JPH0230335B2 - HORIBURENIRUKAGOBUTSUMATAHASONOKONGOBUTSUNOSEIZOHOHO - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing polyprenyl compounds or mixtures thereof. More specifically, the present invention describes the following general formula that can be suitably used as a raw material for the synthesis of dolichol. (In the formula, A represents a hydroxyl group or an acyloxy group, [Formula] represents a trans isoprene unit, [Formula] represents a cis isoprene unit, and n is an integer from 11 to 19.) The present invention relates to a method for producing a polyprenyl compound or a mixture thereof. Dolichols were introduced by JF in 1960.
It was first isolated from pig liver etc. by Pennock et al. [Nature (London), 186 , 470 (1960)]
], and later they described the dolichols using the following general formula: (In the formula, [formula] represents a trans isoprene unit,
[Formula] represents a cis isoprene unit. ) is a mixture of polyprenol homologs having the structure shown in the above formula, in which the number j of cis isoprene units generally ranges from 12 to 18, and j=14,
It was revealed that three homologues of 15 and 16 were the main constituents [RWKeenan et al.,
Biochemical Journal, 165 , 405 (1977)].
Furthermore, 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 test using the white medulla of calves and pigs, and found that exogenous dolichol promoted the incorporation of sugar components such as mannose into lipids, and as a result, sugar that is important for sustaining the life of living organisms It has been revealed that it has the effect of increasing protein formation [Biochemical and
Biophysical Research Communication. 76 ,
1036 (1977)]. Since the effect of dolichol on promoting the incorporation of sugar components into lipids is more pronounced in already mature animals than in living organisms during the growing stage, the role of dolichol in preventing aging is attracting attention. In addition, RWKeenan and colleagues state that it is important for organisms that are rapidly growing, such as during childhood, to ingest dolichol from outside and supplement the dolichol that is biosynthesized within the body. [Archives of Biochemistry
and Biophysics, 179 , 634 (1977)]. Furthermore, Akamatsu et al. quantified dolichol phosphate ester in the regenerated liver of rats and found that the amount was significantly reduced compared to that in the normal liver, indicating that the glycoprotein synthesis function in the liver tissue was greatly reduced. We found that this function was improved by adding exogenous dolichol phosphate [presented at the 54th Annual Meeting of the Japanese Biochemical Society (1981)]. As described above, dolichols are extremely important substances for living organisms, and there is a strong desire for the development of applications in pharmaceuticals, synthetic intermediates thereof, cosmetics, and the like. However, the reality is that it has not been possible to conduct sufficient research on Dolichols because they are difficult to obtain. For example, only about 0.6 g of dolichol can be obtained from 10 kg of pig liver through complicated separation procedures [F. Burgos et al.,
See Biochemical Journal, 88 , 470 (1963)]. On the other hand, it is extremely difficult to fully synthesize dolichols using current synthetic organic chemistry techniques, as evidenced by their complex and unique molecular structures. Therefore, 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.
No such convenient substance has hitherto been found. It has been known that polyprenol compounds can be collected from various plants, and the following polyprenols have been collected. (1) Solanesol (2) Fucaprenols (3) Betulaprenol Betulaprenol, like dolichols,
-It has a structure in which two trans isoprene units are connected to the terminal isoprene unit, but the betulaprenols known so far have only six cis isoprene units at most, as mentioned above. The number of cis isoprene units is 14
In order to synthesize dolichols, which mainly contain 1, 15 and 16 isoprene units, it is necessary to restrict and extend 8 or more isoprene units into the cis form, which is difficult to do using current organic synthesis technology. It's almost impossible. In addition, K. Hannus et al. isolated a polyprenyl fraction of approximately 1% (on a dry weight basis) from the leaves of Pinus sylvestris , and found that this fraction contained 10 to 19 isoprene units mainly in the cis configuration. It has been reported that polyprenylacetate is composed of polyprenylacetate. However, the polyprenyl fraction extracted from Japanese red pine is mainly composed of homologues with 15 and 16 isoprene units, and the main components of mammalian dolichols are 17 and 18 isoprene units. and 19 congeners are present in only trace amounts [see Phytochemistry, 13 , 2563 (1974)]. K.
The report by Hannus et al. does not clarify the details of the trans and cis configurations in the above polyprenyl compound group, but if the polyprenyl compounds had the same trans and cis configuration as mammalian dolichols, However, in order to derive mammalian dolichols, at least two cis isoprene units must be regulated in the cis form and a saturated isoprene unit must be added to the α-terminus. It is clear that this will involve a great deal of difficulty. In addition, D. Zinckel et al. reported the presence of C ₉₀ polyprenols with 18 isoprene units or an average value of 18 isoprene units in extracts from leaves of Pinus strobus [Phytochemistry, 11 , 3387 (1972)].
However, the analysis they are conducting is extremely crude and is based on nuclear magnetic resonance (NMR), and according to the results of the inventors' follow-up tests, it was found that the polyprenol fraction extracted from pine needles It was found that the main component was a homologue with 17 isoprene units. Therefore, even if the polyprenyl fraction isolated from the needles of this pine strobe has the same trans and cis configuration as mammalian dolichols, it is difficult to synthesize mammalian dolichols from this fraction. still has at least 1
It is necessary to introduce two cis-type isoprene units while regulating the cis-type, which still poses great difficulties in synthesis. Therefore, the present inventors discovered that they have the same number of isoprene units as mammalian dolichols, as well as isoprene units in trans and cis configurations. As a result of analyzing extracts from various plants in search of plant sources for polyprenyl compounds that do not require manipulation, we surprisingly found that the polyprenyl fraction (polyprenyl composition) extracted from Himalayan persimmon ( Cedrus deodara ) , exhibiting a polyprenyl homolog distribution very similar to the distribution of polyprenyl homologs in mammalian dolichols, with the only absence of α-terminal saturated isoprene units compared to mammalian dolichols;
It has been found that it is very suitable as an intermediate for the synthesis of mammalian dolichols. According to the present invention, Himalayan cassava leaves are extracted with an organic solvent, a polyprenyl compound or a mixture thereof is separated from the resulting extract, and if necessary, the polyprenyl compound or a mixture thereof is added before or after the separation. A method for producing a polyprenyl compound represented by the general formula () or a mixture thereof is proposed, which comprises subjecting the compound to hydrolysis, esterification, transesterification, or two or more thereof. After extracting Himalayan cucumber leaves with an organic solvent and hydrolyzing the resulting extract as necessary, chromatography, fractional dissolution method, fractional cryoprecipitation method, molecular distillation method, or two or more of these methods are used. In the separation method consisting of the above combination, n-hexane and ethyl acetate were separated using a Merck thin-layer chromatography plate (silica gel 60F ₂₅₄ pre-coated; layer thickness 0.25 mm). The Rf value of solanesyl acetate as a standard substance in thin layer chromatography (10 cm development) using a mixed solvent of 9:1 by volume as the developing solution.
By isolating and collecting fractions exhibiting an Rf value in the range of 0.18 to 0.25 and/or 0.50 to 0.55 under conditions such that A is 0.40 to 0.45, compounds of general formula () and/or A in which A is a hydroxyl group are obtained. consists essentially of a mixture of compounds of general formula () in which n is an acetoxy group, and n is 14, a compound of general formula () in which n is 15 and n
is 16, at least 3 of the compounds of general formula ()
Polyprenyl compositions can be obtained which contain seeds as essential components in substantial amounts and whose total content is at least 70% by weight, based on the weight of the mixture. The Himalayan Sugi used as a raw material is a plant belonging to the Seed Plants, Gymnosperms, Coniferae, Conifera, which is widely distributed in temperate and cold regions.It came to Japan at the beginning of the Meiji period and is now used as a garden tree everywhere. Can be seen. The Himalayan sorcery leaves may be subjected to the treatment according to the invention after drying, or they may be used undried. Dry leaves are generally preferred, and the degree of drying in this case is such that the moisture content is generally about 30% or less, preferably 10% based on the weight of the dry leaves.
It is advantageous that: Furthermore, the leaves are preferably extracted after being crushed, so that
The contact area with the extraction solvent increases, making it possible to improve extraction efficiency. The polyprenyl homologue represented by the above general formula () is generally contained in the leaves of Himalayan cucumbers at a fairly high concentration in the form of free alcohol and/or acetate ester, and the polyprenyl analogue can be extracted from the leaves. For effective extraction, an oil-soluble organic solvent that well dissolves the polyprenyl analog is preferably used. As such oil-soluble organic solvents, those having a dielectric constant (ε) of 32.7 or less, preferably 25.0 or less, and more preferably 20.7 or less are suitable. Specifically, the following solvents may be used alone or in combination. It can be used as a mixed solvent of two or more. (a) Hydrocarbons; for example, petroleum ether, pentane, hexane, heptane, benzene, toluene, xylene, etc. (b) Halogenated hydrocarbons: For example, chloroform, methylene chloride, carbon tetrachloride, ethane tetrachloride, perchloroethylene, 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: e.g. methyl alcohol,
Ethyl alcohol, propyl alcohol, butyl alcohol, etc. When selecting the solvent to be used, it is desirable to use a solvent that selectively extracts the target polyprenyl compound of the general formula () with high efficiency, and extracts as few other substances as possible. Among the above 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 and can be varied widely depending on the type of solvent used, the type and condition of the leaves to be extracted, etc., but in general, 1 part by weight of Himalayan perilla leaves (dry weight) is used. It is advantageous to use within the range of about 1 to about 100 parts by weight, preferably 5 to 50 parts by weight, and more preferably 10 to 30 parts by weight. Extraction can be carried out by immersing the leaves in the above solvent and stirring continuously or intermittently as necessary. The temperature during extraction is also not critical and can be varied over a wide range depending on conditions such as the type and amount of solvent used, but generally a temperature from about 0°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 the leaves and other solid content, and then removing the solvent as necessary. The extract thus obtained is then subjected to a separation process consisting of chromatography, fractional lysis, fractional cryoprecipitation, molecular distillation, or a combination of two or more of these methods to obtain the desired polyprenyl fraction. can be recovered. To confirm the fraction containing polyprenyl compounds in the above separation step, use a Merck thin layer chromatography plate (coated with silica gel 60F ₂₅₄ ; layer thickness 0.25 mm) and compare the volume of n-hexane and ethyl acetate. In thin layer chromatography (10 cm development) using a mixed solvent with a ratio of 9:1 as the developing solvent, the Rf value of solanesyl acetate as a standard substance was
0.40 to 0.45, within the range of 0.18 to 025 [when A represents a hydroxyl group in the above general formula ()] and/or 0.50 to 0.55 [when A represents an acetoxy group in the above general formula ()] of
This can be done depending on whether a spot exists at the Rf value. 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 extract separation process described above are known per se, and the present invention also uses known methods. Therefore, details of each method will be explained by citing literature, and only points to be particularly careful will be described here. (A) Chromatography [e.g. H. Heftman,
“Chromatography”, Reinhold Publishing 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 usable carriers for chromatography include silica gel, alumina, florisil, celite, activated carbon, and cellulose, of which silica gel is preferred. Examples of developing solvents when performing separation operations using a silica gel column include hexane/ethyl acetate (volume ratio 95:5 to 80:20), hexane/ethyl acetate (volume ratio 95:5 to 80:20),
Diisopropyl ether (volume ratio 95:5-80:
20), petroleum ether/methyl acetate (volume ratio 95:5
~80:20), petroleum ether/isopropyl alcohol (volume ratio 99:1~90:10), benzene/diethyl ether (volume ratio 95:5~80:20), benzene/ethyl acetate (volume ratio 98:2~ Examples include mixed solvent systems such as 80:20), chloroform, methylene chloride, etc. (B) Fractional dissolution method [e.g. LCCraig,
“Technique of Organic Chemistry”
Vol. 13, Intersscience, (1951)] The polyprenyl compound of the above general formula () is easily soluble in non-polar solvents such as pentane and hexane, but poorly soluble in polar solvents such as methanol and water. Therefore, by utilizing this difference in solubility, it can be purified by a fractional dissolution method. For example, by dissolving a crude product such as an extract concentrate in the above 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 are largely removed. can do. 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 polyprenyl compound of the general formula () is about -
Solidifies below 10℃. Therefore, by leaving the extract under cooling at -10°C or lower, preferably about -15 to about -30°C, solidifying the target product, and then performing solid-liquid separation, it is possible to prevent it from solidifying at such temperatures. It can be purified from impurities. However, since the polyprenyl compound does not have very good crystallinity and becomes a wax-like solid, it is difficult to completely purify it by this method, so it is preferable to carry out the method in combination with other purification methods. (D) Molecular distillation method [e.g., G.Durrows,
“Molecular Distillation”, Clarendon Press,
Oxford (1960)] Since the compound of the general formula () has a large molecular weight, low molecular weight impurities can be removed by using a molecular distillation method. For example 10 ^-3 ~
Molecular distillation is carried out under a vacuum degree of 10 ^-5 mmHg and heating conditions of 100 to 250° C. to separate 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. If a sufficiently pure polyprenyl fraction cannot be obtained by each of the above separation methods, a combination of two or more of these separation methods may 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 cryoprecipitation can be used. Thus, in thin layer chromatography
Fractions with Rf values of 0.18 to 0.25 and/or 0.50 to 0.55 are isolated and collected. The fraction with an Rf value of 0.18 to 0.25 consists essentially of a mixture of homologues of the general formula () when A represents a hydroxyl group, while the fraction with an Rf value of 0.50 to 0.55 consists of a mixture of homologs of the general formula () )
consists essentially of a mixture of homologues in which A represents an acetoxy group. Individual homolog components can also be isolated by further subjecting the thus obtained fractions to, for example, partition-type high performance liquid chromatography. In addition, in the above separation step, before subjecting the extract to the above separation operation, the extract is hydrolyzed to remove the homologs in the above general formula () where A represents an acetoxy group that may be contained therein. It is possible to convert in advance to the corresponding homologue in which A represents a hydroxyl group. This may simplify the subsequent separation operation. However, such hydrolysis occurs naturally after the separation operation is completed.
It can also be performed on fractions containing components with an Rf value of 0.50 to 0.55. This hydrolysis can be carried out using any conventional method known for hydrolyzing fatty acid esters, for example by dissolving sodium or potassium hydroxide in aqueous methanol or ethanol. solution (the alkali metal hydroxide concentration is preferably about
The above extract or fraction is 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. Bye. In the polyprenyl fraction isolated and recovered by the above-mentioned method, the fraction with an Rf value of 0.18 to 0.25 is essentially a mixture of multiple polyprenol analogues in which A in the general formula () represents a hydroxyl group. The fraction having an Rf value of 0.50 to 0.55 consists essentially of a mixture of a plurality of polyprenylacetate homologs when A in the general formula () represents an acetoxy group. The ratio of the former to the latter in the extract is approximately 20:1 to 5:
1, and the distribution state (distribution pattern) of polyprenol or polyprenylacetate congeners in each fraction is roughly the same, and the distribution state varies depending on the youth of the leaves used as raw materials, the time of collection, and It remains almost constant regardless of factors such as region. The fraction generally contains at least three compounds of the general formula () where n is 14, a compound of the general formula () where n is 15, and a compound of the general formula () where n is 16 as essential components, respectively. in substantial amounts and their total content is at least 70% by weight, usually more than 75% by weight, based on the weight of the fraction. Generally, the fraction contains a maximum content of a compound of the general formula () in which n is 15, usually from 30 to 50% by weight, based on the weight of the fraction, more typically It is within the range of 32-47% by weight. In addition, the fraction generally contains compounds of the general formula () where n is 14, 15 and 16, respectively, in a specific quantitative relationship, and when the respective contents are taken as a, b and c% by weight, The quantitative relationship is b>a>
It is normal that it is c. Further, the fraction generally contains 20 to 35% by weight of compounds of general formula () where n is 14, more typically 23 to 35% by weight.
32% by weight, and generally 10-25% by weight, more typically 11-25% by weight of compounds of general formula () where n is 16.
20% by weight (all based on the weight of the fraction)
Contains. As mentioned above, the polyprenyl composition (fraction) obtained by extraction from Himalayan perilla by the method of the present invention has a distribution pattern of mammalian dolichols and polyprenol congeners, that is, n in general formula () and general formula (A ) is characteristic in that the distribution turns of j are extremely similar, and by comparing this distribution with that of pig dolichols (human dolichols also show almost the same distribution as that of pigs), It is shown as follows. Note that numbers in parentheses indicate more typical ranges. [Table] The average value of n in the polyprenyl composition obtained by the method of the present invention is usually within the range of 14.25 to 15.25. As is clear from the distribution state of polyprenyl homologs shown in Table 1 above and the comparison between general formula () and general formula (A), the polyprenyl composition obtained by the method of the present invention has Mammalian dolichols can be derived by bonding one saturated isoprene unit to the α-terminus. In particular, the saturated isoprene units to be bonded have no problem with the cis- or trans configuration, and the bonding of the saturated isoprene units does not pose any difficulty in reaction drying. Therefore, it can be said that the polyprenyl composition obtained by the method of the present invention is an extremely important substance as a synthetic intermediate for mammalian dolichols. When the polyprenyl composition obtained by the method of the present invention is induced into mammalian dolichols, the composition may be used as it is, or if necessary, each polyprenyl compound constituting the composition may be isolated and then reacted. You can also do that. Furthermore, the composition or the isolated polyprenyl compound, depending on whether it is in the free alcohol or acetate form, is subjected to hydrolysis, esterification or transesterification reactions or reactions of two or more thereof. Polyprenol may be converted into a polyprenonyl ester, or polyprenyl acetate may be converted into another polyprenyl ester or polyprenol represented by the general formula (). As an example of a polyprenyl ester represented by the general formula () produced by the method of the present invention, when A in the general formula () is represented by RCOO-, R is a hydrogen atom, an alkyl group having 1 to 18 carbon atoms ( For example, methyl, ethyl, n-propyl, isopropyl, n
-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isoamyl, n-hexyl, n-octyl, n-decyl, n-dodecyl,
n-undecyl, stearyl, etc.), carbon number 2~
18 alkenyl groups (e.g., 3-butenyl, 3-
pentenyl, 4-pentenyl, geranyl, geranylmethyl, farnesyl, farnesylmethyl, etc.), cycloalkyl groups having 5 to 7 carbon atoms (e.g.
cyclopentyl, cyclohexyl, methylcyclohexyl, cycloheptyl, etc.), carbon number 6-10
aryl group (e.g., phenyl, tolyl, xylyl, naphthyl, etc.), aralkyl group having 7 to 11 carbon atoms (e.g., benzyl, phenethyl, methylbenzyl, dimethylbenzyl, α- or β-naphthylmethyl, etc.), or 1 to 3 carbon atoms Examples include esters which are methyl groups (eg, monofluoromethyl, trifluoromethyl, monochloromethyl, dichloromethyl, etc.) substituted with a fluorine atom or a chlorine atom. The esterification or transesterification reaction of the polyprenol represented by the general formula () or a mixture thereof to obtain these polyprenyl esters or mixtures thereof can be carried out using esterification or transesterification reaction methods and conditions known per se to obtain esters. This can be done using For example, esterification can be carried out by esterifying polyprenols represented by the general formula () or mixtures thereof with formic acid, acetic acid, monofluoroacetic acid, monochloroacetic acid, trifluoroacetic acid, propionic acid, butyric acid in the presence or absence of a catalyst and a solvent. , caproic acid, caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, farnesylic acid, farnesylacetic acid, benzoic acid, 3,5-dimethylbenzoic acid, 4-ethylbenzoic acid This can be easily carried out by mixing with a desired carboxylic acid such as an acid or an acid halide or acid anhydride thereof, and heating and stirring as necessary. Preferably, the above polyprenol or a mixture thereof is dissolved in a solvent such as hexane, benzene, methylene chloride, chloroform, diethyl ether, etc., and 1 to 5 molar equivalents of the above acid halide relative to the polyprenol are added thereto to dissolve the polyprenol. This is carried out by stirring for 1 to 24 hours at an appropriate temperature from room temperature to the reflux temperature of the solvent in the presence of 1 to 5 molar equivalents of pyridine based on lenol. In addition, the transesterification reaction is carried out by combining polyprenol represented by the general formula () or a mixture thereof with a lower alkyl ester (preferably methyl ester or ethyl ester) of a desired carboxylic acid (for example, the various carboxylic acids mentioned above) using a transesterification catalyst. It is carried out by acting in the optional presence of. Preferably, the above polyprenol or mixture thereof and the desired carboxylic acid ester are dissolved in a solvent such as benzene, toluene, xylene, etc., and 0.01 to 0.1 molar equivalent of sodium hydride is added to the polyprenol, and the mixture is heated for 2 hours to 2 hours. The reaction is carried out under conditions of refluxing the solvent for 5 days. The polyprenyl compound represented by the general formula () or a mixture thereof obtained by the method of the present invention can be easily converted into mammalian dolichols, for example, by the following synthetic route. In the above, x is a halogen atom, preferably Cl
or Br, and Z is a hydroxyl-protecting group such as a tetrahydropyranyl group, a methoxymethyl group,
It represents a benzyl group, etc., OCOR represents an acyloxy group, and Q represents the following group. [In the formula, n has the same meaning as that in the general formula (). ] Polyprenyl compounds and mixtures thereof obtained by the method of the present invention are useful in addition to the above-mentioned materials, for example, as cosmetic base materials, ointment base materials, or raw materials for their production. EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited by these Examples. Example 1 10 kg (undried weight) of Himalayan Sugi leaves collected in Kurashiki City in late May were dried with hot air at approximately 40°C for 24 hours, and then dried with chloroform 80 at room temperature (approximately 15°C) for 7 days.
It was extracted by immersion in it. Petroleum ether was added to the concentrate obtained by distilling off chloroform from this extract.
was added to separate insoluble components, the liquid was concentrated, and separated using a silica gel column using chloroform as a developing solvent, and n-hexane/ethyl acetate =
In silica gel thin layer chromatography using a mixed solvent of 9/1 (volume ratio) [using a Merck thin layer chromatography plate (silica gel 60F ₂₅₄ coated; layer thickness 0.25 mm), 10 cm development] Rf value
Approximately 32 g of oil was obtained as a fraction showing 0.52. In the thin layer chromatography described above, solanesyl acetate showed an Rf value of 0.41. Approximately 400 ml of acetone is added to this oil to dissolve the acetone-soluble components, the resulting mixture is filtered, the liquid is concentrated, and the resulting oil is mixed with 400 ml of methanol and 40 ml of water.
After heating at 65°C for 2 hours with 20 g of sodium hydroxide, methanol was distilled off, and the residue was extracted with diethyl ether (500 ml). The ether layer was washed 5 times with about 100 ml of water, and then anhydrous sodium sulfate The solvent was distilled off to obtain 25.3 g of an oil. Next, this oily substance was separated using about 1 kg of silica gel and a mixed liquid of n-hexane/diisopropyl ether = 90/10 (volume ratio),
Rf value 0.19 in the above thin layer chromatography
22.1 g of oil was obtained as a fraction showing . This oil was polyprenol with a purity of 95% or more, and the molecular weight distribution measured for this product was as follows. This value is calculated using a Merck semi-preparative high performance liquid chromatography column for the above oily substance.
Obtained by high performance liquid chromatography using Li Chrosorb RP 18-10 (C ₁₈ type) with a mixed solvent of acetone/methanol = 90/10 (volume ratio) as the eluent and a differential refractometer as the detector. It was calculated from the area ratio of the chromatogram. Value of n Area ratio (%) 11 0.76 12 2.06 13 7.00 14 24.32 15 38.54 16 19.22 17 5.19 18 1.39 19 0.54 Moreover, the average value of n in the polyprenol mixture obtained here was 14.8. Each component was separated from the oily substance using the high performance liquid chromatography described above, 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 in the general formula () where A is a hydroxyl group. Results of field ionization mass spectrometry (FD-MASS) for each component and
The δ values of ¹ H-NMR are summarized in Table 2, and the δ values of ¹³ C-NMR are summarized in Table 3. In Table 2, FD−
The m/e value of MASS analysis is a value corrected for each atomic weight of carbon, hydrogen, and oxygen as ¹² C, ¹ H, and ¹⁶ O. Furthermore, in the ¹ H-NMR data, (b) means a wide signal, (d) a doublet signal, and (t) a triplet signal. [Table] [Table] Example 2 5 kg (undried weight) of Himalayan persimmon leaves collected in Kurashiki City in mid-July were dried with hot air, pulverized into small pieces with a mixer, and then soaked in petroleum ether for 7 days at room temperature (approximately 25°C). / acetone = 1/4 (volume ratio) mixed solvent
40 was used for extraction. After washing the extract with water and drying it with anhydrous sodium sulfate, the solvent was distilled off to give approximately
100 g of residue was obtained. Add 1 part of n-hexane to this to dissolve the n-hexane soluble components,
After concentrating the liquid, the same thin layer chromatography as used in Example 1 was performed using a silica gel column with a mixed solvent of n-hexane/diethyl ether = 95/5 (volume ratio) to obtain an Rf value of 0.50 and
A fraction showing a concentration of 0.19 was collected to obtain about 16 g of an oily substance.
Approximately 200 ml of acetone is added to this oil to dissolve the acetone-soluble components, and the resulting liquid is filtered at a concentration of approximately 500 ml.
Using g of silica gel, it was separated with a mixed solvent of n-hexane/diethyl ether = 95/5 (volume ratio).
13.2 g of oil was obtained. This oil was polyprenylacetate with a purity of 95% or more, and the molecular weight distribution measured for this product was as follows. This molecular weight distribution was determined by using a Waters high performance liquid chromatography column μBondapak/C ₁₈ with a mixed solvent of acetone/methanol = 70/30 (volume ratio) as the eluent.
It was determined from the area ratio of a chromatogram obtained by high performance liquid chromatography using a differential refractometer as a detector. Value of n Area ratio (%) 11 0.82 12 2.01 13 9.82 14 25.48 15 37.24 16 18.20 17 4.65 18 1.00 19 0.78 In addition, the average value of n in the polyprenylacetate mixture obtained here was 14.8. Each component was extracted from the above oil (polyprenylacetate content 95% or more) using the same high performance liquid chromatography as in Example 1, and subjected to mass spectrometry, infrared absorption spectrum, ¹ H-NMR spectrum and ¹³ C-NMR spectrum. It was confirmed that these components were polyprenylacetate in the general formula () in which A was an acetoxy group. Field ionization mass spectrometry (FD) for each component
-MASS) results are shown in Table 4. In Table 4, the m/e values of the FD-MASS analysis are values corrected by setting the atomic weights of carbon, hydrogen, and oxygen as ¹² C, ¹ H, and ¹⁶ O. [Table] Furthermore, polyprenols obtained by similarly hydrolyzing each component according to the hydrolysis reaction performed in Example 1 are the same as those obtained in Example 1 with the same value of n. Same as polyprenols
¹ H-NMR spectrum, ¹³ C-NMR spectrum and infrared absorption spectrum were given. Examples 3 to 23 Himalayan perilla leaves collected in Kurashiki City at the end of October were dried with hot air at about 60°C for 65 hours, then divided into 100g portions and immersed in Solvent 1 shown in Table 5, and left at room temperature for 7 days. (approximately 20°C). The extraction solvent was distilled off from the obtained extract, and the weight of the residue was measured and summarized in Table 5 as the total amount of extract. Dissolve these extracts in 200 ml of hexane, and add the solution to approximately 100 ml of a mixed solution of methanol/water = 9/1 (volume ratio).
After washing twice, it was dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain an oily substance. Add this oil with 50 ml of methanol and 1 g of potassium hydroxide.
After heating at 65°C for an hour, methanol was distilled off, diethyl ether (100ml) was added to the residue for extraction, the ether layer was washed three times with about 50ml of saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain an oil. Using 100g of silica gel, mix this oil with n-hexane/ethyl acetate = 9/
1 (volume ratio), and the Rf value was determined by thin layer chromatography similar to that in Example 1.
A fraction (polyprenol mixture) showing 0.19 was obtained. The weight of this fraction is summarized in Table 5 as the polyprenol content. The composition of the polyprenol mixture obtained was substantially the same as that of the polyprenol mixture obtained in Example 1, regardless of the type of solvent used. [Table] Example 24 In a solution in which 1.24 g of polyprenol of the general formula () with n = 15 and A = OH obtained from Himalayan Sugi in the same manner as in Example 1 and 1.0 g of pyridine were dissolved in dry diethyl ether. acetic anhydride at room temperature.
1.2 g was added dropwise, and after the addition was completed, the mixture was stirred overnight at room temperature. The resulting reaction mixture was washed with saturated brine,
It was then dried over anhydrous magnesium sulfate and diethyl ether was distilled off to obtain a pale yellow viscous liquid. This product was purified by silica gel column chromatography (using hexane/ethyl acetate as a developing solution) to obtain 1.08 g of a pale yellow liquid. When this material was analyzed by IR, the absorption caused by the OH group of the raw material polyprenol at approximately 3300 cm ^-1 disappeared.
-Absorptions of 1745 cm ^-1 and 1255 cm ¹ due to OCOCH ₃ appeared. In addition, when NMR analysis was performed, the signal (doublet, δ = 4.08) attributed to -CH ₂ OH of the raw material polyprenol disappeared, and -
A new signal (doubet, δ=4.55) assigned to C H ₂ OCOCH ₃ was observed. _−CH2OCOC
The signal assigned to H ₃ was observed to overlap with the signal assigned to [Formula] (δ=2.04). In addition, m/e was determined by FD-MASS analysis.
=1284 was obtained. From the above, this liquid is n=
15, it was confirmed that it was a polyprenylacetate of the general formula () where A= _OCOCH3 . n is
Polyprenylacetate other than 15 and n is 11 to
A polyprenylacetate mixture arbitrarily distributed in 19 could also be synthesized by a similar procedure. Example 25 1.24 g of polyprenol of the general formula () with n=15 and A=OH obtained from Himalayan Sugi in the same manner as in Example 1, 0.5 g of methyl oleate and 0.01 g of sodium hydride were added in 50 ml of toluene. and heated at 110°C for 24 hours under a nitrogen gas atmosphere.
After the reaction solution was cooled to room temperature, it was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain a yellow liquid. This product was purified by silica gel column chromatography (using hexane/ethyl acetate as a developing solution).
0.47g of colorless viscous liquid was obtained. IR analysis of this liquid revealed that the absorption of approximately 3300 cm ^-1 due to the OH groups of the raw material polyprenol had disappeared. Also, FD−
MASS analysis gave m/e=1506. From the above, this one is n=15, A=OCOC ₁₇ H ₃₃
It was confirmed that it was a polyprenyl oleate of the general formula (). Polyprenyl oleates with n other than 15 and polyprenyl oleate mixtures with n arbitrarily distributed between 11 and 19 could also be synthesized by similar operations. Example 26 0.28 g of benzoyl chloride was added at room temperature to a mixture of 1.24 g of a polyprenol of the general formula () with n=15 and A=OH obtained from Himalayan Sugi in the same manner as in Example 1 and 10 ml of pyridine. and stirred overnight at room temperature. The reaction mixture was then poured into about 150 ml of water, extracted with diethyl ether, and the resulting ether layer was washed with saturated brine, diluted hydrochloric acid, saturated sodium bicarbonate, and again saturated brine, and then poured over anhydrous magnesium sulfate. The residue was dried and diethyl ether was distilled off to obtain a yellow liquid. This was subjected to silica gel column chromatography (hexane/
(using ethyl acetate as a developing solution)
0.92g of slightly yellow liquid was obtained. IR about this stuff
Upon analysis, the absorption caused by the OH groups of the raw material polyprenol disappeared, and the absorption caused by ester bonds appeared at 1715 cm ^-1 and 1270 cm ^-1 . Also FD−
MASS analysis gave m/e=1346.
From the above, this liquid substance has n=15 and A=
It was confirmed to be polyprenyl benzoate of the general formula (), which is OCOC ₆ H ₅ . Polyprenyl benzoates in which n is other than 15 and polyprenyl benzoate mixtures in which n is arbitrarily distributed between 11 and 19 were also synthesized by similar operations. Example 27 1.24 g of polyprenol of the general formula () where n = 15 and A = OH obtained from Himalayan Sugi in the same manner as in Example 1 and 1.0 g of pyridine were dissolved in 10 ml of methylene chloride, and 0 to 5 After 0.4 g of monochloroacetic anhydride was added dropwise at °C, the mixture was stirred at room temperature overnight. Next, this reaction mixture was poured into water, extracted with diethyl ether, and the resulting ether layer was washed successively with diluted hydrochloric acid, water, and saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off. 1.30 g of a pale yellow liquid was obtained, which was further purified by silica gel column chromatography (using hexane/ethyl acetate as a developing solution) to obtain 1.25 g of a liquid. When this product was subjected to IR analysis, the absorption caused by the OH groups of the raw material polyprenol disappeared, and the absorption caused by C=O appeared at about 1750 cm ^-1 . As a result of NMR analysis, the signal attributed to -C H ₂ OH of the raw material polyprenol disappeared, and a new signal (doublet, δ = 4.57) attributed to -C H ₂ OCOCH ₂ Cl and -OCOC H
₂ Signal assigned to Cl (singlet, δ=3.93)
appeared. Furthermore, m/e was determined by FD-MASS analysis.
=1318 was obtained. From the above, this liquid has n=
15, it was confirmed to be a compound of the general formula () where A=OCOCH ₂ Cl. Polyprenyl monochloroacetates with n other than 15 and polyprenyl monochloroacetate mixtures with n arbitrarily distributed between 11 and 19 were also synthesized by similar operations. Reference example Magnesium strips (0.316 g, 13 mmol), anhydrous tetrahydrofuran (0.5 ml) and 1,2-dibromoethane (0.08 ml) were placed in a three-necked flask that had been purged with argon, and heated with a hair dryer until it bubbled violently. . Next 2-[4-Bromo-
A solution of 3-methylbutoxy]-tetrahydro-2H-pyran (2.51 g, 10 mmol) in anhydrous tetrahydrofuran (3.0 ml) was added dropwise to the activated magnesium at a rate such that the solvent just boiled. After the dropwise addition was completed, the mixture was stirred at 70°C for 15 minutes. Anhydrous tetrahydrofuran (60 ml) was added to this to prepare a Grignard solution. Separately, in a three-necked flask purged with argon, add a solution of polyprenylacetate (6.42 g, 5 mmol) with the general formula () where n = 15 and A = -OCOCH ₃ in anhydrous tetrahydrofuran (15 ml) and a solution of Li ₂ CuCl ₄ in anhydrous tetrahydrofuran. (0.1 molar solution, 20
ml) was added. The previously prepared Grignard solution was added dropwise to this at 0°C over 1 hour, and stirring was continued at 0°C for 2 hours. After that, saturated ammonium chloride water was added to this reaction mixture for hydrolysis.
Extracted with ether. The ether layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was distilled off using a rotary evaporator to obtain 7.95 g of a pale yellow liquid. This product was analyzed using silica gel thin layer chromatography (hexane/ethyl acetate = 97/3
(volume ratio) used as developing solution) Rf = 0.35
It had a main spot in . Furthermore, when this pale yellow liquid was analyzed by FD-MASS, m/e = 1284, which indicates the presence of raw material polyprenylacetate, was not detected at all, and n = 15 and Z =
m/e=1396, which indicates a compound having a tetrahydro-2H-pyranyl group, was detected as a main peak. Next, add this pale yellow liquid to hexane (40ml).
To this were added p-toluenesulfonic acid pyridine (0.13 g, 0.5 mmol) and ethanol (20 ml). This solution was heated and stirred at 55°C for 3 hours.
After cooling, sodium carbonate (0.21 g) was added to neutralize the mixture, and the solvent was distilled off using a rotary evaporator. The obtained concentrate was dissolved in ether, washed with saturated aqueous sodium bicarbonate, then saturated brine, and dried over anhydrous magnesium sulfate. The solvent was removed using a rotary evaporator, and the remaining oily substance was heated at 0.5 Torr. and 150° C. for 30 minutes to remove low-boiling components. The remaining liquid part was subjected to silica gel column chromatography (hexane/ethyl acetate = 9/1 (volume ratio) was used as the developing solution)
A colorless and transparent liquid (5.64 g) was obtained. This product gave a single spot at Rf = 0.19 by silica gel thin layer chromatography (using hexane/ethyl acetate = 9/1 as a developing solution). Furthermore, the analysis results shown below confirmed that this liquid was a compound of general formula (A) where j=15. FD-MASS analysis m/e = 1312 (measured value 1312) IR analysis (cm ^-1 ); 830, 1060, 1376, 1440, 2850,
2920, 3320 ¹³ C—NMR analysis (ppm/intensity) 135.365/430,
135.229/3567, 135.005/349, 134.937/290,
131.210/213, 125.071/5242, 124.993/499,
124.448/505, 124.282/463, 124.214/445,
61.241/551, 40.029/541, 39.757/683,
37.548/582, 32.245/5500, 32.021/456,
29.316/528, 26.825/492, 26.699/548,
26.436/5166, 25.677/542, 25.308/567,
23.430/6330, 19.557/548, 17.679/353,
16.006/640 ¹ H-NMR analysis (ppm, signal shape, proton ratio) 5.10 (b, 18H), 3.66 (m, 2H), 2.03 (b,
70H), 1.68 (s, 48H), 1.60 (s, 9H), 1.80−
1.10 (m, 5H), 0.91 (d, 3H)

Claims (1)

[Scope of Claims] 1. Extracting Himalayan cassava leaves with an organic solvent, separating a polyprenyl compound or mixture thereof from the resulting extract, and hydrolyzing the polyprenyl compound or mixture thereof before or after separation, if necessary. , a general formula characterized by being subjected to esterification or ester conversion reaction, or two or more reactions thereof. (In the formula, A represents a hydroxyl group or an acyloxy group, [Formula] represents a trans-type inprene unit, [Formula] represents a cis-type inprene unit, and n is an integer from 11 to 19.) A method for producing a polyprenyl compound or a mixture thereof. 2. The manufacturing method according to claim 1, wherein the organic solvent is selected from hydrocarbons, halogenated hydrocarbons, ethers, esters, and ketones. 3. Extract Himalayan cucumber leaves with an organic solvent, hydrolyze the resulting extract as necessary, and then perform chromatography, fractional dissolution method, fractional cryoprecipitation method, molecular distillation method, or two or more of these methods. For separation methods consisting of more combinations,
Merck thin layer chromatography plate (silica gel)
60F ₂₅₄ coating; layer thickness 0.25 mm) and thin layer chromatography (10 cm
development), the Rf value of solanesyl acetate as a standard substance is 0.40 to 0.45.
By isolating and collecting fractions exhibiting Rf values in the range of 0.25 and/or 0.50 to 0.55, compounds of general formula () in which A is a hydroxyl group and/or compounds of general formula () in which A is an acetoxy group are obtained. consisting essentially of a mixture of compounds and comprising at least three compounds of the general formula () where n is 14, a compound of the general formula () where n is 15 and a compound of the general formula () where n is 16; 2. A process according to claim 1, for obtaining a polyprenyl composition containing substantial amounts of each of the essential components, the total content of which is at least 70% by weight, based on the weight of the mixture.