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VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY
INTERNATIONAL UNIVERSITY
STUDY ON ANTIOXIDANT AND ANTIMICROBIAL ACTIVITIES
OF ESSENTIAL OILS EXTRACTED FROM PEELS OF POMELOS
GROWN AT DIFFERENT LOCATIONS IN VIETNAM
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Engineering in Biotechnology
Student name: Trieu Thuy Vy – ID: BTIU09048
Supervisor: Dr. Nguyen Thi Lan Phi
February 2014
ACKNOWLEDGMENTS
This report is the summary all my experiences which I have undergone the
very interesting journey in my thesis. The encouragement and the help from a
lot of people have made me confidence.
First of all, I am so grateful to my parents for their unconditional love, help
and support for my study at International University.
Secondly, I sincerely send my profound gratitude to Dr. Nguyen Thi Lan phi,
who is a guide in period my thesis. She has provided profitable knowledge
whenever I need. Again, I want to give my very special thanks for her invaluable
caring and advising.
Besides, I would like to pay my deepest respect to Dr. Pham Van Hung, who
gave me valuable advices and encouraged me to complete this thesis.
Moreover, I would like to give my thanks to all of my professors in School
of
Biotechnology, International University, who trained my devotedly and
imparted me so much valuable knowledge and staffs in the laboratory rooms for
pleasure provide me with all chemicals and equipments needed.
Last but not least, I am very much thankful to my group members and my
close friends, who supported, encouraged me during this course.
TRIEU THUY VY
ABBREVIATION
EO
: Essential oil
GC
: Gas chromatography
TSB
: Tryptone Soybean Broth
TSA
: Tryptone Soybean Agar
S.iniae
: Streptococcus iniae
P.aeruginosa: Pseudomonas aeruginosa
MIC
: Minimum inhibition concentration
DPPH
: 2, 2–diphenyl-1-picrylhydrazyl
CP
: Cold pressing
IC50
: Half inhibition concentration
BDX (BT)
: Da Xanh (Ben Tre) pomelo
BDX (DT)
: Da Xanh (Dong Thap) pomelo
BDC
: Duong Cam pomelo
BL
: Long pomelo
BR
: 5 Roi pomelo
BDX (BT) EO: Da Xanh (Ben Tre) pomelo essential oil
BDX (DT) EO: Da Xanh (Dong Thap) pomelo essential oil
BDC EO
: Duong Cam pomelo essential oil
BL EO
: Long pomelo essential oil
BR EO
: 5 Roi pomelo essential oil
STUDY ON ANTIOXIDANT AND ANTIMICROBIAL
ACTIVITIES OF ESSENTIAL OILS EXTRACTED FROM
PEELS OF POMELOS GROWN AT DIFFERENT LOCATIONS
IN VIETNAM.
Vy T.Trieua, Phi T. L. Nguyenb
a
School of Biotechnology, International University – Vietnam National University
in HCMC.
b
*Faculty of Chemical Engineering, HoChiMinh City University of Technology -
Vietnam National University in HCMC.
Corresponding author’s email address: [email protected]
ABSTRACT
Chemical composition, antioxidant and antimicrobial activities of essential
oils (EOs) extracted by cold pressing method from peel of many pomeloes (Da
Xanh, 5 Roi, Duong Cam, Long pomelo) at different provinces in Vietnam (Dong
Nai, Vinh Long, Ben Tre and Dong Thap) were investigated in this study.
Analysis GC, the chemical compositions of EOs were determined. There were
seventeen components. Among that, each pomelo peels EOs had different
components. However, four components were main components in pomelo peel
EOs including: limonene, γ-terpinene, β-phellandrene and myrcene. In all EOs,
limonene concentration in pomelo peels was highest (67.2%-95.7%) and BL had
highest limonene concentration with 95.7%. To determine antioxidant and
antimicrobial activities, DPPH assay, diffusion and dilution method were applied.
Long
pomelo
EOs
was
the
highest
in
extration
yield,
antioxidant
and
antimicrobial activities. Da Xanh (Ben Tre) pomelo EOs gave the lowest
antioxidant activity. The antimicrobial activity of EOs against S.iniae was lower
than P.aeruginosa. 5 Roi and Duong Cam pomelo EOs had nearly the same
affect on microorganism. This study was shown that differences in chemical
compositions of many pomelos peel EOs lead to their antioxidant and
antimicrobial activities were also different.
Keywords:
pomelo
Essential oil
Gas chromatography
Chemical composition
Antioxidant activity
Antibacterial activity
1
1. Introduction
Nowadays, health is cared more than in public. Diets rich in selected natural
antioxidants such as poly-phenols, flavonoids, vitamin C and vitamin E are
related to reduced risk of incidence of cardiovascular, other chronic diseases and
certain types of cancer which coming from polluted environment and synthetic
preservative agents in food (Choi et al., 2007; Dorman & Hiltunen, 2004;
Majhenic et al., 2007; Mata et al., 2007). The infections risk related to
pathogenic germs increases at the present time, but antibiotics are ineffective to
treat the infectious disease and the antibacterial activities of essential oils (EOs)
from various medicinal plants against microorganisms (Zohra et al., 2011).
Moreover, a great number of scented products are used in our daily life, such as
in cosmetics; household products and EOs have been used in aromatherapy for
relieving bodily and mental distresses (Sawamura et al., 2005).
The genus Citrus of the family Rutaceae includes several important fruits
such as oranges, mandarins, limes, lemons, and grapefruits. Citrus fruits are
one of the important horticultural crops, with worldwide agricultural production
over 80 million tons per year (Marín et al., 2007). Moreover, the fruits are
mainly used for dessert and they have important value for essential oils. Citrus
EOs are the most widely used EOs in the world. At present, approximately 3000
EOs are known, 300 of which are commercially important especially for the
pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries
(Bakkali et al., 2008; Burt, 2004). In recent years, the EOs have attracted a
great deal of scientific interest due to their potential as a source of natural
antioxidants and biologically active compounds (Bozin et al., 2006; Tepe et al.,
2007; Wannissorn et al., 2005).
Citrus maxima (or Citrus grandis), also called pomelo, pummelo or shaddock
is member in the Rutaceae (citrus family). It is a medium-sized tree but the
largest of all Citrus species, with large leaves, flowers, and fruits (Bailey et al.,
1976, Morton, 1987; van Wyk, 2005.). In Vietnam, the production is mostly in
the South; although some cultivars can be grown in the Central areas and even
the North with total area is 5000 ha produce fruit and annual production of
50.000 tonnes (FAO, 2004). Like other citrus fruits, pomelos are high in vitamin
C (Morton, 1987). They are generally eaten as a fresh fruit, and they store well.
Besides that, pomelo EO is a type of essential oil commonly used in
2
aromatherapy. Sourced from the peel of the Citrus maxima fruit, pomelo EO is
said to offer a variety of health benefits. Pomelo EO contains a number of
compounds thought to enhance health, including citronellal and limonene
(Tanida et al., 2005).
There are two methods to extract pomelo EOs: cold pressing and hydro
distillation. Distillation is a method of separating components based on
differences in volatile constituents in a heated mixture. Other methods used to
create pure essential oils are cold pressing, a method where oil is obtained by
using high mechanical pressure to squeeze the oil from peel of pomelo. This
technique is a purely mechanical process while the hydro-distillation use steam
from boiling water for carrying and extracting volatile oils. In comparison with
hydro-distillation method, cold pressed extraction is carried out without applying
heat to avoid the loss, chemical changes in the constituents. Since this method
does not involve the application of heat, citrus essential oils still keep their
superior odor characteristics and some natural antioxidants like tocopherol is
remained.
When
the
process
of
antioxidant
protection
becomes
unbalanced,
deterioration of physiological functions may occur, resulting in diseases and
accelerated aging. Therefore, antioxidant food supplements are used to help the
human body to reduce oxidative damage. Natural antioxidants are being
extensively studied for their capacity to protect organisms and cells from
damage brought on by oxidative stress (Cazzi et al., 1997). The use of essential
oils as functional ingredients in foods, drinks, toiletries and cosmetics is
becoming popular (Reische et al., 1998; Sawamura, 2000). DPPH radical
scavenging assay is the most popular method used for the determination of
antioxidant activity of essential oils. This method is simple and inexpensive.
DPPH is widely used as free radical scavengers or hydrogen donors, to evaluate
antioxidant activity of natural compounds in food (Choi, 2010). This method
used methanol in negative control and BHT (Butylated Hydroxyl Toluene).
Besides the antioxidant activity, EOs also has antimicrobial activity, protect
body to against microorganisms, the origin of disease. Preventing food spoilage,
increasing shelf-life of food. There are two basic techniques used for the
assessment of both antibacterial activity of essential oils (Kalemba and Kunicka,
2003): The agar diffusion method and the agar dilution method. Screening of
EOs for antibacterial activity is often done by the disk diffusion method. This
3
method is mostly applied as a screening method when large numbers of
essential oils and/or large numbers of bacterial isolates (Dorman and Deans,
2000) and selection between EOs. Agar dilution method is also used to
determine
minimum
inhibition
concentrations
(MIC)
of
an
antimicrobial
substance by determining the end-point. The most used methods are that of
optical density OD measurement and the enumeration of colonies by viable
count (Farag et al., 1989; Prudent et al., 1995; Pintore et al., 2002).
In Vietnam, citrus fruit are harvested with huge number and citrus EOs bring
a lot of benefit, but there is little information regarding the detailed evaluation of
antioxidant and antimicrobial activities of citrus essential oils extracted from
pomelo peel. Some previous researches have studied about antimicrobial
activities of pomelo essential oils. This project will provide data and comparisons
for antimicrobial, antioxidant activities of essential oils extracted by cold
pressing method from peels of pomelos grown at different location in Vietnam.
2. Materials and methods
2.1
Plant materials
Pomelo species including Da Xanh and Long pomelo (Dong Thap province);
Duong Cam pomelo (Dong Nai province); Da Xanh pomelo (Ben Tre); 5 Roi
pomelo (Vinh Long) were collected at mature season of pomelo from September,
2013 to December, 2013. This fruits were sent to Southern Horticultural
Research Institute (SOFRI) Vietnam to identify and authenticate scientifically
name.
Pomelos
Da
Xanh
Whole fruit
Cross section
(Dong
Thap)
Duong Cam (Dong
Nai)
4
Da Xanh (Ben Tre)
5 Roi (Vinh Long)
Long (Dong Thap)
Figure 1. Vietnamese pomelo (Citrus maxima) fruits.
After fruits were cleaned with tap water, they were divided into eight equal
portions. For each portions, flesh and albedo layers were removed. Flavedo
layers were collected and extract EO.
2.2
-
Extraction procedure
Extraction by cold pressing method:
By using hand pressing, EOs was extracted from flavedo layers and collected
in brine solution (saturated concentration 40%) kept on ice. The extracts were
centrifuged at 4000g for 15 minutes at 5oC to separate the oils and non-volatile
components. The uppermost supernatants were taken carefully with a Pasteur
pipette then dried with anhydrous sodium sulphate for 24h at 5oC. After
filtration, the pure oils were kept at -21oC until analysis (Lan-Phi et al., 2006).
Yield of extracted EO was calculated base on following formula (Njoroge and
Sawamura, 2010):
Yield (%) =
x 100
5
2.3
Chemical composition analysis
Using system Agilent technologies GC-7890A-MS (USA) to analyze EOs with
flame ionization detector (250oC FID) and a DB-Wax column (60m x 0.25mm,
with film thickness of 0.25µm). Column temperature programming: 70oC at 2
min, increase 2oC/min (70-240oC), stay at 240oC for 20 min. Carrier gas
(helium) rate is 0.8 ml/min.
2.4
Antioxidant activities of EOs
The antioxidant activity of the essential oil was assessed by measuring their
scavenging abilities to 2,2-diphenyl-1-picrylhydrazyl stable radicals (DPPH)
(Bozin et al., 2006). Each EO was diluted with absolute methanol to different
concentrations: 10, 30, 50, and 70 mg/ml. Then, equal volume of methanolic
DPPH solution (100 μM) was added. After incubating sample for 15 min in dark,
scavenging activity on DPPH radical was determined by measuring the
absorbance at 517 nm. The control contained methanol and DPPH solution were
negative control and BHT was used as positive control. DPPH inhibition (%) by
EO was calculated in following way (Ghasemi et al., 2009):
Inhibition (%) =
x 100
Where A control is the absorbance of the negative control and A sample is the
absorbance of the test compound. The concentration of essential oils causing
50% inhibition (IC50) was calculated from the graph-plotted scavenging
percentage against essential oils concentration.
2.5
Antibacterial activities of EOs
2.5.1 Test microorganisms
To determine antimicrobial activities of EOs, using two bacteria. One grampositive
bacteria:
Streptococcus
iniae
and
one
gram
negative
species:
Pseudomonas aeruginosa obtained from Institute of Drug Quality Control – Ho
Chi Minh City (IDQC-HCMC). From stock culture, take a loop of microorganism
intro 10ml tube of broth medium, incubated for 24h at 37oC. Next, take 1 ml
into 9 ml of tryptone Soybean Broth (TSB) to form 10 -1 dilution. Similarly,
dilutions into 10-8. Subsequently, 100µl aliquot of microorganism suspension of
each dilution was spread on plates then incubated at 37oC for 24 hours. Only
the plate which had between 30-300 colonies was picked (Breed and Dotterrer,
6
1916) The stock was adjusted to 1x106colony forming unit (CFU)/ml. CFU was
determined depending on the formula (Benson, 2002):
ρ
Where
N 1
VS D
N:
Number of colonies on plate (Colony forming unit: CFU)
V S:
Volume pipetted onto Petri plate (ml)
D:
Dilution factor for test tube plated out
:
Concentration of cells in original sample (CFU/ml)
2.5.2 Diffusion method
Take 100µL suspension from test microorganisms spread on petri plates
containing TSA medium. Making wells (about 9mm in diameter) in the agar disc.
With 100µl of extracts were diluted in absolute ethanol to obtain a concentration
of 50%. Pump 100ul of extract into wells. Discs without samples were used as a
negative control. The plates were incubated at 37ºC for 24h for bacteria growth.
Antimicrobial activity was assessed by measuring the diameter of the growth
inhibition zone in mm (including well diameter) for the test organisms comparing
to the controls.
2.5.3 Dilution method
Using dilution method to determine MIC.
Absolute ethanol was used to
dilute EOs to obtain concentrations: 0.655, 1.31, 2.63, 5.25, 10.5, 21 to 42
mg/ml. Take 500µl of diluted EOs into the new test tubes. Then, a fixed volume
4ml of liquid culture medium was distributed into the test tubes and add 500µl
of bacterial suspension containing 106 CFU/ml. Experiment included three
controls: one negative control for culture medium and EO only, one positive
control for culture medium and microorganism, and one solvent control for
ethanol, medium and microorganism, ethanol and medium (Takhi et al., 2011).
The test tubes were incubated for 24 hours at 37°C. During the incubation
period, these 10 tubes were agitated continuously using orbital shaker. Finally,
100 µl of each tube was spread on TSB agar medium and incubated for 24hours
7
to determine MIC. The lowest concentration of EO that completely inhibits visual
growth of bacteria.
2.6
Data analysis
In this research, each experiment was tested in triplicate. Means and
standard deviations are calculated by Microsoft excel software. Analysis of
variance (ANOVA) was applied to the data to determine differences (p < 0.05).
Statistical data analysis was undertaken using the Statistical Package for the
Social Sciences (SPSS).
8
3. Results and discussion
3.1
Yield of pomelo essential oils:
Yield of EOs from 200gr pomelo fresh peel extracted by cold pressing method
varied significantly from one type of pomelo to another. Among that, yield of
Long pomelo (BL) was highest with 1.19%. The lowest yield was 5 Roi pomelo
(BR) with 0.50%. That of Da Xanh (Ben Tre) or BDX (BT), Da Xanh (Dong Thap)
or BDX (DT), Duong Cam (BDC) was 0.81%, 0.61%, 0.73%, respectively
(Figure. 2).
The extraction yield of EOs was different for each pomelo, due to many
factors such as weather, soils, time of growing, structure of peel. The peel
sample of BDX (DT) and BDX (BT) were thicker BL; however, yield of EOs
extracted of BL was higher than from BDX (DT) and BDX (BT). The second
reason was extraction method. Cold pressing method had to use force by hand
to extract EO, so the result depends much on personal skills.
1.4
1.19
Extraction yield (%)
1.2
1
0.81
0.8
0.73
0.61
0.6
0.50
0.4
0.2
0
Da Xanh
(Ben Tre)
5 Roi
(Vinh Long)
Long
(Dong Thap)
Da Xanh
(Dong Thap)
Pomelo peel essential oils
Figure 2. Yields of pomelo peel EOs (%w/w).
9
Duong Cam
(Dong Nai)
3.2
Chemical composition analysis
Table 1. Volatile compositions (%w/w) of Vietnamese pomelo peel
EOs
Compounds
RI
Relative concentration (%)
BR
BDX(BT)
BL
BDX(DT)
BDC
1
α-pinene
1035
3.12
3.28
0.572
1.08
2.22
2
β-pinene
1123
0.921
---
---
0.145
0.972
3
sabinene
1132
0.606
0.833
0.106
0.219
---
4
myrcene
1167
1.85
8.57
1.89
1.97
---
5
α-phellandrene
1175
1.43
1.12
---
0.594
2.06
6
α-terpinene
1190
---
---
---
---
0.237
7
limonene
1211
67.2
69.4
95.7
90.1
77.6
8
β-phellandrene
1220
9.21
12.8
0.283
2.95
0.421
9
p-cymene
1279
4.33
1.07
---
0.561
0.908
10
geranial
1744
---
0.384
0.140
0.0846
---
11
bicyclogermacrene
1753
---
0.364
---
---
---
12
cis-carveol
1849
---
0.264
0.082
---
---
13
γ-terpinene
1255
9.85
---
---
1.92
13.5
14
terpinolene
1291
0.413
---
---
---
0.577
15
neral
1690
---
---
0.068
---
---
16
germacrene D
1725
---
---
0.558
---
0.921
17
carvone
1753
---
---
0.106
---
---
98.93
98.09
99.51
99.62
99.42
Total
-
RI, Identification based on Retention Index.
Volatile compounds of BR, BDX (BT), BL, BDX (DT), BDC and their relative
peak percentages are shown in Table 1. By GC-MS system, components of each
pomelo peels EOs was determined. The total number of components found in
BR, BDX (BT), BL, BDX (DT), BDC were 98.93%, 98.09%, 99.51%, 99.62%,
10
99.42% respectively. As can be seen from the table, among components of each
pomelo peel, limonene had highest relative concentration with 67.2%, 69.4%,
95.7%, 90.1% and 77.6% in BR, BDX (BT), BL, BDX (DT), and BDC
respectively. Among that, BL had highest limonene concentration. Besides, γterpinene, β-phellandrene and myrcene were also higher than other compounds
in relative concentration. Easy to see that these above four components were
main components in pomelo peel EOs. Moreover, they also had strong
antioxidant and antimicrobial activities (Sawamura et al, 1991). In details, γterpinene concentrations were different in each pomelo peel, BDC was highest
13.5%, BR > BDX (DT) (9.85%>1.92%). With β-phellandrene concentration,
BDX (BT) > BR (12.8% > 9.21%). BL and BDX (DT) (1.89%, 1.97%) had
myrcene concetrations were lower than BDX (BT) (8.57%). Other compounds
including:
α-pinene,
β-pinene,
sabinene,
cymene,
geranial,
bicyclogermacrene,
α-phellandrene,
cis-carveol,
α-terpinene,
terpinolene,
p-
neral,
germacrene D, carvone were also investigated, so they were a part of role in
antioxidant and antimicrobial activities.
Many research publications have presented data on the composition of the
various EOs. EOs can comprise more than sixty individual components
(Senatore, 1996; Russo et al., 1998). Major components can constitute up to
85%of the EO, while other components are present only as a trace (Senatore,
1996; Bauer et al., 2001). There is some evidence that minor components also
have a part to play in antibacterial activity, by combine effect between other
components. This has been found to be the case for sage (Marino et al., 2001),
and oregano (Paster et al., 1995). The composition of EOs from a particular
species
of
plant
can
differ
between
harvesting
seasons
and
between
geographical sources (Arras and Grella, 1992; Marotti et al., 1994; McGimpsey
et al., 1994; Cosen-tino et al., 1999; Marino et al., 1999; Juliano et al., 2000;
Faleiro et al., 2002). This can be explained that by the formation of antibacterial
substances from their precursors. The composition of EOs from different parts of
the same plant can also differ widely. For example, EO obtained from the seeds
of coriander (Coriandrum sativum L.) has a quite different composition to EO of
cilantro, which is obtained from the immature leaves of the same plant (Delaquis
et al., 2002).
11
3.3
Antioxidant activity of pomelo essential oils
The ability of
EOs to act
as hydrogen
or
electron
donors in
the
transformation of DPPH into its reduced form DPPH-H was investigated. The
antioxidant activity of pomelo peel EOs tested is presented in Figure. 3. Half
inhibition
concentrations
(IC50)
were
calculated
from
the
graph-plotted
scavenging percentage against EOs concentration. The EOs were able to reduce
the stable, purple-colored radical DPPH to the yellow-colored DPPH-H. When the
value of IC50 was high, it means that the antioxidant activity was low. As can be
seen from the figure, IC50 values of BHT control positive was 0.18 mg/ml. It was
very low compare with samples. In that, BL (43.83±1.03 mg/ml) and BDX (DT)
(44.1±0.72 mg/ml) were lower than BDX (BT) (63.1±0.86), BR (53.72±0.96)
and BDC (51.2±0.89) in IC50 value. Therefore, the antioxidant activities of BL
and BDX (DT) were higher than BDC and BR. The lowest antioxidant activity was
BDX (BT) with 63.1±0.86 mg/ml.
Several studies on the chemical composition and bioactivity of different citrus
oils reported strong radical scavenging activity (Seok et al., 2008, Malhotra et
al., 2009, Hamdan et al., 2010, Singh et al., 2010). It is suggested that, even at
low
concentrations,
authentic
flavor
components
such
as
γ-terpinene,
terpinolene, geraniol, β-pinene and myrcene have high antioxidant activities
(Sonbol et al., 1992, Song et al., 2001). Suitable with this study, BDX (BT) EOs
did
not
have
components:
γ-terpinene,
terpinolene,
geraniol,
β-pinene.
Therefore, antioxidant activity of BDX (BT) EOs was lower than other pomelo
peel EOs. Although γ-terpinene, terpinolene, geraniol, β-pinene and myrcene
concentrations of BL and BDX (DT) were low, but they could linkage together
and effect to antioxidant activity of EOs. Moreover, limonene concentration of BL
and BDX (DT) were very high, it was main component in antioxidant activity.
Choi et al., 2000, found that the radical scavenging activity of 34 kinds of citrus
essential oils on DPPH ranged from 17.7% to 64%. These activities were found
to be higher when the oils contained geraniol, terpinolene and γ-terpinene
(Sonbol et al., 1992).
12
70
Antioxidant activity of pomelo essential oil
63.1
60
53.72
IC50 (mg/ml)
50
44.1
43.83
BDX(DT)
BL
51.2
40
30
20
10
0.18
0
BHT
BDX(BT)
BR
BDC
Figure 3. IC50 values (mg/ml) of pomelo peel EOs investigated by DPPH
assay
3.4
Antimicrobial activity of pomelo essential oils against S.iniae
Table 2. Zone of inhibition (mm) of pomelo peel EOs against S. iniae
Samples
S.iniae
BR
23.17±0.35c
BDX(BT)
21.23±0.15a
BL
26.30±0.26d
BDX(DT)
22.17±0.31b
BDC
21.53±0.25ab
Diameter of inhibition zone (mm) including well diameter of 9 mm) are mean
± standard deviation.
Values followed by the same small letter within the column are not
significant different (p>0.05) according to Tukey’s test.
13
The antimicrobial activities of essential oils were determined by both
diffusion method and dilution method. In diffusion method, a comparison of the
inhibition capacity of pomelo peel EOs on S. iniae is shown in Table 2. The table
reveals the zone of inhibition (mm) of pomelo EOs against S.iniae from 21.23
mm to 26.30 mm. To be specific, the highest of inhibition zone was 26.3±0.26
of BL, next were BR and BDX (DT) with 23.17±0.35, 22.17±0.31 respectively.
The zone of inhibition of BDX (BT) and BDC were low (21.23±0.15, 21.53±0.25
respectively). The results were in agreement with previous reports. In result of
Roomiani et al., 2012, inhibition zone also had from 22.2 to 28.2 mm against
S.iniae of Z.multiflora EOs. Besides, from result of Chanthaphon, et al., 2008,
antimicrobial activity of pomelo fresh peels against Staphylococcus aureus and
Escherichia coli had inhibition zone: 12-15 mm. inhibition zone was lower than
S.iniae.
Table 3. MICs values (mg/ml) of pomelo peel EOs of S. iniae
Samples
S.iniae
BR
5.25
BDX(BT)
10.5
BL
2.63
BDX(DT)
10.5
BDC
10.5
The Table 3 compares MIC values of pomelo peel EOs against S.iniae. The
dilution method was found through determine inhibitory concentration (MIC)
values. If the values MIC were low, antimicrobial activities of EOs were high. As
is shown in the table, MIC values of all EOs were ≥ 2.63 mg/ml. In that,
antimicrobial activities of BDX (DT), BDC and BDX (BT) were 10.5 mg/ml. They
were lower than BR (5.25 mg/ml) and BL had highest antimicrobial activity with
2.63 mg/ml MIC value. Moreover, these results were suitable with the above
14
value from zone of inhibition (mm) of EOs. From the result of Roomiani et al.,
2012. At concentration 25 mg/ml, the inhibition zone of EOs affecting on S. iniae
were 22±0.7 mm. Other result from Pirbalouti., et al., 2011, with 27±2.65 mm
of inhibition zone of EOs, the MIC value was 39 mg/ml.
Some earlier reports showed that the changes in chemical composition of an
essential oil directly affected their biological activities (Celiktas et al., 2007; Van
Vuuren et al., 2007). Suppakul et al., 2003, reported that EOs exhibited good
antimicrobial activity against a wide range of microorganisms. Bozin et al., 2006
and Sokovic and Van Griensven, 2006 also reported the antifungal activity of
essential oils with its main component: linalool. Through the report previous,
this result study was suitable. Limonene and α-pinene concentration of BL and
BR were higher than pomelo peels other, so antimicrobial activity against S.iniae
is high. It was shown that, the growth of S. iniae was affected by the
appearance of α-pinene and limonene in the composition of essential oils
(Dorman and Deans, 2000).
The antimicrobial activity of the EO has been studied by many authors, but
its mode of action is still complex and in some cases unknown. The location or
mechanisms thought to be sites of action for EO components are: degradation of
the cell wall, damage to cytoplasm membrane, damage to membrane proteins,
leakage of cell contents, coagulation of cytoplasm and deple of the proton
motive force (ABI-Ayad et al., 2011).
15
3.5
Antimicrobial activity of pomelo essential oils against P.aeruginosa
Table 4.
Zone
of inhibition
(mm)
of pomelo peel
EOs against
P.aeruginosa
Samples
P.aeruginosa
BR
21.23±0.31b
BDX(BT)
20.27±0.21a
BL
25.13±0.05c
BDX(DT)
20.07±0.32a
BDC
21.4±0.26b
Diameter of inhibition zone (mm) including well diameter of 9 mm) are mean
± standard deviation.
Values followed by the same small letter within the column are not
significant different (p>0.05) according to Tukey’s test.
Inhibition zones (mm) of EOs against P.aeruginosa are shown in Table 4. A
close look at table reveals that, the zone of inhibition (mm) of pomelo EOs
against P.aeruginosa from 20.07 to 25.13 mm in diameter. In details, the low of
antimicrobial activities of EOs were BDX (BT) and BDX (DT) (20.27±0.21,
20.07±0.32 respectively). The similarly with inhibition zone against S.iniae, BL
was highest with 25.13±0.05 mm. BR and BDC were almost equal (21.23±0.31
and 21.4±0.26 mm respectively). According to Zohra et al., 2011, no inhibition
zone was observed around discs of bacterial cultures Citrobacter sp (IS) and P.
aeruginosa (IS) of Lavandula stoechas EOs. Therefore, this result study was
better when pomelo peels EOs against P.aeruginosa minimum 20.07 mm.
16
Table 5. MICs values (mg/ml) of pomelo peel EOs of P.aeruginosa
Samples
P.aeruginosa
BR
10.5
BDX(BT)
21
BL
2.63
BDX(DT)
21
BDC
10.5
The comparison MIC values of pomelo peel EOs against P.aeruginosa are
shown in Table 5. BL also had highest antimicrobial activity (2.63 mg/ml MIC
value) both P.aeruginosa and S.iniae. Compares with S.iniae, antimicrobial
activities of EOs were lower than in P.aeruginosa. To be specific, concentration
of BDC and BR against P.aeruginosa were 10.5 mg/ml MIC value. BDX (DT) and
BDX (BT) had antimicrobial activities low with 21 mg/ml value of MIC. In MIC
method, we take a range in essential oils concentration against bacterial, start
with 50% or 42 mg/ml and decrease to 25% (21 mg/ml) and make a series.
Because EOs concentration decrease large in series, so EOs concentration of
each pomelo against microorangism were not significant different.
The chemical composition of EOs affected antibacterial activities on P.
aeruginosa. β-pinene, α-terpinene and geraniol in component of EOs were
effective in P.aeruginosa (Dorman and Deans, 2000). Especially, combined of αpinene, β-pinene, limonene and linalool have a strong antibacterial activity
(Magiatis et al., 1999). That was why BL, BDC and BR had antimicrobial activity
against high P.aeruginosa. Because they contained α-pinene, β-pinene, limonene
higher than EOs other. Several reports investigated the antibacterial activities of
essential oils on P. aeruginosa. Inhibitions zone have not exceeded 20 mm were
recorded against P.aeruginosa in ladaniferus L. and Lavandula EOs (Zohra et al.,
2011). In addition, this microorganism was inhibited by the lemongrass EOs with
MIC value at 1% (v/v) (Hammer et al., 1999).
17
In the conclusion, pomelo peels are a reserve of biologically active
substances. Pomelo peels EOs can be a source of a great diversity with their
antimicrobial capacity, pomelo EOs can have application in therapy of the
infectious diseases or used in synthesis substances.
4. Conclusion
As can be seen from this study, pomelo peel EOs extracted by cold pressing
had high antioxidant and antimicrobial activities. The yield, antioxidant and
antimicrobial activities of BL were highest compare with positive and negative
control. BDX (BT) EOs gave the lowest antioxidant activity. Besides that, the
effects of BDX (BT) EOs and BDX (DT) EOs on microorganism were lower than
other EOs. BR and BDC EOs had nearly the same affect on microorganism. This
result of study gave information in antioxidant and antimicrobial activities from
peels of pomelos grown at different locations in Vietnam. Pomelo peels EOs not
only have application in food industries like preservative of foodstuffs but also
like pharmaceutical.
18
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25
APPENDIX
BR
BDX(BT)
BL
BDX(DT)
BDC
Control
Figure 4: Effect of pomelo peel essential oils on the growth of S. Iniae by
diffusion method
BR
BDX(BT)
BL
BDX(DT)
BDC
Controls
Figure 5: Effect of pomelo peel essential oils on the growth of P.aeruginosa by
diffusion method
BDX(DT)
30
50
70
%24.96
45.37
54.52
64.61
Scavenging (%)
10
80
IC50=44.10
y = 0.6405x + 21.745
R² = 0.9601
60
40
20
0
0
20
40
60
80
concentration EO (mg/ml)
Figure 6: DPPH scavenging (%) in BDX (DT) EOs at different concentration.
26
BL
10
30
50
70
%33.6
42.69
51.14
64.72
IC50=43.83
70
y = 0.509x + 27.676
R² = 0.9872
Scavenging (%)
60
50
40
30
20
10
0
0
20
40
60
80
concentration EO (mg/ml)
Figure 7: DPPH scavenging (%) in BL EOs at different concentration.
BDX(BT)
10
30
50
70
%22.81
32.84
40.3
55.39
IC50=63.10
Scavenging (%)
60
y = 0.526x + 16.795
R² = 0.9795
50
40
30
20
10
0
0
20
40
60
80
concentration EO (mg/ml)
Figure 8: DPPH scavenging (%) in BDX(BT) EOs at different concentration.
27
BR
10
30
50
70
%22.74
34.17
49.39
59.36
IC=53.72
Scavenging (%)
70
y = 0.6254x + 16.399
R² = 0.9941
60
50
40
30
20
10
0
0
20
40
60
80
concentration EO (mg/ml)
Figure 9: DPPH scavenging (%) in BR EOs at different concentration.
BDC
30
50
70
%20.82
34.69
47.24
64.84
Scavenging (%)
10
70
60
50
40
30
20
10
0
IC50=51.2
y = 0.7231x + 12.976
R² = 0.9948
0
20
40
60
80
concentration EO (mg/ml)
Figure 10: DPPH scavenging (%) in BDC EOs at different concentration
28
[ｸﾛﾏﾄｸﾞﾗﾑ] TIC : 813949 - 0
100%
5
6
3
50%
1
24
7
8
910
0%
R.T-->
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
Figure 11. GC of cold pressed BDX (BT) pomelo peels EOs.
[ｸﾛﾏﾄｸﾞﾗﾑ] TIC : 727618 - 0
100%
1
6
789
4
5
50%
2
3
10
0%
R.T-->
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
Figure 12. GC of cold pressed BR pomelo peels EOs.
29
[ｸﾛﾏﾄｸﾞﾗﾑ]
100%
7
6
TIC : 700815 - 0
4
8
50%1
5
9
3
2
10
0%
R.T-->
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
Figure 13. GC of cold pressed BDX (DT) pomelo peels EOs.
[ｸﾛﾏﾄｸﾞﾗﾑ]
100%
4
TIC : 755974 - 0
3
50%
7
1
2
5
8 10
69
0%
R.T-->
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
Figure 14. GC of cold pressed BL pomelo peels EOs.
30
[ｸﾛﾏﾄｸﾞﾗﾑ] TIC : 680869 - 0
100%
1
57
3
10
50%
2
8
6
4
9
0%
R.T-->
10:00
15:00
20:00
25:00
30:00
35:00
40:00
45:00
50:00
Figure 15. GC of cold pressed BDC pomelo peels EOs.
Table 6. Using one way ANOVA (Tukey’s test) to determine not significant
different (p>0.05) zone of inhibition (mm) of pomelo peel EOs against S.iniae
S.iniae
Tukey HSD
Subset for alpha = 0.05
Samples N
1
2
3
21.2333
5
3
21.5333 21.5333
4
3
22.1667
1
3
3
3
Sig.
2
3
4
23.1667
26.3000
.672
.100
1.000
1.000
Means for groups in homogeneous subsets are displayed.
31
Table 7. Using one way ANOVA (Tukey’s test) to determine not significant
different (p>0.05) zone of inhibition (mm) of pomelo peel EOs against
P.aeruginosa
P.aeruginosa
Tukey HSD
Subset for alpha = 0.05
Samples N
1
4
3
20.0667
2
3
20.2667
1
3
21.2333
5
3
21.4000
3
3
Sig.
2
3
25.1333
.859
.920
1.000
Means for groups in homogeneous subsets are displayed.
32
Controls
Samples
Culture tubes
TSB medium
& Bacteria
TSB
medium &
Ethanol &
Bacteria
Concentration (mg/ml)
0.655
1.31
2.63
5.25
10.5
BR
BDX(BT)
BL
BDX(DT)
BDC
Figure 16: Effect of Vietnamese pomelo peel EOs on P.aeruginosa at different EO concentrations by dilution method.
21
42
Controls
Samples
Culture tubes
TSB medium
& Bacteria
TSB
medium &
Ethanol &
Bacteria
Concentration (mg/ml)
0.655
1.31
2.63
5.25
10.5
BR
BDX(BT)
BL
BDX(DT)
BDC
Figure 17: Effect of Vietnamese pomelo peel EOs on S.iniae at different EO concentrations by dilution method.
21
42

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