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Citrus aurantium (Bitter Orange) blossoms essential oil and methanolic extract: Composition and free radical scavenging activity

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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).
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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
Manouba, Tunisia
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
Abstract
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).
INTRODUCTION
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).
aw_mnif@yahoo.fr
Proc. IS on Medicinal and Aromatic Plants – SIPAM 2012
Eds.: M. Neffati and H. Khatteli
Acta Hort. 997, ISHS 2013
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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
freeze-dried.
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.
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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
ingredients.
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
sweet oranges.
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
198
(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.
ACKNOWLEDGMENTS
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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Tables
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.
200
Table 2. Total phenols, flavonoids, tannins and IC50 of the methanolic extract of Citrus
aurantium blossoms.
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.
Figures
62,9
32,6
1,5
Oxygenated monoterpenes
Hydrocarbon monoterpenes
Aliphatic hydrocarbons
Fig. 1. Citrus aurantium blossoms EO composition.
... Such natural compounds own anticarcinogenic properties [3] and supply various health-Citrus aurantium L. (C. aurantium), known as bitter orange or sour orange, is a tree that belongs to the order Geraniales and the family Rutaceae [13]. Sour orange trees in Cyprus is planted around all the fruit gardens to protect fruit trees from the wind and to benefit from their flowers. ...
... Similar findings have been reported by Trabelsi et al. [26], Hsouna et al. [7], Sarrou et al. [20], and Dhifi et al. [13] who reported that C. aurantium EOs consist mainly of linalool, limonene, ␣-terpineol, nerolidol, ␤-pinene, neryl acetate, farnesol and linalyl acetate. As seen on Table 3, Ammar et al. [27], Hsouna et al. [7] and Salma et al. [6] reported that the C. aurantium EO can be characterized by the dominance of limonene (27.5-77.90%), ...
... The EO and hydrosol contained high levels of monoterpene and sesquiterpene constituents which have antioxidant activity close to that of phenolic constituents, break free-radical chain reactions and caused their irreversible oxidation into inert compounds [2]. The results obtained in this study were found to be in agreement with the results of a few authors who declared that Neroli oil and methanol extract have a moderate antioxidant activity [2,13]. ...
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Several parameters mostly affecting the precision and accuracy of vanillin assay were reexamined and optimized. The reexamination was performed both by vanillin reaction with catechins and by vanillin reaction with purified proanthocyanidins. In addition to the acid nature and concentration, the reaction time, the temperature, and the vanillin concentration, other factors such as the water content, the presence of interfering substances, and the standard utilized, for both vanillin reaction with catechins and vanillin reaction with proanthocyanidins, were also important. However, the kinetics of the two types of reactions were markedly different. For estimating accurately catechins or proanthocyanidins that exist simultaneously in plant tissues, it is necessary to preliminarily separate them from each other. Keywords: Catechins; proanthocyanidins; vanillin assay
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The effects of variety, rootstock, and geographical location were studied as to their effects on secondary metabolite production in grapefruit and pummelo. The concentration of the, flavanones narirutin, naringin, and neohesperidin and of the sesquiterpene nootkatone, which is principally responsible for the grapefruit's aroma, varies during fruit development. The highest flavanone levels are detected during the juvenile stages of fruit development, while nootkatone expression is associated with the processes of maturation and senescence, The possibility of increasing the levels of these metabolites by regulating the associated processes of growth and cell differentiation is discussed.
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The kinetic behaviour of polyphenols common in fruits as free radical scavengers was studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH•). After addi-tion of different standard concentrations to DPPH· (0·025 g litre−1), the percentage of remaining DPPH• was determined at different times from the absorbances at 515 nm. The percentage remaining DPPH• against reaction time followed a multiplicative model equation: ln [DPPHREM•]=b ln t+ln a. The slopes of these equations may be useful parameters to define the antioxidant capacity. The steeper the slope, the lower the amount of antioxidant necessary to decrease by 50% the initial DPPH• concentration (EC50). This parameter, EC50, is widely used to measure antioxidant power, but it does not takes into account the reaction time. Time needed to reach the steady state to the concentration corresponding at EC50 (TEC50) was calculated, and antiradical efficiency (AE) was proposed as a new parameter to characterise the antioxidant compounds where AE=1/EC50TEC50. It was shown that AE is more discriminatory than EC50. AE values are more useful because they also take into account the reaction time. The results have shown that the order of the AE (×10−3) in the compounds tested was: ascorbic acid (11·44)>caffeic acid (2·75)⩾gallic acid (2·62)>tannic acid (0·57)⩾DL-α-tocopherol (0·52)>rutin (0·21)⩾quercetin (0·19)>ferulic acid (0·12)⩾3-tert-butyl-4-hydroxyanisole, BHA (0·10)>resveratrol (0·05). © 1998 SCI.
Article
Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one.
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Flavonoids are a group of polyphenolic compounds with health-related properties. Citrus fruits are rich in flavonoids and their extracts are being used as functional ingredients for several industrial products. A new high performance liquid chromatography technique with an UV photodiode-array detector was used to analyze flavonoids of an extract of Citrus species. To our knowledge this is the first study that reports isoquercitrin presence at a level of 77.3 mg/100 g in a sample made of Citrus fruits; four other flavonoids were quantified as rutin (326.59 mg/100 g), naringin (338.36 mg/100 g), quercetin (96.35 mg/100 g) and naringenin (2.35 mg/100 g). Identification was confirmed by a liquid chromatography mass spectrometer system. Method validation was achieved, providing an analytical technique that can be used to detect trace amounts of these compounds in Citrus extracts with an extremely rapid sample preparation.
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The effect of time and temperature on the content of vitamin C, total polyphenols and individual phenolic compounds as well as on the antioxidant activity of two commercial orange juices was studied. The polyphenol content was determined using Folin–Ciocalteu and HPLC methods. The two methods, SPE versus direct injection following a simple treatment of samples, were compared to assess two techniques of sample preparation. For antioxidant capacity determination, DPPH and FRAP assays were used. All analyses were carried out for fresh juices and after storage at 18, 28 and 38 °C for 2, 4 and 6 months. It was found that vitamin C and free and conjugated hydroxycinnamic acids were the most affected by both duration and temperature of storage. The decrease in the content of polyphenols and vitamin C upon storage was reflected by the decrease in the antioxidant capacity of orange juices. Small changes in flavanone content were observed, indicating high stability of these compounds upon storage.