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2.1 Introduction
and cardiovascular illnesses, cancer and diabetes (Konczak and Zhang, 2004).
A good extraction procedure should maximize anthocyanins recovery and
minimize degradation or alteration of the native state. The presence of acids is
an important requirement to stabilize anthocyanins in aqueous environment as
acidic solutions are necessary for obtaining the flavylium cation form, which is
the most stable form. The acids, however, may also change the native form of
anthocyanins in the tissue during extraction. Hydrochloric acid is the most
common mineral acid employed in anthocyanin extraction. High concentration
of acids could cause the hydrolysis of anthocyanins. A concentration of 1%
hydrochloric acid in methanol was found to cause partial hydrolysis of acyl
moieties in acylated anthocyanins although it is helpful to increase the
extraction rate of total monomeric anthocyanins (Revilla et al., 1998).
Extraction with solvents containing Hel may cause pigment degradation during
concentration. That is why acylations with aliphatic acids had been usually
underestimated (Strack and Wray, 1989). To minimize the degradation of
anthocyanins, weaker and volatile organic acids such as trifluoroacetic acid,
about 0.5%-3%, are used in the solvents (Revilla et al., 1998). Many solvents
can be used in extraction, such as methanol, acetone, ethanol and water, due to
the polar character. Most anthocyanins occur naturally as glycosides.
Anthocyanin glycosides have higher solubility in water than the corresponding
aglycones. Giusti and Wrolstad have developed a method to extract
anthocyanins, which involves powdering a sample with liquid nitrogen,
extraction with aqueous acetone (30:70. v/v) followed by partition with
chloroform (Giusti and Wrolstad, 1996).
The young and emerging purple coloured leaves of sweet potato have
been known as a potential source of anthocyanin (Jyothi et al., 2005). For a
commercial production process, it is important to shorten the extraction time
and to increase the extractability from natural sources. This section deals with
29
Section II. �raction, Purification andIsofation of)fotfiocyanins
All the plant materials required for the anthocyanin estimation were
collected from the experimental fields of CTCRI (Central Tuber Crops
Research Institute, Thiruvananthapuram, India). From the germplasm field,
genotypes having purple coloured leaves were selected for anthocyanin
estimation. In majority of cases, only the young and emerging leaves at the
apex of sweet potato vein (4-6 leaves from top) are pink coloured, but in the
case of S-purple, almost all the leaves are purple coloured. Amberlite XAD-7
and Sephadex LH-20 were purchased from Sigma-Aldrich and all other organic
solvents used were of analytical grade. Spectral analyses were done on a
JASCO V-550 spectrophotometer. The HPLC equipment consisted of a liquid
chromatograph system (Gilson, France) equipped with Gilson UV-Vis 156
detector attached to a Gilson 321 binary gradient pump. Extract concentration
/·
was done in a Buchi R-200 (Buchi, Labortechnik, Switzerland) rotary
evaporator. Remi Incubator (India), which operates in a temperature range, 0-
��-C, was used.
The solvent extraction has been the most common method for extraction
of diverse compounds found in plants, including flavonoids. The phenolic
compounds have been extracted by grinding, drying or lyophilizing the samples
30
Section II. P.x:J;raction} Purification andIsofation offlntfwcyanins
31
Section II. ®(traction, Purification andIsofation ofJfotliocyanins
l .L',1 I Sample
Filtration
Freeze dryino/tlash
• i-,
e, aporation
Figure 2.1: Flow chart for the extraction and fractionation of anthocyanin from
sweet potato leaves
32
Section II. p.:{!raction, CFurification atufIsol4tion of)1.ntliocyanins
Among the most common methods are those which use acidified
methanol or ethanol as extractants (Amr and AI-Tamimi, 2007). After
extraction, the extracts of anthocyanins are concentrated under vacuum, and
purified using chromatographic techniques. A flow chart for the extraction and
fractionation of anthocyanin from sweet potato leaves is given in Figure 2.1.
The procedure must allow for recovery of the anthocyanins avoiding any
chemical modification. The effect of various extraction solvent systems at
various pH values and temperatures on anthocyanin extraction is discussed
below.
33
Section II. P.:{Jraction, <Purification an£Isofation of)lntfiocyanins
Fresh leaves were used for all the experiments. The leaves were chopped
and weighed accurately (2.5 g). The sample was then homogenized with 50 ml
of the extracting solvent and extracted as detailed above.
34
Section II. P.:{j;raction, PurifUation atufIsoCation offlntliocyanins
anthocyanin in the sample. If the molar absorptivity of the major pigment is not
available or if the sample composition is unknown, pigment content is
calculated as cyanidin-3-glucoside, where MW = 449.2 and molar absorptivity
= 26,900, the molar absorptivity reported in the literature for the anthocyanin
pigment in acidic aqueous solvent. Quartz cuvettes of 1 cm path length (1) were
used (Francis, 1989).
Fresh young leaves of 8-406 and 8-408 were collected from field. A
fixed weight (2.5 g) of the samples after homogenization with 50 ml of the
extracting solvent (methanol-HCI), were kept overnight at three different
temperatures viz; 30, 35 and 40°C in an incubator. The extracted anthocyanin
. concentration was determined spectrophotometrically. The measurements were
done in triplicate.
1. Liquid-liquid partition
36
Section II. ~raction, (PUrification anaIsoCation of)f.ntfwcyanins
The extracts obtained after the liquid-liquid partition step will also
contain water soluble compounds other than anthocyanins, namely free sugars
and aliphatic acids. These non-aromatic compounds were removed with the use
of Arnberlite XAD-7 column chromatography (Andersen, 1988). Arnberlite
XAD-7 adsorbs the aromatic compounds including anthocyanins and other
flavonoids in aqueous solutions, whereas free sugars and other polar non-
aromatic compounds were removed by washing with distilled water until the
eluted water has a neutral pH. Then, the adsorbed anthocyanins were eluted
using methanol containing 0.5% TFA as mobile phase (Andersen, 1988). The
anthocyanins from sweet potato genotypes S-406 and S-purple were purified
and for comparative studies, grape pomace anthocyanins also were extracted
and purified.
37
Section II. P.:(Jraction, (furi.fieation amiIsofation of;4ntliocyanins
38
Section IL ~raction, (}!urification atufIsofation of)f.ntliocyanins
39
Section II. �raction, Purification anaIsofation of}f.ntnocyanins
flow rate was lml/minute and column pressure 3200 Psi. All solvents were of
HPLC grade filtered through a 0.45-pm Durapore filter (Millipore, Bedford,
MA). Identification of anthocyanins was done by comparing the retention time
of peaks with those of the authentic standard of purple fleshed sweet potato
anthocyanins (Islam et al., 2002; Terahara et al., 2004). The anthocyanin
extracts from two sweet potato genotypes, S-406 and Orissa-3 were analyzed
byHPLC.
Anthocyanin content
Sample
mg/100 g fresh weight
S-406 165.00
S-402 148.55
S-347 98.70
S-480 135.76
S-491 132.23
S-Purple 121.28
Flower(S-31) 80.20
Orissa Elite 28.31
Sree Ratna 121.64
40
Section II. ~raction, Purification adIsoCation of;4.ntfwcyanins
200
,-...
.c
tl!l
'a:;
~
~
a.> 150
..::
tl!l
c:>
c:>
~
E
':: 100
=
.2:l
Q=
<.l
'c=
~
~ 50
-=
Q
.c
~
o
2 3 4 5 6
solvent system used
43
Section II. ~raction, CFuri.fication atufIsofation of;fntfiocyanins
44
Section II. ~ractionl Cl'urification atufIsofation offlntfwcyanins
max is in the range of 0.2-0.3. Harborne found that, when the 5-position in the
flavanoid structure is glycosylated, the value of A 4401 AVis-max is in the range of
0.1-0.2 (Harborne, 1958). From Table 2.4 it can be inferred that for
anthocyanins of S-purple and S-406, the value of A 4401 AVis-max is in the range of
0.1-0.2 showing that the 5-position is glycosylated. For grape pomace
anthocyanins the value of A 4401 Avis-max is 0.36, indicating that the anthocyanin
3-0-glycosides having a free 5-hydroxyl group. The presence of aromatic
acylation can be determined using the ratio Auv-maxl AVis-max. Typical mono-
aromatic acylation of anthocyanins may give an Auv-max 1 AVis-max ratio of ~ 0.6-
1.3. A higher ratio may indicate several aromatic acyl residues (Ando, et ai.,
1999).
From the values presented in Table 2.4, it can be concluded that for
anthocyanins of S-purple and S-406, the value of A uv-max 1 AVis-max is a higher
ratio (more than 10) indicating that several aromatic acyl residues are present in
the sweet potato leaf anthocyanin molecule.
45
Section II. P.tf;raction, <Purification antiIsoCation of)f.ntli.ocyanins
3.5 --S-purple
(A)
--S-406
--grape pomace
3.0
2.5
2.0
.cctl
.cctl 1.5
1.0
0.5
0.0
300 400 500 600
wavelength(nm)
4.0
(B) --S31
-- OrissaElite
3.5
-- Xanthosoma
3.0
Cl) 2.5
ctl
.c 2.0
.c 1.5
ctl
1.0
0.5
0.0
200 300 400 500 600 700 800
wavelength
46
Section II. ~raction, (]!Urifieation atufIsoCation of)f.ntfwcyanins
concluded that stability of foliar anthocyanins of sweet potato are higher than
those of pomace pigments. Since aliphatic groups do not have significant UV-
Vis absorption, their presence can not be directly detected by UV-visible
spectroscopy. Similarly, from Figure 2.3 (B) it can be estimated that A uv-max 1
AVis-max value of anthocyanins from pink Xanthosoma, Orissa Elite and S-31 is
considerably high compared to that of grape pomace anthocyanin, indicating
the higher stabilityof the corresponding pigments.
Three fractions were obtained for S -406, having AVis-max at 527 nm, 528
nm and 529 nm respectively and for S -purple also three fractions were
obtained having AVis-max at 524 nm, 525nm and 526 nm respectively. The value
of A 4401A Vis-max calculated for the first, second and third fractions of the
pigments of S-406 are 0.19, 0.18 and 0.17 respectively, indicating that the 5-
position is glycosylated. The value of A Uv-max I AVis-max for the first, second and
third fractions of S-406 anthocyanin are 2.2, 2.5, 5.6 respectively, showing that
the extent of acylation is more for the third fraction when compared to the other
two fractions. Similarly for S-purple also, the third fraction is more acylated
and in all the three fractions, the 5-position is glycosylated.
47
Section II. f£t!;raction, CJluri.fication atufIsofation ofJfntliocyanins
(A)
5
- - fraction 1
- - fraction 2
- - fraction 3
4
Ql
g 3
I'll
...o
.Q
III
.g 2
o+-----r------.:;::=:::;:=::::::;:=~~-~...,._---r'-_,...._----,
300 400 500 600 700 800
wavelength
(A)
5
- - fraction 1
- - fraction 2
- - fraction 3
4
Ql
g 3
I'll
...o
.Q
III
.Q 2
I'll
o +---,-~:::::::=~::::;:~~!!T-~---,-...,----r------,
300 400 500 600 700 800
wavelength
48
Section II. P.J(f;raction, (}!uri.fieation anaIsofation ofjfntftocyanins
6-0-(E)-p-coumaroyl-~-D-glucopyranoside)-5-0-~-glucopyranosiderespectively by
comparing the peaks with standard peaks (Islam et al., 2002; Terahara et al.,
2004). In the leaves of S-purple, the major pigments are peonidin type and are
acylated with aromatic acids.
Sample SP
2.5
..,.<t t--
..,.
(Il
~
E
0.0
o 10 20 30 40 50 60 70 80 90
Minutes
Figure 2.5: HPLC chromatogram of the pigment of the purple sweet potato
leaves (Ipomoea batatas L.), S-purple
49
Section IL ~raction, fPurification atufIsolation of)f.ntfiocyanins
Sample FS
5.0
2.5
o 10 20 30 40 50 60 70 80 90
Minutes
Figure 2.6: HPLC chromatogram of the pigment of the purple sweet potato
leaves, Orissa-3
From the above results, structures of the major foliar anthocyanins of the
sweet potato genotypes S-purple and Orissa-3 are presented in Figure 2.7. Depending
on the nature of R I , R2 and R3 , there are different types of anthocyanins. Based
on the aglycon present in the structure, anthocyanins can be classified as
peonidin type and cyanidin type. In S-purple, the predominant anthocyanins are
of peonidin type and in S-406; they belong to the cyanidin group.
51
Section II. P.:(f:raction, <Pu,rification anaIsolation of)1.ntfwcyanins
HO
Pigment R1 R2 R3
Cyanidin 3-0-(2-0-(6-0-(E)-p-coumaroyI
-P-D-glucopyranosy1)- 6-0-(E)-p-coumaroyI H p-Coumaric p-Coumaric
-P-D-glucopyranoside)-5-0-P-D-glucopyranoside
Caffeoylated (cyanidin 3-sophoroside-5-glucoside) H Caffeic H
Peonidin 3-(6,6' -caffeoylferuloylsophoroside)
CH3 Ferulic Caffeic
-5-glucoside
based pigments exhibited higher free radical scavenging activity than peonidin
based ones and the acylated anthocyanins showed higher antioxidant ability
compared to nonacylated pigments. Both the anthocyanin extracts studied here,
isolated from S-406 and Orissa-3, are acylated and hence showed extra stability
as a food colourant and as a nutritional additive. The pigment profile of
anthocyanin-rich extracts has been suggested to impact biological activity:
purified cyanidin and delphinidin exhibited superior anticancer activity (Wang
et al., 1997) to other types of anthocyanins such as peonidin. The major
52
Section II. f£:{tractiot; CJ!urification atufIsofation of}f.ntliocyanins
pigments in Orissa-3 are cyanidin based and hence these may prove to be good
chemo preventive agents.
2.4. Conclusion
The results of the present study show that anthocyanins of sweet potato
leaves can be easily extracted with methanolic HCI, preferably at pH levels less
than 2.0 and at an extraction temperature of 40°C. The foliar anthocyanins of
the sweet potato genotypes 8-406 and 8-purple are more stable compared to the
anthocyanins of grape pomace as evidenced by their UV-visible spectra.
Among the two sweet potato anthocyanins studied, 8-purple anthocyanins are
. more acylated and hence more stable than that of 8-406. The foliar anthocyanin
content of 8-406 and 8-purple lies in the range 150-200 mg/lOO g fresh weight,
which is higher than the reported value of about 40--60 mg anthocyanins/g fresh
weight for purple-fleshed sweet potatoes developed in Japan (Furuta et al.,
1998).
53