International Journal of Food Science and Technology 2012, 47, 15931597

Original article
Impact of several variables on the microwave extraction of
Chenopodium Quinoa willd saponins
Vicente Gianna,1,2 Juan Manuel Montes,1 Edgardo Luis Calandri1,2 & Carlos Alberto Guzman2,3
1 School of Chemical Engineering. FCEFyN. UNC, Velez Sarseld Av. 1611., 5015 Cordoba, Argentina
2 Institute of Science and Food Technology. FCEFyN. UNC, Velez Sarseld Av. 1611., 5015 Cordoba, Argentina
3 National Council of Scientic and Technical Research (CONICET), Velez Sarseld Av. 1611., 5015 Cordoba, Argentina
(Received 25 August 2011; Accepted in revised form 23 February 2012)

Summary

Despite their possible applications in diverse elds, saponins are still considered to be industrial waste. The
use of saponins, however, would make seed processing more protable and reduce the pollution of
watercourses. In this work, the microwave extraction method of Chenopodium quinoa Willd saponins was
investigated. The eects of variables such as temperature, time of microwave application, solvent
composition and solvent mass seed ratio were investigated. Solvent mixtures (ethanolwater and
isopropanolwater) were used for the extraction. The Taguchi design methodology was employed to
determine the number of experiments and the optimal conditions for dierent extractions. The eciency of
each assay was determined and the results agreed with the best conditions provided by the Taguchi
experimental design for both solvent mixtures. The isopropanolwater mixture eciency was 91.8% in one
extraction step, and for ethanolwater mixture, it was 57.1%, clearly showing the advantage of the rst one.

Keywords

Extraction, microwave, quinoa, saponins.

Introduction

Quinoa is an annual plant that is native to the Andes

(South America), with Bolivia and Peru, providing 80%
of world production. In Argentina, the production is
targeted at domestic consumption, such as in seed or
our (Vilche et al., 2003).
For the seed to be used for human consumption, the
saponins content must be removed because they impart
bitter taste and are considered to be the main antinutrient of the quinoa. Saponins are known to cause
breakdown in the human small intestine cell membranes
and also negatively aect the assimilation of some
proteins (Moges Woldemichael & Wink, 2001). Saponins are found in quinoa grain pericarp (Taylor &
Parker, 2002), and their presence in the fruits seems to
play a role in defence against pests such as birds and
insects, during physiological maturation of the plant
(Cabieses, 2005).
Quinoa saponins are triterpenoidal glycosides, which
are soluble in methanol and water (Ruales & Nair,
1992). The maximum acceptable level of saponin in
quinoa for human consumption varies between 0.06%
and 0.12% (Bacigalupo & Tapia, 1990). This is consistent
*Correspondent: E-mail: vgianna@efn.uncor.edu

with the results of sensory tests conducted at the

University of Ambato, Ecuador, where it was determined that the maximum tolerance of saponin content
in the cooked grain was 0.1% (Nieto & Soria, 1991).
Saponins also produce foaming in aqueous solutions.
This foam is stable even at very low concentrations (0.1%)
and can be used as a natural emulsier in beverages,
shampoos and soaps, as well as in re extinguishers,
photography and the cosmetics industry. Furthermore,
saponins have been used in the pharmaceutical industry
and agriculture (San Mart n & Briones, 1999).
Another important property of saponins is their
antifungal activity. It has been shown that saponins
inhibit the growth of Candida albicans (Moges Woldemichael & Wink, 2001) and (Reilly et al., 2004) and that
saponins treated with alkali have a signicant antifungal
activity against Botrytis cinerea (Stuardo & San Martin,
2008). In Canada, a commercial product composed
mainly of quinoa saponins called HeadsUp Plant
Protectant has been developed (HeadsUp Plant Protectant Ltd, Kamsack, Canada).
Saponins also have anticarcinogenic properties and
stimulate the immune system (Li et al., 2002). Oleanolic
acid, one of the ve major components of the saponins
from quinoa, showed signicant antitumor activity
when tested in colon cells (Estrada et al., 1998).

doi:10.1111/j.1365-2621.2012.03008.x
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1594

Microwave extraction V. Gianna et al.

The eect of water decit level on saponin composition of quinoa was determinate in a eld experiment
conducted in Mexico. The experiment took place during
the development of Sajama and Chucara cultivars. The
saponin content increases during branching, panicle
initiation and in blooming, followed by a decrease
during the grain lling stage, when the plants under
medium water decit recorded the highest saponin
content (Soliz-Guerrero et al., 2002).
The microwave-assisted extraction (MAE) is a relatively new technique but has been growing rapidly in
recent times. Compounds present in the matrix can
interact with a suitable solvent assisted by microwave
energy, which heats the system and allows for better
extraction. Microwaves have advantages over the rate of
heating of the sample and prevent overheating, avoiding
thermolabile substances denaturation. Therefore, with
this method, can achieve good yields in short time.
As mentioned earlier, the objectives of this research
were to evaluate the extraction eciency of saponins
from quinoa seed by solvent extraction, employing
microwaves through an appropriate combination of the
operating variables (% alcohol, time, temperature,
volume solvent gram seeds).
Materials and methods

The grains were harvested during 2009 in the place La

Poma, located between 65 56 and 66 33 west
longitude and between 23 20 and 24 55 south
latitude, Province of Salta, Argentina.
The tested solvent mixtures were as follows: aethanolwater mixtures, b- isopropanolwater mixtures.
Four variables were studied for the extraction process:
1- temperature, 2- solvent composition, 3 - contact time
and 4- the ratio volume of solvent gram of fruit.
The temperature was varied between 50 and 110 C,
and care was taken not to exceed the molecular
degradation limit for saponins (Chen et al., 2007).
The Taguchi experimental design was employed to
determine the optimal conditions with a minimal number of experiments, for saponins extraction from ethanolwater and isopropanolwater mixtures in a
microwave oven. For the orthogonal array, the design-easy 7.1 software for Windows and a Taguchi
matrix of L16 four factors of four levels each were
used (Montgomery, 2004) and (Anderson & Witcomb,
2007). anova tests were performed using InfoStat, 2010
(statistical software) to analyse the statistical signicance of the results.
To carry out the extractions, a 50 mL glass reactor
(Schott SCHOTT Argentina S.A., Buenos Aires,
Argentina) with a Teon (DUPONT., Buenos Aires,
Argentina) cap was used. It is tted with seals made of
silicone and viton to prevent leakage. A temperature
sensor was xed to the reactor by rubber bands. A

International Journal of Food Science and Technology 2012

900 W Litton BGH 16650 microwave (Argentine industry) with a temperature sensor was used during the
experiments.
The extractions in Soxhlet device were performed with
20% ethanolwater or isopropanolwater mixtures in
both cases, in a ratio of 20 mL of solvent per gram of seed.
Extraction procedure with microwave equipment

The extraction was performed as follows: 1.0000 g of

whole seeds was put into the reactor with the chosen
solvent, and this was weighed and closed. The temperature probe was xed and the reactor is introduced into
the MW oven and started. When the required temperature was reached, the timer was started. At the end, the
oven was stopped and the reactor is cooled with cold
water, opened, weighed at room temperature, and the
extract was recovered, ltering through a 0.2 lm membrane, employing a pressure ltration syringe.
Quantification of saponins

Saponins in the extracts were derivatised by the LibermannBurchard reaction, mainly based on Monje et al.
(2006) although it was taken into account (Hostettmann
& Marston, 2005) and (Abisch & Reichstein, 1960). The
absorbances were measured at 528 nm with a Perkin
Elmer Lambda 25 spectrophotometer. Calibration
curves were determined with oleanolic acid. Linear
regression of data followed the expression:
A 4:5725  S 0:0164

R = 0.9998
A: measured absorbance, [S]: saponin concentration
(mg mL) and R2: the square correlation coecient of
the calibration curve.
The low quantication limit for eqn (1) is
0.05 mg mL and its linearity limit is 0.65 mg mL.
All measurements were performed at least ve times,
and the Q acceptance criteria applied. The condence
interval was established by the Students t test with a
probability of 95%, resulting 0.011 for extractions
with ethanol and 0.010 for isopropanol.
For the extraction eciency (E), the following equation was used:
E = 100 total mass of saponins [g] mass of seed [g]
Results and discussion

Four basic variables were analysed, each one at four

levels, to nd how we can use each variable in a
combination to reach the optimum conditions. Table 1
shows the Taguchi experimental matrix design with the
factors and levels mentioned before. Table 2 shows
Taguchi experimental matrix applied. It was made a
factorial design and found that 44 = 256 assays should

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International Journal of Food Science and Technology  2012 Institute of Food Science and Technology

Microwave extraction V. Gianna et al.

Table 1 Experimental design matrix

Level

Factor A
Vol. Solvent/g seed

Factor B
Time (min)

Factor C
T (C)

Factor D
% alcohol

I
II
III
IV

15
20
25
30

5
15
20
30

50
60
70
90

20
60
80
95

Factors A, B, C and D are independent variables with four levels (levels

can be seen in the table). A: is the volume of solvent (alcoholwater
mixture) g of seeds; B: time to apply microwave; C: the temperature at
which extraction takes place and D: % of alcohol in the solvent.

be necessary, whereas with Taguchi method, only

sixteen experiments must be done to establish the best
extraction condition.
The anova testing for both ethanolwater and
isopropanolwater mixtures has shown that both, factors and model, are signicant with a P value less than
0.05. Values for F have shown results in the same
direction.
Extraction with MW

The matrix of experiments is shown in Table 2, which

includes their respective average yields for both the
ethanolwater and isopropanolwater mixtures.

Experiment number seven showed the maximum

extraction yields for both solvent mixtures (1.65% for
ethanol 20%, and 2.66% for isopropanol 20%). The
numerical analysis of the results following the Taguchi
procedure (Montgomery, 2004) revealed that the optimal extraction conditions for the mixtures of ethanol
water and isopropanolwater were the same, namely
volume of solvent gram of seeds: 20 mL g; time:
20 min; temperature: 90 C; alcohol concentrations:
20%.
The above results show that: (i) The best extractant
solvent was a mixture of isopropanolwater 20%; (ii)
The higher temperature facilitated the diusion of the
solute from the solid to the solvent.
Test for trend

To determine whether the above values corresponded to

those giving the best yields, a new series of experiments
were performed at the best experimental conditions
keeping all the variables constant except one, which was
regularly changed. The results for the isopropanol
water mixtures and ethanolwater mixtures are shown
in Fig. 1ad.
Regarding the eect of solvent composition on
extraction eciency, the tests showed that maximum
extraction of saponins took place at a rather high

Table 2 Taguchi L16 experimental design (4 )

Experimental results(*)
Average efficiency: g saponins/100 g of
seed

Experiment

Factor A
Vol. Solvent/g seed

Factor B
Time (min)

Factor C
T (C)

Factor D
% alcohol

Vacancy

Ethanolwater
mixtures

Isopropanolwater
mixtures

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

I(15)
I
I
I
II(20)
II
II
II
III(25)
III
III
III
IV(30)
IV
IV
IV

I(5)
II(15)
III(20)
IV(30)
I
II
III
IV
I
II
III
IV
I
II
III
IV

I(50)
II(60)
III(70)
IV(90)
II
I
IV
III
III
IV
I
II
IV
III
II
I

I(20)
II(60)
III(80)
IV(95)
III
IV
I
II
IV
III
II
I
II
I
IV
III

1
2
3
4
4
3
2
1
2
1
4
3
3
4
1
2

0.765
0.797
1.070
0.339
0.502
0.107
1.555
1.236
0.065
0.749
0.877
0.933
0.742
0.890
0.073
0.722

0.804
1.008
0.477
0.012
0.196
0.015
2.663
1.380
0.002
0.387
0.898
1.568
0.921
1.565
0.000
0.385

0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011
0.011

0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010
0.010

*Each experiment was performed with factors at the corresponding levels, indicated in roman numbers (see Table 1) and following the proceeding
explained in Materials and Methods.
This table provided the following conditions in the experiment. For example, for the experiment 1: solvent volume is 15 mL g of seeds, applied 5 min
time, temperature 50 C and the percentage of alcohol 20%. The experimentally measured efficiency is 0.804. This value is the average of the efficiencies
of five experiments performed in the same conditions.

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Microwave extraction V. Gianna et al.

Efficiency
(g of saponins per 100 g of seeds)

(a)

Figure 1 (a) Effect of duration of microwave radiation (

isopropanol;
ethanol). In this gure, A: Volume solvent g
seed = 20 mL g, C: Temperature = 90 C, D: % alcohol = 20
remain constant. (b) Eect of alcohol concentration ( isopropanol;
ethanol). In this gure, A: Volume solvent g
seed = 20 mL g, B: Time = 20 min, C: Temperature = 90 C remain constant. (c) Eect of the variable volume of solvent gram of
fruit ( isopropanol;
ethanol). In this gure, B: Time = 20 min, C: Temperature = 90 C, D: % alcohol = 20 remain constant.
(d) Eect of the temperature in the extraction ( isopropanol;
ethanol). In this gure, A: Volume solvent g seed = 20 mL g, B:
Time = 20 min, D: % alcohol = 20 remain constant.

3
2.5
2
1.5
1
0.5
0
0

10

15

20

25

30

35

t (min)
3
2.5
2
1.5
1
0.5
0
0

Efficiency
(g of saponins per 100 g of seeds)

(c)

10

15

20
25
% Alcohol

30

35

40

45

3
2.5
2
1.5
1
0.5
0
0

(d)

polarity, but not the highest one, because pure water

exhibited a lower capacity. This fact could indicate that
the solubility of the saponins does not depend only on
the ability of the solvents to form hydrogen bonds
and or dipoledipole interactions. Due to the fact that
ispropanol gave the best performance, this may indicate
that the carbon chain also participated in the solubilisation process.
As a comparison with MAE, Soxhlet extractions were
performed. After reuxing for 310 min with ethanol
20%, a 1.52% yield in saponins was obtained, while
isopropanol 20% gave 2.57% after 390 min of reux. It
is evident that almost twenty times as much time was
necessary to achieve the same results as with the MW
method, indicating that microwaves had a decisive
participation in the solubilisation of saponins.
It was previously reported that if the extraction
temperature exceeds 90 C, the saponins may be
degraded (Chen et al., 2007). This seems to be conrmed
in Fig. 1d, which shows a decrease in the extraction
eciency at temperatures higher than 90 C.
Figure 2 shows the eciency of the extraction at
several stages, where the same grains were extracted

10
15
20
25
Volume of solvent per gram of seed

30

35

3
2.5
2
1.5
1
0.5

Efficienciy (g of saponin per 100 g of seeds)

Efficincy
(g of saponins per 100 g of seeds)

(b)

Efficiency
(g of saponins per 100 g of seeds)

1596

Efficiency of the extraction in stages

3
2.75
2.5
2.25
2
1.75
1.5
1.25
1
0.75
0.5
0.25
0
1

2
Stages

Figure 2 Efciency of the extraction in stages ( isopropanol;

0
50

60

70
80
Temperature C

90

International Journal of Food Science and Technology 2012

100

ethanol). The three successive extractions were performed in optimal

conditions (A: Volume solvent g seed = 20 mL g, B: Time = 20 min,
C: Temperature = 90 C, D: % alcohol = 20) with the same seeds
and fresh solvent in each extraction.

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Microwave extraction V. Gianna et al.

with successive portions of fresh solvent, keeping the

optimal extractions conditions for each stage. Extraction with the isopropanol mixture in the rst stage
removed 2.663 g of saponins, 0.155 g in the second one
and in the third one, 0.082 g, all expressed by 100 g of
seeds. In the fourth step, the saponins level remained
under quantication limit; therefore, the nal concentration should be the sum of the rst three; it is
2.900 0.010 (g of saponins 100 g of seeds). This
result is within typical values for quinoa saponins
(Repo-Carrasco et al., 2011). In the rst extraction step,
the yield was 91.8%; nevertheless, the extraction with
ethanol is shown lesser eciency, reaching 57.2% yield
for the rst extraction step (Fig. 2).
Conclusion

The present study showed the Taguchi method to be

useful in determining the best saponin extraction conditions.
The eciency of the microwave extraction was
signicantly higher than the Soxhlet extraction, and
the use of alcohol as a solvent enabled an easy saponin
removal.
The MAE extraction time is considerably less than
with the Soxhlet method. Consequently, there is less risk
of gelation of the starch, which makes ltering easier
while avoiding charring by the concentrated sulphuric
acid medium of the LiebermanBurchard reagent.
Acknowledgments

The Science and Technology Ministry of the province of

Cordoba, Argentina for its partial funding of this
research.
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