Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers

Abstract

The aim of this study was to investigate the antioxidant potential of Crataegus pentagyna leaves and flowers in relation to the phenolic content. The total phenolic, flavonoid and proanthocyanidin contents were determined using Folin-Ciocalteu, aluminium chloride and acid butanol assays, respectively. The radical scavenging activity was evaluated by several in vitro assays: Trolox equivalent antioxidant capacity assay, superoxide anion and hydroxyl radicals scavenging assays. In addition, ferrous ion chelating activity and 15-lipoxygenase inhibition were also investigated. Both leaf and flower extracts had high total phenolic (206.94±4.86 and 184.62±1.71 mg/g, respectively), flavonoid (57.08±0.21 and 67.04±0.52 mg/g, respectively) and proanthocyanidin contents (68.92±1.81 and 97.70±3.81 mg/g, respectively), scavenged ABTS (TEAC=0.64±0.01 and 0.65±0.01 µM Trolox equivalent to 1 µg/mL extract, respectively), superoxide (EC50=0.9±0.0 mg/mL) and hydroxyl radicals (EC50=0.9±0.0 and 0.86±0.05 mg/mL, respectively), chelated ferrous ions (EC50=1.3±0.0 and 1.9±0.0 mg/mL, respectively) and inhibited 15-lipoxygenase (EC50=129.63±0.75 and 151.76±1.65 μg/mL, respectively). It is noteworthy that both extracts showed a higher superoxide anion scavenger activity than catechin (EC50=2.2±0.0 mg/mL). The results of the study indicate that Crataegus pentagyna leaves and flowers are a promising source of natural antioxidants.

Full text

Romanian Biotechnological Letters Vol. 19, No.6, 2014
Copyright © 2014 University of Bucharest Printed in Romania. All rights reserved
ORIGINAL PAPER
Romanian Biotechnological Letters, Vol. 19, No. 6, 2014 9859
Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers
Received for publication, July 18, 2014
Accepted, November 20, 2014
CATRINEL FLORENTINA GIURESCU BEDREAG1,2, ADRIANA TRIFAN1, LAURA
ADRIANA BUCUR3, MARIANA ARCUS3, CARMEN TEBRENCU2, ANCA
MIRON1,*, IRINA IULIANA COSTACHE4,5
1 Faculty of Pharmacy, University of Medicine and Pharmacy Grigore T. Popa-Iasi,
Romania
2 Commercial Society for Medicinal Plant Research
and Processing Plantavorel-Piatra Neamt, Romania
3 Faculty of Pharmacy, Ovidius University of Constanta, Romania
4 Faculty of Medicine, University of Medicine and Pharmacy Grigore T. Popa-Iasi, Romania
5 C. I. Negoita 1st Medical Cardiology Clinic, Sf. Spiridon Emergency Clinical Hospital, Iasi,
Romania
*corresponding author: Tel.: 0746917278; Fax: +40-232-211820; e-mail: ancamiron@yahoo.com
Abstract
The aim of this study was to investigate the antioxidant potential of Crataegus pentagyna leaves
and flowers in relation to the phenolic content. The total phenolic, flavonoid and proanthocyanidin
contents were determined using Folin-Ciocalteu, aluminium chloride and acid butanol assays,
respectively. The radical scavenging activity was evaluated by several in vitro assays: Trolox
equivalent antioxidant capacity assay, superoxide anion and hydroxyl radicals scavenging assays. In
addition, ferrous ion chelating activity and 15-lipoxygenase inhibition were also investigated. Both leaf
and flower extracts had high total phenolic (206.94±4.86 and 184.62±1.71 mg/g, respectively),
flavonoid (57.08±0.21 and 67.04±0.52 mg/g, respectively) and proanthocyanidin contents (68.92±1.81
and 97.70±3.81 mg/g, respectively), scavenged ABTS (TEAC=0.64±0.01 and 0.65±0.01 µM Trolox
equivalent to 1 µg/mL extract, respectively), superoxide (EC50=0.9±0.0 mg/mL) and hydroxyl radicals
(EC50=0.9±0.0 and 0.86±0.05 mg/mL, respectively), chelated ferrous ions (EC50=1.3±0.0 and 1.9±0.0
mg/mL, respectively) and inhibited 15-lipoxygenase (EC50=129.63±0.75 and 151.76±1.65 μg/mL,
respectively). It is noteworthy that both extracts showed a higher superoxide anion scavenger activity
than catechin (EC50=2.2±0.0 mg/mL). The results of the study indicate that Crataegus pentagyna leaves
and flowers are a promising source of natural antioxidants.
Keywords: Crataegus pentagyna Willd. ssp. pentagyna, polyphenols, reactive oxygen species,
scavenging activity, chelating activity, 15-lipoxygenase, cardiovascular disease
1. Introduction
The genus Crataegus (Rosaceae, hawthorn) consists of more than 280 species that are
widespread throughout the world. Crataegus monogyna Jacq. (Lindm.) and Crataegus
laevigata (Poiret) D.C. are found and used in Europe, Crataegus pinnatifida Bunge and
Crataegus cuneata Siebold & Zucc. in China, Crataegus sinaica Boiss. in Egypt and
Crataegus aronia syn. azarolus (L.) in Israel (LJUBUNCIC & al. [1], REFAAT & al. [2],
KWOK & al. [3], JURIKOVA & al. [4]).
In Europe extracts of hawthorn leaves, flowers and fruits are used as dietary supplements
and herbal medicines in the treatment of cardiovascular disorders due to their
coronarodilating, positive inotropic, hypotensive and antiarrhythmic effects. Polyphenols,
namely flavonoids and oligomeric proanthocyanidins, have been identified as active
constituents in these extracts (VERMA & al. [5]).

CATRINEL FLORENTINA GIURESCU BEDREAG, ADRIANA TRIFAN, LAURA ADRIANA BUCUR,
MARIANA ARCUS, CARMEN TEBRENCU, ANCA MIRON, IRINA IULIANA COSTACHE
9860 Romanian Biotechnological Letters, Vol. 19, No. 6, 2014
Both in vitro and in vivo studies showed the complexity and unique profile of the activity
exhibited by hawthorn extracts on the cardiovascular system. The most studied extracts were
those isolated from Crataegus laevigata. Hawthorn extracts showed positive inotropic effects
due to the inhibition of the myocardial Na+/K+-ATPase. Hawthorn extracts decreased blood
pressure by inhibition of angiotensin converting enzyme, phosphodiesterase-3 and endothelin-
1 release from endothelium thus causing vasorelaxation. In addition, hawthorn extracts
activated endothelial nitric oxide synthase with a subsequent release of high levels of nitric
oxide, a vasodilating agent. It is worthy to note that hawthorn extracts showed the ability to
regulate not only high, but also low blood pressure. The mechanism of this dual activity has
not been elucidated yet. Hawthorn extracts enhanced coronary flow, increased the ability of
cardiomyocytes to consume oxygen, protected cardiomyocytes from ischemia-reperfusion
injury and prevented reperfusion-induced arrhythmia (JURIKOVA & al. [4]), VERMA & al.
[5]). The protective activity on cardiomyocytes was associated with a significant decrease in
oxidative stress (decrease of xanthine oxidase and NADPH oxidase expressions), upregulation
of anti-apoptotic proteins (Bcl-2, Hsp 70), downregulation of pro-apoptotic proteins
(cytochrome c, caspase-3), decrease in lactate dehydrogenase and creatine kinase activity
(SWAMINATHAN & al. [6]). Apart from cardiac glycosides, hawthorn extracts showed
negative chronotropic effects without causing arrhythmia. On the contrary, they exhibited
antiarrhythmic effects. Recent studies elucidated several mechanisms with possible
implications in the antiarrhythmic activity: prolongation of action potential duration, blockade
of potassium currents, decrease in the number of ventricular ectopic beats (MÜLLER & al.
[7], [8], GARJANI & al. [9]).
Crataegus pentagyna Willd. ssp. pentagyna is spread in the southern and south-eastern
regions of Romania (PARVU & al. [10]). A literature survey revealed only few biological
investigations on the leaves and flowers of Crataegus pentagyna growing in Turkey
(ÖZYÜREK & al. [11]). In this respect, the aim of this work was to evaluate the antioxidant
potential of the leaves and flowers of Crataegus pentagyna growing in Romania in relation to
the phenolic content. The present work is part of a larger study aiming to explore the
possibility of using Crataegus pentagyna leaf and flower extracts as ingredients in dietary
supplements for cardiovascular diseases and other pathological conditions associated with
oxidative stress.
2. Materials and methods
Plant material
Crataegus pentagyna leaves and flowers were collected in Tulcea district in June 2010
and were authenticated in the Department of General Plant and Animal Biology, Faculty of
Pharmacy, Ovidius University of Constanta. Before extraction, the leaves and flowers were
air-dried in dark at ambient temperature (25°C). Herbarium voucher samples (P-l-2010, P-f-
2010) are deposited in the Department of Pharmacognosy, Faculty of Pharmacy, University of
Medicine and Pharmacy Grigore T. Popa-Iasi.
Chemicals
Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid and ferrous chloride were
purchased from Merck (Darmstadt, Germany). 2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS), ethylenediaminetetraacetic acid (EDTA), pyrogallol, Folin-
Ciocalteu's phenol reagent, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4"-disulfonic acid
monosodium salt (ferrozine), linoleic acid, lipoxydase from soybean were from Fluka
(Steinheim, Germany). (+)-Catechin hydrate, gallic acid, sodium carbonate, iron (II) sulfate

Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers
Romanian Biotechnological Letters, Vol. 19, No. 6, 2014 9861
heptahydrate, hydrogen peroxide, sodium salicylate, (R)-(+)-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (Trolox) were from Sigma-Aldrich (Steinheim,
Germany). Ammonium iron (III) sulfate dodecahydrate, potassium persulfate and sodium
nitrite were obtained from Riedel-de Haën (Seelze, Germany). All other chemicals were of
analytical grade.
Extraction
50 g of dried and powdered plant material (leaves and flowers, respectively) were
extracted with 500 mL methanol:water (7:3, v/v) for 6 h at room temperature under
continuous stirring. The extracts were filtered under vacuum and the residues were re-
extracted twice in the same conditions. The extracts were combined, evaporated under
reduced pressure at 40°C and freeze-dried. Both extracts were kept in sealed glass tubes at -
20°C until studied.
Total phenolic content
The total phenolic content was estimated using Folin-Ciocalteu method as described
previously (WANGENSTEEN & al. [12], SINGLETON & ROSSI [13]). Briefly, each extract
(0.04 mL) was mixed with ultrapure water (3.16 mL) and Folin-Ciocalteu's phenol reagent
(0.2 mL). After 5 min, 20% sodium carbonate (0.6 mL) was added followed by vigorous
mixing. The reaction mixture was incubated for 2 h at room temperature before the
absorbance was measured at 765 nm. The total phenolic content was expressed in terms of mg
gallic acid equivalents (GAE)/g of extract.
Flavonoid content
The total flavonoid content was determined by aluminium chloride assay (OZSOY & al.
[14]). Each extract (0.25 mL) was mixed with ultrapure water (1.25 mL) and 5% sodium
nitrite (0.075 mL). After 6 min 10% aluminium chloride (0.15 mL) was added. The mixture
was incubated for another 5 min followed by the addition of 1 M sodium hydroxide (0.5 mL)
and ultrapure water (0.275 mL). After vigorous shaking the absorbance was measured
immediately at 510 nm. The total flavonoid content was expressed in terms of mg (+) catechin
equivalents/g of extract.
Proanthocyanidin content
The total proanthocyanidin content was estimated by acid butanol assay (PORTER & al.
[15]). Briefly, the reaction mixture consisted of extract (0.5 mL), n-butanol-hydrochloric acid
reagent (95:5, v/v, 3.0 mL) and ferric reagent (2% ferric ammonium sulfate dodecahydrate in
2 N hydrochloric acid, 0.1 mL). The mixture was shaken vigorously and kept in the water
bath at 95°C. After 40 min the mixture was cooled and the absorbance was recorded at 550
nm. The total proanthocyanidin content (mg cyanidin/g extract) was calculated using the
molar extinction coefficient of cyanidin ( = 17,360 L·mol-1·cm-1) (QA'DAN & al. [16]).
Trolox equivalent antioxidant capacity assay
The assay was used to evaluate the Trolox equivalent antioxidant capacity (TEAC) of
Crataegus pentagyna extracts. In brief, different concentrations of extracts or Trolox (0.02
mL) were mixed with ABTS radical cation ethanol solution (1.98 mL; A734 nm=0.70±0.02).
The decrease in absorbance at 734 nm was measured after 6 min reaction time at 30°C. (+)-
Catechin hydrate was used as positive control. For calculating TEAC values, the percentage
of absorbance decrease as a function of concentration was plotted for each extract, Trolox and
catechin. TEAC values were calculated as follows: slope of dose-response curve of extracts or
catechin/slope of dose-response curve of Trolox (RE & al. [17]).
Superoxide anion radical scavenging assay
Superoxide anion radical scavenging activity was determined by self-oxidation of 1,2,3-
phentriol (pyrogallol) assay (WANG & al. [18]). Different concentrations of each extract (0.1

CATRINEL FLORENTINA GIURESCU BEDREAG, ADRIANA TRIFAN, LAURA ADRIANA BUCUR,
MARIANA ARCUS, CARMEN TEBRENCU, ANCA MIRON, IRINA IULIANA COSTACHE
9862 Romanian Biotechnological Letters, Vol. 19, No. 6, 2014
mL) were mixed with Tris-HCl buffer (0.05 M, pH 8.0) containing 1 mM EDTA (2.8 mL)
and pyrogallol (6 mM, 0.2 mL). The mixture was vortexed and the absorbance at 325 nm was
recorded every 30 s over a period of 4 min. (+)-Catechin hydrate was used as positive control.
The inhibitory activity on the self-oxidation of 1,2,3-phentriol was calculated from the
absorbance vs. time curves using the following formula: (1-slope of extract or catechin/slope
of control) × 100.
Hydroxyl radical scavenging assay
Hydroxyl radical scavenging activity was evaluated according to a described procedure
with minor changes (JEONG & al. [19]). Different concentrations of each extract (0.225 mL)
were mixed with 20 mM sodium salicylate (0.9 mL), 1.5 mM FeSO4 (0.75 mL) and 6 mM
H2O2 (0.525 mL). After 30 min incubation at 37°C, the absorbance was measured at 562 nm.
(+)-Catechin hydrate was used as positive control. Hydroxyl radical scavenging activity (%)
was calculated using the formula: (1-absorbance in the presence of extracts or
catechin/absorbance of control) × 100.
Ferrous ion chelating assay
The ability to chelate ferrous ions was measured by ferrozine assay (DINIS & al. [20],
TUNG & al. [21]). Various concentrations of each extract (0.4 mL) were mixed with
methanol (1.48 mL), 2 mM ferrous chloride (0.04 mL) and 5 mM ferrozine (0.08 mL). After
vigorous shaking, the mixture was allowed to stand 10 min at room temperature. The
absorbance was measured at 562 nm. EDTA was used as positive control. The ferrous ion
chelating activity (%) was calculated using the equation: (1-absorbance in the presence of
extracts or EDTA/absorbance of control) × 100.
15-Lipoxygenase inhibition assay
Briefly, different concentrations of extracts (0.05 mL) were mixed with 0.2 M borate
buffer at pH 9.0 containing 10,000 U/mL of 15-lipoxygenase (0.95 mL). After 10 min
incubation at 25°C, linoleic acid in borate buffer was added (2 mL). The increase in
absorbance at 234 nm was recorded for 90 s. (+)-Catechin hydrate was used as positive
control. 15-Lipoxygenase inhibition (%) was determined on the basis of the absorbances of
the control and samples after 30 and 90 s reaction time using the formula: 100  [(Acontrol,90-
Acontrol,30)-(Asample,90-Asample,30)/(Acontrol,90-Acontrol,30)] (WANGENSTEEN & al. [12], CRETU &
al. [22]).
Statistical analysis
All experiments were performed in triplicate and all data were presented as mean values
± standard deviations. The EC50 values were calculated by linear interpolation between values
above and below 50% activity.
3. Results and discussions
Contents of total phenols, flavonoids and proanthocyanidins
Previous studies have shown that polyphenols, mainly flavonoids and proanthocyanidins,
are the major bioactive compounds in hawthorn species. They are responsible for many of the
pharmacological effects of hawthorn extracts (antioxidant, cardiovascular, anti-inflammatory
effects) (REFAAT & al. [2], VERMA & al. [5]). Therefore, the total phenolic, flavonoid and
proanthocyanidin contents in Crataegus pentagyna extracts were estimated according to the
methods described by SINGLETON & ROSSI [13], OZSOY & al. [14] and PORTER & al.
[15], respectively. These three methods are simple, rapid and inexpensive. Even if these
methods have a limited specificity, they are routinely used for the quantitative estimation of
the above mentioned phytochemicals and give accurate results (SINGLETON & ROSSI [13],
OZSOY & al. [14], PORTER & al. [15], KELM & al. [23], BLAINSKI & al. [24]). The total

Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers
Romanian Biotechnological Letters, Vol. 19, No. 6, 2014 9863
phenolic content was higher in leaf extract (206.94±4.86 mg/g) in comparison to flower
extract (184.62±1.71 mg/g). Flower extract had a higher flavonoid content than leaf extract
(67.04±0.52 and 57.08±0.21 mg/g, respectively). Likewise, proanthocyanidin content,
calculated as cyanidin equivalents, was higher in flower extract (97.70±3.81 mg/g) in
comparison to leaf extract (68.92±1.81 mg/g) (Table 1).
Table 1. Total phenolic, flavonoid and proanthocyanidin contents in Crataegus pentagyna
extracts.
Extract Total phenolic content
(mg gallic acid/g extract)
Flavonoid content
(mg (+)-catechin/g extract)
Proanthocyanidin
content
(mg cyanidin/g extract)
Leaf extract 206.94 ± 4.86 57.08 ± 0.21 68.92 ± 1.81
Flower extract 184.62 ± 1.71 67.04 ± 0.52 97.70 ± 3.81
Several studies have reported total phenolic contents ranging from 108.65 to 343.54 mg/g
in different extracts from Crataegus monogyna leaves with flowers (ÖZTÜRK & al. [25]),
total phenolic and flavonoid contents of 38.25-396.04 and 2.12-32.62 mg/g, respectively in
Crataegus azarolus leaf extracts (AMEL & al. [26]). In view of these literature data, it may be
concluded that both Crataegus pentagyna extracts contain significant amounts of phenolic
compounds and therefore they are expected to have strong antioxidant activity.
Free radical scavenging activity
Free radical scavenging ability of Crataegus pentagyna extracts was initially evaluated
by TEAC assay. Flower and leaf extracts showed similar TEAC values (0.65±0.01 and
0.64±0.01 µM Trolox equivalent to 1 µg/mL extract, respectively). TEAC value of catechin
was higher (5.46±0.16 µM Trolox equivalent to 1 µg/mL catechin) suggesting a stronger
antioxidant capacity (Table 2). Free radical scavenging effects of Crataegus pentagyna
extracts were further evaluated against superoxide anion and hydroxyl radicals, reactive
oxygen species involved in the pathogenesis of many cardiovascular disorders.
Oxidative stress plays an important role in the development and progression of many
cardiovascular diseases (ischemia, atherosclerosis, cardiomyopathy, cardiac hypertrophy,
congestive heart failure, hypertension) and cardiovascular complications of diabetes mellitus
(pheripheral and coronary artery diseases, diabetic retinopathy) (PHAM-HUY & al. [27],
BADESCU & al. [28]).
Reperfusion of the ischemic myocardial regions is the only way to restore normal blood
flow and prevent myocardial infarction. The increase in blood flow is associated with an
increase of oxygen level in the myocardial tissue, generation of reactive oxygen species and
oxidative stress-induced damage of myocardial tissue (ischemia-reperfusion injury) (MIMIĆ-
OKA & al. [29]). Generation of superoxide anion radical is a key event in ischemia-
reperfusion injury. It is mainly produced by xanthine oxidase, impaired mitochondrial activity
and neutrophil activity. Superoxide anion radical and other reactive oxygen species (hydrogen
peroxide, hydroxyl radical) produce oxidative damage to cardiomyocytes and endothelial cells
with an increase in vascular permeability (GALVEZ & al. [30]).
Superoxide anion radical is indirectly involved in the development of atherosclerosis; it
reduces ferric ions to ferrous ions, the latter generating hydroxyl radical via Fenton reaction.
Hydroxyl radical oxidizes polyunsaturated fatty acids in low-density lipoproteins, a process
which initiates the formation of atherosclerotique plaques. In addition, the final products of
lipid peroxidation (4-hydroxy-nonenal) are cytotoxic leading to tissue injury (GALVEZ & al.
[30]).

CATRINEL FLORENTINA GIURESCU BEDREAG, ADRIANA TRIFAN, LAURA ADRIANA BUCUR,
MARIANA ARCUS, CARMEN TEBRENCU, ANCA MIRON, IRINA IULIANA COSTACHE
9864 Romanian Biotechnological Letters, Vol. 19, No. 6, 2014
In hypertension, circulating leukocytes produce high levels of superoxide anion radical
and hydrogen peroxide. Superoxide anion radical converts the vasodilating nitric oxide into
the highly cytotoxic peroxynitrite anion. The decrease in nitric oxide levels causes arteriolar
vasoconstriction. Lipid peroxidation is enhanced leading to final products with
vasoconstrictor effects such as F2-isoprostanes. Reactive oxygen species promote other events
associated with hypertension (proliferation of vascular smooth muscle cells, deposition of
collagen on arterial wall) (MIMIĆ-OKA & al. [29], GROSSMAN [31]).
Reactive oxygen species induce myocardial damage resulting in cardiomyopathy.
Besides, reactive oxygen species activate enzymes (mitogen-activated protein kinases) and
transcription factors (nuclear factor-kB) which stimulate not only cardiac myocytes to
undergo hypertrophy, but also interstitial and perivascular fibrosis. Both cardiomyopathy and
cardiac hypertrophy increase significantly the risk of heart failure (MAULIK & al. [32]).
All these data clearly indicate that a reduction of oxidative stress might be beneficial in
the management of many cardiovascular diseases.
In superoxide anion radical scavenging assay, both extracts showed similar effects
(EC50=0.9±0.0 mg/mL) being more active than the positive control, catechin (EC50=2.2±0.0
mg/mL). Crataegus pentagyna extracts scavenged hydroxyl radical with comparable EC50
values (0.86±0.05 mg/mL for flower extract; 0.9±0.0 mg/mL for leaf extract); according to
the EC50 values, both extracts were only three times less active than catechin
(EC50=0.27±0.02 mg/mL) (Table 2). As catechin has been reported to be an efficient
scavenger of superoxide and hydroxyl radicals (KASHIMA, [33]), it is obvious that
Crataegus pentagyna extracts exhibited significant scavenging effects against both radicals.
Ferrous ion chelating activity
In living organisms iron exists as ferrous and ferric ions. The ferric ions are relatively
biologically inactive while ferrous ions are involved in the generation of hydroxyl radical
through Fenton reaction (VALKO & al. [34]). Therefore, ferrous ions chelation is one of the
main mechanisms of reduction of oxidative stress. Flower and leaf extracts chelated ferrous
ions with EC50 values of 1.9±0.0 and 1.3±0.0 mg/mL, respectively; EDTA, the positive
control, showed a significantly higher chelating capacity (EC50=6.46±0.25 μg/mL) (Table 2).
15-Lipoxygenase inhibition
15-Lipoxygenase plays a key role in the initiation and development of early
atheroslerotique lesions. The enzyme oxidizes polyunsaturated fatty acids in low density
lipoproteins and induces the expression of vascular cellular adhesion molecule 1 (VCAM-1)
and intercellular adhesion molecule 1 (ICAM-1) thus promoting monocyte chemotaxis and
adhesion to endothelial cells (BOLIK & al. [35]). According to the EC50 values, leaf extract
showed a higher inhibitory activity towards 15-lipoxygenase than flower extract (129.63±0.75
and 151.76±1.65 μg/mL, respectively). Both extracts were less active than the positive
control, catechin (EC50=27.66±0.57 μg/mL) (Table 2).
Table 2. Antioxidant activity of Crataegus pentagyna extracts.
Extract/
Positive control
Trolox
equivalent
antioxidant
capacity
(TEAC)
Superoxide
anion radical
scavenging
activity (EC50)
Hydroxyl radical
scavenging
activity
(EC50)
Ferrous ion
chelating
activity
(EC50)
15-Lipoxygenase
inhibitory
activity
(EC50)
Leaf extract 0.64±0.01* 0.9 ± 0.0*** 0.9 ± 0.0*** 1.3 ± 0.0*** 129.63 ± 0.75**
Flower extract 0.65±0.01* 0.9 ± 0.0*** 0.86 ± 0.05*** 1.9 ± 0.0*** 151.76 ± 1.65**
(+)-Catechin 5.46±0.16* 2.2 ± 0.0*** 0.27 ± 0.02*** - 27.66 ± 0.57**
EDTA - - - 6.46 ± 0.25** -
* µM Trolox equivalent to 1 µg/mL extract/catechin; ** μg/mL; *** mg/mL

Chemical and antioxidant studies on Crataegus pentagyna leaves and flowers
Romanian Biotechnological Letters, Vol. 19, No. 6, 2014 9865
Antioxidant activity has already been reported for other Crataegus species (C.
oxyacantha, C. orientalis, C. aronia syn. azarolus, C. monogyna, C. meyeri, C. pontica). An
ethanol extract of Crataegus orientalis leaves showed good activity in diphenylpicrylhydrazyl
(DPPH) radical scavenging assay (62.91±3.40% at 10 mg/mL) and β-carotene bleaching
assay (42.37% at 1 mg/mL) (BOR & al. [36]). An aqueous extract of Crataegus aronia leaves
inhibited the oxidation of β-carotene, 2,2'-azobis(2-amidino-propan) dihydrochloride
(AAPH)-induced plasma oxidation, iron-induced lipid peroxidation in rat liver homogenate
and increased intracellular glutathione levels; in addition, the extract showed a significant
superoxide anion scavenging activity (50% at 42±6 μg/mL in xanthine-xanthine oxidase
assay) (LJUBUNCIC & al. [1]). The antioxidant activity of methanol extracts of the leaves
and flowers belonging to 14 Crataegus species growing in different regions of Turkey was
investigated by different assays: CUPRAC (cupric reducing antioxidant capacity), FRAP
(ferric reducing antioxidant power), ABTS/persulfate and Folin assays. An extract of
Crataegus pentagyna leaves proved to be the most active among other Crataegus extracts
(TEACCUPRAC=0.378±0.004, TEACFRAP=0.132±0.001, TEACABTS=0.423±0.112 and
TEACFolin=0.752±0.201 mmol TR g-1). To the best of our knowledge, this is the single report
on the antioxidant activity of Crataegus pentagyna leaves and flowers, evaluating only the
ability to reduce cupric and ferric ions, ABTS radical cation and Folin reagent (ÖZYÜREK &
al. [11]). A comparison of the results in ABTS/persulfate assay with our data is difficult due
to different experimental protocols. However, besides ABTS radical cation scavenging
activity, our study evaluated the capacity to scavenge reactive oxygen species, chelate ferrous
ions and inhibit 15-lipoxygenase. In our study, according to the EC50 values, Crataegus
pentagyna extracts proved to be remarkable superoxide anion radical scavengers but also
efficient hydroxyl radical scavengers and inhibitors of 15-lipoxygenase.
4. Conclusions
Our study demonstrated that extracts of Crataegus pentagyna leaves and flowers contain
high levels of polyphenols and possess marked antioxidant effects. These results indicate a
potential health benefit of Crataegus pentagyna leaf and flower extracts in oxidative stress-
related cardiovascular disorders. The in vivo antioxidant and cardioprotective effects of
Crataegus pentagyna extracts need to be evaluated.
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