JP5186628B2 - Method for preparing medicinal composition - Google Patents

Description

The present invention relates to a method for preparing a medicinal composition obtained by extraction from a plant of the family Taceae.
More specifically, it relates to a method for preparing a medicinal composition that is highly likely to be used as a raw material for health foods, and further as a medicament for the prevention or treatment of various diseases. The present invention relates to a pharmaceutical preparation technique.

  The plant belonging to the genus Tadaceae is an annual plant that grows on the watersides of rivers and swamps that are widely distributed from the temperate zone to the tropics of the northern hemisphere including Taiwan and China, from Hokkaido to Okinawa. The cotyledons that have been developed and germinated are called “buds” and are used as sashimi and vinegar.

  These seeds are collected from the whole plant in autumn and used as therapeutic drugs (folk medicines) such as diuresis, antipyretic, insect bites, per meal, and hot air. It is said that there is a descending action.

  On the other hand, it has been clarified that active oxygen and free radicals generated in vivo promote the oxidation of living tissues and are involved in the onset and progression of lifestyle-related diseases, and plant polyphenols and flavonoids having antioxidant activity Is interested.

  In particular, an epidemiological study has revealed that the intake of flavonoids can prevent coronary heart disease. JP-T-2001-51153 (Patent Document 1) mainly contains proanthocyanidins that are water-soluble and highly bioabsorbable. It is shown that the grape seed extract is used as a component of a vascular therapeutic agent.

  In addition, the red pepper that is cultivated in large quantities in the Asakura area of Fukuoka Prefecture contains 41.5 μg / 100 g of anthocyanin-based red pigment and 230 mg / 100 g of polyphenols, which have high antioxidant activity. Therefore, it is expected to work to suppress the onset and progression of lifestyle-related diseases.

Furthermore, as previously described in Japanese Patent No. 3108059 (Patent Document 2), the present applicant can obtain a physiologically active peptide composition by subjecting refined barley powder to protease decomposition after high-concentration acetic acid treatment. I have found that.
JP-T-2001-51153 (Claims) Japanese Patent No. 3108059 (Claims)

  The inventor searches the active ingredients in the seeds in order to pursue the characteristics of the seeds more deeply and effectively use those characteristics. Considered use.

As a result, the inventor found that extraction with acetic acid from high-concentration acetic acid used in the production of a bioactive peptide composition from refined pearl barley powder, The present invention was completed by finding that it is effective for elution of components and acquisition of antioxidant components.

That is, the invention according to claim 1 of the present invention is
After incubating a young bud or a growing leaf of a willow or a cultivar thereof in an acetic acid aqueous solution having an acetic acid concentration of 20 to 30% by mass,
Water is added to the obtained acetic acid aqueous solution to obtain a dilute acetic acid aqueous solution having a concentration of 4% by mass or less,
It is a method for preparing a medicinal composition, wherein a dissolved product of the dilute acetic acid aqueous solution is separated and dried and solidified.

The invention according to claim 2 of the present invention is
After incubating a young bud or a growing leaf of a willow or a cultivar thereof in an acetic acid aqueous solution having an acetic acid concentration of 20 to 30% by mass,
Water is added to the obtained acetic acid aqueous solution to obtain a dilute acetic acid aqueous solution having a concentration of 4% by mass or less,
Upon separation and solidification of the diluted acetic acid aqueous solution,
About the insoluble matter of the dilute acetic acid aqueous solution ,
It is a method for preparing a medicinal composition, wherein ethanol extraction is performed, ethanol is stripped from the obtained extract, and the extract is dried and solidified.

The invention according to claim 3 of the present invention is
In the preparation method of the medicinal composition according to claim 1 or 2,
The incubation process includes
Initiate in an aqueous acetic acid solution with a concentration of 90% by mass, and continue the incubation process while sequentially diluting the aqueous acetic acid solution with water, and finally incubate in an aqueous acetic acid solution with a concentration of 20-30% by mass for 1-2 hours. It is characterized by being.

The invention according to claim 4 of the present invention is
In the preparation method of the medicinal composition in any one of Claims 1-3,
The willow tree or its cultivar is
It is characterized by being red beetle.

The invention according to claim 5 of the present invention is
In the preparation method of the medicinal composition according to claim 2,
The ethanol concentration is
It is 50 to 80% by mass.

The medicinal composition prepared in the present invention is obtained by incubating young shoots or growing leaves of the above-mentioned Yanagita or its cultivars by immersing them in an aqueous acetic acid solution of a specific concentration, adding water to the resulting aqueous acetic acid solution, the following rare aqueous acetic acid concentration, separating the lysate of the rare aqueous acetic acid, obtained by dry solidification those exhibits an excellent medical effects described below, as the health food material, also It is effectively used as a raw material for medicine.
1) Improvement of erythrocyte deformability (improvement of so-called blood freezing condition)
2) Therapeutic effect of mild diabetes 3) Improvement of liver function 4) Activation of immune function 5) Increase of platelets and leukocytes

In particular, since the medicinal composition prepared in the present invention can be used in a powder state or as acetic acid or water or an ethanol solution, it can be used as a health food or a medicine in order to exhibit the effects as described above. It is possible to use it efficiently and effectively.

The present invention, the Polygonum hydropiper or in a process for preparing by extraction from the cultivar, the Polygonum hydropiper or cultivars thereof, usually Matade, Polygonum hydropiper, also called Hontade, preferably, red smartweed (Murasakitade) its cultivar , Azabutade, Hosobatade and the like are used, and particularly preferred is red seed.

In the extraction, the above-mentioned seed leaves are used.
Preferred are the germinated cotyledons, or “buds”, but the grown leaves, or “grown seeds”, can also be used satisfactorily.

For these “buds” and “growth seeds”, lyophilized or air-dried powders are suitable for extraction, but the powders can be used instead of powders.
These fresh leaves are washed with tap water and deionized water, and then extracted, usually acetic acid, after being subjected to high-concentration acetic acid treatment.

Incubation treatment with high-concentration acetic acid is to start with 90% acetic acid such as concentrated acetic acid obtained by concentrating synthetic acetic acid or brewed vinegar, then dilute 3-4 times with water and treat for 1-2 hours.
After this incubation, it is preferable to add a large amount of water to dilute acetic acid, and extract in that state. The acetic acid concentration at that time is preferably 4% or less, particularly preferably 2-3%.

What is extracted in this way is a composition comprising various components, but is soluble in dilute acetic acid, particularly in dilute acetic acid having a concentration of 2 to 3%.
Moreover, since it has the various characteristics mentioned later, it is effectively used as a raw material for health food and further as a raw material for medicine.

The residue remaining after extraction with acetic acid is extracted with 50 to 80% ethanol to obtain an extract that is hardly soluble in dilute acetic acid and soluble in ethanol.
As will be described later, this composition is somewhat different from the above composition, but has appropriate characteristics.

  The extraction with acetic acid and ethanol is preferably performed with stirring at room temperature for 0.5 to 2 hours, whereby the desired extract is obtained.

As described above, the extract prepared in the present invention is obtained mainly by acetic acid extraction and ethanol.
In that case, it is also possible to make the extract mainly ethanol and acetic acid.
However, in that case, since different components become constituent components and there are some differences in characteristics, it is desirable to carry out after careful examination before use.

<Preparation of composition>
To each 100 g of freeze-dried red sprout sprout and growth sprout, 125 ml of 90% acetic acid was added, and then 250 ml of deionized water was added and stirred, followed by incubation at room temperature for 2 hours.
Thereafter, 2.5 liters of deionized water was further added and stirred for about 1 hour, followed by centrifugal filtration using an 80 mesh filter cloth, and 2 liters of deionized water was again added to the residue, followed by stirring and centrifugal filtration.
The obtained filtrate was combined and centrifuged (at 10,000 rpm for 10 minutes) to separate into a supernatant and a precipitate. The obtained supernatant was concentrated under reduced pressure and lyophilized to obtain composition A. .
Composition A was soluble in any of water, dilute acetic acid, and ethanol (80%), and 36.5 g was obtained from the sprouts and 33.7 g from the growth seeds.
On the other hand, the residue in centrifugal filtration and the precipitate in centrifugal separation were combined and freeze-dried, and then 600 ml of 80% ethanol was added and stirred at room temperature for 1 hour.
The supernatant obtained by centrifugation was concentrated under reduced pressure and lyophilized to obtain Composition B.
Composition B is sparingly soluble in dilute acetic acid, soluble in ethanol (80%), partially soluble in water (pH 7), 9.0 g from sprout and 8. 3 g was obtained.

<Analysis of Composition A>
Using 500 mg of the composition A extracted from the sprout, the constituent components were detected and sorted by gel filtration under the following conditions.
The resulting gel filtration elution pattern is shown in FIG. 1, and the results of fractionation in 19 fractions are shown in Table 1.

<Gel filtration conditions>
The lyophilized composition A was dissolved in a 1% acetic acid solution, and the supernatant obtained by centrifugation was applied to a Bio-Gel P-10 (4 × 45 cm) column previously washed with a 1% acetic acid solution. Developed with 1% acetic acid solution.
The eluate was fractionated by a certain amount using a drop counter, and the components were detected by measuring the absorbance at 230 nm and 280 nm.

<Measurement 1 of antioxidant activity>
The antioxidant activity of each of the fractions obtained above was measured by the following method according to the method of Miller et al. Using the action of the linoleic acid oxide to discolor β-carotene. Indicated.
As is clear from the figure, antioxidant activity was observed in many fractions.

<Measurement method of antioxidant activity>
4.9 ml of linoleic acid-β-carotene solution is added to a spectrophotometer test tube cell into which 0.1 ml of the sample solution has been dispensed, and the mixture is stirred and incubated in a constant temperature bath at a temperature of 50 ° C. The degree of fading of β-carotene was determined by absorbance at 470 nm, and the degree of oxidation was determined with the degree of oxidation when no sample was added being 100.

<Immune function measurement>
In order to determine the activation of immune function by the composition A extracted from the sprout, the production amount of TNF (Tumor Necrosis Factor) -α of the composition A was measured.
As is clear from the results shown in FIG. 3, it can be seen that composition A can activate immune function.
The production amount of TNF-α was measured as follows.

<Method for measuring production amount of TNF-α>
To 500 μl of a neutrophil suspension (1.0 × 10 ⁶ cells / ml) separated from human peripheral blood, 10 μl of a solution of composition A obtained from bud seeds with different dilutions is added, and 5% CO ₂ is added. After incubating at 37 ° C. for 6 hours in the incubator containing the mixture, the mixture was centrifuged at 1,500 rpm for 5 minutes, and the amount of TNF-α in 400 μl of the obtained supernatant was quantified by ELISA.
In addition, the density | concentration of the added sample was represented by the dilution.

<Measurement of red blood cell deformability lowering suppression function 1>
The erythrocyte deformability decreases due to the influence of the oxidizing agent (AAPH: 2,2′-azobis (2-amidinopropane) dihydrochloride), but the decrease suppression function by the composition A was measured by the method shown below. .
FIG. 4 shows that the suppression effect by the composition A was remarkably recognized.

<Measurement method of erythrocyte deformability reduction inhibitory function>
Blood 10.0ml was bled blood collection tubes containing 3.8% sodium citrate solution 1.0 ml, then precipitated erythrocytes by centrifugation 2,500 rpm × 10 min, washed, HEPES was added, 6. A 0% erythrocyte suspension was prepared.
HEPES (a 3.0 ml, b 2.40 ml, c 2.34 ml) was added to 3 ml of this 6.0% erythrocyte suspension and preincubated at a temperature of 37.0 ° C., and then 500 mM AAPH was added to b and c. To the solution (0.6 ml), c was added 0.06 ml (10.0 mg / ml) of an acetic acid solution of the composition A, and incubated at a temperature of 37.0 ° C. for 45 minutes.
Then, it cooled on ice until it measured, and the measurement was performed after incubating again for 7 minutes at the temperature of 25.0 degreeC.
A is a control.

This erythrocyte deformability is a nickel mesh (nickel) with significantly improved filter characteristics developed by the inventor as an alternative to the conventional method using a micropore filter that is difficult to quantify and reproducibly. mesh) Measurement was performed by the following method using a filter.
Nickel mesh is a nickel thin film filter made by combining a photoresist method and a special plating method. The number, shape, and distribution of micropores are not only constant, but it is more than 100 times by ultrasonic cleaning for several seconds. Can be reused.
In addition, edge of microporous nickel mesh has a smooth and tapered, thereby contaminating leukocytes receives no mechanical impact, no even fusion or branched in the micropores.
Due to these characteristics, the following method maintains high quantitativeness and reproducibility.

<Erythrocyte deformability measuring method (Nickel mesh filtration method)>
In the test, a nickel mesh holder was connected to a vertically standing glass tube via a Tygon tube, and normal saline solution adjusted with a HEPES buffer from a height of 15 cm (height: h) was used. The prepared erythrocyte suspension is filtered.
Around the glass tube, constant temperature water is refluxed to keep the sample at a constant temperature.
A pressure (P) transducer installed at the zero level of the glass tube continuously detects the pressure drop during the filtration of the sample, and this is taken into a personal computer through an amplifier and an AD converter, and the flow rate (flow: Q) is calculated.
The flow rate is converted from pressure to height (P = ρgh), the differential value (dh / dt) of the height-time (ht) curve is taken, and this is multiplied by the cross-sectional area (a) of the glass tube. (Q = dh / dt · a).
Using the pressure-flow curve of a control solution that does not contain blood cells (HEPES buffer-adjusted saline: Newtonian fluid) as a control, examine the pressure-flow curve of the erythrocyte suspension, and at a certain pressure (usually 100 mm · H ₂ O) The erythrocyte deformability is evaluated based on the flow rate (%) of the erythrocyte suspension relative to the flow rate of the control solution.

<Measurement of red blood cell deformability lowering suppression function 2>
As shown in FIG. 1, the composition A can be roughly divided into an antioxidant phenolic acid-containing fraction eluted in the first half and a red-red flavonoid-containing fraction eluted in the second half. Therefore, each fraction was pooled and the function of suppressing the decrease in erythrocyte deformability was measured.
The sample used here is a 70% ethanol-soluble fraction of composition A.
As shown in FIG. 5, the erythrocyte deformability reduction inhibitory function was remarkably observed in the flavonoid-containing fraction.
Here, a is a control, b is a sample to which only AAPH is added, c is a sample to which AAPH and a phenolic acid-containing fraction are added, and d is a sample to which an AAPH and a flavonoid-containing fraction are added.

<Measurement of antioxidant activity 2>
FIG. 6 shows the results obtained by measuring the antioxidant activity of the two fractions obtained above in accordance with the method described above and showing the amount as a converted amount of the synthetic antioxidant BHA (t-butylhydroxyanisole).
As is apparent from the figure, the antioxidant activity of the phenolic acid-containing fraction was small.
Note that a is the composition A itself, b is a phenolic acid-containing fraction, and c is a flavonoid-containing fraction.

<Measurement of radical scavenging activity>
The radical scavenging activity of the two fractions obtained above and the composition A was measured by the following method.
The result is shown in FIG.
As is clear from FIG. 7, the flavonoid-containing fraction showed remarkable radical scavenging activity.

<Radical scavenging activity measurement method>
To 900 μl of DPPH (1,1-diphenyl-2-picrylhydrazyl) solution (DPPH 400 μM: MES 200 μM: 20% ethanol = 1: 1: 1), add 80% ethanol (300-a) μl and sample a μl, and add room temperature After 20 minutes of reaction, the decrease in absorbance at 520 nm is measured.
A calibration curve of DPPH radical scavenging activity was prepared with ascorbic acid (AsA), and the radical scavenging activity per sample g was expressed in terms of AsA equivalent (μmol).

<Re-fractionation of fractions containing flavonoids>
The flavonoid-containing fraction showing the above-mentioned remarkable characteristics was re-fractionated by reverse phase HPLC, and the result of FIG. 8 was obtained.
The results of measuring the absorption spectrum for the three peaks a ₁ , a ₂ and b are as shown in FIG.
Component peak a ₁ is Idein, components of peak b was estimated to quercitrin.

<Re-fractionation conditions>
After 5.7 mg of the flavonoid-containing fraction was dissolved in 230 μl of 0.1% TFA solution, 200 μl was subjected to reverse phase HPLC (21.5 × 300 mm).
Elution was performed at a temperature of 40 ° C. and a flow rate of 5 ml / min, and detection was performed at 280 nm.
In one reverse phase HPLC, 2.8 mg of the component indicated by peak a ₁ , 1.8 mg of the component indicated by peak a ₂ , and 2.0 mg of the component indicated by peak b were obtained.

<Measurement of red blood cell deformability reduction inhibitory function 3>
The peak a ₁ component, i.e., idein, and peak b component, i.e., quercitrin, was measured by the refractionation of the flavonoid-containing fraction.
As shown in FIG. 10, the function of suppressing the decrease in erythrocyte deformability was recognized in both, but the function of suppressing the peak a ₂ was not recognized.
Incidentally, a is controlled, b is added only AAPH, c is AAPH peak _{a 1-component} additive, d is a sample with the addition of AAPH and peak b component.

<Functional measurement in living body>
The effect of the medicinal composition prepared in this invention on the living body was examined using rats.
As rats, 9 to 10-week-old male Wistar SPF rats were used, and in order to examine the effects of diabetes, rats administered with STZ (streptozotocin) that induces diabetes were also examined.
As the medicinal composition prepared in the present invention, a composition A extracted from sprout seeds and a composition B extracted from growth seeds were selected, and 0.5% and 0.1% thereof were oriental yeast (strain ) And feed MF.
The items to be examined are as follows.
1) Body weight 2) Blood glucose level (tail vein: using Glutest Ace R (Sanwa Chemical Laboratory))
3) Blood test (blood sampling from abdominal aorta: general blood, biochemical test (plasma), erythrocyte deformability)
4) Microscopic observation (microscope)

Table 2 shows the results of examining the effect of the medicinal composition prepared in the present invention in rats administered with STZ and causing abnormalities such as diabetes.
It was confirmed that administration of the medicinal composition prepared in this invention markedly improved the AST and ALT values and was effective in improving liver function.
Furthermore, it was recognized that it also functions to decrease LDL cholesterol.

Table 3 was tested in the same manner as Table 1, but more tests were performed.
From this table, it was confirmed that the administration of the medicinal composition prepared in the present invention significantly improved the erythrocyte deformability and further improved blood.
In addition, the growth tapade group was found to be effective in preventing thrombocytopenia due to STZ administration.

  Tables 4 and 5 show the effects of administering the pharmaceutical composition prepared according to the present invention to normal rats. In normal rats, platelet function was increased, but liver function and other factors were observed. No adverse effects were seen.

The immune function test was performed by tissue observation by immunostaining of TNF-α and pancreatic islets of Langerhans.
By administering composition A (bud spider) , TNF-α increased and an immune function promoting effect was observed.

Tissue observation was performed by injecting STZ 45 mg / Kg intraperitoneally into Wistar rats to prepare diabetes model animals.
These model animals were divided into two groups, a severe diabetes model group and a mild diabetes model group, using weight loss and blood glucose level as indices.
Each group was given a diet containing 0.5% composition A (bud seed), and pancreatic Langerhaus island (Lai island) of a diabetes model animal was observed with a microscope, and the medicinal composition of this invention was prepared. The effect of diabetes on the pancreas was investigated.
For microscopic observation, each animal was sacrificed under ether anesthesia, and then the pancreas was removed, cryostat sections were prepared, immunostained with HE and anti-insulin antibodies, and the primary antibody was mouse anti-insulin monoclonal. The antibody (Lab Vision., Co., CA, USA) and the secondary antibody were used after staining by a fluorescent antibody method using horse anti-mouse IgG-FITC (Vector Labs, CA, USA).

FIG. 11 shows HE / E-stained images of pancreatic islets of normal control rats, and FIG. 12 shows immunostained images of pancreatic islets of normal control rats with an insulin antibody.
FIG. 13 shows an HE-stained image of the pancreatic islet of a severely diabetic model rat, and FIG. 14 shows an immunostained image of the pancreatic islet of the severely diabetic model rat with an insulin antibody.
FIG. 15 shows an HE-stained image of pancreatic islet of a mild diabetic model rat after administration of composition A (bud seed) , and FIG. 16 shows a mild diabetic model rat after administration of composition A (bud seed). The immunostaining image by the insulin antibody of a pancreatic la island is shown.

As is clear from these figures, the severe diabetic model group that showed weight loss and high blood glucose level had cell death caused by remarkable atrophy of La Isle, deformation / degeneration of endocrine cells, and apoptosis.
As a result of immunostaining, the insulin-producing B cells were observed to be deformed and denatured as the number decreased.
The remaining deformed / denatured B cell nuclei were unevenly distributed, and cytoplasmic insulin showed a weak positive immunostaining reaction. No particular effect of the composition A (bud seed) administration was observed on the severe diabetes model group.
On the other hand, in the mild diabetes model group to which the composition A (bud spider) was administered, no particular change was observed in the size of La Island. As a result of immunostaining, a small number of cells showing a weak immunopositive reaction were localized in the vicinity of La Jade Island, but other than this, it was almost normal and showed similar morphology and immunostaining reaction.
From the above results, it was suggested that the administration of the composition A (bud seed) works effectively on the mild diabetes model group, and may suppress or delay the transition from mild diabetes to severe diabetes.

The medicinal composition prepared in the present invention has the above-mentioned excellent characteristics and can be supplied as a non-toxic solvent solution of powder or water, acetic acid or ethanol, so that it is widely used in the health food industry and the pharmaceutical industry. It is likely to be used.

It is an elution pattern figure in the gel filtration method of the medicinal composition prepared by this invention. It is a figure which shows the antioxidant activity of each fraction obtained by fractionating the medicinal composition prepared by this invention. It is the figure which showed the change of the production amount of TNF- (alpha) by addition of the medicinal composition prepared by this invention. It is the figure which showed the red blood cell deformability fall inhibitory function of the medicinal composition prepared by this invention. It is the figure which showed the red blood cell deformability reduction inhibitory function of the fraction of the medicinal composition prepared by this invention. It is the figure which showed the antioxidant activity of the fraction of the medicinal composition prepared by this invention. It is the figure which showed the radical scavenging activity of the medicinal composition prepared by this invention and a fraction. It is a reverse phase HPLC chart of the flavonoid containing fraction of the medicinal composition prepared by this invention. It is an absorption spectrum of the component refractionated by the reverse phase HPLC of the flavonoid containing fraction. It is the figure which showed the red blood cell deformability reduction inhibitory function of a refraction fraction. It is a microscope picture which shows the H * E dyeing | staining image of the pancreas la islet of a normal control rat. It is a microscope picture which shows the immuno-staining image by the insulin antibody of the pancreas la islet of a normal control rat. It is a microscope picture which shows the H * E dyeing | staining image of the pancreatic la island of a severe diabetes model rat. It is a microscope picture which shows the immuno-staining image by the insulin antibody of a severe diabetes model rat pancreas la islet. It is a microscope picture which shows the H * E dyeing | staining image of the pancreas la islet of the mild diabetes model rat after administration of the medicinal composition prepared by this invention. It is a microscope picture which shows the immuno-staining image by the insulin antibody of the mild diabetic model rat pancreas laia after administration of the medicinal composition prepared by this invention.

    None

Claims (5)

After incubating a young bud or a growing leaf of a willow or a cultivar thereof in an acetic acid aqueous solution having an acetic acid concentration of 20 to 30% by mass,
Water is added to the obtained acetic acid aqueous solution to obtain a dilute acetic acid aqueous solution having a concentration of 4% by mass or less,
A method for preparing a medicinal composition comprising separating a solution of the dilute aqueous acetic acid solution and drying and solidifying the solution. After incubating a young bud or a growing leaf of a willow or a cultivar thereof in an acetic acid aqueous solution having an acetic acid concentration of 20 to 30% by mass,
Water is added to the obtained acetic acid aqueous solution to obtain a dilute acetic acid aqueous solution having a concentration of 4% by mass or less,
Upon separation and solidification of the diluted acetic acid aqueous solution,
About the insoluble matter of the dilute acetic acid aqueous solution ,
A method for preparing a medicinal composition comprising subjecting an ethanol extraction, volatilizing ethanol from the obtained extract, and drying and solidifying the extract. The incubation process includes
Initiate in an aqueous acetic acid solution with a concentration of 90% by mass, and continue the incubation process while sequentially diluting the aqueous acetic acid solution with water, and finally incubate in an aqueous acetic acid solution with a concentration of 20-30% by mass for 1-2 hours. The method for preparing a medicinal composition according to claim 1 or 2, wherein The willow tree or its cultivar is
4. The method for preparing a medicinal composition according to any one of claims 1 to 3, wherein the preparation is red. The ethanol concentration is
It is 50-80 mass%, The preparation method of the medicinal composition of Claim 2 characterized by the above-mentioned.