HK1162321B - Burdock fruit extract containing arctigenin at high content and process for producing same - Google Patents

Description

Great burdock fruit extract with high arctigenin content and its preparation method

Technical Field

The present invention relates to a great burdock fruit extract containing arctigenin in high content and its extraction and preparation method.

Background

In the japanese pharmacopoeia 15 th edition, burdock is a fruit of arctumlappaline (compositae), and is essentially classified as a component used exclusively as a pharmaceutical product as a compatible crude drug for a prescription such as yinqiao powder, wind-dispelling and detoxifying decoction, and wind-dispersing powder.

The burdock fruit contains about 7% of arctiin classified into lignan glycosides (lignanglycosides) and about 0.6% of arctigenin as its aglycone.

According to literature reports, in recent years, pancreatic cancer-derived cells such as PANC-1, AsPC-1, BxPC-1 and KP-3 have shown strong resistance even in the state of extreme nutritional deficiency, and elimination of the resistance is a novel biochemical method for cancer therapy (patent document 1).

It has also been reported that arctigenin is found to be effective when a substance capable of eliminating the viability of tumor cells in a low nutritional state is screened using a pancreatic cancer cell line PANC-1 (non-patent document 1).

Prior patent document

Patent document 1: japanese laid-open patent publication No. 2002-065298

Non-patent document

Non-patent document 1: awale, j.lu, s.k.kalauni, y.kurashima, y.tezuka, s.kadota, h.esumi, cancer res, 2006, 66(3), 1751-

Disclosure of Invention

For the currently known burdock, the content of arctigenin in the burdock is low, about 0.6%, and is difficult to dissolve in water. Therefore, it is very difficult to produce an extract containing arctigenin at a high content by a hot water extraction method which has been conventionally performed.

Therefore, it is desired to provide a burdock fruit extract containing arctigenin at a high content in a form that can be actually administered to a living body.

Accordingly, the present invention is directed to provide a great burdock achene extract containing arctigenin at a high content and a method for producing the same.

As a result of intensive studies to solve the above problems, the present inventors have found a technique of converting arctiin into arctigenin by focusing on β -glucosidase, which is an enzyme present in the crude drug itself, and utilizing this enzymatic reaction, a technique of efficiently extracting the converted arctigenin, and a burdock fruit extract containing arctigenin at a high content.

The first invention is a burdock fruit extract with high arctigenin content, and the extract contains more than 3% of arctigenin.

The second invention is a method for producing a burdock fruit extract having a high arctigenin content, characterized in that arctiin is enzymatically converted into arctigenin by using β -glucosidase present in burdock fruit itself, and extraction is performed.

A third aspect of the present invention is a method for producing a burdock fruit extract having a high arctigenin content, wherein in any of the above aspects of the present invention, arctiin is converted into arctigenin by an enzyme, and then ethanol is added to extract the arctigenin.

A fourth aspect of the present invention is a method for producing a burdock fruit extract having a high arctigenin content, characterized in that the extract is produced by extracting, concentrating, and then freeze-drying or spray-drying.

A fifth aspect of the present invention is a method for producing a burdock fruit extract having a high arctigenin content, characterized in that, in any of the above aspects of the present invention, the extract is concentrated after extraction, and then dextrin is added to the concentrate to perform spray drying.

According to the present invention, it is possible to provide a burdock fruit extract containing arctigenin at a high content, which has an antitumor effect. In particular, it is expected that tumor proliferation inhibition and antitumor effects will be expected after administration to patients with pancreatic cancer. Also, the yield in manufacturing can be improved.

Drawings

FIG. 1 is an analytical chromatogram of the results of the effectiveness of the enzymatic conversion;

FIG. 2 is a cytotoxicity evaluation using cultured cells (CAPAN-1);

FIG. 3 is a cytotoxicity evaluation using cultured cells (PANC-1);

FIG. 4 is a cytotoxicity evaluation using cultured cells (PSN-1);

FIG. 5 is an evaluation of antitumor activity in tumor model animals (CAPAN-1 Xenografts (xenorafts));

FIG. 6 is an evaluation of antitumor properties in tumor model animals (PSN-1 xenograft tumors);

FIG. 7 is a graph showing the change in blood concentration of Arctigenin (AG) in human blood;

FIG. 8 is a graph showing the change in blood concentration of glucuronic acid conjugate of Arctigenin (AGG) in human blood.

Detailed Description

The present invention will be described in detail below. The disclosed conditions are only an example and the present invention is not limited thereto.

The burdock fruit extract of the invention is prepared by a crude drug cutting process, an extraction process, a solid-liquid separation process, a concentration process and a drying process.

(crude drug cutting Process)

Cutting fructus Arctii as raw material into pieces suitable for extraction. Crude drugs as raw materials are cut into various parts of plants, minerals, animals, and the like, which have various sizes, shapes, and hardnesses according to their properties. The finer the particle size, the more the enzymatic reaction is promoted and the extract yield is also increased, but on the contrary, the enzymatic reaction is too fast and the process control is difficult or the proper solid-liquid separation is affected in the subsequent step. Therefore, the crude drug used in the present invention is desired to have a particle size such that it is cut into pieces.

(extraction step)

The extraction process is the most important process for quality in the process of preparing the traditional Chinese medicine extract powder, and determines the quality of the traditional Chinese medicine extract powder. In the present invention, the extraction step is carried out in two stages, an enzyme reaction step and an organic solvent extraction step.

(enzyme reaction step)

This step is the most important step found in the present invention, and is a step of converting arctiin contained in burdock into arctigenin by an enzyme.

Adding 7L water into 1kg fructus Arctii pieces prepared in the previous step, stirring at 20-40 deg.C, etc., and standing for about 1 hr. Through the process, the arctiin is converted into the arctigenin through the enzyme, so that the content of the arctigenin is obviously improved. This extraction method is called cold-leaching extraction.

Further, the peak temperature of the enzyme reaction is preferably 37 to 40 ℃ from the viewpoint of the reaction rate, but too high a reaction rate may make process control difficult from the viewpoint of quality control.

Therefore, in the case of small scale, it is desirable to perform extraction at 20 to 25 ℃ for about 1 hour, but in the case of industrial scale, depending on the capability of the stirring apparatus and the capability of temperature control, it is desirable to set the extraction at 30 ℃ for 30 minutes, at 37 ℃ for 15 minutes, or the like. In addition, since enzyme deactivation occurs after exceeding 60 ℃, conventional hot water heating methods at 60 ℃ or higher cannot be used.

(organic solvent extraction step)

The step is a step of extracting the burdock fruit extract by heating and refluxing in a state where the content of arctigenin is high. Here, the arctigenin has a property of being hardly soluble in water, and thus the yield can be improved by adding a solvent.

The specific method comprises the following steps: to the solution (1 kg of burdock fruit pieces + 7 liters of water) to which the enzyme reaction process was completed, 3 liters of the solvent was added, and further heated under reflux for 1 hour. Here, a suitable solvent is ethanol from the viewpoint of safety.

The larger the amount of ethanol, the higher the solubility of arctigenin and the higher the yield, but since many unnecessary oils and fats are dissolved and the load of the concentration step increases, the amount to be charged can be determined as appropriate. In addition, the heating reflux in this step also combines sterilization and disinfection.

(solid-liquid separation step)

This step is a step of separating the crude drug after extraction from the extract solution. The solid-liquid separation method includes a filtration method, a sedimentation method, and the like, and a centrifugal separation method is industrially preferable.

(concentration step)

This step is a step of removing the solvent in the extract liquid before drying. Concentrating under reduced pressure to prevent the extract from further exposing to high temperature for a long time. In this step, the concentrate was concentrated to the following concentrations: drying in the next step can be appropriately carried out, and when the dried extract powder is formulated, appropriate formulation characteristics can be obtained.

In addition, since arctigenin has a characteristic of being hardly soluble in water, the amount of arctigenin adhering to the production apparatus in the drying step is large, and the final yield is greatly reduced. Further, the dextrin is added to prevent the dextrin from adhering to the manufacturing apparatus. The amount of the additive to be added is preferably about 20% based on the solid content of the concentrated solution.

(drying Process)

This step is a step of powdering the extracted extract. Known drying methods are freeze drying and spray drying, the former being generally used if at laboratory level and the latter if at mass production level.

The fructus Arctii extract with high arctigenin content can be obtained by the above preparation process.

(Burdock extract powder preparation)

The extract powder obtained by the above-described operation can be used as it is, but usually, it is produced by adding a common excipient (for example, crystalline cellulose, sucrose fatty acid ester, lactose or the like) used in foods and/or pharmaceuticals and granulating by, for example, a dry granulation method or a wet granulation method, and the granulated product obtained by the above-described operation can be used as it is, or it can be used as a compression molded product by a tablet press.

Further, since the extract powder has a characteristic astringent taste, it is preferable to prepare a preparation masking the extract powder for administration, and a film-coating agent coated with a coating agent may be used. In addition, from the viewpoint of stability of the ingredients, or as a form that can be easily taken, the extract powder or the granulated substance may be directly filled in a hard capsule or a soft capsule and taken.

(test) test of validity of enzyme conversion of arctigenin

In the enzymatic reaction step (cold extraction) of the extraction step, an evaluation was made as to whether arctiin was enzymatically converted into arctigenin.

Comparative example 1

0.1g of coarse burdock fruit powder (sieved through a No. 18 sieve) is taken, 50mL of 50% methanol is added, and after heating for 1 hour, filtration is carried out.

Test example 1

0.1g of burdock fruit coarse powder (sieved through a No. 18 sieve) is taken, 25mL of water is added, the mixture is shaken and mixed, and after the mixture is placed at room temperature (22 ℃) for 1 hour, 25mL of methanol is added, and filtration is carried out.

The concentrated solutions obtained in test example 1 and comparative example 1 were measured by HPLC method under the following analytical conditions.

[ method for measuring arctigenin content ]

A chromatographic column: YMC-PackProC18AS-307-3(3 μm, 4.6 mmID. times.7.5 cm)

Column temperature: 30 deg.C

And (3) detection: UV280nm (the chromatogram outflow curve above each set of chromatograms in FIG. 1), UV230nm (the chromatogram outflow curve below each set of chromatograms in FIG. 1)

Flow rate: 0.8mL/min

Sample introduction amount: 5 μ L, 10 μ L

Mobile phase: solution A/0.05M sodium dihydrogen phosphate solution/acetonitrile mixture (5: 1);

solution B/0.05M sodium dihydrogen phosphate solution/acetonitrile mixture (1: 1)

Gradient elution conditions: 0-10 min/solution B20%, 10-25 min/solution B40%

Comparative example 1 as shown in table 1, the contents of arctiin and arctigenin in the raw material of the crude drug were 6.88% and 0.58%, respectively, and the arctigenin/arctigenin content ratio (hereinafter referred to as AG/a) was 0.08.

Test example 1 as shown in table 1, the arctiin and arctigenin contents were 0.15% and 3.80%, respectively, the AG/a ratio was 25.33, and the arctigenin content was significantly increased. In addition, as shown in fig. 1, arctiin in the crude drug material is converted into arctigenin substantially quantitatively.

TABLE 1

Examples

Example 1 (preparation of Arctium lappa extract by chopping and Cold-soaking extraction)

300g of ground burdock fruit pieces were added to 1.5L of water (22 ℃ C.) and stirred for 1 hour, followed by further heating and refluxing for 1 hour. The same was filtered, washed with 0.5L of water, and the combined extracts (1.5L) were freeze-dried.

As shown in table 2, the arctiin and arctigenin contents of the extract obtained by cold-leaching the ground crude drug pieces were 10.1% and 4.8%, respectively, and the AG/a ratio was 0.48.

Example 2 (preparation of Arctium lappa L extract by cutting, Cold extraction, addition of ethanol)

After 200g of ground burdock fruit pieces were added to 1L of water (22 ℃ C.) and stirred for 1 hour, 0.45L of ethanol was added and further heated under reflux for 1 hour. Filtering with 4 mesh screen (100 mesh metal net), washing with 30% ethanol 0.5L, and freeze drying the mixed extractive solution (1.5L).

As shown in table 2, the arctiin and arctigenin contents of the extract obtained by cold-leaching the cut crude drug were 13.3% and 11.4%, respectively, and the AG/a ratio was 0.86. The arctigenin content was greatly increased compared to example 1, which is presumably due to dissolution of arctigenin by the addition of ethanol.

Examples 3 to 6 (preparation of Arctium lappa L extract by medium-scale spray drying)

After 2kg of ground burdock fruit pieces were added to 14L of water (37 ℃) and stirred for 1 hour, 6L of ethanol was added and further heated under reflux for 1 hour. Centrifuging the liquid, concentrating the obtained extractive solution about 16L under reduced pressure, adding dextrin 0-50% of the solid component of the extractive solution, and spray drying.

Since arctigenin is hardly soluble in water, the loss due to adhesion in the spray drying process is large, and the extract yield is reduced to 5% (example 3).

Therefore, the addition of dextrin (examples 3 to 6) can prevent the adhesion to the equipment, and a spray-dried extract having excellent fluidity can be prepared. The yield of the extract is increased from 5 percent to about 20 percent.

The amount of the additive to be added is preferably about 20% based on the solid content of the concentrated solution.

Example 7 (preparation of Arctium lappa extract by medium-scale, spray drying)

After 2kg of ground burdock fruit pieces were added to 14L of water (22 ℃ C.) and stirred for 1 hour, 6L of ethanol was added and further heated under reflux for 1 hour. The liquid was centrifuged, about 16L of the obtained extract was concentrated under reduced pressure, and dextrin was added thereto in an amount of 20% based on the solid content of the extract to perform spray drying.

The arctiin and arctigenin contents are 7.1% and 6.3%, respectively, and the AG/A ratio is 0.89, so that the results in a laboratory can be reproduced to obtain a large amount of extract.

Example 8 (preparation of Arctium lappa extract by Industrial-Scale, spray drying)

80kg of pieces of burdock fruit are added to 560L of water kept at 30 ℃ and stirred for 30 minutes, and then 265L of ethanol is added and the temperature is raised to 85 ℃ and further heated for 30 minutes to extract. The liquid was centrifuged to obtain a treated extract. Mixing the extractive solutions, concentrating under reduced pressure, adding dextrin 20% of the solid content of the extract, and spray drying.

The contents of arctiin and arctigenin were 6.8% and 5.9%, respectively, and the AG/A ratio was 0.87, which reproduced the results on a medium scale, to obtain 31.5kg of extract powder (containing 20% dextrin).

Comparative example 2 (preparation of Arctium lappa L extract by cutting and Hot Water extraction)

300g of ground burdock fruit pieces (sieved through a No. 8.6 sieve) were added to 1.5L of hot water (80 ℃) and heated under reflux for 1 hour, and then filtered while hot using 4 mesh (100 mesh wire gauze). The extract was washed with 0.5L of water, and the combined extracts (1.4L) were freeze-dried.

The crude drug material is heated and extracted with common water as extraction solvent to obtain hot water extract, wherein the content of arctiin and arctigenin is 29.2% and 0.92%, respectively, and the AG/A ratio is 0.03. Since the AG/a ratio was further decreased, it could be confirmed that arctigenin was difficult to transfer to the extract.

TABLE 2

Example 9 (Arctium lappa extract powder blended granule)

(1) Example 8 Arctium lappa extract powder 33.3%

(2) Lactose 65.2%

(3) Hydroxypropyl cellulose 1.5%

In total: 100 percent

(production method)

The granules are prepared according to the general rule of preparations and the items of granules in Japanese pharmacopoeia. That is, the components described in the above table, from the burdock fruit extract powder to the hydroxypropyl cellulose, were granulated, and the granules of example 9 containing 0.5g of the burdock fruit extract powder per pack were obtained by loading the granules in an aluminum laminated film at 1.5 g.

Example 10 (Arctium lappa extract powder blended tablet)

(1) Example 8 Arctium lappa extract powder 37.0%

(2) 45.1 percent of crystalline cellulose

(3) 10.0 percent of calcium carboxymethyl cellulose

(4) 3.5 percent of crospovidone

(5) 3.4 percent of hydrous silicon dioxide

(6) Magnesium stearate 1.0%

In total: 100 percent

(production method)

Tablets were prepared according to the general rules of the preparation and the tablet project of the Japanese pharmacopoeia. That is, the tablets of example 10 were obtained using the components from the burdock fruit extract powder to magnesium stearate described in the above table.

Test example 2 (evaluation of cytotoxicity Using cultured cells)

(Experimental method)

Pancreatic cancer cell lines CAPAN-1, PANC-1 and PSN-1 were inoculated into 96-well plates, and cultured in DMEM medium in a normal nutrient state at 37 ℃ with 5% CO₂Preincubation for 24 hours in the presence of/95% air. After washing the cells with PBS, the extracts (example 2 and comparative example 2 in the figure) having different extraction methods were added to DMEM medium (end D in the figure) and NDM medium (end I in the figure) in a nutrient-deficient medium in a normal nutrient state while keeping the concentration of arctigenin constant, and the resulting mixture was added to each well, and cultured for 24 hours. The cells were washed again with PBS, 100. mu.L of DMEM medium containing 10% WST-8 was added thereto and reacted for 2 hours, and then absorbance at 450nm was measured with a microplate reader to evaluate the survival state of the cells.

The results of the evaluation were: as shown in fig. 2-4, the burdock fruit extract shows significant selective cytotoxicity to pancreatic cancer cell lines in nutrient-deficient culture medium. In addition, it was also found that the extract had an antitumor effect equivalent to that of purified arctigenin depending on the concentration of arctigenin. In addition, arctiin (in the figure: A), which is a precursor of arctigenin (in the figure: AG), was not observed in the antitumor activity under these conditions.

Test example 3 (evaluation of antitumor Properties in tumor model animals)

(Experimental method)

A human pancreatic cancer cell line CAPAN-1 or PSN-1 was inoculated under the dorsal skin of a donor nude mouse (BALB-cAJnu/nu, Japan クレァ), and the obtained tumor mass of the donor mouse was transplanted under the dorsal skin of a recipient mouse, thereby obtaining a tumor model animal. Arctigenin (AG), arctiin (a) and arctium fruit extract (example 2) were dissolved in DMSO at a concentration of 10mg/ml, the resulting substance was diluted with physiological saline, and 50 μ g was orally administered to each mouse into the stomach 5 times per week. The antitumor properties were evaluated by measuring the size of the subcutaneous tumor mass of the back over time.

Within one month after the start of administration, a significant tumor growth inhibitory effect was observed in the drug-administered group as compared with the control. In addition, although the antitumor effect was also obtained in the group administered with the refined arctigenin, the burdock fruit extract (example 2) containing arctiin as a precursor had a stronger antitumor effect (fig. 5 and 6).

Test example 4 (blood concentration)

(Experimental method)

In a healthy male volunteer 1 as a subject, about 5mL of blood was collected from the vein at 30 minutes before administration and at 0.5, 1, 1.5, 2, 3, 4, 7, and 24 hours after administration of the granule 2 pack (burdock fruit extract powder 1g) of example 9 over time to obtain a plasma sample. To 500. mu.L of the plasma sample obtained by this operation were added 500. mu.L of 0.1mol/L sodium dihydrogenphosphate solution and 100. mu.L of internal standard solution (IS). After transferring the sample to a test tube, 6mL of methanol was added and mixed with shaking, the mixture was centrifuged, the methanol layer was collected and dried under reduced pressure to solidify, and 250. mu.L of 70% acetonitrile was added to the residue to prepare a sample solution. The concentration of Arctigenin (AG) and the concentration of glucuronic acid conjugate of Arctigenin (AGG) were calculated from the peak height ratio to the internal standard substance, respectively, by measuring the concentration by High Performance Liquid Chromatography (HPLC) using the following conditions.

(HPLC conditions)

A chromatographic column: YMC-packODS-A-312

Mobile phase: 0.1mol/L sodium dihydrogen phosphate solution/acetonitrile mixture solution (73.5: 26.5) containing 0.2% phosphoric acid

Column temperature: 40 deg.C

Flow rate: 1.0mL/min

A detector: UV210nm

Sample introduction amount: 10 μ L

Internal standard solution: dissolving isopropyl p-hydroxybenzoate 10mg in 50% methanol to obtain 50mL solution, adding 50% methanol to 1mL solution to obtain 100mL solution as internal standard solution

(test results)

The changes in the concentrations of AG and AGG in human plasma are shown in fig. 7 and 8, respectively. As is clear from fig. 7 and 8, the main component detected in blood by taking the burdock fruit extract powder of the present invention is AGG. AG concentration (G)_AG) Bimodal peak, maximum concentration (C) was detected at 1 hour and 2 hours_max) It was 0.15. mu.g/mL. Further, it is considered that the intestinal-hepatic circulation is influenced by the slow disappearance from blood. The blood concentration (C) of AGG was confirmed_AGG) Peak at 1.5 hours, C_maxIt was 10.7. mu.g/mL. Further, even after 24 hours, the disappearance from the blood was slow, 3.6. mu.g/mL, and the effect of the entero-hepatic circulation was also considered to be present. As described above, by taking the burdock fruit extract powder of the present invention, the concentrations of AG and AGG in blood can be maintained for a long time, and therefore, the effect in vivo can be expected.

Claims (6)

1. A preparation method of a great burdock achene extract with high arctigenin content is characterized in that 7 liters of water is added into 1kg of broken great burdock achene blocks, the mixture is stirred at the temperature of 20-40 ℃, after 1 hour, arctiin is converted into arctigenin through enzyme by utilizing beta-glucosidase existing in great burdock achene, and then the extraction is carried out; alternatively, the first and second electrodes may be,

adding 25mL of water into 0.1g of burdock fruit coarse powder, shaking and mixing, standing at 22 ℃ for 1 hour, converting arctiin into arctigenin through enzyme by utilizing beta-glucosidase existing in burdock fruit, and extracting; alternatively, the first and second electrodes may be,

adding 300g of burdock fruit fragments into 1.5L of water with the temperature of 22 ℃, stirring for 1 hour, converting arctiin into arctigenin through enzyme by utilizing beta-glucosidase existing in the burdock fruit, and extracting; alternatively, the first and second electrodes may be,

adding 2kg of burdock fruit fragments into 14L of water with the temperature of 22 ℃ and stirring for 1 hour, and extracting after converting arctiin into arctigenin through enzyme by utilizing beta-glucosidase existing in the burdock fruit; alternatively, the first and second electrodes may be,

extracting after enzymatically converting arctiin into arctigenin by adding 80kg of crushed burdock into 560L of water kept at 30 ℃ and stirring for 30 minutes by using beta-glucosidase existing in burdock per se.

2. The method for producing the arctigenin-rich burdock fruit extract according to claim 1, wherein the arctiin is converted into arctigenin by an enzyme, and then the arctigenin is extracted by adding ethanol.

3. The method for producing the burdock fruit extract with high arctigenin content according to claim 1 or 2, characterized by concentrating after extraction, and then freeze-drying or spray-drying.

4. The method for producing the arctigenin-rich burdock fruit extract according to claim 1 or 2, wherein the arctigenin-rich burdock fruit extract is obtained by concentrating after extraction, and then adding dextrin to the concentrated arctigenin-rich burdock fruit extract for spray drying.

5. The method for producing the arctigenin-rich burdock fruit extract according to claim 3, wherein the arctigenin-rich burdock fruit extract is obtained by concentrating after extraction, and then adding dextrin to the concentrated arctigenin-rich burdock fruit extract for spray drying.

6. Use of the arctigenin-high arctigenin extract manufactured by the manufacturing method according to any one of claims 1 to 5 in manufacturing a pancreatic cancer-resistant drug, wherein the extract contains 3% or more arctigenin.