Current Research Journal of Biological Sciences 3(1): 52-55, 2011
ISSN: 2041-0778
© M axwell Scientific Organization, 2011
Received: October 28, 2010
Accepted: December 02, 2010
Published: January 15, 2011
Isolation and Structure Elucidation of Phenolic Compounds
of Carob Leaves Grown in Egypt
O.A. Eldahshan
Department of Pharmacognosy, Faculty of Pharmacy, Ain shams University, Cairo, Egypt
Abstract: Carob leaves (Ceratonia siliqua L.), grown in Egypt have not fully identified in Egypt. Therefore,
eight major phenolic compounds were isolated from the aqueous ethanolic extract using different methods of
chromatography; gallic acid, quercetin 3-O- 4 C 1 -B-D-glucoside (Isoquercetin), Kaempferol 3-O- 1 C 4 -"-Lrhamnoside (Afzelin), Quercetin 3-O- 1 C 4 -"-L-rhamno side (Q uercitrin), 1,2,6 tri-O-galloy l-B-D-4 C 1 glucopyranose, (-)-Epigallocatechin-3-O-gallate, kaempferol and quercetin. Their structures established by
conventional methods of analysis. This is the first phytochemical study of polyphenolic content of carob leaves
grow n in Egypt.
Key w ords: Aqueous ethanolic extract, carob leaves, ceratonia ciliqua, phenolics
standpoint, the study describes herein the isolation and
structure elucidation of eight phenolics from the aqueous
ethanolic extract of carob leaves.
INTRODUCTION
The tree of carob is widely cultivated in the
Mediterranean area. It is considered as an important
component of vegetation for economic and environmental
reasons (Batle, 1997 ). Carob fruits are rich in phenolic
compoun ds. The main polyp henolic constituents isolated
from carob pods are insoluble, highly polymerized
condensed tannins containing a flavan nucleus (W ürsch
et al., 1984 ). Degredation experimen ts with hydrochloric
acid yielded (+)-catechin, (-)-epicatichin gallate ester, (-)epicatichin gallate, (-)-epigallocatichin gallate and (-)epiga llocatich in or delphinidin, pelargonidin and
cyaniding, respectively, indicating that the polymers are
composed of flavan-3-ol an d flavan-3, 4-d iol subunits
(Tamir et al., 1971; Marakis et al., 1993, 1997).
W ater extractable polyphe nols of carob pulp were
found to includ e gallic acid, (-)-epigallocatichin, (+)catichin, (-)-epicatichin gallate, and (-)-epigallocatichin
gallate with g allic acid as the main detectable co mpo nents
(Manson et al., 1992; Avallone et al., 2002 ), while
extraction with 9 0% methanol contaning acetic acid
(0.5%) afforded, in addition, quercetin glycosides and
ellagic acid (Sakakibara et al., 2003). HPLC analysis of
carob pod and leaf extracts revealed the presence of gallic
acid, (-) epigallocatich in 3-ga llate and (-) epigallocatichin
3-gallate. These compo unds are w ell know n to exert
antiproliferative effects in a concentration reached 6.28
mg/g in carob leaves and 1.36 mg/g in carob pods extract
(Corsi et al., 2002).
Recent study was performed on carob pods extract
grown in Mo rocco w here the predominant polyphenolics
were gallic acid, gallate glucoside and gallic acid
glucoside (Rakib et al., 2010).
Carob leaves grown in Egypt have not been subjected
to an in-depth p hytoc hem ical inve stigation . From this
MATERIALS AND METHODS
Plant source: Caro b leaves w ere collected from Orman
garden in Giza, Egypt, 2006. They were authenticated by
Prof. Dr Abdel Salam El Noyehy, Prof. of Taxonomy,
Faculty of Science, A in Shams University, C airo, Eg ypt.
Voucher specimen was deposited at the herbarium of
Pharmacogno sy Departm ent, Faculty of Pharmac y, Ain
Shams University, Cairo, Egypt. The leaves w ere dried in
shade and reduc ed to a fine pow der.
Chem icals: Kaemp ferol, quercetin, apigenin an d luteolin
(from NMR department, NRC, Cairo), glucose, rhamnose
and glucu ronic acid (from E.Merk, Darmstadt Germ any),
(-)-Epigallocatechin-3-O-gallate from Pharmacogno sy
Departm ent, Faculty of Pharmacy, Ain Shams University,
Sheets of Whatman paper No.1 and No.3 MM Paper
Chromatography (Whatman Ltd. Maidstone, K ent,
England), Sephadex LH-20 (25-100 Um) (Pharmacia-Fine
Chem icals, Sweden), Polyamid powder polyamide 6S for
CC, Riedel-D e Hae n-AG , Sheeze, Seelze-Hannov er,
Germany.
Extraction protocols: Powders of air dried leaves of
carob (1 kg) w ere extracted by 70 % ethano l on cold till
exhaustion. The solvent was distilled of in rotary
evaporator at 55ºC till dryness. The extract was
concentrated till constant weight 80 g and kept in vacuum
desiccator over anhydrous calcium chloride.
Ultraviolet
spectrophotom etric
Chromatographically, pure materials
52
analysis:
dissolved in
Curr. Res. J. Biol. Sci., 3(1): 52-55, 2010
analy tically pure methanol (definite concen tration), were
subjected to UV spectrophotometric investigation in 4 mL
capa city quartz cells(1 cm thick) using a Carl Zeiss
spectropho tometer PMQ II. AlCl3 , AlCl3 /HCl, fused
NaOA c/H 3 BO 3 and NaOMe reagents w ere separately
added to the methanolic solution of investigated material
and UV measurem ents w ere then carried out.
species (Eldahshan, 2006). Furthermore, polyphenols act
as agonists and/or antagonist with carcinogen-related
receptors
such
as
epidermal growth factor
(Ashida et al., 2000), aryl hydrocarbon receptor
(An
et
al., 2001), and oestrogen receptors
(Agullo et al., 1997), and modulate the expression
of
protein
kinases in tumour cell populations
(Kobuchi et al., 1999). There is a marked cytotoxic
activity of crude aqueous extract of carob on both
mam malian cell lines (Vero and HEp-2) and the
effect is more obvious on the human cell line (HEp-2)
(Nagib et al., 2010).
Carob is an important source of food in tropical
regions, but at present the leaves are discarded. These
waste products which contain a lot of ph enolics app ear to
have real potential low-cost source of antioxidant as we ll
as anticancer drug.
Studies are needed to obtain a more com plete profile
of their anticancer potential. This may be a useful area
for future study.
Nuclear magnetic resonance spectroscopic analysis:
The NM R spectra were recorded on a Varian M ercury
VX-300 NM R sp ectrom eter. 1 H spectra run at 300 and
500 MH z and 1 3 C spectra w ere run at 75.46 M Hz in
deutrated dimethylsulphoxide (DMSO-d 6 ). Chemical
shifts are quoted in * and w ere related to that of the
solvents. The mass spectra were recorded on a Shimadzu
GC MS-Q P-1000E X m ass spectrom eter at 70 e.V.
Isolation and identification: The isolation and
purification of the phenolic components were achieved
through the application of the concentrated extract (60 g)
to a Sephadex LH-20 column (150x4.5 cm) and eluted
with water followed by H 2 O-MeO H mixtures of
decreasing polarities to yield five fractions (I-V).
Compound (1) 22.0 mg and (2) 35.0 m g were isolated
from fraction I (eluted with water) by polyamide column
fractionation using H 2 O/MeO H (decreasing polarity) for
elution then preparative paper chromatography (PPC)
using HOAc-15%. Com pound (3) 8.3 mg and (4) 10.0
mg from fraction II (eluted with 50% M eOH). Compound
(5) was separated from fraction III (eluted by 80% ) by
Sephadex LH-20 column using saturated water butanol
11.7 mg. Purification of the compound achieved through
PPC. Compound (6) 15.2 mg, (7) 8.0 mg and (8) 7.7 mg
from fraction IV paper w ere chromatogra phica lly
analyzed using preparative paper chromatography on
W hatmann paper No. 3 and 6% acetic acid as a
developing system.
Ga llic acid (1): off white am orphous p owder, 8 max
(MeOH) 272 nm, 1 H-NMR (DMSO-d 6 ) 6.91(1H, s, H-2,
6), 1 3 C-NMR (DMSO-d 6 ): 121.0 (C-1), 109.0 (C-2 & C6), 145.9 (C -3 & C -5), 138.3 (C-4 ), and 168.0 (C-7).
Qu ercetin 3-O- 4 C 1 -B-D-glucoside (Iso querc etin) (2):
amorphous yellow pow der, 8 m a x (MeOH) 258, 355 nm,
1
H -N M R (DMSO-d 6 ) 6.20 (1 H, d, J = 2.0 Hz, H-6), 6.41
(1H, d, J = 2.0 Hz, H-8), 6.82 (1H, d, J = 8.5 Hz, H-5'),
7.54 (1H, dd, J = 8.5 and 2.5 H z, H-6 '), 7.76 (1H , d, J =
2.5 Hz, H-2'), 5.345 (d, J= 7.5 Hz, H-1 glucose), 3.083.88 (m, six sugar protons), 1 3 C-NMR(DMSOd 6 )156.164 (C-2), 133.344 (C-3), 177.78 (C-4), 161.241
(C-5), 98.935 (C-6), 164.60 6 (C-7), 93.628 (C-8), 156.40
(C-9), 103.787 (C-10), 121 .106 (C-1 '), 115.254 (C-2'),
144.864 (C-3'), 148.549 (C-4'), 116.212 (C-5'), 121.625
(C-6'), 100.991 (C-1''), 61.027 (C-6''), 69.992 (C -4''),
74.158 (C-2''), 76.561 (C-3''), 77.557 (C-5'').
RESULTS AND DISCUSSION
Kae mp ferol 3-O- 1 C 4 -"-L-rha mn oside (A fzelin) (3):
pale yellow amo rphous po wder, 8 m a x (MeOH) 267, 353,
1
H -N M R (DMSO-d 6 ) 0.9 (d, J = 6 Hz., CH 3 ), 5.29 (1 H,
d, J = 1.5 Hz H-1’’), 6.21 (1 H, d, J = 2.5 Hz, H-6), 6.42
(1H, d, J = 2.5 Hz, H-8), 6.91 (2H , d, J = 8.4 Hz, H-3’
and H-5’), 7.74 (2 H, d, J = 8.4 Hz, H-2’ and H-6’), 1 3 CNMR(DMSO-d 6 ) 17.53(C-6’’), 70.11(C-5’’), 70.35(C2’’), 70.69(C -3’’), 71.13(C-4’’), 93.88 (C-8), 98.92(C-6),
101.77(C-1’’), 103.98(C -10), 11 5.48(C-3’) and (C-5’),
120.49(C-1’), 130.64(C -2’) and (C-6’), 134.15(C-3),
156.56(C-9), 157.24(C-2), 160.14(C -4’), 161.28(C -5),
164.80(C -7), 177 .70(C -4).
The result of this study shows that carob leaves
contain varieties of individual components from several
classes: simple phenols, polyphenols, free flavonoids,
glycosylated flavonoids and g allotannins. Altogether,
eight major individual struc tures w ere identified via
conventional methods o f analysis, these are gallic acid,
que rcetin 3-O - 4 C 1 -B -D-glucoside (Isoquercetin),
Kaempferol 3-O-1 C 4 -"-L-rhamnoside (Afzelin), Quercetin
3-O-1 C 4 - "-L-rhamnoside (Quercitrin), 1,2,6 tri-O-galloylB-D-4 C 1 -glucopyranose, (-)-Epigallocatechin-3-O-gallate,
kaempferol and quercetin.
CONCLUSION
Qu ercetin 3-O- 1 C 4 - "-L-rhamnoside (Quercitrin) (4):
pale yellow amo rphous po wder, 8 m a x (MeOH) 259, 297sh,
348, 1 H-NMR (DMSO-d 6 ) 0.9 (d, J = 6 Hz, CH 3 ), 5.2(d,
J = 1.5 Hz, H-1’’), 6.15(d , J = 2.2 Hz, H-6), 6.36(d , J =
The aqueous ethanolic extract of carob leaves showed
antioxidant activity via scavenging the reactive oxygen
53
Curr. Res. J. Biol. Sci., 3(1): 52-55, 2010
Gallic acid (1)
Kaempferol 3-O- 1 C 4 -"-L-rhamnoside (3 )
Quercetin 3-O-B-4C1-D-glucoside (2)
Quercetin 3-O-1C4-" -L-rhamnoside ( 4)
1, 2, 6-tri-O-galloy l-B-D-glucose (5)
(-)-Epigallocatechin-3-O-gallate (6)
2.2 Hz, H-8), 6.95(d , J = 8.2 Hz, H-5’), 7.25(d d, J = 2.2
Hz and J = 8.2 Hz, H-6’), 7.35(d, J = 2.2 Hz, H-2’), 1 3 CNMR(DMSO-d 6 ) 17.67(C-6’’), 70.21(C-5’’), 70.73(C3’’), 70.50(C -2’’), 71.35(C-4’’), 93.97 (C-8), 99.20(C-6),
101.91(C-1’’), 103.79(C-10), 115.68(C-2’), 115.78(C-5’),
120.79(C-1’), 121.21(C -6’), 134.24(C -3), 145.44(C -3’),
148.78(C-4’), 156.66(C -2), 157.25(C -9), 161.35(C -5),
165.59(C-7), 177.72(C-4).
H-2), 3.7(t, J = 8.5 Hz, H-3), 3.5(t, J = 8.5 H z, H-4),
3.83(m, H-5), 4.43(d , J = 13 Hz, H-6), 4.3(dd , J = 13, 5
Hz, H-6`),6.88(s), 6.92(s), 6.98(s) arom atic galloyl
protons, 1 3 C-NMR(DMSO-d 6 )62.9 (C-6 glc.), 69.7(C-4
glc.), 72.8(C-5 glc.), 73.75(C-3 glc.), 73.82(C-2 g lc.),
92.16(C -1 glc.), 118.0, 119.2, 119.3 (C-1 galloyl), 108.7,
108.8, 109.0 (C-2, 6 galloyl), 145.4, 145.5, 145.6 (C-3, 5,
galloyl), 138.63, 138.69, 139.30(C-4 galloyl), 164.2,
165.0, 165.8 (C = O).
1,2,6 tri-O-galloy l-B-D- 4 C 1 -glucopyranose (5): Cream
colored amorphous powder, 8 m a x (MeOH)277, 1 H -N M R
(DMSO-d 6 )5.88(d, J = 8.5 H z, H-1), 5.02(d, J = 8.5 Hz,
(-)-Ep igall ocatech in-3-O -gallate (6): off-w hite
amorphous pow der, 8 m ax (MeOH) 269, 1 H-NMR (DMSO54
Curr. Res. J. Biol. Sci., 3(1): 52-55, 2010
d 6 ) 4.82 (1 H, d, J = 7.5 H z, H-2), 5.03 (1H, m, H-3), 2.30
(ax.H, dd, J = 16.4, 4.4 H z, H-4 ), 1.91 (eq.H, dd, J = 16.4,
3.5 Hz, H-4), 5.73 (1H, d, J = 1.8 Hz, H-8), 5.62 (1 H, d,
J = 1.8 H z, H-6), 6.07 (2 H, s, H-2', H-6'), 6.67 (2 H, s, H2'', H-6''), 1 3 C-N MR Sp ectral data, 165.69 (C-7'', [C = O]),
157.30 (C-7), 156.32 (C-9), 155.04 (C-5), 146.5 (C-3'', C5''), 145.83 (C-3', C-5'), 139.07 (C-4''), 133.1 (C -4'), 128.9
(C-1'), 119.36 (C -1''), 109.0 (C-2'', C-6''), 105.3 (C-2', C6'), 97.78 (C -10), 95.77 (C-8), 94.39 (C-6), 77.325 (C-2),
69.31 (C-3), 29.2 7 (C-4).
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