JPH08208651A - Production of scoulerine - Google Patents

Abstract

PURPOSE: To readily and stably obtain scoulerine, having antioxidant activities and useful as a sedative, an antispasmodic, a hypotensive agents, etc., from an easily available raw material without using a papaveraceous plant by extracting Nandina domestica Thunb. with a polar solvent. CONSTITUTION: A component soluble in a polar solvent is initially extracted from Nandina domestica Thunb. by a method for allowing the Nandina domestica Thunb. to stand in a polar solvent a whole day and night while occasionally stirring, e.g. the polar solvent and the Nandina domestica Thunb. and then filtering the resultant extract. A basic component is subsequently extracted from the component soluble in the polar solvent according to a fractionation method, etc., utilizing, e.g. dissociating properties due to a difference in pH and finally treated by a chromatography (preferably the high-performance liquid chromatography) to isolate scoulerine. Furthermore, ethanol is preferred as the polar solvent used for extracting the Nandina domestica Thunb. Components, contained in the Nandina domestica Thunb. and soluble in the nonpolar solvent are preferably preremoved with a nonpolar solvent such as hexane before the operation in order to efficiently carry out the extracting operation with the polar solvent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention has scourelin [(R or S) -5,8,13,13a- which has antioxidant activity and can be used as a medicine for sedation, antispasmodic action, hypotensive action and the like.
It relates to a method for producing tetrahydro-3,10-dimethoxy-6H-dibenzo [a, g] quinolizine-2,9-diol].

[0002]

BACKGROUND OF THE INVENTION Scoulerine is one of the plant alkaloids of isoquinolines whose existence is recognized in poppy plants and is a substance having physiological activities such as sedative action, antispasmodic action and antihypertensive action. It is also a useful substance as a synthetic intermediate for various physiologically active substances. Recently, isoquinoline such as scourelin has been expected to be useful as an anticancer agent.

Conventionally, such useful scoulerine has been
Poppy plants, for example, whole grass containing poppy fruits is collected as a raw material, and isolated from the collected plants by known separation methods such as an extraction method, a fractionation method, and a chromatography method,
It is manufactured by purification.

[0004]

However, since the poppy plant contains the narcotics morphine, its cultivation is severely restricted worldwide, and it is extremely difficult to obtain the raw material of the poppy plant. There's a problem.

For this reason, it is possible to produce scourelin by organic synthesis, but it is expected that the number of production steps will be very large and the total yield will be low, and therefore the production cost will be significantly increased.

The present invention is intended to solve the above problems, and an object of the present invention is to make it possible to easily produce scourelin from readily available raw materials.

[0007]

The present inventor has evaluated the antioxidant activity of substances obtained from a huge variety of plants and found that the readily available Nanten (Nandina d
omestica Thunb) leaves contain a substance with strong antioxidant activity, and found that the substance is scourelin, so if nanten is used as a raw material, scourelin can be obtained without the use of poppies. They have found that they can be manufactured and have completed the present invention.

That is, the present invention relates to a method for producing scoulerin: (a) a step of extracting a polar solvent-soluble component from nanten; (b) a step of extracting a basic component from the obtained polar solvent-soluble component; and ( c) A method for producing scourelin, comprising the step of isolating scourelin from the basic component by a chromatography method.

The present invention will be described in detail below.

Step (a) In the method for producing scoulerin of the present invention, scourelin is firstly converted from nanten as a raw material by utilizing the property that scourelin is hardly soluble in a non-polar solvent but easily soluble in a polar solvent. A polar solvent-soluble component containing is extracted.

Here, the raw material, Nanten, is a plant that grows naturally in the warm mountains of Japan and is so widely used that it can be seen as a garden tree. . In the present invention, it is preferable to use the leaf of Nanten as a raw material. This is because the leaves contain scourelin in a relatively high concentration, and even if the leaves are collected to some extent, the leaves can be collected again without dying the nanthene. When Nanten leaves are used as a raw material, either raw leaves or dried leaves (for example, dried in warm air at 50 ° C. or lower or shade dried) can be used. When using fresh leaves, it is preferable to grind them with a homogenizer or a mortar, and when using dry leaves, it is preferable to grind them.

The operation of extracting the polar solvent can be performed by a known operation, for example, by allowing the polar solvent and nanten to stand overnight at room temperature with occasional stirring, and then filtering. The obtained filtrate becomes a polar solvent extract.

It is also possible to perform the polar solvent extraction operation again on the filtered off nanten, and it is usually preferable to perform the extraction operation at least three times.

The obtained polar solvent extract can be used as it is in the liquid state in the next step (b), but normally, the polar solvent is distilled off under reduced pressure and dried to a solid or paste form. Is preferred.

As the polar solvent used in the above-mentioned polar solvent extraction operation, lower alcohols such as methanol and ethanol, lower ketones such as acetone and the like can be used alone or in combination. Especially, it is preferable to use ethanol.

In order to efficiently carry out the above-mentioned polar solvent extraction operation, prior to the polar solvent extraction operation, the non-polar solvent-soluble component contained in Nanten is extracted and removed with a non-polar solvent such as hexane or dichloromethane. Preferably.

(Step b) Next, a basic component containing scourelin is extracted from the polar solvent-soluble component obtained in step (a). This is because the polar solvent-soluble component contains at least a basic component containing scoulerin, a neutral component, and an acidic component, and therefore it is necessary to separate the basic component containing scoulerin from other components. Because there is.

The basic component is extracted from the polar solvent-soluble component obtained in step (a) by, for example, pH.
It can be performed by a fractionation method utilizing dissociation due to the difference between

As a concrete operation of this fractionation method, first,
Water is added to the polar solvent-soluble component to form a uniform solution or dispersion while heating as necessary, and the solution is usually pH 2-5 using an alkali such as sodium hydroxide or an acid such as hydrochloric acid, Preferably pH 2.5-3.
5, more preferably adjusted to pH 3, and the liquid is subjected to extraction treatment using an organic extraction solvent such as chloroform or ethyl acetate. As a result, the basic component containing scoulerin is transferred to the aqueous phase, and the neutral component and the acidic component are transferred to the organic extraction solvent phase.

Next, in order to allow the basic component containing scoulerin to be extracted from the aqueous phase into an organic extraction solvent such as chloroform or ethyl acetate, an alkali such as sodium hydroxide is added to the obtained aqueous phase. The pH of the aqueous phase is alkaline, usually pH 10 to 14, preferably pH 11.5.
˜12.5, more preferably pH 12.

Next, the basic component containing scourelin is extracted from the alkaline aqueous phase with an organic extraction solvent.

The organic solvent extract containing the basic component thus obtained can be used in the next step (c) in a liquid state, but the extract solvent is usually distilled off under reduced pressure to give a solid or paste form. It is preferable to dry it.

Step (c) Next, scourelin is isolated from the basic component obtained in step (b) by a chromatography method. in this case,
As the chromatography method, a known chromatography method can be used, and among them, the high performance liquid chromatography method is preferably used.

For example, the basic component obtained in the step (b) in a concentrated or dried state can be isolated by dissolving it in a small amount of ethanol and applying the solution to high performance liquid chromatography. In this case, the separation conditions such as the type of eluent and packing, the column diameter, the column length, and the flow rate of the eluent can be appropriately determined. In addition, such a chromatographic treatment may be performed a plurality of times instead of once.

The scoulerin produced by the above-mentioned method of the present invention has the same structure and properties as those isolated from poppy plants, and can be used for the same applications.

[0026]

In the method for producing scoulerin of the present invention,
Nanten is used as raw material from which scourelin is isolated. Nanten, which is a raw material, is a plant that is widely grown or cultivated in Japan and can be easily and stably obtained in large quantities. Therefore,
According to the present invention, it becomes possible to easily and stably produce scourelin in large quantities.

[0027]

The present invention will be described in detail below with reference to the following examples.

Example First, polar solvent-soluble components were fractionated from Nanten according to the extraction operation flowchart shown in FIG.

That is, warm air dried Nanten leaf powder 6
55 g was placed in a flask, 1 liter of hexane was further added, and the mixture was left overnight at room temperature with occasional stirring to extract a non-polar soluble component in hexane and then filtered. The residue (the filtered powder of nanten leaves) was subjected to the same operation twice using hexane and then three times using dichloromethane. This removed non-polar solvent soluble components from the leaves of Nanten.

To the leaves of Nanten from which the non-polar solvent-soluble components were removed, 1 liter of ethanol was added, and the mixture was extracted with ethanol three times as in the case of hexane, and the extracts were combined and dried under reduced pressure. As a result, 175.2 g of a solid ethanol-soluble component (polar component) was obtained.

Next, a basic component containing scoulerin was fractionated from the obtained polar components.

That is, about 1 liter of distilled water was added to this solid substance to dissolve it with heating, and then the pH of the solution was adjusted to
The pH was adjusted to 3 while appropriately using sodium hydroxide and hydrochloric acid. The obtained adjusted liquid was placed in a separating funnel, and about 200 ml of chloroform was further added for extraction and then allowed to stand to separate into an aqueous phase A and a chloroform phase B. This operation was repeated 3 times in total, and the chloroform phases B were combined.

The pH of the obtained aqueous phase A was adjusted to pH 12, and the chloroform phases obtained by similarly extracting three times with chloroform were combined and dried under reduced pressure. As a result, 2.01g
The basic component of was able to be fractionated.

On the other hand, a saturated aqueous solution of sodium hydrogencarbonate (about pH 9) was added to the chloroform phase B, and the mixture was extracted with chloroform to separate an aqueous phase C and a chloroform phase D. The aqueous phase C was further extracted twice with chloroform, and the obtained chloroform phase was combined with the above chloroform phase D.

The aqueous phase C was adjusted to pH 3 and then extracted with chloroform, and the chloroform phase was dried under reduced pressure.
As a result, 0.77 g of the acidic component could be fractionated.

The chloroform phase D was adjusted to pH 12 with 1N.
After adjusting with an aqueous solution of caustic soda, the mixture was extracted with chloroform and separated into an aqueous phase E and a chloroform phase F. And
The aqueous phase E was adjusted to pH 6 and then extracted with chloroform, and the chloroform phase was dried under reduced pressure. as a result,
It was possible to fractionate 0.32 g of a weakly acidic component. The chloroform phase F was also dried under reduced pressure. as a result,
11.15 g of neutral component could be fractionated.

The basic component, neutral component, weakly acidic component and acidic component were subjected to the following antioxidant activity test, and the basic component and weakly acidic component had antioxidant activity. It was

(Antioxidant Activity Test) The antioxidant activity test is carried out by the known thiocyanation method as a general antioxidant B.
It was carried out by comparing with HT (Butylated hydroxy toluene) and γ-tocopherol. Specifically, the fractionated component obtained from Nanten was dissolved in ethanol, and 0.1 ml thereof was added to ethanol (99.5%) 1.90 ml.
And ethanol solution of linoleic acid (0.25%) 2.05m
and phosphate buffer (0.05 M, pH 7.0) 4.0
0 ml and 1.95 ml of distilled water were placed in an Erlenmeyer flask having a capacity of 50 ml, placed in an incubator at 40 ° C., and 0.1 ml of a sample was taken after a predetermined period of time, and 75% ethanol was added to the sample. Place in a wide-mouthed vial (33 ml capacity) containing 9.7 ml, and further add 0.1 ml of an aqueous solution of ammonium thiocyanate (30%) and ferrous chloride (2 ×).
0.1 ml of 10 ⁻² M, 3.5% hydrochloric acid aqueous solution) was added to develop color, and 3 minutes later, the absorbance was measured at 500 nm.

By the way, since scoulerine is a kind of alkaloid, it is expected that it is fractionated into basic components. Therefore, scourelin was then isolated from the basic components by high performance liquid chromatography.

That is, the basic component dried under reduced pressure was dissolved in a small amount of 99.5% ethanol, and a part of the solution (2
0 μl, 0.27 mg) as a sample,
High-performance liquid chromatography (“Chromatograph LC800 series” manufactured by JASCO Corporation) using 0% methanol (methanol / water, V / V) as an eluent, used column (Inertsil prep-ODS, 30 × 250 mm), flow rate (2.0 ml
/ min), and detector UV), and 5 fractionation (F-1 to F-2) was performed as shown in FIG.

Next, the fraction component F-2 having a high antioxidant activity
40% methanol (methanol / water, V / V)
Was also used as an eluent and was similarly separated by high performance liquid chromatography, and as shown in FIG.
1 to F-2-5). Among them, the fraction component having particularly high antioxidant activity is the fraction component shown in F-2-1, and this component was analyzed by elemental analysis, MS and NMR. As a result, this component has the formula (1)

[0042]

Embedded image Having a structure of scourelin [(R or S) -5,
It was confirmed that it was 8,13,13a-tetrahydro-3,10-dimethoxy-6Hdibenzo [a, g] quinolizine-2,9-diol]. The following identification data are shown.

High resolution FAB-MS: m / z = 328
(Exact mass 328.1554) Molecular ^{_{formula: C 19 H 22 NO 4 1}} H and ¹³ C-NMR: (Table 1)

[0044]

[Table 1] Attribution of ¹ H Attribution of ¹³ C Hydrogen chemical shift Coupling constant COSY H number HMBC Carbon chemical shift HMQC (ppm) (Hz) (ppm) a 2.63 b, e 1 G A 29.2 b, d b 2.66 a, d 1 I, M B 36.3 c, f c 2.82 f, h 1 G, O C 51.6 a, e d 3.13 b, e 1 D 53.5 g, k e 3.19 a, d 1 G E 55.9 i f 3.25 c 1 F 56.2 j g 3.50 k 1 G 59.2 h h 3.53 c 1 C, L H 109.0 p i 3.87 3 R I 110.7 n j 3.88 3 S J 111.4 q k 4.23 g 1 G, L, N, P K 119.4 o l 5.49 1 L 121.3 m 5.65 1 M 126.2 n 6.60 1 A, O, Q, S N 128.2 o 6.66 8.24 P 1 B, L, R O 130.7 p 6.73 8.24 O 1 N, P P 141.5 q 6.83 1 G, M, Q, S Q 143.9 R 144.0 S 145.1

The direct bond between the proton and carbon is H
MQC (Proton-detected Heteronuclear Multiple Quan
tum Coherence method. The spin-coupled protons are ¹ H- ¹ H DQF-COSY ( ¹ H- ¹ H Double
Quantum Filtered Correlation Spectroscopy) method was used for analysis. The relationship between protons and carbon in the long range is based on HMBC (Proton-detected Heteronuclear Multipl
It was analyzed by the e-Bond Correlation Spectroscopy method.
In addition, the determination of Q and R carbons is based on NOE (Nuclear Overhau
ser effect) method. In addition, carbon E and carbon F may be attributed to each other.

[0046]

EFFECTS OF THE INVENTION According to the production method of the present invention, scourelin having sedative, antispasmodic, hypotensive and antioxidant activities can be easily and easily obtained in a large amount and stably from Nanten which is a plant. It can be manufactured in large quantities and stably.

[Brief description of drawings]

FIG. 1 is a flow chart of an extraction operation for fractionating a basic component containing scoulerin from nanten leaf powder.

FIG. 2 is a chart diagram when the fractionated basic component is separated by high performance liquid chromatography.

FIG. 3 is a chart diagram when the fractionated basic component is separated by high performance liquid chromatography.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location B01D 15/08 // C09K 15/30

Claims (5)

[Claims] 1. A method for producing scoulerin: (a) a step of extracting a polar solvent-soluble component from nanten; (b) a step of extracting a basic component from the obtained polar solvent-soluble component; and (c) this base. A method for producing scourelin, comprising a step of isolating scourelin from a sex component by a chromatography method. 2. The method for producing scourelin according to claim 1, wherein a lower alcohol is used as an extraction solvent in the step (a). 3. The method for producing scourelin according to claim 2, wherein the lower alcohol is ethanol. 4. The method for producing scoulerin according to claim 1, wherein the chromatography method is a high performance liquid chromatography method. 5. The method for producing scourelin according to claim 1, further comprising a step of previously removing a non-polar solvent-soluble component from nanten prior to the polar solvent extraction.