R. Chirinos et al.

/ Separation and Purification Technology 55 (2007) 217225

est recoveries of flavanoids using the
varying from 1 min to 24 h have been acidified solvent mixture
reported. Longer extrac-tion times increase (methanol/acetone/water, 45/45/10) may be
the risk of phenolic oxidation unless reducing attributed mainly to the extraction of
agents are added to the solvent system [14]. proanthocyanidins that may be present in
Thus, 60 min was chosen as the best mashua tubers.
extraction time for phenolic compounds and
OAA from mashua. 3.7. Crude extract purification

3.6. Acidified solvent evaluation Phytochemical purification from natural

sources is a nec-essary step for the use of these
Different acidified solvents were tried out to bioactive compounds in the preparation of
recover antiox-idant compounds from mashua: various products for different industrial sectors.
90% methanol, 90% acetone, 90% ethanol, In addition, their quantification or identification
water and a methanol/acetone/water (45/45/10) is considered to be a quality control parameter.
mixture (Table 1). An average increment in Different crude extracts (0.1% HCl in 90%
phenolic recovery and OAA was observed with methanol and 0.1% HCl in
respect to the use of non-acidified solvents methanol:acetone:water mixture, 45:45:10)
(between 32 and 132% for TP, between 38 and were applied on a C18 Sep-pak cartridge SPE
58% for TA, between 38 and 58% for TFA, and and two fractions were eluted, one with
between 22 and 43% for OAA). In average, the acidified water (0.01% HCl) (Fno-phe) and the
efficiency of the different acid-ified solvents other one with acidified
regarding TP extraction and OAA values can be
classified in the following order: 90% methanol
= solvent mixture > 90% acetone = 90% ethanol
> water. Among the most efficient extraction
solvents, a significantly higher TA recovery was
obtained with 90% methanol, as compared to
the solvent mixture, whereas the reverse was
true for TFA. Our results are in accordance to Ju
and Howard [20] who found, on one hand, that
the methanol/acetone/water/HCl (40/40/20/0.1)
sol-vent mixture recovered almost the same
phenolic content from grape skin than the
methanol/water/HCl (60/40/0.1) mixture, and,
on the other hand, that the solvent mixtures
allowed bet-ter OAA recoveries than acidified
water (0.1% HCl). It has been reported that
aqueous methanol is very efficient in recovering
fla-van 3-ols of low degree of polymerization,
while acidified 70% acetone is widely used to
recover highly polymerized flavan 3-ols, e.g.
procyanidins [13]. Thus, we believe that the
great-

Fig. 2. Distribution of total phenolics (TP) and ORAC

antioxidant activity (OAA) after fractionation of crude
extracts of mashua tubers from the ARB 5241 and DP
0224 genotypes. Fno-phe, non-phenolic fraction; Fphe,
a
phenolic frac-tion. Solvent mixture contains:
methanol/acetone/water (45/45/10). The same letters
indicate that phenolic compounds or OAA values are not
significantly influenced by the solvent used (p value <
0.05).
Fig. 3. HPLC-DAD elution profiles of the ARB 5241 and
DP 0224 mashua genotypes extracts obtained with
acidified 90% methanol. Absorbance results are given at
280 and 520 nm. Fphe fractions were injected at a
concentration of 1.1 mg GAE/ml. Peaks p1p9
correspond to the same compounds for the two mashua
genotypes evaluated.
 R. Chirinos et al. / Separation and Purification Technology 55 (2007) 217225 223

Table 2
HPLC percentage area of the phenolic compounds extracted from the ARB 5241 and DP 0224 mashua genotypes either
a
with acidified 90% methanol or with an acidified solvent mixture
b
Peak area (%) at 280 nm
p1 p2 p3 p4 p5 p6 p7 p8 p9 Others
ARB 5241
c
Acidified 90% methanol 9.5 24.5 3.3 13.7 2.2 4.1 28.4 2.5 3.4 8.1
d,e
Acidified solvent mixture 10.3 20.1 3.0 11.7 1.4 6.8 30.1 2.3 5.1 9.2
DP 0224
c
Acidified 90% methanol 6.7 7.8 2.8 19.8 5.8 12.3 34.2 3.4 2.1 5.1
d,e
Acidified solvent mixture 8.8 5.2 2.3 15.8 4.7 14.3 36.6 3.2 3.6 5.5
b
Peak area (%) at 520 nm
p2 p3 p4 p5 p6 p7 p8 Others
ARB 5241
c
Acidified 90% methanol 28.9 3.4 16.4 6.2 34.6 4.1 3.2 5.2
d,e
Acidified solvent mixture 26.4 2.8 15.9 5.7 38.0 5.1 3.1 3.4
DP 0224
c
Acidified 90% methanol 8.4 2.9 22.9 6.8 14.8 41.2 2.9 0.1
d,e
Acidified solvent mixture 6.4 2.6 18.5 5.6 18.8 44.7 2.9 0.5
a
Mean values of two repetitions.
b
The extracts were purified by SPE before injection.
c
0.1% HCl, pH 2.11.
d
0.1% HCl, pH 2.14.
e
Solvent mixture contains: methanol/acetone/water (45/45/10).
and OAA with respect to those from the DP
methanol (0.01% HCl) (Fphe). The TP and 0224 genotype.
OAA of these two fractions are presented in
Fig. 2 for the two mashua tubers investigated. 3.8. HPLC-DAD
The Fphe fraction showed much higher TP
values (14.418.7 mg GAE/g DM) and OAA The HPLC phenolic profiles for the ARB
values (221 and 359 mmol TE/g DM), as 5241 and DP 0224 mashua genotypes were
compared to the Fno-phe frac-tion (0.871.06 determined on the Fphe frac-tions obtained in
mg GAE/g DM and 5.0 and 8.2 mmol TE/g the previous section. The acidified 90%
DM, respectively). The Fno-phe fraction methanol and acidified solvent mixture
contained between 4.3 and 6.1% of the TP, and
(acetone/methanol/
between 2.1 and 3% of the OAA initially
present in the crude extracts, whereas, the Fphe
fraction con-tained between 70 and 74% of TP,
and between 84 and 89% of OAA from the
initial extract. We found mean losses for TP and
OAA, from 19.9 to 25.0%, and from 8.0 to
13.9%, respectively. Kahkonen et al. [34]
reported that using a Bond elute C18 for
purified berry samples reduced phenolics
between 5 and 11%, whereas Li et al. [18]
found a 76% reduction of phenolics in extracts
purified from citrus peels with respect to their
crude counterpart, using a C18 Sep-pak
cartrigde SPE. On the other hand, the reduction
in OAA values that we found after SPE treat-
ment could be attributed in part to the loss of
some water-soluble constituents (e.g. sugars,
acids, ascorbic acid, glutathione, and other
water-soluble compounds), which also possess
antioxidant activity [21]. Purified extracts
(Fphe) from the ARB 5241 geno-type present
quite higher values in terms of phenolic content
water, 45/45/10) extracts were both used and
gave similar results. Fig. 3 shows the profiles
obtained at 280 and 520 nm for the 90%
methanol extracts. The two mashua genotypes
presented roughly the same phenolic profile but
with differences in the proportions of various
compounds, as revealed by their relative
percentage area (Table 2). A total of nine
representative peaks were obtained at 280 nm
for both mashua genotypes. The HPLC profiles
at 520 nm showed that peaks 28 are
anthocyanins. The DAD-UV/visible spectral
data for peaks 1 and 9 showed maxi-mal
absorbances at 215.0/231.0/279.4 nm, and
253.0/287.0 nm, respectively. Peaks 1 and 9
thus appear to correspond to pheno-lics of the
flavanol and benzoic acid types, respectively.
Peaks 1 and 9 were present in higher
percentages in ARB 5241, as compared to DP
0224. Table 2 allows to compare the extraction
strength of acidified 90% methanol and the
acidified solvent mixture
(methanol/acetone/water, 45/45/10) with regard
to each of the major phenolic compounds
present. The acidified sol-vent mixture
extracted higher quantities (in terms of
percentage area at 280 nm) of peaks 1 and 9
(Table 2). At 520 nm, the acidified 90%
methanol was more efficient for extracting the
anthocyanins with low retention times, whereas
the acidified sol-vent mixture was more
efficient for the recovery of anthocyanins with
longer retention times. These observations
suggest that the rapid elution of some
anthocyanins correspond to glycosylated forms
whereas the lately eluted ones correspond to
acylated forms. Indeed, Durst and Wrolstad [35]
have reported that, dur-ing reverse phase
extraction the release of mono or di-glucoside
anthocyanins is characteristic at early retention
times compared to the acylated anthocyanins,
which appear at later retention times. In
addition, Ju and Howard [20] have observed
that acidi-fied methanol was the most efficient
solvent for extracting most
224 R. Chirinos et al. / Separation and Purification Technology 55 (2007) 217225
anthocyanin monoglycosides from red grape
skin in compar-ison to an acidified solvent
mixture (methanol/acetone/water, 40:40:20).
By contrast, the last mentioned solvent was
very effi-cient in the extraction of acylated
anthocyanins. Thus, peaks 25 in the mashua
purified extracts would correspond to
glycosylated anthocyanins and peaks 68
would correspond to acylated antho-cyanins.
Differences in extraction efficiency for
anthocyanins and the other phenolics are
most likely due to differences in sol-vent
polarity. Finally, under the conditions used, it
is probable that some phenolic compounds
have co-eluted. Thus, a previous fractionation
of the extracts would be necessary for the
identifi-cation and posterior quantification of
all the phenolic compounds present.
4. Conclusion
In conclusion, TP, TA, TFA, and OAA
extracted from the mashua genotypes were
affected by the type of solvent, pH level,
solventwater ratio and extraction time. The
optimal extrac-tion conditions for TP
compounds and ORAC values required 0.1%
HCl in 90% methanol and/or 0.1% HCl in a
solvent mix-ture (methanol/acetone/water,
45/45/10) and consisted in one extraction step
of 60 min at room temperature and using a dried
material/solvent ratio of 60. The solvent
containing 0.1% HCl in 90% methanol
extracted more TA whereas the acidified solvent
mixture extracted the highest levels of TFA. The
purification pro-cess using C18 Sep-pak SPE
appeared to be a good technique for the
elimination of interfering compounds from
mashua crude extracts. The HPLC-DAD
analysis of the best extract revealed the
presence of anthocyanins (seven main peaks)
and other phe-nolic compounds (two main
peaks). These best extracts are bearing a high
antioxidant capacity as measured by the ORAC
assay, and therefore be an economically
interesting phytochem-ical source for the
nutraceutical and functional food market.