JPH09188628A - Separation and purification of pilocarpine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To directly separate and purify highly pure pilocarpine at a low cost from a plant belonging to the genus Pilocarpus by a specific highly simple means. SOLUTION: This method for separating and purifying pilocarpine comprises extracting the leaves of a plant belonging to the genus Pilocarpus in the family Rutaceae with water and/or an organic solvent, concentrating the extract, and subsequently subjecting the concentrated extract to column chromatography at least twice. The plant belonging to the genus Pilocarpus is preferably Pilocarpus jaborandi or Pilocarpus microphyllus. Different kinds of the chromatography are preferably used on the separation and the purification. The first column chromatography is preferably ion exchange chromatography or adsorption chromatography. A filler for the column chromatography is preferably an ion exchange resin, a cellulose ion exchange resin, etc. A filler for the second column chromatography is preferably silica gel, alumina, activated carbon, etc., especially alumina.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for easily separating and purifying pilocarpine.

[0002]

2. Description of the Related Art Plants of the genus Pilocarpus of the citrus family, such as Pilocarpus jabo
randi) is an evergreen shrub that is mostly found in Central and South America, especially Brazil, and its leaves are mainly used as a raw material for the production of pilocarpine (perspirants, miotics).

As a method for producing pilocarpine, an extraction method has been established in which the leaves of Yabolangi leaves are subjected to complicated steps, but the yield as a hydrochloride is low at about 0.31% (11th revision). Japanese Pharmacopoeia Manual C-489-492
Page, Matsuharu Hirayama et al .: Pharmaceutical Journal, 38 (438), 644 (19)
18)). Further, as a method for producing pilocarpine, the methods described in JP-A-4-341192, JP-A-7-258102 and the like are also known, but all of them utilize plant tissue culture, and pilocarpus plants. At present, sufficient isolation and purification of pilocarpine directly from sucrose has not been sufficiently studied.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for easily separating and purifying pilocarpine directly from pilocarpus plants.

[0005]

[Means for Solving the Problems] To achieve the above object,
As a result of intensive studies to develop an excellent method for separating and purifying pilocarpine, the present inventors extracted leaves of a plant belonging to the citrus family Pilocarpus with water and / or an organic solvent, and extracted the extract. The present invention has been completed by finding that pilocarpine can be easily separated and purified by subjecting the extract obtained by concentration to column chromatography at least twice. That is, the present invention includes the following inventions.

(1) Rutaceae Pilocarpus
Separation of pilocarpine characterized by extracting leaves of a genus plant with water and / or an organic solvent, concentrating the extract and subjecting the extract to column chromatography at least twice to separate and purify pilocarpine. -Purification method. (2) The plant of the citrus family Pilocarpus is Pilocarpus jaborandi or Pilocarpus microphyllus.
The method according to (1) above.

(3) The method according to (1) above, characterized in that different types of column chromatography are combined. (4) The method according to (3) above, wherein the first column chromatography is ion exchange chromatography or adsorption chromatography.

(5) The method according to (4) above, wherein the packing material for column chromatography is an ion exchange resin, a cellulose ion exchanger, a Sephadex ion exchanger or a porous polymer. (6) The method according to any one of (3) to (5) above, wherein the packing material for the second column chromatography is silica gel, alumina, activated carbon or Celite. (7) The method according to (6) above, wherein the packing material for the second column chromatography is alumina.

Pilocarpus used in the present invention
The genus plant is not particularly limited as long as it contains pilocarpine in its leaves, for example, Pilocarpus jaborandi (Pilocarpus jaborandi), Pilocarpus microphyllus (Pilocarpus microphyllus), Pilocarpus pen
natifolius (Pilocarpus selloanus), Pilocarpus racem
osus, preferably Pilocarpu
s jaborandi), Pilocarps Microfils
pus microphyllus). As the leaves of the plant, dried leaves or fresh leaves may be used.

A preferred method for extracting the plant is a method in which the plant is appropriately cut or crushed and then extracted with a suitable solvent at room temperature to under heating, which is used. As the extraction solvent, water; lower alcohols (including water) such as methyl alcohol and ethyl alcohol; glycerin, propylene glycol, 1,
Examples include polyhydric alcohols such as 3-butylene glycol; lower carboxylic acid esters such as ethyl acetate; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; and one or more of them can be used. Among these, it is preferable to use water or a water-soluble solvent, particularly water or a mixed solvent of two or more kinds of ethyl alcohol. As the extraction conditions, the above-mentioned extraction solvent is added to the plant in an amount of about 2 to 150 times, preferably 5 to 30 times, and heated at room temperature or for several hours to several days, particularly at room temperature for 1 day or more. If heated, it is preferable to perform extraction while appropriately stirring for 1 hour or more.

The extract obtained under the above-mentioned conditions is concentrated, subjected to a treatment such as filtration, and then subjected to column chromatography to be adsorbed on the packing material. In the present invention, “column chromatography” has a broad meaning and refers to all chromatography using a column as a separation bed.

In the present invention, it is necessary to carry out column chromatography at least twice, and at this time, it is preferable to combine different types of column chromatography. Particularly, the first column chromatography is ion exchange chromatography. More preferably, it is a chromatography or adsorption chromatography, and the packing material for the second column chromatography is silica gel, alumina, activated carbon or celite.

As the packing material for the first column chromatography, there are used ion exchange resins (strongly acidic cation exchange resin, weakly acidic cation exchange resin, strongly basic anion exchange resin, weakly acidic anion exchange resin, porous resin). Ion exchange resins), cellulose ion exchangers, sephadex ion exchangers, and porous polymers are preferable, and porous polymers are particularly preferable. Alumina is particularly preferable as the packing material for the second column chromatography.

[0014]

Preferred embodiments of the present invention will be described below. Pilocarpu
s jaborandi), Pilocarps Microfils
(Pus microphyllus), etc. are appropriately cut or crushed, and water, one or more mixed solvents of ethyl alcohol, etc. are added to the dry leaves in an amount of 5 to 30 times, and the mixture is heated at room temperature or several times. Extract for a few days from the time, particularly 1 day or more at room temperature, and 1 hour or more if heated, with moderate stirring.

Then, the obtained extract is concentrated, and the first column chromatography is carried out using a porous polymer as a packing material which has been subjected to a treatment such as filtration.
Further, the adsorbed substance is eluted with an organic solvent such as ethanol, methanol or acetone. The eluate is concentrated under reduced pressure and subjected to a second column chromatography using alumina as the packing material. The adsorbed product is finally eluted with methanol or the like and separated and purified.

The used porous polymer can be repeatedly used after being washed with a dilute alkaline aqueous solution to remove the insoluble portion and pigments of the adsorbed extract. The use of pilocarpine separated and purified by the method of the present invention is not particularly limited, and it can be used, for example, as a medicine.

[0017]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. (Example 1) Pilocarpus javolandi
borandi) dried leaves 1.5 kg 6 with 10 L water
The mixture was extracted with stirring under heating (70 to 100 ° C.) for an hour. Next, the water extract was distilled under reduced pressure to remove the solvent and freeze-dried to obtain a water extract (hereinafter abbreviated as "YGW", yield 13
(7.1 g, yield 9.14%) was obtained. The YGW (120 g) was dissolved in water (1.2 L) and subjected to the first column chromatography (filler; porous polymer gel (Mitsubishi Chemical, Diaion HP-20) 1.2 kg) for adsorption.
It was eluted with 8 L of methanol, and the eluate was concentrated under reduced pressure to obtain a fraction (hereinafter abbreviated as “YGW-HP”) (weight 31.16 g, recovery rate 26%).

31.16 g of YGW-HP was dissolved in methanol and the second column chromatography (filler;
Alumina (2000 g) was adsorbed. It was eluted with 3.2 L of methanol and 4.75 g of pilocarpine
(0.32% based on the dry leaf of the raw material) was isolated. The used porous polymer gel is washed repeatedly with a 5% aqueous potassium hydroxide solution to remove the insoluble portion and pigments of the adsorbed extract, and then repeatedly used.

[0019]

According to the present invention, pilocarpine can be easily separated and purified, so that pilocarpine with high purity can be provided at low cost.

Claims (7)

[Claims] 1. Extract of leaves of a plant of the genus Pilocarpus belonging to the citrus family Pilocarpus with water and / or an organic solvent, and the extract obtained by concentrating the extract is subjected to column chromatography at least twice to separate pilocarpine. A method for separating and purifying pilocarpine, which comprises purifying. 2. A plant of the citrus family Pilocarpus is Pilocarpus jaborandi.
Or Pilocarpus micropili (Pilocarpus microp
hyllus). 3. Method according to claim 1, characterized in that different types of column chromatography are combined. 4. The method according to claim 3, wherein the first column chromatography is ion exchange chromatography or adsorption chromatography. 5. The method according to claim 4, wherein the packing material for column chromatography is an ion exchange resin, a cellulose ion exchanger, a Sephadex ion exchanger or a porous polymer. 6. The method according to claim 3, wherein the packing material for the second column chromatography is silica gel, alumina, activated carbon or Celite. 7. The method according to claim 6, wherein the packing material for the second column chromatography is alumina.