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Journal of Essential Oil Research

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Comparison of Essential Oils Compositions of Citrus

maxima Merr. Peel Obtained by Cold Press and
Vacuum Stream Distillation Methods and of Its Peel
and Flower Extract Obtained by Supercritical Carbon
Dioxide Extraction Method and Their Antimicrobial
Activity
a a b
Napaporn Thavanapong , Penpun Wetwitayaklung & Juree Charoenteeraboon
a
Department of Pharmacognosy, Faculty of Pharmacy, Sanamchan Palace Campus,
Silpakorn University, Nakhon Pathom, 73000, Thailand
b
Department of Biopharmacy, Faculty of Pharmacy, Sanamchan Palace Campus,
Silpakorn University, Nakhon Pathom, 73000, Thailand
Version of record first published: 09 Dec 2011.

To cite this article: Napaporn Thavanapong , Penpun Wetwitayaklung & Juree Charoenteeraboon (2010): Comparison of
Essential Oils Compositions of Citrus maxima Merr. Peel Obtained by Cold Press and Vacuum Stream Distillation Methods
and of Its Peel and Flower Extract Obtained by Supercritical Carbon Dioxide Extraction Method and Their Antimicrobial
Activity, Journal of Essential Oil Research, 22:1, 71-77

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 C. maxima

Comparison of Essential Oils Compositions of Citrus

maxima Merr. Peel Obtained by Cold Press and
Vacuum Stream Distillation Methods and of Its Peel
and Flower Extract Obtained by Supercritical Carbon
Dioxide Extraction Method and Their Antimicrobial
Activity

Napaporn Thavanapong and Penpun Wetwitayaklung,*

Department of Pharmacognosy, Faculty of Pharmacy, Sanamchan Palace Campus, Silpakorn University,
Downloaded by [University of Arizona] at 19:10 21 January 2013

Nakhon Pathom, 73000, Thailand

Juree Charoenteeraboon,
Department of Biopharmacy, Faculty of Pharmacy, Sanamchan Palace Campus, Silpakorn University,
Nakhon Pathom, 73000, Thailand

Abstract
The oils and extracts of the fruit peels of pomelo (Citrus maxima Merr. cultivar ‘khao–yai’) were obtained by
cold-pressing (CP), vacuum steam distillation (VSD) and supercritical carbon dioxide extraction (SC-CO2) and the
extract of the flowers was obtained by SC-CO2. The composition of the oils and extracts of the peel and flower were
determined by GC and GC/MS. Fifty, 53 and 60 components of the oils and extract from peels by CP, VD and SC-
CO2 were identified, respectively. Their main components were limonene (93.4–95.4%). Fifty-one components of
the flower extract were identified and the main component was also limonene (86.2–86.8%). The oils and extract of
pomelo showed fairly good activity against Staphylococcus aureus and Candida albicans.

Key Word Index

Citrus maxima, Citrus grandis, Rutaceae, essential oil composition, limonene, antimicrobial activity.

Introduction products. They also have been used as fragrance in perfumes,

cosmetic and aromatherapy. The quality of the oils is related
The pomelo (Citrus maxima Merr.) [syn. C. grandis (L.)
to the value of total aldehydes which is around 4–5% (3). The
Osbeck] is a beneficial native plant of Asia, which is best culti-
Chinese, Japanese, Taiwanese, Vietnamese and Italian pomelo
vated in China, southern Japan, Vietnam, Malaysia, Indonesia
peel that was obtained by cold-pressing and steam distillation
and Thailand. The fruit of the pomelo is commonly eaten fresh
and flower oil that was obtained by distillation were analyzed
or made into a juice. In traditional medicine, the fruit peel has
by GC and GC/MS (4–15). The citrus flower oils have been
been used to treat cough, swelling, and epilepsy and the fruit
reported to possess relaxing and hormone balancing effects
pulp has been used for antitoxic, appetizer, cardiac stimulant
which accounts for their use in aromatherapy and perfum-
and stomach tonic (1). The middle layer (albedo) of the fruit
ery (16). The isolation methods for citrus oils are important,
peel is extracted for pectin which is used as dietary fiber for
since they are very sensitive to heat. The traditional method
controlling body weight (2). The Citrus species are famous
that has been used for citrus peel oil production is cold press-
for being a source of essential oils. The Citrus peel oils have a
ing. The scent of cold-pressed oil is natural, but it contains
strong and desirable aroma with refreshing effect. They have
some impurities such as wax, coumarins and carotenoids (17).
been used as flavoring in foods, beverages and pharmaceutical
Another traditional method for essential oil isolation is steam

*Address for correspondence Received: December 2007

Revised: June 2008
1041-2905/10/0001-071$14.00/0­—© 2010 Allured Business Media Accepted: September 2008

Vol. 22, January/February 2010 Journal of Essential Oil Research/71

 Wetwitayaklung et al.
distillation. This method is universal for most oil isolation ex-
cept citrus peel oils. It is an easy method to use yielding an oil
that can be separated from water. However, this method uses
high temperatures at which terpene hydrocarbons of citrus
oils, particularly limonene, are instable and give off-flavors
from their origin (18). At present, supercritical carbon dioxide
extraction (SC-CO2) is appropriate to solve the problems of
the traditional methods. It could extract volatiles with low
operating temperatures that prevent thermal degradation, low
water content that limits hydrolysis, and the absence of solvent
residues. So, SC-CO2 is an advantageous technique for natural
flavor and fragrance production (19).
Neroli is the commonly used oil from Citrus species. This
oil is produced from bitter orange flower (C. aurantium L.) by
distillation or solvent extraction (20). It had been used in high
quality eau de cologne since the seventeenth century. High
quality perfumes are always adding neroli in their recipes.
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In the perfume industry, neroli oil is widely used in blends
with most other floral oils and absolutes (21). The C. maxima
flowers are bigger than bitter orange flowers in size, and they
also contain more oil glands on their petals. However, there
is no commercial oil of C. maxima available. In addition, the
extraction of flower volatiles needs an appropriate technique
that is nontoxic, noncombustible, absent of solvent residue
contamination, nonchemical transformative and inexpen-
sive (22). Therefore, supercritical carbon dioxide extraction
has considerable advantages over the other extraction methods
for flower volatile extraction suitable for pharmaceutical and
perfume industries purposes.
The aim of this work was to compare and characterize the
chemical composition of the oil and extract from different
isolation methods of the peel and flowers of C. maxima cultivar
‘khao–yai.’ In this study, the isolates of C. maxima peel and flow-
ers were investigated for their antimicrobial properties against
Staphylococcus aureus, Escherichia coli and Candida albicans.
Experimental
Plant materials: The fruit of Citrus maxima cultivar
‘khao–yai’ were collected from Bangkontee District, Samut-
songkarm Province, Thailand in December 2005 for their peel,
while their flowers were collected in February 2006. The C.
maxima peels (flavedo) were divided into three portions. The
first portion was prepared for supercritical fluid carbon dioxide
extraction (SC-CO2). It was dried with hot air oven (40°C)
and milled with a cutting mill. The second fresh portion was
distilled by vacuum steam distillation (VSD) and the third
portion was pressed after peeling by a cold-pressing method
(CPM). The fresh flowers, which were extracted by SC-CO2,
were processed as soon as they were collected.
Supercritical fluid carbon dioxide extraction (SC-CO2):
This research used the pilot scale SC-CO2 (Guangzhou Masson
New Separation Technology, China). The evaluated parameters
were temperature and pressure of the extractor (40–60°C and
13–20 MPa) and of two separators (20–40°C and 5–8 MPa).
Other variables that were kept constant were particle size of
plant material, carbon dioxide flow rate (1.0 mL/min), time
for equilibrium condition (30 min), and extraction time (3.0 h
for peel and 24 h for flower extraction). The extracts (called
72/Journal of Essential Oil Research
Table I. The percentage yield of the extract and oils from Citrus
maxima peel and flower
Type of oil / extract % yield (v/w on fresh
peel/flower basis)
SFE-P (extract) 0.4*
CP-P (oil) 1.16
VD-P (oil) 2.39
SFE-F (extract) 0.2
*v/w on dry peel basis; SFE = Supercritical Fluid CO2 extract; CP = Cold-press oil;
VD = Vacuum distilled oil; P = peel; F = flower.
SFE-P for the peel and SFE-F for the flower) were separated
and dried over anhydrous sodium sulfate.
Cold-pressing method (CPM): The fresh C. maxima
peels were cut in small sizes in the range of 1.0–3.0 cm. The
cut peels were fed into the screw pressing instrument (veg-
etable juicer), which is used in the household for producing
vegetable and fruit juices. The mixture of oil and water was
separated and recovered by centrifugation with 3000 rpm for
10 min. The oil was separated from the water and dried over
anhydrous sodium sulfate (oil = CP).
Vacuum steam distillation (VSD): The fresh C. maxima
peels were cut into small sizes in the range of 1.0–3.0 cm and
extracted by VSD apparatus (Pilot scale, Model: T-36, Techni-
cal Business Company). The peels were submitted to steam
distillation for 3.0 h under temperature of 60°C and pressure
of about -600 bar. The isolated oil, (VD), was separated and
dried over anhydrous sodium sulfate.
All of the oil samples were kept in airtight, protected from
light containers and placed in a refrigerator at +4°C prior to
analysis.
Analysis: The chemical compositions of the oils and extract
were analyzed by GC (Agilent Technologies 5890 series II, USA)
and equipped with DB-5 capillary column (30 m × 0.25 mm;
0.25 µm film thickness) and Carbowax 20M capillary column
(60 m × 0.25 mm; 0.25 µm film thickness). The operating
temperature program of DB-5 was 60°C for 5 min, ramp of
3°C/min up to 250°C, 250°C for 15 min; injection temperature,
230°C; detector temperature, 240°C; carrier gas, He (1 mL/
min); detector, FID; split ratio, 1:90; injection of 1.0 µL. The
operating temperature program of Carbowax 20M was 60°C for
5 min, ramp of 3°C/min up to 180°C, 180°C for 30 min; injec-
tion temperature, 230°C; detector temperature, 240°C; carrier
gas, He (1 mL/min); detector, FID; split ratio, 1:90; injection
of 1.0 µL. The identification of individual components was
performed, for both columns, by comparison of their retention
times with some authentic samples and by retention indices
(RI) relative to the series of n-hydrocarbons. The quantitative
data of oils and extracts from DB-5 and Carbowax 20M were
derived from GC-FID.
The GC/MS analyses were performed with Agilent Tech-
nologies GC6890 gas chromatograph (USA) equipped with
DB-5 capillary column (30 m × 0.25 mm; 0.25 µm film thick-
ness) and Carbowax 20M capillary column (60 m × 0.25 mm;
0.25 µm film thickness). The operating temperature program
of DB-5 and Carbowax 20M were the same as that of GC-FID.
Vol. 22, January/February 2010
 C. maxima

Table II. Chemical composition (%) of the oils and extract from Citrus maxima peel analyzed using a DB-5 and Carbowax 20M
capillary column
Compound RI of DB-5 RI of Carbowax % Area
Sample Ref Sample Ref SFE-P CP-P VD-P
N P N P N P
a-thujene 934 930 1039 1038 t nd t 0.2 t nd
a-pinene 940 939 1032 1036 0.4 0.4 0.5 0.4 0.4 0.4
camphene 957 955 1074 1078 t t t t t t
sabinene 978 975 1121 1130 0.4 0.4 0.3 0.4 0.2 0.4
b-pinene 981 979 1112 1120 0.5 0.5 1.1 1.0 0.4 0.3
myrcene 993 991 1155 1156 1.7 2.3 1.9 2.0 1.8 1.3
a-phellandrene 1006 1003 1163 1173 0.4 nd t nd 0.6 1.2
limonene 1025 1029 1219 1210 93.7 93.4 95.3 95.2 95.4 95.2
(E)-b-ocimene 1054 1050 1245 1238 0.1 0.1 t t 0.1 0.1
g-terpinene 1064 1060 t nd t nd t nd
cis-sabinene hydrate 1073 1070 t nd t nd t nd
terpinolene 1091 1089 1277 1279 t t t t t t
linalool 1095 1097 1548 1555 0.2 0.2 0.1 0.1 0.1 0.1
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cis-limonene oxide 1136 1137 t nd t nd t nd

trans-limonene oxide 1141 1142 t nd t nd t nd
citronellal 1155 1153 t nd t nd t nd
a-terpineol 1191 1189 1709 1685 0.2 0.3 0.1 0.1 0.1 0.1
decanal 1206 1202 t nd t nd t nd
trans-carveol 1219 1217 1851 1839 t t t t t t
nerol 1229 1230 1806 1808 t t t t t t
cis-carveol 1232 1229 t nd 0.1 nd t nd
neral 1242 1238 1683 1680 0.1 0.2 t 0.1 0.1 0.1
geraniol 1257 1253 1855 1842 t t 0.1 t t t
geranial 1271 1267 1736 1730 0.2 0.2 t 0.1 0.1 0.1
perillaldehyde 1276 1272 1788 1818 t t t t t t
indole 1292 1291 t nd t nd t nd
methyl anthranilate 1334 1337 2231 2260 t t t nd t nd
a-terpinyl acetate 1342 1349 t nd t nd t nd
neryl acetate 1365 1362 t nd t nd t nd
a-copaene 1381 1377 t nd t nd t nd
geranyl acetate 1383 1381 1752 1754 0.1 0.2 0.1 0.1 t t
b-cubebene 1397 1388 t nd nd nd nd nd
dodecanal 1410 1409 0.1 nd nd nd t nd
b-caryophyllene 1425 1419 1588 1562 0.2 0.2 t 0.1 0.1 0.1
b-copaene 1437 1432 t nd t nd t nd
a-humulene 1460 1455 1660 1672 t t t t t t
germacrene D 1487 1485 1698 1712 1.0 0.2 t t 0.4 1.4
a-selinene 1501 1498 0.1 nd 0.1 nd t nd
bicyclogermacrene 1503 1500 1725 1738 t 0.1 t 0.1 t t
(E,E)-a-farnesene 1512 1506 t nd t nd t nd
d-cadinene 1522 1523 t nd t nd t nd
germacrene B 1564 1561 1819 1864 t t t t t t
(E)-nerolidol 1566 1563 2039 2044 t t t t t t
germacrene D-4-ol 1583 1576 t nd nd nd t nd
tetradecanal 1614 1613 t nd nd nd t nd
(Z,E)-farnesol 1692 1701 2274 Ms t t nd nd nd nd
(Z,Z)-farnesol 1717 1718 t nd nd nd nd nd
(E,E)-farnesol 1726 1725 2411 2371 t t t nd nd nd
(E,Z)-farnesol 1748 1746 t nd nd nd nd nd
nootkatone 1812 1807 2254 2250 0.1 0.1 nd nd nd nd
p-cymene 1263 1272 nd t nd nd nd t
d-elemene 1466 1469 nd t nd t nd t
b-elemene 1558 1591 nd t nd t nd t
linalyl acetate 1561 1569 nd t nd t nd t
terpinen-4-ol 1609 1601 nd t nd t nd t
b-selinene 1753 1756 nd t nd t nd t
citronellol 1775 1765 nd t nd t nd t
spathulenol 2144 2153 nd t nd nd nd nd
The compounds are listed in elution order from DB-5 column, N = DB-5 and P = Carbowax 20M columns. t = trace (< 0.1%), Ms = identification from mass spectra of Wiley
database library and/or Adams (23); nd = not detected; SFE = Supercritical Fluid CO2 extract; CP = Cold-press oil; VD = Vacuum distilled oil; P = peel.

Vol. 22, January/February 2010 Journal of Essential Oil Research/73

 Wetwitayaklung et al.

The detector was Agilent Technologies MSD 5973N mass
spectrometer (USA): EI, 70 eV; mass range 40–450; solvent
delayed 3.0 min.
The identification of individual components was performed,
for both columns, by comparison of their retention times with
some authentic samples, comparing retention indices (RI)
relative to the series of n-hydrocarbons, computer matching
against Wiley 7n, NIST02 and Pesticide and comparing with
the mass spectra reported by Adams (23). The sample RI
data from DB-5 were compared to RI from Adams (23) and
sample RI data from Carbowax 20M were compared to RI
from Njoroge (15) and Davies (24).

Table III. Composition (%) of the flower extract of Citrus maxima flower (SFE-F) analyzed using using a DB-5 and Carbowax 20M
capillary column
Chemical RI of DB-5 RI of Carbowax % Area DB-5
Composition Sample Ref Sample Ref N P
a-thujene 933 930 t nd
a-pinene 941 939 1029 1036 0.6 0.6
camphene 959 955 1072 1078 t t
sabinene 979 975 1120 1130 0.6 0.7
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b-pinene 983 979 1110 1120 0.9 0.9

myrcene 994 991 1154 1156 1.8 1.8
a-phellandrene 1008 1003 1163 1173 1.4 1.6
limonene 1025 1029 1211 1210 86.2 86.8
(Z)-b-ocimene 1045 1037 1223 1228 0.1 0.1
(E)-b-ocimene 1055 1050 1244 1250 2.9 0.1
g-terpinene 1065 1060 t nd
cis-sabinene hydrate 1072 1070 t nd
terpinolene 1091 1089 1275 1279 0.2 t
linalool 1096 1097 1546 1555 0.4 0.4
cis-limonene oxide 1138 1137 t nd
trans-limonene oxide 1148 1142 t nd
citronellal 1156 1153 t nd
a-terpineol 1192 1189 t nd
decanal 1205 1202 t nd
nerol 1230 1230 1806 1808 0.2 0.2
neral 1244 1238 1683 1680 0.7 0.8
geraniol 1257 1253 1856 1842 0.1 0.1
geranial 1273 1267 1736 1730 1.2 1.3
indole 1292 1291 t nd
methyl anthranilate 1343 1337 2231 2260 0.1 0.1
neryl acetate 1366 1362 1712 1699 0.1 0.3
a-copaene 1382 1377 t nd
geranyl acetate 1384 1384 1745 1754 0.1 0.2
b-cubebene 1387 1388 0.1 nd
dodecanal 1410 1409 t nd
b-caryophyllene 1426 1419 1587 1562 0.2 0.3
b-copaene 1435 1437 t nd
a-humulene 1460 1455 1660 1672 t t
germacrene D 1488 1485 1698 1712 0.5 0.6
a-selinene 1497 1498 t nd
bicycolgermacrene 1501 1500 1725 1738 0.3 0.2
(E,E)-a-farnesene 1510 1506 t nd
d-cadinene 1529 1523 t nd
germacrene B 1558 1561 1820 1864 t t
(E)-nerolidol 1567 1563 2039 2044 0.6 0.8
germacrene D-4-ol 1582 1576 t nd
tetradecanal 1614 1613 1922 1967 t t
(Z,E)-farnesol 1700 1701 2274 Ms 0.1 0.4
(Z,Z)-farnesol 1719 1718 2325 Ms 0.1 0.1
(E,E)-farnesol 1737 1725 2336 2371 0.8 0.1
(E,Z)-farnesol 1755 1746 2411 Ms t 1.1
nootkatone 1810 1807 2254 2250 t 0.4
p-cymene 1261 1272 nd t
b-elemene 1558 1591 nd t
perillaldehyde 1787 1818 nd t
The compounds are listed in elution order from DB-5 column, N = DB-5 and P = Carbowax 20M columns. t = trace (< 0.1%); Ms = identification from mass spectra of Wiley
database library and/or Adams (23); nd = not detected.

74/Journal of Essential Oil Research Vol. 22, January/February 2010

 C. maxima

Table IV. The classes of chemical components in Citrus maxima peel oils and extract and flower extract
Components % Relative
SFE-P CP-P VD-P SFE-F
DB-5
Acyclic monoterpene hydrocarbons 1.8 1.9 1.9 4.7
Cyclic monoterpene hydrocarbons 95.5 97.3 97.0 89.6
Acyclic sesquiterpene hydrocarbons t t nd t
Cyclic sesquiterpene hydrocarbons 1.4 0.1 0.5 1.2
Long chain hydrocarbons 0.1 t t t
Total hydrocarbons 98.8 99.4 99.4 95.6
Acyclic oxygenated monoterpenes 0.8 0.2 0.4 3.6
Cyclic oxygenated monoterpenes 0.3 0.3 0.2 0.1
Acyclic oxygenated sesquiterpenes t t nd 0.6
Cyclic oxygenated sesquiterpenes 0.1 nd nd 0.6
Total oxygenated components 1.1 0.6 0.6 4.9
Aromatic compounds 0.1 t t 0.1
Miscellaneous t t t t
Total identified 100 100.0 100 100.0
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Carbowax 20M
Acyclic monoterpene hydrocarbons 2.3 2.0 1.4 2.0
Cyclic monoterpene hydrocarbons 94.8 97.2 97.5 90.6
Acyclic sesquiterpene hydrocarbons nd nd nd nd
Cyclic sesquiterpene hydrocarbons 1.6 0.2 0.6 1.1
Long chain hydrocarbons nd nd nd t
Total hydrocarbons 98.7 99.4 99.5 93.8
Acyclic oxygenated monoterpenes 0.8 0.4 0.4 4.9
Cyclic oxygenated monoterpenes 0.4 0.1 0.1 0.1
Acyclic oxygenated sesquiterpenes t t nd 0.8
Cyclic oxygenated sesquiterpenes 0.2 nd nd 0.4
Total oxygenated components 1.4 0.6 0.5 6.2
Aromatic compounds t nd nd 0.1
Total identified 100.0 100.0 100.0 100.0
a
Classes of essential compounds as relative percentage of sum of detected components; t = trace (< 0.1%); nd = not detected; SFE = Supercritical Fluid CO2 extract; CP =
Cold-press oil; VD = Vacuum distilled oil; P = peel; F = flowers.

Antimicrobial activity: Three test organisms, Staphylo-
coccus aureus ATCC 6538, Escherichia coli ATCC 8739 and
Candida albicans ATCC 17110, were used for the assay. The
microorganisms were maintained in Tryptic soy agar (TSA)
for bacteria and Sabouraud dextrose agar (SDA) for yeast at
37.0°C for 18–24 h. The isolated colony was incubated to broth
and incubated overnight at 37.0°C. One loop of this cultured
broth was transferred to new tube and resuspended in TSB,
then incubated at 37.0°C for 2–5 h in the shaking incubator.
After 2–5 h incubation, the microorganism culture was used
for the assay. In this experiment, Doxycycline and Cotrimazole
were used as standard antibacterial and antifungal chemicals,
respectively.
Microdilution method for antimicrobial assay: The
amounts of S. aureus, E. coli and C. albicans were adjusted
by comparing the turbidity to the McFarland suspension
No. 5 (0.5% BaCl2 in 0.56 N in SO4 v/v) and further diluted to
be 106 CFU/mL using Mueller-Hinton Broth (MHB) as dilu-
ent. To overcome the insolubility of the oils and extracts in the
broth, the assay was performed in 1% DMSO of MHB. The
90.0 µL of five folded dilutions of each oil or extract sample or
standard antimicrobial chemicals were prepared in a 96-well
Vol. 22, January/February 2010
plate. The 10.0 µL of each test organism was added into each
well to make a final concentration of approximately 105 CFU/
mL. In each test, test organism in MHB was used as the positive
growth control, while the oil and extract samples in MHB and
MHB alone were used as negative growth control. The plates
were then incubated at 37°C for 18 h. The minimum inhibitory
concentration (MIC) value was defined as the lowest concen-
tration of the oil or extract inhibiting visible growth by using
microplate reader (primary excitation filter position = Abs 550
and primary emission filter position = Diffuser). To evaluate
whether this MIC of oil or extract could kill the microorgan-
ism, 10 µL of each well at MIC was transferred to a new well
containing 90 µL MHB and incubated at 37°C for 18–48 h. If
the oil or extract could kill microbes, a clear solution was found
in each well. Each experiment was performed in triplicate.
Results and Discussion
The extraction conditions of SC-CO2 that gave highest
yield were as follows: pressure, 13 MPa; temperature, 40°C.
Waxes were entrapped in the first separator set at 8 MPa,
40°C. The extract was recovered in the second separator at
5 MPa, 20°C. The isolation yields are given in Table I. For
Journal of Essential Oil Research/75
 Wetwitayaklung et al.
Table V. Minimum inhibitory concentrations (MIC) of the oils and extract from the peel and flower of Citrus maxima compared with
standard antimicrobial chemicals
Type of essential oils
S. aureus
SFE-P 2.5 [2.20]*
CP-P > 2.5 [2.22]*
VD-P > 2.5 [2.11]*
SFE-F 2.5 [2.13]*
Doxycycline [1.25]
Cotrimazole - -
*calculated with density of oils or extracts; SFE = Supercritical Fluid CO2 extract; CP = Cold-press oil; VD = Vacuum distilled oil; P = peel, F = flowers.
the oil from the peel, VD gave the highest yield, 2.39%, and
CP gave 1.16%, while the extract SFE-P gave 0.4%. The ex-
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traction yield of SFE-F was 0.2%. All oils and extracts were
clear solutions with a dark green color for SFE-P and CP, no
color for VD, and yellow color for SFE-F. The color of the
oils and extracts might indicate the containment of impurities
in the oils. The VD had no color, so it contained no impurity.
The scent of peel and flower oils from all isolation methods
exhibited good quality and were quite similar to their natural
origin. The detailed identification and quantification of chemi-
cal composition found in peel produced by SC-CO2, CPM and
VSD using DB-5 and Carbowax 20M are shown in Table II.
The components were unambiguously identified by compar-
ing their specific retention indices and mass fragmentation
patterns with the report by Adams (2001) and commercial
computer matching against Wiley 7n, NIST02 and Pesticide.
For the DB-5 analysis, 52 components of peel oils and extract
and 48 components of flower extract were identified which
represented ca 100% oils and extracts, respectively. For the
Carbowax 20M analysis, 37 components of peel oils and extracts
and 33 components of flower extract were identified which
also represented ca 100% of total oils and extract, respectively.
The major components of the peel oils and extracts were:
limonene (93.4–95.4%), myrcene (1.3–2.3%), b-pinene (0.3–
1.1%), germacrene D (trace–1.4%), sabinene (0.2–0.5%), a-
pinene (0.4–0.5%), a-terpineol (0.1–0.3%), linalool (0.1–0.2%),
b-caryophyllene (trace–0.2%) and geraniol (trace–0.1%). The
detailed identification and quantification of chemical composi-
tion found in flower extract produced by SC-CO2 using DB-5
and Carbowax are shown in Table III. The main components
of flower oil that were identified by DB-5 and Carbowax were
limonene (86.2–86.8%), (E)-b-ocimene (0.1–2.9%), myrce-
ne (1.8%), a-phellandrene (1.4–1.6%), geranial (1.2–1.3%),
(E,Z)-farnesol (1.1%), b-pinene (0.9%), neral (0.7–0.8%),
(E,E)-farnesol (0.1–0.8%), (E)-nerolidol (0.6–0.8%) and a-
pinene (0.6%). From percentage yield and chemical composi-
tion of peel oils, VSD offered a suitable method for peels oil
isolation. VSD gave high percentage yield with no impurities
and chemical compositions of VD were not much different
from that of CPM which scent was most similar to the starting
material. The major disadvantage of oil obtained by SC-CO2
and CPM were the presence of co-extracted cuticular waxes,
sterols, fatty acid, coloring matters, etc. However, the SFE-P
76/Journal of Essential Oil Research
MIC (µL/mL) [µg/mL]
E. coli C. albicans
> 2.5 [2.20]* 2.5 [2.20]* (50% inhibition)
> 2.5 [2.22]* > 2.5 [2.22]*
> 2.5 [2.11]* > 2.5 [2.11]*
> 2.5 [2.13]* 2.5 [2.13]* (50% inhibition)
[4] -
[20]
recovered a higher number of components than CP and VD.
The chemical compositions of SFE-P and SFE-F were quite
similar. SFE-P and SFE-F had a marked difference in percent-
age of components in the oils. The trans-carveol, cis-carveol,
perillaldehyde, a-terpinyl acetate, d-elemene, terpinen-4-ol,
a-terpineol, b-selinene and spathulenol were the components
found only in SFE-P not in SFE-F. The (Z)-b-ocimene was the
component found only in SFE-F not in SFE-P.
The classes of chemical compositions of C. maxima oils are
shown in Table IV. The oils were presented to contain a mix-
ture of components, mainly cyclic monoterpene hydrocarbons
together with acyclic monoterpene hydrocarbons. The SC-CO2
could extract oxygenated components better than CPM and
VSD. The percentage of total oxygenated components using
DB-5 and Carbowax were 1.13% and 1.35% for SFE-P, 0.59%
and 0.59% for CP, and 0.57% and 0.52% for VD, respectively.
The percentage of total oxygenated components of SFE-F,
which were higher than SFE-P, were 4.9% and 6.2% using
DB-5 and Carbowax, respectively. Thus it may be concluded
that SC-CO2 was a valuable method for oxygenated components
entrapment of C. maxima flower because it contained a higher
proportion of oxygenated components than that of the peel.
In this work, the oil of C. maxima showed fairly good
activity against S. aureus, E. coli and good activity against C.
albicans when compared with Doxycycline and Cotrimazole,
respectively, as shown in Table V. The MIC values of SFE-P
and SFE-F were determined as 2.5 µL/mL (0.25%) against
S. aureus and presented 50% inhibition against C. albicans
at 2.5 µL/mL, while the other oil samples were not active at
this concentration. All the oil and extract samples could not
inhibit E. coli at 2.5 µL/mL, which were different from the
previous report by Ontengo et al. (25). This may be from the
difference in compositions of the oils and extracts. Most mono-
terpenes, which were also the major components in these oils
of C. maxima, had been reported to have a broad spectrum
of antimicrobial activity at a low dose (26) and sesquiterpene
alcohol and fatty acids exhibited weak antimicrobial properties
towards Gram-negative bacteria, whereas the aldehyde and
phenolic compounds were able to inhibit bacteria and fungi at
low concentration (26). So, the antimicrobial activities of the
extract from SC-CO2, which were better than oil product by
cold pressing (CPM) and or steam distillation (VSD), might
be due to their higher amount of oxygenated components
Vol. 22, January/February 2010
 C. maxima
(Table IV). As fairly low concentrations of C. maxima extracts
from SFE-P and SFE-F (0.25%) were bacteriostatic for S.
aureus and C. albicans, it may be possible to use these oils for
disinfection of the skin in cosmetics.
Acknowledgments
The authors wish to thank Somnuek Suchaitanavanit, Thai
Traditional and Herbal Development Center (TTHD), Science Park,
Pathumtani, Thailand, for his permission to use the SC-CO2 extractor
and vacuum steam distillator. This research was supported by a grant
of the faculty of Pharmacy, Silpakorn University.
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