequently, the filtered solution is dried according to a known method, and then acetone and acetonitrile 1: 1 mixed solution may be added to further filter out impurities by precipitation. Precipitates are impurities other than polyprenol. This process is arbitrary.

Polyprenol extraction and purification method according to the present invention includes the step of removing and drying the organic solvent formed during the extraction and purification process, using anhydrous neutral salts such as anhydrous sodium sulfate, anhydrous magnesium sulfate, in particular, anhydrous sodium sulfate It is preferable to dry.

On the other hand, the polyprenol extraction method according to the invention may further comprise the step of powdering the purified polyprenol, the powdering of the polyprenol may be used a general method known in the art. .

The identification of purified polyprenol may be used by known methods known in the art, specifically, the present invention was confirmed using HPLC, NMR, MASS and IR spectral analysis. The polyprenol which can be separated from the plant, preferably cotton, is undecaprenol or dodecaprenol.

In the embodiment of the present invention, a method for extracting and purifying polyprenol according to the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Polyprenol Extraction and Purification from Plant Leaves

The organic solvent solubles were separated by treating 100 g of methylene chloride three times with 10 g of dried cotton leaves. The separated organic solvent soluble material was evaporated using a rotary vaccum evaporator (buchi R-205). Subsequently, 50 ml of ethyl acetate: methanol 1.2: 1.8 mixed solution was added to the dried organic solvent soluble material and left for 6 hours in a freezer at -20 ° C. After confirming that the precipitate was separated from the supernatant in the solution, the precipitate was obtained using Celite 545 (Sigma-Aldrich). The precipitate obtained was well dried, and then 50 ml of isopropyl alcohol was added to dissolve well. A solution in which 0.7 g sodium hydroxide was dissolved in 2 ml of distilled water prepared in advance was subjected to a hydrolysis reaction for 1 hour and 30 minutes while stirring at room temperature while dropping the solution onto the isopropyl alcohol solution. After hydrolysis was completed, 2 g of activated carbon was added thereto, shaken well, and filtered using celite 545. The filter was washed well with 50 ml of isopropyl alcohol.

The solution was concentrated using a rotary vaccum evaporator (buchi R-205), and then stirred well by adding 200 ml of acetone: acetonitrile 1: 1 solution. The well stirred solution was again filtered using celite 545, and the filtered solution was concentrated using a rotary concentrator.

HPLC analysis was performed to determine the polyprenol content of the concentrated material. First, the material was analyzed by high pressure liquid chromatography (instrument: Waters alliance system, column: symmetry shield RP18 5μ, 3.9X150mm, mobile phase: 100% acetonitrile, moving rate: 2.0ml / min), and the results were analyzed. Is shown in FIG.

On the other hand, the above purified material is a method for identifying polyprenol, that is, a liquid chromatography mass spectrometer from a plant extract described in Korean Patent Registration No. 413394 or 405216 (device: VG BIOTECH platform, ion sorce: ESI, Resolution: 1000, mass range: 2-3000 (m / z)) mass spectrometry, NMR (FT-NMR (600MHz), AVANCE 600, Bruker) instrument using proton NMR and carbon ( ¹³ C) NMR Etc., it could be confirmed that the purified material was polyprenol.

According to this example, it was possible to obtain undecaprenol in an amount of 41.9% and dodecaprenol in an amount of 39.3% from cotton leaves. Total polyprenol content was 81.2%

Comparative Example 1 Extraction of Polyprenol from Plant Leaves

The organic solvent solubles were separated by treating 100 g of methylene chloride three times with 10 g of dried cotton leaves. The separated organic solvent soluble material was evaporated using a rotary vaccum evaporator (buchi R-205). Subsequently, HPLC analysis was performed to determine the polyprenol content of the dried organic solvent solubles. First, the material was analyzed by high pressure liquid chromatography (equipment: Waters alliance system, column: symmetry shield RP18 5μ, 3.9X150mm, mobile phase: 100% acetonitrile, moving rate: 2.0ml / min), the result was analyzed Is shown in FIG. 2.

In the above results, in the case of Figure 1, which is the result according to Example 1 after the polyprenol precipitation by the organic solvent mixture, the content (purity) of the undecaprenol and dodecaprenol is high and It can be seen that the content is low. On the contrary, in FIG. 2, which is a result according to Comparative Example 1 without undergoing the precipitation of polyprenol by an organic solvent mixture, it can be seen that the content of impurities on the left side is high.

In the above embodiment, but is an example for the cotton leaf, it is subjected to the precipitation step using two or more solubility difference of the present invention for other plants.

In the method of extracting and purifying polyprenol according to the present invention, the polyprenol is precipitated according to the difference in solubility of two or more organic solvents, and thus, conventional column chromatography or TLC (thin layer chromatograph) It's a huge time saver compared to the same time-consuming and expensive method. In addition, as a commercially available method for mass extraction and purification in the future, there is an advantage that can be produced at a low price because it can be used not only in the laboratory but also in the plant.

1 is a chromatogram showing the results obtained using high-pressure liquid chromatography (HPLC) of the extract extracted from the cotton leaf using the purification method according to the present invention.

2 is a chromatogram showing the results of the extraction liquid extracted from cotton leaves using high pressure liquid chromatography (HPLC) without purification.

Claims (9)

Separating the organic solvent solubles by extracting the polyprenol from a plant containing the polyprenol composed of isoprenoid units; Hydrolyzing the separated organic solvent soluble material; In the method for extracting polyprenol from the plant consisting of the step of separating the hydrolyzed solution using an organic solvent mixture, after separating the organic solvent solubles, using two or more organic solvent mixture Precipitating the polyprenol using the difference in solubility between the organic solvents. The method of claim 1, wherein the plant is any one selected from cotton, horse chestnut, tobacco, Tiannan, birch, ginkgo or soybean. The method of claim 1, wherein the organic solvent in the hydrolysis step is isopropyl alcohol and the base is an aqueous solution of sodium hydroxide. The method of extracting polyprenol according to claim 3, comprising the step of purifying the hydrolyzed solution using active charcoal. The method for extracting polyprenol according to any one of claims 1 to 4, wherein the polyprenol is a compound represented by the following formula (1). [Formula 1] [Wherein m is an integer of 11 to 33 and X is a hydroxy or acetyloxy group. ] Extracting the organic solvent soluble material from the cotton leaf using an organic solvent and separating the organic solvent soluble material by removing the organic solvent; Hydrolyzing the separated material; In the method for extracting polyprenol from the plant consisting of separating the hydrolyzed solution using an organic solvent mixture, after extracting the organic solvent soluble material, the solvent polarity index (4.76) And precipitating the polyprenol using a difference in solubility between the organic solvents using two or more organic solvent mixtures satisfying the conditions of from 5.50 to 5.50. The method of extracting polyprenol according to claim 6, wherein the organic solvent mixture is (ethyl acetate or isopropyl alcohol): (methanol) in a ratio of 1: 1 to 10. 8. The method of claim 7, wherein the polyprenol is undecaprenol or dodecaprenol. The method of extracting polyprenol according to claim 8, wherein the organic solvent mixture has a ratio of (ethyl acetate or isopropyl alcohol) :( methanol) of 1.2: 1.8.