Chiral separation of hesperidin and naringin and its analysis in a butanol extract of Launeae arborescens

Article abstract of DOI:10.1080/14786410903178376

Two flavanone glycosides were isolated from the aerial part of Launeae arborescens (Asteraceae), which were identified as hesperidin and naringin. They are the most abundant flavonoids in the edible parts of many species of citrus fruits. In this study, we were interested in the chiral separation and determination of the diastereomerisation barriers of hesperidin and naringin by HPLC methods. The chiral separation HPLC screening of diastereomers of hesperidin and naringin by HPLC methods was accomplished in the normal-phase mode using 11 chiral stationary phases and various n-hexane/alcohol mobile phases. The rate constants and activation energy of diastereomerisation (G#) of flavanones, naringin and hesperidin were determined, respectively, on Chiralpak IC and Chiralpak IA. The analysis of flavanones isolated in butanol extracts of Launeae arborescens were confirmed by HPLC on Chiralpak IC.

Full text of DOI:10.1080/14786410903178376

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Chiral separation of hesperidin and
naringin and its analysis in a butanol
extract of Launeae arborescens
Nasser Belboukhari a b , Abdelkrim Cheriti a , Christian Roussel b &
Nicolas Vanthuyne b
a Phytochemistry and Organic Synthesis Laboratory, University of
Bechar, 08000, Bechar, Algeria
b UMR Chirotechnologies: Catalyse et Biocatalyse, Université Paul
Cézanne, 13397, Marseille, Cedex 20, France
Published online: 15 Apr 2010.
To cite this article: Nasser Belboukhari , Abdelkrim Cheriti , Christian Roussel & Nicolas Vanthuyne
(2010): Chiral separation of hesperidin and naringin and its analysis in a butanol extract of Launeae
arborescens , Natural Product Research: Formerly Natural Product Letters, 24:7, 669-681
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Natural Product Research
Vol. 24, No. 7, 20 April 2010, 669–681
Chiral separation of hesperidin and naringin and its analysis
in a butanol extract of Launeae arborescens
a
b
Nasser Belboukhariab , Abdelkrim Cheriti , Christian Roussel and
*
Nicolas Vanthuyneb
aPhytochemistry and Organic Synthesis Laboratory, University of Bechar, 08000 Bechar,
b
Algeria; UMR Chirotechnologies: Catalyse et Biocatalyse, Universite Paul Cezanne,
13397 Marseille, Cedex 20, France
´
´
(Received 12 January 2009; final version received 10 July 2009)
Two flavanone glycosides were isolated from the aerial part of Launeae
arborescens (Asteraceae), which were identified as hesperidin and naringin.
They are the most abundant flavonoids in the edible parts of many species
of citrus fruits. In this study, we were interested in the chiral separation and
determination of the diastereomerisation barriers of hesperidin and
naringin by HPLC methods. The chiral separation HPLC screening of
diastereomers of hesperidin and naringin by HPLC methods was accom-
plished in the normal-phase mode using 11 chiral stationary phases and
various n-hexane/alcohol mobile phases. The rate constants and activation
energy of diastereomerisation (DG#) of flavanones, naringin and hesperidin
were determined, respectively, on Chiralpak IC and Chiralpak IA. The
analysis of flavanones isolated in butanol extracts of Launeae arborescens
were confirmed by HPLC on Chiralpak IC.
Keywords: Launeae arborescens; Asteraceae; hesperidin; naringin;
flavanone; diastereomerisation barriers; enantioselective HPLC
1. Introduction
Flavanone-7-O-glycosides, an important class of naturally occurring compounds, are
specifically distributed in citrus fruits (Aturki, Brandi, & Sinibaldi, 2004; Harborne,
T. Mabry & H. Mabry, 1993). Flavanones are chiral compounds because of the
asymmetric C-2 position in their moiety, and thus can exist in two enantiomeric
forms. As their glycosides have an additional optically active sugar residue, they
appear as a pair of diastereomers. Due to their polyphenolic structure, these
compounds have health-related properties, which are based on their antioxidant
activity as well as anticancer, antiviral and anti-inflammatory activities
˜
(Asztemborska & Zukowski, 2006; Yanez, Teng, Roupe, & Davies, 2005).
The flavonoid hesperidin is a flavanone glycoside comprising the flavanone
hesperitin and the disaccharide rutinose. Hesperidin is the predominant flavonoid
*Corresponding author. Email: nasro14@maktoob.com
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786410903178376
http://www.informaworld.com

670
N. Belboukhari et al.
in lemons and oranges. The peel and membranous parts of these fruits have the
highest hesperidin concentrations (Emin, Oliveira, & Lapa 1994). Therefore, orange
juice containing pulp is richer in flavonoids than juice without pulp. Sweet oranges
(Citrus sinensis) and tangelos are the richest dietary sources of hesperidin, classified
as a citrus bioflavonoid (Asztemborska & Zukowski, 2006). Hesperidin, in
combination with a flavone glycoside called diosmin, is used in Europe for the
treatment of venous insufficiency and haemorrhoids (Bok, 1999). Hesperidin, rutin
and other flavonoids thought to reduce capillary permeability and to have anti-
inflammatory action were collectively known as vitamin P. These substances,
however, are not vitamins and are no longer referred to, except in older literature,
as vitamin P (Berkarda, Koyuncu, Soybir, & Baykut, 1998).
In a recent study, hesperidin was separated using normal phase HPLC
in commercial hesperidin and herbal medicine samples, and although the 2S
epimer was predominant, there was a significant amount of 2R hesperidin present in
some samples (Uchiyama, Kim, Kawahara, & Goda, 2005). Finally, the baseline
separation of hesperidin by capillary electrophoresis using sulphobutyl ether
ꢀ-cyclodextrin as the selector was accomplished, and 2S hesperidin was found to
be predominant in lemon and orange juice (Aturki & Sinibaldi, 2003). Furthermore,
the baseline separation of hesperidin by capillary electrophoresis using carbox-
ymethyl-ꢀ-cyclodextrin as the selector has also been accomplished (Wistuba, Trapp,
Gel-Moreto, Galensa, & Schurig, 2006) (Figure 1).
Naringin is a chiral flavanone-7-O-glycoside present in citrus fruits, tomatoes,
cherries, oregano, beans and cocoa; naringin gives the characteristic bitter flavour to
grapefruit. The processors of grapefruit try to select fruits with a low level of
naringin, and often the juices of the mixture use varieties of different grapefruits
to get the required degree of bitterness (Wistuba et al., 2006). Naringin can be used in
the inhibition of cancer and therefore has potential chemotherapeutic value. Several
drugs that are known to be affected by the naringin in grapefruit include blockers of
the calcium channel, estrogen, calmatives, medicines for high blood pressure,
allergies, AIDS, and cholesterol-lowering drugs (Mouly, Arzouyan, Gaydou, &
Estienne, 1994). Caffeine levels and the effects of caffeine can also be spread while
consuming grapefruit or juice of the grapefruit. While the effect of naringin on the
metabolism of a drug can increase the efficiency of the drug, it can also result in
dosages that are accidentally too high. Therefore, it is better not to take any drug
Figure 1. Structures of hesperidin (1) and naringin (2).

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671
with grapefruit juice unless the interaction with the drug is known. Besides, the
effects of drinking grapefruit juice are cumulative, which means, if you drink a glass
of grapefruit juice with your medicine daily for a week, the interaction of the
drug would be stronger at the end of the week compared to the beginning of the week
(Bear & Teel, 2000).
The stereochemistry of naringin changes with the diameter of the fruit, with
greater concentrations of S-naringin in grapefruits with the smallest diameter
(Caccamese, Manna, & Scivoli, 2003). A recent report using ꢀ-cyclodextrin,
dimethyl-ꢀ-cyclodextrin and hydroxypropyl ꢀ-cyclodextrin as mobile phase additives
in capillary electrophoresis resolved the epimers of naringin, although baseline
resolution was not obtained (Gel-Moreto, Streich, & Galensa, 2001). A Chiralpak IA
column was also able to separate naringin directly under normal phase isocratic
conditions, although the baseline resolution was not obtained (Uchiyama, et al.
2005). A more recent study reported the enantiomeric separation of naringin by
capillary electrophoresis using the various cyclodextrins as selectors and demon-
strated the separation with the best resolution of Rs ¼ 4.85 with hydroxypropyl-
ꢁ-cyclodextrin and baseline resolution with methyl-ꢁ-cyclodextrin (Rs ¼ 3.81),
carboxymethyl-ꢀ-cyclodextrin (Rs ¼ 2.26) and sulphato-ꢀ-cyclodextrin (Rs ¼ 3.63)
(Wistuba et al., 2006).
These two glycoside flavanones were detected and isolated from the butanol
fraction of a water–acetone extract of the aerial part of Launeae arborescens
(Asteraceae) in minor quantities by two different methods (naringin at 25ꢀC,
hesperidin at 65ꢀC reflux) (Belboukhari & Cheriti, 2009).
The present work reports the chiral separation screening on 11 chiral stationary
phases (CSPs) and the determination of rate constants and activation energy barriers
of diastereomerisation DG# of naringin and hesperidin using HPLC methods.
2. Experimental
2.1. Reagents
Naringin (97%), hesperidin (97%), TEA, TFA, hexan, ethanol, methanol and
isopropanol were from Acros Organics (New Jersey, USA).
2.2. Apparatus and procedures
The analytical chiral HPLC experiments were performed on a unit composed of
a Merck D-7000 system manager, Merck-Lachrom L-7100 pump, Merck-Lachrom
L-7360 oven, Merck-Lachrom L-7400 UV-detector and a Jasco OR-1590 polari-
meter. The sign given by the online polarimeter for an enantiomer is the sign in the
solvent used for the chromatographic separation. Retention times (Rt) in minutes,
retention factor, ki ¼ (Rti ꢁ Rt0)/Rt0, and enantioselectivity factor, a ¼ k2/k1, are
given.
Hexan, 2-PrOH and ethanol were of HPLC grade, degassed and filtered on
a 0.45 lm membrane before use. Chiralcel OD-H, OD, OJ, OB-H, Chiralpak AD,
AD-H, AD-RH, AS, IA, IC, available from Chiral Technologies Europe (Illkirch,
France), and Ulmo (S, S) (250 3 4.6 mm), Sumichiral OA-2500, Kromasil CHI-TBB,

672
N. Belboukhari et al.
available from Regis (Morton Grove, USA), were used for the analytical
separations.
2.3. Plant materials (extraction and traditional uses)
Launeae arborescens (vernacular name: Oumlbina) is a herbaceous plant belonging
to the Asteraceae family, which is widely distributed in the west Algerian Sahara.
The aerial part of this plant is used in folk medicine for treating diarrhoea fever,
gastrointestinal tracts and various diseases (Belboukhari & Cheriti, 2006; Ozenda
1967). The whole plants were collected in March 2000 from Bechar, south Algeria.
The botanical identification and a voucher specimen are conserved at the
Phytochemical Herbarium of the Phytochemical and Organic Synthesis
Laboratory under accession number CA99/25 (Cheriti, Belboukhari, & Hacini,
2004).
The aerial parts were separated and dried and the plants were ground into
powder using a grinder. Hesperidin and naringin were isolated from a butanol
fraction of water : acetone (2 : 1) extract of an aerial part of the plant by reflux for 3 h
using a Soxhlet apparatus and by steeping to ambient temperature during 24 h
(Belboukhari, Cheriti, & Roussel 2007).
3. Results and discussion
3.1. Chiral separation screening
To optimise the conditions for obtaining the separation of the C-2 diastereomers
(epimers) of hesperidin and naringin present in butanol extracts of L. arborescens,
to assess their identity, and to study their C-2 stereochemistry, we used
commercialised compounds in the chiral separation screening on 11 CSPs and
various (n-hexane/ethanol or isopropanol) mobile phases (Table 1). Typical
separation of the epimers of compounds 1 and 2 is shown in Figures 2 and 3.
Table 1 shows the chromatographic results for the separation of (2R/2S)-
flavanone glycosides using the Chiralcel OD-H as CSP. Epimer selectivity values (R)
ranged from 1.81 for naringin to 1.16 for hesperidin, Chiralpak IA ranged in
different conditions from 1.25 to 1.13 for naringin and hesperidin and Chiralpak
AD-H presented a good chiral separation of naringin and hesperidin with a
selectivity factor towards 1.28.
The Chiralpak AD phase presented only the epimer separation of hesperidin with a
selectivity towards 1.21. Analogously, the resolution factor (Rs) ranged from 2.27 for
naringin to 0.97 for hesperidin. The values of R and Rs obtained for naringin were
much better than those obtained using another polysaccharide-derived CSP
(Chiralpak AD) and a very similar mobile phase (1.51 and 0.7, respectively).
The difference in the chiral recognition ability may be due to the different
volumes of the helical groove of the cellulose derivative (OD-H) and the amylose
derivative (AD), because it is well known that amylose-derived phases possess a
wider and more compact helix (Okamoto & Yashima, 1998).
These two compounds were not resolved into their epimers using the composition
of the mobile phase and Chiracel OJ, Chiracel OBH, Sumi, Ulmo and Kromasil
phases reported in Table 1, as elution times for the epimers of hesperidin on Chiralpak

Natural Product Research
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674
N. Belboukhari et al.

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675
(a)
(b)
(c)
40
50
60
70
80
25
30
35
10
15
20
Retention time (min)
Retention time (min)
Retention time (min)
(d)
(e)
6
10
15
20
8
10
Retention time (min)
Retention time (min)
Figure 2. Chromatograms of chiral separation of epimers of hesperidin on (a) Chiralpak IA
(Entry 2), (b) Chiralpak AD (Entry 10), (c) Chiralpak AD-H (Entry 7), (d) Chiralpak OD-H
(Entry 15), and (e) Chiralpak IC (Entry 23).
IA can be achieved only by using a more polar mobile phase (50% doped ethanol in
n-hexane). Much higher elution times for very broad peaks (460 mn) were obtained
when using a less polar phase.
The strong interaction of hesperidin and naringin with the CSP results in a very
high k1 due to a more polar mobile phase for reasonable elution, and their presence
in the structures of compounds 1 and 2 of two glycosides groups act as hydrogen
donor and hydrogen acceptor groups for hydrogen-bonding interaction with the
carbamate groups of the phase (Caccamese, Caruso, Parrinello, & Savarino, 2005).
The absolute configuration at C-2 of the first and the second peaks of compounds
1 and 2, as reported in Table 1, has been assigned by the knowledge of the Cotton
effects in the circular dichroism (CD) spectra of flavanone glycosides and their
aglycones, because the carbohydrate moiety is CD-inactive (Caccamese et al., 2005;
˜
Yanez, Andrews, & Davies 2007). Moreover, the assignment of the absolute
configuration of the eluting peaks is not automatic in the absence of information
about the CD spectra or if the authentic samples of C-2 diastereomers are not
available. Integration of the peaks shows that the two commercial compounds are
richer in the 2S epimer, which show a diastereomeric purity (dp) 2S/(2S þ 2R),
79.4% of hesperidin and 75.1% of naringin. However, the isolated compounds

676
N. Belboukhari et al.
(a)
(b)
25
30
35
40
45
4
6
8
Retention time (min)
Retention time (min)
(c)
(d)
5
6
7
8
9
10 11
20
25
30
Retention time (min)
Retention time (min)
Figure 3. Chromatograms of chiral separation of epimers of naringin on (a) Chiralpak IA
(Entry 28), (b) Chiralpak AD-H (Entry 34), (c) Chiralpak OD-H (Entry 40), and (d) Chiralpak
IC (Entry 42).
present a diastereomeric purity towards 53% of hesperidin and 68% of naringin,
although the stereochemistry at C-2 is not depicted. The predominance of one
C-2 diastereomer with respect to another one is undoubtedly due to the extraction of
these compounds (Trapp & Schurig, 2001).
3.2. Chiral separation composition of glucoside flavanones (1 and 2)
from L. arborescens
The two glucoside flavanones isolated from the butanol fraction of the water acetone
extract of L. arborescens were identified as hesperidin and naringin, and the structures
were confirmed by chiral separation on eluted Chiralpak IC (hexane/ethanol, 70 : 30).
The results presented in Table 2 demonstrate a racemic composition of hesperidin
(46.96 : 53.04) and naringin (68.45 : 31.55) (Figure 4). The results were explained by the
protocol condition for the isolation of these two compounds, of which the naringin

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677
Table 2. Chiral separation of hesperidin and naringin on
Chiralpak IC.
Compound
Hesperidin
Naringin
CSP
Eluent (%)
FR
k1
Rt1
Chiralpak IC
70
1
5.12
18.67
23.51
1.55
Chiralpak IC
70
1
4.55
16.93
22.75
2.43
Rt2
Rs
ꢂ
Composition (%)
1.31
46.96 : 53.04
1.42
68.45 : 31.55
Note: CSP, Chiral stationary phase.
16 18 20 22 24 26
16
18
20
22
24
26
Time (min)
Time (min)
1
2
Figure 4. Chiral separation of hesperidin and naringin epimers on Chiralpak IC.
was separated at t.a. but hesperidin was extracted with reflux at 100ꢀC (Trapp
& Schurig, 2001).
3.3. Diastereomerisation of hesperidin and naringin
The diastereomerisation of two flavanone glycosides was studied by enantioselective
HPLC combined with the classical kinetic method, enantiomerisation and calculated
from the following equation (Figure 5):
k ¼ ð1=2tÞ lnð½Aꢂ0 ꢁ ½Aꢂeq=½Aꢂt ꢁ ½AꢂeqÞ:
The diastereomerisation barrier DG# was calculated using the Eyring equation:
DG# ¼ RT lnðhk=ꢃkBT Þ,
ð1Þ
ð2Þ

678
N. Belboukhari et al.
16
18
20
22
24
26
1
2
Figure 5. Chromatograms of diastereomerisation of hesperidin (1) and naringin (2) epimers in
MeOH at 40ꢀC by HPLC method.
Hesperidin
3
y = 0.0001x – 0.2174
2.5
2
R
= 0.9599
2
1.5
1
Series 1
Linear (Series 1)
0.5
0
0
5000
10000 15000 20000 25000
–0.5
t (s)
Figure 6. Plot of ln[D] ¼ f(t) for hesperidin obtained by HPLC experiment.
where T is the temperature of diastereomerisation in k, R is the universal gas
constant (R ¼ 8.31441 JKꢁ1 molꢁ1), kB is the Boltzman constant (kB ¼ 1.380662 ꢃ
10ꢁ23 JKꢁ1), h is the Planck’s constant (h ¼ 6.626176 ꢃ 10ꢁ34 Js) and ꢃ is the trans-
mission coefficient (ꢃ ¼ 0.5) (Reich, Trapp, & Schurig, 2000).
The plot of ln([A]0 ꢁ [A]eq)/([A]t ꢁ [A]eq) as a function of time for hesperidin and
naringin are presented, respectively, in Figures 6 and 7, and demonstrate a linear
relationship according to Equation (1). The rate constants were determined from the
plot by linear regression.
In this method, diastereomerisation is performed outside the separation system
at a chosen temperature and time (Asztemborska & Zukowski, 2006). Afterwards,
samples are analysed by enantioselective HPLC at the temperature at which
diastereomerisation is suppressed. This method was applied to determine the rate
constant and Gibbs activation energy of the diastereomerisation of hesperidin and

Natural Product Research
679
Naringin
y = 0.0007x + 0.1572
4
3.5
3
2
R = 0.9694
2.5
2
Series 1
Linear (Series 1)
1.5
1
0.5
0
0
1000
2000
3000
4000
5000
6000
t (s)
Figure 7. Plot of ln[D] ¼ f(t) for naringin obtained by HPLC experiment.
Table 3. The rate constants and Gibbs activation energy of diastereomerisation reaction of
naringin and hesperidin determined by HPLC methods.
Compounds
Hesperidin
Naringin
Method
T (ꢀC)
Solvent
k (sꢁ1
)
DG# (kJ molꢁ1
)
HPLC
HPLC
HPLC
D-HPLC
D-HPLC
40ꢀ
60ꢀ
40ꢀ
50ꢀ
60ꢀ
MeOH : TEA (10 : 0.1) 10ꢁ4
MeOH
MeOH
MeOH : H2O (10 : 90)
MeOH : H2O (10 : 90)
84.00
115.8
78.93
102.5
103.5
2.4 ꢃ 0ꢁ6
7 ꢃ 10ꢁ4
9.218 ꢃ 10ꢁ5
2.078 ꢃ 10ꢁ4
naringin (Table 3). The 0.27 mM solution of hesperidin and naringin in methanol
(added 3 m mL TEA) was stored at 40ꢀC in a thermostat. Every 24 h, samples were
taken and analysed directly by enantioselective HPLC. Additionally, it was
proved that after a few weeks of storage of the solutions at 40ꢀC, the ratio of 2R-
to 2S-diastereomer reached a value very close to 1, which leads to the conclusion
that, for hesperidin as well as for naringin, keq ꢄ 1.
The plot of ln([A]0 ꢁ [A]eq)/([A]t ꢁ [A]eq) as a function of time for hesperidin and
naringin presented in Figures 6 and 7 demonstrates a linear relationship according
to Equation (3).
4. Conclusion
In summary, the developed HPLC assay is stereospecific, reproducible and accurate.
It has been successfully applied to chiral analysis and quantification of diastereomers
of flavanone glucosides in different stages of evolution of the plant, and successfully
used to study the diastereomeric composition of various plant parts (root, stem, twig,
leaf, flower and seed).
Acknowledgements
The authors are grateful to Prof. N. Rayan (Najah University), and would like to express their
gratitude to the MESRS – Algeria for their financial support.

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N. Belboukhari et al.
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