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Section II

Extraction, Purification and Isolation of Anthocyanins

Sweet potato germplasm field showing anthocaynic leaves

2.1 Introduction

The study of natural colourants is an extensive and active area of


investigation due to the growing interest of substituting synthetic colourants
with toxic effects in humans (Chou et al., 2007). Carotenoids and anthocyanins
are amongst the most utilized vegetable colourants in the food. Among the
natmal colourants, anthocyanins are the largest group of water-soluble
pigments in the plant kingdom. Another significant property of anthocyanins is
their antioxidant activity, which plays a vital role in the prevention of neuronal
Section II. f£:{fraction) Purification atufIsofation ofjIntliocyanins

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

the studies undertaken to optimize the conditions for the extraction of


anthocyanin from the young and emerging purple coloured leaves of sweet
potato using different solvent systems and also to purify and fractionate the
pigments. The spectral properties of the pigments are also studied.

2.2 Materials and Methods


2.2.1 Materials

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.

2.2.2 Experimental Methods

2.2.2.1 Extraction of anthocyanin

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

or only by soaking fresh. samples with subsequent solvent extraction (Merken


and Beecher, 2000). The isolation, purification and structure determination of
pure anthocyanins are time consuming processes and because most
anthocyanins are prone to be unstable, these processes must be conducted with
care. During the workup procedure, the anthocyanins may also become more
fragile because of the removal of free sugars and other acylating groups which
stabilize the pigment. Storage of anthocyanins in the dark, at low temperature
and in the dry state, to reduce hydration and degradation, is therefore preferable.
Typical procedures for isolation and characterization of pure anthocyanins·
consist of several steps: (1) extraction of the plant material, followed by a
preliminary purification, (2) fractionation of the mixture followed by isolation
of pure pigments and finally (3) characterization and identification of pure
anthocyanins (Strack and Wray, 1989). Organic solvents efficiently extract
anthocyanins by destroying cell membranes to dissolve the pigments from
vacuoles. Traditionally, acidified organic solvents are used, with methanol
being the most effective solvent. Solvents are typically acidified with weak
organic acids such as formic, acetic or citric acid or low concentrations of
strong acids such as hydrochloric or trifluoroacetic acid. Weak organic acids
denature the cellular membranes to facilitate solubilization of the pigments, but
addition of excess acid can result in hydrolysis of labile, acyl and sugar
residues and may break down complexes with metals and co-pigments (Shahidi
and Naczk, 2004). Furthermore, higher temperatures have been found to
improve efficiency of extraction, but can also increase the rate of anthocyanin
degradation. Anthocyanins are polar molecules, thus the most commonly used
solvents for the preparation of plant extracts are methanol, ethanol and acetone,
at a composition of 70-80% in water (Kahkonen et al., 2001).

31
Section II. ®(traction, Purification andIsofation ofJfotliocyanins

l .L',1 I Sample

Extraction with Methanol-acid

Filtration

I Solvent Extraction "ith Ethyl Acetate/Chloroform


I
Discard the Ethyl +--
Acetate/Chloroform layer

Freeze dryino/tlash
• i-,

e, aporation

I Purification -Amberliie :\.AD-7 chromatography


I

Fnwtio,rntion - SPphafl�,� I J -1-20 �in· f'".fh.'�i(ln fhromatography

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.

As a preliminary work, foliar anthocyanins present in various tuber


crops were estimated. Fresh leaves were used for all the experiments and
pigment was extracted using the following procedure. The leaves were
chopped and weighed accurately (2.5 g). The sample was then homogenized
with 50 ml of the extracting solvent, which was prepared by mixing 80 ml of
methanol, O.lml concentrated HCI and 19.9 ml of water. The homogenized
samples were kept overnight at 4 cC. The extract was then filtered through a
Buchner funnel using Whatman No.1 filter paper and 8 ml of solvent was
added to the residue; this procedure was repeated as many times as required
until all colour had been extracted from the leaf tissues. Finally, the extracts'
were combined and partitioned with chloroform to free it from chlorophyll and
stored at -18 cC.

Sweet potato germplasm varieties like S-402, S-406, S-347, S-480, S-


491, and S-purple were screened. Pigments present in the leaves of sweet potato
variety, Sree Ratna, a released variety from CTCRI, were extracted using the
above solvent. Anthocyanin present in young leaves of Yam, Orissa Elite and
pink Xanthosoma were also extracted using methanolic HCI and were kept at -
18 cc until analysis.

33
Section II. P.:{Jraction, <Purification an£Isofation of)lntfiocyanins

For comparative studies, anthocyanins present in commercial grape seed


skins were also extracted and estimated spectrophotometrically. Flower
samples collected from the sweet potato variety S-31 were also tested for plant
pigments.

2.2.2.2 Extractability using different solvent systems

A comparative study of extraction methods using acetone, methanol,


and isopropanol was made. Six different combinations of solvents were
prepared as follows.

1. 80 ml acetone+ 0.1ml concentrated HCl+ 19.9 ml water


2. 80 ml acetone+ 2ml 20% trifluoroacetic acid + 18 ml water
3. 80 ml methanol+ 0.1ml concentrated HCl+ 19.9 ml water
4. 80 ml methanol+ 2ml 20% trifluoroacetic acid + 18 ml water
5. 80 ml isopropanol+ 0.1ml concentrated HCl+ 19.9 ml water
6. 80 ml isopropanol+ 2ml 20% trifluoroacetic acid + 18 ml water

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.

2.2.2.3 Total monomeric anthocyanin

The measurement of total anthocyanin pigment provides a basis for


comparison and is useful in assessing the colour quality of many foods.
Accurate quantification of anthocyanins and their degradation is of special
interest when comparing fresh and processed fruits and vegetables and can also
be a useful tool in assessing the colour quality of food colourants. The total
anthocyanin content in crude extracts containing other phenolic materials has
been determined by measuring absorptivity of the solution at a single

34
Section II. P.:{j;raction, PurifUation atufIsoCation offlntliocyanins

wavelength. This is possible because anthocyanins have a typical absorption


band in the 490 to 550 run region of the visible spectra. This band is far from
the absorption bands of other phenolics, which have spectral maxima in the UV
range (Fuleki and Francis, 1968a). In many instances, however, this simple
method is inappropriate because of interference from anthocyanin degradation
products or melanoidins from browning reactions (Fuleki and Francis, 1968b).
In those cases, the approach has been to use differential and/or subtractive
methods to quantify anthocyanins and their degradation products. Francis
(1989) suggested pH values of 1.0 and 4.5 when working with cranberries and
these values serve as the basis for the pH-differential method (Wrolstad and
Guisti, 2001; Francis, 1989). The pH-differential method is based on the
reversible transformations that occur in anthocyanins with a change in pH. At
pH 1.0, the flavylium cation is found in the coloured oxonium form, while at a
pH 4.5, the colourless hemiketal form dominates. Methods that utilize
spectroscopy allow for rapid and easy quantification of monomenc
anthocyanins, even in the presence of polymerized degrading pigments and
interfering compounds. Throughout this work, anthocyanins were quantified by
the pH-differential method (Rodriguez-Saona et ai., 2001; Guisti and Wrolstad,
2001). Two dilutions of the sample were prepared, one with potassium chloride
buffer (0.025M), pH 1.0, and the other with sodium acetate buffer (OAM), pH
4.5, and th.ese dilutions were allowed to equilibrate for 15 minutes. The
absorbance of each dilution at the visible maximum (Mmax) and at 700 run (to
correct for haze A 700 ) were measured against a blank cell filled with distilled
water. The absorbance of the diluted sample (A) can be calculated as follows:

Monomeric anthocyanin pigment (mg/litre) = (A x MW x DF x 1000) -+- (£ x 1)

MW is the molecular weight; DF is the dilution factor, and £ the molar


absorptivity. The MW used in this formula corresponds to the predominant
35
Section II. ~raction) Purification arufIsoCation ofJf.ntliocyanins

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).

2.2.2.4 Effect of pH on anthocyanin extraction

This experiment was carried out in methanol-HCI extracting system at


different pH values (1, 2, 3, 4 and 5). The extracted anthocyanin concentration
at different pH values was determined as explained above. Fresh emerging
leaves of 8-406, 8-408 and 8-480 were used as anthocyanin sources. Three
replications were done at each condition.

2.2.2.4 Effect of temperature on anthocyanin extraction

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.

2.2.2.6 Purification of anthocyanins

1. Liquid-liquid partition

The anthocyanins extracted usmg methanolic HCI were purified by


partition against chloroform to remove chlorophylls, stilbenoids, less polar
flavonoids and other non polar compounds from the mixture. The extract was
then concentrated by flash evaporation in rotary evaporator (Buchi R-200).

36
Section II. ~raction, (PUrification anaIsoCation of)f.ntfwcyanins

2. Amberlite XAD-7 adsorption chromatography

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.

2.2.2.7 Sample fractionation and isolation of pure pigments by gel


filtration column chromatography

In this work Sephadex LH-20 was used as column material. This


material permits separation with respect to molecular size. The purified XAD-7
extracts were dissolved in a small amount of the initial mobile phase (MeOH:
H20: TFA; 20:80:0.5, v/v) (Andersen and Francis, 2004). Separation was
achieved by isocratic or gradient elution using increasing amounts of methanol.
Since the column functions on the size-exclusion principle, the anthocyanins
may be mainly eluted in order of decreasing molecular mass. Anthocyanidin
triglycosides are eluted prior to anthocyanidin di-glycosides followed by
anthocyanidin monoglycosides. Acylated anthocyanins are usually more
retarded than non-acylated anthocyanins because of increased adsorption
(Jordheim et al., 2007). The anthocyanin containing fractions were collected on
the basis of observed visual band separation and the spectra were recorded.

37
Section II. P.:(Jraction, (furi.fieation amiIsofation of;4ntliocyanins

2.2.2.8 High performance liquid chromatography

High performance liquid chromatography (HPLC) has been the


method of choice for the qualitative and quantitative analysis of anthocyanins.
This is because of the capability of LC in preparation of samples on the gram
scale using preparative-HPLC and the purification of samples on the milligram
scale with semi preparative-size LC columns and on the microgram scale with
analytical-size column. HPLC is also used for identification of individual
natural compounds in the plant matrices using coupled techniques such as
HPLC with UV- Vis spectrophotometry detection (LC-UV), with mass
spectrometry detection (LCMS), or with nuclear magnetic resonance detection
(LC-NMR). There is not a single standard procedure for the analyses of
anthocyanins using HPLC. Rather, there are a wide variety of columns and
parameters that have been used in anthocyanin characterization from the same
plant source resulting in equivalent separations. A high proportion of isolation
methods for anthocyanins are generally run on reverse-phase columns, such as
octadecyl silane (ODS), polystyrene or phenyl-bonded columns (da Costa et al.,
2000). Also, HPLC methods tend to utilize gradient solvent systems of
acetonitrile-water or methanol-water with a small amount of acid to lower- the
solution pH and increase stability of the anthocyanins. These solvents are most
popular due to their compatibility with gradient methods for isolation and the
various detection methods coupled to the HPLC used for identification. To
obtain reproducible results using this instrumentation, the pH of the mobile
phase and temperature of the column must be controlled due to the instability of
anthocyanin compounds in changing pH and temperature environments. For
optimal results, acidic mobile phases lower than pH 2.0, are employed to ensure
the anthocyanin remains in the more stable flavylium form and to reduce peak
tailing of the chromatogram (da Costa et al., 2000). In reverse-phase
chromatography, the retention time decreases with increasing polarity, which
corresponds to increasing number of hydroxyl moieties on the flavylium ion

38
Section IL ~raction, (}!urification atufIsofation of)f.ntliocyanins

and the elution order of delphinidin, cyanidin, petunidin, pelargonidin, peonidin


and malvidin. Due to reverse-phase elution, diglycosylated anthocyanins will
have the lowest retention time, followed by monoglycosylated anthocyanins,
aglycones and lastly the acylated anthocyanins (Mazza et al., 2004; da Costa et
al., 2000). In the acidic media, the flavylium cation is red coloured and gives an
absorption maximum around 520 nm, which avoids interference from other
flavonoids that may be present in the plant extract. Because of these unique
absorbance maxima, anthocyanins have been accurately identified and
quantified from very crude plant extracts (da Costa et al., 2000). Nonnal phase
systems or those using unmodified silica gel are not effective for the separation
of anthocyanins due to the polarity of these compounds (Andersen et al., 2006).
The main drawback of using HPLC method for anthocyanin identification is the
considerable difficulties in obtaining good quality standard samples of
anthocyanin compounds, even from prominent chemical companies. In the
absence of standards, many researchers identify anthocyanins by comparing the
peaks with authentic standard peaks reported elsewhere. Identification of
anthocyanin compositions by reverse phase HPLC was done by following the
method ofIslam et al. (2002).

The HPLC analysis was carried out on a reversed-phase CI8 column,


Kromasil 100 (5 ~m, 4.6 X 250 mm). 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. The
chromatographic data were analyzed with Gilson Unipoint software. A gradient
solvent system was used for the separation of anthocyanins in a single HPLC
run. The solvents used for separation were, (A) 1.5% phosphoric acid and (B)
1.5 % phosphoric acid, 20% acetic acid and 25 % acetonitrile. The elution
profile was a linear gradient elution with 15-50% solvent B in solvent A for 90
minutes. In all experiments, 20 ~l of the leaf extract was injected and solvent

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.

2.3 Results and Discussion

2.3.1 Preliminary extraction

A wide variability was found to exist in the anthocyanin content of


sweet potato leaves, with values ranging from 50-200 mg/lOOg fresh weight
(FW) of the selected samples (Table 2.1). Among the crops selected for
analysis, the highest value of anthocyanin content now observed was for the
sweet potato variety, S-406 (165.0 mg/lOOg) followed by S-402, S-480, S-491
and S-purple. These results are in conformation with the results of Jyothi et al.
(2005). These results show that sweet potato leaves contain considerable
amount of anthocyanin and hence can be utilized as a cheap natural source of
these pigments.

Table 2.1: Anthocyanin contents in selected tuber crops

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

2.3.2 Extractability of different solvent systems

Figure 2.2 shows the comparison of extractability of anthocyanins from


S-406, S-408 and S- 480 when different solvent systems were used. In all cases,
1.0g of fresh, homogenized leaf samples were taken and extractions were
carried out in triplicate, and it was found that methanol-HCI and isopropanol-
HCI solvent gave good quantitative extraction when compared to the other four
solvents. Methanol-HCI system gave a slightly better extraction than
isopropanol-HCI system, this could be due to higher polarity of methanol and
the ability to diffuse into cell membranes is better for methanol compared to
isopropano 1.

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

Figure 2.2: Comparison of anthocyanin extractability of different solvent


systems for sweet potato genotypes, S-406, S-408 and S- 480. (1-
Acetone and HCI, 2-Acetone and TFA, 3- Methanol and HCI, 4-
Methanol and TFA, 5- Isopropanol and HCI, 6- Isopropanol and
TFA)
41
Section II. ~raction, CPu:ri.fication atufIsofiltion of;f.ntfwcyanins

2.3.3 The effect of pH on anthocyanin extraction

The results obtained after extracting at different pH levels are shown in


a Table 2.2. There was no marked change in the amount of anthocyanin
extracted when pH of the medium was increased from 1 to 2; however, a
noticeable decrease in the pigment extraction was observed when pH was
further increased from 2 to 5. The colour and stability of an anthocyanin in .
solution is highly dependent on pH. Anthocyanins are most stable and most
highly coloured at low pH values and gradually lose colour as the pH increases,
becoming almost colourless between pH 4.0 and 5.0 (Ozela et al., 2007).
Therefore, an increase in pH may lead to a reduction of pigment stability and
results in a decrease in the extraction yield. This is clear from the low
extractability at pH 5.0. Additionally, a low pH may Increase the cell
membrane permeability leading to higher diffusion coefficient values for the
extracting solvent.

Table2.2: Anthocyanin extracted at different pH values

Source of leaf Anthocyanin extracted, mg/100g* (fresh weight) at pH


sample 1.0 2.0 3.0 4.0 5.0
S- 406 192±.23 191 ± .14 150±.11 98±.28 69±.21
S- 408 198 ±.11 197±.15 152±.21 100±.22 71±.15
S- 480 118±.11 116±.16 99 ±.13 79±.24 49±.19
* - values with standard deviation obtained from three replications

2.3.4 The effect of temperature on anthocyanin extraction

The results obtained after extracting anthocyanins at different


temperatures are shown in Table 2.3. There is obviously an increase in
extraction efficiency as temperature is increased. In addition to the effect of
temperature, denaturation of the cell membranes might also occur at higher
42
Section II. P.xJraction, ®lrification anaIsofation ofjIntliocyanins

temperatures, affecting the extraction process. High temperature renders the


plant cell wall permeable and increases solubility and diffusion coefficient of
compounds and decrease the viscosity of solvents, thereby a resulting
improvement of the efficiency of the extraction. However, anthocyanins are
sensitive to heat and easily convert to the colourless chalcone form during
heating (Wrolstad et al., 2002). Hence, it can be inferred that temperature of the
extracting medium cannot be increased indiscriminately to maximize the
process. Temperature and pH of the extraction medium were found to affect the
anthocyanin extraction. Therefore, shortening the extraction time by employing
a low pH (below 2.0) and a high temperature (40°C) may be considered to be
better suited and more feasible for an industrial process.

Table 2.3: The effect of temperature on anthocyanin extraction

Anthocyanin extracted, mg/l00g*


Sample
30°C 35°C 40 °C
S-491 121±.48 129±.21 138±.18
S-492 118±.01 125±.14 129±.49
* - values with standard deviation obtained from three replications

2.3.5 UV-Visible absorption spectra of purified and fractionated


anthocyanins

Figure 2.3 (A and B) shows the UV-Visible absorption spectra of


purified anthocyanins from S-406 and S-purple. The absorption maxima in the
visible region are mainly dependent of the nature of the aglycone, the position
of sugar substituents on the aglycone and the presence of aromatic acyl groups.
Secondly, spectral measurements are of value in determining the position of
attachment of the sugars in the anthocyanin molecular framework. The most
common sugar moieties are glucose, galactose, rhamnose, xylose, arabinose, as
mono-, di-, and tri-glycosides. The sugars may be acylated with aromatic acids,

43
Section II. ~raction, CFuri.fication atufIsofation of;fntfiocyanins

such as p-coumaric, caffeic, ferulic, sinapic, gallic or p-hydroxybenzoic acids


or aliphatic acids, such as malonic, acetic, malic, succinic or oxalic acids
(Robbins, 2003). Confirmation of the presence (or absence) of a hydroxy
aromatic acid as an acyl component can be made by spectral means, as has
already been indicated (Harbome, 1958; Hong and Wrolstad, 1990). Acylated
anthocyanins indicate a low sensitivity to pH changes and possess higher colour
stability in neutral and alkaline solutions. This increased stability is due to the
fact that acylated anthocyanins are more resistant to hydration of the flavylium
ion leading to the equilibrium pushed more towards quinonoidal base forms
(Torskangerpoll and Andersen, 2005). In effect, the acylating groups encourage
the production of the bluer pigmentation that glucosidic moieties bring forth but
also counteract the instability attributed to the sugars. A mechanism called
intramolecular/ intermolecular copigmentation was introduced to illustrate the
increased stability of acylated anthocyanins (Mazza and Miniati, 1993;
Harbome and Williams, 2000). The charged C ring in anthocyanidins can react
with nucleophiles as an electrophilic center. The acyl groups not only affect the
colour but also protect the chromophores from the nucleophilic attack through
an intermolecular stacking of chromophore in a planar or a sandwich spatial
structure for anthocyanins containing two or more aromatic acyl groups (Giusti
and Wrolstad, 2003). The spatial proximity between the hydrogens from
aglycone and acyl groups in radish was observed to be very close to each other
in two-dimensional NMR spectroscopy analyses (Giusti et al., 1998).

The spectra of anthocyanins acylated with p-coumaric acid show two


peaks in the ultraviolet at 289 and 310 nm, due to the superimposition of the
absorption of the cinnamic acid upon that of the pigment absorption. The
spectra of simple anthocyanins exhibit a simple peak at about 270 nm and only
very weak absorption at 310 nm (Harbome, 1958). Figure 2. 3 (A) shows both
S-406 and S-purple show strong peaks around 310 nm showing a high degree

44
Section II. ~ractionl Cl'urification atufIsofation offlntfwcyanins

of acylation and greater stability compared to other anthocyanins. Glycosylation at


the 3 and 5 positions of the anthocyanidin molecule has an important impact on
the spectral characteristics, and extensive research has been published in that
area (Harborne, 1967; Giusti and Wrolstad, 1996). The most important spectral
parameters derived from the UV-Vis absorption spectra of anthocyanins are;
AVis-max, the absorption (A) at A = 440 nm compared to A at AVis -max (A 4401 A Vis-

max), and A at AUv-max compared to A at AVis-max (A Uv- max 1 AVis-max)' For


anthocyanin 3-0-glycosides having a free 5-hydroxyl, the value of A 4401 A Vis -

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.

S-Purple anthocyanins have a greater degree of acylation compared to


the anthocyanins of S-406 and hence more stable and this is confirmed by the
stability studies carried out in our laboratory (Rajeswari et ai., 201 Oa). The
value of Auv-max 1 AVis-max obtained for grape pomace anthocyanins is very low
(1.0) as compared to those extracted from sweet potato leaves. Hence it can be

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

Figure 2.3: The UV-visible absorption spectra of XAD-7 purified anthocyanins


from S-purple, S-406 and grape pomace (A)and Orissa elite, S-31
and Xanthosoma(B) sources

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.

Table2. 4: Spectral details of anthocyanins extracted from S-purple,


S-406 and grape pomace

Absorbance S-purple S-406 Grape pomace


A 440nm 0.04 0.05 0.36
A Vis-max 0.21 0.31 0.82
A Uv-max 2.38 3.37 0.82
A 440lA Vis-max 0.17 0.16 0.44
A Uv-maxlA Vis-max 11.41 10.98 1.0

2.3.6 The fractionation of anthocyanins by Sephadex LH-20

The absorption spectra of fractionated anthocyanins using Sephadex


LH-20 size exclusion chromatography are shown in Figure 2.4.

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

Figure 2.4: The absorption spectra of Sephadex fractionated anthocyanins


extracted from S-purple (A) and S-406 extract (B)

48
Section II. P.J(f;raction, (}!uri.fieation anaIsofation ofjfntftocyanins

2.3.7 The HPLC chromatographic separation and characterization of


anthocyanins

HPLC chromatographic pattern of the anthocyanins of S-purple leaves is


shown in Figure 2.5. Among the peaks in the chromatogram, nine peaks could
be identified as due to known anthocyanins, by comparing with the retention
times with those of authentic standard peaks of purple fleshed sweet potato
anthocyanins (Islam et al., 2002; Terahara et al., 2004) and their chemical
names are shown in Table 2.5.

The major peaks, 8, 14 and 17 were identified as caffeoylated peonidin


3-sophoroside-5-glucoside, peonidin 3-(6,6' -caffeoylferuloylsophoroside)-5-
glucoside and cyanidin 3-0-(2-0-(6-0-(E)-p-coumaroyl-~-D-glucopyranosyl)­

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

The reversed phase-HPLC-chromatogram of the anthocyanins of Orissa-


3 is shown in Figure 2.6. Nine peaks were identified which include the major
peaks and their chemical names are shown in Table 2.5.

The single, major anthocyanin present in Orissa-3 leaves is caffeoylated


(cyanidin 3-sophoroside-5-glucoside), peak No.2. The second largest peak in
terms of peak area is peak No.7, which corresponds to feruloylated (cyanidin 3-
sophoroside-5-glucoside). Thus, we now confirm that the major pigments
present in foliar anthocyanins of the sweet potato genotype Orissa-3 are of
cyanidin based and are acylated with caffeic and ferulic acids.

Table 2.5: Chemical names corresponding to the anthocyanin peaks in the


HPLC chromatograms of foliar anthocyanins of S-purple and Orissa-3

Peak No. Anthocyanin identified


S-purple (S-purple)
1 Cyanidin 3-sophoroside-5-glucoside
3 Peonidin 3-sophoroside-5-glucoside
4 p-Hydroxybenzoylated (cyanidin 3-sophoroside-5-glucoside)
7 p-Hydroxybenzoylated (peonidin 3-sophoroside-5-glucoside)
8 Caffeoylated (peonidin 3-sophoroside-5-glucoside)
9 Feruloylated (cyanidin 3-sophoroside-5-glucoside)
13 Cyanidin 3-(6,6' -caffeoylferuloylsophoroside)-5-glucoside
14 Peonidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside
17 Cyanidin 3-0-(2-0-(6-0-(E)-p-coumaroyl-~-D-glucopyranosyl)­
6-0-(E)-p-coumaroy l-~-D-glucopyranoside)- 5-0-~-D
glucopyranoside(E- p-coumaryl)

Peak No. Anthocyanin identified


Orissa-3 (Orissa-3)
2 Caffeoylated (cyanidin 3-sophoroside-5-glucoside)
3 p-Hydroxybenzoylated (peonidin 3-sophoroside-5-glucoside)
5 Caffeoylated (peonidin 3-sophoroside-5-glucoside)
7 Feruloylated (cyanidin 3-sophoroside-5-glucoside)
11 Cyanidin 3-(6,6'-caffeoyl-p-hydroxybenzoylsophoroside)-5-
glucoside
12 Cyanidin 3-(6-caffeoylsophoroside)-5-glucoside
13 Cyanidin 3-(6,6' -caffeoylferuloylsophoroside)-5-glucoside
14 Peonidin 3-(6,6' -dicaffeoylsophoroside)-5-glucoside
15 Peonidin 3-(6,6'-caffeoylferuloylsophoroside)-5-glucoside
50
Section II. ~raction, (purification arufIsoCation 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.

Terahara et al. studied the stability of anthocyanins having variOUS


degrees of acylation and found that diacylated anthocyanins were more stable
than monoacylated and nonacylated ones (Terahara et al., 2004). The effect of
the substituents on the stabilities of the anthocyanins suggested that the two
aromatic acids protect the aglycon nucleus by a sandwich-type hydrophobic
stacking mechanism and inhibit the attack of a water molecule that leads to a
loss of colour. The studies conducted in our laboratory on the storage stability
of foliar anthocyanins of sweet potato also show similar results (Rajeswari
et al., 2010a). In addition to this, Terahara et al., (2004) reported that cyanidin

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

Figure 2.7: Structures of predominant anthocyanins present in sweet potato


genotypes S-purple and Orissa-3

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).

HPLC studies on the pigments of sweet potato genotypes Orissa-3 and


8-purple indicate that the predominant pigments are cyanidin and peonidin
based anthocyanins acylated with caffeic, ferulic and coumaric acids. This
acylation with aromatic acids imparts greater stability to the pigment. The
results of our study indicate that th~ sweet potato leaves can be used as a cheap
source of anthocyanin pigments and these findings may be useful for further
optimization studies to design a commercial production of anthocyanins from
sweet/potato leaves.

53

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