Citrus aurantium (Bitter Orange) Blossoms Essential Oil and Methanolic
Extract: Composition and Free Radical Scavenging Activity
W. Dhifi1,2, W. Mnif1,3,a, N. Jelali4, M. El Beyrouthy5 and N. Ben Salem4
1Institut Supérieur de Biotechnologie de Sidi Thabet, BiotechPole de Sidi Thabet, 2020,
Université de La Manouba, Tunisia
2UR Ecophysiologie Générale et Comparée, BiotechPole de Sidi Thabet, 2020, Université
de La Manouba, Tunisia
3LR11-ES31 Biotechnologie et Valorisation des Bio-Géo Ressources, Institut Supérieur
de Biotechnologie de Sidi Thabet, BiotechPole de Sidi Thabet, 2020, Université de la
4Centre de Biotechnologie de Borj-Cedria, 2050 Tunis, Tunisia
5Department of Agricultural Engineering, Faculty of Agricultural and Food Sciences, The
Holy Spirit University of Kaslik (USEK), B.P. 446, Jounieh, Lebanon
Keywords: volatiles, macerate, GC-MS, hydrodistillation, Clevenger, antioxydants
In the present work, we were interested on the one hand in the composition of
Tunisian neroli which is essential oil (EO) extracted from Citrus aurantium blossoms
and on the other hand in the total phenolic, total flavonoids and total tannins of
Citrus aurantium blossoms methanolic extract. We evaluated also the free scavenging
activity of this extract. The results showed that the predominant chemical class in
Tunisian neroli was represented by oxygenated monoterpenes accounting for 62.9%
of whole EO. It was followed by hydrocarbon monoterpenes and aliphatic
hydrocarbons whose respective amounts were of 32.6 and 1.5%. The major
compound of Citrus aurantium neroli oil was lianalool with a percentage of 25.7%.
Total polyphenols were present in the methanolic extract of Citrus aurantium
blossom with a quantity of 8.78 mg EAG.g-1 dry matter. Total flavonoids and total
tannins accounted respectively for 4.86 mg EC.g-1 dry matter and 0.06 mg EC.g-1 dry
matter. Furthermore, concerning free radical scavenging activity, Citrus aurantium
blossoms methanolic extract was characterized by a significant IC50 (20 µg/ml).
The genus Citrus includes several important fruits such as oranges, mandarins,
limes, lemons and grapefruits. It is native to the tropical and subtropical regions of Asia
(Chanthaphon et al., 2007). Citrus fruits are produced all around the world. They contain
healthy nutrition content that works wonders for the body. Furthermore, they represent a
fabulous source of vitamin C and a wide range of essential nutrients required for human
health. Citrus EOs are complex mixtures of volatile compounds and mainly composed of
monoterpene hydrocarbons (Sawamura et al., 2004). According to Lanciotti et al. (2004),
these oils could improve the shelf life and the safety of processed fruits. They are also
used in the pharmaceutical, perfumery and food industries (Huet, 1991).
Citrus aurantium belongs to the order Geraniales and family Rutaceae. It is native
to South East Asia. It is a tree 6 to 8 m high bearing fruits with a thick, rugged and easily
detachable cortex. The essential oil obtained from the cortex of C. aurantium amara has
been used to add aroma to beverages and liquors and as an ingredient to give fragrance to
soaps, detergents, cosmetics and perfumes (Quintero et al., 2007). Preparations from peel,
flowers and leaves of Citrus aurantium L. are popularly used in order to treat anxiety and
to minimize central nervous system disorders. (Carvalho-Freitas and Costa, 2002). The
main compound present in the EOs from Citrus aurantium is limonene (97.83%),
followed by myrcene (1.43%), which is present in around one tenth of that amount. Both
compounds have biological activity related to depression (Pultrini et al., 2006).
Proc. IS on Medicinal and Aromatic Plants – SIPAM 2012
Eds.: M. Neffati and H. Khatteli
Acta Hort. 997, ISHS 2013
Despite the pleasant aroma of Citrus blossoms and their uses in the cosmetics
industry and perfumery, few studies have analyzed their volatile composition of intact
Citrus blossoms (Jabalpurwala et al., 2009).
The aim of this work is on one hand the analysis of the composition of Citrus
aurantium blossoms EO and of their methanolic extract and on the other hand the
evaluation of their free radical scavenging activity.
MATERIALS AND METHODS
Citrus aurantium freshly picked blossoms were collected in the region of El fahs
(North-East of Tunisia). 100 g of Citrus aurantium blossoms were crushed and then
submitted to hydro-distillation in a Clevenger apparatus for 4 h with 600 ml of deionized
water. The resulting EO was dried over anhydrous sodium sulphate and stored at 4°C
until further analysis. The GC-MS analyses were performed on a gas chromatograph HP
6890 interfaced with a HP 5973 mass spectrometer (Agilent Technologies, Palo Alto,
California, USA) with electron impact ionization (70 eV). A HP-5MS capillary column
(60 m×0.25 mm i.d.×0.25 mm film thickness) was used. The column temperature was
programmed to rise from 40 to 280°C at a rate of 5°C/min. The carrier gas was helium
with a flow rate of 1.2 ml/min. Scan time and mass range was 1 s and 50-550 m/z,
respectively. The injected volume was 1 L and the total run time was approximately 63
min. The identification of the EO compounds were based on the comparison of their
retention times with those of authentic compounds available in the laboratory, as well as
on the comparison of their retention indices with those of literature. Further identification
was made by matching their recorded mass spectra with those stored in the Wiley/NBS
mass spectral library of the GC-MS data system (Adams, 2001).
Preparation of the Methanolic Extract and Quantification of Total Phenolic
Content, Total Flavonoid Content and Total Condensed Tannin Assay
Citrus aurantium blossoms were dried at room temperature and coarsely ground
before extraction. Dried powdered samples were extracted at room temperature by
percolation with methanol. All extracts were concentrated over a rotary vacuum
evaporator until a solid extract sample was obtained. The resulting crude extract was
Total polyphenol content of marjoram leaf extracts was determined, as described
by Dewanto et al. (2002). An aliquot of 125 µl of leaf extracts was placed in a reaction
test tube to which 500 µl of water and 125 µl of Folin-Ciocalteau reagent were added.
The mixture was shaken before adding 1250 µl Na2CO3 (7%), adjusting with distilled
water to a final volume of 3 ml and mixed thoroughly. After incubation for 90 min at
23°C in the dark, the absorbance versus prepared blank was read at 760 nm. A standard
curve of Gallic acid was used. Total phenolic content was expressed as mg Gallic acid
equivalents per gram of dry weight (mg GAE/g DW) through the calibration curve with
Gallic acid, ranging from 0 to 400 µg/ml. All samples were analysed in triplicate.
Total flavonoids were measured by a colorimetric assay according to Dewanto et
al. (2002). An aliquot of diluted sample or standard solution of (+)-catechin was added to
a 75 µl of NaNO2 solution (5%) and mixed for 6 min before adding 0.15 ml AlCl3 (10%).
After 5 min, 0.5 ml of NaOH was added. The final volume was adjusted to 2.5 ml with
distilled water and thoroughly mixed. Absorbance of the mixture was determined at 510
nm against the blank where the sample was omitted. Total flavonoid content was
expressed as mg catechin per gram of DW (mg CE/g DW), through the calibration curve
of (+)-catechin, ranging from 0 to 400 µg/ml. Analysis was done in triplicate.
The analysis of condensed tannins was carried out according to the method of Sun
et al. (1998). To 50 µl of properly diluted sample, 3 ml of 4% vanillin solution in
methanol and 1.5 ml of concentrated hydrochloric acid were added. The mixture was
allowed to stand for 15 min and the absorption was measured at 500 nm against methanol
as a blank. The amount of total condensed tannins is expressed as mg (+)-catechin/g DW.
The calibration curve range of catechin was established between 0 and 400 µg/ml. All
samples were analysed in triplicate.
Free Radical Scavenging Activity
The antioxidant activity of the orange juices was evaluated by DPPH free radical-
scavenging method. The DPPH free radical-scavenging activity measurements were
carried out according to the procedure of Sanchez-Moreno et al. (1998) with some
modifications. Briefly, 0.1 ml of juice sample (diluted with distilled water and
centrifuged) was added to 2.46 ml of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH;
0.025 g.L-1 in 50% ethanol) and mixed by vortex for 5 min. The absorbance of the
samples was measured at 515 nm every 1 min for 5 min using the spectrophotometer
Genesys 2 (Milton Roy, USA). For each sample, three separate determinations were
carried out. The antioxidant activity was expressed as the percentage of decline of the
absorbance after 1 min, relative to the control, corresponding to the percentage of DPPH
that was scavenged. The percentage of DPPH that was scavenged (%DPPHsc) was
calculated using: % DPPHsc = (Acont _ Asamp) x 100/Acont
where Acont is the absorbance of the control, and Asamp the absorbance of the sample.
RESULTS AND DISCUSSION
Citrus aurantium Blossoms EO Composition
Twenty six volatile compounds were detected in Tunisian neroli oil. These
compounds belong to different chemical classes. The predominant chemical class in this
EO was represented by oxygenated monoterpenes accounting for 62.9% of whole EO
(Fig. 1). It was followed by hydrocarbon monoterpenes and aliphatic hydrocarbons whose
respective amounts were of 32.6 and 1.5%. The major compound of Citrus aurantium
neroli oil was lianalool with a percentage of 25.7%. Appell (1968) reported that the main
constituents of neroli oil include dipentene, pinene, camphene, I-linalool and linalyl
acetate. Poucher (1974) identified the constituents as linalool, linalyl acetate, pinene,
camphene, dipentene, aldehyde C-10, indole and methyl anthranilate and farnesol.
In our sample, esters were relatively abundant as well as reported Jabalpurwala et
al. (2009). They accounted for 26.9%. This characteristic justifies their value as perfume
Our results were in accordance with those of Jabalpurwala et al. (2009) who
reported that oxygenated monoterpenes were the predominant volatiles in sour orange
blossoms and also in pummelo and in contrast, hydrmonoterpenes were the most
abundant volatiles in limes, Volkamer lemons, Kaffir limes, mandarins, grapefruits and
Sour oranges were located between the lemon-lime and pummelo clusters.
Overall, sweet oranges and grapefruits were found to be more closely related to
mandarins, whereas sour oranges were positioned closer to pummelos. This segregation
of varieties based on blossom volatile composition is in agreement with phylogenic
studies based on morphological and biochemical characteristics which identified citron
(C. medica), mandarin (C. reticulata) and pummelo (C. grandis) as the only ‘true’ citrus
species with all others being cultivated biotypes (Moore, 2001; Nicolosi et al., 2000).
According to Table 2, total polyphenols were present in the methanolic extract of
Citrus aurantium blossom with a quantity of 8.78 mg EAG.g-1 dry matter. Phenolic
compounds especially phenolic acids and flavonoids are ubiquitously present in
vegetables and fruits, thus representing integral part of the human diet (Klimczak et al.,
2007). Consumption of these compounds is correlated with a reduced risk of
cardiovascular diseases, stroke and certain forms of cancer. There is no data concerning
polyphenolic content in blossom Citrus extracts. The total phenolic content of sweet
orange (Citrus sinensis) peel extracts ranged from 3 to 105 mg EGA/g dry extract
(Anagnostopoulou et al., 2006) depending on the solvent.
Total Flavonoids, Total Tannins and Free Radical Scavenging Activity
Total flavonoids and total tannins accounted respectively for 4.86 and 0.06 mg
EC.g-1 dry matter (Table 2). Flavonoids are reputed as contributors of beneficial health
effects of fruits and vegetables (Mata Bilbao et al., 2006). Citrus species accumulate
substantial quantities of flavonoids during the development of their different organs
(Benavente-Garcia et al., 1993). Citrus flavonoids were classified into flavanones,
flavones and flavonols. According to Macheix et al. (1990), each Citrus species is
characterized by a particular flavanone glycoside pattern, flavonoids concentration and
composition depend on the fruit development stage (Ortuño et al., 1995).
Table 2 showed that Citrus aurantium blossoms methanolic extract was
characterized by a significant IC50 (20 µg/ml). In Citrus sinensis peel methnolic extracts,
IC50 ranged from 9.7 to 275 µg/ml (Anagnostopoulou et al., 2006). Generally extracts or
fractions with a high radical scavenging activity showed a high phenolic content.
The authors are grateful to Mr. Saber Khammassi (Technopole of Borj-Cedria)
from the Technopole of Borj Cedria for EO extraction.
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Table 1. Chemical composition of Citrus aurantium blossoms essential oil.
Volatile compound Amount
(% of whole volatiles) RI a RI b
-pinene 0.5 938 1031
-pinene 0.03 980 1118
Myrcene 8.1 988 1169
-phellandrene 1.1 1002 1174
-terpinene 0.8 1063 1263
-terpinene 1.9 1018 1192
p-cymene 4.5 1026 1290
Limonene 6.9 1030 1210
Terpinolene 0.4 1088 1301
-elemene 8.5 1391 1600
1,8-cineole 0.3 1032 1212
Linalool 25.7 1097 1453
Linalyl acetate 14.2 1250 1543
Bornyl acetate 5.5 1295 1597
Neryl acetate 0.1 1385 1733
Geranyl acetate 7.1 1383 1765
Camphre 0.2 1125 1510
Nerol 1.8 1228 1797
Citronellol 3.4 1233 1762
p-cymene-8-ol 0.1 1069 1838
Methyl eugenol 0.6 1401 2030
Geraniol 4.0 1255 1857
Tridecane 0.01 1300 1300
Nonadecane 0.02 1900 1900
Tetracosane 1.5 2400 2400
Retention indices relative to n-alkanes on (a) apolar column HP-5MS and (b) polar column HP-Innowax.
Table 2. Total phenols, flavonoids, tannins and IC50 of the methanolic extract of Citrus
Total phenols (mg EAG.g-1 dm) 8.78
Total flavonoids (mg EC.g-1 dm) 4.86
Total tannins (mg EC.g-1 dm) 0.06
IC50 (µg/ml) 20
dm: dry matter.
Fig. 1. Citrus aurantium blossoms EO composition.