Composition containing zizyphi semen extract and method for producing the same

Abstract

To provide a composition containing a Zizyphi Semen extract, and to provide a method for producing the same.SOLUTION: Provided is a method including the steps of: (A) contacting an extract of a Zizyphi Semen fraction containing a Zizyphi Semen extract with an aromatic synthetic adsorbent to adsorb the composition containing the Zizyphi Semen extract onto the aromatic synthetic adsorbent; (B) washing the composition adsorbed onto the aromatic synthetic adsorbent in step (A) with an aqueous solvent; (C) eluting the composition washed in step (B) from the aromatic synthetic adsorbent with an organic solvent to obtain an eluate; and (D) separating the eluate obtained in step (C) using basic functional group-modified chromatographic silica gel to obtain a purified composition containing Zizyphi Semen extract.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition containing jujube seed extract and a method for producing the same. [Background technology]

[0002] The quality of the medicinal herbs that make up Kampo medicines is strictly controlled in accordance with the 18th Revised Japanese Pharmacopoeia (hereinafter referred to as the "Pharmacopoeia Law"). Under the Pharmacopoeia Law, confirmation tests and quantitative methods using thin-layer chromatography to determine the origin of medicinal herbs require specified high-purity standard products called Japanese Pharmacopoeia reference standards. Manufacturers of medicinal herbs purchase these standard products and carry out quality control, including the quantification of the contained ingredients using HPLC or other methods.

[0003] However, in the case of natural products such as herbal medicines, many compounds have complex structural formulas that cannot be produced by chemical synthesis, and it is often difficult to produce reference standards that meet pharmacopoeial specifications.When reference standards cannot be used, quality cannot be determined based solely on characteristic indicators such as the number of spots in thin-layer chromatography or color reactions.

[0004] The jujube seed of the present invention is a herbal medicine used to improve insomnia and peripheral symptoms associated with dementia. Jujube seed is known to contain triterpenes, saponins, and aporphine alkaloids (Non-Patent Documents 1 and 2). Maguroflorin is known to be an aporphine alkaloid contained in jujube seed. Since there are no standard products to determine the origin of the components of jujube seed, quality control has been problematic.

[0005] Because jujube kernels contain compounds such as saponins as their main components, it has been difficult to purify only the aporphine alkaloids. Known methods involve extracting tens of kilograms of jujube kernels with large amounts of methanol and purifying them with silica gel chromatography or ODS column chromatography, but isolating the aporphine alkaloids takes a long time.

[0006] Although aporphine alkaloids were successfully isolated from jujube seeds before 2000, many of these were extracted using benzene or diethyl ether, and due to safety concerns, a simple and efficient purification method was needed (Non-patent documents 3-6). [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Liu SJ. et al.,Phytochem Rev.,2020,20:507-541 [Non-patent document 2] Gao QH.et al.,J Agric Food Chem.,2013,61:3351-3363 [Non-patent document 3] Han BH.et al.,Arch Pharm Res.,1987,10:208-211 [Non-patent document 4] Han BH.et al.,Pure & Appl Chem.,1989,61:443-448 [Non-patent document 5] Yoshikawa M. et al.,Chem Pharm Bull.,1997,45:1186-1192 [Non-patent document 6] Han BH.et al.,Yakhak Hoeji.,1993,37:143-148 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a composition containing jujube seed extract (hereinafter also referred to as "the composition of the present invention") that is suitable for purifying aporphine alkaloids. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve the above problems, and have found that it is possible to obtain hydroxybenzoates represented by the formulas (1), (2) and (3): from jujube kernels using an aromatic synthetic adsorbent and basic functional group-modified chromatographic silica gel: [ka] The present invention has been accomplished by discovering a simple and efficient method for producing a jujube seed extract containing aporphine alkaloids represented by the formulas (1), (2), and (3), which are called fujitin, N,N-dimethyllindocarpine, and magnoflorine, respectively.

[0010] That is, the present invention includes the following embodiments. Section 1. A method for producing a purified composition containing jujube seed extract, comprising: The jujube seed extract is represented by the formulas (1), (2) and (3): [ka] and an additional compound, the content of the additional compound in the entire purified composition is 90% by mass or less, (A) contacting an extract of the jujube seed fraction containing the jujube seed extract with an aromatic synthetic adsorbent to adsorb the composition containing the jujube seed extract onto the aromatic synthetic adsorbent; (B) washing the composition adsorbed onto the aromatic synthetic adsorbent in step (A) with an aqueous solvent; (C) eluting the composition washed in step (B) from the aromatic synthetic adsorbent using an organic solvent to obtain an eluate; (D) obtaining the purified composition from the eluate obtained in step (C) by separating the purified composition using a basic functional group-modified chromatographic silica gel; A manufacturing method comprising: Section 2. The jujube seed fraction is jujube seeds defatted with a degreasing agent, or Jujube seed water extract Item 1. The method for producing the present invention, wherein the extract is an organic solvent extract. Section 3. Item 3. The method according to item 1 or 2, wherein the degreasing agent is hexane. Section 4. Item 4. The method according to any one of Items 1 to 3, wherein the organic solvent is an alcohol. Section 5. Item 5. The method according to Item 4, wherein the alcohol is methanol. Section 6. Item 6. The method according to items 1 to 5, wherein the aqueous solvent is water. Section 7. Item 7. The method according to any one of Items 1 to 6, wherein the aromatic synthetic adsorbent is a styrene-divinylbenzene synthetic adsorbent. Section 8. Item 8. The method according to any one of Items 1 to 7, wherein the terminal of the basic functional group-modified chromatographic silica gel is modified with isopropylamine. Section 9. A composition comprising a jujube seed extract, The jujube seed extract has the formulas (1), (2) and (3): [ka] and an additional compound, A composition, wherein the content of the additional component in the composition is 90 mass % or less. Section 10. Item 10. The composition according to item 9, for purifying at least one aporphine alkaloid selected from the group consisting of compounds represented by formulas (1), (2), and (3). [Effects of the Invention]

[0011] According to the present invention, a composition containing jujube seed extract can be produced simply and efficiently. The jujube seed extract contains a higher proportion of aporphine alkaloids than conventionally produced jujube seed extracts. Therefore, the jujube seed extract is suitable for use in obtaining a purified product of the aporphine alkaloids. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a diagram showing the yield of magnoflorine when each aromatic synthetic adsorbent is used. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a method for producing a purified composition containing jujube seed extract, comprising: The jujube seed extract is represented by the formulas (1), (2) and (3): [ka] and an additional compound, the content of the additional compound in the entire purified composition is 90% by mass or less, (A) contacting the jujube seed fraction containing the jujube seed extract or an extract thereof with an aromatic synthetic adsorbent to adsorb the composition containing the jujube seed extract onto the aromatic synthetic adsorbent; (B) washing the composition adsorbed onto the aromatic synthetic adsorbent in step (A) with an aqueous solvent; (C) eluting the composition washed in step (B) from the aromatic synthetic adsorbent using an organic solvent to obtain an eluate; (D) obtaining the purified composition from the eluate obtained in step (C) by separating the purified composition using a basic functional group-modified chromatographic silica gel; The present invention relates to a manufacturing method comprising the steps of:

[0014] 1. Jujube seed fraction containing jujube seed extract or its extract The form of the jujube seed fraction containing the aporphine alkaloid-containing jujube seed extract used in the present invention is not particularly limited, and may be processed into powder, chips, flakes, fibers, lumber, etc. using a known method. Among these, from the viewpoint of reaction efficiency, processed plant material into powder, chips, flakes, or fibers is preferred. The extract of the jujube seed fraction is not particularly limited, and examples thereof include extracts from water or aqueous ethanol.

[0015] The amount of solvent used to obtain a sour jujube seed extract is preferably 1 mL to 100 mL, more preferably 5 mL to 20 mL, per 1 g of sour jujube seed fraction. The extraction conditions are not particularly limited as long as they allow for sufficient extraction. For example, when ultrasonic extraction is performed, the extraction time is preferably 0.5 hours or more, more preferably 1 hour or more. The extraction temperature is preferably 1°C or higher, more preferably 15°C or higher, and is preferably below the boiling point of the solvent, more preferably 30°C or lower. The extraction method is not particularly limited, but includes conventional methods such as solid-liquid extraction, liquid-liquid extraction, immersion, decoction, percolation, reflux extraction, Soxhlet extraction, ultrasonic extraction, microwave extraction, and stirring.

[0016] When an organic solvent extract is used as an extract of a jujube seed fraction containing an aporphine alkaloid-containing jujube seed extract, it is preferable to perform degreasing using a degreasing agent prior to the step of obtaining the organic solvent extract in order to remove fatty acids and other components. Examples of such degreasing agents include organic solvents that dissolve fats and oils and are liquid at room temperature and have little or no miscibility with water, such as halogenated lower alkanes (chloroform, methylene chloride, carbon tetrachloride, 1,2-dichloroethane, etc.), alkanes having 5 to 17 carbon atoms (hexane, octane, dodecane, etc.), aromatic hydrocarbons (benzene, toluene, xylene, etc.), and mixtures thereof. To prevent the loss of aporphine alkaloids, it is preferable that the degreasing agent have low affinity for aporphine alkaloids. Hexane is a preferred degreasing agent.

[0017] The organic solvent used to obtain the organic solvent extract of the sour jujube seed fraction is not particularly limited, and examples include lower alcohols (e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol), polyhydric alcohols that are liquid at room temperature (e.g., 1,3-butylene glycol, propylene glycol, glycerin), ethers (e.g., diethyl ether, propyl ether), esters (e.g., butyl acetate, ethyl acetate), ketones (e.g., acetone, ethyl methyl ketone), aromatic hydrocarbons (e.g., benzene, toluene, xylene), and mixtures thereof. For the concentration process, organic solvents with low boiling points are preferred. Methanol is particularly preferred from the perspective of environmental impact. These organic solvents may also contain water or salts, but for the concentration process, a high organic solvent content is preferred. For example, the organic solvent accounts for 70% by mass or more, preferably 90% by mass or more, more preferably 95% by mass or more, and even more preferably 99% by mass or more of the total solution containing the organic solvent.

[0018] 2. Process (A) The aromatic synthetic adsorbent used in step (A) is characterized by its strong binding to compounds having double bonds or aromatic rings, and examples of such adsorbents include styrene-divinylbenzene synthetic adsorbents and ethylvinylbenzene synthetic adsorbents. Known synthetic adsorbents include the Diaion (registered trademark) HP series (manufactured by Mitsubishi Chemical Corporation), the Sepabeads (registered trademark) SP series (manufactured by Mitsubishi Chemical Corporation), and the Amberlite (registered trademark) XAD series (Organo Corporation). In particular, it is preferable to use a styrene-divinylbenzene synthetic adsorbent in view of the adsorption ability of the aromatic synthetic adsorbent to aporphine alkaloids.

[0019] The amount of solvent used in step (A) is preferably 1 mL to 20 mL, more preferably 3 mL to 10 mL, per 1 g of aromatic synthetic adsorbent. The adsorption conditions are not particularly limited as long as they allow sufficient adsorption, but from the viewpoint of efficiency, it is preferable to concentrate the adsorption step by vacuum concentration or the like. In such cases, it is preferable to keep the pressure under conditions that do not cause bumping. The extraction temperature is preferably above the boiling point of the solvent, more preferably 25°C or higher, while, to avoid thermal decomposition of the target substance, it is preferably below 60°C, more preferably below 30°C.

[0020] 3. Process (B) The aqueous solvent used in the washing step (B) is not particularly limited and may contain an organic solvent or a salt, but a high water content is preferred to prevent elution of the aporphine alkaloid. For example, the water content of the aqueous solvent is 70% by mass or more, preferably 90% by mass or more, more preferably 95% by mass or more, and even more preferably 99% by mass or more.

[0021] The washing conditions are not particularly limited as long as they allow sufficient washing, but for example, the amount of aqueous solvent used in one washing step is preferably 1 mL to 20 mL, more preferably 5 mL to 15 mL, per 1 g of aromatic synthetic adsorbent. The washing operation is preferably carried out three or more times.

[0022] Furthermore, the washing step may include washing with an organic solvent to remove fat-soluble impurities. The organic solvent used for washing is not particularly limited, but preferably has low affinity with aporphine alkaloids in order to prevent the aporphine alkaloids from leaking out. Examples of such organic solvents include hexane, ethyl acetate, and a mixture thereof.

[0023] 4. Process (C) The organic solvent used in the elution step (C) is not particularly limited, but is preferably one that does not deteriorate the basic functional group-modified chromatographic silica gel used in step (D). Such solvents are those commonly used in chromatography, such as methanol. The amount of solvent used in elution is preferably 1 mL to 3 mL, more preferably 0.5 mL to 10 mL, per 1 g of aromatic synthetic adsorbent. The elution conditions are not particularly limited as long as they ensure sufficient elution. For example, the elution time is preferably 0.1 to 3 hours, more preferably 0.5 to 1 hour. A shorter reaction time reduces costs and also reduces the risk of decomposition of the aporphine alkaloid. The elution temperature is preferably 10°C or higher, more preferably 25°C or higher, and is preferably below the boiling point of the solvent, more preferably 30°C or lower. A higher heating temperature allows for sufficient elution of the aporphine alkaloid, while a lower heating temperature can avoid the risk of decomposition of the aporphine alkaloid. The dissolution means is not particularly limited, but for example, ordinary means such as immersion, ultrasonic waves, microwaves, stirring, etc. can be used.

[0024] In the present invention, the aromatic synthetic adsorbent can be removed from the system by known solid-liquid separation methods, i.e., techniques such as filtration and centrifugation. Filter materials that can be used include filter paper, glass fiber filters, membrane filters, and filter plates. Furthermore, even when the aromatic synthetic adsorbent is packed in a column, the washing step (B) and the elution step (C) can be carried out.

[0025] When the eluate is obtained using methanol in step (C), it can be directly adsorbed onto the basic functional group-modified chromatographic silica gel in step (D). When adsorbed onto the basic functional group-modified chromatographic silica gel in step (D), the eluate may be concentrated for efficiency reasons. Such concentration can be achieved by vacuum concentration or the like.

[0026] 5. Process (D) The basic functional group-modified chromatographic silica gel used in step (D) is silica gel whose surface functional groups are modified with an amine compound, and is suitable for relatively rapid separation of aporphine alkaloids. Examples of such basic functional group-modified chromatographic silica gel include those modified with isopropylamine.

[0027] In the separation step of step (D), although there is no particular limitation, for example, a purified composition can be obtained by using a mixture of dichloromethane and methanol with an appropriate ratio.

[0028] The purified composition obtained by the present invention contains a jujube seed extract, which contains at least one aporphine alkaloid and an additional compound, and the content of the additional compound in the entire purified composition is 90% by mass or less, preferably 80% by mass or less. The additional compound may include, for example, a cyclic peptide alkaloid having a basic functional group, a nucleic acid base, or a basic amino acid.

[0029] 6. Purified composition containing jujube seed extract The present invention also encompasses a purified composition containing the jujube seed extract produced by the production method of the present invention.

[0030] The purified composition of the present invention comprises at least one compound selected from the group consisting of fujitin, N,N-dimethyllindocarpin, and magnoflorine, and an additional compound; The content of the additional component in the composition is 90% by mass or less.

[0031] The purified composition obtained by the production method of the present invention contains a high proportion of at least one aporphine alkaloid selected from the group consisting of fujitin, N,N-dimethyllindocarpine, and magnoflorine, and is therefore suitable for use in obtaining a purified aporphine alkaloid. When the purified composition obtained by the present invention is used, a purified aporphine alkaloid can be obtained more easily and in a shorter time than known methods. The purification method is not particularly limited, and known methods such as column chromatography can be used. Examples of column chromatography include a method using a reversed-phase packing material that achieves reversed-phase partitioning separation by hydrophobic interaction. [Example]

[0032] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples in any way.

[0033] [Example 1] yield 2000 g of jujube kernels (Tochimoto Tenkaido) were defatted with n-hexane at 30°C. The residue was dried and then sonicated in methanol to obtain 119.1 g of extract. The extract was dissolved in methanol, 500 g of Diaion HP20 (Mitsubishi Chemical) was added, and the mixture was concentrated under reduced pressure. 2500 g of HP20 was packed into a column, and the HP20 adsorbed extract was added. The column was washed with 3 L of n-hexane, ethyl acetate, and then 20 L of water. After washing, the column was extracted with 6 L of methanol to obtain 7.3 g of HP20-treated extract. The HP20-treated extract was eluted stepwise with dichloromethane:methanol, 1:0.1 to 1:3, on basic functional group-modified chromatographic silica gel DM1020 (Fuji Silysia), to obtain 3.1 g of purified product. The purified composition was eluted with a reversed phase packing material COSMOSIL 140C18-OPN (Nacalai Tesque) using methanol:water (1:0.25 to 1:1) to isolate 0.12 g of fujitin, 0.22 g of N,N-dimethyllindocarpine, and 0.18 g of magnoflorine. Similarly, 2000 g of jujube kernel (Tochimoto Tenkaido) was defatted with n-hexane at 30°C, and the residue was dried and then ultrasonically treated with methanol to obtain an extract. The contents of fujitin, N,N-dimethyllindocarpine, and magnoflorine in this extract were below the lower limit of quantification.

[0034] [Example 2] Comparison of synthetic aromatic adsorbents The yield was compared with that of aromatic synthetic adsorbents Diaion HP20 and HP21 (Mitsubishi Chemical) and AMBERLITE XAD-2 (Organo). 1 g of jujube kernel (Tochimoto Tenkaido) was sonicated at room temperature in 10 mL of methanol to obtain an extract. The extract was dissolved in methanol, and 1 g of each aromatic synthetic adsorbent was added and concentrated under reduced pressure. The adsorbent with the adsorbed extract was washed with 10 mL of water and eluted with 2 mL of methanol. The methanol eluate from the adsorbent was then adsorbed onto 0.5 g of basic functional group-modified chromatographic silica gel DM1020 (Fuji Silysia), concentrated under reduced pressure, and eluted with a 1:1 mixture of dichloromethane and methanol to obtain a purified composition. The purified composition was dissolved in methanol to a concentration of 10 mg / mL, filtered through a 0.45 μm PTFE filter, and the magnoflorine concentration was measured by HPLC. The magnoflorine concentration was 40 μM or higher with each aromatic synthetic adsorbent.

Claims

1. A method for producing a purified composition containing jujube seed extract, comprising: The jujube seed extract is a compound represented by the formulas (1), (2) and (3): 【Chemical 1】 and an additional compound, the content of the additional compound in the entire purified composition is 90 mass% or less, (A) contacting an extract of the jujube seed fraction containing the jujube seed extract with an aromatic synthetic adsorbent, thereby adsorbing the composition containing the jujube seed extract onto the aromatic synthetic adsorbent; (B) washing the composition adsorbed onto the aromatic synthetic adsorbent in step (A) with an aqueous solvent; (C) a step of eluting the composition washed in the step (B) from the aromatic synthetic adsorbent using an organic solvent to obtain an eluate; (D) obtaining the purified composition from the eluate obtained in step (C) by separating the purified composition using a basic functional group-modified chromatographic silica gel; A manufacturing method comprising:

2. The jujube seed fraction is jujube seeds defatted with a degreasing agent, or Jujube seed water extract The method according to claim 1, wherein the extract is an organic solvent extract of the above.

3. The method of claim 2 wherein the degreasing agent is hexane.

4. The method according to claim 1 , wherein the organic solvent is an alcohol.

5. The method according to claim 4, wherein the alcohol is methanol.

6. The method according to claim 1 , wherein the aqueous solvent is water.

7. The method according to any one of claims 1 to 6, wherein the aromatic synthetic adsorbent is a styrene-divinylbenzene synthetic adsorbent.

8. The method according to any one of claims 1 to 6, wherein the terminal of the basic functional group-modified chromatographic silica gel is modified with isopropylamine.

9. A composition comprising a jujube seed extract, The jujube seed extract is represented by the formulas (1), (2) and (3): 【Chemistry 2】 and an additional compound, A composition, wherein the content of the additional component in the composition is 90 mass % or less.

10. The composition according to claim 9, for purifying at least one aporphine alkaloid selected from the group consisting of compounds represented by the formulas (1), (2), and (3).

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