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 Sarsfield Av. 1611., 5015 Cordoba, Argentina

2 Institute of Science and Food Technology. FCEFyN. UNC, Velez Sarsfield Av. 1611., 5015 Cordoba, Argentina

3 National Council of Scientific and Technical Research (CONICET), Velez Sarsfield Av. 1611., 5015 Cordoba, Argentina

(Received 25 August 2011; Accepted in revised form 23 February 2012)

Summary Despite their possible applications in diverse fields, saponins are still considered to be industrial waste. The

use of saponins, however, would make seed processing more profitable and reduce the pollution of

watercourses. In this work, the microwave extraction method of Chenopodium quinoa Willd saponins was

investigated. The effects of variables such as temperature, time of microwave application, solvent

composition and solvent ⁄mass seed ratio were investigated. Solvent mixtures (ethanol–water and

isopropanol–water) were used for the extraction. The Taguchi design methodology was employed to

determine the number of experiments and the optimal conditions for different extractions. The efficiency of

each assay was determined and the results agreed with the best conditions provided by the Taguchi

experimental design for both solvent mixtures. The isopropanol–water mixture efficiency was 91.8% in one

extraction step, and for ethanol–water mixture, it was 57.1%, clearly showing the advantage of the first 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 istargeted at domestic consumption, such as in seed orflour (Vilche et al., 2003).For the seed to be used for human consumption, the

saponins content must be removed because they impartbitter taste and are considered to be the main anti-nutrient of the quinoa. Saponins are known to causebreakdown in the human small intestine cell membranesand also negatively affect the assimilation of someproteins (Moges Woldemichael & Wink, 2001). Sapo-nins are found in quinoa grain pericarp (Taylor &Parker, 2002), and their presence in the fruits seems toplay a role in defence against pests such as birds andinsects, 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 inquinoa for human consumption varies between 0.06%and 0.12% (Bacigalupo & Tapia, 1990). This is consistent

with the results of sensory tests conducted at theUniversity of Ambato, Ecuador, where it was deter-mined that the maximum tolerance of saponin contentin 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 emulsifier in beverages,shampoos and soaps, as well as in fire extinguishers,photography and the cosmetics industry. Furthermore,saponins have been used in the pharmaceutical industryand agriculture (San Martın & Briones, 1999).Another important property of saponins is their

antifungal activity. It has been shown that saponinsinhibit the growth of Candida albicans (Moges Wolde-michael & Wink, 2001) and (Reilly et al., 2004) and thatsaponins treated with alkali have a significant antifungalactivity against Botrytis cinerea (Stuardo & San Martin,2008). In Canada, a commercial product composedmainly of quinoa saponins called HeadsUp PlantProtectant� has been developed (HeadsUp Plant Pro-tectant Ltd, Kamsack, Canada).Saponins also have anticarcinogenic properties and

stimulate the immune system (Li et al., 2002). Oleanolicacid, one of the five major components of the saponinsfrom quinoa, showed significant antitumor activitywhen tested in colon cells (Estrada et al., 1998).*Correspondent: E-mail: vgianna@efn.uncor.edu

International Journal of Food Science and Technology 2012 1

doi:10.1111/j.1365-2621.2012.03008.x

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The effect of water deficit level on saponin composi-tion of quinoa was determinate in a field experimentconducted in Mexico. The experiment took place duringthe development of Sajama and Chucara cultivars. Thesaponin content increases during branching, panicleinitiation and in blooming, followed by a decreaseduring the grain filling stage, when the plants undermedium water deficit recorded the highest saponincontent (Soliz-Guerrero et al., 2002).The microwave-assisted extraction (MAE) is a rela-

tively new technique but has been growing rapidly inrecent times. Compounds present in the matrix caninteract with a suitable solvent assisted by microwaveenergy, which heats the system and allows for betterextraction. Microwaves have advantages over the rate ofheating of the sample and prevent overheating, avoidingthermolabile substances denaturation. Therefore, withthis method, can achieve good yields in short time.As mentioned earlier, the objectives of this research

were to evaluate the extraction efficiency of saponinsfrom quinoa seed by solvent extraction, employingmicrowaves through an appropriate combination of theoperating variables (% alcohol, time, temperature,volume solvent ⁄gram seeds)

Materials and methods

The grains were harvested during 2009 in the place ‘LaPoma’, located between 65� 56 ‘and 66� 33¢ westlongitude and between 23� 20¢ and 24� 55¢ southlatitude, Province of Salta, Argentina.The tested solvent mixtures were as follows: a-

ethanol–water mixtures, b- isopropanol–water mixtures.Four variables were studied for the extraction process:1- temperature, 2- solvent composition, 3 - contact timeand 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 moleculardegradation limit for saponins (Chen et al., 2007).The Taguchi experimental design was employed to

determine the optimal conditions with a minimal num-ber of experiments, for saponins extraction from etha-nol–water and isopropanol–water mixtures in amicrowave oven. For the orthogonal array, the de-

sign-easy 7.1 software for Windows and a Taguchimatrix of L16 – four factors of four levels each – wereused (Montgomery, 2004) and (Anderson & Witcomb,2007). anova tests were performed using InfoStat, 2010(statistical software) to analyse the statistical signifi-cance of the results.To carry out the extractions, a 50 mL glass reactor

(Schott� SCHOTT Argentina S.A., Buenos Aires,Argentina) with a Teflon� (DUPONT., Buenos Aires,Argentina) cap was used. It is fitted with seals made ofsilicone and viton to prevent leakage. A temperaturesensor was fixed to the reactor by rubber bands. A

900 W Litton BGH 16650 microwave (Argentine indus-try) with a temperature sensor was used during theexperiments.The extractions in Soxhlet device were performed with

20% ethanol–water or isopropanol–water mixtures inboth cases, in a ratio of 20 mLof solvent per gramof seed.

Extraction procedure with microwave equipment

The extraction was performed as follows: 1.0000 g ofwhole seeds was put into the reactor with the chosensolvent, and this was weighed and closed. The temper-ature probe was fixed and the reactor is introduced intothe MW oven and started. When the required temper-ature was reached, the timer was started. At the end, theoven was stopped and the reactor is cooled with coldwater, opened, weighed at room temperature, and theextract was recovered, filtering through a 0.2 lm mem-brane, employing a pressure filtration syringe.

Quantification of saponins

Saponins in the extracts were derivatised by the Liber-mann–Burchard reaction, mainly based on Monje et al.(2006) although it was taken into account (Hostettmann& Marston, 2005) and (Abisch & Reichstein, 1960). Theabsorbances were measured at 528 nm with a PerkinElmer Lambda 25 spectrophotometer. Calibrationcurves were determined with oleanolic acid. Linearregression of data followed the expression:

A ¼ 4:5725� ½S� þ 0:0164 ð1ÞR2 = 0.9998

A: measured absorbance, [S]: saponin concentration(mg ⁄mL) and R2: the square correlation coefficient ofthe calibration curve.The low quantification limit for eqn (1) is

0.05 mg ⁄mL and its linearity limit is 0.65 mg ⁄mL.All measurements were performed at least five times,

and the ‘Q’ acceptance criteria applied. The confidenceinterval was established by the Student’s ‘t’ test with aprobability of 95%, resulting ±0.011 for extractionswith ethanol and ±0.010 for isopropanol.For the extraction efficiency (E), the following equa-

tion was used:E = 100 · total mass of saponins [g] ⁄mass of seed [g]

Results and discussion

Four basic variables were analysed, each one at fourlevels, to find how we can use each variable in acombination to reach the optimum conditions. Table 1shows the Taguchi experimental matrix design with thefactors and levels mentioned before. Table 2 showsTaguchi experimental matrix applied. It was made afactorial design and found that 44 = 256 assays should

Microwave extraction V. Gianna et al.2

International Journal of Food Science and Technology 2012 � 2012 The Authors

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

be necessary, whereas with Taguchi method, onlysixteen experiments must be done to establish the bestextraction condition.The anova testing for both ethanol–water and

isopropanol–water mixtures has shown that both, fac-tors and model, are significant with a ‘P’ value less than0.05. Values for ‘F’ have shown results in the samedirection.

Extraction with MW

The matrix of experiments is shown in Table 2, whichincludes their respective average yields for both theethanol–water and isopropanol–water mixtures.

Experiment number seven showed the maximumextraction yields for both solvent mixtures (1.65% forethanol 20%, and 2.66% for isopropanol 20%). Thenumerical analysis of the results following the Taguchiprocedure (Montgomery, 2004) revealed that the opti-mal extraction conditions for the mixtures of ethanol–water and isopropanol–water were the same, namelyvolume 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 isopropanol–water 20%; (ii)The higher temperature facilitated the diffusion of thesolute from the solid to the solvent.

Test for trend

To determine whether the above values corresponded tothose giving the best yields, a new series of experimentswere performed at the best experimental conditionskeeping all the variables constant except one, which wasregularly changed. The results for the isopropanol–water mixtures and ethanol–water mixtures are shownin Fig. 1a–d.Regarding the effect of solvent composition on

extraction efficiency, the tests showed that maximumextraction of saponins took place at a rather high

Table 1 Experimental design matrix

Level

Factor A

Vol. Solvent/g seed

Factor B

Time (min)

Factor C

T (�C)

Factor D

% alcohol

I 15 5 50 20

II 20 15 60 60

III 25 20 70 80

IV 30 30 90 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 (alcohol–water

mixture) ⁄ g of seeds; B: time to apply microwave; C: the temperature at

which extraction takes place and D: % of alcohol in the solvent.

Table 2 Taguchi L16 experimental design (44)

Experiment

Factor A

Vol. Solvent/g seed

Factor B

Time (min)

Factor C

T (�C)

Factor D

% alcohol Vacancy

Experimental results(*)

Average efficiency: g saponins/100 g of

seed

Ethanol–water

mixtures

Isopropanol–water

mixtures

1 I(15) I(5) I(50) I(20) 1 0.765 ± 0.011 0.804 ± 0.010

2 I II(15) II(60) II(60) 2 0.797 ± 0.011 1.008 ± 0.010

3 I III(20) III(70) III(80) 3 1.070 ± 0.011 0.477 ± 0.010

4 I IV(30) IV(90) IV(95) 4 0.339 ± 0.011 0.012 ± 0.010

5 II(20) I II III 4 0.502 ± 0.011 0.196 ± 0.010

6 II II I IV 3 0.107 ± 0.011 0.015 ± 0.010

7 II III IV I 2 1.555 ± 0.011 2.663 ± 0.010

8 II IV III II 1 1.236 ± 0.011 1.380 ± 0.010

9 III(25) I III IV 2 0.065 ± 0.011 0.002 ± 0.010

10 III II IV III 1 0.749 ± 0.011 0.387 ± 0.010

11 III III I II 4 0.877 ± 0.011 0.898 ± 0.010

12 III IV II I 3 0.933 ± 0.011 1.568 ± 0.010

13 IV(30) I IV II 3 0.742 ± 0.011 0.921 ± 0.010

14 IV II III I 4 0.890 ± 0.011 1.565 ± 0.010

15 IV III II IV 1 0.073 ± 0.011 0.000 ± 0.010

16 IV IV I III 2 0.722 ± 0.011 0.385 ± 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.

Microwave extraction V. Gianna et al. 3

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polarity, but not the highest one, because pure waterexhibited a lower capacity. This fact could indicate thatthe solubility of the saponins does not depend only onthe ability of the solvents to form hydrogen bondsand ⁄or dipole–dipole interactions. Due to the fact thatispropanol gave the best performance, this may indicatethat the carbon chain also participated in the solubili-sation process.As a comparison with MAE, Soxhlet extractions were

performed. After refluxing for 310 min with ethanol20%, a 1.52% yield in saponins was obtained, whileisopropanol 20% gave 2.57% after 390 min of reflux. Itis evident that almost twenty times as much time wasnecessary to achieve the same results as with the MWmethod, indicating that microwaves had a decisiveparticipation in the solubilisation of saponins.It was previously reported that if the extraction

temperature exceeds 90 �C, the saponins may bedegraded (Chen et al., 2007). This seems to be confirmedin Fig. 1d, which shows a decrease in the extractionefficiency at temperatures higher than 90 �C.Figure 2 shows the efficiency of the extraction at

several stages, where the same grains were extracted

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

E

ffic

ien

cy

(g o

f sa

po

nin

s p

er 1

00 g

of

seed

s)

t (min)

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35 40 45

E

ffic

incy

(g

of

sap

on

ins

per

100

g o

f se

eds)

% Alcohol

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

Eff

icie

ncy

(g

of

sap

on

ins

per

100

g o

f se

eds)

Volume of solvent per gram of seed

0

0.5

1

1.5

2

2.5

3

50 60 70 80 90 100

E

ffic

ien

cy (

g o

f sa

po

nin

s p

er 1

00 g

of

seed

s)

Temperature °C

(a)

(b)

(c)

(d)

Figure 1 (a) Effect of duration of microwave radiation (—•—isopropanol; ethanol). In this figure, A: Volume solvent ⁄ gseed = 20 mL ⁄ g, C: Temperature = 90 �C, D: % alcohol = 20

remain constant. (b) Effect of alcohol concentration (—•— isopropa-

nol; ethanol). In this figure, A: Volume solvent ⁄ gseed = 20 mL ⁄ g, B: Time = 20 min, C: Temperature = 90 �C re-

main constant. (c) Effect of the variable volume of solvent ⁄ gram of

fruit (—•— isopropanol; ethanol). In this figure, B: Time = 20 -

min, C: Temperature = 90 �C, D: % alcohol = 20 remain constant.

(d) Effect of the temperature in the extraction (—•— isopropanol;

ethanol). In this figure, A: Volume solvent ⁄ g seed = 20 mL ⁄ g, B:Time = 20 min, D: % alcohol = 20 remain constant.

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

1 2 3

Eff

icie

nci

y (g

of

sap

on

in p

er 1

00 g

of

seed

s)

Stages

Efficiency of the extraction in stages

Figure 2 Efficiency of the extraction in stages (—•— isopropanol;

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.

Microwave extraction V. Gianna et al.4

International Journal of Food Science and Technology 2012 � 2012 The Authors

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with successive portions of fresh solvent, keeping theoptimal extractions conditions for each stage. Extrac-tion with the isopropanol mixture in the first stageremoved 2.663 g of saponins, 0.155 g in the second oneand in the third one, 0.082 g, all expressed by 100 g ofseeds. In the fourth step, the saponins level remainedunder quantification limit; therefore, the final concen-tration should be the sum of the first three; it is2.900 ± 0.010 (g of saponins ⁄100 g of seeds). Thisresult is within typical values for quinoa saponins(Repo-Carrasco et al., 2011). In the first extraction step,the yield was 91.8%; nevertheless, the extraction withethanol is shown lesser efficiency, reaching 57.2% yieldfor the first extraction step (Fig. 2).

Conclusion

The present study showed the Taguchi method to beuseful in determining the best saponin extraction con-ditions.The efficiency of the microwave extraction was

significantly higher than the Soxhlet extraction, andthe use of alcohol as a solvent enabled an easy saponinremoval.The MAE extraction time is considerably less than

with the Soxhlet method. Consequently, there is less riskof gelation of the starch, which makes filtering easierwhile avoiding charring by the concentrated sulphuricacid medium of the Lieberman–Burchard reagent.

Acknowledgments

The Science and Technology Ministry of the province ofCordoba, Argentina for its partial funding of thisresearch.

References

Abisch, E. & Reichstein, T. (1960). Orientierende chemische Untersu-chung einiger Apocynaceen. Helvetica Chimica Acta, 43, 1844–1861.

Anderson, M. & Witcomb, P. (2007). DOE Simplified: Practical Toolsfor Effective Experimentation, 7th edn. New York.: ProductivityPress Inc.

Bacigalupo, A. & Tapia, M. (1990). Potencial agroindustrial de loscultivos andinos subexplotados. In: Cultivos Andinos subexplotados ysu aporte a la alimentacion (edited by M. Tapia). Pp. 136–163.Santiago, Chile: FAO, Ediciones Gegra S.A.

Cabieses, F. (2005). El valor de la Quinua. Chasqui. El correo del Peru.Boletın Cultural del Ministerio de Relaciones Exteriores., 6, p. 8–9.

Chen, Y., Xie, M.-Y. & Gong, X.-F. (2007). Microwave-assistedextraction used for the isolation of total triterpenoid saponins fromGanoderma atrum. Journal of Food Engineering, 81, 162–170.

Estrada, A., Bing, L. & Laarveld, B. (1998). Adjuvant action ofChenopodium quinoa saponins on the induction of antibodyresponses to intragastric and intranasal administeres antigensinmice. Comparative Inmunology Microbiology and Infections Dis-eases, 21, 225–236.

Hostettmann, K. & Marston, A. (2005). Chemistry & Pharmacology ofNatural Products. Saponins. New Work: Cambridge UniversityPress.

InfoStat. (2010). Software estadıstico desarrollado por docentes-investigadores de Estadıstica y Biometrıa y de Diseno deExperimentos de la Universidad Nacional de Cordoba (FCA-UNC).

Li, J., Guo, W.-J. & Yang, Q.-Y. (2002). Effects of ursolic acid andoleanolic acid in human colon carcinoma cell line HCT15. WorldJournal of Gastroenterology, 8, 493–495.

Moges Woldemichael, G. & Wink, M. (2001). Identification andbiological activities of triterpenoids saponins from Chenopodiumquinoa. Journal of Agricultura and foods Chemistry, 49, 2327–2332.

Monje, C., Yarko, A. & Raffaillac, J.P. (2006). Determinacion desaponina total en quinoa (Chenopodium quinoa Willd) metodoespectrofotometrico. Memoria IV Congreso Nacional de la AsociacionBoliviana de Proteccion Vegetal. Oruro, Bolivia: C.E.A.C. – Dpto.Fitotecnia-FCAPV-UTO. ABPV.

Montgomery, D.C. (2004). Design and Analysis of Experiments, 6thedn. New York: Wiley.

Nieto, C.Cultivo, produccion y conservacion de la quinua en Ecuador.http://www.rlc.fao.org/es/agricultura/produ/cdrom/contenido/li-bro14/cap4.3.htm (accessed March 15, 2012)

Reilly, S.K., Hollis, L., Jones, R.S., Peterson, T.A., Greenway, D. &King, K. (2004). Saponins of Chenopodium quinoa (PC Code097094). Pp. 13–23. In: Biopesticides Registration Action Docu-ments Washington DC.: U.S. Environmental Protection AgencyOffice of Pesticide Programs, Biopesticides and Pollution PreventionDivision.

Repo-Carrasco, R., Espinoza, C. & Jacobsen, S. (2011). ValorNutricional y Usos de la Quinua Chenopodium quinoa) y de laKaniwa (Chenopodium pallidicaule) http://www.rlc.fao.org/es/agricultura/produ/cdrom/contenido/libro14/cap5.1.htm (accessedNovember 8, 2011).

Ruales, J. & Nair, B.M. (1992). Quinoa (Chenopodium quinoa willd) animportant Andean food crop. Archivo Latinoamericano de Nutricion,42, 232–241.

San Martın, R. & Briones, R. (1999). Industrial uses and sustainablesupply of Quillaja saponaria (Rosaceae) saponins. Economic Botany,53, 302–311.

Soliz-Guerrero, J.B., Jasso de Rodrıguez, D., Rodrıguez-Garcıa, R.,Angulo-Sanchez, J.L. & Mendez-Padilla, G. (2002) Quinoasaponins: concentration and composition analysis. Book chapter;Conference paper Trends in New Crops and New Uses. Proceed-ings of the Fifth National Symposium, Atlanta, GA, Pp. 110–114.

Stuardo, M. & San Martin, R. (2008). Antifungal properties of quinoa(Chenopodium quinoa Willd) alkali treated saponins against Botrytiscinerea. Industrial Crops and Products, 27, 296–302.

Taylor, J.R.N. & Parker, M.L. 2002. Chap. 3: Quinoa Pp. 93-122.. In:Pseudo Cereals and Less Common Cereals. Grain Properties andUtilization Potential. (edited by P. Belton & J. Taylor). New York:Springer-Verlag, Pp. 98.

Vilche, C., Gely, M. & Santalla, E. (2003). Physical properties ofquinoa seeds. Biosystems Engineering, 86, 59–65.

Microwave extraction V. Gianna et al. 5

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