HK1180552B - Production method of an extract containing hydroxy sanshool - Google Patents

Description

Preparation method of extract containing hydroxy sanshool

Technical Field

The invention relates to a method for effectively extracting hydroxy sanshool from zanthoxylum plants. More specifically, the present invention relates to a method for producing a hydroxysanshool-containing extract containing hydroxysanshool in a high concentration by extracting an extracted oil of zanthoxylum such as a peel oil or a fruit oil of zanthoxylum using an ethanol aqueous solution of a specific concentration.

Background

Hydroxysanshool is a pungent ingredient specifically contained in Zanthoxylum plants and having strong paralytic effect, and is represented by the following formula (1)

Alpha-hydroxy sanshool and the following formula (2)

The N-isobutylalkenylamide has a structure in which a hydroxyl group is bonded to a tertiary carbon of an isobutyl group at the end, and is represented by β -hydroxysanshool.

The pungent taste property of hydroxysanshool is expressed by "pungency (ヒリヒリ) and paralysis" in the case of α -hydroxysanshool and "paralysis, astringency, and bitterness" in the case of β -hydroxysanshool, and is different from the pungent taste property with a pungent feeling called "hot (ピリピリ)" of sanshool (non-patent document 1).

It is known that hydroxysanshool has effects as not only a pungent component but also an anesthetic (patent document 1), a digestive tract motility improving agent (patent document 2), a memory promoter (patent document 3), and the like, and that the effects can be obtained by purifying and adding a small amount of a high-purity extract containing hydroxysanshool.

As a method for purifying hydroxysanshool from an extract oil of zanthoxylum, several methods have been proposed. Examples thereof include: a method of purifying an alcohol extract of a plant belonging to the genus capsicum by appropriately combining normal phase chromatography and reverse phase chromatography (patent document 1); a method of purifying an aqueous extract of dried zanthoxylum bungeanum (zanthoxylum piperitum) powder by column chromatography (patent document 3); a method in which zanthoxylum bungeanum is subjected to steam distillation, and the obtained essential oil is fractionated by column chromatography (non-patent document 2), and the like. However, these methods using column chromatography have problems in terms of yield and complexity of operation, and are not suitable for industrial production.

On the other hand, several methods have been proposed for obtaining an amide compound-containing component by extracting a zanthoxylum plant with an alcohol. For example, a method of extracting zanthoxylum with an aqueous or non-aqueous lower alcohol (monohydric alcohol having 1 to 4 carbon atoms) (patent document 1); a method in which the pericarp of zanthoxylum bungeanum is immersed in ethanol at room temperature for 1 week, and the ethanol is distilled off from the immersion liquid to obtain a zanthoxylum bungeanum extract (patent document 4); a method in which dried ripe pericarp of zanthoxylum bungeanum is cut up, heated to 50 ℃ in ethanol, and extracted for 5 hours (patent document 5); a method in which zanthoxylum bungeanum maxim is treated with a solvent such as hexane or aqueous alcohol to extract aromatic components and pungent components, and then the solvent is removed, thereby obtaining a zanthoxylum bungeanum maxim extract (patent document 6), and the like. However, in these methods, components other than hydroxycamptothecin, for example, high boiling point components such as terpenes and triglycerides are also extracted at the same time, and thus a high-purity hydroxycamptothecin-containing component cannot be obtained with high efficiency.

As described above, the current situation is: a method for efficiently purifying and obtaining a hydroxysanshool component from an extract oil of zanthoxylum plants by a simple and industrially suitable method has not been developed, and development of a new method is strongly desired.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 1-294657

Patent document 2: japanese patent No. 4627574

Patent document 3: japanese patent No. 4421832

Patent document 4: japanese laid-open patent publication No. 2001-131033

Patent document 5: japanese patent laid-open No. 2001 and 288047

Patent document 6: japanese patent application laid-open No. 2010-115118

Non-patent document

Non-patent document 1: perfumes (2006), No.229, 129-

Non-patent document 2: journal of Agricultural and Food Chemistry (2008), 56(5), 1689-.

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a method for efficiently producing a high-purity hydroxysanshool-containing extract free from high-boiling components such as terpenes and triglycerides from an extract oil of zanthoxylum.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that a high-purity hydroxysanshool-containing extract containing hydroxysanshool at a high concentration can be obtained with high efficiency by extracting an extract oil of zanthoxylum with a specific aqueous ethanol solution having an ethanol concentration of 35 to 65 mass%, thereby completing the present invention.

Thus, the present invention provides a method for producing a hydroxysanshool-containing extract, which comprises: extracting hydroxy sanshool component from the extract oil of Zanthoxylum plant with 35-65 mass% ethanol water solution.

The present invention also provides an extract containing hydroxysanshool obtained by the above production method.

Effects of the invention

According to the present invention, a high-purity hydroxysanshool-containing extract containing hydroxysanshool at a high concentration can be provided at low cost by a method having high industrial versatility and high safety.

The present invention is described in further detail below.

Detailed description of the preferred embodiments

The species of zanthoxylum used in the present invention is not particularly limited, and examples thereof include: japanese prickly ash (Zanthoxylum piperitum), pricklyash peel (Zanthoxylum bungeanum), Zanthoxylum schinifolium (Zanthoxylum schinifolium), Zanthoxylum nitidum (Zanthoxylum nitidum). Japanese prickly ash and prickly ash are particularly preferable.

The extracted oil of zanthoxylum used in the present invention can be obtained by treating the pericarp and/or fruit of zanthoxylum, for example, the pericarp of zanthoxylum, the fruit of zanthoxylum, and the like, by a method known per se, for example, squeezing method, steam distillation method, solvent extraction method, supercritical extraction method, carbon dioxide extraction method, oil adsorption method, and the like, and the obtained extracted oil can contain hydroxysanshool at a concentration of usually 5 to 30% by mass, particularly 15 to 30% by mass.

Specifically, for example, dried pericarp or fruit of zanthoxylum is mixed with water, loaded into a supercritical extraction apparatus, extracted at an extraction temperature of 30 to 80 ℃ and a pressure of 10 to 50MPa using supercritical carbon dioxide containing 1 to 5 mass% of 75 to 99.5 mass% of ethanol as an extraction aid at a flow rate of 1 to 10 Kg/hour for about 30 minutes to about 3 hours, and then the carbon dioxide fluid is introduced into a separation tank and separated at a temperature of 15 to 40 ℃ and a pressure of 2 to 10MPa, thereby obtaining an extracted oil of zanthoxylum.

The extraction of the extract containing hydroxysanshool from the oil extracted from zanthoxylum, for example, can be carried out as follows:

mixing the oil extract of Zanthoxylum plant with ethanol water solution, stirring, and standing for a certain period of time. As a result, the oil containing high boiling point substances such as terpenes and triglycerides is separated into the upper layer, and the aqueous ethanol solution is separated into the lower layer. The separated upper layer oil is removed to remove impurities such as terpenes and high boiling point substances, and the lower layer ethanol aqueous solution is distilled off under reduced pressure, for example, to obtain a hydroxysanshool-containing extract.

The mixing and stirring temperature of the extracted oil of zanthoxylum and the aqueous ethanol solution may be usually 0 to 70 c, preferably 5 to 50 c, whereby the concentration of the hydroxysanshool in the extract containing the hydroxysanshool can be increased. The stirring time may be usually 5 to 120 minutes, preferably 10 to 60 minutes, and further, the stirring intensity is usually 400-1000rpm, particularly preferably in the range of 500-800 rpm. The standing time after stirring is usually 20 to 120 minutes, particularly preferably 25 to 60 minutes, and even if the standing time is prolonged, the yield and the concentration of hydroxysanshool in the extract do not differ greatly.

The ethanol concentration of the ethanol aqueous solution used for treating the extract oil of Zanthoxylum may be 35 to 65 mass%, preferably 40 to 60 mass%, and more preferably 45 to 55 mass%. If the ethanol concentration is less than 35% by mass, oil containing high boiling point substances such as terpenes and triglycerides can hardly be separated from the oil extract of Zanthoxylum species, and the yield of the hydroxycamptothecin-containing extract is lowered, and therefore, it is not suitable as an industrial process for extracting the hydroxycamptothecin-containing extract. On the other hand, if the ethanol concentration exceeds 65 mass%, the terpenes cannot be removed although the high boiling point substances contained in the extracted oil of zanthoxylum are precipitated as solids, and the content of hydroxysanshool in the extract is lowered.

The amount of the ethanol aqueous solution to be used may be usually 5 to 50 times, preferably 7.5 to 40 times, and more preferably 10 to 30 times by mass, based on the amount of the extract oil of Zanthoxylum plant.

The hydroxycamptothecin-containing extract obtained by the above method usually contains at least 70 mass%, preferably 70 to 90 mass%, and more preferably 70 to 85 mass% of hydroxycamptothecin based on the mass of the extract, and can be treated in combination with other purification methods as necessary to increase the content of hydroxycamptothecin in the extract. Examples of the purification method include: distillation, silica gel column chromatography, thin layer chromatography, etc.

The extract containing hydroxy sanshool obtained according to the present invention can be used as a food material, particularly a pungent taste enhancing material, as it is, or can be used as a functional material, for example, a sterilizing agent, a crude drug, a physiologically active substance, etc. If necessary, an emulsifier such as lecithin, glycerin fatty acid ester, sucrose fatty acid ester, etc. may be added to the extract to prepare an emulsified state by homogenization; the extract can be mixed with a powdering aid such as gum arabic, starch, dextrin, xanthan gum, cyclodextrin, etc., and dried by a drying method such as spray drying or vacuum drying to give a powder form.

The hydroxysanshool-containing extract obtained in the present invention can be added to foods such as horseradish, mustard, bruised ginger, minced garlic, curry, thick broad-bean sauce, kimchi, ham, sausage, sauce, stew, instant noodle soup, vegetable soup, tomato sauce, pizza, roast chicken, roast pork, ginger-stewed pork, and roasted eel fillet, for example, to impart a pungent taste-enhancing effect.

The present invention is further specifically illustrated by reference examples, examples and comparative examples. It should be noted that the scope of the present invention is not limited to these examples.

Examples

Reference example 1: preparation of zanthoxylum bungeanum extract oil

Mixing 1000g pulverized dried pericarp of Zanthoxyli fructus with 300g water, adding into 5L supercritical extraction device, extracting at 40 deg.C under 20MPa with 2.5% 95% ethanol supercritical carbon dioxide as extraction auxiliary agent at flow rate of 5 Kg/hr for 2 hr. Then, the carbon dioxide fluid containing Zanthoxylum bungeanum extract was introduced into a separation tank, and the separation operation was performed at a temperature of 20 ℃ and a pressure of 5MPa in the separation tank to obtain 130g of Zanthoxylum bungeanum extract oil (reference 1) which is a supercritical carbon dioxide extract.

Example 1: extraction of an extract containing hydroxysanshool (using 40% ethanol aqueous solution as an extraction solvent)

5.0g of Zanthoxylum bungeanum extract oil (reference 1) was mixed with 130g of 40% ethanol aqueous solution while stirring for 30 minutes at room temperature in an open vessel. The resulting stirred mixture was left at room temperature for 30 minutes, resulting in separation into an oil layer (upper layer) and an aqueous ethanol layer (lower layer). This was transferred to a separatory funnel, an aqueous ethanol layer was taken out, and the solvent was distilled off under reduced pressure, whereby 1.4g of a hydroxysanshool-containing extract (inventive product 1) was obtained. The yield thereof was found to be 28%.

Example 2: extraction of an extract containing hydroxysanshool (using 50% aqueous ethanol as an extraction solvent)

In example 1, 1.9g of a hydroxysanshool-containing extract (product 2 of the present invention) was obtained under the same conditions as in example 1 except that 100g of 50% aqueous ethanol was used instead of 130g of 40% aqueous ethanol. The yield thereof was found to be 39%.

Example 3: extraction of an extract containing hydroxysanshool (using 60% aqueous ethanol as an extraction solvent)

In example 1, 2.1g of a hydroxysanshool-containing extract (product 3 of the present invention) was obtained under the same conditions as in example 1 except that 130g of a 60% aqueous ethanol solution was used instead of 130g of a 40% aqueous ethanol solution. The yield thereof was found to be 42%.

Comparative example 1: extraction of an extract containing hydroxysanshool (using 30% aqueous ethanol as an extraction solvent)

In example 1, 0.9g of a hydroxysanshool-containing extract (comparative product 1) was obtained under the same conditions as in example 1, except that 130g of a 30% aqueous ethanol solution was used instead of 130g of a 40% aqueous ethanol solution. The yield thereof was found to be 18%.

Comparative example 2: extraction of an extract containing hydroxysanshool (using 70% ethanol aqueous solution as an extraction solvent)

5.0g of Zanthoxylum bungeanum extract oil (reference 1) was mixed with 100g of 70% ethanol aqueous solution while stirring for 30 minutes at room temperature in an open vessel. The resulting stirred mixture was left at room temperature for 30 minutes, and separated into an aqueous ethanol layer (liquid layer) and a white precipitate (solid layer). The aqueous ethanol layer was removed by decantation, and the solvent was distilled off under reduced pressure, whereby 4.1g of the hydroxycarvitae-containing extract (comparative product 2) was obtained. The yield thereof was found to be 82%.

Comparative example 3: extraction of extract containing hydroxy sanshool (molecular distillation treatment of Zanthoxylum bungeanum extract oil (reference 1))

To 155.5g of zanthoxylum bungeanum extract oil (reference 1), 31.1g of beige salad oil was added, and a total of 3 times of molecular distillation was performed under the conditions shown in table 1 below. In the molecular distillation of the 1 st time, the raw material was supplied as a sample, the distillation residue of the 1 st time was supplied as a sample in the molecular distillation of the 2 nd time, and the distillation residue of the 2 nd time was supplied as a sample in the molecular distillation of the 3 rd time.

TABLE 1 molecular distillation conditions

By the above molecular distillation, 130.6g of the hydroxysanshool-containing extract (comparative product 3) was obtained as a distillation residue. The yield thereof was found to be 64%.

Comparative example 4: extraction of extract containing hydroxy sanshool (steam distillation treatment of Zanthoxylum bungeanum extract oil (reference 1))

500g of ion-exchanged water was added to 133.9g of zanthoxylum bungeanum extract oil (reference 1), and steam distillation was further performed while dropping ion-exchanged water. Terpenes were mainly removed by steam distillation, and 98.3g of the extract containing hydroxysanshool (comparative product 4) was obtained as a distillation residue. The yield thereof was found to be 73%.

Example 4

The content of hydroxysanshool in reference product 1, invention products 1 to 3 and comparative products 2 to 4 was measured by the following HPLC method. Comparative product 1 was not subjected to HPLC measurement because of poor yield. The analysis results are shown in table 2 below.

[ measurement of Hydroxysanshool by HPLC method ]

Preparation of Standard solution

About 0.1g of a hydroxysanshool standard substance was accurately weighed in a 25mL volumetric flask, and the volume was determined with acetonitrile, and then further appropriately and accurately diluted with 50% acetonitrile to prepare a standard solution.

Preparation of HPLC measurement sample

About 0.01g of the extract was precisely weighed in a 50mL volumetric flask, and the volume was determined with methanol, followed by PTFE membrane filtration treatment (ミリポア Co., pore size: 0.45 μm). The preparation was subjected to HPLC analysis.

Conditions for HPLC analysis

Model: SHIMADZU LC20A (Shimadzu corporation)

Column: TSK-GEL ODS-100V (TOSOH)

An inner diameter of 4.6mm, a length of 250mm and a particle diameter of 5 μm

Column temperature: 40 deg.C

Mobile phase: liquid A water acetonitrile phosphoric acid =900:100:1

Liquid B water acetonitrile phosphoric acid =100:900:1

Gradient conditions: (A) (B) =50:50(0 min), 50:50(10 min), -0: 100(30 min), 0:100(45 min)

Flow rate: 0.7 mL/min

Injection amount: 10 μ L

Measuring time: 45 minutes

Retention time: 14-18 minutes (hydroxyl sanshool has 3 peaks)

A detector: PDA (measurement wavelength: 270 nm).

[ Synthesis of Hydroxysanshool Standard substance for HPLC measurement ]

For HPLC measurement, The hydroxysanshool standard was synthesized in non-patent document 2 and The Journal of Organic chemistry (1969), 34(4), 1147-. The synthesized hydroxysanshool is purified by silica gel column chromatography to prepare a hydroxysanshool standard substance for HPLC determination.

TABLE 2 content of hydroxysanshool

	Content (%)
		Product of the invention 1(40% ethanol extract)	83
Product of the invention 2(50% ethanol extract)	79
		Product of the invention 3(60% ethanol extract)	72
Comparative 2(70% ethanol extraction)	30
		Comparative example 3 (molecular distillation treatment)	29
Comparative example 4 (steam distillation treatment)	33
		Reference 1 (Zanthoxylum bungeanum oil)	25

As shown in Table 2, by the extraction methods of examples 1 to 3, a hydroxysanshool-containing extract containing hydroxysanshool at a high concentration with less impurities can be prepared. In addition, as in comparative example 2, when the ethanol concentration of the ethanol aqueous solution used as the extraction solvent is too high, a white precipitate containing high boiling point substances such as triglyceride is formed without forming an oil layer, and terpenes and the like are contained in the layer of the ethanol aqueous solution, so that it is impossible to prepare a hydroxycamptothecin-containing extract containing hydroxycamptothecin in a high purity. Furthermore, the content of hydroxysanshool in the extract obtained by molecular distillation or steam distillation is not greatly different from that of the oil extracted from Zanthoxylum bungeanum as a raw material, and the molecular distillation or steam distillation hardly has an effect of improving the content of hydroxysanshool.

Reference example 2: preparation of Japanese prickly ash extract oil

1000g of pulverized dried pericarp of Zanthoxylum bungeanum Maxim was uniformly mixed with 300g of water, and the mixture was put into a supercritical extraction apparatus having an internal volume of 5L, and extracted at 40 ℃ under 20MPa for 3 hours at a flow rate of 5 Kg/hr using supercritical carbon dioxide containing 2.5% of 95% ethanol as an extraction aid. The carbon dioxide fluid containing Zanthoxylum bungeanum extract was introduced into a separation tank, and separated at 20 deg.C under 5MPa to obtain 118g of Zanthoxylum bungeanum extract oil (reference 2) as supercritical carbon dioxide extract.

Example 5: extraction of an extract containing hydroxysanshool (using 40% ethanol aqueous solution as an extraction solvent)

In an open vessel, 5.0g of zanthoxylum bungeanum extract oil (reference 2) and 130g of 40% ethanol aqueous solution were mixed while stirring for 30 minutes at room temperature. The above stirred mixture was left at room temperature for 30 minutes, resulting in separation into an oil layer (upper layer) and an aqueous ethanol layer (lower layer). This was transferred to a separatory funnel, the aqueous ethanol layer was removed, and the solvent was distilled off under reduced pressure, whereby 1.3g of a hydroxysanshool-containing extract (inventive product 4) was obtained. The yield thereof was found to be 26%.

Example 6: extraction of an extract containing hydroxysanshool (using 50% aqueous ethanol as an extraction solvent)

The same conditions as in example 5 were repeated except that 120g of 50% aqueous ethanol was used instead of 130g of 40% aqueous ethanol in example 5, to obtain 1.4g of a hydroxysanshool-containing extract (product 5 of the present invention). The yield thereof was found to be 28%.

Example 7: extraction of an extract containing hydroxysanshool (using 60% aqueous ethanol as an extraction solvent)

The same conditions as in example 1 were repeated except that 130g of 60% aqueous ethanol was used instead of 130g of 40% aqueous ethanol in example 5, to obtain 1.5g of a hydroxysanshool-containing extract (product 6 of the present invention). The yield thereof was found to be 30%.

Comparative example 5: extraction of an extract containing hydroxysanshool (using 30% aqueous ethanol as an extraction solvent)

The same conditions as in example 1 were repeated except that 130g of 30% aqueous ethanol was used instead of 130g of 40% aqueous ethanol in example 5, to obtain 0.7g of a hydroxysanshool-containing extract (comparative product 5). The yield thereof was found to be 14%.

Comparative example 6: extraction of an extract containing hydroxysanshool (using 70% ethanol aqueous solution as an extraction solvent)

In an open vessel, 5.0g of zanthoxylum bungeanum extract oil (reference 2) and 120g of 70% ethanol aqueous solution were mixed while stirring for 30 minutes at room temperature. The resulting stirred mixture was left at room temperature for 30 minutes, resulting in separation into an aqueous ethanol layer (liquid layer) and a white precipitate (solid layer). The aqueous ethanol layer was removed by decantation, and the solvent was distilled off under reduced pressure, whereby 3.8g of the hydroxysanshool-containing extract (comparative product 6) was obtained. The yield thereof was found to be 76%.

Example 8

The content of hydroxysanshool in reference substance 2, invention products 4 to 6 and comparative product 6 was measured by HPLC in the same manner as in example 4. Comparative product 5 had poor yield, and therefore was not subjected to HPLC measurement. The analysis results are shown in table 3 below.

TABLE 3 content of Hydroxysanshool

	Content ratio
		Product 4 of the invention (40% ethanol extract)	81
Inventive product 5(50% ethanol extract)	74
		Product 6 of the invention (60% ethanol extract)	70
Comparative product 6(70% ethanol extract)	33
		Reference 2 (Japanese pepper oil)	22

As shown in Table 3, by the extraction methods of examples 5 to 7, a high-purity hydroxysanshool-containing extract with less impurities was obtained. In addition, as in comparative example 6, if the ethanol concentration of the ethanol aqueous solution used as the extraction solvent is too high, a hydroxycamptothecin-containing extract containing high-purity hydroxycamptothecin cannot be prepared for the same reason as the method for extracting hydroxycamptothecin from the zanthoxylum extraction oil.

Claims (3)

1. A method for producing a hydroxysanshool-containing extract, characterized by comprising the steps of:

mixing an extract oil of Zanthoxylum containing 5 to 30 mass% of hydroxysanshool with an aqueous ethanol solution having an ethanol concentration of 35 to 65 mass% in an amount of 5 to 50 times the mass of the extract oil of Zanthoxylum at a temperature of 0 to 70 ℃, stirring for 5 to 120 minutes, and standing for 20 to 120 minutes, thereby separating the oil containing a high boiling point substance of terpenes or triglycerides into an upper layer and the aqueous ethanol solution into a lower layer,

removing the separated upper layer oil, removing terpenes or high-boiling point substance impurities, and distilling the lower layer ethanol water solution under reduced pressure to obtain extract containing hydroxy sanshool.

2. The production method according to claim 1, wherein the hydroxycamptothecin-containing extract contains at least 70% by mass of hydroxycamptothecin, based on the mass of the extract.

3. The method according to claim 1 or 2, wherein the Zanthoxylum plant is Zanthoxylum bungeanum (Zanthoxylum piperitum) or Zanthoxylum bungeanum (Zanthoxylum bungeanum).