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Anti-Alzheimer, antioxidant activities and flavonol glycosides of Eryngium

campestre L

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188 Current Chemical Biology, 2013, 7, 188-195

Anti-Alzheimer, Antioxidant Activities and Flavonol Glycosides of

Eryngium campestre L.
Usama W. Hawas*,1, Lamia T. Abou El-Kassem2, Hanem M. Awad3 and Hanan A.A. Taie4

1
Phytochemistry and Plant Systematic Department; 2Department of Pharmacognosy; 3Department of Tanning Materials
and Leather Technology; 4Department of Plant Biochemistry, National Research Centre, El-Bohooth Street, 12311-
Dokki, Cairo, Egypt

Abstract: Phytochemical investigation of the methanolic extract of Eryngium campestre L. aerial parts led to isolation of
eleven known flavonol glycosides. Structures were elucidated by spectroscopic and chemical methods. The methanol
extract of E. campestre and the isolated flavonols exhibited moderate to strong antioxidant activity in DPPH radical
scavenging and reducing power assays. Eryngium campestre extract showed significant inhibition of the -amyloid A 42
(IC50 = 155.75±7.43 ng/ml) without significant reduction in total A (A 40 + A 42) levels in human H4 cell line, using
sensitive sandwich enzyme linked immunosorbent assay (ELISA). The results showed no inhibition activity of the extract
against COX-1 and COX-2 up to 400 ng/ml concentration.

Keywords: Alzheimer's disease, -amyloid peptide (A), antioxidant, reducing power, flavonol glycosides, Eryngium
campestre.

INTRODUCTION consecutive cleavages of - and -secretase to build up

Eryngium is the largest and arguably the most taxonomically
complex genus in the family Apiaceae. The genus is
represented by 317 taxa widespread throughout Central Asia,
northern Africa, America, Central and Southeast Europe [1].
Eryngium campestre (Field eryngo) is a rare perennial
restricted to dry grassy areas near the coast. It is native to
Spain, France, Germany and Greece and other scattered
localities in Europe, and is also found in Africa and Asia [2].
It has been used in European herbal medicine as an infusion
to treat whooping cough, kidney and urinary tract
inflammations [3]. Previous phoytochemial studies on
Eryngo revealed the presence of triterpenoid saponins [4],
flavonoids: kaempferol and quercetin derivatives [5, 6],
coumarins: Aegelinol benzoate, agasyllin, grandivittin, and
aegelinol [7].
Alzheimer's disease (AD) is the leading cause of
dementia in the elderly, presenting itself clinically by
progressive loss of memory and learning and its prevention
is a major public health challenge. The key event in the
progression of AD is the sequential cleavages of -amyloid
precursor protein (-APP) by two proteolytic enzymes, beta-
site APP-cleaving enzyme 1 (BACE-1 or memapsin 2) and
-secretase to produce A 40 and A 42 in the human brain
[8]. The main constituent of the accumulated amyloidic
plaques in the brain is -amyloid peptide (A). The activity
of two aspartic proteases (-secretase and -secretase) results
in A of length from 38 to 42 amino acids. -Secretase
pathway degrades 90% of the APP rest is degraded by the
*Address correspondence to this author at the Phytochemistry and Plant
Systematic Department, National Research Centre, El-Bohooth Street,
12311, Dokki, Cairo, Egypt; Tel 00202-37340064; Fax: 202-33370931;
E-mail; hawasusama@yahoo.com
extracellular A deposits. However, the APP derivative A
42 is thought to be a key factor in the pathogenesis of AD, as
A 42 is more responsible for fibril formation due to its self-
assembly and turned out to be a major A constituent of
amyloid plaques. At present, no causal therapy for AD is
clinically accessible regardless of intense research
endeavours. Anti-oxidant therapy, AChE inhibitors, nicotinic
and muscarinic agonists, nerve growth factor (NGF), low
molecular lipophilic compounds, anti-inflammatory drugs,
drugs which interfere with A formation and deposition, and
drugs that attenuate A toxicity are the existing
pharmacological advances related to AD treatment.
Regardless of the way in which A exerts its deleterious
effects on the brain, the amyloid hypothesis predicts that
decreased production of the A peptide in the brain,
particularly, A 42 will elicit a therapeutic benefit [9].
In this work, we have discussed the isolation and
structure elucidation of eleven flavonoidal compounds from
the aqueous methanol extract of E. campestre aerial parts.
The evaluation of the Eryngo extract and the isolated
flavonol glycosides for their DPPH scavenging activity, and
reducing power was carried out. The inhibition of the A 42
and A 40 formation were measured in the culture medium
of H4 cells, a human neuroglioma cell line expressing the
double Swedish mutation (K595N/M596L) of human APP
(APPsw). The inhibition of the cyclooxygenase activity was
estimated as well for the plant extract.
MATERIAL AND METHODS
NMR spectra were obtained on Varian Unity 300 (1H,
300; 13C, 145 MHz) and Varian Inova 600 (1H, 599.744
MHz; 13C, 150.7 MHz) spectrometers. DMSO-d6 was used
as solvent. The
values reported as ppm relative to TMS in
DMSO-d6 and J values are given in Hz. UV spectra were
recorded on Shimadzu UV-visible spectrophotometer model-

1872-3136/13 $58.00+.00 © 2013 Bentham Science Publishers

Anti-Alzheimer, Antioxidant Activities and Flavonol Glycosides
UV 240 (Tokyo, Japan). Polyamide 6S (Riedel de Haen AG,
Hannover, Germany); Sephadex LH-20 (Pharmacia Fine
Chemicals, Uppsala, Sweden); Solvent mixtures, BAW (n-
butanol:acetic acid:water, 4:1:5 upper phase), and paper
chromatography (PC), Whatman No. 1 & 3 MM (46
57
cm) (Kent, England).
Plant Material
The aerial parts of Eryngium campestre L. were collected
in April 2009 from Borg El-Arab city (50 km East of
Alexandria), Egypt. The plant was identified by Prof.
Ibrahium El-Garf, Botany Department, Cairo University, and
voucher specimens have been deposited at the herbarium of
the National Research Centre, Dokki, Cairo, Egypt.
Extraction and Isolation
Ground aerial parts of E. campestre (500 g) were
extracted three times with 70% aqueous methanol at room
temperature (each 2 L, 12 h). The extract was concentrated
to give a residue (40 g), which was suspended in water and
then partitioned successively with dichloromethane and ethyl
acetate. The ethyl acetate soluble fraction (15 g) was applied
to a polyamide 6S column and eluted with H2O with the
proportional increasing of MeOH. All fractions thus obtained
were recombined according to their paper chromatographic
analysis to give eight major fractions (I-VIII). Fraction I was
subjected to preparative paper chromatography (3 MM)
using 15% acetic acid/water as eluent. The separated bands
were scraped off and eluted with 70% methanol and further
purified by column chromatography (CC) on Sephadex LH-
20 using MeOH to get compounds 1 (6 mg), 5 (7 mg) and 9
(11 mg). Compounds 2 (10 mg), 4 (10 mg) and 6 (7 mg),
were detected as major components in fractions II, III and
IV, respectively, and purified separately by repeated CC on
Sephadex LH-20 using MeOH and MeOH:H2O (8:2) as
eluents. Fraction V was purified on cellulose column with
saturated n-BuOH as eluent to give pure compounds 3 (12
mg) and 7 (8 mg), while compound 8 (23 mg) was
spontaneously precipitated in the concentrated fraction VI.
Fractions VII and VIII, were combined and subjected to
preparative paper chromatography (3 MM) using BAW as
eluent followed by successively CC on Sephadex LH-20
with MeOH as eluent to afford pure samples of 10 (6 mg)
and 11 (7 mg).
Acid Hydrolysis
Compounds 1-11 (2 mg) in a mixture of 8% HCl (1 ml)
and MeOH (4 ml) were separately refluxed for 2 h. The
reaction mixtures were reduced under pressure to dryness,
dissolved in H2O (3 ml) and neutralized with Na2 CO3. The
neutralized products were subjected to PC (eluent,
benzene:n-butanol:pyridine:H2O, 1:5:3:3). The
chromatograms were sprayed with aniline hydrogen
phthalate followed by heating at 100°C. The sugars were
identified after comparison with authentic samples.
Anti-Oxidant Activity
DPPH free Radical Scavenging Assay
The free radical scavenging activity using the 1.1-
diphenyl-2-picrylhyrazil (DPPH) reagent was determined
Current Chemical Biology, 2013, Vol. 7, No. 2 189
according to Brand-Williams (1995) [10]. 0.05 ml of extract
or isolated compound was dissolved in DMSO then added to
0.1 ml of freshly prepared methanolic DPPH solution (20 g
ml-1). The mixture was shaken vigorously and allowed to
stand at room temperature for 30 min. The absorbance was
measured at 517 nm by spectrophotometer and compared
with a blank control. Scavenging of DPPH free radical was
measured using the following equation:
Scavenging activity (%) = (control absorbance – sample
absorbance)/ (control absorbance)
100.
Reducing Power Assay
The reducing power of E. campestre extract, and the
isolated compounds was determined according to Oyizu
[11]. The extract and isolated flavonoids were dissolved in
methanol and mixed with phosphate buffer (2.5 ml, 0.2 M,
pH 6.6) and potassium ferrocyanide (2.5 ml, 1%). The
mixture was incubated at 50°C for 20 min. A portion (2.5
ml) of trichloroacetic acid (10%) was added to the mixture,
which was then centrifuged at 3000 rpm for 10 min. The
upper layer of the solution (2.5 ml) was mixed with distilled
water (2.5 ml) and FeCl3 (0.5 ml, 0.1%) and the absorbance
was measured at 700 nm and compared with standards.
Increased absorbance of the reaction mixture indicated
increased reducing power.
Statistical Analysis
The antioxidant data were performed in triplicate and all
data were expressed with mean standard deviations (SD).
Data obtained were subjected to standard analysis of
variance procedure where values of LSD were obtained at
0.05% as reported by Snedecore and Cochran [12].
Anti-Alzheimer activity
Chemicals
Dimethylsulfoxide (DMSO), nicotinamide adenine
dinucleotide (reduced form) NADH, phosphate buffered
saline (PBS), hydrogen peroxide, fetal bovine serum,
gentamicin sulfate, L-Glutamine,Triton X-100 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), Dulbecco’s modified Eagle’s media (DMEM) and
trypan blue dye were purchased from Sigma Chemical
Company (St. Louis, MO, USA).
Culture of H4 Cells Expressing the Double Swedish
Mutation (K595N/M596L) of Human APP (APPsw).
All cell lines were cultured in DMEM (high glucose)
containing 9% heat-inactivated fetal calf serum, 100-Us/mL
penicillin, 100- g/mL streptomycin, and 2mM L-glutamine.
Stably transfected H4 cells were additionally supplemented
with 200- g/mL G418 [13].
Exposure of H4 Cells to the Extract
Cells were seeded onto 24-well plates (2x105 cell/well)
and allowed to grow to confluence for 24 h, in 5% CO2/95%
air in humidified atmosphere. Increasing concentrations
(from 0 - 300 ng/ml) of the extract were added to the cells
overnight in a final volume of 0.5 ml. R-flurbiprofen was
used as positive control (3 - 1000 M). DMSO (0.5%) was
used as negative control. At the end of the incubation, 100 l
190 Current Chemical Biology, 2013, Vol. 7, No. 2
of each supernatant were removed for measuring -amyloid
peptides.
Detection of A Peptides
Each supernatant was treated with a biotinylated mouse
monoclonal antibody (4G8, Signet Laboratories Inc.,
Dedham, MA, USA), specifically recognizing the 17-24
amino acid region of A and two rabbit polyclonal
antibodies (C-term 42 and C-term 40, BioSource
International, Camarillo, CA, USA), specifically recognizing
the C-terminus of A 42 and A40, respectively. Antigen–
antibodies complexes were recognized by TAG-donkey anti-
rabbit IgG (Jackson Immuno Research Laboratories, Soham,
UK). Streptavidin coated magnetic beads captured the
complexes and the signals were read by an
electrochemiluminescence instrument (Origen M8 Analyzer,
BioVeris Corporation, Gaithersburg, MD, USA). A levels
in cell supernatants were evaluated in comparison to an A
peptide Standard. The total A content was calculated as
sum of A 40 and A 42.
Inhibition of COX-1 and COX-2 Assay
The inhibition of the cyclooxygenase activity was
estimated measuring by PGE2 production form arachidonic
acid according to a modified method [14]. Recombinant
human prostaglandin H2 synthase-1 (PGHS-1) and synthase-
2 (PGHS-2) were expressed in transfected spodoptera
frugiperda (Sf-9) cells (Invitrogen, San Diego, CA, USA).
The microsomal fractions were prepared from the transfected
cells and used to assay the enzymatic activities. Briefly, the
enzymes (2 g) reconstituted in a buffer (100 mM Tris-HCl,
pH 8.0) containing 2 mM phenol, were preincubated with
vehicle (DMSO) or the extract (0 - 300 ng/ml in DMSO) for
20 min at 22ºC. The reaction mixture was completed with
1M hematin. The reaction was initiated adding arachidonic
acid (4 and 2  for COX-1 and COX-2, respectively) and the
mixture was incubated for 5 min at 22ºC for COX-1 assay,
or for 10 min at 25ºC for COX-2 assay. For control
measurements, arachidonic acid was omitted from the
reaction mixture. The reactions were stopped by the
sequential addition of 1 M HCl and 1 M Tris-HCl (pH 8.0),
followed by cooling to 4ºC. The amount of PGE2 present in
the reaction mixture was quantified using an enzyme-
immunoassay.
MTT Cytotoxicity Assay
The anti-proliferative activity against H4 human cell line
was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay, which is
based on the cleavage of the tetrazolium salt by
mitochondrial dehydrogenases in viable cells [15]. Cells
were dispensed in a 96 well sterile microplate (5 x 104
cells/well), and incubated at 37oC with various
concentrations of the extract (0 - 300 ng/ml) or paclitaxel
(taxol, positive control) for 48 h in a serum free medium
prior to the MTT assay. After incubation, media were
carefully removed, 40
L of MTT (5 mg/ml) were added to
each well and then incubated for an additional 4 h. The
purple formazan dye crystals were solubilized by the
addition of 200
L of acidified isopropanol. The absorbance
was measured at 570 nm using a microplate ELISA reader
Hawas et al.
(Biorad, USA). The relative cell viability was expressed as
the mean percentage of viable cells compared to the
untreated control cells, with the half maximal inhibitory
concentration (IC50) calculated from the viability dose
response curve of each cell line.
Lactate Dehydrogenase (LDH) Cytotoxicity Assay
To determine the effect of the extract on membrane
permeability in H4 cells, a lactate dehydrogenase (LDH)
release assay was used. The cells were seeded in 96-well
culture plates at a density of 2  104 cells/well in 100 l
volume and allowed to grow for 18 hours before treatment.
After treatment, the plates were incubated for 24, 48 and 72
hours, respectively. Then, the supernatant (40 l) was
transferred to a new 96 well to determine LDH release and
6% triton X-100 (40 L) was added to the original plate for
determination of total LDH. An aliquot of 0.1 M potassium
phosphate buffer (100 L, pH 7.5) containing 4.6 mM
pyruvic acid was mixed to the supernatant using repeated
pipetting. Then, 0.1 M potassium phosphate buffer (100 L,
pH 7.5) containing 0.4 mg/ml reduced -NADH was added
to the wells. The kinetic changes were read for 1 min using
ELISA microplate reader in absorbance at wavelength 340
nm. This procedure was repeated with 40 l of the total cell
lysate to determine total LDH. The percentage of LDH
release was determined by dividing the LDH released into
the media by the total LDH following cell lysis in the same
well.
Statistical Analysis
All experiments of Alzheimer were conducted in
triplicate (n = 3). All the values were represented as mean ±
SD. Significant differences between the means of parameters
as well as IC50s were determined by probit analysis using
SPSS software program (SPSS Inc., Chicago, IL).
RESULTS
Chemical Characterization of Isolated Compounds
Elven flavonol compounds were isolated and identified
as three quercetin glycosides, quercetin 3-O--glucoside (1),
quercetin 3-O--glucuronide-4'-methylether (2), and rutin
(3); five isorhamnetin glycosides, isorhamnetin 3-O--
glucoside (4), isorhamnetin 3-O--galactoside (5),
isorhamnetin 3-O--rhamnoside (6), isorhamnetin 3-O--
rutinoside (7), and isorhamnetin 3-O--glucoside-7-O--
rhamnoside (8); and three myricetin glycosides, myricetin 3-
O--glucoside-3'-methylether (9), myricetin 3-O--
glucoside-4'-methylether (10), and myricetin 3-O--
galactoside-4'-methylether (11).
The structures of the isolated compounds were
established by means of a UV-visible spectrophotometer,
MS and NMR spectrometer as follows:
Quercetin 3-O--glucoside (1)
Yellow powder, PC Rf 0.54 (BAW), 0.43 (15% HOAc).
UV max (MeOH): 257, 267sh, 324sh 360; (NaOMe): 272,
329, 409; (AlCl3): 272, 430; (AlCl3/HCl): 268, 359sh, 405sh;
(NaOAc): 273, 324, 391; (NaOAc/H3BO3): 262, 379. 1 H
NMR:
= 7.53 (m, H-2'/6'), 6.79 (d, 8.4 Hz, H-5'), 6.34 (d,
2 Hz, H-8), 6.14 (d, 2 Hz, H-6), 5.41 (d, 6.9 Hz, H-1).
Anti-Alzheimer, Antioxidant Activities and Flavonol Glycosides
Quercetin 3-O--glucuronide-4'-methylether (2)
Yellow powder, PC Rf 0.56 (BAW), 0.44 (15% HOAc).
UV max (MeOH): 253, 266sh, 339, 356; (NaOMe): 274,
321, 388 (dec); (AlCl3): 269, 363, 396; (AlCl3/HCl): 268,
360, 364, 371, 400; (NaOAc): 269, 339, 361;
(NaOAc/H3BO3): 255, 358. 1H NMR:
= 8.01 (d, 2 Hz, H-
2'), 7.50 (dd, 8.5 and 2 Hz, H- 6'), 6.9 (d, 8.5 Hz, H-5'), 6.4
(d, 2 Hz, H-8), 6.2 (d, 2 Hz, H-6), 5.6 (d, 7 Hz, H-1''), 3.81
(s, 4'-OCH3). 13C NMR:
= 175 (C-4), 170.2 (C-6), 164 (C-
7), 161.5 (C-2), 156.5 (C-5/9), 149 (C-4'), 146.5 (C-3'),
132.5 (C-3), 121.8 (C-2'), 121 (C-6'), 115 (C-1'), 113 (C-5'),
105 (C-10), 101 (C-1), 98.5 (C-6), 93.7 (C-8), 75.9 (C-2),
75.7 (C-3), 74.0 (C-5), 71.8 (C-4), 55.4 (4'-OCH3).
Rutin (3)
Yellow powder, PC Rf 0.40 (BAW), 0.47 (15% HOAc).
UV max (MeOH): 257, 267sh, 295sh 360; (NaOMe): 275,
320, 415; (AlCl3): 269, 305sh, 335sh, 430; (AlCl3/HCl): 275,
305sh, 345sh, 420; (NaOAc): 270, 320, 385;
(NaOAc/H3BO3): 260, 300sh, 380. 1H NMR:
= 7.50 (dd,
2/8 Hz, H-2'/6'), 6.80 (d, 8 Hz, H-5'), 6.35 (d, 2 Hz, H-8),
6.15 (d, 2 Hz, H-6), 5.31 (d, 7.5 Hz, H-1''). 4.34 (s, H-1'''),
0.95 (d, 6 Hz, CH3-Rh). 13C NMR:
= 177.2 (C-4), 164 (C-
7), 161 (C-5), 156.3 (C-9), 156.1(C-2), 148.2 (C-4'), 144.7
(C-3'), 133.2 (C-3), 121.4 (C-6'), 121.0 (C-1'), 116.1 (C-5'),
115.1 (C-2'), 103.8 (C-10), 101(C-1), 100.6 (C-1'''), 98.6
(C-6), 93.5 (C-8), 76.4 (C-5), 75.8 (C-3), 74.0 (C-2), 71.8
(C-4
), 70.4 (C-3
), 70.2 (C-2
), 69.9 (C-4), 68.1 (C-5
),
66.9 (C-6), 17.5 (C-6
).
Isorhamnetin 3-O--glucoside (4)
Yellow powder, PC Rf 0.56 (BAW), 0.39 (15% HOAc).
UV max (MeOH): 257, 267sh, 324sh 357; (NaOMe): 272,
329, 408; (AlCl3): 265, 385; (AlCl3/HCl): 265, 359sh, 405sh;
(NaOAc): 273, 324, 391; (NaOAc/H3BO3): 262, 379. 1 H
NMR:
= 7.51 (s, H-2'), 7.34 (d, 8.4 Hz, H- 6'), 6.85 (d, 8.4
Hz, H-5'), 6.22 (d, 2 Hz, H-8), 6.03 (d, 2 Hz, H-6), 5.51 (d, 7
Hz, H-1''), 3.78 (s, 3'-OCH3).
Isorhamntin 3-O--galactoside (5)
Yellow powder, PC Rf 0.46 (BAW), 0.35 (15% HOAc).
UV max (MeOH): 257, 267sh, 324sh 359; (NaOMe): 272,
329, 408; (AlCl3): 265, 385; (AlCl3/HCl): 265, 359sh, 405sh;
(NaOAc): 273, 324, 391; (NaOAc/H3BO3): 262, 379. 1 H
NMR:
= 7.99 (d, 2 Hz, H-2'), 7.45 (dd, 8.4 and 2 Hz, H-
6'), 6.88 (d, 8.4 Hz, H-5'), 6.39 (d, 2 Hz, H-8), 6.15 (d, 2 Hz,
H-6), 5.52 (d, 6.9 Hz, H-1''), 3.79 (s, 3'-OCH3).
Isorhamnetin 3-O--rhamnoside (6)
Yellow powder, PC Rf 0.55 (BAW), 0.33 (15% HOAc);
UV max (MeOH): 257, 268, 358; (NaOMe): 272, 324,
420; (AlCl3): 268, 301, 360, 398; (AlCl3/HCl): ) 262, 302,
402; (NaOAc): 270, 322, 408; (NaOAc/H3 BO3): 256, 266,
362. 1H NMR:
= 7.91 (d, 2 Hz, H-2'), 7.53 (d, 8.0 Hz, H-
6'), 6.86 (d, 8.0 Hz, H-5'), 6.41 (d, 2.3 Hz, H-8), 6.18 (d, 2.3
Hz, H-6), 5.53 (br s, H-1''), 3.81 (s, 3'-OCH3), 1.07 (d, 6.1
Hz, CH3-Rh).
Current Chemical Biology, 2013, Vol. 7, No. 2 191
Isorhamnetin 3-O-rutinoside (7)
Yellow powder, PC Rf 0.42 (BAW), 0.46 (15% HOAc).
UV max (MeOH): 254, 266sh, 300sh 352; (NaOMe): 272,
320, 408; (AlCl3): 274, 354, 399; (AlCl3/HCl): 274, 364,
395; (NaOAc): 275, 371; (NaOAc/H3BO3): 255, 339, 354.
1
H NMR:
= 7.85 (d, 2.2 Hz, H-2'), 7.45 (dd, 8 and 2.2 Hz,
H- 6'), 6.90 (d, 8 Hz, H-5'), 6.42 (d, 2 Hz, H-8), 6.20 (d, 2
Hz, H-6), 5.41 (d, 7.4 Hz, H-1''). 4.42 (s, H-1'''), 3.85 (s, 3'-
OCH3), 0.98 (d, 6 Hz, CH3-Rh). 13C NMR:
= 177 (C-4),
163 (C-7), 161.5 (C-5), 158.5 (C-9), 157 (C-2), 147 (C-3'),
146 (C-4'), 133 (C-3), 122 (C-1'), 121.5 (C-6'), 115.5 (C-5'),
113.5 (C-2'), 105.5 (C-10), 102.5 (C-1), 102 (C-1'''), 97 (C-
6), 95 (C-8), 79 (C-5), 76 (C-3), 73.7 (C-4
), 73 (C-2), 71
(C-3
), 70.2 (C-2
), 69.5 (C-4), 68.1 (C-5
), 65.5 (C-6),
56 (CH3O), 17.8 (C-6
).
Isorhamnetin 3-O--glucoside-7-O-
-rhamnoside (8)
Yellow powder, PC Rf 0.43 (BAW), 0.45 (15% HOAc).
UV max (MeOH): 263, 267sh, 354; (NaOMe): 262, 419;
(AlCl3): 272, 358, 407; (AlCl3/HCl): 271, 402, 408;
(NaOAc): 256, 268, 361; (NaOAc/H3BO3): 255, 368, 361.
1
H NMR:
= 7.91 (d, 2.2 Hz, H-2'), 7.63 (d, 8 Hz, H- 6'),
6.90 (d, 8 Hz, H-5'), 6.81 (d, 2 Hz, H-8), 6.43 (d, 2 Hz, H-6),
5.61 (m, H-1''/ H-1'''), 3.85 (s, 3'-OCH3), 1.12 (d, 6.3 Hz,
CH3-Rh). 13C NMR:
= 177 (C-4), 161.5 (C-5), 161 (C-7),
156.5 (C-2), 156 (C-5), 149.5 (C-4'), 146.5 (C-3'), 133.1 (C-
3), 122.0 (C-1'), 121.0 (C-6'), 115.0 (C-5'), 113.5 (C-2'),
105.5 (C-10), 101(C-1), 99.0 (C-6), 94.3 (C-8), 98.5 (C-
1
), 77.1 (C-5), 76.6 (C-3), 74.4 (C-2), 76.9 (C-3), 71.8
(C-4
), 70.6 (C-3
), 70.3 (C-2
), 70.2 (C-4), 61.0 (C-6),
55.0 (3'-OCH3), 17.8 (CH3-Rh).
Myricetin 3-O--glucoside-3'-methylether (9)
Yellow powder, PC Rf 0.41 (BAW), 0.27 (15% HOAc).
UV max (MeOH): 253, 267sh, 356; (NaOMe): 275, 324,
381; (AlCl3): 274, 365, 408; (AlCl3/HCl): 273, 357, 399,
409; (NaOAc): 274, 324, 391; (NaOAc/H3 BO3): 254, 267,
379. 1H NMR:
= 7.59 (s, H-2'), 7.14 (s, H-6'), 6.42 (d, 2
Hz, H-8), 6.20 (d, 2 Hz, H-6), 5.58 (d, 6.8 Hz, H-1''). 3.72 (s,
3'-OCH3). 13C NMR:
= 177 (C-4), 164.5 (C-2), 161.0 (C-
7), 156 (C-5/9), 148 (C-4'), 145.7 (C-5'), 138 (C-3'), 133 (C-
3/2'), 129 (C-6'), 121.0 (C-1'), 121.8 (C-6'), 103.5 (C-10),
101 (C-1), 99.2 (C-6), 93.5 (C-8), 77.4 (C-3), 76.3 (C-5),
74.2 (C-2), 69.8 (C-4), 62.7 (C-6), 55 (3'-OCH3).
Myricetin 3-O--glucoside-4'-methylether (10)
Yellow powder, PC Rf 0.56 (BAW), 0.32 (15% HOAc).
UV max (MeOH): 253, 267sh, 357; (NaOMe): 275, 324,
381(dec); (AlCl3): 277, 365, 412; (AlCl3/HCl): 276, 357,
399, 412; (NaOAc): 274, 324, 391; (NaOAc/H3BO3): 254,
267, 379. 1H NMR:
= 7.53 (s, H-2'/6'), 6.45 (d, 2 Hz, H-8),
6.20 (d, 2 Hz, H-6), 5.62 (d, 6.8 Hz, H-1''). 3.85 (s, 4'-
OCH3).
Myricetin 3-O--galactoside-4'-methylether (11)
Yellow powder, PC Rf 0.55 (BAW), 0.32 (15% HOAc).
UV max (MeOH): 254, 266sh, 339, 358; (NaOMe): 273,
324, 422 (dec); (AlCl3): 273, 306, 396, 403; (AlCl3/HCl):
273, 306, 358, 400; (NaOAc): 274, 324, 366, 422;
(NaOAc/H3BO3): 255, 339, 361. 1H NMR:
= 7.52 (s, H-
192 Current Chemical Biology, 2013, Vol. 7, No. 2 Hawas et al.

100
90

Scavenging Activity (%)

80
70
60
50
40
30
20
10
0
Extract 1 2 3 5 6 7 8 9 10 11 BHT
Compounds

*LSD 0.05%=0.914
Values are means ±S.D. (n=3).

Fig. (1). DPPH radical-scavenging activities (%) of E. campestre extract and isolated compounds (0.5 mg/ml).

2.5

2
Absorbance(700nm)

1.5

0.5

0
Extract 1 2 3 5 6 7 8 9 10 11 BHT

Compounds

*LSD 0.05%=0.105
Values are means ±S.D. (n=3)

Fig. (2). Reducing Power of E. campestre extract and isolated compounds (0.5 mg/ml).

2'/6'), 6.42 (d, 2 Hz, H-8), 6.17 (d, 2 Hz, H-6), 5.58 (d, 6.5
Hz, H-1''), 3.83 (s, 4'-OCH3).
DPPH Free Radical Scavenging Activity
The stable radical DPPH has been used widely for the
determination of primary antioxidant activity. The assay is
based on the reduction of DPPH radicals in methanol which
causes an absorbance drop at 517nm. As shown in Fig. (1),
the antioxidant activity of E. campestre extract exhibited
relatively high DPPH free radical scavenging activity
(66.3%). Among the isolated compounds, rutin (3) showed
the highest DPPH free radical scavenging activity (56.2 %)
followed by isorhamnetin 3-O--glucoside 7-O--
rhamnoside (8) and isorhamnetin 3-O--galactoside (5) with
55.1% and 51.1% free radical scavenging activities,
respectively, whereas myricetin 3-O--glucoside-3'-
methylether (9) exhibited the lowest DPPH free radical
scavenging activity (20.53%).
Reducing Power
Fe (III) reduction is often used as an indicator of
electrondonating activity, which is an important mechanism
of phenolic antioxidant action [16]. From Fig. (2), it was
found that E. campestre extract revealed significant reducing
power (1.8) compared to the isolated flavonol glycosides 1-3
and 5-11. Quercetin 3-O--glucuronide-4'-methylether (2),
isorhamnetin 3-O--glucoside-7-O--rhamnoside (8) and
rutin (3) showed relatively similar reducing power activity
(1.47, 1.43 and1.34 absorbance at 700 nm), respectively.
Anti-Alzheimer Activity
In this study we examined whether the extract E.
campestre alters APP processing and generation of A,
particularly the A 42 species. H4 human neuroglioma cells
expressing the double Swedish mutation (K595N/M596L) of
human APP (APPsw) were used and treated with increasing
concentrations of the E. campestre extract, and analysed for
A 40 and A 42 levels in culture medium by sensitive
sandwich enzyme link immunosorbent assay (ELISA).
In H4 cells, a 73% reduction in the A 42 supernatant
concentration was achieved at concentration of 300 ng/ml
without significant reduction in total A (A 40 + A 42)
level. A dose-dependent inhibition of A 42 secretion was
observed (Fig. 4) with IC50 of (155.75 ± 7.43 ng/ml). No
toxicity was detected by both standard MTT assay and LDH
assay in H 4 cells treated with extract concentration up to
500 ng/ml. In addition, the inhibition activity of different
concentrations of the extract against COX-1 and COX-2 was
Anti-Alzheimer, Antioxidant Activities and Flavonol Glycosides
also investigated. The results showed no inhibition activity
of the extract against COX-1 and COX-2 up to 400 ng/ml
concentration.
DISCUSSION
Dry aerial parts of E. campestre were extracted with 70%
MeOH then subjected to successive fractional partition by
chloroform and ethyl acetate. The ethyl acetate was
chromatographed on a polyamide column followed by
repeated CC on Sephadex LH-20 or cellulose and
preparative paper chromatography, to obtain the pure eleven
flavonoids 1-11.
All compounds appeared as dark purple spots on PC
under UV light, changing to yellow when exposed to
ammonia vapor, except compounds 2 and 10 where no
change in color was observed which indicates flavonoids 2
and 10 with a free 5- and substituted 4´-hydroxyl groups
[17]. Complete acid hydrolysis for isolated flavonol O-
glycosides was carried out, and followed by paper co-
chromatography with authentic samples to identify the
hydrolytic products whether flavonol aglycons and sugar
moieties.
The UV spectra of the isolated compounds 1-11 in
MeOH are characteristic of flavonols showing an intense
band I between 352-361 nm. The bathochromic shift of 42-
55 nm observed for band Ia after addition of the reagent
AlCl3/ HCl indicates 5-hydroxy-3-substituted flavonols. The
absence of a band II bathochromic shift in the presence of
NaOAc is in agreement with a 7-O-substitution as proposed
for flavonol diglycoside (8). A band I hypsochromic shift
with low intensity after addition of NaOMe was observed for
flavonols 2 and 10. The 1H NMR spectra of flavonols 1-11
OH
OR1
HO O
OR2
OH O OH
1: R1 = H, R2 = Glucose
2: R1 = CH3, R2 = Glucuronic acid
3: R1= H, R2 = Rutinoside
OH
O OH
CH3
HO O
OH
OH
O
O
OH O
OH
9 10: R = Glucose
Fig. (3).
Current Chemical Biology, 2013, Vol. 7, No. 2 193
showed only the signals of A- and B-ring, while in 13C NMR
spectra the characteristic carbon resonance of flavonol C-3
(
C 133-135 ppm) was observed. 2D-NMR experiments were
also used to confirm the glycosidic linkage in proposed
structures of flavonol diglycosides (3, 7 and 8) as well as the
di-substituted flavonols (2, 9 and 10).
Phenols are very important plant constituents because of
their scavenging ability due to their hydroxyl groups [18].
The aqueous methanol extract of E. campestre showed the
highest scavenging activity against DPPH. This activity
attributed to the presence of flavonoid contents of this
species. In this study, chemical structure-activity has shown
that quercetin diglycoside (3) are a potent peroxyl radical
scavenger followed by O-methylated derivatives (5). This
finding is in accordance with the results reported by Bors
and co-works (1990) [19] and confirms that the O-dihydroxy
structure in the B-ring is important for enhanced radical
scavenging activity. It has been demonstrated that the spin
distribution during oxidation of quercetin remain entirely on
the B-ring favouring the donation of two electrons leading to
the formation of an ortho-quinone. This has also been related
to the total conjugation of quercetin molecule over the B and
C rings. Flavonols containing a 4'-monohydroxyl group are
less potent antioxidant, the mechanism of action probably via
formation of a phenoxyl radical.
Different studies have indicated that the electron
donation capacity reflects the reducing power of bioactive
compounds is associated with antioxidant activity. In this
study, the reducing power assay using Fe (III) reduction as
an indicator of electron donating showed the presence of
antioxidants in the sample reduced the Fe (III) to Fe (II) by
donating an electron. Amount of Fe (II) complex can be then
CH3
O
OH
R1O O
OR2
O
4: R1 = H, R2 = Glucose
5: R1 = H, R2 = Galactose
6: R1 = H, R2 = Rahmnose
7: R1 = H, R2 = Rutinoside
8: R1 = Rhamnose, R2 = Glucose
O
CH3
HO O
OH
OH OR
OH
OH O
11: R = Galactose
194 Current Chemical Biology, 2013, Vol. 7, No. 2
Fig. (4). Analysis of A from H4 human neuroglioma cultured cells treated with the E. campestre extract by ELISA. A 42 and total A
levels (A 40 + A 42 values) were normalized to values obtained from vehicle-treated cells. Treatment with extract preferentially reduced
A levels in the medium of H4 human neuroglioma cells in a dose-dependent manner. Data are shown as the mean ± s.d. of all experiments.
be monitored by measuring the formation of Perl’s Prussian
blue (Fe4[-Fe(CN)6]3) at 700 nm. The methanol extract of
E. campestre and the isolated flavonol glycosides 1-3 and 5-
11 exhibited moderate reducing power comparable with that
of standard antioxidant butylated hydroxy toluene (BHT).
In this study, we describe the activity of E. campestre
extract that reduced A production by functionally inhibiting
-secretase. Therefore, we examined whether the extract
alters APP processing and generation of A, particularly the
A 42 species using H4 human neuroglioma cells expressing
the double Swedish mutation (K595N/M596L) of human
APP (APPsw). The results indicated that this extract has
reduced the A 42 level at very low concentration without
significant reduction in total A (A 40 + A 42) levels.
The cytotoxicity of E. campestre extract was assessed, in
order to insure that A 42 reduction is not related to
cytotoxicity. Furthermore, the results showed no inhibition
activity of the extract against COX-1, COX-2, and
concomitant reduction in prostaglandin synthesis, indicating
that the reduction in A 42 levels may be independent of
COX activity. Therefore, the E. campestre extract may
affects only the secretion of the A peptide without
significantly affecting the secretion of either  and  forms
of APP as the total A level did not significantly changed.
This finding suggests that the pharmacological action of E.
campestre extract is via the functional inhibition of -
secretase. The results obtained indicate that E. campestre
extract may be used as a potent candidate drug for the
treatment of Alzheimer's disease (AD) or at least ameliorate
or delay the bad effect of this disease. However, further in
vivo and pre-clinical studies will be necessary to assess
whether this extract is capable of altering -amyloidosis and
neuropathology upon prolonged treatment.
CONFLICT OF INTEREST STATEMENT
The author(s) confirm that this article content has no
conflict of interest.
Hawas et al.
ACKNOWLEDGEMENTS
Declared none.
REFERENCES
[1] Wörz A. On the distribution and relationships of the South-West
Asian species of Eryngium L. (Apiaceae-Saniculoideae). Turk J
Bot 2004; 28: 85-92.
[2] Clapham AR, Tutin TG. Flora of the British Isles. 1st ed.
Cambridge: Cambridge University Press 1952.
[3] Gruenwald J, Brendler T, Jaenicke C. PDR for Herbal Medicines.
2nd ed. New Jersey: Medical Economics Company 2000; 729-33.
[4] Kartal M, Mitaine-Offer AC, Paululat T, Abu-Asaker M, Wagner
H, Mirjolet JF, et al. Triterpene saponins from Eryngium
campestre. J Nat Prod 2006; 69(7): 1105-8.
[5] Kartnig T, Wolf J. Flavonoids from the Aboveground Parts of
Eryngium campestre. Planta Med 1993; 59(3): 285.
[6] Hohmann J, Páll Z, Günther G, Máthé I. Flavonol acyl glycosides
of the aerial parts of Eryngium campestre. Planta Med 1997; 63(1):
96.
[7] Erdelmeier CAJ, Sticher O. Coumarin derivatives from Eryngium
campestre. Planta Med 1985; 51: 407-9.
[8] Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease.
Lancet 2006; 368: 387-403.
[9] Schmidt B, Braun HA, Narlawar R. Drug development and PET-
diagnostics for Alzheimer's disease. Curr Med Chem 2005; 12:
1677-95.
[10] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical
method to evaluate antioxidant activity. LWT Food Sci Technol
1995; 28: 25-30.
[11] Oyaizu M. Studies on products of browning reactions: antioxidant
activities of products of browning reaction prepared from glucose
amine. Jap J Nutr 1986; 44: 307-15.
[12] Snedecor GW, Cochran WG. Statistical Methods. 7th Edn. Ames.
Iowa: Lowa State University Press 1980.
[13] Haugabook SJ, Yager DM, Eckman EA. High throughput screens
for the identification of compounds that alter the accumulation of
the Alzheimer's amyloid beta peptide (Abeta). J Neurosci Methods
2001; 108:171-79.
[14] Glaser K, Sung Ml, O'Neill K, Belfast M, Hartman D, Carlson R.
Etodolac selectively inhibits human prostaglandin G/H synthase 2
(PGHS-2) versus human PGHS-1. Eur J Pharmacol 1995; 281:
107-11.
[15] Hansen MB, Nielsen SE, Berg K. Re-examination and further
development of a precise and rapid dye method for measuring cell
growth /cell kill. J Immunol Methods 1989; 119: 203-10.
[16] Nabavi SM, Ebrahimzadeh MA, Nabavi SF, Fazelian M, Eslami B.
In vitro antioxidant and free radical scavenging activity of
 Anti-Alzheimer, Antioxidant Activities and Flavonol Glycosides Current Chemical Biology, 2013, Vol. 7, No. 2 195

Diospyros lotus and Pyrus boissieriana growing in Iran.
Pharmacognosy Magazine 2009; 4(18): 123-27.
[17] Harborne JB, Mabry TJ, Mabry H. The Flavonoids. London:
Chapman and Hall 1975.
[18] Hatano T, Edamatsu R, Mori A, Fujita Y, Yasuhara E. Effect of
interaction of tannins with co-existing substances. VI. Effect of
tannins and related polyphenols on superoxide anion radical and on
DPPH radical. Chem Pharm Bull 1989; 37: 2016-21
[19] Bors W, Heller W, Michel C, Saran M. Flavonoids as antioxidants:
determination of radical scavenging efficiencies. Methods enzymol
1990; 186: 343-55.

Received: June 07, 2012 Revised: July 31, 2012 Accepted: August 01, 2012

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