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Antioxidant activity of Allium tuberosum Rottl. ex Spreng under different

extraction methods

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The  27th  Annual  Meeting  of  the  Thai  Society  for  Biotechnology  and  
International  Conference
INNOVATIVE  BIOTECHNOLOGY
Organized  by

The  Thai  Society  for  Biotechnology  (TSB)

Department  of  Microbiology,  Faculty  of  Science

Chulalongkorn  University

Faculty  of  Science,  Chulalongkorn  University

The  Institute  of  Biotechnology  and  Genetic  Engineering

Chulalongkorn  University
http://www.tsb2015.org
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

Antioxidant activity of Allium tuberosum Rottl. ex Spreng under different extraction

methods

Napatsorn Lawthienchai1, Thanutcha Tongprasan2, Suvaluk Asavasanti3, Aussama Soontrunnarudrungsri1

and Patchanee Yasurin1*
1
Faculty of Biotechnology, Assumption University, Bangkok, Thailand
2
Department of Food Engineering, King Mongkut's University of Technology Thonburi, Bangkok,
Thailand
3
Pilot Plant Development and Training Institute, King Mongkut's University of Technology Thonburi,
Bangkok, Thailand

*e-mail: patchaneeYsr@au.edu

Abstract
The Bang-Phae organic vegetable grower community enterprise group, Ratchaburi province is one of the
big Chinese chives (Allium tuberosum Rottl. ex Spreng) grower in Thailand. However, some Chinese
chives did not meet standard and were sorted out. The Chinese chives essential oil, which contains many
new and known bioactive compounds, is valued product from sorted out Chinese chives. Therefore, this
experiment was aimed to study the antioxidant activity of Chinese chive extracts using 17 different
extraction conditions (Steam distillation for 1.0, 2.0, 2.5, 3.0 hour; Ohmic pretreatment with steam
distillation for 1.0, 2.0, 2.5, 3.0 hour; 95% ethanol, hexane, oil extract using dry, fresh, freeze Chinese
chives). The ferric reducing antioxidant potential assay was used to evaluate antioxidant activity of
Chinese chive extracts. The results showed that the highest antioxidant activity was 1.756 ± 0.008 µmol
Fe2+/ mg sample of freeze Chinese chives extracted with hexane. The antioxidant activity was vary from
0.098 ± 0.005 - 1.756 ± 0.008 µmol Fe2+/ mg sample depend on the extraction condition.

Keywords: antioxidant activity, Allium tuberosum Rottl. ex Spreng, Chinese chives

Introduction
Chinese chives (Allium tuberosum Rottl. ex Spreng) is commonly called garlic chives or Chinese
chives, belonging to the genus Allium and family Liliaceae as same as garlic, white lily and onion.
Chinese chives are widely found in South East Asia, South Asia and some countries in Middle East such
as Iran. (Alizadeh, et al, 2013)
There is antioxidant activity because of flavonoids (flavones and quercetins) and sulfur
containing compounds (allyl-cysteine, diallyl sulfide and allyl trisulfide) that found in Chinese chives by
antioxidant activity mechanism of flavonoids and sulfur containing compounds are different.
Flavonoid is the phenolic compound group. So, the antioxidant activity of flavonoids base on free
radical scavenger. There are 2 mechanisms of free radical scavenger by hydrogen atom (ArOH) is
removed by free radical. (Brewer, 2011)
This mechanism refers to hydrogen atom transfer. (R* + ArOH RH + ArO*) The high inactive
free radical (ArO*) refers to high efficiency of antioxidant activity. This step, the bond dissociation of O-
H bonds is important parameter because weaker O-H bond easier react with free radical inactive. (Brewer,
2011)
Second mechanism, the antioxidant can give an electron to free radical becoming a radical cation.
(R* + ArOH R- + ArOH*+)  The  free  radical  cation  will  be  inactive  free  radical.  So,  it  doesn’t  react  with  
substrate. (Brewer, 2011)

November 17-20, 2015 Mandarin Hotel Bangkok by Centre Point, Bangkok, Thailand
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

Antioxidant activity of sulfur containing compounds refers to metal chelating and oxygen
scavenger by sulfur can bind with transition metal inside the body in order to reduce the oxidation
reaction. (H2O2 + O2*- + Fe2+ OH* +OH- + O2) Moreover, the sulfur containing compound will bind
with oxygen free radical in order to reduce the oxidation reaction. (Brewer, 2011)
According to antioxidant activity in Chinese chives, the extraction method is important factor for
antioxidant efficiency. (Pavia, et al, 2011)
The basic principle of steam distillation is separation of mixed compound by using water as a
solvent. This method is suitable for compound   that   easily   to   volatile,   insoluble   and   doesn’t   react   with  
water. On the other hand, this method uses the high temperature to extract. So, high temperature may
cause antioxidant active compound deterioration. Moreover, there are the higher initial costs for
investment and the initial training is required in order to operate the equipment because this method
involved with high temperature, heat source. (Pavia, et al, 2011)
The principle of ohmic pretreatment based on the alternative electrical current passage through
the product body which serves as an electrical resistance. The mild electroporation mechanism during
ohmic pretreatment at low frequency (50-60 Hz) will help to build up the electrical charge and form pores
across cell wall. According to this principle, the researcher believes that ohmic pretreatment will help the
component in the product will spread out. It may increase the efficiency of extraction method when the
ohmic pretreatment is used as pretreatment. Moreover, ohmic pretreatment will reduce the mechanical
damage and get better nutrient and vitamin retention. On the other hand, the ohmic pretreatment
technique  doesn’t  suit  with  the  product  that  contains  high  fat  globules  because  fat  is  non-conductive. The
fat product will reduce the efficiency to form pore across cell wall of product. (Skudder, 1998)
The principle of solvent extraction is very simple. The needed substance is separated out by using
solvent. The used plants has never passed the chemical and heat process. This technique will minimize the
compound deterioration and use simple equipment. On the other hand, it takes a long time to extract, uses
high volume of solvent and it may have the solvent residue. (Rydberg, et al, 2005)
The effective of solvent extraction depends on the polar properties of solvents. For example, 95%
ethanol is both polar and non-polar solvent because 95% ethanol has both hydroxyl group (polar) and
ethyl group (non-polar). So, 95% ethanol can extract both polar and non-polar compound. Hexane is
strongly non-polar solvent. So, hexane will extract the non-polar compound better than 95% ethanol.
Vegetable oil is non-polar solvent  but  it  is  the  lipid.  Some  compound  can’t  dissolve  in  lipid.  So,  vegetable  
oil may have the lowest efficiency for non-polar extraction than hexane. (Rydberg, et al, 2005)
According to the different limitation of each extraction methods, the effect of extraction method
on antioxidant activity of Chinese chives was studied.

Materials and methods

Chinese chives were obtained from Bang-Phae organic vegetable grower community enterprise
group, Ratchaburi province, Thailand. Steam distillation and Ohmic pretreatment extraction were
obtained from Ms. Thanutcha Tongprasan at King Mongkut's University of Technology Thonburi,
Thailand. Solvent extraction (95% ethanol, hexane, vegetable oil extract using dry, fresh, freeze Chinese
chives and antioxidant activity were conducted at faculty of Biotechnology, Assumption University,
Bangkok, Thailand.

Preparation of dry Chinese chives

Fresh Chinese chives was cleaned and chopped into small pieces. It was weighed into 80 grams
per  1  tray.  It  was  put  into  the  dryer  (model  UM  500,  Memmert)  by  drying  at  45◦C  until  it  dried  or  the  
weight was constant. The dried Chinese chives were ground into powder and kept in tightly container.

Preparation of fresh Chinese chives

Fresh Chinese chives was cleaned and chopped into small pieces before using.

November 17-20, 2015 Mandarin Hotel Bangkok by Centre Point, Bangkok, Thailand
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

Preparation of freeze Chinese chives

Fresh Chinese chives was cleaned and chopped into small pieces. It was put into tightly container
before put into freezer at -20◦C  for  24  hours. It was thawed before using.

Steam distillation
Fresh Chinese chives was cleaned and chopped into small pieces. It was mixed with distilled
water by using 1:3 ratio (mg/mg). After mixing, it was distilled with 10 ml/L of petroleum ether at 100◦C
for 1, 2, 2.5 and 3 hours. After distillation, the water was separated. Then, 10 gram of sodium anhydrous
was added in order to remove the water. The petroleum ether was removed out by evaporating at 50◦C for
838 mPa by these extracted samples were obtained from Ms. Thanutcha T. (King Mongkut's University of
Technology Thonburi, Thailand)

Ohmic pretreatment with steam distillation

Fresh Chinese chives was cleaned and chopped into small pieces. It was mixed with distilled
water by using 1:3 ratio (mg/mg). After mixing, it was treated ohmic at electrical field strength 75 V/cm
and  cut  off  temperature  at  60◦C.  After  that,  it  was  distilled  with  10 ml/L  of  petroleum  ether  at  100◦C  for  
1, 2, 2.5 and 3 hours. After distillation, the water was separated. Then, 10 gram of sodium anhydrous was
added  in  order  to  remove  the  water.  The  petroleum  ether  was  removed  out  by  evaporating  at  50◦C  for  838  
mPa by these extracted samples were obtained from Ms. Thanutcha T. (King Mongkut's University of
Technology Thonburi, Thailand)

Solvent extraction
3 types of Chinese chives which are dry, fresh and freeze were extracted with 3 different solvents
which are 95% alcohol, hexane and vegetable oil. The samples were mixed with each solvent by using 1:5
ratio (g/ml). These  mixtures  were  macerated  at  120  rpm,  25◦C  for  48  hours  in  incubator  shaker.  After  48  
hours, these mixtures were filtered by using Whatman filter paper no.4. These mixtures also called crude
extracts. These crude extracts were concentrated by using rotary evaporator (BUCHI Rota-vapor R-205)
at  45◦C except the samples that macerated in vegetable oil by the samples that macerated in vegetable oil
clearly separated layer. These crude extracts were diluted with dimethyl sulfoxide (DMSO) and kept at -
20◦C.  

Antioxidant activity test

According to Ferric Reducing Antioxidant Potential Assay (Benzie and Strain, 1996), the
antioxidant ability of extracted samples were measured by reaction between FRAP reagent and extracted
sample. The FRAP reagent was freshly prepared by mixing between 300 mM of sodium acetate buffer at
pH 3.6, 20 mM of Ferric chloride and 10 mM of 2,4,6-tri[2-pyridyl]-s-triazine dissolved in 40 mM of
diluted   hydrochloric   acid   at   a   ratio10:1:1   (v:v:v).   The   FRAP   reagent   was   incubated   at   37◦C   for   10
minutes before using. 1000 microliter of FRAP reagent was added in the cuvette that contained 20
microliter of extracted sample. This cuvette was kept in the dark for 30 minutes. This extracted sample
was measured the optical density by using the absorbance at 593 nm. The antioxidant ability of extracted
sample was measured by comparing with standard curve of ferrous sulfate.

Statistical analysis
All  of  experiment  were  analyzed  the  results  by  using  ANOVA  with  Duncan’s  multiple  range  test  
(P < 0.05) by SAS software version 9.3.

November 17-20, 2015 Mandarin Hotel Bangkok by Centre Point, Bangkok, Thailand
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

Results and discussion

Antioxidant activity test

Table 1: Fe2+ concentration in fresh Chinese chives extracted with petroleum ether
Fe2+ concentration (µmol Fe2+/
Time (Hours) Methods of extraction
ml sample)
steam distillation 0.109 ± 0.004L
1.0
ohmic pretreatment + steam distillation 0.102 ± 0.005M
steam distillation 0.181 ± 0.004I
2.0
ohmic pretreatment + steam distillation 0.134 ± 0.005K
steam distillation 0.267 ± 0.004F
2.5
ohmic pretreatment + steam distillation 0.196 ± 0.003H
steam distillation 0.113 ± 0.003L
3.0
ohmic pretreatment + steam distillation 0.131 ± 0.004K

Table 2: Fe2+ concentration in the three forms of Chinese chives extracted with different solvents

Chinese chive Fe2+ concentration (µmol Fe2+/

Solvent
samples ml sample)
95% Ethanol 0.176 ± 0.004J
Dry Hexane 0.355 ± 0.002E
Vegetable oil 0.098 ± 0.005M
95% Ethanol 0.376 ± 0.002D
Fresh Hexane 1.079 ± 0.006B
Vegetable oil 0.229 ± 0.003G
95% Ethanol 0.951 ± 0.005C
Freeze Hexane 1.756 ± 0.008A
Vegetable oil 0.269 ± 0.004F
Note: Different superscript within a column in both 2 tables showed significant different at p <0.05

For antioxidant activity of Chinese chives, ferric reducing antioxidant potential assay was used in
order to compare the activity of antibacterial of extracted samples based on the property of reducing agent
in the redox-linked colorimetric reaction by ferric tripyridyltriazine (Fe 3+- TPTZ) complex. This method
can measure the total reducing power of antioxidant agent that able to transfer the electron from Fe 3+ to
Fe2+. When the Fe3+-TPTZ complex is reduced to Fe2+-TPTZ complex, the color will be changed to
intense blue color. If Fe2+ concentration is high, the antioxidant activity will be high. (Benzie, et al, 1996)
When comparing antioxidant activity between Chinese chives extracted sample both hot and cold
extraction, the result showed that cold extraction has higher antioxidant activity than hot extraction by
freeze Chinese chives extracted sample by hexane (1.756±0.008 µmol Fe2+/ mg sample) has better
antioxidant activity and dry Chinese chives extracted by oil (0.098±0.005 µmol Fe2+/ mg sample) has
lowest antioxidant activity. This result indicated that extraction temperature affected to antioxidant
activity by high extraction temperature has lower antioxidant activity than low extraction temperature. It

November 17-20, 2015 Mandarin Hotel Bangkok by Centre Point, Bangkok, Thailand
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

may cause from active compounds in Chinese chives for antioxidant activity are heat sensitive more than
heat stable. For example, flavonoid that found in Chinese chives as an active compound for antioxidant
activity is the heat sensitive by resisting the temperature up to 60◦C (Andersen, et al, 2006) On the other
hand, sulfur containing compound in Chinese chives is stimulated by heat. (Brewer, 2011) According to
the antioxidant mechanism of flavonoids and sulfur containing compound (Brewer, 2011), flavonoids has
the high efficiency antioxidant activity than sulfur containing compounds because flavonoids react to free
radical directly but sulfur containing compounds just bind with free oxygen and metal that cause
oxidation reaction.
Ohmic pretreatment is the one factor that affected to antioxidant activity by ohmic pretreatment is
the method that using electrical current to heat the sample and break the cell membrane of sample at low
frequency (50-60 Hz). It causes the chemical compounds in cell membrane will spread out. When
chemical compounds stay outside the cell membrane, the chemical compound will be directly heated
during extraction. The heat sensitive chemical compound will easily destroy. The dry samples were
directly heated also (Skudder, 1998). Thus, the ohmic pretreatment and dry samples have the low
antioxidant activity. On the other hand, freeze-thaw is also the one technnique to break the cell. The
chemical compounds in Chinese chives will spread outside the cell. It causes the solvent such as hexane,
95% ethanol and oil will easily to extract the chemical compound (Trombitias, 1993).
The extraction time in hot extraction also affected to antioxidant activity by antioxidant activity
increased when the extraction time increase. On the other hand, the antioxidant activity of Chinese chive
will be decreased when the extraction time equal to 3.0 hours. This result indicated that 2.5 hours for
extraction has the best extraction time for antioxidant activity because long extraction time may denature
sulfur containing compound although alliinase in Chinese chives will transformed into allicin and sulfur
containing compound when it was heated (Shi, et al, 2005).
Moreover, solvent extraction also affected to antioxidant activity by hexane has the highest
antioxidant activity and oil provides lowest antioxidant activity. This indicated that the active chemical
compound for antioxidant that dissolve in non-polar solvent better that polar by hexane, oil and petroleum
ether is the non-polar solvent but 95% ethanol is the semi-polar solvent and water is a polar solvent.
However, some chemical compounds don’t   dissolve   in   oil   although   that   compounds   are   non-polar
(Rydberg, et al, 2005). For example, flavonoid is the semi-polar compound but it can dissolve in non-
polar solvent better than polar solvent but insoluble in oil. Moreover, sulfur containing compound is the
non-polar compound and dissolve well in oil (Brewer, 2011).

Conclusions
The extraction temperature, extraction time, solvent and type of sample are the factors that
affected to antioxidant activity of Chinese chives by cold extraction, 2.5 hours for extraction time, hexane
solvent and freeze Chinese chives have the better antioxidant activity.

Acknowledgement
Used Chinese chives as a raw materials in this research was supported by the Bang-Phae organic
vegetable grower community enterprise group at Ratchaburi province.

References
Alizadeh, B., Royandazagh, S.D., Khawar, K.M. and Ozcan, S. 2013. Micropropagation of garlic chives
using mesocotyl axis. Plant science, 23(2): 543-549.
Brewer, M.S. (2011) Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential
Applications. Comprehensive Reviews in Food Science and Food Safety, 10: 221-247.
Pavia, D.L., Lampman G.M., Kriz, G.S. and Engel R.G. 2011. Steam distillation principle. A small scale
Approach to organic laboratory techniques, 4: 773-779.
Benzie, F.F. and Straint, J. J. 1996. The ferric reducing ability of plasma as a measure of antioxidant
power analytical biochemistry. 239: 70-76.

November 17-20, 2015 Mandarin Hotel Bangkok by Centre Point, Bangkok, Thailand
 The 27th Annual Meeting of the Thai Society for Biotechnology and International Conference

Skudder, P.J. 1998. Ohmic heating process. New alternative for aseptic processing of viscous foods, Food
Engineering, 60: 99–101.
Andersen, Q.M. and Markham, K.R. 2006. Seperation and quantification of flavonoids. Flavonoids
chemistry, biochemistry and applications, 1: 1-36.
Trombitias, K. 1993. Elastic properties of titin filaments demonstrated using freeze-break technique. Cell
motility and the cytoskeleton, 24(4): 274-283.
Rydberg, J., Cox, M., Musikas, C. and Choppin, G.R. (2005). Solvent extraction and properties of solvent
extraction. Solvent extraction principles and practice, 2: 111-140.
Shi, J., Ho, C. and Shahidi, F. 2005. Functional foods from garlic and onion. Asian functional foods.
21:455-464

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