Chemical composition and biological activity of Citrus jambhiri Lush

Article abstract of DOI:10.1016/j.foodchem.2010.12.129

The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by column chromatography employing solvents of different polarity. The chemical structure of the isolated compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,3′,4′- hexamethoxyflavone, 3,5,6,7,8,3′,4′-heptamethoxyflavone, and a mixture of β-sitosterol and stigmasterol. The chloroform fraction afforded 6-demethoxynobiletin, 5,4′-dihydroxy-6,7,8,3′-tetramethoxyflavone, limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and NMR (APT, COSY, HSQC, HMBC, and NOESY). LC-ESI-MS analysis of the ethyl acetate fraction afforded eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven limonoid aglycones. The chloroform fraction exerted the strongest DPPH* free radical scavenging activity in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase indicating an anti-inflammatory action (IC₅₀ 29 ± 1 μg/mL). Some of the isolated polymethoxyflavones exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.

Full text of DOI:10.1016/j.foodchem.2010.12.129

Food Chemistry 127 (2011) 394–403
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Chemical composition and biological activity of Citrus jambhiri Lush
Dalia Hamdan a,b, Mahmoud Zaki El-Readi a, Ahmad Tahrani a, Florian Herrmann a,
Dorothea Kaufmann a, Nawal Farrag b, Assem El-Shazly b, Michael Wink a,
⇑
a Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
b Department of Pharmacognosy, Faculty of Pharmacy, Zagazig University, 44519 Zagazig, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The fresh peel of Citrus jambhiri was extracted with aqueous methanol and the residue was fractionated
using light petroleum, chloroform and ethyl acetate. The constituents of the extracts were separated by
column chromatography employing solvents of different polarity. The chemical structure of the isolated
compounds was then identified by MS and NMR. Column chromatography of the petroleum fraction
resulted in the isolation of nobiletin, 5-O-demethylnobiletin, tangeretin, 5-hydroxy-3,6,7,8,30,40-hexa-
methoxyflavone, 3,5,6,7,8,30,40-heptamethoxyflavone, and a mixture of b-sitosterol and stigmasterol.
The chloroform fraction afforded 6-demethoxynobiletin, 5,40-dihydroxy-6,7,8,30-tetramethoxyflavone,
limonin and nomilin. The flavonoid glycosides naringin, hesperidin and neohesperidin were isolated from
the ethyl acetate fraction. The chemical structure of the isolated compounds was established by MS and
NMR (APT, COSY, HSQC, HMBC, and NOESY). LC–ESI-MS analysis of the ethyl acetate fraction afforded
eight flavonoid glycosides, while the dichloromethane fraction of the defatted seeds contained seven
limonoid aglycones. The chloroform fraction exerted the strongest DPPH⁄ free radical scavenging activity
in comparison to other fractions. The petroleum fraction showed a significant inhibition of lipoxygenase
Received 17 February 2010
Received in revised form 28 October 2010
Accepted 29 December 2010
Available online 8 January 2011
Keywords:
Citrus jambhiri
Polymethoxyflavones
Flavonoid glycosides
Limonoids
NMR
LC–ESI/MS
Antioxidant
Anti-inflammatory
Cytotoxicity
indicating an anti-inflammatory action (IC50 29
exhibited strong cytotoxicity against COS7, HeLa and Caco-2 cell lines.
Ó 2011 Elsevier Ltd. All rights reserved.
1 lg/mL). Some of the isolated polymethoxyflavones
1. Introduction
A review of the literature indicates that only few studies have
been conducted on the fruit peel constituents. Coumarins have
been isolated and detected from different organs of Citrus jambhiri,
such as 6,7-dimethoxycoumarin (Sulistyowati, Keane, & Anderson,
1990), aurapten (Ogawa et al., 2000), bergapten and psoralin
(McCloud, Berenbaum, & Tuveson, 1992). The flavonoid glycosides
hesperidin and neohesperidin have been detected in the fruit juice
using high-performance liquid chromatography (HPLC) (Arriaga &
Rumbero, 1990). Also, the polymethoxyflavones tangeretin, 5-
O-demethyltangeretin and 5-hydroxy-6,7,8,40-tetramethoxyflavone
have been reported from this species (Chaliha, Sastry, & Rao, 1965;
Tatum & Berry, 1972).
The aim of this study has been to investigate the chemical com-
position, as well as the antioxidant, anti-inflammatory and in vitro
cytotoxic activities, of the total methanolic extract, light petro-
leum, chloroform and ethyl acetate fractions and of some second-
ary metabolites isolated from C. jambhiri in order to evaluate its
pharmacological potential.
Citrus fruits (Rutaceae) possess high amounts of bioactive com-
pounds which can influence human health, as e.g. vitamin C,
carotenoids (b-carotene), flavonoids, limonoids, essential oils, cou-
marins, acridone alkaloids, high quality soluble fibre, minerals,
vitamin-B complex and related nutrients such as thiamine, ribofla-
vin, nicotinic acid/niacin, pantothenic acid, pyridoxine, folic acid,
biotin, choline, and inositol (Ladaniya, 2008); health promoting
effects include antioxidant, cardioprotective, anticarcinogenic,
anti-allergic, antiplatelet, antiviral, antibacterial and antifungal
activities (Manners, 2007; Miller, Porter, Binnie, Guo, & Hasegawa,
2004; Tripoli, Guardia, Giammanco, Majo, & Giammanco, 2007).
Lemon juice has had a long tradition in folk medicine as being used
for treating the common cold, and as a diuretic, antiscorbutic,
astringent and fever-reducing remedy.
Flavonoids, in particular polymethoxyflavones, flavanone glyco-
sides and limonoids, are naturally abundant secondary metabolite
compounds in Citrus species, and since these substances bear eco-
logical, biological and chemotaxonomic importance in this genus
(Ladaniya, 2008; Manners, 2007; Tripoli et al., 2007), they have
attracted considerable interest.
2. Materials and methods
2.1. Plant material
⇑
The fresh fruit rind and seeds of rough lemon (C. jambhiri Lush.),
Rutaceae, were separated from the ripe fruits which were collected
Corresponding author. Tel.: +49 6221 54 4880; fax: +49 6221 54 4884.
E-mail address: wink@uni-hd.de (M. Wink).
0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.12.129

D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
395
from the Research Station of the Faculty of Agriculture, Benha
University, Egypt in March 2004. The identity of the plants was
confirmed by Prof. Dr. B. M. Houlyel, Dept. of Pomology, Faculty
of Agriculture, Benha University, Egypt.
developed using the solvent system dichloromethane-acetone,
(9:1, v/v) and eluted to afford compound 3 (8 mg) as a yellow
amorphous powder with Rf an 0.66.
The column fractions 70–85 eluted at a benzene-CHCl3, (1:9, v/v)
were pooled and crystallised in chloroform/acetone producing
100 mg of compound 4 as yellowish-white needle-shaped crystals
with Rf 0.54. In addition, fractions 86–101 eluted with 100% chloro-
form (CHCl3), gave 15 mg of compound 5 as dark yellow needle-
shaped crystals (from hot chloroform/methanol) with Rf 0.35.
Fractions 102–106 eluted at a chloroform/ethylacetate (CHCl3-
EtOAc), (9.5:0.5, v/v) were pooled and crystallised in hot chloro-
form/methanol to afford compound 6 (40 mg) as dark yellow
needle-shaped crystals with Rf 0.28. Fractions 107–115 eluted at a
CHCl3-EtOAc, (9:1, v/v) were collected and crystallised in chloro-
form/methanol to yield compound 7 as yellow granules (25 mg)
with Rf 0.25.
2.2. Chemicals
2,2 Diphenyl-1-picryl-hydrazil radical (DPPH), linoleic acid so-
dium salt, nordihydroguaiaretic acid (NDGA), 3-(4,5 dimethylthia-
zol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and ascorbic
acid were purchased from Sigma–AldrichÒ (Taufkirchen,
Germany). Lipoxydase enzyme from soybean (lyophilised powder)
was supplied from Fluka (Buchs, Switzerland). Cell culture DMEM-
glutamate I media, dimethyl sulphoxide (DMSO) were purchased
from RothÒ (Karlsruhe, Germany). Foetal bovine serum (FBS) from
BiochromeÒ (Berlin, Germany), trypsin–EDTA, penicillin, strepto-
mycin, sodium pyruvate, non-essential amino acids and Hanks’
balanced salt solution were bought from GibcoÒ (Invitrogen;
Karlsruhe, Germany). Authentic compounds; rutin, eriocitrin, neo-
eriocitrin, diosmetin 6-C-glucoside, narirutin, naringin, hesperidin,
neohesperidin, limonin, nomilin, and palmitic acid were obtained
from university of Heidelberg, IPMB. In addition, methanol, dichlo-
romethane, chloroform, petroleum ether, benzene, ethylacetate,
formic acid, acetonitrile and other solvents used for extraction,
separation and/or detection, which were of analytical grade, were
obtained from Merck (Germany).
2.4.2. Compounds 8–11
The dry mixed initial zone was prepared from 8 g of chloroform
fraction and was chromatographed on
a silica gel column
(150 ꢀ 2.5 cm, 200 g) at room temperature. The column was eluted
with a gradient using mixture of benzene–chloroform–ethyl ace-
tate as mobile phase. Fractions of 250 mL each were collected, con-
centrated under vacuum and monitored by TLC using a mixture of
chloroform–ethyl acetate, (8:2, v/v) as a system for TLC develop-
ment. Spots were detected by UV after spraying with H2SO4 10%
(v/v) in water and/or Ehrlich’s reagents. Column fractions 45–50
eluted at a C6H6-CHCl3, (1:9, v/v) were collected and 15 mg of
compound 8 was crystallised as yellowish-white needles from
chloroform–methanol with Rf 0.28. Fractions 66–70 eluted at a
CHCl3-EtOAc, (9.7:0.3, v/v) were concentrated and crystallised in
chloroform/methanol yielding 10 mg of compound 9 as white nee-
dle-shaped crystals with Rf 0.21. Column fractions 71–80 eluted at
a CHCl3-EtOAc, (9.5:0.5, v/v) were crystallised in chloroform/meth-
anol producing 650 mg of compound 10 as a white amorphous
powder with Rf 0.16. The collected fractions 81–91 eluted at a
CHCl3-EtOAc (9:1, v/v) afforded 12 mg of compound 11 as yellow
granules with Rf 0.11.
2.3. Extraction procedure
Fresh peel of C. jambhiri Lush. (5 kg) was exhaustively extracted
with 80% aqueous methanol (3 ꢀ 10 L). The methanolic extract was
filtered and then concentrated under vacuum to yield 1750 g of a
viscous residue. The residue was suspended in water and parti-
tioned against light petroleum (b.p. 60–80 °C), chloroform and
ethyl acetate successively. The organic solvents were evaporated
under vacuum using rotary evaporator at lower temperature to
yield 20, 15 and 20 g of final residue, respectively.
Dried seeds (290 g) were cleaned, further dried, powdered,
defatted with light petroleum and extracted with methanol
(MeOH). The methanolic extract evaporated under vacuum using
rotary evaporator at 55 °C to give 24 g residue which diluted with
water (H2O), extracted with dichloromethane (CH2Cl2) and the sol-
vent was evaporated under vacuum to yield 9 g of residue.
2.4.3. Compounds 12–14
Dry mixed initial zone was done by dissolving 8 g of ethyl ace-
tate fraction in methanol (10 mL), load the solution over silica for
column, triturate the mixture till drying and the dried zone was
chromatographed on a silica gel column (150 ꢀ 4 cm, 300 g) at
room temperature. The column was eluted with a gradient using
mixture of chloroform–methanol as mobile phase. Fractions of
250 mL each were collected, concentrated under vacuum and mon-
itored by TLC using ethyl acetate-formic acid-acetic acid–water,
(10:1.1:1.1:2.6, v/v) as developing system. The TLC was visualised
under UV and H2SO4 10% (v/v) in water spray reagent. Fractions
31–50 eluted at a CHCl3-MeOH, (9:1, v/v) were crystallised in
methanol to give 10 mg of compound 12 as buff granules with Rf
0.77. Crystallisation of column fractions 51–65 eluted at a CHCl3-
MeOH, (8:2, v/v) gave 800 mg of compound 13 as white granules
using hot methanol with Rf 0.72. Column fractions 66–75 eluted
at a CHCl3-MeOH (8:2, v/v) were collected, concentrated and
rechromatographed on a preparative TLC which was developed
using chloroform–methanol–water, (6:4:0.5, v/v) and eluted to af-
ford compound 14 (15 mg) as white granules which obtained by
crystallisation using hot methanol with Rf 0.67.
2.4. Isolation of the compounds
2.4.1. Compounds 1–7
The light petroleum fraction (8 g) redissolved in dichlorometh-
ane and the solution was mixed with 10 g silica for column. The
dry mixed initial zone was chromatographed on a silica gel column
(150 ꢀ 2.5 cm, 200 g) at room temperature. The column was eluted
with a gradient using a mixture of benzene–chloroform–ethyl ace-
tate as mobile phase. Fractions 250 mL were collected and moni-
tored by thin layer chromatography (TLC) using pre-coated silica
gel GF254 (Merck) and a mixture of chloroform–ethyl acetate (8:2,
v/v) for TLC development. Spots were visualised by spraying with
H2SO4 10% (v/v) in water followed by heating at 105 °C for 5 min.
Fractions 23–36 eluted at a benzene/chloroform (C6H6-CHCl3),
(8:2, v/v) were collected, evaporated and after crystallisation in a
chloroform/methanol mixture compound 1 was obtained as a
white amorphous powder (12 mg) with retention factor (Rf) 0.77.
Additionally, fractions 37–50 eluted at a C6H6-CHCl3, (8:2, v/v)
were pooled and compound 2 (15 mg) was collected as white nee-
dle-shaped crystals from chloroform/methanol with Rf 0.74. More-
over, fractions 63–69 eluted at a C6H6-CHCl3, (1:9, v/v) were
collected and chromatographed again on the prep-TLC which was
2.5. Acid hydrolysis
Compounds 12–14 which had been isolated from the ethyl ace-
tate fraction were subjected to acid hydrolysis by dissolving about
5 mg of each compound in 5 mL methanol and refluxing with

396
D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
20 mL aqueous H2SO4 (50%) for 6 h. The reaction mixtures were
extracted twice with chloroform (50 mL). The organic layers were
collected, concentrated and crystallised to yield the corresponding
aglycones. The aqueous hydrolysed was spotted on paper chroma-
tography plates together with authentic sugars (PC, ethyl acetate-
formic acid-acetic acid–water, 10:1.1:1.1:2.6, v/v).
(99.5:0.5, v/v) and solvent B: acetonitrile. The elution was arranged
as follows: 0–5 min, isocratic at 0% B; 5–110 min, gradient from 0%
to 70% B; 110–115 min, gradient from 70% to 100% B; 115–
140 min, isocratic at 100% B. MS analysis was performed using
ESI (positive ion mode) under the same conditions as mentioned
in LC–ESI/MS determination of flavonoid glycosides.
2.6. Spectral data
2.8. Antioxidant activity
NMR spectra (1H and 13C) were recorded on a Mercury 300 and
VARIAN 500 at 300 and 500 MHz for 1H measurements and 75/
125 MHz for 13C measurements, respectively. CD3OD, DMSO-d6
and CDCl3 were used as solvents. Experimental data were pro-
cessed using MestRe-C software. Mass spectra were recorded on
a MAT 8200 instrument with electron energy 70 eV. 3-Nitrobenzyl
alcohol was used as a matrix in fast atom bombardment mass
spectrometry (FAB-MS), while electrospray ionisation mass spectra
of the light petroleum and chloroform fractions were recorded on a
VG QUATTRO II mass spectrometer. Electrospray ionisation mass
spectrometry (ESI-MS) conditions were as following: acquisition
mode, ESI positive; mass scan range, 300–500 m/z; capillary,
3.50 kV; HV lens, 0.50 kV; cone, 45 V; skimmer offset, 5 V;
skimmer, 1.5 V; RF lens, 0.2 V; source temp. 82 °C; pressures for
analyser vacuum and gas cell are 1.8e-5 and 1.5e-4 mbar, respec-
tively. Drying and nebulising gas was nitrogen. 10 mg of light
petroleum and chloroform fractions were separately dissolved in
Stock solutions of the crude methanol extract and fractions
(light petroleum, chloroform and ethyl acetate) were prepared by
dissolving 10 mg in 1 mL methanol. Serial dilution was done from
each stock solutions and 500
individually by adding to 500
rylhydrazyl (DPPH). Standard solutions of 500
l
l
L from each dilution was tested
L of 0.2 mM 2,2-diphenyl-1-pic-
L were prepared
l
from stock solutions of 7 mg/mL of the pure reference compounds:
hesperidin, neohesperidin, naringin, 5-hydroxy-3,6,7,8,30,40-hexa-
methoxyflavone, 5-demethylnobelitin, nobelitin, and 6-demeth-
oxynobelitin. The mixtures were vigorously shaken and allowed
to stand in the dark for 30 min at room temperature. UV/VIS absor-
bance was measured (LKB BIOCHROM ULTROSPEC PLUS 4054) at
517 nm against blanks lacking DPPH (negative controls). The anti-
oxidant activity, expressed as IC50 (lg/mL), was compared with
standard antioxidants such as ascorbic acid (Ricci et al., 2005).
2.9. Lipoxygenase inhibition
MeOH (1 mL) and 100
luted with 500 L formic acid 2% (v/v) in acetonitrile–water (1:1, v/
v). 100 L from each diluted solution was qualitatively analysed by
lL from these solutions were taken and di-
l
Inhibition of soybean 5-lipoxygenase (5-LOX) by total methanol
extract and different fractions such as light petroleum, chloroform
and ethyl acetate (25 mg/mL absolute ethanol, stock solutions)
was determined spectrophotometrically under the following condi-
l
direct introduction into ESI positive ion mode.
2.7. LC–ESI/MS
tions: to 970
LOX (1 mg/mL) and 20
the tested samples (16–250
subsequently incubated at room temperature for 10 min. The enzy-
matic reaction was started by adding 25 L of 62.5 mM sodium lino-
l
L of phosphate buffer (pH 9.0), were added 10
L from each of the nine different dilutions of
g/mL from stock solutions), which was
lL of 5-
l
2.7.1. Flavonoids
l
The chromatographic separation of the ethyl acetate fraction
was carried out by HPLC using a reversed phase C-18 (RP C-18)
l
LiChro
CART
(Merck-Darmstadt)
column
[250 ꢀ 4 mm,
leate and monitored spectrophotometrically at 234 nm every 10 s
for 3 min. The initial reaction rates were determined from the slope
of the straight-line portion of the curve and inhibition of the enzy-
matic activity was calculated from triplicate experiments. Nord-
ihydroguaiaretic acid (NDGA) was used as a positive reference
5 < mu > m)]. The mobile phase consisted of solvent A; water–for-
mic acid (99.5: 0.5, v/v) and solvent B; acetonitrile. The elution was
arranged as follows: 0–60 min, gradient from 0–25% B; 60–
62.5 min, gradient from 25–50% B; 62.5–70 min, isocratic at 50%
B; 70–77 min, gradient from 50–100% B; and 77–87 min, isocratic
at 100% B. The column was equilibrated for 10 min prior to each
analysis. Flow rate 1 mL/min. MS analysis was performed under
the following conditions: acquisition mode, ESI negative; nebuliser
gas, N2, 0.25 l/min; capillary, 3.00 kV, HV lens –0.50 kV; cone, –
35 V; source temp., 120 °C; RF lens, –0.2 V; skimmer, –1.5 V; mass
scan range, 200–800 m/z; interscan time 0.1 s. MS data were ac-
quired and processed using MassLynx V4.0 software. Stock solution
of flavonoid glycoside components of the ethyl acetate fraction
compound (IC50 0.2 0.1
lg/mL) (Ashour et al., 2009).
2.10. Cytotoxicity
2.10.1. Cell culture
Caco-2, COS7 and HeLa cells were obtained from Prof. Dr. G.
Fricker, IPMB, Heidelberg and maintained in DMEM (glutamate I)
supplemented with 10% heat-inactivated foetal bovine serum
(FBS), 100 U/mL penicillin, and 100 U/mL streptomycin in addition
to 1 mM sodium pyruvate, and 10 mM non-essential amino acids
in case of Caco-2 cell lines. Cells were grown at 37 °C in a humid-
ified atmosphere of 5% CO2 at 37 °C.
(10 mg residue dissolved in 1 mL DMSO). 100
solution was diluted with 500
trile–water (1:1, v/v) and 100
l
L from the stock
L formic acid 2% (v/v) in acetoni-
L from the diluted solution was
l
l
qualitatively analysed by direct introduction to liquid chromatog-
raphy–electrospray ionisation mass spectrometry (LC–ESI/MS) in
negative ion mode. Available authentic reference compounds such
as hesperidin, neohesperidin, naringin, and rutin were dissolved in
DMSO and the stock solutions (10 mg/mL) were stored at 4 °C and
protected from daylight. Prior to injection in HPLC, stock solutions
were diluted with acetonitrile–water mixture (1:1, v/v).
2.10.2. MTT assay
Sensitivity of the cells to drugs was determined in triplicate
using the MTT cell viability assay (Marks, Belov, Davey, Davey, &
Kidman, 1992). Exponentially growing Caco-2 (2 ꢀ 104 cells/well),
COS7 and HeLa cell lines were seeded in a 96-well plate (Greiner
LabortechnikÒ) after trypsinisation of a subconfluent culture flask.
After incubation for 48 h the cells were incubated with fresh med-
ium containing various concentrations of compounds at 37 °C for
24 h. The medium was removed and cells were incubated with
fresh medium containing 0.5 mg/mL MTT from a 5 mg/mL stock
solution in PBS. During incubation for further 4 h, MTT is reduced
by mitochondrial dehydrogenases of viable cells to a purple
2.7.2. Limonoids
The chromatographic separation was carried out by HPLC using
RP C-18 LiChro CART (Merck-Darmstadt) column [250 ꢀ 4 mm
(5 < mu > m)]. The column was eluted at a flow rate of 1 mL/min.
The mobile phase consisted of solvent A: water–formic acid

D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
397
formazan product. The medium was discarded and formazan
crystal dissolved in 100 L DMSO. The plates were shaken for
15 min at room temperature, and the spectrophotometric
absorbance was monitored at 570 nm using TecanÒ ELISA plate
reader.
3.3. Compound 3
l
Yellow amorphous powder; FAB-MS [M+1]+ at m/z 373, EI/MS
(rel. int.): m/z 372 [M]+, 357 (M-15) (100), 314 (329–15) (10),
197 (8) and 83 (5). 1H NMR (300 MHz, CDCl3): d (ppm): 7.9 (1H,
d, J = 8.9 Hz, H-20), 7.0 (1H, d, J = 8.9 Hz, H-30), 7.0 (1H, d,
J = 8.9 Hz, H-50), 7.9 (1H, d, J = 8.9 Hz, H-60), 6.6 (1H, s, H-3), 4.1,
4.0, 3.9 ꢀ 2, 3.9 (5 s, OMe at C-5, -6, -7, -8, -40); 13C NMR
(75 MHz, CDCl3): d (ppm): 177.6 (s, C-4), 162.5 (s, C-2), 161.4 (s,
C-40), 151.6 (s, C-7), 148.6 (s, C-9), 147.9 (s, C-8), 144.3 (s, C-5),
138.3 (s, C-6), 127.9 (d, C-20), 127.9 (d, C-60), 124.0 (s, C-10), 115.1
(s, C-10), 114.7 (d, C-30), 114.7 (d, C-50), 106.9 (d, C-3, 62.5, 62.2,
62.0, 61.9, 55.7 (5 q, OMe at C-5, -6, -7, -8, -40). Compound 3 was
identified as 5,6,7,8,40-pentamethoxyflavone (tangeretin) from
these spectral data and physical properties (El-Shafae, 2002;
Weber et al., 2006).
2.11. Statistical analysis
All experiments were repeated at least three times. Results are
reported as means SE. The IC50 was determined as the drug con-
centration which resulted in a 50% reduction in cell viability or
inhibition of the biological activity. IC50 values were calculated
using a four parameter logistic curve (SigmaPlotÒ 11.0).
3. Results and discussion
3.4. Compound 4
The alcoholic extract of C. jambhiri was fractionated using dif-
ferent solvents (e.g. light petroleum, chloroform, and ethyl acetate)
and each fraction was chromatographed on silica gel column. Col-
umn fractions were examined by TLC and fractions having the
same Rf were pooled and purified either by crystallisation or by
preparative TLC. Fractionation of the extracts yielded well-known
substances, most of which, however, had not yet been described
as constituents of C. jambhiri. The chemical structures of the iso-
lated compounds were identified as follow by mass spectrometry
(MS) as well as nuclear magnetic resonance (1D- and 2D-NMR)
including attached proton test (APT), H-correlation spectroscopy
(H-COSY), heteronuclear multiple quantum coherence (HSQC), het-
eronuclear multiple bond coherence (HMBC), and nuclear overha-
user effect spectroscopy (NOESY) (Fig. 1).
Yellowish-white needle-shaped crystals; FAB-MS [M+1]+ at m/z
403, EI/MS (rel. int.): m/z 402 [M]+, 387 (100), 372 (4), 357 (10),
314 (1), 197 (8) 165 (8), 162 (3), 147 (8) and 83 (0.3). 1H NMR
(300 MHz, CDCl3): d (ppm): 7.4 (1H, d, J = 2.1 Hz, H-20), 6.9 (1H,
d, J = 8.5 Hz, H-50) 7.6 (1H, dd, J = 2.1, 8.5 Hz, H-60) 6.6 (1H, s, H-
3), 4.1, 4.06, 3.9, 3.93, 3.92 ꢀ 2 (6 s, OMe at C-5, -6, -7, -8, -30, -
40); 13C NMR (75 MHz, CDCl3): d (ppm): 177.3 (s, C-4), 160.9 (s,
C-2), 151.8 (s, C-40), 151.3 (s, C-7), 149.1 (s, C-30), 148.3 (s, C-9),
147.6 (s, C-8), 143.9 (s, C-5), 137.9 (s, C-6), 123.9 (s, C-10), 119.5
(d, C-60), 114.7 (s, C-10), 111.1 (d, C-50), 108.4 (d, C-20), 106.8 (d,
C-3), 62.2, 61.9, 61.8, 61.6, 55.9, 55.8 (6 q, OMe at C-5, -6, -7, -8,
-30, -40). Compound 4 was identified as 5,6,7,8,30,40-hexamethoxyf-
lavone (nobiletin) from these spectral data and physical properties
(El-Shafae, 2002; Weber et al., 2006).
3.1. Compound 1
3.5. Compound 5
White amorphous powder; EI/MS (rel. int.): m/z 414 [M1, b-
sitosterol]+ (100), 412 [M2, stigmasterol]+ (22), 396 [M1-H2O]+
(26), 381 (13), 255 (17), 213 (12). 1H NMR (500 MHz, CDCl3): d
(ppm): 3.5 (1H, tt, J = 4.5, 11.1 Hz, H-3), 5.4 (1H, br. s, H-6), 0.7
(3H, s, H-18), 1.0 (3H, s, H-19), 0.9 (3H, d, J = 6.6 Hz, H-21), 5.2
(1H, dd, J = 8.6, 15.2 Hz, H-22), 5.0 (1H, dd, J = 8.7, 15.2 Hz, H-23),
0.8 (3H, d, J = 6.8 Hz, H-26), 0.8 (3H, d, J = 7.4 Hz, H-27), 0.8 (3H, t,
J = 7.6 Hz, H-29); 13C NMR (125 MHz, CDCl3): d (ppm): 37.2 (C-1),
31.7 (C-2), 71.8 (C-3), 42.3 (C-4), 140.7 (C-5), 121.7 (C-6), 31.9 (C-
7), 31.9 (C-8), 50.2 (C-9), 36.5 (C-10), 21.1 (C-11), 39.8 (C-12),
42.3 (C-13), 56.8 (C-14), 24.3 (C-15), 28.2 (C-16), 56.1 (C-17), 11.9
(C-18), 19.4 (C-19), 36.1 (C-20), 18.8 (C-21), 33.9, 138.2 (C-22),
26.1, 129.3 (C-23), 45.8, 51.2 (C-24), 29.2, 31.7 (C-25), 19.0 (C-26),
19.8 (C-27), 23.1, 24.3 (C-28), 11.9, 12.2 (C-29). Compound 1 was
identified as a mixture of b-sitosterol and stigmasterol from these
spectral data and physical properties (Goad & Akishisa, 1997).
Dark yellow needle-shaped crystals; FAB-MS [M+1]+ at m/z 433,
EI/MS (rel. int.): m/z 432 [M]+, 417 (100), 403 (6), 389 (4), 373 (6),
358 (5), 343 (3) 197 (10) and 165 (11). 1H NMR (300 MHz, CDCl3): d
(ppm): 6.9 (1H, d, J = 2 Hz, H-20), 6.8 (1H, d, J = 8.5 Hz, H-50), 7.8
(1H, dd, J = 2, 8.5 Hz, H-60), 4.0 ꢀ 2, 3.9 ꢀ 3, 3.8 ꢀ 2 (7 s, OMe at
C-3, -5, -6, -7, -8, -30, -40); 13C NMR (75 MHz, CDCl3): d (ppm):
174.0 (s, C-4), 153.3 (s, C-2), 151.5 (s, C-7), 151.2 (s, C-30), 148.9
(s, C-40), 148.3 (s, C-5), 146.0 (s, C-9), 144.9 (s, C-6), 140.9 (s, C-
3), 138.0 (s, C-8), 123.6 (s, C-10), 122.1 (d, C-60), 115.3 (s, C-10),
111.2 (d, C-50), 111.1 (d, C-20), 62.4, 62.1, 62.0, 61.8, 60.0, 56.2,
56.1 (7 q, OMe at C-3, -5, -6, -7, -8, -30, -40). Compound 5 was iden-
tified as 5,6,7,8,30,40-heptamethoxyflavone from these spectral data
and physical properties (Khatoon, 1995).
3.6. Compound 6
Dark yellow needle-shaped crystals which crystallised (CHCl3/
+
3.2. Compound 2
MeOH); FAB-MS [M+1]+ at m/z 419, EI/MS (rel. int.): m/z 418 [M] ,
403 (100), 373 (6), 359 (1), 209 (5) and 165 (3). 1H NMR
(300 MHz, CDCl3): d (ppm): 7.7 (1H, d, J = 2.2 Hz, H-20), 7.0 (1H, d,
J = 8.7 Hz, H-50) 7.9 (1H, dd, J = 2.2, 8.7 Hz, H-60), 12.4 (OH, s, H-5),
4.1 ꢀ 2, 3.9, 3.94, 3.9, 3,8 (6 s, OMe at C-3,-6,-7,-8,-30,-40); 13C NMR
(75 MHz, CDCl3): d (ppm): 179.2 (s, C-4), 155.8 (s, C-2), 152.9 (s,
C-7), 151.5 (s, C-30), 149.1 (s, C-40), 148.7 (s, C-5), 144.8 (s, C-9),
138.7 (s, C-3), 136.1 (s, C-6), 132.7 (s, C-8), 122.9 (s, C-10), 122.3
(d, C-60), 110.9 (d, C-50), 110.9 (d, C-20), 107.4 (s, C-10), 62.0, 61.7,
61.1, 60.1, 55.9 and 55.8 (6 q, OMe at C-3, -6, -7, -8, -30, -40). Com-
pound 6 was identified as 5-hydroxy-3,6,7,8,30,40-hexamethoxyflav-
one from these spectral data and physical properties (Li, Lo, & Ho,
2006).
White needle-shaped crystals; HR-ESI/MS [M+1]+ at m/z 257, EI/
MS (Rel. Int.): m/z 256 [M]+, 213 [M-43]+ (20), 199 (5), 185 (12),
169 (15), 157 (11), 143 (10), 129 (34), 115 (12), 97 (24), 73 (77),
55 (72), 43 (100) and 29 (30). 1H NMR (300 MHz, CDCl3): d
(ppm): 7.2 (OH), 2.4 (2H, m, H-2), 1.6 (2H, m, H-3), 1.3 (2H, m,
H-4), 1.3 (16H, m, H-5: 12), 1.3 (2H, m, H-13), 1.3 (2H, m, H-14),
1.3 (2H, m, H-15), 0.9 (3H, m, H-16); 13C NMR (75 MHz, CDCl3): d
(ppm): 179.7 (C-1, COOH), 34.2 (C-2), 32.2 (C-14), 29.3: 29.9
(C-4:13) 24.9 (C-3), 22.9 (C-15), 14.3 (C-16). Compound 2 was
identified as palmitic acid from co-TLC with authentic material
and spectral data (Liu et al., 2003).

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D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
Fig. 1. Isolated and identified compounds from C. jambhiri peel and seeds: 1–6, 9, 11, 12, 14, 18, 19 were isolated from the peel by column chromatography; 2–9, 18 detected
in ESI/MS chromatogram (positive ion mode); flavonoid glycosides 10–17 were identified by LC–ESI/MS (negative ion mode); limonoid aglycones 18–24 detected and
preliminarily identified by 2/MS (positive ion mode).
3.7. Compound 7
1H NMR (300 MHz, CDCl3): d (ppm): 7.4 (1H, d, J = 2.2 Hz, H-20), 6.9
(1H, d, J = 8.7 Hz, H-50), 7.6 (1H, dd, J = 2.2, 8.7 Hz, H-60), 6.6 (1H, s,
H-3), 12.5 (OH, s, H-5), 4.1, 3.95, 3.94, 3.9, 3,8, (5 s, OMe at C-6, -7, -
8, -30, -40); 13C NMR (75 MHz, CDCl3): d (ppm): 182.9 (s, C-4), 163.9
Yellow granules; FAB-MS [M+1]+ at m/z 389, EI/MS (rel. int.): m/z
388 [M]+, 373 (100), 343 (5), 327 (6), 211 (14), 183 (15) and 163 (7).

D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
399
(s, C-2), 152.9 (s, C-7), 152.4 (s, C-40), 149.5 (s, C-30), 149.3 (s, C-9),
3.11. Compound 11
145.8 (s, C-5), 136.5 (s, C-6), 132.9 (s, C-8), 123.7 (s, C-10), 120.1 (d,
C-60), 111.7 (d, C-50), 108.8 (d, C-20), 106.9 (s, C-10), 103.9 (d, C-3),
62.0, 61.7, 61.1, 56.1, 55.9 (5 q, OMe at C-6, -7, -8, -30, -40). Com-
pound 7 was identified as 5-O-demethylnobiletin from these spec-
tral data and physical properties (El-Shafae, 2002).
Yellow granules; FAB-MS [M+1]+ at m/z 375, EI/MS (rel. int.):
m/z 374 [M]+, 359 (100), 344 (9), 211 (10), 183 (9), 179 (6), 149
(5), 105 (2). 1H NMR (300 MHz, CDCl3): d (ppm): 12.5 (1H, s, OH),
7.6 (1H, dd, J = 2.1, 8.4 Hz, H-60), 7.4 (1H, d, J = 2.1 Hz, H-20), 7.1
(1H, d, J = 8.4 Hz, H-50), 6.5 (1H, s, H-3), 4.1, 4.0, 3.96, 3.9 (4 s,
OMe at C-6, -7, -8, -30); 13C NMR (75 MHz, CDCl3): d (ppm);
182.9 (s, C-4), 163.9 (s, C-2), 152.9 (s, C-7), 149.5 (s, C-5), 149.4
(s, C-30), 147.9 (s, C-40), 145.7 (s, C-9), 136.5 (s, C-6), 132.9 (s,
C-8), 123.5 (s, C-10), 120.8 (d, C-60), 115.1 (d, C-50), 108.3 (d, C-20),
106.9 (s, C-10), 103.8 (d, C-3), 62.1, 61.7, 61.1, 56.0 (4 q, OMe at
C-6, -7, -8, -30). Compound 11 was identified as 5,40-dihydroxy-
6,7,8,30-tetramethoxyflavone from these spectral data and physical
properties (Li et al., 2006; Weber et al., 2006).
3.8. Compound 8
Yellowish-white needle crystals; FAB-MS [M+1]+ at m/z 373, EI/
MS (rel. int.): m/z 372.1 [M]+, 357 (86), 343 (13), 328 (18), 195 (6),
167 (18), 139 (10) and 91 (3). 1H NMR (300 MHz, CDCl3): d (ppm):
7.6 (1H, dd, J = 2.1, 8.5 Hz, H-60), 7.4 (1H, d, J = 2.1 Hz, H-20), 6.9 (1H,
d, J = 8.5 Hz, H-50), 6.6 (1H, s, H-3), 6.4 (1H, s, H- 6), 3.99, 3.97, 3.95,
3.94 ꢀ 2 (5 s, OMe at C-5, -7, -8, -30, -40); 13C NMR (75 MHz, CDCl3):
d (ppm): 177.8 (s, C-4), 160.5 (s, C-2), 157.8 (s, C-5), 156.6 (s, C-7),
153.1 (s, C-9), 152.9 (s, C-30), 150.1 (s, C-40), 130.4 (s, C-8), 124.8 (s,
C-10), 119.5 (d, C-60), 111.1 (d, C-50), 109.1 (s, C-10), 108.6 (d, C-20),
107.1 (d, C-3), 92.2 (d, C-6), 61.5, 56.5, 56.3, 56.0, 55.9 (5 q, OMe at
C-5, -7, -8, -30, -40). Compound 8 was identified as 6-demethoxyn-
obiletin from these spectral data and physical properties (Weber
et al., 2006).
3.12. Compound 12
Buff granules; EI/MS (rel. int.): m/z 272 [M-308]+ (96), 229 (5),
191 (10), 153 (100), 147 (23), 129 (20), 107 (18) and 91 (26). 1H
NMR (300 MHz, DMSO-d6): d (ppm): 12 (1H, s, hydrogen bonded
5-OH), 9.6 (1H, s, OH). 7.3 (2H, d, J = 8.4 Hz, H-20, 60), 6.8 (2H, d,
J = 8.4 Hz, H-30, 50), 6.3 (1H, d, J = 2.1 Hz, H-8), 6.2 (1H, d, J =
2.1 Hz, H-6), 5.5 (1H, centred t, J = 5.4 Hz, H-2), 5.3 (1H, d,
J = 8.2 Hz, glucosyl, H-100), 5.2 (1H, d, J = 2.2 Hz, rhamnosyl, H-
1000), 3.8 (2H, m, H-600), 3.2 (1H, m, H- 3b), 2.8 (1H, m, H- 3a),
3.2–3.7 (7H, m, rhamnoglucosyl protons) and 1.2 (3H, d,
3.9. Compound 9
White needle-shaped crystals; FAB-MS [M+1]+ at m/z 515, EI/
MS (rel. int.): m/z 514 [M]+, 333 (18), 289 (5), 255 (14), 213 (11),
201 (10), 161 (8), 135 (12), 95 (26) and 43 (53). 1H NMR
J = 6.6 Hz rhamnosyl CH3); 13C NMR (75 MHz, DMSO-d6):
d
(300 MHz, CDCl3):
d
(ppm): 5, (1H, m, H-1), 3.2 (1H, d,
(ppm); 196.7 (C-4), 165.2 (C-7), 162.3 (C-5), 162.2 (C-9), 157.6
(C-40), 128.6 (C-10), 128.1 (C-20), 128.1 (C-60), 115.1 (C-30), 115.1
(C-50), 103.5 (C-10), 100.3 (C-1000), 97.1 (C-100), 96.2 (C-6), 95.0 (C-
8), 78.3 (C-2), 78.2 (C-200), 76.4 (C-300), 76.4 (C-500), 76.0 (C-4000),
72.0 (C-400), 70.2 (C-2000), 68.9 (C-3000), 68 (C-5000), 60.1 (C-600), 42.1
(C-3), 18 (C-Me). Compound 12 was identified as naringin from
these spectral data and physical properties (El-Shafae, 2002;
Maltese, Erkelens, Kooy, Choi, & Verpoorte, 2007).
J = 15.6 Hz, H-2b), 3.1 (1H, dd J = 7.3 and 15.6 Hz, H-2a), 2.8 (1H,
t, J = 15.1 Hz, H-5), 2.6 (2H, m, H-6), 2.5 (1H, dd J = 2.7 and
10.3 Hz, H-9), 1.6 (2H, m, H-11), 1.8 (1H, m, H- 12b), 1.1 (1H, m,
H-12a), 3.8 (1H, m, H-15), 5.4 (1H, s, H-17), 6.3 (1H, s, H-21), 7.4
(1H. br. S, H-22), 7.3 (1H. br. S, H-23), 1.2 (3H, s, 18-Me), 1.3 (3H,
s, 19-Me), 1.1 (3H, s, 24-Me), 1.5 (3H, s, 25a-Me), 1.5 (3H, s, 25b-
Me), 2.0(3H, s, CH3-CO); 13C NMR (75 MHz, CDCl3): d (ppm):
206.9 (C-7), 169.2 (C-3), 169.1 (C-Ac), 166.7 (C-16), 143.2 (C-21),
140.9 (C-23), 120.1 (C-20), 109.1 (C-22), 84.3 (C-4), 77.9 (C-17),
70.7 (C-1), 65.4 (C-14), 53.4 (C-15), 52.9 (C-8), 50.9 (C-5), 44.3
(C-9), 44.2 (C-10), 38.8 (C-6), 37.5 (C-13), 35.3 (C-2), 33.4
(C-25a), 32.4 (C-25b), 32.3 (C-12), 20.8 (C-24), 20.7 (CH3-C@O),
17.2 (C-18), 17.1 (C-19), 16.5 (C-11). Compound 9 was identified
as nomilin from these spectral data and physical properties (Khalil,
Maatooq, & El Sayed, 2003; Nakagawa, Duan, & Takaishi, 2001).
3.13. Compound 13
White granules; FAB-MS [M+1]+ at m/z 611, EI/MS (rel. int.):
302 [M-308]+ (100), 286 (5), 271 (3), 259 (8), 179 (21), 165 (5),
137 (95), 135 (47), 129 (12) and 107 (17). 1H NMR (300 MHz,
DMSO-d6): d (ppm): 12 (1H, s, hydrogen bonded 5-OH), 9.1 (1, H,
s, OH), 6.9 (3H, m, H-20, 50, 60), 6.2 (1H, d, J = 2 Hz, H-8), 6.1 (1H,
d, J = 2 Hz, H-6), 5.5 (1H, dd, J = 7.6, 3.1 Hz, H-2), 4.9 (1H, d,
J = 7.4 Hz, glucosyl H-100), 4.5 (1H, d, J = 2.9 Hz, rhamnosyl, H-1000),
3.8 (3H, s, OMe- 40), 3.7 (2H, m, H-600), 3.2–3.6 (7H, m, rhamnog-
lucosyl protons), 3.2 (1H, m, H- 3b) 2.9 (1H, m, H- 3a) and 1.14
(3H, m, rhamnosyl CH3); 13C NMR (75 MHz, DMSO-d6): d (ppm);
197.7 (C-4), 165.8 (C-7), 163.7 (C-5), 163.1 (C-9), 148.6 (C-40),
147.1 (C-30), 131.6 (C-10), 118.5 (C-60), 114.8 (C-20), 112.7 (C-50),
103.9 (C-10), 101.3 (C-1000), 100.1 (C-100), 97.0 (C-6), 96.2 (C-8),
79.1 (C-2), 76.9 (C-500), 76.1 (C-300), 76.1 (C-5000), 73.6 (C-2000), 72.7
(C-4000), 71.4 (C-200), 70.9 (C-3000), 68.9 (C-400), 66.7 (C-600), 56.1
(C-40-OCH3), 42.7 (C-3), 18.5 (C-CH3). Compound 13 was identified
as hesperidin, from these spectral data and physical properties
(Maltese et al., 2007) .
3.10. Compound 10
White amorphous powder; CI+/MS [M+1]+ at m/z 471, EI/MS (rel.
int.): m/z 470 [M]+, 329 (5), 287 (3), 241 (2), 201 (3), 187 (4), 147 (5),
136 (7), 135 (17), 108 (13) and 95 (20). 1H NMR (300 MHz, CDCl3): d
(ppm): 4.1 (1H, br s, H-1), 2.2 (1H, dd, J = 3.2, 14.8 Hz, H-2a), 2.55
(1H, dd, J = 3.2, 14.8 Hz, H-2b), 2.4 (1H, dd, J = 2.8, 15 Hz, H-5), 2.9
(1H, dd, J = 2.8, 15 Hz, H-6a), 3.4 (1H, t, J = 15 Hz, H-6b), 2.6 (1H,
dd, J = 2, 10 Hz, H-9), 1.8 (1H, m, H-11a), 1.5 (1H, m, H-11b), 1.4
(1H, m, H-12b), 1.8 (1H, m, H-12a), 4.1 (1H, br s, H-15), 5.4 (1H, s,
H-17), 6.3 (1H, br. s, H-21), 7.4 (1H. br s, H-22), 7.2 (1H. s, H-23),
1.0 (3H, s, 18-Me), 4.8 (1H, d, J = 13 Hz, H-19b), 4.4 (1H, d,
J = 13 Hz, H-19a), 1.2 (3H, s, 24-Me), 1.2 (3H, s, 25a-Me), 1.2 (3H,
s, 25b-Me); 13C NMR (75 MHz, CDCl3): d (ppm); 208.3 (C-7), 172.7
(C-3), 168.1 (C-16), 143.2 (C-21), 141.1 (C-23), 120.4 (C-20), 109.9
(C-22), 85.3 (C-1), 85.3 (C-4), 78.6 (C-17), 69.0 (C-19), 66.1 (C-14),
53.6 (C-5), 52.8 (C-15), 50.0 (C-8), 44.8 (C-9), 43.9 (C-10), 39.2
(C-13), 37.7 (C-2), 37.7 (C-6), 32.9 (C-25a), 31.8 (C-12), 23.8
(C-25b), 20.4 (C-11), 17.6 (C-18), 17.1 (C-24). Compound 10 was
identified as limonin from these spectral data and physical proper-
ties (Nakagawa et al., 2001).
3.14. Compound 14
White granules; FAB-MS [M+1]+ at m/z 611, EI/MS (rel. int.):
302 [M-308]+ (28), 272 (1), 259 (3), 231 (2), 217 (2), 191 (9), 173
(11), 153 (58), 147 (21), 137 (100), 129 (18), 124 (20) and 107
(15). 1H NMR (300 MHz, DMSO-d6): d (ppm): 12 (1H, s, hydrogen
bonded, 5-OH), 9.1 (1H, s, OH), 6.95 (1H, d, J = 2.1 Hz, H-20), 6.92
(1H, dd, J = 2.2, 8.7 Hz, H-60), 6.9 (1H, d, J = 8.7 Hz), 6.2 (1H, d,

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D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
J = 2, H-8), 6.1 (1H, d, J = 2, H-6), 5.5 (1H, dd, J = 12.6, 3.2 Hz, H-2),
5.1 (1H, d, J = 7.4 Hz, glucosyl, H-100), 5.1 (1H, d, J = 2.8 Hz, rhamno-
syl, H-1000), 3.8 (3H, s, OMe- 40), 3.6 (2H, m, H-600) 3.2–3.6 (8H, m,
rhamnoglucosyl protons), 3.1 (1H, m, H- 3b), 2.8 (1H, m, H-3a)
and 1.2 (1H, d, J = 6 Hz rhamnosyl CH3); 13C NMR (75 MHz,
DMSO-d6): d (ppm); 197.2 (C-4), 164.8 (C-7), 162.9 (C-5), 162.7
(C-9), 147.9 (C-40), 146.5 (C-30), 130.8 (C-10), 117.9 (C-60), 115.2
(C-20), 114.2 (C-50), 100.4 (C-10), 101.3 (C-1000), 97.4 (C-100), 96.3
(C-6), 95.2 (C-8), 78.7 (C-2), 78.4 (C-500), 77.6 (C-2000), 76.9 (C-4000),
76.2 (C-300), 76.1 (C-5000), 70.4 (C-200), 69.6 (C-3000), 68.3 (C-400),
60.4 (C-600), 55.7 (C-40-OCH3), 42.2 (C-3), 18.0 (C-CH3). Compound
14 was identified as neohesperidin from these spectral data and
physical properties (Maltese et al., 2007) .
LC–ESI/MS (negative ion mode) of the ethyl acetate fraction. It has
been previously reported that rutinosides tend to fragment easier
than the neohesperidoside, and a possible explanation is that the
free hydroxyl group can be involved in breaking the b-bond be-
tween the aglycone and the sugar (Cappiello et al., 1999; Cuyckens,
Ma, Pocsfalvi, & Cleays, 2000). Another trend is that rutinoside iso-
mers elute before neohesperidoside (Cuyckens et al., 2000). Our re-
sults are in agreement with previous reports. A total of eight
flavonoid glycosides, i.e. eriocitrin, neoeriocitrin, rutin, diosmetin
6-C-glucoside, narirutin, naringin, hesperidin and neohesperidin
were identified. Identification of these compounds was based on
comparison with authentic standards, relative retention times and
literature mass data (Cappiello et al., 1999; Cuyckens et al., 2000).
The MS spectra of all rutinosides (i.e. eriocitrin, rutin, narirutin
and hesperidin) showed the addition of a pseudomolecular ion
and a signal corresponding to the aglycone ion, while the neohe-
speridosides (neoeriocitrin, naringin, neohesperidin) show only a
pseudomolecular ion. In the full mass scan analysis of the ethyl ace-
tate fraction, a signal at m/z 595 was found as [M-H]ꢁ. MS-MS pro-
A mixture of sitosterol and stigmasterol (1) and palmatic acid
(2) were isolated and identified from the petroleum ether fraction
of the peel.
A total of seven polymethoxyflavones were isolated and identi-
fied from the petroleum ether and chloroform fractions of the peel
including: tangeretin (3), nobiletin (4), 3,5,6,7,8,30,40-heptameth-
oxyflavone (5), 5-hydroxy-3,6,7,8,30,40-hexamethoxyflavone (6),
5-O-demethylnobeletin (7), 6-demethoxynobiletin (8), and 5,40-
dihydroxy-6,7,8,30-tetramethoxyflavone (11). From this class of
compounds, only tangeretin had previously been reported from
this plant (Chaliha et al., 1965).
Nomilin (9) and limonin (10) were isolated from the chloroform
extract of the peel in a considerable yield. Limonin was the most
abundant natural product. This is the first report of the isolation
of limonoids from this plant, although they are common in others
Citrus species.
Three flavanone glycosides were isolated from the ethyl acetate
fraction including naringin (12), hesperidin (13) and neohesperidin
(14). Hesperidin was the most abundant and naringin was the
least abundant flavanone in the peel. Hesperidin and neohesperi-
din had previously been identified by HPLC in the juice of this
species (Arriaga & Rumbero, 1990).
vided
a
fragment ion at m/z 287 [M-H-rhamnose-glucose]ꢁ
corresponding to the aglycone of eriocitrin in negative ion mode
which had not previously been identified. ESI-MS ion chromato-
gram analysis showed a deprotonated molecule in the negative
mode at m/z 579 and fragment ion by MS-MS at m/z 271. The molec-
ular ion and aglycone correspond to narirutin which is here reported
for the first time in this species. ESI/MS showed two similar depro-
tonated molecules at m/z 609 at different retention times. MS
revealed an aglycone ion at m/z 301 and the (1000?600) interglycosidic
linkage was detected. The one appears to be hesperidin and the
other neohesperidin. Another deprotonated molecule at m/z 609
with a retention time of around 39 min is related to rutin.
LC–ESI/MS analysis of the chloroform extract of the defatted
seeds resulted in the identification of seven limonoid aglycones:
limonin, nomilin, calamine, obacunone acetate, methyl isooba-
cunoate, methyl isoobacunoate diosphenol and isolimonexic acid
methyl ether. Identification of limonin and nomilin was based on
the comparison of the retention times with authentic standards,
chromatographic and reported MS data (Manners & Hasegawa,
1999). High resolution mass spectrometry (HRMS) data (EI mode)
from limonin and nomilin have been reported (Tian & Schwartz,
2003). These data establish that all of these limonoids display a
fragment at m/z 95 that is associated with the formation of a fur-
anal radical from the limonoid furan moiety and oxygen at C-17.
A further fragment ion [M-123]+ arises from the loss of carbon
monoxide at C-16 (28 mass units) from the [M-95]+ fragment
ion. The detection and identification of other limonoid aglycones
in the chromatogram are based on comparison with literature data
(Manners & Hasegawa, 1999).
3.15. ESI/MS
Prior to the isolation of the compounds by column chromatog-
raphy, MS fingerprints of the light petroleum and chloroform frac-
tions were performed using ESI/MS. Fig. 2a reveals five major peaks
in the light petroleum fraction with the following molecular
masses [M+H]+ at m/z 373.1, 389.1, 403.1, 419.1, and 433.3
which are attributed to the following compounds: tangeretin, 5-
O-demethylnobiletin, nobiletin, 5-hydroxy-3,6,7,8,30,40-hexameth-
oxyflavone and 3,5,6,7,8,30,40-heptamethoxyflavone, respectively.
In case of the chloroform fraction, the ESI/MS spectral profile
(Fig. 2b) shows many compounds with the following molecular
masses [M+H]+: 343.1, 359.1, 373.1, 375.1, 389.2, 403.1, 419.2,
433.3, and 471.2. These molecular ions are apparently related to
the following compounds: 6-demethoxytangeretin, 5-hydroxy-
6,7,8,40-tetramethoxyflavone, 6-demethoxynobiletin, 5,40-dihy-
droxy 6,7, 8,30-tetramethoxyflavone, 5-O-demethylnobiletin,
nobiletin, 5-hydroxy-3,6,7,8,30,40-hexamethoxyflavone, 3,5,6,7,8,
30,40-heptamethoxyflavone and limonin, respectively. All of the de-
tected compounds were confirmed later by direct analysis of the
isolated compounds from the column by ESI/MS and by matching
each ion peak in the total ion chromatogram with those of individ-
ual secondary metabolites.
3.17. Antioxidant activity
The DPPH assay is based on measuring the scavenging ability of
antioxidants towards the stable radical 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH). The free radical DPPH⁄ is reduced to the corre-
sponding hydrazine when it reacts with hydrogen donors. The
DPPH assay is considered a valid and easy assay to evaluate the
antioxidant activity of natural products. The IC50 values of the pure
isolated compounds are given in Table 1. Some of the tested com-
pound (Naringin, 7 mg/mL) was not completely soluble in MeOH
and therefore a binary solvent of MeOH and DMSO (1:1, v/v)
(MD) was used instead. In order to compare the activities of
MeOH-soluble and MeOH-insoluble compounds, a relation coeffi-
cient of rutin (1.3) was calculated by determining the IC50 values
of rutin in MeOH (2.8) and in MD (2.2). Table 1 lists data displaying
the ability of the chloroform fraction to scavenge DPPH⁄, with a
stronger scavenging effect than the other fractions. Of the tested
3.16. LC–ESI/MS
Liquid chromatography (LC) combined with electrospray ionisa-
tion mass spectrometry (ESI/MS) proves to be very powerful for fla-
vonoid characterisation (Careri, Elviri, & Mangia, 1999). Fig. 3
shows the total ion current chromatogram (TIC) obtained by

D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
401
Fig. 2. ESI/MS profiles from C. jambhiri (A) petroleum ether fraction: tangeretin (1), 5-O-demethylnobiletin (2), nobiletin (3), 5-hydroxy-3,6,7⁄,8,30,40-hexamethoxyflavone (4),
3,5,6,7,8,30,40-heptamethoxyflavone (5); and (B) chloroform fraction: 6-demethoxytangeretin⁄ (1), 5-hydroxy-6,7,8,40-tetramethoxyflavone (2), 6-demethoxynobiletin (3),
5,40-dihydroxy-6,7,8,30-tetramethoxyflavone (4), 5-O-demethylnobiletin (5), nobiletin (6), 5-hydroxy-3,6,7,8,30,40-hexamethoxyflavone (7), 3,5,6,7,8,30,40-heptamethoxyflav-
one (8), and limonin (9); ⁄tentatively identified by ESI/MS.
compounds, rutin and quercetin exerted the strongest activity and
these compounds were used as positive controls. Neohesperidin is
more active than hesperidin, while naringin has only minor
activity. Total polymethoxyflavones from the petroleum ether frac-
tion (5-hydroxy-3,6,7,8,30,40-hexamethoxyflavone, 5-O-demethyl-
nobelitin, nobelitin and 6-demethoxynobelitin) together exerted

402
D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
Fig. 3. LC–ESI/MS (negative ion mode) of the ethyl acetate fraction from C. jambhiri. Eriocitrin (1), neoeriocitrin (2), rutin (3), diosmetin 6-C-glucoside (4), narirutin
(5), naringin (6), hesperidin (7), and neohesperidin (8).
Bermejo, & Villar, 1995). Table 2 summarises the results obtained
from total extracts and different fractions and IC50 values for
5-LOX: the activity of the studied species of 5-LOX inhibitors
decreased from petroleum ether, followed by chloroform, ethyl
acetate and total extract of the peel as shown.
Table 1
DPPH⁄ scavenging activity of fractions and isolated compounds from C. jambhiri.
Sample
IC50 (lg/mL)
Fractions
Total extract
Petroleum ether fraction
Chloroform fraction
Ethyl acetate fraction
223.5 0.5
166.6 1.1
119.4 0.8
171.3 1.3
3.19. Cytotoxicity
Isolated compounds
Hesperidin
Neohesperidin
5-Hydroxy-3,6,7,8,30,40-hexamethoxyflavone
5-Demethylnobelitin
Nobiletin
Table 3 summarises data on the IC50 of the tested samples
against Caco-2, HeLa and COS7 cell lines. All tested samples show
high IC50 values in the chemotherapy resistant Caco-2 cell line in
comparison to the sensitive HeLa and COS7, which have a low
endogenous ABCG2 and P-gp expression. The Caco-2 cell line
highly expresses multidrug resistance ABC-transporter genes
(El-Readi, Hamdan, Farrag, El-Shazly, & Wink, 2010). In addition,
the polymethoxyflavones are most cytotoxic against COS7, while
5-O-demethylnobiletin shows a stronger cytotoxic effect in COS7
and Caco-2 cell lines than nobiletin. This may be due to the free
phenolic hydroxyl group in 5-O-demethylnobiletin which forms io-
nic bonds with charged amino acid residues. These ionic bonds are
stable and reduce the structural flexibility of proteins. Polyphenols
interfere in an unselective manner with proteins in that the pheno-
lic hydroxyl groups can partly dissociate under physiological con-
ditions resulting in phenolates. These form stable ionic bonds
with the positively charged side chains of basic amino acids. All
polyphenols can also form hydrogen bonds with electronegative
atoms of proteins. One single non-covalent bond is relatively weak,
but if several bonds are formed, a change in protein conformation
is likely to occur and can lead to changes in protein activity (Wink,
2008). In case of the HeLa cell line, nobiletin shows a stronger cyto-
toxic effect than 5-O-demethylnobiletin. As hesperidin and neo-
hesperidin are isomers, it is not surprising that the IC50 values
are very similar in case of COS7, while in case of the HeLa cells hes-
peridin is more cytotoxic than neohesperidin and the reverse being
361.5 0.4
178.9 0.4
735.6 0.4
752.3 0.2
3557.1 0.5
1284.2 0.5
6-Demethoxynobelitin
Positive control
Ascorbic acid
Rutin
16.3 0.2
2.2 0.3
3.7 0.3
Quercetin
Data are means SD from three independent experiments (n = 3).
a lower IC50 than the individual compounds, thus pointing to a syn-
ergistic effect (Table 1).
3.18. Lipoxygenase inhibition
The inhibition of 5-lipoxygenase (5-LOX) is currently a subject
of intense research targeted towards the discovery of novel anti-
allergic and anti-inflammatory agents. The prime objective of this
study was therefore to evaluate the ability of total extracts and dif-
ferent fractions to inhibit 5-LOX in vitro. Previous studies have
shown that nordihydroguaiaretic acid (NDGA) could be used as a
reference compound for studies of 5-LOX inhibition, due to its
widely reported strong inhibitory activity on this enzyme (Abad,

D. Hamdan et al. / Food Chemistry 127 (2011) 394–403
403
Table 2
Cappiello, A., Famiglini, G., Mangani, F., Careri, M., Lombardi, P., & Mucchino, C.
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Inhibition of 5-lipoxygenase by fractions of C. jambhiri (means SD) in %.
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fraction
CHCl3 fraction
Ethyl acetate
fraction
Chromatography A, 855(2), 515–527.
(l
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ionspray mass spectrometry method for the analysis of flavanones, flavones and
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Chaliha, B. P., Sastry, G. P., & Rao, P. R. (1965). Chemical examination of peel of Citrus
jambhiri Lush: Isolation of a new flavone. Tetrahedron, 21, 1441–1443.
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16
4.3 0.1
4.9 0.1
7.2 0.1
20.4 0.5
44.9 0.4
63.1 1.8
81.0 0.8
92.7 1.6
99.6 0.5
99.6 0.5
99.4 0.4
99.7 0.5
9.3 0.5
14.1 0.5
29.0 0.8
43.2 1.5
70.7 1.3
92.8 1.6
99.6 0.5
99.6 0.5
99.6 0.6
4.0 0.8
4.9 1.3
23
31
46
63
94
125
188
250
11.4 1.3
16.9 1.2
30.6 1.3
54.3 2.6
79.3 2.9
97.1 0.8
99.5 1.5
8.5 0.3
14.4 0.4
19.2 0.2
60.7 1.9
97.2 0.5
99.5 0.6
IC50
124.7 2.4
29.0 1.0
48.7 1.8
93.7 2.7
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Cytotoxicity of isolated compounds from C. jambhiri against Caco-2, HeLa and COS7
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lM) mean SD.
Compound
Caco-2
HeLa
22.9 4.8
COS7
4.9 0.2
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172.3 19.2
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5-Hydroxy-3,6,7,8,30,40-
hexamethoxyflavone
5-O-Demethylnobiletin
Neohesperidin
Hesperidin
48.2 28.6
4.2 0.4
44.9 6.1
174.1 19.2 123.5 9.5
194.9 43.9 106.2 12.5
337.8 9.3
294.2 20.1
101.1 16.8
3.8 0.4
51.6 4.9
51.5 3.9
30.3 0.7
144.5 15.5
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114.2 12.3
78.5 3.6
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Acknowledgements
We gratefully acknowledge Theodor C. H. Cole for consultations
regarding the manuscript and for English proofreading. Heiko Rudy
and Tobias Timmermann of IPMB (Dept. of Bioorganic Chemistry),
University of Heidelberg, are thanked for providing MS and
NMR measurements. Astrid Backhous for technical assistance.
D., Hamdan and M. Z., El-Readi are grateful for financial support
by the Egyptian Ministry of Higher Education and Scientific
Research.
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