Iranian Journal of Pharmaceutical Research (2018), 17 (3): 1036-1046
Received: January 2016
Accepted: December 2016
Original Article
Evaluation of Chemical Composition and In-vitro Biological Activities of
Three Endemic Hypericum Species from Anatolia
(H. thymbrifolium, H. spectabile and H. pseudolaeve)
Esra Eroglu Ozkana*, Nurten Ozsoyb, Tugba Yilmaz Ozdenb, Gul Ozhanc and Afife Mata
a
Department of Pharmacognosy, Faculty of Pharmacy, Istanbul University, Istanbul,
Turkey. bDepartment of Biochemistry, Faculty of Pharmacy, Istanbul University, Istanbul,
Turkey. cDepartment of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University,
Istanbul, Turkey.
Abstract
In the present work we carried out a phytochemical and biological investigation on three
endemic Hypericum species, i.e. Hypericum thymbrifolium (H. thymbrifolium), Hypericum
spectabile (H. spectabile) and Hypericum pseudolaeve (H. pseudolaeve) from Anatolia in
order to discover new sources of natural compounds for the treatment of inflammatory and
neurodegenerative disorders. HPLC-DAD analysis indicated that two naphthodianthrones
(pseudohypericin and hypericin) together with chlorogenic acid, rutin, hyperoside, isoquercitrin,
kaempferol, quercitrin, quercetin, amentoflavone, and hyperforin are the main compounds
present in the methanol extracts. After chemical characterization, all extracts were in-vitro
biologically assayed for antioxidant potential by lipid peroxidation inhibitory activity, DPPH,
FRAP assays, and superoxide radical scavenging activity, for AChE inhibitory activity by
Ellman’s method, for COX inhibitory activity by using enzyme immunoassay (EIA) kit, for
cytotoxic activity on HeLa and NRK-52E cell lines by MTT assay. The superoxide radical
scavenging activity and lipid peroxidation inhibitory activity of H. spectabile (EC50 = 0.430
mg/mL) were more remarkable than that of H. thymbrifolium and H. pseudolaeve. The extracts
showed moderate inhibitory activity on AChE (from 49.37% to 63.41%). The best inhibitory
activity against COX-1 (71.77% and 77.04%, respectively) and COX-2 ( 64.14% and 72.23%,
respectively) were shown by H. thymbrifolium and H. spectabile, which may be due to their
richest chlorogenic acid content (0.29576% and 0.23567%, respectively). Cytotoxicity
screening results showed that the extracts did not demonstrate significant cytotoxic activity. It
was concluded that the most promising extract with antioxidant, anti-inflammatory, and AChE
inhibition potential is H. spectabile.
Keywords: Hypericum; Endemic; HPLC; Chemical composition; Cyclooxygenase
inhibition; Alzheimer’s disease.
Introduction
The genus Hypericum L. (Hypericaceae) has
* Corresponding author:
E-mail: esraeroglu@gmail.com
been used for centuries for the treatment of burns,
bruises, swelling, inflammation, and anxiety, as
well as bacterial and viral infections. Locally, it
is traditionally used both externally (as a cream
or oil extract) and internally (as a tea) with many
therapeutic applications (1-4). Hypericum has
Phytochemical and Activity Studies on Hypericum Species
nearly 465 species all over the world and is
represented by nearly 100 taxa grouped under
19 sections in Turkey, among them, 45 taxa are
endemic (5-7).
For the last few years, there has been an
increasing biological activity trend and awareness
in Hypericum research. Quite a significant
amount of research has already been carried out
in exploring the chemistry of different parts of
Hypericum (8-15). The pharmacological studies
showed that this species have several activities,
namely, antidepressant, anti-inflammatory,
antimicrobial, antiviral, antinociceptive, and
wound healing (5). In the recent years, antidepressant applications of Hypericum medical
products have become increasingly popular (16).
The antidepressant activity was first attributed
to hypericins (naphthodianthrone derivatives),
but recent pharmacological and clinical
results focus on hyperforins (phloroglucinol
derivatives) as the main active ingredients of
the extract. Hypericum perforatum L. (St. John’s
wort) preparative forms have recently gained
popularity as an alternative treatment for mild
to moderate depression (17). It is heartening to
see that a traditional plant medicine has now led
to several therapeutically useful preparations,
which encourage the scientists to explore more
information about this medicinal plant. It has
been reported that Hypericum species contain
variety of phenolic compounds and represent
good sources of antioxidants which increase
their usability potential in ethnomedicine (9, 13).
Numerous antioxidant investigations have been
carried out on Hypericum species (18-26).
It has been reported that Hypericum species
have anti-inflammatory activity in-vitro and
in different animal models of edema possibly
due to inhibition of inducible nitric oxide
synthase (iNOS) and cyclooxygenase-2 (COX2) expression (27, 28). COX-2 is responsible
for the production of pro-inflammatory
mediators, prostaglandins, at the inflammatory
site (29). Recognition of COX-2’s key role in
inflammation led to the hypothesis that it may
represent a primary target for non-steroidal antiinflammatory drugs (NSAIDs) in Alzheimer’s
disease (AD), consistent with inflammatory
processes occurring in AD brain (30).
Cholinesterase inhibitors are the first-line
treatment for AD. Acetylcholinesterase (AChE)
inhibitory potential of some Hypericum species
has been reported in previous studies (31-35).
Analysis of the cytotoxicity and anticancer
cell proliferation activity was conducted in
a variety of Hypericum species (36). It was
reported that H. perforatum has no cytotoxic
potential and oral consumption by humans is
safe (37).
Considering the important role of oxidative
stress and inflammation in the pathogenesis
of neurological diseases such as AD, and the
growing evidence of the presence of compounds
with antioxidant, anti-inflammatory and AChE
inhibitory potential in different Hypericum
species; the aim of the present study was to
investigate the chemical profiles and antioxidant,
anti-inflammatory, anti-AChE and cytotoxic
potential of the extracts from three endemic
Hypericum species (Hypericum thymbrifolium
Boiss. and Noë, Hypericum spectabile Jaub. and
Spach., Hypericum pseudolaeve Robson) of the
Turkish flora. Our research is the first report to
study the phytochemical profiles and biological
activities in these species.
Experimental
Chemical agents
Hypericin,
chlorogenic
acid,
rutin,
hyperoside, isoquercitrin, quercitrin, kaempferol,
quercetin, amentoflavon, hyperforin, AlCl3, and
D-galactose were obtained from Sigma-Aldrich
(Taufkirchen, Germany). Pseudohypericin
was obtained from PhytoPlan (Heidelberg,
Germany). Milli-Q ultrapure water was obtained
from Millipore (Billerica, MA, USA), HPLC
grade acetonitrile, methanol, ethyl acetate and
sodium dihydrogen phosphate dihydrate were
obtained from Merck (Darmstadt, Germany) and
ortho-phosphoric acid 85% was obtained from
Fluka (Buchs, Switzerland).
Nitroblue tetrazolium (NBT), β-nicotinamide
adenine dinucleotide reduced (β-NADH),
soybean L-α-phosphatidylcholine type IV-S,
quercetin and catechin were purchased from Fluka
(Buchs, Switzerland). Phenazine methosulphate
(PMS), 2,2-diphenyl-1-picryl-hydrazyl (DPPH),
gallic acid, ascorbic acid, 5,5’-dithiobis-(2nitrobenzoic acid) (DTNB), acetylthiocholine
1037
Eroglu Ozkan E et al. / IJPR (2018), 17 (3): 1036-1046
iodide
(ATChI),
AChE,
galantamine
hydrobromide were obtained from SigmaAldrich (St. Louis, MO, USA). 2,4,6-tripyridyls-triazine (TPTZ), trichloroacetic acid (TCA),
thiobarbituric acid (TBA) and ferric chloride
were obtained from Merck (Darmstadt,
Germany). Enzyme immunoassay (EIA) kit and
aspirin were obtained from Cayman Chemical
(Ann Arbor, MI, USA).
3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reagent
was purchased from Sigma (St. Louis, MO,
USA). Dimethylsulfoxide (DMSO), trypsin
was purchased from (Biomatik, Canada).
Ethylenediamine tetra acetic acid (EDTA),
sodium hydroxide was purchased from Merck
(Darmstadt, Germany). Fetal bovine serum (FBS),
Dulbecco’s Modified Eagle Medium (DMEM),
penicillin-streptomycin, phosphate buffer saline
(PBS) were purchased from Multicell-Wisent
Inc. (Quebec, Canada). Cytotoxicity detection kit
(LDH) which containing catalyst, dye solution
and stop solution were purchased from Roche
(Mannheim, Germany). All other reagents were
of analytical grade.
Plant material
During the field investigations conducted
in June 2010, specimens of flowering aerial
parts of H. thymbrifolium, H. spectabile and H.
pseudolaeve were gathered from their natural
habitats on the roadsides nearby the town of
Malatya located in the East Anatolia Reagion
of Turkey: H. thymbrifolium (Malatya: Malatya
to Darende, 10 km to Gürün, 1425 m), H.
spectabile: (Malatya: Arapgir to Kemaliye, 40
km to Kemaliye, 1157 m) and H. pseudolaeve
(Malatya: Malatya to Arapgir, 20 km to Arapgir,
1264 m). The plant materials were identified by
Prof. Dr. Şükran Kültür and voucher specimens
were deposited in the Herbarium of the Istanbul
University Faculty of Pharmacy, Istanbul, Turkey
(ISTE 93194, 93192 and 93193, respectively).
Preparation of the extracts
The samples were air-dried at room
temperature under shade. The dried flowering
aerial parts (10 g) of the species were macerated
in methanol (100 mL) for 3 days at room
temperature at dark and the resulting extract was
filtered through Whatman No-1. The residue
from the filtration was extracted again twice
using the same procedure. The filtrates were
combined and then evaporated to dryness under
reduced pressure at a temperature below 45 °C.
The crude methanol extract was lyophilized and
stored at -20 °C (38, 39). The extracts prepared
with this procedure were used in the HPLC
analysis and biological activity studies.
HPLC analysis
Preparation of the standards
The calibration curves were prepared with
analytical standards at the different concentration
in methanol. The experiment was conducted
three times providing the same conditions. The
calibration curves were constructed by using
average of peak areas and at least five different
standard concentrations.
Preparation of the samples
The crude methanol extract was dissolved in
mixture of methanol/water (8:2, v/v) (40). All
samples were filtered through a 0.45 µm filter
into a vial for HPLC analysis. Each sample was
prepared and injected three times.
Chromatographic HPLC conditions
The Hypericum species have been analyzed
by reversed phase HPLC coupled with DAD
(HPLC-DAD). The HPLC system consisted of
a Shimadzu 10A model (DAD: SPD-M10A),
pump: LC-10AD and an autosampler: SIL10AD.
The separation was accomplished on an
ACE C18 (250 × 4.6 mm, particle size 5 µm)
(Advanced Chromatography Technologies,
Alberdeen, Scotland) column. The elution
conditions were as follows: flow rate: 1 mL/min;
column temperature: 40 °C; injection volume:
10 µL; detection: 590 nm for pseudohypericin
and hypericin, 360 nm for phenolic compounds
and 275 nm for hyperforin.
The solvent system was used as an isocratic
to identify and quantitate pseudohypericin
and hypericin. Separation was carried out
using solvent A [ethyl acetate/15.6 g/L sodium
dihydrogen phosphate adjusted to pH 2 with
phosphoric acid/methanol (39:41:160, v/v/v)].
The solvent system was used as a gradient to
1038
Phytochemical and Activity Studies on Hypericum Species
identify and quantitate phenolic compounds
and hyperforin. The mobile phase consisted of
solvent A (0.3% formic acid in water (v/v) and
solvent B (0.3% formic acid in acetonitrile (v/v).
The following gradient was applied: 0-8 min,
82% A; 8-18 min, 82-47% A; 18-18.1 min, 473% A; 18.1-29 min, 3% A; 29-40 min, 3-82% A
(European Pharmacopoeia, 2008). All solvents
were filtered through a 0.45 µm filter prior to use
and degassed in an ultrasonic bath.
The control of the system and the data analysis
procedure were performed with Shimadzu LC
Solutions software.
Determination of total phenolic compounds
Total soluble phenolics in the methanolic
extracts were determined with Folin-Ciocalteu
reagent according to the method of Slinkard
and Singleton with some modifications (41).
The amount of total phenolic compounds
was calculated from the calibration curve of
gallic acid standard solution (covering the
concentration range between 0.05 and 0.4 mg/
mL) and expressed as mg gallic acid equivalents
(GAE)/g dry weight (DW) of the plant material.
Determination of total flavonoid content
Total flavonoid content was determined by
using a method described by Sakanaka et al.
(2005). Total flavonoid contents were calculated
from the calibration curve prepared with
catechin standard solution and expressed mg of
(+)-catechin equivalents (CE) per g of DW of the
plant material.
Determination of antioxidant activity
Quercetin was used as reference antioxidant
for the antioxidant activity assays.
Inhibition of lipid peroxidation (LPO)
LPO assay was based on the method
described by Duh et al. (42). The formation of
LPO products was assayed by the measurement
of malondialdehyde (MDA) levels on the basis
of MDA reacted with TBA at 532 nm according
to Buege and Aust (43). The percentage
inhibition of LPO was calculated by comparing
the results of the sample with those of controls
not treated with the extract using the following
Equation:
Inhibition effect (%) = (1 − absorbance of
sample at 532 nm/absorbance of control at 532
nm) × 100.
DPPH radical scavenging activity
The DPPH radical scavenging activity of the
methanolic extracts was measured according to
the procedure described by Brand-Williams et
al. (44). The ability to scavenge DPPH radical
was calculated by the following Equation:
DPPH radical scavenging activity (%) =
(1 − absorbance of sample at 517 nm/
absorbance of control at 517 nm) × 100.
Superoxide radical scavenging activity
The effects of the methanolic extracts
on generation of superoxide radicals were
determined by the NBT reduction method (45).
The abilities to scavenge the superoxide radical
were calculated by comparing the results of the
sample with those of controls not treated with
the extract using the following Equation:
Superoxide radical scavenging activity (%) =
(1 − absorbance of sample at 560 nm/absorbance
of control at 560 nm) × 100.
Ferric reducing antioxidant power (FRAP)
assay
The FRAP assay was carried out according
to the procedure of Benzie and Strain (46).
The standard curve was constructed using iron
sulphate heptahydrate solution (125-2000 µM),
and the results were expressed as mM Fe2+
equivalents.
Determination of AChE inhibitory activity
The extracts were screened for their AChE
inhibitory activity through the modified Ellman›s
spectrophotometric method (47). Galantamine
hydrobromide was used as a standard and tested
in a concentration range between 12.5 to 100
µg/mL (33.75 to 270 µM). Any increase in
absorbance due to the spontaneous hydrolysis of
substrate was corrected by subtracting the rate
of the reaction before adding the enzyme from
the rate after adding the enzyme.
AChE Inhibition (%) = (1 – reaction rate
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Eroglu Ozkan E et al. / IJPR (2018), 17 (3): 1036-1046
of sample at 412 nm/reaction rate of negative
control at 412 nm) × 100.
Determination of COX inhibitory activity
The ability of the extracts to inhibit ovine
COX-1 and COX-2 was determined by
calculating percent inhibition of prostaglandin
production using EIA kit (Catalogue No.
560101, Cayman Chemical) according to the
manufacturer›s instructions. Aspirin was used
as a standard.
In-vitro cytotoxic activity
Cytotoxicity of Hypericum species at various
concentrations was determined on human cervix
adenocarcinoma (HeLa, ATCC® CCL-2™) and
normal rat kidney epithelial (NRK-52E, ATCC®
CRL-6509™) cell lines by the MTT assay,
which is widely used for the measurement of
cell viability (48, 49). Briefly, the cells were
seeded in 96-well plates at a density of 104
cells/well in 100 μL culture medium. Following
24-h incubation and attachment, the cells were
treated with different concentrations of plant
extracts and controls for 24 h. Dry methanolic
extracts were dissolved in DMSO as a solvent
to obtain appropriate stock solutions of the
extracts. Dilution of stock extracts solutions
was made in serum free medium yielding final
extracts concentrations from 0.125 to 2 mg/mL.
DMSO and 5-fluorouracil (5-FU) were used
solvent and positive controls, respectively (32,
50). The concentration range used for 5-FU
was 50 to 1000 μM. The yellow MTT dye
was reduced by succinic dehydrogenase in the
mitochondria of viable cells to purple formazan
crystals. The absorbance was measured by a
microplate reader (BioTek, USA) at 570 nm
with a reference wavelength of 670 nm. The
reduction of absorbance was evaluated the
inhibition of enzyme activity observed in cells
compared to untreated (negative control) cells.
Then, the half maximal inhibitory concentration
(IC50) was expressed as the concentration of
sample caused an inhibition of 50% in enzyme
activities in cells as flows (48, 49). IC50 was
calculated by using following Equation:
IC50 (%) = 100 - [mean absorbance of extract
× 100)/mean absorbance of solvent control]
The results were generated from three
independent experiments; each experiment was
performed in triplicate.
Statistical analysis
Results were expressed as mean ± standard
deviation. Statistical comparisons were
performed with Student’s t-test. Differences
were considered significant at p < 0.05.
Results and Discussion
HPLC analysis
Table 1 shows the main components of the
extracts of Hypericum species analyzed by
HPLC and retention times, the equations and r2
values obtained from calibration curves.
H. thymbrifolium, H. spectabile and H.
pseudolaeve were enriched with chlorogenic
acid. Our results are in an agreement with those
reported in former studies in that the most
common flavonoids present in Hypericum species
under study are rutin, hyperoside, isoquercitrin,
quercitrin, and quercetin (8, 10, 12-15).
Phytochemical composition of Hypericum
species has been reported in various research
works. The main compounds that gave a
significant antioxidant activity from the ethanolic
extract of H. perforatum were identified to be
rutin and isoquercitrin as determined by HPLC,
mass spectrometry, UV/Vis spectroscopy, and
TLC (22, 23 and 51).
Total phenolic and flavonoid compounds
The yields, total phenolics and flavonoids of
methanolic extracts obtained from aerial parts
of Hypericum species are shown in Table 2.
The amount of extractable compounds ranged
from 149.1 to 213.5 mg/g DW. Among the three
extracts, H. spectabile and H. thymbrifolium
contained the highest amount of extractable
compounds while the extract of H. pseudolaeve
contained the lowest one.
The content of total phenols in extracts,
ranged between 13.3 ± 1.70 and 23.1 ± 2.37
mg GAE/g DW. No significant differences (p >
0.05) were found between the amount of total
phenolic compounds in H. thymbrifolium and
H. spectabile, while the amount of phenolics
in H. pseudolaeve were the lowest (p < 0.05).
1040
Phytochemical and Activity Studies on Hypericum Species
Table 1. Chemical compounds of methanolic extracts of Hypericum species.
Retention
time (min)
Calibration
equation values
Linear
regression
(r2)
H. spectabile
(yield%)
H. pseudolaeve
(yield%)
H. thymbrifolium
(yield%)
Pseudohypericin
4.86
y = 2.582269e +
007x + 1741.874
0.9998
0.0015 ± 0.0002
0.0131 ± 0.0004
0.0088 ± 0.0007
Hypericin
13.93
y = 6.03411e +
007x + 297.2292
0.9999
0.0070 ± 0.0001
0.0038 ± 0.0001
0.0044 ± 0.0001
Chlorogenic acid
4.33
y = 5110294x +
1490.398
0.9999
0.2357 ± 0.0269
0.3223 ± 0.0939
0.2957 ± 0.0603
Rutin
8.89
y = 1.383368e +
007x + 5188.182
0.9999
0.0083 ± 0.0004
0.1208 ± 0.0011
0.0100 ± 0.0006
Hyperoside
10.19
y = 2.849917e +
007x + 526.7023
0.9999
0.1138 ± 0.0065
0.2066 ± 0.0652
0.1681 ± 0.0381
Isoquercitrin
10.75
y = 1.671137e +
007x – 3712.788
0.9999
0.1387 ± 0.0126
0.1869 ± 0.0277
0.3038 ± 0.0661
Quercitrin
14.41
y = 1.205178e +
007 – 3518.974
0.9999
1.2863 ± 0.0554
0.2610 ± 0.0384
0.1553 ± 0.0121
Kaempferol
17.09
y = 5.183916e +
007x + 4373.856
0.9999
0.0081 ± 0.0008
0.0036 ± 0.0004
0.0007 ± 0.00003
Quercetin
17.84
y = 3.688175e +
007x + 18905.43
0.9999
0.0567 ± 0.0065
0.0592 ± 0.0052
0.0388 ± 0.0013
Amentoflavon
20.27
y = 2.207879e +
007x + 772.0972
0.9996
0.0030 ± 0.0001
0.0032 ± 0.0001
0.0027 ± 0.0001
Hyperforin
27.75
y = 6212343x
0.9997
0.0041 ± 0.0002
0.0023 ± 0.0002
Nd
Compounds
*
Values were the means of three replicates ± standard deviation, Nd: not determined.
Our results were consistent with the previous
observation on the total phenolic content of
some Hypericum species (24, 25 and 52).
The flavonoid contents varied from 10.3 ±
0.23 to 22.4 ± 0.34 mg CE/g DW which is in
accordance with the previously published data
(53). The amount of flavonoids in H. spectabile,
was higher than that of H. thymbrifolium and H.
pseudolaeve.
Antioxidant activity
In the present study we evaluated the
antioxidant activity of Hypericum species,
measuring their ability of inhibiting LPO,
reducing power, and radical scavenging
activities. For comparison, Table 3 presents the
results of the antioxidant activities, expressed as
half maximal effective concentration (EC50) and
FRAP values.
All the extracts demonstrated the ability
to inhibit LPO, which is in accordance with
the previously published data for Hypericum
species (22, 23, 25, 52, 54 and 55). The antiLPO activities of H. thymbrifolium and H.
pseudolaeve extracts were comparable (p > 0.05)
and less effective than that of H. spectabile.
However, none of the extracts were as effective
LPO inhibitor as the reference antioxidant
quercetin (0.06 ± 0.001 mg/mL).
H. thymbrifolium and H. spectabile did
not differ in their DPPH radical scavenging
activities (p > 0.05), which were higher than that
of H. pseudolaeve. Our results were consistent
with the previous observation that Hypericum
species contain radical-scavenging agents that
could directly react with and quench stable
DPPH radicals (22, 25 and 55). Nonetheless,
when compared to the the EC50 value obtained
for the quercetin (0.034 ± 0.001 mg/mL), the
DPPH scavenging activities of the extracts were
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Eroglu Ozkan E et al. / IJPR (2018), 17 (3): 1036-1046
Table 2. Total extractable compounds (EC), total phenolic compounds (PC) (as gallic acid equivalents) and total flavonoids (as catechin
equivalents) in the extracts.
Extracts
EC (mg/g DW)
PC (mg/g DW)
Flavonoid (mg/g DW)
PC/EC (%)
H. thymbrifolium
172.3
20.7 ± 2.1
a
16.9 ± 0.51
10.4
H. spectabile
213.5
23.1 ± 2.37
b
22.4 ± 0.34
10.8
H. pseudolaeve
149.1
13.3 ± 1.70b
10.3 ± 0.23c
8.9
a
a
Values were the means of three replicates ± standard deviation.
Values with different letters in the same column were significantly (p < 0.05) different.
Table 3. Antioxidant activities (EC50 values), AChE inhibitory and anti-inflammatory activities of the extracts.
EC50 (mg/mL)A
AChE*
Inhibition
(% )
COX-1*
Inhibition
(%)
COX-2*
Inhibition
(%)
Anti-LPO
DPPH
Superoxide
FRAP valueB*
(mM Fe 2+ )
H. thymbrifolium
4.39 ± 0.08a
0.622 ± 0.051a
0.641 ± 0.069a
2.58 0.036a
63.41 ± 3.29a
71.77 ± 2.93a
64.14 ± 2.32a
H. spectabile
2.80 ± 0.28b
0.567 0.028a
0.430 ± 0.006b
2.66 ± 0.031a
59.49 ± 3.14a
77.04 1.55a
72.23 ± 5.41a
H. pseudolaeve
5.41 ± 0.55a
0.916 0.036b
1.730 ± 0.060c
2.21 ± 0.015b
49.37 ± 3.48b
43.27 5.44b
52.66 ± 3.03b
Quercetin
0.06 ± 0.001c
0.034 0.001c
0.513 ± 0.013a, b
2.84 ± 0.01a, γ
Extracts
89.86 ± 0.34c, δ
Galantamine
73.53 3.57a, ε
Aspirin
Values were the means of three replicates ± standard deviation. Values with different letters in the same column were significantly (p <0.05)
different.
a
EC50 value: The effective concentration at which the antioxidant activity was 50%; DPPH and superoxide radicals were scavenged by
50%;
b
Expressed as mM ferrous ions eqivalents. *Determined at 5 mg/mL. γDetermined at 1.25 mg/mL. δDetermined at 0.05 mg/mL. εDetermined
at 0.5 mg/mL.
Antioxidant activities (EC50 values), AChE inhibitory and anti-inflammatory activities of the extracts.
Table 4. Cytotoxic potential of the extracts.
Extracts
IC50 values (mg/mL)
HeLa
NRK-52E
H. thymbrifolium
Na
Na
H. spectabile
Na
Na
1.218
0.964
H. pseudolaeve
Positive Control (5-FU): 48.012 µM for HeLa, 12.645 µM for NRK-52E.
Na: Non active.
1042
Phytochemical and Activity Studies on Hypericum Species
found to be significantly lower (p < 0.05).
The superoxide radical scavenging activities
of H. thymbrifolium and H. spectabile were
comparable to that of quercetin (0.513 ±
0.013 mg/mL). In accordance with Hunt et al.
observations, the results implied that Hypericum
extracts are superoxide scavengers and their
capacity to scavenge superoxide may contribute
to their antioxidant activity (18).
In the FRAP assay, on the basis of the standard
(Fe2+), it was found that the extracts possess
high reducing power (the range between 2.21
and 2.66 mM Fe2+) at 5 mg/mL concentration.
It was comparable (p > 0.05) to that of quercetin
(2.84 ± 0.01 mM Fe2+) at 1.25 mg/mL (Table 3).
This observation was in an agreement with other
reports (25, 53 and 55).
Our results showed that H. spectabile had
a highest degree of potency in inhibiting LPO,
demonstrated strongest reducing power and
scavenging activity against the DPPH and
superoxide radicals, indicating the highest
antioxidant potential amongst the three extract
under study. H. thymbrifolium demonstrated
similar ability to that of H. spectabile to reduce
ferric (III) iron to ferrous (II) iron and scavenge
DPPH radical (p > 0.05) but was less effective
LPO inhibitor together with H. pseudolaeve. The
least effective antioxidant was H. pseudolaeve.
These findings were in agreement with our
observation on phenolic contents of the extracts
and seemed to suggest phenolics to be important
contributors to the antioxidant activity. This result
was in agreement with previous reports that the
phenolic compounds contribute significantly to
the antioxidant activity in different Hypericum
species (19, 21-23, 40, 51, 53 and 56).
AChE inhibitory activity
Hypericum extracts were tested for their
in-vitro AChE inhibitory activities using
galantamine as a positive control. The results,
expressed as percentage inhibitions, are
summarized in Table 3.
The extracts exhibited moderate AChE
inhibitory activities (49.37- 63.41%) at 5 mg/
mL concentration. However, no plant extracts
could have greater inhibitory ability than the
positive control galantamine (89.86 ± 0.34%)
at a concentration of 0.05 mg/mL (135 µM).
Similar results were reported for H. undulatum
by Ferreira et al. (57). Wszelaki et al. reported
the lower (28%) AChE inhibition by methanolic
extract of H. perforatum at a concentration of
400 µg/mL (33).
Anti-inflammatory activity
Methanol extracts of the aerial parts of
Hypericum species under study were tested for
their anti-inflammatory activity in comparison
with aspirin used as the positive control. The
ability of the extracts to inhibit COX-1 and
COX-2 was determined by calculating percent
inhibition of prostaglandin production (Table 3).
It was found that all examined extracts exhibited
inhibiting power against COX-1 and COX-2. The
greatest anti-inflammatory effect was observed
for H. thymbrifolium and H. spectabile. It was
comparable to that of aspirin (73.5 ± 3.5%) at a
concentration of 500 µg/mL.
Because of published evidence that flavonoids
possess anti-inflammatory activity, the extract
with the highest concentrations of flavonoids was
expected to be the most anti-inflammatory (58). It
can be explained with the different phytochemical
composition of the extracts. H. spectabile, H.
thymbrifolium and H. pseudolaeve extracts
contained higher amount of chlorogenic acid. It
was reported that chlorogenic acid contributed
significantly to the anti-inflammatory activity of
the Hypericum species (59). Chlorogenic acid
represents a promising potential drug of natural
anti-inflammatory property for the development
of new drugs that may help to control oxidative
stress and consequently the inflammatory
response (60-62).
In-vitro cytotoxic activity
Hypericum species extracts were evaluated
for their cytotoxic activities in HeLa and NRK52E cell lines.
The extracts did not demonstrate significant
cytotoxic activity against both HeLa and NRK52E cell lines (IC50 ≤ 1.218) (Table 4). Also, the
responses of two cells to 5-FU were showed in
Table 4. Our results were consistent with the
previous observation that Hypericum species did
not show significant cytotoxic activity against
some tumor cell lines (23, 36, 37, 40, 63 and
64).
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Eroglu Ozkan E et al. / IJPR (2018), 17 (3): 1036-1046
Conclusion
HPLC results showed that Hypericum
species from Anatolia represent a good source
of phenolic compounds. In particular, H.
spectabile resulted endowed with high levels
of quercitrin which may support its biological
activities. In addition to this, the high content of
quercitrin has a well known anti-inflammatory
effect. As a conclusion, these results induce to
further investigate the biological properties of H.
spectabile.
Acknowledgment
This work was partially supported by
Research Fund of Istanbul University, Project
No.: 6189 (PhD Thesis Project).
References
(1) Sezik E, Yeşilada E, Honda G, Takaishi Y, Takeda Y
and Tanaka T. Traditional medicine in Turkey X. Folk
medicine in central Anatolia. J. Ethnopharmacol.
(2001) 75: 95-115.
(2) Honda G, Yeşilada E, Tabata M, Sezik E, Fujita
T, Takeda Y, Takaishi Y and Tanaka T. Traditional
medicine in Turkey VI. Folk medicine in West
Anatolia: Afyon, Kütahya, Denizli, Muğla, Aydin
provinces. J. Ethnopharmacol. (1996) 53: 75-87.
(3) Kültür Ş. Medicinal plants used in Kırklareli province
(Turkey). J. Ethnopharmacol. (2007) 111: 341-64.
(4) Tala MF, Tchakam PD, Wabo HK, Talontsi FM, Tane
P, Kuiate JR, Tapondjou LA and Laatsch H. Chemical
constituents, antimicrobial and cytotoxic activities
of Hypericum riparium (Guttiferae). Rec. Nat. Prod.
(2013) 7: 65.
(5) Muller WE. Current St John’s wort research from
mode of action to clinical efficacy. Pharmacol. Res.
(2003) 47: 101-9.
(6) Özkan EE, Özsoy N, Özhan G, Celik BÖ and Mat
A. Chemical composition and biological activities of
Hypericum pamphylicum. Ind. Crops Prod. (2013) 50:
182-9.
(7) Muller WE. St. John’s wort and its active principles
in deppression and anxiety. Birkhäuser Verlag, Basel,
Switzerland (2005) 1-4.
(8) Cirak C. Hypericin in Hypericum lydium Boiss.
growing in Turkey. Biochem. Syst. Ecol. (2006) 34:
897-9.
(9) Smelcerovic A, Spiteller M, Ligon AP, Smelcerovic Z
and Raabe N. Essential oil composition of Hypericum
L. species from Southeastern Serbia and their
chemotaxonomy. Biochem. Syst. Ecol. (2007) 35: 99113.
(10) Smelcerovic A, Zuehlke S, Spiteller M, Raabe N
and Ozen T. Phenolic constituents of 17 Hypericum
species from Turkey. Biochem. Syst. Ecol. (2008) 36:
316-9.
(11) Ayan AK and Cirak C. Hypericin and pseudohypericin
contents in some Hypericum species growing in
Turkey. Pharm. Biol. (2008) 46: 288-91.
(12) Cirak C, Radusiene J and Camas N. Pseudohypericin
and hyperforin in two Turkish Hypericum species:
Variation among plant parts and phenological stages.
Biochem. Syst. Ecol. (2008) 36: 377-82.
(13) Cirak C, Radusiene J, Janulis V, Ivanauskas L, Camas
N and Ayan AK. Phenolic constituents of Hypericum
triquetrifolium Turra (Guttiferae) growing in Turkey:
Variation among populations and plant parts. Turk. J.
Biol. (2011) 35: 449-56.
(14) Cirak C, Radusiene J, Camas N, Caliskan O and
Odabas MS. Changes in the contents of main secondary
metabolites in two Turkish Hypericum species during
plant development. Pharm. Biol. (2013) 51: 391-9.
(15) Camas N, Radusiene J, Ivanauskas L, Jakstas V,
Kayikci S and Cirak C. Chemical composition of
Hypericum species from the Taeniocarpium and
Drosanthe sections. Plant Syst. Evol. (2014) 300: 95360.
(16) Demirkiran O, Mesaik MA, Beynek H, Abbaskhan
A and Choudhary MI. Immunosupressive Phenolic
Constituents from Hypericum montbretii Spach. Rec.
Nat. Prod. (2013) 7: 210-9.
(17) Greeson JM, Sanford B and Monti DA. St. John’s
wort (Hypericum perforatum): A review of the current
pharmacological, toxicological, and clinical literature.
Psychopharmacology (2001) 153: 402-14.
(18) Hunt EJ, Lester CE, Lester EA and Tackett RL. Effect
of St. John’s wort on free radical production. Life Sci.
(2001) 69: 181-90.
(19) Conforti F, Statti GA, Tundis R, Bianchi A, Agrimonti
C, Sacchetti G, Andreotti E, Menichini F and Poli F.
Comparative chemical composition and variability
of biological activity of methanolic extracts from
Hypericum perforatum L. Nat. Prod. Res. (2005) 19:
295-303.
(20) Conforti F, Statti GA, Tundis R, Menichini F and
Houghton P. Antioxidant activity of methanolic
extract of Hypericum triquetrifolium Turra aerial part.
Fitoterapia (2002) 73: 479-83.
(21) Valentao P, Fernandes E, Carvalho F, Andrade PB,
Seabra RM and Bastos MD. Antioxidant activity
of Hypericum androsaemum infusion: Scavenging
activity against superoxide radical, hydroxyl radical
and hypochlorous acid. Biol. Pharm. Bull. (2002) 25:
1320-3.
(22) Zou YP, Lu YH and Wei DZ. Antioxidant activity of a
flavonoid-rich extract of Hypericum perforatum L. invitro. J. Agr. Food Chem. (2004) 52: 5032-9.
(23) Silva BA, Ferreres F, Malva JO and Dias ACP.
Phytochemical and antioxidant characterization of
Hypericum perforatum alcoholic extracts. Food Chem.
(2005) 90: 157-67.
1044
Phytochemical and Activity Studies on Hypericum Species
(24) Wojdylo A, Oszmianski J and Czemerys R. Antioxidant
activity and phenolic compounds in 32 selected herbs.
Food Chem. (2007) 105: 940-9.
(25) Kizil G, Kizil M, Yavuz M, Emen S and Hakimoglu
F. Antioxidant activities of ethanol extracts of
Hypericum triquetrifolium and Hypericum scabroides.
Pharm. Biol. (2008) 46: 231-42.
(26) Del Monte D, De Martino L, Marandino A, Fratianni
F, Nazzaro F and De Feo V. Phenolic content,
antimicrobial and antioxidant activities of Hypericum
perfoliatum L. Ind. Crops Prod. (2015) 74: 342-7.
(27) Savikin K, Dobric S, Tadic V and Zdunic G.
Antiinflammatory activity of ethanol extracts of
Hypericum perforatum L., H-barbatum Jacq.,
H-hirsutum L., H-richeri Vill. and H-androsaemum L.
in rats. Phytother. Res. (2007) 21: 176-80.
(28) Perazzo FF, Lima LM, Padilha MD, Rocha LM,
Sousa PJC and Carvalho JCT. Anti-inflammatory and
analgesic activities of Hypericum brasiliense (Willd)
standardized extract. Rev. Bras. Farmacogn. (2008)
18: 320-5.
(29) Tedeschi E, Menegazzi M, Margotto D, Suzuki H,
Forstermann U and Kleinert H. Anti-inflammatory
actions of St. John’s wort: Inhibition of human
inducible nitric-oxide synthase expression by
down-regulating signal transducer and activator of
transcription-1 alpha (STAT-1 alpha) activation. J.
Pharmacol. Exp. Ther. (2003) 307: 254-61.
(30) Turini ME and DuBois RN. Cyclooxygenase-2: A
therapeutic target. Annu. Rev. Med. (2002) 53: 35-57.
(31) Arruda M, Rainha N, Barreto M, Lima E and Baptista
J. Acetylcholinesterase inhibition properties of
Hypericum foliosum Aiton. Planta Med. (2010) 76:
1211.
(32) Hernandez MF, Fale PLV, Araujo MEM and
Serralheiro MLM. Acetylcholinesterase inhibition and
antioxidant activity of the water extracts of several
Hypericum species. Food Chem. (2010) 120: 1076-82.
(33) Wszelaki N, Kuciun A and Kiss AK. Screening
of traditional European herbal medicines for
acetylcholinesterase
and
butyrylcholinesterase
inhibitory activity. Acta Pharm. (2010) 60: 119-28.
(34) Altun ML, Yilmaz BS, Orhan IE and Citoglu GS.
Assessment of cholinesterase and tyrosinase inhibitory
and antioxidant effects of Hypericum perforatum L.
(St. John’s wort). Ind. Crops Prod. (2013) 43: 87-92.
(35) Zheleva-Dimitrova D, Nedialkov P and Momekov
G. Benzophenones from Hypericum elegans with
antioxidant and acetylcholinesterase inhibitory
potential. Pharmacogn. Mag. (2013) 9: 1-5.
(36) Conforti F, Loizzo MR, Statti AG and Menichini F.
Cytotoxic activity of antioxidant constituents from
Hypericum triquetrifolium Turra. Nat. Prod. Res.
(2007) 21: 42-6.
(37) Peron AP, Mariucci RG, de Almeida IV, Dusman E,
Mantovani MS and Vicentini VEP. Evaluation of the
cytotoxicity, mutagenicity and antimutagenicity of a
natural antidepressant, Hypericum perforatum L. (St.
John’s wort), on vegetal and animal test systems. BMC
Complement. Altern. Med. (2013) 13: 97.
(38) Trovato A, Raneri E, Kouladis M, Tzakou O,
Taviano MF and Galati EM. Anti-inflammatory and
analgesic activity of Hypericum empetrifolium Willd.
(Guttiferae). Farmaco (2001) 56: 455-7.
(39) Rabanal RM, Bonkanka CX, Hernandez-Perez M
and Sanchez-Mateo CC. Analgesic and topical antiinflammatory activity of Hypericum canariense L.
and Hypericum glandulosum Ait. J. Ethnopharmacol.
(2005) 96: 591-6.
(40) Ozkan EE, Ozsoy N, Ozhan G, Celik BO and Mat
A. Chemical composition and biological activities of
Hypericum pamphylicum. Ind. Crops Prod. (2013) 50:
182-9.
(41) Slinkard K and Singleton VL. Total phenol analysis:
Automation and comparison with manual methods.
Am. J. Enology Vitic. (1977) 28: 49-55.
(42) Duh PD, Tu YY and Yen GC. Antioxidant activity
of water extract of Harng Jyur (Chrysanthemum
morifolium Ramat). LWT-Food Sci. Technol. (1999)
32: 269-77.
(43) Buege JA and Aust SD. Microsomal lipid peroxidation.
Methods Enzymol. (1978) 52: 302-10.
(44) Brandwilliams W, Cuvelier ME and Berset C. Use
of a Free-Radical Method to Evaluate Antioxidant
Activity. LWT-Food Sci. Technol. (1995) 28: 25-30.
(45) Nishikimi M, Appaji N and Yagi K. The occurrence of
superoxide anion in the reaction of reduced phenazine
methosulfate and molecular oxygen. Biochem.
Biophys. Res. Commun. (1972) 46: 849-54.
(46) Benzie IFF and Strain JJ. The ferric reducing ability of
plasma (FRAP) as a measure of ‘’antioxidant power’’:
The FRAP assay. Anal. Biochem. (1996) 239: 70-6.
(47) Ellman GL, Courtney KD, Andres VJr and FeatherStone RM. A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem. Pharmacol.
(1961) 7: 88-95.
(48) Mosmann T. Rapid colorimetric assay for cellular
growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods (1983) 65:
55-63.
(49) Alley MC, Scudiero DA, Monks A, Hursey ML,
Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG,
Shoemaker RH and Boyd MR. Feasibility of drug
screening with panels of human-tumor cell-lines using
a microculture tetrazolium assay. Cancer Res. (1988)
48: 589-601.
(50) Mahavorasirikul W, Viyanant V, Chaijaroenkul
W, Itharat A and Na-Bangchang K. Cytotoxic
activity of Thai medicinal plants against human
cholangiocarcinoma, laryngeal and hepatocarcinoma
cells in-vitro. BMC Complement. Altern. Med. (2010)
10: 55.
(51) Silva BA, Malva JO and Dias ACP. St. John’s Wort
(Hypericum perforatum) extracts and isolated phenolic
compounds are effective antioxidants in several invitro models of oxidative stress. Food Chem. (2008)
110: 611-9.
(52) Ozturk N, Tuncel M and Potoglu-Erkara I. Phenolic
1045
Eroglu Ozkan E et al. / IJPR (2018), 17 (3): 1036-1046
compounds and antioxidant activities of some
Hypericum species: A comparative study with H.
perforatum. Pharm. Biol. (2009) 47: 120-7.
(53) Zheleva-Dimitrova D, Nedialkov P and Kitanov
G. Radical scavenging and antioxidant activities of
methanolic extracts from Hypericum species growing
in Bulgaria. Pharmacogn. Mag. (2010) 6: 74-8.
(54) Couladis M, Badisa RB, Baziou P, Chaudhuri
SK, Pilarinou E, Verykokidou E and Harvala C.
Antioxidant and cytotoxic activities of Hypericum
sp. on brine shrimps and human cancer cell lines.
Phytother. Res. (2002) 16: 719-22.
(55) Baris D, Kizil M, Aytekin C, Kizil G, Yavuz M,
Ceken B and Ertekin AS. In-vitro antimicrobial
and antioxidant activity of ethanol extract of three
Hypericum and three Achillea species from Turkey.
Int. J. Food Prop. (2011) 14: 339-55.
(56) Chen CL, Huang CH and Sung JM. Antioxidants in
aerial parts of Hypericum sampsonii, Hypericum
japonicum and Hypericum perforatum. Int. J. Food.
Sci. Tech. (2009) 44: 2249-55.
(57) Ferreira A, Proenca C, Serralheiro MLM and Araujo
MEM. The in-vitro screening for acetylcholinesterase
inhibition and antioxidant activity of medicinal plants
from Portugal. J. Ethnopharmacol. (2006) 108: 31-7.
(58) Calixto JB, Otuki MF and Santos ARS. Antiinflammatory compounds of plant origin. Part I.
Action on arachidonic acid pathway, nitric oxide and
nuclear factor kappa B (NF-kappa B). Planta Med.
(2003) 69: 973-83.
(59) Huang N. Identification of anti-inflammatory
constituents in Hypericum species and unveiling the
underlying mechanism in LPS-stimulated mouse
macrophages and H1N1 influenza virus infected
BALB/c mouse. Ames, Iowa State University (2011)
69.
(60) Farah A, Monteiro M, Donangelo CM and Lafay S.
Chlorogenic acids from green coffee extract are highly
bioavailable in humans. J. Nutr. (2008) 138: 2309-15.
(61) Hwang SJ, Kim YW, Park Y, Lee HJ and Kim KW.
Anti-inflammatory effects of chlorogenic acid in
lipopolysaccharide-stimulated RAW 264.7 cells.
Inflamm. Res. (2014) 63: 81-90.
(62) Azza EM, Yieldez B, Mahmoud K and Abdullatif
M. Chlorogenic acid as potential anti-inflammatory
analgesic agent: An investigation of the possible role
of nitrogen-based radicals in rats. IJPTS (2011) 1:
24-33.
(63) Roscetti G, Franzese O, Comandini A and Bonmassar
E. Cytotoxic activity of Hypericum perforatum L.
on K562 erythroleukemic cells: Differential effects
between methanolic extract and hypericin. Phytother.
Res. (2004) 18: 66-72.
(64) Moon HI. Antiplasmodial and cytotoxic activity of
phloroglucinol derivatives from Hypericum erectum
Thunb. Phytother. Res. (2010) 24: 941-4.
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