JPS629120B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for rapidly producing a single sterol glycoside in large quantities from a sterol glycoside mixture derived from natural products. The substance obtained according to the present invention has a hemostatic effect and a blood vessel reinforcing effect, and is useful as a pharmaceutical. As is already widely known, the constituent sugar of sterol glycosides derived from natural products is generally glucose, but the constituent sterols, like naturally occurring free sterols, usually contain multiple highly similar structures. Its existence as a completely pure single sterol glycoside is not currently believed to exist. Even if something obtained from a certain plant was reported as a single sterol glycoside in the past, recent reexaminations such as gas chromatography-mass spectrometry analysis (hereinafter abbreviated as GC-MS) have revealed that It is becoming clear that all proteins contain heterologous sterol glycosides without exception. As mentioned above, since the structures of the sterols that make up the sterol glycoside mixture are extremely similar, the purification methods that have been applied to date, such as recrystallization, various liquid chromatography methods, and precipitation using alkali metal salts, are extremely similar. Reactions and the like can only separate a sterol glycoside mixture from unrelated contaminants, but are completely powerless to separate sterol glycosides from each other. Therefore, it has been considered difficult to separate sterol glycoside mixtures derived from natural products from each other and to collect even a trace amount of pure sterol glycosides, and there has been no success story so far. As a result of extensive research aimed at overcoming this difficult situation, the present inventors have developed a separation method that can produce sterol glycosides derived from natural products with high purity and on an industrial scale. and completed the present invention. The present invention will be described in detail below, step by step. (1) Separation of the sterol glycoside mixture and separation of the sterol glycoside mixture from the acetylated raw material can be performed by known methods, but
Specific precipitation reaction of sterol glycosides and alkali metal carbonates in lower alcohols by the present inventors
18327) can also be used to easily obtain a highly pure product. Sterol glycosides are commonly handled organic solvents, but by acetylating them to tetraacetate using conventional methods, they can be handled in large quantities with many organic solvents, and they can also be used as mobile phases in liquid chromatography. It is possible to expand the variety of types. (2) Separation of sterol glycoside tetraacetate mixtures by liquid chromatography As mentioned above, until now there has been no effective means for separating sterol glycoside mixtures from each other, making it extremely difficult. The inventors
Based on the results of all preliminary studies on derivatives of sterol glycoside mixtures, we have discovered a means of separating sterol glycoside derivatives from each other by a method using liquid chromatography. That is, we attempted to separate sterol glycoside tetraacetate mixtures by liquid chromatography under various conditions. As a result, effective stationary phases for separation include silica gel, silver nitrate-impregnated silica gel,
Magnesium oxide, alumina, florisil, etc. show a separation ability equivalent to that of free sterols with no sugar attached, and sterol glycosides are also separated extremely effectively based mainly on differences in the structure of the constituent sterols. It became clear. The stationary phase described above can be used for the separation of sterol glycosides in exactly the same way as conventionally applied to free sterols by selecting the appropriate mobile phase solvent. The present inventors investigated further practical and high-performance separation conditions and found that stable fine silica gel-ODS (octadecyl group chemically bonded to silica gel) was used as the stationary phase, and a single solvent was used. For example, a separation method using chromatography (HPLC) using methanol, acetonitrile, etc. as a mobile phase solvent was established. Cholesterol glucoside, brassicasterol glucoside, campesterol glucoside, stigmasterol glucoside, β-sitosterol glucoside, and their corresponding Δ ⁷ -sterol glycosides and reduced forms, etc., which were not possible using other methods, are derived from natural products. It has now become possible to isolate sterol glycosides from a mixture of sterol glycosides. An example of the HPLC chromatogram of sterol glycoside tetraacetate obtained from soybean lecithin under these conditions is shown in FIG. Sterol glycoside mixtures, which are generally found in many plants, show chromatograms similar to this one. In addition, this HPLC separation has all the advantageous conditions necessary for scale-up and can be produced on an industrial scale. (3) Deacetylation If the single sterol glycoside tetraacetate is treated with alkaline alcohol according to a conventional method, the deacetylated poorly soluble sterol glycoside can be quantitatively removed from alcohol. By precipitation, the desired pure sterol glycoside can be easily obtained. Next, Example 1 describes the isolation of the three types of sterol glycosides that exist most abundantly in nature, and Example 2 describes an example of the isolation of trace sterol glycosides. Example 1 Isolation of β-sitosterol glucoside, stigmasterol glucoside, and campesterol glucoside. The free sterols in the acetone extracts of about 10 species were analyzed by GC-MS, and the raw materials suitable for isolation were Kapok seed, soybean, olive, corn, and As a raw material suitable for isolating wheat, which contains almost no ^Δ7 -sterol glycosides, it is suitable for isolating potatoes, which contain almost no Δ7-sterol glycosides, which are difficult to separate by HPLC. Wheat germ, which contains almost no grains, was selected as a raw material. These raw materials are then processed by the following common operations. That is, the raw material that has been defatted with n-hexane and dried is extracted twice with twice the volume of acetone based on its weight, and the acetone is distilled off. Add 20 times the volume of 10% KOH methanol based on the weight of the residue and reflux for 1 hour, then add K ₂ CO ₃ until saturation is reached and continue to reflux for 3 hours. A highly pure sterol glycoside mixture is obtained by washing the extract with methanol and then with water and drying. This is converted into tetraacetate using acetic anhydride in pyridine in a conventional manner and used as a material for separation by HPLC. The conditions for HPLC are as follows. Stationary phase: Silica gel-ODS (5 μ) (column 10 mmφ x 30 cm) Mobile phase: acetonitrile (flow rate 10 ml/min) Under these conditions, the amount of sample processed at one time is 0.1 g, and the required time is within 10 minutes. The target product could be completely isolated from each sample in a single separation without loss. The isolated tetraacetates corresponding to ~ are summarized in Table 1 together with those obtained in Example 2. Tetraacetate was quantitatively precipitated from methanol by hydrolysis with 5% KOH methanol, and pure sterol glycosides were easily obtained. Example 2 Separation of 24-methylenecholesterol glucoside and avenasterol glucoside and are generally present as minor components in sterol glycoside mixtures. Here, commercially available soybean lecithin is used as a raw material, and precipitation reaction with K CO in methanol (Japanese Patent Application No. 50-9996) is carried out.
The sterol glycosides were separated and purified using the method (Japanese Patent Publication No. 54-18327), and the sterol glycosides were further isolated as tetraacetates. Although it is possible to directly apply HPLC to separate the sterols contained in this, the efficiency is low, so column chromatography using silver nitrate-impregnated silica gel as the stationary phase is performed as a pretreatment, and the main sterol that flows out first is separated. A mixture consisting only of tetraacetate with the exception of glycosides was used as a sample for HPLC. For separation by silver nitrate-impregnated silica gel and column chromatography, 5 g of sample was processed at a time using 20% silver nitrate-impregnated silica gel (4 cmφ x 40 cm) as the stationary phase and chloroform/cyclohexane (5:1) as the mobile phase solvent. , a mixture of about 1 g of tetraacetate was obtained from a 25 g sample. Next, using this mixture as a sample, HPLC separation was performed according to the conditions of Example 1, and and were separated from each other. In addition, purity tests using HPLC and GC confirmed that a purity of 99% or higher was obtained, and a purity of 97% or higher was obtained. The separated tetraacetate is summarized in Table 1. The separated tetraacetate was easily deacetylated with 5% KOH-methanol, and a corresponding amount of sterol glycoside was quantitatively precipitated.

【table】

【table】 [Brief explanation of the drawing]

FIG. 1 represents a high performance liquid chromatogram of sterol glucoside tetraacetate of soybean lecithin. The horizontal axis represents elution time, and the vertical axis represents peak height. The column is silica gel-ODS (4 mm x 30
cm), the sample injection amount was 10 mg, the mobile phase was acetonitrile, the pressure was 50 Kg/cm ² , and the flow rate was
A refractometer was used as a detector at a rate of 1.3 ml/min, and the measurement temperature was 25°C. Among the peaks, 1 is 24-methylenecholesterol glucoside tetraacetate, 2 is avenasterol glucoside tetraacetate, 3 is stigmasterol glucoside tetraacetate, 4 is campesterol glucoside tetraacetate, and 5 is β-sitosterol glucoside tetraacetate. and 6 represent stigmastanol glucoside tetraacetate, respectively.

Claims (1)

[Scope of Claims] 1. A sterol glycoside mixture obtained from plant materials is treated with an acetylating agent to form a corresponding acetyl compound mixture, and the resulting acetyl compound mixture is converted into individual tetraacetyl compounds by liquid chromatography. A method for producing a highly pure single sterol glycoside from a raw material containing a sterol glycoside mixture, which comprises separating the acetyl form and hydrolyzing the separated acetyl form under basic conditions. 2. The manufacturing method according to claim 1, wherein the plant material is wheat, rice, corn, soybean, kapok, cottonseed, or coffee beans. 3. The sterol glycoside mixture obtained from plant materials is a group consisting of campesterol glycoside, stigmasterol glycoside, β-sitosterol glycoside, 24-methylene cholesterol glycoside, and avenasterol glycoside. The manufacturing method according to claim 1 or 2, which is a mixture of two or more substances selected from.