JP2019210236A - Methods of making tomatidine or esculeogenin and products using tomatidine or esculeogenin - Google Patents

Abstract

To provide methods of making tomatidine or esculeogenin by a simple industrial technique, and products exploiting the properties of the obtained tomatidine or esculeogenin.SOLUTION: Disclosed herein is a method for producing tomatidine comprising a step of treating tomatine with an acid to obtain an acid-treated material containing tomatidine and a step of purifying the acid-treated material to obtain an acid-treated extract. Also disclosed herein is a method for producing esculeogenin comprising a step of treating esculeoside with an acid to obtain an acid-treated material containing esculeogenin and a step of purifying the acid-treated material to obtain an acid-treated extract.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing tomatidine or escregenin, and an article using tomatidine or escregenin.

Tomatine having a steroid glycoside structure is known to be contained in immature tomatoes, and is particularly abundant in roots, fruits, flowers, leaves and stems. Tomatine is considered a component that plays a defensive role in order to prevent insects from eating it, and its toxicity is not recommended due to its toxicity.
On the other hand, tomatidine, which is an aglycone of tomatine, is not normally contained in tomatoes, and when infected with soil pathogens, it is known that tomatine is converted into non-toxic tomatidine by tomatinase contained in those bacteria. Yes. This tomatidine is known to be effective in lowering blood cholesterol and suppressing arteriosclerosis (Patent Document 1).

On the other hand, mature tomato contains almost no tomatine, and esculeoside A (3-O-β-lycotetraosyl (5S, 22S, 23S, 25S) -23-acetoxy-3β, 27-dihydroxyspirosolan 27 -O-β-D-glucopyranoside) and its analog esculeoside B (3-O-β-lycotetraosyl (5S, 22S, 23R, 25S) -22, 26-epimino-16β, 23- It has been clarified that a steroid glycoside (epoxy-3β, 23,27-trihydroxycholestane 22-O-β-D-glucopyranoside) is contained. It has been clarified that these esculeosides have physiological activities such as anticancer activity (Non-patent Document 1). It has also been reported that edible dry tomato powder contains either or both of esculeoside A and B as a tomato steroid glycoside (Patent Document 2).
Moreover, although esculeogenin, which is an aglycon of esculeoside, is not usually contained in tomatoes, it has been revealed that it has an anti-cancer action and the like as esculeoside.

  Since tomatidine is usually not contained in tomatoes, it can be organically obtained from tomatine, but it takes many steps and time to sufficiently remove toxic tomatine. Not yet practical.

In addition, a method for obtaining esculogenin has not yet been put into practical use.
Therefore, it is also very difficult to verify the effects relating to esculogenin and tomatidine, and to use and evaluate these in cosmetics and the like.

JP 2009-209099 A JP 2010-273593 A

Tetrahedron 60 (2004) 4915-4920

  In view of the above circumstances, the present invention makes use of the method for producing tomatidine or escregenin capable of efficiently producing tomatidine or escregenin by a simple industrial method, and the characteristics of tomatidine or escregenin obtained. It aims at providing the utilization article of a tomatidine or an escreogenin.

  As a result of intensive studies, the present inventors have found a method for efficiently and efficiently producing tomatidine and esculogenin from tomatine and esculeoside, respectively, and have reached the present invention.

  That is, in order to achieve the above-mentioned object, the method for producing tomatidine according to the present invention includes an acid treatment step of obtaining an acid-treated product containing tomatidine by acid-treating tomatine, and an acid-treated extraction by purifying the acid-treated product. To obtain a product. Thereby, tomatidine can be obtained simply and efficiently with few steps and time.

  The manufacturing method of tomatidine which concerns on this invention is the one aspect | mode, and includes the neutralization process in the said acid treatment process.

  Another aspect of the present invention is an article using tomatidine, which includes tomatidine obtained by the method for producing tomatidine according to the present invention. When the used article is a cosmetic, food, or pharmaceutical product, vitamin D-like activity can be obtained, a high moisturizing effect can be obtained, and rough skin can be suppressed.

  In order to achieve the above object, the method for producing esclegenin according to the present invention includes an acid treatment step in which esculeoside is acid-treated to obtain an acid-treated product containing escreogenin A and / or B. Purification step of purifying the acid-treated product to obtain an acid-treated extract. By using escleoside obtained from mature tomatoes that hardly contain toxic tomatine, esculogenin can be obtained more simply and efficiently.

  The manufacturing method of the esclegenin which concerns on this invention is the one aspect | mode, The neutralization process is included in the said acid treatment process.

  The present invention, in another aspect, is an article using esculogenin, and includes esculogenin obtained by the method for producing esculogenin according to the present invention. When the use article is a cosmetic, food, or pharmaceutical, vitamin D-like activity can be obtained, a high moisturizing effect can be obtained, and rough skin can be suppressed.

  In the present specification and claims, tomatine-derived or esculeoside-derived is a deglycosylated product obtained by performing a predetermined treatment on tomatine or esculeoside (a deglycosylated product of tomatine is It means that the deglycosides of tomatidine and esculeoside are derived from esculogenin. The predetermined treatment refers to any treatment for obtaining a deglycosylated substance such as acid treatment and extraction. In addition, the acid-treated extract obtained by the method for producing tomatidine or esculeoside according to the present invention may include only tomatidine or esculoside.

  ADVANTAGE OF THE INVENTION According to this invention, the tomatidine or escregenin manufacturing method which can manufacture tomatidine or esclegenin efficiently with a simple industrial method, and the tomatidine or esclegenin which utilized the characteristic of the tomatidine or escregenin obtained are used. Articles utilizing creogenin are provided.

It is a graph which shows the comparison of the vitamin D like activity of tomatidine and tomatine. It is a graph which shows the vitamin D like activity of an escleogenin. It is a graph which shows the measurement result of the filaggrin gene expression level by an escleogenin.

  In the following, embodiments of the method for producing tomatidine or escregenin according to the present invention and the articles using tomatidine or escreogenin will be described. However, the present invention is not limited to the embodiments described below.

[Method for producing tomatidine]
The method for producing tomatidine according to the present invention comprises an acid treatment step in which tomatine is acid-treated to obtain an acid-treated product containing tomatidine, and a purification step in which the acid-treated product is purified to obtain an acid-treated extract. Including.

  Tomatine as a raw material is a known compound contained in flowers, leaves, stems, roots and immature fruits of immature tomatoes, and may be obtained by extraction from tomatoes or synthesized organically. May be obtained.

The acid treatment step is performed by gently heating a dilute acid aqueous solution containing an organic solvent such as a lower alcohol such as ethanol. Examples of the acid used include hydrochloric acid and acetic acid.
More specifically, tomatine as a raw material is heated at room temperature (24 ° C.) to 60 ° C. in 0.1 to 2N hydrochloric acid containing ethanol and reacted for about 0.5 to 3 hours. The reaction is stopped by adding a dilute alkali to neutralize. Thereby, tomatine can be deglycosylated and an acid-treated product containing tomatidine can be obtained. The inventors of the present invention have intensively studied and have come up with the idea that deglycosylation by such a procedure is the most efficient and suitable for an industrial technique.

In the purification step, precipitates, insoluble residues and the like are removed.
In the purification process, the filtration is performed in stages using a plurality of filters having different mesh sizes. In addition, it can also carry out by using a column besides using a filter.
Thus, according to the method for producing tomatidine according to the present invention, the tomatine is sufficiently deglycosylated efficiently by a simple industrial technique by acid treatment, and the tomatidine is extracted so as not to contain tomatine, Suitable tomatine-derived acid-treated extracts can be produced.

  In the acid-treated extract derived from tomatine of the present invention, the content of residual tomatine is preferably 10% by mass or less, more preferably 5% by mass or less, based on the total amount of the acid-treated extract, More preferably, it is 1 mass% or less.

[Method for producing escleogenin]
The method for producing esculogenin according to the present invention includes an acid treatment step of obtaining an acid-treated product containing esculogenin A and / or B by acid-treating escleoside, and purifying the acid-treated product. Purification step of obtaining an acid-treated extract.

  Esculoside used as a raw material is a known compound and may be obtained by extraction from mature tomatoes or may be obtained by organic chemical synthesis. Esculoside includes esculoside A and B, which are similar to each other, and the content ratio of esculeoside A and B contained varies depending on the production area and type of tomato.

The acid treatment step is performed by gently heating a dilute acid aqueous solution containing an organic solvent such as a lower alcohol such as ethanol. Examples of the acid used include hydrochloric acid and acetic acid.
More specifically, esculeoside as a raw material is heated at room temperature (24 ° C.) to 60 ° C. in 0.1 to 2N hydrochloric acid containing ethanol and allowed to react for 0.5 to 3 hours. The reaction is stopped by adding a dilute alkali to neutralize. Thereby, esculeoside A and / or B can be deglycosylated to obtain an acid-treated product containing escleogenin A and / or B. The inventors of the present invention have intensively studied and have come up with the idea that deglycosylation by such a procedure is the most efficient and suitable for an industrial technique.

In the purification step, precipitates, insoluble residues and the like are removed.
In the purification process, the filtration is performed in stages using a plurality of filters having different mesh sizes. In addition, it can also carry out by using a column besides using a filter.

  As described above, the esculoside used as the raw material includes esculoside A and B, which are similar to each other, and the content ratio of esculoside A and B contained varies depending on the production area and type of tomato. . Therefore, esculeginins A and B deglycosylated from esculeosides A and B are also similar to each other, and the content ratio of esculogenin A and B contained in the acid-treated extract depends on the production area and type of tomato. Is different. Therefore, although the structures of esculeginins A and B are specified, the acid-treated extract derived from esculeoside obtained in the present invention contains escreogenin A and B, and the content ratio is particularly limited. It is not something.

[Used articles of tomatidine or escleogenin]
As a result of intensive studies, the present inventors have found that tomatidine and esculogenin have vitamin D-like effects, particularly a moisturizing effect and a rough skin suppressing effect.
The tomatidine-derived acid-treated extract obtained by the method for producing tomatidine according to the present invention contains tomatidine having such characteristics. Moreover, the acid-treated extract derived from esculeoside obtained by the method for producing esculogenin according to the present invention contains escleogenin having such characteristics.
These acid-treated extracts can be employed in the articles using tomatidine or escleogenin according to the present invention.
The use articles of tomatidine or esculogenin according to the present invention have uses as cosmetics, foods, pharmaceuticals (including oral administration and parenteral administration such as external preparations for skin) and the like.

When the article using tomatidine or esculeginin according to the present invention is employed as a cosmetic, it can be used as a skin care product such as an emulsion, cream or lotion. By using it as a cosmetic, effects such as suppression of dry skin and suppression of rough skin can be obtained.
That is, the utilization article which concerns on this invention includes the following cosmetics.
A cosmetic composition comprising tomatidine.
A cosmetic for expression of vitamin D-like activity containing tomatidine as an active ingredient.
A skin moisturizing cosmetic containing tomatidine as an active ingredient.
A cosmetic for preventing rough skin containing tomatidine as an active ingredient.
A cosmetic composition comprising escreogenin.
A cosmetic for expression of vitamin D-like activity comprising escreogenin as an active ingredient.
A skin moisturizing cosmetic product containing escreogenin as an active ingredient.
Cosmetics for preventing rough skin, containing escreogenin as an active ingredient.

When used as a food, the tomatidine or esculeginin utilization article according to the present invention, in addition to normal foods, nutritional supplements, functional foods, health foods, foods for specified health use, soft drinks, drinks, It can be used as a supplement, feed, and the like, and effects such as a skin beautifying effect such as a moisturizing effect and a rough skin suppressing effect can be obtained.
That is, the utilization article which concerns on this invention includes the following foodstuffs.
Food for expressing vitamin D-like activity containing tomatidine as an active ingredient.
A skin moisturizing food containing tomatidine as an active ingredient.
A food for preventing rough skin containing tomatidine as an active ingredient.
A food for expression of vitamin D-like activity, which contains escreogenin as an active ingredient.
A skin moisturizing food containing escreogenin as an active ingredient.
A food for preventing rough skin that contains escreogenin as an active ingredient.

  The content of esculogenin or tomatidine contained in the use article of tomatidine or esculogenin according to the present invention is preferably 1 to 50% by mass, and 5 to 50% by mass, based on the total amount of the use article. It is more preferable that it is 10-50 mass%.

  In addition to tomatidine or escregenin, the use article of tomatidine or escreogenin according to the present invention can be blended with other components as long as the object of the present invention is not impaired. When used as cosmetics, examples of other ingredients include preservatives, antioxidants, physiologically active ingredients, animal / seaweed / microorganism / plant-derived extract ingredients, fragrances, powders, amino acids, pigments, and natural vitamins. When used as food, other ingredients include various fats and oils, crude drugs, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fibers, surfactants, purified water, excipients, stabilizers, pH Examples include preparation agents, antioxidants, sweeteners, taste ingredients, acidulants such as organic acids, stabilizers, flavors, colorants, and fragrances.

  The tomatidine or escleogenin-use article according to the present invention can take various forms as long as the object of the present invention is not impaired. For example, when used as a cosmetic, an aqueous solution system, a solubilization system, an emulsification system, a powder system, an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil-powder three-layer system, etc. Can take a wide variety of forms. Moreover, when using as a foodstuff, if it is a form which is easy to ingest, it will not specifically limit, A solid, powder, a liquid, a gel form, a slurry form etc. can be mentioned.

  Hereinafter, the method for producing tomatidine or esclegenin according to the present invention and articles using tomatidine or escregenin according to the present invention will be described more specifically with reference to examples. The present invention is not limited to the following examples and comparative examples.

Example 1:
[Manufacture of acid-treated extract containing escragenin derived from red tomato]
The red tomato was homogenized in water, coarsely filtered, and then purified using an HP-20 column. The HP-20 column was first washed with purified water and then the components were eluted with ethanol. The eluted component was acid-treated at 60 ° C. for 1 hour in 50% ethanol containing 1N hydrochloric acid. The solution was neutralized with an aqueous sodium hydroxide solution, purified by filtration and then dried to dryness using an evaporator. The obtained solid was re-dissolved in olive oil to obtain an acid-treated extract containing escleogenin. The presence of esculogenin in the acid-treated extract thus obtained and its content were confirmed using high performance liquid chromatography (HPLC-MS / MS).

Example 2:
[Manufacture of escleogenin derived from red tomato]
The red tomato was homogenized in water, coarsely filtered, and then purified using an HP-20 column. The HP-20 column was first washed with purified water and then the components were eluted with methanol. The eluted component was acid-treated for 1 hour at 60 ° C. in 50% methanol containing 1N hydrochloric acid. After neutralization with an aqueous sodium hydroxide solution, separation and purification were performed using ethyl acetate. The obtained component was purified using a normal phase column with a solution containing chloroform / methanol / water at a ratio of 9/1 / 0.1, whereby escleogenin could be obtained. It was confirmed by using HPLC-MS / MS that escreogenin was obtained.

Example 3:
[Production of acid-treated extract containing tomatidine derived from immature tomatoes]
By treating immature tomatoes in the same procedure as in Example 1, an acid-treated extract containing tomatidine could be obtained. The presence of tomatidine and the content thereof in the obtained acid-treated extract were confirmed using HPLC-MS / MS.

Example 4:
[Manufacture of tomatidine derived from immature tomatoes]
Tomatidine could be obtained by treating immature tomatoes in the same procedure as in Example 2. It was confirmed by HPLC-MS / MS that tomatidine was obtained.

Example 5:
[Confirmation test of tomatidine or escleogenin activity (for effects similar to vitamin D)]
Vitamin D-like effects of tomatidine and escreogenin were confirmed. Specifically, the agonist activity of tomatidine and esculogenin on the vitamin D receptor was confirmed by the following method using a luciferase assay.
First, a vitamin D receptor (VDR) -luciferase expression vector was introduced into HepG2 cells (human hepatoma-derived cell line, hepatocyte cellular carcinoma, JCRB cell bank). In addition, gene introduction was performed using a commercially available Signal VDRE Reporter Assay Kit (CCS-2029L, manufactured by QIAGEN) and Attractene Transfection Reagent (301005, manufactured by QIAGEN) according to the manufacturer's recommended procedure. HepG2 cells were seeded on a 96-well plate at 4 × 10 ⁴ cells / well, and 37 ° C., 5% CO 2 using Opti-MEM (31085-062, Gibco) / 5% fetal bovine serum (FBS) medium. _The culture was performed under the conditions of ₂ .
Subsequently, after 16 hours from gene introduction, the medium was changed to Opti-MEM / 0.5% FBS medium 100 μl / well, followed by further cultivation for 4 hours.
After a total of 20 hours from the gene introduction, the test substance (tomatidine or escleogenin) prepared in Example 2 or 4 was added, and cultured and collected for 18 hours.
Then, intracellular luciferase activity was measured using Dual-Luciferase Reporter Assay System (E1910, manufactured by Promega) according to the procedure recommended by the manufacturer. Both the cell lysate and the luciferase substrate were added to a 96-well white plate for luminescence measurement (MS-8096W, manufactured by Sumitomo Bakelite), and a luciferase was measured using a microplate reader (ARVO MX 1420, manufactured by PerkinElmer).
The measurement results of the luciferase activity of tomatidine are shown in FIG. 1, and the measurement results of the luciferase activity of escleogenin are shown in FIG. In FIG. 1, a sample to which tomatidine and tomatine were not added was used as a control, and the relative value with respect to the control was shown for each sample. Moreover, in FIG. 2, the sample which does not add an escleogenin was made into the control, and the relative value with respect to the said control was shown about each sample (n = 3).

From the results in FIG. 1, it was confirmed that tomatidine showed agonistic activity against vitamin D receptor in a concentration-dependent manner and had a vitamin D-like effect. In particular, the agonist activity was as high as 1.28 at 10 nM, 1.57 at 100 nM, 1.98 at 500 nM, and 2.20 at 1000 nM. In contrast, the agonist activity of tomatine on the vitamin D receptor was similar to the control, and no vitamin D-like effect was observed.
Further, from the results shown in FIG. 2, it was confirmed that escleogenin also showed agonistic activity against vitamin D receptor in a concentration-dependent manner and had a vitamin D-like effect. In particular, it shows a high effect of 2.89 even at a low concentration of 0.01 nM, 4.40 at 0.1 nM, 4.43 at 1 nM, 6.94 at 10 nM, 9.18 at 100 nM, and 9.12 at 1000 nM. It showed very high agonist activity.

Example 6:
[Evaluation of changes in filaggrin gene expression (moisturizing effect)]
It is known that vitamin D can obtain an effect of suppressing rough skin and a high moisturizing effect by ingestion or application to the skin. Therefore, the skin barrier effect and moisturizing effect of esculogenin were measured by an atopic dermatitis model evaluation system. Specifically, we evaluated the expression variation of the filaggrin gene, which is present in keratinocytes of the epidermal granule layer and plays an important role in the barrier function and moisturizing effect of the skin, due to esclegenin.

(Sample C)
PSVK1 cells (human keratinocyte cell line, immortalized human normal keratinocyte, JCRB cell bank) are seeded on a 6-well plate to 1 × 10 ⁵ cells / well and cultured overnight at 37 ° C. and 5% CO _2. did. The medium used was MCDB 153 (M7403, manufactured by Sigma) with an additive (* 1 below) added.
(* 1)
5 μg / ml insulin (insulin), 10 μg / ml transferrin, 0.1 mM ethanolamine (cell culture medium additive RD-1 was used. 551-20300-4, manufactured by Kyokuto Pharmaceutical), 0 0.5 μg / ml hydrocortisone (hydrocortisone, 093-06351, manufactured by Wako), 0.1 mM phosphoethanolamine (phosphorethanolamine, P0503, manufactured by Sigma), 10 ng / ml epidermal growth factor (GF, EGF, 44) Sigma), and 40 μg / ml bovine pituitary extract (13028014, invitrogen)

The next day, the medium was changed. The medium used was MCDB 153 (M7403, manufactured by Sigma) with an additive and 1.35 mM CaCl ₂ (06729-55, manufactured by Nacalai Tesque). Further, esculogenin prepared in Example 2 was added so as to have a concentration of 1 μM (= 1000 nM), and cultured at 37 ° C. under 5% CO ₂ for 2 hours. Thereafter, IL-4 (recombinant human interleukin-4, 200-04, manufactured by PeproTech) was added to a final concentration of 20 ng / ml.
Thereafter, the culture medium was changed every other day, the esculeginin was added, and IL-4 was added after 2 hours for 1 week.
Thereafter, the cells were washed twice with 1 × PBS (−), and the cells were collected (sample C).

(Sample B)
Sample B was obtained by treatment and culture in the same manner as Sample C, except that escreogenin was not added.

(Sample A)
Sample A was obtained by performing treatment and culture in the same manner as Sample C, except that escreogenin and IL-4 were not added.

  RNA was extracted from the collected samples from each cell type of Samples A to C using RNeasy Mini Kit (74106, manufactured by QIAGEN). The operation was performed according to the method recommended by the manufacturer. From the RNA thus obtained, cDNA was prepared using PrimeScript (registered trademark) II 1st strand cDNA Synthesis Kit (6210A, manufactured by Takara Bio Inc.). The operation was performed according to the method recommended by the manufacturer.

  Real-time quantitative PCR (qPCR) by TaqMan method was performed using cDNA as a template, and the expression fluctuation of filaggrin gene was evaluated. In qPCR, Hs00856927_g1 was used as the TaqMan probe of filaggrin, and 18S rRNA Hs9999999901_s1 was used as the endogenous control, and Applied Biosystems (registered trademark) 7500 Real Time PCR System (Thermo Fisher Measurement) was used. All operations were performed according to the manufacturer's recommended method (n = 3).

The measurement result of the filaggrin gene expression level of each sample is shown in FIG. The measurement results of samples B and C are shown by relative values with respect to sample A.
From this result, it was confirmed that the expression level of filaggrin gene was attenuated to 0.29 by adding IL-4 to 1.00 of sample A (sample B).
On the other hand, it was found that by adding escleogenin, the attenuation of the expression level of filaggrin gene due to the addition of IL-4 was suppressed to 0.35 (sample C).
From the above results, it was shown that escleogenin has an effect of suppressing attenuation of the filaggrin gene expression level by the addition of IL-4. Thus, it was found that escleogenin has an effect of improving the skin barrier function and moisturizing function. In addition, in Example 5, tomatidine, which was shown to have a high vitamin D-like effect similar to escleogenin, was also shown to have a high skin barrier function and a moisturizing function.

Example 7:
[Sensory test]
As the use article of tomatidine or escleogenin obtained in Examples 1 and 3, the evaluation on the feeling of use of cosmetics was performed by the following evaluation method.
For each of tomatidine and escleogenin, the articles used were applied to the skin of five subjects, and the finish upon application was self-evaluated. As evaluation items for the feeling of use, the moisturizing feeling and the effect of suppressing rough skin were evaluated on a five-point scale according to the following evaluation criteria, and the average value x of five persons was determined.
Evaluation criteria:
5: Very good 4: Good 3: Normal 2: Somewhat bad 1: Bad

In the article using tomatidine, the average value x of moisturizing feeling was 4.2, and the average value x was 3.8 for the effect of suppressing rough skin.
Moreover, in the article using escleogenin, the average value x of moisturizing feeling was 4.0, and the average value x was 4.2 for the effect of suppressing rough skin.
Thus, it has been found that if tomatidine or escleogenin is used, for example, as a cosmetic, a particularly high moisturizing effect can be obtained, and therefore, there is an effect of suppressing dry skin and suppressing rough skin.

Claims (6)

An acid treatment step of acid-treating tomatine to obtain an acid-treated product containing tomatidine; and
And a purification step of purifying the acid-treated product to obtain an acid-treated extract.   The method for producing tomatidine according to claim 1, wherein the acid treatment step includes a neutralization step.   A tomatidine-use article comprising tomatidine obtained by the production method according to claim 1 or 2. An acid treatment step of acid-treating escleoside to obtain an acid-treated product containing esculogenin A and / or B;
And a purification step of purifying the acid-treated product to obtain an acid-treated extract.   The method for producing esclegenin according to claim 4, wherein the acid treatment step includes a neutralization step.   An esculogenin-use article characterized by containing esculogenin obtained by the production method according to claim 4 or 5.