WO2005086603A2

WO2005086603A2 - Extraction method for polyprenols

Info

Publication number: WO2005086603A2
Application number: PCT/KR2005/000723
Authority: WO
Inventors: Seong-Ryong Kwon
Original assignee: Bio-Plantek Co., Ltd
Priority date: 2004-03-16
Filing date: 2005-03-14
Publication date: 2005-09-22
Also published as: WO2005086603A3; KR20050092648A

Abstract

The present invention relates to a method of extracting and purifying a plant growth regulator for increasing crop yield comprising polyprenols. As apparent from the above description, the method of extracting and purifying polyprenols from a plant, especially a cotton plant, which may be used as a plant growth regulator for increasing crop yield, according to the present invention is capable of saving remarkably much time and cost as compared to the conventional methods.

Description

EXTRACTION METHOD FORPOLYPRENOLS

TECHNICAL FIELD

The present invention relates to a method of extracting and purifying a plant growth regulator for increasing crop yield comprising polyprenols. More particularly, the invention relates to a method of extracting polyprenols by using difference in solubilities in two or more organic solvents.

BACKGROUND ART Polyprenols have been known to be useful as an antiviral agent, an immunomodulator, cosmetics materials and an anticancer agent [U.S. patent no. 4,668,820 to Ibata, et al.; Russian patent no. 2005475; L. L. Danilov, et al., Archivum Immunologiae and Thrapiae Experimentalis, 1996, 44, 395-400; A. V. Sanin, et al. Abstracts of the meeting "Dolichols and Related Lipids", Aug. 1 1-13, 1993, Zakopane, Poland; European patent application no. 0 350 801 ; Japanese patent application publication no. 62-169724; Nature (London), 186, 470 (I960)]. U.S. patent no. 4,613,593 discloses that phosphates of dolicol (i.e. derivatives of polyprenols) are also useful in regenerating liver tissue or treating diabetes.

Korean patent nos. 0405216 and 413394 teach polyprenols as a plant growth regulator.

Polyprenols have been prepared by extraction from a plant or by chemical synthesis. U.S. patent nos. 5,077,046; 5,012,018; and 4,886,904 disclose a method of extracting polyprenols having 14 to 22 isoprenoid units from Ginkgo biloba or Cedrus deodara. This method comprises steps of (i) extracting the leaves of Ginkgo biloba or Cedrus deodara, thus obtaining organic solvent soluble substances; (ii) if required, hydrolyzing the obtained extract with alkaline metal hydroxide such as sodium hydroxide and potassium hydroxide; and (iii) subjecting the extract to chromatography or thin layer chromatography. U.S. patent no. 4,668,820 teaches a method of preparing polyprenols having 13 to 21 isoprenoid units by extracting Pinus

L. of the family Pinaceae and, if required, by further performing a hydrolysis, esterification and/or transesterification. Further, U.S. patent nos. 4,791 ,105 and 4,564,477 disclose a method of extracting polyprenols having isoprenoid units of 15 to

25 and 15 to 24, respectively.

Meanwhile, U.S. patent nos. 5,714,645 and 5,981,81 1 teach chemical synthesis or modification of polyprenols.

However, the aforementioned methods have drawbacks of low yield of polyprenols, complicated process, and low economical efficiency. Further, silica gel column chromatography or thin layer chromatography, which may be used to raise the yield of polyprenols, cannot be utilized in a process of mass (i.e. plant levels) purification.

DISCLOSURE OF THE INVENTION

Therefore, as a result of intensive researches in order to overcome the aforementioned problems, the present inventors have found an efficient method of preparing a plant growth regulator that remarkably increases the yield of polyprenols and reduces costs, thereby completed the present invention Thus, one of the objects of the present invention is to provide an efficient method of extracting or purifying a plant growth regulator capable of increasing germination, yield and growth of crops.

In accordance with one aspect of the present invention, the above and other objects can be accomplished by the provision of a method of extracting polyprenols of the following formula (I) having isoprenoid units from plants.

wherein X is hydroxyl or acetyloxy group, and m is an integer of from 3 to 33, preferably 11 or 12.

Another aspect of the present invention relates to a method of extracting polyprenols from a plant including polyprenols having isoprenol units comprising the steps of:

(a) extracting the leaves of the plant with organic solvents, thus obtaining organic solvent soluble substances comprising the polyprenols; (b) obtaining a precipitate comprising the polyprenols from the organic solvent soluble substances by using the difference in solubilities in two or more organic solvents; (c) optionally hydrolyzing the precipitate by using an organic solvent and a base; and (d) optionally separating the hydrolyzed solution by using a mixture of organic solvents. Due to the process of selectively precipitating polyprenols, it is unnecessary to perform the conventionally essential process of purification such as chromatography. The processes of hydrolysis (c) and separation (d) are not essential steps and may be omitted in the present invention.

Preferable aspect of the method of extracting the polyprenols comprises the steps of:

(a) extracting the leaves of the plant with organic solvents and removing the organic solvents, thus obtaining organic solvent soluble substances;

(b) obtaining a precipitate comprising the polyprenols from the organic (b) obtaining a precipitate comprising the polyprenols from the organic solvent soluble substances by using the difference in solubility between two or more organic solvents;

(c) removing the organic solvents and drying the precipitate; (d) optionally hydrolyzing the precipitate by using an organic solvent such as methanol, ethanol and preferably isopropyl alcohol, and a base such as an aqueous solution of sodium hydroxide, potassium hydroxide and pyrogallol hydroxide ; and

(e) optionally separating the hydrolyzed solution by using a mixture of appropriate organic solvents.

Further, the present invention also provides a method of extracting polyprenols from a cotton plant, comprising the steps of:

(a) extracting the leaves of the cotton plant with organic solvents and removing the organic solvents, thus obtaining organic solvent soluble substances;

(b) obtaining a precipitate comprising the polyprenols from the organic solvent soluble substances by using the difference in solubility between two or more organic solvents having solvent polarity index of between 4.76 and 5.50;

(c) optionally hydrolyzing the precipitate; and

(d) optionally separating the hydrolyzed solution by using a mixture of organic solvents.

The mixture of organic solvents is preferred to be a mixture of (i) ethyl acetate or isopropyl alcohol and (ii) methanol in a volume ratio of 1 : 1-10 and more preferably approximately 1.2 : 1.8, and the polyprenol is preferred to be undecaprenol or dodecaprenol. One of the technical features of the present invention is to obtain organic solvent soluble substance by using a mixture of two or more organic solvents. Thus, conventional methods may be adopted as other steps (a), (c), (d) and (e) such as steps of separating organic solvent soluble substances, hydrolysis and separation. In this regard, the aforementioned patents regarding a method of extracting polyprenols from a plant are incorporated in this application as a reference.

The method of extraction and purification according to the present invention is capable of saving remarkably much time and cost as compared to the conventional methods that require a process of column chromatography or thin layer chromatography. Therefore, the present invention has advantages of being capable of mass production at a low cost.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 is a chromatogram of HPLC for an extract obtained from the leaves of a cotton plant after purification according to the present invention; and

Fig. 2 is a chromatogram of HPLC for an extract obtained from the leaves of a cotton plant without purification.

PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION Hereinafter is provided a detailed explanation of the present invention.

Polyprenols of the following formula (I), existing in nature as a large class of molecules, are polymers of isoprenoids and play diverse biochemical roles.

wherein X and m are defined above.

It is known that such polyprenols serve as constituents for quinones in electron transport systems, cell membranes of microorganisms, pigments of photosynthetic systems, such as carotenoids and chlorophylls, and hormones such as gibberellins and brassinosteroids (Taniguchi et ah, Proc. Natl. Acad. Sci. USA, 97:13177, 2000). Further, polyprenols have characteristics in that they are very soluble in organic solvents such as ethanol, chloroform, hexane and acetone, and are non-toxic. They can also be stored for a long period of 6 to 12 months (Stone et al., Biochem. J., 102: 325- 330, 1967).

As set forth in the formula (I), polyprenols may exist in various size and form. Specifically, polyprenol of formula (I) may have four double bonds, thus forming cis or trans structure. For example, the leaves of a cotton plant contain large amount of undecaprenol or dodecaprenol, which are polyprenol having 11 and 12 isoprenoid units, respectively, the structures of which are as follows: [Undecaprenol]

[Dodecaprenol]

According to an embodiment of the present invention, the plant is preferred to be selected from a cotton plant, horse chestnut, tobacco plant, lords and ladies, silver birch, gingko or soy bean leaves. Their leaves are more preferred and the leaves of a cotton plant are the most preferred for extracting polyprenols according to the present invention. Further, the organic solvents are preferred to be selected from ethanol, acetone, n-hexane, benzene, chloroform or methylene chloride, and methylene chloride is more preferred from an economical point of view.

Sequentially, polyprenols are precipitated by adding a mixture of two or more organic solvents into the organic solvent soluble substances.

In case of undecaprenol or dodecaprenol, it is preferred to utilize two or more organic solvents having solvent polarity index of between 4.76 and 5.50.

Representative examples of this mixture include a mixture of ethyl acetate and methanol and a mixture of isopropyl alcohol and methanol, and specifically, a mixture of (i) ethyl acetate and (ii) methanol in a volume ratio of 1 : 1-10, more preferably 1.2 : 1.8 (solvent polarity index = approximately 4.82) may be used. Precipitation of polyprenols may be induced by adding this mixture of organic solvent and then placing this mixture at a temperature of -30 °C to 0 °C, and preferably -20 °C to - 10 °C, for more than two hours. Examples of ethyl acetate include 1-propanol and 2- propanol. Due to the dependency of the solubility on temperature during the precipitation and the difference in solubilities in different organic solvents, it is important to consider the temperature of precipitation as well as the combination of the organic solvents.

Polyprenols may be precipitated by using the difference in solubilities in solvents because polyprenols easily dissolve in less polar solvent such as n-hexane whereas the solubility of polyprenols decreases with the increase in the polarity of solvent. Therefore, it is possible to precipitate undecaprenol or dodecaprenol by gradually raising the polarity by appropriately mixing two or more organic solvents.

Further, an optimum volume ratio of mixing organic solvents for the highest amount of polyprenols may be found. Meanwhile, it is not easy work to select organic solvents and their mixing ratio because it depends on the kind of polyprenol to be extracted. For example, polyprenols having 10 isoprenoid units might be obtained in larger amount when the volum ratio of ethyl acetate and methanol is 2 : 1 , i.e. when ethyl acetate is greater than methanol in volume ratio. On the contrary, undecaprenol or dodecaprenol, disclosed as a preferable example herein, would hardly be precipitated in a mixture in which ethyl acetate is greater than methanol in volume ratio.

This principle may also be applied to a mixture of more than two organic solvents only if the solvent polarity index complies with the aforementioned requirements.

Sequentially, organic solvent, preferably isopropyl alcohol or ethyl acetate, was added to the precipitate, followed by adding a base, preferably an aqueous solution of sodium hydroxide or potassium hydroxide. The reason for adding the solution of sodium hydroxide is to hydrolyze the polyprenols existing in the form of ester with acetic acid, thus transforming the polyprenols to the form of alcohol. Another reasons are to hydrolyze polyprenols having less isoprenoid unit than undecaprenol or dodecaprenol as well as to hydrolyze impurities of fatty acid.

Active charcoal may be added to the solution hydrolyzed according to known method to adsorb the hydrolyzed impurities, and may be filtered by using celite, diatomite, pearlite, calcium silicate or magnesium silicate.

As an optional process, the filtered solution may be dried and precipitated by adding a mixture of acetone and acetonitrile in a volume ratio of 1 : 1. The precipitate is impurities other than polyprenols. The method of extracting and purifying polyprenols according to the present invention comprises a step of removing organic solvents formed during the processes of extraction and purification. It is preferred to perform the drying step by using anhydrous neutral salts such as anhydrous magnesium sulfate and especially anhydrous sodium sulfate.

Meanwhile, the method of extracting and purifying polyprenols according to the present invention may comprise a step of pulverizing the purified polyprenols by using the conventional methods.

The purified polyprenols may be analyzed according to the conventional method. Particularly, the present invention employed HPLC, NMR, MASS and IR spectroscopy. The polyprenols that may be separated from the aforementioned plants, preferably a cotton plant, is undecaprenol or dodecaprenol.

The invention is further illustrated by the following examples which are not intended to limit the scope of the invention.

Example 1: Extraction and purification of polyprenol from the plant leaves 10 g of dried leaves of a cotton plant were treated with 100 ml ethanol to three times, thus separating organic solvent soluble substances. The organic solvents were evaporated off to yield a concentrated product by using a rotary vacuum evaporator (Buchi R-205 ). The concentrated organic solvent soluble substances were added into 50 ml of a mixture of ethyl acetate and methanol in a volume ratio of 1.2 : 1.8, and placed inside the refrigerator maintained at a temperature of -20 °C for 6 hours. After a precipitate was separated from upper phase liquid, the precipitated was obtained by using Celite 545 (Sigma-Aldrich Co.). The precipitate was dried and dissolved in 50 ml of isopropyl alcohol. After basic solution was prepared by dissolving 0.7 g of sodium hydroxide in 2 ml of distilled water, hydrolysis was performed by adding the basic solution drop wise to the isopropyl alcohol solution for one and half hour with stirring at room temperature. After the hydrolysis was completed, 2 g of active charcoal was added with stirring and filtered by using Celite 545^®. Washing was performed with 50 ml of isopropyl alcohol during the filtration. This solution was concentrated by using Buchi R-205 , and 200 ml of a mixture of acetone and acetonitrile in a volume ratio of 1 : 1 was added in the concentrated solution, followed by stirring. This solution was filtered and concentrated again by using Celite 545^® and Buchi R-205 " , respectively.

Polyprenols in the concentrate were quantified by using HPLC instrumentation

(system: waters alliance system; column: symmetry shield RP18 5 μ, 3.9 x 150 mm; mobile phase: 100% acetonitrile; flow rate: 2.0 ml/min), and the results were shown in

Figure 1. Meanwhile, the purified substance was identified to be polyprenols by using the identification method as set forth in Korean patent nos. 413394 and 405216, i.e. a mass analysis using a liquid chromatography-mass spectrometer (VG BIOTECH platform; ion source, ESI; resolution, 1000; mass range, 2-3000(m/z), and Η-NMR and ¹³C-NMR analysis using a NMR spectrometer (FT-NMR (600 MHz), A VANCE 600, Bruker).

The aforementioned analyses show that the yields of undecaprenol and dodecaprenol are 41.9% and 39.3%, respectively, and the amount of total polyprenols is 81.2%.

Comparative Example 1: Extraction polyprenol from the plant leaves

10 g of dried leaves of a cotton plant were treated with 100 ml ethanol to three times, thus separating organic solvent soluble substances. The organic solvents were evaporated off to yield a concentrated product by using a rotary vacuum evaporator (Buchi R-205^®). Polyprenols in the concentrate was quantified by using HPLC instrumentation

(system: waters alliance system; column: symmetry shield RP18 5 μ, 3.9 x 150 mm; mobile phase: 100% acetonitrile; flow rate: 2.0 ml/min), and the results were shown in Figure 2.

From the Figure 1, it is verified that the yields of undecaprenol and dodecaprenol is high and the amounts of impurities are low as indicated in the left. In contrast, as shown in the Figure 2, the amounts of impurities are relatively high as compared to the Example of the present invention.

Meanwhile, it is noted that the extraction method using the difference in solubilities in two or more organic solvents may be utilized for any plants other than the cotton plant.

INDUSTRIAL APPLICABILITY

As apparent from the above description, the method of extracting and purifying polyprenols from a plant, especially a cotton plant, which may be used as a plant growth regulator for increasing crop yield, according to the present invention is capable of saving remarkably much time and cost as compared to the conventional methods.

Further, the present invention has advantages of being capable of being used in mass production at a low cost because it does not require a process of column chromatography or thin layer chromatography.

Claims

CLAIMSWhat is claimed is:

1. A method of extracting polyprenols from a plant including polyprenols having isoprenol units, the method comprising the steps of:

(a) extracting the leaves of the plant with organic solvents, thus obtaining organic solvent soluble substances comprising the polyprenols;

(b) obtaining a precipitate comprising the polyprenols from the organic solvent soluble substances by using the difference in solubilities in two or more organic solvents;

(c) optionally hydrolyzing the precipitate by using an organic solvent and a base; and

2. The method according to claim 1, wherein the plant is one selected from the group consisting of cotton, horse chestnut, tobacco, lords and ladies, silver birch, gingko and soybean leaves.

3. The method according to claim 1, wherein the organic solvent and the base used in step (c) are isopropyl alcohol and sodium hydroxide aqueous solution, respectively.

4. The method according to claim 3, further comprising a step of purifying the hydrolyzed solution by using active charcoal.

5. The method according to any of claims 1-4, wherein the polyprenols is the compound of following formula (I):

wherein m is an integer of from 1 1 to 33 and X is hydroxyl or acetyloxy group.

6. The method according claim 5, wherein the mixture of organic solvents and mixing ratio thereof may be selected depending on the number of m.

7. The method according to claim 6, wherein m is 1 1 or 12, and the mixture of organic solvents is a mixture of (i) ethyl acetate or isopropyl alcohol and (ii) methanol in a volume ratio of approximately 1.2 : 1.8.

8. The method according to claim 6, wherein m is 10, and the mixture of organic solvents is a mixture of (i) ethyl acetate or isopropyl alcohol and (ii) methanol in a volume ratio of approximately 2 : 1.

9. A method of extracting polyprenols from a cotton plant, the method comprising the steps of:

(c) optionally hydrolyzing the precipitate; and (d) optionally separating the hydrolyzed solution by using a mixture of organic solvents.

10. The method according to claim 9, wherein the mixture of organic solvents is a mixture of (i) ethyl acetate or isopropyl alcohol and (ii) methanol in a volume ratio of 1 : 1-10.

11. The method according to claim 10, wherein the polyprenol is undecaprenol or dodecaprenol.

12. The method according to claim 1 1, wherein the mixture of organic solvents is a mixture of (i) ethyl acetate or isopropyl alcohol and (ii) methanol in a volume ratio of approximately 1.2 : 1.8.

13. The method according to claim 12, wherein the precipitation of the step

(b) is performed at the temperature of -30 °C to 0 °C.