JPS63110259A - Acylated anthocyanin - Google Patents

Abstract

NEW MATERIAL:A compd. of formula I (R1 is H, caffeic acid or ferulic acid residue; R2-R4 are caffeic acid or ferulic acid residue; ANION<-> is anion). EXAMPLE:3-0-{6-0-(2,5-di-O-E-caffeyl-alpha-L-arabinofuranosyl)-beta-D- glycopyranosyl}- 7-0-(6-O-E-caffeyl-beta-D-glucopyranosyl)-3'-0-(6 -O-E-caffeyl-beta-D-glucopyranosyl) cyanidin. USE:A dyestuff for foods, medicines, cosmetics, etc. The compd. of formula I is excellent in acid, alkali, heat and light resistances and is very stable even under neutral - weakly acidic conditions. PREPARATION:A Commelinaceae plant, Zebrina purpusii Brueckn, is extracted with an acid-contg. alcohol (aq.) soln.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) This invention relates to acylated anthocyanins used as pigments in foods, medicines, cosmetics, etc.

(Prior art) Until now, the general formula % formula % (wherein R3 is a hydroxyl group, R5 is a hydroxyl group, R1' is hydrogen, a hydroxyl group, or a methoxy group, and R5= is hydrogen, a hydroxyl group, or a 2-methoxy group.ANION Anthocyanidins represented by - is an anion are known.

(PublicationDevelopments In Food
Co1ours -1Edited by Joh
n Walford (1980) Appli
ed 5ciancs Publishers
Ltd London, or The Fl
avonoidsEdited by J,B
, Harborne, τ, J., Mabry an.
d H, Nabry (1975, 1983) Ch.
See aPman & Hall Ltl.
) to which a sugar is attached has the general formula % formula % (wherein R1 is O-sugar or 0-acylated sugar, R.

is a hydroxyl group or 0-glucose, R, = is hydrogen, a hydroxyl group, or a methoxy group, and RS' is hydrogen, a hydroxyl group, or a methoxy group. ANION- is an anion. ) is andocyanin. Anthocyanins are contained in large amounts in purple corn, berries, grape skins, grape juice, red cabbage, etc., and are produced by soaking the flowers, leaves, or stems of these plants in acid-containing water or alcoholic aqueous solutions. It is used in large quantities as a coloring agent for beverages, foods, confectionery, etc. (Publication Takashi Umeda Sanei News No. 143 15-21 (1983
) See Sanei Chemical Industry, ) (Problem to be solved by the invention) In general, andocyanin exhibits a reddish-purple to blue color in a neutral dilute aqueous solution, but the color is generally very unstable and quickly fades. . However, under acidic conditions it is relatively stable and has a reddish-orange color.

The reason for this is that anthocyanins are Nl0N- (In the formula, R1 is ot* or 0-acylated sugar, R.

is a hydroxyl group or 0-glucose, R8' is hydrogen, a hydroxyl group, or a methoxy group, and R6' is hydrogen, a hydroxyl group, or a methoxy group. ANION- is an anion. ), but it is very stable as a flavylium ion, which occurs at pH 4 to 6. R3' is hydrogen, a hydroxyl group, or a methoxy group; (In the formula, R1 is 0-tang or 0-acylated sugar, R6 is a hydroxyl group or 0-glucose, R3' is hydrogen, hydroxyl group, or methoxy group, and R6' is hydrogen, hydroxyl group, or methoxy group.) This is because it becomes a colorless pseudobase represented by . (R, Brouillard a.
nd B, 5 Laporta, J, A
m, Chew, Sac,, 9913461
(197?), Chikara Hoshino, Chemistry Area, 3723-30
(1983). ) Therefore, there is a problem in that foods, medicines, etc. that utilize anthocyanin pigments easily fade due to the influence of acids or alkalis, and in some cases due to increases in temperature.

In view of these problems, the present inventor has conducted extensive research in order to find more stable anthocyanins from among many plants, and as a result, the paper chromatography method (P P C), or thin layer chromatography (TLC) of cellulose powder
When using either method, tailing is severe and good chromatography cannot be obtained.Furthermore, although HPLC methods are also used in some cases, there is the problem that chromatography with good separation cannot be obtained at pH 3.5 or higher. However, the present inventors have succeeded in discovering a new method for analyzing and preparating andocyanin that completely solves this problem. Through this, stable andocyanin extracted from flowers, leaves, or stems of plants can be obtained. , has a basic structure that is completely different from the conventionally known general andocyanins, and has a structure that is highly acylated with various organic acids (named acylated anthocyanins). It became clear that it had. (Hide Idaka-1
(See JP-A-81-85476, 61-85477) Since the NMR determination of complex acylated anthocyanins was first reported in 1978, four types of complex acylated anthocyanins have been identified: gentioderphin, platyconin, cineralin, and HBA. Only acylated anthocyanins are known (Toshio Goto, Tadao Kondo, Chemistry and Biology 22827 (
It has been revealed that the acylated anthocyanin of the present invention has a structure completely different from those of these four types of anthocyanins, and is a very stable pigment.

That is, this invention provides Nl0N- (wherein R3 is hydrogen, caffeic acid or ferulic acid,
R2 is caffeic acid or ferulic acid, R3 is caffeic acid or ferulic acid, and R4 is caffeic acid or ferulic acid. ANION- is an anion. ) is an acylated anthocyanin. Conventionally, the anthocyanin-based pigments that have been used empirically have not been found to have a method for separating pure anthocyanin and further analyzing this andocyanin.
Stable acylated anthocyanins, in which a sugar is bonded to andocyanidin and an acyl group is bonded to this, cannot be used separately, and a mixture of these has been used as a pigment in foods, medicines, etc.

For this reason, the unstable anthocyanin in anthocyanin-based pigments fades due to the influence of acids or alkalis, and in some cases, due to increases in temperature, and depending on the content of this unstable anthocyanin, the entire pigment may fade or change color. There were problems such as

As a result of intensive research, the present inventor succeeded in discovering a method for fractionating pure andocyanin and an analysis method that can analyze the structure of this anthocyanin, and thus distinguishes between unstable andocyanin and stable anthocyanin. However, only stable andocyanin could be isolated, and this was further analyzed, leading to the structural analysis of acylated anthocyanin having the above-mentioned structure.

Acylated anthocyanin represented by this general formula has anthocyanin core [3-0-[6-O-(a-
L-arabinofuranosyl)-β-D-g
lucopyranosyl)-7-0-(β-D-g
lucopyranasyl)-3'-0-(β-D-
glucopyranasyl) cyanidin]
Each of the two types of organic acids (caffeic acid or ferulic acid) is ester bonded to the two organic acids. Furthermore, these organic acids are coordinated on the A ring or B ring of the anthocyanidin mother nucleus (cyanidin), and the molecule is protected from attack by the hydroxyl group on the C-2 position of the A ring of the anthocyanidin mother nucleus. . Therefore, this acylated anthocyanin has excellent effects on acid resistance, alkali resistance, heat resistance, and light resistance, and is extremely stable. (References regarding the stabilization mechanism of anthocyanins include, for example, (&mm Fuo Kagaku 41559 (1986)). All of the acylated anthocyanins of the present invention are new compounds that have not been described in any literature. Purpusi (Zabrina purPus)
ii) The flowers, leaves, or stems of A. Bruckn.) are crushed, then immersed in an acid-containing alcohol solution or an aqueous solution for extraction, filtered, and vacuum-dried to obtain oily acylated anthocyanins. You can get it. There are three methods for purifying this andocyanin. That is, ether precipitation, purification using an adsorption column, liquid chromatography using a reversed phase distribution column, etc.
For example, in the liquid chromatography method, the anthocyanin is analyzed using a mixed solvent of two or more of acetic acid, acetonitrile, tetrahydrofuran, dioxane, alcohol, and water as the mobile phase, and mineral acid as the acid. ,
Alternatively, add organic acid etc. to about O-5% and adjust the pH to 3°5-0.
Pure acylated anthocyanin can be obtained by further purification by high performance liquid chromatography (HPLC) using a reverse phase partition column [octyl (Cm) column, octadecyl (CIll) column, etc.] with a range of . Analysis of the acylated anthocyanins obtained in step 2 was carried out using acetic acid, acetonitrile, THF,
A mixed solvent of two or more of dioxane, alcohols, and water, and the acids include phosphoric acid, nitric acid,
Mineral acids such as hydrochloric acid and sulfuric acid, or trifluoroacetic acid (TF
Add about 0-5% of organic acids such as A) and adjust the pH to 3.5-
This is carried out by HPLC using a reverse phase partition type column (octyl (C8) column, octadecyl (C+ only) column, etc.) set in the 00 range.

The acylated anthocyanin of this invention was analyzed by HPLC using UV detection sound (column used: osvexost
l 005-5.250 m11! φ4.6 am
1.0. , Column temperature: 40°C1 Mobile phase: Vinegar I
! ! : Acetonitrile: Phosphoric acid: Water = 10:
11:1.5 near 7.5), the retention time (R
t) This acylated anthocyanin (1) (see Figure 1) can be identified by the peak that appears in about 10 minutes.

(Effects of the Invention) As described above, the acylipinthocyanin of the present invention has excellent acid resistance, alkali resistance, heat resistance, and light resistance, and is particularly stable even under neutral to weak acid conditions. When used as a pigment in food, medicine, cosmetics, etc.
The color is extremely stable over long periods of time and does not fade.The acylated anthocyanins of the invention will now be explained in more detail by way of examples.

Example 1 Zebrina purpus
As a result of HPLC analysis, it has become clear that the main component of anthocyanin (I) is contained in fresh leaves. From 10 Kg to pure (1
) 1.2 g was obtained as the hydrochloride. ) The structure was determined as follows.

In order to analyze the partial sideways in (1), an alkaline hydrolysis reaction and an acid hydrolysis reaction were performed. First, in a 2% sodium hydroxide-50% methanol/water solution moat, the reaction temperature was -20
℃ under a nitrogen atmosphere, 4M methyl caffeate and the dye (Product I) [3-Q16-0-
(α-L-arabinofuranasyl)-β-
D-glucopyranosyl-7-0-(β-
D-glucopyran.

5yl)-1'-0-(β-D-glucopyran
cyanidin] was obtained. The reaction formula is shown below.

Alkaline hydrolysis reaction (I) → (II) + 4M methyl caffeate (I
I) is F A B - M S (
Molecular weight 905 from Fast Atom Bo+gbarament ass Sp@ctramstry)
(M'), 6 from its 500MHz 'H-NMR
．． 8-9. Six hydrogen atoms characteristically appear at lppm, and the maximum absorption wavelength in the UV spectrum is 511n (
(0.1% hydrochloric acid in methanol), it was found that the mother nucleus was cyanidin. The UV spectrum is as follows. ^,,x(0,01χHCl- to eOH
,Conc,, 5.3xlO-' mol/l,
r, t,) r++s (6) 511
(25°500), 326 (3,700), 28
0 (17,700); Eaae/E6I+”0°44
, E326/Eill"O, 14 Here, E!11 represents the absorbance at 511ni+. Regarding the absorbance ratio, refer to the literature (Kozo Hayashi Plant Pigments 169 (198
0) 1℃ Kento) Reference, (The entire structure of IHn is 500
It was determined as shown in Fig. 2 by detailed analysis of MHz'HNMR.

Next, an acid partial hydrolysis reaction was performed.

Acid partial hydrolysis reaction CI)→(I I I)+ (IV)+ (V)+ (
VI)+ (VI I) After reaction in 0.7N aqueous hydrochloric acid solution at a reaction temperature of 60°C, HPLC fractionation was performed to obtain four types of acid-hydrolyzed dye products (III-VI). (VII) was obtained as a sugar-organic acid conjugate. From FAB-MS, (
III) is the molecular weight (M”) 1.097 (Cs+
Hsao 2? ), (IV) Molecular weight (M゛)
935 (C4sHaso z2) Also, both (V) and (VI) have a molecular weight (M”) of 611
(C3IM2A0.4), 500 of (III)
From the MHz 'H-NMR spectrum, 6.6-9p
Six hydrogen atoms characteristically appear at p+1, and it is clear that it has a cyanidin skeleton. 5.8-7.
There are two sets of caffeic acid signals extending to 49p■. By decoupling sugar 61 (@ (3-6,8Pps), the three sugars present are glucobylanose rings, and their anomeric hydrogens are all coupled to the coupling constant R(J)7.
．． Since it is 5Hz, it has a β configuration. Also, N0E (Nuclear 0verhauser Ef) at 0℃
fect), the anomeric hydrogen (5,05
PPm, d) is the mother nucleus 4th position (8,40ppm, s) and Δ
The anomeric hydrogen (5,25 ppm, d) is located at the 2' position of the mother nucleus (7,56 ppm, d 1.2 Hz), and the anomeric hydrogen (5,80 PPm, d) is located at the 6 position (6, 78
Since there are negative NOEs at position 8 (6,6'7pp shoulder) and 8th position (6,6'7pp shoulder), the binding site of each part to the mother nucleus is as shown in the structural formula shown in Figure 3 (low temperature negative N in
Regarding OE measurement, refer to the literature, Tadao Kondo et al., 24th NMR
See Abstracts of Symposium Lectures 56-59 (1985). )
. Also, in the sugars marked △ and ・, the methylene hydrogen at the 6th position has been shifted down the magnetic field, so the two molecules of caffeic acid are Δ,
It is connected to the 6th place in the Kaiin section. From the above results, the structural formula of (Technical II) is as shown in Figure 3. (II
I) UV spectrum and 50OMI-(z'H
-All assignments of NMR spectra are shown below.

3-Q-(β-11-gLucopyranosyl)
-7-0-(6-Q-E-caffeyl-β-D-g
lucopyranosyl)-3'-0-(6-0-
E-caffeyl-β-D-glucapyrano
syl) cyanidin (I II I)Cs
+H1Jav”l, 097 υVλ,,, (0,01χHCI/ to eOH,con
c,, 2.7xlO''mol/l, 20"C)
nm (g) 529 (21,200), 330
(21,900).

286 (23,700); Eg, /E, as”0.2
8. E3□/Eq2e Engineering 1.04IH-NMR (50
0 to Hz, 32 CF, GOOD/CD, 00. O
'C), δ (ppm) 8.48 (IH, br,
d, Jlla, 5. H-6°), 8.
40 (LH, s, H-4), 7.56 (IH, d
, Jllll, 2. H-2'), 7.18 (Hl
, d, J=16. H-β), 7.05(il
l, d, J=8.5. H-5″), 6.97
(IH, d, J=16.H-β), 6.78(
IH, d, J-2, 0, H-6), 6.67 (i
ll, d, J-2,0, H-8), 6.58(
IH, d, J=8.5. I(-5”), 6.2
8 (LH, d, J=8.0.H-5"), 6.
15(11(,br,d,Jlla,O,H-6”
), 6.12(1)1゜d, J-16,H-α)
, 6.10 (IH, d, J=1.0.H-2"
).

6.09 (IH, d, J-1,0,1(-2″),
6.00 (ill, br, d, J-8,0
, H-6''), 5.81 (IH, d, J-16
, H-α), S, aO(IH, d, J-1,0
,・-1), 5.25(IH,d, J-7,
51Δ mark 1), 5.19 (IH, br, d, JI1
12. Δ mark 6a), 5.06 (1M, br,,
・-6a), 5.05 (1B, d, Jll
? ,5. Mu-1), 4.04 (3H, br, a
, J, 7.5.・-6b, Mu-6a, 6b
), 4゜03 (IH, m, mu-5), 3.
91 (114, ■, Δ mark 5), 3.86 (IH,
t, J-9, 0, Δ mark 3), 3.79 (IH, t, J-
9,0,@-3), 3.75(l)I, dL
J snow? ,5. 9.0. Mu-2), 3.
71 (18゜dd, J depth 7.5. 9.0. Δ
Mark 2), 3.70 (18, dd, J depth 7.5° 9.0. ・-2), 3.66 (2H, t,
J snow 9. Olmu-3,4).

3.43 (LH, t, J-9, J ・-4),
3.40 (ill, t, J=9.0, Δ mark 4)
, 3.38 (IH, br, Δ mark 6b).

HOE, ・-1→H-6-9z, ・-1-pH
-13-1g, A-1→H-4-B, Δmark 1→
H-2' -101゜However, Δ mark is 3-0-β-D
- Shows the substitution position of hydrogen in the glucopyranosyl group. The * mark indicates the substitution position of hydrogen in the 7-0-β-D-glucopyranosyl group. The Δ mark indicates the substitution position of hydrogen in the 3′-0-β-D-glucopyranosyl group. The shoulder indicates the substitution position of hydrogen in the α-L-arabinofuranosyl group, 6a, 6
b or 5a, 5b indicates the hydrogen signal appearing on the low magnetic field side with a, and the hydrogen signal appearing on the high magnetic field side with b,
, the substitution position of the hydrogen signal of the organic acid is H-2”, H-
5", H-6", H-a, H-11. Substitution position 1 of the hydrogen signal of the mother nucleus (cyanidin) 4.
Toro, H-8, H-2', H-4', H-5'T
Shown. UV7. to') is the maximum absorption wavelength (λ1..)
The molecular extinction coefficient (ε) is shown in 0.The absorbance ratio at each wavelength is also shown.The NMR spectrum shows the chemical shift (δppm), water II, coupling, coupling constant, and attribution in 0. Shown in order of parts.NO
E indicates that, for example, Mu-1 → H-4-4% indicates that when the hydrogen nucleus at Mu-1 position is irradiated, a negative NOE of =4% is observed in the hydrogen signal at H-4 position. There is.

Next, the structure of (IV) was analyzed by 500 MHz 'H-NMR as follows. Δ,・The existence of NOE when irradiating the anomeric hydrogen in the tank, and the fact that the methylene hydrogen at position 6 of the Δ,・inka is shifted down the magnetic field.
(IV) is 3-0-(β-D-glucopyrano
syl)-7-0-(6-0-E-caffsyl-β
-D-glucopyran.

5yl)-3'-0-(6-0-E-caffeyl-
β-D-glucopyranosyl) cyani
From din (I I I ), it is presumed that the sugar bound to the 3rd position of the mother nucleus is missing. This is because when the NOE at the 4th position of the mother nucleus is set to 1, no NOE is absorbed by any anomeric hydrogen, (IV)
was confirmed to have the structural formula shown in FIG. (
All assignments of the 500 MHz 'H-NMR spectrum of IV) are shown below.

7-O-(S-0-E-caffsyl-β-D-gl
ucopyranosyl)-3'-0-(6-(]-
]E-caffsyl-β-D-glucopyran
osyl) cyanidin (IV) C45H,,022-935') I-NMR (500MHz, 3! CF, GOO
D/CD, 00.40” C), 6 (pp ■) 1
1.48(1)1. dd, J-2,0,8,5
, H-6'), 8.10(1)1. s, H
-4), 7.77 (IH, d, J-2,0,
) I-2'), 7゜24 (IH, d, J-11
i, H-β), 7.03 (IH, d, J-1
6,H-β), 7.0L(L)I,d, J-9
,0,I(-5'),6.80(1)1. d.

J-1, 5, H-6), 6.70 (IH, d,
J-1,5,H-8), 8.50(LH,d,
J=8.0. H-5''), 6.31 (18, d
, J=8.0. 11-5”), 6.23(2
H, d, J=2.0. H-2”, dd,
J=2.0. 8. CI, )l-6”), 6.15(
1)! , dd, J=2.0. 8.0. H
-6”), 6.11 (1M, d, J-16,H
-α), 5.79 (IH, d, J-16, H-
α), 5.41 (IH, d, J=7.5.・-
1), 5.15 (IH, d, J-7, 5, Δ mark 1)
, 5.09(1)1. dd, J-2, o,
12. Δ mark 6a).

4.97 (IH, dd, J-2, 5, 12, e-6
a), 4.11 (IH.

dd, J-9,0,12,・-8b), 4.04(
IH, dd, J=9.5. 12, Δmark 6b),
4.0L (IH, ddd, J-2, 5, 9, Q,
9.5.・-5), 3.90 (IH, ddd
, J-2, 0, 9, 0, 9, 5, Δ mark 5).

3.75 (IH, t, J=9.5. -3),
3.71(1)1. t, J=9.0, Δ mark 3
), 3.66 (IH9dd, J-7,5,9,
5, -2), 3.64 (IH, dd, Jl17.
5. 9.0. Δ mark 2), 3.4H1)! ,
t.

1st Army 9.5.・-4), 3.39(IH,t,
J-9,0,Δ-4).

It was revealed from FAB-MS and NMR that (V) and (Vl) had the structures shown in FIGS. 5 and 6, respectively.

(VII) is returned in 50% yield during acid partial hydrolysis. UV spectrum, FAB-MS and NMR
The full spectrum assignments are shown below.

Nethyl 2.5-di-0-caffsyl-α
-L-arabinafuranasid@ (V'I
I) C2a82mOz"488 UV λ ll,, (>1eOH, 20"
C) nm 207. 21F, 333FAB-
MS mHz-4119(M+1)'H-NMR(50
0MHz, CD, 00. -26”C),
δ (ppm) 7.60 (IH, d, J-11i
, H-β), 7.59 (IH, d, J, 16.
H-β), 7.03 (IH, d, J-2,0,
H-2”), 6.92 (IH, dd.

J-2,0,13,0,H-6''), 6.88(l
)I, dd, J-2,0,8,Q.

)1-6”), 6.71i(1)1.d, J-
11,0,11-5'''), 6.72(1)1゜d
, J-8,0,H-5”), 6.29(IH,d
, J-16, H-α).

6.27 (IH, d, J-16, H-α), 5
．． 02 (IH, dd, J wealth 0.8, 2.5. ■-
2), 4.94(1)l, s, ■-1),
4.47(1)1゜dd, J-3,5,12,
■-5a), 4.34 (IH, dd, J-5,
0゜12,--5b), 4.20(L)I, dd
d, J-3,5,5,Q,6. (1°■-4),
4.13 (1M, dd, Jtomi2.5.6.
0. ■-3).

The core sugar is arabinose based on the coupling constant.

Caffeic acid in 2ifl is bound to the 2nd and 5th positions because the hydrogen signals at the 2nd and 5th positions are shifted down the magnetic field in NMR. To determine the anomeric coordination (α, β) and absolute configuration H (D, L) structure of arabinofuranoside,
By alkaline hydrolysis of (VII), ll@thyl a
-L-arabinafuranoside (VI
II) was obtained. This is a specimen obtained by synthesis (Neth
yl a-L-arabinofuranoside)
UV, NMR, and FAB-MS completely matched.

Next, (VIII) was benzoylated to produce Netbyl.
2.1.5-tri-0-b@nzayl-α-L-a
rabinafuranosida (IX),
Standard Methyl 2,3.5-tri-0-bar
H.

yl-a-L-arabinofuranosida and Netbyl 2,3.5-tri-0-he
nzayl-a-0-arabinofuranosi
When comparing a@(X) and CD spectra, it was found that Ne
thyl 2,3.5-tri-0-benzoyl-
It completely matched with α-L-arabfnafuranosids. Therefore, the structure of the pentagonal core is α-L-a
It became clear that it was rabinafuranosida.

Next, we examined the entire structure of (1). According to FAB-MS, the molecular weight (M) is 1,553, and the core is cyanidin. The results of alkaline hydrolysis revealed that 4M caffeic acid was ester bonded to the sugar, and the results of acid partial hydrolysis revealed the bonding positions of 3M glucose and 2M caffeic acid. It was also confirmed that the remaining IM sugar was α-L-arabinose, and that the 2M caffeic acid listed was ester bonded to the second α-17-binose.

CI) UV, FAB-MS and 500MHz'H-
All assignments of the NMR spectra are shown below.

3-Q-+6-0-(2,5-di-0-E-caff
eyl-α-L-arabinofuranosyl)
-β-D-glucopyranosyl-7-0-
(6-0-E-caff@yl-β-D-gLucop
yranosyL)-3'-0-(6-0-E-caf
fsyl-β-D-glucapyranosyl)
cyanidin (I) CyJvt (h7
”l, 553 UV λ ,, (0,01χ )IC1/MeO
H, cone, 2.7xlO-'mal/l,
20” C) nm (ε) 532 (25,0
00), 329 (51,800).

292(47,300):(1/:log phas
Phata buffer, pH6.5, con
c,, 2.7xlO-'++ol/l, 20°
C) 583 (28,800).

543 (27,100), 506 (13,900)
, 470 (5,700), 323 (36,50
0), 306 (39,300), 237 (36
,100) FAB-83mHz 1,553 (M
″)l H-NMR (at 500)IZ, 32C
FtCOOD/CD+OD, -20・C), δ
(ppm) 8.40 (IH, br, d, J
s9.5. H-6'), 8.28 (IH,s,
H-4), 7.41 (IH, d, J Tomi 1
6. H-β), 7J7 (1M, br,
H-2'), 7.28 (IH, d, J-1
6, H-β), 6゜99 (1M, d, J-9
,5,)I-5'), 6.93(2H,d, J
=16. H-β), 6.91 (IH, d,
J=1.3. H-2”), 6.84 (IH, d
, J=1.3. H-2'"), 6.73(
1B, d, J=11. H-6), 6,
65(1)1. dd, J-1,3,8,0,11-
6″), 6.63 (IH, d, ・J-1, 3, H-
8), 6.61 (LH, d, J-8,0,H-
5”), 6.59 (IH, d, J-8, 0,)
I-5''), 6.53 (IH, d, J-8,
0, H-5″), 6.50 (IH, dd, J-1,
3, a, o, H-6”), 6.26 (1) 1.
d, J-1,3,H-2″), 6.21(
IH, d, J snow 8.0. 11-5”), 6.14
(IH, d, JI116.H-cr), 6.06 (
IH, d.

J=1.3. H-2”), 6.055 (LH, dd
, J-1,3,8,0,H-6”), 6.05(I
H, d, J-16, H-α), 5.87 (IH, d
, J=1+i, H-α), 5.76(IH,d,J
x16. H-a), 5.75 (IH, d, J-
16,H-α), 5.313(IH,d,
J-to 7.51 ・-1), 5.20 (IH,s,
■-1), 5.18 (LH, d, J-1, 3, ■
-2), 5.07 (IH, d, J-7, 5, Δ
-1), 5.03 (IH, d.

J=7.5. Mu-1), 4.92 (LH, dd
, J-9,5,12,・-6a), 4.45(
IH, dd, J=2.0.12. Rin-5a), 4
．． 31 (IH, dd, J-5, 0, 12, ■-5b
), 4.25 (IH, m, -14), 4.
18 (IH, br, d, J=10.mu-6
a), 4.02(1)1. dd, J=1.3.
9.0. ■-3), 3.94 (IH, br,
d, J! 12°Δ-6a), 3.92(
IH, dd, J-7, 5, 12, ・-6b),
3.90 (IH, br, d, J=12. Δ
-6b), 3.86 (2H, m, mu-5, mu-
6b), 3.81(1)1. 11.・-5)
, 3.74 (LH, wr.

Δ-5), 3.71 (LH, dd, J-7, 5, 9
,0,mu-2), 3゜71(LH,t, J-9
,0,・-3), 3.67(IH, dd, J-
7.5°9.0.・-2), 3.61i (IH
, dd, J-7, 5, 9, 0, Δ-2), 3.
65 (2H, t, J-9, 0, Mu-3, Δ-3),
3.44(1)1゜t, J-9,0,mu-4)
, 3.38 (IH, t, J-9,0, Δ-4)
.

HOE A-1-*H-4-4L H-4,4-1
-302,・-1→H-6-5L @-14H-! l
-17L Δ-1+H-2' -20L
H-2゜→Δ-1-23χ, Mu1→Mu-3,5,Δ
-1→Δ-3.5゜11-1 facing■-3,41mu-1,6
i, 6b, 14-α (6,05), H
-5" (6, 21), shoe-2 → mu-1, 6a, 6b,
・-1-" H-a (5,76), H-2" (I
i, 06), H-2" (6, 26) → H-α (
6,05), H-β(6,93), H-2″(
S, OS) → H-α (5,76), 11-β (
6,93).

First, the mother nucleus characteristically appears in the low magnetic field, with (H-4 position, H-6 position, H-8 position, H-2' position, H-5' (
Stand, )! -6' position) was found to be cyanidin.Next, four sets of caffeic acid digals were identified (H
-β, H-2-H-6", H-5", H-a)m
The double bonds are all in the trans configuration (E) from J=16Hz.

By IH-NMR decoupling in the line region, the three sugars are D-glucobylanose rings. Furthermore, all of these anomeric hydrogens (@, mu, and Δ sugar) were in the β configuration based on the coupling constant (J = 7.5 Hz). Since the methylene hydrogen at the 6th position of each of the Δ and Mu marks is shifted down the magnetic field, two molecules of caffeic acid are bonded to the 6th position of the Δ and Mu marks. The pentose (■ mark) is an α-L-arabinofuranose ring according to the coupling constant.

Since the hydrogen at the 1st position of arabinose has an HOE with the 16th position of the mu mark, the 1st position of arabinose has a glucosidic bond with the 6th position of the mu mark. The remaining 2M caffeic acid has ester bonds at the 2- and 5-positions because the hydrogens at the 2- and 5-positions of the arabinofuranose ring are shifted down the magnetic field. Based on the above results, the structure of (I) was determined as shown in Figure 1.

Example 2 Zebrina pendula (Japanese name: Hakatakaraksa or Shimahumurasakitsuyukusa, scientific name: Zabrinapen)
dula 5chnitzlain) flowers, leaves, and
The stem also has 3-0-+5-0-(2,5-di-0-E-c
affeyl-a-L-arabinofurano
syl)-β-D-glucopyranosyl-
7-0-(6-0-E-caffeyl-β-D-gl
ucopyranasyl)-3'-0-(6-0-E
-caffeyl-β-D-glucapyranos
yl) cyanidin (I), 3-0-
[6-O-(5-0-E-caffeyl-α-L-a
rabinofuranosyl)-β-D-gluc
opyranosyl-7-0-(6-0-E-ca
ffayl-β-D-glucopyranosyl)
-3'-0-(6-0-E-caffeyl-β-D-
glucopyranosyl) cyanidin
(X I ), and 3-0-+6-O-(
2-0-E-caffeyl-α-L-arabino
furanosyl)-β-D-glucopyran
osyl-7-0-(6-0-E-caffeyl-
β-D-glucopyranosyl)-3'-0-
(6-0-E-caffeyl-β-D-glucap
cyanidin (X I
As a result of HPLC analysis, it was confirmed that acylated anthocyanins such as I) were contained. (XI) and (XrI) were also fractionated by HPLC, and their structures were determined using 500MHz'H-N
Analysis was performed using MRS FAB-MS, UVI:.

From FAB-MS, (XI) and (XII) both have the same molecular weight [+*/zl, 391 (M”) (C
esHa70+4)] was given. As a result of the alkaline hydrolysis reaction, 3-G-+6-O-((
Z-L-arabinafuranosyl)-β-
D-glucopyranosyl-7-Q-(β-
D-glucapyranasyl)-3'-0-(β
-D-glucopyranosyl) cyanid
in (I I ) and 3M caffeic acid were obtained.

As a result of acid partial hydrolysis reaction, in the case of (XI),
(III), (IV), (V), (VI
) and Nethyl 5-O-E-caffeyl
-α-L-arabinofuranaside was obtained. On the other hand, in the case of (XII), (Eng. II),
(IV), (V), (VI) and Net
hyl 2-O-E-caffeyl-α-L-ara
binofuranaside was obtained. From the above results, the structure of (XI) is 3-0-+6-O-(5-0-E
-caffayl-cr -L-arabtnofur
anosyl)-β-D-glucapyranosy
ll-7-0-(6-0-E-caffeyl-β-D
-glucopyranosyl)-3'-0-(6-
0-E-caffayl-β-to11ucopyran
osyl) cyanidin, and the structure of (XII) is 3-O-16-0-(2-0-E-caffey
l-a-L-arabinafuranosyl)-
β-D-glucopyranosyl-7-〇-
(6-0-E-caffeyl-β-D-glucop
yranasyl)-3'-0-(6-0-E-caf
feyl-β-D-glucopyranosyl)
It is cyanidin.

Example 3 Purple goten (scientific name: 5etcreas@a pu)
HPLC analysis revealed that rpureaBOO) also contains several types of acylated anthocyanins, but one of the main components, anthocyanin (XIII), was also analyzed using HPLC. and its structure was similarly determined at 500 MHz 'H-N
Analysis was performed by MR2FAB-MS and UV.

From FAB-MS, molecule 1 (M-) is 1,609 (C?l
UV in 1/30M phosphate buffer solution at pH 6.5
From the spectrum, the maximum absorption wavelength (λ, 8) and molecular extinction coefficient (logε) are each 237 nm (4, 57).

310n+* (4,64), 508nm (4,
16), 543 nm (4,:14) and 58
4 nm (4,44). As a result of the alkaline hydrolysis reaction, (II) and 4M ferulic acid were obtained. As a result of acid partial hydrolysis reaction, 3-
0-β-D-glucopyranosyl-7-0-
(6-0-E-ferulyl-β-D-glucop
yranasyl)-3°-0-(6-0-E-fer
ulyl-β-D-glucopyranosyl)
cyanidin (XIV),
Two-row ([1-O-E-ferulyl-β-D
-glucopyranosyl)-3'-0-(5-
0-E-fsrulyl-β-D-glucopyra
nosyl) cyanidin (XV), 7-0-
(6-0-E-ferulyl-β-D-gluco
pyranosy1) cyanidin (X V
I), 3'-0-(6-0-E-faruly
l-β-D-glucopyranosyl) cyan
idin (XVII) and)l
athyl 2.5-di-0-ferulyl-α-
L-arabinofuranosida (XVIII
) was rejected. Accordingly, the structure of (XIII) is 3
-O-+6-0-(2,5-di-0-E-ferul
yl-cr-L-arabinafuranosyl
)-β-D-glucopyranosyl-7-Q
-(6-0-E-ferulyl-β-D-gluco
pyranosyl)-3'-0-(6-0-E-fa
rulyl-β-D-glucopyranosyl)
It is cyanidin.

Example 4 Murasakiomoto (scientific name: Rhoeo 5patha)
As a result of HPLC analysis, it was revealed that ceaa W, T, 5tearn> also contained several types of acylated anthocyanins, but anthocyanin (XIX), which is the main component of the width, was also separated by HPLC. The structure was determined in the same manner.

FAB-ki (S to molecule 1! (M”) is 1,433 (C
a=HTIOa, ).

UV in 1/30M phosphoric acid mild solution at pH 6.5
From the spectrum, λ1□ and logε are respectively 2]6n
m (4,5:l), 312nm (4,54),
508nm (4,16), 544ni (4,41
) and 588 nm (4,45).

As a result of the alkaline hydrolysis reaction of (XIX), (II)
3M's ferulic acid was returned. In addition, as a result of acid hydrolysis, (XIV), (XV), (XV engineering),
(XVII) and Mstyl 5-O-f
erulyl-α-L-arabinofuranos
ide (X X ) was obtained. Therefore, (XIX
) has the structure 3-016-0-(5-0-E-ferul
yl-cr-L-arabinofuranosyl)
-β-Q-glucopyranosyl-7-0-
(6-0-E-farulyl-β-D-glucop
yranosyl)-3'-0-(6-Q-E-fer
ulyl-β-D-glucopyranosyl)
It is cyanidin.

Comparative Example pH 6,5, (in 1/30M phosphate buffer solution)
I), (II), (III), (XrII
).

The results of comparing the stability of (XIX) under UV light are shown in FIG. From this fact, acylated anthocyanin is considerably more stable than deacyl compound (II), and the stability increases in proportion to the number of acyl groups.

[Brief explanation of the drawing]

Figure 1 shows 3-0-+6-0-(2,5-di-0-E
-caff@yl-a -L-arabinofura
nosyl)-β-D-glucopyranosyl
l-7-0-(6-0-E-caffeyl-β-D-
glucapyranosyl)-3'-0-(6-0
-E-caffayl-β-D-glucopyran
asyl) Structural formula of cyantdin (I), second
The figure shows 3-o-+5-o-(ff-L-arabino
furanosyl)-β-o-glucopyra
nosyll-7-〇-(β-D-glucopyra
nosyl)-3'-0-(β-D-glucopyr
anasyl) cyanldin (II), Figure 3 shows the 3-0- (β-D-glucopyra
nosyl)-7-0-(6-0-E-caf fey
l-β-D-glucapyranosyl)-3'-
0-(Ei-0-E-caffail-β-D-glu
c. pyranosyl) cyanidin (II
The structural formula of I), Figure 4 is 7-O-(6-0-E-caf
feyl-β-D-glucopyranasyl)-
3'-0-(6-0-E-caffeyl-β-D-g
lucapyranosyl) cyanidin (
The structural formula of I V ), Figure 5, is? -0-(6-0-E
-caffeyl-β-D-glucopyranos
yl) The structural formula of cyanidin (V), Figure 6, is 3'-0-(6-0-E-caffeyl-β
-D-glucopyranasyl) cyanid
The structural formula of in (VI), Figure 7, is 3-0-+6
-0-(2,5-di-0-E-caffayl-α-
L-arabinofuranosyl)-β-D-g
lucapyranosyll-7-0-(6-0-E
-caffayl-β-D-glucopyranos
yl)-3'-0-(6-0-E-caffeyl-β
-D-glucapyranosyl) cyani
din (engineering), 3-016-0-(α-L-a
rabinofuranosyl)-β-D-gluc
apyranosyl-7-0-(β-D-gluc
opyranosyl)-3'-o-(β-D-glu
(copyranosyl) cyanidin (I
D), 3-0-(β-D-glucopyrano
syl)-7-0-(6-0-E-caffeyl-β
-D-glucopyranasyl)-3'-0-(
6-0-E-caffayl-β-D-glucopy
ranosyl) cyanidin (I I
I), 3-0-+6-0-(2,5-di-
0-E-ferulyl-α-L-arabinofu
ranosyl)-β-D-glucopyranos
yll-7-0-(6-0-E-ferulyl-β-
D-glucapyranosyl)-3°-0-(6
-0-E-farulyl-β-D-glucopyr
anosyl) cyanidin (X I I
), and 3-0-+6-O-(5-0-E-fs
rulyl-α-L-arabinofuranosy
l)-β-D-glucopyranasyll-7-
0-(6-0-E-faru1411-β-D-glu
capyranosyl)-3'-0-(6-0-E-
ferulyl-β-D-glucapyranosy
1) is a graph showing a comparison of the stability of cyanidin (X I X ). Figure 7 Time

Claims (1)

[Claims] General formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R_1 is hydrogen, caffeic acid or ferulic acid, R_2 is caffeic acid or ferulic acid, R_3 is
Caffeic acid or ferulic acid, R_4 is caffeic acid or ferulic acid. ANION^- is an anion. ) Acylated anthocyanin.