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In vitro prospective effects of various traditional herbal coffees consumed in Anatolia
linked to neurodegeneration
Nazim Sekeroglu
a
, F. Sezer Senol
b
, Ilkay Erdogan Orhan
b,c,
⁎, A. Rifat Gulpinar
d
, Murat Kartal
d
, Bilge Sener
b
a
Department of Biology, Faculty of Art and Sciences, Kilis 7, Aralik University, 79000 Kilis, Turkey
b
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
c
Pharmacognosy and Pharmaceutical Botany Unit, Faculty of Pharmacy, Eastern Mediterranean University, GaziMagosa, North Cyprus, via Mersin10, Turkey
d
Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100 Ankara, Turkey
abstractarticle info
Article history:
Received 24 August 2011
Accepted 5 October 2011
Available online 19 October 2011
Keywords:
Traditional coffees
Cholinesterase inhibition
Antioxidant activity
Fatty oil
Various herbal coffee varieties are consumed traditionally in Turkey. In the current study, the ethanol extracts
of the coffee and seed samples obtained from Gundelia tournefortii (tumble thistle), Nigella sativa (black
cumin), Phoenix dactylifera (date), and Ceratonia siliqua (carob) as well as a sample of instant coffee (Nescafe®,
green blend) were tested against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase,
the enzymes connected to neurodegeneration. Since oxidative stress is strongly associated with neurodegen-
eration, antioxidant activity of the extracts was also determined. Antioxidant activity of the extracts was
measured using anti-radical and metal-related methods. Total phenol and flavonoid contents were calculated
using Folin–Ciocalteau's and AlCl
3
reagents, respectively. Fatty acid compositions of the seed oils of tumble
thistle and black seed were analyzed by GC–MS. Our results showed that the date sample exerted the highest
AChE and BChE inhibition at 300 μgmL
−1
(52.96% and 83.22%, respectively).
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Drinking of coffee is an important part of Turkish culture, one of the
valuable traditions of daily life, and a way of socializing since centuries.
Turkish coffee was first introduced during the Ottoman Empire in 1543
andpeoplearoundtheworldhaveheardofthefameofTurkishcoffee
(Birsel, 1991). It is also a symbol of Turkish hospitality to offer coffee to
the guests. Although the Turkish coffee prepared from the seeds of Coffea
arabica L.(Rubiaceae)isthemostfamous,therearesomeothertypesof
herbal coffee traditionally consumed in Turkey. Among them, “kenger
coffee”obtained from Gundelia tournefortii L. (GT, tumble thistle, tumble-
weed, Asteraceae), “çörek otu coffee”from Nigella sativa L. (NS, black
cumin, Ranunculaceae), “hurma coffee”from Phoenix dactylifera L. (PD,
date, Arecaceae), and “keçiboynuzu coffee”from Ceratonia siliqua L.
(CS, carob, Fabaceae) are some of the herbal coffees consumed in Turkey.
Although P. dactylifera is not grown naturally in Turkey and usually
obtained through import from Arab countries, this plant has been
recorded to be used for memory enhancing purpose traditionally in our
country (I.E.O. —personal communication). The above-mentioned
coffees are obtained in the powdered form after roasting and grinding
of their seeds, which gives extra flavor and aroma. Making style of
these coffees is similar to that of the Turkish coffee, which is explained
as follows. The powdered crude coffees are measured in a certain
amount, mixed with water in a special coffee pot, stirred occasionally,
and let it boil slowly until a foam is occurred on the top, which takes
about a few minutes.
Neurodegeneration is a complex and multifactorial procedure in
human brain and, unfortunately, the prevalence of neurodegenerative
disorders such as Alzheimer's disease (AD) and Parkinson's disease
(PD) is increasing. On the other hand, effective treatments and medica-
tions are still absent to seize the disease. For AD, the most applied medi-
cation class is cholinesterase inhibitors (Orhan, Orhan, Subutay-Oztekin,
Ak, & Sener, 2009). Oxidized dopamine metabolites having a crucial func-
tion in the degeneration of nigrostriatal dopaminergic neurons in PD is
induced by tyrosinase (TYRO) through its oxidase activity and, therefore,
inhibition of TYRO is important in prevention of PD (Hasegawa, 2010).
Another factor contributing to pathology of neurodegenerative diseases
is oxidative stress, which leads to neuronal death (Halliwell, 2006).
In view of the fact that the aforementioned coffees are consumed
commonly in Anatolia, especially in the southern part, and folkloric
use of date is recorded for memory-improvement, we decided to in-
vestigate the ethanol extracts of the aforementioned coffee samples
along with their source seeds for their potential effects in neurode-
generation via enzyme inhibition against acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE), and tyrosinase (TYRO)
using ELISA microplate reader. In addition, a sample of commercial
instant coffee brand (Nescafe®, green blend, GB) was also tested
with other four traditional coffee varieties for a comparative purpose.
Antioxidant activity of the extracts was measured using radical scav-
enging activity tests and metal-related tests including metal-
Food Research International 45 (2012) 197–203
⁎Corresponding author at: Department of Pharmacognosy, Faculty of Pharmacy,
Gazi University, 06330 Ankara, Turkey. Tel.: +90 312 2023186; fax: + 90 312 2235018.
E-mail addresses: iorhan@gazi.edu.tr,ilkay.orhan@emu.edu.tr (I.E. Orhan).
0963-9969/$ –see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.10.008
Contents lists available at SciVerse ScienceDirect
Food Research International
journal homepage: www.elsevier.com/locate/foodres
Author's personal copy
chelation capacity, ferric-reducing antioxidant power (FRAP), and
phosphomolybdenum reducing power (PRAP). Total phenol and
flavonoid contents in the extracts were calculated using Folin–
Ciocalteau and AlCl
3
reagents, respectively. The fatty oils obtained
from the seeds of the tumble thistle and black cumin samples
were analyzed for their fatty acids by gas chromatography–mass
spectrometry (GC–MS).
2. Materials and methods
2.1. Coffee and seed materials
The coffee and seed samples of G. tournefortii (GT, tumble thistle),
N. sativa (NS, black cumin), P. dactylifera (PD, date), and C. siliqua (CS,
carob) were kindly provided by Sekeroglu Baharatcilik Company in
Kilis province (Turkey) in 2011. Instant coffee brand (Nescafe®,
green blend, GB) was purchased from a supermarket in Ankara
province (Turkey), in 2011.
2.2. Extraction procedure
The seed samples were ground by a mechanic grinder and weighed
precisely in a digital balance (Shimadzu). Then, each coffee variety al-
ready in powdered form was also weighed accurately and the coffee
and seed samples were extracted with ethanol (80%) during 3 days at
room temperature by shaking occasionally. Since the solubility of
the powdered seeds and coffee samples was not good in water at all,
the extracts were prepared with ethanol. The ethanolic phases
were filtered and evaporated in vacuo until dryness to give the
crude extracts. Percentage yields of the ethanol extracts (w/w) are
given as follows; GT-seed: 0.97, GT-coffee: 6.44, NS-seed: 2.35, NS-
coffee: 4.33, CS-seed: 4.88, CS-coffee: 9.64, PD-seed: 2.65, PD-coffee:
3.12, GB-coffee: 14.74.
The seed samples belonging to four plant species were separately
subjected to continuous extraction technique using Soxhlet appara-
tus, which was extracted with n-hexane for 8 h. Then, organic phases
were filtrated until dryness to give the fatty oils. Percentage yields of
the fatty oils (w/w) are given as follows; GT: 5.93, NS: 7.31, PD: 0.06,
CS: 0.09.
2.3. Determination of total phenol and flavonoid contents in the extracts
Phenolic compounds were determined in accordance with Folin–
Ciocalteau's method (Singleton & Rossi, 1965). In brief, a number of
dilutions of gallic acid were obtained to prepare a calibration curve.
The extracts and gallic acid dilutions were mixed with 750 μLof
Folin–Ciocalteau's reagent and 600 μL of sodium carbonate in test
tubes. The tubes were then vortexed and incubated at 40 °C for
30 min. Afterward, absorption was measured at 760 nm at a Unico
4802 UV–visible double beam spectrophotometer (USA). Total flavo-
noid content of the extracts was calculated by aluminum chloride col-
orimetric method (Woisky & Salatino, 1998). To sum up, a number of
dilutions of quercetin were obtained to prepare a calibration
curve. Then, the extracts and quercetin dilutions were mixed with
95% ethanol, aluminum chloride reagent, 0.1 mL of sodium acetate
as well as distilled water. Following incubation for 30 min at room
temperature, absorbance of the reaction mixtures was measured at
wavelength of 415 nm with a Unico 4802 UV–visible double beam
spectrophotometer (USA). The total phenol and flavonoid contents
of the extracts were expressed as gallic acid and quercetin equiva-
lents (mg g
−1
extract), respectively.
2.4. AChE and BChE inhibitory activity assays
AChE and BChE inhibitory activity was measured by slightly mod-
ified spectrophotometric method of Ellman, Courtney, Andres, and
Featherstone (1961). Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma,
St. Louis, MO, USA) and horse serum BChE (EC 3.1.1.8, Sigma, St.
Louis, MO, USA) were used, while acetylthiocholine iodide and butyr-
ylthiocholine chloride (Sigma, St. Louis, MO, USA) were employed as
substrates of the reaction, respectively. 5,5′-Dithio-bis(2-nitroben-
zoic)acid (DTNB, Sigma, St. Louis, MO, USA) was used for the mea-
surement of the anticholinesterase activity. All reagents and
conditions were same as described in our previous publication
(Senol et al., 2010). Hydrolysis of acetylthiocholine iodide/butyrylthio-
choline chloride was monitored by the formation of the yellow 5-thio-
2-nitrobenzoate anion as a result of the reaction of DTNB with thiocho-
lines, catalyzed by enzymes at 412 nm utilizing a 96-well microplate
reader (VersaMax Molecular Devices, USA). The measurements and cal-
culations were evaluated by using Softmax PRO 4.3.2.LS software. Per-
centage of inhibition of AChE/BChE was determined by comparison of
rates of reaction of samples relative to blank (ethanol in phosphate
buffer pH= 8) using the formula (E−S)/E×100, whereEis the activity
of enzyme without test sample and Sis the activity of enzyme with test
sample. The experiments were done in triplicate. Galanthamine (Sigma,
St. Louis, MO, USA) was used as the reference.
2.5. Tyrosinase inhibitory activity assay
Inhibition of tyrosinase (EC 1.14.1.8.1, 30 U, mushroom tyrosinase,
Sigma) was determined using the modified dopachrome method with
L-DOPA as substrate (Masuda, Yamashita, Takeda, & Yonemori, 2005).
Assays were conducted in a 96-well microplate using ELISA micro-
plate reader (VersaMax Molecular Devices, USA) to measure absor-
bance at 475 nm. An aliquot of the extracts dissolved in DMSO with
80 μL of phosphate buffer (pH 6.8), 40 μL of tyrosinase, and 40 μLof
L-DOPA were put in each well. Results were compared with
control (DMSO). Baicalein (Sigma, St. Louis, MO, USA) was used as
the reference. The percentage tyrosinase inhibition (I%) was calculated
as follows:
I% ¼Absorbancecontrol −Absorbancesample
=Absorbancecontrol 100:
2.6. Antioxidant activity by radical-formation methods
2.6.1. DPPH radical scavenging activity
The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
activity was determined by the method of Blois (1958). The samples
and references dissolved in methanol (75%) were mixed with DPPH so-
lution (1.5× 10
−4
M). Remaining DPPH amount was measured at
520 nm using a Unico 4802 UV–visible double beam spectrophotometer
(USA). Gallic acid was employed as the reference. Inhibition of DPPH in
percent (I%) was calculated as given below:
I% ¼Ablank −Asample
=Ablank
hi
100;
where A
blank
is the absorbance of the control reaction (containing
all reagents except the test sample), and A
sample
is the absorbance of
the extracts/reference. Analyses were run in triplicate and the results
were expressed as average values with S.E.M. (Standard error mean).
2.6.2. DMPD radical scavenging activity
Principal of the assay is based on reduction of the purple-colored
radical DMPD
+
(N,N-dimethyl-p-phenylenediamine) (Schlesier,
Harvat, Bohm, & Bitsch, 2002). According to the method, a reagent
comprising of 100 mM DMPD, 0.1 M acetate buffer (pH=5.25),
and 0.05 M ferric chloride solution, which led to formation of DMPD
radical, was freshly prepared and the reagent was equilibrated to an
absorbance of 0.900± 0.100 at 505 nm. Then, the reagent was mixed
up with 50 μL of the extract dilutions and absorbance was taken at
198 N. Sekeroglu et al. / Food Research International 45 (2012) 197–203
Author's personal copy
505 nm using a Unico 4802 UV–visible double beam spectrophotometer
(USA). Quercetin was employed as the reference and the experiments
were done in triplicate. The results were calculated according to the
same formula given for DPPH radical scavenging test and expressed as
average values with S.E.M. (Standard error mean).
2.7. Antioxidant activity by metal-related methods
2.7.1. Metal-chelation effect
The metal-chelating effect of the extracts via ferrous ion was esti-
mated by the method of Chua, Tung, and Chang (2008). Briefly, vari-
ous dilutions of the extracts were incubated with 2 mM FeCl
2
solution. The reaction was initiated by the addition of 5 mM ferrozine
into the mixture and left standing at ambient temperature for 10 min.
The absorbance of the reaction mixture was measured at 562 nm
using a Unico 4802 UV–visible double beam spectrophotometer
(USA). The ratio of inhibition of ferrozine–Fe
2+
complex formation
was calculated as follows:
Chelation%¼Ablank−Asample
=Ablank
hi
100;
where A
blank
is the absorbance of the control reaction (containing
only FeCl
2
and ferrozine), and A
sample
is the absorbance of the
extracts/reference. Analyses were run in triplicate and the results
were expressed as average values with S.E.M. The reference was
ethylenediamine tetraacetic acid (EDTA) in this assay.
2.7.2. Ferric-reducing antioxidant power (FRAP) assay
The ferric-reducing power of the extracts was tested using the
assay of Oyaizu (1986). Different concentrations of the extracts
were mixed with 2.5 mL of phosphate buffer (pH 6.6) and 2.5 mL of
potassium ferricyanide. Later, the mixture was incubated at 50 °C
for 20 min and, then, trichloroacetic acid (10%) was added. After the
mixture was shaken vigorously, this solution was mixed with distilled
water and ferric chloride (0.1%). After 30 min of incubation, absor-
bance was read at 700 nm using a Unico 4802 UV–visible double
beam spectrophotometer (USA). Analyses were achieved in triplicate.
Increased absorbance of the reaction meant increased reducing
power and compared to that of chlorogenic acid as the reference.
2.7.3. Phosphomolybdenum-reducing antioxidant power (PRAP) assay
In order to perform PRAP assays on the extracts, each dilution was
mixed 10% phosphomolybdic acid solution in ethanol (w/v) (Falcioni
et al., 2002). The solution was subsequently subjected to incubation
at 80 °C for 30 min and the absorbance was read at 600 nm using a
Unico 4802 UV–visible double beam spectrophotometer (USA). An-
alyses were run in triplicate. Increased absorbance of the reaction
meant increased reducing power and compared to that of querce-
tin as the reference.
2.8. GC and GC–MS conditions for fatty oil analysis
GC analysis was performed on an Agilent 6890N Network GC sys-
tem, under the following conditions; Column: HP Innowax Capillary
(60.0 m×0.25 mm × 0.25 μm); oven temperature program: The column
was held initially at 60 °C for 3 min after injection, then, increased to
185°C with 10°Cmin
−1
heating ramp for 1 min, and increased to
200 °C with 5 °C min
−1
heating ramp for 10 min. Later, the final temper-
ature was increased to 220 °C with 5 °C min
−1
heating ramp for 20 min;
injector temperature: 250 °C; detector (FID) temperature: 275 °C; carri-
er gas: Helium; inlet pressure: 40.65 psi; linear gas velocity: 39 cm s
−1
;
column flow rate: 2.7 mL min
−1
; split ratio: 20:1; injection volume:
1μL.
GC–MS analysis was performed on an Agilent 6890N Network GC sys-
tem combined with Agilent 5973 Network Mass Selective Detector. The
GC conditions were as follows: column: HP Innowax Capillary
(60.0 m× 0.25 mm ×0.25 μm); oven temperature program: The column
held initially at 60 °C for 3 min after injection, then increased to 185 °C
with 10 °C min
−1
heating ramp for 1 min and increased to 200 °C with
5°Cmin
−1
heating ramp for 10 min. Then, the final temperature was in-
creased to 220 °C with 5 °C min
−1
heating ramp for 20 min; injector
temperature: 250 °C; carrier gas: Helium; inlet pressure: 40.65 psi; linear
gas velocity: 44 cm s
−1
;columnflow: 2.9 mL min
−1
;splitratio:20:1;
injection volume: 1.0 μL. MS conditions were regulated as follows:
ionization energy: 70 eV; ion source temperature: 280 °C; interface
temperature: 250 °C; mass range: 35–450 amu.
Identification of the components was assigned by comparison of
their retention times and mass spectra with corresponding data
from reference compounds and by comparison of their mass spectra
with Wiley and Nist libraries.
3. Results
3.1. Enzyme inhibitory activities of the extracts
AChE and BChE inhibitory activity of the ethanol extracts obtained
from the seed and coffee samples of GT, NS, PD, CS, and GB is shown
in Fig. 1. Among the extracts tested at 75, 150, and 300 μgmL
−1
; the
seed extract of PD and the coffee extract of GB were found to have the
highest inhibition against AChE (52.96± 1.72% and 52.26 ± 3.79% at
300 μgmL
−1
, respectively). In BChE inhibitory assay, the most effec-
tive extracts belonged to the seeds of PD and GT (83.22 ± 2.22% and
41.07± 0.55% at 300 μgmL
−1
, respectively). The extracts usually dis-
played either no or weak inhibition towards TYRO. In this assay, the
highest inhibition was caused by the seed extract of NS and the
coffee extract of GT (36.30 ±1.02 and 34.70 ±3.59% at 200 μgmL
−1
,
respectively) (Fig. 2).
3.2. Antioxidant activity of the extracts
The extracts were screened for their anti-radical effect against DPPH
and DMPD radicals as shown in Table 1.Theseedandcoffeeextractsof
PD, CS, and GB exerted a remarkable radical scavenging effect over 90%
against DPPH. However, all of the extracts had a low effect below
40% in DMPD scavenging assay. In FRAP and PRAP tests (Table 2), the
seed extract of PD and the coffee extract of GB gave the highest absor-
bance, in which the higher absorbance is indicative of higher antioxidant
power. Our results obtained from the metal-chelation capacity test
showed that the extracts had moderate level of activity where the seed
extract of GT possessed the best metal-chelation capacity (33.57 ±
1.75% at 3000 μgmL
−1
)amongtheextractstested(Fig. 3).
3.3. Total phenol and flavonoid contents of the extracts
Total phenol content of the extracts was calculated according to
the equation (y= 0.13879x + 0.0508, r
2
=0.9928) as gallic acid
equivalent (mg g
−1
extract), whilst their total flavonoid contents
were determined in accordance with the equation (y = 5.6661x +
0.0903, r
2
=0.9994) obtained by calibration curves as quercetin
equivalent (mg g
−1
extract) (Table 1). According to the results; the
GB-coffee (90.23 ±1.19 mg g
−1
), PD-coffee (76.30 ± 4.59 mg g
−1
),
and PD-seed (74.86 ±2.89 mg g
−1
) extracts were the richest in
total phenol contents.
3.4. Fatty acid compositions
Fatty acid compositions of the seed oils of GT and NS were
analyzed by GC–MS, whereas the seed oils of PD and CS could not
be analyzed to their very low oil yield. Both of the analyzed oils
contained linoleic acid as the dominant fatty acid, followed by
oleic acid (Table 3).
199N. Sekeroglu et al. / Food Research International 45 (2012) 197–203
Author's personal copy
4. Discussion
Our findings revealed that, among the tested extracts, the seed ex-
tracts except for CS showed higher inhibition against AChE and BChE
than their coffee products. However, in metal-chelation test, the re-
sults were vice versa. The seed extract of PD and the coffee extract
of GB were the most active extracts in the antioxidant assays and
also were richest extracts in terms of total phenol content. Therefore,
0
10
20
30
40
50
60
70
80
90
50 100 200
Fig. 2. TYRO inhibitory activity (inhibition% ±S.E.M.) of the ethanol extracts of the coffee and seed samples (GT: Gundelia tournefortii,NS: Nigella sativa, PD: Phoenix dactylifera, CS:
Ceratonia siliqua, GB: Green blend-Nescafe®, concentrations in μgmL
−1
, alpha-kojic acid at 100 μgmL
−1
).
0
10
20
30
40
50
60
70
80
90
100
75 150 300
0
10
20
30
40
50
60
70
80
90
100
75 150 300
A
B
Fig. 1. AChE (A) and BChE (B) inhibitory activity (inhibition% ± S.E.M.) of the ethanol extracts of the coffe e and seed samples (GT: Gundelia tournefortii,NS: Nigella sativa,PD:
Phoenix dactylifera,CS:Ceratonia siliqua, GB: Green blend-Nescafe®, concentrations in μgmL
−1
, galanthamine at 100 μgmL
−1
).
200 N. Sekeroglu et al. / Food Research International 45 (2012) 197–203
Author's personal copy
high antioxidant activity of these extracts could be attributed to their
rich phenol contents. Consistent with our findings, the date was stat-
ed to be rich in antioxidants of phenolic and carotenoid derivatives in
previous studies (Al Farsi & Lee, 2008; Baliga, Baliga, Kandathil, Bhat,
& Vayalil, 2011; Reddy, Sreeramulu, & Raghunath, 2010). The current
results confirmed the local knowledge on PD for memory-enhancement
depending on high cholinesterase inhibitory and antioxidant effects. In a
study by Juhaimi et al. (in press), the seeds of seven P. dactylifera (date)
varieties collected from Saudi Arabia were for analyzed for their fatty
acid contents and the major fatty acid was established as oleic acid,
whereas we found linoleic acid as the main fatty acid in our date seed
oil. In another study on several date fruit cultivars of Tunisian origin
(Chaira et al., 2009), the high antioxidant activity of the cultivars was at-
tributed to their rich total flavonoid contents as quercetin equivalent.
However, in our study, date seeds did not have total flavonoid content
(Table 1). These phytochemical differences might be influenced by
origin of cultivation as well as some other factors. In this regard,
Al-Farsi, Alasalvar, Morris, Baron, and Shahidi (2005) investigated
the antioxidant activity and phenolic ingredients of the fresh and
sun-dried dates of three native varieties from Oman and concluded
that sun-drying process caused a notable loss in antioxidant constit-
uents of the date varieties.
On the other hand, our literature survey indicated that there has
been no report on the cholinesterase inhibitory effect of P. dactylifera
(date) up to date. However, a few studies related to neuroprotection
have been reported on date extracts by different methods, which
support our findings and folkloric use of the date. For instance; Pujari,
Vyawahare, and Kagathara (2011) reported strong neuroprotective
activity of the methanolic extract of the date fruits (purchased in
India) in rats with cerebral ischemia induced by occluding bilateral
common carotid arteries. The aqueous date fruit extract from Iran
was also examined for its neuroprotective action in rats with neuro-
nal damage induced by cerebral ischemia and they concluded that
the efficiency of the extract in focal cerebral ischemia is presumably
due to its antioxidant property (Majid, Marzieh, Shahriar, Zahed, &
Pari, 2008).
C. siliqua (CS) was one of the most effective extracts in the antiox-
idant assays performed herein and its coffee extract also exhibited a
moderate level of inhibition against the enzymes tested. Although
there has been no report on cholinesterase inhibitory activity of this
plant so far, several studies (Custódio et al., 2011; Kumazawa et al.,
2002) have described a remarkable antioxidant effect of different
parts of CS, which is in accordance with our data.
NS is an important food and medicinal plant in Turkey, whose ancient
seeds were found to be used as remedy by the Hittites in Anatolia about
3600 years ago (Salih, Sipahi, & Oybak-Donmez, 2009). The seed extract
of NS had the highest TYRO inhibitory effect in our assay, while its seed
and coffee extracts exerted a moderate level of antioxidant activity,
which was correlated with its low total phenol content. Although various
studies have been performed on antioxidant activity of black cumin ex-
tracts (Ilhan, Gurel, Armutcu, Kamisli, & Iraz, 2005; Mariod, Ibrahim,
Ismail, & Ismail, 2009), we have not encountered any report
Table 1
Total phenol and flavonoid contents and antiradical activity of the ethanol extracts of the seed and coffee samples.
Extracts Total phenol
content
a
±
S.E.M.
b
Total flavonoid
content
c
±S.E.M.
Inhibition%±S.E.M. against DPPH radical Inhibition%±S.E.M. against DMPD radical
750 μgmL
−1
1500 μgmL
−1
3000 μgmL
−1
750 μgmL
−1
1500 μgmL
−1
3000 μgmL
−1
GT-Seed 25.99±2.04 5.89±0.12 27.74±0.83 45.30±1.29 78.52± 3.97 –
d
––
GT-Coffee 12.90 ± 0.17 2.39 ± 0.83 11.75 ± 0.18 16.25 ± 0.46 25.00 ± 3.42 –– –
NS-Seed 19.62±2.21 1.48±0.46 13.05±0.37 17.75±1.11 28.20± 0.74 –– –
NS-Coffee 18.78 ± 0.34 6.66 ± 2.12 14.16 ± 0.09 20.89 ± 1.85 32.51 ± 1.29 –– –
PD-Seed 74.86± 2.89 –93.80 ± 0.09 93.67 ± 0.09 94.06 ± 0.09 9.90 ±2.33 20.57±0.76 35.62 ± 2.65
PD-Coffee 76.30 ± 4.59 3.92 ±0.33 42.69±1.85 73.37±0.97 92.23± 0.28 –– 8.43 ± 2.63
CS-Seed 19.14± 1.53 –35.90 ± 1.66 64.62±1.29 94.26±0.55 9.05 ± 0.44 18.76 ± 2.01 33.90 ± 1.15
CS-Coffee 33.07 ± 2.21 –56.85 ± 0.28 93.34 ± 0.92 94.97 ± 0.09 16.38 ± 1.47 26.00 ± 0.76 38.95 ± 1.35
GB-Coffee 90.23±1.19 15.37 ± 0.12 86.42±2.77 92.36±0.09 93.15± 0.09 27.24 ± 2.29 32.76 ± 0.82 38.67 ± 2.86
Gallic acid (reference for DPPH scavenging activity) 93.05 ±0.27 at 2000 μgmL
−1
Quercetin (reference for DMPD scavenging activity) 68.32±0.67 at 2000 μgmL
−1
a
Data expressed in mg equivalent of gallic acid (GAE) to 1 g of extract.
b
Standard error mean.
c
Data expressed in mg equivalent of quercetin to 1 g of extract.
d
No activity.
Table 2
Ferric- (FRAP) and phosphomolybdenum-reducing power (PRAP) of the ethanol extracts of the seed and coffee samples.
Extracts Ferric-reducing antioxidant power
a
(FRAP) (Absorbance at 700 nm ±S.E.M.
b
)
Phosphomolybdenum-reducing antioxidant power
a
(PRAP) (Absorbance at 600 nm ±S.E.M.)
750 μgmL
−1
1500 μgmL
−1
3000 μgmL
−1
750 μgmL
−1
1500 μgmL
−1
3000 μgmL
−1
GT-Seed 0.312±0.011 0.508±0.04 0.853± 0.011 0.209±0.018 0.215 ±0.06 0.284± 0.027
GT-Coffee 0.214 ± 0.006 0.310 ± 0.02 0.540 ± 0.017 0.120± 0.013 0.173 ± 0.001 0.204± 0.001
NS-Seed 0.218±0.001 0.316±0.003 0.512± 0.016 0.185± 0.027 0.210 ± 0.033 0.218± 0.001
NS-Coffee 0.349 ± 0.006 0.511 ± 0.01 0.877 ± 0.013 0.189± 0.023 0.254 ± 0.004 0.336± 0.014
PD-Seed 0.676± 0.012 0.993 ± 0.008 1.333± 0.004 0.265 ±0.001 0.337 ±0.001 0.552± 0.026
PD-Coffee 0.364 ± 0.001 0.545 ± 0.008 1.245± 0.021 0.188± 0.033 0.212 ± 0.002 0.432± 0.008
CS-Seed 0.263± 0.041 0.415 ± 0.003 0.758±0.035 0.194± 0.008 0.211 ± 0.003 0.248± 0.024
CS-Coffee 0.408±0.014 0.633±0.013 1.050 ± 0.003 0.178±0.011 0.213 ±0.001 0.274 ± 0.010
GB-Coffee 0.685 ± 0.036 1.158 ± 0.021 2.120 ± 0.091 0.297 ±0.025 0.317 ±0.013 0.512 ± 0.024
Chlorogenic acid (reference for FRAP) 3.547±0.006 at 1000 μgmL
−1
Quercetin (reference for PRAP) 0.819±0.001 at 1000 μgmL
−1
a
Higher absorbance indicates greater antioxidant activity.
b
Standard error mean (n=3).
201N. Sekeroglu et al. / Food Research International 45 (2012) 197–203
Author's personal copy
evaluating cholinesterase and tyrosinase inhibitory of the black
cumin. On the other hand, thymoquinone, a main constituent in
NS, was found to protect primary dopaminergic neurons against
MPP(+) and rotenone relevant to Parkinson's disease (Radad,
Moldzio, Taha, & Rausch, 2009) and, thus, TYRO inhibitory activity
of the seed extract of NS could be searched since thymoquinone
might be inhibiting this enzyme and being responsible for inhibitory
effect of this extract.
Previous papers on fatty acid composition of the black cumin oil,
in which linoleic acid was established as the main fatty acid, were
in accordance with our findings (Kokdil & Yilmaz, 2005; Nergiz &
Otles, 1993).
GT is a well-known medicinal and edible plant, particularly in
the southern part of Turkey and also one of the less-searched me-
dicinal plants in the world. In this study, the seed extract of GT
was shown to display enzyme inhibition to some extent and a
notable radical scavenging effect against DPPH. Its coffee extract
had also remarkable TYRO inhibitory and metal-chelation capacity.
The seeds of GT growing in Turkey were earlier investigated by
Coruh, Sagdicoglu-Celep, Ozgokce, and Iscan (2007) using DPPH
radical scavenging and lipid peroxidation inhibition methods and
the authors stated that the seed extract exhibited a noteworthy
antioxidant activity, which is in agreement with our findings. On
the other hand, we report herein the fatty acid composition of the
seed oil of GT for the first time since we have not found any previ-
ous data on its fatty acids. Our results confirm its rich content in
linoleic and oleic acids, which are essential unsaturated fatty acids
beneficial for health.
We also examined the ethanolic coffee extract of a commercial
coffee brand prepared from green coffee beans and found to have
that the extract had a notable AChE inhibiting effect as well as potent
antioxidant activity in the assays applied. The extracts also possessed
the highest total phenol content, which is attributable to its high
antioxidant effect, as reported formerly (Naidu, Sulochanamma,
Sampathu, & Srinivas, 2008).
In the current study, our aim was also to compare the tested bio-
activities and total phenol and flavonoid contents of the seed and cof-
fee extracts and to find out if the roasting process could influence
bioactivity and phytochemical contents. Our evaluation on these ex-
tracts revealed that no clear conclusion could be given according to
these results as in some cases, the seed extracts are more active and
vice versa. In our recent similar study (Erdogan Orhan et al., 2012),
we assessed in vitro neuroprotective activity of various terebinth cof-
fee brands obtained from the fruits of Pistacia terebinthus in Turkey
using the same experimental models herein and found that the tere-
binth coffees produced after roasting of the powdered fruits exhibited
a greater activity in these tests as compared to the fruits per se. The
roasting process caused an elevation especially in the antioxidant ac-
tivity due to the increase in total phenol and flavonoid amounts. Rel-
evantly, Del Castillo, Ames, and Gordon (2002) also stated that a
roasting time of 10 min is ultimate for producing coffee with optimal
oxygen scavenging and chain breaking activities in vitro. However,
the roasting process applied in preparation of the coffee varieties in-
vestigated herein did not have a distinct increasing or decreasing
effect.
5. Conclusion
The results obtained in this study revealed that, among the tested
extracts, the seed extract of PD has a significant effect in cholinester-
ase inhibition and antioxidant assays, which confirms the claimed
folkloric utilization of the plant by the applied methods. To the best
of our knowledge, this is the first study describing cholinesterase
and tyrosinase inhibitory activity of the seed and coffee extracts of
GT, NS, PD, and CS as well as antioxidant activity of the coffee extracts.
We also report the fatty acid composition of the seed oil from GT for
the first time in this work. Our preferential research is in progress
to elucidate active constituents of P. dactylifera with strong anticho-
linesterase and antioxidant activity.
Acknowledgment
F.S. Senol expresses her genuine gratitude to the Scientific and
Technological Research Council of Turkey (TUBITAK) for the scholar-
ship provided for her Ph.D. program.
0
10
20
30
40
50
60
70
80
750 1500 3000
Fig. 3. Metal-chelation capacity (inhibition% ±S.E.M.) of the ethanol extracts of the coffee and seed samples (GT: Gundelia tournefortii,NS: Nigella sativa, PD: Phoenix dactylifera, CS:
Ceratonia siliqua, GB: Green blend-Nescafe®, concentrations in μgmL
−1
, EDTA at 100 μgmL
−1
).
Table 3
Fatty acid composition of the seed oils of GT and NS
a
.
Fatty acid composition (%
b
)
Palmitic
acid
Stearic
acid
Oleic acid Linoleic acid Sum of
areas
Retention time
(min)
22.1 27.5 28.2 30.1
GT 9.71±0.39 2.04±0.18 28.98±0.99 56.25 ± 2.07 95.697
NS 13.31 ± 0.39 3.36 ± 0.15 25.57 ± 0.05 54.347 ±0.42 96.587
a
The seed oils of PD and CS could not be analyzed by GC–MS due to very low oil yield.
b
%calculatedfromFIDdata.
202 N. Sekeroglu et al. / Food Research International 45 (2012) 197–203
Author's personal copy
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