JP4983701B2 - Production method of flavones - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for obtaining flavone at a high yield from sprouts of buckwheat. <P>SOLUTION: This method for obtaining flavone at a high yield comprises removing husks from buckwheat seeds, growing sprouts from the seeds which are sown in the condition, giving ultraviolet ray irradiation to growing buckwheat sprouts, collecting the leaves of the sprouts, extracting the leaves with an organic solvent, and treating the extract thus obtained with an organic acid. <P>COPYRIGHT: (C)2009,JPO&amp;INPIT

Description

  The present invention relates to a method for producing flavones, and specifically relates to a method for obtaining flavones in high yield from buckwheat sprout.

  Buckwheat (buckwheat noodles) is well known to contain ingredients that contribute to health promotion such as rutin, and has long been eaten by many people as a healthy food. In recent years, research on buckwheat sprout has been conducted. Examples of buckwheat sprout include isoorientin (luteolin 6-C-glucoside), orientin (luteolin 6-C-glucoside), flavones such as vitexin and isovitexin. And rutin (flavonols) have been reported.

  For example, in Non-Patent Documents 1 to 3 below, buckwheat sprouts contain flavones such as orientin and isoorientin in addition to rutin. It describes that it can be extracted from buckwheat sprout.

  However, these conventionally known methods have a problem that the yield of flavones is low, which is one of the reasons why the production of buckwheat-derived flavones has not been put into practical use.

  As a method for obtaining flavones from buckwheat sprout in a high yield, the present inventors previously produced flavones from buckwheat sprout in Japanese Patent Application No. 2006-299593 (filed on October 6, 2006). In doing so, ultraviolet rays were applied to the growing buckwheat sprout, the leaves of the sprout were collected, the leaves were extracted with an organic solvent, and the resulting extract was treated with an organic acid to produce flavones. A method to obtain higher yield was proposed.

  According to the above method, flavones can be obtained in a higher yield than before, but if they can be produced in a higher yield, the cost can be further reduced, and buckwheat-derived flavones are used in a wider range. It is expected to become.

  Mitsuru Watanabe et al. “Changes in antioxidant capacity and flavonoid composition depending on the growth stage of buckwheat.” Journal of Japanese Society for Food Science and Technology, 49 (2), p119-125, (2002)   Mitsuru Watanabe et al. “Flavonoid composition of buckwheat seedlings”, Tohoku Agricultural Research, p257-258, (2002)   Mitsuru Watanabe et al. "Effect of light on the amount of phenolic compounds in buckwheat seedlings" Journal of Japanese Society for Food Science and Technology, 50 (1), p32-34, (2003)

  Therefore, an object of the present invention is to provide a method for efficiently obtaining flavones from buckwheat sprout with higher yield.

  The inventors of the present invention have improved the method previously proposed in Japanese Patent Application No. 2006-299593 and conducted further research to further increase the yield of flavones. As a result, the shells were removed from the buckwheat seeds and sown. The inventors have found that the yield is drastically improved by using the grown sprout, and reached the present invention.

The present invention is based on such knowledge, and is a method for obtaining flavones with higher yield from buckwheat sprout as follows.
(1) In producing flavones from buckwheat sprout, sprout is grown from buckwheat seeds from which shells have been removed, and the grown sprout is collected and extracted with an organic solvent. A method for producing flavones, characterized by obtaining flavones by treatment with an acid.
(2) The method for producing flavones according to (1) above, wherein the growing sprout is irradiated with ultraviolet rays for 1 to 6 days.
(3) The method for producing flavones according to (1) above, wherein an aqueous solution of hydrogen peroxide or hypochlorite is applied to the growing sprout before the ultraviolet irradiation.
(4) The method for producing flavones according to (1) above, wherein a flavone-containing component is extracted by treating the collected buckwheat sprout with an organic solvent or an aqueous solution thereof in a crushed state.
(5) The method for producing flavones according to (4) above, wherein the organic solvent used for extraction is at least one organic solvent selected from the group consisting of methanol, ethanol, acetone, and ethyl acetate.
(6) The method for producing flavones according to (1) above, wherein the organic acid for treating the extract is carboxylic acid or an aqueous solution thereof.
(7) The method for producing flavones according to (6) above, wherein the carboxylic acid for treating the extract is malonic acid or acetic acid.
(8) The method for producing flavones according to (1) to (7) above, wherein heat treatment is performed at 60 to 100 ° C. for 0.2 to 3 hours during the organic acid treatment.

  According to the method of the present invention, flavones can be obtained from buckwheat sprout in a much higher yield than conventional. Specifically, luteolin 6-C-glucoside and luteolin 8-C-glucoside (Luteolin 8-C-) have a high yield of about 2 to 4 times that of the conventional method. flavones such as glucoside, apigenin 6-C-glucoside and apigenin 8-C-glucoside can be obtained.

  These flavones have been reported to have various physiological functions such as antioxidant activity and antitumor activity. Therefore, the buckwheat-derived flavones obtained by the method of the present invention can also be used effectively in applications such as pharmaceuticals, health foods, cosmetics and food additives. Further, according to the present invention, there is an advantage that the yield is increased during the preparation of the reagent.

As already reported by Mitsuru Watanabe, buckwheat sprout contains four flavones. That is, flavones contained in buckwheat sprout are luteolin 6-C-glucoside (hereinafter abbreviated as L6CG), luteolin 8-C-glucoside represented by the following chemical formula, depending on the position of the substituent bonded to the flavone skeleton. (Hereinafter abbreviated as L8CG), apigenin 6-C-glucoside (hereinafter abbreviated as A6CG) and apigenin 8-C-glucoside (hereinafter abbreviated as A8CG).
The present invention relates to a method for producing these flavones from buckwheat sprout in a higher yield than conventionally known methods.

  The present invention relates to a method for producing these flavones from buckwheat sprout in a higher yield than conventionally known methods.

<Growth of buckwheat sprout>
In the present invention, in order to grow buckwheat sprout, seeds are first sown in a soil or a medium after removing the shells from the buckwheat seeds. Buckwheat seeds are generally sown in a shelled state, but according to the study by the present inventors, the yield of target flavones and the extraction work efficiency when grown from molted seeds with the shell removed. Was found to improve significantly.

  Here, buckwheat sprout is a seedling plant that has been sown in a culture solution or soil, germinated and grown until leaves emerge, and is usually cultivated for 3 to 10 days. Point to. When buckwheat grows large, the flavone content decreases rapidly and finally disappears. Therefore, the young plant with short leaves as described above is used.

  According to the present invention, when growing buckwheat sprout, first, the shells are removed from the buckwheat seeds, and the unshelled molting seeds are sown in soil or a growth medium. Conventionally, buckwheat seeds are usually sown with a shell, but in the present invention, a molted seed from which the shell has been removed in advance is sown in soil or a suitable medium and grown in the dark to grow. The type of buckwheat used is not limited, but domestic varieties such as kitawase are suitable among buckwheat.

<Addition of stress-imparting agent to the growing medium of buckwheat sprout>
In the present invention, at the time of growth of a sprout that has been sprouted from the buckwheat seeds with the shell removed in this way, a dilute aqueous solution of hydrogen peroxide or hypochlorite is sprayed directly on the sprout, or the growing soil or It is preferable to add stress to the sprout by adding it to the medium. The concentration of hydrogen peroxide and hypochlorite in these aqueous solutions is usually preferably from 0.01 to 5 (weight)%. The preferred amount of the aqueous solution varies depending on the size of the container and the amount of seeds to be sown, but it may be enough to wet the soil or the medium.

<Ultraviolet irradiation treatment>
After growing for about 1 to 2 days as described above, it is effective to further irradiate the growing sprout with ultraviolet rays (UV irradiation). The irradiation intensity is suitably in the range of 0.5 to 100 lux. Such irradiation is preferably performed for 0.1 to 20 hours per day for about 1 to 6 days.

<Collection of sprout>
The buckwheat sprout that has been treated as described above is collected during growth, the collected sprout is placed in a container, an organic solvent for extraction described later is added, and the mixture is crushed by grinding in the solvent. In the present invention, since there is no buckwheat shell, it is not necessary to collect leaves as in the method proposed in Japanese Patent Application No. 2006-299593, and the entire sprout is crushed and extracted.

<Extraction with organic solvent>
In that state, the mixture is left for a while to extract flavone-containing components contained in the leaves in an organic solvent. As the organic solvent used for extraction, methanol, ethanol, ethyl acetate or acetone is suitable. These may be used as an aqueous solution. Usually, the temperature during extraction may be room temperature. The extraction time may be about 3 to 5 minutes. Subsequently, the mixture is sent to a centrifugal separation process or a filtration process of about flash, and the extract (extracted component) and the organic solvent are separated.

<Organic acid treatment>
Next, the extract is treated with an organic acid. As the organic acid used here, a carboxylic acid is suitable, for example, formic acid, acetic acid, succinic acid, citric acid, maleic acid, malonic acid, glucuronic acid, galaculoturonic acid, propionic acid, caffeic acid, ascorbic acid, coumaric acid. At least one carboxylic acid selected from benzoic acid, tartaric acid, gallic acid and lactic acid is preferred. Of these, malonic acid or acetic acid is most suitable. These organic acids can be used in an aqueous solution, and are usually preferably used as an aqueous solution having an acid concentration of about 10 to 30% by weight.
The treatment temperature with the organic acid is from room temperature to 100 ° C., but generally, the higher the treatment temperature, the higher the yield. Therefore, for example, 50 to 90 ° C. is suitable for an acetic acid aqueous solution. The treatment time is preferably 15 to 200 minutes.

<Heat treatment>
In the method of the present invention, heat treatment may be performed during the organic acid treatment, which is usually more effective. In this case, a suitable heating temperature is 60 to 100 ° C., and the heat treatment time is 0.2 to 3 hours.

<Isolation / Purification>
By the method of the present invention described in detail above, a composition in which L6CG, L8CG, A6CG, and A8CG flavones are mixed is obtained. Since this composition is a mixture of flavones, it can be effectively used as a raw material for health foods and cosmetics as it is. However, when it is necessary to isolate and produce each compound, for example, the following The target compound can be separated and purified by the method.

<Preparation of L6CG, L8CG, A6CG, A8CG>
The composition after the organic acid treatment is concentrated with a rotary evaporator, and if necessary, 1 liter of extract liquid substituted with water is filled with ion exchange resin (XAD) in a glass tube (inner diameter 10 cm, height 20 cm). The column is applied to the column chromatography, and water is allowed to flow about 2 to 5 times the column volume. The flavone is recovered using 50-90% methanol as the eluent, and concentrated when the recovered fraction is obtained.
Thereafter, fractionation by HPLC using a photodiode array detector is performed to obtain a target substance. The HPLC conditions at this time are as follows.
Column: Cadenza CD-5C18 (20 mm ID x 150 mm)
Mobile phase: water / methanol = 75/25 (v / v)
Flow rate: 8.0 ml / min
Oven temperature: 40 ° C
Of the fractions purified by preparative HPLC, the A6CG fraction may contain a pectin-like gelling polysaccharide in the same fraction. In this case, the fraction is subjected to heat hydrolysis with a general strong acid as a sugar hydrolysis method, neutralized after hydrolyzing, and filled with water (inner diameter 10 cm, height 20 cm). ). After flowing water about 2 to 5 times the column volume, the recovered fraction is concentrated and dried using 50 to 90% methanol as the eluent to obtain purified A6CG.

Below, the Example and comparative example of this invention method are explained in full detail. However, the scope of the present invention is not limited by these examples. The HPLC conditions used in the examples and comparative examples are as follows.
In the following Examples and Comparative Examples, experiments were conducted using domestic species of Kitawase as buckwheat.

In the examples (samples No. 1 to No. 4), molt seeds from which the shells were removed were used. In the dehulling process, it was processed in the order of brown buckwheat grinder, brown buckwheat stone remover, brown buckwheat sorter, and brown buckwheat mower. In the buckwheat sorter, the seed size was sorted into four types, and each seed was peeled off through a buckwheat sorter with a predetermined opening (sample No. 1 to No. 4). Table 1 shows the seed size of the buckwheat sorter in each of these samples and the opening of the hulling net in the buckwheat hulling net.
On the other hand, in the comparative example (sample No. 0), the same test was performed using seeds with shells.
Sample No. 1-No. 4 molting seeds and sample no. Five seeds of 5 seeds each with 0 shells were seeded on a petri dish, fed with water, and cultivated in the dark for 5 days. At this time, 50 seeds were randomly selected and cultivated in the dark for 4 days. The seeds with roots and buds were germinated, and the germinated seeds were further grown for 1 day. The germination rate for each sample was as shown in Table 2 below.

  In the growth of the above buckwheat sprout, the first two days are cultivated with water, and then for three days, 2% hydrogen peroxide, 0.1 (weight)% hydrochloric acid, 0.1 (weight) are used as stress imparting agents. An aqueous solution of% hypochlorous acid was given to the medium, and then, for 3 days, UV irradiation was performed for 4 hours every day to give further stress, and an experiment was conducted in which cultivation was performed for a total of 5 days.

  In Examples and Comparative Examples (Samples No. 1 to No. 4 and No. 0), 5 days after sowing, respectively, the leaves of sprout are collected, put in a container, and an organic solvent for extraction is added. Triturated and processed. The supernatant after centrifugation was applied to an XAD column to obtain an extract. At this time, extraction with various organic solvents was performed on the sprout treated in the same manner, and it was found that methanol, ethanol, acetone, and ethyl acetate were preferable. Further, as described in the report by Mitsuru Watanabe et al., Extraction by refluxing with ventilated dry methanol was tried, but the result was inferior to the case of immersion in an organic solvent.

Subsequently, each of the extracts extracted under the same conditions was subjected to organic acid treatment. In organic acid treatment, acetic acid, succinic acid, citric acid, maleic acid, malonic acid, glucuronic acid, galacroturonic acid, formic acid, propionic acid, caffeic acid, ascorbic acid, coumaric acid, benzoic acid, tartaric acid, gallic acid, lactic acid, etc. Treatment with organic carboxylic acid or phosphoric acid. The heating conditions during the organic acid treatment were examined at 80 ° C. for 2 hours. As a result, organic carboxylic acids were found to increase the flavone content. Of these, the rate of increase in malonic acid treatment was the highest. In addition, from this test, it was confirmed that the flavones increased when the concentration of the organic acid was about 0.05M. Moreover, in the experiment using acetic acid, it processed by changing temperature and time.
And the analysis by HPLC was performed after various processes.

  In the above series of steps, when the malonic acid treatment was performed as the organic acid treatment, the combination with each treatment before the organic acid treatment was examined. As a result, it was found that the increase amount was highest when malonic acid treatment was performed after applying stress by 0.1 (wt)% sodium hypochlorite and UV irradiation.

  Further, in the examples (samples No. 1 to No. 4), the yield of flavones without UV irradiation is shown as a ratio with the yield of the comparative example (sample No. 0, no UV irradiation). As shown in Table 3, when the molting seeds from which the shell was removed were used, the yield was increased 1.5 to 2.4 times compared to the case with the shell. Among them, sample No. When 2 molting seeds were used, the yield was 2.3 to 2.4 times higher.

  Next, the effect of flavone yield on the presence or absence of UV irradiation in the presence or absence of buckwheat seed husk was compared. The result is shown in FIG. From FIG. 1, it is understood that the yield increases 2.6 to 3.7 times by using the molting particles obtained by removing the shells from buckwheat seeds and performing UV irradiation as compared with the case where they are not performed.

  Next, characteristics such as antioxidant activity of the purified product of luteolin 6-C-glucoside derived from buckwheat produced by the method of the present invention were examined. The results are summarized in Table 4 below. In addition, each measurement was implemented as follows similarly to Japanese Patent Application No. 2006-299593.

A) Lipid peroxidation inhibitory effect 10 ml of 4% methyl linoleate / methanol solution and 10 ml of 0.1M phosphate buffer (pH 7.4) are collected in a centrifuge tube, 1 ml of sample is added, and the mixture is heated in an oven at 55 ° C. for 48 hours. Thus, lipid peroxide was generated. The sample was prepared to a concentration of 1 g / 100 ml of Sasa leaves. Methanol as a blank and 20 ppm tocopherol as a control were added in place of the sample, and similarly heat-treated in an oven at 55 ° C. for 48 hours to produce lipid peroxide.
The peroxide value (POV) of the resulting lipid peroxide was measured according to a conventional method, and the control POV in the absence of antioxidant was taken as 0%, and the lipid peroxidation inhibition rate of the sample was determined.

B) DPPH radical scavenging activity The radical scavenging activity for the stable radical DPPH radical was investigated.
100 μl of 0.5 mM DPPH radical / ethanol solution and 100 μl of sample were collected in a small Wasselman in this order and mixed. The mixture was quickly stirred, sucked up into a flat cell, inserted into the cavity, and after a certain period of time (45 seconds), loaded into an ESR device (Jeol JES-FR30) to start measurement. As the blank, ultrapure water or acetonitrile was used. Each was subjected to ESR under the following conditions, and the radical elimination rate was determined as (1−sample value / blank value) × 100 (%).
Field: 335 ± 5 mT, Power: 4 mW, Modulation Width: 40 μT
Sweep Time: 2 min, Time const: 0.1 sec, Amp: 200

C) Superoxide anion radical scavenging activity (SOD-like activity)
SOD-like activity was measured using hypoxanthine as a substrate and a superoxide anion radical generation system based on xanthine oxidase (XOD) reaction. In this measurement, stock solution DMPO (Labtech NH-687) 15 μl, 5 mM Hypoxanthine (SIGMA H-9377) 50 μl, 5.5 mM DTPA (Dojindo 347-01141) 35 μl, sample 50 μl, 0.4 U / ml XOD ( (SIGMA X-4376) was collected in a small wasselman in the order of 50 μl and mixed. The mixture was quickly stirred, sucked up into a flat cell, inserted into the cavity, and after a certain period of time (45 seconds), loaded into an ESR device (Jeol JES-FR30) to start measurement. As the blank, ultrapure water or acetonitrile was used. Each was subjected to ESR under the following conditions, and the superoxide anion radical elimination rate was determined as (1−sample value / blank value) × 100 (%).
Field: 335 ± 5 mT, Power: 4 mW, Modulation Width: 0.079 mT
Sweep Time: 2 min, Time const: 0.1 sec, Amp: 200

  From the results shown in Table 4, it was confirmed that buckwheat sprout-derived luteolin 6-C-glucoside is excellent in lipid peroxide inhibitory activity in addition to having good DPPH radical scavenging activity and SOD-like activity. It was done.

Furthermore, for the purpose of investigating the inhibitory effect of browning enzyme polyphenol oxidase (PPO) on luteolin 6-C-glucoside, comparative measurements with generally known PPO activity inhibitors and other flavonoids were performed. The measurement method was as follows, and the measurement results were as shown in Table 5 below.
<Measurement of PPO (Browning Enzyme Polyphenol Oxidase) Inhibitory Activity>
An enzyme solution extracted and partially purified from an onion bulb using 0.05M chlorogenic acid as a substrate was used. That is, 0.1 ml of an inhibitor prepared at 1 mM concentration, 2 mM concentration, and 10 mM concentration and 0.1 ml of enzyme solution were added to 1.3 ml of 10 mM phosphate buffer (pH 7.0), mixed, and mixed at 30 ° C. for 10 minutes. After preliminary warming, 0.1 ml of 0.05M chlorogenic acid substrate solution was added and mixed, and the degree of browning at a wavelength of 420 nm after heating at 30 ° C. for 30 minutes was determined. The inhibitory effect was shown as a relative activity, with the addition of 10 mM phosphate buffer (pH 7.0) instead of inhibitor addition as a control and the activity as 100%.

  As a result, it was found that buckwheat sprout-derived luteolin 6-C-glucoside has an excellent PPO inhibitory activity compared to general PPO activity inhibitors and other flavonoids, and can prevent browning even in a small amount of use. . Therefore, the luteolin 6-C-glucoside according to the method of the present invention can be effectively used, for example, as a browning inhibitor for foods.

  Furthermore, the growth inhibitory effect of human gastric cancer cells and leukemia cells was measured for luteolin 6-C-glucoside and similar compounds. As a result, it was confirmed that luteolin 6-C-glucoside derived from buckwheat sprout has an excellent high growth inhibitory effect.

  According to the present invention, the yield when collecting flavones from buckwheat sprout can be increased, which is useful as a flavone production method. And since the flavones obtained by the method of the present invention have an antioxidant action, they are effectively used as materials for pharmaceuticals, health foods, cosmetics and the like. In particular, luteolin 6-C-glucoside has an excellent anti-browning effect and a growth-inhibiting effect, and therefore can be effectively used for each application utilizing these characteristics.

  Graph showing changes in flavone yield from sprout cultivated by UV irradiation with and without buckwheat seed husk

Claims (8)

  In producing flavones from buckwheat sprout, sprout is grown from buckwheat seeds from which shells have been removed, the grown sprout is collected, extracted with an organic solvent, and then the resulting extract is treated with an organic acid. To obtain flavones, a method for producing flavones.   The method for producing flavones according to claim 1, wherein the growing sprout is irradiated with ultraviolet rays for 1 to 6 days.   The method for producing flavones according to claim 1, wherein an aqueous solution of hydrogen peroxide or hypochlorite is applied to the growing sprout before the ultraviolet irradiation.   The method for producing flavones according to claim 1, wherein the flavone-containing component is extracted by treating the collected buckwheat sprout with an organic solvent or an aqueous solution thereof in a crushed state.   The method for producing flavones according to claim 4, wherein the organic solvent used for the extraction is at least one solvent selected from the group consisting of methanol, ethanol, acetone, and ethyl acetate.   The method for producing flavones according to claim 1, wherein the organic acid for treating the extract is a carboxylic acid or an aqueous solution thereof.   The method for producing flavones according to claim 6, wherein the carboxylic acid for treating the extract is malonic acid or acetic acid. The method for producing flavones according to any one of claims 1 to 7, wherein heat treatment is performed at 60 to 100 ° C for 0.2 to 3 hours during the organic acid treatment.

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