Acylated anthocyanin and process for producing the same as well as pigment composition containing the same

A novel acylated anthocyanin of the formula: ##STR1## (wherein R.sup.1 and R.sup.2 may be the same or different and each represents a hydrogen atom, a ferulyl group or a caffeyl group; R.sup.3 and R.sup.4 may be the same or different and each represents a ferulyl group or a caffeyl group; and ANION.sup.- represents an anion) and a process for producing the same, as well as a pigment composition containing said anthocyanin.

FIELD OF THE INVENTION: 
The present invention relates to an acylated anthocyanin of the formula 
(I): 
##STR2## 
(wherein R.sup.1 and R.sup.2 may be the same or different and each 
represents a hydrogen atom, a ferulyl group or a caffeyl group; R.sup.3 
and R.sup.4 may be the same or different and each represents a ferulyl 
group or a caffeyl group; and ANION.sup.- represents an anion) and a 
process for producing the same. The present invention also pertains to a 
pigment composition containing said anthocyanin. 
Acylated anthocyanins according to the present invention are pigments 
derived from a natural source, have markedly superior stability and 
therefore are useful as coloring agents for foods, pharmaceuticals, 
cosmetics, etc. 
PRIOR ART 
Anthocyanidins of the formula (II): 
##STR3## 
(wherein R.sup.5 and R.sup.6 may be the same or different and each 
represents a hydrogen atom, hydroxyl group or methoxy group) are well 
known [see e.g., Developments in Food Colours-I, edited by John Walford 
(1980), Applied Science Publishers Ltd., London, or The Flavonoids, edited 
by J. B. Harborne, T. J. Mabry and H. Mabry (1975, 1983), Chapman & Hall 
Ltd.] 
Anthocyanins of the formula (III): 
##STR4## 
(wherein R.sup.5 and R.sup.6 are as defined above; R.sup.7 represents a 
glycosyl or acyl-glycosyl group; and R.sup.8 represents a hydrogen atom or 
a glycosyl group), which comprise a sugar component attached to the 
anthocyanidins of the formula (II), are contained in large quantities in 
purple corn, berries, the rind of grapes, grape juice, red cabbage and the 
like. Therefore, pigments which are produced by steeping the flowers, 
leaves, stems or fruits of these plants in water or an aqueous alcohol 
solution containing an acid are widely used as coloring agents for 
beverages, foods, candies, etc. see Takeshi Umeda "Sanei News" No. 143 
(1983), Sanei Kagaku Kogyo, pp. 15-21]. 
Athocyanins show generally a purplish red to blue color in a dilute aqueous 
solution at a neutral pH, but they are generally unstable and rapidly 
discolor under neutral to alkaline conditions, although under acidic 
conditions they are relatively stable and assume a red to orange color. 
This is because anthocyanins are present in acidic solutions in the form 
of flavylium ions represented by the aforementioned formula (III). On the 
other hand, in aqueous solutions of pH 4-6, anthocyanins are present in 
the form of anhydro-bases of the formula (IV): 
##STR5## 
(wherein R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are the same as those 
defined above) which are unstable and show a purple or blue color, and the 
anhydro-bases readily hydrate to form colorless pseudo-bases of the 
formula (V): 
##STR6## 
(wherein R.sup.5, R.sup.6, R.sup.7 and R.sup.8 are the same as defined 
above) [see R. Brouillard and B. Delaporte, J. Am. Chem. Soc., 998461 
(1977), or Tsutomu Hoshino "Kagakuno Ryoiki", 37, 23-30 (1983)]. 
Accordingly, foods and pharmaceuticals that utilize anthocyanin pigments 
readily lose their color due to the effect of acids or alkalis or in 
raised temperature conditions under particular circumstances. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a novel acylated 
anthocyanin which has an attractive color which is red in acidic 
conditions and is stable at a wide pH range. 
Another object of the present invention is to provide a method of obtaining 
said acylated anthocyanin from natural sources, that are plants. 
A further object of the present invention is to provide a pigment 
composition for foods, pharmaceuticals or cosmetics, etc. which is stable 
over long periods of time.

DETAILED DESCRIPTION OF THE INVENTION 
In view of the above-described problems, the present inventor made 
exhaustive studies in order to find stable anthocyanins from a wide 
variety of plants. In the conventional analysis of anthocyanins such as 
paper chromatography (PPC) or the thin layer chromatography (TLC) on 
cellulose powder, tailing of the pigments is so heavy that it is difficult 
to separate them satisfactorily. Even the HPLC method which in general 
gives high resolution has not been able to give satisfactory separation 
patterns when applied to anthocyanins. 
In these circumstances the inventor thought that if he could establish a 
method which enables satisfactory separation of anthocyanins he might be 
able to find stable anthocyanins. Motivated by the above assumption, he 
developed a novel method of analyzing or separating anthocyanins. Briefly, 
the method is characterized in that the separation of anthocyanins is 
conducted in acidic conditions which do not exceed pH 3.5. This novel 
method enabled clear cut separation of anthocyanins from various types of 
plants, and the inventor finally found stable types of anthocyanin which 
form the subject matter of the invention and which are expressed by the 
above-described formula (I). 
The structure of the anthocyanins of the invention is completely different 
from that of the known anthocyanins since the former is highly acylated by 
various residues of organic acid (these stable anthocyanins of the 
invention will be hereinafter referred to as "acylated anthocyanins"). The 
present invention has been accomplished on the basis of this finding. As 
to acylated anthocyanins, the only four known ones are gentiodelphin, 
platyconin, cinerarin and HBA (see Toshio Goto and Tadao Kondo "Kagaku to 
Seibutsu", Vol. 22, p. 827, 1984). However, the known acylated 
anthocyanins have very complicated structures which are completely 
different from the basic structure of the novel acylated anthocyanins of 
the formula (I). On top of that, even though HBA is nearly as stable as 
the acylated anthocyanins of the invention, the natural sources of HBA is 
too limited to obtain them on commercial bases. All the other three of the 
known acylated anthocyanins are not stable. 
A novel acylated anthocyanin according to the present invention may be 
produced by the following process. 
For example, the flowers, leaves or stems of Zebrina purpusii Bruechen 
belonging to Commelinaceae are ground. Then, the resulting powder is 
steeped in an alcoholic solvent or aqueous solvent which contains an acid 
to carry out extraction, and the extract thus obtained is filtered and 
then dried in vacuum, thereby obtaining an oily crude acylated 
anthocyanin. There are three different methods of purifying this crude 
acylated anthocyanin, that is, ether precipitation, purification by means 
of an adsorption column, and liquid chromatography employing a counter 
current partition column. For example, in the liquid chromatography 
method, the crude acylated anthocyanin is purified by a high performance 
liquid chromatography (HPLC) that employs a counter current partition 
column [an octyl (C.sub.8) column, octadecyl (C.sub.18) column, etc.] with 
a mobile phase which comprises a mixed solvent composed of two or more 
elements selected from the group consisting of acetic acid, acetonitrile, 
tetrahydrofuran (THF), dioxane, alcohols and water, and which further 
contains about 0-5% of a mineral acid, organic acid or the like as an acid 
so that the pH value of the mobile phase is in the range of 3.5-0, thereby 
obtaining a pure acylated anthocyanin (Ia) [a compound of the formula (I), 
wherein each of R.sup.1, R.sup.2, R.sup.3 and R.sup.4 represents a caffeyl 
group], i.e., 
3-0-{6-0-(2,5-di-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyra 
nosyl}-7-0-(6-0-E-caffeyl-.beta.-D-glucopyranosyl)-3'-0(6-0-E-caffeyl-.beta 
.-D-glucopyranosyl)cyanidin. (Ia) 
This acylated anthocyanin (Ia) is identified by a peak which appears at a 
retention time (RT) of about 10 minutes in the HPLC analysis (column: 
Develosil ODS-C5, manufactured by Nomura Kagaku Kenkyusho Ltd., Japan, 250 
mm.times.4.6 mm I.D.; column temperature: 40.degree. C.; mobile phase: a 
mixed solvent of acetic acid, acetonitrile, phosphoric acid and water 
mixed in the proportion 10:11:1.5:77.5) conducted using a UV detector. 
By similarly extracting, separating and purifying the flowers, leaves or 
stems of the plant Zebrina pendula Schnitzlein, it is possible to isolate 
as the major pigments the above-described acylated anthocyanin (Ia) and 
two other novel acylated anthocyanins, that is, an acylated anthocyanin 
(Ib) [a compound of the formula (I), wherein R.sup.1 represents a hydrogen 
atom; and each of R.sup.2, R.sup.3 and R.sup.4 represents a caffeyl 
group], i.e., 
3-0-{6-0-(5-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosyl 
}-7-0-(6-0-E-caffeyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-caffeyl-.beta.-D- 
glucopyranosyl)cyanidin, (Ib) 
and an acylated anthocyanin (Ic) [a compound of the formula (I), wherein 
R.sup.2 represents a hydrogen atom; and each of R.sup.1, R.sup.3 and 
R.sup.4 represents a caffeyl group], i.e. 
3-0-{6-0-(2-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosyl 
}-7-0-(6-0-E-caffeyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-caffeyl-.beta.-D- 
glucopyranosyl)cyanidin. (Ic) 
By similarly processing the plant Setcreasea purpurea BOOM, it is possible 
to isolate as a main pigment a novel acylated anthocyanin (Id) [a compound 
of the formula (I), wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 
represent ferulyl group], i.e., 
3-0-{6-0-(2,5-di-0-E-ferulyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyra 
nosyl}-7-0-(6-0-E-ferulyl-.beta.-D-glucopyranosyl)-3'-0-(6-0E-ferulyl-.beta 
.-D-glucopyranosyl)cyanidin. (Id) 
By similarly processing the plant Rhoeo spathaceae W. T. Stearn, it is 
possible to isolate a novel acylated anthocyanin (Ie) [a compound of the 
formula (I), wherein R.sup.1 represents a hydrogen atom; and each R.sup.2, 
R.sup.3 and R.sup.4 represents a ferulyl group], i.e., 
3-0-{6-0-(5-0-E-ferulyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosyl 
}-7-0-(6-0-E-ferulyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-ferulyl-.beta.-D- 
glucopyranosyl)cyanidin. (Ie) 
The acylated anthocyanins of the invention cause no health hazards since 
they are components of edible plants. This safeness in combination with 
the clear color and the stability of acylated anthocyanins means that they 
offer great promise for use as pigments in the coloring of 
pharmaceuticals, foods and cosmetics and the like. The amount of pigment 
used in said product may be determined by routine tests, but usually 
0.01-10% of the pigment on the basis of the total volume of the product 
will be sufficient to give it a desired color. 
The acylated anthocyanin of the invention may be used singly or in 
combination with one or more suitable carriers and/or additives 
conventionally used to form a pigment composition. For example, the 
anthocyanin may be dissolved in a suitable medium preferably at pH 3-8. 
The present invention will be explained more specifically below by way of 
Examples. However, the present invention is in no way restricted to these 
Examples. 
EXAMPLE 1 
Fresh leaves (6.4 kg) of Zebrina purpusii was placed in liquid nitrogen, 
and the frozen leaves were immediately ground and then steeped in 5.3 l of 
methanol containing 1% hydrochloric acid for 15 hours at 4.degree. C. to 
extract pigments. The extraction with 3 l of the acidic methanol under the 
same condition was repeated with the extraction residue. The two batches 
of the extracted solution were combined and left to stand overnight at an 
ambient temperature and the precipitates formed were then filtered off. 
The filtrate was mixed with 26.5 l of water and the mixed solution was 
subjected to an Amberlite XAD-7 column (560 mm.times.80 mm I.D.) which had 
been equilibrated with water. The column was washed with 1l of 40% aqueous 
methanol and the successively eluted with 45% aqueous methanol (0.5 l), 
50% aqueous methanol (0.5 l), 55% aqueous methanol (0.5 l), 65% aqueous 
methanol (6 l), 75% aqueous methanol (5 l), 80% aqueous methanol (1 l) and 
methanol (3 l) each of which contains 0.5% TFA. The fraction of 65% 
aqueous methanol containing 0.5% TFA was collected followed by 
concentration under a reduced pressure. The residue was dissolved in a 
small amount of methanol containing 1% HCl. Five volume of ether was 
slowly added to the solution to precipitate the pigments which were then 
collected to obtain about 700 mg of crude anthocyanin. The crude 
anthocyanin was divided into 70 mg portions and each portion was dissolved 
in a small amount of 0.5% aqueous TFA and subjected to a purification step 
by HPLC (column: ODS-C 10/20, 250 mm.times.10 mm I.D.; mobile phase: 
acetic acid/acetonitril/TFA/water=5.4:6.8:0.5:87.3), whereby a main 
pigment component obtained was the acylated anthocyanin of the formula 
(Ia) [a compound of the formula (I), wherein each of R.sup.1, R.sup.2, 
R.sup.3 and R.sup.4 represents a caffeyl group], i.e., 
[3-0-{6-0-(2,5-di-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyr 
anosyl]-7-0-(6-0-E-caffeyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-caffeyl--.b 
eta.-D-glucopyranosyl)cyanidin]. 
Analysis: C.sub.74 H.sub.73,O.sub.37 =1,553 
UV .lambda..sub.max nm (.epsilon.): (0.01% HCl/MeOH, conc., 
2.7.times.10.sup.-5 mol/l, 20.degree. C.); 532(25,000), 329(51,800), 
292(47,300): (1/30 M phosphate buffer, pH 6.5, conc., 2.7.times.10.sup.-5 
mol/l, 20.degree. C.); 583(28,800), 543(27,100), 506(13,900), 470(5,700), 
323(36,500), 306(39,300), 237(36,100) 
FAB-MS (m/z):1,553 (M.sup.+) 
.sup.1 H-NMR (500 MHz, 3% CF.sub.3 COOD/CD.sub.3 OD, -20.degree. C., 
.delta.(ppm): 8.40(1H,br.d,J=9.5), 8.28(1H,s), 7.41(1H,d,J=16), 
7.37(1H,br), 7.28(1H,d,J=16), 6.99(1H,d,J=9.5), 6.93(2H,d,J=16), 
6.91(1H,d,J=1.3), 6.84(1H,d,J=1.3), 6.73(1H,d,J=1.3), 6.65(1H,dd,J=1.3, 
8.0), 6.63(1H,d,J=1.3), 6.61(1H,d,J=8.0), 6.59(1H,d,J=8.0), 
6.53(1H,d,J=8.0), 6.50(1H,dd,J=1.3, 8.0), 6.26(1H,d,J=1.3), 
6.21(1H,d,J=8.0), 6.14(1H,d,J=16), 6.06(1H,d,J=1.3), 6.055(1H,dd,J=1.3, 
8.0), 6.05(1H,d,J=16), 5.87(1H,d,J=16), 5.76(1H,d,J=16), 5.75(1H,d,J=16), 
5.38(1H,d,J=7.5), 5.20(1H,s), 5.18(1H,d,J=1.3), 5.07(1H,d,J=7.5), 
5.03(1H,d,J=7.5), 4.92(1H,dd,J=9.5, 12), 4.45(1H,dd,J=2.0, 12), 
4.31(1H,dd,J=5.0, 12), 4.25(1H,m), 4.18(1H,br.d,J=10), 4.02(1H,dd,J=1.3, 
9.0), 3.94(1H,br.d,J=12), 3.92(1H,dd,J=7.5, 12), 3.90(1H,br.d,J=12), 
3.86(2H,m), 3.81(1H,m), 3.74(1H,m), 3.71(1H,dd,J=7.5, 9.0), 
3.71(1H,t,J=9.0), 3.67(1H,dd,J=7.5, 9.0), 3.66(1H,dd,J=7.5, 9.0), 
3.65(2H,t,J=9.0), 3.44(1H,t,J=9.0), 3.38(1H,t,J=9.0) 
EXAMPLE 2 
One kilogram of fresh leaves of Zebrina pendula Schnitzlein was extracted 
and separated in the same way as in Example 1 to obtain the same acylated 
anthocyanin (Ia) as that obtained in Example 1 and two other novel 
acylated anthocyanins that is (Ib): 
[3-0-{6-0-(5-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosy 
l}-7-0-(6-0-E-caffeyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-caffeyl-.beta.-D 
-glucopyranosyl)cyanidin], 
and (Ic): 
[30-{6-0-(2-0-E-caffeyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosyl 
}-7-0-(6-0E-caffeyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-caffeyl-.beta.-D-g 
lucopyranosyl)cyanidin]. 
The structures of (Ib) and (Ic) were identified from the fact that FAB-MS 
of both of them was m/Z=1.319 (M.sup.+)(C.sub.65 H.sub.67 O.sub.34) from 
the results of alkaline hydrolysis and acidic partial hydrolysis. 
EXAMPLE 3 
One kilogram of fresh leaves of Setcreasea purpurea BOOM was processed in 
the same way as in Example 1 to obtain a novel acylated anthocyanin (Id): 
[3-0-(6-0-(2,5-di-0-E-ferulyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyr 
anosyl}-7-0-(6-0-E-ferulyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-ferulyl-.be 
ta.-D-glucopyranosyl)cyanidin]. 
Analysis: 
FAB-MS (m/Z): 1,609 (M.sup.+) (C.sub.74,H.sub.81 O.sub.37) UV spectrum 
(.lambda..sub.max nm, log .epsilon., pH 6.5): 237(4.57), 310(4.64), 
508(4.16), 543(4.34), 584(4.44) 
The structure of (Id) was determined from the above analysis data and the 
results of alkaline hydrolysis and acidic partial hydrolysis. 
EXAMPLE 4 
One kilogram of fresh leaves of Rhoeo spathaceae W. T. Stearn was processed 
in the same way as in Example 1 to obtain a novel acylated anthocyanin 
(Ie), the principal pigment component of the material: 
[3-0-{6-0-(5-0-E-ferulyl-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosy 
l)-7-0-(6-0-E-ferulyl-.beta.-D-glucopyranosyl)-3'-0-(6-0-E-ferulyl-.beta.-D 
-glucopyranosyl)cyanidin]. 
Analysis: FAB-MS (m/Z): 1,433 (M.sup.+) (C.sub.68 H.sub.73 O.sub.34) 
UV spectrum (.lambda..sub.max nm, log .epsilon., pH 6.5): 236(4.53), 
312(4.54), 508(4.16), 544(4.41), 588(4.45) 
The structure of (Ie) was similarly determined from the analysis data and 
the results of alkaline hydrolysis and acidic partial hydrolysis. 
All products obtained in Examples 1-4 had attractive colors which are 
generally red in acidic conditions. 
EXAMPLE 5 
The stability of the novel acylated anthocyanins of the invention was 
compared with that of 
3-0-{6-0-.alpha.-L-arabinofuranosyl)-.beta.-D-glucopyranosyl}-7-0-(.beta.- 
D-glucopyranosyl)-3'-0-(.beta.-D-glucopyranosyl)cyanidin which has a 
structure corresponding to the compound of Example 1 except that it has no 
acyl group. These anthocyanins were dissolved in a 1/30 M phosphate buffer 
at pH 6.5 and kept at 
20.degree. C. As can be seen from the results given in FIG. 1, the 
anthocyanins of the invention had a superior stability over the 
comparative anthocyanin since the former showed no discoloration as 
measured by the absorbancy at .lambda..sub.max for over 120 minutes.