JP3199948B2 - Cyanidin-3-Zp-coumaroyl-sambubiosides and process for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel anthocyanin dye represented by the following formula (1), which belongs to an anthocyanin dye which is stable to heat, light and the like and does not fade even when stored for a long period of time.

[0002]

Embedded image

In the formula, R represents H or a glycosyl group,
Sam is 2-O-β-D-xylopyranosyl-β-D-
A cyanidin-3 represented by a glucopyranosyl group;
-Zp-coumaroyl-sambubiosides, ie,
Cyanidin-3- (Zp-coumaroyl-sambubioside) wherein R is H in the formula (1) [hereinafter referred to as (A) compound] and cyanidin-3- (where R in the formula (1) is a glucosyl group. Zp-coumaroyl-sambubioside)-
5-glucoside [hereinafter referred to as (B) compound].

[0004] Further, the present invention relates to cyanidin-3- (E
-P-coumaroyl-sambubioside) -5-glucoside [hereinafter referred to as compound (C)]
About 5 langley to about 50 visible light and / or ultraviolet light
The present invention relates to a method for producing a dye solution having a high color value, which comprises producing the compound (A) and / or the compound (B) by irradiating with an irradiation amount within the range of 0 langley.

[0005]

2. Description of the Related Art
Examples of the anthocyanin-based dyes similar in structure to the compounds (A) and (B) include elderberry (Sambuc
us genus) extracted and isolated from the fruits of cyanidin-3-sanbubioside-5-glucoside, cyanidin-3-glucoside-5-glucoside, cyanidin-3-sanbubioside, cyanidin-3-glucoside, cyanidin-3-.
(Ep-coumaroyl-sambubioside) -5-glucoside and the like are known [Phytochemistry 30, (12), 4137-
4141 (1991)].

However, these conventionally known anthocyanin dyes are not always satisfactory in, for example, stability to light, oxygen, heat and the like, and have a drawback in use that they are accompanied by a change in color tone and discoloration over time. .

[0007]

Means for Solving the Problems The present inventors have been conducting research on anthocyanin pigments extracted from elderberry fruits. As a result, a new stable dye, cyanidin-3-E-p-coumaroyl-sambubioside, was found and proposed earlier (Japanese Patent Application No. Hei 5-26).
No. 5530). After that, as a result of further research,
Irradiation of the elderberry extract with visible light and / or ultraviolet light increased the color value of the whole extract. To determine the cause, cyanidin-3- (E-p
-Coumaroyl-sambubioside) -5-glucoside was exposed to visible light and / or ultraviolet light and analyzed by HPLC analysis, NMR, and the like. As a result, the compound (A), which is a novel compound, and ( B) It was found that a compound was formed. Furthermore, they have found that these compounds are stable to light, oxygen, heat, etc., and are useful as a colorant for various products without a change in color tone and discoloration over time, and completed the present invention. .

Accordingly, a main object of the present invention is to provide the compounds (A) and (B) which are stable to light, oxygen, heat and the like, and exhibit little change in color tone and discoloration over time, and a process for producing them. To provide. Further, another object of the present invention is to use the compound, food and drink, cosmetics,
It is to provide a coloring agent useful for coloring various products such as medicines.

According to the present invention, the compound (A) and the compound (B) are derived from Canadian native elderberry ( Sambucu).
s canadensis ) fruit extract, which is present in trace amounts, and / or cyanidin-3- (E-p-
Coumaroyl-sambubioside) -5-glucoside [(C) compound] can be easily isolated by irradiating visible light and / or ultraviolet light with column chromatography, preparative HPLC and the like to separate and purify. it can. Hereinafter, the present invention will be described more specifically. The preparation of the extract from the elderberry fruit is performed, for example, by adding about 3 to about 10 parts by weight of an acidic aqueous solution or acid alcohol solution to 1 part by weight of the shredded fruit, and adding about 10 to 10 parts by weight.
The extraction can be performed by leaving the mixture at a temperature of about 60 ° C. for about 0.5 to about 4 hours or performing an extraction treatment under stirring conditions. Acids that can be used in acidic aqueous solutions or acidic alcohol solutions include, for example, hydrochloric acid, citric acid, tartaric acid, phosphoric acid, acetic acid, and the like.Alcohols include, for example, methanol,
Ethanol and the like can be mentioned.

On the other hand, the compound (C) can be prepared, for example, as follows: First, the extract obtained by the above-mentioned method is recovered as a solvent to obtain a concentrated solution. The concentrate is partitioned between water and an organic solvent such as ethyl acetate, ethyl ether and the like. The aqueous layer containing the water-soluble (C) compound was subjected to MCI gel, SP-207, HP-20
(Mitsubishi Kasei), Amberlite XAD-2, Amberlite XAD-4 (Rohm & Haas)
After adsorption to a chromatographic column packed with a porous synthetic adsorbent such as, it is washed with water to perform desalting and desalting, and then
The crude product containing the compound (C) is eluted with a lower alcohol such as methanol or ethanol or a lower alcohol such as methanol or ethanol containing 0.1 to 1% of an acid such as hydrochloric acid, phosphoric acid or trifluoroacetic acid. The dye solutions can each be separated. In addition, as a preparation method other than the above-mentioned method, as a raw material, a compound constituting the skeleton of the compound (C) is converted by an enzyme or a microorganism.
Alternatively, a pure chemical synthesis method or the like may be used alone or in combination. Next, the irradiation method is described above by using a light source that generates visible light and / or ultraviolet light alone or in combination, such as sunlight, a commercially available ultraviolet light generator (an ultraviolet fade meter manufactured by Suga Test Instruments Co., Ltd.), or the like. The extraction and / or the dye solution containing the compound (C) obtained by the above method can be carried out by irradiating visible light and / or ultraviolet light. The irradiation amount varies depending on the concentration, pH, etc. of the dye, but generally, about 5 la.
ngley to about 500 langley, preferably about 10 langley to
Illumination doses in the range of about 100 langley can be exemplified.

The above irradiation produces the compounds (A) and (B), and these compounds are cis-forms of coumaric acid which binds to the sugar at the 3-position of the anthocyanidin skeleton. All natural acylated anthocyanin dyes reported so far are trans-forms, and no cis-forms have been reported. The cis-form produced by irradiation is surprisingly about 2.4 compared to the trans-form when measured with a solution at pH 3.0.
It has twice the color value, which reduces the overall color value of the extract by about 2
It can be increased by about 30%, which is extremely useful in practical use.

The extract and / or dye solution after irradiation is recovered as a concentrated solution by recovering the solvent by an ordinary method. As in the case of the separation and purification of the compound (C), the concentrated solution is treated with water and ethyl acetate, Partition between organic solvents such as ethyl ether.
The aqueous layer containing the water-soluble compound (A) and / or the compound (B) was subjected to MCI gel, SP-207, HP-2
0 (Mitsubishi Kasei), Amberlite XAD-2, Amberlite XAD-4 (Rohm & Haas
After adsorption on a chromatographic column packed with a porous synthetic adsorbent such as Co., Ltd., washing with water for desalting and desalting, and then 0.1-1% hydrochloric acid, phosphoric acid, trifluoro The crude dye solution containing the compound (A) or (B) is separated by elution with a lower alcohol such as methanol or ethanol containing an acid such as acetic acid, or a lower alcohol such as methanol or ethanol.

Next, the crude dye solution containing the compound (A) is subjected to, for example, Develosil ODS-5.
(A) can be obtained by preparatively purifying by HPLC using a commercially available column such as (Nomura Chemical), Capcell Pak C-18 (Shiseido), Puresil (Waters). . Further, by performing the same operation on the crude dye solution containing the compound (B), a highly purified compound (B) can be obtained. HPLC (φ25 ×
250 mm) column, using a gradient elution method (eluent A;
5% H ₃ PO _4, eluent _{B; 0.5% H 3 PO 4} , 39.5
% H ₂ O, 60% tetrahydrofuran), and the structures of the compounds (A) and (B) can be analyzed by UV-VIS spectrum, NMR measurement and the like.

The compounds (A) and (B) obtained as described above are stable to light, oxygen, heat and the like, and exhibit little change in color tone and little discoloration over time. It is useful as a coloring agent for various products such as food and drink, cosmetics, and medicine. Especially (A)
The compound and the compound (B) are isomers of the respective compounds, cyanidin-3- (Ep-coumaroyl-sambubioside) [(D) the compound and the cyanidin-3- (E
-P-coumaroyl-sambubioside) -5-glucoside has higher thermal stability.

The form of use when the compound (A) and the compound (B) are used as a coloring agent is not particularly limited. For example, the compound (A) and the compound (B) may be used alone, It can also be used in the form of a composition with a suitable diluent or carrier. Examples of such diluents or carriers include solid diluents or carriers such as gum arabic, dextrin, glucose, and sucrose; and liquid diluents or carriers such as water, ethanol, propylene glycol, glycerin, and surfactants. Examples can be given.

The compounds (A) and (B) of the present invention can be used in any dosage form, for example, powder, granule, liquid, emulsified liquid, paste and other appropriate dosage forms. be able to. For example, gum arabic, dextrin, etc. may be added and used in the form of powder, granules or the like, or used as a liquid form by dissolving in ethanol, propylene glycol, glycerin or a mixture thereof. You can also.

The compounds (A) and (B) of the present invention, which are stable at an acidic pH, are stable to light, oxygen, heat and the like. Can be effectively prevented from fading due to light, oxygen, heat, etc., and the color tone can be stably maintained for a long period of time. Such products include food and drink,
Feeds, health and pharmaceuticals, cosmetics, etc. can be mentioned. For example, beverages such as fruitless beverages, fruit-containing beverages, lactic acid beverages, and powdered beverages; ice creams, sorbets, frozen desserts such as frozen desserts; pudding, jelly, bavarois, yogurt, and other desserts; Foods and drinks and luxury goods such as bean jam, processed meat food, grilled meat sauce, pickles; health and pharmaceuticals such as tablets, liquid oral medicines, powdered oral medicines, poultices, etc .; or, for example, soaps, detergents, shampoos It is useful as a coloring agent for cosmetics such as coloring.

Hereinafter, the present invention will be described more specifically with reference to examples.

[0019]

【Example】

Example 1 1.0 kg of frozen fruit of Sambucus canadensis added 0.1% H
1000 ml of Cl-MeOH was added, and the mixture was pulverized with a Waring blender twice in half each. Next, 3000 ml of 0.1% HCl-MeOH was added to the pulverized product, and extraction was performed with stirring at 35 ° C. for 2 hours. Immediately after the extraction, filtration was carried out using powdered cellulose (commercial product: Diafloc) as an aid to obtain 3750 g of a filtrate containing the compound (C).
The filtrate is concentrated under reduced pressure at a temperature of 35 to 45 ° C.
g was obtained.

Example 2 850 g of the concentrated solution obtained in Example 1 was placed in a 2 liter Erlenmeyer flask, and a commercially available ultraviolet ray generator [UV fade meter manufactured by Suga Test Instruments Co., Ltd.] was used for 1 hour.
Irradiation of 250 langley was performed. Table 1 shows the relationship between the irradiation amount and the color value and the amount of each dye compound obtained by analysis. The color value was measured by diluting the concentrated solution after irradiation 250-fold, and the amount of each dye compound was indicated by its peak area in HPLC analysis.

[0021]

[Table 1] Table 1: Changes in irradiation amount and color value Irradiation amount (langley) 0 19 39 97 245 Color value 0.499 0.644 0.631 0.609 0.547 (C) compound 0.109 0.062 0.055 0.050 0.032 (A) compound 0 trace 0.002 0.003 0.006 ( B) Compound 0 0.044 0.040 0.038 0.024 (D) Compound 0 trace 0.004 0.007 0.017 As shown in Table 1, the color value after irradiation with 19 langley was about 30% higher than that without irradiation. . The non-irradiated compound was only the compound (C), but the compounds (A), (B) and (D) were formed by irradiation.

Example 3 Ethyl acetate (500 g) was added to 850 g of the concentrate obtained in Example 1.
Add 3 ml and distribute to remove fat-soluble substances etc.
I went there. The resulting dye solution is converted to SP, a porous synthetic adsorbent.
The solution was passed through a column packed with 300 ml of -207 to adsorb the dye, and then washed with water for desalting and desalting. Then, 1
After elution with 600 ml of% trifluoroacetic acid-MeOH, methanol was distilled off to obtain a crude pigment. Next, the crude pigment is HPL
Preparative purification by C. HPLC is Capcell P
Using a column of ak C ₁₈ (φ25 × 250 mm), gradient elution method (eluent A; 0.5% H ₃ PO ₄ , B; 0.5% H ₃ P)
O ₄ , 39.5% H ₂ O, 60% tetrahydrofuran) to separate the dye solutions containing the compound (A) and the compound (B).

Example 4 When one of the separated dye solutions was examined for UV spectrum, the maximum absorption wavelength in the visible region was around 525 nm.
The shift to the longer wavelength side due to the addition of Cl ₃ suggested that ring B of the anthocyanidin skeleton had an ortho-diphenol structure. From the data of E ₄₄₀ / E _VIS (%), it was presumed that only the third position was bonded to sugar. 315n
Since m also has an absorption, it is suggested that a cinnamic acid type acyl group is present. From the value of E ₃₁₅ / E _VIS (%), it was estimated that one acyl group was present in the molecule. The result of SIMS was considered to have two sugars.

Example 5 Further analysis of the above compound by ¹ H- ¹ HCOSY spectrum revealed that it had a cyanidin skeleton and cis-p-co
one molecule of umaroyl group and one molecule of glucose and one molecule of xylose in the molecule,
It became clear that they were combined. Further, glucose is bound to the 3-position of the cyanidin skeleton, the 2-position is xylosylated, and the 6-position is acylated, ie, cis-p-coumar.
oyl group was found to be bonded, UV-VIS, S
Together with the result of the IMS measurement, the compound
-3-O- (6-Zp-coumaroyl-2-O-β-D-xylopyranosyl) -β-D
The structure was determined to be -glucopyranoside, that is, the compound (A). Table 2 shows the ¹ H-NMR spectrum data.

Example 6 When the UV spectrum of the other compound separated in Example 4 was examined, the maximum absorption wavelength in the visible region was shifted to a longer wavelength side near 525 nm due to the addition of AlCl ₃ .
It was suggested that the ring B of the anthocyanidin skeleton had an orthodiphenol structure. In addition, from the data of E ₄₄₀ / E _VIS (%), it was estimated that the 3rd and 5th positions were bonded to sugar. The absorption at 315 nm also suggests the presence of a cinnamic acid type acyl group, and it was presumed from the value of E ₃₁₅ / E _VIS (%) that the molecule had one acyl group in the molecule.
From SIMS results, this compound was considered to have three sugars.

Example 7 ¹ H- ¹ HCOSY spectrum analysis of the above compound showed that it had a cyanidin skeleton and cis-p-co
It has one molecule of umaroyl group, and two molecules of glucose and one molecule of xylose exist in the molecule, and β
It became clear that they were combined. In addition, one molecule of glucose was bonded to the 5-position of the cyanidin skeleton, and another molecule of glucose was bonded to the 3-position. The 2-position was xylosylated and the 6-position was acylated, that is, the cis-p-coumaroyl group was bonded. It was found that they were bonded, UV-VIS, SIMS
Together with the results of the measurement, the above compound, cyanidin-3-O-
(6-Zp-coumaroyl-2-O-β-D-xylopyranosyl) -β-D-gluc
The structure was determined as opyranosyl-5-O-β-D-glucopyranoside, that is, the compound (B). The ¹ H-NMR spectrum data is shown below.

[0027]

Table 2: ¹ H-NMR spectral data [(CD3) 2SO: CF3COOD = 9: 1; 400 MHz; room temp .; H (ppm) J (Hz)] (A) Compound (B) Compound Cyanidin 4 8.78 s 8.65 s 6 6.75 d 1.8 6.97 d 1.8 8 6.83 d 1.8 7.00 d 1.8 2 '8.00 d 1.8 8.03 d 1.8 5' 7.06 d 8.5 7.06 d 8.6 6 '8.30 dd 8.5,1.8 8.33 dd 8.6,1.8 Glucose-A1 5.69 d 7.9 5.72 d 7.3 2 3.96 dd 8.5,7.9 4.03 dd 8.5,7.3 3 3.71 dd 9.4,8.5 3.72 dd 9.8,8.5 4 3.38 t 9.4 3.37 t 9.8 5 3.9 m 3.97 br dd 9.8,8.6 6a 4.26 dd 12.2,7.9 4.21 dd 12.2,8.6 6b 4.45 br d 12.2 4.43 br d 12.2 Glucose-B 1 5.13 d 7.9 2 3.55 dd 9.2,7.9 3 3.40 t 9.2 4 3.30 t 9.2 5 3.55 dd 9.2,4.9 6a 3.66 dd 11.6,4.9 6b 3.85 br d 11.6 Xylose-C 1 4.71 d 7.3 4.72 d 7.9 2 3.03 dd 8.6,7.3 3.00 dd 8.5,7.9 3 3.16 dd 9.8,8.6 3.14 dd 9.8,8.5 4 3.28 td 9.8,5.5 3.23 td 9.8,5.5 5a 3.52 dd 11.0, 5.5 3.52 dd 11.4,5.5 5b 2.95 dd 11.0,9.8 2.93 dd 11.4,9. 8 Zp-Coumaroyl 2 ", 6" 7.53 d 9.2 7.52 d 9.2 3 ", 5" 6.66 d 9.2 6.62 d 9.2 7 "(α) 6.72 d 12.8 6.85 d 12.8 8" (β) 5.76 d 12.8 5.79 d 12.8 Example 8 500 g of ethyl acetate was added to 850 g of the concentrate obtained in Example 1.
Add 3 ml and distribute to remove fat-soluble substances etc.
I went there. The resulting dye solution is converted to SP, a porous synthetic adsorbent.
The solution was passed through a column packed with 300 ml of -207 to adsorb the dye, and then washed with water for desalting and desalting. Then, 1
After elution with 600 ml of% trifluoroacetic acid-MeOH, methanol was distilled off to obtain a crude pigment. Next, the crude pigment is HPL
Preparative purification by C. HPLC is Capcell P
Using a column of ak C ₁₈ (φ25 × 250 mm), gradient elution method (eluent A; 0.5% H ₃ PO ₄ , B; 0.5% H ₃₎
PO _4, carried out by _{39.5% H 2 O, 60%} Tetrahydrofuran), the dye was isolated. The isolated dye is UV, S
It was confirmed to be the compound (C) from measurement data such as IMS and NMR.

Example 9 The compound (C) isolated in Example 8 was dissolved and diluted in 0.2% citrate buffer (pH 3.0), and Example 2 was used.
Irradiation was performed in the same manner as described above. Table 3 shows the relationship between the irradiation amount and the color value and the amount of each dye compound obtained by analysis. In addition, the amount of each coloring compound is shown by each peak area in HPLC analysis.

[0029]

Table 3: Change in irradiation dose and color value Irradiation dose (langley) 0 20 41 98 243 Color value 0.649 0.842 0.826 0.801 0.723 (C) compound 0.138 0.080 0.071 0.063 0.043 (A) compound 0 trace 0.002 0.004 0.008 ( B) Compound 0 0.060 0.060 0.057 0.034 (D) Compound 0 trace 0.004 0.010 0.022 As shown in Table 3, the color value after irradiation with 20 langley was about 30% higher than that without irradiation. . The non-irradiated compound was only the compound (C), but the compounds (A), (B) and (D) were formed by irradiation.

Example 10 3 kg of the dye solution after irradiation obtained in Example 9 was passed through a column filled with 100 ml of SP-207, which is a porous synthetic adsorbent, to adsorb the dye and washed with water. Desalting and desalting were performed. Next, after elution with 1% trifluoroacetic acid-MeOH (200 ml), methanol was distilled off to obtain a crude pigment. Next, the crude dye was fractionated and purified by HPLC. HPLC is Cap
Using a column of cell Pak C ₁₈ (φ25 × 250 mm), gradient elution method (eluent A; 0.5% H ₃ PO ₄ , B;
0.5% H ₃ PO ₄ , 39.5% H ₂ O, 60% Tetrahydr
ofuran) to separate crude dye solutions containing the compound (A) and the compound (B).

Example 11 The two kinds of crude dye solutions obtained in Example 10 were subjected to the operations shown in Examples 4 to 7 to obtain the separated dye in accordance with the results of UV-VIS, SIMS measurement and NMR. Is (A)
The structure was determined with the compound and the compound (B). Separately from this, HPLC analysis and conventional reports also confirmed the formation of the compound (D) in the dye solution after irradiation. That is, the compound (A), the compound (B),
(D) It was confirmed that three of the compounds were formed.

Example 12 The compound (C) in which the coumaric acid bonded to the 3-position of the anthocyanidin skeleton obtained in Example 8 is bonded in a trans form and the cis isomer obtained in Example 3 The thermal stability of a certain compound (B) was measured. Compound (C) and compound (B) were mixed with 0.2 M citrate buffer (pH 3.
0) and placed in a thermostat at 80 ° C., and the time-dependent change in color value for 60 minutes was measured. Table 4 shows the results.

[0033]

Table 4: Time- dependent change in color value (min) 0 15 30 60 (C) Compound 0.748 0.540 0.535 0.532 (B) Compound 0.752 0.575 0.566 0.563 As shown in Table 4, cis type (B) The color value of the compound was about 5% higher than that of the compound (C) when stored at 80 ° C. for up to 60 minutes, and the thermal stability was excellent.

[0034]

The present invention provides cyanidin-3-Z-p-coumaroyl-sambubiosides, which are novel compounds belonging to anthocyanin-based dyes that are stable against heat, light and the like. The compound can be blended with various products such as foods and drinks, cosmetics, and pharmaceuticals to obtain a colorant that does not fade even after long-term storage.

Continuing from the front page (72) Inventor Hideko Kinoshita 335, Karijuku, Hasegawa-Koryu, Nakahara-ku, Kawasaki-shi, Kanagawa Prefecture (72) Inventor Toshi Tamura, 335, Karijuku, Nakahara-ku, Nakahara-ku, Kawasaki-shi, Kanagawa, Japan R & D Center for Technology (56 References JP-A-62-209173 (JP, A) JP-A-63-234167 (JP, A) JP-A-63-113078 (JP, A) JP-A-5-292989 (JP, A) 57-58871 (JP, A) JP-A-64-74271 (JP, A) JP-A-1-224389 (JP, A) JP-A-3-14596 (JP, A) JP-A-3-223298 (JP, A A) JP-A-6-125750 (JP, A) JP-A-6-271850 (JP, A) JP-A-63-110259 (JP, A) JP-A-63-168464 (JP, A) Phytochemistry, 38 ( 3), 755-7 (1995) (58) Fields investigated (Int. Cl. ⁷ , DB name) C09B 61/00 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] [Claim 1] The following formula (1) In the formula, R represents H or a glycosyl group, and Sam represents 2-
A cyanidin-3-Z-p-coumaroyl-sambubioside represented by the formula, which represents an O-β-D-xylopyranosyl-β-D-glucopyranosyl group. 2. A dye solution containing cyanidin-3- (E-p-coumaroyl-sambubioside) -5-glucoside is irradiated with visible light and / or ultraviolet light at an irradiation dose in the range of about 5 langley to about 500 langley. A process for producing a dye solution having a high color value, thereby producing the compound of the formula (1) according to claim 1.