JPH0314007B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing polyprenol. More specifically, the present invention is based on the following general formula () (In the formula,

[Formula] is a transformer type represents the soprene unit,

[Formula] represents a cis isoprene unit, and n represents an integer from 10 to 19. ) or a mixture thereof. Polyprenols represented by the general formula () and mixtures thereof are useful substances as raw materials for medicines, cosmetics, etc., and are particularly useful as synthetic intermediates for mammalian dolichols. Dolichols were first isolated from pig liver in 1960 by JFPennock et al. [Nature
(London), 186, 470 (1960)] This was later changed to the general formula (A) [During the ceremony,

[Formula] is a transformer type represents the soprene unit,

[Formula] represents a cis isoprene unit. The same shall apply hereinafter in this specification. ] is a mixture of polyprenol analogues having the structure shown in formula (A), where j representing the number of cis isoprene units is generally distributed from 12 to 18, and 3 where j = 14, 15 and 16. It was revealed that species congeners are the main species [RW Keenan et al., Biochemical Journal,
165, 505 (1977)]. Dolichols are widely distributed not only in the liver of pigs but also in the bodies of mammals, and are known to play extremely important functions in maintaining the life of living organisms. For example, JB Harford et al. conducted an in vitro study using the white medulla of calves and pigs, and found that exogenous dolichol promotes the incorporation of sugar components such as mannose into lipids, and as a result, it plays a role in maintaining the normal life of living organisms. It has been revealed that biochemical and
Biophysical Research Communication, 76,
1036 (1977)]. The effect of dolichols on promoting the incorporation of sugar components into lipids is more pronounced in already mature animals than in growing organisms, so the role of dolichols in preventing aging is attracting attention. In addition, RWKeenan and colleagues state that it is important for animals that are rapidly growing, such as during childhood, to ingest dolichol from outside and supplement the dolichol that is biosynthesized within their own bodies. [Archives of Biochemistry and
Biophysics, 179, 634 (1977)]. moreover,
Akamatsu et al. quantified dolichol phosphate in the regenerated rat liver and found that the amount was significantly lower than in the normal liver, indicating that the glycoprotein synthesis function in the liver tissue was significantly reduced. We found that this function was improved when dolichol phosphate was added externally [54th Annual Meeting of the Japanese Biochemical Society (1981)]
Published in 2007]. The dolichols mentioned above are substances that control extremely important functions for living organisms, and are useful as pharmaceuticals or intermediates thereof. Only 0.6 g of dolichol can be obtained through this process [FW Burgos et al., Biochemical
See Journal, 88, 470 (1963)]. Total synthesis of dolichols is extremely difficult using current organic synthesis techniques, as is clear from their complex and unique molecular structures. It would be advantageous if dolichols could be obtained by relying on natural products as synthetic intermediates and adding simple synthetic chemical treatments to them, but such convenient substances have not been found so far. Conventionally, the following general formula (B) [However, it is known that polyprenols represented by k = 4 to 6 (these are called betulaprenols) can be collected from Betula verrucola; It is almost impossible to synthesize dolichols mainly containing 14, 15, and 16 molecules using current organic synthesis techniques. Also
K. Hannus et al.
reported that a polyprenyl component was isolated from leaves of S. sylvestris at a yield of 1% on a dry weight basis, and that this component was a polyprenyl acetate mixture containing 10 to 19 isoprene units primarily in the cis configuration. Phytochemistry, 13, 2563 (1974)], their report does not elucidate the details of the trans and cis configurations in the polyprenyl acetate. Furthermore, DFZinckel et al. reported the presence of C ₉₀ polyprenols with 18 isoprene units or an average number of isoprene units in extracts of leaves of Pinus strobus [ Phytochemistry,
11, 3387 (1972)], this report does not provide a detailed analysis of the trans and cis configurations of the polyprenol. Some of the present inventors and their co-researchers first performed chromatography after saponifying extracts extracted from leaves of Ginkgo biloba and Cedrus deodara with an organic solvent as necessary. A polyprenyl fraction consisting of a polyprenol and/or a mixture of its acetate homologs in which n is distributed from 11 to 19 in the general formula () by processing by graphie, fractional dissolution method, or other suitable separation method. is obtained, and the polyprenyl fraction has α
- the polyprenyl fraction exhibits a distribution of polyprenyl congeners very similar to that in mammalian dolichols only in the absence of terminal saturated isoprene units; the polyprenyl fraction optionally contains its constituent individual ( It can be relatively easily separated into polyprenyl homologs (with a uniform number of isoprene units), and therefore both the polyprenyl fraction and each polyprenyl homologue separated therefrom are highly suitable as synthetic intermediates for mammalian dolichols. I found out that In addition, the present inventors
Black pine (Pinus thunbergii) and Japanese red pine (Pinus thunbergii) belong to the Pinaceae family like Himalayan cedar.
It has been found that polyprenol and/or its ester (generally acetate ester) represented by the general formula () can also be obtained from leaves such as A. desiflora. However, from Japanese black pine and Japanese red pine, polyprenyl homolog mixtures in which n is distributed from 10 to 18 in the general formula () are usually obtained. However, extracts extracted from these plants with organic solvents usually contain components such as chlorophyll, carotenoids, steroids, glycerides, sphingolipids, and flavonoids, which are not always easy to separate from the polyprenols mentioned above. Polyprenol obtained by saponifying the extract and then separating and purifying it by a combination of fractional dissolution and chromatography often contains several percent of these impurities. The present inventors solved the above problems and easily produced polyprenol represented by the general formula () with high purity from extracts of plants containing the polyprenol represented by the general formula () and/or its ester. As a result of extensive research in order to achieve this goal, it was discovered that the desired objective could be achieved by the following method, which includes a step of converting a crude polyprenol fraction into an ester, purifying it, and then returning it to polyprenol. It was completed. That is, according to the present invention, at least one polyprenol represented by the general formula () and/or
Or, the extract obtained by extracting the plant containing the ester with a fat-soluble organic solvent is saponified, and then a fraction containing polyprenol represented by the general formula () as a main component is separated, and the polyprenol is is used as its lower aliphatic monocarboxylic acid ester, the polyprenyl ester is purified and then converted into polyprenol, and the resulting polyprenol represented by the general formula () is recovered, thereby producing the polypropylene represented by the general formula (). Lenol can be easily obtained with high purity. The fat-soluble organic solvent used to extract the polyprenyl component from plants containing at least one type of polyprenol represented by the general formula () and/or its ester generally has a dielectric constant ε of 32.7 or less, It is preferably 25.0 or less, more preferably 20.7 or less, and specifically, the solvents listed below can be used alone or as a mixed solvent of two or more. (a) Hydrocarbons: for example, petroleum ether, pentane, hexane, heptane, benzene, toluene,
such as xylene. (b) Halogenated hydrocarbons: e.g. chloroform,
Methylene chloride, carbon tetrachloride, ethane tetrachloride, perchlorethylene, trichlorethylene, etc. (c) Esters: For example, methyl acetate, ethyl acetate, ethyl propionate, etc. (d) Ethers: For example, diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, etc. (e) Ketones: For example, acetone, methyl ethyl ketone, diethyl ketone, diisopropyl ketone, etc. (f) Alcohols: For example, methyl alcohol, ethyl alcohol, propyl alcohol, butyl alcohol, etc. When selecting the solvent to be used, it is desirable to select a solvent that selectively extracts the polyprenol of the general formula () and its ester with high efficiency, and extracts as little as possible of other substances.From this point of view, the above-mentioned Among the solvents, hydrocarbons, halogenated hydrocarbons, esters, ethers with low polarity such as diethyl ether and diisopropyl ether, and ketones are particularly preferred. The amount of extraction solvent used is not critical; it depends on the type of solvent used, the type and part of the plant to be extracted,
Although it can vary widely depending on the condition, it is generally about 1 to about 100 parts by weight, preferably 5 to 50 parts by weight, per 1 part by weight (dry weight basis) of the plant to be extracted.
It is advantageous to use parts by weight, more preferably in the range from 10 to 30 parts by weight. Extraction can be carried out by immersing the plant in the above solvent and stirring continuously or intermittently as necessary. The temperature during extraction is also not critical and can be varied widely depending on conditions such as the type and amount of solvent used, but generally it is about 0.
Temperatures from °C to the reflux temperature of the solvent can be used, and room temperature is usually sufficient. It is advantageous to carry out the extraction under such conditions, usually for 1 to 10 days. The immersion liquid after the extraction process is made into a concentrated liquid by removing solids such as plants and removing the solvent as necessary. The extract thus obtained can be directly subjected to saponification treatment, but if desired, the extract can also be purified to some extent by appropriate pretreatment before being subjected to saponification treatment. For this pre-purification, the below-mentioned fractional dissolution method and/or chromatography are particularly preferably used. If impurities and/or tar components insoluble in a hydrocarbon solvent (for example, n-hexane) are removed by this pre-purification, subsequent purification will be facilitated. The saponification treatment can be carried out using a method and conditions known per se, for example, by dissolving sodium hydroxide or sodium hydroxide in water-containing methanol or water-containing ethanol with a water content of about 1 to 20% by weight. solution (alkali metal hydroxide concentration preferably approx.
The above extract may be added at a ratio of about 5 to 50 parts by weight to 100 parts by weight (which can be 0.1 to 30% by weight) and reacted at about 25 to 90°C for about 0.5 to 5 hours. After the saponification treatment, a fraction (crude polyprenol fraction) containing polyprenol represented by the general formula () as a main component is separated and obtained from the saponification reaction mixture.
Obtaining this crude polyprenol fraction is usually preferably carried out by the following method. That is, first, the saponification reaction mixture is extracted with an organic solvent substantially immiscible with water in the presence of water, and separated into an organic layer and an aqueous layer. Examples of organic solvents that can be used here include petroleum ether, hydrocarbons such as pentane, hexane, heptane, benzene, toluene, and xylene, chloroform, methylene chloride, carbon tetrachloride, ethane tetrachloride, perchloroethylene, and trichloroethylene. Examples include halogenated hydrocarbons, isopropyl acetate, butyl acetate, ethyl propionate, butyl propionate, diethyl ether, diisopropyl ether, di-n-butyl ether, methyl isobutyl ketone, diisobutyl ketone, and the like. Although the amount of these extraction solvents to be used is not critical, it is generally preferred to use 5 times (by weight) to 100 times (by weight) the saponification reaction mixture. During this extraction, it is sufficient that the amount of water present is at least the minimum amount necessary to cause phase separation between the organic layer and the aqueous layer, but usually the volume ratio of the organic layer and the aqueous layer is 1:10 to 10:1. It is preferable to use it in a suitable proportion. After concentrating the organic layer obtained in this way by distilling off the solvent as necessary,
The crude polyprenol fraction can be recovered by subjecting it to a separation step consisting of chromatography, fractional dissolution, molecular distillation, or a combination of two or more of these methods. To confirm the fraction containing polyprenol in the above separation process, use Merck's thin layer chromatography plate (silica gel 60F ₂₅₄ coated; layer thickness
In thin layer chromatography (10 cm development) using a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 9:1 (0.25 mm), the Rf value of solanesyl acetate as a standard substance was 0.40
This can be done depending on whether or not a spot exists at an Rf value within the range of 0.18 to 0.25 under the condition that the Rf value is 0.45. Therefore, when referring to the Rf value of thin layer chromatography in the following explanation, it should be understood that unless otherwise specified, it means the value measured under the above conditions. The operations of chromatography, fractional dissolution, fractional cryoprecipitation, and molecular distillation methods that can be used in the above separation step are known per se, and in the present invention, they can be carried out in accordance with known methods. Therefore, the details of each method will be explained by citing literature, and only points to be particularly careful about will be described here. (A) Chromatography [e.g. H. Heftman,
“Chromatography”, Reinhold, Publish Co.,
New York (1961)] Thin layer chromatography and liquid chromatography are suitable for small amounts of extracts, but column chromatography is suitable for processing large amounts of extracts. Examples of the chromatography carrier used include silica gel, alumina, florisil, celite, activated carbon, and cererose, with silica gel being preferred. Examples of developing solvents when performing separation operations using a silica gel column include hexane/ethyl acid (volume ratio 95:5 to 80:20), hexane/diisopropyl ether (volume ratio 95:5 to 80:20) ,
Petroleum ether/acetic ether (volume ratio 95:5~
80:20), petroleum ether/isopropyl alcohol (99:1 to 90:10 by volume), benzene/diethyl ether (95:5 to 80:20 by volume), benzene/ethyl acetate (98:2 to 80 by volume) :20), chloroform, methylene chloride, etc. (B) Fractional dissolution method [e.g. LCCraig, “Technique
of Organic Chemistry”, Vol.13, Interscience,
(1951)] The polyprenol of the general formula () is easily soluble in nonpolar solvents such as pentane and hexane,
On the other hand, since it is poorly soluble in polar solvents such as methanol and water, by dissolving the extract concentrate in the non-polar solvent and then washing with a polar solvent that is immiscible with the non-polar solvent, Impurities that are easily soluble in polar solvents can be largely removed. Examples of the nonpolar solvent preferably used in this method include hydrocarbon solvents such as petroleum ether, pentane, hexane, heptane, benzene, and toluene, and halogenated hydrocarbon solvents such as methylene chloride and chloroform. be. Further, as a polar solvent immiscible with such a non-polar solvent, water or methanol is suitable, for example. (C) Fractional cryoprecipitation method [e.g., EW Berg,
“Physical and Chemical Methods of
Separation”Chapter 14, 15, McGraw-Hill,
New York (1963)] The polyprenol of the above general formula () has a temperature of about -10°C.
It solidifies below. Therefore, the extract should be kept at −10°C or lower.
Preferably left to cool at about -15 to about -30°C,
By solidifying the target product and then performing solid-liquid separation, it can be purified from impurities that do not solidify at such temperatures. However, since the polyprenol does not have very good crystallinity and becomes a wax-like solid, it is difficult to completely purify it by this method. Note that, conversely, it is also possible to preferentially precipitate and remove crystalline impurities present in polyprenol. (D) Molecular distillation method [e.g., G.Durrows,
“Molecular Distillation”, Clarendon Press,
Oxford (1960)] Since the polyprenol of the general formula () has a large molecular weight, low molecular weight impurities can be removed by using a molecular distillation method. for example
Molecular distillation is performed under heating conditions of 100 to 200° C. in a vacuum degree of 10 ⁻³ to 10 ⁻⁵ mmHg, and the fraction is divided into a low molecular fraction and a high molecular fraction. At this time, the target substance is retained in the high molecular fraction, and low molecular weight impurities can be largely removed. A combination of two or more of these separation methods can also be used. For example, chromatography and fractional dissolution; chromatography and fractional cryoprecipitation and fractional dissolution; chromatography and fractional cryoprecipitation, fractional dissolution and molecular distillation; chromatography and molecular distillation and fractional dissolution. Combinations of methods such as chromatography and molecular distillation; molecular distillation and fractional dissolution; molecular distillation, fractional dissolution, and fractional freeze precipitation can be used. In one of the preferred embodiments of the invention,
By treating the extract extracted with an organic solvent after the saponification treatment by a separation and purification method mainly based on column chromatography, a fraction having an Rf value of 0.18 to 0.25 in the thin layer chromatography is obtained. (crude polyprenol fraction) is separated and obtained. Once the crude polyprenol fraction obtained in this manner is once converted into a polyprenyl ester and purified in the form of the polyprenyl ester, it can be easily purified to a high purity by a simple operation. To convert polyprenol to polyprenyl ester, polyprenol can be converted into a lower aliphatic monocarboxylic acid such as acetic acid, propionic acid, butyric acid, valeric acid, or a reactive derivative thereof, an acid anhydride such as acetic anhydride, or acetyl chloride. Alternatively, polyprenol may be transesterified with a lower alkyl ester of a lower aliphatic monocarboxylic acid such as methyl acetate, ethyl acetate, or methyl propionate. These esterification reactions and transesterification reactions can be carried out using methods and conditions known per se. For example, the esterification reaction can be carried out as follows. That is, the polyprenol fraction and about 0.8 to 10 mol per mol of polyprenol contained therein,
Preferably, 1 to 5 moles of the acid anhydride or acid halide are combined, preferably in the presence of a suitable solvent such as pentane, hexane, benzene, toluene, diethyl ether, diisopropyl ether, and a suitable base such as pyridine or triethylamine. The reaction may be carried out at about -30 to +80°C, preferably 0 to 30°C. Further, the transesterification reaction can be carried out, for example, as follows. That is, a lower alkyl ester of a lower aliphatic monocarboxylic acid as described above and polyprenol are mixed in the presence of a suitable transesterification catalyst such as sodium hydride or potassium hydride in the presence of a solvent or in the absence of a solvent for 50 to 50 min.
The reaction may be carried out at 200°C, preferably 80 to 150°C. By purifying the polyprenyl ester produced as described above, impurities that are difficult to remove, particularly chlorophyll, carotenoids, steroids, glycerides, sphingolipids, flavonoids, etc., can be easily removed from the polyprenol fraction. Can be done. For this purification, the aforementioned chromatography, fractional dissolution method, fractional cryoprecipitation method, molecular distillation method, etc. can be used alone or in combination of two or more, but the use of chromatography is particularly preferred. be. The preferred embodiment of the chromatography is similar to that described above for the separation method used to obtain the polyprenol fraction. To purify a large amount of polyprenyl ester, for example, a silica gel column and a developing solvent such as hexane/ethyl acetate (volume ratio 99:1 to 90:10), hexane/diisopropyl ether (volume ratio 99:1 to 90:10) are used. Purification by column chromatography using, for example, is simple and particularly preferred. The purified polyprenyl ester is preferably converted to the desired polyprenol by hydrolysis. This hydrolysis reaction can be carried out by various methods known to be useful for hydrolyzing esters of known higher alcohols. Preferably, the polyprenyl ester is prepared by heating and stirring in an alcohol such as methanol, ethanol or isopropanol with about 1 to 10 moles of alkali metal hydroxide, particularly sodium hydroxide or potassium hydroxide, per mole of polyprenyl ester. It will be implemented. Alternatively, polyprenol can be obtained by reacting the polyprenyl ester with lithium aluminum hydride in a solvent such as diethyl ether or tetrahydrofuran with 0.25 to 5.0 molar equivalents of lithium aluminum hydride relative to the polyprenyl ester at or near room temperature. can be transformed. After these reactions, the polyprenol represented by the general formula () can be recovered by separating the organic layer from the reaction mixture and performing simple post-treatments such as washing with water, drying, and concentration. If the polyprenol obtained is a mixture of its homologs, the individual homolog components can be further separated, if desired, by, for example, partition-type high performance liquid chromatography. The polyprenols or mixtures thereof thus obtained contain almost no coloring impurities such as chlorophylls, carotenoids, steroids, glycerides, sphingolipids, flavonoids, etc., and are suitable for use as intermediates in the synthesis of mammalian dolichols, for example. be. Hereinafter, the present invention will be explained in more detail with reference to Examples. In addition, the IR analysis in the examples was measured using a liquid film.
NMR analysis was performed using TMS as an internal standard.
The value of FD-MASS analysis (field ionization mass spectrometry) is
This is a value corrected as ¹ H, ¹² C, and ¹⁶ O. Example 1 10 fig leaves collected in Kurashiki City in mid-October
Kg (undried weight) was dried with hot air at about 50℃ for 6 hours, then at room temperature (about 15℃) n-hexane/acetone =
1 by immersing it in a 4/1 (volume ratio) mixed solvent 80.
Extracted weekly. The solvent was distilled off from this extract to obtain a black liquid. Add this to methanol 400
ml, along with 40 ml of water and 25 g of sodium hydroxide.
After heating and stirring at 65°C for an hour, the mixture was cooled to room temperature, extracted with hexane (500 ml), and the hexane layer was washed five times with about 100 ml of saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. A black and colored liquid was obtained.Then, this liquid was subjected to n-
By processing with a mixed solvent of hexane/ethyl acetate = 95/5 (volume ratio), silica gel thin layer chromatography using a mixed solvent of n-hexane/ethyl acetate = 9/1 (volume ratio) [ Merck TLC Plate (silica gel 60F ₂₅₄ Precoated
Rf=
A fraction with a concentration of 0.21 was separated to obtain 22 g of a red liquid. In this thin layer chromatography, solanesyl acetate showed an Rf value of 0.41. Next, this liquid material was dissolved in 20 g of pyridine and 300 ml of n-hexane, and 25 g of acetic anhydride was added dropwise while stirring at room temperature. After the addition was completed, the mixture was stirred overnight at room temperature. The resulting reaction mixture was washed five times with about 100 ml of saturated brine, dried over anhydrous magnesium sulfate, and n-hexane was distilled off to obtain a red colored liquid. This liquid was filtered through a silica gel column using 1 kg of silica gel to obtain n-hexane/ethyl acetate=
By treating with a mixed solvent of 97/3 (volume ratio), a fraction with Rf = 0.53 was separated in the same silica gel thin layer chromatography as above to obtain 18.2 g of a yellow liquid. Next, add this liquid to 200ml of methanol and 20ml of water.
After heating and stirring at 65°C for 2 hours with 5 g of sodium hydroxide and cooling to room temperature, approximately
The hexane layer was washed five times with about 100 ml of saturated brine, dried over anhydrous magnesium sulfate, and n-hexane was distilled off to obtain a pale yellow liquid. This liquid was treated with a silica gel column containing approximately 1 kg of silica gel using a mixed solvent of n-hexane/ethyl acetate = 95/5 (volume ratio), and then subjected to silica gel thin layer chromatography in the same manner as above. The fraction with Rf=0.21 was separated to obtain 17.1 g of a pale yellow liquid. This pale yellow liquid was mixed with silica gel μ-Porasyl as a filler and n-hexane/ethyl acetate = 97/3.
Adsorption-type high performance liquid chromatography using a mixed solvent (volume ratio) as a developing solution and acetone/silica gel μ-Bondapak-C ₁₈ as a packing material.
As a result of analysis by partition-type high performance liquid chromatography using a mixed solvent of methanol = 70/30 (volume ratio) as a developing solution, almost no peaks other than the target polyprenol represented by the general formula () were present. It was not detected at all, and it was confirmed that the purity was over 99%. In addition, high-performance liquid chromatography was performed using LiChrosorb RP18-10, a semi-preparative high-performance liquid chromatography column manufactured by Merck, using a mixed solvent of acetone/methanol = 90/10 (volume ratio) as the developing solution, and using a differential refractometer as a detector. Chromatographic analysis was performed, and the area ratio of each peak in the obtained chromatogram was determined, and the results were as follows.

【table】

[Table] Using this high-performance liquid chromatography, each component was separated from the above oily substance and analyzed by mass spectrometry, infrared absorption spectrum, ¹ H-NMR spectrum, and
It was confirmed by ¹³ C-NMR spectrum that these components were polyprenol having a structure represented by the general formula (). FD-MASS results for each component and
The δ values of ¹ H-NMR are summarized in Table 1, and the δ values of ¹³ C-NMR are summarized in Table 2.

【table】

【table】

【table】

[Table] Comparative Example 1 In the same manner as in Example 1, the extract was extracted from the leaves of the yam using a mixed solvent of n-hexane/acetone = 4/1 (volume ratio), and the extract was saponified, extracted with hexane, and concentrated. The polyprenol fraction separated by silica gel column chromatography was further purified by silica gel column chromatography twice without being reacted with acetic anhydride, and 20.1 g of a red liquid was obtained. . When this liquid material was analyzed by high performance liquid chromatography in the same manner as in Example 1, impurities were detected.
Polyprenol purity calculated from area intensity is 95%
It was hot. From this, it is clear that by going through the step of converting the polyprenol fraction into polyprenyl ester as in Example 1, it becomes easier to remove impurities, especially coloring components. Example 2 10 kg (undried weight) of Himalayan cedar leaves collected in late May were dried with hot air at about 50°C for 8 hours, and then immersed in chloroform 100 at room temperature (about 20°C) for extraction for one week. The solvent was distilled off from this extract to obtain a black liquid. This product was treated with a mixed solvent of n-hexane/ethyl acetate = 95/5 (volume ratio) through a silica gel column using approximately 1 kg of silica gel, and was then treated with a mixed solvent of n-hexane/ethyl acetate = 9/1 (volume ratio). Silica gel thin layer chromatography using a mixed solvent of
TLC Plate (silica gel 60F ₂₅₄ Precoated, layer thickness
0.25mm) and 10cm expansion] Rf = 0.5
Fractions near and near Rf=0.2 were collected to obtain 58 g of a black and colored liquid. In this thin layer chromatography, solanesyl acetate showed an Rf value of 0.41. Next, mix this black and colored liquid with 400% methanol.
ml, 40 ml of water and 15 g of potassium hydroxide, heated and stirred at 65℃ for 4 hours, cooled to room temperature, extracted with about 800 ml of hexane, and extracted with about 200 ml of hexane layer.
Washed 5 times with saturated saline solution, dried over anhydrous magnesium sulfate, and distilled off the solvent to obtain a black and colored liquid.
Got 49g. By treating this liquid with a silica gel column using approximately 1 kg of silica gel using a mixed solvent of n-hexane/ethyl acetate = 97/3 (volume ratio), Rf = 0.21 in the silica gel thin layer chromatography. The fractions were separated to obtain 23.2 g of a red colored liquid. Next, this liquid material was dissolved in 20 g of pyridine and 300 ml of n-hexane, and 25 g of propionic anhydride was added dropwise to the solution while stirring at room temperature. After the addition was completed, stirring was continued overnight. The obtained reaction solution was diluted with saturated saline solution at about 100%
ml five times, dried over anhydrous magnesium sulfate, and n-hexane was distilled off to obtain a red colored liquid. This liquid was filtered through a silica gel column using approximately 1 kg of silica gel to produce n-hexane/ethyl acetate =
By treating with a mixed solvent of 97/3 (volume ratio), a fraction having Rf=0.51 in the silica gel thin layer chromatography was separated to obtain 20.8 g of a pale yellow liquid. Next, this liquid was dissolved in 200 ml of anhydrous tetrahydrofuran, and 0.5 g of lithium aluminum hydride was added little by little while stirring at room temperature. After the addition was completed, stirring was continued at room temperature for 2 hours, then about 5 ml of water was carefully added, and the mixture was rotated. Tetrahydrofuran was distilled off using an evaporator, and the mixture was extracted with about 500 ml of hexane. The hexane solution was washed five times with about 100 ml of saturated saline, dried over anhydrous magnesium sulfate, and the hexane was distilled off to obtain 18.4 g of a pale yellow liquid. When this liquid material was analyzed by high performance liquid chromatography in the same manner as in Example 1, almost no peaks other than the polyprenol represented by the general formula () were detected, indicating that it has a purity of 99% or more. This was confirmed. Moreover, the molecular weight distribution of this polyprenol was measured using the same method as in Example 1 and was as follows.

[Table] As in Example 1, each component was separated using high-performance liquid chromatography, and the components were analyzed by mass spectrometry, infrared absorption spectrum, ¹ H-NMR spectrum, and ¹³ C-NMR spectrum. It was confirmed that the components were substantially the same as those obtained in Example 1. Comparative Example 2 A crude polyprenol fraction before propionic acid esterification obtained in the same manner as in Example 2 was subjected to silica gel column chromatography [n-hexane/ethyl acetate = 95/ 5 (volume ratio)] was repeated twice to obtain 23.7 g of a red liquid. When this liquid material was analyzed by high performance liquid chromatography as in Example 1, impurities were detected, and the purity calculated from the area ratio was 96%. Examples 3 to 5 and Comparative Examples 3 to 5 10 kg of fig leaves collected in Kurashiki City at the end of October
(undried weight) was dried with hot air at approximately 60°C for 24 hours, then immersed in benzene 100 for 5 days at room temperature (approximately 20°C).
Extracted at ℃). The solvent was distilled off from this extract to obtain a black liquid. methanol
400 ml of water, 40 ml of water, and 30 g of potassium hydroxide, heated to 65°C for 2 hours, stirred, and then cooled to room temperature.
Extraction was carried out by adding 500 ml of diethyl ether, and the diethyl ether layer was washed five times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was distilled off to obtain 75 g of a black liquid. Each 5 g of this black and colored liquid was treated according to the following steps to obtain purified polyprenol (Examples 3 to 5). Black and colored liquid → 1st purification → esterification → 2nd
Purification → saponification → isolation of purified polyprenol. For comparison, purified polyprenol was obtained in the same manner as in Examples 3 to 5, except that esterification after the first purification and saponification after the second purification were not performed (Comparative Examples 3 to 5). The results are summarized in Table 3.

[Table] Example 6 10 red pine leaves collected in Kurashiki City in early December
Kg (undried weight) was dried with hot air at about 60°C for 24 hours, then immersed in chloroform 80 at room temperature (about 10°C) for extraction for one week. Hexane 5 was added to the concentrate obtained by distilling off chloroform from this extract to separate insoluble components, and the liquid was concentrated to obtain a black colored liquid. Add this to 400ml of methanol,
2 hours with 40ml water and 25g sodium hydroxide
After heating to 65°C and stirring, the mixture was cooled to room temperature and extracted with hexane (500 ml). The hexane layer was washed five times with about 100 ml of saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. A black and colored liquid was obtained. Next, this liquid was subjected to n-
By treating with a mixed solvent of hexane/ethyl acetate = 95/5 (volume ratio), n-hexane/
In silica gel thin layer chromatography using a mixed solvent of ethyl acetate = 9/1 (volume ratio)
The fraction with Rf=0.21 was separated to obtain 6.9 g of a red colored liquid. Next, this liquid material was dissolved in 5 g of pyridine and 100 ml of n-hexane, and 5 g of acetic anhydride was added dropwise while stirring at room temperature. After the addition was completed, the mixture was stirred overnight at room temperature. The resulting reaction mixture was diluted with about 20 ml of saturated saline solution.
After washing twice, drying with anhydrous magnesium sulfate,
N-hexane was distilled off to obtain a red colored liquid. This liquid was treated with a silica gel column containing approximately 300 g of silica gel using a mixed solvent of n-hexane/ethyl acetate = 97/3 (volume ratio), and was then subjected to the same silica gel thin layer chromatography as described above. The fraction with Rf=0.53 was separated to obtain 5.18 g of a yellow liquid. Next, this liquid was dissolved in 100 ml of anhydrous tetrahydrofuran, and 0.2 g of lithium aluminum hydride was added little by little while stirring at room temperature. After the addition was complete, stirring was continued for 2 hours at room temperature, and then about 5 ml of water was carefully added. , Tetrahydrofuran was distilled off using a rotary evaporator, and the mixture was extracted with about 100 ml of hexane. The obtained hexane layer was washed five times with about 50 ml of saturated saline, dried over anhydrous magnesium sulfate,
Hexane was distilled off to obtain 5.02 g of a pale yellow liquid. When this liquid material was analyzed by high performance liquid chromatography in the same manner as in Example 1, almost no peaks other than the polyprenol represented by the general formula () were detected, and it was found to have a purity of 99% or more. was confirmed. Moreover, the molecular weight distribution of this polyprenol was measured using the same method as in Example 1 and was as follows.

[Table] Furthermore, as in Example 1, each component was fractionated using high-performance liquid chromatography, and mass spectrometry, infrared absorption spectrum, ¹ H-NMR spectrum, and ¹³ C-NMR spectrum showed that the components were the same. Example 1 having a number of cis isoprene units of
It was confirmed that each component was the same compound as obtained in . The component with peak number 1 above (10 cis isoprene units) gave m/e = 902 in FD-MASS analysis, and ¹ H-NMR and ¹³ C
-NMR analysis gave substantially the same results as those listed in Tables 2 and 3. Comparative Example 6 The crude polyprenol fraction before acetate esterification obtained in the same manner as in Example 6 was subjected to silica gel column chromatography [n-hexane/ethyl acetate = 95/5 (volume When purification was repeated twice using a mixed solvent of (ratio), 5.33 g of a red liquid was obtained. When this liquid material was analyzed by high performance liquid chromatography as in Example 1, impurities were detected.
The purity calculated from the area ratio was 95%.

Claims (1)

[Claims] 1 General formula () (In the formula, [Formula] represents a trans-type isoprene unit, [Formula] represents a cis-type isoprene unit, and n represents an integer of 10 to 19.) After saponifying the extract obtained by extracting ester-containing plants with a fat-soluble organic solvent, a fraction containing polyprenol represented by the general formula () as a main component is separated, and the polyprenol is As its lower aliphatic monocarboxylic acid ester,
A method for producing polyprenol, which comprises purifying the polyprenyl ester, converting it into polyprenol, and recovering the produced polyprenol represented by the general formula (). 2. The method according to claim 1, wherein the plant is Ginkgo biloba. 3 Claim 1 in which the plant is a Pinaceae plant
The method described in section. 4 Pine family plant is Himalayan cedar (Cedrus deodara)
The method according to claim 1. 5. The method according to claim 1, wherein the fat-soluble organic solvent is selected from hydrocarbons, nilogenated hydrocarbons, esters, ethers, ketones, and mixtures thereof. 6. Saponify the extract extracted from the plant,
The saponification reaction mixture is extracted with a substantially water-immiscible organic solvent in the presence of water, the resulting organic layer is concentrated, and a polyprenol fraction is separated and obtained by column chromatography. The method according to claim 1, wherein the polyprenyl ester is converted into an aliphatic monocarboxylic acid ester, purified by column chromatography, and then converted into polyprenol.