Research Article

Extraction methods and varieties affect total anthocyanins

content in acidified extract of papery skin of onion
(Allium cepa L.)
Nyi Mekar Saptarini1*, Irma Erika Herawati2

ABSTRACT

Anthocyanins are water-soluble red pigments. Anthocyanins have anticarcinogenic activity and reduce the risk of stroke and
heart disease. Onion (Allium cepa L.) papery skin contains anthocyanin but rarely used. The aim of this study is to determine
the effect of extraction methods and varieties on total anthocyanin content in acidified extract of onion papery skin varieties
Bima Brebes and Maja Cipanas. Total anthocyanin contents are determinated by differential pH method. The anthocyanin
was extracted with a mixture of 70% ethanol and 2N hydrochloric acid pH 1.0 by maceration, percolation, reflux extraction,
and Soxhlet extraction. Total anthocyanin contents are more extracted in soaking method (maceration and reflux extraction)
than flowing solvent method (percolation and Soxhlet extraction). Total anthocyanin contents of Maja Cipanas are more
stable than Bima Brebes. The extraction method and onion varieties affect total anthocyanin contents.

KEY WORDS: Acidified solvent, Bima brebes, Flowing solvent method, Maja cipanas soaking method

INTRODUCTION (maceration) and 40°C (reflux extraction) and flowing

The production of onion (Allium cepa L., Liliaceae) in
2015 was 1,229,184 tons, decreased slightly compared
to 2014 of 1,233,983 tons.[1] The used part of the onion is
the bulbs, while the papery skin is thrown away. Onion
papery skin has not been utilized, although it contains
anthocyanins.[2] The most anthocyanin in various onions
is cyanidin derivatives.[3] Anthocyanins have typical
chemical structure that can react with free radicals, which
is efficacious as natural antioxidants, anti-inflammatory,
anticancer, anti-Alzheimer, and anti-Parkinson.[4,5]
Onion production is fluctuative, because of the
imbalance of production between in season and off-
season harvest.[6] This is due to the high intensity of
pest and disease if planted in out of season.[7,8] The aim
of this study is to determine the effect of extraction
methods and varieties on total anthocyanin contents
in acidified extract of onion papery skin. Extraction
methods are soaking method at ambient temperature
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solvent method at ambient temperature (percolation)
and 40°C (Soxhlet extraction). This research uses
onion varieties Bima Brebes and Maja Cipanas
because they are superior onion in Indonesia.
MATERIALS AND METHODS
Materials
Onion (A. cepa L.) bulb varieties Bima Brebes and
Maja Cipanas were collected from Gede Bage Market,
West Java Province, Indonesia, in July 2016. Bulbs
were identified by the Department of Biology, Faculty
of Mathematic and Natural Sciences, Universitas
Padjadjaran with No.  198/HB/03/2016. All chemical
reagents are analytical grade (Merck, Germany),
i.e.,  ethanol, hydrochloride acid, sodium hydroxide,
n-butanol, acetic acid, potassium chloride, calcium
chloride, and sodium acetate.
Water Determination
Onion varieties Bima Brebes and Maja Cipanas were
cleaned and peeled, and then, the papery skin were
washed and dried. 5 g of onion papery skin was dried

1
Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, West Java,
Indonesia, 2Department of Pharmacy, Faculty of Pharmacy and Natural Sciences, Universitas Al Ghifari, Bandung, Jawa
Barat, Indonesia

*Corresponding author: Nyi Mekar Saptarini, Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of
Pharmacy, Universitas Padjadjaran, West Java, Indonesia. E-mail: nyi.mekar@unpad.ac.id

Received on: 30-01-2017; Revised on: 26-02-2018; Accepted on: 19-04-2018

Drug Invention Today | Vol 10 • Issue 4 • 2018 471

 Nyi Mekar Saptarini and Irma Erika Herawati
on 105°C at atmospheric pressure for 5 h and then
weighed. Drying and weighing continued, every 1 h,
until a constant weight.[9]
Anthocyanin Extraction
Each of 10 g samples was extracted with 75 mL of mixture
of 70% ethanol and 2N hydrochloride acid pH  1.0 in
macerator, percolator, reflux, and Soxhlet apparatus. The
extraction was repeated for three cycles with solvent
replacement.[10] Each cycle was 24 h for maceration, 8 h
for percolation, and 2 h for reflux and Soxhlet method.
All extracts were collected into a 250 mL volumetric flask
and adjusted with concentrated HCl to a pH of 1.0.[11]
Anthocyanin Qualitative Analysis
(i) Each extract was heated with 2M HCl solution for
5 min at 100°C, the extracts color still red, (ii) Each
extract was added with 2M NaOH solution, and there
was alteration color from red to blue green, then
vanished, and (iii) thin-layer chromatography with
n-buthanol:acetic acid:water (4:5:1) as mobile phase
will produce an Rf value of about 0.40.[12]
Total Anthocyanin Contents Analysis
Determination of anthocyanin maximum wavelength:
Each extract was added 4 mL of 70% ethanol and 2
N HCl pH  1.0 and fulfilled with potassium chloride
buffer pH  1.0 in 10  mL volumetric flask. The
absorbance was measured at 450–700 nm.[11]
Analysis of total anthocyanin contents: Each extract
(1  mL) was buffered, one with 5  mL of potassium
chloride buffer pH 1.0, and the other one with 5 mL
of sodium acetate buffer pH 4.5 in 10 mL volumetric
flask and fulfilled with 70% ethanol. All solutions
were measured at maximum wavelength and 700 nm,
with distilled water as blank. The absorbances were
calculated using formula (1), and total anthocyanin
contents were calculate using formula (2).[11]
A=(Aλmax-A700)pH1.0-(Aλmax-A700)pH4.5 (1)
mg
Total anthocyanin contents ( )=  (2)
100 ml
A×MW×DF×100
( )
µ×1
Total anthocyanin contents were expressed as cyanidin-
3-glucoside (MW 449.2  g/mol, ε 26900  L/mol.cm)
and A=absorbance, Aλmax=absorbance at maximum
wavelength, Aλ700=absorbance at 700 nm, DF=dilution
factor.
Statistical Analysis
Results are presented as the mean ± standard deviation
(SD). One-way ANOVA followed by t-Student test
472
was used to analyze the data comparisons between
groups. Values were considered statistically significant
at P < 0.05.
RESULTS AND DISCUSSION
Onion varieties Bima Brebes and Maja Cipanas are
potential onion growing in Indonesia and resistant
to tuber blight due to Botrytis allii. Papery skin was
the used part of onion, and 4 kg of each onion bulbs
were used to produce 10.021  g of Bima Brebes and
10.038  g of Maja Cipanas papery skin. The papery
skins were 0.25% of gross weight of onion bulbs..
In 2015, the production of Maja Cipanas (10.9 tons/
ha) was higher than Bima Brebes (9.9 tons/ha), with
weight loss of tubers (wet to dry) of Maja Cipanas
(24.9%) was higher than Bima Brebes (21.5%).[1] The
water contents of Bima Brebes and Maja Cipanas were
3.5% and 3.0%, respectively . The values of water
content met the criteria, i.e., 8.0%. The water content
had to be determined, due to the affect the stability
of secondary metabolites.[13] Maja Cipanas grow well
in the low and highlands, while Bima Brebes only
in the lowlands. Maja Cipanas bulb is round, while
Bima Brebes is a small-ringed ring on the neck of the
disc. Both onions have pinky red papery skin[1] which
suggested anthocyanins.[11]
Anthocyanins must be extracted with a solvent
containing acetic acid or hydrochloric acid and the
solution should be stored in a darkened and cooled
place.[12] The mixture of 70% ethanol and HCl pH 1.0
was used as the extraction solvent, due to acid lysis
the plant cells, so anthocyanin extraction would
increase.[11] All acidified extracts of papery skin onion
varieties Bima Brebes and Maja Cipanas are red,
due to oxonium form of anthocyanin,[11] with various
intensities because of different extraction methods.
Extraction methods were soaking method at ambient
temperature (maceration) and 40°C (reflux extraction)
and flowing solvent method at ambient temperature
(percolation) and 40°C (Soxhlet extraction). The
efficacy of soaking method depends on solubility and
effective diffusion.[14] Small particle size allows good
contact between solvent and surface area of simplicia
for proper mixing. There is occasional shaking during
maceration and heating during reflux extraction.
Shaking and heating will increase diffusion and remove
concentrated solution from the simplicia surface
for bringing new solvent to the simplicia for more
extraction yield.[15] The disadvantage of maceration is
long duration of extraction time, so we tried to solve
this problem using reflux extraction, due to shorter
extraction time. Percolation and soxhlet extraction
are repeated cycle extraction with a fresh solvent until
dissolve all the solute in simplicia, while soxhlet
extraction using heating to shorten extraction time.[16]
Distillation is implicated during Soxhlet extraction,
Drug Invention Today | Vol 10 • Issue 4 • 2018
 Nyi Mekar Saptarini and Irma Erika Herawati

due to the solution is heating, then condensed send with maceration, i.e., 0.233 mg/g.[18 ] It was because in
back to the original flask.[10] The disadvantages of this study only red papery skin of onion were used, while
flowing solvent methods are long duration and high Ferreres et al. used all part of onion bulbs. These results
extraction amount of solvent consumed, which leads indicate that the plant part affects the concentration of
economic loss and the environmental problems.[17] extracted solute. Statistical analysis showed that total
Heat labile compounds will degrade in long heating, anthocyanin contents affect by extraction methods and
so we try to solve this problem using percolation, due varieties of onion. Anthocyanins are more extracted
to the extraction at ambient temperature. by soaking methods compared to flowing solvent
methods. Anthocyanins are more stable in ambient
Anthocyanins with BAW as mobile phase showed
temperature than in that of 40°C.
medium Rf value (0.10–0.40).[12] Qualitative
identification shows that all acidified extracts contain
anthocyanins. The chromatogram shows a red spot with CONCLUSION
various Rf values [Table  1]. Anthocyanins that have The extraction method and onion varieties affect total
been identified in onion include cyanidin 3-glucoside, anthocyanin contents.
cyanidin 3-arabinoside, cyanidin 3-malonyl-glucoside,
and cyanidin 3-malonylarabinoside.[18]
ACKNOWLEDGMENTS
Anthocyanin at pH  1.0 has a red oxonium form The authors thank to Juni Astuti Adam, Meilani
[Figure  1]. The maximum wavelength of acidified Syiko, Iin Islamy Toyib, and Arbaini Kurniawaty for
extract is in the range of 511.7–521.0  nm [Table  2].
technical assistance.
These values are convenient to anthocyanins maximum
wavelength, i.e.,  505–535  nm.[12] The determination
of total anthocyanin contents was conducted by pH
differential method [Table 3]. Anthocyanins at pH 1.0 is
in the oxonium form with strong red intensity, whereas
at pH  4.5, it is in the colorless carbinol form.[19] The
extraction process was influenced by simplicia particle
size, simplicia condition, the extracted materials,
solvent, extraction time, and extraction method.[12] The
extraction time for maceration was 72  h, percolation
was 24  h, and reflux and Soxhlet extraction was 6 h.
The highest total anthocyanin contents of Bima Brebes
were obtained by maceration. This result showed that
the longest extraction time (maceration), the highest
extracted solutes.. The highest total anthocyanin
contents of Maja Cipanas were obtained by reflux
extraction. This result indicates that anthocyanins of
Maja Cipanas are more thermostable than Bima Brebes. Figure 1: Maximum wavelength of acidified extract from
Soxhlet extraction, i.e., Bima Brebes at 519 nm and Maja
Total anthocyanin contents from soaking method Cipanas at 521 nm
(maceration and reflux extraction) were higher than
flowing solvent method (percolation and Soxhlet Table 1: Rf value of acidified extract
method). There was a statistical significance for the Extraction Rf value of acidified extract
extraction method on total anthocyanin contents of method
Bima Brebes Maja Cipanas
Bima Brebes (P = 2.67 × 10−6) and Maja Cipanas (P
= 2.62 × 10−6) and between onion varieties (P = 2.78 Maceration 0.48 0.45
Percolation 0.46 0.48
× 10−9). Flowing solvent methods provide lower total Reflux extraction 0.49 0.49
anthocyanin contents due to shorter of contact time Soxhlet extraction 0.46 0.43
between simplicia and solvent compare to soaking
methods.. The solvent flows through the simplicia,
Table 2: Maximum wavelength of acidified extract
and the less solute was extracted. While on soaking
methods, simplicia was soaked in the solvent, so the Extraction Maximum wavelength
contact time was longer. There were shaking and method
Bima Brebes Maja Cipanas
heating, which increased diffusion and removed the Maceration 517.2 511.9
concentrated solution from that of simplicia surface. Percolation 514.6 511.7
Total anthocyanin contents in papery skin onion with Reflux extraction 513.8 509.4
Soxhlet extraction 519.0 521.0
soaking methods were higher than that of fresh onion

Drug Invention Today | Vol 10 • Issue 4 • 2018 473

 Nyi Mekar Saptarini and Irma Erika Herawati
Table 3: Total anthocyanin contents in acidified extract
Acidified extract Extraction method
Bima brebes Maceration
Percolation
Reflux extraction
Soxhlet extraction
Maja cipanas Maceration
Percolation
Reflux extraction
Soxhlet extraction
a
Mean±SD, n=3. SD: Standard deviation
REFERENCES
1. Available from: http://www.jambiprov.go.id/assets/skpd/bpspt-
provinsi jambi/download/11_Keputusan_ Menteri_ Pertanian_
No_131_Thn_2015.pdf. [Last accessed on 2017 Dec 15].
2. Frankly A. Food and Chemistry. 2nd  ed. USA: Mc Graw Hill
Book Camp.; 1983.
3. Slimestad R, Fossen T, Vågen IM. Onions: A source of unique
dietary flavonoids. J Agric Food Chem 2007;55:10067-80.
4. Wang J, Mazza G. Effects of anthocyanins and other phenolic
compounds on the production of tumor necrosis factor alpha in
LPS/IFN-gamma-activated RAW 264.7 macrophages. J Agric
Food Chem 2002;50:4183-9.
5. Hou DX. Potential mechanisms of cancer chemoprevention by
anthocyanins. Curr Mol Med 2003;3:149-59.
6. Baswarsiati L, Rosmahani E, Korlina EP, Kusumainderawati SZ,
Sa’adah SZ. Adaptation of Some Off-Season Onion Varieties.
Proceeding of Seminar of Research Result/Assessment of
BPTP Karangploso. 1996.
7. Duriat AS, Soetiarso TA, Prabaningrum L, Sutarya R.
Application of integrated pest and disease control on red
onion cultivation. In: Onion Production Technology. Jakarta:
Puslitbanghorti; 1994. p. 25-34.
8. Hadisoeganda WW, Suryaningsih S, Moekasa TK. Onion
diseases and pests and how to control them. In: Onion Production
Technology. Jakarta: Puslitbanghorti; 1995. p. 36-44.
9. The United States Pharmacopeial Convention. US
Pharmacopeia. 40th  ed. Water Determination. United Stated,
Rockville: The United States Pharmacopeial Convention;
2010. Available from: https://www.hmc.usp.org/sites/default/
files/documents/HMC/GCs-Pdfs/c921.pdf. [Last cited on
2016 Jul 12].
10. Bart HJ. Extraction of natural products from plant-An
introduction. In: Bart HJ, Pilz S, editors. Industrial Scale
Natural Product Extraction. Weinheim: Wiley-VCH Verlag
474
Absorbancea Total anthocyanin contenta (mg/g)
0.350±0.003 1.463±0.013
0.079±0.002 0.328±0.010
0.339±0.002 1.415±0.008
0.052±0.005 0.218±0.021
0.283±0.002 1.181±0.008
0.143±0.004 0.597±0.015
0.347±0.003 1.449±0.013
0.082±0.005 0.342±0.022
GmbH; 2011. p. 1-25.
11. Wrolstad RE, Acree TE, Decker EA, Penner MH, Reid DS,
Schwartz SJ, et al. Handbook of Food Analytical Chemistry:
Pigments, Colorants, Flavors, Texture, and Bioactive Food
Components. New Jersey: John Wiley and Sons Inc.; 2005.
p. 156-67.
12. Harborne JB. Phytochemical Methods: A  Guide to Modern
Techniques of Plant Analysis. London: Thompson Science;
1998. p. 60-94.
13. Goeswin A. Book of Natural Materials Technology (serial of
Pharmaceutical Industry). Bandung: Penerbit Institut Teknologi
Bandung; 2009. p. 17-79.
14. Wrolstad RE, Giusti MM. Characterization and measurement
of anthocyanins by UV-Vis spectroscopy. Curr Protoc Input
Food Anal Chem 2001; F1:1-9.
15. Cheok CY, Salman HA, Sulaiman R. Extraction and
quantification of saponins: A review. Food Res Int 2014;5:16-40.
16. Azmir J, Zaidul A, Rahman A, Sharif A, Mohamed A, Sahena B,
et al. Techniques for extraction of bioactive compounds from
plant materials: A review. J Food Eng 2013;117:426-36.
17. Grigonis D, Sivik P, Sandahl M, Eskilsson C. Comparison of
different extraction techniques for isolation of antioxidants
from sweet grass (Hierochloe odorata). J  Supercrit Fluids
2005;33:223-33.
18. Wang L, Waller C. Recent advances in extract ion of nut raceut
icals from plants. Trends Food Sci Tech 2006;17:300-12.
19. Ferreres F, Gil MI, Tomas-Barberan FA. Anthocyanins and
flavonoids from shredded red onion and changes during storage
in perforated films. Food Res Int 1996;29:389-95.
20. Wrolstad RE, Giusti MM. Characterization and measurement
of anthocyanins by UV-Vis spectroscopy. Curr Protoc Input
Food Anal Chem 2001; F1:1-9.
Source of support: Nil; Conflict of interest: None Declared
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