JP5583401B2 - Selenium-containing compound and plant and nutrient containing the same - Google Patents

Description

  The present invention relates to a selenium-containing compound and plants and nutrients containing the same.

  Certain cruciferous plants such as radish and mustard are known to accumulate a large amount of selenium-containing amino acids (hereinafter referred to as “selenoamino acids”) and derivatives thereof.

  Selenoamino acids are generally less toxic to animals and are in a form that is easy to use, and not only can be used as a nutrient for selenium, an essential biological element (for example, a dietary supplement), but some selenoamino acids also have anticancer effects. Therefore, it is also expected to be used as a medicine for preventing cancer and anticancer drugs.

  As the main secondary metabolites derived from selenoamino acids accumulated in plants, methylselenocysteine and gamma glutamylmethylselenocysteine have been identified so far.

  However, selenium is a trace element, and it is very difficult to identify the structure of selenium metabolites that are present in very small amounts, and there are only about the above two types of selenoamino acids accumulated in plants or their derivatives. There is no current situation.

  Then, in view of the said subject, this invention aims at providing a more useful novel selenium containing compound, a plant containing this, and a nutrient.

One embodiment of the present invention is selenohomoranthionine represented by the following formula (1), an ester thereof, or a salt thereof.

  The ester of selenohomolanthionine represented by the above formula (1) is a compound obtained by dehydration condensation of the following formula (1) and an alcohol, and is not limited, but has 1 or more carbon atoms. It is a preferable embodiment that the alcohol is 10 or less. The salt of selenohomoranthionine or an ester thereof can be appropriately selected and is not limited, but may be an acid addition salt or a basic salt. Acid addition salts include, but are not limited to, salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid Preferred are salts with organic acids such as citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid and benzenesulfonic acid. The basic salt is not limited, but is a salt with an inorganic base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide or magnesium hydroxide, or an organic base such as caffeine, piperidine, trimethylamine or pyridine. And a salt thereof are preferred. The same applies to the following embodiments.

The plant according to another embodiment of the present invention contains at least one of selenohomoranthionine represented by the following formula (1), an ester thereof, or a salt thereof.

  In addition, in this form, although it is not necessarily limited, it is a particularly preferable aspect that it is a pungent radish.

Moreover, the nutrient which concerns on the other one form of this invention contains at least any one of the selenohomoranthionine or its ester shown by following formula (1), or those salts.

  As described above, an object of the present invention is to provide a more useful novel selenium-containing compound, a plant containing the same, and a nutrient.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. However, the present invention has many different embodiments and is not limited only to the embodiments specifically described below.

(Embodiment)
The selenium-containing compound according to this embodiment (hereinafter also referred to as “the present selenium-containing compound”) is represented by the following formula (1). This selenium-containing compound is a selenoamino acid derivative as represented by the following formula (1).

The selenium-containing compound according to this embodiment can be used as a nutrient for selenium, which is a trace essential nutrient. In recent years, selenium has been sold as a nutritional supplement, but the utilization efficiency and safety of selenium by living bodies depend on the chemical form. The chemical form of selenium currently used as selenium is selenomethionine and other selenoamino acid derivatives, which are safer than inorganic selenite and selenate, but selenomethionine is methionine. It has a disadvantage in terms of utilization efficiency such as being incorporated into the polypeptide chain without distinction. On the other hand, since the present selenium-containing compound is not incorporated into the polypeptide chain in terms of utilization efficiency, it can be expected to be higher than the selenoamino acid. In addition, the selenium-containing compound according to the present embodiment has lower cytotoxicity than the conventionally identified selenoamino acid, and can be used as a nutrient or a pharmaceutical as a more effective selenium-containing compound. When this compound is used as a dietary supplement or pharmaceutical product, it can be formulated using conventional methods, and when it is used as a dietary supplement, it can be added as an additive to foods.

  Further, the selenium-containing compound can be obtained by synthesis or can be obtained by purification from a plant. In the case of plants, it is not limited as long as the above compound can be obtained, but it is particularly preferable because it can be obtained efficiently from pungent radish.

The method for obtaining pungent radish is not limited as long as the above-mentioned compound can be obtained. For example, cultivation and harvesting of a known variety of pungent radish by adding an inorganic selenium compound to the medium and extraction Can be obtained at The inorganic selenium in this case can be appropriately selected and is not limited as long as the above compound can be obtained. For example, barium selenate, barium selenite, sodium selenate and sodium selenite At least one of them can be mentioned. The time for spraying the inorganic selenium compound may be before the seeds are planted, or after some time (about 1 to 2 months) after the growth of pungent radish, but considering the accumulation of selenium in the plant, as much as possible from the harvest It is preferable that it is early. The amount of dispersing an inorganic selenium compound is preferably 100 mg / ^{m 2} or more 10000 mg / ^{m 2} or less, more preferably 200 mg / ^{m 2} or more 5000 mg / ^{m 2} or less. In addition, when spraying an inorganic selenium compound before planting seeds, it is more preferably 500 mg / m ² or more and 1000 mg / m ² or less, and after pungent radish is grown to some extent, it is 200 mg / m ² or more and 5000 mg / m ² or less. More preferably. It should be noted that after the spicy radish has grown to some extent, it is more preferable to spray the above amount several times.

  As described above, the present selenium-containing compound has a lower cytotoxicity and becomes a more useful selenium compound, and a useful plant containing this can also be provided.

  In order to search for a novel selenium compound, the present inventors actually cultivated spicy radish and evaluated it. This will be described below.

Example 1
(Spicy radish harvest)
First, pebbles, sand, and black soil were put in order into a concrete frame having a length of 10 m, a width of 15 m, and a depth of 1 m to obtain a cultivation tank. Next, this cultivation tank and the underground water tank were connected with a pipe to store the permeated water. Furthermore, 500 mg / m ² each of barium selenate and sodium selenate was sprayed on the surface of the soil in the cultivation tank and mixed well with the fertilizer. Then, a cocoon having a width of 50 cm and a height of 10 cm was then erected, and seeds of pungent radish (Raphanus sativus L. longipinnatus L. Bailey Multivar 'Yukibijin') were sown. After 4 months of weeding and controlling weeds, the grown selenium-enriched pungent radish was harvested.

(Confirmation of selenium concentration)
When the selenium concentration of the harvested selenium-enhanced pungent radish was measured by inductively coupled plasma mass spectrometry, it was confirmed that the selenium-enriched pungent radish contained about 30 μg (average of three measurements) per 1 g of pungent radish. In addition, when the same measurement was performed also about the selenium density | concentration in the groundwater tank after a spicy radish harvest, it confirmed that about 1 microgram (average of 3 times of measurement) was contained per 1g of water.

(Selenium compound confirmation test)
The harvested selenium-enhanced pungent radish was grated with grater, added with 4 times the amount of purified water, and sufficiently homogenized using a homogenizer. This was cooled to obtain 6600 g of a supernatant, which was further passed through a 0.22 μm filter to obtain an extract.

  20 μl of this extract was placed in a high performance chromatographic column (Shodex Asahipak GS-320 HQ, Showa Denko KK) and 50 mM ammonium acetate (pH 6.5) was used as an elution buffer under conditions of a flow rate of 0.6 ml / min. Extracted with. The eluate was directly introduced into an inductively coupled plasma mass spectrometer (Agilent 7500, manufactured by Agilent Technologies) having a collision cell, and selenium was detected at m / z = 82.

  As a result, elution of three types of selenium compounds was observed (see FIG. 1). Of these three compounds, it was confirmed that the selenium compound having a retention time of 15.4 minutes was selenic acid, and the compound having a retention time of 19.9 minutes was methylselenocysteine, but the retention time was 17.8 minutes. The selenium compound did not match the known selenium compound.

  Therefore, in order to identify the detected selenium compound having a retention time of 17.8 minutes, the extract obtained above was heat-treated at 95 ° C. for 10 minutes, and 200 μl of the supernatant was added to a high-performance chromatographic column (Shodex Asahipak GS-320). HQ, manufactured by Showa Denko KK) and eluted with 50 mM ammonium acetate (pH 6.5) as an elution buffer under a flow rate of 0.6 ml / min, and an eluate having a retention time of 17.5 minutes to 18.8 minutes. Was sorted. Further, 200 μl of the eluate was put into another high performance chromatographic column (PRP X-100, manufactured by Hamilton), and 10 mM ammonium acetate (pH 9.0) and 200 mM ammonium acetate (pH 9.0) were used as an elution buffer. Gradient elution was performed at a flow rate of 1.2 ml / min, and an eluate having a retention time of 9.5 minutes to 10.5 minutes was collected. This operation was repeated 5 times.

  The collected eluate was pulverized by freeze-drying, then redissolved in a small amount of water, and measured with an electrospray ionization mass spectrometer (API 3000, manufactured by Applied Biosystems). As a result, a mass pattern corresponding to the isotope ratio of selenium was detected only in the vicinity of m / z = 285 in the mass spectrum of the parent ion detected by the first-stage mass spectrometer (see FIG. 2). Further, the mass spectra of fragment ions detected by the tandem mass method can all be attributed to those derived from selenohomolanthionine, and unknown selenium compounds present in the selenium-enhanced pungent radish harvested in this example are It was identified as selenohomolanthionine (see FIG. 3).

(Synthesis of selenohomolanthionine)
Here, in order to confirm that the selenium compound obtained from the selenium-enhanced pungent radish was selenohomolanthionine, selenohomolanthionine was actually synthesized and compared.

First, 2-amino-4-bromobutyric acid shown in the following (2) was reacted with elemental selenium reduced by sodium borohydride to synthesize selenohomolanthionine shown in the above formula (1). The synthesized selenohomolathionin (hereinafter referred to as “synthetic product”) was measured using a proton nuclear magnetic resonance apparatus, and as a result, spectra shown in the following table could be obtained. As a result, it was confirmed that the target compound was synthesized (see Table 1).

(Comparison with electrospray ionization mass spectrometer)
Similar to the selenium compound extracted from the pungent radish (hereinafter also referred to as “extracted product”), the synthetic product is dissolved in a small amount of water, and the electrospray ionization mass spectrometer (API3000, Applied Biosystems) Measurement). As a result, the mass spectra of the fragment ions detected by the tandem mass method completely coincided between the synthesized product and the extracted product (see FIG. 3).

(Comparison with high performance chromatography inductively coupled plasma mass spectrometer)
A synthetic product is added to an aqueous solution containing an extract so as to be 2.0 μg selenium / ml, and 20 μl thereof is placed in a high performance chromatography column (Shodex Asahipak GS-320 HQ, manufactured by Showa Denko KK), and 50 mM acetic acid as an elution buffer. Eluting with ammonium (pH 6.5) at a flow rate of 0.6 ml / min, the eluate was introduced into a high performance chromatography inductively coupled plasma mass spectrometer and selenium was monitored at m / z = 82. .

  On the other hand, 20 μl of the same aqueous solution as before was newly prepared, put into another high performance chromatographic column (PRP X-100, manufactured by Hamilton), and 10 mM ammonium acetate (pH 6.5) and 200 mM ammonium acetate (pH 6.5) as an elution buffer. Gradient elution was performed using pH 6.5) at a flow rate of 1.2 ml / min, and this eluate was introduced into a high performance chromatography inductively coupled plasma mass spectrometer, and selenium was monitored at m / z = 82.

  As a result, it was confirmed that the retention times of both were completely the same.

(Evaluation of cytotoxicity)
The synthetic product was used to evaluate the cytotoxicity against human-derived cultured cells. Toxicity evaluation was conducted by adding human-derived cultured cells HL60 to a RPMI-1640 medium containing 10% fetal bovine serum and adding selenomethionine as a synthetic product or a positive target so that the selenium concentration would be 0 to 1000 μM for 72 hours. It was done by doing. As a result, it was confirmed that the synthetic product had lower cytotoxicity than selenomethionine as a positive target. That is, it was confirmed that selenohomolanthionine is less cytotoxic than selenomethionine.

  As described above, according to this example, it was confirmed that a more useful novel selenium compound can be provided and a plant containing it can be provided.

  The selenium compound according to the present invention can be used in the field of so-called nutritional supplements and pharmaceuticals, and plants containing this compound can be cultivated and can be used in the field of agriculture.

FIG. 1 is a diagram showing the distribution of selenium compounds in a selenium-enhanced pungent radish extract by a high performance liquid chromatography-inductively coupled plasma mass spectrometer. FIG. 2 is a diagram showing the molecular weight of the selenium compound in the selenium-enhanced pungent radish extract by high performance liquid chromatography-electrospray ionization mass spectrometry. FIG. 3 is a diagram showing a molecular structure of a selenium compound in a selenium-enhanced pungent radish extract by a high performance liquid chromatography-electrospray ionization mass spectrometer.

Claims (2)

A selenohomolanthionine represented by the following formula (1), an ester thereof, or a salt thereof.
A nutrient extracted from pungent radish, comprising at least one of selenohomoranthionine represented by the following formula (1), an ester thereof, or a salt thereof as an essential nutrient.